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Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 10. May. 2007 to 09.Nov. 07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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)
Version / remarks:
OECD Guideline fro the Testing of Chemicals, Guideline 471 Bacterial Reverse Mutation, Updated Guideline, 21 July 1997
Qualifier:
according to guideline
Guideline:
other: Directive 2000/32/EC
Version / remarks:
Commission Directive 2000/32/EC of 19 May 2000 adapting to technical progress for the 26th time Council Directive 67/548/ECC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labeling of dangerous substances. Annex 4 D, B. 13/14 Mutagenicity- Bacteria! reverse mutation test.
Qualifier:
according to guideline
Guideline:
other: ICH Topic S2A Genotoxicity
Version / remarks:
ICH Topic S2A, Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for
Pharmaceuticals, July 1995.
Principles of method if other than guideline:
- Principle of test: The aim of this Study was to perform an initial screening for genotoxic activity and, in particular, for point mutation-inducing activity. The mutagenic potential of the test item is assessed in the Ames strains of Sa/mone/la typhimurium and Escherichia coli, in the presence and absence of a metabolic activation system.

- Exprerimental procedure:
Several days before the beginning of the Study, the bacterial strains contained in vials were defrosted and grown on master plates in order to obtain pure cultures. These plates were stored between 2 and 8 °C until required for the test.
The histidine and tryptophan requirements, the absence or presence of the pKM 101 plasmid, the rfa factor (sensitivity to crystal violet in Salmone/la) and the uvrβ factor in Salmonel/a (sensitivity to ultraviolet rays) were checked in the strains used to prepare the inoculum.
Approximately sixteen hours before each test, an inoculum was prepared for each of the five bacterial strains in 20 mL of nutrient broth and was incubated in a bath at 37 ± 1 °C with agitation.


An study plan was performed. See the information of the preliminar study on appendix II (from page 33 to 43) of the study report.
Also an study plan amendment was permormed. See the information of the preliminar study on appendix III (from page 44 to 48) of the study report.
GLP compliance:
yes
Type of assay:
other: Ames strains of Sa/mone/la typhimurium and Escherichia coli, in the presence and absence of a metabolic activation system

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
PREPARATION OF THE TEST ITEM
The solvent used for the test item was distilled water.The following concentrations were tested: 0.63, 1.25, 2.5, 5 and 10µL/plate.

PRELIMINARY TEST
For the preliminary test, the maximum concentration used was 250µL/ /ml. The results obtained in the preliminary test are presented in section 5.4 Preliminary test (page 10 and 11) of the study report.

Method

Target gene:
Not specified
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Sallmonella typhimurium TA-1535 NCTC 12117; Supplier NCTC London
- Suitability of cells:
- Normal cell cycle time (negative control):

For cell lines:
- Absence of Mycoplasma contamination:
- Number of passages if applicable:
- Methods for maintenance in cell culture:
- Cell cycle length, doubling time or proliferation index :
- Modal number of chromosomes:
- Periodically checked for karyotype stability: [yes/no]
- Periodically ‘cleansed’ of spontaneous mutants: [yes/no]

For lymphocytes:
- Sex, age and number of blood donors:
- Whether whole blood or separated lymphocytes were used:
- Whether blood from different donors were pooled or not:
- Mitogen used for lymphocytes:

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Species / strain / cell type:
S. typhimurium TA 1537
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Sallmonella typhimurium TA-1537 NCTC 12118; Supplier NCTC London
- Suitability of cells:
- Normal cell cycle time (negative control):

For cell lines:
- Absence of Mycoplasma contamination:
- Number of passages if applicable:
- Methods for maintenance in cell culture:
- Cell cycle length, doubling time or proliferation index :
- Modal number of chromosomes:
- Periodically checked for karyotype stability: [yes/no]
- Periodically ‘cleansed’ of spontaneous mutants: [yes/no]

For lymphocytes:
- Sex, age and number of blood donors:
- Whether whole blood or separated lymphocytes were used:
- Whether blood from different donors were pooled or not:
- Mitogen used for lymphocytes:

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Sallmonella typhimurium TA-98 NCTC 12116; Supplier NCTC London
- Suitability of cells:
- Normal cell cycle time (negative control):

For cell lines:
- Absence of Mycoplasma contamination:
- Number of passages if applicable:
- Methods for maintenance in cell culture:
- Cell cycle length, doubling time or proliferation index :
- Modal number of chromosomes:
- Periodically checked for karyotype stability: [yes/no]
- Periodically ‘cleansed’ of spontaneous mutants: [yes/no]

For lymphocytes:
- Sex, age and number of blood donors:
- Whether whole blood or separated lymphocytes were used:
- Whether blood from different donors were pooled or not:
- Mitogen used for lymphocytes:

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Sallmonella typhimurium TA-100 NCTC 12116; Supplier NCTC London
- Suitability of cells:
- Normal cell cycle time (negative control):

For cell lines:
- Absence of Mycoplasma contamination:
- Number of passages if applicable:
- Methods for maintenance in cell culture:
- Cell cycle length, doubling time or proliferation index :
- Modal number of chromosomes:
- Periodically checked for karyotype stability: [yes/no]
- Periodically ‘cleansed’ of spontaneous mutants: [yes/no]

For lymphocytes:
- Sex, age and number of blood donors:
- Whether whole blood or separated lymphocytes were used:
- Whether blood from different donors were pooled or not:
- Mitogen used for lymphocytes:

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Escherichia coli WP2 uvrA pKM 101 NCIMB 11703; Supplier NCIMB Ltd Scotland
- Suitability of cells:
- Normal cell cycle time (negative control):

For cell lines:
- Absence of Mycoplasma contamination:
- Number of passages if applicable:
- Methods for maintenance in cell culture:
- Cell cycle length, doubling time or proliferation index :
- Modal number of chromosomes:
- Periodically checked for karyotype stability: [yes/no]
- Periodically ‘cleansed’ of spontaneous mutants: [yes/no]

For lymphocytes:
- Sex, age and number of blood donors:
- Whether whole blood or separated lymphocytes were used:
- Whether blood from different donors were pooled or not:
- Mitogen used for lymphocytes:

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
MP/CAPPEL; Catalog #50412; S9 Manufacturing and Quality Control Certificate
- Lot Nº:9065H
- Sex:Male
- Species:Rat
- Strain: Sprague Dawley
- Tissue: Liver
- Buffer:0.154KCl
- Inducing Agent: Aroclor 1254
- Package Vol: 5ml
- Date of Manufacture: November 8, 2005
- Date of Expiration: November 8, 2007
- quality controls data: "Ames" Assay (Maron&Ames, Mutat Res., v. 113: 173. 1983.)
- Sterility Test
- ProteinAssay(Lowry, el. al., JBC, v. 193:265. 1951):44.4 mglml
- Ethoxyresorufin-o-deethy!ase activity (Burke et al., Biochem. Pharmacol, v. 34: 3337. 1985). 6523.1 picomoles 7-hydroxyresorufin formed per minute per milligram protein. 141.8( - fold increase.)
--------------------------------------------------------------------------------------------------
Lot Nº: 2137J
- Sex:Male
- Species:Rat
- Strain: Sprague Dawley
- Tissue: Liver
- Buffer:0.154KCl
- Inducing Agent: Aroclor 1254
- Package Vol: 5ml
- Date of Manufacture: May 2, 2006
- Date of Expiration: May 2, 2008
- quality controls data: "Ames" Assay (Maron&Ames, Mutat Res., v. 113: 173. 1983.)
- Sterility Test
- ProteinAssay(Lowry, el. al., JBC, v. 193:265. 1951):35mg/ml
- Ethoxyresorufin-o-deethylase activity (Burke et al., Biochem. Pharmacol, v. 34: 3337. 1985). 6523.1 picomoles 7-hydroxyresorufin formed per minute per milligram protein. 141.8( - fold increase.)

*This information it is present on pages 31 and 32 of the study report.
Test concentrations with justification for top dose:
The test concentrations were determined according to the toxicity of the substance. The maximum concentration tested was 10µL/plate.
Vehicle / solvent:
The solvent used for the test item was distilled water.The following concentrations were tested: 0.63, 1.25, 2.5, 5 and 10µL/plate.
Controls
Untreated negative controls:
no
Remarks:
0.1 mL of the suspension of each bacteria! strain was added to 0.5 mL of phosphate buffer or S-9.
Negative solvent / vehicle controls:
yes
Remarks:
The solvent used to dilute the test ítem was distilled water and it was tested as the negative control.
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
other: sodium azide, ≥95.5% AZ; and 2-aminoanthracene, 96% 2AA
Details on test system and experimental conditions:
EXPERIMENTAL PROCEDURE
Preincubation: 0.1 ml of the bacterial culture and 0.1 ml of the dilution of the test item, or 0.1ml of the positive control or 0.1 ml of the solvent, were added to 0.5 ml of S-9 mix with cofactors (or 0.5 ml of phosphate buffer).
This was incubated for 20 minutes at 37 ± 1 °C under agitation. lt was then added to 2 mL of top agar melted at 45 ± 1 °C which contained a sterile solution of 0.5 mM of L-Histidine/0.5 mM Biotin in the proportion of 1/1 O for the Sa/mone//a typhimurium strains.
For the Escherichia coli, 0.25 ml of a sterile solution of tryptophan and 5 ml of nutrient broth were added for every 1 00 ml of agar.
As the metabolizing enzymes of S-9 were not stable at 45 °C, the contents of the test tubes were mixed immediately in a rotamixer, poured over the agar plates which were rotated to spread the layer, and then left to solidify.
Once the agar had solidified, the plates were inverted and incubated at 37 ± 1 °C for 48-72 hours.
After this incubation period, the number of revertant colonies that had grown on each plate was counted.

- OTHER:
POSITIVE CONTROLS
In parallel to the Study of the test item, Batch 7108023, known mutagenic
products were tested in order to check the sensitivity to mutagenic agents of the strains used. See in the section 5.8 "positive controls" (page 13) of the study report the results of the positive controls.
The solvents used were sterile distilled water for AZ and DMSO for 9Ac, 2NF, 4NQO and 2AA.
NEGATIVE CONTROLS
The solvent used to dilute the test ítem was distilled water and it was tested as the negative control.
NONTREATED CONTROLS
0.1 mL of the suspension of each bacteria! strain was added to 0.5 mL of phosphate buffer or S-9.
Rationale for test conditions:
Not specified
Evaluation criteria:
There are several criteria for determining a positive result, such as a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system. Biological relevance of the results should be considerad first. Statistical methods may be used as an aid in evatuating the results; However, statistical significance should not be the only determining factor for a positive response.
A test item for which the results do not meet the above criteria is considerad nonmutagenic in this test.
Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test item. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated.
Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test item is not mutagenic in the tested species.
Statistics:
STATISTICAL ANALYSIS
The results, together with the means and standard deviations for each group, are presented in separate tables for each test, bacterial strain and S-9 concentration.
The comparisons between the results for the standard products and the Control were made using Student's t test, with a level of significance of p < 0.01 and p < 0.05 [4].
The statistical comparison of the different test-item concentrations and the Control was carried out, for all the bacterial strains, both with and without metabolic activation, using a one-way analysis of variance with a level of significance of p < 0.05 [Steel RGD, Torrie JH, Dickey DA. Principies and Procedures of Statistics: A Biometrical Approach. 3"' Edition. McGraw-Hill Book Company, lnc., NewYork, 1996.].

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True 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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
POSITIVE CONTROLS
The response of the five bacterial strains to the substances used as positive Controls are shown in Tables 1 to 10 and 23 (of the study report attach). All the bacterial strains responded positively.
NEGATIVE CONTROLS
Normal values (within the range of historical data for negative Controls, see Appendix IV) were obtained in the revertant colony counts on all the dishes treated only with the solvents.
NONTREATED CONTROLS
Normal values (within the range of bibliographic data) [2,5,6). were obtained in the revertant colony counts on nontreated controls (see Tables 24 and 25, present on the study report attach).

TEST ITEM
The results obtained for the response of the five bacterial strains to the different concentrations of the test item are shown in Tables 11 to 20, 21 and 22 (of the study report attach).
The test item caused toxicity in all the strains with S9 and without S9 at the concentration of 10µL/plate except for the strain TA-98 without S9 that did not show toxicity at any concentration.
No mutagenic activity was detected.

Any other information on results incl. tables

See the tables results from page 16 to 29 present in the report study attach below in "attached full study report"

Applicant's summary and conclusion

Conclusions:
Based on the data obtained in the test performed, it may be concluded that the test item under the experimental conditions used produced no mutagenic activity in any of the five bacterial strains used.
Executive summary:

I. Strains TA-1535, TA-1537, TA-98 and TA-100 of Salmonella typhimurium and Escherichia coli WP2 uvrA pkM101 were treated with the test item, at five different concentrations following the method described by the OECD [Guideline for Testing of Chemicals: Bacteria/ Reverse Mutation Test Protocol no. 471 (Updated Guideline, adopted 21 July 1997)].

Each concentration of the test item was tested in triplicate, with and without the addition of the metabolic activation system, at 10% with standard cofactors. The concentration levels tested varied between 0.63µL/plate and 10 µL/plate.

II. Normal values (within the range of the historical data for negative controls) were obtained in the revertant colony counts on all the plates tretaed with only the solvents (negative controls).

III. Normal values (within the range of the bibliographic data) [2,5,6] were obtained in the revertant colony counts on nontreated controls.

IV. All the substances used as positive controls produced an increase in the number of revertants, both with the metabolic activation system and without it.

V. The test concentrations were determined according to the toxicity of the substance. The maximum concentration tested was 10µL/plate.

VI.The test item caused toxicity in all the strains with S9 and without S9 at the concentration of 10 µL/plate except for the strain TA-98 without S9 that did not show toxicity

at any concentration.

VII. The test item did not produce any mutagenic response in any of the strains used, neither with the metabolic activation system nor without it.