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    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 g/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 g/ml (plasma) and 0.2 g/ml (urine).  OTC-C was
    distributed slowly. The maximum plasma concentration (609 g/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 g/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 g/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 g/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
    g/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 g/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 g/ml
    depending on the formulation involved.  Plasma concentrations
    exceeding 0.5 g/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 g/ml) and milk (1.38 
    0.46 g/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 Ci calcium-45 and 20 g 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 g/pl in DMSO          negative       Andrews et. al.,
                                TA1535, TA1537,                                     (+/- act)      1980
                                TA1538, TA98 and TA100                                             

    Ames test                   S. typhimurium             0-1.0 g pl in DMSO      negative1      NTP, 1987
                                TA100, TA1535,                                      (+/- act)      
                                TA1537, and TA98                                                   

    Mouse lymphoma forward      L5178Y/TK+/-               12.5-800 g/ml toxic     negative       NTP, 1987
    mutation assay              cells                      >400 g/ml2 25-400       (- act)
                                                           g/ml toxic >200         positive
                                                           g/ml3                   (+ act)

    Chromosome aberration       Chinese hamster            80-200 g/ml4            negative       NTP, 1987
    assay                       ovary cells                                         (- act)        
                                                           700-900 g/ml5           negative     
                                                                                    (+ act)        

    Sister chromatid            Chinese hamster            60, 70 and 80 g/ml4     negative       NTP, 1987
    exchange assay              ovary cells                                         (- act)        
                                                           400, 500 and 700         negative       
                                                           g/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 g/ml.  These OTC-concentrations were higher than
    the MIC of the susceptible strain used (MIC=0.5 g/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 g/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 g/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 g
    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)