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Genetic toxicity: in vitro

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
TA98 hisD3052
TA100 hisG46
TA1535 hisG46
TA1537 hisC3076
WP2uvrA trp
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Test concentrations with justification for top dose:
Up to 25 micrograms/plate
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: 2-nitrofluorene, 2-aminoantracene, sodum azide, ICR-191, 4-nitroquinoline-N-oxide
Details on test system and experimental conditions:
In addition to a mutation in either the histidine or tryptophan opérons, the tester strains contain two additional mutations which enhance their sensitivity to some mutagenic compounds. A mutation of either the uvrA gene (Escherichia coli) or the uvrB gene (Salmonella typhimurium), results in a deficient DNA excision repair system which greatly enhances the sensitivity of these strains to some mutagens. Since the uvrB deletion extends through the bio gene, the Salmonella typhimurium tester strains containing this deletion also require the vitamin biotin for growth.

The Salmonella typhimurium tester strains also contain the rfa wall 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 permeability to certain classes of chemicals such, as those containing large ring systems (i.e., benzo[a]pyrene) that would otherwise be excluded by a normal intact cell wall.

Strains TA98 and TA100 also contain the pKMlOl plasmid, which further increases the sensitivity of these strains to some mutagens. The mechanism by which this plasmid increases sensitivity to mutagens has been suggested to be by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process.

Tester strains TA98 and TA 1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strains TA100, TA1535, and WP2«vrA are reverted from auxotrophy to prototrophy by base substitution mutagens.

Source of Tester Strains. The Salmonella typhimurium tester strains in use at Covance were received directly from Dr. Bruce Ames, Department of Biochemistry, University of California, Berkeley. The Escherichia coli tester strain, WP2«vrA, was received from The National Collection of Industrial Bacteria, Torrey Research Station, Scotland (United Kingdom).

Frozen Permanent Stocks. Frozen permanent stocks were prepared by growing fresh overnight cultures, adding DMSO (0.09 mL/mL of culture) and freezing away appropriately vialed aliquots. Frozen permanent stocks of the tester strains were stored at <-70°C.

Master Plates. Master plates of the tester strains were prepared by streaking each tester strain from a frozen permanent stock onto minimal agar appropriately supplemented with either histidine and biotin or tryptophan, and for strains containing the pKMlOl plasmid, ampicillin. Tester strain master plates were stored at 5 ± 3°C.

Preparation of Overnight Cultures
Inoculation. Overnight cultures for use in all testing procedures were inoculated by transferring a colony from the appropriate master plate to a flask containing culture medium. Inoculated flasks were placed in a shaker/incubator which was programmed to begin operation (shaking, 125 ± 25 rpm; incubation, 37 ± 2°C) so that the overnight cultures were in log phase or late log phase when turbidity monitoring began.

Harvest. To ensure that cultures were harvested in late log phase, the length of incubation was determined by spectrophotometric monitoring of culture density. Cultures were harvested once a predetermined density was reached which ensures that cultures had reached a density of at least 0.5 X 109 cells per mL and that the cultures have not overgrown. Overgrown (stationary) cultures may exhibit decreased sensitivity to some mutagens. Cultures were removed from incubation when the target density was reached and were held at 5 ± 3°C until used in the assay.

Confirmation of Tester Strain Genotype. Tester strain cultures were checked for the following genetic markers on the day of their use in the mutagenicity assay:
rfa Wall Mutation. For the Salmonella tester strains, the presence of the rfa wall mutation was confirmed by demonstration of the sensitivity of the culture to crystal violet. An aliquot of an overnight culture of each strain was overlaid onto plates containing selective media and an antibiotic sensitivity disk containing 10 u,g of crystal violet was added. Sensitivity was demonstrated by inhibition of bacterial growth in a zone immediately surrounding the disk.
pKMlOl Plasmid. The presence of the pKMlOl plasmid was confirmed for cultures of tester strains TA98 and TA100 by demonstration of resistance to ampicillin. An aliquot of an overnight culture of each strain was overlaid onto plates containing selective media and an antibiotic sensitivity disk containing 10 jxg of ampicillin was added. Resistance was demonstrated by growth in the zone immediately surrounding the disk.

Characteristic Number of Spontaneous Revertants. The mean number of spontaneous revertants per plate in the vehicle controls that is characteristic of the respective strains was demonstrated by plating 100 jxL aliquots of each culture along with the appropriate vehicle on
selective media.

Culturing Broth. The broth used to grow overnight cultures of the tester strains was Vogel-Bonner salt solution (Vogel and Bonner, 1956) supplemented with 2.5% (w/v) Oxoid Nutrient Broth No. 2 (dry powder).

Minimal Bottom Agar Plates. Bottom agar (25 mL per 15 x 100 mm petri dish) was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956), supplemented with 1.5% (w/v) agar and 0.2% (w/v) glucose.

Top Agar for Selection of Revertants. Top (overlay) agar was prepared with 0.7% agar (w/v) and 0.5% NaCl (w/v) and was supplemented with 10 mLof 1) 0.5 mM histidine/biotin solution per 100 mL agar for selection of histidine revertants, or 2) 0.5 mM tryptophan solution per 100 mL of agar for selection of tryptophan revertants. For the agar overlay, 2.0 mL of the supplemented top agar was used.

Control Articles
Vehicle Controls. Vehicle controls were plated for all tester strains in the presence and absence of S9 mix. The vehicle control was plated, using a 50 uJL aliquot of dimethylsulfoxide (equal to the maximum aliquot of test article dilution plated), along with a 100 uL aliquot of the appropriate tester strain and a 500 uJL aliquot of S9 mix (when necessary), on selective agar.

Sterility Controls. The most concentrated test article dilution was checked for sterility by plating a 50 uL aliquot (the same volume used in the assay) on selective agar. The S9 mix was checked for sterility by plating 0.5 mL on selective agar.

S9 Metabolie Activation System
S9 Homogenate. Liver microsomal enzymes (S9 homogenate) were purchased from Molecular Toxicology, Inc., Batch 1204 (40.0 mg of protein per mL). The homogenate was prepared from male Sprague-Dawley rats that had been injected (i.p.) with Aroclor™ 1254 (200 mg per mLin corn oil) at 500 mg/kg as described by Ames et al., (1975).
S9 Mix. The S9 mix was prepared immediately prior to its use in any experimental procedure.

Rangefinding assay
The growth inhibitory effect (cytotoxicity) of the test article to the test system was determined in order to allow the selection of appropriate concentrations to be tested in the mutagenicity assay.
Design. The rangefinding study was performed using tester strains TA 100 and WP2«vrA in both the presence and absence of S9 mix. Ten concentrations of test article were tested at one plate per concentration. The test article was checked for cytotoxicity up to a maximum concentration
of 5 mg per plate.
Rationale. The cytotoxicity of the test article observed on tester strain TA100 is generally representative of that observed on the other Salmonella typhimurium tester strains and because of the comparatively high number of spontaneous revertants per plate observed with this strain, gradations of cytotoxicity can be readily discerned from routine experimental variation. The Escherichia coli tester strain WP2«vrA does not possess the rfa wall mutation that the Salmonella typhimurium strains have and thus, a different range of cytotoxicity may be observed. Also, the cytotoxicity induced by a test article in the presence of S9 mix may vary greatly from that observed in the absence of S9 mix. Therefore, this would require that different test article concentration ranges be tested in the mutagenicity assay based on the presence or absence of the microsomal enzymes.
Evaluation of the Rangefinding assay. Cytotoxicity is detectable as a decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn.
Selection of the Maximum Concentration for the Mutagenicity Assay. Cytotoxicity was observed in the rangefinding study and the highest concentration level of test article used in the subsequent mutagenicity assay was a concentration which gave a reduction of revertants per plate and/or a thinning or disappearance of the bacterial background lawn.
Mutagenicity Assay
Design. The assay was performed using tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA both in the presence and absence of S9 mix along with the appropriate vehicle and positive controls. The concentrations of test article were selected based on the results of the rangefinding assay. The results of the initial mutagenicity assay were confirmed in an independent experiment.
Frequency and Route of Administration. The tester strains were exposed to the test article via the preincubation modification of the Ames Test originally described by Yahagi et al. (1975) and Maron and Ames (1983). This methodology has been shown to detect a wide range of classes of chemical mutagens. In the preincubation methodology, S9 mix (or phosphate buffer, where appropriate), the tester strain, and the test article were preincubated prior to the addition of molten agar. The agar and the preincubation reaction mixture were mixed and then overlaid onto a minimal agar plate. Following incubation, revertant colonies were counted. All concentrations of the test article, the vehicle controls and the positive controls were plated in triplicate.
Plating Procedures
These procedures were used in both the rangefinding study and the mutagenicity assay. Each plate was labeled with a code which identified the test article, test phase, tester strain, activation condition and concentration level. The S9 mix and dilutions of the test article were
prepared immediately prior to their use.
When S9 mix was required, 500 uL of S9 mix was added to 13 X 100 mm glass culture tubes, which had been pre-heated to 37 ± 2°C. To these tubes was added 100 uL of tester strain and 50 uL of vehicle or test article concentration. When S9 mix was not required, 500 uL of 0.1M
phosphate buffer was substituted for the S9 mix. After the required components had been added, the mixture was vortexed and allowed to incubate for 20 ± 2 minutes at 37 ± 2°C. Two mL of molten selective top agar was then added to each tube, and the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar contained in a 15 x 100 mm petri dish. After the overlay solidified, the plates were inverted and incubated for 52 ± 4 hours at 37 ± 2°C. Positive control articles were plated using a 50 uL plating aliquot.
Evaluation criteria:
Scoring the Plates
Plates which were not evaluated immediately following the incubation period were held at 5 ± 3°C until such time that colony counting and bacterial background lawn evaluation could take place.
Bacterial Background Lawn Evaluation. The condition of the bacterial background lawn was evaluated both macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that concentration level. Lawns were scored as 1) normal, 2) slightly reduced, 3) moderately reduced, 4) extremely reduced, 5) absent, or 6) obscured by precipitate. If present on the plates, macroscopic precipitate was scored as slight, moderate or heavy.
Counting Revertant Colonies. Revertant colonies were counted either by automated colony counter or by hand. If there was sufficient test article precipitate on the plates at any concentration that interferes with automated colony counting, then the plates at that concentration
were counted manually.
Statistics:
For all replicate platings, the mean revertants per plate and the standard deviation were calculated.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
The results of the Salmonella-Escherichia co/i/Mammalian-Microsome Reverse Mutation Assay Preincubation Method with a Confirmatory Assay indicate that under the conditions of this study, the test article, BADGE-2HCL, did not cause a positive increase in the mean number of revenants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor™-induced rat liver (S9). Hence, BADGE-2HCL was considered to be non mutagenic in this assay.
Executive summary:

The objective of this study was to evaluate the test article, Bisphenol A-bis-(3-chlor-2-hydroxypropyl)-ether (BADGE-2HCL), for the ability to induce reverse mutations either in the presence or absence of mammalian microsomal enzymes at 1) the histidine locus in the genome of several strains of Salmonella typhimurium and at 2) the tryptophan locus of Escherichia coli strain WP2«vrA. This assay satisfied the following guidelines: U.S. EPA (1998), EEC (2000), and OECD (1997).

The concentrations tested in the mutagenicity assay were selected based on the results of a , rangefinding assay using tester strains TA 100 and WP2wvrA and ten concentrations of test article ranging from 6.67 to 5000 u,g per plate, one plate per concentration, both in the presence and absence of S9 mix.

The tester strains used in the mutagenicity assay were Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 and Escherichia coli tester strain WP2uvrA. The assay was conducted with a minimum of six concentration levels of test article in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per concentration. The concentrations tested in the mutagenicity assay with the Salmonella tester strains ranged from 1.00 to 1000 \ig per plate in both the presence and absence of S9 mix (0.333 to 1000 ug per plate with TA 1537 in the absence of S9 mix). The concentrations tested in the

mutagenicity assay with Escherichia coli tester strain WP2uvrA ranged from 10.0 to 5000 ug per plate in both the presence and absence of S9 mix. The results of the initial mutagenicity assay were confirmed in an independent experiment.

The results of the Salmonella-Escherichia co/i/Mammalian-Microsome Reverse Mutation Assay Preincubation Method with a Confirmatory Assay indicate that under the conditions of this study, the test article, BADGE-2HCL, did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor™-induced rat liver (S9). Hence, BADGE-2HCL was considered to be non mutagenic in this assay.