Frederick R. Johannsen

Effects of formaldehyde in the rat and dog following oral exposure

Formaldehyde was administered orally (p.o.) to groups of rats and dogs at dosages up to 150 and 100 mg/kg/day, respectively, for 91 consecutive days. Significant body weight changes were observed at the higher dosages tested in both species. Decreased water consumption was observed in a dose-related pattern in all treated groups of rats. Food consumption and feed efficiency were decreased at higher dosage levels in dogs. Results of other clinical studies and microscopic pathology revealed no specific, treatment-related effects on any organ or tissue examined. These findings suggest that formaldehyde possesses little subacute toxicity following oral exposure.

Evaluation of delayed neurotoxicity and dose-response relationships of phosphate esters in the adult hen.

Abstract An oral multipledose technique in hens was developed to determine the neurotoxic potential of a series of triaryl-, trialkyl-, and alkyl-aryl phosphates. This potential was estimated by observation of abnormal behavioral signs and always verified by histological examination. This procedure permits dose titration of an active material to quantify dose-response relationships. Initial evaluation of a series of phosphates for neurotoxicity yielded three active materials: tricresyl phosphate, cresyl diphenyl phosphate, and o-isopropylphenyl diphenyl phosphate. Subsequent experiments demonstrated that their neurotoxic potential differed markedly. The following conclusions regarding structure-activity relationships may be drawn from this study: (1) Phosphates prepared from o-alkyl-substituted phenols frequently are neurotoxic; however, the o-alkyl group must contain at least one hydrogen atom on the α-carbon for activation. (2) The neurotoxic potency of active o-alkyl-substituted phosphates declines with increased mass and branching of the o-substituent. (3) Neurotoxicity is reduced by further substitution in the ring already containing an o-substituent. (4) A series of unsymmetrical phosphates containing an alkyl group in the p-position of one ring are not neurotoxic even though they contain hydrogen atoms on the α-carbon. (5) p-Tertiary-butylphenyl diphenyl phosphate, with no hydrogen on the α-carbon available for activation, does not produced delayed neurotoxicity.

Subchronic inhalation toxicity of p-nitroaniline and p-nitrochlorobenzene in rats☆

For evaluation of subchronic toxicity of the two single-ring nitroaromatics, p-nitroaniline (PNA) and p-nitrochlorobenzene (PNCB), groups of 10 male and 10 female Sprague-Dawley rats were exposed to an aerosol/vapor of PNA in isopropanol at target concentrations of 0, 10, 30, or 90 mg/m3 or to PNCB vaporized from a solution in ethylene glycol monoethyl ether at target concentrations of 0, 5, 15, or 45 mg/m3 for 6 hr/day, 5 days/week for 4 weeks. Clinical signs of toxicity, body weights, results of ophthalmoscopic exam, hematology and clinical chemistry tests, organ weights, gross and histopathological changes were recorded. Exposure to PNA or PNCB resulted in a dose-related increase in blood methemoglobin levels. Mean red blood cell counts, hematocrit, and hemoglobin were significantly decreased in mid and high level animals exposed to PNCB. Mean spleen weights (absolute and relative to body weight) were significantly increased at the high dose levels in the two studies. A slight increase in spleen weights was also observed at the low concentration level in the PNA study. Absolute and relative liver weights also were increased among animals exposed to 45 mg/m3 PNCB. Microscopic changes were observed mainly in the spleen and included an increase in intensity of extramedullary hematopoiesis and hemosiderosis with both compounds. Spleens of animals exposed to PNCB also exhibited congestion. Neither PNA nor PNCB exhibited significant toxicological effects other than those of methemoglobinemia, anemia, and splenic changes classically associated with nitroaromatics at levels significantly above presently accepted occupational standard. Our data suggest that the current TLV for PNA which is 3 mg/m3 will provide adequate protection to the workers. OSHAs PEL of 1 mg/m3 for PNCB is to be preferred over the current TLV of 3 mg/m3 to provide a comparable margin of safety.

Evaluation of the teratogenic potential of three aliphatic nitriles in the rat

Acetonitrile (ACN), adiponitrile (ADN), and propionitrile (PN), were evaluated for embryotoxic and teratogenic potential in rats. Mated Sprague-Dawley rats were administered one of the three nitriles by gavage on gestation Days 6-19, inclusive. Daily dosage levels (mg/kg body wt) were: ACN at 0, 125, 190, and 275; ADN at 0, 20, 40, and 80; PN at 0, 20, 40, and 80. There was evidence of maternal toxicity in each of the high dose groups treated with ACN, ADN, or PN. Some maternal effects also were seen with ADN at the middle dosage. Embryotoxic effects were observed at the highest dosage tested for ACN and middle and high dose levels for PN. Slight fetotoxicity was observed at the highest dosage for ADN. No teratogenic effects were observed at any dosage level with ACN, ADN, or PN.

Chronic Toxicity, Oncogenic Potential, and Reproductive Toxicity of p-Nitroaniline in Rats

Dose levels for these studies were selected mainly on the basis of subchronic studies, although consideration was also given to workplace exposure levels and proposed mechanism of tumor formation with structurally similar compounds. For the chronic study, groups of 60 male and 60 female Sprague-Dawley CD (Registered Trademark of Charles River Breeding Laboratories, Portage, MI) rats were given 0, 0.25, 1.5, or 9.0 mg/kg/day paranitroaniline (PNA) by gavage in corn oil for a period of 2 years. Parameters monitored included clinical observations, ophthalmoscopic exams, body weights, food consumption, hematology, clinical chemistry, and urinalysis at regular intervals throughout the study. All gross lesions and over 40 tissues were examined histologically for all control and high-dosage-level animals. Gross lesions, spleens, and livers of low- and mid-dosage groups were also examined histologically. For the reproduction study, groups of 15 male and 30 female rats, designated as F0 generation, were given PNA at the same levels as the chronic study for 14 weeks prior to mating and during mating, gestation, and lactation. Selected groups of 15 male and 30 female rats of the F1 generation received the same dose of PNA for 18 weeks prior to mating and during mating, gestation, and lactation. F2 pups were observed through weaning at which time they were euthanized. Observations made during the study included body weights, food consumption, mating and fertility indices, pup and litter survival indices, and histopathology of selected tissues. In the chronic study, except for a slight decrease in survival of high-dose male rats late in the study, survival in all treated groups was comparable to controls. Blood methemoglobin levels were elevated in the mid- and high-dosage groups, while slight anemia was observed in the high-dosage group also. Spleen weights were significantly increased in the high-dosage groups. An accumulation of brown pigment was observed in the cytoplasm of the sinusoidal macrophages or littoral cells of the liver and in the reticuloendothelial cells of the spleen. No treatment-related increase in tumor incidence was observed. In the reproduction study, no consistent pattern of effect from treatment between the F0 and F1 generation was seen in mating, pregnancy, or fertility indices. Thus, administration of PNA at levels which produced significant methemoglobinemia and low-level anemia in the rat and histological changes in the spleen produced no tumors or reproducible effects on reproductive performance.

Relationships between Toxicity and Structure of Aliphatic Nitriles

A retrospective structure-activity relationship (SAR) comparison was undertaken of several of the toxicological properties (acute and subchronic toxicity, teratogenicity, and biochemical mechanism studies) of a series of structurally similar aliphatic nitriles based on available published data. Members of this chemical class included several mononitriles (aceto-, propio-, butyro-, and acrylonitrile), dinitriles (succino- and adiponitrile), and cyanohydrin derivatives (lactonitrile and acetone cyanohydrin). Broadly defined, retrospective SAR analysis suggested that the toxicological profiles of this group of aliphatic nitriles were similar; however, unique differences were observed within each of the toxicity categories and nitrile subclasses as the toxicological focus narrowed. Additionally, the toxicological properties associated with acetonitrile, the first of this chemically similar series, were unique and quite different from others within the homologous series. Finally, knowledge regarding the mechanism of toxicological action provided valuable information in relating toxicological properties among the aliphatic nitriles.

Assessment of the Delayed Neurotoxicity of Tributyl Phosphate, Tributoxyethyl Phosphate, and Dibutylphenyl Phosphate:

There industrial organophosphorus compounds were tested for their ability to cause organophosphorus compound-induced delayed neurotoxicity (OPIDN) in the adult hen. The compounds tested were tributyl phosphate (TBP), tributoxyethyl phosphate (TBEP), and dibutylphenyl phosphate (DBPP). The acute oral LD50 of TBP and DBPP were estimated to be 1,863 and 1,500 mg/kg, respectively, and the dose equal to the LD50 was used as a test dose. The acute oral LD50 of TBEP was greater than 5,000 mg/kg and 5,000 mg/kg was used as a test dose. An oral dose of 750 mg tri-o-cresyl phosphate (TOCP) was used as a positive control. For the acute delayed neurotoxicity test, hens were given two test doses of the test materials 21 days apart and killed 21 days after the second dose. None of the hens given TBP, TBEP, or DBPP exhibited nerve damage or clinical signs which distinguished them from untreated control animals. A single dose of TOCP resulted in paralysis and a histopathological profile typical of a distal neuropathy. For the assay of the inhibition of esterases, hens were killed 24 hours after a single dose equal to the greater of either the LD50 or 5000 mg/kg. TOCP administration resulted in over 90% inhibition of brain neurotoxic esterase (NTE), but none of the other three compounds inhibited NTE to an extent (<70%) which would be expected to result in OPIDN. Administration of TOCP, TBEP, or DBPP resulted in approximately a 70% decrease in plasma butyrylchol-inesterase (BuChE) activity. TBP caused a 2–3 fold increase in BuChE activity. TBEP administration resulted in about 45% inhibition of acetycholinesterase (AChE) in brain. These results indicate that TBP, TBEP, and DBPP are all unlikely to cause OPIDN with any single sublethal dose.

A Review of the Subchronic Toxicity of Butyl Benzyl Phthalate

This review compares the subchronic toxicity of butyl benzyl phthalate (BBP) across several species. Data from the published literature as well as previously unpublished studies sponsored by Monsanto are presented. BBP-induced toxicity occurs only at relatively high levels of exposure and is dependent on the species, age and strain of test animals used. These factors should be considered in extrapolating findings from animal toxicology studies to humans when assessing the safety of BBP.

Teratogenic Response of Dimethylacetamide in Rats

Pregnant CD rats (25/group) were used to determine the teratogenic potential of dimethylacetamide (DMAC). DMAC was administered in deionized water once a day by gavage on Days 6 through 19 of gestation at dosages of 0, 65, 160, and 400 mg/kg/day. Cesarean sections were performed on all females on Gestation Day 20. No treatment-related effects were observed in survival, appearance, or behavior at necropsy. Mean maternal body weight gain was reduced significantly only at the 400 mg/kg/day level. Fetotoxicity manifested by increased postimplantation loss was seen at the 400 mg/kg/day level while reduction in mean fetal body weights was noted at the 160 and 400 mg/kg/day test levels. Developmental variations (reduced ossification and unossified skeletal variations) were increased at the 400 mg/kg/day test level and corresponded to the reduced fetal body weights which were observed. Treatment-related malformations of the heart, major vessels and oral cavity, and anasarca were seen at the 400 mg/kg/day DMAC level. No teratogenic effect of DMAC treatment was observed at or below dosage levels of 160 mg/kg/day.

Teratology and multigeneration reproduction studies with maleic anhydride in rats

These studies were initiated to evaluate the effects of maleic anhydride on development and reproduction in CD rats. In the teratology study, pregnant rats (19-23/group) received 0, 30, 90, or 140 mg/kg/day maleic anhydride in corn oil orally from Days 6-15 of gestation and fetuses were examined for gross soft tissue and skeletal defects. A reduced weight gain or weight loss was observed in all maleic anhydride-treated groups between Days 6 and 9; however, mean weights of all groups were within 5% of control on Days 15 and 20. No treatment-related effects on fetal development were observed. In the multigeneration study, rats (10 males and 20 females/group) received 0, 20, 55, or 150 mg/kg/day maleic anhydride in corn oil orally and were mated to produce two generations, each with two litters. Groups of the same size from the second litter were used for subsequent generations and were given the same dose of maleic anhydride as were their parents. The high-dose group was terminated during the second generation due to treatment-related mortality in adults. Renal cortical necrosis occurred in high-dose Fo males and females. Increased kidney weights were observed in low- and mid-dose adult F1 females. No treatment-related effects on reproduction were observed with maleic anhydride at doses up to 55 mg/kg/day over two generations.