Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 225-193-0 | CAS number: 4707-47-5
- 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
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 19 October, 1999 - 19 November, 1999
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 000
- Report date:
- 2000
Materials and methods
Test guideline
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- July 1997
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian erythrocyte micronucleus test
Test material
- Reference substance name:
- Methyl 2,4-dihydroxy-3,6-dimethylbenzoate
- EC Number:
- 225-193-0
- EC Name:
- Methyl 2,4-dihydroxy-3,6-dimethylbenzoate
- Cas Number:
- 4707-47-5
- Molecular formula:
- C10H12O4
- IUPAC Name:
- methyl 2,4-dihydroxy-3,6-dimethylbenzoate
- Test material form:
- other: solid
1
Test animals
- Species:
- mouse
- Strain:
- ICR
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Frederick, MD.
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: Males: 26.7 - 31.2 g; females: 24.8 - 30.0 g
- Assigned to test groups randomly: yes
- Housing: Five per cage in polycarbonate cages
- Diet: Free access to certified laboratory rodent chow
- Water: Free access to tap water
- Acclimation period: At least 5 days
ENVIRONMENTAL CONDITIONS set to maintain
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Administration / exposure
- Route of administration:
- intraperitoneal
- Vehicle:
- - Solvent used: corn oil
- Justification for choice of solvent: Corn oil was determined to be the solvent of choice based on a request by the Sponsor and compatibility of the vehicle with the test system animals.
- Concentration of test material in vehicle: The test article was workable in corn oil at 50 mg/mL, the maximum concentration tested in the study.
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test article was workable in corn oil at 50 mg/mL, the maximum concentration tested in the study. Dosing concentrations at 35 and 50 mg/mL were delivered to the test system as light yellow suspensions and concentrations below 35 mg/mL as light yellow solutions.
VOLUME: 20 mL/kg body weight - Duration of treatment / exposure:
- 24 and 48 hours
- Frequency of treatment:
- Single injection
Doses / concentrationsopen allclose all
- Dose / conc.:
- 0 mg/kg bw (total dose)
- Remarks:
- 24 and 48 hours exposure time
- Dose / conc.:
- 95 mg/kg bw (total dose)
- Remarks:
- 24 hours exposure time
- Dose / conc.:
- 190 mg/kg bw (total dose)
- Remarks:
- 24 hours exposure time
- Dose / conc.:
- 380 mg/kg bw (total dose)
- Remarks:
- 24 and 48 hours exposure time
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide
- Route of administration: intraperitoneal at 20 mL/kg body weight
- Doses / concentrations: Dissolved in sterile distilled water at a concentration of 2.5 mg/mL
Examinations
- Tissues and cell types examined:
- Polychromatic erythrocytes (2000) were scored for the presence of micronuclei
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
Based on the observed mortality in the toxicity assay, dose levels were selected for the micronucleus assay. Intraperitoneal injection was selected to maximize delivery of the test substance to the test system.
DETAILS OF SLIDE PREPARATION:
At the scheduled sacrifice times, mice were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cellw were transferred to a capped centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.
METHOD OF ANALYSIS:
Using oil immersion, 2000 polychromatic erythrocytes were scored for the presence of micronuclei which are defined as round, darkly staing nuclear fragments, having a sharp contour with diameters usually 1/20 to 1/5 of the erythrocyte. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.
- Evaluation criteria:
- Evaluation of the results:
The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and treatment group. Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970). All analysis were performed separately for each sex and sampling time.
In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group.
The test substance was considered to induce a positive response if a dose-reponsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p≤0.05, Kastenbaum-Bowman tables) at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or unconfirmed positive and a repeat assay recommended. The test substance was considered negative if no statistically increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.
Criteria for a valid test:
The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the vehicle control. The incidence of micronucleated polychromatic erythrocyte in the positive control group must be significantly increased relative to the vehicle control group (p≤0.05, Kastenbaum-Bowman tables). - Statistics:
- See evaluation criteria.
Results and discussion
Test results
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: The substance was administered by intraperitoneal injection to male and female mice at 300, 500, 700 or 1000 mg test substance/kg body weight which was administered in a total volume of 20 mL test substance-vehicle mixture/kg body weight. Mortality occurred after dose administration as follows: 2/5 males and 4/5 females at 500 mg/kg bw and in all males and females at 700 and 1000 mg/kg bw. The LD50/3 was calculated by probit analysis to be approximately 487 mg/kg bw for male and female mice. The high dose for the micronucleus test was set at 380 mg/kg bw for male and female mice which was estimated to be approximately 80% of the LD50/3.
- Solubility: The test article was workable in corn oil at 50 mg/mL, the maximum concentration tested in the study.
- Clinical signs of toxicity in test animals: Clinical signs, which were noted after dose administration, included: lethargy and piloerection in males and females at 300 and 500 mg/kg bw.
RESULTS OF DEFINITIVE STUDY
- Mortality/clinical signs: No mortality occurred at any dose level during the course of the micronucleus study. Clinical signs, which were noted after dose administration, included: lethargy and piloerection in males and females at 380 mg/kg bw. All other mice treated with test and control substances appeared normal during the study.
- Induction of micronuclei (for Micronucleus assay): No siginificant increase in micronucleated polychromatic erythrocytes in test substance-treated groups relative to the repsective vehicle control groups was observed in male or female mice at 24 hours and in males at 48 hours after dose administration (p≤0.05, Kastenbaum-Bowman). A statistically significant increase was observed in female mice at 48 hours after treatment with 380 mg/kg bw (p≤0.05, Kastenbaum-Bowman tables). However, this increase was not biologically significant based on the lack of dose response at 24 hour harvest time and the maximum number of micronuclei observed per animal. The number of micronuclei in two females (3/2000 polychromatic erythrocytes) was the same as in the control female animal at 24 hour harvest time and was within the historical solvent control range. Based on these facts, the increase was judged not to be biologically significant. CP induced a siginificant increase in micronucleated polychromatic erythrocytes in both male and female mice (p≤0.05, Kastenbaum-Bowman tables).
- Ratio of PCE/NCE (for Micronucleus assay): Slight reductions of 1% to 17% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test substance treated groups relative to their respective vehicle controls. Reductions were observed in male dose group 24 hours after treatment with 95 mg/kg bw, in female dose group 24 hours after treatment with 190 mg/kg bw and in male and female dose groups 24 and 48 hours after treatment with 380 mg/kg bw. The reductions in the frequency of polychromatic erythrocytes in the bone marrow suggest that the sunstance did not inhibit erythropoiesis.
- Appropriateness of dose levels and route: Intraperitoneal injection was selected to maximize delivery of the test substance to the test system.
Applicant's summary and conclusion
- Conclusions:
- A micronucleus study with the substance was performed equivalent to OECD 474 guideline and GLP principles, in male and female mice. It is concluded that the substance is not mutagenic in the mouse micronucleus assay.
- Executive summary:
In an in vivo micronucleus study, male and female mice were exposed to 95, 190 and 380 mg/kg bw of the substance, performed equivalent to OECD 474 guideline and GLP principles.
No mortality occurred at any dose level during the course of the micronucleus study. Clinical signs, which were noted after dose administration, included: lethargy and piloerection in males and females at 380 mg/kg bw. All other mice treated with test and control substances appeared normal during the study. Reliable positive and negative controls were included.
No siginificant increase in micronucleated polychromatic erythrocytes in test substance-treated groups relative to the repsective vehicle control groups was observed in male or female mice at 24 hours and in males at 48 hours after dose administration (p≤0.05, Kastenbaum-Bowman). A statistically significant increase was observed in female mice at 48 hours after treatment with 380 mg/kg bw (p≤0.05, Kastenbaum-Bowman tables). However, this increase was not biologically significant based on the lack of dose response at 24 hour harvest time and the maximum number of micronuclei observed per animal. The number of micronuclei in two females (3/2000 polychromatic erythrocytes) was the same as in the control female animal at 24 hour harvest time and was within the historical solvent control range. Based on these facts, the increase was judged not to be biologically significant. CP induced a siginificant increase in micronucleated polychromatic erythrocytes in both male and female mice (p≤0.05, Kastenbaum-Bowman tables).
Slight reductions of 1% to 17% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test substance treated groups relative to their respective vehicle controls. Reductions were observed in male dose group 24 hours after treatment with 95 mg/kg bw, in female dose group 24 hours after treatment with 190 mg/kg bw and in male and female dose groups 24 and 48 hours after treatment with 380 mg/kg bw. The reductions in the frequency of polychromatic erythrocytes in the bone marrow suggest that the substance did not inhibit erythropoiesis.
It is concluded that the substance is not mutagenic in the mouse micronucleus assay.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.