2H-1-Benzopyran-2-methanol, alpha,alpha'-[[(phenylmethyl)imino]bis(methylene)]bis[6-fluoro-3,4-dihydro-, (alphaR,alpha'R,2R,2'S)-rel-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-06-07 to 2005-07-04

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

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:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

: Insufficient number of strains tested

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

: Insufficient number of strains tested

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2H-1-Benzopyran-2-methanol, alpha,alpha'-[[(phenylmethyl)imino]bis(methylene)]bis[6-fluoro-3,4-dihydro-, (alphaR,alpha'R,2R,2'S)-rel-

EC Number:

618-043-4

Cas Number:

876666-07-8

Molecular formula:

C29H31F2NO4

IUPAC Name:

2H-1-Benzopyran-2-methanol, alpha,alpha'-[[(phenylmethyl)imino]bis(methylene)]bis[6-fluoro-3,4-dihydro-, (alphaR,alpha'R,2R,2'S)-rel-

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): JNJ-17303806-AAA (T001586)
- Physical state: solid, powder
- Appearance: white, beige

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: RT001586G1B121
- Expiration date of the lot/batch: 31-12-5005
- Purity test date: no data (purity = 100%)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: Not indicated by the sponsor
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data

Method

Target gene:

histidine locus

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: The strain is rfa-; uvrB- and contains the R-factor plasmid pKM 101 which carries the umu DC analogous genes.

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: The strain is rfa-; uvrB- and contains the R-factor plasmid pKM 101 which carries the umu DC analogous genes.

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: The strain contains the multicopy plasmid pAQ1 carrying the hisG428 mutation and a tetracycline resistance gene. The strain is also rfa-; uvrB- and contains the R-factor plasmid pKM 101 which carries the umu DC analogous genes.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and beta-naphthoflavone induced rat liver microsomal fraction (S9)

Test concentrations with justification for top dose:

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

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.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

With metabolic activation (S9): for all strains, 2.5 µg/plate for strains TA98 and TA100 and 10 µg/plate for TA102

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

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

Remarks:

Without metabolic activation (S9): for TA98 at 10 µg/plate

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation (S9): for TA100 at 10 µg/plate

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation (S9): for TA102 at 4 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Experiment I: in agar (plate incorporation)
- In the plate incorporation assay, the following materials were mixed in a test tube and poured onto the selective agar plates: 100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control); 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation); 100 µL bacteria suspension (test system, pre-culture of the strains); and 2000 µL overlay agar.
- Experiment II and IIa: preincubation
- In the pre-incubation assay, 50 µL test solution, solvent or 100 µL positive control, 500 µL S9 mix/S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated 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 from both assays were incubated upside down for at least 48 hours at 37 °C in the dark.

DURATION
- Preincubation period: 1 hour (experiment II)
- Exposure duration: at least 48 hours
- Selection time: 48h (simultaneous with exposure)

SELECTION AGENT (mutation assays):
- tryptophan (E. coli) and histidine (S. typhimurium)

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants

OTHER:
- The stability of the positive control substances in solution was unknown, but a mutagenic response in the expected range is sufficient evidence of biological stability.

Evaluation criteria:

A test substance was considered a mutagen if:
1) 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 was observed;
2) a dose-dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration; and
3) an increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and solvent controls such an increase was not considered biologically relevant.

Statistics:

A statistical analysis of the data was not required.

Results and discussion

Test results

Species / strain:

other: TA98, TA100 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Experiments I, II, and IIa

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see details in section "Additional information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: Precipitation of the test substance in the overlay agar was observed in Experiment I without metabolic activation from 1000 µg/plate up to 5000 µg/plate and in Experiment II with and without metabolic activation from 1000 µg/plate up to 5000 µg/plate.

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 toxic effects were observed in the presence of metabolic activation at the concentration of 1000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA:
-In Experiment I, the data in the negative and solvent control of strain TA 102 were slightly above the historical control range. Since this deviation was rather small, this effect was considered to be based upon biologically irrelevant fluctuations in the number of colonies.

Appropriate mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In Experiment I, toxic effects were observed for all strains at 333-5000 µg/plate with metabolic activation.
- In Experiment II, toxic effects were observed for all strains at 333-1000 µg/plate with metabolic activation and at 1000-5000 µg/plate without metabolic activation for strain TA102 only.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

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

The test substance did not induce gene mutations by base pair changes or frameshifts in Salmonella typhimurium strains TA98, TA100 or TA102 either with or without S9 metabolic activation.