IPCS INCHEM Home


    BENALAXYL

    EXPLANATION

         Residues of benalaxyl, a systemic fungicide, were evaluated by
    the JMPR in 1986 (Annex 1, FAO/WHO, 1986e). Toxicological data on
    benalaxyl is reviewed here.

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    BIOLOGICAL DATA

    Biochemical Aspects

    Absorption, Distribution and Excretion

         Studies reviewed by the 1986 JMPR show that benalaxyl is
    rapidly metabolized and excreted by rats (Annex I, FAO/WHO, 1986e).

    Toxicological Studies

    Special Study on Allergic Sensitisation

         In the induction phase, groups of 20 female Hartley albino
    guinea pigs were injected intradermally (0.1 ml) with Freunds
    Complete adjuvent, p-nitroso dimethylaniline (1%) in petrolatum,
    mercuric chloride (0.1% in saline) or benalaxyl (5% in paraffin
    oil). After 7 days, the respective injection sites were treated
    topically for 48 hours with p-nitroso dimethylalanine (25% in
    petrolatum), mercuric chloride (1% in petrolatum) and benalaxyl
    (25% in petrolatum). The animals were subsequently challenged after
    a further 2 weeks with 5% in petrolatum, mercuric chloride (0.1% in
    saline) or p-nitroso dimethylnitrosamine (10% in petrolatum) and
    results scored 24 hours after removal of the patch. Benalaxyl did
    not induce allergic sensitisation while the positive control,
    p-nitroso dimethylaniline, gave the expected strong response (Vole
    Gera and Vasconi, 1981).

    Special Studies on Carcinogenicity

         See under long-term studies.

    Special Studies on Eye and Skin Irritation

        Table 1.
                                                                                             
    Method              Species     Route             Results          Reference
                                                                                             
    Primary eye         Rabbit      Intraocular       Non-irritant     Dal Re & Vole
    irritation                                                         Gera (1979a)
    (Draize Test)

    Primary skin        Rabbit      Dermal intact     Non-irritant     Dal Re & Vole
    irritation                                                         Gera (1979b)
    (Draize Test)
                                                                                             

    
    Special Studies on Mutagenicity

         See Table 2.

    Special Study on Reproduction

         Groups of 25 male and 25 female Sprague-Dawely rats received
    benalaxyl (purity unspecified) in the diet at 0, 100, 1,000 and
    5,000 ppm for 112 days before intial mating. Similar treatment was
    continued throughout two litter and into a second generation which
    also produced two litters.

         Dietary exposure to benalaxyl had no effect on parental
    mortality or on fertility, length of gestation period, litter size
    or the number of stillborn pups in either the F1 or F2 generations.
    There was a significant reduction in the bodyweight of high dose F1
    males and am irregular reduction for high dose F1, F2A and F2B
    females. F2B pups of the high-dose group had reduced bodyweights at
    irregular intervals.

         At necropsy, the high dose F1 females had significantly
    increased liver weights whilst F1 rats of both sexes had increased
    liver to bodyweight ratios. F2B pups had increased liver to
    bodyweight ratios at 1,000 ppm whilst male and female F2B pups
    exhibited increased liver to bodyweight ratios at
    5,000 ppm. Treatment had no effect on implantation or resorption
    rates or on the number of corpora lutea. There were no
    histopathological findings that could be related to treatment.
    Accordingly, feeding up to 100 ppm benalaxyl in the diet has no
    effect on the reproduction of rats (Johnson and Becci, 1983).

    Special Studies on Teratogenicity

    Rats

         Groups of 20-23 gravid female Charles River CD (SD) BR rats
    received by oral gavage 0, 12.5, 50 and 200 mg/kg benalaxyl (95%
    pure) in methylcellulose (0.5%) from the 6th to 15th day of
    gestation. Twenty days after mating, the animals were sacrificed
    and their uterine contents examined.

         The treatment had no observable effect on the rats and there
    was no mortality. There were no significant differences in the
    numbers of implantations, post-implantation losses, numbers of
    viable fetuses or their sex ratio. No major malformation occurred
    in any group but there was a dose-related increase in the frequency
    of minor skeletal anomalies, arising especially from delayed
    skeletal ossification, which became statistically significant for
    the mid and high-dose groups. Accordingly, the results of this
    study indicate that benalaxyl is not teratogenic to rats at daily
    doses up to 200 mg/kg (Mondino, 1982a).

    Rabbits

         Groups of 16 mated female New Zealand White Rabbits received,
    0, 5, 50 or 250 mg/kg benalaxyl (purity unspecified, in 1% aqueous
    methylcellulose) by daily oral gavage from day 6 to 27 of
    gestation. The animals were sacrificed on day 28 and their uterine
    contents removed and examined.

         Seven of the rabbits died during the study, either from
    intubation error or respiratory disease but these came from all
    groups in similar numbers. High-dose rabbits lost weight during the
    last 5 days of the study. At necropsy, the incidences of pregnancy
    were similar in all groups. Treatment had no effect on the
    implantation rate or post-implantation losses. The mean number of
    fetuses and their sex ratios were unaffected but mean litter weight
    was slightly reduced in the high-dose group. The mean crown-rump
    length was significantly reduced at 250 mg/kg and slightly reduced
    at 50 mg/kg. There were no treatment related effects on the rate of
    minor skeletal defects. The results of this study indicate that
    5 mg/kg daily administered of benalaxyl does not affect the fetal
    development of rabbits (Irvine, 1984).

        Table 2:  Acute Toxicity of Benalaxyl
                                                                                             

    Species    Sex     Route               Purity      LD50/LC50        Reference
                                                                                             

    Mouse      M/F     Oral                92.5%       680 mg/kg        Dal Re & Vola
                                                                        Gera, 1981

    Rat        M/F     Oral                96.5%       4,200 mg/kg      Dal Re & Vola
                                                                        Gera, 1979c

    Rat        M/F     Inhalational        ?           >10 mg/l         Sterner &
                                                                        Pfennig, 1985

    Rat        M/F     Intraperitoneal     ?           1,100 mg/kg      Dal Re & Vola
                                                                        Gera, 1979d

    Rabbit     M/F     Dermal              94%         2,000 mg/kg      Vola Gera &
                                                                        Vasconi, 1983
                                                                                             

    
    SHORT-TERM STUDIES

    Mice

         In a dose-ranging study, groups of 20 male and 20 female Swiss
    mice received benalaxyl (94% pure) in their diet at 0, 500, 1,000,
    2,000, 3,000 and 5,000 ppm for 90 days. There were no apparent
    effects on the behaviour, bodyweight, food and water consumption or
    animal mortality. Half of the mice of each group and sex were
    sacrificed at 40 days for interim evaluation.

         At necropsy, there was a dose-related increase in absolute and
    relative liver weights which occurred at and above 1,000 ppm at
    96 days and at and above 2,000 ppm at 42 days, especially in female
    mice. Histopathology showed no significant differences between
    treated and control groups (Maltoni, 1985).

    Rats

         Groups of Charles River CD(SD) BR rats (20 per sex per group)
    were fed benalaxyl in the diet at levels of 0, 10, 100, 1000 and
    10000 ppm for 13 weeks. A further group (20 per sex) were fed
    benalaxyl in the diet at a level of 12000 ppm for 4 weeks followed
    by a recovery period of 4 weeks with no chemical in the diet.
    Observations included clinical signs, food consumption, body
    weight, organ weight, hematology, urinalysis, gross and microscopic
    pathology.

         There were no treatment related deaths during the study. One
    animal in the recovery group died subsequent to blood sampling
    which was performed by incision of a sublingual vein under light
    ether anaesthesia at 0, 5 and 13 weeks.

         There were no clinical signs of treatment. Body-weight gain
    was reduced slightly between weeks 2 and 13 at the 10000 ppm level,
    and during the treatment period at the 12000 ppm level. Slight
    anemia was seen after 5 weeks in females and males at the 12000 ppm
    level, but values returned to normal during the recovery period.
    Serum cholesterol was increased after 5 and 13 weeks in males and
    females at 10000 ppm. In the recovery groups fed 12000 ppm,
    cholesterol was elevated at 5 weeks but normal by 13 weeks. Liver
    weight was increased at the 1000 and 10000 ppm levels in a dose
    related manner.

         At necropsy the evidence of "lobulated" liver was increased at
    the 10000 ppm level in males. Diffuse hepatic steatosis was
    observed in both sexes at this dose level, but was not seen at
    sacrifice in the recovery group. There were no other treatment
    related histopathological findings. Pituitary adenomas were found
    in one female at both the 10 and 100 ppm levels.

    The study indicated that feeding of benalaxyl at dietary levels up
    to 100 ppm (5 mg/kg b.w./day) is without toxicological effect in
    rats (Mondino, 1982b).

    Dogs

         Groups of 6 male and 6 female Beagle dogs received benalaxyl
    (92.5% pure) in the diet at 0, 10, 200 and 800 ppm for 52 weeks.
    There were no mortalities and no signs were observed that could be
    related to treatment. Ophthalmoscopy, performed at the beginning
    and at termination of exposure, was unremarkable. Treatment had no
    effect on food consumption or bodyweight gain. Hematological and
    clinical chemical parameters and urinalysis, investigated at
    regular intervals throughout the exposure, showed no consistent
    effects and none that were related to treatment.

         At necropsy, there were no remarkable gross pathological
    findings. Histopathology showed atrophy of the seminiferous tubules
    of two high-dose dogs that was not seen in any other group.
    Accordingly, the results of this study indicate that feeding up to
    200 ppm in the diet is without observable effect in dogs (Mondino,
    1982c).

    LONG-TERM STUDIES

    Mice

         Groups of 60 male and female Swiss mice received benalaxyl
    (94% pure) in their diets at 0, 250, 1,000 and 3,000 ppm for 78
    weeks. Benealaxyl exposure had no effect on the survival of female
    mice but reduced the survival of males treated at 1,000 and 3,000
    ppm. The body weight of treated male groups tended to be less than
    controls, but there was no association with dietary concentration
    of benalaxyl. Treatment had no observable effect on behaviour or
    appearance. Clinical chemistry and hematological parameters were
    not influenced by treatment.

         At necropsy, the absolute and relative liver weights of female
    mice exposed to 3,000 ppm benalaxyl was increased. Widespread
    amyloidosis, affecting adrenal and salivary glands, kidney, liver,
    spleen, stomach, intestine and Zymbal gland, was found more
    commonly in treated than control mice, especially mid and high dose
    males. There was also an association between the amyloidosis and
    benalaxyl exposure in those male mice which died spontaneously
    during the study. Three high dose male mice were found to have
    transitional cell carcinoma of the urinary bladder which was not
    seen in other groups. There were no significant increases in other
    tumour types in any group. Due to the increased incidence of
    amyloid deposition in all treated groups, this study did not
    establish a clear no-effect level (Maltoni, 1985).

    Rats

         Groups of Charles River CD(SD) BR rats (65 per sex per group)
    were fed benalaxyl (purity unstated) in the diet at levels of 0, 4,
    100 and 1000 ppm for 104 weeks. A separate group of 30 rats per sex
    were used for the 'pre-study' clinical testing programme. Animals
    were given fresh diet each week and each batch was analysed for
    benalaxyl content.

         All animals were observed daily for clinical symptoms of
    toxicity. Body weight and food consumption were measured weekly for
    14 weeks and twice weekly thereafter. Blood samples for clinical
    chemistry and hematology were collected at 0, 3 (hematology only),
    6, 12, 18 and 24 months. Urinalysis were performed at 12, 18 and'
    24 months; ophthalmological examinations were performed at 0, 1, 6,
    12, 18 and 24 months. An interim sacrifice (10 rats/sex/group) was
    made at 12 months. All animals except the 'pre-study' clinical
    testing group were subject to gross necropsy and histopathological
    examination. Organ weights were determined for brain, heart,
    kidneys, liver, ovaries, testes and thymus.

         No clinical signs of toxicity were apparent during the study.
    Mortality was not treatment related; survival at 104 weeks ranged
    from 34 to 53%. There were no effects of treatment on body weight
    or food consumption. Hematology, clinical chemistry, urinalysis and
    ophthalmoscopy were unremarkable. There were no treatment related
    effects on organ weights, and there was no gross or microscopic
    pathology.

         The study demonstrated no toxic effects of treatment of rats
    with benalaxyl at levels up to 1000 ppm in the diet (equivalent to
    50 mg/kg b.w./day) (Thompson et al., 1983).

    Special Studies on Mutagenicity

         Table 2 summarizes the results of mutagenicity testing of
    benalaxyl.


        Table 2.  Special Studies on Mutagenicity
                                                                                                                                

    Test System             Test Object                Concentrations        Purity       Results         References
                                                                                                                                

    Ames test (with and     Salmonella typhimurium     200, 400, 800,        98%          Negative        de Carneri, 1979a
    without metabolic       TA1535, TA1537, TA1538,    1600 ug/plate                      (1)
    activation)             TA98, TA100

    Gene mutation           Schizosaccharomyces        20, 40, 80,           ?            Negative        de Carneri, 1980
    (without metabolic      pombe P1 (Sp 198,          100 ug/ml                          (2)
    activation) (with       ade 6-60
    metabolic               radio-198, h)              100, 200, 400,                     Negative
    activation)                                        800 ug/ml                          (3)

    Gene mutation           Chinese Hamster            3  10-7, 10-6,       ?            Negative        Monaco, et al.,
    (without metabolic      V79 Cells                  3  10-6, 10-5,                    (3)             1983
    activation) (with                                  3  10-5 M
    metabolic                                                                             Negative
    activation)                                                                           (6)

    Gene conversion         Saccharomyces              8, 40, 200,           ?            Negative        de Carneri, 1979b
    (with and without       cerevisae D4               1000 ug/ml                         (2)
    metabolic
    activation)

    Chromosome              Chinese Hamster            1000, 2000,           ?            Negative        Mondino, 1980a
    Abberation              Ovary Cells                4000 mg/kg                         (5)

    Chromosome              Cultured human                                                                Mondino, 1980b
    aberration              lymphocytes
    (without metabolic                                 0.083, 0.25,          ?            Negative
    activation) (with                                  0.83, 2.5 mg/ml                    (5)
    metabolic                                          0.033, 0.1,                        Negative
    activation)                                        0.033, 1 mg/ml                     (7)
                                                                                                                                
    Table 2. contd.
                                                                                                                                

    Test System             Test Object                Concentrations        Purity       Results         References
                                                                                                                                

    Primary Rat             Rat Hepatocytes            0.5, 1.0              94%          Negative        Myhr and Brusick, 1983
    Hepatocyte
    Unsheduled DNA                                     2.5, 5, 10, 25,
    Synthesis                                          50 ug/ml
                                                                                                                                

    (1)  Positive controls, N-methyl-N-nitro-N-nitrosoquanidine, 2-nitrofluorene and 2-acetylaminofluorine
         (2-AAF) gave expected positive result.
    (2)  Positive control, methyl methanesulphonate, gave expected positive result.
    (3)  Positive control, N-dimethylnitrosamine, gave expected positive result.
    (4)  Positive control, cyclophosphamide, gave expected positive result.
    (5)  Positive control, Mitomycin C, gave expected positive result.
    (6)  Positive control, ethanemethanesulphonate, gave expected positive result.
    (7)  Positive control, phenacetin, gave expected positive result.
    (8)  Positive control, 2-AAF gave expected positive result.

    
    COMMENTS

         Benalaxyl was evaluated toxicologically for the first time by
    the present meeting. It was evaluated by the 1986 JMPR for residues
    only. As noted at that time, benalaxyl is completely metabolized
    and does not accumulate in the tissues of rats. Comprehensive
    short- and long-term dietary administration indicates that the
    toxicity of benalaxyl is low. Hepatic enlargement occurred in rats
    fed benalaxyl (> 1000 ppm) in their diet for 14 weeks and in
    mice fed for 78 weeks (3000 ppm). However, hepatomegaly did not
    occur in rats fed benalaxyl (1000 ppm) for two years or in dogs fed
    for one year (800 ppm).

         The meeting concluded that although the hepatic enlargement
    observed in rodents was of questionable toxicological significance,
    it could be used to establish a no-effect level and estimate an
    ADI.

    TOXICOLOGICAL EVALUATION

    LEVEL CAUSING NO TOXICOLOGICAL EFFECT

          Mouse:   500 ppm in the diet, equivalent to 75 mg/kg bw/day.
          Rat:     100 ppm in the diet, equal to 5 mg/kg bw/day.
          Dog:     200 ppm in the diet, equal to 7 mg/kg bw/day.

    ESTIMATE OF ACCEPTABLE DAILY INTAKE FOR MAN

         0-0.05 mg/kg bw.

    STUDIES WHICH WILL PROVIDE INFORMATION VALUABLE IN THE CONTINUED
    EVALUATION OF THE COMPOUND

         Observations in man.

    REFERENCES

    de Carneri, I., 1979a. Microbiological Mutagenesis Study on CRA 109
    (M9834). Genetic mutation test in Salmonella typhimurium (Ames).
    Farmitalia Carlo Erba Research and Development. Unpublished study
    submitted to WHO by Farmoplant Research and Development.

    de Carneri, I., 1979b. Microbiological Mutagenesis Study on CRA 109
    (M9834). DNA damage and repair test (Mitotic gene conversion in
    Saccharomyces cerevisae D4). Farmitalia Carlo Erba Research and
    Development. Unpublished study submitted to WHO by Farmoplant
    Research and Development.

    de Carneri, I., 1980. Microbiological Mutagenesis Study on CRA 109
    (M9834). In vitro gene mutation test in Schizosaccharomyces pombe
    P1. Farmitalia Carlo Erba Research and Development. Unpublished
    study submitted to WHO by Farmoplant Research and Development.

    Dal Re, U. & Vola Gera, F., 1979a. Primary eye irritation study of
    M9834 in albino rabbits. Montedison Division Agricoltura. Centro
    Ricerche Antiparassitari. Unpublished study submitted to WHO by
    Farmoplant Research and Development.

    Dal Re, U. & Vola Gera, F., 1979b. Primary skin irritation study of
    M9834 in albino rabbits. Montedison Division Agricoltura. Centro
    Ricerche Antiparassitari. Unpublished study submitted to WHO by
    Farmoplant Research and Development.

    Dal Re, U. & Vola Gera, F., 1979c. Acute oral toxicity study of
    M9834 in albino rat. Montedison Division Agricoltura. Centro
    Ricerche Antiparassitari. Unpublished study s Submitted to WHO by
    Farmoplant Research and Development.

    Dal Re, U. & Vola Gera, F., 1979d. Determination of the acute
    intraperitoneal toxicity of M9834, technical grade, in the rat.
    Montedison S.p.A. Agricultural Division, Pesticide Research Centre,
    Milan. Unpublished study submitted to WHO by Farmoplant Research
    and Development.

    Dal Re, U. & Vola Gera, F., 1981. Determination of the acute oral
    toxicity of Galben, technical grade, in albino mice. Gruppo
    Montedison S.p.A. Pesticide Research Centre, Milan. Unpublished
    study submitted to WHO by Farmoplant Research and Development.

    Irvine, L.F.H., 1984. Galben: Oral (Gavage) teratology Study in the
    New Zealand White Rabbit. Hazleton Laboratories Europe Ltd Report
    No. 3596-309/3. Unpublished study submitted to WHO by Farmoplant
    Research and Development.

    Johnson, W.D. & Becci, P.J., 1983. Two generation Reproduction
    Study in Rats with Galben Technical (Final Report). Food and Drug
    Research Laboratories, Inc. Waverly Study No. 7220. Unpublished
    study submitted to WHO by Farmoplant Research and Development.

    Maltoni, C., 1985. Evaluation of Chronic Toxicity and Oncogenic
    Potential of Galben (R) (CAS no. 71626-11-4) in Swiss Mice (Oral
    Dosing Study). Bologna Institute of Oncology Study No. BT 5004.
    Unpublished study submitted to WHO by Farmoplant Research and
    Development.

    Monaco, M., Forster, R. & Nunziata, A., 1983. Gene mutation in
    Chinese Hamster V798 Cells Test substance: Galben TH. Life Sciences
    Research, Roma toxicology Centre Rep. No. LSR-RTC FMP 001.
    Unpublished study submitted to WHO by Farmoplant Research and
    Development.

    Mondino, A., 1980a. In vivo Study of the Induction of Chromosome
    Aberrations in the Chinese Hamster by Compound M 9834 Administered
    Orally. Institute of Biomedical Research Antoine Marxer S.p.A.
    Ivrea. Unpublished study submitted to WHO by Farmoplant Research
    and Development.

    Mondino, A., 1980b. In vitro Study of the Induction of Chromosome
    Aberrations by Compound M 9834 in Human Lymphocyte Cultures.
    Institute of Biomedical Research Antoine Marxer S.p.A. Ivrea.
    Unpublished study submitted to WHO by Farmoplant Research and
    Development.

    Mondino, A., 1982a. Teratogenesis study of the product M 9834 in
    Charles River CD (SD) rats on oral administration from the 6th to
    24th day of gestation, at daily doses of 12.5, 50 and 200 mg/kg.
    Antoine Marxer Institute of Biomedical Research, Ivrea. Unpublished
    study submitted to WHO by Farmoplant Research and Development.

    Mondino, A., 1982b. 13-Week Oral Subacute Toxicity study of the
    Product M 9834 (Galben) administered to Charles River CD (SD) BR
    rats in the diet, at the doses of 10, 100, 1000, 10,000 and
    12,000 ppm. Institute of Biomedical Research 'Antoine Marxer',
    S.p.A. Ivrea. Unpublished study submitted to WHO by Farmoplant
    Research and Development.

    Mondino, A., 1982c. Long term toxicity study with repeated oral
    administration of the product M 9834 (Galben) added to the food of
    Beagle dogs for 52 weeks, at the dosage levels of 10, 200 and
    800 ppm. Antoine Marxer Institute of Biomedical Research, Ivrea.
    Unpublished study submitted to WHO by Farmoplant Research and
    Development.

    Myhr, B.C. & Brusick, D.J., 1983. Evaluation of Galben in the
    Primary Rat Hepatocyte Unscheduled DNA Synthesis Assay. Final
    Report, Litton Bionetics Project No. 20991. Unpublished study
    submitted to WHO by Farmoplant Research and Development.

    Sterner & Pfennig, 1985. Acute Inhalation Toxicity of 'Galben Th'
    in Rats. IBR Forschungs GmBh, Walsrode Proj. No. 1-4-549-81.
    Unpublished study submitted to WHO by Farmoplant Research and
    Development.

    Thompson, S.W., Davidson, T.J. & Becci, P.J., 1983. Lifetime Oral
    Dosing Studies in Rats: Combined Oncogenicity and Chronic Toxicity
    of Galben Technical 9834. Food and Research Laboratories, Inc.
    Report No. 6568-11 (Final Report). Unpublished study submitted to
    WHO by Farmoplant Research and Development.

    Vola Gera, F. & Vasconi, F., 1981. Allergic contact sensitisation
    test on albino guinea pig using the maximisation test: evaluation
    of the allergenic potency of Galben. Farmoplant Centro Ricerche
    Antiparassitari Biological Evaluation Department, Milan.
    Unpublished study submitted to WHO by Farmoplant Research and
    Development.

    Vola Gera, F. & Vasconi, F., 1983. Galben Acute Dermal Toxicity
    Study (LD50) in the Albino Rabbit. Limit Test. Farmoplant Centro
    Ricerche Antiparassitari Biological Evaluation Department, Milan.
    Unpublished study submitted to WHO by Farmoplant Research and
    Development.
    


    See Also:
       Toxicological Abbreviations
       Benalaxyl (JMPR Evaluations 2005 Part II Toxicological)