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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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: 251-311-5 | CAS number: 32961-44-7
- 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:
- weight of evidence
- Study period:
- October 1986 - March 1987
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 987
- Report date:
- 1987
Materials and methods
Test guideline
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- not applicable
- GLP compliance:
- yes
- Remarks:
- OECD-Grundsätze der Guten Laborpraxis (Bundesanzeiger Nr. 42a vom 4. Februar 1983)
- Type of assay:
- other: mammalian soma cell micronucleus assay
Test material
- Reference substance name:
- Isobutyl 4-chloro-3,5-diaminobenzoate
- EC Number:
- 251-311-5
- EC Name:
- Isobutyl 4-chloro-3,5-diaminobenzoate
- Cas Number:
- 32961-44-7
- Molecular formula:
- C11H15ClN2O2
- IUPAC Name:
- 2-methylpropyl 3,5-diamino-4-chlorobenzoate
- Test material form:
- solid: granular
- Remarks:
- brown crystalline granulate
- Details on test material:
- Test item was confirmed to be stable during study duration
Constituent 1
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerated
- Stability under test conditions: confirmed to be stable
Test animals
- Species:
- mouse
- Strain:
- NMRI
- Remarks:
- Bor:NMRI (SPF Han)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Breeder Winkelmann, Borchen
- Age at study initiation: ca. 9-12 weeks
- Weight at study initiation: 31-44 g
- Assigned to test groups randomly: yes
- Fasting period before study: not stated
- Housing: in groups of max. 3 resp. 5 mice, divided according to sex and treatment groups, in macrolone cages type I resp. II, on wooden soft bedding (Fa. Bogner GmbH, 5650 Solingen-Ohligs)
- Diet (e.g. ad libitum): "fixed -formula" ― Standarddiat Altromin 1324; Hersteller Altromin GmbH, Lage) ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 1 week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22°C (set as 22±2°C)
- Humidity (%): 45-54% (set as min. 50%)
- Air changes (per hr): at least 10/h
- Photoperiod (hrs dark / hrs light): 12/12
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: polyethylene glycol (Lutrol)
- Amount of vehicle (if gavage or dermal): 40 mL/kg initially, which were reduced to 24 mL/kg as the first treated animals died due to the inherent toxicity of Lutrol - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Test item was suspended in Lutrol and stirred with a magnetic stirrer until application. - Duration of treatment / exposure:
- single gavage, animals were decapitated either 24, 48, or 72 h after gavage
- Frequency of treatment:
- single gavage
- Post exposure period:
- animals were decapitated either 24, 48, or 72 h after gavage
Doses / concentrations
- Dose / conc.:
- 20 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 5/sex/dose
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide
- Route of administration: oral: gavage
- Doses / concentrations: 20 mg/kg
Examinations
- Tissues and cell types examined:
- femur bone marrow erythroblasts
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: Preliminary toxicity test with doses of 5, 10, 20, and 25 g/kg. All doses led to symptoms as apathy, diminished motility, ruffled fur, face-down position, cramps, difficulties breathing, orange coloured urine, diarrhea and lacking defecation. In the 25 g/kg dose group, 3 of 5 animals died.
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Single treatment, tissue was collected 24, 48, or 72 h after gavage (Controls only after 24h).
Sampling and preparation was done according to Schmid (SCHMID, W. Der Mikrokerntest. Deutsche Forschungsgemeinschaft. Kommission fur Mutagenitätsfragen. Mitteilung II 53—61 1975; Schmid W. The micronucleus—test for cytogenetic analysis. In: Hollander, A. (ed.), Chemical Mutagens, Principles and Methods for their Detection, Vol. 4 31-53 Plenum Press New York (1976)
DETAILS OF SLIDE PREPARATION:
Of each treated animal (no pretreatment with a spindle poison), at least one femur was preparated, in brief, using a syringe with calf serum and purgeing the bone. The collected cells were centrifuged and the sediment was spread onto the slide. Slides were stained with a staining machine Ames Hema-Tek Slide stainer by the company Miles.
METHOD OF ANALYSIS: As a rule, 1000 polychromatic erythrocytes per animals were analysed for micronuclei in a meandering course. In adition it is reasonable to determine the ratio of polychromatic and normochromatic cells. In case there were way more than 3000 normochromatic cells with 1000 polychromatic ones, without similar effects in other animals of the same treatment group, a pathologic, non-substance-related event is detected and the animal is excluded from assessment.
In addition to the number of normochromatic cells the number of normochromatic cells with micronuclei was determined. - Evaluation criteria:
- The treatment group with the highest mean value, provided that this value was above the value of the negative control group, as well as the positive control group, were evaluated with the distribution-free Wilcoxon rank test, with regard to the amount of polychromatic cells with micronuclei and of normochromatic cells. A difference is statistically significantly different if the error probability is <5% that the value of the treatment group is higher than the one of control. Evaluation of the rate of normochromatic cells with micronuclei was performed if already an elevated rate of polychromatic cells with micronuclei was seen. In this case, the group with the highest mean value was compared to control with the one-sided Chi²-Test.
In addition, the 1s ranges for all mean values were calculated. - Statistics:
- See evaluation criteria
Results and discussion
Test results
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- Treated mice showed signs of toxicity incl. apathy, diminished motility, ruffled fur, diarrhea and orange stained urine, 2/30 animals died.
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
In the preliminary toxicity test with doses of 5, 10, 20, and 25 g/kg, all doses led to symptoms as apathy, diminished motility, ruffled fur, face-down position, cramps, difficult breathing, orange coloured urine, diarrhea and lacking defecation. In the 25 g/kg dose group, 3 of 5 animals died.
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Not significantly over control
- Ratio of PCE/NCE (for Micronucleus assay): Treatment with the test item caused an inhibition of erythropoesis, Ratios of PCE/NCE are:
1000 : 1783 (1s = 771) (negative control)
1000 : 3189 (1s = 1741) (treatment group, 24h)
1000 : 2224 (1s = 1077) (treatment group, 48h)
1000 : 1755 (1s = 688) (treatment group, 72h)
- Appropriateness of dose levels and route: Treated mice showed signs of toxicity incl. apathy, diminished motility, ruffled fur, diarrhea and orange stained urine, 2/30 animals died.
Applicant's summary and conclusion
- Conclusions:
- The study was conducted similar to OECD guideline 474 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of Isobutyl 4-chloro-3,5-diaminobenzoate to induce micronuclei in vivo in NMRI mice.
There were no biologically or statistically significant differences between the treatment groups (20 g/kg test item) and the negative control with regard to polychromatic cells with micronuclei, relevant biological differences were not observed.
Hence, Isobutyl 4-chloro-3,5-diaminobenzoate should be regarded as non-clastogenic in vivo. - Executive summary:
Using the micronucleus test similar to OECD TG 474, Isobutyl 4-chloro-3,5-diaminobenzoate was investigated in male and female mice for a possible clastogenic effect on the chromosomes of bone marrow erythroblasts. The known clastogen and cytostatic agent cyclophosphamide served as a positive control.
The animals received a single administration. 24, 48 and 72 hours after the administration the femoral marrow of the Isobutyl 4-chloro-3,5-diaminobenzoate groups was prepared. Negative and positive control were sacrificed after 24 hours only. The dose of Isobutyl 4-chloro-3,5-diaminobenzoate was 20000 mg/kg body weight, and of the positive control cyclophosphamide 20 mg/kg. The test substance and the positive control cyclophosphamide were administered orally.
The animals treated with Isobutyl 4-chloro-3,5-diaminobenzoate showed lasting symptoms of toxicity after the administration. 2 of 30 treated animals died until the end of the test.
Erythrocyte formation, as measured by the ratio polychromatic to normochromatic erythrocytes, was time-dependent affected.
No indications of a clastogenic effect of Isobutyl 4-chloro-3,5-diaminobenzoate were found after a single treatment with 20000 mg/kg per os.
Cyclophosphamide, the positive control, had a clear clastogenic effect, as can be seen from the biologically relevant increase in polychromatic erythrocytes with micronuclei. An inhibition of erythropoiesis was not found here.
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.