NTP-CERHR expert panel report on the developmental toxicity of soy infant formula

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Original Article

NTP-CERHR Expert Panel Report on theDevelopmental Toxicity of Soy Infant Formula

Gail McCarver,1� Jatinder Bhatia,2 Christina Chambers,3 Robert Clarke,4 Ruth Etzel,5 Warren Foster,6

Patricia Hoyer,7 J. Steven Leeder,8 Jeffrey M. Peters,9 Emilie Rissman,10 Michael Rybak,11 ClaireSherman,13 Jorma Toppari,13 and Katie Turner14

1Medical College of Wisconsin, Milwaukee, Wisconsin2Medical College of Georgia, Augusta, Georgia

3University of California San Diego Medical Center, San Diego, California4Georgetown University School of Medicine, Washington, District of Columbia

5George Washington University Washington, District of Columbia6McMaster University, Ontario, Canada7University of Arizona, Tucson, Arizona

8Children’s Mercy Hospitals and Clinics, Kansas City, Missouri9Pennsylvania State University, University Park, Pennsylvania

10University of Virginia School of Medicine, Charlottesville, Virginia11U.S. Centers for Disease Control and Prevention, Atlanta, Georgia

12Cerus Corporation/P-val Gal Consulting, Davis, California13University of Turku/Turku University, Turku, Finland

14RTI International, Research Triangle Park, North Carolina

Soy infant formula contains soy protein isolates and is fed to infants as a supplement to or replacement for human milkor cow milk. Soy protein isolates contains estrogenic isoflavones (phytoestrogens) that occur naturally in some legumes,especially soybeans. Phytoestrogens are nonsteroidal, estrogenic compounds. In plants, nearly all phytoestrogens arebound to sugar molecules and these phytoestrogen–sugar complexes are not generally considered hormonally active.Phytoestrogens are found in many food products in addition to soy infant formula, especially soy-based foods such astofu, soy milk, and in some over-the-counter dietary supplements. Soy infant formula was selected for NationalToxicology Program (NTP) evaluation because of (1) the availability of large number of developmental toxicity studies inlaboratory animals exposed to the isoflavones found in soy infant formula (namely, genistein) or other soy products, aswell as few studies on human infants fed soy infant formula, (2) the availability of information on exposures in infantsfed soy infant formula, and (3) public concern for effects on infant or child development. On October 2, 2008 (73 FR57360), the NTP Center for the Evaluation of Risks to Human Reproduction (CERHR) announced its intention to conductan updated review of soy infant formula to complete a previous evaluation that was initiated in 2005. Both the currentand previous evaluations relied on expert panels to assist the NTP in developing its conclusions on the potentialdevelopmental effects associated with the use of soy infant formula, presented in the NTP Brief on Soy Infant Formula.The initial expert panel met on March 15 to 17, 2006, to reach conclusions on the potential developmental andreproductive toxicities of soy infant formula and its predominant isoflavone constituent genistein. The expert panelreports were released for public comment on May 5, 2006 (71 FR 28368). On November 8, 2006 (71 FR 65537), CERHRstaff released draft NTP Briefs on Genistein and Soy Formula that provided the NTP’s interpretation of the potential forgenistein and soy infant formula to cause adverse reproductive and/or developmental effects in exposed humans.However, CERHR did not complete these evaluations, finalize the briefs, or issue NTP Monographs on these substancesbased on this initial evaluation. Between 2006 and 2009, a substantial number of new publications related to humanexposure or reproductive and/or developmental toxicity were published for these substances. Thus, CERHR determinedthat updated evaluations of genistein and soy infant formula were needed. However, the current evaluation focuses onlyon soy infant formula and the potential developmental toxicity of its major isoflavone components, e.g. genistein,daidzein (and estrogenic metabolite, equol), and glycitein. This updated evaluation does not include an assessment onthe potential reproductive toxicity of genistein following exposures during adulthood as was carried out in the 2006evaluation. CERHR narrowed the scope of the evaluation because the assessment of reproductive effects of genisteinfollowing exposure to adults was not considered relevant to the consideration of soy infant formula use in infants during

Published online in Wiley Online Library (wileyonlinelibrary.com)DOI: 10.1002/bdrb.20314

*Correspondence to: Gail McCarver, Medical College of Wisconsin,Milwaukee, Wisconsin.E-mail: [email protected] 4 March 2011; Accepted 17 May 2011

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the 2006 evaluation. To obtain updated information about soy infant formula for the CERHR evaluation, the PubMed(Medline) database was searched from February 2006 to August 2009 with genistein/genistin, daidzein/daidzin,glycitein/glycitin, equol, soy, and other relevant keywords. References were also identified from the bibliographies ofpublished literature. The updated expert panel report represents the efforts of a 14-member panel of government andnongovernment scientists, and was prepared with assistance from NTP staff. The finalized report, released on January15, 2010 (75 FR 2545), reflects consideration of public comments received on a draft report that was released on October19, 2009, for public comment and discussions that occurred at a public meeting of the expert panel held December 16 to18, 2009 (74 FR 53509). The finalized report presents conclusions on (1) the strength of scientific evidence that soy infantformula or its isoflavone constituents are developmental toxicants based on data from in vitro, animal, or human studies;(2) the extent of exposures in infants fed soy infant formula; (3) the assessment of the scientific evidence that adversedevelopmental health effects may be associated with such exposures; and (4) knowledge gaps that will help establishresearch and testing priorities to reduce uncertainties and increase confidence in future evaluations. The Expert Panelexpressed minimal concern for adverse developmental effects in infants fed soy infant formula. This level of concernrepresents a ‘‘2’’ on the five-level scale of concern used by the NTP that ranges from negligible concern (‘‘1’’) to seriousconcern (‘‘5’’). The Expert Panel Report on Soy Infant Formula was considered extensively by NTP staff in preparing the2010 NTP Brief on Soy Infant Formula, which represents the NTP’s opinion on the potential for exposure to soy infantformula to cause adverse developmental effects in humans. The NTP concurred with the expert panel that there isminimal concern for adverse effects on development in infants who consume soy infant formula. This conclusion wasbased on information about soy infant formula provided in the expert panel report, public comments received during thecourse of the expert panel evaluation, additional scientific information made available since the expert panel meeting,and peer reviewer critiques of the draft NTP Brief by the NTP Board of Scientific Counselors (BSC) on May 10, 2010(Meeting materials are available at http://ntp.niehs.nih.gov/go/9741.). The BSC voted in favor of the minimal concernconclusion with 7 yes votes, 3 no votes, and 0 abstentions. One member thought that the conclusion should be negligibleconcern and two members thought that the level of concern should be higher than minimal concern. The NTP’s responseto the May 10, 2010 review (‘‘peer-review report’’) is available on the NTP website at http://ntp.niehs.nih.gov/go/9741.The monograph includes the NTP Brief on Soy Infant Formula as well as the entire final Expert Panel Report on SoyInfant Formula. Public comments received as part of the NTP’s evaluation of soy infant formula and other backgroundmaterials are available at http://cerhr.niehs.nih.gov/evals/index.html. Reports can be obtained from the web site(http://cerhr.niehs.nih.gov/) or from: Kristina A. Thayer, PhD, NIEHS/NTP K2-04, PO Box 12233, Research TrianglePark, NC 27709. E-mail: [email protected]. Birth Defects Res (Part B) 92:421–468, 2011. r 2011 Wiley Periodicals, Inc.

Key words: endocrine disrupters; nutrition; safety assessment

SUMMARY OF CHEMISTRY, USE,AND HUMAN EXPOSURE

In children and adults, exposures to isoflavones occurthrough consumption of soy foods such as tofu, soy milk,soy flour, textured soy protein, tempeh, and miso (FDA,2000b). Infants can be exposed by consuming soy-basedinfant formula, the breast milk of mothers who consumesoy products, or by the use of soy in weaning or‘‘transition’’ foods. Soy oils or soy sauces contain little-to-no genistein (Setchell, 1998; ILSI, 1999). Soy protein canbe used in baked goods, breakfast cereals, pasta,beverages, toppings, meat, poultry, fish products, anddairy-type products including imitation milk and cheese(Soyfoods Associations of North America, 2009; UnitedSoybean Board, 2009). Soybean derivatives are present in60% of processed foods available from U.K. super-markets (UK-Committee-on-Toxicity, 2003). The percent-age of processed foods containing soybeans in the UnitedStates is not known. Exposure to genistein can also occurthrough soy supplements marketed for the beneficialeffects on health, such as improved cardiovascular healthand treatment of menopausal symptoms (Drugstore.com,2009).

On the basis of the sales of soy products, it appearsthat exposure to soy isoflavones in the United States isincreasing and will continue to increase. The SoyfoodsAssociation of America reported that soy food sales haveincreased from $300 million to over $4 billion between

1992 and 2008, attributing this increase to new soy foodcategories being introduced, soy foods being reposi-tioned in the market place, and new customers selectingsoy for health and philosophical reasons (SoyfoodsAssociations of North America, 2009).

The primary isoflavones detected in soy products,including soybeans and soy formula, are derived fromgenistein, daidzein, and to a smaller extent, glycitein.These isoflavones are often referred to as phytoestrogensbecause of their ability to bind to estrogen receptors(ERs) and display weak estrogenic activity comparedwith estradiol (at least based on in vitro model systems),with a relative estrogenic potency of genistein4daid-zein4glycitein (MAFF, 1998; UK-Committee-on-Toxicity,2003; Choi et al., 2008).

The majority of isoflavones in soy formula andunfermented soy products are conjugated to sugarmolecules to form the glycosides genistin, daidzin, andglycitin (Cassidy, 2006; Nielsen and Williamson, 2007).Glucose groups in glycoside compounds can also beesterified with acetyl or malonyl groups to form acetyl-or malonyl glycosides (UK-Committee-on-Toxicity, 2003)(Fig. 1). The terms genistein, daidzein, and glycitein referspecifically to the unconjugated (aglycone) forms of theisoflavones. Small amounts of genistein and daidzein(3.2–5.8%) are present in soy products in their un-conjugated (aglycone) forms (Chen and Rogan, 2004).As a result of bacterial hydrolysis during fermentation,aglycones represent a larger proportion of the isoflavones

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in fermented soy products such as miso, tempeh, andsoybean paste (ILSI, 1999; UK-Committee-on-Toxicity,2003). Because glycosidic compounds are rapidly decon-jugated in the gut to form the biologically active aglyconecompound, exposure to a particular isoflavone istheoretically the sum of the aglycone and respective

glycoside compound concentrations converted on thebasis of molecular weight (MAFF, 1998; UK-Committee-on-Toxicity, 2003). Unfortunately, there is an inconsistency inthe literature where many studies do not clarify whetherthe presented isoflavone levels were normalized on anaglycone basis. Failure to convert the major glycosides,

Fig. 1. Chemical structures of isoflavones found in soy formula.

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i.e. genistin, daidzin, glycitin, to the appropriate aglyconeequivalents can overestimate isoflavone levels or intake byB1.6-fold. Information in the Expert Panel report isnormalized to aglycone equivalents where feasible.

Isoflavone levels in soybeans can vary as a result ofcrop strain, geographic location, climate, and growingconditions (Setchell et al., 1998; UK-Committee-on-Toxicity, 2003). Heating of soy products can causedecarboxylation, deacetylation, or deglycosylation ofglycosides with decomposition of malonyl compoundsto their respective acetylglycosides (Setchell et al., 1998;UK-Committee-on-Toxicity, 2003). Except for alcoholextraction, processing soybeans does not usually reduceisoflavone content (ILSI, 1999).

Soy formula refers to infant food made using soyprotein isolate and other components such as corn syrup,vegetable oils, and sugar (Drugstore.com, 2009). Decadesago soy formula included soy flour. However, in the1950s and 1960s, cases of altered thyroid function, mostlygoiter, were reported in infants fed soy formula. Thisproblem was eliminated by adding more iodine to theformulas and replacing soy flour with soy protein isolate.Although the early reports of goiter in infants fed soyformula have mostly ceased since manufacturers begansupplementing soy formula with iodine in 1959, there isstill concern that the use of soy formula in infants withcongenital hypothyroidism (CH) may decrease theeffectiveness of thyroid hormone replacement therapy,i.e. L-thyroxin. The soy protein isolate is fortified withL-methionine, L-carnitine, and taurine and other nutrients(FDA, 2000a; Bhatia and Greer, 2008). Soy protein isolateincludes phytates (1.5%), which bind minerals andprotease inhibitors, which have antitrypsin, antichymo-trypsin, and antielastin properties (Bhatia and Greer,2008). Phosphorus, calcium, iron, and zinc are added tosoy formula to compensate for phytate binding ofminerals. Heat applied during the processing of soyprotein removes 80 to 90% of protease inhibitor activity.Aluminum is present in soy formulas because of theaddition of mineral salts.

Many aspects of infant formula use are unknown,including what percent of infants are exclusively fedformula compared with what percent are fed a mixtureof infant formula and breast milk. It is also unknownwhat proportion of formula-fed infants are exclusivelyfed soy formula. A 1998 infant-feeding survey conductedby Ross Products Division indicated that 18% of infantsare fed soy formula during the first year of life (Stromet al., 2001). According to market data and hospitaldischarge records, another estimate was that 25% ofnewborns in the United States are fed soy formula(Badger et al., 2002). A study conducted at YaleUniversity assessed formula changes in 189 breast-fedinfants and 184 formula-fed infants and reported that23% of infants in the study received soy formulasometime during the first 4 months of life (Forsythet al., 1985). Based on recent market data, sales of soyformula in the United States represent B12% of the U.S.infant formula dollar sales (personal communicationwith Robert Rankin, Manager of Regulatory and Techni-cal Affairs at the IFC, October 13, 2009). In the UnitedStates between 1999 and 2009, estimates of total soyinfant formula fed decreased from 22.5 to 12.7%calculated based on total formula sold corrected fordifferences in formula cost, i.e. expressed in equivalent

feeding units (public comment from the InternationalFormula Council (IFC), received December 3, 2009 andpersonal communication with Dr. Haley Curtis Stevens,IFC). When sales are considered as a surrogate measureof actual reported usage, these data provide a lowerindication of usage compared with other frequently citedestimates. The usage and sales of soy formula varygeographically ranging from 2 to 7% of infant formulasales in the United Kingdom, Italy, and France, to 13% inNew Zealand (Agostoni et al., 2006; Turck, 2007), and to31.5% in Israel (Berger-Achituv et al., 2005).

A commonly cited reason for use of soy formula is tofeed infants who are allergic to dairy products or areintolerant of lactose, galactose, or cow-milk protein(Essex, 1996; Tuohy, 2003). In May 2008, the AmericanAcademy of Pediatrics (AAP) released an updated policystatement on the use of soy protein-based formulas(Bhatia and Greer, 2008). The overall conclusion of theAAP was that, although isolated soy protein-basedformulas may be used to provide nutrition for normalgrowth and development in term infants, there are fewindications for their use in place of cow milk-basedformula. The only real indications for use are instanceswhere the family prefers a vegetarian diet or for themanagement of infants with galactosemia or primarylactase deficiency (rare). Soy formula is not currentlyrecommended for preterm infants. Similar conclusionswere reached in 2006 by the European Society forPaediatric Gastroenterology Hepatology and Nutrition(ESPGHAN) Committee on Nutrition (Agostoni et al.,2006).

A number of studies in the United States and abroadhave measured total isoflavone levels in infant formulas(see Table 9 of complete final expert panel reportavailable at http://cerhr.niehs.nih.gov/evals/genistein-soy/soyformula/soyformula.html). For formulas fromthe United States, the range of total isoflavone levelsreported in reconstituted or ‘‘ready-to-feed’’ formulaswas 20.9 to 47 mg/liter formula (Franke et al., 1998;Setchell et al., 1998). When normalized to aglyconeequivalents, genistein is the predominant isoflavonefound in soy formula (B58–67%), followed by daidzein(B29–34%) and glycitein (B5–8%). In contrast to theisoflavone content of soybeans and other soy productssuch as soy supplements or soy protein isolates, theisoflavone content in soy formula is less variable. Therange of total isoflavones content in soy formula samplescollected in the United States and other countries is 10 to47 mg/liter (Table 1) (Setchell et al., 1998; Genovese andLajolo, 2002).

Isoflavone exposure through soy formula intake hasbeen estimated in the United States and other countriesbased on total isoflavone levels measured in soyformulas and assumptions of formula intakes and infantbody weights. In the United States, total isoflavone intakeby infants was estimated at 2.3 to 9.3 mg/kg body weight(bw)/day, depending on age of the infant, the estimatedintake for genistein, expressed in aglycone equivalents,ranges from 1.3 to 6.2 mg/kg bw/day. These intakes areseveral orders of magnitude greater than infants whoconsume breast milk or a cows milk-based formula. Soyformula-fed infants have higher daily intakes of genisteinand other isoflavones compared with other populations(excluding regular consumers of soy supplements)(Table 2). However, differences in strategies used to

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develop the intake estimates and sample representative-ness limit the ability to compare across studies, especiallyfor those based on dietary surveys. In addition, iso-flavone intake seems to be inherently highly variable inadult populations and there is support for the notion thatthis variability cannot solely be attributed to differencesin study methods. Recognizing these caveats, the relativeranking of total isoflavone intake appears to be infantsexclusively fed soy formula4vegans4Japanese consum-ing a traditional diet4vegetarians4soy consumers.

Mean blood-based levels of isoflavones in infants fedsoy formulas are considerably higher than other popula-tions, including vegans and Japanese adults (Table 3).For example, concentrations of total genistein in wholeblood samples from U.S. infants fed soy formula are

1455 ng/ml at the 75th percentile (Cao et al., 2009). Thisvalue is almost five times higher than the maximumgenistein concentration detected in plasma in a smallstudy of Japanese men, n 5 6 (Adlercreutz et al., 1994).The mean level of genistein (757 ng/ml) in the blood ofthe soy formula-fed infants reported by Cao et al. (2009)is almost 20 times higher than the mean level of genisteindetected in a sample of vegetarians and vegans inOxford, England (Peeters et al., 2007). Average bloodlevels of total genistein in the soy formula-fed infants areB160 times higher than the mean levels of total genisteinin omnivorous adults in the United States reported byValentin-Blasini (2003) (757 vs. 4.7 ng/ml). A similarpattern is observed based on urinary concentrations ofisoflavones (Table 4).

Table 2Comparison of Estimated U.S. Intake of Genistein and Total Isoflavones in Infants Fed Soy Formula to Other Populations

Daily intake (mg/kg bw/day)

Population Genistein Total isoflavone Reference

U.S. infants (soy formula) 1.3–6.2 2.3–9.3 Table 9 from unabbreviated final expert panel report(http://cerhr.niehs.nih.gov/evals/genistein-soy/SoyFormulaUpdt/FinalEPReport_508.pdf)

U.S. adults (general) 0.005a–0.056b 0.0097a–0.096b Haytowitz et al. (2009)a; Tseng et al. (2008)b; Kirk et al.(1999)

U.S. vegetarians 0.14 0.21European men Mulligan et al. (2007)

Not soy consumers 0.005 0.009Soy consumers 0.057 0.100

European women Mulligan et al. (2007)Not soy consumers 0.004 0.007Soy consumers 0.062 0.112

Vegans (UK) 1.07 Friar and Walker (1998) as cited in Mortensen et al.(2009)

Japanese 0.077a–0.43b 0.67b Fukutake et al. (1996)a as cited in Fitzpatrick (1998);Arai et al. (2000)b

Table 1Comparison of Isoflavone Content in Infant Soy Formulas from Different Countries

Individual isoflavones, mg/liter formula as fed1

(percent of total)

CountryTotal isoflavones, mg/liter

formula as feda Genistein Daidzein Glycitein Reference

US (n 5 20) 20.9a–47b 12.1a–31.2b 7.1a–13.5b 1.7a–2.4b Franke et al. (1998)a; Setchellet al. (1998)b(57.7–66.3%) (B28.8–34%) (B5–8.3%)

UK (n 5 13) 18a–46.7b 10.4a–31.3b 6.7a–12.5b 0.9a–2.8b UK Ministry of Agriculture,Fisheries, and Food (1998)a;Hoey et al. (2004)b

(58.0–67.1%) (26.8–37%) (5–6.1%)

Australia (n 5 4) 17.2–21.9 – – – Knight et al. (1998)New Zealand 17.1–33 11.2–18 5.9–15 – Irvine et al. (1998)(n 5 5) (55–65.4%) (34.6–45%)Brazil 10–27.4 5.9–16.2 2.4–8.6 1.6–2.4 Genovese and Lajolo (2002)(n 5 7) (59.3–59.8%) (23.9–31.7%) (9–16.3%)

1CERHR converted between mg/kg (or mg/g) and mg/liter because the density of prepared formula is similar to water (specific gravityis 1.03) (personal communication from Mead Johnson Medical Nutrition Affairs). – 5 not specified.

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SUMMARY OF PHARMACOKINETICSAND GENERAL TOXICOLOGY

Pharmacokinetics

Genistein, daidzein, and glycitein exist mainly in theirglycosidic forms in unfermented soy foods. Beforeisoflavone glycosides can be absorbed into the systemiccirculation, they must first be hydrolyzed to theiraglycones, which have greater hydrophobicity and lowermolecular weight. Before systemic availability, mostgenistein and daidzein are conjugated with glucuronicacid by uridine diphosphate (UDP)-glucuronosyltrans-ferases; a smaller amount is conjugated to sulfate bysulfotransferases. Conjugation of isoflavones can alsooccur in liver. The glucuronide and sulfate conjugatesenter the systemic circulation, and the majority ofisoflavone compounds in the circulation are present inconjugated form.

Humans. In humans, a considerable amount ofpharmacokinetic information is available for genisteinand daidzein in adults. Less information is available forequol and very little data have been published onglycitein. Human developmental pharmacokinetic dataare lacking; the available ‘‘pharmacokinetic’’ data inhuman infants are essentially equivalent to biomonitor-ing data. The detection of genistein, daidzein, and equolin serum, urine, amniotic fluid, cord blood, and breastmilk in humans demonstrate fetal exposure and absorp-tion into the systemic circulation of infants. Fullpharmacokinetic data on genistein, daidzein, and/orglycitein among human infants following soy formulafeeding have not been published. Rather, data are limitedto single plasma concentrations, generally after chronicsoy formula feeding, which likely represent steady state(although this is uncertain) and spot urinary concentra-tions (Cruz et al., 1994; Setchell et al., 1997; Irvine et al.,

Table 4Comparison of Urinary levels of Genistein, Daidzein, and Equol in Infants Fed Soy Formula to the General

U.S. Population

Urinary concentration (geometric mean), mg/liter or ng/ml

Population Genistein Daidzein Equol Reference

U.S. infants fed soy formula (n 5 27) 5891 5097 2.3 Cao et al. (2009)U.S. children ages 6–11 years (n 5 721–726) 33.8 88.1 11.5 U.S. CDC (2008)U.S. adults ages 40–59 years (n 5 951) 23.7 52.3 8.39 U.S. CDC (2008)

Table 3Comparison of Blood-Based Levels of Genistein and Daidzein in Infants Fed Soy Formula to Other Populations

Average total isoflavone concentration, nM(ng/ml)

Population Sample Genistein Daidzein Reference

U.S. infants (soy formula, n 5 27) Whole blood(spots)

2801 (757), geometricmean

1007 (256), geometricmean)

Cao et al. (2009)

3296 (891), median 1081 (275), median)5384.5 (1455), 75th

percentile2040 (519), 75th

percentileU.S. infants (soy formula, n 5 7) Plasma 2531 (684) 1160 (295) Setchell et al. (1997)U.S. infants (cow milk formula, n 5 30) Whole blood

(spots)52.5 (14.2), geometric

mean(5.5), geometric mean Cao et al. (2009)

U.S. infants (cow milk formula, n 5 7) Plasma 11.7 (3.16), mean 8.1 (2.06), mean Setchell et al. (1997)U.S. infants (breastfed, n 5 20) Whole blood

(spots)40 (10.8), geometric

mean20.8 (5.3), geometric

meanCao et al. (2009)

U.S. infants (breastfed, n 5 7) Plasma 10.3 (2.77) 5.9 (1.49) Setchell et al. (1997)Omnivorous adults in the United States

(n 5 209)Serum 17.4 (4.7) 15.3 (3.9) Valentin-Blasini et al.

(2003)oLOD–(203), range oLOD–(162), range

Vegetarians, Finland (n 5 14) Plasma 17.1 (4.6) 18.5 (4.7) Adlercreutz et al. (1994)Vegans and vegetarians (Oxford, UK,

n 5 70)Plasma 148 (40) 78.7 (20) Peeters et al. (2007)

Japanese women at delivery (n 5 51a or194b)

Serum 26.6 (7.2)a–116.5 (31.5)b 7.1 (1.8)a–50.2 (12.8)b Todaka et al. (2005)a;Nagata et al. (2006)b

Japanese cord serum at delivery (n 5 51a

or 194b)Serum 71.8 (19.4)a–126.9 (34.3)b 16.9 (4.3)a–38.6 (9.81)b Todaka et al. (2005)a;

Nagata et al. (2006)b

Japanese men (traditional diet, n 5 6) Plasma 90.4 (24)–1204 (325),range

58.3 (15)–924 (235),range

Adlercreutz et al. (1994)

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1998; Hoey et al., 2004; Franke et al., 2006; Halm et al.,2007; Franke et al., 2008a; Halm et al., 2008; Cao et al.,2009). No studies of human infants fed genistein,daidzein, and/or glycitein have been reported. Thus,the human pharmacokinetic data are solely based onstudies conducted in adults.

Relatively, few studies include measurement of glyci-tein and it has not been measured in biomonitoringstudies of the general population conducted by the CDCas part of NHANES (U.S. Centers for Disease Controland Prevention, 2008) or measured in the plasma or urineof soy formula-fed infants (Setchell et al., 1997; Irvineet al., 1998; Franke et al., 2006; Cao et al., 2009). Ifglycitein is measured in humans, it is generally followingintentional dosing of subjects with a soy protein orisoflavone supplement.

Before reaching the systemic circulation, most genis-tein and daidzein is conjugated to glucuronic acid byUDP-glucuronosyltransferases; a much smaller amountis conjugated to sulfate by sulfotransferase enzymes(Joannou et al., 1995; Kurzer and Xu, 1997; UK-Committee-on-Toxicity, 2003). Conjugation of genisteinoccurs in the intestine but also has been reported to occurin liver. Based on in vitro data, the enzymes mostinvolved in glucuronidation are: UGT1A1, 1A8, 1A9,1A10 (Tang et al., 2009), whereas the SULT enzymeslikely responsible for sulfation include SULT1A1 andSULT2A1 (Doerge et al., 2000; Chen et al., 2008).However, interpretation of the in vitro data requiresinformation on tissue-specific expression, particularly forhuman intestine and liver. Moreover, to inform thequestion of disposition of soy formula isoflavones, theontogeny of such tissue-specific expression is needed.Only a limited amount of such data are available (Hines,2008) precluding estimates of developmental differencesamong humans, as well as interspecies differencesamong the young.

The glucuronide and sulfate conjugates can enter thesystemic circulation, and it has been reported that themajority of isoflavone compounds in the circulationare present in conjugated form, thus limiting the bio-availability of the aglycone forms of isoflavones. Instudies in which humans were exposed to genistein orisoflavone aglycones at genistein doses of 1 to 16 mg/kgbw, most of the genistein was present in plasma inconjugated form, while free genistein represented 1 to 3%of total plasma genistein levels in most cases (Setchellet al., 2001; Busby et al., 2002; Bloedon et al., 2002).Conjugated isoflavones undergo enterohepatic circula-tion, and on return to the intestine, they are deconjugatedby bacteria with b-glucuronidase or arylsulfatase activity.The metabolites may be reabsorbed or further metabolizedby gut microflora. Isoflavones can undergo furtherbiotransformation that ultimately leads to the formationof 60-hydroxy-O-desmethylangolensin from genistein andO-desmethylangolensin from daidzein (Joannou et al.,1995; Kurzer and Xu, 1997; UK-Committee-on-Toxicity,2003). The metabolic profile varies among individuals,with some individuals producing little or no O-desmethyl-angolensin or equol, an intermediate metabolite ofdaidzein that is biologically active.

Based on the summary of the studies presentedin Nielsen and Williamson (2007) and Cassidy (2006),pharmacokinetic parameters (cmax, tmax, t1/2, area underthe plasma concentration-time curve [AUC]) are similar

for genistein and daidzein. For genistein, the average tmax

was 5.7 hr (range of 3.5–9.3 hr) and for daidzein, it was6.2 hr (range of 4.0–8.3 hr). These estimates are consistentwith the range of peak values of B6 to 8 hr reported byothers for genistein and daidzein following ingestion ofsoy or isoflavones (Pumford et al. (2002); reviewed inWhitten and Patisaul (2001); UK-Committee-on-Toxicity(2003); Larkin et al. (2008). The maximum concentrations(Cmax) of genistein and daidzein, when normalized toingestion of 1mmol/kg bw, were also similar at 0.49 or0.64 mM or 0.50 or 0.54 mM, respectively. The average half-life of elimination (t1/2) estimates from reviews byNielsen and Williamson (2007) and Cassidy (2006) didnot differ between genistein, 9.5 hr (range 6.1–17 hr), anddaidzein, 7.7 hr (range 4.2–16 hr). These estimates aregenerally similar to those presented in a recent review byLarkin (2008); 3 to 9 hr for daidzein and 8 to 11 hr forgenistein after intake of soy foods or pure isoflavoneglycosides. The AUC values, when normalized toingestion of 1mmol/kg bw, were 11 mmol�hr/liter forgenistein and 18mmol�hr/liter for daidzein. Absorptionhalf-lives for both daidzin and genistin, presumably asglucuronides and sulfates of daidzein and genistein,were reported at B1 to 3 hr following intake of foodscontaining r210 mg of each isoflavone or providingdoses of r2 mg/kg bw/day of each isoflavone (Lu et al.,1995; Lu and Anderson, 1998); reviewed in (Whitten andPatisaul, 2001).

Studies in humans that report the bioavailability andother pharmacokinetic parameters of isoflavones ingestedas glycosides versus aglycones have reported conflictingfindings (reviewed in UK-Committee-on-Toxicity, 2003;Cassidy et al., 2006; Nielsen and Williamson, 2007).Although there is no clear explanation for the conflictingfindings on bioavailability of the aglycone and glucoside,one factor may be the differences in the type of isoflavonepreparations administered to subjects, e.g. soybean extractscontaining mixtures of isoflavones, purified single iso-flavone, ingestion in tablet or liquid form (Cassidy, 2006).In addition, comparing the conclusions on bioavailabilityacross studies is difficult because of the variety of indicesused to assess bioavailability, e.g. relative AUC, cmax, tmax,urinary excretion, and recovery, etc. Prasain and Barnes(2007) discussed the various approaches used to assess thebioavailability of isoflavones. In pharmacology (andtoxicology), the term absolute bioavailability refers to theratio of AUC after oral ingestion to the AUC after systemicadministration, i.e. intravenous injection. This is thefraction of the compound absorbed through nonintrave-nous administration compared with the correspondingintravenous administration of the same drug. Thisapproach may underestimate bioavailability at the tissuelevel if circulating isoflavones in the conjugated form areconverted to aglycones by b-glucuronidases and sulfatasessecreted by cells within target tissues. In the nutritionliterature, bioavailability is often assessed by the percent ofadministered isoflavone that is recovered in the urine.Higher degrees of recovery in urine are interpretedas more bioavailability based on the assumption thatthe isoflavone must have been present in bloodbefore reaching the kidney and ultimately being excretedin urine.

In their review, Nielsen and Williamson (2007) con-cluded that despite the apparently contradictory findings,the data are consistent enough to conclude that (1) at

427NTP-CERHR EXPERT PANEL REPORT

Birth Defects Research (Part B) 92:421–468, 2011

Page 8

equivalent doses, the Cmax is higher for genistein anddaidzein following administration as glucosides comparedwith aglycones and (2) the half-life is not significantlydifferent for aglycone and glucoside. In addition, theyconcluded that deglycosylation is required for absorptionbut does not appear to be a rate-limiting step. As notedabove, studies by Setchell et al. (2001) and Rufer et al.(2008) suggest the percent of genistein and daidzeincirculating in the unconjugated forms do not differ basedon administered form. These conclusions support theassumption used by others that because glycosides arequickly deconjugated in the gut to form the activeaglycones, exposure to a particular isoflavone (e.g.genistein) is theoretically the sum of the aglycone andrespective glycoside compound concentrations convertedon the basis of molecular weight (MAFF; Setchell et al.,1998; UK-Committee-on-Toxicity, 2003; Rozman et al.,2006).

No pharmacokinetic studies, defined as measurementof isoflavone concentrations in serial blood or plasmasamples, have been conducted in infants or children.The only data that are interpretable in this context are thedata presented in Setchell et al. (1997) and Cao et al.(2009). These studies reported genistein and daidzeinconcentrations in plasma or blood samples obtained atunspecified times relative to feeding in infants chroni-cally fed soy formula. Usual feeding behavior wasreported to result in mean genistein concentrations of684 and 757 ng/ml, and mean daidzein concentrations of295 and 256 ng/ml by Setchell et al. (1997) and Cao et al.(2009), respectively. Data obtained by Cao et al. (2009)indicated that total genistein concentrations in wholeblood ranged from approximately 13.5 to 3562.9 ng/ml(range data obtained from personal communication withDr. Cao, December 2009). Assuming that the infants wereconsuming soy formula for more than 2 to 3 days, thesevalues approximate a steady-state condition, and thereported concentrations provide a context in whichanimal data can be prioritized and interpreted.

Limited data suggest low levels of equol are detectedin both whole blood and urine of infants, regardless oftype of feeding (Setchell et al., 1997, 1998; Hoey et al.,2004; Cao et al., 2009). Setchell et al. (1997, 1998) detectedequol in all cow-milk formula-fed infants, four of sevensoy formula-fed infants, and one of seven breast-fedinfants. In contrast, Cao et al. (2009) did not detectequol in the blood of any infant, but reported detectionof equol in urine among infants fed cow milk formula(22%) compared with those fed soy formula (5%) orbreast-fed infants (2%). Also, given that equol is presentin breast milk and cow milk (King et al., 1998; Setchellet al., 1998), it does not appear that soy-based formula isassociated with increased exposure to equol relative tocow milk-based formula or breast milk. Collectively,these data indicate that relatively low exposures toequol can be detected not only in infants fed soy formulabut also in infants fed cow milk-based formula andbreast milk. Thus, this level of equol exposure in humaninfants is markedly lower than that reported in animalsfed soy-based diets or daidzein. These data from humaninfants provide a context for interpreting the animaldata presented in the section ‘‘Summary of Develop-mental Toxicity of Soy Infant Formula, IndividualIsoflavones Found in Soy Infant Formula, or Other SoyTreatments.’’

Experimental animals. Genistein is absorbed inrats and mice following oral or subcutaneous (sc)exposure. According to data in Table 5, maximumgenistein levels in blood are obtained within 2 hr ofexposure. A mass-balance study of rats orally treated bygavage with 14C-genistein 4 mg/kg bw reported Vd at1.27 to 1.47 L (Coldham and Sauer, 2000). The ExpertPanel noted that the reported Vd suggests that most ofthe circulating radioactivity was not genistein but theglucuronide. Plasma protein binding ranged from B80 to90%. Radioactivity was distributed throughout the body,with levels in reproductive organs (vagina, uterus, ovary,and prostate) higher than levels in other organs (brain,fat, thymus, spleen, skeletal muscle, and bone). Somestudies demonstrated higher levels of genistein aglyconeversus conjugates within tissues compared with blood,raising the possibility of accumulation or hydrolysis ofaglycones within tissues (Fritz et al., 1998; Chang et al.,2000; Doerge et al., 2000). The Expert Panel noted thatdifferences between free genistein levels in blood andtissues are probably due to differences in how theaglycone and glucuronide compounds partition betweenfat in blood and tissues.

There are two high-quality studies that togetheraddress the impact of route of administration in youngmice (Doerge et al., 2002; Jefferson et al., 2009a,b). Basedon these reports, the dose-adjusted AUC after oralgenistin in CD-1 mice is about half that of the scgenistein. Although there is some inconsistency in theCmax data, both values are relevant to humans as they arewithin the range of blood and plasma concentrationsreported in human infants.

Studies in adult rats and mice fed soy-containing feedreported that blood total equol levels were 2 to 10 timeshigher than blood total genistein and daidzein levels(Brown and Setchell, 2001; Lund et al., 2001; Weber et al.2001). In most cases, total equol levels were lower than orwithin the same order of magnitude as genistein anddaidzein levels in fetuses or pups of rat dams fed soy-based diets (Brown and Setchell, 2001; Weber et al., 2001).The half-life for genistein and its metabolites followingingestion of soy products by rats was reported at 8.8 hr.No data were found for half-life of daidzein and itsmetabolites following ingestion through soy foods.Urinary levels of total genistein and daidzein werereported to be B25% of total equol levels in mice andB50% of total equol levels in rats.

Studies demonstrated placental transfer of genistein tothe rat fetus (Fritz et al., 1998; Doerge et al., 2001; Soucyet al., 2006) and lactational transfer to the rat pupfollowing dietary administration of genistein to the dam(Chang et al., 2000). A study examining placental transferreported higher concentrations of aglycone in fetusescompared with dams, leading the authors to concludethat placental transfer probably involves the aglycone;the finding was said to be consistent with limitedconjugation ability of the fetal rat (Doerge et al., 2001).One study reported that the percentage of free genisteinin milk from the pup stomach (78–97%) was higher thanin milk from the dams’ nipples (57%), suggesting thatgenistein conjugates may be hydrolyzed in the pupstomach (Fritz et al., 1998).

Studies in rats demonstrated the distribution of iso-flavones and metabolites to fetuses during pregnancy orpups during lactation following ingestion of soy-containing

428 MCCARVER ET AL.

Birth Defects Research (Part B) 92:421–468, 2011

Page 9

Tab

le5

Blo

od

Gen

iste

inL

evel

sin

Ro

den

tsF

edP

hy

toes

tro

gen

-Fre

eD

iets

and

Do

sed

wit

hG

enis

tein

Ser

um

gen

iste

in,

nM

(ng

/m

lo

rmg

/li

ter)

Sp

ecie

san

dst

rain

Ag

ean

dn

um

ber

of

anim

als

Ro

ute

,d

ura

tio

no

fex

po

sure

Do

seT

ota

lA

gly

con

eP

erce

nt

agly

con

eR

efer

ence

Rat

,S

pra

gu

e-D

awle

yD

ams

and

fetu

ses

on

GD

20o

r21

,n

51

dam

(11–

16fe

tuse

s)/

gro

up

Ora

l(g

avag

e),

sin

gle

trea

tmen

to

fd

amo

nG

D20

or

21,

blo

od

coll

ecte

d2

hr

afte

rd

osi

ng

20m

g/

kg

bw

Dam

s:35

40[9

56

]D

ams:

270

[73

]D

ams:

8%D

oer

ge

etal

.(2

001)

Fet

use

s:27

0[7

3]a

Fet

use

s:80

[22

]aF

etu

ses:

31%

34m

g/

kg

bw

Dam

s:54

80[1

48

0]D

ams:

290

[78

]D

ams:

5%F

etu

ses:

190

[51

]F

etu

ses:

60[1

6]

Fet

use

s:34

%75

mg

/k

gb

wD

ams:

4410

[11

91

]D

ams:

780

[211

]D

ams:

18%

Fet

use

s:22

0[5

9]

Fet

use

s:60

[16

]F

etu

ses:

27%

Rat

,S

pra

gu

e-D

awle

yP

ND

1to

2m

ales

and

fem

ales

,n

52

to3

pu

ps

fro

m2

dif

fere

nt

litt

erp

oo

ls

Ora

l(d

iet)

,p

up

sex

po

sed

ind

irec

tly

du

rin

gg

esta

tio

nan

dla

ctat

ion

500

pp

m(B

50m

g/

kg

bw

/d

ay)

tod

ams

1767

307

[48

]47

[13

]53

[27

%b

yC

ER

HR

calc

ula

tio

n]

Do

erg

eet

al.

(200

1)

Rat

,S

pra

gu

e-D

awle

yD

ams

and

PN

D7

and

21p

up

s,n

um

ber

exam

ined

no

tre

po

rted

Ora

l(d

iet)

,d

ams

exp

ose

dd

uri

ng

ges

tati

on

and

lact

atio

n

0D

ams:

6[2

]aD

ams:

6[2

]aD

ams:

100%

Fri

tzet

al.

(199

8)P

ND

7:9

[2]

PN

D7:

9[2

]P

ND

7:10

0%P

ND

21:

6[2

]aP

ND

21:

6[2

]aP

ND

21:

100%

25p

pm

(B2.

2m

g/

kg

bw

/d

ay)

Dam

s:40

[11

]D

ams:

9[2

]D

ams:

23%

PN

D7:

86[2

3]

PN

D7:

16[4

]P

ND

7:19

%P

ND

21:

54[1

5]

PN

D21

:18

[5]

PN

D21

:33

%25

0p

pm

(B22

mg

/k

gb

w/

day

)D

ams:

418

[11

3]

Dam

s:7

[2]

Dam

s:1.

7%P

ND

7:72

6[1

96

]P

ND

7:10

3[2

8]

PN

D7:

14%

PN

D21

:18

10[4

89

]P

ND

21:

120

[32

]P

ND

21:

6.6%

Rat

,S

pra

gu

e-D

awle

yP

ND

21/

PN

D14

0o

ffsp

rin

g,

mal

esan

dfe

mal

es(n

55–

6/g

rou

p)

Ora

l(d

iet)

,d

ams

exp

ose

dd

uri

ng

ges

tati

on

and

lact

atio

n,o

ffsp

rin

gre

ceiv

edd

am’s

die

tat

wea

nin

g

0o

10/o

10[o

3]

–1

to5%

all

do

seg

rou

ps,

bo

thag

esC

han

get

al.

(200

0)5

pp

m[B

0.4

–0.5

mg

/k

gb

w/d

ay]

PN

D21

:–

22[6

](m

ale)

20[5

](f

emal

e)P

ND

140:

–60

[16

](m

ale)

100

[27

](f

emal

e)10

0p

pm

[B8

–1

0m

g/

kg

bw

/day

]P

ND

21:

270

[73

](m

ale)

520

[14

0]

(fem

ale)

PN

D14

0:59

0[1

59

](m

ale)

940

[25

4]

(fem

ale)

500

pp

m[B

40

–50

mg

/k

gb

w/d

ay]

PN

D21

:–

2090

[56

4]

(mal

e)18

70[5

05

](f

emal

e)P

ND

140:

–60

00[1

62

0](m

ale)

7940

[21

44]

(fem

ale)

429NTP-CERHR EXPERT PANEL REPORT

Birth Defects Research (Part B) 92:421–468, 2011

Page 10

Tab

le5

Co

nti

nu

ed

Ser

um

gen

iste

in,

nM

(ng

/m

lo

rmg

/li

ter)

Sp

ecie

san

dst

rain

Ag

ean

dn

um

ber

of

anim

als

Ro

ute

,d

ura

tio

no

fex

po

sure

Do

seT

ota

lA

gly

con

eP

erce

nt

agly

con

eR

efer

ence

Rat

,S

pra

gu

e-D

awle

yP

ND

91,

fem

ale,

n5

4O

ral

(die

t),

21d

ays

star

tin

gat

PN

D70

750

pp

m22

00[5

84

]40

0[1

08

]18

.2%

San

tell

etal

.(1

997)

Rat

,S

pra

gu

e-D

awle

yP

ND

70,

mal

e,n

58/

gro

up

Ora

l(d

iet)

,d

ams

exp

ose

dd

uri

ng

ges

tati

on

and

lact

atio

n,

and

then

dir

ectl

yu

nti

lP

ND

70

0p

pm

18[5

]a0

0%F

ritz

etal

.(2

002)

25p

pm

(B2.

2m

g/

kg

bw

/d

ay)

167

[14

]6

[2]a

3.6%

250

pp

m(B

22m

g/

kg

bw

/d

ay)

1908

[51

5]

20[5

]1.

0%

Rat

,S

pra

gu

e-D

awle

yP

ND

70,

mal

e,n

58/

gro

up

Ora

l(d

iet)

on

PN

D57

–65

and

gav

age

on

PN

D66

–70

028

[8]a

6[2

]a21

.4%

Fri

tzet

al.

(200

2)25

0p

pm

die

t;22

mg

/k

gb

w/

day

gav

ageb

1785

[48

2]

32[7

]1.

8%

1000

pp

md

iet;

88m

g/

kg

bw

/d

ayg

avag

eb96

40[2

60

2]41

[11

]0.

43%

Rat

,S

pra

gu

e-D

awle

yA

du

lt,

fem

ale

dam

s,n

54/

gro

up

Ora

l(d

iet)

on

GD

7–P

ND

2125

0p

pm

2100

[56

7]

30[8

]1.

4%H

old

eret

al.

(199

9)

PN

D63

,fe

mal

eo

ffsp

rin

g,

n5

10/

gro

up

Ora

l(d

iet)

,ex

po

sed

ind

irec

tly

du

rin

gg

esta

tio

n(f

rom

PN

D7)

thro

ug

hla

ctat

ion

(PN

D21

)an

dth

end

irec

tly

on

PN

D21

–63

250

pp

m13

10[3

54

]38

[10

]2.

9%12

50p

pm

5300

[14

31]

150

[40

]2.

8%

Rat

,S

pra

gu

e-D

awle

yA

du

lt,

mal

ean

dfe

mal

e,n

510

/g

rou

p;

n5

7–10

/g

rou

p

Ora

l(d

iet)

,[d

ura

tio

no

ftr

eatm

ent

no

tcl

earl

yre

po

rted

]

25p

pm

(2m

g/

kg

bw

/d

ay)

r25

0[r

68

]c–

–H

old

eret

al.

(199

9)

250

pp

m(2

0m

g/

kg

bw

/d

ay)

1500

[40

5]

(mal

e)–

–20

00[5

40

](f

emal

e)12

50p

pm

(100

mg

/k

gb

w/

day

)60

00[1

62

0](m

ale)

––

9000

[24

30]

(fem

ale)

Rat

,S

pra

gu

e-D

awle

y11

Wee

ks,

fem

ale,

n5

2–8

Ora

l(d

iet)

for

3w

eek

s,b

egin

nin

gat

8w

eek

so

fag

e

049

[13

]aC

otr

on

eoet

al.

(200

1)25

0p

pm

(B16

mg

/k

gb

w/

day

)11

15[3

01

]13

8[3

7]a

12

1000

pp

m20

31[5

48

]44

6[1

20

]23

Rat

,S

pra

gu

e-D

awle

y11

Wee

ks,

fem

ale,

n5

4–5

SC

for

3w

eek

s,b

egin

nin

gat

8w

eek

so

fag

e,b

loo

dco

llec

ted

16–1

8h

raf

ter

last

inje

ctio

n

04

[1]a

––

Co

tro

neo

etal

.(2

001)

5m

g/

kg

bw

/d

ay45

0[1

22

]–

–16

6m

g/

kg

bw

/d

ay13

80[3

73

]66

2[1

79]a

4850

mg

/k

gb

w/

day

5090

[13

74]

a22

43[6

06]a

44R

at,

Sp

rag

ue-

Daw

ley

PN

D21

,50

,an

d10

0,fe

mal

e,n

56–

9S

C,

sin

gle

do

seg

iven

at21

,50

,o

r10

0d

ays

of

age;

blo

od

coll

ecte

d16

–18

hr

afte

rin

ject

ion

500

mg

/k

gb

w21

day

s:55

58[1

50

1]a

21d

ays:

1956

[52

8]a

21d

ays:

[35

]C

otr

on

eoet

al.

(200

1)50

day

s:39

[11

]50

day

s:16

[4]

50d

ays:

[41

]10

0d

ays:

13[4

]10

0d

ays:

6[2

]10

0d

ays:

[46

]

Mo

use

,C

D-1

PN

D1–

5,m

ale/

fem

ale,

n5

3–8/

sex

/ti

me

per

iod

SC

,P

ND

1–5

blo

od

coll

ecte

db

etw

een

0.5

and

24h

rfo

llo

win

gin

ject

ion

50m

g/

kg

bw

/d

ay30

00[8

10

](m

ale)

B14

00[3

78

]m

ale

31D

oer

ge

etal

.(2

002)

5000

[13

50]

(fem

ale)

B23

00[6

21

]fe

mal

e

430 MCCARVER ET AL.

Birth Defects Research (Part B) 92:421–468, 2011

Page 11

feed by the dam (Brown and Setchell, 2001; Weber et al.,2001). In one study, gestation day (GD) 20.5 fetuses andPND 3.5 pups contained about half the total genisteinconcentration and about one-tenth the total equol concen-tration measured in dams; total daidzein concentrationswere about one-third lower in GD 20.5 fetuses and aboutthe same in PND 3.5 pups compared with dams (Weberet al. 2001). A second study reported that in pups born todams fed a soy-containing diet, total genistein levelsremained steady between birth and PND 12, while totaldaidzein levels were reduced by half during the same timeperiod (Brown and Setchell, 2001). On PND 16, the timewhen pups likely began eating food, there was a 5- to10-fold drop in total genistein and daidzein concentrationscompared with earlier time periods. In contrast, total equollevels in pups decreased steadily following birth.

Genistein glucuronide is the most abundant genisteinmetabolite in rat blood (Coldham and Sauer, 2000).Genistein is conjugated with glucuronide in the intestineand liver, and a study in rats demonstrated that themajority of glucuronidation most likely occurs in theintestine (Sfakianos et al., 1997). With the exception of4-hydroxyphenyl-2-propionic acid, all other urinarygenistein metabolites identified in rats were alsoreported for humans, suggesting pathways common tothe two species. Parent compound was the predominantform of genistein in the uterus, while in prostate the mostabundant form was the metabolite 4-hydroxyphenyl-2-propionic acid. One study reported no evidence thatgenistein aglycone or conjugate levels in blood weresaturated following exposure to dietary genistein at up to1250 ppm.

The Expert Panel noted that comparisons of serumaglycone levels in adult versus fetal or neonatal rodentsof the same study can be made from the rat datapresented in Table 5. A sc dosing study conducted in ratsdemonstrated similar percentages of serum aglycone(35–46%) at PND 21, 50, or 100. One study with gavageexposure demonstrated higher aglycone percentages infetuses (27–34%) than dams (5–18%) on GD 20 or 21(Doerge et al., 2001). A dietary study in which dams werefed 25 or 250 ppm genistein did not consistentlydemonstrate higher percentages of aglycone in dams(1.7–23%) compared with pups on PND 7 (14–19%) orPND 21 (6.6–33%) (Fritz et al., 1998). In an evaluationof all the data in Table 5, it was noted that percentagesof free genistein following oral exposure of adult ratswere usually below 10% but sometimes attained levelsof B20%; percentages of aglycone following direct and/or indirect oral exposure to genistein in rat pups r21days old were reported at 1 to 33%.

In a mass-balance study of rats orally treated bygavage with 4 mg/kg bw 14C-genistein, B65% of thedose was excreted in urine and 33% in feces at 166 hrfollowing dosing (Coldham and Sauer, 2000). Approxi-mately, 90% of the dose was recovered within the first48 hr following dosing. Total clearance was 1.18 mL/minin males and 2.0 mL/min in females. Genistein elimina-tion half-lives have been reported at 2 to 9 hr in rats and 5to 8 hr in mice (Coldham and Sauer, 2000). The ExpertPanel noted an apparent contradiction between the half-lives reported by Chang (2000) (B3–4 hr) and Coldhamand Sauer (2000) (B9–12 hr). The differences in half-livesmay have resulted from dosing regimen. Coldham andSauer used a single low dose of 4 mg/kg bw and ChangM

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431NTP-CERHR EXPERT PANEL REPORT

Birth Defects Research (Part B) 92:421–468, 2011

Page 12

et al. (2000) used a high daily dose rate of 50 mg/kg bw.The greatly decreased half-life at the higher dose mayhave resulted in part from saturation of glucuronidationand, hence, reduced enterohepatic circulation. Because itis expected that protein binding is saturated at highgenistein doses, a much smaller portion of the higherdose would be bound to plasma proteins, contributing tothe shorter half-life. In neonatal mice, elimination halflives were reported at 12 to 16 hr for genistein aglyconeand 16 to 19 hr for genistein conjugate.

SUMMARY OF DEVELOPMENTAL TOXICITYOF SOY INFANT FORMULA, INDIVIDUAL

ISOFLAVONES FOUND IN SOY INFANTFORMULA, OR OTHER SOY TREATMENTS

Human Data

No human data were identified on exposures to theindividual isoflavones found in soy formula.

Studies on outcomes after feeding soy formula ininfancy are summarized in Table 6. Most of the studieson growth have not detected differences in growth offull-term infants fed soy and cow-milk formula, but mostof the studies had small sample sizes (Jung and Carr,1977; Steichen and Tsang, 1987; Venkataraman et al.,1992; Mimouni et al., 1993; Lasekan et al., 1999). Agostoniet al. (2007) reported better short-term weight gain ininfants with immunoglobulin E-mediated cow’s milkallergy who were fed a rice or casein hydrolysate formulacompared with soy formula. Some studies reported thatinfants given soy formula had transiently lower bone-mineral content than infants fed cow-milk formula(Kohler et al., 1984) or human milk (Chan et al., 1987);however, other studies reported that serum concentra-tions of bone accretion-related hormones and minerals insoy formula-fed infants demonstrated the appropriatecompensatory response to the vitamin and mineralcontent of the food source (Hillman, 1988; Hillmanet al., 1988; Mimouni et al., 1993). Reports in very lowbirth-weight and preterm babies suggest that soyformula may not support adequate growth and bonedevelopment in these groups (Kulkarni et al., 1984). Soyinfant formula is not marketed for use in preterm infants,and the AAP recommends against its use in this group.

Studies comparing soy- and cow milk-formula feedingin infants with familial predispositions to allergic diseasedid not detect an advantage of one formula over another(Gruskay, 1982; Hillman et al., 1988; Klemola et al., 2002),but most of these studies have demonstrated that breastfeeding was superior to the use of either formula.A series of studies by Chandra (1989, x864; 1989, x765;1991, x865; 1997, x549; 1998, x550) related to allergicdisease were considered by the Expert Panel butclassified as no utility for the evaluation because thevalidity of the articles has been challenged and thescientific integrity cannot be confirmed.

Case reports suggest that hypothyroid infants on oralthyroxine therapy fed soy formula may exhibit lessefficient absorption of the thyroxine from the intestine.This effect has been attributed to fecal wastage withdecreased enterohepatic circulation (Shepard, 1960;Chorazy et al., 1995; Jabbar et al., 1997). Although theaddition of iodine and reduction in fiber content of soyformulas is a partial solution, one article noted that the

problem may occur if formula intolerance increases stoolfrequency (Chorazy et al., 1995). Conrad et al. (2004)reported that infants with CH-fed soy formula had aprolonged elevation of TSH levels compared with infantswith CH-fed nonsoy formula, suggesting that infantswith CH-fed soy formula may require higher levothyr-oxine doses to achieve normal thyroid function tests.

A case–control study of premature thelarche used 130subjects from 552 potentially eligible girls (Freni-Titulaeret al., 1986). Age-matched controls were recruited,consisting in some but not all instances of friends ofthe cases. Parents were interviewed with regard to familyhistory and possible exposures, including the use of soyformula. Multivariate analysis did not show a significantrelationship between premature thelarche and soy-formula feeding. When the analysis was restricted togirls with onset of premature thelarche before 2 years ofage, a significant association with soy-formula feedingwas identified (OR 2.7, 95% CI 1.1–6.8). Other significantfactors included maternal ovarian cysts and consumptionof chicken. Consumption of corn was protective.

Strom et al. (2001) identified 952 adults who hadparticipated as infants in a controlled, nonrandomizedformula trial. There were 248 adults who had been givensoy formula and 563 who had been given cow-milkformula. The subjects were asked about height, weight,sexual maturation, education level, and reproductiveoutcomes. No formula-related differences in height,weight, body-mass index, or sexual maturation wereidentified. Women who had been given soy formula hadlonger menstrual periods (mean 0.37 days longer, 95% CI0.06–0.68 days) and there was a soy formula-associatedincrease in the risk of experiencing severe menstrualdiscomfort (RR 1.7, 95% CI 1.04–3.00 compared with noor mild discomfort, unadjusted). After adjustment formultiple comparisons, these findings were no longerstatistically significant (more than 30 different endpointswere evaluated).

In a study comparing women with and without breastcancer by Boucher et al. (2008), the point estimates of theodds ratios were less than unity. However the variabilityof these estimates indicate that feeding soy formula wasnot statistically significantly associated with developingbreast cancer (soy formula only during first 4 months oflife: OR 5 0.42, 95% CI 5 0.13–1.40; soy formula onlyduring 5–12 months of age: OR 5 0.59, 95% CI 5 0.18–1.90).

Experimental Animal Studies on the IndividualIsoflavones Found in Soy Formula

Studies reporting the most sensitive and apparentlytreatment-related developmental effects are summarizedin Table 7 for oral and parenteral exposures in mice,Table 8 for oral and parenteral exposures in rats, andTable 9 for animal studies in other mammalian species. Inthese tables, dose levels have been converted to mg/kgbw. In general, the most complete information wasavailable from parenteral exposure studies in mice andoral exposure studies in rats. In cases where doses wereconverted to mg/kg bw/day values, ranges were oftenestimated over periods of gestation or lactation or indifferent stages of the offspring’s life.

Pre- and postnatal survival, growth, and gen-eral development endpoints. Oral exposure studiesconducted in rats suggested that genistein exposures can

432 MCCARVER ET AL.

Birth Defects Research (Part B) 92:421–468, 2011

Page 13

Table 6Studies of Human Infants Exposed to Soy Formula

Comparison groups Major findings Reference Utilitya

Growth and NutritionInfants with immunoglobulin E-mediated cow’s milk

allergy fed breastmilk (n 5 32) (reference) andinfants randomly assigned to one of three specialformulas during a feeding period of 6 to 12 monthsof age: soy formula (n 5 32), a casein hydrolysate(n 5 31), and a rice hydrolysate (n 5 30)

The 6- to 12-month change in weight-for-age z-scorefor the soy formula group was significantly lowercompared to the rice (0.18, p 5 0.019) and casein(0.16, p 5 0.029) groups. This indicates better short-term weight gain with hydrolyzed products whencompared with soy formula

Agostoni et al.(2007)

Limited

Infants randomized to one of two different soyformulas (n 5 10/group) and 10 breast-fedinfants (for comparisons to 4 months of age)

k bone mineral density and plasma zinc higher in soyformula-fed infants at 4 months of age; unable todetect difference between soy formula-fed infantsand historical controls in these parameters at 6 and12 months

Chan et al.(1987)

Limited

No effect: on measures of growth and serumchemistries

Infants fed soy infant formula (n 5 28) and infantsfed cow-milk formula (n 5 30) for 6 months. Thesoy formula was not methionine-supplementedas is modern soy formula

Soy-fed infants, especially girls, lagged in volumeconsumed and growth from 2 to 4 months of age;k weight gain per fluid ounce of formula;k vomiting; k number of infants hungry afterfeeding; m loose stools

Cherry et al.(1968)

Limited

No effect: on anemia or hematologic parametersInfants on soy formula (n 5 11), cow-milk formula

(n 5 11), or human milk (n 5 9), followed for 1year

Unable to detect a difference in bone width or bonemineral content; differences in serum and urineminerals and bone-related hormones wereconsistent with homeostatic adjustments todifferent mineral content in the food sources

Hillman et al.(1988, 1988)

Limited

Infants on soy formula (n 5 20) and infants on cow-milk formula (n 5 20) for 16 weeks

Unable to detect a difference between the 2 groups ingrowth parameters, hematology measures, or bloodchemistry

Jung and Carr(1977)

Limited

Infants on soy formula (n 5 13), infants on cow-milk formula (n 5 20), and breast-fed infants(n 5 26). Infants selected at 6 weeks of age andfollowed for 1 year

Weight at birth; unable to detect growth differencesbetween groups after 6 weeks of age; soy-fedinfants showed slower bone mineralization at 3months but not thereafter

Kohler et al.(1984)

Limited

Very low birth-weight infants given soy formula,cow-milk formula, or high-calorie cow-milkformula until 3–4 months of age

k serum phosphorus and serum alkaline phosphataseafter three weeks of age

Kulkarni et al.(1984)

Limited

Infants on soy formula with (n 5 73) or without(n 5 73) supplemental nucleotides, and infantsbreast-fed for 2 months and then given cow-milk formula (n 5 67); infants followed to 12months of age

Groups were not comparable in weight and lengthat birth. No group differences in growth weredetected after 6 months of age

Lasekan et al.(1999)

Limited

Infants given 1 of 2 different soy formulas (n 5 21/group), infants given cow-milk formula (n 5 20),and breast-fed infants (n 5 10), followed for 1year; solid food introduced at 3 months inbreast-fed and 4 months in formula-fed groups

m length gain; m serum 1,25-dihydroxyvitamin D in 1of the soy-formula groups at 8 weeks andthereafter; m serum phosphorus in combinedformula groups at 8 weeks. Unable to detect effectof feeding type on weight and head circumference;bone mineral content and bone width; bloodchemistry

Mimouni et al.(1993)

Limited

Infants being breastfed (n 5 401) and infantsassigned to either cow-milk (n 5 839) or soybased formula (n 5 239) based on family historyof allergy. Evaluated monthly until 6 months ofage and bi-monthly thereafter

No differences in length or weight were detectedbetween the 3 feeding groups.

Sellars et al.(1971)

Limited

Infants fed soy formula (n 5 18) or cow-milkformula (n 5 17) for 12 months

k bone mineral content; Unable to detect differencesin energy intake or growth

Steichen andTsang (1987)

Limited

Infants exclusively fed soy formula (n 5 20), cow-milk formula (n 5 19), or breast-fed (n 5 17) for 4months and followed until 6 months of age

m bone mineral content and bone width in soyformula-fed infants compared to breast-fed infants;Unable to detect group differences in growth andserum measurements related to bone accretion

Venkataramanet al. (1992)

Limited

Allergy, immunology, and gastrointestinal effectsInfants with a family history of major allergy

assigned to soy formula (n 5 79) or cow-milkformula (n 5 201), followed 17 years; 48 infantswith a family history of major allergy werebreast fed

Breast-fed children were significantly less likely todevelop allergy after 3 years than soy or cow-milkformula-fed children

Gruskay (1982) Limited

No significant difference in development of allergicdisease by formula type

433NTP-CERHR EXPERT PANEL REPORT

Birth Defects Research (Part B) 92:421–468, 2011

Page 14

Table 6Continued

Comparison groups Major findings Reference Utilitya

Infants with cow milk allergy diagnosed at age 2–11 months were randomly assigned toextensively hydrolyzed formula (n 5 90) or soyformula (n 5 80) and followed until 2 years ofage

Parents suspected adverse reaction in 28% of subjectson soy formula and 11% of subjects on extensivelyhydrolyzed formula. Among children younger than6 months, 40% were suspected by parents of havingadverse reaction to soy formula

Klemola et al.(2002)

Limited

Infants with milk allergy fed whey formula(n 5 76) or soy formula (n 5 72), examined atage 2, 3, and 4 years

Non-significant increase in soy IgE levels in soyformula-fed infants (OR 2.28, 95% CI 0.90–5.76;p 5 0.082)

Klemola et al.(2005)

Limited

No effect: prevalence of peanut IgE positivity (OR1.27, 95% CI 0.35–4.71; p 5 0.717)

Children with peanut allergy (n 5 49), childrenwith atopy (n 5 70), and non-allergic children(n 5 140) surveyed for infant feeding history

Soy consumption was a significant and independentrisk factor for peanut allergy (adjusted OR 2.61;95% CI 1.31–5.20)

Lack et al. (2003) Limited

Infants given 1 of 2 different soy formulas (n 5 92,94), one of which contained added nucleotides,and breast-fed children who were weaned tocow-milk formula at 2 months (n 5 81); 1-yearfollow-up

Unable to detect a difference by feeding group in antibodyresponse to immunizations (except Hemophilus influenzab), parent-reported diarrhea, or otitis media. Noconsistent differences were detected in immune status,maturation, or level of immunocompetence betweensoy and cow-milk formula-fed infants

Ostrom et al.(2002), Cordleet al. (2002)

Limited

Infants with cow’s milk allergy fed soy formula(n 5 84, mean starting age 5 7.8 mths) or aextensively hydrolyzed whey formula (HWF)(n 5 84, mean starting age 5 7.5 mths),examined until 48 mths of age

m percentage of energy; mintake of zinc and vitamin E;k riboflavin

Seppo et al.(2005)

Limited

No effect: percentages of abnormally low lab values;percentages of high alkaline phosphatase; growthmeasured between 1 to 4 years; nutritional status

Thyroid functionInfant with congenital hypothyroidism who was

fed soy formula (n 5 1)Oral thyroxine therapy did not decrease thyroid-

stimulating hormone until cow-milk formula wassubstituted for soy formula

Chorazy et al.(1995)

Limited

Infants fed soy formula (n 5 8) and infants fednonsoy formula (n 5 70)

Infants with congenital hypothyroidism fed soyformula had prolonged increase of TSH levelscompared to infants with congenitalhypothyroidism fed non-soy formula

Conrad et al.(2004)

Limited

No effect: on total T4 between groups; weight; height;weight for height

Infants with congenital hypothyroidism who werefed soy formula (n 5 3)

Switching from soy formula to cow-milk formularesulted in increased absorption of thyroidreplacement doses

Jabbar et al.(1997)

Limited

Children with goiter related to soy formula-feeding (n 5 3)

Improvement with discontinuation of soy formula(n 5 2) or addition of iodine (n 5 1)

Shepard (1960) Limited

Reproductive function or tissue effectsAdults with breast cancer (n 5 372) and without

breast cancer (n 5 356) who had been fed soyformula, cow-milk formula, or breastmilk

A reduced, but non-significant, association was foundbetween soy formula intake and breast cancer. Soyformula only during fist 4 months of life: OR 5 0.42,95% 5 0.13–1.40. Soy formula only during 5–12months of age: OR 5 0.59, 95% CI 5 0.18–1.90

Boucher et al.(2008)

Limited

Girls with premature thelarche (n 5 130) and age-matched control subjects, retrospectivequestioning of parents about infant feeding

Unable to detect a significant association overallbetween premature thelarche and soy infantformula intake; restriction of multivariate analysisto subjects with thelarche before age 2 yearsshowed significant association (OR 2.7; 95% CI 1.1–6.8, p 5 0.029)

Freni-Titulaeret al. (1986)

Limited

Adults who had been fed soy (n 5 248) or cow-milk formula (n 5 563) during infancy as part ofa controlled trial; interviewed at 20–34 years ofage

Unable to detect infant feeding-related differences inadult height, weight, body-mass index, or sexualmaturation history; duration of menstrual bleedingwas 0.37 days longer and severe menstrualdiscomfort was more common in women fed withsoy formula than with cow-milk formula

Strom et al.(2001)

Limited

Other endpointsInfants on soy formula (n 5 16, some with

cholesterol added), cow-milk formula (n 5 10),or breast-fed with supplemental cow-milkformula (n 5 12)

Breast-fed infants had the highest serum cholesterollevels and lowest fractional cholesterol synthesisrate. There was a significant inverse relationshipbetween cholesterol intake and fractionalcholesterol synthesis

Cruz et al. (1994) Limited

aThe term utility refers to the applicability to the purpose of drawing conclusions on whether or not a chemical adversely affects reproduction.

434 MCCARVER ET AL.

Birth Defects Research (Part B) 92:421–468, 2011

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-1m

ice;

0,0.

5,5,

or

50m

g/

kg

bw

/d

ayg

enis

tein

by

scin

ject

ion

on

PN

D1–

5D

istr

ibu

tio

no

ffe

mal

esin

var

iou

sst

ages

of

estr

ou

scy

cle

at2

mo

nth

so

fag

e–

0.5

Jeff

erso

net

al.

(200

5)H

igh (par

ente

ral)

kn

um

ber

of

dam

sw

ith

litt

ers

at2

and

6m

on

ths

of

age

(au

tho

r’s

rep

ort

that

‘‘a

tren

dte

stsh

ow

eda

stat

isti

call

ysi

gn

ific

ant

dec

reas

ein

the

nu

mb

ero

fm

ice

wit

hli

tter

sat

2an

d6

mo

nth

so

fag

ew

ith

incr

easi

ng

do

seas

det

erm

ined

by

the

Co

chra

n-A

rmit

age

test

[po

0.05

].’’

)

–Z

0.5

(sig

nif

ican

tn

egat

ive

tren

d)

kn

um

ber

of

liv

ep

up

sb

orn

tod

ams

of

2,4

and

6m

on

ths

of

age

(wh

enal

lag

eslo

ok

edat

sim

ult

aneo

usl

y)

0.5

5

kco

rpo

ralu

tea

per

dam

at4

mo

nth

so

fag

e5

50n

oef

fect

on

nu

mb

ero

fp

lug

po

siti

ve

mic

e,n

um

ber

of

ov

ula

ted

oo

cyte

sfo

llo

win

gtr

eatm

ent

of

mic

ew

ith

hu

man

cho

rio

nic

go

nad

otr

op

inat

4m

on

ths

of

age,

ov

eral

ltr

eatm

ent

effe

cts

on

seru

mp

rog

este

ron

e,17b-

estr

adio

l,o

rte

sto

ster

on

ele

vel

s

50(h

igh

do

se;

no

effe

ct)

–

435NTP-CERHR EXPERT PANEL REPORT

Birth Defects Research (Part B) 92:421–468, 2011

Page 16

Tab

le7

Co

nti

nu

ed

An

imal

mo

del

and

stu

dy

des

ign

En

dp

oin

tsN

OE

LL

OE

LR

efer

ence

Uti

lity

a

CD

-1m

ice;

0o

r50

mg

/k

gb

w/

day

gen

iste

inb

ysc

inje

ctio

no

nP

ND

1to

5k

imp

lan

tati

on

site

s(b

oth

nu

mb

ero

fm

ice

and

nu

mb

ero

fsi

tes/

mo

use

);k

per

cen

tp

reg

nan

tm

ice;

kn

um

ber

of

corp

ora

lute

a

–50

(sin

gle

do

sest

ud

y)

Jeff

erso

net

al.

(200

5)L

imit

ed(p

aren

tera

l)

CD

-1m

ice;

0o

r50

mg

/k

gb

w/

day

gen

iste

inb

ysc

inje

ctio

no

nP

ND

1to

5k

nu

mb

ero

fem

bry

os

coll

ecte

dp

erm

ou

sefo

llo

win

gh

CG

adm

inis

trat

ion

;kn

um

ber

and

size

of

imp

lan

tati

on

site

sin

mic

ew

ho

wer

ere

cip

ien

tso

ftr

ansf

erre

db

last

ocy

sts

ob

tain

edfr

om

un

trea

ted

mic

e

–50

(sin

gle

do

sest

ud

y)

Jeff

erso

net

al.

(200

9b)

Lim

ited

(par

ente

ral)

No

effe

cto

nth

en

um

ber

of

ov

ula

ted

egg

s,ti

min

go

fd

evel

op

men

to

rp

erce

nta

ge

of

emb

ryo

sto

reac

hb

last

ocy

stag

e,th

en

um

ber

of

liv

ep

up

s,o

rli

tter

size

50(h

igh

do

se;n

oef

fect

)–

CD

-1;

0,12

.6,

20,

or

25m

g/

kg

bw

/d

ayg

enis

tein

scin

ject

ion

fro

mP

ND

1to

5m

ute

rin

ew

eig

ht

lim

ited

(par

ente

ral)

12.6

20Je

ffer

son

etal

.(2

009a

)sc

po

rtio

n

Lim

ited

(par

ente

ral)

CD

-1m

ice;

0o

r50

mg

/k

gb

w/

day

gen

iste

inb

ysc

inje

ctio

no

nP

ND

1to

5m

nu

mb

ero

fre

pro

du

ctiv

ele

sio

ns:

no

corp

ora

lute

a,ab

no

rmal

ov

idu

cth

isto

log

y,u

teri

ne

squ

amo

us

met

apla

sia,

cyst

icen

do

met

rial

hy

per

pla

sia

and

ute

rin

eca

rcin

om

a

–50

(sin

gle

do

sest

ud

y)

New

bo

ldet

al.

(200

1)L

imit

ed(p

aren

tera

l)

CD

-1m

ice;

0,0.

5,o

r10

mg

/k

gb

w/

day

gen

iste

inb

ysc

inje

ctio

no

nG

D15

to18

mb

od

yw

eig

ht

gai

n;

0.5–

1d

ayea

rlie

ro

nse

to

fv

agin

alo

pen

ing

;m

estr

ou

scy

cle

len

gth

,1.

2–2

day

s;m

len

gth

of

die

stru

s

–0.

5N

ikai

do

etal

.(2

004)

kn

um

ber

of

anim

als

wit

hco

rpo

ralu

tea

at4

wee

ks

of

age,

tran

sien

tef

fect

0.5

10

ICR

mic

e;0

or

1000

mg

/k

gb

w/

day

gen

iste

inb

yin

ject

ion

on

PN

D1

to5

kte

stic

ula

rm

RN

Aex

pre

ssio

no

fE

Ra,

ERb,

AR

and

lam

inin

-g2

–10

00(s

ing

led

ose

stu

dy

)A

dac

hi

etal

.(2

004)

Lim

ited

(par

ente

ral)

No

effe

cto

nb

od

yw

eig

ht,

abso

lute

or

rela

tiv

ete

stis

wei

gh

to

rh

isto

log

ical

chan

ges

inth

ete

stes

at12

wee

ks

of

age

1000

(hig

hd

ose

;n

oef

fect

)–

CD

-1p

reg

nan

tm

ice;

0,0.

2o

r2

mg

/k

gb

w/

day

of

gen

iste

inb

yip

inje

ctio

nfr

om

GD

1to

21N

oef

fect

on

gro

sso

rh

isto

log

ical

ano

mal

ies

of

the

ute

rus,

or

Hox

a10

mR

NA

exp

ress

ion

inth

eu

teru

s2

(hig

hd

ose

;n

oef

fect

)–

Ak

bas

etal

.(2

007)

Lim

ited

(par

ente

ral)

CD

-1p

reg

nan

tm

ice;

0o

r2

mg

/k

gb

w/

day

of

dai

dze

inb

yip

inje

ctio

nfr

om

GD

1to

21N

oef

fect

on

gro

sso

rh

isto

log

ical

ano

mal

ies

of

the

ute

rus,

or

Hox

a10

mR

NA

exp

ress

ion

inth

eu

teru

s2

(hig

hd

ose

;n

oef

fect

)–

CD

-1m

ice;

0,1,

10,

or

100mg

/d

ay(0

,0.

5,5,

or

50m

g/

kg

bw

/d

ay)

gen

iste

inb

ysc

inje

ctio

no

nP

ND

1to

5

mm

ult

i-o

ocy

tefo

llic

les

550

Jeff

erso

net

al.

(200

2)L

imit

ed(p

aren

tera

l)

C57

BL

/6,

ERa

and

ERb

mic

e;0,

1,10

,o

r10

0mg

/d

ay(0

,0.

5,5,

or

50m

g/

kg

bw

/d

ay)

gen

iste

inb

ysc

inje

ctio

no

nP

ND

1–5

mm

ult

i-o

ocy

tefo

llic

les

inE

Ra

and

no

effe

ctin

ERb

0.5

5

CD

-1m

ice;

50m

g/

kg

bw

/d

ayg

enis

tein

by

scin

ject

ion

fro

mP

ND

1to

5m

nu

mb

ero

fm

ult

i-o

ocy

tefo

llic

les;

few

ersi

ng

leo

ocy

tes

–50

(sin

gle

do

sest

ud

y)

Jeff

erso

net

al.

(200

6)L

imit

ed(p

aren

tera

l)C

D-1

mic

e;0

or

10m

g/

kg

bw

/d

ayg

enis

tein

by

scin

ject

ion

on

PN

D15

to18

acce

lera

tio

no

fv

agin

alo

pen

ing

–10

(sin

gle

do

sest

ud

y)

Nik

aid

oet

al.

([20

05)

Lim

ited

(par

ente

ral)

No

effe

ct:

on

bo

dy

wei

gh

t,es

tro

us

cycl

ing

,p

oly

ov

ula

ro

var

ian

foll

icle

s,m

orp

ho

log

ical

abn

orm

alit

ies

inv

agin

alo

ru

teri

ne

epit

hel

ium

,o

rm

amm

ary

gla

nd

dev

elo

pm

ent

10(h

igh

do

se;n

oef

fect

)–

ICR

mic

e;0,

7,71

,an

d71

4m

g/

kg

bw

/d

ayg

enis

tein

by

scin

ject

ion

on

PN

D1

to5

kA

Rm

RN

Aex

pre

ssio

nin

test

is7

71S

hib

ayam

aet

al.

(200

1)L

imit

ed(p

aren

tera

l)k

ERa

mR

NA

exp

ress

ion

inte

stis

7171

4 –

436 MCCARVER ET AL.

Birth Defects Research (Part B) 92:421–468, 2011

Page 17

No

effe

cto

nte

stis

wei

gh

t,sp

erm

cou

nt,

or

sper

mm

oti

lity

at12

wee

ks

of

age

714

(hig

hd

ose

;n

oef

fect

)M

ale

Han

-NM

RI

mic

e;0,

0.1

or

1m

g/

day

(50

or

500

mg

/k

gb

w/

day

)g

enis

tein

by

scin

ject

ion

on

PN

D1

to3

kv

entr

alp

rost

ate

wei

gh

tin

adu

lth

oo

d(h

isto

log

ical

chan

ges

ob

serv

ed)

–50

Str

auss

etal

.(1

998)

Lim

ited

(par

ente

ral)

kco

agu

lati

ng

gla

nd

wei

gh

t;m

his

tolo

gic

abn

orm

alit

ies

(hy

per

pla

sia

and

dis

org

aniz

atio

no

fth

eep

ith

eliu

mo

fth

ep

rost

atic

coll

ecti

ng

du

cts,

ven

tral

lob

es,

and

sem

inal

ves

icle

s);m

fib

rom

usc

ula

rst

rom

aan

din

flam

mat

ory

cell

sin

po

ster

ior

per

iure

thra

lre

gio

n

5050

0

No

effe

cto

nc-

fos

mR

NA

exp

ress

ion

inp

rost

atic

ure

thra

500

(hig

hd

ose

;n

oef

fect

)–

Mam

mar

ygl

and

deve

lopm

ent

and

carc

inog

enes

isC

D-1

mic

e;0

orB

0.7–

0.8

mg

/k

gb

w/

day

gen

iste

inb

yin

ject

ion

on

GD

15to

20m

mam

mar

yg

lan

dep

ith

elia

lar

eao

nP

ND

35(b

ut

no

to

nP

ND

25o

rP

ND

46);m

den

sity

of

TE

Bs

on

PN

D35

and

45;

mP

ND

25b

od

yw

eig

ht;

del

ayed

eye

op

enin

g;

acce

lera

ted

tim

ing

of

vag

inal

op

enin

g

–B

0.7–

0.8

(sin

gle

do

sele

vel

)H

ilak

ivi-

Cla

rke

etal

.(1

998)

Lim

ited

(par

ente

ral)

No

effe

cto

nn

um

ber

of

off

spri

ng

bo

rn,

PN

D1

bo

dy

wei

gh

t,d

iffe

ren

tiat

ion

of

bre

ast

tiss

ue

(ass

esse

du

sin

gd

ensi

tyo

fT

EB

san

dlo

bu

loal

veo

lar

un

its)

,se

rum

17b-

estr

adio

lo

res

tro

us

cycl

icit

y

B0.

7–0.

8(h

igh

do

se;

no

effe

ct)

–

CD

-1m

ice;

0,0.

5,5,

or

50m

g/

kg

bw

/d

ayg

enis

tein

by

scin

ject

ion

on

PN

D1

to5

Mam

mar

yg

lan

dh

orm

on

ere

cep

tor

lev

els:m

PR

pro

tein

exp

ress

ion

at5

wee

ks,

kE

Ra

mR

NA

at5

and

6w

eek

s(c

om

bin

ed)

–0.

5P

adil

la-B

ank

set

al.

(200

6)L

imit

ed(p

aren

tera

l)

md

uct

alel

on

gat

ion

at6

wee

ks;

mE

Rb

mR

NA

inm

amm

ary

gla

nd

at5

and

6w

eek

s(c

om

bin

ed)b

–0.

5b

kn

um

ber

of

TE

Bs

at6

wee

ks;k

nu

mb

ero

fb

ran

chp

oin

tsat

5w

eek

s(b

ut

no

tat

6w

eek

s);

mam

mar

yg

lan

dm

orp

ho

log

yat

9m

on

ths:

kal

veo

lar

dev

elo

pm

ent,

dil

ated

and

/o

rd

ilat

edb

ead

edd

uct

s

0.5

5

kn

um

ber

of

TE

Bs

at5

wee

ks;

km

amm

ary

gla

nd

du

ctal

gro

wth

at5

or

6w

eek

s;k

aver

age

area

of

mam

mar

yg

lan

dat

5o

r6

wee

ks;

alte

red

estr

ou

scy

clic

ity

550

kn

um

ber

of

pu

ps

per

litt

er,

litt

erw

eig

hts

,p

up

wei

gh

ts,

%p

up

ssu

rviv

ing

un

til

wea

nin

g5

50(n

oli

ve

pu

ps

del

iver

ed)

No

effe

cto

nci

rcu

lati

ng

lev

els

of

estr

adio

lo

rp

rog

este

ron

e,li

tter

size

,li

tter

wei

gh

ts,

calc

ula

ted

aver

age

pu

pw

eig

hts

,o

r%

of

pu

ps

surv

ivin

gu

nti

lw

ean

ing

50(h

igh

do

se;n

oef

fect

)–

Sp

rag

ue-

Daw

ley

;50

mg/

day

(au

tho

rses

tim

ated

that

the

do

ses

rece

ived

wer

e1.

25to

3.3

mg

/k

gb

wg

enis

tein

by

scin

ject

ion

on

PN

D8

to20

km

amm

ary

epit

hel

ial

den

sity

;kte

rmin

alen

db

ud

nu

mb

ers;

min

crea

sed

lob

ulo

alv

eola

rst

ruct

ure

s;at

8w

eek

so

fag

e;m

BR

CA

1ex

pre

ssio

n;m

ERa

exp

ress

ion

inlo

bu

les

at3

and

8w

eek

s

–50

(sin

gle

do

sest

ud

y)

Cab

anes

etal

.(2

004)

Lim

ited

(par

ente

ral)

Oth

er/m

echa

nis

tic

Av

ym

ice;

250

mg

/k

gg

enis

tein

ind

iet

bef

ore

mat

ing

thro

ug

hp

reg

nan

cyan

dla

ctat

ion

;as

sess

men

to

fh

eter

ozy

go

us

via

ble

yel

low

ago

uti

(Av

y/a

)o

ffsp

rin

g

Sh

ift

inco

atco

lor

ph

eno

typ

eto

war

ds

the

pse

ud

o-a

go

uti

ph

eno

typ

ean

dre

du

ced

bo

dy

wei

gh

tin

pse

ud

o-a

go

uti

ph

eno

typ

e;h

yp

erm

eth

yla

tio

n

–25

0(s

ing

led

ose

stu

dy

)D

oli

no

yet

al.

(200

6)L

imit

ed(o

ral)

No

effe

cto

nli

tter

size

,m

ean

pu

pw

eig

ht,

per

cen

tsu

rviv

alo

rse

xra

tio

250

(hig

hd

ose

;n

oef

fect

)–

437NTP-CERHR EXPERT PANEL REPORT

Birth Defects Research (Part B) 92:421–468, 2011

Page 18

Tab

le7

Co

nti

nu

ed

An

imal

mo

del

and

stu

dy

des

ign

En

dp

oin

tsN

OE

LL

OE

LR

efer

ence

Uti

lity

a

Av

yfe

mal

em

ice;

wer

eas

sig

ned

too

ne

of

fou

rm

od

ifie

dd

iets

for

two

wee

ks

pri

or

tom

atin

gw

ith

Av

y/a

mal

esan

dth

rou

gh

ou

tp

reg

nan

cyan

dla

ctat

ion

:(1

)m

od

ifie

dA

IN-9

3d

iet

(co

rno

ilsu

bst

itu

ted

for

soy

bea

no

il);

(2)

mo

dif

ied

AIN

-93

die

tsu

pp

lem

ente

dw

ith

50m

gB

PA

/k

gd

iet;

(3)

mo

dif

ied

AIN

-93

die

tsu

pp

lem

ente

dw

ith

50m

gB

PA

and

250

mg

gen

iste

in/

kg

die

t;an

d(4

)m

od

ifie

dA

IN-9

3d

iet

sup

ple

men

ted

wit

h50

mg

BP

A/

kg

die

tan

dm

eth

yl

do

no

rco

mp

ou

nd

s(4

.3m

gfo

lic

acid

,0.

53m

gv

itam

inB

12,

5g

bet

ain

e,an

d7.

97g

cho

lin

ech

lori

de/

kg

die

t)

Mat

ern

ald

ieta

ryB

PA

sig

nif

ican

tly

shif

ted

the

coat

colo

rd

istr

ibu

tio

no

fg

enet

ical

lyid

enti

cal

d22

Av

y/a

off

spri

ng

tow

ard

the

yel

low

coat

colo

rp

hen

oty

pe;

mat

ern

alsu

pp

lem

enta

tio

nw

ith

met

hy

ld

on

ors

or

gen

iste

inre

sto

red

the

coat

colo

rd

istr

ibu

tio

nin

the

BP

A-e

xp

ose

do

ffsp

rin

gto

that

ob

serv

edin

the

con

tro

ls

–25

0(s

ing

led

ose

stu

dy

)D

oli

no

yet

al.

(200

7)L

imit

ed(o

ral)

No

effe

cts:

litt

ersi

ze,

surv

ival

,w

ean

wei

gh

t,g

eno

typ

icra

tio

or

sex

rati

o25

0(h

igh

do

se;

no

effe

ct)

–

C57

Bl/

6m

ice;

0,25

,25

0,o

r12

50p

pm

gen

iste

inin

die

tfr

om

GD

14to

lact

atio

nv

iad

am(m

g/

kg

fee

d;

est

ima

ted

by

the

stu

dy

au

tho

rsto

pro

vid

eg

en

iste

in0

,2

,2

0,

or

10

0m

g/k

gb

w/

da

yto

a2

5-g

mm

ou

se)

Alt

ered

imm

un

ece

llen

dp

oin

ts,

e.g

.,v

ario

us

typ

eso

fth

ym

ocy

tes

and

sple

no

cyte

s(m

any

did

no

td

isp

lay

aco

nsi

sten

tp

atte

rno

fre

spo

nse

);m

mal

ep

up

bo

dy

wei

gh

ts;

msp

leen

wei

gh

tin

mal

es

–2b

Gu

oet

al.

(200

6)L

imit

ed(o

ral)

mm

ater

nal

bo

dy

wei

gh

t;m

mat

ern

alsp

leen

wei

gh

t2

250

ER

E-t

K-L

UC

tran

sgen

icm

ale

mic

e;0

or

50m

g/

kg

gen

iste

into

dam

sb

yo

ral

gav

age

on

PN

D4

mlu

cife

rase

acti

vit

yin

liv

er,

lun

g,

hea

rt,

thy

mu

s,te

stis

,an

db

rain

of

pu

ps

–50

(sin

gle

do

sest

ud

y)

Mo

nta

ni

etal

.(2

008)

Lim

ited

(ora

l)

ER

E-t

K-L

UC

mic

e;0,

0.5,

5,an

d50

mg

/k

gg

enis

tein

on

PN

D4

by

ora

lg

avag

eto

lact

atin

gd

am

mlu

cife

rase

acti

vit

yin

the

test

eso

fm

ale

pu

ps

550

Mo

nta

ni

etal

.(2

009)

Lim

ited

(ora

l)

CD

-1m

ice;

0o

r50

mg

/k

gb

w/

day

gen

iste

inb

ysc

inje

ctio

no

nP

ND

1to

5H

yp

erm

eth

yla

tio

nin

Nsb

p1p

rom

ote

rC

Gis

lan

d(C

G1)

inan

age

dep

end

ent

man

ner

–50

(sin

gle

do

sest

ud

y)

Tan

get

al.

(200

8)L

imit

ed(p

aren

tera

l)

m,k

5S

ign

ific

ant

incr

ease

,d

ecre

ase.

aT

he

term

uti

lity

refe

rsto

the

app

lica

bil

ity

toth

ep

urp

ose

of

dra

win

gco

ncl

usi

on

so

nw

het

her

or

no

ta

chem

ical

adv

erse

lyaf

fect

sre

pro

du

ctio

n.

bD

idn

ot

dis

pla

ya

do

se–r

esp

on

se.

438 MCCARVER ET AL.

Birth Defects Research (Part B) 92:421–468, 2011

Page 19

Tab

le8

Ex

per

imen

tal

Stu

die

sw

ith

Dev

elo

pm

enta

lT

ox

icit

yE

nd

po

ints

inR

ats

Ex

po

sed

toG

enis

tein

,D

aid

zein

,o

rE

qu

ol

An

imal

mo

del

and

stu

dy

des

ign

En

dp

oin

tsN

OE

LL

OE

LR

efer

ence

Uti

lity�

Gro

wth

,re

prod

uct

ive

syst

eman

den

docr

ine-

rela

ted

endp

oin

tsS

pra

gu

e-D

awle

yra

t;0,

5,10

0,o

r50

0p

pm

gen

iste

inin

die

td

uri

ng

pre

gn

ancy

and

lact

atio

n;

on

eh

alf

of

mal

eo

ffsp

rin

gco

nti

nu

edo

nth

eg

enis

tein

die

tsat

wea

nin

g(G

/G

);se

con

dh

alf

of

mal

eo

ffsp

rin

gw

ere

giv

enco

ntr

ol

die

tsat

wea

nin

g(G

/C

);m

ult

igen

erat

ion

des

ign

.(I

nta

ke

sa

ssu

me

dto

be

sim

ila

rto

tho

sein

NC

TR

(NC

TR

,2

00

5)o

fw

hic

hth

isst

ud

yw

as

ap

art

:m

ale

s:0

,0

.3,

7,

35

mg

/kg

bw

/da

y;

fem

ale

s:0

,0

.4,

9,

44

mg

/kg

bw

/day

;fe

mal

es

du

rin

gla

cta

tio

n:

0.7

,1

5,

an

d7

8m

g/k

gb

w/d

ay)

kE

Rb

pro

tein

ind

ors

ola

tera

lp

rost

ate

of

F1

mal

esat

PN

D14

0(G

Cg

rou

po

nly

)–

5p

pm

Dal

uet

al.

(200

2)L

imit

ed(o

ral)

mse

rum

DH

Tle

vel

sin

F1

mal

esat

PN

D14

0(b

oth

G/

Gan

dG

/C

gro

up

s)5

pp

m10

0p

pm

mse

rum

test

ost

ero

ne

lev

els

inF

1m

ales

atP

ND

140

(bo

thG

/G

and

G/

Cg

rou

ps)

100

pp

m50

0p

pm

Sp

rag

ue-

Daw

ley

rat;

0,5,

25,

100,

250,

625,

and

1250

pp

mg

enis

tein

thro

ug

hd

iet

du

rin

gp

reg

nan

cyan

dla

ctat

ion

and

un

til

PN

D50

ino

ffsp

rin

g.

(Me

an

do

ses:

0.3

1,1

.7,

5.7

,1

5,

34

,8

3m

g/k

gb

w/d

ay

inp

reg

na

nt

da

ms;

0.5

6,

2.8

,11

,3

0,

73

,1

38

inla

ctat

ing

da

ms;

B0

.6,

3.0

,1

2,

30

,7

2,

an

d1

80

mg

/kg

bw

/day

inp

up

sa

fte

rw

ea

nin

g.)

Acc

eler

ated

vag

inal

op

enin

g;m

rela

tiv

ev

agin

alw

eig

ht

–S

ign

ific

ant

lin

ear

tren

dD

elcl

os

etal

.(2

001)

Lim

ited

(ora

l)H

yp

ertr

op

hy

of

mam

mar

yal

veo

lian

dd

uct

sin

mal

esat

PN

D50

c;

abn

orm

alit

ies

of

sper

mat

og

enes

is5

pp

m25

pp

m

His

top

ath

olo

gy

inv

agin

aat

PN

D50

250

pp

m62

5p

pm

Alv

eola

rp

roli

fera

tio

nin

mam

mar

yo

ffe

mal

esat

PN

D50

;m

ren

altu

bu

lem

iner

aliz

atio

nin

mal

esan

dfe

mal

es10

0p

pm

250

pp

m

kd

ams

del

iver

ing

litt

ers

andk

litt

erb

od

yw

eig

hts

atb

irth

;kre

lati

ve

ven

tral

pro

stat

ew

eig

ht

atP

ND

50;

pro

stat

ein

flam

mat

ion

;h

isto

pat

ho

log

yin

ov

arie

sat

PN

D50

;k

po

stn

atal

bo

dy

wei

gh

tsin

mal

esan

dfe

mal

es

625

pp

m12

50p

pm

Del

ayed

eye

op

enin

gan

dea

ru

nfo

ldin

g62

5p

pm

1250

pp

m(s

ign

ific

ant

lin

ear

tren

d)

No

effe

cto

nm

ater

nal

bo

dy

wei

gh

td

uri

ng

the

lact

atio

np

erio

d,

ges

tati

on

len

gth

,li

tter

size

,p

rop

ort

ion

of

liv

ep

up

s,se

xra

tio

of

pu

ps,

ano

gen

ital

dis

tan

ceo

nP

ND

2,p

rep

uti

alse

par

atio

n;

or

test

icu

lar

sper

mh

ead

or

epid

idy

mal

sper

mco

un

ts

(hig

hd

ose

;n

oef

fect

)–

Sp

rag

ue-

Daw

ley

rat;

0,5,

100,

500

pp

mg

enis

tein

ind

iet;

mu

ltig

ener

atio

nal

des

ign

(mal

es:

0,0.

3,7,

35m

g/

kg

bw

/d

ay;

fem

ales

,av

erag

een

tire

feed

ing

per

iod

:0,

0.5,

10,

51m

g/

kg

bw

/d

ay;

fem

ales

,n

on

lact

atin

g:

0,0.

4,9,

44;

and

fem

ales

,la

ctat

ing

:0,

0.7,

15,

and

78m

g/

kg

bw

/d

ay)

kp

up

wei

gh

tat

bir

th(F

5)

(no

dir

ect

gen

iste

intr

eatm

ent)

–5

pp

mN

CT

R(2

008)

Lim

ited

(ora

l)M

amm

ary

gla

nd

hy

per

pla

sia

inm

ales

(F1,

F2,

F3);k

mal

ep

up

wei

gh

td

uri

ng

lact

atio

np

erio

d(F

3)

5p

pm

100

pp

m

kli

tter

size

(F2);

dis

rup

ted

estr

ou

scy

cles

,i.

e.,m

len

gth

or

nu

mb

ero

fab

no

rmal

cycl

es(F

1);

incr

ease

dcy

cle

len

gth

(F1,

F2);k

bo

dy

wei

gh

tat

vag

inal

op

enin

g(F

1,

F2,

F3)

and

acce

lera

ted

vag

inal

op

enin

g(F

1,

F2);

del

ayed

test

icu

lar

dec

ent

(F3

);k

ano

gen

ital

dis

tan

cein

fem

ales

(F1,

F2);k

ano

gen

ital

dis

tan

cem

ales

(F1);k

fem

ale

pu

pw

eig

ht

du

rin

gla

ctat

ion

per

iod

(F1,

F3,

F4);k

mal

ep

up

wei

gh

td

uri

ng

lact

atio

np

erio

d(F

2,

F4);k

infe

edco

nsu

mp

tio

nin

adu

ltfe

mal

es(F

0,

F1,

and

F4)

100

pp

m50

0p

pm

439NTP-CERHR EXPERT PANEL REPORT

Birth Defects Research (Part B) 92:421–468, 2011

Page 20

Tab

le8

Co

nti

nu

ed

An

imal

mo

del

and

stu

dy

des

ign

En

dp

oin

tsN

OE

LL

OE

LR

efer

ence

Uti

lity�

kli

tter

size

(F1,

F3)

–S

ign

ific

ant

neg

ativ

eli

nea

rtr

end

kan

og

enit

ald

ista

nce

infe

mal

es(F

3)

–10

0a

km

ale

pu

pw

eig

ht

du

rin

gla

ctat

ion

per

iod

(F1)

?5

pp

mN

oef

fect

:o

nm

atin

g,

fert

ilit

y,o

rp

reg

nan

cyin

dic

esin

any

gen

erat

ion

;o

nd

ura

tio

n;

on

of

ges

tati

on

;o

nre

sorp

tio

ns

site

sin

anim

als

that

did

no

tb

eco

me

pre

gn

ant;

on

ov

aria

nfo

llic

leco

un

to

rsp

erm

par

amet

ers

500

pp

m(h

igh

do

se;

no

effe

ct)

–

CD

sS

DIG

Sra

t;0,

20,2

00,o

r10

00p

pm

gen

iste

inin

die

t(m

ean

:1

.7,

18

,a

nd

90

mg

/kg

bw

/day

)fr

om

GD

15to

PN

D10

kb

od

yw

eig

ht

inm

ales

at11

wee

ks

of

age

–1.

7M

asu

tom

iet

al.

(200

3)

Lim

ited

(ora

l)m

rela

tiv

eb

rain

wei

gh

t;m

rela

tiv

ep

itu

itar

yw

eig

ht

–1.

7a

mre

lati

ve

adre

nal

wei

gh

t1.

718

kab

solu

tep

itu

itar

yw

eig

ht;k

bo

dy

wei

gh

tg

ain

inm

ales

on

PN

D21

–42

1890

Wis

tar

(RO

RO

)ra

ts;

0,20

,15

0,o

r10

00m

g/

kg

bw

/d

ayg

enis

tein

fro

mG

D6–

20b

yg

avag

em

pu

pm

ort

alit

y–

20a

McC

lain

etal

.(2

007)

Lim

ited

(ora

l)V

isce

ral

mal

form

atio

ns

(art

ery

ori

gin

var

ian

t)15

010

00(o

nli

tter

inci

den

ceb

asis

)N

oef

fect

on

nu

mb

ero

fd

eliv

ered

pu

ps,

imp

lan

tati

on

or

reso

rpti

on

site

s,o

rex

tern

alm

alfo

rmat

ion

1000

(hig

hd

ose

;n

oef

fect

)–

Wis

tar

(RO

RO

)ra

ts;

0,5,

50,

100,

500

mg

/k

gb

w/

day

gen

iste

infr

om

GD

5–21

inth

ed

iet

mp

ost

-im

pla

nta

tio

nlo

ss,k

nu

mb

ero

ffe

tuse

sp

erfe

mal

e,k

feta

lb

od

yw

eig

ht

(per

litt

erb

asis

),in

cid

ence

skel

etal

oss

ific

atio

n

100

500

Ald

erle

yP

ark

rat;

0,0.

2,o

r2

mg

/k

gb

w/

day

gen

iste

inb

ysc

inje

ctio

nd

uri

ng

PN

D1–

6an

d4

and

40m

g/

kg

bw

/d

ayb

yg

avag

eo

nP

ND

7–21

(sc

do

ses

wer

ed

eter

min

edto

be

equ

ival

ent

tog

avag

ed

ose

so

f4

and

20m

g/

kg

bw

/d

ay);

on

ep

art

of

the

stu

dy

exam

inin

gS

DN

-PO

Ad

ose

dan

imal

sd

uri

ng

the

sam

ep

erio

dw

ith

scan

do

ral

do

ses

equ

ival

ent

to4

and

40m

g/

kg

bw

/d

ayb

yo

ral

exp

osu

re

Ad

van

ced

vag

inal

op

enin

g,

per

sist

ent

vag

inal

corn

ific

atio

n,

ute

rotr

op

hic

resp

on

se,k

seru

mp

rog

este

ron

ein

fem

ales

,kb

od

yw

eig

ht

infe

mal

es

440

ora

lP

ND

7–21

;20

PN

D1–

6(e

qu

ival

ent

toan

scin

ject

ion

do

seo

f2

mg

/k

gb

w/

day

)

Lew

iset

al.

(200

3)

Nag

aoet

al.

(200

1)

Hig

h(o

ral)

Hig

h(o

ral)

mS

DN

PO

Av

olu

me

infe

mal

es4

40

Sp

rag

ue-

Daw

ley

rat;

0,12

.5,

25,

50,

or

100

mg

/k

gb

w/

day

gen

iste

inb

yg

avag

eo

nP

ND

1–5.

kfe

rtil

ity

ind

ex(g

enis

tein

-tre

ated

fem

ales

mat

edw

ith

un

trea

ted

mal

es);

po

lyo

vu

lar

foll

icle

sin

wea

nli

ng

fem

ales

;k

epid

idy

mal

wei

gh

t;k

bo

dy

wei

gh

tso

fm

ales

at18

wee

ks

of

age;

(kb

od

yw

eig

hts

of

fem

ales

at9

wee

ks

of

age)

–12

.5

kn

orm

ales

tro

us

cycl

e(n

ot

do

sed

epen

den

t)–

12.5

a

kn

um

ber

of

imp

lan

tsp

erli

tter

5010

0k

bo

dy

wei

gh

tso

fb

oth

sex

esat

1,2,

3,5,

7,9

and

18(m

ales

on

ly)

wee

ks

of

age

(kb

od

yw

eig

hts

of

bo

thse

xes

at50

mg

/k

gb

w/

day

fro

m5,

7,9,

and

18(m

ales

on

ly)

wee

ks

of

age)

–10

0a

440 MCCARVER ET AL.

Birth Defects Research (Part B) 92:421–468, 2011

Page 21

Hy

per

tro

ph

yo

fu

teri

ne

my

om

etri

um

–50

(rep

ort

edin

anim

als

atlo

wer

do

sele

vel

s,b

ut

no

tst

atis

tica

lly

anal

yze

d)

mat

reti

cfo

llic

les,k

corp

us

lute

um

18w

eek

s,h

yp

ertr

op

hy

of

lum

inal

and

gla

nd

ula

rep

ith

elia

lce

lls,

andk

inn

um

ber

of

ute

rin

eg

lan

ds

–10

0(r

epo

rted

inan

imal

sat

low

erd

ose

lev

els,

bu

tn

ot

stat

isti

call

yan

aly

zed

)N

oef

fect

:M

ales

-n

oef

fect

inti

me

top

rep

uti

alse

par

atio

n,

cop

ula

tio

n,

or

fert

ilit

y;

inn

um

ber

of

imp

lan

tso

rn

um

ber

of

reso

rpti

on

sin

sire

dp

reg

nan

cies

;se

rum

test

ost

ero

ne;

epid

idy

mal

sper

mco

nce

ntr

atio

n;

or

test

icu

lar

his

tolo

gic

chan

ges

Fem

ales

-n

oef

fect

inag

eat

vag

inal

op

enin

g

100

(hig

hd

ose

;n

oef

fect

)–

Sp

rag

ue-

Daw

ley

rat;

0o

rd

iet

con

tain

ing

0.5

g/

kg

gen

iste

in(5

00

mg

/kg

die

t;a

uth

ors

est

ima

ted

inta

kes

of

10

mg

/da

yd

uri

ng

pre

gn

ancy

an

d1

8.5

mg

/da

yd

uri

ng

lact

ati

on

;th

ise

qu

als

B2

6–

47

mg

/kg

bw

/da

yb

ase

do

nth

ere

po

rte

db

od

yw

eig

ht

ofB

38

0g

ind

am

s)

No

effe

ct:

on

pre

gn

ancy

ou

tco

me;

pu

pb

od

yw

eig

ht

du

rin

gp

reg

nan

cy,

lact

atio

n,

or

afte

rw

ean

ing

;o

rgan

wei

gh

tsin

off

spri

ng

(ute

rus,

ov

ary,

test

es,

epid

idy

mid

esat

PN

D15

,49

and

75)

B26

–47

(hig

hd

ose

;n

oef

fect

)

–T

ou

sen

etal

.(2

006)

Lim

ited

(ora

l)

Sp

rag

ue-

Daw

ley

dam

sw

ere

fed

die

tco

nta

inin

g0

or

5p

pm

gen

iste

infr

om

GD

17th

rou

gh

ou

tth

ela

ctat

ion

per

iod

up

toP

ND

70in

off

spri

ng

.(E

xp

osu

rein

off

spri

ng

est

imate

datB

0.6

8m

g/

kg

bw

/day

ov

er

the

life

tim

e.)

Ch

ang

esin

ov

aria

nh

isto

log

yat

PN

D21

and

70;k

bo

dy

wei

gh

tin

PN

D70

fem

ales

(co

nti

nu

ou

sex

po

sure

toP

ND

70);m

bo

dy

wei

gh

tin

PN

D70

fem

ales

(ex

po

sure

pri

or

toP

ND

21)

–B

0.68

b(s

ing

led

ose

)A

wo

niy

iet

al.

(199

8)

Lim

ited

(ora

l)

No

effe

ctin

fem

ales

on

PN

D70

on

seru

mL

H,

FS

H,

17b-

estr

adio

l,p

rog

este

ron

e,o

var

ian

or

ute

rin

ew

eig

ht

B0.

68b

(sin

gle

do

se;

no

effe

ct)

–

Pre

gn

ant

Sp

rag

ue-

Daw

ley

rats

wer

efe

dd

iets

con

tain

ing

0,20

,o

r10

0p

pm

gen

iste

in(0

,2

0,

or

87

mg

/kg

bw

/day

)fr

om

con

cep

tio

nth

rou

gh

po

stn

atal

life

(GD

1to

PN

D56

).

man

og

enit

ald

ista

nce

infe

mal

es;k

age

and

wei

gh

tat

vag

inal

op

enin

g;m

ute

rus

wei

gh

to

nP

ND

21;m

rela

tiv

ete

stis

wei

gh

to

nP

ND

21;m

rela

tiv

ete

stis

wei

gh

to

nP

ND

56;k

ven

tral

pro

stat

ew

eig

ht

2087

Cas

ano

va

etal

.(1

999)

Lim

ited

(ora

l)

No

effe

ct:

on

imp

lan

tati

on

site

sp

erd

am;x

of

liv

ep

up

sp

erli

tter

;o

rli

tter

wei

gh

tat

bir

th;

pro

po

rtio

no

fm

ales

wit

hre

tain

edn

ipp

les;

age

or

wei

gh

tat

pre

pu

tial

sep

arat

ion

;o

rw

eig

ht

(ab

solu

teo

rre

lati

ve)

of

the

test

is(P

ND

21o

r56

)o

rv

entr

alp

rost

ate

(PN

D56

)as

litt

erm

ean

s

87(h

igh

do

se;n

oef

fect

)–

Lo

ng

Ev

ans

rat;

0o

r15

mg

/k

gb

w/

day

gen

iste

inb

yg

avag

eo

nG

D14

toP

ND

21.

mu

teri

ne

pro

ges

tero

ne

rece

pto

rex

pre

ssio

nin

gla

nd

ula

rep

ith

elia

lce

lls

–15

(sin

gle

do

sest

ud

y)

Hu

gh

eset

al.

(200

4)L

imit

ed(o

ral)

No

effe

ct:

ute

rin

elu

min

alep

ith

elia

lce

llh

eig

ht,

ute

rin

ep

roli

fera

tio

n,

ERa

exp

ress

ion

inu

teri

ne

lum

inal

or

gla

nd

ula

rep

ith

elia

lce

lls,

or

pro

ges

tero

ne

rece

pto

rex

pre

ssio

nin

lum

inal

epit

hel

ial

cell

s

15(h

igh

do

se;n

oef

fect

)–

Sp

rag

ue-

Daw

ley

rat;

at0,

25,

250,

or

1250

pp

mg

enis

tein

inth

ed

iet

fro

mG

D7

tow

ean

ing

via

dam

and

dir

ectl

yto

off

spri

ng

afte

rw

ean

ing

thro

ug

hP

ND

50(B

2,

20

,2

00

mg

/kg

bw

/day

)

Hep

atic

CY

P3A

enzy

me

exp

ress

ion

inm

ales

;al

tere

dfo

rmat

ion

of

test

ost

ero

ne

met

abo

lite

sin

liv

erm

icro

som

es,

i.e.

,D

HT

/3-

dio

lan

d7a

-OH

B2

ZB

20a

Lau

ren

zan

aet

al.

(200

2)

Lim

ited

(ora

l)

441NTP-CERHR EXPERT PANEL REPORT

Birth Defects Research (Part B) 92:421–468, 2011