Felix Lozano Lawsuit

8/7/2019 Felix Lozano Lawsuit

http://slidepdf.com/reader/full/felix-lozano-lawsuit 1/82

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COMPLAINT FOR EQUITABLE RELIEF

AND FOR DAMAGES Page 1 of 25

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HOWARD M. RUBINSTEIN (Fla. SBN: 104108)Attorney at Law914 Waters Avenue, Suite 20Aspen, Colorado 81611 E-mail: [email protected]

Tel.: (832) 715-2788E-mail: [email protected](To apply as Counsel Pro Hac Vice)

HAROLD M. HEWELL (Cal. SBN: 171210)Hewell Law Firm, APC 402 W. Broadway, Fourth Floor San Diego, California 92128Email: [email protected]: (619) 235-6854 • Fax: (619)235-9122

Attorneys for Plaintiff

UNITED STATES DISTRICT COURT

EASTER N DISTRICT OF CALIFORNIA

SACRAMENTODIVISION

LANI FELIX-LOZANO, as an individual

consumer, and on behalf of all otherssimilarly situated,

Plaintiff,

vs.

NALGE NUNC INTERNATIONAL

CORPORATION, a Delaware Corporation,

Defendant .

:

::::::::::::::::::::::

Civil Action No.

COMPLAINT FOR:

EQUITABLE RELIEF

AND FOR DAMAGES

Class Action

Jury Trial Requested

Case 2:08-at-00432 Document 2 Filed 04/22/2008 Page 1 of 82



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Plaintiff alleges:

I. PARTIES

1. Plaintiff LANI FELIX-LOZANO is an individual consumer who resides in, and is a

citizen of, California. She respectfully requests a jury trial.

2. Defendant NALGE NUNC INTERNATIONAL CORPORATION (hereinafter,

“NNIC”) is a corporation organized under the laws of Delaware. NNIC manufactures plastic

products sold under the NALGENE®, NUNC® and I-CHEM® brand names. Its products are

sold through distributors to research laboratories, various types of industrial processing facilities

and camping and backpacking stores. Through its Outdoor Products Division, located at 75

Panorama Creek Drive, Rochester, New York 14625, NNIC manufactures, markets and sells a

variety of reusable beverage containers, including bottles and hydration packs, popular among,

and widely used by, outdoor sports enthusiasts across the United States. NNIC’s consumer

products yield $50-$65 million in estimated annual sales for the company. The principal

executive office for NNIC also is located at 75 Panorama Creek Drive, Rochester, New York

14625, and NNIC lists as its registered agent, with the Delaware Secretary of State, The

Corporation Trust Company, Corporation Trust Center 1209 Orange Street, Wilmington,

Delaware 19801. Thus, for purposes of diversity jurisdiction, NNIC may be considered a

resident of either Delaware or New York. At all times herein, NNIC conducted business in this

judicial district.

II. GENERAL ALLEGATIONS

3. Plaintiff is informed and believes and thereon alleges that, at all times herein

mentioned, the employees of NNIC, its subsidiaries, affiliates and other related entities, were the

agents, servants and employees of NNIC and, at all times herein mentioned, each was acting

within the purpose and scope of said agency and employment. Whenever reference in this

Complaint is made to any act or transaction of NNIC, its subsidiaries, affiliates and/or other

related entities, such allegation shall be deemed to mean that the principals, officers, directors,

employees, agents, and/or representatives of NNIC committed, knew of, performed, authorized,

ratified and/or directed such act or transaction on behalf of NNIC while actively engaged in the




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scope of their duties.

4. All allegations in this Complaint are based on information and belief and/or are likely

to have evidentiary support after reasonable opportunity for further investigation and discovery.

III. VENUE AND JURISDICTION

5. This Court has jurisdiction over the subject matter presented by this Complaint

because it is a class action arising under the Class Action Fairness Act of 2005 (hereinafter,

“CAFA”), Pub. L. No. 109-2, 119 Stat. 4 (2005), which explicitly provides for the original

jurisdiction of the Federal Courts of any class action in which any member of the plaintiff class

is a citizen of a State different from any defendant and in which the matter in controversy

exceeds in the aggregate the sum of $5,000,000, exclusive of interest and costs. Plaintiff alleges

that the total claims of individual class members in this action are in excess of $5,000,000 in the

aggregate, exclusive of interest and costs, as required by 28 U.S.C. § 1332(d)(2), (5). Ms. Felix-

Lozano, the plaintiff, is a citizen of California, whereas, as set forth above, NNIC can be

considered a citizen of either Delaware or New York for the purposes of diversity. Therefore,

diversity of citizenship exists under CAFA, as required by 28 U.S.C. § 1332(d)(2)(A).

Furthermore, plaintiff alleges that more than two-thirds of all of the members of the proposed

Plaintiff Class in the aggregate are citizens of a state other than California, where this action is

originally being filed, and that the total number of members of the proposed Plaintiff Class is

greater than 100, pursuant to 28 U.S.C. § 1332(d)(5)(B).

6. Venue in this district is proper pursuant to 28 U.S.C. §1391(b) because defendant

conducts business within, may be found in, and is subject to personal jurisdiction in this district.

The “Declaration of Harold M. Hewell Pursuant to Civil Code §1780(c) of the Consumer Legal

Remedies Act, Civil Code §§1750 et seq.” regarding venue under the California Consumer

Legal Remedies Act is submitted herewith.

IV. FACTUAL ALLEGATIONS

7. NNIC manufactures, distributes, advertises, labels and sells reusable beverage bottles

widely used by consumers. These come in a variety of types, including, but not limited to, the

NALGENE® “Wide-Mouth” and “Narrow-Mouth Bottles” made of polycarbonate plastic




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(hereinafter, “Bottles”). The popularity of these colorful, durable Bottles, composed specifically

of LEXAN®1

polycarbonate plastic, has increased in recent years, and they are generally the

preferred reusable beverage container found on college campuses, in suburban fitness centers

and elsewhere. A true and correct copy of the Bottles is attached hereto as Exhibit “A” and

incorporated by reference.

8. The durability of the Bottles derives from their polycarbonate composition.

Polycarbonate is a tough, stable thermoplastic that is clear, lightweight and shatter-resistant.

These attributes make polycarbonate the material of choice for a diverse range of products.

9. A General Electric scientist discovered LEXAN polycarbonate resin in 1953 while

seeking to develop a new material for wire insulation. Since then, it has been used to make,

among other things, CDs, automobile parts, computers, mobile phones, outdoor signage, bullet-

resistant shielding and the “Bubble Helmet” worn by astronauts on the Moon. Additionally, it is

widely employed in food and beverage containers such as NNIC’s Bottles.

10. More specifically known as polycarbonate of bisphenol A, LEXAN polycarbonate is

synthesized from bisphenol A and carbonyl chloride.2 Bisphenol A (hereinafter, “BPA”) was

first synthesized in 1891. In the 1930s, it was researched for its ability to mimic estrogen.3

Today, however, BPA is used primarily as an industrial chemical for its monomeric4

properties

in the production of, among other things, polycarbonate and epoxy resins.

11. The ubiquity of polycarbonate plastics has resulted in widespread human exposure to

1LEXAN is a registered trademark for General Electric’s (now SABIC Innovative Plastics’)

brand of polycarbonate resin thermoplastic.

2Carbonyl chloride also is known as phosgene, a colorless, chemically reactive toxic gas

employed in chemical warfare.

3 E. C. Dodds and Wilfrid Lawson, Nature, 137 (1936), 996; E. C. Dodds and W. Lawson,Proceedings of the Royal Society of London, Series B, Biological Sciences, 125, #839 (27-IV-1938), pp. 222–232.

4 A monomer is a relatively light, simple organic molecule that can join in long chains withother molecules to form a more complex molecule or polymer. The term polymer is often usedas a synonym for plastic.




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BPA. In mid-2004, BPA production volume in the United States was reported at approximately

2.3 billion pounds.5

12. BPA can be released into the environment both during the manufacturing process

and by leaching from the final products. BPA degrades into its monomeric form over time, a

process that can be accelerated by exposure to heat; the monomeric form can leach from its

source into adjacent materials, such as water or food products.6

13. Most human exposure is believed to come from ingestion of BPA.7 More than 92.6

percent of urine samples taken from 2,517 participants in the 2003–2004 National Health and

Nutrition Examination Survey who were six years of age or older had detectable BPA

concentrations. In that study, females had statistically higher BPA level than males, and children

had higher concentrations than adolescents who, in turn, had higher concentrations than adults.8

14. Despite this, NNIC and the plastic industry continues to assert that BPA is safe.

NNIC’s website has devoted substantial space to BPA, citing favorable reports and other

information in an attempt to assure customers of the Bottles’ safety. In a section entitled

“Frequently Asked Questions,” NNIC states:

Q. Are polycarbonate bottles safe?

A: Yes. Agencies and researchers worldwide have studied the safety of BPA and

polycarbonate for approximately 50 years; including The Environmental

Protection Agency and The Food and Drug Administration in the USA, The

European Commission Scientific Committee on Food, The German Federal

5 CERHR, 2007. Interm NTP-CERHR report on the reproductive and developmental toxicity of bisphenol A. http://cerhr.niehs.nih.gov/chemicals/bisphenol/BPA Interim DraftRpt.pdf.

6 Lopez-Cervantes J, Paseiro-Losada P. Determination of bisphenol A in, and its migration from,PVC stretch film used for food packaging. Food Addit Contam. 2003;20(6):596–606.

7 Kang JH, Kondo F, Katayama Y. Human exposure to bisphenol A. Toxicology. 2006;226(2– 3):79–89.

8 Calafat AM, Ye X, Wong LY, Reidy JA, Needham LL. Exposure of the U.S. population to bisphenol A and 4-tertiary-octylphenol: 2003–2004. Environ Health Perspect. 2008;116:39–44.




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Institute for Risk Assessment and the Japan Ministry of Health, Labor and

Welfare. Findings of studies from these agencies indicate that food and beverage

containers manufactured from polycarbonate do not pose a health risk to humans.

As a result, polycarbonate is used in a wide variety of consumer products

including baby bottles, water bottles, dental sealants and the lining of most food

& beverage containers [Emphasis added.].9

A true and correct copy of that page is attached hereto as Exhibit “B” and incorporated by

reference.

15. Despite NNIC’s representations, it is widely accepted that BPA exposure affects the

hormonal system, particularly as it relates to estrogen. BPA’s effects have been studied from the

cellular level to whole organisms. Abnormal sperm and reduced fertility found in male rats

exposed to BPA were reversed when exposure stopped.10 One of the few human

epidemiological studies revealed a relationship between BPA exposure and repeated

miscarriage.11 Another study has shown that BPA causes a human breast cancer cell line to

proliferate, indicating that estrogen-sensitive tissues and cells in the body may react similarly.12

16. Other studies involving animal and cell data suggest that many health conditions and

disabilities may be caused, at least in part, by BPA at extremely low doses. These include: early

puberty; reduced sperm count; prostate disease, including prostate tumor proliferation; cellular

causes of spontaneous miscarriage; Down syndrome; increased embryo mortality; breast cancer;

impaired immune function; decreased anti-oxidant enzyme levels; changes in brain chemistry;

9 http://www.nalgene-outdoor.com/technical/bpaInfo.html.

10 Toyama Y, Suzuki-Toyota F, Maekawa M, Ito C, Toshimori K (2004) Adverse effects of

bisphenol A to spermiogenesis in mice and rats. Arch Histol Cytol 67: 373–381.11

Sugiura-Ogasawara M, Ozaki Y, Sonta S, Makino T, Suzumori K (2005) Exposure to bisphenol A is associated with recurrent miscarriage. Hum Reprod 20: 2325–2329.

12 Singleton DW, Feng Y, Chen Y, Busch SJ, Lee AV, et al. (2004) Bisphenol-A and estradiolexert novel gene regulation in human MCF-7 derived breast cancer cells. Mol Cell Endocrinol221: 47–55.




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changes in formation of synapses in the brain; and behavioral changes, including hyperactivity,

increased aggressiveness, impaired learning, and altered sexual behavior.13

17. Yet, the NNIC website is devoid of these and other studies that show health risks

from exposure to BPA.

18. In another statement found on the NNIC website, the U.S. Food and Drug

Administration (“FDA”) asserts that it “sees no reason at this time to ban or otherwise restrict

the uses now authorized. Our conclusion is based on our ongoing review of all available data.”14

A true and correct copy of that page is attached hereto as Exhibit “C” and incorporated by

reference.

19. However, a 2005 review of the literature by Dr. Frederick S. vom Saal, a leading

BPA researcher at the University of Missouri-Columbia’s Division of Biological Sciences,

questions the objectivity of that data:

Source of funding is highly correlated with positive or negative findings in

published articles. For government-funded published studies, 94 of 104 (90%)

report significant effects at doses of BPA < 50 mg/kg/day. No industry-funded

studies (0 of 11, or 0%) report significant effects at these same doses….”15

20. Additionally, the Environmental Working Group,16

and noted researchers and

scientists who study BPA toxicity, have challenged the safety standards set by federal regulatory

agencies such as the FDA and the U.S. Environmental Protection Agency (“EPA”) to evaluate

13vom Saal F, Hughes C. An extensive new literature concerning low-dose effects of bisphenol

A shows the need for a new risk assessment. Environ Health Perspect 113:926–933 (2005).doi:10.1289/ehp.7713 available via http://dx.doi.org/ [Online 13 April 2005].

14“Statement from the FDA, from a written communication dated January 29, 2008,” found on

NNIC website at http://www.nalgene-outdoor.com/technical/FDAstatement.html.

15 vom Saal F, Hughes C. An extensive new literature concerning low-dose effects of bisphenolA shows the need for a new risk assessment. Environ Health Perspect 113:926–933 (2005).doi:10.1289/ehp.7713 available via http://dx.doi.org/ [Online 13 April 2005].

16 The Environmental Working Group (EWG) is a 501(c)(3) non-profit organization founded in1993 which conducts environmental investigations in the areas of toxins, agricultural subsidies, public lands, and corporate accountability.




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BPA risks.17 According to EWG:

(a) The EPA established its generic safety standard for BPA (the “reference

dose,” or “RfD”) in 1987, a decade before low-dose literature was

established for BPA; the vast majority of studies finding BPA toxic at

low doses have been published since 1997. The EPA safety standard (50

ug/kg/d) has not been updated in 20 years and is 25 times the dose now

known to cause birth defects in lab studies (2 ug/kg/d).

(b) The U.S. National Toxicology Program’s 2001 assessment, which found

BPA safe at low doses, relied on industry-sponsored studies published in

2000 or earlier that tested BPA upon animals now known to be resistant

to the effects of estrogen-like chemicals such as BPA. Since then, dozens

of studies have been published that strongly support evidence for low-

dose BPA effects.

(c) The FDA published estimates of infant and adult BPA exposures 10 years

ago. Though it has not established an Acceptable Daily Intake (“ADI”)

for BPA, and has not conducted the agency’s standard, basic toxicology

study to determine a safe dose for humans, an FDA official asserted in

2005 that the agency “sees no reason to change [its] long-held position

that current [BPA] uses with food are safe.”18

17 The FDA, EPA, and other federal agencies have employed the following standard inconsidering the low dose toxicity of BPA: for laboratory animal studies, “low doses” involvedadministration of doses below those used in traditional toxicological studies conducted for risk assessment purposes. For BPA, the lowest dose previously examined for risk assessment purposes was 50mg (kg

í1day

í1) in studies with rats and mice. The 50 mg (kg

í1day

í1) dose is

the currently accepted lowest adverse effect level (LOAEL) that was used to calculate thecurrent EPA reference dose (the daily dose that EPA calculates is safe for humans over thelifetime) of 50 μg (kgí1 dayí1). The current reference dose is thus based on “high dose”experiments conducted in the 1980s. “Low dose” also refers to doses within the range of typicalhuman exposure (excluding occupational exposures).

18 Environmental Working Group. 2007. Bisphenol A: toxic plastics chemical in canned food.www.ewg.org/reports/bisphenola.




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21. The National Institute of Environmental Health Sciences (“NIEHS”) recently issued

two conflicting reports on BPA. The first, issued July 27, 2007, by a panel of 38 BPA

researchers concluded that levels of BPA seen in humans are higher than those that caused

adverse effects in animal studies, and expressed confidence that even low doses of BPA can

have biological effects.19

The report states that, in addition to binding to estrogen receptors:

…more recent evidence has shown that BPA also exhibits other modes of

endocrine disruption…such as alterations in endogenous hormone synthesis,

hormone metabolism and hormone concentrations in blood. BPA also results in

changes in tissue enzymes and hormone receptors, and interacts with other

hormone-response systems, such as the androgen and thyroid hormone receptor

signaling systems. While BPA was initially considered to be a “weak” estrogen

… research shows that BPA is equipotent with estradiol in its ability to activate

responses via recently discovered estrogen receptors associated with the cell

membrane [citations omitted]. It is through these receptors that BPA stimulates

rapid physiological responses at low picogram per ml (parts per trillion)

concentrations.

The published scientific literature on human and animal exposure to low

doses of BPA in relation to in vitro mechanistic studies reveals that human

exposure to BPA is within the range that is predicted to be biologically active in

over 95% of people sampled. The wide range of adverse effects of low doses of

BPA in laboratory animals exposed both during development and in adulthood is

a great cause for concern with regard to the potential for similar adverse effects

in humans. Recent trends in human diseases relate to adverse effects observed in

experimental animals exposed to low doses of BPA. Specific examples include:

19 Chapel Hill bisphenol A expert panel consensus statement: Integration of mechanisms, effectsin animals and potential to impact human health at current levels of exposure. ReproductiveToxicology 24 (2007) 131–138.




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the increase in prostate and breast cancer, uro-genital abnormalities in male

babies, a decline in semen quality in men, early onset of puberty in girls,

metabolic disorders including insulin resistant (type 2) diabetes and obesity, and

neurobehavioral problems such as attention deficit hyperactivity disorder

(ADHD).

There is extensive evidence that outcomes may not become apparent until

long after BPA exposure during development has occurred [Emphasis added].20

A true and correct copy of that report is attached hereto as Exhibit “D” and incorporated by

reference.

22. The second report was issued November 26, 2007, by the Center for the Evaluation

of Risks to Human Reproduction (“CERHR”), National Toxicology Program (“NTP”) of the

U.S. Department of Health and Human Services. CERHR was established in 1998 by the

National Institutes of Health (“NIH”) to study the effect of environmental substances on

reproductive and developmental health. NIH intended for CERHR to use panels of independent

scientists to evaluate the risks and hazards of potentially toxic chemicals, with federal regulatory

agencies as one of its targeted audiences.

23. CERHR convened a 12-member panel of government and non-government scientists

which met publicly in March and August of 2007 to evaluate scientific studies on the potential

reproductive and developmental hazards of BPA. Its November 26, 2007, report, while noting

“some concern” that BPA could cause behavioral and neurological problems in developing

fetuses and young children, concluded that BPA exposure levels for most Americans were well

within the EPA’s standards and found no major health risks associated with exposure.21 A true

and correct copy of that report is attached hereto as Exhibit “E” and incorporated by reference.

CERHR accepted public comments on the report through the end of January 2008; an NTP-

CERHR Monograph for BPA is pending and expected in or around August 2008.

20 Id.

21 Defects Res B Dev Reprod Toxicol. 2006 December; 77(6): 485–638.




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24. NIEHS has attempted to reconcile the two conflicting reports, stating that, “to a large

extent [they] reflect the importance and timeliness of questions regarding the possible health

effects of BPA. …The [Chapel Hill] consensus statement represents the opinions of most of the

attendees and should not be interpreted as the position of the NIEHS.”22

25. In addressing the conflict, however, NIEHS sidestepped controversy that had marked

the CERHR panel and report. A 2007 investigation by EWG found that the consulting company

CERHR was using to conduct its BPA study had substantial ties to BPA manufacturers. NTP

terminated CERHR’s contract with the consulting company, Sciences International, Inc. (“SI”),

on April 13, 2007, after EWG reported, among other things, that SI’s lead manager at CERHR

had co-authored a scientific paper on the application of animal test results to human health risk

with an employee at Dow Chemical Company, one of the world’s largest BPA producers. SI,

however, had already prepared the 300-page briefing document on the risks of BPA used by

CERHR, as well as the first draft of panel’s report, and NTP stood by the quality of SI’s

assessments. However, in a January 25, 2008, comment letter on the final CERHR report, Anila

Jacob, M.D., senior scientist at EWG, referred to the CERHR panel’s continued use of SI’s BPA

review and the fact that CERHR’s final report was derived from the conflicted document. She

noted that in the CERHR interim draft report:

… the expert panel rejected government and independent studies at 3 times the

rate of industry studies. Only some of these issues have been addressed in the

final expert panel report that was released in November of 2007 (CERHR

2007a). … The fact that there are still inconsistencies in the expert panel’s final

draft illustrates how the CERHR expert panel continues to apply arbitrary

standards throughout this evaluation.

A true and correct copy of that letter is attached hereto as Exhibit “F” and incorporated by

reference.

26. In late August 2007, the NIH ordered a review of NIEHS and NTP practices, and

22 “Since You Asked – BPA,” http://www.niehs.nih.gov/news/media/questions/sya-bpa.cfm#13.




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David Schwartz, who had headed NIEHS since 2005, agreed to temporarily step aside. In a

letter dated August 29, 2007, Rep. Henry Waxman, chairman of the House Committee On

Oversight And Government Reform Committee (“Committee”) wrote NTP’s associate director

regarding a report23 prepared by a working group of the Board of Scientific Counselors at NTP

that reviewed the program’s contracts for conflicts of interest. The working group had

determined that no actual or apparent conflict of interest existed in any of the cross-section of

contracts reviewed. In his letter, however, Rep. Waxman stated that the group’s conclusion

appeared “to be based primarily on self-certification by the contractors themselves” and

requested “a plan for conducting an assessment of actual or potential conflicts under existing

NTP contracts that does not rely on the self-certifications of contractors.” A true and correct

copy of that letter is attached hereto as Exhibit “G” and incorporated by reference.

27. On September 20, 2007, Rep. Waxman and Rep. Tom Davis, the Committee’s

ranking member, wrote EPA Administrator Stephen L. Johnson and expressed their “serious

concerns over the agency’s inability to assure the Committee that it is taking adequate and

timely steps to protect the American public from dangerous endocrine-disrupting chemicals.” A

true and correct copy of that letter is attached hereto as Exhibit “H” and incorporated by

reference. The letter stated that the “EPA’s efforts in this area have been characterized by

missed deadlines, prolonged delays, and inadequate incorporation of public input” and had been

“a continued failure to protect the American public from these chemicals.” Citing chemicals

used in plastics, along with dioxin, PCBs, DDT and the drug DES, Reps. Waxman and Davis

reminded Mr. Johnson that the “Safe Drinking Water Act Amendments of 1996 included a

provision authorizing EPA to screen drinking water contaminants for possible endocrine

disrupting properties[,]” and that at an October 2006 Committee meeting on the status of the

endocrine disruptor program, the EPA’s Assistant Administrator Ben Grumbles had testified

23 National Toxicology Program, Report of the Board of Scientific Counselors Working Groupfor the Review of the National Toxicology Program Contracts for Conflicts of Interest (June 22,2007).




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that the agency “would ‘work harder and faster in making more progress’ to implement an

endocrine disruptor screening program.” However, almost a year later, the agency had made

little progress towards the statutory goals of identifying the chemicals. The congressmen wrote

that they were trying to understand why the process was “moving so slowly and what can be

done to speed it up” and stated that they were focusing on: “(l) the agency’s prolonged delay in

initiating a testing program for endocrine disrupting chemicals; (2) the agency’s failure to

establish a plan and timeline for completing each step of the testing program; and (3) potential

flaws in the test batteries to be used to screen chemicals for endocrine disrupting properties.” In

their letter, Reps. Waxman and Davis asserted that the EPA had not yet completed a single step

of the multi-stage process, having just published that summer its first draft list of chemicals to

be screened for endocrine disrupting properties, an initial list of 73 chemicals that was “only a

small fraction of the universe of 1,700 chemicals that the agency has identified for screening

under the FQPA mandate, and a minute percentage of the 75,000 chemicals currently listed on

the Toxic Substances Control Act (TSCA) Chemical Substance Inventory.”

28. Congressional oversight has expanded into other areas of BPA regulation. On

January 17, 2008, Rep. John Dingell, chairman of the House Energy and Commerce Committee

and Rep. Bart Stupak, chairman of the Subcommittee on Oversight and Investigations, began an

inquiry with the FDA and the manufacturers of infant products regarding BPA. The inquiry is

focusing on the use of BPA in plastic baby bottles and the liners of infant formula containers.

The congressmen also are seeking information regarding the FDA’s review of BPA.

29. New studies have continued to demonstrate risks associated with BPA. A study

reported January 30, 2008, found that adding very hot or boiling water to PC drinking bottles

greatly elevates the rate of BPA migration.

While the exposure to elevated temperatures of boiling water is much higher than

the typical home wash water temperatures used by consumers, the storage of

higher temperature (boiling) water or beverages in these PC bottles is a common

practice in cold weather outdoor activities such as alpine snow sports, climbing,

and mountaineering. Such practices would likely result in increased levels of




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BPA in beverages stored and consumed from those bottles. …the contribution of

the concentrations of BPA that contaminate drinking water and foods stored in

PC bottles should be considered as a single, though important component of the

total mixture of EDCs to which humans are acutely and chronically exposed

throughout their life time.24

A true and correct copy of that report is attached hereto as Exhibit “I” and incorporated by

reference.

30. The marketplace has continued to respond to news about BPA and polycarbonate

bottles. Mountain Equipment Cooperative, Canada’s largest consumer cooperative at 2.7 million

members, announced on December 7, 2007, that it would stop selling polycarbonate bottles,

including the NNIC Bottles, “due to regulatory uncertainty surrounding BPA, and because our

members have expressed increasing concern about this potentially harmful chemical.” Patagonia

Inc., the outdoor clothing maker based in Ventura, California, pulled polycarbonate bottles from

its store shelves in December 2005.

31. On April 18, 2008, Canada became the first country to declare BPA unsuitable for

use in baby bottles, setting a ban process in motion that same day. Canadian Health Minister

Tony Clement and Environment Minister John Baird made the announcement in Ottawa, adding

there would be 60 days of public consultations before further steps are taken.

32. That same day, NNIC announced that, “[i]n response to consumer demand,” it “will

phase out production of its Outdoor line of polycarbonate containers that include the chemical

Bisphenol-A (BPA) over the next several months….”

33. Ms. Felix-Lozano has purchased several of the Bottles, including both the “Wide-

Mouth” and “Narrow-Mouth” versions, within the Class period defined below, from local

distributors. She purchased the Bottles, and in addition to using them herself, gave them to her

daughters, ages 11 and 13, who used them in sporting activities. Ms. Felix-Lozano purchased

and used the Bottles, and gave them to her daughters to use, because she believed that they were

24 H.H. Le et al. / Toxicology Letters 176 (2008) 149–156.




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safe, and was given no information or warning by defendant regarding the BPA risks associated

with the Bottles. She had heard of risks associated with water bottled in disposable containers,

and knowing that her tap water was of good quality, she believed that drinking tap water from

the Bottles would provide a safe, risk-free means of hydration for both herself and her

daughters. She did not learn of the BPA-related risks associated with using the Bottles until a

friend notified her of them in mid-April 2008. She and her daughters are discontinuing use of

the Bottles as the result of learning of these risks. She never would have purchased or used the

Bottles, and would not have given them to her daughters to use, had defendant warned her of, or

provided her with accurate and complete information regarding, the Bottles’ BPA-related risks

set forth in detail above. She contends that despite significant and credible scientific and

medical findings regarding those risks, NNIC, in its marketing, advertisement, promotion,

labeling and sale of the Bottles, omitted, withheld and suppressed material information

regarding those risks. She also relied upon NNIC’s good reputation in purchasing Bottles, and

believed that if it was selling an product with known risks, it would have warned or fully

informed her of those risks, but it did not. She contends that NNIC placed profit before

consumer safety in its marketing, advertisement, promotion, labeling and sale of the Bottles by

omitting, suppressing and withholding from consumers material information regarding BPA

risks associated with its Bottles, and by failing to warn consumers of those risks. As a direct and

proximate result, plaintiff suffered actual damages and harm, in an amount to be determined at

trial, in that she was induced to and did spend money on the Bottles, but was deprived of the

benefit of her bargain; she lost the money she paid for the Bottles as the Bottles did not have the

safety qualities she sought, qualities which, as the result of defendant’s omission, suppression

and withholding of material information, she had been led to believe the Bottles had. Had she

known the omitted, suppressed and withheld material information regarding BPA-related risks

associated with the Bottles, she would not have purchased them.

V. CLASS ALLEGATIONS

34. Plaintiff realleges and incorporates by reference the allegations set forth in each of

the preceding paragraphs of this Complaint.




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35. Pursuant to California Code of Civil Procedure § 382 and Fed. R. Civ. P. 23, plaintiff

brings this action on behalf of herself and all other consumers who purchased defendant’s

Bottles during the Class Period, which is defined as the four years preceding the filing date of

this Complaint. Defendant’s practices and omissions were applied uniformly to all members of

the Class and each subclass, so that the questions of law and fact are common to all members of

the Class and each subclass. All putative Class and subclass members were and are similarly

affected by having purchased the Bottles, and the relief sought herein is for the benefit of

plaintiff and members of the putative Class and subclasses. Based on the annual sales of the

Bottles and their popularity, it is apparent that the number of consumers who purchased the

Bottles would at least be in the many tens of thousands, thereby making joinder impossible

36. Questions of law and fact common to the Plaintiff Class and the subclasses exist that

predominate over questions affecting only individual members, including, inter alia:

(a) whether defendant’s practices in connection with the marketing,

advertisement, promotion, labeling and/or sale of the Bottles were

deceptive, unlawful or unfair in any respect, thereby violating California's

Unfair Competition Law (“UCL”), Cal. Bus. & Prof. Code § 17200 et

seq.;

(b) whether defendant’s practices in connection with the marketing,

advertisement, promotion, labeling and/or sale of the Bottles were

deceptive or likely to deceive consumers in any respect, thereby violating

California's False Advertising Law (“FAL”), Cal. Bus. & Prof. Code §

17500 et seq.;

(c) whether defendant fraudulently concealed risks associated with use of the

Bottles in its marketing, advertisement, promotion, labeling and sale of

the Bottles:

(d) whether defendant breached implied warranties in its sale of the Bottles,

thereby causing harm to plaintiff and members of the Class and/or

subclasses;




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(e) whether defendant breached California's Consumer Legal Remedies Act

(“CLRA”), Civil Code §1750 et seq., in its marketing, advertisement,

promotion, labeling and sale of the Bottles, thereby causing harm to

plaintiff and Class members; and

(f) whether defendant’s conduct as set forth above injured consumers and if

so, the extent of the injury.

37. The claims asserted by plaintiff in this action are typical of the claims of the

members of the Plaintiff Class and all subclasses; the claims arise from the same course of

conduct by defendant, and the relief sought is common for the Class and each subclass. Plaintiff

will fairly and adequately represent and protect the interests of the members of the Plaintiff

Class and all subclasses. Plaintiff has retained counsel competent and experienced in both

consumer protection and class action litigation.

38. Certification of this class action is appropriate under Fed. R. Civ. P. 23(b) and

California Code of Civil Procedure § 382 because the questions of law or fact common to the

respective members of the Class and each subclass predominate over questions of law or fact

affecting only individual members. This predominance makes class litigation superior to any

other method available for the fair and efficient adjudication of these claims. Absent a class

action, it would be highly unlikely that the representative plaintiff, or any other member of the

Class and subclasses, would be able to protect his or her own interests, as the cost of litigation

through individual lawsuits might exceed expected recovery. Certification is also appropriate

because defendant acted or refused to act on grounds generally applicable to the Class and each

subclass, thereby making appropriate final injunctive relief with respect to the Class and each

subclass as a respective whole. Further, given the large number of purchasers of the Bottles,

allowing individual actions to proceed in lieu of a class action would run the risk of yielding

inconsistent and conflicting adjudications.

39. A class action is a fair and appropriate method for the adjudication of the

controversy, in that it will permit a large number of claims to be resolved in a single forum

simultaneously, efficiently, and without the unnecessary hardship that would result from the




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prosecution of numerous individual actions and the duplication of discovery, effort, expense and

burden on the courts that such individual actions would engender. The benefits of proceeding as

a class action, including providing a method for obtaining redress for claims that would not be

practical to pursue individually, outweigh any difficulties that might be argued with regard to

the management of this class action.

40. In the aggregate, plaintiff is informed and believes, and thereon alleges, that the

claims of the individual members of the Class exceed the sum of $5,000,000.00, exclusive of

interest and costs.

VI. FIRST CAUSE OF ACTION:

FOR VIOLATION OF BUS & PROF. CODE §17200 ET SEQ.



42. This cause of action is brought on behalf of plaintiff and members of the general

public pursuant to Cal. Bus. & Prof. Code § 17200 et seq., which provides that “unfair

competition shall mean and include any unlawful, unfair or deceptive business act or practice

and unfair, deceptive, untrue or misleading advertising and any act prohibited by Chapter I

(commencing with Section 17500) as Part 3 of Division 7 of the Business and Professions

Code.”

43. Plaintiff contends that defendant committed unfair business acts and/or practices.

Defendant and its related entities represent themselves as being reputable, reliable and safe

manufacturers of reusable beverage containers. The utility of omitting, suppressing and

withholding material information regarding the Bottles’ BPA-related risks in defendant’s

marketing, labeling and sale of the Bottles is negligible, if any, when weighed against the

damages caused by those practices to the general public, plaintiff, and members of the Class and

subclasses as set forth above. Those damages far outweigh any reason or justification by

defendant for omitting, suppressing and withholding material information regarding the Bottles’

BPA-related risks. Defendant had an improper motive, as set forth above, in engaging in these

practices, and the use of these unfair practices was and is under the sole control of defendant,




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and was deceptively hidden from members of the general public in the marketing,

advertisement, promotion, labeling and sale of the Bottles. As a purchaser and consumer of

defendant’s Bottles, and as a member of the general public in California who purchased the

Bottles, plaintiff is entitled to, and does bring, this class action seeking all available remedies

under the UCL, including declaratory, injunctive and other equitable relief, as well as attorneys'

fees and costs.

44. Defendant committed a deceptive act or practice in its marketing, advertisement,

promotion, labeling and sale of the Bottles by omitting, withholding and suppressing material

information regarding the Bottles’ BPA-related risks as set forth in detail above. This had and

continues to have the capacity and tendency to deceive reasonable consumers. Plaintiff and

members of the general public were deceived by these practices, and such deception is likely to

continue until those practices cease.

45. Defendant’s suppression and withholding of material information as set forth in

detail above, constitute unfair and/or deceptive business practices within the meaning of

California Bus. & Prof. Code § 17200 et seq.

46. Defendant’s suppression and withholding of material information, as set forth above,

constitute unlawful business practices within the meaning of California Bus. & Prof. Code §

17200 et seq. in that those practices violate the CLRA, as set forth in detail below. The CLRA

violation serves as a predicate violation of this prong of the UCL.

47. Pursuant to California Bus. & Prof. Code §17203, plaintiff, on behalf of herself and

members of the general public, seeks an order of this Court:

(a) enjoining defendant from continuing to engage, use, or employ any

unfair, unlawful and/or deceptive business acts or practices found by this

Court to be in violation of the UCL; and

(b) restoring all monies that may have been acquired by defendant as a result

of such unfair, unlawful and/or deceptive act or practices.

48. Plaintiff and members of the general public may be irreparably harmed and/or denied

an effective and complete remedy if such an order is not granted.




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49. The unlawful, unfair and/or deceptive acts and practices of defendant, as described

above, present a serious threat to plaintiff and members of the general public.

50. As a result of defendant’s violation of the UCL, plaintiff and the Class are entitled to

restitution for out-of-pocket expenses and economic harm.

51. Pursuant to Civil Code § 3287(a), plaintiff and Class Members are further entitled to

pre-judgment interest as a direct and proximate result of defendant’s wrongful conduct. The

amount of damages suffered as a result is a sum certain and capable of calculation and plaintiff

and Class members are entitled to interest in an amount according to proof.

VII. SECOND CAUSE OF ACTION:

FOR VIOLATION OF BUS. & PROF. CODE §17500 ET SEQ.



53. In violation of California Bus. & Prof. Code §17500, defendant has withheld and

suppressed material information regarding the BPA-related risks associated with its Bottles,

thereby causing, by omission of such information, the dissemination of misleading statements

and representations in its advertisements, promotion, labeling and/or marketing for the Bottles.

54. Defendant’s representations in the advertisements, promotions, labeling and/or

marketing of the Bottles are deceptive in that material information is suppressed and withheld

from the consumer as set forth above. Defendant also has disseminated, or caused to be

disseminated, false and misleading statements and representations in advertisements, promotion

and/or marketing for the Bottles by violating provisions of the CLRA, as set forth below.

55. Defendant disseminated labeling, promotion and advertising regarding the Bottles

that, with its omission of material information regarding the BPA-related risks associated with

the Bottles, is by its very nature unfair, deceptive, untrue or misleading within the meaning of

the FAL.

56. In making and disseminating the statements alleged herein, in which material

information regarding the BPA-related risks associated with the Bottles was omitted, withheld

and suppressed, defendant knew or should have known that the statements were misleading, as




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it had access to reliable, credible and authoritative information regarding the dangers of BPA

and polycarbonate bottles. Therefore it acted in violation of the FAL.

57. As a direct and proximate result of defendant’s wrongful conduct, plaintiff and the

Class/subclass members have suffered substantial monetary damages. Pursuant to the FAL,

plaintiff, on behalf of herself and Class/subclass members, seeks an order of this Court:


misleading statements and representations in advertisements, promotion

and/or marketing for the Bottles this Court finds to be in violation of the

FAL; and

(b) restoring all monies that may have been acquired by defendant as a result

of such practices.

58. Pursuant to Civil Code section 3287(a), plaintiff and Class/subclass members are

further entitled to pre-judgment interest as a direct and proximate result of defendant’s wrongful

conduct. The amount of funds paid by plaintiff and Class/subclass members as a result of those

acts is a sum certain and capable of calculation, and plaintiff and Class/subclass members are

entitled to interest in an amount to be set forth according to proof.

VIII. THIRD CAUSE OF ACTION:

FRAUDULENT CONCEALMENT



60. Plaintiff contends that defendant suppressed and withheld material facts regarding

BPA risks associated with the Bottles, as set forth in detail above.

61. Defendant was bound to disclose to plaintiff and Class members the truth about the

BPA risks associated with the Bottles and all material information related to those risks, as set

forth in detail above, but it has not done so.

62. Plaintiff contends that when defendant omitted, suppressed and withheld material

information regarding BPA risks associated with the Bottles, as set forth in detail above, it knew

and was fully aware of that omitted, suppressed and withheld information and its materiality to




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decisions by plaintiff and Class members in purchasing the Bottles.

63. Plaintiff contends that by omitting, suppressing and withholding material

information regarding BPA risks associated with the Bottles, defendant, as set forth above,

intentionally misled plaintiff and Class members regarding the safety of the Bottles with regard

to BPA risks, and that it did so to induce them to purchase the Bottles.

64. Plaintiff and Class members relied on defendant to provide a safe product, and to

warn them of any inherent risks associated with the Bottles in its marketing, advertising,

promotion and labeling of the Bottles. The reliance by plaintiff and Class members was

reasonable and justified in that defendant appeared to be, and represented itself to be, a

reputable business and distributed its Bottles through reputable companies; that it was not a

company that would sell an product with known risks without warning consumers of those risks.

65. As a direct and proximate result of the fraud and deceit alleged, plaintiff and Class

members suffered actual damages, in an amount to be determined at trial, in that they were

induced to and did spend money on the Bottles, but were deprived of the benefit of their

bargain; they lost the money they paid for the Bottles as the Bottles did not have the safety

qualities they sought, qualities which, as the result of defendant’s omission, suppression and

withholding of material information, they had been led to believe the Bottles had. Had they

known the omitted, suppressed and withheld material information regarding BPA-related risks

associated with the Bottles, they would not have purchased them.

66. Plaintiff contends that when defendant omitted, suppressed and withheld material

information regarding BPA risks associated with the Bottles, as set forth in detail above, it knew

and was fully aware of that omitted, suppressed and withheld information and its materiality

with regard to decisions by plaintiff and Class members in purchasing the Bottles, but it omitted,

suppressed and withheld that material information and the full truth regarding BPA risks

associated with the Bottles intentionally so that plaintiff, Class members and the unknowing

public would buy the Bottles. Plaintiff and other members of the general public, in purchasing

the Bottles as herein alleged, did rely on defendant, as set forth above, in purchasing the Bottles,

all to their damage as herein alleged. In doing these things, defendant was guilty of malice,




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oppression and fraud, and plaintiff and Class members are therefore entitled to recover punitive

damages.

IX. FOURTH CAUSE OF ACTION:

FOR BREACH OF IMPLIED WARRANTY OF FITNESS FOR PURPOSE



68. Plaintiff and other Class members sought to purchase a safe, reusable beverage

container. In doing so, plaintiff and Class Members relied on defendant’s skill and judgment to

select and furnish suitable goods for that purpose; on or about that time, defendant sold to

plaintiff and Class Members the Bottles. By the acts set forth in detail above, defendant

warranted that the Bottles were safe, but intentionally omitted, suppressed and withheld material

information regarding BPA risks associated with the Bottles. Plaintiff and Class members

bought the Bottles from defendant, relying on defendant’s skill, judgment and representations.

However, defendant’s Bottles are not free of risk from harmful exposure to BPA, as set forth in

detail above.

69. At the time of sale, defendant had reason to know the particular purpose for which

the goods were required, and that plaintiff and Class members were relying on defendant’s skill

and judgment to select and furnish suitable and safe goods for that purpose. Accordingly, there

was an implied warranty that the goods were fit for this purpose.

70. However, defendant breached the warranty implied at the time of sale in that plaintiff

and Class members did not receive suitable goods, and the goods were not fit for the particular

purpose for which they were made, as the Bottles are not free of risk from harmful exposure to

BPA, as set forth in detail above.

71. As a direct and proximate result of this breach, plaintiff and Class members sufferedactual damages, in an amount to be determined at trial, in that they were induced to and did

spend money on the Bottles, but were deprived of the benefit of their bargain; they lost the

money they paid for the Bottles as the Bottles did not have the safety qualities they sought,

qualities which, as the result of defendant’s omission, suppression and withholding of material




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information, they had been led to believe the Bottles had. Had they known the omitted,

suppressed and withheld material information regarding BPA-related risks associated with the

Bottles, they would not have purchased them.

X. FIFTH CAUSE OF ACTION:

FOR VIOLATIONS OF CALIFORNIA’S CONSUMER LEGAL REMEDIES ACT



73. The acts and practices described above were undertaken by the defendant in

connection with a “transaction” (as defined in Civil Code § 1761(e)) which was intended to and

did result in the sale of “goods” (as defined in Civil Code § 1761(a)) to “consumers” (as defined

in Civil Code § 1761(d)). Defendant’s acts and practices, as alleged in detail above, violated,

and continue to violate, Section 1770(a)(5) of the CLRA in that in selling the Bottles, defendant

intentionally omitted, suppressed and withheld material information, and otherwise failed to

warn or inform consumers, regarding BPA risks associated with the Bottles.

74. Plaintiff and Class/subclass members seek and are entitled to equitable relief in the

form of an order from this Court:


practices found to violate the CLRA as set forth herein; and

(b) restoring to plaintiff and Class/subclass members all monies that may

have been acquired by defendant as a result of such practices.

75. Pursuant to Section 1782 of the CLRA, plaintiff is notifying defendant in writing of

the particular violations of Section 1770 of the CLRA (the Notice) and is demanding, among

other things, that defendant offer consumers refunds for the purchase of the Bottles. Plaintiff is

sending Notice by means of by certified mail, return-receipt requested, to defendant concurrent

with the service of this Complaint. If defendant fails to respond to plaintiff's demand within

thirty days of receipt of the Notice, pursuant to Section 1782 of the CLRA, plaintiff will amend

this Complaint to request statutory damages, actual damages, plus punitive damages, interest

and attorneys' fees and costs. Regardless of such an amendment to seek damages, however,




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plaintiff seeks, and is entitled to, pursuant to Section 1780(a)(2) of the CLRA, an order, as set

forth herein, enjoining the above-described wrongful acts and practices of defendant, plus costs

and attorneys' fees and any other relief that the Court deems proper.

XI. PRAYER FOR RELIEF

WHEREFORE, plaintiff, on behalf of herself and all others similarly situated, and for

members of the general public as private attorneys general under California Business and

Professions Code § 17204, prays for relief, jointly and severally, pursuant to each cause of

action set forth in this Complaint as follows:

1. For an order certifying that the action may be maintained as a class action.

2. For an award of equitable relief pursuant as follows:


practices found to violate the UCL, FAL and/or CLRA as set forth herein;

and

(b) restoring to plaintiff and Class/subclass members all monies that may

have been acquired by defendant as a result of such practices.

3. For an award of attorney’s fees pursuant to Code of Civil Procedure §1021.5.

4. For actual damages in an amount to be determined at trial for the Third and Fourth

Causes of Action;

5. For punitive damages in an amount to be determined at trial for the for the Third

Cause of Action;

6. For an award of costs and any other award the Court might deem appropriate; and

7. For pre- and post-judgment interest on any amounts awarded.

DATED: April 21, 2008.

Hewell Law Firm, APC

By: ______________________________ Harold M. Hewell

(ELECTRON.SIG)






Exhibit “A”




Advanced Search

All materials on this site are copyright 2006, Thermo Fisher Scientific Inc. If you have any questions or comments, please contact us

Page 1 of 1Welcome to NALGENE Outdoor!

2/15/2008http://www.nalgene-outdoor.com/store/index.html

A - 28




Exhibit “B”




Advanced Search

Home > Specifications and Care > BPA

BPA and NALGENE

As a responsible manufacturer of polycarbonate consumer products, Nalge Nunc International has monitored scientific research

concerning the safety of our products including Bisphenol-A for many years.

Based on the findings of the Food and Drug Administration, The Environmental Protection Agency, The American Plastics Council andother reliable sources from around the world, we continue to firmly believe in the safety of our products.

Nalge Nunc International also believes in providing its customers with the most factual information currently available on this subject.

You can view the most up to date information here

z Statement from the FDA, from a written communication dated January 29, 2008:z http://www.efsa.europa.eu/en/press_room/press_release/pr_bpa.html

z http://www.stats.org/stories/2008/should_baby_bottles_feb9_08.html

The following reference sources are provided for customers wishing to perform additional research.

Frequently Asked Questions:

z View our FAQ for answers to many popular questions.

Downloadable PDF's:

z BPA and NALGENE (PDF 423 KB)

z Polycarbonate Safety Studies (PDF 555 KB)

Additional Websites:

z http://www.bisphenol-a.org/

z http://www.acsh.org/factsfears/newsID.92/news_detail.aspz http://www.plasticsinfo.org/babybottles/index.html

z http://www.epa.gov/endocrine/about.htmlz SNEWS article regarding BPA

Frequently Asked Questions (FAQ)

Nalgene is committed to the well being of everyone that uses our products. Therefore, we’ve compiled the following information to

better inform our consumers on all of our products. We hope you find it useful and reassuring.

Q: Did Mountain Equipment Co-Op (MEC) remove all Nalgene products from its stores?A: No. MEC removed food and beverage containers constructed of polycarbonate. MEC will continue to carry a wide range of Nalgene

hydration products made from other materials, including HDPE, LDPE, PP and PET.

Q: Did Health Canada render a decision that resulted in MEC’s actions?A: No. Health Canada is expected to issue preliminary results of its BPA analysis in May, with their final report due in 2009. We’reconfident that when Health Canada completes its work, they will agree with all the important government agencies worldwide that have

concluded that polycarbonate water bottles pose no health risk to humans.

Q. Are polycarbonate bottles safe?A: Yes. Agencies and researchers worldwide have studied the safety of BPA and polycarbonate for approximately 50 years; includingThe Environmental Protection Agency and The Food and Drug Administration in the USA, The European Commission Scientific

Committee on Food, The German Federal Institute for Risk Assessment and the Japan Ministry of Health, Labor and Welfare. Findingsof studies from these agencies indicate that food and beverage containers manufactured from polycarbonate do not pose a health risk

to humans. As a result, polycarbonate is used in a wide variety of consumer products including baby bottles, water bottles, dental

sealants and the lining of most food & beverage containers.

Furthermore, several scientific panels including the European Union's Scientific Committee on Food, the National Toxicology Programand the Harvard Center for Risk Analysis have concluded that the weight of scientific evidence does not support the hypothesis that low

doses of BPA adversely affects human health. None of the large studies conducted have substantiated the claims made by thoseperforming some of the smaller studies frequently cited.

Q: Where can I find reliable information on polycarbonate and BPA?A: Consumers can visit the following web sites for more information:


2/25/2008http://www.nalgene-outdoor.com/technical/bpaInfo.html

B - 30




z European Food Safety Authority (EFSA) study - www.efsa.europa.eu/en/press_room/press_release/pr_bpa.html

z American Chemistry Council - www.bisphenol-a.orgz Environmental Protection Agency - www.epa.gov/endocrine/about.htmlz American Council on Science and Health - www.acsh.org/search/home_result.asp

z Nalgene - www.nalgene-outdoor.com/technical/bpainfo.html

Q: Which government and regulatory agencies have reviewed polycarbonate?

A: Many government and regulatory agencies, including those listed below, have conducted comprehensive testing and review of

polycarbonate and determined that it poses no health risk to humans.

z

The Environmental Protection Agency (USA)z The Food and Drug Administration (USA)z The European Commission Scientific Committee on Food

z The German Federal Institute for Risk Assessment

z Japan Ministry of Health, Labor and Welfare

Q: What is the latest government-sponsored research conducted on BPA and polycarbonate?A: In early 2007, the European Food Safety Authority (EFSA) announced its findings regarding BPA. The study reviewed all available

data from the last five years and concluded that people’s dietary exposure to BPA is well below the Daily Tolerable Intake Level. In fact,

the study recommended raising the Daily Tolerable Intake Level. Read more on this study at

www.efsa.europa.eu/en/press_room/press_release/pr_bpa.html

Q: Does Nalgene offer products in other materials?A: Yes. Nalgene has, and always will, offer a wide range of materials. Some of these materials include HDPE, LDPE, PP and PET. In aneffort to consistently deliver the most comprehensive product offering, Nalgene will continue to develop both new products and new

materials.

Q: Why does Nalgene use polycarbonate?

A: Many consumers prefer polycarbonate because of its unmatched ability to offer extraordinary durability, glass like clarity andresistance to stains and odors.

Q: Where are Nalgene bottles manufactured?

A: Unlike our major competitors, all Nalgene products are “Made in the USA”. As a US manufacturer, the business meets all applicable

manufacturing standards, including ISO 13485, to ensure the quality and safety of its products.

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2/25/2008http://www.nalgene-outdoor.com/technical/bpaInfo.html

B - 31




Exhibit “C”




Advanced Search

Home > Specifications and Care > BPA

Statement from the FDA, from a written communication dated January 29, 2008The Agency's current position on the presence of BPA impurities in food-contact polymers is as follows. BPA is used in the manufacture

of two types of polymers used for food-contact articles (i.e., polycarbonate (PC) polymers and epoxy-based enamels and coatings) and

is present at very low levels in the finished food contact materials. Typical uses of PC polymers include food processing equipment,

such as popcorn makers, and water and infant baby bottles intended for repeated use. BPA-based epoxy coated cans are used in a

variety of canned food and beverage applications, including cans used to hold infant formula. The Agency is aware of several reportsstating that BPA has estrogen-like activity. However, there are other reports that appear to dispute any reason to expect harm at the

low exposures that humans experience. A March 2007 report from a consumer group included studies showing the levels of BPA foundin canned foods and migrating out of PC baby bottles and included claims that these levels are unsafe. FDA scientists have reviewed

the available information from this report and have concluded that the BPA levels found in canned foods or migrating out of PC baby

bottles are not significantly different than the very low levels previously found by FDA chemists and other laboratories, levels that

result in a dietary exposure that is orders of magnitude below the levels known to not cause toxic effects in animals.

The agency has been actively reviewing the safety of BPA and has completed a review of the available data obtained from animalstudies, and migration studies. Based on the results of the migration studies conducted by FDA chemists, we have determined that the

dietary exposure to BPA is low (3.7 ppb), the level that is orders of magnitude below the levels known to cause toxic effects in animals.Considering the low dietary exposure and the fact that BPA had not demonstrated adverse effects when consumed by animals in

amounts of much higher (orders of magnitude) than humans would consume, FDA sees no reason at this time to ban or otherwiserestrict the uses now authorized. Our conclusion is based on our ongoing review of all available data. We will continue to monitor data

on BPA to determine if its use would raise a safety concern. If such a concern exists, FDA will take the appropriate post-market

regulatory action.

Return to BPA Info Page

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C - 33




Exhibit “D”




Reproductive Toxicology 24 (2007) 131–138

Correspondence

Chapel Hill bisphenol A expert panel consensus statement: Integration of mechanisms, effects in animals and potential toimpact human health at current levels of exposure

Keywords: Bisphenol A; In vitro; In vivo; Rat; Mouse; Aquatic animal; Cancer; Low dose; Non-monotonic dose–response curves; Developmental programming

1. Introduction

This document is a summary statement of the outcome fromthe meeting: “ Bisphenol A: An Examination of the Relevance of

Ecological, In vitro and Laboratory Animal Studies for Assess-

ing Risks to Human Health” sponsored by both the NIEHS and

NIDCR atNIH/DHHS, aswell asthe US-EPA andCommonweal

on the estrogenic environmental chemical bisphenol A (BPA,

2,2-bis(4-hydroxyphenyl)propane; CAS# 80-05-7). The meet-

ing was held in Chapel Hill, NC, 28–30 November 2006 due

to concerns about the potential for a relationship between BPAand negative trends in human health that have occurred in recent

decades. Examples include increases in abnormal penile/urethra

development in males, early sexual maturation in females, an

increase in neurobehavioral problems such as attention deficit

hyperactivity disorder (ADHD) and autism, an increase in child-

hood and adult obesity and type 2 diabetes, a regional decrease

in sperm count, andan increasein hormonally mediated cancers,

such as prostate and breast cancers. Concern has been elevated

by published studies reporting a relationship between treatmentwith “low doses”of BPA andmany of thesesnegative health out-

comes in experimental studies in laboratory animals as well as

in vitrostudies identifying plausible molecular mechanisms that

could mediate such effects. Importantly, muchevidence suggests

that these adverse effects are occurring in animals within the

range of exposure to BPA of the typical human living in a devel-

oped country, where virtually everyone has measurable blood,

tissue and urine levels of BPA that exceed the levels producedby doses used in the “low dose” animal experiments.

Issues relating to BPA were extensively discussed by five

panels of experts prior to and during the meeting, and are sum-

marized in five reports included in this issue:(1) human exposure

to bisphenol A (BPA) [1]; (2) in vitro molecular mechanisms of bisphenol A action [2]; (3) in vivo effects of bisphenol A in

laboratory animals [3]; (4) an ecological assessment of bisphe-

nol A: evidence from comparative biology [4]; (5) an evaluation

Abbreviations: ADHD, attention deficit hyperactivity disorder; BADGE,

bisphenol A diglycidyl ether; BIS-DMA, bisphenol A dimethacrylate; BIS-

GMA,bisphenol A glycerolatedimethacrylate; BPA, bisphenol A; ER, estrogen

receptor

of evidence for the carcinogenic activity of bisphenol A [5].Further discussion occurred at the meeting where participants

from thepanels were reorganized into four breakout groups. The

consensus statements from the meeting are presented below.

The definition of “low dose” of BPA at this meeting used the

same two criteria established at a prior NIH meeting concerning

the lowdose endocrine disruptor issue [6]: (1)for laboratory ani-

mal studies “low doses” involved administration of doses below

those used in traditional toxicological studies conducted for risk assessment purposes. For BPA the lowest dose previously exam-

ined for risk assessment purposes was 50 mg (kg−1 day−1) in

studies with rats and mice. The 50 mg (kg−1 day−1) dose is the

currently accepted lowest adverse effect level (LOAEL) that

was used to calculate the current US-EPA reference dose (the

daily dose that EPA calculates is safe for humans over the life-

time) of 50g(kg−1 day−1). The current reference dose is thus

based on “high dose” experiments conducted in the 1980s [7].

(2) “Low dose” also refers to doses within the range of typicalhuman exposure (excluding occupational exposures). For pur-

poses of this meeting, the published literature that was reviewedmet both of these criteria for being considered within the “low

dose” range.

Hundreds of in vitro and in vivo studies regarding the mech-

anisms and effects of low doses of BPA, as well as studies of

biomonitoring and sources of exposure, have been published in

peer reviewed journals over the last 10 years, since the first “lowdose” BPA in vivo studies were published [8–10]. The meeting

was convened specifically to integrate this relatively new infor-

mation. This task required the combined expertise of scientists

from many different disciplines, and care was taken to ensure

that participants covered these diverse areas.

BPA is a high-volume (>6 billion pounds per year) production

chemical used to make resins and polycarbonate plastic [11]. Of particular concern is the use of BPA in food and beverage plastic

storage and heating containers and to linemetal cans. In addition,potential environmental sources of BPA contamination are due

to usein dental fillings and sealants [12], lossesat theproduction

site [13], leaching from landfill [14,15], and presence in indoors

air [16].

BPA has become a chemical of “high concern” only in recent

years, even though BPA was shown to stimulate the reproductive

0890-6238/$ – see front matter © 2007 Published by Elsevier Inc.doi:10.1016/j.reprotox.2007.07.005

D - 35




132 Correspondence / Reproductive Toxicology 24 (2007) 131–138

system in female rats and thus to be an “environmental estro-

gen” in 1936 [17], long before it was used as the monomer to

synthesize polycarbonate plastic and resins in the early 1950s.

However,morerecent evidencehas shown thatBPAalso exhibits

other modes of endocrine disruption in addition to binding to

estrogen receptors, such as alterations in endogenous hormone

synthesis, hormone metabolism and hormone concentrations

in blood. BPA also results in changes in tissue enzymes andhormone receptors, and interacts with other hormone-response

systems, such as the androgen and thyroid hormone receptor

signaling systems. While BPA was initially considered to be a

“weak” estrogen based on a lower affinity for estrogen recep-

tor alpha relative to estradiol [18], research shows that BPA is

equipotent with estradiol in its ability to activate responses via

recently discovered estrogen receptors associated with the cellmembrane [19–22]. It is through these receptorsthat BPA stimu-

lates rapid physiological responses at lowpicogramper ml (parts

per trillion) concentrations.

2. Purpose and organization of the BPA meeting

2.1. Topic-focused expert panels

To address the strength of the evidence regarding the pub-

lished BPA research, an organizing committee was formed, and

fivepanels of experts fromdifferent disciplines were established.

Each panel had a chair or co-chairs and included a scientist

who agreed to be primarily responsible, along with the chair,

for preparing a preliminary draft of the panel’s report. A web

site was established on which all of the available electronic

files of articles concerning BPA were posted, along with otherpertinent information relating to the meeting. Prior to the meet-

ing, the panel members began working on draft reports and

communicated via electronic media and telephone conference

calls. The resulting preliminary report from each panel was

posted on the web site and distributed at the meeting for all

participants to read. After the meeting, each panel completed

a manuscript that is a part of this meeting report. These five

panel reports were peer reviewed using the normal manuscript

submission process to Reproductive Toxicology.The followingspecific concerns about BPA led to the five expert panels being

established:

(1) Leaching of BPA occurs from the resin lining of metal cans

and from plastic food and beverage containers under con-

ditions of normal use. BPA is also detected in water and airsamples.

(2) Parts per billion (ppb) levels of BPA that are unconjugated(not metabolized and thus biologically active) are detected

in human blood and tissues in different countries, and these

levels appear to be higher than blood levels that would be

present in animals exposed to the US-EPA reference dose.

(3) BPA causes a wide range of adverse effects at “low doses”

that are below the US-EPA reference dose in animals, both

terrestrial and aquatic.

(4) There is evidencefrom in vitro mechanistic studies that indi-cates the potential for disruption of human and animal cell

function at concentrations of BPA far below unconjugated

levels typically found in human blood and tissues.

(5) There is evidence that at very low doses, BPA may be car-

cinogenic or increase susceptibility to cancer in animals.

The five panels each addressed a different topic related to their

specific area of expertise with BPA and prepared a panel report

that included documentation of the relevant published studies:

Panel (1) Sources and amounts of human exposure to BPA as

well as pharmacokinetics.

Panel (2) In vitro studies related to the molecular mechanisms

that mediate responses to BPA with an emphasis on

studies using low doses.

Panel (3) In vivo studies of BPA at “low doses” in laboratoryanimals.

Panel (4) In vivo studies of BPA in aquatic wildlife and labora-

tory animals.

Panel (5) Relationship of BPA to cancers.

The purpose of the 3-day meeting was to provide an oppor-tunity for members of the different panels to interact with each

other to integrate information from different disciplines con-cerning low dose effects of BPA after each panel of experts had

prepared a report in its specific area. The agenda of the meeting

was designed to allow the members of the five panels to have

time to discuss the information in their panel reports and finalize

statements about the strength of the evidence for the literature

that the panel had reviewed.

2.2. Integration of information by breakout groups

For the second part of the meeting the focus was on inte-

grating the information from each of the panel reports. This

was accomplished by assigning panel members to one of four

breakout groups. The four replicate breakout groups were estab-

lished using the following criteria, such that each breakoutgroup

should have

(1) At least two members from each of the five panels.

(2) A person from each panel who had published on BPA.

(3) A person with general knowledge of endocrine disruption

research or endocrinology, but who had not necessarily pub-

lished on BPA.

(4) A person with experience in the process of reaching con-

sensus.(5) A mixture of junior and senior investigators.

The charge to the replicate breakout groups was to individually

integrate the information relating to the following four issues:

Issue (1) Determine the degree to which the findings on BPA

mechanisms of action identify mechanisms and bioac-

tive doses that explain results of the studies reported

by the panel on in vivo laboratory animal studies.

Determine the strength of the evidence for plausiblemechanisms mediating in vivo effects at low doses. In

D - 36




Correspondence / Reproductive Toxicology 24 (2007) 131–138 133

addition, identify any in vivo findings that are unex-

pected based on the in vitro literature.

Issue (2) Assess the degree to which ecological studies with

wildlife are consistent with laboratory studies in sim-

ilar and different species. For example, determine the

similarity of exposure levels and types of responses

seen in wildlife and laboratory animals.

Issue (3) Discuss the degree to which the low doses of BPAused in laboratory animal studies relate to the lev-

els detected in human serum and tissues (including

urine).

Issue (4) Assess the importance of life stage in the pharma-

cokinetics of BPA, levels of exposure to BPA, and the

health effects of BPA in animals and humans.

3. Findings submitted by the four breakout groups

The reports from the breakout groups are presented below.

The four breakout groups conducted a critical examination of

the published research on BPA in relation to the four topics

described above. Each of the breakout groups identified areas of knowledge and research gaps and made suggestions for future

directions of research. In addition, each group identified whichof the following two categories applied to specific outcomes:

• “We are confident of the following”: this category applied

when there were findings reported in multiple papers from

multiple labs that were in agreement. There should have been

no papers reporting conflicting findings, unless there were

flaws in those papers, in which case the flaw(s) should have

been identified.• “We believe the following to be likely but requiring con-

firmation”: This category applied when there were multiple

consistent findings from onelab, or there may have been some

conflicting reports along with reports of significant findings.

4. Levels of confidence for published BPA findings

The responses from the four different breakout groups were

integrated together and organized based on levels of confidence.The criterion for a statement being included in a category was

that there had to be consensus among all four of the breakout

groups about the statement.

4.1. Based on existing data we are confident of the

following

4.1.1. Issue 1: In vitro mechanistic research—laboratory

animal research connection

1. In vitro studies have provided two routes of plausibility for

low dose in vivo effects of BPA. These include binding to

nuclear estrogen receptors that regulate transcription as well

as estrogen receptors associated with the cell membrane that

promote calcium mobilization and intracellular signaling.

Receptors associated with the cell membrane are more sensi-

tive to BPA than the nuclear receptors. Actions mediated bymembrane associated receptor signaling may underlie much

of the low dose BPA phenomena (effects have been reported

at doses as low as 1 pM or 0.23 ppt). This increases the plau-

sibility of effects at low doses, which are within the range of

environmentally relevant doses (human and wildlife levels of

exposure).

2. In vitro mechanistic information has informed us that expos-

ing tissues to only an extremely narrow range of doses of

BPA may lead to erroneous conclusions. Non-monotonicdose–response curves are encountered frequently in basic

endocrinological research, and numerous examples have

been reported for BPA reviewed in Refs. [18,23,24]. Because

of this animal experiments on unstudied systems must avoid

narrow dose ranges, especially the use of only a few very

high doses. Thus, testing one or two doses and concluding

that there are no effects is inappropriate. At somewhat higherdoses than are required for estrogen receptor (ER)-mediated

responses, BPA also interacts with androgen and thyroid hor-

mone receptors, making predictions of effects at different

doses very complex.

3. In vitro studies can dissect mechanisms of complicated

effects observed in vivo. The proposed potential mechanismsacting in vitro and in vivo are the same, involving estro-

gen receptor mediated (nuclear- and membrane-associated)actions. However, specific effects are dose and cell/tissue

specific. In addition, there are in vivo processes that are not

reflective of currently known mechanisms that have been

identified in vitro. This is due to previously unknown mech-

anisms as well as the complexity (due to interactions among

cell and tissue types) of in vivo systems.

4.1.2. Issue 2: Wildlife—laboratory animal research

connection

1. BPA is found in the environment: aquatic, terrestrial and air.

2. Studies of wildlife demonstrate estrogenic responses that are

similar to responses seen in laboratory animals. Specifically,

reductions in spermatogenesis are seen in wildlife at ecolog-

ical concentrations of BPA, and these effects are also seen

in controlled laboratory studies with BPA. In addition, vitel-

logenin response is a common biomarker in non-mammalian

wildlife and laboratory species for BPA-induced estrogenreceptor activation as well as activation by other estrogens.

3. BPA exposure induces similar effects in reproductive sys-

tems in wildlife and experimental animal model systems,

but concentrations used in experiments involving wildlife

species are often higher than environmental exposures. Thereare conditions in the environment, such as landfill leachates

and effluent outflow that cause episodic exposure of field

populations to elevated doses of BPA.4. Responses in a variety of vertebrate wildlife species are qual-

itatively consistent with controlled laboratory studies with

BPA. Thus, animals in the wild show evidence of harm,

and controlled laboratory studies with model aquatic animals

(i.e., medaka, zebrafish, and fathead minnows) are consistent

with observations made in wildlife species. Low dose effects

of BPA (low ppb range) have been observed in many of these

animals.

D - 37





5. The similar effects observed in wildlife and laboratory ani-

mals exposed to BPA predict that similar effects are also

occurring in humans.

4.1.3. Issue 3: Laboratory animal research—human

exposure connection

1. Human exposure to BPA is widespread.2. Human exposure to BPA is variable, and exposure levels

cover a broad range [central tendency for unconjugated BPA:

0.3–4.4 ng ml−1 (ppb)] in tissues and fluids in fetuses, chil-

dren and adults.

3. Because the current published literature states that there is a

linear relationshipbetween administereddose and circulating

levels of BPA in animal studies, this allows circulating levels

at lower administered doses to be predicted in experimentalanimals based on the results from studies in which higher

doses were administered.

4. Allof thecurrently publishedmetabolic studies in rats predict

circulating BPA levels after acute low dose oral exposures at

blood levels less than or equal to 2 ng ml

−1

(ppb), whichis the approximate median and mean unconjugated circulat-

ing BPA level in humans. Therefore, the commonly reported

circulating levels in humans exceed the circulating levelsextrapolated from acute exposure studies in laboratory ani-

mals.

5. BPA levels in the fetal mouse exposed to BPA by maternal

delivery of 25g kg−1, a dose that has produced adverse

effects in multiple experiments, are well within the range of

unconjugated BPA levels observed in human fetal blood.

4.1.4. Issue 4: Life stage—relationship to exposure

pharmacokinetics and health effects

1. Sensitivity to endocrine disruptors, including BPA, variesextensively with life stage, indicating that there are spe-

cific windows of increased sensitivity at multiple life stages.

Therefore, it is essential to assess the impact of life stage on

the response to BPA in studies involving wildlife, laboratory

animals, and humans.

2. Developmental windows of susceptibility are comparable in

vertebrate wildlife species and laboratory animals.3. BPA alters “epigenetic programming” of genes in experimen-

tal animals and wildlife that results in persistent effects that

are expressed later in life [25]. These organizational effects

(functional and structural) in response to exposure to low

doses of BPA during organogenesis persist into adulthood,

long after the period of exposure has ended. Specifically, pre-

natal and/or neonatal exposure to low doses of BPA results

in organizational changes in the prostate, breast, testis, mam-mary glands, body size, brain structure and chemistry, and

behavior of laboratory animals.

4. There are effects due to exposure in adulthood that occurs at

low doses of BPA. Substantial neurobehavioral effects and

reproductive effects in both males and females have been

observed during adult exposures in laboratory animals.

5. Adult exposure studies cannot be presumed to predict the

results of exposure during development.

6. Life stage impacts the pharmacokinetics of BPA.

4.2. We believe the following to be likely but require

confirmation



1. BPA metabolism occursin cell culture systems, and althoughthere are differences between cell types, there is less variabil-

itythan in theentire animal. Metabolism is an important issue

for humans and wildlife field populations with large genetic

variability. Individual differences in BPA pharmacokinetics

allow for underlying variability within a population, and may

allow for the identification of sensitive and insensitive sub-

populations.

2. The activity of various enzymes involved in drug, chemi-cal, and hormone metabolism, as well as protection against

oxidative stress, are programmed by hormone levels during

sensitive periods in development. Developmental alterations

in hormonal programming (activation or inhibition) may thusaffect metabolism of BPA and other hormonesand chemicals.

Directinteraction of BPA with enzymes in cells hasonly been

reported at higher doses than expected for human exposures.

3. Theset of genes regulated by BPA is expectedto differamongdoses. Therefore, different doses of BPA do not produce dif-

ferent effects only due to a quantitative difference in the

expression of the same set of genes.

4. Differential expression of estrogen receptor subtypes ( /ß;

variant isoforms), and protein–protein interactions (estrogen

receptor homo- and hetero-dimer formation, co-regulators,

etc) modulate the cellular response to BPA. Direct actions of

BPA on intracellular signal transduction modulate some cel-

lular responses, which are similarly dependent on differentialexpression and protein–protein interactions.

5. Bioactive doses can be mathematically modeled, but further

model refinement and experimental confirmation is required.

6. Other mechanisms (androgen receptors, thyroid hormone

receptors) may be relevantfor BPA action, but at higherdoses

than for estrogen responsive mechanisms.


connection

1. The effects observed in laboratory animals could be present

in wildlife, because the low doses being studied in labora-

tory animals are now relevant to environmental exposure

levels of wildlife. The similarities in mechanisms that have

been observed between different species suggest that field

populations will respond to the same low levels.

2. Measurements of vitellogenin production in fish have estab-lished that there are exogenous estrogenic signals in the their

environment. BPA may be contributing to this phenomenon

as it entersnatural water systems after leaching from landfills

and due to plastic debris in water.

3. Delayed spawning is seen in male andfemale fish, which may

relate to observed changes in estrous cyclicity in mammals

in laboratory experiments.

D - 38





4. In wildlife and laboratory studies, BPA induces alteration in

steroid biosynthesis/ metabolism/excretion.

5. Wildlife residing in sediment is likely exposed to higher

levels of BPA.


exposure connection

1. Human exposure is likely to be continuous, unlike exposurein most laboratory animal studies of BPA pharmacokinetics.



1. Clearance of BPA in the fetus is reduced compared to

other life stages. Different effects and metabolic clear-ance mechanisms are also observed in neonatal and adult

animals. Conjugation (glucuronidation) and other mecha-

nisms of metabolic clearance of BPA thus vary throughout

life.

2. Exposure to BPA during different life stages differentially

influences reproductive cancer etiology and progression, andexposure during sensitive periods in organogenesis may

increase susceptibility to development of cancers in some

organs, such as the prostate and mammary glands.3. Early life exposure to environmentally relevant BPA doses

may result in persistent adverse effects in humans.

4. The function of the immune system can be altered following

adult exposure to BPA.

5. Effects on insulin metabolism occur following adult expo-

sure.

4.3. Areas of uncertainty and suggestions for future

research



1. Since BPA can act as an agonist or an antagonist in differ-

ent tissues and against different background physiological

states, the specific co-regulators that mediated these dif-

ferent responses of BPA need to be elucidated based onin vitro mechanistic studies, which should be confirmed

in vivo.

2. Research is needed on specific receptor sub-types (i.e., classi-

cal nuclear and non-classical membrane-associated estrogen

receptors) in relation to the potency of BPA in different tis-

sues.

3. The identification of multiple estrogen receptor genes and

variants as well as different co-regulators withdifferent activ-ities reveals that different levels of potency of BPA could

be obtained by complex interactions between these differ-

ent components that would not be predicted in homogeneous

recombinant systems.


connection

1. To directly relate the effects seen in wildlife with BPA expo-sure, biomonitoring dataare needed fromwildlife. In addition

to BPA levels, these studies should assay total estrogenic and

antiandrogenic activity from other contaminants.

2. There is a need to examine sensitive endpoints in wildlife

that have been identified in laboratory animals.

3. There are substantial amounts of plastic debris within marine

and fresh water ecosystems, and studies are needed to

examine the impact of BPA in the environment on aquatic

organisms. Doses used in laboratory experiments involvingwildlife should reflect environmental exposures.

4. More studies need to be done with BPA in invertebrates, and

a fundamental understanding of estrogen action in inverte-

brates is required.

5. Studies shoulddetermine if amplification of BPA through the

food chain occurs, particularly under anaerobic or hypoxic

conditions due to the lack of microbial or photodegrada-tion.

6. Future research emphasis should be placed on populations

of aquatic animals exposed to landfill leachate and sewage

effluent, as these are the primary point sources forBPA expo-

sure.


exposure connection

1. Even though there have been attempts to estimate daily

human intake of BPA, these estimates require many assump-

tions.The best measureswe haveto estimatewhetherhumans

may be affected by current exposures to BPA are levels

in blood (not exposure levels), which can be related to

blood levels in experimental animals after acute exposures.

Known sources of human exposure to BPA do not appearsufficient to explain levels measured in human tissues and

fluids.

2. While BPA is not persistent in the environment or in humans,

biomonitoring surveys indicate that exposure is continuous.

This is problematic because acute animal exposure studies

are used to estimate daily human exposure to BPA, and at

this time, we are not aware of any studies that have examined

BPA pharmacokinetics in animal models following continu-

ous lowlevelexposures.Measurement of BPA levels in serumand other body fluids suggests that either BPA intake is much

higher than accounted for, or that BPA can bioaccumulate in

some conditions such as pregnancy, or both. Research using

both animal models, as well as epidemiology studies, are

needed to address these hypotheses, and this research needs

to better mimic the apparent continuous exposure of humansto BPA.

3. More comprehensive exposure and biomonitoring studies areneeded, especially in developing countries.

4. In both animal and human studies, internal exposure mea-

sures need to be related to health effects. In particular, there

is a need for epidemiological studies relating health out-

comes to BPA exposure, particularly during sensitive periods

in development. These studies should be based on hypothe-

ses from findings in experimental animals. This will require

additional development of appropriate biomarkers in animalstudies that can be used in epidemiological research.

D - 39







1. While there is a great need to continue studying prenatal

and perinatal exposures in laboratory animal studies, many

organs and endpoints continue developing at later stages

(throughout puberty and adolescence). Additional studies

are needed during these later periods of development.

2. Additional research is needed regarding exposure to BPA inadulthood to determine whether post-exposure effects are

temporary or are permanent and associated with subsequent

age-related diseases.

3. Because aging adults lose repair mechanisms, metabolic

enzymes, and imprinted genes, the possibility that adult

exposures (long-term,lowlevel) can increasethe riskof can-

cers and other conditions during aging should be addressedwith additional human research and the development of

appropriate animal models.

4. Epigenetics should be examined as a potential mech-

anism mediating developmental effects as well as the

trans-generational effects of BPA and other contaminants.

Potential effectsof adult exposures alsoneed to be examinedin relation to disruption of epigenetic changes that occur

normally during aging.5. Trans- and multi-generational effects of BPA must be exam-

ined in laboratory animals and humans.

6. There is a need for studies that involve collection of human

blood and urine from humans at several life stages, with

specific emphasis on infants and young children and contin-

ued monitoring throughout adulthood. Additionally, there

is a need to characterize the basis for the variability in

BPA levels in studies examining both human urine andserum.

7. There is a need for research on the genetic basis for differ-

ences in susceptibility to BPA and other contaminants.

8. Studies are needed on comparative BPA pharmacokinet-

ics in invertebrates and vertebrates (non-human primates

included).

9. There is a need to measure total endocrine disrupter load in

humans and wildlife. Therefore, biomarkers of endocrine

disrupter exposure are necessary.10. There is a need for more research directed at examining

human exposure, pharmacokinetics and health effects of

selected BPA precursors (i.e., BADGE, BISGMA, and BIS-

DMA) and metabolites (e.g., halogenated BPAs).

11. There is a need for more studies focused on identification

of other (non-estrogen-receptor mediated) mechanisms of action of BPA.

12. Effects of chemicals on the immune system are life stagedependent, and identifying the life stage dependency for

BPA effects on theimmune systemis necessary. In addition,

studies examining BPA effects on the immune system in

wildlife are necessary.

5. Conclusions

The publishedscientific literature on human and animal expo-sure to low doses of BPA in relation to in vitro mechanistic

studies reveals that human exposure to BPA is within the range

that is predicted to be biologically active in over 95% of peo-

ple sampled. The wide range of adverse effects of low doses

of BPA in laboratory animals exposed both during development

and in adulthood is a great cause for concern with regard to the

potential for similar adverse effects in humans. Recent trends

in human diseases relate to adverse effects observed in experi-

mental animals exposed to low doses of BPA. Specific examplesinclude: the increase in prostate and breast cancer, uro-genital

abnormalities in male babies, a decline in semen quality in men,

early onset of puberty in girls, metabolic disorders including

insulin resistant (type 2) diabetes and obesity, and neurobehav-

ioral problems such as attention deficit hyperactivity disorder

(ADHD).

There is extensive evidence that outcomes may not becomeapparent until long after BPA exposure during development has

occurred. The issue of a very long latency for effects in utero to

be observed is referred to as the developmental origins of adult

health and disease (DOHaD) hypothesis. These developmental

effects are irreversible and can occur due to low dose exposure

during brief sensitive periods in development, even though noBPA may be detected when the damage or disease is expressed.

However, this does not diminish our concern for adult exposure,where many adverse outcomes are observed while exposure is

occurring. Concern regarding exposure throughout life is based

on evidence that there is chronic, low level exposure of virtually

everyone in developed countries to BPA. These findings indicate

that acute studies in animals, particularly traditional toxicolog-

ical studies that only involve the use of high doses of BPA, do

not reflect the situation in humans.

Thefact that very few epidemiologicalstudies have been con-ducted to address the issue of the potential for BPA to impact

human health is a concern, and more research is clearly needed.

This also applies to wildlife,both aquatic andterrestrial.The for-

mulation of hypotheses for the epidemiological and ecological

studies can be greatly facilitated by the extensive evidence from

laboratory animal studies, particularly when common mecha-

nisms that could plausibly mediate the responses are known to

be very similar in the laboratory animal models, wildlife and

humans.

Acknowledgements

Meeting support was provided by NIEHS and NIDCR,

NIH/DHHS, the US-EPA and Commonweal. We thank Paul

French for assistance with the meeting in web site and AlbertKingman for advice during preparation of the manuscript. This

manuscript does not reflect US-EPA, USGS or NIH agency

policy. FvS is supported by NIH grant ES11283.

References

[1] Vandenberg LN, Hauser R, Marcus M, Olea N, Welshons WV. Human

exposure to bisphenol A (BPA). Reprod Toxicol 2007;24:139–77.

[2] Wetherill YB, Akingbemi B, Kanno J, McLachlan JA, Nadal A, Sonnen-

schein C, et al. In vitro molecular mechanisms of bisphenol A action.

Reprod Toxicol 2007;24:178–98.

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[3] RichterCR, Birnbaum LS,Farabollini F, Newbold RR,RubinBS, Talsness

CE,et al.In vivoeffectsof bisphenol A in laboratory rodentstudies.Reprod

Toxicol 2007;24:199–224.

[4] Crain DA, Eriksen M, IguchiT, Jobling S, LauferH, LeBlanc GA, et al.An

ecological assessment of bisphenol A: evidence from comparative biology.

Reprod Toxicol 2007;24:225–39.

[5] Keri RA, Ho S-M, Hunt PA, Knudsen KE, Soto AM, Prins GS. An eval-

uation of evidence for the earcinogenic activity of bisphenol A. Reprod

Toxicol 2007;24:240–52.[6] NTP. Final Report of the Endocrine Disruptors Low Dose Peer

Review Panel. Raleigh; NC. http://ntp.niehs.nih.gov/index.cfm?objectid=

06F5CE98-E82F-8182-7FA81C02D3690D47ml ; 2001 (Access data: June

8, 2007).

[7] IRIS. Bisphenol A (CASRN 80-05-7). US-EPA Integrated Risk Informa-

tion System Substance File. http://www.epa.gov/iris/subst/0356.htm; 1988

(Access data: June 8, 2007).

[8] Nagel SC, vom Saal FS, Thayer KA, Dhar MG, Boechler M, Welshons

WV. Relative binding affinity-serum modified access (RBA-SMA) assay

predicts the relative in vivo bioactivity of the xenoestrogens bisphenol A

and octylphenol. Environ Health Perspect 1997;105(1):70–6.

[9] Colerangle JB, Roy D. Profound effects of the weak environmental

estrogen-like chemical bisphenol A on the growth of the mammary

gland of Noble rats. J Steroid Biochem Mol Biol 1997;60(1–2):

153–60.

[10] Steinmetz R, Brown NG, Allen DL, Bigsby RM, Ben-Jonathan N. The

environmental estrogen bisphenol A stimulates prolactin release in vitro

and in vivo. Endocrinol 1997;138(5):1780–6.

[11] Burridge E. Bisphenol A: product profile. Eur Chem News 2003:17.

[12] Olea N, Pulgar R, Perez P, Olea-Serrano F, Rivas A, Novillo-Fertrell A, et

al. Estrogenicity of resin-based composites and sealants used in dentistry.

Environ Health Perspect 1996;104(3):298–305.

[13] Moore C. Synthetic polymers in the marine environment: what we know,

what we need to know, what can be done? In: Ragaini RC, editor. Proceed-

ings of Conference: International Seminar on Nuclear War and Planetary

Emergencies, 36th Session, Erice, Sicily, August 2006. Singapore: World

Scientific Publishers; in press.

[14] Coors A, Jones PD, Giesy JP, Ratte HT. Removal of estrogenic activ-

ity from municipal waste landfill leachate assessed with a bioassay

based on reporter gene expression. Environ Sci Technol 2003;37(15):

3430–4.[15] Kawagoshi Y, Fujita Y, Kishi I, Fukunaga I. Estrogenic chemicals and

estrogenic activity in leachate frommunicipal waste landfill determined by

yeast two-hybrid assay. J Environ Monit 2003;5(2):269–74.

[16] Rudel RA, Brody JG, Spengler JD, Vallarino J, Geno PW, Sun G, et al.

Identification of selected hormonally active agents and animal mammary

carcinogens in commercialand residential air anddust samples. J Air Waste

Manage Assoc 2001;51:499–513.

[17] Dodds EC, Lawson W. Synthetic oestrogenic agents without the phenan-

threne nucleus. Nature 1936;137:996.

[18] Welshons WV, NagelSC, vomSaal FS.Large effects fromsmall exposures.

III. Endocrine mechanisms mediating effects of bisphenol A at levels of

human exposure. Endocrinology 2006;147(Suppl. 6):S56–69.

[19] Quesada I, Fuentes E, Viso-Leon MC, Soria B, Ripoll C, Nadal A. Low

doses of the endocrine disruptor bisphenol-A and the native hormone

17beta-estradiol rapidly activate transcription factor CREB. FASEB J

2002;16(12):1671–3.

[20] Walsh DE, Dockery P, Doolan CM. Estrogen receptor independent rapid

non-genomic effects of environmental estrogens on [CA2+]i in human

breast cancer cells. Mol Cell Endocrinol 2005;230(1–2):23–30.

[21] Wozniak AL, Bulayeva NN, Watson CS. Xenoestrogens at picomolar to

nanomolar concentrations trigger membrane estrogen receptor-a mediated

Ca++fluxes andprolactin release in GH3/B6 pituitarytumor cells.Environ

Health Perspect 2005;113:431–9.

[22] Zsarnovszky A, Le HH, Wang HS, Belcher SM. Ontogeny of

rapid estrogen-mediated extracellular signal-regulated kinase signal-

ing in the rat cerebellar cortex: potent nongenomic agonist and

endocrine disrupting activity of the xenoestrogen bisphenol A. Endocrinol

2005;146(12):5388–96.

[23] vom Saal FS, Welshons WV. Large effects from small exposures. II. The

importance of positive controls in low-dose research on bisphenol A. Env-

iron Res 2006;100:50–76.

[24] Welshons WV, Thayer KA, Judy BM, Taylor JA, Curran EM, vom Saal

FS. Large effects from small exposures. I. Mechanisms for endocrine-

disrupting chemicals with estrogenic activity. Environ Health Perspect

2003;111(8):994–1006.

[25] Ho SM, Tang WY, Belmonte de Frausto J, Prins GS. Developmental

exposure to estradiol and bisphenol A increases susceptibility to prostatecarcinogenesis and epigenetically regulates phosphodiesterase type 4 vari-

ant 4. Cancer Res 2006;66(11):5624–32.

Frederick S. vom Saal ∗

Division of Biological Sciences, University of

Missouri-Columbia, 105 Lefevre Hall, Columbia, MO 65211,

United States

Benson T. Akingbemi

Department of Anatomy, Physiology and Pharmacology,

Auburn University, Auburn, AL 36849, United States

Scott M. Belcher Department of Pharmacology and Cell Biophysics, Center for

Environmental Genetics, University of Cincinnati, Cincinnati,

OH 45267, United States

Linda S. BirnbaumU.S. Environmental Protection Agency, Research Triangle

Park, NC 27709, United States

D. Andrew Crain

Biology Department, Maryville College, Maryville, TN 37804,

United States

Marcus Eriksen

Algalita Marine Research Foundation, Los Angeles, CA

90034, United States

Francesca Farabollini

Department of Physiology, University of Siena, 53100 Siena,

Italy

Louis J. Guillette Jr. Department of Zoology, University of Florida, Gainesville, FL


Russ Hauser

Department of Environmental Health, Harvard School of

Public Health, Boston, MA 02115, United States

Jerrold J. Heindel

Division of Extramural Research and Training, National

Institute of Environmental Health Sciences, Research Triangle

Park, NC 27709, United States

Shuk-Mei Ho

Department of Environmental Health, University of Cincinnati

Medical School, Cincinnati, OH 45267, United States

Patricia A. Hunt

School of Molecular Biosciences, Washington State

University, Pullman, WA 99164, United States

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Taisen Iguchi

National Institutes of Natural Science, Okazaki Institute For

Integrative Bioscience Bioenvironmental Science, Okazaki,

Aichi 444-8787, Japan

Susan Jobling

Department of Biological Sciences, Brunel University,

Uxbridge, Middlesex, UK Jun Kanno

Division of Cellular & Molecular Toxicology, National

Institute of Health Sciences, Tokyo 158-8501, Japan

Ruth A. Keri

Department of Pharmacology, Case Western Reserve

University School of Medicine, Cleveland, OH 44106,

United States

Karen E. Knudsen Department of Cell and Cancer Biology, University of

Cincinnati College of Medicine, Cincinnati, OH 45267,

United States

Hans Laufer

Department of Molecular and Cell Biology, University of

Connecticut, Storrs, CT 06269, United States

Gerald A. LeBlanc

Department of Environmental and Molecular Toxicology,

North Carolina State University, Raleigh, NC 27695,

United States

Michele Marcus

Department of Epidemiology, Rollins School of Public Health,

Emory University, Atlanta, GA 30322,

United States

John A. McLachlan

Center for Bioenvironmental Research, Tulane and Xavier

Universities, New Orleans, LA 70112, United States

John Peterson Myers

Environmental Health Sciences, Charlottesville, VA 22902,

United States

Angel Nadal

Instituto de Bioingenierı́a, Universidad Miguel Hern´ andez,

Elche 03202, Alicante, Spain

Retha R. Newbold

Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC


Nicolas Olea

CIBERESP Hospital Clinico-University of Granada, 18071

Granada, Spain

Gail S. Prins

Department of Urology, University of Illinois at Chicago,

Chicago, IL 60612, United States

Catherine A. RichterUSGS, Columbia Environmental Research Center, Columbia,

MO 65201, United States

Beverly S. Rubin Department of Anatomy and Cellular Biology, Tufts Medical

School, Boston, MA 02111, United States

Carlos Sonnenschein

Department of Anatomy and Cellular Biology, Tufts University

School of Medicine, Boston, MA 02111, United States

Ana M. Soto

Department of Anatomy and Cell Biology, Tufts University

School of Medicine, Boston, MA 02111, United States

Chris E. TalsnessCharit´ e University Medical School Berlin, Campus Benjamin

Franklin, Institute of Clinical Pharmacology and Toxicology,

Department of Toxicology, 14195 Berlin, Germany

John G. Vandenbergh

Department of Zoology, North Carolina State University,

Raleigh, NC 27695, United States

Laura N. Vandenberg

Tufts University Sackler School of Graduate Biomedical

Sciences, Boston, MA 02111, United States

Debby R. Walser-Kuntz

Carleton College, Department of Biology, Northfield, MN


Cheryl S. Watson

Biochemistry and Molecular Biology Department, Universityof Texas Medical Branch, Galveston, TX 77555, United States

Wade V. Welshons

Department of Biomedical Sciences, University of Missouri,

Columbia, MO 65211, United States

Yelena Wetherill

Department of Epidemiology, Harvard School of Public

Health, Boston, MA 02115, United States

R. Thomas Zoeller

Biology Department, University of Massachusetts, Amherst,

MA 01003, United States

∗ Corresponding author. Tel.: +1 573 882 4367;

fax: +1 573 884 5020.

E-mail address: [email protected] (F.S. vom Saal)

8 June 2007

Available online 27 July 2007

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Exhibit “E”




EWG: THE POWER OF INFORMATION

January 25, 2008

Dr. Michael D. ShelbyDirectorCenter for the Evaluation of Risks to Human ReproductionNational Institute of Environmental Health ServicesDepartment of Health and Human ServicesP.O. Box 12233MD EC-32Research Triangle Park, NC 27709

Re: Comments on the Bisphenol A (BPA) Expert Panel Report

Dear Dr. Shelby:

We are writing to provide comments to the National Toxicology Program (NTP) as theagency develops its final position on the extent to which the toxic plastics chemical bisphenol A (BPA) poses a risk to human reproduction and development. As youconduct your review, we urge you to consider fully the following important data andinformation that is highly relevant to a determination of BPA’s potential impacts onhuman health:

• The objectivity of CERHR’s review of BPA toxicity remains in question.Sciences International, a contractor who was subsequently fired by the National Institutes of Health for potential conflict of interest, prepared the initial BPAreview document for the Center for the Evaluation of Risks to HumanReproduction (CERHR). This document continued to be used by the expert

panel despite the fact that several prominent scientists and public healthadvocates questioned its objectivity. We understand that CERHR’s final versionof this review document (CERHR 2000a) will be used as a basis for NTP’sdetermination. The objectivity of the findings in this document remain inquestion and should be reviewed in full by NTP.

• The CERHR final expert panel report contains many errors andinconsistencies: In their review of the CERHR expert panel interim draft(CERHR 200b), independent BPA experts identified hundreds of errors andinconsistencies (EWG 2007a); review of the final expert panel draft finds thatmany of these errors and inconsistencies were not adequately addressed. NTPmust ensure that its determination is based on accurate information, and mustnot rely on the inconsistent and incorrect findings that still plague the CERHRassessment.

• The CERHR expert panel failed to consider the significant, high exposuresto BPA for formula fed infants (EWG 2007b). NTP’s consideration of theseexposures is essential if the agency is to reach an accurate determination onBPA’s risks.

• New data confirms the relevance of BPA studies that used non-oral routesof administration. These studies were categorically excluded by the expert

1 4 3 6 U S t r e e t N W , S u i t e 1 0 0W a s h i n g t o n , D C 2 0 0 0 9

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panel in reaching their final decision. In light of the new data, NTP shouldincorporate findings from these excluded studies in making its determination.

• BPA experts raise serious concerns about potential human health impacts

from BPA exposures. A NIEHS-sponsored panel of 38 BPA experts, whichconvened in Chapel Hill, North Carolina in November of 2006, published acomprehensive consensus statement regarding BPA toxicity and determinedthat BPA exposure is a risk to human health (vom Saal 2007); the NTP’sthorough consideration of this expert panel’s findings is critical.

Each of these points is described in detail below.

The objectivity of CERHR’s review of BPA toxicity remains in question . In March of 2007, the National Institutes of Health fired the contractor (Sciences International)that was hired to prepare the initial BPA review document, citing potential conflicts of interest when information became available that showed that Sciences International

staff had previously worked with BPA manufacturers. The document that was preparedby this contractor, however, continued to be used by the expert panel, despite the factthat several prominent scientists and public health advocates had questioned theobjectivity of the Sciences International review. CERHR’s final report is derived fromthis original, conflicted document. The objectivity of findings in this final reportremains in question and must be thoroughly reviewed by NTP.

The CERHR final expert panel report contains many errors and inconsistencies . InApril of 2007, CERHR released an interim draft expert panel report (CERHR 2007b);instead of addressing the issues that were brought up regarding the objectivity of theinitial review prepared by Sciences International, the interim draft was even more error-riddled and even less objective that the initial review. In fact, BPA experts who

reviewed this interim draft noted hundreds of errors in documentation, analysis, andinterpretation; they submitted these findings in written comments to the expert panel in June of 2007 (EWG 2007a). These BPA experts found the following:

• 297 potential errors in documentation and analysis of study results, and ininterpretation of the study findings and their significance that are in conflictwith the peer reviewed literature

• 195 instances where the panel assessment is incomplete, including incompletedocumentation of relevant test results or missing justifications for panel assertions

• 48 instances in which the panel inconsistently applied criteria for studyevaluation

Our detailed evaluation of these errors and inconsistencies in the April 2007 draft inincluded as an attachment to this letter (attachment 1). These issues withdocumentation, analysis, and interpretation resulted in an assessment that heavilyfavored industry studies over government and independent studies. In this interimdraft, the expert panel rejected government and independent studies at 3 times therate of industry studies.

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Only some of these issues have been addressed in the final expert panel report that wasreleased in November of 2007 (CERHR 2007a). Significant inconsistencies and errorsremain within this report.

In just one example, the panel reviewed a study from Cagen et al in section 3.2.3.2 inwhich they noted “the lack of much effect with diethylstilbestrol treatment is aweakness”, but they go on to conclude “the panel considered this study adequate andof high utility”. However, in a review of a second study by Cagen et al in section3.2.5.1 in which there were also problems with the positive control, the panel noted“this paper is inadequate for the evaluation process due to absence of response of thepositive control group”. It is unclear why two studies in which there are serious issueswith the positive control are judged so differently.

The expert panel had also noted in the interim draft that they had specific concernswith the use of DMSO as a vehicle for BPA because of its biological activity: in response

to this, we had noted in our public comments from June of 2007 that while the panel singled out DMSO, they did not raise any objections to the use of oil vehicles whichhave been shown to often have background estrogenicity. This issue does not appearto have been addressed at all by the expert panel.

The fact that there are still inconsistencies in the expert panel’s final draft illustrateshow the CERHR expert panel continues to apply arbitrary standards throughout thisevaluation.

The CERHR expert panel failed to consider the significant, high exposures to BPAfor formula fed infants . We would also bring your attention to our recent report onthe presence of BPA in canned infant formula (EWG 2007b). Laboratory studies of

canned infant formula conducted by the Food and Drug Administration (FDA) and acertified commercial laboratory commissioned by EWG reveal that BPA leaches frommetal can linings into formula. EWG analysis of these results revealed the following:

• One of every 16 infants fed ready-to-eat canned formula would be exposed toBPA at doses exceeding those that altered testosterone levels, affectedneurodevelopment, and caused other permanent damage to male and femalereproductive systems (2.0 and 2.4 ug/kg/day- Howdeshell et al 1999, Honma etal 2002- studies cited as ‘adequate’ by the expert panel)

• At the highest BPA levels found in formula (17 parts per billion), nearly two-thirds of all infants fed ready-to-eat formula would be exposed above doses thatproved harmful in animal tests (2.0 and 2.4 ug/kg/day- Howdeshell et al 1999,Honma et al 2002)

Laboratory studies have consistently shown that the most sensitive periods of exposureto BPA are during pregnancy and early life (Maffini 2006). These infant formulafindings reveal that millions of formula fed infants may have daily, sustained exposuresto BPA at levels that have been shown to cause harm in lab animals. These exposurescould be relatively continuous throughout their first 6 months of life and should be

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fully considered by NTP as the agency reaches a determination on BPA’s potential impacts on human health.

New data confirms the relevance of BPA studies that used non-oral routes of administration . At the close of the second expert panel meeting in August of 2007,the CERHR expert panel issued conclusions regarding the potential reproductive anddevelopmental toxicity of BPA. They expressed “some concern” that exposure to BPAmay cause neural and behavioral effects in the developing fetus, infants, and children,but expressed “minimal concern” about other potential health effects. The panel cameto these conclusions by discarding a large number of independent, peer-reviewedstudies linking BPA exposure to mammary and prostate gland lesions, impaired fertility,and ovarian dysfunction in lab animals.

The panel made a decision to include data from only those studies in which BPA wasadministered to lab animals via oral routes of administration. This decision by the

expert panel resulted in the exclusion of many well-conducted studies from academiclabs from across the United States; most importantly, the studies that were excludedhad all passed through the rigors of peer review and had been published in a number of prestigious scientific journals. In addition, this decision to exclude data from studiesin which BPA was administered to lab animals via non-oral routes of administration isnot backed up by the scientific literature and is highly unusual in public healthevaluations.

In fact, a recent study published in the journal Reproductive Toxicology finds that non-oral routes of BPA administration are completely valid in assessing potential healtheffects (Taylor 2008). In this study, scientists administered BPA to neonatal mice byboth oral and subcutaneous routes and found no significant difference in plasma levels

of unconjugated BPA, leading study authors to concluded “the large numbers of BPAstudies that used non-oral administration at very low doses during the neonatal periodshould not be dismissed by scientists or the regulatory community based on route of administration”.

This definitive scientific study refutes the faulty reasoning that the expert panel usedin discarding many valid and scientifically sound studies that linked low dose BPAexposure with adverse health effects such as breast and prostate cancer, infertility, andearly puberty. In fact, the decision by the expert panel to disregard studies that used anon-oral route of administration is just one more in an long list of missteps that hasplagued this review and leads us to question the validity of the conclusions.

BPA experts raise serious concerns about potential human health impacts from BPAexposures . Lastly, we would like to draw your attention to a series of papers that werepublished in the journal Reproductive Toxicology in 2007. These papers were publishedby a group of 38 BPA experts from around the world who systematically reviewed over700 BPA related scientific papers.

In contrast to the CERHR expert panel, none of whom were BPA experts, this group of 38 scientists included many of the world’s most published BPA experts from top

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academic universities and government institutions. These scientists conducted a highlystructured and organized review of the BPA literature that focused on consensusbuilding; their findings were condensed into a consensus statement in which they

concluded:

• The similar effects observed in wildlife and laboratory animals exposed toBPA predict that similar effects are also occurring in humans and

• Much evidence suggests that these adverse effects are occurring in animalswithin the range of exposure to BPA of the typical human living in adeveloped country, where virtually everyone is exposed to measurableblood, tissue and urine levels of BPA that exceed the levels produced bydoses used in the low dose animal experiments” (vom Saal 2007)

The final decision rendered by NTP regarding the reproductive and developmental toxicity of BPA will have repercussions on the public health, and as such, should not be

based on the flawed and biased CERHR evaluation. We urge NTP to recognize the meritsof the review conducted by the 38 BPA experts, in comparison with the CERHRevaluation. The scientific data clearly backs up their concerns about the reproductiveand developmental toxicity of BPA and we urge NTP to recognize the low dosedevelopmental and reproductive toxicity of BPA.

Sincerely,

Anila Jacob, M.D., M.P.H.

Senior ScientistEnvironmental Working Group

References:

1) CERHR 2007a. Expert panel report on Bisphenol A. Bisphenol A evaluation.Center for the Evaluation of Risks to Human Reproduction.

2) CERHR 2007b. Interim draft expert panel report. Bisphenol A evaluation. Centerfor the Evaluation of Risks to Human Reproduction.

3) EWG (Environmental Working Group) 2007a. Failure of CERHR assessment of BPAto meet basic scientific standards. Attachment 1.

4) EWG (Environmental Working Group) 2007b. Toxic plastics chemical in infantformula. Available online at: http://www.ewg.org/node/22233.

5) Honma S, Suzuki A, Buchanan DL, Katsu Y, Watanabe H, Iguchi T. (2002). Lowdose effects of in-utero exposure to bisphenol A and diethylstilbestrol on femalemouse reproduction. Reproductive Toxicology 16: 117-22.

6) Howdeshell K, Hotchkiss AK, Thayer KA, Vandenbergh JG, vom Saal FS. 1999.Plastic bisphenol A speeds growth and puberty. Nature 401: 762-64.

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7) vom Saal FS, Belcher SM, Guillette LJ, Hauser R, Myers JP, Prins GS, WelshonsWV et al. 2007. Chapel Hill Bisphenol A Expert Panel Consensus Statement:Integration of mechanisms, effects in animals and potential impact to human

health at current exposure levels. Reproductive Toxicology 24: 131-38.8) Maffini MV, Sonnenscheim C, Soto AM. 2006. Endocrine disruptors andreproductive health: the case of bisphenol A. Molecular and CellularEndocrinology 8: 254-55.

9) Taylor JA, Welshons WV, vom Saal FS. 2008. No effect of route of exposure (oral;subcutaneous injection) on plasma Bisphenol A throughout 24 hr afteradministration in neonatal female mice. Reproductive Toxicology 25(2): inpress.

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ATTACHMENT 1

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August 6, 2007

Dr. Michael D. ShelbyDirectorCenter for the Evaluation of Risks to Human ReproductionNational Institute of Environmental Health ServicesDepartment of Health and Human ServicesP.O. Box 12233MD EC-32Research Triangle Park, NC 27709

Re: Failure of CERHR Assessment of BPA to Meet Basic Scientific Standards. Supplemental Comments on the Interim Draft NTP-CERHR Report on the Reproductive and Developmental

Toxicity of Bisphenol A.

Dear Dr. Shelby:

You must be aware of the publication last week of a consensus statement on BPA signed by 38independent specialists in BPA toxicity from around the world. These scientists concluded thatBPA presents a clear risk to human health. The statement and the comprehensive review papersthat accompany it underscore, by way of contrast, the hopeless corruption of the ongoingreview of BPA being conducted at your Center (the NIH Center for the Evaluation of Risks toHuman Reproduction, or CERHR).

The Environmental Working Group has conducted a detailed review of the comments by 9

scientists conducting BPA research at 6 laboratories in the U.S. and E.U., submitted to you inresponse to CERHR’s interim draft BPA assessment (Vandenberg et al. 2007; Schonfelder 2007;Prins 2007; vom Saal 2007; Welshons 2007; Zoeller 2007). Our review shows that the CERHRpanel’s review of BPA utterly fails to meet basic, universally understood standards for scientificreviews and data quality, including those laid out in NIH policy and federal law. Thesestandards require that assessments be accurate, unbiased, consistent, complete, and conductedby those with the necessary expertise and independence to ensure objectivity. Instead, ourreview of scientists’ comments reveals that the CERHR assessment may contain nearly 300 errorsof fact and interpretation; is biased, inconsistent, incomplete; and clearly fails to meet themost basic scientific standards. Among our findings, which are detailed in the attached table,are that the CERHR assessment is:

• Inaccurate. 297 errors: Reviewers identified 297 potential errors in documentation and

analysis of study results, and in interpretation of the study findings and theirsignificance, in conflict with the peer reviewed literature.

• Incomplete. 195 instances of incomplete study reviews: Reviewers documented 195instances where the panel assessment is incomplete, including incompletedocumentation of relevant test results or missing justifications for panel assertions.

• Inconsistent. 48 basic inconsistencies: Reviewers documented 48 instances in whichthe panel inconsistently applied criteria for study evaluation.

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• Biased . The assessment heavily favors industry studies over government andindependent studies. In its most recent assessment, the Panel rejected government andindependent studies at 3 times the rate of industry studies (Vandenberg et al. 2007).

Consider also the following, striking differences between the CERHR panel and the BPA panel which released the consensus statement last week (this panel convened in Chapel Hill, NC, andis referred to as the “Chapel Hill panel” for purposes of this document). Both panels are fundedby NIH, but are different in almost every other aspect:

• The objectivity of the CERHR assessment is compromised by the panel’s lack of specialization in BPA science. The CERHR panel contains few with advance knowledgeBPA through their own study, and none for whom the chemical has been a primaryfocus of their work. The panel has just 12 members to assess over 500 BPA-relatedpapers. The Chapel Hill panel includes 38 of the world’s most published BPA expertsfrom top universities and government institutions.

• The accuracy and consistency of the CERHR assessment is compromised by thepanel’s organizational structure and their failure to communicate: Within the CERHRpanel study reviews were conducted independently by each scientist, prompting onpanel member to state in a recent article in Risk Policy Report that “one thing that hasplagued this review is that each reviewer was assigned a bunch of papers, and theyreviewed them without any other input.” Additionally, in the middle of the panel review process CERHR fired the consulting firm managing the project over concernsabout potential conflicts of interest, and a director of the Center was replaced forreasons not disclosed to the public, creating significant changes in management in themidst of the review. In contrast, the Chapel Hill review was conducted in a highlystructured, organized manner: 4 breakout groups were each asked to address 4 critical issues related to BPA, and only if there was consensus among all 4 groups were

responses incorporated into the final consensus statement.

• The objectivity of the CERHR assessment is compromised by not having beensubjected to a standard peer review: The assessment of the CERHR panel has not beensubjected to standard peer review, and contains nearly 300 errors of fact andinterpretation according to BPA specialists. The initial draft was prepared by thecontractor mentioned above who was fired over concerns about possible conflict of interest, calling into question the validity of the contractor’s work on this assessment.In contrast, the work of the Chapel Hill panel was subjected to standard andcomprehensive internal and independent external peer review.

We question the Center’s ability to produce a scientifically sound document from this processwhen the comments you have received from BPA experts include statements calling into

question the ability of the panel to meet the most basic scientific standards:

• “ Is the panel purposefully misrepresenting data or grossly misunderstanding it?"(Vandenberg et al. 2007)

• “There are two general aspects [of the assessment] which to me represent the antithesisof valid science.” (Welshons 2007)

• “The criteria established by the panel are arbitrary .” (Vandenberg et al. 2007)• “highly curious” [comment on an uninformed critique] (Zoeller 2007)

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• “ If one were seeking to establish a mechanism that would be virtually certain tounderestimate the potential for harm to be caused by a chemical, the CERHR mechanism isexactly the process that they would want to establish to achieve that objective.” (vomSaal 2007).

The public has now paid for two assessments of BPA toxicity, the one conducted by your panel,which has failed to meet basic standards for the conduct of scientific reviews; and a peerreviewed assessment by a panel of BPA specialists (the Chapel Hill panel), which issued its final assessments last week. If you proceed with the CERHR panel process the public will have to payfor this assessment four times all told, because your assessment will require both a thoroughpeer review, and a complete revision from top to bottom of the current, corrupted document.

Instead of trying to salvage what you have, we urge you to dissolve your current panel andadopt the recommendations of the Chapel Hill panel issued last week, which is peer reviewedand meets established scientific standards. We would also urge you to invest your Center’sresources in conducting studies and forwarding policies that will help reduce the public’sexposures to this chemical that so clearly poses risks to human reproduction.

EWG has recently requested data from formula manufacturers on BPA levels in their products, tohelp fill data gaps left by FDA’s meager, 14-sample study. We have also completed a newanalysis of infant formula showing, based on available data, that babies who drink ready-to-feed formula can easily be exposed to BPA in amounts that exceed those found harmful inlaboratory studies, and are exposed to BPA at greater levels than any other segment of thepopulation. Our correspondence to formula manufacturers and our new infant formula analysisare available on our website at www.ewg.org.

BPA poses risks to human reproduction and is an urgent public health issue. We urge you tolead on this issue instead of spending energy to rectify a corrupt product from a corruptprocess.

Sincerely,

Anila Jacob, M.D., M.P.H Jane HoulihanSenior Scientist Vice President for Research

References

All references listed below except CHCS (2007) are currently available in pdf form at

http://cerhr.niehs.nih.gov/chemicals/bisphenol/pubcomm-bisphenol.html.

CHCS (Chapel Hill Consensus Statement). 2007. vom Saal FS, Akingbemi BT, Belcher SM,Birnbaum LS, Crain DA, Eriksen M, Farabollini F, Guillette LJ, Hauser R, Heindel JJ, Ho SM, HuntPA, Iguchi T, Jobling S, Kanno J, Keri RA, Knudsen KE, Laufer H, LeBlac GA, Marcus M, McLachlnJA, Myers JP, Nadal A, Newbold RR, Olea N, Prin GS, Richter CA, Rubin BS, Sonnenshein C, SotoAM, Talsness CE, Vandenbergh JG, Vendenberg LN, Walser-Kuntz DR, Watson CS, Welsons WV,Wetherill Y, Zoeller RT. Integration of Mechanisms, Effects in Animals and Potential to ImpactHuman Health at Current Levels of Exposure. Reproductive Toxicology 24(2): 2007, in press.

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Prins GS. 2007. Letter from Dr. Gail S. Prins, College of Medicine, Department of Urology,University of Illinois at Chicago, to Dr. Michael Shelby, National Institute of Environmental Health Sciences. June 19 2007.

Schonfelder G. 2007. Letter from Dr. Gilbert Shonfelder, Institute of Clinical Pharmacology andToxicology, Campus Benjamin Franklin, Charity University Medicine Berlin, to Dr. Michael Shelby, National Institute of Environmental Health Sciences. Received June 20 2007.

Vandenberg LN, Maffini MV, Rubin BS, Soto AM. 2007. Response to the Interim Draft of theNTP-CERHR Report on the Reproductive and Developmental Toxicity of Bisphenol A. TuftsUniversity School of Medicine. Department of Anatomy and Cellular Biology. Boston, MA. June20, 2007.

Vom Saal 2007. Comments on the Interim CERHR Report on Bisphenol A (April 2007). Commentsfrom Dr. Freferick vom Saal, Division of Biological Sciences, University of Missouri, Columbia,MO, to the National Institute of Environmental Health Sciences. Received June 20, 2007.

Welshons WV. 2007. Comments from Dr. Wade V. Welshons, Department of Biological Sciences,Verterinary Medicine, University of Missouri, Columbia MO, to Dr. Michael Shelby, National Institute of Environmental Health Sciences. June 20 2007.

Zoeller RT. 2007. Letter from Dr. R. Thomas Zoeller, Biology Department, Morrell Science Center,University of Massachusetts Amherst, to Dr. Michael Shelby, National Institute of Environmental Health Sciences. May 19 2007.

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ATTACHMENT

Table. CERHR Assessment of BPA fails to meet basic scientific standards

for data quality and objectivity. A summary of comments to CERHR from independent BPA scientists

Scientificstandards for objectivityand dataquality

CERHR assessmentfails to meet thestandard in this

area…

Failure of CERHR assessment according to BPAexpert review comments

Reviewer (reference)

AccurateErrors in interpreting

significance of studyfindings

Panel mistakenly concluded that study effect is of unknown relevance (Ho et al, Cancer Research 2006),when a substantial literature demonstrates itsrelevance, as described in Prins (2007).

Prins 207

AccurateErrors in conclusions

on statistical analysis

Panel deemed a study "inadequate based oninappropriate statistics" with no justification for thisconclusion, even though the study relied on

accepted, rigorous statistical analysis (one-wayanalysis of variance) unlikely to merit criticism if ithad been assessed by the panel (Zoeller et al. 2005).

Zoeller 2007

AccurateErrors in

understanding studyimplications

Panel criticized a study for failing to include apositive control (Zoeller et al. 2005), but failed torecognize that the study provided first-ever data onthe effect measured, so no positive control compound would be available. The reviewer calls thepanel's uninformed critique "highly curious."

Zoeller 2007

AccurateErrors in

understanding studyimplications

Panel failed to understand a major finding of acritical thyroid study, which showed that BPAproduced a profile of effects that were consistentwith the interpretation that BPA acts as a selectiveindirect antagonist on the beta thyroid hormonereceptor (Zoeller et al. 2005).

Zoeller 2007

Accurate

Failure to correct

errors of fact noted bycommenters

Factual errors in prior draft of document, noted inreview comments, were not corrected in this draft. vom Saal 2007

UnbiasedApparent bias of errors

to favor industry-funded assessments

Reviewer notes apparent bias in distribution of errors in panel assessment that would favor industry-funded assessments and would lead to an assessmentunderestimating potential for harm from BPAexposures.

vom Saal 2007

Expertise,Accurate,Complete,Reliable

Exclusion of BPAspecialists from thepanel

Reviewer notes that errors and lack of attention tocritical issues are likely related to the panel's relativelack of expertise in BPA research and lack of familiarity with BPA-related scientific literature.

vom Saal 2007

Consistent,Complete,Accurate

Failure to establishand documentdefensible criteria forassessing the BPA

literature

Reviewer notes that criteria established by thepanel for assessment of study utility are arbitrary,many without explanation or merit.

Vandenberg etal. 2007, pg 2

Accurate,Expertise,Reliable

Failure to followaccepted scientificstandards for reviewand study evaluation

Reviewer notes that two fundmental aspects of theassessment are the "antithesis of valid science:"Failure to institute standard peer review of panel findings that would ensure accuracy; failure to applyestablished criteria for inclusion of and interpretingresults from control animals that are critical tounderstanding BPA study findings.

Welshons

Consistent,Accurate

Inaccurate andincomplete assessmentsof study findings and

Reviewer notes that developmental toxicity datasection of the panel's assessment is "filled withinconsistencies and inaccurate statements."

Vandenberg etal. 2007, page

44

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implications

AccurateInaccurate

representation of study

findings

Reviewer notes that the panel is either"purposefully misrepresenting data or grosslymisunderstanding it," and that summaries provided

for some studies are "completely inaccurate and donot represent the experiments conducted and/or theresults obtained."

Vandenberg et

al. 2007, page 8

Accurate,Reliable

Inaccurateinterpretationsresulting from review of studies by panel without the requisitespecialized knowledge.

Reviewer notes that the panel makes "fundamental errors due to ignorance" in its assessments of BPAstudies, and comments that the panel reviews do notmeet journal standards for peer review.

Welshons

AccurateLack of understanding

of endocrine research

Panel has failed to understand the basicrequirement in endocrine research for study controlsand the implications of results in control animals.The reviewer calls this failing "beyond mycomprehension" given the widely understood needfor negative controls in positive experiments, andpositive controls in negative experiments in

endocrine research. The reviewer notes that inmodern endocrine research, many of the studieswithout controls that the panel considers acceptablewould, in fact, be unpublishable.

Welshons

Complete,Accurate

Lack of scientificallysound reasoning indiscounting importantstudy observations

Reviewer notes that observations in Zoeller et al.(2005) are discounted by the panel for reasons thatare unclear or do not appear to be valid.

Zoeller

AccurateMisrepresentation of

study significanceReviewer states that panel systematically

misrepresented significance of metabolic studies.Vandenberg et

al. 2007, page 8

Accurate

Errors inunderstanding of needfor consistency in

control studies

Panel mistakenly called an industry-funded study(Tyl et al. 2003) a "replication" of an independentstudy (Nagel et al. 1997) which found effects, eventhough the Tyl study used a feed known to becontaminated with estrogenic compounds that maskthe effects of BPA. It is standard scientific practicein replication to use identical feed to the studybeing replicated. This study found no BPA-relatedeffects.

vom Saal 2007

Accurate

Errors in interpretingscientific literature onanimal feedcontamination andinfluence on BPA studyfindings

The panel failed to consider that two industry-funded studies of BPA which failed to find effectsused animal feed known to be contaminated withestrogenic substances that can completely mask theeffects of BPA (Thigpen et al. 2003 (Comp Med)). Thepanel also raised concerns about feed contaminationin a study relying on feed proven to be free of thecontaminants at any levels that would affect studyfindings (vom Saal et al. 1997, PNAS; Timms et al.2005, PNAS; Richter et al. 2007, EHP; Howdeshell etal. 1999, Nature; Palanza et al. 2002, EHP).

vom Saal 2007

Accurate

Errors in interpretingthe role of positivecontrols in studyvalidation

The panel failed to note concerns that an industry-

funded studies showing no effects from BPA alsofound no effects in its positive control animals.Positive control animals are exposed to a substanceknown to produce the same effect scientists areseeking to explore with the study's test substance. Itis standard scientific practice to consider a study ashaving failed if the positive control animals fail toshow a response, because this failure means that thestudy design or test conditions would not allow thestudy to reveal effects from the test substance,either.

vom Saal 2007;Schonfelder

2007

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Reliable,Accurate

Errors ininterpretationstemming from lack of panel members whospecialize in BPAresearch

Reviewer notes that failure to integrate the rangeof avialable data on animal feed contamination, andother related failings of the panel, likely stems fromthe fact that panel composition largely excludes

members who specialize in BPA research.

vom Saal 2007


study results as "falsepositives."

The panel categorized as potential "false positives"findings replicated in many experiments.

vom Saal 2007


relevance of exposureroute.

The panel categorized studies with continuousinstead of episodic exposures useless in theirevaluation of BPA toxicity in the complete absenceof data to support that conclusion, and withoutrecognizing studies which support the continuoussources of exposure for humans, including BPAcontamination in dust and air. The panel justifiedexclusion of these studies in part by expressingconcerns that injection results in excessunmetabolized BPA in the bloodstream relative tooral exposures, but failed to recognize that this form

of exposure mimics human fetal exposures, and alsofailed to note the substantial body of literatureshowing unmetabolized BPA in human tissues andfluids that further support the relevance of continuous exposure studies.

vom Saal 2007

Accurate

Errors in interpretingrelevance of exposureroute and potential for"false positives."

The panel categorized as potential "false positives"findings replicated in many experiments. The panel categorized studies with continuous instead of episodic exposures useless in their evaluation of BPAtoxicity in the complete absence of data to supportthat conclusion, and without recognizing studieswhich support the continuous sources of exposure forhumans, including BPA contamination in dust andair. The panel justified exclusion of these studies inpart by expressing concerns that injection results inexcess unmetabolized BPA in the bloodstream

relative to oral exposures, but failed to recognizethat this form of exposure mimics human fetal exposures, and also failed to note the substantial body of literature showing unmetabolized BPA inhuman tissues and fluids that further support therelevance of continuous exposure studies.

vom Saal 2007

AccurateErrors in analysis of

study findings.

The panel notes a "lack of clarity" in mouse strainin a study deemed of limited usefulness even thoughthe mouse strain is clearly stated in the study ("CF-1mice were purchased from Charles RiverLaboratories…", vom Saal et al 1998).

vom Saal 2007


study findings.

The panel notes a need for consideration of testisweight in a study deemed of limited usefulness, eventhough the study clearly documents the effect of testis weight and uses it as the basis for analysis

(vom Saal et al 1998).

vom Saal 2007


study findings.

The panel inaccurately characterizes study findings,stating that studies did not find statisticallysignificant effects on the prostate at 0.020 mg/kgbw/day, when the studies clearly report statisticallysignificant effects at that dose (Nagel, 1997 #6; vomSaal, 1998 #187). [unclear - did both studies showeffect at this dose?]

vom Saal 2007


study scope

Panel mistakenly concluded that study implicationsare exclusive of estrogenic endpoints when studyauthors clearly state otherwise (Nagel et al. 1997,

vom Saal 2007

E - 57





vom Saal et al. 1998).


the nature of the study

Panel fails to understand the study analysis(Howdeshell et al. 1999, Nature), which relied on alitter-based analysis consistent with a significant

literature on the effects of intrauterine position onpup response, but which was confused by the panel reviewer as potentially a pup-based analysis anddeemed of marginal utility.

vom Saal 2007


study design

The panel noted confusion on whether the pup orlitter was used as the statistical unit for analysis fora study (Gupta 2000 PSEBM) when the author clearlystates that 15 individual pups from 15 separatelitters were used in the analysis.

vom Saal 2007

CompleteExclusion of important

review findings

The panel included findings from a publishedcritique of Gupta (2000, PSEBM) but failed to notethat the model used as the basis for the critique(Elswick et al. 2000) was deemed "misleading","illogical" and "flawed" by the NIH Low Dose ReviewPanel.

vom Saal 2007

Accurate

Lack of understandingof statistical significance inscientific studies

Panel rejected studies for evaluation based solely

on judgment that the studies provided "aninsufficient number of animals for rigorous statistical analysis," reducing "confidence in the results." Thenumber of study animals required is based on poweranalysis and is small when the expected magnitudeof the effect is great, where small numbers of animals can yield statistically significant results. NIHguidance requires the use of the fewest animalspossible to achieve statistical significance. With theirconclusion the panel ignores basic, widely accepted,NIH-endorsed statistical principles behind the designof toxicological studies.

vom Saal 2007;Vandenberg et

al. 2007

Accurate

Errors ininterpretation of toxicological implications andvalidity of study design.

The panel inappropriately excluded some studiesbecause of their use of DMSO as a vehicle toadminister BPA to test animals, failing to note thatDMSO exposures are far below those that are knownto be biologically active, that DMSO did not produceeffects in control animals, that DMSO is notassociated with effects related to those underinvestigation for BPA, and that DMSO has been thevehicle of choice for a wide range of related studies.


ConsistentInconsistent

evaluation of studydesign

The panel erroneously dismissed studies using DMSOas an administration vehicle, but does not similarlydiscuss or assess a wide range of other potentiallyproblematic administration vehicles in other studies,including corn oil that can be contaminated withestrogenic compounds that might mask the effects of BPA.



application of evaluation criteria

The panel proposed a hypothesis that findings in aBPA study they reviewed could be due to interactionsbetween BPA and the administration vehicle (olive

oil), but fails to note the same concern with respectto the other 13 studies evaluated that use olive oil as the administration vehicle.

Vandenberg et

al. 2007, pg 3

Consistent,Complete

Inconsistentapplication of evaluation criteria

The panel found fault with injection as dosingvehicle but failed to note concerns with other dosingvehicles, including stress induced by oral gavage thathas been shown to mask the effects of low doses of hormones, and inaccuracies stemming from non-uniform feed and water exposures.


AccurateArbitrary and

capricious choice of The panel arbitrarily chose 7 as an appropriate

sample size for animal experiments withoutVandenberg etal. 2007, pg 5

E - 58





study evaluationcriterion

justification. The reviewer notes that this "capriciouschoice... is contrary to the understanding of statistical power and sample size analysis, whichshould be done by the experimenter a priori, i.e.before conducting the experiment."


non-monotonic doseresponses

The panel criticized 12 studies as having "non-doserelated" results, demonstrating a lack of understanding of the widely observed non-monotonicresponses characteristic of many endocrine studiesand widely reported in the scientific literature,having been observed in 40% of the studies with adesign that would allow for its detection.


Consistent,Complete

Inconsistentapplication andinadequatedocumentation of evaluation criteria

The panel dramatically changed study evaluationfindings between drafts of the assessment but failedto document changes in evaluation criteria thatresulted in these alterations, and failed to note if evaluation criteria were established at all in advanceof the initial review.


Consistent

Inconsistent


In Section 3 of the assessment the panel criticized43% of all studies reviewed for what the panel

perceived as an inadequate sample size (n<7), butthen inexplicably failed to note this same concern in11% of other studies reviewed that also had an n<7.

Vandenberg etal. 2007, pg 7;

Schonfelder2007



The panel rejected a number of studies fromconsideration because of their use of DMSO as anadministration vehicle, but inexplicably accepted twoother studies that also used DMSO, and for one,supplied by the plastics industry, failed to even noteit as a concern.

Vandenberg etal. 2007, page 8

Accurate,Complete

Inaccurateinterpretation of significance of pharmacokinetic studies

The panel failed to note concentrations of BPA incritical tissues in pharmacokinetic study as thedetermining factor for toxicity, or significance of study for fetal exposures.


Accurate

Inaccurate

representation of studyfindings

In review of a study of anogenital distance, thepanel noted that "study authors concluded that theendpoint was not affected by prenatal, lactional and/or post-wearning exposures to bisphenol A,"when, in fact, the study authors did not draw thisconclusion (Rubin et al. 2001).


Accurate

Lack of understandingof effect of dose andtiming on endocrinestudies

The panel noted a lack of reproducibility associatedwith a studies of LH serum levels, but fails torecognize that BPA scientists would not attempt toreproduce endocrine effects in female rats from fetal development until weaning by studying the sameendocrine effects in male rats during puberty (Rubin2001, Akingbemi 2004).


AccurateLack of understanding

of dose response

The panel cites concerns about a "lack of doseresponse releationships" in a study that found effectsonly at the highest dose (Rubin et al. 2001), asituation in which consideration of dose response isirrelevant.


AccurateMisrepresentation of

study dosesThe panel erroneously lists doses included in a

study of BPA effects in mammary glands (Murray2007).


Accurate,Complete

Misrepresentation of studies and failure todocument

In a discussion of a study for which the panel failedto provide a reference, the panel noted a mammarygland finding that has never been observed in astudy (mammary gland alterations in pubertal andadult mice). They appear to be referencing a studyconducted on gestational day 18 mice, but if so,mistakenly note that the study used subcutaneoussilastic implants for administration (Vandenberg et


E - 59





al. 2007).



Four studies were considered adequate by the panel even though their positive controls failed, a clear,widely accepted sign of a failed experiment.


10

AccurateFailure to recognize

key components of study design

The panel failed to adhere to standard scientificpractice in considering the need for and results fromnegative controls in endocrine studies, and inmultiple instances ignored common potential sourcesof contamination such as phytoestrogens in feed,estrogenic activity of oil vehicles, plastics in theanimal environment, and contamination in tissueculture experiments.


10



The panel inconsistently treated the lack of information about feed in study documentation,listing it as a weakness in only 14% of the studiesreviewed that failed to document it.


10

Accurate,Complete

Failure to recognizekey components of

study design

In studies with oil vehicles the panel failed to noteas a weakness failure to include negative controls,and the panel failed to discuss findings in negativecontrols when they were included. (Note that for

none of the oil vehicle studies including negativecontrols were the results in those animals discussedby the panel.)


10

Accurate,Complete

Failure to recognizekey components of study design

The panel failed to recognize the documentedpotential for contamination from polycarbonatecages, bottles, and other plastics used in BPAexperiments.


10

UnbiasedBias in study

evaluations

In this most recent draft of the assessment, thepanel changed many of their evaluations of studyadequacy, and found many more studies inadequatethan had been proposed in the previous draft, but inmaking these changes they rejected independentlyfunded studies at three times the rate of industryfunded studies.


11

Unbiased,Complete

Bias in studyinclusion, Incompletelist of studies forassessment

In the most recent assessment draft the panel stripped from the report, without explanation, theresults description for 38 studies that were includedin the previous draft. Only one of these was fundedby industry, and 32 of these were originallyconsidered adequate.


Unbiased

Potential bias in studyinterpretation based onperception of quality of the research group

Panel review notes strengths of studies to include"the expertise of the group" and "well conducted bya respected lab," indicating reviewers' preferences forlabs and calling into question the objectivity of thereview


36, 56

Reliable

Reliance onupublished industrystudies and translationsnot subjected to peerreview

Reliance on unpublished industry studies notsubjected to peer review process, and reliance ontranslations of select parts of studies published inJapanese, provided to the panel by the AmericanPlastics Council

Vandenberg etal. 2007, page32, 36, 41, 53,

57

Accurate

166 instances of errorsin documentation,analysis, interpretation,and evaluation of significance

Reviewer identified 166 potential errors in

documentation of study results, and in interpretationof the study findings and their significance, inconflict with the peer reviewed literature. These aredescribed in the detailed section of this reviewer'scomments. Data presented in the comment'ssummary section are, in contrast, documentedindividually in this table.

Vandenberg etal. 2007, pg 16-

60

Complete

95 instances of incompletedocumentation andconsideration of study

Reviewer documented 95 instances where panel assessment appears to be incomplete, includingincomplete documentation of relevant test results or justification for panel assertions. These are


60

E - 60





findings,interpretations, andconclusions

described in the detailed section of this reviewer'scomments. Data presented in the comment'ssummary section are, in contrast, documentedindividually in this table.

Consistent39 instances of

inconsistent applicationof evaluation criteria

Reviewer documented 39 instances in which thepanel inconsistently applied criteria for studyevaluation. These are described in the detailedsection of this reviewer's comments. Data presentedin the comment's summary section are, in contrast,documented individually in this table.


60

Accurate,Complete

88 instances of panel failing to considerabsence of testing forbackgroundcontamination

Reviewer documented 88 instances of the panel failing to consider the potential effect of contamination in studies which did not test forestrogenicity of administration vehicle. These aredescribed in the detailed section of this reviewer'scomments. Data presented in the comment'ssummary section are, in contrast, documentedindividually in this table.


60

Accurate

Misunderstanding of reliable measure for

reproduction status intest animal

Panel criticized study for not documenting anindicator of mating that is not considered reliable for

the test animals (the researcher subsequentlyprovided the panel with the appropriate measure).

Schonfelder

2007

AccurateFailure to find basic

information in studydocumentation

Panel criticized study for not documenting animal numbers when the study documentation clearlystated this information.

Schonfelder2007

AccurateFailure to find basic

information in studydocumentation

Panel noted that they estimated study findings froma graph, when the study findings were provided infull in the study documentation.

Schonfelder2007

AccurateError in understanding

standard statistics usedin study design

Panel criticized a study as having "too few animalsto reach a conclusion with certainty" when the studyidentified statistically significant effects and usedthe appropriate number of animals needed given theexpected magnitude of the effect.

Schonfelder2007

E - 61




Exhibit “F”



http://slidepdf.com/reader/full/felix-lozano-lawsuit 63/82F - 63










Exhibit “G”



http://slidepdf.com/reader/full/felix-lozano-lawsuit 67/82G - 67






















Exhibit “H”




Available online at www.sciencedirect.com

Toxicology Letters 176 (2008) 149–156

Bisphenol A is released from polycarbonate drinking bottles and mimicsthe neurotoxic actions of estrogen in developing cerebellar neurons

Hoa H. Le, Emily M. Carlson, Jason P. Chua, Scott M. Belcher ∗

Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, 231 Albert Sabin Way,

Cincinnati, OH 45267-0575, United States

Received 27 September 2007; received in revised form 9 November 2007; accepted 9 November 2007

Available online 19 November 2007

Abstract

The impact of endocrine disrupting chemical(EDC) exposure on human health is receiving increasingly focused attention. The prototypical EDCbisphenol A (BPA) is an estrogenic high-production chemical used primarily as a monomer for the production of polycarbonate and epoxy resins. It

is now well established that there is ubiquitous human exposure to BPA. In the general population, exposure to BPA occurs mainly by consumption

of contaminated foods and beverages that have contacted epoxy resins or polycarbonate plastics. To test the hypothesis that bioactive BPA was

released from polycarbonate bottles used for consumption of water and other beverages, we evaluated whether BPA migrated into water stored in

new or used high-quality polycarbonate bottles used by consumers. Using a sensitive and quantitative competitive enzyme-linked immunosorbent

assay, BPA was found to migrate from polycarbonate water bottles at rates ranging from 0.20 ng/h to 0.79 ng/h. At room temperature the migration

of BPA was independent of whether or not the bottle had been previously used. Exposure to boiling water (100 ◦C) increased the rate of BPA

migration by up to 55-fold. The estrogenic bioactivity of the BPA-like immunoreactivity released into the water samples was confirmed using

an in vitro assay of rapid estrogen signaling and neurotoxicity in developing cerebellar neurons. The amounts of BPA found to migrate from

polycarbonate drinking bottles should be considered as a contributing source to the total “EDC-burden” to which some individuals are exposed.

© 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: BPA Endocrine disruption; Estrogen; Neurotoxicity; Non-genomic

1. Introduction

Bisphenol A (BPA, 2,2-bis (4-hydroxyphenyl) propane; CAS

RN 80-05-7) is a high-production chemical used in the manu-

facture of numerous consumer goods and products. Bisphenol A

Abbreviations: BPA, 2,2-bis (4-hydroxyphenyl) propane; EDC, endocrine

disrupting chemical; ELISA, enzyme-linked immunosorbent assay; HDPE,

high-density polyethylene; HPLC, high-performance liquid chromatography;

LDH, lactate dehydrogenase; PC, polycarbonate. Thesestudiesweresupported byNIH grant R01-ES015145awarded to SMB;

Emily Carlson, and Jason Chua were fellows of the University of Cincinnati

College of Medicine’s Summer Undergraduate Research Program. The study

sponsors had no involvement in the design of this study, the collection, analysis

or interpretation of data, the writing of this report, nor the decision to submit

this manuscript for publication.∗ Correspondingauthor at: Departmentof Pharmacologyand Cell Biophysics,

University of Cincinnati College of Medicine, 231 Albert Sabin Way, PO Box

670575, Cincinnati, OH 45267-0575, United States. Tel.: +1 513 558 1721;

fax: +1 513 558 4329.

E-mail address: [email protected] (S.M. Belcher).

has well characterized estrogenic and other endocrine disrupting

activities that are mediated via multiple molecular mechanisms

(Wetherill et al., 2007). In 2004, the estimated production vol-

ume of BPA in the United States was ∼2.3 billion pounds

(CERHR, 2007). Of the 1.9 billion pounds of BPA used in the

US in 2003, nearly 3/4 was used to manufacture polycarbonate

resins that were in turn used to manufacture various consumer

products including polycarbonate containers for storage of foodsand beverages (CERHR, 2007).

Because of BPA’s high volume production and extensive use

in plastics, there is widespread environmental contamination and

well-documented human exposure to BPA. While not diminish-ing theimportance of BPA pollutants in marine, aquatic, andsoil

ecosystems (for review see Crain et al., 2007), recent studies

have demonstrated that there is wide-spread BPA contamina-

tion of most individuals in industrialized human populations

(Calafat et al., 2005; Vandenberg et al., 2007). The detection of adverse health effects in a number of laboratory animal models

upon exposure to environmentally relevant doses of BPA that

correspond to those observed in humans, strongly supports the

0378-4274/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.toxlet.2007.11.001

H - 75




150 H.H. Le et al. / Toxicology Letters 176 (2008) 149–156

idea that the endocrine disrupting activities of BPA contribute

to adverse effects on human health (reviewed in Richter et al.,

2007).

In the laboratory setting, biologically active and environ-

mentally relevant levels of BPA were shown to leach from

polycarbonate (PC) flasks upon autoclaving (Krishnan et al.,

1993), from used PC rodent-housing containers (Howdeshell et

al., 2003), and from various other forms of polycarbonate plas-tics and BPA-containing resins (Kang et al., 2006; Vandenberg

et al., 2007). Because the major source of human exposure to

BPA in the general population is likely through consumption

of contaminated foods and beverages that have contacted epoxy

or PC resins (Kang et al., 2006), we evaluated whether BPA

migrated from new or used high-quality PC bottles that are

commonly used by consumers for storage of water and otherbeverages. The exposures and mild treatments used in this study

were designed to mimic conditions representative of normal

consumer usage, including typical use in outdoor recreation set-

tings. Along with characterizing the rate of BPA liberation into

water at room temperature, the effect of short-term exposure to

hot (100 ◦C) water was determined. A sensitive and quantitativecompetitive ELISA employing a BPA-specific monoclonal anti-

body was used to determine relative concentrations of BPA thatwere leached into water samples from newly purchased polycar-

bonate bottles and those subjected to normal use by consumers.

The estrogenic bioactivity of the BPA-like immunoreactivity

released into the water samples was demonstrated with an in

vitro assay of rapid estrogen-signaling and neurotoxicity in

immature cerebellar neurons (Belcher et al., 2005; Wong et al.,

2003).

2. Materials and methods

2.1. Reagents

Bisphenol A (CAS RN 80-05-7; purity grade >99%; 23,965-8; lot no. Cl

03105ES) and dimethyl sulfoxide (Chromasolv Plus, for HPLC≤99.7%; batch

no. 00451HE) were purchased from Sigma–Aldrich (St. Louis, MO). HPLC-

grade water (W5sk , lot no. 056618) and methanol (A452sk lot no. 061495) were

purchased from Fisher Scientific (Fairlawn, NJ) and used for all washes, dilu-

tions, and sample preparations. New PC and high-density polyethylene (HDPE)

bottles (32ounceloop-top; Nalgene,Rochester, NY)were obtaineddirectlyfrom

a national recreation supply retailer (Campmor, Paramus, NJ). Used PC bottles

were the same model as the new PC bottles that were collected from anony-

mous donors from Rockquest Climbing Gym (Cincinnati, OH). All donated

bottles were described as having been used under normal conditions for 1 to

9 years, although specific period of use was undetermined. All used bottles

showed various degrees of visible external and internal wear including superfi-

cial scratches, pitting, and opaque discoloration. All bottles had an approximateinternal surface area of 478 cm2 (bottle dimensions were length 17.2 cm; cir-

cumference 27.8cm; diameter 8.9 cm). Water samples collected were stored in

new 60 ml glass bottles (Fisher) fitted with Teflon® faced polyethylene lined

caps.

2.2. Bottle washing and rinsing

A standardized washing/rinsing procedure was used for all bottles tested.

Initially, each piece was rinsed with at least 1 l of distilled water to remove any

dust or residue, andthenwashed with a soft-nylon bristlelaboratory bottlebrush

with a half-strength solution (5 g/l) of alconox powdered precision cleaner (cat

no. 1104; Alconox White Plains, New York). This wash was followed by six

rinses each with ∼1 l distilled water, and then two rinses with∼100ml HPLC-

grade water. Each piece was then rinsed three times with ∼100ml of HPLC-

grade methanol, inverted and allowed to air dry in a laminar flow hood before

use. To ensure removal of superficial contaminants from previous incubations,

priorto repeated analysis the complete washing/rinsing protocol was performed.

Autoclaved polypropylene pipette tips were used for all liquid transfers

≤1 ml. Larger volume liquid measurements were made in previously unused

glass pipettes or graduated cylinders that were washed and rinsed as described

above. All additional labware that would directly or indirectly come into con-tact with samples was purchased new and pre-rinsed three times with 100%

HPLC-grade methanol.

2.3. Exposure, experimental design and sample collection

Three replicate experiments for each bottle were conducted to determine if

BPA was released into the water stored in new or used PC drinking bottles, or

new HDPE bottles. For each sample, 100 ml of HPLC-grade water was added to

a bottleon day0. Filledbottles were rotated on a cell culture roller bottlesystem

(Wheaton) for indicated times up to 7 days (168 h) at room temperature (22 ◦C).

Bottles were rotated with the purpose of mimicking the motion of water during

normal usage, and to ensure that the bottle’s internal surface was in continuous

contact with the water sample. The water contacted approximately 478 cm2

of the surface area in each of the bottles during rotation. At the same time as

incubationswere initiated, ondays1, 3,and 5,1 ml watersamples were collectedand transferred to new glass collection bottles using polypropylene pipette tips.

On day7, a 50-mlwatersample wascollected foranalysis by direct transfer into

the collection bottle. Immediately following sample collection, all water bottles

were washed and rinsed as described above.

The effects of hot water were assessed as described above except 100 ml of

HPLC-gradewater heatedto 100◦C wasadded to selected bottles. Those bottles

were againrotatedat room temperaturefor 24h during whichtime watersamples

cooled. Samples (50 ml) were then collected; bottles were washed, rinsed and

dried immediately after collection as above. To assess the continued effect of

heating on BPA release, 100 ml of room temperature HPLC-grade water was

then added to each of those bottles with sample collection following 24 h of

incubation at room temperature.

2.4. ELISA analysis of bisphenol A concentrations in water

samples

A supersensitiveBPA ELISA(Ecologiena, JapanEnvironchemicals,Tokyo)

was used to determine the concentration of BPA in water samples collected

from each bottle. With the exception of using additional known BPA stan-

dards to obtain a more accurate fit of the standard curve and performing

triplicate, rather duplicate, determinations for each sample, the competitive

ELISA was performed according to a standard test protocol that results in mini-

mum and maximum quantitative detection limits of 0.05ng/ml and 10 ng/ml,

respectively. Relative cross-reactivity of the BPA ELISA for endocrine dis-

ruptors and chemicals structurally related to BPA (100% reactivity) has been

determined; the most significant cross-reactivity was observed for bisphe-

nol B (15.6%) and bisphenol E (6%). Cross-reactivity for nonylphenol

was 0.19% and <0.05% for diethylhexylphthalate, 17-estradiol and estrone

(http://www.abraxiskits.com/moreinfo/PN590023USER.pdf ).

In addition to the unknown samples, a standard curve from known con-centrations of BPA was generated with non-linear regression analysis of each

assay/analysis performed. Additional BPA standards were prepared for use as

defined concentration standards for generation of each BPA standard curve,

and as internal controls to confirm the performance of each assay. For each

determination the standard curve generated for that experimental assay was

used to calculate the concentration of BPA present in the known standards

and each unknown sample. Additionally, control samples of HPLC water were

included in each of the individual assays. For all control and experimental

samples, triplicate measurements were made at 450 nm with a microprocessor

controlled SPECTRAFluor PLUS microplate reader (Tecan). Data was directly

exported to Excel (Microsoft Corp.), with non-linear regression/sigmoidal

curve fitting and statistical analysis of data performed using Prism 5.0

(GraphPad).

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H.H. Le et al. / Toxicology Letters 176 (2008) 149–156 151

2.5. Preparation of primary cerebellar neurons, treatment and

cytotoxicity assay

Primary cerebellar cultures were prepared from neonatal female

Sprague–Dawley rats (16–17 g) without enzymatic treatment. All animal pro-

cedures were done in accordance with protocols approved by the University

of Cincinnati Institutional Animal Care and Use Committee and followed NIH

guidelines. The resulting primary cerebellar cultures of maturing granule cell

neurons were maintained free of serum and exogenous steroid hormones aspreviously described (Wong et al., 2001, 2003). Dissociated cerebellar cells

were serially diluted in an appropriate volume of culture media and seeded at

2× 105 granule cells/cm2 in 100l of media in 96-well culture plates (TPP;

Basel, Switzerland) that were coated with poly l-lysine (100g/ml; Sigma,

St. Louis, MO). Cultures were incubated in 5% CO2 at 37 ◦C for 4 h to allow

granule cell attachment.Treatments were performed immediatelyfollowing cell

attachment.

For each treatment, attached granule cells were exposed to controls (17-

estradiol, BPA, or appropriate vehicle) or water samples for 15 min and then

washed twice with fresh medium following the exposure period to remove any

residual concentrationsof the test compounds. This 15-min pulsed exposurewas

previously shown to mimic rapid non-transcriptional estrogenic mechanisms in

maturing granule cells (Wong et al., 2003). Treated cultures were maintained

in 5% CO2 at 37 ◦C in a humidified incubator for 24 h, with amounts of lac-

tate dehydrogenase (LDH) released into the media determined as previously

described (Wong et al., 2001, 2003). Briefly, the CytoTox 96® Non-Radioactive

Cytotoxicity Assay (Promega; Madison, WI) was used to quantify LDH lev-

els in accordance with the general guidelines of the manufacturer’s protocol

(Promega, Technical Bulletin #TB163). Following treatments, culture plates

were centrifuged at 250× g for 4 min,and50l of theconditionedmediasuper-

natant from each well was collected and transferred to a new 96-well plate

assay plate. Attached cells or cellular debris settled to the bottom of the wells

were not disturbed during media collection. An equal 50 l volume of CytoTox

96® Non-Radioactive Cytotoxicity Assay substrate mixture was added to each

media sample. Sample/substrate solution was mixed and incubated in the dark

room at temperature for 30 min. Visible wavelength absorbance data was col-

lected at 492 nm immediately following termination of the colorimetric reaction

using a SPECTRAFluor PLUS microplate reader controlled by Genesis Soft-

ware (Tecan). The absorbance values from the conditioned media supernatant

from cells exposed to the test compounds was normalized to mean absorbance

values calculated from control samples.

2.6. Statistical analysis

Unless noted otherwise, all data presented are representative of at least

three experiments or quantitative determinations, and are reported as the mean

Fig. 1. Bisphenol A (BPA) ELISA. (A) The combined standard curve resulting

from a non-linear sigmoidal curve fit from all BPA standard curves used in the

17 experiments presentedin this studywere averagedand are shown graphically.

Data points are mean optical density values±S.E.M. at each concentration. (B)

Shown are the results for a single replicate dilution analysis of sample NPC2 in

which the concentration of undiluted and diluted sample NPC2 was calculated

independently fromthe results shown in Table 1 (i.e. this resultwas not included

in Table 1). Standard curve data points for this assay are indicated with open

circlesand NPC2sample values areindicatedwith filledcircles (mean±S.E.M.;

n = 3). The standard curve used to calculate the indicated BPA concentrations is

shown as a dashed curve.

value±S.D. or S.E.M. As appropriate for the experimental design, statisti-

cal analysis was conducted using an unpaired t -test, or one-way analysis of

variance (ANOVA) with post-test comparison between treatment groups using

Tuckey–Kramer multiple comparison test. A minimal level of statistical signif-

Table 1

Bisphenol A concentrations in drinking bottle water

Bottle description; # Concentration of BPA released into 100ml water at RT (mean (ng/ml)± S.D. (n))

Day 1 Day 3 Day 5 Day 7

New polycarbonate

1 0.08± 0.06 0.25± 0.07 0.28± 0.08 0.73 ± 0.05 (4)

2 0.21± 0.10 (2) 0.35± 0.10 (2) 0.61± 0.05 0.98 ± 0.15 (6)

3 0.36± 0.17 0.79± 0.17 0.68± 0.04 1.33 ± 0.09 (2)

Used polycarbonate

1 0.28± 0.16 (2) 0.33± 0.20 (2) 0.29± 0.10 (6) 0.34 ± 0.06 (6)

2 0.21± 0.05 0.39± 0.14 0.39± 0.12 0.88 ± 0.19

3 ND ND ND 0.93 ± 0.02

4 0.29± 0.05 0.46± 0.06 0.58± 0.14 (5) 0.62 ± 0.12

5 ND 0.76± 0.06 0.72± 0.13 (2) 0.80 ± 0.14 (4)

New high-density polyethylene

1 0.01± 0.02 0.08± 0.03 0.09± 0.04 0.15 ± 0.04

2 0.08± 0.03 0.16± 0.07 0.08± 0.04 0.19 ± 0.13 (6)

3 ND ND ND 0.08 ± 0.09 (2)

Values below the detection limit of the assay are indicated in italics; ND: not determined; S.D.: standard deviation; n = 3, unless specified.

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icance for differences in values was considered to be p < 0.05 and is indicated

with an ‘*’. Data was analyzed with excel (Microsoft) and GraphPad Prism ®

Version 5.0 (GraphPad Software Inc.).

3. Results

Over the course of these experiments, results of the BPA-

specific ELISA were highly reproducible. Demonstrating

the reproducibility of the assay, mean values and variance(S.E.M.) for the combined standard curves generated from

17 different experiments are shown in Fig. 1A. Within that

large data set, ANOVA analysis showed significant differences

between mean values for BPA concentration standards at

0.005 ng/ml and 0.05 ng/ml even though they were outside

the detection range of a typical assay. For each BPA stan-

dard of this assay curve the coefficient of variation ranged

from 4.7% to a maximum of 11.5%. The BPA ELISAlimit of detection for an individual assay with duplicate

sample measurements at each data point was 0.05 ng/ml

(http://www.abraxiskits.com/moreinfo/PN590023USER.pdf ).To confirm the ability to accurately detect reproducible differ-

ences in BPA concentration, a replicate analysis independent

from those included in Table 1 was performed using undiluted

day 7 sample from new PC bottle 2 (NPC2), and the same

sample diluted 1:3, or 1:10 with HPLC water. The results

from the BPA standards and each of the NPC2 samples forthis analysis are shown in Fig. 1B. The mean results of each

diluted sample were calculated from the standard curve and

corresponds well to those predicted from the initial calculated

concentration of 0.98 ng/ml for this sample (Table 1). This

representative experiment also demonstrates the typically low

intra-assay variation of the assay. The maximum % coefficient

of variance (CV) for triplicate measures of the standardswas 7.2%; for the bottle-exposed water sample dilutions themaximal variation was observed in the 1:10 dilution (12.7%).

Analysis of control HPLC-grade water samples was included

for each experiment revealed levels of BPA at the limit of the

assay detection (0.06ng/ml; S.D.± 0.06, n = 28). The concen-

trations of BPA-like immunoreactivity present in all samples

collected from HDPE bottles were very low (Table 1); the

mean value of BPA estimated for water samples collected

Fig. 2. Comparison of bisphenol A (BPA) migration into water from new and

used polycarbonate bottles. Individual values calculated following room tem-

perature incubation for 7 days in new or used polycarbonate bottles are shown.

The calculated mean values are indicated and graphically represented with a

horizontal dashed line. Error bars represent the standard deviation. *Values aresignificantly different fromthe 0.05ng/mldetectionlimit as calculatedwith a one

sample t -testusing a theoreticalmean of 0.05. Valuesfrom newand usedsamples

were not significantly different from one another (unpaired t -test; p = 0.1688).

following 7 days of incubation from the HDPE bottles was

0.14 ng/ml (S.D.± 0.09, n = 11). In contrast to those control val-ues, detectable levels of BPA were identifiedin allwater samples

collected from either the new or used PC bottles ( Table 1).

In general, the concentration of BPA released from polycar-

bonate bottles into water at room temperature increased with

time (Table 1). Following 7 days of room temperature incuba-

tion, the concentrations of BPA released from new PC bottles

were 1.0 ng/ml, and 0.7 ng/ml from used PC bottles (Fig. 2).Mean concentrations of BPA in water samples from new andused bottles were significantly higher than the detection limit of

the assay, but not different from one another. Thus, the amount

of BPA released from the new PC water bottles and ones pre-

viously used by consumers was found to be the same. Based

on the concentrations of BPA at the end of the 168 h incu-

bation period the estimated rate of BPA migration at room

temperature averaged 0.60 ng/h (S.D.± 0.18) for new bottles

Table 2

Effects of heating on BPA migration

Bottle description; # BPA released at RT (day 7) BPA released into 100 ◦C water (24 h) BPA released from heated bottle at RT (24 h)

[BPA] ng/ml Rate (nghr−1) BPA (ng/ml) Rate (ng/h) BPA (ng/ml) Rate (ng/h)

New Polycarbonate

1 0.73± 0.05 (4) 0.44

2 0.98± 0.15 (6) 0.58 7.67 ± 0.57 32.0 4.6 ± 0.59 19.2

3 1.33± 0.09 (2) 0.79 3.84 ± 0.12 16.0 2.3 ± 0.44 9.6

Used Polycarbonate

1 0.34± 0.06 (6) 0.20

2 0.88± 0.19 0.52

3 0.93± 0.02 0.55 1.92 ± 0.40 8.0 0.66 ± 0.01 2.8

4 0.62± 0.12 0.37

5 0.80± 0.14 (4) 0.48

Results are mean± standard deviation; n = 3, unless specified.

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and 0.42 ng/h (S.D.± 0.14) for used bottles (Table 2). Because

thevalues calculated forrates of migration from new or used bot-

tles were not significantly different from each other, the average

rate of migration from all bottles was calculated as 0.49 ng/h

(S.D.± 0.17).

The impact of elevated water temperature on BPA release was

determined for two of the new, and one used PC bottle by addi-

tion of 100 ml of 100◦C HPLC-grade water to each bottle andincubation for 24 h, during which time the water samples slowly

cooled. Theconcentrationof BPA ineach samplewasfoundto be

at least double the value accumulated in unheated samples dur-

ing the entire 7 days incubation period (Table 2). The markedly

Fig. 3. Comparative analysis of BPA-like bioactivity of water samples in devel-

oping cerebellar neurons. (A) Concentration response analysis of LDH released

from granule cells for known concentrations of 17-estradiol and BPA. Results

are expressed as means±S.E.M. (n = 8). (B) Dilutions of water samples from

two polycarbonate bottles stimulate BPA-like increases in LDH release follow-ing brief exposure. After 15 exposure to 17-estradiol, BPA, or concentrations

of diluted water samples corresponding to approximately 10−11 M, 10−10 M, or

10−9 M BPA-like immunoreactivity, the amount of LDH released from granule

cells into culture media at 24 h was determined and compared to LDH levels

released fromvehicle-treated control cultures. Levels of LDH released werenor-

malized to themaximal estrogenic effect induced by10−10 M 17-estradiol(E2)

and compared to the effect induced by known concentrations of BPA. Results

are expressed as means±S.E.M. (n = 4 for BPA and water samples, n =7 for

vehicle and n = 6 for estradiol controls).Levels of significantdifference between

values calculated for each group wasassessedwith a one-way ANOVA. In panel

B, *significant difference in the values between treatment groups exposed to the

same concentration of BPA (0.1nM) and the estimated concentration of BPA in

the experimental water sample.

higher amounts of BPA that leached into the water indicate that

exposure of polycarbonate to heated water greatly increased the

ratesof BPA migration.The increasein therateof release follow-

ing exposure to 100 ◦C water and slow cooling ranged from 15-

fold to 55-fold. Whilethe concentrations of BPA from theheated

water samples were within the range of assay detection, the con-

centrations estimated for both new PC bottles (3.84 ng/ml and

7.67 ng/ml) are at the extreme limit of linearity of the assay. As aresult, the reported concentrations may modestly underestimate

the concentrations of BPA actually present. An elevated rate of

BPA liberation was detectable following subsequent incubation

with room temperature water suggesting that the effects of heat-

ing on BPA migration were not limited to acute effects of heated

water,but thatthere werelonger termeffects uponmigration rate.

The biological activity of liberated BPA in water samples wasexamined using an oncotic cell-death assay for rapid-estrogenic

signaling effects in developing cerebellar granule cells (Wong

et al., 2003). Comparative concentration response results for

the neurotoxic actions of 17-estradiol and BPA are shown in

Fig. 3A. Serially diluted water samples from two new polycar-

bonate bottles calculated to range in BPA concentration fromabout 0.01 nM to 1 nM (0.0023–0.23 ng/ml) were found to sig-

nificantly increase estrogen-like LDH release from immaturegranule cells (Fig. 3B). No detectable increase in LDH release

was observed following exposure of granule cells to parallel

dilutions of water samples from HDPE bottles (not shown).

Effects could be observed at an estimated BPA concentration

of approximately 0.1 nM and 1 nM. Compared to the maximal

low-dose effects of estrogen (0.1 nM), the estrogenic effects

induced by diluted water samples were similar to those observed

for BPA. At the low doses, the estrogen-like effects of sam-ple NPC2 (72± 6%) were significantly reduced compared to

those observed for 1 nM BPA (89± 5% of maximal estradiol

effect). The effects induced with the low dose of sample NPC3

(79± 5%), while slightly less than predicted, were not different

from those induced by BPA. At 1 nM, the BPA positive control

and each diluted water sample elicited maximal estradiol-like

responses.

4. Discussion

Detectable levels of BPA-like immunoreactivity were

observed in all room temperature samples of water following

incubation in polycarbonate water bottles, regardless of whether

or not the bottle was new or previously used by a consumer.

By contrast, water samples collected after identical exposureto negative control water bottles made of HDPE, a polymer

consisting of long chains of the monomer ethylene, containedmuch lower levels of BPA-like immunoreactivity. The water

samples from two of the HDPE bottles were estimated to

contain concentrations of BPA significantly above the theo-

retical mean for the 0.05 ng/ml detection limit of the assay

(bottle no. 1, p = 0.049; bottle no. 2, p = 0.046 as calculated

using a one-sample t -test). The differences between the mean

values for untreated water and water samples incubated in

the HDPE bottles were reproducible and highly significant( p = 0.0025). Neither the HDPE polymers, nor the polypropy-

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lene plastic loop-tops common to tested HDPE and PC bottles,

are expected to contain BPA. However, the presence of non-

polymer additives or coating cannot be ruled out as a source

of the apparently elevated levels of BPA relative to untreated

water.

The bioequivalence of water samples from the PC drinking

bottles and BPA was demonstrated using a rapid and sensi-

tive in vitro assay that detects “non-genomic” rapid estrogenicsignaling effects in developing neurons (Wong et al., 2001,

2003). Specifically, the endpoint of this assay (LDH release due

to oncotic cell death) is dependent on activation of a defined

estrogen signaling mechanism that requires activation of two

signaling pathways; activation of extracellular signal-regulated

kinase (ERK)-dependent signaling at cell surface and a dis-

tinct signaling pathway dependent on intracellular activation of protein phosphatase 2A (PP2A) activity (Belcher et al., 2005;

Wong et al., 2003). Rather than using the traditional MCF7

growth assay to demonstrate estrogen-like activity of BPA and

the BPA-like immunoreactivity present in the collected water

samples, we employed this non-genomic assay because rapid

non-genomic actions are typically more sensitive for detectionof estrogen-like EDC activity (Wetherill et al., 2007). Addition-

ally this assay benefits from being more rapid than the MCF7growth assay. Using the granule cell/LDH system, results from

multiplesamplescan be treated, collectedand analyzed in <36h,

avoiding the much longer incubation periods required to detect

estrogen-like increases in MCF7 cell population growth. The

use of primary cerebellar neurons under defined conditions also

avoids many of the complexities associated with using trans-

formed MCF7 cells. Potentially confounding factors with the

MCF7 growth assay include more complicated culture con-ditions, requirements of serum/hormone withdrawal following

culture expansion, and variability resulting from population het-

erogeneity of MCF7 sub-lines which results in differential ER

expression and variable estrogen growth response characteris-

tics.

The results presented here confirm and extend previous stud-

ies that demonstrated migration of BPA from PC plastics. In the

seminal study of Krishnan and coworkers, biologically active

and environmentally relevant levels of BPA (minimal concen-tration of ∼2.85 ng/ml) were shown to leach into water from

PC flasks used for growth of bakers yeast (Saccharomyces cere-

visiae) upon autoclaving at 120–125 ◦C. While those studies

established clearly that biologically active BPA could migrate

from PC into water at elevated temperatures, a comparison of

the presented results with subsequent studies analyzing migra-tion of BPA under less extreme temperature conditions could be

considered more relevant.The studies of Howdeshell et al. (2003) evaluated whether

BPA was released from plastic animal caging at room temper-

ature and found that BPA was released into water incubated

in new or used PC cages. From a surface area of PC caging

comparable to that of the water bottles analyzed here (478 cm2)

low concentrations (∼0.27 ng/ml) of BPA were detected in the

250 ml water samples following a 7-day exposure period. In

contrast, exposed samples collected from used cages contained

large amounts of BPA, which resulted in BPA concentrations up

to 310 ng/ml of water (Howdeshell et al., 2003). This stands in

apparent contradiction to the findings reported here that demon-

strate no difference in BPA-liberation between new and used

PC bottles. While a number of factors might account for those

apparently different findings, differences in previous treatment

of polycarbonate animal caging and the used water bottles stud-

ied here are a likely explanation for the observed differences

in BPA migration. The used PC cages were acquired follow-ing use in an animal care facility accredited by the Association

for Assessment and Accreditation of Laboratory Animal Care

(Howdeshell et al.,2003).Asaresult,sanitizationofcageswould

have followed the minimal standards outline in the Guide for the

Care and Use of Laboratory Animals (Institute of Laboratory

Animal Research, 1996), which would entail frequent washing

with hot water of at least 180 ◦F (82.2 ◦C) and soap or detergent.While not stated explicitly, these mouse cages were likely used

in a pathogen-freebarrierfacility and would have alsoundergone

repeated rounds of autoclaving at temperatures of 120–122 ◦C.

In comparison to the used PC drinking bottles, which would

have been washed by consumers at a typical household hot

water temperature of ∼50 ◦C, it is likely that the elevated tem-peratures of autoclaving would result in a greater extent of PC

polymer hydrolysis, which could account for increased migra-tion of monomeric BPA. This suggestion is supported by the

finding that brief exposure to 100 ◦C water greatly increased

the rate of BPA migration from the water bottles analyzed

here.

Using a protocol of repeated washing and exposure to 100◦C

waterfor1h Brede etal. (2003)demonstratedthat BPA fromnew

PC baby bottles leached into boiling water (0.23 ± 0.12 ng/ml).

Following simulated use that included dishwashing 51 times,

brushing and boiling, the concentrations of BPA liberated wereelevated to 8.4± 4 ng/ml, a 36.5-fold increase. However, an

additional 118 wash cycles did not change the amount of BPA

released, which was reported to be 6.7± 4 ng/ml. From these

results it was concluded that there was a link between elevated

temperatures and increased BPA migration. It is notable that the

concentration of BPA liberated from the PC baby bottles, and

the increases in migration rateresulting fromexposure to boiling

water were similar to the values reported here. However, becausethe additional variables of washing and brushing were included

in thetreatmentsof thebaby bottles (Brede et al., 2003),it isonly

possible to link increased BPA migration with simulated use. In

contrast, the data presented here clearly uncouple the indepen-

dent variables of new and used, from exposure to elevated water

temperature. As a result, in the presented analysis, the study’streatment design has isolated elevated temperature of water as

a single variable. Thus, the linkage of BPA liberation and ele-vated temperatures is clearly established. While the exposure

to elevated temperatures of boiling water is much higher than

the typical home wash water temperatures used by consumers,

the storage of higher temperature (boiling) water or bever-

ages in these PC bottles is a common practice in cold weather

outdoor activities such as alpine snow sports, climbing, and

mountaineering. Such practices would likely result in increased

levels of BPA in beverages stored and consumed from thosebottles.

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The studies investigating BPA migration from scientific-

grade PC labware (Krishnan et al., 1993) and animal caging

(Howdeshell et al., 2003) directly address potential confound-

ing factors in the laboratory environment. Those studies also

suggest that BPA released from PC plastic could be one point

source of BPA contamination in humans. Studies investigat-

ing migration of BPA from infant feeding bottles have been

especially influential and raised much concern within the gen-eral public (Mountfort et al., 1997; Biles et al., 1997; Brede

et al., 2003; Sun et al., 2000; Wong et al., 2005). To date,

however, few epidemiological studies have been conducted to

investigate the relationship between BPA exposure and human

health. Although, as reviewed by Vandenberg et al. (2007), a

fair number of studies have been conducted that have iden-

tified sources or levels of BPA in humans. The aim of themajority of those studies has been to estimate levels of BPA

migrating from packaging into foods, with most studies focus-

ing on BPA leaching from epoxy resin lining into canned food.

As mentioned above, a subset of studies more directly rele-

vant to those presented here, investigated the release of BPA

from commercial infant formula bottles. In most studies, sig-nificant release of BPA from PC food and beverage containers

was detected, yet based on current acceptable exposure levelsthe potential adverse heath effects were concluded to be mini-

mal (Mountfort et al., 1997; Brede et al., 2003; D’Antuono et

al., 2001; Wong et al., 2005). However, mixtures of different

estrogenic chemicals can have much greater disruptive effects

than those predicted from exposure to low concentrations of

an individual compound (Rajapakse et al., 2002). Studies of

rapid actions of BPA in developing neurons of the rat cerebel-

lum have also demonstrated that BPA alone acts as a highlypotent and efficacious estradiol mimetic; yet, when combine

at very low concentrations (1 pM) with estradiol, BPA acts as

a potent antagonist of estrogen signaling (Zsarnovszky et al.,

2005). Those finding and others have highlighted the complex,

and often unanticipated nature of EDC action. It is now clear

that conclusions from single-compound studies, or association

of healthrisks with a singleEDC, may notfully reflectthe effects

of environmental exposures to EDCs. Thus, the contribution of

the concentrations of BPA that contaminate drinking water andfoods stored in PC bottles should be considered as a single,

though important component of the total mixture of EDCs to

which humans are acutely and chronically exposed through out

their life time.

In summary, in this study it was found that bioactive

BPA was liberated from both new and used PC drinkingbottles and migrated at a similar rate into water at room

temperature. Exposure to elevated temperatures above thosetypically used for washing by consumers, but not outside nor-

mal household practice (e.g. boiling to sterilize infant feeding

bottles), or outdoor applications (addition of very hot or boil-

ing water or beverages to drinking bottles) greatly elevated

the rate of BPA migration. The exposures anticipated from

the BPA drinking bottles are likely one of many sources of

BPA contamination that contributes to the total amount of

endocrine disrupting compounds to which some individuals areexposed.

Conflict of interests

The author’s have no conflicts of interest that would

have inappropriately influenced the work presented in this

manuscript.

References

Belcher, S.M., Le, H.H., Spurling, L., Wong, J.K., 2005. Rapid estrogenic reg-

ulation of extracellular signal-regulated kinase 1/2 signaling in cerebellar

granule cells involves a G protein and protein kinase A-dependent mecha-

nism and intracellular activation of protein phosphatase 2A. Endocrinology

146, 5397–5406.

Biles, J.E., McNeal, T.P., Begley, T.H., Hollifield, H.C., 1997. Determination of

bisphenol-A in reusable polycarbonate food-contact plastics and migration

to food-simulating liquids. J. Agric. Food Chem. 45, 3541–3544.

Brede, C., Fjeldal, P., Skjevrak, I., Herikstad, H., 2003. Increased migration

levels of bisphenol A from polycarbonate baby bottles after dishwashing,

boiling and brushing. Food Addit. Contam. 20, 684–689.

Calafat, A.M., Kuklenyik, Z., Reidy, J.A., Caudill, S.P., Ekong, J., Needham,

L.L., 2005. Urinary concentrations of bisphenol A and 4-nonylphenol in a

human reference population. Environ. Health Perspect. 113, 391–395.

CERHR, 2007. Interm NTP-CERHR report on the reproductive and devel-

opmental toxicity of bisphenol A. http://cerhr.niehs.nih.gov/chemicals/

bisphenol/BPA Interim DraftRpt.pdf .

Crain, D.A., Eriksen, M., Iguchi, T., Jobling, S., Laufer, H., LeBlanc, G.A.,

Guillette Jr., L.J., 2007. An ecological assessment of bisphenol-A: evidence

from comparative biology. Reprod. Toxicol. 24, 225–239.

D’Antuono, A., Dall’Orto, V.C., Balbo, A.L., Sobral, S., Rezzano, I., 2001.

Determination of bisphenol A in food-simulating liquids using LCED with

a chemically modified electrode. J. Agric. Food Chem. 49, 1098–1101.

Howdeshell, K.L., Peterman, P.H., Judy, B.M., Taylor, J.A., Orazio, C.E.,

Ruhlen, R.L.,vom Saal, F.S.,Welshons,W.V., 2003. BisphenolA is released

from used polycarbonate animal cages into water at room temperature. Env-

iron. Health Perspect. 111, 1180–1187.

Institute of Laboratory Animal Research, National Research Council, 1996.

Guide for the Care and Use of Laboratory Animals. National Academy

Press, Washington, DC.Kang, J.-H., Kondo, F., Katayama, Y., 2006. Human exposure to bisphenol A.

Toxicology 226, 79–89.

Krishnan, A.V., Stathis, P., Permuth, S.F., Tokes, L., Feldman, D., 1993.

Bisphenol-A: an estrogenic substance is released from polycarbonate flasks

during autoclaving. Endocrinology 132, 2279–2286.

Mountfort, K., Kelly, J., Jickells, S., Castle, L., 1997. Investigations into the

potential degradation of polycarbonate babybottles during sterilization with

consequent release of bisphenol A. Food Addit. Contam. 14, 737–740.

Rajapakse, N., Silva, E., Kortenkamp, A., 2002. Combining xenoestrogens

at levels below individual no-observed-effect concentrations dramatically

enhances steroid hormone action. Environ. Health Perspect. 110, 917–921.

Richter, C.A., Birnbaum, L.S., Farabollini, F., Newbold, R.R., Rubin, B.S., Tal-

sness, C.E., Vandenbergh, J.G., Walser-Kuntz, D.R., vom Saal, F.S., 2007.

In vivo effects of bisphenol A in laboratory rodent studies. Reprod. Toxicol.

24, 199–224.Sun, Y.W.M., Al-Dirbashi, O., Kuroda, N., Nakazawa, H., Nakashima, K.,

2000. High-performance liquid chromatography with peroxyoxalate chemi-

luminescence detection of bisphenol A migrated from polycarbonate baby

bottles using 4-(4,5-diphenyl-1 H -imidazol-2-yl)benzoyl chloride as a label.

J. Chromatogr. B: Biomed. Sci. Appl. 749, 49–56.

Vandenberg, L.N., Hauser, R., Marcus, M., Olea, N., Welshons, W.V., 2007.

Human exposure to bisphenol A (BPA). Reprod. Toxicol. 24, 139–177.

Wetherill, Y.B., Akingbemi, B.T., Kanno, J., McLachlan, J.A., Nadal, A., Son-

nenschein, C., Watson, C.S., Zoeller, R.T., Belcher, S.M., 2007. In vitro

molecularmechanisms of bisphenolA action. Reprod. Toxicol. 24,178–198.

Wong, J.K., Kennedy, P.R., Belcher, S.M., 2001. Simplified serum- and steroid-

free culture conditions for the high-throughput viability analysis of primary

cultures of cerebellar neurons. J. Neurosci. Methods 110, 45–55.

H - 81





Wong, J.K., Le, H.H., Zsarnovszky, A., Belcher, S.M., 2003. Estrogens and

ICI182,780 (Faslodex) modulate mitosis and cell deathin immature cerebel-

lar neurons via rapidactivation of p44/p42 mitogen-activated protein kinase.

J. Neurosci. 23, 4984–4995.

Wong, K.O., Leo,L.W.,Seah, H.L.,2005.Dietaryexposureassessment ofinfants

to bisphenol A from theuse of polycarbonate baby milk bottles. Food Addit.

Contam. 22, 280–288.

Zsarnovszky, A., Le, H.H., Wong, H.-S., Belcher, S.M., 2005. Ontogeny

of rapid estrogen-mediated ERK1/2 signaling in the rat cerebellar

cortex in vivo: potent non-genomic agonist and endocrine disrupting

activity of the xenoestrogen bisphenol A. Endocrinology 146, 5388–

5396.


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