Regulatory Toxicology and Pharmacology 58 (2010) 18–24

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Regulatory Toxicology and Pharmacology

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Biomonitoring Equivalents for bisphenol A (BPA)

Kannan Krishnan a, Michelle Gagné a, Andy Nong b, Lesa L. Aylward c,*, Sean M. Hays d

a Université de Montréal, Département de santé environnementale et santé au travail, Montréal, QC, Canadab Health Canada, Ottawa, Ont., Canadac Summit Toxicology, LLP, Falls Church, VA, USAd Summit Toxicology, LLP, Lyons, CO, USA

a r t i c l e i n f o

Article history:Received 26 March 2010Available online 10 June 2010

Keywords:Biomonitoring EquivalentsBisphenol ARisk assessmentPharmacokinetics

0273-2300/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.yrtph.2010.06.005

Abbreviations: BE, biomonitoring Equivalent; BEP

point of departure; BPA, bisphenol A; BPA-G, BPA-moconjugate of BPA; CAS, chemical abstracts services; CDand Prevention; CERHR, Center for the Evaluation of R(NTP); EFSA, European Food Safety Authority; LOAeffect level; MOA, mode of action; MRP, multidrug rNOAEL, no observed adverse effect level; NTP, Natiopolycarbonate; POD, point of departure; pTDI, proviRFD, reference dose; USEPA, United States Environmuncertainty factor; t1/2, half-life for elimination; TDIuridine diphosphate-glucuronsyl transferase; WHO, W

* Corresponding author. Address: Summit ToxicoloFalls Church, VA 22044, USA.

E-mail address: laylward@summittoxicology.com

a b s t r a c t

Recent efforts worldwide have resulted in a growing database of measured concentrations of chemicals inblood and urine samples taken from the general population. However, few tools exist to assist in theinterpretation of the measured values in a health risk context. Biomonitoring Equivalents (BEs) aredefined as the concentration or range of concentrations of a chemical or its metabolite in a biologicalmedium (blood, urine, or other medium) that is consistent with an existing health-based exposure guide-line. BE values are derived by integrating available data on pharmacokinetics with existing chemical riskassessments. This study reviews available health-based exposure guidance values for bisphenol A (BPA)from Health Canada, the United States Environmental Protection Agency (USEPA) and the European FoodSafety Authority (EFSA). BE values were derived based on data on BPA urinary excretion in humans. TheBE value corresponding to the oral provisional tolerable daily intake (pTDI) of 25 lg/kg-d from HealthCanada is 1 mg/L (1.3 mg/g creatinine); value corresponding to the US EPA reference dose (RfD) and EFSAtolerable daily intake (TDI) estimates (both of which are equal to 50 lg/kg-d) is 2 mg/L (2.6 mg/g creat-inine). These values are estimates of the 24-h average urinary BPA concentrations that are consistent withsteady-state exposure at the respective exposure guidance values. These BE values may be used asscreening tools for evaluation of central tendency measures of population biomonitoring data for BPAin a risk assessment context and can assist in prioritization of the potential need for additional riskassessment efforts for BPA relative to other chemicals.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction assessments. Such screening criteria would ideally be based on ro-

Interpretation of measurements of concentrations of chemicalsin samples of urine or blood from individuals in the general popu-lation is hampered by the general lack of screening criteria forevaluation of such biomonitoring data in a health risk context.Without such screening criteria, biomonitoring data can be inter-preted in terms of exposure trends, but cannot be used to evaluatewhich chemicals may be of concern in the context of current risk

ll rights reserved.

OD, biomonitoring equivalentnoglucuronide; BPA-S, sulfate

C, Center for Disease Controlisks to Human Reproduction

EL, lowest observed adverseesistance-associated protein;nal Toxicology Program; PC,sional tolerable daily intake;ental Protection Agency; UF,, tolerable daily intake; UGT,

orld Health Organization.gy, LLP, 6343 Carolyn Drive

(L.L. Aylward).

bust datasets relating potential adverse effects to biomarker con-centrations in human populations (see, for example, the U.S.Centers for Disease Control and Prevention (CDC) blood lead levelof concern; see http://www.cdc.gov/nceh/lead/). However, devel-opment of such epidemiologically-based screening criteria is a re-source and time-intensive effort. As an interim approach, thedevelopment of Biomonitoring Equivalents (BEs) has been pro-posed, and guidelines for the derivation and communication ofthese values have been developed (Hays et al., 2008, 2007; LaKindet al., 2008). Such an interpretive tool, in the form of biologicalexposure indices (BEIs), has been developed by the ACGIH for anumber of chemicals (e.g., ACGIH, 2007).

A BE is defined as the concentration or range of concentrationsof chemical in a biological medium (blood, urine, or other medium)that is consistent with an existing health-based exposure guidancevalue such as a reference dose (RfD) or tolerable daily intake (TDI).Its estimation is based on existing chemical-specific pharmacoki-netic data (animal or human) and the point of departure (POD)used in the derivation of an exposure guidance value (such asthe RfD or TDI) (Hays et al., 2008). BEs should be used as screeningtools to allow an assessment of biomonitoring data to evaluate

K. Krishnan et al. / Regulatory Toxicology and Pharmacology 58 (2010) 18–24 19

which chemicals have large, small, or no margins of safety com-pared to existing risk assessments and exposure guidance values.Robustness of a BE value relies on the underlying exposure guid-ance values and pharmacokinetic data used to derive it.

This document presents derivation of BE values for 2,2-bis(4-hydroxyphenyl)propane, also known as bisphenol A (BPA; Chemi-cal Abstracts Services [CAS] Registry number 80-05-7). BPA is amonomer used in the production of polycarbonate (PC) plasticsand epoxy-phenolic resins. Polycarbonates are used extensivelyin food containers: e.g. water bottles, plates, and mugs. Epoxy-phe-nolic resins are used as an internal, protective lining or coating ofcans for foods and beverages (Health Canada, 2008; EFSA, 2006).Because of its wide use in food packaging and food containers,BPA has the potential to leach into foods, and widespread exposureto low levels of BPA in the general population has been confirmedthrough biomonitoring studies (Dekant and Völkel, 2008; Calafatet al., 2005, 2008). Oral exposure is expected to be the predomi-nant route of exposure because of the low vapor pressure of BPAand its use patterns (Dekant and Völkel, 2008).

2. Available data and approach

2.1. Exposure guidance values, critical effects, and mode of action

BPA has been the subject of numerous risk assessment reviews,with increased attention over the last decade related to evaluatingits potential for producing adverse health effects through an endo-crine disruption mechanism (Health Canada, 2008; EFSA, 2006,2008; CERHR, 2008). Table 1 presents the available guidance valuesderived for BPA. In each case, the POD, the toxicological endpoint ofinterest, and the applied uncertainty factors are identified. The pro-visional tolerable daily intake (pTDI) of Health Canada and the RfDof US EPA were both established on the basis of the data reportedby NTP (1982); however, Health Canada used subchronic study re-sults whereas USEPA used the LOAEL associated with the chronicexposures. The TDI from European Food Safety Authority (EFSA,2006) was based on a more recent, three-generation study in therat (Tyl et al., 2002), with supporting data from a report on a fol-low-up two-generation study that examined a wide range of end-points in offspring that are sensitive to endocrine disruption(published as Tyl et al., 2008 following the EFSA evaluation). Otherrecent evaluations have converged on similar tolerable intake esti-mates as those presented in Table 1 (Willhite et al., 2008; METI,2002).

Table 1Health-based exposure guidance values for BPA from various agencies.

Organization,criteria (year ofevaluation)

Study description Critical endpoint an

pTDIa (HealthCanada, 2008)

90-day study in rats (NTP, 1982) Reduced mean bod500 ppm diet (25 m

RfDb, (IRIS US EPA,1993)

Rat Chronic Oral Bioassay (NTP, 1982) Reduced mean bod1000 ppm diet (50

TDIc, (EFSA-EU,2006)

Three-generation study in the rat(Tylet al., 2002)(supported by 2 generationstudy of Tyl et al., 2008)

Reductions in adultand pup body andweightNOAEL of 5 m

Note: LOAEL – lowest observed adverse effect level, NOAEL – no observed adverse effeca pTDI: provisional tolerable daily intake. This value represents an updated review o

recognition by Health Canada that additional toxicological research is ongoing to addreb RfD: reference dose, for chronic exposure.c TDI: tolerable daily intake.

The available guidance values are based on reductions in adultbody weight (and pup body and organ weights in the case of theEFSA’s value), which is a sign of systemic toxicity (Tyl et al.,2002), and not directly linked to the presumed mode of action ofbinding to estrogen receptors. While standard two- and three-gen-eration studies in rats have indicated the potential for BPA to pro-duce endocrine-related effects on rodent offspring, these effectshave typically occurred at far higher dose levels than those thatproduce more non-specific signs of toxicity such as reductions inadult or pup body weights, which may not be mediated throughan endocrine mode of action (Tyl et al., 2002, 2008), or have beendemonstrated in studies relying upon modes of administration thatbypass hepatic first-pass metabolism. BPA is inactive in tests forgenotoxicity and carcinogenicity (Willhite et al., 2008).

The mechanism of action of BPA has been extensively investi-gated. Willhite et al. (2008) and Health Canada (2008) summarizedthe available data on the estrogen-like activity of BPA. Data indi-cate that BPA can bind to estrogen receptors (ER), both a and b,with relatively low affinity and can produce transactivation ofestrogen receptor genes in vitro. Substantial research has demon-strated that the estrogenic potency of BPA is extremely low andthat endocrine-related responses occur only at oral doses wellabove those producing more general signs of toxicity (reviewedby Sharpe, 2010). Research is ongoing regarding potential low-doseendocrine activity of BPA that may involve pathways that are inde-pendent of classical estrogen receptor interactions. Available liter-ature indicates that the conjugates of BPA, including the majormetabolite BPA-monoglucuronide (BPA-G), are essentially devoidof endocrine activity (Snyder et al., 2000; Matthews et al., 2001).The formation of a reactive semi-quinone metabolite at high doseshas been postulated, but lacks further experimental support(Haighton et al., 2002). At the present time, however, the observedtoxicity is thought to arise from free compound rather than metab-olites based on similarity to estradiol as well as its binding andtransactivation of estrogen receptor in vitro (Willhite et al.,2008). Therefore, internal dose metrics relevant to the mode of ac-tion would presumably be related to some measure of free BPAconcentration in the target tissues.

2.2. Available pharmacokinetic data

We identified key studies describing the pharmacokinetics ofBPA through searches of Medline and from recent literature re-views (EFSA, 2008; Health Canada, 2008). Conjugation is the majormetabolic process for BPA clearance, followed by the elimination of

d dose Uncertainty factors Value

y weight NOEL:g/kg/day)

1000– 10 for for subchronic to chronic

extrapolation– 10 for interspecies differences– 10 for inter-individual differences

25 lg/kg-d

y weightLOAEL:mg/kg/day)

1000– 10 for LOAEL-NOAEL extrapolation– 10 for interspecies differences– 10 for inter-individual differences

50 lg/kg-d

body weightorgan

g/kg bw/day

100– 10 for interspecies differences– 10 for inter-individual differences

50 lg/kg-d

t level, NOEL – no observed effect level.f the toxicological literature on BPA, and the provisional designation reflects the

ss issues raised by recent studies.

20 K. Krishnan et al. / Regulatory Toxicology and Pharmacology 58 (2010) 18–24

the conjugates via urine and/or feces. BPA conjugates with UDP-glucuronic acid to form the glucuronide, BPA-G (Dekant and Völkel,2008). This conjugation reaction is mediated by uridine diphos-phate-glucuronsyl transferase (UGT) in the intestine and the liver;a minor amount of sulfate conjugate (BPA-S) is formed as well(Pottenger et al., 2000; Völkel et al., 2002; Kuester and Sipes, 2007).

Distinct differences between mammalian species emerge fromthe available pharmacokinetic studies. In rodents, BPA-G is elimi-nated principally in the bile via MRP2 (Inoue et al., 2004), andundergoes enterohepatic recirculation (Pottenger et al., 2000),while it is mainly eliminated via urine in humans (Völkel et al.,2002). This difference in the route of excretion is likely attributableto the species differences in the threshold of molecular size for bil-iary elimination (rats: 350 Da, humans: 550 Da). Since the molecu-lar weight of BPA-G is 404 Da, below the threshold for biliaryexcretion in humans, it is mainly excreted via urine, as docu-mented by Völkel et al. (2002). In humans, glucoronidation ofBPA occurs rapidly and essentially completely following oral expo-sure (Dekant and Völkel, 2008). In a review of the comparative spe-cies pharmacokinetics in the context of risk assessment, an EFSApanel concluded that ‘‘because of the metabolic differences de-scribed, exposure to free BPA in adult, fetal and neonatal rats willbe greater than in humans and that rats would therefore be moresusceptible to BPA-induced toxic effects than humans on a equiv-alent dose basis” (EFSA, 2008). For these reasons, studies relyingon routes of administration such as intravenous or subcutaneousadministration are also likely to be much less relevant to humanenvironmental exposure, which occurs via the oral route withaccompanying hepatic first-pass metabolism.

2.2.1. HumansHuman pharmacokinetic data, relevant to BE derivation for BPA,

are summarized in Table 2. These data are from two human volun-teer studies carried out by Völkel et al. (2002, 2005). Whereas, the2002 study involved the exposure of 6 volunteers (3 male, 3 fe-male) to 5 mg/person in a hard gelatin capsule, the 2005 study de-scribed the kinetics of d16-BPA in three healthy female and threehealthy male human subjects receiving 25 lg/person orally via50 ml of water. In both studies, nearly the totality of the dosewas collected in the urine, little or no free BPA was found in urine,and the half-life estimates from the two studies were similar. Inthe 2002 study, d16-BPA urine samples were collected in 6 h inter-vals, for up to 96 h. No free d16-BPA was found in urine or plasmasamples collected, confirming the rapid metabolism of this com-pound. The d16-BPA glucuronide was rapidly cleared from theblood (t1/2:5.3 h) and completely eliminated in urine (118 ± 21%;t1/2:5.4 h). In the 2005 study, BPA (25 lg/person) was orallyadministered to six healthy individuals (3 women and 3 men),and urine samples were collected at 0, 1, 3, 5, and 7 h. Determina-tion of total BPA excreted employed hydrolyzation of BPA-G so thatboth conjugated and free BPA were detected in the analysis. Of the

Table 2Kinetic information for Bisphenol A in humans.

Species Study description Excretion rate References

Human Six healthy adult volunteers,orally exposed to 60–80 lg/kgd16-BPA*free BPA was not found in

urine

100% was excretedin urine within 96 h(t1/2 = 5.4 h)

Völkelet al. (2002)

Human 6 healthy adult volunteers,Orally exposed to 0.35–0.45 lg/kg d16-BPA*low levels of free BPAwere found in urine of 2of the subjects, accountingfor 2% of administered dose

84%-97% wasexcretedin urine within 5 h(t1/2 = 4 h)

Völkelet al. (2005)

amount eliminated within 5 h, these two species together ac-counted for 84% and 97% of the administered dose in women andmen, respectively.

2.2.2. Laboratory animalsData on the pharmacokinetics of BPA in rat (the species used in

the derivation of the guidance value presented in Table 1) areavailable (Kurebayashi et al., 2003; Knaak and Sullivan, 1966;Pottenger et al., 2000) and have been recently reviewed (EFSA,2008). These data have been extensively used in the developmentof PBPK models discussed below.

2.2.3. PBPK modelsSeveral PBPK models for BPA have been published (Shin et al.,

2004; Teeguarden et al., 2005; Kawamoto et al., 2007; Edgintonand Ritter, 2009; Mielke and Gundert-Remy, 2009). Whereas thePBPK model developed by Kawamoto et al. (2007) has been evalu-ated in mice and not in rats or humans, those of Edginton and Ritter(2009) and Mielke and Gundert-Remy (2009) have not been evalu-ated for their predictive capacity of free BPA in rats or humans.

The model developed by Teeguarden et al. (2005) consisted ofthe following compartments: blood, uterus, liver, lumen of the GItract and rest of the body compartment (for the remaining per-fused tissues). This model included BPA and BPA-G submodels,such that enterohepatic recirculation and elimination of BPA-Gcould be simulated. The model described the blood kinetics in ratsfollowing intravenous and oral exposures; it was less accurate withrespect to simulations of BPA-G kinetics than BPA kinetics in hu-mans. Given the inadequate consideration and description of glu-curonidation in this model (Willhite et al., 2008) and given theadequacy of the use of a mass-balance approach to urinary excre-tion of BPA-G (vide infra), we elected not to use this PBPK model forthe BE derivation.

The PBPK model developed by Shin et al. (2004) focused on theprediction of the tissue distribution and blood pharmacokinetics ofBPA in rats and humans. The model consisted of the following com-partments: vein, artery, lung, liver, spleen, kidneys, heart, testes,muscle, brain, adipose tissue, stomach, and small intestine. Predic-tions of the model compared well with the experimental data inrats. The model, scaled to humans, predicted the plasma kineticsin humans following single iv and multiple oral doses; the predic-tions in fact were comparable to that of the allometric model ofCho et al. (2002). However, this PBPK model did not describe theurinary elimination kinetics of BPA and its metabolites, and thusis of little use in the context of interpretation of urinary biomarkersfor BPA.

2.3. Potential biomarkers

As discussed above, evidence suggests the parent compound,BPA, is the toxicologically-relevant chemical form. Therefore, theconcentration of free BPA in blood or plasma and available forinteraction with the estrogen receptor would be the dose metricmost relevant to the mechanism of action. As reported by severalauthors, in humans, BPA undergoes extensive first-pass hepaticmetabolism via glucuronidation and sulphate formation after oralexposure. Thus, blood and urine concentrations of free BPA follow-ing exposure to environmentally relevant doses of BPA are verylow, and BPA-G is the major form of BPA present in plasma and ex-creted in urine (Völkel et al., 2002, 2005; Mahalingaiah et al.,2008). However, determination of the very low levels of free BPAin blood or plasma resulting from environmental exposures isnot plausible, at least with the current technology (Dekant andVölkel, 2008), challenging its use as a biomarker at the presenttime. Because essentially all of an orally administered dose ofBPA is recovered in urine as glucuronide and sulfur conjugates

AnimalPOD

UFA

External Relevant MonitoredDose Internal Biomarker

Dose

Animal

K. Krishnan et al. / Regulatory Toxicology and Pharmacology 58 (2010) 18–24 21

within 24 h, the quantification of total urinary BPA after hydrolysisof the conjugates has generally been used as a biomarker of expo-sure to BPA in biomonitoring studies, even though, from a mode ofaction perspective, this biomarker is expected to be much lessinformative than concentrations of free BPA in plasma or serum(Völkel et al., 2008; LaKind and Naiman, 2008) (Table 3).

HumanEquiv.PODHuman

Urinary excretion fraction data Adjust for avg.urinary volume and creatinine excretion

HumanEquiv.BEPOD

UFH

Target UrinaryBPA total

Fig. 1. General schematic for derivation of urinary BE values for BPA (Animal POD:50 mg/kg/d (US EPA, 1993), 25 mg/kg/d (Health Canada, 2008) and 5 mg/kg/d (EFSAEU, 2006).

2.4. BE derivation

Controlled studies have been conducted in which human volun-teers were exposed to BPA via the oral route (Völkel et al., 2002,2005). These and other supporting studies provide the basis forderivation of the BE for the total BPA in urine, based on a mass-balance approach consistent with steady-state exposure to theexposure guidance values. Because BPA has been demonstratedin humans to be eliminated essentially completely in urine (pre-dominantly as the glucuronide conjugate) within 24 h, under con-ditions of steady-state, ongoing exposure, daily excretion willequal daily intake (LaKind and Naiman, 2008; Edginton and Ritter,2009).

The process of BE derivation consists of the following steps foreach of the available exposure guidance values (Fig. 1):

1. Identify the POD used as the basis for the derivation of theguideline exposure reference values (e.g., pTDI or RfD).

2. Apply any uncertainty factors used in the guideline value deri-vation to account for interspecies extrapolation and lowestobserved adverse effect level (LOAEL) (if needed) to NOAELextrapolation, to identify the human-equivalent POD.

3. Estimate the daily urinary excretion of BPAtotal (e.g., LaKind andNaiman, 2008) rationalized by average urine volume and aver-age creatinine excretion at the daily exposure rate equal to thehuman-equivalent POD, assuming 100% elimination of BPA inurine. (Völkel et al. 2002, 2005). The result of this calculationis the urinary BEPOD.

4. Apply the intraspecies uncertainty factors to the BEPOD to derivethe BE.

Step 3 was supported with age- and gender-specific estimatesof average bodyweight, 24 h urinary volumes and 24 h creatinineexcretion. Age-specific estimates of bodyweight and average 24-hurinary volumes or average 24-h creatinine excretion providedan estimate of the 24-h average urinary BPA concentration result-ing from steady-state exposure to a unit dose of BPA for differentgroups (no assessment of values for children under age 6 is pre-sented due to the lack of reliable data on urinary volume and cre-atinine excretion rates). Specifically, the estimated BPA urinaryconcentration on a volume basis was calculated using the follow-ing formula:

Table 3Potential biomarkers of exposure to BPA.

Analyte Medium Advantages

Parent compound BPA Blood Would be expected to be highly relepotential adverse effects

BPA Urine Non-invasive

Metabolite BPA-G Blood –

BPA-G Urine Major urinary metabolite for BPA; spbiomarker of exposure; non-invasive

Metabolite BPA-S Blood –

BPA-S Urine Specific biomarker of exposure; non-

Note: BPA – bisphenol A, BPA-G – BPA-monoglucuronide, BPA-S – sulfate conjugate of B

CV ¼D� BW � FUE

Vð1Þ

where CV is the average urinary concentration on a volume basis ofBPA, D is a unit dose of BPA (1 lg/kg-d) as described in Table 4, BWis the bodyweight for the group, FUE is the urinary excretion fraction(=1 for BPA), i.e., fraction of the applied dose excreted in the urineand V is the 24-h average urinary volume.

It is relevant to note that FUE in the Eqn. above refers to the frac-tion of the BPA dose appearing in the urine. It does not make anyparticular assumptions regarding bioavailability or absorbed frac-tion; rather it represents data-based derivation of the amount ofBPA appearing in the urine in relation to the administered dose.

Similarly, the average creatinine-adjusted concentration wascalculated as follows:

CC ¼D� BW � FUE

CEð2Þ

where CC is the creatinine-adjusted 24-h urinary concentration ofBPA, and CE is the average 24-h creatinine excretion rate. Estimatesof average urinary volume and creatinine excretion rates weredrawn from a variety of studies (see footnotes to Table 4).

Using the estimates of the 24-h average urinary concentrationassociated with a unit dose of BPA (Table 4), the urinary concentra-tions (on both a volume and creatinine-adjusted basis) for the hu-man-equivalent POD (original POD divided by the interspeciesuncertainty factor) and guidance values listed in Table 1 were esti-mated and reported in Table 5. Because the average concentrations

Disadvantages

vant to Short half-life and low levels; invasive sampling required;possible background contamination of the samplesLittle is excreted unchanged in urine; possible backgroundcontamination of the samples

Short half-life in humans; not directly relevant to mode of action;invasive sampling required.

ecific Not directly relevant to mode of action

Not detected in blood in human studies; not directly relevant tomode of action; invasive sampling required

invasive Less present than BPA-G; not directly relevant to mode of action

PA.

Table 4Assumptions for bodyweight, average 24-h urinary volume and creatinine excretion,and estimates of average volume-based and creatinine-adjusted urinary concentra-tion of total BPA (free and conjugated species) consistent with exposure at a unit dose(1 ug/kg-d).

Age Group Bodyweight,kga

Average 24 hurinary volume,Lb (creatinineexcretion, gc)

BPA urinaryconcentration,lg/L (lg/g creatinine)per lg/kg-doral dose

Children, 6–11 32 0.66 48.5(0.5) (64.0)

Adolescents, 11–16 57 1.65 34.5(1.2) (47.5)

Men > 16 70 1.7 41.2(1.5) (46.7)

Women > 16 55 1.6 34.4(1.2) (45.8)

Average, lg/L: 39.6Average: lg/g cr: (51.0)

a Estimated from table 8–1 of US EPA (2008).b Urinary volumes for children from Remer et al. (2006). Volumes for adults from

Perucca et al. (2007). Adolescents were assumed to have urinary volumes similar toaverage values for adults.

c Creatinine excretion for children and adolescents estimated from Remer et al.(2002); average creatinine excretion for boys and girls under age 13, 17 mg/kg BWper day; average creatinine excretion for adolescents, 22 mg/kg BW per day. Cre-atinine excretion for adults estimated based on equations from Mage et al. (2004),average US height, and specified bodyweights.

22 K. Krishnan et al. / Regulatory Toxicology and Pharmacology 58 (2010) 18–24

associated with a unit dose of BPA varied relatively little across ageand gender groups (due to the simultaneous correlation of bothBPA dose rates and urinary volume and creatinine excretion rateswith bodyweight), the average across all age groups was estimatedand carried forward in calculations for the exposure guidance val-ues. This approach allows a direct translation of dose levels ofinterest (for example, Health Canada’s pTDI) into expected averageurinary concentrations.

3. Discussion

3.1. Sources of variability and uncertainty

The BE derived in this document relates to the total BPA in urineand was based on the human data of Völkel et al. (2002, 2005).

Table 5Derivation of BE values consistent with exposure guidance values from Table 1. Seealso Fig. 1 for a schematic representing the process.

BEderivationsteps

Exposure guidance value: pTDI,HealthCanada(2008)

RfD, USEPA(1993)

TDI,EFSA(2006)

1 POD, mg/kg-d: 25 50 52 Uncertainty factors (LOAEL to

NOAEL, subchronic to chronic,and interspecies; see Table 1):

100 100 10

Human-equivalent POD, mg/kg-d:

0.25 0.5 0.5

3 BPA concentration in urine perunit dose, mg/L per mg/kg-d:

39.6 39.6 39.6

(mg/g creatinine per mg/kg-d): (51.0) (51.0) (51.0)BEPOD, mg/L: 10 20 20(mg/g creatinine): (13) (26) (26)

4 UFH: 10 10 10BE, mg/L: 1 2 2(mg/g creatinine): (1.3) (2.6) (2.6)

Note: BE – biomonitoring equivalent, BEPOD – biomonitoring equivalent point ofdeparture, POD – point of departure, pTDI – provisional tolerable daily intake, RfD –reference dose, UFH – inter-individual uncertainty factor, TDI – tolerable dailyintake.

These data and thus the calculated BE values correspond to the to-tal BPA in urine – which reflects the sum total of free BPA and BPA-G. The free BPA in urine would appear to be very low (less than 2%of applied dose) and probably unlikely to be quantitated in a repro-ducible manner.

One of the sources of variability and uncertainty associatedwith the BE values presented in Table 5 is the relatively shorthalf-life for excretion of the BPA (less than 6 h). Because of this ra-pid elimination, substantial variations in urinary concentrationswill be expected over the course of a day following exposure,and therefore, a spot urine sample will not be a good estimatorof the 24 h intake (Arakawa et al., 2004) for an individual. If expo-sures to BPA are expected to occur primarily through food intake,spot urine samples taken in the morning may be expected to haverelatively low concentrations, which might be expected to rise dur-ing the day as exposures occur. The relative magnitude of peaksand troughs of measured urinary concentrations will depend onthe timing of spot urine samples compared to exposure, as wellas on the timing of previous urinary void(s) (Aylward et al.,2009), and the within-day variation in urinary concentrationsmay be substantial. For this reason, the guidelines for the deriva-tion and communication of Biomonitoring Equivalents specify that,for relatively short-lived compounds, only the central tendency ofmeasures from population sampling efforts should be compared toBE values in order to provide a general evaluation of overall popu-lation exposure levels in comparison to exposure guidance values(Hays et al., 2008; LaKind et al., 2008).

Further, the short half-life of BPA means that spot urinary sam-ples can only be interpreted in terms of short-term exposure pat-terns, and can be generalized to longer term exposures only ifother information about exposures is known. Mahalingaiah et al.(2008) evaluated occasional repeated measures of urinary BPA inindividuals over several months and concluded that a single spotsample provided moderate predictive value for subsequent sam-pling events. However, the authors did not control for time ofday of urine sample collection, which may affect the pattern ofmeasured values.

Other sources of potential variation in measured urinary con-centrations and uncertainty in the interpretation of biomonitoringdata include variations in hydration status and creatinine excretionrates. Even under conditions of exposure consistent with the RfD,intra- and inter-individual variations in hydration status and creat-inine excretion rates can impact the measured concentrations inspot urine samples by a factor of 2 to 3. The appropriateness ofadjustment for hydration status using creatinine excretion has beendebated, in general and specifically with respect to BPA (Gardeet al., 2004; Barr et al., 2005; Mahalingaiah et al., 2008; LaKindand Naiman, 2008). Mahalingaiah et al. (2008) concluded that cre-atinine adjustment for evaluation of urinary BPA concentrationscould be inappropriate because organic compounds are eliminatedby active tubular secretion. Furthermore, creatinine concentrationsare influenced by multiple factors such as: rate of glomerular filtra-tion, body mass, age, gender, health status, muscularity, physicalactivity, diets or time of day (LaKind and Naiman, 2008). However,LaKind and Naiman (2008) evaluated the effect of using creatinine-adjusted urinary BPA compared to volume-based estimates of BPAintakes and found that they were highly correlated, suggesting thateither approach will provide comparable estimates.

Variations in urinary concentrations due to the short biologicalhalf-life and due to the variations in hydration status and creatinineexcretion rates can be reduced by collection and analysis of 24 hurine samples (Arakawa et al., 2004; Dekant and Völkel, 2008).

Other sources of uncertainty affect the interpretation of the BEvalues presented here. The pharmacokinetic data on BPA urinaryexcretion rate come from two studies from Völkel et al. (2002,2005), each conducted with controlled exposure of six adult

K. Krishnan et al. / Regulatory Toxicology and Pharmacology 58 (2010) 18–24 23

volunteers. Although the degree of variability among individualsin the studies was considerable, the conclusion that BPA is essen-tially entirely excreted in urine within 24 h holds across all of thetested individuals. For the purposes of the steady-state derivationpresented here, which postulates ongoing exposure and excretionunder steady-state conditions consistent with the definitions ofthe guidance values, variations in the precise kinetic behavior donot affect the overall results of the BE calculation.

Background contamination from use of plastic materials duringsample preparation or use of solvents and reagents during extrac-tion and analysis can affect urine samples and, consequently, inter-fere with the low level concentration quantification (Dekant andVölkel, 2008; Völkel et al., 2005). There has not been any standard-ized quality assurance (QA) study, addressing either holding timeor other parameters which can influence quality control (QC) ofthe BPA biomonitoring data. In this regard, it is important to devel-op standard operating procedures, including QA/QC criteria, foranalysis of BPA and its metabolites in urine and plasma.

3.2. Confidence assessment

The guidelines for derivation of BE values (Hays et al., 2008)specify consideration of two main elements in the assessment ofconfidence in the derived BE values: robustness of the availablepharmacokinetic data and models, and understanding of the rela-tionship between the measured biomarker and the critical or rele-vant target tissue dose metric. As previously discussed, thepharmacokinetic data for BPA come from two studies (6 individu-als each; Völkel et al., 2002, 2005). These data provide a reliable ba-sis for the derivation of urinary BE values based on the assumptionof steady-state exposure using a mass balance assumption. The uri-nary biomarker for BPA is principally derived from the BPA glucu-ronide conjugate, which may not be directly related to the criticalor relevant dose metric for the toxicity of BPA but can still be usefulas a biomarker of exposure.

Thus, the assessment of the confidence level in the derived BEvalues based on these two factors is as follows:

o Robustness of pharmacokinetic data: MEDIUM.o Relevance of biomarker to relevant dose metrics: LOW.

As the analytical methods or PBPK models become available tofacilitate the quantitation of low levels of free BPA levels in plasmawith confidence, plasma-based BEs can be developed. It has beenreported that the limit of detection of the available analytical meth-ods is 2.3 ng/ml which is about 1000 times greater than the plasmaBPA level simulated by the PBPK models for highest measuredexternal exposure (Völkel et al., 2008; Mielke and Gundert-Remy,2009). Moreover, the available PBPK models have not been verifiedfor their predictive ability of free BPA in plasma. Therefore, the cur-rent state of knowledge only permits the development of urine-based BE on the basis of mass balance consideration for total BPAfound in urine (which is essentially reflective of BPA-G since freeBPA is extremely low).

3.3. Interpretation of biomonitoring data using BE values

The BE values presented here represent estimates of the 24-haverage concentrations of BPA in urine that are consistent withthe existing exposure guidance values resulting from the riskassessments conducted by various governmental agencies as listedin Table 1. These BE values were derived based on current under-standing of the pharmacokinetic properties of these compoundsin humans. These BE values do not represent independent riskassessments for BPA, but rather are translations of the existing riskassessments from Table 1. These BE values should be regarded as

interim screening values that can be updated or replaced if theexposure guidance values are updated or if the scientific and regu-latory communities develop additional data on acceptable or toler-able concentrations in human biological media.

Mielke and Gundert-Remy (2009) recently published a model-ing effort for BPA in which they used simple pharmacokinetic ap-proaches to estimate plasma or blood concentrations associatedwith measured urinary excretion rates and estimates of exposure.The urinary BE values presented here could be used in conjunctionwith these simple pharmacokinetic models to estimate plasma orblood concentrations consistent with these BE values and withthe underlying exposure guidance values if blood or plasma bio-monitoring for BPA becomes more common.

The appropriate uses and limitations of urinary BE values havebeen discussed previously (Aylward and Hays, 2008; Aylwardet al., 2009; Hays et al., 2008). As suggested by LaKind and Naiman(2008), additional research is needed to improve estimation of gen-eral population daily intake of BPA, particularly to characterize ef-fect of age and gender on 24 h urinary excretion and to evaluatethe reliability of spot urine sampling when it comes to evaluatedaily or longer-term level of exposure.

These BE values can be used as a screening tool to evaluate pop-ulation- or cohort-based biomonitoring data in the context of exist-ing risk assessments. Concentrations in excess of the BE values, butless than the BEPOD values represent medium priority for risk assess-ment follow-up, while those in excess of the BEPOD indicate high pri-ority for risk assessment follow-up. Based on the results of suchcomparisons, an evaluation can be made of the need for additionalstudies on exposure pathways, potential health effects, other as-pects affecting exposure or risk, or other risk management activities.

BE values do not represent diagnostic criteria and cannot beused to evaluate the likelihood of an adverse health effect in anindividual or even among a population. Measured values in excessof the identified BE values may indicate exposures at or above thecurrent exposure guidance values that are the basis of the BE der-ivations. However, as discussed above, measured concentrationsabove the BE values, which are based on 24-h average urinary con-centrations, would be expected even if exposures do not exceedthe exposure guidance values due to the transient concentrationprofiles in urine expected for these compounds, variations inhydration status, and other factors discussed further above. Thus,interpretation of data for individuals or of tails of the distributionin population-monitoring studies is not appropriate.

In addition, the exposure guidance values for BPA were derivedwith a substantial margin from doses that resulted in no observedeffect in the underlying animal toxicity studies. Thus, these valuesare not ‘‘bright lines” that distinguish safe from unsafe exposurelevels. Chronic exposure guidance values are set at exposure levelsthat are expected to be protective over a lifetime of exposure. Forshort-lived compounds such as BPA, an exceedance of the corre-sponding BE value in a single urine sample may or may not reflectcontinuing elevated exposure. As demonstrated in the limitedavailable datasets and based on the kinetics of urinary elimination,spot urinary concentrations may vary substantially both withinand across days in an individual. Thus, occasional exceedances ofthe BE value in individuals in cross-sectional studies do not implythat adverse health effects are likely to occur, but can serve as anindicator of relative priority for further risk assessment follow-up. Further discussion of interpretation and communication as-pects of the BE values is presented in LaKind et al. (2008) and atwww.biomonitoringequivalents.net.

Conflict of Interest Statement

The authors declare they have no conflicts of interest.

24 K. Krishnan et al. / Regulatory Toxicology and Pharmacology 58 (2010) 18–24

Acknowledgments

Funding for this project was provided under a grant from HealthCanada. The views expressed are those of the authors and do notnecessarily reflect the views or policies of Health Canada. This BEdossier has undergone an independent peer-review to assure themethods employed here are consistent with the guidelines for der-ivation (Hays et al., 2008) and communication (LaKind et al., 2008)of Biomonitoring Equivalents and that the best available chemical-specific data was used in calculating the BEs. We thank the variousreviewers for their insightful suggestions.

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