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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2007-07-23 to 2007-08-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Well documented GLP study performed in accordance with the OECD guideline N°474, Commission Directive 2000/32/EC, Annex 4C, dated May 19, 2000, and ICH-Harmonised Tripartites guidelines S2A and S2B, with minor deviations which do not impact the validity of the study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Remarks:

but data lacking about: some endpoints not recorded for toxic reactions during pre-experiment

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, Annex 4C, dated May 19, 2000.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ICH-Harmonised Tripartite Guideline S2A (CPMP/ICH/141/95), dated April 1996

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH-Harmonised Tripartite Guideline S2B (CPMP/ICH/174/95), dated July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

700-867-1

Molecular formula:

C16H16BrNO3.HCl

IUPAC Name:

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

Test material form:

solid

Details on test material:

- Name of test material (as cited in study reports): T002102
- Physical state: solid
- Appearance: white (SDS)

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH D-33178 Borchen
- Age at study initiation: 7-8 weeks
- Weight at study initiation: males mean value 37.3 g (SD ± 1.4 g) ; females mean value 31.1 g (SD ± 1.7 g)
- Animals were distributed into the test groups at random and identified by cage number.
- Fasting period before study: no data
- Housing: single housing in Makrolon Type I cages, with wire mesh top
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum of five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 30-84%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: 0.9 % NaCl
- Justification for choice of solvent/vehicle: chosen to its relative non-toxicity for the animals
- Concentration of test material in vehicle: 10 mL/kg bw

Details on exposure:

On the day of the experiment, the test item was formulated in 0.9 % NaCl. All animals received a single standard volume of 10 mL/kg body weight orally.

Duration of treatment / exposure:

one single administration

Frequency of treatment:

one single administration

Post exposure period:

Three adequately spaced dose levels spaced by a factor of 2 were administered, and samples were collected at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

24 h preparation interval for the female animals

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

24 h preparation interval for the male and female animals

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

24 h preparation interval for the male and female animals, and 48 h preparation interval for the female animals

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

24 h preparation interval for the male animals, and 48 h preparation interval for the male animals

No. of animals per sex per dose:

Twelve animals, six males and six females, were treated per dose group and sampling time (except highest dose: 12 animals per sex, 6 per time point).

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control(s): no
- Route of administration: orally, once
- Doses / concentrations: dissolved in deionised water, administered at 10 mL/kg bw (dose 40 mg/kg bw)

Examinations

Tissues and cell types examined:

Bone marrow -at least 2000 polychromatic erythrocytes (PCEs) per animal were scored 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 adequately spaced dose levels spaced by a factor of 2 were administered, and samples were collected 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): At the beginning of the treatment, the animals (including the controls) were weighed and the individual volume to be initiated was adjusted to the animal 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 (24 or 48h). The animals of all dose groups were examined for acute toxic symptoms at intervals of around 1h, 2-4h, 6h and 24h after administration of the test item. Sampling of bone marrow was done 24 and 48 hours after treatment.

DETAILS OF SLIDE PREPARATION: The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Merck, D-64293 Darmstadt)/ Giemsa (Merck, D-64293 Darmstadt). Cover slips were mounted with Eukitt (Kindler, D-79110 Freiburg). 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 sampled and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
Twelve animals (6F+6M) per test groups 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 number of micronucleated polychromatic erythrocytes in a single dose group.
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:

The non parametric Mann-Whitney test was used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that T2102 did not have any cytotoxic properties in the bone marrow.
The highest dose (500 mg/kg b.w. for the female animals and 1000 mg/kg b.w. for the male animals) was estimated by pre-experiments to be suitable.
40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Any other information on results incl. tables

Pre-experiment results:

In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 100 mg/kg b.w. T2102 formulated in 0.9 % NaCl. The volume administered was 10 mL/kg b.w.. The animals treated with 100 mg/kg b.w. expressed no toxic reactions.

In a second pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 1000 mg/kg b.w. T2102 formulated in 0.9 % NaCl. The volume administered was 10 mL/kg b.w. A female died 2 -4 hours after administration of the test item.

In a third pre-experiment 2 males received orally a single dose of 1500 mg/kg b.w and 2 females received orally a single dose of 750 mg/kg b.w. T2102 formulated in 0.9 % NaCl. The volume administered was 10 mL/kg b.w. One male and one female died during the hour following the oral administration.

In a fourth pre-experiment 2 males received orally a single dose of 1250 mg/kg b.w and 2 females received orally a single dose of 500 mg/kg b.w. T2102 formulated in 0.9 % NaCl. The volume administered was 10 mL/kg b.w.. One male died during the hour following the oral administration.

On the basis of these data 1000 mg/kg b.w. was estimated to be suitable as the highest dose for the male and 500 mg/kg b.w. for the female animals.

Main experiment:

The animals treated with 1000 mg/kg and 500 mg/kg b.w. respectively expressed toxic reactions as shown in the table (left number: males, rigth number:females):

Toxic reactions following administration	1h	2 -4h	6h	24h	48h*
Reduction of spontaneous activity	9/10	10/12	9/8	0/0	0/0
ruffled fur	12/12	12/12	12/12	7/8	0/0
tremor	8/9	7/9	1/4	0/0	0/0

*data from only 6 animals

Summary of micronucleus test results (main experiment, males)

Test group	dose mg/kg b.w.	sampling time (h)	PCEs with micronuclei (%)	range	PCE per 2000 erythrocytes
Vehicle	0	24	0.175	1 -6	1298
Test item	250	24	0.142	1 -4	1138
Test item	500	24	0.167	1 -6	1236
Test item	1000	24	0.092	0 -4	1204
positive control	40	24	3.492	56 -97	1066
Test item	1000	48	0.120	1 -4	1119

Summary of micronucleus test results (main experiment, females)

Test group	dose mg/kg b.w.	sampling time (h)	PCEs with micronuclei (%)	range	PCE per 2000 erythrocytes
Vehicle	0	24	0.108	1 -4	1249
Test item	125	24	0.142	0 -6	1281
Test item	250	24	0.100	1 -4	1224
Test item	500	24	0.108	0 -3	1265
positive control	40	24	2.758	39 -78	1113
Test item	500	48	0.150	1 -6	1139

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 reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. Based on these results and the criteria in the CLP Regulation, the test substance is considered not classified as mutagenic.