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
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in rats. Unpublished Report No. CTL/P/600 from Central Toxicology
Laboratory, ICI Ltd.
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subchronic toxicity to dogs on oral administration (13-week feeding
study). Unpublished Report No. 12562 from Institute of Toxicology
Bayer AG.
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for subchronic toxicity to rats (three-month feeding study).
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Mihail, F. Triazolylalanine (1982) (THS 2212) Acute toxicity
studies. Unpublished Report No. 11229 from Institute fuer
Toxicology Bayer AG.
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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.