Registration Dossier

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:
2010-07-20 to 2010-09-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed in accordance with OECD principles of GLP with no deviations. Furthermore, the study fulfilled the validity criteria described in the OECD guideline.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2011

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
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
PC2414
IUPAC Name:
PC2414
Details on test material:
- Name of test material (as cited in study report): PC2414
- Lot/batch No.: Y5UB376
- Expiration date of the lot/batch: 2013-03-01

Method

Target gene:
Strain Target mutation Mutation type
TA 1535 hisG46; rfa-;uvrB- Base-pair substitution
TA 100 hisG46; rfa-;uvrB-; R-factor Base-pair substitution
TA 98 hisD3052; rfa-;uvrB- Frame shift
TA 1537 hisC3076; rfa-;uvrB-; R-factor Frame shift
WP2uvrA trp-, urvA- Base-pair substition

All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB-bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortlemans and Zeiger (2000)). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel (1976)).

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
NA
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver homogenate metabolising system (10% liver S9 in standard co-factors prepared from livers of male rats given phenobarbitone/beta-naphthoflavone).
Test concentrations with justification for top dose:
Concentration range in the preliminary toxicity test (with and without metabolic activation): 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

Concentration range in experiment 1 and 2 (with and without metabolic activation): 0, 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: PC2414 was found to be fully soluble in DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
N-ethyl-N-nitro-N-nitrosoguanidine was used without S9-mix at the following concentrations: 2 ug/plate (WP2urvA), 3 ug/plate (TA100), 5 ug/plate (TA1535).
Positive control substance:
9-aminoacridine
Remarks:
9-aminoacridine was used without S9-mix at the following concentration: 80 ug/plate (TA1537).
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
4-nitroquinoline-N-oxide was used without S9-mix at the following concentration: 0.2 ug/plate (TA98).
Positive control substance:
other: 2-aminoanthracene
Remarks:
2-aminoanthracene was used with S9-mix at the following concentrations: 1 ug/plate (TA100), 2 ug/plate (TA1535/TA1537), 10 ug/plate (WP2uvrA).
Positive control substance:
benzo(a)pyrene
Remarks:
benzo(a)pyrene was used with S9-mix at the followng concentration: 5 ug/plate (TA98).
Details on test system and experimental conditions:
Preliminary Toxicity Test:
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of PC2414. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA), 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of PC2414 formulation and 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the PC2414formulation and a vehicle control (dimethyl sulphoxide) were tested. In addition, 0.1 ml of the maximum concentration of PC2414 and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of PC2414. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Mutation Test - Experiment 1:
Five concentrations of PC2414 (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of PC2414 formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of PC2414 both with and without S9-mix. All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Mutation Test - Experiment 2:
The second experiment was performed using fresh bacterial cultures, PC2414 and control solutions, using the pre-incubation method. As it is sound scientific practice to alter one condition in the replicate assay, the exposure condition was changed from plate incorporation to pre-incubation for Experiment 2. The PC2414 dose range was the same as Experiment 1 (50 to 5000 µg/plate). PC2414 formulations and vehicle control were dosed using the pre-incubation method as follows: Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.1 ml of the vehicle or PC2414 formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of PC2414 both with and without S9-mix. The positive and untreated controls were dosed using the standard plate incorporation method. All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.
Evaluation criteria:
Validity criteria:
- negative/positive control data were consistent with historical control data
- positive control showed marked increase over the concurrent negative control
- evaluation was not restricted by loss of plates (e.g. through contamination)
- all bacteria strain must have demonstrated the required charasterics
- the bacterial cell count for each stratin culture should be in the range of 0.9 to 9x109

Evaluation criteria:
- dose-related increases in number of revertant colonies at one or more test points
- increases reproducible between replicate plates
- increases more than twice the corresponding negative control
- biological relevance against in-house historical control ranges
- statistical analysis of data


Statistics:
The numbers of revertant colonies at each treatment test point were compared to the corresponding negative control values using the Analysis of
Variance test. When this test showed statiscal significanct differences in the data, Dunnett´s test was used to determine the statistical significance of increases and decreases in the number of revertant colonies for each set of triplicate plates.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the preliminary Toxicity Test, PC2414 was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). PC2414 formulation and S9-mix used in this experiment were both shown to be sterile.

In the first experiment (plate incorporation method) PC2414 caused no visible reduction in the growth of the bacterial background lawns of any of the tester strains at any dose level in either the presence or absence of S9-mix. In the second experiment (pre-incubation method) PC2414 induced toxicity to the bacterial background lawns of Salmonella tester strains TA100, TA1535 and TA1537 in the absence of S9-mix only at 5000 µg/plate. No toxicity was noted to any of the remaining strains at any PC2414 dose level in either the presence or absence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent PC2414 being tested up to the maximum recommended dose level of 5000 µg/plate. No PC2414 precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of PC2414, either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9‑mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. 

A history profile of vehicle and positive control values for 2008 and 2009 was included in the test report and the results of this study was in accordance with the history profile.

Applicant's summary and conclusion

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

PC2414 was found to be non-mutagenic in the reverse mutation assay (Ames Test) using Salmonella Typhimurium and Escherichia Coli with and
without metabolic activation.
Executive summary:

PC2414 was tested in the reverse mutation assay (Ames Test) using Salmonella Typhimurium strains (TA1535, TA1537, TA98, TA100) and Escherichia Coli strain (WP2uvrA). The test was performed in accordance with OECD test guideline 471 and EU B13/14. Test methods used included plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of rat liver homogenate metabolosing system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 ug/plate in the first experiment.The experiment was repeated on a seperate day (pre-incubatrion method) using the same dose range, fresh cultures of the bacterial strains and fresh PC2414 formulations.

No significant increases in the frequecy of revertant colonies were recorded for any of the bacterial strains, with any dose of PC2414, either with or without metabolic activation per exposure method, hence PC2414 was found to be non-mutagenic.