EPA/635/R-00/006
TOXICOLOGICAL REVIEW
OF
CHLORAL HYDRATE
(CAS No. 302-17-0)
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
August 2000
U.S. Environmental Protection Agency
Washington, DC
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DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental Protection
Agency policy and approved for publication. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
Note: This document may undergo revisions in the future. The most up-to-date version will be
made available electronically via the IRIS Home Page at http://www.epa.gov/iris.
11
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CONTENTSTOXICOLOGICAL REVIEW for CHLORAL HYDRATE
(CAS No. 302-17-0)
FOREWORD v
AUTHORS, CONTRIBUTORS, AND REVIEWERS vi
1. INTRODUCTION 1
2. CHEMICAL AND PHYSICAL INFORMATION RELEVANT TO ASSESSMENTS 2
3. TOXICOKINETICS RELEVANT TO ASSESSMENTS 3
4. HAZARD IDENTIFICATION 6
4.1. STUDIES IN HUMANS - EPIDEMIOLOGY
AND CASE REPORTS 6
4.2. PRECHRONIC AND CHRONIC STUDIES
AND CANCER BIOASSAYS IN ANIMALS 8
4.2.1. Oral 8
4.2.2. Inhalation 12
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES 13
4.4. OTHER STUDIESNEUROLOGICAL, IMMUNOLOGICAL,
GENOTOXICITY, MECHANISTIC 14
4.4.1. Neurological Studies 14
4.4.2. Immunological Studies 15
4.4.3. Genetic Toxicity 16
4.4.4. Mechanistic Studies 17
4.4.4.1. Cell Proliferation 17
4.4.4.2. Oncogene Activation 18
4.4.4.3. Free Radicals and DNA Adduct Formation 18
4.4.4.4. Cell Communication 19
4.4.4.5. Peroxisome Proliferation 19
4.5. SYNTHESIS AND EVALUATION OF MAJOR NONCANCER
EFFECTS AND MODE OF ACTION (IF KNOWN) 19
4.6. WEIGHT-OF-EVIDENCE EVALUATION AND CANCER
CHARACTERIZATIONSYNTHESIS OF HUMAN, ANIMAL,
AND OTHER SUPPORTING EVIDENCE, CONCLUSIONS
ABOUT HUMAN CARCINOGENICITY, AND LIKELY
MODE OF ACTION 20
4.6.1. Susceptible Populations 22
4.6.1.1. Possible Childhood Susceptibility 22
4.6.1.2. Possible Gender Differences 23
in
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CONTENTS (continued)
5. DOSE-RESPONSE ASSESSMENTS 23
5.1. ORAL REFERENCE DOSE (RfD) 23
5.1.1. Choice of Principal Study and Critical Effect
With Rationale and Justification 23
5.1.2. Methods of Analysi sIncluding Model s
(PBPK, BMD, etc.) 23
5.1.3. RfD DerivationIncluding Application
of Uncertainty Factors (UF) and Modifying Factors (MF) 23
5.2. INHALATION REFERENCE CONCENTRATION (RfC) 24
5.3. CANCER ASSESSMENT 24
6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF
HAZARD AND DOSE RESPONSE 24
6.1. HUMANHAZARD POTENTIAL 24
6.2. DOSE RESPONSE 25
7. REFERENCES 25
APPENDIX A. External Peer ReviewSummary of Comments and Disposition 39
APPENDIX B. Toxicokinetics of Chloral Hydrate 42
IV
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FOREWORD
The purpose of this Toxicological Review is to provide scientific support and rationale
for the hazard and dose-response assessment in IRIS pertaining to chronic exposure to chloral
hydrate. It is not intended to be a comprehensive treatise on the chemical or toxicological nature
of chloral hydrate.
In Section 6, EPA has characterized its overall confidence in the quantitative and
qualitative aspects of hazard and dose response. Matters considered in this characterization
include knowledge gaps, uncertainties, quality of data, and scientific controversies. This
characterization is presented in an effort to make apparent the limitations of the assessment and
to aid and guide the risk assessor in the ensuing steps of the risk assessment process.
For other general information about this assessment or other questions relating to IRIS,
the reader is referred to EPA's Risk Information Hotline at 202-566-1676.
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
Chemical Manager/Author
Robert Benson
Region VIII, Denver, CO
Reviewers
This document and summary information on IRIS have received peer review both by EPA
scientists and by independent scientists external to EPA. Subsequent to external review and
incorporation of comments, this assessment has undergone an Agencywide review process
whereby the IRIS Program Manager has achieved a consensus approval among the Office of
Research and Development; Office of Air and Radiation; Office of Prevention, Pesticides, and
Toxic Substances; Office of Solid Waste and Emergency Response; Office of Water; Office of
Policy, Planning, and Evaluation; and the Regional Offices.
Internal EPA Reviewers
National Center for Environmental Assessment
Washington, DC
Jim Cogliano
Cheryl Siegel Scott
Vanessa Vu
National Health and Environmental Effects Research Laboratory
Research Triangle Park, NC
Anthony DeAngelo
Robert Luebke
Office of Water
Washington, DC
AmbikaBathija
External Peer Reviewers
Paul E. Brubaker
Private Consultant
Calvin C. Willhite
Department of Toxic Substances Control, State of California
Jeffrey William Fisher
Operational Toxicology Branch, Wright-Patterson AFB
Summaries of the external peer reviewers' comments and the disposition of their
recommendations are in Appendix A.
vi
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1. INTRODUCTION
This document presents background and justification for the hazard and dose-response
assessment summaries in EPA's Integrated Risk Information System (IRIS). IRIS summaries
may include an oral reference dose (RfD), inhalation reference concentration (RfC), and a
carcinogenicity assessment.
The RfD and RfC provide quantitative information for noncancer dose-response
assessments. The RfD is based on the assumption that thresholds exist for certain toxic effects
such as cellular necrosis but may not exist for other toxic effects such as some carcinogenic
responses. It is expressed in units of mg/kg-day. In general, the RfD is an estimate (with
uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human population
(including sensitive subgroups) that is likely to be without an appreciable risk of deleterious
noncancer effects during a lifetime. The inhalation RfC is analogous to the oral RfD, but
provides a continuous inhalation exposure estimate. The inhalation RfC considers toxic effects
for both the respiratory system (portal-of-entry) and for effects peripheral to the respiratory
system (extrarespiratory or systemic effects). It is generally expressed in units of mg/m3.
The carcinogenicity assessment provides information on the carcinogenic hazard potential
of the substance in question and quantitative estimates of risk from oral exposure and inhalation
exposure. The information includes a weight-of-evidence judgment of the likelihood that the
agent is a human carcinogen and the conditions under which the carcinogenic effects may be
expressed. Quantitative risk estimates are presented in three ways. The slope factor is the result
of application of a low-dose extrapolation procedure and is presented as the risk per mg/kg/day.
The unit risk is the quantitative estimate in terms of either risk per |ig/L drinking water or risk
per |ig/m3 air breathed. Another form in which risk is presented is a drinking water or air
concentration providing cancer risks of 1 in 10,000; 1 in 100,000; or 1 in 1,000,000.
Development of these hazard identification and dose-response assessments for chloral
hydrate has followed the general guidelines for risk assessment as set forth by the National
Research Council (1983). EPA guidelines that were used in the development of this assessment
may include the following: the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1986a),
Guidelines for the Health Risk Assessment of Chemical Mixtures (U.S. EPA, 1986b), Guidelines
for Mutagenicity Risk Assessment (U.S. EPA, 1986c), Guidelines for Developmental Toxicity
Risk Assessment (U.S. EPA, 1991), Proposed Guidelines for Carcinogen Risk Assessment
(1996a), Guidelines for Reproductive Toxicity Risk Assessment (U.S. EPA, 1996b), and
Guidelines for Neurotoxicity Risk Assessment (U.S. EPA, 1998a); Recommendations for and
Documentation of Biological Values for Use in Risk Assessment (U.S. EPA, 1988); (proposed)
Interim Policy for Particle Size and Limit Concentration Issues in Inhalation Toxicity (U.S. EPA,
1994a); Methods for Derivation of Inhalation Reference Concentrations and Application of
Inhalation Dosimetry (U.S. EPA, 1994b); Peer Review and Peer Involvement at the U.S.
Environmental Protection Agency (U.S. EPA, 1994c); Use of the Benchmark Dose Approach in
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Health Risk Assessment (U.S. EPA, 1995); Science Policy Council Handbook: Peer Review (U.S.
EPA, 1998b); and memorandum from EPA Administrator, Carol Browner, dated March 21,
1995, Subject: Guidance on Risk Characterization.
Literature search strategy employed for this compound was based on the CASRN and at
least one common name. At a minimum, the following databases were searched: RTECS,
HSDB, TSCATS, CCRIS, GENETOX, EMIC, EMICBACK, DART, ETICBACK, TOXLINE,
CANCERLINE, MEDLINE, and MEDLINE backfiles. Any pertinent scientific information
submitted by the public to the IRIS Submission Desk was also considered in the development of
this document.
2. CHEMICAL AND PHYSICAL INFORMATION RELEVANT TO ASSESSMENTS
Chloral hydrate is not known to occur as a natural product. For the general public, the
major route of exposure to chloral hydrate is from drinking water. Chloral hydrate and its
metabolites, trichloroacetic acid and dichloroacetic acid, are formed as by-products when water is
disinfected with chlorine. The typical concentration in a public water supply in the United States
is 5 |ig/L (U.S. EPA, 1994d). Additional chloral hydrate can be formed if water containing
chlorine is mixed with food containing humic and fulvic acids (Wu et al., 1998). The low
volatility of chloral hydrate from a water solution precludes significant exposure by inhalation.
Chloral hydrate is also a metabolite of trichloroethylene and tetrachloroethylene. Humans will be
exposed to chloral hydrate if they are exposed to these chemicals. Chloral hydrate is currently
approved by FDA as a habit-forming, central nervous system depressant (Schedule IV, 21 CFR
ง329.1 and ง1308.14) for use in adult and pediatric medicine.
Chloral (CAS # 75-87-6) is the anhydrous form of the chemical. Chloral is used as an
intermediate in the synthesis of the insecticides DDT, methoxychlor, naled, trichlorfon, and
dichlorvos, and the herbicide trichloracetic acid (IARC, 1995). The conversion from chloral to
chloral hydrate occurs spontaneously when chloral is placed in an aqueous media.
Chloral hydrate could be released to the environment from wastewater treatment
facilities, from the manufacture of pharmaceutical-grade chloral hydrate, and from the waste
stream during the manufacture of insecticides and herbicides that use chloral as an intermediate.
Chemical and physical properties of chloral hydrate are presented below (IARC, 1995).
CAS Name 2,2,2-trichloro-1,1 -ethanediol
IUPAC Name Chloral hydrate
Primary Synonyms Chloral monohydrate
Trichloroacetaldehyde hydrate
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CAS Number
Chemical Formula
Molecular Weight
Boiling Point
Melting Point
Specific Gravity
Vapor Pressure
Solubility
Octanol/Water Partition
Coefficient (log Kow)
Trichloroacetaldehyde monohydrate
l,l,l-trichloro-2,2-dihydroxy ethane
Noctec (formulary name)
302-17-0
CC13- CH(OH)2
165.42
96-98 ฐC (decomposes)
57 ฐC
1.908 (20 ฐC)
15mmHg(25ฐC)
soluble in water, acetone, benzene, chloroform, diethylether,
ethanol, and methyl ethyl ketone
0.99
3. TOXICOKINETICS RELEVANT TO ASSESSMENTS
Chloral hydrate is completely absorbed following oral administration. Qualitatively
similar metabolism occurs in mice, rats, dogs, Japanese Medaka, and humans (Abbas et al., 1996;
Abbas and Fisher, 1997; Beland et al., 1998; Breimer, 1977; Elfarra et al., 1998; Fisher et al.,
1998; Goodman and Gilman, 1985; Gorecki et al., 1990; Gosselin et al., 1981; Greenberg et al.,
1999; Henderson et al., 1997; Hindmarsh et al., 1991; Hobara et al., 1986, 1987a,b, 1988a,b;
Lipscomb et al., 1996, 1998; Marshall and Owens, 1954; Mayers et al., 1991; Merdink et al.,
1998, 1999; Owens and Marshall, 1955; Reimche et al., 1989; Stenner et al., 1997, 1998). The
metabolic pathway is shown in Figure 1.
Chloral hydrate is rapidly metabolized in both hepatic and extrahepatic tissues to
trichloroethanol and trichloroacetic acid. The alcohol dehydrogenase responsible for reducing it
to trichloroethanol is located in both liver and erythrocytes. A portion of the trichloroethanol
produced is conjugated with glucuronic acid to form trichloroethanol-* -glucuronide, which is
excreted in the urine. A portion of the trichloroethanol-glucuronide is secreted into the bile and
is subject to enterohepatic circulation. Oxidation of chloral hydrate to trichloroacetic acid occurs
primarily in the liver and kidney via an aldehyde dehydrogenase using nicotinamide adenine
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dinucleotide (NAD) as a cofactor. The major route of excretion of the metabolites of chloral
hydrate is the urine.
Chloral hydrate and its metabolites have been found in milk (Bernstine et al., 1956). As
soon as lactation started, mothers (n=50) were treated with a 1.33 g rectal suppository of chloral
hydrate. Samples of maternal blood and breast milk were taken for analysis from 15 minutes and
at varying intervals up to 24 hours following administration of the drug. The maximum
concentration of the sum of chloral hydrate, trichloroethanol, and trichloroethanol-glucuronide
(the potential pharmacologically active species) in milk occurred within 1 hour after
administration of the drug and averaged 53 mg/L (n=l 1). The amount of chloral hydrate required
for sedation in infants is 10 mg in a single feeding of 100 mL of milk.
In mice and rats, 8% of the administered dose of chloral hydrate is directly eliminated in
urine, 15% is converted to trichloroacetic acid (including the contribution from enterohepatic
circulation), and 77% is converted to trichloroethanol (Beland et al., 1998). In humans 92% of
the administered dose of chloral hydrate is converted to trichloroethanol and 8% is converted
directly to trichloroacetic acid; additional trichloroacetic acid is formed during enterohepatic
circulation of trichloroethanol such that 35% of the initial dose of chloral hydrate is converted to
trichloroacetic acid (Allen and Fisher, 1993).
Although earlier reports claimed detection of substantial quantities of dichloroacetic acid
in blood from studies in rodents (Abbas et al., 1996), data show that the dichloroacetic acid is
most likely formed by an acid-catalyzed dechlorination of trichloroacetic acid in the presence of
reduced hemoglobin (Ketcha et al., 1996). Recent experimental data and pharmacokinetic model
simulations in rodents suggest that dichloroacetic acid occurs only as a short-lived metabolite in
the liver and is rapidly converted to two-carbon, nonchlorinated metabolites and carbon dioxide
(Merdink et al., 1998). Using a different extraction procedure less likely to induce the artifactual
formation of dichloroacetic acid, Henderson et al. (1997) showed the presence of dichloroacetic
acid in children treated with chloral hydrate in a clinic.
Breimer (1977) administered an aqueous solution of chloral hydrate to five human
volunteers. Each volunteer received a single oral dose of 15 mg/kg. Chloral hydrate could not
be detected in the plasma even at the first sampling time of 10 minutes. A method with a limit of
detection of 0.5 mg/L was used. Trichloroethanol and trichloroethanol-glucuronide reached peak
concentrations 20 to 60 minutes after administration of chloral hydrate. The maximum
concentration of trichloroethanol in the plasma was about 5 mg/L. The average half-lives of
trichloroethanol and trichloroethanol-glucuronide were 8 hours (range 7-9.5 hours) and 6.7 hours
(range 6-8 hours), respectively. The half-life of trichloroacetic acid was about 4 days.
Zimmermann et al. (1998) administered a single dose of 250 mg chloral hydrate in 150
mL of drinking water to 18 healthy male volunteers (20 to 28 years of age). Chloral hydrate,
trichloroethanol, and trichloroacetic acid were measured in plasma. Chloral hydrate could only
be detected 8 to 60 minutes after dosing in 15 of 18 plasma samples. The measured
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concentration of chloral hydrate in plasma ranged from 0.1 mg/L (the limit of detection) to 1
mg/L. The mean maximum plasma concentration of trichloroethanol of 3 mg/L was achieved
0.67 hours after dosing. The mean maximum plasma concentration of trichloroacetic acid of 8
mg/L was achieved 32 hours after dosing. The terminal half-life for trichloroethanol was 9.3 to
10.2 hours and for trichloroacetic acid was 89 to 94 hours.
Two toxicokinetic models for chloral hydrate in rats and mice are available (Abbas et al.,
1996; Beland et al., 1998). Beland et al. (1998) treated rats and mice with chloral hydrate by
gavage with 1 or 12 doses using 50 or 200 mg/kg per dose. The maximum concentrations of
chloral hydrate, trichloroethanol, and trichloroethanol-glucuronide in the plasma were observed
at the initial sampling time of 0.25 hour. The half-life of chloral hydrate in the plasma was
approximately 3 minutes. The half-lives of trichloroethanol and trichloroethanol-glucuronide in
the mouse plasma were approximately 5 and 7 minutes, respectively. Trichloroacetic acid was
the major metabolite found in the mouse plasma, with the maximum concentration being reached
1-6 hours after dosing. The half-life of trichloroacetic acid in the mouse plasma was
approximately 8-11 hours. Comparable values were obtained for rats.
Estimates of the concentrations of trichloroacetic acid and trichloroethanol at steady state
under various exposure conditions are in Appendix B.
Several studies have investigated the age-dependence of the metabolism of chloral
hydrate (Gorecki et al., 1990; Hindmarsh et al., 1991; Mayers et al., 1991; Reimche et al., 1989).
These studies were conducted in critically ill patients in neonatal and pediatric intensive care
units and may not be representative of a population of healthy infants. The half-lives for
trichloroethanol and its glucuronide were increased fourfold in preterm and threefold in full-term
infants. The half-life for trichloroethanol in toddlers was similar to that reported for adults. The
reported half-lives for elimination of trichloroethanol were 39.8 hours, 27.8 hours, and 9.67
hours for preterm infants, full-term infants, and toddlers, respectively (Mayers et al., 1991),
compared to 7-9.5 hours reported by Breimer (1977) and 9.3-10.2 hours reported by
Zimmermann et al. (1998). These age-related differences likely are the result of the immaturity
of hepatic metabolism, particularly glucuronidation, and decreased glomerular filtration.
Kaplan et al. (1967) investigated the effect of ethanol consumption on the metabolism of
chloral hydrate in adults. Subjects ingested doses of ethanol (880 mg/kg), chloral hydrate (9 to
14 mg/kg), or both. In subjects consuming both ethanol and chloral hydrate, the concentration of
trichloroethanol in blood rose more rapidly and reached a higher concentration than in subjects
consuming chloral hydrate only. Ethanol promotes the formation of trichloroethanol because the
oxidation of ethanol provides NADH used for the reduction of chloral hydrate (Watanabe et al.,
1998).
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4. HAZARD IDENTIFICATION
4.1. STUDIES IN HUMANS - EPIDEMIOLOGY AND CASE REPORTS
Chloral hydrate has been widely used as a sedative/hypnotic drug in humans. The
recommended dose for an adult as a sedative is 250 mg three times a day (equivalent to 10.7
mg/kg-day); the recommended dose as a hypnotic is 500-1,000 mg (equivalent to 7-14 mg/kg)
(Goodman and Oilman, 1985). The recommended dose for a child as a sedative is 9 mg/kg, three
times a day, to 25 mg/kg in single dose (Hindmarsh et al., 1991). The recommended dose for a
child undergoing a medical or dental procedure is 50 to 100 mg/kg (Badalaty et al., 1990; Fox et
al., 1990). A child is typically given a higher dose than an adult because a deeper level of
sedation is desired to obtain better cooperation from the child during the medical or dental
procedure. There is no evidence that a child is less sensitive than an adult to the sedative effects
of chloral hydrate. Because of the rapid metabolism of chloral hydrate, trichloroethanol is
responsible for the majority of the pharmacological activity (Marshall and Owens, 1954;
Breimer, 1977; Goodman and Oilman, 1985). The concentration of trichloroethanol in the
plasma in the pharmacologically active range is approximately 5 mg/L and above, and in the
toxic range is 100 mg/L and above.
Chloral hydrate is irritating to the skin and mucous membranes and often causes gastric
distress (nausea and vomiting) at recommended doses. There are no reports of sensitization in
humans. Overdoses produce (in order of progression) ataxia, lethargy, deep coma, respiratory
depression, hypotension, and cardiac arrhythmias. The life-threatening effects are from severe
respiratory depression, hypotension, and cardiac arrhythmias. For some representative case
reports, see Anyebuno and Rosenfeld (1991), Ludwigs et al. (1996), Marshall (1977), and Sing et
al. (1996). A potentially life-threatening oral dose for humans is approximately 10 g (143
mg/kg), although death has been reported from as little as 4 g, and some individuals have
survived ingesting 30 g or more. Extended abuse of chloral hydrate may result in development of
paranoid behavior, in tolerance to the pharmacological effect, and in physical dependence or
addiction to chloral hydrate. Sudden withdrawal after habituation can precipitate seizure,
delirium, and death in untreated individuals.
Shapiro et al. (1969) reviewed the medical records of 1,618 patients who had received
chloral hydrate at 1 g (213 patients, 13%), 0.5 g (1,345 patients, 83%), or various other doses (60
patients, 4%). Adverse reactions were reported in 38 patients (2.3%). Of these patients, 4
received 1 g, 1 received 0.75 g, and 33 received 0.5 g. Reported adverse reactions included
gastrointestinal symptoms in 10 patients, depression of the central nervous system in 20 patients,
skin rash in 5 patients, prolonged prothrombin time in 1 patient, and bradycardia in 1 patient. In
all patients the side effects disappeared when chloral hydrate therapy was stopped. There was no
evidence of association between adverse side effects and age, weight, or sex.
Miller and Greenblatt (1979) reviewed medical records of 5,435 hospital patients who
received chloral hydrate at a dose of either 0.5 g (about 7 to 8 mg/kg) or 1 g (about 14 to 16
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mg/kg). Adverse reactions were noted in 119 cases (2.2%). Central nervous system (CNS)
depression was most common (58 patients, or 1.1%), with minor sensitivity reactions, including
rash, pruritus, fever, and eosinophilia, second most common (19 patients, or 0.35%). Other
adverse reactions included gastrointestinal disturbances (0.28%) and CNS excitement (0.22%).
Three individuals (0.05%) were judged to have life-threatening reactions involving CNS
depression, asterixis (flapping tremor characterized by an intermittent lapse of assumed posture
due to involuntary sustained contractions of groups of muscles), or hypotension. The data show
that adverse reactions involving the central nervous system became more frequent with
increasing dosage in patients older than 50 years, in patients who died during hospitalization, in
patients who concurrently received benzodiazepine antianxiety drugs, and in patients with
elevated levels of blood urea nitrogen.
Greenberg et al. (1991) reported various side effects experienced by children receiving
chloral hydrate sedation in preparation for computer tomography (CT) procedures. In a "high-
dose" group, composed of 295 children (average age 2.18 years) that received a single dose of 80
to 100 mg/kg and a maximum total dose of 2 g, adverse reactions occurred in 23 of the patients
(7%) and included vomiting (14 patients), hyperactivity (5 patients), and respiratory symptoms
such as wheezing and secretion aspiration (4 patients). Cardiac monitoring did not reveal any
abnormalities or arrhythmias in any of the children. A second "lower-dose" cohort of 111
children (average age 1.9 years) received 40 to 75 mg/kg chloral hydrate. These patients received
the lower dose because of existing liver or renal impairment, respiratory insufficiency, or CNS
depression. There were no adverse side effects or complications reported in this group.
Children with severe liver or renal disease or affected by severe CNS depression were not treated
with chloral hydrate.
Lambert et al. (1990) conducted a retrospective analysis of hospital medical records to
investigate a possible link between chloral hydrate administration and direct hyperbilirubinemia
(DHB), an increase in the concentration of unconjugated bilirubin in the serum, in neonates
following prolonged administration of chloral hydrate (25 to 50 mg/kg administered for up to 20
days). In the first study, the DHB was of unknown etiology in 10 of the 14 newborns with DHB;
all 10 of these DHB patients had received chloral hydrate. In the second study, among 44
newborns who had received chloral hydrate, 10 patients that developed DHB had received a
mean cumulative dose of 1,035 mg/kg. In contrast, 34 patients whose direct bilirubin levels were
within normal ranges received a mean cumulative dose of 183 mg/kg. As the total bilirubin
levels (free plus conjugated bilirubin) were the same in both groups and within the normal range,
the increased direct bilirubin could result from competition between trichloroethanol and
bilirubin in the glucuronidation pathway, known to function suboptimally in neonates.
Kaplan et al. (1967) investigated whether ethanol ingestion altered the metabolism of
chloral hydrate or increased subjective symptoms. Five male volunteers weighing 70 to 107 kg
consumed ethanol (880 mg/kg), chloral hydrate (1 gram, 9 to 14 mg/kg), or both. Blood pressure
and cardiac rate did not vary significantly among treatments. In the presence of ethanol, the
concentration of trichloroethanol in the blood rose more rapidly and reached a higher
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concentration, but the rate of depletion was not significantly changed. The increase in the
concentration of trichloroethanol was not sufficient to produce a marked enhancement of the
hypnotic effect. The volunteers reported symptoms (drowsiness, dizziness, blurred vision) and
their severity during the 6-hour observation period. At all time points, the rank order of effects
was: ethanol plus chloral hydrate > ethanol > chloral hydrate.
No long-term studies of chloral hydrate in humans were located in the published
literature.
4.2. PRECHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN
ANIMALS
4.2.1. Oral
Sanders et al. (1982) studied the acute toxicity of chloral hydrate in CD-I mice. Groups
of 8 male and 8 female mice were given chloral hydrate by gavage in distilled water at 300, 600,
900, 1,200, 1,500, or 1,800 mg/kg. No deaths occurred at 900 mg/kg or below in either sex. The
calculated LD50 for females was 1,265 mg/kg and for males was 1,442 mg/kg. Effects were seen
within 10 minutes of dosing. The mice became sedated at 300 mg/kg. At 600 and 900 mg/kg,
the animals became lethargic and exhibited loss of righting reflex. Respiration was markedly
inhibited at 1,200, 1,500, and 1,800 mg/kg. Inhibition of respiration appeared to be the
immediate cause of death. Most deaths occurred within 4 hours at 1,800 mg/kg. At 1,200 and
1,500 mg/kg, some deaths occurred after 4 hours, with all deaths occurring within 24 hours.
Goldenthal (1971) reported LD50s of 479 mg/kg and 285 mg/kg in adult and 1-2 day old
Charles River Sprague-Dawley rats, respectively.
Sanders et al. (1982) studied the short-term toxicity of chloral hydrate in mice. Groups of
male CD-I mice were given chloral hydrate by gavage in distilled water at 14.4 or 144 mg/kg-
day for 14 days. No significant effect on body weight was observed. No changes in internal
organs were noted on gross examination. Groups of 11 to 12 mice were evaluated for several
parameters. No significant effects on hematological or serum biochemical parameters were
noted. There was a statistically significant (p<0.05) increase in liver weight (17%) and a
decrease in spleen weight (27%) at 144 mg/kg-day. The NOAEL in this study is 14.4 mg/kg-day;
the LOAEL is 144 mg/kg-day.
Sanders et al. (1982) administered chloral hydrate in drinking water to CD-I mice at 70
or 700 mg/L (equivalent to 16 mg/kg-day or 160 mg/kg-day) for 90 days. In males,
hepatomegaly (an increase in weight of 20% and 34% at the low and high exposure, respectively)
and microsome proliferation (no increase in total microsomal protein, increase in cytochrome b5
of 26% and 40%, increase in aminopyrine N-demethylase of 28% and 20%, and increase in
aniline hydroxylase of 24% and 30% at the low and high exposure, respectively, when reported
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as mg of protein per mg of total liver protein) were observed. There were no biologically
significant changes in serum enzymes. Hepatomegaly was not seen in females, but there were
changes in hepatic microsomal parameters (increase in total microsomal protein of 10%, increase
in aniline hydroxylase of 23%, and decrease in cytochrome b5 of 12%, when reported as mg of
protein per mg of total liver protein) only at the high exposure. No other significant
toxicological changes were observed. Based on hepatomegaly and changes in microsomal
parameters in males at the high exposure, the LOAEL in this study is 160 mg/kg-day; the
NOAEL is 16 mg/kg-day.
Rijhsinghani et al. (1986) evaluated the carcinogenic potential in male C57BL
mice. Groups of 15-day-old mice received a single dose of chloral hydrate by gavage in distilled
water at 0, 5, or 10 mg/kg (26, 15, and 14 mice per group, respectively). Animals were
sacrificed when moribund or at week 78, week 88, or between weeks 89 and 92. Livers were
examined histopathologically using light and electron microscopy. In mice sacrificed 48 to 92
weeks after treatment, the incidence of hepatic nodules (adenomas or trabecular carcinomas) was
3/9 and 6/8 for animals from the 5 and 10 mg/kg-day dose groups, respectively, compared with
2/19 in controls. The increase in tumors was statistically significant (p<0.05) only in the 10
mg/kg group.
NTP (2000a) investigated the ability of a single exposure to chloral hydrate to induce
tumors in female and male B6C3FJ mice. Groups of 15-day-old or 28-day-old female mice (48
animals per dose group) received a single gavage dose of chloral hydrate in distilled water at 0,
10, 25, or 50 mg/kg. An identical study was conducted in 15-day-old male mice. All animals
were sacrificed at 105 weeks of age. No neoplastic or non-neoplastic effects were found in any
organ at any exposure.
Daniel et al. (1992a) exposed 40 male B6C3FJ mice for 104 weeks to drinking water
containing chloral hydrate at 1 g/L (equivalent to 166 mg/kg-day). Untreated control animals (23
in one group and 10 in a second group) received distilled water. Interim sacrifices were
conducted at 30 and 60 weeks of exposure (5 animals per group at each sacrifice interval).
Complete necropsy and microscopic examination were performed. There were no significant
treatment-related effects on survival or body weight. There were no changes in spleen, kidneys,
or testes weights, or histopathological changes in any tissue except the liver. The toxicity in the
liver was characterized by increased absolute liver weight and liver-to-body weight ratio at all
three sacrifice intervals. At week 104, liver weight was 37% higher than controls, and liver-to-
body weight ratio was 42% higher than controls. Hepatocellular necrosis was noted in 10/24
(42%) treated animals; other pathological changes of mild severity reported in the livers of
treated animals included cytoplasmic vacuolization, cytomegaly, and cytoplasmic alteration. The
prevalence of liver tumors at terminal sacrifice was statistically significantly (p<0.05) increased
over controls, with hepatocellular carcinomas in 1 1/24 and hepatocellular adenomas in 7/24; for
carcinomas and adenomas combined, the prevalence was 17/24. In control animals, carcinomas,
adenomas, and carcinomas and adenomas (combined) occurred in 2/20, 1/20, and 3/20,
respectively. At the 60-week sacrifice, there were 2/5 treated animals with hepatocellular
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carcinomas, compared with 0/5 controls. No carcinomas, adenomas, or hyperplastic nodules
were reported in animals sacrificed at week 30.
George et al. (2000) conducted a chronic bioassay for carcinogenicity in male B6C3FJ
mice. Mice were administered chloral hydrate in drinking water for 104 weeks. Mice (72 in
each group) had a mean exposure of 0, 13.5, 65, or 146.6 mg/kg-day. At the termination of the
study, a complete necropsy and histopathological examination of liver, kidney, spleen, and testes
from all animals was conducted. In addition a complete histopathological examination was
conducted on five animals from the high-dose group. There was no change in water
consumption, survival, behavior, body weight, or organ weights (liver, kidney, spleen, and testes)
at any exposure. There was no evidence of hepatocellular necrosis at any exposure and only
minimal changes in the levels of serum enzymes. This study identifies a NOAEL for noncancer
effects in male mice of 146.6 mg/kg-day (the highest exposure tested). There was no increase in
the prevalence of neoplasia at sites other than the liver. The male mice showed an increase of
proliferative lesions in the liver (hyperplasia, adenoma, carcinoma, and combined adenoma and
carcinoma) at all exposures. The prevalence of proliferative lesions in the control, 13.5, 65, or
146.6 mg/kg-day groups was as follows: hyperplasia, 3/42, 15/46, 13/39, 12/32; adenoma, 9/42,
20/46, 20/39, 16/32; carcinoma, 23/42, 25/46, 23/39, 27/32; adenoma or carcinoma, 27/42,
36/46, 31/39, 29/32. All of the changes were statistically significant (p<0.05) except for
carcinoma at the two lower exposures.
NTP (2000a) conducted a chronic bioassay for car cinogeni city in female B6C3FJ mice.
Mice were administered chloral hydrate by gavage in distilled water at 0, 25, 50, or 100 mg/kg 5
days a week for up to 2 years. The calculated exposures are 0, 17.9, 35.7, or 71.4 mg/kg-day.
Additional groups were administered chloral hydrate by gavage for 3, 6, or 12 months and held
without further dosing for the duration of the study (stop-exposure studies). There was no
significant effect on survival, body weight, or organ weights at any exposure. Following
complete necropsy and histopathological examination, the only significant findings were in the
pituitary gland pars distalis. There were no significant effects in the pituitary in the stop-
exposure studies. Following the full exposure regime, the incidence of hyperplasia in the
pituitary gland pars distalis was 4/45, 6/44, 4/50, and 9/50 in the control, 25, 50, and 100 mg/kg
group, respectively. The average severity grade for hyperplasia was 1.5, 1.0, 1.0, and 2.2 in the
control, 25, 50, and 100 mg/kg group, respectively. Only the average severity grade at 100
mg/kg was statistically different from the control (p<0.05). The incidence of adenoma in the
pituitary gland pars distalis was 0/45, 2/44, 0/47, and 5/41, in the control, 25, 50, and 100 mg/kg
group, respectively. Only at 100 mg/kg was the incidence significantly greater from the control
(p=0.0237). For non-neoplastic effects, the NOAEL in this study is 71.4 mg/kg-day (the highest
exposure tested). NTP concluded that this study provided equivocal evidence of carcinogenic
activity for chloral hydrate in female mice.
NTP (2000b) conducted a chronic bioassay for car cinogeni city in male B6C3Fj mice.
Groups of 120 male mice received chloral hydrate by gavage in distilled water at 0, 25, 50, or
100 mg/kg for up to 2 years. The calculated exposures were 0, 17.9, 35.7, or 71.4 mg/kg-day. At
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each exposure 60 mice received feed ad libitum; the other 60 mice received feed in a measured
daily amount calculated to maintain body weight on a previously computed idealized body
weight curve. Twelve mice from each diet and dose group were evaluated after 15 months of
exposure. The remaining 48 animals from each diet and dose group were evaluated at 2 years.
Survival, body weight, organ weights, and serum enzymes in the dosed groups were comparable
to the respective vehicle control. Following complete necropsy and histopathological
examination, no changes were found in any organ except the liver when compared to the
respective vehicle control. The incidence of hepatocellular adenoma or carcinoma in the ad
libitum study was 16/48, 25/48, 23/47, and 22/48 in the control, 25, 50, and 100 mg/kg groups,
respectively. Only in the 25 mg/kg group was the incidence significantly greater from control
(p=0.0437). In the dietary controlled study, the incidence of hepatocellular adenoma or
carcinoma was 11/48, 11/48, 14/48, and 18/48, and the incidence of hepatocellular carcinoma
was 2/48, 5/48, 4/48, and 8/48, in the control, 25, 50, and 100 mg/kg group, respectively. The
only statistically significant increase in incidence was for hepatocellular carcinoma in the 100
mg/kg group (p = 0.042). The NOAEL for non-neoplastic effects in this study is 71.4 mg/kg-day
(the highest exposure tested). NTP concluded that this study provided some evidence of
carcinogenic activity for chloral hydrate in male mice.
Daniel et al. (1992b) exposed male and female Sprague-Dawley rats (10/sex/dose) for 90
days to chloral hydrate in drinking water at a concentration of 300, 600, 1,200, or 2,400 mg/L
(equivalent to 24, 48, 96, or 168 mg/kg-day in males and 33, 72, 132, or 288 mg/kg-day in
females). The tissues of animals from the high-exposure group and liver sections from all treated
males were examined histopathologically. No mortality occurred in any groups prior to sacrifice.
Organ weights, including liver weight, and clinical chemistry values in treated animals were only
sporadically or inconsistently different from control animal values. Focal hepatocellular necrosis
was observed in 2 of 10 males in each of the groups exposed to 96 and 168 mg/kg-day. The
necrotic lesion was minimal at 96 mg/kg-day and was significantly more severe at 168 mg/kg-
day. Necrotic lesions were not reported in any treated females or in any control animals. While
serum enzymes were generally increased in treated animals, dramatic increases were reported in
males in the 168 mg/kg-day group; mean aspartate aminotransferase, alanine aminotransferase,
and lactate dehydrogenase levels in this group were elevated 89%, 54%, and 127% above the
corresponding control values, respectively. Based on the focal hepatocellular necrosis and
accompanying serum enzyme changes, the study identifies a LOAEL of 168 mg/kg-day and a
NOAEL of 96 mg/kg-day. The 96 mg/kg-day exposure is not considered a LOAEL because the
authors reported only minimal microscopic necrosis, there was no corresponding increase in
serum liver enzymes, and Sprague-Dawley rats showed no hepatocellular necrosis after chronic
exposure to a higher daily exposure (Leuschner and Beuscher, 1998; George et al., 2000).
Leuschner and Beuscher (1998) conducted a chronic bioassay for carcinogenicity in
Sprague-Dawley rats. Chloral hydrate was administered in drinking water for 124 weeks (males)
and 128 weeks (females). The rats (50 males and 50 females in each group) received 15, 45, or
135 mg/kg-day. There was no effect on survival, appearance, behavior, body weight, food and
water consumption, or organ weights. There was no increased incidence of tumors in any organ.
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Histopathological examination revealed increased hepatocellular hypertrophy at the highest
exposure in males only (11% in controls versus 28% at the highest exposure, p<0.01 ). This
finding, graded as minimal to slight in severity, was characterized by a diffuse liver cell
enlargement with slight eosinophilic cytoplasm and was considered by the authors as a first sign
of toxicity. The type, incidence, and severity or other non-neoplastic lesions were not increased
in treated animals compared to controls. Based on the evidence of minimal toxicity in the liver,
which is of doubtful biological significance, the NOAEL in this study is 45 mg/kg-day; the
LOAEL is 135 mg/kg-day.
George et al. (2000) conducted a chronic bioassay for carcinogenicity in male F344 rats.
Rats were administered chloral hydrate in drinking water for 104 weeks. Rats (78 in each group)
had a mean daily exposure of 0, 7.4, 37.4, or 162.6 mg/kg-day. At the termination of the study, a
complete necropsy and histopathological examination of liver, kidney, spleen, and testes from all
animals was conducted. In addition, a complete histopathological examination was conducted on
five animals from the high-dose group. There was no change in water consumption, survival,
behavior, body weight, or organ weights (liver, kidney, spleen, and testes) at any exposure.
There was no indication of liver toxicity at any exposure, as shown by the lack of liver necrosis,
hyperplasia, increased mitotic index, and only minimal changes in the levels of serum enzymes.
There was no increase at any exposure in the prevalence or multiplicity of hepatocellular
neoplasia or neoplasia at any other site. The NOAEL in this study is 162.6 mg/kg-day (the
highest exposure tested).
Two of the metabolites of chloral hydrate, trichloroacetic acid and dichloroacetic acid,
have been associated with increased hepatocellular adenomas or carcinomas in rodents. For
example, trichloroacetic acid in drinking water induced hepatocellular adenomas or carcinomas
in male and female mice when the exposure exceeded 200 mg/kg-day (Bull et al., 1990; Herren-
Freund et al., 1987; Pereira, 1996). There was no evidence of increased carcinogenicity,
however, when male rats were exposed to trichloracetic acid at 360 mg/kg-day (DeAngelo et al.,
1997). Dichloroacetic acid in drinking water induced hepatocellular adenomas or carcinomas in
male and female mice when the exposure exceeded 160 mg/kg-day (Bull et al., 1990; Daniel et
al., 1992a; DeAngelo et al., 1991; Ferreira-Gonzalez et al., 1995; Herren-Freund et al., 1987;
Pereira, 1996). Dichloroacetic acid also induced hepatocellular adenomas or carcinomas in male
rats when the exposure exceeded 40 mg/kg-day (DeAngelo et al., 1996; Richmond et al., 1995).
4.2.2. Inhalation
No studies on chloral hydrate were located. One study on chloral was available. Odum et
al. (1992) exposed four female CD-I mice to chloral for 6 hours at a concentration of 100 ppm
(603 mg/m3). This exposure induced deep anesthesia. The mice recovered normally after the
exposure stopped. The effects in the lung included vacuolization of clara cells, alveolar necrosis,
desquamination of the epithelium, and alveolar edema. The lung-to-body weight ratio increased
1.5-fold, most likely because of the alveolar edema.
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4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES
Klinefelter et al. (1995) evaluated sperm morphology and motility in F344 rats
administered chloral hydrate in drinking water for 52 weeks at 0, 55, or 188 mg/kg-day. The
researchers examined cauda epididymal sperm motion parameters and testicular and epididymal
histopathology. Chloral hydrate did not cause any visible systemic toxicity and had no effects on
epididymal or testicular histopathology. However, the percentage of motile sperm was
significantly decreased (p<0.01) from 68% in controls to 58% in rats exposed to 188 mg/kg-day.
The percentage of progressively motile sperm was also significantly decreased (/X0.01) from
63% in controls to 53% in this group. In addition, the frequency distribution of the average
straight-line velocities of sperm at this exposure was significantly shifted (/X0.01) to the lower
ranges when compared to controls. In this study the NOAEL is 55 mg/kg-day; the LOAEL is
188 mg/kg-day.
Kallman et al. (1984) exposed male and female CD-I mice to chloral hydrate in drinking
water at 21.3 or 204.8 mg/kg-day. Animals were exposed for 3 weeks prior to breeding.
Exposure of females (5 per group) continued during gestation and until pups were weaned at 21
days of age. There was no change in drinking water consumption or weight gain in the dams.
No gross malformations were noted in pups, and no significant effects were observed in duration
of gestation, number of pups delivered, pup weight, or number of stillborn pups. All pups (15
per group) showed the same rate of development and level of performance on several
neurobehavioral tests, except that pups exposed to 204.8 mg/kg-day when tested at 23 days of
age showed impaired retention of passive avoidance learning on both the 1-hour and 24-hour
retention tests (p<0.05). This study identified a NOAEL for neurodevelopmental toxicity of 21.3
mg/kg-day and a LOAEL of 204.8 mg/kg-day based on the impairment in passive avoidance
learning. This study also identifies a NOAEL for reproductive and other developmental effects
of 204.8 mg/kg-day (the highest exposure tested).
Johnson et al. (1998) tested the potential for chloral hydrate to cause developmental
toxicity in Sprague-Dawley rats. Chloral hydrate was administered in drinking water to 20 rats
from gestational day 1 to 22 at an average exposure of 151 mg/kg-day. Control animals were
given distilled water. There was no evidence of maternal toxicity, no change in the number of
implantation or resorption sites, no change in the number of live or dead fetuses, no change in
placental or fetal weight, no change in crown-rump length, and no increase in the incidence of
morphological changes. At necropsy there was no evidence of cardiac anomalies. Based on this
study, the NOAEL for developmental toxicity is 151 mg/kg-day (the highest exposure tested).
Johnson et al. (1998) also tested the potential for trichloroethanol and trichloroacetic acid
to cause developmental toxicity in Sprague-Dawley rats. The protocol was identical to the study
with chloral hydrate. Trichloroethanol was administered to 10 rats at an average exposure of 153
mg/kg-day. No evidence of developmental toxicity was found. In contrast, when trichloroacetic
acid was administered to 11 rats at an average exposure of 291 mg/kg-day, developmental
toxicity was observed. The effects included a statistically significant (/X0.05) increase in total
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cardiac defects per litter and an increased number of implantation and resorption sites. The
results with trichloroacetic acid are generally consistent with those reported by Smith et al.
(1989), who reported adverse developmental effects (levocardia) from trichloroacetic acid at an
exposure of 330 mg/kg-day and above.
Saillenfait et al. (1995) tested the potential of chloral hydrate to cause developmental
toxicity in vitro using a rat whole embryo culture system. Embryos (20/dose) from Sprague-
Dawley rats were explanted on gestational day 10 and exposed to chloral hydrate at a
concentration of 0, 0.5, 1, 1.5, 2, or 2.5 mM (equivalent to 83, 165, 248, 331, or 414 mg/L) for
46 hours. At 2.5 mM all embryos died. No lethality was seen at lower exposures. Chloral
hydrate caused concentration-dependent decreases in growth and differentiation and increases in
the incidence of morphologically abnormal embryos. No effects were observed in any parameter
at 0.5 mM. Decreases in crown-rump length, somite (embryonic segment) number, and the
protein or DNA content of embryos were seen at 1 mM and above. At 1, 1.5, and 2 mM chloral
hydrate, respectively, 18%, 68%, and 100% of embryos were malformed. Brain, eye, and ear
malformations were the most prominent effects at these concentrations. Abnormalities in the
trunk and pericardial dilation also occurred at 2 mM. In this in vitro test system, chloral hydrate
was a slightly more potent teratogen than trichloroacetic acid or dichloroacetic acid.
Although chloral hydrate did not cause meiotic delay in the oocytes of adult mice when
administered at the time of resumption of maturation induced by hormones (Mailhes et al.,
1994), it did cause adverse effects in vitro when a synchronized population of oocytes was
exposed prior to resumption of maturation (Eichenlaub-Ritter and Betzendahl, 1995; Eichenlaub-
Ritter et al., 1996). In this test system, chloral hydrate induced lagging of chromosomes during
telophase I, inhibited spindle elongation in anaphase B, and caused chromosome displacement
from the spindle equator in metaphase I and II. Oocytes became irreversibly arrested in
maturation when exposed to chloral hydrate prior to resumption of maturation, or when chloral
hydrate was present during the first or second 8 hours of maturation. Spindle aberrations
(lagging chromosomes and a short interpolar space) were observed when oocytes were treated
with trichloroethanol (Eichenlaub-Ritter et al., 1996).
4.4. OTHER STUDIESNEUROLOGICAL, IMMUNOLOGICAL, GENOTOXICITY,
MECHANISTIC
4.4.1. Neurological Studies
Kallman et al. (1984) administered chloral hydrate by gavage in distilled water at 50, 100,
200, 300, or 400 mg/kg to groups of 12 seven-week-old male CD-I mice. All doses resulted in
the rapid onset of ataxia, with an ED50 of 84.2 mg/kg at 5 minutes (the time of maximal effect).
Animals recovered within 2 to 3 hours. No delayed changes in muscular coordination were
detectable when the mice were tested 24 hours after treatment.
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Kallman et al. (1984) evaluated the potential behavioral toxicity in groups of 12 seven-
week-old male CD-I mice administered chloral hydrate by gavage in distilled water at 14.4 or
144 mg/kg-day for 14 days. When measurements were made 24-48 hours after the 14 day
exposure was terminated, no significant effects were observed on body weight, motor activity,
physical appearance, behavior, muscular coordination, or endurance.
Kallman et al. (1984) exposed groups of 12 five-week-old male CD-I mice to drinking
water containing chloral hydrate at a concentration of 70 or 700 mg/L (equivalent to 15.7 or 160
mg/kg-day) for 90 days. When measurements were made 24 hours after the 90-day exposure was
terminated, no treatment-related effects were observed on mortality, body weight, physical
appearance, behavior, locomotor activity, learning in repetitive tests of coordination, response to
painful stimuli, strength, endurance, or passive avoidance learning. Both exposures resulted in a
decrease of about 1ฐC in mean body temperature (p<0.05). Because of the lack of increased
effect with a tenfold increase in exposure and because hypothermia as a side effect of chloral
hydrate or from an overdose of chloral hydrate has not been reported in humans, the decrease in
body temperature is not considered an adverse effect. This study identifies a NOAEL for
neurobehavioral toxicity of 160 mg/kg-day (the highest exposure tested).
4.4.2. Immunological Studies
Kauffmann et al. (1982) administered chloral hydrate by gavage in distilled water at 14.4
or 144 mg/kg-day to groups of 11 to 12 six-week-old male CD-I mice for 14 days. No effects on
humoral or cell-mediated immunity were detected at either exposure.
Kauffmann et al. (1982) administered chloral hydrate to male and female 4-week-old CD-
1 mice in drinking water at 70 or 700 mg/L (equivalent to 16 or 160 mg/kg-day) for 90 days.
Humoral immunity was assessed by the number of splenic antibody-forming cells produced
against sheep red blood cells (12 mice in the control group and 8 mice in the exposed groups)
and hemagglutination liters (20-21 mice in the control group and 13-16 mice in the exposed
groups). Cell-mediated immunity was assessed by delayed type hypersensitivity to sheep red
blood cells (17-20 mice in the control group and 15-16 mice in the exposed groups).
Lymphocyte response was assessed using a T-cell mitogen (Con A) and a B-cell mitogen (LPS)
(17-22 animals in the control group and 13-16 mice in the exposed groups). In males, no effects
were detected in either humoral or cell-mediated immunity at either exposure. No effects on cell-
mediated immunity were noted in females at either exposure. In females, both exposures
resulted in a statistically significant decrease (p<0.05) in humoral immune function (36% and
40% at the low and high exposure, respectively) when expressed as antibody-forming cells per
spleen. The decrease, however, was statistically significant only at the higher exposure when
expressed as antibody forming cells per million spleen cells (a 32% decrease). There was no
effect on hemagglutination liters or on spleen cell response to the B-cell mitogen at either
exposure. The decrease in antibody-forming cells per million spleen cells at the higher exposure
in female mice is regarded as an adverse response in this study. Accordingly, the NOAEL for
immunotoxicity is 16 mg/kg-day; the LOAEL is 160 mg/kg-day.
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4.4.3. Genetic Toxicity
There is an extensive database on the genotoxicity of chloral hydrate and its metabolites.
A summary is provided in Table 1. The European Union included chloral hydrate in a
collaborative study on aneuploidy (Adler, 1993; Natarajan et al., 1993; Parry, 1993; Parry and
Sors, 1993).
Chloral hydrate did not induce mutation in most strains of Salmonella typhimurium., but
did in some studies with Salmonella typhimurium TA 100 and in a single study with Salmonella
typhimurium TA 104. The latter response was inhibited by free-radical scavengers -tocopherol
and menadione (Ni et al., 1994).
Chloral hydrate did not induce mitotic crossing over in Aspergillus nidulans in the
absence of metabolic activation. Chloral hydrate caused weak induction of meiotic
recombination in the presence of metabolic activation and gene conversion in the absence of
metabolic activation in Saccharomyces cerevisiae. It did not induce reverse mutation in
Saccharomyces cerevisiae. Chloral hydrate clearly induced aneuploidy in various fungi in the
absence of metabolic activation.
Chloral hydrate induced somatic and germ cell mutations in Drosophila melanogaster.
Choral hydrate did not produce DNA-protein cross-links in rat liver nuclei, DNA single-
strand breaks/alkaline-labile sites in primary hepatocytes in vitro, or DNA repair in Escherichia
coli. One study showed induction of single-strand breaks in liver DNA of both rats and mice
treated in vivo; another study in both species using higher concentrations of chloral hydrate
found no such effect.
Chloral hydrate was weakly mutagenic, but did not induce micronuclei in mouse
lymphoma cells in vitro. Chloral hydrate increased the frequency of micronuclei in Chinese
hamster cell lines. Although a single study suggested that chloral hydrate induces chromosomal
aberrations in Chinese hamster CHED cells in vitro, the micronuclei produced probably
contained whole chromosomes and not chromosome fragments, as the micronuclei could all be
labeled with antikinetochore antibodies.
In kangaroo rat kidney epithelial cells, choral hydrate inhibited spindle elongation and
broke down mitotic microtubuli, although it did not inhibit pole-to-pole movement of
chromosomes. It produced multipolar spindles, chromosomal dislocation from the mitotic
spindle, and a total lack of mitotic spindles in Chinese hamster DON:Wg3h cells.
Chloral hydrate weakly induced sister chromatid exchange in cultured human
lymphocytes. It induced micronuclei, aneuploidy, C-mitosis, and polyploidy in human
lymphocytes in vitro. Micronuclei were induced in studies with human whole-blood cultures, but
not in one study with isolated lymphocytes. The differences seen in the micronucleus test have
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been attributed to differences between whole-blood and purified lymphocyte cultures (Vian et al.,
1995), but this hypothesis has not been tested.
Chloral hydrate increased the frequency of chromosomal aberrations in mouse bone
marrow, spermatogonia, and primary and secondary spermatocytes, but not in oocytes, after in
vivo treatment. Chloral hydrate induced chromosomal aberrations in mouse bone-marrow
erythrocytes after treatment in vivo. Chloral hydrate induced micronuclei in the spermatids of
mice treated in vivo in some studies. Chloral hydrate induced aneuploidy in the bone marrow of
mice treated in vivo. Chloral hydrate increased the rate of aneuploidy in mouse secondary
spermatocytes. Chloral hydrate did not produce polyploidy in bone marrow, oocytes, or
gonosomal or autosomal univalents in primary spermatoctyes of mice treated in vivo. Chloral
hydrate, however, induced polyploidy and meiotic delay when a synchronized population of
mouse oocytes were exposed in vitro prior to the resumption of maturation.
Trichloroethanol, a reduction product of chloral hydrate, did not induce prophage in
Escherichia coll or mutation in Salmonella typhimurium TA 100. Trichloroethanol caused
spindle aberrations (lagging of chromosomes and a short interpolar space) when mouse oocytes
were treated in vitro.
Trichloroacetic acid did not induce prophage in Escherichia coli and was not mutagenic
to Salmonella typhimurium in the presence or absence of metabolic activation. Trichloroacetic
acid was weakly positive in the mouse lymphoma assay with metabolic activation.
Trichloroacetic acid also did not induce chromosomal damage in human lymphocytes or
micronuclei in bone marrow in vitro. It is unclear whether trichloroacetic acid can induce
chromosomal damage in vivo because some studies have been positive and other negative.
Dichloroacetic acid did not induce differential toxicity in DNA-repair deficient strains of
Salmonella typhimurium but did induce prophage in Escherichia coli. Dichloroacetic acid
gave equivocal results for gene mutation in Salmonella typhimurium TA100 and TA98.
Dichloroacetic acid was weakly mutagenic in the in vitro mouse lymphoma assay and induced
chromosomal aberrations but not micronuclei or aneuploidy in that test system. Dichloroacetic
acid induced micronuclei in mouse polychromatic erythrocytes in vivo and mutations at the LacI
locus in the transgenic B6C3FJ mouse (Big Blueฎ mouse) in vivo at an exposure that induces
liver tumors in male mice. It is unclear whether dichloroacetic acid can induce primary DNA
damage, as some assays are positive and others negative.
4.4.4. Mechanistic Studies
4.4.4.1. Cell Proliferation
Rijhsinghani et al. (1986) evaluated the acute effects of chloral hydrate on liver cell
proliferation in 15-day-old male C57BL x CSHFj mice. Mice were given a single dose of 0, 5, or
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10 mg/kg chloral hydrate by gavage in distilled water (9, 10, and 6 mice per group, respectively)
and sacrificed after 24 hours. Cell proliferation was evaluated by calculating the mitotic index
(number of mitoses/100 nuclei) from liver sections. The mitotic index in liver cells was
significantly increased (0.9235) in mice receiving 5 mg/kg when compared to the control value
(0.3382), and elevated (0.7433) (although not statistically significantly) in mice receiving 10
mg/kg. Hepatic necrosis was not observed in mice from either treatment group at autopsy.
As part of the chronic bioassay for carcinogenicity, George et al. (2000) evaluated
hepatocyte proliferation in F344 rats and B6C3FJ mice. Exposures are given in Section 8.4.2.
Five days prior to sacrifice at 13, 26, 52, or 72 weeks in rats and 26, 52, or 78 weeks in mice,
animals were given bromodeoxyuridine. Labeled nuclei were identified by chromogen pigment
over the nuclei and the labeling index was calculated. Outside of the areas with tumors in the
livers of male mice, there was no significant evidence of increased hepatocyte proliferation in
rats or mice.
4.4.4.2. Oncogene Activation
Velazquez (1994) investigated the induction of H-ras proto-oncogene mutations in mice.
DNA from normal liver and tumor tissue was obtained from male B6C3Fj mice administered 1
g/L (166 mg/kg-day) chloral hydrate in drinking water for 2 years. H-ras mutations were present
in one out of seven (14%) tumors. The spectrum of mutations was the same as that of
spontaneous liver tumors. Based on these data, it is unlikely that H-ras activation is a
mechanism of potential carcinogenicity relevant to chloral hydrate.
4.4.4.3. Free Radicals and DNA Adduct Formation
Ni et al. (1994, 1995, 1996) studied the metabolism of chloral hydrate in an in vitro
system using microsomes from male B6C3Fj mice. The metabolism of chloral hydrate
generated free radicals as detected by electron spin resonance spectroscopy and caused
endogenous lipid peroxidation, resulting in the production of malondialdehyde, formaldehyde,
and acetaldehyde, all of which are known to produce liver tumors in rodents. Trichloroacetic
acid and trichloroethanol also produced free radicals and induced lipid peroxidation when tested
in this system. The authors speculated that the free radicals were C13CCO2* and/or C13O.
Incubation of chloral hydrate, trichloroethanol, or trichloroacetic acid in the presence of
microsomes and calf thymus DNA resulted in the formation of a malondialdehyde-modified
DNA adduct. This research group further showed that chloral hydrate induced an increase in
mutations at the hprt and tk loci in transgenic human lymphoblastoid cells containing CYP2E1.
In contrast, when the parental cell line lacking CYP2E1 was treated with the same concentration
of chloral hydrate, no mutations were found at either locus. These data implicate CYP2E1 as the
primary cytochrome subfamily involved in the metabolism of chloral hydrate to reactive
intermediates.
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4.4.4.4. Cell Communication
The effects of 1-, 4-, 6-, 24-, 48-, and 168-hour exposures to chloral hydrate (0, 1, 5, or 10
mM) on gap junction intercellular communication in Clone 9 cell cultures (normal rat
hepatocytes) were reported by Benane et al. (1996). No differences in intercellular
communication were seen between the groups treated with 1 mM at 1, 4, and 6 hours of exposure
and controls, as measured by a dye transfer protocol. There were significant differences between
all other groups and the controls. The shortest exposure time and lowest exposure concentration
that reduced dye transfer significantly was in the group treated with 1 mM for 24 hours.
4.4.4.5. Peroxisome Proliferation
As part of the chronic bioassay for carcinogenicity in mice, George et al. (2000) found no
evidence of peroxisome proliferation using cyanide-insensitive palmitoyl CoA oxidase in the
livers of male mice treated with chloral hydrate for 26 weeks. As part of the chronic bioassay for
carcinogenicity in male mice, NTP (2000b) found that chloral hydrate in the 100 mg/kg group
significantly (p<0.05) induced both lauric acid -hydroxylase activity and CYP4A
immunoreactive protein in the dietary-controlled study, but not in the ad libitum study.
4.5. SYNTHESIS AND EVALUATION OF MAJOR NONCANCER EFFECTS AND
MODE OF ACTION (IF KNOWN)
Chloral hydrate has been used for a great many years as a sedative/hypnotic drug in
human and veterinary medicine. The metabolite, trichloroethanol, is responsible for the
pharmacological effect. The proposed mechanisms for the depression of the central nervous
system include potentiating the function of GABAA receptors (Lovinger et al., 1993), inhibition
of excitatory amino acid-activated currents mediated by N-methyl-D-aspartate (Peoples and
Wright, 1998; Scheibler et al., 1999), and allosteric modulation of the 5-hydroxytryptamine3
receptor-mediated depolarization of the vegas nerve (Bentley and Barnes, 1998).
Chloral hydrate is corrosive and irritating to the skin and mucous membranes and can
cause gastric distress, nausea, and vomiting at recommended doses. Acute overdoses produce (in
order of progression) ataxia, lethargy, deep coma, respiratory depression, hypotension, and
cardiac arrhythmias. There is some evidence of hepatic injury in people surviving near lethal,
acute overdoses, but no convincing evidence that hepatic injury results at the recommended
clinical dose. Despite its long use in human medicine, there is no published information on
toxicity in controlled studies in humans following extended exposure.
Chloral hydrate is completely absorbed and rapidly metabolized following oral
administration. The major metabolites are trichloroethanol and its glucuronide and
trichloroacetic acid. Some data suggest a small amount of dichloroacetic acid may be formed. In
humans the half-life of trichloroethanol and its glucuronide is about 8 hours; the half-life of
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trichloroacetic acid is about 4 days. Some data suggest that the half-life of trichloroethanol is
increased several-fold in preterm and full-term infants compared to toddlers and adults. The
major route of excretion of the metabolites of chloral hydrate is the urine. Chloral hydrate and its
metabolites have been found in milk from women treated with chloral hydrate. The
concentration of these chemicals, however, is too low to cause a pharmacological effect in the
nursing infant.
Acute administration of chloral hydrate to mice causes loss of coordination (ataxia) at
about the same exposure as in humans for the same effect. A 90-day study in mice shows no
evidence of behavioral changes or other neurotoxicity. Chronic studies in rats and mice show no
evidence of behavioral changes and no evidence of histopathological changes in nervous tissue.
There is some evidence of mild liver toxicity following chronic exposure in rats and mice. A
slight decrement in humoral immunity was observed in female mice following exposure for 90
days. The antibody-forming cell response is considered an excellent indicator of the status of
humoral immunity because of the complex cellular cooperation required to produce antibody and
because the number of cells that produce antibody can be quantified. A depression in the number
of these cells is considered an adverse response because the production of antibodies is important
to the defense strategy of the organism. However, the quantitative relationship between the
depression in antibody-forming cells in the spleen and the concentration of circulating antibody
is unknown. In this study, because there was no depression in circulating antibodies measured by
the hemagglutination titer, there might be no significant depression in the ability of the host to
mount a protective antibody response. Chloral hydrate has been tested for developmental effects
in rats and mice. No structural abnormalities were observed. A slight effect was observed in
mice in passive avoidance learning when dams were exposed prior to breeding, during gestation
and nursing, and pups were tested at 23 days of age. Although chloral hydrate has not been
tested in a two-generation reproduction study, the data on reproductive performance and on
effects on sperm and oocytes do not suggest that reproductive toxicity is likely to be a critical
effect. In addition, no histopathological effects are observed in reproductive organs of rodents in
subchronic or chronic studies. All of the studies in laboratory animals show noncancer health
effects at an exposure far in excess of the exposure that causes CNS depression and
gastrointestinal irritation in humans. See Table 2.
4.6. WEIGHT-OF-EVIDENCE EVALUATION AND CANCER
CHARACTERIZATIONSYNTHESIS OF HUMAN, ANIMAL, AND OTHER
SUPPORTING EVIDENCE, CONCLUSIONS ABOUT HUMAN
CARCINOGENICITY, AND LIKELY MODE OF ACTION
There are no carcinogenicity data from humans. Two bioassays in rats show no increase
in tumors at any site. Because only minimal toxicity was observed in the livers of the rats in
these bioassays, the tests were not conducted at the maximum tolerated dose. A chronic bioassay
in female mice showed a slight increase in the severity of hyperplasia and a slight increase in the
incidence of adenoma in the pituitary gland pars distalis at the highest exposure tested. There is
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some evidence that chloral hydrate causes hepatocellular tumors in male mice. An earlier study
showing an increase in hepatic adenomas or trabecular carcinomas following a single bolus
exposure could not be confirmed in a study using more animals and higher exposures. Three
separate 2-year bioassays in male mice show an increased incidence of hepatocellular adenoma
or carcinoma. There are no data identifying a lesion that is a precursor to the hepatocellular
tumors. The strain of mice used has a very high spontaneous incidence of hepatocellular tumors.
Two of the metabolites of chloral hydrate, trichloroacetic acid and dichloroacetic acid, have been
shown to cause hepatocellular tumors in rodents. Trichloroacetic acid causes hepatocellular
tumors only in mice. Dichloroacetic acid causes hepatocellular tumors in both rats and mice.
Chloral hydrate has been extensively studied as a genotoxic agent. Chloral hydrate was
positive in some bacterial mutation tests, indicating that it may be capable of inducing point
mutations. It was also positive in the mouse lymphoma assay for mutations at the TK locus.
Chloral hydrate induced somatic and germ cell mutations in Drosophila melanogaster. Some
data also show chloral hydrate to be a very weak clastogen in mammalian cells.
Chloral hydrate has been shown to induce aneuploidy in a variety of cells, including
Saccharomyces cerevisiae, Aspergillus nidulans, Chinese hamster embryonic fibroblasts,
Chinese hamster primary cell lines LUC2 and DON:Wg3h, human peripheral blood lymphocytes,
mouse spermatocytes, and mouse spermatids. Because there is a mixture of positive and
negative in vivo data, with no reason to weigh some studies more than others, it is not clear
whether chloral hydrate is capable of inducing genetic damage in vivo. Additional in vivo
studies using standard protocols would help clarify the relevance of genetic damage to a human
health risk assessment.
The aneugenic effects of chloral hydrate are exposure-dependent and thought to arise via
disruption of the mitotic spindle structure and/or function by inhibition of tubulin and/or
microtubule-associated proteins; both substances are components of the spindle apparatus
(Brunner et al., 1991; Lee at al., 1987; Wallin and Hartley-Asp, 1993). Some data also suggest
that chloral hydrate may act on the spindle apparatus through an increase in the concentration of
intracellular free calcium (Lee et al., 1987).
Although chloral hydrate and its metabolites, trichloroacetic acid and dichloroacetic acid,
can induce a variety of mutational events, they do so with very low potency. Owing to the high
concentration of chloral hydrate and its metabolites required to induce an observable effect in
these assays, it is not likely that a genotoxic mode of action can be held responsible for the
pituitary adenomas found in female mice or the hepatocellular tumors found in male mice.
Several other mechanisms may play a role in the induction of tumors in the liver of male
mice. There is no convincing evidence that chloral hydrate causes direct damage to DNA. In
vitro studies with chloral hydrate, trichloroethanol, and trichloroacetic acid and mouse
microsomes, however, show lipid peroxidation and formation of covalently bound DNA adducts.
These effects appear to be mediated by the formation of free radicals by CYP2E1. Another
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possibility concerns exposure-dependent cytotoxicity leading to compensatory hyperplasia. A
single treatment of mice with chloral hydrate caused an increase in the mitotic index in liver
cells. The increased cell division is hypothesized to either provide additional opportunities for
errors in DNA replication or allow initiated cells to progress to a tumor. Some data suggest a
role for peroxisomal proliferation in the liver of male mice. Another potentially contributing
mechanism of carcinogenesis is disruption of intercellular communication, which has been
shown in one experiment to be influenced by chloral hydrate.
Although the mechanism of chloral hydrate-induced carcinogenicity in mice is unclear,
one mechanism that appears less likely to be responsible is H-ras proto-oncogene activation.
Collectively, these data provide suggestive evidence of carcinogenicity in mice, but the
weight of evidence is not sufficient to conclude that carcinogenesis is the critical effect.
4.6.1. Susceptible Populations
Simultaneous ingestion of ethanol and chloral hydrate increases the sedative and side
effects of chloral hydrate. The mechanism is the increase in the concentration of the
pharmacologically active metabolite, trichloroethanol, in the presence of ethanol. Chronic
abusers of ethanol are, therefore, somewhat more sensitive to the adverse effects of chloral
hydrate.
EPA is not aware of any studies showing increased susceptibility to chloral hydrate in
individuals with genetic polymorphisms in the enzyme that metabolize chloral hydrate. EPA
believes that individuals with deficiencies in alcohol dehydrogenase or aldehyde dehydrogenase
will not be at increased risk from the effects of chloral hydrate when exposure is at or below the
reference dose. Individuals with deficiencies in alcohol dehydrogenase will be less able to
metabolize chloral hydrate to trichloroethanol. However, because chloral hydrate is rapidly
excreted by the kidney, it is unlikely that these individuals will experience greater or more
prolonged central nervous system depression or potential adverse effects from the increase in the
amount of trichloroacetic acid formed. Similarly, individuals with deficiencies in aldehyde
dehydrogenase will be less able to metabolize chloral hydrate to trichloroacetic acid. However,
as 92% of the chloral hydrate is converted to trichloroethanol, the slight increase in the
trichloroethanol concentration in these individuals has no biological significance.
4.6.1.1. Possible Childhood Susceptibility
Because of the immaturity of hepatic metabolism, particularly the glucuronidation
pathway, and decreased glomerular filtration, the half-life of trichloroethanol is longer in infants
(preterm and full-term) than in adults. The half-life of trichloroethanol in toddlers and adults is
similar. Because of the longer half-life of trichloroethanol, pre-term and full term infants will
experience prolonged effects when chloral hydrate is administered. However, at the reference
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dose for chloral hydrate, the steady-state concentration of trichloroethanol in these groups is far
below the concentration required for the pharmacological effect. See Appendix B.
4.6.1.2. Possible Gender Differences
Although male laboratory rodents seem to be more sensitive than female laboratory
rodents to hepatic effects, there is no evidence of a gender effect in humans to the sedative or
side effects of chloral hydrate at the recommended clinical dose.
5. DOSE-RESPONSE ASSESSMENTS
5.1. ORAL REFERENCE DOSE (RfD)
5.1.1. Choice of Principal Study and Critical EffectWith Rationale and Justification
The effect that occurs at the lowest exposure is CNS depression and gastrointestinal
irritation in humans. As these effects would not be intended or desirable in the general
population, EPA considers these responses as adverse effects and uses them to derive the
reference dose.
Acute gavage exposure in mice shows neurological effects (ataxia) at about the same
exposure for the comparable effect in humans. A subchronic study in mice using sensitive tests
for neurobehavioral changes found none. Chronic studies in rats and mice show no evidence of
neurobehavioral changes and no evidence of histopathological changes in nervous tissue. As
with other chlorinated chemicals, there is some evidence of hepatotoxicity in rodent liver
following chronic oral exposure. These effects are of minimal severity, may be related to
precancerous lesions, and occur at an exposure greater than that required for CNS depression and
gastrointestinal irritation following an acute bolus dose.
5.1.2. Methods of AnalysisIncluding Models (PBPK, BMD, etc.)
No data are available to determine a NOAEL in humans. The recommended clinical
dose for sedation in adults is 250 mg, taken 3 times a day (Goodman and Oilman, 1985). The
LOAEL is 10.7 mg/kg-day (assuming a 70 kg body weight). The pharmacokinetic information
shows that chloral hydrate and the pharmacologically active metabolite, trichloroethanol, will not
bioaccumulate. See Appendix B.
5.1.3. RfD DerivationIncluding Application of Uncertainty Factors (UF) and Modifying
Factors (MF)
The reference dose of 0.1 mg/kg-day was estimated from the LOAEL of 10.7 mg/kg-day
using a total uncertainty factor (UF) of 100 and a modifying factor (MF) of 1. An uncertainty
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factor of 10 was used to extrapolate from a LOAEL to NOAEL. An uncertainty factor of 10 was
used for intraspecies variability. An uncertainty factor for chronic duration is not used. Chloral
hydrate and the active metabolite, trichloroethanol, do not bioaccumulate. Therefore, continuous
daily exposure to chloral hydrate at the reference dose will not result in a concentration of
trichloroethanol in the blood required for the pharmacological effect. Developmental toxicity,
including developmental neurotoxicity, and immunotoxicity are not critical effects. Although
there is no two-generation reproduction study, a UF for database limitations is not needed, as
there is evidence from several studies that reproductive toxicity is not likely to be a critical effect.
Although the reference value of 0.1 mg/kg-day derived from the pharmacologically active
dose in humans is an acute RfD, keeping exposure below this level will also be protective for any
noncancer health effect from chronic exposure. For example, chronic exposure to chloral hydrate
does not cause adverse effects in the liver of rats or mice until the exposure approaches 135 or
160 mg/kg-day, respectively. Similarly, there are no reproductive, developmental,
neurobehavioral, or immunological effects following long-term treatment of laboratory animals
until the exposure approaches 160 mg/kg-day. See Table 2. Therefore, it is appropriate to use
the acute RfD also as the chronic RfD.
5.2. INHALATION REFERENCE CONCENTRATION (RfC)
There are no inhalation studies adequate for establishing a reference concentration.
5.3. CANCER ASSESSMENT
No adequate data are available to calculate an oral slope factor or an inhalation unit risk.
6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD
AND DOSE RESPONSE
6.1. HUMAN HAZARD POTENTIAL
Chloral hydrate has a long history of use in human and veterinary medicine as a
sedative/hypnotic drug. Its metabolite, trichloroethanol, is responsible for the pharmacological
action in the central nervous system. Chloral hydrate is completely absorbed following oral
exposure and is rapidly distributed to all major tissues. Chloral hydrate is rapidly metabolized in
the blood and liver. The major route of excretion of metabolites is the urine. Chloral hydrate
and its metabolites have been found in milk from women treated with chloral hydrate, but below
the concentration that would cause sedation in the nursing infant. Chloral hydrate and
trichloroethanol do not bioaccumulate.
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There are no controlled studies on toxicity to humans following extended exposure to
chloral hydrate. Studies in laboratory animals demonstrate that liver is a target tissue and
hepatocellular tumors have been observed in male mice and adenomas in the pituitary gland pars
distalis in female mice after chronic, high dose administration. No tumors occurred in rats after
chronic high-dose administration. Slight effects are also observed in some studies in laboratory
animals on sperm motility, developmental neurotoxicity (passive avoidance learning), and
humoral immunity. All of the adverse effects noted in studies in laboratory animals occur at an
exposure that is greater than the recommended clinical dose for sedation in humans.
6.2. DOSE RESPONSE
The quantitative estimate of human risk for noncancer effects is based on the
recommended clinical dose for sedation in humans. At this exposure the adverse effects are
central nervous system depression and gastrointestinal irritation. The reference dose is 0.1
mg/kg-day. This is 1/100 of the recommended daily dose for sedation in humans.
Although there is suggestive evidence of formation of tumors in mice and some data
showing aneugenicity, the mode of action for the formation of tumors is not known. It is also not
known whether this response is relevant for humans. On the basis of available data, the most
likely mode of action for the formation of tumors in mice involves interaction with cellular
enzymes and proteins in contrast to direct interaction with DNA. These effects are expected to
show a nonlinear response at low exposure.
Millions of people are exposed to chloral hydrate on a daily basis because it is formed
during the disinfection of drinking water with chlorine. The typical concentration in a public
water supply in the United States is 5 |ig/L (US EPA, 1994e). Assuming a water consumption of
2 L/day and a body weight of 70 kg, the exposure is 0.00014 mg/kg-day. This exposure is
approximately 700 times lower than the reference dose.
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APPENDIX A. EXTERNAL PEER REVIEW-
SUMMARY OF COMMENTS AND DISPOSITION
The reviewers made a number of editorial suggestions and other suggestions to clarify
specific portions of the text. These changes were incorporated in the document as appropriate
and are not discussed further.
The reviewers were satisfied that the relevant literature was included. One reviewer
indicated that NCTR had conducted chronic bioassays on chloral hydrate and requested that EPA
incorporate information from these studies if final reports became available in a timely manner.
EPA has incorporated the results of these studies and NTP's conclusions, based on the
peer review conducted on May 18, 2000, in this report.
Although the reviewers were satisfied that the most appropriate critical effect was used to
derive the reference dose, one reviewer requested that more emphases be placed on
gastrointestinal irritation (nausea, vomiting, diarrhea). These effects are directly caused by
chloral hydrate and occur before absorption and metabolism to trichloroethanol. Another
reviewer pointed out that the critical effect is central nervous system depression. The reviewer
further pointed out that although sedation and CNS depression are related, the quantitative
relationship between the effects is not known for chloral hydrate.
EPA agrees with the reviewers and has revised the document to list CNS depression and
gastrointestinal irritation as the critical effects.
Two reviewers questioned why a dated version of Goodman and Oilman was cited as the
source for the LOAEL for chloral hydrate. They suggested consulting an FDA summary on
chloral hydrate, consulting the Physicians Desk Reference, or conducting a critical evaluation of
the human clinical literature.
EPA found no useful information in current versions of the Physicians Desk Reference.
EPA found that the information in Goodman and Oilman corresponded to information found in
the clinical publications cited in the review. EPA therefore did not feel it necessary to evaluate
the publications over the more than 130 years of clinical use of chloral hydrate. EPA was
advised by FDA that because chloral hydrate is a pre-1938 drug product, a summary for chloral
hydrate is not available. FDA advised that old versions of Goodman and Oilman could be
considered to contain authoritative information on chloral hydrate.
Two reviewers asked for more effective use of the toxicokinetic calculations in Appendix
B and the Benchmark Dose calculations in Appendix C.
EPA has incorporated more information from Appendix B, but has decided to delete
Appendix C from the document. Appendix C contained the calculations for the exposure-
39
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response relation for hepatocellular tumors from George et al. (2000). That information was
included in the peer review draft in case reviewers had questions about the exposure-response
relation and thought that chloral hydrate should be assigned to Cancer Group B2 (probable
human carcinogen). As the reviewers agreed that chloral hydrate was properly assigned to
Cancer Group C (possible human carcinogen) under the 1986 Cancer Guidelines [suggestive
evidence of carcinogenicity under the 1996 proposed Cancer Guidelines], information on the
exposure-response relation for tumors is no longer necessary, as it is not appropriate to derive a
cancer slope factor or unit risk for a chemical showing only suggestive evidence of
carcinogenicity.
Two reviewers wanted more scientific justification for the specific values of 10 used for
the uncertainty factors for the LOAEL to NOAEL extrapolation and for intraspecies variability.
One of these reviewers also requested a discussion of the known range of genetic polymorphisms
in different races for the enzymes involved in the metabolism of chloral hydrate, as well as a
discussion of whether individuals with hepatic diseases or infants with respiratory insufficiency
or compromised hepatic and/or renal capabilities are adequately accounted for in the tenfold
factor used for intraspecies variability.
EPA used values of 10 for these two uncertainty factors because there was insufficient
reason to depart from the defaults. A greater justification would have been provided if some
lesser value (3 or 1) had been used. EPA is not aware of any studies with chloral hydrate in
different races that would provide definitive information to answer the reviewer's question.
EPA, however, expanded Section 4.6.1 to discuss the issues raised by the reviewer. EPA has
revised Section 4.6.1.1 to consider the increased half-life for elimination of trichloroethanol in
preterm and full-term infants. The toxicokinetic analysis shows that the increased concentration
of trichloroethanol in these subgroups is below the concentration required for a pharmacological
effect. Finally, it has not been EPA's practice to ensure protection of 100% of the population
from exposure to a chemical from the environment.
One reviewer wanted more discussion, justification, and explanation for using the acute
RfD as the chronic RfD.
EPA expanded the discussion in Section 5.1.3 to clarify the reasoning.
One reviewer wanted a discussion of studies of habitual abuse of chloral hydrate and the
consequences of drug withdrawal after habituation. This reviewer also wanted a more extensive
discussion of the mechanism of depression of the central nervous system.
EPA believes that it is not necessary to include this information in the Toxicological
Review. A discussion of these topics is beyond the scope of the Toxicological Review, which, as
stated in the Foreword, is designed to provide support for the hazard identification and exposure-
response assessment on IRIS.
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One reviewer requested inclusion of information on the organoleptic properties of chloral
hydrate in drinking water and the concentration at which adverse taste and odor become a
problem.
EPA agrees that including this information would be useful. However, such information
was not located in the literature.
One reviewer wanted more discussion of the consequences for humans of the
bioaccumulation of trichloroacetic acid.
EPA acknowledges that because trichloroacetic acid binds fairly strongly to human serum
proteins, its elimination is slow and it is the only metabolite of chloral hydrate that accumulates
with repeated exposure. When exposure to chloral hydrate is at or below the RfD, the
concentration of trichloroacetic acid in humans is below the concentration that will cause adverse
biological effects.
One reviewer suggested that the LOAEL should be based on the 500 mg/day dose rather
than the 750 mg/day dose.
EPA prefers to use the average exposure, rather than the minimum exposure, when
deriving a LOAEL. There is also some anecdotal information that chloral hydrate is not effective
in a large number of patients when the minimum dose is administered.
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APPENDIX B. TOXICOKINETICS OF CHLORAL HYDRATE
This toxicokinetic analysis is used to estimate the steady state concentrations of
trichloroacetic acid (TCA) and trichloroethanol (TCEOH) in mice and humans using a one-
compartment model, assuming that absorption of chloral hydrate (CH) from the gastrointestinal
tract and its metabolism in the blood is very rapid compared to the rate of elimination of TCA
and TCEOH. This assumption is supported by the data of Beland et al. (1998) in mice and
Breimer (1977) and Zimmermann (1998) in humans.
Beland et al. (1998) indicated that 15% of the dose of chloral hydrate is converted directly
to TCA and 77% is converted to TCEOH. In humans Allen and Fisher (1993) estimated that 8%
of a dose of chloral hydrate is converted directly to TCA and 92% is converted to TCEOH.
Additional TCA is formed from TCEOH. The total TCA formed in humans is approximately
35% of the dose of chloral hydrate.
Estimation of TCA in mice at steady state at the clinically recommended dose for humans
[TCA]ss.blood = PK0/VKel = 2.5 mg/L
[TCA]ss,iver = [TCA]ss.blood x PC = 3.0 mg/L
where:
P = proportion of CH converted to TCA = 0.15 (Beland et al; 1998)
K0 = dosing rate for CH = 10.7 mg/kg-day, equivalent to 0.446 mg/kg-hr
V = volume of distribution = 0.321 L/kg (Beland et al; 1998)
Kel = first-order elimination constant for TCA = 0.0819/hr (Beland et al;
1998)
PC = liver-blood partition coefficient =1.18 (Abbas and Fisher, 1997)
Estimation of TCA in humans at steady state at the clinically recommended dose
[TCA]ss.blood = PK0/VKel = 55 mg/L
[TCA]ss,iver = [TCA]ss.blood x PC = 36 mg/L
where:
P = proportion of CH converted to TCA = 0.35 (Allen and Fisher,
1993)
K0 = dosing rate for CH = 10.7 mg/kg-day, equivalent to 0.446 mg/kg-hr
V = volume of distribution = 0.102 L/kg (Allen and Fisher, 1993)
Kel = first-order elimination constant for TCA = 0.028/hr (Allen and
Fisher, 1993)
PC = liver-blood partition coefficient = 0.66 (Fisher et al; 1998)
Estimation of TCA in humans at steady state at the reference dose
[TCA]ss.blood = PK0/VKel = 1.8 mg/L
[TCA]ss,iver = [TCA]ss.blood x PC = 1.2 mg/L
where:
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P = proportion of CH converted to TCA = 0.35 (Allen and Fisher,
1993)
K0 = dosing rate for CH = 0.1 mg/kg-day, equivalent to 0.004 mg/kg-hr
V = volume of distribution = 0.102 L/kg (Allen and Fisher, 1993)
Kel = first-order elimination constant for TCA = 0.0078/hr (Allen and
Fisher, 1993)
PC = liver-blood partition coefficient = 0.66 (Fisher et al; 1998)
Estimation of TCEOH in mice at steady state at 166 mg/kg-day
[TCEOH]ss.blood = PK0/VKel = 0.58 mg/L
where:
P = proportion of CH converted to TCEOH = 0.77 (Beland et al; 1998)
K0 = dosing rate for CH = 166 mg/kg-day, equivalent to 6.917 mg/kg-hr
V = volume of distribution = 1 L/kg (cited in Beland et al; 1998)
Kel = first-order elimination constant for TCEOH = 9.24/hr (Beland et al;
1998)
Chloral hydrate at 160 mg/kg-day was the highest exposure used in the 90-day
neurobehavioral study by Kallman et al. (1984); chloral hydrate at 166 mg/kg-day was the highest
exposure used in the 104-week bioassay of Daniel et al. (1992a). These exposures are a NOAEL
for sedation in mice.
Estimation of TCEOH in humans at steady state at the clinically recommended dose
[TCEOH]ss.blood = PK0/VKel = 5.4 mg/L
where:
P = proportion of CH converted to TCEOH = 0.92 (Allen and Fisher,
1993)
K0 = dosing rate for CH = 10.7 mg/kg-day, equivalent to 0.446 mg/kg-hr
V = volume of distribution = 0.87 L/kg (Fisher et al; 1998)
Kel = first-order elimination constant for TCEOH = 0.087/hr (Breimer,
1977)
Estimation of TCEOH in adult humans at steady state at the reference dose
[TCEOH]ss.blood = PK0/VKel = 0.049 mg/L
where:
P = proportion of CH converted to TCEOH = 0.92 (Allen and Fisher,
1993)
K0 = dosing rate for CH = 0.1 mg/kg-day, equivalent to 0.004 mg/kg-hr
V = volume of distribution = 0.87 L/kg (Fisher et al; 1998)
Kel = first-order elimination constant for TCEOH = 0.087/hr (Breimer,
1977)
43
-------
Estimation of TCEOH in preterm infants at steady state at the reference dose
[TCEOH]ss.blood = PK0/VKel = 0.25 mg/L
where:
P = proportion of CH converted to TCEOH = 0.92 (Allen and Fisher,
1993)
K0 = dosing rate for CH = 0.1 mg/kg-day, equivalent to 0.004 mg/kg-hr
V = volume of distribution = 0.87 L/kg (Fisher et al; 1998)
Kel = first-order elimination constant for TCEOH = 0.017/hr (Mayers et
al; 1991)
Estimation of TCEOH in full term infants at steady state at the reference dose
[TCEOH]ss.blood = PK0/VKel = 0.17 mg/L
where:
P = proportion of CH converted to TCEOH = 0.92 (Allen and Fisher,
1993)
K0 = dosing rate for CH = 0.1 mg/kg-day, equivalent to 0.004 mg/kg-hr
V = volume of distribution = 0.87 L/kg (Fisher et al; 1998)
Kel = first-order elimination constant for TCEOH = 0.025/hr (Mayers et
al; 1991)
44
-------
Figure 1. Metabolism of Chloral Hydrate
OH
C13C-C-H
OH
chloral hydrate
alcohol
dehydrogenase
aldehyde
dehydrogenase
C13C-C-OH
trichloroacetic acid
H
C13C-C-H
OH
trichloroethanol
UDPGA-
transferase
entero-
hepatic
circulation
Cl3C-CH20-glu
trichloroethanol-glucuronide
C12C-C-OH
dichloroacetic acid
45
-------
Table 1. Genetic and related effects of chloral hydrate and its metabolites
Test system
Chloral hydrate
S. typhimurium, TA 100,
reverse mutation
S. typhimurium, TA 100,
reverse mutation
S. typhimurium, TA 100,
reverse mutation
S. typhimurium, TA 100,
reverse mutation
S. typhimurium, TA 100,
reverse mutation
S. typhimurium, TA 100,
reverse mutation
S. typhimurium, TA 104,
reverse mutation
S. typhimurium, TA 98,
reverse mutation
S. typhimurium, TA 98,
reverse mutation
S. typhimurium, TA 98,
reverse mutation
S. typhimurium, TA 98,
reverse mutation
S. typhimurium, TA 1535,
reverse mutation
S. typhimurium, TA 1537,
reverse mutation
Result3
Without With
0 +
+ +
+
+ +
+ +
0 +
Dose"
(LED/HID)
2,500
2,000
500
1,850
300/750
2,000
2,000
2,000
5,000
5,000
1,850
5,000
5,000
Reference
Waskell, 1978
Bruce and Heddle, 1979
Haworth et al., 1983
Leuschner and Leuschner,
1991
Gilleretal., 1995
Metal., 1994
Metal., 1994
Bruce and Heddle, 1979
Waskell, 1978
Haworth et al., 1983
Leuschner and Leuschner,
1991
Haworth et al., 1983
Haworth et al., 1983
46
-------
Table 1. Genetic and related effects of chloral hydrate and its metabolites (continued)
Test system
S. typhimurium, TA 1537,
reverse mutation
S. typhimurium, TA 1538,
reverse mutation
S. cerevisiae D7, reverse
mutation
S. cerevisiae D7, gene
conversion
Gene mutation (small
colony), mouse lymphoma
cells (LSHSY/TK^SJ.l.C)
A. nidulans, diploid stain,
mitotic crossing over
A. nidulans, diploid stain,
mitotic crossing over
A. nidulans, diploid stain,
mitotic crossing over
A. nidulans, diploid stain,
mitotic crossing over
A. nidulans, diploid stain,
haploids and nondisjunctional
diploids
A. nidulans, diploid stain,
aneuploidy
A. nidulans, haploid conidia,
aneuploidy and polyploidy
A. nidulans, diploid stain,
nondisjunctional mitotic
segregants
Result3
Without With
(+)
+
0
0
0
0
+ 0
+ 0
+ 0
+ 0
Dose"
(LED/HID)
1,850
1,850
3,300
2,500
1,000
1,650
6,600
1,000
990
825
825
825
450
Reference
Leuschner and Leuschner,
1991
Leuschner and Leuschner,
1991
Bronzetti etal., 1984
Bronzetti et al., 1984
Harrington-Brock et al.,
1998
Crebilli et al., 1985
Kafer, 1986
Kappas, 1989
Crebilli et al., 1985
Crebilli et al., 1985
Kafer, 1986
Kafer, 1986
Kappas, 1986
47
-------
Table 1. Genetic and related effects of chloral hydrate and its metabolites (continued)
Test system
A. nidulans, diploid stain,
haploids and nondisjunctional
diploids
A. nidulans, haploid stain,
hyperploidy
S. cerevisiae, meiotic
recombination
S. cerevisiae, disomy in
meiosis
S. cerevisiae, diploids in
meiosis
S. cerevisiae, mitotic
chromosomal malsegregation
S. cerevisiae, monosomy
Spring wheat, chromosomal
loss or gain
D. melanogaster, somatic
mutation wing spot test
D. melanogaster, somatic
mutation wing spot test
D. melanogaster, sex-linked
recessive lethal mutation
DNA-protein cross-links, rat
liver nuclei in vitro
DNA single-strand breaks
(alkaline unwinding), rat
primary hepatocytes in vitro
DNA repair, E. coli PQ37,
SOS chromotest
Result3
Without With
+ 0
+ 0
? 0
+ 0
+ 0
+ 0
+ 0
0
+
+
+
0
0
Dose"
(LED/HID)
660
2,640
3,300
2,500
3,300
1,000
1,000
5,000
830
825
1,660
41,250
1,650
10,000
Reference
Crebilli et al., 1991
Crebilli et al., 1991
Sora and Agostini-Carbone,
1987
Sora and Agostini-Carbone,
1987
Sora and Agostini-Carbone,
1987
Albertini, 1990
Parry et al., 1990
Sandhuetal., 1991
Patnaik et al., 1992
Zordan et al., 1994
Patnaik et al., 1992
Keller and Heck, 1988
Chang etal., 1992
Ciller etal., 1995
48
-------
Table 1. Genetic and related effects of chloral hydrate and its metabolites (continued)
Test system
Kinetochore-positive
micronuclei, Chinese hamster
Cl-1 cells, in vitro, with
antikinetochore antibodies
Kinetochore-negative
micronuclei, Chinese hamster
Cl-1 cells, in vitro, with
antikinetochore antibodies
Kinetochore-positive
micronuclei, Chinese hamster
LUC2 cells in vitro
Kinetochore-positive
micronuclei, Chinese hamster
LUC2 cells in vitro
Inhibition of intercellular
communication, B6C3FJ
mouse hepatocytes in vitro
Inhibition of intercellular
communication, F344 rat
hepatocytes in vitro
Chromosomal aberrations,
Chinese hamster CHED cells
in vitro
Aneuploidy, Chinese hamster
CHED cells in vitro
Aneuploidy, primary Chinese
hamster embryonic cells in
vitro
Aneuploidy, Chinese hamster
LUC2 p4 cells in vitro
Result3
Without With
+ 0
0
+ 0
+ 0
0
0
+ 0
+ 0
+ 0
+ 0
Dose"
(LED/HID)
165
250
400
400
83
83
20
10
250
250
Reference
Degrassi and Tanzarella,
1988
Degrassi and Tanzarella,
1988
Parry et al., 1990
Lynch and Parry, 1993
Klaunig et al., 1989
Klaunig et al., 1989
Furnusetal., 1990
Furnusetal., 1990
Natarajanetal., 1993
Warretal., 1993
49
-------
Table 1. Genetic and related effects of chloral hydrate and its metabolites (continued)
Test system
Tetraploidy and
endoreplication, Chinese
hamster LUC2 p4 cells in
vitro
Apolar mitosis, Haemanthus
katherinae endosperm in
vitro
Inhibition of spindle
elongation, PtK2 kangaroo
rat kidney epithelial cells in
vitro
Inhibition of chromosome to
pole movement, PtK2
kangaroo rat kidney epithelial
cells in vitro
Breakdown of mitotic
microtubuli, PtK2 kangaroo
rat kidney epithelial cells in
vitro
Multipolar mitotic spindles,
Chinese hamster DON:Wg3h
cells in vitro
Chromosomal dislocation
from mitotic spindle, Chinese
hamster DON:Wg3h cells in
vitro
Lacking mitotic spindle,
Chinese hamster DON:Wg3h
cells in vitro
Metaphase defects, lacking
mitotic spindle, Chinese
hamster LUC1 cells in vitro
Result3
Without With
+ 0
+ 0
+ 0
+ 0
+ 0
+ 0
+ 0
+ 0
+ 0
Dose"
(LED/HID)
500
200
1,000
1,000
1,000
500
500
250
50
Reference
Warretal., 1993
Mole-Bajer, 1969
Lee et al., 1987
Lee et al., 1987
Lee et al., 1987
Parry et al., 1990
Parry et al., 1990
Parry et al., 1990
Parry et al., 1990
50
-------
Table 1. Genetic and related effects of chloral hydrate and its metabolites (continued)
Test system
Multipolar mitotic spindles,
Chinese hamster DON:Wg3h
cells in vitro
Chromosomal dislocation
from mitotic spindle, Chinese
hamster DON:Wg3h cells in
vitro
DNA single-strand breaks
(alkaline unwinding), human
lymphoblastoid CCRF-CEM
cells in vitro
Sister chromatid exchange,
human lymphocytes in vitro
Micronucleus induction,
isolated human lymphocytes
in vitro
Micronucleus induction,
human lymphocytes in whole
blood in vitro
Micronucleus induction,
human lymphocytes in vitro
Micronucleus induction,
human lymphocytes in vitro
Micronuclei, Chinese
hamster V79 cells in vitro
Micronucleus induction,
newt (Pleurodeles waltl)
larvae, peripheral
erythrocytes in vivo
Micronucleus induction,
mouse lymphoma cells
(LSlTSY/TK^S.T.l.C)
Result3
Without With
+ 0
+ 0
0
(+) 0
+
+ 0
(+) o
+ 0
+ 0
+
Dose"
(LED/HID)
50
500
1,650
54
1,500
100
100
100
316
200
1,250
Reference
Warretal., 1993
Warretal., 1993
Chang etal., 1992
Guetal., 1981
Vianetal., 1995
Migliore and Nieri, 1990
Ferguson et al., 1993
Van Hummelen and
Kirsch-Volders, 1992
Seelbach et al., 1993
Gilleretal., 1995
Harrington-Brock et al.,
1998
51
-------
Table 1. Genetic and related effects of chloral hydrate and its metabolites (continued)
Test system
Chromosomal aberrations,
mouse lymphoma cells
(LSlTSY/TK^S.T.l.C)
Kinetochore-positive
micronuclei, human diploid
LEO fibroblasts in vitro
Aneuploidy, human
lymphocytes in vitro
Aneuploidy, human
lymphocytes in vitro
Polyploidy, human
lymphocytes in vitro
C-Mitosis, human
lymphocytes in vitro
Polyploidy, mice, outbred
MF-1, oocytes in vitro
Polyploidy, mice, outbred
MF-1, oocytes in vitro
Host-mediated assay, S.
cerevisiae D7 recovered from
CD-I mouse lungs
DNA single-strand breaks
(alkaline unwinding), rat liver
in vivo
DNA single-strand breaks
(alkaline unwinding), mouse
liver in vivo
DNA single-strand breaks
(alkaline unwinding), male
Fischer 344 rat liver in vivo
Result3
Without With
+
+ 0
+ 0
+ 0
+ 0
+ 0
+
+
(+)
+
+
Dose"
(LED/HID)
1,250
120
250
50
137
75
50
125
500 po x 1
300 po x 1
100 po x 1
1,650 pox 1
Reference
Harrington-Brock et al.,
1998
Bonatti et al., 1992
Vagnarelli et al., 1990
Sbranaetal., 1993
Sbranaetal., 1993
Sbranaetal., 1993
Eichenlaub-Ritter and
Betzendahl, 1995
Eichenlaub-Ritter et al.,
1996
Bronzetti etal., 1984
Nelson and Bull, 1988
Nelson and Bull, 1988
Chang etal., 1992
52
-------
Table 1. Genetic and related effects of chloral hydrate and its metabolites (continued)
Test system
DNA single-strand breaks
(alkaline unwinding), male
B6C3FJ mouse liver in vivo
Chromosomal aberrations,
(c57Bl/CnexC3H/Cne)Fl
mouse secondary
spermatocytes (staminal
gonia-pachytene treated)
Chromosomal aberrations
(translocations, breaks, and
fragments)
(C57Bl/CnexC3H/Cne)Fl
mouse primary and secondary
spermatocytes (from
differentiating
spermatogonia-pachytene
stages treated)
Chromosomal aberrations,
male and female
(102/ElxC3H/El)Fl mouse
bone marrow cells in vivo
Chromosomal aberrations, rat
bone marrow cells in vivo
Chromosomal aberrations,
BALB/c mouse
spermatogonia treated,
spermatogonia observed in
vivo
Chromosomal aberrations,
ICR mouse oocytes treated in
vivo
Micronuclei, female mice
(C57B16xC3H/He)Fl, bone
marrow erythrocytes
Result3
Without With
+
Dose"
(LED/HID)
825 po x 1
82.7 ip x 1
413 ip x 1
500 ip x 1
1,000 po x 1
83 ip x 1
600 ip x 1
2,500 ip x 5
Reference
Chang etal., 1992
Russoetal., 1984
Liang and Pacchierotti,
1988
XuandAdler, 1990
Leuschner and Leuschner,
1991
Russo and Levis, 1992a
Mailhes et al., 1993
Bruce and Heddle, 1979
53
-------
Table 1. Genetic and related effects of chloral hydrate and its metabolites (continued)
Test system
Micronuclei, male and female
NMRI mice, bone marrow
erythrocytes in vivo
Micronuclei, mouse
spermatids in vivo
(preleptotene spermatocytes
treated)
Micronuclei, male BALB/c
mouse bone marrow
erythrocytes in vivo
Micronuclei (kinetochore-
positive and -negative) male
BALB/c mouse bone marrow
erythrocytes in vivo
Micronuclei, BALB/c mouse
early spermatids in vivo
(diakinesis/metaphase I and
metaphase II stages treated)
Micronuclei, male
(C57Bl/NcexC3H/Cne)Fl
mouse bone marrow
erythrocytes in vivo
Micronuclei, mouse
spermatids in vivo
(spermatogonial stem cells
and preleptotene
spermatocytes treated)
Micronuclei, mice
(102/ElxC3H/El)Fl,
polychromatic bone marrow
erythrocytes
Result3
Without With
+
+
+
+
Dose"
(LED/HID)
500 ip x 1
83 ip x 1
83 ip x 1
200 ip x 1
83 ip x 1
400 ip x 1
41 ip x 1
600 ip x 1
Reference
Leuschner and Leuschner,
1991
Russo and Levis, 1992b
Russo and Levis, 1992a
Russo etal., 1992
Russo and Levis, 1992a
Leopardi et al., 1993
Allen etal., 1994
Adleretal., 1991
54
-------
Table 1. Genetic and related effects of chloral hydrate and its metabolites (continued)
Test system
Micronuclei, mice
(102/ElxC3H/El)Fl,
polychromatic bone marrow
erythrocytes
Micronuclei, mice (E^CSFj),
spermatids in vivo
(spermatogonial stem cells
treated)
Micronuclei, mice (BeCSFj),
spermatids in vivo
(preleptotene and diakinesis
spermatids treated)
Aneuploidy,
(C57Bl/CnexC3H/Cne)Fl,
mouse secondary
spermatocytes in vivo
Aneuploidy,
(C57Bl/CnexC3H/Cne)Fl,
mouse secondary
spermatocytes (from
differentiating
spermatogonia-pachytene
stages treated)
Aneuploidy, ICR mouse,
metaphase II oocytes in vivo
Aneuploidy (hyperploidy).
ICR mouse metaphase II
oocytes in vivo
Polyploidy, male and female
102/ElxC3H/El)Fl mouse
bone marrow cells in vivo
Result3
Without With
+
+
(+)
Dose"
(LED/HID)
200 ip x 1
82.7 ip x 1
413.5ipx 1
82.7 ip x 1
165 ip x 1
200 ip x 1
600 ip x 1
600 ip x 1
Reference
Graweetal., 1997
Nutleyetal., 1996
Nutleyetal., 1996
Russoetal., 1984
Liang and Pacchierotti,
1988
Maihles et al., 1994
Maihles et al., 1994
XuandAdler, 1990
55
-------
Table 1. Genetic and related effects of chloral hydrate and its metabolites (continued)
Test system
Aneuploidy,
(102/ElxC3H/Cne)Fl mouse
secondary spermatocytes in
vivo
Aneuploidy, male
(C57Bl/NcexC3H/Cne)Fl
mouse bone marrow in vivo
Aneuploidy,
(C57Bl/NcexC3H/Cne)Fl
mouse secondary
spermatocytes in vivo
Aneuploidy, male mice (CD-
1), bone marrow erythrocytes
Aneuploidy (micronuclei),
male mice (CD-I), bone
marrow erythrocytes
Binding to DNA, male
B6C3FJ mouse liver in vivo
Gonosomal and autosomal
univalents
(C57Bl/CnexC3h/Cne)Fl
mouse primary spermatocytes
(from differentiating
spermatogonia-pachytene
stages treated)
Porcine brain tubulin
assembly inhibition in vitro
Porcine brain tubulin
assembly inhibition in vitro
Porcine brain tubulin
assembly inhibition in vitro
Result3
Without With
+
+
+
+
+ 0
+ 0
(+) 0
Dose"
(LED/HID)
200 ip x 1
400 ip x 1
400 ip x 1
200 ip x 1
200 ip x 1
800 ip x 1
413 ip x 1
9,900
40
165
Reference
Miller and Adi er, 1992
Leopardi et al., 1993
Leopardi et al., 1993
Gudietal., 1992
Marrazzini etal., 1994
Keller and Heck, 1988
Liang and Pacchierotti,
1988
Brunner et al., 1991
Brunner et al., 1991
Wallin and Hartley -Asp,
1993
56
-------
Table 1. Genetic and related effects of chloral hydrate and its metabolites (continued)
Test system
Centriole migration block,
Chinese hamster cells clone
237 in vitro
Cell transformation, Syrian
hamster embryo
Result3
Without With
+ 0
+
Dose"
(LED/HID)
1,000
350, 1 day
1, 7 day
Reference
Alov and Lyubskii, 1974
Gibson etal., 1995
Trichloroethanol
Prophage induction, E. coli
WP2
S. typhimurium, TA 100,
reverse mutation
Spindle aberrations, mice,
outbred MF-1, oocytes in
vitro
+
155,000
0.5 vapor
125
DeMarini etal., 1994
DeMarini etal., 1994
Eichenlaub-Ritter et al.,
1996
Trichloroacetic acid
prophage induction, E. coli
WP2s
S. typhimurium TA100,
reverse mutation
S. typhimurium TA100,
reverse mutation
S. typhimurium TA100,
reverse mutation
S. typhimurium TA1535,
reverse mutation
S. typhimurium TA1537,
reverse mutation
S. typhimurium TA1538,
reverse mutation
S. typhimurium TA98,
reverse mutation
10,000
225
2,000
4
2,000
1,000
1,000
225
DeMarini etal., 1994
Waskell, 1978
Nestmann et al., 1980
DeMarini etal., 1994
Nestmann et al., 1980
Nestmann et al., 1980
Nestmann et al., 1980
Waskell, 1978
57
-------
Table 1. Genetic and related effects of chloral hydrate and its metabolites (continued)
Test system
S. typhimurium TA98,
reverse mutation
Gene mutation (small and
large colony), mouse
lymphoma cells
(LSlTSY/TK^S.T.l.C)
Chromosomal aberrations,
human lymphocytes in vitro
DNA strand breaks, E6C3Fl
mouse hepatocytes in vitro
DNA strand breaks, Fischer
344 rat hepatocytes in vitro
DNA strand breaks, human
CCRF-CEM cells in vitro
DNA strand breaks, E6C3Fl
mouse hepatic cells in vivo
DNA strand breaks, E6C3Fl
mouse hepatic cells in vivo
DNA strand breaks, E6C3Fl
mouse hepatic cells in vivo
DNA strand breaks, E6C3Fl
mouse hepatic cells and
epithelial cells from stomach
and duodenum in vivo
DNA strand breaks, Sprague-
Dawley rat hepatic cells in
vivo
DNA strand breaks, Fischer
344 rat hepatocyte cells in
vivo
Result3
Without With
+
0
0
0
+
+
+
Dose"
(LED/HID)
1,000
2,250
5,000
1,630
1,630
1,630
1 po x 1
500 po x 1
500 po x 10
1,630 pox 1
100 po x 1
1,630 pox 1
Reference
Nestmann et al., 1980
Harrington-Brock et al.,
1998
Mackay et al., 1995
Chang etal., 1992
Chang etal., 1992
Chang etal., 1992
Nelson and Bull, 1988
Nelson etal., 1989
Nelson etal., 1989
Chang etal., 1992
Nelson and Bull, 1988
Chang etal., 1992
58
-------
Table 1. Genetic and related effects of chloral hydrate and its metabolites (continued)
Test system
Micronucleus induction,
Swiss mice in vivo
Micronucleus induction,
C57Bl/6JfBl 10/Alpk female
mice
Micronucleus induction,
C57Bl/6JfBl 10/Alpk male
mice
Chromosomal aberrations,
Swiss mouse bone-marrow
cells in vivo
Chromosomal aberrations,
Swiss mouse bone-marrow
cells in vivo
Chromosomal aberrations,
Swiss mouse bone-marrow
cells in vivo
Chromosomal aberrations,
chicken bone marrow cells in
vivo
Result3
Without With
+
+
+
+
+
Dose"
(LED/HID)
125 ip x 1
1,300 ipx 2
1,080 ip x 1
125 ip x 1
100 ip x 5
500 po x 1
200 ip x 1
400 po x 1
Reference
Bhunya and Behera, 1987
Mackay etal., 1995
Mackay etal., 1995
Bhunya and Behera, 1987
Bhunya and Behera, 1987
Bhunya and Behera, 1987
Bhunya and Jena, 1996
Dichloroacetic acid
prophage induction, E. coli
WP2s
S. typhimurium, DNA repair-
deficient TS24
S. typhimurium, DNA repair-
deficient TA2322
S. typhimurium, DNA repair-
deficient TA1 950
S. typhimurium, TA100,
reverse mutation
+
(+) (+)
2,500
31,000
31,000
31,000
1
DeMarini etal., 1994
Waskell, 1978
Waskell, 1978
Waskell, 1978
DeMarini etal., 1994
59
-------
Table 1. Genetic and related effects of chloral hydrate and its metabolites (continued)
Test system
S. typhimurium, TA98,
reverse mutation
Gene mutation (small
colony), mouse lymphoma
cells (LSlTSY/TK^S.T.l.C)
Chromosomal aberrations,
mouse lymphoma cells
(LSlTSY/TK^S.T.l.C)
Micronuclei, mouse
lymphoma cells
(LSlTSY/TK^S.T.l.C)
aneuploidy, mouse
lymphoma cells
(LSlTSY/TK^S.T.l.C)
DNA strand breaks, E6C3Fl
mouse hepatocytes in vitro
DNA strand breaks, Fischer
344 rat hepatocytes in vitro
DNA strand breaks, human
CCRF-CEM cells in vitro
DNA strand breaks, E6C3Fl
mouse hepatic cells in vivo
DNA strand breaks, E6C3Fl
mouse hepatic cells in vivo
DNA strand breaks, E6C3Fl
mouse hepatic cells in vivo
DNA strand breaks, E6C3Fl
mouse spleenocytes in vivo
Result3
Without With
(+) (+)
+ 0
+ 0
0
0
0
0
0
+
+
Dose"
(LED/HID)
5
300
600
800
800
2,580
1,290
1,290
13 po x 1
10 po x 1
1,290 po x 1
1,290 po x 1
Reference
Herbert et al., 1980
Harrington-Brock et al.,
1998
Harrington-Brock et al.,
1998
Harrington-Brock et al.,
1998
Harrington-Brock et al.,
1998
Chang etal., 1992
Chang etal., 1992
Chang etal., 1992
Nelson and Bull, 1988
Nelson etal., 1989
Chang etal., 1992
Chang etal., 1992
60
-------
Table 1. Genetic and related effects of chloral hydrate and its metabolites (continued)
Test system
DNA strand breaks, E6C3Fl
mouse epithelial cells from
stomach and duodenum in
vivo
DNA strand breaks, E6C3Fl
mouse hepatic cells in vivo
DNA strand breaks, Sprague-
Dawley rat hepatic cells in
vivo
DNA strand breaks, Fischer
344 rat hepatic cells in vivo
DNA strand breaks, Fischer
344 rat hepatic cells in vivo
Gene mutation, transgenic
B6C3FJ mouse in vivo
Micronucleus induction,
mouse polychromatic
erythrocytes
Result3
Without With
+
+
+
Dose"
(LED/HID)
1,290 po x 1
830 po x 7-
14 d
30 po x 1
645 po x 1
250 po x 30
weeks
160 po x 60
weeks
160 po x 9
Reference
Chang etal., 1992
Chang etal., 1992
Nelson and Bull, 1988
Chang etal., 1992
Chang etal., 1992
Leavitt et al., 1997
Fuscoeetal., 1992
a Without: without an exogenous metabolic activation system; With: with an exogenous metabolic activation system; +:
conclusion considered to be positive; (+): considered to be weakly positive in an inadequate study; : considered to be
negative; ?: considered to be inconclusive (variable responses in several experiments within an inadequate study); 0: not
tested.
b LED, lowest effective dose; HID, highest ineffective dose; in vitro tests, ng/ml; in vivo tests, mg/kg bw; ip,
interperitoneal; po, orally.
61
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Table 2. Summary of nonneoplastic effects
Species
Human
Rat
Rat
Rat
Rat
Rat
Mouse
Mouse
Mouse
Mouse
Mouse
Duration
1 day, 3
doses
90 days
104 weeks
124 weeks
52 weeks
gd 1-22
14 days
90 days
104 weeks
104 weeks
104 weeks
Endpoint
CNS
depression,
GI irritation
Liver
necrosis and
increase in
serum
enzymes
Liver
hypertrophy
Sperm
motility
Development
Increased
liver weight
Increased
liver weight
Increased
liver weight
and necrosis
NOAEL
mg/kg-day
96
162.6
45
55
151
14.4
16
146.6b
71.4C
LOAEL
mg/kg-day
10.7
168
135
188
144
160
166a
Reference
Goodman
and Gilman,
1985
Daniel et al.,
1992b
George et al.,
2000
Leuschner
and
Beuscher,
1998
Klinefelter et
al., 1995
Johnson et
al., 1998
Sanders et
al., 1982
Sanders et
al., 1982
Daniel et al.,
1992a
George et al.,
2000
NTP,
2000a,b
62
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Table 2. Summary of nonneoplastic effects (continued)
Species
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Duration
3 weeks pre-
breeding and
during
gestation
Pre-breeding,
gestation,
and nursing
1 day
14 days
90 days
14 days
90 days
Endpoint
Reproduction
and
development
Passive
avoidance
learning in
pups
Ataxia
Neuro-
behavior
Neuro-
behavior
Immuno-
toxicity
Humoral
immunity
NOAEL
mg/kg-day
204.8
21.3
-
144
160
144
16
LOAEL
mg/kg-day
'
204.8
50
-
-
-
160
Reference
Kallman et
al., 1984
Kallman et
al., 1984
Kallman et
al., 1984
Kallman et
al., 1984
Kallman et
al., 1984
Kauffmann et
al., 1982
Kauffmann et
al., 1982
a Increased incidence of hepatocellular adenomas and carcinomas at 166 mg/kg-day.
b Increased prevalence of hyperplasia and hepatocellular adenomas or carcinomas at 13.5, 65, and 146.6 mg/kg-day.
0 Increased incidence of adenoma in pituitary gland par distalis in females and hepatocellular carcinoma in males.
63
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