IPCS INCHEM Home

    KRESOXIM-METHYL       JMPR 1998

    First draft prepared by 
    K. Fujimori 
    National Institute of Health Sciences, Tokyo, Japan 


         Explanation
         Evaluation for acceptable daily intake
              Biochemical aspects
                   Absorption, distribution, and excretion
                   Biotransformation
              Toxicological studies
                   Acute toxicity
                   Short-term studies of toxicity
                   Long-term studies of toxicity and carcinogenicity 
                   Genotoxicity 
                   Reproductive toxicity 
                        Multigeneration reproductive toxicity
                        Developmental toxicity 
                   Special studies 
                        Tumour initiating potential
                        Tumour promoting potential
                        Hepatic-cell proliferation
                        Morphology of hepatic proliferation
                        Induction of hepatic metabolic enzyme activities 
                        Mechanism of decreased serum enzyme activities 
                   Studies on metabolites 
                        Acute toxicity 
                        Genotoxicity 
         Comments 
         Toxicological evaluation 
         References 

    Explanation 

         Kresoxim-methyl, methyl-(E)-2-methoxyimino-2-[2-(2-
    methylphenoxymethyl)phenyl] acetate, is a broad-spectrum fungicide and
    a member of the strobilurin family, a new class of biologically active
    compounds structurally related to strobilurin A, a natural product of
    the wood-decaying fungus  Strobilurus tenacellus. It is intended for
    use as an agricultural spray in the control and treatment of fungal
    infections on crops and fruits. Strobilurins are known to bind to the
    bcl complex (complex III), one of the oxide reductase proteins of the
    electron transport chain in mitochondria. The ester linkage in
    kresoxim-methyl is essential for its activity. Kresoxim-methyl was
    evaluated for the first time by the present Meeting. 

    Evaluation for Acceptable Daily Intake 

    1.  Biochemical aspects 

     (a)  Absorption, distribution, and excretion 

         Kresoxim-methyl labelled with 14C on the phenyl A ring (phenoxy;
    radiochemical purity, > 98%) or B ring (phenyl; radiochemical purity,
    > 98%) or with 13C on the carbon side-chain was administered to rats
    by gavage as a suspension in 0.5% carboxymethyl cellulose (CMC) or
    intravenously as a 0.9% saline solution. The design of the study
    conformed to good laboratory practice. In groups of five male and five
    female rats given [14C-B ring]kresoxim-methyl by gavage at 50 or 500
    mg/kg bw, with or without pretreatment with unlabelled
    kresoxim-methyl, or [14C-A ring]kresoxim-methyl at a dose of 500
    mg/kg bw, the compound was excreted predominantly in faeces. At the
    low dose of [14C-B ring]-labelled compound, faecal excretion
    represented 65-67% of the administered dose and urinary excretion,
    20-28% of the dose within 48 h; less than 1% of the radiolabel was
    recovered in urine and faeces at this time. Pretreatment with
    unlabelled kresoxim-methyl at the low dose for 14 days did not change
    the excretion pattern. At the high dose, faecal excretion represented
    80-81% of the dose and urinary excretion, 8-13% within 48 h. The total
    radiolabel recovered within 120 h was 97% of the [14C-A ring] and
    90-96% of the [14C-B ring], with 62-78% of the A ring and 81% of the
    B ring excreted in faeces and 17-33% of the A ring and 9-13% of the B
    ring in urine. No radiolabel was detected in exhaled air. 

         In the groups given the [14C-B ring]-labelled material, peak
    concentrations of radiolabel in plasma were reached 0.5-1 h after
    dosing at the low dose and 8 h after dosing at the high dose. The
    plasma level then declined, with a terminal half-life of 17-19 h at
    the low dose and 22-30 h at the high dose. The ratios of the area
    under the curve for the high:low dose (10:1) were 2.3 for males and
    2.1 for females. Radiolabel concentrations were determined in tissues
    0.5, 8, 24, 96, and 120 h after dosing. Except for the
    gastrointestinal tract, the highest residual concentration was found
    in the liver (0.1 g/g at 120 h and 0.3-1.4 g/g at 24 h after dosing at
    50 mg/kg bw). The residual concentrations in other tissues were less
    than 0.1 g/g tissue at 120 h after dosing at 50 mg/kg bw. The
    concentrations of radiolabel in the tissues were comparable in males
    and females, indicating a similar pattern of wide distribution and
    elimination.

         Groups of five male and five female rats given [14C-B
    ring]kresoxim-methyl intravenously as a single dose of 5 mg/kg bw
    excreted 49-66% of the radiolabel in urine and 23-48% in faeces within
    120 h.

         Groups of four male and four female rats with canulated bile
    ducts were given the [14C-B ring]-labelled material as a single oral
    dose of 50 or 500 mg/kg bw. Biliary excretion accounted for 35-43% of
    the radiolabel at the low dose and 14-15% at the high dose within 48

    h. Urinary excretion represented 20-28% at the low dose and 8-13% at
    the high dose, and faecal excretion represented 65-67% at the low dose
    and 80-81% at the high dose within 48 h. Excretion of the [14C-A
    ring]-labelled material in bile was not examined (Gans, 1994).

     (b)  Biotransformation

         The samples collected in the experiments described above (Gans,
    1994) were analysed for metabolites of kresoxim-methyl, in a study
    that conformed to good laboratory practice. After oral administration,
    high proportions of parent compound were found in the faeces (Table
    1), but none was detected in the bile or in tissues (plasma, liver,
    and kidney) sampled about 4 h after administration of the low or high
    dose (Table 2). A total of 34 metabolites, including conjugates, was
    identified by nuclear magnetic resonance spectroscopy and mass
    spectrometry in rat excreta, with 20 in urine, eight in faeces, and 17
    in bile. The major metabolites identified in urine and faeces were M1,
    a hydrolytic product of the acetyl ester; M2, an oxidative metabolite
    of the aryl-methyl moiety of M1; and M9, a hydroxylated metabolite of
    the phenoxy ring of M1. M1 and M9 were the major metabolites
    identified in tissues. Glucuronated conjugates were detected in
    notable quantities in the bile. There was no evidence that the
    metabolic pathways were induced by pretreatment with kresoxim-methyl.
    A small difference in the metabolite pattern in urine and bile was
    observed between males and females, the percentages of M1 and M9 in
    urine from females being greater than in urine from males. In summary,
    the metabolic pathways of kresoxim-methyl consisted of hydrolytic
    cleavages of the ester, the oxime ether, and the benzyl ether bonds;
    hydroxylation at the  para position of the phenoxy ring; oxidation of
    the aryl-methyl group to benzyl alcohol and its subsequent oxidation
    to the corresponding carboxylic acid; and conjugation of the resulting
    hydroxy groups with glucuronate and sulfate (Kohl, 1994). The proposed
    metabolic pathway for kresoxim-methyl in rats is shown in Figure 1. 

         The major metabolites identified in plants were a hydrolytic
    product of the acetyl ester (M1), an oxidative metabolite of the
    aryl-methyl moiety (M2), a hydroxylated metabolite of  para- or
     meta-hydroxylated metabolites of the phenoxy ring of the first
    metabolite (M9 or M54), and their conjugates (Grosshans, 1994a,b;
    Nelsen et al., 1995).

    2.  Toxicological studies

     (a)  Acute toxicity

         Studies of the acute toxicity of kresoxim-methyl are summarized
    in Table 3. Oral administration of 5000 mg/kg bw kresoxim-methyl as a
    suspension of 0.5% CMC produced no deaths or abnormal clinical signs
    in mice or rats, and no abnormal changes in organs were seen at
    necropsy. Dermal application of 2000 mg/kg bw in a suspension of 0.5%
    CMC caused no deaths or signs of clinical toxicity, except for a
    slight but definite erythma at the site of application in some
    animals. Groups of five male and five female Wistar rats were exposed

    FIGURE 1


        Table 1. Percents of a single oral dose of kresoxim-methyl found as parent compound and 
    metabolites in rat excreta and tissues
                                                                                                      

    Substance    Faeces                                      Urine
                                                                                                     
                 50 mg/kg bw           500 mg/kg bw          50 mg/kg bw           500 mg/kg bw
                                                                                                      
                 Male       Female     Male       Female     Male       Female     Male       Female
                                                                                                      

    Parent       49.5       47.1       74.9       39.5                                         
    M1           2.1                   0.1        7.1        0.4        2.7        2.8        2.2
    M2           2.7        0.5        0.5        5.8        2.0        3.4        1.5        2.0
    M4           1.1        0.5        0.3        2.5        mix1       mix1       mix1       mix1
    M6                                                       2.8        1.1        1.9        0.5
    M8                                                       0.1        0.4                   mix3
    M9           5.2        6.0        0.9        13.3       5.5        11.0       2.7        4.9
    M11                                                      mix2                  mix2       mix3
    M12                                                      mix2                  mix2       mix3
    M14                                                      mix1       mix1       mix1       mix1
    M15          1.3        2.7        0.1        3.4                                          
    M16                                                                            0.3        mix3
    M20                                                      mix1       mix1       mix1       mix1
    M24                                0.1                                         0.1        0.4
    M26                                                      mix2                  mix2       mix3
    mix1                                                     1.4        1.6        0.9        1.1
    mix2                                                     0.9                   0.8         
    mix3                                                                                      1.4
    UK1          1.3        0.6        0.4        0.1                   0.1                   0.2
    UK2                     0.2        0.1        1.4                                          
    UK3                                0.1        1.8                                          
    UK4                                0.3                                                     
    UK5                                0.2                                                     
    UK5                                           0.1                                          
    Recovery     83.3       86.7       84.1       86.1       99.2       97.4       100.1      99.4
                                                                                                      

    mix1, mixture of M4 + M14 + M20; mix2, M11 + M12 + M26; mix3, M8 + M11 + M12 + M16 + M26; UK, unknown 
    compound 

    Table 2. Percents of a single oral dose of kresoxim-methyl found as parent compound and 
    metabolites in rat bile and tissues

                                                                                                     

    Substance    Bile                  Plasma                Liver
                                                                                                      
                 50 mg/kg bw           50 mg/kg bw           50 mg/kg bw           500 mg/kg bw
                                                                                                     
                 Male       Female     Male       Female     Male       Female     Male       Female
                                                                                                     

    Parent       0          0          0          0          0          0          0          0
    M1           1.7        1.9        0.386      0.304      0.13       0.07       0.07       0.12
    M2           mix5       mix5       0.095      mix8       0.08       0.04       0.04       0.04
    M4                                 0.041      mix8       0.03       0.02       0.04       0.02
    M6                                 0.027      0.006                                        
    M9           1.1        1.3        0.173      0.164      0.17       0.07       0.06       0.09
    M11                                0.002                 mix4       mix4       mix4       mix4
    mix4                               mix4                  mix4       mix4       mix4       mix4
    M28          0.7        2.9                                                                
    M31          0.5        1.1                                                                
    M35          1.7        0.7                                                                
    M44          0.4        0.3                                                                
    M45          mix5       mix5                                                               
    mix4                               0.115                 0.08       0.02       0.02       0.01
    mix5         1.1        1.2                                                                
    mix6         6.3        3.6                                                                
    mix7         0.4        0.2                                                                
    mix8                                          0.169                                        
    UK1                                                      0.02                              
    UK2                                           0.024      0.02       0.01                   
    Recovery     100.0      100.1                            84.8       82.5       87.0       96.8
                                                                                                     

    mix4, M11 + M12 + M16 + M26; mix5, mixture of M2 + M45; mix6, M25+M26+M29+M33+M39; mix7, 
    M34+M36+M37; mix8, M2 + M4 
    The tissue samples were collected 3.5-4 h after a single oral adminstration. The values in 
    plasma are expressed as microgram equivalent per ml.
    

    to a dust aerosol of kresoxim-methyl at concentrations of 2 and 5.6
    mg/L through a head-nose inhalation system. The mass median
    aerodynamic diameter of the dust aerosol particles was 1.8-2.4 m. No
    deaths occurred; during exposure to either concentration, nonspecific
    clinical signs such as accelerated and intermittent respiration,
    urine-smeared fur, reddish nose, eye discharge, and reddish eyelid
    crust, were observed. These signs disappeared one day after exposure.

         In a study conducted according to good laboratory practice, white
    Vienna rabbits of each sex received 4-h dermal applications of a
    single dose of 0.5 g kresoxim-methyl (purity, 93.7%) as a fine powder
    moistened with distilled water. The skin was examined 1, 24, 48, and
    72 h after removal of the compound: little or no erythema was observed
    (Rossbacher & Kirsch, 1992a).

         In another study conducted according to good laboratory practice,
    a single dose of 39 mg kresoxim-methyl (purity, 93.7%) in a volume of
    0.1 ml was administered to the right eye of white Vienna rabbits of
    each sex. The eyes were examined 1, 24, 48, and 72 h after
    application, without washing. Some conjunctival redness (score, 0.1-4)
    was observed at 1, 24, and 48 h but had disappeared by 72 h after
    application (Rossbacher & Kirsch, 1992b).

         In a further study conducted according to good laboratory
    practice, the skin sensitizing potential of kresoxim-methyl (purity,
    93.7%) was tested in female Dunkin Hartley guinea-pigs by the
    maximization method. For induction, a 5% suspension of kresoxim-methyl
    in 0.5% CMC was applied intradermally, followed by topical application
    of a 50% suspension. At challenge, 50% kresoxim-methyl (20 rabbits) or
    the vehicle (10 rabbits) was applied dermally. No dermal reaction was
    observed in the rabbits challenged with kresoxim-methyl (Rossbacher &
    Kirsch, 1993).

     (b)  Short-term toxicity

     Mice

         In a range-finding study conducted according to good laboratory
    practice, groups of five male and five female B6C3F1(Cr) mice were
    given diets containing kresoxim-methyl (purity, 96.6%) at
    concentrations of 0, 500, 2000, or 8000 ppm for 28 days, equal to 0,
    110, 480, and 2100 mg/kg bw per day for males and 0, 180, 800, and
    3800 mg/kg bw per day for females. The animals were observed for
    clinical signs, deaths, food consumption, body weight, and clinical
    chemical, haematological, and pathological end-points. There were no
    deaths or signs of clinical toxicity. At the highest dose,
    significantly reduced serum concentrations of triglyceride and
    cholesterol were observed in males, and significantly increased
    relative liver weights  (p < 0.05) were observed in animals of each
    sex. No compound-related lesions were observed on histopathological
    examination. The NOAEL was 8000 ppm, equal to 2100 mg/kg bw per day,
    as the increased relative liver weights were not accompanied by
    histopathological changes (Schilling & Hildebrand, 1992b).


        Table 3. Acute toxicity of kresoxim-methyl 

                                                                                                              

    Species     Strain         Sex      Route          LD50 or LC50   Purity     Reference
                                                       (mg/kg bw or   (%)
                                                       mg/L air) 
                                                                                                              

    Mouse       ICR            M/F      Oral           > 5000         94.3       Yamamoto (1994)

    Rat         Chbb Wistar    M/F      Oral           > 5000         93.7       Kirsch & Hildebrand (1993a)

    Rat         Chbb Wistar    M/F      Dermal         > 2000         93.7       Kirsch & Hildebrand (1993b)

    Rat         Chbb Wistar    M/F      Inhalation     > 5.6          96.6       Gamer & Kirsch (1992)
                                                                                                              

    These studies were conducted in accordance with good laboratory practice.
    

         Groups of 10 male and 10 female C57Bl/6N(Cr) mice were given
    diets containing kresoxim-methyl (purity, 98.7%) at concentrations of
    0, 250, 1000, 4000, or 8000 ppm, equal to 0, 57, 230, 910, and 1900
    mg/kg bw per day for males and 0, 80, 330, 1300, and 2600 mg/kg bw per
    day for females, for three months. The study was carried out according
    to good laboratory practice. The animals were observed for clinical
    signs, deaths, food consumption, body weight, clinical chemical
    parameters including the activities of serum alanine (ALAT) and
    aspartate aminotransferases (ASAT), alkaline phosphatase (AP), and
    gamma-glutamyl transferase (GGT), and haematological and pathological
    end-points. There were no deaths, signs of clinical toxicity, or
    changes in haematological or clinical chemical parameters.
    Dose-dependent reductions in terminal body weight, by 4% at 4000 and
    7% at 8000 ppm, and body-weight gain, by 11% at 4000 and 24% at 8000
    ppm, were seen in males; however, these reductions were not
    significant. Significant increases in relative liver weight were
    observed in males at 4000 and 8000 ppm, but no compound-related
    lesions were observed on histopathological examination. The NOAEL was
    8000 ppm, equal to 1900 mg/kg bw per day, on the basis of the absence
    of toxicologically significant changes (Mellert & Hildebrand, 1994b)

     Rats

         In a range-finding study that conformed to good laboratory
    practice, groups of five male and five female Wistar (Cr) rats were
    given diets containing kresoxim-methyl (purity, 96.55%) at a
    concentration of 0, 1000, 4000, or 16 000 ppm for 28 days, equal to 0,
    91, 360, and 1400 mg/kg bw per day for males and 0, 95, 380, and 1500
    mg/kg bw per day for females. The rats were observed for clinical
    signs, deaths, food consumption, body weight, clinical chemical
    parameters including the activities of serum ALAT, ASAT, AP, and GGT,
    and haematological and pathological end-points. There were no deaths,
    signs of clinical toxicity, or changes in haematological parameters.
    The terminal body weights were slightly reduced in animals of each sex
    at 4000 ppm (by 4% in males and 10% in females) and at 16 000 ppm (by
    7% in males and 6% in females), and the absolute liver weights were
    slightly increased in males (by 8%) and females (9%) at 16 000 ppm;
    however, these changes were not statistically significant. Significant
    increases in relative liver weights were observed in females at 16 000
    ppm, and significantly increased serum GGT activity and albumin
    concentration were observed in males at this dose. No compound-related
    lesions were observed on histopathological examination. The NOAEL was
    4000 ppm, equal to 360 mg/kg bw per day, on the basis of increased
    serum enzyme activity in males and increased relative liver weight in
    females (Schilling & Hildebrand, 1992a).

         Groups of 10 male and 10 female Wistar (Chbb) rats were given
    diets containing kresoxim-methyl (purity, 98.7%) at concentrations of
    0, 500, 2000, 8000, or 16 000 ppm, equal to 0, 36, 150, 580, and 1200
    mg/kg bw per day in males and 0, 43, 170, 670, and 1400 mg/kg bw per
    day in females, for 90 days. The rats were observed for clinical
    signs, deaths, food consumption, body weight, clinical chemical
    parameters including the activities of serum ALAT, ASAT, AP, and GGT,

    and haematological and pathological end-points. Food consumption and
    body weights were determined once a week, and enzyme activities were
    determined after six weeks and at the end of the study.

         There were no deaths, signs of clinical toxicity, changes in food
    consumption, or compound-related changes in haematological parameters.
    Slight but significant decreases in terminal body weight (7-8% at 8000
    and 11-13% at 16 000 ppm) and body-weight gain (7-10% at 8000 and
    13-15% at 16 000 ppm) were observed in males. Significant increases in
    relative liver weight were observed in males at 16 000 ppm (10%) and
    in females at 2000 ppm and higher (10% at 2000, 7% at 8000, and 12% at
    16 000 ppm). Significant increases in relative kidney weight were also
    observed in males, but the absolute weights were not increased. No
    compound-related histopathological lesions were observed in these or
    other organs in treated groups. Dose-dependent, statistically
    significantly increased activities of GGT were observed in males at
    8000 ppm and higher, and significantly decreased activities of AP and
    ALAT were observed in males at all doses and in females at 2000 ppm
    and higher. These reductions in enzyme activity were considered to be
    related to the slight decrease in food consumption on the basis of
    mechanistic studies on percent reductions in intestinal and hepatic
    isozymes per total serum AP activity (Moss, 1994; Mellert et al.,
    1997a). The NOAEL was 2000 ppm, equal to 150 mg/kg bw per day, on the
    basis of decreased body weight and body-weight gain and increased GGT
    activity in males at higher doses (Mellert & Hildebrand, 1994a).

         Groups of five male and five female Wistar (Chbb) rats received
    dermal applications of kresoxim-methyl (purity, 94.3%) suspended in
    0.5% CMC at a dose of 0 or 1000 mg/kg bw per day under a
    semi-occlusive dressing (four layers of absorbent gauze and an elastic
    dressing) for 6 h/day for 21 days. The study design corresponded to
    good laboratory practice. The rats were observed for clinical signs,
    deaths, food consumption, body weight, clinical chemical parameters
    including the activities of serum ALAT, ASAT, AP, and GGT,
    haematological parameters including clotting times, and pathological
    end-points. Blood samples for haematological and clotting analysis and
    for clinical chemistry were collected at termination. There were no
    compound-related effects on mortality rates, clinical signs,
    haematological parameters, clotting times, or clinical chemical
    parameters, including serum enzyme activities. There were no
    significant changes in body-weight gain or food consumption in the
    treated group, and no signs of irritation were observed on treated
    skin of test or control animals. No effect on organ weights was
    observed, and histopathological examination revealed no
    treatment-related alterations in the liver or in any other tissue
    examined. The NOAEL was 1000 mg/kg bw per day, the highest dose tested
    (Kirsch & Hildebrand, 1994c).

     Dogs

         Groups of six male and six female beagles, six to nine months
    old, were given diets containing kresoxim-methyl (purity, 94-95.9%) at
    concentrations of 0, 1000, 5000, or 25 000 ppm, equal to 0, 28, 140,

    and 740 mg/kg bw per day for males and 0, 32, 160, and 800 mg/kg bw
    per day for females, for three months. The study was conducted
    according to the principles of good laboratory practice. The animals
    were observed for clinical signs, deaths, food consumption, body
    weight, clinical chemical parameters including the activities of serum
    ALAT, ASAT, AP, and GGT, and haematological and pathological
    end-points. Blood samples for haematological and clinical chemical
    analysis were collected during weeks 4 and 13 of treatment.

         No deaths or ophthalmological abnormalities were observed. During
    the first three weeks, diarrhoea and vomiting were observed frequently
    in most animals at 25 000 ppm, and a slight but significant reduction
    in body-weight gain was observed in females at this dose throughout
    the study. There were no treatment-related changes in haematological
    or urinary parameters; slight but significant decreases in the
    concentration of total protein were observed in males at 25 000 ppm,
    and significant decreases in the concentration of albumin were
    observed in females at 5000 ppm and animals at 25 000 ppm. These
    changes were observed during week 4 of treatment but had disappeared
    by week 13. The changes in albumin and total protein concentration
    might not be related to treatment, because they were slight and
    transient, and may have been a result of the vomiting and diarrhoea
    that occurred during the first weeks of the study. Dose-dependent
    increases in the absolute and relative weights of the liver were
    observed but were not significant. Histopathological examination
    revealed no compound-related lesions in tissues, including the liver.
    The NOAEL was 5000 ppm, equal to 140 mg/kg bw per day, on the basis of
    vomiting and diarrhoea in animals of each sex and reduced body-weight
    gain in females (Mellert & Hildebrand, 1994c).

         Groups of six male and six female beagles, six to nine months
    old, were given diets containing kresoxim-methyl (purity, 93.7% ) at a
    concentration of 0, 1000, 5000, or 25 000 ppm, equal to 0, 27, 140,
    and 710 mg/kg bw per day in males and 0, 30, 150, and 760 mg/kg bw per
    day in females, for 12 months. The study conformed to good laboratory
    practice. The animals were observed for clinical signs, deaths, food
    consumption, body weight, ophthalmological end-points, clinical
    chemical parameters including the activities of serum ALAT, ASAT, AP,
    and GGT, haematological parameters including clotting time, and
    pathological end-points. Blood samples were collected for
    haematological and clotting analysis and clinical chemistry after 3,
    6, and 12 months of treatment.

         No deaths or ophthalmological abnormalities were observed.
    Diarrhoea and vomiting occurred infrequently in animals of each sex at
    25 000 ppm, and the body weights of males at this dose were
    significantly reduced at study termination. There was no reduction in
    body-weight gain or food consumption at any dose. Significant
    increases in the number of platelets were observed in males at all
    doses; the values for males at 25 000 ppm were within the range in
    historical controls, except for the mean value at the third month.
    There were no compound-related changes in clotting time. There were no
    compound-related changes in urinary or clinical chemical parameters or

    in the activities of serum enzymes. Significant increases in relative
    liver weights were observed in males at 5000 ppm, but the absolute
    liver weights were not significantly increased. Histopathological
    examination revealed no treatment-related alterations in the liver or
    in any other tissue examined. The NOAEL was 5000 ppm, equal to 140
    mg/kg per day, on the basis of reduced body weight in males (Hellwig &
    Hildebrand, 1994b).

     (c)  Long-term studies of toxicity and carcinogenicity

     Mice

         Groups of 50 male and 50 female C57Bl/6N (Cr) mice were given
    diets containing kresoxim-methyl (mean purity, 96.3% during the first
    12 months and 93.2% during the following six months) at concentrations
    of 0, 400, 2000, or 8000 ppm for 18 months. Satellite groups of 10
    mice of each sex were treated concurrently for 12 months. The doses
    were equivalent to 0, 60, 300, and 1300 mg/kg bw per day in males and
    0, 81, 400, and 1700 mg/kg bw per day in females in the main groups,
    and 0, 61, 320, and 1400 mg/kg bw per day in males and 0, 84, 410, and
    1900 mg/kg bw per day in females in the satellite groups. The animals
    were observed for clinical signs, deaths, food consumption, body
    weight, and haematological and pathological end-points. Blood samples
    for haematology were collected during months 12 and 18 of treatment.
    The study conformed to good laboratory practice.

         No compound-related effects were observed with respect to
    mortality rates, clinical signs, food consumption, or haematological
    parameters throughout the study. Statistically significant decreases
    in terminal body weights and body-weight gains were observed in the
    main groups in males at 8000 ppm and in females at 2000 and 8000 ppm
    during the final six months. Increased relative liver weights were
    observed in females in the satellite group examined at 12 months and
    in the main groups examined at 18 months at 8000 ppm. Increased
    relative adrenal weights were observed in males at 12 and 18 months
    and in females at 18 months. Histopathological examination at
    12 months revealed no compound-related lesions in any group treated
    for 12 months, but examination at 18 months revealed significantly
    increased incidences of centrilobular fatty infiltration (1/50 at 0
    and 16/50 at 8000 ppm) in the liver, a significantly increased
    incidence and a greater degree of severity of hepatic amyloidosis
    (6/50 at 0 and 16/50 at 8000 ppm), and increased incidences of
    lymphoid infiltration (16/50 at 0 and 27/50 at 8000 ppm) and papillary
    necrosis of the kidney (2/50 at 0 and 13/50 at 8000 ppm) in females.
    There was no treatment-related increase in the incidence of neoplastic
    lesions. The NOAEL was 400 ppm, equal to 81 mg/kg bw per day, on the
    basis of reductions in body weight and body-weight gain in females
    (Mellert & Hildebrand, 1994e).

     Rats

         In a study of toxicity, groups of 20 male and 20 female Wistar
    rats were given diets containing kresoxim-methyl (purity, 92.7-96.6%)
    at concentrations of 0, 200, 800, 8000, or 16 000 ppm, equal to 0, 9,
    36, 370, and 750 mg/kg bw per day in males and 0, 12, 46, 500, and
    1000 mg/kg bw per day in females, for 24 months. The animals were
    observed for clinical signs, deaths, food consumption, body weight,
    ophthalmological end-points, clinical chemical parameters including
    the activities of serum ALAT, ASAT, AP, and GGT, and haematological,
    urinary, and histopathological end-points. Blood samples for
    haematology and clinical chemistry were collected at 3, 6, 12, 18, and
    24 months of the treatment. The design of the study conformed to good
    laboratory practice.

         There were no treatment-related effects on mortality rates,
    clinical signs, or ophthalmoscopic parameters. The terminal body
    weight and body-weight gain were slightly reduced in males at 16 000
    ppm (by 4%) and significantly reduced in females at 8000 ppm (by 9 and
    13%, respectively) and 16 000 ppm (by 6 and 10%, respectively). No
    significant change in food consumption was observed. Slight but
    significant reductions in mean corpuscular volume and mean corpuscular
    haemoglobin were observed in males at 16 000 ppm and in females at
    > 200 ppm; however, these changes were within the background range
    and were not clearly dose-dependent. The activity of serum ALAT was
    significantly decreased in animals of each sex at 8000 and 16 000 ppm
    and that of serum AP in animals of each sex at > 200 ppm. The
    author suggested that these reductions in enzyme activities are not
    toxicologically relevant, which is reasonable (Moss, 1994; Mellert et
    al., 1997a). The relative liver weights were significantly increased
    in males at 8000 and 16 000 ppm, and the absolute liver weights were
    significantly increased in males at the highest dose. Significant,
    dose-related increases in GGT activity were also observed in males at
    > 8000 ppm. 

         Microscopic examination revealed evidence of neoplasia in the
    liver. Increased incidences of hepatocellular carcinoma were observed
    in animals of each sex at 8000 and 16 000 ppm (in males, 0/20 at 0,
    1/20 at 200 ppm, 1/20 at 800 ppm, 3/20 at 8000 ppm, and 8/20 at 16 000
    ppm; in females, 0/20 at 0, 0/20 at 200 ppm, 2/20 at 800 ppm, 6/20 at
    8000 ppm, and 6/20 at 16 000 ppm). No hepatocellular adenomas were
    observed. The incidence and severity of hepatocellular hypertrophy
    were dose-dependent and increased in animals of each sex (males, 0/20
    at 0, 3/20 at 800 ppm, 4/20 at 8000 ppm, and 7/20 at 16 000 ppm;
    females, 1/20 at 0 and 8/20 at 16 000 ppm); however, statistical
    significance was achieved only at 16 000 ppm in animals of each sex.
    Significant increases in the incidence and severity of eosinophilic
    foci (0/20 at 0, 6/20 at 8000 ppm, and 8/20 at 16 000 ppm) and mixed-
    cell foci (0/20 at 0, 4/20 at 8000 ppm, and 5/20 at 16 000 ppm) were
    observed in males. Evidence of a proliferative response in bile-duct
    cells was associated with increased incidences of biliary cysts in
    males at 16 000 ppm (0/20 in controls versus 4/20) and in females at
    8000 and 16 000 ppm (3/20 in controls versus 7/20 and 7/20), bile-duct

    proliferation in females at 8000 and 16 000 ppm (5/20 in controls
    versus 8/20 and 11/20), and pericholangitis of the liver in males at
    16 000 ppm (1/20 in controls versus 4/20). Significantly increased
    incidences of tubular casts of the kidneys (2/20 in controls versus
    10/20) and tubular atrophy of the kidney (4/20 in controls versus
    12/20) were seen in females at 16 000 ppm. Increased incidences of
    lesions in other tissues were age-related or independent of dose and
    were not considered to be toxicologically significant.

         The NOAEL for non-neoplastic alterations was 800 ppm, equal to 36
    mg/kg bw per day, on the basis of increased activity of serum GGT,
    increased relative liver weight, and increased incidence and degree of
    severity of eosinophilic foci in males. The NOAEL for neoplasia was
    also 800 ppm on the basis of an increased incidence of hepatocellular
    carcinoma in animals of each sex (Mellert & Hildebrand, 1994d). 

         In a study of carcinogenicity, groups of 50 male and 50 female
    Wistar rats were fed diets containing kresoxim-methyl (purity,
    92.7-96.6%) at concentrations of 0, 200, 800, 8000, or 16 000 ppm,
    equal to 0, 9, 36, 380, and 770 mg/kg bw per day for males and 0, 12,
    47, 500, and 1000 mg/kg bw per day for females, for 24 months. The
    animals were observed for clinical signs, deaths, food consumption,
    body weight, and haematological and histopathological end-points.
    Blood samples for haematology were collected at the end of the study.
    The study was carried according to the principles of good laboratory
    practice.

         There were no treatment related effects on mortality rates or
    clinical signs. The terminal body weights and body-weight gains were
    significantly reduced in animals of each sex at 8000 ppm              
    (9 and 13% in males and 13 and 20% in females, respectively) and 16
    000 ppm (9 and 12% in males and 14 and 21% in females, respectively).
    No significant change was observed in food consumption. Significantly
    increased relative liver weights were observed in males at 16 000 ppm.
    Microscopic examination revealed hepatic neoplasia: increased
    incidences of hepatocellular carcinoma were observed in animals of
    each sex at 8000 and 16 000 ppm (males, 7/50 at 0, 5/50 at 200 ppm,
    2/50 at 800 ppm, 18/50 at 8000 ppm, and 11/50 at 16 000 ppm; females,
    1/50 at 0, 1/50 at 200 ppm, 2/50 at 800 ppm, 13/50 at 8000 ppm, and
    16/50 at 16 000 ppm). The numbers of animals with adenoma plus
    carcinoma in the liver were significantly increased among males at
    8000 ppm (8/50 at 0, 19/50 at 8000 ppm, and 13/50 at 16 000 ppm) and
    among females at 8000 and 16 000 ppm (1/50 in controls versus 15/50
    and 17/50). The incidence of hepatocellular hypertrophy was increased
    in males at 8000 and 16 000 ppm and in females at 16 000 ppm but
    reached statistical significance only in males at 16 000 ppm (males,
    3/50 at 0, 5/50 at 8000 ppm, and 10/50 at 16 000 ppm; females, 5/50 at
    0, and 7/50 at 16 000 ppm). There were dose-dependent increases in the
    incidences of eosinophilic foci (males, 1/50 at 0, 5/50 at 8000 ppm,
    and 11/50 at 16 000 ppm; females, 3/50 at 0, 8/50 at 8000 ppm, and
    5/50 at 16 000 ppm) and mixed-cell foci in animals of each sex (males,
    4/50 at 0, 9/50 at 8000 ppm, and 12/50 at 16 000 ppm; females, 0/50 at
    0 and 5/50 at 16 000 ppm); however, significant results were observed

    only at 16 000 ppm. There was evidence of alterations in bile-duct
    cells, including an increased incidence of bile-duct proliferation in
    females at 16 000 ppm (10/50 in controls versus 28/50),
    cholangiofibrosis in females at 16 000 ppm (1/50 in controls versus
    7/50), and biliary cysts in males at 8000 ppm (males, 1/50 at 0, 7/50
    at 8000 ppm, and 6/50 at 16 000 ppm; females, 8/50 at 0, 12/50 at 8000
    ppm, and 15/50 at 16 000 ppm). Other non-neoplastic lesions included
    tubular mineralization of the kidneys in males at 16 000 ppm (6/50 in
    controls versus 18/50), and round-cell infiltration of the adrenal
    cortex in males at 8000 ppm (5/50 at 0, 13/50 at 8000 ppm, and 5/50 at
    16 000 ppm). The tubular mineralization was dose-related and
    considered to be related to treatment. The lesions observed in other
    tissues were considered to be independent of dose and age-related. 

         The NOAEL for non-neoplastic alterations was 800 ppm, equal to 36
    mg/kg bw per day, on the basis of reduced body weight and body-weight
    gain and hepatic alterations. The NOAEL for neoplasia was also 800 ppm
    on the basis of increased incidences of hepatocellular carcinoma
    (Mellert & Hildebrand, 1994f).

         A histopathological re-evaluation on the heptocellular tumour
    incidence in the two studies in rats was conducted by a pathology
    working group. The results are shown in Table 4. Concurrent
    reassessment revealed similar dose-response relationships in the
    occurrence of hepatocellular carcinoma, and the statistically
    significant results with the combined data clearly indicate the
    hepatic carcinogenic potential of kresoxim-methyl in rats (van
    Ravenzwaay, 1996).

     (d)  Genotoxicity

         The results of assays for the genotoxicity of kresoxim-methyl are
    summarized in Table 5. No point mutations were observed  in vitro in
    bacterial or mammalian cells. A significantly increased frequency of
    chromosomal damage was observed in Chinese hamster lung cells with an
    exogenous metabolic activation system treated with kresoxim-methyl
    (purity, 93.7%) at > 100 g/ml; however, crystals were observed in
    medium cultured at 100 g/ml for 6 h. No chromosomal damage was
    observed in human lymphocytes  in vitro. Assays for DNA repair and
    damage in rat hepatocytes showed marked cytotoxicty, characterized by
    altered cell morphology and reduced numbers of live cells at > 10
    g/ml . Kresoxim-methyl at these doses also increased extracellular
    lactic dehydrogenase activity. The percent of cells in repair was
    slightly increased at > 1 g/ml (by 1-2% in comparison with 52% in
    the positive control), but the authors considered these percentages to
    be below their evaluation criteria (net grain, > 5%). Kresoxim-methyl
    did not cause DNA damage or repair  ex vivo in hepatocytes isolated
    from treated rats. It did not induce micronucleus formation in mice or
    rats treated  in vivo. 

        Table 4. Incidences of hepatocellular carcinomas and other parameters in 
    rats in the studies of Mellert & Hildebrand (1994e,f)

                                                                                       

    Parameter                             Dose (ppm)
                                                                                       
                                          0        200      800      8000     16 000
                                                                                       

    Study of toxicity

    Males

    Incidence                             0/20     1/20     1/20     3/20     8/20*
    % incidence                           0        5        5        15       40
    Absolute body weight (% of control)   100      106      109      94       96
    Body-weight gain (% of control)       100      109      112      91       96

    Females

    Incidence                             1/20     0/20     2/20     6/20     6/20
    % incidence                           5        0        10       30       30
    Absolute body weight (% of control)   100      105      88       91(*)    94(*)
    Body-weight gain (% of control)       100      107      81       87(*)    90(*)

    Study of carcinogenicity

    Males

    Incidence                             7/50     5/50     2/50     18/50*   13/50*a
    % incidence                           14       10       4        36       26
    Absolute body weight (% of control)   100      101      98       91 (*)   91 (*)
    Body-weight gain (% of control)       100      102      98       87 (*)   79 (*)

    Females

    No of incidence                       1/50     1/50     2/50     13/50*   16/50*
    % of incidence                        2        2        4        26       32
    Absolute body weight (% of control)   100      99       98       87(*)    86(*)
    Body-weight gain (% of control)       100      98       97       80(*)    79(*)
                                                                                       

    Terminal absolute body weight and body-weight gain were expressed as percent of 
    control, but the statistical significance was calculated on the basis of weight. 
    * Statistically significantly different from control.
    a Includes two animals with hepatocholangiocarcinomas
    

        Table 5. Results of assays for the genotoxicity of kresoxim-methyl

                                                                                                                                       

    End-point               Test object                     Concentration                 Purity     Result      Reference
                                                                                          (%)
                                                                                                                                       

    In vitro
    Reverse mutationa,b     S. typhimurium TA98, TA100,     20-5000 g/plate              93.7       Negative    Engelhardt &  
                            TA1535, TA1537; E. coli WP2                                                          Hoffmann (1993a)
                            uvrA

    Reverse mutationa,b     S. typhimurium TA98, TA100,     20-5000 g/plate              94.3       Negative    Engelhardt & 
                            TA1535, TA1537; E. coli WP2                                                          Hildebrandt (1994) 
                             uvrA

    Reverse mutationa,b     S. typhimurium TA98, TA100,     20-5000 g/plate              90.2       Negative    Engelhardt (1996) 
                            TA1535, TA1537; E. coli WP2 
                            uvrA

    Reverse mutationb,c     S. typhimurium TA98, TA100,     51-5000 g/plate              98.6       Negative    Nakajima (1997) 
                            TA1535, TA1537; E. coli WP2 
                            uvrA

    DNA repaird             B. subtilis rec M45+, H17-      191-6100 g/plate (-S9)       98.6       Negative    Nakajima (1997)
                                                            95-3050 g/plate (+S9)                   Negative

    Gene mutatione          Chinese hamster ovary cells,    0.01-100 g/ml (-S9)          94.3       Negative    Polloth & Hoffman 
                             hprt locus                     0.1-100 g/ml (+S9)                                  (1994a)

    Chromosomal             Human lymphocytes               10-40 g/ml                   98.7       Negative    Engelhardt & 
    aberrationb,f                                                                                                Hoffmann  (1993b) 

    Chromosomal             Chinese hamster lung cells      0.45-55 g/ml (-S9)           93.7       Negative    Akanuma et al. (1997)
    aberrationg                                             50-200 g/ml (+S9)                       Positive

    DNA damage and          Wistar rat hepatocytes          0.33-10 g/ml                 94.3       Negative    Polloth & Hoffman 
    repairh                                                                                                      (1994b) 

    Table 5. (continued)

                                                                                                                                       

    End-point               Test object                     Concentration                 Purity     Result      Reference
                                                                                          (%)
                                                                                                                                       

    In vivo

    DNA damage and          Wistar rat hepatocytes          Single oral gavage, 18 h      94.3       Negative    Polloth & Hildebrand 
    repairi                                                 0, 20, 200, 1000 mg/kg bw                            (1994c)

    DNA damage and          Wistar rat hepatocytes          3-week feeding                94.3       Negative    Polloth & Hoffman
    repairi                                                 0, 200, 16 000 ppm                                   (1994b)

    Micronucleus            NMRI mouse bone marrow          Single i.p, 16 and 48 h,      93.7       Negative    Engelhardt & 
    formationj                                              0, 500, 1000, 2000 mg/kg bw                          Hoffmann (1993c)

    Micronucleus            Wistar rat bone marrow          Single i.p, 24 and 48 h,      94.9       Negative    Engelhardt & 
    formationk                                              0, 500, 1000, 2000 mg/kg bw                          Hoffmann (1997) 
                                                                                                                                       

    S9, microsomal fraction of rat hepatocytes; i.p., intraperitoneal
    All of the tests were carried out according to good laboratory practice, and all of the positive controls produced the expected results.
    a In dimethyl sulfoxide; the positive controls were 2-aminoanthracene, N-methyl-N'-nitro-N-nitrosoguanidine, 
       N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine chloride, and 4-nitro-ortho-phenylendiamine.
    b In the presence and absence of S9 
    c In dimethyl sulfoxide; the positive controls were 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide for TA98, TA100, and WP2uvrA; sodium 
       azide for TA1535; and 9-aminoacridine chloride for TA1537 -S9 and 2-aminoanthracene + S9. 
    d Positive controls were mitomycin C -S9 and try-P-1 +S9; negative control was kanamycin -S9.
    e Positive controls were ethylmethanesulfonate -S9 and 3-methylcholanthrene +S9.
    f Positive controls were mitomycin C -S9 and cyclophosphamide +S9.
    g Positive controls were mitomycin C -S9 and benzo[a]pyrene. 
    h Positive control was 2-acetylaminofluorene.
    i Positive control was 2-acetylaminofluorene at a single oral dose of 50 mg/kg bw.
    j Positive controls were cyclophosphamide at a single i.p dose of 20 mg/kg bw and vincristine at a single i.p dose of 0.15 mg/kg bw.
    k Positive control was cyclophosphamide at a single i.p dose of 20 mg/kg bw.
    

     (e)  Reproductive toxicity

    (i)   Multigeneration reproductive toxicity

     Rats

         In a two-generation study of reproductive toxicity, which
    conformed to good laboratory practice, groups of 25 male and 25 female
    Wistar rats were fed diets containing kresoxim-methyl (purity,
    > 93.7%) at concentrations of 0, 50, 1000, 4000, or 16 000 ppm. The
    F0 generation was exposed directly, the F1a and F1b generations
    directly and indirectly, and F2 generation indirectly. The mean daily
    intakes of kresoxim-methyl by the F0 generation were 5, 100, 410, and
    1600 mg/kg bw per day for males and 6, 120, 480, and 2300 mg/kg bw per
    day for females. Female intakes were 6, 110, 440, and 1700 mg/kg bw
    per day during premating; and 4, 87, 360, and 1400 mg/kg bw per day
    during gestation and 7, 150, 600, and 2400 mg/kg bw per day during
    lactation for the F1a and F1b generations. The mean daily intakes by
    the F1 generation were 4, 88, 360, and 1500 mg/kg bw per day for
    males and 5, 110, 440, and 1800 mg/kg bw per day for females. female
    intakes were 5, 100, 420, and 1700 mg/kg bw per day during premating;
    4, 85, 350, and 1300 mg/kg bw per day during gestation for F2a; and
    7, 140, 560, and 2300 mg/kg bw per day during lactation for F2a.

         The parental rats were observed for clinical signs, deaths, food
    consumption, body weight, and clinical chemical, histopathological,
    and reproductive parameters including mating, fertility, gestation,
    and live-birth indices. The litters and pups were observed for
    viability, lactation, behaviour, and developmental indices that
    included pinna unfolding and opening of the auditory canal and eyes.
    The functional tests included grip strength, startle reflex, and
    pupillary reflex. Reproductive organs and the pituitary, liver, and
    kidney were examined histopathologically. The clinical chemical
    end-points included assays for serum ALAT, ASAT, AP, and GGT activity.

         No compound-related clinical signs or deaths were observed in the
    F0, F1, or F2 generation throughout the study. F0 and F1 parental
    animals showed no effects on mating, fertility, gestation, or
    live-birth indices, but significant reductions in food consumption
    were observed in F0 and F1 males during treatment and in F0 and F1
    females during gestation and lactation at 16 000 ppm. Significant
    reductions in body weight were seen at doses of 4000 ppm and higher in
    F0 and F1 males and in F0 and F1 females during gestation and
    lactation of the F1a and F1b generations. Significant reductions in
    body-weight gain were also observed in F0 and F1 males at these
    doses and in F0 females at 16 000 ppm during the premating period
    before the first gestation. Significant reductions in the activities
    of ALAT and AP were observed in F0 and F1 parents of each sex,
    although these reductions may not be toxicologically relevant (Moss,
    1994; Mellert & Hildebrand, 1995). The activity of GGT was
    significantly increased in F0 males at 4000 ppm and higher and in F1
    animals of each sex at 16 000 ppm. Significantly decreased numbers of
    fat storage cells were observed in the livers of F0 and F1 males at

    4000 ppm and higher; however, this change may have occurred as a
    result of the reduced food consumption at higher doses. Significant
    increases in relative kidney weights were observed in F0 males at 16
    000 ppm and in F0 females and F1 males at 4000 ppm and higher. No
    treatment-related morphological lesions were observed in the liver or
    kidney. 

          No compound-related changes in clinical signs, sex ratio,
    viability index, or lactation index were seen in pups of the F1a,
    F1b, and F2a generations. Body weights and body-weight gain during
    lactation were significantly decreased in F1a, F1b, and F2a pups at
    4000 ppm and higher. A significantly lower percentage of F1b pups at
    these doses had pinna unfolding; significant retardations in opening
    of the auditory canal and eyes were also observed in F1b and F2a pups
    at 4000 ppm,but were not dose-dependent. There were no differences in
    the results of reflex tests between controls and treated animals in
    any generation. Necroscopy of pups revealed no external abnormality.

         The NOAEL for parental toxicity was 1000 ppm, equal to 100 mg/kg
    bw per day in F0 males and 88 mg/kg bw per day in F1 males, on the
    basis of reduced body weight and body-weight gain, increased serum GGT
    activity, and increased relative kidney weights. The NOAEL for
    reproductive toxicity was 1000 ppm, equal to an overall mean intake of
    100 mg/kg bw per day for F1 and F2 pups, on the basis of reduced
    body weight and body-weight gain (Hellwig & Hildebrand, 1994a).

     (ii)  Developmental toxicity

     Rats

         Groups of 25 female Wistar rats were given kresoxim-methyl
    (purity, > 93.7%) suspended in 0.5% CMC by gavage at a dose of 0,
    100, 400, or 1000 mg/kg bw per day on days 6-15 of gestation. The
    study was conducted in accordance with good laboratory practice. No
    treatment-related changes in clinical signs, mortality rates, body
    weight, or food consumption were observed in maternal animals. There
    were no differences in conception rate, mean number of corpora lutea,
    total implantations, resorptions, pre- or post-implantation loss, or
    number of live fetuses. No significant differences in fetal sex ratio,
    placental weight, or fetal body weight were observed between control
    and treated groups. External examination revealed three fetuses with
    external malformations: one fetus at 100 mg/kg bw per day had anasarca
    and a cleft palate, one fetus at 400 mg/kg bw per day was acaudate,
    and one fetus at 1000 mg/kg bw per day had meningocele and unilateral
    microphthalmia; however, the incidence of these malformations was
    within the range for historical controls. One fetus at 1000 mg/kg bw
    per day had hydrocephalus, but this incidence was also within the
    historical control range. A significantly increased incidence of
    incompletely ossified thoracic vertebral bodies was seen in 23% of all
    fetuses and 58% of litters at 1000 mg/kg bw per day; the mean
    historical control values were 8% (0-49%) of all fetuses and 23%
    (0-100%) of litters. The NOAEL for maternal toxicity was 1000 mg/kg bw
    per day, and that for embryo and fetal toxicity was 400 mg/kg bw per

    day on the basis of a slight increase in variations in fetuses at 1000
    mg/kg bw per day. There was no evidence of teratogenicity at doses
    < 1000 mg/kg bw per day (Hellwig, 1994).

     Rabbits 

         Groups of 15 female Himalayan rabbits were given kresoxim-methyl
    (purity, 96.6%) suspended in 0.5% CMC by gavage at a dose of 0, 100,
    400, or 1000 mg/kg bw on days 7-19 of gestation. The study was
    conducted in accordance with good laboratory practice. No
    compound-related changes in clinical signs, deaths, body weight, or
    food consumption were observed in maternal animals, and there were no
    compound-related changes in conception rate, mean numbers of corpora
    lutea, total implantations, resorptions, pre- or post-implantation
    loss, or live fetuses. No significant differences in fetal sex ratio,
    placental weight, or fetal body weight were observed between control
    and treated groups. External examination revealed one fetus with
    microcephaly and brachygnathia at 100 mg/kg bw per day, but the
    incidence was within that of historical controls. Eight fetuses (0 /15
    at 0, 2/15 at 100, 2/15 at 400, and 3/15 at 1000 mg/kg bw per day) had
    soft-tissue malformations: one at 100 mg/kg bw per day had a septal
    defect and one had agnesis of the gall-bladder (2.5% incidence); two
    at 400 mg/kg bw per day had a septal defect (1.9%); at 1000 mg/kg bw
    per day, one had a septal defect, dilatation of the aortic arch, and a
    descending aortic, one had hydrocephaly, and one had agnesis of the
    gall-bladder (4.1%). The percent of soft-tissue malformations in
    historical controls was 2.2-3.1%. The incidences of ventricular septal
    defects in the treated groups were comparable to historical values.
    Increased incidences of fused sternebrae were observed in 3/15
    controls, 11/15 at 100 mg/kg bw per day, 7/15 at 400 mg/kg bw per day,
    and 9/15 at 1000 mg/kg bw per day; the increase at 100 mg/kg bw per
    day was significant but was within the historical control range.
    Increased total numbers of fetal malformations were also observed in
    treated groups but again at incidence rates comparable to those of
    historical controls (0% at 0, 4.9% at 100, 3.8% at 400, and 4.1% at
    1000 mg/kg bw per day versus 2.9-3.5% for historical controls). The
    NOAEL for both maternal and developmental toxicity was thus 1000 mg/kg
    bw per day, the highest dose tested (Hellwig & Hildebrand, 1993, GLP)

     (f)  Special studies

    (i)   Tumour initiating potential

         Groups of 10 Wistar rats of each sex were subjected to a partial
    hepatectomy and 14 h later received a single dose of 2388 mg/kg bw
    technical-grade kresoxim-methyl (purity, 92.7-94.3%) suspended in 0.5%
    CMC by gavage to rats. For promotion, phenobarbital was incorporated
    in the diet at a concentration of 500 ppm for eight weeks. Liver
    slices were examined histologically on slides stained with
    haematoxylin and eosin (H&E) or stained immunochemically for the
    placental form of glutathione  S-transferase (GST-P). The study
    conformed to good laboratory practice. The incidences of
    hepatocellular alteration (foci) and of GST-P-positive foci were used

    to estimate initiating potential.  N-Nitrosomorpholine was used as
    the positive control. Hepatocellular hypertrophy was found in almost
    all of the phenobarbital-treated animals, and GST-positive foci and
    foci of hepatocellular alteration were found in nearly all animals
    treated with the positive control. The number of animals with
    GST-P-positive foci in groups treated with kresoxim-methyl was
    comparable to that of vehicle controls. The numbers of foci per liver
    in promoted animals were 0-3 in those given kresoxim-methyl, 0-10 in
    vehicle controls, and 3-100 in positive controls. The results suggest
    that kresoxim-methyl does not have tumour initiating potential in rats
    in this test (Gamer & Hildebrand, 1995).

    (ii)   Tumour promoting potential

         In a medium-term study of promotion, which did not conform to
    good laboratory practice, male Fischer rats were initiated with a
    single intraperitoneal injection of  N-nitrosodiethylamine at a dose
    of 299 mg/kg bw. The animals were then maintained on basal diet
     ad libitum for 14 days. Five groups of 16 male rats were fed diets
    containing 0, 200, 800, 8000, or 16 000 ppm kresoxim-methyl (purity,
    95.4%) for six weeks, with average intakes of 0, 11, 42, 430, and 890
    mg/kg bw per day (not adjusted for purity). The remaining 16 male rats
    were fed a diet containing 500 ppm phenobarbital (28 mg/kg bw per day)
    as a positive control for six weeks. The animals were subjected to a
    two-thirds partial hepatectomy after the first week of feeding with
    kresoxim-methyl or phenobarbital and were observed for clinical signs,
    deaths, food consumption, and body weight. The liver was examined
    grossly and histopathologically. 

         There were no compound-related deaths or clinical signs of
    toxicity. Body weight and food consumption in groups given
    kresoxim-methyl were comparable to those of controls. Significant
    increases in the absolute and relative weights of the liver were
    observed in groups given kresoxim-methyl at 800 ppm and higher.
    Treatment with phenobarbital caused significant increases in body
    weight, food consumption, and relative liver weight. Quantification of
    hepatic foci with a computer-assisted image analyser revealed
    significant, dose-related increases in the number and area of
    GST-P-positive hepatocellular foci in groups given kresoxim-methyl at
    > 8000 ppm, as well as in the phenobarbital-treated positive
    controls. The NOAEL for promotion was 800 ppm (Harada et al., 1997).

     (iii)  Hepatic-cell proliferation

         A series of studies was conducted to investigate the effect of
    kresoxim-methyl on hepatic-cell proliferation in rats, by measuring
    S-phase DNA synthesis, an indicator of cell proliferation.
    Incorporation of bromodeoxyuridine (BrdU) into DNA was measured by
    immunohistochemical staining. 

         In the first study, groups of five young male Wistar rats, 64
    days old, were given diets containing kresoxim-methyl (purity, 94.3%)
    at concentrations of 0, 200, or 16 000 ppm, equal to 0, 15, and 1100
    mg/kg bw per day, for three weeks. Osmotic minipumps filled with 
    BrdU were implanted subcutaneously one week before necroscopy. the 
    animals were observed for clinical signs, deaths, food consumption, 
    and body weight. The livers were examined grossly and
    immunohisto-pathologically. Samples of the hepatic lobule and the
    jejunum were taken as positive tissues for proliferation and were
    stained with H&E and immunochemically with an antibody against BrdU.
    Immunopositive and H&E-counterstained hepatocyte nuclei from 11 fields
    for each of three lobes were counted. No treatment-related changes in
    body weight, food consumption, or clinical signs were seen. A slight
    increase in liver weights was observed at 16 000 ppm, but no
    treatment-related gross lesions or histopathological changes were
    observed in the livers of treated rats. A statistically significant
    increase in the number of hepatocytes in which BrdU was incorporated
    into the DNA of S-phase cells was observed in the periportal zone
    (zone 1) and the intermediate zone (zone 2) of the hepatic lobule in
    the group at 16 000 ppm. No significant increase in cell proliferation
    was observed in the group at 200 ppm (Polloth & Hildebrand, 1994a).

         In a supplementary study with a similar design, groups of five
    young male Wistar rats received kresoxim-methyl (purity, 94.9% ) in
    the diet at a concentration of 0, 800, or 8000 ppm, equal to 0, 61,
    and 600 mg/kg bw per day, for three weeks. Results similar to those
    observed at 16 000 ppm in the first study were observed at 8000 ppm.
    Statistically significant increases in cell proliferation were
    observed in zones 1 and 2 of the hepatic lobule at 8000 ppm, but not
    at 800 ppm (Mellert et al., 1997a).

         The NOAEL from the combined results of these two studies for
    hepatic cell proliferation was 800 ppm, equal to 61 mg/kg bw per day. 

         In a study of the hepatic proliferating activity of
    kresoxim-methyl in the livers of older rats, groups of five male
    Wistar (Chbb) rats aged 16 months were given diets containing
    kresoxim-methyl (purity, 94.3%) at a concentration of 0, 200, or 16
    000 ppm for three weeks. The design of the study was similar to those
    described above. No compound-related changes were seen in clinical
    signs or body weight, and no compound-related lesions in the liver
    were observed by microscopic examination with H&E staining. A
    statistically significant increase in cell proliferation was observed
    in zone 1 of the hepatic lobule at 16 000 ppm, which was comparable to
    that observed in the young rats (Polloth & Hildebrand, 1994d) . 

         In a study of the hepatic proliferating activity of
    kresoxim-methyl in the livers of rats treated for various periods,
    groups of five male Wistar (Chbb) rats, 42 days old, were given diets
    containing kresoxim-methyl (purity, 92.7%) at a concentration of 0 or
    16 000 ppm for 1, 6, or 13 weeks. Groups were were allowed to recover
    for two or three weeks. Significant increases in cell proliferation
    were observed in the treated groups after one week (zones 1, 2, and 3)

    and after six weeks (zone 1). The increase in zone 1 in the group
    treated for one week was greater than that in the group treated for
    six weeks. This compound-related enhancement of cell proliferation was
    significantly reversed in the groups allowed to recover. The zonal
    distribution of increased cell proliferation revealed a selective
    effect of kresoxim-methyl on hepatocytes in zone 1 (Mellert et al.,
    1996a).

         In a study of unscheduled DNA synthesis and S-phase response in
    rat hepatocytes, groups of three male Wistar (Chbb) rats received a
    single oral dose of 0, 20, 200, or 1000 mg/kg bw kresoxim-methyl
    (purity, 94.3%) by gavage. 2-Acetylaminofluorene was used as a
    positive control, at a dose of 50 mg/kg bw in the assay of unscheduled
    DNA synthesis and at 1000 mg/kg bw in the assay of S-phase response.
    Hepatocytes were prepared by in-situ hepatic perfusion 18 h after
    treatment. The isolated hepatocytes were cultured with 3H-thymidine
    for 18 h, and S-phase response and unscheduled DNA synthesis were
    evaluated autoradiographically in the labelled cells. Exposure of rats
    to kresoxim-methyl  in vivo was not cytotoxic to liver cells. Slight
    but dose-dependent increases in the number of cells in S-phase were
    observed in all treated groups, with 1% at 0, 1.37% at 20, 2.78% at
    200, and 2.58% at 1000 mg/kg bw, as well as in the positive control
    group (5.87%). The results suggest that kresoxim-methyl induced a
    moderate increase in S-phase DNA synthesis at 200 mg/kg bw and has a
    weak potential for enhancing hepatic cell proliferation (Polloth &
    Hildebrand, 1994c). 

     (iv)  Morphology of hepatic proliferation

         Groups of three female Wistar (Chbb) rats, 12 weeks old, received
    diets containing kresoxim-methyl (purity, 94.3% ) at concentrations of
    0, 200, or 16 000 ppm, equal to 0, 15, and 1200 mg/kg bw per day, for
    three weeks. At termination, the livers were fixed  in situ by
    perfusion, and the peroxisomes in the liver were examined by light and
    electron microscopy after staining with diaminobenzidine to detect
    catalase activity. There were no compound-related changes in clinical
    signs, body weight, or food consumption; reduced body-weight gain was
    observed at 16 000 ppm. No compound-related lesions were observed in
    the liver, and no difference was seen between treated and control
    animals in the numbers of peroxisomes (Mellert et al., 1995a).

         Groups of three female Wistar (Chbb) rats, 15 months old,
    received diets containing kresoxim-methyl (purity, 94.3%) at
    concentrations of 0, 200, or 16 000 ppm for three weeks and were then
    fixed  in situ by perfusion. Liver samples were examined by light and
    electron microscopy. There were no compound-related changes in
    clinical signs or body weight, and no compound-related lesions were
    observed in the liver on light microscopic examination. Electron
    microscopy showed that the amount, shape, and size of hepatocyte
    mitochondria in the treated group were comparable to those in
    controls. (Mellert et al., 1995b).

     (v)  Induction of hepatic metabolic enzyme activities

         Groups of 10 male and 10 female Wistar rats were fed diets
    containing kresoxim-methyl at concentrations of 0, 200, or 16 000 ppm
    for three weeks, equal to 0, 13, and 1000 mg/kg bw per day for males
    and 0, 15, and 1200 mg/kg bw per day for females. The animals were
    observed for clinical signs, deaths, body weight, and food
    consumption. Indicators of hepatic enzymes were measured, including
    the activities of GGT and drug metabolizing enzymes, the concentration
    of glutathione in liver homogenates, and the content of cytochrome
    P450 in microsomes. Significant increases in the activities of GGT and
    pentoxyresorufin depentylase and in P450 content were observed in
    males at 16 000 ppm. The pattern of induction of drug metabolizing
    enzyme activities resembled that of phenobarbital. In females, only a
    tendency towards induction was observed (Mellert et al., 1996b).

     (vi)  Mechanism of decreased serum enzyme activities

         As marked reductions in the activities of serum AP and ALAT were
    reported in short- and long-term studies of toxicity, a series of
    experiments was conducted in which groups of five males and five
    females were fed diets containing kresoxim-methyl at a concentration
    of 8000 ppm for two weeks. These studies did not conform to good
    laboratory practice. In the first experiment, AP activity was
    determined in serum samples and extracts of liver and small intestine.
    The intestinal activity of AP was not changed by treatment, the
    estimated ratio of intestinal and hepatic or bone AP isozyme
    activities in the serum being 38.5%. The author indicated the
    reduction in serum AP activity observed in the kresoxim-methyl treated
    groups was mostly due to a reduction in intestinal AP activity. In the
    second experiment, serum AP activity was markedly reduced after
    fasting and was increased by feeding a diet supplemented with olive
    oil. In the third experiment, addition of sera collected from treated
    animals to sera collected from untreated animals did not suppress AP
    activity, indicating the absence of an inhibitor. The observed
    reductions in serum AP and ALAT activities was therefore probably due
    to a slight alteration in food absorption in treated rats. (Moss,
    1994). 

         In a second study to investigate the reduced enzyme activities,
    groups of 10 male and 10 female Wistar rats were fed diets containing
    kresoxim-methyl at a concentration of 0 or 16 000 ppm for two weeks,
    equal to 910 mg/kg bw per day for males and 1100 mg/kg bw per day for
    females. The animals were observed for clinical signs, deaths, body
    weight, and food consumption. ALAT and AP activities in serum and
    urine were assayed at the end of the study. There were no
    compound-related changes in clinical signs or mortality rates.
    Significantly decreased food consumption was observed in treated
    animals of each sex. A slight but significant decrease in body weight
    was observed in treated males. Significantly reduced activities of
    ALAT and AP in serum were observed in animals of each sex, but no
    change in the activities of either enzyme was observed in urine. No
    change in urinary creatinine or urinary volume was observed in treated

    animals, indicating no change in renal function. Thus, the reduced
    enzyme activity observed in sera of kresoxim-methyl-treated rats was
    not caused by a change in renal excretion of the enzymes (Mellert et
    al., 1997b).

     (g)  Studies on metabolites

    (i)   Acute toxicity 

         Metabolites M1, M2, and M9 were given orally to rats in a
    suspension of 0.5% CMC. M1 (purity, 98.5%) produced a variety of
    abnormal clinical changes including dyspnoea, staggering gait, and
    tremor at doses of 2000 mg/kg bw and higher. M2 (purity, 97.7%) caused
    no deaths or abnormal symptoms at 5000 mg/kg bw. M9 (purity, 99.6%)
    caused dyspnoea and exhaustion in animals of each sex at 5000 mg/kg bw
    but resulted in no change in general appearance at 3000 mg/kg bw
    (Kirsch & Hildebrand, 1994a,b,c, 1995).

    (ii)   Genotoxicity

         M1, M2, and M9 of the same purities described above did not
    induce reverse mutation in bacteria at a concentration of 5000
    g/plate, whereas the positive controls used gave the expected
    positive responses (Hoffman & Engelhardt, 1995a,b,c)

    Comments

         About 60% of an oral dose of 50 mg/kg bw and 25% of a dose of 500
    mg/kg bw kresoxim-methyl was absorbed. It was excreted mainly in the
    faeces (70% of the low dose and 80% of the high dose), predominantly
    via the bile (about 40% of the low dose and 15% of the high dose
    within 48 h), with lesser amounts in urine (about 20% of the low dose
    and 10% of the high dose). Peak levels of the radiolabel in plasma
    were reached 0.5-1 h after the low dose and 8 h after the high dose.
    The plasma half-life was 17-19 h at the low dose and 22-31 h at the
    high dose. The highest residual concentrations were found in the
    liver, but the concentrations in all tissues, including the liver,
    were less than 0.1 g equivalent/g tissue after 120 h of treatment at
    the low dose. 

         After oral administration of kresoxim-methyl, a high proportion
    of the parent compound was found in the faeces, but none was detected
    in tissues or bile examined 4 h after dosing. In rats, 34 metabolites
    of kresoxim-methyl were identified. The proposed metabolic pathways
    are hydrolytic cleavage of the ester, the oxime ether, and the benzyl
    ether bonds, hydroxylation at the  para position of the phenoxy ring,
    hydroxylation of the aryl-methyl group and its subsequent oxidation to
    form the corresponding carboxylic acid, and conjugation of the
    resulting hydroxy groups with glucuronate or sulfate. The major
    metabolites identified in both rats and plants were the free acid,
    code number 490M1 {(E)-methoxyimino[alpha- (ortho-tolyloxy)- ortho-
    tolyl]acetic acid}, the hydroxy derivative of this, 490M2

    [alpha- (ortho-hydroxymethylphenoxy)- ortho-tolyl
    (methoxyimino)acetic acid] formed by hydroxylation of the aryl-methyl
    group, the  para-hydroxytolyloxy product 490M9
    [alpha- (para-hydroxy- ortho-tolyloxy)- ortho-tolyl
    (methoxyimino)acetic acid], and their conjugates. 490M1, 490M2, and
    490M9 all had low acute toxicity and were not mutagenic.

         WHO has not classified kresoxim-methyl for acute toxicity.

         In a range-finding study in B6C3F1 mice, kresoxim-methyl was
    administered in the diet at concentrations of 0, 500, 2000, or 8000
    ppm for 28 days. The NOAEL was 8000 ppm, equal to 2100 mg/kg bw per
    day. In a three-month study, C57Bl/6N mice received kresoxim-methyl in
    the diet at concentrations of 0, 250, 1000, 4000, or 8000 ppm. The
    NOAEL was 8000 ppm, equal to 1900 mg/kg bw per day.

         In a range-finding study in rats, kresoxim-methyl was
    administered in the diet at concentrations of 0, 1000, 4000, or 16 000
    ppm for 28 days. The NOAEL was 4000 ppm in males, equal to        
    370 mg/kg bw per day, on the basis of increased activities of serum
    gamma-glutamyl transferase at 16 000 ppm, equal to 1500 mg/kg bw per
    day. In a three-week study of toxicity in rats, kresoxim-methyl was
    administered in the diet at concentrations of 0, 10, 50, or 8000 ppm.
    The NOAEL was 50 ppm, equal to 3 mg/kg bw per day, on the basis of
    increased hepatic gamma-glutamyl transferase activity in males at 8000
    ppm. In a 90-day study of toxicity in rats, kresoxim-methyl (purity,
    98.7%) was administered in the diet at concentrations of 0, 500, 2000,
    8000, or 16 000 ppm. The NOAEL was 500 ppm in females, equal to 43
    mg/kg bw per day, based on increased relative liver weight at 2000 ppm
    and above, and 2000 ppm in males, equal to 150 mg/kg bw per day, based
    on decreased body-weight gain and increased activity of serum
    gamma-glutamyl transferase at 8000 ppm and above.

         In a three-month study of toxicity in dogs, kresoxim-methyl was
    administered at dietary concentrations of 0, 1000, 5000, or 25 000
    ppm. The NOAEL was 5000 ppm, equal to 140 mg/kg bw per day, on the
    basis of vomiting, diarrhoea, and reduced body-weight gain in animals
    of each sex at 25 000 ppm. In a 12-month study in dogs,
    kresoxim-methyl was administered at dietary concentrations of 0, 1000,
    5000, or 25 000 ppm. The NOAEL was 5000 ppm, equal to 140 mg/kg bw per
    day, on the basis of a reduction in body weight in males at 25 000
    ppm. No compound-related toxicity was observed in females.

         In an assay for carcinogenicity in mice, kresoxim-methyl was
    administered at dietary concentrations of 0, 400, 2000, or 8000 ppm
    for 18 months. The NOAEL was 400 ppm, equal to 81 mg/kg bw per day, in
    females on the basis of reduction in body weight at 2000 ppm. The
    NOAEL in males was 2000 ppm, equal to 300 mg/kg bw per day, on the
    basis of decreased body weight and increased relative adrenal weight
    at 8000 ppm. At this dose, increased incidences of renal papilliary
    necrosis and hepatic amyloidosis were observed in females. There was
    no evidence of carcinogenicity.

         In a two-year study of toxicity in rats, kresoxim-methyl was
    administered at dietary concentrations of 0, 200, 800, 8000, or 16 000
    ppm. The NOAEL was 800 ppm, equal to 36 mg/kg bw per day, on the basis
    of an increased incidence of hepatocellular carcinoma in animals of
    each sex, increased serum gamma-glutamyl transferase activity,
    increased relative liver weight, an increased incidence and degree of
    severity of eosinophilic foci and mixed-cell foci in males, and a
    decrease in terminal body weight and body-weight gain in females at
    8000 ppm and 16 000 ppm. There was also an increased incidence of
    biliary cysts and bile-duct proliferation.

         In a study of carcinogenicity in rats, kresoxim-methyl was
    administered at dietary concentrations of 0, 200, 800, 8000, or 16 000
    ppm for 24 months. Evidence of biliary alterations included increased
    incidences of biliary cysts and cholangiofibrosis in females at 16 000
    ppm. At this dose, increased relative liver weights and an increased
    incidence of hepatocellular hypertrophy were observed in males. The
    NOAEL was 800 ppm, equal to 36 mg/kg bw per day, on the basis of
    increased incidences of hepatocellular carcinoma, reductions in body
    weight and body-weight gain, and an increased incidence of
    eosinophilic foci and mixed-cell foci in animals of each sex at 8000
    ppm and above. The overall NOAEL for neoplastic and non-neoplastic
    effects was 800 ppm, equal to 36 mg/kg bw per day.

         It is generally recognized that the process of carcinogenesis is
    divided into three stages: initiation, promotion, and progression. A
    series of mechanistic studies was conducted with kresoxim-methyl,
    including tests for tumour initiating and promoting potential. In a
    study on tumour initiating activity, kresoxim-methyl did not increase
    the number of liver-cell foci in rats at a single dose of 2400 mg/kg
    bw. In a study on the promoting potential of kresoxim-methyl, rats
    received an initiating dose of  N-nitrosodiethylamine and then a diet
    containing 0, 200, 800, 8000, or 16 000 ppm kresoxim-methyl for six
    weeks. Quantitative analysis of hepatic foci with a computer-assisted
    image analyser revealed significant, dose-dependent increases in the
    number and area of placental-type glutathione  S-transferase-positive
    hepatocellular foci, indicating a promoting effect of kresoxim-methyl
    on hepatocarcinogenesis at doses of 8000 ppm and above. The NOAEL for
    the promoting effect was 800 ppm, equal to 43 mg/kg bw per day.

         Four studies were conducted to investigate the effect of
    kresoxim-methyl on hepatic-cell proliferation in rat liver by
    measuring bromodeoxyuridine incorporation into hepatocyte DNA during
    S-phase DNA synthesis. The results showed a selective cell
    proliferation effect of kresoxim-methyl on hepatocytes in the
    periportal zone. The NOAEL was 800 ppm, equal to 61 mg/kg bw per day,
    while animals treated with 8000 ppm and above showed a statistically
    significant increase in cell proliferation. There was no difference in
    the sensitivity of young and old rats.

         The genotoxic potential of kresoxim-methyl was investigated in a
    series of tests, including assays for gene mutation in bacteria and
    mammalian cells, unscheduled DNA synthesis, and cytogenicity
     in vitro, an assay for micronucleus formation  in vivo, and an
    assay for unscheduled DNA synthesis  ex vivo. Kresoxim-methyl had
    moderate potential to induce chromosomal aberrations  in vitro with
    exogenous metabolic activation, but positive effects were not observed
    in any other test, including the assay for micronuclei in rat bone
    marrow. The Meeting concluded that kresoxim-methyl is not genotoxic.
    The three major metabolites in rats did not induce reverse mutation in
     Salmonella typhimurium in vitro.

         The increased incidence of liver tumours observed in rats at 8000
    ppm and above was considered to be associated with increased cell
    proliferation. The mechanistic studies indicated that kresoxim-methyl
    has tumour promoting potential at 8000 ppm, which coincides with the
    lowest level at which increased liver-cell proliferation was observed.
    These results indicate a threshold for the neoplastic mode of action.
    The Meeting concluded that a level of 800 ppm kresoxim-methyl has no
    carcinogenic potential.

         In a two-generation study of reproductive toxicity in rats, the
    NOAEL values were 1000 ppm for parental animals of each sex, 100 mg/kg
    bw per day for F0 offspring, and 88 mg/kg bw per day for F1
    offspring; these were based on reductions in body weight and
    body-weight gain and increased serum gamma-glutamyl transferase
    activity and relative kidney weight at 4000 ppm and above. The NOAEL
    for pups was 1000 ppm, equal to 110 mg/kg bw per day for F1 pups and
    97 mg/kg bw per day for F2 pups, on the basis of reductions in body
    weight and body-weight gain at 4000 ppm and above.

         The NOAEL for embryo- and fetotoxicity in a study of
    developmental toxicity in rats was 400 mg/kg bw per day. No maternal
    toxicity or teratogenic effects were observed at doses up to and
    including the highest one of 1000 mg/kg bw per day. Kresoxim-methyl
    did not induce toxicity in a study of developmental toxicity in
    rabbits up to and including the highest dose of 1000 mg/kg bw per day.

         An ADI of 0-0.4 mg/kg bw was established on the basis of the
    NOAEL of 800 ppm, equal to 36 mg/kg bw per day, in the 24-month study
    of toxicity and carcinogenicity in rats, and a 100-fold safety factor.

         An acute RfD was not allocated because kresoxim-methyl has low
    acute toxicity and did not exhibit developmental toxicity. The Meeting
    concluded that the acute intake of residues is unlikely to present a
    risk to consumers.

    Toxicological evaluation

     Levels that cause no toxic effect

         Mouse:    400 ppm, equal to 81 mg/kg bw per day (18-month study
                   of toxicity)

         Rat:      800 ppm, equal to 36 mg/kg bw per day (two-year study
                   of toxicity and carcinogenicity )
                   1000 ppm, equal to 88 mg/kg bw per day (two-generation
                   study of reproductive toxicity)
                   400 mg/kg bw per day (study of developmental toxicity)

         Rabbit:   1000 mg/kg bw per day (developmental toxicity; highest
                   dose tested)

         Dog:      5000 ppm, equal to 140 mg/kg bw per day (12-month study
                   of toxicity) 

     Estimate of acceptable daily intake for humans

         0-0.4 mg/kg bw 

     Estimate of acute reference dose

         Not allocated (unnecessary)

     Studies that would provide information useful for continued 
     evaluation of the compound

         Observations in humans 

        List of end-points relevant for setting guidance values for dietary and non-dietary exposure
                                                                                                 

     Absorption, distribution, excretion, and metabolism in mammals

    Rate and extent of oral absorption         Rapid, 25-60% absorbed
    Dermal absorption                          No data
    Distribution                               Minimum, highest levels in liver
    Potential for accumulation                 Very little 
    Rate and extent of excretion               Rapid/complete, 87-93% within 48 h
    Metabolism in animals                      Extensive. No parent compound in urine, bile, or 
                                               tissues; 34 metabolites identified.
    Toxicologically significant compounds      Parent compound in rat; three major metabolites in 
    (animals, plants and environment)          plants

    Acute toxicity

    Rat: LD50 oral                             > 5000 mg/kg bw
    Rat: LD50 dermal                           > 2000 mg/kg bw
    Rat: LC50 inhalation                       > 5.6 mg/L
    Skin irritation                            Not irritating
    Eye irritation                             Not irritating
    Skin sensitization                         Not sensitizing

    Short-term toxicity

    Target/critical effect                     Liver: increased relative liver weight (mouse, rat)
    Lowest relevant oral NOAEL                 Rat: 28-day, 43 mg/kg bw per day 
    Lowest relevant dermal NOAEL               No data
    Lowest relevant inhalation NOAEL           No data

    Genotoxicity                               Not genotoxic

    Long-term toxicity and carcinogenicity

    Target/critical effect:                    Hepatocellular carcinoma
    Lowest relevant NOAEL                      Rat: 2-year, 36 mg/kg bw per day, diet
    Carcinogenicity                            Non-genotoxic carcinogen, tumour promoter

    Reproductive toxicity

    Reproduction target/critical effect        Reduction in F0 body weight at parenterally toxic 
                                               dose
    Lowest relevant reproductive NOAEL         Rat: 97 mg/kg bw per day, diet
    Developmental target/critical effect       None
    Lowest relevant developmental NOAEL        Rat: 1000 mg/kg bw per day, highest dose tested

    Neurotoxicity/Delayed neurotoxicity        No data

    Other toxicological studies                No data

    Medical data                               No data

    Summary                  Value                Study                     Safety factor
    ADI                      0-0.4 mg/kg bw       2-year study of           100
                                                  toxicity and 
                                                  carcinogenicity, rat
    Acute reference dose     Not allocated 
                             (unnecessary )
                                                                                                 
    
    References

    Akanuma, M. et al. (1997) Report: In vitro cytogenetic investigations
    of Reg. No. 242009 in Chinese hamster cultured cell. Unpublished
    report No. IET 97-0021 from Institute for Environmental Toxicology,
    Kodaira, Tokyo, Japan. Submitted to WHO by BASF AG, Limbergerhof,
    Germany.

    Engelhardt, G. (1996) Report on the study of Reg. No. 242009 in the
    Ames Salmonella/mammalian-microsome mutagenicity test and Escherichia
    coli/mammalian-microsome reverse mutation assay (standard plate test
    preincubation test). Unpublished report No. 93/10249 from BASF
    Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO by BASF
    AG, Limbergerhof, Germany.

    Engelhardt, G. & Hoffmann, H.D. (1993a) Report on the study of Reg.
    No. 242009 in the Ames Salmonella/mammalian-microsome mutagenicity
    test and Escherichia coli/mammalian-microsome reverse mutation assay
    (standard plate test preincubation test). Unpublished report No.
    93/10249 from BASF Aktiengesellschaft, Ludwigshafen, Germany.
    Submitted to WHO by BASF AG, Limbergerhof, Germany.

    Engelhardt, G. & Hoffmann, H.D. (1993b) Report: In vitro cytogenetic
    investigations of Reg. No. 242009 in human lymphocytes. Unpublished
    report No. 93/10493 from BASF Aktiengesellschaft, Ludwigshafen,
    Germany. Submitted to WHO by BASF AG, Limbergerhof, Germany.

    Engelhardt, G. & Hoffmann, H.D. (1993c) Report: Cytogenetic study in
    vivo of Reg. No. 242009 in mice micronucleus test. Single
    intraperitoneal sdministration.Unpublished report No. 93/11104 from
    BASF Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO by
    BASF AG, Limbergerhof, Germany.

    Engelhardt, G. & Hoffmann, H.D. (1994) Report on the study of Reg. No.
    242009 in the Ames test (Salmonella/mammalian-microsome mutagenicity
    test--Standard plate test preincubation test). Unpublished report No.
    93/10249 from BASF Aktiengesellschaft, Ludwigshafen, Germany.
    Submitted to WHO by BASF AG, Limbergerhof, Germany.

    Engelhardt, G. & Hoffmann, H.D. (1997) Report: Cytogenetic study in
    vivo of Reg. No. 242009 in rat micronucleus test. Single
    intraperitoneal administration. Unpublished report No. 06M0180/914384
    from BASF Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO
    by BASF AG, Limbergerhof, Germany.

    Gamer, A.O. & Hildebrand, B. (1995) Report: Study of foci initiating
    activity of Reg. No. 242009 in Wistar rats. Unpublished report No.
    95/10244 from BASF Aktiengesellschaft, Ludwigshafen, Germany.
    Submitted to WHO by BASF AG, Limbergerhof, Germany.

    Gamer, A.O. & Kirsch, P. (1992) Report: Study on the acute oral
    toxicity of Reg. No. 242009 in rats. Unpublished report No. 94/10753
    from BASF Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO
    by BASF AG, Limbergerhof, Germany.

    Gans, G. (1994) Study of the biokinetics of [14C]-Reg. No. 242009
    ([14C]-BAS 490F) in rats. Unpublished report No. 94/10992 from BASF
    Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO by BASF
    AG, Limbergerhof, Germany.

    Grosshans, F. (1994a) The metabolism of 14C-242009 in apples. BASF
    Reg. No. 94/11760. Unpublished report from BASF Aktiengesellshaft,
    Limburgerhof, Germany. Submitted to WHO by BASF AG, Limbergerhof,
    Germany.

    Grosshans, F. (1994b) The metabolism of 14C-242009 in wheat. BASF
    Reg. No. 94/10685. Unpublished report from BASF Aktiengesellshaft,
    Limburgerhof, Germany. Submitted to WHO by BASF AG, Limbergerhof,
    Germany.

    Harada, T. et al. (1997) BAS 490F (Reg. No. 242009): Medium term
    promotion hepato-carcinogenesis study in rats. Unpublished report No.
    97/10918 from Institute for Environmental Toxicology, Kodaira, Tokyo,
    Japan. Submitted to WHO by BASF AG, Limbergerhof, Germany.

    Hellwig, J. (1994) Report: Study of the prenatal toxicity of of Reg.
    No. 242009 in Wistar rats after oral administration (gavage).
    Unpublished report No. 93/10833 from BASF Aktiengesellschaft,
    Ludwigshafen, Germany. Submitted to WHO by BASF AG, Limbergerhof,
    Germany.

    Hellwig, J. & Hildebrand, B. (1993) Report: Study of the prenatal
    toxicity of Reg. No. 242009 in rabbits after oral administration
    (gavage). Unpublished report No. 93/10980 from BASF
    Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO by BASF
    AG, Limbergerhof, Germany.

    Hellwig, J. & Hildebrand, B. (1994a) Report: Reproduction toxicity
    study with Reg. No. 242009 in Wistar rats. Continuous dietary
    administration over 2 generations (2 litters in the first and 1 litter
    in the second generation. Unpublished report No. 94/10950 from BASF
    Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO by BASF
    AG, Limbergerhof, Germany.

    Hellwig, J. & Hildebrand, B. (1994b) Report on the study of the
    toxicity of Reg. No. 242009 in beagle dogs. Administration via the
    diet over 12 months. Unpublished report No. 94/10832 from BASF
    Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO by BASF
    AG, Limbergerhof, Germany.

    Hoffmann, H.D. & Engelhardt, G. (1995a) Report on the study of Reg.
    No. 262451 in the Ames Salmonella/mammalian-microsome mutagenicity
    test and Escherichia coli/mammalian-microsome reverse mutation assay
    (standard plate test preincubation test). Unpublished report No.
    95/10409 from BASF Aktiengesellschaft, Ludwigshafen, Germany.
    Submitted to WHO by BASF AG, Limbergerhof, Germany.

    Hoffmann, H.D. & Engelhardt, G.. (1995b) Report on the study of Reg.
    No. 292932 in the Ames Salmonella/mammalian-microsome mutagenicity
    test and Escherichia coli/mammalian-microsome reverse mutation Assay
    (standard plate test preincubation test). Unpublished report No.
    95/10026 from BASF Aktiengesellschaft, Ludwigshafen, Germany.
    Submitted to WHO by BASF AG, Limbergerhof, Germany.

    Hoffmann, H.D. & Engelhardt, G. (1995c) Report on the study of Reg.
    No. 291685 in the Ames Salmonella/mammalian-microsome mutagenicity
    test and Escherichia coli/mammalian-microsome reverse mutation assay
    (standard plate test preincubation test). Unpublished report No.
    95/10027 from BASF Aktiengesellschaft, Ludwigshafen, Germany.
    Submitted to WHO by BASF AG, Limbergerhof, Germany.

    Kirsch, P. & Hildebrand, B. (1993a) Report: Study on the acute oral
    toxicity of Reg. No. 242009 in rats. Unpublished report No. 93/10730
    from BASF Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO
    by BASF AG, Limbergerhof, Germany.

    Kirsch, P. & Hildebrand, B. (1993b) Report: Study on the acute dermal
    toxicity of Reg. No. 242009 in rats. Unpublished report No. 93/11108
    from BASF Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO
    by BASF AG, Limbergerhof, Germany.

    Kirsch, P. & Hildebrand, B. (1994a) Report: Study on the acute oral
    toxicity of Reg. No. 291685 in rats. Unpublished report No. 94/11172
    prepared by BASF Aktiengesellschaft, Ludwigshafen, Germany. Submitted
    to WHO by BASF AG, Limbergerhof, Germany.

    Kirsch, P. & Hildebrand, B. (1994b) Report: Study on the acute oral
    toxicity of Reg. No. 292932 in rats. Unpublished report No. 94/11171
    from BASF Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO
    by BASF AG, Limbergerhof, Germany.

    Kirsch, P. & Hildebrand, B. (1994c) Report: Study on the acute dermal
    toxicity of Reg. No. 242009 in Wistar rats. Application to the intact
    skin over 3 weeks (21 applications). Unpublished report No. 94/11070
    from BASF Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO
    by BASF AG, Limbergerhof, Germany.

    Kirsch, P. & Hildebrand, B. (1995) Report: Study on the acute oral
    toxicity of Reg. No. 262451 in rats. Unpublished report No. 95/10213
    from BASF Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO
    by BASF AG, Limbergerhof, Germany.

    Kohl, W. (1994) The metabolism of [14C]-Reg.No.242009 ([14C]-BAS
    490F) in rats. Unpublished report No. 94/10981 from BASF
    Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO by BASF
    AG, Limbergerhof, Germany.

    Mellert, W. & Hildebrand, B. (1994a) Report: Study on the oral
    toxicity of Reg. No. 242009 in Wistar rats. Administration in the diet
    over 3 months. Unpublished report No. 94/10954 from BASF
    Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO by BASF
    AG, Limbergerhof, Germany.

    Mellert, W. & Hildebrand, B. (1994b) Report: Study on the oral
    toxicity of Reg. No. 242009 in C57BL mice. Administration in the diet
    for 3 months. Unpublished report No. 94/10496 from BASF
    Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO by BASF
    AG, Limbergerhof, Germany.

    Mellert, W. & Hildebrand, B. (1994c) Report: Study on the oral
    toxicity of Reg. No. 242009 in beagle dogs. Administration via the
    diet over 3 months (Supplementary study to project No.
    83M0180/910149). Unpublished report No. 97/10318 from BASF
    Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO by BASF
    AG, Limbergerhof, Germany.

    Mellert, W. & Hildebrand, B. (1994d) Report: Chronic toxity study with
    Reg. No. 242009 in Wistar rats. Administration in the diet for 24
    months. Unpublished report No. 94/10951 from BASF Aktiengesellschaft,
    Ludwigshafen, Germany. Submitted to WHO by BASF AG, Limbergerhof,
    Germany.

    Mellert, W. & Hildebrand, B. (1994e) Report: Carcinogenicity study
    with Reg. No. 242009 in C57BL mice. Administration in the diet for 24
    months. Unpublished report No. 94/10953 from BASF Aktiengesellschaft,
    Ludwigshafen, Germany. Submitted to WHO by BASF AG, Limbergerhof,
    Germany.

    Mellert, W. & Hildebrand, B. (1994f) Report: Carcinogenicity study
    with Reg. No. 242009 in Wistar rats. Administration in the diet for 24
    months. Unpublished report No. 94/10953 from BASF Aktiengesellschaft,
    Ludwigshafen, Germany. Submitted to WHO by BASF AG, Limbergerhof,
    Germany.

    Mellert, W. & Hildebrand, B. (1995) Test study on enzyme activity
    after treatment with Reg. No. 242009 in Wistar rats. Dietary
    administration for 3 weeks and recovery of 2 weeks. Unpublished report
    No. 95/10853 from BASF Aktiengesellschaft, Ludwigshafen, Germany.
    Submitted to WHO by BASF AG, Limbergerhof, Germany.

    Mellert, W., Kaufmann, W. & Hildebrand, B. (1995a) Reg. No. 242009 --
    Electron microscopic examinations of liver samples to assess
    peroxisomes from Wistar rats treated for 3 weeks in the diet.
    Unpublished report No. 95/11106 from BASF Aktiengesellschaft,
    Ludwigshafen, Germany. Submitted to WHO by BASF AG, Limbergerhof,
    Germany.

    Mellert, W. , Kaufmann, W. & Hildebrand, B. (1995b) Reg. No. 242009 --
    Electron microscopic examinations of liver samples to assess
    mitochondria from old Wistar rats treated for 3 weeks in the diet.
    Unpublished report No. 95/11105 from BASF Aktiengesellschaft,
    Ludwigshafen, Germany. Submitted to WHO by BASF AG, Limbergerhof,
    Germany.

    Mellert, W., Bahnemann, R. & Hildebrand, B. (1996a) Reg. No. 242009 --
    S phase response study in male Wistar rats including reversibility.
    Administration in the diet up to 13 weeks. Unpublished report No.
    96/10053 from BASF Aktiengesellschaft, Ludwigshafen, Germany.
    Submitted to WHO by BASF AG, Limbergerhof, Germany.

    Mellert, W. et al. (1996b) Reg. No. 242009 -- Examination of enzyme
    activities in the liver of Wistar rats. Administration in the diet for
    3 weeks. Unpublished report No. 96/10100 from BASF Aktiengesellschaft,
    Ludwigshafen, Germany. Submitted to WHO by BASF AG, Limbergerhof,
    Germany.

    Mellert, W. et al. (1997a) Report: S-phase response study with BAS
    490F (Reg. No. 242009) in Wistar rats after administration in the diet
    for 3 weeks. Unpublished report No. 94/10496 from BASF
    Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO by BASF
    AG, Limbergerhof, Germany.

    Mellert, W. et al. (1997b) Report: BAS 490F (Reg. No. 242009): Study
    of enzyme excretion in urine of Wistar rats after repeated
    administration in the diet. Unpublished report No. 97/10317 from BASF
    Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO by BASF
    AG, Limbergerhof, Germany.

    Moss, D. (1994) Effects of Reg. No. 242009 on enzyme levels in rat
    serum. Unpublished report No. 94/10578 from Royal Postgraduate Medical
    School, Hammersmith Hospital, London, United Kingdom. Submitted to WHO
    by BASF AG, Limbergerhof, Germany.

    Nakajima, M. (1997) Reverse mutation assay of Reg. No. 279482.
    Unpublished report No.97/11152 from Biosafety Research Center, Foods,
    Drugs and Pesticides, Shizuoka, Japan. Submitted to WHO by BASF AG,
    Limbergerhof, Germany.

    Nelsen, J. et al. (1995) Metabolism of 14C-BAS 490F in grapes. BASF
    Reg. No. 92/11760. Unpublished report from BASF Corporation, ARC,
    Resesarch Triangle Park, North Carolina, USA. Submitted to WHO by BASF
    AG, Limbergerhof, Germany.

    Polloth, C. & Hildebrand, B. (1994a) Report: Ex vivo unscheduled DNA
    synthesis (UDS) assay and S phase response in rat hepatocytes with
    Reg. No. 242009. Unpublished report No. 94/10867 from BASF
    Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO by BASF
    AG, Limbergerhof, Germany.

    Polloth, C. & Hildebrand, B. (1994b) Report: Ex vivo unscheduled DNA
    synthesis (UDS) in rat hepatocytes with Reg. No. 242009. Unpublished
    report No. 94/10894 from BASF Aktiengesellschaft, Ludwigshafen,
    Germany. Submitted to WHO by BASF AG, Limbergerhof, Germany.

    Polloth, C. & Hildebrand, B. (1994c) Report: S phase response with
    Reg. No. 242009 in Wistar rat after administration in the diet for 3
    weeks. Unpublished report No. 94/10922 from BASF Aktiengesellschaft,
    Ludwigshafen, Germany. Submitted to WHO by BASF AG, Limbergerhof,
    Germany.

    Polloth, C. & Hildebrand, B. (1994d) Report: S phase response with
    Reg. No. 242009 in 16 month old Wistar rat after administration in the
    diet for 3 weeks. Unpublished report No. 94/10984 from BASF
    Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO by BASF
    AG, Limbergerhof, Germany.

    Polloth, C. & Hoffmann, H.D. (1994a) Report: Gene mutation test in
    Chinese hamster ovary cells (HPRT locus assay) with Reg. No. 242009.
    Unpublished report No. 94/10350 from BASF Aktiengesellschaft,
    Ludwigshafen, Germany. Submitted to WHO by BASF AG, Limbergerhof,
    Germany.

    Polloth, C. & Hoffmann, H.D. (1994b) Report: In vitro unscheduled DNA
    synthesis (UDS) assay in rat hepatocytes with Reg. No. 242009.
    Unpublished report No. 94/10351 from BASF Aktiengesellschaft,
    Ludwigshafen, Germany. Submitted to WHO by BASF AG, Limbergerhof,
    Germany.

    van Ravenzwaay, B. (1996) Kresoxim-methyl: Mechanism and assessment of
    liver tumor induction. Unpublished report No. 96/10078 from BASF
    Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO by BASF
    AG, Limbergerhof, Germany.

    Rossbacher, R. & Kirsch, P. (1992a) Report: Study on the acute dermal
    irritation/corrosion of Reg. No. 242009 in the rabbit. Unpublished
    report No. 92/11663, BASF Aktiengesellschaft, Ludwigshafen, Germany.
    Submitted to WHO by BASF AG, Limbergerhof, Germany.

    Rossbacher, R. & Kirsch, P. (1992b) Report: Study on the acute eye
    irritation of Reg. No. 242009 in the rabbit. Unpublished report No.
    92/11664, BASF Aktiengesellschaft, Ludwigshafen, Germany. Submitted to
    WHO by BASF AG, Limbergerhof, Germany.

    Rossbacher, R. & Kirsch, P. (1993) Report on the maximization test for
    the sensitizing potential of Reg. No. 242009 in guinea pigs.
    Unpublished report No. 93/10014 from BASF Aktiengesellschaft,
    Ludwigshafen, Germany. Submitted to WHO by BASF AG, Limbergerhof,
    Germany.

    Schilling, K. & Hildebrand, B. (1992a) Report: Study on the oral
    toxicity of Reg. No. 242009 in Wistar rats. Administration in the diet
    over 4 weeks (range finding). Unpublished report No. 92/10551 from
    BASF Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO by
    BASF AG, Limbergerhof, Germany.

    Schilling, K. & Hildebrand, B. (1992b) Report: Study on the oral
    toxicity of Reg. No. 242009 in B6C3F1 mice. Administration in the diet
    over 4 weeks (range finding). Unpublished report No. 92/10539 from
    BASF Aktiengesellschaft, Ludwigshafen, Germany. Submitted to WHO by
    BASF AG, Limbergerhof, Germany.

    Yamamoto, T. (1994) Report: Study on the acute oral toxicity of Reg.
    No. 242009 in mice. Unpublished report No.94/ from Biosafety Research
    Center, Foods, Drugs and Pesticides, Shizuoka, Japan. Submitted to WHO
    by BASF AG, Limbergerhof, Germany.
    


    See Also:
       Toxicological Abbreviations