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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

    WORLD HEALTH ORGANIZATION



    TOXICOLOGICAL EVALUATION OF CERTAIN 
    VETERINARY DRUG RESIDUES IN FOOD



    WHO FOOD ADDITIVES SERIES 45





    Prepared by the
    Fifty-fourth meeting of the Joint FAO/WHO
    Expert Committee on Food Additives (JECFA)



    World Health Organization, Geneva, 2000

    LINCOMYCIN

    First draft prepared by
    Kevin J. Greenlees
    Center for Veterinary Medicine, Food and Drug Administration,
    Rockville, Maryland, USA

    Arturo Anadon
    Department of Toxicology, Faculty of Veterinary Medicine, Universidad
    Complutense de Madrid, Madrid, Spain

    and

    Carl Cerniglia
    National Center for Toxicological Research, Food and Drug
    Administration, Little Rock, Arkansas, USA

            Explanation
            Biological data
                Biochemical aspects 
                    Absorption, distribution, and excretion
                    Biotransformation 
                Toxicological studies 
                    Acute toxicity
                    Short-term studies of toxicity 
                    Long-term studies of toxicity and carcinogenicity 
                    Genotoxicity 
                    Reproductive toxicity 
                        Multigeneration studies 
                        Developmental toxicity 
                    Special studies 
                        Immune responses 
                        Ototoxicity 
                        Microbiological effects 
                Observations in humans 
            Comments 
            Evaluation
            References 


    1.  EXPLANATION

         Lincomycin, like pirlimycin and clindamycin, belongs to a class
    of antibiotics known as lincosaminides. Lincomycin is active mainly
    against Gram-positive bacteria. It exerts its antibiotic action by
    inhibiting RNA-dependent protein synthesis through action on the 50S
    subunit of the ribosome. Lincomycin is used alone or in combination
    with other antimicrobial agents such as spectinomycin, neomycin,
    sulfadiazine, and sulfadimidine. It can be given orally in feed or
    drinking-water, by intramuscular injection, or as an intramammary
    infusion. 

         The recommended doses are: 0.5 mg/kg bw in feed and 3-50 mg/kg bw
    in drinking-water for poultry; 0.2-13 mg/kg bw in feed, 5-10 mg/kg in
    drinking-water, and 5-10 mg/kg bw intramuscularly in pigs; 5 mg/kg bw
    intramuscularly in calves and sheep; and 200-330 mg/quarter of the
    udder as an intramammary infusion, three times after each milking, in
    dairy cows.

         The Committee has not previously evaluated lincomycin.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion

          Dogs 

         A preliminary study, which did not comply with good laboratory
    practice (GLP), conducted in a male and a female dog showed that
    lincomycin is rapidly absorbed after intramuscular injection at 20
    mg/kg bw (Sokolski, 1962). Peak serum concentrations were reached 30
    min after injection of 9 mg of the base per ml. In two beagle dogs
    given a single dose of 300 mg/kg bw by capsule, peak absorption
    occurred within 1-2 h of dosing (Grady & Treick, 1961; Gray &
    Purmalis, 1961a).

          In a 90-day study, lincomycin was determined in lung, liver,
    kidney, muscle, bile, spinal fluid, and serum of beagles dosed orally
    at 0, 400, or 800 mg/kg bw per day. The highest concentrations were
    observed in kidney and bile (66 and 680 mg/g, respectively) and the
    lowest concentrations in spinal fluid (below the limit of detection)
    from animals at the high dose. One control had a concentration of 39
    mg/g in the lung and trace levels in the liver; it was speculated that
    the animal had been inadvertently dosed just before sacrifice (Gray &
    Purmalis, 1964a). 

          Humans 

         The pharmacokinetics of lincomycin in humans has been determined
    after administration by several routes (Table 1). About 72% is bound
    to proteins in human serum. Lincomycin is widely distributed, with a
    volume of distribution approximating total body water, and it is
    excreted in the faeces. Biliary excretion has also been reported to be
    an important route of elimination. Significant concentrations of
    lincomycin are achieved in a number of tissues and fluids regardless
    of the route of administration. These include bile, peritoneal fluid,
    pleural fluid, the eye, brain, bone, bone marrow, joint capsules,
    synovial fluid, and cerebrospinal fluid. It is generally poorly
    distributed into cerebrospinal fluid except in the presence of
    inflammation. Therapeutic concentrations have been achieved in the
    presence of meningitis (Fass, 1981).

        Table 1. Pharmacokinetics of lincomycin in humans

                                                                                                 
    Route           Parameter                             Dose (mg)                Reference
                                                                                                 

    Intramuscular                                         600     1000   1500      Smith et al.
                    Peak serum concentration (g/ml)      12      17     22        (1981)
                       AUCto 24 (mg/mlh)                 82      120    150
                       AUCto infinity (mg/mlh)           92      130    160
                       Time of peak (h)                   1.2     1.5    0.92
                       Elimination t1/2 (h)               4.5     5.3    5.3
                    Peak saliva concentration (mg/ml)     0.86    1.6    2.7
                       Time of peak (h)                   3.7     4.7    3.9
                       AUCto 24 (mg/mlh)                 5.3     10     18

    Intravenous,                                          300     600              Fass (1981)
    2 h             Mean concentration (mg/ml)            7.7-12         16-21

    Oral            Adults                                500     1000             Fass (1981)
                       Peak serum concentration (mg/ml)a  1.8-5.3        2.5-6.7
                       Elimination t1/2 (h)               4.2-5.5
                       Time of peak (h)                   2-6, usually 4

                    Children                              22-33 mg/kg bw
                       Peak serum concentration (mg/ml)   4-9  (maintained above 
                                                          1.0 mg/ml for 15 h)
                                                                                                 

    a  Presence of food in the stomach markedly impairs absorption (Kucers & Bennett, 1979; 
       Fass, 1981). The oral bioavailability is estimated to be 25-50% after fasting but only 5% 
       with a meal (Hornish et al.,1987).
    

         Lincomycin has been shown to cross the placenta, and peak
    concentrations in amniotic fluid of 0.2-3.8 mg/ml are sustained for
    52 h after a single intramuscular injection of 600 mg to pregnant
    women. Lincomycin has been found to be present in milk  post partum 
    (Fass, 1981).

         In a report written to support the safety of the lincosaminide
    pirlimycin, which also addressed the safety of lincosaminides in
    general and of lincomycin in particular, it was noted that only a
    fraction of an oral dose of lincosaminides reaches the lower intestine
    (Kotarski, 1995). While orally administered clindamycin is nearly
    completely absorbed after oral administration (Kapusnik-Uner et al.,
    1996), lincomycin is poorly although rapidly absorbed from the
    gastrointestinal tract (Goodman & Gilman, 1975). The human
    bioavailability of orally administered lincomycin is estimated to be
    25-50% for fasting individuals but only 5% after a meal (Hornish et

    al., 1987). About 10% of orally administered clindamycin is excreted
    unaltered in the urine, and a small fraction is found in the faeces
    (Kapusnik-Uner et al., 1996).

    2.1.2  Biotransformation

         The comparative metabolism of lincomycin was reported in rats,
    cows, pigs, and chickens. Lincomycin was extensively metabolized in
    all tissues but not in cow's milk (after intramammary infusion only).
    Some 16 metabolites were identified, although there were as many as 26
    in pig liver. The principal residues were parent lincomycin,
     N-desmethyl lincomycin, and lincomycin sulfoxide (Hornish et al.,
    1987; Nappier, 1998).

         Approximately 5% of an oral dose administered to rats was
    excreted in urine, where lincomycin and lincomycin sulfone comprised
    97% of the excreted drug; 95% of the drug was found in the
    gastrointestinal tract (Hornish et al., 1987).

         The principal urinary and faecal metabolite in dogs and humans
    after oral and intramuscular administration was unchanged lincomycin,
    representing 40% of the excreted dose; most of the remainder was
    unidentified. There was no evidence of glucuronide or sulfate
    conjugation (Hornish et al., 1987).

         Pig excreta contained markedly less unchanged lincomycin than
    that of other species studied. The urine contained 11-21% of an oral
    dose, and half of this was unchanged parent. Only trace amounts of
     N-desmethyllincomycin were identified. The contents of the
    gastrointestinal tract accounted for 79-86 % of the excreted drug. In
    faecal samples, only 17% of the excreted dose was unchanged parent,
    and the remainder was uncharacterized metabolites (Hornish et al.,
    1987)

    2.2  Toxicological studies

    2.2.1  Acute toxicity

         Lincomycin was toxic in mice and rats when administered
    parenterally and was practically nontoxic after oral administration.
    Lincomycin was toxic to rabbits by all routes of administration.

          Mice 

         The acute LD50 in male mice treated orally was determined for
    USP grade and Premix grade lincomycin (Buller, 1979). No significant
    difference between the LD50 values of 20 000 and 17 000 mg/kg bw was
    determined in this non-GLP study. An LD50 value of 210 mg/kg bw was
    measured in mice treated intravenously, and the signs of toxicity in
    the survivors included severe sedation lasting 1-2 min (Gray &
    Highstrete, 1963a).

          Rats 

         The acute toxicity of lincomycin was determined in a preliminary
    non-GLP study in newborn and adult rats treated by subcutaneous
    injection (Gray & Purmalis, 1962a). The LD50 in newborn rats was 780
    mg/kg bw, while that in adults was 10 000 mg/kg bw. An intravenous
    injection was reported to be more toxic, with an LD50 of 340 mg/kg bw
    (Gray & Highstrete, 1963a).

         The acute toxicity of agricultural-grade lincomycin (Glenn &
    Garza, 1971) and of USP-grade lincomycin (Brown, 1977a,b) was
    determined in a series of non-GLP studies in Sprague-Dawley rats.
    Lincomycin was administered orally to groups of five animals of each
    sex at doses of 5000-16 000 mg/kg bw, and clinical signs and body
    weights were monitored for 2 weeks after treatment. All doses resulted
    in clinical signs of toxicity including diarrhoea and ataxia.
    Depression was observed at doses > 8000 mg/kg bw, and death,
    preceded by coma, was observed at doses of 12 500 and 16 000 mg/kg bw.
    While there was no significant effect on body-weight gain, the
    survivors continued to have diarrhoea for up to 36 h after treatment.
    The LD50 was determined by probit analysis to be about 15 000 mg/kg
    bw for USP lincomycin and 11 000 mg/kg bw for the agricultural-grade
    product. In a separate study, an LD50 of 16 000 mg/kg bw was
    determined for a premix grade preparation of lincomycin (Nielsen,
    1975).

          Rabbits 

         Rabbits have been shown to be quite sensitive to orally
    administered lincomycin (Gray et al., 1965a). After a single
    intravenous injection of 0.5 mg/kg bw, 5 out of 10 rabbits either died
    or were killed for humane reasons within 2 weeks of dosing, and 7 out
    of 10 rabbits had died by 1.5 months. In two studies that did not
    comply with GLP, in which groups of three rabbits were given
    lincomycin, only the lowest dose of 0.5 mg/kg bw was not lethal. All
    the other doses (5, 50, 100, and 150 mg/kg bw) caused death, such that
    by 4 weeks 9 out of 15 and 12 out of 15 rabbits had died. Histological
    examination revealed gastrointestinal stasis and, in those animals
    that died, haemorrhagic suffusion of the serosal surface of the
    caecum. Attempts to modify the toxicity by supplementation with
     Lactobacillus culture or intubation with fresh (rabbit) caecal
    contents were not successful. The observed toxicity was considered to
    result from gastrointestinal Gram-positive floral imbalance.

         The irritability of lincomycin to tissues was investigated in
    rabbits in a series of studies that did not comply with GLP. Doses of
    up to 300 mg/kg bw were injected into the lumbar muscle at pH 4 (Gray
    & Purmalis, 1962b) or pH 7.4 (Gray & Purmalis, 1962c). No difference
    was seen in the minimal to mild muscular irritation after slaughter up
    to 7 days after treatment. Injection of up to 150 mg of lincomycin
    into the stifle joint of New Zealand white rabbits caused no
    treatment-related effects, such as intra-articular irritation (Gray &
    Highstrete, 1965).

          Dogs 

         A series of studies that did not comply with GLP were conducted
    in dogs. In one study of single intrathecal injections of lincomycin,
    two dogs received 15 mg in 1 ml of solution, eight dogs received 50 mg
    in 1 ml of solution, and 10 dogs received the vehicle (isotonic saline
    and benzyl alcohol at 9 mg/ml). No treatment-related clinical effects
    were reported. Cerebrospinal fluid samples taken up to 48 h after
    injection were cloudy and had increased cell counts consisting
    predominantly of lymphocytes. Gross and microscopic examination 24-72
    h after injection did not reveal treatment-related effects, such as
    meningeal irritation (Gray et al., 1965b).

         When intravenous or intramuscular injections of 150 mg/kg bw per
    day were given for 5 and 3 days, respectively (Gray & Purmalis,
    1962d), no treatment-related effects were reported. 

         Lincomycin was injected subcutaneously for 14 days into four pups
    from each of three litters within 24 h of birth at a dose of 0, 30,
    60, or 90 mg/kg bw per day. No significant treatment-related effects
    were reported (Gray et al., 1962).

         In a preliminary study, lincomycin was administered at a dose of
    4000 mg/kg bw orally by gavage for 5 days to two female beagles. While
    both animals vomited for 1-2 h after gavage, no treatment-related
    effects (such as diarrhoea) were reported. In the same study, a third
    dog received an intravenous dose of 940 mg/kg bw in a volume of 230 ml
    as two injections. Transient prostration and slightly increased
    activities of alanine and aspartate aminotransaminases were seen,
    suggesting some hepatic toxicity. The heart rate and respiration
    remained normal, and no histopathological alterations were found in a
    liver biopsy sample 5 days later. The dog appeared clinically normal
    during the subsequent 2 weeks of observation, and no other
    treatment-related effects were reported (Gray & Purmalis, 1963a). 

    2.2.2  Short-term studies of toxicity

          Mice 

         Lincomycin of premix grade was administered for 90 days to groups
    of 15 B6C3F1 mice of each sex at concentrations of 0, 70, 200, 700,
    2000, or 20 000 mg/kg in the feed, equivalent to daily doses of 0, 10,
    30, 100, 300, and 3000 mg/kg bw per day. While the study was completed
    in 1979, before initiation of GLP requirements, a quality assurance
    statement was provided which addressed deviations from GLP. The
    highest dose resulted in a significant suppression of body weight,
    increased food consumption and intestinal weight (with pancreas), a
    decreased serum glucose concentration, and, in females, increased
    serum corticosterone concentration, decreased serum globulin
    concentration, and decreased mean thymus weight. While the mean organ
    weights of animals at the highest dose were the lowest for heart,
    liver, spleen, and kidney (males only), the differences were not
    statistically significantly different from controls. Histologically,

    the lumina of the large and small intestine were found to be distended
    and dilated. The next highest dose of 300 mg/kg bw per day also
    increased intestinal weight (with pancreas) and increased the
    incidence of luminal distention and dilatation of the small and large
    intestines. The serum glucose values were also depressed. The NOEL was
    100 mg/kg bw per day (Platte & Seaman, 1981).

          Rats 

         In a study that did not conform to GLP, groups of five Wistar
    rats of each sex were given lincomycin at a dose of 0, 30, 100, or 300
    mg/kg bw per day by oral gavage for 30 days. Effects on body weight,
    food consumption, organ weights, haematological values, and
    pathological findings were reported, but no significant
    treatment-effects were found at any dose. The NOEL was 300 mg/kg bw
    per day, the highest dose tested (Gray & Purmalis, 1961b).

         In an extension of this study to 3.5 months, lincomycin was
    administered orally by gavage at a dose of 0, 30, 100, or 300 mg/kg bw
    per day to groups of 10 Upjohn Wistar rats of each sex. No
    drug-related effects were observed on body-weight gain, food
    consumption, or pathological findings. The NOEL was 300 mg/kg bw per
    day, the highest dose tested (Gray & Purmalis, 1962e).

         When the study was repeated in groups of 20 rats of each sex at a
    dose of 600 or 1000 mg/kg bw per day for 3 months, the average weight
    of the intestinal tracts of all treated animals was greater than that
    of controls, but it was not clear whether this was due to the tissue
    or the content, as no changes were observed in the intestinal wall or
    mucosa on gross or microscopic examination. The NOEL was 1000 mg/kg bw
    per day, the highest dose tested (Gray & Purmalis, 1964b).

          Dogs 

         In a study that did not conform to GLP, groups of three beagles
    received lincomycin by intramuscular injection at a dose of 0, 15, 30,
    or 60 mg/kg bw per day administered twice daily for 4 weeks. Two of
    the dogs -- one control and one at 60 mg/kg bw per day -- showed mild
    lymphocytic infiltration of the thyroid. Since this effect occurred in
    only two animals and equally in treated and control groups, it was not
    ascribed to lincomycin. Aside from mild inflammatory reactions at the
    injection site observed at necropsy in all groups, including controls,
    no treatment-related effects were reported. The NOEL was 60 mg/kg bw
    per day, the highest dose tested (Gray & Purmalis, 1962f).

         In a further study that did not comply with GLP, lincomycin was
    given to groups of three beagles by capsule three times a day for 30
    days at a dose of 0, 30, 100, or 300 mg/kg bw per day. Daily
    examinations for body weight, haematological and urinary analyses, and
    gross and histopathological examination revealed no treatment-related
    effects (Gray & Purmalis, 1962g).

         In another study that did not comply with GLP, groups of four
    beagles of each sex were given lincomycin at a dose of 0, 400, or 800
    mg/kg bw per day in gelatin capsules administered three times a day
    for 90 days. Transient increases in serum alanine aminotransferase
    activity were observed during the first month of treatment at 800
    mg/kg bw per day and in one animal at the low dose, but the level had
    returned to normal by the end of the study. Bilateral lymphocytic
    thyroiditis was observed in three controls, two animals at 400 mg/kg
    bw per day and two at 800 mg/kg bw per day. This condition had also
    been observed in other beagle colonies. Mild lymphocytic infiltration
    was reported in other organs as well. These lesions did not appear to
    be treatment-related. The NOEL was 800 mg/kg bw per day, the highest
    dose tested (Gray & Purmalis, 1964a). 

         In a study that did not comply with GLP, lincomycin was
    administered in capsules to groups of two beagles of each sex at a
    dose of 0, 30, 100, or 300 mg/kg bw per day for 6 months. No
    treatment-related effects were reported on body weight, organ weights,
    or haematological, clinical chemical, or urinary end-points. The
    summary and conclusions of the report state that further
    histopathological analysis revealed lymphocytic thyroiditis in the
    male and female at the high dose and similar infiltration of the
    kidney in one of the animals (Gray et al., 1963b). Lesions of this
    type were observed at all doses in a later, 90-day study (Gray &
    Purmalis, 1964a). The European Medicines Evaluation Agency (1998)
    identified a NOEL of 100 mg/kg bw per day in the study of Gray et al.
    (1963b) on the basis of an increase in adrenal weight at the high
    dose. However, while a paired  t test showed a significant difference
    in adrenal weights, the relative weights were not significantly
    altered and no significant difference was found in an unpaired  t 
    test. The NOEL was 300 mg/kg bw per day, the highest dose tested.

         Lincomycin was administered orally as the premix grade at a dose
    of 0, 0.38, 0.75, or 1.5 mg/kg bw per day or as the USP grade at 1.5
    mg/kg bw per day in gelatin capsules for 1 year to groups of five
    beagles of each sex in a study that did not conform to GLP. The doses
    were chosen to support a proposed tolerance of lincomycin of 1 mg/kg
    in the edible tissues of poultry, pork, beef, lamb, and dairy
    products, and were based on multiples of 25, 50, and 100 times the
    maximum theoretical human dietary intake. The measured end-points
    included clinical and ophthalmic parameters, food consumption, body
    weights, clinical pathological, chemical, and urinary parameters,
    organ weights, and gross and histological appearance. No differences
    were observed between animals receiving the premix and USP grades, and
    no treatment-related effects were reported. The NOEL was 1.5 mg/kg bw
    per day of each grade of lincomycin, the highest doses tested (Goyings
    et al., 1979a).

    2.2.3  Long-term studies of toxicity and carcinogenicity

          Rats 

         Groups of 10 rats of each sex received lincomycin at a dose of 0,
    30, 100, or 300 mg/kg bw per day by oral gavage for 1 year. The study
    did not comply with GLP. All rats were necropsied, four of each sex
    per group were examined histologically, and haematological parameters,
    body-weight gain, organ weights, and pathological findings were
    reported. No treatment-related effects were found. While there was a
    significant difference ( p = 0.019, two-tailed  t test) in the
    weight of the liver between controls (19  2.3 g) and rats at the high
    dose (24  4.9 g), the weights relative to body weight were not
    significantly different. The NOEL was 300 mg/kg bw per day, the
    highest dose tested (Gray et al., 1963a). 

         In a study that did comply with GLP, groups of pregnant
    Sprague-Dawley rats and groups of 60 of the resulting offspring of
    each sex received field-grade premix lincomycin orally at a dose of 0,
    0.38, 0.75, or 1.5 mg/kg bw per day or USP-grade lincomycin at a dose
    of 1.5 or 100 mg/kg bw per day. Treatment of the offspring was
    continued for 26 months. Food consumption per cage (two rats per cage)
    was monitored weekly; body weights were monitored weekly through week
    56 and during alternate weeks thereafter. Serum chemistry was
    evaluated at 6 and 12 months and at termination, and haematological
    parameters were assessed before treatment, at 3, 6, and 12 months, and
    at termination. Organ weights and urinary parameters were measured at
    the interim kills and at termination. All rats that died or were
    killed were examined grossly and histologically; a full
    histopathological examination was performed on animals in the control
    and the two high-dose groups. 

         The percentage survival, clinical and ophthalmological
    end-points, food consumption, organ weights, and haematological, serum
    chemical, and urinary parameters were unaffected by treatment. A
    statistically significant effect on growth promotion was observed up
    to day 574 of the study in animals given 0.75 mg/kg bw per day of
    premix, but not thereafter. An increased incidence of non-neoplastic
    microscopic lesions of the prostate and seminal vesicles (acute
    prostatitis and seminal vesiculitis) was reported in males at 1.5
    mg/kg bw per day of premix and at 100 mg/kg bw per day of USP grade.
    The frequency of prostatis was 21/59 in controls, 1/35 in rats at 0.38
    mg/kg bw per day, 5/45 in rats at 0.75 mg/kg bw per day, 40/60 in
    those at 1.5 mg/kg bw per day of premix, 3/40 in rats at 1.5 mg/kg bw
    per day of USP grade, and 31/59 in those at 100 mg/kg bw per day of
    USP grade. At the 1-year interim slaughter, the frequency of prostatis
    was 4/10 in controls, 2/10 in rats at 0.75 mg/kg bw per day of premix,
    and 2/10 for those at 100 mg/kg bw per day of USP grade. A review of
    the data for individual animals showed no dose-response relationship,
    and there was no increase in the relative severity of the lesions. The
    prostatis was therefore considered to be unrelated to treatment with
    lincomycin.

         The numbers of benign, malignant, and total tumours in each
    treated group were not statistically significantly different from
    those in the concurrent vehicle control group (Table 2). A
    statistically significant increase in the number of males with
    subcutaneous fibromas was observed at the high dose of USP-grade
    material when compared with concurrent controls, but the total number
    of fibromas was not significantly different. A statistically
    significant increase in the incidence of lymphosarcoma was observed in
    females at 1.5 (6/52) and 100 mg/kg bw per day (7/60) of USP grade
    when compared with control females (1/59). A trend analysis of these
    incidences did not, however, show a significant linear component, and
    it was concluded that the lymphosarcomas were not related to
    treatment. No increase in the incidence of lymphosarcomas was seen in
    males. The incidence of mammary adenomas and cystadenomas in females
    at 1.5 mg/kg bw per day of USP material (10/52) was greater than that
    in concurrent control females (4/59,  p = 0.083) but there was no
    difference in the total number of benign mammary neoplasms. Similarly,
    the incidence of mammary adenocarcinomas and carcinomas in females at
    1.5 mg/kg bw per day of USP material (9/52) exceeded that in
    concurrent female controls (3/59,  p = 0.063). However, the 5.1%
    incidence rate of mammary carcinomas in the concurrent control females
    was well below the 12% incidence rate (23/196) reported for historical
    female controls. While a large number of pituitary adenomas and
    mammary fibroadenomas were observed, these lesions are common in
    Upj:TUC(SD) rats and were not related to treatment.

        Table 2. Numbers of benign, malignant, and total tumours in rats fed diets containing 
             lincomycin of two grades

                                                                                           
    Sex        Tumour       Vehicle    Premix (mg/kg bw per day)    USP (mg/kg bw per day)
                                                                                           
                                       0.38     0.75     1.5        1.5      100
                                                                                           

    Males      Malignant    9          11       13       9          15       10

               Benign       39         25       33       35         22       37

               Total        43         29       38       38         33       40

    Females    Malignant    12         12       15       11         18       15

               Benign       43         39       43       44         40       47

               Total        47         44       47       49         49       51
                                                                                           

    
         Neither premix nor USP-grade lincomycin was carcinogenic under
    the conditions of the assay, but the low maximum dose used and the
    poor survival preclude a definitive conclusion. The NOEL for
    non-neoplastic effects was 100 mg/kg bw per day, the highest dose
    tested. 

    2.2.4  Genotoxicity

         A battery of tests that complied with GLP were conducted to
    address the genetic toxicity of lincomycin (Table 3). The only
    positive result was obtained in an assay for unscheduled DNA synthesis
    in rat primary hepatocytes (Harbarch & Aaron, 1987). While only an
    abstract of this study was available, the positive result was
    duplicated in another assay at the relatively low dose of 0.17 g/ml.
    In these assays, scoring could not be done at doses > 0.17 g/ml
    because of the cytotoxicity of lincomycin. In addition, while the full
    report with raw data is available, it was not made available to the
    Committee. The positive results were addressed in a subsequent report
    to the US Food and Drug Administration (Aaron, 1988b), which refers to
    a similar assay in which negative or equivocal results were obtained
    (Seaman, 1982) after use of an improved procedure for microscope slide
    preparation. The report also noted that negative results were obtained
    in a study that did not conform to GLP in which the same lot of
    lincomycin was used as that in the assay with positive results. In the
    second assay, the toxicity of lincomycin was much lower (> 300
    g/ml), allowing scoring at doses as high as 1000 g/ml. The lower
    toxicity is consistent with that in other assays with negative results
    (Seaman, 1982; Aaron, 1988a). The weight of the evidence suggests that
    lincomycin is not genotoxic.

    2.2.5  Reproductive toxicity

         (a)  Multigeneration studies

         In a three-generation study of reproductive toxicity that did not
    comply with GLP, 30 male and 60 female F0 and 10 male and 20 female
    F2 and F3 Sprague-Dawley rats were treated with lincomycin premix
    grade at 0, 0.38, 0.75, or 1.5 mg/kg bw per day or USP grade at 1.5 or
    100 mg/kg bw per day in the diet, beginning with F0 weanling rats,
    through successive breeding of the F0, F1, and F2 progeny, to
    weaning of the F3a litters. The doses of premix grade were based on
    multiples of 0, 25, 50, 100, and 7000 times the maximum anticipated
    human dietary intake, given a tolerance in edible tissues of 1 mg/kg.
    No treatment-related effects were reported on clinical status,
    fertility, or maintenance of pregnancy in the adults. All other
    variables were only summarized, but the report indicated that litter
    parameters such as pup viability, growth rate, sex ratio, survival
    rates, clinical status, and gross and histological appearance were
    also unaffected. The NOEL was 1.5 mg/kg bw per day of premix-grade
    lincomycin and 100 mg/kg bw per day of USP-grade lincomycin, the
    highest doses tested (Goyings et al., 1979b).

        Table 3. Results of tests for the genotoxicity of lincomycin

                                                                                                 
    End-point          Test object            Concentration       Result        Reference
                                                                                                 

     In vitro 
    Reverse            S. typhimurium         120-1000 g/plate   Negativea,b   Mazurek & 
    mutation           TA98, TA100,                                             Swenson (1981)
                       TA1535, TA1537, 
                       TA1538

    Reverse            S. typhimurium         620-5000 g/plate   Negativea,c   Aaron & Mazurek 
    mutation           TA98, TA100,                                             (1987)
                       TA102, TA1535,
                       TA1537

    Forward            Chinese hamster        30-3000 g/ml       Negatived     Harbach et al. 
    mutation           V79 lung fibroblasts,                                    (1982a)
                       hprt locus

    Forward            Chinese hamster        100-3000 g/ml      Negativee,f   Harbach et al. 
    mutation           V79 lung fibroblasts,                                    (1982b)
                       hprt locus

    DNA damage         Chinese hamster        13-1300 g/ml       Negativea,g   Petzold (1981)
    (alkaline          V79 lung fibroblasts
    elution)

    Unscheduled        Primary rat            10-2500 g/mlh      Negativei     Seaman (1982)
    DNA synthesis      hepatocytes

    Unscheduled        Primary rat            0.17-17 g/mlj      Positive      Harbarch & 
    DNA synthesis      hepatocytes                                              Aaron (1987)k

    DNA repair         Human peripheral       2800-5000 g/ml     Negativea,l   Aaron (1991a)
                       lymphocytes

     In vivo 
    Cytogenicity       Rat bone marrow        1500-3000 mg/kg     Negativen     Trzos & 
                                              bwm                               Swenson (1981)

    Cytogenicity       Mouse bone marrow      150-600 mg/kg bw    Negativeo     Aaron (1991b)

    Sex-linked         Drosophila             25 000 and 50 000   Negative      Aaron (1988a)k
    recessive lethal   melanogaster           g/ml
    mutation
                                                                                                 

    Table 3. (Continued)

    a   With and without rat liver microsomal fraction (S9)
    b   2-Acetylaminofluorene (TA98, TA100, TA1538), cyclophosphamide (TA1535), and 
        9-aminoacridine (TA1537) used as positive controls
    c   2-Aminoanthracene (all strains with S9), 2-nitrofluorene (TA98, TA100 without S9), sodium 
        azide (TA1535 without S9), 9-aminoacridine (TA1437 without S9), and cumene hydroperoxide 
        (TA102 without S9) used as positive controls
    d   Without S9
    e   With S9
    f   7,12-Dimethylbenz[a]anthracene used as positive control
    g    N-Methyl- N'-nitro- N-nitrosoguanidine and epichlorohydrin used as complete carcinogen 
        positive controls and benzo[a]pyrene, 4-nitroguinoline-1-oxide, and 2-acetylaminofluorene 
        as procarcinogen positive controls
    h   Concentrations of 5000 and 10 000 g/ml were also tested but were lethal to the cell 
        cultures. Toxicity was observed at doses as low as 50 g/ml.
    i   2-Aminoathracene used as positive control
    j   Concentrations > 16.7 g/ml were lethal to the cell cultures.
    k   Only abstract provided; full report available from the sponsor upon request
    l   Cyclophosphamide (with S9) and 4-nitroquinoline-1-oxide (without S9) used as positive 
        controls
    m   One-half the dose was administered at 0 and 24 h.  Administration of a single dose of 
        3000 mg/kg (one-half of a 6000 mg/kg dose) was lethal.
    n   Cyclophosphamide used as positive control
    o   Triethylenemelamine used as positive control
    

         In another study that did not conform to GLP, groups of 24
    pregnant rats were given premix-grade lincomycin by gastric gavage at
    a dose of 0, 10, 30, or 100 mg/kg bw per day on days 6-15 of
    gestation. The dams were killed and their fetuses removed on day 20.
    The fetuses were weighed, sexed, and evaluated for gross, visceral,
    and skeletal anomalies. There was no evidence of maternal toxicity at
    any dose. A statistically significant increase in embryolethality was
    reported at the high dose, as indicated by a fetal resorption rate of
    8%, while that of the controls was 2.9% and that of historical
    controls was 5.3%. There was a corresponding decrease in the number of
    live fetuses. No evidence of teratogenicity was seen. The NOEL for
    fetal toxicity was 30 mg/kg bw per day on the basis of increased fetal
    resorptions at the high dose. The NOEL for maternal toxicity was 100
    mg/kg bw per day, the highest dose tested (Morris et al., 1980).

         In a two-generation study of reproductive toxicity that complied
    with GLP, groups of 30 SPF rats of each sex received lincomycin by
    oral gavage at a dose of 0, 100, 300, or 1000 mg/kg bw per day.
    Lincomycin was administered to the F0 generation for 60 days before
    mating until delivery of the F1 generation for males or for 14 days
    before mating until 21 days  post partum for females. All females
    were allowed to deliver and nurse their offspring until weaning. One
    F1 male and one F1 female pup were randomly selected from each
    litter for breeding. Dosing of the F1 pups began on the day that the
    last litter was weaned and followed a similar schedule to that for the

    F0 rats. Gross observations were made on all groups, but only the
    control and high-dose groups were examined microscopically. The only
    treatment-related effect reported was a transient increase in body
    weight and weight gain in all treated females during the first 14 days
    of treatment, but body weight was not affected beyond 21 days of
    treatment. No treatment-related effects were reported on indices of
    reproductive or developmental toxicity, consistent with a maternal and
    fetal NOEL of 1000 mg/kg bw per day (Black et al. 1988). 

         (b)  Developmental toxicity

          Rats 

         A series of studies was conducted before the requirement for GLP.
    In a preliminary study, lincomycin was administered to 10 pregnant
    Upjohn Sprague-Dawley breeder rats by subcutaneous injection at a dose
    of 50 mg/kg bw per day during gestation. Treatment was well tolerated,
    with no apparent effect on the number of young born per dam, birth
    weights, or sex ratios. No effects were observed on body-weight gain
    or at necropsy 3 weeks  post partum. In a related preliminary study,
    the same dose of lincomycin was injected subcutaneously to dams during
    lactation. Antibiotic activity was detectable, although not
    quantifiable, in the tissues of two of three groups of suckling rats.
    The only treatment-related effect was superficial lesions at the
    injection site in 4 of the 11 dams. No treatment-related abnormalities
    were found on physical examination and necropsy (Gray & Purmalis,
    1962a). 

         Lincomycin was administered subcutaneously at a dose of 30 mg/kg
    bw to 65 newborn rats from six litters for 5 weeks beginning 24 h
    after birth. No treatment-related effects on body weight, body-weight
    gain, or pathological end-points were reported (Gray & Purmalis,
    1962h). 

         A subcutaneous dose of 75 mg/kg bw per day was administered to 10
    male and 20 female rats for 60 days and throughout two mating cycles
    (84 days). A concurrent control group received saline injections.
    Litter size, sex ratio, appearance, weight gain, and behaviour of the
    offspring were not affected. No treatment-related effects were
    reported in the parents or either of the two successive litters (Gray
    et al., 1963c). 

         Fifty rat dams were injected subcutaneously once between days 7
    and 16 of gestation with 300 mg/kg bw lincomycin, while 10 received a
    single injection of cyclophosphamide at 10 mg/kg bw as positive
    controls and 15 were untreated. Summarized data were provided for 407
    fetuses from 42 dams. The only  effect reported was an injection-site
    lesion (Mulvihill & Gray, 1965).

          Dogs 

         Six pregnant beagles were given an intramuscular injection of
    lincomycin at 50 mg/kg bw, in a study that did not comply with GLP;
    five controls were available. Individual data were not provided, but
    the memorandum described no significant effects of treatment on the
    bitches or pups. Under the conditions of this study, no NOEL could be
    identified (Gray & Purmalis, 1963b).

    2.2.6  Special studies

         (a)  Immune response

         The ability of lincomycin to initiate hypersensitivity responses
    in humans and animals was assessed in a summary of unpublished reports
    of adverse effects after human use of lincomycin, 61 unpublished
    proprietary reports submitted to the US Food and Drug Administration
    in support of new animal drug applications, and published literature
    (DeGeeter, 1974). During the period 1965-74, 62 incidents of
    sensitization reactions were reported after administration of some 10
    thousand million oral doses. None of the incidents involved persons
    handling lincomycin or lincomycin-medicated feed intended for
    agricultural use. In addition, the published literature emphasized the
    hypoallergenicity of lincomycin. The unpublished proprietary reports
    of testing of lincomycin in 13 species gave no evidence of
    sensitization. 

         (b)  Ototoxicity

         A study that did not conform to GLP was conducted to investigate
    the potential ototoxicity of lincomycin administered by intramuscular
    inejction at 30 or 60 mg/kg bw per day to groups of three cats for 2.5
    months. Two cats were kept as controls and given saline injections.
    Hearing and vestibular function were evaluated from standardized
    hearing responses and post-rotational nystagmus times. No histological
    examinations were performed. Lincomycin had no ototoxic effects (Gray
    & Highstrete, 1963b).

         (c)  Microbiological effects

         Despite the small fraction of lincomycin excreted into the
    intestine (see section 2.1.1), its antimicrobial activity may last
    more than 5 days after parenteral administration (Kapusnik-Uner et
    al., 1996). 

         While no information was available on the formation of
    metabolites of lincosaminides in the gastrointestinal tract of humans,
    parent lincomycin is present in the faeces of persons receiving
    therapeutic doses (Kotarski, 1995). In the absence of other data,
    therefore, faecal recoveries of lincosaminides may be presumed to
    reflect exposure of the gut flora to ingested lincosaminide residues,
    and the doses received therapeutically and from residues are assumed
    to be proportional. The microbiological activity of metabolites of

    lincomycin, evaluated in  Micrococcus luteus, in pig plasma, liver,
    and kidney, and in cows' milk has been reported. Parent lincomycin
    accounted for nearly all of the microbiological activity,
     N-desmethyl lincomycin and lincomycin sulfoxide having 15- and
    100-fold less activity than the parent, respectively (Hornish et al.,
    1987; Nappier, 1998).

         The effect of pirlimycin, a related lincosaminide, on the
    viability of anaerobic bacteria in dense cell suspensions was tested
    by adding the compound at a concentration of 0, 3, or 6 mg/ml to
    suspensions of pure cultures of 39 strains of  Bacteriodes,  
     Bifidoacteria, Clostridium, Fusobacterium, Peptococcus or
     Peptostreptococcus, Lactobacillus, and  Eubacterium. The population
    densities of the cell suspensions were decreased by less than one log
    (average, 0.4 log10) at the highest dose . The cell suspension
    concentrations were considerably lower (107-109 colony-forming
    units [CFU]/ml) than those typically found in the gastrointestinal
    tract (1011 CFU/g) (Greening et al., 1995). Similar data for
    lincomycin are not available. Kotarski (1995) suggested that 6 mg/ml
    is the NOEL for microbiological activity on gut flora. Clindamycin was
    also tested in semi-continuous cultures of composite human faecal
    samples. The drug was added at 0, 0.26, 2.6, 25, or 260 mg/ml culture
    for 7 days. During these treatments and for 7-8 days thereafter,
     Clostridium difficile ATTCC 43255 was added daily at 103 cells/ml.
    The NOEL was 2.6 mg/ml was on the basis of overgrowth of
     C. difficile, changes in pH, and changes in the profile of volatile
    fatty acids (Cerniglia & Kotarski, 1999). 

         Therapeutic daily oral doses of 600 mg of clindamycin (Cerniglia
    & Kotarski, 1999) and > 1500 mg of lincomycin (Kotarski, 1995) to
    an adult (equivalent to 10 mg/kg bw for clindamycin and 25 mg/kg bw
    for lincomycin) can cause marked changes in the gastrointestinal
    flora. One side-effect of the therapeutic use of lincosaminides is
    disruption of the intestinal microflora (Fass, 1981; Kotarski, 1995),
    and therapeutic regimens of lincomycin and clindamycin have been
    associated with marked decreases in anaerobic flora, concurrently with
    increases in aerobic and facultatively anaerobic Gram-negative
    bacilli, enterococci, and yeasts (Fass, 1981). Colitis has been
    reported in 0-2.5% of patients and diarrhoea in 2.6-31% (Fass, 1981;
    Kotarski, 1995). Clindamycin-induced pseudomembranous colitis was
    recognized after the drug had been approved for use in humans, as was
    the role of  C. difficile toxin in the disease process (Tedesco et
    al., 1974; Kucers & Bennett, 1979; Fass, 1981; Jaimes, 1991; Gilbert,
    1994). This condition has been reported at a frequency as high as 10%
    after administration of lincomycin (Kotarski, 1995). Adverse drug
    reactions to clindamycin are essentially limited to rash and
    diarrhoea. Diarrhoea was reported at a frequency of 20-31% in
    susceptible AIDS patients, while diarrhoea associated with
     C. difficile toxin was reported in 0.01-18% of treated patients
    (Gilbert, 1994). A review of the literature indicated that clindamycin
    at a daily oral dose of 150 mg had no adverse effects in 99 adults
    treated for up to 12 months (Kotarski, 1995). The results of studies

    of antibiotic-associated colitis in hamster models, based on a review
    of the published literature and unpublished technical reports, are
    summarized in Table 4.

         The ability of lincomycin to affect the faecal excretion of
    pathogens in food animals was investigated in a study of 32 pigs, 4-5
    weeks of age, which received lincomycin at 0 or 100 g/t, equal to 5.6
    mg/kg bw per day, in the feed for 7 days before bacterial challenge
    and throughout the study. Ten pigs given lincomycin and nine controls
    were challenged by gavage with 1  1011 CFU of nalidixic-resistant
     S. typhimurium in 50 ml trypticase soya broth, while one group
    receiving lincomycin and three further control groups were
    mock-challenged with trypticase soya broth. Faecal samples were
    obtained on days -7, -4, -1, 2, 4, 5, 6, 8, 10, 12, 14, 17, 24, 31,
    39, 46, and 53. When two consecutive negative faecal cultures were
    found after day 31, the animal was slaughtered. All remaining pigs
    were slaughtered on day 53. Colon, liver, spleen, and mesenteric lymph
    node from each animal were cultured for  S. tymphimurium at
    termination. Effective colonization was verified by the presence of an
    average of 1  104 CFU of nalidixic acid-resistant bacteria 2-5 days
    after challenge. Lincomycin had no effect on the quantity, duration,
    or prevalence of excreted  Salmonella spp., and medication for up to
    39 days had no effect on the sensitivity of  S. typhimurium to 10
    antibiotics (DeGeeter & Stahl, 1974).

         The sensitivity of staphylococci isolated from farm animals to a
    number of antibacterial agents was determined over 10 years (DeVriese,
    1980). No consistent trend was observed in the susceptibility to
    lincomycin of  S. aureus strains isolated from pigs (1973-80) or
    poultry (1970-80).

         Human clinical data have been used to determine patterns of
    susceptibility to lincosaminides. The susceptibility of nearly 6
    million bacterial strains from an average of 242 hospitals across the
    United States and of 200 000 strains from the Massachusetts General
    Hospital, Boston, Massachusetts, and the Bronx Lebanon Hospital
    Center, New York, between 1971 and 1984 are shown in Table 5
    (Atkinson, 1986; Atkinson & Lorian, 1984). Yancey (1988) suggested
    that these data indicate that lincomycin has little effect on the
    susceptibility of Gram-positive and anaerobic bacteria isolated from
    humans. The sensitivity of selected human isolates  in vitro to
    lincomycin was similar in 1968 (Kucers & Bennett, 1979). On the basis
    of the data collected in the US hospital survey (Atkinson, 1986;
    Atkinson & Lorian, 1984), the European Medicines Evaluation Agency
    (1998) has proposed use of the reported median inhibitory
    concentration (MIC50) for  Fusobacterium of 0.2 mg/ml (range,
    0.2-0.4 mg/ml) as the NOEC for the antimicrobial effect of lincomycin
    on human gut flora. Additional data on the MIC50 of lincomycin and
    the related lincosaminide, clindamycin, for selected human intestinal
    bacteria are shown in Table 6 (Kotarski, 1995).


        Table 4. Results of studies of antibiotic-associated colitis in the hamster model

                                                                                                                                             
    Drug          Route of                   Weight of     Challenge with   No./dose    LD100       LD50         No effect   Reference
                  administration             animals (g)   C. difficile                 (mg/60 kg)  (mg/60 kg)   (mg/60 kg)
                                                                                                                                             

    Lincomycin    Single subcutaneous        80-100        Yes              10 or 6     NR          174-282      NR          Staepert et al. 
                  injection                                                                                                  (1983, 1991)

                  Single intersca pular,     60-100        No               10 or 15    > 600       6-66         6           Lusk et al. 
                  subcutaneous, or                                                                                           (1978)
                  intraperitoneal injection

    Clindamycin   Single subcutaneous        80-100        Yes              NR          > 750a      240b         NR          Staepert et al.
                  injection                                                                                                  (1983, 1991)

                  Topical, daily for 14      80-100        No               4 or 7 or   . 600a      .6-60        6           Feingold et al.
                  days                                                      NR                                               (1979)

                  Single intraperitoneal     60-90         No               6             > 60      6-60         6           Rifkin et al. 
                  injection                                                                                                  (1978)

                  Single intersca pular,     60-100        No               15 or 10    . 300       32-43        30          Lusk et al.
                  subcutaneous, or                                                                                           (1978)
                  intraperitoneal injection

    Pirlimycin    Single subcutaneous        80-100        Yes              NR          NR          156 mg/kg    NR          Staepert et al.
                  injection                                                                                                  (1983, 1991)
                                                                                                                                             

    Doses reported by authors in mg/kg are presented as mg/60 kg equivalent body weight. For doses reported by the authors as daily dose per
    hamster, it was assumed that each hamster weighed 100 g.

    a   Mortality at 300 mg/60 kg equivalent body weight was not tested.
    b   Mean values of four experiments; range, 302-420 mg/60 kg equivalent body weight

    Table 5. Inhibition of selected bacteria by lincomycin

                                                                                                                                             
    Organism                          No. of    Cumulative percentage of strains inhibited by various               Reference
                                      strains   concentrations of lincomycin (g/ml)
                                                                                                                 
                                                Dose(s)         g/ml    %        g/ml    %        g/ml    %
                                                                                                                                             

    Acinetobacter calcoaceticus 
      var. anitratis                     11     > 400                                                               Finland et al. (1976a)
    Actinomyces spp.                     20     0.12-4.0        0.12     65       0.25     90       2.0      95     Lerner (1968)
    Bacillus anthracis                          0.25-8.0                                                            Barker & Prescott (1973)
    Bacteroides fragilis                195     0.1-12.5        0.8      31       1.6      71       12.5     71     Martin et al. (1972)
    Bacteroides melaninogenicus          29     0.1-0.2         0.1      89       0.2      96                       Martin et al. (1972)
    Bifidobacterium eriksonii             5     0.1-1.6         0.1      40       0.2      80       1.6      100    Martin et al. (1972)
    Bordetalla pertussis                 36     3.1-50          6.25     1        12.5     25       25       93     Bass et al. (1969)
    Brucella abortus, melitensis 
      and suis                                  1.25-> 100      1.25     10       50       65       100      100    Hall & Manion (1970)
    Campylobacter fetus (Vibrio 
      fetus)                             95     0.78-100        6.3      38       12.5     76       50       91     Vanhoof et al. (1978)
    Clostridium perfringens              34     0.1-6.2         0.2      29       1.6      76       3.1      97     Martin et al. (1972)
    Clostridium spp.                     17     0.1-12.5        0.1      23       1.6      53       3.1      76     Martin et al. (1972)
    Corynebacterium diphtheriae          14     0.4-0.8         0.4      93                                         Gordon et al. (1971)
    Edwardsiella tarda                   37     > 5             5        0                                          Nasu et al. (1981)
    Eikenella corrodens                  20     > 100                                                               Robinson & James (1974)
    Enterobacter spp.                    33     > 400                                                               Finland et al. (1976a)
    Eubacterium alactolyticum             2     0.1             0.1      1000                                       Martin et al. (1972)
    Eubacterium lentum                   14     0.1-3.1         0.1      21       0.8      71       1.6      93     Martin et al. (1972)
    Flavobacterium meningosepticum       11     > 100           > 100    100                                        Altman & Bogokovsky (1971)
    Fusobacterium spp.                   18     0.1-6.2         0.1      39       0.8      67       3.1      94     Martin et al. (1972)
    Haemophilus spp.                     68     0.8-12.5        1.6      25       3.1      53       6.3      87     Williams & Andrews (1974)
    Haemophilus influenzae               70     > 20            > 20     100                                        McLinn et al. (1970)
    Klebsiella Pneumoniae                35     > 400                                                               Finland et al. (1976a)
    Mycoplasma pneumoniae                 5     1.6-4.8         1.6a                                                Atkinson & Moore (1977a); 
                                                                                                                    Barker & Prescott (1971)
    Neisseria gonorrhoeae                82     0.5-> 32        4        7        16       44       32       77     Phillips et al. (1970)
                                                                                                                                             

    Table 5. (Cont'd)

                                                                                                                                             
    Organism                          No. of    Cumulative percentage of strains inhibited by various               Reference
                                      strains   concentrations of lincomycin (g/ml)
                                                                                                                 
                                                Dose(s)         g/ml    %        g/ml    %        g/ml    %
                                                                                                                                             

    Neisseria meningitidits              40     16-> 100        46       5        > 100    100                      Devine & Hagerman (1970)
    Nocardia spp.                        25     100-> 400       100      5a       299a     25                       Bach et al. (1973)
    Peptococcus                         145     0.1-> 25        0.1      29       0.4      69       0.8      92     Martin et al. (1972)
    Peptostreptococcus                   72     0.1-3.1         0.1      39       0.4      72       1.6      100    Martin et al. (1972)
    Propionibacterium acnes              16     0.1-1.6         0.1      81       0.4      94       1.6      100    Martin et al. (1972)
    Proteus spp.                         34     > 200                                                               Finland et al. (1976a)
    Providencia stuartii                 34     > 400                                                               Finland et al. (1976a)
    Pseudomonas aeruginosa               35     > 400                                                               Finland et al. (1976a)
    Pseudomonas pseudomonallei           10     > 100                                                               Eikhoff et al. (1970)
    Staphylococcus aureus               106     0.12-2.0        0.25     25       0.5      63       1.0      96     Phillips et al. (1970)
    Staphyloccus epidermidis             35     0.2-> 100       0.2      5        0.4      93                       Sabath et al. (1976)
    Streptococcus pyogenes 
      (Group A)                          35     < 0.01-0.4      0.04     10       0.1      33       0.2      80     Finland et al. (1976b); 
                                                                                                                    Karchmer et al. (1975)
    Streptococus agalaciae 
      (Group B)                          25     0.04-0.19                                                           Karchmer et al. (1975)
    Streptococcus faecalis 
      (enterococcus)                    382     1.6-> 100       25       18       50       55       100      88     Toala et al. (196925.
    Streptococcus (Group D, 
      not enterococcus)                  22     0.04-0.39                                                           Karchmer et al. (1975)
    Streptococcus pneumoniae             25     0.12-1.0        0.12     12       0.25     56       0.5      84     Upjohn Co. (undated)
    Streptococcus viridans               27     0.12-1.0        0.12     37       0.25     85                       Upjohn Co. (undated)
    Viellonella                          13     0.1-6.2         0.1      46       0.2      77       1.6      92     Martin et al. (1972)
    Vibrio alginolyticus                 24     8-32                                                                Hollis et al. (1976)
    Vibrio parahemolticus                24     8-32                                                                Hollis et al. (1976)
    Yeserinia enterocolitica            190     4               4 (3)                                               Raevvori et al. (1978)
                                                                                                                                             

    a Results taken from a graph
    

        Table 6. MIC50 values of lincomycin and clindamycin for selected human intestinal bacteria

                                                                                              

    Bacterial genus             Clindamycin                Lincomycin
                                                                                              
                                No.       MIC50 (g/ml)    No.a       MIC50 (g/ml) 
                                                                                              
                                                                      Median      Range
                                                                                              

    Bacteroides                 15        1                1158       3.1         0.1-12.5
    Bifidobacterium             13        0.03             42         0.4         0.2-1.6
    Eubacterium                 13        0.06             21         0.8         0.1-0.8
    Fusobacterium               6         0.03             91         0.2         < 0.1-12.5
    Peptococcus /               19        0.03             446        0.4         0.2-0.4
      Peptostreptococcus
    Clostridium                 8         0.5              506        1           1-25
    Lactobacillus               2         0.06             124        1           1
    Enterococcus                10        16               27         16          4-32
    Escherichia coli            12        > 128            21         > 128       > 128
                                                                                              

    Modified from Kotarski (1995)
    a Number of strains surveyed in several studies
    

    2.3  Observations in humans

         Gastrointestinal effects are the most commonly reported adverse
    reactions to lincomycin in humans (Fass, 1981; Gilbert, 1994). The
    effects can include nausea, vomiting, abdominal cramps, and diarrhoea.
    Pseudomembranous colitis, when associated with lincomycin or
    clindamycin therapy, usually appears 2-25 days after the start of
    treatment and may occur in up to 20% of patients (Goodman & Gilman,
    1975; Kucers & Bennett, 1979). Rarely, hypersensitivity reactions have
    been reported, most commonly resulting in a rash, although anaphylaxis
    has been reported (Kucers & Bennett, 1979). Patients undergoing
    anaesthesia while receiving lincomycin and clindamycin have been
    reported to show inhibition of neuromuscular transmission, with
    possible potentiation by concurrently administered neuromuscular
    blocking agents (Fass, 1981; Kapusnik-Uner et al., 1996).

         There were no reported effects on fetal development after
    administration of lincomycin to 300 pregnant women (Fass, 1981).

    3.  COMMENTS

         The Committee considered data on the pharmacokinetics,
    metabolism, acute toxicity, short-term and long-term studies of
    toxicity, carcinogenicity, genotoxicity, reproductive toxicity,
    developmental toxicity, immunotoxicity, ototoxicity, and
    microbiological safety of lincomycin. The results of studies on the
    functionally and structurally related drug clindamycin were considered
    in the assessment of the microbiological safety of lincomycin. In all
    of the studies considered, the concentrations of the compound were
    reported as the activity of lincomycin base. While many of the studies
    were conducted prior to the development of GLP, all of the pivotal
    studies were carried out according to appropriate standards for study
    protocol and conduct.

         Dogs given lincomycin intramuscularly or orally showed rapid
    absorption, peak serum concentrations being achieved within 0.5 and
    1.5 h, respectively. In pigs given an oral dose, 53% (with a standard
    deviation of 19%) was bioavailable, and 5-15% was bound to plasma
    proteins. The peak concentrations in serum were reached within 3.6 h
    (standard deviation, 1.2 h), with a half-time of 3.4 h (standard
    deviation, 1.3 h). Pig excreta contained little unchanged lincomycin:
    urine contained 11-21% of the oral dose, half of which was unchanged
    parent compound. Only trace amounts of  N-desmethyllinco-mycin were
    identified. The contents of the gastrointestinal tract accounted for
    79-86% of the excreted drug. In faecal samples, only 17% of the
    excreted dose was unchanged parent compound, and the remainder was
    uncharacterized metabolites. 

         Lincomyin is well distributed in the human body. The tissues and
    fluids that contain significant concentrations include bile, pleural
    fluid, brain, bone marrow, synovial fluid, bone, joint capsule, eye,
    and peritoneal fluid. The distribution of the compound in
    cerebrospinal fluid is generally poor except in the presence of
    inflammation. Lincomycin has been shown to cross the placenta, and
    peak concentrations of 0.2-3.8 g/ml were found in amniotic fluid,
    which were sustained for 52 h after a single intramuscular injection
    of 600 mg to pregnant women. Lincomycin was present in the milk of
    these women. The systemic oral bioavailability of lincomycin in
    persons who had fasted was 25-50%, but this value can be as low as 5%
    in the presence of food; 72% of the amount found in serum was bound.
    Peak serum concentrations are usually reached within 4 h, with a
    half-time of 4.2-5.5 h.

         Lincomycin is extensively metabolized, as less than 10% of
    unchanged parent drug is found in animal tissues. The numerous
    metabolites include  N-desmethyllincomycin and lincomycin sulfoxide,
    which are reported to have 15-100 times less microbiological activity
    than the parent compound.

     Toxicological data

         Lincomycin has high acute toxicity only in rabbits. The LD50
    after oral administration was 17 000-19 000 mg/kg bw in mice and
    11 000-16 000 mg/kg bw in rats, while the lethal dose in 9 of 15
    rabbits was reported to be 50 mg/kg bw.

         Lincomycin was administered to mice for 90 days in the feed to
    provide a dose of 0, 10, 30, 100, 300, or 3000 mg/kg bw per day.
    Animals given the two higher doses showed increased weight of the
    intestine (with pancreas) and an increased incidence of luminal
    distension and dilatation of the small and large intestines. The NOEL
    was 100 mg/kg bw per day.

         In a 90-day study of toxicity, groups of four male and four
    female dogs were given an oral dose of lincomycin at 0, 400, or 800
    mg/kg bw per day. Transient increases in serum alanine
    aminotransferase activity were observed during the first month of
    treatment in dogs at the high dose and in one dog at the low dose, but
    the activity had returned to normal by the end of the study. No other
    treatment-related effects were reported. The NOEL was 800 mg/kg bw per
    day, the highest dose tested.

         Lincomycin was administered to groups of two male and two female
    dogs in capsules for 6 months at a dose of 0, 30, 100, or 300 mg/kg bw
    per day. The NOEL was 300 mg/kg bw per day, the highest dose tested.
    In a 1-year study, lincomycin was administered orally by gelatin
    capsule to groups of five male and five female dogs, as the premix
    grade at a dose of 0, 0.38, 0.75, or 1.5 mg/kg bw per day, or as the
    USP grade at a dose of 1.5 mg/kg bw per day. The NOEL was 1.5 mg/kg bw
    per day, the highest dose tested. 

         Groups of 10 rats of each sex were treated for 1 year with
    lincomycin at a dose of 0, 30, 100, or 300 mg/kg bw per day by oral
    gavage. No treatment-related effects were reported at any dose. The
    NOEL was 300 mg/kg bw per day, the highest dose tested. 

         In a long-term study of toxicity and carcinogenicity, pregnant
    female rats and groups of 60 offspring of each sex were given feed
    containing premix-grade lincomycin to provide a dose of 0, 0.38, 0.75,
    or 1.5 mg/kg bw per day or USP-grade lincomycin to provide a dose of
    1.5 or 100 mg/kg bw per day. Treatment of the offspring was continued
    for 26 months. While lincomycin was not carcinogenic under the
    conditions of the assay, the Committee considered the administered
    dose to be insufficient for assessing the carcinogenicity of
    lincomycin. The NOEL for non-neoplastic lesions was 100 mg/kg bw per
    day, the highest dose tested. 

         Lincomycin was tested for its capacity to induce reverse mutation
    in bacteria, gene mutation in Chinese hamster lung fibroblasts,
    unscheduled DNA synthesis in primary rat hepatocytes, chromosomal
    aberrations in peripheral human lymphocytes  in vitro, DNA damage in
    V79 cells, micronuclei in rat and mouse bone marrow, and sex-linked

    recessive lethal mutations in  Drosophila melanogaster in vivo. The
    only positive finding was the induction of unscheduled DNA synthesis
    in primary rat hepatocytes, but this result could not be replicated.

         Adequate studies of carcinogenicity were not available. However,
    the weight of the evidence indicates that lincomycin is not genotoxic.
    Furthermore, lincomycin is not structurally similar to known
    carcinogens. The Committee therefore concluded that the drug does not
    present a carcinogenic risk, and further carcinogenic studies were
    deemed unnecessary.

         The reproductive and developmental toxicity of lincomycin was
    evaluated in a three-generation study conducted prior to the
    formulation of GLP. Groups of 30 male and 60 female F0 rats and 10
    male and 20 female F1, F2, and F3 animals were given diets
    containing lincomycin of premix grade to provide a dose of 0, 0.38,
    0.75, or 1.5 mg/kg bw per day or the USP grade to provide a dose of
    1.5 or 100 mg/kg bw per day, beginning with F0 weanling rats and
    continuing through successive breeding of the F0, F1, and F2
    progeny to weaning of the F3 litters. No treatment-related effects
    were seen at any dose. The NOEL was 100 mg/kg bw per day for the USP
    grade, the highest dose tested.

         In a two-generation study of reproductive toxicity, lincomycin
    was administered to groups of 30 male and 30 female rats by oral
    gavage at a dose of 0, 100, 300, or 1000 mg/kg bw per day. No
    treatment-related effects were observed at any dose. The NOEL was 1000
    mg/kg bw per day, the highest dose tested.

         In a study of developmental toxicity, pregnant rats were given
    lincomycin by gastric gavage at a dose of 0, 10, 30, or 100 mg/kg bw
    per day on days 6-15 of gestation. An increased incidence of fetal
    resorptions was observed at the highest dose. The NOEL for
    embryotoxicity was 30 mg/kg bw per day.

         The potential ototoxicity of lincomycin was tested in groups of
    three cats that received intramuscular injections of 30 or 60 mg/kg bw
    per day for 2.5 months. Hearing and vestibular function were evaluated
    on the basis of standardized hearing tests and post-rotational
    nystagmus times, respectively. No treatment-related effects were
    reported.

     Microbiological data

         The Syrian hamster is used as an experimental model to evaluate
    antibiotic-associated colitis. In studies in which lincomycin was
    administered by various parenteral routes to hamsters, the NOEL for
    antibotic-associated colitis was 0.1 mg/kg bw per day.

         Administration of lincomycin at an oral therapeutic dose of 25-66
    mg/kg bw daily to 12 patients for periods varying from 6 to 150 days
    caused antibiotic-associated colitis. The condition was also found
    after administration of the structurally and functionally similar

    compound clindamycin to 10 patients for 7 days at a dose of 10 mg/kg
    bw per day. When clindamycin was administered to 99 patients at doses
    up to 2.5 mg/kg bw per day for up to 12 months, the NOEL for adverse
    effects on the gastrointestinal microflora was 2.5 mg/kg bw per day. 

         The ability of lincomycin to affect faecal excretion of pathogens
    was investigated in a study of 32 pigs aged 4-5 weeks that were given
    the drug for periods up to 45 days at a concentration of 100 g/t of
    feed, equivalent to 5.6 mg/kg bw per day. Treatment had no effect on
    the faecal excretion of  S. typhimurium when these animals were
    compared with pigs that received the vehicle alone, nor did it alter
    the susceptibility of the bacteria to lincomycin.

         The sensitivity of staphylococci isolated from farm animals to
    lincomycin was investigated in a 10-year study that ended in 1980. No
    change in the susceptibility of  S. aureus strains isolated from pigs
    or poultry was observed.

         A study was conducted between 1971 and 1982 to examine the
    patterns of susceptibility to antibiotics of more than 5 million
    bacterial strains from hospitals across the USA. The susceptibility of
    Gram-positive aerobic and anaerobic bacteria to lincomycin changed
    little during the survey period. In a separate study, the MIC50 for
    representative bacteria from the human gut was reported for
    lincomycin. The NOEC for the effect of lincomycin on  Fusobacterium, 
    the most sensitive representative species, was 0.2 mg/ml.

         Clindamycin was tested at a concentration of 0, 0.26, 2.6, 25, or
    260 mg/ml of culture for 7 days in semi-continuous cultures of
    composite faecal samples from humans. During these treatments and for
    7-8 days thereafter,  Clostridium difficile was added daily at a
    concentration of 103 cells/ml. The NOEL for clindamycin was 2.6
    mg/ml on the basis of overgrowth of  C. difficile, changes in pH, and
    changes in the profile of volatile fatty acids in the culture medium.

         A 'decision tree' for evaluating the potential of veterinary drug
    residues to affect human intestinal microflora was developed by the
    Committee at its fifty-second meeting (Annex 1, reference  140;
    Figure 1). At its present meeting, the Committee used the decision
    tree to answer the following questions in its assessment of
    lincomycin:

     1.  "Does the ingested residue have antimicrobial properties?" 

         Yes. While the spectrum of activity of lincomycin is essentially
    the same as that of clindamycin, the MIC50 of lincomycin is higher
    (less potent) than that of clindamycin for the relevant species of
    bacteria in the human gastrointestinal tract.

    FIGURE 1a;V45JE01.BMP

    FIGURE 1b;V45JE01B.BMP

     2.  "Does the drug residue enter the lower bowel by any route?" 

         Yes. In humans, 40-50% of an oral dose of lincomycin is excreted
    in faeces. The systemic absorption of lincomycin in humans is
    significantly reduced in the presence of food: while the systemic
    bioavailability of an oral dose of lincomycin is estimated to be
    25-50% in fasting individuals, it may be only 5% with a meal. The
    intestinal bioavailability of lincomycin may therefore be as high as
    95-100%. Measurements of lincomycin activity in the faeces of patients
    receiving therapeutic doses are shown in Table 7. The concentrations
    on day 1 were not included in the calculation below because the full
    concentrations had not been attained. Thus,


       (2.2 + 1.7 + 6.6 + 2.3 + 2.4 + 2.6 + 3.0 + 2.9) mg/g = 23.7 mg/g

                  (23.7 mg/g) / 8 observations = 3.0 mg/g

    Faecal lincomycin therefore accounts for (3.0 mg lincomycin per g
    faeces)  (220 g faeces)/(2000 mg oral dose) or approximately 33% of
    an oral dose. As the drug is extensively metabolized and many of the
    metabolites have little to no antimicrobial activity, a conservative
    estimate of the bioavailability of lincomycin in the gastrointestinal
    tract would be 5%.

     3.  "Is the ingested residue transformed irreversibly to inactive 
         metabolites by chemical transformation, metabolism mediated by 
         the host or intestinal microflora in the bowel and/or by 
         binding to intestinal contents?" 

         Yes, but microbiologically active residues remain. Lincomycin is
    extensively metabolized to about 16 metabolites, three of which have
    been identified as lincomycin sulfoxide,  N-desmethyl lincomyin, and
     N-desmethyllincomycin sulfoxide. None of the metabolites was found
    to have significant microbiological activity.  N-Desmethyl and
    lincomcyin sulfoxide have 15 and 100 times less microbiological
    activity, respectively, than lincomycin. There was no evidence that
    the remaining metabolites have any microbiological activity. 

     4.  "Do data on the effects of the drug on the colonic microflora
         provide a basis to conclude that the ADI derived from 
         toxicological data is sufficiently low to protect the 
         intestinal microflora?" 

         No. A review of studies of the toxicity of orally administered
    lincomycin indicates that the pivotal study is the 26-month study of
    toxicity and carcinogenicity in rats.

        Table 7. Concentrations of lincomycin in faeces of patients receiving therapeutic 
             doses of 2000 mg/person per day orally

                                                                                              

    Dose regimen          No. of      Treatment    Mean concentration of     Reference 
                          patients    day          lincomycin (mg/g)a
                                                                                              

    500 mg 4 times        5           1            1.0                       Upjohn Co.
    daily for 3 days                  2            2.2                       (undated)
                                      3            1.7
                                      4            6.6
                                      5            2.3
    500 mg 4 times        6           1            0.6 (0-3.5)               Rotblatt et al. 
    daily                             2            2.4 (0-4.8)               (1982)
                                      3            2.6 (0-4.4)
                                      4            3.0 (1.6-4.4)
                                      5            2.9 (2.3-3.5)
                                      6            1.9 (0.1-3.5)
                                                                                              

    a The average faecal concentrations in humans given daily doses of 2000 mg may be calculated 
      (Kotarski, personal communication). 
    


                                      NOEL
                        ADI  =                  
                                  Safety factor


                               100 mg/kg bw per day
                   ADI  =                            
                                       100


                         ADI  =  1 mg/kg bw

         The adverse effects of lincomycin on the intestinal microflora
    have been indicated in a number of studies. 

         An evaluation of the MIC50 values for relevant gastrointestinal
    microflora provides a NOEL of 0.2 mg/ml. This value may be used to
    calculate a microbiological ADI as follows:

                                              MIC50  MCC
          Upper limit of ADI (mg/kg bw)  =                 
                                              FA  SF  BW


                                              (0.2 mg/g)  (220 g)
          Upper limit of ADI (mg/kg bw)  =                        
                                               0.5  1  60 kg


          Upper limit of ADI = 1.4 g /kg bw per day

     Fusobacterium was among the most sensitive of the relevant
    gastrointestinal microflora tested. An MIC50 of 0.2 mg/ml was
    established. A conservative factor of 0.5 was determined for
    gastrointestinal bioavailability on the basis of its systemic
    bioavailability in the presence of food (as low as 5%) and the
    measured percentage of an orally administered dose in faeces (as high
    as 15%). A safety factor of 1 was used because sufficient and relevant
    microbiological data were available.

         Clindamycin has also been tested in semi-continuous cultures of
    composite faecal samples from humans. The NOEL was 2.6 mg/ml on the
    basis of overgrowth of  C. difficile, changes in pH, and changes in
    the profile of volatile fatty acids.


                                              MIC50 (mg/g)  MCC(g)
           Upper limit of ADI (mg/kg bw)  =                           
                                                 FA  SF  BW (kg)


                                              (0.26 mg/g)  (220 g)
           Upper limit of ADI (mg/kg bw)  =                         
                                                 0.05  1  60 kg


           Upper limit of ADI = 19 g/kg bw per day

    Faecal excretion of clindamycin represents 5-10% of an oral dose. A
    safety factor of 1 was used because sufficient and relevant
    microbiological data were available.

         The model of antibiotic-associated colitis in hamsters given
    lincomycin intraperitoneally may be used to determine a microbiolgical
    ADI, as follows:

                                      NOEL
                        ADI  =                  
                                  Safety factor


                                0.1 mg/kg bw per day
                      ADI  =                         
                                        10


                         ADI  =  10 g/kg bw

    The NOEL was 0.1 mg/kg bw per day. A safety factor of 10 was used to
    address inter-animal variation. No correction was applied for
    extrapolation from animals to humans because of the sensitivity of the
    hamster model.

         The results of studies with a model of faecal excretion of
     Salmonella by pigs could also be used to evaluate the effects of
    lincomycin on the intestinal microflora. No effects of a single dose
    were seen on the quantity, duration, or prevalence of excreted
     Salmonella spp. or on the sensitivity of the faecally excreted
     Salmonella to 10 antibiotics, including lincomycin. The ADI may be
    calculated as:

                                      NOEL
                        ADI  =                  
                                  Safety factor


                                5.6 mg/kg bw per day
                      ADI  =                         
                                        100


                         ADI  =  56 g/kg bw

    The observed NOEL was 5.6 mg/kg bw per day, the only dose tested. A
    safety factor of 10 was used to address variation between animals, and
    a second safety factor was applied to address the uncertainty in
    extrapolating from data on pig gastrointestinal microflora to that of
    humans.

         Information was available from a clinical study of 99 patients on
    the effects of clindamycin on human gastrointestinal microflora over
    2-4 months. The microbiological ADI may be calculated as follows:

                                      NOEL
                        ADI  =                  
                                  Safety factor


                                2.5 mg/kg bw per day
                      ADI  =                         
                                        100


                         ADI  =  30 g/kg bw


    The observed NOEL was 2.5 mg/kg bw per day. While this was the highest
    dose administered, other studies showed an effect on the
    gastrointestinal microflora of clindamycin at 10 mg/kg bw per day and
    of lincomycin 25 mg/kg bw per day. A safety factor of 10 was used to
    address variation between human subjects, and an additional correction
    factor of 10 was used to address the 10-fold higher bioavailability of
    lincomycin than clindamycin to the colon.

     5.  "Do clinical data from the therapeutic use of the class of 
         drugs in humans or data from in vitro or in vivo model 
         systems indicate that effects could occur in the 
         gastrointestinal flora?"     

         Yes. Gastrointestinal effects are the most commonly reported
    adverse reactions to therapeutic use of lincomycin in humans. The
    effects can include nausea, vomiting, abdominal cramps, and diarrhoea.
    Pseudomembranous colitis associated with lincomycin or clindamycin
    therapy usually appears 2-25 days after the start of treatment and may
    occur in up to 20% of patients. The results of model systems that
    indicate that effects could occur in the gastrointestinal flora are
    discussed above.

     6.  "Determine which is the most sensitive adverse effect of the 
         drug on the human intestinal microflora."

         The available data indicate that disruption of the colonization
    barrier for human gastrointestinal microflora is the major concern,
    rather than the emergence of resistance. Lincosaminides are used
    widely in human medicine and have been shown to cause disruption of
    intestinal microflora. In a study of the magnitude of and trends in
    the development of bacterial resistance to lincosaminides, the pattern
    of susceptibility of human isolates of Gram-positive aerobic and
    anaerobic bacteria changed little over a 12-year period (1971-83).
    Resistance does develop in staphylococci, as seen in humans or
    animals, but most isolates remain susceptible to lincomycin. While no
    data were available on the effects of lincomycin on the metabolic
    activity of the intestinal microflora, disruption of the
    gastrointestinal colonization barrier is the most appropriate
    microbiological end-point for determination of an ADI for lincomycin.

     7.  "If disruption of the colonization barrier is the issue, 
         determine the MIC of the drug against 100 strains of 
         predominant intestinal flora and take the geometric mean MIC 
         of the most sensitive genus or genera to derive an ADI using 
         the formula for estimating an ADI. Other model systems may be 
         used to establish a NOEC to derive an ADI." 

         Disruption of the colonization barrier of the gastrointestinal
    microflora is the microbiological end-point of concern for lincomycin.
    No studies were available to establish a NOEL for effects of
    lincomycin on the human gastrointestinal microflora, but clindamycin,
    a structurally and functionally related lincosaminide, has the same
    spectrum of activity as lincomycin, has the same reported spectrum of
    adverse clinical effects as lincomycin, and is generally considered to
    be a more potent antibacterial agent than lincomycin. The availability
    to the colon of orally administered clindamycin is one-tenth that of
    lincomycin. The study of clinical use of clindamycin is the most
    appropriate one for determining the microbiological safety of
    lincomycin

    4.  EVALUATION

         The Committee could have established a toxicological ADI of 300
    g/kg bw on the basis of the NOEL of 30 mg/kg bw per day for
    embryotoxicity in rats and a safety factor of 100. The Committee
    noted, however, that lincomycin belongs to a group of lincosaminides
    that is active against Gram-positive bacteria and that the human
    gastrointestinal flora are sensitive to therapeutic doses of this
    group of compounds. Because this is the most sensitive end-point, the
    Committee established an ADI of 0-30 g/kg bw on the basis of the NOEL
    of 2.5 mg/kg bw per day for the effects of clindaymcin on the
    gastrointestinal microflora and a safety factor of 100. As is its
    usual practice, the Committee rounded the value of the ADI to one
    significant figure.

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    Gilbert, D.N. (1994) Aspects of the safety profile of oral
         antibacterial agents.  Infect. Dis. Clin. Pract., 3, 236-247.

    Glenn, M.W. & Garza, R. (1971) Lincomycin agricultural grade -- Oral
         LD50 in the rat. Unpublished report No. 519-9610-MWG-71-2.
         Submitted to WHO by Pharmacia & Upjohn, Kalamazoo, Michigan, USA.

    Goodman, L.S. & Gilman, A., eds (1975)  The Pharmacological Basis of 
          Therapeutics, 5th ed., New York: Macmillan Publishing Co., pp.
         1227-1228.

    Gordon, R.C., Yow, M.D., Clark, D.J. & Stevenson, W.B. (1971) In vitro
         susceptibility of  Corynebacterium diphtheriae to thirteen
         antibiotics.  Appl. Microbiol., 21, 548-549.

    Goyings, L.S., Thomas, R.W., VanHuysen, C.M. & Bastianse, R.G. (1979a)
         Lincomycin one year oral tolerance study in the dog. Unpublished
         report No. 768-9610-79-002. Agricultural Research and Development
         Laboratories, The Upjohn Company. Submitted to WHO by Pharmacia &
         Upjohn, Kalamazoo, Michigan, USA.

    Goyings, L.S., Thomas, R.W. VanHuysen, C.N. & Morris, D. (1979b) Three
         generation reproduction study with premix and USP grades of
         lincomycin hydrochloride in Sprague-Dawley rats. Unpublished
         report No. 768-9610-79-001. Agricultural Rresearch and
         Development Laboratories, The Upjohn Company. Submitted to WHO by
         Pharmacia & Upjohn, Kalamazoo, Michigan, USA.

    Grady, J.E & Treick, R.W. (1961) Antibiotic 124a blood levels in dogs
         and monkeys. Unpublished interoffice memo from J.E. Gray and R.W.
         Treick to L.E. Rhuland. Submitted to WHO by Pharmacia & Upjohn,
         Kalamazoo, Michigan, USA. 

    Gray, J.E. & Highstrete, J.. (1963a) Intravenous LD50 mouse, rat.
         Unpublished interoffice memo from J.E. Gray and R.W. Highstrete.
         Submitted to WHO by Pharmacia & Upjohn, Kalamazoo, Michigan, USA.

    Gray, J.E. & Highstrete, J. (1963b) Lincomycin hydrochloride, U-10,
         149a. Ref: 5879 THP 150-1. Ototoxic evaluation in the cat.
         Unpublished interoffice memo from J.E. Gray and J.D. Highstrete
         to E.S. Feenstra. Submitted to WHO by Pharmacia & Upjohn,
         Kalamazoo, Michigan, USA.

    Gray, J.E. & Highstrete, J. (1965) Lincomycin hydrochloride. Lot No.
         14, 121-32 (T. Eble). Intra-articular irritation in the rabbit.
         Unpublished interoffice memo from J.E. Gray and J. Highstrete to
         E.S. Purmalis. Submitted to WHO by Pharmacia & Upjohn, Kalamazoo,
         Michigan, USA.

    Gray, J.E. & Purmalis, A. (1961a) Lincomycin HCl (U-10, 149A).
         Antibiotic 124a, Lot No. 5732-RH. Unpublished interoffice memo
         from J.E. Gray and A Purmalis to E.S. Feenstra. Submitted to WHO
         by Pharmacia & Upjohn, Kalamazoo, Michigan, USA.

    Gray, J.E. & Purmalis, A. (1961b) U-10,149a. Antibiotic 124a. Lot No.
         5449-DMW-63-6. Oral subacute toxicity in the rat. JEG 4538:
         138-140. Unpublished interoffice memo from J.E. Gray and A.
         Purmalis to E.S. Feenstra. Submitted to WHO by Pharmacia &
         Upjohn, Kalamazoo, Michigan, USA.

    Gray, J.E. & Purmalis, A. (1962a) Lincomycin HCl (U-10, 149A).
         Antibiotic 124a, lot No. 5732-RH. I. Subcutaneous LD50
         determination in newborn and adult rats. II. Effect on the rat
         fetus III. Effect on the newborn. IV. Tissue assays for placental
         and milk transfer. Unpublished interoffice memo from J.E. Gray
         and A Purmalis to E.S. Feenstra. Submitted to WHO by Pharmacia &
         Upjohn, Kalamazoo, Michigan, USA.

    Gray, J.E. & Purmalis, A. (1962b) Lincomycin, antibiotic 124a, U-10,
         149a (5686-DJL-139). Intramuscular irritation in rabbits. Ref:
         JEG 5905: 1-9, 11. Unpublished interoffice memo from J.E. Gray
         and A. Purmalis to E.S. Feenstra. Submitted to WHO by Pharmacia
         and Upjohn, Kalamazoo, Michigan, USA.

    Gray, J.E. & Purmalis, A. (1962c) Lincomycin, antibiotic 124a, U-10,
         149a (5686-DJL-25). Intramuscular irritation in rabbits. Effect
         of pH on degree of irritation. Unpublished interoffice memo from
         J.E. Gray and A. Purmalis to E.S. Feenstra. Submitted to WHO by
         Pharmacia and Upjohn, Kalamazoo, Michigan, USA.

    Gray, J.E. & Purmalis, A. (1962d) Antibiotic 124a (10,149A).
         5686-DJL-139). Intramuscular and intravenous tolerance in the
         dog. JEG 4538:21-22, 145. Antibiotic blood levels (J.E. Grady).
         Unpublished interoffice memo from J.E. Gray and A. Purmalis to
         E.S. Feenstra. Submitted to WHO by Pharmacia & Upjohn, Kalamazoo,
         Michigan, USA.

    Gray, J.E. & Purmalis, A. (1962e) Lincomycin hydrochloride. U-10-149.
         Antibiotic 124a. Lot No. 14, 121-1, Ref. 5712 DEG 45-9. Chronic
         (3 month) oral toxicity in rats. JPEG 5905:69-75.. Unpublished
         interoffice memo from J.E. Gray and A. Purmalis to E.S. Feenstra.
         Submitted to WHO by Pharmacia & Upjohn, Kalamazoo, Michigan, USA.

    Gray, J.E. & Purmalis, A. (1962f) U-10, 149a, antibiotic 124a,
         lincomycin hydrochloride. Ref: 5712-DEG-75-9. Subacute
         intramuscular toxicity in the dog. Ref: JEG-5905:54-59, 66-67.
         Unpublished interoffice memo from J.E. Gray and A. Purmalis to
         E.S. Feenstra. Submitted to WHO by Pharmacia and Upjohn,
         Kalamazoo, Michigan, USA.

    Gray, J.E. & Purmalis, A. (1962g) Antibiotic 124A (-10, 149A). Lot No.
         5449-DMW-63-6. Subacute oral toxicity in the dog. JEG-4538:
         142-143, 146-148. Antibiotic levels in body fluids and tissues.
         (W. Sokolski). Unpublished interoffice memo from J.E. Gray and A.
         Purmalis to E.S. Feenstra. Submitted to WHO by Pharmacia &
         Upjohn, Kalamazoo, Michigan, USA.

    Gray, J.E. & Purmalis, A. (1962h) U-10,149a. Res. Lot No. 14, 121-4.
         Notebook ref: 5879 THP 150-1. Multiple dosing (5 weeks) of
         newborn rats with terminal histograms. Unpublished interoffice
         memo from J.E. Gray and A. Purmalis to E.S. Feenstra. Submitted
         to WHO by Pharmacia & Upjohn, Kalamazoo, Michigan, USA.

    Gray, J.E. & Purmalis, A. (1963a) Lincomycin hydrochloride, U-10,
         149a. 5879 THP 150-1. Sublethal oral and intravenous tolerance in
         the dog. Ref: JEG-5890: 16-17. Unpublished interoffice memo from
         J.E. Gray and A. Purmalis to E.S. Feenstra. Submitted to WHO by
         Pharmacia & Upjohn, Kalamazoo, Michigan, USA.

    Gray, J.E. & Purmalis, A. (1963b) Lincomycin U-10, 149a. Teratogenic
         study in the dog (revised report on extended study). Unpublished
         memorandum from J.E. Gray and A. Purmalis to E.S. Feenstra.
         Submitted to WHO by Pharmacia & Upjohn, Kalamazoo, Michigan, USA.

    Gray, J.E. & Purmalis, A. (1964a) Lincomycin hydrochloride, U-10,
         149a. Antibiotic 124a, lot No. 14-121-3. Chronic (3 month) oral
         toxicity in the dog. Special study requested by Food and Drug
         Administration. Ref: JEG 5905: 128-135. Unpublished interoffice
         memo from J.E. Gray and A. Purmalis to E.S. Feenstra. Submitted
         to WHO by Pharmacia & Upjohn, Kalamazoo, Michigan, USA.

    Gray, J.E. & Purmalis, A. (1964b) Lincomycin hydrochloride, U-10,
         149a. Antibiotic 124a, lot No. 14-121-3. Chronic (3 month) oral
         toxicity in rat. Special study requested by Food and Drug
         Administration. Ref: JEG 5905: 117-127. Unpublished interoffice
         memo from J.E. Gray and A. Purmalis to E.S. Feenstra. Submitted
         to WHO by Pharmacia & Upjohn, Kalamazoo, Michigan, USA.

    Gray, J.E., Prestrud, M.C. & Purmalis, A. (1962) Lincomycin
         hydrochloride, U10,149A. Lot No.: 14, 121-4. Notebook ref:
         5876THP150-1. Multiple dosing (2 weeks) of new puppies with
         terminal hemograms. Unpublished interoffice memo from J.E. Gray,
         M.C. Prestrud, and A. Purmalis to E.S. Feenstra. Submitted to WHO
         by Pharmacia & Upjohn, Kalamazoo, Michigan, USA.

    Gray, J.E., Highstrete, J.D. & Purmalis, A. (1963a) Lincomycin
         hydrochloride, U-10, 149a. Antibiotic 124a, Lot No. 14-121-4,
         ref: 5879-THP-150-1. Chronic (1-year) oral toxicity in the rat.
         JEG 5905:105-115. Unpublished interoffice memo from J.E. Gray
         J.D. Highstrete and A. Purmalis to E.S. Feenstra. Submitted to
         WHO by Pharmacia & Upjohn, Kalamazoo, Michigan, USA.

    Gray, J.E., Purmalis, A. Highstrete, J. (1963b) Lincocin (U-10, 149a).
         Lot No. 14, 121-1. Ref. 5712 DEG 45-9. Chronic (6 months) oral
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         JEG 5905: 64m 81-87. Unpublished interoffice memo from J.E. Gray,
         A. Purmalis, and J. Highstrete to E.S. Feenstra. Submitted to WHO
         by Pharmacia & Upjohn, Kalamazoo, Michigan, USA. 

    Gray, J.E., Purmalis, A. & Northam, J.I. (1963c) Lincomycin, U-10,
         149a. Effect on reproduction in the rat. Unpublished interoffice
         memo from J.E. Gray, A. Purmalis and J.I. Northam to E.S.
         Feenstra. Submitted to WHO by Pharmacia & Upjohn, Kalamazoo,
         Michigan, USA.

    Gray, J.E., Purmalis, A. & Lewis, C.N. (1965a) Diarrhea in rabbits
         resulting from treatment with lincocin. Results of studies I, II,
         III (FDA protocol) and studies IV and V. Unpublished interoffice
         memorandum from J.E. Gray, A. Purmalis, and C.N. Lewis to E.S.
         Feenstra. Submitted to WHO by Pharmacia & Upjohn, Kalamazoo,
         Michigan, USA.

    Gray J.E., Purmalis, A. & Whitfield, G.B. (1965b) Lincocin.
         Intrathecal irritation. Ref: JEG 5905: 144-150. JEG 4994: 88-92.
         Unpublished interoffice memo from J.E. Gray, A. Purmalis, and
         G.B. Whitfield to E.S. Feenstra. Submitted to WHO by Pharmacia &
         Upjohn, Kalamazoo, Michigan, USA.

    Greening, R.C., Baker, K.D. & Kotarski, S.F. (1995) Effect of
         pirlimycin on the viability of anaerobic bacterial species found
         in the human gastrointestinal tract. Unpublished report no.:
         782-7922-95-002 from The Upjohn Company. Cited, but not
         submitted, by Pharmaci & Upjohn, Kalamazoo, Michigan, USA.

    Hall, W.H. & Manion, R.E. (1970) In vitro susceptibility of brucella
         to various antibiotics.  Appl. Microbiol., 20, 600-604.

    Harbarch, P.R. & Aaron, C.S. (1987) Evaluation of U-10,149A
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    Harbach, P.R., Johnson, M.A. & Bhuyan, B.K. (1982a) The V-79 mammalian
         cell mutation assay with lincomycin (U-10, 149A). Unpublished
         report No. 9610/82/7263/001 from Agricultural Research and
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         Upjohn, Kalamazoo, Michigan, USA.

    Harbach, P.R., Johnson, M.A. & Bhuyan, B.K. (1982b) The V-79 mammalian
         cell mutation assay with lincomycin (U-10, 149A) using an S9
         activation system. Unpublished report No. 9610/82/7263/007 from
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    Hollis, D.G., Weaver, R.E., Baker, C.N. & Thornsberry, C. (1976)
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         Subacz, C.J. (1985) Pharmacokinetics and oral absorption of
         lincomycin (U-10, 149) in swine. Unpublished report No.
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    Hornish, R.E., Gosline, R.E. & Nappier, J.M. (1987) Comparative
         metabolism of lincomycin in the swine, chicken, and rat.  Drug 
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    Karchmer, A.W., Moellering, R.C., Jr & Watson, B.K. (1975)
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          Comprehensive Review with Clinical Emphasis, 3rd Ed., London,
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    Lerner, P.I. (1968) Susceptibilityof  Actinomyces to cephalosorins
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    Lusk, R.H., Fekety, R., Silva, J., Browne, R.A., Ringler, D.H. &
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    Martin, W.J., Gardner, M. & Washington, J.A., II (1972) In vitro
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    Mazurek, J.H. & Swenson, S.H. (1981) Evaluation of U-10, 149A in the
         Salmonella/microsome test (Ames assay). Unpublished report No.
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    McLinn, S.E., Nelson, J.D. & Haltalin, K.C. (1970) Antimicrobial
         susceptibility of  Haemophilus influenzae. Pediatrics, 45,
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    Morris, D.F., Harris, S.B., Poppe, S.M., Poppe, J.L., Stucinham, J.L.
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    Mulvihill, W.J. & Gray, J.E. (1965). Lincocin. Teratogenic (Wilson).
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    Nappier, J.L. (1998) Additional clarification of lincomycin MRL
         residue file. Unpublished interoffice memo from J.L. Nappier to
         S. Lens. Submitted to WHO by Pharmacia & Upjohn, Kalamazoo,
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    Nasu, M., Maskell, J.P., Williams, R.J. & Williams, J.D. (1981) In
         vitro activity of MK0787 ( N-formidoyl thienameycin) and other
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    Nielsen, R.W. (1975) Acute oral LD50 of salvage lincomycin
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         Upjohn, Kalamazoo, Michigan, USA.

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          Yersinia enterocolitica: in vitro antimicrobial susceptibility.
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    Rifkin, G.D., Silva, J. & Fekety, R. (1978) Gastrointestinal and
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          Bacteroides corrodens and  Eikenella corrodens to ten
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         primary rat hepatocyte unscheduled DNA synthesis assay.
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    Seaman, W.J., Platte, M.J. & Thomas, R.W. (1981) 26-month oral feeding
         study in rats with lincomycin: Complete histopathological
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         report no. 768-9610-81-002 from Agricultural Research and
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         Pharmacia & Upjohn, Kalamazoo, Michigan, USA.

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         Lincomycin serum and saliva concentrations after intramuscular
         injection of high doses.  Clin. Pharmacol., 21, 411-417.

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         Susceptibility of group D sreptococcus (Enterococcus) to 21
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         Submitted to WHO by Pharmacia & Upjohn, Kalamazoo, Michigan, USA.

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    See Also:
       Toxicological Abbreviations
       LINCOMYCIN (JECFA Evaluation)