Fusidic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental work started on 24-Jan-2007 and was completed on 20-Feb-2007.

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1997

Deviations:

yes

Remarks:

plate counts were slightly above the historical ranges, they were all sufficiently comparable to be considered as characteristic and acceptable for demonstrating the correct strain and assay functioning.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: UKEMS Guideline

Version / remarks:

1990

Deviations:

yes

Remarks:

plate counts were slightly above the historical ranges, they were all sufficiently comparable to be considered as characteristic and acceptable for demonstrating the correct strain and assay functioning.

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline

Version / remarks:

1997

Deviations:

yes

Remarks:

plate counts were slightly above the historical ranges, they were all sufficiently comparable to be considered as characteristic and acceptable for demonstrating the correct strain and assay functioning.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fusidic acid

EC Number:

230-256-0

EC Name:

Fusidic acid

Cas Number:

6990-06-3

Molecular formula:

C31H48O6

IUPAC Name:

2-[(1Z,2S,3aS,3bS,5aS,6S,7R,9aS,9bS,10R,11aR)-2-(acetyloxy)-7,10-dihydroxy-3a,3b,6,9a-tetramethyl-hexadecahydro-1H-cyclopenta[a]phenanthren-1-ylidene]-6-methylhept-5-enoic acid

Test material form:

liquid

Details on test material:

Yellow liquid

Specific details on test material used for the study:

Spiked Fusidic acid, batch number 0704514701

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments

Test concentrations with justification for top dose:

Concentrations based on range-finding study where toxicity was observed at a concentration of 200 ug/plate or higher

Experiment 1, using final concentrations of Spiked Fusidic Acid at 0.032, 0.16, 0.8, 4, 20, 100 and 500 μg/plate

Experiment 2, concentration ranges of 5.12 to 500 or 1250 μg/plate

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Negative controls comprised treatments with the vehicle sterile anhydrous analytical grade dimethyl sulphoxide (DMSO).

Controls 2

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Controls 3

Positive controls:

yes

Positive control substance:

sodium azide

Controls 4

Positive controls:

yes

Positive control substance:

9-aminoacridine

Controls 5

Positive controls:

yes

Positive control substance:

mitomycin C

Controls 6

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Controls 7

Positive controls:

yes

Positive control substance:

other:

Remarks:

2-aminoanthracene (AAN)

Details on test system and experimental conditions:

Bacteria Strain Type of mutation in the histidine gene
S. typhimurium TA98 frame-shift
S. typhimurium TA100 base-pair substitution
S. typhimurium TA1535 base-pair substitution
S. typhimurium TA1537 frame-shift
S. typhimurium TA102 base-pair substitution

Statistics:

The m-statistic was calculated to check that the data were Poisson-distributed (10), 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 (10). As multiple regression analysis calculations are
performed (at each concentration level), there is an increased incidence of values which will 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 results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxicity was only observed in experiment 2

Untreated negative controls validity:

valid

Remarks:

The mean numbers of revertant colonies on negative control plates were all considered acceptable,

Positive controls validity:

valid

Remarks:

The mean numbers of revertant colonies were significantly elevated by positive control treatments.

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxicity was only observed at the highest concentration tested (500 ug/plate and/or 1250 ug/plate)

Untreated negative controls validity:

valid

Remarks:

The mean numbers of revertant colonies on negative control plates were all considered acceptable,

Positive controls validity:

valid

Remarks:

The mean numbers of revertant colonies were significantly elevated by positive control treatments.

Test results 3

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxicity was only observed at the highest concentration tested (500 ug/plate and/or 1250 ug/plate)

Untreated negative controls validity:

valid

Remarks:

The mean numbers of revertant colonies on negative control plates were all considered acceptable,

Positive controls validity:

valid

Remarks:

The mean numbers of revertant colonies were significantly elevated by positive control treatments.

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxicity was only observed at the highest concentration tested (500 ug/plate and/or 1250 ug/plate)

Untreated negative controls validity:

valid

Remarks:

The mean numbers of revertant colonies on negative control plates were all considered acceptable,

Positive controls validity:

valid

Remarks:

The mean numbers of revertant colonies were significantly elevated by positive control treatments.

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxicity was only observed at the highest concentration tested (500 ug/plate and/or 1250 ug/plate)

Untreated negative controls validity:

valid

Remarks:

The mean numbers of revertant colonies on negative control plates were all considered acceptable,

Positive controls validity:

valid

Remarks:

The mean numbers of revertant colonies were significantly elevated by positive control treatments.

Any other information on results incl. tables

Experiment 1, using final concentrations of Spiked Fusidic Acid at 0.032, 0.16, 0.8, 4, 20, 100 and 500 μg/plate, plus negative (vehicle) and positive controls. Following these treatments, evidence of toxicity was observed in the absence and presence of S-9 at 500 μg/plate in all strains except TA98. Although no clear evidence of toxicity was seen in strain TA98, treatments in this strain were considered to have been performed at concentrations approaching toxic levels, based on the observations seen in the other test strains.

Following Experiment 2 treatments, clear evidence of toxicity was observed at either 500 μg/plate or 1250 μg/plate in all strains in the absence of S-9, and at either 200 μg/plate and above or 500 μg/plate (and above where applicable) in all strains in the presence of S-9.

Following Spiked Fusidic Acid treatments of all the tester strains, both in the absence and in the presence of a rat liver metabolic activation system (S-9), only Experiment 1 treatments of strain TA102 in the presence of S-9 resulted in any increase in revertant numbers that was statistically significant.

As the increase in Experiment 1 was neither doserelated nor reproducible, it was not considered to have been a biologically relevant or compound-related effect.

As no other strain treatments resulted in an statistically significant increases in revertant numbers, this study was considered to have provided no evidence of any Spiked Fusidic Acid mutagenic activity in this assay system.

Applicant's summary and conclusion

Conclusions:

Spiked Fusidic Acid was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation (S-9) , in two separate experiments. The study was performed in accordance to OECD 471. No toxicological significant increases in the frequecy of revertant colonies were recorded, thus it was concluded that Spiked Fusidic Acid does 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 or approaching the lower limit of toxicity, in the absence and in the presence of a rat liver metabolic activation system (S-9). Hence, Spiked Fusidic Acid was found to be non-mutagenic.

Executive summary:

Spiked Fusidic Acid was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation (S-9) , in two separate experiments. The study was performed in accordance to OECD 471.

Two experiments (refered to as experiment 1 and 2) were conducted:

Experiment 1, using final concentrations of Spiked Fusidic Acid at 0.032, 0.16, 0.8, 4, 20, 100 and 500 μg/plate, plus negative (vehicle) and positive controls. Following these treatments, evidence of toxicity was observed in the absence and presence of S-9 at 500 μg/plate in all strains except TA98. Although no clear evidence of toxicity was seen in strain TA98, treatments in this strain were considered to have been performed at concentrations approaching toxic levels, based on the observations seen in the other tester strains.

Following Experiment 2 treatments, clear evidence of toxicity was observed at either 500 μg/plate or 1250 μg/plate in all strains in the absence of S-9, and at either 200 μg/plate and above or 500 μg/plate (and above where applicable) in all strains in the presence of S-9.

No toxicological significant increases in the frequecy of revertant colonies were recorded, thus it was concluded that Spiked Fusidic Acid does 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 or approaching the lower limit of toxicity, in the absence and in the presence of a rat liver metabolic activation system (S-9). Hence, Spiked Fusidic Acid was found to be non-mutagenic.