Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
The assay was therefore conducted using “a total replacement treat and plate” methodology both in the presence and absence of a rat metabolic activation system (liver S-9 mix) in two separate experiments
Principles of method if other than guideline:
Liraglutide is a polypeptide which may cause artefacts
through growth stimulation in a standard plate incorporation assay. The assay was therefore
conducted using “a total replacement treat and plate” methodology both in the presence and absence
of a rat metabolic activation system (liver S-9 mix) in two separate experiments.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Molecular formula: C172H265N43O51
Molecular weight (MW): 3751.2 Da
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL: Active Pharmaceutical Ingredience (API) Liraglutide
- Source and lot/batch No.of test material: NA
- Expiration date of the lot/batch: NA
- Purity test date: NA



STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Protected from light and moisture, and
refrigerated (5-8°C)
- Stability under test conditions: NA
- Solubility and stability of the test substance in the solvent/vehicle: NA
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: NA

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: NA
- Preliminary purification step (if any): NA
- Final dilution of a dissolved solid, stock liquid or gel:NA
- Final preparation of a solid:NA

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS:

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine-requiring strains
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Additional strain / cell type characteristics:
other: tryptophane-requiring strain
Species / strain / cell type:
other: WP2pKM101
Additional strain / cell type characteristics:
other: tryptophane-requiring strain
Metabolic activation:
with and without
Metabolic activation system:
rat metabolic activation system (liver S-9 mix)
Test concentrations with justification for top dose:
Two experiments were carried out, each in triplicates:

Assay 1 (with and without metabolic activation):
Test concentrations:
-Without Activation: 8, 40, 200, 100 and 5000 ug/mL
-With Activation: 6, 30, 150, 750 and 3750 ug/mL
Assay 2 (with and without metabolic activation):
Test concentrations:
-Without Activation: 50, 158.1, 500, 1581 and 5000 ug/mL
-With Activation: 6, 37.5, 118.6, 375, 1186 and 3750 ug/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 4 mM phosphate buffer
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
4mM phosphate buffer
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
N-ethyl-N-nitro-N-nitrosoguanidine
other: Acridine Mutagen ICR 191 and AAN
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (a total replacement treat and plate methodology)



NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: NA


Assay 1 and 2:

All six strains(4x S. typhimurium and 2x E.coli) were tested in triplicates with and without Aroclor-induced rat liver S9, 10% (4 solvent controls and triplicate positive controls). Plates were investigated for toxicity and where possible revertant colonies were counted.
Rationale for test conditions:
Liraglutide is a polypeptide which may cause artefacts through growth stimulation in a standard plate incorporation assay. The assay was therefore conducted using “a total replacement treat and plate” methodology both in the presence and absence of a rat metabolic activation system (liver S-9 mix) in two separate experiments.
Statistics:
The m-statistic was calculated to check that the data were Poisson-distributed and Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was chekced by linear regression analysis.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: TA98, TA100, TA1535 and TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: not specified
Vehicle controls validity:
other: not specified
Untreated negative controls validity:
other: not specified
Positive controls validity:
valid
Key result
Species / strain:
other: WP2pKM101 and WP2 uvrA pKM101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: not specified
Vehicle controls validity:
other: not specified
Untreated negative controls validity:
other: not specified
Positive controls validity:
valid
Additional information on results:
Liraglutide did not induce mutation in the four strains of Salmonella typhimurium and in the two
strains of Escherichia coli, when tested at concentrations up to 5,000 μg/ml in the absence of rat
liver S-9 mix and up to 3,750 μg/ml in the presence of S-9 mix.
Remarks on result:
other: no mutagenic potential

Applicant's summary and conclusion

Conclusions:
Determination of the mutagenicity of Liraglutide was conducted in accordance to OECD Guideline 471 in four histidine-requiring strains (TA98, TA100, TA1535, TA1537) of salmonella typhimurium and two tryptophane-requiring strains (WP2pKM101 and WP2 uvrA pKM101) of Escherichia coli . Liraglutide did not induce mutation in the four strains of Salmonella typhimurium nor in the two strains of Escherichia coli, when tested at concentrations up to 5,000 μg/ml in the absence of rat liver S-9 mix and up to 3,750 μg/ml in the presence of S-9 mix.
Executive summary:

Determination of the mutagenicity of Liraglutide was conducted in accordance to OECD Guideline 471. Liraglutide was assayed for mutation in four histidine-requiring strains (TA98, TA100, TA1535, TA1537) ofsalmonella typhimurium and two tryptophane-requiring strains (WP2pKM101 and

WP2 uvrA pKM101) of Escherichia coli, both in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9) in two separate experiments.

Liraglutide is a polypeptide which may cause artefacts through growth stimulation in a standard plate incorporation assay. The assay was therefore conducted using “a total replacement treat and plate” methodology both in the presence and absence of a rat metabolic activation system (liver S-9 mix) in two separate experiments.

Liraglutide did not induce mutation in four strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537), nor two tryptophane-requiring strains (WP2pKM101 and

WP2 uvrA pKM101) of Escherichia coliwhen tested under conditions employed in this study. Liraglutide did not induce mutation in the four strains of Salmonella typhimurium and in the two strains of Escherichia coli, when tested at concentrations up to 5,000 μg/ml in the absence of rat liver S-9 mix and up to 3,750 μg/ml in the presence of S-9 mix.