ANTMIicoBRuL AGENTS AND CHEMorHERAPY, June 1973, p. 682-85 Vol. 3, No.

6
Copyright 11973 American Society for Microbiology Printed in U.SA.

Toxicological, Pathological, and Teratological

Studies in Animals with Cephradine
G. L. HASSERT, P. J. DEBAECKE, J. S. KULESZA, V. M. TRAINA, D. P. SINHA, AND E. BERNAL
The Squibb Institute for Medical Research, Princeton, New Jersey 08540
Received for publication 12 March 1973

Cephradine, a semisynthetic cephalosporin antibiotic, has a low order of oral

and parenteral toxicity in animals. The oral LD,0 in mice and rats ranged from 5

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to >8 g/kg, and the intraperitoneal LD50 values in mice and rats were 0.7 to 1.5
g/kg and 4.0 g/kg, respectively. The intravenous LD50 in mice ranged from 3.0 to
3.8 g/kg. In anesthetized dogs, intravenous doses of cephradine (40 and 120
mg/kg, given 45 min apart) had no effect on either the renal or cardiovascular
systems. Single intramuscular injections (0.25 ml or 0.5 ml of a solution
containing 125 to 235 mg of cephradine/ml) elicited no signs of either pain or local
irritation in dogs, and only transient signs of slight-to-moderate irritation were
observed in rabbits. In subacute toxicity studies, cephradine was administered
for 4 weeks to rats (daily intraperitoneal doses of 160, 480, or 1,600 mg/kg) and
dogs (daily intravenous doses of 80, 240, or 800 mg/kg); in addition, over a 2-week
period, monkeys were given daily intravenous doses of 60, 180, or 600 mg/kg. No
clinical, biochemical, gross, or micropathological changes due to cephradine
were observed in these animals; especially notable was the absence of any signs of
nephrotoxicity. In chronic toxicity studies, daily doses of cephradine were
administered orally to rats (100 to 1,000 mg/kg), dogs (50 to 500 mg/kg), and
monkeys (50 to 500 mg/kg) for 26, 26, and 13 weeks, respectively. Significant
responses were observed only in rats, in which grossly enlarged, but histologically
normal, ceca developed, a common finding in rodents dosed with antibiotics; in
addition, there were increases in the relative and absolute weights of the adrenal
glands. None of these effects was observed in rats that were necropsied 3 weeks
after termination of dosage. In reproduction studies in mice and rats given either
daily oral doses (100 or 300 mg/lkg) or daily intraperitoneal doses (rats only; 80 or
320 mg/kg) of cephradine, no drug-related teratogenic changes in the offspring
were observed.

The identification of cephalosporin C by Cephradine, a semisynthetic cephalosporin,

Abraham and Newton (1) led to the synthesis of 7- [-( -)-2-(1, 4-cyclohexadien- 1 -yl)acetamido]
an entirely new series of ,@-lactam antibiotics. 3-metfiyl-8-oxo- 5 -thia-l-azabicyclo [4.2.0]oct-
Abraham (2) found that cephalosporin C could ene-2-carboxylic acid, hydrate, possesses the
be cleaved by acid to provide a low-yield desirable properties noted above. This com-
conversion to the nucleus, 7-aminocephalospo- pound was synthesized in 1969 at The Squibb
ranic acid (7-ACA). When this nucleus became Institute for Medical Research (4). The anti-
available in quantity, many new derivatives microbial activities in vitro of cephradine, and
(semisynthetic cephalosporins) were produced its chemotherapeutic efficacy in animals after
either by acylation of the amino group or by oral administration, have been described re-
replacement of the acetoxy group, or by a cently (H. Gadebusch et al., Progr. Antimicrob.
combination of these processes. It was hoped Anticancer Chemother., in press). Pharma-
that, among these many derivatives, some cological studies, both in animals and in hu-
might possess certain desirable properties that mans, will be reported elsewhere. Various pa-
were lacking in the parent compound. Espe- pers on the evaluation of cephradine in the
cially to be desired were broadening of the treatment of human disease have been pub-
antimicrobial spectrum, more efficient absorp- lished (3, 5, 6, 7, 9, 10), and reports on other
tion after oral administration, and greater re- clinical studies with the drug are in press.
sistance to metabolic degradation. The studies reported here are concerned with
682
VOL. 3, 1973 ANIMAL STUDIES OF CEPHRADINE 683
the toxicological, pathological, and teratological Zealand white rabbits (0.25 ml; 35 to 60 mg of
responses observed in animals after either the cephradine) and beagles (0.5 ml; 115 to 235 mg of
oral administration of bulk cephradine or the cephradine). Some animals were sacrificed on the
parenteral administration of Cephradine for second day postdose, and the remainder 5 to 8 days
Injection, a blend of sodium carbonate and later; the injected muscles were excised and examined
for signs of local irritation.
cephradine; the latter material has been for- Subacute studies in rats, dogs, and monkeys. In
mulated for parenteral administration. The a 4-week study in rats and beagles, and in a 2-week
cephradine component contained in both the study in rhesus monkeys (Macaca mulatta), sterile
oral and parenteral preparations was identical. aqueous solutions of Cephradine for Injection were
administered twice daily, 6 days a week, as follows: to
MATERIALS AND METHODS rats, total daily intraperitoneal doses of 160, 480, or
Acute toxicity. The animals used in these studies 1,600 mg of cephradine/kg, 12 rats per group; to dogs,
and their weight ranges are described below: CD-1, total daily intravenous doses of 80, 240, or 800 mg of
Charles River mice, 18 to 23 g, and Charles River CD cephradine/kg, three dogs per group; and to monkeys,
Sprague-Dawley rats, 125 to 160 g. These animals total daily intravenous doses of 60, 180, or 600 mg of

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were fasted for 1 to 2 h prior to treatment. For oral and cephradine or cephaloridine/kg, two monkeys per
intraperitoneal administration, bulk cephradine was group. A complete postmortem was done on all
usually prepared at a concentration of 10 to 20% in animals, 10 organs from each animal were weighed,
0.15% agar; in oral studies, this suspension was and samples of 50 tissues were taken from each
administered by gavage. For intravenous use, Cephra- animal for histological examination.
dine for Injection was prepared as an aqueous solu- In all the subacute and chronic toxicity studies
tion; the intravenous injection rate was 0.1 ml/5 s. reported here, the criteria for evaluation included
In one study, an aqueous solution of Cephradine for survival data, changes in body weight, food and water
Injection was administered in single intravenous in- consumptions, physical condition, behavior, the re-
jections at doses of 500, 1,000, 2,000, or 4,000 mg of sults of extensive clinical laboratory tests, and gross
cephradine/kg to groups of 10 to 20 male or female and micropathological examinations. The dogs and
mice. In another study, liver or kidney damage was monkeys used in these studies weighed 8.5 to 11.0 kg
established in male mice by pretreatment with either and 3.0 to 5.5 kg, respectively.
oral doses of carbon tetrachloride or intravenous doses Chronic toxicity studies in rats, dogs, and mon-
of uranyl nitrate, respectively. These mice were then keys. Bulk cephradine was suspended in 0.15% agar
given single intravenous doses of cephradine (500, and administered twice daily by gavage to rats, dogs,
1,000, 2,000, or 4,000 mg/kg); there were 10 to 15 mice and monkeys for 26, 26, and 13 weeks, respectively.
per group. The total daily doses administered were as follows:
The LD,0 values were calculated by the method of rats (36 per group), 100, 300, and 1,000 mg of
Miller and Tainter (8), based on 8- to 11-day mortal- cephradine/kg; dogs (8 per group), 50, 150, and 500 mg
ity data. of cephradine/kg; and monkeys (4 per group), 50, 150,
Studies were also carried out to determine the and 500 mg of cephradine/kg. In each study, some
effects of intravenous doses of Cephradine for Injec- animals were sacrificed during treatment, and the
tion on the renal and cardiovascular systems of remainder 3 weeks after treatment had ended; all
anesthetized dogs. Two fasted young-adult male bea- animals were submitted for gross and micropathologi-
gles (10.8 and 10.9 kg) were hydrated with water, cal examinations.
anesthetized with intravenous sodium pentobarbital, Reproduction studies in mice and rats. In rats,
intubated with an endotracheal tube, and maintained the reproduction studies consisted of a fertility and
under anesthesia by a constant intravenous infusion general reproductive performance study (stage I), a
of sodium pentobarbital. Urine was collected from teratology study (stage II), and a peri- and postnatal
cannulae inserted into both ureters. Renal blood flow study (stage III). In each of these experiments, bulk
was determined by means of a Micron blood flowme- cephradine was suspended in 0.15% agar or 1% car-
ter, with a flow probe placed around the exposed left boxymethylcellulose and administered by gavage
renal artery. Glomerular filtration rate was estimated twice daily, 7 days a week, at total daily doses of 100
from the clearance of exogenous creatinine. In addi- or 300 mg of cephradine/kg. In the stage I, II, and III
tion, continuous recordings were made of arterial studies, there were 12, 20, and 22 female rats, respec-
pressure, heart rate, electrocardiogram (ECG), res- tively, per group. In the stage I study, matings were
piration, and rectal temperature. After basal renal made with male rats that had been dosed with
and cardiovascular functions had been established, a cephradine for 10 to 23 weeks on a regimen similar to
20% aqueous solution of Cephradine for Injection was the one described above; at the time of mating, the
injected intravenously into both dogs during 1-min females had been dosed for 2 wcWks. The female rats
periods at doses of 40 and 120 mg of cephradine/kg; in the teratology study were dosed from gestation day
the two doses were given 45 min apart. The poly- 6 until day 15 and then were killed on gestation day
graphic records were examined for evidence of ar- 21. The fetuses were removed, fixed in Bouin's fluid,
rhythmia and for changes in ECG waveforms, arterial and examined for gross malformations and abnor-
pressure, and heart rate. malities; in addition, all pups were eviscerated and
Muscle irritation studies. Cephradine for Injec- prepared for detailed skeletal examinations. In the
tion was dissolved in sterile water, and single injec- peri- and postnatal study, groups of pregnant rats
tions were made into the thigh muscles of New were given the drug daily, beginning on gestation day
684 HASSERT ET AL. ANTIMICROB. AG. CHEMOTHER.
15, and continuing to parturition and through lacta- scopic changes that could be attributed to the
tion until the pups had been weaned. compound. In a similar study, rhesus monkeys
In addition to the teratology study in rats, a similar were given intravenous doses of cephradine or
stage II study was carried out in mice (20 per group); cephaloridine for 2 weeks at total daily doses of
the daily oral doses of cephradine used in the mouse 60, 180, or 600 mg/kg. There were no drug-
study were the same as those given to the rats, i.e., related toxic signs or gross micropathological
either 100 or 300 mg/kg.
changes the monkeys given cephradine; the
in
RESULTS only change noted in these animals was loose
feces during the first 9 days of treatment. In the
Acute toxicity. The acute toxicity of cephra- animals given cephaloridine, one high-dose
dine in mice and rats is shown in Table 1. For all monkey died after 3 days of treatment, and the
routes of administration, the majority of deaths, other high-dose animal and one intermediate-
in both mice and rats, occurred within 24 to 72 h dose animal were sacrificed in poor condition
after dosing. At the highest intraperitoneal and during the first week of dosing. The latter two

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intravenous doses of cephradine (4,000 mg/kg monkeys showed clinical and biochemical evi-
and greater), ataxia was the only sign of toxici- dence of renal damage, and in all three animals
ty. At the highest oral dose (8,000 mg/kg), there there was histological evidence that indicated
was no sign of toxicity. extensive necrosis of the tubules of the kidneys.
In mice with hepatic or renal damage induced As noted above, under similar experimental
experimentally, the intravenous LD,0 was simi- conditions, neither biochemical nor histological
lar to that obtained in normal animals. evidence of nephrotoxicity was found in the
Intravenous doses of cephradine (40 and 120 monkeys given cephradine.
mg/kg, given 45 min apart) had no effect on the Chronic toxicity studies in rats, dogs, and
renal or cardiovascular functions of anesthe- monkeys. The daily oral administration of
tized dogs. cephradine to rats (100, 300, or 1,000 mg/kg),
Muscle irritation studies. Intramuscular in- and to dogs and monkeys (50, 150, or 500 mg/kg)
jections in dogs caused no signs of either pain or for 26, 26, and 13 weeks, respectively, caused no
irritation. In rabbits, there were signs of mild- significant toxic effects. Grossly enlarged, but
to-moderate irritation, slight-to-marked degen- histologically normal, ceca were noted in rats
eration without necrosis, a trace-to-moderate sacrificed during treatment; however, this re-
diffuse hemorrhage, and slight-to-moderate sponse, which is usually seen in rodents dosed
edema; there was no evidence of drug deposi- with an antibiotic, was not observed in animals
tion. necropsied 3 weeks after the cessation of dosage.
Subacute studies in rats, dogs, and mon- In the high-dose rats, sacrificed after 13 to 26
keys. In a 4-week study in rats, Cephradine for weeks of treatment, there was an increase in the
Injection, given intraperitoneally at total daily absolute and relative weights of the adrenal
doses of 160, 480, or 1,600 mg of cephradine/kg, glands; however, this response was not observed
caused no drug-induced toxic signs or histologi- in animals maintained for 3 weeks after the end
cal changes, with the exception of a dose-related of the dosing period. In dogs, the only side
enlargement of the cecum. Enlarged ceca are effects noted (high- and intermediate-dose ani-
commonly seen in rodents dosed with antibiot- mals) were moderate abdominal tenderness,
ics; hence, this effect should not be interpreted emesis, and loose stools; these effects were only
as evidence of toxicity. observed during the first 3 weeks of treatment.
Dogs given Cephradine for Injection intrave- In the monkeys, high- and intermediate-dose
nously for 4 weeks at total daily doses of 80, 240, animals had loose stools throughout the study.
or 800 mg of cephradine/kg showed no signifi- In rats, dogs, and monkeys, hematological and
cant signs of toxicity and no gross or micro- blood chemical values were within the normal
ranges.
TABLE 1. Acute toxicity of cephradine Reproduction studies in mice and rats. The
LD.. (g/kg) daily oral administration of cephradine to rats
at doses of 100 or 300 mg/kg did not induce any
Species Oa" Intra- Intra- adverse signs in (i) a fertility and general
S iOrala peritoneala e reproductive performance study, (ii) a teratol-
Mouse .. 5.0- > 8 0.7-1.5 3.0-3.8 ogy study, and (iii) a peri- and postnatal study.
Rat .. >8.5 4.0 In another teratology study, in which Cephra-
dine for Injection was administered in-
a Bulk cephradine. traperitoneally to rats at daily doses of 80 or 320
b Cephradine for Injection. mg of cephradine/kg, the drug had no effect on
VOL. 3, 1973 ANIMAL STUDIES OF CEPHRADINE 685
fetal development and did not induce terato- ACKNOWLEDGMENTS
genic changes in the offspring. In a teratology Acknowledgement of technical assistance in these studies
study in which mice were given daily oral doses is hereby made to the persons cited below: Mary Miller, P. L.
Sibley, J. Kirpan, and A. E. Peterson. We thank J. W.
of 100 or 300 mg of cephradine/kg, anomalies Poutsiaka, C. H. Keysser, and B. F. Murphy for their advice
were observed in some of the fetuses in both the and guidance in the interpretation of data; in addition, we
treated and the control groups. Since the inci- extend our appreciation to P. Arnow for his assistance in the
dence of these anomalies was within the ex- preparation of this manuscript.
pected limits of the spontaneous changes usu-
ally found in the strain of mice used, it was LITERATURE CITED
concluded that these effects were due to chance, 1. Abraham, E. P., and G. G. F. Newton. 1961. The
structure of cephalosporin C. Biochem. J. 79:377-393.
and were not caused by treatment with cephra- 2. Abraham, E. P. 1967. The cephalosporin group. Q. Rev.
dine. Biol. 21:231-248.
3. de Mendonca, J. S., G. W. Oselka, G. C. Levi, V. A. Neto,
DISCUSSION and H. V. Lopes. 1972. Preliminary observation of the
therapeutic activity of orally administered cephradine,
It has been reported (Gadebusch et al., in

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a new cephalosporin. Rev. Brasil. Clin. Ther. 1:207-
press) that, after oral administration in labora- 210.
tory animals, cephradine is well tolerated, is 4. Dolfimi, J., H. Applegate, G. Bach, J. Bernstein, S.
absorbed efficiently, and is an effective thera- Schwartz, and F. Weisenborn. 1971. A new class of
semisynthetic penicillins and cephalosporins derived
peutic agent against experimental infections. from D-2-(1,4-cyclohexadienyl)glycine. J. Med. Chem.
On the basis of the studies reported here, it is 14:117-119.
also concluded that cephradine has a low order 5. Estrada, F. A., B. D. Alora, and S. L. Lansang. 1972. A
of acute, subacute, and chronic oral toxicity, clinical trial of cephradine, a new cephalosporin deriva-
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and acute and subacute parenteral toxicity. Af- 6. Landa, L. 1972. Cephradine in the treatment of intestinal
ter daily intravenous doses of cephradine had infections caused by Shigella or Salmonella organisms.
been given to monkeys for 2 weeks, these ani- Curr. Ther. Res. Clin. Exp. 14:496-502.
mals showed no signs of nephrotoxicity. 7. Limson, B. M., R. E. Siasoco, and F. P. Dial. 1972. A new
cephalosporin derivative, cephradine, in the treatment
Cephradine did not induce any teratogenic of acute infective diseases. Curr. Ther. Res. Clin. Exp.
changes in the offspring of either mice or rats. 14:101-106.
Heretofore, the route of administration has 8. Miller, L. C. and M. L. Tainter. 1944. Estimation of the
been a determinant in the choice of a cephalo- ED,,, and its error by means of logarithmic-probit
graph paper. Proc. Soc. Exp. Biol. Med. 57:261-264.
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therapeutic advantage that a single cephalo- de sintesis. Evaluacion clinico-bacteriologica. [Cephra-
sporin antibiotic, cephradine, may be used in dine: A new synthetic cephalosporin, clinical-bacterio-
both oral and parenteral preparations. logical evaluation.] Dia Med. 44:152-153.
10. Ravaschino, A. 1972. Experiencia preliminar con ce-
Absorption, excretion, and distribution stud- fradina, un nuevo antibiotico de sintesis. [Preliminary
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