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The effect of maternally inhaled trichloroethylene, perchloroethylene, methyl chloroform, and methylene chloride on embryonal and fetal development in mice and rats
TOXICOLOGYANDAPPLIED
PHARMACOLOGY32,84-96(1975)
The Effect of Maternally Inhaled Trichloroethylene, Perchloroethylene, Methyl Chloroform, and Methylene Chloride on Embryonal and Fetal Development in Mice and Rats1 B. A. SCHWETZ, B. K. J. LEONG,
AND
P. J. GEHRING
Toxicology Research Laboratory, The Dow Chemical Company, Midland, Michigan 48640 Received June 20, 1974; accepted September 16,1974
The Effect of Maternally Inhaled Trichloroethylene, Perchloroethylene, Methyl Chloroform, and Methylene Chloride on Embryonal and Fetal Development in Mice and Rats. SCHWETZ, B. A., LEONG, B. K. J. AND GEHRING, P. J. (1975). Toxicol. Appl. Pharmacol. 32, 84-96. Thesestudies evaluated the effectsof trichloroethylene, perchloroethylene(tetrachloroethylene), methyl chloroform (l,l,l-trichloroethane) and methylene chloride (dichloromethane)on mouseandrat embryonaland fetal development at a concentration two timesthe maximum allowableexcursionlimit for humanindustrial exposureasdefinedby ACGIH, 1973(300,300,875, 1250 ppm, respectively). Groups of pregnant Sprague-Dawley rats and SwissWebstermice were exposedto eachsolvent 7 hr daily on days6-15 of gestation.None of thesesolventscausedsignificantmaternal,embryonal or fetal toxicity and none wasteratogenicin either speciesof animalat the concentrationsstudied.Elevatedcarboxyhemoglobincontent wasobserved in mice and rats exposedto methylenechloride. Trichloroethylene, perchloroethylene (tetrachloroethylene), methyl chloroform (1,I ,ltrichloroethane) and methylene chloride (dichloromethane) are chlorinated, organic solvents that are usedwidely in industry and researchlaboratories for such purposesas degreasingoperations, drycleaning, paint stripping and extraction solvents as well as intermediate chemicals for the synthesis of other chemicals. Some of these usesmay result in the exposure of women of child bearing age. Studies to determine if thesesolvents may affect the developing embryo and fetus have not been reported. This study was performed to determine if these solvents might affect the developing embryo and fetus of mice and rats exposed to a concentration two times the maximum allowable excursion limit for industrial exposure of humans. The threshold limit values for methyl chloroform, methylene chloride, perchloroethylene and trichloroethylene are 350, 500, 100and 100 ppm, respectively; twice the maximum excursion limits are 875,1250,300 and 300 ppm, respectively (ACGIH, 1973). These concentrations are lower than the no-toxic-effect concentrations for adult rats exposed to these solvents (Patty, 1963). Stewart et al. (1972) recently reported significantly elevated carboxyhemoglobin i The animalsusedin this studyweremaintainedin accordance with the directivesin “Guide for care and use of Laboratory Animals,” DHEW Pub. No. (NIH) 73-23. They were housed in facilities accredited by the American Association for Accreditation of Laboratory Animal Care. Copyright 0 1975 by Academic Press, Inc. 84
All rights of reproduction Printed in Great Britain
in any form
reserved.
INHALED
SOLVENT
VAPORS
EMBRYOTOXICITY
85
content in blood of humans exposed to high levels of methylene chloride. Since carbon monoxide has a detrimental effect on fetal development in laboratory animals and.man (Astrup et al., 1972), the carbon monoxide (carboxyhemoglobin) concentration was measured in the blood of nonpregnant mice and rats exposed simultaneously with pregnant females. METHODS
Animals and materials. Adult Sprague-Dawley female rats weighing approximately 250 g were used along with Swiss Webster mice weighing 25 to 30 g.’ The day on which sperm were seen in a vaginal smear of rats or a vaginal plug was observed in mice was considered day zero of pregnancy. Between exposures, animals were housed in wirebottom cages in a room controlled for temperature, humidity and light cycle. Commercial laboratory rat chow3 and water were available free choice. Food and water were not provided during exposure to solvents. Food consumption of each rat or cage of two
rats was measured at 2-day intervals throughout the experimental period. Samples of solvents manufactured by The Dow Chemical Company were used. The purity of these commercial products of methylchloroform, methylene chloride, perchloroethylene and trichloroethylene is indicated in Table 1. Exposures were conducted in 3.7 m3 stainless steel, cubical exposure chambers.
Vapors of the individual
solvents, generated by metering the liquid at a known rate into TABLE PURITY
Component CHLOROTHENEd
1
OF TEST MATERIALS
Volume % in sample0
ppm in chamberb
Nominal concentration,
mm’
VG
l,l,l-trichloroethane inhibitors and impurities”
94.520 5.488
872rt 27 50.60
875
99.992 0.008
304&16 0.02
300
97.860 2.140
1226k 76 27.04
1225
99.240 0.760
306+ 8 2.30
300
DOW-PERd
tetrachloroethylene inhibitors and impurities’ AEROTHENEd
MM
methylenechloride inhibitors and impurities’ NEU-TRId
1,l ,Ztrichloroethylene inhibitors and impurities’
aDeterminedby gas-liquidchromatography. bConcentrationof themajorcomponent wasdetermined by infraredspectrometry.Concentration of theminorcomponents wascalculatedasaproportionof theirconcentrationin theliquidsample relative to the majorcomponent. c Calculatedfrom theratio of therate of liquid introducedto the rateof thedilutionair flow. dRegistered trademarkof The Dow ChemicalCompany,Midland, Mich. eA detaileddescriptionof theminorcomponents of theseinhibitedcommercialsolventsis available from InorganicChemicals R&D, The Dow ChemicalCompany,Midland, Mich. 48640. z Ratsand micewereobtainedfrom SpartanResearch Animals,Inc., Haslett,Mich. 3 Purina Rat Chow,RalstonPurinaCo., St. Louis,MO. 63188.
86
SCHWETZ,
LEONG
AND
GEHRING
a temperature controlled evaporator flask, were diluted with filtered room air at a rate calculated to give the desired concentration. The nominal concentration in each chamber atmosphere, which was calculated from the ratio of the rate of delivery of each solvent to the rate of total air flow through the chamber (350-400 liter/min), was substantiated by analysis three times daily using a Beckman IRlO infrared spectrophotometer with a multipath gas cell. In addition, the concentration in the chamber was monitored continuously using a recording combustion analyzer to assure the absence of significant deviations from the desired level. Because the concentrations determined by analysis were essentially the same as the nominal concentrations, the nominal concentrations will be referred to throughout this paper. Experimental design. In the initial experiment, groups of 20-35 bred rats and 30-40 bred mice were exposed to 875 ppm methyl chloroform or 1250 ppm methylene chloride for 7 hr daily on days 6-15 of gestation. Subsequently, additional groups of rats and mice were exposed to 300 ppm perchloroethylene or 300 ppm trichloroethylene. In each experiment, groups of control animals were exposed concurrently to filtered room air in inhalation chambers. The parameters which were evaluated did not differ significantly in the two control groups exposed to filtered room air. Maternal andfetal observations. All rats and mice were observed daily throughout pregnancy and maternal body weights were recorded on days 6,10 and 16 of gestation as well as on the day on which cesarean sections were performed, gestation days 21 and 18 in rats and mice, respectively. Prior to cesarean section, the dams were sacrificed by exposure to carbon dioxide. After the uterine horns were exteriorized through a mid-line incision in the abdominal wall, the number and position of live, dead and resorbed fetuses were noted. Subsequently, the umbilical cord of each fetus was clamped and severed distally and the fetuses were removed. After being weighed, measured (crown-rump length) the sexes were noted and the fetuses were examined for external anomalies. Each litter was divided equally into two 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 for evidence of skeletal anomalies. One fetus randomly selected from each litter was preserved in buffered 10 % formalin. Saggital sections (6 pm thickness) of the whole body were stained with hematoxylin and eosin for microscopic examination. Carboxyhemoglobin determination. Groups of nonpregnant female mice and rats were exposed simultaneously with the pregnant females. Blood samples for analysis were collected by orbital sinus puncture immediately following the third and tenth (last) exposure as well as 24 hours after the tenth exposure. Carboxyhemoglobin determinations were performed using the spectrophotometric method of Buchwald (1969). StatisticaZ evaluation. The Fisher Exact probability test (Siegel, 1956) was used to evaluate the incidence of anomalies and resorptions among litters. Maternal and fetal body weights and body measurements, food consumption values, liver weights and carboxyhemoglobin values were analyzed statistically by an analysis of variance and the Dunnett test (Steel and Torrie, 1960). In all cases, the chosen level of significance was p < 0.05. The litter was considered the experimental unit of treatment and observation.
INHALED
SOLVENT
VAPORS
EMBRYOTOXICITY
87
RESULTS Exposure to 300 ppm trichloroethylene or perchloroethylene was associated with a slight but statistically significant reduction (4-5 %) in the mean body weights of maternal rats but not mice during and/or following exposure. The body weight of mice and rats exposed to 875 ppm methyl chloroform was not different from control. Exposure to 1250 ppm methylene chloride resulted in a significant increase (1 I-15 %) in the mean body weight of mice during and after exposure but had no effect on the body weight of rats. The food consumption of rats was unaffected by exposure to any of the solvents. The food consumption of mice was not measured. Exposure to trichloroethylene had no effect on either the mean absolute or relative weight of the liver of mice and rats when measured at the time of cesarean section (Table 2). The mean absolute weight of the liver of mice and rats was not altered by 300 ppm perchloroethylene. However, the mean relative weight of the liver of mice but not rats was increased. The mean absolute weight of the liver of rats but not mice exposed to 875 ppm methyl chloroform was significantly increased compared to control values. The mean relative weight of the liver was unaffected by exposure to methyl chloroform in both species. Following exposure to 1250 ppm methylene chloride, the mean absolute weight of the liver was significantly increased in both mice and rats but there was no effect on the mean relative liver weight in either species. Embryonalandfetal toxicity. Exposure to 300 ppm trichloroethylene, 875 ppm methyl chloroform or 1250 ppm methylene chloride had no effect on the average number of implantation sites per litter, litter size, the incidence of fetal resorptions, fetal sex ratios or fetal body measurements among mice (Table 3) or rats (Table 4). These parameters were not altered following exposure to 300 ppm perchloroethylene except for a significant decrease in the fetal body weight of mice (Table 3) and a slight but statistically significant increase in the incidence of resorptions among the rat fetal population (Table 4). The incidence of fetal anomalies among litters of mice and rats is indicated in Tables 5 and 6, respectively. The incidence of gross anomalies observed by external examination of fetuses from rats or mice exposed to any of the solvents was not significantly greater than among control litters. The only soft tissue change which occurred at an incidence significantly greater than that among control litters was subcutaneous edema among the litters of mice exposed to 300 ppm perchloroethylene. Regarding skeletal anomalies, exposure to 300 ppm trichloroethylene or 875 ppm methyl chloroform had no effect on the incidence of skeletal anomalies among the litters of mice or rats. The incidence of skeletal anomalies was not different from that of controls among litters of rats exposed to 300 ppm perchloroethylene but among litters of mice the incidence of delayed ossification of skull bones and the incidence of split sternebrae (unfused centers of ossification) was significantly increased compared to that of controls. Among litters of rats exposed to 1250 ppm methylene chloride, the incidence of lumbar ribs or spurs was significantly decreasedcompared to that of controls while the incidence of delayed ossification of sternebrae was significantly greater than in controls. Among litters of mice, a significant number of litters contained pups with a single extra center of ossification in the sternum. Microscopic examination of saggital sections of whole fetuses revealed no abnormalities of organs, tissues or cells as a result of maternal exposure to any of the solvents.
2
36+4
14.0+ 1.5
30 35f
3
17 13.0 * 1.4
18 13.7 + 1.4 37+4
63 f 8b
52 + 6
2.8 f 0.3
17 3.3 + 0.4
12
30 + 0.5 52 f 7
2.9
300
300
-
4Administeredby inhalation7 hr daily on days6-15 of gestation. bSignifkantlydifferentfrom control by the Dunnett test,p < 0.05.
Rats, number of dams Absolute liver weight (g, mean& SD) Relativeliver weight(mgliver/g body wt, mean+ SD)
Relativeliver weight (mgliver/g body wt, mean+ SD)
Mice, number of darns Absolute liver weight (g, mean+ SD)
Solvent concentration, ppm
Perchloroethylene
Trichloroethylene
Control
36 f 5
25 13.6 f 2.9
52 f 9
24 2.8 f 0.5
-
Control
39Ik 5
38f4
19 14.7 + 1.9b
23 14.7 f 2.6b
13 0.5b 55 + 10
3.4*
1250
Methylene chloride
47 + 6
13 2.6 _+ 0.3
875
Methyl chloroform
EFFECTOFINHALEDSOLVENTS ON THE WEIGHT OF THE LIVER OF PREGNANT MICE AND RATS’
TABLE
3
s g
5
5
E 0
z
OF INHALED
26
-
Control
MADE
300 12 14-t2 13+3 9 (H/168) 75 (9/12) o/12 1.7 (15/9) so:50 1.26+ 0.09 26.2 k 1.0
Trichloroethylene
ON OBSERVATIONS
14f2 12+2 10 (39/369) 69 (18/26) O/26 2.2 (39/l 8) 54~46 1.30 & 0.08 26.2 Z!I0.8
SOLVENTS
LIAdministeredby inhalation7 hoursdaily on days6-15 of gestation. bMean_+SD. c Meanof litter meansf SD. dSignificantlylessthan control by the Dunnett test,p < 0.05.
Solvent concentration, ppm Number of litters Implantation sites/litter” Live fetuses/litterb ‘A, resorptions/implantation sites %, litters with resorptions Litters totally resorbed Resorptions/litters with resorptions Sex ratio, M : F Fetal body weight, gc Fetal crown-rump length, mm=
EFFECT
TABLE
3
17 13f2 12f2 8 (171214) 59 (10/17) o/17 1.7 (17/10) 51:49 1.19+ 0.08d 25.9 + 0.8
24 14+ 3 12+4 14(461325) 75 (18/24) 1124 2.6 (46/l 8) 54146 1.34zko.11 26.2 + 0.7
-
300
SECTION
Control
OF CESAREAN
Perchloroethylene
AT THE TIME
875 13 14&2 12+2 8 (141175) 77 (10/13) o/13 1.4 (14/10) 47:53 1.27+ 0.13 25.6 f 1.0
Methyl chloroform
IN MICE’
1250 13 15f 3 13 +4 14(26/190) 46 (6/13) l/13 4.3 (26/6) 46:54 1.32+ 0.08 26.1 & 0.6
Methylene chloride
s :: ii z
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LEONG
AND
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29 (5) 6 (1) 24 (4) (0) (0)
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12 (2) 6 (1)
(0) (0) (0) (0) (0)
(0) (0)
300 17
Perchloroethylene 25
Control 875 23
Methyl chloroform
30(7) 22(5) 16(3) 4(l) 40)
17(4)
12(3) 20(5) 3203) (0) (0) (0)
g;
4(l) (0)
(0) 0-Q (0)
4 (1) (0) (0) 4 (1)
(0) (0) (0)
Percent of litters affected (number of litters) (0) 6 (1) 3 (1) (0) (0) 12(2) 4 (1) (0)
300 18
Trichloroethylene
0Administeredby inhalation7 hr daily on days6-15 of gestation. bSigni6cantlydifferentfrom control by the FisherExact Probability test,P < 0.05. =p = 0.045by the FisherExact Probabilitytest.
Soft tissue Dilated esophagus Hemorrhagein the esophagus Hemorrhagein cerebral ventricles Dilated renal pelvis Dilated ureters Dilated urinary bladder Displacedkidney Subcutaneousedema Skeletal Delayedossification-skull bones Lumbar ribs or spurs Delayed ossification-sternebra Split sternebra Supernumeraryvertebra (one, thoracic)
3 (1) 3 (1)
Gross Short tail Runts (wt < meanx - 3 SD)
-
Control
INHALED SOLVENTS ON THE INCIDENCE OF FETAL ANOMALIES AMONG RAT LITTERS’
30
OF
Solvent concentration, ppm Numbersof litters examined
EFFECT
TABLE
21 (4) 5(ljb 26(5)b 16(3) (0)
19
1250
Methylene chloride
INHALED
SOLVENT
VAPORS
EMBRYOTOXICITY
93
Curboxyhemoglobin content. Exposure to trichloroethylene. perchloroethylene or methyl chloroform had no effect on the carboxyhemoglobin content in mice and rats. In contrast to this, exposure to 1250 ppm methylene chloride caused a significant increase in carboxyhemoglobin during exposure (Table 7). Twenty-four hours after the last exposure, the carboxyhemoglobin values had returned to control values in both mice and rats.
DISCUSSION The results of these studies indicate that exposure of pregnant mice and rats to trichloroethylene, perchloroethylene, methyl chloroform, or methylene chloride had little effect on embryonal and fetal development at the levels tested. None of these solvents was teratogenic in either species at these concentrations. These responses are summarized in Table 8. Exposure of mice and rats to 300 ppm trichloroethylene or perchloroethylene or 875 ppm methyl chloroform had little or no effect on the dams and their concepti. Exposure to 1250 ppm methylene chloride was associated with significantly increased carboxyhemoglobin content in both species. Despite the high carboxyhemoglobin values, the only effect of this solvent on embryonal and fetal development was an increase in the incidence of variations in the development of the sternum. The effects of inhalation by rats of subanesthetic concentrations of chloroform (30, 100, 300 ppm), carbon tetrachloride (300, 1000 ppm), l,l-dichloroethane (3800. 6000 ppm) and methyl ethyl ketone (1000,300O ppm) have been reported by Schwetz et al. (1974a,b). None of these solvents were teratogenic but chloroform was quite embryotoxic. Relative to their respective threshold limit values, carbon tetrachloride, I,1 dichloroethane and methyl ethyl ketone were much less toxic to the developing embryo and fetus than chloroform. Considering these results plus those of the present study. organic solvents as a group do not appear to be highly embryotoxic but each solvent must be considered individually. In the study reported by Stewart et al. (1972) the carboxyhemoglobin values of three human subjects exposed to 986 -t 104 ppm methylene chloride for 2 hr increased to a mean value of 10% 1 hr after exposure; the pre-exposure mean carboxyhemoglobin value was 1 “/ In the present experiment, comparable carboxyhemoglobin values were observed in mice and rats exposed to 1250 ppm methylene chloride for 7 hr daily for 10 consecutive days. Within 24 hr after the last exposure, carboxyhemoglobin had returned to control values. Kubic et aE. (1973) have found that methylene chloride is metabolized to carbon monoxide following administration of [13C]methylene chloride to rats. In summary, the results of these studies indicate that exposure of pregnant mice and rats to two times to maximum excursion limit of trichloroethylene (300 ppm), perchloroethylene (300 ppm), methyl chloroform (875 ppm) or methylene chloride (1250 ppm) caused little or no maternal, embryonal or fetal toxicity. Elevated carboxyhemoglobin values in both mice and rats followed exposure to methylene chloride. None of the solvents caused a teratogenic response in either mice or rats at the concentrations tested. ACKNOWLEDGMENTS The authors are grateful to Mrs. P. A. Keeler, Mrs. H. C. Pernell, Dr. R. J. Kociba, Dr. G. C. Jersey and Mrs. R. W. Lisowe for their assistance in all aspects of this study.
10 1.7f 1.1 2.6 f 2.3 ND 10 0.4 f 0.5 1.5 f 1.4 ND
-
Control
CONTENTIN
TABLE 7
Perchloroethylene Control
Methyl chloroform
EXPOSEDTO SOLVENTS’
10 1.7 + 1.6 1.7 f 1.4 ND 10 0.3 * 0.5 0.2 f 0.5 ND
10 1.7+ 2.1 1.1 * 1.7 0.5 + 1.0 8 0.4 +_0.7 0.4 f 0.6 1.6 f 1.8
immediately after the
0.5 f 1.1 8 10.3f 2.3c 8.9 + 1.7” 1.0 + 0.9
ND 7 ND 0.4 f 0.3 ND determination
9 12.6+ 3.8” 9.8 + 2.2c
1250
Methylene chloride
8 NDb 1.6 + 1.5
300 875 Percent carboxyhemoglobin, mean+ SD
10 1.3 + 1.6 3.5 f 1.4 ND 10 0.7 -I 0.8 0.9 + 0.9 ND
300
Trichloroethylene
BLENDOF MICE ANDRATS
0 Administered by inhalation 7 hr daily for 10 consecutive days. Blood samples were taken for carboxyhemoglobin third and tenth (last) exposures and 24 hr after the last exposure. b ND = not determined. c Significantly greater than control by the Dunnett test, p c 0.05.
Mice, number After third exposure After tenth exposure 24 hours after last (10th) exposure Rats, number After third exposure After tenth exposure 24 hr after last (10th) exposure)
Solvent concentration, ppm
.-
CARBOXYHEMOGLOBIN
5 $2 E 3
i 24 “i G % o
P
content
OF INHALATION
-
ND -
-b DEC -
Trichloroethylene 300 ppm Mice Rats
OF THE EFFECT
BY MICE
DEC INC -
ND INC DEC INC’ INCd -
Perchloroethylene 300 mm Mice Rats -
OF SOLVENTS
8 AND
PREGNANCY~
-
ND INC -
-
Methyl chloroform 875 ppm Mice Rats
RATS DURING
INC
INC’ -
INC
lNCf -
Methylene chloride 1250 ppm Mice Rats -I__INC ND INC INC -
0 Solvents were inhaled 7 hr daily on days 6-15 of gestation. b Symbols: - means no effect, DEC means decreased, INC means increased relative to control values, ND means not determined. c Subcutaneous edema. d Delayed ossification of skull bones; split sternebrae. p Extra sternebrae. f Delayed ossification of sternebrae.
Maternal weight gain Food consumption Liver weight : absolute (maternal) relative Percent resorptions Average litter size Fetal body measurements Gross anomalies Soft tissue anomalies Skeletal anomalies Microscopic examination Maternal carboxyhemoglobin
__~
SUMMARY
TABLE
2 s 7 4
0
i
$
5
2
2 F z 0 g 5
96
SCHWETZ, LEONG AND GEHRING
REFERENCES ACGIH (1973), American Conferenceof Governmental Industrial Hygienists, “Threshold Limit Valuesfor ChemicalSubstancesand PhysicalAgents in the Workroom Environment with Intended Changesfor 1973.” A~TRUP, P., TROLLE, D., OLSEN, H. M. AND KJELDSEN, K. (1972).Effect of moderatecarbonmonoxide exposureof fetal development.Luncet 2 (7789), 1220-1222. BKJCHWALD, H. (1969). A rapid and sensitivemethod for estimatingcarbon monoxide in blood and its application in problem areas.Amer. Znd.Hyg. Assoc.J. 30, X54-569. DAWSON, A. B. (1926).A note on the stainingof the skeletonof clearedspecimens with alizarin red-S. Stain Tech. 1, 123-124. KUBIC, V. L., ANDERS, M. W., ENGEL, R. R., BARLOW, C. H. AND CAUGHEY, W. S. (1974). Metabolism of dihalomethanesto carbon monoxide. I. In vivo studies.Drug Metabolism and Disposition2, 53-57. PATTY, F. A. (1963).Industrial Hygieneand Toxicology, Secondrevisededition, Vol. II, IntersciencePublishers,New York. SCHWETZ, B. A., LEONG, B. K. J. AND GEHRING, P. J. (1974a).Embryo- and fetotoxicity of inhaled chloroform in rats. Toxicol. Appl. Pharmacol.28,442-451. SCHWETZ, B. A., LEONG, B. K. J. AND GEHRING, P. J. (1974b).Embryo- and fetotoxicity of inhaled carbon tetrachloride, 1,l -dichloroethaneand methyl ethyl ketone in rats. Toxicol. Appl. Pharmacol.28,452-464. SIEGEL, S. (1956).Non-parametricStatisticsfor the Behavioral Sciences.McGraw-Hill, New York. STEEL, R. G. D. AND TORRIE, H. H. (1960). PrinciplesandProceduresof Statistics, McGrawHill, New York. STEWART, R. D., FISHER, T. N., NOSKO, M. J., PETERSON, J. P., BARETTA, E. D. AND DODD, H. C. (1972). Formation of carbon monoxide following exposure to dichloromethane (methylene chloride). Science174, 295-296. WILSON, J. G. (1965). Methods for administering agentsand detecting malformations in experimentalanimals.In TeratologyPrinciplesand Techniques J. G. Wilson and T. Warkany (Eds.), The University of Chicago Press,Chicago.
PHARMACOLOGY32,84-96(1975)
The Effect of Maternally Inhaled Trichloroethylene, Perchloroethylene, Methyl Chloroform, and Methylene Chloride on Embryonal and Fetal Development in Mice and Rats1 B. A. SCHWETZ, B. K. J. LEONG,
AND
P. J. GEHRING
Toxicology Research Laboratory, The Dow Chemical Company, Midland, Michigan 48640 Received June 20, 1974; accepted September 16,1974
The Effect of Maternally Inhaled Trichloroethylene, Perchloroethylene, Methyl Chloroform, and Methylene Chloride on Embryonal and Fetal Development in Mice and Rats. SCHWETZ, B. A., LEONG, B. K. J. AND GEHRING, P. J. (1975). Toxicol. Appl. Pharmacol. 32, 84-96. Thesestudies evaluated the effectsof trichloroethylene, perchloroethylene(tetrachloroethylene), methyl chloroform (l,l,l-trichloroethane) and methylene chloride (dichloromethane)on mouseandrat embryonaland fetal development at a concentration two timesthe maximum allowableexcursionlimit for humanindustrial exposureasdefinedby ACGIH, 1973(300,300,875, 1250 ppm, respectively). Groups of pregnant Sprague-Dawley rats and SwissWebstermice were exposedto eachsolvent 7 hr daily on days6-15 of gestation.None of thesesolventscausedsignificantmaternal,embryonal or fetal toxicity and none wasteratogenicin either speciesof animalat the concentrationsstudied.Elevatedcarboxyhemoglobincontent wasobserved in mice and rats exposedto methylenechloride. Trichloroethylene, perchloroethylene (tetrachloroethylene), methyl chloroform (1,I ,ltrichloroethane) and methylene chloride (dichloromethane) are chlorinated, organic solvents that are usedwidely in industry and researchlaboratories for such purposesas degreasingoperations, drycleaning, paint stripping and extraction solvents as well as intermediate chemicals for the synthesis of other chemicals. Some of these usesmay result in the exposure of women of child bearing age. Studies to determine if thesesolvents may affect the developing embryo and fetus have not been reported. This study was performed to determine if these solvents might affect the developing embryo and fetus of mice and rats exposed to a concentration two times the maximum allowable excursion limit for industrial exposure of humans. The threshold limit values for methyl chloroform, methylene chloride, perchloroethylene and trichloroethylene are 350, 500, 100and 100 ppm, respectively; twice the maximum excursion limits are 875,1250,300 and 300 ppm, respectively (ACGIH, 1973). These concentrations are lower than the no-toxic-effect concentrations for adult rats exposed to these solvents (Patty, 1963). Stewart et al. (1972) recently reported significantly elevated carboxyhemoglobin i The animalsusedin this studyweremaintainedin accordance with the directivesin “Guide for care and use of Laboratory Animals,” DHEW Pub. No. (NIH) 73-23. They were housed in facilities accredited by the American Association for Accreditation of Laboratory Animal Care. Copyright 0 1975 by Academic Press, Inc. 84
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in any form
reserved.
INHALED
SOLVENT
VAPORS
EMBRYOTOXICITY
85
content in blood of humans exposed to high levels of methylene chloride. Since carbon monoxide has a detrimental effect on fetal development in laboratory animals and.man (Astrup et al., 1972), the carbon monoxide (carboxyhemoglobin) concentration was measured in the blood of nonpregnant mice and rats exposed simultaneously with pregnant females. METHODS
Animals and materials. Adult Sprague-Dawley female rats weighing approximately 250 g were used along with Swiss Webster mice weighing 25 to 30 g.’ The day on which sperm were seen in a vaginal smear of rats or a vaginal plug was observed in mice was considered day zero of pregnancy. Between exposures, animals were housed in wirebottom cages in a room controlled for temperature, humidity and light cycle. Commercial laboratory rat chow3 and water were available free choice. Food and water were not provided during exposure to solvents. Food consumption of each rat or cage of two
rats was measured at 2-day intervals throughout the experimental period. Samples of solvents manufactured by The Dow Chemical Company were used. The purity of these commercial products of methylchloroform, methylene chloride, perchloroethylene and trichloroethylene is indicated in Table 1. Exposures were conducted in 3.7 m3 stainless steel, cubical exposure chambers.
Vapors of the individual
solvents, generated by metering the liquid at a known rate into TABLE PURITY
Component CHLOROTHENEd
1
OF TEST MATERIALS
Volume % in sample0
ppm in chamberb
Nominal concentration,
mm’
VG
l,l,l-trichloroethane inhibitors and impurities”
94.520 5.488
872rt 27 50.60
875
99.992 0.008
304&16 0.02
300
97.860 2.140
1226k 76 27.04
1225
99.240 0.760
306+ 8 2.30
300
DOW-PERd
tetrachloroethylene inhibitors and impurities’ AEROTHENEd
MM
methylenechloride inhibitors and impurities’ NEU-TRId
1,l ,Ztrichloroethylene inhibitors and impurities’
aDeterminedby gas-liquidchromatography. bConcentrationof themajorcomponent wasdetermined by infraredspectrometry.Concentration of theminorcomponents wascalculatedasaproportionof theirconcentrationin theliquidsample relative to the majorcomponent. c Calculatedfrom theratio of therate of liquid introducedto the rateof thedilutionair flow. dRegistered trademarkof The Dow ChemicalCompany,Midland, Mich. eA detaileddescriptionof theminorcomponents of theseinhibitedcommercialsolventsis available from InorganicChemicals R&D, The Dow ChemicalCompany,Midland, Mich. 48640. z Ratsand micewereobtainedfrom SpartanResearch Animals,Inc., Haslett,Mich. 3 Purina Rat Chow,RalstonPurinaCo., St. Louis,MO. 63188.
86
SCHWETZ,
LEONG
AND
GEHRING
a temperature controlled evaporator flask, were diluted with filtered room air at a rate calculated to give the desired concentration. The nominal concentration in each chamber atmosphere, which was calculated from the ratio of the rate of delivery of each solvent to the rate of total air flow through the chamber (350-400 liter/min), was substantiated by analysis three times daily using a Beckman IRlO infrared spectrophotometer with a multipath gas cell. In addition, the concentration in the chamber was monitored continuously using a recording combustion analyzer to assure the absence of significant deviations from the desired level. Because the concentrations determined by analysis were essentially the same as the nominal concentrations, the nominal concentrations will be referred to throughout this paper. Experimental design. In the initial experiment, groups of 20-35 bred rats and 30-40 bred mice were exposed to 875 ppm methyl chloroform or 1250 ppm methylene chloride for 7 hr daily on days 6-15 of gestation. Subsequently, additional groups of rats and mice were exposed to 300 ppm perchloroethylene or 300 ppm trichloroethylene. In each experiment, groups of control animals were exposed concurrently to filtered room air in inhalation chambers. The parameters which were evaluated did not differ significantly in the two control groups exposed to filtered room air. Maternal andfetal observations. All rats and mice were observed daily throughout pregnancy and maternal body weights were recorded on days 6,10 and 16 of gestation as well as on the day on which cesarean sections were performed, gestation days 21 and 18 in rats and mice, respectively. Prior to cesarean section, the dams were sacrificed by exposure to carbon dioxide. After the uterine horns were exteriorized through a mid-line incision in the abdominal wall, the number and position of live, dead and resorbed fetuses were noted. Subsequently, the umbilical cord of each fetus was clamped and severed distally and the fetuses were removed. After being weighed, measured (crown-rump length) the sexes were noted and the fetuses were examined for external anomalies. Each litter was divided equally into two 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 for evidence of skeletal anomalies. One fetus randomly selected from each litter was preserved in buffered 10 % formalin. Saggital sections (6 pm thickness) of the whole body were stained with hematoxylin and eosin for microscopic examination. Carboxyhemoglobin determination. Groups of nonpregnant female mice and rats were exposed simultaneously with the pregnant females. Blood samples for analysis were collected by orbital sinus puncture immediately following the third and tenth (last) exposure as well as 24 hours after the tenth exposure. Carboxyhemoglobin determinations were performed using the spectrophotometric method of Buchwald (1969). StatisticaZ evaluation. The Fisher Exact probability test (Siegel, 1956) was used to evaluate the incidence of anomalies and resorptions among litters. Maternal and fetal body weights and body measurements, food consumption values, liver weights and carboxyhemoglobin values were analyzed statistically by an analysis of variance and the Dunnett test (Steel and Torrie, 1960). In all cases, the chosen level of significance was p < 0.05. The litter was considered the experimental unit of treatment and observation.
INHALED
SOLVENT
VAPORS
EMBRYOTOXICITY
87
RESULTS Exposure to 300 ppm trichloroethylene or perchloroethylene was associated with a slight but statistically significant reduction (4-5 %) in the mean body weights of maternal rats but not mice during and/or following exposure. The body weight of mice and rats exposed to 875 ppm methyl chloroform was not different from control. Exposure to 1250 ppm methylene chloride resulted in a significant increase (1 I-15 %) in the mean body weight of mice during and after exposure but had no effect on the body weight of rats. The food consumption of rats was unaffected by exposure to any of the solvents. The food consumption of mice was not measured. Exposure to trichloroethylene had no effect on either the mean absolute or relative weight of the liver of mice and rats when measured at the time of cesarean section (Table 2). The mean absolute weight of the liver of mice and rats was not altered by 300 ppm perchloroethylene. However, the mean relative weight of the liver of mice but not rats was increased. The mean absolute weight of the liver of rats but not mice exposed to 875 ppm methyl chloroform was significantly increased compared to control values. The mean relative weight of the liver was unaffected by exposure to methyl chloroform in both species. Following exposure to 1250 ppm methylene chloride, the mean absolute weight of the liver was significantly increased in both mice and rats but there was no effect on the mean relative liver weight in either species. Embryonalandfetal toxicity. Exposure to 300 ppm trichloroethylene, 875 ppm methyl chloroform or 1250 ppm methylene chloride had no effect on the average number of implantation sites per litter, litter size, the incidence of fetal resorptions, fetal sex ratios or fetal body measurements among mice (Table 3) or rats (Table 4). These parameters were not altered following exposure to 300 ppm perchloroethylene except for a significant decrease in the fetal body weight of mice (Table 3) and a slight but statistically significant increase in the incidence of resorptions among the rat fetal population (Table 4). The incidence of fetal anomalies among litters of mice and rats is indicated in Tables 5 and 6, respectively. The incidence of gross anomalies observed by external examination of fetuses from rats or mice exposed to any of the solvents was not significantly greater than among control litters. The only soft tissue change which occurred at an incidence significantly greater than that among control litters was subcutaneous edema among the litters of mice exposed to 300 ppm perchloroethylene. Regarding skeletal anomalies, exposure to 300 ppm trichloroethylene or 875 ppm methyl chloroform had no effect on the incidence of skeletal anomalies among the litters of mice or rats. The incidence of skeletal anomalies was not different from that of controls among litters of rats exposed to 300 ppm perchloroethylene but among litters of mice the incidence of delayed ossification of skull bones and the incidence of split sternebrae (unfused centers of ossification) was significantly increased compared to that of controls. Among litters of rats exposed to 1250 ppm methylene chloride, the incidence of lumbar ribs or spurs was significantly decreasedcompared to that of controls while the incidence of delayed ossification of sternebrae was significantly greater than in controls. Among litters of mice, a significant number of litters contained pups with a single extra center of ossification in the sternum. Microscopic examination of saggital sections of whole fetuses revealed no abnormalities of organs, tissues or cells as a result of maternal exposure to any of the solvents.
2
36+4
14.0+ 1.5
30 35f
3
17 13.0 * 1.4
18 13.7 + 1.4 37+4
63 f 8b
52 + 6
2.8 f 0.3
17 3.3 + 0.4
12
30 + 0.5 52 f 7
2.9
300
300
-
4Administeredby inhalation7 hr daily on days6-15 of gestation. bSignifkantlydifferentfrom control by the Dunnett test,p < 0.05.
Rats, number of dams Absolute liver weight (g, mean& SD) Relativeliver weight(mgliver/g body wt, mean+ SD)
Relativeliver weight (mgliver/g body wt, mean+ SD)
Mice, number of darns Absolute liver weight (g, mean+ SD)
Solvent concentration, ppm
Perchloroethylene
Trichloroethylene
Control
36 f 5
25 13.6 f 2.9
52 f 9
24 2.8 f 0.5
-
Control
39Ik 5
38f4
19 14.7 + 1.9b
23 14.7 f 2.6b
13 0.5b 55 + 10
3.4*
1250
Methylene chloride
47 + 6
13 2.6 _+ 0.3
875
Methyl chloroform
EFFECTOFINHALEDSOLVENTS ON THE WEIGHT OF THE LIVER OF PREGNANT MICE AND RATS’
TABLE
3
s g
5
5
E 0
z
OF INHALED
26
-
Control
MADE
300 12 14-t2 13+3 9 (H/168) 75 (9/12) o/12 1.7 (15/9) so:50 1.26+ 0.09 26.2 k 1.0
Trichloroethylene
ON OBSERVATIONS
14f2 12+2 10 (39/369) 69 (18/26) O/26 2.2 (39/l 8) 54~46 1.30 & 0.08 26.2 Z!I0.8
SOLVENTS
LIAdministeredby inhalation7 hoursdaily on days6-15 of gestation. bMean_+SD. c Meanof litter meansf SD. dSignificantlylessthan control by the Dunnett test,p < 0.05.
Solvent concentration, ppm Number of litters Implantation sites/litter” Live fetuses/litterb ‘A, resorptions/implantation sites %, litters with resorptions Litters totally resorbed Resorptions/litters with resorptions Sex ratio, M : F Fetal body weight, gc Fetal crown-rump length, mm=
EFFECT
TABLE
3
17 13f2 12f2 8 (171214) 59 (10/17) o/17 1.7 (17/10) 51:49 1.19+ 0.08d 25.9 + 0.8
24 14+ 3 12+4 14(461325) 75 (18/24) 1124 2.6 (46/l 8) 54146 1.34zko.11 26.2 + 0.7
-
300
SECTION
Control
OF CESAREAN
Perchloroethylene
AT THE TIME
875 13 14&2 12+2 8 (141175) 77 (10/13) o/13 1.4 (14/10) 47:53 1.27+ 0.13 25.6 f 1.0
Methyl chloroform
IN MICE’
1250 13 15f 3 13 +4 14(26/190) 46 (6/13) l/13 4.3 (26/6) 46:54 1.32+ 0.08 26.1 & 0.6
Methylene chloride
s :: ii z
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P s J 4 $ 0
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2 2
90
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LEONG
AND
GEHRING
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g; (0)
g; 28 (5) 22 (4) (0) 11(2) (0) (0)
3 (1) (0) (0) 7 (2) (0) 17 (5) 33 (10) 13 (4) 30 (9) (0) (0)
(0)
29 (5) 6 (1) 24 (4) (0) (0)
(0)
12 (2) 6 (1)
(0) (0) (0) (0) (0)
(0) (0)
300 17
Perchloroethylene 25
Control 875 23
Methyl chloroform
30(7) 22(5) 16(3) 4(l) 40)
17(4)
12(3) 20(5) 3203) (0) (0) (0)
g;
4(l) (0)
(0) 0-Q (0)
4 (1) (0) (0) 4 (1)
(0) (0) (0)
Percent of litters affected (number of litters) (0) 6 (1) 3 (1) (0) (0) 12(2) 4 (1) (0)
300 18
Trichloroethylene
0Administeredby inhalation7 hr daily on days6-15 of gestation. bSigni6cantlydifferentfrom control by the FisherExact Probability test,P < 0.05. =p = 0.045by the FisherExact Probabilitytest.
Soft tissue Dilated esophagus Hemorrhagein the esophagus Hemorrhagein cerebral ventricles Dilated renal pelvis Dilated ureters Dilated urinary bladder Displacedkidney Subcutaneousedema Skeletal Delayedossification-skull bones Lumbar ribs or spurs Delayed ossification-sternebra Split sternebra Supernumeraryvertebra (one, thoracic)
3 (1) 3 (1)
Gross Short tail Runts (wt < meanx - 3 SD)
-
Control
INHALED SOLVENTS ON THE INCIDENCE OF FETAL ANOMALIES AMONG RAT LITTERS’
30
OF
Solvent concentration, ppm Numbersof litters examined
EFFECT
TABLE
21 (4) 5(ljb 26(5)b 16(3) (0)
19
1250
Methylene chloride
INHALED
SOLVENT
VAPORS
EMBRYOTOXICITY
93
Curboxyhemoglobin content. Exposure to trichloroethylene. perchloroethylene or methyl chloroform had no effect on the carboxyhemoglobin content in mice and rats. In contrast to this, exposure to 1250 ppm methylene chloride caused a significant increase in carboxyhemoglobin during exposure (Table 7). Twenty-four hours after the last exposure, the carboxyhemoglobin values had returned to control values in both mice and rats.
DISCUSSION The results of these studies indicate that exposure of pregnant mice and rats to trichloroethylene, perchloroethylene, methyl chloroform, or methylene chloride had little effect on embryonal and fetal development at the levels tested. None of these solvents was teratogenic in either species at these concentrations. These responses are summarized in Table 8. Exposure of mice and rats to 300 ppm trichloroethylene or perchloroethylene or 875 ppm methyl chloroform had little or no effect on the dams and their concepti. Exposure to 1250 ppm methylene chloride was associated with significantly increased carboxyhemoglobin content in both species. Despite the high carboxyhemoglobin values, the only effect of this solvent on embryonal and fetal development was an increase in the incidence of variations in the development of the sternum. The effects of inhalation by rats of subanesthetic concentrations of chloroform (30, 100, 300 ppm), carbon tetrachloride (300, 1000 ppm), l,l-dichloroethane (3800. 6000 ppm) and methyl ethyl ketone (1000,300O ppm) have been reported by Schwetz et al. (1974a,b). None of these solvents were teratogenic but chloroform was quite embryotoxic. Relative to their respective threshold limit values, carbon tetrachloride, I,1 dichloroethane and methyl ethyl ketone were much less toxic to the developing embryo and fetus than chloroform. Considering these results plus those of the present study. organic solvents as a group do not appear to be highly embryotoxic but each solvent must be considered individually. In the study reported by Stewart et al. (1972) the carboxyhemoglobin values of three human subjects exposed to 986 -t 104 ppm methylene chloride for 2 hr increased to a mean value of 10% 1 hr after exposure; the pre-exposure mean carboxyhemoglobin value was 1 “/ In the present experiment, comparable carboxyhemoglobin values were observed in mice and rats exposed to 1250 ppm methylene chloride for 7 hr daily for 10 consecutive days. Within 24 hr after the last exposure, carboxyhemoglobin had returned to control values. Kubic et aE. (1973) have found that methylene chloride is metabolized to carbon monoxide following administration of [13C]methylene chloride to rats. In summary, the results of these studies indicate that exposure of pregnant mice and rats to two times to maximum excursion limit of trichloroethylene (300 ppm), perchloroethylene (300 ppm), methyl chloroform (875 ppm) or methylene chloride (1250 ppm) caused little or no maternal, embryonal or fetal toxicity. Elevated carboxyhemoglobin values in both mice and rats followed exposure to methylene chloride. None of the solvents caused a teratogenic response in either mice or rats at the concentrations tested. ACKNOWLEDGMENTS The authors are grateful to Mrs. P. A. Keeler, Mrs. H. C. Pernell, Dr. R. J. Kociba, Dr. G. C. Jersey and Mrs. R. W. Lisowe for their assistance in all aspects of this study.
10 1.7f 1.1 2.6 f 2.3 ND 10 0.4 f 0.5 1.5 f 1.4 ND
-
Control
CONTENTIN
TABLE 7
Perchloroethylene Control
Methyl chloroform
EXPOSEDTO SOLVENTS’
10 1.7 + 1.6 1.7 f 1.4 ND 10 0.3 * 0.5 0.2 f 0.5 ND
10 1.7+ 2.1 1.1 * 1.7 0.5 + 1.0 8 0.4 +_0.7 0.4 f 0.6 1.6 f 1.8
immediately after the
0.5 f 1.1 8 10.3f 2.3c 8.9 + 1.7” 1.0 + 0.9
ND 7 ND 0.4 f 0.3 ND determination
9 12.6+ 3.8” 9.8 + 2.2c
1250
Methylene chloride
8 NDb 1.6 + 1.5
300 875 Percent carboxyhemoglobin, mean+ SD
10 1.3 + 1.6 3.5 f 1.4 ND 10 0.7 -I 0.8 0.9 + 0.9 ND
300
Trichloroethylene
BLENDOF MICE ANDRATS
0 Administered by inhalation 7 hr daily for 10 consecutive days. Blood samples were taken for carboxyhemoglobin third and tenth (last) exposures and 24 hr after the last exposure. b ND = not determined. c Significantly greater than control by the Dunnett test, p c 0.05.
Mice, number After third exposure After tenth exposure 24 hours after last (10th) exposure Rats, number After third exposure After tenth exposure 24 hr after last (10th) exposure)
Solvent concentration, ppm
.-
CARBOXYHEMOGLOBIN
5 $2 E 3
i 24 “i G % o
P
content
OF INHALATION
-
ND -
-b DEC -
Trichloroethylene 300 ppm Mice Rats
OF THE EFFECT
BY MICE
DEC INC -
ND INC DEC INC’ INCd -
Perchloroethylene 300 mm Mice Rats -
OF SOLVENTS
8 AND
PREGNANCY~
-
ND INC -
-
Methyl chloroform 875 ppm Mice Rats
RATS DURING
INC
INC’ -
INC
lNCf -
Methylene chloride 1250 ppm Mice Rats -I__INC ND INC INC -
0 Solvents were inhaled 7 hr daily on days 6-15 of gestation. b Symbols: - means no effect, DEC means decreased, INC means increased relative to control values, ND means not determined. c Subcutaneous edema. d Delayed ossification of skull bones; split sternebrae. p Extra sternebrae. f Delayed ossification of sternebrae.
Maternal weight gain Food consumption Liver weight : absolute (maternal) relative Percent resorptions Average litter size Fetal body measurements Gross anomalies Soft tissue anomalies Skeletal anomalies Microscopic examination Maternal carboxyhemoglobin
__~
SUMMARY
TABLE
2 s 7 4
0
i
$
5
2
2 F z 0 g 5
96
SCHWETZ, LEONG AND GEHRING
REFERENCES ACGIH (1973), American Conferenceof Governmental Industrial Hygienists, “Threshold Limit Valuesfor ChemicalSubstancesand PhysicalAgents in the Workroom Environment with Intended Changesfor 1973.” A~TRUP, P., TROLLE, D., OLSEN, H. M. AND KJELDSEN, K. (1972).Effect of moderatecarbonmonoxide exposureof fetal development.Luncet 2 (7789), 1220-1222. BKJCHWALD, H. (1969). A rapid and sensitivemethod for estimatingcarbon monoxide in blood and its application in problem areas.Amer. Znd.Hyg. Assoc.J. 30, X54-569. DAWSON, A. B. (1926).A note on the stainingof the skeletonof clearedspecimens with alizarin red-S. Stain Tech. 1, 123-124. KUBIC, V. L., ANDERS, M. W., ENGEL, R. R., BARLOW, C. H. AND CAUGHEY, W. S. (1974). Metabolism of dihalomethanesto carbon monoxide. I. In vivo studies.Drug Metabolism and Disposition2, 53-57. PATTY, F. A. (1963).Industrial Hygieneand Toxicology, Secondrevisededition, Vol. II, IntersciencePublishers,New York. SCHWETZ, B. A., LEONG, B. K. J. AND GEHRING, P. J. (1974a).Embryo- and fetotoxicity of inhaled chloroform in rats. Toxicol. Appl. Pharmacol.28,442-451. SCHWETZ, B. A., LEONG, B. K. J. AND GEHRING, P. J. (1974b).Embryo- and fetotoxicity of inhaled carbon tetrachloride, 1,l -dichloroethaneand methyl ethyl ketone in rats. Toxicol. Appl. Pharmacol.28,452-464. SIEGEL, S. (1956).Non-parametricStatisticsfor the Behavioral Sciences.McGraw-Hill, New York. STEEL, R. G. D. AND TORRIE, H. H. (1960). PrinciplesandProceduresof Statistics, McGrawHill, New York. STEWART, R. D., FISHER, T. N., NOSKO, M. J., PETERSON, J. P., BARETTA, E. D. AND DODD, H. C. (1972). Formation of carbon monoxide following exposure to dichloromethane (methylene chloride). Science174, 295-296. WILSON, J. G. (1965). Methods for administering agentsand detecting malformations in experimentalanimals.In TeratologyPrinciplesand Techniques J. G. Wilson and T. Warkany (Eds.), The University of Chicago Press,Chicago.