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EC number: 202-532-0 | CAS number: 96-76-4
- 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
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 008
- Report date:
- 2008
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
Test material
- Reference substance name:
- 2,4-di-tert-butylphenol
- EC Number:
- 202-532-0
- EC Name:
- 2,4-di-tert-butylphenol
- Cas Number:
- 96-76-4
- Molecular formula:
- C14H22O
- IUPAC Name:
- 2,4-di-tert-butylphenol
- Test material form:
- solid
Constituent 1
- Specific details on test material used for the study:
- - Name of test material (as cited in study report): TK 12891/1 (also known as 2,4-di-tert.-butylphenol)
- Physical state: yellow solid
- Analytical purity: 99.1%
- Supplier: Sigma-Aldrich
- Lot/batch No.: S43419-228
- Expiration date of the lot/batch: 24 July 2009
- Storage condition of test material: stored at ambient temperature in the dark when not in use
Test animals
- Species:
- rat
- Strain:
- other: CD rats
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River UK
- Weight at study initiation: mean(males): 266 - 312 g; mean(females): 160 - 193 g
- Housing: in sets of twos and threes
- Diet: SDS Rat and Mouse Maintenance Diet No. 1 which was obtained from Special Diet Services Limited, England. (ad libitum)
- Water: tap water (ad libitum)
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 22
- Humidity (%): 46 - 87
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: corn oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The dose volume used for both the control and test item treated animals was a constant 10 mL/kg body weight.
- Duration of treatment / exposure:
- Bone marrow samples were taken 48 h after the initial 0 h dose.
- Frequency of treatment:
- Twice (Rats were dosed at 0 h and 24 h.)
- Post exposure period:
- Bone marrow samples were taken 48 h after the initial 0 h dose.
Doses / concentrationsopen allclose all
- Dose / conc.:
- 200 mg/kg bw/day (nominal)
- Remarks:
- Females
- Dose / conc.:
- 400 mg/kg bw/day (nominal)
- Remarks:
- Females
- Dose / conc.:
- 800 mg/kg bw/day (nominal)
- Remarks:
- Females
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Remarks:
- Males
- No. of animals per sex per dose:
- - vehicle control: 5 males and 5 females;
- low/mid dose: 5 females each;
- high dose: 10 males and 10 females (The high dose group of rats consisted of an increased group size of 10 males and 10 females of which 5 males and 5 females provided the regular assessment base. The additional rats were processed in normal fashion and the slides labelled with the original animal number. The slides from this spare group were kept as a contingency in case of unscheduled deaths or potential sex differences. In the event of death, the first available animal in the relevant contingency group replaced the missing animal. Preparations were made from remaining contingency group animals and the slides were kept as spares.);
- positive control: 5 males; - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide
- Doses / concentrations: 50 mg/kg bw/day (Cyclophosphamide was prepared fresh as a 5 mg/mL solution in distilled water. It was administered to the positive control animals in dose volumes of 10 mL/kg to give the required target dose of 50 mg/kg)
Examinations
- Tissues and cell types examined:
- bone marrow cells
- Details of tissue and slide preparation:
- DETAILS OF SLIDE PREPARATION:
A drop of the suspension including bone marrow cells was placed at one end of the slide and a smear made by drawing the top of a Pasteur pipette horizontally along the slide. Two slides were prepared from each tube/animal. The smear was left to air dry, fixed in methanol for ca 5 min and then immersed for 15 min in 15% Giemsa stain, prepared in tap water, to give optimum erythrocyte discrimination. The stained smears were finally rinsed in distilled water for ca 1 min and left to air dry overnight. Permanent slide preparations were made by sealing coverslips onto the glass slides using DPX mounting medium.
METHOD OF ANALYSIS:
The better of the 2 prepared slides was selected for examination and the coded slides assessed blind by the same operator. At least two thousand (2000) polychromatic erythrocytes (PCE) per animal were scored for micronuclei and the frequency of micronucleated cells (MN-PCE) determined. As a control against inclusion of artefacts, or action of a mutagen on the G2 and/or mitotic phase of the cell cycle, the numbers of micronucleated normochromatic erythrocytes (MN-NCE) in mature red blood corpuscles were also recorded (Maier and Schmid, 1976; Hamoud et al, 1989). In addition, scored micronuclei were assigned on the basis of size into small or large categories, historically defined as micronuclei occupying less or more than 25% of the visible cellular area. This classification provided a non-specific measure of compound induced spindle dysfunction, as large micronuclei appear to derive from lagging chromosomes caused by damage to the mitotic apparatus during bone marrow erythropoiesis (Yamamoto and Kikuchi, 1980; Vanderkerken et al, 1989). The PCE/NCE ratio, a measure of any induced systemic toxicity, was determined by counting a minimum total of 1000 erythrocytes (PCE + NCE) per marrow preparation.
- Positive Response:
The test would be judged positive if an increase in the number of micronucleated polychromatic erythrocytes (MN-PCE) was obtained for one or more of the test item treated dose groups. That is, an increase greater than 10% over the expected historical control ranges for a group of animals. The increase observed should be biologically relevant and statistically significant relative to concurrent and historical control frequencies for MN-PCE and/or MN-NCE induction.
- Inconclusive Response:
The test would be considered inconclusive if the levels of MN-PCE within any one dose group were increased above the established historical control frequencies for MN-PCE induction, but not high enough to meet the criteria for a positive response. That is an increase up to 10% over the maximum negative control frequency for a group of animals. - Evaluation criteria:
- Acceptance Criteria:
- The prepared slides had uniform staining properties and sufficient number of PCE cells present to allow accurate micronucleus determination.
- The assay was considered acceptable as the MN-PCE frequencies for the vehicle control dosed rats were within the expected historical range. The ranges are defined in accordance with Charles River Laboratories experience of the bone marrow micronucleus test using CD rats.
- An adequate positive control response for at least 2 animals and the dose group as a whole.
Evaluation Criteria:
The average micronucleus incidence in vehicle control dosed and untreated CD rats, has in this laboratory been determined as 0.06±0.05% , a range of 0.01-0.13% per group of 5-6 animals and 0.03-0.11% per group of 10-12 animals. This frequency is in agreement with published data for micronucleus tests with CD rats (Tamura et al, 1990; Salamone and Mavournin, 1994). These historical data have been used in the evaluation of response in this test.
- Negative Response:
The test would be judged negative if no biologically relevant increases in the numbers of MN-PCE were observed, relative to the concurrent and established historical control frequencies for MN-PCE induction. No statistical analysis will be performed if the levels of MN-PCE induction fell within the determined historical control frequencies. A similar biological approach to the data, which avoids the need for statistical evaluations, has recently been described (Ashby and Tinwell, 1995). Variations in the MN-NCE frequencies and PCE/NCE ratios will also not be analysed statistically, unless clearly different from concurrent control values. - Statistics:
- Not required
Results and discussion
Test results
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- There were indications of bone marrow toxicity in the high dose males (PCE/NCE ratio of 0.43 compared to 0.56 in vehicle control group).
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
As toxicity information on the test item was available from the Sponsor, a limit toxicity test (3 males/3 females) was conducted prior to the micronucleus test to establish a suitable dose range for the micronucleus experiment. The limit toxicity study used a starting dose of 1600 mg/kg. Mortality occured at the ranger-finder doses of 1000, 1200, 1400 and 1600 mg/kg bw. At 1600 mg/kg bw each one of three males and females died on day 3 after dosing. Based on the findings of the toxicity study, the maximum tolerated doses were judged to be in the region of 1000 mg/kg/day for males and 800 mg/kg/day for females.
RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
- Induction of micronuclei (for Micronucleus assay): in the range of the negative control
- Ratio of PCE/NCE (for Micronucleus assay): There were indications of bone marrow toxicity in the high dose males (PCE/NCE ratio of 0.43 compared to 0.56 in vehicle control group).
Any other information on results incl. tables
Test group | Sex | No. of rats scored | erythrocytes | ||||
Normochromatic cells (NCE) | Polychromatic cells (PCE) | PCE/NCE Mean +/- S.D. | |||||
No. of MN-NCE | PCE analysed | No. of MN-PCE | % MN-PCE | ||||
vehicle (20 ml corn oil kg/day) | male | 5 | 8 | 10004 | 7 | 0.07 | 0.53 +/- 0.06 |
female | 5 | 4 | 10008 | 4 | 0.04 | 0.58 +/- 0.05 | |
male/female | 10 | 12 | 20012 | 11 | 0.05 | 0.56 +/- 0.06 | |
200 mg /kg/day | female | 5 | 1 | 10009 | 6 | 0.06 | 0.67 +/- 0.12 |
400 mg /kg/day | female | 5 | 3 | 10012 | 5 | 0.05 | 0.58 +/- 0.13 |
1000 mg /kg/day | male | 5 | 9 | 10011 | 3 | 0.03 | 0.43 +/-0.08 |
800 mg /kg/day | female | 5 | 2 | 10008 | 5 | 0.05 | 0.59 +/- 0.12 |
50 mg Cyclophosphamide /kg/day | male | 5 | 53¿ | 10002 | 189¿ | 1.89 | 0.34 +/- 0.07 |
PCE = polychromatic erythrocyts | |||||||
MN-PCE = micronucleated PCE | |||||||
NCE = normochromatic erythrocyts | |||||||
MN-NCE = micronucleated NCE | |||||||
¿= positive response in PCE | |||||||
¿ = evident response in NCE |
No animal deaths occurred following dosing. Clinical signs of hunched, subdued behaviour, wet around anus, wet staining pergenital, wet faeces, piloerection, salivation red discharge (nose), laboured breathing and staggering were observed. There was no indication that the test item induced bone marrow micronuclei in the treated rats. The highest MN-PCE frequency recorded for the test item was in the low dose females where an incidence of 0.06% was observed. There were indications of bone marrow toxicity in the high dose males (PCE/NCE ratio of 0.43 compared to 0.56 in vehicle control group).
Applicant's summary and conclusion
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