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Acute toxicity of cephaloridine, an antibiotic derived from cephalosporin C
TOXICOLOGY
AND
Acute
APPLIED
PHARMACOLOGY
Toxicity
of
ATKINSON,
J. P.
Antibiotic
Limited,
Greenford,
Received
June
Derived
C
B. DAVIS, D. A. H. E. G. TOMICH
CURRIE,
Research
an
Cephalosporin AND
Glaxo
(1966)
Cephaloridine, from
R. M.
398-406
8,
PRATT,
Middlesex,
H.
XI.
SHARPE,
Englaizd
21, 1965
Cephaloridine,l a semisynthetic derivative of cephalosporin C, is 7- [a- (2-thienyl) acetamido] -3- ( I-pyridylmethyl) -3-cephem -4-carboxylic acid betaine (Fig. 1)
FIG.
1.
Structural
formula
-
of cephaloridine.
It is a highly bactericidal, broad-spectrum antibiotic virtually unaffected by penicillinase and not cross-allergenic with penicillin. Its antimicrobial properties have been described by Barber and Waterworth” (1964), Muggleton et al. (1964), and Stewart and Holt (1964)) and its clinical efficacy by Murdoch et aZ. (1964) and Stewart and Holt ( 1964). We report here some laboratory studies of its acute systemic toxicity and local irritant effects. METHODS
Material. Cephaloridine, as a freely water-soluble white crystalline powder of not less than 95% purity by microbiological assay, was administered in freshly prepared aqueous solution at concentrations of 4-4570, w/v. Acute systemic toxicity. Determinations of LD,, values were performed on mice (A2G strain, body weights 16-22 g), rats (PVG strain, 75140 g), and albino guinea pigs (ZOO-500 g). Graded single doses of cephaloridine were administered intravenously (at a rate of 1 ml/30 set), subcutaneously, intramuscularly, or orally to animals of each sex, and mortalities were recorded 4 days after intravenous, and 7 days after subcutaneous, intramuscular or oral administration. The LDno values were sometimes estimated approximately, but usually were calculated, with confidence limits, by the method of Litchfield and Wilcoxon (1949). Rabbits (New Zealand White strain, 2-4 kg), cats (2-5 kg) and monkeys (patas or cynomolgus) were given single intramuscular injections, and mongrel dogs (10-20 kg) were given single subcutaneous injections of 0.2 g/kg of cephaloridine and ob1 Ceporin@, Glaxo Laboratories Ltd. 2 These authors refer to cephaloridine
by
its laboratory 398
code name,
ceph.
87/4
ACUTE
TOXICITY
OF
CEPHALORIDINE
399
served for 7 days. The animals were then killed, and their major organs were removed for histologic examination. Also, one female cat was given a single intramuscular injection of 2.0 g/kg of cephaloridine. :$7ephrotoxkity. In a preliminary experiment, to ascertain the toxic effect of sublethal doses, adult female AZG mice were given single subcutaneous injections of 3-4 g’kg of cephaloridine and killed 48 hours later. Histologic examination revealed necrosis of the proximal convoluted tubules of the kidneys; the glomeruli and distal tubules appeared histologically normal, as did the liver, heart, lungs, small intestine, and ovaries. The time course of the renal changes was investigated by histologic examination of the kidneys of female mice 2 hours to 32 days after they had received single, subcutaneous doses of 1.0 g/kg of cephaloridine. As it was observed that the incidence (as well as the extent) of tubular necrosis was proportional to the logarithm of the dose of cephaloridine, the relative susceptibilities of the males and females of various species were investigated by determining KD,,, values, i.e., the doses producing nephrotoxic changes in 50% of the animals. Mice, rats, guinea pigs, rabbits, cats, dogs, and monkeys of each sex received graded single subcutaneous or intramuscular doses of cephaloridine, and their kidneys were removed 2 days later for ‘histologic examination. To avoid bias, the histologist was not told what dose had been administered to the animals whose kidneys he was examining. ND,,, values, with confidence limits, were calculated by the method of Litchfield and Wilcoxon (1949), or assessed approximately by graphical methods. A-lcute irritancy. Mice and rats surviving acute intravenous toxicity tests, for which IO-30% aqueous solutions of cephaloridine were used, and acute subcutaneous toxicity tests, with 20-4.5s solutions, were examined at intervals for signs of irritation at the injection sites. Guinea pigs surviving acute subcutaneous toxicity tests with S-3070 aqueous solutions and the dogs receiving single subcutaneous injections of 0.2 g/kg of cephaloridine, administered in 3055 aqueous solution, were likewise observed for signs of irritation. Three rabbits received single l.O-ml injections of a 20% aqueous solution of cephaloridine into the left thigh muscles and, for comparison, 1.0 ml of a 20% aqueous solution of sodium penicillin G into the right thigh muscles. They were killed after 4, 11, or 21 days, and the injection sites were examined macroscopically and histologically. Pharmacod~wamic efiects. Seven cats (2-4 kg body weight) were anesthetized with chloralose (80 mg/kg intraperitoneally) and pentobarbitone sodium (10 mg/kg intraperitoneally). Records were made of their arterial blood pressure, respiration, and, in four cats only, ECG (lead II). Recordings were made of the responses of the nictitating membrane of four cats to supramaximal preganglionic stimulation of the cervical sympathetic nerve, and the responses of the cardiovascular system of one cat to stimulation of the peripheral end of the cut vagus nerve were recorded. All the cats received intravenous injections of 0.95% saline, and five of them acetylcholine, histamine, or adrenaline (epinephrine), both before and after intravenous administration of cephaloridine. In two other cats (2.5 and 4.1 kg body weight) the responses of the tibialis and
400
R.
M.
ATKINSON
ET
AL.
soleus muscles to supramaximal stimulation of the peripheral end of the cut sciatic nerve were recorded. The effects of cephaloridine on renal function and the mechanism of urinary excretion of cephaloridine are to be reported by Child and Dodds (1966). RESULTS
Acute Systemic Toxicity Cephaloridine had a low toxicity to mice by all three routes (Table 1). The results of oral administration (five males and five females survived doses of 15 g/kg) suggest that in the mouse, as in man (Muggleton et al., 1964), single doses of cephaloridine are poorly absorbed from the gastrointestinal tract. The female mouse was more sensitive than the male to cephaloridine given subcutaneously, but intravenous dosage revealed no significant difference between the sexes. Intravenously administered cephaloridine was likewise equally toxic to male and female rats, but by the subcutaneous TABLE ACUTE
Species (strain) Mouse (A2G) Mouse Mouse Mouse Mouse Mouse Rat Rat Rat Rat Rat Rat
(A2G) (A2G) (AZG) (A2G) (A2G)
(PVG) (PVG) (PVG) (PVG) (PVG) (PVG)
SYSTEMIC
1
TOXICITY
OF CEPHALORIDINE
Route
Sex
Concentration administered (%)
i.v. i.v.
M F
S.C.
M
S.C.
12
TO VARIOUS
SPECIES -
Number of animals tested
LD,, (g/k) 2.2” (2.0-2.5) 2.2” (2.1-2.3)
12
71 70
F M F
30 30 30 30
40 50 5 5
S.C. p.0. p.0.
M F M F M F
IO-30 10-30 30 30 30-45 30-45
15 18 30 30 8 9
1.4a 1.4a 2.Sd 4.9
25 29
OSP 0.56”
p.0. p.0.
i.v. i.v. S.C.
12.5’~
b
(11.4-13.7)
9.6c (8.4-10.9) >15 >15 (1.0-2.0) (1.0-1.9) (2.4-2.6) (3.4-5.4) 2.5 4
Guinea Guinea
pig (albino) pig (albino)
S.C. S.C.
M F
s-30 S-30
(0.34-0.89) (0.38-0.82)
Rabbit Rabbit
(NZW) (NZW)
i.m. i.m.
M F
20-30 20-30
>0.2 >0.2
Cat Cat
i.m. i.m.
M F
20-30 20-30
>0.2 >0.2
Dog Dog
S.C. S.C.
M F
30 30
>0.2 >0.2
Monkey Monkey
i.m. i.m.
M F
20-30 20-30
>0.2
__
..-.- >02
a Difference between sexes is not statistically significant (P > 0.05) b In parentheses, confidence limits (P = 0.95). When confidence limits are not given, LD,, values are approximate. c The female mouse was 1.30 (95% confidence limits, 1.20-1.40) times as sensitive to the acute toxic effect of cephaloridine as the male. d The male rat was 1.7 (95% confidence limits, 1.3-2.2) times as sensitive to the acute toxic effect of cephaloridine as the female.
ACUTE
TOXICITY
OF
CEPHALORIDINE
401
route the antibiotic was more toxic to the male. The results of the oral tests on rats allowed the determination of only approximate LDno values; 3 of 5 male, but none of 6 female, rats given single oral doses of 3 g/kg of cephaloridine died, a difference just not significant at P = 0.05 (P = 0.061 by an exact test). By subcutaneous administration, cephaloridine was more toxic to guinea pigs than to mice or rats. There was no sex difference. The rabbits, cats, dogs. and monkeys receiving 0.2 g/kg of cephaloridine either intramuscularly or subcutaneously survived the 7-day observation period, appearing healthy and behaving normally. Histologic examination revealed renal tubular changes in the rabbit and monkey, but not in the cat or dog. In the rabbit, many of the proximal convoluted tubules were necrotic, the epithelium lining the tubules being replaced by eosinophilic granular casts and amorphous debris. The interstitial tissue was abnormally cellular, and many round cells were present. There was no evidence of damage to the glomeruli or renal vessels, or to the distal convoluted tubules, but many of them contained casts. Although there was no evidence of tubular necrosis in the monkey kidneys, in one male and one female many of the convoluted tubules were basophilic, and mitotic figures were conspicuous. This observation suggests that tubular necrosis might have occurred during the first few days of the experiment, and that regeneration was almost complete by the end. Eosinophilic colloid casts were present in the lumen of the convoluted tubules of the kidneys of the other two monkeys; in one, some of the tubules were dilated, lined by low cuboidal epithelium, and contained a number of desquamated cells. In one dog the liver cells appeared vacuolated, a finding of uncertain significance, but there was no abnormality in the livers of the other dogs. No abnormalities were observed in the other major organs from these four species. The cat given 2.0 g/kg of cephaloridine intramuscularly appeared to suffer no pain and did not limp. Ten minutes later, it began to shiver and then vomited. Two hours later it vomited again, and the nictitating membranes relaxed. Next morning, when the cat was found dead, the viscera appeared macroscopically normal. Histologic examination showed no evidence of renal damage. The liver was congested, there were areas of recent central zonal necrosis, and the portal tracts were infiltrated with round cells. :Vephrotoxicity In the experiment to determine the time course of renal damage in mice, the earliest histologic evidence was found 12-14 hours after dosing, when the proximal convoluted tubules had become noticeably dilated. The epithelial cells were of a low cuboidal type, the brush border was thinner than normal, and many of the cell nuclei had become pycnotic. By 18 hours, many of the proximal convoluted tubules were necrotic, the epithelial cells being replaced by coarsely granular debris in which all cellular detail was lost. The most sensitive tubules lay immediately beneath the capsule, and with more severe involvement the tubular necrosis spread toward the corticomedullary junction. Although the basement membrane supporting the tubules was usually undamaged, in areas of widespread tubular necrosis it too was destroyed. There was, however, no evidence of any damage to the distal convoluted tubules, the
402
R.
M.
ATKINSON
ET
AL.
loops of Henle, the collecting duct system, or the glomeruli. The picture was essentially the same after 48 hours (Fig. 2)) but by 4 days much of the necrotic tissue had been replaced by basophilic regeneratin, 0 tubular cells with numerous mitoses. After 8 days the regenerating cells had completely replaced the necrotic tubules; mitotic figures were not as plentiful as at 4 days. The healing was accompanied by some increase in cellularity of the interstitial tissue (Fig. 3). The characteristic basophilia of the regenerated tubules was lost by the 14th day, and, apart from some persistent thickening of the interstitial tissue and some small areas of nephrocalcinosis, the kidney
FIG. 2. Female mouse kidney showing of cephaloridine. Hematoxylin and eosin.
tubular necrosis 48 hours Magnification: >( 750.
after
a single
dose
of
1
g/kg
appeared normal. After 32 days, small scattered areas of nephrocalcinosis were present, but no other abnormality was seen. The various species differed markedly in sensitivity to the nephrotoxic effect of subcutaneously or intramuscularly administered cephaloridine (Table 2 ) . In the most sensitive species, the rabbit, cephaloridine in single doses of 0.05 g/kg was not nephrotoxic. Female mice were markedly more sensitive to the nephrotoxic effects of the antibiotic than males, but male rats were more sensitive than females. Though an accurate NDsO value could not be calculated from the results on female guinea pigs, the sex difference in sensitivity (S/6 males and O/4 females had renal abnormality after single doses of 0.5 g/kg) was significant (P = 0.048). The apparent sex difference in rabbits is probably not significant.
ACUTE
TOXICITY
OF CEPHALORIDINE
403
It would appear that the sex differences in mortality reflect sex differences in nephrotoxicity, although the results with guinea pigs do not support this. However, the tubular necrosis resulting from treatment with cephaloridine is not necessarily lethal, as illustrated by the differences between NDR,, and LDGo values. Because the cat treated with 2.0 g/kg of cephaloridine died showing histologic signs of liver necrosis, the livers as well as the kidneys of cats tested for nephrotoxic effects were examined. Two days after dosing there was no histologic abnormality of the livers or kidneys of 3 males and 3 females given 1.0 g/kg of cephaloridine intramuscularly or of a male given 0.5 g/kg.
FIG. 3. Female mouse kidney showing tubular regeneration 8 days after a single dose of 1 g/kg of cephaloridine. Note mitosis in upper left quadrant. Hematoxylin and eosin. Magnification: x 750.
Acute Irritancy Mice and rats showed no sign of thrombophlebitis of the tail veins during the 4 days after single intravenous injections of cephaloridine. Necrosis and ulceration of the skin of mice and rats developed 2-3 days after single subcutaneous injections and did not heal during the next 4-5 days. However the skin of guinea pigs and dogs appeared normal during the week after subcutaneous injections. Rabbit muscles injected with cephaloridine appeared normal to the naked eye. Microscopic examination failed to reveal any evidence of necrosis, the only significant finding being a small area of fascial hemorrhage 4 days after injection and some slight thickening of the connective tissue 11 days after injection. By 21 days the muscle and connective tissue appeared normal. In contrast, the muscles injected with penicillin contained hard discolored areas in which the penicillin had caused localized muscle
404
R.
M.
ATKINSON
ET
TABLE ACUTE TOXICITY
Species Mouse Mouse
(strain)
(A2G) (A2G)
OF CEPHALORIDINE
Route S.C.
Sex
AL.
2
TO THE KIDNEYS
Concentration administered (74)
OF VARIOUS
SPECIES
Number of animals tested
ND,,” (g/k)
S.C.
M F
4-20 4-20
29 25
3.lb (2.4-4.0)c o.6Ob (0.45-0.79)
Rat (PVG) Rat (PVC)
S.C. S.C.
M F
20-25 20-25
22 21
1.0” 1.4d
(0.9-1.2) (1.2-1.6)
Guinea Guinea
pig (albino) pig (albino)
S.C. S.C.
M F
25-30 25-30
16 14
0.4
(0.3-0.5) 0.7
Rabbit Rabbit
(NZW) (NZW)
i.m. i.m.
M F
10-30 IO-30
11 19
Cat Cat
i.m. i.m.
M F
30 30
4 3
>l.O >l.O
Dog Dog
S.C. S.C.
M F
30 30
3 7
>l.O >l.O
Monkey Monkey
i.m. i m.
M F
25 25
10 12
0.09
0.14 (0.06-0.12)
0.3 0.3
a Dose producing nephrotoxic changes after 48 hours in 50% of animals. 1, The female mouse was 5.16 (95% confidence limits, 3.53-7.53) times as sensitive to the nephrotoxic effect of cephaloridine as the male. C In parentheses, confidence limits (P = 0.95). When confidence limits are not given, ND,, values are approximate. d The male rat was 1.35 (95% confidence limits, 1.08-1.69) times as sensitive to the nephrotoxic effect of cephaloridine as the female.
with a brisk fibroblastic 21 days after injection.
necrosis
Pharmacodynamic
reaction. Evidence of muscle damage was still present
Effects
Doses of cephaloridine up to 25 mg/kg given intravenously to anesthetized cats had no effect on any of the systems recorded. Doses of 50 mg/kg transiently depressed the response of the nictitating membrane to preganglionic stimulation, but its response to adrenaline was unchanged. After doses of cephaloridine of 100 mg/kg, the blockade of the superior cervical ganglion was slightly greater. There was also a decreased response to acetylcholine: histamine, and adrenaline. The ganglionic blockade produced by cephaloridine at and above doses of 100 mg/kg was accompanied by a fall in blood pressure lasting several minutes. Given to one cat by intraarterial injection close to the superior cervical sympathetic ganglion, cephaloridine had 0.1% of the activity of hexamethonium administered similarly. Doses of cephaloridine up to 750 mg/kg had no effect on the ECG or on neuromuscular transmission. DISCUSSION
Cephaloridine has a low acute systemic toxicity to laboratory animals, In A2G mice, the intravenous LDBo was marginally greater and the subcutaneous LDjo markedly
ACUTE
TOXICITY
OF
CEPHALORIDINE
405
greater than some values published for sodium benzylpenicillin (Bacharach et al., 1959). In guinea pigs, the LDSo for cephaloridine administered subcutaneously was at least 100 times greater than that reported for sodium benzylpenicillin (Spector, 1957). The kidney, the organ most readily damaged by cephaloridine, is sensitive to many compounds differing widely in structure and pharmacologic activity (Reidenberg et al., 1964). Many antibiotics are nephrotoxic, e.g., bacitracin (Newton et al., 1951), gentamycin (Black et al., 1963), neomycin (Powell and Hooker, 1956), novobiocin (Larson et al., 1956), and the polymyxins (Brownlee et al., 1952). Sex differences in sensitivity to nephrotoxic substances have been encountered previously. Ethionine, serine, carbon tetrachloride, and mercuric chloride are more nephrotoxic to male than to female rats (Wachstein and Meisel, 1951; Fishman and &tom, 1942; Wachstein, 1947; Gyorgy et al., 1946; Harber and Jennings, 1965). The male rat is also more sensitive than the female to the nephrotoxic action of cephaloridine. Likewise, the kidneys of male mice are more sensitive to chloroform than those of females (Eschenbrenner and Miller, 1945; Hewitt, 1956), a finding confirmed for our own A2G mice. It may thus be noteworthy that the female A2G mouseis more sensitive than the male to the acute nephrotoxic action of cephaloridine. Although observed in acute tests, nephrotoxicity was not encountered to the extent expected in subacute tests (Atkinson et aZ., 1966). SUMMARY Cephaloridine, a semisynthetic derivative of cephalosporin C, was administered in single doses to mice, rats, guinea pigs, rabbits, cats, dogs, and monkeys. Acute LD5, values, obtained by variousroutes for mice, rats, and guinea pigs, were high. Rabbits, cats, dogs, and monkeys survived doses of 0.2 g/kg. Sublethal doses of the antibiotic damaged the proximal convoluted tubules of the kidneys of the mice, rats, guinea pigs, rabbits, and, probably, the monkeys. Tubular necrosis in mice was shown to be reversible. The relative sensitivities of the kidneys of various species were assessed by measurement of ND5, values (single doses producing histologic signs of kidney damage after 48 hours in 50% of animals). Sex differences in mortality and nephrotoxicity were observed in some species. -4dministered subcutaneously in large doses at high concentration, cephaloridine ulcerated the skin of mice and rats, but smaller doses were not irritant to the skin of guinea pigs or dogs. Cephaloridine was much less irritant to rabbit muscle than sodium penicillin G, and did not damage the veins of the mouse tail. Doses up to 25 mg/kg, administered intravenously to anesthetized cats, had no demonstrab!e pharmacodynamic effect. ACKNOWLEDGMENTS We thank Miss I. ,4. Byrnes, Mrs. A. Coulthard, Mrs. G. D. Maitland, Mr. R. J. McCulloch, Mr. J. S. Timewell, and Mrs. G. C. Woodford, without whose invaluable assistance these experiments could not have been completed. Dr. .4. C. Laursen kindly supervised the experiments on monkeys. REFERENCES ATKINSON, R. M., CAISEY, J. D., CURRIE, J. P., MIDDLETON, T. R., PRATT, D. A. H., SHARPE, H. M., and TOMICH, E. G. (1966). Subacute toxicity of cephaloridine to various species. Toxicol. Appl. Pharmacol. 8, 407-428. BACHARACH, A. L., CLARK, B. J., MCCULLOCH, M., and TOMICH, E. G. (1959). Comparative toxicity studies on ten antibiotics in current use. /. Pharm. Phavmacol. 11, 737-741.
406
R.
M.
ATKINSON
ET
AL.
BARBER, M., and WATERWORTH, P. M. (1964). Penicillinase-resistant penicillins and cephalosporins. Buit. Med. J. II, 344-349. BLACK, J., CALESNICK, B., WILLIAMS, D., and WEINSTEIN, M. J. (1963). Pharmacology of gentamicin, a new broad-spectrum antibiotic. In: Sntimicrobial llgents and Chemotherapy (J. C. Sylvester, ed.) .4merican Society for Microbiology, .4nn Arbor, Michigan. BROWNLEE, G., BUSHBY, S. R. M., and SHORT, E. I. (1952). The chemotherapy and pharmacology of the polymyxins. &it. J. Pharmacol. 7, 170-188. CHILD, K. J,, and DODDS, M. G. (1966). Mechanism of urinary excretion of cephaloridine and its effects on renal function in animals, Brit. 1. Pharmacol. ‘26, 108-119. ESCHENBRENNER, A. B., and MILLER, E. (1945). Induction of hepatomas in mice by repeated oral administration of chloroform, with observations on sex differences. J. -VVatl.Cuncu Inst.
5,
251-255.
W. H., and ARTO~~, C. (1942). Serine injury. J. Biol. Chem. 145, 345-346. (1946) Influence of dietary P., SEIFTER, J,, TOMARELLI, R. M., and GOLCBLATT, H. and sex on the toxicity of carbon tetrachloride in rats. 1. Exptl. Med. 83, 449-462. M. H., and JENNINGS, R. B. (1965). Renal response of the rat to mercury: the effect and sex hormones. Arch. Puthol. 79, 218-222. H. B. (1956). Renal necrosis in mice after accidental exposure to chloroform. Byit. J. Puthol. 37, 32-39. LARSON, E. J., CONNDR, N. D., SWOAP, 0. F., RUNNELLS, R. A., PRESTRUD, M. C., EBLE, T. E., FREYBURGER, W. A., VELDKAMP, W., and TAYLOR, R. M. (1956). Novobiocin, a new antibiotic. VI. Toxicology. Antibiot. Chemotherapy 6, 226-230. LITCHFIELD, J. T., and WILCOXON, F. (1949) -4 simplified method of evaluating dose-effect experiments. J. Pharmacol. Exptl. Thrrap. 96, 99-113. MUCGLETON, P. W., O’CALLAGHAN, C. H., and STEVENS, WT. K. (1964). Laboratory evaluation of a new antibiotic-cephaloridine (Ceporin). Bvif. Med. J. II, 1234-1237. MURDOCH, J. M., SPEIRS, C. F., GEDDES, .4. M., and W,ZLL;ZCE, E. T. (1964). Clinical trial of cephaloridine (Ceporin), a new broad-spectrum antibiotic derived from cephalosporin C. Bvit. Med. J. 11, 1238-1240. NEWTON, G. G. F., ABRAHAM, E. P., FLOREY, H. W., and SMITH, N. (1951). Some observations on the biological properties of bacitracins A, B, and C. Brit. J. Phurmucol. 6, 417-429. POWUL, L. W., JR., and HOOKER, J. W. (1956). Neomycin nephropathy. J. Am. Med. ASSOC. 160, 557-560. REIDENBERC, M. M., POWERS, D. V., SEVY, R. W., and BELI.O, C. T. (1964). Acute renal failure due to nephrotoxins. Am. J. Med. Sci. 247, 25-29. SPECTOR, W. S. (1957). Handbook of Toxicology, Vol. II, Antibiotics, pp. 140-141. Saunders, Philadelphia. STEWART, G. T., and HOLT, R. J. (1964). Laboratory and clinical results with cephaloridine. Luncet ii, 1305-1309. WACIISTEIN, M. (1947). Nephrotoxic action of dl-serine in the rat. I. The localization of the renal damage, the phosphatase activity and the influence of age, sex, time, and dose. Arch. Pathol. 43, 503-514. WACHSTEIN, M., and MEISEL. E. (1951). Nephrotoxic action of dl-ethionine. Pror. Sot. E.xptl. Biol. Med. 77, 648-651. FISHMAN,
GYORGY, factors HARBER, of sex HEWITT, Exptl.
AND
Acute
APPLIED
PHARMACOLOGY
Toxicity
of
ATKINSON,
J. P.
Antibiotic
Limited,
Greenford,
Received
June
Derived
C
B. DAVIS, D. A. H. E. G. TOMICH
CURRIE,
Research
an
Cephalosporin AND
Glaxo
(1966)
Cephaloridine, from
R. M.
398-406
8,
PRATT,
Middlesex,
H.
XI.
SHARPE,
Englaizd
21, 1965
Cephaloridine,l a semisynthetic derivative of cephalosporin C, is 7- [a- (2-thienyl) acetamido] -3- ( I-pyridylmethyl) -3-cephem -4-carboxylic acid betaine (Fig. 1)
FIG.
1.
Structural
formula
-
of cephaloridine.
It is a highly bactericidal, broad-spectrum antibiotic virtually unaffected by penicillinase and not cross-allergenic with penicillin. Its antimicrobial properties have been described by Barber and Waterworth” (1964), Muggleton et al. (1964), and Stewart and Holt (1964)) and its clinical efficacy by Murdoch et aZ. (1964) and Stewart and Holt ( 1964). We report here some laboratory studies of its acute systemic toxicity and local irritant effects. METHODS
Material. Cephaloridine, as a freely water-soluble white crystalline powder of not less than 95% purity by microbiological assay, was administered in freshly prepared aqueous solution at concentrations of 4-4570, w/v. Acute systemic toxicity. Determinations of LD,, values were performed on mice (A2G strain, body weights 16-22 g), rats (PVG strain, 75140 g), and albino guinea pigs (ZOO-500 g). Graded single doses of cephaloridine were administered intravenously (at a rate of 1 ml/30 set), subcutaneously, intramuscularly, or orally to animals of each sex, and mortalities were recorded 4 days after intravenous, and 7 days after subcutaneous, intramuscular or oral administration. The LDno values were sometimes estimated approximately, but usually were calculated, with confidence limits, by the method of Litchfield and Wilcoxon (1949). Rabbits (New Zealand White strain, 2-4 kg), cats (2-5 kg) and monkeys (patas or cynomolgus) were given single intramuscular injections, and mongrel dogs (10-20 kg) were given single subcutaneous injections of 0.2 g/kg of cephaloridine and ob1 Ceporin@, Glaxo Laboratories Ltd. 2 These authors refer to cephaloridine
by
its laboratory 398
code name,
ceph.
87/4
ACUTE
TOXICITY
OF
CEPHALORIDINE
399
served for 7 days. The animals were then killed, and their major organs were removed for histologic examination. Also, one female cat was given a single intramuscular injection of 2.0 g/kg of cephaloridine. :$7ephrotoxkity. In a preliminary experiment, to ascertain the toxic effect of sublethal doses, adult female AZG mice were given single subcutaneous injections of 3-4 g’kg of cephaloridine and killed 48 hours later. Histologic examination revealed necrosis of the proximal convoluted tubules of the kidneys; the glomeruli and distal tubules appeared histologically normal, as did the liver, heart, lungs, small intestine, and ovaries. The time course of the renal changes was investigated by histologic examination of the kidneys of female mice 2 hours to 32 days after they had received single, subcutaneous doses of 1.0 g/kg of cephaloridine. As it was observed that the incidence (as well as the extent) of tubular necrosis was proportional to the logarithm of the dose of cephaloridine, the relative susceptibilities of the males and females of various species were investigated by determining KD,,, values, i.e., the doses producing nephrotoxic changes in 50% of the animals. Mice, rats, guinea pigs, rabbits, cats, dogs, and monkeys of each sex received graded single subcutaneous or intramuscular doses of cephaloridine, and their kidneys were removed 2 days later for ‘histologic examination. To avoid bias, the histologist was not told what dose had been administered to the animals whose kidneys he was examining. ND,,, values, with confidence limits, were calculated by the method of Litchfield and Wilcoxon (1949), or assessed approximately by graphical methods. A-lcute irritancy. Mice and rats surviving acute intravenous toxicity tests, for which IO-30% aqueous solutions of cephaloridine were used, and acute subcutaneous toxicity tests, with 20-4.5s solutions, were examined at intervals for signs of irritation at the injection sites. Guinea pigs surviving acute subcutaneous toxicity tests with S-3070 aqueous solutions and the dogs receiving single subcutaneous injections of 0.2 g/kg of cephaloridine, administered in 3055 aqueous solution, were likewise observed for signs of irritation. Three rabbits received single l.O-ml injections of a 20% aqueous solution of cephaloridine into the left thigh muscles and, for comparison, 1.0 ml of a 20% aqueous solution of sodium penicillin G into the right thigh muscles. They were killed after 4, 11, or 21 days, and the injection sites were examined macroscopically and histologically. Pharmacod~wamic efiects. Seven cats (2-4 kg body weight) were anesthetized with chloralose (80 mg/kg intraperitoneally) and pentobarbitone sodium (10 mg/kg intraperitoneally). Records were made of their arterial blood pressure, respiration, and, in four cats only, ECG (lead II). Recordings were made of the responses of the nictitating membrane of four cats to supramaximal preganglionic stimulation of the cervical sympathetic nerve, and the responses of the cardiovascular system of one cat to stimulation of the peripheral end of the cut vagus nerve were recorded. All the cats received intravenous injections of 0.95% saline, and five of them acetylcholine, histamine, or adrenaline (epinephrine), both before and after intravenous administration of cephaloridine. In two other cats (2.5 and 4.1 kg body weight) the responses of the tibialis and
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soleus muscles to supramaximal stimulation of the peripheral end of the cut sciatic nerve were recorded. The effects of cephaloridine on renal function and the mechanism of urinary excretion of cephaloridine are to be reported by Child and Dodds (1966). RESULTS
Acute Systemic Toxicity Cephaloridine had a low toxicity to mice by all three routes (Table 1). The results of oral administration (five males and five females survived doses of 15 g/kg) suggest that in the mouse, as in man (Muggleton et al., 1964), single doses of cephaloridine are poorly absorbed from the gastrointestinal tract. The female mouse was more sensitive than the male to cephaloridine given subcutaneously, but intravenous dosage revealed no significant difference between the sexes. Intravenously administered cephaloridine was likewise equally toxic to male and female rats, but by the subcutaneous TABLE ACUTE
Species (strain) Mouse (A2G) Mouse Mouse Mouse Mouse Mouse Rat Rat Rat Rat Rat Rat
(A2G) (A2G) (AZG) (A2G) (A2G)
(PVG) (PVG) (PVG) (PVG) (PVG) (PVG)
SYSTEMIC
1
TOXICITY
OF CEPHALORIDINE
Route
Sex
Concentration administered (%)
i.v. i.v.
M F
S.C.
M
S.C.
12
TO VARIOUS
SPECIES -
Number of animals tested
LD,, (g/k) 2.2” (2.0-2.5) 2.2” (2.1-2.3)
12
71 70
F M F
30 30 30 30
40 50 5 5
S.C. p.0. p.0.
M F M F M F
IO-30 10-30 30 30 30-45 30-45
15 18 30 30 8 9
1.4a 1.4a 2.Sd 4.9
25 29
OSP 0.56”
p.0. p.0.
i.v. i.v. S.C.
12.5’~
b
(11.4-13.7)
9.6c (8.4-10.9) >15 >15 (1.0-2.0) (1.0-1.9) (2.4-2.6) (3.4-5.4) 2.5 4
Guinea Guinea
pig (albino) pig (albino)
S.C. S.C.
M F
s-30 S-30
(0.34-0.89) (0.38-0.82)
Rabbit Rabbit
(NZW) (NZW)
i.m. i.m.
M F
20-30 20-30
>0.2 >0.2
Cat Cat
i.m. i.m.
M F
20-30 20-30
>0.2 >0.2
Dog Dog
S.C. S.C.
M F
30 30
>0.2 >0.2
Monkey Monkey
i.m. i.m.
M F
20-30 20-30
>0.2
__
..-.- >02
a Difference between sexes is not statistically significant (P > 0.05) b In parentheses, confidence limits (P = 0.95). When confidence limits are not given, LD,, values are approximate. c The female mouse was 1.30 (95% confidence limits, 1.20-1.40) times as sensitive to the acute toxic effect of cephaloridine as the male. d The male rat was 1.7 (95% confidence limits, 1.3-2.2) times as sensitive to the acute toxic effect of cephaloridine as the female.
ACUTE
TOXICITY
OF
CEPHALORIDINE
401
route the antibiotic was more toxic to the male. The results of the oral tests on rats allowed the determination of only approximate LDno values; 3 of 5 male, but none of 6 female, rats given single oral doses of 3 g/kg of cephaloridine died, a difference just not significant at P = 0.05 (P = 0.061 by an exact test). By subcutaneous administration, cephaloridine was more toxic to guinea pigs than to mice or rats. There was no sex difference. The rabbits, cats, dogs. and monkeys receiving 0.2 g/kg of cephaloridine either intramuscularly or subcutaneously survived the 7-day observation period, appearing healthy and behaving normally. Histologic examination revealed renal tubular changes in the rabbit and monkey, but not in the cat or dog. In the rabbit, many of the proximal convoluted tubules were necrotic, the epithelium lining the tubules being replaced by eosinophilic granular casts and amorphous debris. The interstitial tissue was abnormally cellular, and many round cells were present. There was no evidence of damage to the glomeruli or renal vessels, or to the distal convoluted tubules, but many of them contained casts. Although there was no evidence of tubular necrosis in the monkey kidneys, in one male and one female many of the convoluted tubules were basophilic, and mitotic figures were conspicuous. This observation suggests that tubular necrosis might have occurred during the first few days of the experiment, and that regeneration was almost complete by the end. Eosinophilic colloid casts were present in the lumen of the convoluted tubules of the kidneys of the other two monkeys; in one, some of the tubules were dilated, lined by low cuboidal epithelium, and contained a number of desquamated cells. In one dog the liver cells appeared vacuolated, a finding of uncertain significance, but there was no abnormality in the livers of the other dogs. No abnormalities were observed in the other major organs from these four species. The cat given 2.0 g/kg of cephaloridine intramuscularly appeared to suffer no pain and did not limp. Ten minutes later, it began to shiver and then vomited. Two hours later it vomited again, and the nictitating membranes relaxed. Next morning, when the cat was found dead, the viscera appeared macroscopically normal. Histologic examination showed no evidence of renal damage. The liver was congested, there were areas of recent central zonal necrosis, and the portal tracts were infiltrated with round cells. :Vephrotoxicity In the experiment to determine the time course of renal damage in mice, the earliest histologic evidence was found 12-14 hours after dosing, when the proximal convoluted tubules had become noticeably dilated. The epithelial cells were of a low cuboidal type, the brush border was thinner than normal, and many of the cell nuclei had become pycnotic. By 18 hours, many of the proximal convoluted tubules were necrotic, the epithelial cells being replaced by coarsely granular debris in which all cellular detail was lost. The most sensitive tubules lay immediately beneath the capsule, and with more severe involvement the tubular necrosis spread toward the corticomedullary junction. Although the basement membrane supporting the tubules was usually undamaged, in areas of widespread tubular necrosis it too was destroyed. There was, however, no evidence of any damage to the distal convoluted tubules, the
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loops of Henle, the collecting duct system, or the glomeruli. The picture was essentially the same after 48 hours (Fig. 2)) but by 4 days much of the necrotic tissue had been replaced by basophilic regeneratin, 0 tubular cells with numerous mitoses. After 8 days the regenerating cells had completely replaced the necrotic tubules; mitotic figures were not as plentiful as at 4 days. The healing was accompanied by some increase in cellularity of the interstitial tissue (Fig. 3). The characteristic basophilia of the regenerated tubules was lost by the 14th day, and, apart from some persistent thickening of the interstitial tissue and some small areas of nephrocalcinosis, the kidney
FIG. 2. Female mouse kidney showing of cephaloridine. Hematoxylin and eosin.
tubular necrosis 48 hours Magnification: >( 750.
after
a single
dose
of
1
g/kg
appeared normal. After 32 days, small scattered areas of nephrocalcinosis were present, but no other abnormality was seen. The various species differed markedly in sensitivity to the nephrotoxic effect of subcutaneously or intramuscularly administered cephaloridine (Table 2 ) . In the most sensitive species, the rabbit, cephaloridine in single doses of 0.05 g/kg was not nephrotoxic. Female mice were markedly more sensitive to the nephrotoxic effects of the antibiotic than males, but male rats were more sensitive than females. Though an accurate NDsO value could not be calculated from the results on female guinea pigs, the sex difference in sensitivity (S/6 males and O/4 females had renal abnormality after single doses of 0.5 g/kg) was significant (P = 0.048). The apparent sex difference in rabbits is probably not significant.
ACUTE
TOXICITY
OF CEPHALORIDINE
403
It would appear that the sex differences in mortality reflect sex differences in nephrotoxicity, although the results with guinea pigs do not support this. However, the tubular necrosis resulting from treatment with cephaloridine is not necessarily lethal, as illustrated by the differences between NDR,, and LDGo values. Because the cat treated with 2.0 g/kg of cephaloridine died showing histologic signs of liver necrosis, the livers as well as the kidneys of cats tested for nephrotoxic effects were examined. Two days after dosing there was no histologic abnormality of the livers or kidneys of 3 males and 3 females given 1.0 g/kg of cephaloridine intramuscularly or of a male given 0.5 g/kg.
FIG. 3. Female mouse kidney showing tubular regeneration 8 days after a single dose of 1 g/kg of cephaloridine. Note mitosis in upper left quadrant. Hematoxylin and eosin. Magnification: x 750.
Acute Irritancy Mice and rats showed no sign of thrombophlebitis of the tail veins during the 4 days after single intravenous injections of cephaloridine. Necrosis and ulceration of the skin of mice and rats developed 2-3 days after single subcutaneous injections and did not heal during the next 4-5 days. However the skin of guinea pigs and dogs appeared normal during the week after subcutaneous injections. Rabbit muscles injected with cephaloridine appeared normal to the naked eye. Microscopic examination failed to reveal any evidence of necrosis, the only significant finding being a small area of fascial hemorrhage 4 days after injection and some slight thickening of the connective tissue 11 days after injection. By 21 days the muscle and connective tissue appeared normal. In contrast, the muscles injected with penicillin contained hard discolored areas in which the penicillin had caused localized muscle
404
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TABLE ACUTE TOXICITY
Species Mouse Mouse
(strain)
(A2G) (A2G)
OF CEPHALORIDINE
Route S.C.
Sex
AL.
2
TO THE KIDNEYS
Concentration administered (74)
OF VARIOUS
SPECIES
Number of animals tested
ND,,” (g/k)
S.C.
M F
4-20 4-20
29 25
3.lb (2.4-4.0)c o.6Ob (0.45-0.79)
Rat (PVG) Rat (PVC)
S.C. S.C.
M F
20-25 20-25
22 21
1.0” 1.4d
(0.9-1.2) (1.2-1.6)
Guinea Guinea
pig (albino) pig (albino)
S.C. S.C.
M F
25-30 25-30
16 14
0.4
(0.3-0.5) 0.7
Rabbit Rabbit
(NZW) (NZW)
i.m. i.m.
M F
10-30 IO-30
11 19
Cat Cat
i.m. i.m.
M F
30 30
4 3
>l.O >l.O
Dog Dog
S.C. S.C.
M F
30 30
3 7
>l.O >l.O
Monkey Monkey
i.m. i m.
M F
25 25
10 12
0.09
0.14 (0.06-0.12)
0.3 0.3
a Dose producing nephrotoxic changes after 48 hours in 50% of animals. 1, The female mouse was 5.16 (95% confidence limits, 3.53-7.53) times as sensitive to the nephrotoxic effect of cephaloridine as the male. C In parentheses, confidence limits (P = 0.95). When confidence limits are not given, ND,, values are approximate. d The male rat was 1.35 (95% confidence limits, 1.08-1.69) times as sensitive to the nephrotoxic effect of cephaloridine as the female.
with a brisk fibroblastic 21 days after injection.
necrosis
Pharmacodynamic
reaction. Evidence of muscle damage was still present
Effects
Doses of cephaloridine up to 25 mg/kg given intravenously to anesthetized cats had no effect on any of the systems recorded. Doses of 50 mg/kg transiently depressed the response of the nictitating membrane to preganglionic stimulation, but its response to adrenaline was unchanged. After doses of cephaloridine of 100 mg/kg, the blockade of the superior cervical ganglion was slightly greater. There was also a decreased response to acetylcholine: histamine, and adrenaline. The ganglionic blockade produced by cephaloridine at and above doses of 100 mg/kg was accompanied by a fall in blood pressure lasting several minutes. Given to one cat by intraarterial injection close to the superior cervical sympathetic ganglion, cephaloridine had 0.1% of the activity of hexamethonium administered similarly. Doses of cephaloridine up to 750 mg/kg had no effect on the ECG or on neuromuscular transmission. DISCUSSION
Cephaloridine has a low acute systemic toxicity to laboratory animals, In A2G mice, the intravenous LDBo was marginally greater and the subcutaneous LDjo markedly
ACUTE
TOXICITY
OF
CEPHALORIDINE
405
greater than some values published for sodium benzylpenicillin (Bacharach et al., 1959). In guinea pigs, the LDSo for cephaloridine administered subcutaneously was at least 100 times greater than that reported for sodium benzylpenicillin (Spector, 1957). The kidney, the organ most readily damaged by cephaloridine, is sensitive to many compounds differing widely in structure and pharmacologic activity (Reidenberg et al., 1964). Many antibiotics are nephrotoxic, e.g., bacitracin (Newton et al., 1951), gentamycin (Black et al., 1963), neomycin (Powell and Hooker, 1956), novobiocin (Larson et al., 1956), and the polymyxins (Brownlee et al., 1952). Sex differences in sensitivity to nephrotoxic substances have been encountered previously. Ethionine, serine, carbon tetrachloride, and mercuric chloride are more nephrotoxic to male than to female rats (Wachstein and Meisel, 1951; Fishman and &tom, 1942; Wachstein, 1947; Gyorgy et al., 1946; Harber and Jennings, 1965). The male rat is also more sensitive than the female to the nephrotoxic action of cephaloridine. Likewise, the kidneys of male mice are more sensitive to chloroform than those of females (Eschenbrenner and Miller, 1945; Hewitt, 1956), a finding confirmed for our own A2G mice. It may thus be noteworthy that the female A2G mouseis more sensitive than the male to the acute nephrotoxic action of cephaloridine. Although observed in acute tests, nephrotoxicity was not encountered to the extent expected in subacute tests (Atkinson et aZ., 1966). SUMMARY Cephaloridine, a semisynthetic derivative of cephalosporin C, was administered in single doses to mice, rats, guinea pigs, rabbits, cats, dogs, and monkeys. Acute LD5, values, obtained by variousroutes for mice, rats, and guinea pigs, were high. Rabbits, cats, dogs, and monkeys survived doses of 0.2 g/kg. Sublethal doses of the antibiotic damaged the proximal convoluted tubules of the kidneys of the mice, rats, guinea pigs, rabbits, and, probably, the monkeys. Tubular necrosis in mice was shown to be reversible. The relative sensitivities of the kidneys of various species were assessed by measurement of ND5, values (single doses producing histologic signs of kidney damage after 48 hours in 50% of animals). Sex differences in mortality and nephrotoxicity were observed in some species. -4dministered subcutaneously in large doses at high concentration, cephaloridine ulcerated the skin of mice and rats, but smaller doses were not irritant to the skin of guinea pigs or dogs. Cephaloridine was much less irritant to rabbit muscle than sodium penicillin G, and did not damage the veins of the mouse tail. Doses up to 25 mg/kg, administered intravenously to anesthetized cats, had no demonstrab!e pharmacodynamic effect. ACKNOWLEDGMENTS We thank Miss I. ,4. Byrnes, Mrs. A. Coulthard, Mrs. G. D. Maitland, Mr. R. J. McCulloch, Mr. J. S. Timewell, and Mrs. G. C. Woodford, without whose invaluable assistance these experiments could not have been completed. Dr. .4. C. Laursen kindly supervised the experiments on monkeys. REFERENCES ATKINSON, R. M., CAISEY, J. D., CURRIE, J. P., MIDDLETON, T. R., PRATT, D. A. H., SHARPE, H. M., and TOMICH, E. G. (1966). Subacute toxicity of cephaloridine to various species. Toxicol. Appl. Pharmacol. 8, 407-428. BACHARACH, A. L., CLARK, B. J., MCCULLOCH, M., and TOMICH, E. G. (1959). Comparative toxicity studies on ten antibiotics in current use. /. Pharm. Phavmacol. 11, 737-741.
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BARBER, M., and WATERWORTH, P. M. (1964). Penicillinase-resistant penicillins and cephalosporins. Buit. Med. J. II, 344-349. BLACK, J., CALESNICK, B., WILLIAMS, D., and WEINSTEIN, M. J. (1963). Pharmacology of gentamicin, a new broad-spectrum antibiotic. In: Sntimicrobial llgents and Chemotherapy (J. C. Sylvester, ed.) .4merican Society for Microbiology, .4nn Arbor, Michigan. BROWNLEE, G., BUSHBY, S. R. M., and SHORT, E. I. (1952). The chemotherapy and pharmacology of the polymyxins. &it. J. Pharmacol. 7, 170-188. CHILD, K. J,, and DODDS, M. G. (1966). Mechanism of urinary excretion of cephaloridine and its effects on renal function in animals, Brit. 1. Pharmacol. ‘26, 108-119. ESCHENBRENNER, A. B., and MILLER, E. (1945). Induction of hepatomas in mice by repeated oral administration of chloroform, with observations on sex differences. J. -VVatl.Cuncu Inst.
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W. H., and ARTO~~, C. (1942). Serine injury. J. Biol. Chem. 145, 345-346. (1946) Influence of dietary P., SEIFTER, J,, TOMARELLI, R. M., and GOLCBLATT, H. and sex on the toxicity of carbon tetrachloride in rats. 1. Exptl. Med. 83, 449-462. M. H., and JENNINGS, R. B. (1965). Renal response of the rat to mercury: the effect and sex hormones. Arch. Puthol. 79, 218-222. H. B. (1956). Renal necrosis in mice after accidental exposure to chloroform. Byit. J. Puthol. 37, 32-39. LARSON, E. J., CONNDR, N. D., SWOAP, 0. F., RUNNELLS, R. A., PRESTRUD, M. C., EBLE, T. E., FREYBURGER, W. A., VELDKAMP, W., and TAYLOR, R. M. (1956). Novobiocin, a new antibiotic. VI. Toxicology. Antibiot. Chemotherapy 6, 226-230. LITCHFIELD, J. T., and WILCOXON, F. (1949) -4 simplified method of evaluating dose-effect experiments. J. Pharmacol. Exptl. Thrrap. 96, 99-113. MUCGLETON, P. W., O’CALLAGHAN, C. H., and STEVENS, WT. K. (1964). Laboratory evaluation of a new antibiotic-cephaloridine (Ceporin). Bvif. Med. J. II, 1234-1237. MURDOCH, J. M., SPEIRS, C. F., GEDDES, .4. M., and W,ZLL;ZCE, E. T. (1964). Clinical trial of cephaloridine (Ceporin), a new broad-spectrum antibiotic derived from cephalosporin C. Bvit. Med. J. 11, 1238-1240. NEWTON, G. G. F., ABRAHAM, E. P., FLOREY, H. W., and SMITH, N. (1951). Some observations on the biological properties of bacitracins A, B, and C. Brit. J. Phurmucol. 6, 417-429. POWUL, L. W., JR., and HOOKER, J. W. (1956). Neomycin nephropathy. J. Am. Med. ASSOC. 160, 557-560. REIDENBERC, M. M., POWERS, D. V., SEVY, R. W., and BELI.O, C. T. (1964). Acute renal failure due to nephrotoxins. Am. J. Med. Sci. 247, 25-29. SPECTOR, W. S. (1957). Handbook of Toxicology, Vol. II, Antibiotics, pp. 140-141. Saunders, Philadelphia. STEWART, G. T., and HOLT, R. J. (1964). Laboratory and clinical results with cephaloridine. Luncet ii, 1305-1309. WACIISTEIN, M. (1947). Nephrotoxic action of dl-serine in the rat. I. The localization of the renal damage, the phosphatase activity and the influence of age, sex, time, and dose. Arch. Pathol. 43, 503-514. WACHSTEIN, M., and MEISEL. E. (1951). Nephrotoxic action of dl-ethionine. Pror. Sot. E.xptl. Biol. Med. 77, 648-651. FISHMAN,
GYORGY, factors HARBER, of sex HEWITT, Exptl.