# Super f und Tec hni cal Suppor t Cent er Na tiona l C enter

for En viron men tal Assessment U

. S. Envi ronme nt al Prot ect i on Agen cy 26 West Mar t i n L ut her Ki ng Dr i ve , MS- AG41 Ci nc i nnat i

, Ohi o 4 5268

Jon Reid/Dire ctor

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. gov

Marc h 19, 2009

Rob e rt Tr ue sd a le RTII nternational Acc epted Reque stor

ASSI STANCE REQUESTED: PPR TV for iron (RfD a

nd sRfD only).

Attachm ent 1: PRO V I S I ONAL PEER REVI EW ED TOXI CI TY

I NF O RM AT I O N FO R I RO N ( CA SRN 7439

- 89- 6) AN D CO M PO UN DS Deri vat i on of S ubc hron i c an d C hr on ic O r al R f

D s

BE ADVI SED: Unless spec ifically indicated to have be en pee r reviewe d, it is t o be noted that the

attached Provisional Toxicity Value Pape r( s) have not be en through the U.S. EPA’ s for ma l re v ie w pr oc ess; the re for e, th e ydo no t re

pr ese nt a U. S. EP

A v e rif ie d a sse ssm e nt.

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# Provisional Peer Reviewed Toxicity Values for

Iron and Compounds (CASRN 7439-89-6)

Derivation of Subchronic and Chronic Oral RfDs

Superfund Health Risk Technical Support Center

National Center for Envi ronmental Assessment Office of Research and Development U.S. Environmental Protection Agency Cincinnati, OH 45268

### Acronyms and Abbreviations

bw body weight

cc cubic cen tim e ters CD Caesarean D e livered CERCLA Com p rehensive Environm en tal Response, Com p ensation and Liability Act of 1980 CNS central nervous system

cu.m cubic m e ter DW EL Drinking Water Equivalent Level FEL frank-effect level FIFRA Federal Inse ctic ide, Fun g icide, and Rodenticid e Act
g gram s GI gastro inte stinal HEC hum a n equivalent concentration Hgb hem oglobin i.m

. intr am uscular i.p. intraperitoneal i.v. intravenous IRIS Integrated R i sk Inform ation System IUR inhala tion un it r i sk kg kilogram L liter LEL lowest-effect level LOAEL lowest-observed-adverse-effect level LOAEL(ADJ) LOAEL adj u sted to continuous exposure duration LOAEL(HEC) LOAEL adj u sted for dosim e tric differences across species to a hum an

m m e ter MCL m a xi m u m c ontam inant level MCLG m a xi m u m c ontam inant level goal MF modifying factor
mg m illigram mg/kg m illigram s per kilogram mg/L m illigram s per lite r MRL m i nim a l risk level MTD m a xi m u m tolerated dose MTL m e dian threshold lim it NAAQS National A mbient Air Quality Stan dards NOAEL no-observed-adverse-effect level NOAE L(ADJ) NOAE L adjusted to continuous exposure duration NOAE L(HE C) NOAE L adjusted for dosim e tric differences across species to a hum an NOEL no-observed-effect level OSF oral slope factor p-IUR provisional inhalation unit risk p-OSF provisional oral slope factor p-RfC provisional inhala tion ref e rence concentration

ip-RfD provisional oral reference dose PBPK physiologically based pharm acokinetic ppb parts per billion
ppm parts pe r m illion PPRTV Provisional Peer Re view ed Toxicity Value RBC red blood cell(s) RCRA Resource Conservation and Recovery Act RDDR Regional deposited dose rati o (for the indicated lung region) REL rela tive exp o sure lev e l RfC inhalation refe rence concentration RfD oral reference dose RGDR Regional gas dose ratio (for the indicated lung region)

s.c. subcutaneou s SCE sister chrom a tid ex chang e SDWA Safe Drinking W a ter Act
sq.cm

. square centim eters TSCA Toxic Substances Control Act UF uncertainty f actor μg m i crogram μm ol m i crom oles VOC volatile organic com pound

ii9-11-2006

PROVISONAL P E E R REVIEW E D TOXICI TY INFORMATION F O R IRON (CASRN 7439-8 9 -6) AND COMPOUNDS Derivation of Subchronic and Chronic Oral RfDs

Background

On Dece m b er 5, 2003, the U.S. Environm enta l Protection Agency' s (EPA's) Office of Superfund Rem e diation and Technology Innovation (OSRTI) revised its hierarchy of hum a n health toxicity values for Superfund risk assessm e n ts, establishing the follow ing three tiers as the new hierarchy:

1. EPA' s Integrated Risk In form ation System (IRIS).

2. Provisional Peer-Reviewed Toxicity Va lues (P PRTV) used in EPA' s Superfund Program

.

3. Other (pe e r-reviewed ) to xicity values, including:

< Minim a l Risk Levels produced by the Agency for Toxic Substances and D i sease Registry (ATSDR),

< California E nvironm ental Protecti on Agency (CalEPA) values, and

< EPA Health Effects Assessm ent Summ ary Table (HEAST) values.

A PPRTV is defined as a toxicity value de rived for use in the Superfund Program when such a valu e is not av ailable in EPA' s Integrated Risk Information Syste m(IRIS). PPRTVs are developed according to a Standard Operating P r ocedure (S OP) and are derived after a review of the relevant scientific literature using the sam e m e thods, sources of data, and Agency guidance for value derivation generally used by the EPA IRIS Program. All provi siona l tox i c ity va lues

rece ive in ter n al rev i ew b y two EPA scientists and external peer review by three independently selec t ed sc ie ntif ic expe rts. PPRTVs differ from IRIS values in that PPRTVs do not receive the multi-p r ogra mconsensus review prov ided f o r IRI S values. T h is is be caus e IRIS value s are generally intended to be used in all E P A program s, while PPRTVs are developed specifically for the Superfund Program

.

Because science and av ailab l e inform ation evolve, PPRTVs are in itially derived witha three-year life-cycle. H o wever, EPA Regi ons or the EPA Headquarters Superfund Program

som e ti m e s request that a frequently used PPR TV be reassessed. Once an IRIS value for a specific chem ical becom e s available for Agencyreview, the analogous PPRTV for that sam e chem ical is retired. It should also be noted that som e PPRTV m a nuscripts conclude that a PPRTV cannot be derived ba sed on inadequate data.

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Disclaimers

Users of this docum ent should first check to se e if any IRIS values exist f o r the chem i c al of concern before proceeding to use a PPRTV. I f no IRIS valu e is ava ilab l e, staf f in th e region al Superfund and RCRA program offices are advised to carefully re view the inform ation provided in this docum e nt to ensure that the P P RTVs us ed are appropriate for the types of exposures and circum stances at the Sup e rfund site o r RCRA f acility in question. PPRTVs are period ically updated; therefore, users should en sure that the values contained in the P P RTV are current at the tim e of use.

It is im portant to rem e mber that a provi sional value alone tells very little about the adverse effects of a chem ical or the quality of evidence on w h ich the v a lue is based. Therefore, users are strongly encouraged to read the entire PPRTV manuscript and understand the strengths and lim itations of the derived provisional valu es. PPRTVs ar e developed by the EPA Office of Research and Development’s Nati onal Center for Environm e n tal Assessm ent, Superfund Health Risk Technical Support Center for OSRTI. Other EPA program s or external parties who m a y choose of their own initi ative to use these PPRTVs are advise d that Superfund resources will not generally be used to respond to challenges of PPR TVs used in a context outside of the Superfund Program

. Questions Regarding PPRTVs

Questions regarding the contents of th e PPRTVs and their ap propriate use (e.g., on chem icals not covered, or whether chem icals have pending IRIS toxicity values) m a y be directed to the EPA Office of Research and Devel opm ent’s National Center for E nvironm ental Assessm ent, Superfund Health Risk Techni cal Support Center (513-569-7300), or OSRTI

INTRODUCTION

A reference dose (RfD) for iron is no t availa ble on the Integrated Risk Inf o rm ation System (IRIS) (U.S. EPA, 2006) or the Drinking W a ter Standards and H ealth Advisories list (U.S. EPA, 2005). The Health Effects Assessm ent Summary Tables (HE A ST) (U.S. EPA, 1997) reported that data regarding iron were inadequate for quantitative risk assessm ent. The Chem ical Assessm ent and Related Activities (CARA) list (1991, 1994) includes a Health Effects Assessm ent (HEA) for Iron and Compounds (U.S. EP A, 1984) that found no reliable quantitative oral toxicity data. Iron has not been the subject of a toxico logical review b y the Agency for Toxic Substances Disease Registry (ATSDR) ( 2005) or the World Health Organization (WHO) (2005). Monographs by the International Agen cy for Research on Cancer (IARC) (1972, 1987), toxicity reviews by Jacobs (1977), B o thwell et al. (1979), Lauffer (1991) and Grim sley (2001), a review on dietary iron by the National Academy of Sciences (NAS) (2001), and the N a tional Toxicology Program (NTP) (2001, 2005) m a nagem e ntstatus report and chem ical repository summary were consulted for relevant inform a tion. The NAS (2001) derived a Tolerable Upper Intake (TUI) level of 45 mg iron/day. The TU I is based on a m i ni m a l LOAEL of 70 mg/day (60 mg iron as ferrous fum e rate plus 11 mg/day of dietary iron) identified by Frykm an et al. (1994) for gastrointestinal effects and an uncertainty fa ctor of 1.5 for use of a m i nim a l LOAEL; a higher

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uncertainty f actor was no t used sin ce the nature of the observed gastro inte s tina l ef f ects was considered to be self-limiting. The U

. S. F ood and Drug Adm i nistration (F DA) prom ulgated a Rule in 199 7 for labelin g of iron-co ntain i ng diet ary supplem ents for the prevention o f accidental poisoning in children (U.S. FDA, 1997). The Rule, as m odifi ed in 2003, does not contain specific exposure lim its (U.S. FDA, 2003). In general, the F DA follows the NAS guidance on exposure lim its for toxicity of essential elem ents, su ch as iron. Previous literature searches were conducted through Septem ber, 2001 as follows: TOXLINE ( o ral and inhalation toxicity and cancer from 1983 - September, 2001); CANC ERLIT (1990 - Septem ber, 2001); MEDLINE (1991 - Septem ber, 2001); TSCATS, RTECS, DART/ETICBACK, EM IC/EMICBACK, HSDB, GENET OX, and CCRIS. Update literature sear ches were perfor m ed in October, 2005 in MEDLINE, TOXLINE ( N TIS subfile), T OXCENTER, TSCATS, CCRI S, DART/ETIC, GENET OX, HSDB, RT ECS and Cu rrent Contents.

REVIEW OF PERTI N ENT LITERATURE

Iron is an essential element and deriving a ri sk assessm ent value for such chem icals poses a special pro b lem in that the dose-adv e rsity curv e is "U-shaped". Thus, th e risk value must be protective against deficiency as well as toxicity. The NAS ( 2001) has esta blished guidelines for iron intake that take into acc ount physiological differences duri ng different life stages. For nonbreast-fed infants aged 0-6 m onths, the NAS (2001) established a daily adequate intake (AI) for iron of 0.27 mg/day (0.04 m g /kg-day for infants 2- 6 m onths old) based on the daily amount of iron secreted in hum an m ilk; breast-fed infants t ypically receive only 0.15 to 0.3 m g Fe/day. The NAS (2001) Dietary Ref e rence Intakes (DRIs) for children are as follows: 11 m g /day (1.2 mg/kg-day) for infants between the ages of 7 and 12 m onths, 7 m g /day (0.54 m g /kg-day) for children aged 1-3 years, 10 m g /day (0.45 m g /kg-da y) for ages 4-8 years, 8 m g /day (0.2 m g /kgday) for ages 9-13 years and 11 m g /day (0.17 m g /kg-day) for boys and 15 mg/day (0.26 m g /kgday) for girls aged 14-18 years. The DRI fo r m e n aged 19 years and above is 8 m g /day (0.11 mg/kg-day). The DRI for non-pregnant wom e n i s 18 m g /day (0.29 m g /kg-day) for ages between 19 and 50 years and 8 m g /day (0.13 mg/kg-day) for ages 51 years and older. The DRI for pregnant wom e n is 27 mg/day (0.37 mg/kg-day for those aged 14-18 years and 0.35 mg/kg-day for those aged 19-50 years). The D R I during la ctation is 10 m g /day (0.18 m g /kg-day) for

wom e n aged 14-18 years and 9 m g /day (0.15 m g /kg-day) for wom e n aged 19-50 years.

According to the Centers for Diseas e Contro l and Prevention (CDC, 1998; CDC, 2005), iron deficiency is one of the m o st common known for m s of nutritional deficiency. Its prevalence is highest among young children and wom e n of childbearing age, pa rticularly pregnant wom e n. In children, iron deficiency causes developm enta l delays and behavioral disturbances, and in pregnant wom e n, it increases the risk for a pr eterm delivery and delivering a low-birthweight baby. Young children are at great ri sk of iron deficiency because of rapid growth and increased iron requirem ents. Iron deficiency can occur due to lack of iron in the diet. If this continues,

anem ia results. Anem ia is a m a nifestation of ir on deficiency when it is relatively severe. Iron deficiency anem ia significantly im pairs m e ntal and psychomotor developm ent in infants and children. Although iron deficiency can be reversed w ith treatm e nt, the revers ibility of the m e ntal and psychomotor im pairm e nt is not yet clearly understood. T hus, prevention and treatm ent need to be em phasized m o re than detection. In addition, iron deficiency increases a child’s

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susceptibility to lead tox i city. Lead replaces iron in the abso r p tive pa thway when iro nis unavailable.

In hum ans a nd other anim als, levels in th e body are regulated prim arily through changes in the am ount of iron absorbed by the gastrointes tinal m u cosa. The absorption of dietary iron is influenced by body stores, by the am ount and chem i cal nature of iron in ingested food and by a variety of dietary factors thatincreas e or decreas e the ava ilab ility of iron f o r absorption (Hillm an, 2001; Santi an d Masters, 2 001). Iron contained in m eat protein (hem oglobin and myoglobin) is absorbed intact without first being broken down to elem ental iron. Non-hem e iron must first be reduced to ferrous iron (Fe 2+ ) before it can be absorbed. Ferrous iron is transported across intes t inal m u cosal cells by active transpo r t w ith the rate of transport inverse l y r e lated to body iron stores. Depending upon the iron status of the body, iron is stored bound to ferritin within m u cosal cells and m acropha ges in the liver, spleen and bone , or is transported in the plasm abound to transferrin. Serum levels of fe rritin and transferrin, along with several red blood cell param e ters, can be used clinically to evaluate iron balance. Although iron absorption is regulated, excessive accum u lation of iron in the body resulting from ch ronic ingestion of high levels of iron cannot be prevented by intestin al regulation and hum a ns do not have a m echanis m to increase excretion of absorbed iron in response to elevated b ody levels (NAS, 1989, 2001).

Human Studies

Acute Exposure

Inform ation on acute oral toxic doses of iron in hu m a ns is av ailab l e from num e rous case reports of ingestion by children, but values vary because it is difficult to obtain accurate estim ates of the am ount taken in m o st overdos e situations. Reviews of these case reports indicate that doses in the range of 200-300 m g iron/kg are generally cons idered lethal (Arena, 1970; Krenzelok and Hoff, 1979; NRC, 1979; E ngle et al., 1987; Ma nn et al., 1989; KleinSchwartz et al., 1990).

Therapeutic Studies

Ferrous salts are adm i nis t ered o r ally for the th e r a p eutic tre a tm ent of iron deficiency. The oral absorption of ferrous iron s upplem e nts is considered to be essentially the sam e for all ferrous salts (e.g., sulfate, fum a rate, succinate and gluconate) and is approxim ately three tim es greater th an that of ferric (Fe 3+ ) salts (Hillm an, 2001); thus, ferric iron is n o t used therapeutically. Constipation a nd other gastrointestinal effect s, including nausea, vom iting, diarrh ea and gastro intestinal pain are commonly associated w ith adm i nistration of oral ferrous salt supplem e nts (Hillm an, 2001; Santi and Master s, 2001). S e verity of effects is variable, ranging from m ild to severe, and depends upon dos e and individual susceptib ility. T h e onset of
sym p tom s t ypically occurs at th e initiation of treatm ent and c ontinues throughout the duration of
treatm ent. Although there is no indi cation that the severity of gastro intestinal effect s varies over the course of treatm e nt, severity is decrease d in som e patients when iron supplem ents are adm i nistered with food (Hillm an, 2001; Santi an d Masters, 2 001). For most patients, iron deficiency is reversed within six m onths of trea tment, thus lim iting th e duration of exposure.

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The m echanism of iron-induced gastrointestinal toxicity is not established, although it is postulated that adverse effects are d u e to irritant effects of the free iron ion on the gastric muscosa (Liguori, 1993). The role of absorbed iron in the developm ent of gastrointestinal adverse effects is unknown. The adverse effects of exposure to oral iron supplem ents has been investigated in several studies (Blot et al., 1981; Brock et al., 1985; Coplin et al., 1991; Fryklm a n et al., 1994; Hallberg et al., 1966; Liguori, 1993).

Frykm an et al. (1994) evaluated the adverse effects of daily oral therapy w ith iron fum a rate in a double-blind, cross over, placebo-controlled study in Swedish m a le [n= 25; m e an age 45 years (range 40-52)] and fe m a le [n=23; mean age 41 years (range 34-45)] adult blood donors. Study subjects were adm i nistered 60 m g elem ental iron as a daily dose of iron fum a rate for one m o nth, with each study subject serving as their own placebo control. Com p ared to the placebo treatm ent period, the percen tage of subjects reporting c onstipation (placebo 20%, ferrous fum a rate 35%, p<0.05) and total ga strointestinal sym p tom s ( n ause a, obstipation, gastric pain and diarrhea (placebo 14%, ferrous fum a rate 25%, p< 0.01) was significantly in creased during ferrous fum a rate treatm ent. Although the severity of ga strointestinal effects was graded as m i nor in most study subjects, four subjects withdrew fr om the study due to seve re gastrointestinal
sym p tom s associa t ed with iron f u m a rate. In a matched group of 49 adults taking a daily

com b ination supplem ent of porcine-derived hem e -i ron and iron fum a rate containing a total daily supplem ent of 18 m g iron/per day, the frequency of gastrointestinal sym p tom s was not increased

com p ared to placebo. N o differences in therap eu tic efficacy, as m easured by serum fe rritin and hem oglobin levels, were observed between the non-hem e iron and hem e -iron treatm ent groups.

Adverse effects of four oral iron preparations were eval uated in 1496 m a le and fem a le adult b l ood donors in a series of dou ble-b lind, pl acebo controlled trials (H allberg et al., 1966). The following treatm ent groups were com p ared: (1 ) placebo (195 subjects) and ferrous sulfate (198 subjects; 222 m g elem ental iron/day); (2 ) p l acebo (199 subjects), ferrous sulfate (120 subjects; 222 m g elem e n tal iron/day), ferrous fum a rate (118 subjects, 222 m g elem e n tal iron/d a y), an d ferrous glu c onate (120 subjects; 22 2 m g elem e n tal iron/d a y ) ; and (3 ) placebo (200 subjects), ferrous sulfate (195 subjects; 180 m g el em ental iron/day), ferrous glycine sulfate (200 subjects; 180 m g elem e n tal iron/day), and ferro us gluconate (196 subjects; 180 m g elem ental iron/day). T r eatm e nts were adm i nistered for two weeks. For all iron treatm ents, the frequency of

adverse g a stro inte stin al ef f ects was significantly increased com p ared to the m a tched placebo group (p<0.05). Adverse effects reported includ e constipation, diarrhea, heartburn, nausea and epigas tric pa in. No statis tica lly s i gnif i cant d i f f e rences in the frequency of adverse effects were observed between iron treatm e nts for subjects r eceiving 222 mg elem ent a l iron/day or between iron treatm e n ts for subjects receiving 180 m g el em ental iron/day. In the seven iron treatm e nt groups, the percentage of subject s reporting gastrointestinal e ffects ranged from 22.9% in the 222 m g ferrous sulfate group to 31.5% in the 222 m g ferrous gluconate group. In the three placebo treatm ent groups, the percentage of subjects reporting ga strointestinal effects ranged from 12.4 to 13.6%. Although statistical com p aris ons were not m a de between the 180 and 222 mg iron/day treatm ents, the frequency of adve rse effects was sim ilar for all iron treatm e nt groups.

Gastroin tes t inal sym p tom s were rep o rted in pregnant wom e n treated daily with oral iron supplem ents containing 105 m g elem ental iron and 500 m g ascorbic acid (55 wom e n) or 105 mg

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elem ental iron, 500 m g ascorbic acid and 350 mg folic acid (54 wom e n) during the third trim ester of pregnancy (Blot etal., 1981). The form of iron was not reported. No placebo control group was included. Gastrointestinal adv e rse effects re ported include nausea, diarrhea, constipation and epigastric pain. Approxi m a tely 16% of all patients reported m i nor gastrointestinal sym p tom s , 14% reported severe effects and 6% stopped treatm e nt due to adverse effects. Adverse effects occurred with approx imately the sa m e f r equency in the two trea tm ent group, although data were not reported.

The tole rability of iron p r ote i n succ inylate and f e rrous su lf ate were com p ared in a double-blind clinical trial in 1095 patients with iron deficiency(Liguori, 1993). Patients received daily treatm e nt with a controlled-release for m ulation of ferrous sulfate containing 105 mg elem ent a l iron (64 m a les and 485 fe m a les) or iron protein succinyl ate containing 120 m g elem ental iron (55 m a les and 491 fem a les) for 60 days. No placebo con trol group was includ ed. In the ferrous sulfate group, 26.3% of patient s reported adverse gastrointestinal effects (heartburn, epigastric pain, constipation and a bdom inal pain), com p ared to 11.5% of patients treated with iron protei n succinylate (p<0.05).

The adverse effects of oral treatm e nt with a conventional ferrous sulfate tablet were

com p ared to a ferrous sulfate wax-matrix ta blet in a single-blind, parallel group study in 543 subjects (Brock et al., 1 985). No placebo control group was includ ed. Subjects were adm i nistered a conventional ferrous sulfate table containing 50 m g elem e n tal iron/day (272 subjects) or a sulfate wax-m a trix tablet contai ning 50 m g elem ental iron/day (271 subjects) for 56 days. Approxim a tely 45% of subjects treate d with conventional ferrous sulfate reported moderate-to

- severe gas t rointes tinal effects, inclu d ing abdom inal dis c omfort, nausea, vom iting, constipation and diarrhea, com p ared to approximat ely 17% of subjects tr e a ted with th e f e rrous sulfate wax-m a trix preparation, a statis tically significant difference (p<0.001).

The tole rability of f

e rrou s sulf ate (50 m g el em ental iron/day ) and bis-g l yc ino iron II ( 50 mg elem ent a l iron/day) was com p ar ed in a double -blind, crossover trial in 42 wom e n (Coplin et al., 1991). The treatm e n t period for each iron su ppl em ent was two weeks. No placebo treatm ent period was included. The frequency of advers e g a stro inte stin al e ffects (abdom i nal pain, bloating, constipation, diarrhea and nausea) was sim ilar for the two treatm ents, with 54% and 59% of subjects reporting gastroin testinal sym p tom s during treatm e nt with bis-glycino iron II and ferrous sulfate, resp ectiv el y. The difference between treatm ents was not sta tis tic a lly significant.

Effects of iron therapy on the upper gastroin testinal tract were evaluated in 14 healthy volunteers [13 wom e n, 1 m a n; m e a nage 29 years (range: 24-48 years)] who were instructed to ingest 325 mg tablets of ferrous sulfate (119.5 mg elem ent a l iron) three tim es/day before m e als (358.5 m g elem ental iron/day) for 2 weeks (Laine et al., 1988). Evaluation consisted of a gastrointestinal sym p tom survey, qualitative (H em occult) and quantitative (Hem oQuant; m g

m e rcury/g stool) testing for fecal bloo d loss, e ndo scopy of the upper gastrointestinal tract and histological exam ination of pi nch biopsies of the gastric body , antrum and duodenum . Based on actual average ingestion of 2.5 ta blets/day (2-week study) and 2.6 tablets/day (1-week study) and a reference hum a n body weight of 70 kg (U.S. EP A, 1987), the estim a ted doses consum ed by the subjects were 4.3 and 4.4 m g iron/k g -day, resp ectiv ely, in addition to diet ary iron. Com p ared to

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baseline m e asurem ents in the two weeks prio r to trea tm ent, all subjects h a d signif i c an tly increased (p <0.05) dark brown-black stools a nd s y m p tom s of nausea and vom iting during th e treatm ent period, but not abdom inal pain. He m o globin levels in stool did not change significantly after iron treatm e nt. Endoscopic exam ination showed a significant (p=0.003) increase in abnorm a lities in the s t omach, but not duodenum , after therapy

. These changes consisted of erythem a , sm all areas of subepithe lial hem o rrhage and solitary antral erosions in nine, six and two subjects, resp ectively, and were considered only m i ni m a lly abnorm al. No
trea tm ent-re lated h i sto l o g ical change s were observed. Although it wa s sp ecula ted tha t the changes in the stom ach could represent a m ild form of iron poisoning, the investigators concluded that the treatm ent caused m ild e ndoscopic abnorm a lities of uncertain clinical significance in the stom a c h. Evidence for iron overload (tissue biopsie s or hem a tologic iron status indices) was not exam ined. Considering addition a l dietary exposur e, an exposur e lev e l of about 4.3 mg/kg-day represents, at w o rst, a m i nim a l LOAEL.

Adverse developm ental effects in hum a ns have not been asso ciated with the ingestion of supplem ental iron during pregnancy. As i ndicated above, NAS (2001) recomm ended that pregnant wom e n supplem ent their diets with 27 mg iron/day (0.35 m g /kg-day). McE l hatton et al. (1991) reported on 49 wom e n who took an overdose of a sim p le iron preparation (53%) or iron with folate preparation (47%). In 48 of the wom e n, the am ount of ir on ingested was known; 28 took > 1.2 g and the rem a inder took

 1.2 g. There were 25 wom e n who received chelation
trea tm ent with desf err i ox am ine (DFO) and 12 w ho received an em etic. Maternal toxicity, consisting of nausea, vom iting, he matoem esis, abdo m i nal pain and diarrhea, was observed in 35 of the wom e n. Two spontaneous abortions occurred and there were three prem ature deliveries. One of the spontaneous abortions and the prem ature deliveries were not related to the iron overdose. It is not know n if the other spontaneo us abortion occurring at 22 weeks (3 weeks after the overdose) was caused by the iron overdose. N o conclusions on the developm ental toxicity of iron can be m a de.

Chronic Exposure

While chronic iron toxicity occurs in people with genetic m e tabolic d i sord ers re sulting in excessive iron absorption or abnormal hem oglobi n synthesis, or who receive frequent blood transfusions (Jacobs, 1977; Both well et al., 1979), ther e is a long-standing controversy as to whether a chronic overload due to oral intake is p o ssible in in dividua ls with a norm a l ability to
contro l iron absorption (Hillm an and Finch, 1985). Neverth e less, "th e cumulative ex perien ce in hum a n subjects suffering from iron overload of va rious etiologies strongl y suggests that iron is noxious to tissues [when]...present in parenchym al cells...for a sufficiently long period of tim e" (Bothwell et al., 1979).

Looker et al. (1988) m a de com p arisons of diet ary iron intake and bi ochem i cal indices of iron status based on values taken from the seco n d National Health and Nutrition Ex am ination Survey (NHANES II) data base 1 . NHANES II wa s a probability sam p le of the noninstitutionalized U.S. population aged 6 m ont hs to 74 years, conducted between 1976 and

1 The l a t e st ver s i on o f th i s dat a base

, NH AN E S I II ( 1 9 8 4

- 1 9 8 8 ) e v al uat e d 30 ,0 0 0 s u bject s age d 2 m onth s an d a b ove ( N A S , 200 1)

. D e sp ite m i n o r d i f f e r e n ces in t h e d a ta sets, t h e con c lu si o n s d r awn b yLooker et al. ( 198 8) b a sed o n NH A N ES II a ppear t o be val i d f o r th e N H A N ES II I dat a

.

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1980 by the National Center for Health Statistics. These data suggest that norm a l intake of iron by m e n 16-74 years old exceeds th e DRI, and that iron intak e is som e what lower thanthe DRI for wom e n younger than 51 years. C oncom itant with the study of diet ary intake, the NHANES II m easured the iron status of these populations . The percent serum tr ansferrin saturation, a

m easure of the residu al capacity of the iron transp ort sys t em to process potential variations in iron from dietary intake or catabolized body stores, ranged fro m 24% saturation for pre- and post-m e nopausal wom e n not using iron supplem ents to 29% satura tion for adult m a le supplem ent users. These values are within the norm al range (20- 40%). The Looker et al. (1988) evaluation of the NHANES II iron status data co ncerned iron deficien cies, only, and did not address iron overload directly. Ho wever, iron overload conditions would likely be evidenced by increased saturation of serum transferrin and incr eased serum ferritin con centration s , which were also within the norm a l range. Therefore, th e corresponding dietary inta kes are presumed to represent chronic NOAELs. Looker et al. (1988) estim ated daily iron intakes ranging from 10.0 for elderly wom e n to 18.7 m g /day for young adu lt m e n in the study population. These daily intakes correspond to a range of about 0.15 to 0.27 m g /kg-day, depending on assum p tions of average body weight. T a king the highest intake level of 18.7 m g /day and a body weight of 70 kg, a NOAE L of 0.27 is established f o r chronic iron toxicity.

He m o siderosis (or siderosis) and iron overloa d are increas es in tis sue iro nor a general increase in iron stores withoutassociated tissue dam a ge (Bot hwell et al., 1979; Jacobs, 1977). He m o chromatosis describes m a ssive iron overload ( 15 g of body iron stores or greater) together with cirrhosis and/or other tissue dam a ge attrib utable to iron. Alt hough focal deposits of iron

m a y occur in any part of the body where red cells are extravasated, the clinical syndrom e of hem o chromatosis typically involves dam a ge to the hepatic parenchym a (particularly fibrosis), heart (cardiac dysfunction includ ing failure) and endocrine glands (particularly hypogonadism ). Pancreatic iron deposition is comm on and m a ssive deposits m a y be associated with fibrosis and diabetes. A num b er of studies i nvolving chronic oral adm i nistration of iron to anim als have been designed in an attem p t to identify an anim al m o del for hem o c h rom a tosis. Most of these studies have been negative (Bothwell et al., 1979; NRC, 1979). Anim al studies involving parenteral adm i nistration of iron have been generally negativ e as well, even though parenteral routes bypass the m echanism s that reg u late absorp tion of iron f r om the gastrointestinal tract.

Chronic iron toxicity has been observed inpeople with idiop a thic hem o chrom a tosis ( a genetic m e tabolic disorder resulting in excessi ve iron absorption), abnormalities of hemoglobin synthesis (e.g., thalassem i a) or various anem ic states (e.g., sidero blastic anem ia), frequent blood transfusions or a com b ination of these conditions (Jacobs, 1977; Bothwell et al., 1979). Chronic hem o chromatosis has also occurred am ong th e South African Bantu population from an excessiv e in take of abso rbable iron in an alcoholic beverage.

Habitual excessive intake of iron by the Bant us is attributed to consum ption of hom e

brewed Kaffir beer, which was contam inated by iron vessels during brew ing (Bothwell and Bradlow, 1960; Bothwell et al., 1964). The beer' s high acidity (pH 3-3.5) enhanced iron leach ing from the vessels. The iron in the beer is readily ass i m ilable (i.e., ionizab le) d u e to the acidity and presence of iron-com p lexing ligands su ch as fructose, and is absorbed to approxim a tely the sam e degree as ferric chloride. The al cohol content of the beer is also believed to contribute to the bioavailability of the ir on (Jacobs, 1977; Finch and Monsen, 1972). Based

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prim arily on drinking habits and analyses of beer sam p les, the estim ated average d i etary iron intake of the Bantu m e n ranged from 50-100 m g / d ay from beer alone (Bothwell et al., 1964). Using a reference body weight of 70 kg (U.S. EPA, 1987), this range corresponds to 0.7-1.4 mg/kg-day. Histological exam inations of the liver of 147 Bantus (129 male, 18 fem a le) ranging in age from 11-70 years (m ost were between 20 and 50 years old) that died from acute traum a tic causes were perform e d (Bothwell and Bradlow, 1960). Varying degrees of hepatic siderosis were observed in 89% of the cases; the degree te nded to increase with age 40-50 years or less. The siderosis was m ild in 59% and severe in 19 % of the cas es, respe c tiv e ly. There was a close correlation between hepatic iron c oncentration and portal fibrosis and cirrhosis. Although the overall prevalence was low (15.6% fibrosis and 1.4% cirrhosis), all 11 subjects with the highest iron concentrations (>2.0% dry weig ht of liver) showed either fibros is or cirrhosis. H i stological

exam ination of the spleen (50 subjects) also showed siderosis and uns pecified histological changes. Malnutrition and alcoholism could have pl ayed a role in the etiology of the hepatic and splenic siderosis in the B a ntus. A NOAEL in th e range of 0.7 - 1.4 m g /k g-day is indicated but

m a y be low given the likely highe r bioavailability of iron in th e beer than for norm a l dietary exposure. Given the generally poor nutritional hea lth status of this popu lation, the relevance of this study for application to the U.S. population is questionable.

Ethiopia reportedly has the hi ghes tp e r cap ita iro nintake in the world, with an ave r ag e daily intake of 471 m g iron/day (range 98- 1418 mg/day; 1.4-20.3 m g iron/kg-day assum i ng 70 kg body weight) (Roe, 1966; Hofvander, 1968). Incr eased stored iron in the liver and adverse health effects have not been observed due to lo wbioavailability of the iron in Ethiopian food.

A few studies have suggested that high iron intake m a y be a risk factor for myocardial infarction (S alonen et al., 1992; Lauffer, 1991; Su lliv an, 1992). Five other large s tudies found no association between serum ferritin levels a nd coronary heart disease (NAS, 2001). Various other m easures of iron status (serum transfer rin saturation, serum iron concentration and total iron-binding capacity) have been exam ined for a possible link to card i ovascular disease in prospective cohort studies, but resu lts overall have been characteri zed as contradictory (Meyers, 1996; NAS, 2001). The NAS (2001) c oncluded that the available evidence “does not provide convincing support for a causal relati onship” between the level of di etary iron intake and the risk for coronary heart disease, although iron cannot be definitively exclude d as a risk factor.

Animal Stu d ies

Repeated-dose oral studies in experim e nt al animals found no si gnificant effect of
treatm ent with inorganic iron com pounds. No tr eatm ent-related adverse changes in clinical signs, body or organ weights, food consum ption or histopathology were observed in m a le Sprague-Dawley rats that had daily dietary intakes of 35, 70 or 140 m g of iron (as FeSO 4 or FeEDTA) per kg for up to 61 days (Appel et al., 2001) . In m a le and fe m a le F344 rats that were exposed to drinking water containi ng 0.25 or 0.5% ferric chloride (FeC l 3 • 6H 2 O) for 104 weeks, there were no dose-related effects other than red u ced water intake (possibly affected by palatability) and body weight gain (Sato et al., 1992). In the latter study, the iron intakes were 58 or 110 m g /kg-day in m a les and 65 or 116 m g /kg-day in fem a l e s.

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No treatm e nt-related teratogenic or embryotoxi c effects were observe d in rats given 2.7 mg iron/kg-day as ferric chloride on gestational days 6-15 (N olen et al., 1972), or in rats and

m i ce given 24-76 m g iron/kg-day as ferrous su lfate for 6 days during gestation (days unspecified) (Tadokoro et al., 1979 ). Som e e m bryonic m o rtality(num bers and species not reported) occurred in the latt er study at 240 m g iron/kg-day.

DERI VATI O N OF PROVISIONAL SUB C HRONIC AND CHRONIC RfDs FOR IRON

Iron is an es sential element, as such, the RfD m u st be protective agai nst both toxicity and deficiency. Using the values for dietary intake and iron status indices taken from the second National Health and Nutrition Exam ination Survey (NHANES II) data ba se, it is possible to establish a NOAE L for chronic toxi city. Looker et al. (1988) m a de co mparisons of dietary iron intake and biochem i cal indices of iron status using data from NHANES II. The average intak e s of iron rang ed from 0.15 to 0.27 m g /kg-day. Th e serum ferritin levels an d percen t serum transferrin saturation were with in the norm a l range. Thus, intake levels of 0.15-0.27 mg/kg-day are sufficient to protect agains t iron deficiency. However, th e NHANES II data do not provide inf o rm ation to iden tif y daily d i etary iron intakes associated with toxicity. Therefore, daily dietary iron intakes were not consid ered as th e basis for the p

- RfD.

Most of the quantitative chr onic oral toxicity data for iron have been obtained from studies of the Bantu population of South Africa. These data indicat e tha tintake s in th e range of 0.7-1.4 m g i r on/kg-day in hom e -brewed beer are associated with hem o siderosis and liver cirrhosis (Bothwell and Bradlow, 1960; Bothwell et al., 1964). However, confounding factors such as m a lnutrition and unusually high iron bioavail ability due to the high acidity and ethanol in the beer preclude use of these da ta for risk assessm ent. Much higher dietary intakes (average 6.7 mg/kg-day) of less soluble form s of iron are to le r a ted in non

- w estern diets as indicated by studies of populations in Ethiopi a. T hus, although toxicity associated with iron overload due to chronic oral intake can be dem ons trated qualita tively or even sem i quantitatively, assignm e nt of a precise LOAEL for nor m a l individuals consum i ng western diets is com p rom i sed by studies containing confounding factors.

Gastroin tes t inal tox i city, whic h is co mmonly associated with the therapeutic use of iron supplem ents, was identified as the critical eff ect for the basis of the provisional subchronic and chronic RfDs. The m o s tfrequently reporte d symptom s include epig astric pain, nausea,

vom iting, constipation and diarrhea. Several prospective clinical trials in healthy subjects and iron-deficient patients identify a LOAEL for gas t ro intestinal toxicity of 50 to 180 m g elem ental iron/day; N OAELs wer e not established (Blot et al., 1981; B r ock et al., 1 985; Coplin et al., 1991; Frykm an et al., 1994; H a llberg et al., 1966; Liguori, 1993). The treatm e n t durations in these studies range from 2 we eks to approxim ately 3 months. Although no chronic exposure studies reporting gastrointestinal toxic ity were identified, clinical experience w ith iron supplem ents indicates that gastrointestinal effects are associated with oral iron therapy, regardless of the duration of treatm e nt and that sym p tom intensit y does not change over the course of treatm e nt (Hillm an, 2001; Santi an d Masters, 2 001). This o b se rvation s uggests th at the respon se is related to the con c e n tra tion of ir on in the intestin al trac t and not to th e tim e-integ r ated dos e. Therefore,

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gastro inte stinal tox i city is conside r ed as the c r itical effect for both th e subchronic and chronic pRfDs.

The lowest LOAEL of 50 m g elem ental iron/d ayf o r gastroin testinal toxicity associated with iron supplem ents was reported in two studies that did not use a placebo-controlled design (Brock et al., 1985; Coplin etal., 1991); therefore, data were not considered suitable for derivation o f the p-RfD. The placeb o-contro ll ed , cross-ov er design studyby Frykm an et al. (1994) reporting a LOAEL of 60 m g /day in Sw edish m e n and wom e n wa s identified as the critical study. Results of this study show that daily treatm e nt with fe rrous fum a rate (60 m g elem ental iron/day) for one m onth produced a statis tic ally signif i cant inc r ease in gas trointestinal effects compared to placebo. To determ ine th e LOAEL for total daily iron intake, the LOAEL f o r daily sup p lem e ntatio n with f e rro us f u m a rate of 60 m g elem ental iron/day was added to the estim ated mean dietary intake for six Europeancountries of 11 m g elem e n tal iron/day (NAS, 2001) for a total daily iron intake of 71 m g elem e n tal iron/day. Based on a reference body weight of 70 kg (U.S. EPA, 1987), the LOAEL for gastrointestinal effects for total daily iron intake is 1 mg elem ent a l iron/kg-day. This LOAEL is considered to be a m i ni m a l LOAEL because gastrointestinal effect s were characterized by m o st st udy particip ants as m i nor in severity.

The provisional subchronic and chronic RfD for iron was derived from the LOAEL of 1 mg/kg-day f o r total da ilyiron in take f o r adverse gastro inte stinal ef f ects a s f o llows:

p-RfD (subchronic and chroni c) = LOAEL ÷ UF = 1 mg/kg-day ÷ 1.5 = 0.7 mg/kg-day

Dividing the LOAEL of 1 m g /kg- day by an uncertainty factor of 1.5 yields a subchronic and chronic p-RfD of 0.7 mg/mg-day. The uncerta inty factor of 1.5 includes the individual uncertainty f actors of 1.5 for use of a m i ni m a l LO AEL, 1 for s e nsitive individuals, 1 for less than lifetim e exposure, and 1 for an adequate data base . An uncertainty factor of 1.5 was applied to account for extrapolation from a m i nim a l LOA EL to a NOAEL for a non-serious effect. A higher uncertainty factor for use of a m i ni m a l LOAEL was not used since the observed gastrointestinal effects are not considered serious and are reversib le when exposure is discontinued. Furtherm ore, gastrointestinal sym p tom s are not associa t ed with dietary intake of sim ilar levels of iron (NAS, 2001). Because ind i viduals sen s itiv e to gastrointestinal sym p tom s are considered to be included in the studies investigating e ffects of therap eutic iron; an uncertainty f actor of 1 for sensi tiv e in dividua ls re s u lts. An uncertainty factor of 1 was used to account for less than lifetim e exposure. Although exposure duration in the Frykm an et al. (1994) study was only one m o nth, there is no evidence to suggest that sym p tom s increase with longer exposure periods. An uncertainty factor of 1 was us ed to reflect an adequate database in hum ans, due to the extensive use of therapeutic iron.

Except f o r individua ls with disord e r s of ir on metabolism

, little inf o rm ation is av aila ble on the long-term system i c toxicity of orally ingested iron. This assessm ent, therefore, f o cuses more on what is known to be a safe oral intake of iron for the general hum a n population (i.e., apparently healthy normal indivi duals). The provisional referen ce dose is estim ated to be an

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intake for the general po pulation that is adequately protectiv e from adverse health effects. Further, it is also im portant to no te that individual requirem ents for, as well as adverse reactions to, iron m a y be highly variable. Some individuals m a y, in fac t , c onsum e a diet that contributes more than the provisional reference dose, without any cause f o r concern. In addition, specific population subgroups may have higher nutritional re quirem ents than the provisional R f D would provide. The p-RfD m a y not be protective of individuals withinherited disorders of iron

m e tabolism or other conditions wh ich affect iron hom e ostasis.

This assessm ent is essentially the sam e as that proposed by Stifelm a n et al. (2005).

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