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EC number: 232-107-5 | CAS number: 7787-20-4
- 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
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- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
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- 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
Skin irritation. Key study. Method according to OECD guideline 439, GLP study. The test item is not irritant since the mean percent viability of the treated tissues was 100.19% in the RHE test.
Eye irritation. Key study. Method according to OECD guideline 437, GLP study. The test item cannot be predicted as not classified for eye irritation/serious eye damage or as causing serious eye damage with the BCOP test method.
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:
- 23 February 2018 - 01 March 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Test system:
- human skin model
- Remarks:
- EpiSkinTM
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other: not specified
- Source strain:
- not specified
- Justification for test system used:
- The assessment of skin irritation has typically involved the use of laboratory animals (OECD TG 404). In relation to animal welfare concerns, TG 404 recommends the use of a tiered testing strategy for the determination of skin corrosion and irritation which includes the use of validated in vitro or ex vivo test methods avoiding pain and suffering of animals.
One of the validated in vitro test methods adopted by the OECD TG 439 makes use of reconstructed human epidermis (RhE) which closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis - Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM kit
- Tissue batch number(s): 18-EKIN-009
- Delivery date: 27 February 2018
- Expiration date: 05 March 2018
- Date of initiation of testing: 23 February 2018
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37ºC
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: PBS, volumen no specified
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours at 37°C, 5% CO2
- Spectrophotometer: TECAN Infinite® M200 Microplate Reader
- Wavelength: 570 nm
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Historical negative control mean OD range = 0.58630-1.13620 (15 min exposure).
- Histology scoring: 21.5 ± 1.2 (CV = 5.7%), specification ≥ 19.5.
- Barrier function: IC50 = 2.1 mg/mL (specification, 1.5 mg/mL ≤ IC50 ≤ 3 mg/mL)
- Morphology: Well-differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination: no
NUMBER OF REPLICATE TISSUES: 3
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: no interference.
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue viability after after exposure and post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the tissue viability after exposure and post-treatment incubation is greater 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 μL (26 μL/cm2)
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% SDS - Duration of treatment / exposure:
- 15 minutes
- Duration of post-treatment incubation (if applicable):
- 42 hours
- Number of replicates:
- 3
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- mean
- Value:
- 100.19
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- (PBS)
- Positive controls validity:
- valid
- Remarks:
- 6.87% viability (5% SDS)
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- - OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no
DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A demonstration of proficiency was performed for the EpiSkinTM kit. Adequate results were obtained for the evaluated chemicals.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, OD mean is 0.69413 (acceptability criteria, 0.6≤OD≤1.5). SD of the negative control group was 0.00378 (acceptablility criteria, SD ≤ 18%).
- Acceptance criteria met for positive control: yes, SD of the positive control group was 0.21% (acceptablility criteria, SD ≤ 18%)
- Acceptance criteria met for variability between replicate measurements: yes. SD of test item was 1.23% (acceptablility criteria, SD ≤ 18%). - Interpretation of results:
- other: No category (CLP Regulation EC no. 1272/2008)
- Conclusions:
- Under in vitro RHE test method performed in Episkin model the test item is predicted to be non-irritant.
- Executive summary:
An in vitro skin irritation test of the test item was performed in a reconstructed human EpiSkin TM model, according to OECD TG 439 (GLP study). Three epidermis units were treated with 10 μL test item for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with PBS. The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol, incubating the tissues at room temperature protected from light for 4 hours, and finally measuring the concentration of formazan by determining the OD at 570 nm. Under the test conditions, the mean percent viability of the treated tissues was 100.19%, versus 6.87% of the positive control (5% Sodium Dodecyl Sulfate) and 100% of the negative control (PBS). Therefore, the test item is predicted to be non-irritant.
Reference
Table 1. Mean OD Values of Individual Epidermis Units
|
Absorption (OD570) |
|
||
R1 |
R2 |
R3 |
Mean |
|
Negative control |
0.73520 |
0.73360 |
0.74080 |
0.73653 |
Positive control |
0.09110 |
0.09080 |
0.08845 |
0.09012 |
Test Item |
0.74580 |
0.73885 |
0.72885 |
0.73783 |
Table 2. True OD Values of Individual Epidermis Units
|
Absorption (OD570) |
||||
R1 |
R2 |
R3 |
Mean |
SD |
|
Negative control |
0.69280 |
0.69120 |
0.69840 |
0.69413 |
0.00378 |
Positive control |
0.04870 |
0.04840 |
0.04605 |
0.04772 |
0.00145 |
Test Item |
0.70340 |
0.69645 |
0.68645 |
0.69543 |
0.00852 |
Blank OD Value (mean of 6 replicate values): 0.04240
True OD value = OD Raw – OD Blank
Table 3. Individual Tissue Viability of Epidermis Units (Relative)
|
% Individual Viability |
||||
R1 |
R2 |
R3 |
Mean |
SD |
|
Positive control |
7.02 |
6.97 |
6.63 |
6.87 |
0.21 |
Test Item |
101.33 |
100.33 |
98.89 |
100.19 |
1.23 |
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:
- 20 February 2018- 25 February 2018
- 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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Species:
- cattle
- Strain:
- not specified
- Details on test animals or tissues and environmental conditions:
- SOURCE OF COLLECTED EYES
- Source: Local abattoir (Slaughter house), Near Frazer town, Bengaluru.
- Characteristics of donor animals (e.g. age, sex, weight): not specified.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The bovine eyes were procured from local abattoir and transported in a jar containing Hank’s Balanced Salt Solution (HBSS) and penicillin-streptomycin (100 IU & 100 µg/mL) in an ice box.
- Time interval prior to initiating testing: Not specified.
- indication of any existing defects or lesions in ocular tissue samples: immediately after receiving the eyes in the lab, all eyes were observed for any evidence of vascularization, pigmentation, opacity or scratches. Corneas from eyes free of visible defects were used. 2 out of 12 corneas exhibited initial opacity more than 7. The corneas which exhibited initial opacity < 7 were considered for further experiment.
- Indication of any antibiotics used: yes, penicillin at 100 IU/mL and streptomycin at 100 μg/mL. - 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 - Duration of treatment / exposure:
- 10 min
- Duration of post- treatment incubation (in vitro):
- 2 h
- Number of animals or in vitro replicates:
- 3 replicates.
- Details on study design:
- SELECTION AND PREPARATION OF CORNEAS: Immediately after receiving the eyes in the lab, all eyes were observed for any evidence of vascularization, pigmentation, opacity or scratches. Post pretest examination, selected eyes were dissected to isolate the corneas from surrounding tissue and then placed in a container with fresh HBSS (Hank’s Balanced Salt Solution). The isolated corneas were mounted on the cornea holder. During mounting, it was ensured that the epithelium of the cornea projected into the anterior chamber. EMEM (Eagle Minimum Essential Medium) without phenol red was added in to both the chambers and kept in an incubator (32°C) for 1 hour and 15 minutes. After pre-test incubation, the EMEM from both the chambers was removed and refilled with the fresh EMEM. The pre-treatment cornea reading (lux) was measured and initial opacity was calculated for all corneas. 2 out of 12 corneas exhibited initial opacity more than 7. The corneas which exhibited initial opacity < 7 were considered for further experiment. Post initial opacity measurement, nine corneas with opacity less than 7 were grouped into Negative control, Positive control and Test item treatment groups with 3 corneas per group.
QUALITY CHECK OF THE ISOLATED CORNEAS: Corneas from eyes free of visible defects and with an opacity value inferior than 7 were used.
NUMBER OF REPLICATES: 3
NEGATIVE CONTROL USED: yes. Distilled water.
POSITIVE CONTROL USED: yes. Acetone.
APPLICATION DOSE AND EXPOSURE TIME: 750 μL of undiluted test item, 10 min exposure.
TREATMENT METHOD: closed chamber.
POST-INCUBATION PERIOD: no.
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Anterior chamber was washed three times with EMEM containing phenol red. After, the anterior chamber was rinsed with EMEM without phenol red.
- POST-EXPOSURE INCUBATION: After rinsing, both the chambers were filled with fresh EMEM and further incubated at 32 (±1) ºC for 2 hours before measuring the final opacity.
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Final opacity of all corneas was measured with fresh EMEM using an OP3.0 Opacitometer (Duratec, Germany)
- Corneal permeability: passage of sodium fluorescein dye (optical density at 490 nm) measured with microplate reader (Tecan, Model: Infinite M200).
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
DECISION CRITERIA: as indicated in the TG (see table below). - Irritation parameter:
- in vitro irritation score
- Run / experiment:
- mean
- Value:
- 18.045
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- 1.429
- Positive controls validity:
- valid
- Remarks:
- 104.32
- Irritation parameter:
- cornea opacity score
- Run / experiment:
- mean
- Value:
- 18
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- 1.33
- Positive controls validity:
- valid
- Remarks:
- 96.34
- Irritation parameter:
- other: permeability
- Run / experiment:
- mean
- Value:
- 0
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- 0.0066
- Positive controls validity:
- valid
- Remarks:
- 0.5320
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no.
DEMONSTRATION OF TECHNICAL PROFICIENCY: yes
ACCEPTANCE OF RESULTS:
- A test is considered acceptable if: the positive control gives an IVIS that falls within two standard deviations of the current historical mean and negative/solvent controls gives values of opacity and permeability lower than upper limits for background values.
- Acceptance criteria met for negative control:yes, distilled water response resulted in opacity and permeability values that are less than established upper limits for negative control opacity (1.33) and permeability values (0.0257)
- Acceptance criteria met for positive control: yes, IVIS score of acetone was 104.32 which falls between two standard deviations of the historical mean i.e. 96-128. - Interpretation of results:
- other: No prediction can be made (CLP Regulation EC no. 1272/2008)
- Conclusions:
- The test item cannot be predicted as not classified for eye irritation/serious eye damage or as causing serious eye damage with the BCOP test method.
- Executive summary:
An in vitro Bovine Corneal Opacity and Permeability (BCOP) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 437 under GLP conditions. Three sets consisting of three corneas each were tested: the first set was the negative control, and was treated with 750 μL distilled water; the second set was the positive control, and was treated with 750 μL of acetone and the third set was treated with 750 μL of test item. The corneas were exposured for 10 min after which the test item was washed and then the corneas were kept in incubation for 2 h at 32ºC. After incubation, opacity of the corneas and fluorescein permeability were measured. Negative control and positive control exhibited expected response as No category (IVIS score 1.429) and Category 1 (IVIS score 104.32) respectively. The test item exhibited an IVIS score of 18.045. Thus, it can be concluded that the test ítem cannot be predicted as not classified for eye irritation/serious eye damage or as causing serious eye damage with the BCOP test method.
Reference
Table 1. Details of Opacity Calculation.
Cornea Holder Number |
Blank value* Io (lux) |
Pre-treatment Cornea reading# I (lux) |
Initial opacity (t0)* |
Treatment group |
Post-treatment Cornea reading$ I (lux) |
Final opacity (10 mins)* |
Change of Opacity |
Corrected Opacity |
Mean Opacity |
2016-123#01 |
502 |
453 |
5 |
Negative control: Distilled water |
448 |
5 |
0 |
NA |
1.33 |
2016-123#02 |
502 |
482 |
2 |
454 |
5 |
3 |
|||
2016-123#04 |
502 |
472 |
3 |
458 |
4 |
1 |
|||
2016-123#21 |
502 |
451 |
5 |
Positive control: Acetone |
140 |
103 |
98 |
96.67 |
96.34 |
2016-123#22 |
502 |
431 |
7 |
130 |
114 |
107 |
105.67 |
||
2016-123#23 |
502 |
472 |
3 |
153 |
91 |
88 |
86.67 |
||
2016-123#11 |
502 |
433 |
7 |
Test item: 3,3-Dimethyl-8,9-Dinorbornan-2-one |
319 |
23 |
16 |
14.67 |
18.00 |
2016-123#13 |
502 |
444 |
6 |
346 |
18 |
12 |
10.67 |
||
2016-123#14 |
502 |
449 |
5 |
268 |
35 |
30 |
28.67 |
Remarks:
* Blank value for each cornea holder with medium but without cornea.
# Reading of each cornea holder with cornea and medium (Post pre-test incubation period).
$ 10 minutes post reference item exposure incubation period
Initial/Final opacity = ((I0/I)-b)/a, where I = individual reading, a=0.0251 & b=0.9894 (a and b values are instrument specific constants).
Change of opacity = Final opacity – Initial opacity
Corrected opacity = Change in opacity of each treated cornea – average change of opacity value of negative corneas i.e. “1.33” of distilled water
Mean opacity = Mean of corrected opacity
Table 2. Details of Permeability Calculation
Cornea Holder Number |
Treatment group |
OD490reading (90 mins) |
Blank corrected OD490* |
Negative control corrected OD490# |
Mean |
|
|
||||
NA |
Blank OD reading
|
0.0387 |
NA |
NA |
0.0390 |
0.0379 |
|||||
0.0385 |
|||||
0.0391 |
|||||
0.0414 |
|||||
0.0382 |
|||||
|
|
||||
2016-123#01 |
Negative control: Distilled water |
0.0471 |
0.0081 |
NA |
0.0066 |
0.0479 |
0.0089 |
||||
2016-123#02 |
0.044 |
0.0050 |
|||
0.046 |
0.0070 |
||||
2016-123#04 |
0.0444 |
0.0054 |
|||
0.0442 |
0.0052 |
||||
|
|
||||
2016-123#21 |
Positive Control: Acetone |
0.5713 |
0.5323 |
0.4867 |
0.5320 |
0.5627 |
0.5237 |
0.4781 |
|||
2016-123#22 |
0.3654 |
0.3264 |
0.2808 |
||
0.3687 |
0.3297 |
0.2841 |
|||
2016-123#23 |
0.9224 |
0.8834 |
0.8378 |
||
0.9088 |
0.8698 |
0.8242 |
|||
|
|||||
2016-123#11 |
Test item: 3,3-Dimethyl-8,9-Dinorbornan-2-one |
0.0765 |
0.0375 |
-0.0081 |
0.0003 |
0.075 |
0.0360 |
-0.0096 |
|||
2016-123#13 |
0.0965 |
0.0575 |
0.0119 |
||
0.0975 |
0.0585 |
0.0129 |
|||
2016-123#14 |
0.0807 |
0.0417 |
-0.0039 |
||
0.0833 |
0.0443 |
-0.0013 |
Remarks:
* Blank corrected OD490 = Individual corneal reading OD490 – Mean OD Blank reading
# Negative control corrected OD490 = Individual blank corrected OD490 – Mean of Negative control OD490 reading (Distilled water)
Table 3. Calculation of In Vitro Irritation Score (IVIS*)
Treatment group |
Mean Opacity value |
Mean Permeability value |
IVIS |
Classification |
Negative control (Distilled water) |
1.33 |
0.0066 |
1.429 |
No Category |
Acetone |
96.34 |
0.5320 |
104.32 |
Category I |
3,3-Dimethyl-8,9-Dinorbornan-2-one |
18 |
0.0003 |
18.045 |
No Prediction can be made |
*IVIS = Mean Opacity value + (15 × Mean Permeability value)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Skin irritation. Key study. An in vitro skin irritation test of the test item was performed in a reconstructed human EpiSkin TM model, according to OECD TG 439 (GLP study). Three epidermis units were treated with 10 μL test item for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with PBS. The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol, incubating the tissues at room temperature protected from light for 4 hours, and finally measuring the concentration of formazan by determining the OD at 570 nm. Under the test conditions, the mean percent viability of the treated tissues was 100.19%, versus 6.87% of the positive control (5% Sodium Dodecyl Sulfate) and 100% of the negative control (PBS). Therefore, the test item is predicted to be non-irritant.
Eye irritation. Key study. An in vitro Bovine Corneal Opacity and Permeability (BCOP) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 437 under GLP conditions. Three sets consisting of three corneas each were tested: the first set was the negative control, and was treated with 750 μL distilled water; the second set was the positive control, and was treated with 750 μL of acetone and the third set was treated with 750 μL of test item. The corneas were exposured for 10 min after which the test item was washed and then the corneas were kept in incubation for 2 h at 32ºC. After incubation, opacity of the corneas and fluorescein permeability were measured. Negative control and positive control exhibited expected response as No category (IVIS score 1.429) and Category 1 (IVIS score 104.32) respectively. The test item exhibited an IVIS score of 18.045. Thus, it can be concluded that the test ítem cannot be predicted as not classified for eye irritation/serious eye damage or as causing serious eye damage with the BCOP test method.
Justification for classification or non-classification
Based on the available information, the test item is not classified for skin irritation/corrosion in accordance with CLP Regulation (EU) No. 1272/2008.
Based on the available information, the test item cannot be predicted as not classified for eye irritation/serious eye damage or as causing serious eye damage in accordance with CLP Regulation (EU) No. 1272/2008.
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