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    ANILAZINE

    EXPLANATION

         Anilazine is a fungicide used in controlling fungus diseases
    which attack lawns and turf, cereals, coffee, and a wide variety of
    vegetables and other crops.  It is also used for the control of
    potato and tomato leafspots.  Anilazine was evaluated for the first
    time by the present meeting.

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    BIOLOGICAL DATA

    Biochemical aspects

    Absorption, Distribution and Excretion

    Rats

         Groups of male albino SD rats received single oral (0.5 or
    5 mg/kg), intravenous (0.5 mg/kg), or intraduodenal (0.5 mg/kg doses
    of [phenyl-UL-14C] anilazine (s.a. = 70.2 uCi/mg).  Female rats
    received only single oral doses (5 mg/kg bw of radioactive (RA)
    material).  Within 48 hours after oral dosing, both sexes eliminated
    100% of the administered RA dose in the feces (85%) and urine (15%). 
    Less than 1 percent of the RA material remained within the body
    after 2 days.  Most of the renal elimination occurred over the first
    6 hours of dosing whereas fecal elimination occurred mainly at the
    6-24 hour time interval.  Less than 0.001% of the administered RA
    material was in expired CO2 over 24 hours. The total RA content of
    the whole animal (including all organs and carcass, but excluding GI
    tract and contents) was 7% of the administered dose at 1 hour, 4% at
    8 hours, 0.5% after 3 days, and 0.2% after 10 days.  The highest
    concentrations of RA were found at 1 hour in blood (4.46 mcg/g),
    kidney (6mcg/g), and liver (1.5 mcg/g), at 4 hours in the GI tract
    (34.8 mcg/g) and at 8 hours in the rest of the carcass (0.28 mcg/g). 
    After 10 days, values of 0.4, 0.06, and 0.11 mcg/g were found in the
    blood, kidneys, and liver, respectively.  In all other organs and
    tissues, the concentration was 0.007 mcg/g or less.  No unusual
    tissue distribution was observed.

         Within 48 hours after i.v. injection, male rats eliminated 85%
    of the administered RA dose in the feces (approximately 33%) and
    urine (51%), while 15% remained in the body.  The finding of RA in
    the feces suggested that biliary excretion occurred.  Most of the
    renal elimination occurred over the first 6 hours, and most of the
    fecal excretion over 6 to 24 hours.

         After intraduodenal administration to male rats with cannulated
    bile ducts, about 30% of the administered RA dose was absorbed from
    the digestive tract within 8 hours, and 14% was excreted in the bile
    over 24 hours (Eubler  et al. 1979).

    FIGURE 1

    Biotransformation

         The metabolism of [phenyl-UL-14C]anilazine was characterized
    over a 24-hour period in the urine and feces of albino SD rats given
    a single oral dose of 5 mg/kg bw.  About 16 to 82% of the
    administered RA dose was excreted in urine and feces, respectively. 
    No parent compound was present in urine or feces.  The major
    identified metabolite was the hydroxylation product,
    2-(2-chloroanilino)-s-triazinodione, which comprised about 25% of
    the RA found in urine but was present only in insignificant amounts
    in feces.  Several other unknown polar metabolites, which were
    predominantly acidic in nature, were found.  In addition, a small
    portion of the RA metabolite became less polar as a result of
    treatment with diazomethane.  The majority of the metabolite could
    also be acetylated (Ecker, 1981).  The proposed metabolic pathway
    for anilazine in dairy goats is shown in Figure 1.

         Another study in male rats comparing the metabolism of
    [phenyl-UL-14C]anilazine and [triazine-UL-14C]anilazine (single
    oral dose of 5 mg/kg) indicated that the urinary and fecal metabolic
    patterns of both labels were identical, suggesting that the basic
    structure of the two rings of the molecule were maintained during
    biotransformation (Ecker, 1983).  


        TABLE 1.  RESULTS OF MUTAGENICITY ASSAYS ON ANILAZINE

                                                                                                                               
                                                     CONCENTRATION
    TEST SYSTEM               TEST OBJECT            OF ANILAZINE             PURITY         RESULTS         REFERENCE
                                                                                                                               

    Ames testa                S. typhimurium         5-12,500 ug/plate        95.9%          Negative        Herbold, 1980a
                                                     TA98, TA100,             in DMSO
                                                     TA1535, TA1537

    Ames testa                S. typhimurium         20-12,500 ug/plate       Technical      Negative        Herbold, 1987
                                                     TA98, TA100,             in DMSO        97.1%
                                                     TA1535, TA1537

    Yeast testa               S. cerevisiae          1-5000                   ?              Positive        Jagannah, 1981
    (Reverse mutation         S138, S211             ug/incubation                           only at
    assay                                            tube in DMSO                            severely
                                                                                             cytoxic
                                                                                             levels

    DNA damage in             E. coli Pol A+/        62.5-1000 ug/plate       Technical      Negative        Herbold, 1981
    E. colia                  Pol A-                 in DMSO                  93.5%

    CH-V79/HGPRT gene         Chinese hamster        0.2-9 ug/mL in           97.3%          Negative        Miltenburger, 1986
    mutation assaya           cell line V79          DMSO

    Mouse lymphoma            L5178Y mouse           2.5-9 ug/mL in           Technical      Positive        Witterland, 1984
    forward mutation          lymphoma cells         DMSO                     95.9%          (nonactivated
    assaya                    (TK+/-)                                                        only)

    Unscheduled DNA           Primary hepatocytes    0.049-9.8 ug/mL          Technical      Negative        Myhr, 1983
    synthesis                 from male F344 rats                             98.6%

    Mouse micronucleus        Bone marrow            7500 mg/kg single        97.1%          Negative        Herbold, 1988a
    test                      erythro-blasts         oral dose
                              (NMRI mice)
                                                                                                                               

    TABLE 1 (contd.)

                                                                                                                               
                                                     CONCENTRATION
    TEST SYSTEM               TEST OBJECT            OF ANILAZINE             PURITY         RESULTS         REFERENCE
                                                                                                                               

    Mouse micronucleus        Bone marrow            100-200 mg/kg            95.9%          Negative        Herbold, 1980b
    test                      erythro-blasts         bw/day for 2 days
                              (NMRI mice)

    Spot test in mice         Coat pigment cells     3-30 mg/kg bw i.p.       97.3%          Negative        Herbold, 1986a
    embryos to evaluate       of crossbred           on gestation day 10                     in F1 mice
    somatic gene changes      C57B1/6J XT mice       to pregnant females

    Dominant lethal test      Mouse (male)           5000 or 7500 mg/kg bw    97.3%          Negative        Herbold, 1988b
                                                     (single or oral dose)

    Dominant lethal test      Mouse (male)           500 mg/kg bw or          94%            Negative        Herbold, 1978
                                                     (single oral dose)

    Chromosome aberration     Chinese hamster        300-3000 mg/kg bw        Technical      Negative        Völkner, 1987
    test                      bone marrow            (single oral dose)

    Cytogenic testa           Human lymphocyte       3-30 ug/ml in DMSO       Technical      Positive        Herbold, 1986b
                              cultures (in vitro)
                                                                                                                               
    a    with and without metabolic activation.
    

    Toxicological studies

    Acute toxicity

         The acute toxicity of anilazine in several species is given in
    Table 2.  After oral administration to rats and cats, the most
    common signs of toxicity were diarrhea and vomiting, respectively. 
    After dermal administration to rabbits, mild skin irritation
    manifested as edema and erythema was observed.  Anilazine was more
    toxic by intraperitoneal injection than by other routes of
    administration, suggesting poor enteral and percutaneous absorption.


        TABLE 2.  ACUTE TOXICITY OF ANILAZINE 1,2
                                                                                            
                                           LD50             LC50
    SPECIES      SEX      ROUTE            mg/kg/bwt        (mg/l)    REFERENCE
                                                                                            

    Mouse        M        oral            approx 5000       -         Flucke & Kimmerle, 1977
                 F                                367       -

    Mouse        M        s.c.                 > 5000       -         Flucke & Kimmerle, 1977
                 F                               3512       -

    Rat          M&F      oral                 > 5000       -         Flucke & Kimmerle, 1977
                                                                      Hixson, 1979, 1980a
                                                                      Heimann, 1985

                 M        oral                   3650       -         Flucke, 1978

                 M&F      i.p.3                71-137       -         Flucke & Kimmerle, 1977
                                                                      Heimann, 1984b

                 M&F      inhal.                    -       > 0.729   Sangha, 1984

                 M&F      percutaneous         > 5000       -         Flucke & Kimmerle, 1977

    Rabbit       M&F      dermal               > 2000       -         Hixson, 1980b, 1980c

    Cat          F        oral                 >  500       -         Flucke & Kimmerle, 1977
                                                                                            
    1   All studies performed using technical grade anilazine except for one LD50 study in
        rats where 50% wettable powder was tested (Hixson, 1979), and two other LD50 where
        formulated anilazine containing 40.5% a.i. was tested (Hixson, 1980a, 1980c).
    2   The acute oral toxicity of dihydroxy-anilazine, a metabolite of anilazine was also
        tested in rats.  The LD50 value was > 5000 mg/kg (Heimann, 1984a).
    3   The acute i.p. toxicity in male rats of anilazine (LD50 = 137 mg/kg) in combination
        with triadimefon (LD50 = 317 mg/kg) was determined to be additive (LD50 of combination =
        194 mg/kg) (Heimann, 1984b)
    

    Short-term studies

    Rats

         SPF Wistar albino rats (20/sex/group) were administered
    anilazine (technical grade, 92% pure) by gavage as an aqueous
    emulsion at dose levels of 0, 30, 100, and 300 mg/kg bw/day for 28
    days.  The treatment period was followed by an additional 4-week
    observation period.  Half of the males in each group were sacrificed
    at the end of the 28-day treatment period and the other half at the
    end of the 4-week observation period.  Hematology, clinical
    chemistry, and urinalysis were performed in selected animals at each
    time period prior to sacrifice.  Animals were necropsied and
    selected organs weighed.  Histopathological examinations were
    performed on 5 rats/sex/group from the 28-day treatment phase of the
    study (16 tissues examined) and from 5 rats/sex/group from the
    post-treatment observation phase of the study (3 tissues examined,
    i.e., stomach, intestines, and esophagus).

         During the 28-day treatment period, eight animals died (2/5
    females at 100 mg/kg bw/day on day 11, and 1/5 males and 5/5 females
    at 300 mg/kg bw/day on days 11-13).  Gross pathology examination of
    these animals indicated the presence of eroded abdominal and
    thoracic viscera and lung lesions.  Other changes seen in treated
    animals were alterations in behaviour (apathy and dyspnea staring on
    days 5-7 and lasting for 3 to 4 weeks) in females given 100 mg/kg
    bw/day and in both sexes of rats at 300 mg/kg bw/day, and reductions
    in body weight in males at 300 mg/kg bw/day on weeks 1 to 4.  The
    only observed treatment-related histological effect was
    hyperkeratosis of the cutaneous mucosa of the proventricular part of
    the stomach.  The severity of the hyperkeratosis was also
    dose-related, ranging from "trace" at the low-dose level; "minimal"
    at the mid-dose level; and "moderate" at the high-dose level.  The
    finding was attributed to a local irritant effect of anilazine on
    the mucosa.  No clear treatment-related hematological, clinical
    chemistry, urinalysis, or organ weight changes were observed during
    the treatment period.  None of the above described adverse effects
    were seen in surviving rats similarly treated with anilazine but
    allowed to recover for 4 additional weeks.  The NOAEL for systemic
    toxicity for anilazine, when administered by gavage to rats, was
    30 mg/kg bw/day.  However, stomach mucosal hyperkeratosis,
    representing a local irritant effect, was seen at all of the tested
    doses of anilazine (Flucke & Kaliner, 1978).

         Groups of Sprague-Dawley CD rats (25 males and 25
    females/group) received dietary concentrations of 0, 500, 2000, or
    8000 ppm anilazine (93.5% pure) for 13 weeks.  Five rats/sex/dose
    group were sacrificed after 6 weeks.

         No treatment-related deaths occurred.  Body weight gain was
    reduced over the study in mid- and high-dose males and in high-dose
    females.  Other changes were seen only in high-dose males and
    females at the end of the study, and in many cases at the 6-week
    interim kill period as well.  These included clinical signs of
    toxicity (piloerection, red-brown staining of fur around head and
    back, rhinorrhea, and exophthalmos), and reduced food consumption,
    increased alkaline phosphatase levels, reduction in lymphocytes,
    creatinine and total protein levels, increases in the relative
    weights of several organs (heart, brain, pituitary, gonads, adrenal
    glands, thyroids, kidneys, and lungs), and various pathological
    findings (pale appearing pancreas, foamy macrophage foci in the
    lungs, testicular atrophy, lymph node hyperplasia, and enlarged
    salivary glands with heightened secretory activity).  As indicated
    by histopathological  analysis, the primary target organ of toxicity
    appeared to be the forestomach.  At the 6-week interim kill period,
    a thickened forestomach mucosa was observed that was characterized
    by dyskeratosis, acanthosis, hyperkeratosis, and edema in most of
    the male and female rats examined at the mid- and high-dose levels. 
    At the 13-week terminal sacrifice period hyperkeratosis/hyperplasia
    of the forestomach occurred at the mid- and high-dose levels at
    incidences of 3/20 and 18/20, respectively, in male rats and 9/20
    and 16/20, respectively, in females.  These changes were accompanied
    in some cases by ulcers and/or erosions of the forestomach, and were
    considered to be a local irritant effect of anilazine on the mucosa. 
    The only finding at the lowest dose level was slight acanthosis and
    hyperkeratosis of skin on the tails of female rats at the 13-week
    sacrifice period (incidence: 2/20 controls; 5/20 low dose; 8/20
    mid-dose, and 10/20 high dose).  The findings occurred in male rats
    only at the mid (1/20) and high (11/20) dose levels.  The tail
    lesions are of questionable biological significance.  The NOAEL was
    considered to be 25 mg/kg bw/day due to the observed reduction in
    body weight gain in males and the local forestomach lesions in both
    sexes (Goodyer, 1981).

    Dogs

         Purebred beagle dogs (4/sex/dose) were administered anilazine
    (92.0% pure) orally (gelatin capsules) at dose levels of 0, 50, 150,
    and 450 mg/kg bw/day for 13 weeks.

         There were no effects of treatment on mortality, or on
    patellar, flexor or extensor reflex activity or body temperature. 
    No adverse effects were observed with respect to ophthalmoscopic
    examinations, or hematological or urinalysis parameters.  Vomiting
    and diarrhea several hours after dosing were prominent signs of
    toxicity in both sexes administered all three dose levels. These
    effects were dose-related and occurred throughout the study.  Test
    compound was frequently seen in vomitus and feces.  The animals also
    were agitated and ran back and forth in their cages before
    defecation (the effect was attributed to possible intestinal spasms

    evoked by the compound).  Additional signs of toxicity were also
    observed.  Changes noted at both the 150 and 450 mg/kg bw/day levels
    included reductions in pulse rate and slight drowsiness, reduced
    serum albumin levels and increased plasma globulin levels (i.e.,
    alpha-1, alpha-2, beta, and gamma globulin levels were increased),
    and (in females), decreases in absolute and relative ovary weights. 
    Changes that occurred only at the 450 mg/kg bw/day dose level were
    dull looking ungroomed hair coats, reductions in body weight gain
    and food consumption, and reductions in total serum protein levels
    and serum calcium levels.  The absolute and relative weights of the
    thymus gland were reduced, and thymus gland atrophy was observed
    grossly, in high-dose males and females.  Upon histological
    examination, thymus gland atrophy was seen at all of the dose levels
    tested in males and the highest dose in females.  Histological
    examination of the esophagus also revealed an increase in the
    incidence of diffuse round cell infiltration of the mucosa at all
    three dose levels.  A NOAEL was not established in this study
    (Hoffmann & Gröning, 1979).

         Beagle dogs (4/sex/group) were administered anilazine
    a.i.(91.5-94.4% pure) orally (gelatin capsules) at dose levels of 0,
    10, 40, and 160 mg/kg bw/day for 18 months.

         Treatment-related toxicological effects were produced by the
    two highest dose levels of anilazine.  Adverse findings that
    occurred at 40 and 160 mg/kg bw/day in both males and females
    included vomiting of the test substance with food, increases in
    alpha-2, beta, and gamma globulins in plasma, and an increase in the
    relative weight of the adrenal glands.  Additional changes that were
    seen in dogs of both sexes only at the highest dose level (160 mg/kg
    bw/day) included salivation, loose-to-liquid feces, reduction in
    bilirubin levels, and (in females) a decrease in the absolute weight
    of the ovaries.  High-dose animals also showed a tendency for a
    decreased body weight gain which was due primarily to one male and
    one female dog, although weight gain was also somewhat low in all of
    the remaining dogs given the high dose level.  One male dog in the
    high-dose group was diagnosed with confluent bronchial pneumonia of
    the right lung and suppurative bronchitis with increased blood
    leukocytes and heightened hematopoiesis in bone marrow. 
    Esophagitis, gastritis, thymus involution, release of splenic
    corpuscles and lymphadenitis were also noted in this animal. The
    bronchial pneumonia was attributed to a bacterial or viral-induced
    infection and was not considered to be compound-induced.

         Anilazine did not produce treatment-related effects on
    survival, vital signs (i.e., reflex activity, temperature, or pulse
    rate) or food consumption, nor were adverse ophthalmological
    findings observed.  Urinalysis parameters were unchanged.  No
    treatment-related non-neoplastic or neoplastic lesions were evident. 
    The NOAEL was 10 mg/kg bw/day anilazine (Hoffmann  et al. 1983;
    Hoffmann  et al. 1987; Hoffmann  et al. 1988).

    Long-term/carcinogenicity studies

    Mice

         Male and female Charles River CD-1 mice (50/sex/dose) were fed
    diets containing 0, 50, 250, or 1250 ppm anilazine (93.5% pure) for
    104 weeks.  Sixteen additional mice/sex group served solely for the
    collection of blood samples, and were not examined
    histopathologically.  The average dietary concentrations were
    equivalent to 7.5, 37.5, and 290.0 mg/kg bw/day. Blood was collected
    at 0, 27, 53, 79, and 103 weeks).  The weights of liver, kidneys,
    gonads, heart, and brain were obtained.

         There were no effects of treatment on mortality, clinical signs
    of toxicity, body weight, food consumption, hematology, or clinical
    chemistry.  Plasma glucose levels were elevated in occasional male
    and female mice in the 250 and 1250 ppm groups.  There were no
    treatment-related effects on organ weights and no remarkable
    findings at gross necropsy or after histopathological examination. 
    The incidences of Harderian gland adenomas were elevated in males
    and females in the high-dose group.  These tumour incidences,
    however, were neither statistically significant nor above historical
    control levels.  On the basis of these findings the NOAEL in this
    study exceeded 1250 ppm in the diet, equal to approximately
    190.0 mg/kg bw/day in males and females (Goodyer, 1984a).

    Rats

         Male and female Sprague-Dawley CD rats (50/sex/group) were fed
    anilazine (93.5% pure) for 104 weeks at levels of 0, 50, 330, or
    2000 ppm.  These concentrations were equivalent to approximately
    2.5, 16.5 and 100 mg/kg bw/day.  Ten additional rats/sex/dose were
    used solely for the collection of blood samples; these animals were
    not examined histopathologically.  Blood was collected at 0, 26/27,
    53, 79 and 103 weeks.  Urinalysis studies, however, were not
    performed.  The weights of liver, kidney gonads, heart, brain,
    adrenals, spleen, lungs, and thyroid glands were obtained.

         There were no clinical signs of toxicity and no effect of
    treatment on mortality.  There were no treatment-related effects on
    body weight or food consumption throughout the study with the
    exception of transient, parallel decreases in these parameters
    during study week number 1 in males and females given 2000 ppm. 
    Hematology and clinical chemistry findings were generally
    unremarkable.  There was no treatment-related effect on organ
    weights and no remarkable findings at gross necropsy or after
    histopathological examination. There were no increases in tumours
    that were treatment-related.  The NOAEL exceeded 2000 ppm in the
    diet, equivalent to more than 100 mg/kg bw/day in males and females
    (Goodyer, 1984b).

         Special carcinogenicity bioassays of anilazine in rodents (rats
    and mice) were also performed by the National Cancer Institute but
    they were not considered for toxicological evaluation (NCI, 1978). 
    However, since these studies were performed by Gulf South Research
    Corporation and were not validated.

    Reproduction study

    Rats

         Groups of male (15/sex/dose) and female (30/sex/dose) Sprague-
    Dawley rats in each of three generations (F0, F1, and F2) were
    fed diets containing 0, 50, 330, or 2000 ppm anilazine (93.5% pure). 
    The dietary concentrations were equivalent to 2.5, 16.5, and 100
    mg/kg bw/day.  After maturation periods of 15 weeks in the F0
    generation and 18 weeks in the F1 and F2 generations, the parental
    animals were allowed to rear their offspring to weaning (F0-F1a;
    F1-F2a; F2-F3a).  The animals for the F1 and F2 generations
    were randomly selected from the F1a and F2a litters, avoiding
    sibling matings.

         No effects of treatment on mortality, clinical signs, food
    consumption, or body weight of parental rats were noted.  Although
    slight inhibition of body weight gain was noted in F0 parental
    males from week 5 until termination, the response was neither
    statistically significant nor dose-related.  The highest dietary
    level of anilazine (2000 ppm) had an apparent adverse effect on the
    libido of F0 males and the fertility (and possible fecundity) of
    F0 females.  This was indicated, respectively, by reductions in the
    number of females that mated and the number that became pregnant in
    comparison to controls.  Although the respective differences were
    not remarkable nor evident in subsequent generations (F1 and F2),
    they were verified by a crossover mating experiment involving rats
    from the control and high-dose groups.  No adverse effects on the
    reproductive parameters were evident at anilazine dietary levels of
    330 and 50 ppm.  No adverse changes occurred with respect to litter
    size, viability, mean pup and litter weights, or physical (i.e.,
    auditory response, pupillary reflex, visual placing response, grip
    strength) and functional (i.e., onset and duration of pinna
    unfolding, onset of hair growth, incisor eruption, and eye opening)
    development of pups.  No unusual pre- or post-weaning clinical
    observations occurred. No treatment-related changes in organ weight
    or pathological (gross and microscopic) findings were noted in
    either parenteral rats or pups at necropsy (Irvine, 1984).  The
    NOAEL is 330 ppm, equivalent to 16.5 mg/kg bw/day.

    Special studies on teratogenicity

    Rats

         Groups of female Long Evans FB-30 strain rats (21-22/dose) were
    administered doses of 0, 30, 100, or 300 mg/kg bw/day anilazine, by
    gavage, from days 6 to 25 of gestation (Day 0 = day of
    insemination).  Doses were based on a preliminary range-finding
    study which demonstrated that a dose of 300 mg/kg bw/day for 10 days
    had no apparent adverse activity.  On gestation day 20, rats were
    sacrificed and fetuses delivered by C-section.  Approximately 30% of
    the fetuses in each group were dissected and examined for visceral
    anomalies and approximately 70% of the fetuses in each group were
    cleared and stained for skeletal observations.

         The 300 mg/kg bw/day dose of anilazine produced a significant
    reduction in body weight gain in the dams during the treatment
    period as well as throughout the entire gestation period.  Body
    weight gain, however, was not influenced by 30 or 100 mg/kg bw/day
    anilazine. None of the doses produced changes in mortality, physical
    appearance, or behaviour, or the fertilization ratio or pregnancy
    ratio in the dams.  Similarly, none of the doses appeared to affect
    embryonic or fetal development and no teratogenic effects were
    observed.  Anilazine was neither fetotoxic, embryotoxic, nor
    teratogenic in rats at oral doses up to 300 mg/kg/day (highest dose
    tested).  Because of maternal toxicity (weight gain  reduction) at
    300 mg/kg bw/day, the NOAEL is 100 mg/kg bw/day by gavage (Machemer,
    1978).

         Groups of female Charles River Crl:CD-BR rats (28/dose) were
    administered oral (gastric intubation) doses of 0, 150, 500, or
    1500 mg/kg bw/day anilazine from days 6 to 15 of gestation.  On
    gestation day 20, rats were sacrificed and fetuses delivered by
    C-section.  Approximately one-half of the fetuses in each group were
    dissected and examined for visceral anomalies, and the other half of
    the fetuses in each group were cleared and stained for skeletal
    observations.

         Anilazine was maternally toxic at doses of 500 and 1500 mg/kg
    bw/day.  The adverse findings were: 1) an increased incidence of
    loose and/or soft stools that often contained a creamy white
    substance (seen in 28.6 and 100% of dams at 500 and 1500 mg/kg
    bw/day, respectively);  2) significant reductions in body weight and
    body weight gain in dams given 1500 mg/kg bw/day on gestation days 6
    to 25 and 0 to 20;  3) a significant reduction in food consumption
    in dams given 500 and 1500 mg/kg bw/day on gestation day 7 only; and
    4) an increased incidence of thickened and coarse mucosa of the
    nonglandular portion of the stomach (seen in 18 and 15% of dams
    given 500 and 1500 mg/kg bw/day, respectively).  This change was
    characterized microscopically as epithelial hyperplasia (acanthosis)
    and hyperkeratosis of the mucus membrane.  In contrast to these

    results, none of the doses of anilazine induced effects on embryo or
    fetal development, and no teratogenic effects were observed.  The
    NOAEL was 150 mg/kg bw/day by intubation (Kowalski  et al. 1988).

    Rabbits

         Groups of female Himalayan rabbits (13/dose) were administered
    0, 5, 15 and 45 mg/kg bw/day anilazine (92.4% pure) from days 6 to
    18 of gestation (Day 0 = day of insemination).  The animals were
    sacrificed on gestation day 29 and fetuses delivered by C-section. 
    All fetuses were necropsied and abdominal and thoracic organs as
    well as the brain examined for visceral malformations.  All fetuses
    were examined for skeletal defects.

         No treatment-related changes in mortality, appearance,
    behaviour or weight gain were observed in the does.  The
    fertilization and pregnancy ratios were not adversely affected. 
    There were no statistically significant dose-related effects on the
    number of implantations or corpora lutea, sex ratio, mean fetal
    weights, mean placental weights, fetuses showing visceral
    malformations, or numbers of fetuses with stunted growth.  No
    treatment-related skeletal abnormalities or malformations were
    observed.  The highest dose tested, 45 mg/kg bw/day by gavage, is a
    NOAEL for maternal, embryo-, and fetotoxicity, and for
    teratogenicity in rabbits (Roetz, 1981).

    Special studies on mutagenicity

         Anilazine was mutagenic in a cytogenetic study in human
    lymphocyte cultures  in vitro (both with and without metabolic
    activation), in a mouse lymphoma forward mutation assay (without
    metabolic activation only), and at severely cytotoxic levels in a
    reverse mutation assay in yeast (both with and without metabolic
    activation).  In contrast, the chemical was negative in a variety of
    the other  in vitro and  in vivo systems.  The weight-of-the-
    evidence indicates that anilazine is not genotoxic.

    Special studies on eye and skin irritation

         Instillation of 100 mg grade anilazine (40.5% a.i. formulation)
    in the rabbit eye for 45 seconds produced reversible iritis, slight
    erythema, chemosis, and discharge which lasted for up to 13 days
    (Hixson, 1980b).

         Anilazine (90 mg of a technical grade formulation) produced a
    strong irritant effect (redness, swelling, and lacrimation) on the
    rabbit eye when placed in the conjunctival sac for 24 hours.  The
    reactions were reversible within 14 days.  No corrosive effect
    occurred (Pauluhn, 1983).

         Slight primary skin irritation (erythema and edema) was
    observed when anilazine (500 mg technical) was applied to shaved
    rabbit skin for 4 hours.  The reactions were reversible within 7
    days.  No corrosive effect occurred (Pauluhn, 1983).

         In a 3-week dermal toxicity study in rabbits, 0%, 1% (10 mg/ml)
    and 4% (40 mg/mL) of anilazine was applied to shaved skin at a
    volume of 0.5 ml/kg for 6 hours/day, 5X/week, for 3 weeks.  In this
    study, a formulation containing 497 grams a.i.(litre (= 38.4% w/w)
    was used.  Both concentrations of the compound produced dermal
    changes consisting of slight erythema and cellular infiltration in
    the subepidermal region;  in addition, slight thickening of the
    epidermis was noted 40 mg/ml.  However, no systemic changes were
    observed as noted from hematological, clinical chemistry and
    urinalysis examinations, by measurements of body weight gain, and by
    histopathological evaluation of selected organs and tissues.  The
    systemic NOAEL was calculated to be approximately 7.7 mg a.i./kg
    bw/day.  (Heimann & Schilde, 1985).

    Special studies on skin sensitizing effect

         Anilazine was shown to be a dermal sensitizer (i.e., to induce
    redness and swelling) when tested in guinea pigs by the Buehler
    Patch test (Heimann, 1988a; Heimann, 1988b), the Magnusson and
    Kligman test (Heimann, 1982a), and in Klecak's open epicutaneous
    test (Heimann, 1982b).  The positive findings were obtained using
    either pure and/or technical anilazine in induction concentrations
    ranging from 0.03 to 5%, and challenge doses ranging from 0.03 to
    1%.  Negative results for sensitizing potential were obtained in one
    study (Klecak's test) with pure anilazine at induction
    concentrations of 0.01 to 0.1% and challenge doses of 0.01 to 0.3%
    (Heimann, 1983).

    Observations in humans

         A group of farm workers employed on a single farm in the United
    States arrived at a hospital emergency room with severe dermatitis
    in July 1980.  All were exposed to three pesticides in a tomato
    field (anilazine, benomyl, and endosulfan).  Seven of the workers
    were available for patch testing to the three pesticides.  Delayed
    hypersensitivity (48-hour eczematous reaction) to 0.1% Dyrene
    (anilazine) was demonstrated in 6 of the 7 affected workers.  Nine
    non-exposed adult volunteers who were used as control subjects had
    negative patch tests to 0.1% Dyrene.  The conclusion was reached
    that anilazine caused allergic contact dermatitis (Schuman  et al.
    1980).

    COMMENTS

         Anilazine administered orally to rats was excreted primarily in
    the feces via biliary excretion, and also in urine.  Anilazine was
    biotransformed in rats primarily to the hydroxylation product,
    2-2-chloroanilino)-s-triazinedione, which was present only in
    urine.  A second biotransformation pathway involving conjugate
    formation via the glutathione - mercapturic acid pathway  was also
    observed in goats and chickens.  No unusual organ or tissue
    localization of (14C)-labelled anilazine was observed.

         Anilazine has a low acute toxicity in the species examined.

         Subchronic studies in rats indicated that the stomach was the
    primary target organ of toxicity;  oral gavage doses of aqueous
    anilazine emulsion of 30 mg/kg bw/day and a dietary level of
    2000 ppm (equivalent to 100 mg/kg bw/day) or greater produced
    forestomach changes characterized by acanthosis, hyperkeratosis, and
    areas of ulceration and erosion.  The NOAEL was 500 ppm (equivalent
    to 25 mg/kg bw/day).

         In an 18-month oral (capsule) toxicity study in dogs, anilazine
    at doses of 40 and 160 mg/kg bw/day produced increases in alpha-1,
    beta-2, and gamma-globulins in plasma, vomiting, and diarrhea in
    males and females.  These findings were also observed with oral
    doses (capsules) of 50 to 450 mg/kg bw/day for 13 weeks in dogs. 
    The NOAEL in the 18-month study was 10 mg/kg bw/day.

         In long-term studies in rats and mice, no treatment-related
    increases in neoplasms were observed.  The NOAELs were 1250 ppm
    (equal to 188 mg/kg bw/day) in Cr CD-1 mice and 2000 ppm (equivalent
    to 100 mg/kg bw/day) in SD-CD rats.

         In a 3-generation, 2 litters per generation, reproduction study
    in rats, reductions in male libido and female fertility occurred in
    F0 generation animals at 2000 ppm in the diet (equivalent to
    100 mg/kg bw/day). Although these effects were confirmed in
    cross-mating experiments, they were not observed in subsequent
    generations.  The NOAEL was 330 ppm (equivalent to 16.5 mg/kg
    bw/day).

         In teratology studies in rats, the NOAEL was 150 mg/kg bw/day
    based on maternal toxicity.  Oral doses of 300 to 1500 mg/kg bw/day
    produced soft/loose stools, weight loss, reduced food consumption
    and acanthosis and hyperkeratosis of the stomach of dams, but had no
    fetotoxic or teratogenic activity.  The NOAEL was 45 mg/kg bw/day
    (highest dose tested) in a rabbit teratology study.

         After reviewing all available  in vitro and  in vivo
    short-term tests, the Meeting concluded that there was no evidence
    of genotoxicity.

    TOXICOLOGICAL EVALUATION

    Level causing no toxicological effect

         Mouse:    1250 ppm in the diet, equal to 188 mg/kg bw/day
         Rat:       330 ppm in the diet, equivalent to 16.5 mg/kg bw/day
         Dog:        10 mg/kg bw/day orally.

    Estimate of acceptable daily intake for humans

         0-0.1 mg/kg bw.

    Studies which will provide information valuable in the continued
    evaluation of the compound

         Further observations in humans.

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    See Also:
       Toxicological Abbreviations