Document kDZVd3GXYpvkddBMxVwzVXa30
PROPOSAL FOR PHARMACOKINETICS STUDY OF ETHYLENE DICHLORIDE
November 22, 1996
Document Control No. OPPTS-42187B, FRL-4869-1
Prepared by: $ ChemRisk --A Division of McLaren/Hart Courtland East Building 29225 Chagrin Boulevard Cleveland, OH 44122
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PROPOSAL FOR PHARMACOKINETICS STUDY OF ETHYLENE DICHLORIDE 1.0 INTRODUCTION
On June 26, 1996, the USEPA proposed a test rule under Section 4(a) of the Toxic Substances Control Act (TSCA) which would require manufacturers and processors of 21 hazardous air pollutants, including ethylene dichloride (EDC), to test these compounds for specific health effects. The tests proposed to be conducted for EDC via inhalation exposures include the following:
acute toxicity, subchronic toxicity, developmental toxicity, reproductive toxicity, and neurotoxicity.
The cost for conducting these tests was estimated by the USEPA to be approximately $2 million. As an alternative to conducting these tests, the USEPA is soliciting proposals regarding the use of pharmacokinetic studies which would permit the extrapolation of toxicity information from other routes of exposure (i.e., oral) to predict risks from inhalation exposures. This document serves as a proposal to conduct pharmacokinetic studies for EDC as a means of filling specific data gaps.
Prior to extrapolating information from one route to another, an evaluation of available information must be made with respect to its adequacy for route-to-route extrapolation. A decision tree for
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conducting route-to-route extrapolations has been proposed (Gerrity and Henry, 1990), and is provided in Figure 1. Following an evaluation of the information available, there are four possible options that can be pursued:
Option 1: Collect data (all routes) Option 2: Use data for a structurally analogous compound Option 3: Collect data for relevant routes (inhalation) Option 4: Route-to-route extrapolation
Route-to-route extrapolation may be conducted using various levels of complexity ranging from the use ofdefault absorption values to physiologically based pharmacokinetics (PBPK) modeling. The approach proposed here centers on the use of a validated PBPK model.
Three general requirements are necessary to conduct proper route-to-route extrapolations using physiologically based pharmacokinetic (PBPK) models:
1. A scientifically plausible (defensible) mechanism of toxicological action is needed.
2. Studies must be available that are considered adequate to assess relevant toxicological endpoints via a route(s) of exposure other than that of interest.
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Page 3 3. A validated PBPK model for the chemical and species of interest must be available
which is capable of predicting the pertinent internal dose measures based on mechanism of action.
When these criteria exist for a compound that also has no toxicological effects on the portal of entry, a route-to-route extrapolation can be performed as follows:
1. The internal dose metric is determined under the experimental conditions for the studies to be extrapolated (i.e. oral) for the NOAEL and/or LOAEL doses.
2. Equivalent NOAEL and/or LOAEL concentrations for route extrapolated to (i.e. inhalation) are determined by estimating an exposure that produces the same internal dose metric as determined in Step 1 above.
For EDC, an evaluation was made of primary and secondary toxicological literature to determine the adequacy ofthe dose-response data for the effects listed above following inhalation, oral, and other relevant exposures. Limited information from inhalation studies for EDC in animals suggests that effects remote from the respiratory tract (i.e., liver, kidney) may be of potential concern. For oral exposures, potential candidate studies were located for route-to-route extrapolation (Option 4) for acute toxicity, subchronic toxicity, neurotoxicity, and reproductive toxicity (only if necessary). A recent inhalation study that evaluated developmental effects appears to fill that data gap (Section 3 4)
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Page 4 The remainder of this proposal will address the status of each of these requirements with regard to EDC, and will describe the specific approaches we propose to use to fill certain data gaps identified in the test rule.
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2.0 MECHANISM OF ACTION
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EDC is metabolized to compounds [2-chloroacetaldehyde, S(2-chloroethyl)gIutathione] that are capable of binding covalently to macromolecules (Fabricant and Chalmers, 1980; Jean and Reed, 1989). EDC has also been shown to promote lipid peroxidation (ATSDR, 1994). Hepatic DNA damage in mice was unaffected by a cytochrome P-450 inhibitor (piperonyl butoxide), but was diminished by the addition of a glutathione depleting agent (diethyl maleate) (Storer and Conolly, 1985). It has been suggested that EDC-induced toxicity occurs when the biotransformation processes (i.e., cytochrome P-450) become saturated, thereby allowing for higher levels of EDC to circulate throughout the body and conjugate with glutathione (activation), instead of being detoxified and eliminated (D'Souza et al. 1987; Reitz et al. 1982). This mechanism of action is also supported by studies on ethylene dibromide (White et al. 1983). It would appear that much of the toxicity associated with EDC exposure could be associated with the formation of glutathione metabolites.
Binding to key cellular molecules by glutathione metabolites of EDC is offered as a potential
mechanism of action for the acute toxicity and subchronic toxicity of EDC. Information regarding
the potential mechanism(s) of action for EDC-induced neurotoxicity were not located. However, the
rapid onset of the anesthetic effects of chlorinated solvents generally precludes the involvement of
a metabolite in the mechanism of action (Cassaret and Doull, 1996), and suggests that certain
neurological effects of EDC and other solvents are likely associated with the parent compound.
Necrosis of the nervous system, however, might be more appropriately associated with glutathione
metabolites of EDC.
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3.0 EVALUATION OF CANDIDATE STUDIES FOR ROUTE-TO-ROUTE EXTRAPOLATION
3.1 Potential for Direct Contact Effects
An important factor to consider prior to conducting a route-to-route extrapolation is the potential i
for direct contact (portal of entry) effects on the lung following inhalation exposures. In general, the
USEPA has classified inhaled chemicals into one of three categories based on water solubility and
reactivity (USEPA, 1994):
Category 1: Do not penetrate to blood (i.e., highly water soluble/very reactive) Category 2: Water soluble/blood accumulation Category 3: Water insoluble/perfusion limited
Portal of entry effects do not appear to be of primary concern for EDC based on the following rationale:
Limited information indicates that effects on the lungs are only of potential concern following acute exposures to very high concentrations in air. Pulmonary congestion was observed in mice, rats, rabbits, and guinea pigs exposed to lethal concentrations ofEDC (3,000 ppm) for 7 hours (Heppel et al. 1945). This effect was not observed in animals exposed to lower concentrations. In addition, pathological changes of the
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lungs were not observed in animals exposed to EDC via inhalation for longer periods (Heppel et al. 1946; Cheever et al. 1990).
Preferential accumulation of EDC in the lungs following inhalation exposures does not appear to occur. Rather, EDC rapidly distributes to other tissues following exposure. For example, following inhalation exposure to 50-250 ppm EDC for 23 hours, the levels of EDC in the lungs were lower than those observed in the blood and much lower than those observed in adipose tissue (Spreafico et al. 1980). The distribution pattern for EDC is very similar for both inhalation and oral exposures (Spreafico et al. 1980).
EDC is described as having a pleasant, sweet odor with an odor threshold of 50100 ppm (ATSDR, 1994), therefore irritation of the respiratory tract from air concentrations at or below these levels is unlikely.
Based on the low reactivity of EDC in the lung (see above), and the fact that EDC has low water solubility (see saline:air partition coefficient, Gargas et al., 1989), this compound should be considered a Category 3 compound (USEPA, 1994) in which direct effects on the portal of entry are not to be expected.
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3.2 Acute Toxicity
A single study was identified as a potential candidate for route-to-route extrapolation for acute toxicity, Daniel et al. (1994). This study is summarized briefly below.
Daniel et al. (1994) - Groups of 10 male and 10 female Sprague-Dawley rats were exposed to 0, 10, 30, 100, or 300 mg/kg-day HDC via com oil gavage for 10 days. Significant mortality (10/10 females, 8/10 males) was noted at the highest dose. Body weight, clinical chemistry, and hematological findings in exposed animals were not significantly different from controls. The main histopathological change noted was inflammation of the forestomach in animals receiving 100 mg/kg-day or more. This endpoint represents a direct contact effect ofEDC, which is attributable in part to the mode of administration (i.e., high concentrations administered as a bolus dose in com oil). Since this effect is not extrapolatable to inhalation exposures, this study identifies a NOAEL of 100 mg/kg-day for histopathological changes in the liver and kidney.
This study is compared with acute toxicity test guidelines from the USEPA (OPPTS) in Table 1.
Based on this comparison, this study was considered to be adequate for route-to-route extrapolation.
As discussed in Section 3.1, the potential for portal-of-entry effects for EDC on the lungs is low.
Based upon the mechanism of action proposed in Section 2.0, the glutathione metabolites of EDC are most likely responsible for any toxic effects. Therefore, it is proposed that some internal measure
ofthese metabolites (i.e., total amount metabolized by the glutathione pathway in a 24 hour period)
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3.3 Subchronic Toxicity
Three studies were identified as potential candidates for route-to-route extrapolation for subchronic toxicity, two by NTP (1991) and one by Daniel et al. (1994). These studies are summarized briefly below.
NTP (1991 - Drinking Water Study in Rats) * Groups of 10-20 male and 10 female F344/N, Osbome-Mendel, and Sprague-Dawley rats were exposed to drinking water containing 0, 500,1,000,2,000,4,000, or 8,000 ppm EDC for 13 weeks. The actual doses received by the animals varied slightly between species and sex, but were generally between 0, 49-82, 86-126, 147-213, 259-428, 515-727 mg/kg-day for the respective water concentrations. Mortality was not significantly affected by exposure in any strain or sex. The remaining findings from this study are discussed according to rat strain below.
F344/N - Water intake and body weights were affected in F344/N rats at the two highest doses. No compound-related clinical signs were noted. The authors attributed slight changes in hematological parameters to mild dehydration. Liver and kidney weights were elevated in all exposed animals compared to controls. Histopathological changes in the liver were not observed While mild renal tubular
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regeneration was comparable to controls in exposed male rats, the incidence of this
effect was increased in a dose-dependent manner in female rats exposed to 1,000 ppm
or more.
Sprague-Dawley - Water intake and body weights were affected in Sprague-Dawley rats at the two highest doses. No compound-related clinical signs were noted. The authors attributed slight changes in hematological parameters to mild dehydration. Liver and kidney weights were elevated in all exposed animals compared to controls. Histopathological changes in the liver were not observed. The incidence of mild renal tubular regeneration was comparable to controls in exposed male and female rats.
Osbome-Mendel - Water intake and body weights were affected in Osbome-Mendel rats exposed to 1,000 ppm (females) or 2,000 ppm (males) or more. No compoundrelated clinical signs were noted. The authors attributed slight changes in hematological parameters to mild dehydration. Kidney weights were elevated in all dosed females. Liver weights were elevated in exposed males receiving 1,0002,000 ppm. Histopathological changes in the liver were not observed. The incidence of mild renal tubular regeneration was not significantly different from controls in exposed male and female rats.
This study identifies a NOAEL and LOAEL of 58 and 102 mg/kg-day, respectively for the effects of EDC on the kidney in F344/N rats
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_____________________ Page 11 NTP (1991 ~ Com Oil Gavage Study in Rats) - Groups of 10-20 male and 10 female F344/N rats were exposed to 0, 30, 60, 120, 240, or 480 mg/kg-day (males), or 0, 18, 37, 75, 150, or 300 mg/kg-day (females) EDC via com oil gavage for 13 weeks. Significant mortality was noted in male rats (all) receiving 240 mg/kg-day or more, and in female rats (9/10) exposed to 300 mg/kg-day. Body weights were decreased in rats exposed to the highest dose. Liver and kidney weights were elevated in all exposed animals, however histopathological changes in these tissues were not observed. Forestomach effects (hyperplasia, inflammation, and mineralization) were noted in animals that died or were moribund. Necrosis of the thymus was observed in males exposed to 240 mg/kg or more, and in females exposed to 300 mg/kg. The incidence of renal tubular regeneration was comparable between exposed and control animals. Histopathological changes in the liver were not observed. Neurological effects were also observed in animals exposed to the highest doses of EDC (see Section 3.3). This study identifies a LOAEL of 240 mg/kg-day and a NOAEL of 120 mg/kg-day for necrosis of the thymus.
NTP (1991 - Drinking Water Study in Mice) - Groups of 10 male and 10 female B6C3F1
mice were exposed to drinking water containing 0, 500, 1,000, 2,000, 4,000, or 8,000 ppm
EDC for 13 weeks. These concentrations corresponded to doses of 0, 249, 448, 781, 2,710,
or 4,207 mg/kg-day (males) and 0, 244, 647, 1,182, 2,478, or 4,926 mg/kg-day (females).
Significant mortality (9/10) limits the interpretation of results from female mice exposed to
the highest dose. All other exposed animals survived the full 13 week exposure. Compound-
related clinical signs were not observed in any dose group. Body weights were lower in all
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exposed males, and in females exposed to 1,000 ppm or more. Liver and kidney weights
were elevated in all exposed animals. In addition, mild-to-moderate tubular regeneration was
noted in males exposed to the two highest doses. Histopathological changes in the liver were
not observed. This study identifies a NOAEL and LOAEL of 781 and 2,710 mg/kgj-day for
renal effects in mice.
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Daniel etal. (1994) - Groups of 10 male and 10 female Sprague-Dawley rats were exposed to 0, 37.5, 75, and 150 mg/kg-day EDC via com oil gavage for 90 days. No treatmentrelated effects were noted regarding mortality, clinical observations, ophthalmology, gross pathology, or histopathology in exposed animals. Body weight gain and food consumption I were significantly decreased in male rats exposed to the highest dose. Statistically significant differences in hemoglobin, hematocrit, red blood cell count, platelets, albumin, and alkaline phosphatase were noted in animals exposed to 75 mg/kg-day or more. Organ weight changes (liver, kidney, brain) were also noted in animals exposed to the two highest doses. This study identifies a LOAEL 75 mg/kg-day and NOAEL of 37.5 mg/kg-day for hematological effects
and organ weight changes.
These studies are compared with toxicity test guidelines from the USEPA (OPPTS) in Table 2. Based on this comparison, all of these studies were considered adequate for route-to-route extrapolation. It is proposed that the NTP (1991) studies using F344/N rats (drinking water and oil gavage) and B6C3F1 mice (drinking water) be used for extrapolation along with the oil gavage study of Daniel et al. (1994) using Sprague Dawley rats. As discussed in Section 3.1 the potential for
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portal-of-entry effects of EDC on the lungs is low. Based upon the mechanism of action proposed in Section 2.0, the glutathione metabolites of EDC are most likely responsible for any toxic effects. Therefore, it is proposed that some internal measure of these metabolites (/.., total amount metabolized by the glutathione pathway in a 24 hour period) be modeled as the appropriate internal dose measure. Equivalent inhalation exposures may be determined, as appropriate, from the NOAELs and LOAELs of the 4 test groups identified above.
3.4 Developmental Toxicity
The USEPA identified developmental toxicity as a data gap for EDC based on an evaluation of the literature available in August, 1995 (USEPA, 1995). However, since the time of that evaluation an inhalation developmental study of EDC has been published. This study is summarized below.
Payan et al. (1995) - Groups of 26 pregnant Sprague-Dawley rats were exposed to either 0, 150, 200, 250, or 300 ppm EDC on days 6 through 21 of gestation. Maternal toxicity (decreased maternal body weight gain, and 2 deaths) was noted in animals exposed to the highest concentration. No treatment-related effects were noted on the number of implantations, resorptions, live fetuses, fetal sex ratio, fetal weight, or malformation incidence. Although the pregnancy rate was significantly lower in animals exposed to 250 ppm, this effect was not observed in animals exposed to the highest dose, and therefore
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Pag was not considered to be treatment related. This study identifies a LOAEL and NOAEL of 300 and 250 ppm, respectively, for maternal toxicity.
This study was compared to USEPA (OPPTS) guidelines for developmental toxicity tests in Table 3. Taken with the results of the rabbit and rat studies (Shell Oil 1979 study also cited as Rao et al. 1980, Schlacter et al. 1979; and Murray et al. 1980), reviewed in the support document for the proposed test rule (USEPA, 1995), this confirms that EDC is neither a teratogen nor a developmental toxicant. This study is also summarized in Table 3.
Lane et al. (1982) have conducted an oral developmental study as summarized below.
Lane et al. (1982) - Groups of 10-18 pregnant female Swiss ICR mice from FI and F2 litters from a multigenerational study were exposed to 0, 5, 15, or 50 mg/kg/day EDC via the drinking water on days 1 through 21 of gestation. No treatment-related effects were noted on pup survival or terata in either groups. This study identifies a NOAEL of 50 mg/kg-day EDC for developmental effects.
This study was compared to USEPA (OPPTS) guidelines for developmental toxicity tests (Table 3). The only potential limitation ofthis study is perhaps in the dose selection since an effect level was not demonstrated. Based on the negative results overall in these inhalation and oral developmental studies, developmental toxicity should not be considered as a data gap for EDC
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3.5 Reproductive Toxicity
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The USEPA identified reproductive toxicity as a data gap for EDC based on the limitations found in a single study (Rao et al 1980). Although the support document for the proposed rule cites the limitation in this study as "No-Observed-Adverse-EfFect Level (NOAEL) was identified", this statement is in error. Although a LOAEL was not identified, the study clearly identifies a NOAEL, below which reproductive effects were not observed. This study is summarized below.
Rao et al (1980, also cited as Murray et al. 1980, Schlacter et al 1979, and Shell Oil 1979) In a single generation study, groups of 20-30 male and 20-30 female rats were exposed via inhalation to 0, 25, 75, or 150 ppm EDC for 6 hours/day beginning 60 days prior to mating, and continuing through gestation (Fla and Fib). No treatment related effects were observed on fertility index, pup survival, gestation length, sex ratio, or organ weights in pups from either the Fla or Fib litters. This study identifies a NOAEL of 150 ppm for the reproductive effects of EDC.
This study is compared to USEPA (OPPTS) guidelines for reproductive toxicity in Table 4. The potential limitation of this study is that it was not a multigenerational study. Although this was not a multi-generation study, as recommended by the guidelines, it still is relevant, particularly since EDC is not a bioaccumulative chemical (bioconcentration factor = 2 ATSDR, 1994) and potential human exposures to EDC are expected to be much lower than those tested by Rao et al (1980).
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Pag Lane et al. (1982) conducted an extensive oral multigeneration study that is summarized below.
Lane et al. (1982) - In a multigeneration study, groups of 10 male and 30 female ICR Swiss mice were exposed to 0, 5, 15, or 50 mg/kg-day EDC via the drinking water. The F0 generation was exposed for 5 weeks prior to mating, whereas the FI generation was exposed to 11 weeks prior to mating. No treatment-related effects on fertility, gestation, terata, pup weight gain, pup survival, or dominant lethal mutations were observed. This study identifies a NOAEL of 50 mg/kg-day for reproductive effects.
In addition to the negative reproductive studies cited above, histopathological evaluation of reproductive tissues from a subchronic study revealed no effects in the mammary gland, ovary, testes, or uterus of rats exposed to 50 ppm EDC via inhalation for 2 years (Cheever et al. 1990). Similarly, histopathological effects of the testes, prostate, tunica vaginalis, uterus, mammary gland, and ovary were not observed in rats exposed orally to 47-95 mg/kg-day or in mice exposed orally to 97299 mg/kg-day for 78 weeks (NCI, 1978). Based on the weight of evidence from these studies, reproductive effects do not appear to be of primary concern for EDC, and would not appear to warrant further study, although the results of Lane et al. (1982) could be extrapolated to inhalation exposures with PBPK modeling.
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3.6 Neurotoxicity
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Two studies were identified as a potential candidates for route-to-route extrapolation, both of which were conducted by NTP (1991). These studies are summarized briefly below.
NTP (1991 - Drinking Water Study) - Groups of 10-20 male and 10 female F344/N, OsbomeMendel and Sprague-Dawley rats, and 10 male and 10 female B6C3F1 mice were exposed to drinking water containing 0, 500, 1,000, 2,000, 4,000, or 8,000 ppm EDC for 13 weeks. Significant mortality (9/10) limits the interpretation of results from female mice exposed to the highest dose. All other exposed animals survived the full 13 week exposure. No compound related clinical signs, changes in brain weight, or histological changes of the central nervous system (brain and spinal cord) were observed in any of the exposed animals. Although no LOAELs were identified, this study identifies a NOAEL of 8,000 ppm for EDC (approximately 492 mg/kg-day in rats and 4,207 mg/kg-day in mice) for neurological effects.*
* NTP (1991 - Com Oil Gavage Study in Pats) - Groups of 10-20 male and 10 female F344/N rats were exposed to 0, 30, 60, 120, 240, or 480 mg/kg-day (males), or 0, 18, 37, 75, 150, or 300 mg/kg-day (females) EDC via com oil gavage for 13 weeks. Significant mortality was noted in male rats (all) receiving 240 mg/kg-day or more, and in female rats (9/10) exposed to 300 mg/kg-day. Clinical signs, including tremors, salivation, emaciation, abnormal posture, ruffled fur, and dyspnea were noted in males exposed to 240 mg/kg-day and in females exposed to 300 mg/kg-day. In addition, necrosis of the cerebellum was observed in males
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exposed to 240 mg/kg or more, and in females exposed to 300 mg/kg. This study identifies
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a LOAEL of 240 mg/kg-day and a NOAEL of 120 mg/kg-day for clinical signs of neurotoxicity and necrosis of the cerebellum.
These studies are compared with toxicity test guidelines from the USEPA (OPPTS) in Table 5. Based on this comparison, these studies were considered adequate for route-to-route extrapolation.
The studies conducted by NTP clearly demonstrate a vehicle effect associated with EDC-induced neurotoxicity. In drinking water, no effects were observed in rats that received up to 492 mg/kg-day, or in mice that received up 4,207 mg/kg-day. On the other hand, significant signs of neurotoxicity
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were noted in rats receiving 240-300 mg/kg-day EDC via com oil gavage. Differences in the kinetics ofuptake ofEDC from the gastrointestinal tract will be explored as an initial means of resolving the observed differences in toxicity.
We propose that the parent chemical in the central nervous system is likely responsible for the effects described above (less necrosis ofthe cerebellum) and that the parent compound in the central nervous system (or proportionately in the blood) be modeled as the appropriate internal dose measure for these studies. For necrosis of the cerebellum, we propose that GSH metabolites are likely involved. Equivalent inhalation exposures will be derived for both of these studies.
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4.0 PBPK MODELS FOR EDC
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This section summarizes the toxicokinetics ofEDC and describes existing and validated PBPK models for this chemical in the rat and mouse.
4.1 Toxicokinetics of EDC
EDC is readily absorbed through the lungs and gastrointestinal tract following exposure (ATSDR, 1994). Absorption of EDC in the gastrointestinal tract is strongly affected by the vehicle in which it is administered. For example, Withey et al. (1983) noted that peak blood concentrations for EDC were four times higher and three times faster following administration in water than when administered in oil. Following absorption, EDC distributes to tissues throughout the body, with a higher preference for adipose tissue and tissues with a higher lipid content. Following oral and inhalation exposures, levels of EDC in liver and lung were lower than those in the blood (Spreafico et al. 1980). There is little difference between oral and inhalation exposure with respect to tissue distribution (ATSDR, 1994).
EDC undergoes biotransformation via two different pathways. EDC is metabolized by cytochrome P-450 to form chlorhydrin and 2-chloroacetaldehyde. Alternatively, EDC can react with glutathione to form S-(2-chloroethyl)glutathione and a glutathione episulfonium ion. Enzymes involved with EDC metabolism appear to become saturated following exposures of 25 mg/kg-day orally, 150 ppm via inhalation, or when blood levels reach 5-10 pg/mL (D'Souza et al 1988, Reitz et al 1982)
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Page 20 EDC is eliminated from the body primarily via urinary excretion and exhaled breath following either oral or inhalation exposures (Reitz et al. 1982; Spreafico et al. 1980).
4.2 Existing PBPK Model for EDC
A PBPK model has been developed for EDC in the rat and mouse (D'Souza et al. 1987). This is a flow-limited model which includes compartments for the lung, liver, richly perfused tissue (i.e., kidney, spleen), poorly perfused tissue {i.e., muscle, skin), and fat (Figure 2). Partition coefficients were available for EDC in Sprague-Dawley rats, F344 rats, and B6C3F1 mice. In addition, a blood:air partition coefficient was available for EDC in humans. The model accounts for the metabolism ofEDC by two competing pathways, (1) cytochrome P-450, and (2) glutathione (GSH). Metabolic rates for these two pathways have been determined by gas uptake measurements (Gargas et al. 1986). Metabolism via the former pathway is a saturable enzymatic oxidation, whereas metabolism via the latter pathway is essentially a first order reaction at low exposure concentrations. At higher exposures to EDC, a first order reaction between EDC and GSH did not adequately describe EDC metabolism and underestimated EDC concentrations, possibly due to a depletion of GSH. For this reason, GSH depletion (D'Souza et al. 1988) has also been incorporated into the model. The PBPK model for EDC has been validated in the rat and mouse by measuring EDC blood concentration and glutathione (GSH) time course concentrations in tissues following various exposures. These exposures included oral gavage doses of 75 and 150 mg/kg, and inhalation exposures to 150 ppm for 7 hours.
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__ Page 21 4.3 Proposed Model Refinement
Based on the toxicity endpoints observed for EDC following oral exposures (see Section 3.0), the following tissue compartments will need to be added to the existing PBPK model (Figure 2):
Kidney Thymus Central nervous system Reproductive tissues (if necessary) Embryo/fetus (if necessary)
For these compartments, the partition coefficients will be assumed to be the same as those reported for the liver since no organ-specific information were available. Blood flow and organ volumes will be obtained from available lab animal physiology literature. A summary of the proposed PBPK model for evaluating the results from oral studies discussed in Section 3.0 are provided in Table 6.
An interesting observation is that the toxicity of EDC by the oral route was affected by the vehicle in which it was administered (i.e., oil > water). There are several potential mechanisms by which an oil vehicle can impact the toxicity of EDC.
Absorption - An oil vehicle can change the rate or extent of EDC absorption from the gastrointestinal tract. In this case, absorption appears to occur more rapidly when
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___ Page 22 EDO is administered in water compared to administration in oil (Withey et al. 1983). However, exposure via oil gavage is usually as a bolus dose, whereas drinking water exposure comes from many episodes spread out through the course of the day. In addition, an oil vehicle may alter the manner in which EDC is absorbed from the gastrointestinal tract. Lipids, such as those present in oil, are absorbed from the gastrointestinal tract via a pathway other than the hepatic portal system. Specifically, lipids are absorbed via chylomicrons to the lymphatic system, where they are transported directly to the venous circulation via the thoracic duct (effectively bypassing first-pass metabolism in the liver). It is conceivable that a significant fraction of the EDC dose administered in oil is absorbed along with the lipids in this manner, however, the extent to which this may occur is not known.
Metabolism - It is possible that an oil vehicle can affect the metabolism of EDC via changes in cytochrome P-450 induction, reduced glutathione levels, or via changes in cellular levels of metabolic cofactors (i.e., NADPH, NADH, or 02). However, information regarding these potential impacts are not available.
Toxic Interaction Finally, it is also possible that a component of the oil interacts with EDC which in some way exacerbates toxicity.
The strong involvement of the thymus in the toxic action of EDC when administered in oil but not when administered in drinking water (NTP, 1991), suggests that the primary effect of the oil vehicle
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______________ ___________________________________ ________________ __________ Page 23 may be on absorption. Under the assumption that a fraction of the EDC dose administered in oil is absorbed via lymphatic uptake, organs in the thoracic region such as the thymus could receive a large fraction of the bolus dose prior to distribution to the rest of the body. It is anticipated that the differences in absorption kinetics between water and oil may partially explain these differences in toxic response and these kinetic differences will be quantitatively accounted for in the PBPK modeling.
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5.0 REFERENCES
ATSDR. 1994. Toxicological Profile for 1,2-dichloroethane. Agency for Toxic Substances and Disease Registry, Atlanta, GA.
Cassaret and Doull. 1996. Toxicology; The basic science of poisons. 5th Edition. New YcJrk, NY;
Macmillan Publishing Co.
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I Cheever KL, Cholakis JM, el-Hawari AM, et al. 1990. Ethylene dichloride; The influence of
disulfiram or ethanol on oncogenicity, metabolism, and DNA covalent binding in rats. Fundam Appl
Toxicol 14;243-261.
Daniel FB, Robinson M, Olson GR, et al. 1994. Ten and ninety-day toxicity studies of 1,2dichloroethane in Sprague-Dawley rats. Drug Chem Toxicol 17:463-477.
D'Souza RW, Francis WR, Bruce RD, et al. 1987. Physiologically based pharmacokinetic model for ethylene dichloride and its application in risk assessment. Drinking Water and Health. Washington, DC: National Research Council, 8:286-301.
i D'Souza RW, Francis WR, Bruce RD, et al. 1988. Physiological model for tissue glutathione depletion and increased resynthesis after ethylene dichloride exposure. J Pharm Exp Therap 345:563568.
Fabricant JD, Chalmers Jr JH. 1980. Evidence of the mutagenicity of ethylene dichloride and structurally related compounds. In: Ames BN, Infante P, Reitz R, eds. Ethylene dichloride: A potential health risk? Banbury report No. 5. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 309-329.
Gargas ML, Andersen ME, Clewell HJ, et al. 1986. A physiologically-based simulation approach for determining metabolic constants from gas uptake data. Toxicol Appl Pharmacol 86:341-352.
Gargas ML, Burgess RJ, Vorsand DJ, et al. 1989. Partition coefficients of low molecular weight volatile chemicals in various liquids and tissues. Toxicol Appl Pharmacol 98:87-99.
Gemty TR, Henry CJ. 1990. Principals of route-to-route extrapolation for risk assessment. New York, NY; Elsevier, p. 3-12.
Heppel LA, Neal PA, Perrin TL, et al. 1945. The toxicology of 1,2-dichloroethane (ethylene): m. Its acute toxicity and the effect of protective agents. J Pharmacol Exp Ther 84:53-63.
Heppel LA, Neal PA, Perrin TL, et al. 1946. The toxicology of 1,2-dichloroethane (ethylene dichloride) . V. The effects of daily inhalations. J Ind Hyg Toxicol 28.113-120.
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November 22. 1996
Page 25
Jean PA, Reed DJ. 1989. In Vitro dipeptide, nucleoside, and glutathione alkylation by S-(2chloroethyl) glutathione and S-(2-chloroethyl)-L-cysteine. Chem Res Toxicol 2:455-460.
Lane RW, Riddle BL, Borzelleca JF. 1982. Effects of 1,2-dichloroethane and 1,1,1-trichloroethane in drinking water on reproduction and development in mice. Toxicol Appl Pharmacol 63:409-421.
NCI. 1978. Bioassay oftechnical grade 1,2-dichloroethane for possible carcinogenicity. Bethesda, MD: National Cancer Institute. NCI-CG-TR 55.
NTP. 1991. Toxicity studies of 1,2-dichloroethane (ethylene-dichloride) (CAS No. 107-06-2) in F344/N rats, Sprague Dawley rats, Osbome-Mendell rats, and B6C3F1 mice (drinking water and gavage studies). Research Triangle Park, NC: U.S. Department of Health and Human Services, Public Health Service, National Institute ofHealth, National Toxicology Program. NIH Publication No. 91-3123.
Payan JP, Saillenfait AM, Bonnet P, et al. 1995. Assessment of the developmental toxicity and placental transfer of 1,2-dichloroethane in rats. Fund Appl Toxicol 28:187-198.
Rao KS, Murray JS, Deacon MM, et al. 1980. Teratogenicity and reproduction studies in animals inhaling ethylene dichloride.
Reitz RH, Fox TR, Ramsey JC, et al. 1982. Pharmacokinetics and macromolecular interactions of ethylene dichloride in rats after inhalation or gavage. Toxicol Appl Pharmacol 62:190-204.
Spreafico F, Zuccato E, Marcucci F, et al. 1980. Pharmacokinetics of ethylene dichloride in rats treated by different routes and its long-term inhalatory toxicity. In: Ames BN, Infante P, Reitz R, eds. Ethylene dichloride: A potential health risk? Banbury report No. 5. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory, 107-133.
Storer RD, Conolly RB. 1985. An investigation of the role of microsomal oxidative metabolism in the in vivo genotoxicity of 1,2-dichloroethane. Toxicol Appl Pharmacol 77:36-46.
USEPA- 1995. Summary oftables on the health effects data dor hazardous air pollutants (HAPs) Group 1.
USEPA. 1994. Methods for derivation of inhalation reference concentrations and application of inhaled dosimetry. Office of Research and Development, Washington, DC. EPA/A600/8-90/066F.
White RD, Gandolfi AJ, Bowden GT, et al. 1983. Deuterium isotope effect on the metabolism and toxicity of 1,2-dibromoethane. Toxicol Appl Pharmacol 69:170-178.
Withey JR, Collins BT, Collins PG 1983 Effect of vehicle on the pharmacokinetics and uptake of four halogenated hydrocarbons from the gastrointestinal tract of the rat. J Appl Toxicol 3 249-253
Nhapsrule.outline.edc
DO 1
B
CONFTDFNTTA1
November 22,1996
Figure 1 Decision Tree for Route-to-Route Extrapolation
Ethylene Dichloride*
OO TZJ O o '0
ZV
-f --
Ethylene Dichloride
Do adequate toxicological data existfor at least one route?
Yes Is toxicity remote from contact site likely?
No Yes
Option 1: Collect data
Option 2: Use data for analogous compound
* Adapted front Gerrity and Henry (1990)
Option 3: Collect data on alt relevant routes
T
Option 4: Candidate
for route-to-route
extrapolation
n:\hapsrule\EDC-TABS.XLS
11/19/96 12:57 PM
Figure 2 Comparison of Current and Proposed PBPK Models for Ethylene Dichloride
Inhaled
Venous emulation
Current Model8
r Alveolor Space Lung Blood Metabolism
P4J0 -^OSII
Liver Metabolism
P450 *-
Richly Perfused Tissue
Exhaled
Oral Dose Arterial Circulation
Poorly Perfused Tissue
Inhaled
Proposed Model11
Alveolor Space > Lung Blood
> Metabolism
P4S0
,, GSII
> Exhaled
>
Thymus
Venom
Circulation
Liver
Metabolism
^<
P450
-^GSH
't
Central Nervous System
Oral Dose
Kidney
Arterial Circulation
n o Cf zo
T1 --k --1
Z> \> vO
Z--1 V
-t o
3>
Richly Perfused
Tissue
^ .. --
-s
<----- ---- ---- --------%
Poorly Perfused
Tissue
>Fat
*Ad.i|nc>l limit ll'Smua el al. (1987) " I Ik- mmId .mild Ite expanded further for Uevelopnienial and reproductive effects if USEPA does not feel that available inhalation studies are sufficient.
h^ zo -1 ^ o^ z-4 'J
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Table 1 Comparison of Candidate Studies to OPPTS Test Guidelines for Acute
Toxicity
Candidate Study for Route Extrapolation
Study: Daniel et al. (1994) Subchronic effects observed: Lack of histopathological effects
LOAEL: None NOAEL: 100
Parameter_______________________ Test species Strain Age Sex Health Status Number of animals Control Groups Concentration level and selection Limit dose i-hr study 8-hr study Exposure environmental conditions Exposure periodicity
Physical measurements Observation period Gross pathology Histopalhology Uronchoalveolar lavage Equipment and test methods reporting Results reporting
Recommended Rat or mouse F344 or B6C3FI Young adult Both Evaluate initially 5 /sex/dose Concurrent sham or vehicle control > =4 (including control) MTD or 5 mg/L If triggered, > =3 If triggered, > =3 Nose only or whole body
'22"C; 40 - 60% humidity 4 hr (1 & 8hr, if triggered) Environmental conditions monitored 24 hr Full necropsy, organ weights Histopalhology, including respiratory tract Provide indicators of lung damage Adequately defined Tabular results, per animal, statistics
Rat Sprague-Dawley Young adult Both Adequate 10/sex/dose Vehicle control 0, 10, 30, 100, 300 mg/kg-day Adequate Not applicable Not applicable Oral (oil gavage) Adequate Ix/d, 10 d Adequate todays Adequate Adequate Not evaluated Adequate Adequate
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Table 2 Comparison of Candidate Studies to OPPTS Test Guidelines for Subchronic Toxicity
Study: NTP (1991) - Drinking water study in rats
Subchronic effects observed: Mild renal tubular
regeneration, increased kidney LOAEL: 102 NOAEL: 58
Candidate Studies for Route Extraonlalion NTP (t99l) - Corn uil gavage NTP (1991) - Drinking water
study in rats
study in mice
Necrosis uf the thymus
MiId-to-moderate renal tubular
regeneration 240 2710
120 781
Daniel ct nl (1994)
Liver uud kidney weight changes, hematological effects 75 37.5
Hum meter
1 cj.1 species
Slldlll
St s Nuinliti nl annuals Jlusliamlry < uiiUiil (J| mj|b ' inn ctiiiuiimi level uml selection
Recommended Rat (other mammal) Common lab
Young, healthy Both 10/sex/duse, more if'interim sacrifice Standard Concurrent sham or vehicle control > =4 (including control)
1 NHlt tli/iC
Maximum tulenible dose achieved
Intel mediate dose
Provides gradtation uf effects
Lowest dose level
Should provide NOAEL
Adiuinisiiuliuii of the substance
6 hr/day, 7 day/week
Observation period
90 days
Exposure spec iflent ions ujttl physical Nose only or whole body
measure incuts
CHrservul u>n of animals
Murtatily (daily), clinical (weekly)
Cliiiical pnlfmlii^y
Hematology and clinical chemistry
Oplitlmlmutogtitit examinutioas
High-dose and control
Rat
Rat
Mouse
Rat
F344/N, Sprague-Dawley,
F344/N
B6C3F1
Sprague-Da wley
Osborne Mendel
Young, healthy
Young, healthy
Young, healthy
Young, healthy
Both Both Both Bmh
10-20/sex/dose
10/sex/duse
10/sex/dose
10/sex/duse
Adequate
Adequate
Adequate
Adequate
Uni rented
Vehicle
Untreated
Vehicle
0,49-82, 86-126, 147-213,259 0, 30,60, 120, 240, 480 mg/kg 0, 249,448, 781.2710, 4207 0, 37.5, 75, 150 mg/kg-day
428, 515-727 mg/kg-day
day (males); 0, 18, 37, 75, mg/kg-duy (inales); 0, 244,
150, 300 mg/kg-day (females) 647, 1182, 2478, 4926 mg/kg-
Adequate
Adequate
day (females) Adequate
Adequate
Adequate
Adequate
Adequate
Adequate
Adequate
Adequate
Adequate
Adequate
Daily
Daily
Daily
Daily
13 weeks
13 weeks
13 weeks
90 days
Oral (drinking water)
Oral (oil gavage)
Oral (drinking water)
Oral (oil gavage)
Adequate
Adequate
Adequate
Adequate
Adequate
Adequate
Adequate
Adequate
Necropsy and histology of eyes Necropsy and histology of eyes Necropsy and histology of eyes
Gross [ititliiili^) HisiopttilHilo^) Kesulls repot
Evaluation
^ ba ^
-Zn> '-a-} 2 '0 --< '0 3-<> \)
Gross necropsy and organ weights
Adequate
Histopalliology, including respiralory tract Adequate
Tubular results, statistics
Tabular means, standard
deviations, incidences
Adequately described
Adequate
Adequate Adequate Tabular means, standard
deviations, incidences . Adequate
Adequate Adequate Tabular means, standard deviations, incidences Adequate
Adequate Adei| units
Table 3 Comparison of Studies to OPPTS Guidelines for Developmental Toxicity
Payan el al. (1995)
Effect: Maternal toxicity LOAEL: 329 ppm N0AEL: 254 ppm
Existing Inhalation Studies
Raoet al. (1980)' Maternal toxicity 300 ppm 100 ppm
Raoet al. (1980)* Maternal toxicity 300 ppm 100 ppm
Candidate for Route Extrapolation Lane et al. 1982 None None 50 mg/kg-day
Parameter l est Species Strain Sex Kuule Number of animals
< 'unlrol group t lii tcenl rations
Recommended >2 Common laboratory Pregnant female Inhalation > 20/group (rats); > 12/group (rabbits) Filtered air/vehicle >4 (including control)
1 lose Sclelioi)
Characterizes dose response
Exposure Duration Observation Period bin v i run men talCond i t i ons Observation Gross Necropsy Data Reporting Evaluation
6 hr/d Gd 6-15 (rat); Gd6-I8 (rabbit) Standard/monitored Daily Uterus/embryo Tabular/detailed Stat istics/desc ribed
Rat Sprague-Dawley Pregnant female Inhalation 26/group
Rat Sprague- Dawley Pregnant female Inhalation 16-30
Rabbit New Zealand White Pregnant female Inhalation 19-21
Filtered air 0, 150, 194, 254, 329 ppm Adequate
6 hr/d Gd 6-20 Adequate Adequate Adequate Adequate Adequate
Filtered air 0, 100, 300 ppm
Significant toxicity at high dose 7 hr/d Gd 6-20 Adequate Adequate Adequate Adequate Adequate
Filtered air 0, 100, 300 ppm
Adequate
7 hr/d Gd 6-18 Adequate Adequate Adequate Adequate Adequate
Mouse Swiss Pregnant female Oral (drinking water) 10-18
U nt rested/Ve h ic le 0, 5, 15, 50 mg/kg-day
No toxicity observed in dose range Daily Gd 1-21 Adequate Adequate Adequate Adequate Adequate
'litis siiiily is also cited as Murray et al. (1980); Shell Oil (1979); Schlacler et al. (1979)
EDC-TABS XLS
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11/19/96 12:57 PM
Table 4 Comparison of Candidate Studies to OPPTS Guidelines for Reproductive Toxicity
_________ Existing Inhalation Study Rao et al. 1980 Effect: None LOAEL: None NOAEL: ISO ppm
Candidate for Route Extrapolation Lane et al. 1982 None None SO mg/kg-day
Parameter____________ Recommended
Species
Rat (other mammal)
Strain
Common laboratoiy
Age Young adult
Number of animals
>20 pregnant females
Doses Route
>4 (including control) Inhalation
Exposure Frequency 7 d/wk
Design
Multigenerational; exposure > 10 wk prior to
Mating Control
mating Random Untreated/vehicle
Observations (adult) Daily/weekly
Observations (litter) PNDO, 4, 7, 14,21
Endpoints
Fertility index, gestation index, sperm
morphology, age at vaginal opening or
preputial separation, gross necropsy, organ
Data reporting
weight, histopathology Tabular
Evaluation
Statistics described
Rat Sprague-Dawley Adequate 20-30/group 0, 25, 75, 150 ppm Inhalation 6 hr/d 5-7 d/wk Single generation; F0 exposed 60 d prior to mating Random Untreated Daily PND 1, 7, 14, 21 Fertility index, gestation index, gross necropsy, terata, limited histopathology
Adequate Adequate
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Mouse Swiss Adequate I0M 30F/group 0, 5, 15, SO mg/kg-day Oral (drinking water) 7 d/wk Multigenerational; Ft exposed 11 wk prior to mating Random Un t rested/veh ic le Weekly PNDO, 4, 7, 14,21 Fertility index, gestation index, gross necropsy, tereta, dominant lethal mutation
Adequate Adequate
EDC-TABS XLS
11/19/96 12:57 PM
Table 5 Comparison of Candidate Studies to OPPTS Test Guidelines for Neurotoxicity
Study Neurological Effects Observed LOAEL NOA EL
Candidate Studies for Route Extrapolation
NTP 1991(drinking water study) NTP 1991 (oil gavage study)
None
Tremors, necrosisof the cerebellum
NA 240 mg/kg-day
492 mg/kg-day
120 mg/kg-day
Parameter Test species Strain
Recommended Rat (mice or dog) Common
Age Sex Number of animals Control Groups Concentration level and selection
Young adult (> 42 d) Both 10/sex/dose Concurrent sham or vehicle control > = 4 (including control)
Dose selection (acute)
MTD; < 2g/kg
Dose selection (subchronic) MTD; < I g/kg
Administration of the subslan Appropriate route of exposure
Combined protocol
Combine with other endpoints
Time of testing
Acute (0, 8hr, 7d, 14 d); subchronic
(0, 4 wk, 8 wk, 13 wk)
Functional observational batt Standard evaluations for appearance,
behavior, and functional integrity
List of measures
Autonomic (unction, abnormal motor
movements, response to general and
sensory stimuli, alertness, grip
strength, landing foot splay, body
Molt ir activity
weight, behavioral changes Individually assessed, automated
Neuropathology
Neuropathological examinations
Kesulls
Tabular, per animal, statistics
(-.valuation
Adequately described
Rat Rat
F344/N, Sprague-Dawley, Osborne F344/N
Mendel
42 d 42 d
Both
Both
10-20/sex/dose
10-20/sex/dose
Vehicle control
Vehicle control
0, 49-82, 86-126, 147-213, 259- Male (0, 30, 60, 120, 240, or 480
428, 515-727 mg/kg-day
mg/kg-day); Female (0, 18, 37, 75,
150, or 300 mg/kg-day) NA NA
Adequate
Adequate
Oral
Oral
NA NA
90 days
90 days
Clinical signs evaluated weekly Clinical signs evaluated weekly
Abnormal body movements (tremor), body weight
Abnormal body movements (tremor), body weight
Not evaluated Brain, sciatic (if signs present) Incidence, mean & SD, Dunn's or Shirley's tests Adequate
Not evaluated Brain, sciatic (if signs present) Incidence, mean Sc SD, Dunn's or Shirley's tests Adequate
Table 6 Summary of Pharmacokinetic Modeling Activities Proposed for Ethylene
Dichloride
Study Species Route Eudpoint
LOAEL NOAEL Dose Metric
Data Gap
Acute
Subchronic
Daniel et al. NTP (1991) NTP (1991) NTP (1991) Daniel et al.
(1994) Rat
Oral (oil
Rat Oral (drinking
Rat Oral (oil
Mouse Oral (drinking
(1994) Rat
Oral (oil
Ravage) Lack of histopathologic
water) Mild renal
tubular
ttavaRe)
water)
Ravage)
Necrosis of the Mild-to-
Liver and
thymus
moderate renal kidney weight
al effects regeneration,
tubular
changes,
increased
regeneration hematological
None
kidney weight
effects
102 mg/kg-day 240 mg/kg-day 2710 mg/kg- 75 mg/kg-day
dav 100 mg/kg-day 58 mg/kg-day 120 mg/kg-day 781 mg/kg-day 37.5 mg/kg-
Total
Total
Total
Total
dav Total
metabolized metabolized metabolized metabolized metabolized
(glutathione) (glutathione) (glutathione) (glutathione) (glutathione)
Target Tissue Liver/kidney
Kidney
Thymus
Kidney
Liver/kidney
Neurotoxicity NTP (1991) NTP (1991)
Rat Rat
Oral (drinking Oral (oil
water) None
eavaee) Necrosis of the
brain, clinical
signs
102 mg/kg-day 240 mg/kg-day
58 mg/kg-day 120 mg/kg-day
EDC and/or total
metabolized (glutathione)
Kidney
EDC and/or total
metabolized (glutathione)
Central nervous system
ino ?9p?
C O N F ID E N T
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