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    Pesticide residues in food -- 1999



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



    Toxicological evaluations




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

    Rome, 20-29 September 1999

    GLUFOSINATE-AMMONIUM (addendum)

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

            Explanation 
            Evaluation for acceptable daily intake 
                Biological data 
                    Absorption, distribution and excretion 
                    Biotransformation 
                    Effects on enzymes and other biochemical parameters
                    Significance of glutamine synthetase inhibition to
                      humans
                Toxicological studies 
                    Short-term studies of toxicity 
                    Long-term studies of toxicity and carcinogenicity
                    Studies on metabolites 
                         N-Acetylglufosinate
                        3-Methylphosphinicopropionic acid 
                Observations in humans 
                        Medical surveillance of manufacturing plant
                          personnel
                        Poisoning incidents 
            Comments 
            Toxicological evaluation 
            References 


    Explanation

         Glufosinate-ammonium was evaluated toxicologically by the 1991
    Joint Meeting (Annex 1, reference  62), when an ADI of 0-0.02 mg/kg
    bw was established on the basis of the NOAEL in a long-term studyof
    toxicity  in rats and a 100-fold safety factor. The 1991 Meeting also
    requested additional observations in humans and information on the
    biological significance of the increased renal glutamine synthetase
    activity observed in rats. 

         The (-) isomer of  N-acetylglufosinate is a major metabolite
    when glufosinate-ammonium is applied to glufosinate-tolerant crops.
    The 1998 JMPR considered residues issues arising from applications of
    glufosinate-ammonium to tolerant crops and proposed that the residue
    be defined as the 'sum of glufosinate-ammonium,
    3-[hydroxy(methyl)phosphinoyl]propionic acid and
     N-acetyl-glufosinate' (Annex 1, reference  83), but it could not
    adopt this definition until  N-acetyl-glufosinate had been evaluated
    toxicologically. The present Meeting reviewed additional data on human
    exposure, glutamine synthetase activity, the toxicity of repeated
    doses, and the toxicokinetics of glufosinate-ammonium, together with
    an extensive dossier on  N-acetyl-glufosinate and a summary of the

    results of studies on 3-[hydroxy(methyl)phosphinoyl]propionic acid,
    another metabolite of glufosinate-ammonium.

    Evaluation for Acceptable Daily Intake

    1.  Biological data

         All of the studies contained statements of compliance with good
    laboratory practice (GLP) and claimed to have been performed to
    applicable guidelines of the OECD, European Union, or US Environmental
    Protection Agency.

    (a)  Absorption, distribution, and excretion

         (i)  Oral administration

          Rats 

         The absorption, distribution, and excretion of
    [3,4-14C]-glufosinate-ammonium was studied after administration by
    oral gavage in aqueous vehicles at doses of 2, 20, or 500 mg/kg bw 
    (Stumpf, 1993a; Lauck-Birkel, 1995a, 1996; Lauck-Birkel & Strunk,
    1999a; Maas & Braun, 1999a). Samples of urine and faeces were
    collected daily for up to 4 days, tissue, organ and excreta samples
    were processed, and total radiolabel was determined by liquid
    scintillation counting. The studies had much in common, and
    representative findings are summarized in Table 1. Less than 10% of an
    oral dose was absorbed, with rapid excretion, mainly as parent
    compound, in the faeces within 24 h. The concentrations of residues
    were markedly higher in kidney and liver than in plasma, while those
    in brain were even lower, indicating limited penetration of the
    blood-brain barrier. There were no marked differences between the
    sexes. Administration at 500 mg/kg bw resulted in a more prolonged
    absorption and excretion phase than after 20 or 2 mg/kg bw.

          Goats 

         A lactating goat weighing about 50 kg was given
    [3,4-14C]glufosinate-ammonium (99% radiochemical purity; specific
    activity, 26 mCi/g) in capsules twice daily for 4 days at a dose of
    ~ 3 mg/kg bw per day. Over 80% of the administered dose was excreted
    in faeces and approximately 3% in urine; only small amounts were found
    in the tissues (< 0.1%) and milk (0.02%). The concentration of
    radiolabel in milk reached a plateau by day 2 (0.02 g/g). The
    concentrations were higher in kidney (0.6 g/g) and liver (0.4 g/g)
    than muscle and fat (< 0.01 g/g) (Huang & Smith, 1995a).

    Table 1. Radiolabel in excreta and tissues of Wistar rats given 
             [3,4-14C]glufosinate ammonium at 2, 20, or 500 mg/kg bw

                                                                          
    Sample          Radiolabel in excreta (% of dose) or tissues (g/g)
                                                                          

                    500 mg/kg bwa        20 mg/kg bwb    2 mg/kg bwc
                                                                          
                    Males     Females    Males           Males    Females
                                                                          

    Urine
       24 h         3         3          5               8        8
       Total        8         5          5               10       9
    Faeces
       24 h         38-71     35-51      92              88       91
       Total        75        89         92              91       95
    Liver, 6 h      15        10         0.7             NA       NA
    Kidney, 6 h     53        49         2.9             NA       NA
    Brain, 6 h      0.6       1          < 0.1           NA       NA
    Plasma, 6 h     1.3       1.4        < 0.1           NA       N
    Spleen, 6 h     9         19         NA              NA       NA
                                                                          

    NA, not analysed 
    a  Results from Lauck-Birkel (1995a); Maas & Braun (1995a)
    b  Results from Lauck-Birkel & Strunk (1999a); Maas & Braun (1999a)
    c  Results from Stumpf (1993a); Lauck-Birkel (1996)


          Chickens 

         Laying hens were given [3,4-14C]glufosinate-ammonium (99%
    radiochemical purity; specific activity, 26 mCi/g) in capsules twice
    daily for 14 days at a dose of ~ 2 mg/kg bw per day. The excreta
    contained over 90% of the administered dose, and < 0.02% of the
    administered radiolabel was present in edible tissues and 0.07% in
    eggs. The peak concentrations were 0.1 g/g in liver, 0.07 g/g in egg
    white, and 0.02 g/g in egg yolk (Huang & Smith, 1995b).

         (ii)  Intravenous administration

          Rats 

         [3,4-14C]Glufosinate-ammonium (99% radiochemical purity;
    specific activity, 5200 MBq/g) was administered in saline to groups of
    five male Wistar rats at a dose of 2.3 mg/kg bw into the tail vein.
    Groups of animals were sacrificed 2 or 24 h after dosing, and samples
    of blood (for plasma), brain, kidney, and liver were processed and
    assayed for total radiolabel by liquid scintillation counting. Urine
    and faecal samples were obtained over 24 h. Excretion was rapid, 78%
    of the administered dose being found in the 24-h urine sample and 2%

    in faeces. The highest tissue concentrations were in the kidney
    (15 g/g at 2 h; equivalent to 5.5% of the administered dose) and
    liver (1 g/g; equivalent to 1.6% of the administered dose), with much
    lower concentrations in brain (0.06 g/g) and plasma (0.1 g/g). The
    half-times could not be determined owing to the limited number of
    samples (Lauck-Birkel & Strunk, 1999b; Maas & Braun, 1999b).

    (b)  Biotransformation

         (i)  Oral administration

          Rats 

         In two similar studies, [3,4-14C]-glufosinate-ammonium (> 93%
    radiochemical purity; specific activity ~ 2400 or 7800 MBq/g) was
    administered to groups of three or five male and female Wistar rats at
    a dose of 2 mg/kg bw by gavage in saline. Urine and faecal samples
    were obtained every 24 h for 2 or 4 days, pooled, and analysed for
    total radiolabel and metabolites by liquid scintillation counting,
    high-performance liquid chromatography (HPLC), and thin-layer
    chromatography with comparison to standards. Less than 10% of the
    administered dose was absorbed. Excretion was rapid (95% within the
    first 24 h) and occurred predominantly as the parent compound in
    faeces (> 70% of the administered dose). The main urinary metabolites
    were 3-[hydroxy(methyl) phosphinoyl]propionic acid and
    4-methylphosphinico-butanoic acid,  N-acetylglufosinate being the
    primary faecal metabolite with hydroxy-4-methylphosphinicobutanoic
    acid. There were no marked differences between males and females. The
    results are presented in Table 2 (Stumpf, 1993a; Lauck-Birkel, 1996).

         [3,4-14C]Glufosinate-ammonium (> 98% radiochemical purity;
    specific activity, 18 MBq/g) was administered to groups of one or five
    male and female Wistar rats at a dose of 500 mg/kg bw by gavage in
    saline. Urine and faecal samples were obtained every 24 h for 4 days,
    pooled, and analysed for total radiolabel and metabolites by liquid
    scintillation counting, HPLC, and thin-layer chromatography with
    comparison to standards. Samples of liver, spleen, kidney, brain, and
    blood were obtained from animals killed at 2, 6, 24, or 96 h, but
    metabolites were not determined. Less than 10% of the administered
    dose was absorbed. Excretion occurred predominantly as parent compound
    in faeces (> 70% of the administered dose). The primary urinary
    metabolite was 3-[hydroxy(methyl) phosphinoyl]propionic acid, and that
    in faeces was  N-acetylglufosinate. There were no marked differences
    between males and females. The results are presented in Table 3
    (Lauck-Birkel, 1995b).

         [3,4-14C]Glufosinate-ammonium (99% radiochemical purity;
    specific activity, ~ 1200 MBq/g) was administered to groups of five
    male Wistar rats at a dose of 20 mg/kg bw by gavage in saline. Groups
    of animals were killed 1, 6, or 24 h after dosing, and samples of
    blood (for plasma), brain, kidney, and liver were pooled, processed,
    and assayed for total radiolabel (liquid scintillation counting) and
    metabolites (HPLC with comparison to standards). Urine and faecal


        Table 2. Glufosinate ammonium and metabolites in pooled samples from Wistar rats given 
             [3,4-14C]-labelled compound at 2 mg/kg bw by gavage

                                                                                                            
    Compound                                     % of administered dose 
                                                                                                            

                                                 Malesa              Femalesa            Malesb
                                                                                                            
                                                 Urine     Faeces    Urine     Faeces    Urine     Faeces 
                                                 0-96 h    0-96 h    0-96 h    0-96 h    0-48 h    0-48 h
                                                                                                            

    Total radiolabel                             10        90        9         95        6         94
    Glufosinate ammonium                         5.1       75        4.5       69        4.3       77
    4-Methylphosphinicobutanoic acid             1.6       < LD      1.8       < LD      0.2       0.4
    Hydroxy-4-methylphosphinicobutanoic acid     < LD      3         < LD      3.5       0.1       4.3
    3-Methylphosphinicopropionic acid            1.9       1         0.7       1         0.8       1.3
    N-Acetylglufosinate                          < LD      7.4       < LD      9.2       0.1       7.5
                                                                                                            

    LD, limit of determination, < 0.001% of the administered dose
    a   From Stumpf (1993a)
    b   From Lauck-Birkel (1996)
    

        Table 3. Glufosinate ammonium and metabolites in pooled 96-h samples from Wistar 
             rats given [3,4-14C]-labelled compound at 500 mg/kg bw by gavage 

                                                                                       
    Compound                                     % of administered dose 
                                                                                       
                                                 Males               Females
                                                                                       
                                                 Urine     Faeces    Urine     Faeces
                                                                                       

    Total radiolabel                             7.7       75        5.2       89
    Glufosinate ammonium                         5.9       72        4.3       84
    4-Methylphosphinicobutanoic acid             0.2       0.3       0.2       0.1
    Hydroxy-4-methylphosphinicobutanoic acid     < LD      0.3       < LD      0.3
    3-Methylphosphinicopropionic acid            1.2       0.6       0.5       0.5
    N-Acetylglufosinate                          0.04      1.2       0.02      1.7
                                                                                       

    LD, limit of determination, < 0.001% of the administered dose
    a  From Stumpf (1993a)
    

    samples were obtained over 24 h. Metabolites were not determined in
    plasma or brain owing to insufficient total radiolabel. Less than 10%
    of the administered dose was absorbed. Excretion was rapid (90% within
    24 h) and occurred predominantly as parent compound in faeces (85% of
    the administered dose). The primary urinary and tissue metabolite was
    3-[hydroxy(methyl) phosphinoyl]propionic acid, and
     N-acetylglufosinate was the primary faecal metabolite. The results
    are presented in Table 4 (Lauck-Birkel & Strunk, 1999b).

          Goats 

         A lactating goat weighing about 50 kg was given
    [3,4-14C]glufosinate-ammonium (99% radiochemical purity; specific
    activity, 26 mCi/g) in capsules, twice daily for 4 days at a dose of
    about 3 mg/kg bw per day. Over 80% of the administered dose was
    excreted in faeces and approximately 3% in urine; only small amounts
    of the administered radiolabel were found in tissues (< 0.1%) and
    milk (0.02%). The concentrations of radiolabel in milk reached a
    plateau by day 2 (0.02 g/g). The concentrations were higher in kidney
    (0.6 g/g) and liver (0.4 g/g) than in muscle and fat (< 0.01 g/g).
    Glufosinate and 3-[hydroxy(methyl) phosphinoyl]propionic acid
    comprised approximately 50% and 30%, respectively, of the radiolabel
    in both kidney and liver. In milk, only 63% or the radiolabel was
    identified, and glufosinate represented about 50% of the total. In
    urine and faeces, glufosinate comprised > 75% and 3-[hydroxy(methyl)
    phosphinoyl]propionic acid > 10% of the total radiolabel. In faeces,
     N-acetylglufosinate represented 8% of the total residue (Huang &
    Smith, 1995a).


        Table 4. Glufosinate ammonium and metabolites in pooled samples from male Wistar rats given 
             [3,4-14C]-labelled compound at 20 mg/kg bw by gavage

                                                                                                                  
    Compound                             % of administered dose    g/g equivalent
                                                                                                                  
                                         Urine      Faeces         Kidney                   Liver
                                                                                                                  
                                         0-24 h     0-24 h         1 h     6 h     24 h     1 h     6 h    24 h
                                                                                                                  

    Total radiolabel                     4.7        92             3.5     2.9     1.4      0.3     0.7    0.6
    Glufosinate ammonium                 3.3        87             3.1     2.5     1.3      0.2     0.3    0.4
    4-Methylphosphinicobutanoic acid     0.2        < LD           0.1     0.2     0.02     0.02    0.06   0.03
    3-Methylphosphinicopropionic acid    0.8        1              0.3     0.2     0.07     0.1     0.3    0.2
    N-Acetylglufosinate                  < LD       3.5            < LD    < LD    < LD     < LD    < LD   < LD
                                                                                                                  

    LD, limit of determination, < 0.001% of the administered dose
    

          Chickens 

         Laying hens were given [3,4-14C]glufosinate-ammonium (99%
    radiochemical purity; specific activity, 26 mCi/g) in capsules twice
    daily for 14 days at a dose of about 2 mg/kg bw per day. The excreta
    contained > 90% of the administered dose, and < 0.02% was present in
    edible tissues and 0.07% in eggs. The peak concentrations were 0.1
    g/g in liver, 0.07 g/g in egg white, and 0.02 g/g in egg yolk.
    Glufosinate was the main residue in eggs, and 3-[hydroxy(methyl)
    phosphinoyl]propionic acid the main residue in liver (Huang & Smith,
    1995b.

         (ii)  Intravenous administration

          Rats 

         [3,4-14C]Glufosinate-ammonium (99% radiochemical purity;
    specific activity, 5200 MBq/g) was administered in saline to groups of
    five male Wistar rats at a dose of 2.3 mg/kg bw into the tail vein.
    Groups of animals were sacrificed at 2 or 24 h after dosing and
    samples of blood (for plasma), brain, kidney, and liver were pooled,
    processed, and assayed for total radiolabel by liquid scintillation
    counting and for  metabolites by HPLC with comparison to standards.
    Urine and faecal samples were obtained over 24 h. Metabolites were not
    determined in plasma owing to insufficient total radiolabel. The
    radiolabel was eliminated rapidly, primarily as the parent in urine.
    The main metabolite, 3-[hydroxy(methyl) phosphinoyl]propionic acid,
    which was excreted in urine, was formed by deamination at C-2. The
    results are presented in Table 5 (Lauck-Birkel & Strunk, 1999b).

         A metabolic pathway for glufosinate-ammonium in various species
    is shown in Figure 1.

    (c)  Effects on enzymes and other biochemical parameters

         The main biological property of glufosinate-ammonium is
    inhibition of the enzyme glutamine synthetase.  N-Acetylglufosinate
    also inhibits this enzyme in a range of tissues, but the
    interpretation of these results is confounded by the presence of
    glufosinate-ammonium in the samples of  N-acetylglufosinate tested.
    In an attempt to determine the degree to which  N-acetylglufosinate
    inhibits glutamine synthetase, comparative studies were performed
     in vitro and  in vivo (Lutkemeier, 1999; Schmid et al., 1999).

         The inhibition of glutamine synthetase by  N-acetylglufosinate
    and glufosinate-ammonium was investigated  in vitro in tissues from
    11-week-old Wistar rats. Samples of liver, kidney, and brain
    (neocortex, medulla oblongata, and hypothallamic region) were removed
    from 10 animals, rapidly cooled, and kept at -20C before preparation.
    Samples were pooled and homogenates prepared. Liver and kidney were
    assayed as homogenates, and brain tissues were assayed as a 1500   g
    supernatant of a homogenate. The assay for glutamine synthetase is
    based on the formation of gamma-glutamyl hydroxamate and ammonia from


        Table 5. Glufosinate ammonium and metabolites in pooled samples from male Wistar rats given
             [3,4-14C]glufosinate ammonium at 2.3 mg/kg bw intravenously

                                                                                                                        
    Compound                             % of administered dose     g/g equivalent
                                                                                                                        
                                         Urine       Faeces         Kidney                     Liver
                                                                                                                        
                                         0-24 h      0-24 h         1 h      6 h      24 h     1 h      6 h      24 h

    Total radiolabel                     78          2.3            0.06     0.04     15       1        1        0.5
    Glufosinate ammonium                 68          2              0.04     0.04     14       1        1        0.5
    3-Methylphosphinicopropionic acid    10          0.05           < 0.01   0.01     1.4      0.1      0.1      0.1
    N-Acetylglufosinate                  < LD        0.2            < LD     < LD     < LD     < LD     < LD     < LD
                                                                                                                        

    LD, limit of determination, < 0.001% of the administered dose
    

    FIGURE 1

    (-)-glutamine and hydroxylamine in the presence of arsenate,
    manganese, and ADP, followed by spectrophotometric measurement of an
    iron compound. A preincubation period of 10 min was used in the main
    assays, as it had been shown that inhibition was not changed by
    extending this period to 60 or 120 min. The samples were incubated for
    20 min in the presence of  N-acetylglufosinate (a 33.8% solution
    containing 0.06% w/w glufosinate-ammonium) at 0-10 000 g/ml or
    glufosinate-ammonium (as a 50.2% solution) at 0-500 g/ml.
    Glufosinate-ammonium induced significant, concentration-related
    inhibition of glutamine synthetase in all tissues at doses > 0.77
    mmol/L (Table 6), the inhibition profile varying with tissue.
     N-Acetylglufosinate induced only marginal inhibition at 13 mmol/L,
    some of which can be attributed directly to the glufosinate-ammonium
    content of the sample. The report did not provide results corrected
    for protein content, and not all of the assays were performed in
    duplicate; however, for the purposes of this comparative exercise, the
    results are considered to be acceptable (Lutkemeier, 1999).

         A comparative study was performed of the inhibition of glutamine
    synthetase in tissues from groups of 10 male Wistar rats given diets
    containing  N-acetylglufosinate (with 0.06% w/w glufosinate-ammonium)
    at 1000 or 10 000 ppm or glufosinate-ammonium at 0, 100, or 1000 ppm
    (Schmid et al., 1999). The animals were exposed for 6, 13, 20, or 90
    days with 91 days plus 31 days for recovery. In addition to standard
    observations and gross necropsy, samples of liver, brain, and kidney
    were removed, rapidly cooled, and stored at -70C prior to processing
    and assaying for glutamine synthetase activity, as described above.

         There were no deaths or treatment-related clinical signs. The
    absolute and relative weights of the kidney were increased by 8-23% in
    all treated groups during the first 20 days of the study, but with no
    associated pathological findings. Statistically significant inhibition
    of glutamine synthetase activity was seen in liver and kidney samples
    by day 6, and, except in liver from animals exposed to 1000 ppm
     N-acetylglufosinate, did not increase markedly up to day 90 (Table
    7). Significant recovery of glutamine synthetase activity occurred
    during the 31-day recovery period. The results indicate that orally
    administered glufosinate-ammonium is approximately 10 times more
    potent at inhibiting glutamine synthetase than is
     N-acetylglufosinate. The extent to which this inhibition is due
    directly to de-acetylation of  N-acetylglufosinate to
    glufosinate-ammonium is uncertain. The activity of glutamine
    synthetase in brain samples was not reduced markedly in animals
    exposed to either  N-acetylglufosinate or glufosinate-ammonium at
    1000 ppm.

    (d)  Significance of glutamine synthetase inhibition to humans

         Glutamine synthetase (E.C.6.3.1.2) is a key enzyme in the
    metabolism of nitrogen and glutamate, catalysing the multi-step
    reaction of 

         (-)-glutamate + ATP + NH3 <=> (-)-glutamine + ADP + P


        Table 6. Inhibition of glutamine synthetase activity  in vitro in tissue samples from rats, in the 
    presence of  N-acetylglufosinate and glufosinate ammonium; in square brackets, absolute activity 
    expressed as mg gamma-glutamylhydroxamate formed per g tissue per 20 min

                                                                                                     
    Compound                Dose        Liver       Kidney      Neocortex   Medulla     Hypothalamus
                            (mmol/L)
                                                                                                     

    Glufosinate ammonium    0           0 [28]      0 [18]      0 [27]      0 [23]      0 [20]
                            0.003       1           0           0           1           
                            0.008       2           1           0           0           0
                            0.026       4           1           3           1           0
                            0.077       14          3           5           6           3
                            0.26        36          5           13          20          11
                            0.77        60          13          32          42          29
                            1.3         72          17          45          53          41

    N-Acetylglufosinate     0.13        1           0           0           0           0
                            0.38        1           0           0           0           0
                            0.63        2           0           0           0           0
                            1.3         2           0           0           0           0
                            6.3         9           1           2           2           1
                            13a         15          2           4           7           5
                                                                                                     

    a Contains approximately 0.03 mmol/L glufosinate ammonium
    

        Table 7. Activity of glutamine synthetase in samples from 10 male Wistar rats that 
             received  N-acetylglufosinate or glufosinate ammonium in the diet or 
             control diet 

                                                                                       
    Day         Tissue      Glutamine synthetase activity (mean % of control value)
                                                                                       
                            Controla    Glufosinate ammonium     N-acetylglufosinate
                                                                                       
                                        100 ppm     1000 ppm    1000 ppm    10 000 ppm
                                                                                       

    6           Liver       24          55          36          96          46
                Brain       28          101         89          94          93
                Kidney      17          60          58          61          54

    13          Liver       30          51          30          74          40
                Brain       25          101         91          102         104
                Kidney      17          61          58          59          54

    90          Liver       24          60          40          58          54
                Brain       22          104         82          99          98
                Kidney      14          67          46          55          53

    91 + 31     Liver       24          97          85          83          94
                Brain       14          98          88          97          97
                Kidney      22          90          97          95          87
                                                                                       

    a  Absolute activity, expressed as mg gamma-glutamylhydroxamate formed per 
       g tissue per 20 min
    

    In plants, glutamine synthetase is the main enzyme involved in the
    control of ammonia concentrations, and its inhibition is the mechanism
    of action of glufosinate-ammonium in plants. In mammals, other
    pathways exist for the homeostatic control of ammonia, such as reverse
    reaction of amino acid dehydrogenases and the carbamoyl phosphate
    synthetase-urea cycle. Glutamate and glutamine can, however, play
    significant roles in other biochemical and physiological processes in
    mammals, such as neurotransmission (glutamate and gamma-aminobutyric
    acid (GABA)). The activity of glutamine synthetase varies between
    tissues and species (see below), but the amino acid sequence is
    reported to be well conserved (LieVenema et al., 1998; Purich, 1998;
    Ernst & Leist, 1999a).

         The liver has two distinct systems for dealing with ammonia. A
    high-capacity, low-affinity system exists in the periportal
    hepatocytes which is based on carbamoyl phosphate synthetase and the
    urea cycle. In central vein hepatocytes, a low-capacity, high-affinity
    system exists which is based on glutamine synthetase and ornithine

    aminotransferase. Hack et al. (1994) showed that doses of
    glufosinate-ammonium did not increase ammonia concentrations in liver
    at a dose (5000 ppm) that inhibited glutamine synthetase activity by
    50%. While a 60% reduction in liver glutamine was seen at day 1, the
    concentration had returned to normal by day 4, indicating the
    induction of alternative pathways. Inhibition of liver glutamine
    synthetase by up to 50% is therefore not considered to be adverse in
    isolation.

         The activity of this enzyme in kidney varies considerably between
    species (LieVenema et al., 1998; see below), with relatively high
    activity in rodents but negligible activity in dogs and humans.
    Inhibition of kidney glutamine synthetase in the absence of
    pathological findings is not considered to be relevant to human risk
    assessment.

         In the brain and central nervous system, ammonia homeostasis is
    controlled by a number of enzymes including glutamine synthetase and
    glutamate dehydrogenase. Under normal conditions (~ 100 mol/L of
    ammonium and 3 mmol/L of glutamate), the flux through glutamine
    synthetase in brain is 2-10% of its theoretical capacity and that of
    glutamate dehydrogenase is approximately 0.1% of its capacity
    (Lie-Venema et al., 1998). With such excess capacity, inhibition of
    brain glutamine synthetase is unlikely to result in significant
    increases in brain ammonia concentrations. This conclusion is
    supported by the finding of Hack et al. (1994) that brain ammonia
    concentrations were not increased at doses of glufosinate-ammonium
    that produced a 40% reduction in brain glutamine synthetase activity
    in rats. However, the glutamine-glutamate shunt between GABA and
    glutamate in neurons and glutamine in astrocytes plays a role in both
    excitatory and inhibitory neurotransmission. The results of Hack et
    al. (1994), although somewhat inconsistent, indicate that significant
    changes in a range of biogenic amines in regions of the dog brain are
    associated with changes of > 8% in glutamine synthetase activity
    after administration of glufosinate-ammonium at 8 mg/kg bw for 28
    days, a dose that produced 'increased gait activity'. It is thus
    proposed that any statistically significant, > 10% inhibition of
    glutamine synthetase activity in brain is a marker of potentially
    adverse effects on brain biochemistry and behaviour.

    2.  Toxicological studies

         All of the studies described below were included statements of
    compliance with GLP and met the basic requirements of the OECD test
    guidelines applicable at the time of study initiation, unless
    otherwise stated.

    (a)  Short-term studies of toxicity

          Mice 

         In response to concern that the doses used in the study of
    carcinogenicity with glufosinate-ammonium in mice evaluated previously

    had not been appropriately high (160 ppm in males, 320 ppm in
    females), a 90-day study was performed in which groups of 10 NMRI mice
    of each sex received diets containing glufosinate-ammonium (purity,
    95.5%) at concentrations of 0, 1750, 3500, or 7000 ppm. The
    homogeneity and achieved concentrations were acceptable, and the
    intakes of animals were 561 and 644 mg/kg bw per day for males and
    females at the intermediate dose and 274 and 356 mg/kg bw per day for
    animals at the low dose, respectively. The investigations included
    clinical signs, body weight, food consumption, haematology, clinical
    chemistry, organ weights, and gross and microscopic pathology. All
    animals at the high dose had died by day 8, 50% of those at 3500 ppm
    had died by day 11, and one female at the low dose died. Clinical
    signs (ruffled fur, sedation, and emaciation), reduced food
    consumption, and initial body-weight loss were seen at all doses,
    although the body-weight gain during the latter part of the study was
    similar in surviving animals. There were no consistent clinical
    chemical or haematological findings and no changes in organ weights or
    on gross examination. Congestion in multiple organs was seen at
    microscopic examination of many animals that died during the study,
    but the cause of death was not determined. No NOAEL could be
    identified, but the design was not optimal for this purpose. The
    lowest dose tested (1750 ppm, equal to 270 mg/kg bw per day) was
    approximately the maximum tolerated dose for a 90-day study, resulting
    in a single death and marked initial effects on body weight. The
    maximum tolerated dose for a 2-year study in mice would be about 600
    ppm if a factor of 3 is used to extrapolate from the approximate dose
    in the 90-day study (Dotti et al., 1994). The results of this study
    indicate that the previous bioassay was performed within a factor of 2
    of the estimated maximum tolerated dose and need not be repeated.

          Rats 

         The toxicity of glufosinate-ammonium (purity, 95.5%) was
    investigated in groups of 10 Wistar rats of each sex given the
    compound in the diet at concentrations of 0, 7500, 10 000, or 20 000
    ppm for 90 days. Routine observations and measurements were made, with
    ophthalmoscopy before treatment and at termination and a basic
    functional observation battery, which was administered before
    treatment and at weeks 1, 2, 3, 4, 8, and 13 and involved observations
    in the home cage, an external area, and in the hand, but no forced
    physical activity such as grip strength or swimming. Blood samples for
    haematological and clinical chemical analyses were taken from fasted
    animals at week 13. At termination, five animals of each sex per group
    were perfused to preserve nervous tissue. Major organs from all
    animals at the highest dose and controls were examined histologically,
    as was nervous tissue from all perfused animals and all gross lesions.
    Organ weights were not determined. The homogeneity, stability, and
    achieved concentrations in the diet were satisfactory, with intakes
    equal to 0, 520, 690, and 1400 mg/kg bw per day in males and 0, 570,
    740, and 1400 mg/kg bw per day in females. 

         Two females at the high dose died within the first 8 days of
    dosing, but there were no other unscheduled deaths. Animals at the
    high dose showed a range of signs during the first 2 weeks of
    treatment, including sedation, dyspnoea, emaciation, and diarrhoea.
    Food consumption was reduced by > 20% in all groups during the first
    two weeks. Body-weight loss was seen in animals at the high dose, and
    reductions in body-weight gain were seen in other groups during the
    first week of dosing. All groups had similar body-weight gains during
    the last weeks of the study. No abnormal ophthalmoscopic findings were
    reported. The haematological findings were similar in test and control
    groups, and an apparent reduction in erythrocyte count in males
    appeared to be related to a high control value. A consistent pattern
    of changes in serum lactate dehydrogenase and creatine kinase activity
    was seen in animals of each sex, with 20% reductions at the low and
    intermediate doses and an increase at the high dose. Small (< 10%)
    but statistically significant ( p < 0.05) increases in serum calcium
    and inorganic phosphorus concentrations were seen in animals at the
    high dose. The functional observational battery identified a similar
    pattern of changes in males and females that included miosis, apathy,
    reduced alertness and grooming, increased body tone, and vocalization.
    These changes were present in all treated groups, with no clear
    pattern over time, but were more prevalent at the high dose. There
    were no significant macroscopic findings, and the only microscopic
    finding of significance was an increased incidence of renal pelvis
    dilatation in males at the high dose; there were no treatment-related
    effects on nervous tissues.

         No NOAEL could be identified owing to alterations in calcium and
    inorganic phosphorus and in the functional observational battery at
    all doses, although there was no indication of irreversible
    neurotoxicity. The effects on food consumption and body weight may
    have been secondary to palatability, as they were mainly transient
    (Dotti et al., 1993).

    (b)  Long-term studies of toxicity and carcinogenicity

          Rats 

         A study was initiated in 1994 (Schmid et al., 1998) to include
    doses above the maximum of 500 ppm used in the first study. Groups of
    60 Wistar rats of each sex received diets containing
    glufosinate-ammonium (purity, 96%) at concentrations of 0, 1000, 5000,
    or 10 000 ppm for 104 weeks. These doses were based on increased
    mortality seen at 20 000 ppm in the 90-day study (Dotti et al., 1993).
    The animals were observed for survival, clinical signs, body-weight
    gain, food consumption, and the presence of nodules or masses. Blood
    smears were prepared from controls and animals at the high dose in
    weeks 52, 78, and 104. At termination, over 30 tissues were removed,
    nine were weighed, and gross and histopathological examination was
    performed on all tissues from all animals. The stability, achieved
    levels, and homogeneity of glufosinate-ammonium in the diets were
    satisfactory, giving intakes equal to 0, 45, 230, and 470 mg/kg bw in
    males and 0, 57, 280, and 580 mg/kg bw per day in females. 

         Survival was similar in all groups, being over 70% at 104 weeks.
    There were no treatment-related changes in clinical signs, food use
    efficiency, the occurrence of nodules or masses, or the appearance of
    blood smears. All treated groups had reduced food consumption and
    body-weight gain over the first month of dosing but these parameters
    were subsequently within 10% of those of controls. The weight of the
    kidney was increased by 15-30% in relation to dose in all treated
    groups, but there were no histological correlates. Macroscopic
    examination showed a decreased incidence of pituitary nodules in all
    treated males but an increased incidence of adrenal gland foci in
    males at the high dose. Microscopic examination revealed a
    statistically significant ( p < 0.05) increase in the incidence of
    retinal atrophy in males and females at 10 000 ppm and in females at
    5000 ppm. This effect was considered to be related to treatment, as a
    dose-response relationship was evident in females and it occurred in
    animals of each sex, even though the incidence was within the range of
    historical controls (males, 20%; females, 38%; Table 8). The incidence
    of a rare skin tumour (trichofolliculoma) was increased in males at
    the high dose, but it was not statistically significant and was not
    seen in females or in males receiving half of the high dose; the
    finding was therefore considered not to provide clear evidence of
    carcinogenic potential. The total number of malignant and benign
    tumours was similar in treated and control groups. The NOAEL was 1000
    ppm, equal to 45 mg/kg bw per day, on the basis of the increased
    incidence of retinal atrophy (Schmid et al., 1998). 


    Table 8. Incidences of lesions in Wistar rats receiving 
             glufosinate ammonium in the diet for 104 weeks

                                                             
    Dose (ppm)     Incidence (%)
                                                             
                   Retinal atrophy       Trichofolliculoma
                                         (males)
                   Males     Females
                                                             

         0          4         3          0
     1 000          3         2          0
     5 000          4        19          0
    10 000         12        29          4
                                                             


    (c)  Studies on metabolites

         (i)   N-Acetylglufosinate

         The (-) isomer of  N-acetylglufosinate is a major metabolite of
    glufosinate-ammonium after its application to glufosinate-tolerant
    crops. In 1998, the Committee proposed that residues arising from
    applications of glufosinate-ammonium to tolerant crops be defined as

    the 'sum of glufosinate-ammonium, 3-[hydroxy(methyl)phosphinoyl]
    propionic acid, and  Nacetylglufosinate' but could not adopt this
    definition until  N-acetylglufosinate had been evaluated
    toxicologically. Extensive toxicokinetic and toxicological studies on
     N-acetylglufosinate have since been submitted. The preparation
    tested was an aqueous solution of the disodium salt, and all of the
    doses cited below have been corrected for the content of the
    technical-grade (-)-isomer. The three batches of  N-acetylglufosinate
    used varied in purity from 74.5% to 99% and in glufosinate-ammonium
    content from 0.06 to 4.5% (Weller, 1994). All of the studies conformed
    to GLP and were claimed to have been performed according to guideline
    85-1 of November 1984 of the US Environmental Protection Agency or to
    meet the basic requirements of the OECD test guidelines applicable at
    the time the study was initiated, unless otherwise stated.

          Absorption, distribution, and excretion: The toxicokinetics of
     N-acetylglufosinate was examined in several studies at doses of
    3 mg/kg bw (Stumpf, 1993b; Kellner et al., 1993) or 1000 mg/kg bw
    (Lauck-Birkel, 1995b; Maas & Braun, 1995b). Groups of five Wistar rats
    of each sex received [3,4-14C] N-acetylglufosinate (purity, > 98%)
    by gavage in saline. Urine and faeces were collected over 24-h periods
    up to 96 h, and up to 12 tissue samples were taken from animals given
    1000 mg/kg bw and killed at 2 and 6 h (two of each sex), 24 h (five of
    each sex), and 96 h (five of each sex). Radiolabel in the
    gastrointestinal tract was determined in one male each killed at 4 or
    24 h after receiving 3 mg/kg bw, and one male at this dose killed at
    96 h was studied by autoradiography (Kellner et al., 1993). Radiolabel
    in tissues and excreta was determined by liquid scintillation counting
    after appropriate processing. The samples were also prepared for
    metabolic investigations (see below).

         The doses administered differed somewhat among animals, but this
    was considered not to have affected the results. Most of each
    administered dose was excreted in the faeces within 48 h (Table 9),
    although excretion was more rapid at the lower dose. The
    concentrations in tissue peaked at 6 h and represented < 1% of the
    administered radiolabel; the highest concentrations were detected in
    kidney, and those in liver and kidney were greater than in plasma. In
    animals at 3 mg/kg bw, the tissue concentration represented < 0.1% of
    the administered dose at day 4, with the highest concentrations in
    kidney (0.06 g/g), a finding confirmed by autoradiography. Analysis
    of the gastrointestinal tract 4 and 24 h after administration of
    3 mg/kg bw showed that 3% and < 0.01% of the dose was in the stomach
    and 91% and 3.5% in the intestine, respectively. Sporadic differences
    by sex were seen but were not consistent between studies or individual
    animals. The tissue concentrations were higher in female than male
    rats given 1000 mg/kg bw, as was the urinary excretion after the dose
    of 3 mg/kg bw. The results of the two studies with 1000 mg/kg bw
    presented a similar profile, although Maas & Braun (1995b) found lower
    tissue concentrations and higher urinary excretion in females (up to
    20%, including cage washes); however, there was a threefold variation
    between individual animals, and the possibility of contamination by
    faeces cannot be dismissed. 

    Table 9. Radiolabel in excreta and tissues of rats given 
             [3,4-14C] N-acetylglufosinate orally at 3 or 1000 mg/kg bw

                                                                            
    Sample              Radiolabel in excreta (% of dose) or tissues (g/g)
                                                                            

                        1000 mg/kg bwa              3 mg/kg bwb
                                                                            
                        Males     Females           Males      Females
                                                                            
    Urine 
       24 h             5         4                 5          8
       48 h             7         6                 5          9
    Faeces 
       24 h             58        63                97         93
       48 h             82        83                100        96
    Liver, 6 h          17        46                NA         NA
    Kidney, 6 h         44        672c              NA         NA
    Brain, 6 h          1         1                 NA         NA
    Plasma, 6 h         3         10                NA         NA
                                                                            

    NA, not analysed
    a   Results from Lauck-Birkel (1995b)
    b   Results from Kellner et al. (1993); Stumpf (1993b)
    c   Possible outlier


         Groups of three Wistar rats of each sex were given
    [3,4-14C] N-acetylglufosinate (purity, 98%; 1400 MBq/g) at 3 mg/kg
    bw in saline, and blood samples were taken from the retro-orbital
    plexus at 15, 30, and 60 min and 2, 4, 6, 8, 24, 48, 72, and 96 h. The
    samples were absorbed onto filter paper and combusted, and radiolabel
    was determined by liquid scintillation counting. The peak
    concentration (0.05 g/g) was seen at 60 min, although significant
    amounts were detected at 15 min, showing rapid initial absorption. The
    concentration in blood declined in a biphasic manner, in an initial
    phase with a half-time of 0.8 h and a second phase with a half-time of
    7 h. The concentration was at the limit of detection by 24 h. The
    integrated area under the curve of concentration-time was ~ 0.2 g
    h/g. Comparison with the results of an identical study in which the
    substance was administered intravenously showed that absorption after
    oral administration represented approximately 5% of the dose over
    24 h. There was no significant difference between the sexes (Kellner &
    Braun, 1993a,b).

         A lactating goat weighing 36 kg was dosed orally twice a day for
    3 consecutive days with capsules containing
    [3,4-14C] N-acetylglufosinate, equivalent to a dose of 3.0 mg/kg bw
    per day. The feed intake was 1.4 kg/day. The animal was milked twice
    daily and was slaughtered 16 h after the final dose. Most of the
    administered radiolabel was excreted in the faeces (68%), with 7.3% in

    urine and 19% in the gastronintestinal tract and its contents. Only
    0.2% of the administered dose was found in the tissues and blood and
    < 0.1% in milk. The concentrations in kidney were higher than in
    other tissues. Those in milk reached a plateau by day 2 (Huang &
    Smith, 1995c).

         Six laying hens weighing 1.3-1.6 kg were dosed orally twice a day
    for 14 consecutive days with capsules containing
    [3,4-14C] N-acetylglufosinate, equivalent to a dose of 2.2 mg/kg bw
    per day. The mean feed intake was 120 g/day. Eggs were collected twice
    daily, and the birds were slaughtered 15 h after the final dose. Most
    of the administered dose was excreted (86%), with 1.0% remaining in
    the gastrointestinal tract; < 0.1% of the administered dose was
    present in edible tissues and blood. The concentrations of radiolabel
    associated with  N-acetyl-L-glufosinate disodium salt were 0.076
    mg/kg in liver, 0.013 mg/kg in muscle, and 0.011 mg/kg in fat; that in
    egg white was only slightly above the level of quantification
    (< 0.009 mg/kg) throughout the study, reaching a peak of 0.014 mg/kg.
    The concentrations in egg yolk increased slowly throughout the 14
    days, with a peak at necropsy of 0.056 mg/kg (Huang & Smith, 1995d).

         Groups of three Wistar rats received an intravenous injection of
    [3,4-14C] N-acetylglufosinate (purity, 98%; 1400 MBq/g) into the
    tail vein at a dose of 3 mg/kg bw as a solution in saline. Blood
    samples were taken from the retro-orbital plexus at 5, 15, 30, and 60
    min and 2, 4, 6, 8, 24, 48, 72, and 96 h. The samples were absorbed
    onto filter paper and combusted, and the radiolabel was determined by
    liquid scintillation counting. The peak concentration (6-7.5 g/g) was
    seen at 5 min, with an initial decline of 4 h, a half-time of 0.3 h,
    and a second phase with a half-time of 14 h. The concentration of
    radiolabel in blood was at the limit of detection at 24 h. The
    integrated area under the curve of concentration-time was ~ 3.8 g
    h/g. There as no significant difference between the sexes (Kellner &
    Braun, 1993a,b).

         The toxicokinetics of  N-acetylglufosinate was investigated in
    groups of five Wistar rats of each sex which received
    [3,4-14C] N-acetylglufosinate (purity, 98%; 1400 MBq/g) in saline
    intravenously at a dose of 3 mg/kg bw. Urine and faeces were collected
    over 0-4, 4-8, 8-24, 24-48, 48-72, and 72-96 h. Tissue samples were
    taken at 96 h. Autoradiography was performed on one male killed at 96
    h. The radiolabel in tissues and excreta was determined by liquid
    scintillation counting after appropriate processing. Excretion was
    rapid, with > 85% of the radiolabel appearing in the 0-4-h urine
    sample. By 96 h, approximately 95% of the dose had been excreted in
    urine; faecal excretion accounted for 4% in females and 2% in males.
    The excretory half-times were slightly longer in males than in
    females. By 96 h, tissue radiolabel accounted for < 0.3% of the dose;
    the highest concentrations were found in kidney, with 0.2 g/g in
    males and 0.07 g/g in females (Kellner et al., 1993)

          Biotransformation: Urine and faeces from Wistar rats given
    [3,4-14C] N-acetylglufosinate at 3 or 1000 mg/kg bw by gavage in
    the studies of Stumpf (1993b) and Lauck-Birkel (1995b), described
    above, were extracted and analysed for metabolites by HPLC or
    thin-layer chromatography with comparison to standards. The tissue
    samples contained insufficient radiolabel for investigation of
    metabolites. The extent of metabolism was greater at 3 mg/kg bw,
    indicating the presence of a saturable reaction pathway. The main
    compound in urine was  N-acetylglufosinate, with low concentrations
    of 3-[hydroxy(methyl) phosphinoyl]propionic acid and
    4-methylphosphinico-butanoic acid. The faeces of animals given the low
    dose contained a significant amount of glufosinate-ammonium, which was
    not seen in those given the high dose. The study of Kellner et al.
    (1993) showed that glufosinate-ammonium is formed in the intestine,
    but the extent to which it is systemically available is not clear. The
    results for male rats are presented in Table 10; female animals showed
    a similar profile.

        Table 10.  N-Acetylglufosinate and metabolites in 96-h samples from male Wistar 
              rats given [3,4-14C]-labelled compound at 3 or 1000 mg/kg bw by gavage

                                                                                        
    Compound                                      Percent administered dose
                                                                                        
                                                  3 mg/kg bwa         1000 mg/kg bwb
                                                                                        
                                                  Urine     Faeces    Urine     Faeces
                                                                                        

    Total radiolabel                              5.3       83        7.6       89
    N-Acetylglufosinate                           4.0       70        7.4       85
    Glufodinate ammonium                          < LD      11        < LD      0.9
    3-Methylphosphinicopropionic acid             0.7       0.6       0.1       0.4
    4-Methylphosphinicobutanoic acid or           0.6       1.2       0.1       0.1
      hydroxy-4-methylphosphinicobutanoic acid 
                                                                                        

    a   From Stumpf (1993b)
    b   From Lauck-Birkel (1995b)
    

         [3,4-14C] N-Acetylglufosinate (98% radiochemical purity;
    specific activity, 830 MBq/g) was dissolved in physiological saline
    and administered to groups of five male Wistar rats at a dose of 30
    mg/kg bw by gavage. Groups of animals were sacrificed 1, 6, or 24 h
    after dosing, and samples of blood (for plasma), brain, kidney, and
    liver were pooled, processed, and assayed for total radioactivity
    (liquid scintillation counting) and metabolites (HPLC). Urine and
    faeces were collected over 24 h. Metabolites were not determined in
    plasma or brain owing to insufficient total radiolabel. There was
    limited metabolism and rapid excretion; > 80% of the recovered

    radiolabel in the faeces was  N-acetylglufosinate. The concentration
    of glufosinate-ammonium in kidney increased with time (Table 11;
    Lauck-Birkel & Strunk, 1999d). A similar pattern of absorption,
    distribution, and excretion was reported by Maas & Braun (1999c).

         Two male Wistar rats received [3,4-14C] N-acetylglufosinate
    (purity, > 98%) by gavage in saline at a dose of 3 mg/kg bw. The
    animals were killed 4 or 24 h later, and the gastrointestinal tract
    was examined for total radiolabel and metabolites. Significant
    deacetylation of  N-acetylglufosinate was found in the intestine,
    giving rise to glufosinate-ammonium (Table 12; Kellner et al., 1993).

         [3,4-14C] N-Acetylglufosinate (98% radiochemical purity;
    specific activity, 7200 MBq/g) was dissolved in physiological saline
    and administered intravenously into the tail vein of groups of five
    male Wistar rats at a dose of 3 mg/kg bw. Groups of animals were
    sacrificed 2 or 24 h after dosing, and samples of blood (for plasma),
    brain, kidney, and liver were pooled, processed, and assayed for total
    radiolabel (liquid scintillation counting) and metabolites (HPLC).
    Urine and faeces were obtained over 24 h. Metabolites were not
    determined in plasma or brain owing to insufficient total radiolabel.
    There was limited metabolism and rapid excretion, and over 95% of the
    radiolabel recovered in urine was  N-acetylglufosinate. At 24 h,
    glufosinate-ammonium was present at higher concentrations than
     N-acetylglufosinate in kidney (Table 13; Lauck-Birkel & Strunk,
    1999c). A similar pattern of distribution and excretion was reported
    by Maas & Braun (1999d).

         Samples from the study of Huang & Smith (1995c) on goats were
    investigated for metabolites.  N-Acetylglufosinate and glufosinate
    accounted for 52% and 34% of the radiolabel in faeces. respectively.
    Glufosinate was the main residue in kidney, liver, and milk, although
     N-acetylglufosinate (the administered material) and
    3-[hydroxy(methyl) phosphinoyl]propionic acid formed a substantial
    proportion of the residue in kidney and liver. The concentration of
    glufosinate-ammonium in the kidney (0.7 ppm) was four times that in
    the liver (0.095 ppm). This study showed that de-acetylation of
     N-acetylglufosinate to glufosinate-ammonium makes a significant
    contribution to tissue residues.

         Samples from the study of Huang & Smith (1995d) on hens were
    investigated for metabolites.  N-Acetyl-L-glufosinate disodium salt
    comprised 73% of the radiolabel in faeces, with glufosinate and
    3-[hydroxy(methyl) phosphinoyl]propionic acid comprising 13% and 8.6%,
    respectively.  N-Acetylglufosinate (the administered material) was
    the main residue identified in liver and egg yolk, and glufosinate and
    3-[hydroxy(methyl) phosphinoyl]propionic acid were also substantial
    components of the liver residue. Glufosinate was the main residue in
    egg white. This study showed that deacetylation of
     N-acetylglufosinate to glufosinate-ammonium makes a significant
    contribution to tissue residues.


        Table 11.  N-Acetylglufosinate and metabolites in pooled samples from male Wistar rats given 
              [3,4-14C]-labelled compound at 30 mg/kg bw by gavage

                                                                                                            
    Compound                                        % of administered dose    g/g equivalent
                                                                                                            
                                                    Urine       Faeces        Kidney           Liver
                                                    0-24 h      0-24 h                                      
                                                                              6 h     24 h     6 h     24 h
                                                                                                            

    Total radiolabel                                2.1         88            1.1     0.7      0.5     0.2
    N-Acetylglufosinate                             1.7         82            0.6     0.04     0.08    0.05
    Glufosinate ammonium                            0.02        5             0.08    0.73     < LD    < LD
    3-Methylphosphinicopropionic acid               0.15        < LD          0.19    0.03     0.3     0.02
    4-Methylphosphinicobutanoic acid (1% in dose)   0.2         1.4           0.09    < LD     0.03    0.01
                                                                                                            

    LD, limit of determination, < 0.001% of the administered dose
    

    Table  12. Residues in stomach and intestine after oral administration 
           of 3 mg/kg bw [3,4-14C] N-acetylglufosinate to two male rats

                                                                            
    Compound                             Percent of radiolabel
                                                                            
                                         Stomach             Intestine
                                                                            
                                         4 h       24 h      4 h       24 h
                                                                            

    Total radiolabel                     3.6       91        < 0.01    3.5
    Glufosinate ammonium                 0.0       2.6       ND        29
    4-Methylphosphinicobutanoic acid     0.0       0.8       ND        0.0
    3-Methylphosphinicopropionic acid    0.0       0.5       ND        4.5
    N-Acetylglufosinate                  99.8      96        ND        66
                                                                            

    ND, not determined


          Effects on enzymes and other biochemical parameters: The main
    biological property of glufosinate-ammonium is inhibition of the
    enzyme glutamine synthetase, and toxicological studies with
     N-acetylglufosinate have also shown inhibition of this enzyme in a
    range of tissues. The interpretation of these results is confounded by
    the presence of glufosinate-ammonium in the samples of
     N-acetylglufosinate tested. In an attempt to determine the degree to
    which  N-acetylglufosinate inhibits glutamine synthetase, comparative
    studies were performed  in vitro and  in vivo (Lutkemeier, 1999;
    Schmid et al., 1999). The studies are summarized in Tables 6 and 7.
     In vitro, N-acetylglufosinate produced only marginal inhibition at
    13 mmol/L, some of which can be attributed directly to the
    glufosinate-ammonium in the sample. When glufosinate-ammonium is
    administered orally, it is approximately 10 times more potent as an
    inhibitor of glutamine synthetase than is  N-acetylglufosinate. The
    amount of this inhibition that is due directly to de-acetylation of
     N-acetylglufosinate to glufosinate-ammonium is uncertain; however,
    the work of Lauck-Birkel & Strunk (1999a,b; Table 11) and the results
     in vitro (Table 6) indicate that most of the inhibition in kidney is
    due to biotransformation of  N-acetylglufosinate to
    glufosinate-ammonium.

          Acute toxicity: N-Acetylglufosinate has little toxicity when
    given as a single oral dose (Table 14). When it was given by
    intraperitoneal injection, deaths occurred at the lowest doses tested
    (580 mg/kg bw) in both rats and mice, but there was no clear
    dose-response relationship in mice. The compound was not tested by
    other routes, as it is formed as a metabolite only in plants, and
    exposure through the skin or by inhalation is unlikely. The clinical
    signs of toxicity seen after oral exposure to  N-acetylglufosinate
    were reduced respiratory rate, reduced activity, contracted flanks,
    and squatting during the first 24 h. The decreased respiratory rate


        Table 13.  N-Acetylglufosinate and metabolites in pooled samples from male Wistar rats given 
              [3,4-14C]-labelled compound at 3 mg/kg bw intravenously

                                                                                                        
    Compound                                        Percent of administered dose
                                                                                                        
                                                    Urine    Faeces   Liver             Kidney
                                                    (24 h)   (24 h)                                     
                                                                      2 h      24 h     2 h      24 h
                                                                                                        

    Total radiolabel                                86       1.8      0.5      0.1      0.9      0.1
    N-Acetylglufosinate                             85       1.7      0.4      0.1      0.8      0.01
    Glufosinate ammonium                            < LD     0.1      0.01     0.01     0.05     0.06
    3-Methylphosphinicopropionic acid               < LD     < LD     0.04     0.01     < LD     0.001
    4-Methylphosphinicobutanoic acid (1% in dose)   1.1      0.02     0.01     < LD     0.01     < LD
                                                                                                        

    LD, limit of determination, < 0.001% of the administered dose
    

        Table 14.  Acute toxicity of  N-acetylglufosinate (purity, 79.4%; containing 
               4.5% glufosinate ammonium)

                                                                                    
    Species (strain)   Route              LD50         Reference
                                          (mg/kg bw)
                                                                                    

    Rat (Wistar)       Oral, gavage          290       Schollmeier & Leist (1989a)
    Mouse (NMRI)       Oral, gavage          290       Schollmeier & Leist (1989b)
    Rat (Wistar)       Intraperitoneal    > 1200       Schollmeier & Leist (1989c)
    Mouse (NMRI)       Intraperitoneal    > 2000       Schollmeier & Leist (1989d)
                                                                                    
    

    persisted for the duration of the study in mice. Gross examination
    showed no abnormalities.

         In a Magnusson and Kligman maximization protocol, groups of 20
    female Pirbright guinea-pigs, 10 weeks of age, received an intradermal
    induction with  N-acetylglufosinate (purity, 79.4%; supplied as a
    57.9% solution) at 5% in saline (equal to 2.9%  N-acetylglufosinate)
    and a 1:1 preparation of Freund's adjuvant. For topical induction and
    challenge, undiluted test material was applied under an occlusive
    dressing. There were no signs of erythema or oedema. Satisfactory,
    contemporary data for positive controls were presented (Hofmann &
    Jung, 1988).  N-Acetylglufosinate was not a skin sensitizer in this
    study (Schollmeier & Leist, 1989e).

          Short-term studies of toxicity: Groups of five NMRI mice of
    each sex were given diets containing  N-acetylglufosinate (purity,
    79.4%; 4.5% glufosinate-ammonium) at concentrations of 0, 120, 580,
    2900, or 5800 ppm for 28 days. The content of  N-acetylglufosinate in
    the diets was routinely below the nominal value, sometimes by as much
    as 30%. The stability and homogeneity of the diets were satisfactory,
    providing intakes equivalent to 0, 19, 100, 520, and 1000 mg/kg bw per
    day. All animals were examined routinely for a range of observations
    and measurements, including basic neurological tests. Samples were
    taken for clinical chemical and haematological examinations from all
    mice (not fasted) at termination. Limited analysis was performed on
    urine samples collected overnight on day 21-22 of the study from
    fasted animals. Heart, lung, liver, kidney, spleen, brain, testis, and
    ovaries were weighed and examined histologically, as was any tissue
    with macroscopic abnormalities. Glutamine synthetase activity was
    measured in brain and liver samples that had been cooled rapidly after
    removal and kept frozen until assay. 

         There were no deaths or clinical signs of toxicity and no effects
    on body-weight gain or food or water consumption, although the latter
    two were very variable. Haematology and clinical chemistry showed no
    treatment-related effects, and no substance-related macroscopic or

    microscopic changes were seen. Glutamine synthetase activity in liver
    and brain was significantly inhibited in animals of each sex at
    5800 ppm, with significant inhibition in brain samples from females
    and liver samples from males receiving 2900 ppm (Table 15). The NOAEL
    was 580 ppm, equivalent to 100 mg/kg bw per day, on the basis of the
    statistically significant, > 10% decrease in glutamine synthetase
    activity in brains of females receiving 2900 ppm (Ebert, 1991a).


    Table 15. Mean glutamine synthetase activity in liver and brain 
              from mice receiving  N-acetylglufosinate in the diet for 
              28 days

                                                                         
    Dose (ppm)    Glutamine synthetase activity (nmol/s per mg protein)
                                                                         
                  Male                        Female
                                                                         
                  Liver         Brain         Liver         Brain
                                                                         

       0          0.43          1.2           0.38          1.5
     120          0.38          1.4           0.5           1.4
     580          0.38          1.5           0.4           1.3
    2900          0.34*         1.1           0.42          0.92*
    5800          0.24*         0.71*         0.22*         0.89*
                                                                         

    * Statistically significant at  p < 0.05


         Groups of 20 NMRI mice of each sex received diets containing
     N-acetylglufosinate (purity, 74.7; 0.5% glufosinate-ammonium) at
    concentrations of  0, 500, 2000, or 8000 ppm for 13 weeks. The content
    of test substance and the homogeneity and stability of the diet were
    satisfactory. The achieved intakes were 0, 82, 320, and 1300 mg/kg bw
    per day for males and 0, 110, 440, and 1700 mg/kg bw per day for
    females at the control, low, intermediate, and high doses,
    respectively. The animals were observed routinely for deaths, general
    condition, clinical signs, behaviour, food consumption, and body
    weight. Blood samples were taken from groups of 10 fasting mice per
    sex per group at the end of the study for haematological and clinical
    chemical investigations. After sacrifice, all animals were examined
    macroscopically, and 10 organs were weighed and more than 30 tissues
    from control and high-dose animals were examined histopathologically.
    Limited histological examinations were performed on nine tissues from
    animals at the low and intermediate doses. Liver, kidney, and brain
    samples (pooled samples from two animals for the last two tissues)
    were rapidly placed in liquid nitrogen before assay for glutamine
    synthetase activity.

         One male at 8000 ppm died after blood sampling. There were no
    treatment-related effects on food consumption, body weight, clinical
    signs, organ weights, macroscopic or macroscopic appearance, or
    haematological parameters. The activity of serum lactate dehydrogenase
    was increased by 180% over controls in males at the two higher doses,
    but the results were within the normal range. Apparent increases in
    the activities of a number of serum enzyme in females at the high dose
    were due to a high value in a single animal and are considered to be
    unrelated to treatment. The main finding was a dose-related decrease
    in glutamine synthetase activity in liver, kidney, and brain (Table
    16). Inhibition of liver and kidney glutamine synthetase activity in
    isolation is not relevant to human risk assessment; however, there was
    > 10% inhibition of glutamine synthetase activity in brain at doses
    > 2000 ppm, and the mean values for animals of each sex were
    outside the control range. The NOAEL was 500 ppm, equal to 82 mg/kg bw
    per day (Tennekes et al., 1992a).


        Table 16. Glutamine synthetase activity in groups of 10 mice receiving 
               N-acetylglufosinate in the diet for 90 days

                                                                                      
    Sex        Dose (ppm)   Glutamine synthetase activitya
                                                                                      
                            Kidney              Liver               Brain
                                                                                      
                            Mean    Range       Mean    Range       Mean    Range
                                                                                      

    Male       0            1.4     1.2-1.7     4.0     3.2-4.8     3.6     3.4-3.8
               500          1.0*    0.7-1.2     3.7     2.7-4.4     3.3*    3.1-3.6
               2000         0.83*   0.6-1.1     3.3*    2.5-4.6     3.2*    2.9-3.4
               8000         0.71*   0.4-0.9     2.9*    2.4-4.2     2.6*    2.4-2.9
    Females    0            1.7     1.5-2.1     5.0     3.9-5.5     3.4     2.9-3.6
               500          1.3*    1.1-1.5     4.9     4.2-5.4     3.3     3.0-3.5
               2000         1.2*    0.9-1.4     4.6     3.8-5.3     2.9*    2.7-3.2
               8000         1.1*    0.7-1.3     4.0*    3.2-5.5     2.2*    1.9-2.5
                                                                                      

    *  p < 0.01 
    a mol gamma-glutamyl hydroxamate formed per ml reaction mixture in 20 min at 37C
    

         Groups of five Wistar rats received diets containing
     N-acetylglufosinate (purity, 79.4%; 4.5% glufosinate-ammonium) at
    concentrations of 0, 120, 580, 2900, or 5800 ppm for 28 days. The
    overall content, stability, and homogeneity of the test diets were
    stated to be acceptable. The achieved intakes (assuming 100% analysis)
    were 0, 12, 59, 310, and 590 mg/kg bw per day for males and 0, 11, 55,
    280, and 560 mg/kg bw per day for females. All animals were examined
    routinely for a range of observations and measurements, and control

    and high-dose animals underwent a basic functional observation
    battery. Samples were taken for extensive clinical chemical and
    haematological examinations from all rats (not fasted) at the end of
    the study. Limited analysis was performed on urine samples collected
    overnight on day 21-22 of the study from fasted animals. Heart, lung,
    liver, kidney, spleen, brain, testis, ovaries, and adrenal, pituitary,
    and thyroid glands were weighed and examined histologically, as was
    any tissue with macroscopic abnormalities. Glutamine synthetase
    activity was measured in brain and liver samples that had been cooled
    rapidly after removal and kept frozen until assay. 

         There were no deaths or clinical signs of toxicity and no effects
    on body-weight gain or food or water consumption. 'Thrombin time' was
    mildly increased (< 13%) in females at doses > 2900 ppm and in
    males at 120, 580, and 5800 ppm; other measures of coagulation were
    unaltered. A statistically significant decrease in lactate
    dehydrogenase activity was found in females at doses > 2900 ppm,
    which may have been related to treatment as there was evidence of a
    dose-response relationship. Slight decreases in the absolute and
    relative weights of the heart in males at 5800 ppm was of no clear
    toxicological significance in the absence of a histological correlate.
    No treatment-related macroscopic or microscopic changes were seen.
    Glutamine synthetase activity in the liver was significantly inhibited
    at doses > 580 ppm in animals of each sex, and was reduced in the
    brains of all treated animals, although there was no clear
    dose-response relationship (Table 17). Decreased activity of glutamine
    synthetase in liver was considered not to be adverse, and the
    alterations in glutamine synthetase activity in brain were not
    consistent. There were no biologically significant changes in any
    other parameter. The NOAEL was 5800 ppm, equal to 560 mg/kg bw per
    day, the highest dose tested (Ebert, 1991b).


    Table 17. Mean glutamine synthetase activity in liver and brain 
              samples from rats receiving  N-acetylglufosinate in the 
              diet for 28 days 

                                                                          
    Dose (ppm)    Glutamine synthetase activity (nmol/s per mg protein)
                                                                          
                  Male                        Female
                                                                          
                  Liver         Brain         Liver         Brain
                                                                          
       0          0.27          1.5           0.31          1.3
     120          0.28          1.1*          0.33          1.1
     580          0.17*         0.9*          0.27          1.1*
    2900          0.17          1.0*          0.23          1.0*
    5800          0.14*         1.3           0.15*         1.1*
                                                                          

    * Statistically significant at  p < 0.05

         Groups of 20 Wistar rats of each sex received diets containing
     N-acetylglufosinate (purity, 74.7%; 0.5% glufosinate-ammonium) at
    concentrations of 0, 2000, or 10 000 ppm for 13 weeks; a group of 10
    animals of each sex received 400 ppm. The content, homogeneity, and
    stability of the test compound in the diet were satisfactory, and the
    achieved intakes were 0, 29, 150, and 740 mg/kg bw per day for males
    and 0, 31, 160, and 800 mg/kg bw per day for females. At 13 weeks, 10
    males and 10 females in each group were killed, and the remainder were
    allowed to recover for 4 weeks. The animals were observed regularly
    for deaths, clinical signs, body weight, food consumption, and tissue
    masses. Ophthalmoscopic examinations were performed before treatment
    and at 11 and 16 weeks. Blood and urine samples were taken from fasted
    animals at weeks 13 and 17 for clinical chemical and haematological
    investigations. At necropsy, 10 organs were weighed and examined
    macroscopically. An extensive range of tissues from control and
    high-dose animals was examined histologically, as were major organs
    from animals at the intermediate and low doses. Glutamine synthetase
    activity was measured in brain, kidney, and liver samples that had
    been rapidly cooled after removal and kept frozen until assay. 

         There were no deaths, clinical signs of toxicity, or effects on
    body weight, the eyes, or urine. Food consumption was reduced during
    the first week of treatment but not subsequently. A number of clinical
    chemical parameters showed variations, but these were generally due to
    values for individual animals or were within normal ranges. A decrease
    in serum sodium concentration in animals at the high dose at 13 weeks
    appeared to be related to treatment. Glutamine synthetase activity was
    inhibited in liver, kidney, and brain (Table 18), and the effect was
    significantly but not completely reversed after 4 weeks. A reversible
    increase in kidney weight (< 15%) was seen in males at all doses, but
    this finding was considered not to be adverse because there was no
    clear dose-response relationship and no associated histological
    change. There were no treatment-related macroscopic or microscopic
    findings. The NOAEL was 2000 ppm, equal to 150 mg/kg bw per day, on
    the basis of inhibition of glutamine synthetase activity in the brain
    (Tennekes et al., 1992b).

         Groups of four beagle dogs of each sex, aged 5-7 months, received
    diets containing  N-acetylglufosinate (purity, 74.7%; 0.5%
    glufosinate-ammonium) at concentrations of 0, 500, 2000, or 8000 ppm
    in 400 g of diet daily for 13 weeks. Additional groups of two animals
    of each sex received the same diets and were then allowed to recover
    for 4 weeks. The content, homogeneity, and stability of the test diet
    were satisfactory. The achieved intakes were 0, 19, 72, and 290 mg/kg
    bw per day for males and 0, 21, 79, and 300 mg/kg bw per day for
    females. The animals were observed routinely for deaths, general
    condition, clinical signs, behaviour, food consumption, and body
    weight. Ophthalmoscopic examinations were performed on all animals
    before treatment, at weeks 4 and 13, and at termination in the group
    allowed to recover. Blood and urine samples were collected from fasted
    animals before treatment, at weeks 4 and 13, and at the end of the
    recovery period. At termination, all dogs were examined
    macroscopically; a range of tissues were weighed, and > 30 tissues


        Table 18. Glutamine synthetase activity in groups of 10 rats receiving  N-acetylglufosinate in 
              the diet for 90 days and after a 4-week recovery period

                                                                                                     

    Sex        Period           Dose        Glutamine synthetase activitya
               (days)           (ppm)                                                                
                                            Liver               Kidney              Brain
                                                                                                     
                                            Mean    Range       Mean    Range       Mean    Range
                                                                                                     

    Male       90               0           3.8     3.3-4.3     2.1     1.6-2.3     3.2     3.1-3.3
                                400         2.8*    2.0-3.4     1.7*    1.5-1.9     3.3     3.1-3.6
                                2000        2.2*    1.9-2.4     1.5*    1.3-1.8     3.0     2.9-3.3
                                10 000      1.8*    1.3-2.2     1.6*    1.4-1.9     2.8*    2.6-3.1
               90 + recovery    0           3.2     2.5-3.8     2.1     1.9-2.3     3.1     2.9-3.4
                                2000        3.5     3.2-3.9     2.1     1.6-2.4     3.0     2.6-3.4
                                10 000      3.4     2.9-4.1     1.9     1.2-2.5     2.9*    2.7-3.1
     Female    90               0           3.7     2.8-4.3     1.2     1.0-1.3     3.1     2.8-3.3
                                400         3.1     2.2-3.5     1.1     1.0-1.2     3.1     2.9-3.2
                                2000        2.6*    1.8-3.2     1.2     1.1-1.5     3.0     2.9-3.2
                                10 000      2.4*    1.9-2.8     1.5*    1.4-1.7     2.8*    2.6-2.9
               90 + recovery    0           3.7     3.2-4.1     1.3     1.2-1.4     3.1     3.0-3.3
                                2000        3.5     3.1-4.0     1.3     1.1-1.5     3.1     2.9-3.4
                                10 000      3.3     2.6-3.9     1.3     1-1.5       2.9     2.7-3.1
                                                                                                     

    *   p < 0.01
    a  mol gamma-glutamyl hydroxamate formed per ml reaction mixture in 20 min at 37C
    

    were preserved and examined histopathologically. Liver, kidney, and
    brain samples were immediately placed in liquid nitrogen and kept
    frozen at -80C before assay for glutamine synthetase activity;
    samples of these tissues were also retained for future analysis.

         There were no deaths or treatment-related clinical signs or
    effects on food consumption, body weight, or ophthalmoscopic or
    haematological parameters. A statistically significant, dose-dependent
    decrease in glutamine synthetase activity was found in liver and brain
    after 13 weeks of treatment (Table 19), which tended to return to
    normal during the recovery period although it was not complete at the
    end of the 4 weeks. The changes in glutamine synthetase activity were
    not consistent in different tissues from the same animal. Activity in
    the brain stem and cerebellum appear to be more sensitive to
    inhibition than that in the cortex. Decreased creatine kinase activity
    was seen at the high dose in males at 13 weeks (17%) and in females at
    weeks 4 and 13 (30%). Lactate dehydrogenase activity was decreased by
    30% in females at this dose at week 13 but in neither males nor
    females after recovery. Exacerbation of the low pretreatment specific
    gravity and osmolality of the urine of females was seen at at 8000 ppm
    in weeks 4 and 13 of treatment and at the end of the recovery period.
    The significance of this finding is unclear as it was not seen in a
    1-year study in dogs. It is of note, however, that the kidney is a
    target organ in rats. 

         A dose-related decrease in prostate gland weight was seen which
    achieved statistical significance at 8000 ppm (40%) at 13 weeks, but
    there was no histological correlate. The prostate weights were similar
    to those of controls after 4 weeks' recovery from the dose of
    2000 ppm, but not after administration of 8000 ppm. Although the
    authors noted that slight differences in the rate of maturity of dogs
    of this age can affect prostate size, the dose-response relationship
    and evidence of recovery at 2000 ppm but not at 8000 ppm indicate an
    association with treatment. No treatment-related effects were seen on
    macroscopic examination. The only histopathological finding of note
    was an increased incidence of pituitary cysts in animals receiving
    8000 ppm for 13 weeks: 3/4 in animals of each sex and 1/4 in male
    controls and 0/4 in female controls. The NOAEL was 500 ppm, equal to
    19 mg/kg bw per day, on the basis of > 10% reductions in glutamine
    synthetase activity in a number of areas of the brain. The effects on
    prostate weight, urinary parameters, and the pituitary indicate that
    8000 ppm was an effect level (Corney et al., 1992)

         Groups of six beagle dogs of each sex received diets containing 
     N-acetylglufosinate (purity, 92.4%; 0.1% glufosinate-ammonium) at
    concentrations of 0, 100, 1000, or 8000 ppm.  Analyses of the diets
    for homogeneity and content were satisfactory, and the overall intakes
    of  N-acetylglufosinate were 0, 4, 44, and 320 mg/kg bw per day for
    males and 0, 4.4, 43, and 350 mg/kg bw per day for females. Two
    animals of each sex per group were killed at 26 weeks and the
    remainder at 52 weeks. The animals were observed routinely for
    clinical signs, deaths, body weight, and food consumption.


        Table 19. Glutamine synthetase activity in dogs receiving  N-acetylglufosinate in the diet for 90 days with or without a 4-week recovery 
              period

                                                                                                                                             
    Sex      Period     No.  Dose    Glutamine synthetase activitya
                             (ppm)                                                                                                           
                                     Liver              Kidney             Mid-brain       Cerebellum       Brain stem       Brain cortex
                                                                                                                                             
                                     Mean    Range      Mean   Range       Mean  Range     Mean   Range     Mean   Range     Mean   Range
                                                                                                                                             

    Male     13 weeks   4    0       2.7     2.6-2.9    0.02   0.01-0.04   2.3   1.8-2.7   1.6    1.5-1.8   1.4    1.2-1.5   3.2    2.8-3.5
                             500     1.9     1.4-2.2    0.04   0.02-0.07   2.6   1.9-2.9   1.4    1.2-1.5   1.3    1.2-1.4   3.4    3.3-3.4
                             2000    1.2     1.0-1.3    0.06   0.03-0.10   2.6   1.8-2.9   1.3    1.0-1.4   0.90   0.8-1.1   2.8    2.3-2.9
                             8000    0.58    0.5-0.7    0.05   0.01-0.14   1.4   1.7-2.6   0.85   0.7-1.0   0.47   0.4-0.5   2.3    1.8-2.8

             Recovery   2    0       2.6                0.07               2.1             1.4              1.3              3.0
                             2000    2.4                0.05               2.3             1.2              0.95             2.4 
                             8000    2.0                0.06               2.1             1.2              0.93             2.5 

    Females  13 weeks   4    0       1.8     1.6-2.1    0.04   0.02-0.06   2.7   2.2-3.0   1.6    1.5-1.7   1.2    1.1-1.3   2.9    2.7-3.3
                             500     1.6     1.2-2.4    0.10   0.05-0.17   2.2   1.9-2.5   1.5    1.4-1.5   1.2    1.0-1.3   3.2    2.9-3.3
                             2000    1.0     0.8-1.5    0.06   0.03-0.09   2.4   1.8-2.9   1.3    1.2-1.4   0.91   0.7-1.0   2.7    2.4-3.3
                             8000    0.68    0.5-0.9    0.09   0.01-0.15   2.0   1.7-2.6   0.87   0.8-0.9   0.73   0.6-0.8   2.5    1.9-2.8

             Recovery   2    0       2.1                0.05               2.1             1.5              1.4              3.1
                             2000    1.7                0.08               2.1             1.3              1.2              2.7 
                             8000    1.3                0.07               1.8             1.1              0.83             2.4 
                                                                                                                                             

    a mol gamma-glutamyl hydroxamate formed per ml reaction mixture in 20 min at 37C
    

    Ophthalmoscopic examinations were performed before treatment and at
    weeks 12, 25, and 51. Samples were taken from fasted animals for
    urinary analysis, haematology, and clinical chemistry before treatment
    and at weeks 13, 26, and 52. Post-mortem examinations were performed
    on all animals; nine organs were weighed and > 30 tissues examined
    histopathologically. Glutamine synthetase activity was not analysed. 

         The only death occurred in a male at the intermediate dose; the
    cause was not found. The incidence of soft faeces was increased in
    animals at the high dose and particularly in males. Reduced
    body-weight gain was seen in animals at this dose, by 17% in males and
    30% in females, during the first half of the study, and the effect
    persisted in females until termination. There was no evidence of
    treatment-related effects in ophthalmoscopic, haematological, or
    urinary examinations and no effects on organ weights or gross or
    histopathological appearance at either the interim or terminal
    sacrifice. Reduced serum lactate dehydrogenase activity was seen
    consistently in animals at the high dose (Table 20). Although there
    was some overlap between the ranges for control and treated animals
    and variation in control values over time, the consistency of the
    effect in males indicates that it is possibly related to treatment.
    The effects at 8000 ppm are not significant in isolation, but the
    combination of findings in males indicates that this dose is an effect
    level. The NOAEL was 1000 ppm, equal to 44 mg/kg bw per day, on the
    basis of reduced body-weight gain, reduced lactate dehydrogenase
    activity, and an increased incidence of soft faeces (Bernier, 1996).


    Table 20. Lactate dehydrogenase activity in dogs fed diets containing 
               N-acetylglufosinate at 8000 ppm

                                                                            
    Group        No.    Week     Serum lactate dehydrogenase activity (U/L)
                                                                            
                                 Males                Females
                                                                            
                                 Mean     Range       Mean     Range
                                                                            

    Controls     6      0        75       47-100      160      120-230
                 6      13       100      66-160      160      52-260
                 6      26       200      87-430      220      99-470
                 4      52       90       66-100      320      160-460

    Treated      6      0        97       57-140      74       32-130
                 6      13       53       43-68       44       31-65
                 6      26       99       39-180      88       48-110
                 4      52       70       50-88       150      110-180
                                                                            

          Long-term studies of toxicity and carcinogenicity: Groups of 90
    Swiss Crl:CD-1 mice received diets containing  N-acetylglufosinate
    (purity, 92.4%; 0.1% glufosinate-ammonium) at concentrations of 100,
    1000, or 8000 ppm. Groups of 20 animals were killed after 1 year and
    the remainder after 2 years. The mice were housed singly, observed
    daily, and monitored routinely for body weight and food consumption.
    The homogeneity, content, and stability of the diet were acceptable,
    and the achieved intakes were 0, 15, 150, and 1200 mg/kg bw per day
    for males and 0, 19 190, and 1500 mg/kg bw per day for females.
    Samples for clinical chemistry and haematology were taken from fasted
    animals at interim sacrifice (all animals) and at termination (10 per
    sex per group). Blood was taken from the tail vein at week 78. At
    autopsy, the animals were examined macroscopially, nine organs were
    weighed, and more than 30 tissues were preserved. Histological
    examinations were conducted on tissues from all controls, animals at
    the high dose, and those that died during the study and on the liver,
    lung, kidney, and adrenals from animals at the low and intermediate
    doses killed after 1 or 2 years. 

         Although the report stated that no treatment-related signs were
    observed, data on individual animals were not presented, as this was
    primarily a study of carcinogenicity. Survival was similar in all
    groups (> 60% at week 90). Fluctuations in food consumption and body
    weight showed no consistent pattern related to dose. There were no
    effects on clinical chemical or haematological parameters or on organ
    weights, any variation in group means being attributable to individual
    values. Macroscopic examination detected no treatment-related effects.
    Histopathological examination showed an increased incidence of
    amyloidosis in various organs in males and females at the high dose
    and testicular necrosis in males, although the only statistically
    significant finding ( p = 0.028) was amyloidosis in the salivary
    gland of females (Table 21). A high incidence of kidney lesions was
    seen in both control and treated animals at 1 and 2 years, with no
    significant differences between groups. Increased incidences of
    uterine adenocarcinoma and thyroid follicular-cell adenoma were seen
    in animals at the high dose; the incidences were not statistically
    significantly increased when compared with concurrent controls but
    were slightly greater than those of historical controls. The overall
    incidences of benign and malignant tumours were similar in treated and
    control groups. As the tumours that occurred at increased incidences
    occur spontaneously in this strain of mouse, the findings were
    considered not to indicate a carcinogenic potential of
     N-acetylglufosinate. The NOAEL was 1000 ppm, equal to 150 mg/kg bw
    per day, on the basis of an increased incidence of amyloidosis
    (Farrell, 1997; Ernst & Stumpf, 1999a).

         Groups of 70 Sprague-Dawley-derived rats of each sex received
    diets containing  N-acetylglufosinate (purity, 92.4%; 0.1%
    glufosinate-ammonium) at concentrations of 0, 200, 2000, or 20 000 ppm
    for 2 years; additional groups of 10 animals of each sex were killed
    at 1 year and 20 at 2 years. The animals were housed singly, observed
    daily, and monitored routinely for body weight and food consumption.
    The homogeneity, content, and stability of the test diet were


        Table 21. Incidences of lesions in mice receiving  N-acetylglufosinate in the diet for 2 years, and ranges for 
              historical controls

                                                                                                                   
    Sex      Lesion                                  Incidence
                                                                                                                   
                                                     0        100 ppm   1000 ppm   8000 ppm   Historical controls
                                                                                                                   

    Male     Testicular degeneration and necrosis    0/70     1/55      2/56       4/70       6-39% (for atrophy) 
             Testicular atrophy                      25/70    28/55     30/56      26/70      6-39% (for atrophy) 
             Thyroid follicular-cell adenoma         0/70     0/69      1/69       3/70       0-2%

    Female   Salivary gland amyloidosis              1/70     1/45      3/43       7/70       No data
             Liver haemangiosarcoma                  0/70     1/70      1/70       2/70       0-4%
             Uterine adenocarcinoma                  0/70     2/69      1/69       3/70       0-2%
                                                                                                                   
    

    acceptable, and the achieved intakes were 0, 9, 91, and 1000 mg/kg bw
    per day for males and 0, 11, 110, and 1200 mg/kg bw per day for
    females. Samples for clinical chemistry and haematology were taken
    from fasted rats in the interim sacrifice and satellite groups at 25,
    51, 78, and 102 weeks. At necropsy, the animals were examined
    macroscopically, nine organs from interim sacrifice animals were
    weighed, and > 30 tissues were preserved. Full histopathological
    examinations were performed on all controls, animals at the high dose,
    and animals that died during the study; only liver, lung, kidney, and
    adrenals from animals at the low and intermediate doses were examined
    histopathologically.

         Survival was similar in all groups (> 50% at week 96). Soft
    faeces were more prevalent in animals at the high dose and especially
    in males from week 8 onwards. Body-weight gain was reduced (~ 5%) in
    males at this dose from week 16 onwards and in females (~10%) from
    week 24, even though the food consumption of these groups increased by
    5-10%. A range of changes in clinical chemistry and haematology were
    seen repeatedly in animals of each sex at the high dose, including
    reduced prothrombin time, potassium concentration, and lactate
    dehydrogenase and creatine kinase activity, and increased sodium,
    calcium, inorganic phosphorus, and glucose concentrations; blood urea
    nitrogen was increased in males from week 25 onwards. In animals
    killed after 1 year, the absolute and relative weights of the kidney
    were increased in males receiving 20 000 ppm and in females receiving
    2000 or 20 000 ppm. Macroscopic examination showed increased
    incidences of a range of abnormalities of the kidney in treated
    animals that were confirmed at histological examination. Renal lesions
    are common in aged rats, and the incidences in treated animals in this
    study were not statistically significant and were within the range
    seen in historical controls; furthermore, they may have been
    exacerbated by the high sodium content induced by
     N-acetylglufosinate. They were therefore considered irrelevant to
    the assessment of the study. Alterations were also seen in the spleen,
    parathyroid, heart, blood vessels and adrenals at the high dose and in
    some animals at the intermediate dose animals (Table 22). The
    incidence of tumours was not statistically significantly increased,
    although low incidences of uncommon tumours of the brain, liver,
    adrenals, skin, and pancreas were seen in animals at the high dose
    (Table 23). Although these incidences are outside the range in
    historical controls, the sporadic nature of the findings and the clear
    NOAEL for tumours at 2000 ppm indicate that  N-acetylglufosinate does
    not have significant carcinogenic potential. The overall incidences of
    benign and malignant tumours were similar in all groups. The NOAEL was
    200 ppm, equal to 9 mg/kg bw per day, on the basis of increased
    incidences of polyarteritis nodosa, adrenal cortical hyperplasia, and
    necrosis and extramedullary haematopoiesis of the spleen (Bernier,
    1997; Ernst & Stumpf, 1999b).


        Table 22. Incidences of non-neoplastic lesions in rats receiving  N-acetylglufosinate in the diet for up to 2 years, 
              and ranges for historical controls

                                                                                                                            
    Sex      Lesion                                                    Incidence
                                                                                                                            
                                                                       0        200 ppm   2000 ppm   20 000 ppm  Historical 
                                                                                                                 controls
                                                                                                                            

    Male     Parathyroid hyperplasia                                   13/62    8/37      16/32      20/64       14-42%
             Adrenal focal cortical hyperplasia                        17/70    18/70     29/70      27/70       0-20%a
             Adrenal  necrosis                                         0/70     0/70      2/70       3/70        0-4%
             Polyarteritis nodosa (blood vessels)                      4/7      5/7       4/9        11/18       No data
             Polyarteritis nodosa (testis; 2 years; main group)        15/70    13/53     13/50      23/70       16-31%
             Polyarteritis nodosa (testis; 2 years; satellite group)   3/20     4/16      8/17       10/20       16-31%

    Female   Parathyroid hyperplasia                                   1/52     0/26      0/31       6/61        1-12%
             Extramedullary splenic haematopoiesis                     7/70     4/34      16/41      20/70       15-36%
             Cardiomyopathy                                            12/70    5/30      6/41       22/70       1-70%
                                                                                                                            

    a  Current study, all > 24%

    Table 23. Incidences of neoplastic lesions in groups of 70 rats receiving  N-acetylglufosinate in the diet 
              for 2 years, and ranges for historical controls

                                                                                                                  
    Sex      Lesion                                  Incidence
                                                                                                                  
                                                     0     200 ppm   2000 ppm   20 000 ppm   Historical controls
                                                                                                                  

    Male     Adrenals: malignant phaeochromocytoma   0     1         1          3            0-3%
             Brain: meningioma                       0     0         0          1            0-1%
             Brain: astrocytoma                      0     1         0          1            0-2% (malignant
                                                                                             glioma)
             Skin: keratocanthoma                    4     5         1          9 (7)*       0-8% 
    Female   Brain: oligodendroglioma                0     0         0          1            No data
             Liver: cholangiocarcinoma               0     0         0          1            No data
             Lung: alveolar/bronchiolar carcinoma    0     0         0          1            No data
             Pancreas: islet-cell carcinoma          1     2         1          4            0-2%
                                                                                                                  

    * Re-evaluation of data indicates that 7 is the correct value.
    

          Genotoxicity: An extensive range of assays for genotoxicity
    have been performed with  N-acetyl-glufosinate both  in vitro and
     in vivo (Table 24). Some of the protocols were less than optimal
    with respect to length of exposure (e.g. for unscheduled DNA
    synthesis) or the highest concentration tested, but these limitations
    are considered not to affect the overall assessment of genotoxic
    potential significantly. The Committee concluded that
     N-acetylglufosinate is not genotoxic.

          Reproductive toxicity: In a single-generation, range-finding
    study in groups of 10 Sprague-Dawley rats of each sex that received
     N-acetylglufosinate (purity, 92.4%; 0.1% glufosinate-ammonium), no
    adverse effects were seen on fertility, reproduction, or development
    at doses of 200, 2000, or 10 000 ppm (equivalent to 14, 140, or 700
    mg/kg bw per day). Exposure was continuous from 21 days before mating
    to day 21  post partum (Beyrouty, 1996a).

         Groups of 30 Sprague-Dawley-derived rats received diets
    containing  N-acetylglufosinate (purity, 92.4%; 0.1%
    glufosinate-ammonium) at concentrations of 0, 200, 2000, or 10 000 ppm
    for two generations. The F0 generation was exposed from 70 days
    before mating until the end of lactation, and F1 pups were exposed
    from weaning (10-12 weeks before mating) until the end of lactation of
    F2 pups. The stability and homogeneity of the diet were
    satisfactory, and the mean achieved intakes were 0, 13, 140, and 700
    mg/kg bw per day for males and 0, 18, 170, and 890 mg/kg bw per day
    for females. Parental animals were observed routinely for clinical
    signs, estrus cycling, body weight, and food consumption, and the
    frequency of observation was increased during gestation and weaning.
    Parturition was observed when possible. Pups were examined for
    malformations, sex, viability, and body weight, and the litters were
    culled to four pups of each sex on day 4. All parental animals, one
    pup of each sex per litter, and any pups that died or were killed when
    moribund were examined  post mortem. The major organs and
    reproductive tissues from all adult animals were examined
    macroscopically, and these tissues from control and high-dose animals
    were examined histologically. Sperm motility, count, and morphology,
    and the number of spermatids were determined for all adult males.

         One male at the intermediate dose and two at the high dose died
    from unknown causes, and males of both generations at the high dose
    had an increased incidence of soft faeces and brown staining of the
    scrotum. There were no effects on body weight, although the food
    consumption of animals at the high dose was slightly increased during
    the first few weeks of treatment. No effects were seen on the weight
    or histological appearance of organs from F0 animals or on mating
    indices, length of gestation, estrus cycling, sperm parameters,
    macroscopic appearance, litter size, pup survival, or pup weight in
    either generation. A dose-related increase in the weight of the
    seminal vesicles was seen in F1 adults, which reached statistical
    significance ( p < 0.05; 14%) in males at the high dose. As there
    were no associated abnormal histological findings and no functional
    deficit, the effect was considered not to be adverse. An increased


        Table 24. Results of studies of the genotoxicity of  N-acetylglufosinate

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

     In vitro 

    Reverse mutation   S. typhimurium TA98,       0, 2, 12, 59, 291, 1455,   79.4     Negative +S9     Muller (1989c)
                       TA100, TA1535,             2910, 5820 g/plate                 Negative - S9
                       TA1537, TA1538; 
                       E. coli WP2 uvrA

    Gene mutation      V79 Chinese hamster        0, 582, 873, 1164,         79.4     Negative +S9     Muller (1989a)
                       lung cells, Hprt locus     1554 g/ml                          Negative - S9

    Gene mutation      V79 Chinese hamster        0, 444, 666, 888,          74.7     Negative +S9     Muller (1991b)
                       lung cells, Hprt locus     1186 g/ml                          Negative - S9

    Unscheduled DNA    Human cell line A549       0, 1, 4, 13, 44, 133,      74.7     Negative +S9     Muller (1991c)
    synthesis                                     444, 1332 g/ml                     Negative - S9

    Unscheduled DNA    Human cell line A549       0, 0.6, 1.8, 5.8, 17, 58,  79.4     Negative +S9     Muller (1989d)
    synthesis                                     175, 582 g/ml, 3-h                 Negative - S9
                                                  exposure

    Chromosomal        Human lymphocytes          0, 0.6, 3.0, 5.0 mg/ml     74.7     Negative +S9     Heidemann & 
    aberrations                                   at 24 h                                              Voelkner (1992)
                                                  0, 5.0 mg/ml at 48 h                Negative - S9

    Chromosomal        V79 Chinese hamster        0, 154, 773, 1546 g/ml    79.4     Negative +S9     Muller (1989b)
    aberrations        lung cells                 for 18 h 
                                                  0, 1546 g/ml for 7 or              Negative - S9
                                                  28 h 
                                                                                                                        

    Table 24. (continued)

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

     In vivo 

    Micronucleus       NMRI mouse bone            0, 222, 1111, 2222         74.7     Negative         Muller (1991a)
    formation          marrow                     mg/kg bw by gavage for 
                                                  24, 48 or 72 h
                                                                                                                        
    


    incidence of extramedullary haematopoiesis in the livers of F1 males
    (8/30  versus 2/30 in controls) was also considered not to be adverse
    as it is a common finding in rats. There were no effects on
    reproduction. These minor effects on seminal vesicle weight and liver
    extramedullary haematopoiesis in F1 males at 10 000 ppm combined
    with the increased incidence of soft stools indicated that this is a
    minimal effect level. The NOAEL was 2000 ppm, equal to 140 mg/kg bw
    per day (Beyrouty, 1996b).

          Developmental toxicity: Twenty mated female Wistar rats
    received  N-acetylglufosinate (purity, 74.7%) by gavage in distilled
    water at the limit dose of 1000 mg/kg bw per day on days 7-16 of
    gestation, whereas 21 controls received starch mucilage in distilled
    water. The dose was based on the results of a preliminary  study. The
    dams were observed routinely for clinical signs, body weight, and food
    consumption. At sacrifice on day 21, they were examined
    macroscopically, and the uterus was opened to determine the numbers of
    live and dead fetuses and resorptions, fetal weights, the sex ratio,
    crown-rump lengths, and placental weights. All fetuses were examined
    for external malformations; half were investigated by Wilson
    sectioning for visceral abnormalities and the remainder stained with
    Alizarin red S for visualization of skeletal defects.

         There were no deaths or treatment-related clinical signs among
    the pregnant dams, but a non-pregnant control was killed during the
    study. The food consumption and body-weight gain of treated animals
    were slightly reduced (~ 5%) on days 7-14 of gestation but were
    similar to those of controls after cessation of dosing. There was no
    effect on pregnancy rate, but a slight increase in pre- and
    post-implantation losses resulted in a marginally reduced litter size
    (13.6 in controls  versus 12.7 in treated animals); the value was
    within the typical range. No malformations were recorded, and there
    was no increase in the incidence of skeletal or external
    abnormalities. The incidence of blood in the pericardium/abdomen was
    increased in the treated group (5/132 fetuses, 4/20 litters) when
    compared with concurrent controls (2/140 fetuses) and historical
    controls (0-1.5%). Fetal weight, crown-rump length, placental weight,
    and sex ratio showed no treatment-related effects. The NOAEL for
    maternal and fetotoxicity was 1000 mg/kg bw per day, the only dose
    tested (Horstmann & Baeder, 1992).

         Groups of 15 mated, 8-10-month-old, female Himalayan rabbits
    received  N-acetylglufosinate (purity, 92.4%) by gavage in distilled
    water on days 6-18 of gestation at doses of 0, 64, 160, or 400 mg/kg
    bw per day, on the basis of the results of a study that found maternal
    and fetal toxicity at 500 mg/kg bw per day. The animals were housed
    under standard conditions and observed daily. Body weights were
    determined on days 0, 6, 13, 19, and 29 of gestation, and the dams
    were killed on day 29 and examined macroscopically. The uteri were
    opened, and the numbers of live and dead fetuses and resorptions were
    determined. Live fetuses were removed to an incubator and their
    survival was monitored for 24 h. Fetal body weight, crown-rump length,
    and sex were determined, and external visceral and skeletal

    examinations were performed by Wilson sectioning and Alizarin red S
    staining. The combined incidences of some lesions were presented only
    in summary tables, but this did not compromise the overall integrity
    of the study.

         There were no deaths during the study, and no consistent clinical
    signs in dams. Abortions by one dam at the low dose and one at the
    intermediate dose were considered not to be related to treatment. Food
    consumption was reduced in relation to dose, by 12% in the groups at
    the low dose, 23% at the intermediate dose, and 37% at the high dose,
    but there were no consistent effects on body weight. Treatment had no
    effect on corpora lutea, pregnancy rate, implantation, resorptions,
    gravid uterine weight, or fetal survival. The pup weights were reduced
    by 7% at 400 mg/kg bw per day. Morphological examination of fetuses
    revealed an increased incidence of a supernumerary thoracic ribs
    (2/90, 0/82, 8/73, and 11/93 in control, low-, intermediate-, and
    high-dose groups, respectively, while the range among historical
    controls was 0-12%). The only malformation reported was a case of
    hydrocephalus in a fetus at the high dose; the incidence in historical
    controls was 0-9%. The NOAEL for maternal toxicity and fetotoxicity
    was 64 mg/kg bw per day on the basis of reduced food consumption in
    dams and extra ribs in fetuses (Baeder & Hofmann, 1994; Ernst & Leist,
    1999b).

          Special studies on neurotoxicity: No overt neurotoxicity was
    seen in basic functional observation batteries of tests with
     N-acetylglufosinate included in short-term studies of toxicity in
    mice and rats, nor does this substance belong to a class of compounds
    with neurotoxic potential.

         (ii) 3-[Hydroxy(methyl) phosphinoyl]propionic acid

         3-[Hydroxy(methyl) phosphinoyl]propionic acid is a metabolite of
    glufosinate-ammonium in rats and represents ~ 30% of the residue in
    liver (Table 4). It may also represent a significant proportion of the
    residue arising from administration of glufosinate-ammonium to plants,
    goats, and hens (Annex 1, reference  83). In 1991, the Meeting
    reviewed a 28-day study in rats and a study of the toxicokinetics of
    3-[hydroxy(methyl) phosphinoyl]propionic acid, which showed that it is
    rapidly and extensively absorbed and excreted, has little toxicity
    (NOAEL, 280 mg/kg bw per day), and does not inhibit hepatic glutamine
    synthetase activity. The 1998 Joint Meeting considered that
    3-[hydroxy(methyl) phosphinoyl]propionic acid should be included in
    the residue definition (Annex 1, reference  83). 

         A summary of various additional studies on 3-[hydroxy(methyl)
    phosphinoyl]propionic acid has been submitted (Bremmer & Leist, 1998),
    although the original reports were not made available. The summary
    indicates that the acute toxicity of the substance is low, it is not a
    skin sensitizer, and had no significant toxicity in studies in rats
    (13 weeks; NOAEL, 560 mg/kg bw per day), mice (13 weeks; NOAEL, 1400
    mg/kg bw per day), or dogs (15 weeks; NOAEL, 110 mg/kg bw per day)
    given repeated doses. An extensive range of tests for genotoxicity was

    reported to show that 3-[hydroxy(methyl) phosphinoyl]propionic acid
    does not induce gene mutation  in vitro, chromosomal aberrations
     in vitro or  in vivo, micronuclei  in vivo, or DNA damage
     in vitro or  in vivo. Studies of developmental toxicity in rats and
    rabbits showed evidence of maternal toxicity and mild fetotoxicity but
    no teratogenicity, with cited NOAELs of 300 mg/kg bw per day in rats
    and 50 mg/kg bw per day in rabbits. 

    3.  Observations in humans

    (a)  Medical surveillance of personnel in manufacturing plants

         No specific health problems were reported in workers during 18
    years of production of glufosinate-ammonium. The exposure of the
    workers was stated to be low, but no details of the extent of
    investigations were available (Kaleja, 1999).

    (b)  Poisoning incidents

         A pesticide formulation containing glufosinate-ammonium was
    reported to have been involved in over 200 cases of attempted suicide
    in Japan (Ernst & Leist, 1999c). The formulation contained 18% w/w of
    glufosinate-ammonium, 30% surfactant, and a glycol ether solvent, and
    it was not clear whether the effects seen were attributable to
    glufosinate-ammonium. The initial signs were vomiting, diarrhoea, and
    nausea. The main clinical concerns were the delayed (hours to 2 days)
    neurological effects, including convulsions, impaired consciousness,
    tremor, and coma, and extensive oedema. Treatment included gastric
    lavage, diuretics, intravenous fluids, sedatives, haemoperfusion, and
    artificial ventilation. After apparent recovery from the physical
    signs, long-lasting amnesia, both retrograde and anterograde was
    reported, although this may have been linked to the use of high-dose
    benzodiazepine therapy (Koyama et al., 1994; Watanabe & Sano, 1998;
    Ernst & Leist, 1999c).

    Comments

    Glufosinate-ammonium

         Orally administered [14C]glufosinate-ammonium is rapidly but
    sparingly (~ 10%) absorbed. Excretion of the absorbed dose was rapid.
    The kidney and liver contained the highest concentrations of residue,
    which were significantly higher than those in plasma. The
    concentrations in brain were lower than those in plasma, indicating
    limited penetration of the blood-brain barrier. There were no marked
    differences between the sexes. Administration of 500 mg/kg bw resulted
    in more prolonged absorption and excretion than with 20 or 2 mg/kg bw.
    The metabolism of glufosinate-ammonium was limited (~ 30% of the
    absorbed dose), and the main urinary and tissue residues were
    3-[hydroxy(methyl)phosphinoyl]propionic acid,
    methylphosphinicobutanoic acid, and
    2-hydroxy-4-methylphosphinicobutanoic acid. In faeces, significant
    concentrations (up to 10%) of  N-acetylglufosinate were detected,

    indicating that acetylation was performed by the gut microflora.
    Metabolites were also found in tissues.

         Glutamine synthetase (E.C.6.3.1.2) is a key enzyme involved in
    the metabolism of nitrogen and glutamate. Inhibition of glutamine
    synthetase, resulting in high levels of ammonia, is the mechanism of
    action of glufosinate-ammonium in plants. The activity of glutamine
    synthetase varies among tissues and species. The Meeting considered
    reports on the relevance of glutamine synthetase activity in the liver
    and kidney of experimental animals and humans, including data reviewed
    by the 1991 JMPR. Because of the presence of alternative pathways for
    the homeostatic control of ammonia, < 50% inhibition of glutamine
    synthetase in rat liver was not associated with increased ammonia
    concentrations and was not considered to be adverse. Glutamine
    synthetase activity in the kidney shows considerable variation between
    species, with relatively high activity in rodents and negligible
    activity in dogs and humans. Inhibition of kidney glutamine synthetase
    in the absence of pathological findings was considered not to be
    relevant to human risk assessment. 

         In the central nervous system, ammonia homeostasis is maintained
    by a number of enzymes, including glutamine synthetase and glutamate
    dehydrogenase. Under normal conditions, the flux through glutamine
    synthetase in brain is reported to be approximately 2-10% of its
    theoretical capacity, and for glutamate dehydrogenase it is
    approximately 0.1% of its capacity. With such excess capacity,
    inhibition of brain glutamine synthetase will not necessarily result
    in significant increases in brain ammonia concentrations; this
    conclusion is confirmed by data showing that animals with decreased
    glutamine synthetase activity do not have increased brain ammonia
    levels. However, the 'glutamine-glutamate shunt', between GABA and
    glutamate in neurons and glutamine in astrocytes, plays a role in both
    excitatory and inhibitory neurotransmission. The results of studies
    considered by the 1991 Meeting indicate that significant changes in a
    range of biogenic amines in regions of the brain in dogs are
    associated with > 8% changes in glutamine synthetase activity after
    administration of glufosinate-ammonium at 8 mg/kg bw for 28 days, a
    dose that produced 'increased gait activity'. Thus, it has been
    proposed that any statistically significant inhibition of glutamine
    synthetase activity in brain by > 10% be considered a marker of
    potentially adverse effects on brain biochemistry and behaviour.

         Studies  in vitro and  in vivo showed that glufosinate-ammonium
    inhibits glutamine synthetase in the brain, kidney, and liver of rats.
    With 100 ppm glufosinate-ammonium in the diet (equivalent to 10 mg/kg
    bw per day), glutamine synthetase activity was inhibited in liver and
    kidney but not in brain, and the Meeting concluded that the NOAEL was
    10 mg/kg bw per day. The inhibition in liver and kidney was evident by
    day 6, did not increase markedly up to day 90, and showed significant
    reversal during a 31-day recovery period. The finding of increased
    renal glutamine synthetase activity in a previous long-term study in
    rats was considered to be a rebound response to continued inhibition
    and to be of no relevance to human risk assessment.

         New studies in which rats and mice received repeated, high doses
    (270-1400 mg/kg bw per day) in the diet for 90 days were designed to
    determine the maximum tolerated doses rather than NOAELs. There was no
    evidence of specific toxicity or of irreversible neurobehavioural
    effects in the rats. The LOAEL in rats was 7500 ppm, equal to 560
    mg/kg bw per day. A new carcinogenicity study in rats showed that
    glufosinate-ammonium had no significant carcinogenic potential at
    doses up to 10 000 ppm, equal to 470 mg/kg bw per day. A significant
    increase in retinal atrophy was seen in this study in females at doses
    > 5000 ppm (equal to 280 mg/kg bw per day) and in males at 10 000 ppm
    (equal to 470 mg/kg bw per day) but not in either sex at 1000 ppm
    (equal to 45 mg/kg bw per day), the NOAEL. The results of these three
    studies were consistent with those of previous investigations and
    supported the overall NOAEL of 40 ppm (2 mg/kg bw per day) identified
    previously in a long-term study in rats that included a more extensive
    range of investigations. 

         No adverse findings were reported in workers in
    glufosinate-ammonium production plants, but their exposure was stated
    to be low. A number of cases of attempted suicide in Japan have
    involved a glufosinate-ammonium-based formulation, but it was not
    clear whether the effects reported were due to glufosinate-ammonium or
    other constituents. The most significant effect was delayed
    neurological symptoms. The available evidence indicates that exposure
    to glufosinate-ammonium under normal conditions of use does not
    present a significant risk to humans.

    N-Acetylglufosinate

         Oral doses of [14C] N-acetylglufosinate are absorbed to a
    limited extent (5-10%), but the absorption is rapid, with peak plasma
    concentrations found 1 h after a dose of 3 mg/kg bw. Excretion of an
    absorbed dose is also rapid and occurs predominantly in the urine as
    the parent compound. The excretory half-time is < 1 h for the initial
    phase and approximately 7 h for the second phase. The residue
    concentrations in the liver and particularly kidneys 4 days after
    dosing were significantly greater than those in the plasma. Absorbed
     N-acetylglufosinate undergoes limited biotransformation, but a
    significant proportion (11%) of a low oral dose (3 mg/kg bw) was
    de-acetylated to glufosinate-ammonium in the intestine. It is not
    clear what proportion of the glufosinate-ammonium present in the
    tissues after oral administration of  N-acetylglufosinate is absorbed
    from the intestine as glufosinate-ammonium. The toxicokinetics of
     N-acetylglufosinate after repeated administration has not been
    investigated.

          N-Acetylglufosinate is of low acute toxicity after oral
    administration to mice and rats (LD50 values > 2000 mg/kg bw) and
    is not a skin sensitizer. Because  N-acetylglufosinate is a plant
    metabolite and is not present in pesticide formulations, it has not
    been studied for acute toxicity by dermal or inhalation administration
    or for ocular or dermal irritancy. 

          N-Acetylglufosinate has not been classified for toxicity by
    WHO. 

          N-Acetylglufosinate is of low toxicity after repeated oral
    administration to mice, rats, or dogs. Some and possibly all of the
    inhibition of glutamine synthetase activity seen in all three species
    was attributable to glufosinate-ammonium. The NOAEL for inhibition of
    glutamine synthetase in the brain in the most sensitive species was
    500 ppm (equal to 19 mg/kg bw per day) in a 90-day study in dogs
    (glutamine synthetase activity was not measured in a 1-year study in
    dogs). In a 2-year study in rats, there was evidence of chronic
    progressive nephropathy and urolithiasis. The Meeting noted the
    absence of pathological changes in the 90-day study, the lack of a
    dose-response relationship for the renal lesions, the high sodium
    concentrations associated with administration of
     N-acetylglufosinate, and the high prevalence of renal lesions in
    aged rats, and concluded that the renal lesions seen in the long-term
    study in rats were not relevant to human risk assessment. Increased
    incidences of adrenal cortical hyperplasia, adrenal necrosis, and
    polyarteritis nodosa were seen in males receiving doses > 2000 ppm
    (equal to > 91 mg/kg bw per day), and an increased incidence of
    extramedullary haematopoiesis of the spleen was seen in females at
    those doses. The NOAEL for pathological findings in rats, the most
    sensitive species, was 200 ppm, equal to 9 mg/kg bw per day.

         The Meeting concluded that  N-acetylglufosinate is not
    carcinogenic at the highest doses tested (equal to 1200 mg/kg bw per
    day in mice and 1000 mg/kg bw per day in rats). 

          N-Acetylglufosinate has been studied in an adequate range of
    tests for genotoxicity. The Meeting concluded, on the basis of the
    results, that  N-acetylglufosinate is not genotoxic.

         Reproductive performance and outcome in a two-generation study of
    reproductive toxicity in rats were not affected by administration of
     N-acetylglufosinate at doses up to 700 mg/kg bw per day. The
    compound was not teratogenic to either rats or rabbits and was not
    fetotoxic to rats. An increased incidence of supernumerary thoracic
    ribs was found in fetuses from rabbits exposed to
     N-acetylglufosinate at > 160 mg/kg bw per day, a finding that may
    be secondary to the maternal toxicity seen at such doses. The NOAEL
    for fetotoxicity and maternal toxicity was 64 mg/kg bw per day.

    3-[Hydroxy(methyl) phosphinoyl]propionic acid

         Summaries of a range of studies on the genotoxicity, acute
    toxicity, short-term toxicity, and teratogenicity of the
    glufosinate-ammonium metabolite 3-[hydroxy(methyl)phosphinoyl]
    propionic acid were available. The lowest NOAEL seen in these studies
    (50 mg/kg bw per day) is 25-fold higher than the NOAEL used to derive
    the ADI for glufosinate-ammonium.

    Overall evaluation

         The present Meeting compared the toxicity of
     N-acetylglufosinate and 3-[hydroxy(methyl) phosphinoyl]propionic
    acid with that of glufosinate-ammonium and concluded that the toxicity
    of the metabolites was comparable to or less than that of the parent
    compound. The Meeting established a group ADI of 0-0.02 mg/kg bw for
    glufosinate-ammonium,  N-acetylglufosinate, and 3-[hydroxy(methyl)
    phosphinoyl]propionic acid (alone or in combination). This is the same
    value as that of the ADI established for glufosinate-ammonium by the
    1991 JMPR on the basis of the NOAEL in the long-term study in rats
    given technical-grade glufosinate-ammonium, and applying a 100-fold
    safety factor. 

         The present Meeting concluded that it was unnecessary to
    establish an acute reference dose because glufosinate-ammonium,
     N-acetylglufosinate, and 3-[hydroxy(methyl) phosphinoyl]propionic
    acid are of low acute toxicity. 

    Toxicological evaluation

     Levels that cause no toxic effect 

     Glufosinate-ammonium  from 1991 JMPR)

    Mouse:    80 ppm, equal to 11 mg/kg bw per day (toxicity in a 2-year
              study of toxicity and carcinogenicity)

    Rat:      40 ppm, equal to 2.1 mg/kg bw per day (toxicity in a 2-year
              study of toxicity and carcinogenicity)

              120 ppm equal to 6 mg/kg bw per day (toxicity in a study of
              reproductive toxicity)

              10 mg/kg bw per day (developmental effects, highest dose
              tested in a study of developmental toxicity)

              2.2 mg/kg bw per day (maternal and fetotoxicity in a study
              of developmental toxicity)

    Rabbit:   6.3 mg/kg bw per day (maternal and fetotoxicity in a study
              of developmental toxicity)

              20 mg/kg bw per day (developmental effects, highest dose
              tested in a study of developmental toxicity)

    Dog:      4.5 mg/kg bw per day (toxicity in a 1-year study)


    N- Acetylglufosinate 

    Mouse:    1000 ppm, equal to 150 mg/kg bw per day (toxicity in a
              2-year study of toxicity and carcinogenicity)

    Rat:      200 ppm, equal to 9 mg/kg bw per day (toxicity in a 2-year
              study of toxicity and carcinogenicity)

              2000 ppm, equal to 140 mg/kg bw per day (parental toxicity
              in a study of reproductive toxicity)

              10 000 ppm equal to 700 mg/kg bw per day (reproductive
              effects, highest dose tested in a study of reproductive
              toxicity)

              1000 mg/kg bw per day (highest dose tested in a study of
              developmental toxicity)

    Rabbit:   400 mg/kg bw per day (developmental effects, highest dose
              tested in a study of developmental toxicity)

              64 mg/kg bw per day (maternal and fetotoxicity in a study of
              developmental toxicity)

    Dog:      500 ppm, equal to 19 mg/kg bw per day (toxicity in a 90-day
              study)

     Estimate of acceptable daily intake for humans 

         0-0.02 mg/kg bw (for glufosinate-ammonium,  N-acetylglufosinate,
         and 3-[hydroxy(methyl) phosphinoyl]propionic acid, alone or in
         combination)

     Estimate of acute reference dose 

         Unnecessary

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

         Further observations in humans


        Toxicological end-points relevant for estimating guidance values for dietary and non-dietary exposure to glufosinate-ammonium

                                            Glufosinate-ammonium                    N-Acetylglufosinate (1991 JMPR)

     Absorption, distribution, excretion and metabolism in mammals 

    Rate and extent of oral absorption      Rapid but limited (5-10%)               Rapid but limited (5-10%)
    Distribution                            Extensive. Higher concentration in      Extensive. Higher concentration in  
                                            kidney and liver                        kidney and liver
    Potential for accumulation              Minimal                                 Minimal
    Rate and extent of excretion            Rats, rapid, > 92% of 30 mg/kg bw       Rats, rapid, >95% of 3 mg/kg bw within 
                                            within 24 h, primarily in faeces        24 h, primarily in  faeces
    Metabolism in animals                   Main metabolite is 3-[hydroxy-(methyl)  Limited. Some de-acetylation 
                                            phosphinoyl] propionic                  to glufosinate-ammonium (rat,
                                            acid (rat, goat, hen)                   goat, hen)
    Toxicologically significant compounds   Parent                                  Parent and glufosinate-ammonium

     Acute toxicity 

    Rat, LD50, oral                         1700 mg/kg bw                           > 3000 mg/kg bw
    Rat, LD50, intraperitoneal              ~ 100 mg/kg bw                          > 1200 mg/kg bw
    Mouse, LD50, oral                       420 mg/kg bw                            > 3000 mg/kg bw
    Skin sensitization, guinea-pigs         Negative (Buehler test)                 Negative (Magnusson & Kigman test)

     Short-term toxicity 

    Target/critical effect                  Glutamine synthetase inhibition;        Glutamine synthetase inhibition in brain 
                                            behaviour in dogs; kidney weights       of dogs, mice, rats
                                            and urinary parameters in rats
    Lowest relevant oral NOAEL              5 mg/kg bw per day in dogs              19 mg/kg bw per day in dogs

    Genotoxicity                            Not genotoxic                           Not genotoxic

     Long-term toxicity and carcinogenicity 

    Target/critical effect                  Glutamine synthetase inhibition;        Adrenal necrosis and hyperplasia; 
                                            increased kidney weight in rats         spleen haematopoiesis in rats
    Lowest relevant NOAEL                   2 mg/kg bw per day in rats              9 mg/kg bw per day
    Carcinogenicity                         Not carcinogenic                        Not carcinogenic 

     Reproductive toxicity 

    Reproductive target/critical effect     Reduced litter size, rats               None
    Developmental target/critical effect    General maternal and fetotoxicity       Extra ribs/maternal toxicity in  rabbits
    Lowest relevant NOAEL for               12 mg/kg bw per day in rats             137 mg/kg bw per day for reproductive 
       general toxicity in rats                                                     toxicity
    Lowest relevant NOAEL for               2 mg/kg bw per day in rats              64 mg/kg bw per day in  rabbits
       developmental toxicity

    Neurotoxicity                           Possibly behavioural, but no            No evidence of specific effectspathological findings 

    Medical data                            Suicidal poisonings producing coma,     No data, not produced commercially
                                            delayed neurological effects, death; 
                                            no findings in  work force

                                                                                                           
    Summary                                 Value                  Study                  Safety factor
                                                                                                           

    Group ADI for glufosinate-ammonium      0-0.02 mg/kg bw        2 years, rat           100
    and  N-acetylglufosinate

    Acute reference dose                    Unnecessary
                                                                                                           
    

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    See Also:
       Toxicological Abbreviations
       Glufosinate-ammonium (Pesticide residues in food: 1991 evaluations Part II Toxicology)