National Toxicology Program

Toxicity Report Series

Number 55

NTP Technical Report

on the Toxicity Studies of

trans-1,2-Dichloroethylene

(CAS No. 156-60-5)

Administered in Microcapsules in Feed

to F344/N Rats and B6C3F1 Mice

April 2002

U.S. Department of Health and Human Services

Public Health Service

National Institutes of Health
FOREWORD

The National Toxicology Program (NTP) is made up of four charter agencies of the U.S. Department of

Health and Human Services (DHHS): the National Cancer Institute (NCI), National Institutes of Health; the

National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health; the National

Center for Toxicological Research (NCTR), Food and Drug Administration; and the National Institute for

Occupational Safety and Health (NIOSH), Centers for Disease Control and Prevention. In July 1981, the

Carcinogenesis Bioassay Testing Program, NCI, was transferred to the NIEHS. The NTP coordinates the

relevant programs, staff, and resources from these Public Health Service agencies relating to basic and

applied research and to biological assay development and validation.

The NTP develops, evaluates, and disseminates scientific information about potentially toxic and hazardous

chemicals. This knowledge is used for protecting the health of the American people and for the primary

prevention of disease.

The studies described in this Toxicity Study Report were performed under the direction of the NIEHS and

were conducted in compliance with NTP laboratory health and safety requirements and must meet or exceed

all applicable federal, state, and local health and safety regulations. Animal care and use were in accordance

with the Public Health Service Policy on Humane Care and Use of Animals.

These studies are designed and conducted to characterize and evaluate the toxicologic potential of selected

chemicals in laboratory animals (usually two species, rats and mice). Chemicals selected for NTP toxicology

studies are chosen primarily on the bases of human exposure, level of production, and chemical structure. 

The interpretive conclusions presented in this Toxicity Study Report are based only on the results of these

NTP studies. Extrapolation of these results to other species and quantitative risk analyses for humans require

wider analyses beyond the purview of these studies. Selection per se is not an indicator of a chemical’s toxic

potential.

Details about ongoing and completed NTP studies are available at the NTP’s World Wide Web site: 

http://ntp-server.niehs.nih.gov. Abstracts of all NTP Toxicity Study Reports and full versions of the most

recent reports and other publications are available from the NIEHS’ Environmental Health Information

Service (EHIS) http://ehis.niehs.nih.gov (800-315-3010 or 919-541-3841). In addition, printed copies of

these reports are available from EHIS as supplies last.
National Toxicology Program

Toxicity Report Series

Number 55

NTP Technical Report

on the Toxicity Studies of

trans-1,2-Dichloroethylene

(CAS No. 156-60-5)

Administered in Microcapsules in Feed

to F344/N Rats and B6C3F1 Mice

Nancy B. Ress, Ph.D., Study Scientist

NATIONAL TOXICOLOGY PROGRAM

P.O. Box 12233

Research Triangle Park, NC 27709

April 2002

NIH Publication No. 02-4410

These studies were supported in part by funds from the Comprehensive Environmental Response, Compensation, and Liability Act trust fund 

(Superfund) by an interagency agreement with the Agency for Toxic Substances and Disease Registry, U.S. Public Health Service.

U.S. Department of Health and Human Services

Public Health Service

National Institutes of Health
2

CONTRIBUTORS

National Toxicology Program

Evaluated and interpreted results and reported findings

N.B. Ress, Ph.D., Study Scientist

J.R. Bucher, Ph.D.

R.S. Chhabra, Ph.D.

J. Mahler, D.V.M.

C.S. Smith, Ph.D.

G.S. Travlos, D.V.M.

M.K. Vallant, B.S., M.T.

K.L. Witt, M.S., ILS, Inc.

Microbiological Associates, Inc.

Conducted studies and evaluated pathology findings

M.L. Wenk, Ph.D., Principal Investigator

A. Allen, D.V.M., Ph.D.

R.M. Kovatch, D.V.M.

J. Miller, D.V.M.

J.M. Pletcher, M.P.H., D.V.M.

Experimental Pathology Laboratories,

Inc.

Provided pathology quality assurance

J.F. Hardisty, D.V.M., Principal Investigator

NTP Pathology Review

Evaluated slides and prepared pathology report

(16 August 1996)

J.C. Seely, D.V.M., Chairperson

PATHCO, Inc.

J. Mahler, D.V.M.

National Toxicology Program

Environmental Health Research

and Testing, Inc.

Provided sperm motility and vaginal cytology evaluations

T. Cocanougher, B.A.

D.K. Gulati, Ph.D.

S. Russell, B.A.

Analytical Sciences, Inc.

Provided statistical analyses

P.W. Crockett, Ph.D., Principal Investigator

L.J. Betz, M.S.

K.P. McGowan, M.B.A.

J.T. Scott, M.S.

Biotechnical Services, Inc.

Prepared Toxicity Study Report

S.R. Gunnels, M.A., Principal Investigator

D.C. Serbus, Ph.D.

W.D. Sharp, B.A., B.S.
 3

PEER REVIEW

The draft report on the toxicity studies of trans-1,2-dichloroethylene was evaluated by the reviewers listed below. 

These reviewers serve as independent scientists, not as representatives of any institution, company, or

governmental agency. In this capacity, reviewers determine if the design and conditions of these NTP studies are

appropriate and ensure that the Toxicity Study Report presents the experimental results and conclusions fully and

clearly.

Michelle Medinsky, Ph.D. Douglas Wolfe, Ph.D.

Durham, NC U.S. Environmental Protection Agency

Research Triangle Park, NC
4

SUMMARY

Background: 1,2-Dichloroethylene is used as a solvent for waxes and resins, in the extraction of

rubber, as a refrigerant, and in the manufacture of artificial pearls. More than 1 million pounds are

used annually in the United States. The chemical exists in two isomeric states, cis and trans; the

trans isomer is used more widely in industry, and thus we tested that form.

Methods: Because 1,2-dichloroethylene is both volatile and insoluble in water, we prepared starch

microcapsules containing the chemical and mixed them into the feed of rats and mice to test the

toxicity of the chemical. For every dose group, 5% of the feed mixture consisted of starch

microcapsules; the doses of trans-1,2-dichloroethylene ranged from 1,470 to 23,500 ppm. The

highest doses were approximately 3,200 mg/kg body weight for rats and approximately 8,000 mg/kg

for mice.

Results: Male rats and male and female mice receiving the highest concentrations of

trans-1,2-dichloroethylene weighed less than the control animals. Liver weights of exposed female

rats were greater than those of the control animals, and kidney weights of exposed rats were

decreased. However, no lesions associated with the chemical were observed in the exposed rats or

mice. trans-1,2-Dichloroethylene was not mutagenic in any of the genetic toxicity tests performed.

Conclusion: Very little toxicity was associated with ingestion of microencapsulated

trans-1,2-dichloroethylene by rats or mice. The animals could have tolerated even higher doses than

those used in this study with no adverse effects.
 5

CONTENTS

ABSTRACT ...................................................................................... 7

INTRODUCTION ................................................................................. 9

Chemical and Physical Properties ................................................................ 9

Production, Use, and Human Exposure ........................................................... 9

Absorption, Distribution, Metabolism, and Excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Reproductive and Developmental Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Genetic Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Study Rationale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Procurement and Characterization of trans-1,2-Dichloroethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Preparation and Analysis of Dose Formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

14-Week Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Statistical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Quality Assurance Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Genetic Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Genetic Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

APPENDIXES

Appendix A Summary of Lesions in Rats and Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1

Appendix B Clinical Pathology Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-1

Appendix C Organ Weights and Organ-Weight-to-Body-Weight Ratios . . . . . . . . . . . . . . . . . . . . . . . . . C-1

Appendix D Reproductive Tissue Evaluations and Estrous Cycle Characterization . . . . . . . . . . . . . . . . D-1

Appendix E Genetic Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-1

Appendix F Chemical Characterization and Dose Formulation Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . F-1
6 trans-1,2-Dichloroethylene, NTP TOX 55
7

ABSTRACT

C C

Cl

H Cl

H

trans-1,2-DICHLOROETHYLENE

CAS Number: 156-60-5

Chemical Formula: C2H2Cl2 Molecular Weight: 96.95

Synonyms: trans-Acetylene dichloride; (E)-1,2-dichloroethene; (E)-(9Cl)-1,2-dichloroethene; trans-1,2-dichloroethene;

(E)-1,2-dichloroethylene; trans-dichloroethylene

1,2-Dichloroethylene exists in two isomeric states: trans-1,2-dichloroethylene and cis-1,2-dichloroethylene. The trans

isomer is used more widely in industry than the cisisomer. trans-1,2-Dichloroethylene is used as a solvent for waxes,

resins, and acetylcellulose. It is also used in the extraction of rubber, as a refrigerant, and in the manufacture of

pharmaceuticals and artificial pearls. F344/N rats and B6C3F1 mice were administered trans-1,2-dichloroethylene in

microcapsules in feed for 14 weeks. Animals were evaluated for clinical pathology, reproductive system effects, and

histopathology. Genetic toxicity studies were conducted in vitro in Salmonella typhimurium and Chinese hamster

ovary (CHO) cells, and in vivo in mouse bone marrow cells and peripheral blood erythrocytes.

In the 14-week feed studies, groups of 10 male and 10 female rats and mice were fed diets containing microcapsules

with a chemical load of 45% trans-1,2-dichloroethylene. Dietary concentrations of 3,125, 6,250, 12,500, 25,000, and

50,000 ppm microencapsulated trans-1,2-dichloroethylene resulted in average daily doses of 190, 380, 770, 1,540, and

3,210 mg/kg for male rats; 190, 395, 780, 1,580, and 3,245 mg/kg for female rats; 480, 920, 1,900, 3,850, and

8,065 mg/kg for male mice; and 450, 915, 1,830, 3,760, and 7,925 mg/kg for female mice. Additional groups of

10 male and 10 female rats and mice served as untreated and vehicle controls. There were no exposure-related deaths

of rats or mice. Mean body weights of male rats and male and female mice in the 50,000 ppm groups were
8

significantly less than those of the vehicle controls. The mean body weight gains of female mice in the 12,500 and

25,000 ppm groups were also significantly less than that of the vehicle controls.

On day 21 and at week 14, there were mild decreases in hematocrit values, hemoglobin concentrations, and

erythrocyte counts in groups of male and female rats in the 25,000 and 50,000 ppm groups. At week 14, these effects

were seen in male rats exposed to 6,250 and 12,500 ppm. There were no exposure-related alterations in clinical

chemistry parameters in rats or mice.

The liver weights of female rats exposed to 6,250 ppm or greater were significantly greater than those of the vehicle

controls. The absolute kidney weights of male rats exposed to 25,000 or 50,000 ppm were significantly decreased.

No gross or microscopic lesions were observed in rats or mice that could be attributed to trans-1,2-dichloroethylene

exposure.

Neither cis-, trans-, nor cis,trans-1,2-dichloroethylene was mutagenic in S. typhimurium strain TA97 (cisisomer only),

TA98, TA100, TA1535, or TA1537, with or without S9 metabolic activation enzymes. In CHO cells in vitro, cis1,2-dichloroethylene induced sister chromatid exchanges (SCEs) in the absence of S9; with S9, the single trial that

was performed yielded equivocal results. The cis,trans isomer induced significant increases in SCEs in cultured CHO

cells with and without S9. In contrast to these positive results, trans-1,2-dichloroethylene gave negative results in the

SCE test, with and without S9. Neither cis-, trans-, nor cis,trans-1,2-dichloroethylene induced chromosomal

aberrations (Abs) in cultured CHO cells, with or without S9. In vivo, no induction of SCEs or Abs was noted in bone

marrow cells of male mice administered cis- or trans-1,2-dichloroethylene by intraperitoneal injection once, with

sampling performed 23 hours (for SCE analyses) or 17 hours (for Abs analyses) after injection. In addition, negative

results were obtained in a peripheral blood micronucleus test in male and female mice administered trans1,2-dichloroethylene in microcapsules in feed for 14 weeks.

Very little toxicity was associated with ingestion of microencapsulated trans-1-2-dichloroethylene. Histopathology and

clinical chemistry data, combined with body and organ weight data, revealed that the maximum tolerated dose was

not reached in these studies.
9

INTRODUCTION

CHEMICAL AND PHYSICAL PROPERTIES

The chemical and physical properties of cis-, trans-, and cis,trans-1,2-dichloroethylene are listed in Table 1.

TABLE 1

Chemical and Physical Properties of cis-, trans-, and cis,trans-1,2-Dichloroethylenea

Isomer

Parameter cis trans cis,trans Mixture

Physical state Liquid Liquid Liquid

(60% cis, 40% trans)

Color Colorless Colorless Colorless

Odor Sweetish Sweetish Ethereal, slightly acrid

Melting point !80.5° C !50° C !50° C

Boiling point 60.3° C at 760 mm Hg 48.35° C at 760 mm Hg Not reported

Specific gravity 1.2743 (25/4° C) 1.2489 (25/4° C) 1.27 (25/4° C)

Solubility 0.35 g per 100 mL water

at 20° C; soluble in

alcohol, ether, benzene,

chloroform, acetone,

other organic solvents

0.63 g per 100 mL water

at 20° C; soluble in

alcohol, ether, benzene,

chloroform, acetone,

other organic solvents

Soluble in alcohol, ether,

benzene, chloroform,

acetone, other organic

solvents

Log octanol/water

partition coefficient

1.86 2.06 Not reported

Threshold limit value 200 ppm 200 ppm 200 ppm

a Threshold limit values were taken from the American Conference of Governmental Industrial Hygienists (2000); all other data

were taken from the Hazardous Substances Data Bank (2000).

PRODUCTION, USE, AND HUMAN EXPOSURE

1,2-Dichloroethylene is synthesized by direct chlorination of acetylene at 40° C, by dehydrochlorination of

1,1,2-trichloroethane at 500° C, and by reduction of 1,1,2,2-tetrachloroethane with steam over an iron catalyst.

The trans isomer is separated from the mixture through fractional distillation (HSDB, 2000).
10 trans-1,2-Dichloroethylene, NTP TOX 55

trans-1,2-Dichloroethylene is listed as a high-production-volume chemical by the U.S. Environmental Protection

Agency (USEPA, 2001) because more than 1 million pounds are produced annually. In 1977, one United States

company produced 10 to 50 million pounds and another produced 1 to 10 million pounds of cis,trans1,2-dichloroethylene. For the mixed isomers, one company reported production to be less than 1,000 pounds,

and another estimated production of 0.1 to 1 million pounds. No production estimates for the trans isomer were

reported. The only manufacturer of the cis isomer reported production of 0.1 to 10 million pounds (ATSDR,

1990).

Industrial use of the 1,2-dichloroethylenes is not widespread because of their high flammability. However, the

isomeric mixture is used as a chemical intermediate for the synthesis of chlorinated solvents. It also is used as

a solvent for waxes, resins, camphor, and acetylcellulose and as an extractant for rubber and for oil and fat from

fish. cis,trans-1,2-Dichloroethylene is used as a refrigerant and in the manufacture of pharmaceuticals and

artificial pearls (HSDB, 2000). The trans isomer has more industrial uses than either the cis isomer or the

mixture, and it has been identified in cleaning agents such as Freon MCA and Freon SMT (Hurtt et al., 1993;

HSDB, 2000). cis-1,2-Dichloroethylene is used in the manufacture of vulcanized rubber and as a retarding agent

in fermentation (HSDB, 2000).

Nonoccupational human exposure to cis-, trans-, and cis,trans-1,2-dichloroethylene may occur through ingestion

of contaminated drinking water and/or inhalation of contaminated air. More than 4 million people are estimated

to receive drinking water contaminated with these isomers (USEPA, 1984a). Current drinking water standards

are 70 μg/L for cis-1,2-dichloroethylene and 100 μg/L for trans-1,2-dichloroethylene (Pontius, 1999).

1,2-Dichloroethylene has been detected in groundwater at concentrations up to 3,900 μg/L and in drinking water

at concentrations ranging from 2 to 120 μg/L (ATSDR, 1990).

In 1997, releases of 17 pounds of 1,2-dichloroethylene in water and 7,757 pounds in air were reported; no

releases on land were reported (USEPA, 1999a). Both cis- and trans-1,2-dichloroethylene have been identified

at concentrations of 1 to 5 μg per dry standard cubic meter in emissions from hazardous waste combustion

(Trenholm, 1998). Additionally, 1,2-dichloroethylene is present at 204 National Priority List (NPL) sites. It has

been detected in the groundwater at 183 NPL sites, in the air at four NPL sites, and in the surface water at

18 NPL sites (USEPA, 1999b). When released in the soil, 1,2-dichloroethylene evaporates or leaches into the

groundwater, where it is relatively stable. 1,2-Dichloroethylene is long lived in the atmosphere but may be lost

through reaction with hydroxyl radicals or scavenged by rain (HSDB, 2000). Thus, there is a potential for human

exposure through ingestion of contaminated water or inhalation of contaminated air.
trans-1,2-Dichloroethylene, NTP TOX 55 11

ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Experimental Animals

1,2-Dichloroethylenes bind to the active site of CYP450, resulting in inhibition of their own metabolism (Costa

and Ivanetich, 1982). Both cis- and trans-1,2-dichloroethylene are metabolized by CYP2E1 to an epoxide

intermediate that has been shown to covalently bind to proteins, forming S-methylcysteine amino acid adducts

(Maiorino et al., 1982). The epoxide intermediate nonenzymatically rearranges to form 2,2-dichloroacetaldehyde

(Costa and Ivanetich, 1982), which is enzymatically converted to 2,2-dichloroethanol or 2,2-dichloroacetate via

aldehyde or alcohol dehydrogenase (Bonseet al., 1975; Costa and Ivanetich, 1982). These metabolites have been

identified in an experiment performed to determine the uptake and metabolism of cis- and

trans-1,2-dichloroethylene (Bonse et al., 1975). In this study, the livers of female Wistar rats were perfused at

a constant rate with cis- or trans-1,2-dichloroethylene. After 60 minutes, the uptake of the trans isomer was

slower than that of the cis isomer, suggesting that the metabolism of the cis isomer is more rapid than that of the

trans isomer. These results are supported by the work of Costa and Ivanetich (1982), who showed that the cis

isomer is metabolized more readily than the trans isomer.

trans-1,2-Dichloroethylene has been shown to selectively inhibit CYP2E1 both in vitro and in vivo. In a study

by Mathews et al. (1998), groups of three male Fischer 344 rats were administered intraperitoneal injections of

25, 50, 100, 400, or 800 mg/kg trans-1,2-dichloroethylene. Maximal CYP2E1 inhibition occurred 2 hours after

administration of 100 mg/kg and extended to 12 hours after treatment; CYP2E1 activity returned to control values

within 24 hours. Total CYP450 and the activities of specific isozymes other than CYP2E1 were not significantly

affected in vivo. However, inhibition of CYP2E1 (80%), CYP1A2, and CYP2C6 was observed in vitro

(Mathews et al., 1998).

Further kinetic characterization of CYP2E1 inhibition by cis- and trans-1,2-dichloroethylene was determined both

in vivo and in vitro, and mechanisms of enzyme inhibition were evaluated with physiological models (Lilly et al.,

1998). Male Fischer 344 rats were pretreated with acetone (50% solution in 10% Emulphor by gavage) for

3 days to enhance CYP2E1 activity. Twenty hours after the last treatment, liver microsomes were isolated and

then incubated with 62.5 to 10,000 ppm cis-1,2-dichloroethylene or 5 to 1,000 ppm trans-1,2-dichloroethylene.

Following incubations, inhibition of p-nitrophenyl hydroxylase, a marker for CYP2E1 activity, was assessed. 

CYP2E1 activity was 30% of controls following incubation with 125 ppm trans-1,2-dichloroethylene; inhibition

was maximal (approximately 19% of controls) in microsomes incubated with 500 ppm trans1,2-dichloroethylene. Higher concentrations of cis-1,2-dichloroethylene (5,000 ppm) were required for

comparable inhibition of CYP2E1 activity. Several mechanistic models were examined; however, the model that
12 trans-1,2-Dichloroethylene, NTP TOX 55

best fit the data incorporated destruction of the enzyme-substrate complex via reaction with a reactive metabolite

of cis- or trans-1,2-dichloroethylene. In a separate experiment, rats were similarly pretreated with acetone but

were then exposed by inhalation to concentrations of 75 to 1,000 ppm cis-1,2-dichloroethylene or 5 to 25 ppm

trans-1,2-dichloroethylene. Samples of the chamber air were taken every 10 minutes for 3 to 6 hours. Gas

uptake results of cis- or trans-1,2-dichloroethylene showed lower chamber concentrations with decreasing

exposure concentrations, indicating inhibition of metabolism at the higher concentrations. In vivo modeling

experiments indicated that the mechanism of CYP2E1 inhibition by cis- or trans-1,2-dichloroethylene occurred

through reaction of the activating enzyme with reactive metabolites formed during the metabolism of the

dichloroethylenes.

Inhalation pharmacokinetics were studied in male Wistar rats exposed to cis- or trans-1,2-dichloroethylene using

a closed inhalation chamber. In this study, a nonphysiologically constrained, two-compartment model was used

to analyze the data (Filser and Bolt, 1979). For the trans and cis isomers, the zero-order Vmax elimination rates

were reported to be 0.67 and 2.4 mg/hour•kg, respectively. The authors suggested that the low maximal

velocities of trans- and cis-1,2-dichloroethylene were due to the inactivation of CYP450 by reactive epoxide

intermediates. In a study to compensate for enzyme inhibition-resynthesis, VmaxC values of 3 and

2.49 mg/hour•kg were reported for cis- and trans-1,2-dichloroethylene, respectively (Gargas et al., 1990). In

a more recent study by Lilly et al. (1998), several physiologically based pharmacokinetic models were tested

based on both in vitro and in vivo experiments (as described previously). Assuming interaction between the

reactive metabolites and the enzyme-substrate complex, it was determined that the in vivo model was the most

biologically relevant upon comparison with VmaxC values for other structurally similar compounds. Using this

model, VmaxC values were determined to be 4.53 mg/hour•kg for the cisisomer and 4.27 mg/hour•kg for the trans

isomer.

Humans

No information on the distribution, metabolism, or excretion of cis-, trans-, or cis,trans-1,2-dichloroethylene in

humans was found in the literature. However, it has been reported that 72% to 75% of inhaled

trans-1,2-dichloroethylene is absorbed in the lungs (ATSDR, 1990). The 1,2-dichloroethylenes are likely

absorbed readily through inhalation, ingestion, and dermal exposure due to their high lipid solubilities and low

molecular weight (USEPA, 1984b).
trans-1,2-Dichloroethylene, NTP TOX 55 13

TOXICITY

Experimental Animals

The LD50 and LC50 values for trans-1,2-dichloroethylene administered to rodents by various routes are provided

in Table 2. 

In an acute toxicity study, groups of four to six adult Holtzman rats were administered either cis- or trans1,2-dichloroethylene in corn oil by gavage at 400 or 1,500 mg/kg (Jenkins et al., 1972). In the 1,500 mg/kg

groups, the cis isomer elicited a greater biochemical response than the trans isomer, although all effects were

minimal. Administration of 400 or 1,500 mg/kg cis-1,2-dichloroethylene caused a significant increase in liver

alkaline phosphatase activity. In animals exposed to 1,500 mg/kg, liver glucose-6-phosphatase, liver tyrosine

transaminase, and plasma alanine aminotransferase activities were significantly increased. Rats exposed to

400 mg/kg of the trans isomer showed increased liver glucose-6-phosphatase activity, while tyrosine

transaminase activity was increased in the 1,500 mg/kg group.

The acute toxicity of trans-1,2-dichloroethylene also was determined in male and female CD-1 mice (Barnes

et al., 1985). For 14 days, groups of 9 or 10 mice were administered daily gavage doses of 21 or 210 mg/kg

trans-1,2-dichloroethylene in a 1:9 solution of Emulphor and deionized water. There were no significant changes

TABLE 2

LD50 and LC50 Values for trans-1,2-Dichloroethylene

Route Species, Strain, Gender LD50/LC50 Reference

Gavage Rats

 Wistar, female

Sprague-Dawley, male

Sprague-Dawley, female

1,275 mg/kga

7,900 mg/kg

9,900 mg/kg

Freundt et al., 1977

Hayes et al., 1987

Hayes et al., 1987

Mice

CD-1, male

CD-1, female

2,221 mg/kg

2,391 mg/kg

Munson et al., 1982

Munson et al., 1982

Intraperitoneal injection Rats

 Wistar, female 7,650 mg/kg Freundt et al., 1977

Mice

NMR, female 4,022 mg/kg Freundt et al., 1977

Inhalation Mice

Unspecified 22,000 mg/m3 ATSDR, 1990

a Possible aspiration of dose, as evidenced by pulmonary damage
14 trans-1,2-Dichloroethylene, NTP TOX 55

in organ or body weights in any dosed group. However, slight decreases in fibrinogen concentrations (12%),

prothrombin times, and lactate dehydrogenase activities were observed in rats administered 210 mg/kg per day.

In another study to assess the subacute effects of oral exposure to the cis isomer, groups of 10 male and

10 female Sprague-Dawley rats were administered 32, 98, 293, or 878 mg/kg in corn oil by gavage daily for 14

days (McCauley et al., 1995). A complete necropsy and hematology and clinical chemistry evaluations were

performed on each animal. Male and female rats administered 878 mg/kg per day were lethargic and had

secretions around the nose and mouth. In addition, a 10% decrease in body weight occurred in males in this

group, while no body weight effects were observed in females. Dose-related increases in liver weights were

observed in males and females in all dosed groups. Kidney weights of females in the 293 and 878 mg/kg groups

and testis weights of males in the 878 mg/kg group were increased.

The short-term toxicity of the trans isomer also was determined in an inhalation study by Freundt et al. (1977).

Adult female SPF Wistar rats and NMRI mice were exposed to 200 ppm trans-1,2-dichloroethylene via inhalation

8 hours per day for 5 or 10 days or 200, 1,000 or 3,000 ppm by inhalation for one 8-hour period. The blood,

lung, heart, liver, kidney, spleen, brain, muscle (quadriceps), and sciatic nerve were collected and examined. In

all exposure groups, increases in fatty degeneration of hepatic lobules and Kupffer cells were observed.

Additionally, a pronounced increase in the appearance of pulmonary capillary hyperemia and distention of the

alveolar septum occurred and became more pronounced with increasing exposure time and concentration.

Exposure to 1,000 ppm for 8 hours resulted in decreased numbers of red and white blood cells in rats. In mice,

exposure to 3,000 ppm for 8 hours resulted in fibrous swelling and hyperemia of the cardiac muscle with barely

maintained striation.

In a subchronic study to assess the oral toxicity of the cis isomer, groups of 10 male and 10 female SpragueDawley rats were administered 10, 32, 98, or 206 mg/kg per day in corn oil by gavage for 90 days (McCauley

et al., 1995). Significant decreases in liver-weight-to-body-weight ratios of males and females and increases in

kidney weights of males occurred in all dosed groups. No significant histopathologic changes were seen in males

or females. Serum phosphorus concentrations in dosed females and serum calcium concentrations in dosed

males were slightly increased. Additionally, males in the 206 mg/kg group exhibited a decreased blood urea

nitrogen concentration. Overall, cis-1,2-dichloroethylene exhibited some toxicity at all doses.

The trans isomer was evaluated for subchronic toxicity in groups of 24 male and 24 female CD-1 mice

administered 0.1, 1.0, or 2.0 mg/mL in drinking water (Barnes et al., 1985). Based on the amounts of water

ingested, males received a daily dose of 17, 175, or 387 mg/kg; females received a daily dose of 23, 244, or

452 mg/kg. No changes in terminal body weights or gross pathology were observed, and clinical chemistry
trans-1,2-Dichloroethylene, NTP TOX 55 15

effects were considered to be minimal. The most noteworthy effects included a 21% decrease in liver glutathione

in males in the 2 mg/mL group and significant decreases in thymus weights of females in the 1 and 2 mg/mL

groups.

In a 90-day study, CD rats were administered trans-1,2-dichloroethylene in drinking water at daily doses of 402,

1,311, or 3,114 mg/kg for males and 353, 1,257, or 2,809 mg/kg for females (Hayes et al., 1987). In female

rats exposed to 1,257 or 2,809 mg/kg, significant decreases in kidney weights were observed. No other

chemical-related effects on body weight or on hematology or clinical chemistry parameters were found.

In a study to assess the immunotoxicity of trans-1,2-dichloroethylene, groups of 10 to 12 male CD-1 mice were

administered 22 or 222 mg/kg by gavage (vehicle not indicated) for 14 days. Treatment did not affect body

weight, organ weights (brain, kidney, liver, lungs, spleen, or thymus), lymphocyte count, hematocrit value, or

hemoglobin concentration at either dose. Immunologically, the trans isomer produced a slight but not dosedependent inhibition in the number of cells forming IgM antibodies to sheep erythrocytes. No change was

observed in cell-mediated immunity evaluated by measuring the delayed hypersensitivity response to sheep

erythrocytes (Munson et al., 1982). 

In another study to assess the immunotoxicity of trans-1,2-dichloroethylene, groups of 12 male and 12 female

CD-1 mice were exposed to drinking water containing 17, 175, or 387 mg/kg (males) and 23, 224, or 452 mg/kg

(females) for 90 days (Shopp et al., 1985). At all doses, the spleen cells of male mice exhibited a decreased

ability to produce antibodies against sheep erythrocytes, indicating a suppression in humoral immune status. In

exposed females, macrophage induction was decreased, as evidenced by a decline in the ability of thioglycolaterecruited peritoneal exudate cells to phagocytize sheep erythrocytes.

Humans

Inhalation of 1,2-dichloroethylene vapors may result in central nervous system depression, nausea, weakness,

tremors, and cramps (HSDB, 2000). Humans exposed by inhalation to 1,715 to 2,220 ppm for 5 minutes or

1,210 ppm for 10 minutes exhibited nausea, drowsiness, fatigue, vertigo, and increased intracranial pressure

(ATSDR, 1990). Inhalation of 1,2-dichloroethylene vapors (duration and concentration not specified) has

resulted in one fatality (Torkelson and Rowe, 1981).
16 trans-1,2-Dichloroethylene, NTP TOX 55

REPRODUCTIVE AND DEVELOPMENTAL TOXICITY

Experimental Animals

Groups of 24 pregnant female Crl:CD Br rats were exposed to 0, 2,000, 6,000, or 12,000 ppm trans1,2-dichloroethylene in air for 6 hours per day on gestation days 7 through 16 (Hurtt et al., 1993). Animals were

killed on gestation day 22 and assessed for toxicity. Maternal body weight loss was observed in dams exposed

to 6,000 ppm on gestation days 11 through 13 and in dams exposed to 12,000 ppm throughout the study; weight

loss paralleled decreased feed consumption in the 6,000 and 12,000 ppm groups. A narcotizing effect occurred

from maternal exposure to 6,000 or 12,000 ppm. Alopecia, lethargy, and salivation were observed in the

12,000 ppm group. Significant increases in the numbers of early and total resorptions were reported in dams

exposed to 6,000 or 12,000 ppm but not 2,000 ppm. Additionally, mean fetal body weights for dams exposed

to 12,000 ppm were significantly decreased. Due to the maternal toxicity in the 6,000 and 12,000 ppm groups,

these data should be evaluated with caution. No differences in incidence or type of malformation were observed

in exposed fetuses, although a slight but nonsignificant increase in the occurrence of hydrocephalus was

observed in the 12,000 ppm group.

Humans

No data were available on the reproductive toxicity of cis-, trans-, or cis,trans-1,2-dichloroethylene in humans.

CARCINOGENICITY

No information was found in a search of the literature regarding the carcinogenicity of cis-, trans-, or cis,trans1,2-dichloroethylene in animals or humans.

GENETIC TOXICITY

The mutagenicity of cis-, trans-, and cis,trans-1,2-dichloroethylene has been tested in the Salmonella typhimurium

microsome assay. Neither the trans isomer nor the isomeric mixture was mutagenic in a preincubation study

using strains TA98, TA100, TA1535, and TA1537, with and without Aroclor 1254-induced rat or hamster

liver S9 (Mortelmans et al., 1986). The cis isomer was similarly tested, and negative results were obtained in

these four strains and strain TA97 (Zeiger et al., 1988). In a study that employed the standard plate incorporation

method, positive results were reported with the trans isomer in strain TA100 with and without S9 and

strains TA97 and TA98 with S9; no mutagenic activity was noted in strain TA104 with or without S9 (Strobel

and Grummt, 1987).
trans-1,2-Dichloroethylene, NTP TOX 55 17

The cis and trans isomers were incubated, in the presence and absence of mouse liver microsomes, with

Escherichia coli K23, a bioauxotrophic strain, to test for back mutations in the gal+, arg+, and nad+ operons and

forward mutations in the MTR operon (confers resistance to 5-methyl-DL-tryptophan). Neither cis- nor trans1,2-dichloroethylene was mutagenic with or without metabolic activation (Greim et al., 1975).

Both the cis and trans isomers induced mitotic recombination in Saccharomyces cerevisiae D7 (Bronzetti et al.,

1984). In addition, cis-1,2-Dichloroethylene induced a small but significant increase in gene mutations in

S. cerevisiae D7 in the absence of S9 activation; neither isomer induced mitotic gene conversion (Bronzetti et al.,

1984). Additionally, negative results were observed in a mitotic gene conversion assay with trans1,2-dichloroethylene in metabolically competent or S9-supplemented lines of S. cerevisiae D7. However, over

the same concentration range, the trans isomer was active in an assay for aneuploidy induction (chromosome

loss) in strain D61.M, with and without S9 activation (Koch et al., 1988).

cis,trans-1,2-Dichloroethylene was tested for micronucleus induction using the cytokinesis block method and

for DNA damage using single-cell gel electrophoresis (COMET assay) in human lymphocytes in vitro (Tafazoli

and Kirsch-Volders, 1996). A small but significant increase in the frequency of micronucleated cells was

observed at the highest concentration tested (20 mM), with and without S9 mix. However, no clear or

reproducible dose-response relationship was indicated. Positive results also were seen in the COMET assay,

indicating induction of DNA damage in isolated lymphocytes, at concentrations of 4 to 8 mM with S9 and 6 mM

without S9. Additionally, in vitro micronucleus studies in lymphoblastoid cell lines with an unspecified

1,2-dichloroethylene isomer showed mixed responses. Micronucleus frequencies were significantly increased

in AHH-1 and h2E1 cell lines but not in MCL-5 cells with high CYP1A activity (Doherty et al., 1996). The

results of special staining techniques used to discern the presence or absence of kinetochores in the induced

micronuclei suggested that the increased frequencies of micronuclei were due both to structural damage and to

induced aneuploidy. In contrast to the positive results observed in in vitro micronucleus assays, earlier tests for

induction of sister chromatid exchanges and chromosomal aberrations in cultured Chinese hamster lung

fibroblasts were negative with both the cis and trans isomers at concentrations up to 2 mg/mL, with and without

induced rat liver S9 (Sawada et al., 1987).

In their published abstract, 1erná and Kyp2nová (1977) reported that intraperitoneal injections of cis1,2-dichloroethylene, administered daily at one-sixth of the LD50 value for 5 days, induced chromosomal

aberrations in the bone marrow cells of female ICR mice sampled 6, 24, and 48 hours after treatment; the trans

isomer had no effect. Furthermore, the authors reported that a dose response was established for induction of

chromosomal aberrations by cis-1,2-dichloroethylene in a follow-up study in which the isomer was administered

in a series of 10 daily injections. No data or protocol details were included in this abstract, precluding
18 trans-1,2-Dichloroethylene, NTP TOX 55

independent evaluation of the results. In contrast to the induction of chromosomal aberrations attributed to cis1,2-dichloroethylene in vivo, male and female CD-1 mice administered intraperitoneal injections of 280 or

490 mg/kg cis,trans-1,2-dichloroethylene (40% or 70% of the LD5 0, respectively) did not have increased

frequencies of micronuclei in bone marrow erythrocytes 24 or 48 hours after injection (Crebelli et al., 1999).

These results were part of a larger, extensive study of 10 halogenated aliphatic hydrocarbons, none of which

induced a micronucleus response in mouse bone marrow.

In conclusion, the limited mutagenicity data for the isomers of 1,2-dichloroethylene show mixed results. Mostly

negative results were obtained in bacterial gene mutation studies, and conflicting results were seen in mammalian

cell studies for chromosomal effects, with some in vitro tests yielding weakly positive results and in vivo study

results suggesting that 1,2-dichloroethylene is not active in mouse bone marrow.

STUDY RATIONALE

1,2-Dichloroethylene was recommended for toxicologic characterization by the National Cancer Institute and the

USEPA under the Superfund program because of possible widespread human exposure due to its presence in

drinking water and at hazardous waste sites. trans-1,2-Dichloroethylene was microencapsulated and administered

in feed to achieve higher concentrations than would be obtainable in drinking water. The trans isomer was

selected for subchronic toxicity studies based on the results of preliminary acute toxicity studies and its

prevalence in industrial applications. In these studies, the most significant effect was a 20% reduction in final

mean body weight of male rats exposed to 50,000 ppm trans-1,2-dichloroethylene. Based on this information,

the highest dose for the 14-week studies was 50,000 ppm. In the 14-week studies, F344/N rats and B6C3F1

mice were exposed to 3,125, 6,250, 12,500, 25,000, or 50,000 ppm microencapsulated trans1,2-dichloroethylene in the diet for 14 weeks. In addition, genetic toxicity studies of cis-, trans-, and cis,trans1,2-dichloroethylene were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, mouse

bone marrow cells, and mouse peripheral blood erythrocytes.
19

MATERIALS AND METHODS

PROCUREMENT AND CHARACTERIZATION OF TRANS-1,2-DICHLOROETHYLENE

trans-1,2-Dichloroethylene was obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI), in one lot

(MP-02224LP). Microencapsulation was performed by the analytical chemistry laboratory, Midwest Research

Institute (Kansas City, MO), and the microcapsules were assigned lot number 343-1B-A. Identity, purity, and

stability analyses were conducted by the analytical chemistry laboratory and the study laboratory (Appendix F).

Reports on analyses performed in support of the trans-1,2-dichloroethylene studies are on file at the National

Institute of Environmental Health Sciences.

Analyses of Neat Chemical

The chemical, a clear, colorless liquid, was identified with infrared, ultraviolet/visible, and nuclear magnetic

resonance spectroscopy. Purity was determined using elemental analyses, Karl Fischer water analysis, free acid

titration, gas chromatography (GC), and GC with mass spectrometry (GC/MS).

Elemental analyses for carbon, hydrogen, and chlorine were in agreement with the theoretical values for trans1,2-dichloroethylene. Karl Fischer water analysis indicated 0.010% ± 0.001% water. Free acid titration indicated

0.0007 ± 0.0002 mEq acid per gram, equivalent to 26 ± 7 ppm as hydrochloric acid. GC by one system indicated

one major peak and five impurities with a combined area of 1.25% relative to the major peak area. GC by a

second system indicated one major peak and three impurities with a combined relative area of 0.69%. The overall

purity of lot MP-02224LP was determined to be approximately 99%. Minor impurities were identified as

chlorinated compounds by GC/MS. The study laboratory confirmed that the purity was 99% or greater.

Stability information supplied by the manufacturer indicated that neat trans-1,2-dichloroethylene is stable when

stored frozen and protected from air, moisture, and light; the chemical was stored under these conditions

throughout the 14-week studies. Stability was monitored with infrared spectroscopy and GC during preliminary

pilot studies conducted by TSI Mason Laboratories (Worcester, MA); no degradation of the bulk chemical was

detected.
20 trans-1,2-Dichloroethylene, NTP TOX 55

Microcapsule Formulation and Analyses

Microcapsules loaded with neat trans-1,2-dichloroethylene and placebos (empty microcapsules) were prepared

by the analytical chemistry laboratory with a proprietary process using food-grade, modified corn starch

(CAPSUL®) and reagent-grade sucrose (80:20) to produce dry microspheres; the outer surfaces of the

microcapsules were dusted with food-grade, hydrophobic, modified corn starch. Following microencapsulation

of trans-1,2-dichloroethylene, the analytical chemistry laboratory tested the microencapsulated chemical for

conformance to specifications. The microcapsules were examined microscopically for appearance.

Conformance to particle size specifications (with no more than 1% of particles having diameters greater than

420 μm) was determined by passing placebo and loaded microcapsules through U.S. standard sieves

(numbers 30, 40, 60, 80, 100, 120, and PAN). The chemical loads (amount of trans-1,2-dichloroethylene in the

starch/sugar matrix) of freshly prepared microcapsules and of microcapsules stored under a variety of conditions

were determined with GC. Major peak comparisons of the neat and microencapsulated chemical and 9-month

stability studies were also performed with GC.

Microscopic examination revealed no unusual characteristics. The particles were within size specifications. The

chemical load was determined to be 45.0% ± 0.3%. Microcapsules stored for 28 days under animal room

conditions (open to air and light), with and without freeze-thaw cycles, or in sealed bottles at 5° C retained 93.8%

of their chemical load by weight. Major peak comparisons indicated that no impurities were introduced by

microencapsulation. Results of the 9-month shelf-life study indicated that microcapsules retained 91% of their

chemical load when stored at 5° C and 89% when stored at room temperature; microcapsules stored at 5° C for

6 months and then at room temperature, open to air and light, for 28 days were stable.

The study laboratory confirmed the identity of the microcapsules with infrared spectroscopy and analyzed the

chemical load of the microcapsules with GC. GC analyses performed at the beginning of the preliminary pilot

studies indicated a chemical load of approximately 43%. Prior to the 14-week studies, GC analyses of trans1,2-dichloroethylene samples from one bottle of microcapsules indicated a concentration of 37.6% ± 2.4%,

which was lower than the expected concentration of 45.3%; microcapsules from six other bottles were combined

and analyzed with GC. Homogeneity was confirmed, and the chemical load of 47.0% was considered to be

acceptable for use in the 14-week studies. The chemical load was monitored during the 14-week studies with

GC; no loss of trans-1,2-dichloroethylene was detected.
trans-1,2-Dichloroethylene, NTP TOX 55 21

PREPARATION AND ANALYSIS OF DOSE FORMULATIONS

The dose formulations were prepared at least every 2 weeks by mixing microencapsulated trans1,2-dichloroethylene with feed (Table F2); placebo microcapsules were added to maintain a starch matrix

concentration of 6% in the diet. Formulations were stored in doubled plastic bags, protected from light, at 5E C

for up to 4 weeks. Homogeneity and stability studies of 0.5% trans-1,2-dichloroethylene formulations were

conducted by the analytical chemistry laboratory using GC. Homogeneity was confirmed. The trans1,2-dichloroethylene formulation prepared with lot 343-1A had losses of 22.0% for samples stored for 7 days

in the dark and 15.5% for samples stored for 1 day at room temperature, open to air and light; dose formulations

prepared with lots 343-10TA, -11TA, and -12TA (not used in the current studies) and lot 343-1B-A were stable

for 4 days when stored at room temperature, open to air and light. Additional analyses performed with GC with

a 0.5% dose formulation prepared with lot 343-1B-A confirmed stability for 7 days for samples stored in a rat

cage, open to air and light, at up to 50% humidity.

The study laboratory conducted homogeneity studies of 0.7% and 11.5% trans-1,2-dichloroethylene formulations

with GC. Prior to the 14-week studies, the study laboratory also performed homogeneity studies of 3,125 and

50,000 ppm dose formulations and stability studies of a 3,125 ppm dose formulation with GC. Homogeneity of

all formulations was confirmed. Stability was confirmed for 28 days for dose formulations stored in doubled

plastic bags at up to 5° C, the storage conditions used during the studies.

Periodic analyses of the dose formulations were conducted by the study laboratory using GC (Table F3). All

dose formulations that were not within 10% of the target concentrations were reblended and reanalyzed; all dose

formulations analyzed and used for dosing were within 10% of the target concentrations.

14-WEEKSTUDIES

Male and female F344/N rats and B6C3F1 mice were obtained from Taconic Farms (Germantown, NY) and were

5 weeks old on receipt. Animals were quarantined for 14 or 15 days (rats) or 12 or 13 days (mice) and were

7 weeks old on the first day of the studies. Before the studies began, five male and five female rats and mice

were randomly selected for parasite evaluation and gross observation for evidence of disease. Blood samples

were collected from five male and five female rats and mice at the beginning of the studies. The sera were

analyzed for antibody titers to rodent viruses (Boorman et al., 1986; Rao et al., 1989a,b). All results were

negative.
22 trans-1,2-Dichloroethylene, NTP TOX 55

The exposure concentrations selected for use in the 14-week studies were based on the results of pilot studies

in which no findings of overt toxicity were observed at exposure concentrations up to 50,000 ppm trans1,2-dichloroethylene (5% in feed). The substitution of greater than 5% of the feed interferes with the availability

of some essential vitamins and minerals and causes nutritional imbalances (USEPA, 1980).

Groups of 10 male and 10 female rats and mice were fed diets containing 3,125, 6,250, 12,500, 25,000, or

50,000 ppm microencapsulated trans-1,2-dichloroethylene for 14 weeks. Additional groups of 10 male and

10 female rats and mice received untreated feed (untreated controls) or feed containing placebo microcapsules

(vehicle controls) for 14 weeks. Feed and water were available ad libitum. Rats and female mice were housed

five per cage, and male mice were housed individually. Clinical findings were recorded weekly. Feed

consumption was recorded weekly by cage. The animals were weighed initially, weekly, and at the end of the

studies. A functional observation battery was performed on rats and mice in the untreated and vehicle control

groups and the 12,500, 25,000, or 50,000 ppm groups during weeks 4 and 13. Details of the study design and

animal maintenance are summarized in Table 3.

Additional groups of 10 male and 10 female rats received the same exposure concentrations of trans1,2-dichloroethylene as the core study animals. These rats were used for clinical pathology testing only and were

not necropsied.

Blood for hematology and clinical chemistry analyses was collected from clinical pathology study rats on days 5

and 21 and from core study rats and mice (clinical chemistry only) at the end of the studies. At all time points,

the animals were anesthetized with a mixture of carbon dioxide and air (70:30), and blood was collected from

the retroorbital sinus. Blood for hematology determinations was placed in tubes containing potassium EDTA as

the anticoagulant. The automated hematocrit value; hemoglobin concentration; erythrocyte, leukocyte, and

platelet counts; mean cell volume; mean cell hemoglobin; and mean cell hemoglobin concentration were

determined with a Serono-Baker 9000 hematology analyzer (Serono-Baker Diagnostics, Allentown, PA). Manual

hematocrit determinations were made with an Adams CT2900 Microhematocrit Centrifuge (Clay Adams, Sparks,

MD). Differential leukocyte counts and erythrocyte, leukocyte, and platelet morphologies were determined by

light microscopy from blood smears stained on an Ames Hema-Tek Slide Stainer (Miles Laboratory, Ames

Division, Elkhart, IN) using a modified Wright’s stain. Reticulocytes were stained with new methylene blue

(Sigma Chemical Company, St. Louis, MO) and counted microscopically. Blood for clinical chemistry analyses

was placed in tubes with no anticoagulant, allowed to clot at room temperature, and centrifuged. The sera were

separated, and clinical chemistry parameters were measured with a Hitachi 717 chemistry analyzer (Boehringer

Mannheim, Indianapolis, IN). Reagents were obtained from the manufacturer with the exception of the reagents
trans-1,2-Dichloroethylene, NTP TOX 55 23

for sorbitol dehydrogenase and total bile acids, which were obtained from Sigma Chemical Company. The

hematology and clinical chemistry parameters measured are listed in Table 3.

At the end of the 14-week studies, samples were collected for sperm count and motility and vaginal cytology

evaluations on rats and mice in the untreated control and vehicle control groups and the 12,500, 25,000, and

50,000 ppm groups. For 12 consecutive days prior to scheduled terminal sacrifice, the vaginal vaults of the

females were moistened with saline, if necessary, and samples of vaginal fluid and cells were stained. Relative

numbers of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used

to ascertain estrous cycle stage (i.e., diestrus, proestrus, estrus, and metestrus). Male animals were evaluated

for sperm count and motility. The left testis and left epididymis were isolated and weighed. The tail of the

epididymis (cauda epididymis) was then removed from the epididymal body (corpus epididymis) and weighed.

Test yolk (rats) or modified Tyrode’s buffer (mice) was applied to slides and a small incision was made at the

distal border of the cauda epididymis. The sperm effluxing from the incision were dispersed in the buffer on the

slides, and the numbers of motile and nonmotile spermatozoa were counted for five fields per slide by two

observers. Following completion of sperm motility estimates, each left cauda epididymis was placed in buffered

saline solution. Caudae were finely minced, and the tissue was incubated in the saline solution and then heat fixed

at 65E C. Sperm density was then determined microscopically with the aid of a hemacytometer. To quantify

spermatogenesis, the testicular spermatid head count was determined by removing the tunica albuginea and

homogenizing the left testis in phosphate-buffered saline containing 10% dimethyl sulfoxide. Homogenizationresistant spermatid nuclei were counted with a hemacytometer.

Necropsies were performed on all core study animals. The heart, right kidney, liver, lung, right testis, and

thymus were weighed. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered

formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with

hematoxylin and eosin. Complete histopathologic examinations were performed on rats and mice in the untreated

control, vehicle control, and 50,000 ppm groups. Table 3 lists the tissues and organs routinely examined.

Upon completion of the laboratory pathologist’s histopathologic evaluation, the slides, paraffin blocks, and

residual wet tissues were sent to the NTP archives for inventory, slide/block match, and wet tissue audit. The

slides, individual animal data records, and pathology tables were sent to an independent pathology laboratory

where quality assessment was performed. Results were reviewed and evaluated by the NTP Pathology Working

Group (PWG); the final diagnoses represent a consensus of contractor pathologists and the PWG. Details of

these review procedures have been described by Maronpot and Boorman (1982) and Boorman et al. (1985).
24 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE 3

Experimental Design and Materials and Methods in the 14-Week Feed Studies of trans-1,2-Dichloroethylene

Study Laboratory

Microbiological Associates, Inc. (Bethesda, MD)

Strain and Species

Rats: F344/N

Mice: B6C3F1

Animal Source

Taconic Farms (Germantown, NY)

Time Held Before Studies

Rats: 14 (males) or 15 days (females)

Mice: 12 (females) or 13 days (males) 

Average Age When Studies Began

7 weeks

Date of First Exposure

Rats: 13 (male) or 14 (female) May 1993

Mice: 11 (female) or 12 (male) May 1993

Duration of Exposure

14 weeks

Date of Last Exposure

Rats: 12 (male) or 13 (female) August 1993

Mice: 10 (female) or 11 (male) August 1993

Necropsy Dates

Rats: 12 (male) or 13 (female) August 1993

Mice: 10 (female) or 11 (male) August 1993

Average Age at Necropsy

20 weeks

Size of Study Groups

10 males and 10 females

Method of Distribution

Animals were distributed randomly into groups of approximately equal initial mean body weights.

Animals per Cage

Rats: 5

Mice: 1 (male) or 5 (female)

Method of Animal Identification

Tail tattoo

Diet

NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed once or twice (female mice)

per week

Water

Tap water (Bethesda municipal supply) via automatic watering system, available ad libitum
trans-1,2-Dichloroethylene, NTP TOX 55 25

TABLE 3

Experimental Design and Materials and Methods in the 14-Week Feed Studies of trans-1,2-Dichloroethylene

Cages

Polycarbonate (Lab Products, Inc., Rochelle Park, NJ), changed once (male mice) or twice per week

Bedding

Heat-treated hardwood chips (P.J. Murphy Forest Products, Montville, NJ), changed once (male mice) or twice per week

Racks

Stainless steel (Lab Products, Inc., Rochelle Park, NJ), changed and rotated every 2 weeks

Animal Room Environment

Temperature: 72° ± 3° F

Relative humidity: 50% ± 15%

Room fluorescent light: 12 hours/day

Room air changes: at least 10/hour

Exposure Concentrations

0, 3,125, 6,250, 12,500, 25,000, or 50,000 ppm, microencapsulated in feed

Type and Frequency of Observation

Animals were observed twice daily and were weighed initially, weekly, and at the end of the studies; clinical findings were recorded

weekly. Feed consumption was recorded weekly by cage.

Method of Sacrifice

Carbon dioxide asphyxiation

Necropsy

Necropsies were performed on all animals. Organs weighed were heart, right kidney, liver, lung, right testis, and thymus.

Clinical Pathology

Blood was collected from the retroorbital sinus of special study rats on days 5 and 21 and from core study rats and mice at the end of

the studies for hematology (rats only) and clinical chemistry.

Hematology: hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; erythrocyte and platelet

morphology; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials

Clinical chemistry: urea nitrogen, creatinine, total protein, albumin, cholesterol, alanine aminotransferase, alkaline phosphatase,

creatine kinase, sorbitol dehydrogenase, 5N-nucleotidase, and bile acids

Histopathology

Complete histopathology was performed on all rats and mice in the untreated control, vehicle control, and 50,000 ppm groups. In

addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland,

esophagus, gallbladder (mice only), heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney,

liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland,

preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal

vesicle, thymus, thyroid gland, trachea, urinary bladder, uterus, and Zymbal’s gland.

Functional Observation Battery

Functional observation batteries were performed on core study rats and mice in the untreated and vehicle control groups and the

12,500, 25,000, and 50,000 ppm groups during weeks 4 and 13. The following parameters were evaluated: body position, activity

level, coordination of movement, gait, general behavior, head flick, head searching, compulsive biting or licking, backward walking,

self-mutilation, circling, convulsions, tremors, lacrimation or chromodacryorrhea, salivation, piloerection, pupillary dilation or

constriction, unusual respiration, diarrhea, excessive or diminished urination, and vocalization.

Sperm Motility and Vaginal Cytology

At the end of the studies, sperm samples were collected from all male animals in the untreated and vehicle control groups and the

12,500, 25,000, and 50,000 ppm groups for sperm motility evaluations. The following parameters were evaluated: spermatid heads

per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration. The left cauda, left

epididymis, and left testis were weighed. Vaginal samples were collected for 12 consecutive days before the end of the studies from all

females in the untreated and vehicle control groups and the 12,500, 25,000, and 50,000 ppm groups for vaginal cytology evaluations. 

The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated.
26 trans-1,2-Dichloroethylene, NTP TOX 55

STATISTICAL METHODS

Calculation and Analysis of Lesion Incidences

The incidences of lesions are presented in Appendix A as the numbers of animals bearing such lesions at a

specific anatomic site and the numbers of animals with that site examined microscopically. The Fisher exact test

(Gart et al., 1979), a procedure based on the overall proportion of affected animals, was used to determine

significance.

Analysis of Continuous Variables

Two approaches were employed to assess the significance of pairwise comparisons between exposed and control

groups in the analysis of continuous variables. Organ and body weight data, which have approximately normal

distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams

(1971, 1972). Hematology, clinical chemistry, spermatid, and epididymal spermatozoal data, which have typically

skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and

Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related

trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for

pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s

test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey (1951) were

examined by NTP personnel, and implausible values were eliminated from the analysis. Average severity values

were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Because vaginal

cytology data are proportions (the proportion of the observation period that an animal was in a given estrous

stage), an arcsine transformation was used to bring the data into closer conformance with a normality

assumption. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976)

to the transformed data to test for simultaneous equality of measurements across exposure concentrations.

QUALITY ASSURANCEMETHODS

The 14-week studies were conducted in compliance with Food and Drug Administration Good Laboratory

Practice Regulations (21 CFR, Part 58). The Quality Assurance Unit of Microbiological Associates, Inc.,

performed audits and inspections of protocols, procedures, data, and reports throughout the course of the

studies.
trans-1,2-Dichloroethylene, NTP TOX 55 27

GENETIC TOXICOLOGY

Salmonella typhimurium Mutagenicity Test Protocol

Testing was performed as reported by Zeiger et al. (1988) (cis isomer) or Mortelmans et al. (1986) (trans and

cis,trans isomers). The three isomers were sent to the laboratories as coded aliquots from Radian Corporation

(Austin, TX). They were incubated with the Salmonella typhimurium tester strains TA97 (cis isomer only),

TA98, TA100, TA1535, and TA1537 either in buffer or S9 mix (metabolic activation enzymes and cofactors

from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver) for 20 minutes at 37E C. Top agar

supplemented with L-histidine and d-biotin was added, and the contents of the tubes were mixed and poured onto

the surfaces of minimal glucose agar plates. Histidine-independent mutant colonies arising on these plates were

counted following incubation for 2 days at 37E C.

Each trial consisted of triplicate plates of concurrent positive and negative controls and five doses of the

chemical. For the study of cis-1,2-dichloroethylene conducted at SRI International and the study of trans1,2-dichloroethylene, 10,000 μg/plate was selected as the high dose. For the study of cis-1,2-dichloroethylene

conducted at Microbiological Associates, Inc., and the study of cis,trans-1,2-dichloroethylene, the high dose was

limited by toxicity. For trans- and cis,trans-1,2-dichloroethylene, all trials were repeated.

In this assay, a positive response is defined as a reproducible, dose-related increase in histidine-independent

(revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase

in revertants that is not dose related, is not reproducible, or is not of sufficient magnitude to support a

determination of mutagenicity. A negative response is obtained when no increase in revertant colonies is

observed following chemical treatment. There is no minimum percentage or fold increase required for a chemical

to be judged positive or weakly positive.

Chinese Hamster Ovary Cell Cytogenetics Protocols

Testing was performed as reported by Galloway et al. (1987). The three isomers were sent to the laboratories

as coded aliquots by Radian Corporation. They were tested in cultured Chinese hamster ovary (CHO) cells for

induction of sister chromatid exchanges (SCEs) and chromosomal aberrations (Abs), both in the presence and

absence of Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix. Cultures were handled

under gold lights to prevent photolysis of bromodeoxyuridine-substituted DNA. Each test consisted of

concurrent solvent and positive controls and of at least three doses of the chemical. For the cis,trans1,2-dichloroethylene trials conducted without S9, the high dose was limited by toxicity; in all other trials of each
28 trans-1,2-Dichloroethylene, NTP TOX 55

chemical, the highest dose was limited by study design to 5,000 μg/mL (cis and trans isomers) or 12,630 μg/mL

(cis,trans isomer). A single flask per dose was used.

Sister Chromatid Exchange Test: In the SCE test without S9, CHO cells were incubated for 25.8 hours with

cis,trans-1,2-dichloroethylene or 26 hours with cis- or trans-1,2-dichloroethylene in supplemented McCoy’s 5A

medium. Bromodeoxyuridine (BrdU) was added 2 hours after culture initiation. After 25.8 or 26 hours, the

medium containing the chemical was replaced with fresh medium plus BrdU and Colcemid, and incubation was

continued for 2 hours. Cells were then harvested by mitotic shake-off, fixed, and stained with Hoechst 33258

and Giemsa. In the SCE test with S9, cells were incubated with cis-, trans-, or cis,trans-1,2-dichloroethylene,

serum-free medium, and S9 for 2 hours. The medium was then replaced with medium containing serum and

BrdU and no test chemical. Incubation proceeded for an additional 25.5 to 26.5 hours, with Colcemid present

for the final 2 hours. Harvesting and staining were the same as for cells treated without S9. All slides were

scored blind, and those from a single test were read by the same person. Fifty second-division metaphase cells

were scored for frequency of SCEs/cell from each dose level.

Statistical analyses were conducted on the slopes of the dose-response curves and the individual dose points

(Galloway et al., 1987). An SCE frequency 20% above the concurrent solvent control value was chosen as a

statistically conservative positive response. The probability of this level of difference occurring by chance at one

dose point is less than 0.01; the probability for such a chance occurrence at two dose points is less than 0.001.

An increase of 20% or greater at any single dose was considered weak evidence of activity; increases at two or

more doses resulted in a determination that the trial was positive. A statistically significant trend (P<0.005) in

the absence of any responses reaching 20% above background led to a call of equivocal.

Chromosomal Aberrations Test: In the Abs test without S9, cells were incubated in McCoy’s 5A medium with

cis- or trans-1,2-dichloroethylene for 10 hours or with cis,trans-1,2-dichloroethylene for 20.5 hours. Colcemid

was added and incubation continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and

stained with Giemsa. For the Abs test with S9, cells were treated with cis-, trans-, or cis,trans1,2-dic hloroethylene S9 for 2 hours, after which the treatment medium was removed and the cells were

incubated for 11 hours (cis and trans isomers) or 8.5 hours (cis,trans mixture) in fresh medium, with Colcemid

present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9. The

harvest time for the Abs test of cis,trans-1,2-dichloroethylene was based on the cell cycle information obtained

in the SCE test: because cell cycle delay was anticipated in the trial conducted without S9, the incubation period

was extended.
trans-1,2-Dichloroethylene, NTP TOX 55 29

Cells were selected for scoring on the basis of good morphology and completeness of karyotype

(21 ± 2 chromosomes). All slides were scored blind, and those from a single test were read by the same person.

One hundred (cis,trans mixture) or two hundred (cis and trans isomers) first-division metaphase cells were

scored at each dose level. Classes of aberrations included simple (breaks and terminal deletions), complex

(rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing

10 or more aberrations).

Chromosomal aberration data are presented as percentage of cells with aberrations. To arrive at a statistical call

for a trial, analyses were conducted on both the dose response curve and individual dose points. For a single

trial, a statistically significant (P#0.05) difference for one dose point and a significant trend (P#0.015) were

considered weak evidence for a positive response; significant differences for two or more doses indicated the

trial was positive. A positive trend test in the absence of a statistically significant increase at any one dose

resulted in an equivocal call (Galloway et al., 1987). Ultimately, the trial calls were based on a consideration of

the statistical analyses as well as the biological information available to the reviewers.

Mouse Bone Marrow Cytogenetic Test Protocols

Sister Chromatid Exchange Test: A dose range-finding study was performed in the absence of adequate toxicity

information from the literature, and the high dose was limited to 2,000 mg/kg. Male B6C3F1 mice (five animals

per dose group) were injected intraperitoneally with cis- or trans-1,2-dichloroethylene dissolved in corn oil.

Vehicle control animals received equivalent injections of corn oil only. The positive control was

dimethylbenzanthracene.

A standard harvest time of 23 hours was used. The animals were implanted subcutaneously with a BrdU tablet

(McFee et al., 1983) 24 hours before harvest (1 hour before cis- or trans-1,2-dichloroethylene treatment). The

use of BrdU allowed selection of the appropriate cell population (cells in the second metaphase following cisor trans-1,2-dichloroethylene treatment) for scoring. Two hours before sacrifice, the animals received an

intraperitoneal injection of colchicine in saline. The animals were killed 23 hours after treatment (24 hours after

BrdU dosing). One or both femurs were removed, and the marrow was flushed out with phosphate-buffered

saline (pH 7.0). The cells were treated with a hypotonic salt solution, fixed, and dropped onto chilled slides.

After a 24-hour drying period, the slides were stained with fluorescence-plus-Giemsa and scored.

Twenty-five second-division metaphase cells were scored from each of four animals per treatment group.

Responses were evaluated as SCEs/cell, and the data were analyzed by a trend test (Margolin et al., 1986).
30 trans-1,2-Dichloroethylene, NTP TOX 55

Chromosomal Aberrations Test: A dose range-finding study was performed in the absence of adequate toxicity

information from the literature, and the high dose was limited to 2,000 mg/kg. A standard harvest time of

17 hours was used.

Male B6C3F1 mice (10 animals per dose group) were injected intraperitoneally with cis- or trans1,2-dichloroethylene dissolved in corn oil. Vehicle control animals received equivalent injections of corn oil only.

The positive control was dimethylbenzanthracene. The animals were subcutaneously implanted with a BrdU tablet

(McFee et al., 1983) 18 hours before the scheduled harvest (1 hour before injection of cis- or trans1,2-dichloroethylene). The use of BrdU allowed selection of the appropriate cell population for scoring. (Abs

induced by chemical administration are present in maximum number at the first metaphase following treatment;

they decline in number during subsequent nuclear divisions due to cell death.) Two hours before sacrifice, the

animals received an intraperitoneal injection of colchicine in saline. The animals were killed 17 hours after cisor trans-1,2-dichloroethylene injection (18 hours after BrdU dosing). One or both femurs were removed, and

the marrow was flushed out with phosphate-buffered saline (pH 7.0). Cells were treated with a hypotonic salt

solution, fixed, and dropped onto chilled slides. After a 24-hour drying period, the slides were stained with

fluorescence-plus-Giemsa and scored.

Fifty first-division metaphase cells were scored from each of seven or eight animals per treatment group.

Responses were evaluated as the percentage of aberrant metaphase cells, excluding gaps. The data were analyzed

by a trend test (Margolin et al., 1986). The trend test P value must be less than or equal to 0.025 for a test to

be significant; pairwise comparisons of each treatment group to the solvent control group are significant when

P is less than or equal to 0.025 divided by the number of groups treated with cis- or trans-1,2-dichloroethylene.

Mouse Peripheral Blood Micronucleus Test Protocol

A detailed discussion of this assay is presented by MacGregor et al. (1990). At the end of the 14-week toxicity

study, peripheral blood samples were obtained from male and female mice. Smears were immediately prepared

and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides

were scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs) in each

of 10 animals per exposure group. In addition, 1,000 polychromatic erythrocytes (PCEs) were scored per animal

to determine the percentage of PCEs in the total erythrocyte population.

The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or

minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by a

statistical software package that tested for increasing trend over exposure groups with a one-tailed Cochran-
trans-1,2-Dichloroethylene, NTP TOX 55 31

Armitage trend test, followed by pairwise comparisons between each exposure group and the untreated control

group (ILS, 1990). In the presence of excess binomial variation, as detected by a binomial dispersion test, the

binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. In

the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to

0.025 or if the P value for any single exposed group is less than or equal to 0.025 divided by the number of

exposed groups. A final call of positive for micronucleus induction is preferably based on reproducibly positive

trials (as noted above). Results of the 14-week studies were accepted without repeat tests, because additional

test data could not be obtained. Ultimately, the final call is determined by the scientific staff after considering

the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects.

Evaluation Protocol

These are the basic guidelines for arriving at an overall assay result for assays performed by the National

Toxicology Program. Statistical as well as biological factors are considered. For an individual assay, the

statistical procedures for data analysis have been described in the preceding protocols. There have been

instances, however, in which multiple aliquots of a chemical were tested in the same assay, and different results

were obtained among aliquots and/or among laboratories. Results from more than one aliquot or from more than

one laboratory are not simply combined into an overall result. Rather, all the data are critically evaluated,

particularly with regard to pertinent protocol variations, in determining the weight of evidence for an overall

conclusion of chemical activity in an assay. In addition to multiple aliquots, the in vitro assays have another

variable that must be considered in arriving at an overall test result. In vitro assays are conducted with and

without exogenous metabolic activation. Results obtained in the absence of activation are not combined with

results obtained in the presence of activation; each testing condition is evaluated separately. The results presented

in the Abstract of this Toxicity Study Report represent a scientific judgement of the overall evidence for activity

of the chemical in an assay.
32 trans-1,2-Dichloroethylene, NTP TOX 55
33

RESULTS

RATS

All rats exposed to trans-1,2-dichloroethylene survived to the end of the study (Table 4). The final mean body

weight and body weight gain of males in the 50,000 ppm group were significantly less than those of the vehicle

controls (Table 4 and Figure 1). Feed consumption by the exposed groups was similar to that by the vehicle

controls. Exposure concentrations of 3,125, 6,250, 12,500, 25,000, and 50,000 ppm resulted in average daily

doses of 190, 380, 770, 1,540, and 3,210 mg trans-1,2-dichloroethylene per kg body weight for males and 190,

395, 780, 1,580, and 3,245 mg/kg for females. There were no clinical findings of toxicity. Results of the

functional observation battery indicated no exposure-related findings of neurotoxicity.

TABLE 4

Survival, Body Weights, and Feed Consumption of Rats in the 14-Week Feed Study 

of trans-1,2-Dichloroethylene

Final Weight

Mean Body Weightb (g) Relative to Feed

Concentration Survival a Initial Final Change Vehicle Consumptionc

(ppm) Controls (%) Week 2 Week 13

Male

Untreated Control 10/10 129 ± 3 362 ± 7 232 ± 5

Vehicle Control 10/10 127 ± 3 360 ± 6 232 ± 5 14.3 15.3

3,125 10/10 128 ± 2 365 ± 5 237 ± 6 101 14.1 15.1

6,250 10/10 128 ± 3 361 ± 3 233 ± 3 100 14.5 15.8

12,500 10/10 127 ± 3 357 ± 5 230 ± 5 99 14.6 15.6

25,000 10/10 128 ± 2 350 ± 6 222 ± 5 97 14.3 15.5

50,000 10/10 127 ± 3 339 ± 4** 213 ± 3** 94 14.2 15.8

Female

Untreated Control 10/10 111 ± 3 196 ± 3 86 ± 1

Vehicle Control 10/10 111 ± 2 190 ± 4 79 ± 3 10.6 9.8

3,125 10/10 111 ± 2 198 ± 3 87 ± 2 104 10.9 9.8

6,250 10/10 110 ± 2 203 ± 2* 93 ± 2** 107 10.6 10.0

12,500 10/10 110 ± 2 198 ± 3 88 ± 2* 104 10.9 10.5

25,000 10/10 110 ± 2 196 ± 3 86 ± 2 103 10.8 9.8

50,000 10/10 110 ± 2 191 ± 2 81 ± 2 101 11.2 9.9

* Significantly different (P#0.05) from the vehicle control group by Dunnett’s test

** Significantly different (P#0.01) from the vehicle control group by Williams’ or Dunnett’s test

a Number of animals surviving at 14 weeks/number initially in group

b Weights and weight changes are given as mean ± standard error. c Feed consumption is expressed as grams per animal per day.
FIGURE 1

Body Weights of Male and Female Rats Exposed to trans-1,2-Dichloroethylene in Feed for 14 Weeks

34 trans-1,2-Dichloroethylene, NTP TOX 55
trans-1,2-Dichloroethylene, NTP TOX 55 35

The hematology data for rats are listed in Tables 5 and B1. On day 21 and at week 14, there were decreases in

the circulating erythroid mass in exposed males and females, as evidenced by decreases in hematocrit values,

hemoglobin concentrations, and erythrocyte counts. On day 21, these erythron effects occurred most

consistently in the 25,000 and 50,000 ppm groups; at week 14, these effects were also observed in males

exposed to 6,250 or 12,500 ppm. At both time points, the decrease in the erythron mass was of minimal severity,

and, generally, the suppression was approximately 5% or less compared to the vehicle control values.

Females exposed to 12,500 ppm or greater had significantly decreased serum alkaline phosphatase activities

compared to the vehicle controls on day 21 (Table B1). These decreases were minimal in severity, were no

greater than approximately 13%, and were transient, with alkaline phosphatase activities in the affected groups

returning to vehicle control levels by week 14. On day 21 and at week 14, there was minimal suppression of

serum 5N-nucleotidase activities in males and females in the 50,000 ppm groups. There were sporadic differences

in clinical chemistry parameters at various time points that generally did not demonstrate an exposure

concentration relationship or were inconsistent between males and females; these differences were not considered

to be toxicologically relevant.

The liver weights of female rats exposed to 6,250 ppm or greater were significantly greater than those of the

vehicle controls (Table C1). Males in the 25,000 and 50,000 ppm groups had significantly lower absolute kidney

weights than the vehicle control group.

Sperm motility and vaginal cytology parameters of exposed rats were generally similar to those of the vehicle

controls (Tables D1 and D2). No gross or microscopic lesions were observed that could be attributed to

trans-1,2-dichloroethylene exposure (Tables A1 and A2).
36 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE 5

Selected Hematology Data for Rats in the 14-Week Feed Study of trans-1,2-Dichloroethylenea

Vehicle

Control 3,125 ppm 6,250 ppm 12,500 ppm 25,000 ppm 50,000 ppm

Male

n

Day 5 10 10 10 10 10 10

Day 21 10 10 9 7 9 10

Week 14 10 10 9 10 10 10

Hematocrit (%)

Day 5 38.8 ± 0.4 39.1 ± 0.6 39.5 ± 0.7 40.5 ± 0.9 39.9 ± 0.6 39.8 ± 0.6

Day 21 43.3 ± 0.4 43.1 ± 0.5 42.4 ± 0.2 42.5 ± 0.6 40.9 ± 1.7 41.6 ± 0.4*

Week 14 41.1 ± 0.3 41.4 ± 0.3 40.4 ± 0.4 39.9 ± 0.4* 39.8 ± 0.3* 39.0 ± 0.8*

Manual hematocrit (%)

Day 5 42.8 ± 0.4 43.2 ± 0.6 43.5 ± 0.7 44.6 ± 0.8 44.1 ± 0.5 43.8 ± 0.6

Day 21 49.1 ± 0.6 48.3 ± 0.8 47.8 ± 0.4 48.4 ± 0.5 46.1 ± 1.8 47.1 ± 0.6

Week 14 45.0 ± 0.4 45.2 ± 0.2 44.2 ± 0.4 44.1 ± 0.4 43.5 ± 0.4* 43.2 ± 0.8*

Hemoglobin (g/dL)

Day 5 14.6 ± 0.2 14.7 ± 0.2 14.8 ± 0.2 15.2 ± 0.3 14.9 ± 0.2 14.8 ± 0.1

Day 21 15.9 ± 0.2 15.7 ± 0.3 15.4 ± 0.1 15.6 ± 0.1 14.9 ± 0.6* 15.1 ± 0.2**

Week 14 14.8 ± 0.1 15.0 ± 0.1 14.7 ± 0.1 14.5 ± 0.1 14.5 ± 0.1 14.3 ± 0.3

Erythrocytes (106/μL)

Day 5 6.50 ± 0.07 6.55 ± 0.09 6.61 ± 0.13 6.78 ± 0.15 6.68 ± 0.09 6.71 ± 0.09

Day 21 7.13 ± 0.08 7.10 ± 0.09 6.91 ± 0.04* 6.96 ± 0.09 6.69 ± 0.25* 6.82 ± 0.07**

Week 14 8.14 ± 0.08 8.17 ± 0.05 7.93 ± 0.10* 7.84 ± 0.09* 7.79 ± 0.08** 7.56 ± 0.15**

Female

n 10 10 10 10 10 10

Hematocrit (%)

Day 5 41.2 ± 0.6 41.1 ± 0.6 40.3 ± 0.6 41.2 ± 0.5 41.0 ± 0.4 40.9 ± 0.5

Day 21 45.4 ± 0.4 45.2 ± 0.3 44.0 ± 0.3 44.6 ± 0.3 43.7 ± 0.5 43.9 ± 0.8

Week 14 41.9 ± 0.3 41.5 ± 0.5 41.2 ± 0.5 41.2 ± 0.2 40.4 ± 0.3** 40.1 ± 0.4**

Manual hematocrit (%)

Day 5 44.8 ± 0.6 45.1 ± 0.6 43.7 ± 0.7 44.6 ± 0.6 44.3 ± 0.4 44.7 ± 0.5

Day 21 49.2 ± 0.5 49.0 ± 0.4 47.5 ± 0.4 48.3 ± 0.4 47.6 ± 0.5 48.0 ± 0.6

Week 14 44.9 ± 0.3 44.6 ± 0.4 44.1 ± 0.4 44.3 ± 0.2 43.6 ± 0.3** 43.3 ± 0.4**

Hemoglobin (g/dL)

Day 5 15.1 ± 0.2 15.3 ± 0.2 14.9 ± 0.2 15.2 ± 0.2 15.1 ± 0.1 15.1 ± 0.2

Day 21 16.6 ± 0.1 16.6 ± 0.2 16.1 ± 0.2* 16.4 ± 0.1 15.9 ± 0.2** 16.1 ± 0.2*

Week 14 15.2 ± 0.1 15.2 ± 0.2 15.0 ± 0.1 15.0 ± 0.1 14.7 ± 0.1** 14.5 ± 0.1**

Erythrocytes (106/μL)

Day 5 7.00 ± 0.13 7.00 ± 0.13 6.83 ± 0.13 6.95 ± 0.10 6.92 ± 0.09 6.92 ± 0.10

Day 21 7.52 ± 0.08 7.48 ± 0.06 7.28 ± 0.07* 7.34 ± 0.06* 7.21 ± 0.08** 7.21 ± 0.14*

Week 14 7.59 ± 0.06 7.58 ± 0.10 7.50 ± 0.08 7.49 ± 0.04 7.34 ± 0.05** 7.20 ± 0.08**

* Significantly different (P#0.05) from the vehicle control group by Dunn’s or Shirley’s test

** Significantly different (P#0.01) from the vehicle control group by Shirley’s test a Mean ± standard error. Statistical tests were performed on unrounded data.
trans-1,2-Dichloroethylene, NTP TOX 55 37

MICE

There were no treatment-related deaths of mice exposed to trans-1,2-dichloroethylene (Table 6). The final mean

body weights and body weight gains of males and females in the 50,000 ppm groups and the body weight gains

of females in the 12,500 and 25,000 ppm groups were significantly less than those of the vehicle controls

(Table 6 and Figure 2). Feed consumption by the exposed groups was similar to that by the vehicle controls.

Exposure concentrations of 3,125, 6,250, 12,500, 25,000, and 50,000 ppm resulted in average daily doses of

480, 920, 1,900, 3,850, and 8,065 mg/kg for males and 450, 915, 1,830, 3,760, and 7,925 mg/kg for females.

There were no clinical findings of toxicity. Results of the functional observation battery indicated no exposurerelated findings of neurotoxicity.

TABLE 6

Survival, Body Weights, and Feed Consumption of Mice in the 14-Week Feed Study 

of trans-1,2-Dichloroethylene

Final Weight

Mean Body Weightb (g) Relative to Feed

Concentration Survival a Initial Final Change Vehicle Consumptionc

(ppm) Controls (%) Week 2 Week 13

Male

Untreated Control 10/10 20.0 ± 0.4 30.6 ± 0.9 10.6 ± 0.5

Vehicle Control 10/10 19.5 ± 0.4 30.3 ± 0.6 10.8 ± 0.5 3.7 4.2

3,125 10/10 20.1 ± 0.3 29.8 ± 0.4 9.7 ± 0.3 98 4.0 4.2

6,250 10/10 19.7 ± 0.4 30.5 ± 0.6 10.8 ± 0.7 101 3.7 3.9

12,500 10/10 20.0 ± 0.4 29.2 ± 1.0 9.2 ± 0.9 96 3.7 4.1

25,000 10/10 19.3 ± 0.6 29.0 ± 0.5 9.7 ± 0.6 96 3.7 4.1

50,000 10/10 20.0 ± 0.3 28.2 ± 0.5* 8.1 ± 0.5** 93 3.8 4.2

Female

Untreated Control 8/10d 16.8 ± 0.4 24.3 ± 0.5 7.1 ± 0.4

Vehicle Control 10/10 15.9 ± 0.4 23.3 ± 0.3 7.4 ± 0.4 3.1 3.1

3,125 10/10 16.2 ± 0.4 23.8 ± 0.5 7.6 ± 0.3 102 2.9 3.2

6,250 9/10d 17.1 ± 0.6 25.0 ± 0.6 8.0 ± 0.5 108 3.1 3.3

12,500 10/10 16.4 ± 0.4 22.0 ± 0.4 5.6 ± 0.2** 94 2.8 3.2

25,000 9/10d 16.1 ± 0.4 22.3 ± 0.4 6.0 ± 0.3** 96 3.0 3.1

50,000 9/10d 16.2 ± 0.4 21.7 ± 0.4* 5.4 ± 0.3** 93 2.6 3.3

* Significantly different (P#0.05) from the vehicle control group by Williams’ test

** P#0.01 a Number of animals surviving at 14 weeks/number initially in group

b Weights and weight changes are given as mean ± standard error. c Feed consumption is expressed as grams per animal per day.

d Week of death: 14 (accidental deaths)
FIGURE 2

Body Weights of Male and Female Mice Exposed to trans-1,2-Dichloroethylene in Feed for 14 Weeks

38 trans-1,2-Dichloroethylene, NTP TOX 55
trans-1,2-Dichloroethylene, NTP TOX 55 39

No alterations in clinical chemistry parameters for mice were attributed to exposure (Table B2). The relative liver

weights of males exposed to 12,500 ppm or greater and females exposed to 25,000 or 50,000 ppm were

significantly greater than those of the vehicle controls (Table C2). Sperm motility and vaginal cytology

parameters of exposed mice were similar to those of the vehicle controls (Tables D3 and D4). No gross or

microscopic lesions were observed that could be attributed to trans-1,2-dichloroethylene exposure (Tables A3

and A4).

GENETIC TOXICOLOGY

cis-1,2-Dichloroethylene, tested at two laboratories, was not mutagenic in any of five strains of Salmonella

typhimurium with or without induced hamster or rat S9 metabolic activation enzymes (Table E1; Zeiger et al.,

1988). Neither trans- nor cis,trans-1,2-dichloroethylene was mutagenic in any of four strains of S. typhimurium

(Tables E2 and E3; Mortelmans et al., 1986). In Chinese hamster ovary (CHO) cells in vitro, cis1,2-dichloroethylene induced sister chromatid exchanges (SCEs) in the absence of S9 at concentrations of 500 to

5,000 μg/mL (Table E4); with S9, results from a single trial were judged to be equivocal, because although a clear

dose-related increase in SCEs was observed (P=0.001), none of the individual dose points differed significantly

from the solvent control value. trans-1,2-Dichloroethylene did not induce SCEs without S9; the results of a

single trial with S9 were judged to be equivocal based on the trend test (P<0.005) and the absence of significant

increases ($20%) at any of the individual dose points (Table E5). A strongly positive response was obtained for

SCE induction by cis,trans-1,2-dichloroethylene at all doses tested, with and without S9 metabolic activation

enzymes (Table E6). No induction of chromosomal aberrations (Abs) by cis-, trans-, or cis,trans1,2-dichloroethylene was observed in cultured CHO cells, with or without S9 (Tables E7, E8, and E9). For the

cis,trans isomer, intermediate toxicity occurred at some of the doses tested in the absence of S9, and etching of

the plastic tissue culture flasks occurred at the higher doses with and without S9. In the first Abs trial with S9,

an increase in Abs was noted at the lowest cis,trans-1,2-dichloroethylene dose tested (772 μg/mL); although the

increase was not statistically significant, a second trial was performed using similar doses. Again, the lowest

dose tested (455 μg/mL) in this trial produced the greatest increase in Abs, but the difference was not significant.

Thus, the Abs test for cis,trans-1,2-dichloroethylene was judged to be negative.

In vivo, neither cis- nor trans-1,2-dichloroethylene administered by intraperitoneal injection at doses of 500 to

2,000 mg/kg induced SCEs or Abs in bone marrow cells of male mice in tests using the standard sampling times

of 23 hours (SCE test) or 17 hours (Abs test) (Tables E10 through E13). Experiments using an extended harvest

time to compensate for special metabolic requirements or induced cell cycle delays are required before the

negative results in the in vivo bone marrow tests may be considered complete. trans-1,2-Dichloroethylene
40 trans-1,2-Dichloroethylene, NTP TOX 55

administered in microcapsules in feed for 14 weeks did not increase the frequency of micronucleated

normochromatic erythrocytes (NCEs) in the peripheral blood of male or female mice (Table E14). In addition,

no effect on the percentage of micronucleated polychromatic erythrocytes among the total erythrocyte population

was observed, indicating no inhibition or stimulation of erythropoiesis in the bone marrow of exposed mice.
41

DISCUSSION

Toxicology studies of trans-1,2-dichloroethylene were conducted by oral administration to F344/N rats and

B6C3F1 mice for 14 weeks. trans-1,2-Dichloroethylene was administered in microcapsules in the feed. This

method prolongs chemical stability in rodent diets and is not toxic (Dieter et al., 1993; NTP, 2000, 2002).

Microencapsulation allowed continual ingestion of trans-1,2-dichloroethylene, was a more appropriate route than

oral gavage, and allowed for higher exposure concentrations than would be obtainable in drinking water. Two

control groups were included in these studies to detect any adverse effects of ingestion of microcapsules. No

biologically significant differences in body or organ weights were observed between the untreated and vehicle

controls. Because there were no differences in response between the control groups, statistical comparisons only

between the vehicle controls and the exposed groups are presented. Previous work has shown that the

bioavailability of chemical administered in microcapsules was comparable to that when administered by gavage

(Yuan et al., 1993; Hébert et al., 1994).

Because 1,2-dichloroethylene occurs in two isomeric forms, cis and trans, 15-day pilot studies using each isomer

and a mixture of the two isomers in microcapsules in feed were performed to identify the most biologically active

isomer and to aid in exposure concentration selection for the 14-week studies. The maximum allowable

concentration of 50,000 ppm 1,2-dichloroethylene was used as the highest exposure concentration in the 15-day

pilot studies. No chemical-related lesions were observed in rats or mice in any exposure group. Kidney and liver

weights were increased in rats and mice exposed to cis- or trans-1,2-dichloroethylene but not in animals exposed

to a mixture of cis- and trans-1,2-dichloroethylene (data not shown). Large variations in initial mean body

weights of rats exposed to cis-1,2-dichloroethylene confounded interpretation of body and organ weight data.

The most significant effect was a 20% reduction in final mean body weights of male rats exposed to

trans-1,2-dichloroethylene. Based on these preliminary data and because of its prevalence in industrial

applications, the trans isomer was selected for 14-week studies. Due to the low toxicity observed in the 15-day

pilot studies, 50,000 ppm trans-1,2-dichloroethylene was selected as the highest concentration for use in the

14-week studies.

In general, toxic effects caused by ingestion of trans-1,2-dichloroethylene for 14 weeks were minimal. The liver

appeared to be the target organ for trans-1,2-dichloroethylene toxicity based on organ weight data and has been

identified as such in several studies (Freundt et al., 1977; Hayes et al., 1987; McCauley et al., 1995). In the
42 trans-1,2-Dichloroethylene, NTP TOX 55

present study and that by Hayes et al. (1987), no gross or microscopic lesions were observed in animals exposed

to trans-1,2-dichloroethylene for 14 weeks. Increased liver weights are often sensitive indicators for toxicity,

especially when accompanied by gross or microscopic lesions. In the case of trans-1,2-dichloroethylene, lesions

were observed only in one study in which inhalation exposure caused fatty accumulation in the liver and Kupffer

cells (Freundt et al., 1977). These lesions were interpreted to be a result of inhibition of cytochrome P450

enzymes by the trans isomer. In the present studies and in those of McCauley et al. (1995) and Hayes et al.

(1987), histopathologic lesions did not accompany increased liver weights. Liver weight changes following

chemical insult are not uncommon and are often associated with proliferation of the smooth endoplasmic

reticulum and increased cytochrome P450 content or drug metabolizing enzyme activity, which can represent

adaptive and/or toxic responses (de la Iglesia et al., 1982).

In the current studies, modest decreases in absolute kidney weights occurred in male rats exposed to 25,000 or

50,000 ppm trans-1,2-dichloroethylene with no accompanying lesions or changes in clinical chemistry

parameters. Changes in kidney weights, with no gross or microscopically visible lesions, also have been

observed by Freundt et al. (1977), Hayes et al. (1987), and McCauley et al. (1995). McCauley et al. (1995)

observed a decrease in blood urea nitrogen levels in male and female rats exposed to cis-1,2-dichloroethylene for

14 days, but this change was not dose related, and its toxicological significance was unknown. Decreased kidney

weights, although observed in the current studies and previous studies, do not provide information on kidney

toxicity in the absence of histopathologic or clinical evidence of toxicity. 

In the current study, slight, but generally exposure concentration-dependent, decreases in red cell mass were

observed in male rats exposed to 6,250 ppm or greater and female rats exposed to 25,000 or 50,000 ppm. In

male and female Sprague-Dawley rats exposed to 872 mg/kg cis-1,2-dichloroethylene by gavage for 90 days,

significant decreases in the circulating erythroid mass were observed; however, this response was not dose

related and was not considered to be biologically relevant (McCauley et al., 1995). No hematologic response was

observed in male or female CD-1 mice exposed to 2,000 mg/L trans-1,2-dichloroethylene (daily dose

approximately 387 mg/kg) in drinking water for 90 days (Barnes et al., 1985). The cis and trans isomers may

have an effect on hematologic endpoints; however, more consistency between studies is necessary before the

biological significance (if any) is known.

trans-1,2-Dichloroethylene is a mechanism-based inhibitor of CYP2E1 and is converted by CYP2E1 to an epoxide

intermediate that subsequently binds to the heme moiety and as such is the rate-limiting step of

trans-1,2-dichloroethylene metabolism in vivo. The exposure concentrations of trans-1,2-dichloroethylene in

the present studies are greater than those required to suppress CYP2E1 after single or repeated gavage
trans-1,2-Dichloroethylene, NTP TOX 55 43

administration (Mathews et al., 1997). Although CYP2E1 activity was not assessed in the present study, it is

likely that its activity was inhibited, thus affecting the metabolism of trans-1,2-dichloroethylene.

trans-1,2-Dichloroethylene has been shown to cause central nervous system effects in humans, characterized

by dizziness, drowsiness, vertigo, and increased intracranial pressure (ATSDR, 1990). To address the possible

central nervous system depression effects, a functional observation battery was included in the current studies.

The results suggest that ingestion of trans-1,2-dichloroethylene does not cause central nervous system depression

in rats or mice. Other investigators have observed ataxia and loss of righting reflex; however, these effects were

observed at lethal doses of cis- or trans-1,2-dichloroethylene (Munson et al., 1982; Hayes et al., 1987; McCauley

et al., 1995).

Symmetrically substituted oxiranes such as 1,2-dichloroethylene and 1,1,2,2-tetrachloroethylene are more stable

and less mutagenic than unsymmetrical chlorinated oxiranes such as 1,1-dichloroethylene, 1,1,2-trichloroethylene,

and vinyl chloride (Henschler et al., 1976; Cantelli-Forti and Bronzetti, 1988). However, the carcinogenicity of

chlorinated hydrocarbons may or may not lie in the reactivity of the epoxide intermediate. Of the chlorinated

hydrocarbons, it has been reported that 1,1-dichloroethylene (Chu and Milman, 1981), vinyl chloride (Apfeldorf

and Infante, 1981; Maltoni et al., 1981; Rice, 1981), trichloroethylene (NCI, 1976; Fukuda et al., 1983; NTP,

1988, 1990), and tetrachloroethylene (NCI, 1977; NTP, 1986) are carcinogens in vivo. From the literature, the

carcinogenicity of 1,1-dichloroethylene is primarily associated with inhalation exposure (Lee et al., 1978; Chu

and Milman, 1981). But, in the case of vinyl chloride (Apfeldorf and Infante, 1981; Infante, 1981; Maltoni et al.,

1981; Rice, 1981), trichloroethylene (NCI, 1976; Fukuda et al., 1983; NTP, 1988, 1990), and tetrachloroethylene

(NTP, 1986), carcinogenicity occurs through both the inhalation and oral routes (NCI, 1977; NTP, 1986).

1,2-Dichloroethylene may be similarly toxic via inhalation. Freundt et al. (1977) reported severe alveolar septal

distention and pulmonary hyperemia in rat lungs after inhalation exposure to trans-1,2-dichloroethylene at

concentrations greater than or equal to the current threshold limit value of 200 ppm (ACGIH, 2000).

In summary, very little toxicity was associated with ingestion of microencapsulated trans-1,2-dichloroethylene.

Histopathology and clinical chemistry data, combined with body and organ weight data, revealed that the

maximum tolerated dose was not reached in these studies. The lowest exposure concentration used in these

studies, 3,125 ppm, exceeds the current drinking water limits of 70 to 100 ppb by a large factor.
44 trans-1,2-Dichloroethylene, NTP TOX 55
45

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54 trans-1,2-Dichloroethylene, NTP TOX 55
A-1

APPENDIX A

SUMMARY OF LESIONS

IN RATS AND MICE

TABLE A1 Summary of the Incidence of Nonneoplastic Lesions in Male Rats 

in the 14-Week Feed Study of trans-1,2-Dichloroethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-2

TABLE A2 Summary of the Incidence of Neoplasms and Nonneoplastic Lesions in Female Rats 

in the 14-Week Feed Study of trans-1,2-Dichloroethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-4

TABLE A3 Summary of the Incidence of Nonneoplastic Lesions in Male Mice 

in the 14-Week Feed Study of trans-1,2-Dichloroethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-6

TABLE A4 Summary of the Incidence of Nonneoplastic Lesions in Female Mice 

in the 14-Week Feed Study of trans-1,2-Dichloroethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-8
A-2 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE A1

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 14-Week Feed Study 

of trans-1,2-Dichloroethylenea

Untreated Vehicle

Control Control 3,125 ppm 6,250 ppm 12,500 ppm 25,000 ppm 50,000 ppm

Disposition Summary

Animals initially in study 10 10 10 10 10 10 10

Survivors

Terminal sacrifice 10 10 10 10 10 10 10

Animals examined microscopically 10 10 1 2 10

Alimentary System

Liver (10) (10) (1) (2) (10)

Hepatodiaphragmatic nodule 1 (100%) 1 (50%)

Inflammation, chronic 1 (10%)

Pancreas (10) (10) (10)

Acinus, atrophy, focal 3 (30%) 2 (20%)

Cardiovascular System

Heart (10) (10) (10)

Cardiomyopathy 9 (90%) 6 (60%) 7 (70%)

Pigmentation 1 (10%)

Endocrine System

Thyroid gland (10) (10) (10)

Ultimobranchial cyst 2 (20%) 2 (20%)

General Body System

None

Genital System

Preputial gland (10) (10) (9)

Inflammation, chronic 6 (60%) 2 (20%) 1 (11%)

Inflammation, chronic active 1 (10%)

Bilateral, inflammation, chronic 2 (20%) 3 (30%) 2 (22%)

Testes (10) (10) (10)

Germinal epithelium, atrophy 1 (10%)

Hematopoietic System

Lymph node, mandibular (10) (10) (10)

Hemorrhage 2 (20%)

Lymph node, mesenteric (10) (10) (10)

Infiltration cellular, histiocyte 1 (10%) 3 (30%)

Spleen (10) (10) (10)

Capsule, inflammation, chronic, 

 focal 1 (10%)

Thymus (10) (10) (9)

Hemorrhage 3 (30%) 1 (10%) 1 (11%)
trans-1,2-Dichloroethylene, NTP TOX 55 A-3

TABLE A1

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 14-Week Feed Study 

of trans-1,2-Dichloroethylene

Untreated Vehicle

Control Control 3,125 ppm 6,250 ppm 12,500 ppm 25,000 ppm 50,000 ppm

Integumentary System

None

Musculoskeletal System

None

Nervous System

None

Respiratory System

Lung (10) (10) (10)

Hemorrhage 2 (20%) 1 (10%) 3 (30%)

Interstitium, inflammation, chronic 1 (10%) 1 (10%) 3 (30%)

Interstitium, inflammation, 

 chronic active 1 (10%)

Nose (10) (10)

Fungus 1 (10%)

Inflammation, chronic active 1 (10%)

Special Senses System

None

Urinary System

Kidney (10) (10) (10)

Nephropathy, chronic 3 (30%) 4 (40%)

Bilateral, nephropathy, chronic 9 (90%) 7 (70%) 4 (40%)

a Number of animals examined microscopically at the site and the number of animals with lesion
A-4 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE A2

Summary of the Incidence of Neoplasms and Nonneoplastic Lesions in Female Rats in the 14-Week Feed Study 

of trans-1,2-Dichloroethylenea

Untreated Vehicle

Control Control 3,125 ppm 6,250 ppm 12,500 ppm 25,000 ppm 50,000 ppm

Disposition Summary

Animals initially in study 10 10 10 10 10 10 10

Survivors

Terminal sacrifice 10 10 10 10 10 10 10

Animals examined microscopically 10 10 5 5 3 2 10

Alimentary System

Liver (10) (10) (2) (1) (1) (10)

Hepatodiaphragmatic nodule 1 (10%) 2 (20%) 1 (50%) 1 (100%) 2 (20%)

Inflammation, chronic 1 (10%)

Pancreas (10) (10) (10)

Acinus, atrophy, focal 2 (20%)

Cardiovascular System

Heart (10) (10) (10)

Cardiomyopathy 2 (20%) 1 (10%) 2 (20%)

Endocrine System

Pituitary gland (10) (10) (10)

Pars distalis, cyst 1 (10%) 1 (10%)

Thyroid gland (10) (10) (10)

Ultimobranchial cyst 1 (10%)

General Body System

None

Genital System

Clitoral gland (10) (10) (10)

Infiltration cellular, chronic 1 (10%)

Inflammation, chronic 1 (10%) 3 (30%) 1 (10%)

Bilateral, inflammation, chronic 2 (20%) 3 (30%) 2 (20%)

Ovary (10) (10) (2) (1) (1) (10)

Cyst 2 (100%) 1 (100%) 1 (100%) 1 (10%)

Uterus (10) (10) (1) (4) (2) (1) (10)

Hydrometra 2 (20%) 3 (30%) 1 (100%) 4 (100%) 2 (100%) 1 (100%) 4 (40%)

Endometrium, polyp stromal 1 (10%)

Hematopoietic System

Bone marrow (10) (10) (10)

Atrophy 1 (10%)

Inflammation, granulomatous 4 (40%) 2 (20%) 2 (20%)

Myelofibrosis 1 (10%) 3 (30%)

Lymph node, mandibular (10) (10) (10)

Hemorrhage 1 (10%)
trans-1,2-Dichloroethylene, NTP TOX 55 A-5

TABLE A2

Summary of the Incidence of Neoplasms and Nonneoplastic Lesions in Female Rats in the 14-Week Feed Study 

of trans-1,2-Dichloroethylene

Untreated Vehicle

Control Control 3,125 ppm 6,250 ppm 12,500 ppm 25,000 ppm 50,000 ppm

Hematopoietic System (continued)

Lymph node, mesenteric (10) (10) (10)

Hyperplasia, lymphoid 1 (10%)

Infiltration cellular, histiocyte 5 (50%) 2 (20%) 3 (30%)

Spleen (10) (10) (1) (10)

Capsule, fibrosis 1 (100%)

Thymus (10) (10) (10)

Hemorrhage 1 (10%) 1 (10%)

Integumentary System

None

Musculoskeletal System

None

Nervous System

None

Respiratory System

Lung (10) (10) (10)

Hemorrhage 1 (10%)

Interstitium, inflammation, chronic 1 (10%) 4 (40%)

Nose (10) (10) (10)

Inflammation, chronic 1 (10%) 1 (10%)

Special Senses System

None

Urinary System

Kidney (10) (10) (10)

Nephropathy, chronic 2 (20%)

Bilateral, mineralization, focal 10 (100%) 10 (100%) 10 (100%)

a Number of animals examined microscopically at the site and the number of animals with lesion
A-6 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE A3

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 14-Week Feed Study 

of trans-1,2-Dichloroethylenea

Untreated Vehicle

Control Control 3,125 ppm 6,250 ppm 12,500 ppm 25,000 ppm 50,000 ppm

Disposition Summary

Animals initially in study 10 10 10 10 10 10 10

Survivors

Terminal sacrifice 10 10 10 10 10 10 10

Animals examined microscopically 10 10 10

Alimentary System

Gallbladder (9) (10) (9)

Cyst 1 (10%)

Stomach, glandular (10) (10) (10)

Cyst 2 (20%) 1 (10%)

Cardiovascular System

Heart (10) (10) (10)

Valve, pigmentation 1 (10%)

Endocrine System

Adrenal cortex (10) (10) (10)

Capsule, hyperplasia 1 (10%) 1 (10%) 1 (10%)

Adrenal medulla (10) (10) (10)

Angiectasis 1 (10%) 2 (20%) 2 (20%)

General Body System

None

Genital System

Preputial gland (10) (10) (10)

Atrophy 9 (90%) 6 (60%) 7 (70%)

Hematopoietic System

Lymph node, mandibular (10) (9) (10)

Hemorrhage 1 (11%)

Spleen (10) (10) (10)

Hematopoietic cell proliferation 1 (10%) 1 (10%)

Integumentary System

None

Musculoskeletal System

None
trans-1,2-Dichloroethylene, NTP TOX 55 A-7

TABLE A3

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 14-Week Feed Study 

of trans-1,2-Dichloroethylene

Untreated Vehicle

Control Control 3,125 ppm 6,250 ppm 12,500 ppm 25,000 ppm 50,000 ppm

Nervous System

None

Respiratory System

None

Special Senses System

None

Urinary System

Kidney (10) (10) (10)

Inflammation, focal 1 (10%)

a Number of animals examined microscopically at the site and the number of animals with lesion
A-8 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE A4

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 14-Week Feed Study 

of trans-1,2-Dichloroethylenea

Untreated Vehicle

Control Control 3,125 ppm 6,250 ppm 12,500 ppm 25,000 ppm 50,000 ppm

Disposition Summary

Animals initially in study 10 10 10 10 10 10 10

Survivors

Accidental deaths 2 1 1 1

Terminal sacrifice 8 10 10 9 10 9 9

Animals examined microscopically 10 10 1 2 10

Alimentary System

Liver (10) (10) (1) (10)

Inflammation, focal 3 (30%) 3 (30%)

Necrosis 1 (100%) 1 (10%)

Stomach, glandular (10) (10) (10)

Cyst 1 (10%)

Cardiovascular System

None

Endocrine System

Adrenal cortex (10) (10) (10)

Capsule, hyperplasia 9 (90%) 9 (90%) 9 (90%)

Adrenal medulla (10) (10) (10)

Angiectasis 1 (10%)

Thyroid gland (10) (10)

Ectopic tissue 1 (10%)

General Body System

None

Genital System

Clitoral gland (7) (9) (6)

Atrophy 4 (57%) 7 (78%) 3 (50%)

Ovary (10) (10) (1) (10)

Hemorrhage 1 (100%)

Hematopoietic System

Lymph node, mandibular (10) (10) (10)

Hemorrhage 1 (10%) 1 (10%) 1 (10%)

Spleen (10) (10) (10)

Hematopoietic cell proliferation 1 (10%) 3 (30%) 6 (60%)

Integumentary System

None
trans-1,2-Dichloroethylene, NTP TOX 55 A-9

TABLE A4

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 14-Week Feed Study 

of trans-1,2-Dichloroethylene

Untreated Vehicle

Control Control 3,125 ppm 6,250 ppm 12,500 ppm 25,000 ppm 50,000 ppm

Musculoskeletal System

None

Nervous System

None

Respiratory System

Lung (10) (10) (10)

Infiltration cellular, focal,

lymphocyte 2 (20%) 5 (50%)

Special Senses System

None

Urinary System

None

a Number of animals examined microscopically at the site and the number of animals with lesion
A-10 trans-1,2-Dichloroethylene, NTP TOX 55
B-1

APPENDIX B

CLINICAL PATHOLOGY RESULTS

TABLE B1 Hematology and Clinical Chemistry Data for Rats in the 14-Week Feed Study 

of trans-1,2-Dichloroethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-2

TABLE B2 Clinical Chemistry Data for Mice in the 14-Week Feed Study 

of trans-1,2-Dichloroethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-8
B-2 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE B1

Hematology and Clinical Chemistry Data for Rats in the 14-Week Feed Study of trans-1,2-Dichloroethylenea

Untreated Vehicle

Control Control 3,125 ppm 6,250 ppm 12,500 ppm 25,000 ppm 50,000 ppm

Male

Hematology

n

Day 5 10 10 10 10 10 10 10

Day 21 9 10 10 9 7 9 10

Week 14 10 10 10 9 10 10 10

Automated hematocrit (%)

Day 5 40.1 ± 1.0 38.8 ± 0.4 39.1 ± 0.6 39.5 ± 0.7 40.5 ± 0.9 39.9 ± 0.6 39.8 ± 0.6

Day 21 43.5 ± 0.5 43.3 ± 0.4 43.1 ± 0.5 42.4 ± 0.2 42.5 ± 0.6 40.9 ± 1.7 41.6 ± 0.4*

Week 14 41.9 ± 0.3 41.1 ± 0.3 41.4 ± 0.3 40.4 ± 0.4 39.9 ± 0.4* 39.8 ± 0.3* 39.0 ± 0.8*

Manual hematocrit (%)

Day 5 44.0 ± 1.1 42.8 ± 0.4 43.2 ± 0.6 43.5 ± 0.7 44.6 ± 0.8 44.1 ± 0.5 43.8 ± 0.6

Day 21 49.0 ± 0.9 49.1 ± 0.6 48.3 ± 0.8 47.8 ± 0.4 48.4 ± 0.5 46.1 ± 1.8 47.1 ± 0.6

Week 14 45.2 ± 0.3 45.0 ± 0.4 45.2 ± 0.2 44.2 ± 0.4 44.1 ± 0.4 43.5 ± 0.4* 43.2 ± 0.8*

Hemoglobin (g/dL)

Day 5 14.9 ± 0.3 14.6 ± 0.2 14.7 ± 0.2 14.8 ± 0.2 15.2 ± 0.3 14.9 ± 0.2 14.8 ± 0.1

Day 21 15.7 ± 0.2 15.9 ± 0.2 15.7 ± 0.3 15.4 ± 0.1 15.6 ± 0.1 14.9 ± 0.6* 15.1 ± 0.2**

Week 14 15.1 ± 0.1 14.8 ± 0.1 15.0 ± 0.1 14.7 ± 0.1 14.5 ± 0.1 14.5 ± 0.1 14.3 ± 0.3

Erythrocytes (106/μL)

Day 5 6.70 ± 0.17 6.50 ± 0.07 6.55 ± 0.09 6.61 ± 0.13 6.78 ± 0.15 6.68 ± 0.09 6.71 ± 0.09

Day 21 7.09 ± 0.09 7.13 ± 0.08 7.10 ± 0.09 6.91 ± 0.04* 6.96 ± 0.09 6.69 ± 0.25* 6.82 ± 0.07**

Week 14 8.13 ± 0.07 8.14 ± 0.08 8.17 ± 0.05 7.93 ± 0.10* 7.84 ± 0.09* 7.79 ± 0.08** 7.56 ± 0.15**

Reticulocytes (106/μL)

Day 5 0.19 ± 0.02 0.21 ± 0.02 0.23 ± 0.01 0.23 ± 0.01 0.24 ± 0.01 0.21 ± 0.02 0.22 ± 0.02

Day 21 0.16 ± 0.01 0.16 ± 0.01 0.15 ± 0.01 0.15 ± 0.01 0.18 ± 0.01 0.18 ± 0.01 0.17 ± 0.01

Week 14 0.10 ± 0.01 0.11 ± 0.01 0.11 ± 0.01 0.10 ± 0.01 0.12 ± 0.01 0.11 ± 0.01 0.11 ± 0.01

Nucleated erythrocytes (103/μL)

Day 5 0.90 ± 0.31 1.10 ± 0.35 1.50 ± 0.40 0.90 ± 0.23 0.90 ± 0.35 1.30 ± 0.21 1.10 ± 0.41

Day 21 0.33 ± 0.33 0.50 ± 0.22 0.20 ± 0.20 1.11 ± 0.31 1.29 ± 0.36 0.67 ± 0.24 0.40 ± 0.22

Week 14 0.30 ± 0.21 0.40 ± 0.27 0.10 ± 0.10 0.11 ± 0.11 0.40 ± 0.22 0.20 ± 0.13 0.20 ± 0.13

Mean cell volume (fL)

Day 5 59.8 ± 0.3 59.6 ± 0.2 59.7 ± 0.4 59.8 ± 0.3 59.8 ± 0.2 59.7 ± 0.3 59.3 ± 0.2

Day 21 61.3 ± 0.3 60.8 ± 0.3 60.8 ± 0.2 61.3 ± 0.3 61.1 ± 0.7 61.2 ± 0.5 61.0 ± 0.3

Week 14 51.5 ± 0.2 50.5 ± 0.1 50.7 ± 0.1 50.9 ± 0.1* 50.9 ± 0.1* 51.1 ± 0.2* 51.6 ± 0.2**

Mean cell hemoglobin (pg)

Day 5 22.3 ± 0.2 22.4 ± 0.1 22.4 ± 0.1 22.4 ± 0.1 22.4 ± 0.1 22.2 ± 0.2 22.0 ± 0.2

Day 21 22.2 ± 0.2 22.3 ± 0.1 22.2 ± 0.1 22.3 ± 0.1 22.4 ± 0.2 22.2 ± 0.2 22.1 ± 0.1

Week 14 18.5 ± 0.1 18.2 ± 0.1 18.3 ± 0.1 18.5 ± 0.2 18.5 ± 0.1 18.6 ± 0.2* 18.9 ± 0.1**

Mean cell hemoglobin concentration (g/dL)

Day 5 37.3 ± 0.2 37.6 ± 0.1 37.5 ± 0.2 37.5 ± 0.2 37.5 ± 0.3 37.2 ± 0.1 37.2 ± 0.2

Day 21 36.2 ± 0.2 36.6 ± 0.2 36.5 ± 0.2 36.4 ± 0.1 36.7 ± 0.3 36.4 ± 0.2 36.3 ± 0.1

Week 14 36.0 ± 0.1 36.0 ± 0.1 36.2 ± 0.2 36.3 ± 0.3 36.3 ± 0.1 36.5 ± 0.2 36.6 ± 0.2*

Platelets (103/μL)

Day 5 946.7 ± 27.7 923.0 ± 9.7 955.5 ± 13.7 887.0 ± 18.4 939.6 ± 24.5 913.8 ± 14.2 930.2 ± 10.4

Day 21 869.4 ± 11.5 863.5 ± 15.3 832.5 ± 14.1 848.6 ± 18.5 865.9 ± 11.9 883.9 ± 18.3 845.2 ± 16.2

Week 14 732.5 ± 12.8 727.1 ± 9.2 744.0 ± 10.5 713.7 ± 18.3 733.3 ± 14.5 724.1 ± 11.2 718.1 ± 17.9
trans-1,2-Dichloroethylene, NTP TOX 55 B-3

TABLE B1

Hematology and Clinical Chemistry Data for Rats in the 14-Week Feed Study of trans-1,2-Dichloroethylene

Untreated Vehicle

Control Control 3,125 ppm 6,250 ppm 12,500 ppm 25,000 ppm 50,000 ppm

Male (continued)

Hematology (continued)

n

Day 5 10 10 10 10 10 10 10

Day 21 9 10 10 9 7 9 10

Week 14 10 10 10 9 10 10 10

Leukocytes (103/μL)

Day 5 8.82 ± 0.59 9.42 ± 0.26 8.76 ± 0.62 8.91 ± 0.54 9.18 ± 0.66 9.46 ± 0.40 9.66 ± 0.36

Day 21 8.79 ± 0.36 8.72 ± 0.57 8.83 ± 0.38 9.10 ± 0.37 9.24 ± 0.43 7.91 ± 0.48 8.79 ± 0.33

Week 14 12.78 ± 0.31 12.77 ± 0.33 12.32 ± 0.40 11.93 ± 0.66 12.27 ± 0.43 11.89 ± 0.32 11.03 ± 0.66

Segmented neutrophils (103/μL)

Day 5 1.37 ± 0.13 1.35 ± 0.10 1.38 ± 0.12 1.33 ± 0.16 1.48 ± 0.09 1.34 ± 0.08 1.84 ± 0.19

Day 21 1.37 ± 0.11 1.10 ± 0.12 1.23 ± 0.22 1.10 ± 0.11 1.55 ± 0.39 0.98 ± 0.15 1.01 ± 0.11

Week 14 1.66 ± 0.12 1.55 ± 0.14 1.80 ± 0.14 1.71 ± 0.26 2.05 ± 0.26 1.63 ± 0.18 1.47 ± 0.13

Bands (103/μL)

Day 5 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

Day 21 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

Week 14 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

Metamyelocytes (103/μL)

Day 5 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000

Day 21 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000

Week 14 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000

Myelocytes (103/μL)

Day 5 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000

Day 21 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000

Week 14 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000

Lymphocytes (103/μL)

Day 5 7.28 ± 0.52 7.87 ± 0.20 7.23 ± 0.54 7.39 ± 0.48 7.62 ± 0.60 7.92 ± 0.38 7.71 ± 0.24

Day 21 7.29 ± 0.31 7.48 ± 0.52 7.48 ± 0.36 7.85 ± 0.31 7.59 ± 0.42 6.81 ± 0.39 7.66 ± 0.32

Week 14 10.84 ± 0.31 10.92 ± 0.39 10.17 ± 0.31 10.01 ± 0.48 9.94 ± 0.38 9.96 ± 0.32 9.36 ± 0.59

Atypical lymphocytes (103/μL)

Day 5 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

Day 21 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

Week 14 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

Monocytes (103/μL)

Day 5 0.18 ± 0.05 0.22 ± 0.06 0.18 ± 0.04 0.19 ± 0.03 0.11 ± 0.05 0.19 ± 0.04 0.15 ± 0.04

Day 21 0.15 ± 0.06 0.12 ± 0.04 0.11 ± 0.03 0.12 ± 0.03 0.13 ± 0.07 0.10 ± 0.04 0.11 ± 0.04

Week 14 0.16 ± 0.04 0.20 ± 0.07 0.27 ± 0.03 0.13 ± 0.05 0.16 ± 0.04 0.22 ± 0.05 0.10 ± 0.04

Basophils (103/μL)

Day 5 0.009 ± 0.009 0.010 ± 0.010 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000

Day 21 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000

Week 14 0.000 ± 0.000 0.000 ± 0.000 0.010 ± 0.010 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000

Eosinophils (103/μL)

Day 5 0.01 ± 0.01 0.00 ± 0.00 0.04 ± 0.02 0.00 ± 0.00 0.01 ± 0.01 0.02 ± 0.02 0.03 ± 0.02

Day 21 0.01 ± 0.01 0.03 ± 0.01 0.03 ± 0.01 0.05 ± 0.04 0.01 ± 0.01 0.02 ± 0.01 0.02 ± 0.01

Week 14 0.12 ± 0.04 0.10 ± 0.04 0.08 ± 0.03 0.08 ± 0.02 0.13 ± 0.04 0.09 ± 0.04 0.10 ± 0.04
B-4 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE B1

Hematology and Clinical Chemistry Data for Rats in the 14-Week Feed Study of trans-1,2-Dichloroethylene

Untreated Vehicle

Control Control 3,125 ppm 6,250 ppm 12,500 ppm 25,000 ppm 50,000 ppm

Male (continued)

Clinical Chemistry

n

Day 5 10 10 10 10 10 10 10

Day 21 9 10 10 9 7 9 10

Week 14 10 10 10 9 9 10 10

Urea nitrogen (mg/dL)

Day 5 17.1 ± 0.5 17.0 ± 0.3 16.9 ± 0.3 16.1 ± 0.4 17.0 ± 0.6 16.2 ± 0.4 15.1 ± 0.5**

Creatinine (mg/dL)

Day 5 0.57 ± 0.02 0.57 ± 0.02 0.54 ± 0.02 0.56 ± 0.02 0.56 ± 0.02 0.56 ± 0.02 0.56 ± 0.02

Day 21 0.67 ± 0.02 0.65 ± 0.02 0.67 ± 0.02 0.66 ± 0.02 0.66 ± 0.02 0.69 ± 0.02 0.67 ± 0.02

Week 14 0.69 ± 0.02 0.70 ± 0.00 0.70 ± 0.02 0.70 ± 0.02 0.69 ± 0.01 0.72 ± 0.01 0.71 ± 0.01

Total protein (g/dL)

Day 5 6.0 ± 0.1 5.8 ± 0.1 5.9 ± 0.1 5.9 ± 0.1 6.1 ± 0.1 5.8 ± 0.1 5.9 ± 0.1

Day 21 6.6 ± 0.1 6.6 ± 0.1 6.4 ± 0.1 6.5 ± 0.1 6.6 ± 0.1 6.6 ± 0.1 6.4 ± 0.1

Week 14 7.0 ± 0.0 7.1 ± 0.1 7.2 ± 0.1 7.1 ± 0.1 7.1 ± 0.0 7.1 ± 0.1 7.2 ± 0.1

Albumin (g/dL)

Day 5 4.3 ± 0.0 4.2 ± 0.0 4.1 ± 0.1 4.1 ± 0.1 4.3 ± 0.0 4.1 ± 0.0 4.2 ± 0.0

Day 21 4.8 ± 0.1 4.8 ± 0.1 4.6 ± 0.1 4.7 ± 0.0 4.7 ± 0.0 4.8 ± 0.0 4.6 ± 0.0*

Week 14 4.9 ± 0.0 4.9 ± 0.0 5.0 ± 0.1 4.9 ± 0.1 4.9 ± 0.1 5.0 ± 0.0 5.0 ± 0.1

Cholesterol (mg/dL)

Day 5 74 ± 2 72 ± 2 72 ± 2 72 ± 2 76 ± 2 74 ± 2 68 ± 2

Day 21 75 ± 2 68 ± 2 68 ± 2 69 ± 2 69 ± 2 67 ± 2 66 ± 1

Week 14 72 ± 2 77 ± 1 77 ± 2 73 ± 1 74 ± 1 74 ± 1 76 ± 3

Alanine aminotransferase (IU/L)

Day 5 39 ± 1 37 ± 2 37 ± 1 40 ± 1 37 ± 1 37 ± 3 34 ± 2

Day 21 43 ± 1 41 ± 2 40 ± 1 39 ± 2 38 ± 0 37 ± 2 36 ± 3**

Week 14 55 ± 2 56 ± 2 55 ± 2 59 ± 3 53 ± 3 51 ± 3 64 ± 2

Alkaline phosphatase (IU/L)

Day 5 652 ± 15 629 ± 12 647 ± 12 644 ± 11 620 ± 13 635 ± 17 645 ± 12

Day 21 499 ± 11 495 ± 5 483 ± 10 497 ± 8 472 ± 13 482 ± 11 464 ± 12

Week 14 236 ± 4 231 ± 6 231 ± 4 224 ± 4 222 ± 6 219 ± 6 225 ± 6

Creatine kinase (IU/L)

Day 21 219 ± 34 343 ± 83 246 ± 29 262 ± 42 290 ± 102 269 ± 48 246 ± 19

Week 14 155 ± 19 155 ± 24 158 ± 14 181 ± 29 151 ± 13 171 ± 27 203 ± 21

Sorbitol dehydrogenase (IU/L)

Day 5 12 ± 1 13 ± 1 14 ± 1 12 ± 1 11 ± 0 13 ± 0 11 ± 1

Day 21 15 ± 1 15 ± 1 17 ± 1 17 ± 1 14 ± 1 18 ± 1 16 ± 1

Week 14 11 ± 1 13 ± 1 13 ± 1 14 ± 2 12 ± 2 13 ± 2 14 ± 1

5N-Nucleotidase (IU/L)

Day 5 27 ± 1 26 ± 1 26 ± 1 26 ± 0 26 ± 0 25 ± 1 25 ± 0

Day 21 28 ± 1 27 ± 1 26 ± 0 27 ± 0 26 ± 1 26 ± 0 25 ± 0**

Week 14 30 ± 1 30 ± 1 29 ± 1 29 ± 0 29 ± 0 28 ± 0* 28 ± 1*

Bile acids (μmol/L)

Day 5 42.3 ± 5.4 38.6 ± 4.1 43.8 ± 4.8 48.4 ± 4.5 40.3 ± 3.6 53.8 ± 3.8 36.3 ± 3.6

Day 21 30.4 ± 3.1 35.9 ± 4.1 34.0 ± 4.3 37.0 ± 4.5 29.5 ± 4.3 40.4 ± 4.4 26.1 ± 2.8

Week 14 18.6 ± 2.3 27.7 ± 4.4 34.0 ± 5.3 26.6 ± 3.8 39.2 ± 7.1 22.9 ± 3.4 31.2 ± 6.6
trans-1,2-Dichloroethylene, NTP TOX 55 B-5

TABLE B1

Hematology and Clinical Chemistry Data for Rats in the 14-Week Feed Study of trans-1,2-Dichloroethylene

Untreated Vehicle

Control Control 3,125 ppm 6,250 ppm 12,500 ppm 25,000 ppm 50,000 ppm

Female

n 10 10 10 10 10 10 10

Hematology

Automated hematocrit (%)

Day 5 42.1 ± 0.7 41.2 ± 0.6 41.1 ± 0.6 40.3 ± 0.6 41.2 ± 0.5 41.0 ± 0.4 40.9 ± 0.5

Day 21 45.4 ± 0.4 45.4 ± 0.4 45.2 ± 0.3 44.0 ± 0.3 44.6 ± 0.3 43.7 ± 0.5 43.9 ± 0.8

Week 14 40.9 ± 0.4 41.9 ± 0.3 41.5 ± 0.5 41.2 ± 0.5 41.2 ± 0.2 40.4 ± 0.3** 40.1 ± 0.4**

Manual hematocrit (%)

Day 5 45.3 ± 0.6 44.8 ± 0.6 45.1 ± 0.6 43.7 ± 0.7 44.6 ± 0.6 44.3 ± 0.4 44.7 ± 0.5

Day 21 48.7 ± 0.4 49.2 ± 0.5 49.0 ± 0.4 47.5 ± 0.4 48.3 ± 0.4 47.6 ± 0.5 48.0 ± 0.6

Week 14 44.2 ± 0.5 44.9 ± 0.3 44.6 ± 0.4 44.1 ± 0.4 44.3 ± 0.2 43.6 ± 0.3** 43.3 ± 0.4**

Hemoglobin (g/dL)

Day 5 15.5 ± 0.2 15.1 ± 0.2 15.3 ± 0.2 14.9 ± 0.2 15.2 ± 0.2 15.1 ± 0.1 15.1 ± 0.2

Day 21 16.6 ± 0.1 16.6 ± 0.1 16.6 ± 0.2 16.1 ± 0.2* 16.4 ± 0.1 15.9 ± 0.2** 16.1 ± 0.2*

Week 14 15.0 ± 0.1 15.2 ± 0.1 15.2 ± 0.2 15.0 ± 0.1 15.0 ± 0.1 14.7 ± 0.1** 14.5 ± 0.1**

Erythrocytes (106/μL)

Day 5 7.14 ± 0.13 7.00 ± 0.13 7.00 ± 0.13 6.83 ± 0.13 6.95 ± 0.10 6.92 ± 0.09 6.92 ± 0.10

Day 21 7.46 ± 0.07 7.52 ± 0.08 7.48 ± 0.06 7.28 ± 0.07* 7.34 ± 0.06* 7.21 ± 0.08** 7.21 ± 0.14*

Week 14 7.41 ± 0.07 7.59 ± 0.06 7.58 ± 0.10 7.50 ± 0.08 7.49 ± 0.04 7.34 ± 0.05** 7.20 ± 0.08**

Reticulocytes (106/μL)

Day 5 0.17 ± 0.02 0.19 ± 0.01 0.17 ± 0.01 0.17 ± 0.01 0.16 ± 0.01 0.16 ± 0.01 0.17 ± 0.01

Day 21 0.09 ± 0.01 0.07 ± 0.01 0.07 ± 0.01 0.09 ± 0.00 0.08 ± 0.01 0.07 ± 0.01 0.08 ± 0.01

Week 14 0.06 ± 0.01 0.07 ± 0.01 0.08 ± 0.01 0.07 ± 0.01 0.07 ± 0.01 0.05 ± 0.01 0.08 ± 0.01

Nucleated erythrocytes (103/μL)

Day 5 1.10 ± 0.31 0.50 ± 0.34 0.70 ± 0.30 0.50 ± 0.31 0.20 ± 0.20 0.80 ± 0.29 0.80 ± 0.29

Day 21 0.10 ± 0.10 0.00 ± 0.00 0.30 ± 0.15 0.20 ± 0.20 0.20 ± 0.13 0.00 ± 0.00 0.20 ± 0.20

Week 14 0.40 ± 0.22 0.60 ± 0.31 0.60 ± 0.16 0.70 ± 0.30 0.60 ± 0.27 0.20 ± 0.13 0.40 ± 0.22

Mean cell volume (fL)

Day 5 59.0 ± 0.2 58.9 ± 0.3 58.8 ± 0.3 59.0 ± 0.3 59.2 ± 0.3 59.3 ± 0.2 59.1 ± 0.3

Day 21 60.8 ± 0.2 60.4 ± 0.2 60.4 ± 0.2 60.4 ± 0.2 60.8 ± 0.2 60.7 ± 0.2 60.9 ± 0.2

Week 14 55.2 ± 0.1 55.1 ± 0.1 54.8 ± 0.1 54.9 ± 0.1 55.0 ± 0.2 55.1 ± 0.1 55.7 ± 0.0**

Mean cell hemoglobin (pg)

Day 5 21.7 ± 0.2 21.6 ± 0.1 21.8 ± 0.1 21.9 ± 0.1 21.9 ± 0.1 21.8 ± 0.1 21.8 ± 0.1

Day 21 22.3 ± 0.2 22.0 ± 0.2 22.2 ± 0.1 22.1 ± 0.1 22.3 ± 0.1 22.1 ± 0.1 22.3 ± 0.2

Week 14 20.2 ± 0.1 20.1 ± 0.0 20.1 ± 0.1 20.0 ± 0.1b 20.0 ± 0.1 20.0 ± 0.1 20.1 ± 0.1

Mean cell hemoglobin concentration (g/dL)

Day 5 36.7 ± 0.2 36.7 ± 0.2 37.1 ± 0.2 37.1 ± 0.2 37.0 ± 0.1 36.8 ± 0.2 37.0 ± 0.2

Day 21 36.6 ± 0.2 36.5 ± 0.2 36.8 ± 0.2 36.6 ± 0.2 36.7 ± 0.2 36.5 ± 0.2 36.7 ± 0.2

Week 14 36.7 ± 0.1 36.4 ± 0.1 36.7 ± 0.1 36.5 ± 0.2 36.4 ± 0.1 36.3 ± 0.2 36.2 ± 0.2

Platelets (103/μL)

Day 5 853.4 ± 14.5 878.6 ± 12.2 854.0 ± 23.1 841.3 ± 18.1 828.5 ± 23.2 856.3 ± 21.2 844.2 ± 19.7

Day 21 804.9 ± 12.3 787.2 ± 20.2 772.8 ± 22.5 748.4 ± 22.0 779.7 ± 10.7 778.7 ± 19.7 769.0 ± 19.7

Week 14 662.0 ± 14.6 664.5 ± 11.5 655.0 ± 13.9 664.4 ± 11.8 681.6 ± 26.7 646.4 ± 9.5 663.9 ± 9.9
B-6 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE B1

Hematology and Clinical Chemistry Data for Rats in the 14-Week Feed Study of trans-1,2-Dichloroethylene

Untreated Vehicle

Control Control 3,125 ppm 6,250 ppm 12,500 ppm 25,000 ppm 50,000 ppm

Female (continued)

n 10 10 10 10 10 10 10

Hematology (continued)

Leukocytes (103/μL)

Day 5 9.43 ± 0.39 9.75 ± 0.42 9.20 ± 0.33 9.06 ± 0.32 9.35 ± 0.25 9.89 ± 0.51 9.22 ± 0.46

Day 21 9.64 ± 0.37 9.53 ± 0.40 9.49 ± 0.42 9.31 ± 0.40 8.87 ± 0.51 8.87 ± 0.36 8.90 ± 0.26

Week 14 10.28 ± 0.38 10.21 ± 0.69 10.01 ± 0.33 9.85 ± 0.46 8.98 ± 0.36 9.32 ± 0.59 9.73 ± 0.49

Segmented neutrophils (103/μL)

Day 5 1.11 ± 0.11 1.05 ± 0.17 1.21 ± 0.13 1.29 ± 0.17 1.05 ± 0.13 1.13 ± 0.26 1.08 ± 0.14

Day 21 1.09 ± 0.10 1.36 ± 0.22 1.35 ± 0.19 1.31 ± 0.20 1.09 ± 0.17 1.04 ± 0.15 0.93 ± 0.10

Week 14 1.60 ± 0.16 1.97 ± 0.50 1.55 ± 0.16 1.63 ± 0.17 1.49 ± 0.16 1.25 ± 0.11 1.74 ± 0.21

Bands (103/μL)

Day 5 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

Day 21 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

Week 14 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

Metamyelocytes (103/μL)

Day 5 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000

Day 21 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000

Week 14 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000

Myelocytes (103/μL)

Day 5 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000

Day 21 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000

Week 14 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000

Lymphocytes (103/μL)

Day 5 8.27 ± 0.38 8.54 ± 0.37 7.91 ± 0.36 7.64 ± 0.25 8.04 ± 0.26 8.57 ± 0.42 8.01 ± 0.43

Day 21 8.42 ± 0.34 8.05 ± 0.33 8.03 ± 0.35 7.90 ± 0.37 7.69 ± 0.38 7.72 ± 0.22 7.77 ± 0.29

Week 14 8.52 ± 0.35 8.04 ± 0.29 8.19 ± 0.32 8.07 ± 0.33 7.28 ± 0.34 7.92 ± 0.53 7.79 ± 0.43

Atypical lymphocytes (103/μL)

Day 5 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

Day 21 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

Week 14 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

Monocytes (103/μL)

Day 5 0.07 ± 0.02 0.17 ± 0.04 0.06 ± 0.02 0.15 ± 0.04 0.24 ± 0.04 0.17 ± 0.04 0.13 ± 0.06

Day 21 0.10 ± 0.03 0.09 ± 0.03 0.06 ± 0.02 0.07 ± 0.02 0.05 ± 0.02 0.08 ± 0.04 0.13 ± 0.04

Week 14 0.08 ± 0.03 0.14 ± 0.04 0.20 ± 0.06 0.10 ± 0.02 0.11 ± 0.04 0.11 ± 0.04 0.15 ± 0.05

Basophils (103/μL)

Day 5 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000

Day 21 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000

Week 14 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000

Eosinophils (103/μL)

Day 5 0.01 ± 0.01 0.02 ± 0.02 0.05 ± 0.02 0.01 ± 0.01 0.02 ± 0.01 0.02 ± 0.02 0.04 ± 0.02

Day 21 0.04 ± 0.02 0.03 ± 0.02 0.05 ± 0.02 0.03 ± 0.01 0.04 ± 0.02 0.03 ± 0.02 0.08 ± 0.03

Week 14 0.09 ± 0.03 0.07 ± 0.03 0.08 ± 0.02 0.06 ± 0.03 0.10 ± 0.03 0.05 ± 0.02 0.04 ± 0.02
trans-1,2-Dichloroethylene, NTP TOX 55 B-7

TABLE B1

Hematology and Clinical Chemistry Data for Rats in the 14-Week Feed Study of trans-1,2-Dichloroethylene

Untreated Vehicle

Control Control 3,125 ppm 6,250 ppm 12,500 ppm 25,000 ppm 50,000 ppm

Female (continued)

n 10 10 10 10 10 10 10

Clinical Chemistry

Urea nitrogen (mg/dL)

Day 5 21.1 ± 0.5 19.1 ± 0.7 20.1 ± 0.7 18.7 ± 1.1 18.6 ± 0.6 18.9 ± 0.7 19.0 ± 0.7

Creatinine (mg/dL)

Day 5 0.60 ± 0.02 0.60 ± 0.00 0.59 ± 0.01 0.62 ± 0.03 0.60 ± 0.02 0.60 ± 0.00 0.60 ± 0.02

Day 21 0.65 ± 0.02 0.67 ± 0.02 0.64 ± 0.02 0.67 ± 0.02 0.68 ± 0.01 0.66 ± 0.02 0.67 ± 0.03

Week 14 0.71 ± 0.01 0.72 ± 0.02 0.71 ± 0.01 0.72 ± 0.01 0.71 ± 0.02 0.70 ± 0.02 0.70 ± 0.02

Total protein (g/dL)

Day 5 5.8 ± 0.1 5.8 ± 0.1 5.6 ± 0.1 5.7 ± 0.1 5.7 ± 0.1b 5.7 ± 0.1 5.8 ± 0.1

Day 21 6.2 ± 0.1 6.1 ± 0.1 6.3 ± 0.1 6.2 ± 0.1 6.2 ± 0.1 6.0 ± 0.1 6.1 ± 0.1

Week 14 7.0 ± 0.1 7.2 ± 0.1 6.9 ± 0.1 7.1 ± 0.1 6.8 ± 0.1 6.8 ± 0.1 6.7 ± 0.1**

Albumin (g/dL)

Day 5 4.3 ± 0.1 4.3 ± 0.1 4.3 ± 0.1 4.2 ± 0.1 4.3 ± 0.1 4.3 ± 0.0 4.3 ± 0.0

Day 21 4.7 ± 0.0 4.8 ± 0.1 4.8 ± 0.0 4.7 ± 0.1 4.8 ± 0.0 4.6 ± 0.1 4.7 ± 0.1

Week 14 5.0 ± 0.1 5.0 ± 0.1 4.9 ± 0.1 5.0 ± 0.0 4.9 ± 0.1 4.9 ± 0.1 4.7 ± 0.0*

Cholesterol (mg/dL)

Day 5 98 ± 3 95 ± 3 100 ± 3 93 ± 3 96 ± 2 96 ± 2 96 ± 3

Day 21 94 ± 2 92 ± 2 95 ± 3 96 ± 3 94 ± 2 97 ± 2 97 ± 2

Week 14 94 ± 3 92 ± 2 95 ± 3 101 ± 2 97 ± 4 94 ± 2 94 ± 3

Alanine aminotransferase (IU/L)

Day 5 37 ± 1 33 ± 1 34 ± 1 31 ± 1 33 ± 1 33 ± 1 33 ± 2

Day 21 34 ± 1 34 ± 2 34 ± 2 33 ± 1 33 ± 2 33 ± 1 34 ± 2

Week 14 42 ± 2 46 ± 4 44 ± 3 36 ± 2 34 ± 2* 38 ± 3 42 ± 2

Alkaline phosphatase (IU/L)

Day 5 530 ± 11 502 ± 7 504 ± 6 494 ± 8 493 ± 7 502 ± 11 476 ± 11

Day 21 404 ± 10 383 ± 8 377 ± 5 367 ± 8 352 ± 7** 356 ± 6* 353 ± 6**

Week 14 191 ± 5 191 ± 10 198 ± 6 203 ± 5 206 ± 4 187 ± 6 176 ± 3

Creatine kinase (IU/L)

Day 21 273 ± 50 257 ± 33b 358 ± 61 248 ± 31 378 ± 85 292 ± 52 274 ± 48

Week 14 217 ± 20 172 ± 15 218 ± 22 201 ± 17 181 ± 19 196 ± 17 186 ± 11

Sorbitol dehydrogenase (IU/L)

Day 5 6 ± 0 6 ± 1 5 ± 0 5 ± 0 5 ± 0 5 ± 1 6 ± 1

Day 21 20 ± 1 21 ± 1 24 ± 1 20 ± 1 19 ± 1 21 ± 1 20 ± 2

Week 14 18 ± 1 19 ± 2 19 ± 1 17 ± 1 17 ± 1 17 ± 1 18 ± 1

5N-Nucleotidase (IU/L)

Day 5 34 ± 1 33 ± 1 36 ± 1 33 ± 1 34 ± 1 32 ± 0 32 ± 1

Day 21 35 ± 1 37 ± 1 37 ± 1 35 ± 1 36 ± 1 34 ± 1 33 ± 1**

Week 14 32 ± 1 34 ± 1 31 ± 1* 32 ± 1* 34 ± 1 32 ± 1 29 ± 1**

Bile acids (μmol/L)

Day 5 27.9 ± 3.5 26.8 ± 2.5 22.6 ± 2.1 28.0 ± 4.7 29.2 ± 2.4 33.6 ± 4.0 21.9 ± 3.0

Day 21 32.2 ± 3.4 27.1 ± 3.5 27.6 ± 2.7 28.5 ± 2.9 35.6 ± 2.9 33.5 ± 3.4 32.1 ± 4.4

Week 14 42.1 ± 4.4 59.2 ± 9.5 64.3 ± 9.5 52.3 ± 6.4 58.7 ± 7.9 55.4 ± 6.1 59.8 ± 6.6

* Significantly different (P#0.05) from the vehicle control group by Dunn’s or Shirley’s test

** Significantly different (P#0.01) from the vehicle control group by Shirley’s test a Mean ± standard error. Statistical tests were performed on unrounded data.

b n=9
B-8 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE B2

Clinical Chemistry Data for Mice in the 14-Week Feed Study of trans-1,2-Dichloroethylenea

Untreated Vehicle

Control Control 3,125 ppm 6,250 ppm 12,500 ppm 25,000 ppm 50,000 ppm

n 10 10 10 10 10 10 10

Male

Creatinine (mg/dL) 0.40 ± 0.02 0.40 ± 0.00 0.40 ± 0.02 0.41 ± 0.01 0.41 ± 0.01 0.41 ± 0.01 0.40 ± 0.00

Total protein (g/dL) 5.7 ± 0.1 5.6 ± 0.1 5.6 ± 0.0 5.6 ± 0.0 5.6 ± 0.1 5.5 ± 0.1 5.5 ± 0.0

Albumin (g/dL) 4.1 ± 0.0 4.1 ± 0.0 4.1 ± 0.0 4.1 ± 0.0 4.1 ± 0.0 4.0 ± 0.1 4.1 ± 0.1

Cholesterol (mg/dL) 141 ± 7 135 ± 7 139 ± 3 135 ± 3 140 ± 7 128 ± 4 128 ± 3

Alanine

 aminotransferase (IU/L) 47 ± 8 35 ± 9 34 ± 9 40 ± 7 46 ± 6 39 ± 7 35 ± 7

Alkaline

 phosphatase (IU/L) 86 ± 3 83 ± 3 88 ± 2 86 ± 2 86 ± 2 90 ± 3 91 ± 2

Creatine kinase (IU/L) 750 ± 228 580 ± 131 653 ± 216 631 ± 174 389 ± 96 269 ± 32 616 ± 156

Sorbitol

dehydrogenase (IU/L) 46 ± 1 45 ± 2 47 ± 2 52 ± 1* 51 ± 3 45 ± 1 51 ± 2

5N-Nucleotidase (IU/L) 18 ± 0 17 ± 1 17 ± 0 17 ± 0 16 ± 0 15 ± 0* 15 ± 0*

Bile acids (μmol/L) 15.6 ± 0.6 16.6 ± 0.8 16.7 ± 0.7 16.6 ± 0.8 17.5 ± 0.8 16.1 ± 0.7 16.0 ± 1.0

Female

Creatinine (mg/dL) 0.46 ± 0.02 0.45 ± 0.02 0.49 ± 0.01 0.48 ± 0.01 0.46 ± 0.02 0.45 ± 0.02 0.46 ± 0.02

Total protein (g/dL) 5.5 ± 0.1 5.7 ± 0.1 5.6 ± 0.1 5.5 ± 0.0 5.8 ± 0.0 5.7 ± 0.1 5.7 ± 0.1

Albumin (g/dL) 4.5 ± 0.0 4.5 ± 0.1 4.4 ± 0.0 4.4 ± 0.0 4.6 ± 0.0 4.6 ± 0.0 4.6 ± 0.1

Cholesterol (mg/dL) 112 ± 3 110 ± 4 115 ± 2 112 ± 3 112 ± 2 114 ± 2 115 ± 3

Alanine

 aminotransferase (IU/L) 29 ± 2 28 ± 3 37 ± 3 55 ± 22 57 ± 14* 45 ± 12 50 ± 14

Alkaline

 phosphatase (IU/L) 132 ± 4 142 ± 5 143 ± 8 124 ± 6 149 ± 3 137 ± 4 144 ± 6

Creatine kinase (IU/L) 356 ± 56 330 ± 55 372 ± 27 508 ± 73 252 ± 39 295 ± 34 455 ± 86

Sorbitol

dehydrogenase (IU/L) 41 ± 1 39 ± 1 42 ± 2 45 ± 2* 44 ± 3b 43 ± 2 44 ± 2

5N-Nucleotidase (IU/L) 53 ± 2 53 ± 2 56 ± 2 55 ± 4 56 ± 2 56 ± 2 54 ± 2

Bile acids (μmol/L) 14.3 ± 0.4 14.8 ± 0.5 17.5 ± 0.6* 15.6 ± 0.7 18.3 ± 2.2 15.1 ± 0.7 16.6 ± 0.7

* Significantly different (P#0.05) from the vehicle control group by Dunn’s or Shirley’s test a Mean ± standard error. Statistical tests were performed on unrounded data.

b n=9
C-1

APPENDIX C

ORGAN WEIGHTS

AND ORGAN-WEIGHT-TO-BODY-WEIGHT RATIOS

TABLE C1 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats 

in the 14-Week Feed Study of trans-1,2-Dichloroethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-2

TABLE C2 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice

in the 14-Week Feed Study of trans-1,2-Dichloroethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-3
C-2 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE C1

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 14-Week Feed Study

of trans-1,2-Dichloroethylenea

Untreated Vehicle

Control Control 3,125 ppm 6,250 ppm 12,500 ppm 25,000 ppm 50,000 ppm

n 10 10 10 10 10 10 10

Male

Necropsy body wt 362 ± 7 360 ± 6 365 ± 5 361 ± 3 357 ± 5 350 ± 6 339 ± 4**

Heart

Absolute 1.056 ± 0.022 1.019 ± 0.018 1.010 ± 0.014 1.024 ± 0.017 0.997 ± 0.021 0.966 ± 0.019 0.968 ± 0.017

Relative 0.292 ± 0.004 0.283 ± 0.004 0.277 ± 0.003 0.284 ± 0.003 0.279 ± 0.005 0.277 ± 0.005 0.285 ± 0.004

R. Kidney

Absolute 1.193 ± 0.028 1.258 ± 0.029 1.254 ± 0.034 1.244 ± 0.026 1.234 ± 0.016 1.167 ± 0.029* 1.172 ± 0.023*

Relative 0.330 ± 0.004 0.350 ± 0.006 0.344 ± 0.006 0.345 ± 0.005 0.345 ± 0.002 0.333 ± 0.004 0.346 ± 0.007

Liver

Absolute 12.376 ± 0.264 12.488 ± 0.389 12.901 ± 0.230 13.210 ± 0.405 12.605 ± 0.314 12.221 ± 0.310 12.326 ± 0.219

Relative 3.427 ± 0.063 3.465 ± 0.058 3.538 ± 0.032 3.658 ± 0.099 3.524 ± 0.050 3.492 ± 0.048 3.634 ± 0.056

Lung

Absolute 1.612 ± 0.047 1.571 ± 0.036 1.719 ± 0.057 1.659 ± 0.087 1.560 ± 0.036 1.467 ± 0.034 1.466 ± 0.031

Relative 0.447 ± 0.012 0.437 ± 0.010 0.471 ± 0.011 0.459 ± 0.022 0.437 ± 0.009 0.420 ± 0.011 0.432 ± 0.009

R. Testis

Absolute 1.452 ± 0.031 1.440 ± 0.034 1.477 ± 0.030 1.361 ± 0.063 1.576 ± 0.104 1.494 ± 0.026 1.460 ± 0.014

Relative 0.402 ± 0.006 0.401 ± 0.011 0.405 ± 0.007 0.378 ± 0.018 0.441 ± 0.027 0.428 ± 0.007 0.431 ± 0.006

Thymus

Absolute 0.362 ± 0.016 0.346 ± 0.010 0.381 ± 0.013 0.363 ± 0.015 0.349 ± 0.013 0.350 ± 0.014 0.331 ± 0.018

Relative 0.100 ± 0.004 0.097 ± 0.004 0.104 ± 0.003 0.100 ± 0.004 0.098 ± 0.004 0.100 ± 0.004 0.097 ± 0.005

Female

Necropsy body wt 196 ± 3 190 ± 4 198 ± 3 203 ± 2* 198 ± 3 196 ± 3 191 ± 2

Heart

Absolute 0.651 ± 0.015 0.644 ± 0.014 0.643 ± 0.013 0.658 ± 0.009 0.638 ± 0.011 0.640 ± 0.013 0.648 ± 0.012

Relative 0.332 ± 0.005 0.339 ± 0.005 0.325 ± 0.005 0.324 ± 0.005 0.322 ± 0.004 0.327 ± 0.007 0.338 ± 0.004

R. Kidney

Absolute 0.672 ± 0.013 0.662 ± 0.010 0.662 ± 0.009 0.707 ± 0.009 0.666 ± 0.015 0.657 ± 0.016 0.683 ± 0.016

Relative 0.343 ± 0.005 0.349 ± 0.006 0.335 ± 0.003 0.349 ± 0.004 0.336 ± 0.004 0.335 ± 0.004 0.356 ± 0.005

Liver

Absolute 5.895 ± 0.148 5.579 ± 0.130 6.011 ± 0.126 6.536 ± 0.145** 6.133 ± 0.108* 6.137 ± 0.146* 6.160 ± 0.139*

Relative 3.005 ± 0.052 2.937 ± 0.038 3.040 ± 0.052 3.220 ± 0.066** 3.100 ± 0.051** 3.132 ± 0.052** 3.216 ± 0.051**

Lung

Absolute 1.104 ± 0.029 1.090 ± 0.028 1.056 ± 0.040 1.064 ± 0.019 1.051 ± 0.030 1.099 ± 0.033 1.062 ± 0.021

Relative 0.563 ± 0.011 0.574 ± 0.011 0.534 ± 0.020 0.524 ± 0.009* 0.530 ± 0.009 0.561 ± 0.014 0.555 ± 0.008

Thymus

Absolute 0.275 ± 0.012 0.253 ± 0.012 0.269 ± 0.006 0.283 ± 0.006 0.287 ± 0.008* 0.266 ± 0.009 0.268 ± 0.008

Relative 0.140 ± 0.006 0.133 ± 0.004 0.136 ± 0.003 0.140 ± 0.002 0.145 ± 0.004 0.136 ± 0.005 0.140 ± 0.004

* Significantly different (P#0.05) from the vehicle control group by Williams’ or Dunnett’s test

** P#0.01 a Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as g organ

weight/g body weight as a percentage (mean ± standard error).
trans-1,2-Dichloroethylene, NTP TOX 55 C-3

TABLE C2

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 14-Week Feed Study

of trans-1,2-Dichloroethylenea

Untreated Vehicle

Control Control 3,125 ppm 6,250 ppm 12,500 ppm 25,000 ppm 50,000 ppm

n 10 10 10 10 10 10 10

Male

Necropsy body wt 30.6 ± 0.9 30.3 ± 0.6 29.8 ± 0.4 30.5 ± 0.6 29.2 ± 1.0 29.0 ± 0.5 28.2 ± 0.5*

Heart

Absolute 0.145 ± 0.003 0.146 ± 0.003 0.144 ± 0.002 0.144 ± 0.005 0.140 ± 0.003 0.147 ± 0.003 0.138 ± 0.002

Relative 0.473 ± 0.009 0.484 ± 0.008 0.482 ± 0.007 0.473 ± 0.011 0.482 ± 0.008 0.505 ± 0.008 0.489 ± 0.004

R. Kidney

Absolute 0.273 ± 0.009 0.275 ± 0.006 0.287 ± 0.009 0.291 ± 0.009 0.263 ± 0.011 0.270 ± 0.005 0.261 ± 0.005

Relative 0.894 ± 0.028 0.908 ± 0.012 0.963 ± 0.024 0.953 ± 0.017 0.902 ± 0.032 0.933 ± 0.016 0.928 ± 0.010

Liver

Absolute 1.397 ± 0.054 1.317 ± 0.027 1.357 ± 0.039 1.403 ± 0.043 1.380 ± 0.040 1.371 ± 0.023 1.401 ± 0.033

Relative 4.554 ± 0.076 4.347 ± 0.056 4.552 ± 0.113 4.597 ± 0.115 4.745 ± 0.084** 4.736 ± 0.079** 4.979 ± 0.111**

Lung

Absolute 0.195 ± 0.008 0.197 ± 0.012 0.193 ± 0.006 0.204 ± 0.009 0.187 ± 0.004 0.201 ± 0.010 0.201 ± 0.006

Relative 0.641 ± 0.030 0.647 ± 0.033 0.648 ± 0.018 0.670 ± 0.030 0.648 ± 0.021 0.699 ± 0.045 0.715 ± 0.018

R. Testis

Absolute 0.120 ± 0.002 0.120 ± 0.004 0.121 ± 0.007 0.117 ± 0.006 0.117 ± 0.003 0.123 ± 0.003 0.124 ± 0.002

Relative 0.396 ± 0.010 0.397 ± 0.014 0.407 ± 0.022 0.387 ± 0.022 0.405 ± 0.011 0.425 ± 0.010 0.439 ± 0.007

Thymus

Absolute 0.042 ± 0.003 0.039 ± 0.002 0.038 ± 0.002 0.039 ± 0.003 0.035 ± 0.002 0.040 ± 0.002 0.036 ± 0.002

Relative 0.139 ± 0.009 0.128 ± 0.005 0.126 ± 0.006 0.126 ± 0.009 0.119 ± 0.005 0.140 ± 0.008 0.127 ± 0.006

Female

Necropsy body wt 24.1 ± 0.5 23.3 ± 0.3 23.8 ± 0.5 25.0 ± 0.6 22.0 ± 0.4 22.4 ± 0.5 21.6 ± 0.4*

Heart

Absolute 0.125 ± 0.003 0.120 ± 0.005 0.119 ± 0.002 0.130 ± 0.003 0.117 ± 0.002 0.112 ± 0.002 0.113 ± 0.001

Relative 0.518 ± 0.009 0.517 ± 0.018 0.499 ± 0.006 0.520 ± 0.013 0.530 ± 0.005 0.502 ± 0.010 0.527 ± 0.011

R. Kidney

Absolute 0.183 ± 0.005 0.175 ± 0.004 0.172 ± 0.005 0.187 ± 0.005 0.167 ± 0.003 0.164 ± 0.004 0.162 ± 0.004*

Relative 0.760 ± 0.012 0.754 ± 0.018 0.723 ± 0.009 0.747 ± 0.010 0.760 ± 0.011 0.732 ± 0.016 0.750 ± 0.015

Liver

Absolute 1.062 ± 0.109 1.076 ± 0.025 1.128 ± 0.035 1.244 ± 0.054** 1.058 ± 0.019 1.141 ± 0.028 1.107 ± 0.028

Relative 4.370 ± 0.429 4.621 ± 0.070 4.738 ± 0.068 4.970 ± 0.127 4.813 ± 0.050 5.115 ± 0.139** 5.117 ± 0.080**

Lung

Absolute 0.199 ± 0.014 0.181 ± 0.010 0.180 ± 0.004 0.189 ± 0.007 0.170 ± 0.004 0.169 ± 0.004 0.172 ± 0.005

Relative 0.832 ± 0.066 0.773 ± 0.034 0.757 ± 0.015 0.758 ± 0.027 0.772 ± 0.012 0.758 ± 0.022 0.798 ± 0.027

Thymus

Absolute 0.042 ± 0.003 0.040 ± 0.002 0.051 ± 0.004** 0.048 ± 0.001 0.041 ± 0.002 0.042 ± 0.002 0.043 ± 0.002

Relative 0.173 ± 0.012 0.172 ± 0.009 0.215 ± 0.014* 0.193 ± 0.004 0.186 ± 0.009 0.187 ± 0.014 0.199 ± 0.008

* Significantly different (P#0.05) from the vehicle control group by Williams’ or Dunnett’s test

** P#0.01 a Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as g organ

weight/g body weight as a percentage (mean ± standard error).
C-4 trans-1,2-Dichloroethylene, NTP TOX 55
D-1

APPENDIX D

REPRODUCTIVE TISSUE EVALUATIONS

AND ESTROUS CYCLE CHARACTERIZATION

TABLE D1 Summary of Reproductive Tissue Evaluations for Male Rats 

in the 14-Week Feed Study of trans-1,2-Dichloroethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2

TABLE D2 Estrous Cycle Characterization for Female Rats 

in the 14-Week Feed Study of trans-1,2-Dichloroethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2

TABLE D3 Summary of Reproductive Tissue Evaluations for Male Mice 

in the 14-Week Feed Study of trans-1,2-Dichloroethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-3

TABLE D4 Estrous Cycle Characterization for Female Mice

in the 14-Week Feed Study of trans-1,2-Dichloroethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-3
D-2 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE D1

Summary of Reproductive Tissue Evaluations for Male Rats in the 14-Week Feed Study

of trans-1,2-Dichloroethylenea

Untreated Vehicle

Control Control 12,500 ppm 25,000 ppm 50,000 ppm

n 10 10 10 10 10

Weights (g)

Necropsy body wt 361 ± 7 360 ± 6 357 ± 5 350 ± 6 339 ± 4*

L. Cauda epididymis 0.1677 ± 0.0058 0.1675 ± 0.0054 0.1719 ± 0.0053 0.1673 ± 0.0025 0.1598 ± 0.0034

L. Epididymis 0.4619 ± 0.0089 0.4737 ± 0.0126 0.4696 ± 0.0095 0.4701 ± 0.0093 0.4554 ± 0.0051

L. Testis 1.5170 ± 0.0244 1.4782 ± 0.0223 1.5200 ± 0.0244b 1.5303 ± 0.0218 1.5039 ± 0.0165

Spermatid measurements

Spermatid heads (107/g testis) 10.413 ± 0.421 10.535 ± 0.247 9.913 ± 0.194b 9.735 ± 0.255* 10.258 ± 0.219

Spermatid heads (107/testis) 15.745 ± 0.538 15.540 ± 0.267 15.180 ± 0.259 14.885 ± 0.401 15.420 ± 0.330

Spermatid count

(mean/10-4 mL suspension) 78.725 ± 2.689 77.700 ± 1.336 75.900 ± 1.294 74.425 ± 2.007 77.100 ± 1.650

Epididymal spermatozoal measurements

Motility (%) 87.60 ± 0.56 87.82 ± 0.54 87.46 ± 0.48 87.69 ± 0.37 86.71 ± 0.48

Concentration

 (106/g cauda epididymal tissue) 382 ± 24 364 ± 43 405 ± 17 410 ± 11 387 ± 24

* Significantly different (P#0.05) from the vehicle control group by Dunnett’s test (body weight) or Dunn’s test (spermatid heads

per g testis) a Data are presented as mean ± standard error. Differences from the vehicle control group are not significant by Dunnett’s test

(tissue weights) or Dunn’s test (spermatid heads per testis, spermatid count, and epididymal spermatozoal measurements).

b n=9

TABLE D2

Estrous Cycle Characterization for Female Rats in the 14-Week Feed Study of trans-1,2-Dichloroethylenea

Untreated Vehicle

Control Control 12,500 ppm 25,000 ppm 50,000 ppm

n 10 10 10 10 10

Necropsy body wt (g) 196 ± 3 190 ± 4 198 ± 3 196 ± 3 191 ± 2

Estrous cycle length (days) 5.000 ± 0.129 5.000 ± 0.000 5.000 ± 0.075 5.150 ± 0.150 5.667 ± 0.373b

Estrous stages (% of cycle)

Diestrus 39.2 40.0 41.7 40.0 46.7

Proestrus 18.3 15.8 20.0 19.2 15.8

Estrus 25.0 24.2 20.0 21.7 20.0

Metestrus 17.5 20.0 18.3 19.2 17.5

a Necropsy body weight and estrous cycle length data are presented as mean ± standard error. Differences from the vehicle control

group are not significant by Dunnett’s test (body weight) or Dunn’s test (estrous cycle length). By multivariate analysis of

variance, exposed females do not differ significantly from the vehicle control females in the relative length of time spent in the

estrous stages.

b Estrous cycle was longer than 12 days or unclear in 1 of 10 animals.
trans-1,2-Dichloroethylene, NTP TOX 55 D-3

TABLE D3

Summary of Reproductive Tissue Evaluations for Male Mice in the 14-Week Feed Study

of trans-1,2-Dichloroethylenea

Untreated Vehicle

Control Control 12,500 ppm 25,000 ppm 50,000 ppm

n 10 10 10 10 10

Weights (g)

Necropsy body wt 30.6 ± 0.8 30.3 ± 0.6 29.2 ± 1.0 29.0 ± 0.5 28.2 ± 0.5

L. Cauda epididymis 0.0214 ± 0.0018 0.0223 ± 0.0010 0.0201 ± 0.0018 0.0215 ± 0.0018 0.0206 ± 0.0017

L. Epididymis 0.0515 ± 0.0023 0.0515 ± 0.0017 0.0521 ± 0.0037 0.0500 ± 0.0030 0.0507 ± 0.0028

L. Testis 0.1175 ± 0.0026 0.1184 ± 0.0041 0.1133 ± 0.0025 0.1206 ± 0.0029 0.1207 ± 0.0033

Spermatid measurements

Spermatid heads (107/g testis) 22.614 ± 0.591 21.614 ± 0.528 23.267 ± 0.513 21.886 ± 0.443 22.289 ± 0.686

Spermatid heads (107/testis) 2.645 ± 0.036 2.546 ± 0.072 2.631 ± 0.068 2.634 ± 0.063 2.674 ± 0.050

Spermatid count

(mean/10-4 mL suspension) 82.675 ± 1.083 79.575 ± 2.238 82.250 ± 2.092 82.325 ± 1.981 83.550 ± 1.550

Epididymal spermatozoal measurements

Motility (%) 86.81 ± 1.05 85.99 ± 1.14 85.28 ± 0.86 76.12 ± 8.49 86.35 ± 1.07

Concentration

 (106/g cauda epididymal tissue) 593 ± 83 546 ± 77 395 ± 62 549 ± 108 574 ± 59

a Data are presented as mean ± standard error. Differences from the vehicle control group are not significant by Dunnett’s test

(body and tissue weights) or Dunn’s test (spermatid and epididymal spermatozoal measurements).

TABLE D4

Estrous Cycle Characterization for Female Mice in the 14-Week Feed Study of trans-1,2-Dichloroethylenea

Untreated Vehicle

Control Control 12,500 ppm 25,000 ppm 50,000 ppm

n 10 10 10 10 10

Necropsy body wt (g) 24.5 ± 0.3 23.3 ± 0.3 22.0 ± 0.4* 22.1 ± 0.5* 21.6 ± 0.4**

Estrous cycle length (days) 4.450 ± 0.398 4.000 ± 0.144b 4.800 ± 0.490 4.500 ± 0.565b 4.120 ± 0.088

Estrous stages (% of cycle)

Diestrus 35.0 37.5 32.5 40.0 25.0

Proestrus 14.2 18.3 12.5 10.8 13.3

Estrus 30.0 28.3 35.0 29.2 37.5

Metestrus 20.8 15.8 20.0 20.0 24.2

* Significantly different (P#0.05) from the vehicle control group by Williams’ test

** P#0.01 a Necropsy body weight and estrous cycle length data are presented as mean ± standard error. Differences from the vehicle control

group for estrous cycle length are not significant by Dunn’s test. By multivariate analysis of variance, exposed females do not

differ significantly from the vehicle control females in the relative length of time spent in the estrous stages.

b Estrous cycle was longer than 12 days or unclear in 1 of 10 animals.
D-4 trans-1,2-Dichloroethylene, NTP TOX 55
E-1

APPENDIX E

GENETIC TOXICOLOGY

TABLE E1 Mutagenicity of cis-1,2-Dichloroethylene in Salmonella typhimurium . . . . . . . . . . . . . . . . . . . . . . . E-2

TABLE E2 Mutagenicity of trans-1,2-Dichloroethylene in Salmonella typhimurium . . . . . . . . . . . . . . . . . . . . . E-5

TABLE E3 Mutagenicity of cis,trans-1,2-Dichloroethylene in Salmonella typhimurium . . . . . . . . . . . . . . . . . . E-6

TABLE E4 Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells 

by cis-1,2-Dichloroethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-7

TABLE E5 Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells 

by trans-1,2-Dichloroethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-9

TABLE E6 Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells 

by cis,trans-1,2-Dichloroethylene ....................................................... E-10

TABLE E7 Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells 

by cis-1,2-Dichloroethylene ............................................................. E-11

TABLE E8 Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells 

by trans-1,2-Dichloroethylene .......................................................... E-12

TABLE E9 Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells 

by cis,trans-1,2-Dichloroethylene ....................................................... E-13

TABLE E10 Induction of Sister Chromatid Exchanges in Bone Marrow Cells of Male Mice

by cis-1,2-Dichloroethylene ............................................................. E-15

TABLE E11 Induction of Sister Chromatid Exchanges in Bone Marrow Cells of Male Mice

by trans-1,2-Dichloroethylene .......................................................... E-15

TABLE E12 Induction of Chromosomal Aberrations in Bone Marrow Cells of Male Mice

by cis-1,2-Dichloroethylene ............................................................. E-16

TABLE E13 Induction of Chromosomal Aberrations in Bone Marrow Cells of Male Mice

by trans-1,2-Dichloroethylene .......................................................... E-16

TABLE E14 Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice

Following Administration of trans-1,2-Dichloroethylene in Feed for 14 Weeks ............... E-17
E-2 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE E1

Mutagenicity of cis-1,2-Dichloroethylene in Salmonella typhimuriuma

Revertants/Plate b

Strain Dose !!S9 +hamster S9 +rat S9

(μg/plate) Trial 1 Trial 2 10% 30% 10% 30%

Study performed at SRI International

TA100 0 110 ± 7.5 130 ± 2.3 101 ± 2.2 121 ± 4.4 95 ± 2.3 127 ± 2.3

100 100 ± 5.6 125 ± 13.0 128 ± 3.3 132 ± 18.4 88 ± 5.0 101 ± 7.0

333 87 ± 3.2 114 ± 8.4 115 ± 5.7 125 ± 6.6 89 ± 1.3 103 ± 6.8

1,000 91 ± 3.5 118 ± 11.9 106 ± 12.9 114 ± 14.5 92 ± 8.4 89 ± 2.7

3,333 97 ± 6.7 116 ± 9.9 95 ± 7.0 125 ± 9.3 102 ± 12.4 84 ± 6.2

6,666 93 ± 3.7 104 ± 13.7

10,000 Toxic 82 ± 4.5c 84 ± 5.8 111 ± 7.0

Trial summary Negative Negative Negative Negative Negative Negative

Positive controld 563 ± 7.9 652 ± 20.9 548 ± 14.0 664 ± 23.0 426 ± 13.9 287 ± 5.4

TA1535 0 18 ± 1.5 34 ± 1.9 12 ± 2.0 15 ± 3.6 11 ± 0.6 27 ± 0.9

100 17 ± 1.9 37 ± 4.6 10 ± 1.2 14 ± 2.7 11 ± 1.5 21 ± 5.0

333 16 ± 0.9 43 ± 3.0 6 ± 1.2 15 ± 1.5 7 ± 1.0 20 ± 2.6

1,000 19 ± 1.5 43 ± 1.0 9 ± 1.5 18 ± 2.3 8 ± 0.9 17 ± 2.3

3,333 15 ± 1.8 38 ± 5.6 7 ± 0.3 14 ± 1.7 7 ± 0.7 18 ± 1.9

6,666 15 ± 1.3 17 ± 1.8

10,000 4 ± 4.0c 5 ± 0.3c 10 ± 0.9 12 ± 2.9

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 378 ± 11.1 588 ± 11.7 270 ± 21.7 484 ± 9.1 165 ± 8.4 91 ± 9.4

TA97 0 147 ± 11.0 179 ± 6.0 155 ± 6.9 185 ± 11.6 150 ± 4.9 155 ± 16.2

100 152 ± 15.9 156 ± 16.6 134 ± 5.2 181 ± 15.8 166 ± 3.1 148 ± 20.9

333 144 ± 4.6 187 ± 8.1 155 ± 15.5 172 ± 26.6 168 ± 4.4 155 ± 4.2

1,000 150 ± 13.7 186 ± 7.3 135 ± 12.9 200 ± 10.9 175 ± 7.3 147 ± 5.6

3,333 127 ± 17.2 170 ± 13.2 132 ± 2.0 202 ± 18.6 165 ± 11.4 176 ± 9.6

6,666 99 ± 19.0c 90 ± 40.7c 175 ± 14.0

10,000 84 ± 2.1c 173 ± 8.1 171 ± 8.7

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 768 ± 102.2 773 ± 52.6 395 ± 17.5 344 ± 6.7 336 ± 3.2 340 ± 18.9

TA98 0 17 ± 0.9 28 ± 5.1 22 ± 2.3 54 ± 0.3 22 ± 1.5 45 ± 1.7

100 17 ± 2.9 29 ± 2.0 30 ± 2.3 39 ± 5.5 21 ± 3.2 36 ± 4.0

333 15 ± 1.2 26 ± 3.5 28 ± 1.5 41 ± 4.8 16 ± 1.5 30 ± 5.5

1,000 15 ± 1.0 29 ± 2.1 29 ± 2.9 28 ± 7.7 19 ± 3.2 28 ± 6.9

3,333 16 ± 0.3 26 ± 0.7 21 ± 3.8c 36 ± 3.3c 20 ± 2.7 23 ± 3.2

6,666 13 ± 1.5 49 ± 2.4c

10,000 Toxic 17 ± 2.3c 17 ± 0.9 39 ± 1.2

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 508 ± 7.0 534 ± 20.5 363 ± 10.8 449 ± 22.3 255 ± 23.2 157 ± 9.3
trans-1,2-Dichloroethylene, NTP TOX 55 E-3

TABLE E1

Mutagenicity of cis-1,2-Dichloroethylene in Salmonella typhimurium

Revertants/Plate

Strain Dose !!S9 +hamster S9 +rat S9

(μg/plate) Trial 1 Trial 2 10% 30% 10% 30%

Study performed at Microbiological Associates, Inc.

TA100 0 127 ± 12.9 95 ± 5.8 85 ± 2.2 117 ± 5.3 101 ± 5.8 142 ± 5.2

33 125 ± 4.2 90 ± 7.5 80 ± 3.2 117 ± 8.8 92 ± 1.5 132 ± 2.3

100 115 ± 6.8 101 ± 4.7 76 ± 2.8 118 ± 3.9 89 ± 1.5 138 ± 3.4

333 132 ± 8.1 91 ± 3.5 82 ± 2.6 108 ± 7.1 98 ± 7.0 134 ± 7.0

1,000 120 ± 4.3 89 ± 6.3 78 ± 3.8 108 ± 5.3 88 ± 4.5 130 ± 0.9

2,000 80 ± 3.8 101 ± 1.2 100 ± 3.2

3,333 80 ± 12.3c 63 ± 2.9c 74 ± 4.1c

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 336 ± 15.1 416 ± 14.8 567 ± 24.8 457 ± 18.6 624 ± 2.7 686 ± 22.0

TA1535 0 19 ± 4.0 19 ± 1.7 8 ± 1.7 10 ± 1.7 8 ± 1.7 11 ± 1.8

33 18 ± 3.8 23 ± 1.5 9 ± 1.5 10 ± 0.7 9 ± 3.3 12 ± 1.0

100 17 ± 3.4 25 ± 4.5 9 ± 0.7 10 ± 1.7 12 ± 3.1 9 ± 0.9

333 19 ± 2.3 22 ± 3.4 7 ± 1.3 9 ± 1.7 11 ± 1.0 9 ± 1.2

1,000 15 ± 0.9 20 ± 0.7 8 ± 2.2 13 ± 0.6 12 ± 1.5 12 ± 2.2

2,500 14 ± 1.2 7 ± 0.3 11 ± 1.9

3,333 14 ± 1.0c 5 ± 2.3c 7 ± 0.9c

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 210 ± 15.3 272 ± 3.0 57 ± 3.2 92 ± 2.0 127 ± 7.0 135 ± 17.4

TA1537 0 6 ± 0.7 6 ± 0.0 4 ± 0.3

33 6 ± 0.6 7 ± 1.0 7 ± 0.6

100 3 ± 1.2 7 ± 0.6 8 ± 1.2

333 4 ± 1.5 3 ± 0.7 6 ± 1.7

1,000 5 ± 2.0 4 ± 1.2 6 ± 2.7

2,500 6 ± 1.2c 6 ± 1.5 6 ± 3.6

Trial summary Negative Negative Negative

Positive control 36 ± 1.5 217 ± 3.4 78 ± 5.5

TA97 0 70 ± 8.4 93 ± 5.6 98 ± 17.0 114 ± 5.2 119 ± 10.8 137 ± 3.0

33 65 ± 10.2 89 ± 5.5 100 ± 4.0 117 ± 3.7 115 ± 12.4 149 ± 8.4

100 74 ± 7.7 79 ± 10.0 105 ± 9.0 124 ± 6.9 117 ± 2.6 152 ± 6.9

333 79 ± 1.2 87 ± 5.0 112 ± 1.7 120 ± 4.7 118 ± 14.9 133 ± 10.2

1,000 81 ± 6.4 81 ± 1.5 111 ± 4.3 125 ± 7.5 117 ± 6.7 129 ± 8.1

2,500 56 ± 2.6c 98 ± 4.0c 97 ± 4.5c

3,333 74 ± 7.6c 78 ± 4.9c 80 ± 7.4c

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 208 ± 26.6 187 ± 9.5 783 ± 36.5 988 ± 40.3 1,409 ± 47.4 577 ± 6.7
E-4 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE E1

Mutagenicity of cis-1,2-Dichloroethylene in Salmonella typhimurium

Revertants/Plate

Strain Dose !!S9 +hamster S9 +rat S9

(μg/plate) Trial 1 Trial 2 10% 30% 10% 30%

TA98 0 15 ± 1.5 15 ± 3.1 33 ± 2.2 21 ± 1.5 26 ± 0.9 21 ± 3.0

33 11 ± 2.3 15 ± 1.5 27 ± 3.1 23 ± 5.8 19 ± 1.7 26 ± 1.0

100 17 ± 4.4 11 ± 3.2 28 ± 2.5 21 ± 2.6 21 ± 4.4 21 ± 1.7

333 14 ± 3.5 14 ± 1.7 26 ± 1.5 24 ± 0.6 22 ± 2.4 23 ± 2.6

1,000 9 ± 1.0 11 ± 1.0 27 ± 2.9 27 ± 1.7 19 ± 4.4 17 ± 2.2

2,000 11 ± 0.3 23 ± 2.7 17 ± 0.6

3,333 13 ± 2.3c 19 ± 0.9c 18 ± 4.2c

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 116 ± 0.6 209 ± 5.0 233 ± 7.1 78 ± 11.6 259 ± 26.9 151 ± 4.2

a The detailed protocol and these data are presented by Zeiger et al. (1988). 0 μg/plate was the solvent control.

b Revertants are presented as mean ± standard error from three plates. c Slight toxicity

d The positive controls in the absence of metabolic activation were sodium azide (TA100 and TA1535), 9-aminoacridine (TA1537

and TA97), and 4-nitro-o-phenylenediamine (TA98). The positive control for metabolic activation with all strains was 2-

aminoanthracene.
trans-1,2-Dichloroethylene, NTP TOX 55 E-5

TABLE E2

Mutagenicity of trans-1,2-Dichloroethylene in Salmonella typhimuriuma

Revertants/Plate b

Strain Dose !!S9 +10% hamster S9 +10% rat S9

(μg/plate) Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2

TA100 0.0 106 ± 4.7 88 ± 4.6 100 ± 4.9 82 ± 8.9 108 ± 7.9 99 ± 4.3

33.3 109 ± 2.6 99 ± 18.2 97 ± 3.5

100.0 113 ± 8.1 99 ± 6.3 104 ± 4.9 92 ± 6.3 97 ± 4.9 109 ± 4.6

333.3 108 ± 9.0 80 ± 6.7 109 ± 5.0 104 ± 7.8 98 ± 5.2 94 ± 2.2

1,000.0 102 ± 12.8 82 ± 8.4 89 ± 5.1 88 ± 5.8 94 ± 8.4 95 ± 11.1

3,333.3 114 ± 5.2 72 ± 2.7c 94 ± 2.2 76 ± 8.1 100 ± 5.7 79 ± 6.5c

10,000.0 Toxic 78 ± 10.0c 85 ± 4.2c

Trial summary Negative Negative Negative Negative Negative Negative

Positive controld 457 ± 14.6 253 ± 7.5 1,423 ± 23.4 1,587 ± 36.5 1,214 ± 55.4 514 ± 8.0

TA1535 0.0 19 ± 2.6 26 ± 2.9 11 ± 1.5 11 ± 1.5 9 ± 0.3 15 ± 1.2

33.3 20 ± 0.3 13 ± 0.7 12 ± 1.8

100.0 18 ± 1.5 20 ± 2.5 12 ± 1.2 13 ± 2.8 14 ± 0.6 15 ± 2.1

333.3 18 ± 1.7 19 ± 4.7 10 ± 1.5 13 ± 0.6 9 ± 2.3 12 ± 3.2

1,000.0 18 ± 2.5 24 ± 4.6 8 ± 2.6 12 ± 2.8 10 ± 1.2 9 ± 2.5

3,333.3 24 ± 4.5 19 ± 3.8 8 ± 0.7 12 ± 1.2 10 ± 1.8 12 ± 0.6

10,000.0 18 ± 1.8 7 ± 1.0 11 ± 1.5

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 385 ± 21.5 542 ± 14.1 513 ± 5.8 639 ± 37.9 479 ± 17.8 303 ± 7.4

TA1537 0.0 7 ± 1.2 9 ± 0.3 29 ± 8.0 21 ± 4.4 24 ± 3.4 15 ± 2.9

33.3 7 ± 1.5 26 ± 4.6 18 ± 0.9

100.0 13 ± 3.4 8 ± 0.0 25 ± 3.3 24 ± 4.0 18 ± 3.8 20 ± 0.6

333.3 11 ± 2.3 12 ± 0.9 22 ± 3.7 23 ± 1.7 16 ± 0.7 13 ± 2.9

1,000.0 8 ± 1.3 13 ± 1.2 28 ± 6.9 22 ± 2.2 16 ± 4.3 17 ± 2.0

3,333.3 6 ± 2.0 9 ± 1.5 21 ± 4.4 18 ± 0.7 13 ± 1.2 13 ± 2.1

10,000.0 7 ± 0.0 18 ± 1.0 18 ± 4.6

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 251 ± 29.2 222 ± 6.1 363 ± 32.1 394 ± 36.0 409 ± 15.6 237 ± 14.1

TA98 0.0 26 ± 3.6 22 ± 4.7 55 ± 7.1 35 ± 2.5 61 ± 2.9 39 ± 2.3

33.3 34 ± 3.2 48 ± 1.5 52 ± 2.1

100.0 34 ± 3.0 16 ± 1.2 56 ± 6.8 32 ± 4.3 56 ± 3.9 39 ± 0.7

333.3 34 ± 2.7 21 ± 2.4 54 ± 1.2 29 ± 2.0 61 ± 6.4 41 ± 0.6

1,000.0 33 ± 3.8 22 ± 2.0 49 ± 3.2 32 ± 5.0 54 ± 5.4 38 ± 3.0

3,333.3 23 ± 4.4 23 ± 6.5 45 ± 3.6 30 ± 1.0 41 ± 2.3 39 ± 1.5

10,000.0 21 ± 3.5 35 ± 1.2 37 ± 4.1 

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 681 ± 18.0 682 ± 36.1 1,513 ± 57.8 1,605 ± 124.9 440 ± 27.3 489 ± 4.7

a Study was performed at SRI International. The detailed protocol and these data are presented by Mortelmans et al. (1986). 

0 μg/plate was the solvent control.

b Revertants are presented as mean ± standard error from three plates. c Slight toxicity

d The positive controls in the absence of metabolic activation were sodium azide (TA100 and TA1535), 9-aminoacridine (TA1537),

and 4-nitro-o-phenylenediamine (TA98). The positive control for metabolic activation with all strains was 2-aminoanthracene.
E-6 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE E3

Mutagenicity of cis,trans-1,2-Dichloroethylene in Salmonella typhimuriuma

Revertants/Plate b

Strain Dose !!S9 +10% hamster S9 +10% rat S9

(μg/plate) Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2

TA100 0.0 85 ± 6.6 107 ± 4.2 84 ± 6.2 78 ± 5.8 96 ± 10.0 94 ± 3.7

33.3 80 ± 4.8 78 ± 1.2 100 ± 7.6 85 ± 4.5 77 ± 5.5 82 ± 3.5

100.0 64 ± 5.2 75 ± 3.8 94 ± 4.4 92 ± 3.5 78 ± 6.9 88 ± 0.9

333.3 83 ± 8.5 85 ± 7.8 85 ± 5.6 83 ± 4.5 78 ± 5.0 82 ± 6.8

1,000.0 72 ± 10.0 63 ± 6.3 82 ± 5.8 66 ± 3.8 79 ± 6.4 72 ± 5.5

3,333.3 73 ± 7.8c 44 ± 2.8c 88 ± 7.1 59 ± 4.3c 66 ± 2.8c 51 ± 1.5c

Trial summary Negative Negative Negative Negative Negative Negative

Positive controld 413 ± 8.8 350 ± 23.3 1,383 ± 66.9 1,991 ± 73.8 528 ± 9.3 807 ± 30.1

TA1535 0.0 10 ± 1.3 13 ± 3.0 6 ± 2.5 7 ± 2.7 9 ± 1.5 7 ± 1.3

33.3 18 ± 1.2 5 ± 0.0 8 ± 0.6 5 ± 0.9 7 ± 1.2 5 ± 2.0

100.0 6 ± 0.9 7 ± 0.7 9 ± 2.7 6 ± 1.3 10 ± 3.5 6 ± 1.8

333.3 11 ± 3.8 6 ± 0.6 7 ± 1.2 4 ± 0.3 9 ± 1.7 4 ± 1.2

1,000.0 9 ± 3.1 7 ± 1.2 5 ± 0.9 5 ± 0.3 6 ± 3.0 4 ± 0.6

3,333.3 6 ± 4.2c 4 ± 1.8 5 ± 1.3 4 ± 1.0 6 ± 0.3 3 ± 0.3

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 350 ± 10.4 353 ± 18.9 470 ± 34.0 413 ± 24.4 313 ± 14.2 288 ± 21.2

TA1537 0.0 7 ± 1.5 3 ± 0.9 7 ± 3.1 5 ± 1.2 9 ± 2.1 6 ± 1.5

33.3 6 ± 0.9 4 ± 0.9 8 ± 0.9 3 ± 0.0 5 ± 0.3 5 ± 1.2

100.0 7 ± 1.0 3 ± 0.3 9 ± 3.3 6 ± 1.0 6 ± 0.0 3 ± 0.6

333.3 5 ± 0.9 3 ± 1.2 6 ± 0.9 4 ± 1.3 5 ± 0.0 4 ± 0.3

1,000.0 4 ± 0.6 2 ± 0.6 5 ± 0.9 4 ± 0.3 7 ± 1.7 4 ± 0.3

3,333.3 3 ± 0.9c 2 ± 0.9c 4 ± 0.3 3 ± 1.7c 4 ± 0.9c 3 ± 0.3c

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 124 ± 40.2 164 ± 42.6 445 ± 13.0 397 ± 37.6 235 ± 5.8 189 ± 15.9

TA98 0.0 28 ± 1.7 20 ± 2.7 24 ± 1.7 31 ± 0.9 35 ± 5.9 30 ± 1.7

33.3 15 ± 0.9 16 ± 1.7 40 ± 9.7 26 ± 2.4 37 ± 4.1 25 ± 1.9

100.0 24 ± 3.8 20 ± 0.6 30 ± 3.4 34 ± 3.6 48 ± 8.0 23 ± 3.4

333.3 21 ± 3.2 20 ± 0.9 31 ± 4.3 36 ± 1.7 34 ± 2.3 26 ± 1.2

1,000.0 16 ± 1.9 11 ± 1.9 26 ± 3.8 30 ± 1.2 23 ± 3.3 13 ± 2.6

3,333.3 7 ± 1.5c 3 ± 1.2c 25 ± 2.9 19 ± 1.9c 14 ± 2.6c 8 ± 2.2c

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 797 ± 18.8 784 ± 38.8 1,177 ± 53.5 1,664 ± 90.1 326 ± 16.1 584 ± 14.2

a Study was performed at SRI International. The detailed protocol and these data are presented by Mortelmans et al. (1986). 

0 μg/plate was the solvent control.

b Revertants are presented as mean ± standard error from three plates. c Slight toxicity

d The positive controls in the absence of metabolic activation were sodium azide (TA100 and TA1535), 9-aminoacridine (TA1537),

and 4-nitro-o-phenylenediamine (TA98). The positive control for metabolic activation with all strains was 2-aminoanthracene.
trans-1,2-Dichloroethylene, NTP TOX 55 E-7

TABLE E4

Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells by cis-1,2-Dichloroethylenea

Total No. of SCEs/ Relative

Concentration Cells Chromo- No. of Chromo- SCEs/ Hrs Change of SCEs/

Compound (μg/mL) Scored somes SCEs some Cell in BrdU Chromosome b

(%)

!!S9

Trial 1

Summary: Positive

Dimethylsulfoxidec 50 1,050 396 0.37 7.9 26.0

cis-1,2-Dichloroethylene

50 50 1,052 452 0.42 9.0 26.0 13.92

160 50 1,050 438 0.41 8.8 26.0 10.61

500 50 1,049 539 0.51 10.8 26.0 36.24*

1,600 50 1,046 521 0.49 10.4 26.0 32.07*

5,000 9d 188 91 0.48 10.1 26.0 28.34*

P<0.001e

Mitomycin-Cf 0.0005 50 1,051 516 0.49 10.3 26.0 30.18

0.005 10 210 311 1.48 31.1 26.0 292.68

Trial 2

Summary: Weakly positive

Dimethylsulfoxide 50 1,050 463 0.44 9.3 26.0

cis-1,2-Dichloroethylene

160 50 1,044 471 0.45 9.4 26.0 2.31

500 50 1,048 525 0.50 10.5 26.0 13.61

1,600 50 1,050 546 0.52 10.9 26.0 17.93

5,000 50 1,046 591 0.56 11.8 26.0 28.13*

P<0.001

Mitomycin-C 0.0007 50 1,046 589 0.56 11.8 26.0 27.70

0.005 10 210 296 1.40 29.6 26.0 219.66
E-8 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE E4

Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells by cis-1,2-Dichloroethylene

Total No. of SCEs/ Relative

Concentration Cells Chromo- No. of Chromo- SCEs/ Hrs Change of SCEs/

Compound (μg/mL) Scored somes SCEs some Cell in BrdU Chromosome

(%)

+S9

Summary: Equivocal

Dimethylsulfoxide 50 1,050 441 0.42 8.8 26.5

cis-1,2-Dichloroethylene

160 50 1,049 450 0.42 9.0 26.5 2.14

500 50 1,050 462 0.44 9.2 26.5 4.76

1,600 50 1,049 495 0.47 9.9 26.5 12.35

5,000 50 1,047 523 0.49 10.5 26.5 18.93

P=0.001

Cyclophosphamidef 0.1 50 1,049 550 0.52 11.0 26.5 24.83

0.6 10 210 207 0.98 20.7 26.5 134.69

* Positive response ($20% increase over solvent control) a Study was performed at Environmental Health Research and Testing, Inc. The detailed protocol is presented by Galloway et al.

(1987). SCE=sister chromatid exchange; BrdU=bromodeoxyuridine

b SCEs/chromosome in treated cells versus SCEs/chromosome in solvent control cells c Solvent control

d Severe toxicity e Significance of SCEs/chromosome tested by the linear regression trend test versus log of the dose

f Positive control
trans-1,2-Dichloroethylene, NTP TOX 55 E-9

TABLE E5

Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells by trans-1,2-Dichloroethylenea

Total No. of SCEs/ Relative

Concentration Cells Chromo- No. of Chromo- SCEs/ Hrs Change of SCEs/

Compound (μg/mL) Scored somes SCEs some Cell in BrdU Chromosome b

(%)

!!S9

Summary: Negative

Dimethylsulfoxidec 50 1,049 428 0.40 8.6 26.0

trans-1,2-Dichloroethylene

160 50 1,042 460 0.44 9.2 26.0 8.20

500 50 1,039 459 0.44 9.2 26.0 8.28

1,600 50 1,039 474 0.45 9.5 26.0 11.81

5,000 50 1,044 416 0.39 8.3 26.0 –2.34

P=0.526d

Mitomycin-Ce 0.0005 50 1,049 561 0.53 11.2 26.0 31.08

0.005 10 210 297 1.41 29.7 26.0 246.64

+S9

Summary: Equivocal

Dimethylsulfoxide 50 1,050 388 0.36 7.8 26.0

trans-1,2-Dichloroethylene

160 50 1,049 414 0.39 8.3 26.0 6.80

500 50 1,050 451 0.42 9.0 26.0 16.24

1,600 50 1,050 457 0.43 9.1 26.0 17.78

5,000 50 1,048 453 0.43 9.1 26.0 16.98

P=0.004

Cyclophosphamidee 0.1 50 1,050 537 0.51 10.7 26.0 38.40

0.6 10 210 226 1.07 22.6 26.0 191.24

a Study was performed at Environmental Health Research and Testing, Inc. The detailed protocol is presented by Galloway et al.

(1987). SCE=sister chromatid exchange; BrdU=bromodeoxyuridine

b SCEs/chromosome in treated cells versus SCEs/chromosome in solvent control cells

c Solvent control

d Significance of SCEs/chromosome tested by the linear regression trend test versus log of the dose e Positive control
E-10 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE E6

Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells by cis,trans-1,2-Dichloroethylenea

Total No. of SCEs/ Relative

Concentration Cells Chromo- No. of Chromo- SCEs/ Hrs Change of SCEs/

Compound (μg/mL) Scored somes SCEs some Cell in BrdU Chromosome b

(%)

!!S9

Summary: Positive

Dimethylsulfoxidec 50 1,041 411 0.39 8.2 25.8

cis,trans-1,2-Dichloroethylene

126 50 1,027 523 0.50 10.5 25.8 28.99*

421 50 1,041 621 0.59 12.4 25.8 51.10*

1,263 50 1,041 871 0.83 17.4 25.8 111.92*

P<0.001d

Mitomycin-Ce 0.005 50 1,036 1,347 1.30 26.9 25.8 229.32

+S9

Summary: Positive

Dimethylsulfoxide 50 1,027 427 0.41 8.5 25.5

cis,trans-1,2-Dichloroethylene

1,263 50 1,037 618 0.59 12.4 25.5 43.33*

4,210 f 50 1,021 1,912 1.87 38.2 25.5 350.41*

12,630 f 50 1,025 2,545 2.48 50.9 25.5 497.19*

P<0.001

Cyclophosphamidee 1.5 50 1,032 2,234 2.16 44.7 25.5 420.65

* Positive response ($20% increase over solvent control) a Study was performed at Litton Bionetics, Inc. The detailed protocol is presented by Galloway et al. (1987). SCE=sister chromatid

exchange; BrdU=bromodeoxyuridine

b SCEs/chromosome in treated cells versus SCEs/chromosome in solvent control cells

c Solvent control

d Significance of SCEs/chromosome tested by the linear regression trend test versus log of the dose e Positive control

f Etching of the plastic tissue culture flasks occurred at this dose.
trans-1,2-Dichloroethylene, NTP TOX 55 E-11

TABLE E7

Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by cis-1,2-Dichloroethylenea

Concentration Total Cells Number Aberrations/ Cells with

Compound (μg/mL) Scored of Aberrations Cell Aberrations (%)

!!S9

Harvest time: 12.0 hours

Summary: Negative

Dimethylsulfoxideb 200 2 0.01 1.0

cis-1,2-Dichloroethylene 500 200 3 0.02 1.5

1,000 200 3 0.02 1.5

1,600 200 3 0.02 1.5

P=0.340c

Mitomycin-Cd 0.0625 200 34 0.17 13.0

0.25 50 20 0.40 36.0

+S9

Harvest time: 13.0 hours

Summary: Negative

Dimethylsulfoxide 200 2 0.01 1.0

cis-1,2-Dichloroethylene 1,600 200 6 0.03 2.5

3,000 200 11 0.06 3.0

5,000 200 2 0.01 1.0

P=0.433

Cyclophosphamide d 2.5 200 34 0.17 14.5

7.5 50 21 0.42 36.0

a Study was performed at Environmental Health Research and Testing, Inc. The detailed protocol is presented by Galloway et al.

(1987).

b Solvent control c Significance of percent cells with aberrations tested by the linear regression trend test versus log of the dose

d Positive control
E-12 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE E8

Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by trans-1,2-Dichloroethylenea

Concentration Total Cells Number Aberrations/ Cells with

Compound (μg/mL) Scored of Aberrations Cell Aberrations (%)

!!S9

Harvest time: 12.0 hours

Summary: Negative

Dimethylsulfoxideb 200 1 0.01 0.5

trans-1,2-Dichloroethylene 1,600 200 0 0.00 0.0

3,000 200 1 0.01 0.5

5,000 200 1 0.01 0.5

P=0.394c

Mitomycin-Cd 0.0625 200 36 0.18 17.0

0.25 50 15 0.30 18.0

+S9

Harvest time: 13.0 hours

Summary: Negative

Dimethylsulfoxide 200 0 0.00 0.0

trans-1,2-Dichloroethylene 1,600 200 1 0.01 0.5

3,000 200 3 0.02 1.5

5,000 200 2 0.01 1.0

P=0.066

Cyclophosphamide d 2.5 200 34 0.17 16.0

7.5 50 21 0.42 22.0

a Study was performed at Environmental Health Research and Testing, Inc. The detailed protocol is presented by Galloway et al.

(1987).

b Solvent control c Significance of percent cells with aberrations tested by the linear regression trend test versus log of the dose

d Positive control
trans-1,2-Dichloroethylene, NTP TOX 55 E-13

TABLE E9

Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by cis,trans-1,2-Dichloroethylenea

Concentration Total Cells Number Aberrations/ Cells with

Compound (μg/mL) Scored of Aberrations Cell Aberrations (%)

!!S9

Harvest time: 22.5 hoursb

Summary: Negative

Dimethylsulfoxidec 100 3 0.03 3.0

cis,trans-1,2-Dichloroethylene 594 100 5 0.05 5.0

1,184 50d 2 0.04 2.0

2,374 e 100 4 0.04 3.0

4,798 e 0 f

9,496 e 100 10 0.10 9.0

P=0.040g

Mitomycin-Ch 500 100 25 0.25 21.0

+S9

Trial 1

Harvest time: 10.5 hours

Summary: Negative

Dimethylsulfoxide 100 5 0.05 5.0

cis,trans-1,2-Dichloroethylene 772 100 24 0.24 13.0

1,543 e 100 6 0.06 6.0

3,086 e 0 f

6,173 e 100 5 0.05 3.0

9,496 e 100 5 0.05 5.0

12,630 e 100 6 0.06 5.0

P=0.941

Cyclophosphamide h 50 100 53 0.53 29.0
E-14 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE E9

Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by cis,trans-1,2-Dichloroethylene

Concentration Total Cells Number Aberrations/ Cells with

Compound (μg/mL) Scored of Aberrations Cell Aberrations (%)

+S9 (continued)

Trial 2

Harvest time: 10.5 hours

Summary: Negative

Dimethylsulfoxide 100 6 0.06 4.0

cis,trans-1,2-Dichloroethylene 455 100 15 0.15 10.0

607 100 7 0.07 6.0

758 100 12 0.12 3.0

910 e 100 6 0.06 6.0

1,210 e 100 12 0.12 8.0

1,516 e 100 10 0.10 8.0

P=0.277

Cyclophosphamide 50 100 45 0.45 28.0

a Study was performed at Litton Bionetics, Inc. The detailed protocol is presented by Galloway et al. (1987).

b Due to cell cycle delay, harvest time was extended to maximize the number of first-division metaphase cells available for analysis. c Solvent control

d Incomplete count due to the poor quality of this culture e Etching of the plastic tissue culture flasks occurred at this dose.

f Severe toxicity

g Significance of percent cells with aberrations tested by the linear regression trend test versus log of the dose

h Positive control
trans-1,2-Dichloroethylene, NTP TOX 55 E-15

TABLE E10

Induction of Sister Chromatid Exchanges in Bone Marrow Cells of Male Mice by cis-1,2-Dichloroethylenea

Compound Dose (mg/kg) SCEs/Cell

Sample time: 23 hours

Corn oilb 3.36 ± 0.43

cis-1,2-Dichloroethylene 500 3.43 ± 0.37

1,000 4.65 ± 0.45

2,000 3.83 ± 0.58

Dimethylbenzanthracenec 100 13.29 ± 0.95

a Study was performed at Oak Ridge Associated Universities. The detailed protocol is presented by Tice et al. (1987); 25 seconddivision metaphase cells were scored from each of four mice per group. Data for SCEs/cell are given as mean ± standard error. 

SCE=sister chromatid exchange

b Vehicle control c Positive control

TABLE E11

Induction of Sister Chromatid Exchanges in Bone Marrow Cells of Male Mice by trans-1,2-Dichloroethylenea

Compound Dose (mg/kg) SCEs/Cell

Sample time: 23 hours

Corn oilb 3.36 ± 0.43

trans-1,2-Dichloroethylene 500 3.55 ± 0.29

1,000 3.38 ± 0.20

2,000 3.00 ± 0.11

Dimethylbenzanthracenec 100 13.29 ± 0.95

a Study was performed at Oak Ridge Associated Universities. The detailed protocol is presented by Tice et al. (1987); 25 seconddivision metaphase cells were scored from each of four mice per group. Data for SCEs/cell are given as mean ± standard error. 

SCE=sister chromatid exchange

b Vehicle control

c Positive control
E-16 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE E12

Induction of Chromosomal Aberrations in Bone Marrow Cells of Male Mice by cis-1,2-Dichloroethylenea

Number of Mice

Dose with Cells Cells with Aberrations b P Value

(mg/kg) Scored

Sample time: 17 hours

Corn oilc 8 2.25 ± 0.45

cis-1,2-Dichloroethylene 500 8 1.50 ± 0.50 0.7829

1,000 8 1.50 ± 0.73 0.7829

2,000 7 2.29 ± 0.81 0.4869

P=0.430d

Dimethylbenzanthracenee 100 8 21.00 ± 2.85 0.0000

a Study was performed at Oak Ridge Associated Universities. The detailed protocol is presented by Tice et al. (1987); 50 firstdivision metaphase cells were scored from each of seven or eight mice per group.

b Mean ± standard error c Vehicle control

d Significance tested by a one-tailed trend test (Margolin et al., 1986) e Positive control

TABLE E13

Induction of Chromosomal Aberrations in Bone Marrow Cells of Male Mice by trans-1,2-Dichloroethylenea

Number of Mice

Dose with Cells Cells with Aberrations b P Value

(mg/kg) Scored

Sample time: 17 hours

Corn oilc 8 2.25 ± 0.45

trans-1,2-Dichloroethylene 500 8 1.75 ± 1.03 0.6367

1,000 8 3.75 ± 1.91 0.1947

2,000 8 1.75 ± 0.80 0.6367

P=0.546d

Dimethylbenzanthracenee 100 8 21.00 ± 2.85 0.0000

a Study was performed at Oak Ridge Associated Universities. The detailed protocol is presented by Tice et al. (1987); 50 firstdivision metaphase cells were scored from each of eight mice per group.

b Mean ± standard error

c Vehicle control

d Significance tested by a one-tailed trend test (Margolin et al., 1986) e Positive control
trans-1,2-Dichloroethylene, NTP TOX 55 E-17

TABLE E14

Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice

Following Administration of trans-1,2-Dichloroethylene in Feed for 14 Weeksa

Number of Mice

Concentration with Erythrocytes Micronucleated NCEs/ P Valuec PCEs (%)

(ppm) Scored 1,000 NCEs b

Male

Untreated Control 10 1.15 ± 0.20 1.6

Vehicle Control 10 1.05 ± 0.16 1.4

3,125 10 1.05 ± 0.23 0.6186 1.4

6,250 10 0.95 ± 0.16 0.7316 1.5

12,500 10 1.05 ± 0.16 0.6186 1.7

25,000 10 0.70 ± 0.17 0.9306 1.7

50,000 10 0.75 ± 0.21 0.9029 1.9

P=0.936d

Female

Untreated Control 10 0.85 ± 0.18 1.7

Vehicle Control 10 0.90 ± 0.21 1.7

3,125 10 0.65 ± 0.11 0.7675 1.9

6,250 10 0.95 ± 0.22 0.3694 1.7

12,500 10 0.85 ± 0.13 0.5000 1.8

25,000 10 0.75 ± 0.19 0.6382 1.8

50,000 10 0.70 ± 0.15 0.7051 1.9

P=0.699

a Study was performed at SITEK Research Laboratories, Inc. The detailed protocol is presented by MacGregor et al. (1990). 

NCE=normochromatic erythrocyte; PCE=polychromatic erythrocyte

b Mean ± standard error c Pairwise comparison with the untreated controls; significant at P#0.005 (ILS, 1990)

d Significance of micronucleated NCEs/1,000 NCEs versus the untreated controls tested by the one-tailed trend test; significant at

P#0.025 (ILS, 1990)
E-18 trans-1,2-Dichloroethylene, NTP TOX 55
F-1

APPENDIX F

CHEMICAL CHARACTERIZATION

AND DOSE FORMULATION STUDIES

PROCUREMENT AND CHARACTERIZATION OF TRANS-1,2-DICHLOROETHYLENE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-2

PREPARATION AND ANALYSIS OF DOSE FORMULATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-3

FIGURE F1 Infrared Absorption Spectrum of trans-1,2-Dichloroethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-5

FIGURE F2 Nuclear Magnetic Resonance Spectrum of trans-1,2-Dichloroethylene . . . . . . . . . . . . . . . . . . . . . . . F-6

TABLE F1 Gas Chromatography Systems Used in the 14-Week Feed Studies

of trans-1,2-Dichloroethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-7

TABLE F2 Preparation and Storage of Dose Formulations in the 14-Week Feed Studies 

of trans-1,2-Dichloroethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-8

TABLE F3 Results of Analyses of Dose Formulations Administered to Rats and Mice

in the 14-Week Feed Studies of trans-1,2-Dichloroethylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-9
F-2 trans-1,2-Dichloroethylene, NTP TOX 55

CHEMICAL CHARACTERIZATION

AND DOSE FORMULATION STUDIES

PROCUREMENT AND CHARACTERIZATION OF TRANS-1,2-DICHLOROETHYLENE

trans-1,2-Dichloroethylene was obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI), in one lot

(MP-02224LP) for use in the 14-week feed studies. The chemical was microencapsulated by the analytical

chemistry laboratory, Midwest Research Institute (Kansas City, MO), and the microcapsules were assigned a

separate lot number (343-1B-A). Identity, purity, and stability analyses of the neat and microencapsulated

chemical were conducted by the analytical chemistry laboratory and the study laboratory. Reports on

analyses performed in support of the trans-1,2-dichloroethylene studies are on file at the National Institute of

Environmental Health Sciences.

Analyses of Neat Chemical

The chemical, a clear, colorless liquid, was identified as trans-1,2-dichloroethylene by the analytical

chemistry laboratory with infrared, ultraviolet/visible, and nuclear magnetic resonance (NMR) spectroscopy

and by the study laboratory with infrared spectroscopy. The infrared and NMR spectra were consistent with

literature references (Sadtler Standard Spectra; High Resolution NMR Spectra Catalog, 1963). All spectra

were consistent with the structure of trans-1,2-dichloroethylene. The infrared and NMR spectra are

presented in Figures F1 and F2.

The purity of trans-1,2-dichloroethylene was determined by the analytical chemistry laboratory using

elemental analyses, Karl Fischer water analysis, free acid titration, and gas chromatography (GC). To

measure the concentration of free acid, trans-1,2-dichloroethylene was dissolved in methanol and titrated

with 0.01 N sodium hydroxide to the phenolphthalein endpoint. GC analyses were performed with systems A

and B as described in Table F1. Additional GC analyses with mass spectrometry (GC/MS) were performed

to determine whether selected chlorinated impurities were present and to quantify any impurities that were

detected. The study laboratory analyzed purity using GC by system C.

Elemental analyses for carbon, hydrogen, and chlorine were in agreement with the theoretical values for

trans-1,2-dichloroethylene. Karl Fischer water analysis indicated 0.010% ± 0.001% water. Free acid

titration indicated 0.0007 ± 0.0002 mEq acid per gram, equivalent to 26 ± 7 ppm as hydrochloric acid. GC

by system A indicated one major peak and five impurities with a combined area of 1.25% relative to the major

peak area. GC by system B indicated one major peak and three impurities with a combined relative area of

0.69%. The overall purity of lot MP-02224LP was determined to be approximately 99%. GC/MS analyses

by systems D and E or similar systems identified nine chlorinated compounds: cis-1,2-dichloroethylene

(1,600 ppm), methylene chloride (300 ppm), 1,1- and 1,2-dichloroethane (300 and approximately 2 ppm,

respectively), chloroform (82 ppm), trichloroethylene (12 ppm), and tetrachloroethylene, chlorobenzene, and

vinyl chloride (less than 1 ppm each). Additionally, carbon tetrachloride and 1,1,2,2-tetrachloroethane were

tentatively identified at concentrations less than 1 ppm. The study laboratory confirmed that the purity was

99% or greater using GC by system C.

Stability information supplied by the manufacturer indicated that neat trans-1,2-dichloroethylene is stable

when stored frozen and protected from air, moisture, and light; the chemical was stored under these

conditions throughout the 14-week studies. Stability was monitored during preliminary pilot studies

conducted by TSI Mason Laboratories (Worcester, MA) with infrared spectroscopy and GC by systems

similar to system C; no degradation of the bulk chemical was detected.
trans-1,2-Dichloroethylene, NTP TOX 55 F-3

Microcapsule Formulation and Analyses

Microcapsules loaded with neat trans-1,2-dichloroethylene and placebos (empty microcapsules) were

prepared by the analytical chemistry laboratory with a proprietary process using food-grade, modified corn

starch (CAPSUL®) and reagent-grade sucrose (80:20) to produce dry microspheres; the outer surfaces of the

microcapsules were dusted with food-grade, hydrophobic, modified corn starch. Following

microencapsulation, the analytical chemistry laboratory tested the chemical for conformance to

specifications. The microcapsules were examined microscopically for appearance. Conformance to particle

size specifications (with no more than 1% of particles having diameters greater than 420 μm) was determined

by passing placebo and loaded microcapsules through U.S. standard sieves (numbers 30, 40, 60, 80, 100,

120, and PAN). The chemical loads (amount of trans-1,2-dichloroethylene in the starch/sugar matrix) of

freshly prepared microcapsules and of microcapsules stored under a variety of conditions were determined

with GC by systems similar to system C. Samples for GC analysis were prepared by dissolving the

microcapsules in 50 mL of a 60:40 methanol:water solution by shaking for 15 minutes; 50 mL of an internal

standard solution (4 mg/mL 1,1,1-trichloroethane in methanol) were added, and the mixtures were shaken

and filtered. Major peak comparisons of the neat and microencapsulated chemicals and 9-month stability

studies were also performed with GC by systems similar to system C.

Microscopic examination revealed no unusual characteristics. The particles were within size specifications. 

The mean chemical load was determined to be 45.0% ± 0.3%. Microcapsules exposed to animal room

conditions (open to air and light) for 28 days retained 93.8% of their chemical load by weight; additional

samples similarly exposed after seven freeze-thaw cycles retained 93% of their chemical load after 28 days. 

Microcapsules stored in sealed bottles at 5° C for 28 days retained 95% of their chemical load by weight. 

Major peak comparisons indicated that no impurities were introduced by microencapsulation; one impurity

occurring in the neat chemical with a relative area of 0.24% was not observed in the encapsulated chemical. 

Results of the 9-month shelf-life study indicated that microcapsules retained 91% of their chemical load when

stored at 5° C and 89% when stored at room temperature; microcapsules stored at 5° C for 6 months and

then at room temperature, open to air and light, for 28 days were stable.

The study laboratory confirmed the identity of the microcapsules with infrared spectroscopy and analyzed

the chemical load of the microcapsules using GC by a system similar to system F. GC analyses performed at

the beginning of the preliminary pilot studies indicated a chemical load of approximately 43%. Prior to the

14-week studies, GC analyses of trans-1,2-dichloroethylene samples from one bottle of microcapsules

indicated a concentration of 37.6% ± 2.4%, which was lower than the expected concentration of 45.3%;

microcapsules from six other bottles were mixed in a twin-shell blender with the intensifier bar on for

5 minutes and off for 10 minutes. Samples were extracted for 15 minutes with 50 mL methanol:water

(60:40); 50 mL methanol were added, and the samples were extracted for an additional minute. The extract

was combined with the internal standard, 200 ppm 1,1,1-trichloroethane in 5% methanol:hexane (1:1), and

diluted to 40 mL with hexane. The mixture was shaken for 1 minute, and an aliquot of the upper hexane

phase was analyzed with GC by system G. Homogeneity was confirmed, and the chemical load of 47.0%

was considered to be acceptable for use in the 14-week studies. The chemical load was monitored during

the 14-week studies with GC by system G; no loss of trans-1,2-dichloroethylene was detected.

PREPARATION AND ANALYSIS OF DOSE FORMULATIONS

The dose formulations were prepared at least every 2 weeks by mixing microencapsulated trans1,2-dichloroethylene with feed (Table F2). Placebo microcapsules were added to maintain a starch matrix

concentration of 6% in the diet. A premix was prepared by hand and then blended with additional feed in a

twin-shell blender for 15 minutes, with the intensifier bar on for the first 5 minutes. The dose formulations

were then kneaded and mixed manually, then mixed for an additional 15 minutes in the blender, with the
F-4 trans-1,2-Dichloroethylene, NTP TOX 55

intensifier bar on for the first 5 minutes. Formulations were stored in doubled plastic bags, protected from

light, at 5E C for up to 4 weeks.

Homogeneity and stability studies of 0.5% trans-1,2-dichloroethylene formulations were conducted by the

analytical chemistry laboratory using GC by system G or similar systems. Homogeneity was confirmed. 

The trans-1,2-dichloroethylene formulation prepared with lot 343-1A had losses of 22.0% for samples stored

for 7 days in the dark and 15.5% for samples stored for 1 day open to air and light; dose formulations

prepared with lots 343-10TA, -11TA, and -12TA (not used in the current studies) and 343-1B-A were stable

for 4 days when stored at room temperature, open to air and light. A loss of 5.2% for the trans isomer was

observed after 3 weeks for dose formulations stored in the dark at room temperature. Additional analyses

performed with GC by system F with a 0.5% dose formulation prepared with lot 343-1B-A confirmed

stability for 7 days for samples stored in a rat cage, open to air and light, at up to 50% humidity.

Prior to the 14-week studies, the study laboratory performed homogeneity studies of the 3,125 and

50,000 ppm dose formulations and stability studies of the 3,125 ppm dose formulation with GC by a system

similar to system G. Homogeneity of all formulations was confirmed. Stability was confirmed for 28 days

for dose formulations stored in plastic bags at up to 5° C. Dose formulations were stored refrigerated in

doubled plastic bags during the studies.

Periodic analyses of the dose formulations were conducted by the study laboratory using GC by systems

similar to system C. The first four sets of dose formulations, as well as those at the midpoint and end of the

studies, were analyzed; animal room samples of the dose formulations taken from feeders on the last dosing

day prior to the expiration date of the batch were also analyzed at the beginning, midpoint, and end of the

studies (Table F3). The results for the initial analyses of the first two sets of dose formulations and the

midpoint dose formulations were high; additional feed was added, and these dose formulations were

reblended and reanalyzed. All were found to be within 10% of the target concentrations. The concentration

of one dose formulation from the fourth set was slightly high; this dose formulation was considered

acceptable for use. Two of the final set of dose formulations also were not within 10% of the target

concentrations; these were remixed, and the remixes were found to be within specifications. The

concentrations of all animal room samples except one sample for rats were more than 10% lower than the

target concentrations; this was considered to be the result of degradation of the microcapsule matrix by

condensation of atmospheric moisture during frozen storage and possibly by contamination of the feed with

urine.
FIGURE F1

Infrared Absorption Spectrum of trans-1,2-Dichloroethylene

trans-1,2-Dichloroethylene, NTP TOX 55 F-5
FIGURE F2

Nuclear Magnetic Resonance Spectrum of trans-1,2-Dichloroethylene

F-6 trans-1,2-Dichloroethylene, NTP TOX 55
trans-1,2-Dichloroethylene, NTP TOX 55 F-7

TABLE F1

Gas Chromatography Systems Used in the 14-Week Feed Studies of trans-1,2-Dichloroethylenea

Detection Oven Temperature

System Column Carrier Gas Program

System A

Flame ionization 10% SP-1000 on 80/100

Supelcoport,

1.8 m × 4 mm

Nitrogen at 70 mL/minute Isothermal at 50° C or

50° C for 5 minutes, then

10° C/minute to 250° C

System B

Flame ionization 80/100 Porapak QS,

1.8 m × 4 mm

Nitrogen at 70 mL/minute Isothermal at 200° C or

50° C for 5 minutes, then

10° C/minute to 225° C

System C

Flame ionization 20% SP-2100/

0.1% Carbowax 1500,

3.6 m × 2 mm

Nitrogen at 30 mL/minute 45° C for 18 minutes, then

20° C/minute to 165° C,

held for 4 minutes

System D

Mass spectrometry with

full mass scan (70 eV; scan

rate 1.05 seconds;

multiplier voltage –1,700

or –1,800 V)

Megabore DB-624,

30 m × 0.53 mm, 3-μm

film (J&W Scientific,

Folsom, CA)

Helium at 10 mL/minute 30° C for 3 minutes, then

8° C/minute to 220° C

System E

Mass spectrometry with

selected ion monitoring

(70 eV; scan rate

0.9 seconds for

identification and

0.756 seconds for

quantitation; multiplier

voltage –2,000 V)

Megabore DB-624,

30 m × 0.53 mm, 3-μm

film (J&W Scientific)

Helium at 10 mL/minute 30° C for 3 minutes, then

8° C/minute to 220° C

System F

Flame ionization 20% SP-2100/

0.1% Carbowax 1500,

3.6 m × 2 mm

Nitrogen at 30 mL/minute 45° C for 5 minutes, then

20° C/minute to 85° C,

held for 2 minutes

System G

Electron capture DB-1 Megabore,

30 m × 0.53 mm, 3-μm

film (J&W Scientific)

Nitrogen at approximately

9 mL/minute

Isothermal at 50° C

a Gas chromatographs were manufactured by Varian, Inc. (Palo Alto, CA) (systems A, B, and F), Perkin Elmer (Norwalk, CT)

(systems C, D, and E), and Hewlett-Packard (Palo Alto, CA) (system G).
F-8 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE F2

Preparation and Storage of Dose Formulations in the 14-Week Feed Studies of trans-1,2-Dichloroethylene

Preparation

A premix of microencapsulated trans-1,2-dichloroethylene and feed was prepared by hand and then blended with additional feed in a

twin-shell blender for 15 minutes, with the intensifier bar on for the first 5 minutes. The dose formulations were placed in a plastic

bag, kneaded and mixed manually, then returned to the blender and mixed for an additional 15 minutes, with the intensifier bar on for

the first 5 minutes. Dose formulations were prepared at least every 2 weeks.

Chemical Lot Number

Neat: MP-02224LP

Microcapsules: 343-1B-A

Maximum Storage Time

4 weeks

Storage Conditions

Stored in doubled plastic bags, protected from light, at 5° C

Study Laboratory

Microbiological Associates, Inc. (Bethesda, MD)
trans-1,2-Dichloroethylene, NTP TOX 55 F-9

TABLE F3

Results of Analyses of Dose Formulations Administered to Rats and Mice 

in the 14-Week Feed Studies of trans-1,2-Dichloroethylene

Target Determined Difference

Date Prepared Date Analyzed Concentration Concentrationa from Target

(ppm) (ppm) ( % )

Rats

3 May 1993 5 May 1993b 3,125 3,870 +24

6,250 7,510 +20

12,500 14,600 +17

25,000 29,600 +18

50,000 59,500 +19

6 May 1993 7 May 1993c 3,125 3,180 +2

6,250 5,970 !4

12,500 12,900 +3

25,000 24,200 !3

50,000 47,700 !5

21-22 June 1993d 3,125 2,860 !8

6,250 4,530 e !28

12,500 9,050 e !28

25,000 19,400 e !22

50,000 41,300 !17

11 May 1993 12 May 1993b 3,125 4,070 +30

6,250 7,890 +26

12,500 14,700 +18

25,000 29,600 +18

50,000 60,000 +20

14 May 1993 14 May 1993c 3,125 3,230 +3

6,250 6,380 +2

12,500 12,100 !3

25,000 25,200 +1

50,000 49,100 !2

17 May 1993 18 May 1993 3,125 3,390 +8

6,250 6,480 +4

12,500 13,300 +6

25,000 25,700 +3

50,000 52,700 +5

25 May 1993 1 June 1993 3,125 3,470 +11

6,250 6,450 +3

12,500 12,700 +2

25,000 25,400 +2

50,000 46,700 !7
F-10 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE F3

Results of Analyses of Dose Formulations Administered to Rats and Mice

in the 14-Week Feed Studies of trans-1,2-Dichloroethylene

Target Determined Difference

Date Prepared Date Analyzed Concentration Concentration from Target

(ppm) (ppm) ( % )

Rats (continued)

22 June 1993 23 June 1993b 3,125 3,790 +21

6,250 2,210 !65

12,500 15,800 +26

25,000 30,000 +20

50,000 58,100 +16

29 June 1993 29 June 1993c 3,125 3,350 +7

6,250 6,540 +5

12,500 12,800 +2

25,000 25,500 +2

50,000 49,000 !2

19 July 1993d 3,125 1,510 !52

6,250 3,460 !45

12,500 6,850 !45

25,000 15,600 !38

50,000 29,600 !41

16 July 1993 19 July 1993 3,125 2,590 f !17

6,250 5,320 f !15

12,500 11,800 !6

25,000 24,900 0

50,000 47,300 !5

22 July 1993 22 July 1993 3,125 2,980g !5

6,250 5,860g !6

28 September 1993d 3,125 1,900 !39

6,250 3,960 !37

12,500 6,640 !47

25,000 14,700 !41

50,000 29,100 !42

Mice

3 May 1993 5 May 1993b 3,125 3,870 +24

6,250 7,510 +20

12,500 14,600 +17

25,000 29,600 +18

50,000 59,500 +19
trans-1,2-Dichloroethylene, NTP TOX 55 F-11

TABLE F3

Results of Analyses of Dose Formulations Administered to Rats and Mice

in the 14-Week Feed Studies of trans-1,2-Dichloroethylene

Target Determined Difference

Date Prepared Date Analyzed Concentration Concentration from Target

(ppm) (ppm) ( % )

Mice (continued)

6 May 1993 7 May 1993c 3,125 3,180 +2

6,250 5,970 !4

12,500 12,900 +3

25,000 24,200 !3

50,000 47,700 !5

23 June 1993d 3,125 1,230 !61

6,250 7,410 +19

12,500 5,300 !58

25,000 7,440 !70

50,000 21,800 !56

24 June 1993h 3,125 1,240 !60

6,250 2,130 !66

12,500 5,460 !56

25,000 7,340 !71

50,000 20,700 !59

11 May 1993 12 May 1993b 3,125 4,070 +30

6,250 7,890 +26

12,500 14,700 +18

25,000 29,600 +18

50,000 60,000 +20

14 May 1993 14 May 1993c 3,125 3,230 +3

6,250 6,380 +2

12,500 12,100 !3

25,000 25,200 +1

50,000 49,100 !2

17 May 1993 18 May 1993 3,125 3,390 +8

6,250 6,480 +4

12,500 13,300 +6

25,000 25,700 +3

50,000 52,700 +5

25 May 1993 1 June 1993 3,125 3,470 +11

6,250 6,450 +3

12,500 12,700 +2

25,000 25,400 +2

50,000 46,700 !7

22 June 1993 23 June 1993b 3,125 3,790 +21

6,250 2,210 !65

12,500 15,800 +26

25,000 30,000 +20

50,000 58,100 +16
F-12 trans-1,2-Dichloroethylene, NTP TOX 55

TABLE F3

Results of Analyses of Dose Formulations Administered to Rats and Mice

in the 14-Week Feed Studies of trans-1,2-Dichloroethylene

Target Determined Difference

Date Prepared Date Analyzed Concentration Concentration from Target

(ppm) (ppm) ( % )

Mice (continued)

29 June 1993 29 June 1993c 3,125 3,350 +7

6,250 6,540 +5

12,500 12,800 +2

25,000 25,500 +2

50,000 49,000 !2

19 July 1993d 3,125 953 !70

6,250 2,170 !65

12,500 4,690 !62

25,000 11,000 !56

50,000 12,500 !75

16 July 1993 19 July 1993 3,125 2,590 f !17

6,250 5,320 f !15

12,500 11,800 !6

25,000 24,900 0

50,000 47,300 !5

22 July 1993 22 July 1993 3,125 2,980g !5

6,250 5,860g !6

28 September 1993d 3,125 1,550 !50

6,250 3,470 !44

12,500 5,900 !53

25,000 12,300 !51

50,000 17,800 !64

a Results of duplicate analyses

b Reblended with additional feed and reanalyzed

c Results for reblended samples

d Animal room samples e Results of triplicate analyses

f Remixed; not used in study

g Results of remix

h Reanalysis of animal room samples