Trimethylolpropane Triacrylate

Trimethylolpropane triacrylate was obtained from Aldrich Chemical Company (Milwaukee, WI) in one lot (01031AW), which was used throughout the studies. Identity, moisture content, purity, and stability analyses were conducted by the analytical chemistry laboratories and the study laboratory (Battelle Columbus Laboratories, Columbus, OH). Reports on analyses performed in support of the trimethylolpropane triacrylate studies are on file at the National Institute of Environmental Health Sciences.

The chemical, a colorless to yellow viscous liquid, was identified as trimethylolpropane triacrylate by the analytical chemistry laboratory using infrared spectroscopy and by proton and carbon-13 nuclear magnetic resonance spectroscopy and by the study laboratory using infrared spectrometry. The analytical laboratories and the study laboratory determined moisture content using Karl Fischer titration and purity using elemental analyses, gas chromatography, high-performance liquid chromatography (HPLC), and HPLC with mass spectrometry (HPLC/MS). Karl Fischer titration indicated approximately 747 ppm water. Elemental analyses for carbon, hydrogen, and oxygen were in agreement with the theoretical values for trimethylolpropane triacrylate. Gas chromatography indicated one major peak and two impurities with areas of 6.5% and 3.4% relative to the major peak area. HPLC indicated a major peak and five impurities with a combined area of 22.2%. HPLC/MS indicated 10 impurities including the five impurities found by HPLC. These impurities included four structurally related acrylates or adducts: trimethylolpropane diacrylate, trimethylolpropane triacrylate acrylic acid adduct, trimethylolpropane triacrylate-trimethylol propane monoacrylate adduct, and trimethylolpropane triacrylate-trimethylolpropane diacrylate adduct. The overall purity of lot 01031AW was estimated to be approximately 80%.

To ensure stability, the bulk chemical was stored at room temperature, protected from light, in amber glass bottles with Teflon^®-lined lids. Stability was monitored throughout the studies with gas chromatography. No degradation of the bulk chemical was detected.

12-O-Tetradecanoylphorbol-13-acetate

12-O-Tetradecanoylphorbol-13-acetate was obtained from Sigma Chemical Company (St. Louis, MO) in one lot (48H1178) for use in the 6-month study. Identity and purity analyses were conducted by the analytical chemistry laboratory, Research Triangle Institute (Research Triangle Park, NC). The bulk chemical was stored in its original containers, protected from light, at −20° C or less.

The chemical was identified as 12-O-tetradecanoylphorbol-13-acetate by infrared and proton NMR spectroscopy. The purity was determined with HPLC, which indicated a major peak, one impurity peak with an area of approximately 0.11% of the total peak area, and two minor impurities with areas less than 0.1% of the total peak area. The overall purity was determined to be greater than 99%.

Acetone

Acetone was obtained in two lots from Honeywell Burdick and Jackson (Muskegon, MI) (lots BK792 and BL631) and in five lots from Spectrum Chemical Manufacturing Corporation (Gardena, CA) (lots JE342, KP206, LS0051, MI0172, and NE0173). Lots BK792, BL631, and JE342 were used in the 2-week studies, lots KP206 and LS0051 were used in the 3-month studies, and lots MI0172 and NE0173 were used in the 6-month study. Identity and purity analyses of lots BL631 and JE342 and all lots used in the 3- and 6-month studies were conducted by the analytical chemistry laboratory (Midwest Research Institute, Kansas City, MO) and the study laboratory.

The chemical, a clear liquid, was identified as acetone by the analytical chemistry laboratory (lots BL631, JE342, KP206, and LS0051) or the study laboratory (lots MI0172 and NE0173) using infrared spectroscopy. The purity was analyzed by the analytical chemistry laboratory (lots BL631, JE342, KP206, and LS0051) or the study laboratory (lots MI0172 and NE0173) using gas chromatography. No significant impurities were detected in any lot. The overall purity of each lot was determined to be greater than 99%.

To ensure stability, the bulk chemical was stored in amber glass bottles at room temperature. Stability was monitored with gas chromatography. No degradation of the acetone was detected.

Study Design

Groups of 15 male and 15 female mice received dermal applications of 0, 0.75, 1.5, 3, 6, or 12 mg/kg in acetone 5 days per week for 28 weeks; the dosing volume was 3.3 mL/kg. Additional groups of 15 male and 15 female mice maintained as positive controls received dermal applications of 1.25 µg 12-O-tetradecanoylphorbol 13-acetate per 100 mL acetone 3 days per week for 28 weeks; the dosing volume was held constant at 100 µL.

Source and Specification of Animals

The foundation colony of FVB/N-TgN(v-Ha-ras) (i.e. Tg.AC) mice was reestablished in 1998 after observation of some Tg.AC mice that were nonresponsive to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate after treatment with a defined exposure regimen known to induce skin papillomas at the site of application. The homozygous FVB/N-TgN(v-Ha-ras) (i.e. Tg.AC) colony was established using homozygous breeders showing an unequivocal pattern of bands of restriction enzyme digests of DNA (Thompson et al., 1998, 2001; Honchel et al., 2001) demonstrating a specific phenotype for the induction of papillomas by 12-O-tetradecanoyl-phorbol-13-acetate. All foundation colony breeders homozygous for the transgene are genotyped and test-mated with wildtype FVB mice and qualified as a homozygous transgenic mouse with a responder phenotype. Homozygous male breeders obtained from the litters of qualified homozygous foundation colonies were further qualified by test mating with wild type FVB/N mice and used to produce hemizygous Tg.AC mice. All Tg.AC hemizygous male and female mice are the product of this continuing quality control of the foundation and production colony.

Male and female Tg.AC hemizygous transgenic mice were obtained from the NIEHS/NTP colony at Taconic Laboratory Animals and Services. On receipt, the mice were 4 weeks old. Animals were quarantined for 11 days and were 6 weeks old on the first day of the studies. Before the study began, five male and five female mice were randomly selected for parasite evaluation and gross observation for evidence of disease. The health of the animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program (Appendix J).

Animal Maintenance

Mice were housed individually. The core study mice were housed in the same room with positive control mice and mice in the pentaerythritol triacrylate study (NTP, 2005). Feed and water were available ad libitum. The feed was irradiated to reduce potential microbial contamination. Cages and racks were rotated every 2 weeks. Further details of animal maintenance are given in Table 1. Information on feed composition and contaminants is provided in Appendix I.

Clinical Examinations and Pathology

The animals were observed twice daily and were weighed initially, weekly, and at the end of the study.

Clinical findings were recorded weekly and at the end of the study.

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.

Necropsies and histopathologic examinations were performed on all core study mice. 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 selected 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. The tissues selected for microscopic evaluation represented gross lesions as well as major organs and tumor target tissues for mice in chronic rodent bioassays. These tissues were examined in all core study mice. For all paired organs (e.g., adrenal gland, kidney, ovary), samples from each organ were examined. Because the Tg.AC model was initially intended to be a skin reporter phenotype, less emphasis was placed on microscopic examination of internal organs. However, with time and for a variety of reasons, more interest developed relative to effects in internal organs. Thus a reduced tissue list (compared to the standard 2-year bioassay) was adopted that included all tissues that are common targets in NTP carcinogenicity studies. While the gross examination would likely detect any significant carcinogenic effects, it is possible that chemically induced nonneoplastic lesions occurred in organs not 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 sent to 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. A quality assessment pathologist evaluated all slides from nine male and nine female mice per group randomly selected from the vehicle control and 12 mg/kg groups and from all mice that died early. Slides of all tumors and of all skin sites of application, which was the primary target tissue, were reviewed. Selected slides of other potential target organs, including the liver, spleen, and lymph nodes, were also reviewed.

The quality assessment report and the reviewed slides were submitted to the NTP Pathology Review 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 examined by the chairperson, an NTP pathologist, and a pathology working group. When the NTP Pathology Review 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, and NTP Pathology Review chairperson. 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).

Survival Analyses

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Animals found dead of other than natural causes 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 of Incidence

The incidences of neoplasms or nonneoplastic lesions are presented in Tables A1, A4, B1, and B4 as the numbers of animals bearing such lesions at a specific anatomic site and the numbers of animals with that site examined microscopically. For calculation of statistical significance, the incidences of most neoplasms (Tables A3 and B3) and all nonneoplastic lesions are given as the numbers of animals affected at each site examined microscopically. However, when macroscopic examination was required to detect neoplasms in certain tissues (e.g., harderian gland, intestine, and mammary gland) before microscopic evaluation, or when neoplasms had multiple potential sites of occurrence (e.g., leukemia or lymphoma), the denominators consist of the number of animals on which a necropsy was performed. Tables A3 and B3 also give the survival-adjusted neoplasm rate for each group and each site-specific neoplasm. This survival-adjusted rate (based on the Poly-3 method described below) accounts for differential mortality by assigning a reduced risk of neoplasm, proportional to the third power of the fraction of time on study, to animals that do not reach terminal sacrifice.

Analysis of Neoplasm and Nonneoplastic Lesion Incidences

The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and nonneoplastic lesion prevalence. This test is a survival-adjusted quantal-response procedure that modifies the Cochran-Armitage linear trend test to take survival differences into account. More specifically, this method modifies the denominator in the quantal estimate of lesion incidence to approximate more closely the total number of animal years at risk. For analysis of a given site, each animal is assigned a risk weight. This value is one if the animal had a lesion at that site or if it survived until terminal sacrifice; if the animal died prior to terminal sacrifice and did not have a lesion at that site, its risk weight is the fraction of the entire study time that it survived, raised to the kth power.

This method yields a lesion prevalence rate that depends only upon the choice of a shape parameter for a Weibull hazard function describing cumulative lesion incidence over time (Bailer and Portier, 1988). Unless otherwise specified, a value of k=3 was used in the analysis of site-specific lesions. This value was recommended by Bailer and Portier (1988) following an evaluation of neoplasm onset time distributions for a variety of site-specific neoplasms in control F344 rats and B6C3F₁ mice (Portier et al., 1986). Bailer and Portier (1988) showed that the Poly-3 test gave valid results if the true value of k was anywhere in the range from 1 to 5. A further advantage of the Poly-3 method is that it does not require lesion lethality assumptions. Variation introduced by the use of risk weights, which reflect differential mortality, was accommodated by adjusting the variance of the Poly-3 statistic as recommended by Bieler and Williams (1993).

Tests of significance included pairwise comparisons of each dosed group with controls and a test for an overall dose-related trend. Continuity-corrected Poly-3 tests were used in the analysis of lesion incidence, and reported P values are one sided. The significance of lower incidence or decreasing trends in lesions is represented as 1–P with the letter N added (e.g., P=0.99 is presented as P=0.01N).

The weekly in-life skin papilloma counts were evaluated by the method of Dunson et al. (2000). The model separates effects on papilloma latency and multiplicity and accommodates important features of the data, including animal-to-animal variability in the expression of the transgene as reflected in the initial tumor counts. The two key parameters are ϒ₁, which measures the dose effect on incidence (number of animals with one or more papillomas during the study), and ϒ₂, which measures the dose effect on multiplicity (rate of appearance of additional papillomas after the initial papilloma has occurred). The model assumes that the rate (number of additional papillomas per time period) is exponentially increasing with respect to dose and that the rate remains constant across time.

More specifically, under the model, the increase in papilloma burden from one week to the next is assumed to be distributed as a Poisson random variable. The Poisson mean is assumed to depend on an animal-specific susceptibility variable, on exposure length, and on the dose. The rate of initial papilloma occurrence is assumed to be log-linear in time. The coefficients for time are levels of dose multiplied by ϒ₁ and the animal-specific susceptibility parameters. This implies that as the dose/time increases, the rate of occurrence for the first papilloma will increase exponentially relative to increases in dose/time. A value of zero for ϒ₁ implies that dose is not associated with incidence (or, equivalently, the length of the latency period prior to initial onset), leaving only animal-specific characteristics to explain any variability.

After the latency period (after the first papilloma occurs), the Poisson mean changes to a rate that is only dependent on dose (that is, no animal-specific rates or dependency with time). More explicitly, the rate of occurrence of additional papillomas is assumed to be log-linear in time. A value of zero for ϒ₂ implies that dose is not associated with rate of additional papilloma occurrence. A non-zero value implies that the rate of additional papillomas increases with dose in a proportional fashion.

Analysis of Continuous Variables

Two approaches were employed to assess the significance of pairwise comparisons between dosed 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, clinical chemistry, spermatid, and epididymal spermatozoal data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) 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). Because vaginal cytology data are proportions (the proportion of the observation period that an animal was in a given estrous stage), an arcsine transformation was used to bring the data into closer conformance with a normality assumption. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across doses.