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:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline Study
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): XU-18838.00
- Physical state: solid
- Lot/batch No.: 20120135-18
- Expiration date of the lot/batch: 18 June 2014
- Storage condition of test material: Ambient (+18 to +36 ºC)

Method

Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell lines (if applicable):
Each S. typhimurium tester strain contains, in addition to a mutation in the histidine
operon, additional mutations that enhance sensitivity to some mutagens. The rfa
mutation results in a cell wall deficiency that increases the permeability of the cell to
certain classes of chemicals such as those containing large ring systems that would
otherwise be excluded. The deletion in the uvrB gene results in a deficient DNA
excision-repair system. Tester strains TA98 and TA100 also contain the pKM101
plasmid (carrying the R-factor). It has been suggested that the plasmid increases
sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase
complex involved with the mismatch-repair process.
TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine
independence (prototrophy) by frameshift mutagens. TA100 is reverted by both
frameshift and base substitution mutagens and TA1535 is reverted only by mutagens
that cause base substitutions.
The E. coli tester strain has an AT base pair at the critical mutation site within the trpE
gene. Tester strain WP2uvrA (pKM101) has a deletion in the
uvrA gene resulting in a deficient DNA excision-repair system. Tryptophan revertants
can arise due to a base change at the originally mutated site or by a base change
elsewhere in the chromosome causing the original mutation to be suppressed. Thus,
the specificity of the reversion mechanism is sensitive to base substitution mutations.
Additional strain characteristics:
other: rfa mutation, uvr8 deletion
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S-9 homogenate
Test concentrations with justification for top dose:
100, 266, 707, 1880 and 5000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: Dimethyl formamide (DMF)
- Justification for choice of solvent/vehicle: Solubility test was conducted to determine the highest soluble concentration of the test
substance in vehicles compatible with this test system in the order of preference,
sterile water, DMSO, Ethanol, Acetone and DMF at 50 mg/mL. Though Acetone is one of the organic vehicles compatible with this test system, since
Advinus did not have any historical control data on using Acetone as the vehicle, in
consultation with the sponsor, it was decided not to use Acetone as vehicle.
DMF is also one of the organic vehicles compatible with this test system. Therefore,
based on the results of the solubility test and in consultation with the Sponsor, DMF
was selected as the vehicle of choice to prepare the stock and dilutions of the test
substance as well as the positive controls.
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-Aminoanthracene
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 48-72 hours
- Exposure duration: 67 hours

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 1-2x109 Colony Forming Units (CFU)/mL

DETERMINATION OF CYTOTOXICITY
- Method: Number of revertants per plate
The revertant colonies will be counted manually and the plates will be examined for bacterial
background lawn. The condition of the bacterial background lawn will be evaluated for evidence
of test substance toxicity and precipitate. Evidence of toxicity will be scored relative to the vehicle
control plate and recorded along with the revertant count for that plate. Toxicity will be evaluated
as a decrease in the number of revertant colonies per plate and/or a thinning or disappearance of
the bacterial background lawn. Precipitation will be evaluated after the incubation period by visual
examination without magnification.
Evaluation criteria:
The Salmonella typhimurium and Escherichia coli reverse mutation test is considered acceptable if
it meets the following criteria:
• Tester strain integrity
All Salmonella typhimurium tester strains must exhibit sensitivity to crystal violet and to
ultraviolet light to demonstrate the presence of rfa mutation and uvrB mutation,
respectively.
The Escherichia coli tester strain must exhibit sensitivity to ultraviolet light to
demonstrate the presence of uvrA mutation.
Salmonella typhimurium strains TA98 and TA100 and Escherichia coli strain WP2uvrA
(pKM101) must exhibit resistance to ampicillin to demonstrate the presence of the
plasmid R-factor.
• The spontaneous reversion rates in the solvent/vehicle control must be in the range of
in-house historical data
• All tester strain culture titers must be in the range of 1-2x109 cells/mL to ensure that
appropriate numbers of bacteria are used for plating.
• Each mean, positive control value must exhibit at least a 3.0-fold increase over the
respective mean vehicle control value for each tester strain.
• Toxicity: A minimum of three non-toxic dose levels will be required to evaluate assay
data. A dose level is considered toxic if it causes a >50% reduction in the mean number
of revertants per plate relative to the mean vehicle control value (this reduction must be
accompanied by an abrupt dose-dependent drop in the revertant count) or a reduction in
the background lawn. In the event that less than three non-toxic dose levels are achieved,
the affected portion of the assay will be repeated with an appropriate change in dose
levels.
Statistics:
none

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:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of the current study, the test substance, XU-18838.00 was negative
(non-mutagenic) in this Salmonella-Escherichia coli/Mammalian-Microsome Reverse
Mutation Assay.
Executive summary:

XU-18838.00 was tested for its mutagenic potential in the bacterial reverse mutation assay. The study was conducted using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli in two phases. In the first phase, an initial toxicity-mutation test was performed. The second phase was an independent confirmatory mutation test. The bacterial tester strains were exposed to the test substance in the presence and absence of a metabolic activation system (S-9 fraction prepared from Aroclor 1254 induced rat liver) using a preincubation procedure. XU-18838.00 was found to be soluble in Dimethyl formamide (DMF) at 50 mg/mL. In the initial toxicity-mutation assay, XU-18838.00 was exposed in duplicate at 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate test doses along with the vehicle and appropriate positive controls. The mean and standard deviation of revertant colonies were calculated for each test dose and the controls for all the tester strains. There was a moderate precipitation of the test substance on the basal agar plates at the top dose of 5000 µg/plate. No toxicity was observed up to 1500 XU-18838.00 was tested for its mutagenic potential in the bacterial reverse mutation assay. The study was conducted using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli in two phases. In the first phase, an initial toxicity-mutation test was performed. The second phase was an independent confirmatory mutation test. The bacterial tester strains were exposed to the test substance in the presence and absence of a metabolic activation system (S-9 fraction prepared from Aroclor 1254 induced rat liver) using a preincubation procedure. XU-18838.00 was found to be soluble in Dimethyl formamide (DMF) at 50 mg/mL. In the initial toxicity-mutation assay, XU-18838.00 was exposed in duplicate at 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate test doses along with the vehicle and appropriate positive controls. The mean and standard deviation of revertant colonies were calculated for each test dose and the controls for all the tester strains. There was a moderate precipitation of the test substance on the basal agar plates at the top dose of 5000 µg/plate. No toxicity was observed up to 1500 µg/plate, either in the presence or absence of metabolic activation as the intensity of the bacterial back-ground lawn was comparable to respective vehicle control plates. However, at the top dose of 5000 µg/plate, a slight thinning of the bacterial background lawn was noticed in the presence and absence of metabolic activation. There was no positive mutagenic response observed in any of the tester strains in any of the tested doses either in the presence or in the absence of metabolic activation.g/plate, either in the presence or absence of metabolic activation as the intensity of the bacterial back-ground lawn was comparable to respective vehicle control plates. However, at the top dose of 5000 µg/plate, a slight thinning of the bacterial background lawn was noticed in the presence and absence of metabolic activation. There was no positive mutagenic response observed in any of the tester strains in any of the tested doses either in the presence or in the absence of metabolic activation.

Based on these initial findings, in the confirmatory mutation assay, XU-18838.00 was exposed in triplicate at 100, 266, 707, 1880, and 5000 µg/plate test doses along with the vehicle and appropriate positive controls. The mean and standard deviation of revertant colonies were calculated for each test dose and the controls for all the tester strains. There was a moderate precipitation of the test substance on the basal agar plates at the top dose of 5000 µg/plate. No toxicity was observed up to 1880 µg/plate, either in the presence or absence of metabolic activation as the intensity of the bacterial back-ground lawn was comparable to respective vehicle control plates. However, at the top dose of 5000 µg/plate, a slight thinning of the bacterial background lawn was noticed in the presence and absence of metabolic activation. There was no positive mutagenic response observed in any of the strains in any of the tested doses either in the presence or in the absence of metabolic activation. In this study, there was a more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay.

All criteria for a valid study were met as described in the protocol. Under the conditions of the current study, the test substance, XU-18838.00 was negative (non-mutagenic) in this Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay.