MATERIALS AND METHODS

Sodium Bromate

Sodium bromate was obtained from Fisher Scientific (Fairlawn, NJ) in one lot (946272A) that was used in the 26- and 39-week dermal studies and in the 27- and 43-week drinking water studies. Identity and purity analyses were conducted by the analytical chemistry laboratories, Research Triangle Institute (RTI) (Research Triangle Park, NC) and Battelle Memorial Institute (Columbus, OH), and by the study laboratory, Battelle Columbus Operations (Columbus, OH) (Appendix G). Reports on analyses performed in support of the sodium bromate studies are on file at the National Institute of Environmental Health Sciences.

Lot 946272A, a white crystalline powder, was identified as sodium bromate by infrared (IR) spectroscopy, elemental analysis, and inductively coupled plasma emission (ICP) spectrometry. The purity of lot 946272A was determined using ion chromatography (IC) and iodometric titration of bromate ions. IC indicated one major peak and no impurities with relative areas greater than 0.1% and purities of 101.2% relative to a frozen reference standard and 97% based on bromate anion recovery. Iodometric titration indicated a purity of approximately 99%. The overall purity of lot 946272A was determined to be 99% or greater.

To ensure stability, the bulk chemical was stored at room temperature, protected from light. Stability was monitored during the 26-, 27-, 39-, and 43-week studies using IC. No degradation of the bulk chemical was detected.

12-O-Tetradecanoylphorbol-13-acetate

12-O-tetradecanoylphorbol-13-acetate (TPA) was obtained from Sigma-Aldrich Chemical Company (St. Louis, MO) in one lot (48H1178) that was used for positive control Tg.AC hemizygous mice in the 26- and 27-week studies.

Lot 48H1178, a white crystalline powder, was identified as 12-O-tetradecanoylphorbol-13-acetate using IR and proton nuclear magnetic resonance spectrometry. The purity of lot 48H1178 was determined using high performance liquid chromatography. The overall purity of lot 48H1178 was determined to be greater than 99%.

Ethanol

USP-grade 95% ethanol was obtained from Spectrum Chemicals and Laboratory Products (Gardena, CA) in one lot (NK0283) that was used in the 26- and 39-week dermal studies.

Lot NK0283, a clear liquid, was identified as ethanol using IR spectroscopy. The purity of lot NK0283 was determined using gas chromatography (GC). GC indicated one major peak and no impurities with areas greater than or equal to 0.1% of the major peak. The overall purity of lot NK0283 was determined to be greater than 99%.

The purity of the bulk chemical was periodically monitored during the studies using GC. No degradation of the ethanol was detected.

Dermal Studies

The dose formulations were prepared every 4 to 5 weeks by mixing sodium bromate and 40% USP-grade 95% ethanol/60% deionized water to give the required concentrations (Table G1). The dose formulations were stored at room temperature in clear glass bottles for up to 35 days. A positive control dose formulation of TPA was prepared twice during the studies by adding the appropriate amount of TPA to acetone; the formulations were stored at approximately 5° C in amber glass bottles for up to 6 months.

Stability studies of a 5.67 mg/mL dose formulation were conducted using IC. Stability was confirmed for at least 35 days for dose formulations stored in 100 mL glass bottles at temperatures up to ambient, for at least 3 hours for dose formulations stored in capped, partially filled clear glass scintillation vials, and for at least 24 hours for dose formulations stored exposed to light and air at 25° C to 30° C followed by resolubilization in 40% USP-grade 95% ethanol/60% deionized water.

Periodic analyses of the dose formulations of sodium bromate were conducted using IC. During the 26- and 39-week studies, dose formulations were analyzed four times. All 12 of the dose formulations used for Tg.AC hemizygous mice were within 10% of the target concentrations (Table G2).

Drinking Water Studies

The dose formulations were prepared every 2 to 5 weeks by adding a specified amount of sodium bromate to tap water (Table G1). The dose formulations were stored for up to 35 days at room temperature in the NALGENE^® tanks in which they were mixed. A positive control dose formulation of TPA was prepared and stored as described for the dermal studies.

Stability studies of a 5.67 mg/mL dose formulation were conducted by the analytical chemistry laboratory using IC. Stability was confirmed for at least 35 days for dose formulations stored in 100 mL glass bottles at temperatures up to ambient, and for at least 7 days in partially filled 500 mL clear glass drinking bottles.

Periodic analyses of the dose formulations of sodium bromate were conducted by the study laboratory using IC. During the 27- and 43-week studies, the dose formulations were analyzed four times. All 13 of the dose formulations used for Tg.AC hemizygous and p53 haploinsufficient mice were within 10% of the target concentrations (Table G3). No sodium bromate was found in control formulations above a method detection limit of 0.45 mg/L.

Dermal Studies

Groups of 15 male and 15 female Tg.AC hemizygous mice received dermal applications of 0, 64, 128, or 256 mg/kg sodium bromate in ethanol/water (40%/60%) 5 days per week for 26 weeks; the dosing volume was 3.3 mL/kg. Groups of 10 male and 10 female Tg.AC hemizygous mice were administered the same doses for 39 weeks. Vehicle control mice were administered the ethanol/water vehicle only. Doses were applied to the clipped dorsal skin from the mid-back to the interscapular area. A high dose of 800 mg/L was selected for the drinking water studies based on a carcinogenicity study of potassium bromate administered in the drinking water to mice (DeAngelo et al., 1998).

Drinking Water Studies

Groups of 15 male and 15 female Tg.AC hemizygous and p53 haploinsufficient mice were exposed to drinking water containing 0, 80, 400, or 800 mg/L sodium bromate for 27 weeks. Groups of 10 male and 10 female Tg.AC hemizygous and p53 haploinsufficient mice were exposed to the same concentrations for 43 weeks. A 14-day range finding study was conducted in FVB/N mice to determine doses for the 26-week study in Tg.AC mice (data not shown). The doses selected were 0, 16, 32, 64, 128, and 256 mg/kg, which were comparable on a weight basis to the concentrations used in the drinking water studies. Because there was no toxicity in the dose range finding study, concentrations of 64, 128, and 256 mg/kg were selected for the 26-week Tg.AC dermal study.

Positive Control Mice

Since it has been found that a subset of Tg.AC mice may revert and become nonresponsive to a tumor promoter (Honchel et al., 2001), positive control groups of 15 male and 15 female Tg.AC hemizygous mice were administered 1.25 µg TPA in 100 µL acetone (12.5 µg TPA/L solution) dermally, three times per week for 26 to 27 weeks to confirm that the mice used in these dermal and drinking water studies of sodium bromate were responsive to carcinogens. The TPA solution was applied to the clipped dorsal skin from the mid-back to the interscapular area. Positive control mice were removed from study after the appearance of 20 or more skin papillomas and discarded.

Source and Specification of Animals

Male and female FVB/N-TgN(v-Ha-ras) (Tg.AC) hemizygous (Tg.AC hemizygous) and B6.129-Trp53^tmlBrd (N5) haploinsufficient (p53 haploinsufficient) mice were obtained from Taconic Laboratory Animals and Services (Germantown, NY) for use in the 26-, 27-, 39-, and 43-week studies. Tg.AC hemizygous mice were quarantined for 16 days before the beginning of the dermal studies and for 13 days before the beginning of the drinking water studies; p53 haploinsufficient mice were quarantined for 14 days before the beginning of the drinking water studies. Five male and five female mice per strain were randomly selected for parasite evaluation and gross observation of disease. Tg.AC hemizygous mice were 6 weeks old and p53 haploinsufficient mice were 6 to 8 weeks old at the beginning of the studies. Blood samples were collected from five male and five female sentinel mice per strain and study group at 4 and 26 weeks, from five male and five female mice from the highest surviving groups at 39 or 43 weeks, and from moribund mice in the 43-week drinking water studies after June 5, 2000. The sera were analyzed for antibody titers to rodent viruses (Boorman et al., 1986; Rao et al., 1989a,b). All results were negative.

Animal Maintenance

Mice were housed individually and feed and water were available ad libitum. Water consumption was measured weekly by cage during the drinking water studies. Cages and racks were rotated every two weeks. Further details of animal maintenance are given in Table 1.

Table 1

Experimental Design and Materials and Methods in the Dermal and Drinking Water Studies of Sodium Bromate.

Clinical Examinations and Pathology

All animals were observed twice daily. Clinical findings and body weights were recorded postdosing on day 1 (dermal studies), initially (drinking water studies), weekly, and at the end of the studies.

In-life observations of papilloma formation on the skin were recorded weekly using the Toxicology Data Management System (TDMS). A papilloma was initially recorded as a mass. The observation “papilloma” was not entered into TDMS for a given animal until the first-observed mass was documented for 3 consecutive weeks. At the third observation, the mass (wart-like in appearance) was entered as a papilloma. Any new mass(es) appearing after the 3-week confirmation period for a given animal at a different site was entered into TDMS first as a mass until the third week, when it was entered as a papilloma. In a few instances, a papilloma that had been previously observed was missing, and therefore not recorded. Reappearance of a mass at a later time was entered into TDMS as a mass until the third observation week, when it was called a papilloma.

At the end of the 26- and 27-week studies, blood was collected from the retroorbital sinus of all mice except the positive control groups for hematology analyses. The mice were anesthetized with a mixture of carbon dioxide and oxygen. Samples for hematology analysis were placed in microcollection tubes (Sarstedt, Inc., Nümbrecht, Germany) coated with potassium EDTA. Hematocrit; erythrocyte, platelet, and leukocyte counts; mean cell hemoglobin; and mean cell hemoglobin concentration were determined with a Cell-Dyn^® hematology analyzer (Abbott Diagnostics, Santa Clara, CA).

Hemoglobin concentrations were determined photometrically using a cyanmethemoglobin procedure. Differential leukocyte counts were determined microscopically from blood smears stained with a modified Wright-Giemsa stain. A Miller Disc was used to determine reticulocyte counts from smears prepared with blood stained with new methylene blue. Mean cell volumes were determined from average red blood cell impedance pulse heights. The parameters measured are listed in Table 1.

Necropsies and microscopic examinations were performed on all mice except the positive controls. The heart, right kidney, liver, lung, right testis, and thymus were weighed. At necropsy, all organs and tissues were examined for grossly visible lesions, and all major tissues were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 µm, and stained with hematoxylin and eosin for microscopic examination. For all paired organs (e.g., adrenal gland, kidney, ovary), samples from each organ were examined. Tissues examined microscopically are listed in Table 1.

Microscopic evaluations were completed by the study laboratory pathologist, and the pathology data were entered into the Toxicology Data Management System. The slides, paraffin blocks, and residual wet tissues were sent to the NTP Archives for inventory, slide/block match, and wet tissue audit. The slides, individual animal data records, and pathology tables were evaluated by an independent quality assessment laboratory. The individual animal records and tables were compared for accuracy, the slide and tissue counts were verified, and the histotechnique was evaluated. For the 39- and 43-week studies, a quality assessment pathologist evaluated slides from all tumors and all potential target organs, which included the kidney and liver of Tg.AC hemizygous and p53 haploinsufficient mice and the pituitary gland, testis, and thyroid gland of Tg.AC hemizygous mice.

For the 39- and 43-week studies, a quality assessment pathologist evaluated slides from all tumors and all potential target organs, which included the kidney and liver of Tg.AC hemizygous and p53 haploinsufficient mice and the pituitary gland, testis, and thyroid gland of Tg.AC hemizygous mice. The quality assessment report and the reviewed slides for the 39- and 43-week studies were submitted to the NTP Pathology Working Group (PWG) chairperson, who reviewed the selected tissues and addressed any inconsistencies in the diagnoses made by the laboratory and quality assessment pathologists. Representative histopathology slides containing examples of lesions related to chemical administration, examples of disagreements in diagnoses between the laboratory and quality assessment pathologists, or lesions of general interest were presented by the chairperson to the PWG for review. The PWG consisted of the quality assessment pathologist and other pathologists experienced in rodent toxicologic pathology. This group examined the tissues without any knowledge of dose groups or previously rendered diagnoses. When the PWG consensus differed from the opinion of the laboratory pathologist, the diagnosis was changed. Final diagnoses for reviewed lesions represent a consensus between the laboratory pathologist, reviewing pathologist(s), and the PWG. Details of these review procedures have been described, in part, by Maronpot and Boorman (1982) and Boorman et al. (1985). For subsequent analyses of the pathology data, the decision of whether to evaluate the diagnosed lesions for each tissue type separately or combined was generally based on the guidelines of McConnell et al. (1986).

The 26- and 27-week studies had not undergone a quality assessment review prior to completion of the pathology review for the 39- and 43-week studies. For these studies, a quality assessment pathologist evaluated all tumor diagnoses from all animals and all potential target organs (both genders, both strains, both routes of administration) which included the lung, thyroid, adrenal cortex, kidney, forestomach, liver, thymus, and skin, using terminology and diagnostic criteria defined by the Pathology Working Group for the 39- and 43-week studies in order to maintain diagnostic consistency between the studies. The quality assessment pathologist and two NTP pathologists met to review selected examples of lesions related to chemical administration, and to address any disagreements in the diagnoses made by the laboratory and quality assessment pathologists. Final diagnoses for reviewed lesions represent a consensus between the laboratory pathologist, the quality assessment pathologist, and the NTP pathologists.

Survival Analyses

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Animals found dead of other than natural causes or missing were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

Calculation and Analysis of Lesion Incidence

The incidences of neoplasms or nonneoplastic lesions are presented in Appendixes A, B, and C as the numbers of animals bearing such lesions at a specific anatomic site and the numbers of animals with that site examined microscopically. The Fisher exact test (Gart et al., 1979), a procedure based on the overall proportion of affected animals, was used to determine significance.

Analysis of Continuous Variables

Two approaches were employed to assess the significance of pairwise comparisons between dosed or exposed and control groups in the analysis of continuous variables. Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology data, which has typically skewed distributions, was analyzed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey (1951) were examined by NTP personnel, and implausible values were eliminated from the analysis. Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973).

Mouse Peripheral Blood Micronucleus Test Protocol

A detailed discussion of this assay is presented by MacGregor et al. (1990). At the end of the 26- and 27-week studies, peripheral blood samples were obtained from male and female mice. Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs) in each of up to 15 animals per group. In addition, the percentage of polychromatic erythrocytes (PCEs) in a population of 1,000 erythrocytes was determined as a measure of bone marrow toxicity.

The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over dose or exposure groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each dosed or exposed group and the control group (ILS, 1990). In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed or exposed group is less than or equal to 0.025 divided by the number of dosed or exposed groups. A final call of positive for micronucleus induction is preferably based on reproducibly positive trials (as noted above). Results of the 26- and 27-week studies were accepted without repeat tests, because additional test data could not be obtained. Ultimately, the final call is determined by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects. The percentage of polychromatic erythrocytes data were analyzed by an analysis of variance (ANOVA) test based on individual animal data.

Evaluation Protocol

These are the basic guidelines for arriving at an overall assay result for assays performed by the National Toxicology Program. Statistical as well as biological factors are considered. For an individual assay, the statistical procedures for data analysis have been described in the preceding protocols. There have been instances, however, in which multiple aliquots of a chemical were tested in the same assay, and different results were obtained among aliquots and/or among laboratories. Results from more than one aliquot or from more than one laboratory are not simply combined into an overall result. Rather, all the data are critically evaluated, particularly with regard to pertinent protocol variations, in determining the weight of evidence for an overall conclusion of chemical activity in an assay. In addition to multiple aliquots, the in vitro assays have another variable that must be considered in arriving at an overall test result. In vitro assays are conducted with and without exogenous metabolic activation. Results obtained in the absence of activation are not combined with results obtained in the presence of activation; each testing condition is evaluated separately. The summary table in the Abstract of this Report presents a result that represents a scientific judgement of the overall evidence for activity of the chemical in an assay.