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    OLAQUINDOX

    1.  EXPLANATION

         Olaquindox (I) is a quinoxaline 1,4-dioxide structurally related
    to carbadox, quindoxin, and cyadox.  The chemical structure is shown
    in Figure 1.

    FIGURE 1

         The compound is used as a growth promoter in pigs and is supplied
    as a 10% premix in feed for admixture in the final feed at rates of
    50-100 ppm for starter rations and 25-50 ppm in grower/fattener feeds
    (Windholz et al., 1983; Anon,1989; Kulczyk et al., 1979; Baars
    et al., 1988).  It has not previously been evaluated by the Joint
    FAO/WHO Expert Committee on Food Additives.

    2.  Biological Data

    2.1  Biochemical Aspects

    2.1.1  Absorption, distribution and excretion.

    2.1.1.1  Rats

         Olaquindox is well absorbed when given orally to rats. 
    Approximately 85% of the radioactivity from a dose of 10 mg/kg b.w. 3-
    14C-olaquindox was excreted in the urine.  Most radioactivity was
    found in the urine within 3 hours of administration.  The remainder
    was eliminated in the faeces.  Less than 1% was recovered in expired
    air as carbon dioxide.  Experiments with 3-14C-olaquindox
    intraduodenally administered to rats with bile duct fistulas suggested
    that around 18% of the dose was excreted in the bile.  Similar
    findings were made after intravenous dosing.  Distribution occurred in
    a generalized manner throughout the body after oral dosing and most of
    the radioactivity had disappeared by 24 hours.  Autoradiography
    revealed the highest amount in the rat kidney at 4 hours, indicative
    of the extent of urinary excretion already noted.  Slightly elevated
    concentrations were also observed in liver, testes, adrenals and hair
    follicles.  When dogs were given 10 mg/kg 3-14C-olaquindox, a similar
    metabolic profile was noted to that seen in the rat (Duhm et al.,
    1970).

    2.1.1.2  Pigs

         Olaquindox was rapidly absorbed when given orally to pigs.  Over
    90% of an oral dose of 2 mg/kg b.w. was eliminated in the urine within
    24 hours, which is indicative of rapid and extensive absorption.  The
    remainder was excreted in the faeces.  Maximum plasma levels were
    attained within 1-2 hours of dosing (1-2 ppm).  This was followed by
    a rapid decline in plasma levels reaching around 0.03 ppm by 24 hours
    and 0.005-0.01 ppm by 48 hours.  Radioactivity was present in all
    tissues when examined 2 days after dosing, but the levels were
    extremely low.  In the kidney and liver, levels of 110 and 52 ppb were
    found, while levels in muscle were only 9 ppb.  After 8 days, levels
    in liver and kidney had fallen to 27 and 12 ppb, respectively, while
    those in muscle were in the range of 2.5 ppb.  By 28 days after dosing
    only low levels were found in kidney and muscle (0.9 and 0.5-0.8 ppb,
    respectively) with slightly higher concentrations in the liver (2 ppb)
    (Duhm et al., 1973).

         When pigs were dosed at levels in the range of those recommended
    in use (up to 100 ppm in the diet) for up to 20 weeks, relatively high
    levels were found in the kidney (around 2000 ppb) with relatively
    moderate levels in the liver (300 ppb) when the animals were killed
    six hours after drug withdrawal.  When killed 2 days after withdrawal,
    levels had fallen to below the limits of detection (50 ppb) in liver,

    kidney and muscle.  Pigs given diets containing olaquindox at levels
    in excess of those recommended (160 or 250 ppm) for up to 4 weeks also
    had high initial levels in kidney, liver and muscle but these had
    fallen to below the limits of detection by day 2 after withdrawal
    (Medenwald, 1974; Medenwald & Gericke, 1974; Bories & Bourdon., 1977).

         Similar findings were made in other studies where pigs were given
    diets containing 100 or 150 ppm olaquindox for 12-30 weeks (Takase &
    Komachi, 1975; 1976).

         After pigs were given diets containing up to 45 ppm olaquindox
    for the duration of the fattening period, the highest levels were
    found in the liver (0.14 ppm) and kidney (0.28 ppm) 6 hours after
    withdrawal.  By 24 hours the levels were below the limit of detection
    (0.1 ppm) (Leibetseder, 1980).  Similar results were noted when pigs
    were given diets containing 10 ppm olaquindox (Anderson & Szabo,
    1982).

    2.1.2  Biotransformation

         The biotransformation of olaquindox has been investigated only in
    the pig.  The majority of an oral dose of olaquindox (70%) was
    excreted in the urine unchanged.  The major metabolites appeared to be
    the reduced compounds, the 1- or 4-mono-N-oxides (16%).  Three other
    compounds thought to be carboxylic acid derivatives made up the
    remainder (Duhm et al., 1973).  Later work led to the elucidation of
    the structures of these metabolites in the pig.  Again the major
    urinary component after oral dosing was olaquindox with about 7%
    present as the 4-mono-N-oxide.  Omega oxidation produced the
    2-carboxymethylaminocarbonyl compound and its 4-mono-N-oxide
    derivative (6%).  Some of the corresponding 1-mono-N-oxide moiety of
    the 2-carboxymethylaminocarbonyl was also noted (1%).  The remaining
    metabolite was the di-desoxy derivative of
    2-carboxymethylaminocarbonyl compound, 2-carboxymethylaminocarbonyl-3-
    methyl quinoxaline (>1%) (Maul et al., 1979a and b).

    2.1.3  Effects on enzymes and other biochemical parameters

         Olaquindox has been shown to cause a decline in plasma
    aldosterone levels in the pig accompanied at higher dietary levels by
    hyponatraemia and hyperkalaemia.  These findings are considered in
    more depth in the section on short-term toxicity studies (van der
    Molen et al, 1989; Baars et al, 1988).

    2.2  Toxicological Studies

    2.2.1  Acute Toxicity

         Acute toxicity studies are summarized in Table 1.

         In a series of experiments, groups of 10 male mice were given
    oral doses of 2500-5000 mg/kg b.w. olaquindox as an aqueous suspension
    in 2% carboxymethylcellulose.  Vehicle controls or undosed groups did
    not appear to have been used.  Only 1/10 mice died at the lowest dose
    used while 100% lethality was noted at the highest dose.  Signs of
    toxicity included decreased activity, lowering of the eyelids, and
    irregular breathing.  Animals died 2-14 days after olaquindox
    administration.  Discolored livers and yellowish-green intestinal
    contents were noted on gross examination.  Similar findings were made
    when groups of male rats were given olaquindox in a similar manner at
    doses of 1400-2000 mg/kg b.w. (Tettenborn, 1969).

    Table 1:  Acute toxicity of olaquindox
                                                                        
    Species/strain Sex   Route       LD50               Reference
                                     (mg/kg b.w.)
                                                                        

    Mouse/CF1      M     oral        3316               Tettenborn, 1969

                   M     s.c.        2237               Tettenborn, 1969

    Rat/Wistar     M     oral        1704               Tettenborn, 1969

                   M     s.c.        1275               Tettenborn, 1969

                   M+F   inhalation  >1751 mg/m3*       Thyssen, 1982

                   F     oral        1657               Steinhoff, 1973

    Rabbit/cross   M+F   oral        1000-2000          Tettenborn, 1969

                   M+F   s.c.        1000-2500          Tettenborn, 1969

    Cat/cross      M+F   oral        1000               Tettenborn, 1969

                   M+F   s.c.        500                Tettenborn, 1969

    Dog/beagle     M+F   oral        emetic**           Tettenborn, 1969

                   M+F   s.c.        ***                Tettenborn, 1969
                                                                        

    *   4 Hour LC50 value.
    **  Lethal doses could not be achieved due to emesis.
    *** 250 mg/kg produced local reaction; temporary inappetence, higher 
        doses were not tried

         Groups of two rabbits, each presumably 1 male and 1 female, were
    dosed orally with olaquindox in 2% methylcarboxycellulose.  No deaths
    occurred at the lowest dose of 500 mg/kg b.w. while in those given
    1000 or 2000 mg/kg b.w., 1/2 animals died.  All the animals given 4000
    mg/kg b.w. died.  Similar observations were made in groups of two cats
    given 500, 1000 or 2000 mg/kg b.w., with both animals given the
    highest dose dying.  Emesis was the main toxicological sign noted. 
    Dogs given oral doses of up to 100 mg/kg b.w. olaquindox showed no
    signs of toxicity but those given 250-2000 vomited; no lethalities
    occurred.  Vehicle controls or undosed animals did not appear to have
    been used in these studies (Tettenborn, 1969).

         When given by the subcutaneous route as a suspension in 2%
    carboxmethylcellulose, the acute toxicity of olaquindox was more
    marked (Table 1).  Doses of 500 to 2500 mg/kg b.w. and above proved
    lethal to mice, rats, rabbits and cats.  For unspecified technical
    reasons, dogs were given only  250 mg/kg b.w. olaquindox, which
    produced a local reaction and temporary inappetence a week later
    (Tettenborn, 1969).

    2.2.2  Short-term studies

    2.2.2.1.  Mice

         Groups of 20 male and 20 female BOM-NMRI mice were fed diets
    containing 0, 300, 600, 1200, 2400 and 4800 ppm olaquindox,
    approximately equivalent to 0, 45, 90, 180, 360 and 720 mg/kg
    b.w./day, for 90 days, as a dose-finding exercise for a
    carcinogenicity study (see Section 2.2.3).

         Signs of toxicity were non-specific and included shaggy fur,
    dyspnoea and reduced motility.  A marked reduction in body weight
    occurred at the highest dietary level in both sexes, and in males
    given 1200 and 2400 ppm.  During the study 1/20 females died at the
    600 ppm level as did 18/20 and 5/20 males and females, respectively,
    at the 1200 ppm level.  All the mice given the two highest doses died. 
    No deaths occurred in other groups.  At necropsy, haemorrhagic lungs
    were the main findings.  Microscopic examination was not conducted
    (Steinhoff & Gunselmann, 1982).

    2.2.2.2  Rats

         Groups of 10 male and 10 female Wistar rats were given oral doses
    of 0, 20, 60 or 180 mg/kg b.w./day olaquindox in 2% aqueous
    carboxymethylcellulose by stomach tube, for 5 days per week for 13
    weeks.

         After 6-8 weeks, signs of toxicity included reddening of the ears
    and plantar surfaces; weakness and emaciation occurred in animals
    given the highest dose.  These animals also developed moist, blood
    encrusted nostrils.  In the eighth week of the study, fatalities began

    to occur, consequently the animals were killed. No clinical signs of
    toxicity or compound-related deaths occurred in the other groups. 
    There were no adverse haematological effects in any groups at 4 weeks
    including the high dose group, nor at 12 weeks in remaining animals. 
    Clinical chemistry was normal at 4 weeks in all dose groups and in
    animals given 0, 20 or 60 mg/kg b.w./day olaquindox at 12 weeks. 
    However, at 8 weeks in the high dose animals (prior to death), blood
    sugar was significantly reduced, while serum aspartate
    aminotransferase was elevated.  Urinalysis was normal in all groups at
    4 weeks and in all but the high dose group (not available for
    examination due to deaths) at 12 weeks.

         Absolute organ weights at 90 days indicated a significant
    splenomegaly and increases in testicular and ovarian weights in
    animals given 60 mg/kg b.w./day olaquindox. These findings were also
    reflected in relative organ weights except for splenic weights in
    females.  There was a significant decrease in relative adrenal weights
    in females given 60 mg/kg b.w./day olaquindox.

         Gross examination revealed reddening of the pyloric area of the
    stomach in the high dose animals and pale and atrophied adrenals.  All
    the females given 60 mg/kg b.w./day and 5/10 of those given 20 mg/kg
    b.w./day had enlarged, reddened ovaries with numerous pin-point
    darkened nodules (corpora lutea).

         Microscopic examination revealed adrenal atrophy in the high and
    mid dose groups, with degenerative changes in the cortical areas. Some
    of the female high dose animals had thyroid atrophy.  Female rats
    given the mid and low dose had no ovarian atrophy but moderate
    atrophic changes were noted in the ovaries of 4/5 high dose females
    (Hoffmann 1969; Urwin & Mawdesly-Thomas, 1969).

         The experiment was later repeated using lower doses: 0, 1, 5 and
    20 mg/kg b.w./day.  All other factors were identical to the original
    study.  No clinical signs were observed during the study and there
    were no effects on body weights.  There were no haematological or
    clinical chemistry abnormalities; urinalyses were normal.

         At necropsy, increases in adrenal weights in males given the two
    highest daily doses were noted.  Increases in ovarian weights occurred
    in females given the two highest doses.  Histopathologic examination
    revealed no changes in any organs in any of the treatment groups.  The
    no-effect level in these studies therefore was 1 mg/kg b.w./day
    (Hoffmann, 1972; Urwin & Spicer, 1971).

         In a 90 day dietary study, olaquindox was given to groups of 20
    male and 20 female Norway rats at levels of 0, 50, 150 and 300 ppm,
    approximately equivalent to oral doses of 0, 5, 15 and 30 mg/kg
    b.w./day.  Haematological and clinical chemistry investigations were
    conducted at days 0, 35, and 63 and at the end of the study.

         No signs of toxicity were noted during the study and there were
    no effects on haematology and clinical chemistry.  Gross and
    microscopic examination revealed no compound-related changes
    (Nastuneak et al, 1986).

         In an inhalation study, groups of 10 male and 10 female Wistar
    rats were exposed to 0, 10, 207 and 541 mg/m3 olaquindox dust, 6
    hours/day, 5 days per week for 3 weeks under dynamic exposure
    conditions.  Based upon number and particle mass median diameter,
    between 30-80% of the particles were inhalable.

         No adverse effects occurred in the rats exposed to the lowest
    concentration of olaquindox nor in the males of the intermediate
    concentration group.  Females in this group showed non-specific
    effects ("sluggishness") for the first 5 days of exposure but after
    this they appeared normal.  Effects of a similar nature and duration 
    occurred in the high concentration males but in females, signs of
    respiratory distress were noted from the 11th day of exposure in
    addition to the non-specific effects seen in other exposed groups.

         No deaths occurred and only minor effects on body weights were
    observed.  There were no effects on haematology nor on clinical
    chemistry.  Urinalyses were normal.  No gross abnormalities were
    evident at autopsy and relative and absolute organ weights were in the
    normal range.  There were no adverse histopathological findings.  The
    non-specific signs and particularly the respiratory effects may have
    been due to the physical effects of the olaquindox particles (Thyssen,
    1983).

    2.2.2.3  Rabbits

         Olaquindox in Lutrol was applied to the shaven intact dorsal
    surface of 3 groups of 6 New Zealand white rabbits (3 male and 3
    female) at doses of 0, 50 or 250 mg/kg b.w./day for 6 hours/day, 5
    days per week for 3 weeks, without an occlusive dressing.  In an
    identical manner, olaquindox was applied to the abraded skin of 3
    groups of 3 male and 3 female rabbits.

         No signs of toxicity were observed in treated animals and skin
    reactions due to olaquindox were not seen in the abraded or intact
    skin groups.  No mortalities occurred.  Clinical chemistry and
    urinalyses at the end of the study were comparable to control values. 
    There were no effects on body weights.  At necropsy, no abnormalities
    were noted and no histopathological changes attributable to olaquindox
    treatment were found (Heimann & Schilde, 1982).

    2.2.2.4  Dogs

         Groups of 2 male and 2 female beagle dogs were given oral doses
    of 0, 20, 60 or 180 mg/kg b.w./day olaquindox in gelatin capsules for
    90 days.


         Vomiting occurred during the first week in dogs given the highest
    dose.  Salivation was noted and food intake declined.  The animals
    became emaciated.  Dogs given the intermediate dose showed inappetence
    and salivation occurred.  No effects were noted in low dose animals.

         All the high dose animals died within 20 days of the start of the
    study.  One dog given the intermediate dose died on day 40 while the
    remainder were killed in extremis after 46 or 56 doses.  No animals
    given the low dose died.

         There were no notable changes in haematology in treated dogs. 
    Clinical chemistry revealed increases in blood urea in all 4 dogs
    given the high dose.  Intermittent elevations of blood urea were noted
    in the other dosed groups.  No abnormalities in urinalyses occurred.

         Gross examination of high dose animals suggested an irritant
    effect on the gastrointestinal tract, with congestion in the lungs. 
    The livers were discolored.  No abnormalities were noted in the low
    dose animals.  Histopathologic examination revealed live cell
    enlargement and fatty degeneration in dogs given 60 or 180 mg/kg
    b.w./day olaquindox, with fatty degeneration of the cells of the
    kidney tubules.  No histopathologic changes were found in low dose
    dogs and the no-effect level in this study appeared to be 20 mg/kg
    b.w./day (Lorke & Tettenborn, 1969; Mawdesley-Thomas & Urwin, 1969).

    2.2.2.5  Pigs

         Groups of 5 castrated male and female German landrace pigs
    weighing 9-10 kg each were fed diets containing 0, 100, 160 and 250
    ppm olaquindox for 20 weeks.

         At the highest dietary level, 5 pigs died and weight gain was
    significantly reduced.  Animals given 100 and 160 ppm in the diet had
    higher rates of weight gain than controls.  No haematological effects
    occurred.  Plasma creatinine and urea were elevated in the
    intermediate and high dietary level groups.  Hyperkalaemia and
    hyponatraemia were noted in pigs given 250 ppm dietary olaquindox. 
    Urinalyses were normal.

         High dose pigs showed a grey-brown discoloration of the renal
    cortex but there were no effects on relative organ weights.  Tubular
    dilatation and flattening of the tubular epithelium occurred in
    intermediate and high dietary level pig kidneys and the adrenals of

    these animals displayed enlarged cortical epithelial cells.  The no-
    effect level in this study was 100 ppm olaquindox in the diet (Gericke
    & Dycka, 1974; Hoffman et al., 1974).

         Groups of 7 hybrid piglets (4 weeks old, at least 3 females per
    group and castrated males) were fed diets containing 0, 25, 50, 100
    and 200 (2 groups) ppm olaquindox for 6 weeks.

         After 2 weeks, dry faeces were produced by piglets given 100 or
    200 ppm olaquindox.  The drinking of urine from the floor of pens or
    directly from urinating pen-mates was noted in pigs given 50 ppm
    olaquindox.  A decrease in abdominal volume occurred in piglets given
    100 or 200 ppm olaquindox after 5 weeks and in the 25 ppm group in
    week 6, but not in animals give 50 ppm.  Significant rises in serum
    albumin values occurred in piglets given 100 or 200 ppm olaquindox
    from week 2 onwards, and marked rises in serum urea values occurred in
    the 200 ppm group from week 4 and in the 100 ppm group from week 5. 
    Gross and microscopic pathology were not conducted (Nabuurs et al,
    1989).

         Groups of 6 female and 6 castrated male hybrid piglets were fed
    diets containing 0, 25, 50, 100 or 200 ppm olaquindox for 6 weeks, in
    a study of the effects of treatment on plasma aldosterone, sodium and
    potassium levels.  There was a gradual decline in plasma aldosterone
    which was significant in all but the 25 ppm group by week 5.  After 6
    weeks the decline was significant in all dosed groups except for that
    given 100 ppm where a small rise was noted.  Hyponatraemia occurred in
    the 25 and 200 ppm groups after weeks 0-2 and a continuous decline in
    the 200 ppm group occurred after week 3.  In the groups given 25 and
    100 ppm olaquindox the levels decreased continuously from 2-3 weeks. 
    Animals in the 50 ppm group were unaffected.  Although elevated
    potassium levels occurred in the 50 and 100 ppm groups, only piglets
    given 200 ppm were considered to be hypokalaemic (van der Molen
    et al, 1989; Baars et al, 1988).

         Similar effects have been noted in pigs given other quinoxaline
    N-oxide drugs, namely cyadox and carbadox (van der Molen et al,
    1985).  The latter drug caused these effects after accidental
    overdosage of pigs (Power et al, 1989).  The toxicity appears to be
    due to specific effects on the aldosterone-releasing zona glomerulosa
    of the adrenals (van der Molen et al, 1986).

    2.2.2.6  Rhesus monkeys

         Olaquindox in gelatin capsules was given orally to two groups of
    3 male and 3 female rhesus monkeys at doses of 0 and 20 mg/kg b.w./day
    and to two groups of 3 males and 5 females at doses of 5 and 40 mg/kg
    b.w./day, 7 days a week for 19 weeks.  Surviving high dose females
    were entered onto a 17 week recovery period.

         Animals given the highest dose showed a general loss of condition
    and loss of body weight.  Suppression of weight gain occurred at the
    intermediate dose while growth promotion occurred at the low dose. 
    Suppression of appetite was evident from week 12 in high dose monkeys.

         Vaginal cytology indicated a suppression of ovulation in the high
    dose females and in 1/3 animals given the intermediate dose. There was
    some evidence of recovery after dosing ceased in the high dose
    females.  Deaths occurred in 2/3 males and 1/5 females form the high
    dose groups during the dosing period and 2 females died during the
    first two weeks of the recovery period.

         Electrocardiography and ophthalmoscopy were normal in all
    animals.  Urinalyses and haematology were essentially normal at 5
    weeks but serum aspartate aminotransferase values in high dose males
    were elevated.  At 8 weeks plasma biochemistry was normal but packed
    cell volume and red cell counts were lowered in high dose animals. 
    Urinary glucose was found in 3 high dose males.  After 15 weeks of
    dosing packed cell volume and haemoglobin showed slight reductions in
    high dose males while plasma glucose levels were reduced but not
    significantly.

         In 7/8 high dose monkeys, urine was positive for glucose and for
    total reducing substances.  One urine showed a lower pH and one was
    positive for ketones.  When examined at 15 weeks, red blood cell
    values were reduced in high dose monkeys.  Plasma glucose values were
    reduced in high dose animals and protein and glucose were present in
    the urines;  pH of urine was lowered in high dose animals.  At the end
    of the 19 week dosing period no haematological examinations were
    performed.  Plasma glucose levels were reduced in high dose animals
    while plasma urea values were increased.  Hypokalaemia was noted in
    high and intermediate dose animals.  Glucose and total reducing
    substances were increased in high dose monkeys while pH was lowered.

         Macroscopic examination revealed pallor of the kidney in high
    dose animals.  Suppression of ovulation occurred in high dose females,
    and abdominal abscesses in high dose males.

         Histopathological examination revealed fatty changes in the
    centrilobular areas of the liver in all high dose animals and
    deposition  of fat in the kidney tubules in all monkeys from this
    group.  Brown pigmentation of the zona reticularis of the adrenals
    occurred in high dose monkeys.  Immature testes were noted in males
    given 20 and 40 mg/kg b.w./day olaquindox while inactivity of the
    ovaries in high dose females and in 1/3 intermediate dose females was
    observed.   The no effect level in this oral study in rhesus monkey
    was 5 mg/kg b.w./day (Heywood et al, 1972).

    2.2.3  Long-term/carcinogenicity studies

    2.2.3.1  Mice

         Groups of 20 male and 20 female NMRI mice were given nominal
    doses of 0, 15 or 75 mg/kg b.w./day olaquindox in the drinking water.
    Low dose mice were given a total of 6.6 g/kg b.w. for 635 days and
    high dose mice were given a total of 32.1 g/kg b.w. for 634 days.  The
    study was terminated when all the mice had died.  Animals were not
    dosed over holidays and no mention was made of week-ends.

         At termination, no excess tumour incidence was noted. 
    Lymphadenosis was reported in 1/40 control mice while 2/40 high dose
    animals had tumours (thymoma and malignant thymus cell tumour) as did
    2/40 low dose mice (pulmonary carcinoma and bronchial carcinoma.

         Only small numbers of mice were used in this study and survival
    was poor, although it was better in the high dose group than in
    controls.  The mean survival had a high standard deviation (mean
    survival 340  187, 338  224 and 403  194 days for mice given 0, 15
    or 75 mg/kg b.w./day, respectively).

         Moreover, even when survival was adjusted for unexplained deaths
    which occurred in the first 82 days in controls, the treatment time
    was still less than is currently viewed as normal for a mouse
    carcinogenicity study.  The short life-span on test may be due to the
    fact that the animals were already around 50 days old when the study
    commenced (Schmael, 1973).

         In a later study, groups of 75 male and 75 female NMRI mice were
    given diets containing 0, 40, 120 or 360 ppm olaquindox, equivalent to
    0, 6, 18 or 57 mg/kg b.w./day, for life (until death or sacrifice in
    a moribund state).

         The only effects on body weights were in males and females given
    the highest dietary level: male weights fell slightly below control
    values from day 50 and female weights declined after day 200. 
    Haematological examination at weeks 4, 13, 26, 52 and 78 revealed no
    abnormalities and survival times were unaffected by olaquindox intake;
    remaining males and females died around day 890 (approximately 29
    months).

         At necropsy there were no differences in liver, kidney, spleen,
    heart, testes, or brain weights, and no increases in non-neoplastic
    findings were found in treated mice.  No increased tumour incidence
    was found in animals given 40 or 120 ppm dietary olaquindox but at 360
    ppm there was an increase in the total number of tumours and in the
    number of animals with benign tumours.  These were due to increases in
    the incidence of pulmonary adenoma and adrenal cortical adenoma in
    males and in pulmonary adenoma and ovarian granulosa cell tumours in

    females (Table 2).  There were no increases in the incidence of any
    malignant tumour types (Steinhoff & Gunselman, 1982).

    Table 2:  Tumor incidence in mice given olaquindox in the diet
                                                                        
                          0 ppm        40 ppm       120 ppm      360 ppm
                                                                        

    Males
    pulmonary adenoma     11 (15%)     17 (23%)     14 (19%)     27 (36%)

    adrenal cortical      5  (7%)      3  (4%)      6  (8%)      13 (17%)
    adenoma

    Females
    pulmonary adenoma     8  (11%)        (7%)      7  (9%)      11 (15%)

    ovarian granulosa     10 (13%)     16 (21%)     15 (20%)     20 (27%)
    cell tumor

                                                                        

    2.2.3.2  Rats

         Groups of 20 male and 20 female Wistar rats were given
    olaquindox, once per week for up to 560 days.  The total dose was 4.7
    g/kg b.w. with individual doses being in the range of 50-150 mg/kg
    b.w.  Each dose was given by gavage as a suspension in physiological
    saline.  Controls were given physiological saline only, but this was
    administered by intraperitoneal injection and not by gavage as they
    simultaneously acted as controls for other studies.

         Survival in treated animals was better than controls (875  105
    days for male and 818  167 days for female rats given olaquindox
    compared with 797  215 and 779  187 days, respectively, for male and
    female controls).

         The incidence of tumours was not presented in a detailed tabular
    form but was given graphically and in separate tables which did not
    allow a full comparison.  However, there was no elevated incidence of
    any tumour type in treated animals when compared with controls and the
    number of tumour-bearing animals was similar in both the treated and
    control groups (Steinhoff, 1973).

         Groups of 80 BR 46 rats were given drinking water containing
    olaquindox at levels to ensure an intake of 15 or 75 mg/kg b.w./day. 
    A group of 49 rats given water only served as controls.  Male and
    female rats were used in the study but the sex ratio was not
    specified.  The treated water was supplied 5 days a week until the
    animals died.

         Survival of animals treated with olaquindox was better than
    controls (704  161 days and 655  229 days in low and high dose rats,
    respectively, compared with 554  248 days in control rats).  The
    carcinogenicity data was presented in an unclear manner and separate
    data for male and female animals were not given.  The only tumour
    which showed an increased incidence was mammary fibroadenoma (1/40,
    2.5%; 3/46, 6.5% and 7/46, 15% in controls and in low and high dose
    rats, respectively).  However, the absence of data on male and female
    incidence of this tumour renders the values uninterpretable (Schmael,
    1973).

         As part of a chronic toxicity/carcinogenicity study with
    in utero exposure, groups of 75 males and 75 females were given
    diets containing 0, 40, 120 or 360 ppm olaquindox.  These doses were
    approximately equivalent to oral doses of 0, 3, 10 and 30 mg/kg
    b.w./day, for one week prior to mating and during a 3 week 1:1 mating
    period.  After mating the males were removed and the females given the
    diets containing olaquindox until the young were 4 weeks old.  At this
    time the young from each treatment group were divided into groups of
    25 males and 25 females and given the same diets as initially given to
    their parents.  The study was continued until the young had been
    treated for 2 years.  Clinical chemistry, haematology, and urinalyses
    were conducted on groups of 5 males and 5 females at 4, 14, 26, 54 and
    102 weeks after commencement of treatment of the F1 generation.

         No overt signs of toxicity were noted in treated animals but
    after day 400 of treatment, male and female animals given the highest
    dietary level showed a marked and statistically significant reduction
    in body weight when compared with control values.

         Clinical chemistry suggested an elevated creatinine level in the
    blood of rats given 360 ppm dietary olaquindox but all the values were
    within the normal range.  The albumen content of the urine was
    generally lower in treated animals than in controls.

         At gross and microscopic examination, there were no increases in
    the incidences of non-neoplastic diseases and no increased incidence
    of any tumour types.  This study employed too few animals to allow an
    assessment of carcinogenic potential (Steinhoff, 1977).

         Olaquindox was tested in a carcinogenicity study using a similar
    protocol on groups of 50 male and 50 female rats remaining in the F1
    generation from the chronic toxicity carcinogenicity test described
    above.  The groups were given diets containing 0, 40, 120 and 360 ppm
    olaquindox, approximately equivalent to oral doses of 0, 3, 10, and 30
    mg/kg b.w./day.  No clinical chemistry or haematological tests were
    conducted, however, and the study was terminated when the duration was
    approximately 3 years (1065 days for males and 1120 days for females),
    at which time 20% of the controls (males and females separately)
    remained alive.

         No signs of toxicity were noted in treated animals except for
    reductions in body weights in rats given 360 ppm after day 500. 
    Reductions occurred despite the fact that in terms of feed consumption
    in g/kg b.w./day these animals had consumed more.

         At termination there was a significant decrease in survival in
    animals given the highest dietary level (98% mortality in males and
    females) and in females given 40 ppm olaquindox (92% mortality)
    compared with controls (80% mortality).  Mortality in the other
    dietary groups was only slightly more than in controls (82-86%).

         At necropsy there were no differences between treated animals and
    controls for the number of animals of each sex with total tumours,
    primary tumours, malignant and benign tumours, malignant tumours with
    metastases and total benign tumours.  Similarly, for particular tumour
    sites (benign and malignant tumours plus metastases) there were no
    excess incidences except for slight increases in the incidence of
    adrenal, reticuloendothelial and seminal vesicle neoplasms, but these
    were due, except for the adrenal tumours, to metastases or
    infiltrations from other organs.  Moreover, the adrenal tumours were
    not increased with respect to any particular histological type and
    again were often of metastatic origin (Steinhoff & Boehme, 1978).

         There were some anomalies in the reporting of this study which
    appear to be due to arithmetical errors.  The summary table of tumour
    incidence cites a benign tumour in female controls which is not listed
    in the detailed histopathology tables.  The summary data also claims
    4 malignant tumours in males given the highest dietary level whereas
    the histopathology table lists 9.  However, these differences do not
    affect the outcome of the study or its assessment.

    2.4  Reproduction Studies

    2.4.1  Mice

         Groups of 20 pregnant NMRI mice were given oral doses of 0, 20,
    60, or 180 mg/kg b.w./day olaquindox as an aqueous suspension in
    tragacanth by gavage from day 6 to day 15 of gestation.  On day 18 of
    gestation the fetuses were delivered by Caesarean section and these
    were weighed and examined for gross malformations and by alizarin
    staining.

         None of the pregnant animals died during the test but animals
    given the highest dose showed reduction in body weight or rate of
    weight gain.  The numbers of implantations, live fetuses and
    resorptions were similar in all dosed groups.  At the highest dose,
    fetal weights were significantly lower than in controls.  The
    incidence of malformations in all treated groups was similar to those
    seen in controls (Lorke, 1971a).

    2.4.2  Rats

         Groups of 20 female pregnant FB 30 rats were given oral doses of
    0, 20, 60 or 180 mg/kg b.w./day olaquindox as an aqueous suspension in
    tragacanth by gavage from day 6 to day 15 of gestation.  Fetuses were
    delivered by Caesarean section on day 20 of gestation and these were
    examined in the same manner as for the mouse study described above.

         The pregnant rats given the highest daily dose showed reductions
    in body weights or rate of weight gain compared with control values. 
    These animals also showed a higher incidence of resorptions and lower
    numbers of live fetuses.  Fetal weights were lower in the high dose
    animals. These indices were similar to controls and to rats given 20
    or 60 mg/kg b.w./day olaquindox.

         The incidence of malformations in fetuses from dams given 20 or
    60 mg/kg b.w./day olaquindox was similar to controls but at the
    highest dose level there was an elevated incidence of malformed
    fetuses, with 5 malformations reported at a dose level of 180 mg/kg
    b.w./day.

         In this study, therefore, there was a teratogenic effect at the
    highest dose level given to pregnant rats (180 mg/kg b.w./day) on days
    6-15 of gestation.  The no-effect level in this study was 60 mg/kg
    b.w./day (Lorke, 1971b).

         A 3-generation study was conducted in groups of 10 male and 20
    female FB30 rats using 0, 20, 100 and 500 ppm olaquindox in the diet,
    equivalent to oral doses of 0, 1, 5 and 25 mg/kg b.w./day.  The F0
    animals were given the diets containing olaquindox throughout the
    study including during the mating periods.  Animals were mated after
    dosing for 70 days.  Animals which died during the study were
    necropsied, while young animals were examined macroscopically after
    birth and subsequently observed during the rearing period for evidence
    of abnormalities. The F3b animals were sacrificed at 3 weeks of age
    and subjected to macroscopic and microscopic examination.

         Treatment had no effect on body weights except that body weight
    of F0 generation females given the highest dietary level of
    olaquindox were slightly higher than weights of controls.

         The fertility rate was also lowered in F0 animals in the first
    and second matings when given the highest level, but there were no
    effects on litter size nor on rearing rates.  F1a and F1b animals did
    not differ from controls in terms of birth weights except for a
    slight, non-statistical increase in those derived from F0 dams given
    the highest dietary level.  After the second F0 mating the average
    number of young in the F1b generation was similar in all treatment
    groups, but at 5 days there was a significant reduction for dams given

    500 ppm olaquindox (6/litter) compared with values in other groups and
    controls (10-12/litter).  Birth weights of the F1b generation were
    unaffected.

         When the F1b generation was mated, the gestation rates in animals
    derived from animals initially given the highest level of olaquindox
    were reduced (80-84%) compared with values for other groups (90-100%). 
    The average numbers of young per litter were also reduced at the high
    dietary level in both the F2a and F2b generations, both at birth and
    5 days after birth (8/litter compared with 11/litter in controls). 
    The F2 birth weights were unaffected and there was some improvement
    in the rearing rates up to 4 weeks.

         In the F3 generation, fertility was again affected in animals
    originally derived from the high dietary level rates, with gestation
    rates of 70-84% compared with 90-100% in other groups.  The average
    number of young per litter was also reduced at the high dietary level
    (5-7/litter) compared with other groups (8.5-10.8/litter).  There were
    no effects on rearing rates nor F3 birth weights.  No malformations
    were noted during the course of the study and no abnormalities were
    found on gross or histopathological examination of three week old F3b
    animals (Loeser, 1974).

         In a fertility test in Wistar rats, olaquindox was administered
    orally by gavage to groups of 10 male rats at doses of 4 and 10 mg/kg
    b.w./kg which were mated with groups of 20 untreated females.  Groups
    of 10 untreated males were mated with groups of 20 females given
    gavage doses of 4 and 10 mg/kg b.w./day.  Males were dosed for 8 weeks
    prior to mating while females were dosed for 3 weeks prior to mating. 
    Males and females were mated in a 1:2 ratio.  Groups of untreated
    males and females were mated as controls.

         Olaquindox administration had no effect on body weights, on
    oestrus cycles, or on copulation and conception rates.  A significant
    reduction in the average number of implantations was noted in the
    group in which females given the lower dose of 4 mg/kg b.w./day
    olaquindox were mated with untreated males.  Pre-implantation losses
    were significantly increased in both the groups where females were
    treated with olaquindox, and post-implantation losses were increased
    in females given the 10 mg/kg b.w./day dose.  There were no effects in
    the groups  where males treated with olaquindox were mated with
    untreated females (Gandalovicova & Sykora, 1986).

    2.2.5  Special studies on pharmacological properties

         Olaquindox has been tested in a number of pharmacological
    screening tests in rats and mice including those for anticonvulsive
    effects, inhibition of defensive reaction, motor coordination,
    analgesia, antihypertensive effects, gastric secretion, bile

    secretion, diuresis, blood sugar and blood lipids and thrombocyte
    aggregation (bovine plasma).  No pharmacological activity was detected
    (Kaller, 1970).

    2.2.6  Special studies on irritancy and hypersensitivity

         Olaquindox as a micronized powder was applied without a vehicle
    to the shaved intact and abraded dorsal skin of 6 New Zealand white
    rabbits using a 24-hour occlusive dressing.  The skin was assessed on
    removal of the dressing, after 48 and 72 hours, and at 7 days.  Slight
    erythema of intact and abraded skin was observed at 24 hours, but not
    at the other time points.  No oedema was noted.  Results indicated a
    mild irritant effect (Murman, 1979).

         Olaquindox was tested for its ability to induce eye irritation by
    application of 15 mg of the micronized substance to the conjunctiva of
    the right eyes of 6 New Zealand white rabbits, and to the left
    conjunctival sacs of a further 6 rabbits.  The material was washed out
    of the left eyes with physiological saline after 1 minute.  Reactions
    were monitored 24, 48, and 72 hours after application and after 7
    days.

         A slight reddening of the conjunctiva was noted in 4/6 eyes where
    olaquindox had been directly applied.  Slight chemosis was noted in
    2/6 eyes.  A slight chemosis was noted in 1/6 rabbit's eyes where
    olaquindox has been applied to the conjunctival sac and then washed
    out.  All reactions subsided within 48 hours.  The result suggest that
    olaquindox has a slight irritant effect but the mechanical effects of
    the dust cannot be ruled out (Murman, 1979).

         Olaquindox was tested in the guinea-pig for its ability to induce
    hypersensitivity.  Olaquindox dissolved in dimethyl sulfoxide or as a
    suspension in phosphate buffered saline was administered
    intracutaneously into the neck region of groups of 10 Pirbright albino
    guinea-pigs on days 1, 3, 6, 9, and 13 of a sensitization schedule. 
    Four days after the last injection, a suspension of olaquindox in 1:1
    acetone/almond oil was applied to the depilated flank and massaged
    gently into the skin.  To allow for any possible effects of light,
    groups of guinea-pigs were treated in a similar manner but were kept
    in darkened cages.  No indications of sensitization were noted in this
    study when assessed by gross examination of the skin for reactions or
    when examined histologically (Schlumberger, 1975).  (This test is not
    widely used and it is unlikely to be as sensitive as those using an
    adjuvant (e.g. Freunds') such as the Magnusson-Kligman or Beuhler
    models).

    2.2.7  Special studies on genotoxicity

         Olaquindox has been tested in a wide range of genotoxicity tests
    and these are summarized in Table 3.  It has produced positive results

    in a number of studies designed to test for reverse mutations in
    bacteria, including the Ames test with Salmonella typhimurium
    strains (Beutin et al, 1981; Yoshimura et al, 1981; Voogd
    et al, 1980; Nunoshiba & Nishioka, 1989).  A positive result has
    been noted in a forward mutation assay with Escherichia coli
    (Nunoshiba & Nishioka, 1989).

         In vitro study with cultured human lymphocytes and a number of
    in vivo assays with mouse bone marrow or Chinese hamster
    spermatogonia as the target tissues have demonstrated the clastogenic
    activity of olaquindox (Tamura, 1977; Cihak & Vontorkova, 1983; Sram
    et al, 1986a; Pokorna, 1986; Herbold, 1983a).  Similarly, olaquindox
    has produced positive results in several micronucleus tests in the
    mouse following oral or inhalation exposure (Herbold, 1983b; Herbold
    & Thyssen, 1982; Cihak & Vontorkova, 1985), and in the rat after
    intraperitoneal injection (Cihak et al, 1983). However, a dermal
    study with a 24 hour exposure was negative (Herbold, 1982a),
    reflecting the poor dermal absorption of the substance.

         Olaquindox has been tested in two dominant lethal assays in the
    male mouse but a weak positive result was observed in only one of
    these when a high dose (1 g/kg b.w) was employed (Machemer, 1977a;
    Herbold, 1982b).  Positive lethal mutations also occurred when female
    mice were treated orally with olaquindox despite the use in one study
    of doses lower than that which effected a positive result in the male
    mouse (200 and 500 mg/kg b.w.) (Machemer, 1977b; Sram et al, 1986b
    & c).  However a toxic effect in female mice could not be excluded.

         Positive results have been obtained in a sister chromatid
    exchange test using Chinese hamster V79 cells indicating that
    olaquindox may induce DNA damage (Scheutwinkel-Reich & von der Hude,
    1984).  Positive results in bacterial assays including the SOS
    chromotest confirm this possibility (Suter et al, 1978; Beutin
    et al, 1981; Yoshimura et al, 1981; Nunoshiba & Nishioka, 1989;
    von der Hude et al, 1988).  However, there is no evidence that
    olaquindox covalently binds to DNA in the rat in vivo (Minini
    et al, 1983).

         The mutagenicity of a number of olaquindox metabolites has also
    been investigated.  The omega oxidation product, its 1 and 4
    monodesoxy derivatives and its didesoxy derivative have been
    investigated in the Ames test using S. typhimurium strains TA 98,
    100, 1535 and 1537 with and without rat liver S9 metabolic activation. 
    All the tests gave negative results (Herbold, 1978; 1979a,b).


        Table 3:  Results of genotoxicity studies with olaquindox
                                                                                                                                    
    Test System              Test Object              Concentration                      Results        Reference
                                                                                                                                    
    
    Ames test1               S. typhimurium           3.8-0.5 nmoles/plate               Positive       Beutin et al., 1981
                             TA98, 100

    Ames test2,4             S. typhimurium           1.9-57 nmoles/plate                Positive       Beutin et al., 1981
                             TA100

    Ames test1               S. typhimurium           1.25-15g/plate                    Positive       Yoshimura et al.,
                             TA98, 100                                                                  1981

    Ames test2               S.typhimurium            0.01-0.1 mmole/1                   Positive       Voogd et al., 1980

    Ames test1               S. typhimurium           0-50 g/plate                      Positive       Nunoshiba & Nishioka, 
                             TA98,100                                                                   1989

    Fluctuation1 test        K. pneumoniae            2x10-4-1x10-2 mmole/1              Positive       Voogd, et al,. 1980

    Fluctuation2 test        K. pneumoniae            2x10-5-1x10-2 mmole/1              Positive       Voogd, et al,. 1980

    Forward1 mutation        E. coli                  0-20 g/plate                      Positive       Nunoshiba & Nishioka,
                             Wp\P2uvrA/pKM101                                                           1989

    In vitro                 Cultured human           3-300 g/ml                        Positive       Tamura, 1977
    cytogenetics             lymphocytes

    In vivo                  Mouse bone marrow        20, 500 or 800  mg/kg              Negative       Sutou, 1977
    cytogenetics                                      b.w. oral

    In vivo                  Mouse bone marrow        200-800 mg/ml b.w. oral            Negative       Cihak & Vontorkova, 1983
    cytogenetics

    In vivo                  Mouse bone marrow        20-500 mg/kg b.w. diet             Positive       Sram, et al., 1986a
    cytogenetics                                      4 and 12 weeks
                                                                                                                                    

    Table 3 (contd)
                                                                                                                                    
    Test System              Test Object              Concentration                      Results        Reference
                                                                                                                                    
    
    In vivo                  Chinese hamster          20 mg/kg b.w. oral, x5             Positive       Pokorna, 1986
    cytogenetics             marrow

    In vivo                  Chinese hamster          2x30-2x1000 mg/kg b.w.             Positive       Herbold, 1983a
    cytogenetics             spermatogonia            oral

    Micronucleus             Mouse bone marrow        500 mg/kg b.w. oral,               Positive       Herbold, 1983b
                                                      sampled at 24, 48 or
                                                      72 hours; 10-300 mg/kg
                                                      b.w. oral, sampled at
                                                      24 hours

    Micronuceus test         Chinese hamster          20 mg/kg b.w. 4.2 and              Positive       Pokorna, 1986
                             bone marrow              100 mg/kg b.w. oral,
                                                      once

    Micronucleus             Mouse bone marrow        6.7 mg/m3 and 161 mg/m3            Positive       Herbold, & Thyssen, 1982
                                                      for 6 hours/day, 2 days,
                                                      inhalation

    Micronucleus             Mouse bone marrow        2034 mg/kg b.w. 30 hours           Negative       Herbold, 1982a
                                                      dermal exposure

    Micronucleus             Mouse bone marrow        100 mg/kg b.w oral or              Positive       Cihak & Vontorkova, 1985
                                                      intraperitoneal

    Micronucleus             Mouse bone marrow        100 mg/kg b.w oral or              Positive       Cihak & Vontorkova, 1985
                                                      intraperitoneal

    Dominant lethal          Mouse (male)             2x1000 mg/kg b.w. week             Positive       Machemer, 1977a
                                                      oral
                                                                                                                                    

    Table 3 (contd)
                                                                                                                                    
    Test System              Test Object              Concentration                      Results        Reference
                                                                                                                                    

    Dominant lethal          Mouse (male)             40, 120 and 360 ppm in             Negative       Herbold, 1982b
                                                      diet for 35 days
                                                      equivalent to 6, 18, and
                                                      54 mg/kg b.w.

    Dominant lethal          Mouse (male)             100, 300 and 500 mg/kg b.w.        Negative       Sram, et al., 1986b
                                                      for 4 weeks, 20, 40, 100,
                                                      200, and 500 mg/kg b.w.
                                                      for 12 weeks, diet

    Dominant3 lethal         Mouse (female)           30, 100, 300 or 1000 mg/kg b.w.    Positive       Machemer, 1977b
                                                      oral once

    Dominant lethal          Mouse (female)           20, 40, 100, 200 and 500 mg/kg     Positive       Sram, et al., 1986c
                                                      b.w. diet, for 4 weeks

    SOS2 Chromotest          E. coli, GE94            0-10 g/plate                      Positive       Nunoshiba & Nishioka, 1989
    (DNA damage)

    SOS2 Chromotest          E. coli, PQ37            0.001-0.1 mM                       Positive       von der Hude, et al., 
    (DNA damage)

    DNA damage               E. coli, K12             Not applicable                     Positive       Suter, et al., 1978

    DNA damage               S. typhimurium           100 g/disc uvr B and recA         Positive       Beutin, et al., 1981

    DNA damage               S. typhimurium           1-100 g/disc                      Positive       Yoshimura, et al., 1981

    Mitotic gene2            S. cerevisiae D4         0.05% w/v                          Positive       Voogd, et al., 1980
    conversion
                                                                                                                                    

    Table 3 (contd)
                                                                                                                                    
    Test System              Test Object              Concentration                      Results        Reference
                                                                                                                                    

    DNA binding              Rat                      500 mg/kg b.w. oral                Negative       Minini, et al., 1983
    in vivo

    Sister chromatid         Chinese hamster          0-200 g/ml V79 cells              Positive       Scheutwinkel-Reich & von 
    exchange                                                                                            der Hude, 1984
                                                                                                                                    

    1 With and without rat liver S-9 fraction. 
    2 In the absence of S-9 fraction.
    3 Positive only at the 1000mg/kg b.w. dose.
    4 Positive in aerobic and anaerobic conditions.
    

         The results obtained with olaquindox are similar to those noted
    with several other quinoxaline di-N-oxides including quindoxin and
    carbadox (Suter et al, 1978; English & Dunegan, 1970; Voogd
    et al, 1980; Negishi et al, 1980; Beutin et al, 1981 (see also
    carbadox)).  The mechanisms of action are unknown although neither
    olaquindox nor quindoxin binds to DNA (Minini et al, 1983, Suter
    et al, 1978).

         Electron spin resonance techniques have demonstrated the
    generation of free radicals during the reduction of quindoxin while a
    related compound 2,3-dihydroxymethyl quinoxaline-1,4-di-N-oxide has
    been shown to inhibit DNA synthesis in E. coli (Suter et al,
    1978).  However, at present the roles of free radicals and inhibition
    of DNA synthesis in the mutagenicity of quinoxaline-1,4-dioxides are
    unknown.

         These studies indicate that olaquindox is genotoxic in a variety
    of test systems.  It induces mutations in bacterial systems and it
    causes chromosome and DNA damage in in vitro and in in vivo
    systems.  Data available from dominant lethal assays and from an
    in vivo cytogenetic study with Chinese hamster spermatogonia suggest
    that olaquindox may have the potential to exert its mutagenic effects
    on germ line cells.

    2.3  Observations in humans

         One of the major routes of exposure to olaquindox is likely to be
    occupational during the preparation of feed and the feeding of the
    final feed to pigs.  In an experimental study, no olaquindox was
    detected in the workplace air (a stall building housing pigs) during
    trough filling operations with a feed containing 50 ppm olaquindox
    (Thyssen et al, 1982).  Olaquindox was only detected at low levels
    in the atmosphere during the preparation of 0.1% feed premix and the
    50 ppm final feed starting with a 10% premix of a proprietary product. 
    Levels in the atmosphere were estimated to be from below 0.4 to below
    0.1 g/m3 air (Inkmann-Koch, 1985a).  Analysis of the urine from a
    single worker engaged in similar preparation work and in the feeding
    of pigs revealed no olaquindox (limit of detection 40 ppb) (Inkmann-
    Koch, 1985b).

         When applied to the skin of two volunteers as a paste in water
    containing 2 g of olaquindox (around 30 mg/kg b.w.) using a 6 hour
    occlusive dressing, no olaquindox was detected in the 48 hour urine
    using an analytical method with a limit of detection of 0.12 g/ml
    (Beermann, 1982).

         There has been one report of allergic contact dermatitis and one
    of photocontact dermatitis following occupational exposure to
    olaquindox (Bedello et al, 1985; Francalanci et al, 1986).  Both
    occurred in pig workers exposed to the substance in the animal feed. 
    There are no reports of systemic toxicity in humans following exposure
    to olaquindox.

    3.  COMMENTS

         The toxicological data considered by the Committee included the
    results of acute and subchronic studies, together with the results of
    studies on mutagenicity, carcinogenicity, and effects on reproduction
    and development.

         Olaquindox is almost completely absorbed from the
    gastrointestinal tract in rats, dogs, and pigs.  In studies using
    radiolabelled olaquindox, the radioactivity was shown to be widely
    distributed in the tissues, with residues in the liver being the most
    persistent.  The compound was primarily eliminated in the urine, with
    lesser amounts being excreted in the faeces and expired air of the
    animals.  In the pig, elimination was virtually complete by 48 hours
    after a single intragastric dose.  Any remaining radioactivity was
    then eliminated with a half-life of 5-9 days, far in excess of that of
    olaquindox (about 3-5 hours).  The parent compound accounted for 70%
    of the radioactivity in the urine, up to 24 hours after
    administration.  There were approximately 16 metabolites detected in
    the urine; six major metabolites have been fully characterized.

         In acute and short-term toxicity studies in rats and mice, rats
    were about twice as sensitive as mice.  In a 90-day study in rats,
    effects were observed in the testes, ovaries, thyroid, and adrenal
    cortex at a dose of 5 mg/kg b.w./day and above.  These lesions were
    described histopathologically as atrophy; and in the adrenal glands
    the effect was greatest in the zona glomerulosa.  No treatment-related
    abnormalities of the pituitary gland were reported.  A 90-day study in
    beagle dogs and a 19-week study in rhesus monkeys also produced
    evidence of toxic effects on the endocrine glands, liver, and kidney. 
    Of five female monkeys dosed at 40 mg/kg b.w./day, one died during
    treatment and two died during the planned 17 week recovery period. The
    two survivors failed to re-establish normal ovarian cycles during the
    recovery period.

         In a 20-week study, groups of five male and five female pigs were
    fed up to 250 mg of olaquindox per kg of diet.  The plasma
    concentrations of urea and creatinine were elevated at 160 and 250 mg
    diet, which suggested an effect on the kidney.  All of the pigs in the
    highest-dose group that survived the study period continued to show
    evidence of this effect during a 16 day-recovery period.  Plasma
    sodium, potassium and chloride concentrations were altered during the
    study, but did not return to normal during the recovery period. 
    Histopathological changes were found at necropsy in kidney and adrenal
    tissues from both groups.  The manufacturers reported a no-observed-
    effect level of 100 mg/kg in feed for this study.

         However, in other studies in piglets in which olaquindox was fed
    at 25, 50, 100 or 200 mg/kg diet for 6 weeks, a dose-dependent fall in
    plasma aldosterone concentration, together with hyponatraemia,

    hypochloraemia, and hyperkalaemia occurred in all groups by the end of
    the study.  Hydropic degeneration of adrenal cortex cells was
    recorded.  There were no effects on weight gain or clinical signs.

         Developmental studies in which the compound was administered
    orally were conducted in mice and rats.  In mice, fetal weights were
    reduced at 180 mg/kg b.w./day, but malformations were absent.  In
    rats, malformations occurred at 180 mg/kg b.w./day, and reductions in
    maternal weight gain, litter size, and fetal weight were also observed
    at this dose.

         A three-generation reproduction study in which olaquindox was
    administered in the diet was conducted in rats.  No malformations were
    observed, the only findings being reductions in fertility rate and
    litter size in the second and third generations at the highest dose of
    25 mg/kg b.w./day.

         The genotoxicity of olaquindox was investigated in a range of
    in vitro and in vivo studies.  Positive findings were reported in
    assays for point mutation and for DNA damage in bacteria, sister-
    chromatid exchange in Chinese hamster V-79 cells, chromosome damage in
    human lymphocytes in vitro,  mammalian bone-marrow cells in vivo,
    and in several micronucleus tests.  Two dominant lethal assays in male
    mice were negative, but a third gave a weak positive response.  Two
    dominant lethal assays in female mice gave positive results, but this
    may have been partly due to the toxicity of the drug.  A weak positive
    result was obtained in an in vivo cytogenicity assay using Chinese
    hamster spermatogonia.  Olaquindox did not bind to rat DNA in vivo.

         The Committee considered data from six long-term studies in
    rodents, but because of poor survival and deficiencies in experimental
    design and reporting, only two carcinogenicity studies, in mice and in 
    rats, were evaluated.

         In the study in mice, doses of olaquindox up to an equivalent of
    54 mg/kg b.w./day were administered in the diet for life (up to 635
    days).  An increase in the incidence of benign adrenal cortical
    adenomas and benign proliferative lesions (nodular hyperplasia and
    adenoma) in the lung was observed in male mice at the highest dose
    level.  There was no effect on the incidence of malignant tumours.

         In the rat carcinogenicity study, which included fetal exposure
    to the drug in utero, olaquindox was administered in the diet at
    levels up to an equivalent of 30 mg/kg b.w./day until 80% of the male
    and female controls had died (about three years).  Survival was
    decreased at 30 mg/kg b.w./day in both sexes, but there was no
    increase in the incidence of benign or malignant tumours.

    4.  EVALUATION

         The Committee considered olaquindox to be a genotoxic agent. 
    There was some evidence to suggest that olaquindox was a germ-line
    mutagen, but more extensive testing in appropriate mammalian studies
    will be required to resolve this issue.  In the carcinogenicity
    studies only the mouse showed an increase in the incidence of tumours,
    and these were benign.  Because of doubts over the mechanism of this
    effect and the results of the genotoxicity studies, the Committee was
    unable to establish an ADI for olaquindox.  However, the Committee
    concluded that residues resulting from the use of olaquindox in food-
    producing animals under conditions of good practice in the use of
    veterinary drugs were temporarily acceptable.

         The results of studies on the nature and availability of residues
    of olaquindox and the results of studies designed to provide an
    indication of the toxic potential of these residues are required by
    1993.

         Depending upon the results of these studies, the following
    additional information may be needed:

         a)   Data to assess the genotoxic potential of olaquindox on
              germ-line cells, which would, at a minimum, necessitate a
              repeat of the Chinese Hamster spermatogonia study.

         b)   Studies designed to assess the effects of olaquindox on
              adrenal function (including sensitive parameters such as
              plasma adrenal hormones and electrolyte levels), sperm
              morphology, and fertility in rats, so that a NOEL can be
              determined for each of these indicators.

         c)   Information on the binding of olaquindox or its metabolites
              to structural proteins such as tubulin, or to enzymes or
              proteins involved in DNA synthesis or repair.  (Such binding
              may help explain why, despite its obvious genotoxic
              potential, olaquindox does not bind to DNA and has given
              equivocal results in carcinogenicity studies).

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    See Also:
       Toxicological Abbreviations
       Olaquindox (WHO Food Additives Series 33)
       OLAQUINDOX (JECFA Evaluation)