TERATOLOGY 42:15-23 (1990)

Developmental Toxicity of Soman in Rats and Rabbits HUDSON K. BATES, JAMES B. LABORDE, JACK C. DACRE, JOHN F. YOUNG Pathology Associates, Inc. (H.K.B.),and Division of Reproductive and Developmental Toxicology (J.B.L., J.F.Y.), National Center for Toxicological Research, Jefferson,Arkansas 72079; US Army Biomedical Bioengineering Research and Development Laboratory, Fort Detrick, Maryland 21701 -5010 (J.C.D.) AND

ABSTRACT Soman (GD; phosphonofluoridic acid, methyl-,1,2,2-trimethylpropyl ester) is a n organophosphate compound with potent anticholinesterase activity. To determine developmental toxicity, soman was administered orally to CD rats on days 6 through 15 of gestation at dose levels of 0,37.5, 75,150, or 165 pglkglday and to New Zealand White (NZW) rabbits on days 6 through 19 of gestation at dose levels of 0, 2.5, 5, 10, or 15 pglkglday. At sacrifice, gravid uteri were weighed and examined for number and status of implants. Individual fetal body weights and external, visceral, and skeletal malformations were recorded. Mean maternal weight changes, fetal implantation statusllitter, fetal weight, and fetal malformationsllitter were compared between dose groups. Monitors for maternal toxicity were net body weight change, treatment weight change, mortality, and clinical signs of toxicity such a s lethargy, ataxia, and tremors. Maternal rats and rabbits in the high-dose groups exhibited statistically significant increases in toxicity and mortality when compared to controls. There were no significant dose-related effects among dose groups in the prevalence of postimplantation loss, malformations, or in average body weight of live fetuses per litter. There was no evidence of increased prenatal mortality or fetal toxicity in the CD rat or NZW rabbit following exposure to soman, even at a dose that produced significant maternal toxicity. When the US. Congress ordered the de- quested by the U S . Army Medical Research struction of certain military chemical muni- and Development Command to determine tions by 1994, they included a mandate to the developmental toxicity of the organodevelop a toxicological data base that would phosphate compound soman in two species incorporate teratology and reproductive of laboratory animals. The CD rat and the studies (Fed. Register, '87). This was to de- New Zealand White rabbit were selected for velop occupational health crtieria and to as- this work. sure public health and safety during the No information concerning the developdemilitarization process. Because some re- mental toxicity of soman administered in lated organophosphate compounds were repeated daily oral doses to pregnant labosuspected to be teratogenic, the Surgeon ratory animals was available; however, limGeneral of the U.S.Army had recommended ited information was available on singlethat women of childbearing age be re- dose acute toxicity (Dacre, '84). Initially, a stricted from work areas where concentra- preliminary toxicity study at the NCTR ustions of these chemical agents would exceed ing multiple doses was necessary to deter3.0 x lop6mg/m3, averaged over a 72-hour mine dose levels for use in the developmenperiod (Department of the Army, '82). The tal toxicity study. This study was conducted destruction of these compounds could influence work schedules due to potential occupational exposure of female (civilian and Received August 9, 1989; accepted December 5 , 1989. military) personnel. The National Center Hudson K. Bates' present address is Research Triangle Instifor Toxicological Research (NCTR) was re- tute,Research Triangle Park, NC 27709. 0 1990 WILEY-LISS, INC.

16

H.K. BATES ET AL.

in both pregnant rats and rabbits (Bates and LaBorde, '86, '87), and the results were used to design our developmental toxicity studies with emphasis on maternal toxicity and mortality, status of uterine implantation sites, live litter size, fetal body weight, and gross morphological abnormalities at various doses.

Animal husbandry Female rats from the National Center for Toxicological Research (NCTR) in-house colony of Charles River CDR [Crl: CDR(SD)BR]strain (Kingston, NY) were 810 weeks old and nulliparous and ranged in weight between 200 and 247 g on GD 0 (day vaginal plug found). Virgin New Zealand White (NZW) rabbits were obtained at 2024 weeks of age (2.7-4.3 kg) from DutchMATERIALS AND METHODS land Laboratories, Inc. (Denver, PA). AniTest material mal care and procedures followed the U S . Department of Health and Human Services Soman (GD; phosphonofluoridic acid, Guide for the Care and Use of Laboratory methyl-,1,2,2-trimethylpropyl ester) was Animals guidelines. Rats were housed indiobtained as dilute chemical surety material vidually in solid-bottom polycarbonate (2 mg/ml) from the United States Medical cages with hardwood chip bedding, and rabResearch Institute of Chemical Defense bits were housed individually in steel cages (Aberdeen Proving Ground, MD). Soman with steel mesh floors. Food and filtered tap was analyzed by the method of Shih and El- water were available ad libitum. Rats were lin ('86). This gas chromatography system fed NIH-31 rodent chow #5022 (Ralston Puwas composed of a Hewlett Packard gas rina Co, Richmond, ID). Rabbits were fed chromatograph model 5880 equipped with a Certified Rabbit Chow #5322 (Ralston Puflame ionization detector (FID), a 10% SP- rina Co., St. Louis, MO). Animal rooms were 1000 6 ft x 4 mm column, helium gas car- equipped with temperature and humidity rier flow rate of 30 ml/min, and a n oven controls and lighting was on a 12-hour light: temperature of 170°C. The sensitivity of dark cycle. Mean values for temperature this analytical method for soman was 200 and relative humidity were 23°C and 58%, nglml. Purity of soman was assayed a t 99%. respectively. The dilution of soman in 4°C distilled water for dose concentration was based on the gas Animal breeding chromatographic analyses. Each concentraRats were mated one ma1e:one female tion was formulated independently in a quantity sufficient to be used throughout overnight in a hanging wire screen cage. the dosing period (10 days for rats; 14 days Each female was examined the following for rabbits) and stored a t -70°C in Teflon- morning for the presence of a copulation sealed glass vials packaged in screw-cap plug and if a plug was found, that day was plastic containers. Once removed from the designated GD 0. Aproximately 2 hours -70°C freezer, dose solutions were allowed prior to insemination, female rabbits were to equilibrate to approximately 4°C and injected intravenously with pituitary were maintained a t 4°C throughout each luteinizing hormone (1mg/kg body weight) day's dosing period (approximately 1 hour). to induce ovulation. Females were insemiAqueous solutions of soman have been nated with 0.25 ml (>18 x lo4 motile shown to be relatively stable when stored at sperm/mm3) of undiluted semen. Day of in4°C for a period of 20 days (Ellin et al., '81). semination was designated GD 0. All operations with soman were conExposure regimen ducted over a spill tray in a chemical fume hood with the ventilation system continuPreliminary dose-response toxicity studously operational a t face air velocity of 150- ies were conducted in both species. The de180 fpm. The hood filter system consisted of velopmental toxicity studies were conducted spunglass filters, HEPA filters, and acti- in three replicates (40-45 rats; 30-36 rabvated charcoal filters. bits per replicate) with four soman-dosed Staff personnel assigned to work with so- groups and a vehicle control (distilled waman wore approved protective clothing and ter) in each replicate. To assure similar were medically monitored for plasma and body weights in each group in the prelimierythrocyte cholinesterase levels prior to nary study and in the developmental toxicand after each operation. ity replicates, the female animals were

DEVELOPMENTAL EFFECTS OF SOMAN

rank-ordered by weight on gestational day (GD) 0 and assigned to dose groups in a sequential manner. To provide exposure throughout major organogenesis, the animals in the preliminary and developmental studies were dosed by gavage on GD 6-15 (rats) or 6-19 (rabbits). This route of administration was chosen in order to expose the developing fetus maximally so as to determine the teratogenic potential of soman. Dams were observed daily for mortality or other clinical signs of toxicity. Rats were weighed on GD 0, 6-16, and 20. Rabbits were weighed on GD 0,6-20, and 29.

Preliminary studies Data were available on the predicted 50% mortality for single oral doses in the rat (400 pglkg) and the rabbit (350-470 pglkg), but the toxicity of a more protracted dose regimen was unknown (Dacre, ’84). A preliminary toxicity study was conducted in pregnant animals of each species to determine maternal lethality over a range of doses. In the rat, 10 animals per group were given 0, 75, 125, 175, 200, 225, 275, 350, or 425 pglkglday of soman. Rabbits (seven or eight per group) were given 0, 5, 7.5,10,15, 25, 30, or 60 pglkglday. Lethality data from the preliminary study were analyzed (probit of lethality vs. log of dose) to calculate a best-fit line. The upper and lower 95% confidence limit was used to calculate a dose that would cause some maternal lethality and thus allow survival of enough maternal animals for statistical analysis of fetal data. This dose and three lower doses plus vehicle control (distilled water) were used in the developmental toxicity studies. Maternal lethality was selected as the endpoint rather than other signs of toxicity because of the steepness of the dose-response curve for lethality. Developmental toxicity studies In the rat developmental toxicity study, 25 “plug-positive” females were given 0, 37.5, 75, 150, or 165 pglkglday of soman. The rationale was to use the predicted 10% mortality dose as the high dose, with lower doses of one-half and one-quarter of that value. Because of the steepness of the doseresponse curve, a higher dose (165 pgkgl day) based on the upper 95% confidence limit of the predicted 10% mortality dose was added to assure that the range was bracketed. The rabbit study had 18 to 23

17

animals per group except for the high-dose group, which had only 14 animals. The doses for the rabbit study were 0, 2.5, 5, 10, or 15 Fglkglday. On GD 20 rats were killed by CO, asphyxiation while rabbits were killed by an intravenous injection of T-61, a rapid-acting euthanasia solution (American Hoechst Corp., Summerville, NJ). Immediately after termination of the animals, gravid uteri were removed and weighed, and the status of uterine implants was tabulated (i.e., resorptions, dead fetuses, and live fetuses). Live fetuses were removed from the uterus, weighed individually, and examined under magnification for gross external defects. All rabbit and rat fetuses were decapitated; heads of half of the fetuses were placed in Bouin’s fixative solution for examination by Wilson’s free-hand razor-blade sectioning method (Wilson and Warkany, ’64) while the heads of the remaining fetuses were processed for skeletal observations. All live fetuses were examined for visceral malformations by using the fresh tissue dissection technique of Staples (’74).Fetal sex was determined at the time of visceral examination. All fetal carcasses were skinned, placed in 95% ethyl alcohol for a minimum of 72 hours, and subsequently stained with alizarin red S and alcian blue to examine bone and cartilage abnormalities (Whitaker and Dix, ’79). In this developmental toxicitylteratology study, no distinction was made between major and minor malformations in the collection and presentation of fetal data. Descriptive terminology, consistent with the scientific literature, with an “is or is not a malformation” approach to observations, was utilized rather than classifying malformations according to degree. Ossification variants such as bipartite centra, rudimentary lumbar rib, irregular or reduced ossification of sternebrae, etc., were not statistically different from controls and were not specifically alluded to in this manuscript.

Statistical analysis A two-way (additive model) analysis of variance was used to test for treatment effects on the variables listed in Tables 1-3. The model used was: DEPENDENT VARIABLE = DOSE + REPLICATE + ERROR. Two F tests, one for an approximate linear trend with 1 degree of freedom and one for a non-linear trend with 2 degrees of free-

18

H.K. BATES ET AL.

TABLE 1. Maternal pregnancy status and mean body weight changes in the developmental toxicity study g t SE No No. gravid Gestational' Gravid Net3 Treatment animals gravidi weight Dose uterine weight weight bred dead change weight change change group1 Rats 0 21125 0 143 i 3 67 i 3 53 f 2 76 i 3 I

37.5 75 150 165

Rabbits 0 2.5

5 10 15

22125 23125 24/25 23125

0 0 0 1

145 i 4 133 t 5 135 i 3 135 i 3

73 i 3 66 t 5 72 i 2 71 2 2

18120 17/18 21121 21/23 12114

0 0 2 7 5

261 _t 42 357 i 37 237 ? 39 307 2 72 279 5 73

439 i 31 449 ? 28 452 i 38 440 t 51 501 i 35

72 67 63 64 -178 -92 -215 -133 -222

t2

53 i 2 46 t 2** 1** 43 I 40 t 3**

2 3* i 2*

t 3" i 59

151 i 32 253 2 30 119 -c 33 182 f 59 40 159 I

i 39

i 63

t 83 i 98

'Dose in pgikgiday. 'Gestational weight change = terminal body weight minus initial body weight. 3Net weight change = gestational weight change minus gravid uterine weight. 'Treatment weight change = GD 16 body weight minus GD 6 body weight (rat); GD 20 body weight minus GD 6 body weight (rabbit). *Statistically different from controls a t P < 0.01. **Statistically different from controls a t P < 0.001.

Dose' group Rats 0 37.5 75 150 165

Rabbits 0 2.5 5 10 15

No. dams gravid at term 21 22 23 24 22 18 17 19 14 7

TABLE 2. Mean fetal data at the time o f lauarotomv Early Late Dead Total Nonlive Viable implants resorbed resorbed fetuses imp1ants implants t SE i SE 2 SE I SE i SE -t SE 12.3 t 0.5 0.6 13.6 I 12.7 2 0.9 13.5 1 0 . 5 14.0 2 0.5 7.6 i 0.7 8.8 I 0.7 9.5 ? 0.8 9.1 i 1.1 9.3 ? 1.0

0.0 0.6 i 0.2 11.7 ? 0.6

0.6 i 0.2 0.0 t 0.0 0.0 2 0.0 0.5 2 0.1 0.9 t 0.3 0.04 i 0.04 0.6 i 0.1 0.04 ? 0.04 0.0 i 0.0 1.2 i 0.4

0.0 0.0 0.0 0.0 0.0

0.1 % 0.1 0.5 t 0.2 0.9 i 0.6 0.4 i 0.2 0.1 t 0.1

0.3 -t 0.3 0.5 i 0.5 0.1 i 0.1 0.0 i 0.0

0.0 i 0.0 0.0 i 0.0 0.2 ? 0.1 0.0 t 0.0 0.3 i 0.2

2

IO.0

0.5 i 0.1 i 0.0 0.9 i 0.3 i 0.0 0.7 i 0.1 t 0.0 1.2 t 0.4

0.4 t 0.3 1.0 t 0.5 1.1t 0.6 0.4 i 0.2 0.0 i 0.0 0.4 ? 0.3

13.0 i 0.7 11.7 t 0.9 12.8 _t 0.5 12.8 t 0.4 7.1 t 0.7 0.6 7.8 I 8.4 ? 0.9 8.7 2 1.1 8.9 i 0.9

Fetal weight(g1 i SE

Sex ratio (M:F)

3.7 3.7 3.5 3.6 3.6

0.1 IO.l* 2 0.1 t 0.1

1:0.74 1:1.01 1:0.09 1:1.05 1:1.08

41.3 2 1.2 38.4 i 1.2 39.2 ? 2.0 40.4 f 1.8 39.8 i 2.4

1:0.92 1:1.18 1:0.98 1:0.66 1:l.OO

i 0.04 rt

'Dose in pglkglday. *Significantly different from control a t P < 0.05.

dom were used. To stabilize the variances and normalize the data, the FreemanTukey-Poisson transformation (Haseman and Kupper, '79) was used on the count variables (e.g., number of resorbed implants). A Freeman-Tukey Binomial arc-sine transformation was used on the proportion variables. Fetal sex ratios were compared by using the chi square test. Variables that were determined t o be significantly different between doses were further analyzed by Duncan's multiple range test (Miller, '66). The level of significance chosen was P < 0.05. RESULTS

Preliminary study Probit analysis of mortality for the rat revealed that mortality rates between 1%and

50% could be expected over the narrow range of 126 to 188 pglkglday. From the probit analysis the dose associated with 10% mortality was 151 pglkglday with 95% confidence limits of 107 and 169 pglkglday (Fig. 1).Based on these calculations, doses of 0, 37.5,75,150, and 165 pglkglday were established for the developmental toxicity study in rats. The rabbit data revealed an expected mortality of 1%to 50% with doses between 3 and 47 pglkglday. The predicted 10% mortality dose was 10 pglkglday, with 95% confidence limits of 2 and 17 pglkglday. The doses for the rabbits in the developmental toxicity segment were set at 0, 2.5, 5, 10, and 15 pglkglday.

19

DEVELOPMENTAL EFFECTS OF SOMAN

No.

TABLE 3. Incidence of malformations per litter’ Visceral Skeletal % No. % No. %

0.00

0.0

37.5

0.00

0.0

75

0.00

0.0

150

0.00

0.0

165

0.05

0.3

Rabbits 0

0.00

0.00

2.5

0.00

0.00

External Dose Rats 0

0.38 (0.14) 0.36 (0.09) 0.09 (0.00) 0.25 (0.04) 0.41 (0.09)

3.1 (1.2) 3.6 (0.7) 0.7 (0.0) 2.3 (0.3) 3.7 (0.6)

1.39 (0.06) 2.53

21.6 (0.6) 33.6 (0.0) 15.3’ (1.6) 20.8 (1.8) 9.5 (0.0)

(0.00)

’( )

5

0.00

0.00

10

0.14

1.00

15

0.00

0.00

=

1.42 (0.16) 1.79 (0.21) 0.86 (0.00)

2.05

16.8

2.68

23.2

1.26

15.7

2.08

16.6

2.50

19.1

0.00

0.0

0.00

0.0

0.00

0.0

0.00

0.0

0.00

0.0

Total No.

%

2.38 (2.19) 2.95 (2.77) 1.30 (1.26) 2.29 (2.08) 2.17 (2.50)

19.5 (17.0) 26.0 (23.9) 16.1 (15.7) 18.7 (16.6) 21.6 (19.1)

1.39 (0.06) 2.53

21.6 (0.6) 33.6

(0.00)

(0.0)

1.42 (0.16) 1.86 (0.29) 0.86 (0.00)

15.3 (1.6) 21.2 (2.3) 9.5 (0.0) -

number and percent values without the findings of hydroureter (rats) and retinal folds (rabbits).

Developmental toxicity study Pregnancy status, body weight changes, and mortality data for both species are listed in Table 1. In the rat, maternal body weight summed by dose across replicates showed significant linear dose-related decreases in net weight change (P < 0.01) and in treatment weight change (P < 0.001) indicating maternal toxicity above the 75 pgt kgtday dose level. In addition, some dams in the high-dose group exhibited clinical signs of toxicity (lethargy, tremors) on GD 9, 12, 14, and 15. There was only one death among the 23 gravid females in the high-dose group (4.3%),slightly lower than the 10% lethality that was anticipated based on the preliminary study. In the rabbit study, maternal body weight change summed by dose group across replicates showed no significant effects (P < 0.05) either by analysis of variance or multiple range test. However, when the data were summed by dose group across replicates for maternal mortality in confirmed gravid dose, the high-dose group had a 42% mortality followed by mortality rates of 10% and 33% in the 5 and 10 pgkgtday dose groups, respectively. Symptoms of organophosphate toxicity including excessive salivation, ataxia, lacrimination, lethargy, tremors, and death were observed especially at the high dose throughout the dose period.

The number and status of fetuses in both species are listed in Table 2. No values are different from control values except the mean fetal weight in the 75 pgtkglday group of rats. For both rats and rabbits, there were no statistically significant increases in the number or percent of malformed fetusestlitter among the treated animals (Table 3). Although there were no significant differences among treatment groups, some findings were of interest. Hydroureter in the rat fetuses was seen at approximately 2%in all groups (Table 4).Since this finding is a relatively subjective observation and may vary with degrees of ureteral dilatation, it was analyzed first as a malformation and then as a variation. This approach affected the number and precent of fetuses with visceral and total malformationstlitter but is still without statistical significance. The prevalence of retinal folds in rabbit fetuses was high in all groups including controls (Tables 3, 4). The number and percent of visceral malformed fetusestlitter and the total number of malformed fetusedlitter were examined statistically with retinal folds included first as a malformation and then as a variation. Removal or inclusion of this finding from the malformation category had no statistically significant effect. Two cartilage defects, “cervical vertebrae arch cartilage fused” and “rib(s) cartilage

20

H.K. BATES ET AL

0

100

80 60 40 20 0

80 60

40 20

0

0

50

100 150 200 Dose (pglkglday)

Calculated Mortality

-..- 95%

Flducial Llmits

250

300

Actual Mortalily

Fig. 1. The results of probit analysis of maternal mortality.

not attached to sternum,” were the only fre- was not a developmental toxicant following quently detected skeletal anomalies in the oral (gavage) administration of maternally fetal rat. They occurred without a dose-re- toxic and non-toxic doses throughout the pesponse relationship (Table 4). riod of major organogenesis in the rat and rabbit. DISCUSSION AND CONCLUSIONS The calculated 50% mortality rate in both This study demonstrated that the organo- animal species indicated that the rabbit was phosphorus cholinesterase inhibitor soman four-fold more sensitive than the rat to the

21

DEVELOPMENTAL EFFECTS OF SOMAN

TABLE 4 . Number of fetuses with specific malformations Rats Dose (pglkglday) External Exencephalus Visceral Microphthalmia Anophthalmia Hydrocephalus Great vessel malformation Ventricular septal defect (VSD) Lobation anomaly of lungs Lobation anomaly of liver Hydronephrosis Hydroureter Undescended testis Skeletal Sternebrae fused Cervical vertebrae arch cartilage fused Cervical vertebrae centra cartilage fused Thoracic vertebrae arches missing Thoracic vertebrae centra cartilage fused Thoracic ribs fused Thoracic ribs decreased or increased number Rib(s) cartilage not attached to sternum Rib discontinuous Rib(s) branched Lumbar ribs decreased or increased number Lumbar vertebrae centra cartilage fused Severe skeletal deformity Rabbits Dose (pgikglday) Exter n a 1 Severely deformed fetus Visceral Retinal folds Cleft palate Brain malformation Hydrocephalus Great vessel malformation Ventricular septal defect (VSD) Rudimentary lungs Diaphragmatic hernia Lobation anomaly in liver Hydronephrosis Testicular malformation Skeletal No findines

0 246 0 246 0 0 0 0

2 0

1 0 5 0 246 0 21 5 0 0 0 1 19 0 0 3 0 0 0 128 0 127l 24 1 0 0 0 1 0 0 0 0 0 128 -

37.5 287 0 287 0 0 0 1 0

75 270 0 270 0 0 0

150 307

0 0

0 0 0

0

307 0 0 0

1 0 1 7 0 287 3 27 3 0 0 1 2 31 2 0 2 0 0

0 0 0 2 0 270 0 19 0

0 1 0

0 0 5 1 307 0 34 1 1 0 1 0 26 0 0 1 0 1

2.5 133 0 133 43 0 1

5 160 0 160 25 0 0 0 1 1 1 1 0 1 1 160 -

10 122 1 121' 23 0 0 1 0 0 0 0 1 1 0 121 -

0 0 0 0 0 0 0 0

133 -

0

0 0 0 16 0 0

165 282 1 281' 1 1 1 1 0 0 0 0 7 0 282 0 31 2 1 1 1 3 20 0 1 0 5 0 15 62 0 62 6 0 0 0 0 0 0 0 0 0 0

62 -

'One fetal finding not recorded

lethal effect of repeated doses of soman administered at 24-hour intervals during major organogenesis (47 pglkglday vs. 188 pgl kglday, respectively). The 10% mortality rate indicated an even greater sensitivity between the two species. The predicted 10% mortality value for the rabbit was 10 pgl kg/day with a fiducial limit (95%) of 8 pgikgiday while the predicted 10% mortality value for the rat was 151 pglkglday with a fiducial limit (95%)of 107 to 169 pglkgl day. Neither species provided a relative advantage over the other in toxic endpoints per litter at the time of laparotomy (Table 2) nor in the incidence of malformations per

*

litter (Table 3) even It maternally toxic doses. The use of alizarin red S stain for the staining of fetal skeletal structues in developmental toxicitylteratology studies appears to be more widely utilized than the double stain method (alizarin red S and alcian blue) which stains bone and cartilage. Methodology describing the usefulness of the double stain and refinements thereof have been made (Inouye, '76; Whitaker and Dix, '79; Kimmel and Trammel, '81, Marr et al., '88). Many of the rat skeletal components develop throughout the perinatal period and therefore ossification is highly

22

H.K. BATES ET AL.

variable at 20 days of gestation (Fritz and Hess, ’70). The utility of the double stain method allows one to detect a n increase i n skeletal malformations as demonstrated by Marr e t al. (’88) and by the authors of this manuscript. The larger number of skeletal abnormalities detected in the rat study resulted from specific examination of cartilage defects and was not a n artifact. Two cartilage defects in the rat (cervical vertebrae arch cartilage fused and rib cartilage not attached to the sternum) were detected a t a rate of 2-3 per litter. Their occurrence was not dose-related and was considered to be unrelated to treatment. The functional significance of cartilage defects such as those reported by Marr et al. (’88), da Cunha Ferreira et al. (’89), and in this study is currently unknown. In the rabbit study, the dose of 2.5 pgt kg/day was a “no observable effect level.” Measures of maternal weight gain (i.e., weight gain during gestation, weight gain during treatment, and net weight gained) were not significantly altered among the dose groups. There was no evidence of doserelated fetal toxicity or teratogenicity, either a t the “no observable effect level” or at the doses that produced overt maternal toxicity. Retinal folding was the only produced fetal soft-tissue anomaly detected. This finding was unrelated to dose and was interpreted to be a spontaneous malformation or a n artifact. Retinal folds have been suggested to be artifacts of fixation in rats (Szczech and Purmalis, ’75). The relationship between maternal toxicity and fetal toxicity remains a critical issue in developmental toxicity studies (Khera, ’84, ’85, ’87; Kavlock et al., ’85; Schardein, ’87). Attempts to better define the association between these two parameters have been made more difficult because the endpoints of toxicity are not adequately defined, and in some studies, not assessed (Khera, ’84, ’85). Data from over 200 published animal teratology studies of chemical and physical agents (Khera,’85) revealed inadequate or unavailable toxicity data in 59, 71, and 29 hamster, rat, and rabbit studies, respectively. Criteria for sufficient evidence t h a t maternal toxicity had occurred were clinical signs of toxicity, significant decreases in body weight gain, pharmacological activity, or death (Khera, ’84). Using these criteria (Khera, ’84) to examine the relationship be-

tween maternal toxicity and fetal abnormalities in 85 mouse teratology studies, four categories were established for the occurrence of maternal and fetal defect in these studies. The categories were: 1) test dose that caused no apparent maternal toxicity, 2) insufficient data on maternal toxicity, 3) maternal toxicity associated with fetal defects, and 4) maternal toxicity accompanied with no fetal malformations. Of these four categories, the greater number of studies were placed in category 2 (39%) followed by category 3 (33%), category 1 (15%),and category 4 (13%). Our study with the organophosphate compound soman demonstrated that the doseresponse curve for maternal lethality in the rat and rabbit was very steep. Either the maternal animal 1)showed clinical signs of organophosphate toxicity or decreased body weight or died during exposure to the chemical or 2 ) it survived with no observable fetal toxicity. Thus, if one uses Khera’s (‘84) indicators of maternal toxicity relative to fetal toxicity, the rat and rabbit developmental toxicity study can be placed in category 4-maternal toxicity accompanied with no significant fetal toxicity or malformation when compared to control. This outcome is also one of four possible events which can occur in teratology Segment I1 studies (Schardein, ’87). In summary, exposure of CD rats and NZW rabbits to soman during the period of major organogenesis for each species did not affect postimplantation loss or average fetal weight in a dose-related manner nor were there detectable effects on fetal external, visceral, or skeletal development. The results of these studies indicate that soman, while toxic to the pregnant rat and rabbit, did not significantly increase fetal toxicity or malformations even when the doses evaluated produced overt maternal toxicity. ACKNOWLEDGMENTS

The authors would like to acknowledge the assistance of the staff of the National Center for Toxicological Research. Particularly, we thank Bobby Gough and John Whatley for preparing the doses, Stacey Dial and Sherri Flumm for their help in completing the teratological assessments, Dr. David Gaylor, Dr. James Chen, and Mr. Richard Allen for their assistance in analyzing the data, and Rose Huber and Kellye

DEVELOPMENTAL EFFECTS OF SOMAN

Luckett for their skillful assistance in manuscript preparation. LITERATURE CITED Bates, H.K., and J.B. LaBorde (1986) Developmental toxicity evaluation of soman in New Zealand white (NZW) rabbits. Teratology, 33:72C. Bates, H.K., and J.B. LaBorde (1987) Developmental toxicity evaluation of soman in CD rats. The Toxicologist, 7:174. da Cunha Ferreira, R.M.C., I.M. Marquiegui, and I.V. Elizaga (1989) Teratogenicity of zinc deficiency in the rat: Study of the fetal skeleton. Teratology, 39:181194. Dacre, J.C. (1984) Toxicology of some anticholinesterases used as chemical warfare agents-A review. In: Cholinesterases: Fundamental and Applied Aspects. M. Brzin, E.A. Barnard, and D. Sket, eds. W. de Gruyter, New York, pp. 415-426. Department of the Army (1982) Special Occupational Safety and Health Standard for the Evaluation and Control of Occupational Exposure to Agent GB. D.A. Pamphlet 40-8. Published by Headquarters, Department of the Army, Washington, D.C., August 15. Ellin, R.I., W.A. Groff, and A. Kaminskis (1981) The stability of sarin and soman in dilute aqueous solutions and the catalytic effect of acetate ion. J. Environ. Sci. Health, B16(6):713-717. Federal Register (1987) Recommendations for protecting the health and safety against potential adverse effects of long-term exposure to low doses of agents GA, GB, VX, mustard (H,HD,T), and lewisite (L). CDCIDHHS, 42:(245):48458-48460. Fritz, H. and R. Hess (1970) Ossification of the rat and mouse skeleton in the rat and mouse skeleton in the prenatal period. Teratology, 3t331-338. Haseman, J.K., and L.L. Kupper (1979) Analysis of dichotomous response data from certain toxicological experiments. Biometric, 35~281-293. Inouye, M. (1976) Differential staining of cartilage and bone in fetal mouse skeleton by alcian blue and alizarin red S. Cong. Anom., 16:171-173.

23

Kavlock, R.J., N. Chernoff, and E.H. Rogers (1985) The effect of acute maternal toxicity of fetal development in the mouse. Teratogenesis Carcinog. Mutagen., 5; 3-13. Khera, K.S. (1984) Maternal toxicity: A possible factor in fetal malformations in mice. Teratology, 29:411416. Khera, K.S. (1985) Maternal toxicity: A possible etiological factor in embryo-fetal deaths and fetal malformations in rodent-rabbit species. Teratology, 31t129153. Khera, K.S. (1987) Maternal toxicity in humans and animals: Effects of fetal development and criteria for detection. Teratogenesis Carcinog. Mutagen., 7t287295. Kimmel, C.A., and C. Trammel (1981) A rapid procedure for routine double staining of cartilage and bone in fetal and adult animals. Stain Technol., 56t271273. Marr, M.C., C.B. Myers, J.D. George, and C.J. Price (1988) Comparison of single and double staining for evaluation of skeletal development: The effects of ethylene glycol (EG) in CD rats. Teratology, 37:476. Miller, R.G. (1966) Simultaneous Statistical Inference. McGraw-Hill, New York. Schardein, J.L. (1987) Approaches to defining the relationship of maternal and developmental toxicity. Teratogenesis Carcinog. Mutagen., 7:255-271. Shih, M.L., and R.I. Ellin (1986) Determination of toxic organophosphorous compounds by specific and nonspecific detectors. Anal. Lett., 19(23 and 24):21972205. Staples, R.E. (1974) Detection of visceral alterations in mammalian fetuses. Teratology, 9:A37-A38. Szczech, G.M., and B.P. Purmalis (1975) Folded retinas in rat fetuses: a n artifact of fixation in alcohol. Teratology, 11:36A. Whitaker, J., and U.M. Dix (1979)Double staining technique for rabbit fetus skeletons in teratologlcal - studies. Lab. Anim., 13t309-310. Wilson, J.G., and J . Warkany, eds. (1964) Teratology: Principles and Techniques. University of Chicago Press, Chicago, pp. 251-277.