EPA Reviewer: Linda L. Taylor, Ph.D.                                                                                                         
Risk Assessment Branch VII, Health Effects Division (7509C)
EPA Secondary Reviewer: Elizabeth Mendez, Ph.D.                                                                                          
Immediate Office, Health Effects Division (7509C)
TXR# 0050697  

                   DATA EVALUATION RECORD  -  SUPPLEMENT # 1
                  see TXR# 0055341 (root) for previous review

STUDY TYPE: F1-Extended One Generation Toxicity Study  -  rat
                          OCSPP 870.3800, 870.6300, 870.7800/OECD 416 
P.C. CODE: 030001	
CAS NO.: 94-75-7
DP Barcode: D386909
TEST MATERIAL (PURITY) : 2, 4-D [97.85- 98.6% a.i.]
SYNONYMS: 2, 4-dichlorophenoxyacetic acid; 2, 4-D acid
SPONSOR: Industry Task Force II on 2, 4-D Research Data
 
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 Unpublished.
                                                            
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 (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.
	
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 pre-mating 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 P1 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 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 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 treatment-related 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 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 (F1 adults) LOAEL is 800/600 ppm (55.6 mg/kg bw/day in males and 46.7 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 300 ppm (20.9/ mg/kg bw/day in males and 23.3 mg/kg/day in females). 

The F1 offspring (PND 22) LOAEL is 800/600 ppm, based on decreased body weight observed throughout lactation. The offspring NOAEL is 300 ppm. The dose on a mg/kg/day basis for the PND 22 F1 offspring was not calculated. 
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). 

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 OCSPP 870.3800 (Reproduction and Fertility Effects), OCSPP 870.6300 (Developmental Neurotoxicity),  OCSPP 870.7800 (Immunotoxicity). The study is in accordance with the OECD extended one-generation reproductive toxicity study guideline (November, 2010).  

COMMENT:  The major change is a change in the offspring (F1 adults) LOAEL from 300 ppm to 600 ppm, making the new NOAEL 300 ppm.  Both the PMRA and EPA do not consider the kidney effects occurring at PND 70 and PND 139 to be adverse at the 300 ppm dose level in either sex.  Although there was some degeneration in the males at 300 ppm on PND 70, this did not correlate with increased kidney weight and was not really different from controls on PND 139.  One would expect the degeneration to be worse after a prolonged exposure.  There is clear correlation in both sexes at 800/600 ppm and at both time points.  

A few typos were identified subsequent to  the finalization of the DER, which include: (1) in the paragraph for P1 adult rats on page 3, it should have indicated that no alterations were observed in estrous cycle pattern in the P1 females rather than F1 females; (2) page 50, first line should read from PND 21-69 (not PND 21-56). 

