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EC number: 207-346-3 | CAS number: 464-06-2
- 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
![](https://echa.europa.eu/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/print_toxicological-information.png)
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
There is no in vitro genetic toxicity data available for 2,2,3-Trimethylbutane. However, data is available for structural analogues, Hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics and 2,2,4-trimethylpentane and presented in the dossier. This data is read across to based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.
The read across genetic toxicity tests listed below had negative results for 2,2,3-Trimethylbutane.
Genetic Toxicity in vitro – Bacterial reverse mutation assay (OECD 471)
Genetic Toxicity in vitro – Mammalian Chromosome Aberration Test (OECD TG 473)
Genetic Toxicity in vitro – Mammalian Cell Gene Mutation Test (OECD TG 476)
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- September - October 1982
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Test procedure according to national standards
- Justification for type of information:
- A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- - limited documentation of cytotoxicity data
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- S. typhimurium strains: his-operon
E. coli strains: tryptophan-operon - Species / strain / cell type:
- S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- E. coli, other: WP2, WP2 uvr A
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver homogenate S9 activation, 10 % S9 liver homogenate from Aroclor treated rats
- Test concentrations with justification for top dose:
- TA 1538 (-S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000; (+S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000 µg/mL
TA 1538 (-S) mix): 0, 15.62, 31.25, 62.5, 125, 250, 500, 1000; (+S9 mix): 0, 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000 µg/mL
TA 1537 (-S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000; (+S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000 µg/mL
TA 1537 (-S9 mix): 0, 3.9, 7.81, 15.62, 31.25, 62.5, 125; (+S9 mix): 0, 31.25, 62.5, 125, 250, 500, 1000, 2000 µg/mL
TA 1535 (-S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000; (+S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000 µg/mL
TA 100 (-S9 mix): 0, 0.31, 0.62, 1.25, 2.5, 5, 10; (+S9 mix): 0, 7.81, 15.62, 31.25, 62.5, 125, 250 µg/mL
TA 100 (-S9 mix): 0, 31.25, 62.5, 125, 250, 500; (+S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000 µg/mL
TA 100 (-S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000; (+S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000 µg/mL
TA 98 (-S9 mix): 0, 0.31, 0.62, 1.25, 2.5, 5, 10; (+S9 mix): 0, 7.81, 15.62, 31.25, 62.5, 125, 250 µg/mL
TA 98 (-S9 mix): 0, 3.9, 7.81, 15.62, 31.25, 62.5, 125; (+S9 mix): 0, 31.25, 62.5, 125, 250, 500, 1000, 2000 µg/mL
TA 98 (-S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000; (+S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000 µg/mL
EC WP2 (-S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000; (+S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000 µg/mL
EC WP2uvrA (-S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000; (+S9 mix): 0, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Tween 80 and ethanol
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: TA 1538, TA 98, TA 100: benzo(a)pyrene; TA 1537: neutral red; TA 1535: Sodium azide; and E. coli strains: 4-nitroquinoline-N-oxide
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 30 min
- Incubation period: 48-72 hours
NUMBER OF REPLICATIONS: All test were carried out in triplicate.
Two replicate assays were carried out on different days to confirm reproducibility of results. - Statistics:
- none
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Concentrations up to 8000 µg/mL in agar layer cultures did not increase the reverse mutation frequency of S. typhimurium strains either in the presence or absence of a rat liver microsomal activation system.
- Cytotoxicity / choice of top concentrations:
- other: There was a differential toxicity of the test material to individual strains of Salmonella and the reduction of toxicity in the presence of an S9 mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Concentrations up to 8000 µg/mL in agar layer cultures did not increase the reverse mutation frequency of S. typhimurium strains either in the presence or absence of a rat liver microsomal activation system.
- Cytotoxicity / choice of top concentrations:
- other: There was a differential toxicity of the test material to individual strains of Salmonella and the reduction of toxicity in the presence of an S9 mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Concentrations up to 8000 µg/mL in agar layer cultures did not increase the reverse mutation frequency of S. typhimurium strains either in the presence or absence of a rat liver microsomal activation system.
- Cytotoxicity / choice of top concentrations:
- other: There was a differential toxicity of the test material to individual strains of Salmonella and the reduction of toxicity in the presence of an S9 mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Concentrations up to 8000 µg/mL in agar layer cultures did not increase the reverse mutation frequency of S. typhimurium strains either in the presence or absence of a rat liver microsomal activation system.
- Cytotoxicity / choice of top concentrations:
- other: There was a differential toxicity of the test material to individual strains of Salmonella and the reduction of toxicity in the presence of an S9 mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Concentrations up to 8000 µg/mL in agar layer cultures did not increase the reverse mutation frequency of S. typhimurium strains either in the presence or absence of a rat liver microsomal activation system.
- Cytotoxicity / choice of top concentrations:
- other: There was a differential toxicity of the test material to individual strains of Salmonella and the reduction of toxicity in the presence of an S9 mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Concentrations up to 8000 µg/mL in agar layer cultures did not increase the reverse mutation frequency of E. coli strains either in the presence or absence of a rat liver microsomal activation system
- Cytotoxicity / choice of top concentrations:
- not specified
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- Concentrations up to 8000 µg/mL in agar layer cultures did not increase the reverse mutation frequency of E. coli strains either in the presence or absence of a rat liver microsomal activation system
- Cytotoxicity / choice of top concentrations:
- not specified
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- There was no evidence of test compound precipitation in the assay system.
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results:
negative with metabolic activation
negative without metabolic activation
The purpose of this study was to determine the mutagenicity of the test substance hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics. An Ames reverse mutation assay was done on S. typhimurium strains, TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and E. coli strains WP2 uvr A and WP2, both with and without metabolic activation. Species were tested at concentrations ranging from 0.31 to 8000 ug/plate. The test plates were incubated for 48 -72 hrs, and then the number of colonies were counted. For all species, there was no significant increase in the number of revertants as compared to negative controls. Positive controls showed a significant increase in mutations. The test substance is not mutagenic. - Executive summary:
The purpose of this study was to determine the mutagenicity of the test substance hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics. An Ames reverse mutation assay was done on S. typhimurium strains, TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and E. coli strains WP2 uvr A and WP2, both with and without metabolic activation. Species were tested at concentrations ranging from 0.31 to 8000 ug/plate. The test plates were incubated for 48 -72 hrs, and then the number of colonies were counted. For all species, there was no significant increase in the number of revertants as compared to negative controls. Positive controls showed a significant increase in mutations. The test substance is not mutagenic.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Study meets generally accepted scientific principles, acceptable for assessment.
- Justification for type of information:
- A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- - no A-T base pair tester strains included (e.g. TA102 or E.coli); limited documentation
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- yes
- Remarks:
- - no A-T base pair tester strains included (e.g. TA102 or E.coli); limited documentation
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his-operon
- Species / strain / cell type:
- S. typhimurium, other: TA 98, TA100, TA1535, TA1537, TA1538
- Details on mammalian cell type (if applicable):
- no data
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 liver microsomes from male Sprague Dawley rats treated with a single intraperitoneal injection of Aroclor 1254 5-days before sacrifice.
- Test concentrations with justification for top dose:
- 20, 60, 560, 1670, and 5000 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: N-methl-N'-nitro-N-nitrosoguanidine
- Remarks:
- positive control for TA100 and TA1535 (5 µg/plate)
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- positive control for TA98 and TA1538 (50 µg/plate) Migrated to IUCLID6: in absence of S9-mix
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- positive control for TA1537 (75 µg/plate)
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 5000 µg/plate
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 5000 µg/plate
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 5000 µg/plate
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 5000 µg/plate
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 5000 µg/plate
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: strain/cell type: TA 98, TA100, TA1535, TA1537, TA1538
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results::
negative
Under the test conditions, the experimental compound, 2,2,4-trimethylpentane, did not exhibit a positive response and is, therefore, considered not be mutagenic in this system. - Executive summary:
Under the test conditions, the experimental compound, 2,2,4 -trimethylpentane, did not exhibit a positive response and is, therefore, considered not be mutagenic in this system.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- September - October 1982
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Test procedure according to national standards.
- Justification for type of information:
- A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- GLP compliance:
- no
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- mammalian cell line, other: Rat liver epithelial-type line (RL4) cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Stock culture media and assay media was Minimal Essential Medium supplemented with 10% fetal calf serum and 1% non-essential amino acids.
- Additional strain / cell type characteristics:
- other: metabolic competent
- Metabolic activation:
- without
- Metabolic activation system:
- RL4 cells are metabolically competent.
- Test concentrations with justification for top dose:
- 0, 2.5, 5.0, and 10 mg/mL
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Tween 80 and ethanol
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Details on test system and experimental conditions:
- see "Any other information on materials and methods"
- Key result
- Species / strain:
- mammalian cell line, other: RL4 cells, metabolic competent
- Metabolic activation:
- not applicable
- Genotoxicity:
- negative
- Remarks:
- There was no significant increase in chromosome damage
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Exposure of RL4 cells to 20 µg/mL test substance resulted in inhibition of cell proliferation by 50 %. Therefore final test concentrations of 2.5, 5, and 10 µg/mL were chosen
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- A maximum dose level of 10 µg/mL was determined in the chromosome assay. There was no significant increase beyond control limits in the frequency of chromatid breaks or total chromatid aberrations in rat liver cells exposed to the test material up to 10 µg/mL indicating that the compound did not induce chromosome damage in rat liver cells. A small increase in the percentage of cells showing gaps in exposed cultures was well within historical control ranges. Cultures exposed to the positive control material, DMBA, showed an increase in the frequency of chromosome damage.
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results: negative
This study examined the potential for the test substance, hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics, to cause chromosomal aberrations in rat liver RL4 cells. Cells were exposed to concentrations of 0, 2.5, 5.0, and 10 ug/mL of test substance, and then examined for chromosomal aberrations including polyploidy, chromatid gaps, and chromatid exchanges. The positive control substance was 7,12 -dimethylbenzanthracene. No significant increase in the frequency of chromosomal damage was observed. Based on the study design the test substance showed no genetic toxicity. - Executive summary:
This study examined the potential for the test substance, hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics, to cause chromosomal aberrations in rat liver RL4 cells. Cells were exposed to concentrations of 0, 2.5, 5.0, and 10 ug/mL of test substance, and then examined for chromosomal aberrations including polyploidy, chromatid gaps, and chromatid exchanges. The positive control substance was 7,12 -dimethylbenzanthracene. No significant increase in the frequency of chromosomal damage was observed. Based on the study design the test substance showed no genetic toxicity.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 1982-1983
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study with acceptable restrictions
- Justification for type of information:
- A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Principles of method if other than guideline:
- No guideline stated; similar to OECD guideline 473; rat hepatocytes with intrinsic metabolic activity, therefore no metabolic activation system added, only 100 cells from each culture analyzed.
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not applicable
- Species / strain / cell type:
- hepatocytes: rat liver (RL4) cells
- Details on mammalian cell type (if applicable):
- The rat liver cell line, RL4, an epithelial-type cell line, was derived in this laboratory following the procedure described by Williams et al. (1971). RL4 was initiated in 1978 from a 10-day-old Wistar rat as described in Dean and Hodson-Walker (1979).
- Metabolic activation:
- not applicable
- Metabolic activation system:
- not necessary, liver cells
- Test concentrations with justification for top dose:
- 2.5, 5, 10 µg/ml
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Tween 80/ethanol
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 22 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours + 2 hours fixation
SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: no data
NUMBER OF CELLS EVALUATED: 100 cells from each culture - Evaluation criteria:
- Significant or dose-related increase in chromosome damage.
- Key result
- Species / strain:
- hepatocytes: Rat liver (RL4) cells
- Metabolic activation:
- not applicable
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results::
negative
This study examined the potential for the test substance Heptane to cause chromosomal aberrations in rat liver RL4 cells. Cells were exposed to concentrations of 0, 2.5, 5, and 10 ug/ml of test substance, and then examined for chromosomal aberrations including polyploidy, chromatid gaps, and chromatid exchanges. The positive control substance was 7,12 -dimethylbenzanthracene. A significant increase in chromatid gaps (7) was seen at the 10 ug/ml concentration. Though greater than the solvent control frequency, this was not accompanied by an increase in any other aberrations and is not considered to be treatment related. Under the conditions of this study, the test material was not clastogenic. - Executive summary:
This study examined the potential for the test substance Normal-Heptane to cause chromosomal aberrations in rat liver RL4 cells. Cells were exposed to concentrations of 0, 2.5, 5, and 10 ug/ml of test substance, and then examined for chromosomal aberrations including polyploidy, chromatid gaps, and chromatid exchanges. The positive control substance was 7,12 -dimethylbenzanthracene. A significant increase in chromatid gaps (7) was seen at the 10 ug/ml concentration. Though greater than the solvent control frequency, this was not accompanied by an increase in any other aberrations and is not considered to be treatment related. Under the conditions of this study, the test material was not clastogenic.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Acceptable, well documented publication which meets basic scientific principles
- Justification for type of information:
- A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- yes
- Remarks:
- - using microtiter plates
- GLP compliance:
- not specified
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- thymidine kinase
- Species / strain / cell type:
- human lymphoblastoid cells (TK6)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI 1640 medium supplemented with 15% heat-inactivated horse serum
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- with Aroclor induced-rat liver homogenate
- Test concentrations with justification for top dose:
- 5 % v/v TMP in DMEM medium, administered undiluted or as 50 % (1:1 saturated to normal medium)
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMEM medium
- Untreated negative controls:
- yes
- Remarks:
- with and without S9
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMEM medium
- Positive controls:
- yes
- Remarks:
- for S9 activated cultures
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- Migrated to IUCLID6: 15 µM
- Untreated negative controls:
- yes
- Remarks:
- with and without S9
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMEM medium
- Positive controls:
- yes
- Remarks:
- for non-activated cultures
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- Migrated to IUCLID6: 0.2 mM
- Details on test system and experimental conditions:
- see "any other information on materials and methods"
- Key result
- Species / strain:
- human lymphoblastoid cells (TK6)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- TMP did not induce significant increases in the mutation frequency at the thymidine kinase locus.
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Cell survival in TMP-saturated medium with and without metabolic activation was greater than 50-60 %.
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- The elevated mutation frequencies of positive control compounds were as expected.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cell survival in positive control treated cultures was 40 or 30% for benzo(a)pyrene and EMS, respectively - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results: negative
Based on the study design there is no incidence of increased genetic toxicity caused by the test substance. - Executive summary:
Based on the study design there is no incidence of increased genetic toxicity caused by the test substance.
Referenceopen allclose all
Table: Relative Mutation Rate – Bacterial Experiment I without Metabolic Activation
Concentration |
E. coli WP2 |
E. coli WP2 uvr A |
S. typhimurium TA 1535 |
S. typhimurium TA 1537 |
S. typhimurium TA 1538 |
S. typhimurium TA 98 |
S. typhimurium TA 100 |
Without S9 |
|||||||
3.91 |
- |
- |
- |
- |
- |
- |
- |
7.81 |
- |
- |
- |
- |
- |
- |
- |
15.6 |
- |
- |
- |
- |
- |
- |
- |
31.3 |
- |
- |
- |
- |
- |
- |
- |
62.5 |
0.8 |
0.7 |
1.2 |
0.1 |
0.7 |
0.2 |
1.4 |
125 |
0.9 |
0.6 |
0.7 |
0 |
0.4 |
0 |
0.7 |
250 |
0.7 |
0.8 |
0.6 |
0 |
0 |
0 |
0.3 |
500 |
0.7 |
0.7 |
0.9 |
0 |
0 |
0 |
0.1 |
1000 |
0.8 |
0.5 |
0.9 |
0 |
0 |
0 |
0 |
2000 |
0.7 |
0.6 |
0.7 |
0 |
0 |
0 |
0 |
4000 |
0.8 |
0.6 |
0.6 |
0 |
0 |
0 |
0 |
8000 |
0.6 |
0.5 |
0.7 |
0 |
0 |
0 |
0 |
Sodium azide 2 µg |
- |
- |
89.8 |
- |
- |
- |
- |
Benzo(a)-pyrene 7 µg |
- |
- |
- |
- |
1.0 |
0.7 |
0.9 |
4-nitroquinoline-N-oxide 3 µg |
39.8 |
3.2 |
- |
- |
- |
- |
- |
Neutral red 7 µg |
- |
- |
- |
1.8 |
- |
- |
- |
Table: Relative Mutation Rate – Bacterial Experiment I with Metabolic Activation
Concentration |
E. coli WP2 |
E. coli WP2 uvr A |
S. typhimurium TA 1535 |
S. typhimurium TA 1537 |
S. typhimurium TA 1538 |
S. typhimurium TA 98 |
S. typhimurium TA 100 |
Without S9 |
|||||||
31.3 |
- |
- |
- |
- |
- |
- |
- |
62.5 |
0.7 |
1.3 |
1.4 |
0.3 |
1.5 |
0.7 |
1.0 |
125 |
0.8 |
1.0 |
1.1 |
0.2 |
1.3 |
1.3 |
0.8 |
250 |
0.9 |
1.0 |
0.8 |
0.6 |
1.5 |
1.2 |
1.0 |
500 |
0.7 |
0.8 |
1.0 |
0.2 |
1.0 |
0.7 |
1.3 |
1000 |
0.7 |
0.7 |
1.0 |
0.4 |
0.7 |
0.2 |
1.4 |
2000 |
1.1 |
0.9 |
1.1 |
0 |
0.4 |
0 |
1.2 |
4000 |
1.0 |
1.0 |
1.0 |
0 |
0.7 |
0 |
1.1 |
8000 |
0.8 |
1.0 |
0.5 |
0 |
0.7 |
0 |
1.1 |
Sodium azide 2 µg |
- |
- |
15.2 |
- |
- |
- |
- |
Benzo(a)-pyrene 7 µg |
- |
- |
- |
- |
3.7 |
5.3 |
3.1 |
4-nitroquinoline-N-oxide 3 µg |
1.2 |
3.6 |
- |
- |
- |
- |
- |
Neutral red 7 µg |
- |
- |
- |
4.4 |
- |
- |
- |
Table: Relative Mutation Rate – Bacterial Experiment II without Metabolic Activation
Concentration |
E. coli WP2 |
E. coli WP2 uvr A |
S. typhimurium TA 1535 |
S. typhimurium TA 1537 |
S. typhimurium TA 1538 |
S. typhimurium TA 98 |
S. typhimurium TA 100 |
Without S9 |
|||||||
3.91 |
- |
- |
- |
1.1 |
- |
0.8 |
- |
7.81 |
- |
- |
- |
0.6 |
- |
0.9 |
- |
15.6 |
- |
- |
- |
0.7 |
0.9 |
0.8 |
- |
31.3 |
- |
- |
- |
0.4 |
1.0 |
0.8 |
1.1 |
62.5 |
1.0 |
1.0 |
1.2 |
0.4 |
0.7 |
0.4 |
1.0 |
125 |
1.0 |
1.0 |
0.9 |
0 |
0.5 |
0 |
0.9 |
250 |
0.7 |
0.8 |
1.4 |
- |
0.2 |
- |
1.0 |
500 |
0.9 |
0.7 |
1.1 |
- |
0 |
- |
0.7 |
1000 |
1.0 |
0.8 |
0.9 |
- |
0 |
- |
- |
2000 |
0.7 |
0.9 |
0.7 |
- |
- |
- |
- |
4000 |
0.9 |
0.8 |
0.9 |
- |
- |
- |
- |
8000 |
0.8 |
0.7 |
0.9 |
- |
- |
- |
- |
Sodium azide 2 µg |
- |
- |
86.6 |
- |
- |
- |
- |
Benzo(a)-pyrene 7 µg |
- |
- |
- |
- |
1.1 |
1.1 |
1.3 |
4-nitroquinoline-N-oxide 3 µg |
36.7 |
5.3 |
- |
- |
- |
- |
- |
Neutral red 7 µg |
- |
- |
- |
1.2 |
- |
- |
- |
Table: Relative Mutation Rate – Bacterial Experiment II with Metabolic Activation
Concentration |
E. coli WP2 |
E. coli WP2 uvr A |
S. typhimurium TA 1535 |
S. typhimurium TA 1537 |
S. typhimurium TA 1538 |
S. typhimurium TA 98 |
S. typhimurium TA 100 |
Without S9 |
|||||||
31.3 |
- |
- |
- |
1.1 |
0.9 |
0.8 |
- |
62.5 |
0.9 |
0.9 |
0.7 |
1.2 |
1.1 |
1.0 |
1.1 |
125 |
1.1 |
1.0 |
1.2 |
1.1 |
1.1 |
0.9 |
1.0 |
250 |
1.4 |
0.8 |
0.8 |
1.4 |
1.1 |
0.8 |
1.1 |
500 |
1.2 |
1.0 |
0.9 |
1.0 |
1.1 |
0.8 |
1.2 |
1000 |
1.0 |
1.1 |
0.8 |
0.5 |
0.9 |
0.4 |
0.9 |
2000 |
0.8 |
1.0 |
1.1 |
0 |
0.9 |
0.1 |
0.9 |
4000 |
0.8 |
0.9 |
0.8 |
- |
0.5 |
- |
0.9 |
8000 |
1.0 |
0.7 |
0.7 |
- |
0.4 |
- |
0.9 |
Sodium azide 2 µg |
- |
- |
18.2 |
- |
- |
- |
- |
Benzo(a)-pyrene 7 µg |
- |
- |
- |
- |
9.7 |
6.1 |
6.5 |
4-nitroquinoline-N-oxide 3 µg |
1.1 |
2.8 |
- |
- |
- |
- |
- |
Neutral red 7 µg |
- |
- |
- |
5.2 |
- |
- |
- |
TA98: Revertants Test
Test substance | Test 1 (without S9) | Test 2 (without S9) | Test 1 (with S9) | Test 2 (with S9) |
solvent control | 22 -26 | 22 -26 | 22 -26 | 18 -20 |
2 -Nitrofluorene | 1508 -1637 | 2131 -2282 | - | - |
2 -Aminoanthracene | - | - | 2213 -2247 | 1913 -2020 |
7 µg/plate 2,2,4 -trimethylpentane | - | 15 -27 | - | 24 -46 |
20 µg/plate 2,2,4 -trimethylpentane | 140 -191 | 19 -23 | 145 -206 | 29 -31 |
60 µg/plate 2,2,4 -trimethylpentane | 112 -124 | 19 -26 | 64 -117 | 24 -33 |
190 µg/plate 2,2,4 -trimethylpentane | 37 -55 | 19 -24 | 17 -28 | 12 -25 |
Retest (test 2) of TA98 without and with S9 at dose levels for 7, 20 and 60 µg/plate evidenced no difference from solvent controls.
Observed 5 -10 fold increase of TA98 revertants without and with S9 at 20 and 60 µg/plate in initial test was not confirmed in retest of TA98 without and with S9 at dose levels 7, 20 and 60 µg/plate.
In all experiments, no increase in revertants for other frame shift strains (TA1537 and TA 1538) was observed. Therefore the in test 1 observed genotoxic effects of 2,2,4 -trimethylpentane in strain TA 98 were rather false positive.
Table: Metaphase chromosome analysis of RL4cells
0 µg/ml |
2.5 µg/ml |
5.0 µg/ml |
10.0 µg/ml |
1.0 µg/ml 7,12-dimethylbenzanthracene |
|
Polyploidy (% cells) |
1.3 |
1.3 |
1.3 |
1.3 |
0.5 |
Chromatid gaps (% cells) |
0.3 |
0.7 |
0.9 |
1.0 |
5.5 |
Multiple aberrations (% cells) |
- |
- |
- |
- |
- |
Severe damage (% cells) |
- |
- |
- |
- |
- |
Chromatid aberrations (% cells) |
0.7 |
- |
0.9 |
- |
4.0 |
Chromosome aberrations (% cells) |
- |
- |
- |
- |
1.5 |
Chromatid gaps (frequency per cell) |
0.003 |
0.007 |
0.009 |
0.010 |
0.060 |
Chromatid breaks (frequency per cell) |
0.007 |
- |
0.004 |
- |
- |
Chromatid exchange (frequency per cell) |
- |
- |
0.004 |
- |
- |
Chromosome breaks (frequency per cell) |
- |
- |
- |
- |
0.015 |
Chromosome exchange (frequency per cell) |
- |
- |
- |
- |
- |
There was no significant or dose-related increase of chromosome damage in any of the cultures exposed.
Table: Metaphase chromosome analysis of RL4cells
0 µg/ml |
2.5 µg/ml |
5.0 µg/ml |
10.0 µg/ml |
1.0 µg/ml 7,12-dimethylbenzanthracene |
|
Polyploidy (% cells) |
3.7 |
2.0 |
2.7 |
1.3 |
1.5 |
Chromatid gaps (% cells) |
1.3 |
1.7 |
1.0 |
2.3 |
6.0 |
Multiple aberrations (% cells) |
- |
- |
- |
- |
0.5 |
Severe damage (% cells) |
- |
- |
- |
- |
0.5 |
Chromatid aberrations (% cells) |
0.3 |
- |
- |
- |
4.0 |
Chromosome aberrations (frequency per cell) |
- |
- |
- |
- |
- |
Chromatid gaps (frequency per cell) |
0.013 |
0.017 |
0.010 |
0.027 |
0.085 |
Chromatid breaks (frequency per cell) |
0.003 |
- |
- |
- |
0.005 |
Chromatid exchange (frequency per cell) |
- |
- |
- |
- |
0.05 |
Chromosome breaks (frequency per cell) |
- |
- |
- |
- |
0.005 |
Chromosome exchange (frequency per cell) |
- |
- |
- |
- |
- |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
There is no in vivogenetic toxicity data available for 2,2,3-Trimethylbutane. However, data is available for structural analogues, Hydrocarbons, C7-C9, isoalkanes and 2,2,4-trimethylpentane and presented in the dossier. This data is read across to based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.
The read across genetic toxicity test listed below had negative results for 2,2,3-Trimethylbutane.
Genetic Toxicity in vivo – Rodent Dominant Lethal Test (OECD TG 478)
Genetic Toxicity in vivo – Unscheduled DNA Synthesis (OECD 486)
Link to relevant study records
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Remarks:
- Type of genotoxicity: DNA damage and/or repair
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study with acceptable restrictions.
- Justification for type of information:
- A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
- Deviations:
- yes
- Remarks:
- - limited documentation
- GLP compliance:
- not specified
- Type of assay:
- unscheduled DNA synthesis
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories (Kingston, N.Y.)
- Weight at study initiation: 200-300 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled
- Humidity (%): controlled
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: none
- Duration of treatment / exposure:
- one single dose
- Frequency of treatment:
- sinlge treatment
- Post exposure period:
- 2, 12, 24 and 48 hours after dosing
- Remarks:
- Doses / Concentrations:
500 mg/kg
Basis:
nominal conc. - No. of animals per sex per dose:
- 3 males/ time period
- Control animals:
- yes
- Positive control(s):
- dimethylnitrosamine (DMN), dissolved in water
- Route of administration: orally
- Doses / concentrations: 10 mg/kg - Tissues and cell types examined:
- hepatocytes
- Details of tissue and slide preparation:
- see "any other information on materials and methods"
- Statistics:
- One-way analysis of variance was performed on UDS and S-phase data with multiple treatment groups. S-phase data were adjusted by square root transformation. Treatment means were compared to control means by Dunnett's multiple comparison test. Level of significance was <0.05.
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- TMP did not induce unscheduled DNA synthesis in hepatocyte cultures from rats treated in vivo.
- Toxicity:
- no effects
- Remarks:
- Mean cell viabilities in preparations of hepatocytes were: 84 % at 2-12 hours after administration of 500 mg/kg TMP.
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- see "Remarks on results"
- Conclusions:
- Interpretation of results: negative
Based on this study design 2, 2, 4- trimethylpentane does not induce unscheduled DNA synthesis in vivo by unchanged application via gavage. - Executive summary:
Based on this study design 2, 2, 4- trimethylpentane does not induce unscheduled DNA synthesis in vivo by unchanged application via gavage.
- Endpoint:
- in vivo mammalian germ cell study: cytogenicity / chromosome aberration
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- July 1978 - January 1980
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline with acceptable restrictions.
- Justification for type of information:
- A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
- Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
- Principles of method if other than guideline:
- Similar to OECD 478, only two dose levels were used.
- GLP compliance:
- not specified
- Type of assay:
- rodent dominant lethal assay
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Massachusetts
- Age at study initiation: males: 10 weeks at initiation of pre-treatment mating period; females: 9 weeks at initiation of week 1 of pre-treatment mating period; females: 8 weeks at initiation of week 1 of post-treatment mating period
- Housing: males: individually housing during the treatment period; females: individually housing during the pre- and post-mating period
- Diet: Purina Laboratory Chow; ad libitum; except chamber-exposed groups during the treatment period
- Water: ad libitum; except chamber-exposed groups during the treatment period
- Route of administration:
- inhalation
- Vehicle:
- none
- Details on exposure:
- TYPE OF INHALATION EXPOSURE: whole body
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The stainless steel and glass chambers had a total volume of one cubic metre with an effective volume of 760 L.
- Air flow rate: 132 L/min
- Air change rate: every 7.6 min
- The test substance was placed in fritted-bottom, gas-washing bottles. Dry air, at various flow rates, was passed through the test material and the vapour-air mixtures entered the chamber air inlets and were further diluted with room air to provide the desired concentrations.
TEST ATMOSPHERE
Atmospheric sampling was performed using a Wilks Scientific Corp., Miran IA Ambient Air Analyzer (long pathlength infrared). On each exposure day three samples were drawn from each exposure chamber (at 1, 3 and 5 hours) and the exposure concentrations calculated by comparing the absorption of this sample to the standard curve. - Duration of treatment / exposure:
- 8 consecutive weeks
- Frequency of treatment:
- 6 hours/day, 5 days/week
- Post exposure period:
- 4 weeks
- Remarks:
- Doses / Concentrations:
400 and 1200 ppm
Basis:
nominal conc. - No. of animals per sex per dose:
- only males were exposed: 10/group
- Control animals:
- yes, sham-exposed
- Positive control(s):
- triethylenemelamine
- Route of administration: intraperitoneally on a single day, at least 2 hours prior to mating
- Doses level/ volume: 0.5 mg/kg body weight; 1 mL/kg body weight
- Vehicle: 0.9 % injectable normal saline solution - Tissues and cell types examined:
- Males: testes, seminal vesicles, epididymides, prostate tissues
Females: uteri - Details of tissue and slide preparation:
- METHOD OF ANALYSIS:
Males: Seminal vesicles, epididymides, prostate and any abnormal lesion or tissue masses were preserved in 10 % neutral buffered formalin. Testes were stored for 48 hours in Bouin´s solution and subsequently placed in formalin. Tissues from 5 randomly chosen males from each group were examined.
Females: The uteri from all females (pregnant and non-pregnant) were appropriately identified and preserved intact in a 10 % neutral buffered formalin solution. - Statistics:
- Comparisons were made during the treatment and post-treatment periods between negative control, positive control and test substance-treated groups by the Chi-square test. Absolute data were compared by using the F-test and Student´s t-test. When variances differed significantly, Student´s t-test was appropriately modified using Cochran´s approximation (t´). Corpora lutea and implantation data were evaluated by the one-tailed test.
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Remarks:
- At dose level of 400 and 1200 ppm the test substance was not mutagenic in rats by the dominant-lethal test.
- Toxicity:
- no effects
- Vehicle controls validity:
- other: no vehicle was used
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Interpretation of results: negative
Under the conditions of the test no negative effects were induced by the test substance, hydrocarbons, C7-C9, isoalkanes. - Executive summary:
Under the conditions of the test no negative effects were induced by the test substance, hydrocarbons, C7 -C9, isoalkanes.
Referenceopen allclose all
Mean cell viabilities in preparations of hepatocytes were: 85 % at 24 hours after administration of water (negative control) and 84 % at 2-12 hours after administration of 500 mg/kg TMP.
No increase in UDS expressed as increase in NG or number of cells in repair was observed in hepatocytes isolated from male rats at 2, 12, 24, or 48 hours post-dose. A strong response was elicited by the DMN positive control, as expected.
Autoradiographic preparations of hepatocytes isolated from rats 24 hr after treatment with TMP revealed a significant increase in the number of cells in S phase (2.1% vs 0.35% in controls); this value dropped back to 0.55% at 48 hours. TMP also significantly increased RDS in mice at 24 hours; no 48 hour values were presented. However low serum alanine transaminase activity were observed at 24 and 48 hours after a single treatment with 500 mg/kg TMP indicating that TMP induced little if any hepatocellular necrosis which could trigger regenerative cell proliferation.
Following 11 consecutive daily treatments with 100 mg/kg/day TMP, a significant increase (p<0.05) in rat hepatic concentration of DNA (mg/g liver) was seen but the total hepatic DNA content per rat was comparable to controls. Liver weight relative to body weight was also comparable to controls. The demonstrated increase in RDS suggests that TMP may stimulate additive rather than regenerative cell proliferation but not of sufficient magnitude to significantly increase the total DNA content of the liver.
TMP was administered at a single 500 mg/kg concentration to evaluate UDS and RDS in conjunction with a multidose study for unleaded gasoline. When RDS was observed, a liver DNA content determination was performed in rats with treatment over 11 days to determine the type of proliferative response.
No treatment-related effects were observed for mortality, in-life physical or necropsy examinations and for body weights in males.
No treatment effect was indicated from microscopic evaluation of testes, seminal vesicles, epididymides and prostate tissues from 5 randomly chosen males from each exposure group.
In one animal of the 400 ppm exposure group a caseous mass was observed in the left epididymis. This caseous mass was seen microscopically to be a spermatic granuloma. Since this was observed in a single animal, this is not considered to be treatment related. Several animals (one in control, one in 100 ppm group, one in 1200 ppm group) presented a purulent prostatitis which was of probable bacterial etiology.
Pregnancy rates, implantation data and implantation efficiency values and fetal death data for females mated to treated males were considered comparable to data for females mated to negative control males for each week of the post-treatment mating period. At dose level of 400 and 1200 ppm the test substance was not mutagenic in rats by the dominant-lethal test.
Positive control:
Females mated to the positive control males had fewer implants and lower implantation efficiency values (indicative of pre-implantation loss) than females mated to the negative control males. Fetal death parameters (mean and expressed as a percentage of total implants) for these same females were significantly increased during the post-treatment mating interval.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
No genetic toxicity data is available for 2,2,3-Trimethylbutane. However, data is available for structural analogues, Hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics, 2,2,4-trimethylpentane, and Hydrocarbons, C7-C9, isoalkanes and presented in the dossier. This data is read across to based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.
In Vitro
In vitro gene mutation study in bacteria
Hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics
A reliable bacterial reverse mutation assay (Ames test) was conducted with hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics following a protocol similar to OECD 471.The pre-incubation procedure was performed with Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and Escherichia coli strains WP2 uvr A and WP2. The strains were exposed to concentrations ranging from 0.31 to 8000 µg/plate for 30 min both with and without metabolic activation prior to plating and further incubation for 48 -72 h. For all strains tested, there was no significant increase in the number of revertants as compared to negative controls. An initial cytotoxicity assay with S. typhimurium TA100 showed that the test substance was extremely cytotoxic, especially in the absence of S9 fraction. Therefore, initial mutation assays were conducted over a low dose range (0.31 -10 µg per mL) with strains TA100 and TA98. Virtually no cytotoxicity was observed at these concentrations, and so more appropriate concentrations were tested for all strains in subsequent assays. Salmonella strains were more sensitive to the cytotoxic effect of the test substance than E. coli strains, and cytotoxicity was greater in the absence of S9-fraction. In conclusion, the test substance was not mutagenic in this assay (Shell Chemicals, 1983; Brooks et al., 1988).
2,2,4 -trimethylpentane
A bacterial reverse mutation assay (Ames test) was conducted with 2,2,4-trimethylpentane following a protocol similar to OECD 471.Salmonella typhimurium strainsTA 98, TA 100, TA 1535, TA 1537 and TA 1538 were exposed to the test substance at 20, 60, 560, 1670, and 5000 µg/plate in the absence and in the presence of a metabolic activator. Cytotoxic effects were observed at 5000 µg/plate. Iso-octane did not induce mutations in the bacterial mutation test in either the absence or presence of metabolic activator in any strain tested. Under the test conditions, the experimental compound, iso-octane, did not exhibit a positive response and is, therefore, considered not be mutagenic in this system (Chevron Phillips, 1982).
In vitro Chromosome Aberration in Mammalian Cells
Hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics
The potential of hydrocarbons, C7-C9, n-alkanes, isoalkanes, cyclics to cause chromosomal aberrations in rat liver RL4 cells was tested with a method comparable to OECD 473. Cells were exposed to concentrations of 0, 2.5, 5, and 10 µg/mL of test substance for 22 h, and then examined for chromosomal aberrations including polyploidy, chromatid gaps, and chromatid exchanges. Exposure of RL4 cells to 20 µg/mL test substance resulted in inhibition of cell proliferation by 50%. Therefore 10 µg/mL was chosen as the maximum dose level. No significant increase in the frequency of chromosomal damage was observed. Under the conditions of this study, the test material was not clastogenic (Shell Chemicals, 1983; Brooks et al., 1988).
Heptane
The potential of n-heptane (CAS No. 142-82-5) to cause chromosomal aberrations in rat liver RL4 cells was tested with a method comparable to OECD 473. Cells were exposed to concentrations of 0, 2.5, 5, and 10 µg/mL of test substance for 22 h, and then examined for chromosomal aberrations including polyploidy, chromatid gaps, and chromatid exchanges. No cytotoxicity was observed. A significant increase in chromatid gaps was seen at 10 µg/mL. However, this effect was neither dose-dependent nor accompanied by an increase in any other aberration type, and therefore not considered to be treatment-related. Under the conditions of this study, the test material was not clastogenic (Shell Chemicals, 1983; Brooks et al., 1988).
In vitro Gene Mutation study in Mammalian Cells
2,2,4-trimethylpentane
2,2,4-trimethylpentane was tested in a mammalian cell gene mutation assay performed according to OECD 476. The test material was prepared by adding 2,2,4 -trimethylpentane at a final concentration of 5 % v/v in culture (DMEM) medium and stirred overnight at room temperature in a foil wrapped, capped parafilm-sealed bottle to saturate the medium. Human lymphoblastoid cells (TK6) were exposed to 100 or 50 % of this saturated DMEM medium with and without metabolic activation for 3 h and allowed for expression for 4 to 8 days. Both with and without metabolic activation, 2,2,4 -trimethylpentane did not induce significant increases in the mutation frequency at the thymidine kinase locus and cell survival in 2,2,4 -trimethylpentane-saturated medium was greater than 50-60 %. Based on the study design there was no incidence of increased genetic toxicity caused by the test substance (Richardson et al., 1986).
2,2,4 -trimethylpentane was also tested in a Mouse Lymphoma Forward Mutation Assay performed according to a protocol similar to OECD TG 476. At concentrations ranging from 20 to 500 µg/mL (limit of solubility), 2,2,4 -trimethylpentane did not induce gene mutations in the mouse lymphoma L5178Y heterozygous TK+/-cells in the presence of metabolic activation, resulting in cytotoxicity at 165 µg/mL and above. Without metabolic activation, cytotoxicity at 500 µg/mL and an equivocal to weakly mutagenic response were observed (Chevron Phillips, 1982).
In Vitro Unscheduled DNA Synthesis in Mammalian Cells
2,2,4-trimethylpentane
The potential of 2,2,4-trimethylpentane to induce unscheduled DNA synthesis (UDS) as an indicator of genotoxicity was tested in rat hepatocytes in a study comparable to OECD TG 482. Primary cultures were prepared from hepatocytes isolated from a rat. After attachment to coverslips and acclimatisation, cells were treated with [3H] thymidine (10 µCi/mL) and undiluted 2,2,4 -trimethylpentane at 0.33, 1.00, 3.33 % (v/v). The cells were then incubated for 18 hours. The highest concentration of 3.33% v/v was lethal to cell cultures. At the lower concentrations, 2,2,4 -trimethylpentane did not induce unscheduled DNA synthesis in vitro by direct application to primary hepatocyte cultures. Based on the study design there was no incidence of increased genetic toxicity caused by the test substance (Loury et al. 1986).
In Vitro Sister Chromatid Exchange Assay in Mammalian Cells
2,2,4 -trimethylpentane
Human lymphoblastoid cells exposed to 2,2,4 -trimethylpentane for 3 h as described above were also tested for the induction of sister chromatid exchange (SCE) according to OECD TG 479. Following exposure, the cells were incubated in the dark in the presence of 5'Bromo-deoxyuridine (10 µM) for 36 h, and metaphases were analysed microscopically thereafter. Both with and without metabolic activation, 2,2,4-trimethylpentane did not induce an increased incidence of SCEs in TK6 cells (Richardson et al., 1986; Wilmer et al., 1981). No statistically significant increase in number of SCEs (with and without metabolic activation) was also observed.
In another study compliant with OECD TG 479, Chinese hamster Ovary (CHO) cells were exposed to 25, 84, 250 µg/mL iso-octane in DMSO and 1400 and 3500 µg/mL iso-octane without vehicle. No cell growth occurred at the 250 µg/mL, without activation. No statistically significant increase in number of SCEs (with and without metabolic activation) was also observed. Based on the study design there was no increased mutagenic response caused by 2,2,4-trimethylpentane in CHO cells with and without metabolic activation. (Chevron Phillips, 1982)
In Vivo
Hydrocarbons, C7-C9, isoalkanes
Hydrocarbons, C7-C9, isoalkanes tested in a dominant lethal study (similar to OECD 478) showed no evidence of genotoxicity in the germ cells of treated male rats exposed to 400 or 1200 ppm by inhalation (ExxonMobil Chemical, 1980).
In Vivo Unscheduled DNA Synthesis Test with Mammalian Liver Cells
2,2,4 -trimethylpentane
As part of an unscheduled DNA synthesis (UDS) study performed in vitro, 2,2,4 -trimethylpentane was administered to rats and mice orally at a single dose of 500 mg/kg. Primary hepatocytes were isolated from livers at sacrifice times 2, 12, 28 or 48 hours post-treatment and cultures were evaluated for unscheduled and replicative DNA synthesis. 2,2,4 -trimethylpentane did not induce unscheduled DNA synthesis in hepatocyte cultures, and increases in replicative DNA synthesis were not of sufficient magnitude to increase the total DNA content of the liver (Loury et al., 1986).
Justification for classification or non-classification
The negative results in in vitro and in vivo genotoxicity assays from structural analogues do not warrant the classification of 2,2,3 -trimethylbutane as genotoxic under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).
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