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EC number: 295-223-5 | CAS number: 91845-48-6 Extractives and their physically modified derivatives such as tinctures, concretes, absolutes, essential oils, oleoresins, terpenes, terpene-free fractions, distillates, residues, etc., obtained from Santalum austro-caledonicum, Santalaceae.
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Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames tests: non mutagenic ( OECD 471, GLP, rel. 1).
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 17 March to 17 April 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP study conducted according to OECD 471 Guideline without any deviation.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- landesamt für umwelt wasserwirtschaft und gewerbeaufsicht (inspected on 29-30 November 2010 / signed on 11 April 2011)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Robertet S.A. / 2259263
- Appearance: Colourless to pale yellow liquid / essential oil
- Production date: 18 December 2013
- Expiration date of the lot/batch: 18 December 2014
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (20 ± 5 °C); test item was stored in the test facility in a closed vessel at room temperature
- Stability under test conditions: Not stated - Target gene:
- Histidine gene for Salmonella typhimurium
- Species / strain / cell type:
- S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535
- Details on mammalian cell type (if applicable):
- not applicable
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- 4% S9; S9 fraction produced from the livers of male Sprague-Dawley rats which were treated with Aroclor 500 mg/kg bw intraperitoneally
- Test concentrations with justification for top dose:
- Cytotoxicity test: 150 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using plate incorporation method
Experiment 1: 0.05, 0.15, 0.5, 1.5, 5 and 150 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using plate incorporation method
Experiment 2: 5, 9, 19, 38, 75 and 150 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using pre-incubation method
Experiment 3: 0.5, 1.5, 5, 15, 50 and 150 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using plate incorporation method - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent doesn’t have any effects on the viability of the bacteria or the number of spontaneous revertants.
- On the day of the start of the first experiment, a stock solution containing 15 g/L of the test item in DMSO was prepared. On the respective day of the start of the second resp. third experiment, a stock solution containing 1.5 g/L of the test item in DMSO was prepared. The stock solution was used to prepare the geometric series of the concentrations to be tested. Each solution was membrane filtrated to accomplish sterility. - Untreated negative controls:
- yes
- Remarks:
- water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- other: 4-Nitro-1,2-phenylene Diamine
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Remarks:
- water
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- other: 2-Amino-Anthracene
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- SOURCE OF TEST SYSTEM: Salmonella typhimurium (all strains used) were obtained from TRINOVA BioChem (batch of the bacteria strains: TA 97a: 4488D, TA 98: 4789D; TA 100: 4569D; TA 102: 4712D; TA 1535: 4717D) and were stored as lyophilisate in the refrigerator at 2 - 8 °C.
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Preincubation period: 37 °C for 20 min
- Exposure duration: 37 °C for 48 h
NUMBER OF REPLICATIONS:
- Cytotoxicity test: 4 plates/dose
- Experiment 1, 2 and 3: 3 plates/dose
DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of growth of the bacterial background lawn. - Evaluation criteria:
- A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain can be observed.
A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity. - Statistics:
- None
- Key result
- Species / strain:
- S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: None
FIRST EXPERIMENT
- Toxicity: After addition of phosphate buffer to the test item solution, the resulting solution showed turbidity in the highest concentration: 150 μg/plate. No signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not reduced.
- Mutagenicity: No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found. To verify this result, a second experiment was performed using the pre-incubation method.
SECOND EXPERIMENT
- No signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not reduced.
- No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed. No concentration-related increase over the tested range was found.
THIRD EXPERIMENT
- A third experiment was performed, because a repetition of the first experiment was necessary. In the first experiment, five concentrations of the test item dilutions had been lower than intended by a factor of ten, because, inadvertently, a calculation error was made when diluting the stock solution.
- No signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not reduced.
- No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed. No concentration-related increase over the tested range was found.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Positive historical control data: 369-908 (577 ± 165), 109-542 (284 ± 172), 466-772 (595 ± 97), 540-976 (726 ± 137), 108-527 (252 ± 160) for TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains (without metabolic activation), respectively; 420-831 (607 ± 137), 95-195 (126 ± 29), 552-1080 (808 ± 155), 449-1332 (780 ± 284), 114-210 (148 ± 30) for TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains (with metabolic activation), respectively.
- Negative historical control data (Water): 99-150 (122 ± 14), 12-18 (14 ± 2), 115-154 (140 ± 12), 138-252 (205 ± 30), 13-23 (19 ± 3) for TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains (without metabolic activation), respectively; 106-152 (131 ± 18), 10-17 (14 ± 2), 119-153 (138 ± 14), 171-254 (207 ± 20), 15-25 (21 ± 3) for TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains (with metabolic activation), respectively.
- Vehicle historical control data (DMSO): 105-141 (127 ± 11), 10-17 (13 ± 2), 118-166 (141± 15), 136-250 (198 ± 28), 16-22 (19 ± 2) for TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains (without metabolic activation), respectively; 105-159 (128 ± 16), 12-18 (14 ± 2), 117-157 (139 ± 14), 183-245 (207 ± 21), 16-22 (19 ± 2) for TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains (with metabolic activation), respectively.
OTHERS:
- Confirmation of genotype is performed once a quarter. The last performance showed no abnormalities.
- Confirmation of the criteria and validity: The treatments for the sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the literature data. All positive controls (diagnostic mutagenes) showed mutagenic effects with and without metabolic activation. - Conclusions:
- Under the test conditions, test substance is not considered as mutagenic in S. typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains).
- Executive summary:
In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (4%S9-mix, rat liver S9-mix induced by Aroclor 1254) for 48 h.
Experiment 1: 0.05, 0.15, 0.5, 1.5, 5 and 150 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using plate incorporation method
Experiment 2: 5, 9, 19, 38, 75 and 150 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using pre-incubation method
Experiment 3: 0.5, 1.5, 5, 15, 50 and 150 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using plate incorporation method
Negative, vehicle and positive control groups were also included in mutagenicity tests.
The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation. Therefore, the sensitivity of the assay and the efficacy of the S9-mix were validated.
No signs of toxicity towards the tested strains could be observed. No visible reduction in the growth of the bacterial background lawn at any dose level. No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed in any of the experiments. No concentration-related increase over the tested range was found.
Under the test condition, the test substance is not mutagenic with and without metabolic activation in Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 24 November to 01 December 2008
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP study conducted in compliance with OECD Guideline No. 471 without any deviation.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- 26 July 2007
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Robertet / 1733624
- Appearance: Yellow liquid
- Expiration date of the lot/batch: May 2010
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: 96 hours - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Details on mammalian cell type (if applicable):
- not applicable
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- 10% v/v S9 fraction; rat liver microsome fraction (S9)
- Test concentrations with justification for top dose:
- Main test: 0.06, 0.19, 0.56, 1.67 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the direct plate incorporation method.
Confirmation test: 0.06, 0.19, 0.56, 1.67 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2 (pKM101) with and without S9 under the pre-incubation method. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: 1% ethanol
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- Remarks:
- without S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene
- Remarks:
- with S9 mix
- Details on test system and experimental conditions:
- BACTERIAL STRAINS: Master and working bacterial banks were prepared from lyophilized bacterial discs. Stock plates were stored at about -80 ± 10 °C.
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 37 °C for 72 h
NUMBER OF REPLICATIONS: 3 plates/dose
DETERMINATION OF CYTOTOXICITY
- Method: A reduction in the number of colonies in a dose-dependent manner compared to negative control for any strain and condition might indicate cytotoxicity.
OTHER:
- After an incubation of about 72 hours at 37 °C, the number of colonies per plate was counted with an automatic colony counter. Data are presented as the number of colonies present per plate (mean ± standard deviation).
The R ratio is calculated as follows:
R = Number of revertant colonies in the presence of the test item / Number of revertant colonies in the absence of the test item
- Sterility test: The sterility of the test item and the metabolic activation system (S9) were tested. For this purpose, the highest concentration of test item and a sample of the S9 mix were added respectively to top agar preheated at about 45 °C and poured over minimal agar medium plates. The plates were incubated for about 72 hours at about 37 °C. Presence or absence of colonies was observed. Bacterial growth would be an indication of microbiological contamination of the test item or S9 mix respectively.
- Solubility test: Solubility was assessed as precipitation in the final mixture under the actual test conditions. Observation of precipitation by naked eye indicates insolubility. - Evaluation criteria:
- Several criteria were used for determining a positive result: a dose-response in the range tested and / or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
Positive results from the bacterial reverse mutation test indicate that a test item induces point mutations or frame shifts in the genome of either Salmonella Typhimurium and/or Escherichia Coli.
Negative results from the test indicate that under the test conditions, the test item is neither mutagenic nor-promutagenic in the tested species. - Statistics:
- None
- Key result
- Species / strain:
- bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: None
CYTOTOXICITY TEST
- No cytotoxic effect was observed.
MUTAGENICITY TEST
- No concentration of the test item showed a biological significant increase (R ≥ 2.5) of the number of revertant either with or without S9 metabolic activation.
- No dose response was observed in none of the tested bacterial strains.
HISTORICAL CONTROL DATA
- Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data.
- See "Attached background material" section for further details. - Conclusions:
- Under the test condition, test substance is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.
- Executive summary:
In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2(pKM101) were exposed to the test substance diluted in ethanol at the following concentrations both in the presence and absence of metabolic activation system (10% v/v S9).
Main test: 0.06, 0.19, 0.56, 1.67 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the direct plate incorporation method.
Confirmation test: 0.06, 0.19, 0.56, 1.67 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2 (pKM101) with and without S9 under the pre-incubation method.
Negative and positive control groups were also included in mutagenicity tests.
Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data. All positive controls performed showed valid ratios (R) above 2.5.
No cytotoxic effect was observed. No concentration of the test item showed a biological significant increase (R ≥ 2.5) of the number of revertant either with or without S9 metabolic activation. No dose response was observed in any of the tested bacterial strains.
Under the test condition, test substance is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.
Referenceopen allclose all
None
None
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
First Ames test:
In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2(pKM101) were exposed to the test substance diluted in ethanol at the following concentrations both in the presence and absence of metabolic activation system (10% v/v S9).
Main test: 0.06, 0.19, 0.56, 1.67 and 5μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the direct plate incorporation method.
Confirmation test: 0.06, 0.19, 0.56, 1.67 and 5μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2 (pKM101) with and without S9 under the pre-incubation method.
Negative and positive control groups were also included in mutagenicity tests.
Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data. All positive controls performed showed valid ratios (R) above 2.5.
No cytotoxic effect was observed. No concentration of the test item showed a biological significant increase (R ≥ 2.5) of the number of revertant either with or without S9 metabolic activation. No dose response was observed in any of the tested bacterial strains.
Under the test condition, test substance is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.
Second Ames test:
In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains ofSalmonella typhimurium(TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (4%S9-mix, rat liver S9-mix induced by Aroclor 1254) for 48 h.
Experiment 1: 0.05, 0.15, 0.5, 1.5, 5 and 150 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using plate incorporation method
Experiment 2: 5, 9, 19, 38, 75 and 150 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using pre-incubation method
Experiment 3: 0.5, 1.5, 5, 15, 50 and 150 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using plate incorporation method
Negative, vehicle and positive control groups were also included in mutagenicity tests.
The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation. Therefore, the sensitivity of the assay and the efficacy of the S9-mix were validated.
No signs of toxicity towards the tested strains could be observed. No visible reduction in the growth of the bacterial background lawn at any dose level. No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed in any of the experiments. No concentration-related increase over the tested range was found.
Under the test condition, thetest substance is not mutagenic with and without metabolic activation inSalmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535.
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, no classification is proposed.
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