IPCS INCHEM Home



    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    cases, based on unpublished proprietary data submitted for the purpose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.



    GUAZATINE

    First draft prepared by
    I. Dewhurst
    Pesticides Safety Directorate, Ministry of Agriculture, Fisheries and
    Food
    York, United Kingdom

         Explanation
         Evaluation for acceptable daily intake 
              Biochemical aspects 
                   Absorption, distribution, and excretion 
                   Biotransformation 
              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 
              Observations in humans 
         Comments 
         Toxicological evaluation 
         References 

    Explanation

    Guazatine was evaluated by the Joint Meeting in 1978 (Annex 1,
    reference 30), when an ADI of 0-0.03 mg/kg bw was established on the
    basis of an NOAEL of 200 ppm (equivalent to 3 mg/kg bw per day) in a
    two-year dietary study in dogs. The compound was reviewed at the
    present Meeting within the CCPR periodic review programme. New data on
    its absorption and metabolism, the toxicity of repeated doses in mice
    and dogs, its long-term toxicity in rats and mice, genotoxicity,
    reproductive toxicity, and developmental toxicity were assessed. Data
    on the pesticide 1,1-imino-di(octamethylene)diguanidine (common name,
    iminoctadine), which constitutes about 1.5% of the guazatine mixture,
    were also considered.

    Evaluation for acceptable daily intake

    Guazatine is a preparation of the triacetates of dimeric and trimeric
    guanidated 1,8-diamino-octane which also contains a range of oligomers
    and reaction products. Pure guazatine reportedly cannot be produced
    industrially; all of the oligomers are necessary for its biological
    activity and are considered together as active ingredients. A coding
    system is used for the compounds that make up guazatine, in which N
    represents any amino group and G represents any guanidated amino

    group; NN represents H2N-(CH2)8-NH2 and GG represents
    H2N-C(NH)NH-(CH2)8-NH-C(NH)-NH2. The stated purity of the
    guazatine tested in toxicology studies was usually about 70%, i.e.
    that of the marketed technical-grade product. The percent purity is
    based on the results of titrimetric analyses, which are normalized to
    one of the constituents (1,1'-iminodi(octamethylene)diguanidine, GNG).
    There are no controls on the levels of individual components. All
    doses and concentrations expressed on a w/w basis in this monograph
    were corrected on the basis of dietary analyses; however, it is not
    clear whether the results are for the technical-grade material or for
    the free base. In some published Japanese studies, the term
    'guazatine' is used synonymously with 'iminoctadine' (> 99% pure
    GNG), which represents about 1.5% of the technical-grade material

    1.  Biochemical aspects

     (a)  Absorption, distribution, and excretion

    In one male Wistar rat that received an unspecified dose of
    14C-guanidino- and 3H-octyl-labelled guazatine, the recoveries were
    83% 14C and 93% 3H; no volatile compounds were trapped. Over 72 h,
    faecal excretion accounted for 64% and urinary excretion for 15% of
    the administered 14C; 39% of the administered 3H was excreted in
    faeces and 42% in urine. The carcass and tissues contained
    approximately 3% of the 14C and 12% of the 3H. It is not clear
    whether these differences in isotope ratios are due to metabolism or
    tritium exchange, as thin-layer chromatography of the faecal and
    urinary extracts showed only spots similar to those for the
    administered material (Leegwater, 1975).

    Two male rats weighing about 200 g received about 10 mg/kg bw of
    [14C- guanidino]-labelled guazatine by gavage as a 0.25% solution of
    Panoctine (purity unspecified; specific activity, 3.8 µCi/mg), and
    faeces and urine were collected over 120 h. Mean recovery was > 90%,
    the majority (about 60%) being found in the urine and about 30% in
    faeces. About 90% of the urinary excretion and 50% of the faecal
    excretion occurred during the first 24 h. The carcass and tissues
    contained 2.5% of the administered dose, with mean values of 0.6% in
    the liver, 0.4% in blood, and 0.08% in kidney. Qualitative thin-layer
    chromatography of faecal and urinary extracts showed one major and
    several minor components, which also appeared in the parent compound.
    The reasons for the apparent difference in the main route of excretion
    in this study and others are unknown (Leegwater, 1980).

    Groups of CD rats received [14C-octyl]-labelled guazatine (purity,
    78%; specific activity not given) by garage in distilled water in a
    variety of protocols, outlined in Table 1. Faeces, urine, expired air,
    and cage wash samples were collected for animals in the first four
    groups. At termination, residues were determined in the carcass and
    selected tissues from animals in groups 1, 2, and 5. The results of
    high-performance liquid chromatography analyses reported in a
    supplementary document (Prout, 1996), showed that the profile of

    radiolabelled guazatine was qualitatively similar to that of
    commercial guazatine (GTA70), although the ratios of the constituents
    were different.

    Mean recoveries were all within the range 95-105%. The results for
    animals in groups 1, 2, and 3 were similar, faecal excretion at 24 h
    accounting for 85-94% and urinary excretion for 3-6% of the
    administered dose, with no sex difference. Less than 1% of the
    administered dose was detected in exhaled air. In groups 1 and 2,
    residues in the carcass and tissues at 96 h accounted for < 2% of the
    administered dose; peak levels, proportionately similar in both
    groups, were seen in the liver (2 and 0.2 g equivalents per g) and
    kidneys (1 and 0.1 g equivalents per g). In bile duct-cannutated
    animals (group 4), urinary excretion at 24 h was similar to that in
    other groups (about 6%), but faecal excretion was reduced to about 55%
    of the administered dose. The levels in bile were low, representing
    less than 0.25% of the administered dose over 24 h. In these animals,
    the residues in the gut accounted for 40% of the administered dose in
    males and 24% in females, and those in the carcass represented 3.5% in
    males and 11% in females. In animals given 14 × 2 mg/kg bw of
    guazatine, there was evidence of accumulation in liver, kidney, and
    fat during treatment, with partial clearance over the next 14 days
    (Table 2).

    Guazatine thus appears to be poorly absorbed after oral administration
    at 2 or 20 mg/kg bw, and faecal excretion apparently represents
    unabsorbed compound. The repeated dosing schedule indicates a limited
    potential for bio-accumulation of guazatine, as clearance was not
    completed 14 days after the last dose (Cameron et al., 1989).
    Subsequent analysis of samples from this study indicated that two
    components, the fully guanidated diamine GG and triamine GGG, are
    absorbed preferentially (Prout, 1996), but the data to support this
    assumption are not considered to be conclusive.

    An extensive investigation of the absorption, distribution, and
    excretion of iminoctadine (used synonymously for guazatine) was
    reported by Kato et al. (1985). Groups of four male Wistar-Imamichi
    rats received [14C-guanidine] - labelled iminoctadine triacetate
    (purity, 99.7%) in saline at 3 or 30 mg/kg bw by gavage. Urine and
    faeces were collected for up to seven days; blood samples were
    collected periodically from animals that received 30 mg/kg bw. Tissues
    were removed from animals killed on day 7.

    The excretion patterns were unaltered by dose; overall excretion of
    the administered dose was 4.6% in urine and 89-91% in faeces,
    primarily during the first 96 h. About 1% of the administered dose was
    found in the carcass after seven days. Peak blood levels (0.13 g
    equivalents per g) were recorded 10 min after treatment, with an
    elimination phase half-life of 27 h and the area under the
    concentration-time curve representing 3.7 g equivalents-h per g. The
    tissue concentrations at seven days were proportional to the
    administered dose, the highest concentrations in animals at 30 mg/kg


        Table 1. Treatment regimens of rats receiving 14C-guazatine

                                                                                                         

    Group       No. of       Dose              Sampling times              Comment
                animals      (mg/kg bw)
                                                                                                         

    1           5/sex        1 × 20            0-96 h
    2           5/sex        1 × 2             0-96 h
    3           5 males      1 × 2             0-96 h                      Fasted
    4           1/sex        1 × 2             0-24 h                      Bile cannulated
    5           15/sex;      1-14 × 2, daily   1, 3, 7, 14, 15, 16, 17,    All doses labelled with
                3 animals/                     20, 23, and 27 days         14C; sequential sacrifices
                sacrifice                      after first dose
                                                                                                         

    From Cameron et al. (1989)
    

    Table 2. Residues (g equivalents per g tissue) of guazatine in 
    three rats receiving up to 14 daily doses of 2 mg/kg bw by gavage

                                                                       

    Time                    Liver          Kidney         Fat
                                                                       

    24 h after dose 1       0.33-0.43      0.08-0.10      0.01-0.02
    24 h after dose 7       1.32-1.97      0.63-0.68      0.04-0.06
    24 h after dose 14      1.75-2.02      1.11-1.36      0.14-0.19
    72 h after dose 14      0.58-1.07      1.05-1.18      0.10-0.12
    168 h after dose 14     0.28-0.50      0.72-0.96      0.06-0.11
    336 h after dose 14     0.11-0.14      0.45-0.64      0.05-0.06
                                                                       

    From Cameron et al. (1989)


    bw being found in kidney (6.2 g equivalents per g), bone marrow (1.1 g
    equivalents per g), liver, spleen, thyroid, salivary gland, and
    pituitary (all 0.47-0.74 g equivalents per g). The finding of high
    residues in kidney is not entirely consistent with the results of
    Cameron et al. (1989), who found the highest levels in liver; they may
    be due to differences in the constituents of the administered
    compounds.

    The distribution of guazatine (purity, 71.7%) containing [14C-octyl]-
    labelled guazatine (specific activity, 6.6 µCi/mg) was investigated in
    one Fresian cow given a single intraruminal injection through the body
    wall and three cows given repeated (21 doses over 10.5 days)
    injections at 0.1 or 1 mg/kg bw per day. The treated animals also
    received the repeated doses. Samples of urine, faeces, expired air,
    milk, blood, and saliva were obtained during the study; at termination
    6 h after the last dose, a range of tissues were removed for analysis
    of residue levels. After single or repeated administration of
    guazatine at 0.1 mg/kg bw per day, the plasma concentrations were at
    or below the level of reliable determination (30 dpm; about 2 ng
    equivalents per ml). The single administration of 1 mg/kg bw per day
    resulted in a peak plasma level of 2.6 ng equivalents per ml at 6-24
    h, whereas the plasma levels after repeated treatment rose to 17.7 ng
    equivalents per ml with time, indicating potential accumulation.
    Guazatine was secreted into milk, with a peak residue of 28 ng
    equivalents per ml after nine days of administration at 1 mg/kg bw per
    day. The relative concentrations in milk fat, curd, and whey were
    about 4:3:1. More than 92% of the single doses were excreted in faeces
    over seven days, with a half-life of 36 h; urinary excretion accounted
    for < 2% of the administered dose. The tissue residues in cows given
    the low dose were near the level of reliable determination; at the
    high dose, the levels of residues were higher than in plasma, with the
    highest levels in liver and kidney (mean concentration, 80 ng
    equivalents per g) but with considerable inter-animal variation, and
    < 15 ng equivalents per g in muscle. These results indicate that

    guazatine may concentrate preferentially in tissues rather than in
    plasma (Cameron & Philips, 1986).

    The fates of 14C-guazatine (as iminoctadine) photo-products and
    14C-iminoctadine residues in apples were investigated in male
    Wistar-Imamichi rats. The photo-products were produced  in vitro by
    irradiation of [14C-guanidino]-labelled iminoctadine triacetate for
    seven weeks with sunlight bulbs (lambdamax, 515 nm). The resulting
    mixture contained 39% unchanged iminoctadine, 35 % of the photo-
    product 4- or 6-methyl-5-oxo-9-azaheptadecane-1,17-diguanadine, and
    eight other photo-products. Three rats received the mixture of photo-
    as an oral dose of 3 mg equivalents of iminoctadine per kg bw in
    water. Two rats received homogenized apples that had been cultivated
    and treated with an aqueous solution of 14C-iminoctadine (specific
    activity, 0.132 µCi/kg) by gavage. The residues in the apples
    consisted of 81% iminoctadine, 4% 4- or 6-methyl-5-oxo-9-
    azaheptadecane-1,17-diguanadine, 7% minor photo-products, and 8% other
    constituents; the administered dose (given as five doses of 10 g
    homogenate per kg bw) was equal to 0.2 mg equivalents of iminoctadine
    per kg bw. Three further rats received 14C-iminoctadine at 3 mg/kg bw
    with apple homogenate at 5.3 g/kg bw. Urine and faeces were sampled
    daily, and tissue samples were taken on day 7 for analysis of
    radiolabel. The photo-product was more readily absorbed (26%) than
    iminoctadine (about 10%) and tended to concentrate in the liver rather
    than the kidney. The residue in apples was less bioavailable than
    iminoctadine administered with apple homogenate, although a
    significant concentration of radiolabel was reported in the kidney
    (Sato et al., 1986).

    Groups of four male Wistar-Imamichi rats with or without bile-duct
    cannulae received 14C-iminoctadine triacetate (purity, 99.7%),
    labelled at either the methylene or the guanidine carbon, at 3 mg/kg
    bw by intravenous injection in saline. Blood, urine, and faeces were
    collected for up to seven days, when tissue samples were removed for
    analysis. The route of excretion varied with the position of the
    radiolabel (Table 3), indicating cleavage of the parent molecule, with
    some retention of the octylamine moieties.

    The relatively high level of faecal excretion with only low levels of
    radiolabel in the bile was attributed to secretion of iminoctadine or
    metabolites by the salivary glands and glands of the stomach (pars
    proventricularis and pars glandularis), which showed high
    concentrations of radiolabel in whole-body autoradiographs.
    Measurements of 14C-guanidine radiolabel in plasma showed an area
    under the curve of 2.7 g equivalents-h per g and a half-life of 33 h,
    whereas the half life of the 14C-methylene radiolabel was 69 h. After
    seven days, the kidney contained the highest levels of radiolabel
    (10-17 g equivalents per g; about 27% of the body burden for both
    labels); salivary, pituitary, and thyroid glands had higher
    concentrations (1.2-3.6 g equivalents per g) than the liver (0.8-1.6
    g-equivalents per g). The radiolabel concentrations in blood and

        Table 3. Excretion of 14C-iminoctadine triacetate after intravenous 
    administration to male rats at 3 mg/kg bw (as % of administered dose)

                                                                                  

    Group               Route of excretion    Time (h)    Position of 14C label
                                                                                  
                                                          Guanidine   Methylene
                                                                                  

    Intact              Urine                 0-96        46          28
                        Urine                 0-168       56          38

                        Faeces                0-96        22          24
                        Faeces                0-168       25          29
                        Carcass               168         21          34

    Bile cannulated     Bile                  0-3         0.3         0.4
                        Bile                  0-24        0.6         1.3
                        Urine                 0-24        20          5
                        Faeces                24          10          9
                                                                                  
    

    plasma were very low (< 0.01 g equivalents per g) at day 7.
    Autoradiography of the kidneys showed that the radiolabel was mainly
    in the malpighian corpuscles (Kato et al., 1985).

     (b)  Biotransformation

    Pooled samples of urine, faeces, liver, and kidneys from groups 1, 2,
    and 5 of the study by Cameron et al. (1989), described above, were
    extracted with methanolic acetate systems and investigated by
    radio-high-performance liquid chromatography. The extraction
    efficiencies were > 80% for urine, liver, and kidney, but only 44%
    for faeces. The findings were similar in all treated animals. In urine
    samples, three peaks were detected, which were tentatively attributed
    to 1,8-diaminooctylacetic acid (NN), 1,8-diaminooctylacetic acid dimer
    (NNN), and diguanidated diaminooctylacetic acid dimer (GNG/GGN). No
    quantitative data were presented, but the largest peak was attributed
    to NNN. Co-chromatography with standards did not confirm the proposed
    identities, and no additional techniques were used to characterize the
    components. Kidney and liver samples contained one major peak,
    attributed to monoguanidated diaminooctylacetic acid dimer (GNN/NGN).
    As NN and NNN are reported to be present at only low levels in the
    parent compound, the results indicate extensive deamidination or
    deguanidation. Given the ill-defined composition of the parent
    guazatine and the lack of definitive confirmation of the metabolites,
    the Meeting decided that no firm conclusions could be drawn about the
    metabolism of guazatine from this study.

    Samples of urine and faeces obtained over 96 h from male
    Wistar-Imamichi rats given 14C-methylene- or guanidino-labelled
    guazatine (as iminoctadine triacetate; purity, 99.7%) intravenously at
    3 mg/kg bw were analysed for metabolites. Most of the urinary
    metabolites were not characterized, but monodeamidino-iminoctadine was
    found to represent 5% of the radiolabel from the guanidine-labelled
    material and 16% from the 14C-methylene compound. No guazatine,
    dideamidino-iminoctadine, or creatinine was found in urine. In faeces,
    the major component was unchanged iminoctadine (78%);
    monodeamidino-iminoctadine represented 3% of the radiolabel, and 15%
    was due to a compound with identical chromatographic properties to the
    main photo-product (4- or 6-methyl-5-oxo-9-azaheptadecane-1,
    17-diguanadine), although its identity was not confirmed by mass
    spectrometry.

    Analyses of kidney samples from rats given the labelled compound
    intravenously at 3 mg/kg bw, orally at 30 mg/kg bw, intraperitoneally
    at 15 mg/kg bw, or intraperitoneally at 4 × 10 mg/kg bw per day showed
    16 metabolites, which were reported to be independent of route of
    administration. The three main constituents characterized were
    iminoctadine, monodeamidino-iminoctadine, and
    dideamidino-iminoctadine; two key metabolites were not identified. The
    proportion of iminoctadine decreased between days 1 and 7 after
    treatment, and the amounts of the four main metabolites increased over
    time. Analyses of liver samples identified a similar pattern to that
    seen in kidney. The results indicate that deamidination is a
    significant step in the metabolism of iminoctadine (Kato et al.,
    1985).

    In the study of Cameron & Philips (1986), described above, samples of
    liver, kidney, urine, and faeces from cows given 21 doses of guazatine
    at 1 mg/kg bw per day for 10.5 days showed that much of the residue
    was similar to some components of guazatine. There was evidence of
    selective absorption, as the peak ratios in the chromatogram of faeces
    differed from those in that compound, which was not present in the
    parent compound. The liver residue contained two polar metabolites in
    addition to guazatine components. Milk samples were not analysed for
    metabolites. Despite the availability of a range of standards,
    individual peaks were not characterized.

    A scheme for the metabolic pathway of guazatine in rats is shown in
    Figure 1. Other components of guazatine would be expected to undergo
    similar deamidination.

    FIGURE 1

    2.  Toxicological studies

     (a)  Acute toxicity

    Guazatine is of moderate toxicity when given orally, of low toxicity
    when applied dermally, but of moderately high toxicity when
    administered by inhalation or intraperitoneally. The results of
    studies of the acute toxicity of guazatine are summarized in Table 4.
    The clinical signs of toxicity after treatment were lethargy or
    sedation, hypothermia, coma, and local irritation. The gross and
    histopathological changes were consistent with a response to a local
    irritant; them was no clear systemic toxicity.

     (b)  Short-term toxicity

     Mice

    In a range-finding study, groups of 10 male and 10 female CD-1 mice
    received guazatine (purity, 70.6%) in the diet at 0, 10, 50, 200, or
    500 ppm for 13 weeks. Samples were taken for limited blood and
    clinical chemical analyses at week 13. A limited range of tissues was
    removed, weighed, and examined macroscopically; only the liver was
    examined histologically. Two male controls, one male at 500 ppm, and
    one at 50 ppm died during the study. Marked reductions in body-weight
    gain (> 25%) were seen in animals of each sex at 200 or 500 ppm. In
    females at the highest dose, aspartate aminotransferase activity was
    increased. The relative liver weights were increased in animals of
    each sex at 500 ppm; reductions in the absolute weights of several
    organs appeared to be secondary to reduced body-weight gain. Altered
    centrilobular hepatocytes were seen in 80% of animals at 200 or 500
    ppm. Given the limited extent of the investigations, no NOAEL was
    identified (Atkinson et al., 1990).


        Table 4. Acute toxicity of guazatine and formulations

                                                                                                                             

    Test material            Species    Route              Purity      LD50/LC50              Reference
                                                           (%)         (mg/kg bw or
                                                                       mg/m3)
                                                                                                                             

    Guazatine GTA            Rat        Oral               69.2        280                    Spanjers & Til (1980)
    Panoctine 42             Cat        Oral               NR          0.38 ml/kg bw          De Grout (1976a)
    Guazatine GTA70          Rat        Dermal             70.9        1050                   Cuthbert & D'Arcy-Burt (1986)
    Panoctine 42             Rabbit     Dermal             NR          2.8 ml/kg bw           van Beck et al. (1976)
    Panoctine Plus 300020    Rabbit     Dermal             NR          2.8-5.6 ml/kg bw       van Beck (1980)
    Paaoctine 42             Rat        Intraperitoneal    NR          0.053 ml/kg bw         De Groot (1976b)
    Guazatine GTA            Rat        Inhalation         69.2        225                    Appelman (1980)
    Panoctine 42             Rat        Inhalation         NR          11                     Kruysse & Immel (1976)
                                                                                                                             
    

     Rats

    Groups of 10 male and 10 female Wistar-derived rats received guazatine
    (54.8% w/w) in the diet at 0, 60, or 200 ppm for 14 weeks.
    Haematological and urinary parameters were measured in samples taken
    at week 13, and serum enzymes and total protein were measured in
    samples taken at termination at week 14. There were no deaths or
    adverse clinical signs. Body weight and food conversion efficiency
    were unaltered by treatment. A slight, dose-related decrease in
    leukocyte count was seen in animals of each sex (up to 13% in males
    and 6% in females), but this was not statistically significant, and
    there was no marked change in differential counts. Serum alkaline
    phosphatase activity was decreased in treated males, by 22% at 200 ppm
    and 7% at 60 ppm. Urinary parameters were unaffected by treatment, but
    the volume and specific gravity were not given. There were no
    significant findings at gross or microscopic examination. The findings
    in this study are of minimal toxicological significance; the NOAEL was
    200 ppm, equivalent to 10 mg/kg bw per day (Sinkeldam & van der
    Heijden, 1974).

    Groups of 10 Wistar-derived rats of each sex received guazatine (54.8%
    w/w) in the diet at 800 ppm for six weeks and then 1200 ppm for eight
    weeks or control diet for 14 weeks. Haematological and urinary
    parameters were measured in samples taken at week 13, and serum
    enzymes and total protein were measured in samples taken at
    termination at week 14. There were no deaths or adverse clinical
    signs. Body-weight gain was reduced in animals of each sex by
    approximately 6% at week 6 and by 8-10% at termination. Food
    efficiency was unaffected in males but reduced in females.
    Haematological parameters were unaltered by treatment. In males, serum
    aspartate aminotransferase activity was increased and alkaline
    phosphatase activity decreased. Females had an increased urine volume
    with an associated decrease in specific gravity; the report does not
    indicate whether samples were collected under fasting conditions.
    Increases in relative adrenal, testis, and heart weights were seen in
    treated animals, without abnormal pathological findings. As data on
    individual animals and absolute organ weights were not presented, the
    significance of these findings is uncertain. In treated females, high
    levels of 'iron' deposition in the spleen were reported. The thyroids
    of two females given guazatine were increased in weight and contained
    small follicles lined with large epithelial cells that had small
    apical nuclei. A re-evaluation of salivary gland tissues from this
    study, reported very briefly by Til & Hendricksen (1976), described
    hyperplasia in the epithelial lining of the excretory ducts of the
    parotid salivary glands, with associated mononuclear cell infiltrates
    in some treated animals. No NOAEL was identified (Til & Feron, 1975).

    Panoctine 42 (purity unspecified) was administered to groups of 10
    Wistar-derived rats of each sex at 0 or 1500/2000 ppm in the diet; the
    level was increased from 1500 to 2000 ppm after week 4. Samples for
    examination by haematology, limited clinical chemistry, and urinalysis
    were taken during week 13. Animals were killed at week 14 and examined
    grossly and histologically. There were no deaths or adverse clinical

    signs. Body-weight gain was reduced by about 10% from week 2 in males
    and by about 8% from week 6 in females. Food efficiency was reduced in
    males for the first 12 weeks of the study. Serum proteins and alkaline
    phosphatase activity were reduced in both treated groups. In treated
    females, the leukocyte counts were increased (by 30%), and
    differential counts indicated an increase in neutrophils and a
    decrease in eosinophils. Increased relative weights of the liver (by
    8%) and kidney (by 14%) were seen in treated males, without associated
    histological findings. In females, an increased relative weight of the
    heart (by 17%) and decreased thyroid weights (by 8%) were reported,
    again without associated histological findings. Urinary aspartate
    aminotransferase activity, proposed as a measure of renal toxicity,
    was increased in treated animals, and urine volume was increased and
    specific gravity decreased in animals of each sex. These findings
    taken together are indicative of renal toxicity, though no associated
    findings were reported at pathological examination. The only
    histopathological finding of note was hyperplasia of the epithelial
    lining of the excretory ducts of the parotid salivary glands with
    associated mononuclear cell infiltrates in six males and six females
    in the treated groups and in none of the controls (rho < 0.01). No
    NOAEL was identified (Til & Hendricksen, 1976).

     Dogs

    In a range-finding study in beagle dogs, guazatine (purity, 67.9%)
    given at 9.4 mg/kg bw per day by gavage for four days induced
    body-weight loss in one of four animals. Subsequent administration of
    14 mg/kg bw per day for four days induced marked body-weight loss and
    reduced food consumption in all animals. The pathological findings
    indicated local irritation of the gastrointestinal tract.
    Administration of 374 ppm guazatine in the diet (12-15 mg/kg bw per
    day) resulted in reduced food consumption, reduced body-weight gain,
    and increased activities of aspartate aminotransferase, alanine
    aminotransferase, and lactate dehydrogenase in serum after two weeks
    (Goburdhun & Carter, 1989).

    Groups of four male and four female beagle dogs received guazatine
    (purity, 54.8%) in the diet at levels of 0, 60, 200, or 300/600 ppm
    for two years; the level of 300 ppm was increased to 600 ppm after 26
    weeks. Extensive observations were made, including haematological,
    clinical chemical, and urinary determinations at 12/13, 26, 52, 78,
    and 104 weeks, and tests for liver function (bromosulfthalein) and
    kidney function (phenol red excretion) at 26, 52, and 104 weeks. Gross
    and histopathological examinations were performed at termination at
    week 104, although the results of histopathology in individual animals
    were not reported. One control male died due to urolithiasis. There
    was no evidence of treatment-related changes in clinical signs.
    Body-weight gain and food consumption were unaffected by exposure. A
    decreased leukocyte count (by 12-25 %) was present in animals at the
    high dose, particularly females, from week 13 onwards, achieving
    statistical significance at week 52, although there were no marked
    alterations in differential counts. Sporadic changes in other
    haematological, clinical chemical, and urinary parameters were not

    consistent or of statistical significance. The results of liver and
    kidney function tests were similar for control and treated groups.
    Increases in absolute and relative ovarian weights were seen in
    females at the high dose, although the significance of these changes
    is unclear as there was considerable inter-animal variation and no
    obviously associated histological findings. Given the uncertainties
    about the findings at the high dose, the NOAEL was 200 ppm, equivalent
    to 5 mg/kg bw per day (Reuzel et al., 1976).

    Groups of four beagle dogs of each sex received guazatine (purity,
    70.6%) in the diet for 52 weeks. Animals were given 400 g of diet per
    day containing 0, 25, 75, or 250 ppm guazatine; the achieved intakes
    were about 0, 0.8, 2.5, or 8 mg/kg bw per day. Extensive observations
    included ophthalmoscopy at 0, 13, 26, and 51 weeks and haematology,
    clinical chemistry, and urinalysis at 0, 6, 13, 26, and 51 weeks.
    Animals were killed at week 52 and examined grossly; 13 tissues were
    weighed, and more than 40 tissues were investigated by histopathology.
    Owing to poor weight gain, one control and two males at the high dose
    received extra food, which reversed the condition. In females, there
    was a clearly dose-related reduction in body-weight gain starting at
    week 10 and continuing throughout the study, with reductions of 30% in
    comparison with controls in the group at the high dose, 20% in those
    at the intermediate dose, and 12% in those at the low dose. Food
    consumption was reduced in females receiving 75 or 250 ppm, with
    occasional reductions in those at 25 ppm. Haematology showed no
    consistent time- or dose-related reactions to treatment. Marked
    increases (up to sixfold) in serum alanine aminotransferase activity
    were seen throughout the study in males and females receiving 250 ppm.
    Serum aspartate aminotransferase activity was increased in males at
    the high dose throughout the study; an apparent increase in alkaline
    phosphatase activity in males appears to be related to differences in
    pretreatment values. A slight increase in liver weight was noted in
    females at the high dose. Reduced prostate weights in males at the low
    dose, increased ovarian weights in all treated females, and increased
    thyroid weights in females at the intermediate dose did not show
    dose-response relationships and were not clearly related to treatment,
    although effects on ovarian weights were also see in the study of
    Reuzel et al. (1976). Gross and microscopic examination showed no
    changes. The reduced body-weight gain of females appeared to be due
    only in part to unpalatability, as food conversion efficiency was also
    reduced. The NOAEL was 25 ppm, equivalent to 0.8 mg/kg bw per day, on
    the basis of marked reductions in body-weight gain in females at 75
    and 250 ppm and clear increases in serum enzyme activities, indicative
    of effects on the liver, in animals of each sex at 250 ppm (Oshodi &
    Thompson, 1993).

     (c)  Long term toxicity and carcinogenicity

     Mice

    Groups of 10 male and 10 female CD-1 mice received guazatine (purity,
    70.6%) in the diet at 0, 50, 120, or 300 ppm for one year; the
    achieved doses were 0, 7.6, 19, or 55 mg/kg bw per day in males and 0,

    10, 25, or 67 mg/kg bw per day in females. All animals were examined
    macroscopically. A wide range of tissues from animals receiving 0 or
    300 ppm and those that died before week 52 were examined
    histologically; the gall-bladder, kidney, liver, lung, salivary gland,
    and grossly abnormal organs from all other animals were also examined
    histologically. One male and one female at 120 ppm and two females at
    300 ppm died during the study. There were no clinical signs associated
    with treatment. Body-weight gain was reduced from week 4 in animals of
    each sex at 300 ppm; by week 52, the weight gain of males was 72% that
    of controls and that of females 66% of control values. Food
    consumption was similar in treated and control animals. The relative
    liver weights were increased by about 10% in all treated males. The
    only notable finding at gross necropsy was an increased incidence of
    ovarian cysts in females receiving 120 or 300 ppm (9/10 versus 6/10 in
    controls). Microscopic examination showed increased incidences of
    lymphocytic infiltration of the kidney and ovarian cysts in females at
    the high dose (the ovaries of those at the intermediate dose were not
    examined), of hepatocellular adenoma in males at the high dose (2/10
    versus 0/10 in controls), and a decreased incidence of lymphoid foci
    of the mandibular salivary gland in females at the intermediate and
    high doses. The finding of hepatocellular adenomas may indicate that
    guazatine reduces the time to onset of the liver turnouts seen
    commonly in mice. Given the small group size, limited examination of
    tissues from animals at low and intermediate doses, and clear effects
    at 300 ppm, there was no NOAEL (Heath et al., 1995).

    In the part of the same study designed to test for carcinogenicity,
    groups of 50 male and 50 female CD-1 mice received guazatine (purity,
    70.6%) in the diet at 0, 50, 120, or 300 ppm for two years; the
    achieved doses were 0, 6.8, 17,or 47 mg/kg bw per day in males and 0,
    8.7, 21, or 57 mg/kg bw per day in females. All animals were examined
    macroscopically, and a wide range of tissues from animals receiving 0
    or 300 ppm or that died before week 104 were examined histologically;
    only the gall-bladder, kidney, liver, lung, salivary glands, and
    grossly abnormal organs from animals in other groups were examined.
    Blood samples for differential leukocyte counts were taken from all
    surviving controls and animals at 300 ppm during weeks 52, 78, and
    103.

    The pattern of deaths was similar in treated and control groups, with
    > 50% survival in all groups until week 100 in males and week gg in
    females. Reduced body-weight gain was seen at the high dose from week
    2 onwards, the deficit being approximately 20% at termination. During
    the second year, males at the intermediate dose lost more weight than
    controls, while females at this dose gained more weight than controls.
    Food consumption, differential leukocyte counts, and findings at gross
    necropsy were unaffected by treatment. Increased absolute and relative
    weights of the kidney and liver were seen in females at the
    intermediate but not the high dose.

    The incidences of a range of neoplastic and non-neoplastic lesions
    showed evidence of treatment-related effects (Table 5). The incidences
    of haemangiosarcoma of the liver in males at the intermediate and high
    doses and of the spleen in males at the high dose were higher than
    those in historical controls, which were 04% in males and 0-3% in
    females for liver and 0-2% in males and 045% in females for spleen.
    The high incidence of haemangiosarcoma in females at the low dose is
    considered to be a chance finding, as them was no dose-response
    relationship. The incidence of hepatocellular carcinoma in females at
    the high dose was greater than that in historical controls (0-4%). The
    incidences of renal adenoma and carcinoma in male historical controls
    were 0-4% and 0-2%, respectively, indicating that the incidence of
    renal adenoma in males at the high dose is a significant finding.

    The presence of rare tumours, including malignant ones, at multiple
    sites was considered by the Meeting to be of concern. The mechanism by
    which these tumours are induced is not evident from the available
    data. Guazatine is not genotoxic (see below), indicating the mechanism
    is probably nongenotoxic, although haemangiosarcomas are generally
    associated with a genotoxic mechanism. As them was no evidence of
    marked toxic or hyperplastic responses in the affected tissues and
    them was evidence of a dose-response relationship, it was not clear
    that these tumours are a direct result of disrupted cell function
    associated with exposure to doses above the maximum tolerated dose.
    Although only a limited range of tissues was examined from animals at
    the low and intermediate doses, it included those with the most
    significant effects at the high dose. The NOAEL was 50 ppm, equal to
    6.8 mg/kg bw per day, on the basis of effects on body-weight gain and
    the occurrence of haemangiosarcomas in males at 120 ppm, marked
    effects on body-weight gain, and a range of neoplastic and
    non-neoplastic changes at 300 ppm (Heath et al., 1995).

    The findings of another two-year study with guazatine in mice were
    summarized in abstract form only. Guazatine (as iminoctadine) was
    given to groups of 80 male and 80 female specific-pathogen-free
    ICR-Crj mice at dietary levels of 0,10, 100, or 300 ppm. Eight animals
    of each sex at each dose were killed at weeks 26 and 52 for
    examination. At 300 ppm (26 mg/kg bw per day in males, 30 mg/kg bw per
    day in females), males and females showed a remarkable depression in
    body-weight gain, decreased food efficiency, and anaemia at weeks 26
    and/or 52, increased plasma urea nitrogen level (only in females),
    increased alkaline phosphatase level (at week 26 in animals of each
    sex and also at week 52 in females), and increased weights of the
    kidney (absolute and relative) and liver (relative). Histological
    examination revealed swelling of hepatocytes and proximal tubular
    cells in animals of each sex. There were increased incidences of
    subcutaneous oedema in females and hydrothorax in males. The
    incidences of splenic atrophy in animals of each sex, atrophy and/or
    brown pigment deposition of the ovary, and glandular epithelial
    atrophy of the Harderian gland in males were significantly increased.
    The incidence of renal epithelial tumours increased slightly in males.
    No toxic changes were seen at the other doses, except for slightly


        Table 5. Lesions (as % of samples examined) seen in CD1 mice exposed to guazatine in the diet 
    for two years

                                                                                                    

    Lesion                                         Guazatine (ppm)
                                                                                                    
                                       Males                           Females
                                                                                                    
                                       0       50      120     300     0       50      120     300
                                                                                                    

    Brain mineralization               22      26      33      20      8       16      10      22*
    Caecal carcinoma                   0       0       0       0       0       0       0       2
    Renal tubular carcinoma            0       0       0       2       0       0       0       0
    Renal tubular adenoma              2       2       2       8       0       0       2       0
    Renal glomerular amyloid           12      8       16      10      24      8       8       0*
    Hepatocellular carcinoma           22      24      22      22      2       2       2       10
    Liver haemangiosarcoma
    Multifocal                         0       0       2       4       0       0       2       0
    Other                              2       2       6       6       0       6       2       0
    Increase in bronchus-associated 
      lymphoid tissue                  10      12      12      20      4       14      16      28*
    Lung lymphoid foci                 0       0       2       0       0       0       4       10*
    Spleen haemangiosarcoraa           0       0       2       4       2       0       2       4
    Vaginal keratinized epithelium     -       -       -       -       15      -       -       43
                                                                                                    

    -, not determined
    * One-tailed p < 0.05, Fisher exact test
    

    increased incidences of swelling of proximal tubular cells in animals
    of each sex at 100 ppm (Maita et al., 1985).

     Rats

    Groups of 60 male and 60 female Wistar-derived rats, in two parallel
    series initiated two months apart, received diets containing guazatine
    (54% solution) at 0, 20, 60, or 200 ppm for two years. A group
    receiving 6 ppm was terminated after a few months, as the results of
    other studies indicated that 20 ppm was probably a NOAEL. Blood and
    urine samples were taken from 10 fasted animals of each sex at each
    dose at various times during the study for haematological, limited
    clinical chemical, and urinary analyses. All animals were examined
    grossly  post mortem. A wide range of organs from surviving animals
    in the first series (11-20) were weighed, but only kidney, spleen, and
    adrenals from rats in the second series were weighed. A full
    histological examination was performed on tissues from 20 rats of each
    sex in the control and high-dose groups in the first series, and gross
    lesions, tumours, adrenals, thyroid, and pituitaries from all animals
    were examined.

    Behaviour and clinical signs were reported to be unaffected by
    treatment. There were more early deaths (before week 72) among treated
    males, but overall survival to week 104 was satisfactory and similar
    in all groups. Body weights were similar in all groups until week 88,
    when all treated females showed a deficit in comparison with controls,
    which lasted until termination. Food consumption and conversion
    efficiency were similar in all groups over the first four weeks.
    Leukocyte counts were decreased (by about 11%) in males at the high
    dose from week 26, with no consistent change in differential counts;
    females showed sporadic changes in leukocyte counts with no consistent
    pattern. Urinalysis gave similar findings in all groups. Reduced
    relative testicular weights (by 11%) were seen in males at the high
    dose, although the absolute weights were similar since the mean body
    weight was 8% higher. In treated females, the relative weights of the
    spleen, brain, and kidney were increased; however, as the absolute
    weights were decreased and there was no evidence of a dose-response
    relationship, this finding is probably secondary to the effects on
    body weight. Occasional increases in the incidences of non-neoplastic
    lesions were reported, but the only statistically significant
    (p < 0.05) increases were for chronic respiratory disease and mammary
    gland inflammation or dilatation in females at the high dose. The
    number of tumour-bearing animals and the total numbers of tumours were
    similar in all groups. The incidence of monocytic leukaemia was
    increased in males and females at the high dose, occurring in four
    males and three females, with none in male and in one female controls.
    As the total numbers of animals examined is unclear, this may be a
    chance finding. Although the study indicates that guazatine has no
    marked toxicity, the limited, investigations performed and the unusual
    design make interpretation of the findings difficult. No NOAEL was
    identified (Til et al., 1976a).

    Groups of 20 male and 20 female Sprague Dawley rats received guazatine
    (purity, 70.6%) in the diet at 0, 50, 150, or 350 ppm for one year.
    The achieved intakes of guazatine were 0, 2.5, 7, or 19 mg/kg bw per
    day for males and 0, 3, 9, or 22 mg/kg bw per day for females. Samples
    for haematological, clinical chemical, and urinary analyses were
    obtained from 10 animals of each sex in each group at weeks 26 and 50
    or 51. At week 52, the animals were killed and examined grossly.
    Selected tissues were weighed, and samples from a wide range of organs
    from controls and animals at the high dose were examined
    histologically; gross lesions, kidneys, liver, lung, and salivary
    glands from animals at the low and intermediate doses were also
    examined histologically.

    Behaviour, clinical signs, body-weight gain, food consumption, urinary
    parameters, and the findings at gross necropsy were similar in all
    groups. None of the controls but one male at the low dose, two each at
    the intermediate and high doses, and one female at 150 ppm died. Total
    leukocyte and lymphocyte counts were reduced in males at 350 ppm at
    both 26 and 51 weeks, but increases were seen in treated females.
    Serum alanine aminotransferase activity was decreased markedly (by
    >40%) at both sampling times in males and females receiving 350
    ppm, but there were occasional deficits in other serum enzyme
    activities (particularly aspartate aminotransferase); total protein
    was similar in all groups. Dose-related decreases in absolute and
    relative prostate weights were significant (rho < 0.05) at both 150
    and 350 ppm and were associated with a low incidence of hyperplasia at
    350 ppm. Females had dose-related increases in the relative and
    absolute weights of the adrenals (by < 10%) and ovary by
    (< 50%). Five males at the high dose but none of the controls had
    testicular atrophy. Mononuclear-cell infiltration of the parotid
    salivary gland was increased in males (6/20 versus 1/20) and females
    (5/20 versus 1/20) receiving 350 ppm. The NOAEL was 150 ppm, equal to
    7 mg/kg bw per day, on the basis of decreased leukocyte count,
    prostatic hyperplasia, decreased alanine aminotransferase activity,
    and effects on the salivary gland at 350 ppm. The minimal changes seen
    at 150 ppm were not consistent or of sufficient magnitude to be
    considered adverse (Heath et al., 1994).

    In the phase of the study that addressed carcinogenicity, groups of 50
    Sprague Dawley rots of each sex received guazatine (purity, 70.6%) in
    the diet at 0, 50, 150, or 350 ppm for two years. The achieved intakes
    were about 0, 2.5, 7, and 19 mg/kg bw per day for males and 0, 3, 9,
    and 22 mg/kg bw per day for females. Samples for haematological,
    clinical chemical, and urinary analyses were obtained from 10 rats of
    each sex per group at weeks 53 (for haematology only), 78, and 101 or
    103. At week 104, the animals were killed and underwent a full gross
    examination. Selected tissues were weighed, and samples from a wide
    range of organs from controls and animals at the high dose were
    examined histologically; gross lesions, kidneys, liver, lung, and
    salivary glands from animals at the low and intermediate doses were
    also examined histologically.

    Behaviour, clinical signs, and organ weights were similar in all
    groups. Survival was acceptable (50% at week 94) and similar in all
    groups. Low body weights were seen consistently in females at the high
    dose from week 9 and in males at this dose from week 20; in the latter
    quarter of the study, females at the low and intermediate doses had
    reduced body-weight gain in comparison with controls. Males at the
    high dose showed consistent reductions in leukocyte and platelet
    counts but increased erythrocyte counts. In females at 350 ppm,
    erythrocyte counts were reduced; an increase in leukocyte counts at
    termination was due to a neurilemmoma in one animal. Reduced
    activities of serum alanine and aspartate aminotransferases (by
    < 50%) were seen consistently in animals of each sex receiving 350
    ppm. Reduced urinary pH was seen at the high dose in animals of each
    sex throughout the study. A slight excess of abnormal findings in
    lymph nodes was seen in females at this dose, but no individual effect
    was significant when compared with the very low background incidence.
    The incidences and severity of a range of lesions in salivary glands,
    lymph nodes, ovaries, spleen, and pituitary were increased in females
    receiving 350 ppm. In males, the only notable finding was increased
    severity and incidence of testicular germinal epithelial degeneration
    at 350 ppm. A rare malignant oligodendroglioma was found in the brain
    of a single female at the high dose, but there was no evidence of
    treatment-related carcinogenicity in any other animal. The NOAEL was
    150 ppm, equal to 7 mg/kg bw per day, on the basis of reduced
    body-weight gain and a range of clinical chemical and histological
    findings at 350 ppm (Heath et al., 1994).

    The findings of a two-year study of guazatine in rats were summarized
    in abstract form only. Guazatine (as iminoctadine) was presented to
    groups of 80 male and 80 female Fischer 344 rats at dietary levels of
    0, 10, 100, or 300 ppm for two years. At 6 and 12 months, eight
    animals of each sex from each group were killed for examination.
    Animals of each sex at 300 ppm (11 mg/kg bw per day for males, 14
    mg/kg bw per day for females) had remarkably depressed body-weight
    gain, decreased food efficiency, higher mortality rates, a tendency to
    anaemia, decreased total protein, and increased kidney and adrenal
    weights; decreased potassium and albumin and increased calcium,
    aspartate aminotransferase and gamma-glutamyltransferase activities,
    and spleen weight were also observed. Histologically, swelling,
    degeneration, and necrosis of renal tubular cells and metaplasia in
    the glandular stomach were observed in animals of each sex. The
    incidence of sperm granuloma in the deferent duct and/or epididymides
    was significantly increased, and the incidences of leukaemia in males
    and of adrenal phaeochromocytoma in animals of each sex were slightly
    increased. At 100 ppm, males had a slightly higher mortality rate, and
    females had depressed body-weight gain; decreased potassium and
    increased calcium and gamma-glutamyltransferase activity were
    occasionally observed in animals of either sex. The kidney and spleen
    weights were increased, and the incidences of intestinal metaplasia
    and sperm granuloma were significantly higher than in controls. At 10
    ppm, no toxic change attributable to guazatine were seen (Hirano et
    al., 1985).

     (d)  Genotoxicity

    Guazatine has been tested for its potential to induce gene mutations,
    sister chromatid exchange, and chromosomal aberrations  in vitro and
    micronuclei  in vivo. Negative results were obtained in all studies.
    The data are summarized in Table 6.

     (e)  Reproductive toxicity

     (i)  Multigeneration reproductive toxicity

     Rats

    Groups of 10 male and 20 female Wistar-derived rats received diets
    containing 0, 60, or 200 ppm guazatine (purity, 54.8%) over four
    generations. The reporting of the study lacks the results for
    individual animals, dietary analyses, time to mating, length of
    gestation, absolute organ weights, and other data. F0 animals
    received the test diets for 12 weeks before the first mating to
    produce the F1a generation; the second mating eight weeks later
    produced the F1b generation. Litters were culled to eight pups on day
    1 after birth, and the F1a litters were discarded at weaning. Animals
    were selected from the F1b litters at weaning to produce the F2
    generation, with mating identical to that for F0 animals, and
    similarly for the F3 generation from the F2b litters. Groups of 10
    animals of each sex from the F3b litters were selected at weaning and
    given test diets for four weeks; they were then killed and subjected
    to gross and histopathological examinations.

    Treatment had no effect on survival, clinical signs, litter size, sex
    ratio, or resorption rates. Minor variations in the body weights of
    pups appeared to be secondary to variations in litter size. F3b
    animals receiving 60 or 200 ppm had increased, relative  kidney
    weights (by 8% at 60 ppm and 12-17% at 200 ppm), and increased
    relative thymus weights (by 19-34%) were seen in all treated groups. A
    dose-related reduction in relative testicular weights (8%) was also
    seen. Histological examination gave no evidence of treatment-related
    effects in any organ. In a summarily described extension of the study,
    it was reported that males in F4b litters given test diets for four
    weeks after weaning had reduced relative weights of the testis (9%)
    and increased relative weights of the thymus (25%) at both doses, and
    females at the high dose had increased relative kidney weights (12%).
    Again, no treatment-related histological findings were reported. This
    study shows that guazatine does not adversely affect reproductive
    outcome in rats. The finding of increased thymus weights may indicate
    that it affects the control of thymic growth or involution; such
    effects may not be detectable in older animals when thymic involution
    is well advanced. Given potential concern about effects on the thymus
    at both 60 and 200 ppm, no NOAEL was identified (Til et al., 1976b).


        Table 6. Results of genotoxicity assays on guazatine

                                                                                                                       

    End-point            Test system              Concentration/          Purity     Results          Reference
                                                  dose                    (%)
                                                                                                                       

    In vitro
    Reverse mutation     S. typhimurium           0.6-50 µg/plate         73         Negativea        Wilmer (1983a)
                         TA98, TA 100.                                               Cytotoxic
                         TA1535. TA1537.                                             at 50 g/plate
                         TA1538

    Reverse mutation     S. typhimurium           To cytotoxic levels     NR         Negative         Moriya et al.
                         TA98, TA100, TA1535,     (numbers not given)                                 (1983)
                         TA1537, TA1538, and
                         E. coli WP2 hcr

    Gene mutation        Chinese hamster          25-100 nl/ml -S9        NR,        Negativea        Davis (1983a)
                         ovary cells (line K1),   50-200 nl/ml +S9        approx.    Cytotoxic at
                         hprt locus                                       70%        highest
                                                                                     concentrations

    Sister chromatid     Chinese hamster          5-30 nl/ml              NR,        Negativea        Davis (1983b)
    exchange             ovary cells (line K1)                            approx.    Cytotoxic
                                                                          70%        at > 35 nl/ml

    Chromosomal          Human                    3.7-100 g/ml -S9        73         Negativea        Wilmer (1983b)
    aberration           lymphocytes              14.8-400 g/ml +S9                  Cytotoxic
                                                                                     at highest
                                                                                     concentrations

    In vivo
    Micronucleus         Mouse (5/sex at          150 mg/kg bw by         73         Negative         Willems (1983)
    formation            each time) bone          gavage in saline                   No effect on
                         marrow                   (approx. 50% of                    P:N ratio
                                                  LD50); 24, 48, and 
                                                  72h
                                                                                                                       

    Table 6 (continued)

    NR, not reported; S9, exogenous metabolic activation system from Aroclor 1254-induced rat liver preparations; 
    P:N, polychromatic:normochromatic erythrocytes 
    a With and without metabolic activation
    

    The reproductive effects of guazatine (purity, 70.6%) were
    investigated in a two-generation (one litter per generation) study in
    which groups of 28 young adult CD rats received diets containing
    guazatine at 0, 50, 150, or 350 ppm, equal to a minimal intake of 0,
    3, 10, or 22 mg/kg bw per day in males and 0, 4, 11, or 25 mg/kg bw
    per day in females. Animals were given the treated diet for 10 weeks
    before F0 mating (1:1) and continuously until sacrifice. Groups of 24
    male and 24 female F1 animals were mated at about 17 weeks; they were
    then killed and examined grossly, and reproductive organs and
    pituitary, liver, adrenals, and salivary glands were weighed and
    prepared for histological examination. Pups that died before weaning
    were examined grossly. On day 21, one pup of each sex per litter was
    examined grossly, and the liver and salivary glands were removed for
    histological examination. All other pups were examined externally.
    Treatment had no effect on clinical signs, survival, time to mating,
    fertility indices, gestation length, litter size, gestation indices,
    or lactation indices. Slight reductions in body-weight gain (5-10%)
    were closely related to reductions in food consumption (4-9%). Slight
    reductions in survival to day 21 (81-83% versus 87% in controls) and
    litter weight at day 21 (about 8%) were seen in F1 pups in all
    treated groups; as there was no dose-response relationship and the
    effects were of small magnitude and were not seen in the F2
    generation, they were considered not to be of biological significance.
    Post-mortem examination showed no treatment-related effects; however,
    the thymus was not weighed or investigated specifically. The NOAEL was
    350 ppm, equal to 22 mg/kg bw per day, the highest dose tested
    (Barton, 1993).

     (ii)  Developmental toxicity

     Rats

    The developmental effects of guazatine were investigated in animals
    selected from the F2b and F3b litters of the study of Til et al
    (1976b), described above Groups of five males and 15 females were
    selected at weaning and maintained on test diets (0, 60, or 200 ppm
    guazatine) until mating (1:3) at week 12; it was not clear whether the
    test diets were given during gestation. On day 21 of gestation, the
    dams were killed, their reproductive tracts investigated, and pups
    examined for gross abnormalities and skeletal defects (with Alizarin
    Red S staining); effects on soft tissues were studied only in controls
    and rats at 200 ppm (by Wilson sectioning). F3b animals showed a
    dose-related reduction in litter size, with 10.7 pups in controls, 10
    at 60 ppm, and 8.9 at 200 ppm; at 200 ppm, this reduction was
    associated with increased pre- and postimplantational losses in two
    dams. Mean fetal weight, and hence litter weight, was reduced at 200
    ppm in the F3b-derived group There were no notable visceral effects;
    although there was an indication of reduced ossification in treated
    animals, this was not consistent with regard to dose, generation, or
    site. Reduced litter size was seen mainly in two animals and is not
    consistent with the results of the main study. The NOAEL was 200 ppm,
    equal to 12 mg/kg bw per day, as guazatine did not directly affect the
    developing fetus or the dam (Til et al., 1976b).

    In a range-finding study, pregnant Sprague Dawley rats died after
    receiving guazatine (purity, 70.9%) at levels of 40, 80, or 120 mg/kg
    bw per day by gavage on days 6-16 of gestation. At 20 mg/kg bw per
    day, there was no evidence of toxicity. Necropsy of animals at doses
    > 40 mg/kg bw per day showed marked irritation of the
    gastrointestinal tract (Hazelden, 1987).

    Four groups of 27 timed-mated Sprague Dawley rats received guazatine
    (purity, 70.9%) in distilled water by garage on days 6-16 of presumed
    gestation at doses of 0, 5, 10, or 20 mg/kg bw per day, on the basis
    of the findings in the range-finding study. An adequate range of
    examinations was performed, and animals were necropsied on day 20
    after nitrogen asphyxiation. About two-thirds of the fetuses from each
    litter were examined for gross external and visceral abnormalities
    before staining with Alizarin Red S for observation of skeletal
    abnormalities and variants. The remainder were examined by free-hand
    dissection (Wilson technique) for soft-tissue abnormalities. One
    control and two animals at the high dose died during the study due to
    dosing accidents. There were no signs of maternal toxicity;
    body-weight gain was similar in all groups, although food consumption
    was decreased marginally (by 1-2%) in rats at 10 and 20 mg/kg bw.
    There were no effects on litter size or fetal weight, or on soft
    tissues or viscera. There was an indication of slightly retarded
    development of some bones (e.g. scapula and pectoral girdle) at 10 and
    20 mg/kg bw, but the findings were not significant, and the overall
    degree of skeletal ossification was unaffected by treatment. The lack
    of maternal toxicity at the high dose may be considered to have
    compromised the study, but use of 20 mg/kg bw per day is considered
    acceptable in view of the 40% mortality rate at 40 mg/kg bw per day in
    the range-finding study. The NOAEL for maternal toxicity,
    fetotoxicity, and teratogenicity was 20 mg/kg bw per day, the highest
    dose tested (Hazelden & Wilson, 1986).

     Rabbits

    Groups of 15 timed-mated New Zealand white rabbits received guazatine
    (purity, 67.9%) in distilled water by gavage on days 6-18 of presumed
    gestation at doses of 0, 2.8. 5.6, or 11 mg/kg bw per day. An adequate
    range of examinations was performed, and necropsy was carried out on
    day 29. About two-thirds of fetuses from each litter were examined for
    gross external and visceral abnormalities before staining with
    Alizarin Red S for observation of skeletal abnormalities and variants.
    The remainder were examined by free-hand dissection for soft-tissue
    abnormalities. After marked weight loss, one animal at each dose was
    killed during the study. One animal at the high dose aborted. Reduced
    body-weight gain (76% of control weight) and reduced food consumption
    (87% of control) were seen during treatment with 11 mg/kg bw per day.
    During days 12-18 of gestation, reduced body-weight gain (87% of
    controls) was seen at 5.6 mg/kg bw per day; them was no associated
    effect on food consumption. Body-weight gain was similar in all groups
    between days 18 and 29. Slight reductions in fetal weight were evident
    at 5.6 mg/kg bw per day (by 3%) and 11 mg/kg bw per day (5%), but
    these findings may have been secondary to the reduced body-weight

    gains of the dams. There were no effects on litter size, fetal
    viability, external abnormalities, skeletal abnormalities or variants,
    extent of ossification, or soft-tissue abnormalities. The overall
    NOAEL was 5.6 mg/kg bw per day on the basis of the markedly decreased
    body-weight gain in dams at 11 mg/kg bw per day. There was no evidence
    that guazatine is fetotoxic or teratogenic to rabbits (Barton &
    Wilson, 1988).

     (f) Special studies: Dermal and ocular irritation and dermal
     sensitization

    Guazatine GTA70 (purity unspecified) was severely irritating when
    applied to rabbit skin for 4 h under occlusive conditions. The lesions
    became more pronounced with time, and severe erythema and moderate
    oedema were seen at the end of the study on day 7 (Cuthbert & Carr,
    1989).

    A 40% guazatine formulation induced ocular lesions that worsened with
    time. A 10% solution of the formulation was only slightly irritating
    to rabbit eyes (van Beek, 1974).

    In a Magnusson and Kligmann maximization protocol, guazatine GTA70
    (purity, 69.2%) did not have sensitizing potential in guinea-pigs at
    induction concentrations of 0.5% intradermally or 1% topically and a
    challenge concentration of 0.5% (Til & Keizer, 1980).

    3.  Observations in humans

    Koyama et al. (1993) reported a case of attempted suicide with a
    formulation containing 25% iminoctadine and 5%
    polyoxyethylenealkylether (Befran). The patient was admitted with
    severe cyanosis and in a stuporous state. Blood pressure and pulse
    could not be measured (the radial arteries were not palpable) until
    noradrenaline (1 mg) was administered.

    [The authors cited a similar case and some experimental work involving
    intravenous administration to dogs, which indicated that severe
    hypotension was produced by iminoctadine. Subsequent work (Koyama et
    al., 1994) on anaesthetized rats showed that iminoctadine administered
    intravenously induced marked (> 10%) tachycardia and hypotension at
    doses > 0.05 mg/kg bw  in vivo. Experiments on isolated rat aorta
    and atria  in vitro showed that the primary effect of iminoctadine
    was vasodilatory.]

    Comments

    Guazatine is a preparation of the triacetates of dimeric and trimeric
    guanidated 1,8-diamino-octane, which also contains a range of
    oligomers and reaction products. The Meeting was concerned that the
    production controls and specifications for guazatine were inadequate.
    The quoted purity of 70% is based on normalization to a component
    which comprises approximately 1.5 % of the mixture and provides no
    control over the levels of the other components. Some data were

    provided to show that the composition of the batch used in the key
    toxicity studies was similar to that of other batches produced at the
    same time, 1990-1991; however, there were no data to confirm that the
    batches used in the studies of toxicity were representative of those
    currently produced.

    Some components of guazatine were absorbed by rats to a limited extent
    after oral administration of 14C-labelled compound and then excreted
    rapidly. Within 24 h, faecal elimination represented 85-94% of the
    dose, with 3-6% in the urine and < 1% in exhaled air. The highest
    levels of radiolabel were found in the kidney and liver; there was
    evidence that the salivary, pituitary, and thyroid glands may also
    contain significant amounts of residue. A study involving treatment
    with 14 doses of 2 mg/kg bw showed limited potential accumulation in
    the liver and kidney. The results of a study by intravenous injection
    showed that some components of guazatine may be secreted back into the
    gastrointestinal tract via the stomach and salivary glands. The
    metabolism of guazatine has not been fully characterized, but
    monodeamidination and dideamidination play significant roles 
     in vivo.

    Guazatine produces severe local irritation, and single oral doses are
    of moderate toxicity, with an oral LD50 value in rats of 280 mg/kg
    bw. WHO has classified guazatine as moderately hazardous (WHO, 1996).

    In a number of short-term studies in rats, guazatine was administered
    at doses of 0, 60, 200, 800/1200, or 1500/2000 ppm in the diet for 14
    weeks. At doses of 60 and 200 ppm, the activity of serum alkaline
    phosphatase was slightly decreased, but no significant changes were
    seen in body-weight gain or in the results of pathological,
    haematological, or urinary examinations. At doses of 800 ppm and
    above, decreased body-weight gain, increased activities of alanine and
    aspartate aminotranferases, and decreased activity of alkaline
    phosphatase were found, together with pathological changes such as
    local irritation of the gut and hyperplasia of the epithelia of the
    excretory ducts of the parotid gland with mononuclear-cell
    infiltration. Increased weights of the kidney, liver, and heart were
    seen without associated histopathological changes. The overall NOAEL
    was 200 ppm, equivalent to 10 mg/kg bw per day.

    In a 13-week range-finding study, mice received guazatine at 0, 10,
    50, 200, or 500 ppm. Significantly reduced body-weight gain was seen
    in animals of each sex at 200 ppm and above. Increased liver weights
    and alterations in centrilobular hepatocytes were seen in both males
    and females at 500 ppm. Alterations in erythrocyte parameters were
    seen in animals at doses of 200 ppm and above. Although no significant
    effects were reported at 10 or 50 ppm, in view of limited histological
    investigations in the study there was no NOAEL.

    In a one-year study in dogs, guazatine was administered at 0, 25, 75,
    or 250 ppm. Reduced body-weight gain in females, increased alanine
    aminotransferase activity in animals of each sex, and increased
    aspartate aminotransferase activity in males were observed at a

    dietary concentration of 250 ppm. In females at 75 ppm, body-weight
    gain was reduced. The NOAEL was 25 ppm, equal to 0.8 mg/kg bw per day.

    Guazatine was not carcinogenic in two two-year studies in rats given
    doses of 0, 20, 60, or 200 ppm or 0, 50, 150, or 350 ppm. The
    non-neoplastic findings included reduced serum alanine and aspartate
    aminotranferases activities, salivary gland hyperplasia, and
    testicular atrophy at 350 ppm. In a two-year study of iminoctadine
    administered at 0, 10, 100, or 300 ppm, there was no reported increase
    in tumour incidence. The overall NOAEL was 150 ppm, equal to 7 mg/kg
    bw per day.

    In a study of carcinogenicity in mice, the animals received 0, 50,
    120, or 300 ppm guazatine. The incidences of malignant tumours were
    increased at 120 and 300 ppm: haemangiosarcoma of the liver and spleen
    was seen in males at 120 and 300 ppm and hepatocellular carcinoma in
    females at 300 ppm. The incidence of renal-tubular tumours (adenoma
    and carcinoma) was increased in males receiving 300 ppm. These are
    rare tumour types in the mouse strain that was used, normally being
    seen in only 0-6% of animals. Although the absolute incidences of
    these tumours in guazatine-treated animals were low and not
    statistically significant, they were clearly greater than those in
    historical controls. No convincing information was available on the
    underlying mechanism of tumour production. The non-neoplastic effects
    seen in this study were increased incidences of lymphoid foci in the
    lung, bronchiole-associated lymphoid tissue, keratinized vaginal
    epithelium, and brain mineralization in females receiving 300 ppm.
    Body-weight gain was reduced by approximately 20% in animals of each
    sex receiving 300 ppm. In addition, an abstract describing a study on
    iminoctadine (at 0, 10, 100, or 300 ppm) reported a slight increase in
    the incidence of renal epithelial tumours in male mice receiving 300
    ppm. The Meeting considered that the production of rare malignant
    tumours by an unknown mechanism is of great concern. The overall NOAEL
    for long-term administration to mice was 50 ppm, equal to 6.8 mg/kg bw
    per day, on the basis of increases in the incidence of
    haemangiosarcoma in males at 120 ppm, equal to 17 mg/kg bw day.

    Guazatine has been tested in an adequate battery of assays for
    genotoxicity. The Meeting concluded that it is not genotoxic.

    In a multigeneration study of reproductive toxicity in rats receiving
    guazatine at 0, 60, or 200 ppm, no significant effects were seen at
    the highest dose, equivalent to 12 mg/kg bw per day. In a
    two-generation study of reproductive toxicity in rats, guazatine
    administered at 0, 50, 150, or 350 ppm did not affect reproductive
    performance at the highest dose, equal to 22 mg/kg bw per day.

    In a study of developmental toxicity in rats, guazatine was
    administered at 0, 5, 10, or 20 mg/kg bw per day. The NOAEL for
    maternal toxicity, teratogenicity, and fetotoxicity was 20 mg/kg bw
    per day, the highest dose tested. In a range-finding study,
    significant mortality was seen at 40 mg/kg bw per day.

    In a study of developmental toxicity in rabbits, guazatine was
    administered at 0, 2.8, 5.6, or 11 mg/kg bw per day. There were no
    signs of fetotoxicity or teratogenicity at the highest dose. The NOAEL
    was 5.6 mg/kg bw per day on the basis of marked decreases in maternal
    body-weight gain.

    The Meeting considered that it could not establish an ADI for
    guazatine owing to the inadequate information on its composition and
    concerns about the production of rare malignant tumours in mice.

    Toxicological evaluation

     Levels that cause no toxic effect

         Mouse:    50 ppm, equal to 6.8 mg/kg bw per day (two-year study
                   of toxicity and carcinogenicity)

         Rat:      150 ppm, equal to 7 mg/kg bw per day (two-year study of
                   toxicity and carcinogenicity)
                   350 ppm, equal to 22 mg/kg bw per day (highest dose
                   tested in a two-generation study of reproductive
                   toxicity)
                   20 mg/kg bw per day (highest dose tested in a study of
                   developmental toxicity)
         Dog:      25 ppm, equal to 0.8 mg/kg bw per day (one-year study
                   of toxicity)

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

    1.   Data on the levels of individual components in batches of
         guazatine from recent production runs
    2.   Investigation of the mechanism of tumour production in mice
    3.   Clarification of the extent of absorption, excretion, and
         metabolism of all components of guazatine
    4.   Clarification as to whether the stated doses used in the studies
         of toxicity were expressed as free base or triacetate

    References

    Atkinson, C., Perry, C.J., Hudson, P. & Robb, D.T (1990) Guazatine 13
    week dietary dose range finding study in mice. Unpublished study from
    Inveresk Research International (report No. 7552). Submitted to WHO by
    Rhône Poulenc Secteur Agro, Lyon, France.

    Appelman, L.M. (1980) Acute inhalation toxicity of guazatine
    triacetate in rats. Unpublished study from CIVO-TNO (report No. R
    6636). Submitted to WHO by Rhône Poulenc Secteur Agro, Lyon France

    Barton, S.J. (1993) Guazatine: Two-generation reproduction study in
    rats. Unpublished study from Inveresk Research International (report
    No. 7854). Submitted to WHO by Rhône Poulenc Secteur Agro, Lyon,
    France.

    Barton, S.J. & Wilson, I.A. (1988) Guazatine: Teratugenicity study in
    rabbits. Unpublished study from Inveresk Research International
    (report No. 5344). Submitted to WHO by Rhône Poulenc Secteur Agro,
    Lyon, France.

    van Beek, L. (1974) Eye irritation test with Panoctine 40 in albino
    rabbits. Unpublished study from CIVO-TNO (report No. R4363). Submitted
    to WHO by Rhône Poulenc Secteur Agro, Lyon, France.

    van Beek, L. (1980) Acute dermal toxicity study with Panoctine plus
    300020 in albino rabbits. Unpublished study from CIVO-TNO (report No.
    R6703). Submitted to WHO by Rhône Poulenc Secteur Agro, Lyon, France.

    van Beek, L., van Oostrum, E.C.M. & Immel, H.R. (1976) Acute dermal
    toxicity of Panoctine 42 in albino rabbits. Unpublished study from
    CIVO-TNO (report No. R4921). Submitted to WHO by Rhône Poulenc Secteur
    Agro, Lyon, France.

    Cameron, B.D. & Phillips, M.W.A. (1986) The disposition of guazatine
    in the lactating cow. Unpublished study from Inveresk Research
    International (report No. 4141 ). Submitted to WHO by Rhône Poulenc
    Secteur Agro, Lyon, France.

    Cameron, B.D., Mutch, P.J. & Scott, G. (1989) The metabolism of
    [14C]-guazatine in the rat. Unpublished study from Inveresk Research
    International (report No. 4826). Submitted to WHO by Rhône Poulenc
    Secteur Agro, Lyon, France.

    Cuthbert, J.A. & Cart, S.M.A. (1989) Guazatine: Acute dermal
    irritation test in rabbits. Unpublished study from Inveresk Research
    International (report No. 5505). Submitted to WHO by Rhône Poulenc
    Secteur Agro, Lyon, France.

    Cuthbert, J.A. & D'Arcy-Burt, K.J. (1986) Guazatine: Acute dermal
    toxicity in rats (limit test) Unpublished study from Inveresk Research
    International (report No. 3461). Submitted to WHO by Rhône Poulenc
    Secteur Agro, Lyon, France.

    Davis, P.B. (1983a) An investigation into the possible induction of
    point mutations at the HGPRT locus of Chinese hamster ovary cells by
    GTA70 (guazatine triacetate) Unpublished study from CIVO-TNO (report
    No. R83/86). Submitted to WHO by Rhône Poulenc Secteur Agro, Lyon,
    France.

    Davis, P.B. (1983b) An investigation into the possible induction of
    sister chromatid exchanges in Chinese hamster ovary cells by GTA 70
    (guazatine triacetate). Unpublished study from CIVO-TNO (report No.
    R83/85). Submitted to WHO by Rhône Poulenc Secteur Agro, Lyon, France.

    De Groot, A.P. (1976a) Determination of the acute oral toxicity of
    Panoctine 42 in cats. Unpublished study from CIVO-TNO. Submitted to
    WHO by Rhône Poulenc Secteur Agro, Lyon, France.

    De Groot, A.P. (1976b) Determination of the intraperitoneal toxicity
    of Panoctine 42 in rats. Unpublished study from CIVO-TNO (report No.
    21-1-76). Submitted to WHO by Rhône Poulenc Secteur Agro, Lyon,
    France.

    Goburdhun, R. & Carter, P.B. (1989) Guazatine: Oral maximum tolerated
    dose study in dogs. Unpublished study from Inveresk Research
    International (report No. 5424). Submitted to WHO by Rhône Poulenc
    Secteur Agro, Lyon, France.

    Hazelden, K. (1987) Guazatine: Dose range finding study in rats,
    preliminary to teratogenicity study. Unpublished study from Inveresk
    Research International (report No. 3441). Submitted to WHO by Rhône
    Poulenc Secteur Agro, Lyon, France.

    Hazelden, K.E & Wilson, J.A. (1986) Guazatine: Teratogenicity study in
    rats. Unpublished study from Inveresk Research International (report
    No. 3540). Submitted to WHO by Rhône Poulenc Secteur Agro, Lyon,
    France.

    Heath, J., Perry, C.J., Hudson, R, Dobb, D. & Millar, P (1994)
    Guazatine 104 week dietary study in rats with 52 week interim kill.
    Unpublished study from Inveresk Research International (report No.
    11006). Submitted to WHO by Rhône Poulenc Secteur Agro, Lyon, France.

    Heath, J., Perry, C.J., Hudson, E & Aitken, R. (1995) Guazatine 104
    week dietary study in mice with 52 week interim kill. Unpublished
    study from Inveresk Research International (report No. 11084).
    Submitted to WHO by Rhône Poulenc Secteur Agro, Lyon, France.

    Hirano, M., Maita, K., Mitsumori, K. & Shirasu, Y. (1985) 24 Month
    chronic toxicity study with guazatine in rats.  J. Toxicol. Sci., 10,
    266.

    Kato, Y., Sato, K., Maki, S., Matano, O. & Goto, K. (1985) Metabolic
    fate including deamidination of guazatine triacetate in male rats. 
     J. Pestic. Sci., 10, 661-675.

    Koyama, K., Yamashita, M., Miyauchi, T. & Goto, K. (1993) A fungicide
    containing iminoctadine causes circulatory failure in acute oral
    poisoning.  Vet. Hum. Toxicol., 35, 512.

    Koyama, K., Goto, K. & Yamashita, M. (1994) Circulatory failure caused
    by a fungicide containing iminoctadine and a surfactant: A
    pharmacological analysis in rats.  Toxicol Appl. Pharmacol., 126,
    197-201.

    Kruysse, A. & Immel, H.R. (1976) Acute inhalation toxicity study with
    Panoctine 42 in rats. Unpublished study from CIVO-TNO (report No.
    R4965). Submitted to WHO by Rhône Poulenc Secteur Agro, Lyon, France.

    Leegwater, D.C. (1975) Study on the fate of [14C, 3H guazatine
    preparation in the rat and in sandy loam. Unpublished study from
    CIVO-TNO (report No. R4823). Submitted to WHO by Rhône Poulenc Secteur
    Agro, Lyon, France.

    Leegwater, D.C. (1980) Study on the metabolic fate of [14C] Panoctine
    preparation in the rat. Unpublished study from CIVO-TNO (report No.
    R6571). Submitted to WHO by Rhône Poulenc Secteur Agro, Lyon, France.

    Maita, K., Mitsumori, K., Hirano, M. & Shirasu, Y. (1985) 24-Month
    chronic toxicity study with guazatine in mice.  J. Toxicol. Sci., 10,
    267.

    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.

    Oshodi, R.O. & Thompson, D.C. (1993) Guazatine: 52 Week dietary study
    in dogs. Unpublished study from Inveresk Research International
    (report No. 7903). Submitted to WHO by Rhône Poulenc Secteur Agro,
    Lyon, France.

    Prout, M.S. (1996) The metabolism of [14C]-guazatine in the rat.
    Unpublished study from Inveresk Research International (Supplement 1
    to report No. 4826: Cameron et al., 1989). Submitted to WHO by Rhône
    Poulenc Secteur Agro, Lyon, France.

    Reuzel, P.G.J, Til, H.P. & Kollen, C.H. (1976) Long term (two year)
    toxicity study with guazatine in beagle dogs. Unpublished study from
    CIVO-TNO (report No. R4983). Submitted to WHO by Rhône Poulenc Secteur
    Agro, Lyon, France.

    Sato, K., Kato, Y., Maki, S., Matano, O. & Goto, S. (1986) Metabolic
    fate of plant guazatine residues in male rats.  J. Pestic. Sci., 11,
    267-270.

    Sinkeldam, E. H. & van der Heijden, C.A. (1974) Sub-chronic (90-day)
    toxicity study with guazatine in albino rats (final report).
    Unpublished study from CIVO-TNO (report No. R4354). Submitted to WHO
    by Rhône Poulenc Secteur Agro, Lyon, France.

    Spanjers, M.T & Til, H.P. (1980) Determination of the acute oral
    toxicity of acetates of guanidated amines (GTA) in rats. Unpublished
    study from CIVO-TNO. Submitted to WHO by Rhône Poulenc Secteur Agro,
    Lyon, France.

    Til, H.P. & Feron, V.J. (1975) Feeding study with guazatine in rats
    for 13 weeks. Unpublished study from CIVO-TNO (report No. R4870).
    Submitted to WHO by Rhône Poulenc Secteur Agro, Lyon, France.

    Til, H.P & Hendriksen, C.F.M. (1976) Toxicity study with Panoctine 42
    (guazatine) in rats for 13 weeks. Unpublished study from CIVO-TNO
    (report No. R5062). Submitted to WHO by Rhône Poulenc Secteur Agro,
    Lyon, France.

    Til, M.P. & Keizer, A.M.M. (1980) Maximisation test with GTA in guinea
    pigs. Unpublished study from CIVO-TNO (report No. R6522). Submitted to
    WHO by Rhône Poulenc Secteur Agro, Lyon, France.

    Til, H.P., Hendriksen, C.F.M. & van der Heijden, C.A. (1976a) Combined
    chronic toxicity and carcinogenicity study with guazatine in rats.
    Unpublished study from CIVO-TNO (report No. R4985). Submitted to WHO
    by Rhône Poulenc Secteur Agro, Lyon, France.

    Til, H.P., Koeter, H.B.W.M., Immel, H.R. & van der Heijden, C.A.
    (1976b) Multigeneration study with guazatine in rats. Unpublished
    study from CIVO-TNO (report No. R5078). Submitted to WHO by Rhône
    Poulenc Secteur Agro, Lyon, France.

    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.

    Willems, M.I. (1983) Examination of GTA in the micronucleus test.
    Unpublished study from CIVO-TNO (report No. V83.118/230201). Submitted
    to WHO by Rhône Poulenc Secteur Agro, Lyon, France.

    Wilmer, J.W.G.M. (1983a) Examination of GTA for mutagenic activity in
    the Ames test. Unpublished study from CIVO-TNO (report No. V83.
    122/230064). Submitted to WHO by Rhône Poulenc Secteur Agro, Lyon,
    France.

    Wilmer, J.W.G.M. (1983b) Chromosome analysis of cultured human
    lymphocytes treated in vitro with GTA. Unpublished study from CIVO-TNO
    (report No. V83. 235.230573). Submitted to WHO by Rhône Poulenc
    Secteur Agro, Lyon, France.
    


    See Also:
       Toxicological Abbreviations
       Guazatine (Pesticide residues in food: 1978 evaluations)
       Guazatine (Pesticide residues in food: 1980 evaluations)