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EC number: 208-217-4 | CAS number: 516-03-0
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Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Two in vitro test (Ames test and in vitro gene mutation study in mammalian cells) show negative results with and without metabolic activation on oxalic acid. Considering the read across approach used for assessment, these results are considered reliable for assessing the mutagenic properties of iron oxalate.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- July, 19 2016 to January 31 2017
- Reliability:
- 1 (reliable without restriction)
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information in a detailed justification report is included as attachment to the same record.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
For the determination of analogue in this read-across approach, the following points have been considered:
- Chemical speciation and valency (common valency of the cation: Fe2+ compared to Mg2+ or Sr2+).
- The water solubility, as it provides a first indication of the availability of the metal ion in the different compartments of interest. The most simplistic approach to hazard evaluation is to assume that the specific metal-containing compound to be evaluated shows the same hazards as the most water-soluble compounds.
- In fluids of organisms and in aqueous media, dissociation of ferrous oxalate di hydrate takes place immediately, resulting in formation of Ferrous cations (Fe2+) and oxalate anions. Thus, any ingestion or absorption of ferrous oxalate di hydrate by living organisms, in case of systemic consideration, will inevitably result of exposure to the dissociation products.
- Iron is an abundant mineral naturally present in the body. Physiologically, it. exists as an ion in the body as Fe2+ (ferrous ion). It is a necessary trace element used by all known living organisms (Williams 2012, NCBI 2019). Iron-containing enzymes, usually containing heme prosthetic groups, participate in catalysis of oxidation reactions in biology, and in transport of a number of soluble gases (Fraùsto da Silva 2001, NCBI 2019). Iron is an essential constituent of hemoglobin, cytochrome, and other components of respiratory enzyme systems. Its chief functions are in the transport of oxygen to tissue (hemoglobin) and in cellular oxidation mechanisms. Inorganic iron involved in redox reactions is also found in the iron-sulfur clusters of many enzymes, such as nitrogenase (involved in the synthesis of ammonia from nitrogen and hydrogen) and hydrogenase (NCBI 2019). A class of non-heme iron proteins is responsible for a wide range of functions such as ribonucleotide reductase (reduces ribose to deoxyribose; DNA biosynthesis) and purple acid phosphatase (hydrolysis of phosphate esters).
- Counter ions: the assumption that the oxalate ion is responsible for the common property or effect implies that the toxicity or ecotoxicity of the counter ion present in the compound will be largely irrelevant in producing the effects to be assessed.
- Likely common breakdown products via physical and/or biological processes for the targeted substance (ferrous oxalate di hydrate) and the analogues identified cannot present strong differences since the structures are very simple and very similar (formation of Fe2+ or oxalate ion).
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical information is provided in the “source” endpoint. No impurity affecting the classification is reported for the source chemical.
Information on the impurities of the target chemical are detailed in the attached report.
3. ANALOGUE APPROACH JUSTIFICATION
The main hypothesis for the analogue approach are verified. They are presented in the detailed report attached. The experimental data performed on the substance (tests performed in this REACH registration) confirms the analogue approach performed (same results on analogues).
4. DATA MATRIX
A data matrix is presented in the detailed report attached.
References :
Fraùsto da Silva J.J.R., Williams R.J.P. 2001 The biological chemistry of the elements, 2nd edition. Oxford University Press, Oxford
NCBI, National Center for Biotechnology Information. PubChem Compound Database; CID=27284, https://pubchem.ncbi.nlm.nih.gov/compound/27284 (accessed Mar. 6, 2019).
Williams R.J.P 2012. Iron in evolution. FEBS Letters. Volume 586, Issue 5, 9 March 2012, Pages 479-484. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Remarks:
- DMSO
- Untreated negative controls validity:
- valid
- Remarks:
- DMEM Medium
- Positive controls validity:
- valid
- Remarks:
- -S9 Ethyl methanesulfonate, +S9 7, 12 Dimethyl benz(a)anthracene
- Conclusions:
- Conclusion
During the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the HPRT locus in V79 cells of the Chinese hamster in the absence and presence of metabolic activation.
Therefore, OXALIC ACID is considered “non-mutagenic” in this HPRT assay, and in a read across approach, magnesium oxalate is also considered as “non-mutagenic”. - Executive summary:
This study was conducted to investigate the potential of OXALIC ACID to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The methods followed were as per OECD guideline No. 476, adopted on 28th July 2015.
The assay was performed in a independent experiment, using two parallel cultures each. The experiment I was performed with and without liver microsomal activation at a treatment period of 4 hours. Experiment II was performed for a treatment period of 4 hours with and 24 hours with out metabolic activation.
500 µg/mL concentration was chosen as the highest dose for the cytotoxicity experiment, based on the solubility and precipitation properties of the test item.
The following concentrations were selected for both Phase - I and Phase - II based on cytotoxicity results.
500 µg/mL, 250 µg/mL, 125 µg/mL, 62.5 µg/mL both in the presence and absence of metabolic activation
No relevant cytotoxic effect as indicated by the relative survival (RS) is cloning efficiency (CE) of cells plated immediately after treatment, adjusted by any loss of cells during treatment, based on cell count and as compared with adjusted cloning efficiency in negative controls (assigned a survival of 100%). The RS for the test item in the other tested concentrations were found to be more than 50% and hence the same doses were selected for the main experiment (Phase-I and Phase-II).
PHASE-I
In culture I, the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (EMS) were 7.5, 8.7, 8.8, 12.9, 17.5, 23.8 and 152.1/106 cells respectively in the absence of metabolic activation and the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (DMBA) were 4.3, 10.3, 11.7, 15.6, 24.7, 28.1 and 1181.2 per 106 cells in presence of metabolic activation.
In culture II, the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (EMS) were 5.8, 9.5, 14.5, 15.7, 17.2, 26.9 and 169.9/106 cells respectively and in the absence of metabolic activation and the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (DMBA) were 9.3, 12.8, 17.6, 17.8, 24.7, 28.7 and 1387.6 per 106 cells in presence of metabolic activation.
PHASE-II
In culture I, the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (EMS) were 6.6, 6.9, 7.8, 9.9, 15.7, 20.9 and 170.6/106 cells respectively in the absence of metabolic activation and the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (DMBA) were 5.2, 6.7, 10.2, 15.1, 19.0, 23.2 and 1022/106 cells in presence of metabolic activation.
In culture II, the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (EMS) were 6.0, 7.5, 10.0, 13.3, 17.4, 23.7 and 155.2/106 cells respectively and in the absence of metabolic activation and the numbers of mutant colonies of NC, VC, T1, T2, T3, T4 and PC (DMBA) were 7.4, 10.7, 12.5, 17.3, 22.1, 25.2 and 1211.6 per 106 cells respectively in presence of metabolic activation.
In both the cultures, there was no distinct increase in the mutant frequency of OXALIC ACID when compared to respective vehicle control and the induction factor not exceeds more than three times the corresponding vehicle controls. No significant and reproducible dose dependent increase in mutant colony numbers was observed in either the Phase I or Phase II of the experiment.
The positive controls used, EMS in the absence of metabolic activation and DMBA in the presence of metabolic activation, revealed significant increase in mutant colonies and induction factor is more than three times of vehicle control indicating that the test system were sensitive and the results are valid
Note: NC: Negative control; VC: Vehicle control; T1: Test concentration1; T2: Test concentration 2; T3: Test concentration 3; T4: Test concentration 4; PC: Positive Control, EMS (ethyl methanesulfonate), DMBA (Dimethyl benz(a)anthracene)
The result of this study is considered reliable in a read across approach for iron oxalate.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 1984
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information in a detailed justification report is included as attachment to the same record.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
For the determination of analogue in this read-across approach, the following points have been considered:
- Chemical speciation and valency (common valency of the cation: Fe2+ compared to Mg2+ or Sr2+).
- The water solubility, as it provides a first indication of the availability of the metal ion in the different compartments of interest. The most simplistic approach to hazard evaluation is to assume that the specific metal-containing compound to be evaluated shows the same hazards as the most water-soluble compounds.
- In fluids of organisms and in aqueous media, dissociation of ferrous oxalate di hydrate takes place immediately, resulting in formation of Ferrous cations (Fe2+) and oxalate anions. Thus, any ingestion or absorption of ferrous oxalate di hydrate by living organisms, in case of systemic consideration, will inevitably result of exposure to the dissociation products.
- Iron is an abundant mineral naturally present in the body. Physiologically, it. exists as an ion in the body as Fe2+ (ferrous ion). It is a necessary trace element used by all known living organisms (Williams 2012, NCBI 2019). Iron-containing enzymes, usually containing heme prosthetic groups, participate in catalysis of oxidation reactions in biology, and in transport of a number of soluble gases (Fraùsto da Silva 2001, NCBI 2019). Iron is an essential constituent of hemoglobin, cytochrome, and other components of respiratory enzyme systems. Its chief functions are in the transport of oxygen to tissue (hemoglobin) and in cellular oxidation mechanisms. Inorganic iron involved in redox reactions is also found in the iron-sulfur clusters of many enzymes, such as nitrogenase (involved in the synthesis of ammonia from nitrogen and hydrogen) and hydrogenase (NCBI 2019). A class of non-heme iron proteins is responsible for a wide range of functions such as ribonucleotide reductase (reduces ribose to deoxyribose; DNA biosynthesis) and purple acid phosphatase (hydrolysis of phosphate esters).
- Counter ions: the assumption that the oxalate ion is responsible for the common property or effect implies that the toxicity or ecotoxicity of the counter ion present in the compound will be largely irrelevant in producing the effects to be assessed.
- Likely common breakdown products via physical and/or biological processes for the targeted substance (ferrous oxalate di hydrate) and the analogues identified cannot present strong differences since the structures are very simple and very similar (formation of Fe2+ or oxalate ion).
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical information is provided in the “source” endpoint. No impurity affecting the classification is reported for the source chemical.
Information on the impurities of the target chemical are detailed in the attached report.
3. ANALOGUE APPROACH JUSTIFICATION
The main hypothesis for the analogue approach are verified. They are presented in the detailed report attached. The experimental data performed on the substance (tests performed in this REACH registration) confirms the analogue approach performed (same results on analogues).
4. DATA MATRIX
A data matrix is presented in the detailed report attached.
References :
Fraùsto da Silva J.J.R., Williams R.J.P. 2001 The biological chemistry of the elements, 2nd edition. Oxford University Press, Oxford
NCBI, National Center for Biotechnology Information. PubChem Compound Database; CID=27284, https://pubchem.ncbi.nlm.nih.gov/compound/27284 (accessed Mar. 6, 2019).
Williams R.J.P 2012. Iron in evolution. FEBS Letters. Volume 586, Issue 5, 9 March 2012, Pages 479-484. - Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- other: S. typhimurium TA92
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Species / strain:
- other: S. typhimurium TA94
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Conclusions:
- Interpretation of results: negative
Oxalic acid does not have mutagenic properties in the Ames test, under the current test conditions. Thus in a read-across approach, it is considered that ferrous oxalate does not have mutagenic properties in the Ames test as well. - Executive summary:
A test published on oxalic acid was used in a read-across approach.
200 food additives used in Japan, including oxalic acid, were tested for mutagenic properties in the Ames test. The strains of Salmonella typhimurium used are the following: TA 92, TA1535, TA100, TA1537, TA94, and TA98, with and without metabolic activation (S-9 prepared from rat liver). Negative results are showed with oxalic acid in all strains, with and without activation. It can be concluded that oxalic acid does not have mutagenic properties in the Ames test, under the current test conditions.
Thus in a read-across approach, it is considered that ferrous oxalate does not have mutagenic properties in the Ames test as well.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Remarks:
- The study is performed in line with OECD guideline 473 (in vitro mammalian chromosomal aberration test), materials and methods are well described. A deviation from this guideline is that the test is not performed with a metabolic activation system. Furthermore, no detailed information is reported on the results.
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information in a detailed justification report is included as attachment to the same record.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
For the determination of analogue in this read-across approach, the following points have been considered:
- Chemical speciation and valency (common valency of the cation: Fe2+ compared to Mg2+ or Sr2+).
- The water solubility, as it provides a first indication of the availability of the metal ion in the different compartments of interest. The most simplistic approach to hazard evaluation is to assume that the specific metal-containing compound to be evaluated shows the same hazards as the most water-soluble compounds.
- In fluids of organisms and in aqueous media, dissociation of ferrous oxalate di hydrate takes place immediately, resulting in formation of Ferrous cations (Fe2+) and oxalate anions. Thus, any ingestion or absorption of ferrous oxalate di hydrate by living organisms, in case of systemic consideration, will inevitably result of exposure to the dissociation products.
- Iron is an abundant mineral naturally present in the body. Physiologically, it. exists as an ion in the body as Fe2+ (ferrous ion). It is a necessary trace element used by all known living organisms (Williams 2012, NCBI 2019). Iron-containing enzymes, usually containing heme prosthetic groups, participate in catalysis of oxidation reactions in biology, and in transport of a number of soluble gases (Fraùsto da Silva 2001, NCBI 2019). Iron is an essential constituent of hemoglobin, cytochrome, and other components of respiratory enzyme systems. Its chief functions are in the transport of oxygen to tissue (hemoglobin) and in cellular oxidation mechanisms. Inorganic iron involved in redox reactions is also found in the iron-sulfur clusters of many enzymes, such as nitrogenase (involved in the synthesis of ammonia from nitrogen and hydrogen) and hydrogenase (NCBI 2019). A class of non-heme iron proteins is responsible for a wide range of functions such as ribonucleotide reductase (reduces ribose to deoxyribose; DNA biosynthesis) and purple acid phosphatase (hydrolysis of phosphate esters).
- Counter ions: the assumption that the oxalate ion is responsible for the common property or effect implies that the toxicity or ecotoxicity of the counter ion present in the compound will be largely irrelevant in producing the effects to be assessed.
- Likely common breakdown products via physical and/or biological processes for the targeted substance (ferrous oxalate di hydrate) and the analogues identified cannot present strong differences since the structures are very simple and very similar (formation of Fe2+ or oxalate ion).
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical information is provided in the “source” endpoint. No impurity affecting the classification is reported for the source chemical.
Information on the impurities of the target chemical are detailed in the attached report.
3. ANALOGUE APPROACH JUSTIFICATION
The main hypothesis for the analogue approach are verified. They are presented in the detailed report attached. The experimental data performed on the substance (tests performed in this REACH registration) confirms the analogue approach performed (same results on analogues).
4. DATA MATRIX
A data matrix is presented in the detailed report attached.
References :
Fraùsto da Silva J.J.R., Williams R.J.P. 2001 The biological chemistry of the elements, 2nd edition. Oxford University Press, Oxford
NCBI, National Center for Biotechnology Information. PubChem Compound Database; CID=27284, https://pubchem.ncbi.nlm.nih.gov/compound/27284 (accessed Mar. 6, 2019).
Williams R.J.P 2012. Iron in evolution. FEBS Letters. Volume 586, Issue 5, 9 March 2012, Pages 479-484. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: The maximum dose (0.125 mg/mL) was determined by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated.
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Additional information on results:
- No data
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information): negative without metabolic activation
Oxalic acid does not have mutagenic properties in the in vitro mammalian chromosomal aberration test, without metabolic activation and under the current test conditions.
This compound is considered for assessing the mutagenic properties of ferrous oxalate in a read-across approach. Consequently, Ferrous Oxalate is considered as not mutagenic according to the in vitro mammalian chromosomal aberration test conditions. - Executive summary:
A publication on oxalic acid mutagen properties was used in a read-across approach.
In this publication, 200 food additives used in Japan, among which oxalic acid, were tested for mutagenic properties in the in vitro mammalian chromosomal aberration test. Chinese hamster lung fibroblast cells were exposed to three doses, without metabolic activation. Oxalic acid tested negative. It can be concluded that oxalic acid does not have mutagenic properties in the in vitro mammalian chromosomal aberration test, without metabolic activation and under the current test conditions.
Thus in a read-across approach, it is considered that ferrous oxalate does not have mutagenic properties in the in vitro mammalian chromosomal aberration test as well.
Referenceopen allclose all
The following table is reproduced from table 1 in the publication (only the part concerning oxalic acid is reproduced):
Additive |
CAS no. |
Purity (%) |
Max dose (mg/mL) |
Polyploid (%) |
Stuct aber (%) |
(hr) |
Result |
Oxalic acid |
144-62-7 |
99.7 |
0.125 |
4.0 |
0.0 |
(48) |
- |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for classification or non-classification
No mutagenic effect was observed on the test item. In a read across approach, these results are considered relevant for assessing the classification of iron oxalate and it is thus considered that there is no need for classification.
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.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.