Developmental toxicity of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). I. Multireplicated dose-response studies in four inbred strains and one outbred stock of mice.

A large-scaled multireplicated developmental toxicity study was conducted in various strains/stocks of mice with the herbicide, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), by gavage on Gestational Days 6 through 14. The most important attributes of the study design were replicated test groups, a minimum of four dose levels per replicate, use of multiple stocks/strains of animals to obtain an estimate of the range in sensitivities due to genotype, complete pathological evaluation of maternal animals, and histopathological as well as teratological evaluation of the fetuses. Developmental toxicity was observed at doses below those producing discernible or measurable maternal toxicity. Regression and/or probit analyses were conducted to determine whether a dose-response relationship existed. Reduced fetal weight and increased incidence of cleft palate and embryolethality were the most significant prenatal effects of 2,4,5-T exposure observed in this study. Each strain/stock exhibited a dose-related decrease in fetal weight with the CD-1 mice having the steepest slope and the A/J mice having the shallowest slope. There was a striking similarity among the slopes of the dose-response curves for the various strains/stocks. The mean incidence of embryolethality in the A/J strain was significantly greater than that of the other strains or stocks. There was substantial variation among replicates within strains. The use of the replicated study design was logistically necessary due to the magnitude of the study and it also served to increase the statistical power of the study.

Developmental toxicity of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). II. Multireplicated dose-response studies with technical and analytical grades of 2,4,5-T in four-way outcross mice.

A series of multireplicated developmental toxicity studies were conducted in four-way outcross mice and CD-1 outbred mice administered either analytical or technical grades of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) by gavage on Gestational Days 6 through 14. The formulations of 2,4,5-T differed by a factor of 10-fold in 2,3,7,8-tetrachlorodibenzo-p-dioxin levels. Reduced fetal weight and increased incidences of cleft palate and embryolethality were the most significant prenatal effects of both formulations of 2,4,5-T observed in all strains/stocks of mice. Both the outcross and outbred mice exhibited a dose-response relationship with each of the above endpoints and the dose-response curves were parallel. There were no embryotoxic or fetotoxic differences between the technical and analytical grades of 2,4,5-T with regard to extent of fetal weight reduction, resorption rate, or cleft palate incidence. There was little difference in the results between the four-way outcross mouse and the CD-1 outbred mouse.

Acute toxicity of pesticides in adult and weanling rats.

LD50 values were determined for 57 pesticides administered by the oral or dermal route to adult male and female Sherman rats. Thirty-six of the chemicals were also tested by the oral route in one sex of weanlings. Nine pesticides tested by the oral route (bufencarb, cacodylic acid, dialifor, deltamethrin, dicamba, diquat, quintozene, phoxim, pyrazon) and four tested by the dermal route (bufencarb, chlordimeform, dichlofenthion, leptophos) were more toxic to females than to males whereas famphur and 2,4,5-T (oral route) were less toxic to females. Eighteen of the test chemicals were more toxic to the adult than to the weanling and four compounds (leptophos, methidathion, pyrazon, and sulfoxide) were more toxic to the weanling. In additional studies the variability of the LD50 value over a 1-year period was examined for two typical insecticides. Six consecutive bimonthly oral LD50 determinations for parathion and DDT in adults of both sexes indicated that the LD50 values were little affected by the time of year that the tests were done.

Retarded development of fetal renal alkaline phosphatase in mice given 2,4,5-trichlorophenoxyacetic acid.

Histologic study of the fetal offspring of maternal mice given 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) suggested that the previously reported fetal "cystic kidneys" were due to a retardation in fetal renal development and downgrowth of the renal papilla into the pelvis. To determine a possible retardation in renal alkaline phosphatase or functional development, maternal mice received by gavage 60-120 mg/kg, 2,4,5-T on days 6-14 of pregnancy. At necropsy on day 17, the fetal kidneys were excised and fixed 24 hr in cold 65% ethanol. Paraffin sections stained by Gomori's method revealed alkaline phosphatase mainly in tubules in the inner renal cortex. Fetal kidneys showing diminished or no alkaline phosphatase were designated subnormal. There was a statistically significant greater incidence of subnormal fetal kidneys in the 2,4,5-T-treated mice than in the untreated controls. In three experiments, some mice were also sacrificed on day 18, and the incidence of subnormal fetal kidneys was significantly lower than on day 17. This retardation in renal alkaline phosphatase development indicates a retardation in renal functional development and indirectly supports the view that 2,4,5-T also retards the morphological development of the fetal kidney and is not a renal teratogen in mice. It also illustrates that selected histochemical studies may be helpful in a teratologic investigation.

Strain differences in histopathologic, hematologic, and blood chemistry changes induced in mice by a technical and a purified preparation of 2,4,5-trichlorphenoxyacetic acid.

Including controls, 978 mice were studied. On days corresponding to days 6 through 14 of pregnancy, groups of pregnant and nonpregnant CD-1 mice and male and nonpregnant female dihybrid cross F2 mice received by gavage 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) ranging in dosage from 30 to 140 mg/kg. Some groups received a technical preparation containing 97.9 +/- 0.4% 2,4,5-T and some a purified preparation containing 99 +/- 0.3% 2,4,5-T. Mice were sacrificed when they became moribund and at 1, 2, 4, 6, 8, and 11 days after beginning treatment. Sick or moribund mice sacrificed after 2-9 doses of 2,4,5-T often showed severe myocardial lesions, hypocellularlity of the bone marrow, and depletion of lymphocytes in the thymus, spleen or lymph nodes. They also showed marked hematologic and blood chemistry changes. Treated mice remaining healthy showed few or no lesions or blood chemistry changes, but often developed a mild anemia attributable to a hemolytic effect of 2,4,5-T. The incidence of animals becoming moribund was less than 1% in the CD-1 mice, including those given 140 mg/kg, and 53-82% in groups of male and female F2 mice receiving 120 mg/kg 2,4,5-T. The incidence of moribund mice tended to be higher in male than in female F2 mice and in those given the purified compound. These findings indicate that impairment of maternal health by severe lesions early in gestation is not the primary cause of an increase in incidence of fetal abnormalities observed in mice given 2,4,5-t. they also indicate that the lesions are due primarily to 2,4,5-T, rather than contaminants in the technical preparation, and illustrate the importance of using more than one strain of mouse in a toxicologic or teratologic study.

Teratological evaluation of FD&C Red No. 2-a collaborative government -industry study. IV. NCTR's study.

Because of recent studies indicating possible embryolethality and teratogenicity of FD&C Red No. 2, an ad hoc committee was convened by the Food and Drug Administration to consider these questions. The committee suggested a collaborative study by three laboratories [Food and Drug Administration (FDA),Industrial Bio- Test Laboratories (IBT), and National Center for Toxicological Research (NCTR)] in which Red No. 2 was given at 200 mg/kg body weight, by gavage during days 0-19, 6-15, or 7-9 of gestation. FD&C Red No. 2 was also given at the same dose level via water bottle. Appropriate controls were utilized. FDA used Osborne-Mendel strain rats, IBT used Charles River, and NCTR used both strains. No significant increases in skeletal or visceral abnormalities were seen. No significant increase in resorptions was seen in the Osborne-Mendel strain, but the Charles River strain at IBT showed a showed a significant increase in litters with two or more resorptions after dams had been given 200 mg/kg at 0-19 days of gestation. The NCTR study on the Charles River strain also showed an increase in the same parameter for the same dose level and in addition showed a significant increase in the percentage of resorptions per litter. It was concluded that because of the inherent variation and the absence of an increase in abnormalities or other indications of embryotoxicity, there is reason to doubt that this effect is either biologically significant or reproducible.

Sequential histopathologic, hematologic, and blood chemistry changes induced in mice by a technical and a purified preparation of 2,4,5-trichlorophenoxyacetic acid.

Maternal mice were given 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) on days 6 through 14 of pregnancy in a tetratologic study at the National Center for Toxicological Research. Sick or moribund mice sacrificed after 4-8 doses of 120 mg/kg 2,4,5-T often showed severe myocardial lesions, hypocellularity of the bone marrow, and depletion of lymphocytes in the thymus, spleen, or lymph nodes. Healthy mice sacrificed on day 17, 11 days after treatment began, showed few or no severe lesions. To determine if lesions earlier in gestation contributed significantly to an increase in fetal abnormalities in the healthy 17-day survivors, dihybrid croos F2 pregnant and nonpregnant mice received by gavage 0, 60, or 120 mg/kg 2,4,5-T on days 6 through 14 of pregnancy. One group received a technical preparation containing 97.9 +/- 0.4% 2,4,5-T; another received a purified preparation containing 99 +/- 0.3% 2,4,5-T. Mice were sacrificed when they became moribund and at 6, 24, and 30 hr, as well as at 4, 6, 8, and 11 days after beginning treatment. Almost all mice given 60 mg/kg and many given 120 mg/kg 2,4,5-T appeared normal at sacrifice either early or late in pregnancy and showed little or no pathologic changes. Mice that became ill or moribund often showed severe lesions; few survived 11 days. Severe myocardial lesions were seen in 26 of 70 moribund mice fiven the technical 2,4,5-T and 24 of 33 given the purified preparation of 2,4,5-T. The moribund mice, particularly those given the purified compound, also showed a high incidence of lesions in other organs and marked hematological and blood chemistry changes. These findings indicate that the lesions are primarily due to 2,4,5-T rather than to impurities in the technical preparation; also impaired maternal health is not the primary cause of the increase in fetal abnormalities.