6H-Benzofuro[3a,3,2-ef][2]benzazepin-6-one, 1-bromo-4a,5,9,10,11,12-hexahydro-3-methoxy-11-methyl-, (4aR,8aR)-rel-

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-01-09 to 2002-04-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Test conducted at 20-80% humidity rather than 30-70% as described in the study plan. This deviation does not affect the validity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, Annex 4C (dated 2000-05-19)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

439-750-2

EC Name:

-

Cas Number:

183487-56-1

Molecular formula:

Hill formula: C17H19BrClNO3 CAS formula: C17H19BrClNO3

IUPAC Name:

(4aR*,8aR*)-1-bromo-4a,5,9,10,11,12- hexahydro-3-methoxy-11-methyl-6H-ben zofuro[3a,3,2-ef][2]benzazepin-6-one hydrochloride

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: RT002113 G3A231
- Expiration date of the lot/batch: 2002-04-08
- Purity: 79.1 - 92.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: not indicated

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

Polychromatic erythrocytes (PCE) in the bone marrow of the mouse are the cell population of choice for mammalian cells in vivo. PCEs are newly formed red blood cells and are easily identifiable by their staining properties. These cells have the advantage that the micronuclei can be readily detected because the nucleus is extruded from the erythroblast after the last cell division.
In addition, mice are recommended in the OECD Guideline 474.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd., Biotechnology and Animal Breeding Division, CH-4414 Füllinsdorf, Switzerland
- Age at study initiation: 8-10 weeks
- Weight at study initiation: Males mean vale 33.2 g (SD+/- 2.6 g), Females mean vale 26.6 (SD+/-2.3 g)
- Assigned to test groups randomly: yes
- Fasting period before study: 18 h
- Housing: single Makrolon Type I, with wire mesh top
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21+/- 4 C
- Humidity (%): 20-80%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12h light (artificial) / 12h dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Deionized water
- Justification for choice of solvent/vehicle: The vehicle was chosen to its non-toxicity for the animals
- Concentration of test material in vehicle: 18.75, 37.5, 75 mg/kg b.w. (24h preparation interval); 75 mg/kg b.w. (48h preparation interval)
- Amount of vehicle (if gavage or dermal): 10 ml/kg b. w.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test item was disolved in deionized water just prior to administration

Duration of treatment / exposure:

24h (18.75, 37.5, 75 mg/kg b.w.); 48h (75 mg/kg b.w.)

Frequency of treatment:

Once

Post exposure period:

Observations recorded at 1 h, 2-4 h, 6 h, 24 h, 30 h and 48 h. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

18.75 mg/kg bw/day (nominal)

Remarks:

deionized water vehicle

Doses / concentrations 2

Dose / conc.:

37.5 mg/kg bw/day (nominal)

Remarks:

deionized water vehicle

Doses / concentrations 3

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

deionized water vehicle

No. of animals per sex per dose:

6 males/6 females for each dose (24h and 48h), negative control and positive control

Control animals:

yes

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): not specified
- Route of administration: oral
- Doses / concentrations: once, 40 mg/kg b.w. (24h preparation interval)

Examinations

Tissues and cell types examined:

bone marrow tissue - at least 2000 polychromatic eurythrocytes per animal for micronuclei.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items. The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours. The volume to be administered should be compatible with physiological space available.
Three adequate spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Approximately 18 h before treatment the animals received no food but water ad libitum. At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test item, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time. The animals of the highest dose group were examined for acute toxic symptoms at intervals of aroun 1h, 2-4h, 6h, 24h, 30h, and 48h after administration of the test item. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

DETAILS OF SLIDE PREPARATION: Animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 min and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and stained with May-Grunwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 2000 PCEs. The analysis was performed with coded slides.
Ten animals (5 males, 5 females) per test group were evaluated as described.

Evaluation criteria:

A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the nupmber of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

Statistical significane at the fice percent level (P < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Remarks:

all concentrations tested

Toxicity:

yes

Remarks:

animals treated with 75 mg/kg bw expressed toxic reactions; 1 death (male) after 1h; no cytotoxic effects were observed.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 75, 100, 236, 36., 500, 1000 and 2000 mg/kg b.w.
- Solubility: soluble in deinised water
- Clinical signs of toxicity in test animals: Reduction of spontaneous activity, abdominal position, eyelid closure, ruffled fur, tumbling, apathy, cramps and death.
1. in the first pre-experiment, 4 animals (2 males / 2 females) received orally a single dose of 2000 mg/kg bw (volume administered 10 mL/kg bw). The animals expressed the following toxic reactions: reduction of spontaneous acivity, adbominal position, apathy and death.
2. in the second pre-experiment, 4 animals (2 males / 2 females) received orally a single dose of 1000 mg/kg bw (volume administered 10 mL/kg bw). The animals expressed the following toxic reactions: death.
3. in the third pre-experiment, 4 animals (2 males / 2 females) received orally a single dose of 500 mg/kg bw (volume administered 10 mL/kg bw). The animals expressed the following toxic reactions: death.
4. in the fourth pre-experiment, 4 animals (2 males / 2 females) received orally a single dose of 250 mg/kg bw (volume administered 10 mL/kg bw). The animals expressed the following toxic reactions: death.
5. in the fifth pre-experiment, 4 animals (2 males / 2 females) received orally a single dose of 125 mg/kg bw (volume administered 10 mL/kg bw). The animals expressed the following toxic reactions: reduction of spontaneous acivity, adbominal position, eyelid closure, ruffled fur, apathy and death.
6. in the sixth pre-experiment, 4 animals (2 males / 2 females) received orally a single dose of 75 mg/kg bw (volume administered 10 mL/kg bw). The animals expressed the following toxic reactions: reduction of spontaneous acivity, adbominal position, eyelid closure, ruffled fur and apathy. Directly after the application cramps were observed by all animals, which subsided after about 3 minutes.
7. in the seventh pre-experiment, 4 animals (2 males / 2 females) received orally a single dose of 100 mg/kg bw (volume administered 10 mL/kg bw). The animals expressed the following toxic reactions: reduction of spontaneous acivity, adbominal position, eyelid closure, ruffled fur, tumbling, apathy, cramps and death.
8. in the eight pre-experiment, 4 animals (2 males / 2 females) received orally a single dose of 75 mg/kg bw (volume administered 10 mL/kg bw). The animals expressed the following toxic reactions: reduction of spontaneous acivity, adbominal position, eyelid closure, ruffled fur, tumbling, apathyand death.
On the basis of these data 75 mg/kg bw were estimated to be suitable for the main experiment.
- Evidence of cytotoxicity in tissue analyzed: Formation of micronuclei; no cytotoxicity was observed
- Rationale for exposure: no data
- Harvest times: 24 and 48 h (for highest dose level)

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with T002113 were below or near to the value of the vehicle control group.
The following percentages of PCEs with micronuclei were calculated:
a) Vehicle (negative control; 24h): 0.080%
b) test item (18.75 mg/kg b.w.; 24h): 0.110%
c) test item (37.5 mg/kg b.w.; 24h): 0.090%
d) test item (75 mg/kg b.w.; 24h): 0.060%
e) CPA (positive control; 40 mg/kg b.w.; 24h): 1.135%
f) test item (75 mg/kg b.w.; 48h): 0.070%
- Ratio of PCE/NCE (for Micronucleus assay):
a) Vehicle (negative control; 24h): 2000/1554
b) test item (18.75 mg/kg b.w.; 24h): 2000/1656
c) test item (37.5 mg/kg b.w.; 24h): 2000/1736
d) test item (75 mg/kg b.w.; 24h): 2000/1913
e) CPA (positive control; 40 mg/kg b.w.; 24h): 2000/2005
f) test item (75 mg/kg b.w.; 48h): 2000/1648
In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test item were below or near to the value of the vehicle control group.
- Appropriateness of dose levels and route: determined via range-finding study
- Statistical evaluation: There was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The CPA (positive control) showed a statistically significant increase of induced micronucleus frequency after administration.

Any other information on results incl. tables

Cytotoxicity:

The mean number of normochromatic erythrocytes was not increased after treatment with the test item as compared to the mean value of NCEs of the vehicle control, indicating that the test item had no cytotoxic properties in the bone marrow.

Positive control:

40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increaseof induced micronucleus frequency.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
In conclusion, it can be stated that during the study described and under the experimental conditions reoported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, T002113 is considered to be non-mutagenic in this micronucleus assay.