TOXICOLOGY AND APPLIED PHARMACOLOGY 48,

19-28 (1979)

Studies on the Developmental Toxicity of Ozone. I. Prenatal Effects 1 ROBERT KAVLOCK, 2 GEORGE DASTON, AND CASIMER T. GRABOWSKI

Department of Biology, University of Miami, Coral Gables, Florida 33124 Received February 8, 1978; accepted October 16, 1978 Studies on the Developmental Toxicity of Ozone. I. Prenatal Effects. KAVLOCK, R., G., AND GRABOWSKI, C. T. (1979). Toxicol. Appl. Pharmacol. 48, 19-28. Ozone, the predominant oxidant in photochemical smog, produces pulmonary and systemic toxicities, and yet possible effects on the fetus have received little attention. Long-Evans rats were exposed during early (Day 6-9), mid (Day 9-12) or late (Day 17-20) gestation, or throughout the period of organogenesis (Day 6-15) to concentrations of ozone varying from 0 to 1297 ppm. No terata were produced by any regimen, but the resorption rate was increased following exposure to concentrations of ozone above 1.26 ppm in midgestation. Exposure to ambient levels (0.44 ppm) for 8 hr/day during the period of organogenesis produced no significant effects. Fetal heart rates, electrocardiograms, hematocrits, and plasma electrolytes were unaltered by exposure to 1 ppm during mid- or late gestation when examined on Day 20. Finally, sodium salicylate (150 mg/kg on Day 10) was shown to be synergistic with ozone (1 ppm in midgestation), in that the combined treatment decreased fetal weight, and to a lesser extent increased the resorption rate. These effects were minimal from either treatment administered alone. DASTON,

P h o t o c h e m i c a l smog has received increasing attention as a potential health h a z a r d ( N a t i o n a l A c a d e m y of Sciences, 1977). Ozone is the p r e d o m i n a n t o x i d a n t in p h o t o chemical smog. It is p r o d u c e d by the reaction of sunlight with h y d r o c a r b o n s and oxides of n i t r o g e n arising from the c o m b u s t i o n o f fuels and is classified as a s e c o n d a r y air p o l l u t a n t (Stephens, 1973; Cadle a n d Allen, 1970). Because o f its a i r b o r n e nature, exposure to ozone is neither elective nor limited to a p a r t i c u l a r segment of the p o p u l a t i o n o f a region. There have been a n u m b e r o f studies con-

cerned with effects o f ozone on r e p r o d u c t i o n in avian a n d m a m m a l i a n species. Chicks hatching in an i n c u b a t o r c o n t a i n i n g 2 p p m ozone showed 8 2 % m o r t a l i t y by the second d a y of life, a l t h o u g h no consistent effects were observed on egg viability or hatching rate (Quilligan et al., 1958). K o t i n a n d T h o m a s (1957) f o u n d that mice exposed to 1.25 p p m ozone c o n t i n u o u s l y from m a t i n g to weaning o f litters had significantly decreased c o n c e p t i o n rates, decreased litter sizes, and an increased frequency o f neonatal deaths. The a u t h o r s a t t r i b u t e d these results to effects on the female. B r i n k m a n et ai. (1964) reached similar conclusions after exposing mice on a daily basis to 0.1 or 0.2 p p m ozone, b u t u n f o r t u n a t e l y the a u t h o r s did n o t specify the precise p e r i o d o f exposure. In a follow-up to the B r i n k m a n study ( B r i n k m a n et al., 1964) Veninga (1967) r e p o r t e d increased b l e p h a r o p h i m o s i s a n d in-

1This paper was presented at the Seventeenth Annual Meeting of the Teratology Society, May 15-18, 1977 at Reston, Va. 2 Please address all reprint requests to Dr. Robert Kavlock, MD-74, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711. 19

0041-008x/79/040019-10 $02.00/0 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

20

KAVLOCK, DASTON, AND GRABOWSKI

c r e a s e d j a w a n o m a l i e s in m i c e p e r i n a t a l l y e x p o s e d to 0.2 p p m o z o n e . H u e t e r et al. (1966) a n d Lewis et al. (1967) used uvi r r a d i a t e d a u t o e x h a u s t to s i m u l a t e s m o g c o n d i t i o n s a n d a g a i n n o t e d d e c r e a s e d fertility a n d i n f a n t s u r v i v a l in m i c e as a result o f a chronic exposure regimen. The latter study i n d i c a t e d t h e effect m i g h t result f r o m the e x p o s u r e o f the m a l e p r i o r to m a t i n g . T h e s e f o r m e r studies w e r e all d e s i g n e d to assess the effects o f c h r o n i c e x p o s u r e to ozone or a simulated smog environment and w e r e n o t i n t e n d e d to e v a l u a t e the t e r a t o g e n i c p o t e n t i a l o f a substance. Since the m a t i n g p a i r o f m i c e was u s u a l l y e x p o s e d t o g e t h e r in these e x p e r i m e n t s a n d t h e exposures continued throughout parturition a n d l a c t a t i o n , it is g e n e r a l l y n o t possible to d e t e r m i n e w h i c h p a r e n t was r e s p o n s i b l e f o r the d e c r e a s e d fertility a n d litter size. F i n a l l y , t h e d e c r e a s e d p o s t n a t a l viability c o u l d be d u e to a l a t e n t in utero effect, a d i r e c t a c t i o n o n the n e o n a t e , or a c o m b i n a tiori o f these factors. T h e p r e s e n t studies were t h e r e f o r e u n d e r t a k e n to f u r t h e r e l u c i d a t e the p o t e n t i a l for o z o n e to i n t e r f e r e w i t h n o r m a l d e v e l o p m e n t processes.

METHODS Animals. Adult, Long-Evans 3 female rats weighing between 225 and 275 g were used. Animals were maintained at constant temperature (22°C), humidity (70~) and lighting (12 hr light) in plastic cages on corncob bedding. Food 4 and water were allowed ad libitum. Females were housed with males overnight and pregnancy was confirmed by the presence of sperm in a vaginal smear. Animals with a positive smear were considered to be at Day 0 of gestation. Exposure apparatus. Rats were exposed to ozone generated from air in a 0400M ozone test chamber? Ozone concentrations were measured by the Saltzman technique (Saltzman and Gilbert, 1959) with the end point determined amperometrically. Intrachamber concentrations were monitored and the output of ozone adjusted to maintain a desired concentration. Such measurements were initially performed on the 3 Blue Spruce Farms, Altamont, N.Y. 4 Purina Lab Chow. s Ozone Research and Equipment Corporation, Phoenix, Ariz.

first and third day of a 72-hr exposure period. The measurements were normally performed only on the second day during later exposures, due to both the consistency of readings from the earlier exposures and a desire to limit the stress placed on the animals by the noise generated by the vacuum pump during periods of air sampling. During the 8-hr-per-day exposures during the period of organogenesis, concentrations were determined every other day. To further determine the stability of the intrachamber zone concentration under normal experimental conditions, replicate readings were taken every 4 hr over one 3-day period. The concentration averaged 0.95 ppm during this period, with a SD of 0.04. No significant fluctuations in ozone concentration were observed in the various areas of the chamber. Teratology. Animals were randomly assigned to treatment groups. Pregnant females were placed in the exposure chamber for one of three exposure periods: continuously during Day 6-9 (Early Term Exposure), continuously during Day 9-12 (Midterm Exposure), or 8 hr/day on Day 6-15 (Organogenesis Exposure). The 3-day exposure period is sufficient to produce full development of the acute inflammatory lesion in the adult rat lung (Schwartz et al., 1976), while the intermittent exposure during the period of organogenesis simulates environmental conditions in terms of the daily duration (National Academy of Sciences, 1977). The initial exposures (conducted during Day 9-12 of gestation) utilized target concentrations of 1.0 ppm with 2 increments of 0.25 ppm. This exposure regimen was later repeated using 0.93 as well as lower (0.64 ppm) and higher (1.97 ppm) concentrations of ozone. In these experiments effects were observed only at doses above 1.26 ppm (see Results)and therefore lower concentrations were used during Early Term or Organogenesis Exposure to determine if the animals were more sensitive during those periods. Animals treated on Day 6-9 were exposed to 0 or 1.04 ppm ozone. Those treated on Day 6-15 were exposed to 0 or 0.44 ppm ozone. Control animals were sham exposed. For all treatment regimens, food and water intake and maternal weight change during the period of exposure were monitored. Between 9 AM and 11 AN on Day 19 of gestation, the pregnant females were anesthetized with an ip injection of 36 mg/kg sodium pentobarbital, and a laparotomy was performed. Females were killed 2 days prior to parturition because several skeletal components (the sternebrae, supraoccipital, and pelvic girdle) are undergoing rapid ossification at this time and hence it was considered that more fetotoxic effects would be observed than if the fetus was allowed further time for development following exposure. The uteri were examined in situ for the number of live fetuses and dead and/or resorbed fetuses. The uterus was then removed and weighed. The maternal weight

OZONE TERATOLOGY gain was calculated as the difference between the overall weight gain during pregnancy and the gravid uterus weight. Following removal from the uterus, the fetuses were blotted, weighed as a group, examined for gross abnormalities, and divided equally for fixation in either a solution of 5 70 formaldehyde, 5 70 glacial acetic acid and 70 70 alcohol, or in 70 % alcohol. A necropsy was performed on the former group using the razor blade technique of Wilson (1965), while the latter were stained with alizarin red S (Dawson, 1926) and examined for skeletal abnormalities and degree of maturity. The maturity of the skeletal system was determined by counting the number of sternal ossification centers, the number of postthoracic vertebral centrums, and the percentage of fetuses in which Meckel's cartilage and the pubis were ossified. For the supraoccipital bone, the degree of ossification was scored on a scale of 1 to 4. A 1 indicated the fusion of the centers had occurred; a 2, that both centers were well formed but did not unite medially; a 3, that some degree of ossification was present; and a 4, that no ossification was present. In the earlier study completed (Experiment 1, Table 3) the scoring was limited to normal or "poorly ossified." This latter designation included all fetuses in which the two ossification centers did not unite medially and is equivalent to a score of 2 or greater in the more comprehensive scoring system (Experiment 2, Table 3). Salicylate synergism. Rats exposed during Day 9-12 of gestation to 0 or 1.0 ppm ozone were further treated with a subteratogenic dose of sodium salicylate 6 on Day 10 (150 mg/kg, Kimmel et al., 1971) and then examined as in the standard teratology studies on Day 19. The salicylate salt was dissolved in water to a concentration of 15 mg/ml and aministered via gastric intubation. Control animals received an identical volume of distilled water (1 ml/100 g body wt). Electrocardiography. Rats were exposed to 1.04 ppm ozone during midgestation (Day 9-12) or to 1.19 ppm during late gestation (Day 17-20) and fetal electrocardiograms were obtained on Day 20 of gestation. The late gestation exposure group was included in the analysis because rapid organ maturation occurs in the rat at this time. For examination, the dam was anesthetized with sodium pentobarbital and a small incision made in the abdomen to allow exposure of the right uterine horn. A small incision was then made on the antimesometrial side of the uterus, starting at the ovarian end. While still maintaining umbilical and placental circulation, the yolk sac and amnion were pulled back to expose a fetus. A hemostat was used to clamp the opened uterus and to hold the remaining fetuses in place. The exposed fetus was then placed on a glass spoon, ventral surface upward. A small patch of skin was removed in the Eastman Kodak Co., Rochester, N.Y.

21

vicinity of the sternum, and a set of electrodes constructed of 0.007-in. silver wire was then inserted into the intercostal muscles with the aid of a micromanipulator. Using a preamplifier, 7 osscilloscope, 8 and chart recorder, 9 electrocardiograms were recorded for no more than the first six fetuses in the right uterine horn so that reduced heart rate due to experimental conditions was not a significant factor. The sampling period required less than 20 min from the time of the initial incision to the completion of the readings. Plasma electrolytes. Rats were exposed on gestation Day 17-20 to either 0 or 1.0 ppm ozone and examined on Day 20. A laparotomy was performed, and fetuses with placenta and yolk sac intact were removed individually from the right uterine horn, starting at the ovarian end. A hemostat was used to close the uterine incision and hold the remaining fetuses in place. Heart rates were immediately measured by visually counting the beats in a 15-sec period. The yolk sac and amnion were then removed and blood was withdrawn from a sinus adjacent to the left ear using suction and a microcapillary tube containing heparin. (Grabowski, 1970). Each fetus was weighed after the sample of blood was removed. No more than six fetuses were sampled and the entire procedure required approximately 20 min. Maternal blood was sampled from the right iliac vein at the end of each experiment. Both maternal and fetal blood samples were then spun for 2 rain in a m!crocentrifuge, the hematocrit recorded, and the concentrations of sodium and potassium in a 5-/11 sample determined using a flame photometer. 1° Statbtics. The litter was regarded as the fundamental unit of comparison for all data with the exception of the food and water consumption studies, where n represents the number of cages studied, Data in the tables is presented as means and standard deviations. Statistical tests utilized in this study included Jonckheere's test for dose-response analysis (Jonckheere, 1954) and a two-way analysis of variance for the evaluation of synergistic effects. In those cases where a significant dose-related effect was found, Student's t test (for parametric data) or the MannWhitney U test (for nonparametric data) was used to define the exposure concentration which was individually significant from the control value. Fisher's Exact Test (Sokal and Rohlf, 1969) was used to evaluate the significance of the occurrence of fully resorbed litters in the various treated groups. A probability o f p < 0.05 was accepted as the overall level of statistical significance. Model 122, Tektronix, Inc., Beaverton, Oreg. 8 Model 514, Tektronix, Inc., Beaverton, Oreg. 9 Model 220, Gould, Inc., Cleveland, O h i o , lo Model 143,Instrumentation Laboratories, Lexington, Mass.

22

KAVLOCK, DASTON, AND GRABOWSKI

RESULTS

Teratology Early term exposure. Maternal weight gain throughout pregnancy was not affected by exposure to 1.04 ppm ozone during D a y 6-9 o f gestation. The treated group had significantly fewer implantation sites (10.7 versus 12.9) and an increased rate o f resorptions ( 1 8 . 5 ~ versus 8 . 1 ~ ) (Table 1). Fetuses in the treated group were generally heavier 2.44 versus 2.20 g) and more advanced than the controls in terms of skeletal development (Table 2). They showed no visceral anomalies. Midterm exposure. Exposure to ozone during D a y 9-12 of gestation (Experiment 1, Tables 1 and 3) significantly reduced maternal weight gain at the two highest doses (t.26 and 1.49 ppm) and increased the resorption rate in the 1.49-ppm dose level (50.3 ~o versus 8 . 9 ~ ) . Both effects were significantly dose

related. There was a significant ( p < 0 . 0 2 ) occurrence of fully resorbed litters in the high dosage group. Seven litters (40 ~ of the total pregnant at term) in this group were fully resorbed, and only 3 2 ~ of the inseminated females in this group were carrying fetuses at term compared with 81 ~ of the controls. The uteri of the animals which showed full resorption did not show the remnants of the metrial gland normally observed at a resorption site, but small yellowish areas were evenly distributed down both uterine horns. Significant dose related trends were noted for reduced fetal weights, for poorly ossified supraoccipitals, and for decreases in the n u m b e r of sternebrae, postthoracic vertebral arches and centrums. However, only the number of sternebrae in the high dosage group was individually significantly different from the control values. Rib malformations (missing, fused, or wavy) were observed in one fetus from an animal

TABLE 1 EFFECTS OF EXPOSURE OF RATS TO OZONE DURING GESTATION--MATERNAL DATA

No. pregnant (term)

Average maternal weight gain" (g)

Average No. implants

No. treated

No. died

No. fully resorbed

21 20

0 0

0 0

15 15

57.3+15.6 53.9+ 15.4

12.9+1.5 10.7+ 3.8b

8.1+8.8 18.5 + 24.9

Day 9-12 (Experiment 1) 0 27 1.00 20 1.26 22 1.49 37

1 0 0 1

0 0 1 7

22 18 17 12

52.7+ 13.3c 45.0+19.0 40.6+12.8 b 44.7+13.7 b

10.6+2.8 11.2+2.7 11.5+2.0 11.4+2.6

8.9 + 9.9c 8.4+12.3 10.5 + 10.5 50.4 + 42.9b

Day 9-12 (Experiment 2) 0 23 0.64 24 0.93 17 1.97 26

0 0 0 I

0 0 0 8

16 17 15 8

45.0___1 5 . 0 42.8+ 1 5 . 4 53.2+18.0 51.1___14.2

12.0+2.2 13.4+2.3 13.5+2.1 10.3+3.7

11.1 +9.2 C 11.8+12.7 12.9+10.0 58,8_+45.8b

Day 6-15 0 0.44

0 0

0 0

11 13

61.2__.21.1 45.6+ 11.3b

10.0+3.3 11.5+2.2

7.3+10.8 9.0+ 16.9

Dose regimen (ppm) Day 6-9 0 1.04

14 14

a Mean + SD. b Significantly different from control value, p < 0.05. c Significant dose-related response, p < 0.05.

Average percentage resorptions

23

OZONE TERATOLOGY TABLE 2 EFFECTS OF EXPOSURE OF RATS TO OZONE DURING GESTATION--FETAL DATA

Treatment regimen Observation

Age

ppm No. litters Average fetal weight (g)

Male/female Visceral anomaliesc Skeletal ossification: Supraoccipital (score) Average No. sternebrae Average No. post-thoracic vertebrae centrums Meckel's cartilage ( 7 0 ) Pubis (~) Ribsc Supernumerary Malformations

Day 6-9

Day 6-15

0

1.04

0

0.44

15

15

11

13

2.20+0.13"

2.44+0.18 b

2.48+0.32

2.32+0.15

43/45

32/37

28/22

32/34

0/0

5.8/20.0a

0/0

0/0

1.7+0.6 0.5+0.6

1.6+0.8 1.3+0.8 b

1.5+0.6 1.2+_0.5

1.5+0.6 0.7+0.7

9.5 + 1.2 70.9+40.2 89.8+26.7

9.5 + 1.4 83.3+ 17.7 87.6+17.9

9.3 + 0.5 73.9+30.3 79.7+27.0

8.7/7.0 0/0

9.1/10.9 0/0

0/0 0/0

8.9 + 0.7 52.4+31.6 62.1+28.5 10.9/8.7 0/0

" Mean + SD. b Significantly different from control value, p < 0.05. c Represented as percentage fetuses affected/percentage litters affected. a Enlarged renal pelvis. receiving the low dose and in one fetus from each of three litters of animals receiving the middle dose. In the second series of experiments during midgestation dosages of 0.64, 0.93, and 1.97 p p m were used. The concurrent control fetuses for these exposures weighed less than in the first series and this data is therefore presented separately (Experiment 2, Tables 1 and 3). The high dose produced a significant elevation in the rate of resorptions (58.8~ versus 11.1~o) and in the occurrence of fully resorbed litters (p < 0.003). Eight litters in this group were fully resorbed, and only 3 1 ~ of the inseminated females (versus 71 ~ in the controls) were carrying fetuses at term. These resorptions resembled those found in the previous series.. The average fetal weight in the highest dose was more variable (F = 4.5, p < 0 . 0 1 ) than in the control group. While some indices of

fetal maturity were affected by the highest dosage, the results were not statistically significant as in the first experiment. One fetus with an extremely enlarged renal pelvis was observed at the highest dosage. Organogenesis exposure. Daily 8-hr exposure to 0 . 4 4 p p m ozone during organogenesis (Day 6-15) reduced maternal weight gain during pregnancy (Tables 1 and 2). N o effects were observed on fetal weight, skeletal ossification, or visceral development. Food and water consumption. Consistent dose-related decreases in daily food and water intake were noted (Table 4). Maternal weight change during the exposure period was correlated with these effects. Food and water intake were depressed by as much as 5 0 ~ in some treated groups as compared with controls, The effects were present at the lowest doses examined, 0.64 p p m during midgestation and 0.44 p p m during the period

24

KAVLOCK~ DASTON~ AND GRABOWSKI

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OZONE TERATOLOGY

25

TABLE 4 EFFECTSOF OZONEON MATERNALDIETARYINTAKEDURINGTHEPERIODoF ExPOSURE

Dose regimen (ppm)

No. pregnant (term)

Day 6-9 0 15 1.04 15 Day 9-12 (Experiment 1) 0 22 1.00 18 1.26 17 1.49 12 Day 9-12 (Experiment 2) 0 16 0.64 17 0.93 15 1.97 8 Day 6-15 0 11 0.44 13

No. cages

Average daily water intake (ml)

Average daily food intake (g)

Average maternal weight change (g)

6 5

34.4+7.0" 26.9+5.4

20.8+2.4 15.9+2.85

9.6+7.5 -5.1+5.1 ~

8 7 6 8

45.1+7.6 c 25.2_+4.2 b 25.1+_3.1 b 24.0_+4.9 b

22.5+2.7 c 14.2+2.5 b 12.2+ 1.6b 11.4+2.2 b

13.2+6.3 c 1.9+7.1 b -4.7+6.9 b 0.0+6.4 ~

5 5 4 5

43.6+3.9 c 30.9_ 3.3b 24.3+4.9 b 22.3 + 1.4b

22.5+1.3 c 16.6+ 1.5b 14.0+ 3.9b 10.4+ 1.2b

15.0+6.3 c 4.7+8.65 -4.7+8.8 b - 9.4 + 5.0b

4 4

39.4+3.1 37.5+2.5

22.7+1.1 20.5+ 1.5b

47.3+12.1 29.2+ 12.9b

"Mean + SD. b Significantly different than control value, p < 0.05. c Significant dose-related response, p < 0.05. of organogenesis. In the latter group, the intermittent exposure regimen produced only a relatively mild anorexia and there was no statistically significant reduction in water consumption. Salicylate synergism. The effects o f exposure to 1 . 0 p p m ozone during Days 9-12 were observed to be minimal (see above). A n increased incidence of supernumerary ribs was the only maternal or fetal effect observed following a single dose of 150 mg/kg sodium salicylate. However, the combination o f ozone and salicylate treatments significantly reduced maternal weight gain and increased the resorption rate. T w o litters were fully resorbed. The decreased fetal weight observed after the combined treatment represents a significant synergistic interaction. The degree of ossification o f the supraoccipital and the sternebrae was significantly lower than in the controls but not significantly different than in the salicylate treatment alone. Twenty-four fetuses (40.7 ~ )

f r o m eight litters (80~o) had supernumerary ribs, a greater involvement o f both fetuses and litters than with the salicylate treatment alone.

Fetal electrocardiography and plasma electrolytes. The rate of heart beat o f D a y 20 rat fetuses subsequent to no treatment (16 litters), 1.04 p p m during Days 9-12 (8 litters), and 1.19 p p m during D a y 17-20 (8 litters) were 157, 159, and 149 beats/min, respectively. Analysis of electrocardiograms f r o m these fetuses demonstrated no changes in the P - Q , QRS, or Q - T intervals. Exposure to 1.0 p p m (8 litters) during D a y 17-20 did not affect fetal weight, hematocrit, plasma sodium or plasma potassium concentrations when compared to similarly handled controls (8 litters). DISCUSSION Exposure of pregnant rats to ozone resulted in embryolethality. The trend towards embryolethality produced by the early term

26

KAVLOCK, DASTON, AND GRABOWSKI TABLE 5 SYNERGISTIC INTERACTIONS OF OZONE AND SODIUM SALICYLATE IN RATS

Group

Maternal data No. females inseminated No pregnant (term) Average maternal weight gain (g) Average No. implantations Average percentage resorptions

Control

Ozone (1 ppm Day 9-12)

Na-salicylate (150 mg/kg, Day 10)

Ozone and salicylate

23 16

17 15

19 14

22 10

45,0+ 15.0" 12.0 + 2.2

53.2+ 18.0 13.5 + 2.1

55.5+ 18.7 11.9 _+2,5

36.0+ 11.9b 12.4 + 2.5

11.1 _ 9.2

12.9+ 10.0

9.9+ 12.3

26.6+ 36.6c

Fetal data Average fetal weight (g) Visceral anomaliesn

2.26 _+0.16 2.4/8.7 e

2.22 _+0.18 0/0

2.31-4-0.23 1.3/7.11

2.02 + 0.14""~ 1.9/10.3s

Supraoccipital (score) Average No. sternebrae

1.59 + 0.69 0.85 + 1.10

1.50 + 0.60 0.64 + 0,50

1.84 _+0.83 0.77 --2_0.78

2.08 + 0.66 c 0.11 +_0.25c

Average No. vertebral centrums Meckel's cartilage ( ~ ) Pubis ( ~ ) Supernumerary ribsn

9.03 _+0.81 76.0 + 32.6 71.4 + 29.1 0/0

9.00 + 0.70 78.3 _+31.0 76.8 _+28.8 1.3/6.6

8.91 + 1.09 46.6 + 30.4 64.8 _+28.0 24.1/50.0

8.36_+ 0.74 42.9 _ 52.5 51.7 ___29.9 30.5]70.0

" Mean + SD. 0 Significant interaction, p < 0.05. c Significantly different from control value, p < 0.05. a Percentage fetuses affected/percentage litters affected. e Enlarged renal pelvis. Unilateral anophthalmia.

t r e a t m e n t c o m b i n e d with a decreased n u m b e r o f i m p l a n t a t i o n s (perhaps due to p r e i m p l a n tation deaths) resulted in smaller litter sizes in this group. The m o r e a d v a n c e d m o r p h o l o g i c a l d e v e l o p m e n t observed in fetuses from this g r o u p c o m p a r e d to their controls m a y have been due to the smaller litter size (van M a r thens et aL, 1975). D o s e s greater than 1.26 p p m d u r i n g m i d g e s t a t i o n p r o d u c e d statistically significant n u m b e r s o f fully r e s o r b e d litters. D a i l y exposure t h r o u g h o u t the p e r i o d o f organogenesis ( D a y s 6-15) to e n v i r o n m e n t a l concentrations (National Academy of Sciences, 1977) o f ozone did n o t affect fetal

development. It is a p p a r e n t that under the exposure regimens utilized in this study ozone is n o t teratogenic in the rat, even at concentrations fivefold that r e p o r t e d to p r o d u c e visceral anomalies in mice (Veninga, 1967). N o reports o f ozone-induced a n o r e x i a are k n o w n in the literature. R e d u c t i o n s in f o o d intake during p r e g n a n c y have been r e p o r t e d to p r o d u c e effects on p r e n a t a l development, but only after p r o l o n g e d periods o f restriction ( E n d o et aL, 1974; S m a r t a n d D o b b i n g , 1971; Z a m e n h o f a n d van M a r t h e n s , 1974; Beall a n d Klein, 1977). T h a t the reduced f o o d intake observed in

27

OZONE TERATOLOGY

this study was neither as severe nor as prolonged (consumption appeared to return to normal levels after exposure) as used by the above-mentioned authors, and that the severity of the embryotoxic effects does not strictly parallel the decreased dietary intake indicate that these two factors are not causally related. A more likely cause (suggested by Menzel, 1970, as the cause of the effects noted by Veninga, 1967, in mice) is that an ozone-induced vitamin E deficiency is responsible for the increased incidence of resorptions. The results of the salicylate synergism study advance the possibility that ozone can potentiate the effect of exogenous compounds. Gardner et al. (1974) first proposed that ozone could interfere with the metabolism of such compounds. Kimmel et al. (197l) demonstrated that the active agent in aspirin teratogenicity is the unmetabolized salicylate structure, and interference with its detoxification could be one explanation for the observed synergistic effect. A second possible explanation is that stress induced in the maternal organism by exposure to ozone may have enhanced the teratogenic effect of sodium salicylate. Goldman and Yakovac (1963, 1964, 1965) presented evidence that systemic stress (induced by maternal immobilization) did p r o d u c e just such an effect. No effects on near-term fetal heart rates or electrocardiograms were observed after Midterm Exposure nor did Late Term Exposure followed by immediate observation affect fetal heart rates, electrocardiograms, hematocrits, or plasma electrolytes. Since all of these parameters have been shown to be sensitive fetal indicators of toxicity (Chernoff and Grabowski, 1971; Lopo, 1974; Grabowski and Tunstall, 1977), it can therefore be assumed that fetal blood flow and oxygenation are not affected by maternal exposure to high levels of ozone. Ozone has thus been shown to produce intrauterine toxicity (embryocidal action and reduced skeletal ossification) but only at

environmentally high concentrations (National Academy of Sciences, 1977). A lower dose, however, was observed to interact with sodium salicylate in a synergistic manner. Physiological indicators of fetal toxicity (electrocardiography and blood chemistry) failed to demonstrate any sign of ozone toxicity.

ACKNOWLEDGMENTS This research was supported by Contract No. 68-02-1778 with the U.S. Environmental Protection Agency and by a gift from the Xerox Corporation.

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