~ 2 - ~ ~~~ ~i 

Task Force 

BY HAND 

6PPl-o2or3-ooi0 

P .O. Box 331, Millwood, -'k 2'2t4B - (540)83?}1602 

July 26, 2010 

Document Control Office 

Office of Pollution Prevention and Toxics 

Room 6428 

U.S . Environmental Protection Agency 

1200 Pennsylvania Ave., N.W. 

Washington, DC 20460 

Re: Testina Consent Order for Ethylene Dichloride; 

Final Report (Docket No. OPPT-2003-0010 ) 

Dear Sirs : 

Enclosed in sextuplicate is a final report entitled "Route-to-Route Extrapolation of 1,2-

Dichloroethane Studies from the Oral Route to Inhalation Using Physiologically Based 

Pharmacokinetic Models: Subchronic Toxicity in the Rat," as required under the Enforceable 

Consent Agreement for Ethylene Dichloride, 68 Fed. Reg. 33125 (June 3, 2003) (the "ECA"). 

Please do not hesitate to contact me if there is any question about this report . 

Sincerely, 

~-- llt~i06~ ~

Peter E. Voytek, Ph.D. 

Manager 

Enclosures 

cc: John E. Schaeffer 

W. Caffey Norman, Esq. 

CONTAINS NO CBI 

5110073 
p. 2 

: 

~o1oooo'o o V13 

Route-to-Route Extrapolation of 1,2-Dichloroethane Studies 

from the Oral Route to Inhalation Using Physiologically 

Based Pharmacokinetic Models: 

Subchronic Toxicity in the Rat 

Prepared for 

RAP Task Force, Millwood, Virginia 

Prepared by 

Lisa M. Sweeney, Ph.D., DABT 

Ann Parker, B.S. 

Toxicology Excellence for Risk Assessment, Cincinnati, Ohio 

July 22, 2010 

CO"'" INS NO CBI 
EDC Subchronic Route-to-Route July 22, 2010 

Page 2 _ 

EXECUTIVE SUMMARY 

A need has been identified for a quantitative understanding of the hazards of 1,2-dichloroethane 

(ethylene dichloride, EDC, CAS No. 107-06-2) exposure by the inhalation route. Under Section 

4 of the Toxic Substances Control Act (TSCA), the U.S . EPA issued a testing consent order 

(Enforceable Consent Agreement, ECA) to perform toxicity testing and mechanistic and 

pharmacokinetic studies to satisfy EPA's data needs for EDC (U.S. EPA, 2003, 2009). As part 

of Tier I testing, pharmacokinetic studies were conducted (Saghir et al ., 2006) and a 

physiologically based pharmacokinetic (PBPK) model was developed that describes the 

disposition of EDC in rats by the oral and inhalation routes (Sweeney et al ., 2008). 

This report serves to fulfill the identified data need for route-to-route extrapolation to the 

inhalation route of results of a subchronic toxicity study previously conducted by the oral route. 

The route-to-route extrapolation of results of the 90-day study of EDC using the PBPK model 

was based on selection of critical endpoints of changes in hematocrit and liver and kidney weight 

changes, the most appropriate internal dose measures, and an appropriate point of departure. 

The available data indicated that the most appropriate dose metric for route to route extrapolation 

of hepatic effects is the minimum post-exposure concentration of glutathione in the liver. For 

extrahepatic effects, a health-protective extrapolation of internal dose based on the amount of 

extrahepatic EDC metabolism was used in route-to-route extrapolation . The specified points of' 

departure were the NOAEL and LOAEL of 37 .5 and 75 mg/kg-day, respectively, which applied 

to both the hepatic and extrahepatic endpoints. Based on PBPK modeling results presented here, 

the rat NOAEL and LOAEL for extrahepatic effects in rats subchronically exposed to EDC by 

corn oil gavage are equivalent to continuous exposure of rats to 4.4 and 6.7 ppm EDC 

respectively, using extrahepatic metabolism as the dose metric that is equivalent in the gavage 

and inhalation scenarios . The hepatic effects, which are of uncertain biological significance, 

yielded higher rat NOAEL and LOAEL equivalents . Sensitivity analyses of the key internal 

dose metric at the oral and inhalation-equivalent NOAEL indicated that in both scenarios, the 

amount of extrahepatic metabolism was sensitive to the ventilation rate, but in opposite 

directions ; the scenarios were otherwise generally sensitive to different parameters. For gavage 

dosing, the simulated amount of extrahepatic metabolism was sensitive to the ventilation rate, 

blood:air and slowly perfused tissues :air partition coefficients and the capacity for extrahepatic 

metabolism. For inhalation exposure, the simulated amount of extrahepatic metabolism most 

sensitive to the ventilation rate and only moderately sensitive to any other physiological or 

biochemical parameters. 
EDC Subchronic Route-to-Route July 22, 2010 

Page - 

INTRODUCTION 

1,2-Dichloroethane (ethylene dichloride, EDC, CAS No . 107-06-2) is used as an intermediate in 

the production of vinyl chloride, trichloroethylene, vinylidene chloride, and l,1,1-

trichloroethane, which are chlorinated solvents used as ingredients in antiknock gasoline, paint 

solvents, coatings, adhesives, and a grain fumigant. A need has been identified for a quantitative 

understanding of the hazards of EDC exposure by the inhalation route. Under Section 4 of the 

Toxic Substances Control Act (TSCA), the U.S. EPA issued a testing consent order (Enforceable 

Consent Agreement, ECA) to perform toxicity testing and mechanistic and pharmacokinetic 

studies to satisfy EPA's data needs for EDC (U.S. EPA, 2003) ; this consent order was 

subsequently modified with respect to timing (U.S. EPA, 2009). 

Summary of Physioloizicallv Based Pharmacokinetic (PBPKMod_el 

A combination of experimental studies (D'Souza et al ., 1987, 1988; Gargas et al . 1986; 

Spreafico et al., 1980; Payan et al., 1995 ; Reitz et al., 1982 ; Withey and Karpinksi, 1985 ; Saghir 

et al ., 2006) and modeling (Sweeney et al., 2008) was used to develop a physiologically based 

pharmacokinetic (PBPK) model that would allow route-to-route extrapolation from existing and 

future data on hazards of oral EDC exposure to assess inhalation hazards . 

The Sweeney et al . (2008) model is an expansion and refinement of previous PBPK models 

(D'Souza et al., 1987, 1988 ; Staats et al., 1991 ; ENVIRON 2004) for disposition of EDC in male 

F344 rats based on EDC kinetics data following oral gavage exposure. Sprague-Dawley rats 

receiving intravenous (iv) doses of EDC (Spreafico et al., 1980) rapidly cleared EDC from the 

blood. However, the D'Souza et al. (1987, 1988) model structure was not able to replicate the 

clearance (even when applying a large rate of metabolism in the liver), suggesting that EDC also 

undergoes extrahepatic metabolism. EDC studies (Spreafico et al ., 1980; Saghir et al., 2006) 

indicate that pulmonary metabolism is not significant as evidenced by the lack of GSH depletion 

in the lung; thus lung extrahepatic metabolism is not needed, although it was included in the 

D'Souza et al. (1987, 1988) model. Oral absorption of EDC was initially described using a 

simple first-order description (D'Souza et al ., 1987, 1988) . However, when uptake from the GI 

tract was measured in rats dosed with radiolabeled EDC, it was found that approximately 50% of 

the administered radiolabeled EDC dose remained in the GI tract at 1 hr post dosing, but only an 

additional 15% was absorbed in the following 3 hrs, indicating bi-phasic absorption (Payan et 

al., 1995). The expanded model (Sweeney et al ., 2008) included a two compartment GI tract 

where EDC was rapidly absorbed from the first compartment then sent to either the portal blood 

or to a second GI compartment where additional absorption occurs, and the extrahepatic 

metabolism was modeled as occurring in a venous blood "pool" fed by the slowly perfused 

tissues, kidney and other richly perfused tissues. Updated model parameters determined from 

previous models and literature were tested against the available data. An iterative approach was 

used to optimize the model parameters (hepatic metabolism, extrahepatic metabolism, exhalation 

rates, and absorption rates); the modified model was able to provide adequate estimates of GSHdependent metabolism. The revised model provided an adequate fit to all the rat data except 
EDC Subchronic Route-to-Route July 22, 2010 

Page 4-- - _ 

fetal tissue (a 3 .3-fold difference was found between modeled and predicted fetal tissue 

concentrations) . The model can be used to calculate specified dose metrics of interest (blood 

concentrations, tissue concentrations, or amounts metabolized) after inhalation or gavage (corn 

oil or water). Model code is provided in Appendix A. 

SUMMARY OF KEY STUDY 

Study Design 

A 90-day oral toxicity study of EDC administered by gavage in corn oil was conducted using 

male and female viral antibody-free Sprague-Dawley rats. Ten animals per sex served as vehicle 

controls . Sprague-Dawley rats (10/sex/dose) received 0 (vehicle only), 37.5, 75, or 150 mg/kgday, or vehicle only were administered to Each rat received a gavage volume of 0.1 ml per 100 g 

body weight calculated from weekly individual body weights (Daniel et al ., 1994) . 

Study Results 

No clinical signs of toxicity were seen during the 90-day study. Some changes were seen in 

hematology and chemistry analysis . Few gross and histological changes were found (single 

incidences) ; none of the changes were dose dependent. Male rats in the high-dose group (150 

mg/kg-day) had a significant decrease in food consumption (data not shown in Daniel et al ., 

1994) and body weight (17%) during the 90-day study compared to the controls . There were no 

significant differences in food consumption or weight gain patterns in the other treated rats 

compared to control rats. However, in a 10-day toxicity study by the same authors, 10/10 

females and 8/10 males dosed at 300 mg/kg-day died before study end (Daniel et al, 1994) . 

Gross pathology examinations of the animals in the 10-day study revealed a diffuse reddening of 

the lungs. 

Changes in relative liver and kidney weights were seen in the mid- and high-dose group for both 

sexes . Female rats had increased liver and kidney weights at the high dose, but only increased 

kidney weights in the mid-dose (75 mg/kg-day) group. Males had significantly increased brain, 

liver and kidney weights at the mid- and high-doses; males also had decreased hemoglobin, 

hematocrit, and alkaline phosphatase levels at the same doses. At the high-dose level, red blood 

cells, lymphocytes, hemoglobin, hematocrit, potassium, and albumin levels were significantly 

decreased while platelets, white blood cells, neutrophils, and monocytes were increased in 

females. Few gross and histological changes were found (single incidences) ; none of the 

changes were dose dependent. Effects for which statistically significant changes were observed 

at the mid-dose are summarized in Table 1. 

Table 1. Organ Weights and Hematology Parameters with Statistically Significant Changes at 

the Middle Dose in a Subchronic Stud of EDC in S ra e-Dawle Rats Daniel et al ., 1994) _ 

Control 37.5 mg/kg- 75 mg/kg-day 150 mg/kg-day 

day a 

Females 

Kidney weight (% of body weight) 0.67 t 0 .09 0.70 f 0.06 0.77 t 0.07a 0.82 f 0.09a 
EDC Subchronic Route-to-Route July 22, 2010 

Page 5 _ 

Males 

Brain wt % of body weight ) 0.40+0.03 0 .40 ± 0.02 0.43 f 0.03a 0.49 t 0.04a 

Kidney weight % of body weight) 0.62 f 0.06 0 .66 t 0 .06 0.73 f 0.05a 0.84 f 0.07a 

Liver weight % of body weight) 2.59 t 0.21 2 .69 t 0.16 3 .10 t 0.42a 3 .40 ± 0.36a 

Hemoglobin H b /dL 16 .2 f 0.06 16.0+0 .6 15 .4 t 0.5a 15 .2 t 0.7a 

Hematocrit (Hct) (%) 46.0 f 2.1 -- 45 .4 t 1 .8 46.7 ± l .la 43 .0 t 2.Oa 

aSignificantly different from the control group (p <_ 0.05) 

SELECTION OF CRITICAL ENDPOINTS AND DOSE MEASURES 

Critical Endpoint 

The critical endpoint in Daniel et al . (1994) was the decrease in hemoglobin in male rats at 75 

mg/kg-day compared to the controls in the 90 day study. Changes in relative liver and kidney 

weight will also be considered because, although these endpoints are considered to be of 

questionable biological significance (changes in relative weight without corresponding 

microscopic lesions), additional dose metrics may be considered relevant to route-to-route 

extrapolation, as discussed in the following paragraphs. 

Critical Dose Measures 

Consistent with U .S . EPA's Guidelines for Carcinogen Risk Assessment and PBPK modeling 

guidance (U.S. EPA, 2005, 2006), the selection of an appropriate dose measure for toxicological 

endpoints of interest (both cancer and non-cancer endpoints) requires a careful consideration of 

the mode of action by which a chemical exerts an effect . Parent compound (peak, average, or 

area under the concentration vs . time curve [AUC]) and amount metabolized were suggested as 

dose metrics in the ECA (U.S . EPA, 2003). In consideration of the toxicological endpoints of 

concern identified in the subchronic study, dose metrics potentially relevant to hepatic effects 

and extrahepatic effects have been identified. 

The dose metrics to be considered were expanded beyond those suggested in the ECA. For 

hepatotoxicity of EDC, the following dose metrics were considered: total amount of EDC 

metabolized in the liver (normalized to liver weight) (AMLVL), peak concentration of 

chloroacetaldehyde (metabolite) in the liver (CML), AUC of chloroacetaldehyde in the liver 

(AUCML), and the minimum concentration of glutathione in the liver (GSHL). For extrahepatic 

effects, the total amount of EDC metabolized (normalized to body weight) (AMETBW) and the 

amount of EDC metabolized in extrahepatic tissues (normalized to body weight) (AMXBW) 

were considered to be the more logical metrics ; the peak concentration of EDC in arterial blood 

(CA) and the AUC of EDC in arterial blood (AUCB) were also evaluated, in order to provide a 

comprehensive assessment. 

The data indicate that the most appropriate dose metric for route to route extrapolation of hepatic 

effects is the minimum post exposure concentration of GSH in the liver (GSHLmin). The reason 

for this choice and supporting data are presented in the following paragraphs. Less information 
EDC Sub_chronic Route-to-Route July 22, 2010 

Page 6 - 

is available for mode(s) of action for extrahepatic effects, so the route-to-route extrapolation for 

extrahepatic effects was based on the dose metric yielding the most conservative equivalent 

inhalation exposure, the amount of extrahepatic metabolism (normalized to body weight). 

Supporting data are provided below. 

Igwe et al . (1986) evaluated the potential interaction of dietary disulfiram (Antabuse) and EDC 

exposure in male Sprague-Dawley rats treated with the compounds for 30 days. Disulfiram acts 

to block the metabolism of aldehydes by aldehyde dehydrogenase. One pathway for EDC 

metabolism, which competes with GSH conjugation, is oxidative metabolism to 

chloroacetaldehyde (Figure 1) ; co-exposure to disulfiram would block the metabolism of 

chloroacetaldehyde by aldehyde dehydrogenase (ALDH), resulting in potentially greater 

accumulation of chloroacetaldehyde, generation of higher levels of the chloroacetaldehyde-GSH 

conjugate, and/or more extensive depletion of GSH. Igwe et al . (1986) found that the combined 

exposure to EDC and disulfiram increased hepatotoxicity (mid-zonal necrosis), decreased spleen 

weight, and resulted in testicular atrophy . These findings support the use of dose metrics related 

to EDC metabolism rather than parent compound as indicators of toxicity . 

CI 

P45i0 

CI 

CI 

ALDFf 

GST S-G 

+ GSH Cl 

5-G 

CI C) '~~~ `Y` O 

6H OH 

Figure 1 . Metabolic scheme for EDC 

Chloroacetaldehyde has been demonstrated to be hepatotoxic when administered directly to mice 

in drinking water (Daniel et al., 1992) and chloroacetaldehyde is cytotoxic to isolated rat 

hepatocytes (Sood and O'Brien, 1993). The studies by Sood and O'Brien (1993) included 

incubations with added antioxidants or GSH-depletion prior to the addition of 

chloroacetaldehyde . The greater susceptibility of hepatocytes when GSH was depleted indicates 

that the toxicity is not likely to be due to the generation of the GSH-chloroacetaldehyde 

conjugate . Incubations of hepatocytes with chloroacetaldehyde and added antioxidants produced 

delays in cytotoxicity, indicating that the overall antioxidant status is the best indicator of 
EDC Subchronic Route-to-Route July 22, 2010 

Page 7 

potential hepatotoxicity in chloroacetaldehyde-exposed liver cells. Thus, GSHLmin (lowest 

GSH concentration after EDC exposure) is supported as the most appropriate dose metric for 

identifying an EDC inhalation concentration that would be expected to produce hepatotoxicity 

equivalent to that observed in gavage-exposed rats. 

In addition to its apparent hepatotoxicity, chloroacetaldehyde has also been implicated as a 

potential nephrotoxicant . The greater nephrotoxicity of ifosfamide vs. cyclophosphamide has 

been attributed to greater generation of chloroacetaldehyde from ifosfamide than from 

cyclosphosphamide . The nephrotoxic potential of chloroacetaldehyde has been demonstrated in 

ex vivo/in vitro systems (isolated perfused rat kidney, renal cortical slices, and cultured proximal 

tubules) (Zamlauski-Tucker et al ., 1994; Yaseen et al ., 2008a, 2008b; Knouzy et al., 2010). 

While these studies and Igwe et al . (1986) are generally supportive of chloroacetaldehyde (or its 

downstream metabolites) as extrahepatic toxicants, the available data cannot provide sufficient 

support for one metabolism-related dose metric over another, so the lowest inhalation-equivalent 

NOAEL was selected for extrahepatic effects . 

ROUTE TO ROUTE EXTRAPOLATION METHODS 

Route-to-route extrapolation of the subchronic NOAEL and LOAEL for rats were performed 

using the PBPK model (Sweeney et al ., 2008). Minor changes were made to the model code to 

convert it from ACSL 11 .8 .4 to acs1X (Version 2 .5 .0 .6) and to facilitate computation of the dose 

metrics to be considered in the subchronic route to route extrapolation, interpretation of 

previously-conducted reproductive and developmental toxicology studies (Alumot et al., 1976; 

Rao et al., 1980), and design of the extended one-generation reproductive toxicity study 

(Appendix A). Internal dose metrics for male and female rats dosed with 37 .5 or 75 mg/kg-day 

EDC in corn oil for 90 consecutive days (per Daniel et al ., 1994 study design) were determined 

from PBPK model simulations (Appendix B, Table B-1) using end-of-study, group-specific body 

weights. Equivalent inhalation exposure concentrations were derived using default body weights 

for male and female rats (U.S. EPA, 1988) and assuming 24 hr/day exposure for 90 consecutive 

days (continuous exposure) . Inhalation exposure concentrations producing NOAEL- and 

LOAEL-equivalent internal dose metrics were derived by iteratively simulating various trial 

concentrations until a trial concentration approximately matching the simulated internal dose 

from the gavage study was derived (Appendix B, Table B-3). The level of precision of the 

equivalent exposure concentrations is two significant figures . 

ROUTE TO ROUTE EXTRAPOLATION RESULTS 

The consideration of an array of dose metrics for both hepatic and extrahepatic effects yielded a 

range of potential NOAEL and LOAEL equivalents, reported in Appendix B and summarized 

here. For hepatic effects, the 24 hr/day NOAEL equivalent inhalation exposures ranged from 20 

to 80 ppm; the most appropriate dose measure, the minimum liver GSH level, yielded a NOAEL, 

of 62 ppm for continuous inhalation exposure of male rats . The hepatotoxicity LOAELequivalents ranged from 32 to 100 ppm continuous inhalation exposure; minimum liver GSH 
EDC Subchronic Route-to-Route July 22, 201.0 

Page 8 _ 

- 

yielded a LOAEL of 100 ppm for male rats. The extrahepatic toxicity 24 hr/day NOAELequivalents ranged from 4 .4 to 200 ppm and the LOAEL-equivalents ranged from 6.7 to 340 

ppm. The metabolism-related NOAEL- and LOAEL-equivalents for extrahepatic effects via 

inhalation exposure were lower than those related to parent compound. 

Of the dose measures considered in this analysis, the amount of extrahepatic metabolism 

(normalized to body weight) (AMXBW) yielded the most conservative NOAEL- and LOAELequivalents . Therefore this dose metric is farther considered in the sensitivity analyses and these 

corresponding inhalation equivalents of 4.4 ppm (NOAEL) and 6.7 ppm (LOAEL) (24 hrs/day) 

are recommended for quantitative evaluation of subehronic inhalation hazards of EDC in rats. 

SENSITIVITY ANALYSIS 

The route-to-route extrapolation of the subchronic oral study of EDC in rats involved the 

consideration of multiple endpoints (hepatic and extrahepatic) and internal dose metrics 

consistent with supportable modes of action, as computed using a PBPK model (Sweeney et al ., 

2008) . 

The sensitivity of the critical dose metric, the simulated amount EDC metabolized in 

extrahepatic tissues of female rats (AMXBW), to changes in model parameter values was 

evaluated for scenarios involving corn oil gavage or continuous inhalation. In the gavage 

scenario, a daily EDC dose of 37 .5 mg/kg-day for 90 days, the NOAEL, was used as the basis of 

the analysis . The continuous inhalation equivalent for female rats, 4.4 ppm for 24 hrs/day for 90 

days, was used as the basis of the inhalation sensitivity analysis . The sensitivity analysis was 

conducted using a relative change ("delta") of 0.0001 % for the input parameter and noting the 

change in the model prediction of interest (total extrahepatic metabolism normalized to body 

weight, AMXBW), normalized to the baseline prediction of dose metric of interest. The 

fractional increase in the prediction divided by the fractional increase in the parameter was 

defined as the normalized sensitivity coefficient (NSC). The results are summarized in Table 2. 

For the gavage scenario, AMXBW was very sensitive to the dose, blood:air and slowly perfused 

tissues :air partition coefficients (PB and PSA), ventilation rate (QPC), and the extrahepatic 

metabolism rate (expressed as a fraction of the rate of oxidative metabolism in the liver, 

XLRATIO) . Under the inhalation scenario, AMXBW was also very sensitive to QPC (but in the 

opposite direction) and to the exposure concentration . 

Table 2. Sensitivity Coefficients for Amount of EDC Metabolized in Extrahepatic Tissues 

(AMXB W) 

Oral dose 

(37.5 mg/kg- Inhalation exposure 

Parameter' day) (4.4 pprn) 

Body weight B -0 .0087 -0.27 

~ Inhaled concentration (CONC) Not 0.96 
EDC Subchronic Route-to-Route _ July 22, 2010 

Page 9 - 

applicable 

Gava e dose ( ODOSEQ 1 .3 Not applicable 

Alveolar ventilation rate PC -1 .00 0.88 

Cardiac output CC 0.27 -0.19 

Fractional blood flow to liver LC -0.062 -0.35 

Fractional volume of adipose fat tissue VFC 0.24 -0.000033 

Blood:air partition coefficient PB 1 .7 0.084 

Fat:air partition coefficient PFA -0.90 -0.000038 

Slowly erfused tissues : air partition coefficient PSA 1 .6 -0.000029 

Michaelis constant for oxidative metabolism of EDC 

KM -0.013 -0.27 

Michaelis constant for EDC for GSH conjugation 

KMG 0.10 0.00071 

Maximum rate of oxidative metabolism of EDC in the 

liver VMAXLC 0.17 0.31 

Maximal rate of extrahepatic EDC metabolism, as a 

fraction of maximum rate of oxidative metabolism of 

EDC in the liver XLRATIO 0.73 0.36 

Maximum rate of GSH conjugation of EDC in the liver 

VMAXLGC -0.39 -0.00071 

a Parameters not listed had INSCI <0.1 for both scenarios. 

In order to interpret the sensitivity analyses, it is helpful to consider the overall disposition of 

orally administered or inhaled dose (ODOSE or AINH, respectively) via elimination by 

exhalation (AEXH), liver metabolism (AML), or extrahepatic metabolism (AMX). (Note: Even 

though AMXBW is equivalent in these scenarios, the amounts [AMX in mg, rather than 

AMXBW in mg/kg] of extrahepatic metabolism are not equal in these scenarios because the 

body weights differ ; the gavage simulations used a study-specific body weight, while the 

inhalation-equivalent simulations used the default body weight specified in U.S. EPA (1988) . 

For the gavage scenario (37.5 mg/kg-day), the cumulative 90-day dose of 1049 mg, 339 mg are 

exhaled, 566 mg metabolized in the liver, and 143 metabolized extrahepatically . For the 

inhalation scenario (lowest NOAEL equivalent of 4.4 ppm, 24 hrs/day), 178 mg are inhaled, 16 

mg exhaled, 68 mg metabolized in the liver, and 94 mg metabolized extrahepatically . Thus, 

while extrahepatic metabolism is the major route of EDC clearance in the inhalation scenario, it 

is the smallest contributor to gavage clearance. Viewed in this light, it is not so surprising that 

the key determinants of AMXBW (i .e., sensitive parameters) differ between routes . 

Under the gavage scenario, a substantial fraction of dosed EDC is cleared by first pass 

metabolism in the liver; of the parent chemical that is not biotransformed by the liver, more is 

exhaled than is metabolized extrahepatically . Thus, an increase in the ventilation rate or 

decrease in PB will increase the amount exhaled, so less EDC will be cleared by extrahepatic 

metabolism. The high sensitivity of AMXBW to XLRATIO (see Table 2 for definition) 
EDC Subchronic Route-to-Route July 22, 2010 

Pa gel0 

demonstrates that, under this scenario, extrahepatic metabolism is limited by the metabolic rate 

constants rather than tissue blood flow. The lack of sensitivity of AMXBW to body weight 

(BW) for the gavage scenario indicates that the choice of using the end-of-study body weights 

reported by Daniel et al. (1994), rather than an estimate of initial weight or average weight did 

not have a significant effect on the point of departure for the route-to-route extrapolation . 

Under the inhalation scenario, the sensitivity of AMXBW to ventilation rate is in the opposite 

direction . The total systemic dose of EDC is increased when ventilation rate is increased, so 

more EDC can then be delivered to extrahepatic tissues for biotransformation to unspecified 

metabolites . Extrahepatic metabolism is decreased if blood flow to the liver increases, is almost 

equally sensitive to VMAXLC, XLRATIO, and KM and is insensitive to the rate of conjugation 

of EDC with GSH in the liver (VMAXLGC and KMG). These findings indicate that liver 

metabolism may be flow limited, while extrahepatic metabolism is limited by the metabolic 

capacity (VMAXLC x XLRATIO/KM, at low concentration) . 

CONCLUSIONS 

The route-to-route extrapolation of results of the gavage subchronic EDC study using the PBP.K 

model was based on selection of critical endpoints, the most appropriate internal dose measures, and 

appropriate points of departure. For hepatotoxicity, a dose metric related to the apparent mode of 

action was chosen. For extrahepatic effects, a conservative dose measure was selected from among 

the options considered because of a lack of information to clearly support selection of another 

metric . The gavage rat NOAEL of 37.5 mg/kg-day was determined to be equivalent to continuous 

(24 hr/day) exposure of rats to 4.4 ppm EDC, when using the conservative dose metric of 

extrahepatic metabolism. 
EDC Subchronic Route-to-Route July 22, 2010 

Page I 1 

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Staats DA, Fisher JW, Connolly RB. ( 1991). Gastrointestinal absorption of xenobiotics in 

physiologically based pharmacokinetic models. A two-compartment description. Drug Metab 

Dispos. 19(1):144-8 . 

Sweeney LM, Saghir SA, Gargas ML. (2008) . Physiologically based pharmacokinetic model 

development and simulations for ethylene dichloride (1,2-dichloroethane) in rats. Regul Toxicol 

Pharmacol. 51 :311-323. 

U.S . EPA (1988) . Recommendations for and Documentation of Biological Values for Use in Risk 

Assessment. EPA/600/6-87/008 . 

U .S . EPA (2003). 1,2-Ethylene dichloride; Final Enforceable Consent Agreement and Testing 

Consent Order. Federal Register 68 No. 106, June 3, 2003, pp 33125-9 

U .S . EPA. (2005) . Guidelines for Carcinogen Risk Assessment U.S . Environmental Protection 

Agency, Washington, DC, EPA/630/P-03/OOlF, 2005. 

U .S . EPA. (2006) Approaches for the Application of Physiologically Based Pharmacokinetic 

(PBPK) Models and Supporting Data in Risk Assessment (Final Report). U.S. Environmental 

Protection Agency, Washington, D.C., EPA/600/R-05/043F. 

U.S. EPA (2009) . 1,2-Ethylene Dichloride; Completion of EPA Program Review. Federal 

Register 74 No. 139, July 22, 2009, pp 36196-36198 . 

http :l/www.re~ulations .fzov/search%Re%!s/contentStrearner?object1d=09000064809t78d9&dispo si 

tion=attachment&contentTxpe=html 

Withey JR, Karpinski K. (1985) . The fetal distribution of some aliphatic chlorinated 

hydrocarbons in the rat after vapor phase exposure. Biol Res Pregnancy Perinatol . 6(2) :79-88 . 
EDC Subchronic Route-to-Route _ July 22, 2010 

Page-13- - 

Yaseen Z, Michoudet C, Baverel G, Dubourg L. (2008a). Mechanisms of the ifosfamide-induced 

inhibition of endocytosis in the rat proximal kidney tubule. Arch Toxicol. 82(9):607-14. 

Yaseen Z, Michoudet C, Baverel G, Dubourg L. (2008b) . In vivo mesna and amifostine do not 

prevent chloroacetaldehyde nephrotoxicity in vitro. Pediatr Nephrol . 23(4):611-8. 

Zamlauski-Tucker MJ, Morris ME, Springate JE . (1994) . Ifosfamide metabolite 

chloroacetaldehyde causes Fanconi syndrome in the perfused rat kidney. Toxicol Appl 

Pharmacol. 129(1):170-5 . 
p. 15 

EDC Subchronic Route-to-Route July 22, 2010 

Page 14 

APPENDIX A : PBPK MODEL CODE 

edc4.cs1 

PROGRAM: GLUTATHIONE DEPLETION MODEL FOR EDC (EDC4 .CSL) 

!Initial parameter values for the rat (DSouza et al . 1987, 1988) 

!Retyped, documentation modified by LMS 7/19/O1 

!revised February 23, 2005 

!Add kidney, update tissue volumes and flows per Brown et al ., 1997 

!Adjust flow and volume calcs to ensure mass balance 

!Add chamber for closed chamber simulations, add iv administration 

!implement GSH delay per Environ report 

!revise GSH parameter calculations to ensure ss in unexposed 

!gsh turnover rates from Potter and Tran, tissue specific values 

!Input pcs as tissue :air and calculate tissue :blood 

!revised February 23, 2006 

!add two compartment GI, repeat gavage dosing, GST conj of parent in liver 

!turn off lung metab for now 

!revised June 2006 extrahepatic metabolism, reformulate reactions 

!for oxidative metabolite! 

!2010-06-11 edited by Lisa M . Sweeney for acslX 

!2010-06-17 added feeding scenario to accommodate simulation of Alumot et al . 

1976 

'.and extended one gen ingestion scenario 

INITIAL 

!SPECIAL FLOW RATES! 

CONSTANT QPC=15 $!alveolar ventilation rate L/hr/kg^0 .74! 

CONSTANT QCC=15 $!Cardiac output L/hr/kg^0 .74! 

!FRACTIONAL BLOOD FLOW TO TISSUES! 

CONSTANT QLC = 0 .18 $!Fractional blood flow to liver! 

CONSTANT QFC = 0 .07 $!Fractional blood flow to fat! 

CONSTANT QSC = 0 .34 $!Fractional blood flow to slow! 

CONSTANT QKC = 0 .14 !Fractional blood flow to kidney 

QRC = 1 .0-(QLC + QFC + QSC + QKC) $!Fractional blood flow to rapid! 

!BODY WEIGHT! 

CONSTANT BW = 0 .22 $!Body weight (kg)! 

!FRACTIONAL TISSUE VOLUMES! 

CONSTANT VPC = 0 .005 $!Fraction lung tissue! 

CONSTANT VLC = 0 .037 $!Fraction liver tissue! 

CONSTANT VFC = 0 .07 $!Fraction fat tissue! 

CONSTANT VSC = 0 .59 $!Fraction slow tissue (skin and muscle)! 

CONSTANT VKC = 0 .0073 !Fraction kidney 

constant vvc = 0 .05 

VRC = 0 .95- (vpc + vlc + vfc + vsc + vkc + vvc) $!Fraction rapid tissue! 

!PARTITION COEFFICIENTS! 

CONSTANT PPA = 33 $!Lung/air partition coefficient! 

CONSTANT PLA = 33 $!Liver/air partition coefficient! 

CONSTANT PFA = 340 $!Fat/air partition coefficient! 
p. 16 

EDC Subchronic Route-to-Route July 22, 2010 

Page 15 

CONSTANT PSA = 23 $!Slowly perfused tissue/air partition coefficient! 

CONSTANT PRA = 33 $!Richly perfused tissue/air partition coefficient! 

CONSTANT PKA = 33 $!Kidney/air partition coefficient! 

CONSTANT PB = 29 $!Blood/air partition coefficient! 

pp=ppa/pb 

pl=pla/pb 

pf=pfa/pb 

ps=psa/pb 

pr=pra/pb 

pk=pka/pb 

CONSTANT MW = 98 .96 $!Molecular weight (g/mol)! 

!KINETIC CONSTANTS! 

CONSTANT VMAXIC = 3 .15 !!Maximum velocity of oxidative metabolism (mg/hrkg^0 .7)! 

CONSTANT KM = 0 .25 $!Michaelis-Menten constant (mg/L)! 

CONSTANT vmaxlgc=480 .0 $!enzy conj rate const gsh w parent(mg/(hr-kg^-0 .7))! 

constant kmg = 2000 !Km with respect to EDC ma/L (not rate limiting at relev 

conc?) 

constant kmgg = 125 !Km with respect to liver GSH in uM (rate limiting when 

gsh depleted) 

CONSTANT KGSM =2 .0 $!gsh conj rate const w metab (1/(uM-hr))! 

CONSTANT KFEE=4600 $!conj rate const w non gsh (1/hr)! 

CONSTANT Kdgsyn=0 .32 $!GSH synthase breakdown (1/hr)! 

CONSTANT Kdgsh1=0 .14 $!GSH turnover rate--liver! 

CONSTANT KDGSHP =0 .011 !Gsh turnover rate--lung 

CONSTANT KSL = 200 $!Maximum GSH production control (liver)! 

CONSTANT KSp = 200 $!Maximum GSH production control (lung)! 

CONSTANT Tau = 2 $!Time delay constant (hr)! 

CONSTANT GSO1 = 5500 $!Initial GSH concentration (uM)! 

constant gslmit = 1200 !mitochondrial GSH, does not conjugate or turnover 

CONSTANT GSOp = 1200 $!Initial GSH concentration (uM)! 

CONSTANT PLRATI0=0.0 $!MFO ratio lung/liver! 

constant racpgp=0 

CONSTANT xLRATI0=0 .0 !MFO ratio extrahepatic/liver 

constant kdet = 0 .025 !P450 turnover constant (1/hr) 

constant kdi = 0 .0 !second order reaction rate for liver P450 inactivation 

by oxid metab 

! 1/((mg-equiv/1)-hr) 

!DOSING INFO! 

CONSTANT CONC = 0 $!Inhaled concentration (ppm)! 

CONSTANT VCHC = 9 .1 !Total chamber volume (L) 

CONSTANT NRATS = 0 . !number of rats in closed chamber 

Vch = vchc-nrats*bw !net chamber volume 

CONSTANT KLC = 0 .0 !loss rate to chamber 

AIO = conc*vch*mw/24450 .0 !initial mass of chemical in chamber 

constant odosec = 0 !gavage dose at t=0 in mg/kg 

constant kas=1 !oral absorption rate from stomach 

constant kt = 1 !transport rate from stom to intestines 

constant kai = 1 ! oral absorption rate from intestines 

constant ivdosec = 0 . !iv dose at t=0 in mg/kg 

constant tinf=0 .01 !duration of iv injection (hr) 
EDC Subchronic Route-to-Route July 22, 2010 

Page 1 6-- 

riv=ivdosec*bw/tinf 

constant FDOSEC =0 . ! daily dose in feed (Alumot study) in mg/kg 

constant CDOSEC = 0 . ! daily dose for continuous ingestion (water or feed) 

mg/kg/d 

constant TING = 12 . ! ingestion duration for continuous ingestion scenario 

(hrs) 

!TIMING PARAMETERS! 

CONSTANT TSTART =0 . $!Start of exposure (hrs)! 

CONSTANT TPER=24 

CONSTANT TSTOP=120 

CONSTANT POINTS=1200 $!Number of points in plot! 

CINT = TSTOP/POINTS $!Communication interval! 

constant TCHNG=6 

!SCALED PARAMETERS! 

QC = QCC*BW**0 .74 

QP = QPC*BW**0.74 

QL = QLC*QC 

QF = QFC*QC 

QS = QSC*QC 

QK = QKC*QC 

qr = qc-(ql+qf+qs+qk) 

VP = VPC*BW 

VL = VLC*BW 

VF = VFC*BW 

VS = VSC*BW 

VR = VRC*BW 

vk = vkc*bw 

vv = vvc*bw 

!Liver metabolism! 

VMAX10=VMAXIC*BW**0 .7 

vmaxlg = vmaxlgc*bw**0 .7 

vmaxp= vmaxl0*plratio 

vmaxx=vmaxl0*xlratio 

AGS01=GSO1*VL 

!Lung metabolism! 

AGSOp=GSOp*VP 

P1=0 

P1R=0 

P2=0 

P2R=0 

P3--100 

P3R=100 

KgshprodLO=agsol*kdgshl !baseline liver gsh synthesis rate umol/hr 

r10=kgshprodl0 

r20=kgshprodl0 

kgshprodtdl0=kgshprodl0 

kgshprodp0=agsop*kdgshp !baseline lung gsh synthesis rate umol/hr 

rpl0=kgshprodp0 

rp20=kgshprodp0 

kgshprodtdp0=kgshprodp0 
EDC Subchronic Route-to-Route July 22, 2010 

Page 17 

kgsynprodl0=kgshprodl0*,kdgsyn !baseline prod gsh synthetase umol/hr/hr 

kgsynprodp0=kgshprodp0*kdgsyn !base prdxn of gsh synthetase in lung 

END $!End of initial! 

DYNAMIC 

ALGORITHM IALG=2 $!Gear method for stiff systems! 

DERIVATIVE 

RAI = nrats*qp*(CA/pb - ci) -klc*ai 

AI = INTEG(RAI, AIO) 

cch= ai/vch 

cp = cch*24450/mw 

!CI=Concentration in inhaled air (mg/1)! 

CI=cch*pulse(tstart, tper, tchng) 

RAinh=qp*ci 

Ainh=INTEG(RAINH,0 .) 

!Algebraic solution for CA1 after gas exchange! 

!CA1 is arterial blood concentration after gas exchange! 

CA1= (QC*CV +QP*CI)/(QC + QP/PB) 

CX = CA1/PB 

raexh=qp*cx 

aexh=integ(raexh,0 .) 

!Mass balance for the lung tissue compartment! 

RAP = QC*(CA1-CA) - RAMpu 

AP = INTEG(RAP,0 .0) 

Clung = AP/VP 

AUCP=INTEG(CP,0 .0) 

CA=C1ung/PP 

AUCB=INTEG(CA,0 .0) 

!UPTAKE BY ORAL ROUTE! 

RFEEDA = 

FDOSEC*BW*(0 .528*(PULSE(0 .0,24 .,1 .))+0.272*(PULSE(1 .,24 .,1 .))+0 .2*PULSE(16 .,24 

.,1 .)) !Alumot study 

RCDOSE= CDOSEC*BW/TING*(PULSE(0 .0, 24 .,TING)) 

RING=RFEEDA+RCDOSE 

AING = INTEG(RING, 0 .) 

RSTOM = -(KAs+kt)*STOM+RING $!dSTOM/dT! 

days=t/24 

ODOSE =odosec*bw*(1 .0 + (int(days))) !repeat dosing 

STOM =INTEG(RSTOM,0 .0) +ODOSE $!amount in stomach (mg)! 

rintest = kt*stom - kai*intest 

intest = integ(rintest, 0 .) 

!AS = Amount in slowly perfused tissues (mg)! 

RAS = QS*(CA-CVS) 

AS = INTEG(RAS,0 .0) 

CVS = AS/(VS*PS) 

CS = AS/VS 

!AR = Amount in richly perfused tissues (mg)! 
EDC Subchronic Route-to-Route July 22, 201.0 

Pd&e-18 

RAR = QR*(CA-CVR) 

AR = INTEG(RAR,0 .0) 

CVR = AR/(VR*PR) 

CR = AR/VR 

!AK = Amount in kidney 

RAk = Qk*(CA-CVk) 

Ak = INTEG(RAk,0 .0) 

CVk = Ak/(Vk*Pk) 

Ck = Ak/Vk 

(mg) ! 

!AF = Amount in fat tissues (mg)' 

RAF = QF*(CA-CVF) . 

AF = INTEG(RAF,0 .0) 

CVF = AF/(VF*PF) 

CF = AF/VF 

!CV = Mixed venous blood concentration (mg/1)! 

rinf=riv*pulse(0 ., 2000 ., tinf) 

ivdose--integ(rinf,0 .) 

RAVx = rinf + (QS*CVS + QR*CVR+ qk*CvK)-(qs+qr+qk)*CVx - ramx 

AV = integ(ravx, 0 .) 

cvx=Av/vv 

cv = QFc*CVF + QLc*CVL + cvx*(qsc + qrc + qkc) 

!AL = Amount in liver tissue (mg)! 

RAL = QL*(CA-CVL)-RAM1 + KAs*STOM+ kai*intest 

AL = INTEG(RAL,0 .0) 

CVL = AL/(VL*PL) 

CL = AL/VL 

AUCL = INTEG(CL,O .) 

!LIVER METABOLISM! 

!AM = Amount metabolized liver (mg)! 

RAM1= RAMox + RAMCon 

RAMox =(VMAXl*CVL)/(KM+CVL) 

ramcon = vmaxlg*(GS1/(gsl+kmgg))*(CVL/(cvl + kmg)) 

AM1=INTEG(RAM1,0 .) 

AMP1=AM1*1000/MW 

RAMP1=RAML*1000/MW 

!time-dependence of hepatic P950 oxidation due to inactivation by oxid 

metabolite 

rvmaxl = (vmaxl0-vmaxl)*kdet - vmaxl*kdi*CML 

vmaxl = INTEG(rvmaxl, vmaxl0) 

!CM1=0XIDATIVE METABOLITE LIVER mg/L)! 

RAMM1= ramox -RACMGl*MW/1000-RACMEE1*MW/1000 

AMMI=INTEG(RAMM1,0 .) 

CML=AMM1/VL 
EDC Subchronic Route-to-Route July 22, 2010 

Page 19 

AUCML=INTEG(CML,O .) 

!ACPG1 = AMT PARENT CONJUGATED WITH GSH LIVER (uMOLES)! 

RACPG1=ramcon*1000/MW 

ACPG1=INTEG(RACPGI,O .) 

!ACMG1=AMT METABOLITE CONJUGATED WITH GSH LIVER (uMOLES)! 

RACMG1=KGSM*GS1*CM1*1000/MW*VL 

ACMG1=INTEG(RACMG1,0 .0) 

!ACMEE1=AMT METABOLITE CONJUGATED WITH EVERYTHING ELSE LIVER! 

!uMOLES! 

RACMEE1=KFEE*VL*CM1*1000/MW 

ACMEE1=INTEG(RACMEE1,0 .) 

!LUNG METABOLISM! 

!AMp=Amount metabolized lung (mg)! 

RAMpu=RAMpox + rampcon 

rampox=(VMAXp*CA)/(KM+CA) 

rampcon=RACPGp*MW/1000 

AMp=INTEG(rampu,0 .) 

AMPp=AMp*1000 ./MW 

RAMPp=RAMpu*1000 ./MW 

!CMp=OXIDATIVE METABOLITE (mg/L)! 

RAMMp= rampox -RACMGp*MW/1000-RACMEEp*MW/1000 

AMMp=INTEG(RAMMp,O .) 

CMp=AMMp/VP 

!ACMGp= AMT METABOLITE CONJUGATED WITH GSH LUNG (uMOLES)! 

RACMGp=KGSM*GSp*CMp*VP*1000/MW 

ACMGp=INTEG(RACMGp,O .) 

!ACMEEp=AMT METABOLITE CONJUGATED WITH EVERYTHING ELSE LUNG (uMOLES)'. 

RACMEEp=KFEE*VP*CMp*1000/MW 

ACMEEp=INTEG(RACMEEp,O .) 

!ACPGp=AMT PARENT CONJUGATED WITH GSH LUNG (uMOLES)! 

! RACPGp=KGS*GSp*Ca*VP*1000/MW 

ACPGPp=INTEG(RACPGp,O .) 

!extrahepatic METABOLISM! 

!AMx = Amount metabolized extrahepatic (mg)! 

RAMx= RAMxox + RAMxCon 

amx=integ(ramx, 0 .) 

RAMxox =(VMAXx*CVx)/(KM+CVx) 

amxox = integ(ramxox, 0 .) 

ramxcon=0 

amxcon=integ(ramxcon, 0 .) 

'.gsh synthesis rate--time dependent 

kgshprodl=integ(kgsynprodl0*(ksl+gsol)/(ksl+gsl) & 

-kdgsyn*kgshprodl,kgshprodl0) 

kgshprodp=integ(kgsynprodp0*(ksp+gsop)/(ksp+gsp) & 

-kdgsyn*kgshprodp,kgshprodp0) 
EDC Subchronic Route-to-Route _ July 22, 2010 

Page 20 - --- 

!time delay (Bishchoff et al . 1971) 

r0=kgshprodl 

rrl=(r0-rl)/tau 

rl=integ(rrl,rl0) 

rr2=(rl-r2)/tau 

r2=integ(rr2,r20) 

rr3=(r2-kgshprodtdl)/tau 

kgshprodtdl=integ(rr3, kgshprodtdl0) 

rp0=kgshprodp 

rrp1=(rp0-rpl)/tau 

rpl=integ(rrpl,rpl0) 

rrp2=(rpl-rp2)/tau 

rp2--integ(rrp2,rp20) 

rrp3=(rp2-kgshprodtdp)/tau 

kgshprodtdp=integ(rrp3, kgshprodtdp0) 

!GS1 = GIUTAHIONE LIVER (uM)! 

RAMGS1=kgshprodtdl-Kdgshl*GS1*VL-RACMGI-RACPG1 

AMGS1=INTEG(RAMGSl,AGS01) 

GS1=AMGS1/VL 

gshl = gsl + gslmit 

!GSp=GLUTAHIONE LUNG (uM)! 

RAMGSp=Kgshprodtdp-Kdgshp*GSp*VP-RACMGp-RACPGp 

AMGSp=INTEG(RAMGSp,AGSOp) 

GSp=AMGSp/VP 

!PCTGSH-PERCENT GSH COMPARED TO CONTROL! 

PCTGSHP=GSp/GSOp*100 

!mass balance 

anet = AING+ odose + ivdose + ainh - aexh 

atissue=ap + as+ ar + al+ ak + af +av 

agi = stom + intest 

amet = aml + amp + amx 

AMETBW = AMET/BW !total amount metabolized, normalized to BW 

AMXBW = AMX/BW !extrahepatic metabolism normalized to BW 

AMLVL = AML/VL !liver metabolism normalized to BW 

termt(t.ge .tstop) 

END $!End of derivative! 

END $!End of dynamic! 

END $!End of program 

Rat.m 

% rat .m 

0 2010-06-11 created from edc4 .cmd proced rat by Lisa M . Sweeney 

o physiol parameters from Brown et al . 1997 

QPC=15 ; QCC=15 ; 

QLC=0 .18 ;QFC=0 .07 ; QSC=0 .34 ; QKC=0 .14 ; 

PB=2 9 ; 

PLA=33 ; PPA=10 ; PFA=340;PRA=33; PSA=23 ; PKA=33 ; 
p. 22 

EDC Subchronic Route-to-Route July 22, 2010 

Page- 21 

VMAXLC=4 .0 ; KM=0 .25 ; PLRATI0=0 .0 ; XLRATI0=0 .3 ; 

%PLRATIO WA0 .026 

VMAXLGC=4 .80 ; KMGG=530 ; KMG=20 .0 ; 

KGSM=2 .O ;KFEE=4600 .0 ; 

KDGSYN=0 .324 ;KDGSHL=0 .14 ; KDGSHP=0 .011 ; 

GSOL=5500;GSOP=1200 ; TAU=0 .89 ; 

KSL=1200 ; KSP=240; 

NRATS=O ; KLC=O ; CONC=O ; IVDOSEC=O ; ODOSEC=O ;FDOSEC=O . ;CDOSEC=O ; 

VCHC=9 .1 ; KAS=8 ; KT=2 ; KAI=0 .15 ; o Default absorption parameters are for corn 

oil gavage 

TSTART=O ; TPER=24; TSTOP=24 ; TCHNG=24 ; TINF=0 .01 ; 

Sdrat.m 

% sdrat .m 

a updated 5/19/2006 by Lisa M . Sweeney 

% updated 6/9/2006 LMS 

% 2010-06-10 sdrat m file created from edc4 .cmd files by Lisa M . Sweeney 

%proced sdrat 

%fat from schoeffner et al . 1999 

opcs from Dsouza et al ., 1987 

use rat 

VFC=0 .065 ; 

PB=28 ; PLA=30 ; PSA=22 ; 

BW=0 .267 ; %default weight for a male SD rat in a subchronic study (EPA, 1988) 

Waterabsparam.m 

%waterabsparam .m absorption parameters for administration in water 

KAS=5 . ; KT=2 . ; KAI=1 .5 ; 

Alumot metrics.m 

oalumot_metrics .m 

%simulate dose metrics for Alumot et al . (1976) assuming absorption from diet is 

similar to absorption from water vehicle 

% body weight estimated as 0 .2 kg per BW after 5 weeks of treatment 

o assume 500 ppm diet is -50 mg/kg/day, since authors state that 250 ppm diet is 

equivalent to 25 mg/kg/day 

% simulate one week and compute daily metrics by dividing by 7 days . 

use sdrat 

BW= .2 

FDOSEC=50 

TSTOP=168 

disp("Alumot dose metrics 50 mg/kg/day)") 

disp("absorption parameters from corn oil") 

start @nocallback 

AMETBW, AMLVL, AUCML, AUCB 

disp("CML max") 

max( cml), 
EDC Subchronic Route-to-Route July 22, 2010 

Page 22 

disp("CA max") 

max( ca) 

use waterabsparam 

disp("absorption parameters from water") 

start @nocallback 

AMETBW, AMLVL, AUCML, AUCB 

disp("CML max^) 

max(_cml), 

disp("CA max") 

max ( ca) 

Rao repro 150.m 

orao_terat_300 

% 150 ppm group, reproductive study of Rao et al . 

use sdrat 

BW=.250 ; o per paper 

TSTOP=168 . ; TCHNG=6 . ; 

CONC=150 .t 

start @nocallback 

AMETBW, AMLVL, AUCML, AUCB 

disp("CML max") 

max(_cml) 

disp("CA max") 

max( ca) 

Rao terat 300.m 

%rao_terat_300 

0 300 ppm group, teratogencity study of Rao et al . 

use sdrat 

BW=.250 ; o per paper 

TSTOP=240 . ; TCHNG=7 . ; 

CONC=300 . ; 

start @nocallback 

AMETBW, AMLVL, AUCML, AUCB 

disp("CML max") 

max(_cml), 

disp("CA max") 

max ( ca) 

Extonegen .m 

%extonegen .m 

eextended one generation reproduction study scenario 

use sdrat 

BW=.25 ; 

TSTOP=24 ; 

CDOSEC=50; 
EDC Subchronic Route-to-Route 

Page 23 

start @nocallback 

AMETBW, AMLVL, AUCML, AUCB 

disp("CML max") 

max(_cml) 

disp("CA max") 

max( ca) 

CDOSEC=100 ; 

start @nocallback 

AMETBW, AMLVL, AUCML, AUCB 

disp("CML max") 

max (_cm1) 

disp("CA max") 

max( ca) 

CDOSEC=200 ; 

start @nocallback 

AMETBW, AMLVL, AUCML, AUCB 

disp("CML max") 

max(_cml) 

disp("CA max") 

max( ca) 

CDOSEC=300 ; 

start @nocallback 

AMETBW, AMLVL, AUCML, AUCB 

disp("CML max") 

max(_cml) 

disp("CA max") 

max( ca) 
EDC Subchronic Route-to-Route _ July 22, 2010 

Page 24 - - - -- 

APPENDIX B 

PBPK-Derived Internal Dosimetry and Route-to-Route Extrapolations for EDC-Exposed 

Rats 

Values of the dose metrics of arterial blood EDC, liver GSH or liver chloroacetaldehyde (metabolite) 

concentration and daily metabolism occurring the liver (normalized to liver weight) or extrahepatic 

metabolism or total metabolism (normalized to body weight) were predicted using the PBPK 

models. Simulations were conducted at the NOAEL (37.5 mg/kg-day) and LOAEL (75 mg/kg-day) 

for both sexes, and the outputs are provided in Table B-1 . 

Table B-1. Internal dose metrics corresponding to the NOAEL and LOAEL in Daniel et al. 

1994 rat subchronic study, as computed using a PBPK model Sweene et al., 2008 

37.5 37.5 75 mg/kg, 75 mg/kg, 

mg/kg, mg/kg, female male 

female male (284 g) (562 g) 

311 590 

Total amount of EDC metabolized, 

normalized by body weight (AMETBW) 

( mg/kg BW 2283 2246 3779 3686 

Total amount of EDC metabolized in 

extrahepatic tissues, normalized by body 

weight AMXB (mg/kg B 463 458 691 682 

Maximum concentration of EDC in arterial 

blood CAmax m /L 10.4 12 .0 24.2 27.2 

AUC for EDC in arterial blood (AUCB) 

m EDC x hrs/L blood 1504 1836 3997 4922 

Amount of EDC metabolized in liver, 

normalized by liver wt (AMLVL) (mg/kg 

liver 4.92 x 104 4.83 x 104 8.34 x 104 8 .12 x 104 

Maximum concentration of EDC 

metabolite in the liver (CMLmax) (mg 

EDC-e uivalent/L liver 0.0117 0.00953 0.0159 0.0126 

Area under the concentration vs. time curve 

(AUC) for EDC metabolite in the liver 

(AUCML) (mg EDC-equivalent x hrs/L 

liver 3.29 3 .20 6.17 5.85 

Minimum concentration of glutathione in 

the liver GSHLmin M 4415 4508 3297 3484 
EDC Subchronic Route-to-Route _ July 22, 2010 

Page 25 -- 

Table B-2. Internal dose metrics corresponding to 24-hr exposure to a range of inhalation 

concentrations m as computed usin g a PBPK model Sweene et al., 2008 

Males 

Concentration 0 1 10 100 1000 

AMETBWa 0 169 1675 14,543 25,882 

AMXBW 0 101 927 3476 3842 

CAmax 0 0 .00965 0.106 2.420 97.531 

AUCB 0 20.8 230 5216 21,0310 

AMLVL 0 1850 20,225 299,110 595,680 

CMLmax 0 5 .43 x 10-5 6.15 x 10 0.0141 0.0284 

AUCML 0 0.117 1 .32 26.7 46.7 

~ GSHLmin 6700 6673 6403 3491 1721 

Females 

Concentration 0 1 10 100 1000 

AMETBWa 0 181 1797 15,657 27,851 

AMXBW 0 108 998 3763 4165 

CAmax 0 0.00960 0 .106 2 .39 97.5 

AUCB 0 20.7 228 5142 210,210 

AMLVL 0 1978 21,608 32,1470 640,160 

CMLmax 0 5 .81 x 10" 6 .59 x 10 0.0158 0.0315 

AUCML 0 0.126 1 .41 29.4 52 .0 

LGSHLmin 6700 6671 6383 3339 1660 

a For definitions and units, see Table B-1 . 

Table B-3 . Inhaled concentrations (ppm) for 24-hr/day exposures to EDC for 90 consecutive days 

that yield simulated internal doses (Sweeney et al ., 2008) equivalent to subchronic NOAEL and 

LOAEL doses 

Subchronic Ora l Dosing Scenario 

Dose Metric' 37.5 

mg/kgday, 

female 

37.5 

mg/kgday, male 

__ 

75 mg/kgday, female 75 mg/kgday, male 

Extrahe atic Effects 

AMETBW 12 13 21 22 

AMXBW 4.4 4 .7 6.7 7.2 

CAmax 200 220 340 360 

AUCB 46 53 88 97 

Hepatic Effects 

AMLVL 21 21 32 33 

CMLmax 80 74 100 90 

AUCML 20 21 12 33 
EDC Subchronic Route-to-Route July 22, 2010 

Page 26 _ 

GSHLmin 61 62 100 10 

'For definitions and units, see Table B-1 . 

The value of the "equivalent" inhaled concentration was highly dependent on the choice of internal 

dose metric. For example, the inhalation "equivalent" of 37.5 mg/kg-day by corn oil gavage to a 

female mouse ranged from 4.4 ppm, based on the amount metabolized in extrahepatic tissues to 200 

ppm, based on peak concentration of EDC in arterial blood. In general, the predicted concentration 

of a compound in a well-perfused, non-metabolizing tissue that is not a portal of entry will track the 

concentration in the blood (i .e ., tissue concentration ;:%- blood concentration x tissue :blood partition 

coefficient) . The choice of AUC vs. peak for a given tissue also affects the results of the route-toroute extrapolation, as shown for both parent compound (EDC in blood) and metabolite 

(chloroacetaldehyde in liver) . Because the inhalation exposures rapidly come to steady-state, 

average concentration = AUC/duration,& peak concentration . In the corn oil gavage studies, average 

concentration < peak concentration . Thus, starting from the gavage study, the choice of AUC rather 

than peak sets a lower target blood or tissue concentration for determining the equivalent inhaled 

concentration . 

Mechanistic evidence indicate that minimum hepatic GSH is the most appropriate dose metric for 

route-to-route extrapolation of liver effects (rationale provided in the main report), but route-to-route 

extrapolation results for other liver-related dose metrics are provided. For extrahepatic effects, the 

dose metric which yields to the lowest inhalation equivalent exposure is recommended, a NOAEL of 

4.4 ppm and LOAEL of 6.7 ppm for AMXBW.