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EC number: 946-569-7 | 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
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- Endpoint summary
- Stability
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- 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 Corrosion/ irritation : Not classified as skin corrosive in an OECD 431 study (Rel.1, GLP, K) and not classified as skin irritant in an OECD 439 study (Rel.1, GLP, K).
Serious eye damage/ eye irritant: Serious eye damage based on the rules of the CLP Regulation for classification of mixtures.
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:
- weight of evidence
- Study period:
- November 2018- April 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Test system:
- human skin model
- Source species:
- other: reconstituted epidermis (epiCS®, CellSystems®)
- Cell type:
- other: reconstituted epidermis (epiCS®, CellSystems®)
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: 0.60 cm² reconstituted epidermis (epiCS®)
EXPOSURE
- The test item was applied as supplied, at the dose of 25 mg, to 2 living Human skin model surfaces (epiCS®, CellSystems®) during 3 minutes and 1 hour.
REMOVAL OF TEST MATERIAL AND CONTROLS
- 3 minutes and 1 hour after the test item application, the human epidermis was washed with 20 mL of PBS.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
The cell viability is quantified by measurement of the cellular mitochondrial dehydrogenases activity. These enzymes are responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; EINECS number 206-069-5, CAS number 298-93-1)] reduction into blue formazan in the viable cells. The skin sample is placed in MTT solution of appropriate concentration (e.g. 0.3 or 1 mg/mL) for 3 hours at 37°C ± 1°C. The precipitated blue formazan product is then extracted using a solvent (e.g. isopropanol), and the concentration of formazan is measured by determining the Optical Density (OD) at a wavelength between 540 and 600 nm (preferably 570 nm). The measured absorbances are proportional to the number of living cells.
The measurement of OD was performed using the ELx800 absorbance microplate reader supplied by BioTek and the validated software Gens ELISA V1.05.11 supplied by BioTek.
VIABILITY
Viability = (OD test item / OD negative control) x 100
For each tissue, OD values and calculated percentage cell viability data for the test item, positive and negative controls, should be reported in tabular form, including data from replicate repeat experiments as appropriate, mean and individual values. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg
- Concentration (if solution): Undiluted - Duration of treatment / exposure:
- 3 minutes and 1 hour
- Number of replicates:
- duplicate
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 3 minutes replicate 1
- Value:
- 83.41
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 3 minutes replicates 2
- Value:
- 74.47
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 1 hour replicate 1
- Value:
- 74.6
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 1 hour replicate 2
- Value:
- 81.82
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- VIABILITY
- 3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 78.94 and 78.21%, respectively.
ACCEPTANCE OF RESULTS:
- The mean optical density obtained with the negative control at 3 minutes and 1 hour was 0.727 and 0.790, respectively, which is within the expected range of 0.3 to 0.9.
- Acceptance criteria met for positive control: Yes; 3 minutes and 1 hour after the positive control application, the viability of the human skin model has been 5.51 and 0.13 %, respectively. - Interpretation of results:
- other: GHS not met for classification as corrosive
- Conclusions:
- In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that test item does not have to be classified in Category 1 “Corrosive”. The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required.
- Executive summary:
An in vitro skin corrosion study was performed according to OECD Guideline 431 and in compliance with GLP to evaluate the possible corrosive effects of the test item after topical administration on in vitro human reconstituted epidermis (epiCS®, CellSystems®). Test item was applied after being reduced in fine powder, during 3 minutes and 1 hour, at the dose of 25 mg to 2 living Human skin model surfaces (epiCS®, supplied by CellSystems®) previously moistened with 25 µL of distilled water. The application wasfollowed by a rinse with 20 mL of DPBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.
The experimental protocol was established in accordance with OECD Test Guideline No. 431 dated 29 July 2016 and method B.40 bis of the Council regulation No. 440/2008 of 30 May 2008.
3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with test item were78.94%and78.21%, versus 5.51% and 0.13%, respectively, with the positive control item (potassium hydroxide 8N).
In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that test item does not have to be classified in Category 1 “Corrosive”. The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required.
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 21 November to 28 November 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP study performed according to OECD Guideline 439 without deviations
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- adopted 18 June 2019
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- 14 October 2019
- Test system:
- human skin model
- Remarks:
- SkinEthic RHE® model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other: foreskin
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- 0.50 cm² reconstructed epidermis (Episkin SA, RHE/S/17 Batch No. 19-RHE-188) were received on 26 November 2019.
- On the same day, the insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in 24 wells culture plate which had been previously filled with 300 µL of growth medium (Episkin SA, batch No. 19 SGM 127) during 2 hours and 25 minutes. Then just before treatment, the inserts were placed in 24 wells culture plate which had been previously filled with 300 μL of maintenance medium (Episkin SA, batch No. 19 SMM 047).
TREATMENT
- The test item was applied, as supplied, during 42 minutes at room temperature, at the dose of 16 mg to the epidermal surface of 3 living skin models previously moistened with 10µL of distilled water.
- In the same experimental conditions, a positive control (16 µL of 5% SDS), and a negative control (16 µL of distilled water – ADL Prochilab - Batch No. 190521) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS (SIGMA Batch No. STBG6142V) in a 10 mL volumetric flask (q.s. 10 mL of distilled water). Then, the preparation was magnetically stirred, just before the treatment. To ensure a good contact with the epidermis, during all the treatment period, the control items were covered with a nylon mesh provided by Episkin SA.
REMOVAL OF TEST MATERIAL AND CONTROLS
- 42 minutes after the test item application, the human epidermis were washed with 25 x 1 mL of DPBS (Dutscher, Batch No. 2190919). The rinsed tissues were checked for any coloration and noted to be whitish, comparable to negative control tissues. They were incubated for a post-treatment incubation period of 42-hour and 46-minute in fresh medium at 37°C, 5% CO2. Then, the epidermis were put in contact with the MTT solution.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- The cell viability was quantified by the measurement of the cell succinate dehydrogenase activity. This enzyme was responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS No. 298-93-1)] reduction into blue formazan crystal.
- The skin samples were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours at 37°C, 5% CO2. The precipitated blue formazan product was then extracted using isopropanol during 2 hours and 02 minutes under gentle agitation in the dark, and the concentration of formazan was measured by determining the Optical Density (OD) at 570 nm, just after dilution of the extracts (1:2 in isopropanol).
- The OD of MTT extract was measured in triplicate. The measured OD was proportional to the number of living cells. The measurement of OD was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.
VIABILITY CALCULATION:
- Data from individual replicate tissues (OD values and calculated percent tissue viability data for the test item and controls), mean percent tissue viability and standard deviation for each individual test item and control were reported in Table 7.3.1/1. The results were expressed as a viability percentage compared with the negative control: viability % = (mean OD test item / mean OD negative control) * 100
PREDICTION MODEL / DECISION CRITERIA
The OD values obtained for each test sample were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%. The cut-off values for the prediction of irritation associated with the RHE models were as follows:
- The test item is considered to be non-irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is >50%.
- The test item is considered to be irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is ≤ 50%. In accordance with Regulation EC No. 1272/2008, the test item has to be classified in Category 2 “Irritant”. The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg
- Concentration (if solution): Undiluted - Duration of treatment / exposure:
- 42 minutes at room temperature
- Duration of post-treatment incubation (if applicable):
- 42-hour and 46-minute post-treatment incubation period in fresh medium at 37°C, 5% CO2
- Number of replicates:
- 3 replicates of living human skin models
- Irritation / corrosion parameter:
- % tissue viability
- Remarks:
- mean
- Run / experiment:
- main test (duration of exposure: 42 minutes)
- Value:
- 91
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- PRELIMINARY TESTS
- Test for direct interaction with MTT:
A yellow solution with the test item on the surface was observed after 3 hours of incubation between 36.1°C and 37.4°C; 5% CO2. Therefore, there is no direct interaction between the test item and MTT.
- Test for the detection of the colouring potential of the test item:
A yellow solution was obtained after 2 hours and 05 minutes of incubation at ambient temperature with gentle shaking. The mean of the corrected OD was 0.029 which is lower than 0.08. Therefore the test item will not interfere with the MTT assay and there is no need to add non-specific coloration controls to the study.
MTT VIABILITY ASSAY RESULTS
The mean percent viability of the treated tissues was 91.0%, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate).
ACCEPTANCE OF RESULTS:
- The mean percent tissue viabilities obtained with the positive control and negative controls were within the range of historical data and therefore validate the experiment.
- The standard deviation of the negative control group (14.7%), the positive control group (0.1%) and the test item group (10.2%) were ≤18%, which are in accordance with the SD acceptability criteria.
- The OD value of the 3 replicates of the negative control should be in the range ≥ 0.8 and ≤ 3.0. However, it was measured after a 1:2 dilution of the formazan extracts in isopropanol; the acceptability criteria should be in the range ≥ 0.4 and ≤ 1.5 for the negative control.
- The mean percent tissue viabilities obtained with the positive control was 1.0%, which is in accordance with the PC acceptability criteria (< 40%). - Interpretation of results:
- GHS criteria not met
- Conclusions:
- Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was considered as non-irritant to skin. It corresponds to UN GHS No Category. No hazard statement or signal word is required.
- Executive summary:
An in vitro skin irritation test using the Reconstructed Human Epidermis (SkinEthic RHE® model) was performed according to the OECD Guideline 439 and in compliance with GLP to predict the acute skin irritation potential of the test item.
The test item was applied as supplied, at the dose of 16 mg, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes, followed by a rinse with 25 mL of PBS and a 42 hours and 46 minutes post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.
The mean corrected percent viability of the treated tissues was 91.0 %, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate).
Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was considered as non-irritant to skin. It corresponds to UN GHS No Category. No hazard statement or signal word is required.
Referenceopen allclose all
Table 7.3.1/1: Skin corrosion assay: Results
|
|
Table 7.3.1/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls
Skin |
OD |
Mean OD / disc (#) |
Mean OD / product |
Viability % |
Mean viability % |
Standard deviation (SD) |
|
Negative control |
1 |
1.204 |
1.232 |
1.058 |
116.4 |
100.0 |
14.7 |
1.229 |
|||||||
1.263 |
|||||||
2 |
0.972 |
1.011 |
95.5 |
||||
1.025 |
|||||||
1.038 |
|||||||
3 |
0.889 |
0.932 |
88.1 |
||||
0.950 |
|||||||
0.958 |
|||||||
Positive control |
1 |
0.010 |
0.010 |
0.011 |
0.9 |
1.0 |
0.1 |
0.011 |
|||||||
0.011 |
|||||||
2 |
0.012 |
0.011 |
1.0 |
||||
0.011 |
|||||||
0.012 |
|||||||
3 |
0.011 |
0.011 |
1.0 |
||||
0.012 |
|||||||
0.012 |
|||||||
Test item |
1 |
0.894 |
0.906 |
0.963 |
85.6 |
91.0 |
10.2 |
0.941 |
|||||||
0.883 |
|||||||
2 |
0.911 |
0.896 |
84.7 |
||||
0.931 |
|||||||
0.848 |
|||||||
3 |
1.104 |
1.088 |
102.8 |
||||
1.122 |
|||||||
1.040 |
#: mean of 3 values (triplicate of the same extract)
OD: optical density; measured after a 1:2 dilution of the formazan extracts in isopropanol.
Acceptability criteria: SD ≤ 18%;values of negative and positive controls were within the range of historical data.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation, other
- Remarks:
- Classification based on calculation rules for mixtures of the CLP Regulation
- Type of information:
- calculation (if not (Q)SAR)
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- accepted calculation method
- Qualifier:
- no guideline required
- Principles of method if other than guideline:
- Classification based on calculation rules for mixtures of the CLP Regulation
- Irritation parameter:
- other: Classification
- Remarks on result:
- other: Eye damage category 1
- Interpretation of results:
- Category 1 (irreversible effects on the eye) based on GHS criteria
- Executive summary:
The NCS is composed of several identified constituents and in that, it can be considered as a mixture according to the definition of the CLP Regulation. The decision logic for classification of mixtures from the ECHA Guidance on the Application of the CLP Criteria (2017) was used to determine the serious eye damage/ eye irritant hazard of the registered substance. The decision of classification as Eye damage category 1 is based on existing data on constituents (additivity principles) the registered substance has more than 3% of its constituents classified as Eye damage Category 1 and should be classified as a
Eye damage Category 1without further testing according to the rules for classification of mixtures of Regulation (EC) No 1272/2008.
Reference
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (irreversible damage)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
SKIN IRRITATION/CORROSION
Skin corrosion
An in vitro skin corrosion study was performed according to OECD Guideline 431 and in compliance with GLP to evaluate the possible corrosive effects of the test item after topical administration on in vitro human reconstituted epidermis (epiCS®, CellSystems®). The test item was applied after being reduced in fine powder, during 3 minutes and 1 hour, at the dose of 25 mg to 2 living Human skin model surfaces (epiCS®, supplied by CellSystems®)previously moistened with 25 µL of distilled water. The application was followed by a rinse with 20 mL of DPBS. The cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.
The experimental protocol was established in accordance with OECD Test Guideline No. 431 dated 29 July 2016 and method B.40 bis of the Council regulation No. 440/2008 of 30 May 2008.
3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with test item were 78.94% and 78.21%, versus 5.51% and 0.13%, respectively, with the positive control item (potassium hydroxide 8N).
In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”. The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required.
Skin irritation
An in vitro skin irritation test using the Reconstructed Human Epidermis (SkinEthic RHE® model) was performed according to the OECD Guideline 439 and in compliance with GLP to predict the acute skin irritation potential of the test item. The test item was applied as supplied, at the dose of 16 mg, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes, followed by a rinse with 25 mL of PBS and a 42 hours and 46 minutes post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.
The mean corrected percent viability of the treated tissues was 91.0 %, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate).
Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was considered as non-irritant to skin. It corresponds to UN GHS No Category. No hazard statement or signal word is required.
EYE IRRITATION
The NCS is composed of several identified constituents and in that, it can be considered as a mixture according to the definition of the CLP Regulation.
The decision logic for classification of mixtures from the ECHA Guidance on the Application of the CLP Criteria (2017) was used to determine the Eye irritation/corrosion hazard of the registered substance. The decision of classification as eye corrosive was based on existing data on constituents (additivity principles) the registered substance has more than 3% classified as Eye damage Category 1 and should be classified as a serious eye damage without further testing according to the rules for classification of mixtures of Regulation (EC) No 1272/2008.
Constituent |
CAS |
Classification |
Source |
|
Benzoic acid |
65 -85 -0 |
H315 - H318 |
Harmonised classification |
Source: ECHA disseminated dossiers or self classification
Justification for classification or non-classification
Harmonized classification:
The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.
Self-classification:
Based on the available information, the substance should not be classified as skin irritant according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the UN-GHS.
Based on the available information and typical composition provided by the Lead Registrant, the registered substance is classified as Serious eye damage Category 1 (H318: Causes serious eye damage) according to the criteria of the Regulation (EC) No. 1272/2008 (CLP).
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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