Regulatory Toxicology and Pharmacology 58 (2010) 10–17

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Biomonitoring Equivalents for triclosan

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, Ontario, 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 EquivalentsTriclosanRisk assessmentPharmacokinetics

0273-2300/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.yrtph.2010.06.004

* 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(s) in a biologicalmedium (blood, urine, or other medium) consistent with an existing health-based exposure guideline,and are derived by integrating available data on pharmacokinetics with existing chemical risk assess-ments. This study reviews available health-based exposure guidance values for triclosan based on recentevaluations from the United States Environmental Protection Agency (US EPA), the European Commis-sion’s Scientific Committee on Consumer Products (EC SCCP) and the Australian National IndustrialChemicals Notification and Assessment Scheme (NICNAS). BE values corresponding to the reference dose(RfD) or margin of safety (MOS) targets from these agencies were derived based on kinetic data (urinaryexcretion and plasma clearance) from human studies and measured blood concentration data in animalstudies. Estimated BE values for urinary total triclosan (free plus conjugates) corresponding to the US EPARfD and the EC-identified margin of safety target from the NOAEL are 6.4 and 2.6 mg/L, respectively (cor-responding to 8.3 and 3.3 mg/g creatinine, respectively). Plasma BE values corresponding to the US EPA,EC, and Australian NICNAS values are 0.3, 0.9, and 0.4 mg/L, respectively. These values may be used asscreening tools for evaluation of population biomonitoring data for triclosan in a risk assessment context.

� 2010 Elsevier Inc. All rights reserved.

1 Abbreviations used: BE, biomonitoring equivalent; BEPOD, biomonitoring equiva-lent point of departure; BMD, benchmark dose, BW, body weight; CAS, chemical

1. Introduction

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 only beinterpreted in terms of exposure trends, but cannot be used toevaluate which chemicals may be of concern in the context of cur-rent risk assessments. Such screening criteria would ideally bebased on robust datasets relating potential adverse effects to bio-marker concentrations in human populations (see, for example,the U.S. Centers for Disease Control and Prevention (CDC) bloodlead level of concern; see http://www.cdc.gov/nceh/lead/). How-ever, development of such epidemiologically-based screening cri-teria is a resource and time-intensive effort. As an interim

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(L.L. Aylward).

approach, the development of Biomonitoring Equivalents (BEs)1

has been proposed, and guidelines for the derivation and communi-cation of these values have been developed (Hays et al., 2007, 2008;LaKind et al., 2008).

A Biomonitoring Equivalent (BE) is defined as the concentrationor range of concentrations of chemical in a biological medium(blood, urine, or other medium) that is consistent with an existinghealth-based exposure guidance value such as a reference dose(RfD) or tolerable daily intake (TDI). BEs are estimated on the basisof existing chemical-specific pharmacokinetic data (animal or hu-man) and the point of departure (e.g., NOAEL, LOAEL, BMD) usedin the derivation of exposure guidance values (Hays et al., 2008).

abstracts services; EC, European Commission, LOAEL, lowest observed adverse effectlevel; MOE, margin of exposure; MOS, margin of safety; NHANES, National Health andNutrition Examination Survey; NICNAS, National Industrial Chemicals Notificationand Assessment Scheme; NOAEL, no observed adverse effect level; POD, point ofdeparture; PK, pharmacokinetic; RED, re-registration eligibility decision; RfD, refer-ence dose; SCCP, scientific committee on consumer products; TDI, tolerable dailyintake; USEPA, United States Environmental Protection Agency.

K. Krishnan et al. / Regulatory Toxicology and Pharmacology 58 (2010) 10–17 11

BEs are intended for use as screening tools to allow an assessmentof biomonitoring data to evaluate which chemicals have large,small, or no margins of safety compared to existing risk assess-ments and exposure guidance values. This document presents der-ivation of BEs for triclosan (Chemical Abstracts Services [CAS]Registry number 3380-34-5).

Triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol) is a lipo-philic (measured log n-octanol:water partition coefficient = 4.8(Ciba-Geigy (1990) cited by NICNAS (2009)), broad-spectrum anti-microbial agent that has found widespread use in a variety of per-sonal care products including toothpaste, mouthwash, bar soap,deodorant, shower gel as well as skin-care and make-up products(Engelhaupt, 2007; McGinnis, 2008; EC, 2009). It is also used inconsumer products such as textiles, toys and plastic kitchenware(e.g., Adolfsson-Erici et al., 2002; Bhargava and Leonard, 1996; Per-encevich et al., 2001; Yazdankhah et al., 2006). There has been in-creased focus on the occurrence of triclosan in biological matricesof individuals without occupational exposures as well as on theevaluation of the potential for endocrine-disrupting effects (Crof-ton et al., 2007; Zorrilla et al., 2009). A number of human biomon-itoring studies have reported the occurrence of triclosan in breastmilk, urine and plasma (Calafat et al., 2008; Hovander et al.,2002; Dayan, 2007; Allmyr et al., 2008; Wolff et al., 2007; Sand-borgh-Englund et al., 2006). The analyses of 2517 single spot urinesamples as part of the National Health and Nutrition ExaminationSurvey (NHANES: 2003-04) indicated that three-quarters of thesamples contained triclosan (Calafat et al., 2008).

The present study focused on establishing BEs for triclosanbased on the available data on point of departure (POD) and guide-line values, as well as the pharmacokinetic information.

2. Available data and approach

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

Based on our review of information from US, Canadian, Austra-lian and European sources, three recent risk assessments and eval-uations for triclosan were found to be available. Of theseassessments, that of the Australian government (NICNAS, 2009)identified a NOAEL of 41 lg/mL (corresponding to the steady-stateplasma level of triclosan in rats administered 40 mg/kg/d) as a ba-sis for estimating MOE based on measured plasma values in hu-mans. In essence, a BE value based on a target minimal margin ofexposure of 100 was derived in the NICNAS (2009) risk assessment.However, we have included this derivation in this article forcompleteness. The US EPA Office of Pesticide Programs completeda recent re-registration eligibility decision (RED) document for tri-closan. In that review, they based the derivation of a chronic refer-ence dose (RfD) on a chronic dietary study conducted in baboonswith a no-observed-adverse-effect-level of 30 mg/kg-d and acomposite uncertainty factor of 100 comprised of interspecies

Table 1Health risk assessments and health-based exposure reference values for triclosan identifie

Organization Study description Critical endpoint and dose

USEPA (2008a) Chronic toxicity study inbaboons (Drake, 1976)

NOAEL = 30 mg/kg based ontoxicity such as vomiting, fail

EC (2009) 2-Year chronic rat bioassay(DeSalva et al., 1989)

NOAEL = 12 mg/kg/d based oand decrease in absolute and

NICNAS (2009)(Australia)

2-Year chronic rat bioassay(DeSalva et al., 1989)

Plasma concentration associa41 mg/L based on liver effect

a Corresponds to values calculated by dividing the point of departure (POD) by the ap

and intraspecies uncertainty factors of 10 each (USEPA, 2008a)(Table 1).

The European Commission (2009) also recently evaluated tri-closan. In that review, the European Commission’s Scientific Com-mittee on Consumer Products (SCCP) identified a point ofdeparture (POD) (NOAEL of 12 mg/kg-d) from a well-conducted ro-dent chronic bioassay and recommended a target margin of safety(MOS) of 100. Based on this assessment, a target daily dose levelanalogous to a reference dose or tolerable daily intake can alsobe derived (Table 1).

The information on mode of action in mammals and associatedrelevant dose metrics is limited for triclosan. The mechanism ofantibacterial action of triclosan has been reported to involve theinhibition of lipid synthesis by blocking the enoyl-acyl reductaseenzyme (McMurry et al., 1998; Heath et al., 1999). Inhibition offatty acid synthesis in parasites has also been documented (Suroliaet al., 2001; Samuel et al., 2003). Further, it has been demonstratedthat this chemical prevents bacterial cell growth and proliferationby interfering with the formation of new cell membranes (Levyet al., 1999). However, the relevance or ability of these mecha-nisms leading to adverse health effects in humans has not beendemonstrated (Sullivan et al., 2003; Sandborgh-Englund et al.,2006).

In mammals, triclosan is reported to alter serum concentrationof thyroxine (Crofton et al., 2007) and to interact with P450-depen-dent enzymes, UDP-glucuronosyltransferases and the human preg-nane X receptor (Hanioka et al., 1996; Jacobs et al., 2005; Wang etal., 2004). The relevance for humans of these interactions andtoxicological endpoints identified in animal studies is not known(Calafat et al., 2008; USEPA, 2008a; Allmyr et al., 2009; EC, 2009).

The effects identified at levels exceeding the NOAEL levels in thebaboon study were relatively non-specific. At doses above theNOAEL in the baboon study, clinical signs of toxicity included vom-iting, failure to eat and diarrhea (Ciba-Geigy, 1977; USEPA, 2008a).The rat NOAEL was based on hematotoxicity as well as decreases inabsolute and relative spleen weights; at higher doses, mild clinicalchemistry and/or hematology changes, together with histopathol-ogical changes in the liver were reported (EC, 2009; Ciba-Geigy,1986; NICNAS, 2009).

Even though there is no definitive information on the mode ofaction or relevant dose metrics for the triclosan-induced effectsin rats and baboons, it would appear that the parent chemical islikely to be the toxic form, given that triclosan does not undergoany oxidative metabolism or bioactivation reaction and has beendocumented to conjugate with UDP-glucuronic acid and sulfate(DeSalva et al., 1989). Thus, plasma concentrations of triclosanwould appear to be relevant to potential toxicity.

2.2. Available pharmacokinetic data

The available data on the pharmacokinetics of triclosan in ani-mals and humans exposed by various routes and vehicles have

d in the current study.

Uncertainty factors Guideline valuea

clinical signs ofure to eat and diarrhea

100– Interspecies UF: 10– Intraspecies UF: 10

0.3 mg/kg-d

n hematoxicityrelative spleen weights

100 - Interspecies UF: 10 -Intraspecies UF: 10

0.12 mg/kg-d

ted with NOAEL:s

Target minimal margin of exposureon a plasma basis: 100– Interspecies UF: 10– Intraspecies UF: 10

0.4 mg/L in plasma

plicable uncertainty factors.

Table 2Summary of urinary excretion data for triclosan in baboons and humans.

Species Study description Excretion rate References

Human – 44–57% of the oral dose was excreted within 48 h Ciba-Geigy Limited (1976) fromNICNAS (2009)

Human Ten volunteers were orally exposed to a single dose (4 mg) 54% of the oral dose was excreted by 96 h Sandborgh-Englund et al. (2006)Baboons Oral administration of approximately 5 mg/kg bw

14C-triclosan to two male baboons53–60% was excreted in urine 20–30% wasexcreted in the feces after 144 h

Ciba-Geigy Limited (1977) fromNICNAS (2009)

12 K. Krishnan et al. / Regulatory Toxicology and Pharmacology 58 (2010) 10–17

been summarized by the National Industrial Chemicals Notifica-tion and Assessment Scheme (NICNAS) of Australia (2009) and EC(2009). Triclosan is extensively conjugated with UDP-glucuronicacid and sulfate, and excreted via urine or feces. In humans, urinaryexcretion is the principal route of elimination; however, in the rat,triclosan is preferentially eliminated via the fecal route. The fol-lowing discussion focuses on data relevant to the derivation ofBE values for triclosan.

DeSalva et al. (1989) summarized the pharmacokinetic dataassociated with the 2-year chronic dietary bioassay in the rat. Spe-cifically, the concentrations of triclosan in blood, liver and kidneyof treated animals were determined at 3, 6, 12, 18 and 24 monthsof treatment. The data indicate: (i) the attainment of steady-stateduring chronic exposure to triclosan, (ii) the predominance of con-jugates rather than free form in the various matrices, and (iii) theproportionality of triclosan concentration with exposure dose inthe rat. The EC SCCP (2009) used these blood data to estimate plas-ma concentrations of total triclosan (free and conjugates) at se-lected time points during the bioassay (see Table 27 in EC (2009)).

A particularly relevant pharmacokinetic study in humans is thatof Sandborgh-Englund et al. (2006). In this study, ten subjects (5males and 5 females) swallowed a single oral dose of 4 mg triclo-san (average estimated dose of 0.06 mg/kg-d). The total triclosanplasma levels were determined at 1, 2, 3, 4, 6, 9, 24, 48, 72, 96and 192 h after dosing, whereas the total urinary levels were deter-mined in 24-h composite samples. The maximal concentration andhalf-lives for plasma clearance and urinary elimination in eachindividual were obtained. Overall, these data indicate median plas-ma and urinary half-lives of 19 and 11 h, respectively. Mediancumulative urinary excretion of triclosan as conjugated speciesand free compound was 54% of the administered dose after 4 days(ranging from 24% to 84%), with the majority of the eliminationoccurring in the first two days. Free parent triclosan accountedfor less than 1% of the eliminated compound. Other pharmacoki-netic data collected by Ciba Geigy in baboons and humans indi-cates similar behavior; these data on urinary kinetics of triclosanwere described by NICNAS and are summarized in Table 2 (Ciba-Geigy, 1986; NICNAS, 2009).

2.3. Potential biomarkers

Triclosan glucuronide is the major metabolite and urinaryexcretion is the principal route of elimination in humans. As such,the total triclosan in urine (conjugates + free form) has been usedas biomarker of exposure (e.g., Calafat et al., 2008; NICNAS,2009). The plasma (or blood) level of total triclosan is also a rele-vant marker, as determined in a 2-year toxicological bioassay(DeSalva et al., 1989). As with other biomonitoring efforts, collec-tion of urinary samples is less invasive and more straightforwardthan collection of blood samples, but sampling of blood or plasmamay provide a more toxicologically relevant measure of exposure.

Fig. 1. Schematic representation of the approach used for deriving BE values forurinary total triclosan concentration (free plus conjugated compound).

3. BE derivation

The BE for triclosan should ideally correspond to a dose mea-sure in a biological matrix that relates most closely to the mode

of action, is reflective of the available pharmacokinetic (PK) data,and is based on the relationship between the biomarker and therelevant internal dose metrics. Triclosan in plasma is likely to bea relevant dose metric for prediction of toxicity, but is only foundin very low levels. Urinary triclosan (conjugates + free form), onthe other hand, exhibits greater uncertainty compared to plasmaconcentrations in its relation to tissue concentrations but it is stilluseful as a biomarker of exposure. Figs. 1, 2a and b, illustrate theapproaches used for computing the BE for urinary and plasma tri-closan, for the two PODs and associated guideline values identifiedfor triclosan (Table 1).

3.1. Urinary BE values

The existing data on elimination kinetics of triclosan (as parentcompound or conjugated) suggest a simple mass-balance ap-proach, with an assumption of steady-state intake and excretion,for derivation of BE values for urinary triclosan (Fig. 1). In this re-gard, the amount of triclosan excreted in urine every day will beapproximately equal to the human-equivalent amount ingestedtimes the urinary excretion fraction. The median of the estimatedfraction of oral triclosan dose excreted via urine from ten individ-uals (54% at 48 h) obtained from Sandborgh-Englund et al. (2006)was combined with age-specific estimates of bodyweight and aver-age 24-h urinary volumes (or average 24-h creatinine excretion) toprovide an estimate of the 24-h average urinary concentrationassociated with a unit dose of triclosan per day (Table 3) for differ-ent age groups. No assessment values for children under age 6were presented due to the lack of reliable data on urinary volumeand creatinine excretion rates. Specifically, the estimated triclosanurinary concentration on a volume associated with a unit dose oftriclosan for a specific population sub-group was calculated usingthe following formula:

CV ¼D� BW� FUE

Vð1Þ

Fig. 2a. Schematic representation of the approach used for deriving BE values fortotal triclosan in plasma based on the rat NOAEL (12 mg/kg-d) identified by EC(2009).

Fig. 2b. Schematic representation of the approach used for deriving BE values fortotal triclosan in plasma based on the baboon study NOAEL (30 mg/kg-d) identifiedby USEPA (2008a).

K. Krishnan et al. / Regulatory Toxicology and Pharmacology 58 (2010) 10–17 13

where CV is the average urinary concentration on a volume basis oftriclosan, D is a unit dose of triclosan (1 lg/kg-d) as shown in Table3, BW is the bodyweight for the group, FUE is the urinary excretionfraction, i.e., fraction of the applied dose excreted in the urine(0.54), and V is the 24-h average urinary volume. Similarly, the cre-atinine-adjusted concentration associated with a unit dose of triclo-san was calculated as follows:

CC ¼D� BW� FUE

CEð2Þ

where CC is the creatinine-adjusted 24-h urinary concentration oftriclosan, and CE is the 24-h creatinine excretion rate. Data on uri-nary volume and creatinine excretion rates were drawn from a vari-ety of studies (see footnotes to Table 3). It is relevant to note thatFUE in Eqs. (1) and (2) above refers to the fraction of the doseappearing in the urine. It does not make any particular assumptionsregarding bioavailability or absorbed fraction; rather it representsthe ratio of the amount appearing in the urine in relation to the

administered dose, based on actual data. The urinary concentrationsof triclosan associated with a unit dose of triclosan (D) for the dif-ferent age groups are presented in Table 3. Because the average con-centrations associated with a unit dose of triclosan varied littleacross age and gender groups, the average across all age groupswas estimated and carried forward in calculations. Using the esti-mates of the 24-h average urinary concentration associated with aunit dose of triclosan, the urinary concentrations (on both a volumeand creatinine-adjusted basis) for the human-equivalent POD (ori-ginal POD divided by the interspecies uncertainty factor) and guid-ance values listed in Table 1 were estimated and reported in Table 4.

3.2. Plasma BE values

For the guidance values described in Table 1, differentapproaches were used in the derivation of BE values for plasmaconcentrations based on the available datasets (see Figs. 2a andb). Because measured blood concentrations and estimates of corre-sponding plasma concentrations were available from the chronicrat bioassay used as the basis of the EC, 2009 risk assessment,we derived a BE starting directly from those measured data usingthe following approach (Fig. 2a):

(1) Estimate the steady-state plasma concentration in theexperimental animals dosed at the POD based on data fromthe underlying study (DeSalva et al., 1989). Based on the val-ues of blood concentration reported in Ciba-Geigy’s 2-yearstudy in Sprague–Dawley rats (see DeSalva et al. 1989),the EC, 2009 converted the measured blood concentrationsto an estimated average steady-state plasma concentrationof 28.16 mg/L at the POD (Table 27 in EC, 2009). However,this value corresponds to the interim measured value forfemale rats (exposed at approximately 17 mg/kd-d), whilethe EC-identified NOAEL was set at the dose level for malerats (12 mg/kg-d). The corresponding interim estimatedplasma concentration in male rats is 21.8 mg/L (Table 27,EC, 2009), and this value is used here for the BE derivation.

(2) Apply the pharmacodynamic component of the interspeciesuncertainty factor (100.5) to derive estimated plasma con-centrations for the human-equivalent BEPOD; and

(3) Apply the intraspecies uncertainty factor to the BEPOD toderive the BE.

A similar approach was used for the NICNAS (2009) risk assess-ment, except that risk assessment was based entirely on the mea-sured plasma concentrations in rats in DeSalva et al. (1989), andexplicitly specified a minimal margin of exposure 100 on a plasmaconcentration basis to identify a target plasma concentration.

For the NOAEL of 30 mg/kg-d in the baboons identified by USEPA(2008a) as the POD, the plasma values associated with the BEPOD andBEwerederivedasfollows(Fig.2b):

(1) Apply the interspecies uncertainty factor to identify thehuman-equivalent POD on an external dose basis.

(2) Estimate the steady-state plasma concentration, CP, in mg/Lin humans associated with the human-equivalent POD (i.e.,BEPOD) by dividing the daily dose, D, in mg/kg-d by the clear-ance, CL (plasma clearance normalized to the fraction ofdose absorbed; 0.041 L/(h kg)), estimated by Sandborgh-Englund et al. (2006) according to this formula:

CP ¼D

CL� 24ð3Þ

(3) Apply the intraspecies uncertainty factor to the BEPOD toderive the BE.

Table 3Assumptions for average bodyweight, 24-h urinary volume, and 24-h creatinine excretion rate and estimates of creatinine-adjusted and volume-based urinary concentration perunit dose of triclosan (lg/kg-d) by age group (considering a urinary excretion fraction of 54%)a.

Age Group Bodyweightb (kg) Average 24 h urinary volumec (L)(creatinine excretion, gd)

Triclosan urinary concentration per lg/kg-dsteady-state dose (lg/L) (lg/g creatinine)e

Children, 6–11 32 0.66 26.2(0.5) (34.6)

Adolescents, 11–16 57 1.65 18.7(1.2) (25.7)

Men, >16 70 1.7 22.2(1.5) (25.2)

Women,>16 55 1.6 18.6(1.2) (24.8)Average, lg/L 21.4(lg/g creatinine) (27.5)

a Urinary excretion fraction of 54% from Sandborgh-Englund et al. (2006).b Estimated from Table 8-1 of USEPA (2008b).c 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 to

average values for adults.d 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 BW per day;

average creatinine excretion for adolescents, 22 mg/kg BW per day. Creatinine excretion for adults estimated based on equations from Mage et al. (2004) and average USheight and specified bodyweights.

e Calculated using equations 1 (for volume-based values) or 2 (for creatinine-adjusted values). For example, using equation 1, for children aged 6–11, the triclosanconcentration in lg/L associated with a dose of 1 lg/kg-d at steady state would be 1 lg/kg-d * 32 kg/0.66 L/d = 26.2 lg/L.

Table 4Derivation of BE values for triclosan urinary concentration (on a volume and creatinine-adjusted basis) consistent with the guideline valuesderived from USEPA (2008a) and EC (2009), according to the scheme presented in Fig. 1. Reported concentrations are the sum of both freeand conjugated triclosan in urine.

BE derivation step USEPA (2008a) RfD EC (2009) risk assessment

Species, endpoint Baboons, general toxicity Rats, hematological endpoint alterationsPOD (NOAEL)a (mg/kg-d) 30 12UF, interspecies 10 10Human-equivalent PODb (mg/kg-d) 3 1.2BEPOD, mg/L in urine 64 26(mg/g creatinine) (83) (33)UF, intraspecies 10 10BE, mg/L in urine 6.4 2.6(mg/g creatinine) (8.3) (3.3)

a From Table 1.b Estimated using the increments in urinary concentrations per unit of steady-state dose reported in Table 3.

14 K. Krishnan et al. / Regulatory Toxicology and Pharmacology 58 (2010) 10–17

The derivation and the resulting values for both guidance valuesare summarized in Table 5.

4. Discussion

4.1. Sources of variability and uncertainty

The urinary and plasma BE values for triclosan derived in thisevaluation were based on the average values of input parameters;however, the resulting values accounted for inter-individual vari-ability by way of the use of uncertainty factors.

For the derivation of urinary BE values, the median urinaryexcretion fraction of 54% of an oral dose of triclosan reported bySandborgh-Englund et al. (2006) was used. This value was obtainedwith 10 study subjects aged between 26 and 42 years (5 femalesand 5 males). The authors reported that the major fraction of triclo-san was excreted within the first 24 h. The lower and upper quar-tile values of fraction excreted were 47% and 61% at 48 h; theindividual values of the fraction excreted, however, varied from24% to 83% after 4 days of exposure (median value = 54%) (Sand-borgh-Englund et al., 2006). In other words, the ratio of the maxi-mal to the median value of excretion fraction for triclosan was lessthan a factor of 2, or well within the inter-individual variability of

100.5 used in risk assessments (to represent PK variability), and thisuncertainty factor component was retained in the derivation of theurinary BE values presented here. The intersubject variability inthe excretion fraction might result from variation of bioavailability,distribution kinetics, metabolic clearance and/or fraction elimi-nated via renal clearance (Sandborgh-Englund et al., 2006). Addi-tional sources of potential variation in measured urinaryconcentrations, even under conditions of exposure consistent withthe RfD, include variations in hydration status and creatinineexcretion rates, which could impact measured concentrations inspot urine sample. The appropriateness of adjustment for hydra-tion status using creatinine excretion has been debated (Gardeet al., 2004; Barr et al., 2005) because creatinine excretion alsocan vary substantially due to variations in dietary pattern as wellas other individual factors (gender, age, muscle mass, seasonaland daily variation, diet) (Garde et al., 2004; Barr et al., 2005).

In the present work, BE values were estimated for triclosan onthe basis of creatinine excretion as well as on the basis of urinaryvolume. Samples collected for a 24-h period would be expectedto be influenced less than spot samples by both variations inhydration status and creatinine excretion. Even though the valueof excretion fraction used in the calculations (54%) was based ona limited dataset from a human volunteer study (Sandborgh-Engl-und et al., 2006), this value is consistent with other available

Table 5Derivation of plasma BE values based on the USEPA (2008b) and EC (2009) risk assessments (Table 1) according to the schemes presented in Fig. 2a (for the EC and NICNAS riskassessments) and Fig. 2b (for the USEPA risk assessment).

BE derivation step USEPA, 2008a,b) RfD EC (2009) risk assessment NICNAS (2009) risk assessment

Species, endpoint Baboons, general toxicity Rats, hematological endpoint alterations Rat, liver toxicityPOD (NOAEL), external dosea (mg/kg-d) 30 12 NAPOD (NOAEL), plasma concentrationb (mg/L) – 21.8 41UF, interspecies 10 2.5d 10e

Human-equivalent POD, external dose (mg/kg-d) 3 –BE_POD, plasma concentrationc mg/L 3.0 8.7 4.1UF, intraspecies 10 10 10e

BE, plasma concentration (mg/L) 0.3 0.9 0.4

a From Table 1.b From DeSalva et al. (1989) as reported by EC (2009).c Estimated using Eq. (3) and median clearance rate measured by Sandborgh-Englund et al. (2006).d Because a relevant internal dose metric is used here, the pharmacokinetic component of the interspecies uncertainty factor is set to 1. We have retained the pharma-

codynamic component of the interspecies uncertainty factor (2.5, according to WHO (1999), guidance used in EC risk assessments),e Specified value on a plasma concentration basis in the NICNAS risk assessment.

K. Krishnan et al. / Regulatory Toxicology and Pharmacology 58 (2010) 10–17 15

information in the literature. In other human studies involving asingle oral dose ranging from 5 to 200 mg, the average cumulativeamount of triclosan excreted in 24-h urine corresponded to about40%, with maximal percent approaching 60% in 4–5 days (DeSalvaet al., 1989). The inter-individual coefficient of variation in urinaryexcretion fraction was on the order of 30% in dermal and oral expo-sure studies (Queckenberg et al., 2009), which is likely to be reflec-tive of differences in rate and extent of absorption as well asmetabolism and renal clearance.

The human plasma clearance data used in deriving the blood-based BE for triclosan (0.041 L/kg h, or 2.9 L/kg d) was also ob-tained from Sandborgh-Englund et al. (2006). In this study, thesubject-specific values of clearance ranged from 0.032 to 0.049 L/h/kg, following a single oral dose of 4 mg/day (which was swal-lowed completely, contrary to 5–40% fraction swallowed undernormal human usage of products containing triclosan – such astoothpaste) (reviewed in Sandborgh-Englund et al., 2006). Theclearance values reported by Sandborgh-Englund et al. (2006)and used in the present study correspond to plasma clearance di-vided by the fraction of dose absorbed, and thus they incorporatevariability in the extent of absorption in study subjects.

The available data do not support any age or gender relatedchanges in the pharmacokinetics of triclosan. For example, theplasma half-lives in adults and children estimated in various stud-ies using toothpaste, dental slurry capsules or aqueous solutionwere comparable, and ranged between 13.4 and 21 h (EC, 2009).The pharmacokinetic study of Sandborgh-Englund et al. (2006)did not find any consistent gender difference among adults, ineither clearance or plasma half-life of triclosan.

Both the plasma- and urine-based BE values for triclosan werederived using clearance and excretion fraction data obtained insingle oral dose studies of Sandborgh-Englund et al. (2006). Oralroute is the route of exposure in the critical toxicity studies; fur-thermore, it the most important route of human exposure to triclo-san based on product use as well as the extent of absorption(Bagley and Lin, 2000; Moss et al., 2000; Allmyr et al., 2008; Quec-kenberg et al., 2009). The use of pharmacokinetic data from singledose studies would appear to be relevant since the dose-normal-ized AUC was similar for ingestion of triclosan following single ormultiple exposures (EC, 2009). This is supported by the animalstudies and repeated human exposure studies with toothpasteand soap use, which indicate that steady-state is reached afterabout 7–10 days (EC, 2009). Accordingly, the uncertainty associ-ated with the use of the single oral dose human study of Sand-borgh-Englund et al. (2006) for deriving BEs for triclosan wouldappear to be low.

For deriving BE associated with the EC (2009) assessment, inaddition to the human clearance data from Sandborgh-Englund

et al. (2006), an estimated steady-state plasma concentration of21.8 mg/L in rats was used. This plasma concentration was ob-tained from EC (2009), based on the interim sacrifice data in malerats from the critical toxicological study. EC (2009) indicated thatthe plasma concentration at interim sacrifice was 21.8 (±9) and28.1 (±12.9) mg/L in male and female rats respectively, whereasat terminal sacrifice it was 26.5 (±18) and 10.6 (±3.4) mg/L in fe-male and male rats. These plasma concentrations were derivedfrom the reported blood concentrations in the original study, basedon volume adjustment (plasma = 40 ml/kg, blood = 64 ml/kg). Not-ing the loss of body weight towards the end of the 2-year rat study,EC (2009) used the estimated plasma values associated with the in-terim sacrifice. Given that the NOAEL in male rats of 12 mg/kg/dused in the EU assessment, we chose the corresponding plasma va-lue of 21.8 mg/L (intrim sacrifice value) as the basis for developingthe BE, with its associated uncertainty as indicated above.

4.2. 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.

For urine-based BE, the cumulative fraction of 54% (at 96 h)used in the present study, based on data obtained in ten volunteersreceiving a single oral dose of 4 mg (Sandborgh-Englund et al.,2006), is comparable to the observations (44–57% at 48 h) in anumber of different clinical and preclinical studies summarizedby DeSalva et al. (1989). However, the relevance of the total triclo-san level in urine to the target tissue exposure to the toxic form ofthe chemical is not known. Thus, the assessment of the confidencelevel in the derived urinary BE values based on these two factors isas follows:

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

The assessment of the confidence level associated with the pla-ma-based BE values is as follows:

� Robustness of pharmacokinetic data: MEDIUM� Relevance of biomarker to relevant dose metrics: MEDIUM

This reflects the relevance of the blood or plasma level of triclo-san to the toxic effects (reviewed in NICNAS, 2009); however itdoes not differentiate between the various forms of triclosan

16 K. Krishnan et al. / Regulatory Toxicology and Pharmacology 58 (2010) 10–17

(free vs. conjugated) and their relevance to the mode of action. Thepharmacokinetic data, specifically plasma clearance, obtained fromten volunteers (Sandborgh-Englund et al., 2006) is considered to befairly robust, given the additional support from the literatureregarding steady-state and inter-individual variability.

4.3. Interpretation of biomonitoring data using BE values

The BE values presented here represent estimates of the 24-haverage concentrations of triclosan in urine that are consistentwith the 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 should be regarded as interim screen-ing values that can be updated or replaced if the exposure guidancevalues are updated or if the scientific and regulatory communitiesdevelop additional data on acceptable or tolerable concentrationsin human biological media.

The appropriate uses and limitations of BE values have been dis-cussed previously (Aylward and Hays, 2008; Hays and Aylward,2008; Hays et al., 2008). These BE values can be used as a screeningtool to evaluation population- or cohort-based biomonitoring datain the context of existing risk assessments. Concentrations in ex-cess of the BE values, but less than the BEPOD values represent med-ium priority for risk assessment follow-up, while those in excess ofthe BEPOD indicate high priority for risk assessment follow-up.Based on the results of such comparisons, an evaluation can bemade of the need for additional studies on exposure pathways, po-tential health effects, other aspects affecting exposure or risk, orother 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 triclosan were de-rived with a substantial margin from doses that resulted in no ob-served effect in the most sensitive animal toxicity studies. Thus,these values are not ‘‘bright lines” that distinguish safe from unsafeexposure levels. Chronic exposure guidance values are set at expo-sure levels that are expected to be protective over a lifetime ofexposure. For short-lived compounds such as triclosan, an exceed-ance of the corresponding BE value in a single urine sample may ormay not reflect continuing elevated exposure. As demonstrated inthe limited available datasets and based on the kinetics of urinaryelimination, spot urinary concentrations may vary substantiallyboth within and across days in an individual. Thus, occasionalexceedances of the BE value in individuals in cross-sectional stud-ies do not imply that adverse health effects are likely to occur, butcan serve as an indicator of relative priority for further risk assess-ment follow-up. Further discussion of interpretation and commu-nication aspects of the BE values is presented in LaKind et al.(2008) and at www.biomonitoringequivalents.net.

5. Conflict of interest statement

The authors declare they have no conflicts of interest.

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. Prepared under HealthCanada Contract 4500195930.

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