Methyl (1R,3S,4R,5R)-3-amino-5-({2,4,6-tri-O-benzoyl-3-O-[(2S)-1-(benzyloxy)-3-cyclohexyl-1-oxopropan-2-yl]-β-D-galactopyranosyl}oxy)-4-[(2,3,4-tri-O-benzyl-6-deoxy-α -L-galactopyranosyl)oxy]cyclohexanecarboxylate hydrochloride

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:

27 August - 14 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

Methyl (1R,3S,4R,5R)-3-amino-5-({2,4,6-tri-O-benzoyl-3-O-[(2S)-1-(benzyloxy)-3-cyclohexyl-1-oxopropan-2-yl]-β-D-galactopyranosyl}oxy)-4-[(2,3,4-tri-O-benzyl-6-deoxy-α -L-galactopyranosyl)oxy]cyclohexanecarboxylate hydrochloride

EC Number:

945-311-0

Molecular formula:

C78H85NO18 HCl

IUPAC Name:

Methyl (1R,3S,4R,5R)-3-amino-5-({2,4,6-tri-O-benzoyl-3-O-[(2S)-1-(benzyloxy)-3-cyclohexyl-1-oxopropan-2-yl]-β-D-galactopyranosyl}oxy)-4-[(2,3,4-tri-O-benzyl-6-deoxy-α -L-galactopyranosyl)oxy]cyclohexanecarboxylate hydrochloride

Details on test material:

- State of aggregation: white solid powder

Specific details on test material used for the study:

No correction was made for the purity/composition of the test item.
PF-06460259-01 was dissolved in dimethyl sulfoxide (Merck). The stock solution was treated with ultrasonic waves until the test item had completely dissolved.

Method

Target gene:

Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor Base-pair substitutions
R-factor = plasmid pKM101 (increases error-prone DNA repair)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

Dose range finding test: 1.7, 5.4, 17, 52, 162, 512, 1600, 5000 μg/plate
First mutation assay: 17, 52, 164, 512 and 1600 μg/plate
Experiment 2 (with and without metabolic activation): 154, 275, 492, 878 and 1568 μg/plate

Vehicle / solvent:

Dimethyl sulfoxide (SeccoSolv, Merck, Darmstadt, Germany)

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Remarks:

without metabolic activation (-S9-mix)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

With metabolic activation (+S9-mix)

Details on test system and experimental conditions:

Test system: Salmonella typhimurium bacteria and Escherichia coli bacteria
The Salmonella typhimurium strains are regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment (Ref.1). The strain is regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.

Stock cultures of the five strains were stored in liquid nitrogen (-196 C)

Rationale for test conditions:

Recommended test system in international guidelines (e.g. OECD, EC)

Evaluation criteria:

Acceptance of the assay

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at WIL Research Europe.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Data evaluation and statistical procedures

No formal hypothesis testing was done.

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up 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 follow up experiment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: TA1535, TA1537, TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Only at highest dose tested (5000 μg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

First mutation experiment
No increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Second mutation experiment
In the second mutation assay, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.
In strain TA100, fluctuations in the number of revertant colonies above the laboratory historical control data range were observed in the absence of S9-mix at the dose levels of 878 and 1568 µg/plate. However, since the increases were not two-fold (a maximum of 1.4-fold was reached), these increases were not considered to be relevant.

Any other information on results incl. tables

Dose range finding test / first mutation experiment

Precipitate

Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 512 µg/plate and upwards. Precipitation of the test item on the plates was observed at the end of the incubation period at 1600 and 5000 µg/plate in TA100 or WP2uvrA (dose range finding test) and at 512 and 1600 µg/plate in the tester strainsTA1535, TA1537 and TA98 (first mutation experiment).

Toxicity

To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. A reduction of the bacterial background lawn was only observed in tester strain WP2uvrA at the highest dose level tested. The number of revertants was not reduced up to the dose level of 1600 µg/plate. Since the test item precipitated heavily on the plates at the test substance concentration of 5000 µg/plate, the number of revertant colonies of this dose level could not be determined.

Experiment 2

Precipitate

Precipitation of the test item on the plates was observed at the start and the end of the incubation period at concentratoins of 878 and 1568 μg/plate.

Toxicity

In the second mutation assay, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

Applicant's summary and conclusion

Conclusions:

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two experiments. 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. Based on the results of this study it is concluded that PF-06460259-01 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Evaluation of the mutagenic activity of PF-06460259 -01 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay.

PF-06460259 -01 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9 -mix (rat liver S9 -mix induced by Aroclor 1254).

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch E010016142 of PF-06460259 -01 was a white powder with a purity of 93.7%. The test item was dissolved in dimethyl sulfoxide.

In the dose range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9 -mix in the strains TA100 and WP2uvrA. PF-06460259 -01 precipitated on the plates at dose levels of 1600 μg/plate and upwards. The bacterial background lawn was reduced at 5000 μg/plate in strain WP2uvrA. In strain TA100, the bacterial background lawn was not reduced at any of the concentrations tested. Since the test item precipitated heavily on the plates at test item concentration of 5000 μg/plate, the number of revertant colonies of this dose level could not be determined.

Based on the results of the dose range finding test, the test item was tested in the first mutation assay at a concentration range of 17 to 1600 μg/plate in the absence and presence of 5% (v/v) S9 -mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 154 to 1568 μg/plate in the absence and presence of 10% (v/v) S9 -mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at dose levels of 512 and 1600 μg/plate in the first mutation experiment and at 878 and 1568 μg/plate in the second mutation experiment. The test item was tested beyond a precipitating dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

PF-06460259 -01 did ont induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9 -metabolic activation. These results were confirmed in a follow-up experiment.

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.

Based on the results of this study it is concluded that PF-06460259 -01 is not mutagenis in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.