Trans-resveratrol

Trans-resveratrol (RES) was obtained from Bayville Chemical Supply Co. Inc. (Deer Park, NY) in a single lot (156AB). Identity, purity, and stability analyses were conducted by the analytical chemistry lab at RTI International (Research Triangle Park, NC) (Appendix A). Reports on analyses performed in support of the RES study are on file at the National Institute of Environmental Health Sciences (NIEHS).

Lot 156AB, a fine off-white powder with a melting point of 253.5°C, was identified as RES using Fourier transform infrared (FT-IR) spectroscopy and ¹H nuclear magnetic resonance (NMR) spectroscopy. Low- and high-resolution mass spectrometry were conducted by the analytical chemistry laboratory at RTI International (Table A-1) and the University of South Carolina (Columbia, SC), respectively. No reference spectra were available for comparison, but all spectra were consistent with the mass and structure of RES.

Purity evaluation using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection did not identify any impurities. Lot 156AB was found to contain the trans-isomer of RES exclusively in an analysis using HPLC with a photodiode array (PDA) detector (Table A-1). Karl Fisher titration performed at Galbraith Laboratories (Knoxville, TN) indicated a water content of 0.24%. The overall purity of lot 156AB was determined to be >99%.

Accelerated stability studies conducted using HPLC/UV confirmed the bulk chemical is stable when protected from light and stored for 2 weeks at refrigerated (5°C), room (25°C), elevated (60°C), and frozen (–20°C) temperatures. The bulk chemical was stored at –20°C and protected from light, per the manufacturer’s recommendations.

Methylcellulose

Methylcellulose used to make the 0.5% aqueous vehicle for gavage formulations was obtained from Spectrum (Gardena, CA) in three lots (UR1026, WL0069, and XB1050). Lot UR1026 was used in the 2-week studies and lots WL0069 and XB1050 were used in the 3-month studies.

The identity of the methylcellulose was confirmed by the study laboratory using FT-IR spectroscopy. Periodic purity analyses were performed by Galbraith Laboratories, Inc. (Knoxville, TN) during the 3-month studies to determine the methoxy group content. The August 27, 2008, sample of lot WL0069, used to prepare the first dose formulation, had methoxy group content (32.2%) outside of the acceptance criteria of 27.5%–31.5%. A replacement lot (XB1050) was procured for the remaining formulations and was within the acceptance criteria for methoxy group content (29.3%).

Deionized water was used to make the 0.5% aqueous methylcellulose vehicle for gavage formulations.

Study Design for Wistar Han Rats

F₀ female Wistar Han rats were 11 to 12 weeks old upon receipt. Gestation day (GD) 1 was defined as the first day with evidence of mating; F₀ females were received from Charles River Laboratories (Raleigh, NC) on GD 2 and held for 4 days. F₀ females were randomly assigned to one of six dose groups on GD 5. To ensure at least 16 females in the 0, 78, 312.5, and 1,250 mg/kg/day groups, 33 females were assigned to each dose group to allow sufficient animals for biological sampling. Randomization was stratified by body weight to produce similar group mean weights using PATH/TOX SYSTEM software (Xybion Medical Systems Corporation, Lawrenceville, NJ).

F₀ Wistar Han females were quarantined for 37 days after receipt. Ten nonmated females received in the same shipment as the time-mated dams were designated for disease monitoring and were used for gross necropsies 2 days after arrival; samples were collected for parasite evaluation and gross observation of disease. The health of the F₁ animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program (Appendix C). All test results were negative.

Beginning on GD 6, F₀ female Wistar Han rats were administered RES in 0.5% aqueous methylcellulose by gavage throughout gestation and lactation at one of five dose levels (78, 156, 312.5, 625, or 1,250 mg/kg/day) or the vehicle control (0.5% aqueous methylcellulose). Formulations were administered daily, except for the day of delivery if the dam was in the process of delivering; dosing volumes were 5 mL/kg. F₀ females were weighed on GD 5 (for randomization) and daily (except for the day of delivery) throughout the perinatal period; the dosing volume was calculated using the dam’s most recent body weight. Feed and water were available ad libitum. F₀ females were housed individually during gestation and with their respective litters during lactation. Cages were changed weekly for pregnant dams before delivery and twice weekly for dams and their litters after postnatal day (PND) 4.

The day of parturition was considered lactation day (LD) 0 for dams and PND 0 for pups. F₀ female Wister Han rats that did not deliver were euthanized on GD 27, and the uteri were examined for evidence of implantation. On PND 1, clinical observations and litter weights by sex were recorded. From PND 2 through PND 12 (when the F₁ pups began receiving the formulations by gavage), the number of pups for each litter was recorded twice daily. F₁ pups were individually weighed on PNDs 4, 7, 12, 15, 18, and 21.

On PND 4, the number of litters was reduced to seven litters per dose group, and litters were standardized to eight Wistar Han pups per litter (four males and four females when possible); litters used had a minimum of at least three pups per sex. On the day the last litter reached PND 18, five litters per group were randomly selected and two pups per sex from each litter were randomly selected for use in the 3-month study. On the day the last litter reached PND 21, the pups were weaned, and dams were removed from the cages and humanely euthanized with carbon dioxide. Weaning marked the beginning of the 3-month study.

After weaning, F₁ pups were housed five per cage. Groups of 10 male and 10 female Wistar Han F₁ pups were administered 0, 78, 156, 312.5, 625, or 1,250 mg/kg/day in 0.5% aqueous methylcellulose by gavage 5 days per week for 3 months. Pups were administered the same dose their respective dam received during gestation and lactation, and F₁ pups began receiving these doses on PND 12 via gavage. Vehicle control animals were administered the 0.5% aqueous methylcellulose vehicle alone. Dosing volumes were 5 mL/kg, using the animal’s most recent body weight, and dosing was completed by noon each day. Feed and water were available ad libitum. Two diets were utilized in the rat studies: (1) NIH-07 during the perinatal phase and (2) NTP-2000 during the postweaning phase. The NIH-07 diet is a higher protein diet that supports reproduction and lactation in rodents, whereas the NTP-2000 diet is a lower protein diet that decreases the incidence of chronic nephropathy in adult rats. Cages were changed twice weekly and rotated every 2 weeks. Details of the study design and animal maintenance are summarized in Table 1. Information on feed composition and contaminants is provided in Appendix B.

Throughout the perinatal phase and at study termination, biological samples were collected from the 0, 78, 312.5, and 1,250 mg/kg/day groups and stored at approximately −20°C before shipment on dry ice to RTI International (Research Triangle Park, NC) for analysis to confirm internal dose (Appendix D). On GD 18, blood was collected from the retroorbital sinus of three randomly selected dams from each dose group at 30 minutes, 60 minutes, or 90 minutes after RES administration (nine dams per dose group in total). Animals were anesthetized with a carbon dioxide/oxygen mixture and blood was collected into tubes containing sodium heparin, centrifuged, and the plasma harvested. The dams were then humanely euthanized with carbon dioxide, and the fetuses were removed and individually flash frozen in liquid nitrogen. On PND 4, following dose administration to dams, 10 male and 10 female randomly selected standardized pups for each dose group were humanely euthanized by decapitation by noon, placed into individual vials (one pup per vial), and flash frozen in liquid nitrogen. On PND 21, following the last dose administration to pups, blood was collected via cardiac puncture from five male and five female Wistar Han rat pups from each dose group. Animals were first anesthetized with a carbon dioxide/oxygen mixture and blood was then collected into tubes containing lithium heparin, centrifuged, and the plasma harvested. After blood collection, the pups were humanely euthanized by carbon dioxide inhalation overdose and were disposed of without further evaluation. At study termination (24 hours after the last dose), blood was collected from the retroorbital sinus of five randomly selected animals per sex for each dose group for biological sampling. Animals were anesthetized with a carbon dioxide/oxygen mixture and blood was collected into tubes containing sodium heparin, centrifuged, and the plasma harvested.

Study Design for B6C3F1/N Mice

Male and female B6C3F1/N mice were 4–5 weeks old on receipt. Animals were quarantined for 11 (females) or 12 (male) days and mice were approximately 5 to 6 weeks old on the first day of the study. Mice were randomly assigned to one of six dose groups before the start of the study. Randomization was stratified by body weight to produce similar group mean weights using PATH/TOX SYSTEM software (Xybion Medical Systems Corporation, Lawrenceville, NJ).

Before the studies began, five male and five female mice were randomly selected for parasite evaluation and gross observation for evidence of disease. In addition, 10 male and 10 female mice were selected for 4-week and end-of-study serologies. The health of the animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program (Appendix C). All test results were negative.

Groups of 10 male and 10 female mice were administered RES at doses of 0, 156, 312, 625, 1,250, or 2,500 mg/kg/day in 0.5% aqueous methylcellulose by gavage 5 days per week for 3 months. Vehicle control animals were administered the 0.5% aqueous methylcellulose vehicle alone. Dosing volumes were 10 mL/kg, using the animal’s most recent body weight, and dosing was completed by noon each day. Animals were administered the dose for at least 2 consecutive days prior to necropsy. Feed and water were available ad libitum. Male mice were housed individually, whereas female mice were housed five per cage. Cages were changed weekly (males) or twice weekly (females) and rotated every 2 weeks. Details of the study design and animal maintenance are summarized in Table 1. Information on feed composition and contaminants is provided in Appendix B.

Clinical Examinations and Pathology

During the 3-month studies, Wistar Han rats and B6C3F1/N mice were observed twice daily for signs of morbidity or moribundity and weighed prior to dosing on day 1, weekly thereafter, and at study termination. Clinical observations were recorded after dosing on day 1, weekly thereafter, and at study termination.

At the end of the 3-month studies, animals were anesthetized with a carbon dioxide/oxygen mixture and bled in random order. Blood was collected from the retroorbital site of all animals for hematology, clinical chemistry (rats only), and erythrocyte micronuclei analyses. Blood for hematology and micronuclei determinations was collected into tubes containing ethylenediaminetetraacetic acid (EDTA). Blood for clinical chemistry measurements was collected into serum separator tubes, centrifuged, and the serum harvested. Hematology parameters were analyzed using an Advia 120 hematology analyzer (Bayer Diagnostics Division, Tarrytown, NY). Clinical chemistry parameters were analyzed using a Roche cobas c501 chemistry analyzer (Roche, Indianapolis, IN). The parameters measured are listed in Table 1. Samples for erythrocyte micronuclei determination were stored at 2°C–8°C immediately after collection and shipped to Integrated Laboratory Systems, LLC (ILS, Durham, NC) for analysis.

At the end of the 3-month studies, samples were collected for sperm motility and vaginal cytology evaluations from F₁ male and female Wistar Han rats in the 0, 312.5, 625, and 1,250 mg/kg/day groups and from male and female B6C3F1/N mice in the 0, 625, 1,250, and 2,500 mg/kg/day groups. The parameters evaluated are listed in Table 1. For 16 consecutive days before scheduled study termination, the vaginal vaults of the females were moistened with saline, if necessary, and samples of vaginal fluid and cells were collected and subsequently stained. Relative numbers of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used to ascertain estrous cycle stage (i.e., diestrus, proestrus, estrus, and metestrus). Measured parameters of cycle length, number of cycles, and time spent in any specific stage of the estrous cycle of female rats and mice are presented in Appendix F. Male animals were evaluated for sperm count and motility. The left testis and left epididymis were isolated and weighed. The tail of the epididymis (cauda epididymis) was then removed from the epididymal body (corpus epididymis) and weighed. Test yolk (rats) or modified Tyrode’s buffer (mice) was applied to slides and a small incision was made at the distal border of the cauda epididymis. The sperm effluxing from the incision were dispersed in the buffer on the slides, and the numbers of motile and nonmotile spermatozoa were counted for five fields per slide by two observers. After completion of sperm motility estimates, each left cauda epididymis was placed in buffered saline solution. Caudae were finely minced, and the tissue was incubated in the saline solution at 34°C–38°C. Sperm density was determined microscopically with the aid of a hemocytometer. To quantify spermatogenesis, the testicular spermatid head count was determined by removing the tunica albuginea and homogenizing the left testis in phosphate-buffered saline containing 10% dimethyl sulfoxide. Homogenization-resistant spermatid nuclei were counted with a hemocytometer.

Necropsies were performed on all animals. Organ weights were determined for the heart, right kidney, liver, lung, right testis, and thymus. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin (except eyes, which were first fixed in Davidson’s solution, and testes, vaginal tunics, and epididymides, which were first fixed in modified Davidson’s solution), processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 μm, and stained with H&E. Complete histopathological examinations were performed by the study laboratory pathologist on all vehicle control and 1,250 mg/kg/day Wistar Han rats and all vehicle control and 2,500 mg/kg/day B6C3F1/N mice. The kidney, intestine (jejunum; rats only), and nose (mice only) were identified as target organs and examined to a no-effect level. Table 1 lists the tissues and organs examined.

After a review of the laboratory reports and selected histopathological slides by a quality assessment (QA) pathologist, the findings and reviewed slides were submitted to an NTP Pathology Working Group (PWG) coordinator for a second independent review. Any inconsistencies in the diagnoses made by the study laboratory and QA pathologists were resolved by the NTP pathology peer-review process. Final diagnoses for reviewed lesions represent a consensus of the PWG or a consensus between the study laboratory pathologist, NTP pathologist, QA pathologist, and the PWG coordinator. Details of these review procedures have been described, in part, by Maronpot and Boorman¹²¹ and Boorman et al.¹²²

Calculation and Analysis of Nonneoplastic Lesion Incidences

Incidences of nonneoplastic lesions are presented as numbers of animals bearing such lesions at a specific anatomical site and the numbers of animals with that site examined microscopically. For the 3-month study in mice, Fisher’s exact test,¹²³ a procedure that uses the overall proportion of affected animals, was used to identify statistically significant differences between animals administered RES and vehicle control animals, and the Cochran-Armitage trend test was used to test for significant trends.¹²⁴

Because up to two pups/sex/litter were present in the perinatal and 3-month study in rats, the Cochran-Armitage test was modified to accommodate litter effects using the Rao-Scott approach.¹²⁵ The Rao-Scott approach accounts for litter effects by estimating the ratio of the variance in the presence of litter effects to the variance in the absence of litter effects. This ratio is then used to adjust the sample size downward to yield the estimated variance in the presence of litter effects. The Rao-Scott approach was implemented in the Cochran-Armitage test as recommended by Fung et al.¹²⁶ formula ₸_RS2.

Analysis of Continuous Variables

Two approaches were employed to assess the significance of pairwise comparisons between dosed and vehicle 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 Dunnett¹²⁷ and Williams.^128,129 Hematology, dam gestation length, litter size and survival, internal dose assessment, spermatid, and epididymal spermatozoal data, which typically have skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley¹³⁰ (as modified by Williams¹³¹ and Dunn¹³²). The Jonckheere test¹³³ was used to assess the significance of dose-related trends and to determine whether a trend-sensitive test (the Williams or Shirley test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (the Dunnett or Dunn test).

For the perinatal and 3-month study in rats, postweaning body weights were measured on two pups/sex/litter; more than two pups/sex/litter were possible in preweaning body weight measurements. The analyses of preweaning pup body weights took litter effects into account by use of mixed-effects regression wherein litters were the random effects.

For some endpoints in the perinatal and 3-month study in rats, such as hematology, clinical chemistry, and internal dose, data were collected for 10 animals across five different source litters. In these cases, the trend across dose groups was analyzed by a permutation test based on the Jonckheere trend test, implemented by randomly permuting whole litters across dose groups and bootstrapping within the litters.¹³⁴ Pairwise comparisons were made using a modified Wilcoxon test that incorporated litter effects.¹³⁵ The Hommel procedure was used to adjust for multiple comparisons.

Before statistical analysis, extreme values identified by the outlier test of Dixon and Massey¹³⁶ were examined by NTP personnel, and implausible values were eliminated from the analysis.

Analysis of Vaginal Cytology Data

Vaginal cytology data consist of daily observations of estrous cycle stages over a 16-day period. Differences from the vehicle control group for cycle length and number of cycles were analyzed using a Datta-Satten modified Wilcoxon test with a Hommel adjustment for multiple comparisons.

To identify disruptions in estrous cyclicity, a continuous-time Markov chain model (multi-state model) was fit using a maximum likelihood approach,¹³⁷ producing estimates of stage lengths for each dose group. Confidence intervals for these estimates were obtained based on bootstrap sampling of the individual animal cycle sequences. Stage lengths that were significantly different from the vehicle control group were identified using permutation testing with a Hommel adjustment for multiple comparisons.

Analysis of Reproductive Performance Data

Reproductive performance data for the perinatal and 3-month study in rats were analyzed using the Cochran-Armitage trend test and the Fisher’s exact pairwise test.

Bacterial Mutagenicity

DNA reactivity combined with Salmonella mutagenicity is highly correlated with induction of carcinogenicity in multiple species/sexes of rodents and at multiple tissue sites.¹⁴² A positive response in the Salmonella test was shown to be the most predictive in vitro indicator for rodent carcinogenicity (89% of the Salmonella mutagens are rodent carcinogens).^143,144 Additionally, no battery of tests that included the Salmonella test improved the predictivity of the Salmonella test alone. However, these other tests can provide useful information on the types of DNA and chromosomal damage induced by the chemical under investigation.

Peripheral Blood Micronucleus Test

Micronuclei (literally “small nuclei” or Howell-Jolly bodies) are biomarkers of induced structural or numerical chromosomal alterations and are formed when acentric fragments or whole chromosomes fail to incorporate into either of two daughter nuclei during cell division.^145,146 The predictivity for carcinogenicity of a positive response in acute in vivo bone marrow chromosome aberration or micronucleus tests appears to be less than that in the Salmonella test.^147,148 However, clearly positive results in long-term peripheral blood micronucleus tests have high predictivity for rodent carcinogenicity; a weak response in one sex only or negative results in both sexes in this assay do not correlate well with either negative or positive results in rodent carcinogenicity studies.¹⁴⁹ Because of the theoretical and observed associations between induced genetic damage and adverse effects in somatic and germ cells, the determination of in vivo genetic effects is important to the overall understanding of the risks associated with exposure to a particular chemical.