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EC number: 931-714-9 | CAS number: 35255-57-3
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
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- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
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- Solubility in organic solvents / fat solubility
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- Flash point
- Auto flammability
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- 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
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- Endpoint summary
- Stability
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- 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
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- 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:
- 1996-06-24 to 1996-09-05
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 996
- Report date:
- 1996
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- (1992)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- micronucleus assay
Test material
- Reference substance name:
- Reaction mass of 2,2,4-trimethylhexane-1,6-diol and 2,4,4-trimethylhexane-1,6-diol
- EC Number:
- 931-714-9
- Cas Number:
- 35255-57-3
- Molecular formula:
- C9H20O2
- IUPAC Name:
- Reaction mass of 2,2,4-trimethylhexane-1,6-diol and 2,4,4-trimethylhexane-1,6-diol
- Test material form:
- other: liquid
- Details on test material:
- 2,2,4(or 2,4,4)-trimethyl-1,6-hexanediol of Hüls AG
purity 94.6 % (GC-FID area)
batch No. Pt. 15570/3033 of August 1995; sample ID 0637/81 770
Constituent 1
Test animals
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ORGANISMS:
- Source: Harlan Winkelmann, Borchen (Germany)
- Age: young adults
- Weight at study initiation: 27.8 +/- 5.6 g (males); 25.9 +/- 5.2 g (females)
- No. of animals per dose: 5 males + 5 females per test duration
- Housing: max 5 animals per sex per cage
- Diet (e.g. ad libitum): Ssniff R 10, complete feed for rats, Ssniff Spezialfutter GmbH, 59494 Soest, Germany
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark rhythm
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle: corn oil
- Details on exposure:
- ADMINISTRATION:
- Vehicle: corn oil
- Control groups and treatment:
negative: vehicle
positive: 100 mg cyclophosphamide (CPA)/kg bw in 0.9 % aqueous NaCl additional treated satellite group to replace mortalities
- Total volume applied: 10 ml/kg bw
- Duration of test: 24 hours; 48 hours
- Sampling times and number of samples: 24 hours; 48 hours - Duration of treatment / exposure:
- Duration of test: 24 hours; 48 hours
- Frequency of treatment:
- single dose
- Post exposure period:
- Sampling times and number of samples: 24 hours; 48 hours
Doses / concentrations
- Dose / conc.:
- 2 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 5 males + 5 females
- Control animals:
- yes
- yes, concurrent vehicle
- Positive control(s):
- positive: 100 mg cyclophosphamide (CPA)/kg bw in 0.9 % aqueous NaCl, oral gavage
Examinations
- Tissues and cell types examined:
- EXAMINATIONS:
- Organs examined at necropsy: femur bone marrow; others not specified in report approx. 2000 PCE (polychromatic erythrocytes) per animal were analysed for micronuclei (5000 in re-evaluation of 48 hour vehicle and test compound slides from males)
- Clinical observations: yes - Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
- Criteria for selection of M.T.D.: maximum dose <= 2000 mg/kg bw without mortalities within 48 hours
TREATMENT AND SAMPLING TIMES:
- Animals were killed by cervical dislocation 24 and 48 hours after test compound administration.
- Both femurs were dissected out from each animal, cleared of tissue and one epiphysis removed from each bone.
- The bone marrow was suspended in fetal calf serum (FCS) and erythrocytes were purified by means of a cellulose chromatography column.
- The eluate (3 x 1.5 ml) was centrifuged (5 min, 750 x g) and the pellet was again suspended in FCS/EDTA.
DETAILS OF SLIDE PREPARATION:
- The pure erythrocyte suspension was used to prepare "flat" cells on glass slides by means of a Shandon Cytospin 3 (2 slides per animal).
- Slides were air dried and stained with MayGrunwald/Giemsa.
METHOD OF ANALYSIS:
- The MIAMED image analyzer equipped with the "Micronucleus Test V 4.00" software was used for fully automated slide scoring.
- Where possible, a total of approximately 2000 PCE (i.e. 10,000 PCE per treatment group) were analyzed for micronuclei.
- The corresponding NCE were also scored for micronuclei. In addition, as an indicator for chemical-induced bone marrow toxicity, for each animal the PCE/NCE ratio was
determined on the basis of 2000 PCE scored.
- Evaluation criteria:
- - Criteria for evaluating results: Statistically significant and biologically relevant increase in frequency of micronucleated polychromatic erythrocytes of at least one test group as
compared to the negative control group of the same sampling time
Results and discussion
Test results
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- MORTALITY:
- Phase 1 of dose finding = 2000 mg/kg bw: No mortalities within 48 hours among 5 males + 5 females. No further phases were required.
- Main test: No mortalities
CLINICAL SIGNS: Predominant signs were hunched posture (only males), slight sedation (only males), piloerection.
NECROPSY FINDINGS: No necropsy reported.
EFFECT ON MITOTIC INDEX OR PCE/NCE RATIO:
- The average PCE/NCE ratio of the positive control groups was significantly lower than that of the corresponding vehicle controls
(0.27 +- 0.09 vs 0.83 +- 0.20 for males, 0.32 +- 0.13 vs 0.96 +- 0.29 for females).
- The PCE/NCE ratio of the male 48 hour dosed group (0.45 +- 0.14) was significantly lower than that of the corresponding vehicle control (0.93 +- 0.30).
GENOTOXIC EFFECTS:
- The micronucleus frequencies of the negative controls were within the range of historical control data of the performing laboratory. - For the positive control a significant increase in the frequency of micronucleated polychromatic erythrocytes was observed
(4.78 +- 0.88 vs 0.22 +- 0.09 for males; 5.90 +- 1.03 vs 0.06 +- 0.07 for females). - No significant increase in the frequency of micronucleated polychromatic erythrocytes over the control was found with the females treated with the test substance. - In males of the 48 hour sampling time a weak but statistically significant increase was observed (0.33 +- 0.12 vs 0.12 +- 0.10; 0.001 < p < 0.01).
- Statistical significance was reduced to 0.01 < p < 0.05 and the increase in micronucleus frequency was reduced to 0.26 +- 0.08 vs 0.16 +- 0.07 by scoring 5000 instead of 2000 PCE. According to Richold et al. (1990), the "statistical significance should increase with increased sample size ... if the response is real and not due to sampling error". Since an opposite trend was observed, and since in addition the final micronucleus frequency of 0.26 % is well within the range of historical negative controls of the performing laboratory (0.00 - 0.40 %; 0.20 +- 0.10), the present observation was not considered of being indicative of a clastogenic activity of the test compound. -The clinical symptoms indicate that the test substance or its metabolites had reached the blood and hence the target organ, i.e. the bone marrow.
Additional evidence for that comes from the reduction of the PCE/NCE ratio in male animals of the 48 hours sampling time, which is indicative of of bone marrow toxicity.
Treatment Sex Time % Micron PCE/NCE
in PCE
2000 T.S. m 24 h 0.25 +- 0.07 0.60 +- 0.26
2000 T.S. m 48 h 0.33 +- 0.12 ** 0.45 +- 0.14 **
2000 T.S. m 48 h 0.26 +- 0.08 * (1)
2000 T.S. f 24 h 0.16 +- 0.19 0.76 +- 0.21
2000 T.S. f 48 h 0.14 +- 0.10 1.21 +- 0.23
Vehicle m 24 h 0.22 +- 0.09 0.83 +- 0.20
Vehicle m 48 h 0.12 +- 0.10 0.93 +- 0.30
Vehicle m 48 h 0.16 +- 0.07 (1)
Vehicle f 24 h 0.06 +- 0.07 0.96 +- 0.29
Vehicle f 48 h 0.13 +- 0.08 1.47 +- 0.68
100 CPA m 24 h 4.78 +- 0.88 *** 0.27 +- 0.09 ***
100 CPA f 24 h 5.90 +- 1.03 *** 0.32 +- 0.13 ***
--------------------------------------------------------
T.S. = test substance (trimethylhexanediol; mg/kg bw)
CPA = cyclophosphamide (mg/kg bw)
* p < 0.05; ** p < 0.01; *** p < 0.001
(1) based on 5000 PCE/animal; other data based on 2000 PCE/animal
Any other information on results incl. tables
no other information
Applicant's summary and conclusion
- Conclusions:
- From the results obtained, it is concluded, that the test item shows no clastogenic activity in this in vivo mouse micronucleus assay.
- Executive summary:
In the in vivo mouse micronucleus assay, test item was tested for its potential to induce micronuclei in polychromatic erythrocytes (PCE) of NMRI mice.
In a preliminary toxicity test, 2000 mg/kg of test item were determined as the maximum tolerable dose.
In the main study, 2000 mg of test item/kg bodyweight were administered to male and female mice as a single oral dose (gavage). The negative control group received the vehicle, corn oil.
Animals of the positive control group were administered cyclophosphamide, at 100 mg/kg bodyweight.
Administration of each control substance was done by oral gavage, too. Five animals/sex/dose/sampling time were employed. Erythrocyte preparations were obtained from the negative and test compound groups at two sampling times, 24 and 48 hours after dosing. Erythrocytes from the positive control group were prepared 24 hours after dosing only. Slides were screened with an automatic image analyzer (LEITZ Miamed). One slide per animal was examined for the presence of micronuclei in at least 2000 PCEs (5000 PCEs in a re-evaluation of vehicle and test compound slides from the 48 hrs sampling time).
The ratio of PCE to normochromatic erythrocytes (NCE) as well as the incidence of micronucleated NCE was determined in parallel. At both sampling times, male and female mice treated with test item did not reveal biologically significant increases in the frequencies of micronucleated PCE.
The positive control compound, cyclophosphamide, induced highly significant increases in the frequency of micronucleated PCE, demonstrating the sensitivity of the test system.
From the results obtained, it is concluded, that the test item shows no clastogenic activity in this in vivo test system.
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