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    PESTICIDE RESIDUES IN FOOD - 1997


    Sponsored jointly by FAO and WHO
    with the support of the International Programme
    on Chemical Safety (IPCS)




    TOXICOLOGICAL AND ENVIRONMENTAL
    EVALUATIONS 1994




    Joint meeting of the
    FAO Panel of Experts on Pesticide Residues
    in Food and the Environment
    and the
    WHO Core Assessment Group 

    Lyon 22 September - 1 October 1997



    The summaries and evaluations contained in this book are, in most
    cases, based on unpublished proprietary data submitted for the purpose
    of the JMPR assessment. A registration authority should not grant a
    registration on the basis of an evaluation unless it has first
    received authorization for such use from the owner who submitted the
    data for JMPR review or has received the data on which the summaries
    are based, either from the owner of the data or from a second party
    that has obtained permission from the owner of the data for this
    purpose.



    TRIFORINE

    First draft prepared by
    D.B. McGregor,
    International Agency for Research on Cancer, Lyon, France

         Explanation
         Evaluation for acceptable daily intake
              Biochemical aspects
                   Absorption, distribution, and excretion
                   Metabolism
                   Effects on enzymes
              Toxicological studies
                   Acute toxicity
                   Short-term toxicity
                   Long-term toxicity and carcinogenicity
                   Genotoxicity
                   Reproductive toxicity
                        Multigeneration reproductive toxicity
                        Developmental toxicity
                   Special studies
                        Dermal and ocular irritation and dermal
                        sensitization 
                        Mode of action
                        Interactions with nitroso compounds
                        Haemolytic anaemia
                   Observations in humans
              Comments
              Toxicological evaluation
              References

    Explanation

    Triforine, a fungicide, was evaluated toxicologically by the 1977 JMPR
    (Annex 1, reference 28), when no ADI was allocated, and again in 1978
    (Annex I, reference 30), when more toxicological data were made
    available and an ADI of 0-0.02 mg/kg bw was established. Subsequently,
    further data have been provided, which were reviewed by the present
    Meeting within the CCPR periodic review programme.

    Evaluation for acceptable daily intake

    1.  Biochemical aspects

     (a)  Absorption, distribution, and excretion

    Triforine labelled with either 3H in the piperazine ring or with 14C
    in the side chains was administered orally to male FW-49 rats at doses
    of 9-200 mg/kg bw (see Table 1) in studies that allowed some aspects
    of their metabolism and disposition to be compared. Kinetic studies by
    intravenous administration were impractical because of the low
    solubility of triforine in water.


        Table 1. Experiments with labelled triforine

                                                                                                                   

    No.     Experiment                        Label              Dose                                 No. of
                                                                                                      rats
                                                                 Triforine           Radioactivity
                                                                 (mg/kg bw)          (µCi/rat)
                                                                                                                   

    1       Blood level                       3H ring            11.5                40.8             9 M
    2a      Urinary and faecal excretion      3H ring            11.5                36.4             10 M
    2b                                        14C side-chain     15.0                38.0             10 M
    3a      Biliary excretion                 3H ring            9.0                 22.5             4 M
    3b                                        14C side-chain     9.0                 38.5             2 M
    4a      Urinary and faecal excretion      3H ring            25, 50, 100, 200    9.2              8 M
    4b                                        14C side-chain     50, 100             3.2              4 M
    5       Urinary, pulmonary and faecal     14C ring           10                                   2 M + 2 F
            excretion, pilot
    6       Urinary, pulmonary and faecal     14C side-chain     10                                   5 M + 5 F
            excretion
    7       Urinary, pulmonary and faecal     14C side-chain     1000                                 5 M + 5 F
            excretion
    8       Urinary, pulmonary and faecal     14C side-chain     10 × 15 daysa                        5 M + 5 F
            excretion
                                                                                                                   
    From Darda (1977); Hawkins et al. (1992)
    a 10 × 14 days unlabelled followed by 10 × 1 day labelled
    

    After dosing with [piperazine-3H]-triforine (experiment 1), the
    maximal blood concentrations of radioactivity were found after 4 h and
    represented 1.3% of the dose. After 96 h, 0.3% of the dose remained in
    blood. In experiment 2, 74% of the tritium was eliminated in the urine
    and 17% in the faeces within 24 h. During the subsequent four days,
    only small amounts were excreted: 3.2% in urine and 1.2% in faeces. A
    total of 1.9% of the administered radiolabel was excreted in the urine
    as tritiated water within six days. After dosing with 14C-triforine
    (experiment 2b), 53% of the dose was eliminated in urine and 39% in
    faeces within the first 24 h. There was little further excretion over
    next 24-72 h. The different patterns of excretion of radiolabel after
    dosing with the two forms of triforine may indicate that metabolism
    occurs at the side chains; however, see the results of experiments 5,
    6, and 7, below. After treatment with higher doses of triforine
    (experiments 4a and 4b), the excretion of radiolabel during the first
    48 h was comparable to that at lower doses, urinary excretion
    accounting for a mean of 73 % of the tritium and 55% of the 14C
    label. Biliary excretion (experiments 3a and 3b) indicated that
    maximum excretion (2.4% of the dose) occurred at 3 h and that a mean
    of 19% was excreted over the first 30 h. Analysis by thin-layer
    chromatography showed that the biliary residue after administration of
    the 14C label consisted of four main components, whereas with the H
    label only two of these components were present, corresponding to the
    two major urinary metabolites, one of which was identified as
    N-[2,2,2-trichloro-1-(piperazin-1-yl)ethyl]formamide (Darda, 1977).
    Its occurrence confirms the metabolism of triforine by elimination of
    one side chain (Boehringer Sohn, 1974a, b).

    The absorption and disposition of 14C-triforine suspended in 5%
    aqueous sodium carboxymethylcellulose was studied in male and female
    CD Sprague-Dawley-derived rats at doses of 10 mg/kg bw, as a single
    dose and after treatment with unlabelled triforine for 14 days, and
    1000 mg/kg bw as a single radiolabelled dose (experiment 5). Both
    side-chain and ring-labelled 14C-triforine were available, but
    piperazine ring-labelled 14C-triforine was used only in a pilot
    experiment in which a single dose of 10 mg/kg bw was administered to
    two male and two female rats. In this experiment, the mean proportions
    of the administered dose excreted during 120 h were: urine, 77% in
    males and 82% in females; faeces, 18% in males and 19% in females;
    expired air, 3.3% in males and 1.5% in females; less than 3% remained
    in the carcass. Most of the radiolabel (73% in males and 76% in
    females) was excreted in the urine within 0-24 h.

    In the main study with side-chain-labelled 14C-triforine, in which
    single doses of 10 mg/kg bw were given to five male and five female
    rats (experiment 6), the mean proportions of the administered dose
    excreted during 120 h were: urine, 78% in males and 79% in females;
    faeces, 12% in males and 14% in females; expired air, 5.2% in males
    and 6.0% in females. Less than 3% remained in the carcass. Most of the
    radiolabel (75% in males and females) was excreted in the urine within
    0-24 h.

    The excretion profile was radically different in experiment 7, in
    which side-chain-labelled 14C-triforine was administered at a single
    dose of 1000 mg/kg bw. The mean proportions of the administered dose
    excreted during 120 h were: urine, 11% in males and 19% in females;
    faeces, 85% in males and 77% in females; expired air, 0.9% in males
    and 1.6% in females. Only about 0.5% remained in the carcass. Most of
    the urinary radiolabel (7.7% in males and 12% in females) was excreted
    within 6-48 h. The delayed urinary excretion (in comparison with
    experiment 6) probably reflects absorption limited by the dissolution
    rate. More than 90% of the radiolabel recovered from the faeces over
    0-72 h was associated with side-chain-labelled 14C-triforine and
    presumably represented unabsorbed material.

    The effect of repeated low doses on the excretion of
    side-chain-labelled 14C-triforine was studied in experiment 8. The
    mean proportions of the administered dose excreted during 120 h were:
    urine, 71% in males and 74% in females; faeces, 17% in males and 15%
    in females; expired air, 6.3% in males and 6.8% in females. About 2%
    remained in the carcass. Most of the radiolabel (68% in males and 69%
    in females) was excreted in the urine within 0-24 h. Thus, in
    comparison with experiments 5 and 6, neither the position of the label
    nor the dosing schedule significantly affected the overall pattern of
    14C excretion The experiments also show no important sex-specific
    differences. Seven days after administration, only low residual
    quantities of triforine were found in the tissues; blood, liver,
    kidney, and skin contained the largest amount of the administered dose
    (about 2.4%).

    These experiments indicate that > 80% of a daily dose of 10 mg/kg bw
    triforine is absorbed by male and female rats, whereas after a single
    dose of 1000 mg/kg bw absorption was about 10% in males and 20% in
    females.

     (b)  Metabolism

    Triforine is rapidly metabolized and excreted in rats; unchanged
    compound accounts for only 0-5 % of the dose (Hawkins et al., 1992).
    Substantial quantities of unchanged triforine were recovered only from
    faeces (Boehringer Sohn, 1974a,b).

    The first metabolite to be identified was  N-[2,2,2-trichloro-1-
    (piperazin-1-yl)ethyl]-formamide, which is formed by the cleavage of
    an entire side chain (Darda, 1977). In later metabolic studies with
    14C labelling in the piperazine ring and aliphatic side chain
    (Hawkins et al., 1992), triforine underwent virtually complete
    metabolism after administration as a single oral dose of 10 mg/kg bw.
     N-[2,2,2-Trichloro-1-(piperazin-1-yl)ethyl]formamide, the major
    radiolabelled urinary component in rats receiving [piperazine
    14C]-triforine, accounted for 46 53% of the dose over 0-24; however,
    in rats receiving side-chain-labelled 14C-triforine, the proportion
    was reduced to 24-27% after a single 10 mg/kg bw dose and 21-24% after
    repeated doses. It was excreted as the glucuronide. The side-chain

    metabolite trichloroethanol and its glucuronide represented 18-21% of
    the dose. Another side-chain metabolite occurring in the urine was the
     N-acetylcysteine conjugate of 2,2,2-trichloroethylamine, which
    represented 13-15% of the administered dose. In faeces collected from
    female rats over 0-48 h, 3.6% of the single 10 mg/kg bw dose and 3.4%
    of the repeated doses was present as  N-[2,2,2-trichloro-1-
    (piperazin-1-yl)ethyl]formamide. This metabolite was not detected in
    the faeces of rats receiving 1000 mg/kg bw. Very little unchanged
    triforine (0-1%) was detected in the faeces of rats given the low
    dose, whereas it represented 70-80% of the dose in rats given 1000
    mg/kg bw (Hawkins et al., 1992). This result suggests that absorption
    of triforine is a saturable process, unless there is extensive biliary
    excretion at the high dose.

    A scheme of the metabolic pathways of triforine is presented in Figure
    1.

     (c) Effects on enzymes

    The potential effects of triforine on hepatic xenobiotic metabolizing
    enzymes were studied by administering the compound for 28 days to six
    male and six female, 35-day-old Sprague-Dawley rats at a dietary
    concentration of 20 000 ppm, equal to 1957 mg/kg bw per day in males
    and 2094 mg/kg bw per day in females, and to eight male and eight
    female, 42-day-old CD-1 mice at a concentration of 7000 ppm, equal to
    1555 mg/kg bw per day in males and 1998 mg/kg bw per day in females.
    Control groups of equal size were included in the study; the positive
    controls received diets containing 500 ppm sodium phenobarbital.
    Relative liver weights were increased in male (23%) and female (26%)
    rats and male (12%) and female (16%) mice. There were no major
    differences between the relevant control and treated groups with
    regard to hepatic homogenate protein or DNA content or in
    cyanide-insensitive palmitoyl-CoA oxidation activity (as a measure of
    peroxisomal fatty-acid oxidizing enzyme activity and peroxisomal
    proliferation). Hepatic microsomal fractions were prepared to measure
    the activities of 7-ethoxyresorufin- O-deethylase (CYP1A subfamily
    marker), 7-pentoxyresorufin- O-depentylase (CYP2B subfamily
    marker),erythromycin- N-demethylase (CYP3A subfamily marker), and
    lauric acid 11- and 12-hydroxylases (CYP4A subfamily markers). The
    microsomal cytochrome P450 content was slightly reduced in male (14%)
    and female (13%) rats and slightly increased only in male (28%) mice
    fed triforine, 7-Ethoxyresorufin- O-deethylase activity was not
    affected in mice but was reduced in both male (54%) and female (60%)
    rats. 7-Pentoxy-resorufin- O-depentylase activity was not affected in
    any group, while erythromycin- N-demethylase activity was increased
    only in male rats (52%) and mice (51%). Lauric acid 12-hydroxylase
    activity was not affected in male rats or female mice, while there was
    a 36% reduction in female rats and a 30% increase in male mice
    (Robbins, 1994).

    FIGURE 1

    2.  Toxicological studies

     (a)  Acute toxicity

    The acute toxicity of triforine has been tested by administration by
    various routes in mice, rats, and dogs (Table 2). The only reported
    observations were reduced activity, swaying, or dyspnoea. There were
    no deaths, and no substance-induced changes in organs were detected
     post mortem.

     (b) Short-term toxicity

     Mice

    Four groups of five male and five female NMRI mice were given
    technical-grade triforine (purity, 98.1%) in the diet at
    concentrations of 0, 200, 1000, or 5000 ppm for four weeks, equal to
    0, 39, 200, or 980 mg/kg bw per day for males and 0, 45, 240, or 1300
    mg/kg bw per day for females. No deaths or clinical signs of toxicity
    were recorded, and there were no effects on food consumption; the
    apparently increased food consumption by females at 5000 ppm was
    possibly due to greater spillage. At the end of the study, males at
    5000 ppm had 8% less body-weight gain than controls. Haematological
    changes included decreased erythrocyte count (by 8-11%,  p < 0.05),
    haemoglobin concentration (by 7-9%, p < 0.05), and packed cell volume
    (by 6-8%  p < 0.05) and increased polychromatic erythrocytes in
    animals of each sex at 5000 ppm. Males at this dose also had
    moderately increased reticulocyte counts and decreased leukocyte
    counts. A significant decrease in mean erythrocyte count was also
    observed in males at 1000 ppm, although most of the values fell within
    the range for the concurrent control group. Males and females at 5000
    ppm showed increased absolute and relative weights of the spleen, and
    females had increased relative liver weights (by about 16%,
     p < 0.01). There were no gross or microscopic changes. The NOAEL
    was 1000 ppm, equal to 196 mg/kg bw per day, on the basis of
    haematological changes in animals of each sex, slightly reduced
    body-weight gain in males, and increased relative liver weight in
    females at 5000 ppm (Tenneker et al., 1988).

    Groups of 10 CD-1 mice of each sex were given technical-grade
    triforine (purity, 99.1%) in the diet at concentrations of 0 or 7000
    ppm for 13 weeks, equal to 1000-1600 mg/kg bw per day in males and
    1900-2500 mg/kg bw per day in females. No deaths or clinical signs of
    toxicity were recorded, and there were no effects on food consumption
    or body-weight gain. The haematological changes included a slight
    reduction in erythrocyte numbers, haemoglobin concentration, and
    haematocrit in animals of each sex. The absolute weights of the spleen
    were increased by 38% in males and 56% in females, and that of the
    liver was increased by 17% in males; the relative liver and spleen
    weights were increased in animals of each sex ( p < 0.01). There
    were no gross pathological findings; tissues were not examined
    microscopically, as the study was designed to select concentrations
    for use in a long-term study of toxicity and carcinogenicity (Atkinson
    et al., 1991a).


        Table 2  Acute toxicity of triforine

                                                                                                                            

    Species, strain               Route of             Sex        LD50L/C50      Length of      Reference
                                  administration                  (mg/kg bw      observation
                                                                  or mg/L)       (days)
                                                                                                                            

    Rat, Wistar                   Oral                 F, M       > 16 000       14             Frohberg et al. (1973)
    Rat, Wistar                   Oral                 F, M       > 5 000        15             Ullman et al. (1986a)
    Rat, Wistar                   Dermal               F, M       > 10 000       14             Frohberg et al. (1973)
    Rat, Wistar                   Dermal               F, M       > 2 000        15             Ulman et al. (1990)
    Rat, Sprague-Dawley           Inhalation (1 h)     F, M       > 4.5          14             Bullock & Narcisse (1973)
    Rat, Wistar                   Inhalation (4 h)     F, M       > 5.1          15             Ullman et al. (1986b)
    Mouse (strain not stated)     Oral                 F, M       > 6 000        7              Muacevic (1968)
    Mouse, NMRI                   Oral                 F, M       > 5 000        15             Ullman et al. (1986c)
    Dog (breed not stated)        Oral                 F, M       > 2 000        28             Muacevic (1969)
                                                                                                                            
    

     Rats

    Groups of five male and five female Wistar rats were given
    technical-grade triforine (purity, 98.1%) in the diet at
    concentrations of 0, 500, 2500, or 12 500 ppm for four weeks, equal to
    0, 50, 240, or 1200 mg/kg bw per day for males and 0, 49, 230, or 1200
    mg/kg bw per day for females. No deaths or clinical signs of toxicity
    were recorded, and there were no effects on food consumption.
    Body-weight gain was reduced by about 15% in males by the end of the
    study ( p < 0.05), whereas them were no consistent, treatment-
    related effects in females. Haematological changes indicative of
    slight anaemia were observed. In rats at 12 500 ppm, slight decreases
    in mean cell haemoglobin concentration ( p < 0.01) and mean cell
    volume were seen in females ( p < 0.05) and increases in
    reticulocyte ( p < 0.01) and polychromatic erythrocyte counts in
    animals of each sex. Increased proportions of circulating immature
    cells were also seen in rats at 2500 ppm, and the increase was
    significant in males ( p < 0.01). Prothrombin time was decreased in
    females at 12 500 ppm. At this dose, a slight increase in total
    protein (by about 6%,  p < 0.05) was seen in animals of each sex and
    a slight increase in cholesterol content (57%,  p < 0.01) in
    females. Urine volume was increased in females at this dose. Changes
    in organ weight were restricted to rats at 12 500 ppm. Absolute spleen
    weights were increased in animals of each sex ( p < 0.05), as were
    the absolute weights of the liver, thyroid, and kidney in females.
    Increased relative weights were observed for liver ( p < 0.01) and
    spleen ( p < 0.05) in animals of each sex and for thyroid in males
    ( p < 0.05). There were no treatment-related gross pathological
    findings; the treatment-related microscopic changes were slight or
    moderate haemosiderin deposition in the spleens of males and females
    at 12 500 ppm and females at 2500 ppm; two females at 500 ppm also had
    increased haemosiderin deposition. No NOAEL was identified (Tenneker
    et al., 1989).

    Groups of 15 male and 15 female Wistar FW-49 rats (25 at the high
    dose) were given triforine (purity not stated) in the diet at
    concentrations of 0, 2500, 7000, or 20 000 ppm for 13 weeks. Ten rats
    of each sex at 20 000 ppm were observed for six weeks after the end of
    treatment. Haematological, clinical chemical, and extended
    histopathological investigations were performed on 10 rats of each sex
    at each dose before treatment and after 6, 13, and 19 weeks.
    Reversible reductions in erythrocyte count and in haemoglobin and
    haematocrit values were seen in all treated groups. These results were
    interpreted as a sign of a slight haemolytic anaemia and correlated
    with haemosiderin deposition in liver, spleen, kidney, lung, and
    heart, which increased in proportion to the dose administered. No
    NOAEL was identified (Stötzer et al., 1971a).

    As there was no no-effect level in the previous study, another study
    was performed in which groups of 25 male and 25 female Wistar FW-49
    rats were given triforine (purity not stated) in the diet at
    concentrations of 0, 100, or 500 ppm for 13 weeks, equivalent to 5 or
    25 mg/kg bw per day. No treatment-related effects were observed. The

    NOAEL was 500 ppm, equivalent to 25 mg/kg bw per day, as no effects
    were observed at 500 ppm, the highest dose tested (Stttzer et al.,
    1971b).

    Groups of 10 male and 10 female Sprague-Dawley rats were given
    technical-grade triforine (purity, 99.1%) in the diet at
    concentrations of 0 or 20 000 ppm for 13 weeks, equal to 1300 mg/kg bw
    per day. No deaths or clinical signs of toxicity were recorded, and
    there were no effects on food consumption or body-weight gain. The
    haematological changes included statistically significant but slight
    reductions in mean cell haemoglobin concentration, by 1.4% in males
    and 2.2% in females, and reduced erythrocyte numbers (by 4-9%) and
    increased mean cell volume (by 3.5%) in females. Increases in the
    absolute ( p < 0.05) and relative ( p < 0.01) weights of the liver
    and spleen were observed in animals of each sex. There were no gross
    pathological findings; the tissues were not examined microscopically
    (Atkinson et al., 1991b).

    Groups of 10 male and 10 female FW-49 rats were given triforine
    (purity not stated) in the diet at concentrations of 0, 25, 120, 620,
    or 3100 ppm for six months, equivalent to 0, 1.3, 6, 31, or 160 mg/kg
    bw per day. Reductions in erythrocyte and haematocrit values and
    increases in reticulocyte numbers were observed in rats at 620 and
    3100 ppm. No consistent, dose-related variations were found in serum
    chemical or urinary parameters. Post-mortem examination did not reveal
    any treatment-related changes, but increased liver weights were found
    in females at all doses, the relative weights being increased by 27%
    in those at 25 ppm, 19% at 120 ppm, 40% at 620 ppm, and 49% at 3100
    ppm (no statistical analysis reported). No such increases were
    observed in males. Increased haemosiderin deposition was found in the
    livers and spleens of male rats at 620 and 3100 ppm and females at
    3100 ppm. No treatment-related histopathological changes were seen in
    the livers of either male or female rats at 25 or 120 ppm. The NOAEL
    was 120 ppm, equivalent to 6 mg/kg bw per day, on the basis of reduced
    erythrocyte numbers and packed cell volume at 620 ppm (Stötzer et al.,
    1972).

    Groups of 10 male and 10 female Sprague-Dawley rats were given topical
    applications on shaved intact, occluded skin of a 0, 0.5, or 1.5%
    aqueous dilution of a 20% triforine emulsion, equivalent to 0, 10, or
    30 mg/kg bw triforine, for 4 h per day daily for 21 days. Five males
    and five females per group were followed up for an additional 21 days.
    Temporary slight reddening and swelling occurred in the covered skin
    areas in all groups, including the controls. No local or systemic
    substance-related reactions were observed (Leuschner et al., 1972).

    Groups of seven Fischer 344 rats of each sex received technical-grade
    triforine in corn oil (3 ml/kg bw) on a shaven area of the back at
    doses of 0, 110, 350, or 1100 mg/kg bw per day on five days per week
    for three weeks. After each application, the treated area was covered
    with gauze and bandage for 6 h, then washed and dried. No deaths or
    treatment-related dermal changes were observed, and there were no
    effects on food consumption or body-weight gain attributable to

    treatment. All animals, including the controls, lost some weight,
    particularly during the first week; this response was attributed to
    the bandaging procedure. No significant, treatment-related variations
    in organ weights or haematological end-points was seen, and them were
    no gross or microscopic pathological findings. Female rats at 350 or
    1100 mg/kg bw showed statistically significant increases in serum
    cholesterol (13% and 22%, respectively), triglycerides (32 and 40%,
    respectively), and bilimbin (27 and 13%, respectively); a 25% decrease
    in serum alkaline phosphatase activity was seen in females at 110
    mg/kg bw. In males, serum alkaline phosphatase activity was reduced by
    15% at 350 mg/kg bw and by 21% at 1 100 mg/kg bw; in rats at 1100
    mg/kg bw, total serum protein was increased by 5.7% and serum albumin
    by 3.8%. Since the changes in bilirubin, cholesterol, and triglyceride
    contents were not accompanied by histopathological changes in the
    liver and were confined to a single sex, their biological significance
    is unclear. The increases in alkaline phosphatase activity may reflect
    a toxicological effect, but the decreases observed in this study are
    not usually considered to be toxicologically significant. The NOAEL
    was 1100 mg/kg bw per day, the highest dose tested, as no toxicity was
    observed (Fokkema, 1992).

     Dogs

    Groups of four male and four female beagle dogs were given triforine
    (purity not stated) in the diet at concentrations of 0, 3500, 10 000,
    or 30 000 ppm (two groups) for 13 weeks. The supplementary group at 30
    000 ppm was observed for an additional six weeks without treatment.
    These concentrations were equal to 0, 83, 230, 690, or 710 mg/kg bw
    per day for males and 0, 85,240,730, or 720 mg/kg bw per day for
    females. Signs of haemolytic anaemia-reduced erythrocyte counts 
    ( p < 0.05) and haemoglobin concentration ( p < 0.05), and,
    occasionally, also in haemocrit--were observed at all doses by week
    13, but were first observed in week 6 in dogs at 10 000 or 30 000 ppm
    ( p < 0.01). These effects were accompanied by a consistent increase
    in the number of reticulocytes in dogs at 10 000 and 30 000 ppm. All
    of the values returned to normal in the group at 30 000 ppm within
    three weeks of observation. Serum chemistry in weeks 6 and 13 showed
    slight increases in alkaline phosphatase activity and bilirubin and
    cholersterol concentrations in all treated groups, but these values
    also returned to normal in dogs at 30 000 ppm within three weeks.
    Urinalysis and ophthalmoscopy showed no differences between the
    groups. Fine, drop-like fatty infiltration of the myocardial fibres
    were seen in 0, 5, 1, 5, and 1 dogs in the five groups, respectively,
    and fatty accumulation in the liver in 0, 3, 0, 4, and 0 dogs. The
    fatty infiltration appeared to be reversible, since it was no longer
    detected in the dogs observed for six weeks. Treatment-related
    siderosis in Kupffer cells in the liver showed a clear tendency
    towards reversibility in the animals allowed to recover. There was no
    NOAEL (Stötzer et al., 1971c).

    Groups of four beagle dogs of each sex were given triforine in the
    diet at concentrations of 0, 100,600, or 3500 ppm for 13 weeks, equal
    to 0, 3.6, 22, or 120 mg/kg bw per day for males and females together.
    The haematological findings in the dogs at 3500 ppm in the previous
    study were essentially confirmed. Furthermore, siderosis was detected
    in the spleen, liver, and bone marrow after administration of 600 ppm,
    and the weight of the spleen was increased in the dogs at 3500 ppm.
    The NOAEL was 100 ppm, equal to 3.6 mg/kg, on the basis of
    haemosiderin deposits in the liver and bone marrow at 600 ppm
    (Leuschner et al., 1971).

    Groups of four male and four female beagle dogs were given
    technical-grade triforine (purity not stated) in the diet at
    concentrations of 0, 10, 40, 100, or 1000 ppm for two years, equal to
    0, 0.23, 0.93, 2.4, or 22 mg/kg bw per day for males and 0, 0.25,
    0.99, 2.6, or 24 mg/kg bw per day for females. Samples were taken for
    haematology, blood chemistry, and urinalysis during weeks 6, 13, 26,
    52, 78, and 104. One male dog at 100 ppm group died of acute
    pneumonia. There were no other deaths, and there were no
    treatment-related signs of toxicity, changes in food consumption,
    changes in body-weight gain, or ophthalmoscopic findings.
    Haematological changes in the dogs at 1000 ppm group included
    increased mean cell volume in males in week 13 (12%,  p < 0.01) and
    females in week 26 (3.5%,  p < 0.05) and decreased mean cell
    haemoglobin concentration in males at week t 3 (3.5 %, p < 0.01).
    The other changes were either not statistically significant or were
    inconsistent with respect to time interval, sex, or dose. Examination
    of femoral bone-marrow smears at termination showed a shift in the
    granulopoietic:erythropoietic ratio towards erythropoiesis in two
    males and three females at 1000 ppm. The erythropoietic mitotic index
    was also increased in one female in this group. No treatment-related
    changes were observed on blood chemistry or urinalysis. The absolute
    and relative organ weights were comparable in all groups, and there
    were no treatment-related gross pathological findings.
    Microscopically, haemosiderin deposition of moderate severity was
    observed in Kupffer cells in four dogs at 1000 ppm and one control.
    Haemosiderosis of the bone marrow was also observed in two dogs at
    1000 ppm. The NOAEL was 100 ppm, equal to 2.4 mg/kg bw per day, on the
    basis of haematological changes, increased erythropoiesis, and
    haemosiderin deposition in the liver and bone marrow in animals of
    each sex at 1000 ppm (von Sandersleben et al., 1974; Goburdhun &
    Greenough, 1990; Greenough, 1994).

     (c)  Long-term toxicity and carcinogenicity

     Mice

    Groups of 40 NMRI mice of each sex were given triforine (purity not
    stated) in the diet at concentrations of 0, 30, 150, or 750 ppm for 81
    weeks, equal to 0, 4.7, 24, or 120 mg/kg bw for males and 0, 5.2, 28,
    and 140 mg/kg bw for females. Mortality at 81 weeks was 32% of control
    males, 20% of those at 30 ppm, 35% at 150 ppm, and 50% at 750 ppm, and
    30% of control females, 40% of those at 30 ppm, 26% at 150 ppm, and

    33% at 750 ppm. Survival time, clinical symptoms, body-weight gain,
    and food consumption were not affected by treatment. As morphological
    findings were described only if they were considered to be relevant to
    an evaluation of the carcinogenic effects of the substance, no NOAEL
    was identified. Treatment did not increase the total number of
    neoplasms (mainly lymphocytic leukaemias and lymphosarcomas), and the
    latent periods remained unchanged. The frequencies of specific, less
    common tumours in the treated groups were usually not higher than in
    the concurrent controls or in the literature (Hofmann et al., 1975).

    Four groups of 50 CD-1 mice of each sex were given technical-grade
    triforine (purity, 98.9%) in the diet at concentrations of 0, 70, 700,
    or 7000 ppm for 105 weeks, equal to 0, 11, 120, or 1200 mg/kg bw per
    day for males and 0, 16, 160, or 1600 mg/kg per day for females. Blood
    samples were collected for analysis at weeks 52, 77, and 103. The
    mortality at 105 weeks was 14% of control males, 19% of those at 70
    ppm, 35% at 700 ppm, and 28% at 7000 ppm; for females, 27% of controls
    were dead at this time and 27% of those at 70 ppm, 32% at 700 ppm, and
    30% at 7000 ppm. The mortality rate in the control group of males was
    considered to be unusually low for CD-1 mice at 105 weeks. There were
    no treatment-related clinical signs of toxicity or changes in food
    consumption. Body-weight gain at the end of the study was reduced by
    11% in males at 700 ppm and 16% in those at 7000 ppm. No significant
    changes in body-weight gain were seen in females. No haematological
    changes were observed. The absolute and relative weights of the liver
    were increased by 21% ( p < 0.01) in females at 7000 ppm. At
    autopsy, thickening or enlargement of the large intestine was observed
    in males that died before the end of the study in the groups receiving
    700 ppm (17%) or 7000 ppm (36%), whereas none was seen in the
    controls. The large intestine was ulcerated, and inflammation was
    observed microscopically, predominantly in male mice that died before
    the end of the study after receiving 700 ppm (23%) or 7000 ppm (21%).
    The overall occurrence of these findings was none in male controls, 6%
    at 70 ppm, 16% at 700 ppm, and 12% at 7000 ppm.

    The incidences of hepatocellular adenoma were 9/50 in control males,
    12/50 at 70 ppm, 8/50 at 700 ppm, and 8/50 at 7000 ppm; the incidences
    of hepatocellular carcinoma were 5/50 in control males, 7/50 at 70
    ppm, 8/50 at 700 ppm, and 10/50 at 7000 ppm. There were no significant
    differences by either Fisher's exact test or the Cochrane-Armitage
    test for trend, for adenomas or carcinomas independently or combined
    (Fisher's exact test for comparison of carcinomas in males at 0 and
    7000 ppm gave  p = 0.263). All of the incidences of liver tumours
    were within the ranges found in a compilation of five control groups
    of male CD-1 mice in the same laboratory: adenomas, 0-32%; carcinomas,
    0-21%. Females had no increase in the incidences of liver tumours. The
    incidences of alveolar or bronchiolar adenomas in males were 14/50 in
    controls, 13/50 at 70 ppm, 8/50 at 700 ppm, and 17/50 at 7000 ppm, and
    the incidences of alveolar or bronchiolar carcinomas were 5/50 in
    controls, 2/50 at 70 ppm, 4/50 at 700 ppm, and 7/50 at 7000 ppm. These
    differences were not statistically significant. The incidences of
    alveolar or bronchiolar adenomas in females were 5/50 in controls,
    6/50 at 70 ppm, 7/50 at 700 ppm, and 21/50 at 7000 ppm (Fisher's exact

    test,  p < 0.001), and the incidences of alveolar or bronchiolar
    carcinomas were 1/50 in controls, 2/50 at 70 ppm, 1/50 at 700 ppm, and
    6/49 at 7000 ppm (Fisher's exact test,  p = 0.059). The incidences of
    lung tumours were beyond the ranges observed in five other control
    groups of female CD- 1 mice at this laboratory: adenomas, 4-18%;
    carcinomas, 0-8% The Meeting concluded that triforine increased the
    incidence of alveolar or bronchiolar adenomas fit female mice. The
    NOAEL for carcinogenicity was 700 ppm, equal to 160 mg/kg bw per day,
    on the basis of an increased incidence of adenomas and/or carcinomas
    in females at 7000 ppm. The NOAEL for toxicity was 70 ppm, equal to 11
    mg/kg bw per day, on the basis of a slight decrease in body-weight
    gain and changes in the large intestine of males fed 700 ppm (Heath et
    al., 1992).

     Rats

    Groups of 35 Wistar (CHBB: THOM-SPF) rats of each sex, with 50 of each
    sex in the group of controls and that at the high dose, were given
    triforine (purity, 96.6%) in the diet at concentrations of 0, 25,
    125,625, or 3120 ppm for two years, equivalent to 0, 1.3, 6.3, 31, or
    160 mg/kg per day. Samples were taken for haematological and blood
    chemical investigations during weeks 0, 6, 13, 26, 52, 78, and 104
    from 20 male and female rats per group. Urine was analysed before and
    at the end of the study in 10 animals of each sex from the control
    group and that at the highest dose. All animals were examined  post 
     mortem. Complete histopathological examinations were performed at
    the end of the study on 20 rats of each sex from the control group and
    that at the highest dose, on 15 rats of each sex from the other
    groups, on rats that died before the end of the study, and on all
    tumour-bearing rats. Mortality, clinical signs, body-weight
    development, and parameters of clinical chemisstry and the urinalyses
    were not affected by the treatment. Temporary reductions in the number
    of erythrocytes ( p < 0.01 for males) and haematocrit values
    ( p< 0.01 for males) and increased numbers of mticulocytes (39% in
    males, 29% in females) were seen in rots at 3125 ppm during week 6,
    the variations being more pronounced among male rats. There were no
    significant differences in organ weights. Haemosiderosis was observed
    in the spleens of both control and treated rats, and the frequency and
    intensity of the condition did not differ significantly between the
    groups. The sinuses of the adrenals of females in all groups showed
    cavernous dilatation, and some were thrombosed. These changes occurred
    more frequently in treated than in control animals, but they are
    common findings in Wistar rats. No significant difference in tumour
    incidence between the groups was found. The NOAEL was 625 ppm,
    equivalent to 31 mg/kg bw per day, on the basis of signs of anaemia at
    3120 ppm (Hill, 1974).

    Groups of 70 Sprague-Dawley rats of each sex were given
    technical-grade triforine (purity, 99.1%) in the diet at
    concentrations of 0, 200, 2000, or 20 000 ppm for two years, equal to
    an average of 0, 10, 100, or 1000 mg/kg bw per day for males and 0,
    13, 140, or 1400 mg/kg bw per day for females. Twenty rats of each sex
    per group were killed at one year and the survivors at two years.

    Blood and urine samples were collected for analysis at weeks 26
    (females) or 28 (males), 52, and 104. Mortality at two years among the
    remaining 50 animals in each group was 25% of control males, 24% of
    those at 200 ppm, 19% at 2000 ppm, and 19% at 20 000 ppm and 22% of
    control females, 20% of those at 200 ppm, 21% of those at 2000 ppm,,
    and 23% of those at 20 000 ppm. There were no treatment-related
    clinical signs of toxicity and no changes on ophthalmoscopy or
    urinalysis. There were no significant differences in food or water
    consumption and only slight differences in body weight between the
    groups at the end of the study.

    A number of findings could be attributed to the treatment with
    triforine. At one year, the body weights of females were moderately
    but not statistically significantly lower than those of controls, by
    11% in those at 200 ppm, 13% at 2000 ppm, and 21% at 20 000 ppm.
    Haemoglobin concentrations were 4-6% lower in male rats at 2000 ppm
    ( p < 0.05) and 20 000 ppm ( p < 0.01 ) at weeks 28 and 51 and in
    those at 200 ppm at week 51. Although the changes were consistent in
    males, in female rats the haemoglobin concentrations were slightly and
    nonsignificantly lower only at 20 000 ppm at week 26. The mean
    corpuscular haemoglobin count was 2% lower in both male and female
    rats at 20 000 ppm at weeks 28 and 26, respectively, but not at week
    51. The values in the control group were, however, clearly higher than
    normally expected for rats of this age, sex, and strain in this
    laboratory. Changes in blood chemistry in the group at 20 000 ppm
    included slightly raised concentrations of sodium in males at week 28,
    of calcium in males at week 28 and females at week 104 (by 4%,
     p < 0.01 ), of cholesterol in females at week 104 (by 39%,
     p < 0.01), and of total protein in males at week 104 (by 9%,
     p < 0.01 ), and a slightly lower concentration of glucose in males
    at week 52. females at 2000 ppm also had raised calcium levels at week
    104. There were no gross pathological findings. Liver weights were
    increased at week 52 in males (11%) and females (15%) at 20 000 ppm
    and in all females at week 104; both absolute and relative liver
    weights were increased in females at week 104, by 18% in those at 200
    ppm ( p < 0.05), 24% at 2000 ppm ( p < 0.01), and 43 % at 20 000
    ppm ( p < 0.001 ). Although not significant, the relative weights of
    the liver were increased in males, by 19% at 200 ppm, 11% at 2000 ppm,
    and 18% at 20 000 ppm. The absence of a dose-related response in the
    males calls into question the biological significance of the response
    in females at the low dose. Significant, dose-related increases in
    absolute and relative spleen weights were seen in females at 2000 and
    20 000 ppm that were killed at one year. The relative spleen weights
    were also increased in females at 20 000 ppm at week 104. The
    increased weights of the liver and spleen correlated with
    histopathological findings (described below) and are considered to be
    related to treatment. Increased kidney weights were observed at week
    52, but only in females, by 9% in those at 200 ppm, 12% at 2000 ppm,
    and 17% at 20 000 ppm. Adrenal weights were reduced by 13-15% in all
    treated males but were increased by 35% in females at 2000 ppm and by
    40% in those at 20 000 ppm. Microscopic examination showed increased
    deposition of haemosiderin in the spleen of females at 2000 and 20 000
    ppm at two years, in males at these doses at one year, and in females

    at 20 000 ppm at one year. The incidence of haemosiderin deposition in
    Kupffer cells and macrophages of the liver was increased in male and
    female rats at 2000 and 20 000 ppm at one year. The severity was also
    increased in these groups of males but only in females at 2000 ppm. In
    the latter group, there was an increased incidence of pale-cell foci
    in males and bile-duct hyperplasia in females. There were no
    treatment-related changes in the proportions of tumour-bearing animals
    or in the incidence of any particular tumour type. The Meeting
    concluded that triforine is not carcinogenic to rats. The NOAEL was
    200 ppm, equal to 10 mg/kg bw per day, on the basis of slight
    reductions in body-weight gain in males, haematological changes in
    animals of each sex, increased absolute and relative weights of the
    spleen in females at one year and of the liver at two years, and
    haemosiderin deposition in the spleens of males and females at one
    year and in the livers of females after one year at 2000 ppm (Everett
    et al., 1992; Perry et at., 1992).

     (d)  Genotoxicity

    Triforine has been tested for genetic damaging activity in an adequate
    range of assays, most of which were conducted to currently acceptable
    standards (see Table 3 for salient features and references). Mutations
    were not induced by triforine in either bacteria or at the  hprt 
    locus of cultured mammalian cell lines, and excision repairable DNA
    damage was not increased in treated primary cultures of rat
    hepatocytes. Gene conversion was not induced in either yeast or fungal
    cells. The potential of triforine for inducing chromosomal damage was
    tested in mammalian cell lines  in vitro by examining treated
    metaphase cells for gross aberrations and  in vivo by examining
    polychromatic erythrocytes taken from the bone marrow of mice treated
    orally for an increase in the proportion of micronucleated cells.
    There was no indication of activity  in vitro. In one test, an
    increase in the proportion of micronucleated cells was observed in
    groups of five female mice sampled 48 h after dosing (controls: 0.4 ±
    0.55/103; treated with 5000 µg/kg bw: 2.4 ± 1.14/103) but not at 24
    or 72 h and not in male mice at any sampling time. The increase was
    not confirmed when the study was partially repeated with female mice
    sampled only 48 h after dosing (controls: 1.6 ± 1.14/103; treated with
    5000 µg/kg bw: 1.2 ± 1.10/103). The Meeting concluded that triforine
    is not genotoxic.

     (e)  Reproductive toxicity

     (i)  Multigeneration reproductive toxicity

     Rats

    Groups of 10 male and 20 female CHBB-THOM rats were given triforine
    (purity not stated) in the diet at concentrations of 0, 100, 500, or
    2500 ppm in a three-generation study in which the rats were mated to
    produce two litters in each generation. The only sign of toxicity in
    parent rats and their offspring was temporarily, slightly reduced
    body-weight gain of male rats, particularly at the high dose.


        Table 3. Genetic activity of triforine

                                                                                                                    

    Test system          Test object           Concentration/    Purity    Results        References
                                               dose              (%)
                                                                                                                    

    In vitro
    Reverse mutation     S. typhimurium        100 µg/plate      NR        Negativea      Rohrborn (1977)
                         TA 100, TA1535

    Reverse mutation     S. typhimurium        5000 µg/plate     NR        Negativea      Moriya et al. (1983)
                         TA98, TA 100,
                         TA1535, TA1537,
                         TA1538, E. coli
                         WP2hcr

    Reverse mutation     S. typhimurium        5000 µg/plate     99.9      Negativea      Kramer (1985)
                         TA98, TA 100,
                         TA1535, TA1537,
                         TA1538

    Gene conversion,     Saccharomycess        1000 µg/ml        NR        Negativea      De Bertoldi et al. (1980)
    adc2, trp5 loci      cerevisiae D4

    Gene conversion,     Aspergillus           1000 µg/ml        NR        Negativeb      De Bertoldi et al. (1980)
    pabaA locus          nidulans

    Unscheduled          Rat hepatocyte        63 µg/ml          99.8      Negativeb      Proudlock et al. (1993)
    DNA synthesis        primary culture

    Cell mutation,       Chinese hamster       50 µg/ml          NR        Negativea      Miltenburger (1984)
    hprt locus           V79 cells

    Cell mutation,       Chinese hamster       200 µg/ml         99.8      Negativea      Adams et al. (1993)
    hprt locus           ovary cells

    Table 3. (continued)

                                                                                                                    

    Test system          Test object           Concentration/    Purity    Results        References
                                               dose              (%)
                                                                                                                    

    Chromosomal          Chinese hamster       400 µg/ml         99.8      Negativea      Brooks & Wiggins (1994)
    aberration           ovary cells (24 and
                         48 h sampling)

    Chromosomal          Chinese hamster       50 µg/ml          NR        Negativea      Miltenburger (1985)
    aberration           V79 cells (7, 18,
                         and 28 h sampling)

    In vivo
    Micronucleus         Mouse bone            5000 mg/kg bw     98.8      Weakly         Guenard (1984a)
    induction            marrow (24, 48,       by gavage         positive
                         and 72 h sampling)

    Micronucleus         Mouse bone            5000 mg/kg bw     98.8      Negative       Guenard (1984b)
    induction            (females only,        by gavage
                         48 h sampling)
                                                                                                                    

    NR, not reported
    a With and without metabolic activation
    b Without metabolic activation
    

    Reproductive performance, duration of pregnancy, malformation
    frequency, and postnatal development were unaffected by treatment.
    When the F3b rats were subjected to post-mortem and histopathological
    examinations, no treatment-related change was observed. Since this
    study has been superseded by one at higher doses, a full description
    is not given; however, the dietary concentration of 2500 ppm had no
    adverse effect on reproduction (Niggeschulze et al., 1974).

    In a range-finding study, groups of 10 male and 10 female
    Sprague-Dawley rats were given technical-grade triforine (purity,
    99.1%) in the diet at concentrations of 0, 1250, 5000, or 20 000 ppm.
    The parental (FA) rats received the diets for 10 weeks before mating
    and throughout mating, gestataon, and lactation. These concentrations
    were equal to 0, 100, 410, or 1600 mg/kg bw per day for males and 0,
    100, 400, and 1600 mg/kg per day for females during the premating and
    mating periods; for F0 females, they were equal to 0, 97, 400, or
    1600 mg/kg bw per day during gestation and 0, 150, 650, or 2900 mg/kg
    bw per day during lactation. All F1 rats were weaned onto the same
    diets as their parents and continued on this treatment until they were
    six to seven weeks old, when they were killedś The only death occurred
    in a male rat in the control group. No signs of toxicity were noted.
    Food consumption was slightly reduced in the group at 20 000 ppm,
    among males during weeks 1-10 and among females during weeks 1-3.
    Body-weight gain deficits at these doses during these periods resulted
    in a reduction in body weight of about 11% in males throughout the
    experiment (17 weeks), whereas females had recovered by the end of the
    premating period. The relative liver weights of F0 males and females
    at 5000 and 20 000 ppm were increased, and females at these doses also
    had increased absolute weights of both liver and spleen. No gross
    pathological changes were found at autopsy of the F0 rats; no
    histological examinations were performed. Treatment did not affect
    mating performance, fertility, duration of gestation, litter size, or
    F1 pup survival, and no clinical signs of toxicity were seen among
    the F1 pups. From lactation day 14, the body-weight gain of male and
    female pups was reduced by 7% in those at 5000 ppm and 11 and 10%,
    respectively, at 20 000 ppm. These reductions persisted after weaning
    (day 24) and were present at six weeks in males and five weeks in
    females. The overall depression in mean weight gain after weaning was
    11% for males and 4% for females at 5000 ppm and 19% for males and 10%
    for females at 20 000 ppm. Food consumption after weaning was also
    reduced at this dose, by 15% in males and 12% in females. There were
    no gross pathological findings at autopsy of the F1 rats. The effects
    observed at the dietary concentration of 20 000 ppm triforine were
    considered not to contraindicate its use as the high dose in a
    two-generation study. The NOAEL for reproductive toxicity was 1000 ppm
    (McCay & Hazelden, 1990).

    Groups of 29 male and 28 female Sprague-Dawley rats were given
    technical-grade triforine (purity, 98.9-99.1%) in the diet at
    concentrations of 0, 500, 3000, or 20 000 ppm in a two-generation
    study. The parental (F0) rats received the diets for 10 weeks before
    mating and throughout mating, gestation, and lactation. At weaning of

    the F1 litters, pups were selected to provide the F2 generation
    litters and continued on the same diets as had been offered to their
    parents. The study was completed after weaning of the F2 rats. The
    dietary concentrations were equal to 0, 38, 230, or 1500 mg/kg bw per
    day for F0 males and 0, 48, 290, or 1900 mg/kg per day for F0
    females during the premating and mating periods. For F0 females,
    these concentrations were equal to 0, 41,240, or 1700 mg/kg bw per day
    during gestation and 0, 63, 380, or 2600 mg/kg bw per day during
    lactation. The dietary concentrations of 0,500, 3000, and 20 000 ppm
    were equal to 0, 40, 280, and 2000 mg/kg bw per day for F2 males and
    0, 61,360, and 2500 mg/kg, per day for F1 females during the
    premating and mating periods. For F1 females, these concentrations
    were equal to 0, 40,230, and 1800 mg/kg bw per day during gestation
    and 0, 65, 420, and 2900 mg/kg bw per day during lactation. Pups were
    not culled during the study.

    One F0 male and one F0 female at 500 ppm died during the study, and
    one F1 female at 20 000 ppm was killed prematurely because of
    dystocia. None of these deaths was related to treatment. No clinical
    signs of toxicity related to triforine were noted in either the F0 or
    F1 generations There were slight reductions in food consumption among
    F0 rats at 20 000 ppm and F0 females at 3000 ppm during the first
    week of treatment. A decrease of 9% was observed in the body-weight
    gain of F0 females, but not males, at 500 ppm during the premating
    period, at the beginning of which there had been a 3% body-weight gain
    in these rats. Slight deficits in body-weight gain were seen
    throughout the treatment period for F0 males at 20 000 ppm, resulting
    in an overall reduction in body-weight gain of 9%. During the
    premating period, F0 females show body-weight gain deficits of 19% at
    3000 ppm and 20% at 20 000 ppm. Overall weight gain was decreased by
    9% in F0 females at 20 000 ppm during gestation, whereas there was no
    deficit in these rats during, lactation. Food consumption was reduced
    among F1 males and females at 3000 ppm (10%) and 20 000 ppm (11 and
    12%, respectively), mostly during the first three weeks after weaning.
    Corresponding deficits in body-weight gain were seen during this
    period, of 9% in males and females at 3000 ppm and 13% in males and 9%
    in females at 20 000 ppm. Between weeks 6 and 23, the deficits in body
    weight were 5% for males and 10% for females at 20 000 ppm. There were
    no effects on the body-weight gain of F1 females at any dose during
    gestation or lactation.

    There were no gross pathological findings attributable to treatment.
    Kidney weights were significantly higher (5-9%), in F0 males and
    females at 3000 and 20 000 ppm and in F1 males at 20 000 ppm than in
    controls. Liver weights were significantly higher (11-45% and
    increasing with dose) in F0 females, F1 males, and F1 females at
    3000 and 20 000 ppm; a marginal increase in liver weight was also seen
    in F1 males at 500 ppm (6.4%,  p < (0.05). Significant increases
    were observed in the weight of the spleen in F1 males at 20 000 ppm
    (16%) and F1 females at 3000 (20%) and 20 000 ppm (42%). Triforine
    treatment did not affect mating performance, fertility, duration of
    gestation, gestation index, postimplantation loss, litter size, or pup
    survival in either the F0 or F1 generation.

    The clinical signs of toxicity in pups that could be attributed to
    treatment included a reduction in the mean weights of F1 and F2 pups
    at 20 000 ppm from day 4 of lactation, so that, by day 21, the body
    weights were reduced by 17% for F1 males, 18% for F1 females, 20%
    for F2 males, and 19% for F2 females. Decreases were also observed
    in F1 pups at 3000 ppm on days 14-21 of lactation, by 9% in males and
    8% in females.

    The liver, kidney, spleen, and thyroid of parental F0 and F1 rats at
    all doses and a number of other organs of parental F0 and F1 rats at
    0 and 20 000 ppm were examined histologically. Dose-related increases
    in haemosiderin deposition in the spleen, accompanied by dose-related
    increases in extramedullary haematopoeisis and in spleen weight were
    observed. There was also a dose-related increase in the severity of
    spontaneous nephropathy in males and nephrocalcinosis in females.
    Small increases in kidney weight were observed in this study,
    particularly in the F0 generation, but with no change at 500 ppm.
    Liver size increased with dose and was accompanied by sporadic
    centrilobular hepatocyte hypertrophy. The hepatic changes were
    regarded by the authors as an adaptive response to increased metabolic
    activity. The thyroids of female rats of both generations at 20 000
    ppm had the histological appearance of very active secreting glands,
    consisting of numerous very small acini with little or no lumina,
    scant stored secretion, and lined by cuboidal or columnar epithelium
    with pale, foamy cytoplasm. The severity of these effects did not
    appear to be increased in the F1 generation. The NOAEL for
    reproductive toxicity was 20 000 ppm, equal to 1500 mg/kg per day, the
    highest dose tested. The NOAEL for parental toxicity and growth and
    development of the offspring was 500 ppm, equal to 40 mg/kg bw per
    day, on the basis of decreased food consumption in F0 females, F1
    males, and F1 females, decreased body-weight gain in F0 females, F1
    males, and F1 females, decreased F1 pup weight, and increased
    relative spleen weight in F1 females at 3000 ppm (McCay & Hazelden,
    2992; Hazelden & Aitken, 1992; Hazelden, 1994).

     (ii)  Developmental toxicity

     Rats

    Groups of 20 inseminated female Sprague-Dawley rats were given 0, 100,
    400, 800, or 1600 mg/kg bw per day triforine (purity not stated)
    suspended in 1% carboxymethylcellulose by gavage on days 6-15 of
    pregnancy. The rates of resorption and variation were increased in
    animals at 1600 mg/kg bw per day. Furthermore, the number of fetuses
    was reduced, dur to significantly increased postimplantation loss at
    this dose. Because the frequency of variations was also slightly
    increased at 800 mg/kg bw per day, the NOAEL for fetotoxicity was 400
    mg/kg bw per day. Although toxic effects were observed at 800 and 1600
    mg/kg bw per day, there were no signs of a teratogenic effect
    (Leuschner, 1972).

    In a preliminary study, groups of eight inseminated Sprague-Dawley
    rats were given triforine (purity, >97%) by garage at doses of 250,
    500, or 1000 mg/kg bw per day on days 6-15 of gestation. There were no
    deaths or treatment-related clinical signs of toxicity. Mean body
    weight was slightly reduced on gestation days 9-12 in animals at 500
    and 1000 mg/kg bw per day, but overall body-weight gain in the four
    groups was comparable throughout treatment. No gross pathological
    changes were found in fetuses removed on day 20 of gestation. No
    reproductive parameters were affected by treatment, and them were no
    indications of treatment-related embryotoxicity or teratogenicity
    (Fuchs, 1992).

    Groups of 30 inseminated Sprague-Dawley rats were given triforine
    (purity not stated) by garage at doses of 200, 500, or 1000 mg/kg bw
    per day on days 6-15 of gestation. There were no deaths or
    treatment-related clinical signs of toxicity. Food consumption was
    reduced in animals at 1000 mg/kg bw per day, but this was not
    accompanied by a significant decrease in body-weight gain. No gross
    pathological changes were seen in fetuses removed on day 20 of
    gestation. No reproductive parameters were affected by treatment, and
    there were no indications of treatment-related embryotoxicity or
    teratogenicity. The NOAEL for maternal and developmental toxicity was
    1000 mg/kg bw per day (Fuchs, 1993a).

     Rabbits

    Groups of 15 inseminated Himalayan rabbits were given technical-grade
    triforine (purity not stated) in 0.5% carboxymethylcellulose by gavage
    on days 6-18 of gestation at doses of 0, 5, 25, or 125 mg/kg bw per
    day. Them were no deaths or treatment-related clinical signs of
    toxicity. Dose-related decreases in food consumption were observed in
    rabbits at 25 and 125 mg/kg bw per day on days 7-14 of treatment. Food
    consumption was also reduced in the animals at 5 mg/kg bw per day, but
    this had no effect on body-weight gain and was considered to be
    toxicologically insignificant. For most of the treatment period, body
    weight was reduced in rabbits at 25 and 125 mg/kg bw per day, as a
    result of reductions during gestation days 6-9. Towards the end of
    treatment, these groups regained weight. The does were killed and
    their fetuses removed on day 29 of gestation. No gross pathological
    findings were observed. No reproductive parameters were affected by
    treatment, and them were no indications of treatment-related
    embryotoxicity or fetotoxicity at any dose. The NOAEL for maternal
    toxicity was 5 mg/kg bw per day, on the basis of decreased food
    consumption and body weight at 25 mg/kg bw per day. The NOAEL for
    fetal effects was 125 mg/kg bw per day, the highest dose tested
    (Gleich et al., 1981).

    Groups of 18 inseminated New Zealand rabbits were given
    technical-grade triforine (purity not stated) in 0.5%
    carboxymethylcellulose by garage on days 6-18 of gestation at doses of
    0, 6, 30, or 150 mg/kg bw per day. There were no deaths or
    treatment-related clinical signs of toxicity. Decreased food
    consumption were observed in does at 150 mg/kg bw per day on days

    12-18, although no statistically significant reduction in body-weight
    gain was observed. The does were killed and their fetuses removed on
    day 28 of gestation. No gross pathological findings were observed. No
    reproductive parameters were affected by treatment, and there were no
    indications of treatment-related embryotoxicity or fetotoxicity at any
    dose. The NOAEL for maternal toxicity was 30 mg/kg bw per day, on the
    basis of decreased food consumption at 150 mg/kg bw per day. The NOAEL
    for fetal effects was 150 mg/kg bw per day, the highest dose tested
    (Müller, 1989).

    Since maternal effects were minimal in the previous study, triforine
    was tested at higher doses. In a preliminary study, four groups of
    eight inseminated New Zealand rabbits were given technical-grade
    triforine (purity not stated) in distilled water by gavage on days
    6-18 of gestation at doses of 0, 250, 500, or 1000 mg/kg bw per day.
    There were no deaths or treatment-related clinical signs of toxicity.
    Body-weight gain was reduced in all treated groups, mainly due to
    body-weight losses on days 6-9 of gestation; the reduction (64%) was
    statistically significant in does at 1000 mg/kg bw per day. At the end
    of treatment, the body-weight gain in the treated groups exceeded that
    of the controls. When the does were killed and their fetuses removed
    on day 28 of gestation, no gross pathological changes were found, and
    reproductive parameters were not affected. The group mean fetal
    weights were slightly reduced at 500 and 1000 mg/kg bw per day. There
    were no indications of teratogenicity at any dose (Müller, 1991).

    Groups of 18 inseminated New Zealand rabbits were given
    technical-grade triforine (purity not stated) in distilled water by
    gavage on days 6-18 of gestation at doses of 0 or 1000 mg/kg bw per
    day. There were no deaths or treatment-related clinical signs of
    toxicity. Reductions in food consumption and body-weight gain were
    observed in does at 1000 mg/kg bw per day, mainly due to body-weight
    losses on days 6-9 of gestation. At the end of treatment, food
    consumption and body-weight gain in the treated group were comparable
    to those of the control group. The does were killed and their fetuses
    removed on day 28 of gestation. No gross pathological changes were
    observed, and reproductive parameters were not affected by treatment.
    The group mean fetal weights were slightly reduced, and this
    indication of fetotoxicity was accompanied by reduced ossification of
    the carpals and/or tarsals, sternum, and pelvis. There were no
    indications of teratogenicity at any dose. No NOAEL for maternal
    toxicity was identified (Fuchs, 1993b).

     (f)  Special studies

     (i)  Dermal and ocular irritation and dermal sensitization

    Triforine, moistened with water and left on the shaven skin of
    Sprague-Dawley rats for 24 h, was tolerated with no irritation up to a
    dose of 10 000 mg/kg bw (Frohberg et al., 1973; Ullmann et al., 1990).
    When 0.5 g of moistened triforine (purity, 98.1%) was applied to
    shaven skin areas of three male and three female New Zealand whim
    rabbits and left on the skin for 4 h under a semi-occlusive bandage,

    no erythema or oedema occurred within the 72-h follow-up (Ullmann &
    Porricello, 1988a).

    No reactions occurred in mucosa or eyes of New Zealand white rabbits
    after insertion of 0.1 g triforine into the conjunctival sac (Frohberg
    et al., 1973). When triforine (purity, 98.1%) was inserted into the
    conjunctival sac of three male and three female New Zealand whim
    rabbits, all rabbits showed mild local reactions (reddening, chemosis,
    discharge) 1 h after application. The reaction disappeared almost
    entirely within 24 h, and only one female still had minor conjunctival
    reddening (mean cumulative score, 0.17). No changes were observed in
    the eyes with a slit-lamp ophthalmoscope 48 and 72 h after
    application. No systemic clinical reactions occurred during the
    testing period, and body weight was not affected. The overall
    irritation score was 0.83 out of a maximum attainable score of 13
    (Ullmann & Porricello, 1988b).

    In a Maurer optimization test involving aggravation of testing
    conditions by additional injection of Freund's adjuvant in groups of
    12 male and 12 female Dunkin-Hartley albino guinea-pigs, triforine
    (purity, 99.9%) showed no sensitizing potential after epidermal and
    intradermal challenge (Ullmann & Suter, 1984).

     (ii)  Mode of action

    Triforine is regarded as an inhibitor of sterol biosynthesis; in
    particular, it inhibits microsomal sterol demethylation, as was shown
    by means of gas chromatographic-mass spectrometric measurements of
    sterol accumulation in  Neurospora crassa and  Aspergillus 
     fumigatus. Because triforine has only weak or no antifungal
    properties  in vitro, metabolic activation can be assumed (Langcake
    et al., 1983).

     (iii)  Interactions with nitroso compounds

    Groups of 25 or 50 male and 25 or 50 female Swiss mice were given
    0.05% sodium nitrate in drinking-water, triforine suspended in water
    at 300 mg/kg bw by garage twice each week, or a combination of the two
    treatments for up to 180 days. Tumour incidences were compared with
    those in a control group of 184 males and 117 females. Triforine alone
    did not increase the number of tumours; however, the combination
    increased the frequencies of lymphomas (including thymoma) and of
    epithelial adenomas and carcinomas of the gastrointestinal tract, the
    lung, and, in males, the liver. Incubation of triforine with 4%
    acidified sodium nitrite solution for 24 h formed dinitrosopiperazine,
    which is known to be carcinogenic and was suspected to be the
    substance that induced the tumours (Börzsönyi et al., 1978).

     (iv)  Haemolytic anaemia

    The main toxic effect observed in the experiments with triforine was
    anaemia, which is a reduction in the concentration of haemoglobin in
    the blood below the normal range for the species studied and is

    usually accompanied by a reduction in the number of erythrocytes. This
    condition occurs when the rate of erythyrocyte production is
    outstripped by the rate of erythrocyte destruction. In many cases of
    anaemia, there is both a reduced rate of production and an increased
    rate of destruction. Nevertheless, because of the greater importance
    of one or other of these processes, anaemias are classified as (i)
    those involving excessive loss (post-haemorrhagic anaemia) or
    destruction (haemolytic anaemias) of erythrocytes and (ii) those
    involving failure of production of erythrocytes, diminished production
    with bone-marrow hyperplasia being dyserythropoietic anaemia and
    diminished production with marrow hypoplasia being hypoplastic or
    aplastic anaemia.

    Haemolytic anaemia in adult experimental animals can result from an
    immune reaction in which the antibody is directed against the chemical
    or chemical-plasma protein complex, which can bind to the erythrocyte
    cell surface; alternatively, the chemical induces formation of an
    antibody which cross-reacts with a normal erythrocytic antigen. In the
    first case, haemolysis ceases immediately when exposure to the
    chemical ceases; in the second case, evidence of an immune reaction
    against erythrocytes may persist for some months after exposure to the
    chemical has ceased.

    Many chemicals cause haemolysis, e.g. phenylhydrazine, lead, and
    arsenic and copper compounds; lead may also interfere with haemoglobin
    synthesis. Hypoplastic and aplastic anaemias can be produced by a
    number of chemicals, including cytostatic drugs, which generally
    depress the bone marrow. In addition, there may be idiosyncratic
    reactions to certain drugs, e.g. chloramphenicol, sulfonamides,
    phenylbutazone, and other antirheumatic and antithyroid drugs.
    Aplastic anaemia can also be induced by some hair dyes and industrial
    chemicals, such as benzene.

    The effect of triforine is mild, occurs in all or many exposed animals
    (depending on the dose), and is characterized by haemosiderin
    deposition in several organs, no bone-marrow depression, the entry of
    increased numbers of immature cells into peripheral circulation, and
    the absence of evidence of immunotoxicty. At least in dogs, there is
    actually a (presumably) compensatory increase in erythropoiesis. Of
    the three common circumstances in which haemolytic anaemia is induced
    by chemicals, two, therefore, are unlikely to be important: an immune
    response and increased sensitivity of erythrocytes to oxidative stress
    because of glutathione depletion, since glutathione is not
    significantly involved in triforine metabolism. The third, oxidative
    haemolysis in animals: with normally functioning erythrocytes, would
    appear to be the most likely mechanism and one which would probably
    cease as soon as exposure ceased. This appears to be the situation in
    triforine-exposed rats and dogs. Also, there is no evidence of
    methaemoglobin formation or of increased numbers of Heinz
    body-containing cells or deformed erythrocytes in the circulation.

    3.  Observations in humans

    Medical reports from three companies that synthesize triforine were
    available. Boehringer Ingelheim, Germany, examined 12 workers
    repeatedly between 1970 and 1983 and reported no haematological or
    blood chemical results considered to be related to exposure to
    triforine (Celamerck, 1983a,b). The industrial medical service of E.
    Merck Co., Darmstadt, Germany, found no adverse systemic, dermal, or
    mucosal effects of exposure to triforine among production workers
    during routine examinations performed in conformity with the
    requirements of the German Chemical Trade Union (Merck, 1984). No
    adverse effects were observed during triforine production at Shell
    Agrar GmbH in Spain between 1987 and 1993 (Toubes, 1993).

    Comments

    A battery of tests for acute toxicity with technical-grade triforine
    showed that it is slightly hazardous by both the oral and dermal
    routes, with respective LD50 values of > 5000 and > 2000 mg/kg bw.
    The LC in rats exposed by inhalation was > 5.1 mg/L. Triforine was
    not irritating or sensitizing to the skin of rodents and was minimally
    irritating to the eyes of rabbits. WHO has classified triforine as
    unlikely to present an acute hazard in normal use (WHO, 1996).

    Dietary administration of technical-grade triforine for 4 or 13 weeks
    showed that the haematopoietic system is a target, as indicated by
    mild haemolytic anaemia with associated secondary effects in the
    spleen and liver. Similar results were found in two-year studies of
    toxicity in mice, rats, and dogs, in which the haematological changes
    were accompanied by increased weights of the spleen and liver. In
    addition, in a 105-week dietary study in mice, changes in the large
    intestine characterized by thickening, enlargement, inflammation, and
    ulceration were observed; in a two-year dietary study in dogs,
    increased erythropoiesis and increased haemosiderin deposition were
    found in the liver and bone marrow. In a four-week dietary study in
    rats at 0, 500, 2500, or 12 500 ppm, there was no NOAEL. In a
    four-week study in mice at 0, 200, 1000, or 5000 ppm, the NOAEL was
    1000 ppm, equal to 200.mg/kg bw per day. The NOAEL in a 13-week study
    in rats at 0, 100, or 500 ppm was 500 ppm, equivalent to 25 mg/kg bw
    per day. In a six-month study in rats fed diets giving 0, 25, 120,
    620, or 3100 ppm, the NOAEL was 120 ppm, equivalent to 6 mg/kg bw per
    day. The two-year dietary studies in rats (0, 200, 2000, or 20 000
    ppm), mice (0, 70, 700, or 7000 ppm), and dogs (0, 10, 40, 100, or
    1000 ppm) showed NOAELs of 200 ppm, equal to 10 mg/kg bw per day, 70
    ppm, equal to 11 mg/kg bw per day, and 100 ppm, equal to 2.4 mg/kg bw
    per day, respectively.

    The major toxic effect observed in the experiments summarized above
    was anaemia. The effect was mild, occurring in all or many of the
    exposed animals (depending on the dose); the effects were reversible
    in rats and dogs and were characterized by haemosiderin deposition in
    several organs, the absence of evidence of bone-marrow depression,
    entry of increased numbers of immature cells into the peripheral

    circulation, and the absence of effects on organs of the immune
    system. In dogs, there was actually an increase in erythropoiesis.
    Oxidative haemolysis in animals with normally functioning erythrocytes
    would appear to be the most likely mechanism and one that would cease
    as soon as exposure ceased. Also, there was no evidence of
    methaemoglobin formation or of any increase in Heinz body-containing
    cells or deformed erythrocytes in the circulation.

    No genotoxic activity was observed in an adequate battery of tests for
    mutagenicity and clastogenicity  in vitro and  in vivo. The Meeting
    concluded that triforine is not genotoxic.

    The results of the studies critical to derivation of an ADI are shown
    below; the list does not include an 81-week study in mice treated
    orally, which was considered inadequate for evaluation since
    histopathological examination was limited to lesions that were judged
    at autopsy to be neoplastic.

    No carcinogenic effect was observed in rats given dietary
    concentrations of 0, 25, 125,625, or 3120 ppm in one study and 0, 200,
    2000, or 20 000 ppm in another. Triforine increased the incidence of
    pulmonary tumours in female mice given 7000 ppm; the NOAEL for
    carcinogenicity was 700 ppm, equal to 160 mg/kg bw per day. The
    Meeting concluded that the murine response involves an unidentified
    nongenotoxic mechanism and that the carcinogenic activity seen in mice
    is unlikely to be indicative or a human carcinogenic risk at the
    expected levels of exposure to triforine.

    Triforine at dietary concentrations of 0, 500, 3000, or 20 000 ppm did
    not affect reproductive performance in rats over the course of a
    two-generation study, the NOAEL being the highest dose tested, 20 000
    ppm, equal to 1500 mg/kg bw per day. The NOAEL for parental toxicity
    and for the growth and development of the offspring was 500 ppm, equal
    to 40 mg/kg bw per day, on the basis of decreased food consumption,
    body-weight gain, and F1 pup weight and increased relative spleen
    weight at the next highest dose of 3000 ppm.

    In a study of developmental toxicity in rabbits given oral doses of 0,
    5, 25, or 125 mg/kg bw per day, the maternal NOAEL was 5 mg/kg bw per
    day, on the basis of decreased food consumption and body weight at 25
    mg/kg bw per day; the NOAEL for developmental toxicity was 125 mg/kg
    bw per day. In a later study of rabbits given oral doses of 0, 6, 30,
    or 150 mg/kg bw per day, the NOAEL for maternal toxicity was 30 mg/kg
    bw per day, on the basis of decreased food consumption and body-weight
    gain at 150 mg/kg bw per day; the NOAEL for developmental toxicity was
    150 mg/kg bw per day. In two subsequent studies in which triforine was
    given orally to rabbits at doses of 0, 250, 500, or 1000 mg/kg bw per
    day and 0 or 1000 mg/kg bw per day, maternal and embryotoxicity (as
    shown by reduced fetal weight) occurred at 1000 mg/kg bw per day.
    Decreased fetal weights and delayed ossification were observed in a
    study of developmental toxicity in rabbits at the maternally toxic
    dose of 1000 mg/kg bw per day. Thus, developmental toxicity in rabbits
    occurred only at doses that were also maternally toxic. A study of

    developmental toxicity in rats given triforine orally at doses of 0,
    200, 500, or 1000 mg/kg bw per day did not show adverse effects in
    either dams or fetuses at doses up to 1000 mg/kg bw per day. The
    Meeting concluded that triforine has no specific developmental or
    reproductive toxicity.

    Monitoring of workers in three manufacturing plants did not reveal any
    health effects that might be associated with exposure to triforine.

    An ADI of 0-0.02 mg/kg bw was established on the basis of the NOAEL of
    2.4 mg/kg bw per day in the two-year study of toxicity in dogs, with a
    safety factor of 100.

    Toxicological evaluation

     Levels that cause no toxic effect

         Mouse:    70 ppm, equal to 11 mg/kg bw per day (105-week study of
                   toxicity and carcinogenicity)
         Rat:      200 ppm, equal to 10 mg/kg bw per day (two-year study
                   of toxicity and carcinogenicity)
                   500 ppm, equal to 40 mg/kg bw per day (parental and
                   fetal toxicity in a study of reproductive toxicity)
                   1000 mg/kg bw per day (study of developmental toxicity)

         Rabbit:   5 mg/kg bw per day (maternal toxicity in a study of
                   developmental toxicity)

         Dog:      100 ppm, equal to 2.4 mg/kg bw per day (two-year study
                   of toxicity)

     Estimate of acceptable daily intake

              0-0.02 mg/kg bw

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

    Further observations in humans

    References

    Adams, K., Ransome, S., Anderson, A. & Dawe, I.S. (1993) Chinese
    hamster ovary/hprt locus assay: Triforine. Unpublished report from
    Huntingdon Research Centre Ltd. (Study No. SLL282/931168). Submitted
    to WHO by American Cyanamid, Princeton, NJ, USA.

    Atkinson, C., Perry, C.J. & Hudson, P. (1991 a) Triforine: 13-Week
    dietary maximum tolerated dose study in mice. Unpublished report from
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    American Cyanamid, Princeton, NJ, USA.


        Toxicological criteria for estimation of guidance values for dietary and non-dietary exposure to triforine

                                                                                                                                     

    Human exposure       Relevant route, study type, species                Results, remarks
                                                                                                                                     

    Short-term           Oral, single dose, rat                             LD50 > 5000 mg/kg bw
    (17 days)            Dermal, single dose, rat                           LD50 > 2000 mg/kg bw
                         Inhalation (4 h), rat                              LC50 5.1 mg/L
                         Dermal, irritation, rabbit                         Not irritating
                         Ocural, irritation, rabbit                         Minimally irritating
                         Dermal, irritation, rat                            Not irritating
                         Dermal, sensitization, guinea-pig                  Non-sensitizing

    Medium-term          Dermal, 3 weeks, rat                               NOAEL = 1100 mg/kg bw per day (highest dose tested)
    (1-26 weeks)         Oral, 13 weeks, dog                                NOAEL = 3.6 mg/kg bw per day: haemosiderin deposition
                         Oral, two-generation, reproductive oxicity, rat    NOAEL = 1500 mg/kg bw per day: reproductive toxicity;
                                                                            NOAEL = 49 mg/kg bw per day: parental and offspring
                                                                            toxicity
                         Oral, developmental toxicity, rabbit               NOAEL = 5 mg/kg bw per day: maternal toxicity;
                                                                            NOAEL = 125 mg/kg bw per day: fetal and developmental
                                                                            toxicity

    Long-term            Oral, 2 years, dog                                 NOAEL = 2.4 mg/kg bw per day: haematological changes;
    (> 1 year)                                                              haemosiderin deposition; haematopoiesis
                                                                                                                                     
    

    Atkinson, C., Perry, C.J. & Hudson, P .(1991b) Triforine: 13-Week
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    Boehringer Sohn (1974a) The pharmacokinetics of the systemic fungicide
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    Princeton, NJ, USA.

    Langcake, P., Kuhn, P.J. & Wade, M. (1983) The mode of action of
    systemic fungicides. In: Hudson, D.H. & Roberts, T.R., eds, 
     Progress in Pesticide Biochemistry and Toxicology, Vol. 3, pp.
    1-109.

    Leuschner, F. (1972) The effect of W-524, lot 1, on pregnant rats and
    their fetuses, following oral administration. Unpublished report from
    Laboratorium für Pharmakologie und Toxikologie, Hamburg, Germany.
    Submitted to WHO by American Cyanamid, Princeton, NJ, USA.

    Leuschner, F., Leuschner, A., Schwerdtfeser, W. & Dontenwill, W. (1971
    ) 13 Weeks oral toxicity study in beagle dogs with W-524. Unpublished
    report from Laboratorium für Pharmakologie und Toxikologie, Hamburg;,
    Germany. Submitted to WHO by American Cyanamid, Princeton, NJ, USA.

    Leuschner, F., Leuschner, A., Schwerdtfeser, W. & Dontenwill, W.
    (1972) 21-Day toxicity tests with the compounds W-524 in
    Sprague-Dawley rats using dermal application. Unpublished report from
    Laboratorium für Pharmakologie und Toxikologie, Hamburg, Germany.
    Submitted to WHO by American Cyanamid, Princeton, NJ, USA.

    McCay, C. & Hazelden, K.P. (1990) Triforine dose range finding
    reproductive assay in rats. Unpublished report from Inveresk Research
    International (Report No. 7073). Submitted to WHO by American
    Cyanamid, Princeton, NJ, USA.

    McCay, C. & Hazelden, K.P. (1991 ) Triforine, two generation
    reproduction study in rats. Unpublished report from Inveresk Research
    International (Report No. 7368). Submitted to WHO by American
    Cyanamid, Princeton, NJ, USA.

    Merck (1984) [Triforine: Medical/occupational health position paper.]
    Unpublished report dated 28 March 1984, Darmstadt, Germany. Submitted
    to WHO by American Cyanamid, Princeton, NJ, USA (in German).

    Miltenburger, H.G. (1984) Triforine tech., test report of study LMP
    76A (mutations affecting the hypoxanthine-guanine phosphoribosyl
    transferase locus in V79 cells: HGPRT-test). Unpublished report from
    LMP, Darmstadt, Germany (Study No. LMP 076A). Submitted to WHO by
    American Cyanamid, Princeton, NJ, USA.

    Miltenburger, H.G. (1985) Triforine tech., test report of study LMP
    136 (chromosome aberrations in cells of Chinese hamster cell line
    V79). Unpublished report from LMP Darmstadt, Germany (Study No. LMP
    136). Submitted to WHO by American Cyanamid, Princeton, NJ, USA.

    Moriya, M., Ohta, T., Watanabe, K., Miyazawa, T., Kato, K. & Shirasu,
    Y. (1983) Further mutagenicity studies on pesticides in bacterial
    reversion assay systems.  Mutat. Res., 116, 185-216.

    Muacevic, G. (1968) Pharmacological investigation, acute oral
    toxicity, W-524, male and female albino mice. Unpublished report from
    Boehringer Sohn, Ingelheim, Germany. Submitted to WHO by Celamerck
    GmbH & Co., Ingelheim/Rhine, Germany.

    Muacevic, G. (1969) Pharmacological investigation, acute oral
    tolerance in dogs. Unpublished report from Boehringer Sohn, Ingelheim,
    Germany. Submitted to WHO by Celamerck GmbH & Co., Ingelheim/Rhine,
    Germany.

    Müller, W. (1989) Triforine: Oral (gavage) teratogenicity study in the
    rabbit. Unpublished report from Hazleton Deutschland GmbH (Project No.
    460/29). Submitted to WHO by American Cyanamid, Princeton, NJ, USA.

    Müller, W. ( 1991 ) Triforine: Preliminary oral (gavage)
    embryotoxicity study in the rabbit. Unpublished report from Hazleton
    Deutschland GmbH (Report No. 951-121-003). Submitted to WHO by
    American Cyanamid, Princeton, NI, USA.

    Niggeschulze, A., Hill, K. & Stötzer, H. (1974) Three generation study
    with the fungicide W-524 in rats. Unpublished report from Boehringer
    Sohn, Ingelheim, Germany. Submitted to WHO by American Cyanamid,
    Princeton, NJ, USA.

    Perry, C.J., Mulhern, M. & Finch, L (1992b) Triforine: 104 Week
    dietary carcinogenicity study in rats, incorporating 52 week toxicity
    study. Unpublished report from Inveresk Research International (Report
    No. 7745). Submitted to WHO by American Cyanamid, Princeton, NJ, USA.

    Proudlock, R.J., Stocker, K.J., Howard, W.R., Anderson, A. & Dawe,
    I.S. (1993) Triforine: in vitro DNA repair test using rat hepatocytes.
    Unpublished report from Huntingdon Research Centre Ltd (Study Report
    No. SLL 281/931152). Submitted to WHO by American Cyanamid, Princeton,
    NJ, USA.

    Robbins, M.C. (1994) An investigation of the effect of triforine on
    rat and mouse hepatic xenobiotic metabolising enzymes. Unpublished
    report from BIBRA Toxicology International (Report No. 1356/2/ 2/94).
    Submitted to WHO by American Cyanamid, Princeton, NJ, USA.

    Röhrborn, G. (1977) [Scientific comment on the mutagenicity of
    triforine using the liver microsome test of Ames.] Unpublished report.
    Submitted to WHO by American Cyanamid, Princeton, NJ, USA (in German).

    von Sandersleben, J.H., Herbst, M., Weisse, I., Frölke, W., Gutnard,
    J. & Stötzer, H. (1974) Chronic toxicity test of the substance
    W-524-XX on beagles, oral application, over 104 weeks. Unpublished
    report from Boehringer Sohn, Ingelheim, Germany. Submitted to WHO by
    American Cyanamid, Princeton, NJ, USA.

    Stötzer, H., Herbst, M., Köllmer, H., Weisse, I., Guénard, J. & Tiler,
    T. (1971a) Testing of the subacute toxicity of the substance W-524 in
    rats following oral administration. Unpublished report from Boehringer
    Sohn, Ingelheim, Germany. Submitted to WHO by American Cyanamid,
    Princeton, NJ, USA.

    Stötzer, H., Herbst, M., Köllmer, H., Weisse, I., Frölke, W., Guénard,
    J. & Tilov, T. (1971b) Testing of the subacute toxicity of the
    substance W-524 in rats following oral administration. Unpublished
    report from Boehringer Sohn, Ingelheim, Germany. Submitted to WHO by
    American Cyanamid, Princeton, NJ, USA.

    Stötzer, H., Herbst, M., Ganz, H., Weisse, I., Frölke, W., Guénard, J.
    & von Sandersleben, J. (1971c) Testing of the subacute toxicity of the
    substance W-524 in dogs following oral administration. Unpublished
    report from Boehringer Sohn, Ingelheim, Germany. Submitted to WHO by
    American Cyanamid, Princeton, NJ, USA.

    Stötzer, H., Köllmer, H., Herbst, M., Weisse, I., Frölke, W., Guénard,
    J. & Tilov, T. (1972) Chronic toxicity studies with the compound
    W-524-XX in rats using oral administration; duration 26 weeks.
    Unpublished report from Boehringer Seth, Ingelheim, Germany. Submitted
    to WHO by American Cyanamid, Princeton, NJ, USA.

    Toubes, G.P. (1993) Medical certificate, Malgrat de Mer. Unpublished
    report. Note of 6.4.1993 from Dr Inchaurrondo to Dr Dammermann, Shell
    Agrar GmbH. Submitted to WHO by American Cyanamid, Princeton, NJ, USA.

    Tenneker, H., Stucki, H.P., Luetkemeier, H., Vogel, O., Terrier, C.,
    Pappritz, G., Mladenovic, P. & Janiak, T. (1988d) 4-Week toxicity
    (feeding) study with triforine techn, in the mouse. Unpublished report
    from Research & Consulting Co., Itingen, Switzerland (Project No.
    097751). Submitted to WHO by American Cyanamid, Princeton, NJ, USA.

    Tenneker, H., Stucki, H.P., Luetkemeier, H., Vogel, O., Terrier, C.,
    Pappritz, G., Mladenovic, P. & Janiak, T. (1989) 4-Week toxicity
    (feeding) study with triforine technical in the rat. Unpublished
    report from Research & Consulting Co., Itingen, Switzerland (Project
    No. 097740). Submitted to WHO by American Cyanamid, Princeton, NJ,
    USA.

    Ullmann, L. & Porricello, T. (1988a) Primary skin irritation study
    with triforine technical in rabbits (4-hour semi-occlusive
    application). Unpublished report from Research & Consulting Co.,
    Itingen, Switzerland (Project No. 213300). Submitted to WHO by
    American Cyanamid, Princeton, NJ, USA.

    Ullmann, L. & Porricello, T. (1988b) Primary eye irritation study with
    triforine technical in rabbits. Unpublished report from Research &
    Consulting Co., Itingen, Switzerland (Project No. 213311). Submitted
    to WHO by American Cyanamid, Princeton, NJ. USA.

    Ullmann, L. & Surer, B. (1984) Delayed contact hypersensitivity to
    triforine technical in albino guinea pigs, the Maurer optimization
    test. Unpublished report from Research & Consulting Co., Itingen,
    Switzerland (Project No, 33276). Submitted to WHO by American
    Cyanamid, Princeton, NJ, USA.

    Ullmann, L., Sacher, R., Mohler, H. & Pappritz, G. (1986a) Acute oral
    toxicity study with triforine technical in rats. Unpublished report
    from Research & Consulting Co., Itingen, Switzerland (Project No.
    076151). Submitted to WHO by American Cyanamid, Princeton, NJ, USA.

    Ullmann, L. Sacher, R., Mohler, H. & Vogel, O. (1986b) 4-Hour acute
    inhalation toxicity study with triforine technical in rats.
    Unpublished report from Research & Consulting Co., Itingen,
    Switzerland (Project No. 076162). Submitted to WHO by American
    Cyanamid, Princeton, NJ, USA.

    Ullmann, L., Sacher, R., Mohler, H. & Vogel, O. (1986c) Acute oral
    toxicity study with triforine technical in mice. Unpublished report
    from Research & Consulting Co., Itingen, Switzerland (Project No.
    077433). Submitted to WHO by American Cyanamid, Princeton, NJ, USA.

    Ullmann, L., Althaus, P., Janiak, T. & Vogel, D. (1990) Acute dermal
    toxicity study with triforine techn. (SAG 102) in rats. Unpublished
    report from Research & Consulting Co., Itingen, Switzerland (Project
    No. 33276, 278774). Submitted to WHO by American Cyanamid, Princeton,
    NJ, USA.

    WHO (1996)  The WHO Recommended Classification of Pesticides by 
     Hazard and Guidelines to Classification 1996-1997 (WHO/PCS/96.3),
    Geneva, International Programme on Chemical Safety.
    


    See Also:
       Toxicological Abbreviations
       Triforine (Pesticide residues in food: 1978 evaluations)