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    TRIAZOLYL ALANINE

    EXPLANATION

         Triazolyl alanine is a plant metabolite of various fungicides
    of the triazole family.  Since the formation of triazolyl alanine is
    not observed in animals, it became necessary to examine the
    toxicology of this compound separately in order to assess the
    safetycof possible residues in edible crops.  Triazolyl alanine is
    considered for the first time by the present meeting.

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    BIOLOGICAL DATA

    Biochemical aspects

    Absorption, distribution and excretion

         [3,5-14C]-Labelled D,L-triazolyl alanine was administered
    orally by gavage at single doses of 0.5 and 50 mg/kg bw to 2 groups
    of 4 male and 4 female Tif:RAIf rats.  Administration resulted in
    rapid, nearly complete absorption from the gastro-intestinal tract
    and very rapid elimination mainly via the kidneys.  Within 24 hours
    (at both dose levels) 95-105% of the radioactivity applied was
    eliminated from the animal, 85-103% of which was found in the urine.
    Seven days after dosing 88-108% and 2-12% of the dose was recovered
    with the urine and faeces, respectively.  Less than 0.5% was found
    in the expired air.  Residues in organs and tissues 7 days after
    administration of 0.5 mg/kg bw  were all below the detection limit. 
    After a dose of 50 mg/kg bw, residues were only found in liver,
    kidneys, blood, stomach, muscle and remaining carcass and were below
    0.02 mg/kg in each case (Hamboeck, 1983a).

    Biotransformation

         The excreta of rats obtained in the previously described study
    after a single oral dose of 0.5 or 50 mg/kg bw 14C D,L-triazolyl
    alanine were used for isolation of metabolites.  The major
    radioactive urinary component (69-86% of dose applied) was
    identified as unchanged D,L-triazolyl alanine, 8-19% of the dose was
    eliminated in the urine after metabolic transformation to N-acetyl-
    D,L-triazolyl alanine.  The pattern of metabolites in feces was
    quite similar with 1-2% found as unchanged parent compound and <1%
    as the N-acetyl metabolite. The remaining radioactivity consisted of
    4 polar metabolite fractions (1-3% of dose each) which were not
    further investigated (Hamboeck, 1983b).

    Short-term studies

         In a two weeks-range finding study groups of 10 male rats were
    administered 0, 3000 or 10000 ppm triazolyl alanine (purity ca 100%)
    in the drinking water.  At all dose levels no treatment related
    effects were observed on appearance and behaviour, food and water
    consumption, body weight, organ weight and macroscopy. A slight but
    not significant increase was observed in liver and kidney weight at
    10000 ppm (Bomhard, 1982).

        Toxicological studies

    Acute toxicity

    TABLE 1.  ACUTE TOXICITY OF TRIAZOLYL ALANINE

                                                                                     

    SPECIES   SEX      ROUTE     PURITY    LD50     LC50      REFERENCE

                                                                                     

    Mouse     M&F      oral      ?         >5000              Mihail, 1982

    Rat       ?        oral      ?         >2000              Henderson & Parkinson,
                                                              1981

    Rat       M&F      oral      ?         >5000              Mihail, 1982

    Rat       M&F      i.p.      ?         >5000              Mihail, 1982
                                                                                     
    
         Groups of 20 male and 20 female rats (Bor: Wisw SPF/Cpb) were
    administered by gavage triazolyl alanine for 28 days at dose levels
    of 0, 25, 100 or 400 mg/kg bw.  Groups of 10 male and 10 female rats
    were kept for a 4 week recovery period.  Observations included
    mortality and signs of toxicity, body weight, food and water
    consumption, haematology, clinical chemistry and urinalysis,
    microsomal N-demethylase and O-demethylase and P-450 in the liver,
    organ weight, macroscopy and histopathology.  There were no effects
    on mortality, appearance, body weight, food and water consumption,
    urinalysis, liver biochemistry, macroscopy and histopathology during
    dosing and recovery period.  Significantly decreased values for
    creatinine and urea (males only) were observed at 400 mg/kg bw after
    the dosing as well as after the post-observation period (urea values
    only).  Liver weight was significantly decreased in females after 4
    weeks at 400 mg/kg bw.  The NOAEL in this study is 100 mg/kg bw
    (Mihail & Vogel, 1983).

         Groups of rats [Bor:WisW (SPF CPB)] (20/sex/group) were orally
    administered 0, 1250, 5000 or 20000 ppm triazolyl alanine (purity
    97.5%) in the diet (equal to 90, 370 or 1510 and 100, 400 or
    1680 mg/kg bw/day for males and females, respectively) for 97 days. 
    All animals were inspected twice daily for mortality and appearance
    and behaviour, while body weight, food intake and water consumption
    were recorded weekly.  Ophthalmoscopical observations were performed
    before the start and at the end of the study while haematology,
    clinical chemistry and urinalysis were carried out on 10 male and 10
    female rats from each group after 1 month and at the end of the
    study. At the end of the study all animals were killed, selected
    organs were weighed, and complete gross and histopathological
    examinations were performed.  No toxic symptoms or treatment related
    deaths occurred.  No differences between control and treated animals
    were noted in food and water consumption, ophthalmoscopy,
    haematology and urinalysis.  Body weight was slightly decreased in
    males at 20000 ppm from week 2 to the end of the study.  Clinical
    biochemistry revealed statistically significant decreases in
    triglyceride, bilbirubin and urea values in males at 20000 ppm and
    in triglyceride values in females at 5000 and 20000 ppm.  ASAT
    values were significantly increased in males at 20000 ppm.  Relative
    heart weight was significantly decreased and relative kidney weight
    was significantly increased in high dosed males.  Based on the
    effect in females the NOAEL in this study is 1250 ppm in the diet,
    equal to 100 mg/kg bw (Maruhn & Bomhard, 1984).

    Dogs

         Groups of 4 male and 4 female Beagle dogs received triazolyl
    alanine (purity 97.5%) in the diet at 0, 3200, 8000 or 20000 ppm for
    13 weeks.  No treatment related effects were observed on mortality,
    clinical signs, body temperature, pulse rate, ophthalmoscopy and
    neurological findings, water consumption, hematological and clinical
    chemical examinations, urinalysis, gross pathology and

    histopathological liver findings.  Female body weight and food
    consumption were decreased at 20000 ppm.  Spleen weight was
    increased in males at the same dose. The NOAEL in this study is
    8000 ppm in the diet, equivalent to 200 mg/kg bw (von Keutz &
    Groning, 1984).

    Reproduction study

    Rats

         In a preliminary reproduction study groups of 12 female and 6
    male rats were fed 0, 150, 625, 2500 or 10000 ppm triazolyl alanine
    in the diet prior to mating.  Male rats were killed after mating,
    while in females treatment was continued throughout pregnancy,
    lactation and weaning of offspring, whereupon all females and
    offspring were killed.  No treatment related effects were observed
    except for an increase in mean pre-coital period and a significantly
    decreased mean litter weight at day 1 at 10000 ppm (Birtley, 1983).

         Groups of 15 male and 30 female ALpk:AP rats received triazolyl
    alanine (purity 97.6-97.8%) in the diet at 0, 500, 2000 or
    10000 ppm.  After 12 weeks of treatment the rats were paired to
    start a 2-generation (2 litters/generation) study. Diets were
    maintained during mating, gestation and lactation for two successive
    litters (F1a and F1b) and repeated for 2 generations.  F1 parents
    selected from F1b offspring were mated after 11 weeks. No adverse
    effects were observed on clinical signs and food consumption,
    parental bodyweight, male and female fertility, pre-coital interval,
    gestation period, number of viable litters, live and dead fetuses,
    macroscopy in parental rats and offspring, microscopy in offspring.

         Initial pup weight was slightly decreased in F1b, F2a and F2b
    pups at 10000 ppm and the total weight of the F2b litter was
    significantly decreased at the same dose.  No compound related pup
    abnormalities were observed (Milburn  et al. 1986).

    Special studies on mutagenicity

         See Table 2


        TABLE 2.  RESULTS OF MUTAGENICITY ASSAYS ON TRIAZOLYL ALANINE

                                                                                                                          

                                                      CONCENTRATION
    TEST SYSTEM             TEST OBJECT               OF TRIAZOLYL             PURITY    RESULTS      REFERENCE

                                                                                                                          

    Ames test (both         S. tyhimurium             20, 100, 500,            ?         Negative     Herbold, 1983a
    with and without        TA1535, TA100, TA1537     2500 and 12500                     (1)
    activation)             TA1538, TA98              ug/pl dissoved
                                                      in DMSO

    Ames test (both         S. tyhimurium             20, 78, 313, 1250        97.4      Negative     Deparade, 1986
    with and without        TA1535, TA100, TA1537     and 5000 ug/0.1                    (1,2)
    metabolic activation)   TA98, TA102               ml in DMSO

    Gene mutation test      Chinese hamster cells     500, 1000, 2000,         97.4      Negative     Dollenmeier, 1986
    (both with and          V79                       4000, 6000, 8000 and               (1)
    without metabolic                                 10000 ug/ml in
    activation)                                       bidist. water

    Transformation assay    BHK 21 C13 cells          500, 1000, 2000          ?         Negative     Richold et al. 1981
    (without metabolic                                4000 and 8000 ug/pl                (1)
    activation)                                       in DMSO

    Transformation assay    BHK 21 C13 cells          1000, 1000, 4000         ?         Positive     Richold et al. 1981
    (with metabolic                                   8000 and 16000                     at toxic
    activation)                                       ug/pl in DMSO                      doses
                                                      toxic >= 16000                     (1)

    Transformation assay    Mouse                     62.5, 125, 250, 500      ?         Negative     Beilstein, 1984
    (with and without       embryofibroblasts         and 1000 ug/ml in                  (1)
    metabolic activation)   BALB/3T)                  bidistilled water
                                                                                                                          

    TABLE 2 (contd.)

                                                                                                                          

                                                      CONCENTRATION
    TEST SYSTEM             TEST OBJECT               OF TRIAZOLYL             PURITY    RESULTS      REFERENCE

                                                                                                                          

    Micronucleus test       Male mice (CBC F1)        2500 and 5000 mg/kg      ?         Negative     Watkins, 1982
                                                      i.p. in 0.5% Tween                 (1)
                                                      80 (administered
                                                      twice, 24 hr apart)

    Micronucleus test       Mice (NMRI strain)        8000 mg/kg bw            ?         Negative     Herbold, 1982, 1983b
                                                      orally in 0.5%                     (1)
                                                      cremophor emulsion

    Micronucleus test       Chinese hamster           5000 mg/kg oral          97.4      Negative     Strasser, 1986
                                                      in 0.5% CMC                        (1)

    DNA repair test         E. coli pol A1            62.5, 125, 250           ?         Negative     Herbold, 1983c
                                                      500 and 1000 ug/pl                 (1)
                                                      in DMSO

    DNA repair test         Rat hepatotes             0.08, 0,4, 2 and         97.4      Negative     Puri, 1986
                                                      10 mg/ml                           (1)
                                                                                                                          

    1)   Positive control yielded positive results.
    2)   Precipitations occurred at concentrations of 78 µg/0.1 ml and above.
    

    Special study on teratogenicity

    Rats

         Groups of 24 female rats (Alderley Park AlpK/AP) received 0,
    100, 300 or 1000 mg triazolyl alanine (purity 84,8%)/kg bw by gavage
    from day 7-16 of gestation.  Dams were observed for clinical
    observations, bodyweights and food consumption.  On day 22 of
    pregnancy the animals were sacrificed and their uteri weighed and
    examined for deaths and live fetuses.  The fetuses were sexed,
    weighed and examined for developmental abnormalities.  No clinical
    signs of maternal toxicity were noted.  Maternal body weight, food
    consumption and reproductive parameters (pregnancy rate, survival
    and growth of the foetuses in utero) were comparable to those of the
    controls.  At 1000 mg/kg bw ossification was delayed (7th cervicial
    vertebrae, 13th thoracic centrum and 5th sternebrae). The incidence
    of not ossified odontoid processes was significantly increased at
    300 and 1000 mg/kg bw.  The effect at 300 mg.kg bw is minimal and
    100 mg/kg b.w is a no observed adverse effect level (Clapp  et al.
    1983).

    COMMENTS

         Triazolyl alanine is rapidly absorbed and excreted, mainly as
    the unchanged parent compound in the urine.  A small proportion is
    excreted as the N-acetyl metabolite.

         The substance has a low acute toxicity in the species examined.

         In a 90-day study in rats at 20 000 ppm, growth inhibition was
    found.  In addition, triglyceride and urea in blood were decreased,
    and aspartate aminotransferase was increased.  A decrease in
    triglyceride was also observed in females at 5000 ppm.  The NOAEL
    was 1250 ppm, which is equal to 100 mg/kg bw.  In a 90-day study in
    dogs, 20 000 ppm in the diet resulted in decreased body weight and
    decreased food consumption.  The NOAEL was 8000 ppm, equivalent to
    200mg/kg bw/day.

         In a 2-generation 2-litter per generation reproduction study in
    rats, pup and litter weights were reduced at the highest dose level
    of 10 000 ppm (equivalent to 500 mg/kg bw day).  No teratogenic
    effects were induced in rats, but a delay in ossification at the
    highest dose levels was reported.  The NOAEL was 100 mg/kg bw/day.

         After reviewing all available  in vitro and  in vivo
    short-term tests, the Meeting concluded that there was no evidence
    of genotoxicity.

         The Meeting concluded from the available data that residues of
    triazoylalanine arising from the use of triazole fungicides do not
    present a toxicological hazard.

    REFERENCES

    All reports are send to WHO on behalf of the triazolyl alanine group
    (Bayer, Ciba-Geigy, ICI and Rohm & Haas) by Ciba-Geigy AG, Basel,
    Switzerland.

    Beilstein, P. (1984)  Transformation/liver-microsome test (in vitro
    test for transformation-inducing properties in mammalian
    fibroblasts).  Unpublished Report Test No. 840324 from Ciba-Geigy
    Ltd.

    Birtley, R.D.N. (1983)  Triazole alanine:  Preliminary reproduction
    study in the rat. Unpublished Report No. CTL/L/470 from Central
    Toxicology Laboratory, ICI PLC.

    Bomhard, E. (1982)  THS 2212, Preliminary subacute toxicity study on
    male rats, administration in the drinking water.  Unpublished Report
    No. 11253 from Institut für Toxicology Bayer AG.

    Clapp, M.J.L. & Killick, M.E. Hollis, K.J. & Godley, M.J. (1983) 
    Triazole alanine. Teratogenicity study in the rat.  Unpublished
    Report No. CTL/P/875  from Central Toxicology Laboratory ICI PLC.

    Deparade, E. (1986)   Salmonella/mammalian-microsome mutagenicity
    test.  Unpublished Report Test No. 860187 from Ciba-Geigy Ltd.

    Dollenmeier, P. (1986)  Point mutation test with chinese hamster
    cells V79 (OECD conform).  Unpublished Report Test No. 860258 from
    Ciba-Geigy Ltd.

    Hamboeck, H. 1983a)  Distribution, degradation and excretion of
    D,L-2-amino-3-(1-H-1,2,4-triazol-1-yl)-propanoic acid
    (D,L-triazolylalanine) in the rat. Unpublished project report 1/83
    from Biochemistry Department R & D Plant  Protection Agricultural
    Division Ciba-Geigy Limited.

    Hamboeck, H. (1983b)  The metabolism of D,L-2-amino-3-(1H-1,2,4-
    triazol-1-yl)- propanoic acid (D,L-triazolylalanine) in the rat. 
    Unpublished project report 11/83 from Biochemistry Department R & D
    Plant Protection Agricultural Division Ciba-Geigy Ltd.

    Herbold, B. (1982)  THS 2212 Triazolylalanine, Micronucleus test for
    mutagenic effect on mice.  Unpublished Report No. 11054 from
    Toxicological Institute Bayer AG.

    Herbold, B. (1983a)  THS 2212 Triazolylalanine,  Salmonella/
    Microsome test for point mutagenic effect.  Unpublished Report No.
    11388 from Toxicological Institute Bayer AG.

    Herbold, B. (1983b)  THS 2212 Triazolylalanine, Micronucleus test
    for mutagenic effect on mice.  Supplement to unpublished Bayer
    Report No. 11054: 11054 A from Toxicological Institute, Bayer AG.

    Herbold, B. (1983b)  THS 2212 Triazolylalanine, Pol A1 test on
     E.Coli during testing for effects harmful to DNA.  Unpublished
    Report No. 11390 from Toxicological Institute Bayer AG.

    Henderson, C. & Parkinson, G.R. (1981)  R152056: Acute oral toxicity
    in rats. Unpublished Report No. CTL/P/600 from Central Toxicology
    Laboratory, ICI Ltd.

    Keutz von, E. & Groening, P. (1984)  THS 2212 (tiazolylalanine),
    subchronic toxicity to dogs on oral administration (13-week feeding
    study).  Unpublished  Report No. 12562 from Institute of Toxicology
    Bayer AG.

    Maruhn, D. & Bomhard, E. (1984)  Triazolylalanine (THS 2212) study
    for subchronic toxicity to rats (three-month feeding study). 
    Unpublished Report No. 12397 from Institut of Toxicilogy Bayer AG.

    Mihail, F. Triazolylalanine (1982)  (THS 2212) Acute toxicity
    studies.  Unpublished Report No. 11229 from Institute fuer
    Toxicology Bayer AG.

    Mihail, F. & Vogel, O. (1983)  Triazolylalanine (THS 2212) subacute
    oral  toxicity study on rats.  Unpublished Report No. 11491 from
    Institute of Toxicology Bayer AG.

    Milburn, G.M. & Birtley, R.D.N. & Pate, I., Hollis, K. & Moreland,
    S. (1986) Triazole alanine: Two generation reproduction study in the
    rat.  Unpublished Report No. CTL/P/1168 (revised) from Central
    Toxicology Laboratory, ICI PLC.

    Puri, E. (1986)  Autoradiographic DNA repair test on rat
    hepatocytes.  Unpublished Report Test No. 860184 from Ciba-Geigy
    Ltd.

    Richold, M. & Allen, A., Williams, A. & Ransome, S.J. (1981) 
    CTL/C/1085, Cell transformation test for potential carcinogenicity
    of R152056.  Unpublished Report No. 394A/81153 from Huntingdon
    Research Centre.

    Strasser, F. (1986)  Micronucleus test (chinese hamster). 
    Unpublished Report Test No. 860185 from Ciba-Geigy Ltd.

    Watkins, P.A. (1982)  R152056: 3-(1,2,4-triazol-1-yl) alanine,
    Micronucleus test in CBC F1 mice-TOM/4.  Unpublished Report 156,342
    from Central Toxicological Laboratory, ICI PLC.


    See Also:
       Toxicological Abbreviations