Cyclopenta[c]cyclopropa[g][1,6]diazacyclotetradecine-12a(1H)-carboxylic acid, 2,3,3a,4,5,6,7,8,9,11a,12,13,14,14a-tetradecahydro-2-[[7-methoxy-8-methyl-2-[4-(1-methylethyl)-2-thiazolyl]-4-quinolinyl]oxy]-5-methyl-4,14-dioxo-, (2R,3aR,10Z,11aS,12aR,14aR)-

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:

From 2011-08-01 to 2011-08-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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: the OECD principles of Good Laboratory Practice (1998) and the Application of the Principles of GLP to In Vitro Studies (2004)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: FDA Toxicological Principles for the Safety Assessment of Direct Food Ingredients. Section IV.C1.a:Bacterial Reverse Mutation Test. “Redbook 2000”. U.S. FDA Washington DC 2000

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Ministry of Health and Welfare (MHW), Japanese Guidelines for Nonclinical Studies of Drugs Manual (1995), Section 5. Reverse Mutation Test in Bacteria.

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human use

Version / remarks:

Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. S2A document recommended for adoption at step 4 of the ICH process on July 19, 1995. Federal Register 61:18198-18202, April 24, 1996.

Deviations:

no

Test guideline 6

Qualifier:

according to guideline

Guideline:

other: International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human use.

Version / remarks:

Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. S2B document recommended for adoption at step 4 of the ICH process on July 16, 1997. Federal Register 62:16026-16030, November 21, 1997

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:

(1R,4R,6S,7Z,15R,17R)-17-({7-methoxy-8-methyl-2-[4-(propan-2-yl)-1,3-thiazol-2-yl]quinolin-4-yl}oxy)-13-methyl-2,14-dioxo-3,13-diazatricyclo[13.3.0.0^{4,6}]octadec-7-ene-4-carboxylic acid

EC Number:

618-844-9

Cas Number:

923604-58-4

Molecular formula:

C35H42N4O6S

IUPAC Name:

(1R,4R,6S,7Z,15R,17R)-17-({7-methoxy-8-methyl-2-[4-(propan-2-yl)-1,3-thiazol-2-yl]quinolin-4-yl}oxy)-13-methyl-2,14-dioxo-3,13-diazatricyclo[13.3.0.0^{4,6}]octadec-7-ene-4-carboxylic acid

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): JNJ-38940642-AAA (T003010)
- Physical state: solid
- Appearance: white powder

Specific details on test material used for the study:

Batch number: RT003010PFA121
Conversion factor: 1.04
Purity: 96%
Storage conditions: At room temperature in a closed and labelled container
Retest date: 2012-12-01

Method

Target gene:

histidine locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced S9-mix from male Sprague-Dawley rat liver

Test concentrations with justification for top dose:

Based on the solubility findings, 5000 μg/plate was selected as the maximum final concentration.

First mutation experiment: 0, 78.13, 156.25, 312.5, 625, 1250, 2500, 5000 μg/plate (with and without S9)
Second mutation experiment: 0, 6.86, 20.58, 61.73, 185.19, 555.56, 1666.67, 5000 µg/plate (with and without S9)

Vehicle / solvent:

According to the solubility determination step of the study, the test substance was found to be soluble in DMSO, and then was used as the concurrent vehicle control for the test item.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9, at 5 µg/plate for TA98

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9, at 1 µg/plate for TA1535 and TA 100

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9, at 50 µg/plate for TA1537

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9, at 5 µg/plate for TA102

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9, at 2.5 µg/plate for TA98, TA100, TA1535 and TA1537 and at 7.5 µg/plate for TA102

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

EXPERIMENTAL PERFORMANCE:
The following solutions were added to 2 ml histidine-biotin supplemented top agar:
- 0.1 ml of an overnight bacterial culture of the tester strain
- 0.1 ml of a dilution of the test item, vehicle control or positive control
- 0.5 ml of S9-mix for the activation portion or 0.5 ml phosphate buffer for the non-activation portion.
The content of the tube was then mixed and poured onto minimal glucose agar plates. The plates were incubated in the dark at 37°C for 48 to 72 hours.

DURATION
- Exposure duration: 48-72 hours
- Selection time: 48-72h (simultaneous with exposure)

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: All concentration levels of JNJ- 38970191-AAA, vehicle controls and positive controls were plated in triplicate.

Approximate No. of Bacteria Assayed/Dose: 10^9

DETERMINATION OF CYTOTOXICITY:
reduced number of revertants and/or thinning of the bacterial background lawn

Rationale for test conditions:

The Ames reverse mutation test has been shown to be a sensitive, rapid and accurate indicator of the mutagenic activity of a wide range of chemical classes (Ames et al., 1973a, 1973b; McCann et al., 1975a, 1975b).

Evaluation criteria:

CRITERIA FOR A POSITIVE RESPONSE:
According to Brusick (1980), a test item is considered positive (mutagenic) if all of the following criteria are met:
- The test substance produces at least a two-fold increase in the mean number of revertants with one of the strains TA98, TA102 or TA100, or a three-fold increase in the mean number of revertants with one of the strains TA1535 or TA1537 at one or more concentration levels in comparison to the mean concurrent vehicle control value;
- a concentration-related effect is observed;
- these effects can be reproduced in an additional experiment.

CRITERIA FOR A NEGATIVE RESPONSE:
If the test item does not produce (1) a concentration-dependent increase in the number of revertant colonies and (2) a two-fold increase in the mean number of revertants with one of the strains TA98, TA102 or TA100, or a three-fold increase in the mean number of revertants with one of the strains TA1535 and TA1537 in comparison to the mean concurrent vehicle control value, it will be considered as negative (non-mutagenic) in this test system under the current test conditions. Furthermore, the negative response should be reproducible.

CRITERIA FOR AN EQUIVOCAL RESPONSE
When criteria for a clear positive or a clear negative are not satisfied (e.g., concentration-dependent increase that fails to reach two-fold in the mean number of revertants for the strains TA98, TA102 or TA100, or three-fold in the mean number of revertants for the strains TA1535 and TA1537; or biological significant increase in the reversion rate that does not appear to be concentration-dependent), more tests may be required, in order to evaluate the mutagenic potential of the test item. If the test item produces a positive response in a single test that cannot be reproduced in additional testing, the initial positive data will be discounted. If still the test item cannot be judged to be positive or negative, the results may be classified as equivocal.

Statistics:

No data

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation:

RANGE-FINDING/SCREENING STUDIES: No dose range finding study.

HISTORICAL CONTROL DATA:
- Positive historical control data: The positive controls induced a biologically significant increase in th e mean number of revertant colonies in comparison to the mean concurrent vehicle control value, indicating the capacity of the test system to identify mutagens.
- Negative (solvent/vehicle) historical control data: The mean number of spontaneous and vehicle control revertant colonies in the absence and in the presence of S9-mix fell within the range of the laboratory historical data

ADDITIONAL INFORMATION ON CYTOTXICITY:
First mutation experiment: Bacteriotoxic effects, visualised by a reduction in the number of revertant colonies were observed with the strain TA1537 in the absence of S9-mix from 1250 μg/plate onwards. From 625 μg/plate onwards at the start and from 2500 μg/plate onwards at the end of treatment, a concentration-related increase in milky suspension was observed with the five strains in the absence and/or in the presence of S9-mix.
Second mutation experiment: Bacteriotoxic effects, visualised by a reduction in the number of revertant colonies were observed with the strain TA98 in the absence of S9-mix and with the strain TA1537 in the absence and in the presence of S9-mix from 1666.67 μg/plate onwards. From 555.56 μg/plate onwards at the start and from 1666.67 μg/plate onwards at the end of treatment, a concentration-related increase in milky suspension was observed with the five strains in the absence and/or in the presence of S9-mix.

OTHERS:
Sterility checks and bacterial titre of the five strains were according to the criteria.

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Solubility determination

The test substance was found to be insoluble in water at 50 mg/ml. In DMSO, the test item was soluble at 50 mg/ml (= 5000 μg/plate) after 10 minutes at 37ºC, but a strong milky suspension was obtained upon mixing with water. Sequential dilutions from this maximum concentration resulted in moderate to slight milky suspension upon mixing with water, until at 3.125 mg/ml (= 312.5 μg/plate) a clear solution was obtained upon mixing with water. Based on these solubility findings, DMSO was selected as vehicle and 5000 μg/plate was selected as the maximum final concentration for the first mutation experiment (plate incorporation method).

Concentration analysis

Concentration analysis of the vehicle control (DMSO) revealed that there were no traces of the test substance present and that the concentration of test item in the stock formulation fell within the predefined acceptance criteria (85% - 115%). This stock formulation was used to prepare further dilutions.

Applicant's summary and conclusion

Conclusions:

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

Based on this study, it is concluded that the test substance has no mutagenic properties towards the various Salmonella typhimurium strains in the absence and in the presence of S9-mix under the test conditions described in this report, up to the maximum test concentration of 5000 μg/plate.