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 vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-11-13 to 2001-12-13

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 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annexe 4D (dated 2000-05-19)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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: 2001-12-01
- Purity test date: no data
- Purity: 92.9 %

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

Method

Target gene:

Histidine (S. typhimurium strains) and tryptophan (E. coli strains) loci

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-experiment: TA 98 and TA 100: 3, 10, 33, 100, 333, 1000 and 2500 µg/plate (based on solubility findings)
Experiment 1: TA 1535, TA 1537, WP2uvrA: 3, 10, 33, 100, 333 and 1000 µg/plate; TA 98, TA 100: 3, 10, 33, 100, 333, 1000 and 2500 µg/plate. (based on the results of the pre-experiment)
Experiment 2: all strains: 3, 10, 33, 100, 333 and 1000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionized water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

untreated

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation, TA 1535 and TA 100, 10 µg/plate, dissolved in water deionised

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

untreated

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

Without metabolic activation, TA 1537 and TA 98, 10 µg/plate in TA98, 50 µg/plate in TA1537, dissolved in DMSO

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

untreated

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation, WP2 uvrA, 5 µL/plate, dissolved in water deionised

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

untreated

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation, All strains, 2.5 µg/plate in TA strains, 10 µg/plate in WP2 uvrA strain, dissolved in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation (experiment 1), pre-incubation (experiment 2)

EXPERIMENTAL PERFORMANCE:
The following materials were mixed in a test tube and poured onto the minimal agar plates:
- 100 μL of test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
- 500 μL of S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without me tabolic activation)
- 100 μL of bacteria suspension (c.f. test system, pre-culture of the strains)
- 2000 μL overlay agar
In the pre-incubation assay 100 μL test solution, 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and shaken at 37°C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45°C) was added to each tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 h at 37°C in the dark.

DURATION
- Preincubation period: 60 minutes (experiment II)
- Exposure duration: at least 48 h
- Fixation time (start of exposure up to fixation or harvest of cells): 48 h (simultaneously with exposure)

SELECTION AGENT (mutation assays): Histidine (S. typhimurium); Tryptophan (E. coli)

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY:
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA98, TA100 and WP2 uvrA) or thrice (strains TA1535 and TA1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

No statistical evaluation of the data is required.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item occurred up to the highest investigated dose

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item a pre-experiment was performed with strains TA98 and TA100. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as those of Experiment I (plate incorporation test). Due to toxic effects at 2500 and 500 µg/plate, 1000 µg/plate were chosen as maximal concentration for experiment I with strains TA 1535, TA 1537 and WP2 uvrA, as well as for experiment II with all strains.

COMPARISON WITH HISTORICAL CONTROL DATA: In experiment I, without metabolic activation, the number of colonies in the positive control were above the historical control data in strain TA1535. In experiment II, with S9 mix, the number of colonies did not quite reach the lower limit of the historical control data in strain TA1537 in the negative and solvent control. Since these deviations are rather small, these effects are judged to be based upon statistical fluctuations and have no detrimental impact on the outcome of the study.
Appropriate reference substances were used as positive controls. They showed distinct increase of induce revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No visible reduction of the background growth was observed up to the highest concentration.
However, toxic effects were observed in all strains, in the conditions escribed in the table below (see section "any other information on results").

Any other information on results incl. tables

Toxic effects were observed at the following concentrations:

Strains	Experiment I		Experiment II
	-S9	+S9	-S9	+S9
TA1535	/	/	333, 1000	1000
TA1537	1000	1000	333, 1000	/
TA98	1000	/	1000	1000
TA100	1000, 2500	2500	333, 1000	/
WP2 uvrA	/	/	1000	/

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.