Monograph on the Potential Human Reproductive and ... - OEHHA

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production at all dose levels, and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane reduced testosterone production at concentrations Z100 nM. Some statistically significant effects were observed in the mechanistic studies in which cells were exposed to 0.01 nM bisphenol A. In one study, LH-stimulated but not basal testosterone production was reduced by bisphenol A exposure. A second study demonstrated a decrease in basal testosterone production following bisphenol A exposure, but no decrease in testosterone level was observed following incubation of cells with bisphenol A in combination with ICI 182,270. 17b-Estradiol production was decreased in cells exposed to bisphenol A. Changes in mRNA expression following bisphenol A exposure included reduced expression of mRNA for the steroidogenic enzymes P45017b-hydroxylase and aromatase. ERb was not detected in Leydig cells, and expression of ERb mRNA was not affected. The study authors concluded that environmentally relevant concentrations of bisphenol A act directly on Leydig cells to inhibit steroidogenesis, presumably via the ER. Strengths/Weaknesses: This study appears to have been very well-conducted. The study used a wide dose range and showed decreased testosterone production in in vitro Leydig cell cultures at low (0.1 nM) but not at higher concentrations. The response of multiple endpoints provides compelling evidence of a biological effect at 0.01 nM. An explanation for the selective effect of bisphenol A at this single low concentration (0.1 nM) was not provided, nor was the dose range of this effect explored. Utility (Adequacy) for CERHR Evaluation Process:. This study is adequate but of limited utility for the evaluation process. Song et al. (2002), supported by the Hormone Research Center and the Korean Andrological Society, examined the role of bisphenol A in inducing expression of orphan nuclear receptor Nur77, a receptor that plays an important role in the regulation of LH-induced steroidogenesis in Leydig cells. Methods used in this study are described in conjunction with the results. [It does not appear that statistical analyses were conducted in this study.] Following treatment of the mouse Leydig cell line K28 with bisphenol A [purity not indicated] atZ0.01 mM, expression of Nur77 mRNA was increased in a dose-related manner, with saturation of expression observed at 1 mM [0.23 mg/L] [culture ware not indicated]. In a time-response study with 1 mM [0.23 mg/L] bisphenol A, maximal expression of Nur77 mRNA was observed at 30 min following treatment, basal levels of expression were observed from 2–12 hr following treatment, and expression was again increased at 24 hr following treatment. When K28 cells were pretreated with the protein kinase inhibitor H89 or the mitogen-activated protein kinase (MAPK) inhibitor PD98059, induction of Nur77 mRNA by bisphenol A was reduced by 40–45%. Induction of c-fos and c-jun mRNA occurred concurrently with induction of Nur77 mRNA. Bisphenol A-induced increases in Nur77 promotor activity were greater following transfection of cells with Nur77 promoter reporter and c-jun but not with cfos. Possible activation of MAPK by bisphenol A was examined using an immunoblot method with an antibody specific for phosphorylated MAPK. Phosphorylation of MAPK reached a maximum level at 10 min Birth Defects Research (Part B) 83:157–395, 2008 BISPHENOL A 355 following bisphenol A treatment. No changes in bisphenol A-induced induction of Nur77 were observed following pretreatment with a protein kinase C inhibitor or P13 K inhibitor. The study authors stated that together these results suggest possible involvement of the protein kinase A and MAPK pathways in bisphenol A-induced induction of Nur77. In K28 cells transfected with Nur77 promoter or monomer binding site-luciferase reporters, gene promoter activities and transactivation were increased following treatment with Z0.1 mM [0.023 mg/L] bisphenol A, thus suggesting similar responses between promotor activity and mRNA induction. In a yeast assay, bisphenol A had no effect on interactions between Nur77 and its corepressor, silencing mediator of retinoid and thyroid receptor. Exposure of K28 cells to 1 mM [0.23 mg/L] bisphenol A resulted in increased progesterone production, which was inhibited 25% by the overexpression of dominant negative Nur77, which reduces the transactivation activity of Nur77. Expression of mRNA for steroidogenic enzymes was investigated and it was found that bisphenol A treatment increased expression of steroidogenic acute regulatory mRNA, cholesterol side-chain cleavage enzyme, and 3b-hydroxysteroid dehydrogenase. Effects of bisphenol A on expression of mRNA for Nur77 and steroidogenesis enzymes was tested in prepubertal mice (18 days old). Injection of 5 mice/ group with 125 mg/kg bw/day bisphenol A resulted in increased expression of Nur77 mRNA and testosterone levels in mouse testis from 1–6 hr following exposure. [Very few details were provided for the in vivo experiment.] The study authors concluded that the results of these studies indicate that bisphenol A induces Nur77 gene expression and alters steroidogenesis in Leydig cells, indicating a possible novel mechanism of toxicity. Strengths/Weaknesses: This study appears to have been well conducted and links in vitro bisphenol A administration to dose-related (classic, not inverted) activation of Nur77 and subsequent downstream signal transducing proteins. Various confirmatory experiments supported this relationship. These data strongly suggest that bisphenol A (40.1 mM) activates Nur77. The toxicological implications of these findings were not addressed. Utility (Adequacy) for CERHR Evaluation Process: This study was not considered useful for the evaluation process. Hughes et al. (2000), supported by the Medical Research Council, the British Heart Fund, and the European Chemical Industry Council, examined the effects of bisphenol A on rat testicular calcium pumps. Other phenolic compounds were examined, some in greater detail than bisphenol A, but this discussion is limited to bisphenol A. Studies were conducted to determine the effects of bisphenol A exposure on calcium ATPase pump activity, calcium uptake in testicular microsomes, calcium levels in the TM4 Sertoli cell line, and TM4 cell viability [culture ware not indicated]. In the cell-viability study, cells were exposed to bisphenol A [purity not indicated] at 0, 100, 300, or 600 mM [0, 23, 68, or 137 mg/L] for 16 hr. In each study, 2–12 samples/group were analyzed. [For most studies, very few details were provided about procedures such as exposure
356 CHAPIN ET AL. concentrations used and time that cells were incubated. There was no discussion of statistical procedures, and it was not clear if statistical analyses were conducted for some endpoints.] Bisphenol A inhibited calcium ATPase activity in rat testis microsomes. Mean7SEM median inhibitory concentration (IC50) values were reported at 0.4070.15 mM [91734 lg/L] for inhibition of calcium ATPase activity and 2.571.0 mM [5717228 mg/L] for calcium uptake. Exposure to 200 mM [47 mg/L] bisphenol A increased intracellular calcium levels in TM4 cells. A viability study was conducted to determine if increased intracellular calcium levels resulted in cell death. Bisphenol A exposure resulted in reduced TM4 cell viability (percent viability compared to control cells was 93, 64, and 17% at concentrations of 100, 300, and 600 mM). The study authors concluded that these results provide evidence that environmental estrogens may induce toxicity in male reproductive development by disrupting calcium homeostasis. Strengths/Weaknesses: This interesting mechanistic study examined the role of bisphenol A in modulating intracellular calcium levels. It is difficult to interpret the relationship between microsomal and intact cell effects of bisphenol A given the large difference in concentrations needed to produce an effect. Moreover, it is not clear if bisphenol A caused cytotoxicity by a calcium-dependent or non-calcium-mediated process. Utility (Adequacy) for CERHR Evaluation Process: This study was not considered useful for the evaluation process. Tabuchi et al. (2002), supported by the Japanese Ministry of Education, Culture, Sports, Science, and Technology and Takeda Science Foundation, examined the effects of bisphenol A exposure on viability and gene expression in TTE3 cells, a mouse Sertoli cell line. The cells were incubated for 24 hr in media containing 0 or 24–400 mM [5.5–91 mg/L] bisphenol A (99.7% purity) in a DMSO vehicle [culture ware not indicated]. Cell viability was determined, and gene expression changes were examined using microarray and PCR techniques. Data were analyzed by Dunnett multiple conversion test or Student t-test. Compared to values in control cells, bisphenol A exposure reduced cell viability by 25% at 100 mM [23 mg/L], 33% at 200 mM [46 mg/L], and 96% at 400 mM [91 mg/L]. Based on the results of the cell-viability studies, a bisphenol A concentration of 200 mM [46 mg/L] was selected for the gene expression studies. Of 1081 genes examined by microarray, mRNA was downregulated in 3 cases and upregulated in 10 cases. Six genes were selected for evaluation of mRNA expression by PCR, and of those genes, 1 was downregulated (ERa) and 5 were upregulated (iNOS, chop-10, odc, BipGRP78, and osip). The study authors concluded that microarray analysis is a useful tool for investing molecular mechanisms of bisphenol A-induced toxicity in testicular cells. Strengths/Weaknesses: This interesting mechanistic study appears to have been well conducted, but it is unclear from the data if bisphenol A-related changes in chop-10 are a primary (or secondary) effect or are the result of cytotoxicity. Utility (Adequacy) for CERHR Evaluation Process: This study is not useful in the evaluation. Tabuchi and Kondo (2003), supported by Japanese Ministry of Education, Culture, Sports, Science, and Technology, Takeda Science Foundation, and Toyama Daiichi Bank Foundation, conducted a series of experiments to examine the effects of in vitro bisphenol A exposure on gene expression in mouse Sertoli cells. The experiments used TTE3 cells, an immortalized Sertoli cell line established from transgenic mice expressing temperature-sensitive simian virus large T-antigen. Cells were exposed to bisphenol A (99.7% purity) in a DMSO vehicle [culture ware not discussed]. The majority of experiments were repeated 2–4 times, and data were analyzed by Student t-test. [Statistical significance was not reported in the results section of the study.] Before conducting gene expression studies, cells were exposed to 25–400 mM [5.7–91 mg/L] bisphenol A for 3– 24 hr, and viability was determined using a tetrazolium compound. Cell viability was reduced at bisphenol A concentrationsZ200 mM [46 mg/L], and reductions in viability were increased with longer durations of exposure. Intracellular calcium levels were measured using a fluorescence imaging technique over a 15-min period in cells exposed to 0–400 mM [0–91 mg/L] bisphenol A, and a dose-related increase in calcium influx was observed at Z100 mM [23 mg/L]. Based on results for cell viability and calcium influx studies, a concentration of 200 mM [46 mg/L] was selected for the gene-expression experiments. Using a PCR technique, it was determined that expression of mRNA for transferrin was decreased and glucose-regulated protein mRNA was increased by bisphenol A exposure of up to 24 hr. Observations of increased intracellular calcium concentration and upregulated glucose-regulated protein mRNA expression led the study authors to conclude that bisphenol A stresses the endoplasmic reticulum. Gene expression was analyzed by a cDNA microarray technique after exposure for 3, 6, 12, and 24 hr, and it was determined that 31 of 865 genes examined were upregulated by exposure to bisphenol A; no downregulation of genes was observed. The greatest change in gene expression was observed for chop-10, a stress-response gene. Upregulation of 4 genes, c-myc, fra-2, odc, and chop-10, were confirmed by quantitative PCR. Chop-10 was determined to be the most responsive gene. To determine if chop-10 was required for development of endoplasmic reticulum stress and cell injury, a stably transfected cell line expressing chop-10 antisense RNA (chopR14) was developed. Mock cells were used as negative controls in studies where cells were exposed to 200 mM [46 mg/L] bisphenol A for up to 24 hr. Production of chop-10 protein, as determined by Western blot analysis, was reduced in the chopR14 cells compared to the mock cells following exposure to bisphenol A. In contrast to the mock cells, no reductions in cell viability or transferrin mRNA expression were observed in the chopR14 cells following bisphenol A exposure. There were no changes in glucose-regulated protein mRNA expression in chopR14 versus mock cells. The study authors postulated that bisphenol A may disrupt the male reproductive system by altering calcium homeostasis in Sertoli cell endoplasmic reticulum without interacting with the ER and that genes such as chop-10 may be involved in the process. Strengths/Weaknesses: This mechanistic study appears to have been well conducted, but it is unclear from the data if bisphenol A-related changes in chop-10 Birth Defects Research (Part B) 83:157–395, 2008
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National Toxicology Program U.S. De
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NTP.will.transmit.the.NTP-CERHR.<st
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National Toxicology Program U.S. De
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nTp brief tainers.with.internal.epo
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Population Adult General. Populatio
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Table 3. Blood and Breast Milk Biom
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Figure 2b. The weight of evidence t
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in.considering.the.biological.plaus
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isphenol.A.as.efficiently.as.adult.
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isphenol.A..For.example,.this.raise
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laboRaToRy aNImal STudIeS In.contra
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death.(168),.synaptic.plasticity.(1
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gland.carcinogen.or.that.bisphenol.
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concentrations.from.maternal,.fetal
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appendix i. nTp-Cerhr bisphenol a e
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158 CHAPIN ET AL. the CERHR Core Co
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160 CHAPIN ET AL. referred to as 4,
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162 CHAPIN ET AL. concentrations in
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164 CHAPIN ET AL. Food (no. sampled
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166 CHAPIN ET AL. Table 6 Continued
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168 CHAPIN ET AL. comparison of the
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170 CHAPIN ET AL. Table 8 Urinary C
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172 CHAPIN ET AL. the blood of male
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174 CHAPIN ET AL. Table 11 Bispheno
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176 CHAPIN ET AL. Table 13 Summarie
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178 CHAPIN ET AL. Table 15 Estimate
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180 CHAPIN ET AL. Table 17 Maximum
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182 CHAPIN ET AL. 2.0 GENERAL TOXIC
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184 CHAPIN ET AL. fetuses (3.572.7
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186 CHAPIN ET AL. Secondary sources
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188 CHAPIN ET AL. Table 25 Maternal
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190 CHAPIN ET AL. Table 27 Bispheno
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192 CHAPIN ET AL. Table 31 Qualitat
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194 CHAPIN ET AL. Table 33 Toxicoki
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198 CHAPIN ET AL. appeared to occur
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200 CHAPIN ET AL. Table 43 Biliary
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202 CHAPIN ET AL. suggesting that 4
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204 CHAPIN ET AL. low following ora
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206 CHAPIN ET AL. scaling and speci
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208 CHAPIN ET AL. 60-100% in each d
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210 CHAPIN ET AL. related. No histo
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212 CHAPIN ET AL. Table 52 Continue
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214 CHAPIN ET AL. Model and exposur
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216 CHAPIN ET AL. Model and exposur
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218 Model and exposure Husbandry a
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220 CHAPIN ET AL. Table 54 Differen
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222 CHAPIN ET AL. Table 56 Anti-And
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224 Table 57 In Vitro Genetic Toxic
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226 CHAPIN ET AL. Table 58 In Vivo
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228 CHAPIN ET AL. Table 59 Developm
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232 CHAPIN ET AL. Table 64 Tissue R
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236 CHAPIN ET AL. treatment conditi
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238 CHAPIN ET AL. clinical signs, b
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240 CHAPIN ET AL. Table 71 Benchmar
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242 CHAPIN ET AL. group, 5 from 1 l
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244 CHAPIN ET AL. least significant
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246 CHAPIN ET AL. group was a reduc
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248 CHAPIN ET AL. 100-151 g for fem
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250 CHAPIN ET AL. 3.2.3.2 Developme
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252 CHAPIN ET AL. weights, bispheno
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254 CHAPIN ET AL. 120 mg/kg bw/day
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256 CHAPIN ET AL. comparisons condu
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258 CHAPIN ET AL. the findings of t
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260 CHAPIN ET AL. analyzed.] Anogen
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262 CHAPIN ET AL. purposeful confou
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264 CHAPIN ET AL. increased lordosi
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266 CHAPIN ET AL. that there was a
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268 CHAPIN ET AL. duration of adver
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270 CHAPIN ET AL. doses of potent e
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272 CHAPIN ET AL. Table 74 Effects
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274 CHAPIN ET AL. reproductive orga
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276 CHAPIN ET AL. tyrosine-hydroxly
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278 CHAPIN ET AL. include small sam
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280 CHAPIN ET AL. males/group. [It
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282 CHAPIN ET AL. termination, whic
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284 CHAPIN ET AL. provided a reliab
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286 CHAPIN ET AL. grooming, and out
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288 CHAPIN ET AL. method of oral do
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290 CHAPIN ET AL. reared by their d
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292 CHAPIN ET AL. has been informed
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294 CHAPIN ET AL. buffered paraform
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296 CHAPIN ET AL. unit. Further, th
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298 CHAPIN ET AL. PND 21 and housed
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300 CHAPIN ET AL. Tando et al. (200
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302 CHAPIN ET AL. Purina Certified
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Page 229 and 230: 306 CHAPIN ET AL. born to those dam
Page 231 and 232: 308 CHAPIN ET AL. average weight we
Page 233 and 234: 310 CHAPIN ET AL. study authors con
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Page 239 and 240: 316 CHAPIN ET AL. of testes and com
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Page 243 and 244: 320 CHAPIN ET AL. Table 81 Summary
Page 245 and 246: 322 CHAPIN ET AL. Table 82 Continue
Page 251 and 252: 328 CHAPIN ET AL. 600 mg/kg bw/day)
Page 253 and 254: 330 CHAPIN ET AL. (Nagao et al., 19
Page 255 and 256: 332 CHAPIN ET AL. antinuclear antib
Page 257 and 258: 334 CHAPIN ET AL. least 6 animals.
Page 259 and 260: 336 CHAPIN ET AL. Utility (Adequacy
Page 261 and 262: 338 CHAPIN ET AL. stomach weight wa
Page 263 and 264: 340 CHAPIN ET AL. Schirling et al.
Page 265 and 266: 342 CHAPIN ET AL. Endpoint Table 88
Page 267 and 268: 344 CHAPIN ET AL. different diets b
Page 269 and 270: 346 CHAPIN ET AL. with 40 mg vitami
Page 271 and 272: 348 CHAPIN ET AL. the bisphenol A v
Page 273 and 274: 350 CHAPIN ET AL. and determining t
Page 275 and 276: 352 CHAPIN ET AL. expression [to B6
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Page 287 and 288: 364 CHAPIN ET AL. Table 97 Effects
Page 289 and 290: 366 CHAPIN ET AL. Table 99 Treatmen
Page 291 and 292: 368 CHAPIN ET AL. Therefore the NTP
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Page 297 and 298: 374 Table 101 Summary of High Utili
Page 299 and 300: 376 Table 102 Summary of Limited Ut
Page 303 and 304: 380 CHAPIN ET AL. from other suppli
Page 305 and 306: 382 CHAPIN ET AL. U.S. populations.
Page 307 and 308: 384 CHAPIN ET AL. 10. Critical desi
Page 309 and 310: 386 CHAPIN ET AL. Engel SM, Levy B,
Page 311 and 312: 388 CHAPIN ET AL. alpha and beta ex
Page 313 and 314: 390 CHAPIN ET AL. Murray TJ, Maffin
Page 315 and 316: 392 CHAPIN ET AL. Ryan BC, Vandenbe
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