Teratological evaluation of trimethyl phosphite in the rat

TOXICOLOGY

AND APPLIED

72, 119-123 ( 1984)

PHARMACOLOGY

Teratological

Evaluation of Trimethyl Phosphite in the Rat

MYRON A. MEHLMAN, PETER H. CRAIG, AND MICHAEL A. GALLO* Department of Environmental Affairs and Toxicology, Mobil Oil Corp., P.O. Box 1029, Princeton. New Jersey 08540, and *Department of Environmental and Community Medicine, UMDNJ-Rutgers Medical School, Piscatawav. New Jersey 08854

Received

Teratological

January

27, 1983; accepled

August

15. 1983

Evaluation of Trimethyl

Phosphite in the Rat. MEHLMAN, M. A., CRAIG, Pharmacol. 72, 119-123. Trimethyl phosphite (TMP) is an organophosphorus alkylating agent used primarily in the synthesis of organophosphate compounds. To evaluate teratogenic potential, TMP was administered by gavage to pregnant rats at rates of 16, 49, or 164 mg/kg/day, on gestation Days 6 through 15. Acetyl salicyhc acid (250 mg/kg/day) was also administered to a group of rats as a positive control. Teratologic evaluation revealed gross fetal abnormalities, skeletal defects, and soft tissue defects at a dose rate of 164 mg/kg/day of trimethyl phosphite, but not at the two lesser rates. An increased frequency of fetal resorption was also observed at 164 mg/kg/day. P. H.. AND GALLO,

M. A. (1984).

Toxicol,

Appl.

The adverse effects of trimethyl phosphite (TMP) on man are largely unknown. There have been no reported casesof TMP poisoning in humans, nor is there documentation of specific target organs which are influenced by the injection of toxic doses of this chemical into laboratory animals. In limited studies on rats, the nervous system has been identified as a target organ for TMP toxicity. Death in rats from intragastric administration of the compound is preceded by signs of stimulation and depression of the central nervous system (Kodama et al., 1955). TMP is an organophosphorus alkylating agent used primarily in the synthesis of organophosphate (OP) insecticides. These insecticides are irreversible inhibitors of acetyl cholinesterase, and some of them inhibit the less specific carboxylesterase (Brauer, 1948; Wills, 1972). Most OP insecticides are not teratogenic, with notable exceptions being dichlorvos, diazinon, chlorophos, and phthalophos (Kimbrough and Gaines, 1968; Martson and Veronina, 1976). TMP is an alkylating agent, and alkylating agents adversely af-

feet rapidly dividing differentiating tissues (Kimbrough and Gaines, 1968). This feature alerted us to the possible teratogenic potential of TMP. The present study was undertaken to determine the teratologic potential of TMP in the rat and to establish the nature of the effects on organogenesis and intrauterine development. METHODS Pregnant Sprague-Dawley rats [BLU:(SO)BRJ (Charles River Breeding Lab., Wilmington, Mass.), weighing ap proximately 250 g on Day 3 of pregnancy (confirmation of sperm plug as Day 0). were randomly distributed into five groups of 25 each. Animals were individually housed in suspended wire-mesh cages with tap water and Purina Laboratory Chow (St. Louis, MO.) available ad libitum. Artificial light in the animal room was cycled 12 hr on. 12 hr off. Temperature was maintained at 21 to 23°C and relative humidity at 65 to 85%. Mating was conducted in stages to produce a total of I25 pregnant females over a period of 3 days. The purity of trimethyl phosphite, 98.6% was established by gas chromatography and by iodine titration. With the Dohrman nitrogen analyzer, nitrogen content was 235 ppm. 119

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120

MEHLMAN,

CRAIG,

Body weights and feed consumption were recorded on Days 6, 10, 15, and 20 of pregnancy. TMP dosages of 164, 49, and 16 mg/kg/day were selected based on the acute intragastric LD50 of 1640 mg/kg in the rat (unpublished data). TMP in corn oil, a corn oil control, and an aqueous solution of acetyl salicylate (positive control) were administered by gavage at a rate of 10 ml/kg of body weight from Days 6 through 15. Volumes administered were based on the body weight of each animal determined on Day 6 of pregnancy. All dams were killed on Day 20. Laparotomies were performed, and the dams and pups were examined for gross abnormalities. One-third of the pups from each litter was selected at random and fixed in Bouin’s solution for visceral examination (Wilson, 1965). The remaining fetuses were eviscerated, fixed, macerated, and stained with Alizarin Red S for skeletal examination (Dawson, 1962). Noncategorical data were subjected to analysis of variance. When necessary to support interpretation, group means were compared by nonpaired t tests.

RESULTS Maternal Changes All dams survived the treatment and observation period. No toxic signs were observed in dams treated with corn oil or TMP, during the in-life phase of the study. No differences were observed in feed consumption except in the aspirin group, which showed an increased consumption during organogenesis (p < 0.0 1). Decreased body weight gain in the aspirintreated group of animals from Days 15 through 20 of gestation also occurred (p < 0.0 1). Correction of pregnant dam weights for the weight of the gravid uterus revealed that virtually all of the decrement occurred in the uterus, with little or no evidence of maternal weight loss. In the high TMP dose group (164 mg/kg/day), there was an apparent decrement in pregnant dam body weight gain, although not statistically significant. After correction for uterus weight, a decrement of less than 4% in comparison to the control mean body weight was not regarded as evidence of maternal toxicity of TMP. On the other hand, an 11% reduction in average gravid uterus weight in the 164 mg/kg group suggested fetal toxicity from TMP at the highest rate of administration.

AND GALL0

The number of live fetuses per dam was similar in all except the aspirin-treated group, where there was a nonsignificant decrease in this parameter. Total resorptions were increased in the 164 mg/kg TMP group (42 vs 15 for control) and in the aspirin group (41 vs 15 for control). These findings are indicative of effects on the fetus at high dose rates of both chemicals. Fetal Morphologic

Changes

Gross abnormalities were observed only in the aspirin and 164 mg/kg TMP groups. The major defects involved the neural tube. Exencephaly with or without spina bifida occurred in two litters of the 164 mg/kg TMP group and four litters of the aspirin group. In addition, the 164 mg/kg group had an incidence of cleft palate and agnathia in 40 to 50% of the litters (Table 1). Skeletal defects, TABLE 1 SUMMARYOFGROSSABNORMALITIESOFPWSFROM DAMSTREATEDWITHTMPDURINGPREGNANC~ Number at&ted/ number observed Group findings Control Aspirin (250 mg/kg) Exencephaly Spina bitida Scoliosis (curvature of spine) Umbilical Hernia

Fetuses

Litter

No gross abnormality 5125 1 41251 l/25 1 2125 1

4123 3123 l/23 2123

TMP ( 16 mg./kg)

No gross abnormality

TMP (49 mg/kg)

No gross abnormality

TMP ( 164 mg/kg) Exencephaly Spina bifida Scoliosis Cleft palate Agnathia (jaw recessed)

31265 121265 121265 66/265 551265

2123 l/23 l/23 12/23 1 l/23

TERATOLOGY

STUDY

OF TRIMETHYL

manifested as decreased ossification of bone centers throughout the skeleton, were more prevalent in the 164 mg/kg TMP and aspirin groups than in the control or lower dose groups. Variations in the ossification patterns of skull and stemebrae showed peak incidences in the high dose TMP and aspirin groups. Sporadic partial or widened cranial sutures were also observed at 16 and 49 mg/kg/day (Table 2) but were regarded as subtoxic man-

121

PHOSPHITE

ifestations, there being no other evidence of fetal effect at these rates. Hydronephrosis, hydroureters, and dilated ventricles of the brain were observed sporadically throughout the study population. However, the incidence of dilated ventricles was markedly increased in the aspirin and 164 mg/ kg groups (6 to 7X). Undescended testes and edematous pups were not observed in the control, 16, or 49 mg/kg groups. These anomalies were observed in approximately 20 and 10%

TABLE 2 SUMMARY OF SELECTEDSKELETAL FINDINGS OF PUPS FROM DAMS TREATED WITH TMP DURING PREGNANCY’ Groups Fetuses examined

Control

Aspirin 250 mg/kg

TMP 16 mg/kg

TMP 49 mg/kg

TMP 164 mg/kg

119123

164123

196124

169122

174123”

Skull Wide cranial suture

-

108/21

-

913

95114

Clavicle Partial

-

3118

-

-

39/X

Partial scapula, humerus, ulna, and radius

-

212

-

-

62110

Ribs Rudimentary Partial

713 -

54116 4/l

-

211

814 -

3219

Ilium, ischium, and pubis Partial Abnormal

-

3919

-

211

-

W

512

3717

Abnormal femur, Tibia and fibula

42/l

Metacarpals Less than 6 completely ossified

l/l

53115

-

2/l

71/12

Metatarsals Less than 6 completely ossified

-

22111

-

-

5619

Second stemebra’ Vertebra’ Cervical Thoracic

26/l 1

81121

20117

l/l 8/3

62118 I16123

l/l

l/l

615

613

a Numerator = number of fetuses affected; denominator b One pup lost. ’ Partially ossified, absent, or abnormal.

= number of litters affected.

23112

79118 3819 lOl/lO

122

MEHLMAN.

CRAIG,

respectively, of the pups examined in the aspirin and 164 mg/kg/day groups (Table 3). DISCUSSION The results of this study indicate that trimethyl phosphite at a daily intragastric administration rate of 164 mg/kg (approximately 10% of the LD50) is fetotoxic and teratogenic in pregnant Sprague-Dawley rats. The responses to TMP were similar to those seen in dams and fetuses treated with aspirin. No teratogenic or other toxic effects were observed in the pups or the dams administered TMP at rates of 49 or 16 mg/kg/day. Slight variations in the rate of closure of skull sutures were seen in fetuses from the low and middle dose TMP group. When such variations are TABLE

3

SUMMARY OF SOFT TISSUEABNORMALITIES OF Pups FROM DAMS TREATED WITH TMP DURING PREGNANCY

G~OUp/fidings

WIUXS Number a&ted/ number observed

Litter Number al&ted/ number ohserved

Control Hydmnephmis Hydmmter Dilated ventricle

IO/87 I l/87 l/87

7123 7123 l/23

Aspirin (250 mg/kg) Hydronephmis Hydmmter Dilated ventricle Undescended testes Enlarged auricle Edema Cranial hemorrhage

I l/86’ 8186 65186 l/86 IO/86 l/86 2186

9123 5123 I9123 l/23 5123 l/23 l/23

7199 6199 4199

6124 4124 4124

8184 I4184 2184

7122 s/22 2122

12190 9190 78190 17190

8123 4123 21123 I t/23 2123 2123

TMP (16 m@g) Hydronephrosis Hydroureter Dilated ventricle TMP(49 m&g) Hydmnephmsis Hydmureter Dilated ventricle

TMP(I64 m&g) Hydmnephmis Hydmureter Dilated ventricle Undexendcd testes Edema Cranial hemorrhage oSwd

tissue in one dead fetus.

WI 2l90

AND

GALL0

severe they may be taken as a manifestation of an adverse effect (Fritz and Hess, 1970) in conjunction with other evidence of fetal damage. However, sporadic occurrence of minor variations in skull sutures is very common and should not be regarded as significant evidence of fetal toxicity (Fritz and Hess, 1970; Palmer, 1972; I&era, 198 1; Kimmel and Wilson, 1973). Reports on the nature and extent of maternal and fetal effects of OP insecticides vary from compound to compound. Kimbrough and Gaines (1968) record malformations and fetal toxicity after a single maternal toxic dose of organic phosphorus compounds, including parathion and dichlorvos. With maternally tolerated single doses, parathion produced only fetal toxicity; dichlorvos was without effect. Similar variations with organic phosphorus compounds have been reported by Mattson and Veronina (1976). In the present instance, terata were produced by TMP at a level that was maternally tolerated but which produced fetal toxicity in a lo-day multiple dosage regimen. An inflection in the relationship between dosage and effects of TMP, both fetal toxicity and teratogenicity, was implied by the absence of any significant effect at 49 mg/kg day or less. Hence, under the conditions of this experiment, a no observed effect dose rate of at least 49 mg/kg/day was established. REFERENCES BRA~JER,R. W. (1948). Inhibition of cholinesterase activity of human blood plasma and erythrocyte stromata by alkylated phosphorus compounds. J. Pharmacol. Ther. 92, 162-72. COLLINS, T. F. X., AND COLLINS, E. V. (1976). Current methodology in teratology. In New Concepts in Safety Evaluation (M. A. Mehlman, R. E. Shapiro, and H. Blumenthal, eds.), Vol. 1, pp. 155-175. Hemisphere. Publishing, Washington, D.C. DAWSON, A. B. (1962). A note on the staining of the skeleton of cleared specimens with alizarin red s. Stain Technol. 1, 123-124. FRITZ, H., AND HESS, R. (1970). Ossification of the rat and mouse skeleton in the p&natal period. Teratology 3, 331-338. hERA, K. S. (198 1). Common fetal aberrations and their

TERATOLOGY

STUDY

OF

teratologic significance: A review. Fund. Appi. Toxicol. 1, 13-18. K~MBROUGH, R. D., AND GAINES, T. B. (1968). Effect of organic phosphorus compounds and alkylating agents on the rat fetus. Arch. Environ. Health 16, 805-808. KIMMEL, C. A., AND WILSON, J. G. (1973). Skeletal deviations in the rat: Malformations or variations? Teratology 8, 309-3 16. KODAMA, J. K., ANDERSON, H. H., DUNLAP, M. K., AND HINE, C. H. (1955). Toxicity of organophosphorus compounds, structure-action relationships in laboratory animals and man. A.M.A. Arch. Indust. Health 11,487493.

MARTSON, L. V., AND VERONINA, V. M. (1976). Exper-

TRIMETHYL

123

PHOSPHITE

imental study of the effect of a series of phosphoroorganic pesticides (Dipterex and Imidan) on embryogenesis. Environ. Health Perspect. 13, 121-125. PALMER, A. K. (1972). Sporadic malformations in lab oratory animals and their influence on drug testing. Adv.

Exp.

Med.

Biol.

1, 45-60.

WILLS, J. H. (1972). The measurement and significance of changes in cholin-esterase of erythrocytes and plasma in man and animals. CRC Crit. Rev. Toxicol. 1, 153202.

WILSON, J. G. (1965). Embryological considerations in teratology. In Teratology: Principles and Techniques (J. G. Wilson and J. Warkany, eds.), pp. 251-261. University of Chicago Press, Chicago.