Document 37ro5nwEy76LQ8NkZ6dXK6wrJ
PROPOSAL FOR PHARMACOKINETICS STUDY OF 1,1,2-TRICHLOROETHANE 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 1,1,2-TRICHLOROETHANE
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 1,1,2-trichloroethane, to test these compounds for specific health effects. The tests proposed to be conducted for 1,1,2-trichloroethane via inhalation exposures include the following:
acute toxicity, subchronic toxicity, developmental toxicity, reproductive toxicity, neurotoxicity, carcinogenicity, in vivo cytogenicity, and immunotoxicity.
The cost for conducting these tests was estimated by the USEPA to be approximately $3.8 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
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a proposal to conduct pharmacokinetic studies for 1,1,2-trichloroethane as a means of filling specific data gaps and as a means of offering additional evidence for other 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 conducting route-to-route extrapolations has been proposed (Gerrity and Henry, 1990), and is provided in Figure 1. Following an evaluation ofthe 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 of default absorption values to physiologically based pharmacokinetic (PBPK) modeling. The approach proposed here centers on the use of validated PBPK models.
Three general requirements are necessary to conduct proper route to route extrapolations using PBPK models:
1. A scientifically plausible (defensible) mechanism of toxicological action is needed
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Page 3 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.
3. A validated PBPK model for the chemical and species of interest must be ivailable
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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:
I
1. The internal dose metric is determined under the experimental condition^ 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 1,1,2-trichloroethane, 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 1,1,2trichloroethane in animals suggest that effects remote from the respiratory tract (i.e., the central nervous system, liver) may be of potential concern. However, adequate information for inhalation exposures were not located for any of the effect categories listed above. For oral exposures, potential
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candidate studies were located for route-to-route extrapolation (Option 4) for subchronic toxicity, neurotoxicity, carcinogenicity, and immunotoxicity. No adequate inhalation or oral studies were identified for acute toxicity, developmental toxicity, reproductive toxicity, nor in vivo cytogenicity. However, as will be presented in later sections, it is proposed that only limited (if any) inhalation studies will be needed to evaluate acute toxicity (Sections 3.1 and 3.2), in vivo cytogenicity studies will not be required (Section 3.7), and weight of evidence arguments, which include a summary of data for structurally analogous compounds (Option 2), will be presented indicating that developmental and reproductive (Section 3.4) studies are also not necessary for 1,1,2-trichloroethane.
The remainder of this proposal will address the status of each of these requirements with regard to 1,1,2-trichloroethane, and will describe the specific approaches we propose to use to fill the data gaps identified in the test rule (both PBPK modeling and otherwise).
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2.0 MECHANISM OF ACTION
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1.1.2- Trichloroethane is metabolized to reactive intermediates via two potential pathways. Along one pathway free radicals and acyl chlorides (reactive metabolites) are generated from metabolism of 1.1.2- trichloroethane by cytochrome P-450. 1,1,2-Trichloroethane and its metabolites can also react with glutathione which may possibly give rise to a glutathione episulfonium ion, a reactive intermediate which has been implicated in the toxicity of chemicals like 1,2-dichloroethane (NTP, 1991). The reactive metabolites generated by both pathways are capable of binding to cellular macromolecules. Following exposure of rats and mice to a single dose of radiolabeled 1,1,2trichloroethane, the radiolabel was covalently bound to DNA, RNA, and protein of the liver, kidney, lung, and stomach (Mazzulo et al. 1986). Binding to DNA was greatest in the liver, and was greater in mice than rats, correlating with the carcinogenic potency of 1,1,2-trichloroethane (NCI, 1978). In vitro studies demonstrate that binding of the radiolabel is decreased by the addition of glutathione, suggesting that cytochrome P-450-mediated metabolism is the primary activating pathway for metabolites to bind to these macromolecules (Mazzulo etal. 1986).
Binding to key cellular molecules following metabolic activation is offered as a potential mechanism of action for the subchronic toxicity and carcinogenic effects of 1,1,2-trichloroethane on the liver. The rapid onset ofthe anesthetic effects of chlorinated solvents in general precludes the involvement of a metabolite in the mechanism of action (Cassaret and Doull, 1996), and suggests that the neurological effects of 1,1,2-trichloroethane are associated with the parent compound. Information regarding the potential mechanism(s) of action for 1,1,2-trichloroethane-induced immunotoxicity
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were not located,: but we propose to use parent chemical present in "the lymphoid tissue as an appropriate internal dose metric (see Section 3.9).
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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 for direct contact (i.e. portal of entry) effects on the lung following inhalation exposures (see Figure 1). 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 1,1,2-trichloroethane based on the following rationale:
Macroscopic evaluation of lungs revealed no effects in rats acutely exposed to concentrations exceeding 1,000 ppm (Bonnet etal. 1980).
Preferential accumulation of 1,1,2-trichloroethane in the lungs following inhalation exposures does not appear to occur. Rather, 1,1,2-trichloroethane rapidly distributes to other tissues following exposure. For example, following inhalation exposure of
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__________________________________ Pag mice to 1,000 ppm 1,1,2-trichloroethane for 1 hour, the levels of 1,1,2-trichIoroethane in the lungs (20-35 pg/g) were less than those observed in the blood and brain (4560 pg/g), kidney and liver (80 pg/g), and adipose tissue (600 pg/g) in mice (Takahara 1986).
Although evidence of lung damage (hemorrhagic, reddened areas) were observed following a single gavage dose above the LD50 (500 - 600 mg/kg-day) in mice (White et al 1985), this effect was not observed in mice exposed to lower doses, or in mice exposed to up to 38 mg/kg for 14 days, or in mice exposed to up to 384 mg/kg-day 1,1,2-trichloroethane in the drinking water (White et al. 1985). Furthermore, histopathological changes of respiratory tract tissues were not observed in mice exposed to up to 390 mg/kg-day 1,1,2-trichloroethane via gavage for 78 weeks (NCI, 1978).
One of the metabolically activating pathways for 1,1,2-trichloroethane in the liver {i.e., reductive dechlorination to a free radical) is not applicable for the lungs. Reductive dechlorination of chlorinated compounds by cytochrome P-450 requires conditions of low oxygen content, a condition that is not likely to be found in the lung.
Based on the low reactivity of 1,1,2-trichloroethane in the lung and the fact that 1,1,2-trichloroethane has low water solubility (see saline:air partition coefficient, Gargas et al, 1989b), this compound
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should be considered a Category 3 compound (USEPA, 1994) in which direct effects on the portal of entry are not expected.
3.2 Acute Toxicity
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I As described in the introduction, no adequate inhalation or oral studies were identified that assessed
acute toxicity. Since 1,1,2-trichloroethane is a Category 3 compound and based on the data
presented in Section 3.1, it would seem unlikely that 1,1,2-trichloroethane will have direct
toxicological effects on the lung (portal of entry). However, it is proposed that appropriate acute
toxicity data (including for the lung) be collected during both the oral and inhalation studies! needed
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to support PBPK model development and validation as described in Section 4.0. It is further
proposed that evaluation of acute toxicity be conducted as per OPPTS Guidelines for acute studies,
including the lung (i.e. gross necropsy and histopathology, [if deemed necessary]) as part of the
studies described in Section 4.0.
3.3 Subchronic Toxicity
Three studies were identified as potential candidates for route-to-route extrapolation, one study by White et al. (1985) and two (rats and mice) by NCI (1978). These studies are summarized briefly below.
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Page 10 White et al. (1985) - Groups of 32-48 male and 32-48 female CD-I mice were exposed to doses of 0, 4,4, 46, or 305 mg/kg-day (males), or 0, 3.9, 44, or 384 (females) 1,1,2-trichloroethane in the drinking water for 90 days. Drinking water consumption and body weight gain were reduced in concentration-dependent manner in male mice, but not in female mice. Absolute liver and kidney weights were decreased in male mice receiving 46 or 305 mg/kg-day; however, this was not observed when organ weights were expressed relative to body weight. Liver weights were significantly increased (absolutely and relatively) in female mice exposed to the highest dose. Absolute spleen and kidney weights were also elevated in this group. Hematological changes in male mice did not appear to be significant. However, hemoglobin and hematocrit values were significantly decreased in female mice exposed to the highest dose. Fibrinogen was increased in all exposed females, however this was not in a dose-dependent manner. Prothrombin time was significantly decreased in a dose-dependent manner in female mice exposed to 44 or 384 mg/kg-day. In male mice, serum cholesterol and serum alkaline phosphatase activity was significantly increased at the highest dose. In female mice, serum cholesterol and SGPT activity was significantly increased at the highest dose. Liver glutathione was significantly decreased in a dose-dependent manner in males exposed to the two highest doses, but was increased in females exposed to the highest dose. The dose-response information from female mice were used to base a LOAEL and NOAEL since they present a more consistent pattern of toxicity. This study identifies a LOAEL of 384 and a NOAEL of 44 mg/kg-day for increased liver weight, serum
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Page 11 cholesterol, and SGPT activity, and decreased (10%) hemoglobin and hematocrit in female mice.
NCI (1978) - Groups of 50 male and 50 female rats (Osbome-Mendel) were exposed to each of two dose levels via com oil gavage, 5 days/week for 78 weeks. Two control groups (vehicle and untreated) of 20 animals/sex were also included in the study. Initial doses of 0,30, and 70 mg/kg-day were raised to 0, 50, and 100 mg/kgday after week 8 since animals seemed to tolerate exposure. Time-weighted average doses of 0, 46, and 92 mg/kg-day were calculated by the study authors. There was no appreciable difference in mortality or body weight gain from controls. However, high mortality was noted in the vehicle control group. During the first 6 months, the incidence of clinical signs (i.e., appearance and behavior changes) were comparable between exposed and control animals. However, after 6 months, exposed animals exhibited a higher frequency of hunched appearance, rough fur, urine stains, wheezing, dyspnea, and squinted eye (sometimes with reddish exudate). Histopathological examinations revealed no significant differences between exposed and control animals for noncancer effects in any tissue site (including the respiratory tract). This study identifies a NOAEL of 92 mg/kg/day for histopathological changes.
NCI (1978) - Groups of 50 male and 50 female mice (B6C3F1) were exposed to each of two dose levels via com oil gavage, 5 days/week for 78 weeks. Two control groups (vehicle and untreated) of 20 animals/sex were also included in the study
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Page 12 Initial doses of 0, 150, and 300 mg/kg-day were raised to 0, 200, and 400 mg/kg-day after week 20 since the animals seemed to tolerate exposure. Time-weighted average doses of 0,195, and 390 mg/kg-day were calculated by the study authors. Mortality was significantly increased in exposed female mice, although this may not have been dose-related (mortality was greater in the low dose group compared to the high dose group). In any event, the number of animals surviving the entire exposure exceeded the requirement of the OPPTS guideline for number of animals/dose group (10/sex/group). For males, there was no appreciable difference in mortality from controls. Body weight gain was not affected in either sex. Clinical signs (/.e., appearance and behavior changes) were observed at comparable rates in exposed and control groups. Histopathological examinations revealed no significant differences between exposed and control animals for noncancer effects in any tissue site (including the respiratory tract). This study identifies a NOAEL of 390 mg/kg-day for noncancer histopathological effects in all tissues examined.
These two studies are compared with toxicity test guidelines from the USEPA (OPPTS) in Table 1. Based on this comparison, the study by White et al. (1985) appears to be adequate, however this study lacks a histopathological examination of tissues. For this reason, the liver histopathology results ofthe NCI (1978) study are used to supplement this study. As discussed in Section 3.1, direct contact (portal of entry) effects do not appear to be of concern for subchronic exposures to 1,1,2trichloroethane.
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Page 13 Based upon ther mechanism of action proposed in Section 2.0, it appears that cytochrome P-450 metabolites of 1,1,2-trichIoroethane are likely to be involved in the hepatic effects described above for both studies. Therefore, it is proposed that some internal measure of these metabolites (i.e., total amount metabolized in a 24 hour period) be modeled as the appropriate internal dose measure. Final choice of the internal dose measure will be decided after the internal dose/response relationship is evaluated.
3.4 Developmental and Reproductive Toxicity
The USEPA identified developmental and reproductive toxicity as a data gap for 1,1,2trichloroethane, based on the availability of only a single study (Seidenberg et al. 1986). This study is summarized below.
* Seidenberg et al. (1986) - Groups of 30 ICR/SIM mice were exposed to 0 (vehicle control) or 350 mg/kg-day 1,1,2-trichloroethane on days 8 through 12 of gestation via com oil gavage. Significant toxicity (3 deaths) were observed in exposed animals. However, no effects were observed on pup viability, pup weight, litter size, or terata. The dose level of 350 mg/kg-day serves as a LOAEL for maternal toxicity and a NOAEL for developmental effects.
This study is compared with USEPA (OPPTS) guidelines for developmental toxicity tests in Table 2. Although there are limitations to this study (single dose tested, short exposure duration), the study
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__Page 14 demonstrates a lack of developmental effects for 1,1,2-trichloroethane at a dose approximate to the maximum tolerable dose. For this reason, little information would be obtained from additional study since the testing of higher doses would only result in greater maternal toxicity, while the testing of lower doses would only result in the identification of lower NOAELs for developmental effects.
A weight-of-evidence argument is presented below for the developmental and reproductive effects of 1,1,2-trichloroethane. This argument includes issues regarding the toxicity, potential for exposure, and current regulations for 1,1,2-trichloroethane.
Toxicity
Although there is very little information regarding the potential for developmental effects of 1,1,2trichloroethane, data are available which suggest that developmental effects are not of primary concern for chlorinated ethanes in general. A summary of the available information regarding the developmental effects of chlorinated ethanes is provided in the table below (1,1,2-TCA = 1,1,2trichloroethane; 1,1-DCA= 1,1-dichloroethane; 1,1,1-TCA = 1,1,1-trichloroethane; 1,2-DCA= 1,2dichloroethane).
Summary of Developmental Toxicity Data for Chlorinated Ethanes
Study Seidcnberg et al. 1986
Species
Route
Mouse Oral
Duration Gd 8-12
EFTect None
LOAEL
NOAEL
None
350 mg/kg*
day
Chemical l.IJMCA
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Study Schwetz et al.
Species Route Rat Inhalation
Duration Gd 6-15
Effect Skeletal anomalies
LOAEL
NOAEL
6000
3800 ppm
Chemical 1,1-DCA
1974 BRRC 1987
Rat, rabbit
Inhalation Gd 6-15
Schwetz et al. Rat,
Inhalation Gd 6-15
Decreased fetal
ppm 6000
weight, delayed
ppm
ossification, extra rib
None
None
3000 ppm 875 ppm
1 Jl.l-TCA
1
1
l,i,l-TCA
1975 York et al.
mouse Rat
Inhalation 2 wk premating; Delayed ossification, 2100
None
1,1,1-TCA
1982 NTP 1988 Lane et al. 1982 Maurissen et
Rat Mouse
Oral Oral
Rat Oral
Gd 1-20 70 d Multigeneration
reduced clavicle None None
Gd 6-10
None
ppm None None
None
3.5 mg/kg-day 1,1,1-TCA
1000 mg/kgday 750 mg/kg-
1,1,1-TCA
1 1
1,1,1-TCA
al. 1994 George et al.
Rat
Oral
2 wk premating None
None
day 30 ppm
1,1,1-TCA
1989
through PND 21
(drinking
Payan et al.
Rat
Inhalation Gd 6-21
None
None
water) 300 ppm
1,2-DCA
1995 Rao et al.
Rat, Inhalation Gd 1-21
None
None
150 ppm
1,2-DCA
1980 Lane et al.
rabbit Mouse
Oral
Multigeneiation None
None
50 mg/kg-day 1,2-DCA
1982
In general, most studies have reported either no developmental effects or effects only at very high concentrations of chlorinated ethanes. Therefore, when the negative results of the Seidenberg et al. (1986) study are considered along with the weight*of-negative evidence for structurally related compounds, additional study on the developmental effects of 1,1,2-trichloroethane do not appear to
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be necessary, particularly if potential human exposures are minimal and are likely to be less than minimal potential risks estimated for carcinogenicity (see below).
Reproductive toxicity does not appear to be a sensitive endpoint for 1,1,2-trichloroethane based on the following rationale. In general, there are three mechanisms by which chemicals may affect reproduction:
(1) Direct toxic action on reproductive tissues;
(2) Indirect action on reproductive tissues through endocrine system modulation; and
(3) Indirect action on reproductive behavior through central nervous system effects.
Under the assumption that 1,1,2-trichloroethane is capable of producing reproductive effects, endocrine system modulation does not appear to be a likely mechanism for 1,1,2-trichloroethane since there is no evidence which suggests that 1,1,2-trichloroethane is capable of interacting with hormone receptors. In fact, the chemical structure for 1,1,2-trichloroethane suggests that such interactions are unlikely. Additionally, since CNS effects (anesthesia) are generally only observed following relatively high exposures to 1,1,2-trichloroethane (ATSDR, 1989), reproductive behavior effects are unlikely as well. Instead, the mechanism by which 1,1,2-trichloroethane may impact reproduction most likely involves the direct toxic action of 1,1,2-trichloroethane on reproductive tissues. However, studies in which reproductive tissues were histopathologically evaluated have generally reported negative
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Pag results. For example, no histopathological effects were observed in the testes, prostate, tunica vaginalis, uterus, mammary gland, and ovary in rats exposed orally to 46-92 mg/kg-day or in mice exposed orally to 195-390 mg/kg-day for 78 weeks (NCI, 1978).
Additionally, available data suggest that reproductive effects are not of primary concern for chlorinated ethanes in general. A summary of the available information regarding the reproductive effects ofchlorinated ethanes is provided in the table below (1,1,2-TCA =1,1,2-trichloroethane; 1,1DCA = 1,1-dichloroethane; 1,1,1-TCA = 1,1,1-trichloroethane; 1,1,2,2-TCA = 1,1,2,2tetrachloroethane; 1,2-DCA= 1,2-dichloroethane).
Summary of Reproductive Toxicity Data for Chlorinated Ethanes
Study NCI 1978
NCI 1978
Lane et al. 1982 Rao et al. 1980 NTP 1988
Species Rat, mouse
Rat, mouse
Mouse
Rat
Rat
Route Oral
Oral
Oral
Duration 78 wk
Effect No
LOAEL None
NOAEL 92-390
Chemical l.U-TCA
histopathological
mg/kg-day
78 wk
changes No
None
284 mg/kg-
1,1,2,2-TCA
histopathological
day
Multigeneration
changes None
None
SO mg/kg-day 1,2-DCA
Inhalation Single generation
Oral 70 d
None None
None
150 ppm
1,2-DCA
None
3.5 mg/kg-day 1,1,1-TCA
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Study NCI 1977
Species Rat, mouse
Route Oral
Duration 78 wk
Effect No
LOAEL None
NOAEL 1500-5615
histopathological
mg/kg-day
changes
Chemical 1,1,1-TCA
Based on the lack ofhistopathological effects of 1,1,2-trichloroethane on reproductive tissues, along with the negative available data on structurally related compounds and very low human exposure potential (see below), additional study on the reproductive effects of 1,1,2-trichloroethane does not appear to be necessary.
Exposure The extremely low human exposure to 1,1,2-trichloroethane is due to the fact that virtually the entire U.S. production of 1,1,2-trichloroethane is as a captive or "site-limited" intermediate in the production ofvinylidene chloride (VDC) or as a byproduct of the production of ethylene dichloride (EDC). Thus, 1,1,2-trichloroethane is found primarily in the vicinity of VDC and EDC production facilities, where in many cases the 1,1,2-trichloroethane is not isolated. The one manufacturer that markets 1,1,2-trichloroethane sells less than 3 million pounds annually. The small group (fewer than 10) of its customers use 1,1,2-trichloroethane as a carrier solvent in the manufacture of other materials. Two of these customers have indicated that they intend to switch to alternative carriers. These users also recycle/recover as much 1,1,2-trichloroethane in their operations as possible.
Available data on the concentrations of 1,1,2-trichloroethane in the environment are consistent with the foregoing paragraph. The Agency for Toxic Substances and Disease Registry (ATSDR) has
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_Page 19 stated that "whfcre 1,1,2-trichloroethane is found, levels appear to be about 10-50 ppt [parts per trillion] " Toxicological Profilefor 1,1,2-Trichloroethane (1989), 63. These concentrations would have been undetectable without advances in detection capability. The low environmental exposure is consistent with available information as to use. ATSDR found that "no use with significant consumer and general population exposures has been identified." Id., 61. Moreover, there is some doubt as to whether 1,1,2-trichloroethane meets the criteria articulated in EPA's so-called "B policy" for "substantial release" and "substantial human exposure," as will be addressed in greater detail in the comments of the HAP Task Force on the proposed test rule. For present purposes, it may be noted that the preamble to the proposed test rule does not identify any exposure as meeting the threshold for "substantial human exposure" except for 1,036 workers -- the threshold being 1,000 workers. The HAP Task Force will be providing a better estimate of the number ofworkers actually exposed to 1,1,2-trichloroethane as part of its comments.
Regulations and Guidelines
On top of the fact that human exposure to this site-limited intermediate is extremely low, EPA's regulatory structure would appear to make superfluous any determination as to the reproductive or developmental toxicity of 1,1,2-trichloroethane. As discussed above, the most significant potential exposure would appear to be from production facilities for VDC or EDC. VDC, EDC, and 1,1,2trichloroethane obviously are subject to regulation under the MACT standards for chemical production facilities, and any additional controls imposed as part of the residual risk determination for VDC and EDC will reduce exposure to 1,1,2-trichloroethane. The Agency's Integrated Risk
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Page 20 Information System (IRIS) lists a unit risk estimate or cancer potency for 1,1,2-trichloroethane of 1.6xl0'5 (pg/m3)*1. The maximum 8-hour time-weighted-average (TWA) exposure allowed for 1,1,2-trichloroethane is 10 parts per million (1.8 mg/m3), 29 C.F.R. 1910.1000, Table Z, and the American Conference of Governmental Industrial Hygienists (ACGIH) lists it as an animal carcinogen (A3). In drinking water, the maximum contaminant level goal (MCLG) is 3 pg/1, which according to EPA corresponds to a theoretical cancer risk of 10'5, and the maximum contaminant level (the enforceable limit) is 5 pg/1, the same as for organic contaminants which have an MCLG of2ero. The HAP Task Force is aware of no instance where the allowable exposure level for a substance so regulated has ever been reduced on account of such substance being identified as a reproductive or developmental toxin.
A specific illustration of these points is provided by an evaluation of 1,1,2-trichloroethane outside of one of the fewer than 20 U S. facilities where VDC or EDC is produced, specifically, the Dow Chemical Company facility in Lake Jackson, Texas. Actual monitoring data in the community near the facility showed that out of 306 samples, 97% were non-detects at 0.55 pg/m3 (0.0001 ppm). Only three values exceeded the detection limit. Put another way, only three samples exceeded an upper bound cancer risk of 9x10"6 (using the EPA unit risk estimate of 1.6x10_5). It is therefore inconceivable that any additional reproductive/developmental studies would result in setting exposure levels below those established for carcinogenic effects.
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3.5 Neurotoxicity
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Three studies were identified as potential candidates for route-to-route extrapolation, one fj>r acute
effects. White etal. (1985), and two for longer-term effects, both of which were conductedjby NCI
(1978). These studies are summarized briefly below.
I
White et al. (1985) - Seven groups of 8 male and female CD-I mice were administered a single dose of 200 to 600 mg/kg 1,1,2-trichloroethane via gavage. Sedation and loss of righting reflex were noted in mice soon after receiving a single gavage dose of 450 mg/kg 1,1,2-trichloroethane or more. These animals recovered
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four hours after exposure. No gross changes to the central nervous system were noted upon necropsy. This study identifies an acute LOAEL of 450 mg/kg for the anesthetic effects of 1,1,2-trichloroethane.
NCI (1978)- Groups of 50 male and 50 female rats (Osbome-Mendel) were exposed to each of two dose levels via com oil gavage, 5 days/week for 78 weeks. Two control groups (vehicle and untreated) of 20 animals/sex were also included in the study. Initial doses of 0, 30, and 70 mg/kg-day were raised to 0, 50, and 100 mg/kgday after week 8 since animals seemed to tolerate exposure. Time-weighted average doses of 0, 46, and 92 mg/kg-day were calculated by the study authors. During the first 6 months, the incidence clinical signs (i.e., appearance and behavior changes) were comparable between exposed and control animals. However, after 6 months.
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Page 22 exposed animals exhibited a higher frequency of hunched appearance, rough fur, urine stains, dyspnea, and squinted eye (sometimes with reddish exudate). Histopathological examinations revealed no significant differences between exposed and control animals for noncancer effects in the brain or nerves. This study identifies a NOAEL of 92 mg/kg-day for histopathological effects on the central nervous system.
NCI (1978) - Groups of 50 male and 50 female mice (B6C3F1) were exposed to each of two dose levels via com oil gavage, 5 days/week for 78 weeks. Two control groups (vehicle and untreated) of 20 animals/sex were also included in the study. Initial doses of 0, 150, and 300 mg/kg-day were raised to 0, 200, and 400 mg/kg-day after week 20 since the animals seemed to tolerate exposure. Time-weighted average doses of 0, 195, and 390 mg/kg-day were calculated by the study authors. Clinical signs (i.e., appearance and behavior changes) were observed at comparable rates in exposed and control groups. Histopathological examinations revealed no significant differences between exposed and control animals for noncancer effects in the brain and nerves. This study identifies a NOAEL of 390 mg/kg-day for histopathological changes.
These studies are compared with toxicity test guidelines from the USEPA (OPPTS) in Table 3. Based on this comparison, there seems to be a lack of evaluation of subtle neurological effects of
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1,1,2-trichloroetKane. However, we still believe that these studies should be considered adequate for evaluating neurological effects.
We propose that the parent chemical in the central nervous system is likely responsible for the effects described above and that the parent compound in the CNS (or proportionately in the blood) be modeled as the appropriate dose measure.
3.6 Carcinogenicity
Two studies were identified as potential candidates for route-to-route extrapolation, both of which were conducted by NCI (1978). These studies are summarized briefly below.
NCI (1978) - Groups of 50 male and 50 female rats (Osbome-Mendel) were exposed to each of two dose levels via com oil gavage, 5 days/week for 78 weeks. Two control groups (vehicle and untreated) of 20 animals/sex were also included in the study. Initial doses of 0, 30, and 70 mg/kg-day were raised to 0, 50, and 100 mg/kgday after week 8 of exposure. Time-weighted average doses of 0, 46, and 92 mg/kgday were calculated by the study authors. High mortality was noted in the vehicle control group. Animals were followed up to 34 weeks after exposure. No statistically significant increase in tumor incidence was observed in exposed rats. The authors concluded that there was no convincing evidence for the carcinogenicity of
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Page 24 b 1,2-trichloroethane in rats. However, the maximum tolerable dose may not have been achieved in this study
NCI (1978) - Groups of 50 male and 50 female mice (B6C3F1) were exposed to each of two dose levels via com oil gavage, 5 days/week for 78 weeks. Two control groups (vehicle and untreated) of 20 animals/sex were also included in the study. Initial doses of 0, 150, and 300 mg/kg-day were raised to 0, 200, and 400 mg/kg-day after week 20 of exposure. Time-weighted average doses of 0, 195, and 390 mg/kgday were calculated by the study authors. Animals were followed for up to 12 weeks following exposure. Mortality was significantly increased in exposed female mice, however this may not have been dose-related (mortality was greater in the low dose group compared to the high dose group). The incidence of hepatocellular carcinomas was significantly increased in exposed mice of both sexes. Additionally, adrenal pheochromocytomas were elevated in female mice. The authors concluded that 1,1,2trichloroethane was carcinogenic to mice under the conditions of this bioassay.
These studies are compared with toxicity test guidelines from the USEPA (OPPTS) in Table 4 and are considered adequate. Although there are limitations in these studies (i.e., less than lifetime exposure, mortality in vehicle control group in rats, and in low dose female mice), these limitations do not detract from the results of the mouse study, which are clearly positive for liver tumors. Furthermore, the USEPA has placed sufficient confidence in this study as to base both the oral and inhalation cancer potency factors on its results (IRIS, 1996) For this reason, a repetition of the
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Page 25 cancer bioassay for 1,1,2-trichloroethane does not appear to be a justified use of laboratory animals, particularly in light of the fact that a chronic bioassay (negative results reported following chronic subcutaneous injection) was also recently conducted for this chemical (Norpoth et al 1988). Extrapolation of the NCI (1978) studies to inhalation exposures using PBPK modeling is offered as an appropriate alternative to repeating the cancer bioassay.
Based upon the mechanism ofaction proposed in Section 2.0, it appears likely that cytochrome P-450 metabolites of 1,1,2-trichloroethane are likely to be involved in the hepatic effects described above for both studies. Therefore, it is proposed that some internal measure of these metabolites (/.., total amount metabolized in a 24 hour period) in the liver be modeled as the appropriate internal dose measure.
3.7 In Vivo Cytogenicity
Two key studies were identified regarding the potential cytogenicity of 1,1,2-trichloroethane, an in vivo study (Mazzulo et al, 1986), and an in vitro study (Doherty et al, 1996). These studies are summarized below.
Mazzulo et al (1986) - A group of 4 Wistar rats and 12 BALB/c mice were administered a single dose of 6.35 umol/kg of radiolabeled l, 1,2-trichloroethane via intraperitoneal injection. Twenty-two hours after exposure, radiolabel was found covalently bound to DNA, RNA, and proteins of the liver, kidney, lung, and stomach
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Page 26 The DNA binding in all tissues was greater in mice than rats, thus correlating with observations on the relative susceptibility of these species to 1,1,2-trichloroethaneinduced carcinogenesis (NCI, 1978).
Doherty et al. (1996) - In this in vitro study, metabolically competent human cell lines (AHH-1 MCL-5 and h2El) were exposed to 1,1,2-trichloroethane. These cell lines differed with respect to the cytochrome P-450 isozyme expressed. Micronuclei induction (3.5-4 fold) was noted in 2 of the 3 cell lines.
These studies clearly demonstrate a genotoxic effect for 1,1,2-trichloroethane in vivo, as well as a cytogenetic effect for 1,1,2-trichloroethane in vitro. Therefore, it is reasonably safe to assume that both genotoxic and cytogenetic effects would be observed following inhalation exposures to 1,1,2trichloroethane. In fact, we will assume a genotoxic mechanism when the positive oral NCI (1978) bioassay data are extrapolated to equivalent inhalation exposures (see Section 3.6), Since further study on the cytogenicity of 1,1,2-trichloroethane would provide little additional insight regarding the mechanism of action for tumor induction, additional study is not recommended.
3,8 Immunotoxicity
Two studies were identified as potential candidates for route-to-route extrapolation, Sanders et al. (1985) and NCI (1978). These two studies are summarized briefly below.
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Page 27 Sanders et al. (1985) - Groups of 32-48 male and 32-48 female CD-I mice were exposed to doses of 0, 4.4, 46, or 305 mg/kg-day (males), or 0, 3.9, 44, or 384 (females) 1,1,2-trichloroethane in the drinking water for 90 days. Drinking water
jconsumption and body weight gain were reduced in concentration-dependent manner
in male mice, but not in female mice. Spleen weights were decreased in 'a dosedependent manner in male mice, but were significantly increased in females exposed to the highest dose. Bone marrow status as indicated by DNA synthesis was not affected by exposure. Cell-mediated immunity, as measured by delayed-type hypersensitivity and popliteal lymph node proliferation responses to sheep erythrocytes, was unaffected in both sexes. However, humoral immune status was
I
significantly depressed in both sexes in a dose dependent manner. For example, hemagglutination titers were significantly depressed in both sexes at the two highest doses. In addition, lymphocyte responsiveness to Con A and LPS was significantly depressed in female mice exposed to the highest dose, but unaffected in exposed male mice. This study identifies a LOAEL of 44-46 mg/kg-day and a NOAEL of 3.94.4 mg/kg-day for depression of humoral immunity.
NCI (1978) - Groups of 50 male and 50 female mice (B6C3F1) were exposed to each of two dose levels via com oil gavage, 5 days/week for 78 weeks. Two control groups (vehicle and untreated) of 20 animals/sex were also included in the study. Initial doses of 0, 150, and 300 mg/kg-day were raised to 0, 200, and 400 mg/kg-day after week 20 since the animals seemed to tolerate exposure. Time-weighted average
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closes of 0, 195, and 390 mg/kg-day were calculated by the study authors. Histopathological examinations revealed no significant differences between exposed and control animals for noncancer effects in immunological tissues (i.e., spleen, bone marrow, lymph nodes). This study identifies a NOAEL of 390 mg/kg-day for histopathological effects.
These two studies are compared with guidelines for conducting immunotoxicity tests from the USEPA (OPPTS) in Table 5. Based on this comparison, the study by Sanders et al. (1985) was considered adequate for route-to-route extrapolation. The primary limitation with this study is the lack of histopathological examination of immune system organs (spleen, thymus, lymph nodes). For this reason, the histopathology results from the NCI (1978) could be used to supplement this study.
In the absence of specific information regarding the potential mechanisms of action for 1,1,2trichloroethane-induced immunological effects, we propose that parent chemical present in the lymphoid tissue (i.e., spleen) is the appropriate dose metric leading to these effects.
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4.0 PBPK MODELS FOR 1,l,2-TRICHLOROETHANE
Page 29
This section summarizes the toxicokinetics of 1,1,2-trichloroethane and describes an existing PBPK model for this chemical in the rat. Studies needed to validate the rat model and to develop and validate a mouse model necessary to fill the data gaps described in Section 3.0 are proposed.
4.1 Toxicokinetics of l,l2-Trichloroethane
Available studies indicate that 1,1,2-trichloroethane is readily absorbed following oral and inhalation exposures (ATSDR, 1989). Following absorption, 1,1,2-trichloroethane distributes to all organs of the body, with a tendency for higher concentrations in adipose tissue and tissues with a high lipid content (ATSDR, 1989).
1,1,2-Trichloroethane is metabolized by the cytochrome P-450 enzyme system via oxidative and
reductive pathways. Chloroacetic acid is generated through oxidative metabolism and reductive
metabolism generates free radicals. 1,1,2-Trichloroethane also reacts with glutathione, yielding
S-carboxymethyl cysteine and thiodiacetic acid. Mice appear to have a greater capacity than rats in
metabolizing 1,1,2-trichloroethane (Mitoma et at. 1985) and a greater capacity to generate reactive
intermediates capable ofbinding DNA, RNA, and proteins (Mazzulo et at. 1986). Since cytochrome
P-450 and glutathione are found in the greatest amounts in the liver, there is likely to be a first-pass
metabolic effect for 1,1,2-trichloroethane following oral exposure which may alter its distribution and
toxicity.
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____________________________________________ Following exposure to an inhaled dose of radiolabeled 1,1,2-trichloroethane, approximately 2.9% of the radiolabel was excreted in air after 1 hour in a volunteer (Morgan et al. 1970). Urinary excretion was approximately 0.01%/min. The half-life of 1,1,2-trichloroethane in various tissues has been determined in mice following inhalation exposure (Takahara, 1986) with overall half-life reported to be 49.3 minutes. Following oral exposure, approximately 7-10% ofthe dose was excreted unchanged by the lungs, 3-7% as exhaled C02, 72-87% as urinary metabolites and 1% in the feces (Mitoma et al, 1985; Yllner etal. 1971).
4.2 Existing PBPK Model for l,l*2-TrichIoroethane
A PBPK model for 1,1,2-trichloroethane dosimetry in the rat has been described (Gargas et al., 1989a; Gargas et al., 1990). This model accounted for inhalation of 1,1,2-trichloroethane, distribution to liver, fat, richly perfused and slowly perfused tissues, and elimination via exhalation and P-450 mediated metabolism in the liver. Partition coefficients were determined by vial equilibration techniques and the kinetic constants of P-450 metabolism were determined in vivo by modeling exhaled breath concentrations following inhalation exposures at constant concentrations of 1,1,2-trichloroethane.
Unfortunately, the model was not validated for inhalation exposure and oral absorption was not part of the original model description. In addition, no inhalation or oral model currently exists for 1,1,2-trichloroethane in mice. Validated PBPK models capable of describing inhalation and oral
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Page 31 exposures in rats and mice are necessary to perform the route-to-route extrapolations described in Section 3 above.
4.3 Proposed Model Development and Validation
The following studies are proposed to develop and validate the models described in the previous section. Detailed protocols are not described here, but rather, a general description ofthe types of studies necessary to develop and validate rat and mouse models are presented for USEPA consideration.
4.3.1 Validation of Rat Inhalation Route
Validation of a PBPK model involves testing how well a particular model performs at predicting experimental data not used for model development. Ideally, predictions of time courses of internal dose measures during and following exposure are preferable, although this is quite impractible for most reactive metabolites. Usually, blood and/or tissue concentration time course data for the parent chemical have been used for model validation (Corley et al, 1990; D'Souza et al, 1987; Reitz et al, 1996) and are appropriate if it can be demonstrated that the dose measure of interest (i.e., reactive metabolites) is dependent on the kinetic behavior of parent chemical.
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Page 32 To validate the inhalation portion of the rat PBPK model, the following experiments are proposed:
Measure 1,1,2-trichloroethane blood concentration time courses during and following 4-6 hour inhalation exposures at several constant inhaled concentrations.
Use existing inhalation rat model to predict these time courses.
Compare model output to actual data
4.3.2 Develop Model and Validate Oral Exposures
Uptake of chemicals such as 1,1,2-trichloroethane from the gastrointestinal tract have been described using a first-order rate constant for absorption. The rate constants vary when chemical is delivered in water or in com oil (Corley et al., 1990; Gargas, 1990; Ramsey and Andersen, 1984). However, the rate constants seem to be about 1.0 hr'1 and 5.0 hr'1 for water and oil vehicles, respectively, for compounds similar to 1,1,2-trichloroethane (i.e., chloroform, methylene chloride, 1,1,1trichloroethane). Therefore, we propose to use these constants as default values for oral absorption and test their ability to predict oral uptake in the following validation experiments. If validation of this model fails, it will be necessary to more formally measure the rate constants, possibly using an exhaled breath technique (Gargas et al. 1989a).
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Page 33 To validate the rat oral absorption portion of the model, the following experiments are proposed:
Deliver several concentrations of 1,1,2-trichloroethane in com oil by gavage and
measure blood time course concentrations of parent compounds.
j
I
Compare data to model predictions.
Deliver several concentrations of 1,1,2-trichloroethane in water by gavage and
measure blood time course concentrations of parent chemical if technically feasible
(i.eif enough chemical can be dissolved in water to result in measurably blood
I
concentrations).
Compare to model predictions.
4.3.3 Develop Initial Mouse Model
In vivo metabolic constants and in vitro blood:air partition coefficients are needed to develop an initial PBPK model for the mouse. It is proposed that metabolic constants be derived using the exhaled breath technique developed by Gargas et al. (1989a) and that blood:air partition coefficients be determined by vial equilibration techniques (Gargas et al. 1989b). Tissue, air partition coefficients are not usually necessary for mouse tissues if rat tissue:air values are available since it is technically difficult to obtain "clean" mouse tissue samples, and rat and mouse tissue-air partition coefficients
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have been shown to be quite similar. The rat tissue:air partition coefficients can be divided by the mouse blood:air values to estimate the tissue:blood coefficients used in the model. This approach has been used successfully in previous work (Corley et at., 1990; Reitz et at., 1996).
4.3.4 Inhalation and Oral Route Validation
The experiments necessary to conduct the model validation for the mouse will be essentially the same as described above for the rat (Sections 4.3.1 and 4.3 .2). Selection of inhalation concentration ranges and oral dose ranges may vary and will be determined using preliminary PBPK models as aids to experimental design.
It is anticipated that all model development and validation studies will be described in manuscripts that will be submitted to peer-reviewed journals for publication. These published models will then be used to conduct the dose-route extrapolations necessary to fill the data gaps for 1,1,2trichloroethane identified in the test rule for subchronic toxicity, neurotoxicity, carcinogenicity and immunotoxicity. The studies, species, toxicological endpoints and internal dose measures proposed to fill the data gaps via PBPK modeling are summarized in Table 6.
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Page 35
5.0 REFERENCES
ATSDR. 1989. Toxicological Profile for 1,1,2-trichloroethane. Agency for Toxic Substances and Disease Registry, Atlanta, GA,
Bonnet P, Francin JM, Gradiski D, et al. 1980. [Determination of the median lethal concentration of principle chlorinated aliphatic hydrocarbons in the rat ] Arch Mai Prof Med Trav Secur Soc 41:317-321. (French).
Cassaret and Doull. 1996. Toxicology: The basic science of poisons. 5th Edition. New York, NY: Macmillan Publishing Co.
Corley RA, Mendrala AL, Smith FA, et al. 1990. Development of a physiologically-based pharmacokinetic model for chloroform. Toxicol Appl Pharmacol 103:512-527.
Doherty AT, Ellard S, Parry EM, et al. 1996. An investigation into the activation and deactivation of chlorinated hydrocarbons to genotoxins in metabolically competent human cells. Mutagenesis 11(3):247-274.
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.
Gargas ML. 1990. An exhaled breath chamber system for assessing rates of metabolism and rates of gastrointestinal absorption with volatile compounds. J Am Coll Toxicol 9:447-453.
Gargas ML, Andersen ME. 1989a. Determining kinetic constants of chlorinated ethane metabolism in the rat from rates of exhalation. Toxicol Appl Pharmacol 99:344-353.
Gargas ML, Burgess RJ, Voisard DJ, et al. 1989b. Partition coefficients of low molecular weight volatile chemicals in various liquids and tissues. Toxicol Appl Pharmacol 98: 87-99.
Gargas ML, Clewell HJ, Andersen ME. 1990. Gas uptake techniques and the rates of metabolism of chloromethanes, chloroethanes, and chloroethylenes in the rat. Inhal Toxicol 2:295-319.
Gerrity TR, Henry CJ. 1990. Principals of route-to-route extrapolation for risk assessment. New York, NY: Elsevier, p. 3-12.
IRIS. 1996. Integrated Risk Information System. U.S. Environmental Protection Agency.
Mazzulo M, Colacci A, Grilli S, et al. 1986. 1,1,2-Trichloroethane: Evidence of genotoxicitv from short-term tests. Jpn J Cane Res 77:532-539.
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________________________________________________ Pag
Mitoma C, Tyson CA, Riccio ES. 1985. Investigation of the species sensitivity and mechanism of carcinogenicity of halogenated hydro-carbons final report EPA Contract 68-01-5079. EPA/OTS; Document #40-842-8424225.
Morgan A, Black A, Belcher DR. 1970. The excretion in breath of some aliphatic halogenated hydrocarbons following administration by inhalation. Ann Occup Hyg 15:273-282.
NCI. 1978. Bioassay of 1,1,2-trichloroethane for possible carcinogenicity. Report. ISS DHEW/PUB/NIH-78-1324. NCI-CG-TR-74. PB-283337.
Norpoth K, Heger M, Muller G, et al. 1988. Investigations on the metabolism and carcinogenicity of 1,1,2-trichloroethane. J Cancer Res Clin Oncol 114:158-162.
NTP. 1991. Toxicity studies of 1,2-dichloroethane (ethylene-dichloride) in F344/N rats, Sprague Dawley rats, Osbome-Mendell rats, and B6C3F1 mice. Research Triangle Park, NC. National Toxicology Program. Pub. 91-3123.
Ramsey JC, Andersen ME. 1984. A physiologically based description of the inhalation pharmacokinetics of styrene monomer in rats and humans. Toxicol Appl Pharmacol 72:159-175.
Reitz RH, Gargas ML, Andersen ME, et al. 1996. Predicting cancer risk from vinyl chloride exposure with a physiologically based pharmacokinetic model. Toxicol Appl Pharmacol 137:253267.
Sanders VM, White KL Jr, Shopp GM Jr, et al. 1985. Humoral and cell-mediated immune status of mice exposed to 1,1,2-trichloroethane. Drug Chem Toxicol 8:357-372.
Seidenberg JM, Anderson DG, Becker RA. 1986. Teratogenesis, carcinogenesis, and mutagenesis. 6:361-374.
Takahara K. 1986. [Experimental study on toxicity of trichloroethane: Part 1. Organ distribution of 1,1,1- and 1,1,2-trichloroethanes in exposed mice ] Okayama Igakkai Zasshi 98:1079-1090. (Japanese).
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 KL Jr, Sanders VM, Barnes DW, et al. 1985. Toxicology of 1,1,2-trichloroethane in the mouse. Drug Chem Toxicol 8:333-356.
Yllner S. 1971. Metabolism of 1,1,2-trichloroethane-l,2-(14)C in the mouse Acta Pharmacol Toxicol 30:248-256
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00 1?91B5
00NFTDFNTTA1
Figure 1 Decision Tree for Route-to-Route Extrapolation
1,1,2-Trichloroethane*
1,1,2-Trichloroethane
Do adequate toxicological data exist for at least one route?
No
Can SAR or analogy be used? No
Yes
Option I: Collect data
Option 2: Use data for analogous compound
Adapted from Gerrity and Henry (1990)
Yes
Is toxicity remote from contact site likely?
No Yes
Option 3: Collect data on all relevant routes
T Option 4: Candidate
for route-to-route extrapolation
n:\hapsrule\112-TABS.XLS
11/19/96 12:53 PM
Figure 2 Comparison of Current and Proposed PBPK Models for 1,1,2-
Trichloroethane
Current Model
Proposed Model
Inhaled
Alveolor Space ---- Exhaled
^ Lung Blood
>|
Venous Circulation
^Richly Perfused ' Tissue
Arterial Circulation
^ Metabolism
Inhaled
> Alveolor Space
> Lung Blood
> Exhaled ->
Fat
Venous Circulation
Richly Perfused Tissue
's
Poorly Perfused Tissue
------------------- ----- 1 Central Nervous
System
Arterial Circulation
Spleen
OONFTDFNT T
Liver
Metabolism
Oral Dose
Table 1 Comparison of Candidate Studies to OPPTS Test Guidelines for Subchronic Toxicity
Candidate Studies for Route Extrapolation
Study: White et al. I9SS
NCI t97R
Subchronic effects observed: Liver, hematological
Lack of histopathological
LOAEL: 44-46 mg/kg-day
changes None
NOAEL: 3.9-4.4 mg/kg-day
400 mg/kg-day [CHECK]
Parameter___________________________Recommended
Test species
Rat (other mammal)
Mouse
Mouse
Strain
Common lab
CD-I
B6C3FI
Age
Young, healthy
Young, healthy
Young, healthy
Sex Both
Both
Both
Number of animals
10/sex/dose, more if interim sacrifice
32-48/sex/group
50/sex/dose
Husbandry
Standard
Adequate
Some limitations, but adequle
Control Groups
Concurrent sham or vehicle control
Vehicle control
overall Vehicle and untreated controls
Concentration level and selection
> =4 (including control)
Male (0, 4.4, 46, and 305
0, 195, 390 mg/kg-day
mg/kg-day); Female (0, 3.9, 44,
Limit dose
Maximum tolerable dose achieved
and 384 mg/kg-day) Range determined based on 1- Range determined based on 6
day, and 14-day range finding week study
Intermediate dose
Provides gradiation of effects
studies Adequate
NA
Lowest dose level
Should provide NOAEL
Adequate
Adequate
Administration of the substance
6 hr/day, 7 day/week
Daily, 90 days
5 d/wk, 78 wks
Observation period
90 days
90 days
630 days
Exposure specifications and physical Nose only or whole body
Oral (drinking water)
Oral (oil gavage)
measurements
Observation of animals Clinical pathology
Mortality (daily), clinical (weekly) Hematology and clinical chemistry
Adequate Adequate
Adequate Not evaluated
Ophthahnological examinations
High-dose and control
Not applicable
Histopathological examination
< irois pathology Hi stops! hology
Gross necropsy and organ weights ------- ^Adequate Histopathology, including respiratory tract Not evaluated
of ocular tissues Adequate"Dated methods, but adequate
Kcstills rcjxirting
Tabular results, statistics
overall Tabular means & SDs, Duncan's Tabular incidences
Evaluation
Adequately described
multiple range test Adequate
Adequate
ft h it
DO 1 ? 9 1 0 7
GONF TDFNT T
bz zo>
3et V,0 z-
-I 33
-h * >
Table 2 Comparison of Studies to OPPTS Guidelines for Developmental Toxicity
_________ Candidate for Route Extrapolation Seidenberg et al. (1986) Effect: No developmental effects observed LOAEL: None NOAEL: 350 mg/kg-day
Parameter________________________________ Recommended
Test Species
>2
Strain
Common laboratory
Sex Pregnant female
Route
Inhalation
Number of animals
>20/group (rats); > 12/group (rabbits)
Control group
Filtered air/vehicle
Concentrations
>4 (including control)
Dose Seletion
Characterizes dose response
Exposure Duration
6 hr/d
Observation Period
Gd 6-15 (rat); Gd6-18 (rabbit)
EnvironmentalConditions
Standard/monitored
Observation
Daily
Gross Necropsy
Utenis/embryo
Data Reporting
Tabular/detailed
Evaluation
Statistics/described
Mouse ICR/SIM Pregnant female Oral (oil gavage) 30/group Vehicle 0, 350 mg/kg-day Maternal toxicity observed (3/30 deaths) Daily Gd 8-12 Adequate Adequate Adequate Adequate Adequate
112 TABS XI.S
11/19/96 12:51 PM
Table 3 Comparison of Candidate Studies to OPPTS Test Guidelines for Neurotoxicity
Parameter Test species Age Sen Number of animals Control Groups
Concentration level and selection
Acute studies Sutx.ltionic and chrunic studies
Administration of the substance Combined protocol Time of testing
(`unciional observational battery
List ot measures
Molm atm u y Neuropathology Results Evaluation
n
oV zo
T| --( 1--*
o \> 2^
2S
Candidate Studies for Route Extrapolation
Study: White et al. (1985) - Acute NCI (1978) - Longer term NCI (1978) - Longer term
Neurological cITects observed: Sedation, loss of righting Lack of histopathological Lack of htslopatliologicai
reflex
changes for brain and nerves changes for brain and
LOAEL: 450 mg/kg-day
None
nerves None
NOAEL: 400 mg/kg-day
390
92
Recommended
Rat (mice or dog)
Mouse
Mouse
Young adult (>42 d)
Young adult
Young adult
Both
Both
Both
10/sex/dose
8/sex/dose
50/sex/dose
Concurrent sham or vehicle control
None
Vehicle and untreated
controls
> =4 (including control)
7 dose groups between 200 0, 195, 390 mg/kg-day
and 600 mg/kg
MTD; < 2g/kg
Adequate
NA
MTD; < 1 g/kg
NA Range determined by 6
week study
Appropriate route of exposure
Oral (water gavage)
Oral (oil gavage)
Combine with other endpoints
NA
NA
Acute (0, 8hr, 7d, 14 d); subchronic (0, 4 wk, 8 up to 14 days
Functional tests not
wk, 13 wk)
evaluated
Standard evaluations for appearance, behavior, Appearance and behavior Appearance and behavior
and functional integrity
evaluated grossly
Autonomic function, abnormal motor
Abnormal motor movements Not evaluated
movements, response to general and sensory
stimuli, alertness, grip strength, landing foot
splay, body weight, behavioral changes Individually assessed, automated
Not evaluated
Not evaluated
Neuropathological examinations
Gross necropsy only
Adequate
Tabular, per animal
Text only
Tabular incidences
Adequately described, statistics
Limited
Adequate
Rat Young adult Both 50/sex/dose Vehicle and untreated controls 0, 46, 92 mg/kg-day
NA Range determined by 6 week study Oral (oil gavage) NA Functional tests not evaluated Appearance and behavior evaluated grossly Not evaluated
Not evaluated Adequate Tabular incidences Adequate
Table 4 Comparison of Candidate Studies to OPPTS Test Guidelines for Carcinogenicity
n oo zn o
--i --
OV
3z v>O-* --I ~C
o
I>
Parameter Test species Strain Age Sex Number of animals Contra) Groups
Concentration level and selection Limit dose Intermediate dose Ix)west dose level Route exposure
Observation period Observation of animals Clinical pathology linmunoioxicity screen Grass necropsy llistopathology Results reporting Evaluation reporting
Candidate Studies for Route Extrapolation
Study: NCI (1978) - Mouse study
NCI (1978) - Rat study
Carcinogenic effects observed: Liver, adrenal tumors
None
Recommended Rats and mice Common lab strain Young, healthy (<8 wks) Both 50/sex/group Concurrent sham or vehicle control
Mouse B6C3F1 Young Both 50/sex/group Vehicle and untreated controls
Rat Osbome-Mendel Young
50/sex/group Vehicle and untreated controls
> =4 (including control) MTD; < 1000 mg/kg-day Provides gradation of effects Provides NOAEL Inhalation 6 hr (inhalation), 5-7 d/wk for 18 (mice) or 24 (rat) months Lifespan Morbidity (daily), clinical (weekly) at 12 and 18 months Optional Complete Complete Tabular, per animal, statistics Adequately reported
0, 195, 390 mg/kg-day Adequate Adequate NA Oral (oil gavage) 5 d/wk, 78 wk
630 days Adequate Not evaluated NA Adequate Adequate Incidence Adequate
0, 46, 92 mg/kg-day May not have been achieved Adequate NA Oral (oil gavage) 5 d/wk, 78 wk
777 days Adequate Not evaluated NA Adequate Adequate Incidence Adequate
' M I'M
Table 5 Comparison of Candidate Studies to OPPTS Test Guidelines for Immunotoxicity
Candidate Studies for Route Extrapolation
Study: Sanders et al. (198S);
NCI (1978)
White el at. 1985
Immunological effects observed: Humoral immune system Lack or histopathology
depression LOAEL: 44-46 mg/kg-day
None
NOAEL: 3.9-4.4 mg/kg-day
390 mg/kg-day
Parameter Test species Strain Age Sex Number of animals Husbandry Control & Test Substances Control Groups
Dose administration Concentration level and selection
Limit dose
t .owest dose level Administration of the substance Observation period Immunotoxicity tests
Results reporting
I s .ilu.ition reputing
Recommended Rat or mouse Common lab strain Young, healthy (6-8 wks) Both 6 - 10/sex/group Standard Purity and vehicle evaluated Concurrent sham or vehicle control
Inhalation > =4 (including control)
MTD
Provides NOAEL 30-90 days; 5-7 days/week 30-90 days Functional (antibody plaque forming cell assay or immunoglobin quantification); Enumeration of splenic or peripheral blood T cells, B cells and NK cells Tabular, statistics
Adequately described
Mouse CD-I Young, healthy Both 32-48/sex/dose Adequate 95%
Mouse B6C3F1 Young, healthy Both 50/sex/dose Adequate 92.70%
Vehicle control
Vehicle and untreated
Oral (drinking water)
controls Oral (oil gavage)
Male (0, 4.4,46, and 305 0, 195, 390 mg/kg-day
mg/kg-day); Female (0,
3.9, 44, and 384 mg/kgRange determined based on Range determined based
l-day, and 14-day range finding studies Adequate 90 days 90 days
on 6 week study
Adequate 5 d/wk, 78 wks 630 days
Complete
Histopathology of
lymph nodes, spleen,
bone marrow
Tabular means & SD,
Duncan's multiple range
lest Adequate
Tabular incidences Adequate
n I e t lifter t'iri ia
C O N FID E N T TAl,
Table 6 Summary of Pharmacokinetic Modeling Activities Proposed for l,l2-TrichIoroethane
Data Gap
Subchronic
Neurotoxicity
Carcinogenicity
Immunotoxiclty
Study
While et a). NCI (1978) NCI (1978) NCI (1978) NCI (1978) NCI (1978) White et al. NCI (1978)
(1985)
(1985)
Species
Mouse
Mouse
Mouse
Rat
Mouse
Rat
Mouse
Mouse
Route
Oral (drinking Oral (oil
Oral (oil
Oral (oil
Oral (oil
Oral (oil Oral (drinking Oral (oil
water)
eavage)
savage)
Ravage)
Ravage)
Ravage)
water)
Ravage)
Endpoint
Liver,
Lack of
Lack of
Lack of
Liver, adrenal Lack of tumors Humoral
Lack of
hematological histopathologic histopalhology histopalhology
tumors
immune system histopathologic
al changes to
to CNS
to CNS
depression
al changes
LOAEL
44-46 mg/kg-
the liver None
None
None
195 mg/kg-day
None
44-46 mg/kg-
None
dav day
NOAEL
3.9-4.4 mg/kg- 390 mg/kg-day 390 mg/kg-day 92 mg/kg-day
NA
NA 3.9-4.4 mg/kg- 390 mg/kg-day
dav day
Dose Metric
Total
Total
Parent
Parent
Total
Total
Parent
Parent
metabolized (P metabolized (P compound
compound metabolized (P metabolized (P compound
compound
450)
450)
450)
450)
Target Tissue
Liver
Liver
Central Central nervous nervous system system
Liver
Liver
Lymphoid
tissue (i.e.,
Lymphoid tissue (i.e.,
ioleen)
soleen)
DO 1 P 9 1 9 ?
OONFTDFNT T