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A mixture of: trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(4-(4-nitro-2-sulfonatoanilino)phenylazo)phenolato)ferrate(1-); trisodium bis(2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)ferrate(1-); trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(4-nitro-2-sulfonatophenylazo)phenolato)ferrate(1-); trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(3-sulfonatophenylazo)phenolato)ferrate(1-); disodium 3,3'-(2,4-dihydroxy-1,3(or 1,5 or 3,5)-phenylenediazo)dibenzenesulfonate
EC number: 406-870-1 | CAS number: 115100-55-5
- 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
Repeated dose toxicity: oral
Administrative data
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 23 October, 1990 to 19 November 1990
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 990
- Report date:
- 1991
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
- Version / remarks:
- 1981
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
- Version / remarks:
- 1984
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
Test material
- Reference substance name:
- A mixture of: trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(4-(4-nitro-2-sulfonatoanilino)phenylazo)phenolato)ferrate(1-); trisodium bis(2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)ferrate(1-); trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(4-nitro-2-sulfonatophenylazo)phenolato)ferrate(1-); trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(3-sulfonatophenylazo)phenolato)ferrate(1-); disodium 3,3'-(2,4-dihydroxy-1,3(or 1,5 or 3,5)-phenylenediazo)dibenzenesulfonate
- EC Number:
- 406-870-1
- EC Name:
- A mixture of: trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(4-(4-nitro-2-sulfonatoanilino)phenylazo)phenolato)ferrate(1-); trisodium bis(2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)ferrate(1-); trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(4-nitro-2-sulfonatophenylazo)phenolato)ferrate(1-); trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(3-sulfonatophenylazo)phenolato)ferrate(1-); disodium 3,3'-(2,4-dihydroxy-1,3(or 1,5 or 3,5)-phenylenediazo)dibenzenesulfonate
- Cas Number:
- 115100-55-5
- Molecular formula:
- not applicable
- IUPAC Name:
- triiron(3+) nonasodium 2-(2-{4-hydroxy-2-oxido-5-[2-(3-sulfophenyl)diazen-1-yl]phenyl}diazen-1-yl)-4,6-dinitrobenzen-1-olate 2-(2-{5-[2-(3,5-dinitro-2-oxidophenyl)diazen-1-yl]-2-hydroxy-4-oxidophenyl}diazen-1-yl)-4,6-dinitrobenzen-1-olate tris(2-(2-{5-[2-(3,5-dinitro-2-oxidophenyl)diazen-1-yl]-4-hydroxy-2-oxidophenyl}diazen-1-yl)-4,6-dinitrobenzen-1-olate) 2-{2-[4-hydroxy-5-(2-{4-[(4-nitro-2-sulfophenyl)amino]phenyl}diazen-1-yl)-2-oxidophenyl]diazen-1-yl}-4,6-dinitrobenzen-1-olate
- Test material form:
- solid: particulate/powder
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
-Source: BRL Ltd., Basel, Switzerland
-Age at study initiation: approximately 6 weeks
-Weight at study initiation:
males : 158 to 177 grams
females: 122 to 152 grams
-Fasting period before study:
-Housing: Animals were housed 5 to a cage (same sex) in stainless steel suspended cages with wire mesh floors.
-Diet: Free access to standard pelleted laboratory animal diet (Kliba, Klingentalmueble AG, 4303, Kaiseraugst, Switzerland). Each batch was analysed for contaminants and results were examined and archived.
-Water: Tap water, ad libitum. Results of chemical and contaminants analyses are examined and archived quarterly.
-Acclimation period: At least 7 days. Veterinary examinatlon was performed prior to commencement of treatment to ensure that the animáis were in a good State of health.
ENVIRONMENTAL CONDITIONS
-Temperature (°C): 21 °C
-Humidity (%): 55 %
-Air changes (per hr): Air-conditioned room 15 air changes per hour
-Photoperiod (hrs dark / hrs light): Lighting was 12 hours artificial fluorescent light and 12 hours dark per day.
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- water
- Remarks:
- Distilled water
- Details on oral exposure:
- -PREPARATION OF DOSING SOLUTIONS:
The test article was weighed into a glass flask on an analyticaí balance and the vehicle (w/w) added.
-VEHICLE
-Amount of vehicle (if gavage): 5 ml/kg body weight. - Analytical verification of doses or concentrations:
- not specified
- Details on analytical verification of doses or concentrations:
- Samples of formulations prepared during week 1 were analysed to check the accuracy of preparation.
- Duration of treatment / exposure:
- 28 days
- Frequency of treatment:
- Once daily, approximately the same time each day, 7 days per week
Doses / concentrationsopen allclose all
- Dose / conc.:
- 50 mg/kg bw/day (nominal)
- Dose / conc.:
- 200 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 5 per sex x doses
- Control animals:
- yes, concurrent no treatment
Examinations
- Observations and examinations performed and frequency:
- DETAILED CLINICAL OBSERVATIONS: Yes
At least once daily. Severity of observations were graded.
BODY WEIGHT: Yes
FOOD CONSUMPTION: yes
OPHTHALMOSCOPIC EXAMINATION: Yes
HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Blood samples were collected under light ether anaesthesia on day 29, between 8.00 and 10.00 a.m.. The animals were fasted overnight before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0.6 ml) and untreated tubes for dinical biochemistry parameters (>2.0 ml). All samples were dispatched to "Bergschot Centrum voor Onderzoek" (BCO), Breda, The Netherlands for analysis.
The following haematology parameters were determined from blood containing EDTA as an anti-coagulant using the methods listed:
Erythrocyte count/RBC
Haemoglobin/HB
Haematocrit/HCT
Mean corpuscular
volume/MCV
Mean corpuscular
haemoglobin/MCH
Mean corpuscular
haemoglobin concentration/MCHC
Red cell distribution
width/RDW
Platelet count/ PLATELETS
Total leucocyte count/ WBC
Differential leucocyte count/(Neutrophi1s/SEG,
Eosinophils/EO, Basophils /BASO, Lymphocytes/LYMPH
Monocytes/MONO, Large Unstained Cells/L.UNST.CEL)
Key to observed quantitative parameters and scores in haematology
a) OPM. DlFf, (Remark.Differentiation)
Code/Differentiation
343/No Abnormalities
58/Platelet agregates
40/Atypical Lymphocytes
b) OPM. GRAD. (Remark.Grading)
Code/Description
0000/Not Applicable
2055/Many
2063/Occasional
c) THROMBOCYTES
Score/Description
2/normal
CLINICAL BIOCHEMISTRY: Yes
The following clinical biochemistry parameters were determined from serum samples after clotting and centrifugation using the methods Usted:
Parameter/Abbreviation
Glucose/GLUCOSE
Urea/UREA
Creatinine/CREATININE
Bilirubin, total/ BILI T.
Aspartate aminotransferase/(ASAT/GOT)
Alanine aminotransferase/(ALAT/GPT)
Gamma-glutamyltransferase/G-GT
Sodium/SODIUM
Potassium/POTASSIUM
Chloride/CHLORIOE
Calclum/CALCIUM
Phosphorus/INORG PHOSPH
Protein, total/ PROTEIN T.
Protein, albumin/ ALBUMIN
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY:No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
All animals surviving to the end of the observation period (day 30) were deeply anaesthetised by Intraperitoneal injection of sodium pentobarbital and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution:
Adrenal glands
Aorta
Brain
Cecum
Cervix
Clitoral gland
Colon
Duodenum
Epididymides
Esophagus
Eyes with optic nerve and Harderian gland Female mammary gland area
Femur including joint
Heart
Ileum
Jejunum
Kldneys
Larynx
Lacrimal gland, exorbital
Liver
Lung, infused with formalin
Lymph nodes - mandibular, mesenteric
Nasopharynx
Ovarles
Páncreas
Pituitary gland
Preputial gland
Prostate gland
Rectum
Salivary glands - mandibular, sublingual
Sciatic nerve
Seminal vesicles
Skeletal muscle
Skin
Spinal cord -cervical, midthoracic, lumbar
Spleen
Sternum with bone marrow
Stomach
Testes
Thymus
Thyroid including parathyroid
Tongue
Trechea
Urinary bladder
Uterus
Vagina
All gross lesions
ORGAN WEIGHTS
The following organ weights (and terminal body weight) were recorded at termination:
Adrenal glands
Brain
Heart
Kidneys
Liver
Spleen
Testes
HISTOTECHNOLOGY
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometres and stained with haematoxylin and eosin. Sections of the spleen of all animals were stained with Perl’s staining method for iron pigment.
All slides were histopathologically examined at RCC NOTOX B,V. instead of at RCC (UK) Ltd., Hereford, England, as per original protocol.
HISTOPATHOLOGY : yes
Slides of adrenals, heart, kidneys, liver and spleen, collected at termination from all animals of the control and high dose group as well as from all gross lesions of all animals were examined by a pathologist.
Based on the treatment related morphologic changes, spleen was also examined from all rats of group 2 {50 mg/kg/day) and 3 (200 mg/kg/day). - Statistics:
- The following statistical methods were used to analyse the body weight, organ weights and clinical laboratory data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
All tests were two-sided and in all cases p<0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled valances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values
Results and discussion
Results of examinations
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Dark coloured faeces was noted in males and females treated at 200 or 1000 mg/kg/day from day 3 of treatment onwards until termination of the study.
Excessive salivation was noted intermittently in animals receiving 1000 mg/kg/day and on one occasion in males receiving 200 mg/kg/day. Since excessive salivation may be attributed to a bad taste of the test substance, it was considered not to represent a sign of toxicity.
Alopecia was noted at various locations and over transient periods in 3/5 males receiving 1000 mg/kg/day. As alopecia is commonly noted in rats of this age and strain, this finding was considered not to be an adverse effect.
Other incidental findings noted among animals receiving 1000 mg/kg/day, i.e. hunched posture, rales, laboured breathing, and regurgitation of test substance, were considered not to be of toxicological significance. - Mortality:
- no mortality observed
- Description (incidence):
- No mortality occurred during the study period
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Body weights and body weight gain of treated animals were considered to be in the same range as controls over the 4 week study period.
Statistically significantly decreased body weights between males receiving 50 mg/kg/day and control males were, in the absence of a dose-related response, considered not to be a sign of toxicity. - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- There were no differences in food consumption before or after allowance for body weight between treated and control animals.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- There were no ophthalmoscopical changes noted at the pretest examination and the examination at week 4.
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- Red blood cell parameters of treated animals were all in the same range as control animals.
White blood cell counts of males receiving 1000 mg/kg/day were statistically significantly increased when compared to controls. White blood cell counts of females receiving 1000 mg/kg/day were also slightly higher than controls, but this difference did not attain statistical significance.
The number of white blood cells in animals receiving 200 or 50 mg/kg/day were comparable to control animals.
Differential white blood cell profiles of treated males or females did not differ from the white blood cell profile of control animals. Statistically significantly decreased lymphocytes among females receiving 1000 mg/kg/day were, as this was not supported by a clear shift in other white blood cell types, considered to have arisen fortuitously. - Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- There were no differences noted between control and treated rats that could be related to treatment with test substance.
Sodium values of females receiving 1000 mg/kg/day were noted to be statistically significantly decreased in comparison with control females.
However, as sodium values were still within the normal biological range for rats of this age and strain, it was considered to have occurred by chance. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Spleen weights of animals receiving 1000 mg/kg/day were noted to be higher than controls before and after correction for body weight (although this difference did not achieve a level of statistical significance between controls and males receiving 1000 mg/kg/day before adjustment for body weight). Spleen weights of animals receiving 200 or 50 mg/kg/day did not differ from those of control animals,
Kidney weights, which are related to body weight, were slight but statistically significantly higher in males receiving 1000 mg/kg/day when compared to control weights. Kidney weights of females receiving 1000
mg/kg/day or animals receiving 200 or 50 mg/kg/day were not thus affected. Other organ weights and relative organ weights of treated animals were indistinguishable from those of control animals. - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as an effect of treatment. The incidentally noted crateriform lesions and thickening of the caecum wall in 1 female receiving 1000 mg/kg/day, was considered not to have arisen as an effect of treatment.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Compared to controls, a slightly increased incidence of haemopoietic foci was noted in the spleen of animals receiving 1000 mg/kg/day. This was not apparent in the spleen of animals receiving 200 or 50 mg/kg/day.
Spleens of animals receiving 200 or 1000 mg/kg/day showed an increase in accumulated iron pigments, which was dose-related.
There were no other microscopic findings noted that were considered to be treatment-related. The small number of changes recorded in treated animals were within the range commonly seen for rats of this age and strain.
Effect levels
- Dose descriptor:
- NOEL
- Effect level:
- 50 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
Target system / organ toxicity
- Critical effects observed:
- no
Any other information on results incl. tables
Principle toxicologically significant changes noted as a result of repeated oral treatment were located in the spleen of animals receiving 200 or 1000 mg/kg/day. At microscopic examination, spleens of animals receiving 1000 mg/kg/day were noted with an increased incidence of haemopoietic foci. In addition, animals receiving 200 or 1000 mg/kg/day showed a dose dependent increase in accumulated iron pigments.
These microscopically observed changes may be associated with the increase of spleen weights which was apparent in animals receiving 1000 mg/kg/day.
Increased white blood cells, as noted in these high dose animals, also may be correlated with increased production of myeloid cells. However, as there was no apparent shift in differential white blood cell profile, a clear biological explanation can not be given.
Increased relative kidney weights were noted among males receiving 1000 mg/kg/day only. Given the isolated occurrence and lack of clinicopathological and histopathological evidence, the toxicological
significance of this finding was considered doubtful.
Dark appearance of the faeces was noted in animals receiving 1000 or 200 mg/kg/day. No microscopic correlates were observed in the gastrointestinal tract of these animals, leading to the hypothesis that this finding can be attributed to the staining properties of the test substance and may not be considered a sign of toxicity.
There were no changes in body weights, food consumption, ophthalmoscopic examination and acroscopic examination that were considered to be an effect of treatment.
Applicant's summary and conclusion
- Conclusions:
- NOEL = 50 mg/kg/day
- Executive summary:
The repeated dose toxicity of the substance was evaluated in a subacute 28-day toxicity study, according to the OECD Guideline 407 (1981) and method B.7 of the EEC-Directive 92/69 EEC.
A 5-day range finding study was performed (with 3 rats/sex/group at dose levels of 50, 200 and 1000 mg/kg/day) to provide a basis for selection of dose levels for a study of longer duration.
With the exception of dark appearance of the feces, no differences of biological significance were apparent between animals of the 3 treatment groups.
Based on these observations, a high treatment level of 1000 mg/kg/day was selected for a study of 28 days duration.
In the main study, the test item was administered dally by gavage to SPF-bred Wistar rats. The study comprised of four groups. The number of rats assigned to toxicity testing per group as well as the dose levels administered were as follows:
group Dose level mg/kg males females 1 0 1-5 21-25 2 50 6-10 26-30 3 200 11-15 31-35 4 1000 16-20 36-40 The following findins were observed.
At 50 mg/kg/day: No treatment-related changes detected.
At 200 mg/kg/day:
1) Dark appearance of the faeces was noted in males and females.
2) Microscopically observed accumulatlon of iron pigments were noted in the spleen of males and females.
At 1000 mg/kg/day:
1) Dark appearance of the faeces was noted in males and females
2) Increased total white blood cell counts were noted in males and females,
3) Increased spleen weights and relative spleen weights were noted in males and females and increased relative kidney weights were noted in males only.
3) Microscopically observed accumulatlon of iron pigments and increased number of haemopoletic foci were noted in the spleen of males and females.
From the results presented in this report a definitive no observed effect level (NOEL) of 50 mg/kg/day was established.
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