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:
April 9 to May 8, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
in compliance with the UK Good Laboratory Practice Regulations 1999, Statutory Instrument No. 3 I06 as amended by the Good Laboratory Practice (Codification Amendments Etc.) Regulations 2004 and the OECD Principles on Good Laboratory Practice.
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Prednisolone mesylate (also known as Deltahydrocortisone mesylate)
- Substance type: organic
- Physical state: off-white powder
- Analytical purity: 99%
- Lot/batch No.: 0704742707
- Storage condition of test material: at 1-10ºC in the dark

Method

Target gene:
Histidine
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9)
Species / strain:
S. typhimurium TA 102
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9)
Test concentrations with justification for top dose:
Range-finding study: 1.6, 8, 40, 200, 1000 and 5000 μg/plate
Experiment 1: 0.32, 1.6, 8, 40, 200, 1000 and 5000 μg/plate
Experiment 2: 39.06, 78.13, 156.3, 312.5, 625, 1250, 2500 and 5000 μg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
Negative controls:
yes
Remarks:
vehicle
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA98 without S-9
Negative controls:
yes
Remarks:
vehicle
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100, TA1535 without S-9
Negative controls:
yes
Remarks:
vehicle
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537 without S-9
Negative controls:
yes
Remarks:
vehicle
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
TA102 without S-9
Negative controls:
yes
Remarks:
vehicle
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
TA98 with S-9
Negative controls:
yes
Remarks:
vehicle
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA100, TA1535, TA1537, TA102 with S-9
Evaluation criteria:
Acceptance criteria
The assay was considered valid if the following criteria were met:
1. the negative control counts fell within the normal ranges
2. the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9 preparation
3. no more than 5% of the plates were lost through contamination or some other unforeseen event.

Evaluation criteria
For valid data, the test article was considered to be mutagenic if:
1. Dunnett's test gave a significant response (p ≤ 0.01) and the data set(s) showed a significant concentration correlation
2. the positive responses described above were reproducible. The test article was considered as positive in this assay if all of the above criteria were met.
The test article was considered as negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.
Statistics:
The m statistic was calculated to check that the data are Poisson distributed, and Dunnett's test was used to compare the counts at each concentration with the control.
The presence or otherwise of a concentration response was checked by linear regression analysis. As multiple regression analysis calculations are performed (at each concentration), there is an increased incidence of values which fall outside the 95 or 99% probability range, but are not due to compound related
increases. Therefore, a statistically significant regression analysis is not considered as a biologically relevant event unless accompanied by a statistically significant Dunnett’s test.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
reduction in the number of revertants was observed at 1000 μg/plate and above
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: S. typhimurium TA 100 (Range-finding test)
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
reduction in the number of revertants at 1000 and/or 5000 µg/plate
Cytotoxicity:
yes
Remarks:
slight thinning of the background bacterial lawn at 1000 and/or 5000 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: TA98, TA1535, TA1537, TA102 (Experiment 1)
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
reduction in the number of revertants at 1000 and/or 5000 µg/plate
Cytotoxicity:
yes
Remarks:
slight thinning of the background bacterial lawn at 1000 and/or 5000 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: TA98, TA1535, TA1537, TA102 (Experiment 1)
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
reduction in the number of revertants at 1000 and/or 5000 µg/plate
Cytotoxicity:
yes
Remarks:
slight thinning of the background bacterial lawn at 1000 and/or 5000 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: TA98, TA1535, TA1537, TA102 (Experiment 1)
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
reduction in the number of revertants at 312.5 µg/plate and above or 625 µg/plate and above
Cytotoxicity:
yes
Remarks:
slight thinning of the background bacterial lawn at 312.5 µg/plate and above or 625 µg/plate and above
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: TA 1535, TA 1537, TA 98, TA 100 and TA102 (Experiment 2)
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
reduction in the number of revertants at 312.5 µg/plate and above or 625 µg/plate and above
Cytotoxicity:
yes
Remarks:
slight thinning of the background bacterial lawn at 312.5 µg/plate and above or 625 µg/plate and above
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: TA 1535, TA 1537, TA 98, TA 100 and TA102 (Experiment 2)
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
reduction in the number of revertants at 1250 μg/plate and above
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: TA 1535, TA 1537, TA 98, TA 100 and TA102 (Experiment 2)
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
reduction in the number of revertants at 1250 μg/plate and above
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: TA 1535, TA 1537, TA 98, TA 100 and TA102 (Experiment 2)
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: observed at the time of treatment at the highest treatment concentration (5000 μg/plate) in the Range-Finder and Experiment 1, and at 1250 μg/plate and above in Experiment 2. Precipitate was also observed following incubation at 5000 μg/plate in the Range-Finder Experiment, at 1000 μg/plate and above in Experiment 1 and at 1250 μg/plate and above in Experiment 2.

Any other information on results incl. tables

The individual plate counts were averaged to give mean values. From the data it can be seen that mean vehicle control counts fell within the normal historical ranges. The positive control chemicals all induced large increases in revertant numbers in the appropriate strains, which fell within the normal historical ranges. Less than 5% of plates were lost, leaving adequate numbers of plates at all treatments. The study therefore demonstrated correct strain and assay functioning and was accepted as valid.

No statistically significant, concentration-related and reproducible increases in revertant numbers were observed when the data were analysed at the 1% level using Dunnett’s test following any strain treatments in the absence or presence of metabolic activation.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Prednisolone mesylate did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg/plate, in the absence and in the presence of a rat liver metabolic activation system (S-9).