L-Leucine, 4-fluoro-N-[(1S)-2,2,2-trifluoro-1-[4'-(methylsulfonyl)[1,1'-biphenyl]-4-yl]ethyl]-, compd. with N-cyclohexylcyclohexanamine (1:1)

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:

02-Jan-2012 to 09-feb-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

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:

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

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:

N-cyclohexylcyclohexanaminium (2S)-4-fluoro-4-methyl-2-{[(1S)-2,2,2-trifluoro-1-{4'-methanesulfonyl-[1,1'-biphenyl]-4-yl}ethyl]amino}pentanoate

EC Number:

700-778-8

Cas Number:

1064076-86-3

Molecular formula:

C21H23F4NO4S.C12H23N

IUPAC Name:

N-cyclohexylcyclohexanaminium (2S)-4-fluoro-4-methyl-2-{[(1S)-2,2,2-trifluoro-1-{4'-methanesulfonyl-[1,1'-biphenyl]-4-yl}ethyl]amino}pentanoate

Details on test material:

- Name of test material (as cited in study report): Biphenyl DCHA Salt
- Substance type: White powder
- Physical state: Solid
- Storage condition of test material: At room temperature protected from light in desiccator

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA97 and TA98:
Without and with S9-mix: 10, 33, 100, 333 and 1000 µg/plate
Experiment 2:
Without and with S9-mix: 10, 33, 100, 333 and 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
A homogeneous suspension could be obtained in DMSO and DMSO is accepted and approved by authorities and international guidelines

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9

Migrated to IUCLID6: 650 µg/plate in DMSO for TA100

Controls 2

Positive control substance:

2-nitrofluorene

Remarks:

without S9

Migrated to IUCLID6: 10 µg/plate in DMSO for TA98

Controls 3

Positive control substance:

other: ICR-191: 1 µg/plate in DMSO for TA97

Controls 4

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9

Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA

Controls 5

Positive control substance:

sodium azide

Remarks:

without S9

Migrated to IUCLID6: 5 µg/plate in saline for TA1535

Controls 6

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA97 and TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA97 and TA100) or more or a three-fold (TA1535, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Results and discussion

Test results

Test results 1

Species / strain:

other: TA1535, TA97, TA98, TA100

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
DRF: Slight precipitation was observed at dose levels of 1000 µg/plate and above
Mutation experiment 1: Slight precipitation was observed at the dose level of 1000 µg/plate
Mutation experiment 2: Slight precipitation was observed at the dose level of 1000 µg/plate, except in the tester strains TA1535, TA98 and WP2uvrA (absence of S9-mix) where no precipitation of Biphenyl DCHA Salt was observed

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Toxicity was only observed at tester strain TA97 in the absence of S9-mix in the first experiment.

Any other information on results incl. tables

In the second experiment, no dose level with precipitate or toxicity was tested in the tester strains TA1535, TA98 and WP₂uvrA in the absence of S9-mix. Since in the dose range finding test, precipitate was observed at 1000 µg/plate, this dose level was selected as top dose in the mutation experiments. Although the dose level of 1000 µg/plate showed no toxicity or precipitate in these three tester strains, clear negative responses over the entire dose range was observed. Furthermore in the first experiment and the other tester strains in experiment 2, no increase in the number of revertants was observed up to the precipitating dose level of 1000µg/plate, therefore the testing of a top dose level of 3330 µg/plate in these tester strains in the second experiment would have given limited additional information.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Biphenyl DCHA Salt is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Executive summary:

Biphenyl DCHA Salt did not induce a significant dose-related increase in the number of revertant (His⁺) colonies in each of the four tester strains (TA1535, TA97, TA98 and TA100) and in the number of revertant (Trp⁺) colonies in tester strain WP₂uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.