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EC number: 269-041-1 | CAS number: 68186-45-8
- 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
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From July 29, 2011 to January 04, 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 012
- Report date:
- 2012
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: in vitro mammalian chromosome aberration test (migrated information)
Test material
- Reference substance name:
- Phosphoric acid, mono- and di-C6-10-alkyl esters
- EC Number:
- 269-616-7
- EC Name:
- Phosphoric acid, mono- and di-C6-10-alkyl esters
- Cas Number:
- 68307-94-8
- Molecular formula:
- No information available
- IUPAC Name:
- Esterification Products of Phosphorus Pentoxide and Alcohols C6-C10 (even numbered)
- Test material form:
- liquid
Constituent 1
Method
- Target gene:
- Not applicable.
Species / strain
- Species / strain / cell type:
- lymphocytes: human
- Details on mammalian cell type (if applicable):
- For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 16 h under typical experimental exposure conditions. Cell Culture: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at 37°C with 5% CO2 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbitone and beta-naphthoflavone induced rat liver, S9
- Test concentrations with justification for top dose:
- Preliminary Toxicity Test (Cell Growth Inhibition Test): The dose range of test substance used was 19.53 to 5000 µg/mL
Experiment 1: 4 (20) h without S9: 0*,6.25, 12.5, 25, 50*, 75*, 100*, 150, 200 µg/mL, 4 (20) h with S9: 0*, 6.25, 12.5, 25*, 50*, 75*, 100*, 150, 200, µg/mL
Experiment 2: 24 h without S9: 0*,12.5, 25, 50*, 75*, 100*, 200 µg/mL, 4 (20) h with S9: 0*, 12.5, 25, 50*, 75*, 100*, 200, µg/mL
*Dose levels selected for metaphase analysis - Vehicle / solvent:
- Dimethyl sulphoxide
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Details on test system and experimental conditions:
- Methods of application: In medium
Duration- Pre-incubation period: 48 h Exposure duration:
Experiment 1 – 4 h with and without S9.
Experiment 2 – 24 h without S9, 4 h with S9.
Expression time (cells in growth medium): 20 h for 4 h exposure
Selection time (in incubation with a selective agent): Not applicable
Fixation time (start of exposure up to fixation or harvest of cells): 24 h
Spindle inhibitor (Cytogenetic assays): Demecolcine
Stain (for cytogenetic assays): When slides were dry they were stained in 5% giemsa for 5 minutes, rinsed, dried and cover slipped using mounting medium.
Number of replications: Duplicate cultures
Number of cells evaluated: 100/culture
Determination of cytotoxicity - Method
Mitotic index – A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index as a percentage of the vehicle control value.
Scoring of Chromosome Damage
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
Other examinations
Determination of polyploidy: In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. - Evaluation criteria:
- A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
- Statistics:
- The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's exact test.
Results and discussion
Test results
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Refer to information on results and attached tables.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Results
Preliminary toxicity test (cell growth inhibition test): The mitotic index data are presented in Appendix 1 (5) and (6) (see attached background material - Appendix 1). The test substance exhibited clear evidence of dose-related toxicity in all three exposure groups. A precipitate was observed in the parallel blood-free cultures at the end of the exposure period initially at 156.25 µg/mL and 312.5 µg/mL without and with S9-mix, respectively. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 78.13 µg/mL in the continuous exposure and 4(20) h with S9, whereas metaphase cells were present at 156.25 µg/mL in the 4(20) h without S9 exposure group. Therefore, the dose selection for Experiments 1 and 2 was based on toxicity in accordance with the OECD 473 test guideline.
Chromosome aberration test - Experiment 1
The dose levels of the controls and the test substance are given in the table below:
Group Final concentration of Esterification products of Phosphorus Pentoxide and Alcohols C6-C10 (Even numbered) (µg/mL)
4 (20) h without S9: 0*, 6.25, 12.5, 25, 50*, 75*, 100*, 150, 200, MMC 0.4*
4 (20) h with S9: 0*, 6.25, 12.5, 25*, 50*, 75*, 100*, 150, 200, CP 5**
Dose levels selected for metaphase analysis
MMC: Mitomycin CCP: Cyclophosphamide
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to the test substance dose level of 100 µg/mL in the absence and presence of metabolic activation (S9). The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in Form 1, Appendix 2 (see attached background material - Appendix 2). These data show an approximate 50% growth inhibition was achieved at 100 µg/mL in the presence of S9. However, in the absence of S9, an 18% growth inhibition was observed at 100 µg/mL but there were no metaphases present above this dose level. The selection of the maximum dose level for metaphase analysis was based on toxicity, and was 100 µg/mL both in the absence and presence of S9, because there were no metaphases present above this dose level. The chromosome aberration data are given in Form 1, Appendix 2 (see attached background material - Appendix 2). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control substances induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test substance did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation (S9). The polyploid cell frequency data are given in Form 1, Appendix 2. The test substance did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.
Chromosome aberration test - Experiment 2:
The dose levels of the controls and the test substance are given in the table below:
Group Final concentration of Esterification products of Phosphorus Pentoxide and Alcohols C6-C10 (Even numbered) (µg/mL)
24 h without S9: 0*, 12.5, 25, 50*, 75*, 100*, 200, MMC 0.2*
4 (20) h with S9: 0*, 12.5, 25, 50*, 75*, 100*, 200, CP 5**
Dose levels selected for metaphase analysis
MMC: Mitomycin CCP: Cyclophosphamide
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test substance dose level of 100 µg/mL in the presence and absence of S9. The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in Form 2, Appendix 2 (see attached background material - Appendix 2). These data show 29% and 21% growth inhibition was achieved at 100 µg/mL in the absence and presence of S9, respectively. The selection of the maximum dose level for metaphase analysis was similar to Experiment 1, and was based on toxicity. The chromosome aberration data are given in Form 2, Appendix 2 (see attached background material - Appendix 2). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control substances induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test substance did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of metabolic activation. The polyploid cell frequency data are given in Form 2, Appendix 2 (see attached background material - Appendix 2). The test substance did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.
Applicant's summary and conclusion
- Conclusions:
- Under study conditions, the test substance was considered to be non-clastogenic to human lymphocytes in the chromosomal aberration assay, with and without metabolic activation
- Executive summary:
An in vitro study was conducted to determine the clastogenic potential of the test substance, mono-, di- and tri- C6-10 PSE, in cultured human lymphocytes, according to the OECD Guideline 473, EU Method B10, EPA OPPTS 870.5375, in compliance with GLP. Duplicate cultures of human lymphocytes, treated with the test substance, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, a 4 h exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20 h expression period (tested at 0, 6.25, 12.5, 25, 50, 75, 100, 150, 200 µg/mL test concentrations) and a 4 h exposure in the absence of metabolic activation (S9) with a 20 h expression period (tested at: 0, 6.25, 12.5, 25, 50, 75, 100, 150, 200 µg/mL test concentrations). In Experiment 2, the 4 h exposure with addition of S9 was repeated (using a 1% final S9 concentration) (tested at: 0, 12.5, 25, 50, 75, 100, 200 µg/mL test concentrations, mitomycin C 0.2 µg/mL); whilst in the absence of metabolic activation the exposure time was increased to 24 h (tested at: 0, 12.5, 25, 50, 75, 100, 200 µg/mL test concentrations, cyclophosphamide 5 µg/mL). The dose levels used in the main experiments were selected using data from the preliminary toxicity test. All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control substances induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. The test substance did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced or exceeded approximately 50% mitotic inhibition. Under study conditions, the test substance was considered to be non-clastogenic to human lymphocytes in the chromosomal aberration assay, with and without metabolic activation (XXXX, 2012).
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