- Email: [email protected]
Comparative disposition and elimination of chlordane in rats and mice
Toxicology
Letters,
233
26 (1985) 233-239
Elsevier
TOXLett.
1450
COMPARATIVE DISPOSITION RATS AND MICE (cis-[14C]Chlordane; excretion rates) ALAN
D. EWING,
Department
chlordane
ABDEL
of Entomology
AND ELIMINATION
toxicokinetics;
M. KADRY
tissue distribution
and H. WYMAN
and Graduate
OF CHLORDANE
IN
and dissipation;
DOROUGH*
Center for Toxicology,
University
of Kentucky,
Lexington,
KY 40546 (U.S.A.) (Received
August
(Accepted
June 7th,
lOth, 1984) 1985)
SUMMARY The absorption, was determined
distribution
slower in mice, but total ceeded the maximum observed
which occurred
equivalents
the total body burden
in both species were
by the findings
that peak tissue
rate was higher
than in rats. At
rates of tissue residues
resulting
somewhat at 8 h) ex-
for the latent peak blood levels in mice
in mouse and rat feces, respectively;
83% of the dose in the feces. Clearance
(113 ng/ml
at 2 h). Peak tissue residues responsible
(1 .O mg/kg)
appeared
concentration
by the tissues. This was supported
12 h, 34% and 7% of the doses were excreted rat, and consequently,
cis-[‘4C]chlordane
mice. Absorption
at peak blood
in rats (81 ng/ml
that the radiocarbon
rather than sequestered
administered
levels were lower in mice, and that the initial fecal elimination
had voided greater
of orally
rats and C57BL/6JX
[“‘Clchlordane
within 4 h, suggesting
was eliminated residue
and elimination
in male Sprague-Dawley
from chronic
by 3 days, both species
were markedly
exposure
to chlordane
faster in the will be far
in mice than in rats.
INTRODUCTION
Chlordane(l,2,4,5,6,7,8,8-octachloro-2,3,3a,4,7,7a-hexahydro-4,7-methano-lHindene) is an organochlorine insecticide which has been used extensively for the control of numerous pests since its introduction in the 1940s. The technical material is a complex mixture, whose insecticidal components are primarily the cis and truns isomers of chlordane [I]. Like other organochlorine pesticides, chlordane has been scrutinized closely by various regulatory agencies over the past several years, and
*To whom correspondence 39762, U.S.A. 0378-4274/85/$
03.30
should
0 Elsevier
be addressed;
Science
Publishers
present
B.V.
address
Drawer
GY, Mississippi
State,
MS
234
in 1978 a decision was made to phase out all use by 1983, except that for the control of underground termites [2]. The concern for the continued widespread use of chlordane is centered around its carcinogenicity in mice. Hepatocellular carcinoma is induced in mice given chlordane, but not in rats [3,4],. This apparent species-specificity provided the impetus for the present investigation. Attempts were made to determine if the general toxicokinetics of chlordane in rats and mice were sufficiently different to suggest that metaboIic peculiarities were reponsible for the selective tumor induction in mice. MaIe rats and mice were treated orally with c&chlordane, as our earlier work on the metaboIism of chlordane in rats [S] showed that either the cis or tram isomer would be satisfactory for comparing the fate of this insecticide in rats and mice. MATERIALS
AND
METHODS
Male Sprague-Dawley rats (22.5-27.5 g) were purchased from Harlan Industries (Indianapolis, IN) and male C57BL/6JX mice (20-25 g) from Jackson Labs (Bar Harbor, ME). The animals were maintained on a 12-h light cycle (21-23”C, 45-50% RH) in Nalgene metabolism cages (Nalge Co., Rochester, NY) designed for the separate collection of feces and urine. c~~-[~4~]~hlordane (28.1 mCi/mmoI) was obtained from Midwest Research Institute (Kansas City, MO) and was > 99% pure, as determined by GLC analysis, as described by Barnett and Dorough [5]. For treatment, the radiolabeled chlordane
Fig. 1. Mean levels of radiocarbon treatment
with 1 mg/kg
on day 14.
in the blood of male rats and mice over a 7 day period
ci#C]chlordane
(IV = 3-5). No radiocarbon
was detected
foltowing
in blood
oral
sampled
235
was diluted with sufficient analytical grade cis-chlordane (Velsicol Chemical Corp., Chicago, IL) for each animal treated with 1.0 mg/kg to receive approx. 1.5 x lo6 dpm. The chlordane was administered to the animals as a single oral dose in 0.2 ml of corn oil. Rodent Chow (Ralston Purina Co., St. Louis, MO) and tap water were provided ad lib; the animals were not fasted prior to treatment. Feces and urine were collected at 12-h intervals for 3 days and at 24-h intervals thereafter. Animals were killed at various times after treatment, at least 3/species/time, and tissue and/or blood samples taken for radioassay. Urine was counted directly (Beckman LS 9000 scintillation counter) while feces, tissues and whole blood were combusted (Packard 306 oxidizer) and the trapped 14C02 was assayed. Using 2 x background as the cut-off point, the limit of detection was approximately 10 ng of [14C]chlordane equivalents per g of the sample. RESULTS
AND DISCUSSION
Total [14C]chlordane equivalents in the blood of rats and mice following oral administration of cis-chlordane, 1 .O mg/kg, are shown in Fig. 1. The initial absorption, O-2 h, was similar in both groups of animals, although at each sampling time the levels were higher in the rat. Even at 15 min (data not shown) the blood of rats contained 14.1 ? 2.4 ng/ml and that of mice only 3.4 f 0.9 (mean f SEM) [14C]chlordane equivalents. The major difference between the species was that peak blood levels in the rat (81 ng/ml) occurred at 2 h, while the peak concentration in mouse blood (113 ng/ml) did not occur until 8 h. Once the peak concentrations were reached, the rates of clearance of the residues from the blood was greater in rats. For example, the mean [‘4C]chlordane equivalents in rat blood at 24 h was 36% of the peak level, whereas for mice the
TABLE
1
[r+C]CHLORDANE WITH
1 .O mg/kg
EQUIVALENTS
IN TISSUES
OF RATS
AND
MICE
TREATED
ORALLY
CIS-[‘4C]CHLORDANE
Tissue
O-24-h Tissue/blood Rats
ratio” Mice
Brain
1.7
0.6
Liver
17.6
11 .o
Peak concentrationb Rats 221 k
(ng/g) Mice
23
1959 + 114
68*
I
1180 + 74
Kidney
8.1
3.2
129 t
57
349 + 30
Lungs
2.6
3.0
217 +
4
248 + 21
Testes
1.6
0.9
135 *
12
92 + 17
Muscle
1.2
1.9
130 +
29
164 k 42
1239 & 299
808 + 42
Fat _ aMean
of determinations
bMean
+ SEM. Peak concentrations
at 2 h.
19.8
8.8
made at 2, 4, 12 and 24 h. N = 3-5. occurred
at 4 h, except for rat brain,
liver, and muscle,
which was
236
level was 52% of the maximum, which occurred at 8 h. While the patterns of accumulation in the blood suggested that absorption of chlordane was slower in mice, the difference in clearance rates indicated that the material absorbed was, at least in part, different in the 2 species. The same inference was evident from the results of tissue analysis (Table 1). Of the 7 tissues assayed, the peak concentrations occurred either at 2 or 4 h. Such an accumulation pattern was consistent with the rat blood data, but inconsistent with the latent peak blood levels in the mouse. Had the material absorbed during the 2-8 h period by the mouse been the same as that absorbed during the first 2 h, then peak tissue residues would have occurred at the 8- or 12 h sampling intervals. Since they peaked at 4 h, as did rat tissue residues, it appears that the material absorbed initially was the same in rats and mice, and that the nature of the sequestered residues was largely the same. In rats, the major tissue residue following chlordane treatment was the metabolite oxychlordane [5]. The findings that the 2 h blood of mice contained less [14C]chlordane equivalents than rats (Fig. 1) and that mice also had lower peak residues in tissues (Table I) are in agreement with the supposition that the initial absorption and deposition of cam-chlordane was essentially the same in rats and mice. Evidence that later differences occurred was the latent peak blood residues in mice, which failed to increase tissue residue levels, and the difference in the tissue/blood ratios between the 2 groups of animals (Table I). The ratios were consistently higher in the rat, the greatest difference being in liver and fat.
TABLE
II
CLEARANCE TREATED
OF [14CJCHL~RDANE ORALLY
WITH
Percent Tissue Brain
EQUIVALENTS
1 .O mg/kg
Animal
12 h
FROM
of peak concentration
Rats
23+
4
62~
7
OF RATS
AND
after”
1 Day
Mice
TISSUES
Cam-~‘~C]CHLORDANE.
14 Days
7 Days 1
0
0
34 + 12
0
0
5+
Rats
40 z?z 14
II?
3
3-t
0
4*
1
Mice
78rt
9
19rt_
9
17rt
5
lo+
3
Kidney
Rats
47t
7
26 +- 10
si
3
82 i
IS
44 + 12
17-+
5
Lung
Mice Rats
26 I
IO
lo+
2
41
0
Mice
79t
9
68i
4
3oi
7
Testes
Rats
46 rt 15
30*
9
0
Mice
7 11
58 + 13 15i 2
38& 0
29+ 0
5
Rats
79rt 31 I
5
Muscle Fat
Mice Rats Mice
801 4 76 i 10 72 i: 14
60 + 14 71* 3 68 ? 17
45* 6 IO* 1 37 + I1
26-+8i: 19t
9 0 7
Liver
aData
presented
.--
as means
..__.
+ SEM of peak concentration
shown
in Table
s-+182
1 1
0 13i
3 0
I.
MICE
237
The clearance of the tissue residues was faster in rats than in mice (Table II) which might further suggest that the nature of the deposited residues were partially dissimilar. However, this could well be a function of blood-flow differences in the 2 species or of other inherent physiological differences. Nonetheless, a marked difference was evident, especially at 12 h, when the mean dissipation from peak levels, considering all tissues, was 59% for rats and only 24% for mice. From the standpoint of chlordane-induced hepatocellular carcinoma in mice but not rats, it was particularly interesting that the mean liver residue level in mice after 12 h was 78% of the peak concentration, and only 40% in rats. It must be pointed out, however, that differences of a similar magnitude were apparent with other tissues and, unlike the liver, the differences (rat vs. mouse) in some tissues were maintained throughout the lbday test period (Table II). Higher [14C]chlordane equivalents in mouse blood, but lower tissue residues as compared to rats, might be explained by greater plasma protein binding in the mouse or by more rapid elimination in this species. As seen in Fig. 1, the dissipation of residues from the blood did not indicate that protein binding was appreciably greater in mice than in rats. However, the elimination of [‘4C]chlordane equivalents in the feces and urine was initially much higher in mice (Table III). Mice designated as high excretors in Table III represented the vast majority of individuals of this species used in the study and it was from these animals that data were obtained for
TABLE
III
ELIMINATION
OF
ORALLY
1.0 mg/kg
WITH
[‘“CICHLORDANE
Days after treatment Animal
EQUIVALENTS
BY
RATS
AND
MICE
TREATED
C1.S-[‘4C]CHLORDANE. - cumulative
1
0.5
percent
of dosea
3
7
14
Mice Low excretorsb Feces
1.1 * 0.8
f 2.8
41.1 k 3.2
1.2 * 0.1
4.4 + 0.1
7.7 + 0.4
10.4 * 1.2
1.1 + 0.8
3.7 + 0.8
24.5 + 2.8
48.8 + 3.2
70.9 + 4.6
Feces
34.4 + 1.4
69.3 -t 2.0
83.1 rf- 5.4
86.8 * 5.6
89.1 t
Urine
3.3 * 0.5
7.3 + 1.1
10.2 f
11.0 + 1.1
11.9 + 0.8
Total
37.7 * 1.4
Feces
7.3 & 3.2
Urine
2.5 * 0.9
0
Total
20.1
60.5 + 4.6
High excretors
76.6
1.2
+ 2.0
93.3 + 5.4
97.8 f
54.9 * 4.5
82.5 + 7.6
90.8
1.3 * 0.1
2.0 * 0.1
56.2 ? 4.5
84.5 f 7.6
3.9
5.6
101.0 f 3.9
+ 5.9
95.5 +- 4.8
Rats Urine
0
Total aData
are mean
7.3 f
data
93.5
f
5.9
3.2 f 0.2 98.7
+ 4.8
+ SEM. N = 3-5.
bOf the approximately cretion.
3.2
2.7 + 0.2
30 mice used at various
The low excretors
in Fig. 1 and Tables
were not included I and 11.
times during in studies
these studies,
of blood
6 exhibited
and tissue residues
this pattern
of ex-
which yielded
the
238
Fig. 1 and Tables I and IX. As shown in Table III, these mice voided 34% of the dose in the feces and none in the urine during the same period. By 3 days, the cumulative excretion via the feces was 83% for both species, although urinary excretion remained lower in rats, 2% of the dose, than in mice where 10% of the dose was eliminated. The high fecal excretion of [*4C]chlordane residues probably signifies that biliary excretion predominates in both the rat and mouse. Additional evidence for this was obtained when 2 rats and 2 mice were treated by i.p. injection with I.0 mg/kg cis~14C~chlordane. Fecal excretion after 7 days was 47% of the dose for rats and 67% for mice; urine values were 2% and lo%, respectively. While fecal elimination was reduced in ‘both species relative to the oral doses, the faster initial elimination observed in orally treated mice was not evident following i.p. administration of the insecticide. This may suggest that the rapidly excreted material following oral administration in mice resulted from bioalteration of chlordane in the gut. Since the blood levels were higher in mice, it is possible that the gut-derived metabolites were absorbed, further metabolized, and then returned to the gut via the bile, where they were subsequently voided in the feces. Mice designated as low excretors in Table III were not used in other phases of this study, but the elimination data are presented because they may have some significance. After 24 h, these animals had excreted less than 4% of the oral chlordane dose and only 25% after 3 days (Table III). It is impossible to estimate the frequency of occurrence of the low excretors from the small number of mice used in this study. However, we did establish that mice designated as high or low excretors demonstrated the same patterns of elimination when again treated with cis[‘4C]chlordane. It was also determined that the excretion pattern was not related to feed/water intake, amount of excreta produced, liver weight, or to any disease state evidenced by visual observations of the whole animal or its internal organs. The importance of the low excretors is that they almost certainly would respond differently to chronic chemical exposure than would the high or normal excretors. Consequently, the chronic toxicological information obtained with these low excretors would not be representative of the total test animal population. Where low incidence of response may be of great significance, such as in tumor induction, a few low excretors may have a profound effect on the conclusions derived from a chronic toxicity study. In conclusion, the results of the present study strongly suggest that the fate of chlordane is different in rats and mice, and that further study of the intricacies of metabolism is in order. It may be that one of these species is a better animal model for humans than the other. However, this can be determined only after the metabolism of chlordane in both species has been more fully compared and the results evaluated carefully in regard to what is known about the metabolism of chlordane and other chemicals in man.
239
ACKNOWLEDGEMENTS
This study was supported by funds from Regional Research Project S-146 and from a grant from Velsicol Chemical Corporation. REFERENCES I G.T.
Brooks,
2 U.S.
Environmental
ceedings, 3 National
Chlorinated
Fed. Reg., Cancer
national
Agency
5 J.B. Barnett 612-619.
Vol. I, CRC Press,
Agency,
Consolidated
Cleveland,
OH,
pp.
1974,
heptachlor/chlordane
140-158.
cancellation
pro-
43 (1978) 12372-12375.
Institute,
Series No. 8., NIH, 4 IARC Monographs
Insecticides,
Protection
Bioassay
Bethesda,
MD,
on the evaluation for Research
and H.W.
of chlordane
of the carcinogenic
on Cancer,
Dorough,
for possible
carcinogenicity,
Technical
Report
1977. risk of chemicals
to humans,
Vol. 20, Inter-
1979, pp. 45-65.
Metabolism
of chlordane
in rats, J. Agr. Food Chem.,
22 (1974)
Letters,
233
26 (1985) 233-239
Elsevier
TOXLett.
1450
COMPARATIVE DISPOSITION RATS AND MICE (cis-[14C]Chlordane; excretion rates) ALAN
D. EWING,
Department
chlordane
ABDEL
of Entomology
AND ELIMINATION
toxicokinetics;
M. KADRY
tissue distribution
and H. WYMAN
and Graduate
OF CHLORDANE
IN
and dissipation;
DOROUGH*
Center for Toxicology,
University
of Kentucky,
Lexington,
KY 40546 (U.S.A.) (Received
August
(Accepted
June 7th,
lOth, 1984) 1985)
SUMMARY The absorption, was determined
distribution
slower in mice, but total ceeded the maximum observed
which occurred
equivalents
the total body burden
in both species were
by the findings
that peak tissue
rate was higher
than in rats. At
rates of tissue residues
resulting
somewhat at 8 h) ex-
for the latent peak blood levels in mice
in mouse and rat feces, respectively;
83% of the dose in the feces. Clearance
(113 ng/ml
at 2 h). Peak tissue residues responsible
(1 .O mg/kg)
appeared
concentration
by the tissues. This was supported
12 h, 34% and 7% of the doses were excreted rat, and consequently,
cis-[‘4C]chlordane
mice. Absorption
at peak blood
in rats (81 ng/ml
that the radiocarbon
rather than sequestered
administered
levels were lower in mice, and that the initial fecal elimination
had voided greater
of orally
rats and C57BL/6JX
[“‘Clchlordane
within 4 h, suggesting
was eliminated residue
and elimination
in male Sprague-Dawley
from chronic
by 3 days, both species
were markedly
exposure
to chlordane
faster in the will be far
in mice than in rats.
INTRODUCTION
Chlordane(l,2,4,5,6,7,8,8-octachloro-2,3,3a,4,7,7a-hexahydro-4,7-methano-lHindene) is an organochlorine insecticide which has been used extensively for the control of numerous pests since its introduction in the 1940s. The technical material is a complex mixture, whose insecticidal components are primarily the cis and truns isomers of chlordane [I]. Like other organochlorine pesticides, chlordane has been scrutinized closely by various regulatory agencies over the past several years, and
*To whom correspondence 39762, U.S.A. 0378-4274/85/$
03.30
should
0 Elsevier
be addressed;
Science
Publishers
present
B.V.
address
Drawer
GY, Mississippi
State,
MS
234
in 1978 a decision was made to phase out all use by 1983, except that for the control of underground termites [2]. The concern for the continued widespread use of chlordane is centered around its carcinogenicity in mice. Hepatocellular carcinoma is induced in mice given chlordane, but not in rats [3,4],. This apparent species-specificity provided the impetus for the present investigation. Attempts were made to determine if the general toxicokinetics of chlordane in rats and mice were sufficiently different to suggest that metaboIic peculiarities were reponsible for the selective tumor induction in mice. MaIe rats and mice were treated orally with c&chlordane, as our earlier work on the metaboIism of chlordane in rats [S] showed that either the cis or tram isomer would be satisfactory for comparing the fate of this insecticide in rats and mice. MATERIALS
AND
METHODS
Male Sprague-Dawley rats (22.5-27.5 g) were purchased from Harlan Industries (Indianapolis, IN) and male C57BL/6JX mice (20-25 g) from Jackson Labs (Bar Harbor, ME). The animals were maintained on a 12-h light cycle (21-23”C, 45-50% RH) in Nalgene metabolism cages (Nalge Co., Rochester, NY) designed for the separate collection of feces and urine. c~~-[~4~]~hlordane (28.1 mCi/mmoI) was obtained from Midwest Research Institute (Kansas City, MO) and was > 99% pure, as determined by GLC analysis, as described by Barnett and Dorough [5]. For treatment, the radiolabeled chlordane
Fig. 1. Mean levels of radiocarbon treatment
with 1 mg/kg
on day 14.
in the blood of male rats and mice over a 7 day period
ci#C]chlordane
(IV = 3-5). No radiocarbon
was detected
foltowing
in blood
oral
sampled
235
was diluted with sufficient analytical grade cis-chlordane (Velsicol Chemical Corp., Chicago, IL) for each animal treated with 1.0 mg/kg to receive approx. 1.5 x lo6 dpm. The chlordane was administered to the animals as a single oral dose in 0.2 ml of corn oil. Rodent Chow (Ralston Purina Co., St. Louis, MO) and tap water were provided ad lib; the animals were not fasted prior to treatment. Feces and urine were collected at 12-h intervals for 3 days and at 24-h intervals thereafter. Animals were killed at various times after treatment, at least 3/species/time, and tissue and/or blood samples taken for radioassay. Urine was counted directly (Beckman LS 9000 scintillation counter) while feces, tissues and whole blood were combusted (Packard 306 oxidizer) and the trapped 14C02 was assayed. Using 2 x background as the cut-off point, the limit of detection was approximately 10 ng of [14C]chlordane equivalents per g of the sample. RESULTS
AND DISCUSSION
Total [14C]chlordane equivalents in the blood of rats and mice following oral administration of cis-chlordane, 1 .O mg/kg, are shown in Fig. 1. The initial absorption, O-2 h, was similar in both groups of animals, although at each sampling time the levels were higher in the rat. Even at 15 min (data not shown) the blood of rats contained 14.1 ? 2.4 ng/ml and that of mice only 3.4 f 0.9 (mean f SEM) [14C]chlordane equivalents. The major difference between the species was that peak blood levels in the rat (81 ng/ml) occurred at 2 h, while the peak concentration in mouse blood (113 ng/ml) did not occur until 8 h. Once the peak concentrations were reached, the rates of clearance of the residues from the blood was greater in rats. For example, the mean [‘4C]chlordane equivalents in rat blood at 24 h was 36% of the peak level, whereas for mice the
TABLE
1
[r+C]CHLORDANE WITH
1 .O mg/kg
EQUIVALENTS
IN TISSUES
OF RATS
AND
MICE
TREATED
ORALLY
CIS-[‘4C]CHLORDANE
Tissue
O-24-h Tissue/blood Rats
ratio” Mice
Brain
1.7
0.6
Liver
17.6
11 .o
Peak concentrationb Rats 221 k
(ng/g) Mice
23
1959 + 114
68*
I
1180 + 74
Kidney
8.1
3.2
129 t
57
349 + 30
Lungs
2.6
3.0
217 +
4
248 + 21
Testes
1.6
0.9
135 *
12
92 + 17
Muscle
1.2
1.9
130 +
29
164 k 42
1239 & 299
808 + 42
Fat _ aMean
of determinations
bMean
+ SEM. Peak concentrations
at 2 h.
19.8
8.8
made at 2, 4, 12 and 24 h. N = 3-5. occurred
at 4 h, except for rat brain,
liver, and muscle,
which was
236
level was 52% of the maximum, which occurred at 8 h. While the patterns of accumulation in the blood suggested that absorption of chlordane was slower in mice, the difference in clearance rates indicated that the material absorbed was, at least in part, different in the 2 species. The same inference was evident from the results of tissue analysis (Table 1). Of the 7 tissues assayed, the peak concentrations occurred either at 2 or 4 h. Such an accumulation pattern was consistent with the rat blood data, but inconsistent with the latent peak blood levels in the mouse. Had the material absorbed during the 2-8 h period by the mouse been the same as that absorbed during the first 2 h, then peak tissue residues would have occurred at the 8- or 12 h sampling intervals. Since they peaked at 4 h, as did rat tissue residues, it appears that the material absorbed initially was the same in rats and mice, and that the nature of the sequestered residues was largely the same. In rats, the major tissue residue following chlordane treatment was the metabolite oxychlordane [5]. The findings that the 2 h blood of mice contained less [14C]chlordane equivalents than rats (Fig. 1) and that mice also had lower peak residues in tissues (Table I) are in agreement with the supposition that the initial absorption and deposition of cam-chlordane was essentially the same in rats and mice. Evidence that later differences occurred was the latent peak blood residues in mice, which failed to increase tissue residue levels, and the difference in the tissue/blood ratios between the 2 groups of animals (Table I). The ratios were consistently higher in the rat, the greatest difference being in liver and fat.
TABLE
II
CLEARANCE TREATED
OF [14CJCHL~RDANE ORALLY
WITH
Percent Tissue Brain
EQUIVALENTS
1 .O mg/kg
Animal
12 h
FROM
of peak concentration
Rats
23+
4
62~
7
OF RATS
AND
after”
1 Day
Mice
TISSUES
Cam-~‘~C]CHLORDANE.
14 Days
7 Days 1
0
0
34 + 12
0
0
5+
Rats
40 z?z 14
II?
3
3-t
0
4*
1
Mice
78rt
9
19rt_
9
17rt
5
lo+
3
Kidney
Rats
47t
7
26 +- 10
si
3
82 i
IS
44 + 12
17-+
5
Lung
Mice Rats
26 I
IO
lo+
2
41
0
Mice
79t
9
68i
4
3oi
7
Testes
Rats
46 rt 15
30*
9
0
Mice
7 11
58 + 13 15i 2
38& 0
29+ 0
5
Rats
79rt 31 I
5
Muscle Fat
Mice Rats Mice
801 4 76 i 10 72 i: 14
60 + 14 71* 3 68 ? 17
45* 6 IO* 1 37 + I1
26-+8i: 19t
9 0 7
Liver
aData
presented
.--
as means
..__.
+ SEM of peak concentration
shown
in Table
s-+182
1 1
0 13i
3 0
I.
MICE
237
The clearance of the tissue residues was faster in rats than in mice (Table II) which might further suggest that the nature of the deposited residues were partially dissimilar. However, this could well be a function of blood-flow differences in the 2 species or of other inherent physiological differences. Nonetheless, a marked difference was evident, especially at 12 h, when the mean dissipation from peak levels, considering all tissues, was 59% for rats and only 24% for mice. From the standpoint of chlordane-induced hepatocellular carcinoma in mice but not rats, it was particularly interesting that the mean liver residue level in mice after 12 h was 78% of the peak concentration, and only 40% in rats. It must be pointed out, however, that differences of a similar magnitude were apparent with other tissues and, unlike the liver, the differences (rat vs. mouse) in some tissues were maintained throughout the lbday test period (Table II). Higher [14C]chlordane equivalents in mouse blood, but lower tissue residues as compared to rats, might be explained by greater plasma protein binding in the mouse or by more rapid elimination in this species. As seen in Fig. 1, the dissipation of residues from the blood did not indicate that protein binding was appreciably greater in mice than in rats. However, the elimination of [‘4C]chlordane equivalents in the feces and urine was initially much higher in mice (Table III). Mice designated as high excretors in Table III represented the vast majority of individuals of this species used in the study and it was from these animals that data were obtained for
TABLE
III
ELIMINATION
OF
ORALLY
1.0 mg/kg
WITH
[‘“CICHLORDANE
Days after treatment Animal
EQUIVALENTS
BY
RATS
AND
MICE
TREATED
C1.S-[‘4C]CHLORDANE. - cumulative
1
0.5
percent
of dosea
3
7
14
Mice Low excretorsb Feces
1.1 * 0.8
f 2.8
41.1 k 3.2
1.2 * 0.1
4.4 + 0.1
7.7 + 0.4
10.4 * 1.2
1.1 + 0.8
3.7 + 0.8
24.5 + 2.8
48.8 + 3.2
70.9 + 4.6
Feces
34.4 + 1.4
69.3 -t 2.0
83.1 rf- 5.4
86.8 * 5.6
89.1 t
Urine
3.3 * 0.5
7.3 + 1.1
10.2 f
11.0 + 1.1
11.9 + 0.8
Total
37.7 * 1.4
Feces
7.3 & 3.2
Urine
2.5 * 0.9
0
Total
20.1
60.5 + 4.6
High excretors
76.6
1.2
+ 2.0
93.3 + 5.4
97.8 f
54.9 * 4.5
82.5 + 7.6
90.8
1.3 * 0.1
2.0 * 0.1
56.2 ? 4.5
84.5 f 7.6
3.9
5.6
101.0 f 3.9
+ 5.9
95.5 +- 4.8
Rats Urine
0
Total aData
are mean
7.3 f
data
93.5
f
5.9
3.2 f 0.2 98.7
+ 4.8
+ SEM. N = 3-5.
bOf the approximately cretion.
3.2
2.7 + 0.2
30 mice used at various
The low excretors
in Fig. 1 and Tables
were not included I and 11.
times during in studies
these studies,
of blood
6 exhibited
and tissue residues
this pattern
of ex-
which yielded
the
238
Fig. 1 and Tables I and IX. As shown in Table III, these mice voided 34% of the dose in the feces and none in the urine during the same period. By 3 days, the cumulative excretion via the feces was 83% for both species, although urinary excretion remained lower in rats, 2% of the dose, than in mice where 10% of the dose was eliminated. The high fecal excretion of [*4C]chlordane residues probably signifies that biliary excretion predominates in both the rat and mouse. Additional evidence for this was obtained when 2 rats and 2 mice were treated by i.p. injection with I.0 mg/kg cis~14C~chlordane. Fecal excretion after 7 days was 47% of the dose for rats and 67% for mice; urine values were 2% and lo%, respectively. While fecal elimination was reduced in ‘both species relative to the oral doses, the faster initial elimination observed in orally treated mice was not evident following i.p. administration of the insecticide. This may suggest that the rapidly excreted material following oral administration in mice resulted from bioalteration of chlordane in the gut. Since the blood levels were higher in mice, it is possible that the gut-derived metabolites were absorbed, further metabolized, and then returned to the gut via the bile, where they were subsequently voided in the feces. Mice designated as low excretors in Table III were not used in other phases of this study, but the elimination data are presented because they may have some significance. After 24 h, these animals had excreted less than 4% of the oral chlordane dose and only 25% after 3 days (Table III). It is impossible to estimate the frequency of occurrence of the low excretors from the small number of mice used in this study. However, we did establish that mice designated as high or low excretors demonstrated the same patterns of elimination when again treated with cis[‘4C]chlordane. It was also determined that the excretion pattern was not related to feed/water intake, amount of excreta produced, liver weight, or to any disease state evidenced by visual observations of the whole animal or its internal organs. The importance of the low excretors is that they almost certainly would respond differently to chronic chemical exposure than would the high or normal excretors. Consequently, the chronic toxicological information obtained with these low excretors would not be representative of the total test animal population. Where low incidence of response may be of great significance, such as in tumor induction, a few low excretors may have a profound effect on the conclusions derived from a chronic toxicity study. In conclusion, the results of the present study strongly suggest that the fate of chlordane is different in rats and mice, and that further study of the intricacies of metabolism is in order. It may be that one of these species is a better animal model for humans than the other. However, this can be determined only after the metabolism of chlordane in both species has been more fully compared and the results evaluated carefully in regard to what is known about the metabolism of chlordane and other chemicals in man.
239
ACKNOWLEDGEMENTS
This study was supported by funds from Regional Research Project S-146 and from a grant from Velsicol Chemical Corporation. REFERENCES I G.T.
Brooks,
2 U.S.
Environmental
ceedings, 3 National
Chlorinated
Fed. Reg., Cancer
national
Agency
5 J.B. Barnett 612-619.
Vol. I, CRC Press,
Agency,
Consolidated
Cleveland,
OH,
pp.
1974,
heptachlor/chlordane
140-158.
cancellation
pro-
43 (1978) 12372-12375.
Institute,
Series No. 8., NIH, 4 IARC Monographs
Insecticides,
Protection
Bioassay
Bethesda,
MD,
on the evaluation for Research
and H.W.
of chlordane
of the carcinogenic
on Cancer,
Dorough,
for possible
carcinogenicity,
Technical
Report
1977. risk of chemicals
to humans,
Vol. 20, Inter-
1979, pp. 45-65.
Metabolism
of chlordane
in rats, J. Agr. Food Chem.,
22 (1974)