1 Internal Report

Title: The distribution of atrazine in the Wistar rat following gestational/lactational exposures. By: Tammy E. Stoker, Leah Zorrilla, Lilian Strader , Emily Gibson, Alaa Kamel, Yaorong Qian, and Lynda Podhorniak. Endocrine Toxicology Branch, Toxicity Assessment Di vision (TAD),

NHEERL, ORD, RTP, NC Analytical Chemistry Branch (ACB), Biological and E conomic Analysis Division (BEAD),

Office of Pesticide Programs, USEPA, Ft. Meade, MD.

Please address all correspondence to Tammy E. Stoke r, ETB, TAD, NHEERL, ORD, USEPA< RTP< NC 27711. Phone: 919-541-2783, Fax: 919-541- 5138, E-mail: stoker.tammy@epa.gov

This Internal Report was prepared for the use by th e Office of Pesticide Programs, Office of Chemical Safety and Pollution Prevention. July 14, 2010

Disclaimer : This manuscript has been reviewed in accordance with the policy of the National Health and Environmental Effects Research Laborator y, Office of Research and Development, U.S. EPA, and approved for publication. Approval d oes not signify that the contents necessarily reflect the views or policy of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation f or use.2 

### Abstract Several studies have examined the potential health effects of a gestational or lactational exposure to the chlorotriazine herbicide, atrazine. Such exposure to atrazine and/or its

metabolites have been linked to a number of effects on reproductive function and development, including delays in puberty onset, altered testoste rone secretion, altered mammary and prostate gland development. To our knowledge, the transfer of atrazine and/or metabolites into the fetus during gestation has not been examined. In a previ ous study in our laboratory, lactational

transfer of C14-labeled atrazine was evaluated. Ho wever, that study examined only total chlorotriazines following the administration of rad iolabeled atrazine, so it could not identify the transfer of the specific atrazine metabolites. For this reason, TAD, NHEERL in collaboration with the BEAD lab, conducted a study to examine the gestational and postnatal lactational transfer of maternally administered atrazine and it s metabolites into plasma, milk and tissues (brain, adrenal, mammary glands and gonads). Three exposure paradigms were used, including (1) gestation day (GD) 18 to 20, (2) GD 14 to 20 an d (3) GD 14 to postnatal day (PND) 10. At the end of the designated treatment period (two hou rs after last dose), blood was drawn by cardiac puncture and the dam was perfused with sali ne to clear the blood from the tissues. When the pregnant dams were killed, two males and two fe male fetuses were collected from the uterus on GD 20. For those rats killed on PND 10, the pup s were removed from the dam three hours prior to kill. One hour after separation, the dams were dosed. The pups were then returned to

the dam and allowed to nurse for 30 minutes before collection of samples. This fresh milk was in the pup stomach for collection at the time of th e kill. Plasma, brain, adrenal and gonads were

also collected from the neonates at kill. The resu lts show that DACT accounted for the highest concentration in the plasma and tissues, followed b y DIA and DEA. Interestingly, the parent3

compound, atrazine, was present in only very low am ounts. The OH- metabolites of DEA and DIA were not quantifiable and the OH- metabolite of atrazine was not detected in the majority of the samples. It appeared that the amount of DACT p resent in the neonates on PND 10 following 18 days exposure to the dam (8 gestational and 10 p ostnatal) was due to the amount in the milk, which was much higher than the amount present in th e plasma in the neonate. None of the other metabolites were present in the neonate. There did appear to be a transfer of atrazine from the dam to the fetus on GD 20 following either 3 or 7 d ays of exposure.4 Introduction During the last decade, a number of studies have e xamined the potential adverse health

effects of atrazine and related chlorotriazines on human health and wildlife. One of the more consistent observations has been that these compoun ds do influence reproductive development in laboratory rodents. For example, atrazine treatme nt to the dam during the perinatal period has been reported to result in prostatitis in the male offspring (Stoker et al., 1999; Raynor et al., 2007). Others have reported that prenatal (Rosenbe rg et al. 2009) and peripubertal (Stoker et al., 2000 Trentacosta et al., 2000, Friedman et al., 200 2; Pogrmic et al., 2009) exposure to atrazine can lead to altered male reproductive development, decreased testosterone secretion and a decrease in testosterone dependent target tissues.

These studies and studies in the adult female indic ate that the brain may be an important target organ for atrazines effects on the neuroendo crine control of reproduction. For example, atrazine has been shown to impair the hypothalamic control of luteinizing hormone (LH) (Cooper et al., 2000; McMullin et al, 2003 ; Forado ri et al., 2009) and stimulate the release of adrenocorticotropic hormone (ACTH) (Fraites et al., 2009; Laws et al., 2009). As a consequence, these neuroendocrine alterations lead to altered gonadal and adrenal hormonal regulation. In contrast to these well-documented changes in th e neuroendocrine control of reproductive and adrenal function, there is relativ ely little information available describing the

blood levels of atrazine and its metabolites follow ing exposure to this herbicide. In vitro studies indicate that ATR is metabolized primarily by cytoc hromes P450 (P450s). The major P450-

derived metabolites of ATR detected in these in vit ro studies are the N -dealkylated products desethyl atrazine (DEA) and desisopropyl atrazine ( DIA). However, didealkyl atrazine (DACT)5

is the major in vivo metabolite of ATR detected in rat plasma (Brzezick i et al., 2003; McMullin et al., 2003) and in mouse plasma and urine (Ross a nd Filipov, 2006). In humans, major ATR metabolites detected in urine of occupationally exp osed subjects are DE, DIP, and DACT (Catenacci et al., 1993, Ross et al, 2009).

While the above research on serum and tissue concen trations of atrazine and its metabolites have focused on exposure to the adult a nimal, very little is known about the effects of gestational and lactational exposures to the off spring. Stoker and Cooper (2007) examined

lactational transfer of C14-labelled atrazine from dams to pups. Although lactational transfer was confirmed, it was not possible to discern how m uch of the parent compound or its metabolites were actually transferred. We are unaw are of any studies in which the transplacental

transfer of atrazine and/or metabolites into the fe tus has been evaluated. The present study is the result of a cooperative ef fort between TAD, ORD and BEAD, OPPTS. The purpose was to examine the gestational and lactational transfer of atrazine and its

metabolites into the fetus and neonate. As atrazin e has been reported to target the brain, another objective of this study was to examine the ability of atrazine and its metabolites to cross the blood-brain barrier. Finally, we examined the conc entration of this herbicide and its metabolites in the adrenal and the mammary glands, two other ti ssues of concern for atrazine toxicity. The target analytes of interest were the parent compoun d ATR, along with metabolites desethyl atrazine (DEA) and OH-DEA, desisopropyl atrazine (D IA) and OH-DIA, desethyl-desisopropyl atrazine (DACT), and hydroxyl atrazine (OH-ATR).



### Methods6

This work was performed by both NHEERL, ORD in RTP, NC and Analytical Chemistry Branch, BEAD, OPP in the Environmental Science Cent er in Ft. Meade, MD. Dosing Solutions Atrazine (97% purity, Novartis, Greensboro, NC) was administered as a suspension in 1.0% methyl cellulose (M-7140, Sigma Chemical Co., St. L ouis, MO) in distilled water. The doses included 0 (vehicle), 5 and 25 mg/kg body weight, d elivered by oral gavage in 5.0 ml dosing solution per kg body weight.

Animals Timed pregnant Wistar rats were obtained from Charl es Rivers Laboratories (Raleigh, NC) on GD 6 and maintained under controlled temperature (2 0-24 o C), humidity (40-50%), light (14 h light/10 h dark; on at 6 am off at 8 pm EST) condit ions with Purina Laboratory Rat Chow 5001

and water available ad libitum in an AAALAC accredited facility. Polycarbonate ca ges with heat-treated pine shavings were used for housing th e dams.7

Three different dosing paradigms were employed, inc luding dosing from (1) GD 14 to 20, 2) dosing from GD 18 to 20, and 3) GD 14 to PND 10. T hree dams from each treatment period were dosed with 0 (vehicle), 5 or 25 mg/kg. Two ho urs following the last dose on GD 20, blood was collected from dams (from the GD 14 to 20 and GD 18 to 20 exposure periods) under halothane anesthesia, followed by perfusion o f the tissues with saline. Two males and two female fetuses per dose group were then collect ed from the dams and weighed. In addition, the brain, adrenal and mammary glands fro m each dam were collected and weighed. In the GD 14 to PND 10 group, the neonate s were removed from the dam for 3

hours before kill and then allowed to nurse for thi rty minutes just prior to kill. Following this nursing period, one male and one female PND 10 pup were perfused and plasma, milk from the stomachs, adrenal gland and the gonads (ovaries and testes) from each was collected and weighed. All milk, plasma and tissues collected wer e frozen prior to and during shipment for analysis by the BEAD lab. Sample Analysis Chlorotriazine concentrations in the milk, plasma a nd tissue samples were determined using a modified protocol from Ross et al. (2006, 2 009) using the Waters Acuity/Xevo LC/MS/MS for concentrations of atrazine, DEA and OH -DEA, DIA and OH-DIA, DACT, and OH-ATR. The plasma concentrations were determined on a per volume basis, and the tissue, whole fetus and milk samples were determined on a p er gram basis. Sample sets included a method blank (procedural blank), matrix blank (cont rol), and a matrix spike at 2x LOQ with all target analytes. All sample sets were calibrated w ith standard reference compounds. Standards

were obtained the standards from the US EPA Nationa l Pesticide Standard Repository in Ft. Meade, MD. An instrument calibration curve, with a minimum of four concentration levels,8

(typically between estimated limit of detection (LO D) to 10x limit of quantitation (LOQ) was established for each analyte prior to the analysis of actual samples. The LOQ was estimated as 3 X LOD, so that any value between the LOD and LOQ is considered an estimation. The proper controls and spiked samples were used to determine recovery, bias and precision. The correlation coefficient (R 2 ) of the calibration curve, for each quantitated an alyte, was > 0.990. Confirmation of the presence of each analyte was ac hieved by monitoring a second precursor/product ion transition. The ratio of bot h ion transitions was within 20% of the ratio of

the same ion transitions found in standards. 

### Results The results presented in this section are limited to those metabolites which demonstrated reliable measures in this study. The hydroxymetabo lites of DIA and DEA were only semiquantifiable as they were present in low concentrat ions and the extraction efficiencies in plasma and tissues were low. For this reason, they will n ot be included in the results and tables. The hydroxyatrazine metabolite had extraction efficienc ies of acceptable limits, but the majority of the samples analyzed revealed undetectable hydroxyl atrazine values. These values were included in the results and tables to compare the l evels of OH-ATR to the concentrations of the other non-hydroxylated metabolites. To summarize a nd compare the data, the LOQ (level of quantitation) was inserted as the default concentra tion when the concentrations were either nondetectable or lower than the LOQ.

Gestational Exposure

Dam9

In the dam, the most prominent chlorotriazine meta bolite detected in all tissues examined was DACT, (Table 1 and 2). DACT accounted for the l argest percent of chlorotriazines detected in dams treated for 3- and 7-days prior to kill. F or example, in the GD 20 plasma from both dosing periods , DACT accounted for 74% and 82% of the total chlorotriazines (atrazine plus all metabolites) detected. Atrazine itself represented only 0.8 % or less of the total chlorotriazines in both treatment periods. It is also of note that the concentration of this metabolite was relatively similar in all tissues examined. For ex ample, examination of the brains of the treated dams revealed that DACT accounted for approximately 85% of the total chlorotriazines and

atrazine less than 1.0 % of the total. The two int ermediate metabolites, DIA and DEA, were detected in concentrations that were higher than th at of atrazine itself, but they were still present

at concentrations lower than that observed for DACT

. A dose-dependent increase for atrazine and its thre e metabolites was consistent in the dams under both dosing regimens and in all tissues examined. For example, there was approximately 700 and 2500 ng/g of DACT in the brai n following the exposure to 5 and 25 mg/kg atrazine, respectively, in both the GD 18 to 20 and GD 14 to 20 dosing periods. In the mammary gland, there was approximately 500 ng/g and 1650 ng/g following the 5 and 25 mg/kg in the two dosing periods. In contrast, in the adr enal, there was less DACT found following the 7-day exposure period (90 and 260 ng/g) as compared to the 3-day exposure in the dam (200 and 750 ng/g). To summarize both dosing paradigms, DACT, DIA and DEA accounted for 85%, 10% and 2% of the total in brain tissue, respectively. Atrazine accounted for less than 1% of the total

chlorotriazines in the brain of the GD 20 dam. How ever, in the adrenal and mammary tissue of the dam, atrazine was present in higher amounts, ac counting for approximately 13% of the total10

in the adrenal and 5% of the total in the mammary g land. DACT, DIA and DEA accounted for 65%, 19% and 7% in the adrenal and 65%, 22%, and 6% in the mammary gland. The amounts of the hydroxy-chlorotriazines in the tissues and plas ma were negligible (<0.1% or undetectable).

Fetus

The amount of chlorotriazines found in the whole f etus was similar whether the dams were treated for 3 or 7 days (Table 1 and 2). Agai n, DACT accounted for approximately 78% of the total chlorotriazines in the fetus, with atrazi ne, DIA and DEA accounting for less than 1%, 17%, and 3% of the total, respectively. Again, the hydroxyl-chlorotriazines in the tissues and plasma were negligible (<0.1% or undetectable). Gestational/Lactational exposure

Dam

The concentrations and distribution of the chlortr iazine metabolites in the plasma, brain, adrenal and mammary gland of dams exposed from GD 1 4 to PND 10 and killed on PND 10 (Table 3) were similar to those observed in the dam s killed on GD20 (Table 1 and 2) in the plasma, brain adrenal and mammary gland (Table 3). There did appear to be less chlorotriazines detected in the adrenal gland as compared to the am ount detected after just 3 days of exposure, similar to the decreased amount in the 7 day exposu re, as mentioned earlier. DACT accounted for the majority of the chlorotriaz ines in the tissues and plasma collected. For example, in the plasma, DACT repres ented 90% of the chlorotriazine metabolites detected. In contrast, atrazine accounted for only 0.3 % of the total at both doses. DIA and DEA represented 6 and 2% of the total, respectively. A similar amount was observed in the brain of the dam, with DACT accounting for 95% of the total and atrazine only 0.5% of the total. DIA

and DEA accounted for 3 % and 1% of the total in br ain tissue, respectively. However, in the11

adrenal atrazine was present in higher amounts, acc ounting for 10% of the total. DACT

accounted for 75% of the total chlorotriazines dete cted in the adrenal, with DIA and DEA accounting for 8 and 5%, respectively. Unlike the previous exposures, atrazine only accounted for 0.7% of the total in the mammary gland, with DA CT at 90%, DIA at 6% and DEA at 2%. The amount of OH-ATR was increased in this exposure as compared to the previous exposures, accounting for 0.7% in the adrenal gland and 0.5% i n the mammary tissue. However, the amounts of the other two hydroxy-chlorotriazines (O H-DIA and OH-DEA) were still negligible (<0.1% or undetectable) following this exposure.

Neonate The neonatal plasma, milk and tissues contained pri marily DACT (Table 4). For example, in the plasma, DACT accounted for 99% of t he total chlorotriazines detected with no atrazine and 0.6 and 0.2% of DIA and DEA, respectiv ely. The milk obtained from the neonatal stomachs following the 30 minute nursing period con tained primarily DACT (91% of total), but also contained DIA, DEA, Atrazine and OH-atrazine a t 6, 2, 0.4 and 0.3%, respectively. The adrenal gland contained only DACT. DACT accounted for 96% of the brain chlorotriazine levels, with relatively small amounts of atrazine ( 2.5%), DIA (0.4%), DEA (0.1%) and OHatrazine (1%) present. Again, the hydroxyl-chlorot riazines in the tissues and plasma were negligible (<0.1% or undetectable).

The gonads of the neonates were analyzed for all m etabolites, but only DACT was detected (Table 5). The testes showed a dose respo nse increase of the concentration of DACT, with 77 and 278 ng/g in the 5 and 25 mg/kg dose gro up. However, in the ovary DACT was only detected at the 25 mg/kg dose.12 

### Summary This study demonstrates that the primary metabolite found in the dam and fetus/neonate following a gestational or gestational/lactational exposure to atrazine in the dam is diaminochlorotriazine (DACT). However, atrazine an d the metabolites DIA and DEA were also detected. The hydroxyl metabolites were not detect ed in most cases, and when they were, the

contribution to the total chlorotriazines observed was negligible. DIA was present to a greater extent in the tissues and plasma, as compared to DE A or atrazine. It appeared that atrazine was able to cross the placenta to get into the fetal ci rculation, as the whole fetus had appreciable levels of most of the metabolites, including DACT, DIA, DEA and atrazine. In the neonate, the plasma only contained significant concentrations of DACT, reflecting the presence of significant

concentrations of DACT in the dam’s milk . It was also interesting that the neonatal brains contained both DACT and atrazine, demonstrating the ability of these compounds to cross the

blood-brain barrier in the neonates.13 References

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. Toxicol. Lett. 69, 217–222, (1993). Cooper RL, Laws SC, Das PC, Narotsky MG, Tyree, L., Goldman JM, Stoker TE. 2006.Hormonal control of ovarian function following chlorotriazine exposure: Effect on reproductive function and mammary tumor development . Birth Defects Research. Part B: Developmental and Reproductive Toxicology. 76.

Foradori, C.D., Hinds, L.R., Hanneman, W.H., Handa, R.J. (2009) Effects of atrazine and its withdrawal on gonadotropin-releasing hormone neuroe ndocrine function in the adult female Wistar rat. Biol. Reprod. Fraites, M.J., Cooper, R.L., Buckalew, A., Jayarama n, S., Mills, L. and Laws, S.C. (2009) Characterizaion of the hypothalamic-pituitary-adren al axix response to atrazine and metabolites in the female rat. Toxicol. Sci. 112(1):88-99. Laws, S.C. Hotchkiss, M. Ferrell, J. Jayaraman, S. Mills, L. Modic, W. Tinfo, N. Fraites, M. Stoker, T. and Cooper. R. Chlorotriazine Herbicides and Metabolites Activate an ACTHdependent Release of Corticosterone in Male Wistar Rats. Toxicological Sciences 112(1), 78–87 (2009). doi:10.1093/toxsci/kfp190

McMullin, T. S., Brzezicki, J. M., Cranmer, B. K., Tessari, J. D., and Andersen, M. E.. Pharmacokinetic modeling of dispos ition and timecourse studies with [14C]atrazine. J. Toxicol. Environ. Health A 66, 941–964, (2003). Pogrmic, K., Fa, S., Dakic, V., Kaisarevic, S. & Ko vacevic, R. (2009). Atrazine oral exposure of peripubertal male rats downregulates steroidogenesi s gene expression in Leydig cells. Toxicol. Sci., 111:1, 189-197.

Raynor, J.L., Enoch, R.R., Wolf, D.C., Fenton, S.E. (2007) Atrazine-induced reproductive tract alterations after transplacental and/or lactational exposure in male Long-Evans rat. Toxicology and Applied Pharmacology 218(3):238-48.14

Rosenberg, B.G., Chen, H., Folmer, J., Liu, J., Pap adopoulos, V. and Zirkin, B.R. (2008) Gestational exposure to atrazine: effects on the p ostnatal development of the male offspring. J. Androl. 29(3):304-11. Ross, M. K., Jones, T. L., and Filipov, N. M. Dispo sition of the Herbicide 2-Chloro-4- (ethylamino)-6-(isopropylamino)- s -triazine (Atrazine) and Its Major Metabolites in M ice: A Liquid Chromatography/Mass Spectrometry Analysis of Urine, Plasma, and Tissue Levels Drug Metabolism and Disposition, Vol. 37, No. 4 (2008). Ross, M. K. and Filipov, N. M. Determination of at razine and its metabolites in mouse urine and plasma by LC-MS analysis. Analytical Biochemistry 351, 161-173 (2006). Ross, M. K., Jones, T. L., and Filipov, N. M. Dispo sition of the herbicide atrazine and its major metabolites in mic e: An LC-MS analysis of urine, plasma, and tissue levels. Drug Metab. Dispo s. 37, 776–786, (2009).

Stoker, T.E., Robinette, C.L. and Cooper, R.L. (19 99). Maternal exposure to atrazine during lactation suppresses suckling-induced prolactin rel ease and results in prostatitis in the adult offspring. Toxicological Sciences. 52(1):68-79.

Stoker, T.E. (2007) Distribution of 14C-Atrazine Following an Acute Lactational Exposure in the Wistar Rat. Reproductive Toxicology 23(4):607- 1015Table 1. Chlorotriazine concentrations in the damfollowing oral exposure of the dam to Atrazinefrom GD 18 to 20 (3 day exposure).AtrdoseATR OH-ATR DACT DIA DEAPlasmang/ml 00.2 ^ 1.0 ^ 3.0 ^ 1.0 ^ 0.5^52.9 ± 0.8 1.4 ± 0.2 980.0 ± 88.6 108.3 ± 27.0 24.7± 6.22537.3 ± 9.5 7.3 ± 1.5 3333.3 ± 540.1 953.3 ± 155.2190.0 ± 25.5Adrenalng/g 00.2 ^ 1.0 ^ 3.0 ^ 1.0 ^ 0.5^518.6 ± 6.1 4.0 ± 3.7 192.6 ± 61.7 23.7 ± 1.5 13.3± 1.825194.9 ± 3.7 5.7 ± 5.7 746.7 ± 328.8 286.8 ± 91.0106.3 ± 29.8Brainng/g 00.2 ^ 1.0 ^ 3.0 ^ 1.0 ^ 0.5^52.8 ± 0.9 1.0 ^ 776.7 ± 96.5 31.0 ± 5.1 10.6 ± 2.42531.0 ± 4.3 1.4 ± 0.3 2700.0 ± 561.3 373.3 ± 137.9 93.0 ± 25.5Mammaryng/g 00.2 ^ 1.0 ^ 6.0 ± 3.7 1.8 ± 1.0 0.5^515.2 ± 5.1 1.0 ^ 466.7 ± 71.5 66.0 ± 14.2 24.0 ± 6.525210.0 ± 44.2 4.6 ± 1.0 1763.3 ± 660.0 720.0 ± 237.9206.7 ± 64.2GD 20Fetuses00.2 ^ 1.0 ^ 3.0 ^ 1.0 ^ 0.5^54.3 ± 0.9 1.0 ^ 1094.2 ± 70.5 102.7 ± 25.6 21.0 ±4.22577.9 ± 14.5 1.3 ± 0.1 4070.0 ± 582.5 1075.0 ±195.5220.0 ± 43.6Mean ± SEM (standard error of the mean). One mammary gland, 5th^ LOQ or limit of quantitation was used if the sample concentration was undetected or below the LOQ16Table 2. Chlorotriazine concentrations in the damfollowing oral exposure of the dam to Atrazine fromGD 14 to 20 (7 day exposure).AtrdoseATR OH-ATR DACT DIA DEAPlasmang/ml 00.2 ^ 1.0 ^ 19.1 ± 17.1 2.2 ± 1.1 0.6 ± 0.251.5 ± 0.6 1.4 ± 0.2 883.4 ± 108.0 108.3 ± 27.0 21.0± 5.0258.0 ± 3.5 4.3 ± 0.9 3100.0 ± 717.7 573.3 ± 290.2 88.7 ± 40.9Adrenalng/g 00.2 ^ 1. 0 ^ 9.3 ± 7.8 1.0^ 0.5 ^517.4 ± 6.6 3.5 ± 3.1 93.3 ± 19.5 19.0 ± 3.2 10.9 ±1.92555.3 ± 16.3 4.7 ± 4.5 260.1 ± 37.4 71.7 ± 27.5 27.7 ± 10.8Brainng/g 00.2 ^ 1.0^ 13.2 ± 10.9 1.5 ± 0.6 0.5 ^52.3 ± 0.9 1.0 ^ 630.0 ± 129.6 34.7 ± 14.9 10.0 ± 3.92511.1 ± 4.1 1.0 ^ 2366.7 ± 549.3 218.3 ± 118.7 43.3± 20.9Mammaryng/g 00.2 ^ 1.0 ^ 9.6 ± 7.0 1.5 ± 0.6 0.6 ± 0.61516.1 ± 5.5 1.0^ 416.5 ± 82.7 73.0 ± 23.1 27.7 ± 7.42579.0 ± 27.1 3.9 ± 0.5 1600.0 ± 255.0 406.7 ± 180.9108.0 ± 41.7GD 20fetuses00.2 ^ 1.0 ^ 17.8 ± 9.9 1.8 ± 0.7 0.6 ± 0.153.7 ± 0.9 1.0 ^ 875.0 ± 69.5 100.8 ± 22.9 21.8 ± 4.12518.5 ± 5.6 1.2 ± 0.1 3466.7 ± 563.2 639.2 ± 225.8 108.0 ± 36.4Mean ± SEM. One mammary gland, 5th.^ LOQ or limit of quantitation was used if the sample concentration was undetected or below the LOQ17Table 3. Chlorotriazine concentrations in the damfollowing oral exposure of the dam to Atrazinefrom GD 14 to PND10.ATRdosemg/kgATR OH-ATR DACT DIA DEAPlasmang/ml 00.2 ^ 1.0 ^ 3.0 ^ 1.0 ^ 0.5 ^50.9 ± 0.4 1.1 ± 0.1 953.3 ± 29.4 38.0 ± 6.7 11.2 ±1.9259.8 ± 8.3 3.3 ± 1.7 2500.0 ± 860.4 174.0 ± 112.8 64.4 ± 43.2Adrenalng/g 00.2 ^ 1.0 ^ 3.0 ^ 1.0 ^ 0.5 ^510.2 ± 4.2 2.0 ± 1.3 333.3 ± 152.7 11.0 ± 6.56.2 ± 3.62558.6 ± 45.6 4.3 ± 4.1 426.7 ± 172.4 44.5 ± 28.5 27.0 ± 15.3Brainng/g 00.2 ^ 1.0 ^ 3.0 ^ 1.0 ^ 0.5 ^51.2 ± 0.2 1.0 ^ 713.3 ± 40.7 10.9 ± 1.9 4.5 ± 0.72510.6 ± 8.9 1.0 ^ 1953.3 ± 673.4 55.6 ± 37.3 27.7 ±20.0Mammaryng/g 00.2 ^ 1.0 ^ 3.0 ^ 1.0 ^ 0.5 ^53.1 ± 0.5 1.8 ± 0.3 736.7 ± 68.0 27.0 ± 2.4 11.5 ±1.22515.5 ± 8.1 11.8 ± 7.0 2180.0 ± 821.5 140.3 ± 78.4 54.0 ± 31.5Mean ± SEM. One mammary gland, 5th.^ LOQ or limit of quantitation was used if the sample concentration was undetected or below the LOQ18Table 4. Postnatal Day 10 neonatal chlorotriazineconcentrations following exposure of the damto Atrazine from GD 14 to PND 10.AtrdoseATR OH-ATR DACT DIA DEAPlasmang/ml 00.2 ^ 1.0 ^ 3.0 ^ 1.0 ^ 0.5 ^50.2 ^ 1.0 ^ 95.8 ± 5.6 1.0 ^ 0.5 ^250.2 ^ 1.0 ^ 330.0 ± 126.5 1.8 ± 0.5 0.8 ± 0.3Milkng/g 00.2 ^ 1.0 ^ 3.0 ^ 1.0 ^ 0.5 ^51.0 ± 0.7 1.9 ± 0.5 383.5 ± 179.1 18.5 ± 12.3 5.2 ±3.5255.9 ± 2.8 4.6 ± 1.1 1365.0 ± 438.6 91.6 ± 24.0 30.0 ± 13.6Adrenalng/g 00.2 ^ 1.0 ^ 3.0 ^ 1.0 ^ 0.5 ^53.0 ± 3.1 1.0 ^ 3.0 ^ 1.0 ^ 0.5 ^250.2 ^ 1.0 ^ 86.9 ± 79.1 1.0 ^ 0.5 ^Brainng/g 00.2 ^ 1.0 ^ 3.0 ^ 1.0 ^ 0.5 ^55.7 ± 8.5 1.4 ± 0.5 100.4 ± 16.0 1.1 ± 0.2 0.5 ^259.7 ± 16.0 4.5 ± 6.1 382.1 ± 162.5 1.6 ± 0.5 0.7 ±0.3Mean ± SEM.^ LOQ or limit of quantitation was used if the sample concentration was undetected or below the LOQ19Table 5. Concentration of DACT in PND pup gonads from dams dosed GD 14 to PND 10.ATR dose Ovary (ng/g) Testes (ng/g)03.0 ^ 3.0 ^53.0 ^ 77.3 ± 5.32558.8 ± 41.3 277.7 ± 120.6Mean ± SEM.^ LOQ or limit of quantitation was used if the sample concentration was undetected or below the LOQ