Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 August - 03 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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
ethyl 2,4,6-tri-O-benzoyl-3-O-[(2S)-1-(benzyloxy)-3-cyclohexyl-1-oxopropan-2-yl]-1-thio--D-galactopyranoside
EC Number:
805-702-8
Cas Number:
322761-81-9
Molecular formula:
C45H48O10S
IUPAC Name:
ethyl 2,4,6-tri-O-benzoyl-3-O-[(2S)-1-(benzyloxy)-3-cyclohexyl-1-oxopropan-2-yl]-1-thio--D-galactopyranoside
Test material form:
solid: particulate/powder

Method

Target gene:
The objective of this study is to evaluate the test item for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidine-independent Salmonella typhimurium strains, and in a gene of tryptophan-requiring Escherichia coli bacterial strain resulting in a tryptophan-independent strain.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate. The highest concentration of PF-06460246 used in the subsequent mutation assay was the level at which the test item exhibited limited solubility.
Vehicle / solvent:
Dimethyl sulfoxide
Controlsopen allclose all
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 S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
The Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay have been shown to be rapid and adequate indicators of the mutagenic activity of a wide range of chemical compounds.
The assays will be conducted in the absence and in the presence of a metabolic system (S9-mix). The Salmonella typhimurium strains used in this study will be TÁ98, TA100, TA1535 and TA1537, and the Escherichia coli strain used will be WP2uvГА. The strains TÁ98 and TA1537 are capable of detecting frameshift mutagens, and the strains TAlIO, TA1535 and WP2uvгА are capable of detecting base-pair substitution mutagens (1-5).
All incubations will be carried out in the dark at 37.0 ± 1.0 °C. The temperature will be continuously monitored throughout the experiment. Due to addition of plates (which were at room temperature) to
the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Any variation will be evaluated and maintained in the raw data.
Rationale for test conditions:
Recommended test system in international guidelines (e.g. OECD, EC).
Evaluation criteria:
No formal hypothesis testing will be done.
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvГА is not greater than two (2) times the concurrent control, and the total number of revertants in tester strain TAl 535, TA1537 or TÁ98 is not greater than three (3) times the concurrent 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 tester strain TA100 or WP2uvгА is greater than two (2) timesthe concurrent control, or the total number of revertants In tester strain TAl 535, TA1537 or ТА98 is greater than three (3) times the concurrent control.
b) In case a follow up experiment is performed when a positive response is observed in one of thetester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
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
Species / strain:
S. typhimurium TA 1537
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
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Cytotoxicity was only observed in the presence of S9-mix at the highest concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
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
Species / strain:
E. coli WP2 uvr A
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

Applicant's summary and conclusion

Conclusions:
All bacterial strains showed negative responses over the entire dose range, i.e. no significant doserelated increase in the number of revertants in two experiments.
The negative control values were within the laboratory historical control data ranges, except the response for TA100 in the presence of S9-mix, second experiment. However since the mean number of revertant colonies showed a characteristic number of revertant colonies (69 revertant colonies) when compared against relevant historical control data (70 relevant colonies), the validity of the test was considered to be not affected.
The 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-06460246 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.