ToxSci Advance Access originally published online on April 19, 2006
Toxicological Sciences 2006 92(1):295-310; doi:10.1093/toxsci/kfj203
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Three-Generation Evaluation of Dietary para-Nonylphenol in CD (Sprague-Dawley) Rats
* RTI International and Experimental Pathology Laboratories, Research Triangle Park, North Carolina 27709; General Electric Company, Pittsfield, Massachusetts 01201; Toxicology/Regulatory Services, Charlottesville, Virginia 22911; and ¶ Rhodia Inc., Raleigh, North Carolina 27615
1 To whom correspondence should be addressed at RTI International, 3040 Cornwallis Road, PO Box 12194, HLB-150, Research Triangle Park, NC 27709. Fax: (919) 541-5956. E-mail: rwt{at}rti.org.
Received January 18, 2006; accepted April 13, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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This study evaluated the potential for dietary para-nonylphenol (NP; CAS No. 84852-15-3) to affect parental fertility and growth and development of three offspring generations in CD (Sprague-Dawley [SD]) rats, including sperm counts across generations to determine the validity of equivocal reductions observed in the F2 generation by R. E. Chapin et al. (1999, Toxicol. Sci. 52, 80–91). Male rat kidney toxicity was also examined based on inconsistent observations in NP-exposed rats at 2000 ppm but not at 200 or 650 ppm in Purina 5002 (H. C. Cunny et al., 1997, Regul. Toxicol. Pharmacol. 26, 172–178) and at all of these NP concentrations in NIH-07 diet (R. E. Chapin et al., 1999, Toxicol. Sci. 52, 80–91). Concentrations were 0, 20, 200, 650, and 2000 ppm NP in Purina 5002 diet and 0 and 650 ppm NP in NIH-07 diet. 17ß-estradiol (E2) was used as a positive control at 2.5 ppm in Purina 5002 diet. There were no NP effects on any reproductive parameters in any generation, including sperm counts. Kidney toxicity (histopathology) occurred at 650 and 2000 ppm with no clear difference for the two diets. Ovarian weight was decreased at 2000 ppm NP in all generations, with no effect on reproduction. Dietary E2 at 2.5 ppm caused renal, reproductive, and developmental (lactational and peripubertal) toxicity in all generations. This study confirmed that dietary NP is not a selective reproductive toxicant with an no observable adverse effect level (NOAEL) of > 2000 ppm (
> 150 mg/kg/day) and provided an NOAEL for male rat kidney toxicity of 200 ppm NP ( 15 mg/kg/day). Key Words: p-nonylphenol; 17ß-estradiol; reproduction; kidney; CD (SD) rats; Purina Certified 5002 Diet; NIH-07 diet.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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In a previous three-generation study of dietary p-nonylphenol (NP) in rats, Chapin et al. (1999)
reported reduced sperm counts in the mid- and high-dose groups in the F2 (but not in F0 or F1) generation, although no reproductive effects were observed. The extent of these changes, the lack of effects in other generations, and the relationship to other control groups led Chapin et al. to conclude that the results required confirmation prior to conclusion of a transgenerational effect of NP on sperm count. In addition, Chapin et al. (1999) observed kidney toxicity in F0, F1, and F2 males at all NP dietary concentrations (200, 650, and 2000 ppm, equivalent to 9–35, 30–100, and 100–350 mg/NP/kg/day, respectively), while a 90-day study with the same NP concentrations and route in rats found kidney toxicity in males only at the highest dietary concentration of 2000 ppm ( 150 mg/kg/day; Cunny et al., 1997). The kidney lesions were medullary cysts and mineralization at the corticomedullary junction. Since Chapin et al. observed the kidney effects at the lower doses in the F0 animals (as well as the F1 and F2 males), the only clear difference (other than breeding) between the male treatment in the Cunny et al. and Chapin et al. studies was the diet used. Cunny et al. used Purina 5002 diet, while NIH-07 diet was used in the Chapin et al. study. The present study was therefore initiated to confirm the lack of reproductive effects in rats including the potential for transgenerational sperm count effects and to evaluate the kidney toxicity resulting from multiple generations of dietary exposure to NP. NP has been shown to have weak estrogen-like activity following high doses, including uterotrophic responses (Odum et al., 1997) and premature vaginal opening in offspring (Chapin et al., 1999; Nagao et al., 2001). Since potential reproductive effects have been suggested to result from weak estrogen activity, 17ß-estradiol (E2) was used as a positive dietary control (at 2.5 ppm equivalent to 0.17 mg/kg/day [170 µg/kg/day]; based on data at this dietary dose from a one-generation study in CD (Sprague-Dawley [SD]) rats by Biegel et al., 1998a,b; Cook et al., 1998) to define the effects expected from an estrogen on the critical endpoints. Other reproductive endpoints (including full andrological assessment) were recorded as necessary to monitor reproductive functional capacity, but no effort was made to evaluate the full range of multigeneration endpoints evaluated by Chapin et al. (1999) or Nagao et al. (2001). Dietary concentrations of NP at 0, 20, 200, 650, and 2000 ppm (equivalent to 0, 1.5, 15, 45, and 150 mg/kg/day) in Purina Certified 5002 were selected to reproduce the dietary doses (and effects) observed in previous study designs and with the 20-ppm group to ensure that an NOAEL for male kidney toxicity could be determined. In addition, a second rodent diet (NIH-07) was evaluated at 0 and 650 ppm (0 and 45 g/kg/day) because dietary differences were identified as a potential confounder in the differences in kidney toxicity observed in the Cunny (Purina) versus the Chapin (NIH-07) studies. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Where appropriate, the study met or exceeded the U.S. EPA Office of Prevention, Pesticides, and Toxic Substances (OPPTS), Health Effects Test Guidelines, OPPTS 870.3800, Reproduction and Fertility Effects (U.S. EPA, 1998
). Some endpoints required to meet full Guideline compliance (e.g., vaginal patency [VP] determinations in NP-treated animals) were not conducted because previous studies adequately defined the effects and doses for these responses. The study exceeded the Guideline requirements in other cases (e.g., by examining a third generation). All facets of the study were conducted in compliance with EPA Toxic Substances Control Act, Good Laboratory Practice Standards (U. S. EPA, 1989), and the NRC (1996) Guide for the Care and Use of Laboratory Animals.
Test chemicals.
para-NP (CAS No. 84852-15-3) was supplied by Schenectady International (Schenectady, NY) with specified purity of 94.25%. E2 (CAS No. 50-28-2) was obtained from Sigma-Aldrich (St Louis, MO) with a specified purity of 100.0%.
Animals and husbandry.
The animals used for this study were CD (SD) rats (Charles River Laboratories, Raleigh, NC) as used in previous studies (Chapin et al., 1999; Cunny et al., 1997). The two diets used were Purina Certified Rodent Chow (No. 5002, PMI Feeds, Inc., St Louis, MO) and NIH-07 (No. 7022 CM, Harlan Teklad, Madison, WI). (Examination of the phytoestrogen content of the batches of the two feeds [Certified Purina 5002 and Harlan Tekland NIH-07] used in this study produced the following information. The five lot numbers of the Purina Certified 5002 meal used in this study were MAY 01 01 2C, SEP 01 01 3A, NOV 13 01 1C, MAR 04 02 3C, and APR 11 01 3A [no data available from Purina on phytoestrogen from this last 5002 lot number]. For the four lot numbers with data in above order: total daidzein [in aglycone units] were 129, 159, 155, and 171 ppm; total genistein [in aglycone units] were 157, 178, 1841, and 166 ppm; total glycitein [in aglycone units] were 36, 43, 43, and 41 ppm; and total isoflavones [in aglycone equivalents] were 322, 380, 382, and 378 ppm. To put these values in context, the averages ± SD [minimum-maximum] for all Purina 5002 lot analyses for 2001–2002 [the time of in-life for this study] were: daidzein, 149 ± 21 ppm [99–209 ppm]; genistein, 164 ± 18 ppm [108–222 ppm]; glycitein, 43 ± 9 ppm [30–71 ppm]; and total isoflavones, 356 ± 42 ppm [238–475 ppm] [analytical information provided by Dr D. G. Haught, Technical Director, Purina Mills, LLC, Gray Summit, MO; personal communication to R.W.T]. The five NIH-07 feed batches were 7022 C-051801 MA, 7022 CM – 091001 MA, 7022 CM – 112601 MA, 7022CM – 011002 MB, and 7022 CM – 040102 MA, all from the same 7022 lot number. NIH-07 is an "open formula" diet which means that the components and their amounts are fixed. With the NIH-07 7022 diet containing 12% soybean meal, the daidzein content in the feed is estimated at 98–127 ppm and the genistein content in the feed at 102–138 ppm [estimates provided by Dr Charles E. Benton, Nutritionist, Harlan Tekland, Madison, WI; personal communication to R.W.T].). The NP concentrations and resulting doses (NP intake) for each diet are defined in Table 1.
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Diet preparation, analysis, and dosing.
Dosed diet preparations were formulated by dissolving NP or E2 in acetone, adding to premix with drying (to drive off the acetone), and adding to the final diet followed by mixing. Diet analyses by high-performance liquid chromatography with a fluorescence detector showed that NP and E2 were mixed homogeneously, were stable frozen for at least 49 days and stable for at least 9 days under cageside conditions, and were administered at the desired feed concentrations throughout the study. Dosed feed formulations were made approximately every month and stored frozen. Feed jars were changed weekly. There was no NP detected in the control diets, with an estimated limit of detection (ELD) of
1.2 ppm in Purina 5002 and 1.1 ppm in NIH-07 feed. There was no E2 in the control Purina diet with an ELD of 0.0062 ppm.
Study design, animal observations, and measurements.
A graphic representation of the study design is presented in Figure 1. The dietary concentrations are specified in Table 1. A total of 400 animals (25/sex/group in eight groups) were assigned to the study at the initiation of the treatment period. The study was conducted in two cohorts, staggered by 1 week, to optimize scheduling of technical staff, animal rooms, equipment, and major study phases (e.g., deliveries, necropsies, etc.) with 13/sex/group for cohort 1 and 12/sex/group for cohort 2. For each cohort, the animals were 6 weeks of age on the scheduled animal receipt date (approximate weights upon arrival: 120–180 g for males and 100–160 g for females). Exposure began for each cohort when the animals were 7 weeks old (males 175–275 g, females 150–225 g). Clinical signs for toxicity, body weights, and feed consumption were monitored according to the OPPTS guideline. The F0 animals were given the experimental diets ad libitum for a 10-week prebreed exposure period.
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The animals were mated (1:1) following the 10-week prebreed exposure for a period of 14 days, with no change in mating partners. The day sperm was found in the vaginal tract was designated gestational day 0. On the day of birth, designated postnatal day (pnd) 0, the pups in each litter were counted, weighed, sexed, and examined thoroughly, and anogenital distance was measured in all pups at 0 and 2.5 ppm E2 in Purina 5002 diet. On pnd 4, the size of each F1 litter was adjusted to 10 pups by eliminating extra pups by random selection to yield, as nearly as possible, five males and five females per litter. On pnd 21, each litter was weaned, and at least one F1 male and one F1 female pup per litter, if possible, were randomly selected (25/sex/group) to produce the F2 generation. Control and E2-treated F1 females (both in Purina) were individually identified on pnd 18 for examination for puberty (VP) beginning on pnd 18 prior to weaning (since acceleration was anticipated). Control and E2-treated F1 males at 0 and 2.5 ppm E2 were also evaluated for puberty (preputial separation [PPS]) beginning on pnd 35.
Selected animals of the F1 generation were administered NP in the diet at their respective formulations for 10 weeks and then mated to produce the F2 generation following the same study design as described for the F0 generation. Following lactation, the same procedures were used to select the F2 generation to produce the F3 generation. To allow for evaluation of sperm parameters in the third offspring generation, selected F3 weanling males (25/group) were administered NP in the diet at 0, 20, 650, and 2000 ppm and E2 at 2.5 ppm in Purina until 111 ± 5 days of age (Purina diet only).
Parental F0, F1, and F2 males and females and retained F3 male offspring were subjected to a complete gross necropsy. Kidneys, testes, and epididymides were weighed for all males and ovaries were weighed for females. At the time of sacrifice of the adult males in each generation, testicular homogenization-resistant spermatid head count and calculation of daily sperm production (DSP) and efficiency of DSP were determined from one frozen testis/male for all males. In addition, number, motility, and morphology of sperm from one cauda epididymis were evaluated in these same animals at necropsy within 2 min of sacrifice. Epididymal sperm motility and number were determined using an HTM TOX-IVOS Automated Sperm Analysis System (Hamilton-Thorne Research, Beverly, MA). Epididymal sperm morphology was examined manually based on at least 200 sperm/male, if possible. The numbers of animals used for the andrology and histopathologic evaluation of the kidneys and testes are shown in Table 2. Histopathologic evaluation of the testes followed the procedures and terminology of Russell et al. (1990).
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Data analysis.
The unit of comparison was the male, the female, the pregnant female, or the litter, as appropriate. Values for parameters in NP groups in Purina were compared to the values for the 0 ppm Purina control group values. Values for parameters in the NP groups in NIH-07 were compared to the values in the 0 ppm NIH-07 control group. Values for parameters in the E2 group in Purina were compared to the values in the 0 ppm Purina control group. Quantitative continuous data were analyzed using Levene's Test for homogeneity of variances (Levene, 1960
). If variances were homogeneous, standard ANOVA tests (Snedecor and Cochran, 1967) and Dunnett's test for pairwise comparisons (Dunnett, 1955, 1964) were used. Frequency data were analyzed for differences among treatment groups by Chi-Square Tests for independence (Snedecor and Cochran, 1967), Cochran-Armitage test for linear trend (Agresti, 1990; Armitage, 1955; Cochran, 1954), followed by Fisher's Exact Test for intergroup comparisons (SAS Institute, 1989a,b, 1990a,b,c, 1996, 1997). If Levene's test indicated lack of homogeneity of variances, robust linear regression methods (which make no assumptions regarding homogeneity of variance or normality of the data) were used (Huber, 1967; Royall, 1986; Shah et al., 1997; Zeger and Liang, 1986), including Wald Chi-Square tests for overall treatment group differences, tests for the presence of linear trends, and individual t-tests for exposed versus the appropriate control group comparisons when the overall treatment effect was significant. A test for statistical outliers (SAS Institute, 1997) was performed on body weights, feed consumption (in g/day), and organ weights. If examination of data did not provide a plausible, biologically sound reason for inclusion, the data were designated "outliers" and excluded from summarization and analyses. For all statistical tests, the significance limit of 0.05 (one- or two tailed) was used as the criterion for significance. |
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Treatment-related effects for the 2000-ppm (males and females) and 650-ppm (females only) NP groups and for the 2.5-ppm E2-exposed group in life were limited to reduced body weights (Fig. 2) and, in some cases, associated reduced feed consumption (data not shown), primarily in the prebreed exposure period. These effects were similar to those observed by Cunny et al. (1997)
and Chapin et al. (1999) at these dietary doses. Dietary intakes of NP and E2 during the prebreed period are also shown in Figure 2, which are the bases for the actual intake values presented in Table 1.
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For males fed Purina 5002 feed, absolute weights (weights of the organ(s) per se) of the kidneys were statistically significantly increased in F1 generation at 200, 650, and 2000 ppm and in the F0 and F2 generations only at 650 ppm NP. Absolute kidney weight was unaffected in F3 males at 2000 ppm. Absolute kidney weights were also increased at 650 ppm NP in NIH-07 diet for F0 and F2 males. For males fed Purina 5002 feed, relative (to terminal body weight) kidney weights were increased in F0 and F2 generations at 650 and 2000 ppm NP and in the F1 generation at 200, 650, and at 2000 ppm NP. Relative kidney weight was increased for all three generations in males given NP at 650 ppm in NIH-07 diet. Kidney toxicity was observed histopathologically, manifested as medullary cysts at 2000 ppm NP in Purina and at 650 ppm NP in NIH-07 feed, mineralization at the corticomedullary junction at 650 ppm NP in Purina and NIH-07, and at 2000 ppm NP, and tubular nephropathy at 2000 ppm NP in Purina and at 650 ppm NP in NIH-07 (Tables 3 and 4).
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No treatment-related effects were observed in the F1, F2, or F3 males for absolute and relative testes weights, percent motile cauda epididymal sperm, percent progressively motile sperm, or percent abnormal sperm. Relative (but not absolute) paired epididymal weights were significantly increased for F2 and F3 (but not for F0 or F1) males at 2000 ppm NP (Table 3). Testicular homogenization-resistant spermatid head counts were also unaffected, as were DSP and efficiency of DSP (calculated from the spermatid head counts). No treatment-related effects were observed for cauda epididymal sperm parameters including concentration, motility, or morphology (Table 5). Reduced absolute and relative paired ovarian weights were observed at 2000 ppm NP for F0, F1, and F2 females, at 650 ppm in Purina feed for F1 females, and at 650 ppm NP in NIH-07 feed for F0 and F2 females (Table 6).
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For the NP-exposed groups, reproductive indices, precoital interval, offspring lactational survival indices, and litter sizes (pre- and postcull) were unaffected. Gestational length in days was significantly reduced in F1 and F2 females (but not in F0 females) at 650 NP in NIH-07, relative to the NIH-07 control but not to the Purina 5002 control group value. This finding is because the values at 0 ppm NP in NIH-07 in F1 and F2 females were, in fact, increased relative to concurrent and historical control values in Purina 5002. Pup body weight per litter was reduced for all F1, F2, and F3 pups at 2000 ppm NP at pnd 21 but not at earlier time points. There were no treatment-related effects on sex ratios or pup deaths. The reproductive and offspring data are summarized in Tables 7 and 8.
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For E2-exposed males, absolute and relative paired kidney weights were significantly reduced for F0 and F1 males. Absolute (but not relative) paired kidney weights were significantly reduced for F2 and F3 males. Relative (but not absolute) paired testes weights were significantly increased for E2-exposed F0, F1, F2, and F3 males (Table 3). Increased incidences of mineralization at the corticomedullary junction were present in F0, F1, and F2 males, while the incidence of nephropathy was decreased in the E2-treated males compared to the controls (Table 4). Absolute and relative ovarian weights were reduced in F0, F1, and F2 adult females (Table 6). There were reduced epididymal sperm concentrations in adult F1, F2, and F3 males (Table 5). Reduced fertility, gestational and pregnancy indices, and reduced numbers of total and live pups/litter on pnd 0 were also observed (Table 7). Reduced numbers of F1, F2, and F3 litters and reduced numbers of live pups/litter at birth were also observed (Table 8). Anogenital distance at birth was unaffected in both male and female offspring; acquisition of puberty was significantly accelerated by 8.7 days in female offspring (VP) and significantly delayed by 7.0 days in male offspring (PPS; Table 9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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This study was designed to confirm and extend the findings of Chapin et al. (1999)
for potential reproductive toxicity, including transgenerational sperm effects (effects in the second generation that did not occur in the first), as well as those of Cunny et al. (1997) and Chapin et al. (1999) for adult male kidney toxicity resulting from continued exposure to NP over multiple generations. In addition, following the initiation of this study, Nagao et al. (2001) reported a slight decrease in the number of uterine implantation sites and of live pups per litter at birth at 50 mg/kg/day of NP administered by gavage (approximately equivalent to 670 ppm in the diet, but administered once daily as a bolus dose in the Nagao et al. 2001 study) in a two-generation reproduction study, findings which were not observed in the Chapin et al. (1999) study. The present study was extended to examine three offspring generations, with the F3 males raised to adulthood with continued dietary exposure. The study by Cunny et al. (1997) employed NP in Purina 5002 at 0, 200, 650, and 2000 ppm. The study by Chapin et al. (1999) employed the same dietary concentrations in NIH-07 feed. The need to establish an NOAEL for the renal lesions in the present study necessitated adding a low NP dose (20 ppm). In addition, the possible effect of diet on kidneys was evaluated by employing 0 and 650 ppm NP in both Purina 5002 and NIH-07 diets. A reference compound (positive control) group was also added, 2.5 ppm dietary E2 in Purina 5002, dietary concentration based on the results by Biegel et al., 1998a,b; Cook et al., 1998, because NP has been shown to have weak estrogen-like activity (Chapin et al., 1999).
Adult Systemic Toxicity
Two renal findings, mineralization at the corticomedullary junction and medullary cysts, were observed after exposure of male rats to NP in Purina 5002 (Cunny et al., 1997), with higher incidence and severity in NIH-07 (Chapin et al., 1999), with the renal pathology from both studies reviewed by Hard (1998, Expert Panel Report on Renal Histopathological Changes in Rat Dietary Studies with Nonylphenol. Unpublished report. Prepared for Alkylphenols and Ethoxylates (APE) Research Council, Washington, DC., pp. 1–12.). The interpretation of the renal lesions in NP-exposed males from these studies was confounded by differences in the diets (e.g., Meyer et al., 1978) and by sex differences in the incidence and severity of spontaneous corticomedullary junction mineralization (typically high incidence in females, with low or no incidence in males) and in renal tubular nephropathy (typically high incidence in males, with low incidence in females; Geary and Cousins, 1969; Ritskes-Hoitinga and Beynen, 1992). In the present study (consistent with previous reports of NP effects on male kidneys), there were no treatment-related increases in medullary cysts at 200 and 650 ppm NP in Purina 5002 or at 650 ppm NP in NIH-07 in F0, F1, or F2 males. A treatment-related increase in the incidence of medullary cysts was observed in the F0 (2/10), F1 (4/10), and F2 (8/10) males at 2000 ppm NP in Purina 5002. (F3 retained male kidneys were not evaluated histopathologically in this study.)
Also in the present study, mineralization at the corticomedullary junction was not observed in the Purina 5002 control group in F0, F1, or F2 males and in one animal for the NIH-07 control group only in the F1 generation males. The incidence was increased at 650 and 2000 ppm NP in Purina 5002 and at 650 ppm NP in NIH-07 in all three male generations evaluated (F0, F1, and F2), with no increased incidence at 200 ppm NP in Purina 5002 (0, 2, and 0 in F0, F1, and F2 males, respectively).
E2 exposure resulted in male kidney findings that included increased incidence of mineralization at the corticomedullary junction (common in female rats) and reduced incidence of nephropathy (common in male rats). Although the histological changes in the kidneys were similar for NP and E2 in this study, there is no evidence to suggest a common mechanism. In fact, there are a number of differences that suggest a different mechanism including: increases in kidney weight for NP-treated animals with decreases in kidney weight for E2-treated animals; NP did not affect the incidence of nephropathy as observed for E2; and NP-related kidney effects occurred at doses below those for which estrogen-like activity was observed.
This study verified renal toxicity in F0 adult males at 650 and 2000 ppm (Cunny et al., 1997) and in F1 and F2 adult male offspring at these dietary concentrations (Chapin et al., 1999) but not the limited effects observed in some animals at 200 ppm in the Chapin et al. study. Although increased absolute and relative kidney weights were observed in F1 males at 200 ppm NP, they were not associated with increased incidence of the two microscopic findings (medullary cysts and mineralization at the cortico-medullary junction) and there were no renal effects (organ weights or histopathology) in F0 or F2 males at 200 ppm NP. The NOAEL for adult male renal toxicity, based on absence of histopathology at 200 ppm NP, was 200 ppm NP in Purina.
Hard (1998) reviewed the renal histopathology in both the Cunny and Chapin studies of dietary exposure to NP in rats. He considered in both studies the primary treatment-related renal effect in male rats was the formation of renal tubular mineralization "of the so-called corticomedullary type (actually not corticomedullary in the rat but OSOM/ISOM junction)" (Hard, 1998, p. 7). This was the only renal finding in the 90-day study (Cunny et al., 1997). Hard (1998) considered that the mineralization represents calcium phosphate formation and is most frequently associated with a decrease in the dietary calcium/phosphorus ratio below 1.0. The rat is considered less able than other species to cope with disturbance in calcium homeostasis, with female rats more prone to renal tubular mineralization than male rats, "as estrogen levels may play a role in the process" (Hard, 1998, p. 8) consistent with the role of estrogen. The incidence of renal tubular mineralization in female rats in both studies was high in the control groups and similarly high in both incidence and severity in all the NP-exposed groups. In the present study, mineralization of the corticomedullary junction in the renal tubule was not present in F0, F1, or F2 males at 0 ppm Purina (and at very low incidence at 0 ppm in NIH-07). This finding did increase in incidence in males at 650 ppm NP (in Purina and NIH-07 diets) and at 2000 ppm NP and in males at 2.5 ppm E2. Hard (1998) further stated that tubular mineralization can be treatment related when the test material interferes with systemic availability or absorption of calcium or when the test material adds to the phosphate load in the diet. Hard (1998) also indicated that the extent of mineralization in male rats appeared to be greater in the multigeneration study (Chapin et al., 1999) than in the 90-day study (Cunny et al., 1997), which he (Hard, 1998) viewed as possibly due to the difference in diets (Purina 5002 in the Cunny study, NIH-07 in the Chapin study), "perhaps" related to the calcium/phosphorus ratio. In the present study, the Ca/P ratio for the Purina 5002 was 1.21, 1.25, 1.45, 1.10, and 1.20 (0.807%/0.669%, 0.866%/0.693%, 103%/0.712%, 0.729%/0.661%, and 0.776%/0.644%, respectively, for the five lots used, as mentioned in "Animals and Husbandry" section). The Ca/P ratio for the Teklad NIH-07 feed was 1.26 (1.20%/0.95% for the single lot, No. 7022, used). The diets were therefore essentially equivalent in Ca/P ratio. No clear effect of diet on NP-induced toxicity of the male kidney was observed in the present study.
The second major renal finding in the Chapin study (and in the present study) was medullary cysts, with Hard suggesting that "they are causally related to the mineralization process" (Hard, 1998, p. 8). Medullary cysts were also observed in a range-finding study of dietary NP in CD (SD) rats (Latendresse et al., 2001). In that study, NP was administered in a soy-free, casein-containing diet with NP at 0, 5, 25, 200, 500, 1000, and 2000 ppm. "Severe polycystic kidney disease (PKD)" was observed in all adult offspring of both sexes at 2000 ppm and in 67% of the males and 53% of the females at 1000 ppm. There was no evidence of PKD observed in animals in the control or lower dietary dose groups of NP (Latendresse et al., 2001). The authors concluded that "the renal toxicity of NP is highly dependent on the diet on which the animals are maintained" (Latendresse et al., 2001, p. 140). Tomobe et al. (1998) have also reported exacerbation effects of dietary soy and genistein on disease progression in mice with PKD. Therefore, the five batches/lots each of Purina 5002 and Harlan Tekland NIH-07 feeds used in this study were assessed for phytoestrogen content by the suppliers (as mentioned in "Animals and Husbandry" section). For both feeds, the diadzein and genistein concentrations in ppm (mg/kg feed) were consistent across all feed lots. For Purina feed, glycitein was also assayed with consistent values across lots. These values were also within the range of concentrations analyzed for the Purina lots for 2001–2002 (the time of performance of this study).
Reproductive Toxicity
There were no treatment-related effects on reproductive parameters in this study, which is consistent with the conclusions of earlier studies (Chapin et al., 1999; Nagao et al., 2001). In the Nagao et al. (2001) study, Crj:CD (SD) IGS rats (25/sex/group) were dosed with NP by gavage at 0, 2, 10, or 50 mg/kg/day. They reported that NP did not affect sperm parameters or estrous cyclicity at any dose; at 50 mg/kg/day, significant increases in liver and kidney weights in males were associated with increased histopathologic findings in livers of both sexes and in male kidneys, and ovarian weights were decreased in females at 50 mg/kg/day. Also, at 50 mg/kg/day, there were significant decreases in the number of implantation sites and live pups per litter at birth, with reduced survival at this dose on pnd 0-4. NP did not affect acquisition of PPS, but it did accelerate acquisition of VP at 50 mg/kg/day (Nagao et al., 2001). Chapin et al. (1999) found similar effects on kidneys and VP but found no effect on implantation sites (consistent with the results of the present study).
Relative (but not absolute) paired epididymal weights were significantly increased for F2 and F3 (but not for F0 or F1) males at 2000 ppm NP. This is most likely due to the significant reductions in male body weights during in-life and at termination at this dietary dose. The absolute values were equivalent across all groups, and the relative weights are the absolute organ weights relative to terminal body weights (which were reduced). There were no effects on absolute epididymal weights in any NP group for any generation. Importantly, andrological assessments were unaffected across all NP groups and generations, thus showing that the equivocal effects on sperm count at 650 and 2000 ppm in the F2 generation observed in the Chapin et al. (1999) study were not substantiated.
In this study, ovarian weights were reduced at 2000 ppm in F0 females, at 650 and 2000 ppm NP in F1 females in Purina, and at 650 ppm NP in F0 females in NIH-07. This finding is consistent with those of Nagao et al. (2001), although in the Chapin et al. (1999) study, changes in ovarian weight were less definitive. There were no effects on reproduction in the studies by Chapin et al. (1999), Nagao et al. (2001), or in the present study as a result of these ovarian weight changes, suggesting that the mechanism is unrelated to key reproductive processes performed by the ovary. In the present study, E2 also reduced ovarian weights consistent with effects reported by Biegel et al. (1998a,b). The relationship of these E2-induced ovarian weight changes to the reproductive effects of E2 is uncertain.
The effects of E2, including reduced fertility, gestational, and pregnancy indices, reduced number of implantation sites per litter, reduced numbers of litters, reduced numbers of total and live pups per litter at birth, reduced adult male testes and epididymal weights, and reduced epididymal sperm counts, were consistent with those reported by Biegel et al. (1998a,b) and Cook et al. (1998). Since no similar findings occurred even at the highest concentration of NP, it is concluded that the weak estrogen-like activity of NP at these dietary doses, up through 2000 ppm, does not result in reproductive effects.
Offspring Toxicity
The only treatment-related effect on the offspring in the present study was a decrease in pup body weight at weaning at the highest dietary concentration. The decrease in weight was not present earlier in the lactational period. Therefore, it was concluded that the reduced body weight resulted from the high intake on a mg NP/kg/day basis (as much as 2.5 times the adult intake) that occurs when the pups begin to self-feed on pnd 14 (as well as possible translactational exposure and/or coprophagia) resulting in a direct toxic effect from overexposure to NP (Tyl et al., 2002).
The effects observed on reproductive development at 2.5 ppm E2 in CD (SD) rats in this study closely matched the effects reported in CD (SD) rats by Biegel et al. (1998a,b) and Cook et al. (1998), also at 2.5 ppm E2 in the diet (their next highest dietary dose [10.0 ppm] resulted in total infertility, and their next lowest dietary dose [0.05 ppm] resulted in little or no biologically or statistically significant effects). In the present study, VP was significantly accelerated by 8.7 days, with a significant reduction in female body weight at acquisition, since they were 9 days younger than controls. In the study by Biegel et al., (1998b), VP was accelerated by 8.8 days at 2.5 ppm. This acceleration was expected since female puberty is under E2 control. Also in this study, PPS in males was significantly delayed by 7.0 days at 2.5 ppm, with a significant increase in body weight at acquisition since they were 7 days older than controls. In the study by Biegel et al. (1998b), PPS was also delayed (by 8.2 days) at 2.5 ppm. Since acquisition of puberty in males is under testosterone (T) control, it is less obvious (although it is clear and confirmed) how dietary E2 delays acquisition of puberty in males. The delay in acquisition of PPS in offspring males may be due to excess systemic E2 in males (from the dietary E2) causing negative feedback to the pituitary or hypothalamus (Brawer et al., 1983), which in turn downregulates hypothalamic gonadotropin releasing hormone, pituitary follicle stimulating hormone and luteinizing hormone, and finally T biosynthesis in the testis; the androgen receptor may also be downregulated in the presence of E2 in males (Ewing et al., 1979; Robaire et al., 1979). This delay in acquisition of puberty from E2 in male CD (SD) rats has since been confirmed from E2 exposure in CD-1 (Swiss) male mice (Myers et al., 2004).
Because this study was designed and performed to confirm (or not) the effects on sperm parameters in the F2 (but not F1) generation in the NP study by Chapin et al. (1999) and to define an NOAEL for the kidney toxicity identified in the Chapin et al. (1999) and Cunny et al. (1997) studies, the dietary concentrations were the same as those they used plus 20 ppm, the diets were the same as those they used, and the endpoints were the same as those they used (plus we added a positive control group of 2.5 ppm E2 with additional endpoints to demonstrate the sensitivity of this rat strain to an estrogen and to demonstrate the ability of the performing laboratory to detect estrogenic effects were they to occur). The study was not designed or performed to determine mechanisms of action by including endpoints such as plasma or urine analyses for the kidney effects, circulating hormone assays, sperm molecular endpoints for the reproductive effects, or assessment of the developmental genes which regulate urogenital development.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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This study demonstrates a lack of transgenerational effects (effects in the second generation that did not occur in the first) on epididymal sperm counts or on any other reproductive endpoints and confirms the conclusions of Chapin et al. (1999)
and Nagao et al. (2001) that NP is not a selective reproductive toxicant with a reproductive toxicity NOAEL of > 2000 ppm (> 150 mg/kg/day) in the diet. It also provides an NOAEL for male rat kidney toxicity of 200 ppm NP ( 15 mg/kg/day) in the diet. |
ACKNOWLEDGMENTS |
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This work was supported by the Alkylphenols and Ethoxylates Research Council (APERC), Washington, DC.
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REFERENCES |
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