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EC number: 235-285-2 | CAS number: 12158-74-6
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
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- Nanomaterial 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
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In vitro and in vivo studies are available.
The in vitro data performed on dicopper hydroxide phosphate has been
conducted in accordance with recommended guidelines and under the
conditions of GLP and is therefore considered to be reliable for use in
assessing the genotoxic potential of the material to be registered. In
addition two in vivo studies on the analogous material copper sulphate
are also available and as such the genotoxicity endpoint has been
fulfilled by applying a weight of evidence approach.
All available studies indicate that copper and copper compounds are not
genotoxic.
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:
- 29 November 2010 - 17 December 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- Sponsor's identification: Dicopper hydroxide phosphate
Description: light green powder
CAS number: 12158-74-6
Lot number: 90101
Date received: 28 August 2009
Storage conditions: room temperature in the dark - Target gene:
- Histidine for Salmonella.
- Species / strain / cell type:
- S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102, TA 1535
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- Conc 1: 50 mg/ml
Conc 2: 15.82 mg/ml
Conc 3: 5.01 mg/ml
Conc 4: 1.58 mg/ml
Conc 5: 0.5 mg/ml - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: phosphate buffered saline
- Justification for choice of solvent/vehicle: no data - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-acetylaminofluorene
- sodium azide
- benzo(a)pyrene
- mitomycin C
- other: Daunomycin, ICR 191
- Remarks:
- See table 1 for details on the use of positive controls
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: incubation period was 48 hours. - Key result
- Species / strain:
- S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102, TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- The results of the negative and positive controls confirm the sensitivity and accuracy of the test system, according to the requirements of the Moltox-kit. In general, the positive control frequencies should be at least 2.5 times the negative counts (spontaneous frequency).
- Conclusions:
- The test material was determined to be non-genotoxic under the conditions of the study.
- Executive summary:
The test material was determined to be non-genotoxic under the conditions of the study.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2016-06-28 to 2016-08-04
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- 2014
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- LPT Laboratory of Pharmacology and toxicology GmbH & Co. KG, Redderweg 8, 21147 Hamburg
- Type of assay:
- in vitro mammalian cell micronucleus test
- Specific details on test material used for the study:
- Batch no.: 9000023447
- Species / strain / cell type:
- lymphocytes: human
- Details on mammalian cell type (if applicable):
- Human peripheral blood was obtained by venipuncture from young (approximately 18 to 35 years of age), healthy, non-smoking male or female individuals with no known recent exposures to genotoxic chemicals or radiation.
- Cytokinesis block (if used):
- CytoB (Cytochalasin B) 5 μg/mL
- Metabolic activation:
- with and without
- Metabolic activation system:
- post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- 6.25, 12.5, 25, 50 and 100 μg/mL
In a preliminary experiment precipitate and/or cytotoxicity were detected with higher concentrations. - Vehicle / solvent:
- - Solvent used: 0.05 M HCl solution and culture medium
- Justification for choice of solvent: The test item was completely soluble at 1.25 mg per mL 0.05 M HCl solution. This solution was diluted with culture medium to obtain a final concentration of 100 μg/mL medium and was then further diluted to the appropriate lower concentrations. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: colchicine 0.02 μg/mL without metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 48 h
- Exposure duration: 4 and 24 h exposure
- Incubation time with inhibitor: 20 hours
- Fixation time: 24 or 44 h
SPINDLE INHIBITOR: CytoB (Cytochalasin B) 5 μg/mL
STAIN: 10 % Giemsa
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The cultures were centrifuged for 10 minutes at 800 rpm, the supernatant was discarded and the cells resuspended in KCl (0.56%). After incubation for 17 minutes at 37°C, the cell suspensions were centrifuged for 10 minutes at 800 rpm. The supernatant was discarded and 5 mL of freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid v/v) added. The cells were left in fixative for 30 minutes followed by centrifugation at 800 rpm. The supernatant was carefully removed and discarded, and the cell pellet was resuspended in about 0.5 mL of fresh fixative and 30% glacial acetic acid by repeated aspiration through a Pasteur pipette. Two drops of this cell suspension were dropped onto a prewarmed, pre-cleaned microscope slide and left to air-dry at room temperature.
NUMBER OF CELLS EVALUATED: at least 500 cells per replicate cell culture
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: micronucleus frequencies were analysed in at least 2000 binucleated cells per concentration
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Care was taken not to score binucleate cells with irregular shapes or where the two nuclei differ greatly in size; neither would binucleate cells be confused with poorly spread multi-nucleate cells. Cells containing more than two main nuclei were not analysed for micronuclei, as the baseline micronucleus frequency might be higher in these cells. Scoring of mononucleate cells is acceptable if the test item is shown to interfere with CytoB activity.
DETERMINATION OF CYTOTOXICITY
- Method: The evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI). The CBPI indicates the average number of cell cycles per cell during the period of exposure to CytoB, and is used to calculate cell proliferation. The RI indicates the relative number of nuclei in treated cultures compared to control cultures and can be used to calculate the % cytostasis. - Evaluation criteria:
- Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative control data (Poisson-based 95% control limits)
When all of these criteria are met, the test chemical is then considered able to induce chromosome breaks and/or gain or loss in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data (Poisson-based 95% control limits).
The test chemical is then considered unable to induce chromosome breaks and/or gain or loss in this test system. - Statistics:
- No details provided.
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 100 μg/mL and higher
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None
- Precipitation: only in pre-experiment 316.2 and 1000 μg/mL
RANGE-FINDING/SCREENING STUDIES:
A preliminary experiment without and with metabolic activation concentrations of 1.0, 3.162, 10.0, 31.62, 100, 316.2 and 1000 μg/mL was done.
CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells:
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: Test substance: 3.0 to 11.5 micronucleated cells per 1000 binucleated cells without activation, 9.0 to 12.0 micronucleated cells per 1000 binucleated cells with metabolic activation; Vehicle controls: 13.0 or 6.5 micronucleated cells per 1000 binucleated cells for the 4-hour and 24- hour exposure without metabolic activation, 2.5 micronucleated cells per 1000 binucleated cells with metabolic activation; positive control: 59.5 or 42.0 micronucleated cells per 1000 binucleate cells for the 4-hour and 24-hour without metabolic activation, 39.5 micronucleated cells per 1000 binucleate cells for the 4-hour exposure with metabolic activation
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI or RI - Conclusions:
- The test substance revealed no indications of chromosomal damage in the in vitro micronucleus test.
- Executive summary:
Test samples of Dicopper hydroxide phosphate were assayed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals. The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. The harvesting time was 20 hours after the end of exposure. The cytokinesis-block technique was applied. The test item was not soluble in any of the solvents recommended: aqua ad iniectabilia, dimethylsulfoxide (DMSO), ethanol or acetone. However, Dicopper hydroxide phosphate was completely soluble at 1.25 mg per mL 0.05 M HCl solution. This solution was diluted with culture medium to obtain a final concentration of 100 μg/mL medium and was then further diluted to the appropriate lower concentrations. 3.95 or 12.5 mg Dicopper hydroxide phosphate were suspended in 1 mL 0.05 M HCl solution. These suspensions were diluted with culture medium to obtain final concentrations of 316.2 or 1000 μg/mL medium, respectively, for the highest tested doses in the preliminary test for determination of cytotoxicity. The vehicle 0.05 M HCl solution served as the negative control. No correction factor was used. The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 1.0, 3.162, 10.0, 31.62, 100, 316.2 and 1000 μg Dicopper hydroxide phosphate/mL medium were employed. Test item precipitation was noted at concentrations of 316.2 and 1000 μg/mL in both experiments. In the experiments without and with S9 mix (24- or 4-hour exposure, respectively) cytotoxicity was noted at concentrations of 100 μg/mL medium and higher. Hence, 100 μg/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation with a 4-hour or 24-hour exposure. In the main study pronounced to complete cytotoxicity was noted at the top concentration of 100 μg Dicopper hydroxide phosphate/mL medium in the experiments without and with metabolic activation (4- or 24-hour exposure). Hence, the top concentration of 100 μg/mL was not scored for micronuclei. Mitomycin C (at 0.2 μg/mL) and colchicine (at 0.02 μg/mL) were employed as positive controls in the absence and cyclophosphamide (at 20 μg/mL) in the presence of metabolic activation.
Tests without metabolic activation (4- and 24-hour exposure)
The micronucleus frequencies of cultures treated with the concentrations of 6.25, 12.5, 25 and 50 μg Dicopper hydroxide phosphate/mL medium in the absence of metabolic activation (4- and 24-hour exposure) ranged from 3.0 to 11.5 micronucleated cells per 1000 binucleated cells. There was no dose-related increase in micronuclei up to the top concentration of 50 μg/mL medium. The frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls. Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test the following frequencies were observed: vehicle control: 13.0 or 6.5 micronucleated cells per 1000 binucleated cells for the 4-hour and 24- hour exposure, respectively. The vehicle result was within the historical control ranges. In the positive control cultures the micronucleus frequencies were increased to 59.5 or 42.0 micronucleated cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively. This demonstrated that Mitomycin C induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus.
Test with metabolic activation (4-hour exposure)
The micronucleus frequencies of cultures treated with the concentrations of 6.25, 12.5, 25 and 50 μg Dicopper hydroxide phosphate/mL medium (4-h exposure) in the presence of metabolic activation ranged from 9.0 to 12.0 micronucleated cells per 1000 binucleated cells. There was no dose-related increase in micronuclei up to the top concentration of 50 μg/mL medium. The frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls. Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test a mean frequency of 12.5 micronucleated cells per 1000 binucleated cells was observed. The vehicle result was within the historical control ranges. In the positive control culture the micronucleus frequency was increased to 39.5 micronucleated cells per 1000 binucleate cells for the 4-hour exposure. This demonstrated that cyclophosphamide induced significant chromosomal damage.
Conclusion
Under the present test conditions, Dicopper hydroxide phosphate tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test. The results for the vehicle controls were within historical control range. In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 07 March 2011 - 24 March 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: DIN EN ISO 10993-3, 4-09-SOP-11-153
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- Guideline 490 was not available when the study was performed.
- Deviations:
- not specified
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: in vitro mammalian cell gene mutation
- Specific details on test material used for the study:
- Sponsor's identification: Dicopper hydroxide phosphate
Description: light green powder
CAS number: 12158-74-6
Lot number: 90101
Date received: 28 August 2009
Storage conditions: room temperature in the dark - Target gene:
- Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI with 10% horse serum
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix. Supplier: Moltex USA
- Test concentrations with justification for top dose:
- Conc 1: 0.05 mg/ml
Conc 2: 0.025 mg/ml
Conc 3: 0.01 mg/ml
Conc 4: 0.005 mg/ml - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: cell culture medium
- Justification for choice of solvent/vehicle: no data - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Cell culture medium: RPMI = 10% horse serum
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- methylmethanesulfonate
- Remarks:
- - Methylmethane sufonate: 10 µg/ml without S9-mix, Benzo(a)pyrene: 1.5 µg/ml without S9-mix.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Cells were incubated with test material.
DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 48 hours
A part of the cells was used for the determination of cloning efficiency (CE 1). After 48 hours the cloning efficiency was determined again (CE 2). These determinations were made to give information on the cytotoxicity of the test material.
The mutation frequencies (MF) of each test group were defined after 14 days.
Cell culture conditions: 37°C, 5% CO2 , water saturated atmosphere - Evaluation criteria:
- The following parameters were determined after incubation:
- number of wells without colonies
- number of wells without big colonies
- number of wells without small colonies
The mutation frequency was calculated using the equation in Attachment 1. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- slight decreases in CE were seen.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- A decrease in the cloning efficiency (CE 1) of test material solutions in comparison with negative controls was found in test solution concentrations 1, 2 and 3. The positive control shows a slight decrease of CE 1.
A decrease in the cloning efficiency (CE 2) of test material solutions in comparison with negative controls was found in test solution concentrations 1, 2 and 3. The positive control shows a slight decrease of CE 2.
A significant increase of mutation frequencies was found only I the positive controls.
The presence of liver extract as a metabolic activation system (S9) has no effect on the test results.
The distribution of small and big colonies shows neither chromosome nor gene mutations.
The results conform to literature data and test validation. - Conclusions:
- No genotoxic activity was observed under the test conditions of the mouse lymphoma assay. The results of the negative and positive controls confirm the validity of the test.
- Executive summary:
No genotoxic activity was observed under the test conditions of the mouse lymphoma assay. The results of the negative and positive controls confirm the validity of the test.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- other: OECD 487 (In Vitro Mammalian Cell Micronucleus Test)
- Deviations:
- yes
- Remarks:
- Extended exposure, no metabolic activation system. Extracts of the test item were used.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Specific details on test material used for the study:
- Sponsor's identification: Dicopper hydroxide phosphate
Description: light green powder
CAS number: 12158-74-6
EC Number: 235-285-2
Sample number: 2011040713
Date received: 19 April 2011
Storage conditions: room temperature in the dark
Sample prepared under laminar flow. - Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- not applicable
- Test concentrations with justification for top dose:
- Dilutions of the test material extract: 8-fold, 24-fold and 72-fold
- Vehicle / solvent:
- - Preparation of extracts of test material (DIN ISO 10993-12)
0.563 g of test material was suspended in 13 ml extraction medium.
Extraction medium: Cell culture medium (RPMI1640 supplemented with 10% fetal calf serum) was used for extraction. The extraction was carried out by gentle agitation (about 20 rpm) in a shaker for 37°C for 24 hours. The saturated and particle containing suspension was centrifuged (3000 x g, 15 min), passed through a filter (0.2 µm pore size) and after finishing pH value of extract was controlled. Cell culture medium was used as a negative control and cell culture medium supplemented with mytomycin C acted as a positive control. - Untreated negative controls:
- yes
- Remarks:
- The negative controls (and the vehicle controls) were the cell cultures with cell culture medium RPMI 1640 supplemented with 10 % foetal calf serum.
- Negative solvent / vehicle controls:
- yes
- Remarks:
- The negative controls (and the vehicle controls) were the cell cultures with cell culture medium RPMI 1640 supplemented with 10 % foetal calf serum.
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in suspension
DURATION
- Preincubation period: 1 day
- Exposure duration: 24 hours
Cells were harvested after a further day of incubation.
STAIN (for cytogenetic assays): Giemsa solution
NUMBER OF REPLICATIONS: no data
NUMBER OF CELLS EVALUATED: 2 x 1000 cells per extract concentration and controls.
DETERMINATION OF CYTOTOXICITY
- Method: no data. The 8-fold dilution of the extract caused a permitted cytotoxicity between 50-60%.
DETERMINATION OF COMPELTED CELL CYCLE
- The cells were counted by staff member (before start and after ending of the assay). At least a doubling of cell counts per day was observed and is an evidence of passing a complete cell cycle run (mitosis) per day. - Evaluation criteria:
- The total number of cells and cells with none, one, two, three, four and more than four micronuclei were counted and logged. Cells incubated with the cell culture medium only (negative control) were the basis for the identification of a genotoxic potential. A 3-fold increase in the number of micronuclei in relation to the negative control was the threshold value.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- not specified
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- the 8-fold concentration produced between 50-60% cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- The acceptable rates of micronuclei in the negative controls show that the used cells at this time point were able to build micronuclei after an exemplary incubation with a genotoxic substance as such the results of this study are considered to be valid for use.
- Conclusions:
- Under the conditions of the study, the extracts of dicopper hydroxide phosphate were found to be non-genotoxic.
- Executive summary:
Under the conditions of the study, the extracts of dicopper hydroxide phosphate were found to be non-genotoxic.
Referenceopen allclose all
RESULTS
Table 2: Experiments with test strain TA 97a
Control mutagen: ICR 191 acridine
Experiment without S9-mix |
Experiment with S9-mix |
||
Mutagen µg/ml |
Number of revertants |
Mutagen µg/ml |
Number of revertants |
10 |
1191 |
100 |
493 |
Spontaneous revertants:131 |
Spontaneous revertants:127 |
||
Test preparation concentration |
Number of revertants |
Test preparation concentration |
Number of revertants |
50 mg/ml |
94 |
50 mg/ml |
80 |
15.82 mg/ml |
127 |
15.82 mg/ml |
93 |
5.01 mg/ml |
132 |
5.01 mg/ml |
88 |
1.58 mg/ml |
93 |
1.58 mg/ml |
109 |
0.5 mg/ml |
68 |
0.5 mg/ml |
76 |
No genotoxic activity observed.
Table 3: Experiments with test strain TA 98
Control mutagen: Daunomycin
Experiment without S9-mix |
Experiment with S9-mix |
||
Mutagen µg/ml |
Number of revertants |
Mutagen µg/ml |
Number of revertants |
60 |
555 |
100 |
338 |
Spontaneous revertants:21 |
Spontaneous revertants:21 |
||
Test preparation concentration |
Number of revertants |
Test preparation concentration |
Number of revertants |
50 mg/ml |
31 |
50 mg/ml |
33 |
15.82 mg/ml |
27 |
15.82 mg/ml |
25 |
5.01 mg/ml |
27 |
5.01 mg/ml |
21 |
1.58 mg/ml |
15 |
1.58 mg/ml |
25 |
0.5 mg/ml |
16 |
0.5 mg/ml |
20 |
No genotoxic activity observed.
Table 4: Experiments with test strain TA 100
Control mutagen: Sodium azide
Experiment without S9-mix |
Experiment with S9-mix |
||
Mutagen µg/ml |
Number of revertants |
Mutagen µg/ml |
Number of revertants |
15 |
>300 |
100 |
>300 |
Spontaneous revertants:65 |
Spontaneous revertants:79 |
||
Test preparation concentration |
Number of revertants |
Test preparation concentration |
Number of revertants |
50 mg/ml |
63 |
50 mg/ml |
68 |
15.82 mg/ml |
50 |
15.82 mg/ml |
60 |
5.01 mg/ml |
36 |
5.01 mg/ml |
41 |
1.58 mg/ml |
31 |
1.58 mg/ml |
44 |
0.5 mg/ml |
17 |
0.5 mg/ml |
47 |
No genotoxic activity observed.
Table 5: Experiments with test strain TA 102
Control mutagen: Mytomycin C
Experiment without S9-mix |
Experiment with S9-mix |
||
Mutagen µg/ml |
Number of revertants |
Mutagen µg/ml |
Number of revertants |
5 |
754 |
100 |
874 |
Spontaneous revertants:221 |
Spontaneous revertants:277 |
||
Test preparation concentration |
Number of revertants |
Test preparation concentration |
Number of revertants |
50 mg/ml |
213 |
50 mg/ml |
216 |
15.82 mg/ml |
192 |
15.82 mg/ml |
250 |
5.01 mg/ml |
211 |
5.01 mg/ml |
256 |
1.58 mg/ml |
205 |
1.58 mg/ml |
272 |
0.5 mg/ml |
145 |
0.5 mg/ml |
217 |
No genotoxic activity observed.
Table 6: Experiments with test strain TA 1535
Control mutagen: sodium azide
Experiment without S9-mix |
Experiment with S9-mix |
||
Mutagen µg/ml |
Number of revertants |
Mutagen µg/ml |
Number of revertants |
15 |
339 |
100 |
381 |
Spontaneous revertants:7 |
Spontaneous revertants:8 |
||
Test preparation concentration |
Number of revertants |
Test preparation concentration |
Number of revertants |
50 mg/ml |
10 |
50 mg/ml |
7 |
15.82 mg/ml |
7 |
15.82 mg/ml |
6 |
5.01 mg/ml |
7 |
5.01 mg/ml |
6 |
1.58 mg/ml |
7 |
1.58 mg/ml |
10 |
0.5 mg/ml |
4 |
0.5 mg/ml |
6 |
No genotoxic activity observed.
4 h exposure without metabolic activation
Concentration of test item [µg/mL] |
CBPI |
RI [%] |
Number of binucleate cells scored |
Number of micronucleated cells per 1000 binucleate cells |
0.05 M HCl |
1.43 |
100 |
2000 |
13 |
6.25 |
1.37 |
85 |
2000 |
11.5 |
12.5 |
1.4 |
93 |
2000 |
10.5 |
25 |
1.44 |
101 |
2000 |
9 |
50 |
1.24 |
55 |
2000 |
10.5 |
100 |
1.08 |
19 |
n.d. |
|
Mitomycin (0.2) |
1.27 |
62 |
2000 |
59.5 |
24 h exposure without metabolic activation
Concentration of test item [µg/mL] |
CBPI |
RI [%] |
Number of binucleate cells scored |
Number of micronucleated cells per 1000 binucleate cells |
0 |
1.22 |
80 |
2000 |
5.5 |
0.05 M HCl |
1.29 |
100 |
2000 |
6.5 |
6.25 |
1.32 |
110 |
2000 |
3 |
12.5 |
1.29 |
98 |
2000 |
3 |
25 |
1.28 |
96 |
2000 |
6.5 |
50 |
1.17 |
57 |
2000 |
5.5 |
100 |
n.d. |
n.d. |
n.d. |
|
Colchicine (0.02) |
1.25 |
88 |
2000 |
42 |
4 h exposure with metabolic activation
Concentration of test item [µg/mL] |
CBPI |
RI [%] |
Number of binucleate cells scored |
Number of micronucleated cells per 1000 binucleate cells |
0.05 M HCl |
1.26 |
100 |
2000 |
12.5 |
6.25 |
1.17 |
66 |
2000 |
12 |
12.5 |
1.21 |
79 |
2000 |
9 |
25 |
1.18 |
69 |
2000 |
10.5 |
50 |
1.14 |
52 |
2000 |
11 |
100 |
1.06 |
23 |
n.d. |
|
Cyclophosphamide (20) |
1.2 |
77 |
2000 |
39.5 |
n.d. = not determined due to cytotoxicity
Table 1: Mean of CE 1 and CE 2
Test item |
CE 1 |
CE 2 |
Positive control – S9 |
0.84 |
0.88 |
Positive control + S9 |
0.80 |
0.83 |
Negative control – S9 |
1.80 |
1.04 |
Negative control + S9 |
0.92 |
1.06 |
Concentration 1 – S9 |
0.56 |
0.71 |
Concentration 1 + S9 |
0.50 |
0.70 |
Concentration 2 – S9 |
0.54 |
0.83 |
Concentration 2 + S9 |
0.57 |
0.83 |
Concentration 3 – S9 |
0.82 |
0.94 |
Concentration 3 + S9 |
0.75 |
0.94 |
Concentration 4 – S9 |
1.06 |
0.99 |
Concentration 4 + S9 |
1.00 |
0.99 |
Table 2: Mutation Frequency (MF) for big and small colonies
Test item |
MF (big colonies) |
MF (small colonies) |
Positive control – S9 |
343.08 |
301.34 |
Positive control + S9 |
355.65 |
336.14 |
Negative control – S9 |
47.37 |
50.14 |
Negative control + S9 |
50.69 |
51.39 |
Concentration 1 – S9 |
78.80 |
73.67 |
Concentration 1 + S9 |
77.32 |
76.30 |
Concentration 2 – S9 |
68.98 |
61.11 |
Concentration 2 + S9 |
62.42 |
56.39 |
Concentration 3 – S9 |
58.03 |
56.50 |
Concentration 3 + S9 |
56.05 |
49.94 |
Concentration 4 – S9 |
50.51 |
49.06 |
Concentration 4 + S9 |
49.07 |
50.51 |
Table 1: Number of micronuclei detected
|
0 |
1 |
2 |
3 |
4 |
>4 |
number/1000 cells |
Negative Control (I) |
974 |
21 |
5 |
|
|
|
26 |
Negative Control (I) |
1039 |
23 |
3 |
|
|
|
25 |
Average of negative controls (I) and (II) |
25.5 |
||||||
Threshold value (based on average of negative controls (I) and (II)) |
76.5 |
||||||
8-fold dilution (saturated) extract (I) |
976 |
23 |
1 |
|
|
|
24 |
8-fold dilution (saturated) extract (II) |
970 |
24 |
5 |
1 |
|
|
30 |
24-fold dilution (saturated) extract (I) |
983 |
16 |
1 |
|
|
|
17 |
24-fold dilution (saturated) extract (II) |
971 |
22 |
7 |
|
|
|
29 |
72-fold dilution (saturated) extract (I) |
987 |
12 |
1 |
|
|
|
13 |
72-fold dilution (saturated) extract (II) |
982 |
18 |
6 |
|
|
|
24 |
0.8 µM Mitomycin C (positive control) (I) |
809 |
136 |
24 |
18 |
7 |
6 |
191 |
0.8 µM Mitomycin C (positive control) (II) |
814 |
147 |
13 |
19 |
2 |
5 |
186 |
Table 2: number of cells counted
SAMPLE |
NUMBER OF ALL CELLS COUNTED |
Negative Control (I) |
1000 |
Negative Control (I) |
1065 |
8-fold dilution (saturated) extract (I) |
1000 |
8-fold dilution (saturated) extract (II) |
1000 |
24-fold dilution (saturated) extract (I) |
1000 |
24-fold dilution (saturated) extract (II) |
1000 |
72-fold dilution (saturated) extract (I) |
1000 |
72-fold dilution (saturated) extract (II) |
1006 |
0.8 µM Mitomycin C (positive control) (I) |
1000 |
0.8 µM Mitomycin C (positive control) (II) |
1000 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Link to relevant study records
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
For further information please refer to read across justification in IUCLID section 13. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- not examined
Reference
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for classification or non-classification
The data set is considered to be adequate and reliable for the purpose of classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP) and as such dicopper hydroxide phosphate is not considered to be genotoxic.
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