2-[(5A,7A)-17-(CYCLOPROPYLMETHYL)-3,6-DIMETHOXY-18,19-DIHYDRO-4,5-EPOXY-6,14-ETHENOMORPHINAN-7-YL]-3,3-DIMETHYLBUTAN-2-OL

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:

2006-05-11 to 2006-05-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Non-GLP study performed according to the OECD Guideline 471 (Bacterial Reverse Mutation Assay) with the following deviations: only three strains are tested. However, an expert statement is added in the field "any other remarks" to justify the fact that no further testing is necessary.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only 3 bacterial strains tested instead of 5

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(2S)-2-[(1S,2R,6S,14R,15R,16R)-3-(cyclopropylmethyl)-11,15-dimethoxy-13-oxa-3-azahexacyclo[13.2.2.1²,⁸.0¹,⁶.0⁶,¹⁴.0⁷,¹²]icosa-7,9,11-trien-16-yl]-3,3-dimethylbutan-2-ol

Cas Number:

16524-65-5

Molecular formula:

C30H43NO4

IUPAC Name:

(2S)-2-[(1S,2R,6S,14R,15R,16R)-3-(cyclopropylmethyl)-11,15-dimethoxy-13-oxa-3-azahexacyclo[13.2.2.1²,⁸.0¹,⁶.0⁶,¹⁴.0⁷,¹²]icosa-7,9,11-trien-16-yl]-3,3-dimethylbutan-2-ol

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): JNJ-18163678-AAA (T002713)
- Physical state: solid (powder)
- Appearance: White powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.: 00477910 RT002713G4A051

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Not indicated by the sponsor

OTHER SPECIFICS:
- Name of test material (as cited in study report): T2713
- Molecular formula (if other than submission substance): C30H43NO4
- Molecular weight (if other than submission substance): 481.68 g/mol
- Substance type: White
- Physical state: Solid
- Analytical purity: 100%
- Other: Chemical name: 6,14-Ethenomorphinan-7-methanol, 17-(cyclopropylmethyl)-a-(1,1-dimethylethyl)-4,5-epoxy-18,19-dihydro-2,6-dimethoxy-amethyl-,(aS,5a,7a)-

Method

Target gene:

The histidine dependent strains were derived from S. typhimurium strain LT2 through a mutation in the histidine locus.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-Naphtoflavone induced rat liver S9 from male Wistar Hanlbm rats

Test concentrations with justification for top dose:

Experiment I (pre-experiment): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without S9-mix
Experiment II: 33, 100, 333, 1000, 2500 and 5000 µg/platewith and without S9-mix

In the pre-experiment, the maximum recommended dose of 5000 µg/plate was chosen as the highest dose level. Based on the results of the pre-experiment, 5000 µg/plate was chosen as the highest dose level for Experiment II.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria. On the day of the experiment, the test item T2713 was dissolved in DMSO (purity > 99 %)

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, without metabolic activation, 10 µg/plate

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine (4-NOPD)

Remarks:

TA98, without metabolic activation, 10 µg/plate

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

TA102, without metabolic activation, 4.0 µL/plate

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

All strains, with metabolic activation,2.5 µg/plate (TA98 and TA100), 10.0 µg/plate (TA102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment I: plate incorporation; Experiment II: preincubation

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours
- Selection time (if incubation with a selection agent): 48 hours (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls three plates were used.

DETERMINATION OF CYTOTOXICITY: To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for experiment I (plate incorporation test).
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

OTHER: The colonies were counted using the Petri Viewer Mk2 with the software program Ames Study Manager. The counter was connected to an IBM AT compatible PC with printer which printed out both the individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to precipitation of the test item some plates were counted manually.

Evaluation criteria:

Evaluation of the results:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) 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:

A statistical analysis of the data is not required

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no 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

Remarks:

minor toxic effects at 5000µg/plate in Experiment I without S9-mix

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

Remarks:

minor toxic effects at 2500µg/plate in Experiment I without S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation occurred at 1000, 2500 and 5000 µg/plate with and without S9, for all strains, in both experiment I and experiment II

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I since no relevant toxic effects were observed six concentrations were tested in experiment II and 5000 µg/plate were chosen as maximum concentration.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

Any other information on results incl. tables

No substantial increase in revertant colony numbers of any of the three tester strains was observed following treatment with T2713 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with and 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.