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    PESTICIDE RESIDUES IN FOOD - 1997


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




    TOXICOLOGICAL AND ENVIRONMENTAL
    EVALUATIONS 1994




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

    Lyon 22 September - 1 October 1997



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    cases, based on unpublished proprietary data submitted for the purpose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



    PHOSALONE (addendum)

    First draft prepared by
    T.C. Marrs
    Medical Toxicology and Environmental Health
    Department of Health, London, United Kingdom

         Explanation
         Evaluation for acceptable daily intake 
              Long-term toxicity and carcinogenicity 
         Comments 
         Toxicological evaluation 
         References 

    Explanation

         Phosalone was evaluated toxicologically by the 1972 JMPR (Annex
    1, reference 18), when an ADI of 0-0.006 mg/kg bw was established; it
    was re-evaluated in 1993 (Annex 1, reference 68), when an ADI of
    0-0.001 mg/kg bw was established on the basis of the lowest dose
    tested (5 ppm), equal to 0.2 mg/kg bw per day, in a two-year study in
    rats, with a safety factor of 200 because of concern about the
    possibly significant occurrence of testicular atrophy and reduction in
    testicular weight. More information on this study was supplied for
    consideration at the present Meeting. 

    Evaluation for acceptable daily intake

    Long-term toxicity and carcinogenicity

         A study was completed by Barker and Sortwell in 1993 in which
    groups of 50 Sprague-Dawley CD rats of each sex were given phosalone
    (purity, 94.5%) in the diet for two years. Volume 1 of the study was
    evaluated at the 1993 meeting (Annex 1, reference 68). The entire
    report became available, with an addendum containing a review of the
    testicular pathology. Phosalone was administered in the diet at
    concentrations of 0, 5, 50, or 1000 ppm, the highest dietary
    concentration being reduced to 500 ppm at week 27. These
    concentrations were equal to 0, 0.2, 1.8, or 20 mg/kg bw per day for
    males and 0, 0.2, 2.5, or 31 mg/kg bw per day for females, on the
    basis of consumption from week 27 until the end of the study.
    Satellite groups of 15 rats of each sex at each dose except the high
    dose, which contained 25 animals, were killed at 52 weeks. 

         Phosalone did not affect mortality; in fact, the rate was lower
    in the group at the high dose, especially among males, than in the
    other groups. The clinical effects were mainly confined to rats at the
    high dose, which showed hypersensitivity, abnormal posture, and poor
    grooming; females at this dose also had a marked impairment in
    body-weight gain during the first 26 weeks, and the effect was seen to
    a lesser extent in males. Body weight recovered after the dose of
    phosalone was reduced. Depression of plasma and erythrocyte

    cholinesterase activity was found in rats at the highest dietary
    concentration and to a lesser degree in those at 50 ppm at various
    times throughout the study. Biologically significant depression of
    brain acetylcholinesterase activity was found only at the highest dose
    (see Table 1). No evidence of tumorigenesis was found. Some
    treatment-related changes were seen in the adrenals, consisting of
    enlargement and foamy change in the zona glomerulosa in animals at the
    high dose at the interim kill and vacuolation of adrenal cortical
    cells at the terminal kill. 

         Statistically significant atrophy of the testes was seen in rats
    at the high and intermediate doses. Moreover, there appeared to be a
    dose-response relationship in the prevalence of testicular atrophy:
    2/27, 3/34, 8/30, and 12/42 in survivors at the time of terminal kill.
    A dose-response relationship was seen even when only the incidence of
    'marked' testicular atrophy was considered: 1/27, 3/34, 6/30, 10/42;
    however, the incidence in each group was not statistically
    significantly greater than that in the controls, when only those
    animals surviving to the terminal kill were considered. At the highest
    doses, the findings were significant when decedents were included; in
    view of the bias that could be introduced as a result of longer
    survival in this group resulting from the inclusion of decedents, the
    use of the figures for animals killed at the end of the study is more
    appropriate. Thus, it can be concluded that no clear dose-response
    relationship was demonstrated. No evaluation was made of whether the
    atrophy was bilateral or unilateral at this stage. 

         The slides were re-examined by a consultant who concluded that
    the apparent dose-response relationship was largely a product of
    increased survival of animals at the high dose, although this is
    clearly not the case when only data on survivors are considered. The
    data could be interpreted as showing a trend for bilateral or
    unilateral testicular atrophy, but the consultant argued that
    bilateral atrophy is a more reliable indicator of treatment-related
    change than unilateral atrophy. On this basis, there was no clear
    dose-response relationship (Table 2; Isaacs, 1995). The number of
    animals used was slightly different because of the inclusion of those
    that died in week 106 of the study but were analysed as part of the
    terminal kill. 

         The findings were re-examined at the laboratory which originally
    performed the study (Barker, 1996; Table 3). The incidences of
    unilateral or bilateral testicular atrophy in the survivors to
    termination were 26% in controls, 24% at the low dose, 40% at the
    intermediate dose, and 40% at the high dose; these percentages were
    stated to be within the background range in the laboratory. The
    incidences of bilateral atrophy only in the survivors to termination
    of the bioassay were 7% in controls, 3% at the low dose, 7% at the
    intermediate dose, and 12% at the high dose. Since testicular atrophy
    in the rat is commonly an age-related phenomenon, the prevalence of
    lesions in animals killed at termination is the appropriate variable;
    an exception would be analysis of the data in an age-to-occurrence
    fashion. This was not the case.


        Table 1. Erythrocyte and brain acetylcholinesterase activity (% concurrent controls) in rats given 
    phosalone in the diet for two years

                                                                                                            

    Dose           Erythrocytes                                                               Brain
    (ppm)                                                                                     (week 105/6)
                   Week 13        Week 26        Week 52        Week 78        Week 104
                                                                                                            

    Male
    5              101            70             120            99             101            98
    50             77             58             < 93           61             98             92
    1000/500       < 35           < 36           < 45           < 24           34             27

    Female
    5              107            118            100            103            105            147
    50             68             67             52             < 65           78             140
    1000/500       < 40           < 42           < 33           43             37             40
                                                                                                            

    Table 2. Testicular lesions in decedents (D) and terminally-killed animals
    (T) in rats given phosalone in the diet for two years (Isaacs, 1995)

                                                                                                        

                             Dose (ppm)
                                                                                                        
                             0                   5                   50                  1000/500
                                                                                                        
                             D         T         D         T         D         T         D         T
                                                                                                        

    No. of animals           21        29        16        34        20        30        7         43

    Lesion
    Unilateral atrophy       1         5         1         6         4         11        1         10
    Bilateral atrophy        4         2         4         4         4         3         1         8
    Total atrophy            5         7         5         10        8         14        2         18
                                                                                                        

    Table 3. Testicular lesions in decedents (D) and terminally-killed animals
    (T) in rats given phosalone in the diet for two years (Barker, 1996)

                                                                                               

                                  Dose (ppm)
                                                                                               
                                  0               5               50              1000/500
                                                                                               
                                  D       T       D       T       D       T       D       T
                                                                                               
    No. of animals                23      27      16      34      20      30      8       42
    Lesion                                                                                
      Reduced spermiogeuesis      0       0       1       1       1       2       0       1
      Unilateral atrophy          3       5       1       7       6       10      1       12
      Bilateral atrophy           2       2       3       1       3       2       1       5
      Total atroplxy              5       7       4       8       9       12      2       17
                                                                                               
    

         Testicular weight was reduced in these groups at the terminal but
    not the interim kill, the mean testicular weights being 5.03 g in
    controls, 4.66 g at the low dose, 4.51 g at the intermediate dose, and
    4.22 g at the high dose. These reductions were statistically
    significant at the high and intermediate doses. Nonetheless, these
    values were stated to be within the range in historical controls at
    the institution where the study was performed. 

         If it is assumed that only bilateral testicular atrophy is
    toxicologically significant, the incidence figures of neither Isaacs
    (1995) nor Barker (1996) show any clear dose-response relationship
    (see Table 4). Furthermore, there appeared to be no effects of
    treatment in the testes examined at 52 weeks. The first long-term
    studies in rats evaluated by the 1993 JMPR showed no evidence of
    testicular effects. The Meeting was satisfied that no
    treatment-related testicular effects had been induced. Accordingly,
    the Meeting concluded that the NOAEL was 50 ppm, equal to 1.8 mg/kg bw
    per day, on the basis of inhibition of brain acetylcholinesterase
    activity at the highest dose. 

         No data from the other studies reviewed in 1993 support a
    specific treatment-related effect on the testis.

    Comments

         Dietary concentrations of 0, 5, 50, or 1000 ppm phosalone were
    administered to rats for two years, the highest dose being reduced to
    500 ppm later in the study. Significant depression of brain
    acetylcholinesterase activity was found only at the highest
    concentration. There was a statistically significant increase in the
    prevalence of testicular atrophy, a reduction in testicular weights in
    animals at both the high and intermediate doses, and a dose-response
    relationship across all groups for both effects. All testicular
    weights were within the range in historical controls at the institute
    where the study was performed. The slides were re-examined by a
    consultant, who argued that the apparent dose-response relationship
    for testicular atrophy was largely a product of increased survival
    among the rats at the highest dose. Furthermore, it was argued that
    bilateral atrophy is a more reliable indicator of treatment-related
    change than unilateral atrophy; on this basis, there was no clear
    dose-response relationship. The findings were also re-examined at the
    laboratory where the study was originally performed; no clear
    dose-response relationship was found. In view of the fact that neither
    an earlier long-term study in rats nor studies in mice and dogs gave
    evidence of changes in the male reproductive system, the Meeting
    considered the NOAEL to be 50 ppm, equal to 1.8 mg/kg bw per day, on
    the basis of inhibition of brain acetylcholinesterase activity at the
    highest dose.

         The rat remained the most sensitive species examined, and on the
    basis of the NOAEL of 1.8 mg/kg bw per day in the above study the
    Meeting established an ADI of 0-0.02 mg/kg bw, using a safety factor
    of 100. 

        Table 4. Incidence of bilateral testicular atrophy (%) in rats given phosalone in the diet 
    for two years, according to Isaacs (1995) and Barker (1996)

                                                                                                  

    Reference           Dose (ppm)
                                                                                                  
                        0                   5                   50                  1000/500
                                                                                                  
                        No.       %         No.       %         No.       %         No.       %
                                                                                                  

    Isaacs (1995)       2/29      7         4/34      12        3/30      10        7/43      16
    Barker (1996)       2/27      7         1/34      3         2/30      7         5/42      12
                                                                                                  
    

    Toxicological evaluation

     Levels that cause no toxic effect

         Mouse:    150 ppm, equal to 23 mg/kg bw per day (two-year study
                   of toxicity and carcinogenicity)

         Rat:      50 ppm, equal to 1.8 mg/kg bw per day (two-year study
                   of toxicity and carcinogenicity)

                   50 ppm, equivalent to 2.5 mg/kg bw per day
                   (multigeneration study of reproductive toxicity)

         Rabbit:   10 mg/kg bw per day (study of developmental toxicity)

         Dog:      200 ppm, equivalent to 5 mg/kg bw per day (several
                   studies)

     Estimate of acceptable daily intake for humans

         0-0.02 mg/kg bw

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

         Observations in humans

    References

    Barker, M.H. (1996) Report amendment RPN375/930643 dated 14 June 1996
    to: Barker, M.H. & Sortwell, R.J. (1993) Unpublished study. Phosalone
    (11974 RP). Potential tumorigenic and toxic effects in prolonged
    dietary administration to rats. Huntingdon Research Centre Ltd,
    Huntingdon, Cambridgeshire, United Kingdom. Submitted to WHO by
    Rhone-Poulenc, United Kingdom.

    Barker, M.H. & Sortwell, R.J. (1993) Unpublished study. Phosalone
    (11974 RP). Potential tumorigenic and toxic effects in prolonged
    dietary administration to rat. Huntingdon Research Centre Ltd,
    Huntingdon, Cambridgeshire, United Kingdom. Submitted to WHO by
    Rhone-Poulenc, United Kingdom.

    Isaacs, K.R. (1995) Unpublished report. Evaluation of the testes from
    study RNP/375 (Project No. K94/001). Submitted to WHO by Rhone
    Poulenc, Research Triangle Park, North Carolina, USA.
    


    See Also:
       Toxicological Abbreviations
       Phosalone (ICSC)
       Phosalone (WHO Pesticide Residues Series 2)
       Phosalone (WHO Pesticide Residues Series 5)
       Phosalone (Pesticide residues in food: 1976 evaluations)
       Phosalone (Pesticide residues in food: 1993 evaluations Part II Toxicology)
       Phosalone (JMPR Evaluations 2001 Part II Toxicological)