Weight-of-the-evidence evaluation of 2,4-D potential for interactions with the estrogen, androgen and thyroid pathways and steroidogenesis

Figures & data

Table 1. Modified Klimisch criteria.

Score	Description	Comments
1	Reliable without restriction	Generally guideline and GLP compliant studies using validated methodology; study is transparently reported and report internally consistent.
2	Reliable with restriction	Generally studies from the published literature, often not GLP compliant but sufficient to accept the data and “scientifically acceptable,” or GLP studies that do not follow a specific guideline or cover limited components of a guideline.
3	Not reliable	Studies generated by a method that is not acceptable, insufficiently documented, or not convincing using expert judgment.
4	Not assignable	Studies reported only in abstracts or as part of book chapters with insufficient detail to evaluate study quality.

Table 2. Examples of endpoints in mammalian toxicological studies indicating potential interactions with the estrogen or androgen pathways.

Mechanism	Potential effects in males	Potential effects in females
Estrogenicity	•Delayed preputial separation (marked, or in absence of significant body weight decreases)•↓ sperm counts•↓ fertility•Presence of hypospadias/epispadias•↓ reproductive organ weight (particularly prostate, seminal vesicles)•Histopathological findings of the testes (e.g. leydig cell proliferation and/or tumors)•↑ incidence, growth of pituitary tumors	•Accelerated vaginal opening•Persistent estrus•↑ time to mating•↓ gestation duration•↑ uterine weights (particularly indicative in immature or ovariectomized animals)•Histopathological findings of the female reproductive organs (e.g. vaginal cornification, uterine hypertrophy and hyperplasia)•↑ mammary tumors•↑ incidence, growth of pituitary tumors•Ovarian, uterine and vaginal tumors in female offspring
Anti-estrogenicity	•↓Height of epithelium in testicular tubules•↑testicular weight (short term)•Testicular atrophy (long term)•Infertility and testicular atrophy (moderate term)	•Delayed vaginal opening (VO)•Delayed start of estrous cycling•Irregular or absent estrous cyclicity•↓ fertility•↓ corpora lutea, implantations•↓ female reproductive organ weights•↓ mammary tumor incidence
Androgenicity	•↑ or ↓ male reproductive organ weights•↓ sperm counts•Testicular atrophy	•↑ Anogenital distance•Accelerated vaginal opening•↓ fertility•Altered differential follicle count•Histopathological findings of the female reproductive organs (e.g. vaginal agenesis)•Induced male sex accessory tissues
Anti-androgenicity	•Delayed preputial separation•↓ anogenital distance•Ectopic testes (pre-natal exposure)•Hypospadias/epispadias (pre-natal exposure)•↓ fertility•↓ reproductive organ weight (particularly prostate, seminal vesicles)•Retained nipples/areolas in male pups•Histopathological findings of the reproductive organs (e.g. epididymal agenesis, testicular tumors)	•Altered pup sex ratios between external and internal sexing
Aromatase inhibition	•↑ time to mating•↓ male mounting behavior•↓ body weight (chronic)•↑ testis weight (chronic)	•↑ Body weight•↓ or ↑uterine weight•↑ ovary size•Polycystic ovaries•Stromal hyperplasia in ovary (chronic)•Hyalinization in ovary (chronic)•↑ ureter and bladder infection•↓ mammary tumor incidence (S-D rats)
Reduced steroid biosynthesis	•Similar to anti-androgenicity•Possible increased serum cholesterol levels•↑ incidence Leydig cell tumors	•Similar to anti-estrogenicity/aromatase inhibition

↑ Increase

↓ Decrease

Table 3. Results from EDSP in vitro studies of 2,4-D (data from individual study reports; published in Coady et al. 2014).

Study	Assay and test system	Purity 2,4-D	Concentration range tested	Result	Klimischscore	Rationale for Klimisch score
LeBaron et al. 2011a	Estrogen receptor (ER) binding assay (US EPA 2009a) with uterine cytosol from Sprague-Dawley rats	98.5%	10^−11 –10^−4 M	Negative, non-binder to ER	1	Guideline compliant study; dose range based on mammalian TK data to be below the TSRC
LeBaron & Kan 2011	Estrogen transcriptional activation assay (US EPA 2009b) with human ERα-HeLa-9903 cells	98.5%	10^−11–10^−4 M	Negative; no ER transactivation	1	Guideline compliant study; dose range based on mammalian TK data to be below the TSRC
LeBaron et al. 2011b	Androgen receptor (AR) binding assay (US EPA 2009c) with ventral prostate cytosol from Sprague-Dawley rats	98.5%	10^−11–10^−4 M	Negative, non-binder to AR	1	Guideline compliant study; dose range based on mammalian TK data to be below the TSRC
Coady & Sosinski 2011	Aromatase assay (US EPA 2009d) with human recombinant aromatase and titrated androstenedione	98.5%	10^−10–10^−4 M	Negative, non-inhibitor of aromatase activity	1	Guideline compliant study; dose range based on mammalian TK data to be below the TSRC
LeBaron et al. 2011c	Steroidogenesis assay (US EPA 2009e) with H295R cells	98.5%	10^−10–10^−4 M	Small, significant ↑ estradiol at 10^−4 M; magnitude of change less than criterion used to define a positive response in validation studies (Hecker et al. 2008); considered negative	1	Guideline compliant study

Table 4. Published in vitro assays relating to potential endocrine activity for 2,4-D.

Study	Assay and Test system	Purity 2,4-D	Concentration range tested	Result	Klimisch score	Rationale for Klimisch score
Blair et al. 2000	Competitive rat ER binding assay in uterine tissue homogenates from ovariectomized Sprague-Dawley rats	99%	“2 high concentrations spanning 3 log concentrations”	Negative	3	Strengths: Well documented; method close to validated Guideline design; Weaknesses: 2,4-D specific data not provided; Only tested at 2 concentrations which were not reported
Fang et al. 2003	Binding to recombinant rat AR	Purity not specified; Supplier (Supelco) produces analytical standards but also mixtures	4.28 × 10^−9–4.28 × 10^−4 M	Negative	3	Adequate study but lack of information on purity of 2,4-D
Jung et al. 2004	ER antagonist activity in yeast-based reporter system	“Highest grade commercially available”	Not specified	Negative	3	Weaknesses: concentrations tested not reported; possibly commercial formulation tested and purity unspecified. Not relevant/reliable: Yeast two-hybrid detection system
Jungbauer & Beck 2002	ER antagonist activity in yeast two-hybrid system	Not specified	Not specified	Negative	3	Weaknesses: concentrations tested not reported; purity unspecified. Not relevant/reliable: Yeast two-hybrid detection system
Kim et al. 2005	AR+ 22Rv1 cell proliferation with 2,4-D and its metabolite DCP	>98%	10^−13–10^−5 M	Negative with 2,4-D or DCP alone, but positive with 2,4-D or DCP with dihydrotestos-terone (DHT) added	3	Weakness: Lack of solvent only control; data for DHT alone appears to have been generated for a single subset of tests only (set A in graphs); no rationale for 10 nM concentration of DHT added (possibly supra physiological); measured cell proliferation, which may be stimulated by ER-independent factors, including epidermal growth factor (based on information in the ATCC website (www.atcc.org) for this particular strain of cells. See also Sramkoski et al. 1999)
	Transactivation reporter assays with AR+ 22Rv1 and AR- PC3 cells	>98%	10^−12–10^−6 M	Negative with 2,4-D or DCP alone, but positive with 2,4-D or DCP with DHT	3	Weakness: Lack of solvent only control; no rationale for concentration of DHT added (possibly supra physiological); only one concentration of 2,4-D tested in AR-PC3 cells.
	AR expression (mRNA and total protein levels) in 22Rv1 cells	>98%	10^−7 M 2,4-D; 10^−10 M DCP	Negative	3	Weakness: Single concentration of 2,4-D tested; Lack of solvent only control
	AR binding assay in monkey kidney COS cells	>98%	5 × 10^−9 M to 5 × 10^−7 M	Positive (50% inhibition with both 2,4-D and DCP)	3	Weakness: Lack of solvent only control; did not use reagent to separate unbound from bound ligand; evaluated limited number of test compound concentrations; typical dose-response curves for inhibition of receptor binding were not observed, even for the positive control, suggesting a potential solvent (vehicle) effect; biological plausibility of findings poor given lack of concordant findings in in vivo studies
	Nuclear translocation in PC3 cells	>98%	10^−9 M	Negative with 2,4-D or DCP alone, but positive with 2,4-D or DCP with DHT	3	Weaknesses: Lack of solvent only control; only single concentration 2,4-D tested; no rationale for concentration of DHT added
Kojima et al. 2004	Estrogenic and anti-estrogenic activity in CHO cells transiently transfected with human ERβ	≥95%	10^−8–10^−5 M	Negative	2	Strengths: Method very close to validated guideline; well documented; positive control used; weakness: specific response data for 2,4-D not provided.
	Androgenic and anti-androgenic activity in CHO cells transiently transfected with human AR	≥95%	10^−8–10^−5 M	Negative	2	Strengths: Method very close to validated guideline; well documented; positive control used; weakness: specific response data for 2,4-D not provided
	Dual activity as ER agonists and AR antagonists	≥95%	10^−8–10^−5 M	Negative	2	Well documented but method not formally validated. Weakness: specific response data for 2,4-D not provided
Lee et al. 2006	Estrogenic activity in yeast one-hybrid and two-hybrid systems	NR	10^−7–10^−4 M	Negative in one-hybrid system; Positive in two hybrid system	3	Weakness: test material uncharacterized. Not relevant: Yeast one-hybrid and two-hybrid detection system
Lemaire et al. 2006	Estrogenic and anti-estrogenic activity in HeLa cells stably transfected with human ERα or human ER-β	>95%	10^−6 M	Negative	3	Weakness: Only one concentration tested; otherwise adequate quality
Lin & Garry 2000	MCF-7 proliferation	2,4-D (reagent grade) 2,4-D isooctyl ester (reagent grade)	0.1–10 μg/mL	Negative	2	Weakness: MCF-7 cell line may provide variable responses and result may not be specific for estrogenicity (Odum et al. 1998)
	MCF-7 proliferation	2,4-D LV4 (commercial grade 66.24% 2,4-D isooctyl ester)); 2,4-D amine (commercial grade) 46.5% 2,4-D dimethylamine salt	0.1–10 μg/mL	Positive	3	Weaknesses: Results in commercial grade materials appear confounded due to formulation excipients as reagent grade materials did not show effects; MCF-7 proliferation may be a non-estrogen specific response and cell line may provide markedly variable responses (Odum et al. 1998)
Nishihara et al. 2000	Estrogenic activity using the yeast two-hybrid system	“Highest grade commercially available”	NR	Negative	3	Weaknesses: formulation and test material purity not characterized concentrations tested not reported; Not relevant: Yeast two-hybrid detection system
Orton et al. 2009	Xenopus laevis ovulation and ovarian steroidogenesis (production of progesterone, testosterone and estradiol)	>97%	6.25 × 10^−6 and 62.5 × 10^−6 M	Negative	3	Weaknesses: Methods not specific regarding stage of oocytes used (ranges given) or how these were distributed to the test wells; methods not specific regarding number of replicates or criteria for accepting or rejecting replicate findings; non-validated assay; only two concentrations tested
	ER and AR agonist and antagonist activity in yeast	>97%	4.9 × 10^−7–1 × 10^−3 M	Negative	3	Weaknesses: Not relevant: Yeast assay; otherwise well reported and conducted
Petit et al. 1997	VTG in mRNA expression in primary hepatocyte cultures derived from male rainbow trout	NR	10^−4 M	Equivocal	3	Weaknesses: Only tested at a single concentration; test material uncharacterized. VTG mRNA expression only 8% increase compared to control (cannot determine if ineffective or weakly responsive).
	β-galactosidase activity in yeast cells stably transfected with rainbow trout ER	NR	10^−8–10^−4 M	Negative	3	Weakness: test material uncharacterized: Not relevant Yeast-based system
	ER competitive binding assay in yeast cells stably transfected with rainbow trout ER	NR	>10^−4 M	Negative	3	Weaknesses: only tested at a single concentration; test material uncharacterized: Not relevant: Yeast-based system
Soto et al. 1995	Estrogenic activity by measuring MCF-7 proliferation	NR	NR	Negative	3	Weaknesses: test material uncharacterized and concentrations tested not reported; MCF-7 cell line may provide markedly variable responses (Odum et al. 1998)
Sun et al. 2012	Estrogenic and anti-estrogenic activity in Vero cells	≥99%	0.003–3.0 mg/L	Negative	2	Well-conducted and reported assay. Rationale for dose selection questionable; high dose exceeds potential human exposure although it falls within the linear TK range
	Androgenic and anti-androgenic activity in Vero cells	≥99%	0.003–3.0 mg/L	Negative for androgenicity or anti-androgenicity; at 3.0 mg/L increased the effects of testosterone (in anti-androgenic assay); other concentrations negative	2	Well-conducted and reported assay. Rationale for dose selection questionable; effect seen only at 3.0 mg/L which exceeds predicted concentrations for potential human exposure although it falls within the linear TK range; biological relevance of the finding questionable as the test was designed to measure anti-androgenic activity rather than potentiation or androgenic activity
	Agonist and antagonist activity to TR in Vero cells	≥99%	0.003–3.0 mg/L	Negative	3	Well-conducted and reported assay; however In vitro thyroid assay model not validated
Vonier et al. 1996	Competitive binding to ER and PR extracted from the oviduct tissues of adult female alligators	≥99%	NR	Negative	3	Well-conducted study with positive control; Weaknesses: concentrations tested not reported.

Table 5. Summary: fish short-term reproduction assay with 2,4-D (Marino et al. 2010).

Nominal concentration of 2,4-D (mg a.e./L)	0.4	4.0	40	100
Mean measured concentration of 2,4-D (mg a.e./L)	0.245	3.14	34.0	96.5	Interpretation
Systemic toxicity
Survival	N	N	N	N	No effect. One high dose death, not likely to be exposure-related.
Clinical signs	N	N	N	N	No effect. No abnormal behavior or coloration was observed.
Body weight M	N	N	N	N	No effect.
Body weight F	N	N	N	N	No effect.
Feed consumption	N	N	N	N	No observations noted.
Body length M	N	N	N	N	No effect.
Body length F	N	N	N	N	No effect.
Fertilization success	N	N	N	N	No effect; no difference between groups in numbers of fertilized eggs.
Fecundity	N	N	N	↓	May be associated with stress; high concentration exceeds larval maximum acceptable toxicant concentration (MATC) in an early life stage study with P. promelas
Estrogen pathway – potentially indicative endpoints
Nuptial tubercles M	N	N	N	N	No effect in M; present in males in equivalent numbers between control and dosed groups.
Gonadal somatic index (GSI) M	N	N	N	N	No effect.
GSI F	N	N	N	N	No effect.
VTG M	N	N	N	N	No increase VTG in males.
VTG F	N	N	N	N	No increase or decrease VTG in females.
Gonadal histopathology	N	N	N	N	No effect in M or F. The pattern of predominant germ cell distribution (staging) in testes and ovaries was comparable between the controls and all dosed groups.
Androgen pathway – potentially indicative endpoints
Nuptial tubercles M	N	N	N	N	No effect in M; present in males in equivalent numbers between control and dosed groups
Nuptial tubercles F	N	N	N	N	Not present in females normally; none observed.
GSI M	N	N	N	N	No effect.
GSI F	N	N	N	N	No effect.
VTG M	N	N	N	N	No alteration in M VTG.
VTG F	N	N	N	N	No alteration in F VTG.
Gonadal histopathology	N	N	N	N	No effect in M or F. The pattern of predominant germ cell distribution (staging) in ovaries and testes was comparable between the controls and all dosed groups

N: no effect; M: male; F: female.

↓ Statistically significant decrease (p ≤ 0.05) compared to control.

Table 6. Results in one-generation quail reproductive toxicity (Mitchell et al. 2000).

Dose (ppm)	Control	160	400	1000
Number of replicates	16	16	16	14
Total eggs laid	746	788	768	638
Eggs laid/maximum laid (%)	75	80	77	74
Viable embryos/eggs set (%)	91	76	95	91
Live 3-week embryos/viable embryos (%)	99	98	99	99
Hatchlings/live 3-week embryos (%)	95	91	92	94
14-Day survivors/hatchlings (%)	94	96	95	93
Hatchlings/eggs set (%)	85	68*	86	86
14-Day survivors/eggs set (%)	80	65*	82	80
Hatchlings/maximum set (%)	64	55	67	60
14-Day survivors/maximum set (%)	60	52	64	56
Mean (± SD) eggshell thickness (mm)	0.226 ± 0.016	0.228 ± 0.010	0.239*± 0.015	0.232 ± 0.232
Eggs cracked/eggs laid (%)	1	2	1	3
Mean (± SD) body weight hatchlings (g)	6 ± 0	6 ± 1	6 ± 0	6 ± 0
Mean (± SD) body weight 14-day survivors (g)	26 ± 2	26 ± 3	26 ± 2	26 ± 2

* p ≤ 0.05

Table 7. 2,4-D ecotoxicological studies possibly relevant to assessment of potential endocrine interactions.

Reference	Assay and test system	Purity 2,4-D	Concentration range tested	Result	Klimisch score	Klimisch score rationale
Amphibians
Coady et al. 2010; (Coady et al. 2013)	Amphibian metamorphosis assay (US EPA 2009f, OECD 231). African clawed frog tadpoles exposed in continuous flow-through system for 21 days.	98.6%	0.4–100 mg a.e./L	No exposure-related effects	1	Guideline and GLP compliant study; tested to limit concentration
Aronzon et al. 2011	South American toad exposed to 2,4-D DBE or formulated product either through embryogenesis or in pulsed exposures	99% 2,4-D di-butyl ether	1–15 mg/L 2,4-D DBE for continuous exposure	“Teratogenic” to toads	3	No controls were included in this study; results cannot be interpreted
Heggstrom 2009	Wood frog tadpoles in microcosms exposed to 2,4-D dimethylamine	99% 2,4-D dimethylamine	0.1–100 μg/L	Negative for survival, deformities, effects on time to metamorphic climax; total length decreased in mid dose group only; no effect on plasma corticosterone levels	3	High non-exposure related mortality
Heggstrom 2009	Wood frog tadpoles in field ponds (agricultural and forested) exposed to 2,4-D dimethylamine	99% 2,4-D dimethylamine	10 μg/L	Tadpoles from the agricultural pond in the absence of 2,4-D dimethylamine and tadpoles from the agricultural pond applied with 10 μg/L 2,4-D dimethylamine gave similar results in the measured endpoints	3	Control tadpoles from the two ponds showed some marked differences; only one concentration level evaluated
Stebbins-Boaz et al. 2004	Xenopus oocytes exposed to 2,4-D sodium salt	NR	0.6–2.43 g/L (2.5–10 mM)	Germinal vesicle breakdown inhibited	3	Mechanistic study; extremely high doses; purity not reported
LaChapelle et al. 2007	Xenopus oocytes exposed to 2,4-D	NR	2.43 g/L (10 mM)	Irreversible dysfunction of meiotic signaling	3	Mechanistic study; only one extremely high dose evaluated; purity not reported
Morgan et al. 1996	Frog embryo teratogenic assay in Xenopus	Commercialformulation (99%)	180–270 mg/L	Teratogenic only at high concentrations	2	Adequate study but irrelevant to evaluation of potential endocrine effects; formulation tested
Vardia et al. 1984	Indian tadpoles exposed to 2,4-D in a static renewal exposure system	NR	7.5–11 mg/L	96-h LC50: 8.05 mg/L	4	Experiment not described in detail; lacks information on potential endocrine effects
Fish
Marino et al. 2010; (Coady et al. 2013)	Fish short term reproduction assay (OPPTS 890.1350, OECD 229, US EPA 2009g). Adult fat head minnows were exposed via continuous flow-through for 21 days.	98.6%	0.4–100 mg a.e./L	No interaction with endocrine pathways; decreased fecundity at highest concentration tested attributed to systemic toxicity	1	Guideline and GLP compliant study; tested to limit concentration
Holcombe et al. 1995	Acute larval survival and growth tests in Japanese Medaka exposed to 2,4-D acid	99% 2,4-D acid	567–8970 mg/L	96-h LC50: 2780 mg/L 2,4-D Acid	2	Adequate study but no specific endocrine endpoints evaluated
Holcombe et al. 1995	Chronic larval survival and growth tests in Japanese Medaka exposed to 2,4-D acid	99% 2,4-D acid	27.2–425 mg/L	Survival and growth reduced at 56.5 mg/L	2	Adequate study but no specific endocrine endpoints evaluated
Holcombe et al. 1995	Chronic larval survival and growth tests in Japanese Medaka exposed to 2,4-D acid	99% 2,4-D acid	2.37–60.2 mg/L	Survival and growth reduced at 60.2 mg/L	2	Adequate study but no specific endocrine endpoints evaluated
Koç & Akbulut 2012	Acute toxicity to ovary of zebrafish	NR	0.1–1 mg/L	Ovarian histopathological changes and atretic follicles following 5 days exposure	3	Source and purity not defined; report says 2,4-D “available as a formulation” so possible a formulation was tested; methods not well defined; potential sectioning artifacts in slides
Padilla et al. 2012	Zebrafish developmental screening assay in zebrafish embryos exposed to 2,4-D	>90%	0.001–80 μM	Negative in concentration range study; weak positive in single high concentration study	2	Reported findings too non-specific to be useful for WoE
Rehwold et al. 1977	Acute toxicity tests with striped bass, the banded killifish, pumpkinseed, white perch, American eel, carp, and guppy exposed to 2,4-D	NR	NR	96-h LC50 ranged from 26.7 mg/L (banded killifish) to 300.6 mg/L (American eel)	4	Experimental detail not provided; fish field collected
Rehwold et al. 1977	Chronic toxicity tests with striped bass, the banded killifish, pumpkinseed, white perch, American eel, carp, and guppy exposed to 2,4-D	NR	0.1 mg/L	No observable physiological symptoms	4	Experimental detail not provided; fish field collected
Rehwold et al. 1977	Breeding effects on guppy chronically exposed to 2,4-D	NR	0.1 mg/L	No observable physiological symptoms	4	Experimental detail not provided
Xie et al. 2005	Juvenile rainbow trout exposed to 2,4-D dimethylamine in static test system	NR	0.00164–1.64 mg/L	VTG levels significantly greater at 0.164 and 1.64 mg/L	3	High variability; small sample size, sex of fish not determined, purity of test material not reported
Avian
Mitchell et al. 2000	Avian single generation reproductive study in Northern Bobwhite quail. US EPA OPP 71-4; OECD Guideline 206; Subjects exposed via diet for 21 weeks.	96.9%	160, 400, 1000 ppm (No mg/kg/d dose calculated)	No treatment-related effects including on potentially endocrine related parameters	1	Guideline compliant
Somers et al. 1974	Fertilized hen’s eggs exposed to 2,4-D by spraying	NR	2.8–3.4 kg/ha and 44.8 kg/ha	No mortalities in ovo, at pip, or at hatch; no weight gain effects	3	Non-conventional method of application; purity not reported; no positive control; evidence of poor absorption into the egg content
Somers et al. 1978a	Hens and cockerels exposed to 2,4-D by spraying	Formulation purity NR	111.2 kg/ha	No effect on chicken reproduction in various endpoints measured	3	Non-conventional method of application; formulation; single concentration and purity not reported; no positive control; evidence of poor absorption into the egg content
Somers et al. 1978b	Hens eggs exposed to 2,4-D by spraying	Formulation purity NR	111.2 kg/ha	No effect on hatching success, weight gain, or mortality;	3	Non-conventional method of application; formulation; single concentration; purity not reported; no positive control; evidence of poor absorption into the egg content
Reptiles
Crain et al. 1997	American alligator eggs exposed topically to 2,4-D prior to sexual differentiation	97.6%	0.14–14 ppm	No effects on sex reversal, plasma steroid concentrations, gonadal aromatase activity	2	Non-conventional study design; validated with estradiol positive control
Crain et al. 1999	American alligator eggs exposed topically to 2,4-D prior to sexual differentiation	97.6%	0.14–14 ppm	No effects on hepatic aromatase activity or testicular histopathology	2	Non-conventional study design; validated with estradiol positive control
Spiteri et al. 1999	American alligator eggs exposed topically to 2,4-D prior to sexual differentiation, at two temperatures	97.6%	0.14–14 ppm	No effects on sex reversal, hepatic aromatase activity or gonadal histopathology	2	Non-conventional study design; validated with estradiol positive control
Mixed species
Relyea 2005	Algal microcosms and other microcosms containing 25 species exposed to 2,4-D	Formulation (44.5%)	0.117 mL/m^2	No effect on community diversity, survival, or biomass	3	Formulation tested and single concentration; No specific endocrine endpoints evaluated

NR: not reported.

Table 8. F1-extended one generation dietary toxicity study summary table (Marty et al. 2010).

Dose (ppm)	100	300	600F/800
Targeted mg/kg/day doses	5	15	30F/40M	Interpretation
Systemic toxicity
Mortality	N	N	N	No exposure-related mortality in any group
Live birth index	N	N	N	No effect
Mean number of pups	N	N	N	No effect
Clinical signs	N	N	N	No effect
Body weight	N	N	↓	Decreased P1 F bw during lactation; decreased F1 pup weight
Feed and water consumption	N	N	↓P1 F	Decreased P1 F feed consumption during lactation
Liver weight	N	N	N	No effect
Kidney weight	N	↑F1 F	↑P1 M and F; F1 F	Exposure related; very slight and considered non-adverse at 300 ppm
Kidney histopathology	N	F1 M	P M; F1 M and F	Exposure related; very slight and considered non-adverse at 300 ppm
Estrogen pathway-potentially mediated endpoints
Sexual maturation (vaginal opening)	N	N	N	No effect F1
Ano-genital distance	N	N	N	No effect F1
Nipple retention (male)	N	N	N	No effect F1
Estrous cycling	N	N	N	No effect P or F1
Female mating	N	N	N	No effect P
Mean duration of gestation	N	N	N	No effect P
Gestation index	N	N	N	No effect P
Pup sex ratio	N	N	N	No effect F1
Ovaries (paired) weights	N	N	N	No effect P or F1
Differential ovarian follicle count	N	N	N	No effect F1
Uterus weight w oviducts and cervix	N	N	↑(N)	Non-statistically significant; increase within HCD; stage of estrus uncontrolled at necropsy; estrous cyclicity not changed; conclusion no effect P or F1
Uterine histopathology	N	N	N	No effect P or F1
Ovarian histopathology	N	N	N	No effect P or F1
Vaginal histopathology	N	N	N	No effect P or F1
Androgen pathway-potentially mediated endpoints
Sexual maturation (preputial separation)	N	N	↓(N)	Slight delay F1 M attributed to decreased body weight before and post weaning (artifact from group assignments)
Sperm parameters	N	N	N	No effect P or F1
Male fertility	N	N	N	No effect P
Epididymis weight	N	N	N	No effect P or F1
Testicular weight	N	N	N P ↓(N) P F1 pups; N F1 adults	No effect P or F1 adults Decreased testis weights in F1 PND 21 pups at high dose strongly correlated with decreased body weight; did not persist in adults.
Prostate weight	N	N	↓(N) P1; N F1	P1 prostate weights not statistically different from control; decreases not considered exposure-related because the absolute and relative prostate weights in the control group were atypical, exceeding the laboratory HCD ranges; not seen in F1
Seminal vesicles with coagulating glands weight	N	N	↓(N) P1; N F1	P1 males decreased seminal vesicle weight not considered exposure-related because the absolute and relative seminal vesicle weight in the control group were atypical, exceeding the laboratory HCD ranges; not seen in F1
Testicular histopathology	N	N	N	No effect P or F1
Prostate histopathology	N	N	N	No effect P or F1
Epididymides histopathology	N	N	N	No effect P or F1
Seminal vesicle histopathology	N	N	N	No effect P or F1
Coagulation gland histopathology	N	N	N	No effect P or F1
Thyroid pathway-potentially mediated endpoints
T3	N	N	↓GD17 satellite F N Other life stages	No adverse findings; response in dams considered adaptive
T4	N	N	↓GD17 satellite F N Other life stages	No adverse findings; response in dams considered adaptive
TSH	N	N	↑GD17 satellite F N Other life stages	No adverse findings; response in dams considered adaptive
Thyroid weight	N	N	N	No adverse findings
Thyroid histopathology	N	N	Colloid depletion GD 17 F N Other life stages	Adaptive response in GD 17 satellite female dams consistent with decreased thyroxine at high dose; not considered adverse
DNT effects (Functional observational battery, motor activity, startle, histopathology)	N	N	N	No effects (assessed in F1 cohort)
Brain morphometry or myelination	N	N	N	No effects (assessed in F1 cohort)
Other potentially endocrine-mediated endpoints
Pituitary weights	N	N	↓(N) F1 Set 3 M	Not considered exposure related; no findings in any other set or correlating effects, including the absence of histopathological changes
Pituitary histopathology	N	N	N	No effect P or F1
Adrenal (paired) weights	N	N	N	No effect P or F1
Adrenal histopathology	N	N	N	No effect P or F1

NA: not applicable; N: no effect; (N): finding not considered related to potential endocrine pathway interaction, see interpretation; M:male; F: female; P1: parental generation; F1: first generation (P offspring); GD: gestation day.

↑Increase relative to control

↓Decrease relative to control

Table 9. 2,4-D: two-generation reproductive toxicity study summary table (Rodwell & Brown 1985).

Dose (mg/kg/day)	Vehicle control	5	20	80*	Interpretation
Systemic toxicity
Mortality	N	N	N	Y	Pup mortality increased in the F1b litters (overdosed during mating, gestation and lactation).
Mean number of pups	NA	N	N	N	No effect
Live birth index	NA	N	N	Y	Increased stillbirths in F1b litters (overdosed during mating, gestation and lactation).
Clinical signs	NA	N	N	N	No effect
Body weight	NA	N	N	↓	Decreased body weights in F1a and F1b litters at ≥80 mg/kg/day
Feed consumption	NA	N	N	↓	Decreased in dams producing F1b litters (overdosed during mating, gestation and lactation).
Liver weight	NA	N	N	N	No dose related effect
Kidney weight	NA	N	N	N	No dose-related effect
Estrogen pathway –potentially indicative endpoints
Estrous cycling (extrapolated)	NA	N	N	N	No effect (extrapolated from time to mating)
Mating index	NA	N	N	N	No effect
Fertility index	NA	N	N	↓	Decreased (not statistically significantly) in mating to produce F1b litters at ≥80 mg/kg/day (overdosed during mating, gestation and lactation).
Mean duration of gestation	NA	N	N	↑	Increased 1 day in F1b litters at ≥80 mg/kg/day (may be hormone mediated but not due to estrogen or androgen interactions; may be secondary to systemic toxicity at exaggerated high dose due to mis-dosing)
Gestation index	NA	N	N	N	No effect
Pup sex ratio	NA	N	N	(N)	Increased number of male pups at 80 mg/kg/day in F1a litters; no effect on F1b litters dosed at a higher level; therefore, considered incidental
Uterine histopathology	NA	N	N	N	No effect (weanlings)
Ovarian histopathology	NA	N	N	N	No effect
Androgen pathway –potentially indicative endpoints
Fertility index	NA	N	N	↓	Decreased (not statistically significantly) in mating to produce F1b litters at ≥80 mg/kg/day
Pup sex ratio	NA	N	N	(N)	Increased number of male pups at 80 mg/kg/day in F1a litters; no effect on F1b litters dosed at higher level; therefore, considered incidental
Testes weight	NA	N	N	N	No effect
Testes histopathology	NA	N	N	N	No effect
Prostate histopathology	NA	N	N	N	No effect (weanlings)
Epididymides histopathology	NA	N	N	N	No effect
Seminal vesicle histopathology	NA	N	N	N	No effect (weanlings)

* Mis-dosing during F1b gestation probably to a dose approximating 100 mg/kg/day.

NA: not applicable; N: no effect; (N): finding but not likely exposure related.

↑Increase

↓Decrease

Table 10. Regulatory 2,4-D developmental, subchronic and chronic mammalian studies and evaluation of study quality.

Study	Assay and test system	Purity 2,4-D	Concentration range or doses tested (mg/kg/day)	Result	Klimisch score	Weaknesses
Developmental studies
Rodwell 1983	OPP 83-3 Guideline developmental toxicity evaluation in F344 rats. Dosing by oral gavage from GD 6-15.	97.5%	8, 25, 75	Maternal: 75 mg/kg/day produced slight toxicity Developmental: No adverse effects observed	1	None noted; however note current Guideline requires a longer exposure period; high dose exceeds TSRC
Hoberman 1990	OPP 83-3 Guideline developmental toxicity evaluation in New Zealand white rabbits. Dosing by oral gavage from GD 6-18.	97.5%	10, 30, 90	Maternal: High dose produced two abortions Developmental: No toxicity observed.	1	None noted; however note current Guideline requires a longer exposure period and more animals per dose level (20 vs 12); high dose exceeds TSRC in rabbits
Subchronic and chronic toxicity studies
Schulze 1991a	OPP 82-1 guideline subchronic study with F344 rats. Dosing via the diet for 13 weeks.	96.1%	1, 15, 100, 300	Systemic toxicity: 300 mg/kg/day: marked (excessive) systemic toxicity, ↓ body weight (28%), renal toxicity and effects on eyes, hearts, and lungs. 100 mg/kg/day: renal toxicity Endocrine endpoints: 300 mg/kg/day (F): ↓ T3, T4, ↑thyroid/parathyroid weights; thyroid follicular cell hypertrophy ↓ pituitary weights. 100 mg/kg/day (F): ↓ T3 and T4 300 mg/kg/day (M): ↓ T4, ↑ thyroid/parathyroid weights, ↓testes weight and atrophy; ↓ pituitary weights 100 mg/kg/day (M): ↓T4; ↑ thyroid/parathyroid weights No histopathological changes: pituitary, epididymides, ovary, uterus, and vagina.	1	High dose excessive; exceeded MTD; doses ≥100 mg/kg/day exceed TSRC.
Gorzinski et al. 1981a	OPP 82-1 non-Guideline study with F344 rats. Dosing via the diet for 13 weeks.	97.3%	15, 60, 100, 150	150 mg/kg/day: marked Systemic toxicity in both genders. ≥100 mg/kg/day (F): ↓T4 . ≥100 mg/kg/day (M): ↓testes weight	2	Not all guideline endpoints evaluated. Doses ≥60 mg/kg/day exceed TSRC.
Gorzinski et al. 1981b	Non-guideline subchronic study with F344 rats. Dosing via the diet for 13 weeks.	100%	15, 60, 100, 150	Systemic toxicity in both genders at ≥100 mg/kg/day. ≥ 60 mg/kg/day (F): ↓T4. (M): No effects on endocrine relevant endpoints including testes weights	2	Doses ≥60 mg/kg/day exceed TSRC. Very limited endpoints evaluated. Study done primarily to evaluate testes weight change in prior study
Jeffries et al. 1995	Guideline chronic toxicity/ oncogenicity study with F344 rats. Dosing via the diet for 12 or 24 months.	96.45%	5, 75, 150	150 mg/kg/day (F): ↓body weight, renal and other systemic toxicity; ↓secretory material in thyroidal epithelial cells, ↓ ovary weight, ↓ incidence of benign adenomas in pituitary, and ↓ mammary gland hyperplasia. ≥ 75 mg/kg/day: renal tox, ↓T4 levels, ↑ thyroid weights, ↑focal cystic dilatation. 150 mg/kg/day (M): ↑ thyroid weights, ↓testes weights. ≥75 mg/kg/day (M); ↓ T4	1	Doses ≥75 mg/kg/day exceed TSRC; guideline study; thyroid tissue accountability issue in females at terminal sacrifice.
Schulze 1991b	Guideline B6C3F1 mice subchronic toxicity study. Dosing via the diet for 13 weeks.	96.1%	1, 15, 100, 300	≥100 mg/kg/day: ↓ T4 levels at 100 and 300 mg/kg/day, no effect on testes or ovary weights. No histopath. findings: pituitary, adrenal, thyroid, parathyroid, testes, epididymides, ovary, uterus.	1	Doses ≥100 mg/kg/day likely exceed TSRC
Stott 1995a	Oncogenicity study B6C3F1 mice (females only). Dosing via diet for 24 months; 12-month interim sacrifice. Guideline when considered in conjunction with Stott 1995b study	96.4%	5, 150, 300	≥150 mg/kg/day: systemic toxicity (renal). No histopath. findings: pituitary, adrenal, thyroid, ovary, uterus, vagina, mammary.	1	Doses ≥150 mg/kg/day likely exceed TSRC
Stott 1995b	Oncogenicity study with B6C3F1 mice (males only). Dosing via the diet for12 or 24 months. Guideline when considered in conjunction with Stott 1995a study.	96.4%	5, 62.5, 125	No significant exposure-related effects. No histopath. findings: testes, epididymides, prostate, seminal vesicles, adrenal, thyroid, pituitary	1	125 mg/kg/day likely exceeds TSRC
Schulze 1990	Guideline subchronic study with dogs (82-1). Dosing via capsule for 13 weeks.	96.1%	0.3, 1, 3, 10	10 mg/kg/day: Severe systemic toxicity including lethality; ↓ testes weight; testicular atrophy. No effects thyroid: T3, T4, weight and hIstopath; no effect ovary weight; no histopath findings: ovary, uterus, epididymides, pituitary.	1	Guideline study; 10 mg/kg/day dose exceeds MTD; ≥ 3 mg/kg/day exceeds dog TSRC; immaturity of dogs at study initiation limits ability to detect any exposure-related testes lesions due to very high background incidence in published HCD
Dalgard 1993a	Guideline subchronic study with dogs. Dosing via diet for >13 weeks.	96.7%	0.5, 1, 3.75, 10 (lowered after 8 weeks to 7.5)	↓ body weight at mid and high dose. ↑ thyroid weight not considered exposure-related. No clearly dose-related effects on testes or prostate histopath; no effects thyroid histopath.	1	Guideline study; doses ≥3.75 mg/kg/day exceed dog TSRC; immaturity of dogs at study initiation limits ability to detect any exposure-related testes and prostate lesions due to very high background incidence in HCD
Dalgard 1993b	Guideline chronic study with dogs. Dosing via diet for 1 year.	96.7%	1, 5, 10 (lowered after 8 weeks to 7.5)	Body weight ↓ in high dose group. Renal pathology and altered BUN and creatinine levels at ≥5 mg/kg/day. No effects on thyroid or testes histopath. Absence of testes findings support that findings in subchronic dog studies were not exposure related or at most may reflect delayed development.	1	Guideline study; doses ≥5 mg/kg/day exceed dog TSRC.

Table 11. Published literature references for mammalian studies and evaluation of study quality.

Study	Assay and test system	Purity 2,4-D	Concentration range or doses tested (mg/kg/day)	Result	Klimisch score	Weaknesses
Developmental studies
Bage et al. 1973	Pregnant NMRI mice dosed subcutaneously from GD 6–14	Mixture of formulated products, purity of 2,4-D not defined	50 and 110 (2:1 2,4-D and 2,4,5-T)	Increases in fetal resorptions, cleft palate. Decreased fetal body weights; no malformations characteristic of endocrine modulators	3	Weaknesses: 2,4-D not tested separately (although 2,4,5-T was); purity of 2,4-D not defined; maternal toxicity not evaluated; subcutaneous dosing not relevant for risk assessment; Strength; formulation excipients were added to the control group
Cavieres et al. 2002	ND4 mice dosed orally by gavage from GD 6–15	Formulation (Purity unspecified)	0.01–100	Decreases in litter sizes and purported decreases in implantations	3	Weaknesses: Formulation; purity of 2,4-D unspecified; data discrepancies; lack of appropriate concurrent controls; lack of biological plausibility for reported findings
Charles et al. 2001	OPP 83-3 guideline study. Sprague-Dawley rats dosed orally by gavage with 2,4-D 2-butoxyethyl ester (BEE) from GD 6–15	2,4-D BEE 95.6%	25, 75, 185 ai (17, 51, 125 ae)	Maternal toxicity at 125 mg/kg/day ae; ↑ skeletal variations at 125 mg/kg/day ae; No dose related visceral malformations	2 (for publication)	Overall high quality study; minor limitations in amount of detail reported due to multiple studies and compounds, e.g. nature of visceral malformations not specified; ae doses ≥51 mg/kg/day exceed TSRC.
	OPP 83-3 guideline study. Sprague-Dawley rats dosed orally by gavage with 2,4-D 2-ethylhexyl ester (EHE) from GD 6–15	2,4-D EHE 95.0%	14.1, 45.2, 135.7 ai (10, 30, 90 ae)	Maternal toxicity (slight) at 30 mg/kg/day ae; ↓fetal-body weights (90 mg/kg/day ae); ↑ skeletal variations at 30 mg/kg/day ae; No visceral malformations; wavy ribs at 10 and 30 mg/kg/day ae not dose related.	2 (for publication)	Overall high quality study; minor limitations in amount of detail reported due to multiple studies and compounds; e.g. skeletal variations at 30 mg/kg/day ae not shown in Table; Table identifies wavy ribs as malformations; typically classified as deviations (Kimmel et al. 2014); doses ≥90 mg/kg/day ae exceed TSRC.
	OPP 83-3 guideline study. Sprague-Dawley rats dosed orally by gavage with 2,4-D isopropylamine (IPE) from GD 6–15	2,4-D IPE 97.1%	12.3, 36.9, 123 ai (10, 30, 100 ae)	Maternal toxicity ≥30 mg/kg/day ae; ↓ fetal-body weights and ↑skeletal var. (100 mg/kg/day ae); No dose related visceral malformations	2 (for publication)	Overall high quality study; minor limitations in amount of detail reported due to multiple studies and compounds;
	OPP 83-3 guideline study. Sprague-Dawley rats dosed orally by gavage with 2,4-D diethanolamine salt (DEA) from GD 6–15	2,4-D DEA Aqueous based manufacturing concentrate (73.1%)	15, 75, 150 ai (10.2, 50.8, 101.6 ae)	Maternal toxicity ≥50.8 mg/kg/day ae; ↓ fetal-body weights;↑fetal skeletal variations (101.6 mg/kg/day ae); no exposure related visceral malformations	2 (for publication)	Overall high quality study; minor limitations in amount of detail reported due to multiple studies and compounds;
	OPP 83-3 guideline study. Sprague-Dawley rats dosed orally by gavage with 2,4-D dimethyl-amine (DMA) from GD 6–15	2,4-D DMA Aqueous based manufacturing concentrate (66.2%)	15, 60.2, 120.4 ai (12.5, 50, 100 ae)	Maternal toxicity ≥50 mg/kg/day ae; ↓ fetal-body weights and ↑ skeletal changes 100 mg/kg/day ae); No exposure related visceral malformations	2 (for publication)	Overall high quality study; minor limitations in amount of detail reported due to multiple studies and compounds;
	OPP 83-3 guideline study. Sprague-Dawley rats dosed orally by gavage with 2,4-D isopropylamine (IPA) from GD 6–15	2,4-D IPA Aqueous based manufacturing concentrate (50.2%)	22, 65, 190 ai (17, 51, 150 ae)	Slight maternal toxicity (150 mg/kg/day ae); No exposure related visceral malformations	2 (for publication)	Overall high quality study; minor limitations in amount of detail reported due to multiple studies and compounds
	OPP 83-3 guideline study. Sprague-Dawley rats dosed orally by gavage with 2,4-D triisopropylamine (TIPA) from GD 6–15	2,4-D TIPA Aqueous based manufacturing concentrate (72.2%)	32.5, 100, 325 ai (17, 51, 175 ae)	Maternal toxicity (severe) 175 mg/kg/day ae; ↓ fetal-body weights, ↑skeletal changes and malformations 175 mg/kg/day ae. No exposure related visceral malformations	2 (for publication)	Overall high quality study; minor limitations in amount of detail reported due to multiple studies and compounds
	OPP 83-3 guideline study. New Zealand white rabbits dosed orally by gavage with 2,4-D BEE from GD 7–19	2,4-D BEE 95.6%	15, 45, 110 ai (10, 30, 75 ae)	Severe maternal toxicity at ≥30 mg/kg/day ae; ↑ resorptions at 30 mg/kg/day ae; no exposure related visceral malformations	2 (for publication)	Overall high quality study; minor limitations in amount of detail reported due to multiple studies and compounds; weakness: excessive maternal toxicity at mid and high dose
	OPP 83-3 guideline study. New Zealand white rabbits dosed orally by gavage with 2,4-D EHE from GD 6–18	2,4-D EHE 95.0%	15.1, 45.2, 113.1 ai (10, 30, 75 ae)	Maternal toxicity at 75 mg/kg/day ae; no exposure related visceral malformations	2 (for publication)	Overall high quality study; minor limitations in amount of detail reported due to multiple studies and compounds
	OPP 83-3 guideline study. New Zealand white rabbits dosed orally by gavage with 2,4-D DEA from GD 6–18	2,4-D DEA Aqueous based manufacturing concentrate (73.1%)	15, 30, 60 ai (10.2, 20.3, 40.6 ae)	Maternal toxicity and ↑ resorptions and skeletal variations at 40.6 mg/kg/day ae; no exposure related visceral malformations	2 (for publication)	Overall high quality study; minor limitations in amount of detail reported due to multiple studies and compounds
	OPP 83-3 guideline study. New Zealand white rabbits dosed orally by gavage with 2,4-D DMA from GD 6–18	2,4-D DMA Aqueous based manufacturing concentrate (66.2%)	12, 36.1, 108.4 ai (10, 30, 90 ae)	Equivocal maternal toxicity; ↑ deaths at mid dose; ↓ litter size low and high dose; no evidence exposure related; no exposure related visceral malformations	2 (for publication)	Overall high quality study; minor limitations in amount of detail reported due to multiple studies and compounds Weaknesses: Equivocal maternal toxicity at high dose; decreased number of litters available for evaluation
	OPP 83-3 guideline study. New Zealand white rabbits dosed orally by gavage with 2,4-D IPA from GD 7–19	2,4-D IPA Aqueous based manufacturing concentrate (50.2%)	13, 38, 95 ai (10, 30, 75 ae)	Severe maternal toxicity at 75 mg/kg/day ae; no exposure related visceral malformations	2 (for publication)	Overall high quality study; minor limitations in amount of detail reported due to multiple studies and compounds; Weakness: excessive maternal toxicity at high dose but sufficient litters for evaluation
	OPP 83-3 guideline study. New Zealand white rabbits dosed orally by gavage with 2,4-D TIPA from GD 7–19	2,4-D TIPA Aqueous based manufacturing concentrate (72.2%)	19, 56, 140 ai (10, 30, 75 ae)	Severe maternal toxicity at 75 mg/kg/day ae; no exposure related visceral malformations	2 (for publication)	Overall high quality study; minor limitations in amount of detail reported due to multiple studies and compounds; Weakness: excessive maternal toxicity at high dose but sufficient litters for evaluation
Collins & Williams 1971	Syrian golden hamsters dosed orally by gavage from GD 6–10	Three different lots of technical 2,4-D	20–100	Increased incidence of fetal abnormalities at high doses; decreased fetal viability	3	Poor methodology (use of different lots of test material), purity undefined; and reporting deficiencies; additionally hamster is a poor model for developmental toxicity evaluations due to multiple spontaneous malformations
Dinamarca et al. 2007	Pregnant mice dosed with technical and formulated 2,4-D in drinking water from GD 0–9	Purity unspecified	0.01–100	No changes in maternal toxicity, body weight gain, implantation sites, resorptions; no endocrine related effects	2	Purity unspecified for technical and formulated 2,4-D; no analytical confirmation of doses; otherwise study quality seemed adequate; refuted findings of Cavieres et al. (2002)
Duffard et al. 1996	Wistar rats orally exposed to 2,4-D DBE	Formulation; concentration and purity of 2,4-D unspecified	70	Increased serotonin levels	3	Unclear methodologies; insufficient data provided; results not specific to endocrine activity
Pochettino et al. 2013	Prenatal and postnatal 2,4-D exposure in pregnant Wistar rats evaluated at PND 45, 60, and 90	Purity unspecified	70	Evidence of potential oxidative stress in various tissues	3	Source of 2,4-D not identified; purity not stated; single dose; no evidence dose analysis conducted; no evidence potential litter effects were accounted for
Maternal nursing behavior and pup body weight effects
Stürtz et al. 2000	Wistar rats exposed to 2,4-D via intraperitoneal injection	Purity unspecified	50–100	Decreased pup body weights; detectable 2,4-D residues in stomach, blood, brain, and kidney of breast-fed neonates	3	Irrelevant route of exposure (intraperitoneal); purity not specified; no evidence litter effects accounted for
Stürtz et al. 2006	Wistar rats dosed orally with 2,4-D	98%	15–70	Decreased pup body weight gains; decreased lipid content of milk; altered fatty acid content	3	Unclear methodology; no evidence litter effects accounted for; dose formulation procedures questionable; neither effects nor internal dosimetry consistent with other 2,4-D studies
Stürtz et al. 2008	Wistar rats dosed orally with 2,4-D	98%	15–50	Altered dam-pup interactions; increased catecholamine levels; decreased indolamine and prolactin levels	3	Unclear methodology; no evidence litter effects accounted for; dose formulation procedures questionable; limited assessment of maternal nursing behavior, pup observations inconsistent with typical findings in pups suffering from maternal neglect
Stürtz et al. 2010	Wistar rats dosed orally or IP with 2,4-D	98%	2.5–70	Decreased pup weight gain; decreases in amount of milk ejected, plasma prolactin and oxytocin altered	3	Unclear methodology; no evidence litter effects accounted for; dose formulation procedures questionable; no bedding material mentioned- absence could stress dams; procedure for evaluation of milk production flawed; incorrect statistical procedures
Male reproductive toxicity
Lamb et al. 1981a	Male C57BL/6N mice dosed orally via diet mixture of 2,4-D and 2,4,5-T; mated with untreated females	98.5% (2,4-D Purity specified in Lamb et al. 1981c toxicity study)	20 and 40 mg/kg/day 2,4-D	No changes in fertility, sperm number, motility, or morphology	2	2,4-D not tested in isolation, but purity and doses of 2,4-D defined; a negative response reported; provides useful information re potential male repro. toxicity
Lamb et al. 1981b	Male C57BL/6N mice dosed orally via diet mixture of 2,4-D and 2,4,5-T; mated with untreated females	98.5% (2,4-D Purity specified in Lamb et al. 1981c toxicity study)	20 and 40 mg/kg/day 2,4-D	No impact of on reproductive performance of males; no changes in development and survival of fetuses and pups	2	2,4-D not tested in isolation, but a negative response reported and purity and doses of 2,4-D defined; provides useful information regarding potential male repro. toxicity
Blakley et al. 1989	CD-1 male mice exposed to a mixture of 2,4-D and picloram via drinking water mated to untreated females	Mixture of chemicals; purity of 2,4-D not specified	84–336	High mortality in males at high concentration; no changes in resorptions, implantations; ↓fetal weight at highest dose; ↑ malformations all doses	3	2,4-D not tested in isolation; types of malformations not reported; small group size
Hassanein 2012	Male Sprague-Dawley rats exposed via gavage	Purity unspecified	30	Congestion of blood vessels in testicles and epididymis after two months; necrosis and sloughing seminiferous tubules, necrobiotic changes in epithelial lining of epididymis	3	Unknown purity; single dose exposure
Kim et al. 2002	Hershberger assay in CD rats	Purity unspecified	50	Increased ventral prostate, Cowper’s gland and glans penis weights; in testosterone (T) supplemented phase of assay; authors hypothesize 2,4-D synergizes with T in increasing accessory sex tissue weights or inhibited T metabolizing enzymes’ activity	3	T dose reporting discrepant (different doses reported in different sections of paper); only one 2,4-D dose tested; inconsistencies in reported methods; T-supplemented Hershberger phase tests anti-androgenicity; not validated to assess androgenicity; organ weights not similar to weights for the same organs reported by other investigators (note article is in Korean; no translation)
Oakes et al. 2002a	Male Sprague-Dawley rats dosed orally via gavage	Formulation; 2,4-D concentration and purity unspecified	37.5–150	No effects on plasma testosterone; decreased weight gain and decreased testicular weight at high dose	3	Formulation; concentration and purity unspecified poor study design; small sample size; insufficient reporting of data
Stoker et al. 2007	Male Wistar rats exposed to 2,4-D orally by gavage	Purity unspecified	100 and 200 reported in abstract; per Stoker and Zorilla (2010) book chapter 3 and 30 were also evaluated	Delayed PPS at high dose; decreased ventral prostate weight; T and androstenedione decreased at high dose; no change in LH and prolactin; T3 decreased at both high doses; no effects at 30 mg/kg/day based on Stoker and Zorilla 2010 book chapter	4	Insufficient information provided (only reported as abstract and as limited information in book chapter); doses reported in abstract exceed threshold for renal clearance
Subchronic and chronic toxicity
Charles et al. 1996a	OPP 82-1 Guideline study; Fischer 344 rats exposed to 2,4-D in the diet for 13 weeks	96.1%	1, 15, 100, 300	Reviewed above with regulatory toxicology studies on 2,4-D based on study report (Schulze 1991a)	2 (for publication)	Limited detail due to multiple studies in one publication
	OPP 82-1 Guideline study Fischer 344 rats exposed to 2,4-D dimethyl amine (DMA), 2,4-D ethylhexyl amine (EHE) in the diet for 13 weeks	66.2% a I (95.3% dry wt) 2,4-D DMA	1–300 mg/kg/day a.e. (acid equivalent)	300 mg/kg/day a.e.: ↓red cell mass, T3 and T4 levels, ↓ ovary and testes weight. ↑ liver, kidney and thyroid weights and cataracts and retinal degeneration (high-dose females).	2 (for publication)	Limited detail due to multiple studies in one publication
	OPP 82-1 Guideline study Fischer 344 rats exposed to 2,4-D ethylhexyl amine (EHE) in the diet for 13 weeks	95.1% a i 2,4-D EHE	1–300 mg/kg/day a.e. (acid equivalent)	300 mg/kg/day a.e.: ↓red cell mass, T3 and T4 levels, ↓ ovary and testes weight. ↑ in liver, kidney and thyroid weights and cataracts and retinal degeneration (high-dose females).	2 (for publication)	Limited detail due to multiple studies in one publication
Oakes et al. 2002b	Male Sprague-Dawley rats dosed orally with gavage with a formulation of 2,4-D and picloram	Formulation with multiple active ingredients; 2,4-D concentration and purity not specified	37.5–150 (2,4-D)	No effects on plasma T; ↓ weight gain and ↓ testicular weight at 150 mg/kg/day	3	Formulation; 2,4-D concentration and purity not specified; 2,4-D not tested in isolation, poor study design; small sample size; insufficient reporting of data
Kobal et al. 2000	Wistar rats dosed by gavage for ten days	Formulation (2,4-D in formulation 98% pure, but inerts in formulation unspecified)	11 and 110	↓ serum T4 at 110 mg/kg/day; ↓T3 males day 6 at 110 mg/kg/day. Statistically significant differences in the low-dose group (↑ in females and ↓ in males); examination of the figures suggests that these changes were not biologically significant and may reflect inherent variability of the assays. No other thyroid-related parameters were assessed in this study; the biological significance of the findings cannot be assessed	3	T4 pre-test values showed considerable variance; no data are presented for thyroid hormone measurements, only figures, which show mean values only without confidence limits or standard errors. Formulated commercial material with unknown formulation excipients. High dose exceeds TSRC; other toxicity to animals not monitored. TSH, thyroid weight, and thyroid histopathology not evaluated.
Charles et al. 1996b	OPP 82-1 subchronic and 83-1 chronic guideline studies in purebred beagle dogs exposed to 2,4-D in diet for 90 days or one year	96.7% 2,4-D	0, 0.5, 1.0, 3.75 and 7.5 in subchronic study; 0, 1, 5 and 10/7.5 in chronic study	Studies (Dalgard 1993a, 1993b) reviewed above with regulatory toxicology studies based on study reports	2 (for publication)	Studies reviewed above based on actual study reports
	OPP 82-1 guideline subchronic study in purebred beagle dogs exposed to 2,4-D dimethylamine salt (DMA) for 90 days	66.7% aqueous solution (93.5% dry weight basis)	0, 1, 3.75 and 7.5 (a.e. basis)	↓ body weight gains at high dose; no effects on thyroids; slightly ↓ testes weight at 7.5 mg/kg/day ae; non-exposure related testicular histopathology; slightly ↑incidence of “juvenile/inactive” prostate at 7.5 mg/kg/day ae	2 (for publication)	Limited details included due to multiple studies presented
	OPP 82-1 guideline subchronic study in purebred beagle dogs exposed to 2,4-D 2-ethylhexyl ester (2-EHE) for 90 days	95.1%	0, 1, 3.75 and 7.5 (a.e. basis)	↓body weight gains at mid and high dose; thyroid weights ↑; not statistically significant or dose related; no thyroid histopath; no effects testes weights; no testicular path.	2 (for publication)	Limited details included due to multiple studies presented
Obidike et al. 2012	West African Dwarf goats dosed via diet	Formulation; purity of 2,4-D not specified	75–125 mg/kg of 720 g/L formulation of 2,4-D	Decreases in testicular sperm reserves and in epididymal sperm reserves; hyperemia and edema of the stroma; decrease in Sertoli cells	3	Non-conventional test system; formulation tested; small number of animals of varying ages obtained from different sources; no assurance control animals representative
Rawlings et al. 1998	Female ewes dosed directly to the rumen via gelatin capsules	Formulation; concentration and purity of 2,4-D not defined	10 mg/kg; 3 times per week	Decreased serum T4; no effect on LH, FSH, P, E2, cortisol, insulin; no changes in histopathology of any organs tested	3	Small sample size; formulation tested; concentration and purity of test material not defined; only one dose tested; direct administration into rumen of questionable relevance
Thyroid mechanism related
Florsheim & Velcoff 1962	Sprague-Dawley rats on low Iodine diet subcutaneously injected with 2,4-D	Formulation	80	Decrease in serum protein-bound iodine; decrease in thyroid to serum radioiodide ratio; no changes in pituitary TSH concentrations, thyroidal cell height, or thyroid histopathology	“5”	Subcutaneous injection, formulation, unspecified purity of test material; dose likely exceeds TSRC; mechanistic study
Florsheim et al. 1963	Sprague-Dawley rats	Formulation	80	Decreased serum thyroxine concentration; increased brain and liver thyroxine concentration; no change in thyroxine half-life, thyroxine distribution or in thyroid histopathology	“5”	Route of administration not specified (probably subcutaneous injection); small sample size formulation, unspecified purity of test material; mechanistic study; dose likely exceeds TSCR
Van den Berg et al. 1991	Male WAG/MBL rats exposed to 2,4-DB in diet; 2,4-D tested in vitro	“Highest purity available”	0.06 mmol/kg 2,4-DB	Lower plasma T4 levels in vivo following 2,4-D B exposure; decreased T4 binding to serum protein in vitro after 2,4-D exposure	“5”	2,4-D not tested in vivo; intraperitoneal route irrelevant; only one concentration tested. In vitro mechanistic study: 2,4-D demonstrated a high (70-100%) competition for binding of T4 to transthyretin at a concentration of 100 uM (22 ug/ml – a concentration well above TSRC).

Table 12. Results for 2,4-D from EDSP tier 1 in vitro and ecotoxicological assays relevant to potential interaction with the estrogen pathway.

Study*	ERbinding	ERtransactivation	Fishfecundity	Fishfertility	Nuptialtubercles (M)	Nuptialtubercles (F)	Fish gonadsomaticindex (M)	Fish gonadsomaticindex (F)	Fish gonadhistopathology (M)	Fish gonadhistopathology (F)	VTG (M)	VTG (F)
ERB	N(2)	–	–	–	–	–	–	–	–	–	–	–
ERTA	–	N(2)	–	–	–	–	–	–	–	–	–	–
FSTRA	–	–	L↓(3)^†	N(2)	N(2)	N(2)	N(3)	N(3)	N(2)	N(2)	N(1)	N(2)

* ERB: OPPTS 890.1250 ER-binding assay (LeBaron et al. 2011a); ERTA: OPPTS 890.1300 ERα transcriptional activation assay (LeBaron & Kan 2011); FSTRA: OPPTS 890.1350 Fish short-term reproduction assay (Marino et al. 2010).

† Marino et al. 2010 decreased fecundity observed only at high (limit dose) dose; non-specific finding; equivocal relationship to pathway interaction

All studies:

NNo evidence of pathway interaction

LPotential evidence of pathway interaction at limit dose only

–Endpoint not evaluated

↓Decreased relative to control

Endpoint scores in parentheses based on Borgert et al. (2014)

(1) specific and sensitive to the hypothesis

(2) potentially sensitive for the hypothesis; stronger if correlated with Rank 1 data

(3) relevant, but useful only if corroborating Rank (1) or (2) endpoints

Table 13. Results for 2,4-D from regulatory reproductive/developmental toxicity studies relevant to potential interaction with the estrogen pathway.

Study*	Time tomating	Gestationduration	Gestationindex	Matingindex	Fertilityindex	Implantations	Pupsex ratio	Anogenitaldistance (AGD)	Vaginalopening	Estrouscyclicity	Urogenitalmalformations	Uterusweight	Uterushistopathology	Ovariesweight	Ovarieshistopathology	Vaginalhistopathology
EOGRT	N (2)	N (3)	N (3)	N (3)	N (3)	N (3)	N (3)	N (2)	N (2)	N (3)	N (2)	O↑ (3)^†	N (2)	N (2)	N (2)	N (2)
Two Gen	N (2)	O↑ (3)^‡	N (3)	N (3)	N (3)	N (3)	O (3)^¶	–	–	–	–	–	N (2)^§	–	N (2)	–
Dev Tox Rat	–	–	–	–	–	–^ǁ	–^ǁ	–	–	–	N (2)	–	–	–	–	–
Dev Tox Rabbit	–	–	–	–	–	–^#	–^#	–	–	–	N (2)	–	–	–	–	–

* EOGRT: F1-extended one-generation reproductive toxicity study (Marty et al. 2010); Two Gen: OPP 83-4 Two-generation reproductive toxicity study (Rodwell & Brown 1985); Dev Tox Rat: OPP 83-3 Rat Developmental toxicity study (Rodwell 1983); Dev Tox Rabbit: OPP 83-3 Rabbit Developmental toxicity study (Hoberman 1990).

† Marty et al. 2010 Uterine weight differences were not statistically different from control and attributable to non-exposure related difference from control in stage of estrous cycle at termination; uterine weights were within HCD.

‡ Rodwell and Brown 1985 The length of gestation was statistically significantly prolonged (by 1 day) in the production of the F1b pups at ≥80 mg/kg/day, compared with controls; may be attributable to the very excessive dose of 2,4-D to the dams during production of the F1b litters; high pup mortality was seen at this excessive dose.

¶ 80 mg/kg/day F1a pups showed a statistically significant change in sex ratio (increased M pups), compared with the controls. This finding was not repeated in the F1b pups (at a higher dose) and is considered unlikely to be exposure-related because of the lack of consistency.

§ The gross and microscopic evaluation of uteri in the F1b PND 28 offspring showed no evidence of imbibed uteri or uterine lining proliferation.

ǁ Evaluated but not sensitive; as implantations complete and offspring sex determined prior to initiation of dosing.

# Change in fetal sex ratio present; not attributed to test article as offspring sex determined prior to initiation of dosing; implantations complete prior to dosing; no evidence selective loss of females

All studies

NNo evidence of potential interaction

OFinding over the TSRC

↑Increased relative to control

–Endpoint not evaluated

Endpoint scores in parentheses based on Borgert et al. 2014 (modified in some cases based on context or strength of response)

(1) specific and sensitive to the hypothesis

(2) potentially sensitive for the hypothesis; stronger if correlated with Rank 1 data

(3) relevant, but useful only if corroborating Rank (1) or (2) endpoints

Table 14. Results for 2,4-D from regulatory toxicity subchronic and chronic toxicity studies relevant to potential interaction with the estrogen pathway.

Study*	Uterus histopathology	Ovaries weight	Ovaries histopathology	Mammary histopathology	Vaginal histopathology
Rat SC (1)	N(2)	O↑ (2)^†	N(2)	–	N(2)
Rat SC (2)	N(2)	–	N(2)	N(2)	–
Rat C	N(3)	O↓ (3)^‡	N(2)	O(3)^¶	N(2)
Mouse SC	N(2)	N(2)	N(2)	–	–
Mouse C (F)	N(3)	–	N(3)	N(2)	N(3)
Dog SC (1)	N(2)	N(2)	N(2)	–	–
Dog SC (2)	N(2)	N(2)	N(2)	N(2)	N(2)
Dog C	N(2)	N(2)	N(2)	N(2)	N(2)

* Rat SC (1): OPP 82-1 13-week rat subchronic toxicity study (Schulze 1991a); Rat SC (2): OPP 82-1 13-week rat subchronic toxicity study (Gorzinski et al. 1981a); Rat C: OPP 83-5 two year rat chronic/oncogenicity study (Jeffries et al. 1995); Mouse SC: OPP 82-1 13 week mouse subchronic toxicity study (Schulze 1991b); Mouse C(F): OPP 83-2 mouse oncogenicity study females (Stott 1995a); Dog SC (1): OPP 82-1: 13-week dog subchronic toxicity study (Schulze 1990); Dog SC (2): OPP 82-1: 13-week dog subchronic toxicity study (Dalgard 1993a) and Dog C (1): OPP 83-1: dog chronic toxicity study (Dalgard 1993b).

† Schulze 1991a Ovary weight increase at 300 mg/kg/day high dose; no correlating histopathological findings and stage of estrous cycle not controlled at necropsy.

‡ Jeffries et al. 1995 Ovary weight decrease at 150 mg/kg/day high dose terminal sacrifice attributable to body weight loss, no correlating histopathological findings.

¶ Decreased incidence of mammary gland hyperplasia at 150 mg/kg/day terminal sacrifice; likely attributable to body weight loss

All studies

NNo evidence of potential interaction

OFinding over the threshold for renal saturation

↑Increased relative to control

↓Decreased relative to control

–Endpoint not evaluated

Endpoint scores in parentheses based on Borgert et al. 2014 (modified in some cases based on context or strength of response)

(1) specific and sensitive to the hypothesis

(2) potentially sensitive for the hypothesis; stronger if correlated with Rank 1 data

(3) relevant, but useful only if corroborating Rank (1) or (2) endpoints

Table 15. Results for 2,4-D from EDSP tier 1 in vitro and ecotoxicological assays relevant to potential interaction with the androgen pathway.

Study*	ARbinding	Fish behavior(sex-linked)^†	Fishfertility	Fishfecundity	Fish nuptialtubercles (M)	Fishnuptialtubercles (F)	FishGSI (M)	FishGSI (F)	Fish gonadhistopathology (M)	Fish gonadhistopathology (F)	VTG (M)	VTG (F)
ARB	N(2)	–	–	–	–	–	–	–	–	–	–	–
FSTRA	–	N(3)	N(2)	N(3)	N(2)	N(1)	N(3)	N(3)	N(2)	N(2)	N(2)	N(2)

* ARB: OPPTS 890.1250 AR-binding assay (LeBaron et al. 2011b); FSTRA: OPPTS 890.1350 fish short-term reproduction assay (Marino et al. 2010).

† Based on impression of typical male nest-guarding behavior which may be associated with testosterone concentrations (O’Connor et al. 2011); no detailed assessment conducted

All studies

NNo evidence of potential interaction

–Endpoint not evaluated

Endpoint scores in parentheses based on Borgert et al. (2014) (modified in some cases based on context or strength of response)

(1) specific and sensitive to the hypothesis

(2) potentially sensitive for the hypothesis; stronger if correlated with Rank 1 data

(3) relevant, but useful only if corroborating Rank (1) or (2) endpoints

Table 16. Results for 2,4-D from regulatory reproductive and developmental toxicity studies relevant to potential interaction with the androgen pathway.

Study*	Matingindices	Fertilityindices	Anogenitaldistance (AGD)	Nippleretention	Pupsex ratio	Developmental(e.g. urogenital)malformations	Preputialseparation	Spermparameters	Testesweight	Testeshistopathology	Epididymidesweight	Epididymideshistopathology	Seminalvesiclesweight	Seminalvesicleshistopathology	Prostateweight	Prostatehistopathology
EOGRT	N(2)	N(2)	N(1)	N(1)	N(3)	–	O(2)^†	N(2)	O↓(2)^‡	O(2)^¶	N(2)	N(2)	N(2)^§	N(2)	N(2)^§	N(2)
Two Gen Repro	N(2)	–	–	–	O (3)^ǁ	N(2)	–	–	N(2)	N(2)	–	N(2)	–	N(2)	–	N(2)
Dev Tox Rat	–	–	–	–	–^#	N(2)	–	–	–	–	–	–	–	–	–	–
Dev Tox Rabbit	–	–	–	–	^--**	N (2)	–	–	–	–	–	–	–	–	–	–

* EOGRT: Tier 2 equivalent F1-extended one-generation reproductive toxicity study (Marty et al. 2010); Two Gen: OPPTS 83-4 Two-generation reproductive toxicity study (Rodwell & Brown 1985); Dev Tox Rat: 83-3 developmental toxicity study in rats (Rodwell 1983); Dev Tox Rabbit: OPP 83-3 developmental toxicity study in rabbits (Hoberman 1990).

† Marty et al. 2010 Slight delay in preputial separation considered associated with decreased pup weight post weaning and not endocrine related.

‡ Decreased testes weight F1 pups at weaning, attributable to decreased body weight, not apparent in F1 adults.

¶ Testicular atrophy in 2 high-dose P adults, considered spontaneous, not seen in F1 dosed adults and incidence within HCD.

§ Seminal vesicle and prostate weights decreased compared to control at ≥300 ppm in P1; not statistically significant, within HCD and controls below HCD; conclusion no exposure-related effect.

ǁ Rodwell and Brown 1985 Pup sex ratio statistically significantly different in F1a high dose only; but not in F1b which received a higher dose (due to mis-dosing) during gestation; altered ratio considered incidental.

# Evaluated but not sensitive; as offspring sex determined prior to initiation of dosing.

** Change in fetal sex ratio present; not attributed to test article as offspring sex determined prior to initiation of dosing; no evidence selective loss of females

All studies:

NNo evidence of potential interaction

OFinding over the threshold for renal saturation

↓Decreased relative to control

–Endpoint not evaluated

Endpoint scores in parentheses based on Borgert et al. 2014 (modified in some cases based on context or strength of response)

(1) specific and sensitive to the hypothesis

(2) potentially sensitive for the hypothesis; stronger if correlated with Rank 1 data

(3) relevant, but useful only if corroborating Rank (1) or (2) endpoints

Table 17. Results for 2,4-D from regulatory subchronic and chronic toxicity studies in rat, mouse and dog relevant to potential interaction with the androgen pathway.

Study*	Testes weight	Testes histopathology	Epididymides weight	Epididymides histopathology	Seminal vesicles histopathology	Prostate histopathology	Coagulating glands histopathology
Rat SC (1)	O↓ (2)^†	O (2)^†	N (2)	N(2)	–	–	–
Rat SC (2)	O↓ (2)^†	N(2)	–	N(2)	N(2)	N (2)	N(2)
Rat SC (3)	N(2)	N(2)	–	–	–	–	–
Rat C	O↓ (2)^‡	O (2)^‡	–	N(2)	N(2)	N(2)	–
Mouse SC	N(2)	N(2)	–	N(2)	–	–	–
Mouse C (M)	N(2)^¶	N(2)	–	N(2)	N(2)	N(2)	N(2)
Dog SC (1)	O↓ (3)^§	O (3)^ǁ	O↓(3)^#	N(2)	–	–	–
Dog SC (2)	O↓ (3)**	N(2)	–	N(2)	–	N(2)^††	–
Dog C	N(2)	N(2)	–	N(2)	–	N(2)	–

* Rat SC (1): 13 week rat subchronic toxicity study (Schulze 1991a); SC (2): 13 week rat subchronic toxicity study 82-1 (Gorzinski et al. 1981a); SC (3): 13 week rat subchronic toxicity study (Gorzinski et al. 1981b); Rat C: OPP 83-5. Two year rat chronic/oncogenicity study (Jeffries et al. 1995); Mouse SC: OPP 82-1 13 week mouse subchronic toxicity study (Schulze 1991b); Mouse C (M): OPP 83-2 mouse oncogenicity study (males) (Stott 1995b); Dog SC (1): OPP 82-1: 13-week dog subchronic toxicity study (Schulze 1990); Dog SC (2): OPP 82-1: 13-week dog subchronic toxicity study (Dalgard 1993a) and Dog C: OPP 83-1: dog chronic toxicity study (Dalgard 1993b).

† Schulze 1991a and Gorzinski et al. 1981a Testes weight: decrease at ≥100 mg/kg/day; atrophy at 300 mg/kg/day (doses far exceeded TSRC and systemically toxic).

‡ Jeffries et al. 1995 Atrophy in 2/10 animals at 150 mg/kg/day in interim sac; not evident at termination (dose far exceeded TSRC and systemically toxic).

¶ Stott 1995b Relative weight increased but not absolute weight at 24 month only; attributed to body weight decrease.

§ Schulze 1990 Testes weight decreased at 10 mg/kg/day (dose exceeded TSRC and MTD); juvenile animals tested.

ǁ Hypospermia in 2 of the 10 mg/kg/day dose group surviving dogs; equivocal relationship to exposure (dose exceeded MTD) and juvenile animals tested.

# Epididymal weight taken with testes weight; see comments on testes; no histopathological correlates in epididymides.

** Dalgard 1993a Testes weight decreased at 7.5 mg/kg/day dose (dose systemically toxic and exceeded TSRC); juvenile animals tested

†† “Juvenile” prostate noted in all groups; not exposure-related

All studies:

NNo evidence of potential interaction

OFinding over the threshold for renal saturation

↑Increased relative to control

↓Decreased relative to control

–Endpoint not evaluated

Endpoint scores in parenthesis based on Borgert et al. 2014 (modified in some cases based on context or strength of response)

(1) specific and sensitive to the hypothesis

(2) potentially sensitive for the hypothesis; stronger if correlated with Rank 1 data

(3) relevant, but useful only if corroborating Rank (1) or (2) endpoints

Table 18. Results for 2,4-D from EDSP tier 1 in vitro toxicology and ecotoxicology assays relevant to potential interaction with steroidogenesis.

Study*	Steroidogenesis	Aromataseinhibition	Fishfecundity	Fishfertility	Fishnuptialtubercles (M)	Fishnuptialtubercles (F)	Fishgonadsomaticindex (M)	Fishgonadsomaticindex (F)	Fishgonadhistopathology(M)	Fish gonadhistopathology(F)	VTG(M)	VTG(F)
Steroidogenesis	N(3)^†	–	–	–	–	–	–	–	–	–	–	–
Aromatase	–	N(3)	–	–	–	–	–	–	–	–	–	–
FSTRA	–	–	L↓ (3)^‡	N(2)	N(2)	N(1)	N(3)	N(3)	N(2)	N(2)	N(1)	N(2)

* Steroidogenesis: OPPTS 890.1550 Steroidogenesis (LeBaron et al. 2011c); Aromatase: OPPTS 890.1200 Aromatase (Coady & Sosinski 2011); FSTRA: OPPTS 890.1350 Fish short-term reproduction assay (Marino et al. 2010).

† LeBaron et al. 2011c Steroidogenesis assay showed slight increased estradiol in all runs at the high concentration. The fold increase was below that established in the EDSP validation assays as a positive response (Hecker et al. 2008), and is therefore not considered biologically meaningful.

‡ Marino et al. 2010 Decreased fecundity observed only at high (limit dose) concentration; non-specific finding

All studies

NNo evidence of interaction

LEquivocal evidence of potential interactionat the limit dose only

–Endpoint not evaluated

↓Decreased relative to control

MMale

FFemale

Endpoint scores in parentheses based on Borgert et al. 2014 (modified in some cases based on context or strength of response)

(1) specific and sensitive to the hypothesis

(2) potentially sensitive for the hypothesis; stronger if correlated with Rank 1 data

(3) relevant, but useful only if corroborating Rank (1) or (2) endpoints

Table 19. Results for 2,4-D from regulatory reproductive and developmental toxicity studies relevant to potential interaction with steroidogenesis.

EOGRT	N (2)	N (2)	–	N (3)	N (2)	N (2)	N (2)	–	N (2)	N (2)	O↑ (3)^‡	N (2)	N (2)	N (2)	N (2)	O↓ (3)^¶	O (3)^§	N (2)	N (2)	N (2)^ǁ	N (2)	N (2)^ǁ	N (2)	N (2)	O (2)^#	N (2)	N (2)
Two-Gen	–	–	N (2)	O↑ (3)**	N (2)	N (2)	O (2)^††^,**	–	–	N (2)	–	N (2)	–	–	–	N (2)	N (2)	–	N (2)	–	N (2)	–	N (2)	–	–	–	–
Dev Tox Rat	–	–	–	–	–	–	–^‡‡	N (1)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
Dev Tox Rabbit	–	–	–	–	–	–	–^¶¶	N (1)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–

* EOGRT: EOGRT study (Marty et al. 2010); Two Gen: OPP 83-4 Two-generation reproductive toxicity study (Rodwell & Brown 1985); Dev Tox Rat: OPP 83-3 developmental toxicity study in rats (Rodwell 1983): Dev Tox Rabbit: OPP 83-3 developmental toxicity study in rabbit (Hoberman 1990).

†With coagulating gland.

‡ Marty et al. 2010 High dose uterine weight increases not considered exposure related (non-statistically significant change in cycling females; no correlating histopathology except normal estrous cycle related changes; within HCD).

¶ Decreased testes weight F1 pups at weaning, attributable to decreased body weight, not apparent in F1 adults.

§ Testicular atrophy in 2 high-dose P1 males, considered spontaneous, not present in F1 dosed animals.

ǁ Seminal vesical and prostate weights decreased compared to control at ≥300 ppm in P1; not statistically significant, within HCD and controls below HCD; conclusion no effect.

# Increased age at preputial separation; considered associated with decreased growth and not endocrine related.

** Rodwell and Brown 1985 The length of gestation was statistically significantly prolonged (by 1 day) in the production of the F1b pups at ≥80 mg/kg/day, compared with controls; likely attributable to the very excessive dose of 2,4-D to the dams during production of the F1b litters; high pup mortality was seen at this excessive dose (≥100 mg/kg/day).

†† 80 mg/kg/day F1a pups showed a statistically significant change in sex ratio (increased M pups), compared with the controls. This finding was not repeated in the F1b pups (at a higher dose) and is considered unlikely to be exposure related because of the lack of consistency across generations or with other 2,4-D studies.

‡‡ Evaluated but not sensitive; as offspring sex was determined prior to initiation of dosing.

¶¶ Change in fetal sex ratio present; not attributed to test article as offspring sex determined prior to initiation of dosing; no selective loss of females

All studies:

NNo evidence of potential interaction

OFinding over the threshold for renal saturation

–Endpoint not evaluated

↑Increased relative to control

↓Decreased relative to control

Endpoint scores in parentheses based on Borgert et al. 2014 (modified in some cases based on context or strength of response)

(1) specific and sensitive to the hypothesis

(2) potentially sensitive for the hypothesis; stronger if correlated with Rank 1 data

(3) relevant, but useful only if corroborating Rank (1) or (2) endpoints

Table 20. Results for 2,4-D from regulatory subchronic (SC) and chronic (C) toxicity studies relevant to potential interaction with steroidogenesis.

Study*	Ovaryweight	Ovaryhistopathology	Uterinehistopathology	Vaginalhistopathology	Mammaryhistopathology	Testesweight	Testeshistopathology	Epididymidesweight	Epididymideshistopathology	Prostatehistopathology	Seminalvesicleshistopathology	Adrenalweight	Adrenalhistopathology
Rat SC (1)	O↑ (2)^†	N(2)	N(2)	N(3)	–	O↓ (2)^‡	O (2)^‡	N(3)	N(2)	–	–	O(2)^¶M↑F↓	O (2)^¶ MF
Rat SC (2)	–	N(3)	N(2)	–	N(2)	O↓ (2)^‡	N(2)	–	N(2)	N(2)	N(2)	–	N(2)
Rat SC (3)	–	–	–	–	–	N(2)	N(2)	–	–	–	–	–	–
Rat C	O↓ (3)^§	N(2)	N(2)	N(3)	N(3)	O↓ (2)^ǁ	O (2)^#	–	N(2)	N(2)	N(2)	O↓ (2)**	N(2)
Mouse SC	N(2)	N(2)	N(2)	–	–	N(2)	N(2)	–	N(2)	–	–	O↑(2)^††	N(2)
Mouse C (1 F)	–	N(3	N(2)	N(3)	N(3)	–	–	–	–	–	–	–	N(2)
Mouse C (2 M)	–	–	–	–	–	O↑ (2)^‡‡	N(2)	–	N(2)	N(2)	N(2)	–	N(2)
Dog SC (1)	N(2)	N(2)	N(2)	–	–	O↓ (3)^¶¶	O (3)^§§	O↓(3)^ǁǁ	N(2)	–	–	–	N(2)
Dog SC (2)	N(2)	N(2)	N(2)	N(3)	N(2)	O↓ (3)^##	N(2)	–	N(2)	N(2)	–	N(2)	N(2)
Dog C	N(2)	–	N(2)	N(3)	N(2)	N(2)	N(2)	–	N(2)	N(2)	–	N(2)	N(2)

* Rat SC (1): OPP 82-1 13-week rat subchronic toxicity study (Schulze 1991a); Rat SC (2): 13 week rat subchronic toxicity study 82-1 (Gorzinski et al. 1981a); Rat SC (3): 13 week rat subchronic toxicity study (Gorzinski et al. 1981b); Rat C: OPP 83-5 Two year rat chronic/oncogenicity study (Jeffries et al. 1995); Mouse SC: OPP 82-1 13 week mouse subchronic toxicity study (Schulze 1991b); Mouse C(1 F): OPP 83-2 mouse oncogenicity study (females) (Stott 1995a); Mouse C(2 M): OPP 83-2 mouse oncogenicity study (males) (Stott 1995b); Dog SC(1): OPP 82-1: 13-week dog subchronic toxicity study (Schulze 1990); Dog SC(2): OPP 82-1: 13-week dog subchronic toxicity study (Dalgard 1993a); Dog C: OPP 83-1: dog chronic toxicity study (Dalgard 1993b).

† Schulze 1991a Increased ovary weight at 300 mg/kg/day (far exceeded TSRC and systemically toxic), no histopathological correlate.

‡ Schulze 1991a and Gorzinski et al. 1981a Testes weight: decrease at ≥100 mg/kg/day; atrophy at 300 mg/kg/day (far exceeded TSRC and systemically toxic).

¶ Schulze 1991a Adrenal: weight changes and histopathology (hypertrophy zona glomerulosa) at 300 mg/kg/day (far exceeded TSRC and systemically toxic); zona glomerulosa does not respond to HPA axis.

§ Jeffries et al. 1995 Ovary: weight decrease at high dose attributable to body weight decrease; no correlating histopathological changes.

ǁ Testes: absolute and relative testes weights decreased at 150 mg/kg/day at both interim and terminal sacrifices; no corresponding histopathological findings at terminal sacrifice.

# Testes: 2/10 with atrophy at intermediate (1 year) sacrifice in high-dose group; no exposure related findings at 2 yr sacrifice.

** Adrenal: weights in females decreased at 75 and 150 mg/kg/day at 2 yr sac.; may be attributable to body weight decreases.

†† Schulze 1991b Adrenal weights increased; no dose response; considered unlikely to be exposure related.

‡‡ Stott 1995b Relative testes weight increased but not absolute weight at 24 month only; attributed to body weight decrease.

¶¶ Schulze 1990 Testes weight decreased at 10 mg/kg/day (dose exceeded MTD).

§§ Hypospermia in 2 of the 10 mg/kg/day dose group surviving dogs; equivocal relationship to exposure (dose exceeded MTD).

ǁǁ Weight taken with testes weight; see comments on testes; no histopathological correlates in epididymides.

## Dalgard 1993a Testes weight decreased at 7.5 mg/kg/day; no correlating histopathological changes

All studies:

NNo evidence of potential interaction

OFinding over the threshold for renal saturation

–Endpoint not evaluated

↓Decreased relative to control

↑Increased relative to control

Endpoint scores in parentheses based on Borgert et al. 2014 (modified in some cases based on context or strength of response)

(1) specific and sensitive to the hypothesis

(2) potentially sensitive for the hypothesis; stronger if correlated with Rank 1 data

(3) relevant, but useful only if corroborating Rank (1) or (2) endpoints

Table 21. Data from 2,4-D tier 1 EDSP and regulatory toxicity studies relevant to potential interactions with the HPT axis.

Study*	Tadpole developmental stage (day 7 and 21)	Tadpole normalized hind limb length (day 7 and 21)	Asynchronous development (day 7 and 21)	Thyroid weight	Thyroid histopathology	Thyroid hormone (T4)	Thyroid hormone (T3)	Thyroid hormone (TSH)	Pituitary weight	Pituitary histopathology
AMA	N(2)	N(2)	N(1)	–	N(1)	–	–	–	–	–
EOGRTS	–	–	–	N(2)^†	O (1)^‡	O↓ (2)^¶	O↓(2)^¶	O↑(2)^§	O↓ (3)^ǁ	N(3)
Rat SC (1)	–	–	–	O↑ (2)^#	O (1)**	O↓ (2)	O↓(2)	–	O M ↑; F↓ (3)^††	N(3) MF
Rat SC (2)	–	–	–	–	N(1)	O↓ (2)	–	–	–	N(3) MF
Rat SC (3)	–	–	–	–	–	O↓ (2)	–	–	–	–
Rat C	–	–	–	O↑ (2)^‡‡	O (1)^¶¶	O↓ (2)^§§	–	–	–	N(3)
Mouse SC	–	–	–	N(2)	N(1)	O↓ (2)^ǁǁ	–	–	N(3)	N(3)
Mouse C (1 F)	–	–	–	–	N(1)	–	–	–	–	N(3)
Mouse C (2 M)	–	–	–	–	N(1)	–	–	–	–	–
Dog SC (1)	–	–	–	N(2)	N(1)	N(2)	N(2)	–	–	N(3)
Dog SC (2)	–	–	–	O↓(2)^##	N(1)	–	–	–	N(3)	N(3)
Dog C	–	–	–	N(2)	N(1)	–	–	–	N(3)	N(3)

* AMA: amphibian metamorphosis assay (Coady et al. 2013); EOGRTS: F1-extended one generation dietary toxicity study (Marty et al. 2010); Rat SC (1): OPP 82-1 13-week rat subchronic toxicity study (Schulze 1991a); Rat SC (2): OPP 82-1 13-week rat subchronic toxicity study (Gorzinski et al. 1981a); Rat SC 3: 13-week rat subchronic study (non-guideline) (Gorzinski et al. 1981b); Rat C: OPP 83-5 two year rat chronic/oncogenicity study (Jeffries et al. 1995); Mouse SC: OPP 82-1 13 week mouse subchronic toxicity study (Schulze 1991b); Mouse C(1 F): OPP 83-2 mouse oncogenicity study (females) (Stott 1995a); Mouse C(2 M): OPP 83-2 mouse oncogenicity study (males) (Stott 1995b); Dog SC (1): OPP 82-1: 13-week dog subchronic toxicity study (Schulze 1990); Dog SC (2): OPP 82-1: 13-week dog subchronic toxicity study (Dalgard 1993a) and Dog C: OPP 83-1: dog chronic toxicity study (Dalgard 1993b).

† Marty et al. 2010 Thyroid weight change not considered exposure related; no dose response, no consistency in direction or biologically significant magnitude of change and no correlating histopathology.

‡ Thyroid histopathology slight adaptive change (depleted colloid) in GD 17 females at 600 ppm.

¶ Non-statistically significant decrease T3 and T4 at 600 ppm; considered likely exposure related in GD 17 females because of pattern of findings.

§ Non-statistically significant increase TSH at 600 ppm; considered likely exposure related in GD 17 females because of pattern of findings.

ǁ Decreased pituitary weight in set 3 males only; no correlating histopathology; considered unlikely to be exposure related.

# Schulze 1991a thyroid weight: Males had higher absolute thyroid/parathyroid weights at 300 mg/kg/day and relative weights at 100 and 300 mg/kg/day. Females at 300 mg/kg/day had higher relative thyroid/parathyroid weights.

** Follicular cell hypertrophy (adaptive change) in females at high and systemically toxic 300 mg/kg/day dose exceeding MTD and renal clearance saturation threshold.

†† Females had lower absolute and relative pituitary weights at 300 mg/kg/day. Males had higher relative pituitary weights at 300 mg/kg/day.

‡‡ Jeffries et al. 1995 Thyroid weights (absolute and relative) increased in males at 150 mg/kg/day, and in females at 75 and 150 mg/kg/day at both sacrifices.

¶¶ Decreased colloid (adaptive change) in thyroid of females at 1 year at 150 mg/kg/day; parafollicular hyperplasia non-statistically significantly increased at 150 mg/kg/day.

§§ Decreased T4 at 150 mg/kg/day at 1 year interval.

ǁǁ Schulze 1991b Non statistically significant decreased T4 at 100 and 150 mg/kg/day.

## Dalgard 1993a Relative but not absolute weight increase at high dose (7.5 mg/kg/day) attributed to body weight loss

All studies

NNo evidence of potential interaction

OFinding over the threshold for renal saturation

–Endpoint not evaluated

↑Increased relative to control

↓Decreased relative to control

MMale

FFemale

Endpoint scores in parentheses based on Borgert et al. 2014 (modified in some cases based on context or strength of response)

(1) specific and sensitive to the hypothesis

(2) potentially sensitive for the hypothesis; stronger if correlated with Rank 1 data

(3) relevant, but useful only if corroborating Rank (1) or (2) endpoints

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