2,4-D (Pesticides)

Summary

Abstract

Food Safety Commission of Japan (FSCJ) conducted a risk assessment of 2,4-D¹⁾ (CAS No. 94-75-7), a phenoxy herbicide, based on results from various studies. Major adverse effects of 2,4-D observed were suppressed body weight, renal tubular degeneration, hypertrophy of hepatocytes, reduced weight of testis and retinal degeneration in rats. No adverse effects were detected in carcinogenicity, reproductive toxicity, teratogenicity and genotoxicity relevant to human health. The relevant substance to the residue definition for dietary risk assessment was identified as 2,4-D and metabolite C²⁾ in agricultural products and 2,4-D (parent compound only) in livestock products. The lowest no-observed-effect level (NOAEL) obtained from all the studies was 0.99 mg/kg bw/day in a two-year combined chronic toxicity/carcinogenicity study (the 1^st study in Table 1) in rats. FSCJ specified an acceptable daily intake (ADI) of 0.0099 mg/kg bw/day by applying a safety factor of 100 to the NOAEL. The lowest NOAEL for adverse effects likely to be elicited by a single oral administration of 2,4-D was 15 mg/kg bw/day obtained from the acute neurotoxicity study in rats. Consequently, FSCJ specified an acute reference dose (ARfD) of 0.15 mg/kg bw by applying a safety factor of 100 to the NOAEL.

Conclusion in Brief

Table 1. Levels relevant to toxicological evaluation of 2,4-D

Species	Study	Dose (mg/kg bw/day)	NOAEL (mg/kg bw/day) and critical endpoints1)
Rat	90-day subacute toxicity study (the 1st study)	0, 1, 5, 15, 45	M/F: 1 M/F: Renal lesion
	90-day subacute toxicity study (the 2nd study)	0, 1, 15, 100, 300 M: 0, 0.93, 14.0, 93.9, 278 F: 0, 0.96, 14.4, 96.2, 293	M: 14.0 F: 14.4 M/F: Supressed body weight, etc.
	90-day subacute toxicity study (the 3rd study)	0, 15, 60, 100, 150	M/F: 15 M/F: Renal tubular lesion, etc.
	Two-year combined chronic toxicity/carcinogenicity study (the 1st study)	0, 1, 5, 15, 45 M: 0, 0.99, 4.95, 14.8, 44.5 F: 0, 0.99, 4.96, 14.9, 44.7	M/F: 0.99 M/F: Brown pigmentation of renal tubule, etc. (Not carcinogenic)
	Two-year combined chronic toxicity/carcinogenicity study (the 2nd study)	0, 5, 75, 150 M: 0, 4.77, 73.2, 145 F: 0, 4.89, 73.1, 144	M: 4.77 F: 4.89 M/F: Increase in ALP, etc. (Not carcinogenic)
	One-year chronic neurotoxicity study	0, 5, 75, 150 M: 0, 4.77, 73.2, 145 F: 0, 4.89, 73.1, 144	M: 4.77 F: 4.89 M/F: Supressed body weight (No chronic neurotoxicity)
	Two-generation reproductive toxicity study	0, 5, 20, 80 PM: 0, 5.0, 20.1, 79.8 PF: 0, 5.0, 19.9, 78.5 F1M: 0, 5.0, 19.2 F1F: 0, 5.0, 20.2	Parent PM: 5.0 PF: 19.9 F1M: 5.0 F1F: 20.2 Offspring PM: 5.0 PF: 5.0 F1M: 5.0 F1F: 5.0 Parent M: Localized tubular degeneration in renal medulla F: Supressed body weight, etc. Offspring: Lower body weight (No adverse effect on fertility)
	Extended one-generation reproductive toxicity study	0, 100, 300, 600(F)/800(M) ppm	P generation M: 16.6 (300 ppm) F: 40.2 (600 ppm) M: Increased absolute/relative renal weight, etc. F: No toxicity F1 generation M: 20.9 (300 ppm) F: 23.3 (300 ppm) M/F: Supressed body weight (During lactation period) F1 generation F(Adult): Proximal tubular degeneration in kidney (No adverse effects on fertility, developmental neurotoxicity, and developmental immunotoxicity)
	Developmental toxicity study (the 1st study)	0, 12.5, 25, 50, 75, 88	Maternal: 88 Fetus: 25 Maternal: No toxicity Fetus: Lower body weight, increased skeletal variations (lumbar rib, wavy rib), etc. (Not teratogenic)
	Developmental toxicity study (the 2nd study)	0, 8, 25, 75	Maternal: 25 Fetus: 25 Maternal: Supressed body weight Fetus: Increased skeletal variations (Not teratogenic)
Mouse	90-day subacute toxicity study (the 1st study)	0, 5, 15, 45, 90	- M/F: Renal lesion
	90-day subacute toxicity study (the 2nd study)	0, 1.0, 15.0, 100, 300 M: 0, 0.98, 14.7, 98.2, 293 F: 0, 0.99, 14.8, 98.9, 296	M: 14.7 F: 14.8 M: Decrease in T4F: Decrease in Glu, etc.
	Two-year carcinogenicity study (the 1st study)	0, 1, 15, 45 M: 0, 0.98, 14.9, 44.8 F: 0, 1.00, 14.9, 44.8	M: 0.98 F: 14.9 M: Homogenous change of renal tublar epitherlium F: Increased absolute/relative renal weight (Not carcinogenic)
	Two-year carcinogenicity study (the 2nd study)	F: 0, 5, 150, 300 F: 0, 5.01, 150, 310	F: 5.01 F: Increased absolute/relative renal weight, etc. (Not carcinogenic)
	Two-year carcinogenicity study (the 3rd study)	M: 0, 5, 62.5, 125 M: 0, 5.0, 61.9, 129	M: 5.0 M: Proximal tubular degeneration/regeneration in kidney, etc. (Not carcinogenic)
Rabbit	Developmental toxicity study	0, 10, 30, 90	Maternal: 30 Fetus: 90 Maternal: Supressed body weight, etc. Fetus: No toxicity (Not teratogenic)
Dog	90-day subacute toxicity study (the 1st study)	0, 0.3, 1, 3, 10	M: 1 F: 3 M/F: Change of renal convoluted proximal tubule, etc.
	90-day subacute toxicity study (the 2nd study)	0, 0.5, 1, 3.75, 7.5 M: 0, 0.5, 1.0, 3.8, 7.8 F: 0, 0.5, 1.0, 3.8, 7.7	M/F: 1.0 M/F: Supressed body weight, etc.
	One-year chronic toxicity study	0, 1, 5, 10/7.5* M: 0, 1.0, 5.2, 8.2 F: 0, 1.0, 5.0, 7.9 *: In the 8th week after administration, the dose was cut to 7.5 mg/kg bw/day	M/F: 1.0 M/F: Supressed body weight, etc.
ADI (cRfD)			NOAEL: 0.99 SF: 100 ADI: 0.0099
The critical study for setting ADI			Two-year combined chronic toxicity/carcinogenicity study in rats (the 1st study)

M, Male; F, Female; M/F, both sexes; PM, Male in P (Parent) generation; PF, Female in P generation;

F₁M, Male in F₁ generation; F₁F, Female in F₁ generation; -, NOAEL could not be specified; ADI, Acceptable daily intake;

cRfD, Chronic reference dose; SF, Safety factor; UF, Uncertainty factor; NOAEL, No-observed-adverse-effect level;

NOEL, No-observed-effect level; -, NOAEL could not be specified

¹⁾ The adverse effect observed at the lowest-observed-adverse-effect level (LOAEL)

Food Safety Commission of Japan (FSCJ) conducted a risk assessment of 2,4-D¹⁾ (CAS No.94-75-7), a phenoxy herbicide, based on results from various studies.

The data used in the assessment include the fate in animals (rats, mice, goats, chickens and humans), fate in plants (paddy rice, wheat and others), residues in crops, acute neurotoxicity (rats), subacute toxicity (rats, mice and dogs), chronic toxicity (dogs), combined chronic toxicity/carcinogenicity (rats), carcinogenicity (mice), two-generation reproductive toxicity (rats), developmental toxicity (rats and rabbits), genotoxicity, and also on their mechanisms.

Major adverse effects of 2,4-D observed were suppressed body weight, renal tubular degeneration, hypertrophy of hepatocytes, reduced weight of testis and retinal degeneration in rats. No adverse effects were detected in carcinogenicity, reproductive toxicity, teratogenicity and genotoxicity relevant to human health.

Based on the results from various studies, the relevant substance to the residue definition for dietary risk assessment was identified as 2,4-D and metabolite C²⁾ in agricultural products and 2,4-D (parent compound only) in livestock products.

The lowest no-observed-effect level (NOAEL) obtained from all the studies was 0.99 mg/kg bw/day in a two-year combined chronic toxicity/carcinogenicity study (the 1^st study in Table 1) in rats. FSCJ specified an acceptable daily intake (ADI) of 0.0099 mg/kg bw/day by applying a safety factor of 100 to the NOAEL.

The lowest NOAEL for adverse effects likely to be elicited by a single oral administration of 2,4-D was 15 mg/kg bw/day obtained from the acute neurotoxicity study in rats. Consequently, FSCJ specified an acute reference dose (ARfD) of 0.15 mg/kg bw by applying a safety factor of 100 to the NOAEL.

Table 2. Potential adverse effects of a single oral administration of 2,4-D

Species	Study	Dose (mg/kg bw/day)	NOAEL (mg/kg bw/day) and critical endpoints1)
Rat	Acute toxicity study	0, 197(M), 250, 318, 403, 512, 650, 826	M/F: - M/F: Hypoactivity, abnormal gait (1 hour after the treatment)
	Acute neurotoxicity study	0, 15, 75, 250	M: 75 F: 15 M: Motor coordination ataxia, abnormal gait, decreased locomotor activity (5–6 hours after the treatment) F: Abnormal gait (5–6 hours after the treatment)
Mouse	General pharmacology study (General state)	0, 30, 100, 300	30 Abnormal/atatic gait (0.5–5 hours after the treatment)
	Acute toxicity study	0, 200, 264, 348, 460, 670, 801	M/F: - M/F: Hypoactivity, abnormal gait (1 hour after the treatment)
Rabbit	General pharmacology study (General state)	M:0, 30, 100, 300	M: 100 Abnormal gait, decreased locomotor activity (3–4 hours after the treatment)
	General pharmacology study (Body temperature)	M:0, 30, 100, 300	M: 100 Elevated body temperature (2–3 hours after the treatment)
ARfD			NOAEL: 15 SF: 100 ARfD: 0.15
The critical study for setting ARfD			Acute neurotoxicity study in rats

ARfD, Acute reference dose; SF, Safety factor; NOAEL, No-observed-adverse-effect level; -, NOAEL or LOAEL could not be specified

¹⁾ The adverse effect observed at LOAEL

Table 3. Levels relevant to toxicological evaluation of 2,4-D

Species	Study	Test substances	Dose (mg/kg bw/day)	NOAEL(mg/kg bw/day) and critical endpoints1)
Rat	90-day subacute toxicity study	DEA salt	0, 1.5, 27, 150, 440 [0, 1, 18, 100, 300]	27 [18] Elevated mortality rate,etc.
		DMA salt	0, 1.2, 18, 120, 360 [0, 1, 15, 100, 300]	18 [15] Supressed body weight, etc.
		IPA salt	0, 1, 19, 130, 380 [0, 1, 15, 100, 300]	19 [15] Supressed body weight, etc.
		TIPA salt	0, 2, 28, 190, 560 [0, 1, 15, 100, 300]	28 [15] Histopathological change in kidney, etc.
		BEH ester	0, 1.5, 22, 140, 440 [0, 1, 15, 100, 300]	22 [15] Supressed body weight, etc.
		EH ester	0, 1.5, 23, 150, 450 [0, 1, 15, 100, 300]	23 [15] Supressed body weight, etc.
	Developmental toxicity study	DEA salt	0, 15, 75, 150 [0, 11, 55, 110]	Maternal: 15 [11] Fetus: 15 [11] Maternal: Supressed body weight Fetus: Increased skeletal variations (Not teratogenic)
		DMA salt	[0, 12, 50, 100]	Maternal: [12] Fetus: [50] Maternal: Supressed body weight Fetus: Lower body weight, etc. (Not teratogenic)
		IPA salt	0, 22, 65, 190 [0, 9, 25, 74]	Maternal: 65 [25] Fetus: 190 [74] Maternal: Supressed body weight, etc. Fetus: No toxicity (Not teratogenic)
		TIPA salt	0, 32, 100, 320 [0, 12, 37, 120]	Maternal: 100 [37] Fetus: 32 [12] Maternal: Death, etc. Fetus: Increase in skeletal variations
		BEH ester	0, 25, 75, 180 [0, 17, 50, 120]	Maternal: 75 [50] Fetus: 75 [50] Maternal: Supressed body weight Fetus: Delayed ossification (Not teratogenic)
		EH ester	[0, 10, 30, 90]	Maternal: [30] Fetus: [30] Maternal: Supressed body weight, etc. Fetus: Delayed ossification (Not teratogenic)
Rabbit	Developmental toxicity study	DEA salt	[0, 15, 30, 60]	Maternal: [15] Fetus: [30] Maternal: Supressed body weight, etc. Fetus: Increased skeletal variations (Not teratogenic)
		DMA salt	[0, 10, 30, 90]	Maternal: [30] Fetus: [90] Maternal: Reduced motor activity, etc. Fetus: No toxicity (Not teratogenic)
		IPA salt	0, 13, 38, 95 [0, 10, 30, 75]	Maternal: 13 [10] Fetus: 95 [75] Maternal: Death, etc. Fetus: No toxicity (Not teratogenic)
		TIPA salt	0, 19, 56, 140 [0, 10, 30, 75]	Maternal: 19 [10] Fetus: 140 [75] Maternal: Death, etc. Fetus: No toxicity (Not teratogenic)
		BEH ester	0, 15, 45, 110 [0, 10, 30, 75]	Maternal: 15 [10] Fetus: 110 [75] Maternal: Death, etc. Fetus: No toxicity (Not teratogenic)
		EH ester	[0, 10, 30, 75]	Maternal: [30] Fetus: [75] Maternal: Death, etc. Fetus: No toxicity (Not teratogenic)
Dog	90-day subacute toxicity study	DMA salt	[0, 1, 3.8, 7.5]	M/F: [1] M/F: Supressed body weight, etc.
		EH ester	[0, 1, 3.8, 7.5]	M/F: [1] M/F: Supressed body weight, etc.

The values given in parentheses [ ] are the corresponding values in acid

DEA, dethanolamine; DMA, dimethylamine; IPA, Isopropylamine; TIPA, Triisopropanolamine; BEH, butoxyethyl; EH, ethylhexyl

¹⁾ The adverse effect observed at LOAEL

Table 4. Potential adverse effects of a single oral administration of 2,4-D

Test substances	Species	Study	Dose (mg/kg bw/day)	NOAEL (mg/kg bw/day) and critical endpoints1)
2,4-D ethyl	Rat	Acute toxicity study	250, 350, 500	M/F: - M/F: Hypoactivity, etc. (1 hour after the treatment)
	Mouse	Acute toxicity study	0, 125, 250, 290(M), 335(M), 375, 500	M/F: - M/F: Hypoactivity, fece/soil (1 hour after the treatment)
Na salt	Rat	Acute toxicity study	250, 500, 1 000, 2 000	M/F: - M/F: Ataxia, hypoactivity, etc. (1 hour after the treatment)
	Mouse	Acute toxicity study	125, 250, 375, 500, 1 000	M: - F: 125 M/F: Hypoactivity, etc. (1 hour after the treatment)
DMA salt	Rat	Acute toxicity study	500, 710, 1 000	M/F: - M/F: Ataxia, hypoactivity, etc. (1 hour after the treatment)
	Mouse	Acute toxicity study	250, 500, 1 000, 2 000, 4 000	M/F: - M/F: Hypoactivity, ataxia, etc. (1 hour after the treatment)
IPA salt	Rat	Acute toxicity study	500, 750, 1 000, 5 000	M/F: - M/F: Stiffness in handling (1 day after the treatment)
DEA salt	Rat	Developmental toxicity study	0, 15, 75, 150 [0, 11, 55, 110]	Maternal: 15 [11] Maternal: Suppressed body weight (6–9 days after GD)
TIPA salt	Rat	Developmental toxicity study	0, 32, 100, 320 [0, 12, 37, 120]	Maternal: 100 [37] Embryo/fetus: 32 [12] Maternal: Death, rigid limbs, salivation, suppressed body weight (During the treatment) Embryo/fetus: Increased skeletal variation (wavy rib)
BEH ester	Rat	Developmental toxicity study	0, 25, 75, 180 [0, 17, 50, 120]	Maternal: 75 [50] Maternal: Suppressed body weight (6–9 days after GD)
EH ester	Rat	Developmental toxicity study	[0, 10, 30, 90]	Maternal: [30] Maternal: Suppressed body weight (6–9 days after GD)

The values given in parentheses [ ] are the corresponding values in acid

GD, gestation day; Na salt, sodium salt; other abbreviation are described in Table3.

¹⁾ The adverse effect observed at LOAEL

References

• 1.  2,4-dichlorophenoxy acetic acid
• 2.  2,4-dichlorophenol