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Hexachlorobenzene (HCB) deposition in maternal and fetal tissues of rat and mouse
ENVIRONMENTAL
RFSEARCH
19, 1 - 13 (1979)
Hexachlorobenzene (HCB) Deposition in Maternal and Fetal Tissues of Rat and Mouse I. Chemical K. DIANE COURTNEY,
Quantification
of HCB in Tissues
JAMES E. ANDREWS,
AND DAVID
J. SVENDSGAARD
U.S. Environmental Protection Agency, Health Effects Research Laboratory, Research Triangle Park, North Carolina 2771 I Received July 27, 1978 CD-I mice received a single treatment of hexachlorobenzene (HCB) on Day 11 or 16 of gestation or daily treatments on Days 6- 11 or 6- 16 of gestation at levels of 10, 50, or 100 m&kg. CD rats received daily treatments of 10 or 50 mg/kg HCB on Days 6- 12 or 12- 16 of gestation. Animals were sacrificed 24 hr after last dose and maternal and fetal tissues were assayed for HCB content. Rat and mouse fetuses and placentas showed a dose-dependent concentration of HCB, and generally, the fetuses had a lower concentration than their respective placentas. Multiple low doses of HCB resulted in higher concentrations of HCB in mouse maternal and fetal tissues than single doses of equivalent total dose. Late gestational rats received fewer doses of HCB than midgestational rats, yet their fetuses and placentas had significantly higher concentrations of HCB suggesting a gestational effect. Resorptions had higher average HCB concentrations than their living counterparts at all dose regimens.
INTRODUCTION Hexachlorobenzene (HCB) was widely used as a fungicide until 1959 when an incident in Turkey produced numerous cases of HCB toxicity, a result of people eating HCB-treated wheat instead of using it for its intended agricultural purpose (Schmid, 1960; Cam et al., 1963). Currently, HCB is an industrial chemical used in the production of chlorinated solvents. It occurs as an unwanted by-product in the pesticides PCNB and Dachthal (Courtney et al., 1976; Burns et al., 1974). It also occurs as an unwanted by-product in other manufacturing procedures from which it is often reclaimed. A contamination problem occurred in 1972 when HCB was detected in the fat of cattle in an industrialized area of Louisiana (Burns et al., 1975). HCB is now found in many wild animals (Koss et al., 1976), birds (Gilbertson et al., 1972), soil (Laska et al., 1976), human fat and milk (Bakke et al., 1976; Brady et al., 1972; Siyali, 1972; Acker et al., 1970), dairy products (Smyth, 1972), and cow milk (Goursaud et al., 1972). Reports of the incident in Turkey suggested that infants and young people may have been affected more than adults and an increased incidence of stillbirths was noted. Reproduction studies in rats have shown a high mortality in neonates during lactation (Somers et al., 1973; Kimbrough et al., 1974; Grant et al., 1977). HCB has been shown to cross the placenta in rats and rabbits (Villeneuve et al., 1974; Villeneuve et al., 1975). No teratogenic activity was detected in rats (Khera et al., 1974)) but there was a report that HCB was teratogenic in mice (Courtney et al., 1976). Therefore, this study was undertaken to determine the placental trans0013-9351/79/030001-13$02.00/O Copyright0 1579by AcademicFTCSS, Inc. AU ridtts of rcproductioain anyformreserved.
2
COURTNEY,
ANDREWS,
port of HCB in the mouse compared gestation.
SVENDSGAARD
to the rat during early and late stages of
METHODS
Pregnant CD rats with known insemination dates were purchased from Charles River Lab., Inc., Wilmington, Mass. The CD-l mice (Charles River Lab.) were bred at our laboratory. Detection of sperm or vaginal plugs denoted Day zero of gestation. HCB in corn oil was administered by gastric intubation at a daily volume of 0.4 ml/rat or 0.2 ml/mouse. At the highest dose level, 100 mg/kg, HCB was partly in suspension. HCB was administered at various times during gestation and the animals sacrificed 24 hr after the last treatment according to the information in the tables. Doses are in mg/kg/day. Samples of maternal tissues were weighed and frozen until assayed. The mouse fetus and its respective placenta and yolk sac were each weighed, stored, and analyzed separately. The yolk sacs of the rat were left attached to the placentas for analyses. The Day 17 mouse fetuses were dissected and the liver, brain, and remaining carcass analyzed separately. The gestational Day 12 mouse fetuses and the rat fetuses of Days 13 and 17 were analyzed whole. Tissues from nontreated pregnant mice and rats were assayed for HCB. Since none was detected, the data from these animals have not been included in the tables. The HCB was obtained from the British Drug House Chemicals, Ltd., Poole, England, with a purity not less than 99.5%. HCB was extracted from tissue by a modification of the procedures of Kuchar (1969) and Enos ef al. (1971) in which tissues were homogenized in 20 vol of acetonitriie to which 2.0 ml of 2% sodium sulfate was added. The HCB was extracted into hexane. The extract was assayed for HCB using GLC with an electron-capture tritium detector and a column of 1.5% OV-17/1*950/o OV-210 at 195°C. Rat liver was spiked with 0.35, 0.58, 1.14, 2.88, or 57.5 ppm HCB and each concentration assayed in quintuplet. For all concentrations the recovery was 85.8% t 3.0 (SE). The results of the experiments are presented in parts per million based on the wet weight of the tissues. Results are not corrected for recovery. The following conventions were observed in compiling the data. If a fetus was either dead or resorbed, it was regarded as a resorption. The data are presented as mean values with standard errors. The litter was considered the experimental unit. In addition, the values for the HCB content of the fetuses and placentas of the mice are presented using both the litter and the individual as the experimental unit for comparison. The data were analyzed statistically using the Student’s t test, both paired and nonpaired. It is recognized that the Type II error can become excessive when large numbers oft tests are done. This problem will be addressed by Svendsgaard (1979) in a separate paper. RESULTS
AND DISCUSSION
HCB Deposition in Rats The rats were studied at two dose levels at both middle and late gestation. The midgestational rats were sacrificed on Day 13 after six treatments and the late
HCB
IN
FETAL
RAT
AND
TABLE BODY AND ORGAN
3
MOUSE
1
WEIGHTS AND HCB LEVELS IN PREGNANT DAILY ORAL ADMINISTRATION OF HCB”
RATS AFTER
Treated Gestational Days 7- 12 Treated Gestational Days 12- 16 Dose (mg/kg) No. rats total no. fetuses
50 4 27
10 4 41
Row 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Column
Body (db Liver (g) Thymus (mg) Lung (n-d Fetus (mg) Placenta (mg)’
263 f 13 15 2 1 181 k 8 82 f 15 120 k 16
Liver Thymus Fat Urinary bladder Lung Fetus Placenta’ Amniotic fluid”
18 r7 22 k 4 420 2 106 451 0.9 2 0.1 2.9 f 0.1 7?2 A
P
Value 0.05
9B:9D 12A: 12B
Weight 266 2 7 14 k 1 145 * 11 90 2 12 124 + 18
309 16 217 187 853 442
10 5 41
50 5 49
f 2 2 2 2 k
313 * 10 16 + 1 204 k 20 167 + 26 925 2 5 406 2 7
6 1 10 32 15 11
HCB content (ppm) 42 2 3 18 2 7 22 2 8 15 23 1054 + 163 364 ” 61 38 k 13 33 f 14 Sk1 2.0 t 0.2 3.7 2 1.1 4.8 + 0.1 4.9 f 0.1 12 + 3 18 24 B C P
Value 0.01
9A:9B 13A: 13B 13A: 13C 13B: 13D 13C: 13D 14B: 14C 14B: 14D 14C: 14D
26 2 2 26 k 8 627 -+ 141 43 2 14 18 t 2 9.1 2 0.3 10.2 f 1.0 60 + 11 D P
Value 0.001
7A:7B 7B:7D 10A: 10B 1IC:ilD 12A: 12C 12B: 12D 12C: 12D 12A: 13A 12B:13B 12c: 13c
” Results are means ? SE. b Maternal weight at sacrifice minus gravid uterus weight. c Placenta, membranes, and yolk sac. ’ 100 ~1 assayed for each rat.
gestational rats were sacrificed on Day 17 after five treatments. The values from these animals are presented in Table 1. Body weight. There was no significant difference in the maternal body weight of the treated rats at either dose level or gestational period compared to the values of 266 + 3 and 292 + 16 g for the nontreated rats for the mid- and late gestational periods, respectively. Liver. There was no significant difference in the maternal liver weight of the
4
COURTNEY,
ANDREWS,
SVENDSGAARD
HCB-treated rats and the value 13.5 + 1 g for the nontreated rats at mid and late gestation. The HCB content of the midgestational rats (six treatments) did show a significant dose-dependent deposition while that of the late gestational rats (five treatments) did not. Thymus. There was no significant difference in the thymus weight between the HCB-treated rats and the nontreated rats (162 & 14 mg). There was no significant difference in the HCB content at the two dose levels at either stage of gestation. Lungs. Lungs were studied only in the later stage of gestation. There was no significant difference between the lung weight of the treated rats and the nontreated rats (133 + 16 mg). However, there was a significantly greater deposition of HCB in the lungs of the rats receiving 50 mg/kg of HCB compared to those receiving 10 mg/kg HCB. Urinary bladder. There was a significant difference in the HCB concentration of the urinary bladder between the two dose level groups at midgestation. However, at the later stage of gestation, there was no significant difference in the HCB concentration at the two dose levels. Far. HCB is readily deposited in the fat at both stages of gestation in a dosedependent manner although only the rats at midgestation showed a significant difference between the two dose levels. The lesser deposition of HCB in the fat in the late gestation rats probably reflects both the fewer treatments and the mobilization of fat as the end of gestation approached. Fetus. There was no significant difference between the fetal weight of the HCB-treated rats and the nontreated rats at either stage of gestation. (The fetal rat weight of nontreated rats was 90 t 6 and 90.4 2 23 mg for gestational days 13 and 17, respectively). The fetuses receiving the higher dose of HCB had a significantly greater deposition of HCB than those receiving the lower dose. This was noted at both stages of gestation. The fetuses at the later stage of gestation (five treatments) had a significantly greater concentration of HCB than those at comparable dose levels at midgestation (six treatments). A late-gestation fetus has more fat than an earlier gestation fetus which might facilitate a build up of HCB. In many studies, a few fetuses from a litter are selected as being representative of that litter. It can be shown that only a 5% increase in the variance of the mean fetal HCB concentration would result if four fetuses had been sampled rather than analyzing the entire litter. Placenta and yolk sac. The placentas were weighed and assayed with the membranes and yolk sacs attached. There was no significant difference between the weight of the placentas of the nontreated rats (106 + 10 and 528 + 84 mg) and the HCB-treated rats at both stages of gestation. There was a significant increase in the deposition of HCB at the higher dose level compared to the lower dose level at both stages of gestation. The pattern and dose-dependent deposition of HCB in the placentas was similar to that seen in the fetuses. There was a greater deposition of HCB in the placentas at the later stage of gestation than the earlier stage even though these rats received one less treatment. This was also observed in the fetuses. At the earlier stage of gestation, the placentas always had a greater HCB concentration than their respective fetuses, while at the later stage of gestation, a few
HCB
IN
FETAL
RAT
AND
MOUSE
5
of the placentas had a lower HCB concentration than the fetuses. Specifically, at the 50 mg/kg dose level, in three of the five litters, all the placentas had greater HCB concentrations than the fetuses, and in the other two litters most placental values exceeded the fetal values. At the 10 mg/kg dose level, almost all of the placental values exceeded the corresponding fetal values. It can be shown that only a 5% increase in the variance of the treatment mean placental HCB concentration would result if four placentas had been sampled rather than analyzing the entire litter. Amniotic fluid. Aliquots of 100 ~1 of amniotic fluid per fetus were assayed for HCB content. There was a greater amount of HCB in the amniotic fluid at the higher dose level compared to the lower dose level at both stages of gestation. The difference was not significant in the midgestational rats but was significant in the later gestational animals. In these later gestational rats, the amount of HCB in the amniotic fluid was much greater than that in the earlier gestational rats even though they received less total HCB. This was the same pattern of HCB deposition as was observed in the fetuses and placentas. HCB Deposition
in the Mouse Maternal
Tissues
Tissues from pregnant CD-l mice were assayed for HCB content. These values are presented in Table 2. The mice received single or multiple treatments of HCB and were sacrificed at midgestation or late gestation. The midgestational mice were treated on Days 7- 11 and killed on Day 12 of gestation, a day earlier than the rats, in order to have the fetuses of the rats and mice at similar stages of development. By gross observation of the liver, it appeared that the rat and mouse fetuses were in the same stage of development. There was no significant difference in the maternal body and organ weights of the treated compared to the nontreated mice. Blood and plasma. HCB was readily detected in whole blood at all times and at all dose levels. Although HCB was detected in the plasma, the concentrations did not necessarily reflect the dose level or the number of treatments. The plasma level was always less than the level in whole blood. Those groups receiving 11 administrations of HCB had the highest plasma levels. Yang (1975) had suggested that HCB was bound to the red blood cell and Pederson (1976) suggested that it could be bound to one of the porphyrin compounds due to its similarity to porphyrin in size, polarizability, and planarity. Liver. There was a dose-related deposition of HCB in the liver at both stages of gestation with single and multiple treatments. The concentration of HCB in the livers of the mice receiving 6 administrations did not differ greatly from those receiving 11, suggesting that the liver was not a major depot for HCB. Thymus. In general, there was a dose-dependent accumulation of HCB with slightly more accumulation during the later stage of gestation than the earlier stage. It should be noted that there was a marked accumulation in the multipletreatment mice at the later stage of gestation. It is not known if there is any change in the fat content of this organ during gestation. Lung. The concentration of HCB in the lung was generally low in most of the groups and the deposition was dose dependent. Urinary bladder. The mice receiving a single treatment of HCB had very low
column
4:6:710 6:8
7:8
3:6 3:9 3:lO
6:9 7:9 7:lO 8:9 8:lO 910
* 0.05, ** 0.01,
-e SE in ppm wet-tissue *** 0.001.
bP dues:
are means
** 1:8 I:9 4:9 l:lO
7: 10 8:9 6: IO
*** I:5 2: 10 7:8
weight.
5:8: 10 10
9: 10
7:9
5:-l 5:9
B
valuesb
3 4 5
2
4 3 3
4 6 3
NO. mice
3:lO 5:8
* 2~9
B
by rows-P
50 10 50
10
10 50 100
” Results
17 17 17
17
12 12 12
10
50 100
12 12
4: 10
1:;
2:lO 4:6
* 3:4
*** l:6 1:8 2: 10 2:6 3:6
A
A
comparisons
Dose
m&t)
12
Kill
1:lO
Within
Column
16
8 9 IO
6-16 6-16
16
7
6-11 6-11 6-11
II 11 11
1 2 3
4 5 6
Treat
Gestation
ROW
Day
HEXACHLOROBENZENE
C
3: 10 4: 10 5: 10 8: 10 7:
3:9
2:;
2:6
1:8 2~4
* 1:3
A
9 -c 0.6 25 2 5.0 87 ” 16.1
3 -c 0.7
13 -+ 5.0 59 + 4.6
2 z 0.5 8 2 2.9 3 ‘- 1.7
Whole blood
CONTENT
TABLE
2
k 0.03
7:9 910
2: 10
l:lO 2:9
*** l:9
C
B
0.80 -t 0.03 1.94 2 0.26 7.01 + 1.20
0.19
1.02 2 0.39 0.66 -t 0.04 0.91 k 0.65
0.13 + 0.04 0.60 + 0.37 1.04 k 0.83
Plasma
8: 10
7:8
5:8 5:lO 6:7 6:8 6:lO
4:6
2~9 3:4 3: 10
l:4 1:7 2:5
* 1:3
D
C
18 *4 14 f 1 47 f 5
421
14 f I 12 -c 7 61 238
1 k2 452 7&l
Liver
OF TISSUES OF PREGNANT
2:8
*** I:6 2~6 1:8
3:8 4: 10 5~6 910
I:10 2:lO 3:6
** 1:5
D
D
E
8: 10 910
218 3: 10 4:9 5:9 6: 10 7: 10 7:9
2:6
2~3
l:7 1:8 I:9
* l:3
57 f 6 59 2 25 362 + 97
17 f 2
38 k 3 27 f 6 97 f 11
422 12 f 3 16 2 3
Thymus
2:9 2: 10 410 5: 10 8:lO
** 1:lO
E
E
10 * 2.9 26 f 7.5 118 -t 22.4
4 f 0.7
62 1.5 6 f 2.3 30 f 13.3
1 k 0.5 3 r 0.8 11 -c 4.0
Lung
CD- 1 MICE 24 HR AFTER LAST DOSES
* 10
2: *** 10
** 1:lO 3: 10 4:lO 5:lO 8: 10
7: 10 9: 10
6:
F
F
221 423 339 i 72
G *
719
1:8 3:lO 5:lO
**
4:lO 9: 10
2:lO a:9
6:IO
*** I:9 l:lO 2:9
2.8
7:lO 8: 10
3:9 4:9 5:9
2:6
1:6
G
230 + 46 416 2 36 + 216 1842 G
68 + 25
150 k 75 169 k 66 303 * 107
14 r 6 22 + 9 53 r 25 211
28 + 11 82 + 29 167 ? 87
Fat
222 924 28 ‘-c 10
UlitlalY bladder
lz
%
$
g
c”
2 “Y
iz
5
“2
2
s
2
HCB
IN
FETAL
RAT
AND
MOUSE
7
concentrations in the bladder while those receiving multiple treatments showed a definite increase in HCB content. Of note is the group administered 11 treatments of HCB at 50 mg/kg which had a markedly increased deposition compared to all the other groups. Since HCB is not excreted in the urine (Courtney er al., 1976), the compound was probably in the tissue of the bladder. Fur. In the multiple treatment mice, there was proportionately more HCB in the fat of the mice in late gestation compared to those in midgestation. This was also seen in the mice receiving a single treatment of 50 mg/kg. At this time, it is not known whether the kinetics of HCB deposition in the fat change during gestation or whether the maternal fat is mobilized towards the end of gestation creating a concentration of HCB in the fat such as reported in food-deprived rats (Villeneuve, 1975). HCB Deposition in Mice-Fetus, Placenta, and Yolk Sac The values for the weights of the fetuses, placentas, and yolk sacs are presented using the litter as the experimental unit. The values for the HCB content of these tissues are presented in two ways; first, by using the litter as the experimental unit and second, by using the individual fetus, placenta, or yolk sac as the experimental unit. This approach was generated by the current discussions concerning the statistical treatment of data from polytocous animals (Kalter, 1974; Staples et a/. , 1974; Becker, 1974; Abbey et al., 1973; Weil, 1970; Palmer, 1974; Haseman et al., 1975). These data are presented in Table 3. Fetus. The fetal body weights of the mice receiving 50 or 100 mg/kg on Days 6 through 11 were smaller than the other Day 12 fetuses; however, there was no statistically significant difference between the weight of these fetuses and that of nontreated mice (87 _t 8 mg). The large standard errors are due to variation between litters and not within the litters. There was no significant difference in the fetal weight of the Day 17 mice either between groups or compared to fetuses of nontreated mice (985 + 32 mg). The HCB content of the fetuses of the single-treatment Day-12 mice was dose related. As the dose increased fivefold from 10 to 50 mg/kg, the content in the fetuses increased two- to threefold. Increasing the dose again from 50 to 100 mg/kg produced another two- to threefold increase. These differences were statistically significant when calculated on a per fetus basis but not on a litter basis. The Day 17 fetuses of the single-treatment mice receiving 10 or 50 mg/kg HCB had significantly greater HCB concentrations than the Day 12 fetuses at comparable dose levels. This might reflect the greater amount of fat in these older fetuses. The fetuses of mice treated with 50 mg/kg had about four times the concentration of HCB found in the 10 mg/kg group. A four- to fivefold increase was seen in the fetuses of the multiple-treatment mice. Thus, the HCB concentration in the fetus is related to dose level, total amount received, and stage of gestation. PIucentn. There was no significant difference between the placental weights of the treated mice and the nontreated mice at either stage of gestation. The HCB content of the placentas showed the same dose-dependent relationship as that seen in the fetuses. In all groups, the concentration of HCB was greater in almost all the placentas compared to their respective fetuses. Occasionally, in the Day 12 group receiving 100 mg/kg for 6 treatments and in the Day 17
11 11 11
1
17 17
9
10 Column
6-16 6-16
16 16
17 17
17 17
12 12 12
12 12 12
11 11 11
2 3
6-11 6-11 6-11
Kill
Treat
Day gestation
6-16 6-16
17
17
38 36
20 36
10
10 50
50
100
35 48
37 40 36
10
50
100
50
34 55 21
No.
4 5
E
98?5 107 + 10
82 f 7
A
t O.% -c 2.39
1.12 2 0.078 4.99 IL 0.62
+ 0.37 + 1.82 + 1.27
2.06 2.92 15.37
By litter
F
-t 0.70 -+ 0.25
B
3.69 + 0.50 18.45 +- 2.39
0.72 2.62
+ 0.21 f 0.49 f 1.77
+ 0.08 f 0.05 kO.33
6.75 23.69
+ 0.47 L 1.51 G
1.11 f 0.08 5.14 + 0.20
2.00 2.46 14.54
0.60 0.91 3.66
By placenta
f 0.11
f 0.32
C
37 47
19 36
37 24
33 44 10
DURING
50 2 3 56 k 2 H
37 * 1 34 e 2
10 IO
10 10 10
wt. ow)
J
4.7 zi 0.6 20.7 rt: 3.3
0.5 zk 0.1 3.0 2 0.4
2.0 2 0.5 12.8 + 6.8
0.4 I!I 0.4 0.7 z!i 0.2 4.0 _' 2.0
By litter
1.9 * 0.1
0.5 f 0.2 0.6 k 0.1 6.0 -t 0.5
By sac
K
4.8 f 0.4 23.0 2 1.7
0.5 2 0.1 3.0 f 0.2
12.4 L 1.7
2 ppm HCB/yolk
sack
21.3 L 8.7 D
611 ? 10
2 P
;
$
2 4 7 2
3.0 2 1.1 6.7 + 1.6 31.5 k 1.6
64 231 20 -t 10 881 T 43 32 2 25 14 f 4
2
2
z
,? 4.4 * 0.3
2.2 k- 0.1 11.5 ? 1.8
7.3 f 0.8
1.4 -c 0.1
1.0 * 0.2
ppdesorption
1.1 k 0.1
28 ?- 6 2.5 + 4
17 22
30 -t 2 28 r 2
Wt. (mg)
Resorptions
2 2
40 2
4
3 2
No.
GESTATIONS
Yolk sac@ No.
3.68 -c 0.07 19.09 -c O.%
0.73 2.69
-tf 0.09 0.29 20.69
1.38 + 0.16 0.74 1.49 4.56
1.09 rr 0.49
2 0.03
0.19 f 0.04
0.76 1.46 It?z 0.13 0.84 4.16 _c 2.52
0.15 2 0.11 0.52 rt 0.16
By fetus
TREATED
0.39
2 ppm HCB/fetus
OF CD- 1 MICE
x ppm HCB/placenta
t 0.25 f 0.14 f 0.75
7.18 23.45
3
By litter
SACS
TABLE AND YOLK
0.46 0.80 3.54
980 k 34 1097 +38
+ 117
1103 + 110
1130
66 46 +? 519 45 _t 13
18 i 5
69 -t 5 79 + 2
wt em-)
Fetus
PLACENTAS
96 -+ 11
51 2 2 40?8 41 -c5
59 -c 4 55 k 4 62 f 5
wt. bt?)
Placenta
34 44
20 36
22
3
44 3
2 3
34 56
No.
OF FETUSES,
4 6
No. litt.
10
uose @v&z)
50
10
10 50
100
10 50
12 12
12
10 50 100
hdkg)
Dose
CONTENT
12 12 12
Kill
Row
Column
10
9
16
8
6
16
6-11
5
I
6-11
6-11
4
2 3
Treat
ROW
Day gestation
HCB
***
1:9 1:lO 2:8 2:9 2:lO 4:8 4:9 410 5:10
*
5:9 7:8 8~9
**a I:2 1:3 I:4 1:5 I:6 1:7
** 3~6 3:7 4:s 5:8
2:8 5:7 6:8
*
C
1:8 1:9 1:lO 2:3 2~4 2:5 2:6 2:7 2:9 2:lO 3:4 3:6 3:8 3:9 3:lO 4~6 4:8
***
C
4:9 4: 10 5:6 5:9 5: 10 6:7 6:lO 7~8 7:9 7: 10 8:9 8:10 910
***
C
a Mice were sacriiiced 24 hr after last dose. b Assumed weight of 10 mg/yolk sac. c P values: * 0.05, ** 0.01, *** 0.001.
I:7 1:8
***
1:4 3:9 3:lO 6:lO 7:9 7:lO 8:lO 910
**
B
B
Results
** 4:7 5:7 8:9
*** 1:8 1:lO 2:8 2:lO 4: 10 5: 10
? SE in ppm
I:2 1:3 1:4 1:5 I:6 1:7 1:8 1:9
***
2~7 4:5
*
G
***
G
wet tissue
1: 10 2:3 2:4 2:s 2:6 218 2:9 2: 10 3:4 3~6 3:? 3:8 3:9 3: 10 4~6 4:8 4:9
by rows
8: 10 910
**
F
P VALUES comparisons
are means
** I:3 2:3 2~4 2:5 2:9 3: 10 4~8 7: 10
1:4 1:5 1:6 1:9 2:6 4:9 7~8
*
F
within-column
wt.
4: 10 5~6 5~8 5:9 5: 10 6~7 6:8 6~9 6: 10 7:8 7:9 7: 10 8: 10 910
**I:
G
*** 1:9
** 1:8 1:lO 2: 10 410 7:9 910
I:4 1:6 4:9 7:8 7:lO 8: 10
1
J
*** 1:3 1:4 I:5 1:6 1:8 I:9 1:lO 2~3 2:4 2:6 2~8 2~9 2: 10 3:4
6: 10
**
K
3:7 3:8 3:lO 4:6 4:7 4:8 4:9 4: 10 6:7 6:8 6:9 7~8 7:9 7: 10 8:9 8: 10 910
***
K
*** 2:2
3:3 44 8:8
**
5:5 9:9 10: 10
*
B:F
1:l 2:2 3:3 4:4 5:5 6:6 8:8 9:9
***
7:7 lo:10
**
C:G
* 6:6
* 1:l 7:7 9:9 lo:10
3:3 4:4 616 8:8
***
2:2
**
G:K
C:K
P VALUES between-column comparison
**
F:J
W
10
COURTNEY,
ANDREWS,
SVENDSGAARD
fetuses receiving 50 mgikg for 11 treatments, the fetal values exceeded the placental values. Yolk sac. The yolk sacs of the Day 12 fetuses were taken with the membranes attached and were analyzed as such without weighing. It was found that these structures needed to be blotted before weighing and that it was difficult to retrieve them from the blotting paper. Therefore, a weight of 10 mg/sac was assumed, a value close to the median weight based on the samples which were weighed. For the gestational Day 17 fetuses, the yolk sacs were blotted and weighed. The variation in weights probably reflects the amount of membrane left attached. The HCB content of the yolk sacs from the days 12 and 17 fetuses showed a clear dose-related response. It is interesting to note that the sacs from the singletreatment mice at 10 mg/kg contained the same concentration of HCB at both 12 and 17 days of gestation. Upon multiple treatments at both time periods, there was an increase in HCB content. Resorptions. All resorptions had detectable levels of HCB. It can be seen that the concentration appeared to increase in the resorbing tissue, i.e., the smaller the weight of the tissue, the greater the concentration of HCB. This suggests that HCB was not readily transported back across the placenta and was being concentrated in the remaining material. HCB Content of Fetal Tissues The Day 17 mouse fetuses were dissected in order to assay the fetal liver, brain, and carcass. These results are presented in Table 4. At each dose level and for each regimen, the liver had the greatest concentration followed by the carcass and the brain. The HCB concentration of the fetal carcass of the single-treatment mice was 65 and 60% of that of the fetal liver for the 10 and 50 mg/kg groups, respectively. For the multiple-treatment mice, the HCB concentration of carcass was 53 and 74% of the liver concentration for the 10 and 50 mg/kg groups, respectively. Villeneuve et al. (1975) reported a similar distribution of HCB in fetal rats. Fetal HCB Accumulation Comparison of muuse to rat. The midgestational mice and rat fetuses are very similar in their HCB concentration. The rat fetuses at Day 13 had values of 0.9 and 2.0 ppm for the 10 and 50 mg/kg groups, respectively, and the values for the Day 12 mice fetuses were 0.8 and 1.5 ppm for comparable dose-level groups. These values indicated that there is very little difference between the mouse and rat fetus at midgestation when the liver is just developed and the palate is beginning closure. Thus, the concentration of HCB in the fetuses of these two species is the same and probably would not account for any variation in fetal effects produced by the compound. The placental values for these animals were also very similar. The values from the 10 and 50 mg/kg groups of the mice were 2.9 and 4.8 ppm, respectively, and for the rat the values were 2.1 and 2.9 ppm, respectively. Thus, there is very little difference between the placental values for these two species. A direct comparison of the late-gestation animals of the two species is not possible since the mice received 11 treatments and the rats 5 treatments. However, it should be noted that the Day 17 mouse and rat fetuses were of comparable
HCB
IN
FETAL
RAT
TABLE HEXACHLOROBENZENE MICE
Row
Sample
CONTENT TREATED
Days gestation treat
OF DAY
ON DAY
Dose bdk)
16 or
AND
4 17 FETUSES AND DAYS 6- 16 OF
No. fetuses
11
MOUSE
FETAL
TISSUE
OF
CD- 1
GESTATION”
HCB ppm -+ by fetus
No. litters
HCB ppm +- by litter
1 Fetus 2 Carcass 3 Liver 4 Brain
16
10
20
0.73 0.74 0.94 0.28
2 2 i ‘-
0.07 0.08 0.13 0.05
2
0.72 0.73 1:13 0.29
2 ? ” k
0.02 0.03 0.05 0.03
5 Fetus 6 Carcass 7 Liver 8 Brain
16
50
36
2.69 2.63 4.39 1.55
2 r + f
0.11 0.12 0.19 0.08
3
2.62 2.57 4.25 1.52
f t ” 2
0.07 0.08 0.16 0.05
9 Fetus 10 Carcass 11 Liver 12 Brain
6-16
10
34
3.69 3.60 6:58 2.65
f f 2 +
0.16 0.16 0.53 0.28
4
3.68 3.54 6.65 2.72
k k 2 +
0.08 0.07 0.36 0.14
13 Fetus 14 Carcass 15 Liver 16 Brain
6-16
50
44
19.09 -t 19.90 ? 26.21 f 11.35 f
0.96 1.12 1.57 0.60
5
18.45 18.22 24.48 11.32
2 k + t
0.76 0.84 0.81 0.33
HCB ppm (x by litter) within column comparison by rows P Value = 0.05
P Value = 0.01
P Value = 0.001
1:5 2:6 317 4:8 5:9 6: 10
9:13 10: 14 5:13 6:14
12: 16 11:15 8:16 7: 15
a Results are means 2 SE in ppm wet-tissue weight.
weight and that the fetuses of the 10 mg/kg dose levels had 3.69 and 3.7 ppm HCB, respectively. Both the rat and mouse fetuses were capable of greater HCB concentrations as seen in the higher dose-level groups which suggests some regulation of fetal concentration. Comparison of mouse with rabbit. Villeneuve et al. (1974) reported data for the rabbit treated with 10 mg/kg HCB from the 1st to the 28th day of gestation. The number of treatments of the rabbit (28) was greater than the 11 treatments of the mouse in these studies but they were both term animals which permits some comparisons. The rabbit had only 350 ppm of HCB in the maternal fat compared to 416 ppm for the mouse. Since the mouse had fewer than half the treatments of HCB compared to the rabbit, it appears that HCB is more readily accumulated in the mouse compared to the rabbit. From the information in Villeneuve’s report, it could be calculated that the whole rabbit fetus had 10.9 ppm while in the mouse studies the whole fetus had 3.7
12
COURTNEY,
ANDREWS, SVENDSGAARD
ppm HCB. These values are proportional to the number of treatments and in this regard, the rabbit fetus may not differ greatly from the mouse fetus. However, the livers of these fetuses showed marked differences. The fetal rabbit liver had 123 ppm while the fetal mouse liver had 6.6 ppm. Thus, the pattern of deposition of the mouse and rabbit fetus may differ very much. Based on the information on the rat, mouse, and rabbit, it is apparent that HCB crosses placentas and is deposited in fetuses. More studies are needed to determine the magnitude of the fetal accumulation, pattern of fetal deposition, and the kinetics of placental transport of these species. This information is needed to extrapolate this type of data from experimental animals to human beings, REFERENCES Abbey, H., and Howard, E. (1973). Statistical procedure in developmental studies on species with multiple offspring. Dev. Psychobiol. 6, 329-335. Acker, L., and Schulte, E. (1970). Uber das Vorkommen von chlorierten Biphenylen and Hexachlorobenzol neben chlorierten Insektiziden in Humanmilch and menschlickem Fettgewebe. Naturwissenschaften 57, 497-499. Avrahami, M., and Steel, R. T. (1972). Hexachlorobenzene. I. Accumulation and elimination of HCB in sheep after oral dosing. N. Z. J. Agr. Res. 15, 476-481. Bakken, A. F., and Seip, M. (1976). Insecticides in human breast milk. Acta Pediatr. Stand. 65, 535-539. Becker, B. A. (1974). The statistics of teratology. Teratology 9, 261-262. Brady, M. N., and Siyali, D. S. (1972). Hexachlorobenzene in human body fat. Med. J. Aust. 1, 158-163. Bums, J. E. (1974). Hexachlorobenzene exposure from contaminated DCPA in vegetable spraymen. Arch. Environ. Health 29, 192-194. Bums, J. E., and Miller, F. M. (1975). Hexachlorobenzene contamination: Its effects in a Louisiana population. Arch. Environ. Health 30, 44-48. Cam, C., and Nigogosyan, G. (1963). Acquired toxic porphyria cutanea tarda due to hexachlorobenzene. Report of 348 cases caused by this fungicide. .I. Amer. Med. Assoc. 184, 88-92. Courtney, K. D., Copeland, M. F., and Robbins, A. (1976). The effects of pentachloronitrobenzene, hexachlorobenzene, and related compounds on fetal development. Toxicol. Appl. Pharmacol. 35, 239-256. Gilbertson, M., and Reynolds, L. M. (1972). Hexachlorobenzene in the eggs of common terns in Hamilton Harbour, Ontario. BUN. Environ. Contam. Toxicol. 7, 371-373. Grant, D. L., Phillips, W. E. J., and Hatina, G. V. (1977). Effect of hexachlorobenzene on reproduction in the rat. Arch. Environ. Contam. Toxicol. 5, 207-216. Goursaud, J., Luquet, F. M., Boudier, J. F., and Casalis, J. (1972). Sur la pollution du lait par les residus d’hexachlorobenzene (HCB). Industr. Alim. Agr. 89, 31-35. Haseman, J. K., and Hogan, M. D. (1975). Selection of the experimental unit in teratology studies. Teratology 12, 165- 171. Kalter, H. (1974). The choice of the number of sampling units in teratology. Teratology 9, 257-258. Khera, K. S. (1974). Teratogenicity and dominant lethal studies on hexachlorobenzene in rats. Food Cosmet. Toxicol. 12, 471-477. Kimbrough, R. D., and Linder, R. E. (1974). The toxicity of technical hexachlorobenzene in the Sherman strain rat. A preliminary study. Res. Commun. Chem. Pathol. Pharmacol. 8,653-656. Koss, G., and Manz, D. (1976). Residues of hexachlorobenzene in wild mammals of Germany. Bull. Environ. Contam. Toxicol. 15, 189-191. Laska, A. L., Bartell, C. K., and Laseter, J. L. (1976). Distribution of hexachlorobenzene and hexachlorobutadiene in water, soil and selected aquatic organisms along the lower Mississippi River, Louisiana. Bull. Environ. Contam. Toxicol. 15, 535-542. Leoni, V., Puccetti, G., and D’Alessandro De Luca, E. (1976). Indagini sul contenuto di pesticidi cloroorganici in diete totale in Italia. IV. L’esachlorobenzene negli alimenti non preparati. Nuovi Ann. lg. Microbial. 26, 34-43.
HCB
IN FETAL
RAT
AND
MOUSE
13
Luquet, F. M., Goursaud, J., and Casalis, J. (1975). Pollution des laits de femme par les residus de pesticides organochlores en France. Lair 55, 207-211. From Pestic. Abstr., 76-1406. Melnikov, N. N. (1971). Chemistry of pesticides. Residue Rev. 36, l-208. Palmer, A. K. (1974). Statistical analysis and choice of sampling units. Teratology 10, 301-302. Pederson, L. G., and Carlson, G. L. (1975). The planarity of hexachlorobenzene: An ab inifio investigation. J. Chem. Physics 62, 2009-2010. Schmid, R. (1960). Cutaneous porphyria in Turkey. N. Eng. J. Med. 263, 397-399. Siyah, D. S. (1972). Hexachlorobenzene and other organochloride pesticides in human blood. Med. J. Aust. 2, 1063-1066. Smyth, Robert J. (1972). Detection of hexachlorobenzene residues in dairy products, meat, fat, and eggs. J. Assoc.
Ofic.
Anal.
Chem.
55, 806-808.
Somers, E., Grant, D. L., and Phillips, W. E. J. (1973). Toxicodynamics of organochlorine compounds. In “Pesticides in the Environment” (W. B. Deichmann Ed.), pp. 33-41. Intercontinental Med. Book, New York. Staples, R. E., and Haseman, J. K. (1974). Selection of appropriate experimental units in teratology. Teratology
9, 259-260.
Svendsgaard, D. J., Courtney, K. D., and Andrews, J. E. (1979). HCB deposition in maternal and fetal tissues of rat and mouse. II. Statistical quantification of HCB in tissues. Environ. Res., submitted. Villeneuve, D. C. (1975). The effect of food restriction on the redistribution of hexachlorobenzene in the rat. Toxic. Appl. Pharmacol. 31, 313-319. Villeneuve, D. C., and Hierlihy, S. L. (1975). Placental transfer of hexachlorobenzene in the rat. Bull. Environ. Contam. Toxicol. 13, 489-491. Villeneuve, D. C., Panopio, L. G., and Grant, D. L. (1974). Placental transfer of hexachlorobenzene in the rabbit. Environ. Physiol. Biochem. 4, 112- 115. Vos, J. G., van der Maas, H. L., Musch, A., and Ram, E. (1971). Toxicity of hexachlorobenzene in japanese quail with special reference to porphyria, liver damage, reproduction, and tissue residues. Toxicol. Appl. Pharmacol. 18, 944-957, We& C. S. (1970). Selection of the valid number of sampling units and a consideration of their combination in toxicologic studies involving reproduction, teratogenesis, or carcinogenesis, Food Cosmet. Toxicol. 8, 177-182. Yang, R. S. H., Coulston, F., and Golberg, L. (1975). Binding of hexachlorobenzene to erythrocytes: Species variation. Life Sci. 17, 545-550.
RFSEARCH
19, 1 - 13 (1979)
Hexachlorobenzene (HCB) Deposition in Maternal and Fetal Tissues of Rat and Mouse I. Chemical K. DIANE COURTNEY,
Quantification
of HCB in Tissues
JAMES E. ANDREWS,
AND DAVID
J. SVENDSGAARD
U.S. Environmental Protection Agency, Health Effects Research Laboratory, Research Triangle Park, North Carolina 2771 I Received July 27, 1978 CD-I mice received a single treatment of hexachlorobenzene (HCB) on Day 11 or 16 of gestation or daily treatments on Days 6- 11 or 6- 16 of gestation at levels of 10, 50, or 100 m&kg. CD rats received daily treatments of 10 or 50 mg/kg HCB on Days 6- 12 or 12- 16 of gestation. Animals were sacrificed 24 hr after last dose and maternal and fetal tissues were assayed for HCB content. Rat and mouse fetuses and placentas showed a dose-dependent concentration of HCB, and generally, the fetuses had a lower concentration than their respective placentas. Multiple low doses of HCB resulted in higher concentrations of HCB in mouse maternal and fetal tissues than single doses of equivalent total dose. Late gestational rats received fewer doses of HCB than midgestational rats, yet their fetuses and placentas had significantly higher concentrations of HCB suggesting a gestational effect. Resorptions had higher average HCB concentrations than their living counterparts at all dose regimens.
INTRODUCTION Hexachlorobenzene (HCB) was widely used as a fungicide until 1959 when an incident in Turkey produced numerous cases of HCB toxicity, a result of people eating HCB-treated wheat instead of using it for its intended agricultural purpose (Schmid, 1960; Cam et al., 1963). Currently, HCB is an industrial chemical used in the production of chlorinated solvents. It occurs as an unwanted by-product in the pesticides PCNB and Dachthal (Courtney et al., 1976; Burns et al., 1974). It also occurs as an unwanted by-product in other manufacturing procedures from which it is often reclaimed. A contamination problem occurred in 1972 when HCB was detected in the fat of cattle in an industrialized area of Louisiana (Burns et al., 1975). HCB is now found in many wild animals (Koss et al., 1976), birds (Gilbertson et al., 1972), soil (Laska et al., 1976), human fat and milk (Bakke et al., 1976; Brady et al., 1972; Siyali, 1972; Acker et al., 1970), dairy products (Smyth, 1972), and cow milk (Goursaud et al., 1972). Reports of the incident in Turkey suggested that infants and young people may have been affected more than adults and an increased incidence of stillbirths was noted. Reproduction studies in rats have shown a high mortality in neonates during lactation (Somers et al., 1973; Kimbrough et al., 1974; Grant et al., 1977). HCB has been shown to cross the placenta in rats and rabbits (Villeneuve et al., 1974; Villeneuve et al., 1975). No teratogenic activity was detected in rats (Khera et al., 1974)) but there was a report that HCB was teratogenic in mice (Courtney et al., 1976). Therefore, this study was undertaken to determine the placental trans0013-9351/79/030001-13$02.00/O Copyright0 1579by AcademicFTCSS, Inc. AU ridtts of rcproductioain anyformreserved.
2
COURTNEY,
ANDREWS,
port of HCB in the mouse compared gestation.
SVENDSGAARD
to the rat during early and late stages of
METHODS
Pregnant CD rats with known insemination dates were purchased from Charles River Lab., Inc., Wilmington, Mass. The CD-l mice (Charles River Lab.) were bred at our laboratory. Detection of sperm or vaginal plugs denoted Day zero of gestation. HCB in corn oil was administered by gastric intubation at a daily volume of 0.4 ml/rat or 0.2 ml/mouse. At the highest dose level, 100 mg/kg, HCB was partly in suspension. HCB was administered at various times during gestation and the animals sacrificed 24 hr after the last treatment according to the information in the tables. Doses are in mg/kg/day. Samples of maternal tissues were weighed and frozen until assayed. The mouse fetus and its respective placenta and yolk sac were each weighed, stored, and analyzed separately. The yolk sacs of the rat were left attached to the placentas for analyses. The Day 17 mouse fetuses were dissected and the liver, brain, and remaining carcass analyzed separately. The gestational Day 12 mouse fetuses and the rat fetuses of Days 13 and 17 were analyzed whole. Tissues from nontreated pregnant mice and rats were assayed for HCB. Since none was detected, the data from these animals have not been included in the tables. The HCB was obtained from the British Drug House Chemicals, Ltd., Poole, England, with a purity not less than 99.5%. HCB was extracted from tissue by a modification of the procedures of Kuchar (1969) and Enos ef al. (1971) in which tissues were homogenized in 20 vol of acetonitriie to which 2.0 ml of 2% sodium sulfate was added. The HCB was extracted into hexane. The extract was assayed for HCB using GLC with an electron-capture tritium detector and a column of 1.5% OV-17/1*950/o OV-210 at 195°C. Rat liver was spiked with 0.35, 0.58, 1.14, 2.88, or 57.5 ppm HCB and each concentration assayed in quintuplet. For all concentrations the recovery was 85.8% t 3.0 (SE). The results of the experiments are presented in parts per million based on the wet weight of the tissues. Results are not corrected for recovery. The following conventions were observed in compiling the data. If a fetus was either dead or resorbed, it was regarded as a resorption. The data are presented as mean values with standard errors. The litter was considered the experimental unit. In addition, the values for the HCB content of the fetuses and placentas of the mice are presented using both the litter and the individual as the experimental unit for comparison. The data were analyzed statistically using the Student’s t test, both paired and nonpaired. It is recognized that the Type II error can become excessive when large numbers oft tests are done. This problem will be addressed by Svendsgaard (1979) in a separate paper. RESULTS
AND DISCUSSION
HCB Deposition in Rats The rats were studied at two dose levels at both middle and late gestation. The midgestational rats were sacrificed on Day 13 after six treatments and the late
HCB
IN
FETAL
RAT
AND
TABLE BODY AND ORGAN
3
MOUSE
1
WEIGHTS AND HCB LEVELS IN PREGNANT DAILY ORAL ADMINISTRATION OF HCB”
RATS AFTER
Treated Gestational Days 7- 12 Treated Gestational Days 12- 16 Dose (mg/kg) No. rats total no. fetuses
50 4 27
10 4 41
Row 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Column
Body (db Liver (g) Thymus (mg) Lung (n-d Fetus (mg) Placenta (mg)’
263 f 13 15 2 1 181 k 8 82 f 15 120 k 16
Liver Thymus Fat Urinary bladder Lung Fetus Placenta’ Amniotic fluid”
18 r7 22 k 4 420 2 106 451 0.9 2 0.1 2.9 f 0.1 7?2 A
P
Value 0.05
9B:9D 12A: 12B
Weight 266 2 7 14 k 1 145 * 11 90 2 12 124 + 18
309 16 217 187 853 442
10 5 41
50 5 49
f 2 2 2 2 k
313 * 10 16 + 1 204 k 20 167 + 26 925 2 5 406 2 7
6 1 10 32 15 11
HCB content (ppm) 42 2 3 18 2 7 22 2 8 15 23 1054 + 163 364 ” 61 38 k 13 33 f 14 Sk1 2.0 t 0.2 3.7 2 1.1 4.8 + 0.1 4.9 f 0.1 12 + 3 18 24 B C P
Value 0.01
9A:9B 13A: 13B 13A: 13C 13B: 13D 13C: 13D 14B: 14C 14B: 14D 14C: 14D
26 2 2 26 k 8 627 -+ 141 43 2 14 18 t 2 9.1 2 0.3 10.2 f 1.0 60 + 11 D P
Value 0.001
7A:7B 7B:7D 10A: 10B 1IC:ilD 12A: 12C 12B: 12D 12C: 12D 12A: 13A 12B:13B 12c: 13c
” Results are means ? SE. b Maternal weight at sacrifice minus gravid uterus weight. c Placenta, membranes, and yolk sac. ’ 100 ~1 assayed for each rat.
gestational rats were sacrificed on Day 17 after five treatments. The values from these animals are presented in Table 1. Body weight. There was no significant difference in the maternal body weight of the treated rats at either dose level or gestational period compared to the values of 266 + 3 and 292 + 16 g for the nontreated rats for the mid- and late gestational periods, respectively. Liver. There was no significant difference in the maternal liver weight of the
4
COURTNEY,
ANDREWS,
SVENDSGAARD
HCB-treated rats and the value 13.5 + 1 g for the nontreated rats at mid and late gestation. The HCB content of the midgestational rats (six treatments) did show a significant dose-dependent deposition while that of the late gestational rats (five treatments) did not. Thymus. There was no significant difference in the thymus weight between the HCB-treated rats and the nontreated rats (162 & 14 mg). There was no significant difference in the HCB content at the two dose levels at either stage of gestation. Lungs. Lungs were studied only in the later stage of gestation. There was no significant difference between the lung weight of the treated rats and the nontreated rats (133 + 16 mg). However, there was a significantly greater deposition of HCB in the lungs of the rats receiving 50 mg/kg of HCB compared to those receiving 10 mg/kg HCB. Urinary bladder. There was a significant difference in the HCB concentration of the urinary bladder between the two dose level groups at midgestation. However, at the later stage of gestation, there was no significant difference in the HCB concentration at the two dose levels. Far. HCB is readily deposited in the fat at both stages of gestation in a dosedependent manner although only the rats at midgestation showed a significant difference between the two dose levels. The lesser deposition of HCB in the fat in the late gestation rats probably reflects both the fewer treatments and the mobilization of fat as the end of gestation approached. Fetus. There was no significant difference between the fetal weight of the HCB-treated rats and the nontreated rats at either stage of gestation. (The fetal rat weight of nontreated rats was 90 t 6 and 90.4 2 23 mg for gestational days 13 and 17, respectively). The fetuses receiving the higher dose of HCB had a significantly greater deposition of HCB than those receiving the lower dose. This was noted at both stages of gestation. The fetuses at the later stage of gestation (five treatments) had a significantly greater concentration of HCB than those at comparable dose levels at midgestation (six treatments). A late-gestation fetus has more fat than an earlier gestation fetus which might facilitate a build up of HCB. In many studies, a few fetuses from a litter are selected as being representative of that litter. It can be shown that only a 5% increase in the variance of the mean fetal HCB concentration would result if four fetuses had been sampled rather than analyzing the entire litter. Placenta and yolk sac. The placentas were weighed and assayed with the membranes and yolk sacs attached. There was no significant difference between the weight of the placentas of the nontreated rats (106 + 10 and 528 + 84 mg) and the HCB-treated rats at both stages of gestation. There was a significant increase in the deposition of HCB at the higher dose level compared to the lower dose level at both stages of gestation. The pattern and dose-dependent deposition of HCB in the placentas was similar to that seen in the fetuses. There was a greater deposition of HCB in the placentas at the later stage of gestation than the earlier stage even though these rats received one less treatment. This was also observed in the fetuses. At the earlier stage of gestation, the placentas always had a greater HCB concentration than their respective fetuses, while at the later stage of gestation, a few
HCB
IN
FETAL
RAT
AND
MOUSE
5
of the placentas had a lower HCB concentration than the fetuses. Specifically, at the 50 mg/kg dose level, in three of the five litters, all the placentas had greater HCB concentrations than the fetuses, and in the other two litters most placental values exceeded the fetal values. At the 10 mg/kg dose level, almost all of the placental values exceeded the corresponding fetal values. It can be shown that only a 5% increase in the variance of the treatment mean placental HCB concentration would result if four placentas had been sampled rather than analyzing the entire litter. Amniotic fluid. Aliquots of 100 ~1 of amniotic fluid per fetus were assayed for HCB content. There was a greater amount of HCB in the amniotic fluid at the higher dose level compared to the lower dose level at both stages of gestation. The difference was not significant in the midgestational rats but was significant in the later gestational animals. In these later gestational rats, the amount of HCB in the amniotic fluid was much greater than that in the earlier gestational rats even though they received less total HCB. This was the same pattern of HCB deposition as was observed in the fetuses and placentas. HCB Deposition
in the Mouse Maternal
Tissues
Tissues from pregnant CD-l mice were assayed for HCB content. These values are presented in Table 2. The mice received single or multiple treatments of HCB and were sacrificed at midgestation or late gestation. The midgestational mice were treated on Days 7- 11 and killed on Day 12 of gestation, a day earlier than the rats, in order to have the fetuses of the rats and mice at similar stages of development. By gross observation of the liver, it appeared that the rat and mouse fetuses were in the same stage of development. There was no significant difference in the maternal body and organ weights of the treated compared to the nontreated mice. Blood and plasma. HCB was readily detected in whole blood at all times and at all dose levels. Although HCB was detected in the plasma, the concentrations did not necessarily reflect the dose level or the number of treatments. The plasma level was always less than the level in whole blood. Those groups receiving 11 administrations of HCB had the highest plasma levels. Yang (1975) had suggested that HCB was bound to the red blood cell and Pederson (1976) suggested that it could be bound to one of the porphyrin compounds due to its similarity to porphyrin in size, polarizability, and planarity. Liver. There was a dose-related deposition of HCB in the liver at both stages of gestation with single and multiple treatments. The concentration of HCB in the livers of the mice receiving 6 administrations did not differ greatly from those receiving 11, suggesting that the liver was not a major depot for HCB. Thymus. In general, there was a dose-dependent accumulation of HCB with slightly more accumulation during the later stage of gestation than the earlier stage. It should be noted that there was a marked accumulation in the multipletreatment mice at the later stage of gestation. It is not known if there is any change in the fat content of this organ during gestation. Lung. The concentration of HCB in the lung was generally low in most of the groups and the deposition was dose dependent. Urinary bladder. The mice receiving a single treatment of HCB had very low
column
4:6:710 6:8
7:8
3:6 3:9 3:lO
6:9 7:9 7:lO 8:9 8:lO 910
* 0.05, ** 0.01,
-e SE in ppm wet-tissue *** 0.001.
bP dues:
are means
** 1:8 I:9 4:9 l:lO
7: 10 8:9 6: IO
*** I:5 2: 10 7:8
weight.
5:8: 10 10
9: 10
7:9
5:-l 5:9
B
valuesb
3 4 5
2
4 3 3
4 6 3
NO. mice
3:lO 5:8
* 2~9
B
by rows-P
50 10 50
10
10 50 100
” Results
17 17 17
17
12 12 12
10
50 100
12 12
4: 10
1:;
2:lO 4:6
* 3:4
*** l:6 1:8 2: 10 2:6 3:6
A
A
comparisons
Dose
m&t)
12
Kill
1:lO
Within
Column
16
8 9 IO
6-16 6-16
16
7
6-11 6-11 6-11
II 11 11
1 2 3
4 5 6
Treat
Gestation
ROW
Day
HEXACHLOROBENZENE
C
3: 10 4: 10 5: 10 8: 10 7:
3:9
2:;
2:6
1:8 2~4
* 1:3
A
9 -c 0.6 25 2 5.0 87 ” 16.1
3 -c 0.7
13 -+ 5.0 59 + 4.6
2 z 0.5 8 2 2.9 3 ‘- 1.7
Whole blood
CONTENT
TABLE
2
k 0.03
7:9 910
2: 10
l:lO 2:9
*** l:9
C
B
0.80 -t 0.03 1.94 2 0.26 7.01 + 1.20
0.19
1.02 2 0.39 0.66 -t 0.04 0.91 k 0.65
0.13 + 0.04 0.60 + 0.37 1.04 k 0.83
Plasma
8: 10
7:8
5:8 5:lO 6:7 6:8 6:lO
4:6
2~9 3:4 3: 10
l:4 1:7 2:5
* 1:3
D
C
18 *4 14 f 1 47 f 5
421
14 f I 12 -c 7 61 238
1 k2 452 7&l
Liver
OF TISSUES OF PREGNANT
2:8
*** I:6 2~6 1:8
3:8 4: 10 5~6 910
I:10 2:lO 3:6
** 1:5
D
D
E
8: 10 910
218 3: 10 4:9 5:9 6: 10 7: 10 7:9
2:6
2~3
l:7 1:8 I:9
* l:3
57 f 6 59 2 25 362 + 97
17 f 2
38 k 3 27 f 6 97 f 11
422 12 f 3 16 2 3
Thymus
2:9 2: 10 410 5: 10 8:lO
** 1:lO
E
E
10 * 2.9 26 f 7.5 118 -t 22.4
4 f 0.7
62 1.5 6 f 2.3 30 f 13.3
1 k 0.5 3 r 0.8 11 -c 4.0
Lung
CD- 1 MICE 24 HR AFTER LAST DOSES
* 10
2: *** 10
** 1:lO 3: 10 4:lO 5:lO 8: 10
7: 10 9: 10
6:
F
F
221 423 339 i 72
G *
719
1:8 3:lO 5:lO
**
4:lO 9: 10
2:lO a:9
6:IO
*** I:9 l:lO 2:9
2.8
7:lO 8: 10
3:9 4:9 5:9
2:6
1:6
G
230 + 46 416 2 36 + 216 1842 G
68 + 25
150 k 75 169 k 66 303 * 107
14 r 6 22 + 9 53 r 25 211
28 + 11 82 + 29 167 ? 87
Fat
222 924 28 ‘-c 10
UlitlalY bladder
lz
%
$
g
c”
2 “Y
iz
5
“2
2
s
2
HCB
IN
FETAL
RAT
AND
MOUSE
7
concentrations in the bladder while those receiving multiple treatments showed a definite increase in HCB content. Of note is the group administered 11 treatments of HCB at 50 mg/kg which had a markedly increased deposition compared to all the other groups. Since HCB is not excreted in the urine (Courtney er al., 1976), the compound was probably in the tissue of the bladder. Fur. In the multiple treatment mice, there was proportionately more HCB in the fat of the mice in late gestation compared to those in midgestation. This was also seen in the mice receiving a single treatment of 50 mg/kg. At this time, it is not known whether the kinetics of HCB deposition in the fat change during gestation or whether the maternal fat is mobilized towards the end of gestation creating a concentration of HCB in the fat such as reported in food-deprived rats (Villeneuve, 1975). HCB Deposition in Mice-Fetus, Placenta, and Yolk Sac The values for the weights of the fetuses, placentas, and yolk sacs are presented using the litter as the experimental unit. The values for the HCB content of these tissues are presented in two ways; first, by using the litter as the experimental unit and second, by using the individual fetus, placenta, or yolk sac as the experimental unit. This approach was generated by the current discussions concerning the statistical treatment of data from polytocous animals (Kalter, 1974; Staples et a/. , 1974; Becker, 1974; Abbey et al., 1973; Weil, 1970; Palmer, 1974; Haseman et al., 1975). These data are presented in Table 3. Fetus. The fetal body weights of the mice receiving 50 or 100 mg/kg on Days 6 through 11 were smaller than the other Day 12 fetuses; however, there was no statistically significant difference between the weight of these fetuses and that of nontreated mice (87 _t 8 mg). The large standard errors are due to variation between litters and not within the litters. There was no significant difference in the fetal weight of the Day 17 mice either between groups or compared to fetuses of nontreated mice (985 + 32 mg). The HCB content of the fetuses of the single-treatment Day-12 mice was dose related. As the dose increased fivefold from 10 to 50 mg/kg, the content in the fetuses increased two- to threefold. Increasing the dose again from 50 to 100 mg/kg produced another two- to threefold increase. These differences were statistically significant when calculated on a per fetus basis but not on a litter basis. The Day 17 fetuses of the single-treatment mice receiving 10 or 50 mg/kg HCB had significantly greater HCB concentrations than the Day 12 fetuses at comparable dose levels. This might reflect the greater amount of fat in these older fetuses. The fetuses of mice treated with 50 mg/kg had about four times the concentration of HCB found in the 10 mg/kg group. A four- to fivefold increase was seen in the fetuses of the multiple-treatment mice. Thus, the HCB concentration in the fetus is related to dose level, total amount received, and stage of gestation. PIucentn. There was no significant difference between the placental weights of the treated mice and the nontreated mice at either stage of gestation. The HCB content of the placentas showed the same dose-dependent relationship as that seen in the fetuses. In all groups, the concentration of HCB was greater in almost all the placentas compared to their respective fetuses. Occasionally, in the Day 12 group receiving 100 mg/kg for 6 treatments and in the Day 17
11 11 11
1
17 17
9
10 Column
6-16 6-16
16 16
17 17
17 17
12 12 12
12 12 12
11 11 11
2 3
6-11 6-11 6-11
Kill
Treat
Day gestation
6-16 6-16
17
17
38 36
20 36
10
10 50
50
100
35 48
37 40 36
10
50
100
50
34 55 21
No.
4 5
E
98?5 107 + 10
82 f 7
A
t O.% -c 2.39
1.12 2 0.078 4.99 IL 0.62
+ 0.37 + 1.82 + 1.27
2.06 2.92 15.37
By litter
F
-t 0.70 -+ 0.25
B
3.69 + 0.50 18.45 +- 2.39
0.72 2.62
+ 0.21 f 0.49 f 1.77
+ 0.08 f 0.05 kO.33
6.75 23.69
+ 0.47 L 1.51 G
1.11 f 0.08 5.14 + 0.20
2.00 2.46 14.54
0.60 0.91 3.66
By placenta
f 0.11
f 0.32
C
37 47
19 36
37 24
33 44 10
DURING
50 2 3 56 k 2 H
37 * 1 34 e 2
10 IO
10 10 10
wt. ow)
J
4.7 zi 0.6 20.7 rt: 3.3
0.5 zk 0.1 3.0 2 0.4
2.0 2 0.5 12.8 + 6.8
0.4 I!I 0.4 0.7 z!i 0.2 4.0 _' 2.0
By litter
1.9 * 0.1
0.5 f 0.2 0.6 k 0.1 6.0 -t 0.5
By sac
K
4.8 f 0.4 23.0 2 1.7
0.5 2 0.1 3.0 f 0.2
12.4 L 1.7
2 ppm HCB/yolk
sack
21.3 L 8.7 D
611 ? 10
2 P
;
$
2 4 7 2
3.0 2 1.1 6.7 + 1.6 31.5 k 1.6
64 231 20 -t 10 881 T 43 32 2 25 14 f 4
2
2
z
,? 4.4 * 0.3
2.2 k- 0.1 11.5 ? 1.8
7.3 f 0.8
1.4 -c 0.1
1.0 * 0.2
ppdesorption
1.1 k 0.1
28 ?- 6 2.5 + 4
17 22
30 -t 2 28 r 2
Wt. (mg)
Resorptions
2 2
40 2
4
3 2
No.
GESTATIONS
Yolk sac@ No.
3.68 -c 0.07 19.09 -c O.%
0.73 2.69
-tf 0.09 0.29 20.69
1.38 + 0.16 0.74 1.49 4.56
1.09 rr 0.49
2 0.03
0.19 f 0.04
0.76 1.46 It?z 0.13 0.84 4.16 _c 2.52
0.15 2 0.11 0.52 rt 0.16
By fetus
TREATED
0.39
2 ppm HCB/fetus
OF CD- 1 MICE
x ppm HCB/placenta
t 0.25 f 0.14 f 0.75
7.18 23.45
3
By litter
SACS
TABLE AND YOLK
0.46 0.80 3.54
980 k 34 1097 +38
+ 117
1103 + 110
1130
66 46 +? 519 45 _t 13
18 i 5
69 -t 5 79 + 2
wt em-)
Fetus
PLACENTAS
96 -+ 11
51 2 2 40?8 41 -c5
59 -c 4 55 k 4 62 f 5
wt. bt?)
Placenta
34 44
20 36
22
3
44 3
2 3
34 56
No.
OF FETUSES,
4 6
No. litt.
10
uose @v&z)
50
10
10 50
100
10 50
12 12
12
10 50 100
hdkg)
Dose
CONTENT
12 12 12
Kill
Row
Column
10
9
16
8
6
16
6-11
5
I
6-11
6-11
4
2 3
Treat
ROW
Day gestation
HCB
***
1:9 1:lO 2:8 2:9 2:lO 4:8 4:9 410 5:10
*
5:9 7:8 8~9
**a I:2 1:3 I:4 1:5 I:6 1:7
** 3~6 3:7 4:s 5:8
2:8 5:7 6:8
*
C
1:8 1:9 1:lO 2:3 2~4 2:5 2:6 2:7 2:9 2:lO 3:4 3:6 3:8 3:9 3:lO 4~6 4:8
***
C
4:9 4: 10 5:6 5:9 5: 10 6:7 6:lO 7~8 7:9 7: 10 8:9 8:10 910
***
C
a Mice were sacriiiced 24 hr after last dose. b Assumed weight of 10 mg/yolk sac. c P values: * 0.05, ** 0.01, *** 0.001.
I:7 1:8
***
1:4 3:9 3:lO 6:lO 7:9 7:lO 8:lO 910
**
B
B
Results
** 4:7 5:7 8:9
*** 1:8 1:lO 2:8 2:lO 4: 10 5: 10
? SE in ppm
I:2 1:3 1:4 1:5 I:6 1:7 1:8 1:9
***
2~7 4:5
*
G
***
G
wet tissue
1: 10 2:3 2:4 2:s 2:6 218 2:9 2: 10 3:4 3~6 3:? 3:8 3:9 3: 10 4~6 4:8 4:9
by rows
8: 10 910
**
F
P VALUES comparisons
are means
** I:3 2:3 2~4 2:5 2:9 3: 10 4~8 7: 10
1:4 1:5 1:6 1:9 2:6 4:9 7~8
*
F
within-column
wt.
4: 10 5~6 5~8 5:9 5: 10 6~7 6:8 6~9 6: 10 7:8 7:9 7: 10 8: 10 910
**I:
G
*** 1:9
** 1:8 1:lO 2: 10 410 7:9 910
I:4 1:6 4:9 7:8 7:lO 8: 10
1
J
*** 1:3 1:4 I:5 1:6 1:8 I:9 1:lO 2~3 2:4 2:6 2~8 2~9 2: 10 3:4
6: 10
**
K
3:7 3:8 3:lO 4:6 4:7 4:8 4:9 4: 10 6:7 6:8 6:9 7~8 7:9 7: 10 8:9 8: 10 910
***
K
*** 2:2
3:3 44 8:8
**
5:5 9:9 10: 10
*
B:F
1:l 2:2 3:3 4:4 5:5 6:6 8:8 9:9
***
7:7 lo:10
**
C:G
* 6:6
* 1:l 7:7 9:9 lo:10
3:3 4:4 616 8:8
***
2:2
**
G:K
C:K
P VALUES between-column comparison
**
F:J
W
10
COURTNEY,
ANDREWS,
SVENDSGAARD
fetuses receiving 50 mgikg for 11 treatments, the fetal values exceeded the placental values. Yolk sac. The yolk sacs of the Day 12 fetuses were taken with the membranes attached and were analyzed as such without weighing. It was found that these structures needed to be blotted before weighing and that it was difficult to retrieve them from the blotting paper. Therefore, a weight of 10 mg/sac was assumed, a value close to the median weight based on the samples which were weighed. For the gestational Day 17 fetuses, the yolk sacs were blotted and weighed. The variation in weights probably reflects the amount of membrane left attached. The HCB content of the yolk sacs from the days 12 and 17 fetuses showed a clear dose-related response. It is interesting to note that the sacs from the singletreatment mice at 10 mg/kg contained the same concentration of HCB at both 12 and 17 days of gestation. Upon multiple treatments at both time periods, there was an increase in HCB content. Resorptions. All resorptions had detectable levels of HCB. It can be seen that the concentration appeared to increase in the resorbing tissue, i.e., the smaller the weight of the tissue, the greater the concentration of HCB. This suggests that HCB was not readily transported back across the placenta and was being concentrated in the remaining material. HCB Content of Fetal Tissues The Day 17 mouse fetuses were dissected in order to assay the fetal liver, brain, and carcass. These results are presented in Table 4. At each dose level and for each regimen, the liver had the greatest concentration followed by the carcass and the brain. The HCB concentration of the fetal carcass of the single-treatment mice was 65 and 60% of that of the fetal liver for the 10 and 50 mg/kg groups, respectively. For the multiple-treatment mice, the HCB concentration of carcass was 53 and 74% of the liver concentration for the 10 and 50 mg/kg groups, respectively. Villeneuve et al. (1975) reported a similar distribution of HCB in fetal rats. Fetal HCB Accumulation Comparison of muuse to rat. The midgestational mice and rat fetuses are very similar in their HCB concentration. The rat fetuses at Day 13 had values of 0.9 and 2.0 ppm for the 10 and 50 mg/kg groups, respectively, and the values for the Day 12 mice fetuses were 0.8 and 1.5 ppm for comparable dose-level groups. These values indicated that there is very little difference between the mouse and rat fetus at midgestation when the liver is just developed and the palate is beginning closure. Thus, the concentration of HCB in the fetuses of these two species is the same and probably would not account for any variation in fetal effects produced by the compound. The placental values for these animals were also very similar. The values from the 10 and 50 mg/kg groups of the mice were 2.9 and 4.8 ppm, respectively, and for the rat the values were 2.1 and 2.9 ppm, respectively. Thus, there is very little difference between the placental values for these two species. A direct comparison of the late-gestation animals of the two species is not possible since the mice received 11 treatments and the rats 5 treatments. However, it should be noted that the Day 17 mouse and rat fetuses were of comparable
HCB
IN
FETAL
RAT
TABLE HEXACHLOROBENZENE MICE
Row
Sample
CONTENT TREATED
Days gestation treat
OF DAY
ON DAY
Dose bdk)
16 or
AND
4 17 FETUSES AND DAYS 6- 16 OF
No. fetuses
11
MOUSE
FETAL
TISSUE
OF
CD- 1
GESTATION”
HCB ppm -+ by fetus
No. litters
HCB ppm +- by litter
1 Fetus 2 Carcass 3 Liver 4 Brain
16
10
20
0.73 0.74 0.94 0.28
2 2 i ‘-
0.07 0.08 0.13 0.05
2
0.72 0.73 1:13 0.29
2 ? ” k
0.02 0.03 0.05 0.03
5 Fetus 6 Carcass 7 Liver 8 Brain
16
50
36
2.69 2.63 4.39 1.55
2 r + f
0.11 0.12 0.19 0.08
3
2.62 2.57 4.25 1.52
f t ” 2
0.07 0.08 0.16 0.05
9 Fetus 10 Carcass 11 Liver 12 Brain
6-16
10
34
3.69 3.60 6:58 2.65
f f 2 +
0.16 0.16 0.53 0.28
4
3.68 3.54 6.65 2.72
k k 2 +
0.08 0.07 0.36 0.14
13 Fetus 14 Carcass 15 Liver 16 Brain
6-16
50
44
19.09 -t 19.90 ? 26.21 f 11.35 f
0.96 1.12 1.57 0.60
5
18.45 18.22 24.48 11.32
2 k + t
0.76 0.84 0.81 0.33
HCB ppm (x by litter) within column comparison by rows P Value = 0.05
P Value = 0.01
P Value = 0.001
1:5 2:6 317 4:8 5:9 6: 10
9:13 10: 14 5:13 6:14
12: 16 11:15 8:16 7: 15
a Results are means 2 SE in ppm wet-tissue weight.
weight and that the fetuses of the 10 mg/kg dose levels had 3.69 and 3.7 ppm HCB, respectively. Both the rat and mouse fetuses were capable of greater HCB concentrations as seen in the higher dose-level groups which suggests some regulation of fetal concentration. Comparison of mouse with rabbit. Villeneuve et al. (1974) reported data for the rabbit treated with 10 mg/kg HCB from the 1st to the 28th day of gestation. The number of treatments of the rabbit (28) was greater than the 11 treatments of the mouse in these studies but they were both term animals which permits some comparisons. The rabbit had only 350 ppm of HCB in the maternal fat compared to 416 ppm for the mouse. Since the mouse had fewer than half the treatments of HCB compared to the rabbit, it appears that HCB is more readily accumulated in the mouse compared to the rabbit. From the information in Villeneuve’s report, it could be calculated that the whole rabbit fetus had 10.9 ppm while in the mouse studies the whole fetus had 3.7
12
COURTNEY,
ANDREWS, SVENDSGAARD
ppm HCB. These values are proportional to the number of treatments and in this regard, the rabbit fetus may not differ greatly from the mouse fetus. However, the livers of these fetuses showed marked differences. The fetal rabbit liver had 123 ppm while the fetal mouse liver had 6.6 ppm. Thus, the pattern of deposition of the mouse and rabbit fetus may differ very much. Based on the information on the rat, mouse, and rabbit, it is apparent that HCB crosses placentas and is deposited in fetuses. More studies are needed to determine the magnitude of the fetal accumulation, pattern of fetal deposition, and the kinetics of placental transport of these species. This information is needed to extrapolate this type of data from experimental animals to human beings, REFERENCES Abbey, H., and Howard, E. (1973). Statistical procedure in developmental studies on species with multiple offspring. Dev. Psychobiol. 6, 329-335. Acker, L., and Schulte, E. (1970). Uber das Vorkommen von chlorierten Biphenylen and Hexachlorobenzol neben chlorierten Insektiziden in Humanmilch and menschlickem Fettgewebe. Naturwissenschaften 57, 497-499. Avrahami, M., and Steel, R. T. (1972). Hexachlorobenzene. I. Accumulation and elimination of HCB in sheep after oral dosing. N. Z. J. Agr. Res. 15, 476-481. Bakken, A. F., and Seip, M. (1976). Insecticides in human breast milk. Acta Pediatr. Stand. 65, 535-539. Becker, B. A. (1974). The statistics of teratology. Teratology 9, 261-262. Brady, M. N., and Siyali, D. S. (1972). Hexachlorobenzene in human body fat. Med. J. Aust. 1, 158-163. Bums, J. E. (1974). Hexachlorobenzene exposure from contaminated DCPA in vegetable spraymen. Arch. Environ. Health 29, 192-194. Bums, J. E., and Miller, F. M. (1975). Hexachlorobenzene contamination: Its effects in a Louisiana population. Arch. Environ. Health 30, 44-48. Cam, C., and Nigogosyan, G. (1963). Acquired toxic porphyria cutanea tarda due to hexachlorobenzene. Report of 348 cases caused by this fungicide. .I. Amer. Med. Assoc. 184, 88-92. Courtney, K. D., Copeland, M. F., and Robbins, A. (1976). The effects of pentachloronitrobenzene, hexachlorobenzene, and related compounds on fetal development. Toxicol. Appl. Pharmacol. 35, 239-256. Gilbertson, M., and Reynolds, L. M. (1972). Hexachlorobenzene in the eggs of common terns in Hamilton Harbour, Ontario. BUN. Environ. Contam. Toxicol. 7, 371-373. Grant, D. L., Phillips, W. E. J., and Hatina, G. V. (1977). Effect of hexachlorobenzene on reproduction in the rat. Arch. Environ. Contam. Toxicol. 5, 207-216. Goursaud, J., Luquet, F. M., Boudier, J. F., and Casalis, J. (1972). Sur la pollution du lait par les residus d’hexachlorobenzene (HCB). Industr. Alim. Agr. 89, 31-35. Haseman, J. K., and Hogan, M. D. (1975). Selection of the experimental unit in teratology studies. Teratology 12, 165- 171. Kalter, H. (1974). The choice of the number of sampling units in teratology. Teratology 9, 257-258. Khera, K. S. (1974). Teratogenicity and dominant lethal studies on hexachlorobenzene in rats. Food Cosmet. Toxicol. 12, 471-477. Kimbrough, R. D., and Linder, R. E. (1974). The toxicity of technical hexachlorobenzene in the Sherman strain rat. A preliminary study. Res. Commun. Chem. Pathol. Pharmacol. 8,653-656. Koss, G., and Manz, D. (1976). Residues of hexachlorobenzene in wild mammals of Germany. Bull. Environ. Contam. Toxicol. 15, 189-191. Laska, A. L., Bartell, C. K., and Laseter, J. L. (1976). Distribution of hexachlorobenzene and hexachlorobutadiene in water, soil and selected aquatic organisms along the lower Mississippi River, Louisiana. Bull. Environ. Contam. Toxicol. 15, 535-542. Leoni, V., Puccetti, G., and D’Alessandro De Luca, E. (1976). Indagini sul contenuto di pesticidi cloroorganici in diete totale in Italia. IV. L’esachlorobenzene negli alimenti non preparati. Nuovi Ann. lg. Microbial. 26, 34-43.
HCB
IN FETAL
RAT
AND
MOUSE
13
Luquet, F. M., Goursaud, J., and Casalis, J. (1975). Pollution des laits de femme par les residus de pesticides organochlores en France. Lair 55, 207-211. From Pestic. Abstr., 76-1406. Melnikov, N. N. (1971). Chemistry of pesticides. Residue Rev. 36, l-208. Palmer, A. K. (1974). Statistical analysis and choice of sampling units. Teratology 10, 301-302. Pederson, L. G., and Carlson, G. L. (1975). The planarity of hexachlorobenzene: An ab inifio investigation. J. Chem. Physics 62, 2009-2010. Schmid, R. (1960). Cutaneous porphyria in Turkey. N. Eng. J. Med. 263, 397-399. Siyah, D. S. (1972). Hexachlorobenzene and other organochloride pesticides in human blood. Med. J. Aust. 2, 1063-1066. Smyth, Robert J. (1972). Detection of hexachlorobenzene residues in dairy products, meat, fat, and eggs. J. Assoc.
Ofic.
Anal.
Chem.
55, 806-808.
Somers, E., Grant, D. L., and Phillips, W. E. J. (1973). Toxicodynamics of organochlorine compounds. In “Pesticides in the Environment” (W. B. Deichmann Ed.), pp. 33-41. Intercontinental Med. Book, New York. Staples, R. E., and Haseman, J. K. (1974). Selection of appropriate experimental units in teratology. Teratology
9, 259-260.
Svendsgaard, D. J., Courtney, K. D., and Andrews, J. E. (1979). HCB deposition in maternal and fetal tissues of rat and mouse. II. Statistical quantification of HCB in tissues. Environ. Res., submitted. Villeneuve, D. C. (1975). The effect of food restriction on the redistribution of hexachlorobenzene in the rat. Toxic. Appl. Pharmacol. 31, 313-319. Villeneuve, D. C., and Hierlihy, S. L. (1975). Placental transfer of hexachlorobenzene in the rat. Bull. Environ. Contam. Toxicol. 13, 489-491. Villeneuve, D. C., Panopio, L. G., and Grant, D. L. (1974). Placental transfer of hexachlorobenzene in the rabbit. Environ. Physiol. Biochem. 4, 112- 115. Vos, J. G., van der Maas, H. L., Musch, A., and Ram, E. (1971). Toxicity of hexachlorobenzene in japanese quail with special reference to porphyria, liver damage, reproduction, and tissue residues. Toxicol. Appl. Pharmacol. 18, 944-957, We& C. S. (1970). Selection of the valid number of sampling units and a consideration of their combination in toxicologic studies involving reproduction, teratogenesis, or carcinogenesis, Food Cosmet. Toxicol. 8, 177-182. Yang, R. S. H., Coulston, F., and Golberg, L. (1975). Binding of hexachlorobenzene to erythrocytes: Species variation. Life Sci. 17, 545-550.