Reproductive Toxicology Review - Science Direct

Reproductive Toxicology, Vol. 8, No. 4, pp. 283-295, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0890-6238/94 $6.00 + .00

Pergamon 0890-6238(94) E0026-R

• Reproductive Toxicology Review

MATERNAL

TOXICITY AND THE IDENTIFICATION OF INORGANIC ARSENIC AS A DEVELOPMENTAL TOXICANT MARI S. GOLUB Office of Environmental Health Hazard Assessment, California Environmental Protection Agency, Sacramento

Abstract - - Assessment of the potential developmental toxicity of arsenic in humans must be based entirely on the extensive animal literature; no appropriate human data are available. Hazard identification of developmental toxicity of arsenic in animal studies is complicated by the co-occurence of maternal and developmental toxicity when the pregnant dam is exposed to the toxicant. Current regulatory guidance requires that, when maternal and developmental toxicity occur at the same or similar doses, detailed consideration needs to be given to whether developmental toxicity is secondary to maternal toxicity or whether it represents a distinct hazard. In this review, these principles were applied to the relatively large database of animal studies available for hazard identification of inorganic arsenic as a developmental toxicant. It is concluded that maternal and developmental toxicity occur in the same dose range for this potent cytotoxicant, although differential no observed adverse effect levels can be identified depending on the endpoints used. Various evidence from the basic science literature indicates that developmental toxicity is not secondary to maternal toxicity. Current regulatory guidance falls short of defining effective approaches to resolving the difficulties posed by cooccurence of maternal and developmental toxicity. Key Words: arsenic; risk assessment; malformation; toxicity; fetus.

In contrast, information on arsenic developmental toxicity is derived almost entirely from animal studies. The human data are extremely limited. There are two case reports of poisoning of pregnant women (2,3). There is also an analysis of reproductive outcomes in areas around a copper smelter in Scandinavia that produced environmental contamination by arsenic, as well as a number of other metals, due to use of ores with high arsenic content (4). In addition, some associations between arsenic and adverse reproductive effects were identified in studies that correlated pregnancy outcome with drinking water contaminant analysis (5,6). Conversely, a large animal literature has more than adequately demonstrated the teratogenicity of arsenic in a variety of species (7). From what is known of arsenic toxicokinetics (8), there is no reason to conclude that humans would be uniquely resistant to this effect. Nonetheless, the status of arsenic as a potential human developmental toxicant is unresolved despite a high degree of perceived risk to human populations. Arsenic was among the 30 chemicals identi-

RELEVANCE TO HUMAN HEALTH ASSESSMENT Arsenic is well known as a lethal toxicant, as a human carcinogen, and as a human neurotoxicant. The use of arsenic for poisoning people and pests has a long history documented in folk stories, literature, and ancient and medieval medical texts. Perhaps because of the extensive information available from human poisonings, animal models of acute arsenic toxicity are not well developed. The evidence for arsenic as a carcinogen and neurotoxicant is also derived entirely from studies of human populations. Risk assessments for arsenic are based on its carcinogenic properties as demonstrated in populations exposed at low levels via environmental media (1). Opinions and conclusions are those of the author and do not necessarily represent the California Environmental Protection Agency, Address correspondence to Mari S. Golub, PhD, Cal/EPAOEHHA, 601 North 7th Street, P.O. Box 942732, Sacramento, CA 94234-7320. E-Mail: [email protected]. 283

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Table 1. Recent statements by regulatory agencies concerning the developmental toxicity of inorganic arsenic USPHS/ATSDR, 1991 (77): "Studies in animals, however, do support the view that high doses of ingested arsenic may be fetotoxic and weakly teratogenic. . . . These studies . . . indicate that the fetus may be affected by ingested arsenic, but suggest that the fetus is not more susceptible to arsenic than is the mother." CDHS/ATES, 1990 (68): "Although in some animal experiments the optimal doses used to produce malformations were near doses which are associated with maternal toxicity and mortality (Hood, 1983), this was not always the case (Willhite, 1986). . . . Furthermore to discount the teratogenicity of arsenic compounds on the basis of maternal toxicity would be inappropriate because the compounds appear to have specific targets in the developing organisms (Hanlon and Ferm, 1974). . . . Arsenic may have teratogenic or other adverse reproductive effects at high doses in humans. Such effects would be unlikely to occur at ambient arsenic levels because in animals they have only been observed at much higher levels (more than three orders of magnitude higher). However, existing data are inadequate to rule this possibility out definitively." USPHS/ATSDR, 1989 (78): "It should be noted that these dose levels may cause maternal lethality in exposed animals and are considerably higher than levels which may cause lethality in humans. On this basis, it seems likely that developmental end points are not of primary concern at exposure levels lower than those which cause maternal toxicity." Cal/EPA/PETS, 1992 (79): "Arsenic compounds are fetotoxic and teratogenic in mice, rats and hamsters. Generally these effects are seen only at dose levels which also result in maternal toxicity." USPHS/ATSDR, 1989 (78): "Developmental and reproductive effects in mammalian species have been induced using parenteral administration. However, by the oral route, fetotoxic effects have been produced only in the presence of notable maternal toxicity."

fled as of most c o n c e r n in the recent G A O report on regulation of reproductive toxicants (9). Further, arsenic was ranked first in the prioritization of chemicals for consideration under California's Proposition 65 as developmental or reproductive toxicants (10). Survey of regulatory documents indicates that the major issue in resolution of the status of arsenic as a potential h u m a n developmental toxicant is maternal toxicity (Table 1). Thus, appropriate analysis of this issue is critical to answering the concern about arsenic as a developmental toxicant in humans.

GUIDANCE OF REGULATORY AGENCIES O N E V A L U A T I N G DEVELOPMENTAL TOXICITY THAT OCCURS IN THE PRESENCE OF MATERNAL TOXICITY H a z a r d identification of developmental toxicants in animal studies is complicated by the issue of maternal toxicity. If an agent produces developmental toxicity (malformations, growth retardation, deficits in function, embryo/fetal lethality) but is also toxic to the dam, should the agent be considered a developmental toxicant? Several groups responsible for hazard identification of developmental toxicants have developed guidance on this issue. Current regulatory guidance in California and at the Federal level (Table 2) indicates that adverse effects on d e v e l o p m e n t can lead to identification of an agent as a developmental toxicant even when maternal toxicity also occurs at the same dose. H o w e v e r , undesirable o u t c o m e s can be anticipated if this default policy is always followed:

• A n y agent that is toxic is de facto a developmental toxicant at some dose since the pregnant organism will be c o m p r o m i s e d to the point that the pregnancy can no longer be supported and toxicity to the fetus will be identified. • If maternal and developmental toxicity occur at the same doses, reference doses established for adults will protect the developing organism. Risk assessment based on developmental endpoints is u n n e c e s s a r y and wasteful of resources. Thus, application of a default assumption should only take place if necessary after thorough review of the data and use of scientific judgment. This point is also emphasized in discussions of this issue outside of regulatory documents (11-14). This review deals with application of current thinking in this area to the use o f animal studies for hazard identification o f inorganic arsenic. In reviewing available information, the following questions were considered: 1. Is developmental toxicity secondary to maternal toxicity? 2. Does developmental toxicity o c c u r at doses below those producing maternal toxicity?

DESCRIPTION OF DATABASE AVAILABLE FOR REVIEW There are over 30 research reports in the open literature addressing arsenic-induced teratogenesis in laboratory animals. Most studies address hypotheses c o n c e r n e d with malformations; evaluation of postnatal function has not been conducted. Little useful information on the issue of co-occurence of maternal and developmental toxicity has been pro-

Arsenic and maternal toxicity • M. S. GOLUB

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Table 2. Current guidance for evaluating developmental toxicity that occurs in the presence of maternal toxicity Cal/EPA/RCHAS, 1992 (67): "Developmental effects which occur in the presence of maternal toxicity can be used as evidence of developmental toxicity; however, the possible influence of the maternal effects on developmental outcome should always be considered. In some contexts it may be necessary or desirable to distinguish those agents which are specific developmental toxicants (i.e., those which cause developmental toxicity at exposure levels which do not cause maternal or adult toxicity), from agents which can potentially cause adverse developmental effects via severe maternal toxicity at high exposure levels. If adverse developmental effects are entirely secondary to maternal toxicity, then prevention of the maternal toxicity will clearly also prevent developmental toxicity. As stated earlier, acceptable exposure should be based on the most sensitive endpoints of toxicity. In some cases it may therefore be inappropriate to base estimates of acceptable exposure levels on developmental endpoints, if other toxic effects occur at lower exposure levels. Conversely, there may be situations in which developmental effects are of concern even when maternal toxicity is occurring at the same or lower levels of exposure. The decision as to whether or not to identify a hazard or establish an acceptable exposure level based on developmental toxicity should be made on a case-by-case basis with the general proviso that such action should be taken if the developmental effect is the most sensitive endpoint of toxicity." IEHR, 1992 (70): "Agents that produce developmental toxicity at a dose that is not toxic to the maternal animal are of greatest concern because the developing organism appears to be more sensitive than the adult. However, when adverse developmental effects are produced only at doses that cause minimal maternal toxicity, they are still considered to represent developmental toxicity and should not be discounted as secondary to maternal toxicity. If a dose causes maternal toxicity that is significantly greater than the minimal, toxic dose developmental effects may be difficult to interpret." USEPA, 1990 (69): "Agents that produce developmental toxicity at a dose that is not toxic to the maternal animal are especially of concern because the developing organism is affected but toxicity is not apparent in the adult. However, the more common situation is when adverse developmental effects are produced only at doses that cause minimal maternal toxicity; in these cases the developmental effects are still considered to represent developmental toxicity and should not be discounted as being secondary to maternal toxicity. At doses causing excessive maternal toxicity (that is, significantly greater than the minimal toxic dose), information on developmental effects may be difficult to interpret and of limited value. Current information is inadequate to assume that developmental effects at maternally toxic doses result only from maternal toxicity; rather, when the LOAEL is the same for the adult and developing organism, it may simply indicate that both are sensitive at that dose level. Moreover, whether developmental effects are secondary to maternal toxicity or not, the maternal effects may be reversible while effects on the offspring may be permanent. These are important considerations for agents to which humans may be exposed at minimally toxic levels whether voluntarily or involuntarily, since several agents are known to produce adverse developmental effects at minimally toxic doses in adult humans (e.g., smoking, alcohol, isotretinoin)."

d u c e d b y s t u d i e s in the o p e n l i t e r a t u r e b e c a u s e dosing a n d a s s e s s m e n t p r o t o c o l s w e r e p l a n n e d to ans w e r o t h e r q u e s t i o n s , as d i s c u s s e d b e l o w . S t a n d a r d F I F R A S e g m e n t II studies c o n d u c t e d in c o n n e c t i o n w i t h r e g i s t r a t i o n of a n a r s e n i c - b a s e d h e r b i c i d e ( a r s e n i c acid) are a v a i l a b l e in the a r c h i v e s o f r e g u l a t o r y a g e n c i e s as p r o v i d e d in the F r e e d o m of I n f o r m a t i o n A c t . S o m e d a t a f r o m these studies r e l e v a n t to the c o - o c c u r e n c e o f m a t e r n a l a n d develo p m e n t a l t o x i c i t y are p r e s e n t e d b e l o w . This r e v i e w is l i m i t e d to i n o r g a n i c a r s e n i c salts ( a r s e n i t e , A s +3, a n d a r s e n a t e , As+5). L i m i t e d inform a t i o n a v a i l a b l e o n a r s i n e (15), a r s e n i c trioxide (16,17), a n d c a c o d y l i c acid (18,19) w e r e n o t inc l u d e d i n this r e v i e w .

T h e v e r y l i m i t e d i n f o r m a t i o n a v a i l a b l e o n the d e v e l o p m e n t a l t o x i c i t y o f a r s e n i c in h u m a n s is n o t i n c l u d e d in this r e v i e w .

FINDINGS OF STUDIES IN THE OPEN LITERATURE

Syndromes of arsenic-induced teratogenesis E a r l y s t u d i e s (20-23) s o u g h t to d e s c r i b e arsen i c - i n d u c e d m a l f o r m a t i o n s in v a r i o u s species (Table 3). A s y n d r o m e c h a r a c t e r i z e d b y e x e n c e p h a l y / e n c e p h a l o c e l e , r e n a l a g e n e s i s , a n d axial skeletal m a l f o r m a t i o n s w a s identified. E x e n c e p h a l y w a s freq u e n t l y a c c o m p a n i e d b y facial a b n o r m a l i t i e s s u c h as a n o p h t h a l m i a / m i c r o p t h a l m i a , s h o r t e n e d j a w ,

Table 3. Doses used in arsenic acid registration studies

Species

Dosing route

Agent

Low dose

Dose (mg/kg/day) Mid-dose

High dose

Mouse

Gavage; embryogenesis Gavage; embryogenesis Diet; two generations

Arsenic acid Arsenic a Arsenic acid Arsenic Arsenic acid Arsenic b

10 3.96 0.25 0.10 1 0.53

32 12.67 1 0.40 5 2.65

64 25.34 4 ! .58 50 13.25

Rabbit Mouse

aEstimates based on Cal/EPA, 1992. bEstimates based on USEPA, DPR, 1991; arsenic acid was added to diet at concentrations of 20, 100, and 500 ppm.

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and facial cleft. The renal agenesis syndrome included genital tract manifestations such as absence of the oviduct and seminal vesicles, absence of uterine horn, and incomplete testes descent. Axial skeletal abnormalities included fused ribs and vertebrae. These studies were conducted primarily with intraperitoneal (i.p.) sodium arsenate (As÷5). A subsequent focus was on the origin and development of arsenic-induced malformation (2427). In such experiments, a common approach was to conduct a dose-finding study using single i.p. injections on different gestation days to identify a dose that produced a high incidence of the malformation of interest. Subsequently, embryos were examined grossly or microscopically at various times after dosing to describe the disruption of development that led to the malformation. In addition, one study investigated the role of the embryonic kidney in producing amniotic fluid via the use of arsenicinduced renal agenesis (28). These studies demonstrated that cytotoxicity and delay of embryonic development occur as early as 6 h after arsenic administration (28,27). Pathogenesis of the cephalic neural tube closure defect (exencephaly/encephalocele) was seen as early as 10 h after treatment (27). Observations included a failure of elevation and apposition of the neural folds in the head area and the presence of cytoplasmic inclusions in both mesoderm and epithelium. An unresolved issue has been whether the primary lesion occurred in the mesoderm or the neuroepithelium. Pathogenesis of the renal agenesis syndrome was first seen 48 h after arsenic was administered on embryonic day 10 (25). It consisted of growth retardation of the mesonephric duct, which failed to extend to the appropriate somite and produce the ureteric bud and metanephric blastema that are kidney precursors. Pathogenesis of skeletal defects has not been studied. Doses used in these studies were sometimes characterized relative to maternal toxicity. Hood and Bishop (22) described the dose used in their study (45 mg/kg i.p, in mice) as "barely sublethal." Morrissey and Mottet (24) identified a maternal LD50 at 69.2 mg/kg i.p. and selected a dose of 45 mg/kg i.p. in mice that was reported as "without apparent maternal effects." Willhite (27) stated that doses of 20 mg/kg arsenate or 10 mg/kg arsenite "failed to provoke signs of overt maternal arsenic poisoning" in hamsters. Group sizes in studies characterizing the development of teratologic lesions were typically small (4 to 10), making a low incidence of maternal mortality difficult to demonstrate. Because of a readily identified acute toxicity

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syndrome ending fairly rapidly in death, mortality was the main maternal toxic endpoint assessed in these and most later studies. These studies bear on the issue of hazard identification in that they demonstrated that arsenic produces a distinctive and reproducible pattern of malformation that is dose, developmental stage, species, and organ specific. These findings strongly suggest a distinct developmental toxicity and not a nonspecific pattern of fetal damage due to failure of maternal homeostasis (29-32). However, it should be noted that some components of the arsenic syndrome (exencephaly/encephalocele, fused ribs/vertebrae) were identified by Khera (33,34) as commonly occurring in mice and hamsters administered maternally toxic doses of several different teratogens. Regarding the minimum doses at which maternal and developmental toxicity occur (Lowest Observable Adverse Effect Levels, LOAELs), these studies produce little useful information. Arsenic forms and routes of administration In a series of studies more relevant to risk assessment, Hood and collaborators compared the teratogenic action of arsenate (As+5), arsenite (As+3), and methylated arsenic metabolites (22,3537), the relative potency of intraperitoneal versus oral dosing (35,37,38), and the relative sensitivity of mice and hamsters (35,36). Further studies compared maternal-fetal transfer of arsenate and arsenite after administration by the intraperitoneal and oral routes (39,40). To make these comparisons, standardized dosing and assessment protocols were necessary. Maternal toxicity was routinely measured as maternal mortality occurring between the time of dosing (mid-embryogenesis) and the time of assessment (end of gestation). Arsenite was found to produce both maternal mortality and fetal mortality at about fourfold lower doses than arsenate; however, malformation incidence was higher with arsenate at doses producing similar maternal/fetal lethality (35,41). This finding helps explain the preference for use of arsenate in teratology studies. Organic arsenic (methyl or dimethyl) was much less toxic in terms of effective dose (two orders of magnitude greater than inorganic arsenic) but did produce maternal mortality and was embryolethal and teratogenic (37). Both arsenate and arsenite were about fourfold less potent by the oral route than by the intraperitoneal route (42). Hamsters were more sensitive than mice, with about half the effective dose in mice required to produce dam mortality, embryolethality, and teratogenesis in hamsters (36). About threefold


40

mg/kg p.o.

10 mg/kg i.p.

1 00

9080-' 70. 60. 50'

b

0

damn

mortality

. . . . . O ........

fetal mortality

.... • ....

malformation

1

7

I

8

11"

T

T

T

T

T

9 10 11 12 13 14 15 days gestation

45 100j 90, 80 -~ 0 ~7o60== 50-" 40o30. ~D . 2 0 10" 0 7

100 90~= 80(U

4oi 302010' 0

mg/kg

287

70-

60¢= 50e= 40o 3020Q. 100 6 8

"\/

i

I

10 12 days gestation

12

p.o.

mg/kg

100 90- oN, o......x(/ ~= 80~70~- 60" == 50-'

1

o 30-~

20-t I

8

I

I

v

1IF

T

'IF

9 10 11 12 13 14 15 days gestation

0_h

8

15

i.p.

o

er'"" " /

/

g'\ /

,

-

12 days gestation

10

I

15

Fig. 1. Comparison of maternal and developmental toxicity of arsenite administered at different doses, by different routes (p.o. = per os, gavage administration; i.p. = intraperitoneal injection) and on different gestation days. Data from Hood (35) and Baxley et al. (38). Group sizes (dams treated per time point and dose) were 8.2 -- 1.4 (mean - SEM) for the intraperitoneal studies, 8.2 -+ 2.2 for the 45 mg/kg p.o. studies, and 19.7 --- 3.9 for the 40 mg/kg p.o. studies. Gross and soft tissue malformations are included; skeletal defects were not uniformly assessed. Fetal mortality included early and late resorptions and fetal death. lower levels o f arsenic were found in the fetus after oral (gavage) versus intraperitoneal administration; patterns o f distribution and clearance also differed (39,40). Because o f the consistent dosing and assessment protocols, this set of studies offers one of the best opportunities for comparing some measures of maternal and developmental toxicity. Taken together, the data suggest that fetal and maternal toxicity, as represented by dam mortality, fetal mortality, and malformation rate, can be dissociated and do not necessarily c o v a r y quantitatively when different forms o f arsenic, times o f treatment, and routes of administration are used.

In Figure 1, data are plotted for three endpoints (maternal mortality, fetal death, and malformation) by day o f gestation when arsenic was administered. The curves for intraperitoneal administration (right hand panels) demonstrate that fetal mortality covaried with maternal mortality. Fetal and maternal deaths both occurred more frequently later in embryogenesis (days 11 and 12) possibly due to the higher total dose administered due to increased maternal body weights. In contrast, malformation incidence did not covary with maternal mortality. Malformation incidence was greatest after arsenite administration on days 9 and 10. A high incidence of malformation

288


with no maternal mortality was seen on these days. However, the same doses clearly produced dam mortality on other days. With oral dosing (left hand panels), few malformations were recorded. Administration via the oral route led to a closer correspondence between maternal and fetal lethality; by the i.p. route, fetal lethality was more marked than dam mortality.

Dose-response, duration-response, and effective internal dose In another series of studies related to risk assessment, Hanlon and Ferm (43-45) used subchronic (4 day) administration of arsenate (via minipump) to determine dose-response curves and duration-response curves for various developmental toxicity measures (embryolethality, malformation, growth retardation). Speciation of arsenic and estimation of internal dose were also studied. Dose was found to be more critical in determining malformation incidence than was duration of treatment, and a minimal internal dose (plasma concentration) for producing malformation was identified. Both dose and duration were important in determining embryolethality and growth retardation. These studies did not mention maternal toxicity; however, Dr. Hanlon (personal communication) has stated that the doses used were one quarter of those producing reduced food intake during dosing.

Maternal-fetal distribution of arsenic Other studies have investigated the distribution of maternally administered arsenic to the fetus (39,40,45-47). Arsenic is readily transmitted to fetal tissues. It has been shown that distribution to fetal neuroepithelium is prominent in early embryogenesis whereas a more general distribution is seen later in gestation. In the maternal organism, the highest arsenic concentrations were found in intestine and kidney after i.p. injection. For these studies, dams were typically killed at various short intervals (30 min to 24 h) after arsenic administrations and maternal toxic effects were not reported.

Chelator protective effects Several studies have investigated the effectiveness of various chelators in protecting against arsenic-induced malformations (48,49). A single i.p. dose of arsenite that produced high and reliable rates of malformation was used. One study (49) ineluded several measures of maternal toxicity and demonstrated about 12 to 32% maternal mortality at doses of sodium arsenite that produced a 25 to 55%


gross malformation rate. One of the chelators studied (dimercapto-propanesulfonic acid) appeared to protect against embryotoxic effects at doses below those that protected the dam from death, hemorrhage, and total resorption.

Interaction of arsenic with other teratogens Finally, several studies have looked into the effect of arsenic combined with other teratogens in terms of potential interactions indicative of common mechanisms of action. Selenium was found to antagonize arsenic teratogenesis (50) whereas chromium enhanced this effect (51). Cadmium (50) and copper (51) did not interact with arsenic. Hyperthermia acted synergistically with arsenic to produce exencephaly and encephalocele in hamsters (52) possibly due to the increased arsenic body burden associated with decreased kidney function in heat-stressed animals (44). Some information on co-occurrence of maternal and developmental toxicity is found in these reports. The selenium interaction study characterized the arsenic (sodium arsenate) dose as "barely sublethal" but did not report maternal toxicity data. Interestingly, in the chromium interaction study, the dose of arsenate used (5 mg/kg, i.p. in rats) produced an increase in malformation rate without any effect on maternal toxicity as measured by weight gain during pregnancy. This is the lowest effective dose administered in developmental toxicity studies in rats. Arsenic and chromium together led to a marked increase in malformation rate as well as a dramatic decrease in maternal weight gain. There was no mention of maternal toxicity in the hyperthermia interaction studies, which were conducted using hamsters. In general, these studies are not well enough developed to indicate whether interactions suggest a direct developmental effect of arsenic or an effect secondary to maternal toxicity. Such studies would be useful if a common mechanism at the level of the embryo could be identified as the site of interaction.

Multigeneration study In an early study (53), arsenic was administered to mice as 5 ppm As +3 (approximately 1 mg/ kg/day) in drinking water. The only adverse effect reported (maternal or developmental) was reduced litter size in all three generations of the arsenictreated groups.

In vitro studies In vitro experiments, although not sufficient for hazard identification, help clarify the role of maternal toxicity in producing developmental toxicity by


determining if developmental effects occur without the maternal system being present. Studies with arsenic have been done with both pre- and postimplantation mouse embryos. When added to preimplantation mouse embryo culture, arsenite inhibited blastocyst formation, cell proliferation, and hatching, and increased micronucleus formation in a dose-dependent manner (54). More important to the purposes of this review, when postimplantation mouse embryos (day 8 gestation) were cultured for 48 h in the presence of various concentrations of sodium arsenite and sodium arsenate, dose-dependent patterns of growth retardation, malformation, and embryolethality were observed (55). Growth retardation (yolk sac diameter, crown rump length, head length) and developmental delay (somite number) were the most sensitive parameters. Doses producing growth retardation were 10 times higher for arsenate than for arsenite. However, arsenate led to developmental delay at lower doses than arsenite. Malformations were seen at the same doses that produced growth retardation and developmental delay while embryolethal effects occurred at approximately fourfold higher doses. Malformations produced at lower doses were characterized as prosencephalon hypoplasia, somite formation alterations, hydropericardium (arsenate only), and failure of closure of the neural tube. More recent studies conducted with day 9 and 10 embryos and using shorter exposure times have produced consistent results (56). Another study used day 10 rat embryos cultured for 24 h in various concentrations of sodium arsenite (57). Dysmorphology included hypoplastic prosencephalon, abnormal somites, and abnormal flexion of the tail in addition to growth retardation and developmental delay. Abnormal tail flexion has previously been reported in association with arsenate teratogenesis (41). Malformation syndromes identified after a short embryo culture period are difficult to compare with those detected later in gestation or at term. However, hydropericardium seen in the embryo culture studies had previously been noted as a common finding in hamster embryos examined shortly after early in vivo embryonic arsenate treatment (26). Anterior neural tube closure failure, also seen in embryo culture, has been reported as the most characteristic feature of arsenic-induced teratologic syndromes in mice, rats, and hamsters where it is manifest at later stages of development as exencephaly and encephalocele. Other types of defects that develop after day 10, such as axial skeletal defects and renal agenesis, would not be detected in short-term embryo culture experiments.

289

Embryo culture assays can be interpreted as demonstrating direct developmental toxicity of inorganic arsenic independent of maternal toxicity. The embryo culture syndrome is compatible with malformations seen after in vivo dosing. No information on NOAELs for maternal and developmental effects are produced in this type of study. Recent results from the in vitro hydra system (58) are consistent with in vivo mammalian studies concerning the relative potency of arsenite, arsenate, and methylated arsenic in producing developmental toxicity. Arsenite was 4.5 times more potent than arsenate and 100 times more potent than cacodylic acid in producing developmental toxicity. The ratio of doses required to produce adult versus developmental toxicity was 10:1 for arsenite and 11 : 1 for arsenate in this system. The following are conclusions from studies in the open literature. • Arsenic can be identified as a developmental toxicant in that it produces a characteristic syndrome of malformation in a dose and stage specific manner. Specific developmental processes affected by arsenic have been identified for neural tube closure defects (exencephaly and encephalocele) and renal agenesis. Selective cytotoxicity has been suggested as the mechanism of action for developmental toxicity. Mechanisms are not known at the cellular level. • Developmental toxicity as manifested in malformations and embryotoxicity is not secondary to maternal toxicity, as evidenced by its occurrence in the absence of maternal toxicity under in vitro and some in vivo dosing situations. Ready distribution of maternally administered arsenic to the conceptus also supports a direct effect. • In most reports, maternal and developmental toxicity co-occur at the doses studied; no studies allow determination of both a maternal and a developmental L O A E L and N O A E L so that the issue of relative sensitivity can be addressed. • In general, maternal toxicity has not been well characterized (dam mortality is the major endpoint measured). No hypotheses concerning mechanisms by which maternal toxicity might cause developmental toxicity have been developed. FINDINGS FROM REGISTRATION STUDIES In response to registration data-gap identifications, standard Segment II studies were conducted for arsenic acid ( A s H 3 0 4 ) , a registered herbicide based on arsenate. Developmental toxicity studies were performed in mice (59) and rabbits (60) using gavage, and a two-generation study was performed in mice

290


using oral administration of arsenic acid in food (61). Mice rather than rats were used as a test species because the pharmacokinetics of arsenic are different in rats from humans, mice, and hamsters (62). These studies are important to risk assessment because they use oral administration of arsenic, which is valuable in extrapolating to human exposures. Most basic research studies in the open literature used intraperitoneal administration. Human populations are exposed to arsenic primarily through drinking water and seafood (63). The developmental toxicity studies (59,60) assessed embryolethality, malformation, and fetal weight as developmental endpoints. The two-generation study assessed birthweight, postnatal mortality, and postnatal growth retardation. Maternal toxicity was recorded in both types of studies as mortality, weight gain, food intake, and gross and histopathology at sacrifice. Each study utilized four doses, including a maternally toxic dose (MTD) and a negative control (Table 3).

Results of the mouse developmental toxicity study A low incidence of malformation was recorded in the mouse teratology study (2.03% in the highdose group versus 0.98% in controls (59). However, rare malformations (exencephaly, thoracogastroschisis) not seen in control but reported in other previous mouse studies using the intraperitoneal route of administration (22) were recorded in arsenic acid groups. Two occurrences (1 litter) of exencephaly were recorded in the high-dose group. This group also demonstrated maternal toxicity in terms of reduced dam body weights after dosing (days 15 and 18 gestation) and mortality (3/23 dams died during pregnancy) in addition to reproductive toxicity in the form of fewer corpora lutea and viable fetuses and more early resorptions and postimplantation loss than controls. Reduced fetal weights were also recorded at the high dose. One instance of exencephaly and two instances of thoracogastroschisis occurred in the low and mid-dose groups, which did not exhibit maternal or reproductive toxicity. The fetus with exencephaly also had a facial cleft, a defect identified in connection with arsenic-induced exencephaly in hamsters (26). Thoracogastroschisis has not been widely identified as a component of arsenic-induced malformation syndromes in the open literature. However, few of these reports present a complete tally of anomalies, focusing instead on a single type of malformation or reporting summed incidence. An early report of a study using mice (22) nonetheless con-


tained complete information on individual malformations and reported two incidences of "eventration" and 15 instances of umbilical hernia out of 340 treated fetuses examined. The similarity between "eventration" and thoracogastroschisis is apparent in the description of the eventration anomalies as "defects of the ventral body wall allowing certain thoracic and abdominal organs such as the heart and liver to protrude to the outside" (22, p. 64). Thoracogastroschisis, omphalocele, umbilical hernia, and eventration defects are sometimes described as a class as "midline closure defects" although no mechanistic basis for this classification is available (64). Further, contemporary and historical controls from WlL Research Laboratories recorded no incidence of this anomaly. It should be noted that this was a large study using 22 to 25 litters per group and examining 146 to 307 fetuses per dose group. It thus enabled detection of malformations occurring at a low incidence at low doses. This study is taken to support occurrence of malformation in the absence of maternal toxicity. However, the low rate of malformation precludes statistical confirmation. The maternal NOAEL could be set at 32 mg/kg/day based on reduced weight gain at 64 mg/kg/day while the developmental L O A E L could be set at 10 mg/kg/day based on the occurrence of exencephaly with facial cleft at this dose.

Results of rabbit developmental toxicity study Rabbits were more sensitive to arsenic-induced acute toxicity than were mice (60). As indicated in Table 3, the MTD for rabbits was only 4 mg/kg/day as compared to 64 mg/kg/day in mice. Interestingly, there are no studies in rabbits of arsenic-induced developmental toxicity in the open literature to confirm the impression that rabbits are the most sensitive species. As was the case within the mouse teratology study, the malformation rate was very low in all groups, but rare malformations characteristic of arsenic occurred in the arsenic acid groups. Renal agenesis was seen in the highest dose group (1 pup in 1 litter) whereas fused ribs and sternebrae were seen in 3 pups of 1 litter at the mid-dose. The incidence of these abnormalities in 738 historical control litters was 2 for renal agenesis and 19 for fused sternebrae/ribs. The high-dose group in this study exhibited maternal toxicity in terms of mortality (7/ 18 deaths) and reduced weight gain during dosing (-179 +_ 264 g versus +25 + 163 g in controls), while no maternal toxicity was suggested in the mid-dose group. Fetal weights were not affected by arsenic acid at any dose; maternal weight effects

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Fig. 2. Comparison of maternal and offspring weights during the postnatal period in the arsenic acid mouse multigeneration study (59). Weights are expressed as the mean of the treatment group as a percent of the mean of the control group. For offspring, percents were calculated separately for males and females and averaged. The number of litters represented at weaning for the control, 20, 100, and 500 ppm groups was 22, 23, 26, and 12 for the F0 generation and 26, 19, 25, and 2 for the F~ generation. *Statistically significant differences from controls as stated in the study report. were confined to the dosing period, and maternal weights had recovered by term. The NOAEL for both maternal toxicity and fetal toxicity could be identified at 1 mg/kg/day. Although fused ribs in the mid-dose group (1 mg/kg/day) are characteristic of arsenic-induced teratogenesis, all instances occurred in a single litter, thus complicating interpretation of this dose level as a LOAEL.

Results of the mouse two-generation study The mouse two-generation study (61) demonstrated severe maternal and developmental toxicity at the high dose (500 ppm in diet). Increased maternal mortality (13% prebreeding, 38% during lactation) and reduced weights (-10% below controls during lactation) were seen at the high dose in the

first generation dams (F0). Smaller litter sizes were also seen in this group as well as an increased rate of resorption and lower birth weights. F0 offspring were severely growth retarded (