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Biomonitoring Equivalents for molybdenum
Regulatory Toxicology and Pharmacology 77 (2016) 223e229
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Biomonitoring Equivalents for molybdenum Sean M. Hays a, *, Kristin Macey b, Devika Poddalgoda b, Ming Lu b, Andy Nong b, Lesa L. Aylward c a
Summit Toxicology, LLP, Lyons, CO 90540, USA Health Canada, Ottawa, ON, Canada c Summit Toxicology, LLP, Falls Church, VA 22044, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 December 2015 Received in revised form 4 March 2016 Accepted 6 March 2016 Available online 10 March 2016
Molybdenum is an essential trace element for mammalian, plant, and other animal systems. The Institute of Medicine (IOM) has established an Estimated Average Requirement (EAR) to assure sufficient molybdenum intakes for human populations; however excessive exposures can cause toxicity. As a result, several agencies have established exposure guidance values to protect against molybdenum toxicity, including a Reference Dose (RfD), Tolerable Daily Intake (TDI) and a Tolerable Upper Intake Level (UL). Biomonitoring for molybdenum in blood or urine in the general population is being conducted by the Canadian Health Measures Survey (CHMS) and the U.S. National Health and Nutrition Examination Survey (NHANES). Using pharmacokinetic data from controlled human dosing studies, Biomonitoring Equivalents (BEs) were calculated for molybdenum in plasma, whole blood, and urine associated with exposure guidance values set to protect against both nutritional deficits and toxicity. The BEEAR values in plasma, whole blood and urine are 0.5, 0.45 and 22 mg/L, respectively. The BEs associated with toxicity range from 0.9 to 31 mg/L in plasma, 0.8e28 mg/L in whole blood and 200e7500 mg/L in urine. These values can be used to interpret molybdenum biomonitoring data from a nutritional and toxicity perspective. © 2016 Published by Elsevier Inc.
Keywords: Biomonitoring Biomonitoring Equivalents Molybdenum Risk assessment
1. Introduction Molybdenum (Mo) is a naturally occurring element and is found in trace levels in all environmental media, food and drinking water. Mo is an essential trace element for mammalian, plant, and other animal systems. In humans, Mo is an activator of three enzymes: sulfite oxidase, xanthine oxidase, and aldehyde oxidase, which are involved in the oxidation of sulfite to sulfate, the production of uric acid, and the oxidation of aldehydes (EFSA, 2013; IOM, 2001; Vyskocil and Viau, 1999; Turnlund et al., 1995a). However, at elevated exposures, Mo toxicity has been observed in human and animal studies (US EPA, 1992; IOM, 2001; OECD SIDS, 2013). All humans are exposed to Mo through their diet. Concentrations of Mo are higher in legumes, pulses, cereal grains and nuts than in dairy and meat (EFSA, 2013; Vyskocil and Viau, 1999). In Europe, cereals and cereal-based products including bread are the major contributors to dietary intake (EFSA, 2013). The main
* Corresponding author. 165 Valley Rd., Lyons, CO 80540, USA. E-mail address: [email protected] (S.M. Hays). http://dx.doi.org/10.1016/j.yrtph.2016.03.004 0273-2300/© 2016 Published by Elsevier Inc.
contributors of Mo in North American diet are legumes, grain products and nuts (IOM, 2001). Dietary intakes in adults range from 58 to 157 mg/day (0.97e2.61 mg/kg-bw per day based on a 60 kg BW) in Europe (EFSA, 2013), while the IOM (2001) reports dietary intakes ranging from 120 to 240 mg/day (1.71e3.42 mg/kg-bw per day based on a 70 kg BW in adults in the United States. In Canada, average dietary intake is 2.6 mg/kg-bw per day (Health Canada, 2003). In addition to naturally occurring Mo, there is anthropogenic contribution of Mo to environmental media from mining and related use industries. The primary use of Mo is as a component of steel alloys to increase strength and durability and aid in corrosion resistance (IMOA, 2015). Mo is also found in a number of products available to consumers including nutritional supplements, plant care products, household cleaning products, and products available to children (e.g. toys, clothing) (US Household Products Database, 2015; State of Washington, (2015)). Large human biomonitoring surveys, such as the Canadian Health Measures Survey (CHMS) and the U.S. National Health and Nutrition Examination Survey (NHANES) are providing valuable data on the prevalence and concentration of many environmental
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chemicals and essential elements, including Mo, in the general population. Biomonitoring in urine or blood allows for an assessment of integrated exposures to molybdenum across different sources and routes of exposure. However, measured concentrations of Mo in blood or urine cannot be directly interpreted using the available exposure guidance values as the latter are presented in terms of daily intake levels (e.g. mg/kg-bw per day). The purpose of this evaluation is to derive Biomonitoring Equivalent (BE) values for interpretation of population biomonitoring data for Mo. BE values are estimates of the concentration of a chemical or its metabolite in blood or urine that are consistent with defined exposure guidance values such as RfDs or TDIs (Hays et al., 2007, 2008; Angerer et al., 2011). BE values can be used as screening values for the assessment of biomonitoring data in order to provide an initial evaluation of whether the detected concentrations are below, near, or above the concentrations consistent with exposure guidance values. The BEs developed here provide tools for interpreting Mo biomonitoring levels in the context of exposure guidance values designed to protect for nutritional adequacy and to protect against toxicity associated with excessive exposures. 2. Methods Derivation of BE values for evaluation of biomonitoring data in a risk assessment context generally requires three main elements: an assessment of toxicity with a corresponding derived exposure guidance value such as a RfD or a TDI; the availability of a biomarker that is specific to the exposure of interest; and sufficient toxicokinetic data to predict the biomarker concentrations in urine or blood under steady-state exposure conditions consistent with the chronic exposure guidance value(s). The following sections describe the available information for molybdenum and outline the approaches taken to derive BE values for Mo in blood and urine. 2.1. Nutrition and toxicity data and risk assessments Mo is an essential trace element for humans, animals and plants, which acts as a cofactor (molybdopterin) for certain enzymes (IOM, 2001; WHO, 2011; Turnlund et al., 1995a). The clinical signs of Mo deficiency associated with low dietary intakes are not commonly observed in experimental animals or in healthy people (IOM, 2001). However, a genetic defect called molybdenum cofactor deficiency that results in decreases in the activities of Mo-dependent enzymes has been identified (Kikuchi et al., 2012). Deficiency in these enzymes usually leads to fatal neurological toxicity in infants born with this condition, or irreversible neurological damage due to the inability of the body to convert sulfite to sulfate (reviewed in IOM, 2001). Excess intakes of Mo appear to have low toxicity in humans (IOM, 2001; Turnlund et al., 1995a). Animals, especially ruminants, are known to be more susceptible Mo toxicity than humans (IOM, 2001). Ruminants show adverse effects of Mo under copper deficiency and marginal sulfur amino acid intake (Underwood, 1977). Hence, the basis of toxicity in ruminants is not considered relevant to humans (IOM, 2001). In female rats, dietary supplementation of Mo resulted in prolonged estrus cycle, decreased gestational weight gain of pups and several adverse effects in embryogenesis at doses of 1.6 mg Mo/kg-bw per day, but not at 0.9 mg Mo/kg-bw per day (Fungwe et al., 1990). Similarly, Schroeder and Mitchener (1971) reported adverse reproductive effects at 1.5 mg Mo/kg-bw per day in a drinking water three-generation reproductive toxicity study in mice. However, a 90-day oral repeated dose toxicity study (Murray et al., 2014a) reviewed by the Organization for Economic Co-operation and Development (OECD) where male and female
rats were exposed to Mo in disodium molybdate dehydrate via feed did not report any adverse effects in reproductive parameters in male or female rats at the highest dose tested (60 mg Mo/kg-bw per day). The same study (Murray et al., 2014a) reported reduced body weight gains and kidney effects at 60 mg Mo/kg-bw per day and the No Observed Adverse Effects Level (NOAEL) was identified as 17 mg Mo/kg-bw per day. Another guideline compliant pre-natal developmental toxicity study reviewed by the OECD (Murray et al., 2014b), where female rats were exposed up to 40 mg Mo/kg-bw per day via feed to same form of Mo as above during gestation day 6e20, did not report any maternal toxicity or dose-related adverse effects on development of the offspring. Based on the evidence from a 2-year inhalation carcinogenicity study and in vitro genotoxicity studies by NTP (1997), Mo is not considered to have mutagenic or carcinogenic potential (RIVM, 2001). Several international agencies have conducted risk assessments on Mo and have established exposure guidance values for general population to protect against both nutrient deficiency and toxicity. A series of regulatory guidance values were established by the Institute of Medicine (IOM), European Commission Scientific Committee on Food (EC SCF), US Environmental Protection Agency (US EPA) and The Netherlands National Institute for Public Health and the Environment (RIVM). The IOM (2001) has established an Estimated Average Requirement (EAR) of 0.034 mg/day (0.00049 mg/kg-bw per day assuming 70 kg BW) based on the results from Turnlund et al. (1995b) to ensure population level nutritional adequacy, and a Recommended Daily Allowance (RDA) of 0.045 mg/day (0.00064 mg/kg-bw per day) as the target intake for individuals. IOM (2001) also estimated a Tolerable Upper Intake Level (UL) of 2 mg/day (0.03 mg/kg-bw per day assuming 68 kg BW) based on the results from Fungwe et al. (1990) to protect against toxicity. EC SCF (2000) also used the same study as IOM (2001) (Fungwe et al., 1990) to derive a UL of 0.6 mg/day (0.01 mg/kgbw per day assuming 60 kg BW). RIVM (2001) established a TDI of 0.01 mg/kg-bw per day for oral exposures based on kidney effects in rats (van Esch, 1978) and a tolerable concentration in air (TCA) for inhalation exposures. The US EPA (1992) derived a RfD of 0.005 mg/ kg-bw per day for Mo based on a human epidemiology study (Koval'skiy et al., 1961) in which the most sensitive endpoint was increased serum uric acid levels. While some of these international agencies have developed guidance values for different age groups and sub-populations (e.g. pregnant and lactating women), the guidance values presented here are for adults in the general population and these values are summarized in Table 1. In addition, the OCED conducted a review of molybdenum in 2013, however they did not establish an exposure guidance value, rather the lowest NOAEL in the assessment profile was identified based on effects on body weight and kidney. 2.2. Biomarker selection Biomonitoring for Mo exposures involves quantifying Mo in urine and/or blood (plasma and/or whole blood). Mo in both plasma and urine has been shown to be well correlated with dietary intake (Turnlund and Keyes, 2004). Urine is the predominant route of elimination, with upwards of 90% of an oral dose being eliminated via urine with a half-life of less than 12 h (Turnlund and Keyes, 2004), indicating urine is a good matrix for assessing exposures to Mo. In plasma, elimination has been described with a biexponential function with mean half-lives of 30 min and 6.6 h, respectively (Werner et al., 2000). Generally, biomarkers with a longer half-life of elimination have less daily fluctuation around the chronic-steady state average concentrations and are more suitable biomarkers to estimate the exposure (Hays et al., 2007). The halflife of elimination of Mo following an oral dose is short in both
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Table 1 Available exposure guidance values for molybdenum for both nutritional requirements and to protect against toxicity. Organization, criteria Critical endpoint (year of evaluation) EAR, IOM (2001)
Dose level
Uncertainty factors
Value 0.034 mg/d (adults) (0.0005 mg/kg-d)
EAR*1.3 to account for population variation in requirement
0.045 mg/d (adults) (0.00064 mg/kg-d)
UL, IOM (2001)
Mo balance without any clinical Based on the balance data, the minimum requirement signs of deficiency in humans plus increment for miscellaneous loss is 0.025 mg/d. EAR is estimated assuming 75% bioavailability from food Turnlund et al., 1995b Estimated average requirement (EAR) of Mo for adults Essential trace element for is 0.034 mg/d. selected enzyme function in EAR values for other age groups calculated based on humans bodyweight0.75 scaling Turnlund et al., 1995b Reproductive effects in rats NOAEL, 0.9 mg/kg/day Fungwe et al., 1990
30
EC SCF (2000)
Reproductive effects in rats
100
Chronic oral RfD, U.S. EPA (1992) Chronic oral TDI, RIVM (2000) 90-day toxicity study OECD SIDS (2013)
Increased serum uric acid levels LOAEL, 0.14 mg/kg-d Koval'skiy et al., 1961 in humans. Kidney effects in rats NOAEL 1.0 mg/kg-d van Esch, 1978
30
2 mg/d (0.03 mg/kg-d) 0.6 mg/d (0.01 mg/kg-d) 0.005 mg/kg-d
100
0.01 mg/kg-d
100
1.2 e2 mg/m3
RDA, IOM (2001)
NOAEL, 0.9 mg/kg/day Fungwe et al., 1990
Reduced body weight and kidney NOAEL 17 mg/kg-d Murray et al., 2014a effects at 60 mg/kg-d
a a
A summary of the study was published in 2014 following review by the OECD in Murray et al. (2014a).
Several informative pharmacokinetic (PK) studies have been conducted in humans. Turnlund et al. (1995a) conducted an extremely valuable study in which human (healthy male) volunteers were confined to a metabolic research station for 120 days and Mo exposures were controlled. They administered five increasing levels of Mo via the diet for 24 days each (22, 72, 121, 467, 1490 mg/day). Daily urinary and fecal excretion of Mo was measured in the middle and end of each dosing regimen. Blood samples were also drawn on the same days, but analytical techniques employed at that time did not allow the researchers to quantify Mo in plasma (Turnlund et al., 1995b). The researchers later developed an analytical approach that allowed for quantifying low levels of Mo in plasma and subsequently analyzed plasma samples from the earlier study (Turnlund and Keyes, 2004). The results suggest highly linear relationships between both plasma and urinary Mo and daily dose via the diet (Figs. 1 and 2 and Table 2). As part of their experimental
design, isotope enriched Mo was administered via i.v. to one group while the other two groups were administered Mo as ammonium molybdate in a liquid, to drink, in addition to their basic diet. Analysis of Mo isotopes in blood, urine and feces throughout the study supported development of a PK model (Novotny and Turnlund, 2007), which allowed the researchers to better characterize tissue storage and turnover of Mo. The half-life of Mo in blood was less than 24 h, and likely to be on the order of 1e3 h (independent analysis of Table 3 in Turnlund and Keyes, 2004). Giussani et al. (2007) conducted PK studies in which male volunteers received single injections (i.v.) of enriched 96Mo isotopes and 95Mo was administered simultaneously orally in various foods (Werner et al., 1998). Urine samples were collected post-dosing in three 4-h periods, followed by collection over a 12-h period and 24h composites over the following 6 days. The majority of the 96Mo excreted in urine occurred within the first couple of hours and was almost completed by 12 h post-dosing. Giussani et al. (2007) did not provide enough data to be able to accurately estimate percent excretion in urine following oral doses. All that can be determined from their data is that greater than 65% appears to have been excreted within 24 h post dosing. A series of studies were conducted to assess the dietary needs of Mo in infants and preterm infants (Sievers et al., 2001a, 2001b). Mo
Fig. 1. Relationship between daily dietary administration of Mo and plasma Mo (data from Table 4, group 3 in Turnlund and Keyes, 2004).
Fig. 2. Relationship between daily dietary administration of Mo and daily urinary excretion of Mo (data from Table 5, group 3 in Turnlund and Keyes, 2004).
blood and urine and therefore, neither Mo in blood or urine holds an advantage with respect to half-life of elimination. Analysis of urine Mo concentration has the advantage of being less invasive. BEs are derived here for Mo in plasma, whole blood, and urine. 2.3. Available pharmacokinetic data to support calculations
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Table 2 Summary of available average urinary excretion fraction (Fue) estimates for molybdenum. Study
Age of Participants
Dose (mg/kg)
Media
Duration of dosing
Fue
Sievers et al., 2001a Sievers et al., 2001b
20 days post-term 37.4 (34.1e40.6) weeks posteconception
30.3 2.2 3.0 23.2 27.6 44.7 46.7
Human milk or formula Preterm infant formula Preterm infant formula Preterm infant formula Preterm infant formula Preterm infant formula Preterm infant formula
Turnlund and Keyes (2004)
28 years (average)
0.3 0.9 1.5 5.7 18.1
Solid Solid Solid Solid Solid
Single dose Single dose Single dose Single dose single dose Single dose single dose Average of Infant Studies 24 days 24 days 24 days 24 days 24 days Average of Adult Studies Average of all studies
0.57 0.89 0.62 0.96 0.75 0.62 0.78 0.74 0.97 0.83 0.83 0.90 0.88 0.88 0.80
and and and and and
liquid liquid liquid liquid liquid
diet diet diet diet diet
Table 3 Derivation of Biomonitoring Equivalents for molybdenum.
Basis of guidance value, Nutritional or Toxicity Animal POD, mg/kg-d - Interspecies UF Human POD, mg/kg-d - LOAEL to NOAEL UF Human Equiv. POD, mg/kg-d: Human Equiv. POD, mg/d: Blood BE values: BEPOD Steady-state plasma & serum concentration, mg/L:a BEPOD Steady-state whole blood concentration, mg/L:b - Intraspecies UF BE in plasma & serum, mg/L: BE in whole blood, mg/L: Urinary BE Values Urinary molybdenum excretion, mg/d:c BEPOD, mg/L & mg/g crd - Intraspecies UF BE, mg/L & mg/g cr
IOM, EAR
IOM, RDA
US EPA RfD
RIVM, TDI
IOM, UL
OECD
Nutritional NA NA 0.00049 NA 0.00049 34
Nutritional NA NA 0.00064 NA 0.00064 45
Toxicity NA NA 0.14 3 0.047 3267
Toxicity 1.0 10 NA NA 0.1 7000
Toxicity 0.9 10 NA NA 0.09 6300
Toxicity 17 10 NA NA 1.7 119000
0.50 0.45 1 0.50 0.45
0.52 0.47 1 0.5 0.47
8.9 8.01 10 0.9 0.80
18.6 16.75 10 1.9 1.67
16.8 15.11 3 5.6 5.04
310 279 10 31.0 27.9
30.3 21.7 1 21.7
39.8 28.4 1 28.4
2888.4 2063 10 206
6189.4 4421 10 442
5570.5 3979 3 1326
105220 75157 10 7516
NA - Not Applicable. a - Calculated using Equation (1) (see text). b - Calculated as BE in plasma* 0.9. c - Calculated using Equation (2) (see text). d Urinary flow rate assumed to be 20 ml/kg-d and creatinine excretion rate 20 mg/kg-d, BW ¼ 70 kg (Aylward et al., 2015).
was administered in single doses to infants in either human milk or infant formula and elimination of Mo in urine and feces was determined. It was found that between 57% and 96% of the administered dose of Mo was eliminated in urine (Table 2). The data from Turnlund and Keyes (2004) are the ideal data required for derivation of a BE. They carried out controlled exposure dosing for a duration sufficient to achieve steady-state and for a reasonable range of doses. They measured plasma Mo concentrations (Figure 1) and 24-h urinary excretion rate (UER e Fig. 2; Table 2) as a function of daily dose, yielding linear regressions of;
Mo plasmaðmg=LÞ ¼ 0:0026DDðmg=dayÞ þ 0:4068 ðR2 ¼ 0:992
(1) And
Mo UER ðmg=dayÞ ¼ 0:8842DDðmg=dayÞ
ðR2 ¼ 0:999
(2)
Where DD is daily dose in units (mg/day). Conversion to molar concentration is conducted using the conversion of 1 mg Mo ¼ 0.0104 mmol.
As can be seen from these regression equations and from Figs. 1 and 2, the proportional increase in Mo in plasma is small compared to a large increase in dietary intake of Mo. In contrast, the increase in Mo urinary excretion is proportional to increases in Mo dietary intake (larger slope in the regression equation). This indicates the body is efficient in maintaining homeostasis by controlling plasma Mo and efficiently excretes excess Mo in urine (Giussani, 2008). Mo binds to a2-macroglobulins in plasma in the form of molybdate and to the protein spectrin on erythrocytes (Schultze et al., 2014). No data could be found on the differences in concentrations of Mo in plasma and serum. However, based on the proteins it binds to, it is expected that plasma and serum Mo concentrations should be nearly identical. The whole blood and serum data from both Schultze et al. (2014) and INSPQ (2004) indicated that the ratio of Mo in whole blood to serum (B/S) is approximately 0.9. Therefore, BEs derived in plasma using the data of Turnlund and Keyes (2004) will be assumed to be identical for interpreting Mo in serum and scaled by a factor of 0.9 for deriving a BE in whole blood. The average urinary excretion fraction (Fue) for the studies in infants is 0.74, compared to an average of 0.88 for all the dose groups in the Turnlund and Keyes (2004) study of adults (Table 2).
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Differences are potentially due to 1) single dose administration in infants versus 24 days of dosing, and 2) method of collecting urine (diapers versus complete collections of all urine voids in the adult volunteers). PK studies of inhalation exposures to Mo were not identified. Since inhalation exposures to Mo are considered to be negligible relative to food exposures in individuals exposed from the general environment, and since effects for which guidance values have been established are systemic effects (thus route of administration may not impact toxicity), a BE will not be derived for the RIVM TCA. BEs associated with the oral guidance values should be applicable for interpreting biomonitoring data resulting from inhalation exposures (e.g., systemic effects occur at the same internal/absorbed doses regardless of the route of exposure). 2.4. BE derivation approach The BEs were derived for the EAR, RDA and UL from IOM, RfD from EPA, TDI from RIVM, UL from EC SCF, and the lowest NOAEL identified for Mo in the OECD SIDS assessment profile from a 90day toxicity study (Murray et al., 2014a), herein referred to as the OECD SIDS NOAEL (Table 1). The OECD SIDS NOAEL was included for BE derivation because the OECD SIDS assessment profile includes newer OECD test guideline compliant studies, which were not available at the time of the EPA, IOM, EC SCF or RIVM evaluations of Mo. Additionally, these studies have higher reliability than the studies used in above mentioned exposure guidance values because they were conducted according OECD test guidelines and good laboratory practices. The BEs associated with the IOM EAR, RDA and EPA RfD were derived using the convention of deriving BEs for guidance values which rely on human studies (Fig. 3). While the BEs associated with RIVM TDI, EC SCF UL, IOM UL and the OECD SIDS NOAEL were derived using the convention of deriving BEs associated with guidance values based on animal toxicity studies (Fig. 4). The linear relationship observed in Turnlund and Keyes (2004) (equations (1) and (2)) were used to convert the daily doses associated with the points of departure (PODs) from the exposure guidance values into resulting plasma Mo concentrations (BEPOD) and urinary Mo excretion rate (UER) (eqs. (1) and (2)). The BE for Mo in urine was calculated by dividing the corresponding UER by the daily urinary volume (V24) or daily creatinine excretion (Cr24). Daily urinary volume and creatinine excretion were estimated using urinary flow rate and creatinine excretion rates and
Fig. 3. Schematic of approach to derivation of urinary molybdenum concentrations consistent with current exposure guidance values based on human epidemiology studies.
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standard body weight as these parameters were not reported in Turnland and Keys (2004).
BEPOD ¼
UER V24 or Cr24
(3)
Where V24 ¼ urinary flow 20 ml/kg-day * 70 kg-bw and Cr24 ¼ creatinine excretion rate of 20 mg/kg-day * 70 kg-bw (Aylward et al., 2015). BEs corresponding to the final exposure guidance values were calculated by dividing the respective BEPOD by the corresponding intraspecies uncertainty factor(s) (UF). 3. Results and discussion The calculated BE values corresponding to each guidance value and the OECD SIDS NOAEL are presented in Table 3. The plasma/ serum and whole blood BEs for the EAR are 0.5 and 0.5 mg/L and for the RDA are 0.45 and 0.47 mg/L, respectively. The urinary BE for EAR and RDA are 28 and 22 mg/L, respectively. The plasma, whole blood and urine BEs range from 0.9e31, 0.8e28 and 200 to 7500 mg/L, respectively for the guidance values (e.g., RfD, TDI, UL) and the OECD SIDS NOAEL, which are designed to protect against toxicity. The human equivalent PODs associated with the RfD, TDI and UL ranged from 0.047 to 0.1 mg/kg-bw per day, above the maximum daily dose of 0.018 mg/kg-bw per day tested in Turnlund and Keyes (2004). Similarly, the human equivalent POD derived from the OECD SIDS NOAEL was 1.7 mg/kg-bw per day, which was almost two orders of magnitude higher than the maximum daily dose tested by Turnlund and Keyes (2004). This is not expected to be an area of high uncertainty given the relationships between Mo dose and Mo in plasma and urine were highly linear. All of the actual guidance values, are within the dose range tested in Turnlund and Keyes (2004). Therefore, to examine potential impacts on the derived BEs, we derived BEs using two approaches, 1) use the regressions of fits to Turnlund and Keyes (2004) to convert the human equivalent POD into the respective BEPOD and then divide the BEPOD by the intraspecies UF to derive the BE, and 2) use the regressions from Turnlund and Keyes (2004) to convert the human equivalent POD into the BEPOD and the guidance values into the respective BE (results not presented). While slightly different results were obtained for the BEs for Mo in plasma, the two approaches yielded the same BEs when reduced to 1 significant figure (the same significant figures as the underlying guidance values). The differences in
Fig. 4. Schematic of approach to derivation of urinary molybdenum concentrations consistent with current exposure guidance values based on animal toxicology studies.
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values for Mo in urine were negligible (since the slope of the regression line is near 1). The BE values for Mo in urine for the guidance values related to toxic effects (e.g., RfD, TDI, and UL) and the OECD SIDS NOAEL ranged from approximately 200 to more than 7500 mg/L, while the BE in urine associated with the EAR was approximately 22 mg/L. This range reflects a clear ability to discern nutritionally sufficient Mo exposure levels from those associated with overt toxicity. In contrast to the BEs for Mo in urine, the absolute difference between BEs for Mo in plasma associated with the EAR and the guidance values for toxicity (e.g., RfD, TDI, UL) and the OECD SIDS NOAEL are not that large (0.5 versus 0.9e31). This narrow range likely reflects the physiological regulation of Mo, with the body regulating retained Mo. As a result, discerning nutritionally sufficient from excess Mo exposures on the basis of plasma concentrations is more difficult than on the basis of the urinary concentrations. This issue is compounded by the short half-life in Mo in plasma following acute exposures, which can lead to within-day fluctuations in plasma concentrations following intake episodes. Likewise, the rapid elimination kinetics of Mo in urine, compounded by variations in hydration status and urine flow may add substantial variability to Mo in urine. Both should be considered when interpreting Mo in blood or urine. The BE values presented here can assist in the evaluation of population based biomonitoring data in the context of existing exposure guidance values. Concentrations below the BE values could be considered a lower priority for risk assessment or management follow-up while concentrations in excess of the BE values could be considered a higher priority for follow-up. 3.1. Sources of variability and uncertainty The appropriate uses and limitations of BE values have been discussed previously (Hays et al., 2008). BEs do not address any underlying limitations or uncertainties in existing exposure guidance values. These BEs should be regarded as interim screening values that can be updated or replaced if the exposure guidance values are updated. The user may want to review the underlying existing exposure guidance value prior to use of the BE. These BE values do not represent diagnostic criteria and cannot be used to evaluate the likelihood of an adverse health effect in an individual or even among a population. There have been several papers, which have investigated the sources of variability in biomarker concentrations and the impacts on interpreting biomonitoring data (Hays et al., 2008; Aylward et al., 2012, 2014). A key assumption in developing these BEs is that the biomarker concentrations are at steady state. For chemicals with short half-lives and infrequent exposure, it is unlikely that the biomarker concentrations are at steady state. In those cases, comparing BEs to the tails of the biomarker concentration distribution is not appropriate. However, in the case of Mo, there are multiple exposure events per day resulting from dietary intake. Thus, the half-lives of Mo in blood or urine relative to exposure frequency are not considered to be short and the biomarker concentrations are more likely to approach steady-state, as per the analysis by Aylward et al. (2014). The Mo blood and urine biomarker concentration distributions generated from surveys such as NHANES and CHMS should be reflective of the magnitude and variation of external exposure to Mo in the general population. The data from Turnlund and Keyes (2004) are ideal for deriving the BEs as the study involved durations of exposure sufficiently long to reach steady-state metabolic balance for Mo. It has also been suggested that the extent of Mo absorption is a factor of the medium in which it is administered (Guissani, 2008), with percent absorption being lower when naturally present in foods (range 0.3e0.6) versus added to liquids (>0.85). However, the Fue
associated with the lowest dose in Turnlund and Keyes (2004) was 0.97, which was the only dose in that study and the exposure to this dose was entirely via food. The data of Turnlund and Keyes (2004) likely provide the most reliable means of estimating both plasma concentration and urinary excretion of Mo following oral doses of Mo in the general population. There is higher uncertainty in using Turnland and Keys (2004) data when interpreting urinary Mo concentrations in infants, as infants have a lower Fue than adults as observed by Sievers et al. (2001b). Since Turnlund and Keyes (2004) study was based on healthy males, there is some uncertainty in interpreting blood or urinary Mo levels associated with subpopulations such as pregnant women. While there are limited toxicokinetic data in human or animals to characterize the potential kinetic differences in these subpopulations, the intra-species uncertainty factor of 10 is assumed to account for uncertainties in the database. There is also some uncertainty whether the elimination kinetics will remain linear for high Mo intake levels. 3.2. Confidence assessment Although some studies used in deriving exposure guidance values were dated or with various limitations in study design or result interpretation, the overall database for derivation of BE values for Mo is robust and provides a high-confidence BE value for use in the assessment of both urinary and plasma Mo biomonitoring data. The methods used to convert daily dose into plasma Mo concentrations and Mo urinary excretion rate based on a robust human volunteer study (Turnlund et al., 1995b) in which several volunteers were maintained in a controlled metabolic research station for 120 days, and were exposed to several different daily doses of Mo. They were maintained on each daily dose for sufficient time to reach steady-state kinetics. These data are ideal for deriving a BE as such the confidence in the BEs derived here are considered to be high. However, there is less confidence that similar linear kinetics would exist at higher intakes levels than the above study. The relatively small absolute difference in BEs in blood associated with the nutrition (EAR) and toxicity (RfD, TDI, UL and the OECD SIDS NOAEL), though, may indicate blood is less reliable as a metric for chronic Mo exposures. When blood Mo concentrations exceed the guideline values related to toxicity based BEs, urinary Mo concentrations (if available) should be interpreted as well to confirm conclusions. The BEs derived here can be used to interpret biomonitoring data of Mo in blood or urine in the context of both minimal nutrition requirements (>22 mg/L in urine) and for potential toxicity (200e7500 mg/L in urine). Mo levels in blood should be interpreted with greater caution because homeostasis effective modulates blood concentrations under conditions of excess Mo intake. Acknowledgments SMH and LLA received funding to prepare this analysis and preparation of manuscript from Health Canada (Contract No. 4500323309). Authors thank Scott Hancock and Michelle Deveau for their careful review and comments on this manuscript. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.yrtph.2016.03.004. References Angerer, J., Aylward, L.L., Hays, S.M., Heinzow, B., Wilhelm, M., 2011 Sep. Human biomonitoring assessment values: approaches and data requirements. Int. J.
S.M. Hays et al. / Regulatory Toxicology and Pharmacology 77 (2016) 223e229 Hyg. Environ. Health 214 (5), 348e360. Aylward, L.L., Kirman, C.R., Adgate, J.L., McKenzie, L.M., Hays, S.M., 2012. Interpreting variability in population biomonitoring data: role of elimination kinetics. J. Expos Sci. Environ. Epidemiol. 22, 398e408. Aylward, L.L., Hays, S.M., Smolders, R., Holger, Koch, Cocker, J., Jones, K., Warren, N., Levy, L., Bevan, R., 2014. Sources of variability in biomarker concentrations. J. Toxicol. Environ. Health B Crit. Rev. 17 (1), 45e61. Aylward, L.L., Hays, S.M., Vezina, A., Deveau, M., St-Amand, A., Nong, A., 2015 Jun. Biomonitoring Equivalents for interpretation of urinary fluoride. Regul. Toxicol. Pharmacol. 72 (1), 158e167. European Commission, Scientific Committee on Food, 2000. Opinion of the Scientific Committee on Food on the Tolerable Upper Intake Level of Molybdenum. European Commission, Brussels. SCF/CS/NUT/UPPLEV/22 Final Report. EFSA (European Food Safety Authority), 2013. Scientific opinion on dietary reference values for molybdenum. EFSA J. 2013 11 (8), 3333. Fungwe, T.V., Buddingh, F., Demick, D.S., Lox, C.D., Yang, M.T., Yang, S.P., 1990. The role of dietary molybdenum on estrous activity, fertility, reproduction and molybdenum and copper enzyme activities of female rats. Nutr. Res. 10, 515e524. Giussani, A., 2008. A recycling systemic model for the biokinetics of molybdenum radionuclides. Sci. Total Environ. 404 (1), 44e55. € llriegl, V., Oeh, U., Tavola, F., Veronese, I., 2007. Giussani, A., Cantone, M.C., Ho Modelling urinary excretion of molybdenum after oral and intravenous administration of stable tracers. Radiat. Prot. Dosim. 127 (1e4), 136e139. Epub 2007 Jun 8. PubMed PMID: 17561520. Hays, S.M., Becker, R.A., Leung, H.W., Aylward, L.L., Pyatt, D.W., 2007 Feb. Biomonitoring equivalents: a screening approach for interpreting biomonitoring results from a public health risk perspective. Regul. Toxicol. Pharmacol. 47 (1), 96e109. Hays, S.M., Aylward, L.L., LaKind, J.S., Bartels, M.J., Barton, H.A., Boogaard, P.J., Brunk, C., DiZio, S., Dourson, M., Goldstein, D.A., Lipscomb, J., Kilpatrick, M.E., Krewski, D., Krishnan, K., Nordberg, M., Okino, M., Tan, Y.M., Viau, C., Yager, J.W., 2008 Aug. Biomonitoring equivalents expert workshop. Guidelines for the derivation of biomonitoring equivalents: report from the biomonitoring equivalents expert workshop. Regul. Toxicol. Pharmacol. 51 (3 Suppl. l), S4eS15. Health Canada, 2003. Canadian Total Diet Study [Internet]. Average Dietary Intakes (mg.Kg Bw/day) of Trace Elements for Canadians in Different Age/sex Groups for Total Diet Study from 1993 to 1999. Health Canada, Ottawa (ON) [cited: 2016 Jan]. Available from: http://www.hc-sc.gc.ca/fn-an/surveill/total-diet/intakeapport/metal_intake-plomb_apport_93-99-eng.php. publique du Que bec), 2004. Etude INSPQ (Institut national de sante sur tablissement de valeurs de re fe rence d'e l e ments traces et de me taux dans le l'e rum et l'urine de la population de la grande re gion de Que bec. Institut sang, le se publique du Que bec. Que bec, Que. Cote: INSPQ-2004-030. national de sante IMOA (International Molybdenum Association), 2015. Molybdenum Uses. http:// www.imoa.info/molybdenum-uses/molybdenum-uses.php. IOM (Institute of Medicine), 2001. Dietary Reference Intakes for Vitamin a, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicone, Vanadium, Zinc. http://www.nap.edu/catalog/10026/dietary-referenceintakes-for-vitamin-a-vitamin-k-arsenic-boron-chromium-copper-iodine-ironmanganese-molybdenum-nickel-silicon-vanadium-and-zinc. Kikuchi, K., Hamano, S., Mochizuki, H., Ichida, K., Ida, H., 2012 Aug. Molybdenum cofactor deficiency mimics cerebral palsy: differentiating factors for diagnosis. Pediatr. Neurol. 47 (2), 147e149. Koval'skiy, V.V., Yarovaya, G.A., Shmavonyan, D.M., 1961. Changes of purine metabolism in man and animals under conditions of molybdenum biogeochemical provinces. Zh. Obshch. Biol. 22, 179e191 (Russian trans.). Murray, F.J., Sullivan, F.M., Tiwary, A.K., Carey, S., 2014a. 90-Day subchronic toxicity study of sodium molybdate dihydrate in rats. Regul. Toxicol. Pharmacol. 70 (3), 579e588. Murray, F.J., Tyl, R.W., Sullivan, F.M., Tiwary, A.K., Carey, S., 2014b. Developmental toxicity study of sodium molybdate dihydrate administered in the diet to Sprague Dawley rats. Reprod. Toxicol. 49, 202e208. NTP (National Toxicology Program), 1997. NTP Technical Report on the Toxicology
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and Carcinogenesis Studies of Molybdenum Trioxide (Cas No. 1313-27-5) in F344/n Rats and B6C3F1 Mice (Inhalation Studies). 1997. NTP TR 462. US Department of Health and Human Services. NIH Publication No. 97-3378. Novotny, J.A., Turnlund, J.R., 2007 Jan. Molybdenum intake influences molybdenum kinetics in men. J. Nutr. 137 (1), 37e42. PubMed PMID: 17182798. OECD SIDS (Organisation for Economic Cooperation and Development Screening Information Dataset), 2013. Highly Soluble Molybdenum Salts. Avaialbel from. http://webnet.oecd.org/HPV/UI/SIDS_Details.aspx?id¼5c88d62f-4401-4cadb521-521a4bd710f3. RIVM. (Rijksinstituut voor volksgezondheid en milieu), 2001. Baars AJ et al. 2001. Re-evaluation of human-toxicological maximum permissible risk levels. RIVM report no. 711701025, National Institute of Public Health and the Environment, Bilthoven, The Netherlands, March 2001. Available at:, pp. 75e77. or at. http:// www.rivm.nl/bibliotheek/rapporten/711701025.pdf. http://www.rivm.nl/en/. http://www.rivm.nl/bibliotheek/rapporten/711701025.pdf (click on Search, type “711701025”, then click on document). Schroeder, Mitchener, 1971. Toxic effects of trace elements on the reproduction of mice and rats. Archiv. Environ. Health 23, 102e106. €rner, K., Garbe-Scho €nberg, D., Schaub, J., 2001a. Molybdenum metaSievers, E., Do bolism: stable isotope studies in infancy. J. Trace Elem. Med. Biol. 15 (2e3), 185e191. € rner, K., Kollmann, M., Schaub, J., 2001b. Molybdenum Sievers, E., Oldigs, H.D., Do balance studies in premature male infants. Eur. J. Pediatr. 160 (2), 109e113. Schultze, B., Lind, P.M., Larsson, A., Lind, L., 2014 Mar. Whole blood and serum concentrations of metals in a Swedish population-based sample. Scand. J. Clin. Lab. Invest. 74 (2), 143e148. State of Washington, 2015. State of Washington Department of Ecology Product Testing Data. Online Database. Available at: https://fortress.wa.gov/ecy/ ptdbpublicreporting/. Turnlund, J.R., Keyes, W.R., 2004 Feb. Plasma molybdenum reflects dietary molybdenum intake. J. Nutr. Biochem. 15 (2), 90e95. PubMed PMID: 14972348. Turnlund, J.R., Keyes, W.R., Peiffer, G.L., 1995a. Molybdenum absorption, excretion, and retention studied with stable isotopes in young men at five intakes of dietary molybdenum. Am. J. Clin. Nutr. Oct;62 (4), 790e796. PubMed PMID: 7572711. Turnlund, J.R., Keyes, W.R., Peiffer, G.L., Chiang, G., 1995b. Molybdenum absorption, excretion, and retention studied with stable isotopes in young men during depletion and repletion. Am. J. Clin. Nutr. 61, 1102e1109. Underwood, E.J., 1977. Trace Elements in Human and Animal Nutrition, fourth ed. Academic Press, New York, pp. 109e131. U.S. EPA (United States Environmental Protection Agency), 1992. Integrated Risk Information System (IRIS). Online. National Center for Environmental Assessment, Washington, DC (Available on IRIS). US HPD (United States Household Products Database), 2015. Online Database. Department of Health and Human Services. Available at: http:// householdproducts.nlm.nih.gov/index.htm. van Esch, G.J., 1978. Letter to Dr. a. Betz Dd 2 October 1978. RIVM Letter No. U 200/ 78 Tox VI VE/1b. National Institute of Public Health and the Environment, Bilthoven, The Netherlands. Vyskocil, A., Viau, C., 1999 MayeJun. Assessment of molybdenum toxicity in humans. J. Appl. Toxicol. 19 (3), 185e192. Werner, E., Giussani, A., Heinrichs, U., Roth, P., Greim, H., 1998. Biokinetic studies in humans with stable isotopes as tracers. Part 2: uptake of molybdenum from aqueous solutions and labelled foodstuffs. Isot. Environ. Health Stud. 34 (3), 297e301. Werner, E., Roth, P., Heinrichs, U., Giussani, A., Cantone, M.C., Zilker, T.H., Felgenhauer, N., Greim, H., 2000. Internal biokinetic behaviour of molybdenum in humans studied with stable isotopes as tracers. Isot. Environ. Health Stud. 36 (2), 123e132. WHO (World Health Organisation), 2011. Molybdenum in Drinking-water. Background Document for Development of WHO Guidelines for Drinking-water Quality. Document No. WHO/SDE/WSH/03.04/11/Rev/1. World Health Organisation.
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Biomonitoring Equivalents for molybdenum Sean M. Hays a, *, Kristin Macey b, Devika Poddalgoda b, Ming Lu b, Andy Nong b, Lesa L. Aylward c a
Summit Toxicology, LLP, Lyons, CO 90540, USA Health Canada, Ottawa, ON, Canada c Summit Toxicology, LLP, Falls Church, VA 22044, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 December 2015 Received in revised form 4 March 2016 Accepted 6 March 2016 Available online 10 March 2016
Molybdenum is an essential trace element for mammalian, plant, and other animal systems. The Institute of Medicine (IOM) has established an Estimated Average Requirement (EAR) to assure sufficient molybdenum intakes for human populations; however excessive exposures can cause toxicity. As a result, several agencies have established exposure guidance values to protect against molybdenum toxicity, including a Reference Dose (RfD), Tolerable Daily Intake (TDI) and a Tolerable Upper Intake Level (UL). Biomonitoring for molybdenum in blood or urine in the general population is being conducted by the Canadian Health Measures Survey (CHMS) and the U.S. National Health and Nutrition Examination Survey (NHANES). Using pharmacokinetic data from controlled human dosing studies, Biomonitoring Equivalents (BEs) were calculated for molybdenum in plasma, whole blood, and urine associated with exposure guidance values set to protect against both nutritional deficits and toxicity. The BEEAR values in plasma, whole blood and urine are 0.5, 0.45 and 22 mg/L, respectively. The BEs associated with toxicity range from 0.9 to 31 mg/L in plasma, 0.8e28 mg/L in whole blood and 200e7500 mg/L in urine. These values can be used to interpret molybdenum biomonitoring data from a nutritional and toxicity perspective. © 2016 Published by Elsevier Inc.
Keywords: Biomonitoring Biomonitoring Equivalents Molybdenum Risk assessment
1. Introduction Molybdenum (Mo) is a naturally occurring element and is found in trace levels in all environmental media, food and drinking water. Mo is an essential trace element for mammalian, plant, and other animal systems. In humans, Mo is an activator of three enzymes: sulfite oxidase, xanthine oxidase, and aldehyde oxidase, which are involved in the oxidation of sulfite to sulfate, the production of uric acid, and the oxidation of aldehydes (EFSA, 2013; IOM, 2001; Vyskocil and Viau, 1999; Turnlund et al., 1995a). However, at elevated exposures, Mo toxicity has been observed in human and animal studies (US EPA, 1992; IOM, 2001; OECD SIDS, 2013). All humans are exposed to Mo through their diet. Concentrations of Mo are higher in legumes, pulses, cereal grains and nuts than in dairy and meat (EFSA, 2013; Vyskocil and Viau, 1999). In Europe, cereals and cereal-based products including bread are the major contributors to dietary intake (EFSA, 2013). The main
* Corresponding author. 165 Valley Rd., Lyons, CO 80540, USA. E-mail address: [email protected] (S.M. Hays). http://dx.doi.org/10.1016/j.yrtph.2016.03.004 0273-2300/© 2016 Published by Elsevier Inc.
contributors of Mo in North American diet are legumes, grain products and nuts (IOM, 2001). Dietary intakes in adults range from 58 to 157 mg/day (0.97e2.61 mg/kg-bw per day based on a 60 kg BW) in Europe (EFSA, 2013), while the IOM (2001) reports dietary intakes ranging from 120 to 240 mg/day (1.71e3.42 mg/kg-bw per day based on a 70 kg BW in adults in the United States. In Canada, average dietary intake is 2.6 mg/kg-bw per day (Health Canada, 2003). In addition to naturally occurring Mo, there is anthropogenic contribution of Mo to environmental media from mining and related use industries. The primary use of Mo is as a component of steel alloys to increase strength and durability and aid in corrosion resistance (IMOA, 2015). Mo is also found in a number of products available to consumers including nutritional supplements, plant care products, household cleaning products, and products available to children (e.g. toys, clothing) (US Household Products Database, 2015; State of Washington, (2015)). Large human biomonitoring surveys, such as the Canadian Health Measures Survey (CHMS) and the U.S. National Health and Nutrition Examination Survey (NHANES) are providing valuable data on the prevalence and concentration of many environmental
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chemicals and essential elements, including Mo, in the general population. Biomonitoring in urine or blood allows for an assessment of integrated exposures to molybdenum across different sources and routes of exposure. However, measured concentrations of Mo in blood or urine cannot be directly interpreted using the available exposure guidance values as the latter are presented in terms of daily intake levels (e.g. mg/kg-bw per day). The purpose of this evaluation is to derive Biomonitoring Equivalent (BE) values for interpretation of population biomonitoring data for Mo. BE values are estimates of the concentration of a chemical or its metabolite in blood or urine that are consistent with defined exposure guidance values such as RfDs or TDIs (Hays et al., 2007, 2008; Angerer et al., 2011). BE values can be used as screening values for the assessment of biomonitoring data in order to provide an initial evaluation of whether the detected concentrations are below, near, or above the concentrations consistent with exposure guidance values. The BEs developed here provide tools for interpreting Mo biomonitoring levels in the context of exposure guidance values designed to protect for nutritional adequacy and to protect against toxicity associated with excessive exposures. 2. Methods Derivation of BE values for evaluation of biomonitoring data in a risk assessment context generally requires three main elements: an assessment of toxicity with a corresponding derived exposure guidance value such as a RfD or a TDI; the availability of a biomarker that is specific to the exposure of interest; and sufficient toxicokinetic data to predict the biomarker concentrations in urine or blood under steady-state exposure conditions consistent with the chronic exposure guidance value(s). The following sections describe the available information for molybdenum and outline the approaches taken to derive BE values for Mo in blood and urine. 2.1. Nutrition and toxicity data and risk assessments Mo is an essential trace element for humans, animals and plants, which acts as a cofactor (molybdopterin) for certain enzymes (IOM, 2001; WHO, 2011; Turnlund et al., 1995a). The clinical signs of Mo deficiency associated with low dietary intakes are not commonly observed in experimental animals or in healthy people (IOM, 2001). However, a genetic defect called molybdenum cofactor deficiency that results in decreases in the activities of Mo-dependent enzymes has been identified (Kikuchi et al., 2012). Deficiency in these enzymes usually leads to fatal neurological toxicity in infants born with this condition, or irreversible neurological damage due to the inability of the body to convert sulfite to sulfate (reviewed in IOM, 2001). Excess intakes of Mo appear to have low toxicity in humans (IOM, 2001; Turnlund et al., 1995a). Animals, especially ruminants, are known to be more susceptible Mo toxicity than humans (IOM, 2001). Ruminants show adverse effects of Mo under copper deficiency and marginal sulfur amino acid intake (Underwood, 1977). Hence, the basis of toxicity in ruminants is not considered relevant to humans (IOM, 2001). In female rats, dietary supplementation of Mo resulted in prolonged estrus cycle, decreased gestational weight gain of pups and several adverse effects in embryogenesis at doses of 1.6 mg Mo/kg-bw per day, but not at 0.9 mg Mo/kg-bw per day (Fungwe et al., 1990). Similarly, Schroeder and Mitchener (1971) reported adverse reproductive effects at 1.5 mg Mo/kg-bw per day in a drinking water three-generation reproductive toxicity study in mice. However, a 90-day oral repeated dose toxicity study (Murray et al., 2014a) reviewed by the Organization for Economic Co-operation and Development (OECD) where male and female
rats were exposed to Mo in disodium molybdate dehydrate via feed did not report any adverse effects in reproductive parameters in male or female rats at the highest dose tested (60 mg Mo/kg-bw per day). The same study (Murray et al., 2014a) reported reduced body weight gains and kidney effects at 60 mg Mo/kg-bw per day and the No Observed Adverse Effects Level (NOAEL) was identified as 17 mg Mo/kg-bw per day. Another guideline compliant pre-natal developmental toxicity study reviewed by the OECD (Murray et al., 2014b), where female rats were exposed up to 40 mg Mo/kg-bw per day via feed to same form of Mo as above during gestation day 6e20, did not report any maternal toxicity or dose-related adverse effects on development of the offspring. Based on the evidence from a 2-year inhalation carcinogenicity study and in vitro genotoxicity studies by NTP (1997), Mo is not considered to have mutagenic or carcinogenic potential (RIVM, 2001). Several international agencies have conducted risk assessments on Mo and have established exposure guidance values for general population to protect against both nutrient deficiency and toxicity. A series of regulatory guidance values were established by the Institute of Medicine (IOM), European Commission Scientific Committee on Food (EC SCF), US Environmental Protection Agency (US EPA) and The Netherlands National Institute for Public Health and the Environment (RIVM). The IOM (2001) has established an Estimated Average Requirement (EAR) of 0.034 mg/day (0.00049 mg/kg-bw per day assuming 70 kg BW) based on the results from Turnlund et al. (1995b) to ensure population level nutritional adequacy, and a Recommended Daily Allowance (RDA) of 0.045 mg/day (0.00064 mg/kg-bw per day) as the target intake for individuals. IOM (2001) also estimated a Tolerable Upper Intake Level (UL) of 2 mg/day (0.03 mg/kg-bw per day assuming 68 kg BW) based on the results from Fungwe et al. (1990) to protect against toxicity. EC SCF (2000) also used the same study as IOM (2001) (Fungwe et al., 1990) to derive a UL of 0.6 mg/day (0.01 mg/kgbw per day assuming 60 kg BW). RIVM (2001) established a TDI of 0.01 mg/kg-bw per day for oral exposures based on kidney effects in rats (van Esch, 1978) and a tolerable concentration in air (TCA) for inhalation exposures. The US EPA (1992) derived a RfD of 0.005 mg/ kg-bw per day for Mo based on a human epidemiology study (Koval'skiy et al., 1961) in which the most sensitive endpoint was increased serum uric acid levels. While some of these international agencies have developed guidance values for different age groups and sub-populations (e.g. pregnant and lactating women), the guidance values presented here are for adults in the general population and these values are summarized in Table 1. In addition, the OCED conducted a review of molybdenum in 2013, however they did not establish an exposure guidance value, rather the lowest NOAEL in the assessment profile was identified based on effects on body weight and kidney. 2.2. Biomarker selection Biomonitoring for Mo exposures involves quantifying Mo in urine and/or blood (plasma and/or whole blood). Mo in both plasma and urine has been shown to be well correlated with dietary intake (Turnlund and Keyes, 2004). Urine is the predominant route of elimination, with upwards of 90% of an oral dose being eliminated via urine with a half-life of less than 12 h (Turnlund and Keyes, 2004), indicating urine is a good matrix for assessing exposures to Mo. In plasma, elimination has been described with a biexponential function with mean half-lives of 30 min and 6.6 h, respectively (Werner et al., 2000). Generally, biomarkers with a longer half-life of elimination have less daily fluctuation around the chronic-steady state average concentrations and are more suitable biomarkers to estimate the exposure (Hays et al., 2007). The halflife of elimination of Mo following an oral dose is short in both
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Table 1 Available exposure guidance values for molybdenum for both nutritional requirements and to protect against toxicity. Organization, criteria Critical endpoint (year of evaluation) EAR, IOM (2001)
Dose level
Uncertainty factors
Value 0.034 mg/d (adults) (0.0005 mg/kg-d)
EAR*1.3 to account for population variation in requirement
0.045 mg/d (adults) (0.00064 mg/kg-d)
UL, IOM (2001)
Mo balance without any clinical Based on the balance data, the minimum requirement signs of deficiency in humans plus increment for miscellaneous loss is 0.025 mg/d. EAR is estimated assuming 75% bioavailability from food Turnlund et al., 1995b Estimated average requirement (EAR) of Mo for adults Essential trace element for is 0.034 mg/d. selected enzyme function in EAR values for other age groups calculated based on humans bodyweight0.75 scaling Turnlund et al., 1995b Reproductive effects in rats NOAEL, 0.9 mg/kg/day Fungwe et al., 1990
30
EC SCF (2000)
Reproductive effects in rats
100
Chronic oral RfD, U.S. EPA (1992) Chronic oral TDI, RIVM (2000) 90-day toxicity study OECD SIDS (2013)
Increased serum uric acid levels LOAEL, 0.14 mg/kg-d Koval'skiy et al., 1961 in humans. Kidney effects in rats NOAEL 1.0 mg/kg-d van Esch, 1978
30
2 mg/d (0.03 mg/kg-d) 0.6 mg/d (0.01 mg/kg-d) 0.005 mg/kg-d
100
0.01 mg/kg-d
100
1.2 e2 mg/m3
RDA, IOM (2001)
NOAEL, 0.9 mg/kg/day Fungwe et al., 1990
Reduced body weight and kidney NOAEL 17 mg/kg-d Murray et al., 2014a effects at 60 mg/kg-d
a a
A summary of the study was published in 2014 following review by the OECD in Murray et al. (2014a).
Several informative pharmacokinetic (PK) studies have been conducted in humans. Turnlund et al. (1995a) conducted an extremely valuable study in which human (healthy male) volunteers were confined to a metabolic research station for 120 days and Mo exposures were controlled. They administered five increasing levels of Mo via the diet for 24 days each (22, 72, 121, 467, 1490 mg/day). Daily urinary and fecal excretion of Mo was measured in the middle and end of each dosing regimen. Blood samples were also drawn on the same days, but analytical techniques employed at that time did not allow the researchers to quantify Mo in plasma (Turnlund et al., 1995b). The researchers later developed an analytical approach that allowed for quantifying low levels of Mo in plasma and subsequently analyzed plasma samples from the earlier study (Turnlund and Keyes, 2004). The results suggest highly linear relationships between both plasma and urinary Mo and daily dose via the diet (Figs. 1 and 2 and Table 2). As part of their experimental
design, isotope enriched Mo was administered via i.v. to one group while the other two groups were administered Mo as ammonium molybdate in a liquid, to drink, in addition to their basic diet. Analysis of Mo isotopes in blood, urine and feces throughout the study supported development of a PK model (Novotny and Turnlund, 2007), which allowed the researchers to better characterize tissue storage and turnover of Mo. The half-life of Mo in blood was less than 24 h, and likely to be on the order of 1e3 h (independent analysis of Table 3 in Turnlund and Keyes, 2004). Giussani et al. (2007) conducted PK studies in which male volunteers received single injections (i.v.) of enriched 96Mo isotopes and 95Mo was administered simultaneously orally in various foods (Werner et al., 1998). Urine samples were collected post-dosing in three 4-h periods, followed by collection over a 12-h period and 24h composites over the following 6 days. The majority of the 96Mo excreted in urine occurred within the first couple of hours and was almost completed by 12 h post-dosing. Giussani et al. (2007) did not provide enough data to be able to accurately estimate percent excretion in urine following oral doses. All that can be determined from their data is that greater than 65% appears to have been excreted within 24 h post dosing. A series of studies were conducted to assess the dietary needs of Mo in infants and preterm infants (Sievers et al., 2001a, 2001b). Mo
Fig. 1. Relationship between daily dietary administration of Mo and plasma Mo (data from Table 4, group 3 in Turnlund and Keyes, 2004).
Fig. 2. Relationship between daily dietary administration of Mo and daily urinary excretion of Mo (data from Table 5, group 3 in Turnlund and Keyes, 2004).
blood and urine and therefore, neither Mo in blood or urine holds an advantage with respect to half-life of elimination. Analysis of urine Mo concentration has the advantage of being less invasive. BEs are derived here for Mo in plasma, whole blood, and urine. 2.3. Available pharmacokinetic data to support calculations
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Table 2 Summary of available average urinary excretion fraction (Fue) estimates for molybdenum. Study
Age of Participants
Dose (mg/kg)
Media
Duration of dosing
Fue
Sievers et al., 2001a Sievers et al., 2001b
20 days post-term 37.4 (34.1e40.6) weeks posteconception
30.3 2.2 3.0 23.2 27.6 44.7 46.7
Human milk or formula Preterm infant formula Preterm infant formula Preterm infant formula Preterm infant formula Preterm infant formula Preterm infant formula
Turnlund and Keyes (2004)
28 years (average)
0.3 0.9 1.5 5.7 18.1
Solid Solid Solid Solid Solid
Single dose Single dose Single dose Single dose single dose Single dose single dose Average of Infant Studies 24 days 24 days 24 days 24 days 24 days Average of Adult Studies Average of all studies
0.57 0.89 0.62 0.96 0.75 0.62 0.78 0.74 0.97 0.83 0.83 0.90 0.88 0.88 0.80
and and and and and
liquid liquid liquid liquid liquid
diet diet diet diet diet
Table 3 Derivation of Biomonitoring Equivalents for molybdenum.
Basis of guidance value, Nutritional or Toxicity Animal POD, mg/kg-d - Interspecies UF Human POD, mg/kg-d - LOAEL to NOAEL UF Human Equiv. POD, mg/kg-d: Human Equiv. POD, mg/d: Blood BE values: BEPOD Steady-state plasma & serum concentration, mg/L:a BEPOD Steady-state whole blood concentration, mg/L:b - Intraspecies UF BE in plasma & serum, mg/L: BE in whole blood, mg/L: Urinary BE Values Urinary molybdenum excretion, mg/d:c BEPOD, mg/L & mg/g crd - Intraspecies UF BE, mg/L & mg/g cr
IOM, EAR
IOM, RDA
US EPA RfD
RIVM, TDI
IOM, UL
OECD
Nutritional NA NA 0.00049 NA 0.00049 34
Nutritional NA NA 0.00064 NA 0.00064 45
Toxicity NA NA 0.14 3 0.047 3267
Toxicity 1.0 10 NA NA 0.1 7000
Toxicity 0.9 10 NA NA 0.09 6300
Toxicity 17 10 NA NA 1.7 119000
0.50 0.45 1 0.50 0.45
0.52 0.47 1 0.5 0.47
8.9 8.01 10 0.9 0.80
18.6 16.75 10 1.9 1.67
16.8 15.11 3 5.6 5.04
310 279 10 31.0 27.9
30.3 21.7 1 21.7
39.8 28.4 1 28.4
2888.4 2063 10 206
6189.4 4421 10 442
5570.5 3979 3 1326
105220 75157 10 7516
NA - Not Applicable. a - Calculated using Equation (1) (see text). b - Calculated as BE in plasma* 0.9. c - Calculated using Equation (2) (see text). d Urinary flow rate assumed to be 20 ml/kg-d and creatinine excretion rate 20 mg/kg-d, BW ¼ 70 kg (Aylward et al., 2015).
was administered in single doses to infants in either human milk or infant formula and elimination of Mo in urine and feces was determined. It was found that between 57% and 96% of the administered dose of Mo was eliminated in urine (Table 2). The data from Turnlund and Keyes (2004) are the ideal data required for derivation of a BE. They carried out controlled exposure dosing for a duration sufficient to achieve steady-state and for a reasonable range of doses. They measured plasma Mo concentrations (Figure 1) and 24-h urinary excretion rate (UER e Fig. 2; Table 2) as a function of daily dose, yielding linear regressions of;
Mo plasmaðmg=LÞ ¼ 0:0026DDðmg=dayÞ þ 0:4068 ðR2 ¼ 0:992
(1) And
Mo UER ðmg=dayÞ ¼ 0:8842DDðmg=dayÞ
ðR2 ¼ 0:999
(2)
Where DD is daily dose in units (mg/day). Conversion to molar concentration is conducted using the conversion of 1 mg Mo ¼ 0.0104 mmol.
As can be seen from these regression equations and from Figs. 1 and 2, the proportional increase in Mo in plasma is small compared to a large increase in dietary intake of Mo. In contrast, the increase in Mo urinary excretion is proportional to increases in Mo dietary intake (larger slope in the regression equation). This indicates the body is efficient in maintaining homeostasis by controlling plasma Mo and efficiently excretes excess Mo in urine (Giussani, 2008). Mo binds to a2-macroglobulins in plasma in the form of molybdate and to the protein spectrin on erythrocytes (Schultze et al., 2014). No data could be found on the differences in concentrations of Mo in plasma and serum. However, based on the proteins it binds to, it is expected that plasma and serum Mo concentrations should be nearly identical. The whole blood and serum data from both Schultze et al. (2014) and INSPQ (2004) indicated that the ratio of Mo in whole blood to serum (B/S) is approximately 0.9. Therefore, BEs derived in plasma using the data of Turnlund and Keyes (2004) will be assumed to be identical for interpreting Mo in serum and scaled by a factor of 0.9 for deriving a BE in whole blood. The average urinary excretion fraction (Fue) for the studies in infants is 0.74, compared to an average of 0.88 for all the dose groups in the Turnlund and Keyes (2004) study of adults (Table 2).
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Differences are potentially due to 1) single dose administration in infants versus 24 days of dosing, and 2) method of collecting urine (diapers versus complete collections of all urine voids in the adult volunteers). PK studies of inhalation exposures to Mo were not identified. Since inhalation exposures to Mo are considered to be negligible relative to food exposures in individuals exposed from the general environment, and since effects for which guidance values have been established are systemic effects (thus route of administration may not impact toxicity), a BE will not be derived for the RIVM TCA. BEs associated with the oral guidance values should be applicable for interpreting biomonitoring data resulting from inhalation exposures (e.g., systemic effects occur at the same internal/absorbed doses regardless of the route of exposure). 2.4. BE derivation approach The BEs were derived for the EAR, RDA and UL from IOM, RfD from EPA, TDI from RIVM, UL from EC SCF, and the lowest NOAEL identified for Mo in the OECD SIDS assessment profile from a 90day toxicity study (Murray et al., 2014a), herein referred to as the OECD SIDS NOAEL (Table 1). The OECD SIDS NOAEL was included for BE derivation because the OECD SIDS assessment profile includes newer OECD test guideline compliant studies, which were not available at the time of the EPA, IOM, EC SCF or RIVM evaluations of Mo. Additionally, these studies have higher reliability than the studies used in above mentioned exposure guidance values because they were conducted according OECD test guidelines and good laboratory practices. The BEs associated with the IOM EAR, RDA and EPA RfD were derived using the convention of deriving BEs for guidance values which rely on human studies (Fig. 3). While the BEs associated with RIVM TDI, EC SCF UL, IOM UL and the OECD SIDS NOAEL were derived using the convention of deriving BEs associated with guidance values based on animal toxicity studies (Fig. 4). The linear relationship observed in Turnlund and Keyes (2004) (equations (1) and (2)) were used to convert the daily doses associated with the points of departure (PODs) from the exposure guidance values into resulting plasma Mo concentrations (BEPOD) and urinary Mo excretion rate (UER) (eqs. (1) and (2)). The BE for Mo in urine was calculated by dividing the corresponding UER by the daily urinary volume (V24) or daily creatinine excretion (Cr24). Daily urinary volume and creatinine excretion were estimated using urinary flow rate and creatinine excretion rates and
Fig. 3. Schematic of approach to derivation of urinary molybdenum concentrations consistent with current exposure guidance values based on human epidemiology studies.
227
standard body weight as these parameters were not reported in Turnland and Keys (2004).
BEPOD ¼
UER V24 or Cr24
(3)
Where V24 ¼ urinary flow 20 ml/kg-day * 70 kg-bw and Cr24 ¼ creatinine excretion rate of 20 mg/kg-day * 70 kg-bw (Aylward et al., 2015). BEs corresponding to the final exposure guidance values were calculated by dividing the respective BEPOD by the corresponding intraspecies uncertainty factor(s) (UF). 3. Results and discussion The calculated BE values corresponding to each guidance value and the OECD SIDS NOAEL are presented in Table 3. The plasma/ serum and whole blood BEs for the EAR are 0.5 and 0.5 mg/L and for the RDA are 0.45 and 0.47 mg/L, respectively. The urinary BE for EAR and RDA are 28 and 22 mg/L, respectively. The plasma, whole blood and urine BEs range from 0.9e31, 0.8e28 and 200 to 7500 mg/L, respectively for the guidance values (e.g., RfD, TDI, UL) and the OECD SIDS NOAEL, which are designed to protect against toxicity. The human equivalent PODs associated with the RfD, TDI and UL ranged from 0.047 to 0.1 mg/kg-bw per day, above the maximum daily dose of 0.018 mg/kg-bw per day tested in Turnlund and Keyes (2004). Similarly, the human equivalent POD derived from the OECD SIDS NOAEL was 1.7 mg/kg-bw per day, which was almost two orders of magnitude higher than the maximum daily dose tested by Turnlund and Keyes (2004). This is not expected to be an area of high uncertainty given the relationships between Mo dose and Mo in plasma and urine were highly linear. All of the actual guidance values, are within the dose range tested in Turnlund and Keyes (2004). Therefore, to examine potential impacts on the derived BEs, we derived BEs using two approaches, 1) use the regressions of fits to Turnlund and Keyes (2004) to convert the human equivalent POD into the respective BEPOD and then divide the BEPOD by the intraspecies UF to derive the BE, and 2) use the regressions from Turnlund and Keyes (2004) to convert the human equivalent POD into the BEPOD and the guidance values into the respective BE (results not presented). While slightly different results were obtained for the BEs for Mo in plasma, the two approaches yielded the same BEs when reduced to 1 significant figure (the same significant figures as the underlying guidance values). The differences in
Fig. 4. Schematic of approach to derivation of urinary molybdenum concentrations consistent with current exposure guidance values based on animal toxicology studies.
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values for Mo in urine were negligible (since the slope of the regression line is near 1). The BE values for Mo in urine for the guidance values related to toxic effects (e.g., RfD, TDI, and UL) and the OECD SIDS NOAEL ranged from approximately 200 to more than 7500 mg/L, while the BE in urine associated with the EAR was approximately 22 mg/L. This range reflects a clear ability to discern nutritionally sufficient Mo exposure levels from those associated with overt toxicity. In contrast to the BEs for Mo in urine, the absolute difference between BEs for Mo in plasma associated with the EAR and the guidance values for toxicity (e.g., RfD, TDI, UL) and the OECD SIDS NOAEL are not that large (0.5 versus 0.9e31). This narrow range likely reflects the physiological regulation of Mo, with the body regulating retained Mo. As a result, discerning nutritionally sufficient from excess Mo exposures on the basis of plasma concentrations is more difficult than on the basis of the urinary concentrations. This issue is compounded by the short half-life in Mo in plasma following acute exposures, which can lead to within-day fluctuations in plasma concentrations following intake episodes. Likewise, the rapid elimination kinetics of Mo in urine, compounded by variations in hydration status and urine flow may add substantial variability to Mo in urine. Both should be considered when interpreting Mo in blood or urine. The BE values presented here can assist in the evaluation of population based biomonitoring data in the context of existing exposure guidance values. Concentrations below the BE values could be considered a lower priority for risk assessment or management follow-up while concentrations in excess of the BE values could be considered a higher priority for follow-up. 3.1. Sources of variability and uncertainty The appropriate uses and limitations of BE values have been discussed previously (Hays et al., 2008). BEs do not address any underlying limitations or uncertainties in existing exposure guidance values. These BEs should be regarded as interim screening values that can be updated or replaced if the exposure guidance values are updated. The user may want to review the underlying existing exposure guidance value prior to use of the BE. These BE values do not represent diagnostic criteria and cannot be used to evaluate the likelihood of an adverse health effect in an individual or even among a population. There have been several papers, which have investigated the sources of variability in biomarker concentrations and the impacts on interpreting biomonitoring data (Hays et al., 2008; Aylward et al., 2012, 2014). A key assumption in developing these BEs is that the biomarker concentrations are at steady state. For chemicals with short half-lives and infrequent exposure, it is unlikely that the biomarker concentrations are at steady state. In those cases, comparing BEs to the tails of the biomarker concentration distribution is not appropriate. However, in the case of Mo, there are multiple exposure events per day resulting from dietary intake. Thus, the half-lives of Mo in blood or urine relative to exposure frequency are not considered to be short and the biomarker concentrations are more likely to approach steady-state, as per the analysis by Aylward et al. (2014). The Mo blood and urine biomarker concentration distributions generated from surveys such as NHANES and CHMS should be reflective of the magnitude and variation of external exposure to Mo in the general population. The data from Turnlund and Keyes (2004) are ideal for deriving the BEs as the study involved durations of exposure sufficiently long to reach steady-state metabolic balance for Mo. It has also been suggested that the extent of Mo absorption is a factor of the medium in which it is administered (Guissani, 2008), with percent absorption being lower when naturally present in foods (range 0.3e0.6) versus added to liquids (>0.85). However, the Fue
associated with the lowest dose in Turnlund and Keyes (2004) was 0.97, which was the only dose in that study and the exposure to this dose was entirely via food. The data of Turnlund and Keyes (2004) likely provide the most reliable means of estimating both plasma concentration and urinary excretion of Mo following oral doses of Mo in the general population. There is higher uncertainty in using Turnland and Keys (2004) data when interpreting urinary Mo concentrations in infants, as infants have a lower Fue than adults as observed by Sievers et al. (2001b). Since Turnlund and Keyes (2004) study was based on healthy males, there is some uncertainty in interpreting blood or urinary Mo levels associated with subpopulations such as pregnant women. While there are limited toxicokinetic data in human or animals to characterize the potential kinetic differences in these subpopulations, the intra-species uncertainty factor of 10 is assumed to account for uncertainties in the database. There is also some uncertainty whether the elimination kinetics will remain linear for high Mo intake levels. 3.2. Confidence assessment Although some studies used in deriving exposure guidance values were dated or with various limitations in study design or result interpretation, the overall database for derivation of BE values for Mo is robust and provides a high-confidence BE value for use in the assessment of both urinary and plasma Mo biomonitoring data. The methods used to convert daily dose into plasma Mo concentrations and Mo urinary excretion rate based on a robust human volunteer study (Turnlund et al., 1995b) in which several volunteers were maintained in a controlled metabolic research station for 120 days, and were exposed to several different daily doses of Mo. They were maintained on each daily dose for sufficient time to reach steady-state kinetics. These data are ideal for deriving a BE as such the confidence in the BEs derived here are considered to be high. However, there is less confidence that similar linear kinetics would exist at higher intakes levels than the above study. The relatively small absolute difference in BEs in blood associated with the nutrition (EAR) and toxicity (RfD, TDI, UL and the OECD SIDS NOAEL), though, may indicate blood is less reliable as a metric for chronic Mo exposures. When blood Mo concentrations exceed the guideline values related to toxicity based BEs, urinary Mo concentrations (if available) should be interpreted as well to confirm conclusions. The BEs derived here can be used to interpret biomonitoring data of Mo in blood or urine in the context of both minimal nutrition requirements (>22 mg/L in urine) and for potential toxicity (200e7500 mg/L in urine). Mo levels in blood should be interpreted with greater caution because homeostasis effective modulates blood concentrations under conditions of excess Mo intake. Acknowledgments SMH and LLA received funding to prepare this analysis and preparation of manuscript from Health Canada (Contract No. 4500323309). Authors thank Scott Hancock and Michelle Deveau for their careful review and comments on this manuscript. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.yrtph.2016.03.004. References Angerer, J., Aylward, L.L., Hays, S.M., Heinzow, B., Wilhelm, M., 2011 Sep. Human biomonitoring assessment values: approaches and data requirements. Int. J.
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