.alpha.-D-Glucopyranoside, methyl 1-C-[4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl]-, compd. with 2-butyne-1,4-diol (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:

March 15 to May 16, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in 2006 according to OECD Method 471 and EU Annex V test B 13 and B14 and in accordance with GLP. Study material is well characterized.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

Solvate of (2S,3R,4S,5S,6R)-2-(4-chloro-3-(4-ethoxybenzyl)phenyl)-6-(hydroxymethyl)-2-methoxytetrahydro-2H-pyran-3,4,5-triol with 2-butyne-1, 4-diol (1:1)

Cas Number:

960404-59-5

Molecular formula:

C26 H33 Cl O9

IUPAC Name:

Solvate of (2S,3R,4S,5S,6R)-2-(4-chloro-3-(4-ethoxybenzyl)phenyl)-6-(hydroxymethyl)-2-methoxytetrahydro-2H-pyran-3,4,5-triol with 2-butyne-1, 4-diol (1:1)

Details on test material:

Test material is a white solid which was received at testing laboratory on January 11, 2006 and stored at room temperature in the dark.

Method

Target gene:

four histidine-requiring strains and one tryptophan-requiring strain

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver post mitochondrial fraction (rat-liver S-9)

Test concentrations with justification for top dose:

Range finding experiment and main experiment carried out at concentrations from 15 to 5000 ug/plate for salmonella and 50 to 50000 ug/plate for e coli strain. For the main study doses of 15, 150 ,500, 1500 and 5000 ug/plate for salmonella and 50, 150, 500, 1500 and 5000 ug/plate for e coli strain where used. An additional dose had been added to main test to allow for test material induced toxicity.

Vehicle / solvent:

Test solutions were prepared by dissolving BMS 587319-03 in sterile anhydrous grade dimethyl sulphoxide (DMSO).

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

(see solvent controls)

Negative solvent / vehicle controls:

yes

Remarks:

treatment with solvent DMSO)

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Migrated to IUCLID6: use strain TA98 without S-9)

Controls 2

Untreated negative controls:

yes

Remarks:

(see solvent controls)

Negative solvent / vehicle controls:

yes

Remarks:

treatment with solvent DMSO

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Migrated to IUCLID6: use strain TA100, TA 1535 without S-9)

Controls 3

Untreated negative controls:

yes

Remarks:

(see solvent controls)

Negative solvent / vehicle controls:

yes

Remarks:

treatment with solvent DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: (use strain TA 1537 without S-9)

Controls 4

Untreated negative controls:

yes

Remarks:

(see solvent controls)

Negative solvent / vehicle controls:

yes

Remarks:

treatment with solvent DMSO

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: use strain TA98 with S-9

Controls 5

Untreated negative controls:

yes

Remarks:

(see solvent controls)

Negative solvent / vehicle controls:

yes

Remarks:

treatment with solvent DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: use strain WP2uvrA without S-9

Controls 6

Untreated negative controls:

yes

Remarks:

(see solvent controls)

Negative solvent / vehicle controls:

yes

Remarks:

treatment with solvent DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

use strain TA100,TA1535,TA1537, WP2uvrA with S-9

Details on test system and experimental conditions:

A preliminary range finding experiment was conducted with strain TA100 and WP2uvrA at 10 concentrations from 15 to 5000 ug/plate plus negative (solvent) controls. There was a minor error in were the plates were left in incubator for 18 hours longer but no variations were noted and they were deemed acceptable. No evidence of toxicity was observed following this treatment for WP2uvrA but weakened lawns for TA100 at high doses of 5000 ug/plate were noted. This test was acceptable.For experiment 1 and 2 the Experiment 2 involved the five strains with and without metabolic activation using the same concentrations as preliminary but adding a dose for samonella strains to allow for test material induced toxicity. Normal plating treatment procedures followed. No evidence of toxicity was observed following this treatment. Negative and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates were all acceptable.

Evaluation criteria:

Acceptance criteria for validity of assay: the mean negative control counts fell within normal ranges, the positive control chemical induced clear increases in revertant numbers confirming discrimination between different strains and an active S-9 preparation, and no more than 5 % of the plates were lost through contamination or some other unforeseen event. Evaluation criteria: test article would be considered mutagenic if: the assay was valid, and an increase in frequency in revertant colonies in excess of 2 or 3 fold depending on the tester strain was noted verses the concurrent solvent controls.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

>5000ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

>5000ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

>5000ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

>5000ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

(> 5000 ug/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

It was concluded that BMS 587319-03 did not induce mutation in four histidine-requiring strains of Salmonella typhimurium and one tryptophan-requiring strain of e-coli when tested under conditions of this study. These conditions included treatment concentrations up to 5000 ug/plate in the absence and in the presence of a rat liver metabolic activation system (S-9).