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    CHLORDIMEFORM

    EXPLANATION

         Chlordimeform was reviewed and evaluated for an acceptable daily
    intake by the Joint Meetings in 1971, 1975, 1978, 1979 and 1980 (Annex
    1, FAO/WHO, 1972a, 1976a, 1979a, 1980a and 1981a). A toxicological
    monograph was prepared by the Joint Meeting in 1971 (Annex 1, FAO/WHO,
    1972b) and monograph addenda were prepared in 1975, 1978, 1979, and
    1980 (Annex 1, FAO/WHO, 1976b, 1979b, 1980b, and 1981b). A temporary
    acceptable daily intake (TADI) of 0-0.01 mg/kg b.w. was established in
    1971 and reduced to 0-0.0001 in 1978. Further evaluations carried out
    in 1979 and 1980 did not modify the TADI. The 1980 Meeting required
    for review at the present meeting a report on continued surveillance
    and epidemiological studies of occupationally exposed workers in both
    industry and agriculture. A confirmatory long-term animal bioassay
    using a third species for evaluation of potential carcinogenic hazard
    was considered desirable. A considerable amount of additional
    information, not all relevant to the above requests, was evaluated by
    the Meeting and is presented in this monograph addendum.

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    BIOLOGICAL DATA

    Biochemical Aspects

    Effects on enzymes and other biochemical parameters

         In a study to investigate the effects of chlordimeform on hepatic
    enzymes in rats and mice, groups of eight male and eight female
    Tif:Magf (SPF) mice and Sprague-Dawley-derived Tif:RAI f (SPF) rats
    were treated by gastric intubation with 0, 50, 100 and 150 mg/kg b.w.
    chlordimeform in 0.9% saline solution (1 ml/100 g b.w.) for seven
    consecutive days. The animals were then fasted for 24 hours before
    sacrifice. The following parameters were investigated in hepatic
    microsomal fractions: benzo(a)pyrene hydroxylase, ethoxycoumarin
    O-de-ethylase ethylmorphine N-demethylase, cytochrome P-450 content,
    NADPH cytochrome P-450 reductase, UDP-glucuronyl transferase, and
    microsomal epoxide hydrolase. Glutathione S-transferase was measured
    in 100,000xg supernatants.

         Chlordimeform treatment induced several hepatic drug-metabolising
    enzymes with some species and/or sex specificity. Relative liver
    weights were increased in female rats and mice in a dose-dependent
    manner. Cytochrome P-450 content was increased in all cases but
    cytochrome P-450 reductase activity was increased only in the highest
    dose in male rats and mice and in the lowest and highest doses only
    in female mice. Ethylmorphine N-demethylase was induced in a dose-

    dependent manner in male mice but only at the highest dose in female
    mice. Ethoxycoumarin O-de-ethylase was induced in dose-dependent
    manner in male and female rats but only at high doses in both sexes of
    mice. Benzo(a)pyrene hydroxylase was increased in a dose-dependent
    manner in female rats and mice but only at the mid-dose in male mice.
    UDP-glucuronyl transferase was induced in rats of both sexes and
    female mice in a dose-dependent manner but male mice exhibited no
    induction.

         Dose-dependent increases of expoxide hydrolase and glutathione
    S-transferase occurred in male and female rats and female mice.
    (Bentley et al., 1985a). The results of these investigations clearly
    indicate that chlordimeform induces the activity of several hepatic
    enzymes but reveal no clear distinctions between rats and mice.

         The effect of acute and repeated chlordimeform treatment on rat
    hepatic microsomal enzymes has been examined following acute or
    repeated dosing. Groups of 6 male and 6 female Sprague-Dawley rats
    received either a single intraperitoneal injection of chlordimeform
    (100 mg/kg) 60 minutes prior to sacrifice or daily injections
    (75 mg/kg) for 4 days. Control animals received equivolume injections
    of normal saline (1 ml/kg). The duration of zoxazolamine-induced
    paralysis (70 mg/kg i.p.) or pentobarbitone-induced hypnosis (40 mg/kg
    i.p.) were assessed in the latter group.

         Hepatic microsomal preparations were utilized to assay the
    following enzymatic activities: ethylmorphine N-demethylase, aniline
    hydroxylase, p-nitroanisole-O-demethylase and NADPH cytochrome C
    reductase. Hepatic microsomal cytochrome P-450 content was estimated
    and the spectral binding of chlordimeform (2.1 mM), aniline (20 mM)
    and hexobarbital (10 mM) to hepatic microsomes was studied.

         It was found that acute chlordimeform exposure prolonged
    zoxazolamine paralysis and pentobarbitone hypnosis. However, after
    repeated exposure, the duration of zoxazolamine paralysis was
    significantly reduced whilst the duration of pentabarbitone hypnosis
    remained unchanged.

         Significant sex-related difference in hepatic metabolism were
    found. In male rats, ethylmorphine N-demethylation was significantly
    reduced in acutely treated animals and to a lesser extent in the
    repeatedly exposed group. Repeated exposure caused a modest reduction
    in aniline hydroxylation but did not affect the metabolism of
    p-nitranisole. For female rats, however, either acute or repeated
    chlordimeform treatment had little effect on ethylmorphine
    N-demethylation and p-nitroanisole demethylation whilst aniline was
    metabolised to a lesser extent following both treatment regimens.
    These results suggest that the hepatic microsomal enzymes of male rats
    are significantly more responsive to chlordimeform exposure than are
    those of females.

         In acutely-treated rats, hepatic microsomal NADPH cytochrome C
    reductase activity, cytochrome P-450 content and the spectral binding
    of hexobarbitone and aniline were significantly reduced. However, only
    the spectral binding of hexobarbitone was significantly reduced in
    rats repeatedly exposed to chlordimeform. Chlordimeform was found to
    produce a characteristic Type I difference spectrum when added to rat
    liver microsomes. The authors concluded that chlordimeform bound to
    cytochrome P-450 at non-active sites, producing a change in protein
    conformation (Budris et al., 1983).

    Special studies on mutagenicity

         Chlordimeform and its principal metabolites, 4-chloro-2-toluidine
    and N-formyl-2-toluidine, were variably active in a range of
    in vitro assays. In vivo, however, chlordimeform was inactive but
    the metabolites were each positive in one assay. Results are
    summarised in Table 1.

    Special studies on macromolecular binding

         Species difference in the DNA-binding of the metabolite
    4-chloro-2-toluidine were investigated in rats and mice in vivo and
    in vitro.  l4C-Ring labelled metabolite was dosed daily at 25 mg/kg
    to male Tif:MAG f (SPF) mice, and Sprague-Dawley derived Tif:RAI f
    (SPF) rats, by gastric intubation. Animals were held for 20 hours
    before sacrifice. The radioactivity bound to purified DNA isolated
    from liver homogenates was found to increase with total dose in both
    rats and mice. DNA binding in mice was about twice that in rats.

         Covalent binding of 4-chloro-2-toluidine to calf thymus DNA was
    studied in vitro after incubation with liver fractions from rats and
    mice. Mouse liver fractions produced more metabolites which bound to
    DNA than did those prepared from rat liver. Binding was catalysed
    by active liver homogenates and microsomal fractions and was
    NADPH-dpendent. Pretreatment with known inducers of cytochrome P-450
    (phenobarbitone, 3-methylcholanthrene, and arochlor 1254) was without
    effect.

         The patterns of adducts produced in rat and mouse liver in vivo
    and by incubation of calf thymus DNA in vitro with hepatic
    subcellular fractions of each species were analysed
    chromatographically. The patterns were qualitatively similar, the same
    metabolites being formed in each case. Significant quantitative
    differences occurred with two metabolites only (Bentley et al.,
    1985b).

         Species differences in the toxicity of 4-chloro-2-toluidine were
    further investigated. 14C-4-Chloro-2-toluidine (25 mg/kg) was
    administered in aqueous solution by gastric intubation to male Tif:MAG
    f(SPF) mice and Sprague-Dawley derived Tif:RAI f (SPF) rats. Urinary

        Table 1:  Results of mutagenicity assays on chlordimeform and its metabolites
                                                                                                                                              

    Test system                 Test              Test substance      Concentrations                    Results             Reference
                                Object            (purity)            used
                                                                                                                                              

    Studies on microorganisms

    Salmonella typhimurium/     TA98              chlordimeform       -S9: 0.1,1,10.100,1000 µg/0.1 ml  -                   Arni & Müller,
    microsome                   TA100             HCl*                +S9: 0.1,1,10,100,1000,2000       -                   1976a
                                TA1535                                µg/0.1 ml                         -
                                TA1537                                                                  -

                                TA98              N-formyl-4-         ±S9: 0.1,1,10,100,1000 µg/O.1 ml  -                   Arni & Müller,
                                TA100             chloro-2-                                             +(with S9 only)     1976b
                                TA1535            toluidine*                                            -
                                TA1537                                                                  -

                                TA98              4-chloro-2-         ±S9: 0.1,1,10,100,1000 µg/0.1 ml  +(with S9 only)     Arni & Müller,
                                TA100             toluidine                                             +(with S9 only)     1976b
                                TA1535            HCl*                                                  -
                                TA1537                                                                  -

    Salmonella typhimurium/     TA98              chlordimeform       single oral dose of 50, 100,      -                   Arni & Müller,
    Intrasanguine host-         TA100             HCl (99.9%)         200 mg/kg in mice                 -                   1983a
    mediated assay              TA1535                                                                  -

                                TA98              4-chloro-2-         single oral dose of 250,500,      -                   Arni & Müller,
                                TAlO0             toluidine           1000 mg/kg in mice                -                   1983b
                                TA1535            HCl (99.9%)                                           -
                                                                                                                                              

    Table 1:  (Con't)
                                                                                                                                              

    Test system                 Test              Test substance      Concentrations                    Results             Reference
                                Object            (purity)            used
                                                                                                                                              

    Saccharomyces cerevisiae/   D7                chlordimeform       ±S9: 80,400,2000,10000 µg/ml      inhibition of       Arni & Müller,
    microsome                                     HCl (99.9%)         ±S9: 15,45,125,405 µg/ml          growth above        1983c
                                                                                                        400 µg/ml

                                D7                4-chloro-o-         ±S9: 80,400,2000,10000 µg/ml      inhibition of       Arni & Müller,
                                                  toluidine HCl       ±S9: 3.2,10,30,90 µg/ml           growth at all       1983d
                                                  (99.9%)                                               conc. +(at 30,
                                                                                                        90 µg/ml)

    Salmonella typhimurium/     TA98              chlordimeform*      ±S9: 100 µg/ml                    -                   Konopka &
    microsome                   TA100                                                                   -                   Heymann,
                                TA1535                                                                  -                   1977
                                TA1537                                                                  -
                                TA1538                                                                  -

                                TA98              4-chloro-2-         -S9: 500 µg/ml                    +(with S9 at 250    Konopka &
                                                  toluidine                                             µg/ml)              Heymann, 1977
                                TA100                                 +S9: 10,250,500 µg/ml             +(with S9 at 10
                                                                                                        µg/ml)
                                TA1535                                                                  -
                                TA1537                                                                  -
                                TA1538                                                                  -

                                TA98              2-chloro-N-         -S9: 500 µg/ml                    -                   Konopka &
                                TA100             formyl-2-           +S9: 100, 500 µg/ml               +(with S9 at        Heymann, 1977
                                                  toluidine*                                            100 µg/ml)
                                TA1535                                                                  -
                                TA1537                                                                  -
                                TA1538                                                                  -
                                                                                                                                              

    Table 1:  (Con't)
                                                                                                                                              

    Test system                 Test              Test substance      Concentrations                    Results             Reference
                                Object            (purity)            used
                                                                                                                                              

                                TA98              chlordimeform       ±S9: 100,500,1000,2000 µg/plate   -                   Meucke
                                TA100             HCl                                                   -                   et el., 1979

                                TA98              4-chloro-2-         ±S9: 0.1,1,5,10,25,50,100,1000    +(with S9 only)     Meucke
                                TA100             toluidine HCl       2000 µg/plate                     +(with S9 above     et al., 1979
                                                                                                        100 µg only)

                                TA100             N-formyl-4-         ±S9: 0.1-2000 µg/plate            -                   Meucke
                                                  chloro-2-                                                                 et el., 1979
                                                  toluidine

                                TAl00             4-chloro-2-         ±S9: 10-2000 µg/plate             -                   Meucke
                                                  toluidyl-N,N-                                                             et el., 1979
                                                  dimethy
                                                  iormamide

    Salmonella typhimurium/     TA100             4-chloro-2-         ±S9: 10-2000 µg/plate             -                   Meucke
    microsome                                     toluidyl-N,N-                                                             et al., 1979
                                                  methyl
                                                  formamide

                                TA100             4-chloro-2-         ±S9: 10-2000 µg/plate             -                   Meucke
                                                  toluidyl-                                                                 et al., 1979
                                                  formamide

                                TA100             4-chloro-2-         ±S9: 0.1-2000 µg/plate            *(above 25 µg       Meucke
                                                  methylphenyl-                                         with S9)            et al., 1979
                                                  hydroxylamine                                         +(above 50 µg
                                                                                                        without S9)
                                                                                                                                              

    Table 1:  (Con't)
                                                                                                                                              

    Test system                 Test              Test substance      Concentrations                    Results             Reference
                                Object            (purity)            used
                                                                                                                                              

                                TA100             5-chloro-2-         ±S9: 0.1-2000 µg/plate            *(above l0 µg       Meucke
                                                  nitroso-                                              with S9 only)       et al., 1979
                                                  toluene

                                TA100             4-chloro-acetyl-    ±S9: 0.1-2000 µg/plate            -                   Meucke
                                                  2-toluidine                                                               et al., 1979

                                TA100             4-chloro-acetyl-    ±S9: 0.1-2000 µg/plate            -                   Meucke
                                                  N-hydroxy-o-                                                              et al., 1979
                                                  toluidine

                                TA100             4,4'-dichloro-      ±S9: 0.1-2000 µg/plate            -                   Meucke
                                                  2,2'-dimethyl-                                                            et al., 1979
                                                  azobenzene

                                TA100             4,4'-dichloro-      ±S9: 0.1-2000 µg/plate            -                   Meucke
                                                  2,2'-dimethyl-                                                            et al., 1979
                                                  azobenzene-
                                                  N-oxide

                                TA98              chlordimeform       -S9: 1,5,25,125,625 µg/plate      -                   Rashid
                                TA100             (analytical         +S9: 5,10,50,250,1250 µg/plate    -                   et al., 1984
                                TA1535            grade)                                                -
                                TA1537                                                                  -
                                TA1538                                                                  -

                                TA98              4-chloro-N-         -S9: 1,5,25,125,325 µg/plate      -                   Rashid
                                TAlO0             formyl-2-                                             -                   et al. 1984
                                TA1535            toluidine*                                            +(325 µg/plate)
                                TA1537                                                                  -
                                TA1538                                                                  -
                                                                                                                                              

    Table 1:  (Con't)
                                                                                                                                              

    Test system                 Test              Test substance      Concentrations                    Results             Reference
                                Object            (purity)            used
                                                                                                                                              

                                TA98              4-chloro-2          -S9: 1,5,25,125,325 µg/plate      -                   Rashid
                                TA100             toluidine                                             -                   et al. 1984
                                TA1535            (99%)                                                 -
                                TA1537                                                                  -
                                TA1538                                                                  -

    Eschericia coli/            WP2               chlordimefom        ±S9: 250,500,1000,                -                   Rashid
    microsome                   WP2uvrA           (analytical         2000 µg/plate                     -                   et al., 1984
                                WP67              grade)                                                -
                                CM611                                                                   -
                                CM571                                                                   -

                                WP2               4-chloro-N-         ±S9: 250,500,1000,                -                   Rashid
                                WP2uvrA           formyl-2-           2000 µg/plate                     -                   et al., 1984
                                WP67              toluidine*                                            -
                                CM611                                                                   -
                                CM571                                                                   -

                                WP2               4-chloro-2-         ±S9: 250,500,1000,                -                   Rashid
                                WP2uvrA           toluidine           2000 µg/plate                     -                   et al., 1984
                                WP67              (99%)                                                 -
                                CM611                                                                   -
                                CM571                                                                   -
                                                                                                                                              

    Table 1:  (Con't)
                                                                                                                                              

    Test system                 Test              Test substance      Concentrations                    Results             Reference
                                Object            (purity)            used
                                                                                                                                              

    In vitro assays

    Cell transformation         BALB/             chlordimeform       0.063,0.125,0.25,0.5,1.0 µg/ml    +(not dose-         Beilstein &
    assay                       3T3               HCL (99.9%)                                           related)            Müller, 1983

    Mouse lymphoma test         L5178Y/           chlordimeform       -S9: 42.5-1700 µg/ml              -(cytotoxicity      Beilstein &
                                TK+/-             HCl*                                                  above 850 µg/ml)    Müller, 1984a
                                                                      +S9: 75-3000 µg/ml                -(cytotoxicity
                                                                                                        above 1800 µg/ml)

                                L5178Y/           4-chloro-2-         -S9: 37.5-600 µg/ml               -(cytotoxicity      Bellserin &
                                TK+/-             toluidine HCl                                         above 450 µg/ml)    Müller, 1984b
                                                  *                   +S9: 31.3-500 µg/ml               +

    Cell transformation         BALB/             4-chloro-2-         2.25, 4.5, 9, 18, 36 µg/ml        +                   Bellstein &
    assay                       3T3               toluidine HCl                                                             Müller, 1984c
                                                  (99.9%)

    DNA repair test             rat               chlordimeform       5, 25, 125, 625 µg/ml             -                   Puri & Müller,
                                hepatocytes       HCl (99.9%)                                                               1983a

                                rat               4-chloro-2-         0.61, 3.1, 15.7, 78.5 µg/ml       +(dose-related)     Puri & Müller,
                                hepatocytes       toluidine HCl                                                             1983b
                                                  (99.9%)

    DNA repair test             CRL               chlordimeform       2, 10, 50, 250 µg/ml              -                   Puri & Müller,
    human lymphocytes           1121              HCl (99-9%)                                                               1983c

                                CRL               4-chloro-2-         1.25, 6.25, 31.25, 156.25 µg/ml   -                   Puri & Müller,
                                1121              toluidine HCl                                                             1983d
                                                  (99.9%)
                                                                                                                                              

    Table 1:  (Con't)
                                                                                                                                              

    Test system                 Test              Test substance      Concentrations                    Results             Reference
                                Object            (purity)            used
                                                                                                                                              

    Mouse lymphoma cells/       L5178Y            4-chloro-2-         single oral dose of 330 mg/kg     - (no positive      Strasser &
    host-mediated                                 toluidine HCl*      in mice                           control)            Müller, 1983a
    assay

                                L5178Y            N-formyl-4-         single oral dose of 300 mg/kg     - (no positive      Strasser &
                                                  chloro-2-           in mice                           control             Müller, 1983b
                                                  toluidine*

    Mouse lymphoma cells/       L5178Y            4-chloro-o-         111, 255 µg/ml                    + (no positive      Strasser &
    point mutation                                toluidine HCl*                                        control)            Müller, 1984a

                                L5178Y            N-formyl-4-         213,640 µg/ml                     + (no positive      Strasser &
                                                  chloro-2-                                             control)            Müller, 1984b
                                                  toluidine*

    In vivo assays

    Chromosomal/germinal        spermatogonia     N-formyl-4-         1 oral dose o£ 80, 160, or        -                   Arni
    epithelium                                    chloro-2-           320 mg/kg in mice                                     et al., 1983a
                                                  totuidine*

                                spermatocytes     chlordimeform       5 oral doses of 18, 36, or        -                   Arni
                                                  HCl*                72 mg/kg in mice                                      et al., 1983b

                                spermatocytes     N-formyl-4-         5 oral doses of 80, 160, or       + (chromosomal      Arni & Müller,
                                                  chloro-2-           320 mg/kg in mice                 aberration, not     1983e
                                                  toluidine*                                            dose-related)
                                                                                                                                              

    Table 1:  (Con't)
                                                                                                                                              

    Test system                 Test              Test substance      Concentrations                    Results             Reference
                                Object            (purity)            used
                                                                                                                                              

    Dominant lethal                               4-chloro-2-         single oral dose of               -                   Fritz
                                                  toluidine HCl*      110 or 330 mg/kg in mice                              et al., 1978

                                                  chlordimeform *     single oral dose of               -                   Fritz, 1978a
                                                                      22 or 66 mg/kg in mice

                                                  N-formyl-4-         single oral dose of               -                   Fritz, 1978b
                                                  chloro-2-           105 or 315 mg/kg in mice
                                                  toluidine *

    Chromosomal/germinal        spermatogonia     chlordimeform       5 oral doses of 9, 18, 22, 36,    -                   Hool
    epithelium                                    HCL*                and 66 mg/kg in mice                                  et al., 1983

    Chromosomal/somatic         bone              N-formyl-4-         2 oral doses of 300, 600, and     + (not seen in      Hool & Arni,
    cells                       marrow            chloro-2-           1200 mg/kg in Chinese hamster     repeat              1983a
                                                  toluidine*                                            experiment)

                                bone              4-chloro-2-         2 oral doses of 100,200,237,356,  +                   Hool & Arni,
                                marrow            toluidine HCl*      533,800 mg/kg in Chinese hamster                      1983b

    Chromosomal/germinal        spermatogonia     4-chloro-2-         5 oral doses of 85, 125, 170,     -                   Hool & Arni,
    epithelium                                    toluidine HCI*      250, 340, 500 mg/kg in mice                           1983c

                                spermatocytes     4-chloro-2-         5 oral doses of 85, 125, 170,     -                   Hool & Arni,
                                                  toluidine HCl*      250, 340, 500 mg/kg in mice                           1983d
                                                                                                                                              

    Table 1:  (Con't)
                                                                                                                                              

    Test system                 Test              Test substance      Concentrations                    Results             Reference
                                Object            (purity)            used
                                                                                                                                              

    Sister chromatid            bone              chlordimeform       single oral dose of 31, 62, 324   -                   Hool & Armi,
    exchange                    marrow            HCl (99.9%)         mg/kg in Chinese hamster                              1983e

                                bone              4-chloro-2-         single oral dose of 100, 200,     -                   Hool & Arni,
                                marrow            toluidine HCl       400 mg/kg in Chinese hamsters                         1983f
                                                  (99.9%)

    Chromosomal/somatic         bone              chlordimeform       2 oral doses of 60, 120, 240      -                   Hool & Müller,
    cells                       marrow            HCl*                mg/kg in Chinese hamster                              1978

    Heritable translocation     male              chlordimeform*      49 daily oral doses of 120 mg/kg  -                   Lang & Adler,
                                mice                                                                                        1982

                                male              N-formyl-4-         49 daily oral doses of 100 mg/kg  -                   Lang & Adler,
                                mice              chloro-2-                                                                 1982
                                                  toluidine*

                                male              4-chloro-2-         49 daily oral doses of 200 mg/kg  -                   Lang & Adler,
                                mice              toluidine*                                                                1982

    Mammalian spot test         C57BL/            chlordimeform       3 oral doses of 160 mg/kg         -                   Lang, 1984
                                6J mice
                                genotype
                                a/a

                                b/+;cch           N-formyl-4-         3 oral doses of 100 mg/kg         -                   Lang, 1984
                                p/++;             chloro-2-
                                dse/++;           toluidine*
                                s/+
                                                                                                                                              

    Table 1:  (Con't)
                                                                                                                                              

    Test system                 Test              Test substance      Concentrations                    Results             Reference
                                Object            (purity)            used
                                                                                                                                              

                                                  4-chloro-2-         3 oral doses of 100 mg/kg         +(spots of          Lang, 1984
                                                  toluidine*                                            genetic
                                                                                                        relevance)

    Nucleus anomaly/            bone              chlordimeform       2 oral doses of 60, 120, 240      -                   Langauer &
    somatic nuclei              marrow            HCI*                mg/kg in Chinese hamsters                             Müller, 1977

                                bone              N-formyl-4-         2 oral doses of 300, 600, 1200    -                   Langauer &
                                marrow            chloro-2-           mg/kg in Chinese hamsters                             Müller, 1978a
                                                  toluidine*

                                bone              4-chloro-2-         2 oral doses of 100, 200, 400     -                   Langauer &
                                marrow            toluidine*          mg/kg in Chinese hamsters                             Müller, 1978b
                                                                                                                                              

    *    Purity not specified
        excretion of radioactivity was monitored daily until sacrifice.
    Covalent binding of radioactivity to hepatic DNA, RNA and protein was
    measured 6, 12, 20 and 68 hours after treatment. In each case, the
    extent of binding to mouse DNA significantly exceeded that to rat DNA
    but binding to mouse RNA and protein exceeded that to rat DNA and
    protein.

         The effect of 4-chloro-2-toluidine upon the incorporation of
    3H-thymidine into capillary endothelial cells was also studied.
    Male rats and mice were dosed orally with 4-chloro-2-toluidine
    (25 mg/kg/day) and received subsequently 3H-thymidine by i.p.
    administration at varying intervals. The incorporation of
    radioactivity into blood capillary endothelial cell nuclei was studied
    by autoradiography. Between 500 to 1500 nuclei per animal were
    assessed. It was found that the incorporation of 3H-thymidine was not
    elevated in treated animals when compared to controls (Bentley
    et al., 1985c).

    Observations in humans

         Information was submitted on the results of monitoring the urine
    of more than 100 workers engaged in chlorimeform production and
    packaging in 1976. In more than 800 individual urine samples, total
    urinary levels ranged from 0.05-50 ppm. Microscopic haematuria was not
    detectable, suggesting that it may not occur at these levels of
    excretion (Barnett, 1979). Information was also submitted on the
    results of urinary monitoring of workers engaged in chlordimeform
    application to cotton in seven countries (Limmer, 1985). As the
    temporal relationship between exposure and urine sampling was not
    established, it was not possible to relate this information to actual
    levels of exposure.

         In the period 1980-1984, four separate incidents resulting in 7
    cases of frank haematuria following industrial exposure occurred in
    the USA. Chemical cystitis, confirmed by cystoscopy and biopsy, was
    diagnosed in one case while non-specific bladder mucosal lesions was
    found in another. Six cases required hospitalisation but all resolved
    after cessation of exposure (Barnett, 1985).

         In the same period, no cases of chlordimeform-induced haematuria
    occurred at manufacturing plants in Switzerland and West Germany or
    formulation plants in Australia, Columbia, Central America, Mexico and
    the USA. No cases of haematuria reportedly resulted from application
    or use of chlordimeform in the field. (Reckefus and Kossman, 1985;
    Pfister and Dubach, 1985).

         Twenty-one cases of haemorrhagic cystitis have been reported in
    workers exposed to chlordimeform wastes and in those drinking
    contaminated water (Morgan and Gillen, 1982).

    COMMENTS

         Chlordimeform has been found to induce haemangiosarcomas in mice
    but not in rats (JMPR, 1980; Annex 1, FAO/WHO, 1981a). 4-Chloro-2-
    toluidine (previously termed 4-chloro-O-toluidine), a metabolite of
    chlordimeform, also produces haemangiosarcomas in mice but not in rats
    (IARC, 1983).

         Data reviewed by the present meeting indicated that chlordimeform
    induced the activity of several hepatic drug metabolising enzymes but
    the observed biological differences could not be attributed to the
    results of either in vivo or in vitro studies.

         Studies of macromolecular binding of 4-chloro-2-toluidine to RNA
    and DNA of rat and mouse tissues in vitro and in vivo also could
    not explain the observed differences in response to this compound, nor
    could a study of its effect on the incorporation of 3H-thymidine into
    capillary endothelial cells.

         Chlordimeform was without mutagenic activity in various cellular
    systems but gave positive results in a cell transformation assay. The
    metabolites 4-chloro-2-toluidine and N-formyl-4-chloro-2-toluidine
    were positive in several reverse mutation assays but only with
    metabolic activation; the former metabolite was also positive in mouse
    lymphoma, cell transformation and DNA repair assays. Both produced
    chromosomal anomalies in at least one in vivo assay.

         Occupational studies indicate that haematuria and cystitis have
    occurred following incidents of relatively high exposure. Exposed
    workers readily absorb chlordimeform and the excretion of 4-chloro-2-
    toluidine can be monitored.

         Data were submitted on the results of extensive urinary
    monitoring of chlordimeform applicators in seven countries. Results
    were variable but could not be related to exposure adequately, as
    information on the timing of exposure and sample collection was not
    provided. It is of interest to note that a daily excretion of
    1.5 litres of urine containing 0.5 mg/1 of 4-chloro-2-toluidine by a
    60 kg individual approximates to absorption of 0.017 mg/kg body weight
    of chlordimeform.

         Epidemiological data were not submitted.

         The Meeting was advised by the submitters of the data that
    chlordimeform is used only on cotton and that no other uses are
    anticipated. The Meeting recommended that the use of chlordimeform be
    restricted to cotton production.

         As the Meeting considered that the available information
    partially met the requirement of the 1980 Joint Meeting, it decided to
    extend the temporary ADI.

    TOXICOLOGICAL EVALUATION

    LEVEL CAUSING NO TOXICOLOGICAL EFFECT

         Rat: 2 ppm in the diet, equivalent to 0.1 mg/kg b.w.

         Dog: 250 ppm in the diet, equivalent to 6.25 mg/kg b.w.

    ESTIMATE OF TEMPORARY ACCEPTABLE DAILY INTAKE FOR MAN

         0.0001 mg/kg b.w.

    REQUIRED (by 1987)

         Interpretable epidemiological and urinary monitoring data on
    occupationally-exposed workers.

    DESIRED

    1.   Confirmatory long-term animal bioassay using a third species
         for evaluating the potential carcinogenic hazard.

    2.   Further observations in man.

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    See Also:
       Toxicological Abbreviations
       Chlordimeform (EHC 199, 1998)
       Chlordimeform (ICSC)
       Chlordimeform (WHO Pesticide Residues Series 1)
       Chlordimeform (WHO Pesticide Residues Series 5)
       Chlordimeform (Pesticide residues in food: 1978 evaluations)
       Chlordimeform (Pesticide residues in food: 1979 evaluations)
       Chlordimeform (Pesticide residues in food: 1980 evaluations)
       Chlordimeform (Pesticide residues in food: 1987 evaluations Part II Toxicology)
       Chlordimeform (IARC Summary & Evaluation, Volume 30, 1983)