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2, 4-Dichlorophenoxyacetic Acid/PC Code 030001 OPPTS 870.3800; 870.6300, 870.7800/ DACO 4.5.1/ OECD 416

EPA Reviewer: Linda Taylor, Ph.D. Signature: 

Risk Assessment Branch VII, Health Effects Division (7509P) Date: 

EPA Secondary Reviewer: Elizabeth Mendez, Ph.D. Signature: 

Risk Assessment Branch VII, Health Effects Division (7509P) Date:

Template version 02/06

TXR#: 0055341

DATA EVALUATION RECORD

STUDY TYPE: F1-Extended One Generation Toxicity Study – rat 

OPPTS 870.3800, 870.6300, 870.7800/OECD 416

PC CODE: 030001 DP BARCODE: D376556

TEST MATERIAL (PURITY): 2, 4 D 

SYNONYMS: (2,4-Dichlorophenoxy) acetic acid, 2,4-D Acid

CITATION: Marty, MS; Zablotny, CL; Andrus, AK; et al. (2010) 2, 4-D: An Extended One 

Generation Dietary Toxicity Study in Crl:CD(SD) Rats. Toxicology &

Environmental Research and Consulting. The Dow Chemical Company. Laboratory

Project Study ID 081104, January 30, 2010. MRID 47972101. Unpublished.

Saghir, S. A., Zablotny, C. L. Marty, M. S. Perala, A. W. and Yano, B. L. (2008). A 

Dietary Dose Range-Finding and Pharmacokinetic Study of 2,4-

Dichlorophenoxyacetic Acid (2,4-D) in the Pregnant CRL:CD(SD) Rat and Its 

Offspring in Preparation for a Subsequent F1-Extended One-Generation Toxicity

Study in Rats. Toxicology & Environmental Research and Consulting, The Dow 

Chemical Company. Laboratory Project Study IDs 071153 and 071153A, May 2, 

2008. MRID 47417901. Unpublished.

Saghir, S. A., Perala, A. W., and Clark, A. J. (2008). A dietary titration study of 2,4-

dichlorophenoxyacetic acid (2,4-D) pharmacokinetics in female CRL:CD(SD) rats. 

Toxicology & Environmental Research and Consulting, The Dow Chemical 

Company. Laboratory Project Study ID 071210, April 29, 2008. MRID 47417902. 

Unpublished.

SPONSOR: Industry Task Force II on 2, 4-D Research Data

EXECUTIVE SUMMARY: In an extended dietary one-generation reproductive toxicity study

(MRID 47972101), 2,4-dichloro phenoxyacetic acid (2,4-D; 97.85%-98.6% a.i.; lot # 2006 2433 

8006-USA) was administered to 27 Crl:CD(SD) young adult rats/sex/dose via the diet at dose levels 

of 0, 100, 300, or 600 (females)/800 (males) ppm [equivalent to 0, ≈5, 15, or 30 (females)/40 (males) 

mg/kg bw/day] for approximately four weeks prior to mating and continuing through mating (up to 2 

weeks), gestation, and lactation. P1 males were exposed for a minimum of 11 weeks including 7 

weeks from the initiation of the mating phase. P1 females were exposed until lactation day 22 
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2, 4-Dichlorophenoxyacetic Acid/PC Code 030001 OPPTS 870.3800; 870.6300, 870.7800/ DACO 4.5.1/ OECD 416

(LD22). A satellite group of P1 females (12/dose) were subject to the same exposures as the P1 

females on the main study (exposure for 4 weeks during the pre-mating, up to 2 weeks during the 

mating period and during gestation until termination on gestation day 17 (GD 17). Satellite males 

were not exposed to dietary 2, 4-D except during co-housing with satellite females during the mating 

period. 

P1 Generation: A comprehensive evaluation of P1 male and P1 female reproductive system was 

conducted, including an evaluation of gonadal function, the estrous cycle, sperm parameters, mating 

performance, conception, gestation, parturition and lactation, as well as survival, growth and 

development of the offspring. Selected systemic toxicity parameters were also evaluated in the P1 

males and P1 females. 

Satellite GD 17 Females: A satellite group of P1 females (12/dose) was included for assessments of 

selected systemic toxicity parameters, clinical chemistry/hematology, thyroid hormone levels, thyroid 

weights, plasma 2, 4-D levels, histopathology, and selected reproductive parameters during gestation

(corpora lutea and implantation numbers). 

F1 Generation: F1 offspring were evaluated for potential effects on the nervous system, immune 

system, reproductive and endocrine systems, thyroid function, and other systemic toxicity

parameters. 2, 4-D plasma levels were also assessed in the F1 offspring. In-life parameters in all F1 

offspring included clinical observations, body weights, feed consumption, anogenital distance, nipple 

retention and puberty onset. Selected F1 offspring were divided into three different groups (Sets 1, 2, 

and 3) at weaning (postnatal day 21; PND 21). Each set of F1 offspring was maintained on the test 

diet until PND 60 (Set 1b F1 offspring), ≈PND 70 (Sets 1a and 2a F1 offspring), or ≈PND 90-139 

(Sets 2b and 3 F1 offspring). 

Set 1a (10/sex/dose): assessment of general systemic and thyroid toxicity, which included 

clinical chemistry/hematology parameters, thyroid hormone assessment, and urinalysis (males 

only). Post-mortem evaluations in Set 1a (PND70) included gross pathology, organ weights and 

histopathology on a wide range of tissues, including thyroids.

Set 1b (10/sex/dose): developmental neurotoxicity (DNT) assessment, which included functional 

observational battery (FOB), motor activity and acoustic startle response (ASR). On PND 60, Set 

1b animals were perfused for central nervous system (CNS) and peripheral nerve neuropathology

evaluation and brain morphometry. A special stain (Luxol Fast Blue) was used to evaluate brain 

myelination. 

Set 2a (10sex/dose): assessment of potential developmental immunotoxicity (DIT): examination 

of humoral immune function using the sheep red blood cell (SRBC) antibody-forming cell (AFC) 

assay on PND 70-74.

Set 2b (10/sex/dose): assessment of potential developmental immunotoxicity (DIT): examination 

of innate cellular immunity using the natural killer cell (NK) assay on PND 87-93.

Set 3 (23-27/sex/dose): assessment of reproductive/endocrine toxicity, which included estrous 

cycle evaluation and post-mortem evaluations that focused on reproductive organs, sperm 

assessment, and ovarian follicle counts on PND 139. TK analyses were conducted on Set 3 males 

and females on PND 63 and 84 to determine plasma 2, 4-D levels.
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In addition, selected pups culled on PND 4 were used to assess thyroid hormone levels. Additional 

data were gathered from F1 offspring not assigned to Sets 1-3. On PND 22, unselected weanlings 

were either perfused for examination of neuropathology (12/sex/dose) or euthanized for assessment 

of systemic toxicity, which included thyroid hormone assessment, organ weights, and post-mortem 

examinations (gross pathology and histopathology) in 10/sex/dose. 

Reproductive and selected data from the F1 generation were used to assess whether a second 

generation would be produced. None of the criteria were met (Table 1), and a second generation was 

not assessed in this study. 

P1 Adult Rats: There were no treatment-related deaths or clinical signs of toxicity in either sex of 

P1 adults. Body weights and body-weight gains were comparable among the groups during the premating and mating phases (both sexes) and during gestation and the latter part of lactation (dams). 

Prior to dietary adjustment of 2, 4-D concentration during the second week of lactation, the 600 ppm 

dams displayed a decrease in body weight (LD 7; ↓5%) and body-weight gain (LD 1-4; ↓64%), 

which is consistent with reduced food intake during the first week of lactation. The reduction in food 

intake can be attributed to the increase in the actual dose (≈65 mg/kg/day) above the targeted level 

(30 mg/kg/day) during this time. After dietary adjustment, food intake for the 600 ppm dams was 

above control levels. 

There were no apparent treatment-related effects on hematology, differential white blood cell counts, 

and prothrombin time, and clinical chemistry and urinalysis parameters were comparable among the 

groups (both sexes). P1 males displayed increased kidney weights (absolute and relative) at 800 ppm, 

which were accompanied by histopathological findings (degenerative lesion in the proximal 

convoluted tubules in the outer zone of the medulla) and are consistent with previous findings that 

the kidney is a target organ. There were no treatment-related findings in the P1 female kidney. 

Decreased reproductive and accessory sex gland weights were observed at 300 ppm and/or 800 ppm. 

These changes, however, are related to the concurrent control being outside of the laboratory

historical control range. P1 females at 600 ppm displayed increased uterine weights (↑17%, both 

absolute and relative), although statistical significance was not attained. There were no alterations in 

estrous cycle pattern in the 600 ppm F1 females compared to the control, and no significant 

difference in mean estrous cycle length in P1 females at any dose level compared to the control. 

There were no significant, treatment-related effects on sperm motility or progressive motility, no 

differences in testicular spermatid and epididymal sperm counts, and no differences in the proportion 

of abnormal sperm. Male and female mating, conception, fertility, and gestation indices were 

comparable among the groups, and post-implantation loss was comparable among the groups. Both 

the time to mating and gestation length were comparable among the groups. 

GD 17 Satellite Females: All P1 satellite females survived to scheduled sacrifice, and body weights 

were comparable among the groups. Hematology and clinical chemistry parameters were comparable 

among the groups. Reproductive indices and the numbers of corpora lutea and implantations were 

comparable among the groups. There was a slight increase in resorptions at 600 ppm (0.9 vs 1.5), 

although there was wide variability (standard deviations exceed the means). There was a slight 

increase in post-implantation loss at 600 ppm (9.2 vs 5.5). It should be noted that this observation 

was not corroborated since post-implantation losses in the P1 adults of the definitive study were 

comparable amongst all dose groups. Both the 100 ppm and 600 ppm females displayed an increase 
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2, 4-Dichlorophenoxyacetic Acid/PC Code 030001 OPPTS 870.3800; 870.6300, 870.7800/ DACO 4.5.1/ OECD 416

in thyroid weight (↑9%), but there was no dose-response. There were no statistically significant, 

treatment-related differences in serum T3, T4, or TSH in the GD 17 satellite females. Although the 

600 ppm GD 17satellite females displayed the predicted pattern of thyroid hormone changes (↓ T3 

and ↓ T4 with ↑TSH levels) that suggest 2, 4-D exposure may adversely affect thyroid function at 

doses above the renal saturation clearance, the thyroid effects noted below renal saturation are not 

considered sufficiently robust to be adverse. 

F1 Offspring: There were no treatment-related effects on the numbers of live or dead F1 pups 

born/litter or on pup survival or sex ratio. Slightly lower body weights were observed in the 600 ppm 

pups during early lactation, which coincided with the dams decreased food intake LD 1-4 and LD 4-

7). Pup body weight (600 ppm) remained lower in the 600 ppm pups (↓6%) during PND 14-21. 

There was no significant, treatment-related difference in absolute or relative anogenital distance in 

either sex and no differences in nipple/areolae retention between control and high-dose groups in 

either sex. F1 males at 800 ppm displayed a 1.6 days delay in preputial separation (well within 

normal variability), which was accompanied by a very slight reduction in body weight compared to 

the control (↓2.1 grams; 99% of control). The age at vaginal opening was comparable among the 

groups of F1 females.

F1 Offspring Thyroid Assessments: PND 4 - There were no statistically-significant differences in 

serum T3, T4, or TSH in PND 4 culled pups. T4 was reduced to a similar extent in both sexes at the 

300 ppm (↓14%-15%) and 600 ppm/800 ppm (↓12%-14%) dose levels, and female PND 4 pups 

showed an increase in TSH (↑19%) at 600 ppm. F1 PND 22 Weanlings - F1 PND 22 males 

displayed a statistically-significant reduction (↓28%) in T4 at 800 ppm, and F1 PND 22 females 

displayed a non-statistically significant reduction (↓20%) in T4 at 600 ppm. T3 was reduced in the 

males at 300 ppm (↓19%) and 800 ppm (↓13%), but there was no dose response. F1 PND 62-64 -

Both sexes displayed increased TSH at 300 ppm (↑26%) and at 800 ppm (males ↑23%))/600 ppm 

(females ↑24%)), although the increase in males was not dose-related and none of the differences in 

thyroid hormone levels were statistically significant. T4 was decreased at 800 ppm in males (↓13%). 

Though these findings suggest that 2, 4-D exposure may adversely affect thyroid function at doses 

above the renal saturation clearance, the thyroid effects noted below renal saturation are not 

considered sufficiently robust to be adverse.

F1 Unselected Offspring (PND 22 weanlings): There were no effects on survival of the unselected 

weanlings used for systemic toxicity (non-perfused). All treated males displayed a decrease in body

weight (↓9%-10%) compared to the control males. Decreased adrenal weights were observed in 

males at 800 ppm (absolute ↓37% and relative ↓29%). The decreases in kidney (↓15%), liver (↓18%), 

testes (↓15%), and thyroid (↓14%) weights observed in males at 800 ppm were slightly greater than 

the body-weight deficit of 10%. Organ weights were comparable among the groups of females. There 

were no significant differences in perfused absolute brain weights, cerebral lengths and widths or 

cerebellar lengths and widths in perfused F1 PND 22 weanlings of either sex. There were no 

neuropathological observations attributed to treatment in the perfused F1 PND 22 weanlings, and no 

treatment-related changes in myelin in either males at 800 ppm or females at 600 ppm.

F1 Offspring Set 1a (PND 70): All Set 1a pups survived to scheduled sacrifice. Males at 800 ppm 

displayed decreased body weight (↓11%-17%) and body-weight gains (↓11%-25%) throughout the 

study period, with the magnitude of the reduction lessening with time of exposure. Females 

displayed comparable body weight/gain among the groups. Platelet counts were reduced in the 800 
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2, 4-Dichlorophenoxyacetic Acid/PC Code 030001 OPPTS 870.3800; 870.6300, 870.7800/ DACO 4.5.1/ OECD 416

ppm males but not in the females at any dose level. Both sexes displayed a slight increase in ALT 

(↑18%/25%) and an increase in triglyceride (↑31%/43%) levels. Although some of the decreases in 

organ weights observed in the 800 ppm males may be attributed to the 10% decrease in body weight 

at termination, the decreases in liver (↓16%), pituitary (↓14%), and adrenal glands (↓12%) might be 

related to treatment. Increased uterine weights (↑31% absolute and ↑32% relative) were observed at 

600 ppm. Although statistical significance was not attained, the finding is considered treatmentrelated since a similar increase was observed at 600 ppm in the P1 and Set 3 F1 females. Increased 

ovarian weight (↑9%) was observed in the 600 ppm F1 Set 1a females, although statistical 

significance was not attained. Increased kidney weights (↑9% absolute and ↑11% relative) were 

observed in the females at 300 ppm and 600 ppm, although there was no dose-response and kidney

weights were comparable among the male groups. Decreased thymus weights (↓12% absolute and 

↓10% relative) were observed in females at 600 ppm and in Set 3 females at 600 ppm (↓14% 

absolute and ↓13% relative). An increased incidence of degeneration of the proximal convoluted 

tubule in the kidney was observed in males at 300 ppm and 800 ppm and in females at 600 ppm. 

Regarding the terminal stage of estrous, 2 of 10 females at 300 ppm and 3 of 10 females at 600 ppm 

displayed proestrus, whereas none of the 10 females in the control and 100 ppm groups displayed 

proestrus.

F1 Offspring Set 1b (PND 54-56): There were no significant differences in body weight/gain in 

either sex. There was an increase in the level of urination in all treated male groups compared to the 

control group, but there was no dose response. There was a 10% reduction in hind limb grip strength 

at 800 ppm in males and at 600 ppm in females. Males at 800 ppm displayed a decrease in total 

motor activity (↓10%), whereas females at 600 ppm showed an increase (↑12%). During the first half 

of the session, all male groups displayed a similar motor activity level (were within 6%), whereas the 

800 ppm males showed a progressive lessening of activity with increased time; i. e., the 800 ppm 

males displayed decreased activity compared to the control (↓11%, ↓16%, ↓30%, and ↓34% in 

Epochs 5, 6, 7, and 8, respectively). Males at 800 ppm displayed a different acoustic startle response 

(ASR) initially compared to the control males. There was no apparent difference in ASR in females. 

There were no significant differences in perfused absolute brain weights, cerebral lengths and widths, 

or cerebellar lengths and widths in either sex (PND 60). There were no treatment-related (1) 

microscopic changes in the central or peripheral nervous system in the perfused offspring; (2) 

changes in myelin; or (3) changes in microscopic measurements of structures in the cerebral cortex, 

cerebellum, thalamus, or hippocampus.

F1 Offspring Set 2a (PND 67-73): Developmental Immunotoxicity (Primary Immune Response to 

Sheep Red Blood Cells): There were no deaths. Slight decreases in body weights and body-weight 

gains were observed in males at 800 ppm (↓6%-10% and ↓15%) and females at 600 ppm (↓8%-9%

and ↓10%). Terminal body weights were comparable among the male and female groups. Both 

absolute (↓10%) and relative (↓8%) thymus weight decreases were observed in the males at 800 ppm 

and in the females at 600 ppm [absolute (↓13%) and relative (↓10%)]. Males at 300 ppm showed a 

17% decrease in thymus weight but no dose response. Spleen weights were slightly lower in females 

at 600 ppm [absolute (↓13%) and relative (↓14%)]. There was no significant difference in response 

for AFC/spleen and AFC/106 splenocytes among the male groups. Females at 600 ppm displayed a 

non-significant decrease of 54% for AFC/spleen and 27% for the AFC/106 splenocytes. 

F1 Offspring Set 2b (PND 67-73): Developmental Immunotoxicity (Natural Killer Cell Activity): 

There were no deaths, and body weights/gains showed a similar slight reduction in males at 800 ppm 
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2, 4-Dichlorophenoxyacetic Acid/PC Code 030001 OPPTS 870.3800; 870.6300, 870.7800/ DACO 4.5.1/ OECD 416

as observed in the other offspring groups. Female body weights/gains were comparable among the 

groups. Terminal body weights (PND 87-93) were comparable among the groups (both sexes). There 

were no significant treatment-related effects on absolute or relative spleen or testes weights in males, 

and no significant treatment-related effects on spleen weights in females (only organs weighed). 

There were no significant, treatment-related differences in the percent target cell cytotoxicity at any

dose level compared to control (both sexes), and 2, 4-D did not alter the cytotoxic ability of splenic 

NK-cells in male or female rats at any dose level. 

F1 Offspring Set 3 (PND 90 or 139): Reproductive Toxicity: There were no treatment-related 

deaths or clinical signs of toxicity. Terminal body weights were comparable among the groups (both 

sexes). No significant differences were observed in mean estrous cycle length at any dose level 

compared to the control. There were no significant, treatment-related effects on the numbers of small 

follicles, growing follicles, or total follicles. There were no significant, treatment-related effects on 

sperm motility or progressive motility, no differences in testicular spermatid and epididymal sperm 

counts, and no differences in the proportion of abnormal sperm between the control and 800 ppm 

males. Absolute (↓9%) and relative (↓8%) pituitary gland weights were significantly lower in the 800 

ppm males and absolute (↓9%) and relative (↓10%) pituitary gland weights were non-significantly

lower in the 600 ppm females. There was no associated histopathology in the pituitary glands. 

Uterine weights were increased at 300 ppm (↑10% absolute and ↑10% relative) and 600 ppm (↑10% 

absolute and ↑11% relative) compared to the controls. Thymus weights were decreased (↓14% 

absolute and ↓13% relative) in females at 600 ppm, although statistical significance was not attained. 

No histopathological changes were observed in the pituitary or thymus in either sex. A degenerative 

lesion was observed in the kidney (proximal convoluted tubule) in both sexes at 300 ppm and at 600 

ppm/800 ppm. Ovarian follicle counts were comparable between the control and 600 ppm females 

(PND 139).

The parental systemic LOAEL is 800 ppm (45.3 mg/kg bw/day in males), based on 

nephrotoxicity manifested as increased kidney weights, and degenerative lesions in the 

proximal convoluted tubules in the main study P1 rats. The parental systemic NOAEL is 300 

ppm (16.6 mg/kg bw/day in males). No toxicologically relevant effects were identified in P1 

females or in the GD 17 satellite female groups at the highest dose tested (600 ppm; 40.2 

mg/kg/day).

The thyroid toxicity NOAEL is established at 800/600 ppm (45.3 mg/kg/day in males and /40.2 

mg/kg/day in females), the highest dose tested. The thyroid effects noted in the database were 

considered to be adaptive. 

The offspring LOAEL is 300 ppm (20.9/ mg/kg bw/day in males and 23.3 mg/kg/day in 

females), based on kidney toxicity manifested as increased kidney weights and increased 

incidence of degeneration of the proximal convoluted tubules. The offspring NOAEL is 100 

ppm (6.83 mg/kg bw/day in males and 7.59 mg/kg/day in females). 

The DNT offspring (PND 21-60) LOAEL is >800/600 ppm (81.7 mg/kg bw/day in males, 59.2

mg/kg bw/day in females), based on the lack of evidence of DNT (FOB parameters, motor 

activity, and acoustic startle response). The DNT offspring NOAEL is 800 ppm/600 ppm (81.7

mg/kg bw/day in males, 59.2 mg/kg bw/day in females). 
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The DIT offspring (PND 139) LOAEL is >800/600 ppm (71.8 mg/kg bw/day in males, 55.3

mg/kg bw/day in females), based on the lack of evidence of DIT [SRBC antibody-forming cell 

assay (PND 66-70) and Natural Killer Cell assay (PND 87-93)]. The DIT offspring NOAEL is 

800/600 ppm (71.8 mg/kg bw/day in males and 55.3 mg/kg bw/day in females), the highest dose 

tested. 

The reproductive LOAEL is > 800/600 ppm (45.3 mg/kg bw/day in males, 40.2 mg/kg bw/day 

in females), based on the lack of effect on estrous cyclicity, (P1 females, satellite GD 17 dams, 

Set 3 F1 offspring) or reproductive indices (mating, fertility, time to mating, gestation length, 

pre-and post-implantation loss, number of corpora lutea (satellite GD 17 dams), sperm 

parameters, ovarian follicle counts, and reproductive organ histopathology). The reproductive 

NOAEL is 800/600 ppm (45.3 mg/kg bw/day in males, 40.2 mg/kg bw/day in females), the 

highest dose tested. 

This study is classified acceptable/non-guideline. The study does not satisfy a guideline requirement 

for 2, 4-D. It satisfies the data call-in requirements for 2, 4-D for OPPTS 870.3800 (Reproduction 

and Fertility Effects), OPPTS 870.6300 (Developmental Neurotoxicity), OPPTS 870.7800 

(Immunotoxicity). The study is in accordance with the draft OECD extended one-generation 

reproductive toxicity study guideline. 

COMPLIANCE: Signed and dated Good Laboratory Practices (GLP), Quality Assurance, and 

Data Confidentiality statements were provided. 
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2, 4-Dichlorophenoxyacetic Acid/PC Code 030001 OPPTS 870.3800; 870.6300, 870.7800/ DACO 4.5.1/ OECD 416

I. MATERIALS AND METHODS:

A. MATERIALS:

1. Test material: 2,4-D

Description: Solid, off-white flakes (prior to milling)

Lot #: 2006 2433 8006-USA

Purity: 97.85%-98.6% a.i.

Compound stability: Stable in diet for 27 days

CAS # of TGAI: 94-75-7

Structure:

2. Vehicle and/or positive control: none (test material was milled and mixed with certified feed 

flour, then diluted with ground feed). 

3. Test animals:

Species: Rat

Strain: Crl:CD(SD)

Age at study initiation: (P) 10 wks; (F1

) wks

Wt. at study initiation: (P) Males: 340-345 g; Females: 231-233 g

(F1) Males: g; Females: g

Source: Charles River Laboratories Inc. (Portage, Michigan)

Housing: Singly in stainless steel cages, except during mating (1 ♂/1 ♀), littering, early postweaning phases of study; during littering, dams (and their litters) were housed in plastic 

cages provided with ground corn cob nesting material from ≈GD 19 until PND 21.

Diet: LabDiet Certified Rodent Diet #5002, ad libitum (PMI Nutrition International, MO)

Water: Municipal water, ad libitum

Environmental conditions: Temperature:

Humidity:

Air changes:

Photoperiod:

22 ± 1°C

40-70%

12-15/hr

6 hrs dark/6 hrs light

Acclimation period: ≥ one week

B. PROCEDURES AND STUDY DESIGN

1. Mating Procedure: In the main study, each P1 female was placed with a single P1 male from 

the same dose level (1:1 mating). High-dose males had access to the female high-dose diet 

during this period to avoid overdosing the high-dose females. In the satellite groups, each female 

was placed with an unexposed male that had access to a 2,4-D-containing feed crock during the 

mating period only. The rats were paired until mating occurred or two weeks elapsed. During the 

mating period, daily vaginal lavage samples were evaluated for the presence of sperm as an 

indication of mating. The day on which sperm were detected or a vaginal copulatory plug was 

observed in situ was considered gestation day 0 (GD 0). Each sperm- or plug-positive (presumed 

pregnant) female was then separated from the male and returned to her home cage. If mating had 

not occurred after two weeks, the rats were separated without further opportunity for mating. 

After the mating phase, satellite males were euthanized with no further data collection.
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2. Study Schedule: Mating of the P1 adults (main study animals and females from a satellite 

group) commenced after approximately 4 weeks of dietary exposure to 2,4-D. P1 males from the 

satellite group were not exposed to 2,4-D except during the mating period. Main study P1 male 

rats were maintained on the test diets through breeding and for an additional 5-7 weeks (11 

weeks total exposure). P1 females (main study and satellite females) continued on the test diets 

through breeding, gestation (satellite females through GD 17 only), and lactation (study report 

Figures 1 and 2, reproduced below). Triggers for breeding a second generation were identified 

prior to study initiation (Table 1). Since these criteria were not met, breeding of a second 

generation was not conducted.

Table 1. Triggers for Decision Making on the Need for a Second Generation

P1 Estrous Cycle Evaluation Trigger1

P1 Fertility Trigger2

F1 Litter Parameters Trigger3

F1 Developmental Landmarks (AGD, nipple retention, puberty onset) Trigger4

F1 Estrous Cycle Evaluation Trigger1

1 If biologically relevant, dose-related changes in estrous cycle length without overt toxicity in dams

2 In the absence of corresponding, exposure-related reproductive organ histopathology

3 If significant or biologically relevant, exposure-related decreases in litter size/pup survival are seen 

in the absence of severe maternal toxicity or lethality

4 Dose-related effects; in the absence of body weight-mediated changes in these parameters

3. Animal Assignment: Prior to dose initiation, rats were stratified by body weight and randomly

assigned to exposure groups (Table 2) using a computer program designed to increase the 

probability of uniform group mean body weights and standard deviations at the start of the study.

TABLE 2. Animal Assignment (Study Phases)

Dose 

(ppm)

MALES Dose 

(ppm)

FEMALES

0

100

300

800

P1 males (main study; n=27)

0

100

300

600

P1 females (main study; n=27) B

P1 males (satellite; n=12) A

P1 females (satellite; n=12)

F1 Set 1a (systemic toxicity; n=10) CF1 Set 1a (systemic toxicity; n=10) D

F1 Set 1b (developmental neurotoxicity; 

n=10) E

F1 Set 1b (developmental neurotoxicity; 

n=10) F

F1 Set 2a (developmental immunotoxicity; 

SRBC; n=10) E

F1 Set 2a (developmental immunotoxicity; 

SRBC; n=10) F

F1 Set 2b (developmental immunotoxicity; 

NK cell assay; n=10) E

F1 Set 2b (developmental immunotoxicity; 

NK cell assay; n=10) F

F1 Set 3 (reproductive toxicity; n=24-27)EF1 Set 3 (reproductive toxicity; n=24-27) F

* Ano data collected since these rats were not treated with 2, 4-D except during mating; 

B

On LD 7-14 the dietary concentrations 

adjusted to 0, 50, 150, and 300 ppm; on LD 14-21 to 0, 33, 100, and 200 ppm.; 

C

On PND 21-28 and PND 28-35 the dietary 

concentrations were adjusted to 0, 50, 150, and 300 ppm and 0, 50, 150, and 400 ppm, respectively. DOn PND 21-28 and PND 28-

35 the dietary concentrations were adjusted to 0, 50, 150, and 300 ppm; 

E

On PND 21-28 and PND 28-35 the dietary concentrations 

were adjusted to 0, 50, 150, and 300 ppm, and 0, 50, 150, and 400 ppm, respectively; FOn PND 21-28 and PND 28-35 the dietary 

concentrations were adjusted to 0, 50, 150, and 300 ppm.
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4. Dose Selection Rationale: Doses were selected (Table 3) based on a combined analysis of 

toxicity and toxicokinetic (TK) data collected from the 2,4-D range-finding/TK study

(APPENDIX A - MRID 47417901; abbreviated DER ) and titration studies (APPENDIX B -

MRID 47417902; abbreviated DER). The high dose was set at or slightly above the threshold for 

nonlinear kinetics. As stated in the report, the range-finding study confirmed a gender-based 

difference in the renal clearance of 2,4-D in adult rats, and different high-dose levels were 

selected for the sexes. The male high dose was 40 mg/kg/day (dietary concentration of 800 ppm), 

slightly higher than the inflection point for nonlinear TK in male pups from PND 35 to 

adulthood. The female high dose was 30 mg/kg/day (600 ppm), which was clearly higher than 

the inflection point in female pups and adults throughout the entire (range-finding) study. The 

mid- and low-dose levels were the same for both sexes. The low dose of 5 mg/kg/day (100 ppm) 

was predicted to identify a clear NOAEL and was consistent with the dose identified in a 

previously conducted 2,4-D dietary 2- generation reproduction study (MRID 00150557, MRID 

00163996). The mid-dose level of 15 mg/kg/day (300 ppm) would provide dose-response data. It

was anticipated that the 300 ppm mid-dose level would be slightly above the inflection point for 

non-linear TK in female adults and pups, and within the range of linear TK for adult males. The 

dose range-finding and toxicokinetic studies conducted in pregnant rats indicated that 2,4-D was 

adequately transferred through maternal milk to pups (MRIDs 47417901 and 47417902). The 

2,4-D pup/dam plasma ratios on LD 4 were 0.29, 0.30, and 0.48 for dams treated with 100, 400 

and 800 ppm 2,4-D, respectively. By LD 14, the pup/dam plasma 2,4-D ratios increased to 0.75, 

1.25 and 1.70 for the respective dietary doses. Because there was evidence of significant transfer 

of 2,4-D to neonatal pups through milk (μg 2,4-D/g milk on LD 4 were: 1.08 at 100 ppm, 9.27 at 

400 ppm and 30.84 at 800 ppm), it was not necessary to directly gavage dose neonates with 2,4-

D.

Table 3. Study Design. 

Dietary Concentration (ppm) ≈ Doses (mg/kg/day) P1 

#rats/sex/dose

Satellite group 

(targeted # pregnant 

females/dose) male female Male female

0 0 0 0 27 12

100 100 5 5 27 12

300 300 15 15 27 12

800 600 40 30 27 12

5. Dietary Concentration Adjustment: To avoid potential overdosing of females during mating, 

co-housed rats were fed the female diet. No dietary concentration adjustments were performed 

during gestation or during the first week of lactation. The dietary concentration adjustments 

during lactation and post-weaning were designed to provide a relatively constant mg/kg body

weight/day dose throughout all life phases, these are outlined in Table 4.

Table 4. 2,4-D Dietary Concentration Adjustments During Lactation/Post-Weaning

Exposure period TMI Increase1 Unadjusted (ppm) Adjustment Factor Adjusted (ppm)

LD 7-14 3.1X 100, 300, 600 2X 50, 150, 300

LD 14-21 3.8X 100, 300, 600 3X 33, 100, 200

PND 21-28 2.4X 100, 300, 600 2X 50, 150, 300

PND 28-35 1.9X F: 100, 300, 600

M: 100, 300, 800

2X F: 50, 150, 300

M: 50, 150, 400

 1

relative to non-pregnant adult females; TMI= test material intake based on feed consumption data derived

 from male Crl:CD(SD) rats at PND 23-28 from Marty et al., 2003.
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During lactation, dietary concentrations were adjusted using historical control feed consumption 

data for lactating females to account for the large and rapid increase in feed consumption (2-3-

fold) typical for dams in mid to late lactation. Dams awaiting necropsy received a diet containing 

the same concentration of 2, 4-D that was given during breeding. To account for the large 

amount of feed consumed per kg body weight in weanling pups from PND 21-28, male and 

female weanlings received a diet containing the same concentration of 2, 4-D that was given to 

the P1 females during the second week of lactation (one-half the adult female concentration). 

From PND 28-35, female weanlings received a diet containing the same concentration of 2, 4-D 

that was given to the P1 females during the second week of lactation (one-half the adult female 

concentration). Male weanlings received a diet containing one-half the adult male concentration 

of 2, 4-D during PND 28-35; i.e., concentrations in the mid- and low-dose groups for male 

weanlings during this interval paralleled that of the female weanlings, but the high-dose male 

weanlings received a higher dose than female weanlings of the same age. F1 male and female 

offspring were transitioned to adult dietary concentrations of 2, 4-D on PND 35, which continued 

until termination.

6. Dosage Preparation and Analysis: Prior to diet mixing, the test material was air milled (particle 

size: ≈149 micron) and used for preparation of a concentrated test material-feed mixture (premix). Each pre-mix was prepared by mixing test material with certified feed flour to minimize 

clumping, then diluting this mixture with ground feed to achieve the targeted pre-mix 

concentration. Diets were prepared by diluting pre-mixes with ground feed. Control diets were 

prepared by mixing feed flour with ground diet using a similar procedure. Pre-mixes and test 

diets were not adjusted for purity and were prepared every 3 weeks based on stability data. 

Analysis: Homogeneity: Representative samples of the low- and high-dose test diets were 

evaluated concurrently with the concentration verification analyses to ensure homogeneous 

distribution of the test material in the feed.

Stability: In the range-finding study (MRID 47417901), stability of 2, 4-D in the diet was 

determined to be 27 days. Fresh diets were prepared at least once every 3 weeks for the main 

study. Additional stability work was conducted to verify that the lowest concentration in the main

study was stable for the 3-week feeding period.

Concentration Verification: Dose confirmation analyses of all dose levels, plus control and 

premix, were determined pre-exposure. Dose confirmation included sampling during the 

following study phases: 4 weeks (end of pre-breeding period), 8 weeks (approximately the end of 

gestation), 12 weeks (at weaning), 15 weeks, and 19 weeks. The homogeneity of the low-dose 

and high-dose diets was determined concurrent with dose confirmation. The method used for 

analyzing the test material in the diet was a solvent extraction followed by analysis using liquid 

chromatography-mass spectrometry (LCMS) with internal standards.

Results: Homogeneity analysis: Analyses of low-dose and high-dose diets indicated that 2, 4-D 

was homogenously distributed in the feed based on the mean and relative standard deviations, 

ranging from 2.6 to 11.2%. Stability analysis: Previously, 2, 4-D was shown to be stable in the 

diet for < 27 days at concentrations ranging from 0.01 to 0.5% (Marty and Andrus, 2007). 

Additional stability work verified that lower dietary concentrations used during the study

(0.0033%) were stable. Diets were used within 3 weeks of preparation. Concentration analysis:
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Analyses of the test diets for concentration verification were conducted on nine different 

occasions during the study. The average concentrations of 2, 4-D in the diets were 86.7% to 

106.8% of the targeted concentrations. Of the 55 samples analyzed over the course of the study, 

all but four samples were within + 10% of nominal and all analyses were within + 15%.

C. METHODS

PARENTAL ANIMALS

1. Observations: Observations and the schedule for those observations are shown in Figures 1 and 

2 (reproduced from study report pages 190-191).

a. Cageside Observations: All rats were observed for morbidity and mortality at least twice 

daily. Cageside examinations (rats not hand-held) included an assessment for 

decreased/increased activity, repetitive behavior, vocalization, in-coordination/limping, 

injury, neuromuscular function (convulsion, fasciculation, tremor, and twitches), altered 

respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in 

fecal consistency, and fecal/urinary quantity. 

b. Clinical Examinations: Clinical observations were conducted on all males prior to exposure 

and weekly thereafter; on all females pre-exposure and weekly throughout the pre-breeding 

and breeding periods. Mated (sperm-positive or plug-positive) females received clinical 

examinations on GD 0, 7, 14, and 20. Females that delivered litters were subsequently

evaluated on LD 0, 1, 4, 7, 14, and 21. Females were observed for signs of parturition (≈GD 

20), and dams were examined for signs of abnormal nursing behavior. Examinations 

included a careful, hand-held examination of the rat with an evaluation of abnormalities in 

the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive 

system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of 

general behavior, injuries, or palpable mass/swellings. 

c. Detailed clinical observations (DCO): The DCO evaluations were performed on the P1 rats 

of both sexes pre-exposure and once during the last week of the pre-mating period and 

included cage-side, hand-held, and open-field observations (Table 5).

Table 5. DCO Parameters

Cage-Side Observations

Abnormal movements or behaviors 

Resistance to removal from cage
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Hand-Held Observations 

Eye observations 

- Palpebral closure 

- Pupil Size 

- Lacrimation (non-colored periocular wetness) 

Salivation (non-colored perioral wetness) 

Muscle tone 

Extensor-thrust response 

Reactivity to stimuli 

Abnormal behavior 

Abnormalities of the eye Description

Abnormal urine or feces Description

Abnormalities of the gastrointestinal (GI) tract 

Injury 

Missing extremity 

Abnormal muscle movements 

Palpable mass/swellings 

Abnormal posture 

Abnormalities of the reproductive system 

Abnormal respiration 

Abnormal skin or hair-coat/mucous membranes 

Excessive soiling 

General abnormalities

Open-Field Observations 

Responsiveness to touch 

Gait evaluation

 From Table 1, page 227 of the report

d. Dam observations: Dams were observed periodically for signs of parturition beginning on 

or about GD 20. When possible, parturition was observed for signs of difficulty or unusual 

duration. The day of parturition was recorded as the day one or more delivered fetuses were 

noted (designated lactation day 0; LD 0).

2. Body weight: All parental rats were weighed during the pre-exposure period and weekly during 

the 4-week pre-mating period. Males were weighed weekly after mating until termination. 

Mated females were weighed on GD 0, 7, 14, and 15 (satellite group)/20 (main study). Lactating 

females were weighed on LD 1, 4, 7, 14, and 21. 

3. Food consumption and compound intake: Food consumption was measured at intervals 

similar to body-weight measurements. For females delivering litters, food consumption was 

measured every 2-4 days. 

4. Estrous Cycle Evaluation: Vaginal lavage samples from all P1 females were collected daily

beginning one week after the initiation of dosing. Estrous cycles were evaluated for 3 weeks 

prior to mating, and during cohabitation until each female was sperm-or plug-positive or until the 

two week mating period had elapsed. Vaginal lavage slides collected during the 3-week period 

prior to mating were examined microscopically to determine estrous cycle length and pattern. On 

the day of scheduled necropsy, the stage of the estrous cycle was also determined. 

5. Sperm Analyses: Sperm parameters were evaluated in P1 and Set 3 F1 males at termination. 

The left and right epididymides and testes were allocated as follows: right epididymis: motility

and histopathology; left epididymis: counts; right testis: histopathology; left; testis: counts.
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a. Motility: Sperm motility was evaluated in P1 males from all dose groups. Immediately after 

euthanasia and isolation of their epididymides, a small sample of sperm from the right cauda 

epididymis was processed (placed in chamber of the HTM Integrated Visual Optical System) for 

the determination of total percent motile (showing any motion) and percent progressively motile 

(showing net forward motion) sperm. After sperm were released, the epididymis was placed in 

Bouin’s fixative for histopathological examination. 

b. Counts: The left testis and cauda epididymis were weighed and then frozen at ≈ -20°C for 

subsequent determination of the number of homogenization-resistant spermatids and sperm per 

testis/cauda epididymis and per gram of testicular/epididymal tissue. Spermatid/sperm counts 

were evaluated from the control and high-dose groups, as well as any males that failed to mate 

successfully during the mating period. Since treatment–related effects were not observed, sperm 

from the lower-dose levels were not evaluated. 

c. Morphology: An aliquot of sperm suspension was placed on a slide, and a smear prepared and 

air-dried for subsequent evaluation of sperm morphology. At least 200 sperm per male were 

evaluated and classified as normal or abnormal. Morphological evaluation of sperm was 

conducted using samples from the control and high-dose groups and any male that failed to mate 

successfully during the mating period. Sperm morphology was scored “blinded” with respect to 

exposure group. Since treatment-related effects were not observed, sperm from the lower-dose 

levels were not evaluated.

OFFSPRING

1. Litter Observations: Litters were examined as soon as possible after delivery (Table 6). Dead 

pups were sexed, examined grossly for external and internal abnormalities, and preserved. Any

visible physical abnormalities or demeanor changes in the neonates were recorded as they were 

observed during the lactation period on LD 0, 1, 4, 7, 14 and 21. Any pup found dead or 

sacrificed in moribund condition was sexed and examined grossly, to the extent possible, for 

external and visceral defects. These pups were preserved in neutral, phosphate-buffered 10% 

formalin.

TABLE 6. Litter Observations a

Observation

Time of observation (lactation day)

Day 

0

Day 1 Day 4 Day 7 Day 14 Day 21

Number of live and dead pups X X X X X X

Pup sex and body weight X X

b , c X X X

External alterations or demeanor changes X X X X X X

a

Data obtained from page 57 in the study report. bBefore standardization (culling). cAfter standardization (culling)

Pups that died or appeared moribund were examined for the presence of milk bands (i.e., milk in 

the pup’s stomach that was visible on external examination). F1 offspring received weekly

clinical observations until necropsy. Examinations included a careful, hand-held examination of 

the rat with an evaluation of abnormalities in the eyes, urine, feces, gastrointestinal tract, 

extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous 

membranes, as well as an assessment of general behavior, injuries, or palpable mass/swellings.
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2. Body Weight: The F1 offspring were weighed weekly beginning on PND 21, on the day of 

puberty onset (i.e., vaginal patency or preputial separation), and at termination. The Set 1b F1 

offspring also were weighed on the day of the FOB and acoustic startle measurement. 

3. Food Consumption: Feed consumption was determined weekly beginning when the F1 

offspring were individually housed on PND 28 until the day prior to necropsy. Feed consumption 

was calculated as above for adults.

4. Anogenital Distance: Anogenital distance (absolute and relative to the cube root of body

weight) was measured in all F1 pups on PND 1.

5. Culling and Culled Pups: All litters were standardized to ten pups per litter on PND 4 by

randomly ordering the pups in each litter by sex. Pups culled were randomly selected using a 

computer generated randomization procedure, so that five males and five females remained in 

each litter (where possible), or unequal numbers were retained; e.g., six males, four females. 

Litters with fewer than ten pups were not culled. Preferential culling of runts was not performed. 

Culled pups that were not used for thyroid sampling (see below) were euthanized, examined 

grossly for external and visceral defects, and discarded.

6. F1 Culled Pup Sampling for Thyroid Analysis: PND 4 culled pups were anesthetized, 

examined grossly, and blood was collected (heart). To ensure a sufficient volume for hormone 

analyses, blood was pooled by sex for each litter until a total of 10 samples/sex/dose were 

obtained. Thyroid glands were harvested from the same subset of the pups used for thyroid 

hormone analyses. The trachea with the thyroid gland were removed and preserved for possible 

histopathological examination. Following thyroid gland removal, the animals were euthanized 

via decapitation. 

7. Nipple/Areolae Retention: All offspring were evaluated for the presence of nipple/areolae on 

PND 12. The average number of nipples/areolae in male and female offspring in each litter was 

determined. The mean number of nipples/areolae for males and females in each dose group was 

calculated from these litter means. Observers were blind to exposure group. Since there was no 

significant increase in retained nipples/areolae in males on PND 12, Set 3 males were not 

examined for this endpoint at necropsy. 

8. Weaning and Set Assignment: All litters were weaned on PND 21. Three male and three 

female F1 pups/litter were randomly selected, and one male and one female each were assigned 

to Set 1, 2, or 3. For Set 1 (Figure 4; reproduced from study report page 193), one male or one 

female per litter were assigned to Set 1b: the Developmental Neurotoxicity group (n = 10 

males + 10 females/dose level with 20 litters represented). Unselected F1 males or F1 females 

from each litter designated for Set 1 were assigned to Set 1a: the Systemic Toxicity group (n = 

10 males + 10 females/dose level with 20 litters represented). Similarly, 20 F1 males and 20 F1 

females/dose level were assigned to Set 2 (Figure 5; reproduced from study report page 194):

the immunotoxicity group. One male and one female per litter were assigned to Set 2a for the

initial evaluation of immunotoxicity with the SRBC AFC assay (n = 10 males + 10 

females/dose level with 10 litters represented). One male and one female from the remaining 

litters designated for Set 2 were used for Set 2b (n = 10 males + 10 females/dose level with 10 
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litters represented). This group was used for a secondary evaluation of immunotoxicity (i.e., 

natural killer (NK) cell assay). One male and one female per litter were randomly selected for 

assignment to Set 3 (Figure 6; reproduced from study report page 195): the reproductive 

endpoints group (n > 20 males and 20 females/dose level). If there were insufficient litters from 

which to select both male and female offspring for groups 1, 2, and 3, additional animals were 

randomly selected from available litters as needed in order to obtain the required number of 

animals/dose level. Use of same sex littermates in the same Set (1, 2, 3) was avoided whenever 

possible. The assignment of pups from each litter is shown in study report Appendix Table 161. 

Pups were transpondered on ~ PND 17 for individual identification after weaning. Selected F1 

pups were housed in plastic cages with same sex littermates from PND 21-28, then individually

thereafter. Non-selected pups (Figure 1 above and Figure 3 below): All non-selected F1 

weanlings (designated for pathological evaluation as described below) were given a gross 

necropsy on PND 22. When possible, one pup/sex/litter (n = 12/sex/dose) at weaning was 

perfused for neuropathology evaluations. Additionally, one pup/sex/litter (n = 10/sex/dose) 

was randomly selected for a necropsy examination with collection of blood for thyroid hormone 

analyses, organ weights, and preservation of tissues for subsequent histopathological 

examination. Details of weanling necropsies are described below. Any excess weanlings were 

discarded.
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9. Puberty Onset: All F1 animals (Sets 1-3) were observed daily for vaginal opening beginning on 

PND 26 or for balano-preputial separation beginning on PND 35. The presence of vaginal and 

preputial threads (if any) was noted. Age and body weight of the rats were recorded on the day

these markers of puberty onset were acquired. The age and body weight at puberty onset were 

averaged by litter for data analysis. 

10. Estrous Cycle Evaluation: The estrous cycle was evaluated in Set 3 F1 females for 4 weeks 

from ~ PND 40-68 (initiated after vaginal patency). On the day of scheduled necropsy, the stage 

of the estrous cycle was also determined for the adult F1 female rats (Sets 1a and 3). 

ASSESSMENTS COMMON AMONG AGE GROUPS

1. Hematology, Clinical Chemistry, and Urinalysis: Blood was collected from 10 rats/sex/dose 

from P1 males and P1 main study females and Set 1a F1 males and females (both sexes fasted 

overnight) and GD 17 Satellite dams (not fasted because pregnant). Blood samples were obtained 

from the orbital sinus following anesthesia with O2/CO2 at the scheduled necropsy (after ~11 

weeks of exposure in P1 males; on LD 22 in P1 females; on ≈PND 70 in Set 1a F1 offspring). 

The CHECKED (X) hematological, coagulation, and clinical chemistry parameters were 

examined. 
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a. Hematology:

X Hematocrit (HCT)* X Leukocyte differential count*

X Hemoglobin (HGB)* X Mean corpuscular HGB (MCH)*

X Leukocyte count (WBC)* X Mean corpusc. HGB conc.(MCHC)*

X Erythrocyte count (RBC)* X Mean corpusc. Volume (MCV)*

X Platelet count* X Reticulocyte count

Blood clotting measurements*

(Thromboplastin time)

(Clotting time)

X (Prothrombin time)

b. Clinical chemistry:

X ELECTROLYTES X OTHER

X Calcium X Albumin

X Chloride X Creatinine

Magnesium X Urea nitrogen

X Phosphorus X Total Cholesterol

X Potassium X Globulins

X Sodium X Glucose

ENZYMES X Total bilirubin

X Alkaline phosphatase (ALK) X Total protein (TP)

Cholinesterase (ChE) X Triglycerides

Creatine phosphokinase Serum protein electrophoresis

Lactic acid dehydrogenase (LDH)

X Alanine aminotransferase (ALT/also SGPT) X TSH

X Aspartate aminotransferase (AST/also SGOT X T3

Sorbitol dehydrogenase X T4

X Gamma glutamyl transferase (GGT)

Glutamate dehydrogenase

c. Urinalysis: Urine samples were obtained from P1 males and Set 1a F1 offspring (PND 64-66 

males and females) during the week prior to the scheduled necropsy. Rats were housed in 

metabolism cages, and urine was collected overnight (≈16 hours). Feed and water were available 

during this procedure. The CHECKED (X) parameters were examined. 

X Appearance/color X Glucose

X Volume X Ketones

X Specific gravity/osmolality X Bilirubin

X PH X Blood/blood cells

X Sediment (microscopic) Nitrate

X Protein X Urobilinogen

2. Toxicokinetics (TK) (GD 17 Satellite dams, Set 3 F1) Blood samples (5/dose group) collected 

from non-fasted GD 17 dams at necropsy were used for single time point TK analyses to 

determine blood levels of 2,4-D. These samples were a subset of samples taken for clinical 

pathology and thyroid hormone assessment. For Set 3 adults, three blood samples (~200 μl) were 

collected from randomly selected non-fasted animals (5 rats/sex/dose) on PND 63 and PND 84. 

Blood was collected from the jugular vein without the use of any anesthesia. The first blood 

sample was collected at 6:00 AM (at the time when lights were turned on), the second blood 
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sample was collected 3 hours later (9:00 AM) and the final blood sample was collected ~1 hour 

prior to the start of the dark phase of the photocycle (5:00 PM). As demonstrated by Saghir et al., 

(2006), these samples were used to estimate Cmax, Cmin, and AUC. Blood was collected from 

different Set 3 rats at the PND 63 and 84 sampling time points. Immediately after collection, 

blood samples were centrifuged to separate plasma from red blood cells. Plasma was carefully

removed, placed in glass vials and stored in the –80°C freezer until analysis. Stability of 2, 4-D 

in plasma was established in the previous range-finding study (MRID 47417902).

3. Hormone Measurements, TK Samples, and Clinical Pathology: Serum for Thyroid Hormone 

Analyses (Satellite dams, Culled pups, PND 22 Nonperfused Weanlings, Set 1a F1). Blood from 

PND 4 culled pups (first 10 litters/dose, if possible; male and female samples separate), PND 22 

assigned for termination and standard histopathology (nonperfused), GD 17 satellite dams, and 

Set 1a F1 offspring (10/sex/dose) was collected under isoflurane anesthesia and placed on ice 

immediately after collection. Blood from PND 4 same sex pups culled from the same litter were 

pooled in plastic serum separator tubes and placed on ice. A terminal blood sample from nonfasted GD 17 satellite dams, non-fasted PND 22 weanlings assigned for termination and standard 

histopathology (non-perfused), and culled pups were used, whereas Set 1a F1 offspring were 

anesthetized a few days prior to necropsy (~PND 65, non-fasted) for in-life sample collection to 

avoid the potential effects of fasting on thyroid hormone levels. Serum was separated from cells 

as soon as possible following blood collection. All sera samples for hormone assays were stored 

at –80°C. Samples were shipped on dry ice to AniLytics Incorporated (Gaithersburg, Maryland) 

for analyses of triiodothyronine (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH) 

using radioimmunoassays. If the volume of serum samples was limited, the priority for hormone 

analyses was T4, TSH, then T3. Blood samples were not collected from satellite dams that were 

not noticeably pregnant on GD 17, nor from any PND 22 weanlings not assigned for termination 

and standard histopathology (nonperfused). 

4. Postmortem Observations (Anatomic Pathology)
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a. P1 Adult and Set 3 F1 Offspring: P1 males (fasted) were necropsied after ~ 11 weeks of 

exposure. Adult P1 females (fasted) were terminated on LD 22 after weaning of their litters, 

or at least 24 days after the end of the mating period for females with no evidence of mating. 

F1 male and female adults (fasted) from Set 3 were necropsied on ~ PND 139. Rats were 

weighed and vaginal lavage smears were prepared from surviving P1 and Set 3 F1 females 

prior to necropsy. The rats were anesthetized, blood was collected from the orbital sinus (P1 

males and females only), and the rats were euthanized by decapitation. A complete necropsy

was conducted on all rats. The uteri of all P1 females were stained with an aqueous solution 

of 10% sodium sulfide stain after removal of the ovaries and examined for the presence and 

number of implantation sites (data not provided). After evaluation, uteri were preserved. 

Weights of the left testis and left cauda epididymis were collected in P1 males and Set 3 F1 

males for use in calculating sperm count parameters. Histopathological examinations were 

conducted on 10 control and 10 high-dose animals/sex, as well as males and females that 

failed to mate and/or deliver offspring. The following tissues (X) were prepared for 

microscopic examination and weighed (XX): 

DIGESTIVE SYSTEM CARDIOVASC./HEMAT. NEUROLOGIC

- Tongue X Aorta XX Brain (multiple sections)*

X Salivary glands X Heart X Peripheral nerve (tibial)

X Esophagus X Bone marrow X Spinal cord (3 levels)

X Stomach X Lymph nodes XX Pituitary*

X Duodenum XX Spleen* X Eyes 

X Jejunum XX Thymus♪ X Cranial nerve (optic nerve)

X Ileum GLANDULAR

X Cecum XX Adrenal gland*

X Colon UROGENITAL X Lacrimal gland

X Rectum XX Kidneys* XX Thyroid* b

XX Liver* X Urinary bladder XX Parathyroids* b

Gall bladder XX Testes*

X Pancreas XX Epididymides* OTHER

XX Prostate* X Bone (including joint)

XX Seminal vesicle*a X Skeletal muscle

RESPIRATORY XX Ovaries*+ X Skin

X Trachea XX Uterus* c X All gross lesions and masses*

X Lungs X Mammary gland* X Oral tissues

X Nose X Vagina* X Mesenteric tissues

- Pharynx X Oviducts* X Mediastinal tissues

X Larynx X Cervix*

Data from Table 4, page 230 of the report

* tissues for histopathological examination in P1 rats (both sexes) & Set 3 F1 offspring; ♪ examined in Set 3 F1 offspring

a with coagulating glands and fluids

b Thyroid and parathyroids were weighed together

c with oviducts and cervix

b. Satellite Females: Satellite females (not fasted) were necropsied on GD 17. Rats were 

weighed, anesthetized, and blood samples were collected by orbital sinus. The tracheas were 

exposed and clamped, and the animals were euthanized by decapitation using caution not to 

damage the thyroid glands. A complete necropsy was conducted on all rats. The necropsy
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included an examination of the external tissues and all orifices. The head was removed, the 

cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. 

The eyes were examined in situ, and the thoracic and abdominal cavities were opened and the 

viscera examined. All visceral tissues were dissected from the carcass, re-examined and 

selected tissues were incised. Weights of the thyroid/parathyroid glands (post-fixation) were 

recorded, and the thyroid/parathyroid glands were preserved. No other tissues were weighed 

or saved. Histopathological evaluation of thyroids from control and all dose groups of the 

GD 17 dams was performed. Gross observations and the number of corpora lutea, 

implantation sites, and resorptions were recorded. Fetuses were humanely euthanized, 

examined grossly, and discarded.

c. Culls and Pups Dying or Sacrificed Moribund Before Weaning: Some pups selected for 

culling on PND 4 were used for thyroid hormone assessment (T3, T4, and TSH). These pups 

were anesthetized, and blood was collected (heart nick) for serum thyroid hormone analyses. 

Blood samples were pooled as needed for same sex pups from the same litter. After blood 

collection, thyroids/parathyroids were collected and preserved for possible future 

histopathological evaluation. Pups were euthanized by decapitation. Cull pups not selected 

for thyroid evaluation were euthanized, examined grossly for external and visceral defects, 

then discarded. Any pup found dead or sacrificed in moribund condition was sexed and 

examined grossly, to the extent possible, for external and visceral defects, and preserved.

d. PND 22 Weanlings: On PND 22, weanlings (non-fasted) not assigned to Sets 1, 2, or 3 were 

assigned to a non-perfusion group (1 pup/sex/litter from 10 litters/dose) or a perfusion group 

(1pup/sex/litter from 12 litters/dose). Gross pathological examination was performed on both 

non-perfused and perfused selected weanlings. The gross necropsy was performed as 

described for adults, except that the weanlings were not fasted overnight. Any remaining 

weanlings not assigned to any of the sets or groups were anesthetized, euthanized by cervical 

dislocation or decapitation and discarded.

Non-Perfusion Gross Necropsy/Histopathology: PND 22 pups randomly selected at the 

time of weaning were subjected to a complete necropsy. Pups were anesthetized, weighed, 

blood collected by orbital sinus or cardiac puncture, and euthanized by decapitation. Organ 

weights (Table 7) were recorded from 10/sex/dose (representing 10 litters/dose, if possible). 

The listed organs, and the epididymides and ovaries, as well as relevant gross lesions, were 

preserved. Slides of kidney, liver, spleen, thyroid, and testis were processed by standard 

histologic procedures, and histopathological examination of these organs was performed in 

high-dose and control rats. Relevant gross lesions at all dose levels also were examined. 

Based on a lack of treatment-related findings at the high dose, no tissues were examined 

from the lower-dose groups. 
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Table 7. Organs Weights of PND 22 Non-Perfused Weanlings

Adrenals Liver

Brain Spleen

Pituitary (post-fixation) Thyroid/parathyroid (post-fixation)

Kidney Testes

Perfusion Neurotoxicity Group (weanlings): Necropsy: Selected weanlings (non-fasted) 

were injected (i.p.) with heparin ≈10 minutes prior to perfusion and anesthetized. While 

under deep anesthesia, the heart was exposed, the left ventricle cannulated, and the right 

atrium was incised. Rats were perfused by gravity pressure with phosphate buffer containing 

sodium nitrite followed by a phosphate-buffered solution of glutaraldehyde – formaldehyde 

and tissues were examined for gross pathologic findings. The brain (excluding the olfactory

lobes), head, spinal column with spinal cord, fore- and hind limbs, and tail were trimmed to 

remove excessive skin and muscle as needed; muscles from the hind limbs were reflected to 

further expose the nerves. Tissues listed in Table 7 were saved. Neuropathology: Tissues 

(Table 9 below under DNT Set 1f offspring) were prepared from all rats in all dose groups. 

Nine cross-sections of the brain were prepared from the following structures: olfactory bulb, 

cerebrum (frontal, parietal, temporal and occipital lobes), thalamus/hypothalamus, midbrain, 

pons, medulla oblongata, and cerebellum. In addition, sections were prepared from the 

trigeminal ganglion and nerve, pituitary gland, eyes with optic nerves, spinal cord (cervical 

and lumbar), olfactory epithelium, and skeletal muscles (gastrocnemius and anterior tibial). 

Additionally, sections containing representative sections of the cerebellum and corpus 

callosum were stained (Luxol Fast Blue) to assess myelin. Spinal nerve roots (cervical and 

lumbar), dorsal root ganglia (cervical and lumbar), and peripheral nerves (sciatic, tibial 

(proximal and distal (muscular); at the knee and calf muscle branches) and sural) were 

osmicated, embedded in epoxy resin, sectioned ≈ 2 to 3 μm thick and stained with toluidine 

blue. Tissues were evaluated using a light microscope. Histopathologic findings were 

subjectively graded as appropriate to assess the potential effects of exposure with regard to 

the contribution of a specific lesion to the health status of an animal. Histopathological 

examination was performed in high-dose and control rats only, since no relevant gross 

lesions or treatment-related histopathologic effects were found at the high dose. Brain 

Weight and Gross Measurement: Brain weight and gross linear measurements were 

recorded on all dose groups. Brains (excluding the olfactory lobes) were weighed following 

7-15 days of fixation. Linear measurements consisted of the: 1) cerebral length (L2 – anterior 

to posterior, excluding olfactory lobes) and width (L3 – maximum), and 2) cerebellar length 

(L10 – anterior to posterior) and width (L5 – maximum), which were obtained using a handheld, electronic digital slide caliper (daily calibration).

5. Sperm Analyses: Sperm parameters were evaluated in Set 3 F1 males at termination 

(same procedures as for P1 males). 

DEVELOPMENTAL NEUROTOXICITY (F1 offspring Set 1b)

1. Functional observational battery (FOB): The FOB was conducted between PND 54 and 

PND 56 under red light conditions by the same observer (“blinded” to the exposure status of 

the rat) on randomly selected Set 1b F1 rats (10 males and 10 females representing 20 litters) 

at approximately the same time each test day. The FOB included cage-side, hand-held, and 
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open-field observations, and measurements of body weight, rectal temperature, grip 

performance, and landing foot splay (Table 8). 

Table 8. FOB Parameters 

Cage-side Observations 

Abnormal movements or behavior 

Resistance to removal from cage 

Open-field Observations

Level of activity (ambulatory and rearing) 

Responsiveness to sharp noise 

Responsiveness to touch 

Responsiveness to tail pinch 

Gait evaluation 

Urination 

Defecation 

Measurements

Rectal temperature 

Hind limb grip performance 

Forelimb grip performance grams

Landing foot splay

Hand-held Observations 

Palpebral closure 

Lacrimation (non-colored periocular wetness) 

Pupil size 

Pupil reactivity 

Salivation (non-colored perioral wetness) 

Muscle tone 

Extensor-thrust response 

Reactivity to handling 

Categorical Observations

Abnormal behavior 

Abnormalities of the eye 

Abnormal urine or feces 

Abnormalities of the gastrointestinal (GI) tract 

Injury 

Missing extremity 

Abnormal muscle movements 

Palpable mass/swellings 

Abnormal posture 

Abnormalities of the reproductive system 

Abnormal respiration 

Abnormal skin or hair-coat/mucous membranes 

Excessive soiling 

General abnormalities 

a. Hand-Held and Open-Field Observations: Hand-held and open-field observations 

included a careful physical examination and sensory evaluation according to an 

established format. Open-field observations and sensory evaluations were made in a clear 

plastic box (50 cm × 50 cm). The rat was placed in the center of a 20-cm diameter circle 

marked on the bottom of the open-field box. Activity was defined as ambulatory activity

including rearing. Non-ambulatory movements, such as grooming and exploratory

sniffing, were not a part of this evaluation. If present, abnormal movements (i.e., tremors, 

convulsions), or behaviors (i.e., stereotypes), were recorded. 

b. Rectal Temperature: Rectal temperature was measured by carefully placing a rectal 

thermistor ≈ 4 cm into the rectum for ≈10 seconds to record temperature. The thermistor 

was validated at 37°C before, during and after the study. 

c. Grip Performance: Hind limb grip performance was tested according to the procedure 

described by Mattsson et al. (1986). Briefly, the observer placed the rat’s forepaws on a 

plastic bench and the hind paws were set on a horizontal screen attached to an electronic 

strain gauge. The observer then smoothly but firmly pulled backward on the tail until the 

rat’s grip on the screen was broken. An electronic strain gauge was used to record the 

rat’s resistance to the pull in grams. The average of three trials was used for statistical 

analysis. Forelimb grip performance was similarly tested. In this application, a bench was 

not used, and the rats were placed so that the forepaws were on the screen and the hind 
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paws were suspended ≈10 cm above the plastic platform. 

d. Landing Foot Splay: The outermost toe of each hind paw was marked with ink, and the 

rat was dropped from a height of 30 cm onto the recording sheet. This procedure was 

repeated three times (toes re-inked, as necessary). The distance from center-to-center of 

the ink marks, for each trial, were measured (cm) and the average of the three landing 

splay values was used for statistical analysis.

e. Motor Activity: An automated system was used for motor activity (MA) data collection 

on 10 Set 1b rats/sex/dose between PND 54 and 56. Each test session consisted of eight 

8-minute epochs, totaling 64 minutes of testing per rat per test session. This duration was 

chosen based on the results of a validation study indicating that performance of control 

rats approached asymptote in 50-60 minutes in Crl:CD(SD) rats (Marty and Andrus, 

2007). Activity counts for each epoch were recorded. Data were presented as total motor 

activity counts and counts/epoch. Rats were allocated to the motor activity cages in such 

a way that the counterbalancing of exposure groups and sexes across cages and test times 

was maximized.

f. Acoustic Startle Response (ASR) Habituation System Description: The ASR of the 

F1 Set 1b rats was tested on PND 57-59. Body weights were collected on the day of ASR 

assessment. The ASR system was a commercially available package consisting of 8 

acoustically insulated chambers and hardware to generate the auditory stimuli and 

measure the resulting startle responses. Each chamber contained a dual-speaker enclosure 

(for the generation of background noise and audio stimuli), a load-sensing platform, and 

an adjustable gain preamplifier. The startle stimulus speaker had a frequency response of 

1.8 kHz - 30 kHz, and the background noise speaker was had a frequency response of 

40Hz - 8kHz. Each acrylic animal holder was attached to a load-sensing platform that 

was connected to the data acquisition system. Each chamber was equipped with a red 

house light, which was kept on during all experimental procedures.

On the day of testing, each rat was allowed to acclimate (≈ 30 minutes) in the testing area 

in a transfer cage before being placed into an acrylic animal holder, which was then 

attached to a load cell platform inside the chamber. Following a 15-minute acclimation 

period in the presence of background noise (~65 dB[A]), rats received 50 startle trials. 

Each trial consisted of a burst of white noise (~120 dB[A], 50-msec duration, 2-msec 

rise/fall time), and trials were separated by a variable inter-trial interval (10-20 seconds). 

The first positive peak occurring within 250 msec following the startle stimulus 

(minimum latency 20 msec, minimum peak amplitude 20) was defined as the peak startle 

response. The trials were analyzed in blocks of 10 to examine habituation.

2. Necropsy/Neuropathology: On PND 60, F1 offspring Set 1b male and female rats 

selected for perfusion were counterbalanced across dose groups and given an ip injection 

of heparin ≈10 minutes prior to perfusion. Rats were perfused by gravity pressure with 

phosphate buffer containing sodium nitrite followed by a phosphate-buffered solution of 

1.5% glutaraldehyde–4% formaldehyde. Tissues were examined for pathologic 

alterations. The brain (excluding the olfactory lobes), head, spinal column with spinal 

cord, fore-and hind limbs, and tail were trimmed to remove excessive skin and muscle; 
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muscles from the hind limbs were reflected to further expose the nerves. 

Tissues for neuropathologic evaluation (Table 9) were prepared from all rats in the 

control and high-dose groups. In addition, appropriate brain sections from the low- and 

intermediate-dose groups were taken to slide for possible morphometric evaluation. Nine 

cross-sections of the brain were prepared using a hand-held, single-edged industrial blade 

from the following structures: olfactory bulb, cerebrum (frontal, parietal, temporal and 

occipital lobes), thalamus/hypothalamus, midbrain, pons, medulla oblongata, and 

cerebellum. For morphometric purposes, two transverse tissue blocks were cut through 

the cerebrum and midbrain (block #3 and #4). A longitudinal (anterior to posterior) cut 

was made midway through the cerebellum after it was removed from the midbrain (block 

#10). The following gross rostral landmarks for each block were used: block #3 –optic 

chiasm, block #4 – anterior edge of infundibulum, and block #10 dorsal midline apex of 

the cerebellum. Blocks #3 and #4 would contain the following structures: cerebrum 

(frontal and parietal lobes), thalamus/hypothalamus, and midbrain. These tissues were 

processed by standard histologic procedures, embedded in paraffin, sectioned 

approximately 6-μm thick and stained with hematoxylin and eosin, and coverslipped.

Table 9. Nervous System Tissue Collection

Brain

►Olfactory bulb 

►Cerebrum, frontal, parietal, temporal, occipital 

►Thalamus/Hypothalamus 

►Midbrain 

►Pons 

►Cerebellum 

►Medulla including nucleus gracilis/cuneatus 

Pituitary gland 

Trigeminal ganglia with nerve 

Spinal cord (cross and oblique section)

►Cervical Swelling (C3-C6) 

►Lumbar Swelling (L1-L4)

Dorsal Root Ganglia

►Cervical and Lumbar

Dorsal and Ventral Roots

►Cervical and Lumbar

Peripheral nerves (cross and longitudinal section)*

►Proximal sciatic

►Proximal & distal tibial (at knee & calf muscle branches)

►Peroneal (saved) 

►Sural 

►Caudal (saved) 

Nasal tissue with olfactory epithelium

Eyes - with optic nerve (longitudinal section only)

Skeletal muscle

►Anterior tibial and Gastrocnemius 

Data from Table 5, page 233 of the report. *Fore- and hind limbs saved, but nerves collected only from the 

right hind limb. 

A sufficient number of sections were cut from each block to meet the criteria of finding 

the appropriate microscopic landmarks for the purpose of measuring specific microscopic 

structures. Microscopic landmarks were as follows: block #3 (Figure 8) – first occurrence 

of the anterior commissure, block #4 (Figure 9) – contact/close proximity of the dentate 

gyrus and the polymorph layer of the dentate gyrus, and block #10 (Figure 10) – midline 

longitudinal section that contains the medial cerebellar nucleus. The most appropriate 

section from each block was used for the microscopic measurements. Additional sections 

from blocks #3, #4, and #10 from the brain were stained with Luxol Fast Blue and coverslipped to assess myelin. In addition, sections were prepared from the trigeminal ganglion 

and nerve, pituitary gland, eyes with optic nerves, spinal cord (cervical and lumbar), 

olfactory epithelium, and skeletal muscles (gastrocnemius and anterior tibial). These 

tissues were processed by standard histologic procedures, embedded in paraffin, 

sectioned approximately 6-μm thick and stained with hematoxylin and eosin, and 
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coverslipped. Spinal nerve roots (cervical and lumbar), dorsal root ganglia (cervical and 

lumbar), and peripheral nerves (sciatic, tibial (proximal and distal (muscular) – at the 

knee and calf muscle branches) and sural) were osmicated, embedded in epoxy resin, 

sectioned ≈ 2 to 3 μm thick and stained with toluidine blue. Tissues were evaluated using 

a light microscope. Histopathologic findings were subjectively graded as appropriate to 

assess the potential effects of exposure with regard to the contribution of a specific lesion 

to the health status of an animal. The report indicated that treatment-related effects were 

not observed in any neurological tissues in high-dose rats; therefore, slides from 

peripheral nervous system tissues were not prepared from the low- and intermediate-dose 

level rats. Aside from sections #3, #4, and #10, slides from other brain sections (Figure 7 

in report) were not prepared due to the lack of treatment-related effects in the high-dose 

group. Sections #3, #4, and #10 (brain morphometry sections) were prepared for low- and 

intermediate-dose level rats to avoid potential temporal artifacts from retention in 

paraffin blocks. 

a. Brain Weight and Gross Measurements - Brain weight and gross linear measurements 

were recorded on all dose groups. Brains (excluding the olfactory lobes) were weighed 

following 7-15 days of fixation. Linear measurements consisted of the: 1) cerebral length 

(L2 – anterior to posterior, excluding olfactory lobes) and width (L3 – maximum), and 2) 

cerebellar length (L10 – anterior to posterior) and width (L5 – maximum), which were 

obtained using a hand-held, electronic digital slide caliper, whose calibration was 

checked prior to use each day (Figures 11 and 12).

b. Morphometrics - Microscopic Brain Measurements. Microscopic brain measurements 

were recorded on all rats in the control and high-dose groups. Microscopic brain 

measurements were obtained from a number of anatomical structures in tissue sections 

stained with H&E from blocks #3, 4, and 10 having the appropriate landmarks. Images of 

each section of brain were digitally captured using an Olympus DP70 or a SPOT RT 

camera mounted on a Leitz dissecting microscope (blocks # 3 and 4; Figures 8 and 9) or 

a Leitz light microscope (block #10; Figure 10) with image capture software (Diagnostic 

Instruments, Inc., Sterling Heights, Michigan). Simple morphometric measurements were 

obtained using the Image-Pro Plus image analysis software (Media Cybernetics, Silver 

Spring, Maryland) as follows: 1) distance measurement - creates a measurement of the 

distance between two user-selected features, and 2) curve measurement - creates a 

measurement of the average distance between two user-selected traces in any direction. 

Measurements were excluded as necessary due to microscopic artifacts, such as missing 

portion(s) of tissue, tears in tissue etc. Morphometric data obtained using the Image-Pro 

Plus was electronically transferred to Microsoft Excel spreadsheets.

DEVELOPMENTAL IMMUNOTOXICITY (F1 offspring Set 2) 

1. Sheep Red Blood Cell (SRBC) Antibody-Forming Cell (AFC) Assay 

Immunotoxicity evaluations: To obtain data on the functional responsiveness of the 

immune system, Set 2a F1 rats (10/sex/dose from 10 litters) were immunized four days 

prior to necropsy (PND 66 to 70) with a single, 0.5 ml intravenous (i.v.) injection of 2 x 

108SRBC, via the lateral tail vein. In addition, five rats/sex from similarly aged satellite 

rats (extra rats in laboratory) served as a concurrent positive control (IPC) group, which 
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was also immunized four days prior to necropsy. These latter rats were administered a 

daily dose of 20 mg/kg/day cyclophosphamide (CP; causes reduction in the anti-SRBC 

antibody forming cells (AFC) response) via intraperitoneal (i.p.) injection for the four 

days prior to necropsy. On the day of necropsy (~PND 67-73), all rats were euthanized 

via CO2 anesthesia followed by cervical dislocation and removal of the spleens. The 

spleens were trimmed, weighed and placed into homogenization bags containing RPMI. 

A single cell suspension of the spleen cells was prepared by gentle disaggregation using a 

tissue homogenizer. The homogenized spleen suspensions were filtered through a 100 

μm cell strainer (VWR catalog #21008-952) into labeled 50 ml centrifuge tubes. Each 

bag was rinsed with ≈2 ml of 5% RPMI and the rinse transferred to the appropriate 

centrifuge tube. The tubes containing the spleen cell suspensions were centrifuged at 300 

x g for 10 minutes at 4°C. The supernatant was discarded. The cells were washed once by

re-suspending the pellet in 10 ml of 5% FBS/RPMI and centrifuged (as above) and the 

supernatant again discarded. The cells were then re-suspended in 5 ml of 5% FBS/RPMI. 

A 1:1000 dilution of the spleen cells was prepared in 10 ml of Isoton II with three drops 

of Zap-Oglobin II lytic reagent and the number of cells was counted using a Coulter Z1 

cell counter (Beckman Coulter, Miami, Florida). Three additional dilutions of spleen 

cells were prepared (e.g., 1:50, 1:100, or 1:200) in 5% FBS/RPMI. The diluted cells were 

mixed with washed SRBC (same lot used for immunization), guinea pig complement, 

and warm agar with DEAE-dextran. The mixture was poured into the middle of a Petri 

dish. A humidified (breath) coverslip was placed on the mixture and after one or two 

minutes, the dish was placed into a humidified incubator set at 37°C for 3 hours. The 

dishes were removed from the incubator and the plaques were counted. Plaques are 

formed as a result of complement-mediated lysis of the SRBC from antibodies secreted by 

the plasma cells (differentiated B cells).

2. Natural Killer Cell (NK) Assay: Immunotoxicity assessment of the remaining rats in 

Set 2 (Set 2b: 10/sex/dose from 10 litters) was determined after evaluation of the 

preliminary AFC data. Set 2b rats were evaluated using the natural killer cell (NK) assay

on PND 87-93 for effects on the immune system that are more cellular in nature. 

Mononuclear lymphoid cells from the spleen were obtained from Set 2b rats maintained 

on 2, 4-D diet continuously through PND 87-93. The spleens were surgically removed 

and a single cell suspension was prepared by gentle disaggregation using a tissue 

homogenizer. Mouse lymphoma cell line YAC-1 (ATCC) was used as target cells. YAC1 cells were subcultivated prior (~48 hours) to NK incubation to develop a growth phase 

population of target cells. YAC-1 cells were labeled with carboxy-fluorescein 

succinimidyl ester (CFSE) just prior to mixing with spleen cells (effectors) at 

effector:target ratios that range from 6.25:1 to 200:1. Note: The effector to target ratios in 

this study ranged from 50:1 to 800:1. The samples were incubated for 4 hours at 37°C 

with 5% CO2. At the end of incubation, propidium iodide (PI) was added and incubated 

at 4°C for 5 minutes to identify dead cells. Rabbit anti-mouse/rat Asialo GM1 polyclonal 

antibody was used as a positive control. A separate group of age-matched rats received a 

single intravenous injection of the positive control 24 hours prior to necropsy. The 

samples were analyzed by flow cytometry within one hour after adding PI. Comparative 

assessments of the flow cytometry and chromium release approach indicate a similar 

sensitivity in their ability to detect NK cell activity and inhibition. The present evaluation 

revealed linear increases in cytotoxicity with increasing effector to target cell ratios (from 
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5 to 40% cytotoxicity) and significant inhibition with the positive control anti asailoGM1 (greater than 50% inhibition); responses that were similar to that reported for the 

chromium release approach. The study authors stated that advantages of the flow 

cytometry approach to the NK-cell assay include higher signal, lower spontaneous release 

of signal, and avoidance of isotope waste and expense. The use of flow cytometry for the 

NK-cell evaluation as a non-guideline study was approved by the USEPA prior to the 

initiation of this procedure. It is to be noted that the flow cytometry for NK cell activity

assay has not been approved as an alternative assay vs 51Cr-release NK assay for the 

Guideline Immunotoxicity Study (OPPTS 870.7800) until further validation has been 

completed. 

3. Necropsy/Anatomic Pathology (Set 2 F1 Offspring): A limited necropsy was 

conducted on Set 2a rats (10 rats/sex/dose). Terminal body weights, spleen, brain and 

thymus weights were collected and organ:body weight ratios were calculated. Spleens 

were used for the SRBC AFC assay. The thymus and mesenteric and mediastinal lymph 

nodes were collected and preserved for possible future histopathological examination. In

addition, the brain also was preserved. The remaining F1 Set 2 rats (Set 2b; 10/sex/dose) 

used for an NK assay on PND 87-93 were weighed and anesthetized (CO2 inhalation) 

followed by decapitation. Only terminal body weights, spleen, and testes weights were 

collected. Spleens were used for the NK assay. The testes were preserved for possible 

future histopathological examination.

D. DATA ANALYSIS

1. Statistical Analyses: Appendix Table A summarizes the statistical analyses performed. 

Averages and standard deviations were calculated for the brain weights and morphometric 

measurements using Microsoft Excel spreadsheets and databases in full precision mode (15 

digits of accuracy). Means and standard deviations were calculated for all continuous data. 

Descriptive statistical (i.e., mean ± standard deviation) analyses of toxicokinetic data were 

performed using Microsoft Excel (Microsoft Corporation, Redmond, Washington) spreadsheets 

in the full precision mode (15 digits of accuracy). Individual values obtained were analyzed for 

AUC0→t by trapezoidal method using PK Functions for Microsoft Excel. (J. I. Usansky, A. Desai, 

D. Tang-Liu, Department of Pharmacokinetics and Drug Metabolism, Allergan, Irvine, 

California; Pizarro et al., 2004; Beringer et al., 2005). Whenever statistical analysis was needed 

to compare trends among doses, data was analyzed using SAS statistical analysis computer 

software (SAS Institute Inc., Cary, North Carolina) through a two-way analysis of variance. 

Linearity was determined graphically via regression analysis (as per Sweatman and Renwick, 

1980; Roberts and Renwick, 1989) as well as by comparing ratios of plasma 2, 4-D and AUC24h. 

If deemed necessary, additional statistical tests were performed. Because numerous 

measurements were statistically compared in the same group of animals, the overall false positive 

rate (Type I errors) was greater than the nominal alpha levels. Therefore, the final toxicologic 

interpretation of the data considered other factors, such as dose-response relationships, biological 

plausibility and consistency, and historical control values. 

2. Indices: Reproductive indices (all dose levels in P1 rats) and offspring viability indices were 

calculated as follows (as provided in the study report):
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a. Reproductive indices:

• Female mating index = (No. females with evidence of mating/No. paired) x 100

• Male mating index = (No. males with evidence of mating/No. paired) x 100

• Female conception index = (No. females with evidence of pregnancy/No. mated) x 100

• Male conception index = (No. males siring a litter/No. mated) x 100

• Female fertility index = (No. females with evidence of pregnancy/No. paired) x 100

• Male fertility index = (No. males siring a litter/No. paired) x 100

• Gestation index = (No. females delivering viable litter/No. females with evidence of pregnancy) x 

100

• Gestation survival index = percentage of delivered pups alive at birth

b. Offspring viability indices: 

• Post-implantation loss = (No. implants – No. viable offspring)/(No. implants) x 100

• Day 1 or 4 pup survival index = (No. viable pups on day 1 or 4/No. born live) x 100

• Day 7, 14, or 21 pup survival index = (No. viable pups on day 7, 14 or 21/No. after culling) x 100

3. Historical control data: Historical control data were provided for Acoustic Startle Response 

(ASR) from recent control data sets from performing laboratory in male rats of the same strain, 

age, and body weight; for male reproductive and sex accessory gland weights; for male pituitary

weights; testis size, bladder calculi. A dose range-finding study (MRID 47417901) was 

submitted that provides additional data, and a separate abbreviated DER is appended.

II. RESULTS

A. PARENTAL ANIMALS

1. Mortality and clinical signs: There were no treatment-related deaths or clinical signs of 

toxicity in either sex. All P1 rats survived to scheduled necropsy.

2. Body weight and food consumption: Body weight was not adversely affected in the P1 male 

and female rats during the pre-mating period (Table 10), and body-weight gains were comparable 

among the groups (both sexes). During gestation, there were no treatment-related effects on body

weight or body-weight gain (Table 11) in the main study females. During lactation, female body

weights were slightly but significantly decreased (↓5%) on lactation day 7 (LD7) in the high-dose 

group just prior to the diet adjustment of 2,4-D concentrations (1/2 concentration female diets 

given on LD 7-14 to account for the large increase in maternal feed consumption during this 

period). By LD 14, body weights were similar to the control group. Body-weight gain was

reduced at the 600 ppm dose level during LD 1-4 (↓64%*) and LD 4-7 (↓50%) but was greater 

than control during LD 7-14 (Table 11). NOTE: The standard deviation in body-weight gain 

values during lactation are greater than the mean in several instances (all groups), and the 300 

ppm dose group displayed a greater reduction in body-weight gain during LD 4-7 (↓79%) than 

the 600 ppm dose group at either time point. Food consumption was comparable among the 

groups during pre-mating (both sexes) and in females during gestation. During the first week of 
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lactation, the high-dose dams consumed less food (LD 1-4: ↓10%; LD 4-7: ↓7%) than the control 

dams. 

TABLE 10. Mean (SD) Body weight, body-weight gain, and food consumption - pre-mating a

Observations/study week

Dose group

Control 100 ppm 300 ppm 800/600 ppm

P1 Males - Pre-mating

Mean body weight (g)

Day 1

 Day 78

339.7±12.4

548.2±43.9

344.2±10.6

548.2±49.2

344.9±11.9

550.3±51.3

343.1±11.0

560.7±45.2

Mean weight gain (g)

 Days 1-8

 Days 1-78

38.5±9.3

208.5±37.1

36.0±8.0

204.0±42.4

38.8±9.8

205.3±42.7

40.5±9.9

217.6±39.4

P1 Females – Pre-mating

Mean body weight (g)

Day 1

 Day 29

231.1±9.5

266.3±19.2

231.3±8.1

269.9±17.1

232.9±9.4

272.1±21.0

232.1±11.0

267.8±19.1

Mean weight gain (g)

 Days 1-8

 Days 1-29

13.6±7.1

35.2±13.3

16.0±7.6

38.7±12.0

12.2±9.0 (↓10%)

39.2±14.5

12.3±7.5 (↓10%)

35.7±13.0

a

Data obtained from Table 17 (pages 248-249) and Table 18 (page 250) in the study report.

* p<0.05; ** p<0.01. n = 27 (males)/39 (females; main +satellite)

TABLE 11. Mean (SD) Body weight and body-weight gain – Gestation and Lactationa

Observations/study week

Dose group

Control 100 ppm 300 ppm 800 ppm

P1 Females – Gestation

Mean body weight (g)

Day 0

 Day 7

 Day 20

n = 26

268.4±19.9

306.0±20.8

414.3±31.4

n = 27

273.4±16.8

308.7±16.6

416.6±30.2

n = 24

276.7±23.1

310.0±25.8

417.5±34.5

n = 24

269.4±18.6

302.4±19.4

416.0±32.9

Mean weight gain (g)

 Days 0-7

 Days 0-20

37.6±12.6

145.9±25.8

35.3±7.6

143.2±24.0

33.3±8.3

140.8±18.0

32.9±6.3 ↓12%

146.6±20.4

P1 Females – Lactation

Mean body weight (g)

Day 1

 Day 7

 Day 21

n = 27

317.4±21.8

336.6±14.8

335.2±19.0

n = 28

318.3±20.8

333.3±20.2

336.8±18.4

n = 24

317.6±23.8

330.9±23.8

341.5±22.2

n = 24

312.7±23.1

320.2±21.7*↓5%

335.6±25.0

Mean weight gain (g)

 Days 1-4

 Days 4-7

 Days 7-14 

 Days 1-21

15.4±10.3

3.8±7.5

12.8±12.5

17.8±20.8

10.9±7.7↓29%

4.2±7.6

14.3±13.5

18.5±15.4

12.5±9.6 ↓19%

0.8±7.9 ↓79%

14.0±13.4

23.8±12.3

5.5±11.6* ↓64%

1.9±9.8 ↓50%

26.5±14.7*

22.9±12.1

a

Data obtained from Tables 19, 21, 23, 24 (pages 251, 253, 255, 256 ) in the study report; * p<0.05; ** p<0.01. 

3. Test material intake: Test material intake (TMI, expressed as mg/kg/day) was calculated using 

feed concentrations, body weights, and feed consumption data and is shown in Table 12. The 

TMI values for the P1 males were fairly close to the predicted values (i.e., 5.5 vs. 5 mg/kg/day; 

16.6 vs. 15 mg/kg/day and 45.3 vs. 40 mg/kg/day). However, in the P1females, TMI exceeded 

the targeted low and high dose levels of 5 and 30 mg/kg/day, particularly during the early

lactation period (LD 1-7), when TMIs were double these targeted dose levels.
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TABLE 12. Mean test substance intake during premating, gestation, and lactation (mg/kg body weight/day) a

P1 Males P1 Females

100 ppm 300 ppm 800 ppm 100 ppm 300 ppm 600 ppm

Premating 5.51±0.68 16.6±2.08 45.3±5.73 7.0±0.42 20.6±0.65 40.2±1.18

Gestation

●Main study

●Satellite

- - - 7.37±0.77

7.63±0.55

21.9±2.14

22.9±1.17

43.7±4.02

45.3±2.65

Lactation (main)

●LD 1-7

B

●LD 7-14B

- - -

10.7

8.4

32.4

25.5

64.5

51.5

a

Data obtained from Text Table 24 (page 136), Table 32 (males), page 264, Tables 34-35 (females), pages 266-267 in study report. 

B interval standard deviation not provided

4. Hematology – P1 Males and Main Study Females

There were no apparent treatment-related effects on hematology, differential white blood cell 

counts, and prothrombin time in either sex at any dose level. Table 13 provides the platelet count 

findings for comparison to those of the F1 offspring Set 1a males who displayed a reduction in 

platelet counts (↓14%) on PND 70.

Table 13. Platelet Counts – P1 Adults

Parameter 0 ppm 100 ppm 300 ppm 600/800 ppm

Males

Platelet counts (E3/UL) 1130146 1060102 116599 1058150 ↓6%

WBC (E3/UL) 10.87±2.10 10.05±2.39 9.42±1.74 ↓13% 11.39±2.17

Females

Platelet counts (E3/UL) 1250271 1225246 1234152 1237159

WBC (E3/UL) 9.98±3.44 10.27±3.49 10.25±1.80 8.32±2.30 ↓17%

 Data from Tables 39 and 40, pages 272 and 273; Tables 43 and 44, pages 276 and 277 of report; n=10 

5. Clinical chemistry – P1 Males and Main Study Females

At 800 ppm, P1 males displayed statistically significant (slight) decreases in total protein (↓6%) 

and albumin (↓5%) levels compared with controls and non-significant increases in ALT (↑80%) 

and AST (↑26%). The increases in ALT and AST were due to one outlier. P1 females did not 

display any treatment-related changes in any of the clinical chemistry parameters monitored. 

6. Urinalysis – P1 Males

There was a slight, dose-related increase in white blood cells per microscopic field, which was 

noted at 300 and 800 ppm during the urine microscopic examinations. All other parameters were 

comparable among the groups. 
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7. Macroscopic examination – P1 Males and Main Study Females

Males: Two males [one at 300 ppm (#4310) and one at 800 ppm (#4334)] had decreased testis 

size (bilateral) and no motile sperm. The 300 ppm male also had a decreased seminal vesicle size 

(unilateral). Two males [one at 300 ppm (#4311) and one at 800 ppm (#4338)] had calculi in the 

urinary bladder. This resulted in diffuse thickening of the bladder wall in these rats. Females: 

There were no gross observations attributed to 2, 4-D in the P1 females. 

8. Organ weights – P1 Males and Main Study Females

Males: There were no effects on terminal body weights in the P1 males following ≈11 weeks of 

exposure (Table 14). Similarly, there were no significant, treatment-related effects on absolute or 

relative liver, brain, spleen, thyroid, pituitary or adrenal gland weights. There was a significant 

increase in absolute (13%) and relative (11%) kidney weights of P1 males at 800 ppm. Kidney

weights were not significantly affected at 100 ppm or 300 ppm. The kidney-weight changes were 

accompanied by histopathological findings in the kidneys (see below). Absolute seminal vesicle 

weights were significantly decreased at both 300 and 800 ppm (↓12%). Relative seminal vesicle 

weights also were significantly decreased by 12% and 14%, respectively. There was a doserelated decrease in absolute and relative prostate weights in males at the 800 ppm (10% and 11%, 

respectively) and 300 ppm (9% and 8%, respectively), but these decreases were not statistically

significant. It is noteworthy that the control values for relative organ weights of the seminal 

vesicles, prostate, testes and epididymides in the P1 males were outside of the laboratory

historical control range (Table 15), suggesting that the control values were atypical. The organ 

weights for the 800 ppm-treated males were within the laboratory historical control range. 

Absolute and relative prostate and seminal vesicle weights also were within historical control 

values for the 300 ppm-exposed males. There were no treatment-related effects on testes or 

epididymal weights. Additionally, there was no associated histopathology in P1 male 

reproductive or accessory sex gland tissues. These changes in male reproductive organ weight 

were considered spurious because no consistent pattern of altered androgenicity was observed in 

male rats treated with 2, 4-D, no significant changes on sperm parameters (spermatid/sperm 

counts, sperm motility and sperm morphology) were found, and lower accessory sex gland 

weights were not observed in the F1 offspring (Set 1a or Set 3) with longer 2, 4-D exposures (in 

utero, during lactation and into adulthood). Females (LD 22): No treatment-related effect was 

observed on terminal body weight in P1 females. There were no significant, treatment-related 

effects on absolute or relative liver, kidney, brain, spleen, ovary, pituitary, or adrenal gland 

weights. Uterine weight (↑17% both absolute and relative) was increased at 600 ppm, but 

statistical significance was not attained. However, increased uterine weights were observed in Set 

1a and Set 3 F1 offspring also. Relative thyroid weights were statistically significantly decreased 

(↓11%) at 300 ppm, but absolute thyroid weights (↓11%) were not statistically significant, and no 

dose response was evident. NOTE: The thymus was not weighed in this group of rats, but 

decreased thymus weights were observed in the offspring at PND 70 and PND 139.
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TABLE 14. Organ WeightsaP1 Main Study Rats

Organ Dose group (ppm)

Control 100 300 600/800

P1 MALES

Terminal Body Weight (g)

N=

529.7±45.4

27

528.8±50.8

27

530.627±51.0

27

540.7±46.8

27

Kidneys

●absolute

●relative

3.513±0.532

0.662±0.070

3.621±0.440

0.686±0.064

3.629±0.496

0.685±0.083

3.965±0.488* ↑13%

0.734±0.068* ↑11%

Liver

●absolute

●relative

14.006±1.794

2.638±0.154

14.002±2.048

2.641±0.217

26

14.035±1.635

2.661±0.184

14.403±2.084

2.655±0.206

Thyroid

●absolute

●relative

0.0240±0.0048

0.0045±0.0009

0.0235±0.0033

0.0045±0.006

0.0234±0.0031

0.0044±0.0005

0.0253±0.0044

0.0047±0.0008

Testes

●absolute

●relative

3.718±0.229

0.706±0.060

3.654±0.247

0.696±0.075

3.595±0.551

0.686±0.127

3.528±0.501 ↓5%

0.653±0.086 ↓8%

Prostate 

●absolute

●relative

1.313±0.174

0.248±0.029

1.274±0.232

0.242±0.047

1.198±0.228 ↓9%

0.228±0.052 ↓8%

1.188±0.257 ↓10%

0.221±0.053 ↓11%

Seminal vesicles

●absolute

●relative

1.887±0.221

0.358±0.045

1.811±0.345

0.347±0.078

1.653±0.297* ↓12%

0.315±0.067* ↓12%

1.655±0.297* ↓12%

0.308±0.059* ↓14%

Epididymides

●absolute

●relative

1.498±0.106

0.285±0.033

1.428±0.110

0.272±0.029

1.405±0.165 ↓6%

0.268±0.042 ↓6%

1.418±0.154 ↓5%

0.263±0.030 ↓8%

Brain

●absolute

●relative

2.126±0.106

0.404±0.037

2.129±0.085

0.406±0.040

2.160±0.091

0.410±0.036

2.154±0.089

0.401±0.031

Adrenal

●absolute

●relative

0.055±0.010

0.010±0.002

0.059±0.010

0.011±0.002

26

0.056±0.008

0.011±0.002

0.058±0.009

0.011±0.002

Pituitary

●absolute

●relative

0.0136±0.0015

0.0026±0.0002

0.0137±0.0015

0.0026±0.0003

0.0132±0.0020

0.0025±0.0003

0.0135±0.0014

0.0025±0.0003

P1 FEMALES (LD 22)

Terminal Body Weight (g)

n=

295.8±15.1

27

297.5±16.6

28

300.5±23.6

24

296.5±19.3

24

Kidneys

●absolute

●relative

2.328±0.192

0.789±0.078

2.275±0.215

0.766±0.077

2.417±0.159

0.807±0.062

2.395±0.272

0.809±0.086

Liver

●absolute

●relative

12.538±1.241

4.244±0.423

12.081±1.303

4.061±0.375

12.784±1.352

4.253±0.264

12.130±1.179

4.089±0.258

Thyroid

●absolute

●relative

0.0180±0.0029

0.0061±0.0010

0.0168±0.0021

0.0057±0.0007

0.0162±0.0028

0.0054±0.0009*↓12%

0.0172±0.0021

0.0058±0.0008

Ovaries

●absolute

●relative

0.113±0.024

0.038±0.008

0.118±0.027

0.040±0.009

0.119±0.029

0.039±0.009

0.111±0.023

0.037±0.008

Uterus

●absolute

●relative

0.677±0.169

0.229±0.056

0.668±0.179

0.225±0.058

0.657±0.128

0.219±0.039

0.793±0.260 ↑17%

0.269±0.092 ↑17%

Brain

●absolute

●relative

2.011±0.105

0.681±0.040

1.961±0.083

0.660±0.034

1.970±0.096

0.658±0.047

1.959±0.072

0.663±0.041

Pituitary

●absolute 0.0164±0.0022 0.0159±0.0018 0.0163±0.0022 0.0161±0.0020
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TABLE 14. Organ WeightsaP1 Main Study Rats

Organ Dose group (ppm)

Control 100 300 600/800

●relative 0.0055±0.0007 0.0053±0.0006 0.0054±0.0005 0.0054±0.0006

Adrenal

●absolute

●relative

0.081±0.009

0.028±0.003

0.082±0.011

0.027±0.004

0.082±0.011

0.027±0.004

0.081±0.008

0.027±0.002

a

Data obtained from Tables 51-52, page 284-289 in the study report. * α = 0.05; mean SD

Table 15. Historical control data for male reproductive and sex accessory gland weights

Parameter Historical range A

Absolute prostate (g) 1.115-1.392

Relative prostate (g/100 g BW) 0.181-0.235

Absolute testes (g) 3.439-3.936

Relative testes (g/100 g BW) 0.571-0.682

Absolute seminal vesicles (g) 1.558-2.109

Relative seminal vesicles (g/100 g BW) 0.266-0.356

Absolute epididymides (g) 1.335-1.545

Relative epididymides (g/100 g BW) 0.215-0.277

 Data from Text Table 50, page 165 of the study report; 

 

A mean ranges from 8 two-generation reproduction studies (2003-2008)

9. Histopathological Observations – P1 Males and Main Study Females

Microscopic examination: P1 males: Consistent with the kidney weight increases observed in 

P1 males at 800 ppm, treatment-related kidney histopathology findings were also observed at 800 

ppm (Table 16). The lesion was described as a degenerative lesion involving the proximal 

convoluted tubules in the outer strip of the outer zone of the medulla, multifocal in distribution, 

and slight in degree. This lesion was primarily characterized by tubular epithelial cells, which 

were basophilic staining and had nuclei that were crowded together due to a decrease in the 

amount of cytoplasm (eosinophilic staining). Additionally, pyknotic nuclei were occasionally

noted in these tubules. The remaining portions of these tubular profiles were considered normal. 

Affected tubules also had focally thickened basement membranes, adjacent interstitial fibrous 

connective tissue proliferation, and a mononuclear inflammatory cell infiltrate. P1 Main Study 

Females: There were no histopathological observations attributed to 2, 4-D exposure in the P1 

females. Seven main study P1 females were non-pregnant (1 at 0 ppm, 3 at 300 ppm, and 3 at 

600 ppm). For 3 of the 7 females, the reason for the failure to become pregnant could be 

attributed to effects on their mating partners (e.g., testicular atrophy, decreased spermatic 

elements, or prostate inflammation). There were no histopathological changes in the remaining 

females that explained their failure to mate or conceive. The terminal stage of estrous of the 

control and 600 ppm females was determined by histopathological examination of the vagina and 

uterus. There were no alterations in estrous cycle patterns in these rats. With the exception of 

preselected control and high-dose rats and the females that failed to mate or deliver litters, the 

remaining P1 females were not examined because of the lack of treatment-related effects in the 

subset of rats that were examined.
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TABLE 16. Terminal Stage of Estrous (females)Band Microscopic Kidney Findings (both sexes)a

Observation Dose group (ppm)

Control 100 300 600/800

P1

MALES

Degeneration, proximal convoluted tubule, outer stripe, outer zone n=

● focal, very slight

● multifocal, very slight

● multifocal, slight

10

0

9

1

10

4

4

0

10

1

6

1

11

0

3

8

FEMALES

Terminal stage of estrous n=

● estrus

● diestrus

● metestrus

● proestrus

28

0

6

3

19

-

-

-

-

-

-

-

-

-

-

27

1

7

5

14

Degeneration, proximal convoluted tubule, outer stripe, outer zone n=

● focal, very slight

● multifocal, very slight

● multifocal, slight

10

0

0

0

0

-

-

-

0

-

-

-

10

0

0

0

a

Data obtained from Text Table 16, page 99 in the study report. Bdetermined by histopathological exam of vagina and uterus, data 

from Appendix Table 39 (pages 750-751 of study report

10. Reproductive function - P1 Males and Main Study Females

a. Estrous cycle length and periodicity: No significant difference was observed in mean 

estrous cycle length in P1 females at any dose level compared to the control (Table 17). 

There was no indication of persistent estrus; i.e., greater than 2 consecutive days in estrus, 

and no apparent difference in the percentage of time spent in estrus or diestrus (see Figure 13 

from study report in Appendix B).

TABLE 17. Estrous cycle dataaP1 Main Study and Satellite Females

Mean days per cycle

Dose group

Control 100 ppm 300 ppm 600 ppm

3.8 1 - - -

4.0 31 34 32 33

4.3 2 2 3 3

4.5 - 2 - -

4.7 3 - 1 -

4.8 - - 1 -

5.0 2 - 2 3

5.3 - 1 - -

Mean±sd 4.1±0.3 4.1±0.2 4.1±0.3 4.1±0.3

 a Data obtained from pages 688-695 in the study report; n=39 

b. Sperm measures: P1 males: Sperm Motility: There were no significant, treatment-related, 

effects on sperm motility or progressive motility (Table 18). Although sperm motility was 

slightly lower in the 300 and 800 ppm males (95% vs. 98% in controls), the magnitude of the 

effect was minimal and could be attributed to a single outlier in each of the dose groups,. 

Removal of these two outlier values generated mean sperm motility values that were the 

same as the control (98%). Sperm/Spermatid Counts: With respect to testicular spermatid 
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and epididymal sperm counts between control and 800 ppm males, there were no significant, 

treatment-related, differences (Table 18). Sperm/spermatid counts were not conducted in the 

100 ppm and 300 ppm groups due to the lack of effect at 800 ppm. Sperm Morphology: The 

proportion of abnormal sperm was not significantly different between control and 800 ppm 

males (Table 18). The slight increase in abnormal sperm at the 800 ppm dose level was due 

to a single outlier. When this one outlier was removed from the data, both the control and 

high-dose males had 1.4% abnormal sperm. Sperm morphology was not examined in the 100 

ppm and 300 ppm dose groups due to the lack of effect at 800 ppm.

TABLE 18. Sperm dataa(P1 males)

Dose group

Parameter Control 100 ppm 300 ppm 800 ppm

Motile (%)

rangeC

97.9±1.4

93-100

97.7±1.5

94-100

94.6±19.0

94-100

94.7±19.0

96-100

Progressively Motile (%)

Range

89.0±3.9

80-95

86.9±3.5

81-93

85.2±17.3

82-94

85.4±17.4

83-96

Testicular spermatid counts

Total Sperm (1006)

rangeC

222.8±62.3

98.8-365.4

-

-

-

-

238.9±62.9

67.4-393.7

Conc/g (1006)

rangeC

119.7±30.0

50.5-179.9

-

-

-

-

135.1±30.7

72.7-217.9

Epididymal sperm counts

Total Sperm (1006)

rangeC

235.3±70.8

112.9-445.4

- - 226.3±77.5

116.1-382.7B

Conc/g (1006)

rangeC

669.2±186.6

370.3-1113.6

- - 681.1±207.3

441.3-1060.1 B

Proportion of abnormal sperm summary

Abnormal sperm/total 0.014±0.015 - - 0.020±0.036A

 a Data obtained from pages 290-293 in the study report; n=27, except A

(n=26); Bone male with no sperm; 

 

C

ranges from Appendix Tables 42-44, pages 778-783 of the study report

11. Reproductive Performance: There were no significant effects on any of the reproductive 

indices in the P1 rats at any dose level. Male and female mating, conception, fertility, and 

gestation indices and the percent post-implantation loss were comparable among the groups. 

Fertility index was decreased slightly (88.9%) at the mid- and high-dose levels, compared to the 

concurrent control (96.3%) but was within the range of historical control data (fertility male: 84-

100%/female: 85.2-100%). Time-to-mating and gestation length were comparable among the 

groups. Results are summarized from the report in Table 19. 

TABLE 19. Reproductive Data a Main Study P1 rats

Observation Dose group (ppm)

Control 100 300 600

P1 

Number of males/females 27/27 27/27 27/27 27/27

Mean days per cycle (premating) B

4.130.32 4.090.27 4.150.32 4.100.27

Time to mating (days) 3.02.4 2.61.0 2.61.5 2.60.9

Mating index (%) 100 100 96.3 92.6

Conception index (%) 96.3 100 92.3 96

Fertility index (%) 96.3 100 88.9 88.9

Gestation index (%) 100 100 100 100

Gestation length (days) 21.70.5 21.40.5 21.60.5 21.50.5

 a

Data obtained from Table 38 (pages 270-271) and Appendix Table 20 (pages 688-695) in the study report; Bcalculated 
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 by reviewer (report combined Satellite GD 17 dams in their calculation)

B. GD 17 SATELLITE FEMALES

1. Reproductive Indices and Litter Data: Time to mating was comparable among the groups, 

although one 300 ppm GD 17 satellite dam was an outlier (14 days to mate). Post-implantation 

loss was slightly increased at 600 ppm compared to the control, although the standard deviation 

exceeded the mean in all cases. The mean number of fetuses per litter was comparable among 

the groups (Table 20).

TABLE 20. Reproductive Data aSatellite Females (GD 17)

Observation Dose group (ppm)

0 100 300 600

P1 Satellite Females

Mating index (%) 100 100 100 100

Conception index (%) 100 91.7 83.3 100

Fertility index (%) 100 91.7 83.3 100

Number of pregnant females 11 10 10 12

Number of corpora lutea (meanSD) 16.91.3 17.42.5 15.81.4 16.31.8

Number of implantations (meanSD) 16.61.5 16.41.6 15.51.2 15.81.7

Number of resorptions (meanSD)

Resorptions/litters w/ resorptions

0.91.1

2.2 (13/6) B1

0.81.1

2.0 (8/4)

0.50.7

1.3 (5/4)

1.52.2

2.0 (14/7) B1

Mean days per cycle (premating) 4.080.21 4.040.14 4.030.09 4.110.29

Time to mating (days) 2.71.0 2.21.0 2.93.6 2.91.0

Preimplantation loss (%) 1.682.88 4.819.44 1.772.85 2.296.46

Postimplantation loss (%) 5.546.94 4.796.93 3.314.59 9.2212.67

Total number of fetuses 167 156 150 178

B2Fetuses/litter (meanSD) 15.22.1 B2 15.61.8 15.01.5 14.82.5 B2

Total number of resorptions 13 E 7E, 1L 5E 14E

 a

Data obtained from Table 68 (page 318-319), Appendix Tables 20 (pages 688-695), 36 (pages 740-741), and 155 (pages 1426-

1471) in the study report; BTable 68 lists: (

B12.0 (10/5) for control and 2.3 (18/8) for 600 ppm groups) and (B215.72.0 for the 

control and 14.32.4 for 600 ppm groups); one 100 ppm female and two 300 ppm females were not pregnant; E = early 

resorption; L = late resorption; 

2. Thyroid Hormones –GD 17 Satellite Females: There were no statistically significant, 

treatment-related, differences in serum T3, T4, or TSH in the GD 17 satellite females (Table 21 

and Figure 14, reproduced from page 203 of the study report). However, it is to be noted that the 

600 ppm females displayed the predicted pattern of thyroid hormone changes that could signify a

thyroid effect; i.e., ↓ T3 and T4 with ↑TSH levels, and the thyroid was examined microscopically

in the P1 GD 17 satellite females (see below). These changes in the thyroid hormones along with 

the histopathological findings were considered treatment-related, although not adverse.

Table 21. Thyroid Hormone – GD 17 Satellite Females

Parameter 0 ppm 100 ppm 300 ppm 600 ppm

Females

N= 11 10 10 12

T3 (ng/dL) 73.1214.17 71.6911.42 69.6410.67 ↓5% 68.1219.04 ↓7%

Range 52.83-95.78 53.99-93.33 47.94-89.35 35.95-104.12

T4 (μg/dL) 1.260.28 1.220.43 1.160.64 ↓8% 1.150.42 ↓9%

Range 0.82-1.66 0.77-1.42 0.46-2.17 0.61-1.87

TSH (ng/mL) 2.921.56 2.760.84 2.601.37 3.651.59 ↑25%

range 1.03-5.62 1.47-4.02 1.17-3.45 (6.01)A

1.25-6.23B
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Table 21. Thyroid Hormone – GD 17 Satellite Females

Parameter 0 ppm 100 ppm 300 ppm 600 ppm

#>4 2 1 1

A

5

Data from Table 59, page 305 of study report; Aidentified as outlier in report; Bnot identified as outlier in report

3. Hematology Parameters – P1 Satellite Females: No treatment-related changes in hematology

parameters, including differential white blood cell counts and prothrombin time, were reported. 

Table 22 provides the platelet count findings since F1 offspring Set 1a males displayed a 

reduction in platelet counts (↓14%) on PND 70.

Table 22. – P1 Satellite Females

Parameter 0 ppm 100 ppm 300 ppm 600 ppm

Females

N= 11 10 10 12

Platelet count (E3/UL) 1246165 1228148 1248148 1199248 ↓4%

Data from Tables 60 and 62, pages 306 and 308 of study report

4. Clinical Chemistry– P1 Satellite Females: Clinical chemistry parameters were comparable 

among the groups of pregnant females.

5. Survival – P1 Satellite Females: All P1 satellite females survived to scheduled necropsy.

6. Organ Weights P1 Satellite Females on GD 17: Terminal body weights were comparable 

among the groups. The thyroid was the only organ weighed in the P1 satellite females. Although 

there were minor differences in thyroid weights, there was no dose-response (Table 23). 
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Table 23. – P1 Satellite Females Terminal Body and Organ Weights

0 ppm 100 ppm 300 ppm 600 ppm

Females

n= 11 10 10 12

Body Weight (g) 398.214.6 388.019.2 383.925.7 397.317.5

Thyroid 

Absolute (g)

Relative (g/100)

0.01720.0022

0.00430.0006

0.01880.0038 ↑9%

0.00480.0008

0.01670.0027

0.00430.0006

0.01870.0026 ↑9%

0.00470.0006

Data from Table 65, page 311 of study report

7. Gross Pathology Observations – P1 Satellite Females: There were no gross findings attributed 

to treatment in the P1 satellite females. None of the non-pregnant females (#4439 at 100 ppm and

#4478 and #4484 at 300 ppm) showed gross findings associated with the failure to conceive. 

8. Histopathological Observations – P1 Satellite Females: Although changes in thyroid 

hormones were not significant, the pattern of thyroid changes at the high dose was consistent 

with a potential perturbation in thyroid function (i.e., decreased T4 [9%] and T3 [7%] with 

increased TSH [25%]); therefore, thyroid glands from GD 17 satellite females were examined 

histopathologically. Three of 12 satellite females at 600 ppm had follicles that were smaller than 

those of the controls, and the colloid within the smaller follicles frequently contained clear 

vacuoles along the periphery of the follicular epithelial cells suggestive of colloid resorption. 

This change was graded as very slight. There were no adverse pathologic observations (i.e., 

degeneration, necrosis etc.) associated with the smaller follicles, suggesting that these alterations 

were adaptive rather than pathologic and consistent with a more rapid colloid turnover. 

Consequently, these thyroid alterations were considered treatment-related but not adverse. There 

were no effects on thyroid histopathology at 100 ppm or 300 ppm, consistent with the lack of 

thyroid hormone changes at these dose levels (Table 24). 
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TABLE 24. Microscopic Thyroid FindingsaP1 Satellite Females

Observation Dose group (ppm)

Control 100 300 600

P1 Satellite Females

Number of dams examined 11 11 12 12

Cyst; with keratinous debris; focal/multifocal; very slight 8 8 7 4

Aggregates of mononuclear cells, multifocal, very slight 0 0 0 1

Decreased follicle size, multifocal, very slight 0 0 0 3

a

Data obtained from Appendix Table 155 pages 1426-1471 in the study report. 

C. OFFSPRING: Main Study F1

1. Viability and clinical signs: The following observations were reported: There was no treatmentrelated effect on the number of live F1 pups born/litter (or the number of dead pups) or on pup 

survival or sex ratio (Table 25). There was no mention of clinical observations of the offspring 

during lactation. The number dying initially (PND 0-4) was slightly greater at 600 ppm than in 

the other groups.

TABLE 25. Litter Data a Main Study (F1 litters)

Observation Dose group (ppm)

Control 100 300 600

Number of litters 27 28 24 24

Number of implantations 407 423 373 374

Implantations/litter 15.7 15.1 15.5 15.6

Gestation survival index (%) 98.6 99.5 98.5 99.7

Number of fetuses born 369 376 325 344

Number born live (meanSD) 364 (13.52.7) 374 (13.43.3) 320 (13.32.1) 343 (14.32.6)

Number born dead (meanSD) 5 (0.20.8) 2 (0.10.4) 5 (0.20.7) 1 (0.00.2)

# Deaths days 0-4 (% of live born) 5 (1.37) 4 (1.07) 5 (1.56) 8 (2.33)

 Mean litter size Day 1 13.32.6 13.23.4 13.11.9 14.12.6

Mean litter size Day 4 b

13.32.6 13.13.3 13.11.9 14.02.5

Mean litter size Day 4 c

9.81.2 9.51.5 10.00.2 9.90.9

Mean litter size Day 7 9.81.2 9.51.5 9.90.3 9.80.8

Mean litter size Day 14 9.71.2 9.51.5 9.90.3 9.80.8

Mean litter size Day 21 9.71.2 9.41.5 9.90.3 9.80.8

Total number of resorptionsd

- - - -

Postimplantation loss (%) 10.039.91 12.1715.39 13.6312.34 8.6412.14

Sex ratio (M:F; day 1) 54:46 47:53 50:50 55:45

Survival

Day 1

Day 4

Day 7

Day 21

98.6

98.6

100

99.6

98.7

98.4

99.3

98.9

98.4

98.4

99.6

99.2

98.5

97.7

99.6

99.2

 a

Data obtained from Tables 38 (pages 270-271), 69 (page 320) in the study report; 

 d could not determine (data not provided); Gestation index (% newborn pups alive at birth); b Before standardization (culling); c 

After standardization 

2. Body weight (F1 offspring – Main Study): Litter weight data were not provided. On PND 1, 

male pups (↓3%) and female pups (↓4%) in the 600 ppm group weighed slightly less than control 

pups (not statistically significant), which may be the result of the slightly larger litter sizes in the 

600 ppm group (14.1 pups/litter at 600 ppm and 13.3 pups/litter at 0 ppm). By PND 4, pre-
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culling body weights in 600 ppm pups were 8% lower than controls and this decrease was 

sustained on PND 7 (Table 26). These decreases in pup body weight were not statistically

significant, but coincided with a statistically significant decrease in feed consumption in the 

dams prior to adjustments of dietary concentrations and lower body weights/gains in 600 ppm 

dams on PND 1-4 and PND 4-7. At time points after dietary concentration adjustment (PND 14 

and PND 21), 600 ppm dams had body weights and feed consumption values similar to control 

dams. Pup body weights in the 600 ppm group remained ↓5%-6% below control values during 

the PND 14-21 interval likely related to combined 2, 4-D intake through diet and milk. Pup body

weights were comparable to the control at 100 ppm and 300 ppm during lactation. 

TABLE 26. Mean (SD) F1 Pup Body Weights (g) a (litter means)

Dose group (ppm)

Lactation

Day

0 100 300 800 0 100 300 600

F1 Pups – male F1 Pups – female

1 7.30.6 7.20.8 7.40.7 7.10.7 ↓3% 7.00.7 6.90.8 6.90.6 6.70.6 ↓4%

4 b 10.41.1 10.21.6 10.41.0 9.61.2 10.01.3 9.81.5 9.81.0 9.21.0

4 c 10.41.1 10.31.7 10.41.0 9.71.2 10.01.3 9.81.5 9.81.0 9.11.0

7 16.61.4 16.52.3 16.41.4 15.51.8↓7% 15.91.6 15.82.1 15.61.4 14.71.4 ↓8%

21 52.54.5 51.45.4 50.34.2 49.45.4↓6% 50.64.7 49.65.3 48.74.1 47.64.0 ↓6%

a

Data from Table 70, page 321 in the study report; n = 27 (control), 28 (100 ppm), and 24 (300 ppm and 800 ppm/600 ppm)

b Before standardization (culling); c After standardization (culling)

3. Body Weight F1 Weanlings - PND 21 and PND 22: Because of assignment of pups to different 

groups on PND 21, multiple sets of pup body weights were collected during the PND 21-22 

interval. Pups were assigned to the groups based on day of birth (not body weight), so no 

stratification by body weight was possible. PND 22 male pups selected for organ weight 

measurements displayed a statistically significant (↓10%) decrease in body weight compared to 

control, while those selected for perfusion displayed a non-significant (↓6%) decrease (Table 27). 
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TABLE 27. Mean (SD) F1 Weanlings - Pup Body Weights (g) a– PND 21 and PND 22

Dose group (ppm)

Group

0 100 300 800 0 100 300 600

F1 Pups – male F1 Pups – female

PND 22 (n=10 

pups/dose)

Organ wts

57.93.8 52.23.8* 52.83.9 51.97.4*↓10% 52.76.1 50.47.9 50.54.4 51.25.9

PND 22 (n=12 

pups/dose)

Perfusions

57.13.9 56.45.7 52.54.8 53.76.1 ↓6% 54.15.4 53.14.5 49.93.8 50.04.3 ↓8%

PND 22 

combined (n=22 

pups/dose)

57.5±3.8 54.5±5.3 52.6±4.3* 52.8±6.6* ↓8% - - - -

PND 21 (24-28 

litters/dose)

all pups

52.5±4.5 51.4±5.4 50.3±4.2 49.4±5.4 ↓6% 50.6±4.7 49.6±5.3 48.7±4.1 47.6±4.0 ↓6%

a

Data obtained from Text Tables 32 and 33, pages 146-147 in the study report. *α = 0.05

4. Achieved Test Material Intake: Test material intake (expressed as mg/kg/day) is shown in 

Table 28. For F1 offspring, doses were nearly twice the targeted dose levels of 5, 15 and 30 or 

40 mg/kg/day in Sets 1a, 1b and 2a, all of which were euthanized by PND 70. As the F1 rats 

were maintained on test diet beyond PND 70, feed consumption decreased and TMI approached 

the targeted dose levels. Although closest to the target, TMI in Set 3 male and female F1 

offspring were reported as still exceeding targeted dose levels by 37-56% across all dose levels.

TABLE 28. Mean Test Material Intake (TMI) for F1 Male and Female Offspring (mg/kg body weight/day) a

Male Female

100 ppm 300 ppm 800 ppm♪ 100 ppm 300 ppm 600 ppm♪

Set 1a 

PND 28-69 9.242.25 28.47.18 76.618.4 9.561.99 28.86.10 57.912.7

Set 1b

PND 28-56 9.882.31 29.57.43 81.719.8 10.12.35 30.06.90 59.214.3

Set 2a

PND 28-70 9.152.16 28.46.66 75.318.8 9.661.91 28.75.69 58.411.5

Set 2b

PND 28-84 8.672.34 25.87.27 71.819.1 9.051.91 26.75.65 55.311.4

Set 3

PND 28-133 6.832.43 20.97.14 55.619.3 7.591.89 23.35.94 46.711.6

a

Data (means only) obtained from page 137 in the study report (s.d. were obtained from each Set’s TMI data tables by reviewer). 

♪ did not receive adult dietary concentration until PND 35; TMI calculations corrected for adjusted diets given on PND 28-35

5. Anogenital Distance – F1 Offspring: There were no significant, treatment-related, differences 

in absolute or relative anogenital distance in either sex (Table 29). 
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Table 29. Anogenital Distance – F1 Offspring

Parameter 0 ppm 100 ppm 300 ppm 600/800 ppm

Males

AGD (mm) 3.920.27 3.780.38 3.830.23 3.740.28

Relative AGD B

2.020.13 1.950.16 1.970.11 1.950.12

Body weight (g) 7.30.6 7.20.8 7.40.7 7.10.7

Females

AGD (mm) 2.080.25 2.030.24 2.060.21 2.010.27

Relative AGD B

1.090.13 1.060.11 1.080.12 1.060.13

Body weight (g) 7.00.7 6.90.8 6.90.6 6.70.6

Data from Table 71, page 322 of report; B mm AGD/cubed root of BW in grams; n=27 (control); 28 (100 ppm); 24 (300 

ppm and 600/800 ppm)

6. Nipple Retention: There was no difference in nipple/areolae retention between the control and 

high-dose group in either sex (Table 30). Consequently, the lower dose levels were not assessed 

for this endpoint. 

Table 30. Nipple/Areolae Retention

parameter 0 ppm 100 ppm 300 ppm 600/800 ppm

Males

# of nipples 0 NA NA 0

Females

# of nipples 120.1 120.0 120.1 12.00.1

Data from Table 72, page 323 of report; n=27 (control); 28 (100 ppm); 25 (300 ppm and 600/800 ppm)

7. Hormone Measurements - Thyroid

a. Thyroid Hormones - F1 offspring on PND 4: There were no statistically-significant differences 

in serum T3, T4, or TSH in PND 4 culled pups (Table 31; Figure 15 reproduced from page 204 

of the study report). T4 was reduced (↓12%) in both sexes at their high-dose level, and female 

PND 4 pups showed an increase in TSH (↑19%) at 600 ppm. However, a dose response was not 

evident for any of the alterations in these parameters

Table 31. Thyroid Hormone – F1 PND 4 (culled) pups

parameter 0 ppm 100 ppm 300 ppm 600/800 ppm

Males

T3 (ng/dL) 34.515.83 (9) 35.905.99 (8) 35.467.07 (8) 32.198.17 ↓7%

T4 (μg/dL) 0.640.25 0.620.32 (8) 0.550.21 ↓14% 0.560.19 ↓12%

TSH (ng/mL) 1.120.31 0.980.26 (7) 1.060.27 (9) 1.090.20

Females

T3 (ng/dL) 41.999.78 38.648.28 ↓8% 36.597.15 (6) ↓13% 40.298.06 (9)

T4 (μg/dL) 0.850.16 0.990.16 0.720.32 (9) ↓15% 0.730.23 ↓14%

TSH (ng/mL) 0.970.24 1.060.35 ↑9% 0.900.18 (9) 1.150.34 ↑19%

Data from Tables 73 and 74, pages 324 and 325 of report; n=10, unless ( )
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b. Thyroid Hormones - F1 offspring (PND 22 Weanlings): F1 PND 22 males displayed a 

statistically-significant reduction (↓28%) in T4 at 800 ppm, and F1 PND 22 females displayed a 

non-statistically significant reduction (↓20%) in T4 at 600 ppm. T3 was reduced in the males at 

300 ppm (↓19%) and 800 ppm (↓13%), but there was no dose response (Table 32; Figure 16 

reproduced below from page 205 of the study report). 

Table 32. Thyroid Hormone – F1 PND 22 Weanlings

Parameter 0 ppm 100 ppm 300 ppm 600/800 ppm

Males

T3 (ng/dL) 107.2216.59 100.8212.88 86.569.16* ↓19% 93.4615.06 ↓13%

T4 (μg/dL) 3.620.84 4.400.54 2.980.75 2.591.04* ↓28%

TSH (ng/mL) 1.320.24 1.250.59 1.480.73 ↑12% 1.270.37

Females

T3 (ng/dL) 99.1414.68 110.4320.01 99.4215.02 107.4211.68

T4 (μg/dL) 3.570.75 3.991.19 3.550.77 2.850.66 ↓20%

TSH (ng/mL) 0.990.26 1.130.33 0.940.22 1.020.16

Data from Tables 75 and 76, pages 326 and 327 of report; n=10; * α = 0.05

Males not given adult dietary concentration until PND 35.
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c. Thyroid Hormones – Set 1a F1 Offspring (PND 62-64): Both sexes displayed increased TSH 

at 300 ppm and at 800 ppm (males)/600 ppm (females), although the increase in males was not 

dose-related and none of the differences in thyroid hormone levels were statistically significant. 

T4 was decreased at 800 ppm in males (Table 33; Figure 17 reproduced below from page 207 of 

the study report). 

Table 33. Thyroid Hormone – F1 Set 1a Males (PND 62-64)

Parameter 0 ppm 100 ppm 300 ppm 600/800 ppm

Males

T3 (ng/dL)

range

78.6912.07

63.03-102.07

69.787.91

58.34-80.05

66.779.69

46.97-84.54

72.0317.40

43.51-94.29

T4 (μg/dL)

range

4.750.92

3.50-6.44

4.461.23

2.93-6.56

5.311.09 ↑12%

2.79-6.35

4.110.85 ↓13%

2.63-5.45

TSH (ng/mL)

range

2.950.74

2.12-3.42 (4.73)A

3.211.29

1.91-5.66

3.720.97 ↑26%

2.77-5.26

3.621.20 ↑23%

2.11-5.77

Females

T3 (ng/dL)

range

67.0817.71

51.49-81.31 (109.79) A

66.8910.71

46.65-79.02

70.4512.99

57.25-96.84

74.2814.67 ↑11%

58.13-100.28

T4 (μg/dL)

range

2.351.05

1.00-4.72

2.270.85

0.99-3.36

2.801.43 ↑19%

1.54-5.65

2.791.08 ↑19%

1.29-5.03

TSH (ng/mL)

range

1.890.53

0.93-2.60

2.050.61

1.24-3.00

2.100.42 ↑11%

1.84-2.46 (1.03) A

2.340.67 ↑24%

1.66-2.41 (4.15) A

Data from Tables 77 and 78, pages 328 and 329 of report; n=10; Aoutlier
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8. Sexual maturation (F1): F1 males at 800 ppm displayed a slight delay in preputial separation 

(1.6 days), which was accompanied by a very slight reduction in body weight compared to the 

control (↓2.1 grams; 99% of control). This reduction is within the normal variability for this 

measure and is not considered to be toxicologically relevant or indicative of adverse effect on 

sexual maturation. There was no evidence of retained preputial threads. The age at vaginal 

opening was comparable among the groups of F1 females (Table 34). 

Table 34. Puberty Onset – F1 Offspring (Sets 1, 2, 3)

Parameter 0 ppm 100 ppm 300 ppm 600/800 ppm

Preputial separation Males

Age (days) 43.01.3 43.61.5 43.31.5 44.62.3* (↑1.6 days)

Body weight (g) 223.213.7 230.314.9 221.015.7 (↓2.2 g) 221.112.3 (↓2.1 g)

#≥ 43.0

#≥ 44.6

18

3

17

5

11

7

17

11

Vaginal opening Females

Age (days) 32.00.9 31.90.7 31.81.0 32.30.8

Body weight (g) 108.86.3 106.56.3 106.07.7 104.19.9 (↓4.7 g)

 Data from Tables 87 and 88, pages 353 and 354 of report; n=27 (control); 28♂/27♀ (100 ppm); 

 24 (300 ppm and 600/800 ppm); * α = 0.05

C. OFFSPRING: F1 Offspring Set 1a Males and Females (PND 70) – General Toxicity

1. Survival: There was no treatment-related effect on F1 Set 1a male or female survival. All 

offspring survived to scheduled necropsy (PND 70).

2. Body weight (F1 Set 1a Males and Females): Males: Males displayed significant decreases in 

body weight at 800 ppm (↓11%-17%) throughout the study period, with the magnitude of the 

reduction lessening with time of exposure (Table 35). Consistent with these body weight 

decrements, body weight gains were significantly decreased in 800 ppm males at all intervals 
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from PND 21-56 (↓11%-25%). Body weights/body weight gains were not significantly altered at 

100 ppm and 300 ppm. Female: There were no treatment-related effects on body weights/body

weight gains in females at any dose level. 

TABLE 35. Mean (SD) F1 Body Weights/Gains of Set 1a (PND 70) (g) a

Dose group (ppm)

Day

0 100 300 800 0 100 300 600

F1 Set 1a – male F1 Set 1a – female

21 53.75.4 50.25.2 48.75.5 47.65.0 ↓11% 50.94.7 49.44.8 48.03.7 48.05.1

28 92.56.5 88.78.7 86.59.6 76.97.8* ↓17% 85.15.7 83.64.4 81.95.9 79.36.9 ↓7%

69 412.828.1 403.438.7 390.746.5 366.325.2* ↓11% 247.821.5 243.213.6 246.922.4 243.722.5

21-28 38.93.5 38.54.1 37.85.7 29.36.6* ↓25% 34.23.3 34.23.0 33.92.8 31.22.8 ↓9%

21-69 359.230.0 353.335.6 343.345.0 318.822.6 ↓11% 197.018.9 193.616.8 198.822.0 195.621.2

a

Data obtained from Tables 91-92, pages 357-360 in the study report. n=10, except day 69 [female 100 ppm (n=8); female 300 ppm 

(n=9)] due to BW inadvertently not collected

3. Hematology: Platelet counts were reduced (↓14%) in the 800 ppm males compared to the 

control males, with all of the 800 ppm values being less than the mean control value (Table 36). 

A similar reduction was not observed in the F1 Set 1a females. There were no other changes in 

the hematological parameters monitored, and the differential white blood cell counts and 

prothrombin times were similar among the groups (both sexes). 

Table 36. Platelet Counts – F1 Offspring (Set 1a)

Parameter 0 ppm 100 ppm 300 ppm 600/800 ppm

Males

Platelet counts (E3/UL) 1441234 1386181 1354172 1236151 ↓14%

Range 1165-1908 1147-1711 1089-1629 1015-1426

Females

Platelet counts (E3/UL) 1365182 1361156 1388166 1341204

 Data from Tables 97 and 98, pages 365 and 366 of report; n=10 

4. Clinical Chemistry: F1 Males: In the Set 1a 800 ppm males, there was a slight, but statistically

significant, increase (18%) in ALT levels (Table 37) but a lack of corresponding liver 

histopathology (see below). Triglyceride levels were increased (↑23% and 31%) at 300 ppm and 

800 ppm, respectively, compared to the control. No significant, treatment-related, changes in 

glucose or electrolyte levels were reported. F1 Females: Increased ALT ((↑25%) and 

triglycerides ((↑43%) values were observed in females at 600 ppm compared to the control 

(Table 36). No significant, treatment-related changes in glucose or electrolyte levels were noted. 

Table 37. Clinical Chemistry – F1 Offspring (Set 1a) – PND 70

Parameter 0 ppm 100 ppm 300 ppm 600/800 ppm

Males

ALT (U/L) 405 375 427 477* ↑18%

Triglycerides (mg/dL) 3914 4112 4817 ↑23% 5114 ↑31%

Females

ALT (U/L) 327 347 365 409 ↑25%

Triglycerides (mg/dL) 4012 345 4111 5724 ↑43%

 Data from Tables 103 and 104, pages 371 and 372 of report; n=10, except 100 ppm females (n=9) 
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5. Urinalysis: PND 64-66: There were no treatment-related changes in urinalysis parameters at any

dose level. The females at 300 ppm and 600 ppm displayed higher urine volumes (not doserelated) and lower specific gravity but these minor differences, which were not present in the 

males, were considered to be due to normal variation and not treatment related.

6. Organ and Organ/Body Weights Summary (F1 Set 1a Males and Females, PND 70): Males:

Terminal body weights were significantly decreased by 10% in males at 800 ppm, but were not 

significantly affected at 100 ppm or 300 ppm. Decreased absolute and related spleen weights 

were observed at all dose level, with the 800 ppm dose group displaying a 17% deficit (not 

statistically significant) compared to the control. Slight, but significantly, decreased absolute 

liver (↓16%) weight, relative liver weight (↓ 6%, not significant), absolute brain (↓4%) weight, 

and increased relative brain weight (↑7%, not significant) were observed at 800 ppm. 

Additionally, absolute thyroid weight was decreased (↓11%) and pituitary weight was deceased 

(absolute ↓14%/relative ↓6%) in males at 800 ppm, although statistical significance was not 

attained. Other decreases in absolute organ weights at 800 ppm may be attributed to the 10% 

lower body weight at 800 ppm (Table 38). Organ weights were not significantly altered at 100 

ppm and 300 ppm. Females: Terminal body weights were not significantly affected. At 600 ppm, 

uterine weights (absolute ↑31%; relative ↑32%) were increased and thymus weights were 

decreased (absolute ↓12%; relative ↓10%) compared to the control, although statistical 

significance was not attained. Relative kidney weights were significantly increased (↑11%) at 

300 and 600 ppm, which was similar to the increase in kidney weights observed in females in the 

2,4-D range-finding study (5% and 16% increase in relative kidney weights at 400 and 800 ppm, 

respectively; MRID 47417901). Non-significant increases in absolute kidney weights also were 

seen (11% and 9% at 300 and 600 ppm, respectively). Kidney weights were not significantly

altered in females at 100 ppm. These kidney findings were considered to be treatment-related but 

were not considered adverse in the absence of corresponding histopathological findings. It is to 

be noted that the kidney appears to be a target organ and histopathology was observed in the 

kidney in other sets. There were no significant, treatment-related effects on absolute or relative 

adrenal, heart, liver, brain, spleen, pituitary, or thyroid gland weights at any dose level in the Set 

1a females (Table 38). 

TABLE 38. Organ Weightsa F1 Set 1a Offspring – General Toxicity (PND 70)

Organ Dose group (ppm)

Control 100 300 600/800

F1 Set 1a MALES (PND 70)

Terminal Body Weight (g)

N=

379.4±24.4

10

373.21±31.6

10

379.3±52.1

10

340.1±22.7* ↓10%

10

Kidneys

●absolute

●relative

3.108±0.302

0.819±0.064

3.004±0.395

0.803±0.056

3.208±0.412

0.850±0.075

3.050±0.334

0.899±0.102

Liver

●absolute

●relative

12.076±1.343

3.177±0.202

11.538±1.076

3.090±0.059

12.353±2.269

3.244±0.185

10.169±0.809* ↓16%

2.994±0.191 ↓6%

Thyroid

●absolute

●relative

0.0195±0.0031

0.0051±0.0008

0.0204±0.0028

0.0055±0.0007

0.0190±0.0035

0.0050±0.0009

0.0174±0.0019 ↓11%

0.0052±0.008

Testes

●absolute

●relative

3.306±0.115

0.875±0.074

3.366±0.239

0.904±0.060

3.201±0.358

0.849±0.077

3.237±0.277

0.954±0.088

Prostate 
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TABLE 38. Organ Weightsa F1 Set 1a Offspring – General Toxicity (PND 70)

Organ Dose group (ppm)

Control 100 300 600/800

●absolute

●relative

0.753±0.139

0.198±0.035

0.776±0.164

0.207±0.035

0.791±0.129

0.209±0.021

0.707±0.144 ↓6%

0.208±0.043

Seminal vesicles w/cg

●absolute

●relative

1.072±0.180

0.282±0.041

1.147±0.196

0.307±0.042

1.141±0.218

0.301±0.045

1.010±0.163

0.298±0.054

Epididymides

●absolute

●relative

0.895±0.063

0.237±0.028

0.862±0.053

0.232±0.014

0.870±0.085

0.232±0.027

0.842±0.062 ↓6%

0.248±0.021

Brain

●absolute

●relative

2.008±0.076

0.532±0.047

1.978±0.089

0.532±0.035

2.023±0.054

0.541±0.068

1.927±0.035* ↓4%

0.569±0.035

Pituitary

●absolute

●relative

0.0116±0.0010

0.0031±0.0003

0.0114±0.0013

0.0031±0.0003

0.0109±0.0019

0.0029±0.0004

0.0100±0.0012 ↓14%

0.0029±0.0002

Thymus

●absolute

●relative

0.545±0.080

0.144±0.024

0.561±0.099

0.150±0.022

0.599±0.074

0.160±0.022

0.525±0.070 ↓4%

0.155±0.023

Adrenals

●absolute

●relative

0.064±0.013

0.017±0.004

0.066±0.011

0.018±0.004

0.061±0.011

0.016±0.003

0.056±0.009 ↓12%

0.016±0.003

Spleen

●absolute

●relative

0.7660.194

0.2010.044

0.7140.098

0.1920.026

0.7000.082

0.1860.023

0.6350.071 ↓12%

0.1870.020 ↓7%

F1 Set 1a Females (PND 70)

Terminal Body Weight (g)

N=

229.6±21.1

10

228.3±13.4

10

230.9±21.6

10

224.6±19.0

10

Kidneys

●absolute

●relative

1.749±0.162

0.765±0.078

1.812±0.173

0.793±0.041

1.946±0.108 ↑11%

0.846±0.053* ↑11%

1.905±0.272 ↑9%

0.846±0.068* ↑11%

Liver

●absolute

●relative

7.226±0.606

3.152±0.132

7.259±0.767

3.179±0.258

7.194±0.842

3.114±0.186

7.091±0.760

3.154±0.133

Thyroid

●absolute

●relative

0.0159±0.0012

0.0070±0.0007

0.0157±0.0026

0.0069±0.0010

0.0144±0.0018

0.0063±0.0012

0.0151±0.0024

0.0067±0.0007

Ovaries

●absolute

●relative

0.099±0.018

0.044±0.011

0.105±0.022

0.046±0.010

0.105±0.018

0.045±0.007

0.108±0.014 ↑9%

0.048±0.007

Uterus

●absolute

●relative

0.5229±0.1082

0.2307±0.0596

0.5354±0.1114

0.2351±0.0491

0.5521±0.1540

0.2436±0.0840

0.6861±0.2367 ↑31%

0.3039±0.1003 ↑32%

Brain

●absolute

●relative

1.887±0.072

0.826±0.057

1.850±0.076

0.812±0.037

1.859±0.068

0.812±0.086

1.864±0.083

0.835±0.079

Pituitary

●absolute

●relative

0.0116±0.0011

0.0051±0.0007

0.0125±0.0022

0.0055±0.0009

0.0116±0.0012

0.0050±0.0004

0.0126±0.0024 ↑9%

0.0056±0.0007

Adrenals

●absolute

●relative

0.063±0.006

0.028±0.004

0.066±0.011

0.029±0.004

0.067±0.010

0.029±0.004

0.063±0.010

0.028±0.004

Thymus

●absolute

●relative

0.522±0.109

0.227±0.042

 0.490±0.116

0.214±0.045

0.508±0.133

0.220±0.053

0.4600.104 ↓12%

0.2040.036 ↓10%

Spleen

●absolute

●relative

0.493±0.081

0.215±0.027

0.489±0.082

0.213±0.029

0.508±0.074

0.220±0.025

0.465±0.104 ↓7%

0.206±0.033 ↓4%

a

Data obtained from Tables 111-112, page 379-384 in the study report. * α = 0.05; mean SD
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7. Gross Pathology Observations (F1 Set 1a Males and Females): There were no gross 

observations attributed to dietary 2, 4-D exposure in the F1 Set 1a rats. 

8. Histopathological Observations (F1 Set 1a Males and Females): Males: The kidney appears 

to be a target organ. There was a dose-related increase in microscopic findings in the kidney in 

males. A degenerative lesion involving the proximal convoluted tubules in the outer stripe of the 

outer zone of the medulla, which was multifocal in distribution and very slight or slight in 

degree, was observed in the kidney in males at the 300 ppm and 800 ppm dose levels (Table 39). 

There were no histopathological alterations in the kidneys at 100 ppm. With the exception of 

kidneys, other tissues from animals in the low- and middle-dose groups were not examined, 

because of the lack of treatment-related effects in the high-dose animals. Females: At 600 ppm, 

females displayed microscopic, treatment-related, kidney effects similar in nature to those 

observed in the males; however, these effects were very slight in degree (Table 39). There were 

no histopathological alterations in the kidneys at 100 ppm or 300 ppm. Regarding the terminal 

stage of estrous, 2 of 10 females at 300 ppm and 3 of 10 females at 600 ppm displayed proestrus 

whereas none of the 10 females in the control and 100 ppm groups displayed proestrus.

TABLE 39. Terminal Stage of Estrous and Microscopic Kidney Findingsa F1 Set 1a Males and Females (PND 70)

Observation Dose group (ppm)

Control 100 300 600/800

F1 Set 1a

MALES

Degeneration, proximal convoluted tubule, outer strip, outer zone n=

● multifocal, very slight

 ● multifocal, slight

 ● focal, very slight

10

1

0

1

10

1

0

2

11

6

0

1

11

4

5

0

FEMALES

Terminal stage of estrous n=

● estrus

● diestrus

● proestrus

10 

3

7

0

10

3

7

0

10

1

7

2

10

3

4

3

Degeneration, proximal convoluted tubule, outer stripe, outer zone

● multifocal, very slight

 ● multifocal, slight

 ● focal, very slight

10

1

0

1

10

0

0

1

10

1

0

2

10

5

0

1

a

Data obtained from Text Table 19, page 113 and Appendix Table 87, pages 892-895 in the study report. 

C. OFFSPRING: F1 Offspring Set 1b (PND 54-56) – Developmental Neurotoxicity 

1. Body weight (Set 1b F1 Males and Females): There were no significant differences in body

weight or body-weight gain in either sex (Table 40).
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TABLE 40. Mean (SD) F1 Body Weights/Gains of Set 1b (PND 54-56) (g) a

Dose group (ppm)

Day

0 100 300 B800 0 100 300 600

F1 Set 1b – male F1 Set 1b – female

21 51.23.5 52.46.0 51.64.0 48.24.7 ↓6% 50.74.8 48.56.3 48.34.6 46.04.1

28 89.74.7 91.77.3 89.77.7 81.77.8* ↓9% 83.86.5 81.910.4 80.27.3 76.46.6 ↓9%

56 332.423.4 343.122.0 337.930.1 318.324.8 213.219.6 214.718.8 201.922.2 201.713.0

21-28 38.52.0 39.33.7 38.15.2 33.43.8* ↓13% 33.13.2 33.44.4 31.95.4 30.43.4 ↓8%

21-56 281.225.1 290.717.9 286.327.1 270.122.2 ↓4% 162.518.9 166.216.5 153.622.6 155.710.5 ↓4%

a

Data obtained from Tables 117-118, pages 399-400 in the study report. n=10; Bincludes one outlier; * α = 0.05; mean SD 

2. Functional Observational Battery (FOB): There was no significant, treatment-related, effect 

on body weight at any dose level in either sex (Table 41). The only reported difference in FOB 

observations was in the level of urination in males during the open-field evaluation. There was 

an increase in the level of urination in all treated male groups compared to the control but there 

was no dose-response. 

a. Rectal Temperature: There were no treatment-related differences observed in either sex.

b. Grip Performance: Although there were no statistically significant differences among the 

groups in either forelimb or hind limb grip performance, both sexes displayed a 10% reduction in 

hind limb grip performance compared to their respective controls (Table 41). NOTE: Forelimb 

grip strength appears to be similar between the sexes, whereas hind limb grip strength is greater 

in the males than in the females.

c. Landing Foot Splay: At the high-dose level in both sexes, males displayed a 17% increase 

and females displayed a 13% decrease in landing foot splay (Table 41). 

Table 41. FOB Parameters – F1 Offspring Set 1b

Parameter 0 ppm 100 ppm 300 ppm 600/800 ppm

Males

Body weight (g) 332.723.3 337.321.2 332.226.1 319.423.8 (↓4%)

Rectal temperature (°C) 38.20.5 38.30.5 38.40.6 37.90.7

Hind limb grip (g) 1002.3154.0 921.8167.4 (↓8%) 991.1116.8 904.8151.2 (↓10%)

Forelimb grip (g) 622.9140.2 659.2142.4 662.8251.6 (↑6%)B

608.7119.4 (↓2%)

Landing foot splay (cm) 9.621.71 10.471.61 10.491.51 11.262.33 (↑17%)

Urination

none

minimal

moderate

1.1

9

1

0

2.1

2*

5

3

1.8

4*

4

2

1.7

5

3

2

Females

Body weight (g) 212.120.6 214.818.5 200.721.1 199.214.6 (↓4%)

Rectal temperature (°C) 38.50.4 38.60.3 38.30.6 38.40.6

Hind limb grip (g) 714.8135.1 699.1121.0 689.8136.4 649.5124.1 (↓10%)

Forelimb grip (g) 635.5145.8 635.9106.2 584.6133.7 647.879.3

Landing foot splay (cm) 10.311.79 9.881.83 8.971.65 8.942.16 (↓13%)

Urination 1.2 1.2 1.3 1.6
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Table 41. FOB Parameters – F1 Offspring Set 1b

Parameter 0 ppm 100 ppm 300 ppm 600/800 ppm

none

minimal

moderate

8

2

0

8

2

0

8

1

1

6

2

1

Data from Tables 123-139, pages 405-422 of report; n= 10; α = 0.02; B606.2187.5 (↓3%) without outlier (1172.3), 

d. Motor Activity: Males: During the first half of the session, all male groups displayed a 

similar motor activity (MA) level (were within 6%), whereas the 800 ppm males showed a 

progressive lessening of activity with increased time (Epochs 5 through 8); i. e., the 800 ppm 

males displayed decreased activity compared to the control (↓11%, ↓16%, ↓30%, and ↓34% in 

Epochs 5, 6, 7, and 8, respectively, compared to control activity). Total MA counts were reduced 

(↓10%) in the 800 ppm males compared to the control (Table 42). Females: The 600 ppm 

females displayed a 12% increase in total motor activity compared to the control females. All 

treated groups displayed slightly greater activity (↑5%-8%) than the control during the 3 initial 

epochs, although there was no dose-response. 

Table 42. Motor Activity Counts per Epoch (square root) and Total MA Counts – Set 1b F1

Dose 

(ppm)

Epoch 1 Epoch 2 Epoch 3 Epoch 4 Epoch 5 Epoch 6 Epoch 7 Epoch 8 Total

Males

0 7.750.80 6.090.95 5.670.88 4.810.96 4.911.25 4.361.16 4.271.83 3.801.92 41.666.99

100 7.710.58 6.211.05 4.741.82 4.461.83 4.641.03 3.421.92 4.301.61 3.501.96 38.985.71

300 7.400.74 6.101.10 5.320.96 5.111.20 4.372.02 4.331.83 3.712.44 3.442.32 39.768.90

800 7.280.69 6.160.72 5.461.03 5.091.40 4.362.08 3.681.91 2.992.74 2.492.21 37.509.80 ↓10%

Females

0 6.920.82 5.210.81 5.121.07 4.940.81 4.990.76 3.901.63 4.301.23 4.340.93 39.713.39

100 7.290.50 6.460.82 5.470.87 5.870.78 5.451.05 4.572.21 4.632.01 4.202.09 43.935.57

300 7.540.80 6.521.07 5.901.38 4.411.84 5.440.73 5.041.19 4.511.08 3.732.05 43.106.52

600 7.450.65 6.280.50 5.871.09 5.520.82 4.941.73 4.940.94 5.070.97 4.370.67 44.434.40 ↑12%

Data from Tables 141-144, pages 424-427; means.d.; n=10
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3. Acoustic Startle Response (ASR): Males: Exposure did not affect the ASR in F1 male (p = 

0.5888) Set 1b rats. The interaction of Exposure × Block was statistically significant in males (p 

= 0.0022), indicating that the habituation of the ASR was significantly affected by exposure. 
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However, subsequent linear contrasts of control and treated groups for the interaction of 

Exposure × Block were not statistically significant (Block × 0 ppm vs. 100 ppm, p = 0.1701; 

Block × 0 ppm vs. 300 ppm, p = 0.0702; Block × 0 ppm vs. 800 ppm, p = 0.0456). The 

significant Exposure × Block interaction in males was thought to be due to the slightly higher 

ASR in the first block for the 800 ppm males when compared to controls (Table 43; Figure 26 

from study report). This was not interpreted to be a treatment-related effect due to the lack of 

effect on habituation, lack of statistical significance in the linear contrast when compared to 

controls, and because the ASR of the 800 ppm males was similar to two recent control data sets 

from the performing laboratory in male rats of the same strain, age and body weight (Andrus, 

2007). Females: There were no apparent treatment-related differences in the acoustic startle 

response in females (Table 43). 

In assessing whether the high-dose males displayed a different response compared to the control, 

it is noted that one of the 10 control males (the outlier) displayed a value greater than 1000 in 

Block 1 compared to 3/10, 2/10, and 6/10 males at the 100 ppm, 300 ppm and 600 ppm dose 

levels, respectively. One 800 ppm male had a value in Block 5 (1327) that was comparable to 

that in Block 1 (1396). Another 800 ppm male displayed values greater than 1100 in Blocks 1-4.

Table 43. Acoustic Startle Response– F1 Offspring Set 1b

Block # 0 ppm 100 ppm 300 ppm 600/800 ppm

Males

1 717.6339.4♪ 885.8446.1 738.6357.5 1111.6475.2

2 426.3257.4♪ 639.7349.0 468.0265.1 496.1351.5

3 438.2245.0♪ 485.8200.5 356.1243.8♪ 509.5381.5

4 360.0268.5♪ 420.4197.2 415.0271.9♪ 486.6349.0

5 371.1249.5 340.7159.7 433.8195.6 480.9415.6

Females

1 808.9280.5 903.1401.1♪ 999.6358.8 881.9313.2

2 417.6256.1 454.9331.2 692.7377.4 517.3288.5♪

3 355.3228.5 409.6282.3 551.3366.3 417.6245.3♪

4 333.2125.6 400.7307.7 484.2275.4 424.3204.7

5 269.395.5 367.2293.6 451.6281.0 353.6134.7

 Data from Tables 146-147, pages 429-430 of the study report; n= 10; ♪ includes statistical outlier 
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4. Necropsy/Neuropathology

a. Brain Weight/Gross Measurements: Male and female terminal body weights were comparable 

among the groups (Table 44). There were no significant differences in perfused absolute brain 
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weights, cerebral lengths and widths, or cerebellar lengths and widths in perfused Set 1b males or 

females (PND 60).

Table 44. Brain Weight and Gross Measurements – F1 Offspring Set 1b (perfused)

0 ppm 100 ppm 300 ppm 600/800 ppm

Males

Body weight (g) 320.024.0 332.825.4 325.232.3 310.423.7 ↓3%

Brain weight (g) 2.3480.133 2.3950.134 2.3350.109 2.3850.133

CRL (mm) 16.380.33 16.050.39 16.170.28 16.200.28

CRW (mm) 16.580.37 16.680.39 16.560.18 16.620.27

CBL (mm) 7.140.30 7.210.38 7.280.321 7.090.45

CBW (mm) 12.400.49 12.510.48 12.520.23 12.400.31

Females

Body weight (g) 203.517.2 207.717.6 195.121.1 194.211.4 ↓5%

Brain weight (g) 2.2280.115 2.1880.157 2.2090.104 2.1520.083

CRL (mm) 15.590.31 15.300.42 15.400.27 15.300.27

CRW (mm) 16.020.35 15.960.39 16.190.30 15.990.29

CBL (mm) 7.600.36 7.350.26 7.560.29 7.430.33

CBW (mm) 12.180.17 12.210.36 12.120.27 12.070.33

Data from Tables 149-150, pages 432-433 of the study report; n= 10; CRL=cerebrum length; CCRW= cerebrum width; 

CBL=cerebellum length; CBW= cerebellum width

b. Gross Pathology Observations: There were no gross observations attributed to 2, 4-D in 

perfused F1 Set 1b male or female rats.

c. Neuropathological Observations: There were no treatment-related microscopic changesin the 

central or peripheral nervous system in F1 Set 1b perfused male and female rats. This assessment 

included an evaluation of the cerebellum and corpus collosum for signs of myelin alterations 

using Luxol Fast Blue. There were no treatment-related changes in myelin. Histopathological 

observations were interpreted to be spontaneous occurrences common to this strain/age of rat and 

unrelated to 2, 4-D. Neuropathology was not assessed in the 100 and 300 ppm animals, because 

of the lack of treatment-related effects at 600 ppm and 800 ppm.

d. Brain Morphometrics (Perfused F1 Set 1b rats): There were no treatment-related changes in 

microscopic measurements of structures in the cerebral cortex, thalamus, or hippocampus. Due to

slight damage of some sections in the cerebellum during dissection, an accurate measurement of 

one parameter (cerebellar width) was not possible in all samples. However, sample sizes of 

undamaged cerebellum were considered adequate for evaluation (n > 6). There were no 

significant differences in microscopic cerebellar width measurements and no indications of 

significant morphometric alterations based on the two additional microscopic measurements in 

the cerebellum with larger sample sizes. Furthermore, gross measurements of cerebellar length 

and width did not indicate any significant changes. It was determined that there were no 

significant treatment-related effects on brain morphometry measurements in F1 Set 1b 800 ppm 

males and 600 ppm females compared with controls, and morphometric analysis of brain 

structures was not conducted in the 100 and 300 ppm rats.

C. OFFSPRING: F1 Unselected Offspring (PND 22 Weanlings) 

1. Survival: Non-perfused F1 PND 22 Weanlings: There were no effects on the survival of 
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unselected F1 male or female weanlings used for systemic toxicity (non-perfused weanlings). 

Weanlings designated as unselected F1 weanlings survived to scheduled necropsy. 

2. Organ and Organ/Body Weights: F1 PND 22 Weanlings: Males: Terminal body weights 

were decreased in all dose groups (9%-10%), although the decrease at the 300 ppm dose level 

was not statistically significant (Table 45). Decreased absolute (↓37%*) and relative (↓29%) 

adrenal weights were observed at 600 ppm. Testes weight (absolute) was significantly decreased 

at all dose levels (14%*-15%*), and relative testes weight was slightly (↓5%) lower at all dose 

level compared to the control. Absolute spleen weights were significantly decreased (↓18%-26%) 

at all dose levels, although there was no dose-response. The organ weight effects in both the 100 

ppm and 300 ppm males were considered secondary to lower body weights and not treatment 

related. Additionally, the body weight decrements related to systemic toxicity at 800 ppm 

contributed to the magnitude of the organ weight decreases in relative liver weights and absolute 

adrenal, kidney, liver, spleen and testes weights observed at 800 ppm. There were no effects on 

pituitary, thyroid or brain weights in male weanlings at any dose level. NOTE: The prostate, 

seminal vesicles, epididymides, and thymus were not weighed in this group. Females: There 

were no significant effects reported on terminal body weights or absolute or relative organ 

weights (adrenal glands, kidneys, liver, brain, spleen, pituitary, and thyroid) at any dose level. It

is to be noted that decreased (↓31%) adrenal weights (absolute and relative) were observed at 600

ppm, which is similar in magnitude to the response noted in the males at 800 ppm. NOTE: The 

ovaries, uterus, and thymus were not weighed in this group. 

TABLE 45. Organ Weightsa F1 PND 22 Weanlings (Unselected – General Toxicity)

Organ Dose group (ppm)

Control 100 300 600/800

F1 PND 22 MALES 

Terminal Body Weight (g)

N=

57.9±3.8

10

52.2±3.8*↓10%

10

52.8±3.9 ↓9%

10

51.9±7.4*↓10%

10

Kidneys

●absolute

●relative

0.695±0.085

1.200±0.114

0.582±0.052*↓16%

1.117±0.066

0.614±0.078 ↓12%

1.159±0.081

0.589±0.088*↓15%

1.137±0.088

Liver

●absolute

●relative

2.451±0.322

4.218±0.324

2.046±0.266* ↓17%

3.910±0.282

2.129±0.231 ↓13%

4.035±0.339

1.998±0.389*↓18%

3.832±0.273*↓9%

Thyroid

●absolute

●relative

0.0063±0.0014

0.0109±0.0022

0.0054±0.0012 ↓14%

0.0104±0.0019

0.0051±0.0009 ↓19%

0.0096±0.0013

0.0054±0.0011 ↓14%

0.0105±0.0015

Testes

●absolute

●relative

0.278±0.044

0.479±0.067

0.237±0.024* ↓15%

0.454±0.039 ↓5%

0.239±0.027* ↓14%

0.453±0.035 ↓5%

0.236±0.031*↓15%

0.457±0.041 ↓5%

Brain

●absolute

●relative

1.549±0.091

2.681±0.186

1.511±0.053 ↓2%

2.905±0.176

1.492±0.074 ↓4%

2.840±0.237

1.486±0.070 ↓4%

2.912±0.386

Pituitary

●absolute

●relative

0.0030±0.0008

0.0052±0.0013

0.0027±0.0006 ↓10%

0.0051±0.0010

0.0027±0.0009 ↓10%

0.0051±0.0017

0.0027±0.0008 ↓10%

0.0052±0.0016

Adrenal glands

●absolute

●relative

0.0261±0.0080

0.0449±0.0124

0.0220±0.0066 ↓16%

0.0422±0.0117

0.0257±0.0490

0.0490±0.0192

0.0165±0.0037*↓37%

0.0318±0.0054↓29%

Spleen

●absolute

●relative

0.282±0.047

0.485±0.060

0.209±0.045* ↓26%

0.398±0.071* ↓18%

0.232±0.028* ↓18%

0.440±0.040 ↓9%

0.222±0.050* ↓21%

0.424±0.053 ↓13%

F1 PND 22 Females

Terminal Body Weight (g) 52.7±6.1 50.4±7.9 50.5±4.4 51.2±5.9
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TABLE 45. Organ Weightsa F1 PND 22 Weanlings (Unselected – General Toxicity)

Organ Dose group (ppm)

Control 100 300 600/800

N= 10 10 10 10

Kidneys

●absolute

●relative

0.629±0.097

1.191±0.081

0.612±0.099

1.219±0.093

0.607±0.048

1.214±0.042

0.599±0.087 ↓5%

1.169±0.070

Liver

●absolute

●relative

2.138±0.335

4.046±0.229

2.018±0.419

3.985±0.331

2.064±0.265

4.077±0.249

2.059±0.362

4.002±0.328

Thyroid

●absolute

●relative

0.0059±0.0011

0.0112±0.0015

0.0055±0.0015 ↓7%

0.0109±0.0023

0.0051±0.0006 ↓14%

0.0100±0.0014

0.0055±0.0008 ↓7%

0.0108±0.0015

Brain

●absolute

●relative

1.491±0.064

2.862±0.316

1.439±0.065

2.918±0.439

1.458±0.064

2.903±0.234

1.438±0.057

2.842±0.309

Pituitary

●absolute

●relative

0.0030±0.0007

0.0057±0.0009

0.0026±0.0004↓14%

0.0052±0.0009

0.0025±0.0003 ↓17%

0.0050±0.0005

0.0027±0.0003 ↓10%

0.0052±0.0003

Adrenal

●absolute

●relative

0.0264±0.0081

0.0511±0.0178

0.0247±0.0087

0.0493±0.0165

0.0251±0.0086

0.0502±0.0191

0.0181±0.0046 ↓31%

0.0353±0.0066 ↓31%

Spleen

●absolute

●relative

0.253±0.034

0.481±0.038

0.245±0.065

0.484±0.100

0.213±0.044 ↓16%

0.421±0.072 ↓12%

0.230±0.050 ↓9%

0.446±0.063 ↓7%

a

Data obtained from Tables 79-80, pages 330-333 in the study report. * α = 0.05; mean SD

3. Gross Pathology Observations (F1 PND 22 Weanlings): There were no gross observations 

attributed to 2, 4-D exposure in the unselected F1 weanlings (both sexes). 

4. Histopathological Observations (F1 PND 22 Weanlings): There were no treatment-related 

histopathological observations in the weanling rats at 600 ppm/800 ppm (only group examined). 

5. Survival (Perfused F1 PND 22 Weanlings): There was no treatment-related effect on the 

survival of unselected weanlings used for perfusions. All unselected F1 weanlings survived to 

scheduled necropsy.

6. Brain Weight and Gross Brain Measurements (Perfused F1 PND 22 Weanlings): Males:

Terminal body weights were decreased slightly at the 300 ppm (↓8%) and 800 ppm (↓6%) dose 

levels, but there was no dose-response. There were no significant differences in perfused 

absolute brain weights, cerebral lengths and widths, or cerebellar lengths and widths in perfused 

F1 male weanlings. Females: Terminal body weights were not significantly altered in the 

perfused F1 female weanlings. According to the report, gross brain measures (CRL, CRW, CBL, 

CBW) in the 100 ppm group were statistically different from controls (multivariate analysis 

across measures followed by linear contrasts), but because this finding did not follow a doseresponse relationship, the result was considered spurious and unrelated to 2, 4-D exposure (Table 

46). NOTE: It is not evident to this reviewer how, for example, 15.830.32 (control CRW) is 

statistically different from 15.820.34 (100 ppm CRW). The CRW for the 600 ppm females is 

15.660.43 (↓1%).
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Table 46. Brain Weight and Gross Measurements – F1 Weanlings PND 22 (perfused)

0 ppm 100 ppm 300 ppm 600/800 ppm

Males

Body weight (g) 57.13.9 56.45.7 52.54.8 ↓8% 53.76.1 ↓6%

Brain weight (g) 1.8380.092 1.8460.063 1.8290.108 1.8030.085 ↓2%

CRL (mm) 14.840.35 14.830.28 14.730.34 14.550.27

CRW (mm) 16.010.31 16.080.30 16.000.37 15.910.34

CBL (mm) 6.440.27 6.060.44 ↓6% 6.110.33 5.980.26 ↓7%

CBW (mm) 11.920.29 11.870.32 11.590.26 11.750.22

Females

Body weight (g) 54.15.4 53.14.5 49.93.8 ↓8% 50.04.3 ↓8%

Brain weight (g) 1.8220.127 1.7400.071 ↓5% 1.7740.075 1.7440.101

CRL (mm) 14.740.41 14.370.14 ↓3% 14.400.35 14.390.29

CRW (mm) 15.830.32 15.820.34 15.890.29 15.660.43 ↓1%

CBL (mm) 7.020.38 6.820.42 ↓3% 6.990.23 6.840.25

CBW (mm) 11.720.30 11.620.26 ↓1% 11.670.24 11.440.37 ↓2%

Data from Tables 83-84, pages 342-343 of the study report; n= 12; CRL=cerebrum length; CCRW= cerebrum width; 

CBL=cerebellum length; CBW= cerebellum width; linear contrast for multivariate analysis of female 100 ppm group CRL, CRW, 

CBL, CBW statistically different from control at α = 0.02

7. Gross Pathology Observations: Perfused F1 PND 22 Weanlings: There were no gross 

observations attributed to 2, 4-D exposure in either male or female perfused F1 weanlings.

8. Neuropathological Observations: Perfused F1 PND 22 Weanlings: There were no 

neuropathological observations attributed to treatment with 2, 4-D in the perfused F1 PND 22 

weanlings. This assessment included an evaluation of myelin using Luxol Fast Blue. The 100 

ppm and 300 ppm groups were not examined due to the lack of treatment-related changes in 

myelin in the 600 ppm/800 ppm PND 22 weanlings.

D. F1 OFFSPRING: Set 2a (PND 67-73) – Developmental Immunotoxicity: Primary Immune 

Response to Sheep Red Blood Cells (SRBCs)

 

1. Survival and clinical signs: There were no effects on survival. All Set 2a F1 rats survived to 

scheduled necropsy. 

2. Body Weights/Body Weight Gains: Set 2a F1: Males: A slight decrease in body weight was 

observed initially in males at the 300 ppm (7%) and 800 ppm (6%) on PND 21, and the 800 ppm 

males displayed a statistically significant decrease (10%) on PND 28. Body-weight gain during 

PND 21-28 was decreased by 15% at 800 ppm but was comparable to control thereafter (Table 

47). Females: Body weights in the 600 ppm females were decreased by 8% on PND 21 and 9% 

on PND 28. Consistent with these body weight decrements, body weight gains were decreased by

11% in the 600 ppm females from PND 21-28, but were not markedly different from control 

values thereafter (PND 21-70). There were no exposure-related effects on body weights/body

weight gains at 100 ppm and 300 ppm.
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TABLE 47. Mean (SD) F1 Body Weights/Gains of Set 2a (PND 70) (g) a Immunotoxicity SRBC

Dose group (ppm)

Day

0 100 300 800 0 100 300 600

F1 Set 2a – male F1 Set 2a – female

21 52.73.9 51.95.3 47.84.2 

\\\\↓↓6%↓6%

49.66.2 ↓6% 51.44.6 48.74.9 50.16.3 47.33.8 ↓8%

28 90.83.9 90.26.8 83.18.1 81.910.4* ↓10% 85.85.8 81.55.8 84.48.1 78.06.7 ↓9%

70 410.333.2 415.125.0 394.038.5 398.817.2 ↓3% 256.623.4 257.723.6 254.738.8 242.513.6 ↓5%

21-28 38.12.5 38.43.6 35.35.5 32.48.6 ↓15% 34.44.3 32.83.0 34.33.1 30.83.5 ↓10%

21-70 357.631.9 363.322.4 346.237.0 349.316.7 ↓2% 205.221.3 209.024.4 204.633.7 195.316.1 ↓5

a

Data obtained from Tables 157-158, pages 447-450 in the study report’ n=10

3. Food consumption (F1 Set 2a Males and Females): There were no significant differences in 

Set 2a male (PND 28-70, ↓2%-5%) and female (PND 28-35, ↓9%) food consumption values at 

any interval examined, which is consistent with the minimal effects on body weights/body

weight gains.

4. Organ and Organ/Body Weights Summary (F1 Set 2a Males and Females): Set 2a rats were 

euthanized on PND 71-73 to minimize inter-assay variability for the SRBC assay. Terminal body

weights were comparable among the groups (both sexes). Males: Both absolute and relative 

thymus weights were 17% lower in the 100 ppm males (only relative weight was statistically

significant), but there was no dose response relationship and the finding was considered spurious. 

There were no significant, treatment-related effects on absolute or relative brain, spleen or 

thymus weights compared to the controls. The positive control displayed a significant loss in 

absolute (↓44%) and relative (↓41%) spleen weights and absolute (↓73%) and relative (↓72%) 

thymus weights, consistent with the expected response of a positive control. Females: Body

weights/gains were comparable among the groups, and there was no effect on terminal body

weights at any dose level. There were no statistically significant effects on absolute or relative 

brain, spleen, or thymus weights, although the thymus weight was decreased at 600 ppm 

(absolute ↓13% and relative ↓10%), and spleen weights were decreased at 300 ppm (↓13%) and 

600 ppm (↓14%/10%). The positive control (cyclophosphamide) showed a significantly

decreased terminal body weight (13%), a significant decrease in absolute (47%) and relative 

(39%) spleen weights, and a significant decrease in absolute (72%) and relative (68%) thymus 

weights (Table 48).

TABLE 48. Mean (SD) BodyWeight and Organ Weight – Set 2a F1 Offspringa

Weight (grams) Dose group

Control +Control 100 ppm 300 ppm 800/600 ppm

F1 Males – PND 67-73

Final body weight 424.3±36.1 406.3±30.0 ↓4% 431.1±24.4 408.5±34.6 ↓4% 418.7±17.1

Brain

●absolute

●relative

2.078±0.095

0.492±0.035

2.052±0.072

0.507±0.036

2.013±0.063

0.468±0.024

1.997±0.093 ↓4%

0.491±0.038

2.024±0.085

0.484±0.029

Spleen

●absolute

●relative

0.889±0.171

0.209±0.029

0.498±0.021* ↓44%

0.123±0.010 ↓41%

0.847±0.139

0.196±0.024

0.827±0.130

0.202±0.026

0.836±0.138

0.199±0.028

Thymus
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TABLE 48. Mean (SD) BodyWeight and Organ Weight – Set 2a F1 Offspringa

Weight (grams) Dose group

Control +Control 100 ppm 300 ppm 800/600 ppm

●absolute

●relative

0.614±0.109

0.144±0.019

0.165±0.035* ↓73%

0.041±0.009* ↓72%

0.512±0.046↓17%

0.119±0.010* ↓17%

0.625±0.140

0.153±0.031

0.554±0.060 ↓10%

0.132±0.013 ↓8%

F1 Females – PND 67-73

Final body weight 259.6±21.9 225.8±21.1*↓13% 261.2±22.4 259.0±38.1 252.5±18.2 ↓3%

Brain

●absolute

●relative

1.925±0.070

0.746±0.063

1.853±0.146

0.828±0.108

1.910±0.074

0.736±0.067

1.867±0.086

0.731±0.084

1.845±0.057 ↓4%

0.734±0.061

Spleen

●absolute

●relative

0.644±0.099

0.249±0.043

0.343±0.059* ↓47%

0.151±0.016*↓39%

0.638±0.094

0.245±0.036

0.563±0.102↓13%

0.217±0.018↓13%

0.556±0.118 ↓14%

0.219±0.036 ↓12%

Thymus

●absolute

●relative

0.542±0.097

0.208±0.033

0.153±0.041* ↓72%

0.067±0.014* ↓68%

0.475±0.092

0.182±0.035

0.511±0.108

0.197±0.030

0.473±0.079 ↓13%

0.187±0.025 ↓10%

a

Data obtained from Tables 163-164, pages 455-456 in the study report; n=10; +positive control (n=8); α = 0.05

5. Primary Immune Response to Sheep Red Blood Cells (SRBCs) Antibody Forming Cell 

(AFC) assay (F1 Set 2a Rats): Males: There was no significant difference in response for 

AFC/spleen and AFC/106splenocytes among the groups of male rats (Table 49). The results for 

the positive control cyclophosphamide (>95% suppression in both AFC/spleen and AFC/106 

splenocytes) showed that the test system was working properly. Females: At the 600 ppm dose 

level, females displayed a non-statistically significant decrease of 54% for the AFC/spleen and 

27% for the AFC/106splenocytes. No significant difference in response for AFC/spleen and 

AFC/106 splenocytes was observed is the 100 ppm and 300 ppm dose females compared to the 

vehicle control females. This change was not considered to be toxicologically relevant since it 

occurred at a dose beyond linear toxicokinetics and may have been confounded by the temporal 

variability introduced by the need to conduct the assay over several days. Analysis of the data by

day revealed that days 1 and 2 of the assay yielded a stronger AFC response when compared to 

days 3 and 4 (dose groups not balanced across days for the assay due to adherence to predefined 

age restrictions for study conduct). The positive control cyclophosphamide produced >99% 

suppression in both AFC/spleen and AFC/106 splenocytes. 

TABLE 49. SRBC AFC Response – Set 2a F1 OffspringaPND 67-73)

Parameter

Dose group

Control +Control (CYP) 100 ppm 300 ppm 800 ppm

F1 Males – PND 67-73

PFC/spleen 528022±645310 3017±7211*

↓>99%

615920±454496 513870±619679 555200±410656

PFC/million 580±524 18±44*↓97% 844±641 684±824 680±473

N= 9 8 10 10 9

F1 Females – PND 67-73

PFC/spleen 339200±448969 3125±2979*

↓>99%

230330±198225 

↓36%

355950±345431 156020±112661 

↓54%

PFC/million 491±466 19±16* ↓96% 502±398 809±788 360±312 ↓27%

N= 10 8 10 10 10

a

Data obtained from Tables 165-166, pages 457-458 in the study report; +control; n=8); α = 0.05
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E. F1 OFFSPRING Set 2b (PND 87-93) – Developmental Immunotoxicity: Natural Killer Cell 

Activity 

1. Survival and clinical signs: There were no effects on survival. All Set 2b F1 rats survived to 

scheduled necropsy. 

2. Body Weights/Body Weight Gains: Males: A slight decrease in body weight was observed initially

in Set 2b males at 800 ppm (8%) on PND 21. Thereafter, body weights were comparable among 

the groups (Table 50). Body-weight gain during PND 21-28 was comparable to the control.

Females: Body weights and body-weight gains were comparable among the groups (Table 50).
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TABLE 50. Mean (SD) F1 Body Weights/Gains of Set 2b (PND 84) (g) a Immunotoxicity NK Cell

Dose group (ppm)

Day

0 100 300 800 0 100 300 600

F1 Set 2b – male F1 Set 2b – female

21 50.04.9 52.06.4 49.84.1 45.85.2 ↓8% 48.64.8 52.16.6 47.53.5 46.74.0 ↓4%

28 84.011.6 90.39.5 89.56.1 79.87.7 ↓5% 81.26.1 84.99.9 81.06.6 77.55.7 ↓5%

70 407.324.2 427.324.3 423.827.6 404.337.5 238.917.1 243.516.9 238.121.4 244.619.2

84 461.029.6 488.831.6 490.031.0 468.948.7 262.017.5 266.614.9 263.118.4 269.527.3

21-28 34.07.6 38.33.7 38.83.7* 34.03.9 32.61.6 32.85.0 33.55.9 30.92.8 ↓5%

21-84 411.029.3 436.828.7 440.230.3 423.148.1 213.416.0 214.512.6 215.618.9 222.828.1

a

Data obtained from Tables 169-170, pages 461-464 in the study report.

3. Food consumption: Food consumption was not adversely affected.

4. Organ and Organ/Body Weights Summary (F1 Set 2b Males and Females): Rats were 

euthanized over a range of days (PND 87-93) to facilitate a balanced study design for each NK 

cell assay. Terminal body weights were comparable among the groups (both sexes). There were 

no significant effects on absolute or relative spleen or testes weights (only organs weighed) in the 

positive control and treated males, and no significant effects on absolute or relative spleen weight 

(only organ weighed) in the positive control and treated females (Table 51).

TABLE 51. Mean (SD) Body Weight and Organ Weight – F1 Set 2b Offspringa

Weight (grams)

Dose group

Control +Control 100 ppm 300 ppm 800 ppm

F1 Males – PND 87-93

Final body weight 483.0±33.0 449.0±62.1 ↓7% 513.8±33.0 519.4±29.5 495.2±57.7

Spleen

●absolute

●relative

0.749±0.155

0.154±0.025

0.709±0.100 ↓5%

0.159±0.017

0.773±0.071

0.151±0.015

0.804±0.139

0.155±0.024

0.741±0.135

0.149±0.017

Testes

●absolute

●relative

3.575±0.168

0.743±0.062

3.416±0.097 ↓4%

0.772±0.096

3.476±0.387

0.677±0.073

3.560±0.239

0.686±0.045

3.267±0.326 ↓9%

0.664±0.073

F1 Females – PND 87-93

Final body weight 269.3±16.8 253.0±22.1 ↓6% 278.7±16.9 272.1±17.0 281.1±30.1

Spleen

●absolute

●relative

0.495±0.096

0.184±0.032

0.467±0.036 ↓6%

0.185±0.013

0.505±0.066

0.182±0.025

0.517±0.048

0.190±0.019

0.507±0.071

0.181±0.020

 a

Data obtained from Tables 175-176, pages 469-470 in the study report; n=10; +positive control (n=8)

5. Natural Killer Cell Activity – F1 Set 2b Males and Females: There was no significant 

difference in percent target cell cytotoxicity at any dose level compared to the control (both 

sexes), whereas anti-asialo-GM1 (positive control) produced a significant 40–60% suppression 

of NK cell activity at the 400:1 and 200:1 effector-to-target ratios in both males and females 

(Table 52; Figure 33, copied from the study report, page 222), demonstrating the expected 

response. Based on these results, 2, 4-D did not alter the cytotoxic ability of splenic NK-cells in 

male or female rats at any dose levels.
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TABLE 52. NK Cell Activity – F1 et 2b Offspringa(PND 87-93) - percent target cell cytotoxicity

Effector-to-target cell 

ratios

Dose group

Control +Control (1:5GM1) 100 ppm 300 ppm 800 ppm

F1 Males – PND 87-93

NK % 50:1 RATIO 7.8±2.7 7.8±3.1 7.6±2.8 7.4±2.2 6.5±1.7 ↓17%

NK % 100:1 RATIO 9.9±2.5 8.5±2.5 10.6±3.6 9.9±2.3 8.0±1.3 ↓19%

NK % 200:1 RATIO 18.5±8.2 9.7±2.8* 19.2±8.5 17.8±7.1 16.5±9.9 ↓11%

NK % 400:1 RATIO 32.4±12.7 13.3±4.6* 36.5±10.3 30.9±7.5 31.8±13.4

NK % 800:1 RATIO 39.0±11.3 19.1±6.6* 42.3±9.7 36.8±5.7 39.6±11.6

F1 Females – PND 87-93

NK % 50:1 RATIO 4.3±3.2 3.7±0.7 5.3±2.5 4.4±2.2 3.5±2.1 ↓19%

NK % 100:1 RATIO 6.7±2.9 5.3±1.2 8.1±3.3 7.7±2.7 6.5±3.1

NK % 200:1 RATIO 14.3±4.5 8.7±3.2* 14.5±2.0 17.5±5.0 15.1±6.4

NK % 400:1 RATIO 29.7±8.7 13.6±5.3* 31.4±5.8 33.5±10.0 33.8±12.0

NK % 800:1 RATIO 37.7±6.7 21.2±13.4* 38.7±5.4 39.0±9.6 40.8±10.8

a

Data obtained from Tables 177-178, pages 471-472 in the study report; n=10; +control (anti asialo GM1; n=8); α = 0.05
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F. F1 OFFSPRING: Set 3 (PND 139) - Reproductive Toxicity

1. Survival and clinical signs: The were no compound-related effects on survival or clinical signs..
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2. Body weight (F1 Set 3 Males and Females): Males: After weaning, body weights in the 800 

ppm males were slightly decreased (≈↓7%) initially but were similar to control body weights at 

study termination (PND 139). Consistent with these reductions in body weight, body weight 

gains were decreased (↓≈6%-12%) from PND 21-49 but were similar to control values overall. 

Body weight/body weight gains were comparable to control values at 300 ppm, but significant 

increases in body weight (↑5%-7%) and body-weight gain (↑6%-7%) were observed at 100 ppm 

during PND 84-137. Females: Initially, a slight decrease in body weight (PND 21 ↓7%; PND 28

↓8%) was observed at 600 ppm compared to the control, and body weight gains were decreased 

by ↓10% (PND 21-28). Comparable body weights/gains were observed at 600 ppm compared to 

control during the rest of the treatment period. There were no significant treatment-related effects 

on body weights/body weight gains at 100 ppm and 300 ppm (Table 53). 

TABLE 53. Mean (SD) Body weight – F1 Offspring Set 3 Post-Weaning a

Observations/study week

Dose group

Control 100 ppm 300 ppm 800/600 ppm

F1 males – Set 3

Mean body weight (g)

 Day 21

 Day 28

 Day 35

 Day 137

52.0±4.9

89.6±9.8

150.7±12.3

606.7±50.4

51.7±5.3

90.0±7.4

150.5±10.7

644.0±50.7*

50.3±6.1

88.0±8.6

147.4±12.3

614.4±64.3

49.9±5.4 ↓4%

83.1±10.8*↓7%

140.0±15.1*↓7%

605.9±57.6

Mean weight gain (g)

 Days 21-28

 Days 21-137

37.6±6.4

554.7±48.6

38.3±3.2

592.2±49.4*

37.8±4.6

563.8±62.8

33.2±8.1*↓12%

556.0±59.0

F1 females – Set 3

Mean body weight (g)

 Day 21

 Day 28

 Day 35

 Day 137

49.7±5.2

83.4±6.2

128.0±9.4

316.6±29.5

49.4±4.9

82.3±5.4

127.1±6.5

316.0±23.6

48.7±5.7

81.3±7.7

126.4±10.1

322.3±29.2

46.0±4.2*↓7%

76.7±6.6*↓8%

121.6±9.9 ↓5%

316.9±35.4

Mean weight gain (g)

 Days 21-28

 Days 21-137

33.7±3.5

266.9±28.6

32.8±2.3

266.6±22.5

32.7±3.3

273.8±28.9

30.6±3.8*↓10%

270.8±33.6

a

Data obtained from Tables 181-182, pages 476-481in the study report.

* α <0.05. ** α <0.01. n = 27 (males)/39 (females; main +satellite)

3. Feed Consumption – F1 Set 3 Males and Females: Decreased food consumption was observed 

in the 800 ppm males (6%-8%) over the interval from PND 28-49, which is consistent with the 

decreased body weight observed in this group. There was no significant difference in food 

consumption among the female groups. 

4. Estrous cycle length: F1 Set 3 Females. No significant difference was observed in mean estrous 

cycle length in females at any dose level compared to the control (Table 54). There was no 

indication of persistent estrus; i.e., greater than 2 consecutive days in estrus, and no apparent 

difference in the percentage of time spent in estrus or diestrus (see Figure 13 in Appendix B).
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TABLE 54. Estrous cycle dataa(F1 Set 3 Females)

Mean days per cycle

Dose group

Control 100 ppm 300 ppm 600 ppm

4.0 8 15 10 15

4.2 4 3 1 2

4.3 1 2 3 -

4.4 1 1 1 1

4.5 2 1 - -

4.6 2 2 3 -

4.8 5 3 2 4

5.0 4 - - 2

5.2 - - 1 -

5.3 - - 1 -

5.7 - - 1# -

mean±sd 4.4±0.4 4.2±0.3 4.4±0.5 (23) 4.3±0.4 (24)

 a Data obtained from Table 187, page 488 and Appendix Table 144, pages 1136-1139 in study report.

 n=27, except (); # statistical outlier

5. Ovarian follicle counts – F1 Offspring Set 3 females: There were no significant, treatmentrelated effects on the numbers of small follicles, growing follicles, or total follicles (small + 

growing) at 600 ppm. Due to the lack of effect at 600 ppm, ovarian follicle counts were not 

conducted for the 100 ppm and 300 ppm samples (Table 55).

Table 55. Ovarian Follicle Counts – F1 Set 3 Females

Dose (ppm) Small Growing Total

0 66±27 25±10 92±26

600 66±26 28±7 94±28

 Data from Table 194, page 499 of the study report; n=15

6. Sperm Analyses – F1 Offspring Set 3: There were no significant, treatment-related effects on 

sperm motility or progressive motility (Table 56). With respect to epididymal sperm and 

testicular spermatid counts, there were no significant, treatment-related differences between 0 

and 800 ppm males. Due to the lack of effect at 800 ppm, sperm/spermatid counts were not 

conducted for the 100 and 300 ppm groups. Similarly, there were no significant, treatmentrelated, changes in sperm morphology in the 800 ppm group compared with the control group. 

While the proportion of abnormal sperm was slightly higher in the 800 ppm males (0.025 vs. 

0.016 in controls), this result was due to a single outlier value in the 800 ppm group (0.24). 

Removal of this outlier value resulted in an equivalent value for mean proportion of abnormal 

sperm between the 800 ppm and control groups (abnormal sperm = 0.016 in both groups). Sperm 

morphology was not examined in samples from the 100 ppm and 300 ppm groups due to the lack 

of effect at 800 ppm.

TABLE 56. Sperm dataa(F1 Set 3 males)

Dose group

Parameter Control 100 ppm 300 ppm 800 ppm

Motile (%) 96.8±4.6 97.4±2.6 97.4±2.6 A

97.4±2.3 B

Progressively Motile (%) 88.0±7.5 86.5±4.8 86.6±4.9 A

87.4±4.0 B

Testicular spermatid counts

Total Sperm (1006) 344.2±106.3 - - 322.5±72.4 B

Conc/g (1006) 184.7±49.5 - - 176.2±44.8 B
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TABLE 56. Sperm dataa(F1 Set 3 males)

Dose group

Parameter Control 100 ppm 300 ppm 800 ppm

Epididymal sperm counts

Total Sperm (1006) 278.5±92.9 - - 292.7±87.1 B

Conc/g (1006) 833.7±263.9 - - 895.6±228.1 B

Proportion of abnormal sperm

Abnormal sperm/total 0.016±0.012 - - 0.025±0.047 B

 a Data obtained from Tables 190-193, pages 495-498 in the study report; n=27, except A

(n=23); B(24)

7. Organ weights (F1 Set 3): Males – There was no adverse effect on terminal body weights in the 

males. There were no significant, treatment-related effects on absolute or relative weights of the 

adrenal glands, brain, kidneys, liver, prostate, spleen, testes, thymus, epididymides, seminal 

vesicle or thyroid glands. Absolute (↓9%) and relative (↓8%) pituitary gland weights were 

significantly lower at 800 ppm (Table 57). The magnitude of the differences from pituitary

weights in control rats are minimal (800 ppm: absolute 0.0127 g and relative 0.0022 g/100 g body

weight; control: absolute 0.0139 g and relative 0.0024 g/100 g body weight), and both the 

absolute and relative pituitary weights were within the historical control range for the testing 

facility. Additionally, there was no associated histopathology in the pituitary glands. 

Females - There was no adverse effect on terminal body weights in the females. Absolute and 

relative kidney weights were increased in 600 ppm females (↑8%), although the relative 

difference did not attain statistical significance. This finding was consistent with kidney weight 

increases in other groups, had a histopathological correlate, and was considered treatment 

related, although not adverse. Increased uterine weight (absolute ↑10% and relative ↑11%) was 

observed in the 600 ppm females, which is consistent with the increase observed in the P1 

females (LD 22) and F1 Set 1a females (PND 70). Pituitary weights were decreased (absolute 

↓9% and relative ↓10%) in the 600 ppm Set 3 females, but statistical significance was not 

attained. Additionally, thymus weight (absolute ↓14% and relative ↓13%) was decreased in the

600 ppm Set 3 females, but statistical significance was not attained. There were no significant, 

treatment-related, effects on absolute or relative weights of the adrenal glands, liver, brain, 

ovaries, spleen, or thyroid glands (Table 57). 

TABLE 57. Organ Weightsa F1 Set 3 Offspring

Organ Dose group (ppm)

Control 100 300 600/800

F1 Set 3 Males (PND 139)

Terminal Body Weight (g)

N=

578.7±48.2

27

616.1±49.3*

27

585.3±64.6

23

577.4±55.8

24

Kidneys

●absolute

●relative

4.005±0.415

0.693±0.060

4.171±0.404

0.678±0.059

4.167±0.401

0.717±0.081

4.094±0.392

0.712±0.064

Liver

●absolute

●relative

16.176±2.144

2.788±0.190

17.296±2.425

2.801±0.256

16.043±2.395

2.734±0.202

15.491±1.832 ↓4%

2.683±0.197

Thyroid

●absolute

●relative

0.0269±0.0040

0.0046±0.0006

0.0279±0.0047

0.0045±0.0007

0.0265±0.0048

0.0045±0.0007

0.0259±0.0049↓4%

0.0045±0.0008

Testes

●absolute

●relative

3.743±0.305

0.651±0.075

3.801±0.387

0.620±0.071

3.684±0.287

0.636±0.079

3.725±0.527

0.645±0.057

Prostate 
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TABLE 57. Organ Weightsa F1 Set 3 Offspring

Organ Dose group (ppm)

Control 100 300 600/800

●absolute

●relative

1.041±0.199

0.181±0.038

1.075±0.241

0.174±0.035

1.081±0.215

0.186±0.036

1.070±0.181

0.187±0.037

Seminal vesicles

●absolute

●relative

1.807±0.287

0.313±0.047

1.741±0.247

0.284±0.045

1.760±0.376

0.304±0.072

1.761±0.286

0.308±0.060

Epididymides

●absolute

●relative

1.399±0.125

0.244±0.031

1.386±0.113

0.226±0.023

1.375±0.091

0.237±0.029

1.388±0.088

0.243±0.028

Brain

●absolute

●relative

2.158±0.091

0.375±0.030

2.146±0.069

0.350±0.030*

2.139±0.085

0.369±0.039

2.123±0.093

0.370±0.033

Pituitary

●absolute

●relative

0.0139±0.0018

0.0024±0.0002

0.0143±0.0015

0.0023±0.0003

0.0137±0.0012

0.0024±0.0003

0.0127±0.0014* ↓9%

0.0022±0.0002* ↓8%

Thymus

●absolute

●relative

0.322±0.058

0.056±0.010

0.323±0.084

0.053±0.012

0.331±0.125

0.056±0.020

0.305±0.060 ↓5%

0.053±0.010

Adrenal

●absolute

●relative

0.060±0.010

0.010±0.002

0.067±0.009

0.011±0.001

0.066±0.010

0.011±0.002

0.061±0.011

0.011±0.002

F1 Set 3 Females (PND 139)

Terminal Body Weight (g)

N=

299.5±27.3

27

299.0±23.3

27

304.3±27.7

23

300.0±33.6

24

Kidneys

●absolute

●relative

2.080±0.212

0.698±0.084

2.146±0.173

0.720±0.061

2.199±0.192

0.726±0.067

2.241±0.218* ↑8%

0.751±0.069 ↑8%

Liver

●absolute

●relative

8.063±1.023

2.693±0.252

7.999±0.760

2.676±0.157

8.033±0.862

2.641±0.183

8.047±0.773

2.698±0.237

Thyroid

●absolute

●relative

0.0177±0.0030

0.0059±0.0009

0.0191±0.0029

0.0064±0.0010

0.0188±0.0035

0.0062±0.0010

0.0187±0.0029

0.0063±0.0011

Ovaries

●absolute

●relative

0.089±0.023

0.030±0.008

0.096±0.020

0.032±0.007

0.094±0.015

0.031±0.005

0.091±0.014

0.031±0.005

Uterus

●absolute

●relative

0.679±0.245

0.228±0.082

0.734±0.280

0.245±0.090

0.750±0.309 ↑10%

0.250±0.109 ↑10%

0.750±0.244 ↑10%

0.252±0.084 ↑11%

Brain

●absolute

●relative

1.971±0.084

0.662±0.055

1.933±0.064

0.650±0.047

1.978±0.080

0.654±0.056

1.925±0.066

0.649±0.069

Pituitary

●absolute

●relative

0.0152±0.0035

0.0051±0.0012

0.0149±0.0021

0.0050±0.0007

0.0145±0.0031

0.0048±0.0011

0.0139±0.0022 ↓9%

0.0046±0.0006 ↓10%

Thymus

●absolute

●relative

0.280±0.060

0.093±0.016

0.262±0.062

0.088±0.020

0.296±0.059

0.097±0.018

0.242±0.043 ↓14%

0.081±0.013 ↓13%

Adrenal

●absolute

●relative

0.066±0.010

0.022±0.004

0.070±0.009

0.024±0.004

0.068±0.011

0.023±0.004

0.065±0.013

0.022±0.004

a

Data obtained from Tables 188-189, pages 489-494 in the study report. * α = 0.05; mean SD

8. Gross Pathology - F1 Set 3 Males and Females

There were no treatment-related findings at necropsy. 

9. Histopathological Observations (F1 Set 3 Males and Females)
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Microscopic examination: Histopathological examinations were conducted on 10 randomly

selected control and high-dose rats/sex, with the exception of the testis and epididymis, which 

were examined in all F1 Set 3 males of the control and high-dose groups (PND 139). Effects 

were observed on the kidney in male rats at 300 ppm and 800 ppm and in females at 600 ppm 

(Table 58). The effect was described as a degenerative lesion involving the proximal convoluted 

tubules in the outer stripe of the outer zone of the medulla, which was multifocal in distribution, 

and very slight degree in females and slight degree in males. No histopathological changes were 

observed in the thymus or pituitary in either sex. There were no apparent treatment-related 

lesions in the reproductive organs/tissues.

TABLE 58. Microscopic Kidney Findingsa F1 Set 3 Males and Females

Observation Dose group (ppm)

Control 100 300 600/800

F1 Set 3

MALES

Number of kidneys examined 10 10 11 11

Degeneration, proximal convoluted tubule, outer strip, outer zone

● multifocal, very slight

 ● multifocal, slight

10

0

10

0

8

3

4

6

FEMALES

Number of kidneys examined 10 10 10 10

Degeneration, proximal convoluted tubule, outer stripe, outer zone

● multifocal, very slight

 ● multifocal, slight

0

0

0

0

1

0

7

0

a

Data obtained from Text Table 20, page 130 in the study report. 

III. DISCUSSION AND CONCLUSIONS:

INVESTIGATORS’ CONCLUSIONS: Systemic toxicity was assessed across life stages. In the 

parental generation, high-dose females had decreased body weight on LD 7, which coincided with 

decreased feed consumption (LD 1-7) and decreased high-dose F1 pup body weights. The F1 pup 

body weights in the high-dose group recovered to control values by ~ PND 42 (females) and 56 

(males). The study confirmed that the kidneys were a target organ for 2, 4-D toxicity. P1 males had 

increased kidney weights and very slight to slight degeneration of the proximal convoluted tubules in 

the outer zone of the medulla at 800 ppm. Similar exposure-related histopathological renal lesions 

occurred in the 300 ppm and 800 ppm F1 adult males and 600 ppm F1 adult females. No exposurerelated kidney lesions were seen in PND 22 F1 pups. The higher incidence of the kidney

histopathology effects in the F1 adults compared to P1 adults was likely related to higher 2, 4-D 

doses in F1 offspring and was associated with non-linear TK. These kidney lesions were slight to 

very slight in severity and would not be expected to alter renal function, confirmed by the lack of 

exposure-related effects on clinical pathology (urea nitrogen and creatinine) or urinalysis (volume 

and specific gravity). 

With respect to reproductive toxicity, 2,4-D had no effects on estrous cyclicity (P1, satellite GD 17 

dams, F1 Set 3 offspring) or reproductive indices, including mating, fertility, time to mating, 

gestation length, pre- and post-implantation loss and corpora lutea number (satellite GD 17 dams). 

Litter sizes, pup survival, sperm parameters, ovarian follicle counts, and reproductive organ 

histopathology were unaffected by 2, 4-D. One P1 male in each of the 300 and 800-ppm groups had 

decreased bilateral testis size, but this incidence rate was within the laboratory historical control data. 
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A similar unilateral finding was noted in one control F1 male. These findings were not reproduced in 

F1 offspring Set 1a or Set 3 with longer 2, 4-D exposures, which included higher mg/kg/day

exposures during critical life stages. There were no exposure-related histopathological changes in 

reproductive organs. Overall, there was no indication of reproductive toxicity by 2, 4-D in this study. 

Based on a priori established criteria, mating of a second generation was not triggered. 

In P1 males, decreased seminal vesicle and prostate weights were seen at > 300 ppm; however, there 

was no associated histopathology, and absolute and relative control organ weights exceeded the 

laboratory historical control data. These findings were not reproduced in Set 1a or Set 3 F1 offspring 

with longer 2, 4-D exposures, which included higher mg/kg/day exposures during critical life stages. 

Therefore, these organ weight differences were not attributed to 2, 4-D exposure. Decreased testis 

weights in PND 22 F1 weanlings at all exposure levels, which lacked corresponding histopathology, 

were attributed to decreased body weights, which were artifactual differences in PND 22 male pup 

body weights that were introduced during group assignment at weaning. These body weight 

differences may have been exacerbated by 2, 4-D-related toxicity and/or palatability issues in the 600 

ppm group. Available data predicted that this dose group would have exceeded saturation kinetics, 

despite adjustment of dietary concentrations during lactation, due to the presence of 2, 4-D in milk 

and in the diet, and the high dietary intakes on a mg/kg basis for this life stage. There were no 

significant, exposure-related changes in reproductive organ weights in P1 or F1 PND 22 females, or 

in F1 Set 1a, Set 2 (testis weights only), or Set 3 males and females. There was a slight delay in F1 

preputial separation at 800 ppm, which was attributed to high-dose body weight decrements. Other 

androgen-sensitive endpoints, including anogenital distance (AGD) and nipple retention, which are 

considered very sensitive endpoints, sperm parameters and testicular and accessory sex gland 

histopathology, were not altered. There were no effects on estrogen-sensitive endpoints including 

vaginal opening, estrous cyclicity, uterine weights or histopathology, or quantitative ovarian follicle 

counts. There was no consistent pattern of effects on thyroid parameters. High-dose satellite GD 17 

dams given 600 ppm 2, 4-D had non-significant decreases in T4 and T3 with a corresponding 

increase in TSH. Thyroid histopathological alterations were seen in three of 12 dams, comprised of 

smaller thyroid follicles with small vacuoles in the colloid that were suggestive of colloid resorption. 

There were no adverse pathological alterations (e.g., degeneration); thus, these thyroid changes were 

considered adaptive. There were no thyroid effects at lower-dose levels in GD 17 dams and no 

biologically significant effects on thyroid endpoints at the other life stages examined. Thus, 2, 4-D 

did not alter estrogen or androgen-sensitive endpoints, and altered thyroid endpoints only at an 

exposure level (600 ppm) that clearly resulted in nonlinear TK. 

With respect to developmental neurotoxicity (DNT), there were no exposure-related effects on FOB 

parameters, motor activity, or acoustic startle response (ASR). There was a significant EXPOSURE 

x BLOCK interaction for ASR in high-dose males, which was attributed to a slightly higher ASR in 

the first block. Habituation was not affected at any dose of 2, 4-D. Gross and morphometric brain 

measurements also were not affected. There was no exposure-related neuropathology in PND 22 or 

PND 60 animals, including no effects on brain myelin (assessed by special staining). Related 

neurotoxicity endpoints were examined in P1 adults and other F1 offspring, including brain weights 

and/or histopathology in non-perfused animals (P1 adults and PND 22, Set 1a on PND 70, and Set 3 

on PND 139). Terminal body weights and absolute brain weights were significantly decreased in 

PND 70 high-dose males; however, this group had a smaller sample size (n=10/sex/dose) than other 

groups with no brain weight changes (P1 adults and F1 Set 3 on PND 139; both with n>23/sex/dose). 

There were no exposure-related effects on brain histopathology at any age. Thus, there was no 
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evidence of DNT related to 2, 4-D exposure. 

With respect to developmental immunotoxicity, 2, 4-D had no effect on Antibody Forming Cell 

(AFC) assay responses in males. High-dose females had non-significant decreases in AFC/spleen and 

AFC/106 splenocytes (54% and 27%, respectively). However, this observation appeared to be due to 

temporal variability over the 4-day span of the evaluations. In addition, this result occurred at 600 

ppm, a dose level that demonstrated non-linear TK. The AFC response was not altered at < 300 ppm 

in females. The F1 Natural Killer Cell activity (NK) assay showed no effects from 2, 4-D exposure. 

The assay showed linear cytotoxicity with increasing E:T cell ratios (from 5 to 40% cytotoxicity), 

which were identical across all doses. Overall, 2, 4-D exposure had no biologically significant effect 

on the development of immune function. 

The 2, 4-D F1-extended one generation study has established across life stages a no observed adverse 

effect level (NOAEL) based on systemic toxicity of 100 ppm in males (5.5 mg/kg/day) and 300 ppm 

in females (20.6 mg/kg/day in non-pregnant P1 adults). The male NOAEL is ~13,000-fold higher 

than 2, 4-D exposures reported in human biomonitoring studies. Very slight to slight, renal tubular 

degeneration was seen in P1 males at 800 ppm and in F1 males exposed >300 ppm 2, 4-D. The 

increased incidence of renal changes in the F1 adult males compared to the P1 males was likely

related to their higher feed consumption and consequent test material intake prior to adulthood. For 

females, toxicity was limited to the 600 ppm group, where nonlinear TK prevailed, making these 

results irrelevant for 2, 4-D risk assessment.

B. REVIEWER COMMENTS: Potential Estrogenic Effects: There was no treatment-related 

effect on developmental landmarks. AGD, which was measured in all pups, nipple retention (all nonculled pups), and age at vaginal opening (all Set 1-3 offspring) were comparable among the groups. 

There were no adverse effects on estrous cycle length or estrous cycle pattern, including a lack of 

persistent estrus (assessed in all P1 main study and satellite GD 17 females and all F1 Set3 females). 

Reproductive indices were comparable among the groups [mating, fertility, time to mating, gestation 

length, pre-implantation loss, number of corpora lutea (satellite GD 17 dams), sperm parameters, 

ovarian follicle counts (F1 Set 3 females), and reproductive organ weights (P1, F1 offspring Sets 1a 

or 3) and histopathology]. There were no signs of dystocia in P1 dams and no adverse effects on 

post-implantation loss, litter size, and pup survival. In all three age groups in which uterine weights 

were measured [P1 (LD 22), F1 Set 1a (PND 70), and F1 Set 3 (PND 139)], increased absolute and 

relative uterine weights (10%-32%) were observed at 600 ppm. There were no significant, treatmentrelated changes in other reproductive organ weights in P1, PND 22, Set 1a or Set 3 females.

Androgen-Sensitive Endpoints: There were no adverse effects on anogenital distance or nipple 

retention in any F1 males exposed in utero, during lactation and into adulthood. There was no 

evidence of hypospadias, ectopic testes, or treatment-related testicular, prostate or seminal vesicle 

histopathology. In P1 and F1 Set 3 (PND 139) males, there were no significant changes in sperm 

parameters (spermatid/sperm counts, sperm motility and sperm morphology). Decreased accessory

sex gland weights were observed in the P1 males, but there were no effects on reproductive or 

accessory sex gland weights in the F1 generation males when assessed at PND 70 (Set 1a), PND 87-

93 (Set 2b) or PND 139 (Set 3). There were no treatment-related effects on reproductive organ 

histopathology (P1, Set 1a, Set 3, F1 weanlings). 
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Anti-Androgenic Effects: Decreased male reproductive organ weights (testes and seminal vesicle) 

were observed in P1 males, although statistical significance was not attained. Flaccid testes were 

observed in one 300 ppm P1 male and one 800 ppm P1 male. Decreased testes weight was observed 

at all dose levels (same magnitude) in one subset of weanlings (F1 PND 22) and was associated with 

decreased body weight. There were no effects on reproductive or accessory sex gland weights in the 

F1 offspring when assessed at PND 70 (Set 1a), PND 87-93 (Set 2b), or PND 139 (Set 3). Delayed 

preputial separation (1.6 days) was observed in F1 weanlings, which is within normal variability and 

was accompanied by a slight reduction in body weight. There was no associated histopathology in P1 

male reproductive or accessory sex gland tissues, and reproductive function was not affected. In F1 

males, exposed in utero, during lactation and into adulthood, there were no effects on anogenital 

distance or nipple retention, two endpoints that are considered highly sensitive to altered androgen 

status. There were no treatment-related effects on reproductive organ histopathology (Set 1a, Set 3, 

F1 weanlings). 

Thyroid: GD 17 satellite dams displayed the expected hormone response consistent with a 

perturbation in thyroid function. These findings correlated with the thyroid alterations observed 

histologically. There was a comparable increase (↑9%) in thyroid weights at the 100 ppm and 600 

ppm dose levels but no change in thyroid weight at 300 ppm. F1 PND 4 pups displayed a slight 

decrease in T3 (males) and T4 (both sexes; no dose response) and an increase in TSH (females at 600

ppm). Thyroid weight and histopathology data were not collected for this age group. F1 offspring 

PND 22 displayed decrease T3 (300 ppm and 800 ppm males) and T4 (both sexes 800 ppm/600 

ppm) and increased TSH (300 ppm males). Thyroid weight and histopathology data were not 

collected for this age group. F1 Offspring PND 62-64 displayed increased TSH at 300 ppm and 800 

ppm/600 ppm (both sexes), although a dose-response was observed only in the females. Decreased 

T4 was observed only in the 800 ppm males, and decreased T3 was observed at all dose levels in 

males but there was no dose-response. F1 Set 1a male offspring (PND 70) displayed decreased 

absolute thyroid weights (↓11%) at 800 ppm, but terminal body weight was also decreased (↓10%). 

F1 Set 1a female offspring (PND 70) displayed decreased absolute thyroid weights at 300 ppm 

(↓9%) and 600 ppm (↓5%). There were no histopathological findings reported in the thyroids. These 

findings suggest that 2, 4-D exposure may adversely affect thyroid function at doses above the renal 

saturation clearance; however, the thyroid effects noted below renal saturation are not considered 

sufficiently robust to be adverse.

Developmental Neurotoxicity (DNT): F1 Set 1b offspring were examined on PND 54-59. There 

was a lack of evidence of DNT (FOB parameters, motor activity, and acoustic startle response). 

PND 22 pups and PND 60 F1 adult rats were perfused for central and peripheral nervous system 

neuropathology, and brain weights, gross brain measurements (PND 22, PND 60) and morphometric 

measurements (PND 60) were evaluated. There were no treatment-related findings on brain weights, 

gross and morphometric brain measurements in perfused rats, neuropathologic effects in the CNS or 

PNS, and no effects on brain myelin. In non-perfused rats (P1 adults, PND 22, PND 70, PND 139 F1 

offspring), there were no adverse effects on brain weight and brain histopathology.

Developmental Immunotoxicity (DIT): Based on the SRBC antibody-forming cell assay (PND 66-

70) and Natural Killer Cell assay (PND 87-93), there was no evidence of developmental 

immunotoxicity. 
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The parental systemic LOAEL is 800 ppm (40 mg/kg bw/day in males), based on 

nephrotoxicity manifested as increased kidney weights, and degenerative lesions in the 

proximal convoluted tubules in the main study P1 rats. The parental systemic NOAEL is 300 

ppm (15 mg/kg bw/day in males). No toxicologically relevant effects were identified in P1 

females or in the GD 17 satellite female groups

The thyroid toxicity NOAEL is established at 800/600 ppm (40/30 mg/kg/day in males and 

females, respectively), the highest dose tested. The thyroid effects noted in the database were 

considered to be adaptive.

The offspring LOAEL is 300 ppm ( 15 mg/kg bw/day), based on kidney toxicity manifested as 

increased kidney weights and increased incidence of degeneration of the proximal convoluted 

tubules. The offspring NOAEL is 100 ppm (5 mg/kg bw/day). 

The DNT offspring (PND 21-60) LOAEL is >800/600 ppm (40 mg/kg bw/day in males, 30 

mg/kg bw/day in females), based on the lack of evidence of DNT (FOB parameters, motor 

activity, and acoustic startle response). The DNT offspring NOAEL is 800 ppm/600 ppm (40 

mg/kg bw/day in males, 30 mg/kg bw/day in females), the highest dose tested. 

The DIT offspring (PND 139) LOAEL is >800/600 ppm (40 mg/kg bw/day in males, 30 mg/kg 

bw/day in females), based on the lack of evidence of DIT [SRBC antibody-forming cell assay 

(PND 66-70) and Natural Killer Cell assay (PND 87-93)]. The DIT offspring NOAEL is 

800/600 ppm (40 mg/kg bw/day in males, 30 mg/kg bw/day in females), the highest dose tested. 

The reproductive LOAEL is > 800/600 ppm (40 mg/kg bw/day in males, 30 mg/kg bw/day in 

females), based on the lack of effect on estrous cyclicity, (P1 females, satellite GD 17 dams, Set 

3 F1 offspring) or reproductive indices (mating, fertility, time to mating, gestation length, preand post-implantation loss, number of corpora lutea (satellite GD 17 dams), sperm parameters, 

ovarian follicle counts, and reproductive organ histopathology). The reproductive NOAEL is 

800/600 ppm (40 mg/kg bw/day in males, 30 mg/kg bw/day in females), the highest dose tested. 

This study is classified acceptable/non-guideline. The study does not satisfy a guideline requirement 

for 2, 4-D. It satisfies the data call-in requirements for 2, 4-D for OPPTS 870.3800 (Reproduction 

and Fertility Effects), OPPTS 870.6300 (Developmental Neurotoxicity), OPPTS 870.7800 

(Immunotoxicity). 

C. STUDY DEFICIENCIES: None that would adversely affect study interpretation.
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APPENDIX A

Saghir, S. A., Zablotny, C. L. Marty, M. S. Perala, A. W. and Yano, B. L. (2008). A 

Dietary Dose Range-Finding and Pharmacokinetic Study of 2,4-

Dichlorophenoxyacetic Acid (2,4-D) in the Pregnant CRL:CD(SD) Rat and Its 

Offspring in Preparation for a Subsequent F1-Extended One-Generation Toxicity

Study in Rats. Toxicology & Environmental Research and Consulting, The Dow 

Chemical Company. Laboratory Project Study IDs 071153 and 071153A, May 2, 

2008. MRID 47417901. Unpublished.
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APPENDIX B

Saghir, S. A., Perala, A. W., and Clark, A. J. (2008). A dietary titration study of 2,4-

dichlorophenoxyacetic acid (2,4-D) pharmacokinetics in female CRL:CD(SD) rats. 

Toxicology & Environmental Research and Consulting, The Dow Chemical 

Company. Laboratory Project Study ID 071210, April 29, 2008. MRID 47417902. 

Unpublished.
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APPENDIX C

REFERENCES:

Marty, M. S. and Andrus, A. K. (2007). Motor Activity Validation Study Using Positive 

Controls: Effects of Amphetamine and Chlorpromazine. Report of Toxicology & Environmental 

Research and Consulting, The Dow Chemical Company, Midland, Michigan.

Saghir, S. A., Mendrala, A. L., Bartels, M. J., Day, S. J., Hansen, S. C., Sushynski, J. M., and 

Bus, J. S. (2006). Strategies to assess systemic exposure of chemicals in subchronic/chronic diet 

and drinking water studies. Toxicol. Appl. Pharmacol. 211, 245-260.