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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2008 - February 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Test Material generically named as : FR 930

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
FR-930 was evaluated in the initial mutagenicity assay, in all five tester strains, at doses of 1.00, 3.33, 10.0, 33.3, 100, 333, 10007 and 5000 microg/plate with S9 and 10.0, 33.3, 100, 333, 1000, 3330, and 5000 microg/plate without S9.
Vehicle / solvent:
DMSO
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
other: ICR-191
Remarks:
without metabolic activation
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-aminoantracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
Test System Rationale
The bacterial reverse mutation assay has been shown to be a sensitive, rapid and accurate indicator of the mutagenic activity of many materials including a wide range of chemical classes. By using several different tester strains, both base pair substitution and frameshift mutations can be detected. Salmonella and E. coli strains used in this assay are histidine and tryptophan auxotrophs, respectively, by virtue of conditionally lethal mutations in the appropriate operons. When these histidine (his-) or tryptophan (tm) dependent cells are exposed to the test article and grown under selective conditions (minimal media with a trace amount of histidine or tryptophan), only those cells which revert to histidine (his) or tryptophan (trp+) independence are able to form colonies. Trace amounts of histidine or tryptophan added to the media allow all the plated bacteria to undergo a few cell divisions, which is essential for mutagenesis to be fully expressed. his* or trp revertants are readily discernable as colonies against the limited backyound growth of his- or trp- cells.

Tester Strains
The tester strains used were the Salmonella histidine auxotrophs TA98, TA 100, TA 1535, and TA 1537 (Ames et al. 1975) and the E. coli tryptophan auxotroph WP2uvrA (Green and Muriel 1976). Specific genotypes of the strains are shown below (Table Ill).


In addition to a mutation in either the histidine or tryptophan operons, the tester strains contain additional mutations that enhance their sensitivity to some mutagenic compounds. Mutation of the uvrA gene (E. coli) or the uvrB gene (Salmonella) results in a deficient DNA excision repair system that greatly enhances the sensitivity of these strains to some mutagens. Since the uvrB deletion extends through the bio gene, Salmonella tester strains containing this deletion also require the vitamin biotin for gowth.

Salmonella tester strains also contain the rfa walk mutation, which results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide (LPS) barrier that forms the surface of the bacterial cell wall. The resulting cell wall deficiency increases penneability to certain classes of chemicals, such as those containing large ring systems (i.e., benzo[a]pyrene), that otherwise would be excluded by a normal cell wall.

Tester strains TA98 and TAI 00 also contain the pKM101 plasmid, which further increases the sensitivity of these strains to some mutagens. The suggested mechanism for this increased sensitivity is modification of an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process.
Tester strains TA98 and TAI 537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. In contrast, tester strains TAI 00, TAl 535, and WP2uvrA are reverted from auxofrophy to prototrophy by base substitution mutagens.

Source of Tester Strains
Salmonella tester strains were received from Dr. Bruce Ames, Department of
Biochemistry, University of California (Berkeley, CA). E. coli tester strain WP2uvrA
Rationale for test conditions:
Cytotoxicity of the test article observed in tester stain TAl 00 is generally representative of that observed in the other Salmonella tester strains. Because ofTA100's comparatively high spontaneous revertant frequency (revertant colonies/plate), gadations of cytotoxicity can be readily discerned from routine experimental variation. E. coli tester strain WP2uvrA does not have the rfa wall mutation possessed by the Salmonella strains; therefore a different range of cytotoxicity may be observed. Also, cytotoxicity of a test article in the presence of S9 may vary greatly from that observed in its absence, requiring that different dose ranges be evaluated in the mutagenicity assay with and without S9.
Evaluation criteria:
Criteria for a Positive Response
A test article is considered to have produced a positive response if it induces a dosedependent increase in revertant frequency that is 22.0-fold vehicle control values for tester strains TA98, TAI 00, and WP2uvrA, or 23.0-fold vehicle control values for tester strains TAl 535 and •TA1537. In addition, any response should be reproducible.

Criteriafor a Negative Response
A test article is considered to have produced a negative response if no dose-dependent,
22.0-fold or 23.0-fold increases are observed in tester strains TA98, TAI 00, and WP2uvrA, or TAI 535 and TA1537, respectively.

Criteria for an Equivocal Response
Even after repeated trials, a test article may produce results that are neither clearly positive nor clearly negative (e.g., responses that do not meet the dose-dependency or fold increase requirements but are reproducible). In those rare instances, the test article may be considered to have produced an equivocal response.

Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director will use sound scientific judgrnent and clearly report and describe any such considerations.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Additional information on results:
Test Article Handling
The test article was found to form an opaque, white, heterogenous, non-viscous suspension in deionized water at a concentration of approximately 100 mg/mL. The test article was found to fom a transparent, colorless, non-viscous solution in DMSO at a concentration of approximately 100 mg/mL, which was the most concentrated dose formulation prepared for treatment; it remained freely soluble at all succeeding lower dilutions prepared for the assay.

Dose Analyses
Determination of stability, homogeneity and concentration of the dosing preparations was not conducted.

Dose Range-finding Assay
FR-930 was evaluated in the dose range-finding assay in tester strains TA 100 and WP2uvrA. Ten doses of test article, from 6.67 to 5000 microg/plate, were evaluated with and without S9 (one plate per dose; Trial 29938-A1). No cytotoxicity was observed with either tester strain in the presence or absence of S9 asevidenced by no dose-related decreases in revertant frequency. Normal bacterial background lawns were observed up to 667 microg/plate with both tester strains in the absence of S9, and with TA 100 in the presence of S9. Normal bacterial backgound lawns were observed up to 1000 microg/plate with WP2uvrA in the presence of S9. Test article precipitate was observed on the plates with both tester sfrains at doses >= 1000 microg/plate with and without S9. The test article precipitate obscured the bacterial backgound lawns at >= 1000 microg/plate (>= 3330 microg/plate for WP2uvrA with S9). Dose-related increases in revertant frequency were observed with TA 100 in the presence and absence ofS9.

Mutagenicity Assay
Based upon the results of the dose range-finding assay, FR930 was evaluated in the initial mutagenicity assay, in all five tester strains, at doses of 1.00, 3.33, 10.0, 33.3, 100, 333, 1000 and 5000 microg/plate with S9 and 10.0, 33.3, 100, 333, 1000, 3330, and 5000 microg/plate without S9 (Trial 29938-B1. All doses of the test article, as well as the concurrent positive and vehicle controls, were evaluated in triplicate plates. Normal growth again was observed in all five tester strains evaluated with and without S9 except at the highest 1-2 doses where the background lawns were obscured by precipitate. Revertant frequencies for all doses of FR-930 in tester strains TA98, TA 1537, and WP2uvrA with and without S9 approximated or were less than those observed in the concurrent vehicle control cultures. In contrast, dose-dependent increases in revertant frequencies were observed in TA 100 at doses of >= 100 microg/plate with S9 and at
> 3330 microg/plate without S9 and in TA 1535 at doses of >= 100 microg/plate with S9 and at >= 1000 ug/plate without S9. In addition, the test article again was found to be incompletely soluble in the aqueous top agar at all doses evaluated >= 333 microg/plate with and without S9.
FR-930 was re-evaluated in the confimatory mutagenicity assay under identical conditions, and similar results were observed (Trial 29938-C). Normal growth again was observed in all five tester strains evaluated with and without S9 except at 5000 microg/plate where the background lawns were obscured by precipitate. Revertant frequencies for all doses of FR-930 in tester strains TA98 and WP2uvrA with and without S9 approximated or were less than those observed in the concurrent vehicle control cultures. In contrast, dose-dependent increases in revertant frequencies were observed in TA 100 at doses of >= 100 microg/plate with S9 and at >= 1000 pg/plate without S9 and in TA 1535 at doses of >= 10.0 microg/plate with S9 and at >= 3330 gg/plate without S9. In addition, the test article again was found to be incompletely soluble in the aqueous top agar at the top 3 doses with and without S9. The mean positive control value for TA 1537 without S9 did not meet the minimum protocol criteria for an acceptable positive control (3-fold increase over the vehicle control). For this reason, a repeat confirmatory assay was conducted in TA 1537 with and without S9.
FR-930 was re-evaluated in a repeat confirmatory mutagenicity assay under identical conditions, and similar results to the initial mutagenicity assay were observed (Trial 29938-DI). Normal growth again was observed with and without S9 except at 5000 gg/plate where the background lawns were obscured by precipitate. Revertant frequencies for all doses of FR-930 in TA 1537 with and without S9 approximated or were less than those observed in the concurrent vehicre control cultures.
All positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met.

Applicant's summary and conclusion

Conclusions:
SUMMARY
The objective of this study is to evaluate the test article, FR-930, and/or its metabolites for their ability to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (Salmonella; TA98, TA 100, TA 1535, and TA 1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).
FR-930 was evaluated in the dose range-finding assay in tester strains TA 100 and WP2uvrA. Ten doses of test article, from 6.67 to 5000 microg/plate, were evaluated with and without S9 (one plate per dose). No cytotoxicity was observed with either tester strain in the presence or absence of S9 as evidenced by no dose-related decreases in revertant frequency. Normal bacterial background lawns were observed up to 667 microg/plate with both tester strains in the absence of S9, and with TA 100 in the presence of S9. Normal bacterial background lawns were observed up to 1000 microg/p]ate with WP2uvrA in the presence of S9. Test article precipitate was observed on the plates with both tester strains at doses >= 1000 microg/plate with and without S9. The test article precipitate obscured the bacterial background lawns at >= 1000 ug/plate (>= 3330 pg/plate for WP2uvrA with S9). Dose-related increases in revertant frequency were observed with TA 100 in the presence and absence of S9.
Based upon the results of the dose range-finding assay, FR-930 was evaluated in the initial mutagenicity assay, in all five tester strains, at doses of 1.00, 3.33, 10.0, 33.3, 100, 333, 1000 and 5000 microg/plate with S9 and 10.0, 33.3, 100, 333, 1000, 3330, and
5000 microg/plate without S9. All doses of the test article, as well as the concurrent positive and vehicle controls, were evaluated in triplicate plates. Normal growth again was observed in all five tester strains evaluated with and without S9 except at the highest 1-2 doses where the background lawns were obscured by precipitate. Revertant frequencies for all doses of FR-930 in tester strains TA98, TA 1537, and WP2uvrA with and without S9 approximated or were less than those observed in the concurrent vehicle control cultures. In contrast, dose-dependent increases in revertant frequencies were observed in TA 100 at doses of >= 100 microg/plate with S9 and at >= 3330 microg/plate without S9 and in TA 1535 at doses of >= 100 microg/piate with S9 and at >= 1000 microg/plate without S9. In addition, the test article again was found to be incompletely soluble in the aqueous top agar at all doses evaluated >= 333 microg/plate with and without S9.
FR-930 was re-evaluated in the confirmatory mutagenicity assay under identical conditions, and similar results were observed. Nonnal growth again was observed in all five tester strains evaluated with and without S9 except at 5000 microg/plate where the background lawns were obscured by precipitate. Revertant frequencies for all doses of FR-930 in tester strains TA98 and WP2uvrA with and without S9 approximated or were less than those observed in the concurrent vehicle control cultures. In contrast, dosedependent increases in revertant frequencies were observed in TA 100 at doses of >= 100 microg/plate with S9 and at >= 1000 microg/plate without S9 and in TA 1535 at doses of >= 10.0 gg/plate with S9 and at >= 3330 microg/plate without S9. In addition, the test article again was found to be incompletely soluble in the aqueous top agar at the top 3 doses with and without S9. The mean positive control value for TA 1537 without S9 did not meet the minimum protocol criteria for an acceptable positive control (3-fold increase over the vehicle control). For this reason, a repeat confirmatory assay was conducted in TA1537 with and without S9.
FR-930 was re-evaluated in a repeat confirmatory mutagenicity assay under identical conditions, and similar results to the initial mutagenicity assay were observed. Norma! growth again was observed with and without S9 except at 5000 microg/plate where the background lawns were obscured by precipitate. Revertant frequencies for all doses of FR-930 in TA 1537 with and without S9 approximated or were less than those observed in the concurrent vehicle control cultures.
Except as noted, all positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met.
These results indicate FR-930 was positive with tester strains TA 100 and TA 1535 both with and without S9 in the Bacterial Reverse Mutation Assay with a Confirmatory Assay under the conditions, and according to the criteria, of the test protocol.

CONCLUSION
These results indicate FR-930 was positive with tester strains TA 100 and TA 1535 both with and without S9 in the Bacterial Reverse Mutation Assay with a Confirmatory Assay under the conditions, and according to the citeria, of the test protocol.