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Embryo- and fetotoxicity of inhaled chloroform in rats
TOXICOLOGY
AND
Embryo-
APPLIED
and
PHARMACOLOGY
Fetotoxicity
28,
442451
(1974)
of Inhaled
Chloroform
B. A. SCHWETZ, B. K. J. LEONG AND P. J.
in Rats
GEHRING
Chemical Biology Research, l%e Dow Chemical Company, Midland, Michigan 48640 Received September 14, 1973; accepted November 20, 1973
Embryo- and Fetotoxicity of Inhaled Chloroform in Rats. SCHWETZ, LEONG, B. K. J. AND GEHRING, P. J. (1974). Toxicol. Appl. Phurmacol., 28,442-451. This study evaluated the effects of inhalation of subanesthetic concentrations of chloroform on rat embryo& and fetal development. Pregnant Sprague-Dawley rats were exposed to 30,100 or 300 ppm chloroform for 7 hr/day on days 6 through 15 ofgestation. Exposure to chloroform caused an apparent decreasein the conception rate and a high incidence of fetal resorption (300 ppm), retarded fetal development (30, 100,300 ppm), decreased fetal body measurements (30, 300 ppm) and a low incidence of acaudate fetuses with imperforate anus (100 ppm). Chloroform was not highly teratogenic but was highly embryotoxic. The results of this study disclosed no relationship between maternal toxicity and embryo or fetotoxicity as the result of exposure to chloroform by inhalation. B.A.,
Exposure of mice or rats for up to 1 hr to anesthetic concentrations of chloroform (25,000-37,000 ppm) during organogenesis has been demonstrated to be highly embryotoxic (Schwetz and Becker, 1974). Although chloroform is no longer widely used as an anesthetic agent in the United States, it is used extensively as a starting material and a chemical intermediate in industry and laboratories. This study was performed to determine whether exposure of pregnant rats to levels of chloroform more like those which may be encountered in industry or laboratories may have an adverse effect on the development of the embryo or fetus. Three Ievels of exposure were studied, 30, 100 and 300 ppm for 7 hr/day on days 6 through 15 of gestation. Maternal toxicity, including hepatotoxicity, was also determined in order to reveal any relationships between the effects of chloroform on the embryo or fetus and the mother. METHODS Animals. Adult Sprague-Dawley (Spartan) female rats weighing approximately 250 g were used. The day on which sperm were seen in a vaginal smear was considered day 0 of pregnancy. Between daily exposures, animals were housed in wire-bottom cages and were maintained on commercial laboratory rat chow1 and water ad libitum in a room controlled for temperature, humidity and light cycle. Food or water were not provided during exposure to chloroform. Food consumption of each animal or group of 2 animals was measured at 2-day intervals throughout the experimental period. 1 Purina Rat Chow@,Ralston Purina Co., St. Louis, Missouri. Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
442
EMBRYOTOXICITYOFINHALED
443
CHLOROFORM
Materials and exposure procedure. Reagent grade chloroform” was used in these experiments. Exposures were conducted in 3.7 m3 stainless steel, cubical dynamic
exposure chambers. Vapors of chloroform,
generated by metering the liquid at known
rates into a temperature-controlled evaporating flask, were diluted with filtered room air at a rate calculated to give the desired concentration. The nominal concentrations in the chamber atmospherewhich were calculated from the ratio of the rate of delivery of chloroform to the rate of total air flow through the chamber were substantiated by analysis 3 times daily using a Beckman IRlO infrared spectrophotometer with a multipath gascell (Table 1). In addition, the concentration in the chamberswas continuously monitored using combustion analysis to assure the absence of significant deviations from the desired level. Becausethe concentrations determined by analyseswere essentially the same as the nominal concentrations, the nominal concentrations will be referred to throughout this paper.
TABLE 1 SAMPLE DESCRIPTION AND CONCENTRATIONS OF COMPONENTS IN EXPOSURE ATMOSPHERES
Component Chloroform Carbon tetrachloride Unknown
Volume % in sample” 99.30
ppm in ChambeP Nominal : Analytical:
0.67 0.03
30 30 + 1 0.1 0.03
100 95 & 29 0.5 0.1
300 291& 22 i .4 0.3
aDeterminedby gas-liquidchromatography. ’ Thenominalchloroformconcentrationwascalculatedfromtheratio of therateof liquidintroduced to the rate of the dilution air flow. Theanalyticalconcentrationwasdeterminedby infraredspectrometry, meanf SD of 3 measurements on eachof 10consecutive daysof exposure.The concentration of minorcomponents wascalculatedasa proportionof their concentrationin theliquidsamplerelative to the majorcomponent. Experimentaldesign. In the initial experiment, pregnant rats were exposed to 300 ppm chloroform 7 hr/day on days 6 through 15 of gestation. Subsequently, additional groups of rats were exposed to 100 or 30 ppm. Nonpregnant female rats were exposed simultaneously with the pregnant rats at the 2 higher concentrations to allow for evaluation
of treatment-related changes in serum glutamic-pyruvic during and after exposure. Control animals for each currently to filtered room air. The parameters which significantly in the 3 air-control groups used. Therefore,
transaminase (SGPT) activity experiment were exposed conwere evaluated did not differ for statistical comparisons, the
data for the control groups were combined.
Marked anorexia was observed in rats exposed to 300 ppm chloroform in the initial experiment. Therefore, another control group, “starved control”, was included in the secondexperiment to simulate the anorexia observed in the first experiment. This group was handled similarly except that each rat was allowed to eat only 3.7 g of food/day on days 6 through 15 of gestation. Maternal andfetal examination. All rats were observed daily throughout pregnancy and maternal body weights were recorded on days 6, 13 and 21 of gestation. The dams ’ Lot No. 9649,Burdick & JacksonLab., Inc., Muskegon,Michigan.
444
SCHWETZ,
LEONG
AND
GEHRING
were sacrificed by COZ anesthesia on day 21 of gestation. After the uterine horns had been exteriorized through a mid-line incision in the abdominal wall, the number and position of live, dead and resorbed fetuses were noted. The umbilical cord was clamped and severed distally. After being weighed, measured (crown-rump length) and the sexes recorded, the fetuses were examined for external anomalies. Each litter was divided equally into 2 subgroups for preservation and subsequent examination. One subgroup, preserved in Bouin’s solution, was examined by the method of Wilson (1965) for evidence of soft tissue anomalies. The second subgroup, preserved in alcohol, was cleared and stained with Alizarin Red-S (Dawson, 1926) for examination of the skeletons for evidence of abnormalities. Hepatotoxicity. SGPT activity was determined in nonpregnant rats following the second, fourth, seventh, and tenth (final) exposure to 100 or 300 ppm chloroform. After the last exposure, 4 nonpregnant rats were sacrificed for gross examination of the liver. Six days later, the day the pregnant females were subjected to a cesarean section, the SGPT activity was determined in the remaining nonpregnant rats and 10 pregnant rats. All livers were weighed and examined grossly. SGPT activity was determined fluorometrically using the Technicon Auto Analyzer (Method N-54). Statistical evaluation. The Fisher Exact Probability Test (Siegel, 1956) was used to evaluate the frequency of anomalies and resorptions between litters. Maternal and fetal body weights and body measurements, liver weights and SGPT activities were analyzed statistically by an analysis of variance and Dunnett’s or Tukey’s test (Steel and Torrie, 1960). In all cases, the level of significance was chosen asp < 0.05. The litter was considered the experimental unit of treatment and observation. RESULTS Embryo- and Fetotoxicity Exposure to 300 ppm chloroform on days 6 through 15 of gestation caused a significant increase in the incidence of fetal resorptions and a decrease in fetal body measurements (Table 2). At this concentration, the conception rate was only 15 % (3 pregnant animals/20 animals bred) compared to 88 % (68 pregnant/77 bred) in the control group (Table 2). In 1 of 3 pregnant females exposed to 300 ppm chloroform, the remaining metrial glands were only about 1 mm in diameter compared to the usual 4-6 mm after an early resorption, indicating that the conceptus had been completely resorbed very soon after implantation. In 7 of the 17 nonpregnant bred females, there was vascular evidence in the mesometrium of implantation but there was no intrauterine evidence of implantation. In contrast, exposure to 100 or 30 ppm chloroform did not alter the incidence of fetal resorptions, fetal body weight, the conception rate, the number of implantations or the average litter size. Fetal crown-rump length was statistically significantly less than control at 30 ppm but not at 100 ppm. At 30 ppm chloroform, the incidence of gross and soft tissue anomalies was not different from control (Table 3). The incidence of delayed ossification of skull bones and of wavy ribs was significantly greater than among control litters. Examination of the fetuses of dams exposed to 100 ppm revealed a significant incidence of acaudia (absence of tail) or short tails, imperforate anus, subcutaneous edema, missing ribs and delayed ossification of sternebrae (Table 3). At 300 ppm chloroform, subcutaneous edema and anomalies of the skull and sternum were observed, but
2
7 (6/87) 25 (2/8)
8 (63/769) 57 (39/68) O/68
5.19 * 0.29’ 42.1 -t 1.1’
3.0 (6/2) 45:55 42.5+ 0.6’
5.51 + 0.20
53:47
1.6 (24/l 5)
68 (15/22) o/22
13+2 12f2 8 (24/291)
16f3e
30 71 (22/31)
55:45 5.59 + 0.24 43.b + 0.7
1.3 (16/12)
6 (16/278) 52 (12/23) O/23
12+2 11_+2
loo 82 (23/28) 14_+ 2
Chloroform
THE TIME OF CESAREANSECTION~
4f 7p 61 (20/33)d loo (3/3) l/3 6.7 (20/3) 34 : 66’ 3.42 & 0.02’ 36.9 + 0.2’
11+4
14+ 1
300 15 (3/20)d
n Administered by inhalation 7 hr daily on days 6-15 of gestation. Starved controls were fed 3.7 g of food daily on days 6-15 of gestation. * Mean + SD. c Mean of litter means + SD. d Significantly different from control by the Fisher Exact Probability test, p < 0.05. e Significantly different from control by an analysis of variance and Dunnett’s test, p < 0.05.
53:47 5.69 + 0.36 43.5 + 1.1
W3
11f4 10+4
11+3 10+4
1.6 (63/39)
100 W) 14+2
-
-
Chloroform concentration (ppm) Percentpregnancy(pregnant/bred) Corpora lutes/dam* Implantations/littel Live fetuses/We? ‘A Resorptions/implantations % Litters with resorptions Litters totally resorbed Resorptions/litterswith resorptions Sexratio, M :F Fetal body weight (g) Fetal crown-rump length (mm) 88 (68/77) 14f2
Air control starved
Air control
Statistic
EFFECTOF INHALED CHLOROFORMON OBSERVATIONS MADEAT
TABLE
iz s kz 3 E
E F B
$
2
ij
2
;s
E
446
SCHWETZ,
LEONG
AND
GEHRING
a significant increase in incidence was not statistically demonstrable because of the small number of litters involved. TABLE 3 EFFECT OF INHALED CHLOROFORM ON THE INCIDENCE OF FETAL ANOMALIES AMONG RAT LITTERS’
Air control
Air control starved
68
-
Chloroform concentration, ppm Number of litters examined
Chloroform 30 22
8
loo 23
Percent of Litters A&ted Gross Acaudia or short tail Imperforate anus Total gross Skeletal Delayed ossification,skull Missing ribs Wavy ribs Split sternebrae Delayed ossification,sternebrae Total skeletal Soft tissue Subcutaneousedema Total soft tissue
300 3
(No. of litters)
(0) (0) 1.5 (1)
(0) (0) (0)
(0) (0) (0)
13 (3)b 13 (3)b 13 (3)b
21 (14) (0) (0) 1.5 (1) 22 (15) 68 (46)
(0) (0) g; 38 (3) 38 (3)
73 (16)b (0) 18 (4)b 9 (2) (0) 90 (20)b
30 (7) 13(3)b (0) 9 (2) 74 (17)b 74 (17)
50 (1) (0) (0) 50 (1) 100(2) loo (2)
34 (23) 48 (33)
38 (3) 38 (3)
41 (9) 45 (10)
61 (14)b 65 (15)
100 (l!
(0) (0) (0)
100(1)
a Administered by inhalation 7 hr daily on days 6-15 of gestation. Starved controls were fed 3.7 g of food daily on days 6-15 of gestation. b Significantly different from control by the Fisher Exact Probability test, p -C0.05.
Maternal Toxicity
Exposure of rats to 30 or 100 ppm chloroform crease in the rate of maternal weight gain and (Tables 4 and 5). Exposure of females to 300 ppm in the rate of weight gain and food consumption
caused a statistically significant defood consumption during exposure chloroform caused a drastic decrease during pregnancy.
TABLE 4 EFFECT OF INHALED CHLOROFORM ON MATERNAL BODY WEIGHT DURING GESTATION”
Air control Chloroform concentration (mm) Number of dams Gestation day 6 13 21
68 275+21” 310* 17 389 + 28
Air control starved 8 274+ 13 223a 13’ 326k 24”
Chloroform 30
100
22 266 + 14 280+ 14” 381+ 23
23 274+ 17 274 f 18’ 365&-22”
300 3 284f 9 192+9= 241 k 29”
a Administered by inhalation 7 hr daily on days 6-15 of gestation. Starved controls were fed 3.7 g of food daily on days 6-15 of gestation. b Body weight, g, mean + SD. CSignificantly different from control by an analysis of variance and Dunnett’s test, p < 0.05.
20 &- 3
20 _+ 2 18+ 1
8
23 3
-z
13f4d 1 + Id
5 + 3d
19f3
67
i5f2d 4 f 2d
18+ 1
__
20 lk 3
8-9
Food consumption
23 + 2
lo-11
16_+2d 1 * Id
18k2
12-13
day numbe?
15_+2d 1 f Id
205 1
19f 2d 1 rlr Id
21+2
23 rf: 3
14-15
CONSUMPTION
22 +- 2 ___
on gestation
STARVED
’ Administered by inhalation 7 hr/day on days 6-15 of gestation. ND, not determined. b g/rat/day, mean t SD. ’ Each rat was given 3.7 g of food daily on days 6 through 15 of gestation. d Significantly different from air control by Dunnett’s test, p < 0.05.
19+2
8
Starvation controP Chloroform 30 mm 100 mm 300 mm
19*4
4-5
53
N
Air control
Test material and concentration”
5
EFFECTOF INHALED CHLOROFORMON RAT MATERNALFOOD
TABLE
t
30 * 3d 12f2d
27 f 3
21_+2d
25 L- 4
16-17
--
33 f 3d ND”
29 zk 5
24 + 8d
261k 3
18-19
--
E
2 z
3 F B
4 8
Y
2 x
E E 2
448
SCHWETZ,
LEONG
AND
GEHRING
In order to assessthe degree of liver damage which may have been incurred by rats exposed to 100 or 300 ppm chloroform, the SGPT activity was determined during and after exposure (Table 6). In all groups, the total weight of the liver and its weight relative to total body weight (mg/g) were determined and the gross appearance of the liver was recorded (Table 7). Exposure to 100 or 300 ppm chloroform did not increase the SGPT activity of nonpregnant rats during exposure or at 6 days after the last exposure. The SGPT activity of pregnant rats 6 days after the last exposure to chloroform, day 21 of gestation, was the same as that of controls. TABLE 6 EFFECT OF INHALED CHLOROFORM ON SGPT ACTIVITY IN RATS
SGPT activity, Karmen units, mean + SE Chloroform” Day of exposure 2nd 4th 7th 10th (last) Six days after last exposure Nonpregnant Pregnant
N
Control
10 10 10 10
63 + 3 57+2 57 4 5 56+2
6
57If: 3 72 + 3
10
100 mm
300 PPm
ND* 45 + 2
52+4 49f3 54+3 54 + 6
59+4 ND
52+3 61 +4
58 IL 3
ND
“Administered by inhalation 7 br/day for 10 consecutive days (days 6-15 of pregnancy for the pregnant rats). Blood samples were taken immediately upon removal from the chamber on the indicated days of exposure. Pregnant rats were bled at the time of cesarean section on day 21 of gestation. All other values are for nonpregnant rats. No values were significantly different from control by Tukey’s test, p < 0.05. b ND, not determined.
In nonpregnant rats, minimal changes in the gross appearance of the liver were observed following the last exposure to 300 ppm chloroform-pale, mottled liver, in 4/4 rats. Six days after the last exposure, the gross appearance of the livers of pregnant and nonpregnant control and chloroform-exposed rats was normal. Neither the absolute nor relative liver weight was altered following exposure to 30ppm chloroform. When measured immediately after the last daily exposure to 100 ppm chloroform, both the absolute and relative weights of the liver of nonpregnant rats were increased significantly. Similar increases were not detected 6 days after the last exposure. A slight but statistically significant increase in the relative, but not the absolute, weight of the liver of pregnant rats exposed to 100 ppm chloroform was observed at the time of cesarean section, 6 days after the last exposure. In contrast to the increase in the total weight of the liver of nonpregnant rats exposed to 100 ppm chloroform at the end of the exposure period, exposure to 300 ppm caused a significant
decrease.
However,
the weight
of the liver relative
to total body weight
was
unchanged, probably reflecting an effect of anorexia in rats exposed to 300 ppm chloroform. At the time of cesarean section, 6 days after the last exposure, the total weight of the liver of pregnant rats exposed to 300ppm chloroform was decreased while the weight
EMBRYOTOXICITY
OF INHALED
CHLOROFORM
449
450
SCHWETZ,
LEONG
AND
GEHRING
of the liver relative to the total body weight was increased, The changes in liver weight just described very likely reflect the anorexic effects of exposure to chloroform and perhaps a direct effect of chloroform on the liver. In order to determine whether the anorexic effects of chloroform may have been responsible for the maternal, embryonal and fetal changes described herein, a group of pregnant rats was fed only 3.7 g of food daily on days 6 through 15 of gestation. This degree of starvation caused a decrease in fetal body measurements but had no effect on the incidence of fetal resorptions (Table 2). In addition, the conception rate, number of implantations and litter size were not different from those of air-exposed control animals. With regard to weight of the maternal liver at the time of cesarean section, starvation caused a slight but significant decrease in the total weight of the liver and a slight but significant increase in the weight of the liver relative to the total body weight. The liver weight changes observed in the starvation-controls explain in part similar changes observed in pregnant rats exposed to chloroform. However, the increase in the relative liver weight of pregnant rats exposed to 300 ppm was much greater, suggesting that this effect may not have been due solely to anorexia. DISCUSSION In these studies, a high degree of embryo and fetal toxicity was associated with the exposure of pregnant rats to 100 or 300 ppm chloroform daily on days 6 through 15 of gestation, A conception rate of only 15 % for rats exposed to 300 ppm chloroform is unquestionably lower than the rate observed in control rats or rats exposed to lower levels of chloroform in the present study. The absence of metrial glands in 17 of 20 bred females exposed to 300 ppm chloroform indicates that one of the effects of chloroform on conception occurs very early in gestation. In these rats, the only evidence of implantation was a focal increase in the vascularity of the mesometrium. Thus, chloroform may have interfered with the process of implantation. Other effects which occurred in a manner related to the concentration of chloroform include the incidence of resorptions, an increased incidence of anomalies of the vertebrae and sternebrae and the occurrence of subcutaneous edema. In contrast to these anomalies which are evidence of retarded fetal development, true terata (acaudia and imperforate anus) were observed in fetuses of rats exposed to 100 ppm chloroform 7 hr/day on days 6 through 15 of gestation. These findings are similar to those of Schwetz and Becker (1974) in which exposure to 25,000 to 37,000 ppm chloroform (initial chamber concentration) for up to 1 hr on 1 of 3 consecutive days of gestation was highly embryotoxic in mice and rats. The deleterious effect of 1 hr of chloroform anesthesia on days 8 through 10 or 12 through 14 of gestation was quantitatively and qualitatively similar to the effect of exposure to 100 ppm 7 hr/day on days 6 through 15 of gestation in the present study. In both studies, anorexia and severe weight loss were associated with exposure to chloroform; the untoward effects on the embryos and fetuses were not attributable to the anorexia, since the same degree of starvation without exposure to chloroform was not embryo- or fetotoxic. Thus, the observed effect must have been due primarily to the chloroform.3 3A report by Thompson et al., “Teratology studies on orally administered chloroform in the rat and rabbit” has been accepted for publication in a later issue of this journal.
EMBRYOTOXICITY
OF INHALED
CHLOROFORM
451
Exposure to chloroform caused some maternal toxicity but resulted in considerably greater embryo- and fetotoxicity. The largest factor of the maternal toxicity, anorexia and its resultant weight loss, was not responsible for the embryo- and fetotoxicity associated with chloroform exposure. In addition, there was no positive correlation between the degree of hepatotoxicity and embryo- or fetotoxicity. The observed effect of chloroform on the absolute and relative liver weight is usually due to an increase in the absolute liver weight, a decrease in body weight or a combination of the two. Among the pregnant rats exposed to chloroform, the relative liver weight was increased in spite of a decreased absolute liver weight because of the considerable loss of body weight. Another point of interest is the physiological increase in liver size associated with pregnancy. The increase in liver weight is proportional to the increase in body weight during pregnancy, since the relative liver weight remains constant while the absolute liver weight nearly doubles by the end of gestation. Hope (1970) has reported that liver enlargement during pregnancy is due to an initial hypertrophy and subsequent hyperplasia of liver parenchymal cells. In summary, the following conclusions are based on the results of exposure of pregnant rats to chloroform for 7 hr/day on days 6 through 15 of gestation: (a) at 30ppm chloroform, minor toxicity to the rat embryo and fetus was observed; 100 ppm was highly embryotoxic and fetotoxic; at 300 ppm, chloroform was embryocidal as well as highly embryotoxic and fetotoxic. Serious malformations as well as retarded fetal development were found; (b) these effects could not be correlated with maternal toxicity. ACKNOWLEDGMENTS The authors are grateful to P. A. Keeler and H. C. Pernell for their assistance in all aspects of this study. REFERENCES DAWSON, A. B. (1926). A note on the staining of the skeleton of cleared specimens with Alizarin Red-S. Stain Technol. 1, 123. HOPE, J. (1970). Stereological analysis of the ultrastructure of liver parenchymal cells during pregnancy and lactation. J. Ultrastruct. Res. 33, 292-305. SCHWETZ, B. A. AND BECKER, B. A. (1974). Embryotoxicity and fetal malformations of rats and mice due to maternally administered volatile anesthetics. Anesthesiology in press. SIEGEL, S. (1956). Nonparametric Statistics for the Behavioral Sciences, pp. 96-104. McGrawHill, New York. STEEL, R. G. D. AND TORRIE, H. H. (1960). Principles andProcedures of Statistics, pp. 101-l 15, 194-205. McGraw-Hill, New York. WILSON, J. G. (1965). Methods for administering agents and detecting malformations in experimental animals. In: Teratology Principles and Techniques (J. G. Wilson and T. Warkany, eds.), pp. 262-277. Univ. of Chicago Press, Chicago, Illinois.
AND
Embryo-
APPLIED
and
PHARMACOLOGY
Fetotoxicity
28,
442451
(1974)
of Inhaled
Chloroform
B. A. SCHWETZ, B. K. J. LEONG AND P. J.
in Rats
GEHRING
Chemical Biology Research, l%e Dow Chemical Company, Midland, Michigan 48640 Received September 14, 1973; accepted November 20, 1973
Embryo- and Fetotoxicity of Inhaled Chloroform in Rats. SCHWETZ, LEONG, B. K. J. AND GEHRING, P. J. (1974). Toxicol. Appl. Phurmacol., 28,442-451. This study evaluated the effects of inhalation of subanesthetic concentrations of chloroform on rat embryo& and fetal development. Pregnant Sprague-Dawley rats were exposed to 30,100 or 300 ppm chloroform for 7 hr/day on days 6 through 15 ofgestation. Exposure to chloroform caused an apparent decreasein the conception rate and a high incidence of fetal resorption (300 ppm), retarded fetal development (30, 100,300 ppm), decreased fetal body measurements (30, 300 ppm) and a low incidence of acaudate fetuses with imperforate anus (100 ppm). Chloroform was not highly teratogenic but was highly embryotoxic. The results of this study disclosed no relationship between maternal toxicity and embryo or fetotoxicity as the result of exposure to chloroform by inhalation. B.A.,
Exposure of mice or rats for up to 1 hr to anesthetic concentrations of chloroform (25,000-37,000 ppm) during organogenesis has been demonstrated to be highly embryotoxic (Schwetz and Becker, 1974). Although chloroform is no longer widely used as an anesthetic agent in the United States, it is used extensively as a starting material and a chemical intermediate in industry and laboratories. This study was performed to determine whether exposure of pregnant rats to levels of chloroform more like those which may be encountered in industry or laboratories may have an adverse effect on the development of the embryo or fetus. Three Ievels of exposure were studied, 30, 100 and 300 ppm for 7 hr/day on days 6 through 15 of gestation. Maternal toxicity, including hepatotoxicity, was also determined in order to reveal any relationships between the effects of chloroform on the embryo or fetus and the mother. METHODS Animals. Adult Sprague-Dawley (Spartan) female rats weighing approximately 250 g were used. The day on which sperm were seen in a vaginal smear was considered day 0 of pregnancy. Between daily exposures, animals were housed in wire-bottom cages and were maintained on commercial laboratory rat chow1 and water ad libitum in a room controlled for temperature, humidity and light cycle. Food or water were not provided during exposure to chloroform. Food consumption of each animal or group of 2 animals was measured at 2-day intervals throughout the experimental period. 1 Purina Rat Chow@,Ralston Purina Co., St. Louis, Missouri. Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
442
EMBRYOTOXICITYOFINHALED
443
CHLOROFORM
Materials and exposure procedure. Reagent grade chloroform” was used in these experiments. Exposures were conducted in 3.7 m3 stainless steel, cubical dynamic
exposure chambers. Vapors of chloroform,
generated by metering the liquid at known
rates into a temperature-controlled evaporating flask, were diluted with filtered room air at a rate calculated to give the desired concentration. The nominal concentrations in the chamber atmospherewhich were calculated from the ratio of the rate of delivery of chloroform to the rate of total air flow through the chamber were substantiated by analysis 3 times daily using a Beckman IRlO infrared spectrophotometer with a multipath gascell (Table 1). In addition, the concentration in the chamberswas continuously monitored using combustion analysis to assure the absence of significant deviations from the desired level. Becausethe concentrations determined by analyseswere essentially the same as the nominal concentrations, the nominal concentrations will be referred to throughout this paper.
TABLE 1 SAMPLE DESCRIPTION AND CONCENTRATIONS OF COMPONENTS IN EXPOSURE ATMOSPHERES
Component Chloroform Carbon tetrachloride Unknown
Volume % in sample” 99.30
ppm in ChambeP Nominal : Analytical:
0.67 0.03
30 30 + 1 0.1 0.03
100 95 & 29 0.5 0.1
300 291& 22 i .4 0.3
aDeterminedby gas-liquidchromatography. ’ Thenominalchloroformconcentrationwascalculatedfromtheratio of therateof liquidintroduced to the rate of the dilution air flow. Theanalyticalconcentrationwasdeterminedby infraredspectrometry, meanf SD of 3 measurements on eachof 10consecutive daysof exposure.The concentration of minorcomponents wascalculatedasa proportionof their concentrationin theliquidsamplerelative to the majorcomponent. Experimentaldesign. In the initial experiment, pregnant rats were exposed to 300 ppm chloroform 7 hr/day on days 6 through 15 of gestation. Subsequently, additional groups of rats were exposed to 100 or 30 ppm. Nonpregnant female rats were exposed simultaneously with the pregnant rats at the 2 higher concentrations to allow for evaluation
of treatment-related changes in serum glutamic-pyruvic during and after exposure. Control animals for each currently to filtered room air. The parameters which significantly in the 3 air-control groups used. Therefore,
transaminase (SGPT) activity experiment were exposed conwere evaluated did not differ for statistical comparisons, the
data for the control groups were combined.
Marked anorexia was observed in rats exposed to 300 ppm chloroform in the initial experiment. Therefore, another control group, “starved control”, was included in the secondexperiment to simulate the anorexia observed in the first experiment. This group was handled similarly except that each rat was allowed to eat only 3.7 g of food/day on days 6 through 15 of gestation. Maternal andfetal examination. All rats were observed daily throughout pregnancy and maternal body weights were recorded on days 6, 13 and 21 of gestation. The dams ’ Lot No. 9649,Burdick & JacksonLab., Inc., Muskegon,Michigan.
444
SCHWETZ,
LEONG
AND
GEHRING
were sacrificed by COZ anesthesia on day 21 of gestation. After the uterine horns had been exteriorized through a mid-line incision in the abdominal wall, the number and position of live, dead and resorbed fetuses were noted. The umbilical cord was clamped and severed distally. After being weighed, measured (crown-rump length) and the sexes recorded, the fetuses were examined for external anomalies. Each litter was divided equally into 2 subgroups for preservation and subsequent examination. One subgroup, preserved in Bouin’s solution, was examined by the method of Wilson (1965) for evidence of soft tissue anomalies. The second subgroup, preserved in alcohol, was cleared and stained with Alizarin Red-S (Dawson, 1926) for examination of the skeletons for evidence of abnormalities. Hepatotoxicity. SGPT activity was determined in nonpregnant rats following the second, fourth, seventh, and tenth (final) exposure to 100 or 300 ppm chloroform. After the last exposure, 4 nonpregnant rats were sacrificed for gross examination of the liver. Six days later, the day the pregnant females were subjected to a cesarean section, the SGPT activity was determined in the remaining nonpregnant rats and 10 pregnant rats. All livers were weighed and examined grossly. SGPT activity was determined fluorometrically using the Technicon Auto Analyzer (Method N-54). Statistical evaluation. The Fisher Exact Probability Test (Siegel, 1956) was used to evaluate the frequency of anomalies and resorptions between litters. Maternal and fetal body weights and body measurements, liver weights and SGPT activities were analyzed statistically by an analysis of variance and Dunnett’s or Tukey’s test (Steel and Torrie, 1960). In all cases, the level of significance was chosen asp < 0.05. The litter was considered the experimental unit of treatment and observation. RESULTS Embryo- and Fetotoxicity Exposure to 300 ppm chloroform on days 6 through 15 of gestation caused a significant increase in the incidence of fetal resorptions and a decrease in fetal body measurements (Table 2). At this concentration, the conception rate was only 15 % (3 pregnant animals/20 animals bred) compared to 88 % (68 pregnant/77 bred) in the control group (Table 2). In 1 of 3 pregnant females exposed to 300 ppm chloroform, the remaining metrial glands were only about 1 mm in diameter compared to the usual 4-6 mm after an early resorption, indicating that the conceptus had been completely resorbed very soon after implantation. In 7 of the 17 nonpregnant bred females, there was vascular evidence in the mesometrium of implantation but there was no intrauterine evidence of implantation. In contrast, exposure to 100 or 30 ppm chloroform did not alter the incidence of fetal resorptions, fetal body weight, the conception rate, the number of implantations or the average litter size. Fetal crown-rump length was statistically significantly less than control at 30 ppm but not at 100 ppm. At 30 ppm chloroform, the incidence of gross and soft tissue anomalies was not different from control (Table 3). The incidence of delayed ossification of skull bones and of wavy ribs was significantly greater than among control litters. Examination of the fetuses of dams exposed to 100 ppm revealed a significant incidence of acaudia (absence of tail) or short tails, imperforate anus, subcutaneous edema, missing ribs and delayed ossification of sternebrae (Table 3). At 300 ppm chloroform, subcutaneous edema and anomalies of the skull and sternum were observed, but
2
7 (6/87) 25 (2/8)
8 (63/769) 57 (39/68) O/68
5.19 * 0.29’ 42.1 -t 1.1’
3.0 (6/2) 45:55 42.5+ 0.6’
5.51 + 0.20
53:47
1.6 (24/l 5)
68 (15/22) o/22
13+2 12f2 8 (24/291)
16f3e
30 71 (22/31)
55:45 5.59 + 0.24 43.b + 0.7
1.3 (16/12)
6 (16/278) 52 (12/23) O/23
12+2 11_+2
loo 82 (23/28) 14_+ 2
Chloroform
THE TIME OF CESAREANSECTION~
4f 7p 61 (20/33)d loo (3/3) l/3 6.7 (20/3) 34 : 66’ 3.42 & 0.02’ 36.9 + 0.2’
11+4
14+ 1
300 15 (3/20)d
n Administered by inhalation 7 hr daily on days 6-15 of gestation. Starved controls were fed 3.7 g of food daily on days 6-15 of gestation. * Mean + SD. c Mean of litter means + SD. d Significantly different from control by the Fisher Exact Probability test, p < 0.05. e Significantly different from control by an analysis of variance and Dunnett’s test, p < 0.05.
53:47 5.69 + 0.36 43.5 + 1.1
W3
11f4 10+4
11+3 10+4
1.6 (63/39)
100 W) 14+2
-
-
Chloroform concentration (ppm) Percentpregnancy(pregnant/bred) Corpora lutes/dam* Implantations/littel Live fetuses/We? ‘A Resorptions/implantations % Litters with resorptions Litters totally resorbed Resorptions/litterswith resorptions Sexratio, M :F Fetal body weight (g) Fetal crown-rump length (mm) 88 (68/77) 14f2
Air control starved
Air control
Statistic
EFFECTOF INHALED CHLOROFORMON OBSERVATIONS MADEAT
TABLE
iz s kz 3 E
E F B
$
2
ij
2
;s
E
446
SCHWETZ,
LEONG
AND
GEHRING
a significant increase in incidence was not statistically demonstrable because of the small number of litters involved. TABLE 3 EFFECT OF INHALED CHLOROFORM ON THE INCIDENCE OF FETAL ANOMALIES AMONG RAT LITTERS’
Air control
Air control starved
68
-
Chloroform concentration, ppm Number of litters examined
Chloroform 30 22
8
loo 23
Percent of Litters A&ted Gross Acaudia or short tail Imperforate anus Total gross Skeletal Delayed ossification,skull Missing ribs Wavy ribs Split sternebrae Delayed ossification,sternebrae Total skeletal Soft tissue Subcutaneousedema Total soft tissue
300 3
(No. of litters)
(0) (0) 1.5 (1)
(0) (0) (0)
(0) (0) (0)
13 (3)b 13 (3)b 13 (3)b
21 (14) (0) (0) 1.5 (1) 22 (15) 68 (46)
(0) (0) g; 38 (3) 38 (3)
73 (16)b (0) 18 (4)b 9 (2) (0) 90 (20)b
30 (7) 13(3)b (0) 9 (2) 74 (17)b 74 (17)
50 (1) (0) (0) 50 (1) 100(2) loo (2)
34 (23) 48 (33)
38 (3) 38 (3)
41 (9) 45 (10)
61 (14)b 65 (15)
100 (l!
(0) (0) (0)
100(1)
a Administered by inhalation 7 hr daily on days 6-15 of gestation. Starved controls were fed 3.7 g of food daily on days 6-15 of gestation. b Significantly different from control by the Fisher Exact Probability test, p -C0.05.
Maternal Toxicity
Exposure of rats to 30 or 100 ppm chloroform crease in the rate of maternal weight gain and (Tables 4 and 5). Exposure of females to 300 ppm in the rate of weight gain and food consumption
caused a statistically significant defood consumption during exposure chloroform caused a drastic decrease during pregnancy.
TABLE 4 EFFECT OF INHALED CHLOROFORM ON MATERNAL BODY WEIGHT DURING GESTATION”
Air control Chloroform concentration (mm) Number of dams Gestation day 6 13 21
68 275+21” 310* 17 389 + 28
Air control starved 8 274+ 13 223a 13’ 326k 24”
Chloroform 30
100
22 266 + 14 280+ 14” 381+ 23
23 274+ 17 274 f 18’ 365&-22”
300 3 284f 9 192+9= 241 k 29”
a Administered by inhalation 7 hr daily on days 6-15 of gestation. Starved controls were fed 3.7 g of food daily on days 6-15 of gestation. b Body weight, g, mean + SD. CSignificantly different from control by an analysis of variance and Dunnett’s test, p < 0.05.
20 &- 3
20 _+ 2 18+ 1
8
23 3
-z
13f4d 1 + Id
5 + 3d
19f3
67
i5f2d 4 f 2d
18+ 1
__
20 lk 3
8-9
Food consumption
23 + 2
lo-11
16_+2d 1 * Id
18k2
12-13
day numbe?
15_+2d 1 f Id
205 1
19f 2d 1 rlr Id
21+2
23 rf: 3
14-15
CONSUMPTION
22 +- 2 ___
on gestation
STARVED
’ Administered by inhalation 7 hr/day on days 6-15 of gestation. ND, not determined. b g/rat/day, mean t SD. ’ Each rat was given 3.7 g of food daily on days 6 through 15 of gestation. d Significantly different from air control by Dunnett’s test, p < 0.05.
19+2
8
Starvation controP Chloroform 30 mm 100 mm 300 mm
19*4
4-5
53
N
Air control
Test material and concentration”
5
EFFECTOF INHALED CHLOROFORMON RAT MATERNALFOOD
TABLE
t
30 * 3d 12f2d
27 f 3
21_+2d
25 L- 4
16-17
--
33 f 3d ND”
29 zk 5
24 + 8d
261k 3
18-19
--
E
2 z
3 F B
4 8
Y
2 x
E E 2
448
SCHWETZ,
LEONG
AND
GEHRING
In order to assessthe degree of liver damage which may have been incurred by rats exposed to 100 or 300 ppm chloroform, the SGPT activity was determined during and after exposure (Table 6). In all groups, the total weight of the liver and its weight relative to total body weight (mg/g) were determined and the gross appearance of the liver was recorded (Table 7). Exposure to 100 or 300 ppm chloroform did not increase the SGPT activity of nonpregnant rats during exposure or at 6 days after the last exposure. The SGPT activity of pregnant rats 6 days after the last exposure to chloroform, day 21 of gestation, was the same as that of controls. TABLE 6 EFFECT OF INHALED CHLOROFORM ON SGPT ACTIVITY IN RATS
SGPT activity, Karmen units, mean + SE Chloroform” Day of exposure 2nd 4th 7th 10th (last) Six days after last exposure Nonpregnant Pregnant
N
Control
10 10 10 10
63 + 3 57+2 57 4 5 56+2
6
57If: 3 72 + 3
10
100 mm
300 PPm
ND* 45 + 2
52+4 49f3 54+3 54 + 6
59+4 ND
52+3 61 +4
58 IL 3
ND
“Administered by inhalation 7 br/day for 10 consecutive days (days 6-15 of pregnancy for the pregnant rats). Blood samples were taken immediately upon removal from the chamber on the indicated days of exposure. Pregnant rats were bled at the time of cesarean section on day 21 of gestation. All other values are for nonpregnant rats. No values were significantly different from control by Tukey’s test, p < 0.05. b ND, not determined.
In nonpregnant rats, minimal changes in the gross appearance of the liver were observed following the last exposure to 300 ppm chloroform-pale, mottled liver, in 4/4 rats. Six days after the last exposure, the gross appearance of the livers of pregnant and nonpregnant control and chloroform-exposed rats was normal. Neither the absolute nor relative liver weight was altered following exposure to 30ppm chloroform. When measured immediately after the last daily exposure to 100 ppm chloroform, both the absolute and relative weights of the liver of nonpregnant rats were increased significantly. Similar increases were not detected 6 days after the last exposure. A slight but statistically significant increase in the relative, but not the absolute, weight of the liver of pregnant rats exposed to 100 ppm chloroform was observed at the time of cesarean section, 6 days after the last exposure. In contrast to the increase in the total weight of the liver of nonpregnant rats exposed to 100 ppm chloroform at the end of the exposure period, exposure to 300 ppm caused a significant
decrease.
However,
the weight
of the liver relative
to total body weight
was
unchanged, probably reflecting an effect of anorexia in rats exposed to 300 ppm chloroform. At the time of cesarean section, 6 days after the last exposure, the total weight of the liver of pregnant rats exposed to 300ppm chloroform was decreased while the weight
EMBRYOTOXICITY
OF INHALED
CHLOROFORM
449
450
SCHWETZ,
LEONG
AND
GEHRING
of the liver relative to the total body weight was increased, The changes in liver weight just described very likely reflect the anorexic effects of exposure to chloroform and perhaps a direct effect of chloroform on the liver. In order to determine whether the anorexic effects of chloroform may have been responsible for the maternal, embryonal and fetal changes described herein, a group of pregnant rats was fed only 3.7 g of food daily on days 6 through 15 of gestation. This degree of starvation caused a decrease in fetal body measurements but had no effect on the incidence of fetal resorptions (Table 2). In addition, the conception rate, number of implantations and litter size were not different from those of air-exposed control animals. With regard to weight of the maternal liver at the time of cesarean section, starvation caused a slight but significant decrease in the total weight of the liver and a slight but significant increase in the weight of the liver relative to the total body weight. The liver weight changes observed in the starvation-controls explain in part similar changes observed in pregnant rats exposed to chloroform. However, the increase in the relative liver weight of pregnant rats exposed to 300 ppm was much greater, suggesting that this effect may not have been due solely to anorexia. DISCUSSION In these studies, a high degree of embryo and fetal toxicity was associated with the exposure of pregnant rats to 100 or 300 ppm chloroform daily on days 6 through 15 of gestation, A conception rate of only 15 % for rats exposed to 300 ppm chloroform is unquestionably lower than the rate observed in control rats or rats exposed to lower levels of chloroform in the present study. The absence of metrial glands in 17 of 20 bred females exposed to 300 ppm chloroform indicates that one of the effects of chloroform on conception occurs very early in gestation. In these rats, the only evidence of implantation was a focal increase in the vascularity of the mesometrium. Thus, chloroform may have interfered with the process of implantation. Other effects which occurred in a manner related to the concentration of chloroform include the incidence of resorptions, an increased incidence of anomalies of the vertebrae and sternebrae and the occurrence of subcutaneous edema. In contrast to these anomalies which are evidence of retarded fetal development, true terata (acaudia and imperforate anus) were observed in fetuses of rats exposed to 100 ppm chloroform 7 hr/day on days 6 through 15 of gestation. These findings are similar to those of Schwetz and Becker (1974) in which exposure to 25,000 to 37,000 ppm chloroform (initial chamber concentration) for up to 1 hr on 1 of 3 consecutive days of gestation was highly embryotoxic in mice and rats. The deleterious effect of 1 hr of chloroform anesthesia on days 8 through 10 or 12 through 14 of gestation was quantitatively and qualitatively similar to the effect of exposure to 100 ppm 7 hr/day on days 6 through 15 of gestation in the present study. In both studies, anorexia and severe weight loss were associated with exposure to chloroform; the untoward effects on the embryos and fetuses were not attributable to the anorexia, since the same degree of starvation without exposure to chloroform was not embryo- or fetotoxic. Thus, the observed effect must have been due primarily to the chloroform.3 3A report by Thompson et al., “Teratology studies on orally administered chloroform in the rat and rabbit” has been accepted for publication in a later issue of this journal.
EMBRYOTOXICITY
OF INHALED
CHLOROFORM
451
Exposure to chloroform caused some maternal toxicity but resulted in considerably greater embryo- and fetotoxicity. The largest factor of the maternal toxicity, anorexia and its resultant weight loss, was not responsible for the embryo- and fetotoxicity associated with chloroform exposure. In addition, there was no positive correlation between the degree of hepatotoxicity and embryo- or fetotoxicity. The observed effect of chloroform on the absolute and relative liver weight is usually due to an increase in the absolute liver weight, a decrease in body weight or a combination of the two. Among the pregnant rats exposed to chloroform, the relative liver weight was increased in spite of a decreased absolute liver weight because of the considerable loss of body weight. Another point of interest is the physiological increase in liver size associated with pregnancy. The increase in liver weight is proportional to the increase in body weight during pregnancy, since the relative liver weight remains constant while the absolute liver weight nearly doubles by the end of gestation. Hope (1970) has reported that liver enlargement during pregnancy is due to an initial hypertrophy and subsequent hyperplasia of liver parenchymal cells. In summary, the following conclusions are based on the results of exposure of pregnant rats to chloroform for 7 hr/day on days 6 through 15 of gestation: (a) at 30ppm chloroform, minor toxicity to the rat embryo and fetus was observed; 100 ppm was highly embryotoxic and fetotoxic; at 300 ppm, chloroform was embryocidal as well as highly embryotoxic and fetotoxic. Serious malformations as well as retarded fetal development were found; (b) these effects could not be correlated with maternal toxicity. ACKNOWLEDGMENTS The authors are grateful to P. A. Keeler and H. C. Pernell for their assistance in all aspects of this study. REFERENCES DAWSON, A. B. (1926). A note on the staining of the skeleton of cleared specimens with Alizarin Red-S. Stain Technol. 1, 123. HOPE, J. (1970). Stereological analysis of the ultrastructure of liver parenchymal cells during pregnancy and lactation. J. Ultrastruct. Res. 33, 292-305. SCHWETZ, B. A. AND BECKER, B. A. (1974). Embryotoxicity and fetal malformations of rats and mice due to maternally administered volatile anesthetics. Anesthesiology in press. SIEGEL, S. (1956). Nonparametric Statistics for the Behavioral Sciences, pp. 96-104. McGrawHill, New York. STEEL, R. G. D. AND TORRIE, H. H. (1960). Principles andProcedures of Statistics, pp. 101-l 15, 194-205. McGraw-Hill, New York. WILSON, J. G. (1965). Methods for administering agents and detecting malformations in experimental animals. In: Teratology Principles and Techniques (J. G. Wilson and T. Warkany, eds.), pp. 262-277. Univ. of Chicago Press, Chicago, Illinois.