trans-Nonachlor and cis-nonachlor toxicity in Sprague-Dawley rats: comparison with technical chlordane.

Abstract

cis-Nonachlor and trans-nonachlor are bioaccumulating components of the pesticide chlordane, which can be detected in various environmental biota and in humans. Existing studies have focused on the potential adverse health effects of the parent chlordane mixture. Comparable toxicity data are nonexistent for individual chlordane constituents such as trans-nonachlor, cis-nonachlor, or oxychlordane, which are among the most common chlordane-related environmental contaminants and tissue residues. In this study, rats were administered cis-nonachlor, trans-nonachlor, or technical chlordane by gavage for 28 days at doses of 0.25 to 25 mg/kg body weight. Residue analyses indicated that trans-nonachlor accumulation in adipose was greater than cis-nonachlor when rats were administered each chemical under identical conditions of dose and exposure. For all test chemicals, the major metabolite oxychlordane accumulated in adipose tissue. Adipose tissue residue levels of all test chemicals and the major metabolite were higher in female rats. The liver was a target organ in male and female rats, indicated by increased liver weight and histopathological changes consistent with microsomal enzyme induction. Hepatic changes were most pronounced in rats treated with trans-nonachlor. Elevated kidney weights and depressed organic ion transport were observed in males treated with trans-nonachlor and chlordane. Although in general, changes in target organs and clinical chemistry endpoints were similar for all 3 test chemicals, the approximate toxicity ranking from most to least toxic was trans-nonachlor > technical chlordane > cis-nonachlor.

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trans-Nonachlor and cis-Nonachlor Toxicity in Sprague-Dawley Rats:
Comparison with Technical Chlordane
G. S. Bondy,*,1W. H. Newsome,† C. L. Armstrong,* C. A. M. Suzuki,* J. Doucet,† S. Fernie,* S. L. Hierlihy,*
M. M. Feeley,‡ and M. G. Barker*
*Toxicology Research Division, †Food Research Division and ‡Chemical Health Hazard Assessment Division, Food Directorate,
Health Protection Branch, Health Canada, Ottawa, Ontario K1A 0L2, Canada
Received June 14, 2000; accepted September 5, 2000
cis-Nonachlor and trans-nonachlor are bioaccumulating com-
ponents of the pesticide chlordane, which can be detected in
various environmental biota and in humans. Existing studies have
focused on the potential adverse health effects of the parent
chlordane mixture. Comparable toxicity data are nonexistent for
individual chlordane constituents such as trans-nonachlor, cis-
nonachlor, or oxychlordane, which are among the most common
chlordane-related environmental contaminants and tissue resi-
dues. In this study, rats were administered cis-nonachlor, trans-
nonachlor, or technical chlordane by gavage for 28 days at doses
of 0.25 to 25 mg/kg body weight. Residue analyses indicated that
trans-nonachlor accumulation in adipose was greater than cis-
nonachlor when rats were administered each chemical under iden-
tical conditions of dose and exposure. For all test chemicals, the
major metabolite oxychlordane accumulated in adipose tissue.
Adipose tissue residue levels of all test chemicals and the major
metabolite were higher in female rats. The liver was a target organ
in male and female rats, indicated by increased liver weight and
histopathological changes consistent with microsomal enzyme in-
duction. Hepatic changes were most pronounced in rats treated
with trans-nonachlor. Elevated kidney weights and depressed or-
ganic ion transport were observed in males treated with trans-
nonachlor and chlordane. Although in general, changes in target
organs and clinical chemistry endpoints were similar for all 3 test
chemicals, the approximate toxicity ranking from most to least
toxic was trans-nonachlor > technical chlordane > cis-nonachlor.
Key Words: trans-nonachlor; cis-nonachlor; chlordane; oxy-
chlordane; toxicity.
The organochlorine pesticide chlordane was used in North
America until the late 1980?s as a termiticide, a seed dressing
or coating, and an insecticide on crops, lawns, and gardens.
Technical grade chlordane is a mixture of over 120 structurally
related compounds. The most abundant constituents are cis
(?)-chlordane (CAS No. 5103–71–9), trans (?)-chlordane
(CAS No. 5103–74–2), heptachlor (CAS No. 76–44–8),
trans-nonachlor (CAS No. 39765–80–5), and cis-nonachlor
(CAS No. 5103–73–1) (Dearth and Hites, 1991a). As a result
of the widespread usage of chlordane, which has an environ-
mental half-life of 10 to 20 years (Bennett et al., 1974), this
toxic and persistent mixture has accumulated in the food chain.
This is particularly the case in polar regions, where chlordane-
related compounds have been detected in fish and marine
mammals at levels comparable to polychlorinated biphenyls
(PCBs) and DDT isomers (sum of p,p?-DDE, DDD, DDT and
o,p?-DDT) (Muir et al., 1988). Oxychlordane, cis- and trans-
nonachlor, and cis- and trans-chlordane have been detected in
three trophic levels of the Arctic marine food chain, including
polar bear fat, ringed seal blubber and Arctic cod muscle (Muir
et al., 1988). Humans have also been exposed to chlordane-
related contaminants. In the Arctic, consumption of traditional
foods contributes to chlordane exposure (Kinloch et al., 1992;
Kuhnlein, 1995; Kuhnlein et al., 1995; ). trans-Nonachlor and
oxychlordane have been detected in human milk from residents
of northern Canada (Newsome and Ryan, 1999). cis-Nonachlor
and trans-nonachlor, oxychlordane, and trans-chlordane were
detected in human breast adipose tissue samples obtained from
Bloomington, IN (Dearth and Hites, 1991b). trans-Nonachlor
and oxychlordane were detected in human adipose tissue au-
topsy samples from 6 Ontario municipalities in the Great Lakes
Basin (Williams et al., 1988). In samples from Japan, human
abdominal adipose tissue was shown to contain (in order of
increasing concentration) chlordane, oxychlordane, and nona-
chlor (Hirai and Tomokuni, 1991).
The unofficial Canadian tolerated daily intake value for
chlordane is 0.05 ?g/kg body weight (bw)/day, with an official
maximum residue limit of 0.1 ppm for chlordane and oxychlor-
dane (calculated on fat content) for a variety of dairy and meat
products (Dr. M. Feeley, personal communication; Canada
Food and Drugs Act). The U.S. Food and Drug Administration
has established that chlordane and its metabolites should not be
present at levels higher than 300 ppb in fruits and vegetables or
100 ppb in animal fat and fish (U.S. Department of Health and
Parts of this manuscript were presented at Experimental Biology ‘99, April
17–21, Washington, DC.
1To whom correspondence should be addressed at the Toxicology Research
Division, Postal Locator 2204D2, Food Directorate, HPB, Health Canada, 2E
Banting Building, Ottawa, Ontario K1A 0L2, Canada. Fax: (613) 941-6959.
E-mail: genevieve_bondy@hc-sc.gc.ca.
TOXICOLOGICAL SCIENCES 58, 386–398 (2000)
Copyright © 2000 by the Society of Toxicology
386

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Human Services [U.S. DHHS], 1994). The primary focus of
the majority of toxicological studies supporting these recom-
mendations is on the potential adverse health effects of the
parent chlordane mixture, for which a toxicological profile has
been compiled (U.S. DHHS, 1994). However, toxicity data for
individual chlordane constituents or metabolites such as trans-
nonachlor, cis-nonachlor, or oxychlordane, which are among
the most common chlordane-related environmental contami-
nants and tissue residues, are nonexistent. This study addresses
the toxicological data gap for trans-nonachlor and cis-nona-
chlor. Male and female rats were exposed to trans- and cis-
nonachlor by gavage for 28 consecutive days, and multiple
toxicological endpoints were examined. The clinical chemistry,
hematology, urinalysis, tissue residue analyses and histopathol-
ogy results have been summarized for these chemicals. Also
included in this study were male and female rats exposed to the
technical chlordane mixture for comparison to rats treated with
cis- and trans-nonachlor.
MATERIALS AND METHODS
Test chemicals.
pure) and technical chlordane were purchased from Radian International
(Austin, TX). The major constituents of the technical chlordane used in this
study were trans-chlordane, 13.2%; cis-chlordane, 11.3%; trans-nonachlor,
5.4%; cis-nonachlor, 1.9%; and heptachlor, 2.1%. In comparison, the major
constituents of an EPA technical chlordane standard (Lot No. R704) were
trans-chlordane, 13.6%; cis-chlordane, 11.5%; trans-nonachlor, 5.7%; cis-
nonachlor, 1.9%; and heptachlor, 6.4% (Dr. Terry Bidleman, personal com-
munication). To prepare stock solutions, the test chemicals were dissolved in
acetone and diluted with corn oil. Corn oil vehicle for control animals was
prepared by adding acetone to corn oil in amounts equivalent to those used for
dissolving the test chemicals. The acetone was removed from the oil by
trans-Nonachlor (99% pure) and cis-nonachlor (99%
evaporation under vacuum. Dosing solutions were prepared from stock solu-
tions by further dilution with corn oil. Standards for residue analyses were
purchased from the following sources: trans-nonachlor (99% pure), oxychlor-
dane (98% pure), and heptachlor (99% pure) from Radian International;
trans-chlordane (99.98% pure) from Velsicol Chemical Corporation (Rose-
mont, IL); cis-chlordane (100% pure) and cis-nonachlor (99.9% pure) from the
U.S. Environmental Protection Agency (Research Triangle Park, NC).
Animals.
obtained from Charles River Canada, Inc. (Montreal, Quebec). Upon receipt,
rats were housed individually in plastic cages (Health Guard System, Research
Equipment Company, Inc., Bryan, TX) under conditions meeting the require-
ments of the Canadian Council for Animal Care. All rats were acclimatized for
a minimum of one week before the study. Purina Rodent Chow (Woodstock,
Ontario) and water were provided ad libitum throughout the study.
Male and female Sprague-Dawley rats (45 to 50 days old) were
Experimental design.
into 6 test groups, designated A through F, as indicated in Table 1. Within each
group, rats were randomly assigned to receive 1 of 4 test chemical doses:
controls (0 mg/kg test chemical; corn oil vehicle only), 0.25, 2.5, or 25 mg test
chemical/kg body weight (bw)/day. The highest test doses were chosen based
on studies indicating that 50 mg chlordane/kg bw administered by gavage to
rats caused deaths after 9 to 12 days, whereas no deaths occurred in rats treated
with 25 mg chlordane/kg for 15 days (Ambrose et al., 1953). Mean starting
bws are indicated in Table 1. For all groups, the number of rats at each dose
level was 7. Body weight data were analyzed prior to the beginning of the
study to confirm that within each group there were no significant differences in
starting body weights between rats assigned to each dose level. For 28
consecutive days, each rat received a single daily gavage dose of test chemical
in a volume of 0.5 ml corn oil/100 g bw. Body weights were monitored daily
throughout the studies; food and water consumption were monitored biweekly.
For urine collection, rats were transferred to Nalgene metabolic cages for 24 h
before the first dose and for 24 h after the last dose. On the final day of the
study (24 h after the last dose), each rat was anesthetized with isoflurane
(Janssen, Toronto, Ontario, Canada), exsanguinated and necropsied. Organ
weights were recorded for liver, kidneys, spleen, thymus, adrenals, brain,
ovaries, and testes.
Male and female rats were randomized and divided
TABLE 1
Summary of Dose Group Designations, Mean Daily Food Consumption (g) and Mean Daily Water Consumption (g)
in Rats Receiving cis-Nonachlor, trans-Nonachlor or Technical Chlordane by Gavage for 28 Days
GroupTest chemical SexMean daily consumption
Test chemical dose (mg/kg bw/day)
0 0.252.5 25
A
cis-NonachlorF Food
Water
Food
Water
Food
Water
Food
Water
Food
Water
Food
Water
14.4 ? 0.2
27.0 ? 0.5
14.4 ? 0.2
33.2 ? 1.0
14.4 ? 0.3
23.9 ? 0.6
18.2 ? 0.3
31.8 ? 0.6
18.4 ? 0.4
34.6 ? 0.7
18.2 ? 0.2
31.9 ? 0.5
13.9?0.3
27.1 ? 0.9
13.9 ? 0.3
25.8 ? 0.9**
13.9 ? 0.2
28.1 ? 1.1**
19.4 ? 0.3*
33.7 ? 0.4**
20.8 ? 0.3*
39.3 ? 1.0**
19.2 ? 0.3
37.2 ? 1.1**
14.4 ? 0.2
28.1 ? 0.9
13.8 ? 0.3
27.8 ? 0.5**
14.1 ? 0.2
30.1 ? 0.6**
20.3 ? 0.5*
33.1 ? 0.7
19.6 ? 0.3*
33.3 ? 0.8
18.0 ? 0.3
33.0 ? 0.7**
13.2 ? 0.3*
27.9 ? 0.8
12.6 ? 0.3*
25.4 ? 0.9**
12.8 ? 0.3*
27.3 ? 0.8*
19.2 ? 0.2*
31.3 ? 0.5
19.2 ? 0.2
34.7 ? 0.5
17.4 ? 0.4*
35.2 ? 0.8**
B
trans-NonachlorF
C Technical chlordaneF
D
cis-NonachlorM
E
trans-NonachlorM
F Technical chlordaneM
Note. All data are expressed as mean ? SE. Data are mean food or water consumption per day over the 29-day study period, except for Group B at the 25
mg/kg dose level, where the rats were necropsied at day 24.
*Significantly different from mean daily food consumption in corresponding control rats (p ? 0.05).
**Significantly different from mean daily water consumption in corresponding control rats (p ? 0.05).
387
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Hematology and clinical chemistry.
drawn from the abdominal aorta into EDTA tubes for hematology and serum
separator tubes for clinical chemistry (Becton Dickinson Vacutainers). For
hematology, blood was processed with a Coulter Counter Model S-PLUS IV
system (Coulter Electronics Inc., Hialeah, FL). Smears for leukocyte differ-
entials were stained with Wright’s Giemsa. The following hematological
parameters were measured: red blood cell numbers, hemoglobin, hematocrit,
mean corpuscular volume, mean corpuscular hemoglobin concentration, mean
corpuscular hemoglobin, red cell distribution width, platelet numbers, mean
platelet volume (MPV), platelet distribution width and white blood cell num-
bers (including lymphocytes, neutrophils, eosinophils and basophils).
For clinical chemistry, clotted blood was centrifuged at 700 ? g for 20 min
to prepare serum. A Beckman Synchron CX5 clinical system (Beckman
Instruments Canada, Inc., Mississauga, ON) and Beckman reagent kits were
used to measure clinical endpoints. The following clinical chemistry parame-
ters were measured in serum from each rat: glucose, blood urea nitrogen
(BUN), creatinine, uric acid, total protein, albumin, immunoglobulin A (IgA),
IgM, IgG1, IgG2a, IgG2b, IgG2c, total bilirubin, cholesterol, triglycerides, as-
partate aminotransferase (AST), alanine aminotransferase (ALT), alkaline
phosphatase (ALP), ?-glutamyltransferase (GGT), ornithine carbamyl trans-
ferase (OCT), lactate dehydrogenase (LDH), creatine kinase, amylase, thyrox-
ine (T4), thyroxine uptake, calcium, sodium, potassium, magnesium, chloride,
phosphorus, and osmolality.
Renal toxicology.
The methodology for urinalysis has been described
previously (Suzuki et al., 1995). Briefly, urine volume, osmolality, and total
protein were measured in whole urine. A 1-ml aliquot of each urine sample was
applied to Sephadex G-25 PD-10 columns (Pharmacia LKB, Baie D’Urfe,
Canada) and eluted through with 8 ml of saline to remove low molecular
weight enzyme inhibitors. The first 4-ml fraction collected from the column
was analyzed for creatinine using the method of Heinegard and Tiderstrom
(1973). The enzymes, N-acetyl-?-D-glucosaminidase (NAG) and ?-glutamyl-
transpeptidase (GGT) were measured in the next 4-ml fraction by the methods
of Leaback and Walker (1961) and Dierickx (1980), respectively. Transport of
the organic anion, p-aminohippuric acid (PAH), and the cation, tetraethylam-
monium (TEA) in kidney slices was measured as described previously (Suzuki
et al., 1995). Kidneys were weighed and prepared for transport assays imme-
diately after the rat was exsanguinated. Transport was expressed as a ratio
between the amount of ion transported into the slice and the amount remaining
in the medium (S/M). A decrease in this ratio represents reduced transport
function.
Pathology.
For light microscopy, tissues were fixed in 10% neutral buff-
ered formalin (pH 7.0). Paraffin sections (4 ?m) were stained with hematox-
ylin and eosin. The following tissues were examined: adrenals, bone marrow,
esophagus, heart, kidneys, liver, lungs, pancreas, skeletal muscle, spleen,
thymus, thyroid, ovaries, uterus, testes, epididymis, prostate, coagulatory
gland, and seminal vesicles. To identify hepatocellular vacuole contents, liver
sections were stained with periodic acid Schiff (PAS) for water soluble
polysaccharides or oil red O for lipids (Luna, 1968).
Residue analyses.
Whole blood was collected from male rats at necropsy
for residue analyses; female rats were too small to provide sufficient blood for
residue analyses in addition to clinical chemistry and hematology. Liver (1 g)
and adipose tissue from the abdominal fat pad (1 g) were collected from male
and female rats. All tissues, including whole blood, were frozen in tightly
sealed vials at –20°C until extraction. All samples were analyzed for the
following residues: cis-nonachlor, trans-nonachlor, cis-chlordane, trans-chlor-
dane, oxychlordane, and heptachlor. Adipose, liver, or blood samples were
extracted with acetone:hexane (2:1) and the extract filtered through glass wool.
The solvent was concentrated, dried over sodium sulfate, and evaporated to
dryness. The residue was then chromatographed on 2% water deactivated
Florisil (Fisher Scientific, Fair Lawn, NJ) to remove lipid. Analytes were
eluted with 2% dichloromethane in hexane and quantified by gas chromatog-
raphy on a DB-5 capillary column using a Varian Star 3400 chromatograph
fitted with an electron capture detector (Varian, Walnut Creek, CA). Recov-
eries of oxychlordane, cis-nonachlor, trans-nonachlor, cis-chlordane and trans-
At the time of necropsy, blood waschlordane spiked into corn oil were 95% or greater, while that of heptachlor
was 82%. Corn oil, spiked at from 2 to 4 ppm, was run with each set of 10
samples to verify satisfactory recoveries.
Statistics.
Statistical analyses were done using SigmaStat (Jandel Scien-
tific, San Rafael, CA). For each test chemical, data from control and treated
rats were compared using one-way analysis of variance (ANOVA) for multiple
comparisons, followed by Dunnett’s test for pairwise comparisons if neces-
sary. For nonparametric data, multiple comparisons were made using the
Kruskal-Wallis ANOVA on ranks, followed by Dunnett’s (for equal sample
sizes) or Dunn’s (for unequal sample sizes) pairwise tests if necessary. Data
comparisons were considered significant if p ? 0.05.
RESULTS
General Observations
Some female rats (3 out of 7) receiving 25 mg trans-
nonachlor/kg bw began to lose weight between days 21 and 23
of the dosing period, losing an average of 9.4 g on the first day
of apparent illness and 14.7 g on the second day. Affected rats
had a hunched posture and unkempt appearance. When han-
dled, they showed signs of abdominal tenderness. Death oc-
curred overnight between 48 and 60 h after the first recorded
day of weight loss. Since these 3 rats were necropsied several
h after death, variable degrees of autolysis were seen in many
organs. The remaining 4 rats in this dose group were exsan-
guinated and necropsied on day 24 and were apparently healthy
at the time of necropsy. A corresponding female control rat
was also necropsied on day 24 to verify (for statistical analy-
ses) that clinical chemistry and hematological endpoints fell
within the range of control rats necropsied on day 29. There
were no other overt signs of illness in rats from any other
group.
Body Weight and Food and Water Consumption
In female rats receiving cis-nonachlor (Group A), daily food
consumption was significantly lower at the 25 mg/kg dose level
(Table 1), but this did not translate into changes in total weight
gain or final bw (Table 2). In female rats receiving trans-
nonachlor (Group B), daily food consumption was significantly
depressed in the 25 mg/kg dose group (Table 1). In conjunction
with the rapid weight loss and death of 3 rats at this dose level,
total weight gain and final bw in these rats were significantly
lower than in corresponding controls (Table 2). Technical
chlordane had no effect on weight gain or final bw in female
rats (Table 2, Group C), even though food consumption was
significantly lower than controls at the 25 mg/kg dose level
(Table 1). In males treated with cis-nonachlor (Group D), both
weight gain and food consumption were significantly higher
for all doses compared to controls (Tables 1 and 2), but this did
not result in significantly higher final bws (Table 2). In trans-
nonachlor-treated males (Group E), food consumption was
significantly higher in the 0.25 and 2.5 mg/kg dose groups
(Table 1), but weight gain and final bw were unaffected (Table
2). Food consumption was significantly depressed in male rats
388
BONDY ET AL.

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receiving 25 mg/kg technical chlordane (Table 1, Group F), but
the apparent drop in bw at this dose level was not significant
(Table 2).
Daily water consumption over the entire dosing period is
summarized in Table 1. cis-Nonachlor-treated female rats were
unaffected (Group A), and male rats had significantly elevated
daily water consumption only at the 0.25 mg/kg dose level
(Group D). Daily water consumption in trans-nonachlor-
treated males was unchanged (Group E), but females drank
significantly less water per day compared to control rats, at
every dose level (Group B). Female and male rats receiving
technical chlordane (Groups C and F, respectively) consumed
significantly more water than control rats at every dose level
(Table 1).
Organ weights
Liver weight was consistently elevated in Groups A through
F at the 25 mg/kg dose level, regardless of whether the data
were expressed as total liver wet weight (g) or as % final bw
(Table 3). In male rats treated with trans-nonachlor (Group E),
both liver wet weight and liver % bw were also elevated at the
2.5 mg/kg dose level. In male rats treated with technical
chlordane (Group F), liver % bw was elevated in the 2.5 mg/kg
dose group. Overall, trans-nonachlor had the greatest effect on
liver weight. In both females and males, liver wet weights were
2.1 ? higher than comparable controls, whereas livers from
rats treated with cis-nonachlor and technical chlordane ranged
from 1.5? to 1.8? larger than controls. Kidney weights were
elevated in male rats but not in female rats (Table 3). cis-
Nonachlor treatment (Group D) resulted in significantly ele-
vated kidney weights at the 2.5 mg/kg dose level, and both
elevated kidney weight and kidney % bw at the 25 mg/kg dose
level. Kidney weight (Group E) or kidney % bw (Group F)
were significantly elevated in males treated with trans-nona-
chlor or technical chlordane, respectively, at the 25 mg/kg dose
level. Spleen, thymus, adrenal, brain, ovary, and testis weights
were unaffected in all groups (data not shown).
Hematology and Clinical Chemistry
None of the test chemicals caused consistent changes in any
of the hematological parameters measured (data not shown).
Erythrocyte mean corpuscular volume was significantly re-
duced in female rats treated with 25 mg cis-nonachlor/kg
(Group A) compared to control rats, whereas male rats (Group
D) had significantly lower MPVs at the same dose. Female rats
treated with technical chlordane (Group C) also had signifi-
cantly lower MPVs at the highest dose compared to control
rats, and male rats treated with technical chlordane (Group F)
TABLE 2
Mean Starting Body Weight, Body Weight Gain, and Final Body Weight in Rats Receiving cis-Nonachlor, trans-Nonachlor
or Technical Chlordane by Gavage for 28 Days
Groupa
Mean starting bwb
Total wt gain/final bwc
Test chemical dose (mg/kg body weight/day)
0 0.252.5 25
A 225.0 ? 2.4 Total wt gain
Final bw
Total wt gain (day 24)
Final bw (day 24)
Total wt gain (day 29)
Final bw (day 29)
Total wt gain
Final bw
Total wt gain
Final bw
Total wt gain
Final bw
Total wt gain
Final bw
29.1 ? 2.5
259.8 ? 3.4
21.7 ? 3.7
248.8 ? 4.1
23.1 ? 4.5
248.8 ? 6.6
19.8 ? 1.9
250.4 ? 7.1
55.6 ? 4.7
429.2 ? 13.7
62.0 ? 6.4
442.7 ? 16.4
61.0 ? 4.8
429.8 ? 12.3
26.4 ? 4.0
248.7 ? 10.1
28.8 ? 2.4
257.6 ? 5.4
18.0 ? 3.3
236.8 ? 6.4
10.6 ? 2.1*
225.3 ? 3.9**
B222.6 ? 2.7
21.6 ? 5.1
243.7 ? 9.8
24.4 ? 1.4
249.1 ? 6.1
72.2 ? 5.2*
452.3 ? 13.5
77.5 ? 5.4
465.4 ? 8.0
62.9 ? 6.5
446.4 ? 15.7
21.5 ? 2.1
239.7 ? 5.2
27.1 ? 2.9
255.1 ? 8.2
85.6 ? 10.9*
465.6 ? 24.1
65.7 ? 4.7
439.7 ? 12.3
53.5 ? 6.8
417.8 ? 16.7
C227.8 ? 2.9 13.6 ? 2.9
243.7 ? 9.2
69.4 ? 4.3*
444.2 ? 6.9
48.4 ? 6.5
420.6 ? 9.5
43.0 ? 7.1
413.1 ? 15.5
D377.1 ? 4.9
E 378.6 ? 3.9
F372.2 ? 4.7
Note. Body weight, bw; weight, wt. All weight/weight gain values are given in g.
aGroup designations are for the following: A, females, cis-nonachlor; B, females, trans-nonachlor; C, females, technical chlordane; D, males, cis-nonachlor;
E, males, trans-nonachlor; F, males, technical chlordane.
bAll data are expressed as mean ? SE for 6 ? n ? 7 rats, except for Group B rats in the 25 mg/kg dose group where n ? 4. Mean starting bws were calculated
for all dose groups combined (n ? 28 rats).
cFor all rats except those in Group B (25 mg/kg dose group) mean weight gain was calculated by subtracting the starting bw (day 1) from the final bw (day
29) for each rat. In Group B rats, mean weight gain in control rats at day 24 has been included for comparison to rats in the 25 mg/kg dose group, which were
necropsied on day 24.
*Significantly different from the weight gain in corresponding control rats (p ? 0.05).
**Significantly different from the final bw of corresponding control rats (p ? 0.05).
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had lower hemoglobin and hematocrit values at the 25 mg/kg
dose level. There were no changes in male or female rats
treated with trans-nonachlor.
Statistically significant serum clinical chemistry changes due
to cis-nonachlor, trans-nonachlor, and technical chlordane are
summarized in Tables 4, 5, and 6, respectively. Most clinical
chemistry changes were observed in the 25 mg/kg dose group
for all of the test chemicals. One of the most consistent changes
was elevated serum cholesterol at the 25 mg/kg dose level in
male and female rats. Alanine aminotransferase (ALT) was
elevated in trans-nonachlor-treated rats only (Table 5),
whereas ?-glutamyltransferase (GGT) and ornithine carbamyl
transferase (OCT) were unaffected in all rats (data not shown).
Serum triglycerides were significantly depressed in male rats
treated with technical chlordane (Table 6). Total serum protein
was significantly elevated by all of the test chemicals, in
conjunction with depressed albumin/globulin (A/G) ratios (Ta-
bles 4–6). Serum calcium and/or magnesium levels were ele-
vated in males treated with cis-nonachlor (Table 4) and tech-
nical chlordane (Table 6), and in males and females treated
with trans-nonachlor (Table 5). Thyroxine uptake was signif-
icantly depressed in trans-nonachlor-treated rats (Table 5), and
both thyroxine uptake and thyroxine levels were depressed in
rats treated with technical chlordane (Table 6).
Urinalysis and Renal Function
Urinalysis endpoints, including urine volume, protein lev-
els, creatinine, GGT, and osmolality were unaffected by
treatment with cis-nonachlor, trans-nonachlor, or technical
chlordane (data not shown). In trans-nonachlor-treated fe-
male rats, urine NAG was significantly decreased at the 25
mg/kg dose level (2.04 ? 0.15 ?M/min over a 24-h sample
period compared to 1.32 ? 0.07 ?M/min, respectively; p ?
0.05). Urine NAG was increased compared to control rats in
male rats receiving 25 mg technical chlordane/kg bw/day
(2.83 ? 0.25 ?M/min compared to 4.58 ? 0.23 ?M/min,
respectively; p ? 0.05). Para-aminohippuric acid transport
was unaffected in kidney slices from both male and female
rats, as was TEA transport in female rats. The effects of the
TABLE 3
Summary of Final Liver and Kidney Weights in g and % BW for Male and Female Rats Treated with cis-Nonachlor,
trans-Nonachlor or Technical Chlordane by Gavage for 28 Days
Groupa,b
Parameter
Test chemical dose (mg/kg bw/day)
0 0.252.5 25
A Mean final liver weight (g)
Liver weight/ bw (%)
Mean final kidney weight (g)
Kidney weight/bw (%)
Mean final liver weight (g)
Liver weight/bw (%)
Mean final kidney weight (g)
Kidney weight/ bw (%)
Mean final liver weight (g)
Liver weight/bw (%)
Mean final kidney weight (g)
Kidney weight/ bw (%)
Mean final liver weight (g)
Liver weight/ bw (%)
Mean final kidney weight (g)
Kidney weight/ bw (%)
Mean final liver weight (g)
Liver weight/ bw (%)
Mean final kidney weight (g)
Kidney weight/ bw (%)
Mean final liver weight (g)
Liver weight/ bw (%)
Mean final kidney weight (g)
Kidney weight/ bw (%)
8.6 ? 0.4
3.3 ? 0.1
1.87 ? 0.03
0.73 ? 0.01
8.3 ? 0.2
3.3 ? 0.1
1.83 ? 0.12
0.73 ? 0.05
8.5 ? 0.4
3.3 ? 0.1
1.87 ? 0.07
0.73 ? 0.02
12.6 ? 0.7
3.0 ? 0.1
2.67 ? 0.08
0.64 ? 0.01
12.7 ? 0.6
3.0 ? 0.1
2.84 ? 0.16
0.67 ? 0.02
12.7 ? 0.4
3.0 ? 0.1
2.68 ? 0.07
0.64 ? 0.02
8.8 ? 0.7
3.5 ? 0.2
1.76 ? 0.08
0.70 ? 0.01
8.5 ? 0.4
3.5 ? 0.1
1.79 ? 0.09
0.73 ? 0.03
8.6 ? 0.3
3.5 ? 0.1
1.95 ? 0.06
0.79 ? 0.03
14.0 ? 0.7
3.1 ? 0.1
2.74 ? 0.12
0.62 ? 0.02
14.4 ? 0.7
3.2 ? 0.1
2.80 ? 0.07
0.61 ? 0.02
13.5 ? 0.7
3.1 ? 0.1
2.87 ? 0.07
0.66 ? 0.01
9.3 ? 0.3
3.6 ? 0.1
1.92 ? 0.05
0.75 ? 0.03
9.3 ? 0.3
3.9 ? 0.1
1.81 ? 0.08
0.75 ? 0.03
9.4 ? 0.3
3.7 ? 0.1
1.85 ? 0.06
0.73 ? 0.02
16.2 ? 1.0
3.5 ? 0.1
3.05 ? 0.12*
0.67 ? 0.03
15.5 ? 0.7*
3.6 ? 0.1*
2.90 ? 0.14
0.67 ? 0.03
14.1 ? 0.6
3.4 ? 0.1*
2.79 ? 0.10
0.68 ? 0.02
12.7 ? 0.4*
5.2 ? 0.1*
1.76 ? 0.8
0.73 ? 0.02
17.4 ? 0.4c,*
7.7 ? 0.1*
1.70 ? 0.02
0.76 ? 0.01
13.5 ? 0.6*
5.6 ? 0.2*
1.90 ? 0.06
0.78 ? 0.01
21.7 ? 0.8*
5.0 ? 0.2*
3.13 ? 0.07*
0.72 ? 0.01*
26.7 ? 1.0*
6.4 ? 0.2*
3.21 ? 0.15
0.77 ? 0.03*
23.3 ? 1.2*
5.6 ? 0.3*
3.22 ? 0.23*
0.77 ? 0.04
B
C
D
E
F
aGroup designations are for the following: A ? females, cis-nonachlor; B ? females, trans-nonachlor; C ? females, technical chlordane; D ? males,
cis-nonachlor; E ? males, trans-nonachlor; F ? males, technical chlordane.
bAll data are expressed as mean ? SE for 6 ? n ? 7 rats, except for Group B rats in the 25 mg/kg dose group where n ? 4.
cRats in this dose group were necropsied on day 24; all other rats were necropsied on day 29.
*Significantly different from corresponding control (p ? 0.05).
390
BONDY ET AL.

Page 6

test chemicals on TEA transport in kidney slices from male
rats are summarized in Figure 1. Tetraethylammonium
transport was significantly depressed in both cis- and trans-
nonachlor-treated rats at the 2.5 and 25 mg/kg dose levels.
The apparent drop in TEA transport in chlordane-treated
rats was not statistically significant.
Pathology
Changes in the livers of male and female rats treated with
cis-nonachlor, trans-nonachlor, and technical chlordane are
summarized in Table 7. For all test chemicals, there were
dose-related increases in the extent and incidence of hepato-
TABLE 4
Clinical Changes in Serum from Male and Female Rats Receiving cis-Nonachlor by Gavage for 28 Days
Parameter
Females (Group A)Males (Group D)
0 mg/kg0.252.52500.25 2.525
Glucose
Cholesterol
Amylase
T. protein
A/G ratio
Ca
Osmolality
12.0 ? 0.4
0.80 ? 0.05
946 ? 45
59.7 ? 1.3
0.41 ? 0.01
2.62 ? 0.04
286.2 ? 0.7
11.9 ? 0.4
0.77 ? 0.07
993 ? 88
58.3 ? 1.0
0.40 ? 0.01
2.63 ? 0.03
288.1 ? 1.3
12.0 ? 0.4
0.86 ? 0.06
1133 ? 121
61.3 ? 1.4
0.41 ? 0.01
2.74 ? 0.01
290.3 ? 1.8
10.4 ? 0.3*
1.30 ? 0.10*
1458 ? 168*
65.3 ? 1.9*
0.36 ? 0.03
2.70 ? 0.05
287.2 ? 2.0
12.3 ? 0.3
0.76 ? 0.03
1480 ? 131
58.0 ? 1.4
0.38 ? 0.01
2.60 ? 0.03
290.6 ? 0.6
12.6 ? 0.6
0.80 ? 0.07
1571 ? 141
60.6 ? 0.9
0.37 ? 0.01
2.73 ? 0.02**
289.7 ? 0.9
13.1 ? 0.6
0.82 ? 0.06
1841 ? 224
61.1 ? 2.0
0.36 ? 0.01**
2.75 ? 0.04**
290.8 ? 1.0
11.4 ? 0.5
1.35 ? 0.09**
1810 ? 146
64.9 ? 1.1**
0.35 ? 0.01**
2.77 ? 0.02**
287.6 ? 0.4**
Note. cis-Nonachlor dose in units of mg/kg bw/day. Control rats (0 mg/kg dose group) received corn oil vehicle at comparable volumes to treated rats (0.5
ml/100 g bw). All data are expressed as mean ? SE for 6 ? n ? 7 rats. Units and abbreviations for clinical parameters: glucose (mM); cholesterol (mM); amylase
(International Units or IU/l); total protein (g/l); A/G or albumin/globulin ratio (no units); Ca or calcium (mM); osmolality (milliosmoles/kg). The following
parameters were not significantly altered by treatment with cis-nonachlor: blood urea nitrogen, creatinine, uric acid, albumin, immunoglobulins A, M, G1, G2a,
G2b, G2c, total bilirubin, triglycerides, AST, ALT, ALP, GGT, OCT, LDH, creatine kinase, thyroxine, thyroxine uptake, sodium, potassium, magnesium,
chloride, phosphorus.
*Significantly different from corresponding female control rats (p ? 0.05).
**Significantly different from corresponding male control rats (p ? 0.05).
TABLE 5
Clinical Changes in Serum from Male and Female Rats Receiving trans-Nonachlor by Gavage for 28 Days
Parameter
Females (Group B)Males (Group E)
0 mg/kg0.25 2.5 25a
00.252.5 25
Cholesterol
ALT
GGT
Amylase
BUN
T. protein
Albumin
A/G ratio
Ca
Mg
TU
0.78 ? 0.08
28.7 ? 2.3
1.9 ? 0.5
998 ? 56
14.7 ? 1.1
60.7 ? 0.8
17.3 ? 0.4
0.41 ? 0.01
2.58 ? 0.02
2.39 ? 0.06
46.7 ? 0.4
0.88 ? 0.03
28.1 ? 1.6
1.9 ? 0.7
1011 ? 29
16.6 ? 0.8
60.3 ? 1.4
18.1 ? 0.7
0.43 ? 0.01
2.69 ? 0.04
2.41 ? 0.06
46.4 ? 0.3
1.02 ? 0.06
26.9 ? 0.5
3.0 ? 0.7
1242 ? 96
15.6 ? 0.7
62.4 ? 0.5
17.1 ? 0.3
0.38 ? 0.01*
2.71 ? 0.02
2.44 ? 0.04
45.3 ? 0.2
2.78 ? 0.40*
44.3 ? 2.4
12.5 ? 5.6
1839 ? 43*
24.8 ? 1.7*
80.5 ? 1.9*
21.5 ? 0.7*
0.36 ? 0.01*
2.92 ? 0.05*
2.83 ? 0.03*
43.3 ? 0.9*
0.86 ? 0.08
22.1 ? 1.3
1.0 ? 0.2
1317 ? 86
15.3 ? 1.7
57.6 ? 1.3
15.1 ? 0.6
0.35 ? 0.01
2.64 ? 0.03
2.27 ? 0.05
47.9 ? 0.6
0.77 ? 0.06
24.4 ? 2.1
1.3 ? 0.5
1404 ? 83
14.0 ? 1.1
59.3 ? 1.6
16.1 ? 0.4
0.37 ? 0.01
2.66 ? 0.03
2.24 ? 0.06
47.9 ? 0.3
0.89 ? 0.07
28.9 ? 2.5
0.7 ? 0.3
1557 ? 68
16.0 ? 1.0
62.4 ? 1.4
16.6 ? 0.4
0.36 ? 0.01
2.82 ? 0.03**
2.21 ? 0.04
47.2 ? 0.3
1.86 ? 0.09**
32.6 ? 2.7**
3.4 ? 0.8**
1974 ? 87**
16.6 ? 0.3
73.3 ? 1.7**
18.6 ? 0.6**
0.34 ? 0.01
2.95 ? 0.05**
2.50 ? 0.06**
44.7 ? 0.3
Note. trans-Nonachlor dose in units of mg/kg bw/day. Control rats (0 mg/kg dose group) received corn oil vehicle at comparable volumes to treated rats (0.5
ml/100 g bw). All data are expressed as mean ? SE for 6 ? n ? 7 rats, except for females in the 25 mg/kg dose group where n ? 4.Units and abbreviations
for clinical parameters: cholesterol (mM); ALT or alanine aminotransferase (International Units or IU/L); GGT or ?-glutamytransferase (IU/L); amylase (IU/L);
BUN, or blood urea nitrogen (mg/dL); total protein (g/L); albumin (g/L); A/G or albumin/globulin ratio (no units); Ca or calcium (mM); Mg or magnesium (mM);
TU or thyroxine uptake (%). The following parameters were not significantly altered by treatment with trans-nonachlor: glucose, creatinine, uric acid,
immunoglobulins A, M, G1, G2a, G2b, G2c, total bilirubin, triglycerides, AST, ALP, OCT, LDH, creatine kinase, thyroxine, sodium, potassium, chloride,
phosphorus, osmolality.
aRats in this dose group were necropsied on day 24; all other rats were necropsied on day 29.
*Significantly different from corresponding female control rats (p ? 0.05).
**Significantly different from corresponding male control rats (p ? 0.05).
391
TRANS- AND CIS-NONACHLOR TOXICITY

Page 7

cellular hypertrophy and paler, more homogenous hepatocyte
cytoplasm. Similarly, cis-nonachlor, trans-nonachlor, and
chlordane caused dose-related increases in the extent and in-
cidence of anisokaryosis. These changes were evident at lower
doses in male and female rats treated with trans-nonachlor
compared to rats treated with cis-nonachlor or technical chlor-
dane. In general, there was a dose-related progression of le-
sions from zone 3 only, to zone 3 and/or zones 2 and 3, to
zones 2 and 3 in the most affected dose. Vacuolation (PAS
negative and oil red O positive), producing a foamy appearance
of the cytoplasm, was evident in occasional to numerous zone
2 hepatocytes, occasionally extending into zone 3 hepatocytes.
The incidence of cytoplasmic vacuolation was highest in male
rats treated with 25 mg cis-nonachlor/kg bw and in males and
females treated with 25 mg trans-nonachlor/kg bw (Table 7).
Kidney lesions were observed only in male rats. In males
treated with technical chlordane at 25 mg/kg bw/day, all rats
had occasional to numerous tubule segments in the cortex and
outer stripe of the outer medulla in which there was hyperplasia
of epithelial cells and rare to occasional necrosis and sloughing
of epithelial cells. Also observed in this dose group were rare
to numerous tubule luminae near the junction of the inner and
outer stripes of the outer medullas containing proteinaceous
material or cell debris. Similar but less severe changes were
observed in 2 rats receiving 0.25 mg chlordane/kg and in 3 rats
receiving 2.5 mg chlordane/kg (3/7 rats). In the kidneys of rats
treated with trans-nonachlor, similar changes were seen in one
rat in the 2.5 mg/kg dose group and in 3 rats in the 25 mg/kg
dose group. Kidney lesion incidence is summarized in Table 7.
In the thyroids of control rats and of rats treated with 0.25
mg/kg cis-nonachlor, trans-nonachlor, or technical chlordane,
spherical follicles with squamous to cuboidal epithelium were
evident. In 2 male control rats (Group D), in a few male rats at
the 2.5 mg/kg dose level for trans-nonachlor and chlordane,
FIG. 1.
ney slices from male rats gavaged with cis-nonachlor (Group D), trans-
nonachlor (Group E), and technical chlordane (Group F) for 28 days. Each bar
represents the mean ? SE from a group of 4 rats. *Significantly different from
corresponding control group.
Uptake of the organic cation tetraethylammonium (TEA) in kid-
TABLE 6
Clinical Changes in Serum from Male and Female Rats Receiving Technical Chlordane by Gavage for 28 Days
Parameter
Females (Group C)Males (Group F)
0 mg/kg 0.252.5 2500.25 2.525
Cholesterol
Triglycerides
AST
Amylase
BUN
CK
T. protein
A/G ratio
Ca
Mg
T4
TU
0.82 ? 0.06
0.68 ? 0.08
77.3 ? 5.3
1429 ? 262
16.6 ? 1.2
301 ? 33
62.4 ? 1.7
0.43 ? 0.01
2.73 ? 0.05
2.51 ? 0.08
41.0 ? 3.9
46.0 ? 0.4
0.77 ? 0.02
0.66 ? 0.13
77.6 ? 8.2
1386 ? 275
17.0 ? 0.7
363 ? 62
59.3 ? 1.2
0.39 ? 0.02
2.59 ? 0.03
2.31 ? 0.03
36.6 ? 5.3
46.3 ? 0.6
0.83 ? 0.04
0.52 ? 0.07
78.0 ? 4.2
1195 ? 159
15.7 ? 1.2
436 ? 49
60.0 ? 1.0
0.41 ? 0.00
2.63 ? 0.01
2.34 ? 0.07
42.0 ? 5.4
46.1 ? 0.3
1.33 ? 0.05*
0.50 ? 0.07
74.7 ? 4.2
1334 ? 72
16.2 ? 0.6
362 ? 44
66.7 ? 0.3
0.37 ? 0.00*
2.71 ? 0.03
2.45 ? 0.05
40.3 ? 4.7
43.4 ? 0.4*
0.75 ? 0.04
0.74 ? 0.11
103.4 ? 2.8
1629 ? 227
14.4 ? 0.5
589 ? 52
60.0 ? 1.2
0.36 ? 0.01
2.70 ? 0.03
2.36 ? 0.07
70.1 ? 2.6
48.0 ? 0.3
0.74 ? 0.05
0.73 ? 0.08
92.3 ? 5.9
1377 ? 88
13.1 ? 0.7
526 ? 58
58.1 ? 1.4
0.35 ? 0.01
2.67 ? 0.03
2.17 ? 0.06
59.4 ? 4.7
47.2 ? 0.4
0.78 ? 0.04
0.83 ? 0.15
87.7 ? 8.5
1932 ? 278**
14.9 ? 1.0
493 ? 81
58.7 ? 1.0
0.36 ? 0.01
2.65 ? 0.03
2.29 ? 0.06
66.4 ? 3.5
48.3 ? 0.3
1.33 ? 0.07**
0.46 ? 0.06**
72.4 ? 6.9**
1879 ? 57**
17.6 ? 0.9**
263 ? 70**
65.4 ? 1.2**
0.32 ? 0.01**
2.82 ? 0.02**
2.54 ? 0.06**
42.9 ? 3.5**
46.1 ? 0.6**
Note. Technical chlordane dose in units of mg/kg bw/day. Control rats (0 mg/kg dose group) received corn oil vehicle at comparable volumes to treated rats
(0.5 ml/100 g bw). All data are expressed as mean ? SE for 6 ? n ? 7 rats.Units and abbreviations for clinical parameters: cholesterol (mM); triglycerides (mM);
AST or aspartate aminotransferase (International Units or IU/L); amylase (IU/L); BUN or blood urea nitrogen (mg/dL); CK or creatine kinase (IU/L); total protein
(g/L); A/G or albumin/globulin ratio (no units); Ca or calcium (mM); Mg or magnesium (mM); T4or thyroxine (nM); TU or thyroxine uptake (%). The following
parameters were not significantly altered by treatment with technical chlordane: glucose, creatinine, uric acid, albumin, immunoglobulins A, M, G1, G2a, G2b,
G2c, total bilirubin, ALT, ALP, GGT, OCT, LDH, sodium, potassium, chloride, phosphorus, osmolality.
*Significantly different from corresponding female control rats (p ? 0.05).
**Significantly different from corresponding male control rats (p ? 0.05).
392
BONDY ET AL.

Page 8

and in male and female rats at the 25 mg/kg dose level for
cis-nonachlor, trans-nonachlor, and chlordane, numerous as-
pherical follicles with cuboidal to columnar epithelium were
evident in the thyroid. In some high-dose rats, the aspherical
follicles dominated. Follicular changes in the thyroids of rats
treated with 25 mg/kg trans-nonachlor can be seen in Figure 2.
Thyroid lesion incidence is summarized in Table 7.
Residue Analyses
Residue analyses confirmed that there was dose-dependent
accumulation of the test chemicals and their metabolites in the
tissues of treated rats. In cis-nonachlor-treated rats the primary
residues in adipose tissue and liver were cis-nonachlor and
oxychlordane (Table 8). In adipose tissue from males and
females there was approximately 3? to 4?more cis-nonachlor
than oxychlordane. In liver cis-nonachlor and oxychlordane
levels were similar, with the exception of males in the 0.25
mg/kg dose group which had 4.5? more cis-nonachlor than
oxychlordane in liver. In male rats small amounts of trans-
nonachlor were detected in adipose tissues, at levels approxi-
mately 400–500? lower than cis-nonachlor (Table 8). Overall,
female rats accumulated more cis-nonachlor and oxychlordane
in adipose tissue than male rats (Table 8). Residues in blood
were measured only in cis-nonachlor-treated male rats. At the
25 mg cis-nonachlor/kg dose level, mean blood levels of cis-
nonachlor and oxychlordane were 0.2 p.p.m. and 0.3 p.p.m.,
respectively. cis-Chlordane, trans-chlordane and heptachlor
were not detected in adipose tissue, liver or blood of rats
treated with cis-nonachlor.
In trans-nonachlor-treated rats, trans-nonachlor and oxy-
TABLE 7
Summary of the Incidence of Liver, Kidney and Thyroid Lesions in Rats Receiving cis-Nonachlor, trans-Nonachlor,
or Technical Chlordane by Gavage for 28 Days
Lesion
Females Males
00.25 2.52500.25 2.525
cis-Nonachlor
Liver
Hypertrophy
Pale, homogenous cytoplasm
Anisokaryosis
Fat vacuoles
Kidneya
Thyroid
Aspherical follicles
trans-Nonachlor
Liver
Hypertrophy
Pale, homogenous cytoplasm
Anisokaryosis
Fat vacuoles
Kidneya
Thyroid
Aspherical follicles
Technical chlordane
Liver
Hypertrophy
Pale, homogenous cytoplasm
Anisokaryosis
Fat vacuoles
Kidneya
Thyroid
Aspherical follicles
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
6/6
6/6
5/6
1/6
0/6
0/7
0/7
0/7
0/7
0/7
1/7
0/7
0/7
0/7
0/7
3/7
3/7
0/7
1/7
0/7
7/7
7/7
2/7
7/7
0/7
0/70/70/7 2/62/70/70/7 1/7
0/7
0/7
0/7
0/7
0/7
2/7
2/7
0/7
0/7
0/7
7/7
7/7
2/7
0/7
0/7
4/4b
4/4
4/4
4/4
0/4
0/7
0/7
0/7
0/7
0/7
2/7
0/7
0/7
0/7
0/7
4/7
0/7
0/7
2/7
1/7
7/7
7/7
4/7
7/7
3/7
0/70/7 0/71/4 0/70/75/7 5/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
1/7
0/7
1/7
0/7
7/7
7/7
7/7
1/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
0/7
2/7
0/7
0/7
0/7
0/7
3/7
7/7
7/7
2/7
3/7
7/7
0/70/70/7 3/6 0/70/7 3/74/7
Note. Test chemical dose values given in mg test chemical/kg bw/day. Incidence of lesion (# animals affected/# animals assessed). Assessment of lesion
severity was not undertaken because some slides were compromised by nonspecific artefacts associated with tissue manipulation at necropsies and processing.
This did not interfere with the identification of morphological changes in treated rats or with the enumeration of lesion incidence in groups of treated rats, as
described in the above table.
aKidney lesions: occasional to numerous tubule segments in the cortex and outer stripe of the outer medulla in which there was hyperplasia of epithelial cells
and rare to occasional sloughing of epithelial cells; rare to numerous tubule luminae near the junction of the inner and outer stripes of the outer medulla containing
proteinaceaous material or cell debris.
bRats in this dose group were necropsied on day 24; all other rats were necropsied on day 29.
393
TRANS- AND CIS-NONACHLOR TOXICITY

Page 9

chlordane were detected in adipose tissue and liver (Table 9).
In adipose tissue, trans-nonachlor levels ranged between 2.5?
to 6.9? higher than oxychlordane levels between sexes and
dose groups. In liver, trans-nonachlor levels ranged from 0.6?
to 3.9? oxychlordane levels. Female rats accumulated higher
residue levels than males, particularly of trans-nonachlor (Ta-
ble 9). Residues in blood were measured only in trans-nona-
chlor-treated male rats. At the 25 mg trans-nonachlor/kg dose,
mean blood levels of trans-nonachlor and oxychlordane were
3.7 and 2.5 ppm, respectively. cis-Chlordane, trans-chlordane,
cis-nonachlor, and heptachlor were not detected in adipose
tissue, liver, or blood of rats treated with trans-nonachlor.
Tissues from rats treated with technical chlordane were
screened for oxychlordane, cis-nonachlor, trans-nonachlor,
heptachlor, cis-chlordane and trans-chlordane. Residue levels
in adipose tissues are summarized in Table 10. Oxychlordane
and trans-nonachlor were present in the greatest quantities,
with lesser amounts of cis-nonachlor, trans-chlordane, and
heptachlor. Adipose tissue from both males and females had
approximately 2? more oxychlordane than trans-nonachlor.
Female rats had approximately 2? more oxychlordane than
trans-nonachlor in adipose tissue than male rats (Table 10).
Liver residues consisted primarily of trans-nonachlor and oxy-
chlordane. Liver from females receiving 25 mg technical chlor-
dane/kg had 2.1 and 5.9 ppm trans-nonachlor and oxychlor-
dane, respectively. Liver from males at the same dose level had
1.2 and 7.7 ppm trans-nonachlor and oxychlordane, respec-
tively. Residues in blood were measured only in chlordane-
treated male rats. At the 25 mg chlordane/kg dose, mean blood
levels of trans-nonachlor and oxychlordane were and 0.1 and
0.4 ppm, respectively. cis-Chlordane residues were not de-
tected in adipose tissue, liver or blood of rats treated with
technical chlordane.
DISCUSSION
Only trans-nonachlor was overtly toxic at the levels tested in
this study, with 43% mortality by day 23 in female rats receiv-
ing 25 mg trans-nonachlor/kg bw/day. Gross and histopathol-
ogy of these and the remaining female rats in this dose group
FIG. 2.
treated with corn oil (A) or 25 mg/kg trans-nonachlor (B) by gavage for 28
days. Numerous aspherical follicles are evident in (B).
Periodic acid Schiff (PAS)-stained sections of male rat thyroid
TABLE 8
Residues in Adipose Tissues and Liver of Rats Receiving cis-Nonachlor for 28 Days by Gavage
Tissue Residuea
Females (Group A)Males (Group D)
00.25 2.5250 0.252.5 25
AdiposeOxy
Cis
Trans
Oxy
Cis
0.0 ? 0.0
0.1 ? 0.0
0.0 ? 0.0
0.0 ? 0.0
0.0 ? 0.0
3.5 ? 0.2
14.3 ? 3.1
0.0 ? 0.0
0.2 ? 0.0
0.4 ? 0.1
28.6 ? 2.2
90.5 ? 9.2
0.0 ? 0.0
1.4 ? 0.1
2.1 ? 0.3
326.9 ? 36.7
868.8 ? 89.4
0.0 ? 0.0
15.6 ? 1.7
15.8 ? 4.1
0.0 ? 0.0
0.1 ? 0.0
0.0 ? 0.0
0.0 ? 0.0
0.1 ? 0.0
2.7 ? 0.2
7.6 ? 0.8
0.0 ? 0.0
0.2 ? 0.0
0.9 ? 0.7
13.2 ? 1.6
42.8 ? 5.1
0.1 ? 0.1
1.3 ? 0.1
0.9 ? 0.3
136.1 ? 9.5
542.6 ? 99.6
1.1 ? 0.3
12.3 ? 0.4
11.8 ? 5.7
Liver
Note. cis-Nonachlor dose in mg/kg bw/day. Data for adipose tissue are expressed as ?g/g lipid; data for liver are expressed as ?g/g tissue wet weight. All data
are expressed as mean ? SE for 6 ? n ? 7 rats. All tissue samples were analysed for oxychlordane, cis-nonachlor, trans-nonachlor, cis-chlordane and
trans-chlordane as described in the Materials and Methods section; only detectable residues are included in the table. Abbreviations: Oxy ? oxychlordane; Cis ?
cis-nonachlor; Trans ? trans-nonachlor.
aThe following residues were not detected in adipose tissue and liver of cis-nonachlor-treated rats: cis-chlordane, trans-chlordane, heptachlor.
394
BONDY ET AL.

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indicated that the primary target organ was the liver, but did
not indicate a definitive cause of death. Further studies are
planned to assess the possible cause(s) of death and the severity
of lesions described in this study. Test chemical and metabolite
residue levels in adipose were higher in females treated with
trans-nonachlor than in any other test group, pointing to a
connection between chemical burden and toxicity.
Mean daily food and water consumption were significantly
lower in female rats treated with 25 mg/kg trans-nonachlor,
which was consistent with a significant decrease in both mean
weight gain and mean final bw. In all other groups, significant
changes in food consumption, water consumption, or weight
gain did not result in significant changes in the final bws of
treated rats compared to control rats. It is likely that the
cumulative effects of changes in food and water consumption
over a longer treatment period would have eventually resulted
in decreased bws, but these effects did not appear within the
time frame of the present study.
Technical chlordane causes increased liver weights in rats
(Khasawinah and Grutsch, 1989; Ogata and Izushi, 1991). as
was confirmed in this study. This change is considered to be an
adaptive response associated with increased liver microsomal
enzyme activity and not an adverse effect (U.S. DHHS, 1994).
The present study confirmed that oral exposure to trans- and
cis-nonachlor had similar effects on rat livers, although trans-
nonachlor was more potent. Both chemicals caused increased
liver weights, which were highest in rats exposed to trans-
nonachlor. The observation of liver cell hypertrophy in treated
rats was consistent with increased liver weight, as was the
observation of hepatic drug metabolizing enzyme induction in
treated rats in the present study (I. Curran, unpublished data).
Technical chlordane, oxychlordane, trans-chlordane and cis-
chlordane have previously been shown to induce rat hepatic
microsomal enzymes (Campbell et al., 1983). The increased
incidence of lipid-filled vacuoles in livers of treated rats in the
present study is consistent with increases in liver total lipids,
triglycerides, and phospholipids measured in rats gavaged with
100 mg chlordane/kg bw for 4 days (Ogata and Izushi, 1991).
Increased serum cholesterol, which is associated with hepatic
changes, was more pronounced in rats treated with trans-
nonachlor than in those treated with cis-nonachlor or technical
chlordane. Increased serum alanine aminotransferase (ALT),
TABLE 9
Residues in Adipose Tissue and Liver of Rats Receiving trans-Nonachlor for 28 Days by Gavage
Tissue Residuea
Females (Group B) Males (Group E)
0 0.25 2.525b
0 0.252.5 25
AdiposeOxy
Trans
Oxy
Trans
0.0 ? 0.0
0.0 ? 0.0
0.0 ? 0.0
0.0 ? 0.0
5.9 ? 0.8
30.6 ? 5.0
0.2 ? 0.0
0.7 ? 0.1
48.6 ? 4.5
158.9 ? 31.0
2.4 ? 0.1
2.5 ? 0.4
527.1 ? 40.0
3613.7 ? 1022.1
24.3 ? 0.9
95.7 ? 29.4
0.0 ? 0.0
0.1 ? 0.0
0.0 ? 0.0
0.0 ? 0.0
5.0 ? 0.3
13.7 ? 1.1
0.4 ? 0.0
0.7 ? 0.1
29.2 ? 16.3
72.2 ? 6.7
3.6 ? 0.3
2.2 ? 0.5
428.2 ? 16.1
1367.7 ? 98.8
31.5 ? 1.5
57.2 ? 7.4
Liver
Note. trans-Nonachlor dose in mg/kg bw/day. Data for adipose tissue are expressed as ?g/g lipid; data for liver are expressed as ?g/g tissue wet weight. All
data are expressed as mean ? SE for 6 ? n ? 7 rats, except for females in the 25 mg/kg dose group where n ? 4. All tissue samples were analysed for
oxychlordane, cis-nonachlor, trans-nonachlor, cis-chlordane and trans-chlordane as described in the Methods section; only detectable residues are included in
the table. Abbreviations: Oxy ? oxychlordane; Trans ? trans-nonachlor.
aThe following residues were not detected in adipose tissue and liver of trans-nonachlor-treated rats: cis-nonachlor, cis-chlordane, trans-chlordane, heptachlor.
bRats in this dose group were necropsied on day 24; all other rats were necropsied on day 29.
TABLE 10
Residues in Adipose Tissue of Rats Receiving Technical Chlordane for 28 Days by Gavage
Residuea
Females (Group C)Males (Group F)
00.25 2.52500.25 2.525
Oxy
Cis
Trans
Hepta
Gamma
0.0 ? 0.0
0.0 ? 0.0
0.0 ? 0.0
0.0 ? 0.0
0.0 ? 0.0
3.1 ? 0.3
0.2 ? 0.0
1.7 ? 0.1
0.0 ? 0.0
0.1 ? 0.01
19.6 ? 1.7
2.1 ? 0.4
11.9 ? 2.5
0.1 ? 0.0
1.8 ? 0.4
226.9 ? 36.8
13.8 ? 1.7
106.5 ? 21.7
0.5 ? 0.1
12.6 ? 1.1
0.0 ? 0.0
0.0 ? 0.0
0.0 ? 0.0
0.0 ? 0.0
0.0 ? 0.0
1.2 ? 0.1
0.1 ? 0.0
0.8 ? 0.1
0.0 ? 0.0
0.0 ? 0.0
8.4 ? 0.5
0.9 ? 0.1
4.0 ? 0.2
0.0 ? 0.0
0.8 ? 0.1
97.6 ? 3.4
9.0 ? 0.5
49.2 ? 2.3
0.6 ? 0.0
11.4 ? 0.7
Note. Technical chlordane dose in mg/kg bw/day. Data for adipose tissue are expressed as ?g/g lipid. All data are expressed as mean ? SE for 6 ? n ? 7
rats. Abbreviations: Oxy ? oxychlordane; Cis ? cis-nonachlor; Trans ? trans-nonachlor; Hepta ? heptachlor; Gamma ? trans-chlordane.
acis-Chlordane was not detected in adipose tissues of rats receiving technical chlordane.
395
TRANS- AND CIS-NONACHLOR TOXICITY

Page 11

which can be indicative of hepatotoxicity, was observed in
male rats treated with trans-nonachlor. This increase was sta-
tistically significant but the degree of elevation was modest and
there was no corresponding hepatocellular necrosis in liver
sections.
Renal changes were sex-specific and were statistically sig-
nificant in male, but not female rats. Elevated kidney weights
in male rats were a general index of renal changes. Standard
urinalysis endpoints such as protein, NAG, and osmolality
were not sufficiently sensitive to indicate any corresponding
functional changes. Changes in blood urea nitrogen were in-
consistent and did not correspond to changes in kidney
weights. However, changes in organic ion transport in renal
cortical slices have been used extensively to study nephrotoxic
compounds (Berndt, 1987) and significant depression of this
parameter in male rats confirmed that changes in kidney
weights were accompanied by altered kidney function. Uptake
of the organic cation TEA was inhibited by 32, 24, and 17% in
trans-nonachlor, cis-nonachlor, and chlordane-treated male
rats, respectively. Since uptake of the organic anion PAH was
unaffected, the results suggest that damage to renal tubular
cells was specific to the cationic transport system and was not
due to nonspecific, membrane-related perturbations.
Morphological changes were observed in kidneys from male
rats but not females. This was consistent with the sex-related
nature of renal functional changes. However, a comparison of
changes in kidney morphology and other indices of renal
toxicity in rats treated with cis-nonachlor, trans-nonachlor, or
chlordane revealed several inconsistencies. First, there were no
visible renal lesions in male rats treated with cis-nonachlor, in
spite of increased kidney weights and decreased TEA transport
in these animals. This implies that there was not necessarily a
direct relationship between the renal toxicity endpoints used in
this study and the observed structural changes. Second, chlor-
dane-treated rats had the highest incidence of visible kidney
lesions, indicating that components of chlordane other than
cis-nonachlor and trans-nonachlor were partly responsible for
renal changes. Overall, the consequences of renal changes were
not immediately apparent from the healthy outward appearance
of male rats and from the lack of consistent changes in urine
volume and water consumption.
Treatment with each of the test chemicals resulted in ele-
vated serum total protein at the highest test dose, with trans-
nonachlor causing the most pronounced changes. The lack of
significant or consistent changes in serum and urine osmolality
and urine output indicates that dehydration was not responsible
for increased serum total proteins. Serum immunoglobulins
can be ruled out as contributors to increased serum total protein
levels, as they were not significantly altered by any of the test
chemicals. In trans-nonachlor-treated rats, increased serum
albumin appears to account in part for elevated total protein,
but this is not the case for rats treated with cis-nonachlor and
technical chlordane, indicating that more in-depth analyses are
necessary to determine which proteins are elevated in treated
rats. Serum magnesium, and to a greater extent serum calcium,
were elevated in some treatment groups. Both of these ions can
be elevated by increases in plasma carrying proteins (Riley and
Cornelius, 1989), so it is possible that elevated serum total
protein levels play a role in the observed increases in serum
calcium and magnesium.
The induction of hepatic microsomal enzymes by chlori-
nated hydrocarbons, including chlordane, has been shown to
disrupt thyroid hormone metabolism (Capen, 1994). It is pos-
sible that microsomal enzyme induction played a part in the
reduction of serum thyroxine (T4) and thyroxine uptake in
chlordane-treated rats and in reduced T4uptake in trans-nona-
chlor-treated rats. However, altered protein levels may also
have had an effect on serum T4and T4uptake as albumin is an
important T4binding protein in rats (Capen, 1992; Do ¨hler et
al., 1979). The interaction of these factors and their relation-
ship to the increased incidence of irregularly shaped follicles in
the thyroids of rats given cis-nonachlor, trans-nonachlor, or
chlordane remains to be determined. It is possible that the
thyroid changes observed in the present study were transient.
Barrass et al (1993) used bromodeoxyuridine labeling to mea-
sure increased cellular proliferation in the rat thyroid, which
peaked after 5 days of receiving 50 ppm chlordane in the diet,
but which was not evident after day 99. Increased thyroid cell
proliferation was not accompanied by histopathological
changes in sections stained with hematoxylin and eosin. In
addition, there were no histopathological changes in the thy-
roids of rats ingesting chlordane in the diet at levels up to 25
ppm for 130 weeks (Khasawinah and Grutsch, 1989).
One of the most consistent changes in rats treated with
cis-nonachlor, trans-nonachlor, and technical chlordane was
increased serum amylase, primarily at the 25 mg/kg dose level.
This effect was most pronounced in rats treated with trans-
nonachlor. Increased circulating amylase is associated with
pancreatic damage, which results in leakage of pancreatic
enzymes into the peripancreatic area and their subsequent
absorption into the general circulation (Short, 1961). The ab-
sence of histopathological changes in the pancreas of treated
rats rules out pancreatitis, which is the condition most often
associated with elevated serum amylase. Other possibilities for
future consideration include direct effects on serum amylase at
the level of enzyme production, release, and degradation that
may affect the half-life of circulating amylase.
The primary metabolite of cis-nonachlor, trans-nonachlor,
and technical chlordane was oxychlordane, as indicated by
tissue residues. In cis- and trans-nonachlor-treated rats the
parent compounds also accumulated in adipose tissue and liver
at higher levels than oxychlordane. This was also observed by
Hirasawa and Takizawa (1989), who showed that in mice,
nonachlor metabolism is slower than chlordane metabolism
and large amounts of unchanged nonachlors are retained in the
tissues. Since trans-nonachlor has been shown to be rapidly
metabolized in rat liver microsomes in vitro (Tashiro and
Matsumura, 1978), it is possible that the proportion of cis- or
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BONDY ET AL.

Page 12

trans-nonachlor to oxychlordane residues would have de-
creased in rat tissues over a longer treatment period. In addition
to cis- and trans-nonachlor, oxychlordane is an important
metabolite of other components of the technical chlordane
mixture, including cis- and trans-chlordane (Barnett and Dor-
ough, 1974). This supports the observation that oxychlordane
was the most abundant tissue residue in liver and adipose tissue
in rats treated with technical chlordane.
The pattern of residue accumulation in rats treated with cis-
and trans-nonachlor in this study was similar in several re-
spects to the pattern of accumulation observed in rats treated
with cis- and trans-chlordane (Barnett and Dorough, 1974).
First, residue levels were highest in adipose tissue. Second,
treatment with trans-nonachlor resulted in higher tissue oxy-
chlordane levels than treatment with cis-nonachlor. Finally,
residue levels were higher per gram of adipose tissue in fe-
males than in males, whereas residue levels per gram of liver
were similar for both sexes. In the present study the mean final
bws of female rats reached a maximum of 259.8 g, compared
to 465.6 g in male rats. Assuming that male rats had more total
body fat at necropsy than females and that chlordane-related
compounds preferentially localize to adipose tissues, it is pos-
sible that increased fat residues in females were the result of
test chemical localization into a smaller pool of fat tissue. This
cannot be confirmed because total body fat percentages in male
versus female rats were not determined in this study. High
adipose tissue residue levels appeared to correlate with overt
toxicity, as the highest levels were measured in trans-nona-
chlor-treated female rats. Since organochlorine compounds can
be transferred to the fetus via the placenta and to the infant via
breast milk (Skaare et al., 1988), a possible sex-related differ-
ence in tissue organochlorine accumulation merits further at-
tention. Although dietary surveys indicate that women in their
childbearing years in aboriginal communities are not always
the maximum consumers of organochlorine contaminants in
wildlife food (Kuhnlein et al., 1995), the potential for greater
residue accumulation in body fat of females based on sex-
related differences in size and percentage of body fat are
factors to be considered when estimating the impact of dietary
organochlorine exposure.
Based on the present study, the target organs and effects of
cis-nonachlor, trans-nonachlor, and technical chlordane in rats
were generally similar. However, trans-nonachlor accumula-
tion in adipose tissue was greater than cis-nonachlor when rats
were administered each chemical under identical conditions of
dose and exposure. This is consistent with the observation that
in the Arctic food chain, trans-nonachlor has been measured in
fat or muscle at levels 5 to 8 times higher than cis-nonachlor
levels in fish, seal, and polar bears (Muir et al., 1988). In
addition, human milk and breast adipose tissue has been shown
to be contaminated with trans-nonachlor at levels ranging from
2 to 12 times higher than cis-nonachlor levels (Dearth and
Hites, 1991b; Polder et al., 1998). Furthermore, in the present
study trans-nonachlor was overtly toxic to female rats and had
more pronounced effects than cis-nonachlor on some clinical
chemistry and histopathological endpoints. These results indi-
cate a need for further characterization of the long-term effects
of trans-nonachlor exposure as well as a need for further
examination of sex-related differences in responses to trans-
nonachlor.
ACKNOWLEDGMENTS
The authors would like to thank the following individuals for their valuable
contributions to this work: Ian Greer, Andre ´ Masse ´, Peter Smyth, and the staff
of the Animal Resources Division (Food Directorate, Health Canada). Special
thanks to Liisa Jantunen and Terry Bidleman (Atmospheric Environment
Service, Environment Canada) for providing analyses of the technical chlor-
dane mixture. Special thanks also to Dr. Ruedi Mueller for assistance with
preparation of photomicrographs.
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