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EC number: 247-063-2 | CAS number: 25513-64-8
- 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
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- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
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- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
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- Endpoint summary
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- 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
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- 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
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- January 1985
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study with acceptable restrictions: TA 102 or E.coli WP2 were not tested
- Principles of method if other than guideline:
- Ames BN et al. (1975). Mutat. Res. 31, 347-364
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- mutated gene loci resposible for histidine auxotrophy
- Species / strain / cell type:
- other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100
- Additional strain / cell type characteristics:
- other: histidine auxotroph
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix based on S9 fraction from Aroclor 1254 induced male Wistar rat liver
- Test concentrations with justification for top dose:
- 1 to 5000 µg/plate
- Vehicle / solvent:
- DMSO dimethyl sulfoxide (CAS No. 67-68-5)
- Untreated negative controls:
- yes
- Remarks:
- untreated
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: without metabolic activation: 2-nitrofluorene, sodium azide, 9-aminoacridine, picrolonic acid; with metabolic activation: 2-aminoanthracene, 2-aminoanthracene, ethidium bromide
- Remarks:
- details see below
- Details on test system and experimental conditions:
- Ames test
SYSTEM OF TESTING
- Metabolic activation system: S9 mix based on S9 fraction from Aroclor 1254 induced male Wistar rat liver (Robens Institute, University of Surrey, 50 µl S9 fraction/plate
ADMINISTRATION:
- Dosing:
Test 1: 15.6; 31.2; 62.5; 125; 250; 500; 1000; 2500; 5000 µg/plate
Test 2: 1; 10; 50; 250; 1000; 5000 µg/plate
- Number of replicates: 3 replicates x 2 tests
- Application: Solvent dimethyl sulfoxide (CAS No. 67-68-5)
- Positive and negative control groups and treatment:
Positive (TA 1535, TA 100, -S9): Sodium azide
Positive (TA 1537, -S9): 9-Aminoacridine
Positive (TA 1538, -S9): 2-Nitrofluorene
Positive (TA 98, -S9): Picrolonic acid
Positive (TA 1535, TA 100, +S9): 2-Aminoanthracene
Positive (TA 1537, +S9): Neutral red or 2-Aminoanthracene
Positive (TA 1535, TA 98, +S9): Ethidium bromide
Negative: Untreated plus solvent
- Incubation time: 72 hours at 37 °C; no pre-incubation - Evaluation criteria:
- CRITERIA FOR EVALUATING RESULTS: Ratio of revertant rates treated/control >= 2 at any concentration with generally positive dose-response
relationship in any strain - Statistics:
- not reported
- Species / strain:
- other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: Onset at 5000 µg/plate (only -S9)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- GENOTOXIC EFFECTS:
- With metabolic activation: None
- Without metabolic activation: None
The positive controls were functional.
PRECIPITATION CONCENTRATION: No precipitation reported - Remarks on result:
- other: other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
The test substance 2,2,4-trimethylhexamethylenediamine showed no evidence of genetic activity when tested against Salmonella typhimurium TA1535, TA1537, TA1538, TA 98, TA100, both in the presence and in absence of rat liver S9. - Executive summary:
The test substance 2,2,4-trimethylhexamethylenediamine was tested in the Ames Salmonella/microsomes mutagenicity test for any mutagenic activity using the five histidine-auxotrophic Salmonella typhimurium strains TA 153, TA 1537, TA 1538, TA 98 and TA 100. The test substance concentrations were in the range between 1 and 5000 µg/plate. 2,2,4-trimethylhexamethylenediamine proved to be non-mutagenic under the conditions of this study, both in the absence and in the presence of metabolic activation system, for all the test strains. At the maximum concentration tested without metabolic activation the test substance produced a thinning of the background lawn indicating the onset of toxicity towards the bacterial strain.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1992-05-26 to 1992-09-11
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- mammalian cell system( Chinese hamster Ovary cells)
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CHO-K1 BH4
cell cycle length of 12 hours - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- male Sprague-Dawley rat liver S9 from Aroclor 1254 induced animals
- Test concentrations with justification for top dose:
- Preliminary toxicity test: 0; 19.5; 39.1; 78.13; 156.25; 312.5; 625; 1250; 2500; 5000 mg/l
Experiment 1: 156.25; 312.5; 625 mg/l with and without S9
Experiment 2: 156.25; 312.5; 625 mg/l (without S9, 12 hour harvest); 312.5; 625; 937.5 mg/l (other) - Vehicle / solvent:
- Solvent Ham's Media
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Solvent Ham's Media
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- positive with metabolic activation: cyclophosphamide
Migrated to IUCLID6: without metabolic activation - Details on test system and experimental conditions:
- SYSTEM OF TESTING
- Species/cell type: CHO-K1 BH4 cell line, cell cycle length 12 hours
- Metabolic activation system: male Sprague-Dawley rat liver S9 from Aroclor 1254 induced animals, lots Aro. S9/08/05/92 and Aro. S9/08/06/92
from British Industrial Biological Research Association
- No. of metaphases analyzed: first 100 consecutive from each culture if possible; mitotic index based on 1000 cell nuclei counted
ADMINISTRATION:
- Dosing:
Preliminary toxicity test: 0; 19.5; 39.1; 78.13; 156.25; 312.5; 625; 1250; 2500; 5000 mg/l
Experiment 1: 156.25; 312.5; 625 mg/l with and without S9
Experiment 2: 156.25; 312.5; 625 mg/l (without S9, 12 hour harvest); 312.5; 625; 937.5 mg/l (other)
Confirmatory experiment: 800, 937.5, 1000 mg/l with S9, both with and without Hepes buffer, evaluated for 937.5 mg/l, only 20 hour duration performed
- Number of replicates: 2
- Application: Solvent Ham's Media with S9: 4 hours exposure + 8 or 16 h culture period without S9: continuous for 12 or 20 hours
- Positive and negative control groups and treatment:
negative: solvent
positive with S9: cyclophosphamide, 10 mg/l (experiment 1, 12 hours) or 5 mg/l (other)
positive without S9: mitomycin C, 0.075 mg/l (experiment 1, 12 hours) or 0.05 mg/l (other) - Evaluation criteria:
- CRITERIA FOR EVALUATING RESULTS: significant increase in the frequency of aberrations, Fisher's Exact test
- Statistics:
- The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared with the concurrent vehicle control value using Fisher's Exact Test for the hypothesis of equal means.
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: complete at >= 1250 mg/l (with and without metabolic activation)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- GENOTOXIC EFFECTS:
- With metabolic activation: none
The increase in the frequency of cells with aberrations at 12 hours / 312.5 mg/l in experiment 1 was not considered to be toxicologically
significant because the frequency of cells with aberrations was well within historical ranges for this cell line and similar to the maximum vehicle
control value in this study. The increase at 20 hours / 937.5 mg/l in experiment 2 was investigated further in the confirmatory experiment and
identified to be an artefact caused by the interaction of a high pH value and the S9 metabolic activation system.
- Without metabolic activation: none
- Positive controls: highly significant response
CYTOTOXIC CONCENTRATION:
- With metabolic activation: observed at >= 625 mg/l nearly total absence of metaphase cells at >= 1250 mg/l
- Without metabolic activation: observed at >= 156.25 (20 hour culture) or 312.5 (12 hour culture) mg/l total absence of metaphase cells
at >= 1250 mg/l
- A dose-related increase of cytotoxicity was noted. A color change observed in the culture medium indicated that the test substance caused a
dose-related increase in the pH. Since addition of a buffer in the confirmatory experiment reduced toxicity, pH seems to be decisive for
cytotoxicity. - Remarks on result:
- other: strain/cell type: CHO (Chinese hamster ovary) cells
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
The test substance 2,2,4-(2,4,4)-trimethyl-1,6-hexanediamine was shown to be non-clastogenic to CHO cells in vitro. - Executive summary:
The substance 2,2,4-(2,4,4)-trimethyl-1,6-hexanediamine was tested for its ability to induce structural chromosome aberrations in an in vitro mammalian cell system (CHO Chinese hamster ovary cells). Two independent experiments were carried out with and without the addition of Arochlor 1254 -induced rat liver S9 mix. 2,2,4-(2,4,4)-trimethyl-1,6-hexanediamine produced a highly statistically significant increase in the frequency of cells with chromosome aberrations both including and excluding gaps in 20-hour with metabolic activation treatment group at the maximum dose level only, in Experiment 2. However, this was demonstrated to be an artefact caused by the interaction of a high ph value and the S9 metabolic activation systhem. 2,2,4-(2,4,4)-trimethyl-1,6-hexanediamine is therefore considered to be non-clastogenic to CHO cells in vitro.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- disregarded due to major methodological deficiencies
- Study period:
- 1993-01-08 to 1993-03-15
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- other: Guideline study, but the number of three analyzable doses without accompanied cytotoxicity was not achieved. Therefore a dose response related increase or reproducible increase could not be evaluated.
- Qualifier:
- according to guideline
- Guideline:
- other: Japanese guidlines No. 700, No. 1039, No. 1014 (1986) and No. 143 (1987)
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- mammalian cell system( Chinese hamster Ovary cells)
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CHL/IU, optained from Dainippon Pharmaceutical Co., Ltd.
doubling time: about 16 h - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- male Sprague-Dawley rat liver S9 from Phenobarbital (PB), 5,6-Benzoflavone (BF) induced animals
- Test concentrations with justification for top dose:
- Preliminary toxicity test: 0; 62.5, 125, 250, 500, 1000 µg/ml , 24 and 48 hours without metabolic activation, 6 hours with metabolic activation
Experiment 1: 75, 150, 300 µg/ml, 24 hours and 65, 130, 260 µg/ml, 48 hours, without S9
Experiment 2: 163, 325, 650 µg/ml (+/- S9, 6 hours) - Vehicle / solvent:
- Physiological saline
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Physiological saline
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- mitomycin C
- Remarks:
- Migrated to IUCLID6: without metabolic activation
- Details on test system and experimental conditions:
- SYSTEM OF TESTING
- Species/cell type: CHL/IU, optained from Dainippon Pharmaceutical Co., Ltd.
- Metabolic activation system: male Sprague-Dawley rat liver S9 from Phenobarbital (PB), 5,6-Benzoflavone (BF) induced animals,
amount of S9 protein: 1.39 mg/ml
- No. of metaphases analyzed: first 100 consecutive from each culture if possible; mitotic index based on 1000 cell nuclei counted
ADMINISTRATION:
- Dosing:
Cell growth inhibition test
0 (solvent); 62.5, 125, 250, 500, 1000 µg/ml , 24 and 48 hours without metabolic activation, 6 hours with metabolic activation
Chromosomal aberration test
Experiment without S9 : 75, 150, 300 µg/ml, 24 hours and 65, 130, 260 µg/ml, 48 hours,
Experiment with S9: 163, 325, 650 µg/ml (+/- S9, 6 hours)
- Number of plates: 2
- Application: Solvent Physiological saline with S9: 6 hours exposure + 18 h cell recovery period, without S9: continuous for 24 or 48 hours
- Positive and negative control groups and treatment:
negative: solvent
positive with S9: benzo(a)pyrene, 20 µg/ml (6 hours)
positive without S9: mitomycin C, 0.03 µg/ml ( 24 and 48 hours) - Evaluation criteria:
- CRITERIA FOR EVALUATING RESULTS: significant increase in the frequency of aberrations
- Statistics:
- A significant difference was tested using X² test
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 285, and 227 µg/ml at 24 and 48 hours without metabolic activation, 622 µg/ml with metabolic acvtivation at 6 hours
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- GENOTOXIC EFFECTS:
- With metabolic activation:
- Positive controls: highly significant response
CYTOTOXIC CONCENTRATION:
- with metabolic activation: 285 and 227 µg/ml at 24 and 48 hours
- without metabolic activation: 622 µg/ml with metabolic acvtivation at 6 hours - Remarks on result:
- other: strain/cell type: CHO (Chinese hamster ovary) cells
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Although there was a positive response after 24 h exposure without S9 activation at 300 ug/l and after 6 h exposure with S9 activation at 625 ug/l, these concentrations have been shown to be cytotoxic. A positive response without accompanied cytotoxicity was observed after 6 h exposure with S9 activation at 325 ug/l. However, it is requested to have three analyzable doses. This has not been achieved in this study. Therefore the outcome of this study is questionable.
- Executive summary:
The test item was tested for its ability to induce structural chromosome aberrations in an in vitro mammalian cell system (CHO Chinese hamster ovary cells). Two independent experiments were carried out with and without the addition of Phenobarbital (PB), 5,6-Benzoflavone (BF) induced rat liver S9 mix.
In the cell growth inhibition test, the concentrations of the test substance showing 50% cell growth inhibition were 285, 227 and 622 µg/mL at 24 and 48 hours treatments without metabolic activation and at treatment with metabolic activation, respectively. Therefore, for the chromosomal aberration test, the following 3 concentrations each were established: 75, 150 and 300 µg/mL at 24 hours treatment without metabolic activation, 65, 130 and 260 µg/mL at 48 hours treatment without metabolic activation and 163, 325 and 650 µg/ml at treatment with metabolic activation in the absence and presence of S9Mix. Since there was no metaphase because of cytotoxicity at the highest concentration with metabolic activation in the absence of S9Mix after preparation of chromosome sample, the remaining 2 concentrations were observed. As for the results of observation, the significant difference between the negative control group and each treatment group was examined using X² test. As for the structural chromosomal aberration, only the totalized value without the gap was used for the test. Because the incidence of the cell with structural chromosomal aberration, in which a significant increase was observed, showed to be clearly positive as 18% and 13.5% at 24 hours treatment without metabolic activation and at treatment with metabolic activation, respectively, no identification test was performed.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1992-11-17 to
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: TA 102 was not tested
- Qualifier:
- according to guideline
- Guideline:
- other: Japanese guidlines No. 700, No. 1039, No. 1014 (1986) and No. 143 (1987)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- mutated gene loci responsible for histidine auxotrophy
- Species / strain / cell type:
- other: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2 uvrA
- Details on mammalian cell type (if applicable):
- Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 obtained fromProfessor B.N. Ames, California Univerisity
E.coli WP2 uvrA obtained from Professor Matsushima, University of Tokio - Additional strain / cell type characteristics:
- other: histidine auxotroph
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital, 5,6-Benzoflavone induced rat S9 liver, male rat, SD strain
- Test concentrations with justification for top dose:
- main test: 156, 313, 625, 1250, 2500, 5000 µg/plate (+/- metabolic activation)
pre-incubation test: 156, 313, 625, 1250, 2500, 5000 µg/plate (+/- metabolic activation) - Vehicle / solvent:
- Destilled water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Destilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- benzo(a)pyrene
- other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, N-Ethyl-N'-nitro-N-nitrosoguanidine, 2-Aminoanthracene
- Details on test system and experimental conditions:
- Ames test
SYSTEM OF TESTING
- Metabolic activation system: Phenobarbital, 5,6-Benzoflavone induced rat S9 liver, male rat, SD strain
ADMINISTRATION:
- Dosing:
main test: 156, 313, 625, 1250, 2500, 5000 µg/plate (+/- metabolic activation)
pre-incubation test: 156, 313, 625, 1250, 2500, 5000 µg/plate (+/- metabolic activation)
- Number of replicates: 2
- Positive and negative control groups and treatment:
TA100: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), -S9Mix, 0.01 µg/plate
TA100: Benz(a)pyrene (BP), +S9Mix, 5 µg/plate
TA1535: Sodium azide (NaN3), -S9Mix, 0.5 µg/plate
TA1535: 2-Aminoanthracene (2-AA), +S9Mix, 2 µg/plate
WP2 uvrA: N-Ethyl-N'-nitro-N-nitrosoguanidine (ENNG), -S9Mix, 2 µg/plate
WP2 uvrA: 2-Aminoanthracene (2-AA), +S9Mix, 10 µg/plate
TA98: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), -S9Mix, 0.1 µg/plate
TA98: Benz(a)pyrene (BP), +S9Mix, 5 µg/plate
TA1537: 9-Aminoacridene (9-AA), -S9Mix, 80 µg/plate
TA1537: Benz(a)pyrene (BP), +S9Mix, 5 µg/plate
negative: solvent
- Pre-incubation: 20 minutes at 37 °C, incubation 48 hours at ca. 37 °C - Evaluation criteria:
- CRITERIA FOR EVALUATING RESULTS: mutagenic effects (i.e ratio of revertant rates treated/control >= 2) at <= 5000 µg/plate with generally
positive dose-response relationship in any strain - Statistics:
- No statistical method was used for judgement of test results.
- Species / strain:
- other:
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: at 2500 and 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- GENOTOXIC EFFECTS:
- With metabolic activation: None
- Without metabolic activation: None
The positive controls were functional.
PRECIPITATION CONCENTRATION: No precipitation.
CYTOTOXIC CONCENTRATION:
In all bacterial strains in the absence and presence of S9 Mix, antimicrobial effect by the test substance was observed at the concentrations of
2500 and 5000 µg/plate. - Remarks on result:
- other: other: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2 uvrA
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
The test substance proved to be non-mutagenic under the conditions of this study, both in the presence and in absence of
Phenobarbiturate/ 5,6-Benzoflavone -induced liver microsomes for all the test strains - Executive summary:
The objective of the study was to perform a reversion test using Salmonella strain and Escherichia coli strain to examine the mutagenicity of test substance.
The test substance concentrations were in the range between 156 and 5,000 µg/plate. In all bacterial strains in the absence and presence of S9 Mix, antimicrobial effect by the test substance was observed at the concentrations of 2500 and 5000 µg/plate.
This test substance did not increase the revertant colonies not less than twice of the solvent control value in any bacterial strain. On the other hand, the positive control induced the revertant colonies not less than twice of the solvent control value in each bacterial strain, indicating that the test was adequately performed. From the above results, the mutagenicity of this test substance was judged to be negative.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1993-01-05 to 1993-02-08
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Guideline study with acceptable restrictions: Both TA 102 and E.coli WP2 were not tested (not required by applied version of guideline).
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- Cited as Directive 84/449/EEC, B.14
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- mutated gene loci responsible for histidine auxotrophy
- Species / strain / cell type:
- other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100
- Additional strain / cell type characteristics:
- other: histidine auxotroph
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbiturate induced rat S9 liver, male Bor: WISW (SPF/Cpb) rats
- Test concentrations with justification for top dose:
- main test: 8; 40; 200; 1000; 5000 µg/plate (+/- metabolic activation)
pre-incubation test: 125; 250; 500; 1000; 2000 µg/plate (+/- metabolic activation) - Vehicle / solvent:
- Water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Water
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- enzymatic activity of S9 homogenate was checked on strain TA100 with cyclophosphamid
- Positive control substance:
- other: positive control: TA 98 and TA 1538: nitrofluorene / TA 100 and TA 1535: sodium azide / TA 1537: aminoacridine
- Remarks:
- without metabolic activation
- Details on test system and experimental conditions:
- Ames test
SYSTEM OF TESTING
- Metabolic activation system: Phenobarbiturate induced rat S9 liver, male Bor: WISW (SPF/Cpb) rats
ADMINISTRATION:
- Dosing:
main test: 8/40/200/1000/5000 µg/plate (+/- metabolic activation)
pre-incubation test: 125/250/500/1000/2000 µg/plate (+/- metabolic activation)
- Number of replicates: 3
- Application: solvent water main test: 50 g/l; pre-incubation test: 20 g/l
- Positive and negative control groups and treatment:
positive, TA 98 and TA 1538: nitrofluorene
positive, TA 100 and TA 1535: sodium azide positive, TA 1537: aminoacridine
negative: untreated
activity of metabolic system: cyclophosphamide / TA 100
- Pre-incubation: 30 minutes at 30 +/- 1 °C incubation ca. 96 hours at ca. 37 °C - Evaluation criteria:
- CRITERIA FOR EVALUATING RESULTS: mutagenic effects (i.e ratio of revertant rates treated/control >= 2) at <= 5000 µg/plate with generally
positive dose-response relationship in any strain - Statistics:
- usual statistical methods (standard deviation; means; factors) were calculated through a computer program by Messrs BIOSYS.
- Species / strain:
- other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: at >= 2000 or >= 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- GENOTOXIC EFFECTS:
- With metabolic activation: None
- Without metabolic activation: None
The positive controls were functional.
PRECIPITATION CONCENTRATION: No precipitation.
CYTOTOXIC CONCENTRATION (including effects on background lawn):
TA 98: 5000 µg/plate (+ S9), 2000 µg/plate (- S9) TA 100: none (+ S9), 2000 µg/plate (- S9)
TA 1535: 5000 µg/plate (+ S9), 2000 µg/plate (- S9)
TA 1537: 2000 µg/plate (+/- S9)
TA 1538: 5000 µg/plate (+ S9), 2000 µg/plate (- S9) - Remarks on result:
- other: other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
The test substance 2,2,4(or 2,4,4)-trimethylhexane-1, 6-diamine proved to be non-mutagenic under the conditions of this study, both in the presence and in absence of Phenobarbiturate-induced liver microsomes for all the test strains even in addition of 5000 µg/plate and when the pre-incubation test was used. - Executive summary:
The test substance 2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine was tested in the Ames Salmonella/microsomes mutagenicity test for any mutagenic activity using the five histidine-auxotrophic Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 . The test substance concentrations were in the range between 8 and 5,000 µg/plate.
2,2,4(or 2,4,4)-Trimethylhexane-1,6-diamine proved to be non-mutagenic under the conditions of this study, both in the presence and in the absence of Phenobarbiturate-induced liver microsomes for all the test strains even in addition of 5,000 µg of test substance per plate and when the pre-incubation test was used.- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1992-04-27 to 1992-07-20
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- hypoxanthine-guanine phophoribosyl-transferase (HPRT) locus
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CHO cells, substrain K1
- Additional strain / cell type characteristics:
- other: duobling time about 12-16 hours; high plating efficiency (about 90%); karyotype mith a modal number of 20 chromosomes
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix based on S9 fraction from Aroclor 1254 induced Wistar rat liver
- Test concentrations with justification for top dose:
- Preliminary toxicity test: 0; 0.02; 0.03; 0.06; 0.12; 0.2; 0.3; 0.6; 1.2; 2.0 mg/ml (+/- metabolic activation)
Main study Test 1 and 2: 0; 0.02; 0.06; 0.2; 0.6; 2.0 mg/ml ( each +/- metabolic activation) - Vehicle / solvent:
- HO medium
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- HO medium (with / without S9 mix)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- positive with metabolic activation: 3-methylcholanthrene
Migrated to IUCLID6: without metabolic activation - Details on test system and experimental conditions:
- HGPRT assay
SYSTEM OF TESTING
- Species/cell type: CHO-K1 cells (Flow Laboratories, Meckenheim, Germany)
- Metabolic activation system: S9 mix based on S9 fraction from Aroclor 1254 induced Wistar rat liver (Cytotest Cell Research, Rossdorf, Germany, lots 061191 and 240292)
ADMINISTRATION:
- Dosing:
(1) Preliminary toxicity test: 0; 0.02; 0.03; 0.06; 0.12; 0.2; 0.3; 0.6; 1.2; 2.0 mg/ml (+/- metabolic activation)
(2) Main study: 0; 0.02; 0.06; 0.2; 0.6; 2.0 mg/ml (2 tests, each +/- metabolic activation)
- Number of replicates: 2
- Application: 2E+05 cells/25 cm2 flask, incubated at 37 °C for approx. 20 hours, thereafter exposure for 4 hours
- Positive and negative control groups and treatment:
positive, without metabolic activation: 300 mg ethyl methanesulfonate (EMS)/l in HO medium
positive, with metabolic activation: 10 mg 3-methylcholanthrene (MCA)/l in dimethyl sulfoxide
negative: HO medium (with / without S9 mix) - Evaluation criteria:
- CRITERIA FOR EVALUATING RESULTS:
(1) Statistically significant (t-test), dose related increase in mutant frequency at concentrations of the test substance resulting in > 20 % cell
survival. And
(2) Mean mutant frequency in treated cultures > maximum spontaneous mutant frequency (approx. 20/1 million viable cells)
(3) Results reproduced in a second, independent experiment. - Statistics:
- The number of mutant colonies in each flask of the dose groups and the positive controls were compared with that of the solvent control group.
Statistical significance is determined on the basis of a t-test ("Two-sample analysis") - Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: no cytotoxicity observed below solubility limit (2.0 mg/l)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- GENOTOXIC EFFECTS:
- With metabolic activation: None
- Without metabolic activation: None
The spontaneous mutation frequency of the negative control in the first test (- S9) was twice as high as usual (40 +/- 16/1 million instead of
20/1 million). In view of the complete absence of effects in the other cultures, this isolated finding was judged to be within the biological variability
of the test system.
The positive controls were functional.
PRECIPITATION CONCENTRATION: Solubility limit 2.0 mg/l (in HO medium)
CYTOTOXIC CONCENTRATION: No effects on the viability of treated cells were detectable over the whole concentration range tested. - Remarks on result:
- other: other: Chinese hamster ovary (CHO) K1 cells
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative in the absence and in the presence of metabolic activation
The results of both tests indicate, that 2,2,4(or 2,4,4)-trimethylhexamethylenediamine, under the experimental conditions described, is not mutagenic in this in vitro mammalian cell system. - Executive summary:
The substance 2,2,4(or 2,4,4)-trimethylhexamethylenediamine was tested for its ability to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out with and without the addition of Aroclor-induced rat liver S9 mix. On the basis from the results of the present study, the test substance did not cause any increase in the mutant frequencies without and with S9 mix in two experiments performed independently of each other. Thus, under the experimental conditions of this study, 2,2,4(or 2,4,4)-trimethylhexamethylenediamine has no mutagenic activity in vitro in the CHO/HPRT forward mutation assay neither in the absence nor in the presence of metabolic activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Adequacy of study:
- other information
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- other: Abstract
- Principles of method if other than guideline:
- Method: other: Ames BN et al. (1975). Mutat. Res. 31, 347-364
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100
- Metabolic activation:
- with and without
- Test concentrations with justification for top dose:
- 10 to 1000 µg/plate
- Details on test system and experimental conditions:
- IUCLID4 Type: Ames test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: not reported
- Remarks on result:
- other: other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100
- Remarks:
- Migrated from field 'Test system'.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- in 1979
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study with acceptable restrictions: TA 102 or E.coli WP2 were not tested, no test with metabolic activation
- Principles of method if other than guideline:
- Method: Ames BN et al. (1975). Mutat. Res. 31, 347-364
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- mutated gene loci responsible for histidine auxotrophy
- Species / strain / cell type:
- other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538
- Additional strain / cell type characteristics:
- other: histidine auxotroph
- Metabolic activation:
- without
- Test concentrations with justification for top dose:
- 15; 30; 60; 120; 250; 500 µg/plate; 2-4 non-cytotoxic concentrations per strain tested
- Vehicle / solvent:
- DMSO dimethyl sulfoxide (CAS No. 67-68-5)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- no
- Remarks:
- None positive control substance but simultaneous test of several substances (including one positive result)
- Details on test system and experimental conditions:
- Ames test
ADMINISTRATION:
- Number of replicates: 3
- Application: Solvent dimethyl sulfoxide (CAS No. 67-68-5)
- Positive and negative control groups and treatment:
Negative: Blank
Positive: None (except simultaneous test of several substances including one positive result)
- Incubation: 72 hours at 37 °C - Evaluation criteria:
- CRITERIA FOR EVALUATING RESULTS:
Mutagenic effects (i.e ratio of revertant rates treated/control >= 2) at <= 500 µg/plate - Statistics:
- usual statistical methods (standard deviation; means; factors)
- Species / strain:
- other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: depending on strain, see Results for details
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- not applicable
- Additional information on results:
- GENOTOXIC EFFECTS: None
CYTOTOXIC CONCENTRATION:
TA 98: >= 250 µg/plate
TA 100: > 500 µg/plate
TA 1535: >= 250 µg/plate
TA 1537: >= 120 µg/plate
TA 1538: >= 120 µg/plate - Remarks on result:
- other: other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
The test substance trimethyl-1,6-hexanediamine proved to be non-mutagenic under the conditions of this study, in absence of metabolic activation system for all test strains. - Executive summary:
The test substance trimethyl-1,6-hexanediamine was tested in the Ames Salmonella/microsomes mutagenicity test for any mutagenic activity using the five histidine-auxotrophic Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 . The test substance concentrations were in the range between 15 and 500 µg/plate. Trimethyl-1,6 -hexanediamine proved to be non-mutagenic under the conditions of this study, in the absence of metabolic activation system, for all the test strains.
Referenceopen allclose all
no further remarks
MITOTIC INDEX: mean of 2 replicate cultures each
- With metabolic activation:
------------------------------------------
Experiment Concn. 12 hours 20 hours
------------------------------------------
No. 1 0 8.15 7.75
No. 1 156.25 9.55 8.1
No. 1 312.5 8.45 11.2
No. 1 625 6.05 10.1
No. 1 pos. control 2.7 6.3
No. 2 0 9.9 9.25
No. 2 312.5 7.55 8.45
No. 2 625 6.9 7.15
No. 2 937.5 3.3 4.55
No. 2 pos. control 1.35 10.6
Confirm. 0 with buffer 10.3
Confirm. 937.5 with buffer 17.2
Confirm. 0 without buffer 12.2
Confirm. 937.5 without buffer 2.85
------------------------------------------
- Without metabolic activation:
------------------------------------------
Experiment Concn. 12 hours 20 hours
------------------------------------------
No. 1 0 9.4 3.4
No. 1 156.25 8.45 2.2
No. 1 312.5 9.55 4.15
No. 1 625 7.65 8.0
No. 1 pos. control 3.05 2.5
No. 2 0 8.45 4.1
No. 2 156.25 10.6 7.5
No. 2 312.5 9.9 8.0
No. 2 625 4.1 1.6
No. 2 pos. control 6.05 4.8
------------------------------------------
Higher concentrations (see test conditions) are reported for 20 hours without metabolic activation in Experiment No. 2. The increase of the mitotic index observed at higher concentrations particularly in the 20-hour cultures may indicate a certain degree of treatment induced cell-cycle synchrony.
------------------------------------------
CHROMOSOMAL ABERRATIONS (cells with aberrations excluding gaps): mean of 2 replicates each
- With metabolic activation:
------------------------------------------
Experiment Concn. 12 hours 20 hours
------------------------------------------
No. 1 0 0.5 % 0.5 %
No. 1 156.25 2.5 % 0.5 %
No. 1 312.5 3.5 % * 1.0 %
No. 1 625 4.0 % 2.0 %
No. 1 pos. control 9.5 % *** 11.5 % ***
No. 2 0 3.5 % 1.5 %
No. 2 312.5 3.0 % 2.0 %
No. 2 625 0.5 % 2.5 %
No. 2 937.5 1.5 % 7.5 % **
No. 2 pos. control 12.5 % *** 21.3 % ***
Confirm. 0 with buffer 1.5 %
Confirm. 937.5 with buffer 0.0 %
Confirm. 0 without buffer 1.5 %
Confirm. 937.5 without buffer 12.4 % **
------------------------------------------
- Without metabolic activation:
------------------------------------------
Experiment Concn. 12 hours 20 hours
------------------------------------------
No. 1 0 2.5 % 1.5 %
No. 1 156.25 1.0 % 0.0 %
No. 1 312.5 0.0 % 1.0 %
No. 1 625 1.0 % 3.0 %
No. 1 pos. control 11.0 % *** 14.5 % ***
No. 2 0 0.5 % 1.0 %
No. 2 156.25 3.0 % 1.5 %
No. 2 312.5 0.0 % 2.0 %
No. 2 625 1.4 % 3.0 %
No. 2 pos. control 10.5 % *** 20.0 % ***
------------------------------------------
*** p<0.001; ** p<0.01; * p<0.05
Higher concentrations (see test conditions) are reported for 20 hours without metabolic activation in Experiment No. 2.
------------------------------------------
see tables Annex 1
no further remarks
no further remarks
no further remarks
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1987-03-02 to 1987-10-23
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test)
- GLP compliance:
- yes
- Type of assay:
- chromosome aberration assay
- Species:
- hamster, Chinese
- Strain:
- other: Han-Chinese
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ORGANISMS:
- Age: 3 to 4 months
- Source: Zentralinstitut für Versuchstierkunde, Hannover (Germany)
- Weight at study initiation: males 30 to 40 g; females 27 to 55 g
- No. of animals per dose: 5 males + 5 females
- Diet: Ssniff R pelleted diet, ad libitum
- Water: ad libitum, controlled visually
- Acclimation period: 4 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21,5 C +- 1,5 C
- Humidity (%): 65 % +- 10 %
- Air changes (per hr): 16 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours daily from 7.00 a.m. to 7.00 p.m. - Route of administration:
- intraperitoneal
- Vehicle:
- water
- Details on exposure:
- ADMINISTRATION:
- Vehicle: water (aqua ad injectabilia); 10 ml/kg bw applied
- Duration of test: 24 hours; 22 hours after application: Treatment with 3.3 mg colcemid/kg bw
- Control groups and treatment:
Positive: 20 mg cyclophosphamide/kg bw
Negative: Vehicle - Duration of treatment / exposure:
- single dose
- Frequency of treatment:
- 1 time
- Post exposure period:
- 24 hours
- Remarks:
- Doses / Concentrations:
3.75; 15; 60 mg/kg bw
Basis:
nominal conc. - No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- 20 mg cyclophosphamide/kg bw
- Tissues and cell types examined:
- EXAMINATIONS:
Evaluation of 50 metaphase cells/animal (= 500/dose level)
Mitotic index determined from 1000 cells/animal - Details of tissue and slide preparation:
- Sampling times and number of samples: 24 hours after application: Killing by cervical dislocation, removal of femurs, dislocation of epiphysis,
rinsing with Hanks solution (37 °C) from diaphysis to obtain bone marrow, centrifugation, incubation for 20 minutes at 37 °C in 1 % trisodium
citrate, two times centrifugation / fixation in methanol/acetic acid (3:1), drying, staining with Giemsa. - Evaluation criteria:
- - Criteria for evaluating results: Significant increase of aberration rate (as compared with the negative control) with at least one dose level.
- Statistics:
- The mitotic index was statistically compared using a one factorial analysis of variance with subsequent Scheffe-test.
Group mean values might be compared employing the U-test of Mann-Whitney (chromosomale aberrations) - Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- Animals of the 60 mg/kg bw dose group showed transient and slight loss of activity.
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- MORTALITY: No animal died at any dose level.
CLINICAL SIGNS: Animals of the 60 mg/kg bw dose group showed transient and slight loss of activity.
-------------------------------------------
MITOTIC INDEX AND CHROMOSOMAL ABERRATIONS (excl. gaps):
-------------------------------------------
Dose level % M.I. % C.A.
-------------------------------------------
neg. control 3.78 1.6
pos. control 1.88 p<0.05 3.8 *
3.75 mg/kg bw 4.02 1.0
15 mg/kg bw 3.30 1.0
60 mg/kg bw 3.98 0.6
* Borderline of significance; highly significant (p<0.001) result including gaps. - Conclusions:
- Interpretation of results (migrated information): negative
The results of this study indicate that under the test conditions, the test substance 2,2,4(or 2,4,4)-trimethylhexamethylenediamine did not induce chromosome aberrations in the bone-marrow of male and female Chinese Hamster. - Executive summary:
2,2,4(or 2,4,4)-trimethylhexamethylenediamine was tested for its ability to induce in vivo chromosomal aberrations in the bone marrow of Chinese Hamsters. Each 5 male and 5 female Chinese Hamsters were exposed to 3.75, 15.0, and 60.0 mg/kg/bw by intraperitoneal injection. Bone marrow were collected 24 hours after single treatment and metaphase cells were examined microscopically for chromosomal aberrations. The analyse of metaphase cells showed that the test substance 2,2,4(or 2,4,4)- trimethylhexamethylene diamine did not induce chromosome aberration in the bone-marrow of Chinese Hamsters. Therefore, 2,2,4(or 2,4,4)-trimethylhexa methylenediamine is considered to be non-mutagenic in this chromosome aberration assay.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1992-10-19 to 1992-12-10
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ORGANISMS:
- Age: young adults
- Source: Winkelmann, Borchen (Germany)
- Weight at study initiation: 33.0 +/- 6.6 g (males); 27.5 +/- 5.5 g (females)
- No. of animals per dose: 5 males + 5 females per test duration
- Fasting period before study: 16 hours
- Housing: 5 mice per cage
- Diet: SSniff R 10 ad libitum
- Water: tab water ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 °C +/- 1 °C
- Humidity (%): 50 - 70 %
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark/light - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- ADMINISTRATION:
- Vehicle: corn oil
- Control groups and treatment:
negative: vehicle
positive: 100 mg cyclophosphamide (CPA)/kg bw in physiological NaCl solution
additional treated satellite group to replace mortalities
- Total volume applied: 10 ml/kg bw
- Duration of test: 24 hours; 48 hours
- Sampling times and number of samples: 24 hours; 48 hours - Duration of treatment / exposure:
- single dose
- Frequency of treatment:
- 1 time
- Post exposure period:
- 24 and 48 hours
- Remarks:
- Doses / Concentrations:
825 (female) or 1000 (male) mg/kg bw
Basis:
nominal conc. - No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- 100 mg cyclophosphamide (CPA)/kg bw in physiological NaCl solution per oral gavage
- Tissues and cell types examined:
- polychromatic erythrocytes of the bone marrow from femur
- Details of tissue and slide preparation:
- - Criteria for selection of M.T.D.: maximum dose <= 2000 mg/kg bw without mortalities within 48 hours
EXAMINATIONS:
- Clinical observations: yes
DETAILS OF SLIDE PREPARATION:
Animals were sacrified at appropriate sampling times. Femurs were removed and bone marrow cells obtained by flushing with foetal calf serum.
The cells were centrifuged and a concentrated suspension prepared to make smears on slides. Slides were air-dried and then stained with
May-Gruenwald and Giemsa. Three slides were made from each animal, >= 2000 PCE (polychromatic erythrocytes) per animal were analysed for
micronuclei - Evaluation criteria:
- Criteria for evaluating results: statistically significant and biologically relevant increase in frequency of micronucleated polychromatic
erythrocytes of at least one test group as compared to the negative control group of the same sampling time - Statistics:
- - Degree of heterogeneity within each group was first calculated and in case all the groups are homogenous, comparisons can be made
between the control and test groups
- a modified chi-squared calculation was employed to compare treated and control groups
- Chi-squared values are taken to show the significance of any difference between each treated group and the controls - Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- see "Additional information on results"
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- MORTALITY:
- Phase 1 of dose finding
2000 mg/kg bw: 2/2 males and 2/2 females died within 24 hours.
- Phase 2 of dose finding
1000 mg/kg bw: 0/2 males and 1/2 females died (at 4 hours).
464; 215; 100 mg/kg bw: 2/2 males and 2/2 females each survived.
- Phase 3 of dose finding
950 mg/kg bw: 1/5 females was sacrificed 24 hours after treatment due to severe clinical signs. 5/5 males survived.
800 mg/kg bw: 5/5 males and 5/5 females survived.
700 mg/kg bw: 1/5 females died probably due to application failure. 5/5 males survived.
- Main test 1 female of the test group died within 6 hours and was replaced by an animal from the satellite group.
CLINICAL SIGNS (test group): Mainly piloerection and squatting position, sporadically slight sedation and staggering. Animals from the dose finding study that died showed abdominal or lateral position, tremor, spasms, convulsions, closed eyes. 4/5 males and 4/5 females from the 48 hour test
groups as well as most surviving animals from the dose finding study were free from symptoms within 48 hours.
NECROPSY FINDINGS:
Phase 1: 1 male was necropsied: Severe irritation of the gastro-intestinal tract, swelling of the spleen.
Phase 2: The female that died was necropsied: Distinct changes in appearance of liver, spleen, and gastro-intestinal tract.
Phase 3: The female that died was necropsied: Swelling of liver, sanguineous changes in gastric and intestinal mucosa.
Main test: The female that died was necropsied: Sanguineous gastric mucosa, pale liver with mottling.
EFFECT ON MITOTIC INDEX OR PCE/NCE RATIO:
The average PCE/NCE ratio of the positive control groups was significantly lower than that of the corresponding vehicle controls
(0.19 +- 0.05 vs 0.38 +- 0.16 for males, 0.51 +- 0.10 vs 0.98 +- 0.23 for females).
The PCE/NCE ratio of the male 24 hour vehicle control group was 0.38 +- 0.16 and thus below the range of approx. 0.6 - 1.2 which is considered
normal in the literature. The PCE/NCE of the other vehicle groups were within the literature range.
The PCE/NCE ratio of the male 48 hour treatment group (0.35 +- 0.16) was significantly lower than that of the corresponding vehicle control
(0.81 +- 0.23).
GENOTOXIC EFFECTS: For the positive control a significant increase in the frequency of micronucleated polychromatic erythrocytes was observed
(2.63 +- 1.07 vs 0.18 +- 0.10 for males; 1.85 +- 0.16 vs 0.09 +- 0.02 for females). No significant increase over the control was found with any
group treated with the test substance.
The necropsy findings, particularly in liver and spleen, indicate that the test substance or its metabolites had reached the blood and hence the
target organ, i.e. the bone marrow. - Conclusions:
- Interpretation of results (migrated information): negative
The results of this study indicate that under the test conditions, 2,2,4(or 2,4,4)-trimethylhexamethylenediamine did not induce micronucleated polychromatic erythrocytes in male and female mice. - Executive summary:
In this in vivo mouse micronucleus assay 825 (female) or 1000 (male) mg/kg bw of the test substance 2,2,4(or 2,4,4)-trimethylhexamethylenediamine was administered oral to 5 male and 5 female NMRI mice per group. This doses were selected as the maximum tolerated dose (MTD) based upon a preliminary toxicity study. Bone marrow polychromatic erythrocytes, collected 24, and 48 hours after single treatment, were examined microscopically for micronucleated polychromatic erythrocytes (PCE). No significant increase in the frequency of PCE over the control was found with any group treated with the test substance. For the positive control (cyclophosphamid, CPA) a significant increase in the frequency of PCE was observed.
Therefore, the conclusion is drawn, that 2,2,4(or 2,4,4)-trimethylhexamethylenediamine is not a mutagenic substance under the in vivo conditions in this micronucleus assay using male and female NMRI mice.
Referenceopen allclose all
Results:
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Treatment Sex Time % Micron. in PCE PCE/NCE
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100 CPA m 24 h 2.63 +- 1.07 * 0.19 +- 0.05 *
100 CPA f 24 h 1.85 +- 0.16 * 0.51 +- 0.10 *
Vehicle m 24 h 0.18 +- 0.10 0.38 +- 0.16
Vehicle f 24 h 0.09 +- 0.02 0.98 +- 0.23
Vehicle m 48 h 0.19 +- 0.11 0.81 +- 0.23
Vehicle f 48 h 0.18 +- 0.12 0.84 +- 0.26
1000 TMD m 24 h 0.18 +- 0.08 0.52 +- 0.19
825 TMD f 24 h 0.08 +- 0.08 0.72 +- 0.30
1000 TMD m 48 h 0.17 +- 0.10 0.35 +- 0.16 *
825 TMD f 48 h 0.14 +- 0.08 0.73 +- 0.27
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CPA = cyclophosphamide (mg/kg bw)
TMD = trimethylhexanediamine (mg/kg bw)
* p < 0.05
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Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In vitro Studies
2,2,4-(2,4,4)-trimethyl-1,6-hexanediamine did not induce gene mutation in bacteria (EU Method B.13/14; Hüls AG, 1993), or in mammalian cells (OECD TG 476; Hüls AG, 1992) and demonstrate no potential to induce chromosome aberrations in Chinese Hamster Ovary cells in vitro (OECD 473; SafePharm, 1992) either with or without metabolic activation.
In vivo Studies
The results of the micronucleus assay (OECD TG 474; Hüls AG, 1993) and of the chromosome aberration assay (OECD TG 475; IBR, 1987) indicate that under the test conditions, 2,2,4(or 2,4,4)-trimethyl-1,6-hexanediamine neither induce micronucleated polychromatic erythrocytes in male and female mice nor chromosome aberrations in the bone-marrow of male and female Chinese Hamster.
Short description of key information:
In several in vitro studies conducted with bacteria and mammalian cell cultures as well as in in vivo animal studies the test substance 2,2,4-(2,4,4)-trimethyl-1,6-hexanediamine did not show a genotoxic effect. Therefore, it can be assumed that 2,2,4-(2,4,4)-trimethyl-1,6-hexanediamine is not genotoxic.
Endpoint Conclusion: No adverse effect observed (negative)
Justification for classification or non-classification
Because all in vitro and in vivo genotoxicity studies revealed clearly negative results, it can be concluded that 2,2,4-(2,4,4)-trimethyl-1,6-hexanediamine is not genotoxic in vitro and in vivo and therefore must not be classified according to the criteria of EC Directive 67/548/EEC and EC Regulation 1272/2008.
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