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Teratogenicity and neonatal toxicity of clindamycin 2-phosphate in laboratory animals
TOXlCOLOGY
AND APPLIED
PHARMACOLOGY
27, 322-329
(1974)
Teratogenicity and Neonatal Toxicity of Clindamycin 2-Phosphate in Laboratory Animals J. A. BOLLERT, J. E. GRAY, J. D. HIGHSTRETE, J. MORAN, B. P. PURMALIS AND R. N. WEAVER Research Division, The Upjohn Company, Kulamazoo, Michigan 49001 Received April 10,1973; accepted June 18,1973
Teratogenicity and Neonatal Toxicity of Clindamycin-2-Phosphate in Laboratory Animals. BOLLERT, J. A., GRAY, J. E., HIGHSTRETE, J. D., MORAN, J., PURMALIS, B. P. and WEAVER, R. N. (1974). Toxicol. Appl. Pharmacol. 27, 322-329. Subcutaneous injections of clindamycin 2-phosphate at 100 and 180 mg/kg on gestation days 6 through 15 in Upjohn SpragueDawley rats, Upjohn ICR and CFl mice had no detremental effect on reproductive performance. Teratogenic effects were not observed. A concurrent low incidence of spontaneous cleft palate was confirmed in the Upjohn ICR strain of mouse. This condition was not eliminated by vitamin B supplementation. The SCLD50 of clindamycin-2-phosphate has been shown to be 179 mg/kg in the neonatal rat and ~2000 mg/kg in the adult rat. The newborn/adult toxicity ratio was calculated at 1 :11.2, which is comparable to potassium penicillin G. Subcutaneous injections of clindamycin-2-phosphate at 120 mg/kg in nearly mature and recently weaned Gunn rats did not alter the partially impaired bilirubin metabolism in this neonate model. Simultaneous treatment of immature Gunn rats with 120 mg/kg of clindamycin-2-phosphate and therapeutic levels of sulfadiazine and sulfamerazine did not produce sufficient displacement of bilirubin from albumin binding in the serum to produce clinical signs of kernicterus. Teratological studies in rats and mice, comparative LD50 determinations in newborn and adult rats and toxicopathologic studies in the Gunn rat with impaired bilirubin metabolism were conducted with clindamycin-2-phosphate as part of the safety evaluation program prior to clinical testing in man. Several antibiotics have been found to be teratogenic in various laboratory species. They include tetracycline (Filippi and Mela, 1957a), penicillin and streptomycin in combination (Filippi and Mela, 1957b), hadacidin (Chaube and Murphy 1963), actinomycin D (Tuchmann-Duplessis and MercierParot, 1958; Wilson, 1965; Winfield, 1966), mitomycin C (Nishimura, 1963), streptonigrin (Warkany and Takacs, 1965) and streptomycin (Ericson-Strandvik and Gyllensten 1963). These studies emphasize the need for critical teratogenic evaluation of antibiotics. The study of acute toxicity of drugs in neonates was prompted by the knowledge that newborn animals are deficient in the ability to detoxify certain drugs due to a combination of factors including immature metabolic systems, renal function and bloodCopyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
322
TOXICITY
OF CLINDAMYCIN
323
Z-PHOSPHATE
brain barrier. Neonate human and other animal species have been found to be particularly sensitive to chloramphenicol (Sutherland, 1959), morphine (Chester et al., 1942) progesterone (Karnofsky er al., 1952), compounds with vitamin K activity (Allison, 1955; Laurance, 1955; Crosse et al., 1955), sulfonamides (Silverman et al., 1956), sodium glutamate (Lucas and Newhouse, 1957) and novobiocin (Sutherland and Keller, 1961). The comparison of LD50 values in newborn and adult animals is one method of assessing the potential for excessive toxicity in a neonate (Michael and Sutherland, 1961). In addition, the Gunn rat, which exhibits a genetic deficiency of glucuronyltransferase, can be used as a model of the newborn. Human fetal liver does not have glucuronyltransferase activity, and newborn babies do not approach adult values of glucuronyltransferase activity until 2-3 weeks after birth (Brown and Zuelzer, 1958). This condition results in a deficiency in an important detoxification mechanism by which compounds are conjugated with glucuronic acid (Vest, 1959). Thus the Gunn rat would appear to be a likely “neonate” model for detecting potential toxicity of compounds which are metabolized in this manner. In addition, sulfonamides have produced kernicterus in premature human infants (Silverman et al., 1956) and Gunn rats (Johnson et al., 1959) by competing with bilirubin for binding sites on the serum albumin. This results in decreasing serum bilirubin concentrations and diffusion of the bilirubin into the tissues. Used also in this conjunction, the Gunn rat would appear to be useful in neonatal toxicology studies. METHODS TeratoZogy studies. Pregnant Upjohn Sprague-Dawley (TUC/SD) rats, Upjohn ICR (UPJ/ICR) and CFl mice were given clindamycin-2-phosphate by SCinjection on gestation days 6 through 15 (counting the day of finding sperm in the vagina as day 0) at 100 and 180 mg/kg of body weight/day. Control animals received equivalent volumes of sterile water vehicle. On gestation day 20 in the rat and 18 in the mouse, the dams were sacrificed by CO, asphyxiation and cervical dislocation, respectively. Laparotomy was performed, the fetuses were recovered and weighed and the sex was noted. The uterus was also examined for resorption sites. All fetuses were examined for gross external malformations. Then each litter was divided into 2 subgroups : one-third of each litter was fixed in Bouin’s solution and examined for visceral malformations by a modification of the Wilson (1965) method and two-thirds of each litter were fixed in 70% ethanol, cleared, stained with alizarin red S and examined for skeletal malformations. This protocol was executed 3 times in TUC/ICR mice and once in CFl mice and UPJjSD rats. In another experiment following the same protocol in TUC/IUR mice, daily SCinjections of Solu-B@’ containing 0.05 mg riboflavin, 0.05 mg thiamine hydrochloride, 0.025 mg pyridoxine hydrochloride, 0.25 mg calcium pantothenate and 1.25 mg niacinamide were administered concurrent with clindamycin-2-phosphate. Control mice received Solu-B and sterile water vehicle injections. This dose of vitamin B complex was selected because it approximated the required level of riboflavin in the diet of the mouse (Fenton and Cowgill, 1947). LD50 studies in neonatal and adult rats. Neonate Upjohn Sprague-Dawley (TUC/’ SD) rats were treated by SCinjection within 24 hr after birth. Ten pups of each litter were 1 Solu-Ba is a registered trademark of The Up,john Company, Kalamazoo,
Michigan.
324
BOLLERT ET AL.
injected and the remaining rats were used as controls. Survivors were kept with the dam for 1 week. Ten neonates were treated with the following doses: 100, 125, 160, 200, 250 and 320 mg/kg. Groups of 5 male adult rats of the same strain were treated by SC injection at 1600 and 2000 mg/kg and observed for 7 days. The Spearman-Karber method (Finney, 1952) was employed to calculate the LD50 values. Toxicopathologic studies in the Gum rat. This rat is congenitally deficient in bilirubin glucuronyltransferase in the homozygous state, jj. Homozygous animals used in these studies were bred from homozygous jj male x heterozygous Jj female matings in the colony maintained by our department. Three studies were conducted on the Gunn rat with SCinjections of clindamycin-2-phosphate at 120 mg/kg/day to determine effects on serum bilirubin concentrations. In study I rats, 77-109 days old, were paired genetically (homozygous with homozygous and heterozygous with heterozygous) and as closely as possible according to age. Injections were made for 9 days in 1 rat of each pair. In study II rats, 21-39 day old, were paired and injected as in study I for 8 days. In study III immature, homozygous rats were divided into 3 groups and treated for 9 days as follows: 5 controls (untreated), 8 treated po with Citrasulfas MB2 (2.0 ml/kg; each fluid ounce containing 20 grains sulfadiazine, 20 grains sulfamerazine and 93 grains sodium citrate), and 8 treated po with Citrasulfas M and SCwith clindamycin-2-phosphate. Both compounds were dissolved in sterile water. The genetic characteristic and number of rats used was : Study I II III
Homozygous (jj) 14 9
21
Determinations of total bilirubin (Malloy at the termination of each study. Complete liver, spleen, pancreas, stomach, duodenum, bular salivary gland, heart, thyroid, lung, brain and pituitary were carried out.
Heterozygous (Jj) 14 -
9
and Evelyn, 1937) were made prior to and necropsies and histologic examinations of jejunum, mesenteric lymph node, mandithymus, kidney, adrenal, urocyst, testis,
RESULTS Teratology Studies
Subcutaneous injections of clindamycin-2-phosphate at 100 and 180 mg/kg/day on gestation days 6 through 15 in the TUCjSD rat had no detrimental effect on the reproductive parameters studied (Table 1). There was no indication of teratogenic activity in 112, 118 and 108 fetuses examined for gross, visceral and skeletal malformations at the 0, 100 and 180 mg/kg dose levels, respectively. In TUC/ICR mice SCinjections of clindamycin-2-phosphate at 100 and 180 mg/kg/ day on gestation days 6 through 15 did not adversely affect the reproductive parameters studied (Table 2). The study was conducted 3 times in order to clarify the presence of a low incidence of cleft palate at both the 100 and 180 mg/kg dose levels in study A. None of the control litters contained a fetus with cleft palate in study A; however, in studies B ’ Citrasulfas M@ is a registered trademark of The Upjohn Company, Kalamazoo,
Michigan.
TOXICITY
OF CLINDAMYCIN
TABLE
325
Z-PHOSPHATE
1
SUMMARY 0~ REPRODUCTION DATA FROM CONTROL AND CLINDAMYCIN-~-PHOSPHATETREATED PREGNANT TUC/SD RATS
No. of pregnant females
Dose (w/kg)
Avg. litter
weight (d
Avg. live pup weight cd
Avg. No. live pups/ litter
Avg. No. dead pups/ litter
Avg. No. resorptions/ litter
9 10 10
0 100 180
48.8 45.4 41.8
3.9 3.9 3.9
12.4 11.8 10.8
0.0 0.0 0.0
0.9 0.7 0.5
TABLE 2 SUMMARY 0~ REPRODUCTION DATA FROM CONTROL AND CLIN~~~M~~IN-~-PH~SPHATETREATED PREGNANT TUC/ICR MICE
Study
No. pregnant females
Avg. litter weight (d
A B C
10 8 9
13.4 8.5 11.9
A B C
8 8 10
14.1
A
10 6 10
B C
Avg. live pup weight (g)
Avg. No. live pups/ litter
Avg. No. dead pups/ litter
Avg. No. resorptions/ litter
Control 1.2 0.8 1.2
9.4 10.9 10.0
1.4 0.4 0.0
1.8 0.8 1.0
11.1 13.7
100 mg/kg 1.3 1.0 1.4
8.4 11.9 10.1
2.5 0.4 0.0
0.5 1.8 0.9
15.6 11.6 15.1
180 mg/kg 1.4 1.1 1.4
9.8 11.2 10.6
2.3 0.3 0.0
0.7 1.0 0.6
and C a similar low incidence of cleft palate occurred in the controls. Comparison of the incidences of litters containing fetuses with cleft palates (Table 3) highlights the inconsistencies in the number of litters affected in studies A, B and C. There was no significant difference in the percentages of litters with cleft-palate fetuses for control and clindamycin-2-phosphate-treated litters (Table 4). In a further attempt to characterize any teratogenic activity of clindamycin 2-phosphate the same protocol was run using the CFl strain of mouse. Reproductive parameters were not affected by treatment (Table 5). No evidence of teratogenic activity was indicated by gross, visceral and skeletal examinations of the 116,78 and 92 fetuses at the 0, 100 and 180 mg/kg dose levels, respectively. In a final study with TUC/ICR mice a vitamin B complex supplementation (Solu-B) was administered daily by SCinjection concurrently with clindamycin 2-phosphate under the protocol previously described. This was done to determine whether the sporadic cases of cleft palate might be related to vitamin B deficiency following antibiotic treatment (Filippi, 1967). Reproductive parameters in mice treated with clindamycin 2phosphate and vitamin B complex were not different fromcontrol micereceiving vitamin
326
BOLLERT ETAL. TABLE
3
INCIDENCE OF LITTERS WITH FETUSES EXHIBITING CLEFT PALATE IN CONTROL AND CLINDAMYCIN Z-PHOSPHATE-TREATED TUC/ICR MICE Study
Dose (mg/kg)
A
B
C
0
o/10 218
l/S 218 l/6
l/9 l/10 o/10
100 180
2110 TABLE
4
COMPARISON OF LITTERS WITH AND WITHOUT FETUSES EXHIBITING CLEFTPALATES (CPF) IN CONTROL AND CLINDAMYCINZPHOSPHATETREATED TUC/ICR MICE(~OMBINED STUDIES A,B, AND C)
Dose (mg/kg)
No. litters with CPF
No. litters without CPF
Total No. litters
% Litters with CPF
9 loo 180
2 5 3
25 21 23
27 26 26
7.4 19.2 11.5”
0 Chi Square= 1.9 (2 df). No significant difference between groups (p = 0.38).
B complex and sterile water injections (Table 6). Gross, visceral and skeletal examinations of the fetuses revealed 1 fetus with cleft palate in the control group and 1 at the 180-mg/kg dose level. This incidence of cleft palate is similar to those seen in the above reported TUC/ICR studies without vitamin B supplementation.
L D50 studies in Neonatal and Adult Rats. The SCLD50 value for clindamycin 2-phosphate in the neonatal rat was 179 mg/kg. In the adult rat no deaths occurred at either 1600 or 2000 mg/kg; therefore, the LD50 value is ~2000 mg/kg. Treatment at doses greater than 2000 mg/kg was prevented by the insolubility of the aqueous preparation. The newborn/adult toxicity ratio was calculated to be 1 :11.2. TABLE
5
SUMMARYOF REPRODUCTION DATAFROM CONTROL AND CLINDAMYCIN Z-PHOSPHATETREATED PREGNANT CFl MICE
No. pregnant females 12 9 10
Dose bdkd 0 100 180
Avg. litter weight Cd 13.1 11.6 12.7
Avg. live
Avg. No.
Avg. No.
Avg. No.
pup weight k>
live pups/ litter
dead pups/ litter
resorptionsl litter
1.4 1.4 1.5
9.7 8.7 9.0
0.0 0.0 0.2
0.8 0.8 0.9
TOXICITY
OF CLINDAMYCIN
327
Z-PHOSPHATE
TABLE 6 SUMMARY OF REPRODUCTION DATA FROM CLINDAMYCIN ~-PHOSPHATE- AND VITAMIN B-TREATED TUC/ICR MICE
No. pregnant females 8 8 8
Dose” bm/kg)
Avg. litter weight (EC>
Avg. live pup weight (d
Avg. No. live pups/ litter
Avg. No. dead pups/ litter
Avg. No. resorptions/ litter
0 100
13.5
14.0
1.3
10.4
11.4 9.0
0.3 0.4
0.9
1.3
180
12.0
1.4
1.1
0.4 1.3
a Dose refers to clindamycin 2-phosphate.All micereceivedvitamin B complex during doseperiod. SeeMethods section. Toxicopathologic
Studies in the Gum Rat.
The SCadministration of clindamycin 2-phosphate at 120 mg/kg to 77-109-day-old rats (study I) and recently weaned (study II) Gunn rats did not alter the partially impaired metabolism of bilirubin in these rats. No significant changes in serum bilirubin concentrations or morphologic evidence of increased deposition of bile pigment and destruction of brain tissue was noted. Simultaneous treatment of homozygous rats with sulfa drugs (Citrasulfas M) and clindamycin 2-phosphate (study III) did not afford sufficient displacement of bilirubin from albumin binding in the serum to produce clinical manifestations. DISCUSSION The sporadic occurrence of cleft palate seen in the teratology studies with the TUC/ ICR mice illustrates an important necessity for teratology testing procedures, namely, adequate information on the incidence of the various spontaneous malformations which characterize any particular strain of laboratory animal. Several factors indicate that the low incidence of cleft palate found in the TUC/ICR mice was spontaneous or the result of some as yet undetermined environmental alteration other than treatment with clindamycin 2-phosphate. They are: (1) the inconsistencies in the occurrence of cleft palate in successive studies with clindamycin 2-phosphate-treated litters; (2) the occurrence of cleft palate in all control groups except study A; (3) the failure of vitamin B administration to prevent the occurrence of cleft palate when one established mechanism for antibiotic induction of cleft palate is vitamin B deficiency (Filippi, 1967) ; (4) the absence of any cleft palate induction in another strain of mouse (CFI); and (5) subsequent studies with another compound in the TUC/ICR mouse also revealed similar incidences of cleft palate in control and treated fetuses (Bollert, unpublished observations). The newborn/adult toxicity ratio of 1 :l 1.2 for clindamycin 2-phosphate is comparable to potassium penicillin G at I : 12 and less than chloramphenicol at 1:16 (Raynsford, 1963). Regardless of these ratios, particular caution and careful consideration of dose levels should be exercised in treating neonatal humans because of their potential increased susceptibility to any drug.
328
BOLLERT
ET AL.
The relevance of the Gunn rat in toxicology screening as a neonate model in glucuronidation deficiency is predicated, in part, upon the assumption that the drug being tested is conjugated with glucuronic acid and that this mechanism represents an important detoxification pathway in this animal and in man. The current studies indicating no toxic manifestations of clindamycin 2-phosphate in the Gunn rat were initiated before we were aware that Sun (1970) had found glucuronide not to be a metabolite of clindamycin in the Sprague-Dawley rat. The displacement of bilirubin from albumin binding in the serum resulting in kernicterus would not appear to be a potential problem following clindamycin 2-phosphate treatment. This finding appears to be compatible with the results of W. Sokolski (unpublished observations) that clindamycin base to which the phosphate is hydrolyzed in vivo binds to a glycoprotein, not albumin in the serum.
REFERENCES A. C. (1955). Danger of vitamin K to newborn. Lancer 1,669. A. K. AND ZUELZER, W. W. (1958). Studies on the neonatal development of the glucuronide conjugating system. J. Clin. Invest. 37, 332-340. CHAUBE, S. AND MURPHY, M. L. (1963). Teratogenic effect of hadacidin (a new growth inhibitory chemical) on the rat fetus. J. Exp. Zool. 152,67-73. CHESTER, A., LABELLE, G. C. AND HIMWICH, H. E. (1942). A study of the comparative toxic effects of morphine on the fetal, newborn and adult rats. J. Pharmacol. Exp. Ther. 75, ALLISON, BROWN,
363-366.
V. M., MEYER, T. C. AND GERRARD, J. W. (1955). Kernicterus and prematurity. Arch. Dis. Childhood 30, 501-508. ERICSON~TRANDUIK, B. AND GYLLENSTEN, L. (1963). The central nervous system of foetal mice after administration of streptomycin. Acta Pathol. Microbial. &and. 59,292-300. FENTON, P. F. AND COWGILL, G. R. (1947). The nutrition of the mouse. J. Nutr. 34,273-283. FILIPPI, B. (1967). Antibiotics and congenital malformations: evaluation of the teratogenicity of antibiotics. In: Advances in Teratology (D. H. M. Woollam, ed.), Vol. II, pp. 239-256. Logos Press,London. FILIPPI, B. AND MELA, V. (1957a). Malformazioni congenite facciali e degli arti da tetracyclina. Minerva Chir. 12, 1106-l 110. FILIPPI, B. AND MELA, V. (1957b). Malformazioni congenite degli arti ottenute sperimentalmente in embrioni de ratto in seguito a trattamento con penicillina e streptomicina. Minerva Chir. 12, 1047-52. FINNEY, D. J. (1952). Statistical Methods of Biological Assay, p. 524. Hafner, New York. JOHNSON, L., SARMIENTO, F. AND BLANC, W. A. (1959). Kernicterus in rats with an inherited deficiency of glucuronyl transferase. A.M.A. J. Dis. Childhood 97,591-608. KARNOFSKY, D. A., HAMRE, P. J. AND HYSOM, G. (1952). Toxicity of progesterone in the newborn mouse. Proc. Sot. Exp. Biol. Med. 79,641~643. LAIJRANCE, B. (1955). Danger of vitamin K analogues to newborn. Lancet 1, 819. LUCAS, D. R. AND NEWHOUSE, J. P. (1957). The toxiceffect of sodium-l-glutamate on the inner layers of the retina. A.M.A. Arch. Ophthalmal. 58, 193-201. MALLOY, H. T. AND EVELYN, K. A. (1937). The determination of bilirubin with the photoelectric calorimeter. J. Biol. Chem. 119,481-490. MICHAEL, A. F. AND SUTHERLAND, J. M. (1961). Antibiotic toxicity in newborn and adult rats. A.M.A. J. Dis. Childhood 101,4424I6. NISHIMLJRA, H. (1963). Interstrain differences in susceptibility to the teratogenic effects of mitomycin in mice. Abstr. Teratol. Sot. 3, 16. RAYNSFORD, G. (1963). Technique of comparing acute toxicity in infant vs. adult rats. Amer. J. Dis. Childhood 105,323-328. CROSSE,
TOXICITYOF CLINDAMYCIN 2-PHOSPHATE
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SILVERMAN, W. A., ANDERSEN, D. H., BLANC,W. A. ANDCROZIER,D. N. (1956). A difference
in mortality rate and incidence of kernicterus among premature infants allotted to two prophylactic antibacterial regimens. Pediatrics 18, 614-625. SUN, F. (1970). Disposition of clindamycin in rat and dog. Fed. Proc., Fed. Amer. Sot. Exp. Biol. 29, 677.
SUTHERLAND, J. M. (1959). Fatal cardiovascular collapse of infants receiving large amounts of chloramphenicol. A.M.A. J. Dis. Childhood 97, 761-767. SUTHERLAND, J. M. AND KELLER,W. H. (1961). Novobiocin and neonatal hyperbilirubinemia A.M.A. J. Dis. Childhood 101,447-453. TUCHMANN-DUPLESSIS, H. AND MERCIER-PAROT, L. (1958). Sur l’activite teratogene chez le rat de l’actinomycine. C.R. Acad. Sci. 247, 2200-2203. VEST,M. (1959). Studien zur Entwicklung des Glucuronidbildungsvermiigens der leber beim Neugeboren; das Verhalten des Aminophenol-Glukuronids im Blut nach Verabreichung von Acetanilid. Schweiz. Med. Wochenshr. 89, 102-105. WARKANY,J. AND TAKACS,E. (1965). Congenital malformations in rats from streptonigrin. Arch, Pathol. 79,65-79.
WILSON,J. G. (1965). Embryological considerations in teratology. Ann. IV. Y. Acad. Sci. 123, 219-227. WINFIELD, J. B. (1966). Actinomycin D teratogenesis in the young mouse embryo. Amer. Zool. 6, 551 (Abstr.).
AND APPLIED
PHARMACOLOGY
27, 322-329
(1974)
Teratogenicity and Neonatal Toxicity of Clindamycin 2-Phosphate in Laboratory Animals J. A. BOLLERT, J. E. GRAY, J. D. HIGHSTRETE, J. MORAN, B. P. PURMALIS AND R. N. WEAVER Research Division, The Upjohn Company, Kulamazoo, Michigan 49001 Received April 10,1973; accepted June 18,1973
Teratogenicity and Neonatal Toxicity of Clindamycin-2-Phosphate in Laboratory Animals. BOLLERT, J. A., GRAY, J. E., HIGHSTRETE, J. D., MORAN, J., PURMALIS, B. P. and WEAVER, R. N. (1974). Toxicol. Appl. Pharmacol. 27, 322-329. Subcutaneous injections of clindamycin 2-phosphate at 100 and 180 mg/kg on gestation days 6 through 15 in Upjohn SpragueDawley rats, Upjohn ICR and CFl mice had no detremental effect on reproductive performance. Teratogenic effects were not observed. A concurrent low incidence of spontaneous cleft palate was confirmed in the Upjohn ICR strain of mouse. This condition was not eliminated by vitamin B supplementation. The SCLD50 of clindamycin-2-phosphate has been shown to be 179 mg/kg in the neonatal rat and ~2000 mg/kg in the adult rat. The newborn/adult toxicity ratio was calculated at 1 :11.2, which is comparable to potassium penicillin G. Subcutaneous injections of clindamycin-2-phosphate at 120 mg/kg in nearly mature and recently weaned Gunn rats did not alter the partially impaired bilirubin metabolism in this neonate model. Simultaneous treatment of immature Gunn rats with 120 mg/kg of clindamycin-2-phosphate and therapeutic levels of sulfadiazine and sulfamerazine did not produce sufficient displacement of bilirubin from albumin binding in the serum to produce clinical signs of kernicterus. Teratological studies in rats and mice, comparative LD50 determinations in newborn and adult rats and toxicopathologic studies in the Gunn rat with impaired bilirubin metabolism were conducted with clindamycin-2-phosphate as part of the safety evaluation program prior to clinical testing in man. Several antibiotics have been found to be teratogenic in various laboratory species. They include tetracycline (Filippi and Mela, 1957a), penicillin and streptomycin in combination (Filippi and Mela, 1957b), hadacidin (Chaube and Murphy 1963), actinomycin D (Tuchmann-Duplessis and MercierParot, 1958; Wilson, 1965; Winfield, 1966), mitomycin C (Nishimura, 1963), streptonigrin (Warkany and Takacs, 1965) and streptomycin (Ericson-Strandvik and Gyllensten 1963). These studies emphasize the need for critical teratogenic evaluation of antibiotics. The study of acute toxicity of drugs in neonates was prompted by the knowledge that newborn animals are deficient in the ability to detoxify certain drugs due to a combination of factors including immature metabolic systems, renal function and bloodCopyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
322
TOXICITY
OF CLINDAMYCIN
323
Z-PHOSPHATE
brain barrier. Neonate human and other animal species have been found to be particularly sensitive to chloramphenicol (Sutherland, 1959), morphine (Chester et al., 1942) progesterone (Karnofsky er al., 1952), compounds with vitamin K activity (Allison, 1955; Laurance, 1955; Crosse et al., 1955), sulfonamides (Silverman et al., 1956), sodium glutamate (Lucas and Newhouse, 1957) and novobiocin (Sutherland and Keller, 1961). The comparison of LD50 values in newborn and adult animals is one method of assessing the potential for excessive toxicity in a neonate (Michael and Sutherland, 1961). In addition, the Gunn rat, which exhibits a genetic deficiency of glucuronyltransferase, can be used as a model of the newborn. Human fetal liver does not have glucuronyltransferase activity, and newborn babies do not approach adult values of glucuronyltransferase activity until 2-3 weeks after birth (Brown and Zuelzer, 1958). This condition results in a deficiency in an important detoxification mechanism by which compounds are conjugated with glucuronic acid (Vest, 1959). Thus the Gunn rat would appear to be a likely “neonate” model for detecting potential toxicity of compounds which are metabolized in this manner. In addition, sulfonamides have produced kernicterus in premature human infants (Silverman et al., 1956) and Gunn rats (Johnson et al., 1959) by competing with bilirubin for binding sites on the serum albumin. This results in decreasing serum bilirubin concentrations and diffusion of the bilirubin into the tissues. Used also in this conjunction, the Gunn rat would appear to be useful in neonatal toxicology studies. METHODS TeratoZogy studies. Pregnant Upjohn Sprague-Dawley (TUC/SD) rats, Upjohn ICR (UPJ/ICR) and CFl mice were given clindamycin-2-phosphate by SCinjection on gestation days 6 through 15 (counting the day of finding sperm in the vagina as day 0) at 100 and 180 mg/kg of body weight/day. Control animals received equivalent volumes of sterile water vehicle. On gestation day 20 in the rat and 18 in the mouse, the dams were sacrificed by CO, asphyxiation and cervical dislocation, respectively. Laparotomy was performed, the fetuses were recovered and weighed and the sex was noted. The uterus was also examined for resorption sites. All fetuses were examined for gross external malformations. Then each litter was divided into 2 subgroups : one-third of each litter was fixed in Bouin’s solution and examined for visceral malformations by a modification of the Wilson (1965) method and two-thirds of each litter were fixed in 70% ethanol, cleared, stained with alizarin red S and examined for skeletal malformations. This protocol was executed 3 times in TUC/ICR mice and once in CFl mice and UPJjSD rats. In another experiment following the same protocol in TUC/IUR mice, daily SCinjections of Solu-B@’ containing 0.05 mg riboflavin, 0.05 mg thiamine hydrochloride, 0.025 mg pyridoxine hydrochloride, 0.25 mg calcium pantothenate and 1.25 mg niacinamide were administered concurrent with clindamycin-2-phosphate. Control mice received Solu-B and sterile water vehicle injections. This dose of vitamin B complex was selected because it approximated the required level of riboflavin in the diet of the mouse (Fenton and Cowgill, 1947). LD50 studies in neonatal and adult rats. Neonate Upjohn Sprague-Dawley (TUC/’ SD) rats were treated by SCinjection within 24 hr after birth. Ten pups of each litter were 1 Solu-Ba is a registered trademark of The Up,john Company, Kalamazoo,
Michigan.
324
BOLLERT ET AL.
injected and the remaining rats were used as controls. Survivors were kept with the dam for 1 week. Ten neonates were treated with the following doses: 100, 125, 160, 200, 250 and 320 mg/kg. Groups of 5 male adult rats of the same strain were treated by SC injection at 1600 and 2000 mg/kg and observed for 7 days. The Spearman-Karber method (Finney, 1952) was employed to calculate the LD50 values. Toxicopathologic studies in the Gum rat. This rat is congenitally deficient in bilirubin glucuronyltransferase in the homozygous state, jj. Homozygous animals used in these studies were bred from homozygous jj male x heterozygous Jj female matings in the colony maintained by our department. Three studies were conducted on the Gunn rat with SCinjections of clindamycin-2-phosphate at 120 mg/kg/day to determine effects on serum bilirubin concentrations. In study I rats, 77-109 days old, were paired genetically (homozygous with homozygous and heterozygous with heterozygous) and as closely as possible according to age. Injections were made for 9 days in 1 rat of each pair. In study II rats, 21-39 day old, were paired and injected as in study I for 8 days. In study III immature, homozygous rats were divided into 3 groups and treated for 9 days as follows: 5 controls (untreated), 8 treated po with Citrasulfas MB2 (2.0 ml/kg; each fluid ounce containing 20 grains sulfadiazine, 20 grains sulfamerazine and 93 grains sodium citrate), and 8 treated po with Citrasulfas M and SCwith clindamycin-2-phosphate. Both compounds were dissolved in sterile water. The genetic characteristic and number of rats used was : Study I II III
Homozygous (jj) 14 9
21
Determinations of total bilirubin (Malloy at the termination of each study. Complete liver, spleen, pancreas, stomach, duodenum, bular salivary gland, heart, thyroid, lung, brain and pituitary were carried out.
Heterozygous (Jj) 14 -
9
and Evelyn, 1937) were made prior to and necropsies and histologic examinations of jejunum, mesenteric lymph node, mandithymus, kidney, adrenal, urocyst, testis,
RESULTS Teratology Studies
Subcutaneous injections of clindamycin-2-phosphate at 100 and 180 mg/kg/day on gestation days 6 through 15 in the TUCjSD rat had no detrimental effect on the reproductive parameters studied (Table 1). There was no indication of teratogenic activity in 112, 118 and 108 fetuses examined for gross, visceral and skeletal malformations at the 0, 100 and 180 mg/kg dose levels, respectively. In TUC/ICR mice SCinjections of clindamycin-2-phosphate at 100 and 180 mg/kg/ day on gestation days 6 through 15 did not adversely affect the reproductive parameters studied (Table 2). The study was conducted 3 times in order to clarify the presence of a low incidence of cleft palate at both the 100 and 180 mg/kg dose levels in study A. None of the control litters contained a fetus with cleft palate in study A; however, in studies B ’ Citrasulfas M@ is a registered trademark of The Upjohn Company, Kalamazoo,
Michigan.
TOXICITY
OF CLINDAMYCIN
TABLE
325
Z-PHOSPHATE
1
SUMMARY 0~ REPRODUCTION DATA FROM CONTROL AND CLINDAMYCIN-~-PHOSPHATETREATED PREGNANT TUC/SD RATS
No. of pregnant females
Dose (w/kg)
Avg. litter
weight (d
Avg. live pup weight cd
Avg. No. live pups/ litter
Avg. No. dead pups/ litter
Avg. No. resorptions/ litter
9 10 10
0 100 180
48.8 45.4 41.8
3.9 3.9 3.9
12.4 11.8 10.8
0.0 0.0 0.0
0.9 0.7 0.5
TABLE 2 SUMMARY 0~ REPRODUCTION DATA FROM CONTROL AND CLIN~~~M~~IN-~-PH~SPHATETREATED PREGNANT TUC/ICR MICE
Study
No. pregnant females
Avg. litter weight (d
A B C
10 8 9
13.4 8.5 11.9
A B C
8 8 10
14.1
A
10 6 10
B C
Avg. live pup weight (g)
Avg. No. live pups/ litter
Avg. No. dead pups/ litter
Avg. No. resorptions/ litter
Control 1.2 0.8 1.2
9.4 10.9 10.0
1.4 0.4 0.0
1.8 0.8 1.0
11.1 13.7
100 mg/kg 1.3 1.0 1.4
8.4 11.9 10.1
2.5 0.4 0.0
0.5 1.8 0.9
15.6 11.6 15.1
180 mg/kg 1.4 1.1 1.4
9.8 11.2 10.6
2.3 0.3 0.0
0.7 1.0 0.6
and C a similar low incidence of cleft palate occurred in the controls. Comparison of the incidences of litters containing fetuses with cleft palates (Table 3) highlights the inconsistencies in the number of litters affected in studies A, B and C. There was no significant difference in the percentages of litters with cleft-palate fetuses for control and clindamycin-2-phosphate-treated litters (Table 4). In a further attempt to characterize any teratogenic activity of clindamycin 2-phosphate the same protocol was run using the CFl strain of mouse. Reproductive parameters were not affected by treatment (Table 5). No evidence of teratogenic activity was indicated by gross, visceral and skeletal examinations of the 116,78 and 92 fetuses at the 0, 100 and 180 mg/kg dose levels, respectively. In a final study with TUC/ICR mice a vitamin B complex supplementation (Solu-B) was administered daily by SCinjection concurrently with clindamycin 2-phosphate under the protocol previously described. This was done to determine whether the sporadic cases of cleft palate might be related to vitamin B deficiency following antibiotic treatment (Filippi, 1967). Reproductive parameters in mice treated with clindamycin 2phosphate and vitamin B complex were not different fromcontrol micereceiving vitamin
326
BOLLERT ETAL. TABLE
3
INCIDENCE OF LITTERS WITH FETUSES EXHIBITING CLEFT PALATE IN CONTROL AND CLINDAMYCIN Z-PHOSPHATE-TREATED TUC/ICR MICE Study
Dose (mg/kg)
A
B
C
0
o/10 218
l/S 218 l/6
l/9 l/10 o/10
100 180
2110 TABLE
4
COMPARISON OF LITTERS WITH AND WITHOUT FETUSES EXHIBITING CLEFTPALATES (CPF) IN CONTROL AND CLINDAMYCINZPHOSPHATETREATED TUC/ICR MICE(~OMBINED STUDIES A,B, AND C)
Dose (mg/kg)
No. litters with CPF
No. litters without CPF
Total No. litters
% Litters with CPF
9 loo 180
2 5 3
25 21 23
27 26 26
7.4 19.2 11.5”
0 Chi Square= 1.9 (2 df). No significant difference between groups (p = 0.38).
B complex and sterile water injections (Table 6). Gross, visceral and skeletal examinations of the fetuses revealed 1 fetus with cleft palate in the control group and 1 at the 180-mg/kg dose level. This incidence of cleft palate is similar to those seen in the above reported TUC/ICR studies without vitamin B supplementation.
L D50 studies in Neonatal and Adult Rats. The SCLD50 value for clindamycin 2-phosphate in the neonatal rat was 179 mg/kg. In the adult rat no deaths occurred at either 1600 or 2000 mg/kg; therefore, the LD50 value is ~2000 mg/kg. Treatment at doses greater than 2000 mg/kg was prevented by the insolubility of the aqueous preparation. The newborn/adult toxicity ratio was calculated to be 1 :11.2. TABLE
5
SUMMARYOF REPRODUCTION DATAFROM CONTROL AND CLINDAMYCIN Z-PHOSPHATETREATED PREGNANT CFl MICE
No. pregnant females 12 9 10
Dose bdkd 0 100 180
Avg. litter weight Cd 13.1 11.6 12.7
Avg. live
Avg. No.
Avg. No.
Avg. No.
pup weight k>
live pups/ litter
dead pups/ litter
resorptionsl litter
1.4 1.4 1.5
9.7 8.7 9.0
0.0 0.0 0.2
0.8 0.8 0.9
TOXICITY
OF CLINDAMYCIN
327
Z-PHOSPHATE
TABLE 6 SUMMARY OF REPRODUCTION DATA FROM CLINDAMYCIN ~-PHOSPHATE- AND VITAMIN B-TREATED TUC/ICR MICE
No. pregnant females 8 8 8
Dose” bm/kg)
Avg. litter weight (EC>
Avg. live pup weight (d
Avg. No. live pups/ litter
Avg. No. dead pups/ litter
Avg. No. resorptions/ litter
0 100
13.5
14.0
1.3
10.4
11.4 9.0
0.3 0.4
0.9
1.3
180
12.0
1.4
1.1
0.4 1.3
a Dose refers to clindamycin 2-phosphate.All micereceivedvitamin B complex during doseperiod. SeeMethods section. Toxicopathologic
Studies in the Gum Rat.
The SCadministration of clindamycin 2-phosphate at 120 mg/kg to 77-109-day-old rats (study I) and recently weaned (study II) Gunn rats did not alter the partially impaired metabolism of bilirubin in these rats. No significant changes in serum bilirubin concentrations or morphologic evidence of increased deposition of bile pigment and destruction of brain tissue was noted. Simultaneous treatment of homozygous rats with sulfa drugs (Citrasulfas M) and clindamycin 2-phosphate (study III) did not afford sufficient displacement of bilirubin from albumin binding in the serum to produce clinical manifestations. DISCUSSION The sporadic occurrence of cleft palate seen in the teratology studies with the TUC/ ICR mice illustrates an important necessity for teratology testing procedures, namely, adequate information on the incidence of the various spontaneous malformations which characterize any particular strain of laboratory animal. Several factors indicate that the low incidence of cleft palate found in the TUC/ICR mice was spontaneous or the result of some as yet undetermined environmental alteration other than treatment with clindamycin 2-phosphate. They are: (1) the inconsistencies in the occurrence of cleft palate in successive studies with clindamycin 2-phosphate-treated litters; (2) the occurrence of cleft palate in all control groups except study A; (3) the failure of vitamin B administration to prevent the occurrence of cleft palate when one established mechanism for antibiotic induction of cleft palate is vitamin B deficiency (Filippi, 1967) ; (4) the absence of any cleft palate induction in another strain of mouse (CFI); and (5) subsequent studies with another compound in the TUC/ICR mouse also revealed similar incidences of cleft palate in control and treated fetuses (Bollert, unpublished observations). The newborn/adult toxicity ratio of 1 :l 1.2 for clindamycin 2-phosphate is comparable to potassium penicillin G at I : 12 and less than chloramphenicol at 1:16 (Raynsford, 1963). Regardless of these ratios, particular caution and careful consideration of dose levels should be exercised in treating neonatal humans because of their potential increased susceptibility to any drug.
328
BOLLERT
ET AL.
The relevance of the Gunn rat in toxicology screening as a neonate model in glucuronidation deficiency is predicated, in part, upon the assumption that the drug being tested is conjugated with glucuronic acid and that this mechanism represents an important detoxification pathway in this animal and in man. The current studies indicating no toxic manifestations of clindamycin 2-phosphate in the Gunn rat were initiated before we were aware that Sun (1970) had found glucuronide not to be a metabolite of clindamycin in the Sprague-Dawley rat. The displacement of bilirubin from albumin binding in the serum resulting in kernicterus would not appear to be a potential problem following clindamycin 2-phosphate treatment. This finding appears to be compatible with the results of W. Sokolski (unpublished observations) that clindamycin base to which the phosphate is hydrolyzed in vivo binds to a glycoprotein, not albumin in the serum.
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