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    cases, based on unpublished proprietary data submitted for the purpose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



    AMINOMETHYLPHOSPHONIC ACID (AMPA)

    First draft prepared by 
    E. van Apeldoorn and P.H. van Hoeven
    National Institute of Public Health and Environment, 
    Bilthoven, The Netherlands

         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
                   Toxicological profiles of AMPA and glyphosate
         Comments
         Toxicological evaluation
         References

    Explanation

         Glyphosate was evaluated toxicologically by the 1986 JMPR, which
    allocated an ADI of 0-0.3 mg/kg bw (Annex I, reference 47). The
    primary degradation product of glyphosate in plants, soil, and water
    is aminomethylphosphonic acid (AMPA), whose chemical structure is very
    similar to that of glyphosate (Figure 1). AMPA itself has no
    commercial use. 

    FIGURE 1

         On the basis of the low residual levels of AMPA in crops which
    are susceptible to glyphosate, the 1986 Joint Meeting concluded that
    AMPA could be omitted from the residue in recommendations for MRLs,
    but recent supervised trials on the application of glyphosate to
    genetically modified crops have shown AMPA to be the main residue. As
    residues of AMPA may therefore be of toxicological concern, the
    compound was evaluated by the present Meeting.

    Evaluation for acceptable daily intake

    1.  Biochemical aspects

    (a)  Absorption, distribution, and excretion

         Eight male Wistar rats weighing about 150 g were fasted for 4 h
    and then given a single oral dose of 6.7 mg/kg bw 14C-AMPA by gavage.
    Urine, faeces, and expired carbon dioxide (in 1 N NaOH) were analysed
    for radiolabel 12, 24, 48, 72, 96, and 120 h after treatment (see
    Table 1). After 120 h, a blood sample was taken and the animals were
    killed; cage washes and tissues (see Table 2) were then analysed for
    radiolabel. AMPA was moderately absorbed from the gut, as demonstrated
    by excretion of 13% of the radiolabel in urine within 12 h, 18% within
    24 h, and 20% within 120 h. Consequently, the major route of excretion
    is the faeces: > 50% of the administered radiolabel was excreted
    within 24 h and 74% by 120 h. In tissues, 0.06% of the radiolabel was
    recovered after 120 h; < 0.1% was recovered as expired CO2 within 24
    h. Minor residues were found in liver, kidneys, and muscle (Colvin et
    al., 1973). Bone, bone-marrow, and eyes, which contain relatively high
    residues of radiolabel (0.2-0.6% of the dose in comparison with
    0.02-0.05% in other tissues) after oral administration of
    14C-glyphosate to rats (Howe et al., 1988; Ridley & Mirly, 1988),
    were not analysed in the study of Colvin et al. (1973).

    (b)  Biotransformation

         Two groups of five Wistar rats of each sex received 10 mg/kg bw
    14C-glyphosate orally, one group after preconditioning with
    unlabelled glyphosate. Only 0.2-0.4% of the radiolabel recovered in
    the excreta appeared to be AMPA (Howe et al., 1988).

        Table 1.  Recovery of radiolabel after a single oral dose of 6.7 mg/kg bw 14C-AMPA

                                                                                                
    Matrix      Time after treatment (h)
                                                                                                
                0-12        12-24       24-48       48-72       72-96       96-120      0-120
                                                                                                

    Urine       13          4.5         1.5         0.66        02.3        0.05        20

    Faeces      5.9         47          19          0.91        0.12        0.06        73

    CO2         0.06        0.01        0           0           0           0           0.07

    Cage wash                                                                           0.13

    Tissues                                                                             0.06

    Total       19          52          21          1.6         0.35        0.11        94
                                                                                                
    


    Table 2. Radiolabelled AMPA in tissues 120 h after a single oral
    dose of 6.7 mg/kg bw

                                                                
    Tissue              % of dose           AMPA equivalent
                                            (g/kg fresh weight)
                                                                

    Liver                  0.01                    6
    Kidney                <0.01                    6
    Muscle                 0.02                    3
    Fat                    0.01                    4
    Gut                    0.02                    8
    Spleen                <0.01                    4
    Heart                 <0.01                    4
    Brain                 <0.01                    1
    Testis                <0.01                    3
    Blood                 <0.01                    3
                                                                

         Colvin et al. (1973) showed that most orally absorbed 14C-APMA
    is excreted as unchanged compound in the urine of rats, as measured by
    thin-layer and gas-liquid chromatography and nuclear magnetic
    resonance and mass spectral analyses. The fact that < 0.1% of the
    administered activity was expired as carbon dioxide also demonstrated
    that only very small amounts of the absorbed material were
    metabolized.

    2.  Toxicological studies

    (a)  Acute toxicity

         Groups of two male and three female Sprague-Dawley rats were
    given AMPA (purity unspecified) as a 40% solution in corn oil at one
    of four doses by gavage and observed for seven days. Deaths occurred
    at a dose of 8300 mg/kg bw after one to two days. Reduced appetite and
    activity, increasing weakness, slight diarrhoea, collapse, and death
    were observed. At autopsy, slight liver discolouration and acute
    gastrointestinal inflammation were seen; the viscera appeared normal
    macroscopically (Birch, 1973).

    (b)  Short-term toxicity

     Rats

         In a 14-day range-finding study available only in summary, groups
    of five male and five female rats received AMPA at 0, 1000, 2000, or
    4000 mg/kg bw per day in the diet. Moderately reduced body-weight gain
    and food consumption (no further details) were seen at 4000 mg/kg bw
    per day. No other effects were observed. The NOAEL was 2000 mg/kg bw
    per day (Goldenthal, 1978).

         In a 13-week study of toxicity, groups of 20 male and 20 female
    weanling Charles River CD rats (aged four weeks) received AMPA
    (purity, 99.9%) at 0, 400, 1200, or 4800 mg/kg bw per day in the diet.
    The animals were observed for death and signs of overt toxicity twice
    a day on seven days a week. Body weights and food consumption were
    monitored weekly. Haematological, biochemical, and urinary parameters
    were measured in 10 rats of each sex per group after 45 and 88 days;
    the baseline values for these parameters were measured in an
    additional 10 male and 10 female rats that were sacrificed for this
    purpose. At the end of the study, the absolute and relative (to body
    weight) weights of the liver, kidneys, testes/ovaries, heart, and
    brain were determined in each animal, and macroscopy was performed.
    About 30 tissues from controls and rats at the highest dose and liver,
    kidneys, heart, urinary bladder, and tissues with gross lesions from
    all rats at 400 and 1200 mg/kg bw per day were examined
    microscopically.

         One male and five female rats at 4800 mg/kg bw per day were found
    dead after blood collection on day 45, and one female rat in the
    control group and two female rats at 1200 mg/kg bw per day were dead
    after blood collection on day 88. One male control rat found moribund
    after observation of soft stools, distended abdomen, and morbidity was
    killed at week 12. Body-weight gain was significantly decreased in
    males and females at 4800 mg/kg bw per day (difference for females at
    termination, < 10%) and in males at 1200 mg/kg bw per day
    (difference, < 10%). The food consumption of treated males was lower
    than that of control males, but the differences were not statistically
    signifcant. No treatment-related changes in haematological parameters
    were observed after 45 or 88 days. Biochemical analyses showed
    increased lactic dehydrogenase activity in males and females at 4800
    mg/kg bw per day, which was statistically significant for males after
    45 and 88 days and for females after 88 days only. Males at 1200 mg/kg
    bw per day also showed statistically significant increases in lactic
    dehydrogenase activity after 88 days; females at 400 or 1200 mg/kg bw
    per day showed significant increases in the activity of this enzyme
    after 88 days, but the biological significance of these observations
    is questionable in view of the magnitude of the increases and the low
    control values in females. Glucose levels were significantly decreased
    in males and females at 4800 mg/kg bw per day after 88 days in
    comparison with control values, but the levels in animals at 400 or
    1200 mg/kg bw per day were significantly increased after 45 days. At
    4800 mg/kg bw per day, males and females showed increased activity of
    aspartate aminotransferase, which was significant in females after 45
    days and in both males and females after 88 days, and males showed
    increased serum cholesterol levels, which were significant after both
    45 and 88 days. The occasionally significant changes in other
    biochemical parameters (albumin and bilirubin) did not show a
    dose-response relationship and were considered not to be biologically
    significant. Urinalysis revealed significant decreases in pH and
    increased calcium oxalate crystals in males at 4800 mg/kg bw per day
    after 88 days and in females after 45 and 88 days. The weights of
    several organs showed significant changes from control values, but the
    absence of any dose-response relationship or of morphological changes
    in these organs make their biological significance questionable.
    Macroscopy revealed no treatment-related changes. Microscopy showed
    dose-related increases in the incidence and severity of irritation of
    the mucosal and submucosal layers of the urinary tract in males and
    females at 1200 or 4800 mg/kg bw per day, corresponding to hyperplasia
    of the urinary bladder, which was more marked in males than in
    females. Epithelial hyperplasia in the pelvic section was seen in the
    kidneys of several rats at 4800 mg/kg bw per day, and some of the
    hyperplastic epithelial cells contained a hyaline-like cytoplasmic
    material. The NOAEL was 400 mg/kg bw per day (Estes et al., 1979).

     Dogs

         In a one-month range-finding study, groups of two male and two
    female beagle dogs (aged about six months and weighing 7.3-9.8 kg for
    males and 5.9-8.4 kg for females) received AMPA (purity, 94.4%) at 0,
    10, 30, 100, 300, or 1000 mg/kg bw per day orally in gelatin capsules.

    The animals were observed for mortality, moribundity, and signs of
    toxicity twice daily and for detailed signs of toxicity once weekly.
    Body weight and food consumption were determined weekly.
    Haematological and biochemical analyses were carried out in all dogs
    at termination; no urinalysis was performed. The absolute and relative
    weights of the liver, kidneys, spleen, heart, brain, thyroid,
    adrenals, and testes of all dogs were determined. Macroscopy was
    performed on all animals, but no microscopy was done. 

         No unscheduled death occurred. Diarrhoea was seen in both males
    and one female dog at 1000 mg/kg bw per day (total, 18 incidents), and
    emesis was observed in both females and one male dog at the same dose
    (four incidents). No effects on body-weight gain or food consumption
    were seen. Males and females at 1000 mg/kg bw per day had decreased
    haemoglobin and haematocrit values (significant in females), decreased
    erythrocyte counts (significant in males and females), and increased
    reticulocyte counts (significant in females). In addition, an increase
    in mean corpuscular haemoglobin concentration was seen in males at
    1000 mg/kg bw per day (significant) and 300 mg/kg bw per day, but with
    no dose-response relationship. Females at 300 mg/kg bw per day had
    significantly decreased haemoglobin and haematocrit values and a
    nonsignificant decrease in reticulocyte count. Activated partial
    prothrombin times were significantly decreased in females at all
    doses, but these decreases were considered not to be biologically
    significant because of their small size and the lack of a clear
    dose-response relationship. No effects on biochemical parameters were
    seen. Organ weights were unchanged, and macroscopy revealed no
    treatment-related abnormalities. No adverse effects were seen at
    100 mg/kg bw per day. There was no NOAEL owing to the limited number
    of animals. The authors stated that no adverse effects were observed
    at 300 mg/kg bw per day in males and 100 mg/kg bw per day in females
    (Stout, 1991).

         Groups of five male and five female beagle dogs (males weighing
    8.1-12 kg and females, 6.8-11 kg) received AMPA (purity, 87.8%) at
    doses of 0, 10, 30 100, or 300 mg/kg bw per day orally in gelatin
    capsules for 91-92 days. The dogs were observed twice daily for
    mortality, moribundity, and overt signs of toxicity. Ophthalmoscopy
    was conducted on all dogs before the study and at week 12. Body
    weights were recorded weekly and food consumption daily.
    Haematological, biochemical, and urinary analyses were carried out in
    all dogs before the study and during weeks 6 and 13. The absolute and
    relative weights of the liver, kidneys, brain, thyroid, adrenals, and
    ovaries/testes of all dogs were determined. All dogs were examined
    macroscopically, and about 40 tissues and all gross lesions were
    examined microscopically. 

         Treatment had no effect on survival, and ophthalmoscopy revealed
    no abnormalities. Several clinical signs were seen at all doses and in
    controls, but there was generally no clear dose-response relationship.
    Only the incidence of scabbed, reddened, and/or swollen ears in males
    at 300 mg/kg bw per day was increased; in females at this dose, the
    incidence of these symptoms was below that in controls, the latter

    being the same as that in males at 300 mg/kg bw per day. The incidence
    of soft stools varied from group to group, with considerable
    individual variability; no dose-response relationship was seen. No
    significant treatment-related effects on body-weight gain or food
    consumption were observed. Haematology showed occasionally significant
    decreases in erythrocyte count, haemoglobin and haematocrit values,
    mean corpuscular volume, mean corpuscular haemoglobin, or mean
    corpuscular haemoglobin concentration at low doses. No relationship
    with dose or time was seen, and these changes were considered not to
    be biologically significant. No treatment-related effects on
    biochemical or urinary parameters or organ weights were seen, and
    there were no macroscopic or microscopic changes. The NOAEL was 300
    mg/kg bw per day, the highest dose tested (Tompkins, 1991).

    (c)  Long-term toxicity and carcinogenicity

         No long-term toxicity studies have been performed with AMPA
    itself, but two-year toxicity studies are available in mice and rats
    treated with technical-grade glyphosate contaminated at a low level
    with AMPA (WHO, 1994).

         In a two-year study of toxicity and carcinogenicity in which
    technical-grade glyphosate (AMPA content not specified) was given to
    mice at dietary levels of 0, 0.1, 0.5, or 3%, no treatment-related
    increase in tumour incidence was seen. The NOAEL was 0.5%, equal to
    810 mg/kg bw per day (Bio/Dynamics Inc., 1983).

         In a 26-month study of toxicity and carcinogenicity in which
    technical-grade glyphosate (AMPA content not specified) was given to
    rats at dietary levels of 0.006, 0.02, or 0.06%, equal to about 3, 10,
    or 32 mg/kg bw per day, no treatment-related increase in tumour
    incidence was seen. The NOAEL was 32 mg/kg bw per day, the highest
    dose tested (Bio/Dynamics Inc., 1981a).

         In a two-year study of toxicity and carcinogenicity, rats were
    given technical-grade glyphosate containing 0.68% AMPAin the diet to
    provide doses of 0.2, 0.8, or 2% glyphosate, equivalent to 100, 400,
    or 1000 mg/kg bw per day. The diets thus contained 14, 54, and 135 mg
    of AMPA per kg, corresponding to 0.69, 2.8, and 7.2 mg/kg bw per day.
    No increase in tumour incidence was seen. At the highest dose, female
    body-weight gain was reduced, and cataracts were found in males. The
    NOAEL was 0.8% in the diet, equivalent to 400 mg/kg bw per day
    glyphosate and 2.8 mg/kg bw per day AMPA (Monsanto 1990a; Stout &
    Ruecker, 1990).

    (d)  Genotoxicity

         The results of tests for the genotoxicity of AMPA are shown in
    Table 3. In the test for micronucleus formation, groups of 15 male and
    15 female mice were used. Five animals of each sex per dose were
    killed 24, 48, and 72 h after treatment. Toxicity was observed at 500
    and 1000 mg/kg bw, as demonstrated by weight loss and listlessness. No
    significant decrease in mean polychromatic erythrocytes:total

    erythrocytes was seen at any dose. At 100 mg/kg bw, a significant
    increase in mean nucleated polychromatic erythrocyte frequency was
    seen after 72 h in females only; however, the increased frequency was
    within the range in historical vehicle controls in the same
    laboratory, and the frequency was not increased in males at 100 mg/kg
    bw or in males or females at higher doses. The increase in mean
    nucleated polychromatic erythrocyte frequency in females at 100 mg/kg
    bw was thus considered not to be related to treatment (Kier &
    Stegeman, 1993).

    (e)  Reproductive toxicity

    (i)  Multigeneration reproductive toxicity

         No multigeneration studies of reproductive toxicity have been
    performed with AMPA itself, but two studies were reported in rats
    treated with technical-grade glyphosate contaminated at low levels
    with AMPA (WHO, 1994).

         In a three-generation study of reproductive toxicity, rats were
    given diets providing technical-grade glyphosate (AMPA content not
    specified) at doses of 0, 3, 10, or 30 mg/kg bw per day. The only
    effect was an increased incidence of unilateral renal tubular
    dilatation in male F3b pups at 30 mg/kg bw per day (6/10 versus 0/10
    in controls); F1 and F2 pups were not examined (Bio/Dynamics Inc.,
    1981b).

         In a two-generation study of reproductive toxicity, groups of 30
    male and 30 female Sprague-Dawley rats received diets containing
    technical-grade glyphosate with 0.61% AMPA (Reyna, 1990), providing 0,
    0.2, 1, or 3% glyphosate and 0, 12, 61, or 180 ppm AMPA. Parental
    animals receiving 3% glyphosate had soft stools, decreased body
    weights, and slightly decreased litter sizes; pup weights were
    decreased on days 14 and 21 of lactation. Parental and pup body
    weights were also slightly decreased after treatment with 1.0%. The
    NOAEL was 1.0% in the diet, corresponding to 740 mg/kg bw per day
    glyphosate and 3 mg/kg bw per day AMPA (Monsanto, 1990b). The renal
    changes seen in the three-generation study were thus not reproduced;
    however, the numbers of pups examined histologically was limited in
    both studies.

    (ii)  Developmental toxicity

         In a range-finding study, groups of eight pregnant Sprague-Dawley
    rats received AMPA (purity, 94.4%) in corn oil at doses of 0, 125,
    250, 500, 750, or 1000 mg/kg bw by gavage daily on days 6-15 of
    gestation. All animals were killed on day 20 of gestation. Clinical
    observations, body weights, net body-weight changes, and gravid
    uterine weights of the dams were recorded, and the weights of the
    liver, kidney, and spleen were determined. Uteri and ovaries were
    examined, and the numbers of fetuses, early and late resorptions,
    implantation sites, and corpora lutea were recorded. Fetuses were


        Table 3. Results of tests for the genotoxicity of AMPA

                                                                                                                                      

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

    In vitro
    Reverse mutationa       S. typhimurium          10-5000 g/plate            99              Negativeb       Shirasu et al. (1980)
                            TA98, TA100,            in distilled water
                            TA1535, TA1537,
                            TA1538, and
                            E. coli WP2

    DNA repairc             B. subtilis H17,        20-2000 g/disc             99              Negatived       Shirasu et al. (1980)
                            M45 (rec+/-)            in distilled water

    Unscheduled DNA         Primary hepatocytes     5-5000 g/ml in             94.4            Negative        Bakke (1991)
    synthesise              from male Fischer       culture medium
                            344 rats

    In vivo
    Micronucleus            CD-1 mice               Single intraperitoneal      94.4            Negative        Kier & Stegeman
    formulationf                                    injection of 100, 500,                                      (1993)
                                                    or 1000 mg/kg bw in
                                                    corn oil
                                                                                                                                      

    a   No independent duplicate study but duplicate plates at each concentration; distilled water used as solvent; solvent and
        positive controls included
    b   With and without metabolic activation
    c   No independent duplicate study; distilled water used as solvent; solvent and positive controls included
    d   Only without metabolic activation
    e   Cytotoxicity at 3800 and 5000 g/ml
    f   No characterization of test or control substances or their concentrations, homogeneity in carrier, or stability of test 
        and control substances neat and after mixing with carrier
    

    weighed, sexed, and examined externally for developmental
    abnormalities and variations. No effect on survival or body or organ
    weights was seen in the dams. Greyish faeces were observed
    infrequently in animals at doses > 125 mg/kg bw per day. The
    predominant findings 1 h after treatment were red staining around the
    nose in one or two animals per treated group and a low incidence of
    salivation in those at 250, 500, 750, or 1000 mg/kg bw per day. In the
    absence of a clear dose-response relationship and the limited
    findings, they did not appear to be treatment-related. Fetuses had no
    treatment-related effects (Holson, 1991a). 

         Groups of 25 pregnant Sprague-Dawley rats received AMPA (purity,
    94.4%) in corn oil at doses of 0, 150, 400, or 1000 mg/kg bw by
    gavage, daily on days 6-15 of gestation. All animals were killed on
    day 20 of gestation. The dams were observed twice daily for
    moribundity and mortality, and signs of toxicity about 1 h after
    treatment and clinical observations on days 0-20 of gestation were
    recorded. The body weights and food consumption of the dams were
    determined on gestation days 0, 6, 9, 12, 16, and 20. Gravid uterine
    weights and net body-weight changes were recorded, and the liver,
    kidney, and spleen of all dams were weighed. Uteri and ovaries were
    examined, and the numbers of fetuses, early and late resorptions,
    implantation sites, and corpora lutea were recorded. Fetuses were
    weighed, sexed, and examined for external, visceral, and skeletal
    abnormalities or variations. 

         Maternal survival was not affected. Dams at 400 or 1000 mg/kg bw
    per day had dose-related increased incidences of mucoid faeces, hair
    loss, and soft stools. Dams at 150 mg/kg bw per day had only an
    increased incidence of soft stools. The total occurrence of soft
    stools/no. of animals was 49/15 in controls, 89/19 in dams at 150
    mg/kg bw per day, 102/22 at 400 mg/kg bw per day, and 135/23 at 1000
    mg/kg bw per day. At the highest dose, the dams had short periods of
    slightly decreased body-weight gain and food consumption. No effect
    was seen on the weights of the liver, kidney, spleen, or gravid
    uterus. Fetal survival was not affected. A slight but significant
    decrease in mean fetal body weight was seen at 1000 mg/kg bw per day.
    No further developmental effect was seen, and there were no
    teratogenic effects. Because the increased incidence of soft stools at
    150 mg/kg bw per day was not accompanied by possibly associated
    effects such as hair loss and mucoid faeces, this dose is the NOAEL
    for maternal toxicity. The NOAEL for developmental toxicity was 400
    mg/kg bw per day, as the authors stated that the only possible
    maternal responses at this dose were mucoid faeces, soft stools, and
    hair loss (Holson, 1991b).

    (f) Special studies: Dermal and ocular irritation

         Two male and one female albino rabbits (strain not specified)
    received an application of 0.5 g finely ground AMPA moistened with
    water on the clipped intact skin (6.5 cm2), which was kept under
    cover for 24 h. Observations were made after 1, 24, 48, 72, 120, and

    168 h. No skin irritation was seen (Birch, 1973). Only a limited
    report was available.

         One male and two female albino rabbits (strain not specified)
    received an application of 0.1 g finely ground AMPA into one eye. Both
    the control and the treated eye were rinsed with warm isotonic saline
    after 24 h. Observations were made after 1, 24, 48, 72, 120, and 168 h
    and scored according to Draize. Slight erythema, very slight oedema,
    and copious discharge were seen after 10 min and after 1 h, and
    slight-to-moderate erythema, a moderate discharge containing a white
    exudate, and no oedema were seen after 24 h. After 72 h, slight
    erythema was seen in one animal, but without oedema or discharge.
    After 120 and 168 h, the treated eyes were normal. The average scores
    were 10 out of 110 after 1 h and 7.3, 2.6, and 0.6 after 24, 48, and
    72 h, respectively. AMPA was considered by the author to be a slight
    ocular irritant; however, the average score after 24, 48, and 72 h was
    3.5, indicating that AMPA is not an ocular irritant (Birch, 1973). In
    the limited report, separate scores were not given for conjunctivae,
    iris, and cornea. 

    3.  Toxicological profiles of AMPA and glyphosate

    The results of toxicological studies of the metabolite AMPA and of its
    parent compound glyphosate, as previously evaluated (Annex 1,
    reference 47; WHO, 1994), are compared in Table 4.  AMPA was no more
    toxic than glyphosate.  Similar effects were often found,e xcept for
    the lesions in the salivary gland seen in the 90-day studies of
    toxicity and carcinogenicity with glyphosate in mice nad rats from the
    US national Toxicology Program and the cataracts induced by glyphosate
    in a two-year toxicity study in rats.  Such effects were not seen in
    other studies with glyphosate or AMPA.

    Comments

         After oral administration of AMPA to rats, 20% of the dose was
    absorbed and excreted unmetabolized in the urine within 120 h (17% of
    the dose within 24 h), and 73% of the dose was eliminated in the
    faeces. Only 0.07% of the dose was excreted as expired carbon dioxide
    within 24 h, and 0.06% of the dose was recovered from tissues after
    120 h. Minor amounts (1-6 g/kg) were found in tissues after 120 h.

         AMPA is slightly hazardous to rats given a single oral dose, with
    an LD50 of 8300 mg/kg bw (WHO, 1996).

         In a 90-day study of toxicity, rats received AMPA in the diet at
    0, 400, 1200, or 4800 mg/kg bw per day. A significant, dose-related
    decrease in body-weight gain was seen in males at the two highest
    doses and in females at the highest dose. The two highest doses also
    resulted in significantly increased lactate dehydrogenase activity,
    whereas aspartate aminotransferase activity and cholesterol levels
    were significantly increased only at the highest dose. Urinalysis
    showed a significant decrease in urinary pH and increased amounts of
    calcium oxalate crystals in the urine of animals at the highest dose.

        Table 4. Toxicity of AMPA and technical-grade glyphosate

                                                                                                                                      

    Type of study                           AMPA                                            Glyphosatea
                                                                                                                                      

    Metabolism                              20% absorption after oral exposure; excreted    36% absorption after oral exposure;
                                            unchanged in urine; only 0.07% expired as       essentially no biotransformation (0.2-
                                            CO2; minor residues in tissues                  0.4% AMPA)

    Acute oral toxicity                     LD50 = 8300 mg/kg bw                            LD50 > 5000 mg/kg bw

    90-day toxicity, diet, mice                                                             NOAEL = 10 000 ppm, equal to 1890
                                                                                            me/kg bw.  Effects at LOAEL of 50 000
                                                                                            ppm: decreased growth, increased brain,
                                                                                            heart, and kidney weights

    90-day toxicity, diet, mice                                                             NOAEL = 3125 ppm, equal to 505 mg/
                                                                                            kg bw.  Effects at LOAEL of 6250 ppm:
                                                                                            lesions of salivary gland

    90-day toxicity, diet, rats             NOAEL = 400 mg/kg bw.  Effects at LOAEL         No effect at highest dose of 20 000 ppm,
                                            of 1200 mg.kg bw: biochemical changes,          equal to 1300 mg/kg bw
                                            changes in urinary bladder.  At 4800 mg/kg bw,
                                            also renal changes

    90-day toxicity, diet, rats                                                             NOAEL < 3100 ppm, equal to < 200 mg/
                                                                                            kg bw; lesions of salivary gland

    90-day toxicity, diet, rats                                                             No effect at highest dose of 12 500 ppm,
                                                                                            equal to 1300 mg/kg bw

    90-day toxicity, gelatin capsule, 
      dogs                                  No effect at highest dose of 300 mg/kg bw

    52-week toxicity, gelatin capsule, 
      dogs                                  No effect at highest dose of 500 mg/kg bw

    Table 4. (continued)

                                                                                                                                      

    Type of study                           AMPA                                            Glyphosatea
                                                                                                                                      

    Two-year toxicity and carcinogenicity,                                                  NOAEL = 5000 ppm, equal to 810 mg/
    diet, mice                                                                              kg bw. Effects at LOAEL of 30 000
                                                                                            ppm: decreased growth, changes in
                                                                                            urinary bladder and kidneys

    Two-year toxicity and carcinogenicity,                                                  No effect at highest dose of 600 ppm,
    diet, rats                                                                              equal to 32 mg/kg bw; slight decrease in
                                                                                            growth considered not to be relevant

    Two-year toxicity and carcinogenicity,                                                  NOAEL = 8000 ppm, equivalent to 400
    diet, rats                                                                              mg/kg bw.  Effects at LOAEL of 20 000
                                                                                            ppm: decreased growth and cataracts.
                                                                                            Technical-grade glyphosate containing
                                                                                            0.68% AMPA

    Three-generation reproductive                                                           Increased incidence (6/10 versus 1/10 in
    toxicity, diet, rats                                                                    controls) of unilateral renal tubular
                                                                                            dilatation in F3b pups at highest dose of
                                                                                            30 mg/kg bw; not seen in more recent
                                                                                            study (below)

    Two-generation reproductive                                                             NOAEL = 10 000 ppm, equivalent to
    toxicity, diet, rats                                                                    500 mg/kg bw.  Effects at LOAEL of
                                                                                            30 000 ppm: soft stools, decreased
                                                                                            weights of parental animals and pups.
                                                                                            Technical-grade glyphosate containing
                                                                                            0.61% AMPA

    Developmental toxicity, rats            NOAEL for maternal toxicity = 150 mg/kg         NOAEL = 1000 mg/kg bw.  Effects at
                                            bw; NOAEL for developmental toxicity = 400      LOAEL of 3500 mg/kg bw: deaths,
                                            mg/kg bw.  At 400 mg/kg bw, increased           clinical signs, decreased growth of dams,
                                            incidences of soft stools, mucoid faeces, and   resorptions, decreased implantations and
                                            hair loss in dams. At 1000 mg/kg bw, slight     visible fetuses, decreased ossification of
                                            decrease in fetal body weight                   fetal sternebrae; no fetal malformations

    Table 4. (continued)

                                                                                                                                      

    Type of study                           AMPA                                            Glyphosatea
                                                                                                                                      

    Developmental toxicity, rabbits                                                         NOAEL = 175 mg/kg bw. Effects at
                                                                                            LOAEL of 350 mg/kg bw: diarrhoea,
                                                                                            soft stools, and nasal discharge in dams

    Genotoxicity
    Reverse mutation, S. typhimurium        Negative                                        Negative

    Reverse mutation, E. coli               Negative                                        Negative

    DNA repair, B. subtilis rec             Negative                                        Negative

    Gene mutation, Chinese hamster ovary                                                    Negative
    cells

    Unscheduled DNA synthesis, rat          Negative                                        Negative
    hepatocytes

    Chromosomal aberration in vivo, rat                                                     Negative
    bone marrow

    Micronucleus formation in vivo,         Negative                                        Negative
    mouse bone marrow

    Dominant lethal mutation in vivo,                                                       Negative
    mice

    Recessive sex-linked lethal mutation,                                                   Negative
    Drosophila melanogaster
                                                                                                                                      

    a From WHO (1994) and Annex I, reference 47
    

    Dose-related irritation of the mucosal and submucosal layers of the
    urinary tract, corresponding to hyperplasia of the urinary bladder,
    was seen in rats at 1200 and 4800 mg/kg bw per day, the effect being
    more marked in males than in females. In addition, epithelial
    hyperplasia in the renal pelvis was observed at the highest dose. The
    NOAEL was 400 mg/kg bw per day.

         In a 90-day study of toxicity in dogs receiving AMPA at 0, 10,
    30, 100, or 300 mg/kg bw per day in gelatin capsules, no statistically
    significant treatment-related changes were observed. The NOAEL was
    thus the highest dose, 300 mg/kg bw per day. It should be noted that
    in a one-month range-finding study with groups of only two male and
    two female dogs, changes in some haematological parameters (e.g.
    decreased haemoglobin, packed cell volume, and erythrocyte counts)
    were seen in animals at 300 or 1000 mg/kg bw per day. These effects
    were not reproduced in the 90-day study. 

         No indication of genotoxic activity was seen in studies of gene
    mutation in bacteria, of DNA repair in bacteria and mammalian cells
     in vitro, or of micronucleus formation  in vivo. No assays for gene
    mutation were performed in mammalian cells  in vitro, but the
    structural similarity of AMPA to glyphosate and the lack of
    genotoxicity of glyphosate, including in an assay for gene mutation in
    mammalian cells  in vitro, indicate that such an assay with AMPA
    would be redundant.

         In a study of developmental toxicity, rats received AMPA at 0,
    150, 400, or 1000 mg/kg bw per day in corn oil by gavage. Dose-related
    increases in the incidences of soft stools, mucoid faeces, and hair
    loss were seen in dams at the two higher doses. Dams at the highest
    dose also had short periods of decreased body-weight gain and food
    consumption. Fetal body weight was decreased at 1000 mg/kg bw per day.
    No teratogenic effects were observed. Dams at 150 mg/kg bw per day
    also had an increased incidence of soft stools; however, in the
    absence of any associated effects, such as hair loss or mucoid faeces,
    the Meeting considered this dose to be the NOAEL for maternal
    toxicity. The NOAEL for developmental toxicity was 400 mg/kg bw per
    day.

         AMPA did not induce dermal or ocular irritation in rabbits.

         No long-term study of the toxicity or carcinogenicity of AMPA has
    been carried out, but in the more recent of two such studies with
    technical-grade glyphosate in rats at dietary levels of 0.2, 0.8, or
    2%, the AMPA content of the test compound was given, namely 0.68%. At
    the highest dose of 2% glyphosate in the diet, females showed
    decreased body-weight gain and males showed an increased incidence of
    degenerative lenticular changes. The NOAEL for technical-grade
    glyphosate was 0.8% in the diet, corresponding to 400 mg/kg bw per day
    for glyphosate and 2.7 mg/kg bw per day for AMPA. No increase in
    tumour incidence was seen in this study. 

         No multigeneration study of the reproductive toxicity of AMPA has
    been reported, but in a recent two-generation study in rats with
    technical-grade glyphosate at dietary levels of 0.2, 1, or 3%, the
    test compound contained 0.61% AMPA. At the highest dose, soft stools,
    decreased parental body weights, slightly decreased litter sizes, and
    decreased pup weights were observed. The NOAEL was 1% in the diet,
    corresponding to 740 mg/kg bw per day glyphosate and 4.5 mg/kg bw per
    day AMPA.

         Glyphosate and AMPA have very similar chemical structures.
    Studies of the metabolism of glyphosate in experimental animals
    indicate that essentially none is biotransformed into AMPA.
    Toxicological data on the metabolite are therefore essential for risk
    assessment. The Meeting compared the toxicity profile of AMPA with
    that of glyphosate and concluded that the major targets of the
    toxicity of AMPA had been investigated. The results showed little
    toxicity. The Meeting concluded that the two compounds have similar
    toxicological profiles and concluded that a full database on AMPA is
    unnecessary. AMPA was considered to be of no greater toxicological
    concern than its parent compound. The Meeting established a group ADI
    for AMPA alone or in combination with glyphosate of 0-0.3 mg/kg bw on
    the basis of the 26-month study of toxicity in rats fed
    technical-grade glyphosate, using a safety factor of 100.

         Since the last JMPR evaluation fof glyphosate or toxicity in
    1986, new data have become available, some of which are evaluated in
    WHO (1994). The Meeting recommended that glyphosate be re-evaluated by
    the JMPR.

    Toxicological evaluation

    Levels that cause no toxic effect

     AMPA
         Rat:      400 mg/kg bw per day (90-day study of toxicity)
                   150 mg/kg bw per day (maternal toxicity in a study of
                   developmental toxicity)
                   400 mg/kg bw per day (fetal toxicity in a study of
                   developmental toxicity)
         Dog:      300 mg/kg bw per day (highest dose in 90-day study of
                   toxicity)

     Glyphosate (from Annex I, reference 47):

         Mouse:    0.5% in the diet, equal to 810 mg/kg bw per day (two-
                   year study of toxicity and carcinogenicity)
         Rat:      31 mg/kg bw per day (26-month study of toxicity and
                   carcinogenicity)
         Dog:      500 mg/kg bw per day (one-year study of toxicity)

    Estimate of acceptable daily intake for humans

         0-0.3 mg/kg bw (sum of glyphosate and AMPA)


        Toxicological criteria for setting guidance values for dietary and non-dietary exposure to aminomethylphosphonic acid (AMPA)

                                                                                                                                 

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

    Short-term          Oral toxicity, rat                                LD50 = 8300 mg/kg bw
    (1-7 days)          Skin irritation, rabbit                           Not irritating
                        Eye irritation, rabbit                            Not irritating
                        Skin sensitization                                No data

    Medium-term         Repeated oral, 90 days, toxicity, rat             NOAEL = 400 mg/kg bw per day: urinary tract changes
    (1-26 weeks)        Repeated oral, developmental toxicity, rat        NOAEL = 150 mg/kg bw per day: maternal toxicity 
                                                                          NOAEL = 400 mg/kg bw per day: developmental 
                                                                          toxicity
                        Repeated oral, reproductive toxicity              No data

    Long-term           Repeated oral, toxicity                           No data
    (> 1 year)
                                                                                                                                 
    

    References

    Bakke, J.P. (1991) Evaluation of the potential of AMPA to induce
    unscheduled DNA synthesis in the  in vitro hepatocyte DNA repair
    assay using the male F-344 rat. Unpublished study No. 2495-V01-91 from
    SRI International, Menlo Park, California, USA (SRI Project LSC-2495;
    Monsanto study No. SR-91-234, dated 4 December 1991). Submitted to WHO
    by Monsanto, Agricultural Group, St Louis, Missouri, USA.

    Bio/Dynamics Inc. (1981a) A lifetime feeding study of glyphosate
    (Roundup technical) in rats (Project N0. 410/77 [BDN-77-416]).
    Bio/Dynamics Inc., Division of Biology and Safety Evaluation, East
    Millstone, New Jersey, USA. Submitted to WHO by Monsanto, St Louis,
    Missouri, USA.

    Bio/Dynamics Inc. (1981b) A three generation reproduction study in
    rats with glyphosate (Project No. 77-2063 [BDN-77-147]). Final report.
    Bio/Dynamics Inc., Division of Biology and Safety Evaluation, East
    Millstone, New Jersey, USA. Submitted to WHO by Monsanto, St Louis,
    Missouri, USA.

    Bio/Dynamics Inc. (1983) A chronic feeding study of glyphosate
    (Roundup technical) in mice (Project No. 77-2061 [BDN-77-420]).
    Bio/Dynamics Inc., Division of Biology and Safety Evaluation, East
    Millstone, New Jersey, USA. Submitted to WHO by Monsanto, St Louis,
    Missouri, USA.

    Birch, M.D. (1973) Toxicological investigation of CP 50435 Lot:
    XHD-16. Unpublished report from Younger Laboratories Inc., St Louis,
    Missouri, USA, dated 7 March 1973. Submitted to WHO by Monsanto,
    Agricultural Group, St Louis, Missouri, USA.

    Colvin, L.B., Moran, S.J. & Miller, J.A. (1973) Final Report on CP
    67573 residue and metabolism. Part 11: The metabolism of
    aminomethylphosphonic acid-14C (CP 50435-14C) in the laboratory rat.
    Job No. 9-23-760.06-7863. Unpublished report from Monsanto Commercial
    Products Co., Agricultural Research Report No. 303 dated August 1973.
    Submitted to WHO by Monsanto, Agricultural Group, St Louis, Missouri,
    USA.

    Estes, F.L., Jefferson, N.D., Blair, M. & Goldenthal, E.I. (1979)
    90-Day subacute rat toxicity study (IRD-78-174). Test article: CP
    50435. Unpublished report from International Research and Development
    Corporation, Mattawan, Michigan, USA, dated 15 August 1979. Submitted
    to WHO by Monsanto, Agricultural Group, St Louis, Missouri, USA.

    Goldenthal, E.I. (1978) CP 50435 (aminomethyl phosphonic acid)
    fourteen day rat feeding study. Unpublished report No. IRD-77-319 from
    Monsanto. Submitted to WHO by Monsanto, St Louis, Missouri, USA.

    Holson, J.F. (1991a) A dose range-finding developmental toxicity study
    of AMPA in rats. Final report. Unpublished report from WIL Research
    Laboratories Inc., Ashland, Ohio, USA. WIL Project No.: WIL-50146.
    Sponsor No.: WI-90-247, dated 21 May 1991. Submitted to WHO by
    Monsanto, Agricultural Group, St Louis, Missouri, USA.

    Holson, J.F. (1991b) A developmental toxicity study of AMPA in rats.
    Final report. Unpublished report from WIL Research Laboratories Inc.,
    Ashland, Ohio, USA. WIL Project No.: WIL-50159. Sponsor No.:
    WI-90-266, dated 6 August 1991. Submitted to WHO by Monsanto,
    Agricultural Group, St Louis, Missouri, USA.

    Howe, R.K., Chott, R.C. & McClanahan, R.H. (1988) Volume 2: Metabolism
    of glyphosate in Sprague-Dawley rats. Part II. Identification,
    characterization, and quantitation of glyphosate and its metabolites
    after intravenous and oral administration. Unpublished report from
    Monsanto Co., St Louis, Missouri, USA. Laboratory project no.
    MSL-7206, dated February 1988. Submitted to WHO by Monsanto,
    Agricultural Group, St Louis, Missouri, USA.

    Kier, L.D. & Stegeman, S.D. (1993) Final report. Mouse micronucleus
    study of AMPA. Unpublished report from Environmental Health Laboratory
    of Monsanto Co., St Louis, Missouri, USA. EHL Study Number: 90170.
    Sponsor Project Number: ML-90-404. Submitted to WHO by Monsanto,
    Agricultural Group, St Louis, Missouri, USA.

    Monsanto (1990a) Chronic study of glyphosate administered in feed to
    albino rats (Project No. MSL-10495). Monsanto Environmental Health
    Laboratory, St Louis, Missouri, USA. Unpublished report submitted
    to WHO by Monsanto, St Louis, Missouri, USA.

    Monsanto (1990b) Two generation reproduction feeding study with
    glyphosate in Sprague-Dawley rats (Study No. MSL-10387). Monsanto
    Environmental Health Laboratory, St Louis, Missouri, USA. Unpublished
    report submitted to WHO by Monsanto, St Louis, Missouri, USA.

    Reyna, M.S. (1990) Two generation reproduction study with glyphosate
    in Sprague-Dawley rats. Unpublished Report no. ML-88-106. Submitted to
    WHO by Monsanto, St Louis, Missouri, USA.

    Ridley, W.P. & Mirly (1988) Volume 1: The metabolism of glyphosate in
    Sprague-Dawley rats. Part I. Excretion and tissue distribution of
    glyphosate and its metabolites following intravenous and oral
    administration. Unpublished report MSL-7215 from Monsanto Co.,
    Environmental Health Laboratory, St Louis, Missouri, USA. Submitted to
    WHO by Monsanto, Agricultural Group, St Louis, Missouri, USA.

    Shirasu, Y., Moriya, M. & Ohta, T. (1980) CP 50435: Microbial
    mutagenicity study. Unpublished report from the Institute of
    Environmental Toxicology Kodaira, Tokyo, Japan, dated November 1980.
    Submitted to WHO by Monsanto, Agricultural Group, St Louis, Missouri,
    USA.

    Stout, L.D. (1991) One month study of AMPA administered by capsule to
    beagle dogs. Unpublished report from Environmental Health Laboratory
    of Monsanto Co., St Louis, Missouri, USA. Study Number 90074. Project
    Number: ML-90-186. Submitted to WHO by Monsanto, Agricultural Group,
    St Louis, Missouri, USA.

    Stout, L.D. & Ruecker, F.A. (1990) Chronic study of glyphosate
    administered in feed to albino rats. Unpublished report no. ML-90-186.
    Submitted to WHO by Monsanto, St Louis, Missouri, USA.

    Tompkins, E.C. (1991) 90-Day oral (capsule) toxicity study in dogs
    with AMPA. Final report. Unpublished report from WIL Research
    Laboratories Inc., Ashland, Ohio, USA. Project No.: WIL-50173. Sponsor
    No.: WI-90-354, dated 16 July 1991. Submitted to WHO by Monsanto,
    Agricultural Group, St Louis, Missouri, USA.

    WHO (1994)  Environmental Health Criteria 159. Glyphosate. Geneva,
    International Programme on Chemical Safety.

    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