Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
477-080-2
EC Name:
-
Cas Number:
103121-85-3
Molecular formula:
C13 H19 N3 O3 S . HCl (Hill Formula) C13 H20 N3 O3 S . Cl (CAS Formula)
IUPAC Name:
1-{[(6R,7R)-7-amino-2-carboxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methyl}-1-methylpyrrolidin-1-ium chloride
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: MPI-ACA
Chemical name: Pyrrolidinium, 1-[(7 -amino-2-carboxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl )methyl]-1-methyl-, chloride
Molecular formula: C13H19N3O3S.HCI
CAS No.: 103121-85-3.
Supplier: Sandoz GmbH
Batch-No. 49900408.
Appearance: White to yellowish powder.
Purity: 85.5 %
Solubility: Soluble in water: 120 g/L at 20 °C, poorly soluble in non-polar organic solvents
Melting point: 165 °C (decomposition).
pH = 2.86 (1% solution in deionised water, w/v)
Conditions of storage: In the refrigerator.
Stability at conditions of storage: Stable for 12 months.
Stability in aqueous solutions/suspensions: Not defined.
Date of expiry: 27 April 2008 (retest).
Date of receipt: 16 August 2007.

Method

Target gene:
his- (S. typhimurium)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
other: S. typhimurium TA97a
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from Aroclor 1254 induced microsomes of rat liver
Test concentrations with justification for top dose:
The test substance was dissolved in water. The following concentrations were tested:
7, 21, 62, 185, 556 and 1667 µg per plate in the first experiment.
2.3, 7, 21, 62, 185 and 556 µg per plate in the second experiment.
Vehicle / solvent:
Deion. water.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene; 7,12-Dimethylbenz[a]anthracene ; 1,8-Dihydroxy-anthraquinone; 2-Nitrofluorene; Sodium azide; 4-Nitro-o-phenylenediamine; t-Butyl-hydroperoxide
Details on test system and experimental conditions:
A preliminary toxicity test was performed with strain TA100. The test substance was toxic to the bacteria at 5000 and 1667 µg/plate, resulting in a missing bacterial background lawn. Therefore 1667 µg/plate was chosen as highest concentration which could be in the toxic range and 6 concentrations were tested. The concentrations for the second experiment were changed due to the results of the first experiment: The test substance was toxic to the bacteria at 1667 µg/plate, resulting in a missing bacterial background lawn or a reduced number of colonies, and at 556 µg/plate resulting in a reduced number of colonies. Therefore the concentrations were decreased one step.

The exposure for the first experiment was performed according to the 'Plate Incorporation Assay', in which bacteria, test substance (and microsomes) are in contact on the plate without preceding incubation in the liquid state.
For each sample the following solutions were combined:
• 0.1 mL of the overnight culture of the bacteria,
• 0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation),
• 0.1 mL of the appropriate test- or reference substance solution and
• 2 mL of top agar.
The combined solutions were mixed and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).


The exposure for the second experiment was performed according to the 'Preincubation Assay', in which bacteria, test substance (and microsomes) are in contact on the plate with preceding incubation in the liquid state.
For each sample the following solutions were combined:
• 0.1 mL of the overnight culture of the bacteria,
• 0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation),
• 0.1 mL of the appropriate test- or reference substance solution.
The solutions were preincubated for 20 minutes at 37 °C using a shaker, afterwards combined with 2 mL of top agar and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).
Evaluation criteria:
The criteria for a positive result are:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
• For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2½ fold of the amount of the spontaneous revertants.
• For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 1.67 fold of the amount of the spontaneous revertants.

Statistics:
Means and standard deviations were calculated for the number of mutants in every concentration group.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA 97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle 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
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Solubility:
No precipitation of the test substance was seen in any of the concentration groups.

Toxicity
In the preliminary test the test substance was toxic to the bacteria at 5000 and 1667 µ/plate, resulting in a missing bacterial background lawn.
In the main test the test substance was again toxic to the bacteria at 1667 µ/plate, resulting in a missing bacterial background lawn or a reduced number of colonies, and at 556 µg/plate resulting in a reduced number of colonies.

Mutagenicity
There was no increase in the number of mutants in any of the tested bacterial strains at any of the tested concentrations. The addition of an external metabolising system did not change these results.

Properties of the bacteria:
The used strains of Salmonella typhimurium showed the expected genetic properties and were sensitive against several mutagenic chemicals. The numbers of spontaneous revertants were comparable with the historic control data for the negative controls.

Positive control substances:
All positive control substances increased the mutation frequency to more than the threshold values stated above. As 2-aminoanthracene, 1 ,8-dihydroxy-anthraquinone and 7, 12-dimethyl-benz[a]anthracene require metabolic activation for mutagenicity, the results of these substances demonstrate also the efficiency of the metabolising system.

Applicant's summary and conclusion

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

According to these results, MPI-ACA is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 with and without an external metabolising system up to 1667 µ/plate, which is the limit of toxicity.
Executive summary:

MPI-ACA was tested for mutagenic activity with the "Salmonella typhimurium Reverse Mutation Test" (Ames Test). The study was conducted in accordance with the OECD-guideline 471 and directive 2000/32/EC, part 8.13/14.

Two independent experiments were performed with and without an external metabolising system. The first experiment was performed according to the "direct plate incorporation method", the second experiment according to the ''preincubation method". As test system the bacterial strains Salmonella typhimurium TA97a, TA98, TA100, TA1 02 and TA1535 were used. Negative and positive controls were included.

Results:

Positive controls: All positive control groups showed significantly increased mutation frequencies which demonstrate the sensitivity of the test system.

Toxicity: The test substance was toxic to the bacteria at 1667 µg/plate, resulting in a missing bacterial background lawn or a reduced number of colonies, and at 556 µg/plate resulting in a reduced number of colonies.

Solubility: No precipitation of the test substance was seen in any of the concentration groups.

Mutagenicity: In none of the concentrations tested and with none of the strains used an increase of the mutation frequency to more than the threshold values (250 % of the controls for strains TA98 and TA 1535, 167 % of the controls for strains TA97a, TA100 and TA102) was obtained. Metabolic activation did not change these results.

According to these results, MPI-ACA is not mutagenic in the Ames test.