** Evaluation in CCPR periodic review programme
TOXICOLOGY
2,4-D, 2,4-dichlorophenoxyacetic acid, was evaluated for toxicological effects by the JMPR in 1970, 1971, 1974, and 1975. The 1970 Joint Meeting did not establish an ADI because of the absence of long-term studies. The 1971 Meeting established an ADI of 0-0.3 mg/kg bw on the basis of an NOAEL of 31 mg/kg bw per day in a two-year dietary study in rats. The ADI was not changed by the 1974 Joint Meeting and was reaffirmed by the 1975 Meeting. The compound was reviewed at the present Meeting within the CCPR periodic review programme.
2,4-D was rapidly absorbed, distributed, and excreted after oral administration to mice, rats, and goats. At least 86-94% of an oral dose was absorbed from the gastrointestinal tract in rats. Once absorbed, 2,4-D was widely distributed throughout the body, but did not accumulate because of its rapid clearance from the plasma and rapid urinary excretion. 2,4-D was excreted rapidly and almost exclusively (85-94%) in urine by 48 h after treatment, primarily as unchanged 2,4-D. No metabolites have been reported apart from conjugates. Pharmacokinetic studies with salts and esters of 2,4-D have shown that the salts dissociate and the esters are rapidly hydrolysed to 2,4-D. The similarity in the fate of 2,4-D and its salts and esters explains their similar toxicities.
In humans who have ingested 2,4-D, it was quickly absorbed and excreted rapidly in the urine; about 73% of the administered dose was found in the urine after 48 h. No metabolites were detected.
After dermal applications of 2,4-D to volunteers, 5.8% of the dose was absorbed within 120 h. When the acid and its dimethylamine (DMA) salt were applied, 4.5% of the acid and 1.8% of the salt were absorbed, and of this 85% of the acid and 77% of the salt were recovered in the urine 96 h after application.
2,4-D, its amine salts and its esters are slightly toxic when administered orally or dermally, the oral LD50 values being 400-2000 mg/kg bw and the dermal LD50 value generally exceeding 2000 mg/kg bw. In rats exposed to 2,4-D at the maximum attainable concentration (up to 5.4 mg/litre) by inhalation for 4 h, no deaths were seen. While 2,4-D and its amine salts and esters do not induce dermal irritation in rabbits or dermal sensitization in guinea-pigs, they cause severe eye irritation in rabbits. WHO has classified 2,4-D as 'moderately hazardous'.
In mice fed diets that provided 2,4-D at doses of 0, 5, 15, 45, or 90 mg/kg bw per day for three months, renal lesions were observed in animals of both sexes at all doses. An NOAEL was not identified.
In mice fed diets that provided doses of 2,4-D of 0, 1, 15, 100, or 300 mg/kg bw per day for 90 days, treatment-related changes were observed in animals of both sexes at 100 mg/kg bw per day and above. These effects included decreases in glucose level in females, decreases in thyroxine activity in males, and increases in absolute and relative kidney weights in males. The NOAEL was 15 mg/kg bw per day.
In rats fed diets providing doses of 2,4-D of 0, 1, 5, 15 or 45 mg/kg bw per day for 90 days, renal lesions were observed at 5 mg/kg bw per day and above. The NOAEL was 1 mg/kg bw per day.
In rats fed diets providing doses of 2,4-D of 0, 1, 15, 100, or 300 mg/kg bw per day for 90 days, treatment-related changes were observed in animals of both sexes at 100 mg/kg bw per day and above. These effects included decreases in body-weight gain, haematological and clinical chemical alterations, changes in organ weights, and histopathological lesions in the adrenals, liver, and kidneys. The NOAEL was 15 mg/kg bw per day.
In six studies of toxicity rats fed diets containing the diethanolamine (DEA), DMA, isopropylamine (IPA), or tri-isopropanolamine (TIPA) salt or the butoxyethylhexyl (BEH) or 2-ethylhexyl (EH) ester at acid-equivalent doses of 0, 1, 15, 100, or 300 mg/kg bw per day for 13 weeks, the results demonstrated the comparable toxicity of the acid, salts and esters. The NOAEL was 15 mg acid equivalent per kg bw per day for all six compounds.
Dogs were given gelatin capsules containing 2,4-D at 0, 0.3, 1, 3, or 10 mg/kg bw per day or diets containing 2,4-D, the DMA salt, or the EH ester at acid-equivalent doses of 0, 0.5, 1, 3.8, or 7.5 mg/kg bw per day for 13 weeks. Treatment-related findings were observed in the three studies at 3 mg/kg bw per day and above. The NOAEL was 1 mg acid equivalent per kg bw per day in all three studies.
In a two-year study of toxicity and carcinogenicity, mice were fed diets providing doses of 2,4-D of 1, 15, or 45 mg/kg bw per day. Increases in absolute and/or relative kidney weights and renal lesions were observed at 15 and 45 mg/kg bw per day. There was no evidence of carcinogenicity. The NOAEL was 1 mg/kg bw per day.
In another two-year study of toxicity and carcinogenicity, mice were fed diets providing doses of 2,4-D of 0, 5, 62, or 120 mg/kg bw per day (males) or 0, 5, 150, or 300 mg/kg bw per day (females). Dose-related increases in absolute and/or relative kidney weights and renal lesions were observed in animals of both sexes at 62 mg/kg bw per day and above. There was no evidence of carcinogenicity. The NOAEL was 5 mg/kg bw per day.
In another two-year study, rats received diets providing doses of 2,4-D of 0, 1, 5, 15, or 45 mg/kg bw per day. Renal lesions were observed in animals of both sexes at 5 mg/kg bw per day and above. There was no evidence of carcinogenicity. The NOAEL was 1 mg/kg bw per day.
In a further two-year study, rats were fed diets providing doses of 2,4-D of 0, 5, 75, or 150 mg/kg bw per day. Treatment-related effects were observed in animals of both sexes at 75 mg/kg bw per day and above. The effects included decreases in body-weight gain and food consumption, increases in serum alanine and aspartate aminotransferase activities, decreased thyroxine concentrations, increases in absolute and relative thyroid weights and histopathological lesions in the eyes, kidneys, liver, lungs, and mesenteric fat. There was no evidence of carcinogenicity. The NOAEL was 75 mg/kg bw per day in males and 5 mg/kg bw per day in females.
Dogs were fed diets providing doses of 2,4-D of 0, 1, 5, or 7.5 mg/kg bw per day for 52 weeks. At 5 and 7.5 mg/kg bw per day body-weight gain was decreased, increases were observed in blood urea nitrogen, creatinine, alanine aminotransferase activity, and cholesterol, and histopathological lesions were observed in the kidneys and liver. The NOAEL was 1 mg/kg bw per day.
In a two-generation study of reproductive toxicity, rats received dietary doses of 2,4-D of 0, 5, 20, or 80 mg/kg bw per day. Reduced body weight in F1 dams and renal lesions in F0 and F1 adults were observed at 20 and 80 mg/kg bw per day. The NOAEL for parental and reproductive toxicity was 5 mg/kg bw per day.
In order to evaluate the dermal toxicity of 2,4-D and its salts and esters, rabbits received 15 dermal applications of the acid, the DEA, DMA, IPA, or TIPA salt or the BEH or EH ester at acid-equivalent doses of 0, 10, 100, or 1000 mg/kg bw per day for 6 h per day on five days per week for 21 days. No systemic toxicity was observed at any dose, and no dermal toxicity was observed with the acid, the TIPA salt, or the BEH ester. Dermal lesions were observed in rabbits treated with the DEA, DMA, or IPA salt, or the EH ester at 100 mg/kg bw per day and above. The lesions were characterized as acanthosis, hyperkeratosis, oedema, inflammation, and epidermal hyperplasia. The NOAEL was 10 mg acid equivalent per kg bw per day for dermal toxicity and 1000 mg acid equivalent per kg bw per day (the highest dose tested) for systemic toxicity.
In a study of developmental toxicity, pregnant Sprague-Dawley rats were given 2,4-D in corn oil by gavage at doses of 12, 25, 50, 75, or 88 mg/kg bw per day during days 6-15 of gestation. There was no maternal toxicity. Fetotoxicity was manifested as decreased fetal body weights at 50 mg/kg bw per day and above. The NOAELs were 88 mg/kg bw per day for maternal toxicity and 25 mg/kg bw per day for developmental toxicity.
In a further study, pregnant Fischer 344 rats received 2,4-D in corn oil by gavage at doses of 8, 25, or 75 mg/kg bw per day during days 6-15 of gestation. Decreased body-weight gain of the dams during the dosing period and increased incidences of skeletal variations (7th cervical and 14th rudimentary ribs and missing sternebrae) were observed at 75 mg/kg bw per day. The NOAEL was 25 mg/kg bw per day for both maternal and developmental toxicity.
The developmental toxicity of the DEA, DMA, IPA, and TIPA salts and the BEH and EH esters was evaluated in pregnant rats after oral administration during days 6-15 of gestation. The acid-equivalent doses were 11, 55, or 110 mg/kg bw per day for the DEA salt; 12, 50, or 100 mg/kg bw per day for the DMA salt; 9, 25, or 74 mg/kg bw per day for the IPA salt; 12, 37, or 120 mg/kg bw per day for the TIPA salt; 17, 50, or 120 mg/kg bw per day for the BEH ester; and 10, 30, or 90 mg/kg bw per day for the EH ester. The maternal and developmental toxicities of the salts and esters of 2,4-D were comparable to those of the acid. Maternal toxicity, as evidenced by reduced body-weight gain during treatment, was observed in all dams at the high dose of each compound; in addition, mortality, clinical signs, and reduced food consumption were observed in dams given 120 mg/kg bw TIPA salt per day. Although embryo- and fetotoxicity and teratogenicity were observed with the high dose of the TIPA salt, this may be attributed to maternal toxicity; none of the other compounds had such effects. No external gross or visceral anomalies (malformations or variations) were observed in any of the fetuses, but skeletal variations were observed at the high dose of each compound except the IPA salt which were similar to those seen in the fetuses of dams given the acid. The overall NOAELs were approximately 10 mg acid equivalent per kg bw per day for maternal toxicity and 50 mg acid equivalent per kg bw per day for developmental toxicity.
In a study of developmental toxicity, pregnant rabbits were given 2,4-D orally at 0, 10, 30, or 90 mg/kg bw per day during days 6-18 of gestation. Maternal toxicity, which included clinical signs, abortions, and reduced body-weight gain during and after the treatment period, was observed only at the high dose. No gross, visceral, or skeletal malformations or variations were observed in the fetuses at any dose. The NOAELs were 30 mg/kg bw per day for maternal toxicity and 90 mg/kg bw per day (the highest dose tested) for developmental toxicity,
The developmental toxicity of the DEA, DMA, IPA, and TIPA salts and the BEH and EH esters was evaluated in rabbits after oral administration during days 6-18 of gestation. The acid-equivalent doses were 10, 30, or 60 mg/kg bw per day for the DEA salt; 10, 30, or 90 mg/kg bw per day for the DMA salt; 13, 38, or 95 mg/kg bw per day for the IPA salt; and 10, 30, or 75 mg/kg bw per day for the TIPA salt and the BEH and EH esters. Unlike 2,4-D, which produced maternal toxicity only at the high dose, most of the amine salts and the esters were maternally toxic at the middle and high doses, as evidenced by mortality, clinical signs of neurotoxicity, abortions, and decreases in body-weight gain. No gross, visceral, or skeletal malformations or variations were observed in the fetuses at any dose. The overall NOAELs were approximately 10 mg acid equivalent per kg bw per day for maternal toxicity and 90 mg acid equivalent per kg bw per day (the highest dose tested) for developmental toxicity.
In summary, of the four salts tested for developmental toxicity only the TIPA salt exhibited developmental toxicity in rats and only at a maternally toxic dose; no developmental toxicity was observed in rabbits with this or the other salts. Consequently, the Meeting concluded that the developmental toxicity of the TIPA salt is of little concern.
The genotoxic potential of 2,4-D has been adequately evaluated in a range of assays in vivo and in vitro. Overall, the responses observed indicate that 2,4-D is not genotoxic, although conflicting results were obtained for mutation in Drosophila. In a more limited range of assays, the DEA, DMA, IPA, and TIPA salts and the BEH and the EH esters were not genotoxic in vivo or in vitro. The Meeting concluded that 2,4-D and its salts and esters are not genotoxic.
In rats given single doses of 2,4-D of 0, 15, 75, or 250 mg/kg bw by gavage, there were no treatment-related gross or neuropathological changes at any dose. Animals of both sexes at the highest dose exhibited inco-ordination and gait abnormalities on day 1, but the signs disappeared by day 5. The NOAEL was 75 mg/kg bw. When rats were fed diets containing 2,4-D at doses of 0, 5, 75, or 150 mg/kg bw per day for 12 months neurotoxicity, manifested as increased relative forelimb grip strength, was observed in animals of both sexes at 150 mg/kg bw per day. The NOAEL was 75 mg/kg bw per day.
Epidemiological studies have suggested an association between the development of soft-tissue sarcoma and non-Hodgkin's lymphoma and exposure to chlorophenoxy herbicides, including 2,4-D. The results of these studies are not, however, consistent; the associations found are weak, and conflicting conclusions have been reached by the investigators. Most of the studies did not provide information on exposure specifically to 2,4-D, and the risk was related to the general category of phenoxy herbicides, a group that includes 2,4,5-T which can be contaminated with dioxins. Case-control studies provide little evidence of an association between the use of 2,4-D and soft-tissue sarcomas. Although some case-control studies have shown a relationship with non-Hodgkin's lymphoma others (even the positive studies) have produced inconsistent results, raising doubt about the causality of the relationship. Cohort studies of exposed workers have not confirmed the hypothesis that 2,4-D causes either neoplasm.
The Meeting was informed of the on-going "Agricultural Health Study" initiated in North Carolina and Iowa, and of a study of pesticide applicators in Finland. The Agricultural Health Study addresses both cancer and non-cancer risks, including neurotoxicity, reproductive effects, immunological effects, kidney disease, non-malignant respiratory disease, and growth and development of children, in men and women directly exposed to pesticides and other agricultural agents.
The Meeting concluded that the toxicities of the salts and esters of 2,4-D were comparable to that of the acid. An ADI was therefore established for the sum of 2,4-D and its salts and esters, expressed as 2,4-D. An ADI of 0-0.01 mg/kg bw was established on the basis of the NOAEL of 1 mg/kg bw per day in the one-year study of toxicity in dogs and the two-year study in rats, using a safety factor of 100.
A toxicological monograph was prepared, summarizing the data received since the previous evaluation and including summaries from the previous monograph and monograph addenda.
TOXICOLOGICAL EVALUATION
Levels that cause no toxic effect
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Mouse: |
15 mg/kg bw per day (13-week study of toxicity) |
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5 mg/kg bw per day (two-year study of toxicity and carcinogenicity) |
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Rat: |
1 mg/kg bw per day (two-year study of toxicity and carcinogenicity) |
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5 mg/kg bw per day (two-generation study of reproductive toxicity) |
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10 mg acid-equivalent/kg bw per day (maternal toxicity in a series of studies of developmental toxicity with salts and esters) |
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15 mg acid-equivalent/kg bw per day (series of 13-week studies of toxicity with salts and esters) |
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25 mg/kg bw per day (maternal and developmental toxicity in a study of developmental toxicity) |
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Rabbit: |
10 mg acid-equivalent/kg bw per day (maternal toxicity in a series of studies of developmental toxicity with salts and esters) |
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30 mg/kg bw per day (maternal toxicity in a study of developmental toxicity) |
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90 mg acid-equivalent/kg bw per day (highest dose tested in studies of developmental toxicity with the acid and its salts and esters) |
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Dog: |
1 mg/kg bw per day (13-week and one-year studies of toxicity) |
Estimate of acceptable daily intake for humans
0-0.01 mg/kg bw (sum of 2,4-D and its salts and esters expressed as 2,4-D)
Studies that would provide information useful for the continued evaluation of the compound
1. Follow-up of the Agricultural Health Study in North Carolina and Iowa in the USA.
2. Follow-up of the study of pesticide applicators in Finland.
Toxicological criteria for setting guidance values for dietary and non-dietary exposure to 2,4-dichlorophenoxyacetic acid and its amine salts and esters.
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EXPOSURE |
RELEVANT ROUTE, STUDY TYPE, SPECIES |
RESULTS, REMARKS |
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Short-term (1-7 days)
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Oral toxicity, rat (acid, salts and esters) |
LD50 = 400-2000 mg/kg bw |
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Dermal toxicity, rabbit (acid, salts and esters) |
LD50 > 2000 mg/kg bw |
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Inhalation toxicity, rat (acid, salts and esters) |
LC50 > 0.84-5.4 mg/litre |
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Dermal irritation, rabbit (acid, salts and esters) |
Not irritating |
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Ocular irritation, rabbit (acid, salts and esters) |
Severely irritating |
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Dermal sensitization, guinea-pig (acid, salts and esters) |
Not sensitizing |
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Oral, single dose, neurotoxicity, rat (acid) |
NOAEL = 75 mg/kg bw |
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Medium-term (1-26 weeks)
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Dietary, three months, toxicity, mouse |
NOAEL = 15 mg/kg bw per day, renal toxicity |
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Dietary, three months, toxicity, rat |
NOAEL = 1 mg/kg bw per day, renal lesions |
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Dietary, three months, toxicity, rat (salts and esters) |
NOAEL = 15 mg/kg acid-equivalent/kg bw per day, renal toxicity |
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Dietary or capsule, three months, toxicity, dog |
NOAEL = 1 mg acid-equivalent/kg bw per day, reduced body-weight gain and other systemic toxicity |
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Dermal, 21 days, repeated dose, rabbit (acid, salts and esters) |
NOAEL = 1000 mg acid-equivalent/kg bw per day, highest dose tested |
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Dietary, two generations, reproductive toxicity, rat |
NOAEL = 5 mg/kg bw per day, reduced body weights in F1 dams and renal lesions in F0 and F1 adults |
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Oral, gavage, developmental toxicity, rat |
NOAEL = 25 mg/kg bw per day, maternal and developmental toxicity |
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Oral, gavage, developmental toxicity, rat (salts and esters) |
NOAEL = 10 mg acid-equivalent/kg bw per day for maternal toxicity and 50 mg acid-equivalent/kg bw per day for developmental toxicity |
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Oral, gavage, developmental toxicity, rabbit |
NOAEL = 30 mg/kg bw per day, maternal toxicity; >90 mg/kg bw per day, developmental toxicity |
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Oral, gavage, developmental toxicity, rabbit (salts and esters) |
NOAEL = 10 mg acid-equivalent/kg bw per day for maternal toxicity; 90 mg acid-equivalent/kg bw per day (highest dose tested) for developmental toxicity |
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Long-term (> one year) |
Dietary, two years, toxicity and carcinogenicity, mouse |
NOAEL = 5 mg/kg bw per day, renal effects; no evidence of carcinogenicity |
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Dietary, two years, toxicity and carcinogenicity, rat |
NOAEL = 1 mg/kg bw per day, renal lesions; no evidence of carcinogenicity |
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Dietary, one year, toxicity, dog |
NOAEL = 1 mg/kg bw per day, changes in serum chemistry and lesions in kidneys and liver |
