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Genetic toxicity: in vivo

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in vivo mammalian germ cell study: gene mutation
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well-documented
Reason / purpose:
reference to same study

Data source

Reference Type:
Quantification of Kras mutant fraction in the lung DNA of mice exposed to aerosolized particulate vanadium pentoxide by inhalation
Banda, M. et al.
Bibliographic source:
Mutation Research 789-790: 53–60

Materials and methods

Test guideline
no guideline available
Principles of method if other than guideline:
This study investigated whether Kras mutation is an early event in the development of lung tumours induced by inhalation of particulate vanadium pentoxide aerosols. This study sought to: 1) characterize any Kras mutational response with respect to vanadium pentoxide exposure concentration, and 2) investigate the possibility that amplification of preexisting Kras mutation is an early event in vanadium pentoxide-induced mouse lung tumorigenesis. Male Big Blue B6C3F1 mice (6 mice/group) were exposed to aerosolized particulate vanadium pentoxide by inhalation, six hours/day, five days/week for four or eight weeks, using vanadium pentoxide exposure concentrations of 0, 0.1, and 1 mg/m³. The levels of two different Kras codon 12 mutations [GGT→GAT (G12D) and GGT→GTT (G12V)] were measured in lung DNAs by Allele-specific Competitive Blocker PCR (ACB-PCR).
GLP compliance:
Type of assay:
other: Kras mutation in the development of lung tumours

Test material

Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Vanadium pentoxide (milled test substance; supplied by EvrazStratcor, Inc. (Hot Springs, AR)
- Molecular formula: V2O5
- Physical state: yellow powder
Purity: 100.4 % (based on analytical quantification of vanadium, including measurement uncertainty)
- Lot no.: H091608:62
- Expiration date: January 2023
- Storage condition of test material: stored at room temperature

Test animals

other: Big Blue (BB) B6C3F1
Details on test animals and environmental conditions:
- Source: BioReliance, Rockville, MD (supplied through Taconic Farms, Germantown, New York)
- Age: eight weeks old
- Weight: control: 27.1 ± 1.02 g; 0.1 mg/m³ dose: 27.3 ± 1.01 g; 1.0 mg/m³ dose: 27.6 ± 1.53 g
- Housing: during the quarantine period (prior to group assignment), animals were housed in whole body stainless steel mouse cages used in Hazleton-2000 chambers. After randomization, animals were housed in Hazleton-2000 chambers as racks for the whole body stainless steel 40 compartment mouse cages.
- Diet (ad libitum, except during inhalation exposures): certified irradiated NTP-2000 Diet (Zeigler Brothers, Inc. Gardeners, PA)
- Water (ad libitum, except during inhalation exposures): drinking water (City of Chicago)
- Acclimation period: at least one week

- Temperature: 20-29ºC
- Relative humidity: 17-70%
- Air changes: minimum of 10/hour
- Photoperiod (hrs dark / hrs light): 12/12

The study employed currently acceptable practices of good animal husbandry (Guide for the Care and Use of Laboratory Animals; National Research Council,2011) and all animal care and use procedures used in the study were approved by IIT Research Institute Institutional Animal Care and Use Committee.

Administration / exposure

Route of administration:
inhalation: aerosol
- Vehicle(s)/solvent(s) used: air
Details on exposure:

- Method of holding animals in test chamber: animals were restrained in holding tubes (CH Technologies, Westwood, NJ, USA) connected to the exposure chamber ports. Animals were acclimated to the tubes over the course of three days (1.5, 3 and 4.5 hours/day, respectively) prior to the beginning of the test period.
- System of generating particulates/aerosols: the vanadium pentoxide test atmosphere was generated by aerosolization of the test substance using compressed air-operated Wright Dust Feeder Aerosol Generation systems (BGI Incorporated, Waltham, MA), which were positioned over each chamber. The test substance was weighed and packed into a reservoir using a hydraulic shop press, forming a cake. A thin film of material was removed from the cake by a scraper rotating at a constant speed and dispersed as an aerosol with the aid of compressed air. The dispersed material was mixed with filtered, breathable quality compressed air in a mixing plenum to the appropriate vanadium pentoxide concentration prior to introduction to the inhalation chambers.
- Method of particle size determination: aerosol particle size distribution in the inhalations chambers were measured at preselected intervals using quartz crystal microbalance (QCM) cascade impactor (California Measurements Inc., Sierra Madre, CA) equipped with 10 stages to collect size-segregated samples.

- Brief description of analytical method used: test material concentration in the exposure atmosphere was determined by collecting samples on mixed cellulose ester filters followed by chemical analysis to determine vanadium content.
Duration of treatment / exposure:
4 and 8 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrationsopen allclose all
Doses / Concentrations:
0.1 and 1.0 ppm V2O5
nominal conc.
Doses / Concentrations:
0.093 and 1.178 mg/m³ V2O5
analytical conc.
4 week exposure
Doses / Concentrations:
0.103 and 1.041 mg/m³ V2O5
analytical conc.
8 week exposure
No. of animals per sex per dose:
6 male mice per dose and exposure duration
Control animals:
yes, sham-exposed
Positive control(s):


Tissues and cell types examined:
Please refer to the field "Details of tissue and slide preparation" below.
Details of tissue and slide preparation:
Sham-exposed and vanadium pentoxide-exposed mice were sacrificed, then lungs were removed and snap-frozen in liquid nitrogen. The left lobe of each mouse lung was placed on dry ice until ACB-PCR analysis of Kras mutation.

Clinical signs were evaluated twice a day and any abnormal findings were recorded.

- lung tissues were minced
- lung tissue sample were homogenized in 1 mL of extraction buffer (0.5 mg/mL proteinase K, 20 mM NaCl, 1 mM CaCl2, pH 8.0, and 10 mM Tris pH 8.0).
- samples were incubated for 3 hours at 37°C
- samples were extracted with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1), and ethanol-precipitated.
- samples were resuspended in 400 μL of RNase buffer (10 mg/mL RNase A, 600 units/mL Ribonuclease T1, 100 mM sodium acetate, and 50 mM Tris-HCl (pH 8))
- samples were incubated ~16 hours at 37°C, then re-extracted with phenol/chloroform/isoamyl alcohol as described above.
- each ethanol-precipitated sample was resuspended in 50 μL of TE buffer (5 mM Tris, 0.5 mM EDTA, pH 7.5).
- DNA was digested with HindIII according to the manufacturer’s instructions (New England Biolabs).
- DNA was phenol/chloroform/isoamyl alcohol extracted and ethanol-precipitated, as described above, then resuspended in 20 μL of TE buffer.
- DNA concentrations were measured spectrophotometrically, diluted to ~0.5 μg/uL, and then the diluted DNA concentration was measured again.

- first-round PCR products were prepared from standards and unknowns as previously described [Parsons, 2013]*.
- first-round PCR amplification, employing the PfuUltra hotstart high-fidelity DNA polymerase amplified a Kras gene sequence from linearized plasmid DNAs, in order to synthesize the mutant frequency standards.
- similarly, HindIII-digested, lung DNA samples were used to amplify Kras gene sequence from each DNA sample.
- optimized PCR conditions were used to ensure any PCR-induced errors would occur at a level below that measurable by ACB-PCR.
- HindIII-digested, lung DNA was used for first-round PCR amplification of a 170 bp gene segment encompassing part of the 5’ untranslated region, exon 1, and part of intron 1 (NC_000072 Region: 29,950 to 30,119).
- 200 μL PCR reaction contained: 1 μg genomic DNA, 200 nM primer TR67 (TR67, 5’-TGGCTGCCGTCCTTTACAA-3’), 200 nM primer TR68 (TR68, 5’-GGCCTGCTGAAAATGACTGAGTATAAACTTGT-3’), 200 nM dNTPs, 1X PfuUltra reaction buffer, and 10 units PfuUltra hotstart high-fidelity DNA Polymerase.
- cycling conditions were 94°C for 2 minutes, followed by 28 cycles of 94°C for 1 minute, 58°C for 2 minutes, 72°C for 1 minute, followed by a 7 minutes extension at 72°C.
- primers were purchased from Integrated DNA Technologies.

- PCR products (standards and unknowns) were purified by ion-pair reverse phase chromatography using a WAVE Nucleic Acid Fragment Analysis System.
- PCR products were complexed with 0.1 M triethylammonium acetate (Buffer A: 0.1M TEAA)
- PCR products were bound to a DNASep column (containing C18 alkylated PS/DVB polymer).
- PCR products, input template, unincorporated nucleotides, and primers were eluted using a gradient of increasing acetonitrile concentration (Buffer B: 0.1 M TEAA, 25% acetonitrile), thereby separating nucleic acids by size/column retention time.
- threshold collection method was used to collect the 170 bp PCR products based on their absorbance at 260 (using a UV detector at the appropriate retention time) into individual tubes in a chilled fraction collector.
- PCR products were evaporated to dryness using a Savant Speed-VacConcentrator (Model ISS110).
- PCR products were resuspended in TE buffer
- multiple 2-μl aliquots were prepared (stored at -80°C).
- multiple aliquots were repeatedly quantified using an Epoch Micro-Volume Spectrophotometer System with a Take3 Microplate Reader, until three measurements that varied by <10% from the group mean were obtained.

- purified mutant and wild type first-round PCR products (generated using plasmid templates) were mixed to generate standards with mutant frequencies of 10^-1, 10^-2, 10^-3, 10^-4, 10^-5, and 0.
- duplicate mutant frequency standards and a no-DNA control were analysed in parallel with equal numbers of copies of first-round PCR products generated from mouse lung DNA samples.
- ACB-PCR analysis of both mutations: a total of 5 x 10^8 copies were analysed in 50 μL reactions performed in 96-well plates using a DNA Engine Tetrad 2.

1) Quantification of Kras codon 12 GAT mutation:
- each ACB-PCR reaction contained: 1X Standard Taq (Mg-free) reaction buffer, 0.1 mg/mL gelatin, 1 mg/mL Triton X-100, 40 μM dNTPs, 1.6 mM MgCl2, 150 nM mutant-specific primer (TR76, 5’-fluorescein-CTTGTGGTGGTTGGAGCTAA-3’), 520 nM blocker primer (TR77, 5’-CTTGTGGTGGTTGGAGCTAdG-3’), and 150 nM upstream primer (TR73, 5’-TCGTAGGGTCGTACTCATC-3’).
- each reaction was initiated with the addition of 1.2 units of Extreme Thermostable Single-stranded DNA Binding Protein, 0.33 mUnits of PerfectMatch PCR Enhancer, and 70 mUnits of Hemo KlenTaq DNA polymerase.
- cycling conditions were 2 minutes at 94°C, followed by 36 cycles of 94°C for 30 seconds, 45°C for 45 seconds, and 72°C for 1 minute.
- Kras codon 12 GAT ACB-PCR product is 91 bp in length.

2) Quantification of Kras codon 12 GTT mutation:
- each ACB-PCR reaction contained: 1X Standard Taq (Mg-free) reaction buffer, 0.1 mg/mL gelatin, 1 mg/mL Triton X-100, 40 μM dNTPs, 1.5 mM MgCl2, 400 nM mutant-specific primer (TR87, 5’-fluorescein-CTTGTGGTGGTTGGAGCTAT-3’), 440 nM blocker primer (TR113, 5’-CTTGTGGTGGTTGGAGCTTG-3’-phosporylation), and 400 nM upstream primer (TR110, 5’-TCGTAGGGTCATACTCATC-3’).
- each reaction was initiated with the addition of 160 mUnits of PerfectMatch PCR Enhancer (Stratagene), and 80 mUnits of Hemo KlenTaq DNA polymerase.
- cycling conditions were 2 minutes at 94°C, followed by 36 cycles of 94°C for 30 seconds, 41°C for 45 seconds, and 72°C for 1 minute.
- Kras codon 12 GTT ACB-PCR product is 91 bp in length.

- following ACB-PCR, 10 μL of bromophenol blue/xylene cyanol-containing 6X ficol loading dye were added to each well of the 96-well plate.
- 15 μL of each Kras codon 12 GAT ACB-PCR reaction or 10 μL of each Kras codon 12 GTT ACB-PCR reaction were analysed on 8% nondenaturing polyacrylamide gels.
- fluorescent DNA length marker was used to confirm the identity of the 91-bp products.
- fluorescent bands were visualized using a PharosFX scanner with an external blue laser.
- pixel intensities of the bands were quantified using Quantity One® software and a locally-averaged background correction.

- B.L. Parsons, M.G. Manjanatha, M.B. Myers, K.L. McKim, S. D. Shelton, Y. Wang, B.B. Gollapudi, N.P. Moore, L.T. Haber, M.M. Moore, Temporal Changes in K-ras Mutant Fraction in Lung Tissue of Big Blue B6C3F1 Mice Exposed to Ethylene Oxide, Toxicological Sciences, 136 (2013) 26-38.
Evaluation criteria:
no data
The pixel intensities determined for the mutant frequency standards were plotted against their mutant frequencies on log-log plots. A trend line (power function) was fitted to the data and the formula of the function was used to calculate the mutant frequency in each unknown sample based on its pixel intensity. The arithmetic average of the three independent mutant frequency measurements was calculated.The mean of the three independent measurements and the standard error of the mean were calculated and plotted using GraphPad Prism Version 5. The average mutant frequency in each lung DNA sample was log-transformed and the average log-transformed mutant frequency for the six mice in each treatment group was calculated. This value is the geometric mean mutant frequency for each treatment group.
Because a significant portion of the ACB-PCR measurements was below the limit of accurate ACB-PCR quantification (10^-5), the statistical significance of treatment effects was performed by comparing the distribution of samples above and below 10^-5 among treatment groups, using a Fisher’s exact test. The Spearman correlation coefficient was used to determine whether the Kras codon 12 GAT and GTT mutant frequencies within particular mice were correlated. Relationships between these variables were also visualized using linear regression analyses. All statistical analyses were performed using GraphPad Prism Version 5 (GraphPad Software, Inc., La Jolla, CA), two-sided tests, and a significance level of 0.05.

Results and discussion

Test results
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
- wet inguinal fur was seen in all mice including controls, likely resulting from being restrained in the nose-only exposure tubes. No other clinical signs were observed and all animals survived the 4 and 8 week exposure periods.
- a statistically significant increase (P ≤ 0.05) in the lung weights was observed in the mice exposed to vanadium pentoxide at the 1 mg/m³ level after 4 and 8 weeks; however, no effect on lung weights was apparent in the 0.1 mg/m³ animals at either time [Manjanatha, in press].

- following DNA isolation, first-round PCR products encompassing Kras exon 2 sequences were generated from the 36 study samples, which were produced with similar yields.
- three independent ACB-PCR measurements were collected on each sample.

- average correlation coefficient found were as follows:
Kras codon 12 GAT standard curves: 0.9496 (range 0.9435 to 0.9528).
Kras codon 12 GTT standard curves: 0.9594 (range 0.9579 to 0.962)

- coefficient of determination were as follows:
for triplicate GAT mutant frequency measurements: 0.71.
for the triplicate GTT mutant frequency measurements: 0.82.

- all ACB-PCR mutant frequency data were log-transformed.

- 8-week Kras codon 12 GAT and 4- and 8-week Kras codon 12 GTT datasets included measurements below the limit of accurate ACB-PCR quantification (10^-5). Therefore, the significance of potential treatment effects within the datasets was analysed by comparing the number of samples with mutant frequencies greater than and less than 10^-5, using Fisher’s exact test. No significant differences were observed among treatment groups.

- a correlation analysis was performed to determine whether the levels of the two Kras mutations within individual mice were correlated. Only seven samples had measureable levels of both mutations (i.e., >10^-5). The two mutations were correlated at the 0.1, but not the 0.05, confidence level (Spearman r = 0.7143, P = 0.0881).

- M.G. Manjanatha, S.D. Shelton, L.T. Haber, B. Gollapudi, M. J.A., N. Rajendran, M.M. Moore, Evaluation of cII mutations in lung of male Big Blue mice exposed by inhalation to vanadium pentoxide for up to 8 weeks, Mutation Research - Genetic Toxicology and Environmental Mutagenesis, (in press).

Applicant's summary and conclusion

Interpretation of results (migrated information): negative
Inhalation of aerosols of particulate divanadium pentaoxide for 4 or 8 weeks did not result in significant changes in levels of Kras codon 12 GAT or GTT mutation. The data support the idea that the accumulation of additional Kras mutants is not an early event, and/or that the proliferative advantage of Kras mutant clones requires either longer expression times or larger cumulative divanadium pentaoxide exposures. Furthermore, the data do not provide support for either a direct genotoxic effect of divanadium pentaoxide on Kras in the context of the exposure conditions used, or early amplification of preexisting mutation as being involved in the genesis of divanadium pentaoxide-induced mouse lung tumours.