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EC number: 204-661-8 | CAS number: 123-91-1
- 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
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
1,4 -dioxane was clearly negative in all available in vitro mutagenicity studies: Ames test (OECD 471); Mammalian gene mutation assay (OECD 476) and Chromosomal aberration test (OECD 473).
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 21 Mar 1979 - 23 Mar 1979
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- (neither Escherichia coli strain WP2 uvrA pKM101a nor S. typhimurium strain TA102 tested)
- Principles of method if other than guideline:
- The study was conducted according to the method described by Ames BN et al. (1975). Mutation Research 31: 347-364.
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- His -
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced rat liver S-9 mix
- Test concentrations with justification for top dose:
- 4, 20, 100, 500, 2500 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: aqua dest.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- other: With S-9: 2-aminoanthracene (TA100, TA98, TA1538, TA1537, TA1535); without S-9: N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) (TA100, TA98, TA1537, TA1535)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 4 plates
DETERMINATION OF CYTOTOXICITY
- Method: clearing or diminution of the background lawn (= reduced his- background growth) - Evaluation criteria:
- Positive results
A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S 9 mix or after adding a metabolizing system.
A test substance is generally considered nonmutagenic in this test if: The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other. - Statistics:
- No statistics were performed.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: presumably (up to 40 % test substance over 48 h test period)
- Water solubility: yes
- Precipitation: none - Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 20 Mar 1979 - 22 Mar 1979
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- (neither Escherichia coli strain WP2 uvrA pKM101a nor S. typhimurium strain TA102 tested)
- Principles of method if other than guideline:
- The study was conducted according to the method described by Ames BN et al. (1975). Mutation Research 31: 347-364.
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- His -
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced rat liver S-9 mix
- Test concentrations with justification for top dose:
- 4, 20, 100, 500, 2500 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: aqua dest.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- other: With S-9: 2-aminoanthracene (TA100, TA98, TA1538, TA1537, TA1535); without S-9: N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) (TA100, TA98, TA1537, TA1535)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 4 plates
DETERMINATION OF CYTOTOXICITY
- Method: clearing or diminution of the background lawn (= reduced his- background growth) - Evaluation criteria:
- Positive results
A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S 9 mix or after adding a metabolizing system.
A test substance is generally considered nonmutagenic in this test if: The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other. - Statistics:
- No statistics were performed.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: presumably (up to 40 % test substance over 48 h test period)
- Water solubility: yes
- Precipitation: none - Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 14 Mar 1979 - 16 Mar 1979
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- (neither Escherichia coli strain WP2 uvrA pKM101a nor S. typhimurium strain TA102 tested)
- Principles of method if other than guideline:
- The study was conducted according to the method described by Ames BN et al. (1975). Mutation Research 31: 347-364.
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- His -
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced rat liver S-9 mix
- Test concentrations with justification for top dose:
- 4, 20, 100, 500, 2500 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: aqua dest.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- other: +S9: 2-aminoanthracene (TA100, TA98, TA1538, TA1537, TA1535); -S9: N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) (TA100, TA98, TA1537, TA1535)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 4 plates
DETERMINATION OF CYTOTOXICITY
- Method: clearing or diminution of the background lawn (= reduced his- background growth)
- Evaluation criteria:
- Positive results
A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S 9 mix or after adding a metabolizing system.
A test substance is generally considered nonmutagenic in this test if: The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other. - Statistics:
- No statistics were performed.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: presumably (up to 40 % test substance over 48 h test period)
- Water solubility: yes
- Precipitation: none - Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- His -
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A pKM 101
- Species / strain / cell type:
- E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 from SD rat liver
- Test concentrations with justification for top dose:
- 0, 156, 313, 625, 1250, 2500, 5000 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene in the presence of S9 for TA1535, TA100, E.coli WP2, and E.coli WP2 urvA pKM101)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Details on test system and experimental conditions:
- Standard plate and pre-incubation test.
The plates were incubated at 37 degrees Celsius for 2 days and revertant colonies were counted. Triplicate plates were used for treatments and positive controls, five plates were used for vehicle controls. - Evaluation criteria:
- Positive results:
A dose-related and reproducible increase in the number of revertant colonies, either without S 9 mix or after adding a metabolizing system. In comparison to negative/ and or positive control, using Dunnet's test. - Statistics:
- The data were analyzed statistically by Dunnet’s test.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative 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:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative 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:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative 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:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A pKM 101
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- valid
- Positive controls validity:
- valid
Referenceopen allclose all
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
In vivo, there were positive and negative findings observed in both, gene mutation and chromosomal aberration tests.
However, positive results, i. e. genotoxicity, were only observed at dose levels clearly exceeding the linear toxicokinetic range of 1,4-dioxane.
Therefore, and although it cannot be completely excluded that 1,4-dioxane may induce micronuclei via the oral route with a low potency and at high doses only, 1,4-dioxane is considered a non-genotoxic compound based on the total weight of evidence evaluation of all relevant in vivo and in vitro studies.
Link to relevant study records
- Endpoint:
- in vivo mammalian germ cell study: cytogenicity / chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- July - October 1976
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The test procedure was based on the method described by Roehrborn G and Vogel F (1967). Deutsche Medizinische Wochenschrift 92: 2315-2321
- GLP compliance:
- no
- Type of assay:
- rodent dominant lethal assay
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: WIGA, Sulzfeld
- Age at study initiation: approx. 14-16 weeks old
- Weight at study initiation: 28.4 g (mean)
- Assigned to test groups randomly: yes
- Housing: 1 male/cage (except during mating: 1 male and 3 females/cage)
- Diet (ad libitum): Altromin-R (Altromin GmbH, Lage/Lippe)
- Water (ad libitum): tap water
ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55 ± 5 %
- Photoperiod (hrs dark / hrs light): 12 h / 12 h - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: aqua bidest.
- Details on exposure:
- - Applied volume: 10 mL/kg bw
- Duration of treatment / exposure:
- single administration
- Frequency of treatment:
- single administration
- Post exposure period:
- not applicable
- Dose / conc.:
- 2 575 mg/kg bw/day (actual dose received)
- Remarks:
- corresponding to 2500 µL/kg bw (Recalculation based on relative density = 1.03)
- No. of animals per sex per dose:
- 20
- Control animals:
- yes, concurrent no treatment
- Positive control(s):
- none
- Evaluation criteria:
- Mutagenicity index:
MI = ((dead foetuses + deciduomata + early resorptions) / total implants) x 100 - Statistics:
- Chi-square-test (* >= 95 %, ** >= 99 %, compared to the untreated control)
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- not applicable
- Endpoint:
- in vivo mammalian somatic cell study: gene mutation
- Remarks:
- gpt delta transgenic F344 rat
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- gpt delta transgenic F344 rats as well as wildetype F344 rats were administered 1,4-dioxane at different doses in the drinking water for 16 weeks.
- GLP compliance:
- not specified
- Type of assay:
- transgenic rodent mutagenicity assay
- Species:
- rat
- Strain:
- Fischer 344
- Details on species / strain selection:
- gpt delta transgenic and wildtype
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: transgenic animals: Japan SLC Inc.; wildtype animals: Charles River Japan, Inc.
- Age at study initiation: 6 weeks
- Fasting period before study: no
- Housing: polycarbonate cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: not specified
ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C
- Humidity: 55 +/-5 %
- Air changes: not specified
- Photoperiod: 12 / 12 hrs dark / hrs light
- Route of administration:
- oral: drinking water
- Vehicle:
- - Vehicle/solvent used: distilled water
- Details on exposure:
- - fresh distilled water containing the test item was changed three times a week
- Duration of treatment / exposure:
- daily
- Frequency of treatment:
- 16 weeks
- Post exposure period:
- not applicable
- Dose / conc.:
- 0 ppm
- Remarks:
- Experiment 1; control group
- Dose / conc.:
- 200 ppm
- Remarks:
- Experiment 1
corresponding to 18.7 mg/kg bw/d - Dose / conc.:
- 1 000 ppm
- Remarks:
- Experiment 1
corresponding to 92.3 mg/kg bw/d - Dose / conc.:
- 5 000 ppm
- Remarks:
- Experiment 1
corresponding to 440.2 mg/kg bw/d - Dose / conc.:
- 0 ppm
- Remarks:
- Experiment 2; control group
- Dose / conc.:
- 0.2 ppm
- Remarks:
- Experiment 2
corresponding to 0.02 mg/kg bw/d - Dose / conc.:
- 2 ppm
- Remarks:
- Experiment 2
corresponding to 0.2 mg/kg bw/d - Dose / conc.:
- 20 ppm
- Remarks:
- Experiment 2
corresponding to 1.9 mg/kg bw/d - Dose / conc.:
- 0 ppm
- Remarks:
- Experiment 3; control group
- Dose / conc.:
- 2 ppm
- Remarks:
- Experiment 3
- Dose / conc.:
- 20 ppm
- Remarks:
- Experiment 3
- Dose / conc.:
- 200 ppm
- Remarks:
- Experiment 3
- Dose / conc.:
- 2 000 ppm
- Remarks:
- Experiment 3
- Dose / conc.:
- 5 000 ppm
- Remarks:
- Experiment 3
- No. of animals per sex per dose:
- Experiment 1
Dose group number of animals (gpt delta transgenic)
0 8 males
200 7 males
1000 7 males
5000 8 males
Experiment 2
5 gpt delta transgenic males per dose group
Experiment 3
30 wildtype males per dose group - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- not applicable
- Tissues and cell types examined:
- liver
- Details of tissue and slide preparation:
- Experiment 1, 2 and 3:
At necropsy, livers were excised and weighed. A total of 3 sections of liver tissue (one section each from the left lateral lobe, right middle lobe, and caudate lobe) were fixed in phosphate buffered formalin, embedded in paraffin, and processed for hematoxylin/eosin and immunohistochemical staining. The remaining liver tissues were snap frozen with liquid nitrogen and stored at − 80 °C for mutation assays, oxidative DNA damage, and gene expression analyses.
In addition,for Experiment 3: One hour prior to sacrifice, ten rats from each group were injected i.p. with 100 mg BrdU/kg body weight for evaluation of cell proliferative activity in their livers. - Key result
- Sex:
- male
- Genotoxicity:
- positive
- Toxicity:
- yes
- Remarks:
- reduced body weight and relative liver weight increase (at 5000 ppm); histopathological changes in the liver (hypertrophy, swelling, necrosis, apoptosis, or fatty changes)
- Vehicle controls validity:
- not specified
- Negative controls validity:
- not applicable
- Positive controls validity:
- not applicable
- Remarks on result:
- other: Experiment 1
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- not specified
- Negative controls validity:
- not applicable
- Positive controls validity:
- not applicable
- Remarks on result:
- other: Experiment 2
- Key result
- Sex:
- male
- Genotoxicity:
- not determined
- Toxicity:
- yes
- Remarks:
- body weight decrease at 5000 ppm
- Vehicle controls validity:
- not specified
- Negative controls validity:
- not applicable
- Positive controls validity:
- not applicable
- Remarks on result:
- other: Experiment 3
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with national standard methods with acceptable restrictions
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- Method:
- according to Butterworth, B.E. et al. (1987). A protocol and guide for the in vivo rat hepatocyte DNA repair assay. Mutation Research 189, 123-133 (hepatocyte DNA-repair assay)
- according to Bermudez and Allen (1984). The assessment of DNA damage and repair in rat nasal epithelial cells. Carcinogenesis 5: 1453-1458 (respiratory epithelial cells assay) - 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 Lab. Inc. Raleigh NY
- Age at study initiation: 10-12 weeks
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72+/- 2
- Humidity (%): 50 +/- 10
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- other: oral: drinking water in combination with gavage
- Vehicle:
- water
- Duration of treatment / exposure:
- - liver hepatocytes: a single application of 1000 mg/kg bw or treatment with 1% 1,4-dioxane in drinking water for 2 weeks or treatment with 2% 1,4-dioxane for 1 week
- respiratory epithelial cells: 1% 1,4-dioxane in drinking water for 8 days, or 1% in drinking water for 8 days with an additional single gavage dose of 0, 10, 100 and 1000 mg/kg 12 h prior to sacrifice - Frequency of treatment:
- continuous and once by gavage
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- 1% in drinking water with an additional single gavage
- Dose / conc.:
- 10 mg/kg bw/day (actual dose received)
- Remarks:
- 1% in drinking water with an additional single gavage
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Remarks:
- 1% in drinking water with an additional single gavage
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Remarks:
- 1% in drinking water with an additional single gavage
- Dose / conc.:
- 1 other: %
- Remarks:
- concentration in drinking water
- Dose / conc.:
- 2 other: %
- Remarks:
- concentration in drinking water
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent no treatment
- Positive control(s):
- - hepatocyte DNA repair assay: DMN and 2-AAF
- nasal epithelial cell DNA repair assay: MMS - Tissues and cell types examined:
- liver hepatocytes and nasal epithelial cells
- Evaluation criteria:
- Labelled cells were recognized by by the dense pattern of silver grains overlying the nuclei, cells with a nuclei density of >= 10 were scored as undergoing replicative DNA-synthesis. The labeling index(LI) was calculated by dividing the number of labeled nuclei by the total counted.
The autoradiograms were stained and scored for the number of grains over the nucleus for 100 cells. - Statistics:
- for the hepatocytes : P <= 0.05 by the Newman-Keuls multicomparison test.
for the nasal epithelium: unpaired t-test at significance level of P<= 0.05 - Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- not specified
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Neither a single application of 1000 mg/kg bw, nor treatment with 1% 1,4-dioxane in drinking water for 2 weeks or with 2% 1,4-dioxane for 1 week, induced unscheduled DNA synthesis in primary rat hepatocytes. Negative results for unscheduled DNA synthesis were also found in rat nasal respiratory epithelial cells (from the nasoturbinate or the maxilloturbinate) after treatment of rats with 1% 1,4-dioxane in drinking water for 8 days, or after treatment with 1% in the drinking water for 8 days with an additional single gavage dose of up to 1000 mg/kg bw 1,4-dioxane.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
- Remarks:
- bone marrow and liver cell micronucleus (cytogenicity) and in vivo mutagenicity in Pig-a gene mutation assay in peripheral blood
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with national standard methods with acceptable restrictions
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The ability of 1,4-dioxane to induce micronuclei in the liver and bone marrow of rats was investigated.
The juvenile animal method and two methods using partial hepatectomy (PH), dosing before PH or dosing after PH were applied for the liver micronucleus test.
In vivo mutagenicity of 1,4-dioxane was also studied by Pig-a gene mutation assay using rat peripheral blood. - GLP compliance:
- not specified
- Type of assay:
- other: bone marrow and liver cell micronucleus (cytogenicity) and in vivo mutagenicity in Pig-a gene mutation assay in peripheral blood
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc. (Yokohama, Japan)
- Age at study initiation: 4 - 8 weeks (liver micronucleus); 6 - 7 weeks (bone marrow micronucleus and Pig-a assay)
- Assigned to test groups randomly: not specified
- Fasting period before study: not specified
- Housing: Econ PC cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1week
ENVIRONMENTAL CONDITIONS
- Temperature: 23 +/- 3 °C
- Humidity: 30-70 %
- Air changes: not specified
- Photoperiod: 12 / 12 hrs dark / hrs light - Route of administration:
- oral: unspecified
- Vehicle:
- - Vehicle/solvent used: Water
- Duration of treatment / exposure:
- Bone marrow MNT: Once on day 1
Liver MNT:
Juvenile rat method: Dosing on day 1 and 2
Dosing before partial hepatectomy (PH) method: Once on day 1 (PH on day 2)
Dosing after PH method: Once on day 1 (PH on day -1)
Pig-a Mutation assay: Once on day 1 only - Frequency of treatment:
- once a day
- Post exposure period:
- Bone marrow MNT: treatment on day 1; bone marrow sampling on day 3
Liver MNT:
Juvenile rat method: treatment on day 1 and 2; liver sampling on day 6
Dosing before partial hepatectomy (PH) method: treatment on day 1 (PH on day 2); liver sampling on day 6 (4 days after PH)
Dosing after PH method: treatment on day 1 (PH on day -1); liver sampling on day 4 (4 days after PH)
Pig-a Mutation assay: treatment on day 1; blood sampling on days 15 and 30 - Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 2 000 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 3 000 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- Bone marrow MNT: 5 males per dose group
Juvenile rat method: 4 males per dose group
Dosing before partial hepatectomy (PH) method: 4 males per dose group
Dosing after PH method: 4 males per dose group
Pig-a Mutation assay: 5 males per dose group - Control animals:
- yes
- Positive control(s):
- Bone marrow MNT: Cyclophosphamide (CP)
Juvenile rat and PH methods: diethylnitrosamine (DEN) & carbendazim
Pig-a Mutation assay: 7,12-dimethylbenz[a]anthracene (DMBA) - Tissues and cell types examined:
- liver, bone marrow and peripheral blood
- Evaluation criteria:
- not specified
- Statistics:
- Statistical analyses were performed by EXSUS Ver. 7.7 with significance level at 5%.
In the liver micronucleus tests (juvenile rat and PH methods), the incidence of MNH and dose dependency across all groups including the vehicle control group were analyzed by two-tailed Fisher’s exact test and two-tailed Cochran-Armitage trend test, respectively.
The relative liver weight was analyzed by a tree-type algorithm for quantitative data.
In the bone marrow micronucleus test, the incidence of MNIE and dose dependency across all groups including the vehicle control group were analyzed by two-tailed Fisher’s exact test and two-tailed Cochran-Armitage trend test, respectively. For the percentage of IE, two-tailed Wilcoxon’s rank sum test was used. The body weight was analyzed by two-tailed Dunnett’s test.
In the Pig-a assay, the incidence of CD59 negative red blood cells was analyzed by Bartlett’s test to evaluate the homogeneity of variance. The parameter was further analyzed by a parametric Dunnett’s test when the variance was homogeneous or by a Steel’s (nonparametric Dunnett’s) test when it was not. - Key result
- Sex:
- male
- Genotoxicity:
- positive
- Toxicity:
- no effects
- Vehicle controls validity:
- not specified
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: Liver micronucleus test by juvenile rat method
- Key result
- Sex:
- male
- Genotoxicity:
- positive
- Toxicity:
- no effects
- Vehicle controls validity:
- not specified
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: liver micronucleus test by both PH methods
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- decreased body weight at 3000 mg/kg bw/d
- Vehicle controls validity:
- not specified
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: bone marrow micronucleus
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- not specified
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: Pig-a assay
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
- GLP compliance:
- no
- Type of assay:
- mammalian erythrocyte micronucleus test
- Species:
- mouse
- Strain:
- B6C3F1
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 10-16 weeks
- Weight at study initiation: within +/-2 gm of a common weight
ENVIRONMENTAL CONDITIONS
No data - Route of administration:
- intraperitoneal
- Vehicle:
- saline (phosphate buffered saline)
- Duration of treatment / exposure:
- - three daily injections
- single injection - Frequency of treatment:
- once daily
- Post exposure period:
- - three daily injections: 24 hrs after the last injection
- single injection: 24 and 48 hrs after the last injection - Dose / conc.:
- 500 mg/kg bw/day (actual dose received)
- Remarks:
- ip injected
(three daily injections) - Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Remarks:
- ip injected
(three daily injections) - Dose / conc.:
- 2 000 mg/kg bw/day (actual dose received)
- Remarks:
- ip injected
(three daily injections) - Dose / conc.:
- 2 000 mg/kg bw (total dose)
- Remarks:
- ip injected (single injection)
- Dose / conc.:
- 3 000 mg/kg bw (total dose)
- Remarks:
- ip injected (single injection)
- Dose / conc.:
- 4 000 mg/kg bw (total dose)
- Remarks:
- ip injected (single injection)
- No. of animals per sex per dose:
- n=5 and n=6
- Control animals:
- yes
- Positive control(s):
- mitomycin C
- Tissues and cell types examined:
- bone marrow erythrocytes
- Details of tissue and slide preparation:
- Bone marrow smears were stained in acridine orange and examined under fluorescent microscopy.
- Evaluation criteria:
- Two observers each scored 1000 PCEs from each mouse on separate, coded slides, and determined the proportion of PCEs among 200 randomly selected erythrocytes. Data for the number of micronucleated PCEs were analyzed by a 1-tailed trend test (Margolin et al ., 1986), while values for the proportion of PCEs in the erythrocyte population were subjected to analysis of variance . Pairwise comparisons between each treatment group and their concurrent controls were also made using the t-test.
- Statistics:
- Statistical significance was set at an alpha level of 0 .05 but to adjust for multiple statistical analyses, i.e., a trend analysis and multiple pairwise comparisons, a p value of 0.025 is required for significance in the trend test and 0 .008 is required for significance of a pairwise comparison when three dosed groups are included in a trial.
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- the ratio PCE/NCE was decreased
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
- GLP compliance:
- no
- Type of assay:
- mammalian erythrocyte micronucleus test
- Species:
- mouse
- Strain:
- other: C57BL6 and Balb/c
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- Male and female C57BL6 mice and male BALB/c mice were used and maintained as described earlier (Ashby and Mirkova, 1987).
- Route of administration:
- oral: gavage
- Vehicle:
- distilled water
- Duration of treatment / exposure:
- once
- Frequency of treatment:
- once
- Post exposure period:
- 24 or 48 hrs after treatment
- Dose / conc.:
- 450 mg/kg bw (total dose)
- Dose / conc.:
- 900 mg/kg bw (total dose)
- Dose / conc.:
- 1 800 mg/kg bw (total dose)
- Dose / conc.:
- 3 600 mg/kg bw (total dose)
- Dose / conc.:
- 5 000 mg/kg bw (total dose)
- No. of animals per sex per dose:
- 0, 3600 mg/kg: males 10 per dose (24 hr sampling time)
0, 5000 mg/kg: females 10 per dose (5 for 24 hr sampling time and 5 for 48 hr sampling time)
0, 450, 900, 1800, 3600 mg/kg, 24 hr sampling time: males 10 per dose (for 3600 mg/kg 48 hr sampling time 5 males)
0, 900, 1800, 3600 mg/kg, 24 hr sampling time: males 4 per dose (for 3600 mg/kg 48 hr sampling time 4 males) - Control animals:
- yes
- Positive control(s):
- cyclophosphamide
- Tissues and cell types examined:
- bone marrow erythrocytes
- Evaluation criteria:
- Bone marrow smears were prepared and assessed for the incidence of micronucleated polychromatic erythrocytes (MPE) among polychromatic
erythrocytes (PE) 24 h and 48 h after dosing as described in detail earlier (Ashby and Mirkova, 1987). 2000 PE were assessed for MPE per animal. - Statistics:
- one-sided Student 's t- test
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Remarks:
- Balb/c mice
- Toxicity:
- not specified
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Sex:
- male/female
- Genotoxicity:
- positive
- Remarks:
- C57BL6 mice
- Toxicity:
- not specified
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- GLP compliance:
- no
- Type of assay:
- mammalian bone marrow chromosome aberration test
- Species:
- mouse
- Strain:
- CD-1
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Japan.
- Age at study initiation: 9 weeks
- Weight at study initiation: 32.6 - 39.7 gram
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 1 week
ENVIRONMENTAL CONDITIONS
No data - Route of administration:
- intraperitoneal
- Vehicle:
- saline for injection
- Duration of treatment / exposure:
- 96 hours
- Frequency of treatment:
- animals were dosed twice (24 hr apart)
- Dose / conc.:
- 500 mg/kg bw (total dose)
- Dose / conc.:
- 1 000 mg/kg bw (total dose)
- Dose / conc.:
- 2 000 mg/kg bw (total dose)
- Dose / conc.:
- 3 200 mg/kg bw (total dose)
- No. of animals per sex per dose:
- 5 males per dose
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- mitomycin C
- Tissues and cell types examined:
- peripheral erythocytes
- Details of tissue and slide preparation:
- Peripheral blood preparations were made just before treatment and 24, 48, and 72 h after the second treatment, using a supravital acridine orange staining method.
- Evaluation criteria:
- not reported
- Statistics:
- Kastenbaum and Boman (1970). Tables for determining the statistical significance of mutation frequencies. Muta. Res. , 9; 527-549.
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- one highest dose (3200 mg/kg) male died after 72 hrs
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
- GLP compliance:
- no
- Type of assay:
- mammalian erythrocyte micronucleus test
- Species:
- mouse
- Strain:
- CD-1
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Harlan (Indianapolis, Indiana, USA)
- Age at study initiation: 21 days
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22–25
- Humidity (%): 50–54%,
- Photoperiod (hrs dark / hrs light): 12/12
- Route of administration:
- oral: gavage
- Duration of treatment / exposure:
- once per day
- Frequency of treatment:
- 5 days
- Post exposure period:
- 24 hr after the last exposure
- Dose / conc.:
- 1 500 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 2 500 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 3 500 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 5 per group
- Control animals:
- yes
- Positive control(s):
- vinblastine sulphate (ip injection)
- Tissues and cell types examined:
- hepatocytes and bone marrow erythrocytes
- Evaluation criteria:
- The frequency of micronucleated eryhtrocytes among total erythrocytes were (2000/animal) was determined from the CREST stained bone-marrow slide. The proliferation-index was calculated as the number of nuclei incorporating BrdU divided by the total nuclei counted. Dose-related increases in the frequency of micronucleated eryhtrocytes and hepatocytes induced by the test chemicals were determened using ANOVA or regression analysis on the transformated data.
- Statistics:
- Following a positive result, Fisher's protected least significant difference (PLSD) was used as a post-hoc test.
- Key result
- Sex:
- male
- Genotoxicity:
- positive
- Toxicity:
- yes
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Significant dose-related increases in micronuclei were seen in both the liver and the bone-marrow with significant increases being detected at all the tested doses in the bone marrow and at the 2500 and 3500 mg/kg doses in the liver. Using CREST staining or pancentromeric FISH to determine the origin of the induced micronuclei, it was determined that 80-90% of the micronuclei in both tissues originated from chromosomal breakage. Small increases in centromere-containing micronuclei were also seen in the hepatocytes. Decreases in hepatocyte proliferation as well as in the ratio of bone marrow PCE:NCE were also observed.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
- GLP compliance:
- no
- Type of assay:
- mammalian erythrocyte micronucleus test
- Species:
- mouse
- Strain:
- other: C57BL6 and CBA
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 8-12 weeks
no further data
ENVIRONMENTAL CONDITIONS
no data - Route of administration:
- oral: gavage
- Vehicle:
- distilled water
- Duration of treatment / exposure:
- once
- Frequency of treatment:
- once
- Post exposure period:
- 24 hours
- Dose / conc.:
- 1 800 mg/kg bw (total dose)
- Dose / conc.:
- 3 600 mg/kg bw (total dose)
- No. of animals per sex per dose:
- CBA mice: 1800 mg/kg: 12 animals
C57BL6: 3600 mg/kg: 4 animals - Control animals:
- yes
- Positive control(s):
- cyclophosphamide
- Tissues and cell types examined:
- bone marrow erythrocytes
- Details of tissue and slide preparation:
- Bone marrow smears were prepared as described previously (Tinwell and Ashby, 1989). As the present studies were performed during our transition from the use of Giemsa to acridine orange (AO) as our stain of choice for bone marrow smears, one study was analysed using Giemsa stained slides.
- Evaluation criteria:
- The incidence of micro nucleated polychromatic erythrocytes (MPE) was determined among 2000 PE for each animal . The ratio of PEs to normocytes (PE/NE) was based on 1000 erythrocytes per smear .
- Statistics:
- one-sided Students t-test
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- ratio PCE/NCE was decreased in CBA mice
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- Method: according to Hayashi et al., 1990 (periphal blood), Igarashi, Shimada, 1994 (liver)
- GLP compliance:
- no
- Type of assay:
- mammalian erythrocyte micronucleus test
- Species:
- mouse
- Strain:
- CD-1
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River, Japan, Inc . (T'sukuba, Japan),
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 30.3-38.5 g)
- Assigned to test groups randomly: five animals were randomly allocated to each group except for the positive control group, which had four animals
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: one week
ENVIRONMENTAL CONDITIONS
no data
- Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: physiol. saline
- Duration of treatment / exposure:
- once
- Frequency of treatment:
- once
- Dose / conc.:
- 1 000 mg/kg bw (total dose)
- Dose / conc.:
- 2 000 mg/kg bw (total dose)
- Dose / conc.:
- 3 000 mg/kg bw (total dose)
- No. of animals per sex per dose:
- 5/dose
- Control animals:
- yes
- Positive control(s):
- mitomycin C
- Tissues and cell types examined:
- peripheral blood and liver
- Evaluation criteria:
- The selection and scoring of hepatocytes for micronucleated hepatocytes was according to published criteria [Braithwaite and Ashby,1988; Cliet et al ., 1989]. For the peripheral blood micronucleus assay the results were analyzed with Kastenbaum and Bowman's [1970] table.
- Statistics:
- Not reported.
- Key result
- Sex:
- male
- Genotoxicity:
- positive
- Toxicity:
- not specified
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: liver
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- not specified
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: peripheral blood
- Additional information on results:
- The blood micronucleus assay was negative. The mouse liver micronucleus assay, on the other hand, was positive.
Referenceopen allclose all
Male mice:
- Clinical signs of toxicity:
The animals showed immediately after i.p. application of the test substance an increase in spontaneous motor function for a period of approx. 15 seconds. Apart from these unspecific symptoms no test substance-related clinical signs were observed. Untreated and vehicle-control animals did not demonstrate any clinical symptoms.
- Body weights:
Treated males showed body weight development comparable to untreated and vehicle-control animals.
- Gross-pathological examination:
At necropsy treated, untreated, and vehicle-control animals did not show any abnormalities.
Female mice:
- Conception rate:
The conception rate varied among test groups. No substance-related alteration could be detected.
- Mutagenicity Index (implants, dead implants):
No substance-related difference between test and control groups could be observed.
Control untreated:
Mating week |
Conception rate |
Implantations (mean no.) |
Living fetuses (%) |
Dead fetuses (%) |
Deciduomata (%) |
Early resorptions (%) |
Dead implantations (%) |
Corpora lutea |
Mutagenicity |
1 |
83.33 |
12.24 |
91.67 |
0.65 |
7.68 |
0.00 |
8.33 |
0.0 |
8.33 |
2 |
88.33 |
11.89 |
87.62 |
0.48 |
10.95 |
0.95 |
12.38 |
0.0 |
12.38 |
3 |
93.33 |
12.91 |
91.42 |
0.55 |
7.88 |
0.14 |
8.58 |
0.0 |
8.58 |
4 |
93.33 |
13.00 |
89.70 |
0.82 |
9.48 |
0.00 |
10.30 |
0.0 |
10.30 |
5 |
88.33 |
12.72 |
88.58 |
1.04 |
10.09 |
0.30 |
11.42 |
0.0 |
11.42 |
6 |
88.33 |
13.47 |
90.34 |
0.14 |
9.10 |
0.42 |
9.66 |
0.0 |
9.66 |
7 |
90.00 |
13.46 |
89.27 |
0.55 |
10.18 |
0.00 |
10.73 |
0.0 |
10.73 |
8 |
81.67 |
13.47 |
88.48 |
1.06 |
8.48 |
1.97 |
11.52 |
0.0 |
11.52 |
Control water:
Mating week |
Conception rate |
Implantations (mean no.) |
Living fetuses (%) |
Dead fetuses (%) |
Deciduomata (%) |
Early resorptions (%) |
Dead implantations (%) |
Corpora lutea |
Mutagenicity |
1 |
90.00 |
12.46 |
90.04 |
1.49 |
8.32 |
0.15 |
9.96 |
0.0 |
9.96 |
2 |
91.67 |
13.49 |
91.24 |
0.94 |
7.82 |
0.00 |
8.76 |
0.0 |
8.76 |
3 |
95.00 |
13.81 |
87.93 |
0.89 |
11.18 |
0.00 |
12.07 |
0.0 |
12.07 |
4 |
100.00 |
13.63 |
89.49 |
0.61 |
9.66 |
0.24 |
10.51 |
0.0 |
10.51 |
5 |
95.00 |
13.25 |
90.46 |
0.79 |
8.74 |
0.00 |
9.54 |
0.0 |
9.54 |
6 |
95.00 |
14.07 |
92.14 |
0.12 |
7.73 |
0.00 |
7.86 |
0.0 |
7.86 |
7 |
93.33 |
13.71 |
89.71 |
0.52 |
9.77 |
0.00 |
10.29 |
0.0 |
10.29 |
8 |
91.67 |
13.18 |
93.93 |
0.28 |
5.79 |
0.00 |
6.07 |
0.0 |
6.07* |
* Significant
Treatment group: 2500 µL/kg b.w. Dioxane
Mating week |
Conception rate |
Implantations (mean no.) |
Living fetuses (%) |
Dead fetuses (%) |
Deciduomata (%) |
Early resorptions (%) |
Dead implantations (%) |
Corpora lutea |
Mutagenicity |
1 |
85.00 |
11.80 |
90.37 |
0.33 |
9.14 |
0.17 |
9.63 |
0.0 |
9.63 |
2 |
88.33 |
13.09 |
89.63 |
0.72 |
9.65 |
0.00 |
10.37 |
0.0 |
10.37 |
3 |
90.00 |
13.44 |
90.63 |
0.14 |
9.23 |
0.00 |
9.37 |
0.0 |
9.37 |
4 |
96.67 |
13.57 |
90.98 |
0.51 |
8.51 |
0.00 |
9.02 |
0.0 |
9.02 |
5 |
93.33 |
13.46 |
93.24 |
0.13 |
6.63 |
0.00 |
6.76 |
0.0 |
6.76* |
6 |
93.33 |
12.84 |
91.38 |
0.70 |
7.93 |
0.00 |
8.62 |
0.0 |
8.62 |
7 |
95.00 |
13.12 |
89.97 |
0.13 |
9.89 |
0.00 |
10.03 |
0.0 |
10.03 |
8 |
86.67 |
13.56 |
89.79 |
0.85 |
9.36 |
0.00 |
10.21 |
0.0 |
10.21 |
* Significant
The overall mutation frequency (MF) and A:T- to -G:C transitions and A:T- to -T:A transversions in the gpt transgene were significantly increased by administration of 5000 ppm 1,4 -dioxane (experiment 1).
A:T- to -T:A transversions were also significantly increased by administration of 1000 ppm 1,4 -dioxane (experiment 1).
Furthermore, the DNA repair enzyme MGMT was significantly induced at 5000 ppm 1,4-dioxane, implying that extensive genetic damage exceeded the repair capacity of the cells in the liver and consequently led to liver carcinogenesis (experiment 1).
No evidence supporting other MOAs, including induction of oxidative stress, cytotoxicity, or nuclear receptor activation, that could contribute to the carcinogenic effects of 1,4-dioxane were found.
Table 1 gpt transgene MFs and induction of GST-P positive foci in the livers of gpt delta transgenic F344 rats administered high doses of 1,4-dioxane for 16 weeks (Experiment 1; n=8 for 0 and 5000 ppm group; n=7 for 200 and 1000 ppm group)
1,4-dioxane (ppm) |
Average MF (x10-5) |
GST-P positive foci (No./cm2) |
0 |
0.43 +/- 0.26 |
1.3 +/- 1.4 |
200 |
0.65 +/- 0.31 |
1.6 +/- 1.0 |
1000 |
0.79 +/- 0.29 |
2.5 +/- 1.5 |
5000 |
1.32 +/- 0.71a |
13 +/- 5.5b |
aSignificantly different from the control group (0 ppm) at p < 0.05
bSignificantly different from the control group (0 ppm) at p < 0.001
In addition, the authors describe a negative result when using 5000 mg/kg for Balb/c mice (a dose at which death occured 1/6).
Table 1 Liver MN-test
Dose (mg/kg bw) |
Hepatocytes with MN/2000 hepatocytes |
0 |
0.43 +/- 0.14 |
1000 |
0.49 +/- 0.19 |
2000 |
1.02 +/- 1.04* |
3000 |
1.24 +/- 0.33* |
Mitomycin C |
2.65 +/- 1.80* |
(*p < 0.01)
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In vitro studies
Bacterial reverse mutation assay (Ames test)
There are four valid Ames tests available with 1,4-dioxane that are considered reliable and sufficient for evaluation (BASF AG, 1997a, b, c; Morita 1998). Table 1 summarises respective test conditions and results.
Table 1. Summary of Ames tests with 1,4 -dioxane
Strains tested |
Concentrations tested |
Vehicle |
Metabolic activation (S9) |
Outcome |
Source |
S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 1538 |
4, 20, 100, 500, 2500 µg/plate |
Aqua. Dest. |
With and without |
Negative |
BASF AG, 1997a |
S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 1538 |
4, 20, 100, 500, 2500 µg/plate |
Aqua. Dest. |
With and without |
Negative |
BASF AG, 1997b |
S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 1538 |
4, 20, 100, 500, 2500 µg/plate |
Aqua. Dest. |
With and without |
Negative |
BASF AG, 1997c |
S. typhimurium TA 1535, TA 1537, TA 98, TA 100,
E. coli WP2, WP2 uvr A pKM 101 |
0, 156, 313, 625, 1250, 2500, 5000 µg/plate |
Water |
With and without |
Negative |
Morita, 1998 |
In summary, all four bacterial reverse mutation tests revealed negative results.
Gene mutation in mammalian cells (HPRT)
A test on gene mutation in mammalian cells according to OECD TG 476 was conducted with 1,4-dioxane applying concentrations ranging from 0.05 to 10 mg/mL with and without metabolic activation. Under the conditions of this assay, 1,4-dioxane was found to be non-mutagenic.
Chromosome aberration
1,4-dioxane was negative in an in vitro chromosome aberration assay conducted in Chinese hamster ovary cells with and without metabolic activation system at concentrations ranging from 1050 to 10500 µg/mL (Galloway, 1987).
In vivo
A rodent dominant lethal test in mice was conducted with 1,4-dioxane applying a single dose of 2575 mg/kg bw via intraperitoneal injection (BASF AG, 1991). Under the conditions of this study no germ cell mutagenicity could be detected.
An unscheduled DNA synthesis test in male rats administered 1,4-dioxane orally by gavage with dose levels ranging from 10 to 1000 mg/kg bw/d and additionally exposed via drinking water at a concentration of 1 % revealed no positive findings in both liver hepatocytes and nasal epithelial cells (Goldsworthy et al., 1991).
In a transgenic rodent mutagenicity assay somatic gene mutation activity of 1,4-dioxane was investigated (Gi et al., 2018). Male gpt delta transgenic rats were administered 1,4-dioxane at doses ranging from 0.2 to 5000 ppm in drinking water for 16 weeks. Under the conditions of this study indications of mutagenicity were observed at doses levels of 2000 and 5000 ppm in drinking water.
Mutation of Pig-A gene in peripheral blood of male Fischer 344 rats was investigated after single oral exposure with 1,4-dioxane at doses of 1000, 2000, 3000 mg/kg bw/d (Itoh &Hattori, 2019). Under the conditions of this study, no indications for mutagenicity were observed.
Micronucleus tests
There are numerous in vivo micruncleus tests (MNT) available conducted with 1,4-dioxane. Only relevant and reliable studies are included and discussed in this dossier. MNT test conditions and results are summarized in table 2.
Table 2. Summary of in vivo Micronucleus Tests (MNT) conditions and results.
Species |
Dose level (mg/kg bw) |
Vehicle |
Route & frequency of exposure |
Outcome (tissue examined) |
Source |
Rat (Fischer 344) |
1000, 2000, 3000 |
water |
Oral (once)
|
Positive (liver)
Negative (bone marrow) |
Itoh & Hattori, 2019 |
Mouse (B6C3F1) |
500, 1000, 2000, 3000, 4000 |
Saline (PBS) |
i.p. (once or daily for 3 days) |
Negative (bone marrow) |
McFee et al., 1994 |
Mouse (C57BL6) |
450, 900, 1800, 3600, 5000 |
Dist. water |
Oral (once)
|
Positive (bone marrow) |
Mirkova, 1994 |
Mouse (BALB/c) |
450, 900, 1800, 3600, 5000 |
Dist. water |
Oral (once)
|
Negative (bone marrow) |
|
Mouse (C57BL6 & CBA) |
1800, 3600 |
Dist. water |
Oral (once)
|
Negative (bone marrow) |
Tinwell & Ashby, 1994 |
Mouse (CD-1) |
500, 1000, 2000, 3200 |
saline |
i.p. (daily for 2 days) |
Negative (peripheral blood) |
Morita, 1994 |
Mouse (CD-1) |
1500, 2500, 3500 |
n. a. |
Oral (daily for 5 days) |
Positive (bone marrow)
Positive (hepatocytes) |
Roy et al., 2005 |
Mouse (CD-1) |
1000, 2000, 3000 |
saline |
Oral (once) |
Negative (peripheral blood)
Positive (liver) |
Morita, 1998 |
Discussion
Gi et al. (2018) reported indications for a
mutagenic potential of 1,4-dioxane under the conditions of the TRM study
conducted. It is noted that the treatment period of 16 weeks applied in
this study is unusually long for this type of study and it raises
concerns of false positive findings. In particular, according to the
validated OECD test guideline 488 (2013) treatment periods longer than 8
weeks in transgenic rodent models “may produce an apparent increase in
mutant frequency through clonal expansion” (OECD TG 488, 2013; Thybaud
et al., 2003).
Further, it is noteworthy that the two highest doses applied at which
positive findings were obtained only, i. e. 2000 and 5000 ppm
(equivalent to approx. 176 and 440 mg/kg bw/d) exceeded the linear
toxicokinetic range of 1,4-dioxane in rats by far (please also refer to
IUCLID section 7.1). The OECD TG 488 states that “chemicals which
exhibit saturation of toxicokinetic properties may be exceptions to the
dose-setting criteria and should be evaluated on a case-by-case basis”.
Therefore, positive results obtained above the toxicokinetic linearity
must be treated with caution.
Itoh & Hattori (2019) reported positive
findings in the liver micronucleus test conducted in rats applying the
juvenile rat method as well as two different partial hepatectomy
methods. As already discussed above, partial hepatectomy may display an
additional primary event cofounding genotoxicity findings by means of
induction of regenerative cell proliferation. Hepatectomy is thus
considered an abnormal physiological condition which might lower the
informative value of the study´s results.
Further, it is noted that again all three study designs applied doses
being far beyond the toxicokinetic linear range. Though it is recognized
that only single or two time treatments were conducted, the dose levels
administered highly likely led to over proportional high internal doses.
This in turn, questions the value of the resulting findings at the dose
levels tested.
Mirkova (1994) reported increased micronuclei findings in bone marrow of one (C57BL6) but not in another (BALB/c) mouse strain exposed to the same dose levels of 1,4-dioxane and tested under the same conditions. Tinwell & Ashby (1994) could not re-produce this finding when testing 1,4-dioxane in the same mouse strain at same dose levels in a bone marrow micronucleus test applying same sampling times. Against this background, the positive finding reported by Mirkova is regarded as rather equivocal.
Regarding the results of Roy et al. (2005) in CD-1 mice, it should be recognized that particularly high doses were applied (1500, 2500, and 3500 mg/kg bw/d) for five consecutive days exceeding the normally used maximum dose of 2000 mg/kg bw for these type of studies. Furthermore, the effect appeared to be stronger in the study due to the low background of very young mice used (only 3 weeks old). Therefore, the result of this study is questionable.
In the oral MNT study by Morita (1998), negative effects were observed in blood, while positive effects were observed in the liver. As partial hepatectomy was performed, the positive results in the liver may have been caused by another primary stimulus. Partial hepatectomy will cause proliferation in the liver and is considered an abnormal physiological condition. The overall test result of the Morita study therefore has to be considered as negative.
Overall, it is noteworthy that in vivo micronucleus studies using the i. p. route (McFee et al., 1994; Morita, 1994 (B6C3F1 and CD-1 mice)) showed clearly negative results.
Conclusion
Based on in vitro data, there are no indications for a genotoxic potential of 1,4-dioxane. In vivo studies revealed both positive and negative results in regards to point mutation as well as cytogenicity.
Where the negative results obtained in
in vivo studies are consistent with in vitro results, it is noteworthy
that positive results, were found at high dose levels only. Considering
that 1,4-dioxane is known to follow a non-linear toxicokinetic and its
elimination becomes saturated at doses of approx. 9.6 – 42 mg/kg bw/d in
rats and 57 – 66 mg/kg bw/d in mice (Dourson et al., 2017; IUCLID
Section 7.1 & 7.12), any effects observed beyond this kinetic MTD must
be regarded to be of questionable relevance for the assessment of
1,4-dioxane. Moreover, respective study designs did not always follow
generally agreed methods, i. e. exposure duration or dose levels were
exceeded where positive findings were obtained.
It can be concluded that the weight of
scientific evidence supports that 1,4-dioxane is not mutagenic. It is
noted that exposure to 1,4-dioxane may elicit clastogenicity in vivo at
high doses exceeding the linear range of toxicokinetics.
References not provided as RSS in this dossier:
- OECD TG 488, OECD Guidelines for the testing of chemicals: Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays, 2013
- Thybaud, V., S. Dean, T. Nohmi, J. de Boer, G.R. Douglas, B.W. Glickman, N.J. Gorelick, J.A. Heddle, R.H. Heflich, I. Lambert, H.-J. Martus, J.C. Mirsalis, T. Suzuki and N. Yajima (2003), “In vivo Transgenic Mutation Assays”, Mutation Res., 540: 141-151.
Justification for classification or non-classification
Classification,
Labelling, and Packaging Regulation (EC) No 1272/2008
The
available experimental test data are reliable and suitable for
classification purposes under Regulation (EC) No 1272/2008. Based
on the total weight of evidence of all available data, classification as
a genotoxic substance is not triggered according to EU Classification,
Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC)
No. 1272/2008.
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