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

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isphenol A exposure on the development of Xenopus laevis embryos. Three different sets of experiments were conducted. Data were analyzed by ANOVA followed by Fisher protected least significant difference test. From 3– 96 hr following fertilization, embryos were exposed to bisphenol A [purity not indicated] at 1, 2.5, 5, 10, 15, 20, 25, or 30 mM (0.3, 0.6, 1.1, 2.3, 3.4, 4.6, 5.7, or 6.8 mg/L). Each exposure was replicated 3 times. Negative control groups consisted of the ethanol vehicle, medium alone, or dilution medium. Rates of normal embryo development were equivalent in the 3 different negative control groups. In groups exposed to Z20 mM bisphenol A, there was a significant decrease in normal embryos and a nonsignificant increase in mortality rate. Teratogenicity was characterized by short body length, microcephaly, flexure, edema, and abnormal gut coiling. Increases in embryo abnormalities were also observed following exposure to Z10 mM 17b-estradiol or nonylphenol. To determine sensitive stages, embryos were exposed to control media or 20 mM [4.6 mg/L] bisphenol A for 45– 48-hour periods ranging from 3–48 hr post-fertilization, 12–60 hr post-fertilization, 24–72 hr post-fertilization, 36– 84 hr post-fertilization, or 48–96 hr post-fertilization. Body length, gross malformations, and distance between eyes were measured at 96 hr following exposure. [The Methods section indicated that 59–71 embryos were examined in the bisphenol A group for each time period of exposure. However, a figure in the study reported the sample size as 3/time period.] During the period of 3–48 hr following fertilization, statistically significant effects in the bisphenol A group included decreased body length and increased incidences of microcephaly, flexure, edema, and abnormal gut coiling. No increases in abnormal effects were observed following exposure at later time periods. Abnormalities were observed following exposure to 17b-estradiol or nonylphenol at early or late stages. In the third part of the study, embryos were exposed to 20 mM [4.6 mg/L] bisphenol A from 3–96 hr following fertilization. RNA was isolated from whole embryos and subjected to analysis by cDNA microarray. Results obtained in microarray analyses were confirmed by PCR analysis. The sample size was reported as 2. The microarray analysis revealed 179 upregulated and 103 downregulated genes following exposure of embryos to bisphenol A. The study authors identified 27 genes in which expression was changed following exposure to bisphenol A, nonylphenol, or 17b-estradiol. The identified genes included: KNP-Ia, CmaB, XIRG, a-skeletal tropomyosin, apelin, cyclin G1, Ube213, HGF, toponin C2, ribosomal protein L9, and Rattus norvegicus similar to CG10042-PA. The other genes were not identified. The study authors concluded that these findings might provide clues to deciphering mechanisms of teratogenic effects associated with bisphenol A and the other compounds examined in this study. Strengths/Weaknesses: The inclusion of 17b-estradiol as a comparator was a strength and the high bisphenol A concentration is a weakness. Utility (Adequacy) for CERHR Evaluation Process: This study is not useful for the evaluation process. Pickford et al. (2003), supported by the Bisphenol A Global Industry group, the Society of the Plastics Industry, the Bisphenol A Sector Group of the European Chemical Industry Council, and the Japan Chemical Birth Defects Research (Part B) 83:157–395, 2008 BISPHENOL A 311 Industry Association, examined the effects of bisphenol A exposure on development of frog gonads. Beginning at Stage 43/45 (B2 days post-hatching, 4 days postfertilization, exposure day 0) and continuing through Stage 66, Xenopus laevis larvae were exposed to bisphenol A [purity not indicated] at nominal concentrations of 0 (water control), 1.0, 2.3, 10, 23, 100, or 500 mg/L in a flowthrough test system [culture ware not discussed]. Actual concentrations were verified as 0.83, 2.1, 9.5, 23.8, 100, and 497 mg/L. A positive control group was exposed to 2.7 mg/L 17b-estradiol. There were 4 replicate test vessels/dose, with each containing 40 larvae (i.e., 160 larvae/test condition). Larvae were observed daily for mortality, behavior, and appearance. Growth and development were assessed on all larvae of a replicate tank on exposure days 32 and 62 (36 and 68 [66?] days postfertilization). Froglets were killed and observed at completion of metamorphosis (Stage 66). Total length was measured, sex was determined, and testes and ovaries were assessed for abnormalities such as asymmetry, complete absence, presence of melanocytes, irregular shape, segmentation or fragmentation, vacuoles, and ambiguous sexual morphology. Data were analyzed by Fisher exact test, ANOVA, Wilcoxon rank sum test, G test, and w 2 test. Following exposure to bisphenol A, there were no significant differences in survival, distribution of developmental stages on Day 32 or 62, time to completion of metamorphosis (Stage 66), or length of Stage 66 froglets. Bisphenol A exposure did not affect sex ratio or abnormalities in testis or ovary [data were not shown by authors for testis and ovary effects]. In contrast, exposure to 17b-estradiol resulted in an increase in ratio of females to males and testicular and ovarian abnormalities. The study authors identified a no-observed-effect concentration of 500 mg/L for bisphenol A. Strengths/Weaknesses: The use of a wide range of exposure levels is a strength, but the incomplete data presentation with missing organ weight data and the lack of histological evaluations are weaknesses. Utility (Adequacy) for CERHR Evaluation Process: This study may have utility for environmental assessment, but is not useful for human risk assessment. Levy et al. (2004), supported by the Ministry of Environment and Traffic of Baden-Württemberg, evaluated the effect of bisphenol A on gonad development in Xenopus laevis tadpoles. Tadpoles (n 5 40/group) were exposed beginning at Stages 42/43 to ethanol vehicle or to bisphenol A (499% purity) or 17b-estradiol, both at concentrations of 10 -8 or 10 -7 M [bisphenol A concentrations 2.3 and 23 lg/L. Actual concentrations were 90–105% of target concentrations after addition of bisphenol A to the media but decreased to low levels by the end of the 48-hr period between media changes. Culture ware was not discussed.] After completion of metamorphosis, froglets were killed for examination of gonads. Tadpoles not completing metamorphosis were killed after 120 days of chemical exposure for examination of gonads. In a second experiment, bisphenol A concentrations were 10 -8 ,10 -7 ,or10 -6 M [2.3, 23, or 228 lg/L] and the 17b-estradiol positive control used a concentration of 10 -7 M. In a third experiment, 50 tadpoles/group were treated for 2 weeks with ethanol vehicle, bisphenol A 10 -7 M [23 lg/L], or10 -7 M17bestradiol after which whole-body homogenates were
312 CHAPIN ET AL. used for extraction of RNA and determination of ER by RT-PCR. Statistical analyses were performed with Kruskal–Wallis H test followed by Mann–Whitney U test. The gonadal sex of control animals was 56% male and 44% female. 17b-Estradiol treatment increased the female ratio to 81% at 10 -7 M and 84% at 10 -8 M. Bisphenol A treatment resulted in a significant increase in females (69%) at 10 -7 M [23 lg/L]. At10 -8 M bisphenol A, there were 65% females, which did not reach statistical significance. In the second experiment, a significant increase in females was seen after treatment with 10 -7 M [23 lg/L] (70%, compared to 48% in controls and 96% with 17b-estradiol treatment). There was no significant effect of bisphenol A at 10 -8 M [2.3 lg/L] (51% female) or 10 -6 M [228 lg/L] (53% female). Bisphenol A and 17bestradiol both resulted in increased ER mRNA. The authors concluded that bisphenol A affects the sexual development of Xenopus laevis, probably through an estrogenic mechanism. Strengths/Weaknesses: The measurement of bisphenol A in the media is a strength, but its lack of stability is a weakness. Utility (Adequacy) for CERHR Evaluation Process: This study is not useful for the evaluation process. Yang et al. (2005), supported by the Chinese Ministry of Science and Technology, examined the effects of bisphenol A exposure in black-spotted pond frog tadpoles. Thirty tadpoles/tank were exposed in duplicate to bisphenol A (Z95% purity) at concentrations of 0, 0 (1DMSO vehicle), 2, 20, or 200 mg/L [ppb] for up to 60 days [culture ware not discussed]. Tadpoles were also exposed to mixtures containing bisphenol A1 nonylphenol at 212, 20120, or 2001200 mg/L. Additional tadpoles were exposed to mixtures containing the same bisphenol A/nonylphenol mixtures in addition to p,p0-DDE 21210.5, 2012015, or 2001200150 mg/L. Five tadpoles/tank were pooled at 15, 30, 45, and 60 days. The tadpoles were homogenized for measurement of testosterone and thyroxin levels by radioimmunoassay. Alkaline-labile phosphate was measured as a biomarker for vitellogenin. Data were analyzed by ANOVA. Malformations of tail flexure were observed in 10% of tadpoles exposed to 200 mg/L bisphenol for 45 days, and similar rates of malformation (13.3%) were observed in the mixtures containing 200 mg/L bisphenol A. A ‘‘decrease’’ (not statistically significant) in thyroxine levels was observed following 60 days of exposure to all bisphenol A doses (Z2 mg/L). ‘‘Increases’’ (not statistically significant) in testosterone levels were reported with all bisphenol A doses at 30 days of exposure. p,p0-DDE at Z5 mg/L inhibited increases in testosterone level observed with mixtures of bisphenol A and nonylphenol [not statistically analyzed]. ‘‘Increases’’ (not statistically significant) in alkaline-labile phosphate levels were reported following 30 or more days of exposure to all bisphenol A doses. In animals exposed to bisphenol A and nonylphenol in combination compared to either compound alone, alkaline-labile phosphate levels were increased at 15 days of exposure but decreased at 60 days of exposure [not statistically analyzed]. p,p0-DDE inhibited the increase in alkalinelabile phosphate levels induced by the bisphenol A1 nonylphenol mixture on Day 15 of exposure [not statistically analyzed]. Strengths/Weaknesses: The lack of attention to statistical analysis is a weakness and makes the authors’ conclusions unreliable. Utility (Adequacy) for CERHR Evaluation Process: This study is not useful in the evaluation process. Imaoka et al. (2007), supported by the Japanese Ministry of Education, Science, Culture, Sports, and Technology, evaluated the effects of bisphenol A on development of the African clawed frog, Xenopus laevis. Embryos were cultured with bisphenol A from Stage 10.5, formation of the neural plate, to Stage 35 at a bisphenol A (in DMSO) concentration of 25, 50, or 100 mM [5.8, 11, or 23 mg/L]. Tadpoles were morphologically evaluated at Stages 28–35. Total RNA was extracted and reversed transcribed and RT-PCR used to quantify the expression of specific genes. Expression levels relative to b-actin or histone H4 were compared with Student t-test. Abnormalities in the head and eye region were described with a ‘‘minor effect’’ at 25 mM and a ‘‘major effect’’ at 50 mM bisphenol A. [Data were not shown.] There were no treatment-related effects on expression of sox-2, nrp-1, myoD, sox17a, or notch. Relative expression levels of pax 6 declined in a concentration-related manner to about 56% if control at the high concentration [estimated from a graph]. Relative expression levels of esr-1 decreased in a concentration-dependent manner to about 22% of control at the high concentration [estimated from a graph]. Microinjection into blastomeres of plasmids containing NICD (the intracellular domain of notch), but not of X-delta-1 (a notch ligand) corrected the decreased expression of esr-1. The authors concluded that bisphenol A decreased esr-1 expression by disrupting notch signaling. Strengths/Weaknesses: This is an interesting study on the molecular alterations induced in frog embryos exposed to BPA. The study demonstrated alterations in several key developmental genes and malformed development at high concentrations. The high concentrations are, however, weaknesses and the effects of uncertain concern to human health because humans would not be exposed in this manner. Utility (Adequacy) for CERHR Evaluation Process: This study is not useful in the evaluation process. 3.2.10.3 Fish: Kishida et al. (2001), supported by the National Science Foundation and U.S. EPA, included bisphenol A in a study to test the utility of changes in CYP450 aromatase mRNA expression as a marker of xenoestrogen effects in the CNS of zebrafish (Danio rerio). Fish embryos were incubated in solutions containing bisphenol A [purity not indicated] at 0 (DMSO vehicle), 0.01, 0.1, or 10 mM [0, 2.3, 23, or 228 lg/L] from 2–48 hr post-fertilization [culture ware not discussed]. Expression of the CYP450 aromatase gene was determined in 50 embryos/treatment group using an RT-PCR/Southern blot technique. [There was no mention of statistical analyses of data.] The Southern blot analysis revealed a B3-fold increase in the band intensity of CYP450 aromatase at the high concentration (10 mM) of bisphenol A. The potency of bisphenol A was determined to be lower than those of 17b-estradiol and diethylstilbestrol, which induced B3–4-fold increases in band intensity at concentrations up to 3 orders of magnitude lower than bisphenol A. In additional experiments with exposure to bisphenol at 2–48 hr post-fertilization, embryo mortality was increased by exposure to 10 and 20 mM [228 and Birth Defects Research (Part B) 83:157–395, 2008
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National Toxicology Program U.S. De
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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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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
Page 183 and 184: 260 CHAPIN ET AL. analyzed.] Anogen
Page 185 and 186: 262 CHAPIN ET AL. purposeful confou
Page 187 and 188: 264 CHAPIN ET AL. increased lordosi
Page 189 and 190: 266 CHAPIN ET AL. that there was a
Page 191 and 192: 268 CHAPIN ET AL. duration of adver
Page 193 and 194: 270 CHAPIN ET AL. doses of potent e
Page 195 and 196: 272 CHAPIN ET AL. Table 74 Effects
Page 197 and 198: 274 CHAPIN ET AL. reproductive orga
Page 199 and 200: 276 CHAPIN ET AL. tyrosine-hydroxly
Page 201 and 202: 278 CHAPIN ET AL. include small sam
Page 203 and 204: 280 CHAPIN ET AL. males/group. [It
Page 205 and 206: 282 CHAPIN ET AL. termination, whic
Page 207 and 208: 284 CHAPIN ET AL. provided a reliab
Page 209 and 210: 286 CHAPIN ET AL. grooming, and out
Page 211 and 212: 288 CHAPIN ET AL. method of oral do
Page 213 and 214: 290 CHAPIN ET AL. reared by their d
Page 215 and 216: 292 CHAPIN ET AL. has been informed
Page 217 and 218: 294 CHAPIN ET AL. buffered paraform
Page 219 and 220: 296 CHAPIN ET AL. unit. Further, th
Page 221 and 222: 298 CHAPIN ET AL. PND 21 and housed
Page 223 and 224: 300 CHAPIN ET AL. Tando et al. (200
Page 225 and 226: 302 CHAPIN ET AL. Purina Certified
Page 227 and 228: 304 CHAPIN ET AL. high-doses of bis
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: 310 CHAPIN ET AL. study authors con
Page 237 and 238: 314 CHAPIN ET AL. jar, and there we
Page 239 and 240: 316 CHAPIN ET AL. of testes and com
Page 241 and 242: 318 CHAPIN ET AL. differentiation o
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
Page 277 and 278: 354 CHAPIN ET AL. indicated] at 0 (
Page 279 and 280: 356 CHAPIN ET AL. concentrations us
Page 281 and 282: 358 CHAPIN ET AL. animals were non-
Page 283 and 284: 360 CHAPIN ET AL. following adjustm
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362 CHAPIN ET AL. Table 94 Treatmen
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364 CHAPIN ET AL. Table 97 Effects
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366 CHAPIN ET AL. Table 99 Treatmen
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368 CHAPIN ET AL. Therefore the NTP
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370 CHAPIN ET AL. weeks before mati
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372 CHAPIN ET AL. Table 100 Summary
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374 Table 101 Summary of High Utili
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376 Table 102 Summary of Limited Ut
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378 CHAPIN ET AL. Table 103 Summary
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380 CHAPIN ET AL. from other suppli
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382 CHAPIN ET AL. U.S. populations.
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384 CHAPIN ET AL. 10. Critical desi
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386 CHAPIN ET AL. Engel SM, Levy B,
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388 CHAPIN ET AL. alpha and beta ex
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390 CHAPIN ET AL. Murray TJ, Maffin
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392 CHAPIN ET AL. Ryan BC, Vandenbe
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394 CHAPIN ET AL. Thomas P, Dong J.
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appendix iii. publiC CommenTs and p
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