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Genetic toxicity in vitro

Description of key information

Genetic toxicity "in vitro" is discussed below.

Link to relevant study records
Reference
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:
December 17th 2002 to March 4th 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, carried out according to recognised guideline.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Metabolic activation:
with and without
Metabolic activation system:
Liver microsomal enzymes (S9 homogenate) prepared from male Sprague-Dawley rats injected with Arochlor 1254.
Test concentrations with justification for top dose:
33.3, 100, 333, 1000, 3330 and 5000 µg of Phosflex TXP per plate in the presence and absence of the S9 mix.
Vehicle / solvent:
DMSO

The test article was immiscible in water at concentrations of approximately 116.1, 58.1, and 29.1 mg per mL. The test article was miscible in ethanol, forming a solution at a concentration of442 mg per mL. The test article was miscible in dimethylsulfoxide (DMSO), forming a solution at concentrations of 445, 223, 112, 56.0, 28.0, and 14.0 mg per mL. Based on these results, DMSO (Acros Organics, Lot Nos. A015334901 and AO 17325001) was selected as the vehicle. At 100 mg/mL, which was the most concentrated stock dilution prepared, the test article was observed to form a transparent, non-viscous, colorless solution. The test article remained in solution at all succeeding lower dilutions prepared for the mutagenicity assay.
Negative solvent / vehicle controls:
yes
Remarks:
dimethylsulfoxide
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Positive controls:
yes
Positive control substance:
sodium azide
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (CAS# 613-13-8)
Positive controls:
yes
Positive control substance:
other: ICR-191 (CAS# 17070-45-0)
Details on test system and experimental conditions:
MATERIALS AND METHODS
The experimental materials, methods and procedures are based on those described by Ames et al. (1975) and Green and Muriel (1976). The assay design is based on the OECD Guideline 471, updated and adopted 21 July 1997.

Test System
Tester Strains. Tester strains used were Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 (Ames et al., 1975) and Escherichia coli tryptophan auxotroph WP2wvrA (Green and Muriel, 1976). Specific genotypes of the strains were documented.

In addition to a mutation in the histidine or tryptophan operons, tester strains contain additional mutations that enhance their sensitivity to some mutagenic compounds. Mutation of 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, Salmonella typhimurium tester strains containing this deletion also require the vitamin biotin for growth.

Salmonella typhimurium tester strains also contain the rfa wall mutation, which results in loss of one of the enzymes responsible for synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall. Resulting cell wall deficiency increases permeability to certain classes of chemicals such as those containing large ring systems (i.e., benzo[a]pyrene) otherwise excluded by a normal intact cell wall.
Tester strains TA98 and TA100 also contain the pKMlOl plasmid, which further increases the sensitivity of these strains to some mutagens. The suggested mechanism by which this plasmid increases sensitivity to mutagens is by modification of an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process.

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strains TA100, TA1535 and WP2wv/A are reverted from auxotrophy to prototrophy by base substitution mutagens.

Source of Tester Strains. 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, WP2wvrA, 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 tester strains were stored at -60°C to -80°C.

Master Plates. Master plates of 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 >0°C to 10°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 programmed to begin operation (shaking, 125 + 25 rpm; incubation, 37 ± 2°C) so overnight cultures were in late log phase when density monitoring began.

Harvest. To ensure that cultures were harvested in late log phase, length of incubation was determined by spectrophotometric monitoring of culture density. Cultures were harvested once a predetermined density was reached which ensured that cultures had reached a density of at least
0.5 x 109 cells/mL and had not overgrown. Overgrown (stationary) cultures may exhibit decreased sensitivity to some mutagens. Cultures were removed from incubation when target density was reached and were held at >0°C to 10°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 use in the mutagenicity assay:

rfa Wall Mutation. For Salmonella tester strains, presence of the rfa wall mutation was confirmed by demonstration of 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 ug of crystal violet was added. Sensitivity was demonstrated by inhibition of bacterial growth in a zone immediately surrounding the disk.

pKMlOl Plasmid. 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 ug 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 characteristic of the respective strains was demonstrated by plating 100 uL 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% (w/v) NaCl and was supplemented with 10 mL of 0.5 mM histidine/biotin solution per 100 mL agar for selection of histidine revertants or 0.5 mM tryptophan solution per 100 mL of agar for selection of tryptophan revertants.
When S9 was required, 2.0 mL of supplemented top agar was used in the overlay. However, when S9 was not required, water was added to supplemented top agar (0.5 mL of water per 2.0 mL of supplemented top agar), and the resulting 2.5 mL of diluted supplemented top agar was used for the overlay. This dilution ensured that final top agar and amino acid supplement concentrations remained the same in the presence and absence of S9.

Test Article
The Sponsor was responsible for the determination and documentation of the analytical purity and composition of the test article and the stability and strength of the dosing solutions. The test and control article dosing solutions were not analyzed for stability, homogeneity or accuracy of preparation.

Control Articles
Vehicle Controls. Vehicle controls were plated for all tester strains in the presence and absence of S9. The vehicle control was plated, using a 50 uL 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 uL aliquot of S9 mix (when necessary), on selective agar.

Positive Controls. The combinations of positive controls, activation conditions and tester strains plated concurrently with the assay are as indicated below in Table II

The sources and grades of positive control articles are as follows:
benzo[a]pyrene (CAS #50-32-8), Sigma Chemical Co., purity >97% 2-aminoanthracene (CAS #613-13-8), Sigma Chemical Co., purity >90% 2-nitrofluorene (CAS #607-57-8), Aldrich Chemical Co., purity >98% sodium azide (CAS #26628-22-8), Sigma Chemical Co., purity >99% ICR-191 (CAS #17070-45-0), Sigma Chemical Co., purity >90% 4-nitroquinoline-N-oxide (CAS #56-57-5), Sigma Chemical Co., purity >99%.
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 Metabolic Activation System
S9 Homogenate. Liver microsomal enzymes (S9 homogenate) were purchased from Molecular Toxicology, Inc., Lot 1471 (39.1 mg/mL protein) and Lot 1488 (37.4 mg/mL protein). The homogenate was prepared from male Sprague-Dawley rats that had been injected (i.p.) with Aroclor™ 1254 (200 mg/mL in corn oil) at 500 mg/kg as described by Ames et al, (1975).

S9 Mix. The S9 mix was prepared immediately prior to use and contained the components indicated in Table III below.

Dose 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 doses to be tested in the mutagenicity assay.

Design. The dose rangefinding study was performed using tester strains TA100 and WP2wvrA in the presence and absence of S9. Ten doses of test article, up to 5000 pg/plate, were tested for cytotoxicity (one plate per dose).

Rationale. Cytotoxicity of test article observed on tester strain TA100 is generally representative of that observed on other Salmonella typhimurium tester strains. 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. Escherichia coli tester strain WP2«vrA does not have the rfa wall mutation possessed by Salmonella typhimurium strains; thus, a different range of cytotoxicity may be observed. Also, 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 dose ranges be tested in the mutagenicity assay based on the presence or absence of the microsomal enzymes.

Evaluation of the Dose 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 Dose for the Mutagenicity Assay. Since no cytotoxicity was observed in the dose rangefinding study, the highest dose level of test article used in the mutagenicity assay was the same dose as that tested in the rangefinding study.

Mutagenicity Assay
Design. The assay was performed using tester strains TA98, TA100, TA1535, TA1537 and WPluvrA in the presence and absence of S9. Doses of test article were selected based on results of the dose rangefinding assay. Results of the initial mutagenicity assay were confirmed in an independent experiment.

Frequency and Route of Administration. Tester strains were exposed to test article via the plate incorporation methodology originally described by Ames 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 plate incorporation methodology, test article, tester strain, and S9 mix (when appropriate) were combined in molten agar, which was then overlaid onto a minimal agar plate. Following incubation, revertant colonies were counted. All doses of test article, vehicle controls, and positive controls were plated in triplicate.

Plating Procedures
These procedures were used in the dose rangefinding study and the mutagenicity assay.

Each plate was labeled with a code identifying the test article, test phase, tester strain, activation condition, and dose level. S9 mix and dilutions of test article were prepared immediately prior to use.

When S9 was not required, 100 uL of tester strain and 50 uL of test or control article were added to 2.5 mL of molten selective top agar (maintained at 45 ± 2°C). When S9 was required, 500 uL of S9 mix, 100 uL of tester strain and 50 uL of test or control article were added to 2.0 mL of molten selective top agar. After required components had been added, 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, plates were inverted and incubated for 52 ± 4 hours at 37±2°C.

Scoring the Plates
Plates not evaluated immediately following the incubation period were held at >0°C to 10°C until colony counting and bacterial background lawn evaluation could take place.

Bacterial Background Lawn Evaluation. Condition of the bacterial background lawn was evaluated 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 dose level. Lawns were scored as normal (N), reduced (R), obscured by precipitate (O), macroscopic precipitate present (P), absent (A), or enhanced (E); contaminated plates (C) also were noted.

Counting Revertant Colonies. Revertant colonies were counted by automated colony counter or by hand.

Data Presentation
For all replicate platings, mean revertants per plate and standard deviation were calculated. Results of these calculations are presented in tabular form in the Data Tables section of this report. Historical control data are presented after the data tables.

Assay Acceptance Criteria
Before assay data were evaluated, criteria for a valid assay had to be met. The following criteria were used to determine a valid assay:

Tester Strain Integrity. rfa Wall Mutation. All Salmonella typhimurium tester strain cultures exhibited sensitivity to crystal violet, demonstrating presence of the rfa wall mutation.

pKMlOl Plasmid. Tester strains TA98 and TA100 exhibited resistance to ampicillin, demonstrating presence of the pKMlOl plasmid.

Characteristic Number of Spontaneous Revertants. All vehicle control cultures, with the exception of tester strain TA1537 in the presence of S9 mix in Experiment 24695-B1, exhibited their characteristic number of spontaneous revertants per plate, demonstrating the requirement for histidine {Salmonella typhimurium) or tryptophan (Escherichia coli). acceptable ranges for the mean vehicle controls were as follows:

Tester Strain Culture Density. The cell densities of all tester strain cultures were >0.5 x 109 bacteria/mL, and/or the optical densities of these cultures reached a target value demonstrated to produce cultures with >0.5 x 109 bacteria/mL, demonstrating that appropriate numbers of bacteria were plated.

Positive Control Values in the Absence of S9 Mix. The mean value of the positive control for each tester strain exhibited at least a 3-fold increase over the mean value of the vehicle control for that strain, demonstrating that the tester strains were capable of identifying a mutagen.

Positive Control Values in the Presence of S9 Mix (S9 Mix Integrity). The mean value of the positive control for each tester strain exhibited at least a 3-fold increase over the mean value of the vehicle control for that strain, demonstrating that the S9 mix was capable of metabolizing a promutagen to its mutagenic form(s).

An acceptable positive control in the presence of S9 for a specific strain was evaluated as having demonstrated the integrity of the S9 mix and the ability of the tester strain to detect a mutagen.

Cytotoxicity. A minimum of three non-toxic doses was required to evaluate assay data. 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. A thinning of the bacterial background lawn that was not accompanied by a reduction in the number of revertants per plate was not evaluated as an indication of cytotoxicity.

Assay Evaluation Criteria
Once the criteria for a valid assay had been met, responses observed in the assay were evaluated.

Tester Strains TA98, TA100 and WP2«vrA. For a test article to be considered positive, it had to produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.

Tester Strains TA1535 and TA1537. For a test article to be considered positive, it had to produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.
Evaluation criteria:
Before assay data were evaluated, criteria for a valid assay had to be met. The following criteria were used to determine a valid assay:
tester strain integrity
tester strain culture density
positive control values in the absence of S9 mix
positive control values in the presence of S9 mix (S9 mix integrity)
cytotoxicity
Key result
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Dose rangefinding assay
Doses tested in the mutagenicity assay were selected based on results of the dose rangefinding assay conducted with the test article using tester strains TA100 and WP2uvrA in the presence and absence of S9 (one dose per plate). Ten doses of test article, from 6.67 to 5000 µg per plate were tested in trial 24695 -A1. No cytotoxicity (as evidenced by no decrease in the number of revertants per plate and normal bacterial background lawns) was observed with either tester strain at any dose tested in the presence or absence of the S9 mix.

Mutagenicity Assay
Results of the dose rangefinding study were used to select doses tested in the mutagenicity assay. Doses tested with all tester strains were 33.3, 100, 333, 1000, 3330 and 5000 µg per plate in the presence and absence of the S9 mix.
In the initial mutagenicity assay, all data generated with tester strains TA98, TA100, TA1535 and WP2uvrA were acceptable and no positive increases in the mean number of revertants per plate were observed with any of these tester strains in the presence or absence of S9 mix. The mean control vehicle value for tester strain TA1537 was not within the acceptable range in the presence of S9 mix. In addition, the mean vehicle control value for tester strain TA1537 in the absence of S9 mix was higher than routinely observed in this laboratory. For these reasons, the test article was retested with tester strain TA1537 in the presence and absence of S9 mix. In the confirmatory mutagenicity assay all dat were acceptable and no positive increases in the number of revertants per plate were observed with tester strain TA1537 in either the presence or absence of S9 mix.
All criteria for a valid study were met.
Remarks on result:
other: all strains/cell types tested
Conclusions:
The results of the Salmonella- Escherichia coli/Mammalian microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test article, Phosflex TXP, did not cause a positive increase in the mean number of revertants per plate with any of the tester strains in the presence or absence of Arochlor-induced rat liver S9.
Executive summary:

The results of the Salmonella- Escherichia coli/Mammalian microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test article, Phosflex TXP, did not cause a positive increase in the mean number of revertants per plate with any of the tester strains in the presence or absence of Arochlor-induced rat liver S9.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The substance was negative in the following tests:

1) Ames Test - salmonella

2) Chromosome aberration test.

3) Sister chromatid exchange assay in mammalian cells ( This study was read across to the analogue Tris(methylphenyl) phosphate, CAS 1330-78-5 which is considered an appropriate structural analogue.)

Justification for selection of genetic toxicity endpoint

GLP, carried out according to recognised guideline.

Justification for classification or non-classification

The above studies have all been ranked reliability 1 or 2 according to the Klimish et al system. This ranking was deemed appropriate because the studies were not conducted to GLP or in compliance with agreed protocols. The reports do not detail a specific method; however it documents dose levels and responses in detail, so is deemed appropriate for use in the support of a formal registration. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds.

Justification for classification or non classification

The above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008). No classification for mutagenic effects is therefore required.