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EC number: 421-750-9 | CAS number: 57280-22-5 TRIOXABICYCLOOCTAN; TRIOXABICYCLOOCTANE
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Study conducted according to OECD test guideline 471; strains TA 98, TA 100, TA 1535, and TA 1537, with and without metabolic activation using the plate incorporation method, result: positive
Study conducted according to OECD test guideline 471; strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538, with and without metabolic activation using the pre-incubation method, result: positive
Study conducted according to OECD test guideline 473; human peripheral lymphocytes, with and without metabolic activation, result: positive
In vivo UDS study in rat liver cells (OECD test guideline 486), result: negative
In silico prediction (DEREK) for mutagenicity in bacteria: positive
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1995-09-25 to 1996-02-27
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
- Version / remarks:
- 26 May 1983
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- lymphocytes: human
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: human lymphocytes from peripheral blood
For lymphocytes:
- Sex, age and number of blood donors: not specified
- Whether whole blood or separated lymphocytes were used: yes, whole blood
- Whether blood from different donors were pooled or not: two different donors, male in experiment 1 and female in experiment 2
- Mitogen used for lymphocytes: Phytohaemagglutinin (PHA, reagent grade) 10 µg/mL - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system: S9-Mix
- source of S9: The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats induced with Aroclor 1254 and obtained from Molecular Toxicology Incorporated, Annapolis, Maryland, USA.
- concentration or volume of S9 mix and S9 in the final culture medium: Glucose-phosphate (180 mg/mL), NADP (25 mg/mL), 150 mM KCl and rat liver S-9 (3.3) were mixed in the ratio 1:1:1:2. An aliquot of the resulting S-9 mix was added to each cell culture containing ,the test article to achieve the required final concentration in a total of 10 mL. The final concentration of liver homogenate in the test system was 2 %.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each batch was checked by the manufacturer for sterility, protein content (minimum 32 mg/mL), ability to convert known promutagens to bacterial mutagens and cytochrome P-450-catalyzed enzyme activities (alkoxyresorufin-O-dealkylase activities). - Test concentrations with justification for top dose:
- Experiment 1: 0, 28.51, 40.73, 58.19, 83.13, 118.8, 169.7, 242.4, 346.2, 494.6, 706.6, 1009, 1443 µg/mL
Experiment 2: 0, 342.2, 456.3, 608.3, 811.1, 1082, 1442 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- cyclophosphamide
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration : duplicate, controls quadruplicate
- Number of independent experiments: 2
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 h
- Exposure duration/duration of treatment: 3h and 20h
- Harvest time after the end of treatment (sampling/recovery times): 17h and 41h
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Approximately 1 ½ hours prior to harvest, colchicine was added to give a final concentration of approximately 1 µg/mL to arrest dividing cells in metaphase.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): Cultures were then centrifuged at 200 x g for 10 minutes,. the supernatant was carefully removed and cells were resuspended in 4 mL hypotonic (0.075 M) KCl and incubated at 37°C for 15 minutes to allow cell swelling to occur. Cells were then fixed by dropping the KCl suspension into an equal volume of fresh, ice-cold methanol/glacial acetic acid (3:1, v/v). The fixative was changed by centrifugation (200 x g, 10 minutes) and resuspension. This procedure was repeated several times (centrifuging at 1250 x g, 2-3 minutes) until the supernatants were clean. Lymphocytes were kept in fixative in the refrigerator before slides were prepared but slides were not made on the day of harvest to ensure cells were adequately fixed. Cells were pelleted and resuspended in a minimal amount of fresh fixative so as to give a milky suspension. Several drops of 45 % (v/v) aqueous acetic acid were added to each suspension to enhance chromosome spreading, and several drops of suspension were transferred to clean microscope slides. After the slides bad dried the cells were stained for 5 minutes in 4% (v/v) filtered
Giemsa stain in pH 6.8 buffer. The slides were rinsed, dried and mounted with coverslips.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Twenty-five cells from each of the selected NQO and CPA positive control cultures were analysed to ensure that the system was operating satisfactorily.
Slides from the selected treatments and from solvent controls were coded using randomly generated letters by a person not connected with the scoring of the slides. Labels bearing only the study reference number, the sex of the donor and the code
were used to cover treatment details on the slides. Each experiment was analysed by two analysts only. One hundred metaphases from each code were analysed for chromosome aberrations. Only cells with 44-46 chromosomes were considered acceptable for analysis of structural aberrations. Any cell with more than 46 chromosomes, that is polyploid, endoreduplicated and hyperdiploid cells, observed during this search was noted and recorded separately. Classification of structural aberrations was based on the scheme described by ISCN. Observations were recorded on raw data sheets
with the microscope stage coordinates of any aberrant cell. After completion of microscopic analysis, data were decoded. The aberrant cells in each culture were categorised as follows:
1) cells with structural aberrations including gaps
2) cells with structural aberrations excluding gaps
3) polyploid, endoreduplicated or hyperdiploid cells.
The totals for category 2 in negative control cultures were used to determine whether the assay was acceptable or not. The proportions of aberrant cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test. The proportion of cells in category 2 for each test treatment condition, was compared with the proportion in concurrent negative controls using Fisher's exact test. Probability values of p ≥ 0.05 were accepted as significant.
The proportions of cells in categories 1, 2 and 3 were examined in relation to historical negative control (normal) ranges.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: mitotic index (MI) - Rationale for test conditions:
- As recommended by the test guideline
- Evaluation criteria:
- Evaluation criteria
The test article was to be considered as positive in this assay if:
1) a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurred at one or more concentrations, and
2) . the proportion of cells with structural aberrations at such doses exceeded the normal range, and
3) the results were confirmed in the second experiment.
A positive result only at the delayed harvest or pulse -S-9 treatment in Experiment 2 was to be taken as evidence of clastogenicity provided criteria 1 and 2 were met. Increases in numbers of cells with gaps or increases in the proportions of cells with structural aberrations, not exceeding the normal range or occurring only at very high or very toxic concentrations, were likely to be concluded as "equivocal". Full assessment of the biological importance of such increases is likely only to be possible with reference to data from other test systems. Cells with exchange aberrations or cells with greater than one aberration were to be considered of particular biological significance. - Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Conclusions:
- It is concluded that Trioxabicyclooctan induced chromosome aberrations in cultured human peripheral blood lymphocytes when tested to 10 mM, and this effect was only seen in the absence of S-9 under the conditions of the test.
- Executive summary:
In a mammalian cell cytogenetics assay (Chromosome aberration) according to OECD test guideline 473 (1983), human peripheral lymphocytes were exposed to Trioxabicyclooctan, (100 % a.i.), in DMSO at concentrations of Experiment 1: 0, 28.51, 40.73, 58.19, 83.13, 118.8, 169.7, 242.4, 346.2, 494.6, 706.6, 1009, 1443 µg/mL in the first experiment for 3h and 20 h and at 0, 342.2, 456.3, 608.3, 811.1, 1082, 1442 µg/mL for the 3h treatment with 17h and 41 h post-treatment recovery with and without metabolic activation [S9-liver mix].
Trioxabicyclooctan was tested up to the limit concentration. Cultures treated with the test item in the absence of S9 mix showed statistically significant and biologically relevant increases of numbers of metaphases with aberrations, starting at 706.6 µg/mL without S9 mix. Positive controls induced the appropriate response. There was evidence of Chromosome aberrations induced over background.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1995-09-08 to 1995-12-14
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 26 May 1983
- Deviations:
- yes
- Remarks:
- Only preincubation method
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- His locus
- Species / strain / cell type:
- S. typhimurium TA 102
- Remarks:
- or E.coli WP2 uvr, were not tested as recommended by the former guideline
- Species / strain / cell type:
- S. typhimurium TA 1538
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 1537
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 1535
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 100
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 98
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system: S9 mix
- source of S9: S9, derived from male Sprague-Dawley rats pretreated with Aroclor 1254, was obtained from Organon Teknika Co., Durham, NC, USA, [S9 batch no. 38273; protein content 32 mg/mL; activity (37°C; pmoles/min/mg S9 protein) 7-hydroxyresorufin, 2813]. S9, derived from Wistar rats pretreated with Aroclor 1254, was obtained from Cytotest Cell Research GmbH & Co .. KG, Roßdorf, Germany (S9 batch no. 220595; protein content 33.2 mg/mL)
- concentration or volume of S9 mix and S9 in the final culture medium: The components of the standard S9 mix were 8 mM MgCl2, 33 mM KCI, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM sodium phosphate, pH 7.4 and S9 at a concentration of 0.3 mL per mL of mix. - Test concentrations with justification for top dose:
- 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0 mg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- N-dimethylnitrosamine
- benzo(a)pyrene
- cyclophosphamide
- other: 2-AA
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 1
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): E-06 dilution per plate
- Test substance added in preincubation
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 72 h
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition - Rationale for test conditions:
- as recommended by the respective OECD test guideline
- Evaluation criteria:
- The plates were scored for the number of mutant colonies with an automated colony counter (Artek M 9828, Artek Systems Corporation, Farmingdale, NY, USA). In exceptional cases where reliable automatic counting is not possible, e.g. due to distinct precipitates of the test compound, the colonies are scored manually. The arithmetic means of the number of mutant colonies of the 3 parallel plates in the negative control groups were compared with those of the compound groups. A positive response was considered if at least 5 mg/plate or up to a toxic dose had been tested (or the compound formed precipitates in the agar) and if the number of induced revertants compared to the number of spontaneous ones was higher than 2-fold. A dose-dependent increase in the number of revertants was also considered to indicate a mutagenic effect.
A toxic effect of the substance on the background lawn of non-revertant bacteria and precipitates in the agar were examined stereomicroscopically. - Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- 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 nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- 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 nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- 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 nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- In conclusion it can be stated that under the conditions reported, Trioxabicyclooctan induces gene mutations by base-pair substitutions in the genome of strains TA1535 and TA100. The mutagenic effect was dose-dependent but never exceeded thrice that observed in the negative control even when tested up to the maximum recommended dose of 5 mg/plate, Trioxabicyclooctan has to be classified as a mutagen in the Ames test.
- Executive summary:
In a reverse gene mutation assay in bacteria according to OECD test guideline 471 (1983), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Trioxabicyclooctan (100 % a.i.), in DMSO at concentrations of 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, and 5.0 mg/plate in the presence and absence of mammalian metabolic activation using the pre-incubation method.
Trioxabicyclooctan was tested up to limit concentration (5000 µg/plate). There were gene mutations by base-pair substitutions in the genome of strains TA1535 and TA100. Although the mutagenic effect was dose-dependent but never exceeded thrice that observed in the negative control even when tested up to the maximum recommended dose of 5.0 mg/plates. The positive control substance provided the expected increase in revertants numbers. Based on these results Trioxabicyclooctan is a mutagen under the conditions of the test.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1990-10-25 to 1990-11-15
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 26 July 1983
- Deviations:
- yes
- Remarks:
- only four strains used as recommended by the former guideline
- Principles of method if other than guideline:
- plate incorporation method
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his locus
- Species / strain / cell type:
- S. typhimurium TA 102
- Remarks:
- or E.coli WP2 uvr, were not tested as recommended by the former guideline
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 1537
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 1535
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 100
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 98
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system: S9-Mix
- method of preparation of S9 mix: SPF Wistar rats of the strain Mol:WIST were obtained from the Mollegaard Breeding Center Ltd, Ejby, DK-4623 Lille Skensved. Rats weighing approximately 200 g were used for induction of liver enzymes. A single intraperitoneal injection of Aroclor® 1254 at a dose of 500 mg/kg body weight was given to each rat. The animals were killed by gassing with CO2 5 days after being injected and following a 16 hour period of fasting. All steps in preparation of the liver homogenate were performed on ice using aseptic techniques and cold sterile solutions. The livers were removed and minced in 0.15 M KCI solution (3.0 mL KCI solution per gram wet liver). After homogenization, the homogenate was centrifuged at 9000 g for 15 minutes. The supernatant (S-9 fraction) was decanted, frozen and stored at -196°C until use.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S-9 mix/plate:
S-9 fraction (rat liver homogenate) 2.4 mL, Phosphate buffer (0.2 M, pH 7.4) 30.0 mL, Salt solution (0.4 M MgCl2, 1.65 M KCI) 1.2 mL, Glucose-6-phosphate solution (0.1 M) 03 mL, NADP solution (0.1 M) 2.4 mL, Distilled water 24.0 mL - Test concentrations with justification for top dose:
- 0, 0.31, 0.63, 1.3, 2.5 and 5.0 mg/plate
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- sodium azide
- other: 2-Aminoanthracene
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): E+08 - E+09 bact/mL
- Test substance added in agar (plate incorporation)
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 72 h
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition - Rationale for test conditions:
- As recommended by the test guideline
- Statistics:
- Statistical analysis of the negative control versus test data was performed using the Analysis of Variance method (general linear model, least square mean). Statistical analysis of the negative versus positive control data was performed using the Student’s. t-test.
- Key result
- Species / strain:
- S. typhimurium TA 102
- Remarks:
- or E. coli WP2 uvr not tested
- Metabolic activation:
- not applicable
- Genotoxicity:
- not determined
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- 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 nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Conclusions:
- The test article, Trioxabicyclooctan, was found to be mutagenic in the Ames Test.
- Executive summary:
In a reverse gene mutation assay in bacteria according to OECD test guideline 471 (1983), strains TA 98, TA 100, TA 1535, and TA 1537 of S. typhimurium were exposed to Trioxabicyclooctan (100 % a.i.), at concentrations of 0, 0.31, 0.63, 1.3, 2.5, and 5.0 mg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method.
Trioxabicyclooctan was tested up to limit concentration (5000 µg/plate). The test article induced statistically significant, dose-related and reproducible increases in the number of reyertants in TA 100 with and without S-9 mix. The increases were consistent but relatively small, i.e. below 1.5 1.5 times of the concurrent controls. There was no major difference in effect between the test series with or without S-9 mix. In TA 1535 similar increases in revertants were found in both test series but only with S-9 mix present. However, in this strain the largest increases were more than twice the control levels. The single statistically significant increase in TA 1535 without S-9 mix was only marginal, and not reproducible although the same tendency was evident in the 1. test series. The positive control substance provided the expected increase in revertants numbers. Based on these results Trioxabicyclooctan is a mutagen under the conditions of the test.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.
Referenceopen allclose all
ZK 39.294: summary of the numbers and types of structural aberrations observed | ||||||||||
20+0 hours, -S9, Experiment 1 | ||||||||||
Treatment (µg/mL) | Rep | Cells* | g | Chr del | Chr exch | Ctd del | Ctd exch | Other | Abs +g | Abs -g |
Solvent | A | 100 | 2 | 0 | 1 | 1 | 0 | 0 | 4 | 2 |
| B | 100 | 5 | 1 | 0 | 2 | 0 | 0 | 8 | 3 |
| A+B | 200 | 7 | 1 | 1 | 3 | 0 | 0 | 12 | 5 |
706.6 | A | 100 | 8 | 2 | 0 | 7 | 1 | 0 | 18 | 10 |
| B | 100 | 5 | 3 | 0 | 9 | 0 | 0 | 17 | 12 |
| A+B | 200 | 13 | 5 | 0 | 16 | 1 | 0 | 35 | 22 |
1009 | A | 100 | 0 | 1 | 0 | 6 | 0 | 0 | 7 | 7 |
| B | 100 | 7 | 1 | 0 | 9 | 0 | 0 | 17 | 10 |
| A+B | 200 | 7 | 2 | 0 | 15 | 0 | 0 | 24 | 17 |
1442 | A | 100 | 6 | 7 | 0 | 14 | 1 | 0 | 28 | 22 |
| B | 100 | 8 | 2 | 0 | 17 | 0 | 0 | 27 | 19 |
| A+B | 200 | 14 | 9 | 0 | 31 | 1 | 0 | 55 | 41 |
NQO, 2.5 | A | 25 | 0 | 2 | 0 | 19 | 4 | 1 | 26 | 26 |
| B | 25 | 1 | 1 | 1 | 9 | 4 | 0 | 16 | 15 |
| A+B | 50 | 1 | 3 | 1 | 28 | 8 | 1 | 42 | 41 |
| ||||||||||
ZK 39.294: summary of the numbers and types of structural aberrations observed | ||||||||||
3+17 hours, +S9, Experiment 1 | ||||||||||
Treatment (µg/mL) | Rep | Cells* | g | Chr del | Chr exch | Ctd del | Ctd exch | Other | Abs +g | Abs -g |
Solvent | A | 100 | 1 | 0 | 0 | 1 | 0 | 0 | 2 | 1 |
| B | 100 | 2 | 2 | 0 | 1 | 0 | 0 | 5 | 3 |
| A+B | 200 | 3 | 2 | 0 | 2 | 0 | 0 | 7 | 4 |
706.6 | A | 100 | 3 | 0 | 0 | 2 | 0 | 0 | 5 | 2 |
| B | 100 | 3 | 2 | 0 | 1 | 0 | 0 | 6 | 3 |
| A+B | 200 | 6 | 2 | 0 | 3 | 0 | 0 | 11 | 5 |
1009 | A | 100 | 6 | 0 | 0 | 1 | 0 | 0 | 7 | 1 |
| B | 100 | 4 | 0 | 0 | 0 | 0 | 0 | 4 | 0 |
| A+B | 200 | 10 | 0 | 0 | 1 | 0 | 0 | 11 | 1 |
1442 | A | 100 | 2 | 0 | 0 | 1 | 0 | 0 | 3 | 1 |
| B | 100 | 4 | 0 | 0 | 2 | 0 | 0 | 6 | 2 |
| A+B | 200 | 6 | 0 | 0 | 3 | 0 | 0 | 9 | 3 |
CP, 25 | A | 25 | 4 | 4 | 0 | 20 | 0 | 0 | 28 | 24 |
| B | 25 | 8 | 5 | 0 | 15 | 2 | 3 | 33 | 25 |
| A+B | 50 | 12 | 9 | 0 | 35 | 2 | 3 | 61 | 49 |
ZK 39.294: summary of the numbers and types of structural aberrations observed | ||||||||||
.20+0 hours, -S9, Experiment 2 | ||||||||||
Treatment (µg/mL) | Rep | Cells* | g | Chr del | Chr exch | Ctd del | Ctd exch | Other | Abs +g | Abs -g |
Solvent | A | 100 | 0 | 1 | 0 | 0 | 0 | 0 | 1 | 1 |
| B | 100 | 3 | 0 | 0 | 1 | 0 | 0 | 4 | 1 |
| A+B | 200 | 3 | 1 | 0 | 1 | 0 | 0 | 5 | 2 |
811.1 | A | 100 | 2 | 0 | 0 | 3 | 0 | 0 | 5 | 3 |
| B | 100 | 10 | 7 | 0 | 20 | 0 | 0 | 37 | 27 |
| A+B | 200 | 12 | 7 | 0 | 23 | 0 | 0 | 42 | 30 |
1082 | A | 100 | 2 | 2 | 0 | 4 | 0 | 0 | 8 | 6 |
| B | 100 | 24 | 4 | 0 | 52 | 0 | 0 | 80 | 56 |
| A+B | 200 | 26 | 6 | 0 | 56 | 0 | 0 | 88 | 62 |
1442 | A | 100 | 7 | 5 | 0 | 14 | 0 | 1 | 27 | 20 |
| B | 100 | 17 | 2 | 0 | 30 | 1 | 2 | 52 | 35 |
| A+B | 200 | 24 | 7 | 0 | 44 | 1 | 3 | 79 | 55 |
NQO, 2.5 | A | 25 | 2 | 2 | 0 | 1 | 5 | 0 | 10 | 8 |
| B | 25 | 5 | 1 | 0 | 5 | 2 | 1 | 14 | 9 |
| A+B | 50 | 7 | 3 | 0 | 6 | 7 | 1 | 24 | 17 |
ZK 39.294: summary of the numbers and types of structural aberrations observed | ||||||||||
3 + 17 hours, +S9, Experiment 2 | ||||||||||
Treatment (µg/mL) | Rep | Cells* | g | Chr del | Chr exch | Ctd del | Ctd exch | Other | Abs +g | Abs -g |
Solvent | A | 100 | 1 | 1 | 0 | 2 | 0 | 0 | 4 | 3 |
| B | 100 | 0 | 0 | 1 | 1 | 0 | 0 | 2 | 2 |
| A+B | 200 | 1 | 1 | 1 | 3 | 0 | 0 | 6 | 5 |
811.1 | A | 100 | 3 | 0 | 0 | 3 | 0 | 0 | 6 | 3 |
| B | 100 | 1 | 1 | 0 | 3 | 1 | 0 | 6 | 5 |
| A+B | 200 | 4 | 1 | 0 | 6 | 1 | 0 | 12 | 8 |
1082 | A | 100 | 0 | 4 | 0 | 2 | 0 | 0 | 6 | 6 |
| B | 100 | 0 | 1 | 0 | 1 | 0 | 0 | 2 | 2 |
| A+B | 200 | 0 | 5 | 0 | 3 | 0 | 0 | 8 | 8 |
1442 | A | 100 | 2 | 0 | 0 | 1 | 0 | 0 | 3 | 1 |
| B | 100 | 1 | 1 | 0 | 1 | 0 | 0 | 3 | 2 |
| A+B | 200 | 3 | 1 | 0 | 2 | 0 | 0 | 6 | 3 |
CPA, 25 | A | 25 | 3 | 7 | 0 | 3 | 1 | 0 | 14 | 11 |
| B | 25 | 4 | 4 | 0 | 10 | 2 | 0 | 20 | 16 |
| A+B | 50 | 7 | 11 | 0 | 13 | 3 | 0 | 34 | 27 |
ZK 39.294: summary of the numbers and types of structural aberrations observed | ||||||||||
3+41 hours, +S9, Experiment 2 | ||||||||||
Treatment (µg/mL) | Rep | Cells* | g | Chr del | Chr exch | Ctd del | Ctd exch | Other | Abs +g | Abs -g |
Solvent | A | 100 | 2 | 0 | 0 | 1 | 0 | 0 | 3 | 1 |
| B | 100 | 0 | 0 | 1 | 0 | 0 | 0 | 1 | 1 |
| A+B | 200 | 2 | 0 | 1 | 1 | 0 | 0 | 4 | 2 |
1442 | A | 100 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 |
| B | 100 | 0 | 2 | 0 | 1 | 0 | 0 | 3 | 3 |
| A+B | 200 | 1 | 2 | 0 | 1 | 0 | 0 | 4 | 3 |
ZK 39.294: summary of the numbers and types of numerical aberrations observed | ||||||||||
20+0 hours, -S9, Experiment 1 Donor sex: Male | ||||||||||
Treatment (µg/mL) | Rep | Cells** | H | E | P | Tot abs | % with num abs |
| ||
Solvent | A | 100 | 0 | 0 | 0 | 0 | 0 | |||
| B | 101 | 0 | 0 | 1 | 1 | 1.0 | |||
| A+B | 201 | 0 | 0 | 1 | 1 | 0.5 | |||
706.6 | A | 100 | 0 | 0 | 0 | 0 | 0 | |||
| B | 101 | 0 | 0 | 1 | 1 | 1.0 | |||
| A+B | 201 | 0 | 0 | 1 | 1 | 0.5 | |||
1009 | A | 100 | 0 | 0 | 0 | 0 | 0 | |||
| B | 101 | 0 | 1 | 0 | 1 | 1.0 | |||
| A+B | 201 | 0 | 1 | 0 | 1 | 0.5 | |||
1442 | A | 100 | 0 | 0 | 0 | 0 | 0 | |||
| B | 100 | 0 | 0 | 0 | 0 | 0 | |||
| A+B | 200 | 0 | 0 | 0 | 0 | 0 | |||
NQO, 2.5 | A | 25 | 0 | 0 | 0 | 0 | 0 | |||
| B | 25 | 0 | 0 | 0 | 0 | 0 | |||
| A+B | 50 | 0 | 0 | 0 | 0 | 0 | |||
| ||||||||||
3+ 17 hours, +S9, Experiment 1 Donor sex: Male | ||||||||||
Treatment (µg/mL) | Rep | Cells** | H | E | P | Tot abs | % with num abs |
| ||
Solvent | A | 100 | 0 | 0 | 0 | 0 | 0 | |||
| B | 103 | 2 | 0 | 1 | 3 | 2.9 | |||
| A+B | 203 | 2 | 0 | 1 | 3 | 1.5 | |||
706.6 | A | 101 | 1 | 0 | 0 | 1 | 1.0 | |||
| B | 100 | 0 | 0 | 0 | 0 | 0 | |||
| A+B | 201 | 1 | 0 | 0 | 1 | 0.5 | |||
1009 | A | 100 | 0 | 0 | 0 | 0 | 0 | |||
| B | 100 | 0 | 0 | 0 | 0 | 0 | |||
| A+B | 200 | 0 | 0 | 0 | 0 | 0 | |||
1442 | A | 101 | 0 | 0 | 1 | 1 | 1.0 | |||
| B | 101 | 1 | 0 | 0 | 1 | 1.0 | |||
| A+B | 202 | 1 | 0 | 1 | 2 | 1.0 | |||
CPA, 25 | A | 25 | 0 | 0 | 0 | 0 | 0 | |||
| B | 25 | 0 | 0 | 0 | 0 | 0 | |||
| A+B | 50 | 0 | 0 | 0 | 0 | 0 | |||
ZK 39.294: Summary of the numbers and types of numerical aberrations observed | ||||||||||
20+0 hours, -S9, Experiment 2 Donor sex: Female | ||||||||||
Treatment (µg/mL) | Rep | Cells** | H | E | P | Tot abs | % with num abs |
| ||
Solvent | A | 100 | 0 | 0 | 0 | 0 | 0 | |||
| B | 102 | 2 | 0 | 0 | 2 | 2.0 | |||
| A+B | 202 | 2 | 0 | 0 | 2 | 1.0 | |||
811.1 | A | 100 | 0 | 0 | 0 | 0 | 0 | |||
| B | 100 | 0 | 0 | 0 | 0 | 0 | |||
| A+B | 200 | 0 | 0 | 0 | 0 | 0 | |||
1082 | A | 100 | 0 | 0 | 0 | 0 | 0 | |||
| B | 100 | 0 | 0 | 0 | 0 | 0 | |||
| A+B | 200 | 0 | 0 | 0 | 0 | 0 | |||
1442 | A | 100 | 0 | 0 | 0 | 0 | 0 | |||
| B | 100 | 0 | 0 | 0 | 0 | 0 | |||
| A+B | 200 | 0 | 0 | 0 | 0 | 0 | |||
NQO, 2.5 | A | 25 | 0 | 0 | 0 | 0 | 0 | |||
| B | 25 | 0 | 0 | 0 | 0 | 0 | |||
| A+B | 50 | 0 | 0 | 0 | 0 | 0 | |||
ZK 39.294: Summary of the numbers and types of numerical aberrations observed | ||||||||||
3+ 17 hours, +S9, Experiment 2 Donor sex: Female | ||||||||||
Treatment (µg/mL) | Rep | Cells** | H | E | P | Tot abs | % with num abs |
| ||
Solvent | A | 100 | 0 | 0 | 0 | 0 | 0 | |||
| B | 101 | 0 | 1 | 0 | 1 | 1.0 | |||
| A+B | 201 | 0 | 1 | 0 | 1 | 0.5 | |||
811.1 | A | 101 | 1 | 0 | 0 | 1 | 1.0 | |||
| B | 101 | 0 | 0 | 1 | 1 | 1.0 | |||
| A+B | 202 | 1 | 0 | 1 | 2 | 1.0 | |||
1082 | A | 100 | 0 | 0 | 0 | 0 | 0 | |||
| B | 100 | 0 | 0 | 0 | 0 | 0 | |||
| A+B | 200 | 0 | 0 | 0 | 0 | 0 | |||
1442 | A | 100 | 0 | 0 | 0 | 0 | 0 | |||
| B | 100 | 0 | 0 | 0 | 0 | 0 | |||
| A+B | 200 | 0 | 0 | 0 | 0 | 0 | |||
CPA, 25 | A | 25 | 0 | 0 | 0 | 0 | 0 | |||
| B | 25 | 0 | 0 | 0 | 0 | 0 | |||
| A+B | 50 | 0 | 0 | 0 | 0 | 0 | |||
ZK 39.294: Summary of the numbers and types of numerical aberrations observed | ||||||||||
Donor sex: Female 3+41 hours, +S9, Experiment 2 | ||||||||||
Treatment (µg/mL) | Rep | Cells** | H | E | P | Tot abs | % with num abs |
| ||
Solvent | A | 100 | 0 | 0 | 0 | 0 | 0 | |||
| B | 100 | 0 | 0 | 0 | 0 | 0 | |||
| A+B | 200 | 0 | 0 | 0 | 0 | 0 | |||
1442 | A | 100 | 0 | 0 | 0 | 0 | 0 | |||
| B | 100 | 1 | 0 | 1 | 2 | 2.0 | |||
| A+B | 200 | 1 | 0 | 1 | 2 | 1.0 | |||
* = Total cells examined for structural aberrations ** = Total cells examined for numerical aberrations | ||||||||||
TA 1535 | |||||||||
Dose/Plate | REVERTANTS PER PLATE | QUOTIENT | |||||||
|
| -S9 | M | SD | +S9 | M | SD | -S9 | +S9 |
Phosphate | buf. 50 µL | 23 | 29 | 6 | 20 | 16 | 4 | 1.0 | 1.0 |
29 | 15 | ||||||||
34 | 12 | ||||||||
DMSO | 50 µL | 38 | 29 | 8 | 11 | 10 | 1 | 1.0 | 0.7 |
24 | 9 | ||||||||
26 | 11 | ||||||||
ZK39294 | 0.5 mg | 38 | 39 | 2 | 14 | 16 | 2 | 1.4 | 1.0 |
38 | 18 | ||||||||
41 | 15 | ||||||||
1.0 mg | 38 | 35 | 3 | 18 | 16 | 3 | 1.2 | 1.0 | |
33 | 13 | ||||||||
35 | 18 | ||||||||
1.5 mg | 31 | 32 | 2 | 24 | 22 | 5 | 1.1 | 1.4 | |
31 | 16 | ||||||||
34 | 26 | ||||||||
2.0 mg | 50 | 39 | 10 | 23 | 27 | 5 | 1.3 | 1.7 | |
33 | 27 | ||||||||
33 | 32 | ||||||||
2.5 mg | 35 | 41 | 7 | 33 | 29 | 6 | 1.4 | 1.8 | |
40 | 22 | ||||||||
49 | 31 | ||||||||
3.0 mg | 46 | 43 | 5 | 39 | 33 | 6 | 1.5 | 2.1 | |
46 | 34 | ||||||||
38 | 27 | ||||||||
4.0 mg | 47 | 50 | 6 | 35 | 32 | 3 | 1.7 | 2.0 | |
56 | 32 | ||||||||
46 | 29 | ||||||||
5.0 mg | 52 | 48 | 5 | 46 | 42 | 5 | 1.7 | 2.7 | |
43 | 36 | ||||||||
49 | 44 | ||||||||
Anthracen-2-ami ne 5 µg | 29 | 30 | 2 | 71 | 75 | 5 | 1.1 | 4.8 | |
29 | 80 | ||||||||
33 | 74 | ||||||||
Cyclophosphamide 400 µg | 66 | 63 | 10 | 588 | 581 | 26 | 2.2 | 37.l | |
71 | 603 | ||||||||
51 | 552 | ||||||||
Sodium azide 5 µg
| 690 | 669 | 30 | 75 | 69 | 7 | 23.3 | 4.4 | |
635 | 70 | ||||||||
681 |
|
| 61 |
|
|
|
| ||
TA 100 | |||||||||
| REVERTANTS PER PLATE | QUOTIENT SD | |||||||
Dose/Plate | -S9 | M | SD | S9 | M | SD | -S9 | +S9 | |
Phosphate buf. 50 µL | 113 | 113 | 3 | 99 | 101 | 5 | 1.0 | 1.0 | |
115 | 97 | ||||||||
110 | 106 | ||||||||
DMSO 50 µL | 98 | 99 | 1 | 73 | 83 | 9 | 0.9 | 0.8 | |
100 | 89 | ||||||||
100 | 86 | ||||||||
ZK39294 0.5 mg | 133 | 126 | 12 | 96 | 97 | 2 | 1.1 | 1.0 | |
112 | 100 | ||||||||
133 | 96 | ||||||||
1.0 mg | 150 | 138 | 12 | 123 | 117 | 5 | 1.2 | 1.2 | |
138 | 115 | ||||||||
127 | 114 | ||||||||
1.5 mg | 144 | 143 | 7 | 112 | 123 | 10 | 1.3 | 1.2 | |
149 | 131 | ||||||||
135 | 125 | ||||||||
2.0 mg | 165 | 152 | 11 | 149 | 141 | 8 | 1.3 | 1.4 | |
146 | 133 | ||||||||
145 | 142 | ||||||||
2.5 mg | 186 | 164 | 19 | 157 | 151 | 12 | 1.5 | 1.5 | |
153 | 159 | ||||||||
154 | 137 | ||||||||
3.0 mg | 170 | 174 | 3 | 164 | 171 | 6 | 1.5 | 1.7 | |
176 | 174 | ||||||||
!76 | 174 | ||||||||
4.0 mg | 161 | 168 | 9 | 164 | 170 | 9 | 1.5 | 1.7 | |
178 | 167 | ||||||||
164 | 180 | ||||||||
5.0 mg | 173 | 184 | 9 | 155 | 181 | 22 | 1.6 | 1.8 | |
190 | 196 | ||||||||
188 | 191 | ||||||||
Anthracen-2-ami ne | 157 | 149 | 9 | 526 | 525 | 8 | 1.3 | 5.2 | |
5 µg | 149 | 532 | |||||||
140 | 516 | ||||||||
DMNA | 147 | 135 | 16 | 847 | 854 | 76 | 1.2 | 8.5 | |
5 µL | 142 | 781 | |||||||
117 | 933 | ||||||||
Sodium azide | 835 | 821 | 21 | 142 | 135 | 23 | 7.3 | 1.3 | |
5 µg | 831 | 109 | |||||||
| 797 |
|
| 154 |
|
|
|
| |
TA 1537 | ||||||
Dose/ Plate | -S9 | REVERTANTS PER PLATE | QUOTIENT | |||
| -S9 | M | SD | -S9 | ||
Phosphate buf. | 50 µL | 16 | 18 | 2 | 1.0 | |
19 | ||||||
18 | ||||||
DMSO | 50 µL | 13 | 12 | 1 | 0.7 | |
12 | ||||||
12 | ||||||
ZK39294 | 0.5 mg | 14 | 16 | 3 | 0.9 | |
20 | ||||||
15 | ||||||
1.0 mg | 12 | 10 | 2 | 0.5 | ||
9 | ||||||
8 | ||||||
1.5 mg | 15 | 13 | 2 | 0.8 | ||
12 | ||||||
13 | ||||||
2.0 mg | 11 | 11 | 3 | 0.6 | ||
14 | ||||||
8 | ||||||
2.5 mg | 10 | 13 | 4 | 0.8 | ||
17 | ||||||
13 | ||||||
3.0 mg | 12 | 13 | 2 | 0.8 | ||
16 | ||||||
12 | ||||||
4.0 mg | 10 | 10 | 4 | 0.5 | ||
13 | ||||||
6 | ||||||
5.0 mg | 8 | 10 | 3 | 0.5 | ||
13 | ||||||
8 | ||||||
9-Acridinamine | 103 | 103 | 3 | 5.8 | ||
40 µg | 106 | |||||
|
| 100 |
|
|
| |
TA 1537 | |||||||||||
Dose/Plate | REVERTANTS PER PLATE | QUOTIENT | |||||||||
|
|
| +S9 | M | SD |
| +S9 | ||||
Phosphate buf. 50 µL |
|
| 17 | 23 | 6 |
| 1.0 | ||||
26 | |||||||||||
27 | |||||||||||
DMSO 50 µL | 22 | 21 | 1 | 0.9 | |||||||
21 | |||||||||||
20 | |||||||||||
ZK39294 0.5 mg | 24 | 20 | 4 | 0.8 | |||||||
17 | |||||||||||
18 | |||||||||||
1.0 mg | 24 | 22 | 3 | 0.9 | |||||||
19 | |||||||||||
23 | |||||||||||
1.5 mg | 23 | 24 | 3 | 1.0 | |||||||
27 | |||||||||||
21 | |||||||||||
2.0 mg | 22 | 21 | 2 | 0.9 | |||||||
23 | |||||||||||
19 | |||||||||||
2.5 mg | 24 | 21 | 3 | 0.9 | |||||||
22 | |||||||||||
18 | |||||||||||
3.0 mg | 27 | 25 | 2 | 1.1 | |||||||
23 | |||||||||||
24 | |||||||||||
4.0 mg | 22 | 21 | 2 | 0.9 | |||||||
19 | |||||||||||
22 | |||||||||||
5.0 mg | 24 | 22 | 2 | 1.0 | |||||||
20 | |||||||||||
23 | |||||||||||
Anthracen-2-amine 5 µg | 133 | 140 | 8 | 6.0 | |||||||
| 139 | ||||||||||
| 149 | ||||||||||
Benzo[a]pyrene 5 µg
|
| 126 | 116 | 19 | 5.0 | ||||||
| 128 | ||||||||||
|
| 94 |
|
|
| ||||||
TA 1538 |
| |||||||||||||||||||
Dose/Plate | REVERTANTS PER PLATE | QUOTIENT |
| |||||||||||||||||
| -S9 | M | SD | +S9 | M | SD | -S9 | +S9 |
| |||||||||||
Phosphate buf. 50 µL | 9 | 9 | 4 | 26 | 26 | 2 | 1.0 | 1.0 |
| |||||||||||
5 | 28 |
| ||||||||||||||||||
12 | 25 |
| ||||||||||||||||||
DMSO 50 µL | 10 | 12 | 5 | 26 | 26 | 2 | 1.4 | 1.0 |
| |||||||||||
18 | 27 |
| ||||||||||||||||||
9 | 24 |
| ||||||||||||||||||
ZK39294 | 0.5 mg | 18 | 13 | 4 | 24 | 21 | 3 | 1.5 | 0.8 |
| ||||||||||
12 | 18 |
| ||||||||||||||||||
10 | 22 |
| ||||||||||||||||||
1.0 mg | 7 | 12 | 6 | 15 | 18 | 6 | 1.3 | 0.7 |
| |||||||||||
18 | 24 |
| ||||||||||||||||||
10 | 14 |
| ||||||||||||||||||
1.5 mg | 9 | 9 | 6 | 24 | 22 | 3 | 1.0 | 0.8 |
| |||||||||||
3 | 24 |
| ||||||||||||||||||
14 | 19 | |||||||||||||||||||
2.0 mg | 17 | 16 | 4 | 33 | 25 | 7 | 1.8 | 0.9 | ||||||||||||
19 | 20 | |||||||||||||||||||
11 | 22 | |||||||||||||||||||
2.5 mg | 15 | 12 | 5, | 21 | 21 | 9 | 1.4 | 0.8 | ||||||||||||
15 | 12 | |||||||||||||||||||
6 | 30 | |||||||||||||||||||
3.0 mg | 10 | 9 | 3 | 29 | 23 | 6 | 1.1 | 0.9 | ||||||||||||
6 | 23 | |||||||||||||||||||
12 | 17 | |||||||||||||||||||
4.0 mg | 14 | 16 | 4 | 21 | 22 | 10 | 1.8 | 0.8 | ||||||||||||
13 | 32 | |||||||||||||||||||
20 | 13 | |||||||||||||||||||
5.0 mg | 12 | 11 | 2 | 24 | 21 | 4 | 1.2 | 0.8 | ||||||||||||
8 | 22 | |||||||||||||||||||
12 | 16 | |||||||||||||||||||
Anthracen-2-amine |
|
| 20 | 15 | 5 | 562 | 613 | 54 | 1.8 | 23.3 | ||||||||||
5 µg |
|
| 11 | 607 | ||||||||||||||||
|
| 15 | 670 | |||||||||||||||||
Benzo[a]pyrene 10 µg |
| 15 | 12 | 4 | 67 | 62 | 8 | 1.4 | 2.3 | |||||||||||
| 13 |
| 53 | |||||||||||||||||
| 8 |
| 65 | |||||||||||||||||
2-Nitrofluorene 10 µg |
| 908 | 918 | 21 | 294 | 279 | 22 | 106.0 | 10.6 | |||||||||||
| 905 | 289 | ||||||||||||||||||
942 | 254 |
|
|
|
| |||||||||||||||
TA 1538 | |||||||
Dose/Plate | REVERTANTS PER PLATE | QUOTIENT | |||||
|
|
| +S9 | M | SD |
| +S9 |
Phosphate buf. 50 µL | 50 µL |
| 32 | 35 | 3 |
| 1.0 |
38 | |||||||
35 | |||||||
DMSOMSO 50 µL | 50 µL | 40 | 36 | 6 | 1.0 | ||
39 | |||||||
30 | |||||||
ZK39294 0.5 mg | 0.5 mg | 27 | 31 | 6 | 0.9 | ||
27 | |||||||
38 | |||||||
1.0 mg | 1.0 mg | 32 | 33 | 7 | 0.9 | ||
40 | |||||||
27 | |||||||
1.5 mg | 1.5 mg | 39 | 38 | 3 | 1.1 | ||
41 | |||||||
35 | |||||||
2.0 mg | 2.0 mg | 41 | 33 | 9 | 1.0 | ||
23 | |||||||
36 | |||||||
2.5 mg | 2.5 mg | 44 | 40 | 9 | 1.1 | ||
46 | |||||||
30 | |||||||
3.0 mg | 3.0 mg | 27 | 31 | 5 | 0.9 | ||
29 | |||||||
36 | |||||||
4.0 mg | 4.0 mg | 49 | 43 | 11 | 1.2 | ||
30 | |||||||
49 | |||||||
5.0 mg | 5.0 mg | 44 | 43 | 4 | 1.2 | ||
47 | |||||||
39 | |||||||
Anthracen-2-amine | 5 µg |
| 739 |
|
|
|
|
|
|
| 811 |
|
|
|
|
|
|
| 796 | 782 | 38 |
| 22.3 |
Benzo[a]pyrene | 10 µg |
| 282 |
|
|
|
|
|
|
| 277 |
|
|
|
|
|
|
| 278 | 279 | 3 |
| 8.0
|
TA 1538 | ||||||
|
|
| REVERTANTS PER PLATE | QUOTIENT | ||
Dose/Plate |
| -S9 | M | SD | -S9 | |
Phosphate | buf. 50 µL | 9 | 10 | 1 | 1.0 | |
10 | ||||||
10 | ||||||
DMSO | 50 µL | 14 | 12 | 4 | 1.3 | |
8 | ||||||
15 | ||||||
ZK39294 | 0.5 mg | 12 | 12 | 2 | 1.3 | |
14 | ||||||
11 | ||||||
1.0 mg | 16 | 11 | 4 | 1.2 | ||
8 | ||||||
10 | ||||||
1.5 mg | 7 | 9 | 2 | 1.0 | ||
10 | ||||||
11 | ||||||
2.0 mg | 12 | 11 | 2 | 1.1 | ||
9 | ||||||
11 | ||||||
2.5 mg | 15 | 11 | 3 | 1.1 | ||
9 | ||||||
9 | ||||||
3.0 mg | 12 | 14 | 3 | 1.4 | ||
16 | ||||||
|
|
| C |
|
|
|
4.0 mg | 5 | 10 | 8 | 1.1 | ||
6 | ||||||
20 | ||||||
5.0 mg | 14 | 12 | 2 | 1.3 | ||
12 | ||||||
11 | ||||||
| 2-Nitrofluorene | 10 µg | 976 | 933 | 60 | 96.5 |
|
|
| 864 |
|
|
|
|
|
| 958 |
|
|
|
TA98 | |||||||||
Dose/Plate | REVERTANTS PER PLATE | QUOTIENT | |||||||
|
| -S9 | M | SD | +S9 | M | SD | -S9 | +S9 |
Phosphate | buf. 50 µL | 23 | 25 | 2 | 39 | 42 | 6 | 1.0 | 1.0 |
27 | 38 | ||||||||
25 | 48 | ||||||||
DMSO | 50 µL | 33 | 29 | 5 | 42 | 35 | 8 | 1.1 | 0.8 |
30 | 26 | ||||||||
23 | 37 | ||||||||
ZK39294 | 0.5 mg | 40 | 32 | 7 | 40 | 42 | 2 | 1.3 | 1.0 |
29 | 44 | ||||||||
28 | 41 | ||||||||
1.0 mg | 40 | 30 | 10 | 45 | 41 | 5 | 1.2 | 1.0 | |
21 | 41 | ||||||||
29 | 36 | ||||||||
1.5 mg | 32 | 30 | 2 | 34 | 39 | 7 | 1.2 | 0.9 | |
30 | 36 | ||||||||
29 | 47 | ||||||||
2.0 mg | 35 | 34 | 6 | 36 | 40 | 3 | 1.4 | 1.0 | |
28 | 41 | ||||||||
40 | 42 | ||||||||
2.5 mg | 30 | 29 | 6 | 46 | 45 | 8 | 1.1 | 1.1 | |
22 | 36 | ||||||||
34 | 52 | ||||||||
3.0 mg | 32 | 28 | 4 | 36 | 40 | 4 | 1.1 | 1.0 | |
28 | 43 | ||||||||
25 | 41 | ||||||||
4.0 mg | 21 | 30 | 8 | 44 | 48 | 5 | 1.2 | 1.2 | |
37 | 54 | ||||||||
31 | 47 | ||||||||
5.0 mg | 44 | 40 | 5 | 46 | 43 | 4 | 1.6 | 1.0 | |
41 | 44 | ||||||||
35 | 39 | ||||||||
Anthracen-2-amine | 5 µg | 22 | 21 | 3 | 528 | 532 | 16 | 0.8 | 12.8 |
|
| 23 |
|
| 550 |
|
|
|
|
|
| 18 |
|
| 518 |
|
|
|
|
Benz[a]pyrene | 10 µg | 24 | 30 | 7 | 115 | 109 | 10 | 1.2 | 2.6 |
|
| 38 |
|
| 97 |
|
|
|
|
|
| 27 |
|
| 114 |
|
|
|
|
2-Nitrofluorene | 10 µg | 615 | 648 | 46 | 167 | 214 | 41 | 25.9 | 5.1 |
|
| 680 |
|
| 240 |
|
|
|
|
|
| * |
|
| 234 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
TA 100 | -S9 |
|
|
|
|
|
Dose | Plate 1 | Plate 2 | Plate 3 | Mean | STD | Ratio |
Control | 235 | 191 | 193 | 206.3 | 24.8 |
|
5.0 mg | 257 | 259 | 283 | 226.3 | 14.5 | 1.29** |
2.5 mg | 227 | 219 | 215 | 220.3 | 6.1 | 1.07 |
1.3 mg | 217 | 216 | 217 | 216.7 | 0.6 | 1.05 |
0.63 mg | 212 | 208 | 189 | 203.0 | 12.3 | 0.98 |
0.31 mg | 212 | 208 | 180 | 200.0 | 17.4 | 0.97 |
Na-azide 0.5 µg | 740 | 830 | 990 | 853.3 | 126.6 | 4.14** |
|
|
|
|
|
|
|
TA 100 | -S9 |
|
|
|
|
|
Dose | Plate 1 | Plate 2 | Plate 3 | Mean | STD | Ratio |
Control | 192 | 232 | 209 | 211.0 | 20.1 |
|
5.0 mg | 325 | 285 | 276 | 295.3 | 26.1 | 1.40** |
2.5 mg | 275 | 300 | 331 | 302.0 | 28.1 | 1.43** |
1.3 mg | 296 | 242 | 228 | 255.2 | 35.9 | 1.21* |
0.63 mg | 202 | 224 | 222 | 216.0 | 12.2 | 1.02 |
0.31 mg | 198 | 196 | 206 | 200.0 | 5.3 | 0.95 |
Na-azide 0.5 µg | 990 | 710 | 1020 | 906.7 | 171.0 | 4.30** |
|
|
|
|
|
|
|
TA 100 | +S9 |
|
|
|
|
|
Dose | Plate 1 | Plate 2 | Plate 3 | Mean | STD | Ratio |
Control | 190 | 204 | 223 | 205.7 | 16.6 |
|
5.0 mg | 248 | 247 | 276 | 257.0 | 16.5 | 1.25** |
2.5 mg | 248 | 235 | 245 | 242.7 | 6.8 | 1.18* |
1.3 mg | 233 | 236 | 271 | 246.7 | 21.1 | 1.20** |
0.63 mg | 249 | 228 | 237 | 238.0 | 10.5 | 1.16* |
0.31 mg | 205 | 185 | - | 195.0 | 14.1 | 0.95 |
2-AA 1.25 µg | 800 | 930 | 640 | 790.0 | 145.3 | 3.84** |
|
|
|
|
|
|
|
TA 100 | +S9 |
|
|
|
|
|
Dose | Plate 1 | Plate 2 | Plate 3 | Mean | STD | Ratio |
Control | 246 | 203 | 237 | 228.7 | 22.7 |
|
5.0 mg | 314 | 330 | 331 | 325.0 | 9.5 | 1.42** |
2.5 mg | 274 | 284 | 235 | 264.3 | 25.9 | 1.16* |
1.3 mg | 261 | 256 | 241 | 252.7 | 10.4 | 1.10 |
0.63 mg | 234 | 216 | 224 | 224.7 | 9.0 | 0.98 |
0.31 mg | 222 | 218 | 235 | 225.0 | 8.9 | 0.98 |
2-AA 1.25 µg | 800 | 800 | 720 | 773.3 | 46.2 | 3.38** |
|
|
|
|
|
|
|
TA 98 | -S9 |
|
|
|
|
|
Dose | Plate 1 | Plate 2 | Plate 3 | Mean | STD | Ratio |
Control | 30 | 36 | 38 | 34.7 | 4.2 |
|
5.0 mg | 28 | 33 | 27 | 29.3 | 3.2 | 0.85 |
2.5 mg | 31 | 30 | - | 30.5 | 0.7 | 0.88 |
1.3 mg | 33 | 33 | 27 | 31.0 | 3.5 | 0.89 |
0.63 mg | 33 | 31 | 27 | 30.3 | 3.1 | 0.88 |
0.31 mg | 33 | 31 | 30 | 31.3 | 1.5 | 0.90 |
2-NF 0.6 µg | 240 | 256 | 384 | 293.3 | 78.9 | 8.46** |
|
|
|
|
|
|
|
TA 98 | -S9 |
|
|
|
|
|
Dose | Plate 1 | Plate 2 | Plate 3 | Mean | STD | Ratio |
Control | 48 | 46 | 47 | 47.0 | 1.0 |
|
5.0 mg | 49 | 51 | 59 | 53.0 | 5.3 | 1.13 |
2.5 mg | 57 | 40 | 33 | 43.3 | 12.3 | 0.92 |
1.3 mg | 60 | 51 | 36 | 49.0 | 12.1 | 1.04 |
0.63 mg | 50 | 45 | 46 | 47.0 | 2.6 | 1.00 |
0.31 mg | 46 | 53 | 46 | 48.3 | 4.0 | 1.03 |
2-NF 0.6 µg | 480 | 380 | 210 | 356.7 | 136.5 | 7.59** |
|
|
|
|
|
|
|
TA 98 | +S9 |
|
|
|
|
|
Dose | Plate 1 | Plate 2 | Plate 3 | Mean | STD | Ratio |
Control | 31 | 32 | 36 | 33.0 | 2.6 |
|
5.0 mg | 27 | 41 | 28 | 32.0 | 7.8 | 0.97 |
2.5 mg | 34 | 36 | 38 | 36.0 | 2.0 | 1.09 |
1.3 mg | 32 | 33 | 38 | 34.3 | 3.2 | 1.04 |
0.63 mg | 33 | 41 | 37 | 37.0 | 4.0 | 1.12 |
0.31 mg | 39 | 28 | 31 | 32.7 | 5.7 | 0.99 |
2-AA 1.25 µg | 1090 | 1120 | 1220 | 1143.0 | 68.1 | 34.65 |
|
|
|
|
|
|
|
TA 98 | +S9 |
|
|
|
|
|
Dose | Plate 1 | Plate 2 | Plate 3 | Mean | STD | Ratio |
Control | 62 | 57 | 60 | 59.7 | 2.5 |
|
5.0 mg | 57 | 53 | 60 | 56.7 | 3.5 | 0.95 |
2.5 mg | 68 | 67 | 58 | 64.3 | 5.5 | 1.08 |
1.3 mg | 59 | 40 | 53 | 50.7 | 9.7 | 0.85 |
0.63 mg | 50 | 41 | 50 | 47.0 | 5.2 | 0.79 |
0.31 mg | 52 | 64 | 47 | 54.3 | 8.7 | 0.91 |
2-AA 1.25 µg | 1400 | 1000 | 1000 | 1133.3 | 230.9 | 18.99** |
|
|
|
|
|
|
|
TA 1537 | -S9 |
|
|
|
|
|
Dose | Plate 1 | Plate 2 | Plate 3 | Mean | STD | Ratio |
Control | 8 | 7 | 8 | 7.7 | 0.6 |
|
5.0 mg | 9 | 9 | 9 | 9.0 | 0.0 | 1.17 |
2.5 mg | 8 | 7 | 6 | 7.0 | 1.0 | 0.91 |
1.3 mg | 6 | 8 | 7 | 7.0 | 1.0 | 0.91 |
0.63 mg | 10 | 11 | 6 | 9.0 | 2.6 | 1.17 |
0.31 mg | 5 | 12 | 8 | 8.3 | 3.5 | 1.09 |
2-NF 0.6 µg | 152 | 150 | 144 | 148.7 | 4.2 | 19.39** |
|
|
|
|
|
|
|
TA 1537 | -S9 |
|
|
|
|
|
Dose | Plate 1 | Plate 2 | Plate 3 | Mean | STD | Ratio |
Control | 10 | 10 | 10 | 10.0 | 0.0 |
|
5.0 mg | 15 | 12 | 14 | 13.7 | 1.5 | 1.37 |
2.5 mg | 16 | 13 | 8 | 12.3 | 4.0 | 1.23 |
1.3 mg | 12 | 15 | 8 | 11.7 | 3.5 | 1.17 |
0.63 mg | 10 | 9 | 13 | 10.7 | 2.1 | 1.07 |
0.31 mg | 9 | 10 | 9 | 9.3 | 0.6 | 0.93 |
2-NF 0.6 µg | 150 | 152 | 148 | 150.0 | 2.0 | 15.00** |
|
|
|
|
|
|
|
TA 1537 | +S9 |
|
|
|
|
|
Dose | Plate 1 | Plate 2 | Plate 3 | Mean | STD | Ratio |
Control | 10 | 8 | 12 | 10.0 | 2.0 |
|
5.0 mg | 12 | 10 | 11 | 11.0 | 1.0 | 1.10 |
2.5 mg | 15 | 12 | 11 | 12.7 | 2.1 | 1.27 |
1.3 mg | 11 | 9 | 15 | 11.7 | 3.1 | 1.17 |
0.63 mg | 9 | 9 | 10 | 9.3 | 0.6 | 0.93 |
0.31 mg | 10 | 14 | 9 | 11.0 | 2.6 | 1.10 |
2-AA 1.25 µg | 68 | 73 | 82 | 74.3 | 7.1 | 7.43** |
|
|
|
|
|
|
|
TA 1537 | +S9 |
|
|
|
|
|
Dose | Plate 1 | Plate 2 | Plate 3 | Mean | STD | Ratio |
Control | 12 | 13 | 14 | 13.0 | 1.0 |
|
5.0 mg | 14 | 15 | 23 | 17.3 | 4.9 | 1.33 |
2.5 mg | 16 | 9 | - | 12.5 | 4.9 | 0.96 |
1.3 mg | 12 | 11 | 16 | 13.0 | 2.6 | 1.00 |
0.63 mg | 9 | 11 | 12 | 10.7 | 1.5 | 0.82 |
0.31 mg | 16 | 9 | 14 | 13.0 | 3.6 | 1.00 |
2-AA 1.25 µg | 76 | 60 | 56 | 64.0 | 10.6 | 4.92** |
|
|
|
|
|
|
|
TA 1535 | -S9 |
|
|
|
|
|
Dose | Plate 1 | Plate 2 | Plate 3 | Mean | STD | Ratio |
Control | 21 | 23 | 18 | 20.7 | 2.5 |
|
5.0 mg | 27 | 30 | 20 | 25.7 | 5.1 | 1.24 |
2.5 mg | 35 | 21 | 20 | 25.3 | 8.4 | 1.23 |
1.3 mg | 25 | 21 | 16 | 20.7 | 4.5 | 1.00 |
0.63 mg | 24 | 25 | 27 | 25.3 | 1.5 | 1.23 |
0.31 mg | 23 | 24 | 30 | 25.7 | 3.8 | 1.24 |
Na-azide 1.0 µg | 1280 | 1280 | 1320 | 1293.3 | 23.1 | 62.58** |
|
|
|
|
|
|
|
TA 1535 | -S9 |
|
|
|
|
|
Dose | Plate 1 | Plate 2 | Plate 3 | Mean | STD | Ratio |
Control | 29 | 31 | 30 | 30.0 | 1.0 |
|
5.0 mg | 35 | 39 | 39 | 37.7 | 2.3 | 1.26* |
2.5 mg | 40 | 31 | 34 | 35.0 | 4.6 | 1.17 |
1.3 mg | 25 | 32 | 30 | 29.0 | 3.6 | 0.97 |
0.63 mg | 31 | 29 | 28 | 29.0 | 1.0 | 0.97 |
0.31 mg | 21 | 26 | 30 | 25.7 | 4.5 | 0.86 |
Na-azide 1.0 µg | 1310 | 1280 | 1500 | 1363.3 | 119.3 | 45.44** |
|
|
|
|
|
|
|
TA 1535 | +S9 |
|
|
|
|
|
Dose | Plate 1 | Plate 2 | Plate 3 | Mean | STD | Ratio |
Control | 18 | 16 | 19 | 17.7 | 1.5 |
|
5.0 mg | 35 | 35 | 43 | 37.7 | 4.6 | 2.13** |
2.5 mg | 30 | 30 | 29 | 29.7 | 0.6 | 1.68** |
1.3 mg | 16 | 26 | 21 | 21.0 | 5.0 | 1.19 |
0.63 mg | 25 | 18 | 32 | 25.0 | 7.0 | 1.42 |
0.31 mg | 19 | 20 | 13 | 17.3 | 3.8 | 0.98 |
2-AA 1.25 µg | 160 | 180 | 156 | 165.3 | 12.9 | 9.36** |
|
|
|
|
|
|
|
TA 1535 | +S9 |
|
|
|
|
|
Dose | Plate 1 | Plate 2 | Plate 3 | Mean | STD | Ratio |
Control | 33 | 27 | 27 | 29.0 | 3.5 |
|
5.0 mg | 88 | 68 | 88 | 81.3 | 11.5 | 2.80** |
2.5 mg | 55 | 48 | 38 | 47.0 | 8.5 | 1.62** |
1.3 mg | 33 | 41 | 41 | 38.3 | 4.6 | 1.32 |
0.63 mg | 35 | 30 | 33 | 32.7 | 2.5 | 1.13 |
0.31 mg | 23 | 33 | 32 | 29.3 | 5.5 | 1.01 |
2-AA 1.25 µg | 206 | 200 | 216 | 207.3 | 8.1 | 7.15** |
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Description of key information
Study conducted according to OECD test guideline 486; male Wistar rats (3 animals per group) were treated with Trioxabicyclooctan at doses of 0, 1000 and 2000 mg/kg bw once by oral gavage. Hepatocytes were isolated after 2h and after 16h after administration and incubated with 3HTdR for 4 h. Subsequently the hepatocytes were cultured over night with unlabelled thymidine. After the last incubation the DNA repair was measured as incorporation of 3HTdR into the DNA of the treated cells, result: negative
Link to relevant study records
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2003-01-24 to 2003-06-24
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
- Version / remarks:
- adopter 21 July 1997
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- unscheduled DNA synthesis
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- Hanlbm: WIST (SPF)
- Details on species / strain selection:
- The rat is an animal which has been used for many years as suitable experimental animal in genotoxicity investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the UDS test. In addition, the rat is an experimental animal in many physiological, pharmacological, and toxicological studies. Data from such experiments may also be useful for the design and the performance of the UDS test.
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division CH-4414 Füllinsdorf
- Age at study initiation: 6-10 weeks
- Weight at study initiation: Mean value 183.6 g (SD*± 15.1 g)
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: single, Makrolon Type II, with wire mesh top (Ehret, D-79312 Emmendingen) with granulated soft wood bedding (Altromin, D-32791 Lage/Lippe)
- Diet (e.g. ad libitum): pelleted standard diet (Altromin, D-32791 Lage/Lippe) ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: minimum 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21+/-3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12
- Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: physiol. saline
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment the test item was formulated in 0.9% NaCl solution. The vehicle was chosen to itsnon-toxicity for the animals. The animals received a single standard volume of 10 mL/kg body weight orally - Duration of treatment / exposure:
- treatment once and isolation of hepatocytes after 2 and 16 h
- Frequency of treatment:
- once
- Post exposure period:
- 2 and 16 h
- Dose / conc.:
- 2 000 mg/kg bw/day
- Dose / conc.:
- 1 000 mg/kg bw/day
- Dose / conc.:
- 0 mg/kg bw/day
- No. of animals per sex per dose:
- 8
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- none; no data; 2-acetylaminofluorene; N, N`-dimethylhydrazinehydrochloride
- Route of administration: oral
- Doses / concentrations: 40 mg/kg bw and 100 mg/kg bw, respectively - Tissues and cell types examined:
- liver, hepatocytes
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: A preliminary study on acute toxicity was performed with two animals per group and sex under identical conditions as in the UDS study concerning: starvation period, animal strain, vehicle, route, frequency, and volume of administration. The animals were treated orally (gavage) and examined for acute toxic symptoms at intervals of 1 h, 2-4 h, 6 h, 24 h, and 48 h after administration of the test item.
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The animals were starved overnight (2 hours treatment) or approximately 6 hours (16 hours treatment} before receiving the test item, the positive or the vehicle control substance. Water was available ad libitum. Before the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the body weigiht of the animals. The animals received the test item once. Four animals (males) were treated per dose group. After anaesthetizing the rats with Na-thiopental (Trapanal, Byk Gulden, D-78467 Konstanz) the liver was perfused through the vena portae with Hanks' balanced salt solution (HBSS, Gibco/BRL, D-76344 Eggenstein} supplemented with collagenase (0.05 % (w/v), Boehringer Mannheim, D-68305 Mannheim) adjusted to pH 7.4 and maintained at 37° C
DETAILS OF SLIDE PREPARATION: The washed hepatocytes were centrifuged and transferred into Williams medium E. At least three cultures were established from each animal. After an attachment period of approximately 1.5 h in a 95 % air/ 5 % CO 2 humidified incubator at 37° C the culture medium was discarded. Then, the cell layer was rinsed once with PBS to remove non-adherent cells. Subsequently, 3HTdR (5 µCi/mL, specific activity 20 Ci/mmol; New England Nuclear, D-63033 Dreieich) in 2.0 mL culture medium (WME, 1 % (v/v), FCS) was added to the cultures. After a labelling time of 4 h the cells were washed twice with WME supplemented with 1 % (v/v) FCS and 0.25 mM unlabelled thymidine. Cultures were incubated overnight using the same medium. To prepare for autoradiography the medium was replaced by a hypotonic solution of 1 % (w/v) sodium citrate for 10 minutes to swell the nuclei for better grain detection. The cells on the coverslips were then fixed by three changes of methanol:acetic acid (3+1 v/v) for 20 minutes each, rinsed with 96 % (v/v) ethanol, and air-dried. The cover slips were mounted the side carrying the cells up on glass slides and coated with KODAK NTB2 (Tecnomara, D-35463 Fernwald) photographic emulsion in the dark. The coated slides were stored in light-proof boxes in the presence of a drying agent for 14 days at 4° C. The photographic emulsion was then developed with KODAK Dektol Developer (Tecnomara, D-35463 Fernwald) at room temperature, fixed in TETENAL (Tetenal, D-22844 Norderstedt) and stained with hematoxylin/eosin.
METHOD OF ANALYSIS: Evaluation was performed microscopically on coded slides using NIKON microscopes with oil immersion objectives. The cells for scoring were randomly selected according to a fixed scheme. The number of silver grains in the nuclear area was counted automatically using the ARTEK 880 or 982 counter. In addition, the number of grains of the most heavily lableled nuclear-sized cytoplasm area adjacent to the nucleus was counted. At least two slides per animal; and 50 cells per slide were evaluated. Heavily radiolabeled cells undergoing replicative DNA synthesis were excluded from counting. Three animals per group were evaluated as described above.
Nuclear and net grain counts are estimated together. Increased net grains should be based on enhanced nuclear grain counts rather than on decreased cytoplasmic grain counts. A test item is classified as positive if the mean number of net grains is higher than five per nucleus at one of the test points. A group average between 0 and 5 net grains is considered as a marginal response. A dose-related increase in nuclear and net grains and/or a substantial shift of the percentage distribution of the nuclear grain counts to higher values provide additional information to confirm a positive response with less than 5 net grains. Statistical significance may give further evidence for a positive evaluation. Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test. A test item producing net grains not greater than 0 at anyone of the test points is considered non-effective in this system. - Evaluation criteria:
- see above
- Statistics:
- non-parametric Mann-Whitney test
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Solubility: not reported
- Clinical signs of toxicity in test animals: reduction of spontaneous activity, abdominal position, eyelid closure, ruffled fur.
- Evidence of cytotoxicity in tissue analysed: No
- Rationale for exposure: Based on preliminary acute toxicity testing
- Conclusions:
- In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item Trioxabicyclooctan did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats.
- Executive summary:
In a test conducted according to OECD test guideline 486 (1995) male Wistar rats (3 animals per group) were treated with Trioxabicyclooctan at doses of 0, 1000 and 2000 mg/kg bw once by oral gavage. Hepatocytes were isolated after 2h and after 16h after administration and incubated with 3HTdR for 4 h. Subsequently the hepatocytes were cultured over night with unlabelled thymidine. After the last incubation the DNA repair was measured as incorporation of 3HTdR into the DNA of the treated cells. The amount of incorporated radioactivity is determined by silver grain counting.
The viability of the hepatocytes was not substantially affected due to the in vivo treatment with the test item at any of the treatment periods or dose groups.
No dose level of the test item revealed UDS induction in the hepatocytes of the treated animals. as compared to the current vehicle controls.
Neither the nuclear grains nor the resulting net grains were distinctly enhanced due to the in vivo treatment of the animals with the test item for 2 hours or 16 hours, respectively. Therefore, the net grain values obtained after treatment with the test item were consistently negative. Appropriate reference mutagens (DMH, 40 mg/kg b.w. and 2-AAF, 100 mg/kg b.w.) were used as positive controls. In vivo treatment with DMH or 2-AAF revealed distinct increases in the number of nuclear and net grain counts as indication of induced DNA-repair.
Reference
Treatment | Period | Animal No. | Viability* [%] | Number of isolated cells [x E+06] |
Vehicle control 0.9% NaCl solution | 2 h | 2 | 75 | 341 |
3 | 80 | 216 | ||
4 | 90 | 576 | ||
500 mg/kg bw | 2 h | 5 | 79 | 111 |
6 | 75 | 229 | ||
7 | 92 | 354 | ||
2000 mg/kg bw | 2 h | 9 | 72 | 310 |
10 | 70 | 294 | ||
11 | 81 | 126 | ||
40 mg/kg bw DMH | 2 h | 13 | 84 | 185 |
15 | 76 | 247 | ||
16 | 84 | 139 | ||
Vehicle control 0.9% NaCl solution | 16 h | 17 | 77 | 370 |
18 | 83 | 593 | ||
19 | 90 | 171 | ||
500 mg/kg bw | 16 h | 22 | 76 | 365 |
23 | 91 | 578 | ||
24 | 75 | 420 | ||
2000 mg/kg bw | 16 h | 25 | 71 | 540 |
26 | 78 | 270 | ||
27 | 86 | 357 | ||
100 mg/kg bw 2-AAF | 16 h | 29 | 72 | 266 |
30 | 81 | 150 | ||
31 | 80 | 164 |
Treatment | Period | Grains per nucleus | Grains per cytoplasmic area | Net grains per nucleus | |||
Mean * | ± SD** | Mean * | ± SD** | Mean * | ± SD** | ||
Vehicle control 0.9% NaCl solution | 2 h | 9.41 | 4.69 | 14.58 | 5.29 | -5.17 | 5.13 |
1000 mg/kg bw | 2 h | 10.68 | 5.00 | 17.65 | 7.24 | -6.97 | 5.80 |
2000 mg/kg bw | 2 h | 13.77 | 7.01 | 18.44 | 7.27 | -4.67 | 6.69 |
40 mg/kg bw DMH | 2 h | 17.91 | 11.19 | 12.02 | 6.83 | 5.89 | 7.82 |
Vehicle control 0.9% NaCl solution | 16 h | 8.86 | 4.30 | 11.40 | 4.49 | -2.54 | 4.69 |
1000 mg/kg bw | 16 h | 10.20 | 5.40 | 15.47 | 6.29 | -5.27 | 5.05 |
2000 mg/kg bw | 16 h | 10.23 | 4.14 | 17.25 | 5.42 | -7.02 | 5.57 |
100 mg/kg bw 2-AAF | 16 h | 33.98 | 13.96 | 16.90 | 7.41 | 17.08 | 11.80 |
* Mean of 3 animals ** Standard deviation | |||||||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
- Derek Version: Derek Nexus: 6.1.0, Nexus: 2.3.0, Derek KB 2020 1.0
- Leadscope Version: 3.1.0-40
In a reverse gene mutation assay in bacteria according to OECD test guideline 471 (1983), strains TA 98, TA 100, TA 1535, and TA 1537 of S. typhimurium were exposed to Trioxabicyclooctan (100 % a.i.), at concentrations of 0, 0.31, 0.63, 1.3, 2.5, and 5.0 mg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method.
Trioxabicyclooctan was tested up to limit concentration (5000 µg/plate). The test article induced statistically significant, dose-related and reproducible increases in the number of reyertants in TA 100 with and without S-9 mix. The increases were consistent but relatively small, i.e. below 1.5 1.5 times of the concurrent controls. There was no major difference in effect between the test series with or without S-9 mix. In TA 1535 similar increases in revertants were found in both test series but only with S-9 mix present. However, m this strain the largest increases were more than twice the control levels. The single statistically significant increase in TA 1535 without S-9 mix was only marginal, and not reproducible although the same tendency was evident in the 1. test series. The positive control substance provided the expected increase in revertants numbers. Based on these results Trioxabicyclooctan is a weak mutgen under the conditions of the test.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.
In a reverse gene mutation assay in bacteria according to OECD test guideline 471 (1983), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Trioxabicyclooctan (100 % a.i.), in DMSO at concentrations of 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, and 5.0 mg/plate in the presence and absence of mammalian metabolic activation using the pre-incubation method.
Trioxabicyclooctan was tested up to limit concentration (5000 µg/plate). There were gene mutations by base-pair substitutions in the genome of strains TA1535 and TA100. Although the mutagenic effect was dose-dependent but never exceeded thrice that observed in the negative control even when tested up to the maximum recommended dose of 5.0 mg/plates. The positive control substance provided the expected increase in revertants numbers. Based on these results Trioxabicyclooctan is a weak mutagen under the conditions of the test.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.
In a mammalian cell cytogenetics assay (Chromosome aberration) according to OECD test guideline 473 (1983), human peripheral lymphocytes were exposed to Trioxabicyclooctan, (100 % a.i.), in DMSO at concentrations of Experiment 1: 0, 28.51, 40.73, 58.19, 83.13, 118.8, 169.7, 242.4, 346.2, 494.6, 706.6, 1009, 1443 µg/mL in the first experiment for 3h and 20 h and at 0, 342.2, 456.3, 608.3, 811.1, 1082, 1442 µg/mL for the 3h treatment with 17h and 41 h post-treatment recovery with and without metabolic activation [S9-liver mix].
Trioxabicyclooctan was tested up to the limit concentration. Cultures treated with the test item in the absence of S9 mix showed statistically significant and biologically relevant increases of numbers of metaphases with aberrations, starting at 706.6 µg/mL without S9 mix. Positive controls induced the appropriate response. There was evidence of Chromosome aberrations induced over background.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data.
In a test conducted according to OECD test guideline 486 (1995) male Wistar rats (3 animals per group) were treated with Trioxabicyclooctan at doses of 0, 1000 and 2000 mg/kg bw once by oral gavage. Hepatocytes were isolated after 2h and after 16h after administration and incubated with 3HTdR for 4 h. Subsequently the hepatocytes were cultured over night with unlabelled thymidine. After the last incubation the DNA repair was measured as incorporation of 3HTdR into the DNA of the treated cells. The amount of incorporated radioactivity is determined by silver grain counting.
The viability of the hepatocytes was not substantially affected due to the in vivo treatment with the test item at any of the treatment periods or dose groups. No dose level of the test item revealed UDS induction in the hepatocytes of the treated animals. as compared to the current vehicle controls. Neither the nuclear grains nor the resulting net grains were distinctly enhanced due to the in vivo treatment of the animals with the test item for 2 hours or 16 hours, respectively. Therefore, the net grain values obtained after treatment with the test item were consistently negative. Appropriate reference mutagens (DMH, 40 mg/kg b.w. and 2-AAF, 100 mg/kg b.w.) were used as positive controls. In vivo treatment with DMH or 2-AAF revealed distinct increases in the number of nuclear and net grain counts as indication of induced DNA-repair.
In addition, an in silico assessment on the mutagenic potential using (Q)SAR methologies was performed (V. Ziegler, Sept 2022) with the following tools:
Results:
Alert based on Epoxide in DEREK: PLAUSIBLE
Leadscope predicted value (model result): Positive Prediction Probability = 0.509; Indeterminate
Thus, a mutagenic potential cannot be excluded for the substance.
Justification for classification or non-classification
Both Ames tests resulted in similar findings identifying the test item as a mutagen in bacteria. Also the chromosomal abberation test in vitro in mammalian cells was positive indicating potential clastogenicity of the test item. Although the results of the in vivo study might hint to negative genotoxicity, the particular test used (UDS, Unscheduled DNA-Synthesis) is meanwhile regarded as less relevant for a correct assessment.
Based on the available information from studies conducted according to OECD test guidelines 471, 473 and 486 with Trioxabicyclooctan it is not possible to classify the test item according to Regulation (EC) No. 1272/2008 (CLP). Further data are needed (data lacking).
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