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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-10-03 to 1997-10-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
- Principle of test: Evaluation of the mutagenic potential in vitro of Alendronate sodium.
- Short description of test conditions: Alendronate sodium was evaluated for mutagenic potential in a microbial mutagenesis test system using mutant strains of Salmonella typhimurium (TA1535, TA97a, TA98, and TAlOO) and Escherichia coli (WP2, WP2 uvrA, and WP2 uvrA pKMlOI). In this test system, mutation is measured as reversion to histidine prototrophy of Salmonella test strains which are histidine auxotrophs and as reversion to tryptophan proto trophy of E. coli test strains which are tryptophan auxotrophs. The compound was tested with and without a liver microsomal enzyme activation system (S-9) prepared from xenobiotic treated rats. The protocol for this test is well-documented in the literature.
- Parameters analysed / observed: Revertants per plate.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: Soluble up to 4000 and 5000 active ingredient per plate, as those showed a precipitate up to


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Diluted with water so that 0.1 mL contained the final concentration per plate. The final concentrations were 100, 500, 1000, 2000, 4000, and 5000 ug active ingredient per plate.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Concentrations of 100, 500, 1000, 2000, 4000, and 5000 ug active ingredient per plate.

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
E. coli WP2
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
xenobiotic-induced rat liver S-9;
Test concentrations with justification for top dose:
Concentrations: 100, 500, 1000, 2000, 4000, and 5000 ug active ingredient per plate.
Based on the limited solubility of the compound, 5000 ug per plate was the highest dose chosen for testing
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The compound is soluble in water up to the desired testing concentration
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: see below for details
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar

DURATION
- Preincubation period: 12-15 hours
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours

SELECTION AGENT (mutation assays):
No selection agent was used.

NUMBER OF REPLICATIONS: 3 + one supplement palte for each strain and control with and without metabolic activation as relevant.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Slides were not stained, and instead were counted using an Artek colony counter

NUMBER OF CELLS EVALUATED: All supplemental plates were evaluated for lawn, and all minimal test plates were evalutated for colony growth. The exact number of cells evaluated is not relevant.

DETERMINATION OF CYTOTOXICITY
- Any supplementary information relevant to cytotoxicity: Supplemental plates were examined for evidence of inhibition or contamination.

- OTHER:
Five-tenths mL of the activation system or buffered saline (control) was added to the various concentrations of the compound and controls, followed by 2 mL of soft agar containing 0.1 mL of the suspension of the organism under study. The mixture was gently agitated for even dispersion and poured onto a base layer of agar. Triplicate plates (minimal) containing only a trace of histidine or tryptophan and one plate (supplemental) with sufficient histidine and tryptophan for growth of auxotrophs were prepared for each treatment group with and/or without metabolic activation.
After incubation for 48 hours at 37°C, revertant colonies on the histidine or tryptophan deficient plates were counted and the supplemental plates examined for
evidence of inhibition or contamination. Revertant colony counts on the test plates were averaged and compared with the appropriate control plate average.

Microsomal Activation System
Liver homogenates (S-9) are prepared from rats treated with suitable inducers of liver enzymes (see b below) and used at a dose of 35 ul/test
plate or other satisfactory dose as determined by S-9 testing.

Positive and diagnostic Controls:
Positive Controls and Diagnostic Mutagens:
1) Positive Controls
Salmonella strains, E. coli strains WP2 uvrA and WP2 uvrA pKMIOl:
2-aminoanthracene 2 and 5 µg per plate or 10 and 15 µg/plate
With and without S-9 metabolic activation
E. coliWP2:
Hydrazine sulfate 500 and 1000 µg/plate
With S-9 metabolic activation

2) Diagnostic Mutagens
Salmonella strains:
Sodium azide, 1.5 µg/plate.
Methyl methanesulfonate (MMS), 2 µg/plate.
ICR-191, 1.0 and 4.0 µg/plate.
2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), O.01 and 0.05 µg/plate

E. coli strains:
2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), 0.01 and 0.05 µg!plate
Methyl methanesulfonate (MMS), 2 µg/plate.
Rationale for test conditions:
The E. coli trp- reversion utagenesis assay detects mutagens by means of their ability to damage the DNA in specific E. coli strains which causes reversion from tryptophan dependence to tryptophan independence. The bacterial strains are tested in the presence of liver tissue homogenate thus incorporating an important aspect of mammalian metabolism into the in vitro test. The compound under test, the histidine-requiring or tryptophan- requiring mutant, and the liver homogenate are combined on an agar medium plate without histidine and tryptophan, and after incubation for two days at 37°C, the number of histidine or tryptophan revertants are scored. Trace histidine or tryptophan in medium allows expression of a background lawn. This permits detection of toxicity and/or suppressed revertant growth.

These procedures are in line with referenced documents unless speified otherwise.
Evaluation criteria:
The assay is positive if the number of revertant colonies induced is at least twofold higher than the solvent negative control with an evident dose-related
increase.
Statistics:
None

Results and discussion

Test resultsopen allclose all
Key result
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
Key result
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
Key result
Species / strain:
S. typhimurium TA 97
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
E. coli WP2
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
Key result
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
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: While there was precipitation at the 4000 and 5000 ug active ingredient / plate concentrations, the colonies were still able to be counted via eye couting rather than the Antrak colony counter.

RANGE-FINDING/SCREENING STUDIES: None

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Criteria for the present postive controls matched that of the historical postivie controls, and they were therefore valid.
- Negative (solvent/vehicle) historical control data: The negative control for DMSO was intially assessed historically, but was then tested and determined to be independently valid as well.

Any other information on results incl. tables

A supplmenetal study with DMSO as a negative vehicle control and solvent was run as it was missing from the original test and was required in the approved procedure. The results were similar to the tests done in water, and did not change classification or the analysis of the results.

Applicant's summary and conclusion

Conclusions:
Alendronate sodium does not induce mutation in any of the Salmonella or E. coli
strains under these testing conditions. This conclusion is based on the fact that, in two
separate assays, at the dose levels tested with and without S-9 activation, Alendronate
sodium did not meet the criteria for a positive test compound which are: (1) a twofold
or greater increase in number of revertant colonies and, (2) a dose-related increase in
number of revertant colonies.