"Acute Toxicity

Results of acute oral and dermal toxicity studies with a series of aryl and alkyl phosphate esters are presented in Table 2. This series of phosphates exhibits a very low order of oral toxicity to rats and chickens and dermal toxicity to rabbits. Those phosphates containing at least two aryl groups are considered to be practically nontoxic by either route of exposure. Ester substitution to form a dialkyl-aryl or a trialkyl phosphate increased the degree of toxicity. However, these dialkyl-aryl and trialkyl phosphates are still considered to be only slightly toxic following oral or dermal administration.

Neurotoxicologic Evaluations

Table 3 presents results of experiments depicting the potential for a series of phosphate esters to produce delayed neurotoxicity. The neurotoxicologic potential of each phosphate was estimated by observation of abnormal behavioral signs in treated hens. Final evaluation of delayed neurotoxicity was based on the results of histologic examination. Tricresyl phosphate, whether derived from a mixture of o-, m-, and p-isomers of cresol or from a cresol with a high o-isomer content, was neurotoxic. Cresyl diphenyl phosphate, made from a mixture containing the three isomers of cresol, was neurotoxic at a cumulative dosage of 1.2 g/kg. In contrast, a loo-fold higher dosage of cresyl diphenyl phosphate, made from cresol containing only m- and p-isomers, was not neurotoxic. o-Isopropylphenyl diphenyl phosphate was neurotoxic at a cumulative dosage of 12 g/kg. A similar 12-g/kg dosage of other esters containing tris-osubstituents failed to induce neurotoxicity, i.e., tris-(o,o-dimethylphenyl) phosphate, tris- (o-ethylphenyl) phosphate, tris-(o-isopropylphenyl) phosphate, and tris-(o,p-dimethylphenyl) phosphate. A cumulative dosage of 120 g/kg of o-xenyl diphenyl phosphate failed to produce signs of neurotoxicity. Two alkyl-diary1 phosphates, isodecyl diphenyl phosphate and 2-ethylhexyl diphenyl phosphate, also were not neurotoxic under our test conditions. Unsymmetrical, p-substituted phosphates which produced no delayed neurotoxicity at cumulative dosages of 120 g/kg were p-isopropylphenyl diphenyl phosphate, p-tertiary-butylphenyl diphenyl phosphate, p-hexylphenyl diphenyl phosphate, and mixed p-nonylphenyl, p-cumylphenyl diphenyl phosphate. An unsubstituted ester, triphenyl phosphate, was not neurotoxic at 60 g/kg, the maximum dosage it was feasible to administer. Dibutylphenyl phosphate and tributyl phosphate were treated at lower dosages because of their acute toxicity to hens. The cumulative dosage for each, however, was greater than their respective single oral LD50 values. Three neurotoxic esters, tri-o-cresyl phosphate, cresyl diphenyl phosphate, and oisopropylphenyl diphenyl phosphate (Table 3), were evaluated further by means of the multiple-dose regimen. This procedure permitted titration of the amount of the ester required to produce neurotoxicity. Results are presented in Fig. 1. A median effective dose (ED50), based on histologic evaluation of nerve tissue, was calculated for each material. The ED50 values were: 0.42 g/kg for tri-o-cresyl phosphate, 1.23 g/kg for cresyl diphenyl phosphate, and 10.28 g/kg for o-isopropylphenyl diphenyl phosphate. No-effect dose levels for each ester can also be estimated from the dose-response curves. Signs of peripheral polyneuropathy occur on a delayed basis. Gross observation of treated birds revealed treatment-related effects, generally beginning 8-14 days following initial administration of an active phosphate. Overt signs of neurotoxicity exhibited by hens were graded according to the general scheme shown in the legend in Table 4. An initial generalized weakness of the legs was followed by an unsteadiness of gait (ataxia). These abnormal signs increased in intensity until a partial paralysis of the legs had developed. Finally, there was full immobilization so that affected birds were unable to stand. On occasion, severe cases also showed weakness of the wings and wingdrop was noted. Table 4 presents the gradation and duration of abnormal behavioral signs observed in groups of hens treated at two dosages of tri-o-cresyl phosphate. No hens dosed with 0.01 g/kg exhibited abnormal signs, while 6 of 10 hens treated with 0.025 g/kg exhibited characteristic signs of neurotoxicity. Initial signs of neurotoxicity were observed between Days 8 and 14 in four treated hens. However, the appearance of similar signs was delayed until Days 35 and 39 in the other two birds. The severity of abnormal signs exhibited by hens at this dosage level did not exceed a grade of 2. Adverse behavioral signs increased in intensity through day 22 of the test period, following which the intensity decreased. All hens showed an absence of abnormal signs between test Days 26 and 29, even though all birds had been redosed on test Days 21-23. Abnormal signs reappeared in these birds 9- 11 days after the second series of doses and continued until termination of the study. All 10 hens treated with 0.25 g/kg of tri-o-cresyl phosphate exhibited abnormal signs 6-13 days after initial treatment. Severity of signs increased rapidly and most hens exhibited a grade of 3 or 4 prior to sacrifice. Histopathologic evaluation of tissues obtained from treated hens revealed an apparent sequence in the development of neural lesions as seen by light microscopy. The morphological characteristics of lesions produced by all three active phosphates in this study were similar. Figures 2 and 3 show normal sections from sciatic nerve and spinal cord, respectively, of untreated control hens. The development of the neural lesion observed in sciatic nerve and spinal cord sections is shown in Figs. 4-7. Axonal swelling is the first histopathologic lesion observed. This progresses to axonal degeneration during which vacuolation, clumping, and fragmentation of axons are evident (Figs. 4 and 5). Finally, degradation or loss of the myelin sheath occurs at sites of earlier damage on the sciatic nerve (Fig. 6). In severe cases, loss of myelin also occurred in sections of the spinal cord (Fig. 7). In general, the relative severity and degree of tissue involvement observed histopathologically were dose related. With active phosphates, there was good correlation between gross behavioral signs graded as moderate and more severe and the presence of histopathologic changes in neural tissue. When hens exhibited a slight to mild degree of abnormal behavioral signs, the duration of the signs appeared to be a factor in determining whether neural lesions would be observed microscopically. "

Female White Leghorn Chickens