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### Atrazine Activates an ACTH-Dependent Release of Cor ticosterone and

Progresterone in Male Wistar Rats Following Acute E xposure

Internal Report Prepared for the use of the Office of Pesticide Program, Office of Chemical Safety and Pollution Prevention, U.S. Environmental Protection Agency,

Washington, DC

July 15, 2010

U.S. EPA/ORD Contributors

Michelle Gatien Hotchkiss, Deborah S. Best, Ralph L . Cooper, Susan C. Laws

Endocrine Toxicology Branch, Toxicity Assessment Di vision, National Health and Environmental Effects Research Laboratory,

Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711

This document has been reviewed in accordance with the policy of the National Health and Environmental Effects Research Laboratory, U.S. Env ironmental Protection Agency, and approved for publication. Approval does not signif y that the contents necessarily reflect the views or policy of the Agency nor does mention of t rade names or commercial products constitute endorsement or recommendation for use

.Page 2 of 14 Table of Contents

Executive Summary............................................................................3

Background.......................................................................................4

Materials and Methods..........................................................................5

Results........................................................................... ..................7

Discussion......................................................... ................................8

References.................................................................................... ...10

Figure 1....................................................................................... ....13

Table 1...........................................................................................14Page 3 of 14



### Executive Summary:

Previous studies conducted in the laboratory of Dr. Susan C. Laws have shown that a single oral administration of atrazine (ATR; 50-200 mg/kg, oral gavage), a chlorotriazine herbicide, induces dose-dependent increases in plas ma adrenocorticotropic hormone (ACTH), serum corticosterone (CORT) and progesterone in mal e Wistar rats(Toxicol Sci., 112(1):78-87). The clear and rapid increase in ACTH followed by an increase in the adrenal steroid hormones (corticosterone and progesterone) have defined a no vel endocrine effect of the chlorotriazines not previously characterized. Additional studies in ou r laboratory have indicated that this chemicalinduced change in the hypothalamic-pituitary-adrena l axis (HPA-axis) activation is not due to a generalized stress response to animal handling or g astrointestinal distress (Laws et al ., 2009;

Fraites et al ., 2009; unpublished observations in ). While the cellular events leading to the activation of the HPA-axis are not known, extended elevation of adrenal steroids can adversely impact a number of homeostatic mechanisms including normal functioning of the hypothalamicpituitary-gonadal (HPG)-axes. As part of an extensi ve re-evaluation of the human health effects of atrazine, the Office of Chemical Safety and Poll ution Prevention, U.S. Environmental Protection Agency (U.S. EPA), is reviewing studies that have characterized the effects of atrazine and its dealkylated metabolites on adrenal steroid synthesis. During the new evaluation, U.S. EPA will consider the potential for cancer and non-cancer effects of atrazine, and will include data generated since 2003 from laboratory a nd population studies. To be certain that the best science possible is used in its assessment of the atrazine human health risk and to ensure transparency, U.S. EPA is seeking advice from an in dependent Scientific Advisory Panel (SAP) established under the Federal Insecticide, Fungicid e and Rodenticide Act. The data in this report are provided to support the third meeting of the SA P peer review with its evaluation of cancer

and non-cancer effects of atrazine based on animal toxicology studies.

A fundamental requirement for a better understandin g of the effects of atrazine on the HPA-axis is the characterization of the dose-respon se relationship between exposure to the herbicide and the hormonal response. Although a pa rtial description of acute (single dose) atrazine-induced changes in ACTH, corticosterone, a nd progesterone in the male Wistar rat was published by Laws et al . (2009); however a better characterization of the lower end of the doseresponse curve was needed (5-50 mg/ kg). In respo nse to this need, the current reportPage 4 of 14

summarizes subsequent experiments conducted within National Health and Environmental Effects Research Laboratory (NHEERL) to confirm and extend the results reported in Laws et al ., 2009. The 15 min post-dosing collection time poi nt was chosen for these additional experiments based on the previous observation that elevations in plasma ACTH concentrations after atrazine treatment peak at approximately 15 m in and begin to decline by 30 min (Laws et al ., 2009; Figure 1). For the current study, data dem onstrate that the LOEL for elevations in plasma concentrations of ACTH 15 min following a si ngle dose of atrazine (oral gavage), is 12.5 mg/kg, and 5 mg/kg for both serum corticosterone an d progesterone. To evaluate the reproducibility and robustness of the response, the study was conducted in two separate blocks, with each block containing a full dose-response foratrazine (Table 1). An additional diamino-schlorotriazine (DACT) group was included to replica te the earlier observation that an equal molar dose of this di-dealkylated metabolite of atr azine produces an effect on the HPA-axis of much smaller magnitude than that produced by the pa rent compound. These data are in agreement with previously published results (Laws et al. , 2009), and are provided as supplemental data that can be further evaluated usi ng a benchmark analysis.



### Background Atrazine (ATR), a chlorotriazine herbicide, is wide ly used in the United States for broadleaf weed control in several commercial crops and landscape vegetation. Biotransformation by-products of ATR, as well as th e parent compound, are relatively persistent in water and mobile in soil (Seiler et al ., 1992) and have been detected in surface and grou nd water in areas of major usage, with highest levels detected most commonly in the Midwestern states during the growing season (Baker, 1998; Balu et al ., 1998). Environmental exposure to ATR and its biotransformation by-products has gener ated much concern about the potential adverse effects of these chemicals, resulting in a large body of research on this subject over the past 10 years. Primarily, exposure to ATR and its biotransformation by-products has been observed to disrupt the development and function of the mammalian reproductive system by causing neuroendocrine effects (Cooper et al ., 1996, 2000, 2007; Foradori et al .,2009a,b), delays in pubertal development in male and female rats (La ws et al ., 2000, 2003; Stoker et al ., 2000, 2002), and altered gene expression in the steroidog enic pathway (Sanderson et al ., 2000; Suzawa and Ingraham 2008). ATR administration has also be en observed to increase serumPage 5 of 14

concentrations of estradiol and estrone (Stoker et al ., 2000), progesterone, corticosterone (CORT), and plasma adrenocorticotropic hormone (ACT H; Fraites et al ., 2009; Laws et al

., 2009; Pruit et al ., 2003). This report provides data from a study conducted to better characterize the lowest observed effect level (LOEL) for the effects of atr azine on plasma ACTH, corticosterone and progesterone concentrations in male Wistar rats 15 min post-dosing. The 15 min post-dosing time point was chosen for these additional experime nts based on the previous observation that elevations in plasma ACTH concentrations after atra zine treatment peak at approximately 15 min and begin to decline by 30 min (Laws et al ., 2009, Figure 1). To evaluate the reproducibility and robustness of the response, the study was conducted in two separate blocks, with each block containing a full dose response for atrazine (5-200 mg/kg). Data from the blocks were analyzed separately, and then combined and tested for block and treatment effects, and block x treatment

interactions. An additional diamino-s-chlorotriazi ne (DACT, 135 mg/kg) group was included as a replicate of the earlier study by Laws et al. 200 9. These data are submitted to the Office of Chemical Safety and Pollution Prevention, U.S. EPA. , Washington, DC, as supplemental data for further review and benchmark analysis.



### Materials and Methods

Animals Male Wistar rats, 60 days of age were obtained from Charles River, Inc, (Raleigh, NC) and were maintained under controlled temperature (2 0–24°C) and humidity (40–50%). To acclimate the animals to the dosing procedure, all rats were pre-dosed daily by oral gavage with 1% methylcellulose vehicle for one week prior to ad ministration of the test chemical. Previous experience has shown that such handling of the anim als prior to actual exposure to the test chemical provides more consistent ACTH and corticos terone concentrations during the morning

hours when the circadian fluctuation of these hormo nes is the lowest (Laws et al., 2009). Animals were ranked by body weight and placed into treatment groups such that the mean body weights + SE for all groups were similar. All experiments we re approved by the U.S. EPA,

NHEERL’s Institutional Animal Care and Use Committe e (IACUC) and was conducted in

accordance with National Institute of Health standa rds for laboratory animal research (LAPR# 12-06-005).Page 6 of 14

Dosing solutions and procedures. ATR (2-chloro-4-ethylamino-6-isopropylamino-s-triaz ine, CAS 1912-24-9, purity 97.1%; a gift from Syngenta Crop Protection, Greensboro, N C), and DACT (diamino-s-chlorotriazine, CAS 3397-62-4, purity 97%, ACROS Organics, Belgium; A017756301) were administered by oral gavage in a suspension of 1% methyl cellulose/ distilled water (CAS 9004-67-5, M-7140, Sigma-Aldrich Chemical Company, St. Louis, MO) in a volume of 5.0 ml dosing solution/ kg body weight. Control animals received 1% methylcel lulose only (5.0 ml dosing solution/ kg body weight). To facilitate the comparison of the potency of each test chemical with that of ATR, the dose selected for DACT is the molar equiva lent of ATR 200mg/kg. Doses of ATR and DACT were selected based on results reported in pre vious studies of ATR on female pubertal development and HPA-axis activation (Laws et al., 2000, 2003, 2009).

Experimental Design: Characterization of the HPA-ax is response to ATR 15 min after a single administration. In order to further characterize the HPA-axis respo nse to ATR across a lower range of doses, a dose-response experiment was conducted to measure plasma ACTH and serum CORT, progesterone, and body temperature 15 min after a s ingle administration of atrazine. Animals were housed under controlled lighting conditions (1 2 hr light/12 hr dark; lights on at 0700 EST and off at 1900 EST) and given ad libitum access to food (Purina Laboratory Rat Chow, 5001) and filtered tap water. On the day of necropsy, an imals were given one dose of either vehicle (methyl cellulose), ATR (5, 12.5, 25, 50, 100, or 2 00 mg/kg), or DACT (135 mg/kg), by oral gavage at 0900 EST and killed by decapitation 15 mi n post-dosing. Animals were housed in a room immediately adjacent to the necropsy room to l imit the transfer time of the animals from the home cage to the necropsy room to within 15 sec onds. Upon decapitation, blood was processed for serum and plasma, and aliquots were s tored at -80°C until measurement of ACTH, CORT, and progesterone by radioimmunoassay (RIA). T o evaluate the reproducibility and robustness of the effects, the study was conducted in two experimental blocks with each treatment group containing ten animals/ treatment g roup.Page 7 of 14

Radioimmunoassays: Total serum CORT and progesterone were measured usi ng Coat-A-Count RIA kits from Siemens Medical Solutions Diagnostics (TKRC and TKP G; Los Angeles, CA). The limits of sensitivity for these assays were 5.7ng/ml and 0.02 ng/ml, respectively. Total plasma ACTH was measured using an RIA kit from MP Biomedicals (07-1 06102, Orangeburg, NY). Crossreactivity of human ACTH antibody with rat ACTH was tested using serial dilutions of rat plasma with and without spikes of ACTH. Linearity of ACTH concentration was confirmed across all dilutions of rat plasma and in the prese nce of rat plasma samples spiked with ACTH. The limit of sensitivity for this assay was 5.7 pg/ ml.

Statistical Analysis Treatment means for each dose group were tested for homogeneity of variance using the Bartlett test (Graph Pad Instat; Graph Pad Software , San Diego, CA). As heterogeneity of variance was evident for each hormone tested, data were log transformed prior to further analysis. Data were analyzed by ANOVA using the ge neral linear model (GLM) procedure (Statistical Analysis System; SAS Institute, Inc., Cary, NC) for block and treatment effects, and treatment x block interactions. Subsequently, sinc e no significant block or block x treatment interactions were observed for any of the hormones, data from both blocks were combined for statistical analysis. In cases where a significant treatment effect (p< 0.05) was observed, the ATR dose-response data were further analyzed using Least Squared Means post-hoc tests to compare control values to those for each treatment group. A two-tailed Mann Whitney test (Graph Pad Instat; Graph Pad Software, San Diego, C A) was used to compare the DACT group to the control group. All post-hoc and Mann Whitney results were considered significant when p < 0.05. Data are reported as mean + SEM, (n=10/treatment for each block unless otherwi se specified).



### Results Effects of a Single Dose of ATR or DACT on ACTH, CO RT, and Progesterone

Concentrations ACTH, CORT, and progesterone concentrations 15 min following a single dose of ATR or DACT are shown in Table 1 for each experimental block. Hormone concentrations for thePage 8 of 14

vehicle control animals were within range of those typically observed in untreated control animals in our laboratory, suggesting that the expe rimental design had no impact on these measures (Laws et al, 2009). A dose-dependent incr ease in plasma ACTH concentrations was observed following ATR administration, with signifi cant increases observed at doses of 12.5mg/kg and above (Table 1, NOEL= 5mg/kg). Simil arly, ATR treatment resulted in dosedependent increases in serum CORT and progesterone concentrations with significant increases observed at all doses tested (5-200 mg/kg; Table 1 , LOEL= 5mg/kg). Although a single administration of DACT (135 mg/ kg) caused a signif icant increase in ACTH (Block 1 only), CORT and progesterone concentrations, this increase was substantially less than that observed for an equal molar dose of ATR (200 mg/kg, Table 1) . This observed difference between the potency of equal molar doses of ATR and DACT agrees with previous reports from this laboratory (Laws et al ., 2009).



### Discussion The data reported here confirm previous findings in this laboratory that a single administration of ATR causes a significant elevatio n in plasma ACTH and serum CORT and progesterone concentrations 15 min post-administrat ion (50-200 mg/kg; Laws et al ., 2009;

Fraites et al ., 2009). The current data also extend previous fi ndings on the effects of atrazine on ACTH, CORT, and progesterone by adding additional d oses at the lower end of the dose response curve (5-50 mg/kg). Previous work (Laws et al ., 2009) and the current studies demonstrate that a single dose of ATR produces a re producible and robust elevation in ACTH (12.5-200 mg/ kg), CORT (5-200 mg/kg), and progeste rone (5-200 mg/kg) concentrations that is both dose-and time-responsive. In all studies to d ate, peak elevations of ACTH concentrations following a single dose of ATR occur approximately 15 min before those for CORT and progesterone, suggesting that the observed activati on of the HPA-axis is likely mediated through a neuroendocrine pathway (e.g., corticotrophin rele asing hormone release from the hypothalamus). However, this does not rule out the possibility that ATR may have effects at multiple levels of the HPA-axis simultaneously (i.e . the hypothalamus, pituitary and/or adrenal)

to achieve these effects. Additional in vitro and in vivo studies to address these questions are

needed.Page 9 of 14

The current data also confirm previous findings (La ws et al ., 2009) that though the primary ATR metabolite DACT (135 mg/kg; molar equiv alent to ATR 200 mg/kg) significantly elevates ACTH, CORT, and progesterone as compared t o control values at 15 min; however, it does so at a magnitude approximately 6-fold less th an ATR (200 mg/kg) for ACTH and progesterone, and nearly 3-fold less for CORT. Thi s difference in the magnitude of hormone changes occurs in spite of the fact that over 60-fo ld more DACT is found in blood plasma 15 minutes after a single dose of DACT (135 mg/ kg) th an after a single dose of ATR (200 mg/kg;

Laws et al ., 2009). Thus, although it has been postulated th at DACT is the active metabolite responsible for HPG-axis effects following exposure to ATR, it is unlikely to be responsible for the HPA-axis activation observed in this study and others (Laws et al ., 2009; Fraites et al

., 2009). Given the rapid metabolism of ATR to DACT, it may be that the parent compound ATR and/or its mono-dealkylated intermediary metabolite s deisopropyl atrazine (DIA) or deethyl atrazine (DEA) are primarily responsible for the ef fects observed on the HPA-axis after an acute exposure, while DACT may play a role in producing t he effects on the hypothalamic-pituitarygonadal axis (HPG-axis). In conclusion, the current data confirm and extend previous findings that demonstrate that ATR increases plasma ACTH (LOEL= 12.5mg/kg), s erum CORT (LOEL= 5mg/kg), and progesterone (LOEL 5mg/kg) concentrations in a dose -dependent fashion 15 min following a single dose. These changes in hormones are robust and reproducible. Though the exact mode/ mechanism of action for these changes is unknown, t he rapid release of ACTH and subsequent activation of adrenal steroidogenesis (i.e. CORT an d progesterone release) may reflect chlorotriazine-induced changes at the level of the brain and/ or pituitary.Page 10 of 14



### References

Baker, D. B. (1998). Herbicides in drinking water: A challenge for risk communication. Triazine Herbicides Risk Assessment. L. F. Ballantine, McFar land, J.E., and Hackett, D.S., American Chemical Society, Washinton D.C.: 303-321.

Balu, K., Holden, P.W., Johnson, L.C., and Cheung, M.W. (1998). Summary of Ciba Crop Protection groundwater monitoring study for atrazin e and its degradation products in the United

States. Triazine Herbicides Risk Assessment. L. G. Ballantine, McFarland, J.E., and Hackett, D.S., American Chemical Society, Washington D.C.: 2 27-238.

Cooper, R. L., S. C. Laws, et al. (2007). "Atrazine and reproductive function: mode and

mechanism of action studies." Birth Defects Res B D ev Reprod Toxicol 80(2): 98-112.

Cooper, R. L., T. E. Stoker, et al. (1996). "Effect of atrazine on ovarian function in the rat." Reprod Toxicol 10(4): 257-264.

Cooper, R. L., T. E. Stoker, et al. (2000). "Atrazi ne disrupts the hypothalamic control of pituitary-ovarian function." Toxicol Sci 53(2): 297 -307.

Fan, W., T. Yanase et al. (2007), “Herbicide atrazi ne activates SF-1 by direct affinity and

concomitant co-activators recruitments to induce ar omatase expression via promoter II.” Biochem Biophys Res Commun 355(4): 1012-1018.

Foradori, C. D., L. R. Hinds, et al. (2009). "Effec ts of atrazine and its withdrawal on gonadotropin-releasing hormone neuroendocrine funct ion in the adult female Wistar rat." Biol Reprod 81(6): 1099-1105.

Foradori, C. D., L. R. Hinds, et al. (2009). "Atraz ine inhibits pulsatile luteinizing hormone release without altering pituitary sensitivity to a gonadotropin-releasing hormone receptor agonist in female Wistar rats." Biol Reprod 81(1): 40-45.Page 11 of 14

Fraites, M. J., R. L. Cooper, et al. (2009). "Chara cterization of the hypothalamic-pituitaryadrenal axis response to atrazine and metabolites i n the female rat." Toxicol Sci 112(1): 88-99.

Laws, S. C., J. M. Ferrell, et al. (2003). "Puberta l development in female Wistar rats following exposure to propazine and atrazine biotransformatio n by-products, diamino-S-chlorotriazine and hydroxyatrazine." Toxicol Sci 76(1): 190-200.

Laws, S. C., J. M. Ferrell, et al. (2000). "The eff ects of atrazine on female wistar rats: an evaluation of the protocol for assessing pubertal d evelopment and thyroid function." Toxicol Sci 58(2): 366-376.

Laws, S. C., M. Hotchkiss, et al. (2009). "Chlorotr iazine herbicides and metabolites activate an ACTH-dependent release of corticosterone in male Wi star rats." Toxicol Sci 112(1): 78-87.

McMullin, T. S., M. E. Andersen, et al. (2004). "Ev idence that atrazine and diaminochlorotriazine inhibit the estrogen/progeste rone induced surge of luteinizing hormone in female Sprague-Dawley rats without changing estroge n receptor action." Toxicol Sci 79(2): 278-

286.

Pruett, S. B., R. Fan, et al. (2003). "Modeling and predicting immunological effects of chemical stressors: characterization of a quantitative bioma rker for immunological changes caused by atrazine and ethanol." Toxicol Sci 75(2): 343-354.

Sanderson, J. T., W. Seinen, et al. (2000). "2-Chlo ro-s-triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carc inoma cells: a novel mechanism for estrogenicity?" Toxicol Sci 54(1): 121-127.

Seiler, A., Brenneisen, P., and Breen, D.H. (1992). "Benefits and risks of plant protection products- possibilities of protecting drinking wate r: Case atrazine." Water Supply 10: 31-42.Page 12 of 14

Stoker, T. E., D. L. Guidici, et al. (2002). "The e ffects of atrazine metabolites on puberty and thyroid function in the male Wistar rat." Toxicol S ci 67(2): 198-206.

Stoker, T. E., S. C. Laws, et al. (2000). "The effe ct of atrazine on puberty in male wistar rats: an evaluation in the protocol for the assessment of pu bertal development and thyroid function." Toxicol Sci 58(1): 50-59.

Suzawa, M. and H. A. Ingraham (2008). "The herbicid e atrazine activates endocrine gene networks via non-steroidal NR5A nuclear receptors i n fish and mammalian cells." PLoS One 3(5): e2117.Page 13 of 14

Figure 1 Dose and time response for ACTH, corticosterone an d progesterone concentrations following a single dose of atrazine. Male Wistar ra ts were pre-dosed with vehicle control for 1 week prior to receiving a single dose of vehicle (m ethyl cellulose) or), atrazine (5, 50, 100 or 200 mg/kg, oral gavage). Groups of animals were killed at 5, 15, 30, 60 and or 180 min. post-dosing, and ACTH, cortcosterone, and progesterone were meas ured by RIA. Mean ± SEM (n=10). *Significantly different from control at each time point by Kruskal-Wallis Test (non-parametric ANOVA) and Dunn’s Multiple Comparison Test (p < 0.0 5). Mean ACTH ± SEM for controls over all time points: 49.51 ±4.25 pg/ml; corticoste orne: 31.08 ±6.04 ng/ml; P4: 0.55 ± 0.07 ng/ml.

From: Laws et al., 2009, Toxicol Sci., 112(1):78-87Page 14 of 14

Table 1: Hormone Concentrations 15min after a Singl e Administration of Atrazine or DACT

a Mean + SE (n=10 for single block, n=20 for combined block s unless otherwise noted ) b Dose is equimolar to ATR 200mg/kg

c Indicates different from vehicle control using the LS Means post-hoc test (ANOVA, GLM Procedure, p< 0.05). d n=9

e n=8 f n=19 g n=18 Dose

(mg/kg) ACTH (pg/ml) Corticosterone (ng/ml) Progesterone

(ng/ml) Block 1 ATR 0 64.67 + 3.64 a 46.34 + 5.81 0.58 + 0.06

ATR 5 75.54 + 5.91 117.64 + 19.56 c 1.09 + 0.16 c ATR 12.5 96.29 + 7.55 216.90 + 31.55 c 1.90 + 0.24 c ATR 25 127.69 + 11.97 c 281.69 + 27.66 c 2.95 + 0.39 c ATR 50 135.40 + 18.28 c 284.87 + 33.11 c 3.53 + 0.65 c ATR 100 332.14 + 76.96 c 364.32 + 29.78 c 5.60 + 0.73 c ATR 200 479.14 + 99.07 c 356.08 + 23.07 c 6.42 + 0.98 c DACT 135 b 89.41 + 6.55 c 129.28 + 21.46 c 1.07 + 0.12 c Block 2

ATR 0 59.84 + 7.73 d 36.51 + 7.76 0.65 + 0.10 ATR 5 58.25 + 5.06 d 83.29 + 21.81 c 1.00 + 0.12 ATR 12.5 99.93 + 9.43 c d 230.24 + 40.12 c 2.29 + 0.39 c ATR 25 128.66 + 32.67 c d 249.05 + 44.21 c 3.19 + 0.84 c ATR 50 163.87 + 26.04 c d 339.05 + 35.31 c 4.61 + 0.91 c ATR 100 318.82 + 89.87 c d 413.09 + 32.58 c 7.28 + 1.06 c ATR 200 439.87 + 93.50 c d 396.60 + 19.47 c 7.60 + 0.55 c DACT 135 b 64.98 + 5.77 e 113.04 + 27.36 c 1.26 + 0.11 c Blocks 1 & 2 Combined ATR 0 62.38 + 4.05 f 41.43 + 4.85 0.62 + 0.06

ATR 5 67.35 + 4.32 f 100.46 + 14.79 c 1.05 + 0.10 c ATR 12.5 98.01 + 5.82 c f 223.57 + 24.89 c 2.09 + 0.23 c ATR 25 128.15 + 16.20 c f 265.37 + 25.66 c 3.07 + 0.45 c ATR 50 148.89 + 15.55 c f 311.96 + 24.36 c 4.07 + 0.56 c ATR 100 325.83 + 57.11 c f 388.71 + 22.20 c 6.44 + 0.66 c ATR 200 460.54 + 66.67 c f 376.34 + 15.41 c 7.01 + 0.57 c DACT 135 b 78.55 + 5.23 c g 121.16 + 17.02 c 1.17 + 0.08 c