IPCS INCHEM Home


    CHLORTETRACYCLINE AND TETRACYCLINE

    First draft prepared by

    M.F.A. Wouters
    J.E.M. van Koten-Vermeulen
    F.X.R. van Leeuwen

    Toxicology Advisory Centre
    National Institute of Public Health and
    Environmental Protection
    Bilthoven, Netherlands

    Explanation

    Biological data
         Biochemical aspects
         Absorption, distribution, and excretion
         Interactions with bones and teeth
         Observations in humans
         Biotransformation

    Toxicological studies
         Acute toxicity studies
         Short-term toxicity studies
         Long-term toxicity/carcinogenicity studies
         Reproductive toxicity studies
         Special studies on embryotoxicity/teratogenicity
         Special studies on genotoxicity
         Special studies on microbiological effects
         Special studies on eye irritation and sensitization
         Other studies
         Observations in humans

    Comments

    Evaluation

    References

    1.  EXPLANATION

         Chlortetracycline (CTC) and tetracycline (TC) are broad-spectrum
    antimicrobial drugs with a long history of use in humans and animals.
    TC is used primarily for the short-term oral treatment of clinical
    diseases, while CTC is usually administered in the feed or water for
    prophylactic purposes.

         The tetracyclines (TCs) as a group were previously evaluated at
    the twelfth meeting of the Committee (Annex 1, reference 17), when a
    temporary ADI of 0-0.15 mg/kg bw was established. Oxytetracycline
    (OTC) was re-evaluated at the thirty-sixth meeting of the Committee
    (Annex 1, reference 91), when an ADI of 0-3 g/kg bw was established,
    based on effects on the human gut flora. Additional data have become
    available on CTC and TC since the twelfth meeting, which were
    evaluated at the present meeting.

         CTC and TC are closely related in structure (see Figure 1). The
    toxicological profiles and spectra of antimicrobial and biological
    activity are similar and therefore the Committee considered data on
    the two compounds together in evaluating their toxicological
    potential.

    Figure 1. Structure of tetracycline and chlortetracycline

    CHEMICAL STRUCTURE 5

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion

    2.1.1.1  Tetracycline (TC)

    Rats

         Fasted white rats were given a single oral dose of TC
    hydrochloride by gavage corresponding to 75 mg/kg bw TC base. The rats
    were sacrificed 1, 2, 3, 4 or 6 h after treatment and plasma and
    tissue levels recorded.

         Peak plasma concentrations of 3.6 mg/l were observed 2 h after
    treatment, declining to 0.5 mg/l after 6 h. Peak tissue levels were
    found 2 h after treatment with highest levels in the liver and kidney
    (Bert & Vandoni, 1962).

         To investigate the importance of the biliary route of excretion,
    4 rats (2 with ligated bile ducts) were administered single i.v. doses
    of 15 mg/kg bw tritium- labelled TC. Radioactivity was measured in
    urine, bile and GI tract 24 h following treatment.

         In non-ligated rats, 85-92% of the total radioactivity was
    recovered of which 67-72% was excreted in urine and 18-20% in faeces.
    About 70-85% of the total radioactivity was recovered in the rats with
    ligated bile ducts, 68% and 88% being recovered in the urine of each
    rat, and 30% and 9% in the bile. Only very small amounts (on average
    2.5%) were recovered in the GI tract. When the same dose of TC was
    administered to rats with ligated ureters, no increase in the
    excretion of TC in the faeces was observed (Wulf & Eisner, 1961).

         Male Sprague-Dawley rats were injected with 10 mg/kg bw 3H-7-TC
    hydrochloride (98% pure) in the femoral vein over 5 min. The
    intestinal absorption of TC hydrochloride excreted in bile was
    evaluated using  in situ intestinal preparations. About 73% of TC
    excreted in the bile was reabsorbed in the intestinal lumen,
    indicating enterohepatic circulation (Adir, 1975).

         Single i.v. doses of 15 or 4 mg/kg bw tritium-labelled TC
    hydrochloride were administered to 2 rats and 1 dog, respectively.
    Within 72 h, 69.2% and 19.5% of the radioactivity was recovered in the
    urine and faeces of rats, respectively. Within 168 h, 71% of the
    radioactive dose was recovered in the urine and 9% in the faeces of
    the dog (Eisner & Wulf, 1962).

    Dogs

         Tritium-labeled TC hydrochloride was administered i.v. to 2
    beagle dogs at 10 mg TC/kg bw to study the distribution of TC
    throughout the body. The animals were sacrificed after 4 h and the
    various organs and tissues examined for radioactivity. The organs
    containing the highest radioactivity were liver and kidney, containing
    on average 15 and 45 mg/kg, respectively. A large proportion of the
    recovered TC activity was found in the urine, intestinal contents and
    bile. No radioactivity was measured in subcutaneous fat (Kelly, 1964).

         Beagle dogs given single oral doses of 25 mg TC/kg bw showed peak
    TC levels averaging 3 g/ml 2 h after dosing, declining to an average
    of 0.27 g/ml by 24 h post-dosing. Excretion in the urine accounted
    for 10% of the administered dose within 72 h after dosing.

         When dogs were given a single i.v. dose of 10 mg TC/kg bw,
    average serum TC levels of 10.6 g/ml were found at 24 h, and
    0.14 g/ml at 48 h after dosing. Excretion in the urine was 58% of the
    administered dose by 72 h after dosing (microbiological assay;
    sensitivity 0.05-0.1 g/ml) (Kanegis, 1958).

    Pigs

         The bioavailability of TC hydrochloride administered orally to
    fasted gilts was calculated from the area under the plasma
    concentration in time curve (AUC) and estimated to be 23%. After i.v.
    administration the disposition kinetics of TC in plasma were best
    described by a tri-exponential equation. The drug had a rapid
    distribution phase followed by a relatively slow elimination phase,
    with half-life of 16 h (Kniffen  et al, 1989).

    2.1.1.2  Chlortetracycline (CTC)

    Rats

         14C-Labelled CTC was orally administered to rats at a dose of
    60 mg/kg bw. Radioactivity was measured in urine and faeces after
    24, 48 and 72 h. Radioactivity was found primarily in the faeces
    (92% within 72 h, the major part being excreted within 24 h). About 5%
    of the radioactivity was recovered in urine.

         When 30 mg/kg bw 14C-CTC was administered i.p., 33% of the
    radioactivity was excreted in the urine within the first 24 h, and 5%
    in the faeces. Between 24 and 72 h, 7% was excreted in the urine and
    40% in the faeces.

         To investigate the importance of the biliary route of excretion,
    rats (2 with ligated bile ducts) were administered single i.v. doses
    of 15 mg/kg bw 14C-CTC. Radioactivity was measured in urine, bile
    and GI tract, 24 h following treatment.

         In non-ligated rats, 75-79% of the total radioactivity was
    recovered of which 35-37% was excreted in urine and 44-38% in faeces.
    In the rats with ligated bile ducts, about 47-63% of the total
    radioactivity was recovered, with 66% and 43% being recovered in the
    urine of each rat, and 22% and 51% in the bile. Only very small
    amounts (on average 5%) were recovered in the GI tract (Wulf &
    Eisner, 1961).

         Groups of 6 rats given single oral doses of 75 mg CTC/kg bw had
    plasma levels of 2.1 mg/l at 1 h after administration, declining to
    0.8 mg/l after 6 h. The rats were sacrificed at 1, 2, 3, 4 or 6 h
    after treatment. Tissue levels were highest in liver and kidneys at
    all observations. Maximum concentration was found after 2 h in the
    liver, and after 1 h in the kidney (Berte & Vandoni, 1962).

         Oral doses ranging from 6 to 800 mg CTC/kg bw administered to
    female rats and male guinea-pigs gave no proportional increases in
    serum CTC concentrations. When guinea-pigs received the same doses
    each day for 9 days, higher serum levels were found than with single
    doses. Serum CTC levels were increased by the simultaneous
    administration of various adjuvants, such as citric acid. This effect
    (observed in doses of CTC up to 200 mg/kg bw) was apparent within 1 h
    of administration and lasted at least 8 h (Eisner et al., 1953)

    Rabbits

         Ten adult white Californian rabbits (males and females) received
    a single oral dose of 20 mg/kg bw of technical CTC or CTC-chloride.
    Serum CTC levels averaged 2.3 mg/l 3 h after dosing. The concentration
    of CTC declined to a mean value of 0.09 mg/l by 12 h, and to 0.08 mg/l
    by 24 h after dosing. The highest concentrations were found in the
    liver (1.53 mg/kg) 24-h post-dosing, followed by kidney, lung and
    heart. No measurable levels were found in muscle (detection limit:
    37.5 g/kg) (Neuschl, 1991)

    Dogs

         Four beagle dogs were given a single oral dose of 25 mg
    CTC/kg bw. Peak serum CTC levels ranged from 0.40-1.9 mg/l 2 h after
    dosing, declining to an average of 0.21 mg/l by 24 h. When the dogs
    were given an i.v. injection of 10 mg CTC/kg bw, serum levels averaged
    6.6 mg/l 1 h after administration, declining to 2.4 g/ml at 8 h,
    0.29 g/ml at 24 h and 0.06 g/ml at 48 h (Kanegis, 1958).

         Two female beagle dogs received a single i.v. injection of 10 mg
    14C-CTC/kg bw. The dogs were sacrificed 4 h after dosing. The organ
    containing the highest radioactivity was the liver (30 mg/kg),
    followed by kidney (25 mg/kg), ileum (15 mg/kg), jejunum (12 mg/kg)

    and heart (10 mg/kg). A large proportion of the recovered
    radioactivity was found in urine, intestinal contents and bile. With
    the exception of subcutaneous fat, radioactivity was found throughout
    all tissues and fluids examined (Kelly, 1964).

    2.1.2  Interactions with bones and teeth

         Tissues from mice, rats, guinea-pigs, rabbits and dogs
    parenterally treated with TCs (TC, CTC or OTC, doses varying from
    0.1 - 50 mg/kg bw) were examined in U.V. light. In all tissues with
    the exception of the brain, a brilliant yellow-gold fluorescence
    was apparent within 30 min, irrespective of the dose. The induced
    fluorescence disappeared from all tissues (except bone) within 6 h
    after a single parenteral injection. However, bone fluorescence
    persisted throughout a 10-week observation period after dosing
    (Milch et al., 1967).

         Single oral doses of 250 mg/kg bw tritium-labelled TC or single
    oral dose of 250 mg 14C-CTC/kg bw were administered to male rats
    (Sherman strain). Radioactive residues in the femur of the rats
    treated with TC were 9.6, 1.9 and 0.4 mg/kg of bone measured after
    4 h, 24 h, and 4 weeks, respectively. The radioactivity averaged
    12 mg/kg of bone 4 h after CTC treatment, and 2.3 mg/kg after 4 weeks.

         After a lifetime diet containing 0.5 to 1000 mg CTC/kg of feed,
    the maximum radioactivity in femur was 570 mg/kg. After i.p.
    injections with 10-150 mg TC/kg bw, the concentrations found in the
    femur were directly related to the administered dose and were much
    higher than the concentrations measured after oral treatment with
    250 mg TC/kg bw (Buyske  et al., 1960).

    2.1.3  Observations in humans

         In humans, about 30% of an oral therapeutic dose of CTC was
    absorbed on an empty stomach. For TC and OTC, the absorption was
    60-80% (Sande & Mandell, 1990).

         Oral doses of 250 to 500 mg every 6 h produced plasma
    concentrations of TC ranging from 1 to 5 mg/l. Intravenous injections
    of 250-500 mg produced plasma concentrations of about 15-20 mg/l at
    0.5 h, falling to 4-10 mg/l at 1 to 2 h. At 12 h, 1-3 mg/l was still
    present. In circulation, TCs are bound to plasma proteins in varying
    degrees (24-65% for TC, and about 47% for CTC). TCs appeared in the
    milk of nursing mothers where concentrations were be 60% or more of
    those in the plasma. TCs diffused across the placenta and appeared in
    the fetal circulation at concentrations of about 25 to 75% of those in
    the maternal blood. The plasma half-lives of CTC and TC were reported
    to be about 8-10 h and 5.5 h, respectively (Martindale, 1989).

         Absorption of TCs was impaired by milk products, sodium
    bicarbonate, aluminium hydroxide and iron preparations due to
    chelation and increase in gastric pH (Sande & Mandell, 1990).

    2.1.4  Biotransformation

    2.1.4.1  Tetracycline

         The metabolism of 14C-TC was examined in rats following single
    i.p. or oral administration of 60 mg/kg bw, and in dogs after single
    oral administration of 25 mg/kg bw tritium-labelled TC. Approximately
    90% of the administered radioactivity in the rat was eliminated either
    by urinary or faecal route. A significant portion of the remaining
    activity was bound as chelated TC in the skeleton of the animal. With
    the exception of this chelate, TC was chemically unaltered by the rat.
    Urine of dogs contained only unchanged TC (Kelly & Buyske, 1960).

    2.1.4.2  Chlortetracycline

         Male Wistar rats (n=6) and male beagle dogs (n=2) were
    administered 14C-labelled CTC via different routes (oral:
    60 mg/kg bw; i.p.: 30 mg/kg bw; i.v.: 15-60 mg/kg bw). The
    recovery of antimicrobial activity was significantly lower than the
    recovery of radioactivity by all routes of administration and
    excretion. The major 'presumed' metabolite was identified as
    4-epichlortetracycline, which accounted for 23-35% of the
    radioactivity in the urine of rats, and 31-60% in dogs. It was
    essentially inactive in microbiological assays. It was not clear
    whether this represented a true metabolite or was a degradation
    product due to alkaline treatment. The presence of small amounts
    (5-10%) of isochlortetracycline in the urine and faeces of some
    animals was also demonstrated (Wulf & Eisner, 1961; Eisner & Wulf,
    1963).

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

         The results of acute toxicity studies with TC and CTC after oral,
    i.v. or s.c. administration are summarized in Tables 1 and 2,
    respectively.

         Dogs developed transient hyperpnea, weakness and anorexia
    following an i.v. dose of 50 and 100 mg CTC/kg bw, and a dose of
    150 mg/kg bw caused respiratory distress, general paresis, somnolence
    and death within a few hours (Hines, 1956).

        Table 1.  Acute toxicity of tetracycline
                                                                                                             

    Species         Sex       Route      Substance     Purity      LD50            Reference
                                                                   (mg/kg bw)
                                                                                                             

    mouse           n.s.      oral       TC            n.s.        2550            Tubaro et al., 1964

    mouse           n.s.      oral       TC            n.s.        >3000           Cunningham et al., 1954

    mouse (42d)     n.s.      oral       TC            n.s.        808             Goldenthal, 1971
          (3d)                                                     300

    mouse           n.s.      i.v.       TC            n.s.        160             Tubaro et al., 1964

    mouse           n.s       i.v.       TC            n.s.        157             Maffii & Mainardi, 1955

    mouse           n.s.      i.v.       TC            n.s.        170             Cunningham et al., 1954

    mouse           n.s.      i.p.       TC            n.s.        340             Tubaro et al., 1964

    mouse           n.s.      i.p.       TC            n.s.        330             Cunningham et al., 1954

    rat             M         oral       TC HCl        n.s.        >4000           Diermeier, 1959

    rat             n.s.      oral       TC            n.s.        >3000           Cunningham et al., 1954

    rat   (49(d)    n.s.      oral       TC            n.s.        807             Goldenthal, 1971
          (3d)                                                     360
                                                                                                             

    Table 1.  Acute toxicity of tetracycline (cont'd)
                                                                                                             

    Species         Sex       Route      Substance     Purity      LD50            Reference
                                                                   (mg/kg bw)
                                                                                                             

    rat   (adult)   n.s.      oral       TC HCl        n.s.        6443            Goldenthal, 1971
          (<2d)                                                    3827

    rat             n.s.      i.v.       TC            n.s.        220             Cunningham et al., 1954

    rat             n.s.      i.v.       TC            n.s.        128             Maffii & Mainardi, 1955

    rat             n.s.      i.v.       TC HCl        n.s.        375             Diermeier, 1959

    rat             n.s.      i.p.       TC            n.s.        320             Cunningham et al., 1954
                                                                                                             

    n.s.     not specified
    d        age of animal in days
        2.2.2  Short-term toxicity studies

    2.2.2.1  Tetracycline

    Mice

         Groups of male mice (10/sex/group) were given by gavage 0, 20 or
    100 mg/kg bw TC hydrochloride once daily, 5 days/week for 6 weeks as a
    2% starch suspension containing 4-6% test substance. At termination of
    the study a complete blood count was performed in the high-dose group
    and no significant haematological alterations were observed.
    Accelerated growth rates were observed in dosed mice when compared to
    the control mice (Cunningham et al., 1954).

         In a range-finding study, groups of B6C3F1 mice (6-7 week old;
    10-15/sex/group) were fed diets containing 0, 0.31, 0.63, 1.25, 2.5 or
    5% TC hydrochloride for 13 weeks (equivalent to 470, 950, 1800, 3700
    or 7500 mg/kg bw/day, respectively). No mortalities occurred. Final
    mean body weight was slightly decreased in males (16%) and females
    (6%) at the 5% level. The TC concentration in bone increased with
    increasing dose of TC hydrochloride (NTP, 1989).

    Rats

         In a range-finding study, groups of F344/N rats (7-8 week old;
    10-15/sex/group) were fed diets containing 0, 0.31, 0.63, 1.25, 2.5 or
    5% TC hydrochloride for 13 weeks (equivalent to 155, 315, 625, 1250 or
    2500 mg/kg bw/day, respectively). No mortalities occurred. Cytoplasmic
    vacuolization was observed in the livers of male rats at the two
    highest dose groups. Bone marrow atrophy was seen in both female and
    male rats at 1250 and 2500 mg/kg bw/day. The concentration of TC in
    bone increased with increasing dose of TC hydrochloride (NTP, 1989)

    Dogs

         Mongrel dogs (8-4/group) were given a capsule of 0 or
    250 mg/kg bw TC once daily, 6 days/week for 98 days. No mortality
    occurred. No effects were seen on peripheral blood and no pathological
    changes were observed (Nelson & Radomski, 1954).

         Groups of 4 mongrel dogs were orally administered 10 or
    100 mg/kg bw TC twice daily, 5 days/week for 3 months. No effects were
    observed on body weight, liver function (bromosulfophthalein
    clearance), kidney function (phenolsulfophtalein clearance),
    blood-clotting time, non-protein nitrogen levels, blood sugar values
    or complete blood counts (Cunningham et al., 1954).

        Table 2.  Acute toxicity of chlortetracycline
                                                                                                             

    Species         Sex       Route      Substance        Purity      LD50            Reference
                                                                      (mg/kg bw)
                                                                                                             

    mouse           M & F     oral       CTC HCl          n.s.        >3000           Cunningham, 1954

    mouse           F         oral       CTC HCl          n.s.        2150            Tubaro & Banci, 1964

    mouse           M & F     oral       CTC HCl          n.s.        3350 (M)        Bacharach, 1959
                                                                      4200 (F)

    mouse           n.s.      oral       duomycin         n.s.        >3000           Harned, 1948

    mouse           M & F     i.v.       CTC HCl          n.s.        155             Cunningham, 1954

    mouse           F         i.v.       CTC HCl          n.s.        93              Tubaro & Banci, 1964

    mouse           n.s.      i.v.       duomycin         n.s.        102             Harned, 1948

    mouse           n.s.      i.v.       duomycin         n.s.        134             Harned, 1948

    mouse           M & F     i.v.       CTC HCl          n.s.        102 (M)         Bacharach, 1959
                                                                      108 (F)

    mouse           F         i.v.       CTC bisulfate    n.s.        113             Levinskas 1963

    mouse           n.s.      s.c.       duomycin         n.s.        >600            Harned, 1948

    mouse           M & F     s.c.       CTC HCl          n.s.        5500 (M)        Bacharach, 1959
                                                                      8250 (F)
                                                                                                             

    Table 2.  Acute toxicity of chlortetracycline (cont'd)
                                                                                                             

    Species         Sex       Route      Substance        Purity      LD50            Reference
                                                                      (mg/kg bw)
                                                                                                             

    mouse           n.s.      i.p.       CTC HCl          n.s.        192             Cunningham, 1954

    mouse           M & F     i.p.       CTC HCl          n.s.        128 (M)         Bacharach, 1959
                                                                      168 (F)

    mouse           F         i.p.       CTC HCl          n.s.        214             Tubaro & Banci, 1964

    rat             M & F     oral       CTC HCl          n.s.        >3000           Cunningham, 1954

    rat             F         oral       CTC HCl          n.s.        >4000           Diermeier, 1959

    rat             n.s.      oral       CTC-HCl          n.s.        55001           Goldenthal, 1971
                                                                      103002

    rat             F         oral       calcium CTC      n.s.        >10000          Levinskas, 1963

    rat             n.s.      oral       Aureo Biomass    n.s.        >5000           Lowe & Fisher, 1993
                                                                                      (summary only)

    rat             n.s.      oral       duomycin         n.s.        >3000           Harned, 1948

    rat             M & F     i.v.       CTC HCl          n.s.        160             Cunningham, 1954
                                                                                                             

    Table 2.  Acute toxicity of chlortetracycline (cont'd)
                                                                                                             

    Species         Sex       Route      Substance        Purity      LD50            Reference
                                                                      (mg/kg bw)
                                                                                                             

    rat             M         i.v.       CTC HCl          n.s.        167             Diermeier, 1959

    rat             n.s.      i.v.       duomycin         n.s.        118             Harned, 1948

    rat             n.s.      i.p.       CTC HCl          n.s.        335             Cunningham, 1954

    guinea pig      n.s.      i.v.       duomycin         n.s.        100             Harned, 1948

    guinea pig      n.s.      s.c.       duomycin         n.s.        >300            Harned, 1948
                                                                                                             

    n.s.     not specified
    1        < 2 days old
    2        adult
    
         Groups of male dogs (4 mongrel dogs and 4 beagle dogs) were fed
    diets containing 0.1, 0.3 or 1% TC hydrochloride for 24 months
    (equivalent to 25, 75 or 250 mg/kg bw/day, respectively). An interim
    sacrifice of 1 beagle and 1 mongrel dog of each group was performed at
    12 months for microscopic examination. No effects were observed on
    clinical signs, mortality, body weight, food consumption, haematology,
    ALP, bromosulfophtalein clearance, urea nitrogen determinations,
    testes and epididymides weight, macroscopy, concentration and motility
    of the spermatozoa, or appearance of semen. The bony structures of all
    dosed dogs showed a yellowish coloration. The intensity of the colour
    was related to the dose fed. A brownish-black pigmentation with a
    dose-related intensity was observed in the thyroid glands of the dogs
    of all groups. Microscopic examination of the thyroids revealed
    intracytoplasmic granules in the follicular epithelial cells of most
    of the treated dogs. This effect might be caused by deposits of TC or
    its metabolites. Histopathological changes were not observed
    (Deichmann et al., 1964).

    2.2.2.2  Chlortetracycline

    Mice

         Groups of 10 male mice were given 0, 20 or 100 mg CTC/kg bw/day,
    once daily by gavage, 5 days/week for 6 weeks. At 100 mg/kg bw/day,
    complete blood count indicated that there were no significant
    haematological alterations. Beneficial effects such as increased
    growth were observed at 20 and 100 mg/kg bw/day (Cunningham, 1954).

         Two groups of 20 mice received 20 or 100 mg/kg bw/day of CTC for
    3 months (route not specified). Final body weight of both groups
    exceeded those of the controls by 15%. No evidence of toxicity was
    seen and no difference in blood counts was found at the end of
    experiment (Hines, 1956).

         Groups of 20 male mice (strain not specified) received orally
    0, 40 or 100 mg CTC/kg bw/day, 5 days/week for 14 weeks. From the
    fourth week onwards, the high dose was increased to 200 mg/kg bw/day
    (administration of 100 mg/kg bw, twice daily). There were no
    significant effects on mortality, clinical signs, body-weight gain,
    haematology, macroscopy or microscopy. The NOEL in this study was
    200 mg/kg bw/day (Harned et al., 1948).

    Rats

         Sherman rats (6/sex/group) received diets containing 0, 2.0 or
    5.0% CTC for 28 days (equivalent to 2000 or 5000 mg/kg bw/day).
    Body-weight gain was significantly decreased at 5000 mg/kg bw/day. No
    other examinations were performed (Halliday & Fanelli, 1960).

         Two groups of 20 rats each were given oral doses of 150 or
    300 mg/kg bw/day CTC for 3 months. In both groups, body weight of the
    animals was higher than the controls. No difference in blood counts
    was found at the end of the experiment (Hines, 1956).

         Groups of 20 male rats (strain not specified) received by gavage
    0, 10, 40 or 100 mg CTC/kg bw/day, 5 days/week for 14 weeks. From the
    fourth week onwards, the high dose was increased to 200 mg/kg bw/day
    (administration of 100 mg/kg bw twice daily). No treatment-related
    effects were seen on mortality, clinical signs, body weight,
    haemoglobin, blood pressure, macroscopy or microscopy. The NOEL in
    this study was 200 mg/kg bw/day (Harned et al., 1948).

    Dogs

         Oral administration of 100 mg CTC/kg bw/day to dogs (number and
    strain not specified) for 17 days, followed by 100 mg/kg bw twice
    daily for 14 days, produced no unfavourable effects on general
    appearance, growth rate, haemoglobin, complete blood count, gross or
    microscopic examinations (Hines, 1956).

         Dogs (4/group) received 10, 50 or 100 mg of CTC/kg bw/day for 14
    weeks (route not specified). At the end of this period, liver and
    kidney function tests, blood sugar values, blood coagulation time,
    blood non-protein nitrogen and complete blood counts did not differ
    significantly from pre-test values (Hines, 1956)

         Ten dogs (strain not specified) were given CTC twice daily
    (morning and afternoon) by capsule at a total dose of 100 mg/kg
    bw/day, 5 days/week for 9 to 15 weeks. CTC treatment had no effect on
    general appearance, body weight, haematology, clotting time, liver and
    kidney function (Bromosulfophtalein and phenolsulfophtalein
    clearance), urinalysis, gross or microscopic examinations
    (Harned, 1948).

         Ten mongrel dogs (8-19 kg; 2-5 years old) received capsules
    containing 250 mg CTC/kg bw/day, 6 days/week, for 98 days. Another 4
    dogs received the same dose intermittently, 2 weeks on the drug and 3
    weeks off, for 121 days in total. Five dogs of the continuously
    treated group died. These animals had progressive weight loss, apathy
    and anorexia. Haemoglobin, RBC, granulocyte and total leucocyte counts
    were slightly depressed. The pathologic changes observed included
    emaciation, fatty liver, excess fat in the kidney, depletion of the
    bone marrow and atrophic changes in the spleen, lymph nodes and
    skeletal muscle (Nelson & Radomski, 1954).

         Beagle and basenji dogs (2/sex/group; 9-12 months old) received
    daily a single oral dose of 10 or 100 mg CTC-HCl/kg bw in capsules for
    54 weeks; a third group consisting of 1 female beagle dog, 2 male and
    1 female basenji dogs were given 50 mg/kg bw/day. No control group was
    used. Observations included haematology, clinical chemistry,
    urinalysis, faecal smears, macroscopy, organ weight, microscopy, and
    terminal tissue concentration.

         Treatment-related GI disorders (vomiting, diarrhea, appetite loss
    and swelling of the anal glands) were observed, and particularly in
    the first half of the experiment in all groups, most often in basenji
    dogs. The average blood concentration 4 h after administration of the
    dose (calculated for each group from 12-14 determinations carried out
    over a period of 1 year) were 0.34 g/ml, 0.58 g/ml and 0.94 g/ml at
    10, 50 and 100 mg/kg bw/day, respectively. Thyroid weight was high in
    one male at 100 mg/kg bw/day, but the shape and appearance of the
    gland were normal. Slight yellow discolouration in the bones was
    observed at necropsy in the high-dose dogs, and 2 dogs at each dose
    level showed evidence of chronic gastritis. Measurable tissue
    concentrations (no details given) were found in bone > kidney >
    liver but none in brain, heart or spleen. The NOEL in this study was
    100 mg/kg bw/day (Dessau & Sisson, 1957).

    2.2.3  Long-term toxicity/carcinogenicity studies

    2.2.3.1  Tetracycline

    Mice

         Groups of B6C3F1 mice (50/sex/group) were fed diets for 103
    weeks containing 0, 1.25 or 2.5% TC-HCl (purity 91%), equal to 1500 or
    3000 mg/kg bw/day for males, and 1500 or 3500 mg/kg bw/day for
    females. Observations included clinical signs, body weight, feed
    consumption, macroscopy, and histopathology.

         Survival was increased for male mice (31/50, 43/50, 43/50); no
    differences were observed in females (37/50, 35/50, 38/50). Mean body
    weights in both dose groups were slightly lower for both sexes
    compared to control mice. Dosing had no effects on feed consumption.
    The tumour incidence was not significantly increased in either sex. On
    the contrary, dosed female mice did not develop hepatocellular
    adenomas or carcinomas (combined incidence: 10/49 for controls; 0/48
    at 1.25%; 0/50 at 2.5%) (NTP, 1989; Dietz et al., 1991).

    Rats

         Groups of weanling male rats (Osborne-Mendel) were fed diets
    containing 0 (180 rats), 100 (100 rats), 1000 (130 rats) or 3000
    (100 rats) mg TC-HCl/kg of feed, equivalent to 5, 50 or 150 mg/kg
    bw/day for 2 years. Ten Rats/group were killed at frequent intervals.
    Sacrificed and moribund rats were macroscopically and histologically
    examined. No effects were seen on clinical signs, body weight, food
    consumption or haematological examinations. Beneficial effects were
    observed during the first 18-19 months, all dosed rats appearing more
    vigorous and gaining weight more rapidly than the control rats.
    Mortality rates were lower in the treated rats than in the control
    rats. At histopathology, yellow discoloration of long bones and
    calvarium was observed at the highest dose. Tumour incidence was not
    enhanced (Deichmann et al., 1964).

         Groups of F344/N rats (50/sex/group) were fed diets containing
    0, 1.25% or 2.5% mg TC-HCl (purity 91%), equal to 440 or 910 mg/kg
    bw/day for males, and 510 or 1060 mg/kg bw/day for females for
    103 weeks. Observations included clinical signs, body weight, feed
    consumption, macroscopy and histopathology.

         The survival of female rats of both the low- and high-dose groups
    was significantly greater than that of the controls (control 27/50,
    low-dose 39/50, high-dose 38/50). No compound-related effects on body
    weight or feed consumption were observed. A dose-related increased
    incidence of basophilic cytoplasmic changes and clear cell changes
    were observed in the livers of male rats. The incidence and severity
    of nephropathy were reduced in dosed male rats (control 48/50,
    severity 2.8; mid-dose 35/50, severity 1.6; high-dose 36/50, severity
    1.5). The tumour incidence was not enhanced (NTP, 1989; Dietz et al.,
    1991).

    2.2.3.2  Chlortetracycline

    Rats

         Forty rats (sex or strain not specified) were maintained for 52
    weeks on a diet containing 1% CTC, equivalent to 1000 mg/kg bw/day.
    There was no significant difference in body weight and no evidence of
    toxicity was seen.

         In a parallel experiment another group of 40 rats (sex or strain
    not specified) received 5% CTC, equivalent to 5000 mg/kg bw/day. Ten
    animals died within the first 10 weeks. The remaining 30 rats survived
    for the 52-week test period and had a 33% lower weight than the
    controls, and an apparent yellow staining of all tissues (Hines, 1956).

         Groups of Sherman rats (20/sex/group) were fed diets containing
    CTC at concentrations of 0, 1, 5, 20, 100, 500, 2000, 10000 or
    50000 mg/kg of feed, equal to 0, 0.07, 0.35, 1.3, 7, 34, 130, 700 or
    5200 mg/kg bw/day, respectively. Observations were made on mortality,
    clinical signs, body-weight gain, food consumption, haematology,
    macroscopy, organ weight, and microscopy.

         During the first 12 weeks of the study, 8 male rats in the
    highest dose group died compared to none in the other groups. At
    termination, the total mortality for males was not different from
    controls. Both males and females in the 50000 mg/kg group showed signs
    of GI irritation which included abdominal distension, nose and mouth
    encrustation and salivation. Body-weight gain was reduced in both
    sexes at 50000 mg/kg. WBC was reduced in both sexes at 50000 mg/kg.
    Microscopic changes at 50000 mg/kg included accumulation of yellow
    pigment in splenic lymph follicles for both sexes, testicular atrophy
    occasionally with degenerative changes in seminiferous tubulus, fatty
    infiltration of the liver in males and infiltration of monocytes
    sometimes coupled with interstitial fibrosis in the lungs in both
    sexes. Fluorometric determinations showed the presence of significant
    amounts of CTC at dose levels of 500 mg/kg of feed and higher. No
    blood or urine biochemistry was carried out. The tumour incidence was
    not enhanced. The NOEL in this study was 10000 mg/kg diet, equal to
    700 mg/kg bw/day (Dessau & Sullivan, 1958; Dessau & Sullivan, 1961).

    2.2.4  Reproductive toxicity studies

    2.2.4.1  Chlortetracycline

    Mice

         Mice maintained through 4 reproductive cycles on diets containing
    175 mg CTC/kg of feed (equivalent to 25 mg/kg bw/day) did not differ
    significantly from parallel control groups in the number of young born
    per litter, the number of surviving pups or the average body weight at
    the end of 4 weeks (Hines, 1956).

    Rats

         Rats maintained through 2 reproductive cycles on a diet
    containing 45 mg CTC/kg of feed (equivalent to 2 mg/kg bw/day) did not
    differ significantly from parallel control groups in the number of
    young per litter, the number of surviving pups or the average body
    weight at the end of 4 weeks (Hines, 1956).

         A 2-generation reproductive toxicity study was carried out with 2
    groups of 5 male and 15 female rats (Sherman strain, approximately
    21-day old). One group received the control diet and the other
    received the same diet containing 1% CTC, equivalent to 500 mg/kg
    bw/day. When the rats were approximately 100-day old, the animals were

    mated to produce the first generation. All litters were reduced to 8-9
    rats. Pups were weaned to the diet given to their parents. Second
    generation breeding was carried out with 21 female and 7 male rats in
    the 1% group and a few more rats per group in the control group.
    Observations were made for clinical signs, body weight, food
    consumption, fertility, reproductive performance and pup growth. Male
    body weights were slightly lower than controls for both parental
    generations. No further toxicity, nor effects on male or female
    reproduction were observed (Hallesy & Hine, 1964).

    2.2.5  Special studies on embryotoxicity/teratogenicity

    2.2.5.1  Tetracycline

         Groups of 16 pregnant Wistar rats were given 0 or 150-200 mg TC
    chlorhydrate/rat/day from days 1 to 18 of gestation (Group I) and
    another group received the same treatment from days 1 to 28 after
    delivery (Group II). At day 20 of gestation, 3 females of group I and
    3 control females were sacrificed and the uterine contents examined.
    The remaining rats were allowed to deliver their litters and the
    offspring were killed and developmental effects investigated. No
    effects were observed on gestation duration, body size or weight, and
    no malformations were observed. Pups of group II exhibited a 15%
    reduction in the length of the legs in comparison with the controls at
    28 days of age. Examination by UV light of the pups skeleton revealed
    typical TC-associated fluorescence, indicating that TC had been
    absorbed in the bones (Hurley & Tuchmann-Duplessis, 1963).

         Sprague-Dawley male and female rats were given 500 mg/kg TC-HCl
    in the feed, equivalent to 25 mg/kg bw/day, starting 3 days prior to
    mating. Groups of 15-20 pregnant rats continued to receive the
    treatment throughout gestation. Some of the pregnant rats (number not
    given) were sacrificed on day 21 of gestation and the contents of the
    uteri were examined. The remainder of the dams were allowed to deliver
    their litters. No significant effects were observed on resorptions,
    pregnancy rate, offspring mortality, litter size or weight. At
    external skeletal and visceral examinations, the only treatment-
    related effects observed were in animals delivered by section and
    included an increase in the occurrence of hydroureter (14%;
    control:0.8%) and fragmented lumbar (3%; control:0%). In animals
    delivered naturally, the incidence for hydroureter was 15% and 12% for
    control and treated rats, respectively (McColl et al., 1965).

         Pregnant Wistar rats (number not given) were orally dosed from
    days 1 to 21 of gestation with TC or TC-HCl at doses of 54, 270 or
    540 mg/kg bw/day, and 40, 200 or 400 mg/kg bw/day, respectively. All
    rats were sacrificed on day 21, and fetuses were removed and examined
    for skeletal anomalies. A reduction of ossification in the anterior
    extremities, but not in the posterior ones was observed, more
    frequently in the fetuses of the highest dose groups (Szumigowska-
    Szrajber & Jeske, 1970).

         The development of rat embryonic tissue, transplanted in athymic
    (nude) mice was studied for several weeks. Limb buds of fetal rats
    were cut into small pieces and transplanted into the subcutaneous
    tissue of the back of female nude mice. On the 7th, 9th and 11th days
    after grafting, the host mice were treated intraperitoneally with 1500
    or 3000 mg/kg TC suspended in 0.55% CMC. Control mice were treated
    with the vehicle only. On day 20, the grafted tissue was examined
    macroscopically and histologically. No treatment-related effects were
    observed on the growth of the grafted limbs or on the differentiation
    of the grafts (Shiota et al., 1990).

         Groups of 5 female rabbits (strain unspecified) and groups of 5
    female Wistar rats were injected i.v. from days 10 to 20 of pregnancy
    with 10 mg/kg bw/day of TC or CTC. Rabbit and rat neonates did not
    show significant variations in weight between the groups or any
    malformation (examinations in UV-light), however, the study was very
    limited (Tubaro, 1964).

    2.2.6  Special studies on genotoxicity

         The results of the genotoxicity studies with TC and CTC are given
    in Tables 3 and 4, respectively.

    2.2.7  Special studies on microbiological effects

         The TCs are primarily bacteriostatic antibiotics inhibiting
    protein synthesis in susceptible microorganisms. Following transport
    through the cytoplasmic membrane, TCs bind, predominantly reversibly
    to the 30 S sub-unit of bacterial ribosomes, thereby preventing access
    of aminoacyl tRNA to the acceptor site on the mRNA-ribosome complex
    (Sande & Mandell, 1990; Pratt & Fekety, 1986).

         The TCs possess a wide range of antimicrobial activity against
    Gram-positive and Gram-negative bacteria. They are also active against
    some microorganisms that are resistant to agents that exert their
    effects on the bacterial cell wall such as  Rickettsiae, Mycoplasma,
     Chlamydia, some  atypical mycobacteria and amebae. They have little
    activity against fungi (Sande & Mandell, 1985).

         Up to 50% of the antimicrobial activity of CTC can be inhibited
    by the presence of magnesium sulfate. The relationship between CTC
    inhibition and the magnesium sulfate concentration (50-200 mM) is
    linear (Chiang, 1982)

        Table 3.  Results of genotoxicity studies on tetracycline
                                                                                                             

    Test system          Test object           Concentration            Result           Reference
                                                                                                             

    In vitro

    Ames test            S.typhimurium         0-10 g/plate            negative1        NTP, 1989
                         TA100, TA98           toxic > 3 g/plate
                         TA1535, TA1537

    Mouse lymphoma       L5178Y/TK+/-          -S9:25-300 g/ml         negative1        NTP, 1989
    assay                cells                 toxic > 200 g/ml
                                               +S92:10-120 g/ml        negative1
                                               +S93:20-120 g/ml        weakly
                                                                        positive1

    Sister chromatid     Chinese hamster       -S9:5-49.9 g/ml         negative1        NTP, 1989
    exchange assay       ovary cells           toxic >40.2 g/ml
                                               +S9:302-600 g/ml        negative1
                                               toxic 600 g/ml

    Chromosome           Human                 10 g/ml                 equivocal        Meisner et al.,
    aberration assay     lymphocytes                                                     1977

    Chromosome           Chinese hamster       -S9:39.9-400 g/ml       negative1        NTP, 1989
    aberration assay     ovary cells           toxic 400 g/ml
                                               +S9:1000-2750 g/ml      negative1
                                                                                                             

    Table 3.  Results of genotoxicity studies on tetracycline (cont'd)
                                                                                                             

    Test system          Test object           Concentration            Result           Reference
                                                                                                             

    Gene mutation        FM3A cells from       10-100 g/ml             positive4        Tsutsui, et al.,
                         C3H mice                                                        1976

    In vivo

    Chromosome           Allium cepa           12-20 g/ml5             positive         Mann, 1978
    aberration assay

    SLRL assay           Drosophila            injection:                                NTP, 1989
                         melanogaster          5000-5300 ppm            negative
                                               feeding:
                                               9005 ppm                 negative
                                                                                                             

    1    with and without metabolic activation
    2    S9 prepared from the liver of Aroclor 1254-induced F344 rats
    3    S9 prepared from liver of non-induced F344 rats
    4    induction of an 8-azaguanine-resistant mutation chromosomal damage
         inhibition of protein and DNA synthesis
    5    TC pure was used as test substance
    
         Therapy with TCs is associated with a high rate of development of
    supra-infections with bacteria, yeast or fungi not sensitive to these
    agents, particularly in patients with diabetes and other conditions
    that reduce the natural host defense mechanisms
    (Pratt & Fekety, 1986).

    2.2.7.1  In vitro studies

         MIC values for TC for different bacterial strains of animal
    origin are given in Table 5.

         MICs for TC, CTC and OTC, ranging from 0.1- > 100 g/ml, have
    been reported for a number of clinical isolates, including
     Staphylococcus spp.,  Proteus spp. and  E. coli. The data indicate
    that the antimicrobial potency of the three TCs for various
    micro-organisms is quite similar (Walter & Heilmeyer, 1975).

         The antimicrobial potency of CTC in relation to that of OTC was
    established in 34 isolates of 3 different animal pathogens  (P.
    hemolytica, P. multocida and B. bronchiseptica). The geometric mean
    for the MICs was 0.32 g/ml for CTC and 0.52 g/ml for OTC. For
     E. coli (only one isolate), the MIC was 4.8 g/ml for CTC, and
    2.8 g/ml for OTC (Rogalski, 1985; Gustafson, 1995).

         The MIC observed for  S. aureus in vitro was 0.19 g/ml for CTC,
    0.21 g/ml for TC, and 0.55 g/ml for OTC (Fourtillan & Lefebvre,
    1985).

         In  in vitro studies with  E. coli of animal and human origin,
    MIC50 values of 2 g/ml for CTC and 4 g/ml for OTC were found. MIC
    values for  E. faecalis and  E. faecium also indicated a 2-fold
    higher potency for CTC than for OTC (Devriese, 1994).

         The antimicrobial activity of TC was compared with that of OTC in
    a range of bacterial species (10 strains/species) isolated from faeces
    of healthy humans. The concentrations of TC and OTC in the MIC
    determinations ranged from 0.06 to 32 g/ml. The bacterial density was
    107 colony forming units/ml. The results are presented in Table 6.
    The sensitivity of the strains of most bacterial species was similar
    for TC and OTC, whereas TC was active at lower concentrations for
     Bifidobacterium spp.,  Eubacterium spp. (p<0.05), and  Fusobacterium spp.
    (p<0.1), and at higher concentrations for  Spectrococcus spp. (p<0.05).
    The geometric mean MIC, taking into consideration all tested strains
    (using a value of 32 g/ml for resistant strains) was 3.2 g/ml for
    TC and 3.8 g/ml for OTC (Richez, 1994).

        Table 4.  Results of genotoxicity studies on chlortetracycline
                                                                                                             

    Test system      Test object            Concentration             Result           Reference
                                                                                                             

    In vitro

    Ames test        S. typhimurium         0.1-15 g/plate;          negative1,2      Mulligan, 1989
                     TA100,TA98,TA1535      conc > 1.0 g
                     TA 1337, TA1538        were toxic
                     E. coli WP-2uvrA-

    Amest test       S. typhimurium         up to 10 g/plate;        negative1        Sharma et al., 19893
                     TA100, TA98,           toxicity was
                     TA1535, TA1537         seen
                     E. coli WP-2uvrA-

    HGPRT assay      CHO cells              +act: 20-100 g/ml        negative         Harbell, 1988
                                            -act: 25-125 g/ml
                                            toxicity was
                                            observed at the
                                            highest conc.

    UDS              rat hepatocytes        25-75 g/ml;              negative         Thilagar, 1988
                                            toxicity was seen
                                            at the highest dose
                                                                                                             

    Table 4.  Results of genotoxicity studies on chlortetracycline (cont'd)
                                                                                                             

    Test system      Test object            Concentration             Result           Reference
                                                                                                             

    In vivo

    chromosome       rat                    orally 500, 2500,         negative         Putman, 1988
    aberrations                             5000 mg/kg bw4

    chromosome       Vicia sativa           1% solution               negative         Sharma &
    aberrations      Lens esculenta            "                      equivocal        Bhattacharyya, 1967
                                                                                                             

    1    with and without metabolic activation
    2    no positive controls were used; background was not given
    3    abstract only
    4    no effects on mitotic index
    
    Table 5.  Summary of MIC values for different bacterial species for
              tetracycline (Pratt & Fakety, 1986)
                                                                        

    Species                      MIC (g/ml)1
                                                                        

    Escherichia coli             12.5 (3.1-500)

    Proteus mirabilis            >100 (50->100)

    Enterobacter                 25 (6.3-50)
                                                                        

    1    Values given are the mean (range in parenthesis)

    2.2.7.2  In vivo studies

         The  in vivo activity of  S. aureus in mice was comparable for
    the three different TCs; ED50 values were 7.6, 7.2 and 5.8 mg/kg bw,
    for CTC, TC and OTC, respectively (Fourtillan & Lefebvre, 1985)

    2.2.8  Special studies on eye irritation and sensitization

         CTC produced local irritation when injected i.p., i.m. or s.c. or
    when administered directly to the rabbit eye. Eye irritation from 1%
    solutions was mild and completely reversible within 48 h (Harned et
    al., 1948).

    2.2.9  Other studies

    2.2.9.1  Cardiovascular effects

         Six anaesthetized male rabbits were given i.v. injections of 1, 2
    or 5 mg TC-HCl/kg bw dissolved in 0.5 ml saline. The injections were
    performed in 3, 10, 20 or 60 seconds. A dose- and injection
    time-dependent slowing of the heart rate (from 270-300 (normal) to 100
    beats/min or less) was observed 1-3 min after the injection. No effect
    on arterial blood pressure was found.

         Respiration was depressed which led at high doses to respiratory
    arrest or to a slow, shallow respiration for 1-2 min (Gyrd-Hansen,
    1980).

    Table 6.  MIC values (g/ml) for tetracycline and oxytetracycline
              against bacterial species obtained from human isolates.
                                                                        

                               TETRACYCLINE             OXYTETRACYCLINE
                                                                        

    Bacterial species          MIC50     geometric      MIC50    geometric
                                         mean                    mean
                                                                        

    Escherichia coli           > 32      > 32           > 32     > 32

    Bifidobacterium spp.       16        8.6            > 32     > 17.1

    Bacteroides fragilis       4         2.5            4        2.5

    Eubacterium spp.           2*        1.6            4        2.6

    Clostridium spp.           0.062     0.2            0.25     0.2

    Streptococcus spp.         16        > 19.7         8        > 13.9

    Fusobacterium spp.         0.125     0.1            0.25     0.2

    Lactobacillus spp.         2*        1.9            2        2.3

    Proteus spp.               > 32      > 32           > 32     > 32

    Peptostreptococcus         2         3.2            4        4.0
    spp.
                                                                        

    *    MIC50 = MIC90

    2.2.9.2  Hepatotoxicity

         TC-HCl (>0.25 mM) inhibited reversibly the -oxidation of
    fatty acids as well as the tricarboxylic acid cycle activity of mouse
    and human liver mitochondria  in vitro. After i.p. administration of
    1 mmol/kg bw to mice following an oral administration of a tracer dose
    of [U-14C]palmitic acid, an increase in the levels of hepatic
    triglycerides was observed and liver histology showed microvesicular
    steatosis (Freneaux et al., 1988).

         The effects of TC on the synthesis and secretion of triglycerides
    and proteins were studied in isolated rat hepatocytes. TC produced a
    concentration-dependent inhibition of 14C-triglyceride secretion
    without affecting triglyceride synthesis and protein secretion
    (Deboyser et al., 1989).

    2.2.9.3  Nephrotoxicity

         Groups of Wistar albino rats were given single i.v. injections of
    150 mg TC/kg bw into the tail vein. Different experiments failed to
    demonstrate any significant ischaemia in the kidneys (Tapp & Lowe,
    1966)

    2.3  Observations in humans

    2.3.1  Effects after medical treatment

         In human therapy, the usual oral dose for TC-HCl is 250 or 500 mg
    every 6 h for adults as specified in the US National Formulary and
    British Pharmacopoeia. CTC is approved as capsules (50, 100 and
    250 mg) and by injection (100, 200 and 500 mg).

         A variety of adverse effects in humans have been reported from
    the use of TCs. The TCs are irritating substances. When given i.v.
    they can cause thrombophlebitis. Intramuscular administration is
    painful and not recommended. Given orally, TCs can cause epigastric
    burning, abdominal discomfort, nausea and vomiting (Schindel, 1965;
    NTP, 1989; Sande & Mandell, 1990).

         Bone and teeth discoloration are known to occur in humans under
    clinical treatment with high levels of TC drugs. Children receiving
    long- or short-term therapy with a TC may develop a yellow or brownish
    discoloration of the enamel. The duration of therapy appears to be
    less important than the total quantity of antibiotic administered. The
    risk of this effect is highest when the TC is given to neonates and
    babies prior to the first dentition. However, pigmentation of the
    permanent dentition may develop if the drug is given between the ages
    of 2 months and 8 years, when the teeth are being calcified. An early
    characteristic of this effect is a yellow fluorescence of the dental
    pigment.

         Treatment of pregnant patients with TCs may produce
    discolouration of the teeth in their children, but exposure also
    occurs during breast feeding. (Schindel, 1965; Piper et al., 1988;
    Friedman, et al., 1990; Sande & Mandell, 1990).

         TCs are deposited in the skeleton during gestation and throughout
    childhood. A 40% reduction in bone growth, as determined by
    measurements of fibulas, was demonstrated in premature infants treated
    with these agents. This effect is readily reversible if the period of
    exposure to the drug is short (Sande & Mandell, 1990; Pratt & Fekety,
    1986).

         No toxic effects were reported from treatment of premature
    infants, children, adults or elderly with CTC. Effects after short-
    and long-term exposure included increased body-weight gain, and
    prophylactic activity against acute and chronic infections and
    diarrhea (Hines, 1956).

         All the TCs (dose levels not specified) can cause phototoxicity,
    manifested by mild to severe skin reactions when the skin is exposed
    to direct sunlight (Sande & Mandell, 1990; Schindel, 1965).

         Liver effects, characterized by fatty accumulation have been
    observed, predominantly following repeated parenteral administration
    of TCs (dose levels were not specified). TCs can aggravate uremia in
    patients with impaired renal function. For unknown reasons, pregnant
    woman appear to be more susceptible to the development of hepatic
    damage (Pratt & Fekety, 1986; Sande & Mandell, 1990; Friedman,
    et al., 1990).

         TCs rarely cause allergic reactions. Hypersensitivity to TCs have
    been described as allergic manifestations of mucosal tissues. Central
    nervous system side effects and effects on blood cells were rarely
    described. Long-term therapy (dose levels not specified) with TCs may
    produce changes in the peripheral blood. Leucocytosis, atypical
    lymphocytes, toxic granulation of granulocytes, and thrombocytopenic
    purpura have been observed.

         The TCs may cause increased intracranial pressure and tense
    bulging of the fontanels (pseudotumor cerebri) in young infants, when
    given in therapeutic doses. Young adults complain of headache, nausea,
    vomiting and diplopia.

         TC is given orally in low doses (usually 100 mg/person/day) for
    the treatment of chronic severe acne vulgaris. A common complication
    of treatment with TCs is minor GI irritation (Sande & Mandell, 1990;
    Pratt & Fekety, 1986).

    3.  COMMENTS

    Toxicological data

         The Committee considered data from studies on pharmacokinetics,
    biotransformation, acute and short-term toxicity, reproductive
    toxicity, teratogenicity, long-term toxicity/carcinogenicity,
    genotoxicity, antimicrobial effects and observations in humans.
    Aspects of the comparative toxicity of these compounds with OTC were
    also considered.

         Pharmacokinetic studies showed that CTC and TC were rapidly
    absorbed following oral administration. Peak plasma concentrations
    were higher in rats receiving TC than in those receiving the same dose
    of CTC. In humans receiving oral therapeutic doses, 30% of the dose of
    CTC was absorbed, as compared with 60-80% of the dose of TC. TC is
    excreted mainly in urine, whereas CTC is excreted in urine and faeces
    in comparable amounts. After absorption following administration by
    various routes, CTC and TC are widely distributed in the body, the
    highest levels are present in the liver and kidney. Detectable levels
    of TCs in bone persist for more than 28 weeks after dosing. The plasma
    half-lives of CTC and TC in humans are about 5 and 8 h, respectively.
    TCs have been found in the breast milk of women receiving therapeutic
    doses and can cross the placenta.

         Orally administered CTC and TC have low acute toxicity in rats
    and mice, LD50s ranging from 2150 to >5000 mg/kg bw.

         In a 13-week range-finding study with mice in which TC was
    incorporated in the diet at concentrations equivalent to 470, 950,
    1800, 3700 or 7500 mg/kg bw/day, the only sign of toxicity observed
    was a decrease in body weight in the highest-dosed group. In a 13-week
    range-finding study in which rats were dosed at 155, 315, 625, 1250 or
    2500 mg TC/kg bw/day, vacuolization of liver cells and bone marrow
    atrophy were observed at 1250 and 2500 mg/kg bw/day. No toxicity was
    observed in dogs given TC orally at dose levels of up to
    250 mg/kg bw/day for 24 months.

         CTC did not cause any toxic effects in short-term toxicity
    studies with rats or mice at doses of up to 200 mg/kg bw/day. In dogs
    given CTC orally at a dose of 250 mg/kg bw/day for 98 days, increased
    incidences of mortality, fatty liver and bone marrow atrophy were
    observed. In another experiment in dogs which received CTC orally at
    doses of up to 100 mg/kg bw/day for 54 weeks, no effects were
    observed, except for signs of GI disorders; these were considered of
    minor importance because the increase in body-weight gain was normal.

         In carcinogenicity studies with mice and rats, TC was administered
    in the diet at doses of up to 3500 and 1060 mg/kg bw/day, respectively.
    Survival rates were increased in male mice, while body weights were
    slightly lower than controls for both sexes. Survival rates were also
    increased in female rats, but male rats showed a dose-related increase
    in basophilic cytoplasmic changes and clear cell changes in the liver.
    There were no increases in tumour incidence in mice or rats.

         In a chronic toxicity/carcinogenicity study, CTC was administered
    in the diet of rats at doses varying from 0.07 to 5200 mg/kg bw/day.
    In the highest dose group, GI irritation, decreased body-weight gain
    and a decreased number of white blood cells were observed in both
    sexes, while testicular atrophy was observed in males. Microscopic
    changes in the highest-dose group included infiltration of monocytes
    in the lungs of males and females, and fatty changes in the liver in
    males. Tumour incidence was not increased. The NOEL was 700 mg/kg
    bw/day. The Committee concluded that there was no evidence that CTC or
    TC is carcinogenic.

         In a two-generation reproductive toxicity study in which CTC was
    administered to rats in the diet at a dose equivalent to 500 mg/kg
    bw/day, no effects on reproductive performance were observed.
    Reproductive toxicity data were not available on TC. In a number of
    teratogenicity studies with rats, TC and TC hydrochloride caused a
    reduction in ossification at the highest oral dose tested, 540 or
    400 mg/kg bw/day, respectively. No irreversible structural changes
    were found on skeletal or visceral examination. In studies with a
    limited number (5 per group) of rats and rabbits, no evidence of
    teratogenicity was found at intravenous doses of 10 mg of CTC or TC/kg
    bw/day, administered on days 10-20 of pregnancy. The Committee
    concluded that CTC and TC did not cause significant toxic effects on
    either reproduction or development.

         The genotoxicity of CTC and TC was investigated in a wide range
    of  in vitro and  in vivo test systems. CTC gave negative results in
    all tests. TC was negative in the majority of  in vitro and  in vivo
    assays, but induced gene mutations in one  in vitro test in mammalian
    cells and chromosomal aberrations in plants. The Committee considered
    these effects to be due to the inhibiting effect of TCs on protein
    synthesis, based on their binding to ribosomes, and concluded that CTC
    and TC do not pose a genotoxic hazard.

    Microbiological data

          In vitro studies of the antimicrobial effect of TCs on
    pathogenic microorganisms of animal origin indicate that the
    activities of CTC and OTC are not significantly different. MIC values
    (geometric mean) for CTC and OTC were 0.32 and 0.52 g/ml,
    respectively.

         In a limited number of microorganisms representative of human gut
    flora, the antimicrobial activity of CTC was of the same order as that
    of OTC.

         The antimicrobial activity of TC was compared with that of OTC
    using a wide range of bacterial species isolated from the faeces of
    healthy human volunteers. The sensitivity of most species was similar
    for TC and OTC except for  Bifidobacterium, Fusobacterium and
     Eubacterium species which were slightly more sensitive to TC, and
     Streptococcus spp. which were slightly more sensitive to OTC. The
    geometric mean MICs for all tested strains were 3.2 g/ml for TC and
    3.8 g/ml for OTC.

    4.  EVALUATION

         After reviewing the available toxicological and antimicrobial
    data, the Committee concluded that the antimicrobial data provided the
    most appropriate end-point for the evaluation of CTC and TC.

         In view of the similarity of the antimicrobial activity of TC,
    CTC and OTC, the Committee established a group ADI of 0-3 g/kg bw for
    the three drugs separately or in combination. This ADI was established
    for OTC at the thirty-sixth meeting of the Committee (Annex 1,
    reference 91), and is based on the NOEL of 2 mg/person/day for the
    effects of OTC on the gut flora in human volunteers and a safety
    factor of 10.

         The Committee noted that this ADI provides an adequate margin of
    safety when compared with the lowest NOEL for toxicological effects of
    100 mg/kg bw/day for CTC in dogs.

    5.  REFERENCES

    Adir, J. (1975) Enterohepatic circulation of tetracycline in rats.
     J. Pharm. Sci, 64(11) 1847-1850.

    Bacharach, A.L., Clark, B.J., McCulloch, m. & Tomich, E.G. (1959)
    Comparative toxicity studies on ten antibiotics in current use.
     J. Pharm. Toxicol. 11: 737-741.

    Bert, F. & Vandoni, G. (1962) On the intestinal absorption and
    organotropism of some tertacyclines.  Chemotherapia 5, 219-230.

    Buyske, D.A., Eisner, H.J. & Kelly, R.G. (1960) Concentration and
    persistence of tetracycline and chlortetracycline in bone.
     J. Pharm. Exper. Therap. 130, 150-156.

    Chiang, T (1982) Inhibition of chlortetracycline activity by magnesium
    ions.  J. Assoc. Off. Anal. Chem., 65 (5), 1044-1047.

    Cunningham, R.W., Hines, L.R., Stokey, E.H., Vessey, R.E. & Yuda, N.N.
    (1954) Pharmacology of tetracycline.  Antibiotics Annual, 63-69.

    Deboyser, D., Goethals, F., Krack, G. & Roberfroid, M. (1989)
    Investigation into the mechnism of tetracycline-induced steatosis:
    study in isolated hepatocytes.  Tox.appl.pharmacol., 97, 473-479.

    Deichmann, W.B., Bernal, E., Anderson, W.A.D., Keplinger, M.,
    Landeen, K., Mcdonald, W., Mahon, R. & Stebbins, R. (1964) The chronic
    oral toxicity of oxytetracycline HCl and tetracycline HCl in the rat,
    dog and pig.  Ind.Med.Surg., 787-806.

    Dessau, F.I. & Sisson, G., (1957) Aureomycinr life studies. Studies
    of chlortetracycline hydrochloride (food grade in dogs). Project
    number 77601. Unpublished report of American Cynamid Company.
    P.R. 3, 440-493. Research Division. Pearl River Laboratories.
    Submitted to WHO by American Cyanamid Company, Princeton, USA.

    Dessau, F.I. & Sullivan, W.J., (1958) Aureomycinr life studies.
    Studies of chronic toxicity of CL 13,555: Chlortetracycline
    hydrochloride (food grade) in rats. Project number 77601 American
    Cyanamid Company, Experimental Pathology Research Division, Pearl
    River Laboratories. Submitted to WHO by American Cyanamid Company,
    Princeton, USA.

    Dessau, F.I. & Sullivan, W.J. (1961) A two year study of the toxicity
    of chlotetracycline hydrochloride in rats.  Toxicol and Appl.
     Pharmacol., 3, 654-677.

    Devriese, L.A. (1994) Comparative minimal inhibitory concentration
    determinations with chlortetracycline and oxytetracycline on gut
    bacteria of animal and human origin. Faculty of Veterinary Medicine.
    University of Gent, Belgium. Submitted to WHO by American Cyanamid
    Company, Princeton, USA.

    Diermeier, H.F. (1959) Acute oral and intravenous toxicity of rats of
    a A-VIII hydrochloride (CL 22,416), chlortetracycline hydrocholoride
    (CL 13,555), and tetracycline hydrochloride (CL13,554). P.R. 5,
    899-907. Unpublished report of American Cyanamid Company, Lederle
    laboratories, Pearl River, New York. Submitted to WHO by American
    Cyanamid Company, Princeton, USA.

    Dietz, D.D., Abdo, K.M., Haseman, J.K., Eustis, S.L. & Huff, J.E.
    (1991) Comparative toxicity and carcinogenicity studies of
    tetracycline and oxytetracycline in rats and mice.  Fund. Appl.
     Toxicol. 17 (2), 335-346.

    Eisner, H.J.  et al., (1953) The enhancement of serum levels of
    Aureomycin experimental animals.  J. Pharmacol. Exp. Ther.
    108, 442-449.

    Eisner, H.J. & Wulf, R.J. (1962) The metabolic fate of
    chlortetracycline and some comparisons with other tetracyclines.
     J. pharmacol, exp. therap., 142, 122-11.

    Fourtillan & Lefebvre (1985) cited in Rogalski, W. (1985) Chemical
    modifications of the tetracyclines. In: The tetracyclines. Eds.
    Hlavka and Boothe. Springer Verlag, Berlin.

    Freneaux, E., Labbe, G., Letteron, P. The Le Dinh, T.L., Degott, C.
    Geneve, J. Larrey, D. & Pessayre, D. (1988) Inhibition of the
    mitochondrial oxidation of fatty acids by tetracycline in mice and in
    man: possible role in microvesicular steatosis induced by this
    antibiotic.  Hepatology, 8(5), 1056-1062.

    Friedman, J.M., Little, B.B., Brent, R.L., Cordero, J.F., Hanson,
    J.W., & Shepard, T.H., (1990) Potential human teratogenicity of
    frequently prescribed drugs.  Obstetics and Gynecol, 75, 594-599.

    Goldenthal, E.I. (1971) A compilation of LD50 values in newborn and
    adult animals.  Tox. and Appl. Pharm, 18, 185-207.

    Gustafson, R.H. (1995) MIC tests of veterinary clinical isolates,
    tetracycline. Memo to D.L. Ingle, d.d. january 11, 1995. Submitted to
    WHO by American Cyanamid Company, Princeton, USA.

    Gyrd-Hansen, N (1980) The effect of tetracyclines on the rabbit heart.
     Zbl.vet.med. A, 27, 228-237.

    Halliday, S.L. & Fanelli, G. (1960) Antibacterial tetracycline:
    relative toxicity of CL 13,555, chlortetracycline hydrochloride and
    CL 30,782, sodium benzoate orally in rats. Project number 14797.
    P.R. 6, 1428-1439. Unpublished report of American Cyanamid Company,
    Lederle Laboratories, Pearl River, New York, Submitted to WHO by
    American Cyanamid Company, Princeton, USA.

    Harned, B.K. Cunningham, R.W. Clark, M.C., Cosgrove, P., Hine, C.H.,
    Mccauley, W.J., Stokey, E., Vessey R.E., Yuda, N.N. & Subbarow, Y.
    (1948) The pharmacology of Duomycin.  Ann. NYAS 51, 182-210.ALLESY,
    D.W. & HINE, C.H., (1964) The effect of chlortetracycline
    hydrochloride on the fertility and growth of rats.
     Tox. and Appl. Pharm. 6, 9-14.

    Harbell, J.W. (1988) CHO/HGPRT mutation assay. Study number
    T8133.332021. Unpublished report of Microbilogical Associates, 1988.
    Submitted to WHO by American Cyanamid Company, Princeton, USA.

    Hines, L.R. (1956) An apprasal of the effects of long-term
    chlortetracycline administration.  Antibiotics. chemother.,
    6 (11), 623-641.

    Hurley, L.S. & Tuchmann-Duplessis, H (1963) Influence de la
    tetracycline sur le developpement pr- et post-natal du rat.
     Compt. rend. acad. sci., 257, 302-304.

    Kanegis, L. (1958) The comarative pharmacology of tetracyclines:
    initial studies on serum levels and urinary excretion of antibiotic
    A-VIII following single oral and intravenous doses in the dog. Report
    P.R. 4:884-921. American Cyanamid Company (Pearl River). Submitted to
    WHO by American Cyanamid Company, Princeton, USA.

    Kelly, R.G. (1964) Antibacterial tetracyclines. In tissue distribution
    of tetracycline and chlortetracycline. American Cyanamid Company
    Report P.R. 9:485-492 (Pearl River). Submitted to WHO by American
    Cyanamid Company, Princeton, USA.

    Kelly, R.G. & Buyske, D.A. (1960) Metabolism of tetracycline in the
    rat and the dog.  J. Pharmacol. Exp. Therap., 130, 144-149. Submitted
    to the WHO by American Cyanamid Company, Princeton, USA.

    Kniffen, T.S., Bane, D.P., Hall, W.F., Koritz, G.D. & Bevill, R.F.
    (1989) Bioavailability, pharmacokinetics, and plasma concentration of
    tetracycline hydrochloride fed to swine.  Am. J. Vet. Res.
    50, 518-521.

    Levinskas, C.J. (1963) Auromycinr and formulation: Acute oral and
    intravenous toxicity. Central Mediacal Department Report
    No. 6305, 53-5,. Submitted to the WHO by American Cyanamid Company,
    Princeton, USA.

    Lowe C.A. & Fisher J. (1993) Rat oral LD50 study with Aureo Biomass.
    Report number A93-61. Data from American Cyanamid Company, Agricultural
    Research Division. Submitted to WHO by American Cyanamid Company,
    Princeton, USA. (summary only)

    Maffii, G. & Mainardi, L. (1955) Studio Farmacologico su un nuovo
    antibiotico: la tetraciclina.  Il Farmaco Ed. Sci 10 (4), 197-210.

    Mann, S.K. (1978) Interaction of tetracycline (TC) with chromosomes in
    Allium cepa  Environm. exp. botany. 18, 201-205.

    Martindale (1989) The Extra pharmacopoeia. 29th edition. Eds. J.E.F.
    Reynolds. London, The Pharmaceutical Press.

    Mccoll, J.D. Globus, M. & Robinson, S (1965) Effect of some therapeutic
    agents on the developing rat fetus.  Tox. appl. pharmacol. 7,
    409-417.

    Meisner, L.F., Chuprevich, T.W. & Inhorn, S.L. (1977) Mechanisms of
    chromatid breakage in human lymphocyte cultures.  Acta. cytol.
    21(4), 555-558.

    Milch, R.A., Rall, D.P. & Tobie, J.E. (1967) Bone localization of the
    tetracyclines.  J. Nat. Canc. Inst. 19, 87-91.

    Mulligan, E. (1989) Microbial mutagenicity test report for screening
    study MMR89-026. American Cyanamid Company, Report number MMR89-026,
    20 April 1989. Submitted to WHO by American Cyanamid Company,
    Princeton, USA.

    Nelson, A.A. & Radomski, J.L. (1954) Comparative pathological study in
    dogs of feeding of six broad-spectrum antibiotics.  Antibiotics and
     Chemotherapy IV, (11), 1174-1180.

    Neuschl, J. (1991) Comparison of some pharmacokinetic parameters of
    tetracyclines which are most frequently used in veterinary medicine.
     Arch. Exp. Veterinarmed. 45, 105-112.

    NTP (1989) NTP technical report on the toxicology and carcinogenesis
    studies of tetracycline hydrochloride (Cas no. 64-75-5) in F344/N rats
    and B6C3F1 mice (feed studies) NTP toxicology program P.O. Box 12233.
    NTP TR 344 NIH Publication No. 89-2600. U.S. Department of health and
    human services.

    Piper, J.M., Baum, C., Kennedy, D.L. & Price, P. (1988) Maternal use
    of prescribed drugs associated with recognized fetal adverse drug
    reactions.  Am. J. Obstet. and Gynecol. 159 (5) 1173-1177.

    Pratt, W.B. & Fekety, R. (1986) The antimicrobial drugs. Oxford
    University Press, New York, pp 205-228.

    Putman, D.L. (1988) Acute in vivo cytogenetics assay in rats. Final
    report. Report number T8133.105003. Unpublished report of
    Microbiological Associates, 11-7-1988. Submitted to WHO by American
    Cyanamid Company, Princeton, USA.

    Richez, P. (1994) Antibacterial activity of tetracycline and
    oxytetracycline against human gut flora. Study No. SVL 029.
    Unpublished report of Datavet Veterinary Drug Consultants,
    May 31 1994. Submitted to WHO by Intervet International,
    Boxmeer, Netherlands.

    Rogalski, W. (1985) Chemical modifications of the tetracyclines.
    Chapter 5, pp 233-. In: The tetracyclines. Eds. Hlavka and Boothe.
    Springer Verlag, Berlin.

    Sande, M.A. & Mandell, G.L. (1990) Antimicrobial agents:
    tetracyclines, chloramphenicol, erythromycin and miscellaneous
    antibacterial agents. In: Goodman and Gilman's The pharmacological
    basis of therapeutics, 8th edition. Gilman, A.G., Rall, T.W., Nies,
    A.S. and Taylor, P. eds. Pergamon Press, New York, (1990),
    pp 1117-1145.

    Schindel, L.E. (1965) Clinical side-effects of the tetracyclines.
     Antibiotica et Chemotherapia Advances 13, pp. 300-316.

    Sharma, A.K. & Bhattacharyya, G.N. (1967) A study on the respomse of
    chromosomes to antibiotic treatment.  Acta Biol. Acad. Sc. Hung.
    18 (1), 67-75.

    Sharma, R.K., Traul, K.A., Caterson, C.R., Harbell, J., Thilagar, A. &
    Putman, D. (1989) Genotoxicity evaluation of chlortetracycline.
     Environ. Mol. Mutagen 14 (suppl. 15), 183. (summary only).

    Shiota, K., Uwabe, C. Yamamoto, M. & Arishima, K. (1990) Teratogenic
    drugs inhibit the differentiation of fetal rat limb buds grafted in
    athymic (nude) mice.  Reprod. toxicol. 4, 95-103.

    Szumigowska-Szrajber, G. & Jeske J. (1970) Effect of tetracycline
    base, tetracycline hydrochloride and oxytetracycline base on
    developmental changes in metatarsal bones of extremities of rat
    fetuses.  Acta polon. pharm. 27 (I), 85-90.

    Tapp, E. & Lowe, M.B. (1966) Tetracycline toxicity.  Experientia
    22, 530-531.

    Thilagar, A. (1988) Test for chemical induction of unscheduled DNA
    synthesis in rat primary hepatocyte cultures by autoradiography. Test
    report 0082-5100. Unpublished report of Sitek Research Laboratories,
    22-7-1988. Submitted to WHO by American Cyanamid Company, Princeton,
    USA.

    Tsutsui, T., Umeda, M., Sou, M. & Maizumi, H. (1976) Effect of
    tetracycline on cultured mouse cells.  Mutat. res. 40, 261-268.

    Tubaro, E. (1964) Possible relationship between tetracycline stability
    and effect on foetal skeleton.  Brit. J. Pharmacol. Chemotherap.
    23, 445-448.

    Tubaro, E. & Banci, F. (1964) The pharmacology of chlormethylene-
    cycline, a new chlortetracycline.  Arzneimittel-Forschung, 14,
    95-100.

    Tubaro, E., Barletta, M. & Banci, F. (1964) Some pharmacological
    aspects of a new water-soluble tetracycline.  J.pharm. pharmacol.
    16, 33-37.

    Walter, A.M. & Heilmeyer, L. (1975) Antibiotika-Fibel. Antibiotika und
    Chemotherapeutila Therapie mikrobieller infektionen, pp.323-324.
    Georg Thieme Verlag Stuttgart.

    Wulf, R. & Eisner, H. (1961) Antibacterial tetracyclines: Metabolism
    of chlortetracycline in the rat and in the dog. American Cyanamid
    Company Report C.P. 1, 626-683. Experiment P-70-21-FT. Submitted to
    WHO by American Cyanamid Company, Princeton, USA.
    


    See Also:
       Toxicological Abbreviations