Assessing children's exposure to manganese in drinking water using a PBPK model

Toxicology and Applied Pharmacology 380 (2019) 114695

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Assessing children's exposure to manganese in drinking water using a PBPK model

T

M. Yoona, , C. Ringb, C.B. Van Landinghamc, M. Suhd, G. Songd, T. Antonijevice, P.R. Gentryc, M.D. Taylorf, A.M. Keeneg, M.E. Andersenh, H.J. Clewelli ⁎

a

ToxStrategies, Inc., RTP, NC, USA ToxStrategies, Inc., Austin, TX, USA c Ramboll, Monroe, LA, USA d ToxStrategies, Inc., Orange County, CA, USA e ToxStrategies, Inc., Katy, TX, USA f NiPERA, Durham, NC, USA g Afton Chemical Corporation, Richmond, VA, USA h Andersen ToxConsulting LLC, NC, USA i Ramboll, RTP, NC, USA b

ARTICLE INFO

ABSTRACT

Keywords: Manganese Drinking water exposure Bioavailability Children Infant PBPK

A previously published human PBPK model for manganese (Mn) in infants and children has been updated with Mn in drinking water as an additional exposure source. Built upon the ability to capture differences in Mn source-specific regulation of intestinal uptake in nursing infants who are breast-fed and formula-fed, the updated model now describes the bioavailability of Mn from drinking water in children of ages 0–18. The age-related features, including the recommended age-specific Mn dietary intake, age-specific water consumption rates, and age-specific homeostasis of Mn, are based on the available human data and knowledge of the biology of essential-metal homeostasis. Model simulations suggest that the impact of adding drinking-water exposure to daily Mn exposure via dietary intake and ambient air inhalation in children is not greater than the impacts in adults, even at a drinking-water concentration that is 2 times higher than the USEPA's lifetime health advisory value. This conclusion was also valid for formula-fed infants who are considered at the highest potential exposure to Mn from drinking water compared to all other age groups. Our multi-route, multi-source Mn PBPK model for infants and children provides insights about the potential for Mn-related health effects on growing children and will thereby improve the level of confidence in properly interpreting Mn exposure-health effects relationships in children in human epidemiological studies.

1. Introduction Manganese (Mn) occurs naturally in the environment and is an essential element required for proper development and growth (ATSDR, 2012; EFSA, 2013; Health Canada, 2016). Human exposures to Mn occur primarily through ingestion of food containing Mn (USEPA, 2003a). Mn exposures can also occur via ingestion of drinking water, ingestion of soil, and inhalation of air or dust containing Mn (USEPA, 2003a). For children and infants, ingestion of food and drinking water containing Mn are the main sources of Mn exposure. However, Mn exposure characterization for children is limited due to the lack of data on concentration levels, timing, and duration of exposure (Coetzee et al., 2016).

⁎

Several studies have suggested potential neurodevelopmental and neurobehavioral effects of Mn in children, especially concerning drinking water as a significant source of Mn (Coetzee et al., 2016; Bouchard et al., 2011; Khan et al., 2011; Roels et al., 2012; Oulhote et al., 2014a). However, many of these epidemiologic studies evaluating the neurological effects of Mn in children have significant limitations (ATSDR, 2012; Health Canada, 2016) including that Mn exposure in those studies are usually not measured, estimated or reported. The absence of information on exposures makes it difficult to draw any quantitative conclusions on Mn exposure-response relationships. In addition, covariate information, such as the presence of lead, arsenic and other heavy metals in drinking water are not measured, estimated, or reported. More accurate characterization and assessment of Mn

Corresponding author at: 1249 Kildaire Farm Road #134, Cary, NC 27511, USA E-mail address: [email protected] (M. Yoon).

https://doi.org/10.1016/j.taap.2019.114695 Received 10 May 2019; Received in revised form 20 July 2019; Accepted 30 July 2019 Available online 05 August 2019 0041-008X/ © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).

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exposures are necessary to evaluate the possibility of neurological effects in children. Concerns for increased vulnerability to Mn exposure in children arise mainly due to increased needs for Mn during intrauterine and postnatal growth. Several differences between children and adults in regards to Mn homeostasis have been interpreted as predisposing children to enhanced Mn uptake and associated toxic effects compared to adults. These differences include higher intestinal absorption of ingested Mn in children than in adults (Keen et al., 1986; Dorner et al., 1989) and a lower basal biliary excretion rate observed in neonatal animals (Ballatori et al., 1987). Enhanced delivery of Mn to brain has also been reported (Takeda et al., 1999). However, homeostasis mechanisms are functioning and effectively responding to excess Mn exposure during early life periods (Yoon et al., 2011). In addition, sources of exposure to Mn in early life periods are different from those for adults. Infants receive Mn through breast milk or infant formula fortified with Mn rather than the normal dietary exposure in adults. In fact, the bioavailability of Mn from breast-milk appears to be much higher than that of solid food, a difference likely necessary to cope with limited Mn supplies from maternal milk during lactation (Davidson and Lönnerdal, 1988; Davidson and Lönnerdal, 1989). The scarcity of Mn in milk also appears to be responsible for the observed reduced excretion of Mn in neonatal animals (Aschner, 2000), even though biliary excretion is still inducible under conditions of excess Mn exposure (Sampson et al., 1983; Stastny et al., 1984; Zlotkin and Buchanan, 1986; Ballatori et al., 1987; Hambidge et al., 1989; Kostial et al., 2005). There are two major questions raising concerns for Mn exposure via drinking water consumption in children: 1) Is the bioavailability of Mn from drinking water enhanced compared to the bioavailability of Mn from the diet? and 2) Do higher water consumption rates during early life periods result in increased uptake of Mn? Mn bioavailability from drinking water had been regarded to be much higher than from diet. However, Foster et al. (2015) demonstrated in rats that there are no significant differences in Mn bioavailability between diet and drinking water. Based on this study, a PBPK model-based analysis was designed which demonstrated that Mn bioavailability from drinking water is similar to that from diet (Song et al., 2018). However, potentially increased bioavailability of Mn in infants and young children, if exposed via drinking water, still remains to be addressed given the immature gastrointestinal tract during this early life period (Pacha, 2000; Walthall et al., 2005). In addition, children can be at a higher risk than adults to compounds in drinking water because they consume more water per unit of body weight than adults (USEPA, 2011). To obtain a reliable estimate of Mn exposure in children, it is necessary to account for age-related differences in exposure factors such as water consumption rates, for Mn concentrations in various exposure media (e.g. drinking water) and for age-dependent differences in patterns of Mn-homeostasis. While PBPK models for Mn in infants and children (Yoon et al., 2011) have included age-related differences in Mn homeostasis during early life periods under various exposure scenarios, they have not accounted for Mn exposure through drinking water. In this study, we update the Mn-PBPK model focusing on Mn exposure in infants and children and evaluate the different factors that determine tissue uptake in early life stages versus the adult.

updated version of the lactation portion of the model reported by Yoon et al. (2011). It includes both a lactating mother and a nursing infant and was used to simulate both breast-fed and formula-fed infants for the first 6 months of life after birth. This model was also used to simulate exposure of a child from age 0.5 to age 3. The second model (the “child/adolescent/adult model”) was based on the average adult model reported by Song et al. (2018), describing intake-dependent fractional uptake of ingested Mn (Fdietup). The published model was modified to describe children and adolescents by using age-dependent growth curves for tissue volumes, blood flows, and respiratory parameters. The model also has the option to “switch off” the age-dependent growth curves, and instead, assign constant, usually sex-specific values for tissue volumes, blood flows, and respiratory parameters. The modified model was used to simulate the child and adolescent scenarios (ages 3–18) (using the age curves) and the adult scenarios, i.e., simulations of an individual at constant body weight with parameters set for a standard female or male adult (Schroeter et al., 2011; Yoon et al., 2011). Both the lactation/infant and child/adolescent/adult models were modified to provide more detail about Mn in drinking-water with agedependent drinking-water intake rates. The lactation/infant model was also modified to describe intake-dependent uptake of ingested Mn (variable values of Fdietup) in both the infant after weaning and in the mother using the methods described in Song et al. (2018). These updates were made to the gestation model to determine if drinking water exposure during gestation period would affect tissue Mn in the newborn. We simulated the effect of drinking water exposure on tissue Mn in the pregnant mother and fetus using the updated gestation model from Yoon et al. (2011). All the parameters for the gestation model except the drinking water exposure related parameters (Table 1) were kept the same as the previous work (Yoon et al., 2011). 2.2. Exposure scenarios Several exposure scenarios were simulated (Table 1). (1) A male infant exclusively breast-fed for 6 months after birth (2) A male infant exclusively formula-fed for 6 months after birth, assuming the average daily Mn intake from formula powder of 1.145 mg Mn/day from Stastny et al. (1984) (3) A male infant exclusively formula-fed for 6 months after birth, assuming a daily Mn intake from formula powder of approximately 0.05 mg Mn/day (Table 1) as calculated in Brown and Foos (2009) (4) A 3-year old male toddler (5) A 10-year old male child (6) An 18-year old male teenager (7) A male adult (8) A female adult (9) A pregnant female and a male fetus All scenarios included exposure to background levels of air Mn (0.015 μg/m3) consistent with midpoint for the range of respirable ambient air concentrations in the US reported by Gentry et al. (2017). For the breast-fed infant, both mother and baby were exposed to background air Mn. All post-weaning scenarios included dietary intake of Mn at levels equal to the age- and sex-specific recommended daily intake (FNB, 2001). The breast-fed infant scenario included dietary intake for the mother at 3 mg/day Mn as in the original lactation model (Yoon et al., 2011). The same dietary intake was used for the pregnant mother. All children scenarios were simulated for male as the sex differences in fractional uptake in the gut and biliary excretion are applicable for women of childbearing age (Yoon et al., 2011). All post-weaning scenarios included intake of drinking water containing Mn at age-specific rates or a rate during pregnancy (USEPA, 2011). The breast-fed infant scenario included drinking-water intake

2. Materials and methods 2.1. Overview of Mn PBPK models for simulation of different ages To simulate Mn kinetics in children and adults, the two previously published Mn PBPK models for adults and children (Schroeter et al., 2011; Yoon et al., 2011; Song et al., 2018) were updated and used for the simulation of different age groups as described below. The first model, referred to as “lactation/infant model,” was an 2

Toxicology and Applied Pharmacology 380 (2019) 114695

for the mother only; the baby consumed no water. The formula-fed infant scenarios included drinking-water intake for the baby, under the assumption that formula was prepared with Mn-containing drinking water. Each scenario was run with multiple drinking-water Mn concentrations: 0, 0.05, 0.3, and 0.58 ppm or mg/L. These drinking-water concentrations were based on the following criteria: the US EPA's Secondary Maximum Contaminant Level (SMCL) of 0.05 ppm for Mn based on taste and staining considerations; the US EPA's lifetime health advisory value (LHA) of 0.3 ppm; and 0.58 ppm, which is the 95th percentile of the acceptable drinking-water concentration in Iowa (USEPA, 2003b). The toddler, child, teenager, and adult scenarios were run with initial values (i.e., free and bound tissue Mn levels at the beginning of simulation age) taken from the end of each of the three infant-feeding scenarios and helped to determine whether the infant feeding scenario had any lingering effects on body Mn after weaning. However, we found that by age 7 months (i.e., 1 month after weaning), infants in all three infant-feeding scenarios had reached the same state. Therefore, results presented here for the toddler, child, teenager, and adult scenarios begin with the formula-feeding scenario in the infant. The formula Mn level of 0.05 mg Mn/day that was used is based on Brown and Foos (2009). 2.3. PBPK model parameters Parameters of each model are summarized in Tables 2–5, including physiological parameters and Mn-specific parameters. Exposure parameters — including daily dietary intake, drinking-water intake, and formula-milk intake — are summarized in Table 6 through Table 7 as well as in Table 1. 2.3.1. Age-dependent parameters Age-dependent changes in physiological parameters were included as described in Yoon et al. (2011) to perform simulations using the lactation/infant model, whereas they were extended to age 18 to simulate child/adolescent scenarios. The data for physiological parameters for ages over 3 were adopted from the same references that were used in Yoon et al. (2011). Further details of the descriptions of agedependent physiological parameters including brain regional volumes, respiratory parameters such as alveolar ventilation rates, and Mn respiratory tract fractional deposition parameters are in Supplementary Materials. Respiratory parameters are not just age-dependent but are also influenced by the activity levels across ages. In this study, alveolar ventilation rates (L/day) for children were derived from activity-adjusted daily inhalation rate reported in Table 6-36 of the USEPA's ChildSpecific Exposure Factors Handbook (USEPA, 2008). The EPA-derived inhalation rates were used as alveolar ventilation rates in this study. To be conservative, resting respiratory values were used for adults as in Yoon et al. (2011). 2.4. Source-specific Mn exposure 2.4.1. Drinking-water Mn exposure In general, drinking-water Mn exposure rate in mg/kg/day was calculated by multiplying the Mn concentration in drinking water (in mg/L) by an age-dependent drinking-water ingestion rate or a rate during pregnancy (in L/day). Multiple drinking-water Mn concentrations were considered, including 0.05 ppm which represents USEPA's non-enforceable guideline; 0.3 ppm which represents USEPA's Health Reference Level (HRL; same as the lifetime health advisory value); and, 0.58 ppm which represents Iowa's drinking water Mn concentration that is the 95th percentile in USEPA's National Inorganics and Radionuclides Survey (USEPA, 2003b). For comparison, we also ran a 0 ppm scenario (i.e., no Mn in drinking water). To investigate the dose-response relationship for

e

d

c

IOM RDI = IOM recommended daily intake (age- and sex-specific). See Table 7 for details. Formula-powder Mn dose calculated following Brown and Foos (2009). See Equation 1 and Table S5 for details. Age-dependent drinking-water intake rate is the age-specific mean consumers-only intake rate from USEPA Exposure Factors Handbook (2011), Table 3-1 (relevant data reproduced in Table 10). Fdietup in ages 0 to 0.5 = FDP * Fdietup of the adult male. Beyond age 0.5, Fdietup is intake dependent and sex-specific. Standard adult male or female was used as in the previous models (Schroeter et al., 2011; Yoon et al., 2011). b

RDI) RDI) RDI) RDI) RDI) (IOM (IOM (IOM (IOM (IOM – – Age-dependent Age-dependent Age-dependent Age-dependent Age-dependent 3 1.5e-5 1.5e-5 1.5e-5 1.5e-5 1.5e-5 1.5e-5 1.5e-5 1.5e-5 Formula-fed infant (Stastny et al., 1984) Formula-fed infant (Brown and Foos, 2009) Toddler (male) Child (male) Teenager (male) Adult male Adult female Pregnant female

0–0.5 0–0.5 0.5–3 3–10 10–18 —e —e —e

– 1.5e-5 0–0.5 Breast-fed infant

a

FDP x 0.06, FDP = 0.01 FDP x 0.06, FDP = 0.01 Intake-dependent Intake-dependent Intake-dependent Intake-dependent Intake-dependent Intake-dependent 1.145 Approx. 0.05b – – – – – –

–

mother only: 0.58 0.58 0.58 0.58 0.58 0.58 0.58 0.58 0.58

(0.470–0.467) (0.470–0.467) (0.467–0.382) (0.382–0.612) (0.612–0.816) Baby: 0 Mother: 1.227 Age-dependent Age-dependent Age-dependent Age-dependent Age-dependent 1.227 1.227 0.872 Modeled for 0, 0.05, 0.3, 0, 0.05, 0.3, 0, 0.05, 0.3, 0, 0.05, 0.3, 0, 0.05, 0.3, 0, 0.05, 0.3, 0, 0.05, 0.3, 0, 0.05, 0.3, 0, 0.05, 0.3,

FDP x 0.06, FDP = 10

Drinking water intake rate (L/day)c Air Mn (ppm) Age (years) Scenario

Table 1 Summary of exposure scenarios.

Dietary Mn (mg/day)a

Formula milk Mn (mg/day)

Drinking water Mn (ppm)

Fdietupd

M. Yoon, et al.

3

4

0.022 0.0238 0.007 Pre-pregnancy: 0.0062 Time-varying increase during lactation (Yoon et al., 2011) Pre-pregnancy: 0.316 Time-varying increase during lactation (Yoon et al., 2011)

Brain

Liver

Lung

Mammary –

0.05 0.12 0.27 0.01 Pre-pregnancy: 0.027 Scales up with mammary tissue volume during lactation (Yoon et al., 2011) Pre-pregnancy: 0.052 Scales up with fat tissue volume during lactation (Yoon et al., 2011)

Tissue blood flows (fraction of total cardiac output) Bone

Brain

Liver

Nose Mammary

c

b

a

–

–

Age-dependent (Yoon et al., 2011)

Age-dependent (Yoon et al., 2011)

Age-dependent (Yoon et al., 2011)

Age-dependent (Yoon et al., 2011)

Age-dependent (Yoon et al., 2011)

Age-dependent (Table S3)

–

0.079 (male) 0.068 (female) 0.12 (male) 0.127 (female) 0.02 (male) 0.022 (female) 0.026 (male) 0.023 (female) 0.008 (male) 0.007 (female) –

–

0.01 –

Age-dependent (Yoon et al., 2011)

Age-dependent (Yoon et al., 2011)

–

0.01 –

Age-dependent (Yoon et al., 2011)

Age-dependent (Yoon et al., 2011)

0.042c

0.52 9.81 375.0 20

Age-dependent (Table S2) Age-dependent (Table S2) Age-dependent (Table S2)

–

0.042 (male) 0.05 (female) 0.114 (male) 0.12 (female) 0.227 (male) 0.2 (female) 0.01 –

0.52 9.81 375.0 20

0.0015 0.00055 0.08

Adult male and female parameter values are from Schroeter et al. (2011) and Yoon et al. (2011), respectively. All the physiological parameters for pregnant mother and fetus were kept the same as in Yoon et al. (2011). This represents the brain blood volume. Same as adults.

Fat

0.52 9.81 375.0 20

thickness of the nasal tissue) 0.52 9.81 375.0 20

Nose tissue volume (calculated from surface area and Surface area, respiratory nasal cavity (cm2/kg0.75) Surface area, olfactory nasal cavity (cm2/kg0.75) Average tissue thickness in nasal cavity (um) Gallbladder volume, mL

0.042c

0.002 0.00055 0.131

Regional brain volumes (fraction of total brain volume) Globus pallidus 0.0015 Olfactory bulb 0.00055 Cerebellum 0.08

Tissue volume (L) Rest of body/other

–

Age-dependent (ICRP, 2003)

Age-dependent (Yoon et al., 2011)

Age-dependent (Yoon et al., 2011)

Age-dependent (Yoon et al., 2011)

Age-dependent (Yoon et al., 2011)

Age-dependent (Table 8)

13 (male) 16.42 (female) 18 (male) 13.25 (female)

70 (male) 60 (female)

Male and female adulta

Difference between total body weight and sum of the following volumes: (liver, bone, Difference between total body weight and sum of the following volumes: (liver, bone, lung, blood, lung, blood, nose, mammary, globus pallidus, olfactory bulb, cerebellum, brain*0.03) nose, globus pallidus, olfactory bulb, cerebellum, brain*0.03b)

0.127

Bone

Fat

0.068

13.25

Age-dependent (Yoon et al., 2011)

Age-dependent (Yoon et al., 2011)

Child/teenager

Nursing mother Varies with post-birth time-varying changes in mammary and fat compartment masses. Age-dependent (Yoon et al., 2011) Pre-pregnancy BW (BWpre) = 60 kg. Immediately post-birth BW = BWpre x (1 + (0.0836 + 0.005929)) 16.42 Age-dependent (Yoon et al., 2011)

Modification of Song et al. (2018)

Modification of Yoon et al. (2011) Infant/toddler

Child/Adolescent/Adult model

Lactation/Infant model

Tissue volume (fraction of BW) Blood

Alveolar ventilation (L/h/ kg BW0.75)

Cardiac output, scaled (L/h/kg BW0.75)

Body weight (kg)

Table 2 Physiological pparameters.

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Table 3 Mn tissue binding parameters. Tissue

Tissue binding

Partition coefficient, tissue:blood

BmaxC (ug/kg tissue)

ka (1/ug/h)

kd (1/h)

Nursing mother Bone Cerebellum Olfactory bulb Globus pallidus Liver Lung Mammary Rest of body

80 370 620 470 1250 460 85 85

0.00464 0.001 0.35 0.2 0.0011 0.01 0.093 0.0093

0.024 0.003 0.005 0.02 0.00048 0.038 0.00464 0.0045

0.5 – – – 1 1 1 1

Infant/toddler Bone Cerebellum Olfactory bulb Globus pallidus Liver Lung Rest of body

850 × xbo a 250 500 450 1250 460 100 × xothb

0.00464 0.002 0.4 0.7 0.0011 0.01 0.0092

0.024 0.003 0.005 0.02 0.00048 0.038 0.0047

0.5 – – – 1 1 1

Child/adolescent/adult Bone Cerebellum Olfactory bulb Globus pallidus Liver Lung Pituitary Rest of body

78 (male), 80 (female) 370 620 500 (male), 470 (female) 1245 (male), 1250 (female) 460 620 84 (male), 85 (female)

0.00464 0.001 0.35 0.2 0.0011 0.01 0.3 0.00927

0.0238 0.003 0.005 0.02 0.00048 0.038 0.036 0.00464

0.5 – – – 0.85 1 – 1

Note: All the Mn-specific parameters for pregnant mother and fetus were kept the same as in Yoon et al. (2011). a xbo represents changes in the maximal bound-Mn capacity in bone during early life. BmaxC rapidly decreases during nursing, reaching adult value around 1 year of age. xbo = 0.8995 × exp (−0.01462 × days) + 0.1. b xoth represents changes in the maximal bound-Mn capacity of other tissues during early life. Before weaning, xoth = 1.373 × exp (−0.01992 × days) + 0.1. Between 0 and 10 days after weaning, xoth = − 6.376 + 0.03619 × days. More than 10 days after weaning, xoth = 0.5.

drinking water Mn, the models were also run for each scenario with drinking-water Mn concentrations between 0 and 100 ppm. The drinking-water ingestion rate in each model was computed as follows using the age-dependent water ingestion rates from USEPA Exposure Factors Handbook Table 3-1 (USEPA, 2011):

pregnant mother, the drinking water ingestion rate was 0.872 L/day (USEPA, 2011). For the nursing mother, the drinking-water ingestion rate was constant 1.227 L/day, which is the mean consumersonly value for adults ≥21 years old from the USEPA Exposure Factors Handbook (USEPA, 2011). For the baby, the drinking-water ingestion rate was zero if breastfeeding. If formula-fed, the drinkingwater ingestion rate was linearly interpolated from the age-dependent data in the USEPA Exposure Factors Handbook using mean consumers-only water ingestion rates (Table 6). Drinking-water ingestion rate in the child/adolescent/adult model: For child and adolescent scenarios, the drinking-water ingestion rates were linearly interpolated from the age-dependent data in the USEPA Exposure Factors Handbook using mean consumers-only water ingestion rates (Table 6). For adult scenarios, the drinkingwater ingestion rate was constant 1.227 L/day, which is the mean consumers-only value for adults ≥21 years old from the USEPA Exposure Factors Handbook (Table 6).

Drinking-water ingestion rate in the lactation/infant model: For the Table 4 Brain diffusion parameters for Mn. Tissue

kinC (1/h/kg0.25)a

koutC (1/h/kg0.25)a

kinmax (unitless)

kin50 (μg)

Nursing mother Globus pallidus Olfactory bulb Cerebellum

0.08 3 2.4

0.4 7.5 0.08

4.6 – –

0.16 – –

Infant/toddler Globus pallidus Olfactory bulb Cerebellum

0.08 3 2.4

0.8 7.5 0.08

3.5 – –

0.16 – –

0.4 7.5 0.08 0.3

4.6 – – 3.5

0.16 – – 0.16

Child/adolescent/adult Globus pallidus 0.08 Olfactory bulb 3 Cerebellum 2.4 Pituitaryb 0.079

2.4.2. Milk Mn exposure Mn exposure through breastmilk or formula milk was simulated for the first 6 months after birth. For the first 6 months of life infants are fed exclusively breastmilk or formula milk as recommended by The American Academy of Pediatrics and IOM FNB. Lactational exposure via breast-feeding was simulated as described by Yoon et al. (2011), which shows the Mn concentrations in human breastmilk during lactation is about 3 μg/L resulting in approximately 1.5 μg/day Mn exposure to the breast-fed infant. There were two different scenarios regarding formula-milk exposure. The first had the average daily Mn intake from formula powder reported in Stastny et al. (1984), i.e., 1.145 mg/day Mn. The second had a daily Mn intake from

Note: All the Mn-specific parameters for pregnant mother and fetus were kept the same as in Yoon et al. (2011). a scaled to tissue weight0.25 (kg) b Due to lack of data during lactation and post-weaning development, pituitary was not described in infant/toddler and nursing mother. 5

6

Scaled basal biliary excretion rate constant Maximal biliary induction factor Biliary affinity rate constant Slope factor for biliary induction Rate of (1-Fent) fraction of unabsorbed Mn movement into lower gut lumen for fecal excretion Delayed transport to systemic circulation in nursing infant Fraction of unabsorbed Mn trapped in enterocytes before moving into lower gut lumen for fecal excretion Rate of Mn trapped in enterocytes moving into lower gut lumen due to sloughing of gut epithelium

kbileC (1/h/BW0.75) kBinduc km (μg/g) n kGI (1/h)

0.0022

0.005

–

0.1 2.5 0.027 3 0.026

kbileCBW0.75 factor

0.0022k ent, factor See note (d)

Before weaning: 0.013 After weaning: 0 0.005

See note (b) kbileCBW0.75kbile, See note (c) 0.051 2.5 0.065 3 0.026

kbile ×

kbile ×

n ) (1 + kBinduc Cart n + Cn ) (km art

– Before weaning: FDP × 0.06 See note (a) After weaning: Depends on Mn intake (dietary + drinking water). See Eq. 2 n ) (kBapp + kBinduc Cart n + Cn ) (km art

Infant/toddler

0.00065 Depends on Mn intake (dietary + drinking water). See Eq. 2

Nursing mother

n ) (1 + kBinduc Cart n + Cn ) (km art

0.002222

0.005

–

0.051 (male), 0.1 (female) 2.5 0.027 3 0.026

kbileCBW0.75

kbile ×

– Depends on Mn intake (dietary + drinking water). See Equaiton 2

Child/adolescent/adult

Note: Bold italic text indicates changes from the previously-published models (i.e., Yoon et al. (2011) for the lactation/infant model, and Song et al. (2018) for the child/adolescent and average adult models). Parameters governing uptake and excretion of Mn in the pregnant mother and fetus were kept the same as in Yoon et al. (2011). a FDP represents relative uptake of Mn from breastmilk or formula milk, compared to an average baseline fractional uptake for solid food of 0.06 (equivalent to intake-dependent Fdietup at an Mn intake of approximately 2.8 mg/day). For breastmilk, FDP = 10. For formula milk, FDP = 0.01. For drinking water used to prepare formula, FDP = 0.01. b kBapp is a factor representing apparent low biliary excretion during nursing. It is set at a value of 0.001 during nursing, and a value of 1 after weaning. Cart represents arterial blood concentration in μg/g. c kbile, factor represents changing biliary excretion before weaning: 50% at birth, reaching 100% of adult at weaning. Before weaning, it is given by 0.15 + (1.18 × days)/(180 + days). After weaning, it is 1. d kent, factor represents changing enterocyte turnover during early ages: 20% at birth, reaching adult rate around weaning. It is given by 0.2 + (1.009 − 0.2) × (1 − exp [−0.3451 × (months − 1)]).

kent (1/h)

Fent

DelGI (1/h)

Basal biliary excretion rate

kbile (1/h)

Rate of clearance of Mn into breastmilk Fractional gut absorption of Mn

Description

Biliary excretion rate including free-Mn-dependent biliary induction

)

0.75

kbilex (1/h)

CLmilkC (L/h/kgBW Fdietup

Parameter

Table 5 Parameters governing uptake and excretion of Mn.

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2.5. Intake-dependent oral Mn bioavailability

Table 6 Age-dependent drinking water ingestion rates. Age (months)

Drinking water ingestion rate (L/day)

0 1 3 6 12 (1 year) 24 (2 years) 36 (3 years) 72 (6 years) 132 (11 years) 192 (16 years) 216 (18 years) 252+ (21+ years)

0.470 0.552 0.556 0.467 0.308 0.356 0.382 0.511 0.637 0.702 0.816 1.227

In the current model, there are three different Mn sources that can potentially contribute to the total ingested Mn daily intake: milk, diet and drinking water. As shown by the recent analysis by Song et al. (2018), Mn bioavailability from the gut is only dependent on the total amount ingested, not on the sources, i.e., either diet or drinking water. Total daily ingestion of Mn, as well as the relative contribution of each to total ingested amount, vary across ages. To model the regulation of fractional uptake of ingested Mn in response to changing Mn intake rates, the following equation was used (Song et al., 2018):

Fdietup = Sex × 0.00627 exp(3.081281 exp( 0.1097×mDiet))

Where: Fdietup = fractional uptake of ingested Mn; with a value of 1 assumed for males and a value of 2 assumed for females as Fdietup is 2fold higher for females (Yoon et al., 2011); and mDiet = Total Mn ingestion rate from non-milk sources, i.e., sum of dietary Mn ingestion rate and drinking-water ingestion rate in mg/day. In the lactation/infant model, Eq. (2) was used to estimate the mother's Fdietup throughout gestation and lactation, and it was used for the baby's Fdietup only after weaning, i.e., after the baby is no longer consuming either breastmilk or formula milk and shifts to consuming a solid diet instead. Eq. (2) was also used to estimate Fdietup in the child/ adolescent/adult model. During periods of exclusive breastfeeding or formula-feeding, i.e., in the first 6 months of life, the baby's Fdietup was constant, independent of Mn intake. This decision reflects evidence that the mechanism by which Mn uptake is regulated based on intake appears to be immature in infants (Collard, 2009; Kelleher, 2006). Instead, Mn uptake is controlled by the sources of Mn, i.e., milk vs. non-milk in infants (Erikson et al., 2007). Accordingly, the baby's Fdietup was computed using a factor FDP, which represents relative uptake of Mn from breastmilk or formula milk, compared to an average baseline fractional uptake for solid food of 0.06 (equivalent to intake-dependent Fdietup at an Mn intake of approximately 2.8 mg/day estimated in adults, according to Eq. (2). For breastmilk, FDP = 10. For formula milk, FDP = 0.01 (Yoon et al., 2011). It was assumed that all the Mn in formula milk are not bound to milk proteins such as lactoferrin. When drinking water was used to prepare formula, FDP = 0.01.

Table 7 Age-dependent daily dietary mn intake as recommended daily intake (RDI). Age (months)

RDI for males (mg/day)

RDI for females (mg/day)

0 7 12 48 108 168 228+

0.003 0.6 1.2 1.5 1.9 1.9 2.3

0.003 0.6 1.2 1.5 1.6 1.6 1.8

These age-dependent values were used in child/adolescent model only. Source: IOM FNB (2001).

formula powder calculated (Eq. (1)) following the approach of Brown and Foos (2009). Brown and Foos (2009) found that median Mn concentration in prepared formula milk was 15 μg/148 mL – the average intake per feeding (Brown and Foos, 2009). To calculate Mn intake on a daily basis requires estimation of the number of formula servings per day. Brown and Foos (2009) estimated number of formula servings per day from the daily water intake rate, reasoning that in an exclusively formula-fed infant, all drinking-water intake would be from formula.

Mn daily intake from formula powder (mg/day) (IRDW × 1000 mL/L) = × 15 µ g/serving × 0.001 mg/ µ g 148 mL/serving

(2)

(1)

2.6. Model runs and analyses

where IRDW is the daily drinking water ingestion rate in L/day during formula-feeding, interpolated from the data in Table 6. The estimated daily Mn intake from formula powder containing Mn at 15 μg/ serving is generally on the order of 0.05 mg/day (Table S5). The Brown and Foos value of 0.05 mg/day is in the range of Mn exposures that would be predicted based on formula intake recommendations for 0–6 month-old infant provided by the American Academy of Pediatrics (American Academy of Pediatrics, 2018).

All simulations and analyses for the early age and adult human Mn PBPK models were performed using GNU MCSim (Bois, 2009) and R (R Core Team, 2018). The R package for this work is available from the corresponding author on request. 3. Results To determine if drinking water exposure during gestation period would affect tissue Mn in the newborn, i.e., age 0, we simulated the effect of drinking water exposure in the pregnant mother on fetal tissue Mn. The effect of adding drinking water exposure was shown in Supplementary Material (Figs. S1). There were no noticeable increases in placental or fetal tissue Mn concentrations even at 0.58 ppm Mn in drinking water. Therefore, we decided that it was not necessary to account for in-utero exposure due to maternal drinking water Mn consumption to simulate infants tissue Mn concentrations at birth. To evaluate the effect of drinking water Mn exposure in infants on blood and tissue concentrations of Mn, two different milk Mn exposure scenarios, breastmilk and formula milk, were simulated with and without drinking water exposure. First, the blood concentration of Mn was simulated and compared to the results of Stastny et al. (1984), in which the details of the Mn exposure infants for both a breast-fed and a formula-fed infant at age 90 days without drinking water Mn were

2.4.3. Dietary Mn exposure In the lactation/infant model, daily dietary Mn intake for the mother was assumed to be a constant 3.0 mg/day consistent with Yoon et al. (2011). The same value was used for the pregnant mother model. For the baby in the lactation/infant model, before weaning at age 6 months, dietary Mn intake was assumed to be zero, i.e., the baby was exclusively fed on breastmilk or formula milk. After weaning, dietary Mn intake was set to 0.6 mg/day up to age 1 year and was then 1.5 mg/ day between ages 1–5 years. In the child/adolescent scenarios, dietary Mn intake was modeled by linear interpolation from the IOM's recommended daily dietary intakes (RDI) by age and sex (Table 7). In the adult scenarios, dietary Mn intake was constant at the sex-specific IOM RDI values for age 228+ months, i.e., 19+ years: 2.3 mg/day for males and 1.8 mg/day for females. 7

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reported together with the corresponding serum Mn concentrations. In these simulations, the formula-fed infant had a Mn intake from formula powder equal to the mean formula Mn intake reported by Stastny et al. (1984), i.e., 1.154 mg Mn/day. Blood Mn concentrations were used to calculate the ratio of the serum Mn concentrations between breast-fed and formula-fed infants (Fig. 1). A full tissue Mn dose-response curve for a range of formula Mn doses in comparison to the breastmilk Mn dose is presented in Supplementary Material (Fig. S2). To determine whether the difference between breastfed and formula-fed infants was affected by drinking-water Mn, exposure scenarios were simulated for exposures where the nursing mother consumed Mncontaining drinking water at 0.58 ppm, and the same Mn-containing drinking water was used to prepare formula. Simulated blood concentrations, as a percentage of the blood concentration in the breastfed infant whose mother consumed drinking water with 0 ppm Mn, for both 0 ppm and 0.58 ppm drinking water were compared to the results of Stastny et al. (1984). For breast-fed infants, these results suggest that maternal consumption of Mn-containing drinking water even at a high concentration (0.58 ppm) had negligible effect on infant serum Mn (Fig. 1). For formula-fed infants, the results for both 0 ppm and 0.58 ppm drinking water are within one standard deviation of the mean value reported by Stastny et al. (1984), suggesting that the use of Mncontaining drinking water to prepare formula does not substantially elevate infant serum Mn compared to the use of non-Mn-containing drinking water, even at a relatively high Mn level of 0.58 ppm. For an exclusively breastfed infant, the drinking-water Mn concentration consumed by the mother had little effect on the infant's brain, blood, or liver Mn concentrations (Fig. 2). For a formula-fed infant, if the formula were prepared with drinking water with 0 or

Fig. 1. Comparison of simulated serum Mn in infants fed with breastmilk or formula milk. Note that the simulated serum Mn concentrations in the breastfed and formulafed infants without drinking water exposure are presented in in the gray columns, which are expressed relative to the breastfed scenario with 0 ppm drinking water at age 90 days after birth. The reported mean and SD of the serum Mn concentrations in breastfed and formula-fed infants are shown in hollow columns (Stastny et al., 1984). The effect of adding drinking water Mn exposure is shown in black columns. Note that for breastfed scenarios, the drinking water exposure to Mn is through lactational exposure resulting from the drinking water exposure in the mother, whereas the drinking water exposure to Mn in formula-fed infants is from the drinking water exposure in the infant.

Fig. 2. Time course of tissue Mn concentration in infants with or without drinking water Mn exposure. Simulated Mn concentrations in globus pallidus (top row), whole blood (second row), and liver (bottom row) are shown for ages 0–6 months, for scenarios of breastfed infant (left column) and formula-fed infant (right column), for maternal (for breastfed infantscenarios) and infant (for formula-fed infant scenarios) drinking-water concentrations of 0, 0.05, 0.3, and 0.58 ppm (lines in each panel). The formula-fed infant is fed with formula containing Mn based on the Brown and Foos (2009) scenario. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3. Time course of tissue Mn concentration in young children. Predicted Mn concentrations in globus pallidus (top row), whole blood (second row), and liver (bottom row) are shown for ages 6–36 months, for scenarios of breastfed infant (left column) and formula-fed infant (right column), for drinking-water concentrations of 0, 0.05, 0.3, and 0.58 ppm (lines in each panel). The formula-fed infant is the Brown and Foos (2009) scenario. Note that “breastfed” and “formula-fed” refer to the infant's feeding before weaning at 6 months; after weaning, both scenarios have identical dietary and drinking-water intake rates. Differences in internal dose metrics disappear quickly after weaning; there is no long-term effect of having been breastfed or formula-fed. The sharp change at 6 months is an artifact introduced by the fact that weaning is modeled as instantaneous rather than gradual. The sharp change at 12 months is introduced by modeling an abrupt change in dietary intake jumping from 0.6 mg/day to 1.5 mg/day at age 12 months. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

0.05 ppm Mn, the tissue concentrations would be the same or lower than for the breastfed infant (Fig. 2). However, formula prepared with drinking water containing 0.3 or 0.58 ppm Mn results in simulations showing noticeable increases in tissue concentrations compared to 0 ppm drinking water — although even 0.58 ppm predicted tissue concentrations are only slightly higher than those in the breastfeeding scenario. The time courses of tissue concentrations shown from ages 0–6 months in Fig. 2 are continued in Fig. 3 after weaning, up to age 36 months. Note that very soon after weaning at 6 months, differences between breastfed and formula-fed scenarios disappear, indicating that there is no long-term effect on tissue Mn concentrations when comparing breastfeeding vs. formula-feeding. Differences in internal dose metrics between water with 0 ppm Mn and 0.3 or 0.58 ppm Mn were present before the age of 1 year, and much less noticeable after that age. The effects of various drinking-water Mn concentrations on predicted tissue concentrations are shown for 7 discrete age groups in Fig. 4. The values for the infant scenarios correspond to the values shown in Fig. 2 at 6 months; the values for the toddler scenario in Fig. 4 corresponds to the values shown in Fig. 3 at 36 months. Overall, the effect of adding drinking water exposure to a total daily dietary exposure appears to be similar across ages. There were no noticeable differences in globus pallidus concentrations expected from drinking water containing Mn between children and adults. Simulated globus pallidus Mn concentrations were higher in the 3- and 10-year-old child scenarios than the teenager and adult scenarios, a set of differences that may be fraught with some uncertainty since there is some question about age-related changes in tissue volumes across ages after age 3. In addition, the relative levels of daily Mn intakes used for children versus adults in the published model (Yoon et al., 2011) are different from those used in the current study indicating that the time-profiles of basal

brain Mn concentrations are influenced by how we set dietary intake across ages. It should be noted that the daily intake values we used are recommended values, not the actual intake values reported. Nonetheless, all the simulated brain concentrations in the globus pallidus region were within the range observed in human cadavers (Tingey, 1937). The age-profiles of whole-blood Mn concentrations are consistent with the reported human data (Henn et al., 2010; Spencer, 1999). Liver Mn concentrations are similar across all age groups. The relative contribution of dietary Mn and drinking-water Mn to the total ingested Mn is illustrated in Fig. 5, on both a mg/day basis (top row) and a mg/kg/day basis (bottom row). On a mg/day basis, for the formula-fed infant, drinking-water Mn contributes almost all ingested Mn. For toddlers, drinking-water Mn does not change much from formula-fed infancy, but dietary Mn is much higher; thus, drinking-water Mn contributes proportionally much less to total ingested Mn. The relative contribution of drinking-water Mn tends to increase through childhood then adolescence and on to adulthood. On a mg/kg/day basis, the picture is somewhat different. On this weight adjusted basis, the 3-year-old toddler has the highest total ingested Mn, compared to the other age groups. Nonetheless, the relative contribution of drinking-water Mn tends to increase through childhood and adolescence to adulthood, even as the total per-kilogram ingested Mn decreases. For the same seven age groups shown in Figs. 4 and 5, a “doseresponse” depiction of globus pallidus Mn concentrations for different drinking-water Mn concentrations is illustrated in Fig. 6. For the breastfed infant, globus pallidus Mn concentrations remain effectively constant over all drinking-water Mn concentrations. Globus pallidus Mn concentrations for all age groups remain fairly constant for drinkingwater Mn up to about 0.1 ppm, which is twice the USEPA secondary maximum contaminant level [SMCL] for Mn. For child and toddler age groups, globus pallidus Mn concentrations remain fairly constant for 9

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Fig. 4. Tissue Mn concentrations simulated for infants, children of various ages and 35-year old adults. Simulated Mn concentrations in globus pallidus (top row), whole blood (second row), and liver (bottom row) are shown for 7 different age scenarios (horizontal axis), for drinking-water concentrations of 0, 0.05, 0.3, and 0.58 ppm (greyscale bars). Here, the formula-fed infant is the Brown and Foos (2009) scenario. The toddler, child, teenager, and adult scenarios are all those that began with the Brown and Foos (2009) formula-feeding scenario; as shown in Fig. 3, the differences in Mn concentrations seen in the infant feeding scenarios disappear quickly after weaning, so these results would be effectively identical for those that began with breastfeeding or with the Stastny et al. (1984) formula-feeding scenarios. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Comparison of Mn daily doses across ages with and without drinking water exposure. Daily ingested Mn (upper panel: mg/day; lower panel: mg/kg/day), for a range of drinking-water Mn concentrations (horizontal axis) are shown for 7 different age scenarios (panels). Daily ingested Mn is split into dietary intake (gray bars) and drinking-water intake (black bars). Here, “dietary” includes breastmilk or formula intake, for the two nursing-infant scenarios (breastfed infant and formula-fed infant). Total height of the stacked bars indicates total daily ingested Mn. Note that the intake of the breastfed infant (leftmost panels) is not actually zero; it is very small compared to the vertical scale of the plot.

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Fig. 6. Changes in globus pallidus Mn with a range of drinking-water Mn concentrations. Simulations were performed with a range of drinking water concentrations (horizontal axis, log10 scale) for 7 different age scenarios (solid and dashed curves). Arrows pointing to the x-axis mark the drinkingwater Mn concentrations highlighted in Fig. 4 and Fig. 5 (0.05, 0.3, and 0.58 ppm), which are based on US EPA's Secondary Maximum Contaminant Level (SMCL) for Mn based on taste & staining considerations, US EPA's lifetime health advisory value (LHA), and the 95th percentile of the drinking-water concentration in Iowa (US EPA's National Inorganics and Radionuclides Survey, 2003), respectively.

drinking-water Mn up to 0.3 ppm (USEPA, 2004). These simulations indicate that drinking-water Mn at levels commonly encountered is expected to have minimal effect on globus pallidus Mn concentrations in very young children through age 18. Interestingly, Fig. 6 shows that globus pallitus Mn concentrations in the formula-fed infant do not reach that of the breastfed infant until drinking-water Mn concentrations exceed approximately 0.5 ppm (i.e., 0.5 μg/mL). Drinking-water Mn, at levels commonly encountered when used to prepare infant formula, is unlikely to meaningfully affect the brain Mn concentration in infants fed using that formula.

to sequester Mn to build up tissue levels. The model simulations also showed that the internal exposure to Mn in the target brain region in children was comparable to that in adults even after incorporating the child-specific respiratory parameters and age-specific deposition patterns for the respiratory tract (Yoon et al., 2011). While the inhalation model has proven valuable in comparing internal exposure to Mn across ages, it did not include drinking water Mn exposures and only modeled childhood exposures through age 3. Here, we extended the model beyond age 3 covering children of ages ranging from 0 to 18 and added several additional age-specific parameters that are critical in reducing uncertainties in the evaluation of age-related sensitivity to Mn, particularly via drinking water exposure. Formula-fed infants are regarded as the highest risk group to drinking water contaminants because > 75% of infants are formula-fed either exclusively or along with breastfeeding at 6 months of age in the United States (CDC, 2018). In addition, the drinking water consumption rates are the highest during the first year of life (Table 6), and if formula is prepared with Mn containing drinking water, their Mn exposure may be much higher than adults, considering that Mn concentrations in infant formulas are 3 to > 100-fold higher than in breastmilk (Sievers, 2005; Collipp et al., 1983; Stastny et al., 1984; Lönnerdal et al., 1994). The formula Mn content/concentration used in the current study as a realistic scenario is still approximately 34-fold greater than average breast milk Mn concentrations of ~3 μg/L (Brown and Foos, 2009), even without the addition of Mn from drinking water. In fact, the estimated external exposure to Mn per kg body weight was the highest in formula-fed infants (Fig. 4) with drinking water Mn exposure. However, the relative importance of drinking water exposure was reduced when expressed as internal exposure even at a high concentration in water of 0.58 ppm (Fig. 4). This difference occurs because the bioavailability of Mn in drinking water (0.06%) is much lower than breastmilk Mn (60%). The lower bioavailability was also used for the Mn in formula milk. Clearly, bioavailability in drinking water in formula-fed infants versus that from breast milk is a critical parameter in assessing expected tissue concentrations of Mn in early life. Potential differences in Mn bioavailability between drinking water

4. Discussion Several studies have raised concerns for increased sensitivity to Mn neurotoxic effects in children, particularly when exposed through drinking water (Bouchard et al., 2010; Khan et al., 2012; Oulhote et al., 2014b). The sources of these concerns are 1) the possibility of immature homeostasis in children based on animal studies, 2) enhanced delivery of Mn to the developing brain due to increased needs for Mn during intrauterine and postnatal development, and 3) exposure factors in children, including higher water consumption and higher body-weight adjusted ventilation rates compared to the adults. Previously, we developed a PBPK model for Mn during lactation and early childhood periods with a focus on inhalation exposure (Yoon et al., 2011) and showed that the normal processes for Mn homeostasis are active during development for both Mn-maintenance and in the face of Mn excess (Yoon et al., 2011). The higher percentage retention of dietary Mn with lower biliary excretion does lead to higher percentage uptake from the gut. However, this enhanced uptake compared to the adult is still a part of normal Mn homeostasis in the young and is largely attributable to very low Mn content in breast milk compared to other foods rather than poorly developed homeostatic mechanisms for Mn absorption (Anderson, 1992). The PBPK modeling analysis strongly indicated that the apparent difference in uptake in neonates is not due to immature homeostatic mechanisms as previously inferred from earlier studies (Ballatori et al., 1987; Keen et al., 1986) but rather the need 11

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and diet have long been a major uncertainty in risk assessment considerations for different human populations including children. When experimental data on Mn uptake from drinking water in adult rats (Foster et al., 2015) were examined using a PBPK model (Song et al., 2018), no significant differences were found for oral bioavailability of Mn from drinking water or from dietary sources of Mn. After scaling the adult model to include age-appropriate body weight and physiological parameters and similar uptake characteristics for dietary and drinking water sources of Mn, blood Mn concentrations in older children were reasonably well simulated (Song et al., 2018). The success of this simulation suggested that the bioavailability of drinking water Mn is similar to that of dietary Mn in these older children. This conclusion was confirmed using a model construct that that incorporated a variety of age-specific factors. In general, trace elements in human milk are often more bioavailable than when present in infant formula (Saarinen et al., 1977; Johnson and Evans, 1978), possibly as a result of difference in molecular forms of Mn in milk and formula (Lönnerdal et al., 1985). A receptor for milk protein, believed to be lactoferrin, expressed during lactation in the neonatal gut is considered responsible for the enhanced bioavailability of Mn and other essential elements like iron, zinc and copper from human breast milk (Lönnerdal et al., 1985; Davidson and Lönnerdal, 1988, 1989; Pacha, 2000). This protein-based recognition of Mn in the infant gut is less important as the child ages and divalent metal transporter-1 (DMT-1) mediated absorption appears more important once the child starts on a solid food diet. DMT-1 is more specific to Mn concentrations/amounts in food, rather than the complexing proteins to which Mn binds (Chan et al., 1982; Lönnerdal et al., 1985). Because of the similarity of bioavailability of Mn from drinking water and diet as found by Song et al. (2018), absorption of Mn from drinking water is presumably mediated largely by DMT-1 and a similar process is likely to control Mn uptake from constituted/Mn-fortified non-milk diet, whether solid or formula based. None of these other sources of Mn are expected to be as bioavailable in infants as is the Mn in breastmilk. Results from various experimental studies are consistent with this conclusion. For instance, despite > 100-fold difference in total Mn contents between breastmilk and formula-milk chemically fortified with Mn, serum Mn concentrations in formula-fed infants were within the range of variation of the concentrations in breastfed infants (Stastny et al., 1984). Our model reproduced the observed serum Mn concentrations in formula-fed infants relative to those in breastfed infants (Yoon et al., 2011 and Fig. 1). The previous analysis (Yoon et al., 2011) showed a higher bioavailability of Mn from cow-milk based formula (Hatano et al., 1985) than from formula containing Mn by chemical fortification (Stastny et al., 1984). However, the bioavailability of Mn from cow-milk based formula was still not as high as that from human breastmilk likely due to the difference in Mn-binding milk-proteins between species. This further supports the critical role of Mn binding to milk proteins mediating Mn absorption in young infants and the presumed lower bioavailability of drinking water Mn. Simulations with this expanded multi-dose route, multi-age model structure indicate that the effect of including drinking-water exposure along with dietary intake and ambient air inhalation on tissue Mn concentrations in children is not expected to be any greater than the effects in adults, even at a drinking-water concentration approximately twice the Lifetime Health Advisory value set by the USEPA. When the relative contribution of dietary Mn and drinking water Mn to the total ingested Mn was assessed on both a mg/day basis and mg/kg/day basis, the relative contribution of drinking water Mn to total uptake increases through childhood and adolescence to adulthood. The higher water consumption rates in children were not a major factor in increasing internal exposure of Mn in children (Fig. 5). In addition, simulation of globus pallidus Mn concentrations at a range of different drinking water Mn concentrations demonstrated that globus pallidus Mn concentrations would remain fairly constant for drinking water Mn up to 0.3 ppm for the toddler and child age groups. This suggest that the conclusion of

the air concentration-internal exposure in brain to Mn relationship analysis performed previously both for children and adults will still holds true even when typical drinking water exposure is included together with the dietary exposure (Yoon et al., 2011; Gentry et al., 2017). The current study is consistent with our previous study (Yoon et al., 2011) indicating that the internal exposure to Mn in the brain of nursing infants and young children ages up to age 3 is not expected to differ from that in the adult brain. This conclusion is also consistent with the biology of essential element brain delivery mechanisms during development in early ages. Transport of essential elements including Mn is mediated by active transporters, not by simple diffusion through the blood brain barrier. The expression of transporter proteins responsible for active uptake of Mn and other essential elements, in particular DMT1 and transferrin receptor proteins, are reported to be comparable to or greater in fetuses/neonates than in adults (Erikson et al., 2007; Siddappa et al., 2002; Garcia et al., 2006). 5. Conclusion This expanded Mn PBPK model describes age-dependent Mn homeostasis at dietary steady state, introduces environmentally relevant inhalation and drinking water exposure conditions, and accounts for differences in oral bioavailability of Mn for infants ingesting breast milk, formula milk, or drinking water. This biologically-based model provides internal dose information for children exposed by both inhalation and drinking-water and allows a better understanding of the question of whether there is age-related sensitivity to Mn exposure via drinking water. Based on the results of the model, infants and children are not expected to be at greater exposure than adults to Mn in drinking water. Funding This work and publication are based on studies sponsored and funded by Afton Chemical Corporation in satisfaction of registration requirements arising under Section 211(a) and (b) of the Clean Air Act and corresponding regulations at 40 CFR Substance 79.50 et seq. Transparency document The Transparency document associated to this article can be found, in the online version. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.taap.2019.114695. References American Academy of Pediatrics, 2018. Amount and Schedule of Formula Feedings. Last updated July 24. https://www.healthychildren.org/English/ages-stages/baby/ formula-feeding/Pages/Amount-and-Schedule-of-Formula-Feedings.aspx. Anderson, R.R., 1992. Comparison of trace elements in milk of four species. J. Dairy Sci. 75 (11), 3050–3055 Nov 1. Aschner, M., 2000. Manganese: brain transport and emerging research needs. Environ. Health Perspect. 108 (Suppl. 3), 429–432. ATSDR, 2012. Toxicological Profile for Manganese. U.S. Department of Health and Human Services, Public Service, Atlanta, GA. Ballatori, N., Miles, E., Clarkson, T.W., 1987. Homeostatic control of manganese excretion in the neonatal rat. Am. J. Phys. 252, R842–R847. Bois, F.Y., 2009. Bayesian statistical inference for SBML-coded systems biology models. Bioinformatics 25, 1453–1454. Bouchard, M.F., Sauvé, S., Barbeau, B., Legrand, M., Brodeur, M.È., Bouffard, T., Limoges, E., Bellinger, D.C., Mergler, D., 2010 Sep 20. Intellectual impairment in school-age children exposed to manganese from drinking water. Environ. Health Perspect. 119 (1), 138–143. Bouchard, M.F., Sauve, S., Barbeau, B., Legrand, M., Brodeur, M.E., Bouffard, T., Limoges, E., Bellinger, D.C., Mergler, D., 2011. Intellectual impairment in school-age children

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