TOXICOLOGY
Triforine, a fungicide, was evaluated toxicologically by the 1977 JMPR, when no ADI was allocated, and again in 1978, when more toxicological data were made available and an ADI of 0-0.02 mg/kg bw was established. Subsequently, more data have been produced, which were reviewed at the present Meeting within the CCPR periodic review programme.
A battery of tests for acute toxicity with technical-grade triforine showed that it is slightly hazardous by both the oral and dermal routes, with respective LD50 values of >5000 and >2000 mg/kg bw. The LC50 in rats exposed by inhalation was >5.1 mg/l. Triforine was not irritating or sensitizing to the skin of rodents and was minimally irritating to the eyes of rabbits. WHO has classified triforine as unlikely to present an acute hazard in normal use.
Dietary administration of technical-grade triforine for 4 or 13 weeks showed that the haematopoietic system is a target, as indicated by mild haemolytic anaemia with associated secondary effects in the spleen and liver. Similar results were found in two-year studies of toxicity in mice, rats, and dogs, in which the haematological changes were accompanied by increased weights of the spleen and liver. In addition, in a 105-week dietary study in mice, changes in the large intestine characterized by thickening, enlargement, inflammation, and ulceration were observed; in a two-year dietary study in dogs, increased erythropoiesis and increased haemosiderin deposition were found in the liver and bone marrow. In a four-week dietary study in rats at 0, 500, 2500, or 12,500 ppm, an NOAEL was not identified. In a four-week study in mice at 0, 200, 1000, or 5000 ppm, the NOAEL was 1000 ppm, equal to 200 mg/kg bw per day. The NOAEL in a 13-week study in rats at 0, 100, or 500 ppm was 500 ppm, equivalent to 25 mg/kg bw per day. In a six-month study in rats fed diets giving 0, 25, 120, 620, or 3100 ppm, the NOAEL was 120 ppm, equivalent to 6 mg/kg bw per day. Two-year dietary studies in rats (0, 200, 2000, or 20,000 ppm), mice (0, 70, 700, or 7000 ppm), and dogs (0, 10, 40, 100, or 1000 ppm) showed NOAELs of 200 ppm, equal to 10 mg/kg bw per day, 70 ppm, equal to 11 mg/kg bw per day, and 100 ppm, equal to 2.4 mg/kg bw per day, respectively.
The major toxic effect observed in the experiments summarized above was anaemia. The effect was mild, occurring in all or many of the exposed animals (depending upon the dose); the effects were reversible in rats and dogs and were characterized by haemosiderin deposition in several organs, the absence of evidence of bone-marrow depression, entry of increased numbers of immature cells into the peripheral circulation, and the absence of effects on organs of the immune system. In dogs, there was actually an increase in erythropoiesis. Oxidative haemolysis in animals with normally functioning erythrocytes would appear to be the most likely mechanism and one that would cease as soon as exposure ceases. Also, there was no evidence of methaemoglobin formation or of any increase in Heinz body-containing cells or deformed erythrocytes in the circulation.
No genotoxic activity was observed in an adequate battery of tests for mutagenicity and clastogenicity in vitro and in vivo. The Meeting concluded that triforine is not genotoxic.
The results of the studies critical to the derivation of an ADI are shown below; the list does not include an 81-week study in mice treated orally, which was considered inadequate for evaluation since histopathological examination was limited to lesions that were judged at autopsy to be neoplastic.
No carcinogenic effect was observed in rats given dietary concentrations of 0, 25, 125, 625, or 3120 ppm in one study and 0, 200, 2000, or 20,000 ppm in another. Triforine increased the incidence of pulmonary tumours in female mice given 7000 ppm; the NOAEL for carcinogenicity was 700 ppm, equal to 160 mg/kg bw per day. The Meeting concluded that the murine response involves an unidentified non-genotoxic mechanism and that the carcinogenic activity seen in mice was unlikely to be indicative of a human carcinogenic risk at the expected levels of exposure to triforine.
Triforine at dietary concentrations of 0, 500, 3000, or 20,000 ppm did not affect reproductive performance in rats over the course of a two-generation study, with the NOAEL being the highest dose tested, 20,000 ppm, equal to 1500 mg/kg bw per day. The NOAEL for parental toxicity and for the growth and development of the offspring was 500 ppm, equal to 40 mg/kg bw per day, on the basis of decreases in food consumption, body-weight gain, and Fi pup weight and increases in relative spleen weight at the next highest dose of 3000 ppm.
In a study of developmental toxicity in rabbits given oral doses of 0, 5, 25, or 125 mg/kg bw per day the maternal NOAEL was 5 mg/kg bw per day, on the basis of decreased food consumption and body weight at 25 mg/kg bw per day; the NOAEL for developmental toxicity was 125 mg/kg bw per day. In a later study of rabbits given oral doses of 0, 6, 30, or 150 mg/kg bw per day the NOAEL for maternal toxicity was 30 mg/kg bw per day, on the basis of decreased food consumption and body-weight gain at 150 mg/kg bw per day; the NOAEL for developmental toxicity was 150 mg/kg bw per day. In two subsequent studies in which triforine was given orally to rabbits at doses of 0, 250, 500, or 1000 mg/kg bw per day and 0 or 1000 mg/kg bw per day, maternal and embryotoxicity (as shown by reduced fetal weight) occurred at 1000 mg/kg bw per day. Decreased fetal weights and delayed ossification were observed in a study of developmental toxicity in rabbits at the maternally toxic dose of 1000 mg/kg bw per day. Thus, developmental toxicity in rabbits occurred only at doses that were also maternally toxic. A study of developmental toxicity in rats given triforine orally at doses of 0, 200, 500, or 1000 mg/kg bw per day did not show adverse effects in either dams or fetuses at doses up to 1000 mg/kg bw per day. The Meeting concluded that triforine has no specific developmental or reproductive toxicity.
The monitoring of workers in three manufacturing plants did not reveal any health effects that might be associated with exposure to triforine.
An ADI of 0-0.02 mg/kg bw was established on the basis of the NOAEL of 2.4 mg/kg bw per day in the two-year study of toxicity in dogs, with a safety factor of 100.
A toxicological monograph was prepared.
TOXICOLOGICAL EVALUATION
Levels that cause no toxic effect
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Mouse: |
70 ppm, equal to 11 mg/kg bw per day (105-week study of toxicity and carcinogenicity) |
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Rat: |
200 ppm, equal to 10 mg/kg bw per day (two-year study of toxicity and carcinogenicity) |
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1000 mg/kg bw per day (study of developmental toxicity) 500 ppm, equal to 40 mg/kg bw per day (parental and fetal toxicity in a study of reproductive toxicity) |
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Rabbit: |
5 mg/kg bw per day (maternal toxicity in a study of developmental toxicity) |
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Dog: |
100 ppm, equal to 2.4 mg/kg bw per day (two-year study of toxicity) |
Estimate of acceptable daily intake for humans
0-0.02 mg/kg bw
Studies that would provide information useful for the continued evaluation of the compound
Further observations in humans.
Toxicological criteria for estimation of guidance values for dietary and non-dietary exposure to triforine
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Human exposure |
Relevant route, study type, species |
Results, remarks |
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Short-term |
Oral, single dose, rat |
LD50 > 5000 mg/kg bw |
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Dermal, single dose, rat |
LD50 > 2000 mg/kg bw |
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Inhalation (4h), rat |
LC50 5.1 mg/l |
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Dermal, irritation, rabbit |
Not irritating |
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Ocular, irritation, rabbit |
Minimally irritating |
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Dermal, irritation, rat |
Not irritating |
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Dermal, sensitization, guinea-pig |
Non-sensitizing |
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Medium-term |
Dermal, 3 weeks, rat |
NOAEL = 1100 mg/kg bw per day (highest dose tested) |
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Oral, 13 weeks, dog |
NOAEL = 3.6 mg/kg bw per day: haemosiderin deposition |
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NOAEL = 1500 mg/kg bw per day: reproductive toxicity |
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Oral, two-generation, reproductive toxicity, rat |
NOAEL = 49 mg/kg bw per day: parental and offspring toxicity |
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NOAEL = 5 mg/kg bw per day: maternal toxicity |
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Oral, developmental toxicity, rabbit |
NOAEL = 125 mg/kg bw per day: fetal and developmental toxicity |
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Long-term |
Oral, 2 years, dog |
NOAEL = 2.4 mg/kg bw per day: haematological changes; haemosiderin deposition; haematopoiesis |
