OXYTETRACYCLINE
1. EXPLANATION
Oxytetracycline (OTC) was evaluated at the twelfth meeting of the
Joint FAO/WHO Expert Committee (Annex 1, Reference 17), at which a
temporary ADI of 0-0.15 mg/kg b.w. was established.
Since that time additional data have become available; they are
summarized and discussed in the following monograph. The previously-
published monograph has been expanded and is incorporated into this
monograph.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution and excretion
2.1.1.1 Mice
After oral administration of 47.6 mg 14C-labelled OTC-HCl/kg b.w.
to mice, 72% of the applied dose was found in the large intestine
after 2 hours; only 5% was absorbed, of which the major portion (3.6%)
was excreted in the urine. In the liver 1.9% and 1.1% of the dose
applied was recovered after 1 and 2 hours, respectively (Snell et
al., 1957).
2.1.1.2 Dogs
Dogs received 10, 50 or 100 mg OTC/kg b.w. as a single oral dose
or 2 oral doses 12 hours apart of 10 or 50 mg OTC/kg b.w. OTC
concentrations in plasma were determined by a fluorometric method. A
single administration resulted in peak blood levels 2 hours after
dosing of 0.88, 1.01 and 2.51 µg/ml, respectively. These levels
dropped to about 60% after 12 hours. Slightly higher blood levels
were attained after administration of a second dose (Immelman, 1977).
2.1.1.3 Pigs
Six and 4 pigs received 20 mg/kg b.w. i.m. of OTC as long-acting
(OTC-LA) formulation or as conventional formulation (OTC-C),
respectively. Blood and urine samples were taken and OTC
concentrations were determined spectrofluorometrically. The levels of
sensitivity were 0.1 µg/ml (plasma) and 0.2 µg/ml (urine). OTC-C was
distributed slowly. The maximum plasma concentration (609 µg/ml) was
obtained about 4 hours after dosing. About 60% of the administered
dose was excreted in the urine during the first 24 hours and a total
of 69% was recovered in the urine within 1 week.
After injection with OTC-LA the initial absorption rate was
faster and the maximum plasma concentration was reached within 1 hour
after dosing. Although the excretion rate was lower with OTC-LA than
after administration of OTC-C, the total amounts excreted in urine
were comparable. In 3 days 60-75% of the total dose was excreted in
the urine (Xia et al., 1983).
Oral administration of 50 mg OTC-HCl/kg b.w. to 21 Yorkshire
swine produced detectable OTC residues in the kidney (highest
amounts), liver, lung, adrenal, heart, bile, fat, lymph node, spleen,
thyroid and urine. The highest residue levels (441 µg/ml) were
observed in the urine 3 hours after dosing and were still detectable
at 48 hours. Mean peak plasma concentrations of 6.3 (range 4.2-8.7)
were observed after 3 hours (Black & Gentry, 1984).
Weaned piglets were given a single oral dose of 20 mg OTC/kg b.w.
as a drench or were given a diet with 400 mg OTC/kg feed during 3
consecutive days. The drench route of administration revealed a
maximum plasma concentration 6x higher than that of the feed route
(1.27 versus 0.2 µg/ml). A peak plasma concentration was reached
after 3 ± 2 hours by the drench route, while the feed route revealed
a steady state concentration of 0.2 µg/ml beyond 30 hours after the
onset of administration until administration stopped. Within 48 hours
after cessation plasma OTC levels were below the detection limit (0.06
µg/ml). Estimated OTC bioavailabilities were low: 9.0% and 3.7%
after the drench and the feed route, respectively (Mevius, et al.,
1986a).
After i.v. administration of 20 mg OTC/kg b.w., OTC was well
distributed in the body (distribution volume 1.62 ± 0.83 l/kg). The
elimination half-life ranged between 11.6 and 17.2 hours and the mean
overall body clearance was estimated to be 0.249 l/kg/hour. Urinary
recovery of OTC within 72 hours post injection ranged between 42 and
62% of the administered dose (Mevius, et al., 1986a).
2.1.1.4 Cattle
Three groups of calves (3, 12, or 14 weeks old) received i.v.
doses of 7.54, 6.88 or 17.00 mg OTC/kg b.w., respectively, and 2
groups of cows (lactating and non-lactating) received 3.32 and 7.94 mg
OTC kg b.w., respectively. Blood samples were collected for
determination of OTC concentrations by the agar-plate diffusion method
(detection limit not reported). Distribution volume in 3-week old
calves was 2.48 1/kg which was 2 to 3 times higher than in the cows.
Half-life was 13.5 ± 3.6 hours and 8.8 ± 0.52 for the 3- and 12-week
old calves, respectively. The dose and state of lactation did not
affect the distribution volume or the half-life time in cows (Nouws
et al., 1983).
Dairy cows were injected i.v. and i.m. with 3 different 10% OTC-
formulations (dose rates about 5 mg/kg b.w.). Serial blood and urine
samples were collected. Distribution volume was 1.00 ± 0.18 1/kg and
did not differ for the various formulations. Peak plasma
concentrations of 2.28 ± 0.15 µg/ml were reached at 7 hours after i.m.
administration. Plasma half life was 9.02 ± 0.88 hours. Most of the
OTC was excreted by the kidney (85-86%) and a very small portion (2%)
via the bile (Nouws et al., 1985).
Five dairy cows were treated with single i.m. injections of 5
different 20% OTC formulations at a dose rate of 10 mg/kg b.w. OTC
concentrations in plasma and the renal clearance of OTC and creatinine
were determined (sensitivity of determination by microbiological
assay: 0.05 mg/l). Maximum plasma concentrations were achieved 5 to
10 hours after treatment and varied between 4.6 and 6.8 µg/ml
depending on the formulation involved. Plasma concentrations
exceeding 0.5 µg/ml were maintained for 48 to 72 hours depending on
the formulation involved. Mean renal clearance was 0.062 l/kg/hour.
The urinary recovery of OTC within 72 hours after treatment ranged
between 61.7 and 88% of the dose administered (Mevius et al.,
1986b).
Newborn calves (aged from 1 to 42 days) and older calves (250
days) were administered OTC at an i.v. dose rate of 10 mg/kg b.w. on
day 2 and at weeks 1, 2, 4 and 6 of the study. Blood samples were
collected for determination of OTC concentrations (detection limit not
reported). The elimination of OTC was significantly slower in newborn
calves. The half-life of elimination decreased from 11.2 ± 1.7 hours
in newborn calves to 6.4 ± 1.3 hour at 6 weeks of age, to 6.3 ± 0.7
hours in the 250-day old calves (Burrows et al., 1987).
Five Jersey cows received single i.m. doses of 5 mg OTC/kg b.w.
Peak concentrations in plasma (1.67 ± 0.66 µg/ml) and milk (1.38 ±
0.46 µg/ml) were attained after 6 and 12 hours, respectively. The
elimination half-life was 7.99 ± 2.20 hours (Prasad et al., 1987).
2.1.1.5 Humans
OTC is incompletely absorbed from the gastrointestinal tract of
humans. After oral administration about 60% of the ingested dose is
absorbed (Fabre et al., 1971). After a single oral dose to
humans, peak plasma concentrations are attained within 2 to 4 hours
and within 2.5 hours after repeated dosing (Sande & Mandell, 1985).
In humans given 7 daily oral doses of 500 mg OTC the volume of
distribution appeared to be 4.07 1/kg (Green et al., 1979).
Absorption of oxytetracycline is impaired by milk products, aluminum
hydroxide gels, sodium bicarbonate, calcium and magnesium salts and
iron preparations due to chelation and an increase in gastric pH
(Sande & Mandell, 1985).
2.1.2 Effects on enzymes and other biochemical parameters
2.1.2.1 Rats
In three trials, groups of 6 male Sprague-Dawley rats were
treated daily for 14 days with i.p. injections of 0, 20, 40 and 100 mg
OTC/kg b.w. in a sterile physiological saline solution. In the first
trial, rats were killed after 2, 4, 6, 8, 10 and 14 days of treatment.
In trials 2 and 3, rats were killed after 1, 2, 4, 6, 8, 10 and 14
days of treatment. At 100 mg/kg b.w. weight gain was significantly
reduced and rats showed a decreased activity in microsomal O
dealkylation and in epoxidation. Gross pathology revealed enlarged
pale kidneys and at histopathology focal interstitial nephritis with
mixed infiltration of neutrophils and lymphocytes was observed (Tarara
et al., 1976).
2.1.3 Interactions with bones and teeth
2.1.3.1 Rats
Fifteen Hebrew University Sabra rats (15 days old) received 6
consecutive injections of 100 mg OTC-HCl/kg b.w. every 12 hours during
a period of 72 hours. Five rats served as controls. Rats were
sacrificed 4 hours after the last injection and tibial bones were
removed and epiphyseal plates were examined by either transmission or
scanning microscopy to establish the influence of OTC-HCl on matrix
vesicle production and initial calcification of epiphyseal cartilage.
Degeneration of chondrocytes in the proliferating and hypertrophic
zones was observed. Chondrocytes had short processes with only few
matrix vesicles covering their surface. There were fewer matrix
vesicles in the hypertrophic and calcifying cartilage as compared to
controls and their ability to aggregate and form mineralized
calcospherites was impaired. In ashed bones, minerals containing
calcospherites were hardly seen (Levy et al., 1980).
2.2 Toxicological studies
2.2.1 Acute toxicity studies
Table 1 summarizes the results of acute toxicity studies with
OTC.
2.2.2 Short-term studies
2.2.2.1 Mice
In a range finding study groups of B6C3F1 mice (10/sex/group)
were fed diets containing 0, 3100, 6300, 12500, 25000 or 50000 ppm
OTC-HCl for 13 weeks. These dose levels are equal to an intake of
392, 741, 1845, 3821 or 8300 mg/kg of body weight for males and 459,
845, 1850, 3860 or 7990 mg/kg body weight for females. No dose
related effects were observed on mortality, food consumption,
macroscopy and histology. Body weights were decreased from 3 to 15%
at 25000 ppm and at 50000 ppm. OTC concentrations in bone were
measurable fluorometrically in high-dosed females (NTP, 1987).
Table 1: Acute toxicity of Oxytetracycline
Species Sex Route Chemical LD50 Reference
form (mg pure OTC/
kg b.w.)
Mouse M&F oral pure >5200 P'An et al., 1950
M&F oral OTC-HCl 7200 P'An et al., 1950
M&F oral OTC-HCl 3600-4400 Bacharach et al., 1959
M&F i.p. OTC-HCl 285-420 Bacharach et al., 1959
M&F i.v. OTC-HCl 192 P'An et al., 1950
M&F oral OTC-HCl 154-189 Bacharach et al., 1959
M&F s.c. pure >3500 P'An et al., 1950
M&F s.c. OTC-HCl 892 P'An et al., 1950
M&F s.c. OTC-HCl 243-330 Bacharach et al., 1959
Rat M&F i.v. OTC-HCl 280 P'An et al., 1950
Rabbit M&F i.v. OTC-HCl 75-112 P'An et al., 1950
Dog M&F i.v. OTC-HCl 150 P'An et al., 1950
2.2.2.2 Rats
In a range finding study, groups of F344/N rats (10/sex/group)
were fed diets containing 0, 3100, 6300, 12500, 25000 or 50000 ppm
OTC-HCl for 13 weeks. These dose levels were equal to intakes of 198,
394, 778, 1576 or 3352 mg/kg of body weight for males and 210, 431,
854, 1780 or 3494 mg/kg of body weight for females. No dose related
effects were observed on mortality, food consumption, body weight or
macroscopy. Minimal periacinar fatty metamorphosis in the liver of
male rats was observed at all dose levels (no dose relation, control
values not given). Measurable OTC concentrations in bones were
detected in both sexes and increased with the dose. The OTC
concentration in bone was significantly increased in females from
12500 ppm and up and in males at 50000 ppm only (NTP, 1987).
2.2.2.3 Dogs
Groups of mongrel dogs (2/sex/group) were fed diets containing 0,
5000 or 10000 ppm OTC-HCl for 12 months. Observations included
clinical signs, mortality, body weight, food consumption, haematology,
organ weights, macroscopy and histopathology. No dose related effects
were observed except for a degenerating germinal epithelium in the
testicular tubules in high-dosed male dogs. The NOAEL in this study
was 5000 ppm in the diet, equivalent to 125 mg/kg b.w.
Groups of 8 male dogs, four beagle dogs and four mongrel dogs per
group, were fed diets containing 0, 1000, 3000 or 10000 ppm OTC-HCl
for 24 months. An interim sacrifice of 1 beagle and 1 mongrel
dog/group was performed after 12 months. Observations included
clinical signs, mortality, body weight, food consumption, haematology,
alkaline phosphatase (ALP), bromosulphophthalein (BSP) clearance, urea
nitrogen determinations, organ weight macroscopy, histopathology and
semen examination. Two dogs died after 12 and 24 months, respectively
(1 because of filaria and 1 because of gastroenteritis). No dose-
related effects were observed. Atrophy of testes and epididymus
occurred more frequently in control dogs than in treated ones. The
NOAEL was 10000 ppm in the diet (the highest dose tested), equivalent
to 250 mg/kg b.w. (Deichmann et al., 1964).
2.2.3 Long-term/carcinogenicity studies
2.2.3.1 Mice
Groups of B6C3F1 mice (50/sex/group) were fed diets containing 0,
6300 or 12500 ppm OTC-HCl (purity 98.8%) for 103 weeks. Observations
included clinical signs, mortality, body weight, food consumption,
macroscopy and histopathology. Mean body weights of high dosed mice
were 5-9% lower than those in the control group only after the first
half year of the study. The tumour incidence was not significantly
increased in either sex. The NOAEL in this study was 12500 ppm in the
diet (the highest dose tested), equal to 1372 mg/kg b.w. (NTP, 1987).
2.2.3.2 Rats
Groups of Osborne-Mendel male rats were fed diets containing 0
(180 rats), 100 (100 rats), 1000 (130 rats) or 3000 ppm (100 rats)
OTC-HCl for 24 months. Observations included clinical signs,
mortality, food consumption, body weight, haematology, macroscopy, and
histopathology.
After 24 months the mortality rates were 43, 23, 23 and 13% for
the control and experimental groups, respectively. Treated rats
gained weight more rapidly than controls. Body weight and haematology
were not affected. At macroscopy pale kidneys were observed in 4, 7,
16 and 16% in the control and treated groups, respectively. A slight
to moderate brownish pigmentation of the thyroid gland was seen in
treated rats, but it was not dose-related. Tumour incidences were not
enhanced. The NOAEL in this study was 3000 ppm (highest dose tested),
equivalent to 150 mg/kg b.w. (Diechmann et al., 1964).
Groups of F344/N rats (50/sex/group) were fed diets containing 0,
25000, or 50000 ppm OTC-HCl (purity 98.8%) for 103 weeks.
Observations included clinical signs, mortality, body weight, food
consumption, macroscopy and histopathology. Mean male body weights
were 5-8% lower during the first year of the study at 50000 ppm.
Histological examination showed a dose related increase in the
incidence of benign phaeochromocytomas in the adrenal gland of male
rats. In females an increase in the incidence of adenomas of the
pituitary gland was found in the highest dose group (see Tables 2 and
3, respectively) (NTP, 1987).
Table 2: Adrenal gland lesions in male rats
Oxytetracycline in the diet
0 ppm 25000 ppm 50000 ppm
Adrenal medullary 7/50 14/50 9/50
hyperplasia
Phaeochromocytoma 10/50 18/50 25/50
(benign)
Phaeochromocytoma 2/50 1/50 0/50
(malignant)
Table 3: Pituitary gland lesions in female rats
Oxytetracycline in the diet
0 ppm 25000 ppm 50000 ppm
Hyperplasia 16/50 10/50 11/50
Adenoma 19/50 17/50 30/50
Adenocarcinoma 2/50 7/50 3/50
2.2.4 Reproduction studies
2.2.4.1 Rats
Groups of 30 female and 10 male Wistar rats were fed diets
containing 0 or 360 ppm OTC-HCl beginning at weaning (23 days of age).
Animals were first mated at 120 days of age. A second mating was
performed one month after the weaning of the first litter. One male
and one female of these second litters were mated and effects on
reproduction and lactation were determined in the second generation.
Growth rate was not significantly affected. Reproductive parameters
such as litter size, litter and pup weight, and the number and percent
of live or dead fetuses did not show significant differences in the
first and second generations. Dosed pups of both generations gained
significantly more weight from days 3-21 post partum compared to
controls. The NOAEL in this study was 360 ppm OTC-HCl, equivalent to
18 mg/kg b.w. (Uram, et al., 1954)
2.2.4.2 Cattle
Nine beef bulls were treated with OTC-HCl by a single dose of
26.4 mg/kg b.w. administered subcutaneously followed by 6 doses of
17.6 mg/kg b.w. each (12 hours between the doses). Another 9 bulls
were kept as controls. Semen was collected by electroejaculation twice
daily beginning on day 3 and then every 4 days. None of the dosed
bulls obtained palpable penile engorgement or protrusion during
electroejaculation on day 3. However, there were no adverse effects
on spermatogenesis, seminal pH, ejaculate volume, percentage of motile
spermatozoa, rate of spermatozoal motility or concentration of
spermatozoa in ejaculates harvested on days 3 or 7 (Abbitt et al.,
1984).
2.2.5 Special studies on cardiovascular effects
2.2.5.1 Rabbits
Six anaesthetized male rabbits were injected i.v. with 2 or 5 OTC
mg/kg b.w. dissolved in 0.5 ml saline. The injections were performed
in 3, 10, 20 or 60 seconds. One to 3 minutes after rapid injection,
slowing of the heart rate was observed (normal between 270 and 310 per
min.) to 100 per min. or less. This effect increased with higher
doses and shorter injection time. No effect on arterial blood
pressure was found. A depressive effect on respiration was seen which
led, at high doses, to respiratory arrest of up to 60 seconds and
otherwise to a slow, shallow respiration for 1-2 minutes (Gyrd-Hansen,
1980).
2.2.6 Special studies on embryotoxicity and/or teratogenicity
2.2.6.1 Mice
Pregnant CD-1 mice (42/group) were orally dosed at 0, 1325, 1670
or 2100 mg OTC-HCl in corn oil/kg b.w. from days 6-15 of gestation.
On day 17 all animals were sacrificed. Mortalities were 0/42, 1/42,
3/41 and 3/39, respectively. Gravid uterine weight and maternal
absolute liver weight were significantly reduced at 2100 mg/kg b.w.
There were no significant differences among treated and control groups
with respect to maternal body weight, number of implantations,
resorptions, dead and live fetuses, fetus weight and gross external,
visceral and skeletal abnormalities. The NOAEL for maternal toxicity
was 1670 mg/kg b.w. (Morrissey et al., 1986).
2.2.6.2 Rats
Groups of pregnant Sprague-Dawley rats received a diet containing
0, 250, 1000 or 2000 ppm OTC and all rats were given intragastrically
1.0 ml of a solution containing 0.7 µCi calcium-45 and 20 µg calcium
twice daily from days 1 to 20 of gestation. Fetuses were delivered by
Caesarean section on day 21 and weighed. Maternal femurs and 3
individual fetuses of each litter were incinerated and analyzed for
radiocalcium. No compound-related effects were observed on food
consumption, body weight, number of fetuses/litter and mean fetal
weight. All fetuses were viable at delivery and showed no external
developmental anomalies. Maternal as well as fetal radiocalcium
uptake in the bones increased in a dose-related manner with the OTC
dose in the diet (Likins & Pakis, 1965).
In a limited study 17 pregnant rats received daily i.m.
injections of 41.5 mg OTC/kg b.w. from days 7 to 18 of gestation. No
effects were observed on the number of implantations, the number of
live and normal fetuses, the number and percentage of resorptions or
fetal body weight; no macroscopic malformations were observed (Savini
et al., 1968).
Pregnant Wistar rats were orally dosed with 48, 240 or 480 mg
OTC/kg b.w. from days 1 to 21 of gestation. On day 21 all rats were
sacrificed. Fetuses were removed and skeletal anomalies were examined
by staining their bones with alizarin red. Compared to the control
group, ossification in the anterior extremities of fetuses was reduced
and an increase in fetal resorptions was observed in all dose groups.
The disturbances were more frequently observed in rats at the highest
dose (Szumigowska-Szrajber & Jeske, 1970).
Pregnant CD rats (36/group) were orally dosed with 0, 1200, 1350,
or 1500 mg OTC-HCl in corn oil/kg b.w. by gavage from days 6-15 of
gestation. On day 20 all animals were sacrificed. A dose-related
increase in mortality rate was observed (0, 5.6, 15.2 and 24.2% for
groups treated with 0, 1200, 1350 or 1500 mg/kg b.w., respectively).
Clinical signs such as respiratory difficulties and rough coat
occurred with increased incidence in treated dams. Maternal body
weight gain and maternal absolute liver weight and fetal body weight
were significantly reduced in all treated groups. No teratogenic
effects were observed (Morrissey et al., 1986).
2.2.6.3 Rabbits
In a limited study 12 pregnant rabbits received daily i.m.
injections with 41.5 mg OTC/kg b.w. from days 10 to 28 of gestation.
The number and percentage of partial and total resorptions were
significantly increased compared to the controls (54% to 25%,
respectively). No effects on fetal body weight were observed. No
abnormalities were found at macroscopy (Savini et al., 1968).
2.2.6.4 Dogs
In a limited study, 10 pregnant dogs of unspecified origin,
received daily i.m. injections of 20.75 mg OTC/kg b.w. from days 18 to
48 of gestation. Laparotomy was performed on day 18 and hysterectomy
was performed on day 58. Eight dogs served as controls (4 of these
delivered spontaneously, the other 4 dogs were operated in the same
way as the experimental animals). No resorptions or abnormalities
were observed in 8/8 control dogs or in 1/10 treated dogs. The number
of resorptions in treated dogs was high (9/10 dogs); from a total of
69 observed implantations 30 were resorbed. Malformations were found
in 5/10 treated dogs; 12/39 treated pups showed skeletal
malformations; displacia of the hind paws (5), angled tail (4),
monstrum with general oedema and cranio-faciale anomaly (2),
omphalocele (1) and 1 macerated fetus were observed. Visceral
malformations included shortened digestive tract, enlarged stomach
with thin walls and dilated and shortened intestinal tract (1 dog),
polycystic kidneys (1 dog) and absence of pulmonary development (2
dogs) (Savini et al., 1968).
2.2.7 Special studies on genotoxicity
Table 4 summarizes the results of genotoxicity studies that have
been performed on OTC.
2.2.8 Special studies on the effect of combined treatment of OTC and
nitrite
Groups of 15 male and 15 female Sprague-Dawley rats received 0.1%
OTC or 0.1% OTC + 0.1% sodium nitrite in the drinking water for 60
weeks. Surviving animals were killed at 130 weeks. No differences
were observed in the mortality rates of the two groups. No liver
tumours were observed in the OTC group. In the group receiving
combined treatment, 3 hepatocellular tumours and 1 hepatic cholangioma
were found (Taylor & Lijinsky, 1975).
Table 4: Results of genotoxicity assays on oxytetracycline-HCl
Test system Test Object Concentration Results Reference
In vitro
Ames test S. typhimurium 1 µg/pl in DMSO negative Andrews et. al.,
TA1535, TA1537, (+/- act) 1980
TA1538, TA98 and TA100
Ames test S. typhimurium 0-1.0 µg pl in DMSO negative1 NTP, 1987
TA100, TA1535, (+/- act)
TA1537, and TA98
Mouse lymphoma forward L5178Y/TK+/- 12.5-800 µg/ml toxic negative NTP, 1987
mutation assay cells >400 µg/ml2 25-400 (- act)
µg/ml toxic >200 positive
µg/ml3 (+ act)
Chromosome aberration Chinese hamster 80-200 µg/ml4 negative NTP, 1987
assay ovary cells (- act)
700-900 µg/ml5 negative
(+ act)
Sister chromatid Chinese hamster 60, 70 and 80 µg/ml4 negative NTP, 1987
exchange assay ovary cells (- act)
400, 500 and 700 negative
µg/ml5 (+ act)
In vivo
Micronucleus assay Mouse 2 times 50, 250 and positive6 Blitek et. al.,
500 mg/kg b.w., 1983
24 hours apart
Host mediated assay Mouse S. typhimurium 100 mg/kg b.w. negative7 Blitek et. al.,
G46 1983
Table 4 (continued)
1 Both with and without rat and hamster liver S9 fraction.
2 Ethylmethanesulfonate was used as a positive control.
3 Methylcholanthrene was used as a positive control.
4 Mitomycin C was used as a positive control.
5 Cyclophosphamide was used as a positive control.
6 There was no dose relationship, increases in micronuclei were 5.5, 46.2 and 5.6 times, respectively.
DMNA was used as a positive control.
7 DMNA was used as a positive control.
The nitrosation product of OTC and sodium nitrite was tested for
mutagenicity in the Ames Salmonella assay. Positive results were
obtained in Salmonella typhimurium strains TA1537, TA1538, TA98
and A100 with and without metabolic activation (Andrews et al.,
1980).
OTC simultaneously administered with potassium nitrite (150 mg/kg
b.w.) was positive in a micronucleus assay and in a host mediated
assay with mouse/S. typhimurium G46 (Blitek et al., 1983).
2.2.9 Special studies on microbiological effects
2.2.9.1 Mice
Three clones of E. coli K-12 strains were introduced into germ-
free mice. The organisms were allowed to multiply and establish a
stable population. OTC was then administered through the drinking
water and it was shown that the susceptible strains remained dominant
in number throughout. The minimal selecting dose of OTC in this mouse
model was 8 to 12 µg/ml. These OTC-concentrations were higher than
the MIC of the susceptible strain used (MIC=0.5 µg/ml) (Corpet &
Lumeau, 1987).
In vitro findings with the same clones and OTC showed a minimal
selecting dose of OTC of 1/10 of the MIC, 0.05 µg/ml (Lebek & Egger,
1983).
2.2.9.2 Rats
Mature albino rats (3 controls, 9 treated) were fed a diet
containing 0 or 10 mg OTC/kg diet for 6 weeks. After that period the
OTC concentration was raised to 50 mg/kg diet and administration was
continued for 2 weeks. No evidence was found for the development of
OTC-resistant organisms in the faeces of treated rats (Rollins et
al., 1975).
2.2.9.3 Dogs
Mature beagles (5/group) were fed a diet containing 0, 2 or 10 mg
OTC/kg diet for 44 days. Faecal samples from individual animals were
collected during the experimental period and examined for resistant
coliforms by a comparative plate counting technique. A shift to drug
resistant organisms was observed at 10 mg OTC/kg diet. No effect was
found at 2 mg/kg diet, equivalent to 50 µg/kg b.w. (Rollins et
al., 1975).
2.2.9.4 Turkeys
Groups of Nicholas strain male poults received 0, 50, or 100 mg
OTC/kg feed for 18 weeks. The turkeys were sacrificed after 8, 16 or
18 weeks and bacteria were isolated from blood and liver tissue. The
isolates were tested for resistance against 8 antibiotics. The
antibiotic resistance increased with increasing OTC levels (Swezey
et al., 1981).
2.3 Observations in humans
2.3.1 Effects after medical treatment
A variety of toxic and irritative effects in humans have been
reported from the use of OTC. OTC may cause gastrointestinal
irritation. Epigastric burning and distress, abdominal discomfort,
nausea, vomiting, and diarrhea may occur. I.V. administration may
produce thrombophlebitis. Long-term therapy may produce changes in
the peripheral blood. Leukocytosis, atypical lymphocytes, toxic
granulation of granulocytes and thrombocytopenia purpura may occur.
A phytotoxic reaction may occur, sometimes accompanied by oncholysis
and pigmentation of the nails. Liver injury and delayed blood
coagulation may occur. Children under 7 years of age may develop a
brown discoloration of the teeth. Infants of mothers treated with OTC
during pregnancy may develop discoloration of the teeth. OTC is
deposited in the skeleton in fetuses and children which can produce
depression of bone growth (which is readily reversible when the period
of exposure to OTC is short) (EPA, 1988).
2.3.2 Hypersensitivity
In a 4 year old girl and a 6 year old boy sensitization was
observed after treatment with OTC for otitis and a urinary tract
infection, respectively (Walczynski & Stengel, 1968).
The presence of eczematous contact allergy to OTC was established
in 3 patients by patch testing, which was negative in all controls
(Bojs & Moller, 1974).
In a patch test 31 patients were used as negative controls and 10
patients were sensitized with 3% OTC. In 7/10 treated patients a
strong reaction was observed and in 2/10 patients a weak reaction was
observed; negative reactions were seen in 30 control patients and in
1/10 treated patients (Moller, 1976).
2.3.3 Special studies on microbiological effects
The ecological impact of low doses of OTC on faecal microflora
was studied in normal adult volunteers. Thirty subjects were
controlled once a week during 4 consecutive weeks for the total
population of faecal Enterobacteriaceae and for OTC-resistant
Enterobacteriaceae. Fourteen other volunteers received OTC orally
during 7 consecutive days: 2 received 2 x 1 g/day, 6 received 2 x 10
mg/day and another 6 volunteers received 2 x 1 mg/day.
Faecal OTC concentrations, total count of anaerobes, and
morphologic and physiologic characterization of the dominant strains
of anaerobes were described. Determination of the MIC of OTC on these
dominant anaerobes and the count of total and OTC resistant
Enterobacteriaceae were measured before treatment, on day 7 of
treatment and 7 days after the end of treatment. At 2 g OTC/day the
dominant anaerobes and the OTC-susceptible Enterobacteriaceae were
effectively eliminated while an overwhelming growth of OTC-resistant
Enterobacteriaceae was observed and colonization by yeasts occurred.
At 20 mg OTC/day the composition of the dominant anaerobic flora
was not affected and no colonization by exogenous microorganisms was
observed. However, if the OTC susceptible Enterobacteriaceae were not
eliminated, the most OTC susceptible anaerobes disappeared, indicating
that OTC at this dosage may have an ecological impact in the digestive
tract. At 2 mg OTC/day the composition and the OTC susceptibility of
faecal flora were not modified. The no-effect dose in this study was
2 mg OTC/day (Tancrede & Barakat, 1987).
3. COMMENTS
The Committee considered pharmacokinetic data and results from
short-term studies in rats, mice, and dogs, a multigeneration study in
rats, teratogenicity studies in mice, rats, rabbits, and dogs, long-
term/carcinogenicity studies in mice and rats, mutagenicity tests and
studies on microbiological effects in laboratory animals and humans.
Pharmacokinetic studies demonstrated that about 60% of ingested
oxytetracycline was absorbed from the gastrointestinal tract in
humans, compared to 4-9% in mice and swine. Following absorption by
various routes of administration, oxytetracycline was widely
distributed in the body, particularly in the liver, kidney, bones, and
teeth. Systemically available oxytetracycline was primarily excreted
in the urine, as parent drug.
In the short-term toxicity studies, oxytetracycline was
incorporated into the diets of mice and rats at levels up to the
equivalent of 7500 and 2500 mg/kg b.w./day, respectively. Decreased
body weights were observed in mice at 3750 and 7500 mg/kg b.w./day and
a non-dose-related incidence of minimal periacinar fatty metamorphosis
of the liver was observed in male rats at all dose levels.
In a study in dogs that received oxytetracycline at 0, 5 or 10
g/kg in the diet degenerative change in the germinal epithelium of the
testes were noted at 10 g/kg. However, these findings were not
confirmed in a second study. The no-observed-effect level was
equivalent to 250 mg/kg b.w./day. No effects on reproductive
performance were observed in a two-generation study in rats in which
the compound was incorporated at a level of 360 mg/kg in the diet,
equivalent to 18 mg/kg b.w./day. In studies in rats which received
the compound orally at 48, 240, and 480 mg/kg b.w./day on days 1 to 21
of gestation, teratogenic effects were observed. However, an increase
in mortality rate and number of fetal resorptions and a decrease in
fetal ossification were noted at all dose levels tested. In a study
in mice in which the compound was administered orally, the highest
dose of 2100 mg/kg b.w./day caused maternal toxicity. There was no
evidence of a teratogenic effect.
In teratogenicity studies in rats and rabbits in which
oxytetracycline was administered intramuscularly at 41 mg per kg of
body weight, there was no evidence of a teratogenic effect. However,
an increased number of fetal resorptions was noted in rabbits.
Intramuscular administration of the compound in dogs, at approximately
20 mg/kg b.w./day, caused skeletal and visceral malformation in the
pups. The Committee noted that this study was difficult to evaluate
since it was poorly reported.
In a carcinogenicity study in mice and a similar study in rats,
in which oxytetracycline was administered in the diet at doses up to
1372 and 150 mg/kg b.w./day respectively, there was no evidence of an
increase in the incidence of tumours. In a second study, rats were
fed diets containing up to 2000 mg of oxytetracycline per kg of body
weight per day for 103 weeks. A dose-related increase in the
incidence of benign phaeochromocytomas was observed in males, but
because the survival rate of male rats in the control group was low,
this increase was not considered to be significant. Although there
was an increase in the incidence of benign neoplasms of the pituitary
gland in female rats in the highest-dose group, there was a lower
incidence of pituitary-gland hyperplasia than in controls. The
Committee concluded that there was no evidence of a carcinogenic
effect in rats or mice.
The mutagenic potential of oxytetracycline was investigated in a
range of studies. Negative results were recorded in bacterial tests,
a chromosomal aberration test, a sister chromatid exchange test (with
and without metabolic activation), and in a mouse lymphoma test
without metabolic activation. The Committee noted that a positive
effect in the mouse lymphoma test with metabolic activation was
obtained using dose levels close to toxic concentrations and that the
positive effect in the in vivo micronucleus assay in mice was not
dose-related.
In assessing the microbiological effects of oxytetracycline, the
Committee considered the results of studies on the induction of drug-
resistant organisms in dogs and humans. In a 6-week study in dogs,
which received oxytetracycline, there was no increase in the level of
resistant faecal coliforms at 2 mg/kg in the diet (equivalent to 50 µg
per kg of body weight per day). In humans receiving oral treatment
with oxytetracycline at 2 g, 20 mg, or 2 mg per day for 7 consecutive
days, there was no evidence of resistant bacteria of the family
enterobacteriaceae in the faeces at the lowest dose. The data on the
induction of bacterial resistance in dogs, when recalculated on the
basis of a 60-kg person, yielded a similar no-effect dose of 3 mg per
day.
4. EVALUATION
In view of the results of studies on the toxicological and
microbiological effects of oxytetracycline, the Committee concluded
that information about the induction of resistant coliforms in the
human intestine was most appropriate for the safety assessment of
oxytetracycline. The Committee adopted this conservative approach,
although it recognized that the no-effect levels in toxicological
studies were 18 mg/kg b.w./day or higher.
An ADI of 0-0.003 mg per kg of body weight was established for
oxytetracycline, based on a no-observed-effect level of 2 mg per
person per day from the study with human volunteers and a safety
factor of 10. It should be noted that the next dose tested was 20 mg
per person per day, so the true no-effect level may be significantly
higher than suggested by this study. A repeat study using doses
between these values may result in a higher no-observed-effect level.
The safety factor was selected by the Committee to account for
the variation in the intestinal microbial flora among humans. This
factor was viewed as being conservative because the people in the
study were selected because they had an oxytetracycline-sensitive
microbial flora. Furthermore, the MRL's derived from the established
ADI would be similar to those derived from in vitro 50% minimal
inhibitory concentration data for many species of microorganisms.
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See Also:
Toxicological Abbreviations
OXYTETRACYCLINE (JECFA Evaluation)