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EC number: 421-750-9 | CAS number: 57280-22-5 TRIOXABICYCLOOCTAN; TRIOXABICYCLOOCTANE
Basic toxicokinetics
Administrative data
- Endpoint:
- basic toxicokinetics, other
- Remarks:
- expert statement
- Type of information:
- other: expert statement based on physico-chemical and toxicological data
- Adequacy of study:
- other information
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- data from handbook or collection of data
Cross-referenceopen allclose all
- Reason / purpose for cross-reference:
- reference to other study
Reference
- 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.
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 | |||||||
- Reason / purpose for cross-reference:
- reference to other study
Reference
- 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.
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 | ||||||||||
- Reason / purpose for cross-reference:
- reference to other study
Reference
- 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.
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 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
- Reason / purpose for cross-reference:
- reference to other study
Reference
- 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.
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** |
- Reason / purpose for cross-reference:
- reference to other study
Reference
- Endpoint:
- skin sensitisation: in vivo (non-LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 406 (Skin Sensitisation)
- Version / remarks:
- 17 July 1992
- GLP compliance:
- yes
- Type of study:
- guinea pig maximisation test
- Justification for non-LLNA method:
- The study was conducted prior to implementation of the new requirement to use as in vivo method the LLNA.
- Species:
- guinea pig
- Strain:
- Pirbright-Hartley
- Sex:
- male/female
- Details on test animals and environmental conditions:
- TEST ANIMALS
- Source: Hagemann
- Females (if applicable) nulliparous and non-pregnant: not specified
- Weight at study initiation: 391-528 g (males); 368-462 g (females)
- Housing: conventional; 1 or 2 animals per cage Makrolon®, type IV with embedding
- Diet (e.g. ad libitum): pell. Altromin® MS, hay and apple ad libitum
- Water (e.g. ad libitum): acidified, demineralized water (pH 2-3) ad libitum
- Acclimation period: 12 days
- Indication of any skin lesions: No
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24
- Humidity (%): 52-68
- Photoperiod (hrs dark / hrs light): 12/12 - Route:
- epicutaneous, occlusive
- Vehicle:
- unchanged (no vehicle)
- Concentration / amount:
- 100%
- Day(s)/duration:
- 48 h
- Adequacy of induction:
- non-irritant substance, but skin pre-treated with 10% SDS
- Route:
- intradermal
- Vehicle:
- physiological saline
- Concentration / amount:
- a) 0.1 ml FCA + vehicle (1 + 1)
b) 0.1 ml ZK 39294;. 5% (w/v)
c) 0.1 ml ZK 39294; 10% (w/v) + FCA (1 +·1) = 5% (w/v) - Adequacy of induction:
- highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
- No.:
- #1
- Route:
- epicutaneous, occlusive
- Vehicle:
- unchanged (no vehicle)
- Concentration / amount:
- 100%
- Day(s)/duration:
- 24 h
- Adequacy of challenge:
- highest non-irritant concentration
- No. of animals per dose:
- 10
- Details on study design:
- RANGE FINDING TESTS: A local tolerance study (pretest) was performed to ensure an intracutaneously tolerable concentration for the induction procedure which does not lead to necrosis (reddenings and swellings are allowed). The high concentration of 5% used in the present test for intracutaneous application is a recommended concentration for induction procedure, provided that the treatment does not produce excessive irritation. The reactions were recorded 24 hours after application. Furthermore, an epicutaneously tolerable concentration for the epicutaneous challenge was determined under occlusive conditiqns. Since no irritation after single epidermal application of the test item was observed in the local tolerance study on the skin of rabbits, the original substance was also tested epidermally for its local tolerance. Two concentrations, namely ca. 25%, and pure substance, were applied to the lateral backs.
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: 8 days and 48 h for intradermal and epicutaneous exposure, respectively.
- Test groups:
Intradermal:
A. Test group
a) 0.1 mL FCA + vehicle (1 + 1)
b) 0.1 mL ZK 39294;. 5% (w/v)
c) 0.1 mL ZK 39294; 10% (w/v) + FCA (1 +·1) = 5% (w/v)
epicutaneous:
The same area of skin (4 x 6 cm) in which intradermal injections were performed was reshaved on day 8 of the test and epidermally treated with approximately 1 mL sodium lauryl sulphate (10% (w/v)] in liquid paraffin. 24 hours later, on day 9, 0.2 mL of the original liquid compound was spread over a 2 x 4 cm filter paper {Whatmann, no. 3M) for use in the test group, whereas for the control group only 0.9% (w/v) NaCl-solution was used.
This filter paper was subsequently applied to the above-mentioned area pretreated with sodium lauryl sulphate. The patch was bandaged occlusively for 48 hours. The test item (pure) had previously been proved not to provoke any irritation on the skin atter epidermal administration under occlusion.
- Control group:
Intradermal:
B. Control group
a) 0.1 mL FCA + vehicle (1, + 1)
b) 0.1 mL vehicle
c) 0.1 mL FCA + vehicle (1 + 1)
Epicutaneous:
0.9% NaCl
- Site: In the neck region (right to left side of the spine) of each animal an overall area of 4 x 6 cm
- Frequency of applications: each once
- Duration: 8 days and 48h
- Concentrations: Intradermal 5%, Epicutaneously: 100%
B. CHALLENGE EXPOSURE
- No. of exposures: once
- Day(s) of challenge: on day 23
- Exposure period: 24 h
- Test groups: Approximately 0.1 mL ZK 39294 (original substance)., which previously proved to be well tolerated locally,. was spread over a 2 x 2 cm piece of filter paper. This patch was placed in the middle of the shorn area of skin and bandaged occlusively for 24 hours.
- Control group: 0.9% NaCl
- Site: a skin area of 5 x 5 cm on the right flank - Positive control substance(s):
- not required
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- positive control
- Dose level:
- mercaptobenzothiazol
- No. with + reactions:
- 3
- Total no. in group:
- 10
- Remarks on result:
- other: In the test more than 30% of the animals reacted positively to the challenge with mercaptobenzothiazoL
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- negative control
- Dose level:
- 0.9% NaCl
- No. with + reactions:
- 0
- Total no. in group:
- 10
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- negative control
- Dose level:
- 0.9% NaCl
- No. with + reactions:
- 2
- Total no. in group:
- 10
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- test chemical
- Dose level:
- 100%
- No. with + reactions:
- 7
- Total no. in group:
- 10
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- test chemical
- Dose level:
- 100%
- No. with + reactions:
- 9
- Total no. in group:
- 10
- Interpretation of results:
- Category 1 (skin sensitising) based on GHS criteria
- Conclusions:
- The results of this study indicate that Trioxabicyclooctan has contact-sentitizing potential in the guinea-pig in the maximization test.
- Executive summary:
In a dermal sensitisation study according to OECD TG 406, (1992) with Trioxabicyclooctan (a.i. 100 %) in physiological saline (5%) solution for epicutaneous apllication, young adult Pribright guinea pigs were tested using the Maximization test method. Positive control substance was 2-mercaptobenzothiazole with a sensitisation rate of over 30 %.
Since the test substance was found not to cause a primary skin irritation in the dose range - finding study, the treated areas of the animals were irritated with sodium lauryl sulfate prior to the topical induction. After the challenge, 9 (90%) of the test substance animals responded with redness and swelling to the 100% test substance. There were no skin reactions in the control group. The controls exhibited no dermal reactions following the challenge with 100% test substance formulation. Therefore, the sensitisation rate was 0 %.
For the challenge concentration, which is the highest non-irritant dose, 100 % of the test item was used. In this study, Trioxabicyclooctan (a.i. 100 %), is a dermal sensitiser.
- Reason / purpose for cross-reference:
- reference to other study
Reference
- Endpoint:
- eye irritation: in vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- March to June 1996
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 405 (Acute Eye Irritation / Corrosion)
- Version / remarks:
- 24 February 1987
- GLP compliance:
- yes
- Species:
- rabbit
- Strain:
- New Zealand White
- Details on test animals or tissues and environmental conditions:
- TEST ANIMALS
- Source: Schriever
- Age at study initiation: adults
- Weight at study initiation: male: 2.6 kg; females: 3.1 kg
- Housing: individually in conventional metal cages
- Diet (e.g. ad libitum): pell. Altromin® K; ad libitum
- Water (e.g. ad libitum): demineralized water; ad libitum
- Acclimation period: 18 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%):52-58
- Photoperiod (hrs dark / hrs light): 12/12 - Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent no treatment
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): 100 % a.i.
- Duration of treatment / exposure:
- single exposure and observation 0.5, 1 and 2 h after application and once daily afterwards until day 16
- Observation period (in vivo):
- 16 days
- Number of animals or in vitro replicates:
- 3
- Details on study design:
- SCORING SYSTEM: According to OECD test guideline 405
TOOL USED TO ASSESS SCORE: hand-slit lamp / biomicroscope / fluorescein: not specified - Irritation parameter:
- chemosis score
- Basis:
- animal: #2 #3
- Time point:
- 24/48/72 h
- Score:
- 3
- Max. score:
- 4
- Reversibility:
- fully reversible within: 16 days
- Irritation parameter:
- chemosis score
- Basis:
- animal #1
- Time point:
- 24/48/72 h
- Score:
- 2.67
- Max. score:
- 4
- Reversibility:
- fully reversible within: 16 days
- Irritation parameter:
- conjunctivae score
- Basis:
- animal: #2 #3
- Time point:
- 24/48/72 h
- Score:
- 2.67
- Max. score:
- 3
- Reversibility:
- fully reversible within: 16 days
- Irritation parameter:
- conjunctivae score
- Basis:
- animal #1
- Time point:
- 24/48/72 h
- Score:
- 1.3
- Max. score:
- 3
- Reversibility:
- fully reversible within: 16 days
- Irritation parameter:
- iris score
- Basis:
- animal: #1 - #3
- Time point:
- 24/48/72 h
- Score:
- 1
- Max. score:
- 2
- Reversibility:
- fully reversible within: 16 days
- Irritation parameter:
- cornea opacity score
- Basis:
- animal: #2 #3
- Time point:
- 24/48/72 h
- Score:
- 1
- Max. score:
- 4
- Reversibility:
- fully reversible within: 16 days
- Irritation parameter:
- cornea opacity score
- Basis:
- animal #1
- Time point:
- 24/48/72 h
- Score:
- 1.67
- Max. score:
- 4
- Reversibility:
- fully reversible within: 16 days
- Interpretation of results:
- Category 2 (irritating to eyes) based on GHS criteria
- Conclusions:
- On the whole, a single application of Trioxabicyclooctan into the conjunctival sac of the rabbit eye provoked severe irritation which lasted for several days after application and which only very slowly normalized over a period of up to 16 days.
Although the rabbit eye reacts more sensitively than the human eye due to anatomical and physiological. differences, similar effects have to be expected on the human eye. Therefore, inadvertent contact of the human eye with this substance should be strictly avoided. - Executive summary:
In a primary eye irritation study according to OECD test guideline 405 (1987), 100 µL Trioxabicyclooctan (100 % a.i.) were instilled into the conjunctival sac of one eye of 3 young adult New Zealand White rabbits. Animals then were observed for 16 days. Irritation was scored by the method recommended by the test guideline.
After application of 100 µL of the test sample into the conjunctival sac, there were slight reactions in the cornea and initially strong changes in the conjunctivae, which are completely reversible within 16 days. According to Regulation (EU) No. 1272/2008 (CLP) the test item is classified as Category 2 (irritating to eyes) based on the results obtained under the conditions described.
Test substance/ Calculation for EEC classification | ||||
Location (finding) | Time point after end of exposure time | 690M | 702F | 704F |
Cornea (opacity | 24 h | 1.0 | 1.0 | 1.0 |
48 h | 2.0 | 1.0 | 1.0 | |
72 h | 2.0 | 1.0 | 1.0 | |
Individual mean values |
| 1.67 | 1.0 | 1.0 |
| ||||
iris | 24 h | 1.0 | 1.0 | 1.0 |
48 h | 1.0 | 1.0 | 1.0 | |
72 h | 1.0 | 1.0 | 1.0 | |
Individual mean values |
| 1.0 | 1.0 | 1.0 |
| ||||
Conjunctivae (reddening) | 24 h | 2.0 | 3.0 | 3.0 |
48 h | 3.0 | 3.0 | 3.0 | |
72 h | 3.0 | 3.0 | 3.0 | |
Individual mean values |
| 2.67 | 3.0 | 3.0 |
| ||||
Conjunctivae (swelling) | 24 h | 2.0 | 3.0 | 3.0 |
48 h | 1.0 | 3.0 | 3.0 | |
72 h | 1.0 | 2.0 | 2.0 | |
Individual mean values |
| 1.3 | 2.67 | 2.67 |
| ||||
- Reason / purpose for cross-reference:
- reference to other study
Reference
- Endpoint:
- skin irritation: in vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1995-09-08 to 1996-01-09
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
- Version / remarks:
- 31. July 1992
- GLP compliance:
- yes
- Species:
- rabbit
- Strain:
- New Zealand White
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Schriever
- Age at study initiation: adults
- Weight at study initiation: males: 2.7 - 2.8 kg; females: 2.6 - 2.8 kg
- Housing: individually in conventional metal cages
- Diet (e.g. ad libitum): pell. Altromin® K ad libitum
- Water (e.g. ad libitum): demineralized water ad libitum
- Acclimation period: > 14 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 64-70
- Photoperiod (hrs dark / hrs light): 12/12
- Type of coverage:
- semiocclusive
- Preparation of test site:
- shaved
- Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent no treatment
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL
- Concentration (if solution): 100% a.i.
- Duration of treatment / exposure:
- 4 h
- Observation period:
- 7 days
- Number of animals:
- 4
- Details on study design:
- TEST SITE
- Area of exposure: 6 cm² each site of the spine
- Type of wrap if used: Both sites (treated and untreated) were covered with a piece of gauze (semi-occlusive) fixed on the skin with Leukoflex®.
REMOVAL OF TEST SUBSTANCE
- Washing (if done): After exposure the gauze was removed and the treated and untreated skin areas were wiped carefully with lukewarm tap water and cotton wool.
- Time after start of exposure: 4 h
OBSERVATION TIME POINTS
(indicate if minutes, hours or days): Clinical observations were performed 30-60 minutes and 24 hours after end of exposure and thereafter daily until day 7 of the test and findings were recorded. Deviating from the method referred to in the study protocol the interval between daily clinical observations between days 4-5 for animal no. 548 and days 2-3 for the other animals was only 16 hours instead of 24 hours. This deviation is not considered to have influenced the result of the study since the animals were without findings on the respective days. Body weights were recorded on day 1 and day 7 of the test. Necropsy was not performed.
SCORING SYSTEM: According to guideline - Irritation parameter:
- edema score
- Basis:
- animal: #1 - #4
- Time point:
- 24/48/72 h
- Score:
- 0
- Max. score:
- 4
- Reversibility:
- other: not applicable
- Irritation parameter:
- erythema score
- Basis:
- animal: #1 - #4
- Time point:
- 24/48/72 h
- Score:
- 0
- Max. score:
- 4
- Reversibility:
- other: not applicable
- Irritant / corrosive response data:
- The test item provoked on the intact skin of the rabbit after single application only very slight to slight reddening in two males and one female animal only on the appl!ication day. No other findings were observed during the observation period of 7 days.
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- According to the system of evaluation recommended by EEC (1 ), the mean values of findings relevant for classification as ".irritant" (reddening· and scab formation, swelling) at the time points 24, 48 and 72 hours after application were 0. Also based to the classification criteria of Regulation (EU) No. 1272/2008 (CLP) the test item does not need to be classified with respect to skin irritation.
- Executive summary:
In a primary dermal irritation study according to Annex to EEC directive 92/69/EWG, dated 31 Jul. 1992, for the 17th adaption of the EEC directive 67/548/EWG, 8.4. Acute Toxicity (Skin lrritation), young adult New Zealand White rabbits (4 animals) were dermally exposed to 500 µL of Trioxabicyclooctan (100 % a.i.) for 4 hours to 6.5 cm² skin. Animals then were observed for 7 days. Irritation was scored by the method recommended by the guideline.
Trioxabicyclooctan provoked on the intact skin of the rabbit after single application only very slight to slight reddening in two males and one female animal only on the application day. No other findings were observed during the observation period of 7 days. According to the system of evaluation recommended by EEC (1 ), the mean values of findings relevant for classification as ".irritant" (reddening· and scab formation, swelling) at the time points 24, 48 and 72 hours after application were 0.
Based on the test results described above, the test item is not considered to be irritating to the skin according to Regulation (EU) No. 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).
- Reason / purpose for cross-reference:
- reference to other study
Reference
- Endpoint:
- acute toxicity: dermal
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2002-02-18 to 2002-06-21
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 402 (Acute Dermal Toxicity)
- Version / remarks:
- 24 February 1987
- GLP compliance:
- yes
- Test type:
- standard acute method
- Limit test:
- yes
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- Shoe: WIST (SPF)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Tierzucht Schönwalde GmbH, Schönwalde, Germany
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: not specified
- Weight at study initiation: males: 262-265 g; females: 198-209 g
- Housing: individually in conventional cages
- Diet (e.g. ad libitum): pell. Ssniff M-H ad libitum 24 hours per day
- Water (e.g. ad libitum): demineralized, acidified water, pH 2-3 ad libitum
- Acclimation period: at least 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 52-62
- Photoperiod (hrs dark / hrs light): 12/12
- Type of coverage:
- semiocclusive
- Vehicle:
- physiological saline
- Details on dermal exposure:
- TEST SITE
- Type of wrap if used: patches
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): dermal administration of the substance solution at a dose of 2000 mg/kg using an administration volume of 0.53 - 0.72 mL.
- Concentration (if solution): 750 mg/mL - Duration of exposure:
- 24 h
- Doses:
- 2000 mg/kg bw
- No. of animals per sex per dose:
- 3
- Control animals:
- no
- Details on study design:
- - Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observations were performed 30 min, 1 hour, 3 hours and after > 6 hours after administration and then once daily until day 14. On day 14 a complete post mortem examination was performed. The application sites were evaluated 1, 24, 48 and 72 hours after removal of the patches. Additionally, in the three male animals the application site was also evaluated on day 6 of the study.
- Necropsy of survivors performed: yes
- Clinical signs including body weight
- Other examinations performed: clinical signs, body weight, gross pathology - Key result
- Sex:
- male/female
- Dose descriptor:
- LD50
- Effect level:
- > 2 000 mg/kg bw
- Based on:
- test mat.
- Clinical signs:
- other:
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- According to the system of evaluation recommended for EU, the mean values of findings relevant for classification at the time-points 1, 24, 48 and 72 hours after administration were 0 for swelling, reddening and scab formation.
- Executive summary:
In an acute dermal toxicity study according to OECD test guideline 402, groups of young adult Wistar rats (3/sex) were dermally exposed to Trioxabicyclooctan (100 % a.i) in 0.9% NaCl (750 mg/mL) for 24 hours at dose of 2000 mg/kg bw. Animals then were observed for 14 days.
Dermal LD50 Females/Males > 2000 mg/kg bw
Trioxabicyclooctan is of low to moderate Toxicity based on LD50 values in males and females. Based on these results the test item does not need to be classified according to Regulation (EU) No. 1272/2008 (CLP).
- Reason / purpose for cross-reference:
- reference to other study
Reference
- Endpoint:
- acute toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- June 1988
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 401 (Acute Oral Toxicity)
- Version / remarks:
- February 1987
- GLP compliance:
- yes
- Test type:
- standard acute method
- Limit test:
- yes
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- Mol:WIST
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Mollegaard Breeding Centre Ltd, Ejby, DK-4623 Ll. Skensved.
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 144-151 g
- Fasting period before study: 18 h
- Housing: The rats were individually ear-tagged and kept in Macrolone cages Type III (42 x 26 x 15 cm) 2 or 3 to a cage,· males and females separated. The bedding was softwood sawdust "Spanvall' Special White 11 from Spanvall Ltd, Jorlose, DK-4490 Jerslev.
- Diet (e.g. ad libitum): ad libitum, complete rodent diet ."Altromin 1314" from Chr. Petersen Ltd, DK-4100 Ringsted
- Water (e.g. ad libitum): tap water acidified with hydrochloric acid to pH 2.5, ad libitum
- Acclimation period: None
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21+/- 2
- Humidity (%): 55+/-15
- Air changes (per hr): 6
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on oral exposure:
- VEHICLE
- Concentration in vehicle: 200 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw
- Doses:
- 2000 mg/kg bw
- No. of animals per sex per dose:
- 5
- Control animals:
- no
- Details on study design:
- - Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observations: 1, 3 and 6 hours after administration and thereafter daily; weighing on day 0, 7, 14
- Necropsy of survivors performed: yes
- Clinical signs including body weight
- Other examinations performed: clinical signs, body weight - Key result
- Sex:
- male/female
- Dose descriptor:
- LD50
- Effect level:
- > 2 000 mg/kg bw
- Based on:
- test mat.
- Clinical signs:
- other:
- Interpretation of results:
- Category 5 based on GHS criteria
- Conclusions:
- Since no rats died of the treatment the oral LD50 must exceed 2000 mg ",4,4-dimethyl-3,5;8-trioxabicyclo(5.1.0)octane"/kg b. wt.. According to Regulation (EU) No. 1727/2008 (CLP) the substance does not need to be classified with respect to acute oral toxicity.
- Executive summary:
In an acute oral toxicity study according to OECD test guideline 401 (1987), groups of fasted, Wistar rats (5/sex) were given a single oral dose of 4,4-dimethyl-3,5;8-trioxabicyclo(5.1.0)octane in water at a dose of 2000 mg/kg bw and observed for 14 days.
Oral LD50 Females/Males > 2000 mg/kg bw
After single oral administration of 4,4-dimethyl-3,5;8-trioxabicyclo(5.1.0)octane at 2000 mg/kg bw to male and female rats (5/sex/group) sedation and piloerection were found as clinical signs. At autopsy, no gross pathological organ changes were observed.
4,4-dimethyl-3,5;8-trioxabicyclo(5.1.0)octane is of LOW Toxicity based on the LD50 in female and male Wistar rats, thus, 4,4-dimethyl-3,5;8-trioxabicyclo(5.1.0)octane is not classified for acute oral toxicity according to Regulation (EU) No. 1272/2008 (CLP).
Data source
Reference
- Reference Type:
- other: expert statement
- Title:
- Unnamed
- Year:
- 2 002
- Report date:
- 2002
Materials and methods
- Objective of study:
- absorption
Test guideline
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- expert statement based on physico-chemical and toxicological data according to REACH Guidance R.7
- GLP compliance:
- no
Test material
- Reference substance name:
- 4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane
- EC Number:
- 421-750-9
- EC Name:
- 4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane
- Cas Number:
- 57280-22-5
- Molecular formula:
- C7H12O3
- IUPAC Name:
- 4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane
Constituent 1
Results and discussion
Toxicokinetic / pharmacokinetic studies
- Details on absorption:
- At >1000 g/L at 20°C, ZK 39294 is readily soluble in water and not very lipophilic, which is reflected in the octanol/water partition coefficient (log Pow) of -0.07 at 25°C.
Due to this low fat solubility, absorption of the substance through the outer skin and mucous membranes is considered to be rather low.
The substance is expected to be absorbed unchanged from the gastrointestinal tract, however only to a rather small extent. This is indicated by the results of the 28-day study in rats, in which no findings were reported up to 40 mg/kg daily. Even the daily administration of 200 mg/kg over 4 weeks caused only minor impairment of the general condition, which indicates that the limit of general tolerability has been reached.
The test results for mutagenicity are negative. It is assumed that either rapid deactivation of the epoxide function by epoxide hydrolase or the absence of cellular absorption is responsible for this result.
The vapor pressure with a value of 30.5 Pa (20°C) is rather high, Thus, absorption by inhalation must be expected.
Because of its low lipophilicity, the substance will most likely be distributed primarily in the extracellular space and hardly in organs and tissues.
Because of the small molecular size and the high hydrophilicity, predominantly renal excretion is to be expected. In principle, possible metabolic reactions, such as hydroxylation and various conjugations may further increase the water solubility and subsequently stimulating the renal excretion.
No toxicologically significant systemic effects of ZK 39294 after single or multiple oral or single dermal administration were detected. These results indicate either a lack of adverse effects based on low bioavailability or a good systemic availability and tolerability, the latter one can be explained by a rapid deactivation of the epoxide function in first place.
Applicant's summary and conclusion
- Conclusions:
- Treatment with the test item does not result in a major impairment of the general condition in the available toxicological data. These results in correlation with its physico-chemical properties rather indicate a reduced bioavailability via the gastrointestinal tract and the skin. However, a rapid metabolism due to its hydrophilic structure, i.e. the epoxid moiety can also not be excluded. Based on the high vapour pressure also absorption via inhalation may be possible.
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