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EC number: 219-330-3 | CAS number: 2416-94-6
- 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
- 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 somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2003, July-November, day of application: 2003-08-04
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 003
- Report date:
- 2003
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- July 21, 1997
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- May 19, 2000
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- micronucleus assay
Test material
- Reference substance name:
- 2,3,6-trimethylphenol
- EC Number:
- 219-330-3
- EC Name:
- 2,3,6-trimethylphenol
- Cas Number:
- 2416-94-6
- Molecular formula:
- C9H12O
- IUPAC Name:
- 2,3,6-trimethylphenol
- Details on test material:
- - Name of test material (as cited in study report): Trimethylphenol
- Physical state: solid
- Analytical purity: 99.0 area% (analytical report dated 2003-07-25)
- Impurities (identity and concentrations): no data
- Purity test date: 2003-07-25
- Lot/batch No.: B 3020/03.06.2003
- Expiration date of the lot/batch: no data
- Stability under test conditions: yes, determined analytically
- Storage condition of test material: room temperature, N2 conditions
- Other: yellowish solidified melt
Constituent 1
Test animals
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: 29 g (mean)
- Assigned to test groups randomly: yes, computerized randomization plan
- Fasting period before study: no data
- Housing: individually in Makrolon cages, type MI
- Diet: standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water ad libitum; from bottles
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): no data; the animals were accommodated in fully air-conditioned rooms in which central air conditioning
- Photoperiod (hrs dark / hrs light): 12/12 (6.00 - 18.00 hours light/18.00 - 6.00 hours dark)
Administration / exposure
- Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: olive oil
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, olive oil was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
- Concentration of test material in vehicle: 10, 20, 30 g/l
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
- Lot/batch no. (if required): no data
- Purity: no data - Duration of treatment / exposure:
- single dose
- Frequency of treatment:
- single dose
- Post exposure period:
- one
Doses / concentrations
- Remarks:
- Doses / Concentrations:
100, 200, 300 mg/kg bw
Basis:
analytical conc.
- No. of animals per sex per dose:
- 5 males/group
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide ((Endoxan®, ASTA MEDICA, Reg. Nr. E 432-1) for clastogenic effects; solvent: water
- Justification for choice of positive control(s): The stability of CPP is well-defined under the selected conditions, since the positive control article is a well-established reference clastogen.
- Route of administration: i.p.
- Doses / concentrations: 20 mg/kg bw
vincristine sulphate (SIGMA - V 8879) for aneugenic effects; solvent water
- Justification for choice of positive control(s): The stability of VCR is well-defined under the selected conditions, since the positive control article is a well-established reference aneugen.
- Route of administration: i.p.
- Doses / concentrations: 0.15 mg/kg bw
Examinations
- Tissues and cell types examined:
- bone marrow
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute intraperitoneal toxicity, deaths were observed down to a dose of 450 mg/kg body weight . 400 mg/kg bw were survived by all animals, but led to clinical signs such as piloerection, squatting posture and the general state of the animals was poor. However, there were no distinct differences in the symptoms between males and females. Thus, only male animals were used for the cytogenetic investigations.
A dose of 400 mg/kg bw was selected as the highest dose in the present cytogenetic study. However, in the main experiment, 3 out of 10 animals died unexpectedly after test substance administration. Therefore the following doses were finally selected for the cytogenetic study: 300 mg/kg, 200 mg/kg and 100 mg/kg body weight.
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
At the beginning of the study, the animals were weighed and the substance to be administered or the amount of volume was related to the specific weight of the individual animals on the day of the experiment. All test substance formulations were prepared immediately before administration.
Groups of 5 males were given a single intraperitoneal dose if the test substance, the vehicle or of the positive control substances and were sacrificed at 24 and/or 48 hours after dosing (see freetext below).
The bone marrow was prepared according to the method described by SCHMID W [The micronucleus test for cytogenetic analysis. In : Hollaender, A. (ed), Chemical Mutagens, Principles and Methods for their Detection, Volume 4, Plenum Press, New York (1976 )] and SALAMONE M et al [Mutat Res 74: 347-356 (1980)]
• The two femora of the animals sacrificed by cervical dislocation were prepared by dissection and removing all soft tissues.
• After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 ml/femur).
• The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for ~ 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µl fresh FCS.
• 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained .
DETAILS OF SLIDE PREPARATION:
• The slides were stained in eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes.
• After having briefly been rinsed in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
• Subsequently, the slides were stained in Giemsa solution (15 ml Giemsa, 185 ml purified water) for about 15 minutes.
• After having been rinsed twice in purified water and clarified in xylene, the preparations were mounted in Corbit-Balsam.
METHOD OF ANALYSIS:
Microscopic evaluation
In general, 2000 polychromatic erythrocytes (PCEs) from each of the male animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) which occur are also scored. The following parameters are recorded :
• Number of polychromatic erythrocytes
• Number of polychromatic erythrocytes containing micronuclei
• Number of normochromatic erythrocytes
• Number of normochromatic erythrocytes containing micronuclei
• Ratio of polychromatic to normochromatic erythrocytes
• Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D = cell diameter)
Slides were coded before microscopic analysis. Since the absolute values shown were rounded but the calculations were made using the unedited values, there may be deviations in the given relative values. - Evaluation criteria:
- Acceptance criteria:
The mouse micronucleus test is considered valid if the following criteria are met:
• The quality of the slides must allow the identification and evaluation of a sufficient number of analyzable cells, i.e. => 2 000 PCEs and a clear differentiation between PCEs and NCEs.
• The ratio of PCEs/NCEs in the untreated animals (negative control) has to be within the normal range for the animal strain selected.
• The number of cells containing micronuclei in negative control animals has to be within the range of the historical control data both for PCEs and for NCEs.
• The two positive control substances have to induce a significant increase in the number of PCEs containing small and large micronuclei within the range of the historical control data or above.
Assessment criteria:
A finding is considered positive if the following criteria are met:
• Significant and dose-related increase in the number of PCEs containing micronuclei.
• The number of PCEs containing micronuclei has to exceed both the concurrent negative control and the highest value of the historical control range.
A test substance is considered negative if the following criteria are met:
• The number of cells containing micronuclei in the dose groups is not significantly above the negative control and is within the historical control data. - Statistics:
- The statistical evaluation of the data was carried out using the program system MUKERN (BASF Aktiengesellschaft).
The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there were significant differences between the control group and dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal was used as a criterion for the rank determination for the U test. Significances were identified as follows:
* p >=0.0 5; ** p <=0.0 1
Results and discussion
Test results
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
Any other information on results incl. tables
1. MICROSCOPIC EVALUATION
The single intraperitoneal administration of olive oil in a volume of 10 ml/kg body weight led to 1.0 ‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 1.0 ‰ after the 48-hour sacrifice interval.
After the single administration of the highest dose of 300 mg/kg body weight, 2.0 ‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 1 .2 ‰ after 48 hours.
In the two lower dose groups, rates of micronuclei of about 1.6 ‰ (200 mg/kg group) and 1.2 ‰ (100 mg/kg group) were detected after a sacrifice interval of 24 hours in each case.
With 19.5 ‰ the positive control substance cyclophosphamide for clastogenicity led to the expected clear increase in the number of polychromatic erythrocytes containing exclusively small micronuclei.
With 76.7 ‰ the positive control vincristine for spindle poison effects also led to a clearly enhanced number of polychromatic erythrocytes containing micronuclei with the expected amount of large micronuclei, i.e. 18.1 ‰.
The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals.
Thus, the test substance 2,3,6-trimethylphenol did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d > D/4) did not deviate from the vehicle control value at any of the sacrifice intervals and was within the historical control range.
No inhibition of erythropoiesis induced by the treatment of mice with 2,3,6 -trimethylphenol
was detected.
2 . CLINICAL EXAMINATIONS
The single intraperitoneal administration of the vehicle in a volume of 10 ml/kg body weight was tolerated by all animals without any signs or symptoms.
The administration of the test substance led to evident signs of toxicity.
Neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg body weight nor that of vincristine in a dose of 0 .15 mg/kg body weight caused any signs of toxicity.
Applicant's summary and conclusion
- Executive summary:
REPORT SUMMARY
The substance 2,3,6 -trimethylphenol was tested for chromosomal damage (clastogenicity) and for the ability to induce spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. For this purpose, the test substance, dissolved in olive oil, was administered once intraperitoneally to male animals at dose levels of 100 mg/kg, 200 mg/kg and 300 mg/kg body weight in a volume of 10 ml/kg body weight in each case. As a negative control, male mice were administered merely the vehicle, olive oil, by the same route, which gave frequencies of micronucleated polychromatic erythrocytes within the historical control range.
Both of the positive control chemicals, i.e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.
Animals which were administered the vehicle or the positive control substances cyclophosphamide or vincristine did not show any clinical signs of toxicity. The administration of the test substance led to evident signs of toxicity.
The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 300 mg/kg body weight and in the vehicle controls. In the test groups of 200 mg/kg and 100 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded.
According to the results of the present study, the single intraperitoneal administration of 2,3,6 -trimethylphenol did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the historical control data. No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.
Thus, under the experimental conditions chosen here, the test substance 2,3,6 -
trimethylphenol does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.
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