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EC number: 208-144-8 | CAS number: 512-56-1
- 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 vivo
Administrative data
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
Data source
Reference
- Reference Type:
- publication
- Title:
- In vivo Comet assay - statistical analysis and power calculations of mice testicular cells
- Author:
- Merete Kjær Hansen, Anoop Kumar Sharma, Marianne Dybdahl, Julie Boberg, Murat Kulahci
- Year:
- 2 014
- Bibliographic source:
- Mutation Research 774 (2014) 29–40
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
- GLP compliance:
- not specified
- Remarks:
- The animal study was performed under conditions approved by The Danish Agency of Protection of Experimental Animals and the in-house Animal Welfare Committee.
- Type of assay:
- mammalian comet assay
Test material
- Reference substance name:
- Trimethyl phosphate
- EC Number:
- 208-144-8
- EC Name:
- Trimethyl phosphate
- Cas Number:
- 512-56-1
- Molecular formula:
- C3H9O4P
- IUPAC Name:
- trimethyl phosphate
- Test material form:
- liquid
Constituent 1
Test animals
- Species:
- mouse
- Strain:
- CD-1
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Taconic MB, DK-4623, Lille Skensved, Denmark
- Age at study initiation: 5 weeks old
- Weight at study initiation: mean 29.8 +/- 1.2 g
- Assigned to test groups randomly: [no/yes, under following basis: ] The mice were randomly divided into dose groups.
- Housing: individually
- Diet (e.g. ad libitum): free access to feed (Altromin 1324,Lage, Germany)
- Water (e.g. ad libitum): tap water acidified with citric acid, pH = 3.5 (to prevent growthof microorganisms)
- Acclimation period: one week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1
- Humidity (%): 55 ± 5
- Air changes (per hr): 10 times/h
- Photoperiod (hrs dark / hrs light): 12 h/12h
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- Dosing suspensions were freshly prepared prior to each dosing occasion and given in a volume of 1 ml/100 g bw. All doses were placed on a magnetic stirrer until dosing. All animals were fasted overnight before the first dosing.
- Duration of treatment / exposure:
- 2 days
- Frequency of treatment:
- once per day
Doses / concentrationsopen allclose all
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 125 mg/kg bw/day (nominal)
- Dose / conc.:
- 250 mg/kg bw/day (nominal)
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- ethylmethanesulphonate (EMS)
- Doses / concentrations: 300 mg/kg bw
Examinations
- Tissues and cell types examined:
- right testicles
the DNA isolated from the testicular tissue origins from a mixture of different celltypes. - Details of tissue and slide preparation:
- SAMPLING TIMES (in addition to information in specific fields): Two to four hours after the second dosing the animals were anaesthetized in CO2/O2 and decapitated. After macroscopic examination, the testicles were excised and weighed. After removing the capsule the right testicles were put in cryotubes and used later in the Comet assay. Freezing medium was not added because it was an organ that was frozen and not cell suspensions. The cryotubes were immediately frozen in Mr. Frosty for about 1 h and then transferred to −80°C freezer until analyzed in the Comet assay.
DETAILS OF SLIDE PREPARATION: The cryotubes with the testicules from each mouse were added with 1.5 ml ice cold mincing solution (Hank’s balanced salt solution (Ca2+, Mg2+free) with 20 mM EDTA and 10% dimethyl sulfoxide). The testicules were gently crushed 4–5 times with a pastil. The solution was filtered through a 100 µm nylon filter (BD Falcon, Sigma–Aldrich, Denmark). Then, the solution was centrifuged at 1200 rpm for 5 min at 4°C (Eppendorf Centrifuge 5810R, Buch & Holm, Herlev, Denmark). The supernatant was removed and the pellet was resuspended in 1.5 ml mincing solution. This solution was filtered through a 100 µm nylon filter. Three microlitres of this suspension was mixed with 150 µl of the molten CometAssay TM LM Agarose (Trevigen, Gaithersburg, Maryland, US).Thirty microlitres of this mixture was applied onto one sample area of two gels ontwo different slides (one gel on one slide) consisting of 20 gels (CometSlideTMHT,Trevigen, Gaithersburg, Maryland, US). After solidification the embedded cells werelysed werelysed in a cold alkaline lysis buffer for 60 min. For DNA unwinding the slides were placed in the alkaline electrophoresis solution (pH > 13) in the electrophoresis jarat 4°C for 40 min, and electrophoresis was run in the same buffer for 30 min at 4°C(1 V/cm and 270 mA). After neutralization, fixation in 96% ethanol and DNA staining with 10 µl SYBR Green on all gels of the slides and a drop of antifade solution was added to each gel to avoid fading. Negative (Caco-2 cells in culture medium) and positive (Caco-2 cells exposed to 200 and 400 µM ethyl methanesulfonate for 30 min) controls were included with each 20 gels slide for each electrophoresis run. Testicule samples from each mouse were analyzed in two gels on two different slides.
METHOD OF ANALYSIS: Fully automatic Comet assay scoring was performed using the PathfinderTM Cellscan Comet imaging system (IMSTAR, Paris, France). Tail intensity (% tail DNA) of each comet was used. The number of cells scored on the gels depended on the cell density of the gels. In this study 100 random cells were subsequently selected from each gel unless it was explicitly stated otherwise, hence for each mouse 200 cells were scored (100 cells per gel and two gels per mouse). - Statistics:
- For each gel the following summary statistics were calcu-lated: the mean, median (50th), 55th, 60th, 65th, 70th, 75th, 80th, 85th, 90th and 95th percentile. The % tail DNA measured for each cell is naturally restricted to be non-negative and the distribution of the % tail DNA within each sample are strongly positively skewed. A popular endeavour to normalize the data and/or stabilize the variance of such data is to take the natural logarithm. Each of the statistics was thus calculated from the raw data as well as from data subjected to the natural logarithm. As some observations were recorded as zero, a small constant (0.001) was added todata when calculating the mean of the log-transformed data to avoid taken the logarithm of zero. Additionally, one summary statistic was calculated as the mean ofthe raw data and subsequently log-transformed. This measure will be referred to as the log(mean). In total, 23 candidate summary statistics were extracted. The summarized data was fitted using a linear mixed-effects model with dose as a fixed effect and animal as a random effect. The animals in one dose group are different from the animals in other dose groups, and this induces a nested structure in data.
Results and discussion
Test results
- Key result
- Sex:
- male
- Genotoxicity:
- positive
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
Applicant's summary and conclusion
- Conclusions:
- Trimethyl phosphate induced a significant positive effect at 500 mg/kg bw in the present study.
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