DRAFT, DO NOT CITE

Re-Evaluation of Human Health Eff ects of Atrazine: Review of Experimental Animal and In vitro Studies and Drinking Water Monitoring Frequency

Presented Jointly To The FIFRA Sc ientific Advisory Panel By:

U.S. Environmental Protection Agency Office of Pesticide Programs

Health Effects Division

and

Environmental Fate and Effects Division

in collaboration with the

Office of Water

and

Office of Research and Development

Presented On:

April 26-30, 2010Page 2 of 263 TABLE OF CONTENTS

1. INTR ODUCTION

.................................................................................. 12

2. 2003 HUMAN HEALTH RISK ASS ESSM EN T .................................... 16

3. MAMMALIAN TOXICITY

..................................................................... 18

3.2 M ODE OF A CTION (MOA)

......................................................................................................... 21

3.2.1 Background

.................................................................................................................. 21

3.2.2 2003 Key Events

– HPG axis

................................................................................... 22

3.2.3 Effect of Atrazine on the hypothalamic

‐ pituitary

‐ gonadal function.

................... 23

3.2.4 2010 Key Events

– HPA axis

................................................................................... 24

3.2.5 Effect of Atrazine on the regulation of the hypothalamic

‐ pituitary

‐ adrenal axis 25

3.2.6 Linking the hypothalamic

‐ pituitary

‐ adrenal and hypothalamic pituitary

‐ ovarian

responses.

............................................................................................................................. 26

3.3 R EPRODUCTIVE /D EVELOPMENTAL T OXICITY

......................................................................... 30

3.3.1 Female Reproductive/Developmental Toxicity

.......................................................... 30

3.3.2 Male Reproductive/Developmental Toxicity

......................................................... 32

3.3.2.1 Delayed Puberty Onset

...................................................................................................... 32

3.3.2.2 Prostatitis

............................................................................................................................ 35

3.3.2.3 Effects on Aromatase and steroidogenesis

........................................................................ 37

3.3.2.4 Andrology

........................................................................................................................... 39

3.4 N EUROTOXICITY

............................................................................................................... 41

3.5 I MMUNOTOXICITY

............................................................................................................ 44

3.6 G ENOTOXICITY AND C ARCINOGENESIS

................................................................................. 48

3.7 I NSULIN R ESISTANCE AND M ITOCHONDRIAL F UNCTION I MPAIRMENT

........................................ 51

4. SUMMARY OF MA MMALIAN TO XICITY

............................................ 51

5. APPROACHES TO EVALUATING WATER SAMPLING
STRATEGIES AND FREQUE NCY OF M O NITORING............................ 53

5.1. I NTRODUCTION

....................................................................................................................... 53

5.2 T HE N ATURE OF P ESTICIDE O CCURRENCE IN S URFACE W ATER

........................................................... 54

5.3 S AMPLING F REQUENCY IN M ONITORING : I MPLICATIONS FOR P ESTICIDE E XPOSURE A SSESSMENTS

............ 57

5.3.1 General Monitoring Strategies..................................................................................... 57

5.3.2 Data Quality Objectives................................................................................................ 58

5.3.3 Monitoring Strategies Related to Pesticides ................................................................ 59

5.4 A PPROACHES TO A NALYZING AND I NTERPRETING M ONITORING D ATA C OLLECTED AT D IFFERENT S AMPLING

F REQUENCIES

................................................................................................................................. 61

5.4.1 Characterizing Exposure Time Series From Less Frequent Monitoring Data................ 61

5.4.2 Determining Confidence Bounds on Exposure Estimates From Monitoring Data........ 65

5.5 E XAMINING THE P ERFORMANCE OF V ARIOUS S AMPLING S TRATEGIES WITH D ATA FROM THE A TRAZINE

M ONITORING P ROGRAM (AMP)....................................................................................................... 66

5.5.1 Profiles of Community Water Systems in the AMP ...................................................... 66

5.5.2. Approaches from the 2007 and 2009 SAP Meetings on Monitoring in Surface Water

Streams in the Corn and Sorghum Growing Region of the US .............................................. 68

5.5.3 Current Methods .......................................................................................................... 69

5.5.3.1 Comparing Upper Percentile Values from Different Sampling Strategies with those of the

“True” Prof ile ........................................................................................................................

........... 69

5.5.3.2. Examining the Ranges of Maximum Running Averages of Different Durations for Various
Sampling Strategies.......................................................................................................................... 74Page 3 of 263 5.5.3.3 Results........................................................................................................................

.......... 76

5.5.4. Summary of Initial Analyses ........................................................................................ 93

5.6. A RTIFICIAL N EURAL N ETWORK (ANN) M ODELING OF A TRAZINE O CCURRENCE P ATTERNS

..................... 95

5.6.1 Time Series Plots Using Frequent Monitoring Data...................................................... 95

5.6.2 Artificial Neural Network Modeling ............................................................................. 98

5.6.3. Predictive Analytics ................................................................................................... 100

5.6.4. Simulating Daily Levels.............................................................................................. 102

5.6.5 Evaluating a Monitoring Strategy .............................................................................. 103

5.6.7. Summary

– Advantages and Disadvantages of Using the ANN Models.................... 105

5.7 S UMMARY OF W ATER M ONITORING F REQUENCY M ETHODS

........................................................... 105

5.7.1. Planned Approach to Evaluate the Methods and Models for Estimating Pesticide

Concentrations From Less Frequent Monitoring Data ........................................................ 106

APPENDIX A ......................................................................................... 110

EVALUATION OF ARTICLES ON ATRAZINE AND ATRAZINE METABOLITES RELATED TO

MAMMALIAN TOXICITY STUDIES SUGGESTING POTENTIAL ADVERSE HUMAN HEALTH

OUTCOMES

.............................................................................................................................. 110

A.1 CONTENT OF TH IS APPE NDIX

.......................................... 111

A.2 EFFECTS ON FEMALES

.................................................... 112

A.2.1 Dooley, G.P., Reardon, K.F., Prenni, J.E., Tjalkens, R.B., Legare, M.E., Foradori,

C.D., Tessari, J.E., Hanneman, W.H. (2008). Proteomic analysis of diaminochlorotriazine

adducts in Wistar rat pituitary glands and L β T2 rat pituitary cells. Chem Res Toxicol

21:844

‐ 851.

......................................................................................................................... 112

A.2.2 Fraites, M.J.P., Cooper, R.L., Buckalew, A., Jayaraman, S., Mills, L., and Laws, S.C. (2009). Characterization of the Hypothalamic

‐ Pituitary

‐ Adrenal Axis Response to Atrazine

and Metabolites in the Female Rat. Tox. Sci. 112(1):88

‐ 99.

............................................. 113

A.2.3 Gojmerac, T., Pleadin, J., Zuric, M., Rajkovic

‐ Janje, R., and Korsic, M., 2004.

Serum Luteinizing Hormone Response to Administration of Gonadotropin

‐ Releasing

Hormone to Atrazine

‐ Treated Gilts. Vet Human Toxicol. 46(5): 245

‐ 247.

........................ 116

A.2.4 Juliani, C., Silva

‐ Zacarin, E., Santos, D. and Boer, P. 2008. Effects of Atrazine on
Female Wistar Rats: Morphological Alterations in Ovarian Follicles and
Immunocytochemical Labeling of 90 kDa Heat Shock Protein. Micron. 39:607

‐ 616.

...... 117

A.2.5 Laws, S., Farrell, J., Stoker, T. and Cooper, R. (2003) Pubertal Development in

Female Wistar Rats following Exposure to Propazine and Atrazine Biotransformation By

‐ Products, Diamino

‐ S

‐ Chlorotriazine and Hydroxyatrazine. Toxicological Sciences 76:190

‐ 200.

.............................................................................................................................. 119

A.2.6 McMullin, T.S., Andersen, M.E., Nagahara, A. Lund, T.D., Pak, T., Handa, R.J.,

Hanneman, W.H. (2004). Evidence that atrazine and diaminochlorotriazine inhibit the estrogen/progesterone induced surge of luteinizing hormone in female Sprague Dawley

rats without changing estrogen receptor action. Tox. Sci. 79:278

‐ 286.

........................... 120

A.2.7 Shibayama, H. Kotera, T., Shinoda, Y., Hanada, T. Kajihara, T., Ueda, M.,

Tamura, H., Ishibashi, S., Ymashita, Y., and Ochi, S. 2009. Collaborative Work on
Evaluation of Ovarian Toxicity Two ‐ or Four

‐ week Repeated Dose Studies and Fertility
Study of Atrazine in Female Rats.

..................................................................................... 121

A.3 REPRODUCTIVE EFFE CTS IN MALES

............................. 124

A.3.1 Betancourt, M., Reséndiz, A., Casas, E. and Fierro, R. (2006) Effect of two

insecticides and two herbicides on the porcine sperm motility patterns using computer

‐ assisted semen analysis (CASA) in vitro. Repro. Tox. 22:508

‐ 512.

.................................... 124Page 4 of 263 A.3.2 Friedmann, AS. 2002. Atrazine inhibition of testosterone production in rat males
following peripubertal exposure. Reproductive Toxicology 16: 275

‐ 279.

........................ 125

A.3.3 Hase Y, Tatsuno M, Nishi T, Kataoka K, Kabe Y, Yamaguchi Y, Ozawa N, Natori

M, Handa H and Watanabe H. (2008) Atrazine binds to F 1 F

0 ‐

ATP synthase and inhibits

microchondrial function in sperm. Biochem Biophy Research Comm 366: 66

‐ 72.

.......... 126

A.3.4 Kandori, H, Suzuki S, Asamoto M, et al. 2005. Influence of atrazine

administration and reduction of calorie intake on prostate carcinogenesis in probasin/SV40 T antigen transgenic rats. Cancer Sci: 96: 221

– 226.

............................ 127

A.3.6 Laws, SC, Stoker, TE, Ferrell, JM, Hotchkiss, MG, and Cooper RL. 2007. Effects of

Altered Food Intake During Pubertal Development in Male and Female Wistar Rats.

Toxicol. Sci. 100(1): 194

‐ 202.

.............................................................................................. 130

A.3.7 Maravilla

‐ Galvan, R., Fierro, R., Gonzáles

‐ Marquez, H. Gómez

‐ Arroyo, S.,
Jiménez, Ir and Betancourt, M. 2009. Effects of Atrazine and Fenoxaprop

‐ Ethyl on Capacitation and the Acrosomal Reaction in Boar Sperm. International Journal of
Toxicology. Volume 2, Number 1. pp. 24

‐ 32.

..................................................................... 132

A.3.9 Pogrmic K, Fa S, Dakic V, Kaisarevic S, and Kovacevic R. 2009. Atrazine Oral

Exposure of Peripubertal Male Rats Downregulates Steroidogenesis Gene Expression in
Leydig Cells. Toxicological Sciences 111(1):189

‐ 197.

......................................................... 134

A.3.10 Rayner J L., Enoch R R, et al. 2007. Atrazine

‐ induced reproductive tract

alterations after transplacental and/or lactational exposure in male Long

‐ Evans rats. Tox Appl Pharmacol: 218: 238

– 248.

...................................................................................... 136

A.3.11 Rosenberg, B.G., Chen, H., Folmer, J., Liu, J., Papadopoulos, V., and Zirkin,

B.R. (2008). Gestational Exposure to Atrazine: Effects on the Postnatal Development of

Male Offspring. J. Andrology 29(3):304

‐ 311.

.................................................................... 138

A.3.12 Stoker TE, Guidici DL, Laws SC, Cooper RL. 2002. The Effects of Atrazine

Metabolites on Puberty and Thyroid Function in the Male Wistar Rat. Toxicological

Sciences 67: 198

‐ 206.

.......................................................................................................... 139

A.3.13 Stoker, T.E., Laws, S.C., Guidici, D., and Cooper, R.L. 2000. The effects of

atrazine on puberty in male wistar rats: An evaluation of a protocol for the assessment of
pubertal development and thyroid function. Toxicological Sciences. 58: 50

‐ 59.

.......... 141

A.3.14 Trentacoste, S V.. Friedmann, AS, Youker, RT, Breckenridge, CB, and Zirkin,

BR. 2001. Atrazine Effects on Testosterone Levels and Androgen

‐ Dependent
Reproductive Organs in Peripubertal Male Rats. Journal of Andrology, Vol. 22, No. 1, January/February 2001 ; pp. 142

‐ 148.

............................................................................... 142

A.3.15 Victor

‐ Costa, AB, Bandeira SMC, Oliveira AG, Mahecha GAB, Oliveira, CA.

2010. Changes in Testicular Morphology and Steroidogenesis in Adult Rats Exposed to

Atrazine. Reproductive Toxicology. oi10.1016/j.reprotox.2009.12.006.

......................... 143

A.4 AROMATA SE

...................................................................... 145

A.4.1 Albanito L, Lappano R, Madeo A, Chimento A, Prossnitz ER, Cappello AR, Dolce V,

Abonante S, Pezzi V, Maggiolini M. (2008). G

‐ protein

‐ coupled receptor 30 and estrogen

receptor ‐α are involved in the proliferative effects induced by atrazine in ovarian cancer
cells. Environ Health Perspect. 116(12):1648

‐ 55.

.............................................................. 145

A.4.2 Benachour N, Moslemi S, Sipahutar H, Seralini GE. (2007). Cytotoxic effects of

aromatase inhibition by xenobiotic endocrine disrupters alone and in combination.
Toxicology and Applied Pharmacology. 222:129

‐ 140.

.................................................... 147

A.4.3 Fan W, Yanase T, Morinaga H, Gonda S, Okabe T, Nomura M, Hayes T, Takayanagi R, Nawata H. (2007a). Herbicide atrazine activates SF

‐ 1 by direct affinity and

concomitant co

‐ activators recruitments to induce aromatase expression via promoter II.

Biochemical and Biophysical Research Communications. 355:1012

‐ 1018.

..................... 148

A.4.4 Fan W, Yanase T, Morinaga H, Gondo S, Okabe T, Nomura M, Komatsu T,

Morohashi K, Hayes TB, Takayanagi R, Nawata H. (2007b). G Atrazine

‐ induced aromatasePage 5 of 263 expression is SF

‐ 1 dependent: implications for endocrine disruption in wildlife and
reproductive cancers in humans. Environ Health Perspect. 115(5):720

‐ 7.

....................... 149

A.4.5 Heneweer M, van den Berg M, Sanderson JT. (2004). A comparison of human

H295R and rat R2C cell lines as in vitro screening tolls for effects on aromatase. Toxicol.

Lett. 149(2):183

‐ 194.

.......................................................................................................... 150

A.4.6 Higley EB, Newsted JL, Zhang X, Giesy JP, Hecker M. (2010). Assessment of

chemicals effects on aromatase activity using the H295R cell line. Environ Sci Pollut Res

Int. Jan 20. (Epub ahead of print).

...................................................................................... 152

A.4.7 Holloway, A. Anger, D., Crankshaw, D., Wu, M, and Foster, W. 2008. Atrazine

‐ induced Changes in Aromatase Activity in Estrogen Sensitive Target Tissues. Journal of
Applied Toxicology. 28:260

‐ 270.

........................................................................................ 154

A.4.8 Laville N, Balaguer P, Brion F, Hinfray N, Casellas C, Porcher JM, Ait

‐ Aissa S. (2006). Modulation of aromatase activity and mRNA by various selected pesticides in the

human choriocarcinoma JEG

‐ 3 cell line. Toxicology. 228:98

‐ 108.

................................... 155

A.4.9 Rayner, J., Wood, C., and Fenton, S. (2003) Exposure Parameters Necessary for
Delayed Puberty and Mammary Gland Development in Long

‐ Evans Rats Exposed in Utero

to Atrazine. Toxicology and Applied Pharmacology 195:23

‐ 34.

....................................... 156

A.4.10 Suzawa, M and Ingraham, HA. 2008. The herbicide atrazine activates

endocrine gene networks via non

‐ steroidal NR5A nuclear receptors in fish and

mammalian cells. PLoS ONE 3(5): e2117. doi:10.1371/journal.pone.0002117.

............ 157

A.4.11 Yi D K, Kim P J, et al. 2010. Dose ‐ and time

‐ dependent effect of atrazine or

its metabolites on aromatase mRNA in vitro. Only a preprint of article available.

....... 159

A.4.12 Yi, KD, Perez, EJ, Breckenridge, CB, and Simpkins, JW (2010?) Ex vivo effects
of atrazines or metabolites on phosphodiesterase activity.

............................................. 160

A.5. NEUROTOX ICITY

................................................................ 161

A.5.1 Belloni V, Alleva E, et al. 2007. Effects of low doses of atrazine on the

neurobehavioural development of mice. Ethology Ecology & Evolution 19: 309

– 322. 161

A.5.2 Coban A, and Filipov, N M. 2007. Dopaminergic toxicity associated with oral

exposure to the herbicide atrazine in juvenile male C57BL/6 mice. Journal of
Neurochemistry 100: 1177

‐ 1187.

...................................................................................... 163

A.5.3 Filipov N M, Stewart M A et al. 2007. Dopaminergic toxicity of the herbicide

atrazine in rat striatal slices. Toxicology 232: 68

– 78.

.................................................... 165

A.5.4 Foradori, C D, Hinds, L R, et al. 2009. Atrazine inhibits pulsatile luteinizing

hormone release without altering pituitary sensitivity to a gonadotropin

‐ releasing

hormone receptor agonist in female wistar rats. Biology of Reproduction. 81: 40

– 45.

....

........................................................................................................................................... 166

A.5.5 Foradori, C D, Hinds, L R, et al. 2009. Effects of atrazine on GnRH

neuroendocrine function after its withdrawal in the adult female wistar rat. Biology of
Reproduction 81(6): 1099

– 1105.

..................................................................................... 167

A.5.6 Giusi G, Facciolo RM, et al. 2006. The endocrine disruptor atrazine accounts for

a dimorphic somatostatinergic neuronal expression pattern in mice. Toxicological

Sciences 89(1): 257

– 264.

.................................................................................................. 169

A.5.7 Hossain M M and Filipov N M. 2008. Alteration of dopamine uptake into rat

striatal vesicles and synaptosomes caused by an in vitro exposure to atrazine and some of

its metabolites. Toxicology 248: 52

– 58.

......................................................................... 171

A.5.8 Rodriguez V M, Thiruchelvam M, Cory

‐ Slechta D A. 2005. Sustained Exposure to

the widely used herbicide atrazine: altered function and loss of neurons in brain

monoamine systems. Environmental Health Perspectives. 113(6): 708

‐ 715.

................ 172Page 6 of 263 A.6 IMMUNOT OXICITY.............................................................. 174

A.6.1 Cimino

‐ Reale, G., Ferrario, D., Casati, B., Brustio, R. Diodovich, C., Collotta, A.,
Vahter, M., Gribaldo, L. (2008). Combined in utero and juvenile exposure of mice and

atrazine in drinking water modulates gene expression and clonogenicity of myeloid
progenitors. Toxicol. Letters 180:59

‐ 66.

........................................................................... 174

A.6.2 Devos S, Bosscher K De, et al. 2003. Inhibition of cytokine production by the

herbicide atrazine

– Search for nuclear receptor targets. Biochemical Pharmacology 65:

303

– 308.

............................................................................................................................ 175

A.6.3 Devos S, Van Den Heuvel R, et al. 2004. Limited effect of selected organic
pollutants on cytokine production by peripheral blood leukocytes. Eur. Cytokine Netw:

15: 145

– 151.

..................................................................................................................... 177

A.6.4 Filipov, N M, Pinchuk, L M, et. al. 2005. Immunotoxic effects of short

‐ term

atrazine exposure in young male C57BL/6 mice. Toxicological Sciences: 86(2): 324

‐ 332.

..

........................................................................................................................................... 177

A.6.5 Karrow, N A, McCay, J A, et. al. 2005. Oral exposure to atrazine modulates cell

‐ mediated immune function and decreases host resistance to the B16F10 tumor model in
female B6C3F1 mice. Toxicology: 15

‐ 28.

.......................................................................... 178

A.6.6 Mizota K and Ueda H. 2006. Endocrine disrupting chemical atrazine causes degranulation through G q/11 protein

‐ coupled neurosteroid receptor in mast cells.
Toxicological Sciences 90(2): 362

– 368.

........................................................................... 179

A.6.7 Pinchuk L M, Lee S

‐ R, and Filipov N M. 2007. In vitro atrazine exposure affects

the phenotypic and functional maturation of dendritic cells. Toxicology and Applied
Pharmacology 223: 206

– 217.

.......................................................................................... 181

A.6.8 Pruett, S B, Fan, R, et al. 2003. Modeling and predicting immunological effects
of chemical stressors: characterization of a quantitative biomarker for immunological
changes caused by atrazine and ethanol. Toxicological Sciences: 75: 343

‐ 354.

........... 182

A.6.9 Pruett, S B, Fan, R, et al. 2009. Patterns of immunotoxicity associated with

chronic as compared with acute exposure to chemical or physical stressors and their

relevance with regard to the role of stress and with regard to immunotoxicity testing.
Toxicological Sciences: 109(2): 265

‐ 275.

.......................................................................... 183

A.6.10 Rooney, A A, Matulka R, and Luebke, R. 2003. Developmental atrazine

exposure suppresses immune function in male, but not female Sprague

‐ Dawley rats. Toxicological Sciences 76: 366

‐ 375.

.................................................................................. 184

A.6.11 Rowe, A M, Brundage, K M, Schafer, R, and Barnett J B. 2006.
Immunomodulatory effects of maternal atrazine exposure on male Balb/c mice.
Toxicology and applied Pharmacology 21: 69

– 77.

......................................................... 185

A.6.12. Rowe A M, Brundage K M and Barnett J B. 2007. In vitro atrazine

‐ exposure
inhibits human natural killer cell lytic granule release. Toxicology and Applied
Pharmacology 221: 179

– 188.

.......................................................................................... 187

A.6.13 Schwab, C L, Fan R, et al. 2005. Modeling and predicting stress

‐ induced
immunosuppression in mice using blood parameters. Toxicological Sciences 83: 101

– 113.

......................................................................................................................... 188

A.6.14 Whalen M M, Loganathan B G, et al. 2003. Immunomodulation of human

natural killer cell cytotoxic function by triazine and carbamate pesticides. Chemico

‐ Biological Interactions 145: 311

– 319.

............................................................................. 188

A.7 CARCINOGENESIS/ GENOTOXICITY

................................. 189Page 7 of 263 A.7.1 Fujimoto, N and Honda, H. (2003). Effects of Environmental Estrogenic
Compounds on Growth of a Transplanted Estrogen Responsive Pituitary Tumor Cell Line in

Rats. Food Chem. Toxicol. 41:1711

‐ 1717.

......................................................................... 189

A.7.2 Fukamachi, K., Han, B.S., Kim, C.K., Takasuka, N., Matsuoka, Y., Matsuda, E.,
Yamasaki, T., and Tsuda, H. (2004). Possible enhancing effects of atrazine and
nonylphenol on 7,2

‐ dimethylbenz[a]anthracene

‐ induced mammary tumor development

in human c

‐ Ha

‐ ras proto

‐ oncogene transgenic rats. Cancer Sci. 95(5):404

‐ 410.

............. 190

A.7.3 Garaj

‐ Vrhovac, V. and Zeijezic, D. (2004) Genotoxicity evaluation of pesticide
formulations containing alachlor and atrazine in multiple mouse tissues (blood, kidney,
liver, bone marrow, spleen) by comet assay. Neoplasma 51.3:198

‐ 203.

......................... 191

A.7.4 Kmetic, I., Srcek, G., Slivac, I., Simic, B., Knlewald, Z., Kniewald, J. 2008. Atrazine

Exposure Decreases Cell Proliferation in Chinese Hamster Ovary (CHO

‐ K1) Cell Line. Bull

Environ Contam Toxicol. 81: 205

‐ 209.

............................................................................... 193

A.7.5 Malik, S. I., Terzoudi, G.I., and Pantelias, G.E. (2004). SCE analysis in G2 lymphocytes prematurely condensed chromosomes after exposure to atrazine: the non

‐ dose

‐ dependent increase in homologous recombinational events does not support its
genotoxic mode of action. Cytogenet Genome Res 104: 315

‐ 319.

.................................. 194

A.7.6 Manske, M., Beltz, L, and Dhanwada, K. 2004. Low

‐ Level Atrazine Exposure

Decreases Cell Proliferation in Human Fibroblasts. Arch. Environ. Contam. Toxicol. 46:438

‐
444.

.............................................................................................................................. 194

A.7.7 Mastrangelo, S., Tomassetti, M, Carratu, M.R., Evandri, M.G., and Bolle, P. (2006). Quercetin reduces chromosome aberrations induced by atrazine in the Allium

cepa test. Environ and Mol Mutagen 47:254

‐ 259.

........................................................... 197

A.7.8 Singh, M., Sandhir, R., and Kiran, R. 2008. Atrazine

‐ Induced Alterations in Rat Erythrocyte Membranes: Ameliorating Effect of Vitamin E. J. Biochem. Molecular
Toxicology. Vol 22, No. 5: pgs. 363

‐ 369.

............................................................................ 198

A.7.9 Singh, M., Kaur, P., Sandhir, R., Kiran, R. (2008). Protective effect of vitamin E
against atrazine

‐ induced genotoxicity in rats. Mutat Res 654:145

‐ 149.

......................... 199

A.7.10 Son, H

‐ Y., Nishikawa, A., Okazaki, K., Lee, K

‐ Y., Imazawa, T., and Hirose, M. (2003). Lack of Modifying Effects of Atrazine and/or Tamoxifen on Thyroid
Carcinogenesis in Rats Pretreated with N

‐ bis(2

‐ hydroxypropyl)nitrosamine (DHNP). Food

Chem. Toxicol. 41:1811

‐ 1816.

............................................................................................ 200

A.7.11 Tanaka, T., Kohno, H., Suzuki, R., Sugie, S. (2004) Lack of modifying effects of

an estrogenic compound atrazine on 7,12

‐ dimethylbenz(a)anthracene

‐ induced ovarian
carcinogenesis in rats. Cancer Lett. 210:129

‐ 137.

............................................................ 201

A.7.12 Ueda, M., Imai, T., Takizawa, T., Onodera, H., Mitsumori, K., Mtsui, T. and

Hirose, M. (2005) Possible enhancing effects of atrazine on growth of 7,12

‐ dimethylbenz(a)anthracene

‐ induced mammary tumors in ovariectomized Sprague

‐ Dawley rats. Cancer Sci. 96(1):19

‐ 25.

................................................................................ 202

A.7.13 Yi, Kun Don, Breckenridge, Charles B, and Simpkins, James W. 2010.
Cytotoxicity of Atrazine and its Metabolites in vitro. Unpublished data. Syngenta Crop

Protection Inc.

..................................................................................................................... 206

A.8 INSULIN/MITOCHO NDRIA FUNC TION

.............................. 207

A.8.1 Lim, S., Ahn, S.Y., Song, I.C., Chung, M.H., Jang, H.C., Park, K.S., Lee, K

‐ U, Pak,

Y.K., Lee, H.K. (2009). Chronic Exposure to the Herbicide, Atrazine, Causes Mitochondrial
Dysfunction and Insulin Resistance. PLoS ONE 4(4): e5186, p.1

‐ 11.

................................. 207Page 8 of 263 A.9 PHARMACOKINETICS: INTE RNAL DOSIMETRY, METABOLISM,

& TIME COURSE................................................................................... 209

A.9.1 Dooley, G.P, Prenni, J.E., Prentiss, P.L., Cranmer, B.K., Anderson, M.E., Tessari,

J.D. (2006). Identification of novel hemoglobin adduct in Sprague Dawley rats exposed to

atrazine. Chem Res Toxicol 19:692

‐ 700.

........................................................................... 209

A.9.2 Dooley, G.P., Hanneman, W.H., Carbone, D.L., Legare, M.E., Anderson, M.E.,
Tessari, J.D., (2007). Development of an immunochemistry detection method for

atrazine

‐ induced albumin adducts. Chem Res Toxicol 20:1061

‐ 1066.

............................. 210

A.9.3 Kim D. and Breckenridge C. (Syngenta). Derivation of the short

‐ term inhalation

NOAEL using a physiologically based pharmacokinetic model. January 29, 2010.

.......... 211

A.9.4 McMullin T.S. (2003) Pharmacokinetic modeling of disposition and time

‐ course
studies with [ 14 C]

‐ atrazine. J. Toxicol. Environ. Health, 66(A):941

‐ 964.

........................... 212

A.9.5 McMullin, T.S., Hanneman, W.H., Cranmer, B.K., Tessari, J.D., and Andersen,

M.E. (2007). Oral absorption and oxidative metabolism of atrazine in rats evaluated by
physiologically modeling approaches. Toxicology 240:1

‐ 14.

............................................ 213

A.9.6 Ross, M.K., Jones, T.L., and Filipov, M.N. (2009). Disposition of the herbicide 2

‐ chloro

‐ 4

‐ (ethylamino)

‐ 6

‐ (isopropylamino)

‐ s

‐ triazine (atrazine) and its major metabolites

in mice: a liquid chromatography/mass spectrometry analysis of urine, plasma, and

tissue levels. Drug Metab. Dispos. 37(4): 776

‐ 786.

........................................................... 215

APPENDIX B ......................................................................................... 217

A TRAZINE IMMUNOTOXICITY DATA

.................................................................................................. 217

APPENDIX C ......................................................................................... 220

A RTIFICIAL N EURAL N ETWORK M ODELING OF A TRAZINE O CCURRENCE P ATTERNS

.................................. 220

A RTIFICIAL N EURAL N ETWORK M ODELING OF A TRAZINE O CCURRENCE P ATTERNS

.................................. 221

C.1 ABS TRACT

.................................................................................... 222

C.2 BACK GROUND

............................................................................. 223

C.2.1 M ONITORING S TRATEGIES

................................................................................................ 223

C.2.2 D ATA Q UALITY O BJECTIVES

.............................................................................................. 223

C.2.3 A TRAZINE O CCURRENCE P ATTERNS

.................................................................................... 224

C.3 OCCURRE NCE DAT A

................................................................... 226

C.3.1 F REQUENT M ONITORING D ATA

......................................................................................... 226

C.3.2 T IME S ERIES P LOTS

. ........................................................................................................ 227

C.3.3 D ATASETS S UITABLE FOR M ODELING

.................................................................................. 231

C.4 ARTIFICIAL NEURAL NETWORK MODELING

............................ 233

C.4.1 ANN MODELS

................................................................................................................ 233

C.4.2 ANN MODELING FOR THE M AUMEE R IVER

, 1998. ............................................................... 235

C.4.3 ANN MODELING FOR THE S ANDUSKY R IVER

, 1995............................................................... 239

C.5 PREDICTI VE ANALYT ICS

............................................................. 242

C.5.1 D IRECT D ERIVATION OF THE E XPOSURE

............................................................................... 242

C.5.2 S CALING THE O CCURRENCE P ATTERNS

................................................................................ 243

C.5.3 S IMULATING D AILY L EVELS

............................................................................................... 243

C.5.4 E VALUATING A M ONITORING S TRATEGY

.............................................................................. 246Page 9 of 263 C.6 SUMMARY – ADVANTAGES AN D DISADVANTAGES OF USING

THE ANN MODELS............................................................................... 247

BIBLIOGRAPHY.................................................................................... 248

P OST 2003 R EFERENCES

............................................................................................................... 248

P RE

‐ 2003 R EFERENCES

................................................................................................................. 259Page 10 of 263 Figure 1: Atrazine and it s chlorinated metabo lites........................................................ 20 Figure 2: Relationship Between Duration of Expos ure and No Observed Effect Levels 27 Figure 3: PROPOSED MOA SCHE MATIC

.................................................................... 30 Figure 4: Concentrations of atrazine and chlorpyrifos over time in the White River,

Indiana. Source: G illiom et al, 2006.

...................................................................... 56 Figure 5: Comparison of daily time series for pesticide concentrations (dark blue line) to linear (red l ines) and stair-step (light blue lin es) extrapolations for samples taken at 14- or 30-day interv als. .......................................................................................... 62 Figure 6: Conceptual illustration of the nearest neighb or approach for estimating daily pesticide concentrations at le ss frequent sampli ng interv als.................................. 64 Figure 7: (a-c) Profiles fo r Chemographs #2 2, #8, and #24

......................................... 73 Figure 8: Illustration of a CWS chemograph with a single, relatively short duration (narrow) peak

......................................................................................................... 77 Figure 9: Illustration of a CWS ch emograph with a moderat e duration peak and

ex tended tail........................................................................................................... 81 Figure 10: Illustration of a Chemograph with a Broad or Ex tended Series of Peaks .... 85 Figure 11: Data fo r Maumee Riv er 2001

...................................................................... 96 Figure 12: Data fo r Maumee Riv er 1998

...................................................................... 97 Figure 13: Maumee 1998 and A NN Model with One N ode

.......................................... 98 Figure 14: Maumee 1998 and Max Li kelihood Three- Node ANN Model

.................... 100 Figure 15: Simulation Ba sed on the M aumee 1998 Model

......................................... 103 Figure 16: Effect of Atra zine Exposure on Aromatase Activity and Hormone Levels

. 153 Figure 17: Tr eatment Groups

..................................................................................... 203 Figure 18 Atrazine Occurrence at Missouri-American Water In take............................ 225 Figure 19: Source Wate r with Low Va riabilit y............................................................. 226 Figure 20: Source Water with Mo derate Variabi lity .................................................... 226 Figure 21: Data for Maumee River 2001

................................................................... 228 Figure 22: Data fo r Maumee Riv er 1998

.................................................................... 229 Figure 23: Data for a Headwate r Stream

.................................................................... 230 Figure 24: Data fo r Sandusky Riv er 1995

.................................................................. 232 Figure 25: Maumee 1998 and A NN Model with One Node

........................................ 234 Figure 26: Maumee 1998 and Max Li kelihood Four-N ode ANN Model

...................... 237 Figure 27: Maumee 1998 and Max Li kelihood Three- Node ANN Model

.................... 238 Figure 28: Residuals for M aumee 1998 Three- Node Mo del....................................... 239 Figure 29: Data Contributing to Small Likelihood fo r Two-N ode Model

...................... 241 Figure 30: Simulation Ba sed on the M aumee 1998 Model

......................................... 244 Figure 31: Simulation Ba sed on the S andusky 1995 Model

....................................... 245 Figure 32 Utility of ANN M odeling

.............................................................................. 247Page 11 of 263 LIST OF TABLES

Table 1: HPG Axis Perturbations : Summary of Te mporal and Dose

............................ 24

Table 2: HPA Axis Activation: Summary o f Tem poral an d Dose .................................. 26

Table 3: HPA/HPG Axes Nexus: Summa ry of Temporal and Dose Concordance

....... 29 Table 4: Strain and Dose Comparison in Male Rats dosed peripubertally with atrazine 34 Table 5: Summary of Atrazine Monitoring Program (AMP) Data for Atrazine in Raw

Water*

.................................................................................................................... 67 Table 6: Atrazine Monitoring Progra m Data for Atrazi ne in Raw Water*

...................... 68 Table 7: Upper Percentile Values Derived from Different Sampling Strategies for 3 Example CWS

........................................................................................................ 71

Table 8: Percentiles of Maximum Concentrati ons for C WS #4 Sampled at 4-, 7-, or 14- day Inte rvals........................................................................................................... 78 Table 9: Percentiles of Maximum 3-Day Average Concentrations for CWS #4 Sampled at 4-, 7-, or 14-day Inte rvals ................................................................................... 79

Table 10: Percentiles of Maximum 7- Day Average Co ncentrations for CWS #4 Sampled at 4-, 7-, or 14-day In tervals.................................................................... 80 Table 11: Percentiles of Maximum Daily Co ncentrations for CWS #21 Sampled at 4-, 7- , or 14-day Intervals

............................................................................................... 82

Table 12: Percentiles of Maximum 3- Day Average Co ncentrations for CWS #21 Sampled at 4-, 7-, or 14-day In tervals.................................................................... 83

Table 13: Percentiles of Maximum 7- Day Average Co ncentrations for CWS #21 Sampled at 4-, 7-, or 14-day In tervals.................................................................... 84 Table 14: Percentiles of Maximum Daily Co ncentrations for CWS #30 Sampled at 4-, 7- , or 14-day Intervals

............................................................................................... 86

Table 15: Percentiles of Maximum 3- Day Average Co ncentrations for CWS #30 Sampled at 4-, 7-, or 14-day In tervals.................................................................... 87

Table 16: Percentiles of Maximum 7- Day Average Co ncentrations for CWS #30 Sampled at 4-, 7-, or 14-day In tervals.................................................................... 88 Table 17: Maximum 3-Day Average Concentr ations for All CWS Sampled at 4-day

Intervals ................................................................................................................. 89 Table 18: Single Day Concentrations fo r All CWS Sampled at 7-day Intervals

............ 90 Table 19: Maximum 3-Day Averages for All CWS Sampling Every Day (April thru August); E very 14 Days (balance of year)............................................................. 91 Table 20: 90-Day Averages for All CWS Sa mpling Ev ery 7 Days (April thru August); Every 14 Days (bal ance of year)............................................................................ 92

Table 21: 75th Percentiles and M eans for the Th ree Node Model

............................. 101

Table 22: 75th Percentiles and Means for Sele cted M odels

...................................... 242Page 12 of 263 1. INTRODUCTION

Atrazine – a chlorotriazine herbicide – is currently one of the most widely used agricultural herbicides in the United Stat es, with approximately 70 million pounds of active ingredient applied domestically per ye ar. The Interim Reregi stration Eligibility Document (IRED) for atrazine was fina lized in January 2003 with an addendum added in October 2003 (US EPA 2003). The Agency co mpleted the reregistration eligibility document (RED) process for atrazine in Ap ril 2006. In recent years, numerous experimental toxicology and epi demiologic studies evaluating the toxicity profile and/or mode of action (MOA) of atra zine have become available. In order to consider the

extent to which these new studies ma y impact the Agency’s human health risk characterization for atrazine, the Office of Pesticide Programs (O PP) in collaboration with the Office of Research and Development (ORD) and the Office of Water is in the

midst of a re-evaluation of atrazine and its metabolites’ [deet hyl-atrazine (DEA), deisopropyl-atrazine (DIA), and diam ino-s-chlorotriazine (DACT)]. 1

As part of the re-evaluation of the health e ffects of atrazine, three meetings of the FIFRA Scientific Advisory Panel (SAP) ar e scheduled in 2010. The first of these was held February 2-4, 2010. The Februar y, 2010 SAP meeting focused on a number of

broad scientific issues related to the use of epidemiology and hum an incident data in risk assessment. As part of the February meeting, the Agency presented two case studies involving atrazine. The first ca se study considered se lected epidemiology studies associating atrazine exposure with birth outcomes. The second case study

described a collaborative effort between the A gency and investigators at the Agricultural Health Study ( http://aghealth.nci.nih.gov ) to compare exposure assessment approaches used to evaluate pesticide applicator exposure 2 . At the February SAP, the Agency also presented a draft framework for incorporat ing epidemiology and hu man incident data into risk assessment (USEPA, 2010). Th is draft framework described a proposed approach for developing a weight of the evidence (WOE) analysis using data from multiple sources (e.g., in vitro and in vivo experimental studies along with epidemiology studies) and from multiple levels of biologic al organization. The draft framework relies heavily on existing Agency and OPP guidance docum ents in addition to the concept of problem formulation in the 2009 National Re search Council (NRC) of the National Academy of Science (NAS), “Science and Decisions" and the concept of understanding

toxic ity pathways in the 2007 “Toxicity Testing in the 21st Century: A Vision and A Strategy (2007)”.

OPP’s proposed WOE approach for int egrating epidemiologic data with experimental toxicology studies emphasiz es pharmacokinetics (PK) and MOA as the scientific foundation for evaluating dose-re sponse, species differences, lifestage

1 Henceforth mention of atrazine in this document also infers its chlorinated metabolites (DEA, DIA, and DACT) unless otherwise specified.

2 More detailed information on the Agency’s atrazine case studies from the February SAP meeting can be

found at http://www.regulations.gov (Docket EPA-HQ-OPP-2009-0851).Page 13 of 263 sensitivity, route to route differences, and biological plausi bility of reported outcomes. Specifically, the draft framework describes t he use of modified Bradford Hill criteria like

that in the MOA Framework as a tool to organize, review, and interpret data (U.S. EPA, 1999, 2005; Boobis et al, 2008; Sonich-Mullin et al., 2001; Meek et al., 2003; Seed et al, 2005). Briefly, the concept of MOA involves the identification of a biologically plausible sequence of key events that are obligatory and quant ifiable leading to an adverse outcome. The MOA analysis takes into acc ount factors such as dose-response and temporal concordance, coherence, and consist ency. In this analysis, data from humans and experimental in vitro and in vivo studies can be evaluated together for biological plausibility, to identify areas of confidence in addition to areas of uncertainty and where further research may be helpful. The Agency’s re-evaluation of the human health effects of atrazine is following OPP’s draft framework (USEPA, 2010). Specifically, prior to the fall, 2010 SAP meeting, the Agency will develop a draft WOE analysis for atrazine integrating data and information from epidemiology studies wit h experimental toxicology studies. The draft WOE analysis is also ex pected to include proposed, updated points of departure (PoDs) for ex trapolating risk to atrazine along with an updated evaluation of the potential sensitivity to infants and children and proposed FQPA Safety Factor.

Consistent with recommendations by t he NRC in its 2009 report, OPP’s draft

framework also describes the importance of using problem formulation at the beginning of a complex scientific analysis. The problem formulation stage starts with planning dialogue with risk managers to identify goals for the analysis and possible risk management strategies. This initial dialogue provides the regulatory context for the scientific analysis and helps define the sc ope of such an analysis. The problem formulation stage also involves consideratio n of the available information regarding the pesticide use/usage, toxicological effect s of concern and exposure pathways and duration along with key gaps in data or scientific information. At present time, the Agency is still in the problem formulation stage with respect to both hazard and exposure assessment for atrazine. Agency scientists and risk managers have determined that, consistent wit h the RED, exposure in drinking water continues to be the major exposure pathway of concern for at razine. In addition, it has been determined that the two key goals for the cu rrent re-evaluation are: 1) to determine the extent to

which new science indicates a need for the A gency to develop a new human health risk assessment for atrazine and 2) to re-consider , as appropriate, the frequency of drinking water monitoring conducted by Syngenta, the atrazine registrant, as required by the Agency in the RED. To achieve these goals, so far, the A gency has compiled and reviewed approximately 100 atrazine in vivo and in vitro experimental toxicology studies published since 2003. With respect to drinking water exposure, the Agency is considering the performance of water sa mpling strategies for assessing human exposure from drinking water.

The Agency’s scientific review of toxi cology studies includes experimental toxicology studies availabl e by January 30, 2010. Experim ental toxicology studies available after January 30, 2010 will be integr ated into the Agency’s draft WOE analysis scheduled for review by the FIFRA SAP in fall 2010. Not included in the current issuePage 14 of 263 paper are studies on epidemiology and those evaluating the effect of atrazine on mammary gland development. Epidemiology studies will be evaluated in fall 2010 SAP meeting as part of the WOE analysis. A review of the liter ature on mammary gland development has been deferred to the fall 2010 meeting. Currently , there are several research efforts underway to further characte rize the impact of atrazine exposure, if any, on mammary gland developm ent. The Agency anticipates that these studies will be published in the spring of 2010 . In order to perform an evaluation of these studies together with those already publ ished, the Agency has decided to evaluate them as part of the fall, 2010 SAP m eeting. Thus, the available data (Rayner et al., 2003 & 2005,

Enoch et al., 2007) on the development of the ma mmary gland are not discussed in detail in this issue paper.

This issue paper includes extensive di scussion of the MOA of atrazine—this discussion involves data on the key event s identified in the 2003 risk assessment related to the hypothalamic-pituitary-gonadal axis (HPG) in addition to new data on the hypothalamic-pituitary-adrenal (HPA) axis. As discussed in Section 2 (Previous Human Health Risk Assessment) below, in the 2003 IRED, the A gency identified perturbations of the neuroendocrine system leading to reproducti ve toxicity as the most biologically plausible and sensitive effects attributable to atrazine exposure. Specifically, in 2003, the Agency identified disruption of estrous cyclicity and delays in puberty onset (males and females) occurring as a consequence of di sruptions to the HPG axis as the major endpoints of concern. Sections 3 and 4 (Mammalian Toxicity & Summary of Mammalian Toxicity) of this paper describe how the complex interaction between the

HPA and HPG axes leads to a host of neur oendocrine-related effects – it is this

cascade of events that are believed to result in the most sensitive effects of atrazine,

i.e. ,reproductive and developmental effects. The Agency believes that neuroendocrine related reproductive and developmental effects continue to be most important toxicological endpoints of concern for atrazine.

This issue paper discusses new dat a on the immune and nervous systems, prostate, and the enzyme aromatase (Section 3, Mammalian Toxicity) . Evidence of neurotoxicity and immunotoxicity either occur at doses higher than those eliciting the reproductive/developmental e ffects or are related to t he neuroendocrine perturbations described by the MOA. The only tumorigenic response reported in the atrazine data base is the mammary gland tumors seen in Sprague Dawley rats after chronic exposure

to atrazine. This tumor res ponse is not considered relevant to humans as supported by the 2000 SAP review. Reproductive aging in the rat and human are driven by two

different series of events. In rats, atrazine exposure le ads to an early reproductive senescence marked by increases in estrogen and prolactin. Constant stimulation by these hormones, in turn, results in mamma ry tumor development. On the other hand, human reproductive senescence (menopause) is marked by a decrease in estrogen levels. Thus, the stimulus needed to cause the proliferative process in the mammary gland is not present. In the males, atra zine induces prostatitis and early postnatal exposure is a critical window of susceptibility to this effect. Atra zine exposure had also been linked to changes in aromatase activity in vitro. However, no evidence of atrazine-Page 15 of 263 induced changes in aromatase have been seen in vivo. Reported effects on the prostate occur at doses higher than t hose associated with the neuroendocrine-related alterations described by the MOA.

It is prudent for the Agency to evaluate the hazard potential of atrazine and the drinking water monitoring strat egy together. Namely, the frequency of drinking water monitoring is related to the temporal pattern of the toxicologica l endpoint of concern used for risk assessment. Generally, longer dur ations of toxicological concern (e.g., a long-term chronic effect) require a less frequent sampling design to approximate longer term exposures. However, as the durati on of concern shortens, the frequency and timing of sampling become more important in determining how well the sample data capture these short-duration exposures. Depending on the aquatic resource being sampled, the likelihood increases that a peak exposure may be missed by less frequentsampling. This is most critical when the Agency is concerned about acute or very shortterm effects resulting from a single day (peak) or only a few days of exposure.

Section 5 (Approaches for Evaluating Wate r Sampling Strategies & Frequency of Monitoring) considers several approaches for examining the issue of monitoring frequency for assessing exposure durations of interest in human health risk

assessments. Two methods are discussed in greater detail than the others. The first approach discussed in detail l ooks at upper percentiles of single day estimates and maximum running averages of several days’ duration and compares the performance of different sampling strategies for deriving esti mates of the “true” c oncentrations for these durations of interest. The second appr oach discussed in detail offers a modeling approach that can be applied to pesticide o ccurrence data and combined with different sampling strategies to evaluate their per formance in meeting various data quality objectives.

The current drinking water monitori ng frequency being conducted by Syngenta and required by EPA, involves weekly sa mpling during the growing season and biweekly sampling for the remainder of the year. As discussed in Section 5 , preliminary indications provided in this issue paper sugg est that the current monitoring frequency is sufficient to assess the 90-day rolling averag e used in the most recent human health risk assessment (IRED, 2003). For the risk assessment, the 90-day rolling average was selected to correspond to: (i) the period within the year during which most peak concentrations are likely to occur; and (ii) the most sensitive effect (LH surge attenuation) occurring 4-5 months after daily dos ing in a 6 month toxicity study. This combination represented the mo st conservative pairing of exposure duration with LH

effects as seen in the available animal studi es. The previous atrazine human health risk assessment was based on a strong science f oundation, supported by significant external peer review, and represented the stat e-of-the-science at the time. The 90-day rolling average was believed to be a health pr otective duration of exposure given the

state-of-the-science for atrazine prior to 2006. As such, t he drinking water monitoring frequency required by the Agency in the RE D was appropriate for protecting health given that knowledge.Page 16 of 263 As part of the re-evaluation of atrazine, the Agency is placing particular emphasis on evaluating the PK time course and temporal pattern of key events in the MOA in addition to all other reported toxicity endpoints. At this time, the Agency has not made any conclusions on the appropria te duration of toxicological concern given the new science or the appropriate frequency of drinki ng water monitoring. However, if the current re-evaluation leads to a shortening of the duration of toxicological concern from 90 days to a shorter duration (e.g., 3 or 7 days), then more frequent monitoring than the current sampling frequency may be needed to accurately assess atrazine concentrations. This issue paper concludes in Section 6 (Implications of MOA & Mammalian Toxicology Findi ngs on Sampling Frequency) with a discussion how new knowledge of the MOA of atra zine may potentially impact drinking water monitoring frequency. The Agency will be soliciting comment from the SAP at t he April meeting on the temporal aspects of atrazine toxicity and on the proposed statistical approaches for evaluating drinking water monitoring. The A gency will use the input from the SAP at the April meeting on these im portant science issues to inform fu rther review and analysis in preparation for the fall 2010 meeting.

This science issue paper describes the status of the Agency’s effo rts to review of the experimental toxicology literature and to evaluate drinking water monitoring strategies. As such, the April SAP meeti ng on atrazine will focus on scientific issues related to the toxicity, MOA, and drinking water exposure profile.

2. 2003 HUMAN HEALTH RISK ASSESSMENT

The 2003 human health risk assessment for atrazine is complex and incorporated significant data developm ent, regulatory evaluation, and FIFRA SAP review. The Agency has sought advice fr om the SAP on human health issues during three previous meetings (FIFRA SAP, 1988, 2000, 2003). Atrazine was first taken to the SAP for evaluation of rat mammary gl and tumor response seen in adult cancer studies in 1988 (FIFRA SAP, 1988). At that time, the SAP not ed that a “hormonal influence” might be an important considerat ion in the development of these mammary gland tumors in adult rats. The Agency retu rned to the SAP in 2000 (FIFRA SAP, 2000) for advice on atrazine’s MOA leading to mammary gland tumors, reproductive and developmental effects in rats , as well as the human relev ance of these findings.

The SAP agreed with the Agency’s proposal for atrazine’s neuroendocrine MOA, and they further concluded that it is unlikel y that the mechanism by which atrazine induces mammary tumors in adult female Sp rague Dawley rats could be operational in humans. Nevertheless, the SAP further concluded that it is not unreasonable to expect that atrazine might cause adverse effects on hypothalamic-pituitary function in humans if exposures were sufficiently high and that the MOA was relevant for developmental and reproductive effects (FIFRA SAP, 2000). Based on the 2000 SAP guidance, EPA changed its position on atrazine and reclassified it from a “possible carcinogen” to “not likely to be carcinogenic in humans”.Page 17 of 263 In 2003, the Agency presented its evaluatio n on prostate cancer to the FIFRA SAP. At that meeting, the SAP evaluat ed the available epidemiology studies suggesting an association between atrazine and prostate cancer in workers at an atrazine manufacturing plant in St. Gabr iel Louisiana. The SAP concurred with EPA’s conclusion that the data did not suggest an association between atrazine exposure and prostate cancer, but that the in crease in incidence of prosta te cancer in workers at the plant could likely be explained by the in crease in Prostate Serum Antigen (PSA) screening at the plant (FIFRA SAP, 2003). T he SAP also stated that a more thorough and systematic review of the biologic and epi demiologic literature on the topic of the potential or observed carcinogenic effects of atrazine exposure was required before concluding that atrazine exposure is an unlik ely explanation for at least part of the excess of prostate cancer in the St. Gabr iel manufacturing plant. Review of the epidemiologic studies with atrazine is on- going and will be consider ed as part of the draft WOE analysis and review ed by the SAP in fall 2010.

The 2003 EPA human health risk assessment identified non-cancer effects of atrazine following acute and chronic diet ary exposures, short- and intermediate incidental oral exposures, and short- , intermediate-, and long-term dermal and inhalation exposures. Effects observed in developmental toxicity studies ( i.e. , delayed

ossification of certain cranial bones in fetuses, and decreased body weight gain in adults that are not likely due to atrazine’s MOA but rather to general toxicity) were selected as the endpoint of concern for ac ute dietary exposure (NOAEL/LOAEL of 10/70 mg/kg/day). Delayed prepu tial separation in male rats (which is associated with atrazine’s neuroendocrine MOA) wa s the basis for assessing the short-term oral, dermal and inhalation exposures (NOAEL/L OAEL of 6.25/12.5 mg/kg/day). Effects indicative of disruption of hypot halamic function ( i.e. , attenuation of pre-ovulatory luteinizing hormone surge) were determined to be appropr iate toxicity endpoints of concern for chronic dietary, intermediate incidental oral, and intermediate- and long-term dermal and inhalation exposures (NOAEL/LO AEL of 1.8/3.65 mg/kg/day).

The review of atrazine described in this issue paper focuses on the toxicological

and in vitro data generated since the IRED was fi nalized in 2003 and thus builds upon the findings and conclusions drawn by Agency scientists at that time. The following

section will describe the evol ution of the stat e-of-the science since the IRED was finalized in 2003. Furthermore, it will descr ibe how these developments have enhanced our understanding of the MOA that serves as the basis for the atrazine risk assessment. This issue paper does not propose updated PoDs for use in extrapolating human risk to atrazine; these values will be included in the draft WOE scheduled for review by the SAP in September, 2010. The Agency is, how ever, soliciting comment from the Panel on important scientific issues which will help inform the selection of such updated PoDs.Page 18 of 263 3. Mammalian Toxicity

The current evaluation has investigated t he array of adverse effects reported in the literature over the past 7 y ears including reproductive/devel opmental toxicity in both males and females, neurotoxicity, immunotoxic ity, carcinogenicity and most recently

insulin resistance and mitochondrial function impairment as well as the hypotheses on atrazine’s mode of toxicity put forth by num erous researchers. In the 2003 IRED, the Agency identified perturbations of the neuroendocrine system leading to reproductive toxicity as the most biologically plausible and sensitive effects attributable to atrazine exposure. These adverse outcomes include di sruption of estrous cyclicity and delays in puberty onset (males and females) occurring as a consequence of disruptions to the

HPG axis. The other toxicities that have been implicated in atrazine-exposure ( e.g

. neurotoxicity, immunotoxicity) occur at doses higher than those eliciting the reproductive/developmental effe cts. Although it is reasonabl e to postulate that these effects may be related to the neuroendocrine pertu rbations described in the MOA, there are no atrazine specific data to conclusively establish this.

The current evaluation of the post-2003 data, supports the neuroendocrine MOA and key events identified in the 2003 IRED but also expands our understanding byidentifying another disrupted pathway in the MOA: activation of the hypothalamicpituitary-adrenal (HPA) axis manifested as incr eases in corticosterone levels. Activation of the HPA axis in turn leads to perturbati ons of the HPG axis. It is this cascade of

events that results in the effects on LH and consequent reproductive and developmental effects observed after atrazine exposure. Howe ver, in the case of the aromatase data,

while there are indications that aromatase activity increases in vitro there is no evidence that atrazine alters aromatase activity following in vivo exposure. Andrology experiments also fail to demonstrate a link between atrazine exposure and adverse reproductive performance.

As was the case in the 2003 assessm ent, the new data on neurotoxicity and immunotoxicity indicate that effects reported after atrazine exposure are either linked to or occur at higher doses than the neuroendocrine perturbat ions in the MOA. No compelling evidence of structural or functional deficits was observed in the neurotoxicity. For carcinogenicity, none of the new data suggest that atrazine exposure contributes to tumor progression or that it makes the tumors already present in the system more aggressive. With regards to the suggestion that atrazine exposure may lead to mitochondrial dysfunction, insulin resistance, and perhaps obesity, there is only one published article suggesting this hypothesis. This article, however, is significantly

flawed with numerous inconsistencies and deficiencies in the reporting and interpretation of the data thus renderi ng it of limited utility in understanding the toxicological profile of atrazine.

In conclusion, the research conducted in the last seven years supports the

neuroendocrine MOA identif ied in the 2003 IRED. The cr itical and most sensitive

adverse effects continue to be related to the reproductive consequences ofPage 19 of 263 perturbations to the HPG axis. The new data, however, point to a connection between activation of the HPA axis and HPG axis per turbations. From t he available data, it appears that HPA activation occurs shortly after atrazine exposure. However, the toxicological significance of this event rema ins unclear. In order to better characterize the temporal and dose-response relationship of changes in various events in the HPA and HPG axes and potential adverse health outcomes, it is critical to consider internal dosimetry and the temporal relationship between atrazine exposure and observable physiological effects. The following se ction will describe the most recent pharmacokinetic data available. This will be followed by a description and discussion of atrazine’s operative neuroendocri ne MOA (including HPA axis activation) in the context of the pharmacokinetic data. This will, in turn, se rve to frame the discussion and description of the various toxic effe cts observed after atrazine exposure.

3.1 Pharmacokinetics: Internal Dosi metry, Metabolism, & Time Course

Upon entering the body via the oral route, atrazine is qu ickly metabolized to its dealkylated chlorinated metabolites deethyl- atrazine (DEA), deisopropyl-atrazine (DIA), and diamino-s-chlorotriazine (DACT) as illustrated in Figure 1. Following a single dose of 90 mg/kg atrazine administered via oral gavage, atrazine was barely detectable at 24-h in rat plasma while DACT was the pr edominant species accounting for >98% of total chlorotriazine levels (McMullin, 2003). A plasma half-life of about 11 hours has been estimated for atrazine in rats (Timchalk, 1990). Oral administration of atrazine in rat toxicity studies has resulted in a double- peak behavior in plasma-time courses that

have been attributed to discontinuous absorption from limited solubility of atrazine in the gut (McMullin, 2007). The primary routes of excretion have been identified to be the urinary and fecal excretion, accounting for about 67 and 18% of administered dose, respectively (Timchalk, 1990).Page 20 of 263 Figure 1: Atrazine and its chlorinated metabolites

Efforts to develop a physiologically bas ed pharmacokinetic (PBPK) model for atrazine using rat information have focused on the liver as the primary site of metabolism and brain as a potential target organ for toxicity. An estimated 1.5 % of the administered dose has been reported to be bound to hemoglobin in red blood cells, although the specific interactions have not been characterized (Timchalk, 1990 and McMullin, 2003).

More detailed disposition studies hav e been carried out in mice. Ross et al.

examined the metabolism and dis position of atrazine in mice and how tissue levels may

correlate with levels of atrazine and its me tabolites found in plasma and urine (Ross, 2009). Plasma levels of atrazine exhibited a biphasic behavior similar to that observed in rats with maximum levels seen at 0.5 hours post dose. Atrazine levels were below the limit of detection by 24 hrs. In contrast, le vels of DACT, the major metabolite identified (~ 10-fold higher levels than mono-dealkylated metabolites), peaked at 2 hrs at all dose

levels. The clearance of DACT was dose-dependent and relatively slow during the first 24 hours (plasma half-lives of 7.5 and 42 hrs for 5 and 250 mg/kg bw atrazine, respectively). When plasma area under the curves (AUCs) for DACT were plotted versus atrazine dose, a significant linear correlation was observed (r 2 = 0.987). In urine, DACT levels were 2 orders of magnitude hi gher (as high as 5400 μM) than atrazine or its mono-dealkylated metabolites in the 0-24 hr time period. In liver and kidneys,

atrazine and its metabolites were detectabl e at 4 hrs. DACT was the most abundantPage 21 of 263 species followed by atrazine. Lower levels of each compound were found in the kidneys as compared to the liver. Significant levels of DACT (as much as 50 μM at the highest dose) were found in the brain of mice at 4 hours along with low levels of atrazine (~1 μM at the highest dose). Atrazine wa s still detectable in the brain at all dose levels at 24 hrs, while DACT levels remained high at this time point. At the low doses of 5 and 25 mg/kg, maximum levels of atrazine (~2-3 μM ) were detected in the spleen and thymus at 1 hr, whereas at the two highest doses (125 and 250 mg/kg atrazine), maximum

ATRA levels were seen at 4 hrs. Peak le vels of DACT, the major species detected,

were seen at 4 hrs at the tw o highest doses of atrazine.

Stoker et al. (2007) examined the potential lactational exposure of atrazine in the developing suckling rat neonate following a si ngle oral gavage dose of 2 or 4 mg/kg radiolabeled atrazine to the lactating dam on postnatal day 3. Following 2 1/2 hours post-dose, the neonates were allowed to nurse for 30 minutes. At t he end of the nursing period, radioactivity was measured in differ ent tissues of the dam and in the stomach and brains of the pups. Radioactivity accounting approximately for 0.007% and 0.0002% of the administered dose was measured in the pup stomachs and brains, respectively. In the dam (at 3 hours post-dose) , the highest levels of radioactivity were found in the liver and kidneys, and only very low levels (0.003%) were present in the

brain.

The kinetic profile of this pesticide is cr itical to understanding its toxicity profile. Particularly, in light of recent data indicati ng that corticosterone levels can be affected within 15 minutes of atrazine exposure. Thus, the rapid absorption and metabolism demonstrated in the majority of the kinetic da ta are consistent with the apparent time-toeffect profile seen throughout the database t hat are key components of the temporal concordance analyses supporting t he neuroendocrine MOA for atrazine.

3.2 Mode of Action (MOA)

3.2.1 Background

In describing and analyzing a MOA for any chemical, the Agency applies a framework for organizing and anal yzing the available data. This framework developed by a working group convened by the Internati onal Program on Chemical Safety (IPCS) takes a systematic approach to analyzing the data (Boobis et al. , 2008). This process allows for a more transparent assessment of a chemical’s pot ential to elicit an adverse effect. The first step in this approach is to a scertain if it is possible to establish a MOA by describing the key events – including an analysis of the dose-response and temporal

concordance of the events – that lead to the adverse outcome. Following the description of the key events, the data are scrutinized to determine the strength, consistency, and specificity of the relati onship between the key events and the toxic response. Finally, the data are analyzed to det ermine if the key events are biologically plausible in humans both on a qualitative and quantit ative basis. Once the data have been subjected to this evaluat ion, other potential MOAs c onsidered and shown to bePage 22 of 263 unlikely, the analysis culminates in a statement of confidenc e, analysis, and implications that clearly articulates the uncertainties, st rengths, weaknesses, and implications of the analysis.

3.2.2 2003 Key Events – HPG axis

The review of atrazine described in this issue paper focuses on the toxicological

and in vitro data generated since the IRED was finalized in 2003. In the 2003

evaluation of atrazine, the disruption of the LH surge was used for setting the points of departure for the intermediate and chronic assessm ents. Similarly, t he effect of atrazine on the neuroendocrine control of reproduction was considered a key step in the atrazine-induced delay in pubertal devel opment in the both sexes (Stoker et al. , 2000;

Laws et al. , 2000) and the disruption of prostate f unction in the male offspring when the dam is exposed immediatel y following birth.

The goal of this new analysis is to a scertain if the new data impact the conclusions drawn in atrazine’s previous risk assessment (2003). In itially postulated to elucidate the physiological events and endocrine changes l eading to mammary tumor formation in the Sprague-Dawley (SD) rats, the operative MOA for atrazine involves a series of key events that ultimately lead to early reproductive senescence in SD rats resulting in mammary gland tumor developmen t. The key events described in the 2003

IRED are:

• Hypothalamic effects resulting in changes in catecholamine function and regulation of the pulsat ile release of GnRH.

• Attenuation of the LH surge

• Cessation of ovulation with the ens uing persistent release of estrogen

• Increased prolactin release by the pituitary as a secondary consequence resulting from the el evated estrogen levels

• Prolactin and estrogen-induc ed proliferative processe s in the mammary gland leading to tumorigenesis.

In addition to identifying the key events in the MOA, a dose-response and time concordance analysis was also part of the 2003 evaluation. The attenuation of the LH surge was observed at a dose of 3.65 mg/kg/day after 26 weeks of exposure (Morseth et al., 1996c). The dose-response and time-to-effe ct profile for the LH surge attenuation closely parallel the profile for estrous cycl e disruption and ultimately mammary tumor formation which occurred after two years of atrazine exposure at a dose level of 3.1 mg/kg/day. Therefore, in general t here was good dose- and time- concordance between the key events in the MOA and mammary tumors as well as the LH surge attenuation and reproductive effects with the key events occurring at doses ≤ to those

where these effects were observed.Page 23 of 263 One of the objectives of the current review is to ascertain if the neuroendocrine MOA leading to disruption of hypothalamic-pituitary-gonadal (HPG) axis described in 2003 is still atrazine’s primary mode of action leading to adverse consequences in light of the new data produced over the last 6-7 years. The lines of evidence that have emerged from the new data support the neuroendocrine MOA and its key events but also identify important atrazine-induced ch anges in HPA axis namely increases in corticosterone levels. It is proposed that the activation of the HPA axis and its subsequent impact on the HPG axis provide a potential point of convergence for most –

but not all – the toxic effects associat ed with atrazine exposure ranging from reproductive effects, and perhaps to neurotoxic and imm unotoxic effects.

3.2.3 Effect of Atrazine on th e hypothalamic-pituitary-gonadal

function.

Several investigators have reported t hat atrazine impairs the neuroendocrine regulation of ovarian function. The disruption of ovarian cycles has been attributed to an atrazine-induced interruption of the ovulatory surge of luteinizing hormone (LH) that occurs on the day of vaginal proestrus (Cooper et al. , 1996; Eldridge et al. , 1999; Cooper et al. , 2000; Cooper et al. , 2007, McMullin, 2004; Foradori et al. , 2009). During the last decade, a number of studies were co mpleted attempting to further characterize the cellular and neuroendocrine changes responsib le for how atrazine interferes with the regulation of LH secretion. T he preponderance of evidence provides added support for the hypothesis that atrazine targets the brain to modify the neuronal control of pituitary function. For example, eval uating the secretion of gonadotropin releasing hormone (GnRH) in rats revealed that atrazi ne alters the release of this neuropeptide

from the brain. GnRH is essential for LH re lease from the pituitary (Cooper et al., 2007,

Foradori et al. , 2009a).

Importantly, the HPG axis in humans and rodents is maintained by the activity of GnRH neurons, whose cells are distributed th roughout the preoptic region including the medial preoptic area (mPO A) of the anterior hypothalamus (Rivest and Rivier, 1995). The nerve terminals of these neurons projec t to the median emi nence, where GnRH is released into the hypophysial portal circulation. The pulsatile secretion and the ovulatory surge of LH depend on the activity of GnRH neurons (Kal ra and Kalra, 1983).

Research from two laboratories has shown t hat atrazine interferes with the pattern and amplitude of GnRH release by slowing the frequency of the pulses and lowering the overall amount of GnRH released into the portal system (Cooper et al. , 2007; Foradori

et al. , 2009). In contrast, the ab ility of the pituitary LH-secreting cells (gonadotrophs) to respond to GnRH does not appear to be affected by atrazine, either in vivo or ex-vivo (Cooper et al. , 2000; Foradori et al. , 2009b). Further evidence supporting this observation comes from the work done on pigs demonstrating that a single intravenous injection of GnRH (100 μ g) was able to partially reverse or counteract atrazine-induced suppression of the LH surge (Gojmerac et al., 2004). There have been some reports indicating that atrazine, or its metabolites, may interfere directly with GnRH activity ( e.g.

, Zorrilla et al. , 2010) as well as the activity of other neurons (Das et al. , 2001). WhilePage 24 of 263 these new data enhance our under standing of the processes involved in the HPG axismediated attenuation of the LH surge and subsequent toxic effect s, they do not – at this time – suggest a lower effect level than the one used in the 2003 IRED.

Table 1: HPG Axis Perturbations: Summary of Temporal and Dose

Concordance

Axis Key Event Time Dose (NOEL/LOEL) Reference
Hypothalamus ↓ GnRH release 4 days N.A./25 mg/kg/day ‡ Cooper et al.

2007

1 day N.A./300 mg/kg/day Cooper et al

. 2000

3 days N.A./50 mg/kg/day Cooper et al.

2000

4 days N.A./6.25 mg/kg/day Cooper et al.

2007 Pituitary

Attenuation of LH surge

26weeks 1.8/3.65 mg/kg/day Morseth et al.,

1996c

1-day N.A./ 300 mg/kg/day Cooper et al. (2000)

21 days N.A./75 mg/kg/day Cooper et al. (1996) HPG Axis

Ovary Estrous cyclicity disruption

26 weeks 1.8/3.65 mg/kg/day Morseth (1996c)

‡ NOEL not available

3.2.4 2010 Key Events – HPA axis

The physiological interaction between the hormones in the HPA axis ( e.g., corticotrophin releasing hormone (CRH) and adrenal corticoids) and hormones in the HPG axis ( e.g., GnRH and LH) was described by Rivier and Rivest (1991) and Kirby et

al. (2009). The key events in the acti vation of the HPA axis include:

• Hypothalamic changes resulting in an increase in CRH release

• Increased release of adrenocorticotropi n hormone (ACTH) from the pituitary

• Increased production of coticoster one and progesterone by the adrenals

• Decrease in GnRH release from the hypothalamus as a result of one, or all of the above, noted HPA changes.

Given that decreased GnRH release from the hypothal amus is one of the key

events in atrazine’s neuroendocrine MOA, it is plausible that activation of the HPA axis may play a role in atrazine’s neuroendocrine MOA. In vitro and in vivo studies conducted over the last seven years support this hypothesis (Sanderson et al., 2000 &Page 25 of 263 2001; Laws et al. , 2009; Fraites et al. , 2009; Pruett et al. , 2003; Holloway and Foster; and Tinfo and Laws, 2009).

3.2.5 Effect of Atrazine on th e regulation of the hypothalamicpituitary-adrenal axis

Research in male and female rats (Laws et al. , 2009; Fraites et al. , 2009) and male mice (Pruett et al. , 2003) has shown that atrazi ne and its metabolites DIA and DEA (but not DACT) have a clear effect on t he secretion of pituitary ACTH and adrenal steroid hormone secretion ( i.e. , corticosterone and progesterone ). In the rat, this response is rapid ( i.e. , blood concentrations are signi ficantly increased within 15 minutes after oral gavage) after exposure to either 75 mg/kg/day of atrazine or 60.2 mg/kg/day DIA [exposure to 50.2 mg/kg/day of DACT had no effect on pituitary-adrenal hormone release]. The corticosterone and progesterone response was observed after each daily dose for at least four days in the female (Fraites et al. , 2009) and for at least 21 days in the male (Laws et al. , 2009) indicating that the animal does not habituate to this exposure. Furthermore, this respons e occurred following either gavage of atrazine

or an intravenous administration of DIA (7.8 mM solution) (Fraites et al. , 2009). These observations strongly suggest that the atrazi ne-induced endocrine response is a direct, chemical-induced change in the hypothalamic-pituitary-adrenal tissue and not the result of a non-specific gastrointestinal stress re sponse. Indeed, it has been reported that atrazine, DIA and DEA (but not DACT) can in crease the synthesis of steroid hormones in vitro, including progesterone, cortisol, estradiol and estrone in human adrenocortical carcinoma cells (H295R) and granulosa cell s obtained from humans or rats further supporting the argument t hat the chlorotriazines increase steroidogenesis at the cellular level (Sanderson et al., 2000 & 2001; Holloway and Foster; Tinfo and Laws, 2009).Page 26 of 263 Table 2: HPA Axis Activation: Summary of Temporal and Dose

Concordance

Axis Key Event Time Dose (NOEL/LOEL) Reference

Hypothalamus ↑ CRH Not measured

15 minutes N.A./50 mg/kg ‡ Laws et al.,

2009

15 minutes N.A./75 mg/kg

atrazine 60.2 mg/kg DIA Fraites et al.,

2009

1 hour N.A./200 mg/kg

atrazine Pruett et al.,

2009

6 hours N.A./100 mg/kg

atrazine Pruett et al.,

2003 Adrenals ↑ progesterone,

corticosterone

4 days N.A./12.5 mg/kg/day atrazine or 10 mg/kg/day

DIA Fraites et al.,

2009

15 minutes N.A./75 mg/kg

atrazine 60.2 mg/kg DIA Fraites et al.,

2009 Pituitary ↑ ACTH 4 days N.A./12.5 mg/kg/day atrazine or 10 mg/kg/day

DIA Fraites et al.,

2009 HPA Axis

Hypothalamus ↓ GnRH release 4 days N.A./25 mg/kg/day Cooper et al.

2007

‡ NOEL not available

3.2.6 Linking the hypothalamic-pit uitary-adrenal and hypothalamic pituitary-ovarian responses.

The studies described in the preceding sect ions demonstrate a clear disruption of both the brain-pituitary-gonadal and brain-pituitary-adrenal function. The fact that both these important neuroendocrine ax es are disrupted, often wit h a similar dose response, has led to the hypothesis that the mode of action (MOA) for the loss of reproductive function begins with an atrazine-induced disrupti on of the pituitary-adrenal axis (Fraites

et al. , 2009; Laws et al. , 2009). In this case, atrazine or its metabolites influence these two axes sequentially rather than in parallel with the changes in adrenal hormone secretion driving many of the changes initially observed in the gonadal axis. The evidence behind this hypothesis includes the following:

Time to effect : The increase in pituitary ACTH and adrenal hormone secretion occurs immediately after a single dose whereas significant changes in pituitary-gonadalPage 27 of 263 function, especially the LH surge, requires a longer exposure time ( e.g. , three daily doses) (Cooper et al., 2000 and 2007). Furthermore, t he longer exposure regimens typically have a lower LOAEL (Figure 2). In fact, the current LOAE L for the effect of atrazine on the LH surge is based on a 6-month exposure period.

Figure 2: Relationship Between Duration of Exposure and No Observed Effect

Levels

a Cooper et al., Foradori at al., and Morseth et al. NOAELs based on suppression of LH and prolactin surge. Stoker et al. NOAEL based on delay in preputial separation. (From Simpkins, presentation to EPA, Jan 2010).

Adrenal steroids impair the LH surge :

The influence of the adrenal hormones on the function of t he pituitary-gonadal axis is well documented. Thus, it should not be surprising that atrazine, through its stimulatory effect on the adr enal axis, can impair control of reproductive development and function. Consider the following:

• Activation of the pituitar y-adrenal axis has short-term and long-term effects on many aspects of reproductive physiolog y and behavior in a variety of species, including rats and humans (Selye, 1939; Rivier and Rivest, 1991).

• In the rat, repeated increases in cortic osterone affect the gonadal axis at the level of the brain, pitu itary and gonads including:Page 28 of 263 o Brief increases in ACTH and corticosterone for four days induced by other means ( i.e. , restraint stress for 5 minutes) at 0900 h suppresses

LH secretion to the same extent t hat atrazine or DIA administered at 0900 h does (Fraites et al. , 2009).

o Increases in corticosterone alter t he activity of the neurotransmitter and neuropeptide of those neurons controlling GnRH (Kirby et al. , 2009), including an increase in gonadotr opin inhibiting factor, CRH and norepinephrine all of which are known to decrease GnRH pulses.

o Decreased pituitary gonadotropin response to GnRH (Rivier and Rivest, 1991)

o Decreased steroidogenesis within the gonads ( e.g. , activation of the glucocorticoid receptors within t he Leydig cell are known to lower testosterone synthesis, 11 ß-HSD is protective, Gao et al. , 1996).

The postulated nexus between activation of the HPA axis and perturbations of

the HPG axis is summarized in Table 3. Fi gure 3 is a graphical representation of the sequence of events leading to t he disruption of estrous cyc licity that is the most

sensitive adverse health outcome attributed to atrazine exposure. Although, it is wellunderstood that a continuous 3 to 4 day elevation of corticosterone leads to perturbations of the HPG axis, the toxicologi cal relevance of this rapid increase is currently unclear ( i.e., at what duration and at what doses does this perturbation lead to an adverse outcome). Current ly, the Agency is in the process of conducting BMD analyses for these endpoints to more clearly define the dose-response relationship between atrazine exposure and the observed e ffects. Unlike LOELs which reflect different levels of response and are limited by dose selection, BMD analysis will allow the Agency to more accurately illustrate the dose-response relationships as they relate to exposure durations.

This re-evaluation attempts to dete rmine the link, if any, between atrazineinduced activation of the HPA axis and the su ite of toxicological effects reported after atrazine exposure. To that effect, a brief summary of the various toxic effects observed after atrazine exposure follows.Page 29 of 263 Table 3: HPA/HPG Axes Nexus: Summary of Temporal and Dose Concordance

Axis Key Event Time

Concordance Dose Concordance (LOEL) Reference

Hypothalamus ↑ CRH Not measured

15 minutes N.A./75 mg/kg

atrazine ‡
60.2 mg/kg DIA Fraites et al.,

2009 Pituitary ↑ ACTH

4 days N.A./12.5 mg/kg/day

atrazine or 10 mg/kg/day DIA Fraites et al.,

2009

15 minutes N.A./50 mg/kg Laws et al., 2009

15 minutes N.A./75 mg/kg

atrazine 60.2 mg/kg DIA Fraites et al.,

2009

1 hour N.A./200 mg/kg

atrazine Pruett et al.,

2009

6 hours N.A./100 mg/kg

atrazine Pruett et al.,

2003 HPA Axis

Adrenals ↑ progesterone,

corticosterone

4 days N.A./12.5 mg/kg/day

atrazine or 10 mg/kg/day DIA Fraites et al.,

2009

HPA/HPG

Axis Hypothalamus ↓ GnRH release 4 days N.A./25 mg/kg/day Cooper et al.

2007

1 day N.A./300 mg/kg/day Cooper et al

. 2000

3 days N.A./50 mg/kg/day Cooper et al.

2000

4 days N.A./6.25 mg/kg/day Cooper et al.

2007 Pituitary

Attenuation of LH surge

26weeks 1.8/3.65 mg/kg/day Morseth et al.,

1996c

1-day N.A./ 300 mg/kg/day Cooper et al. (2000)

21 days N.A./75 mg/kg/day ‡ Cooper et al. (1996) HPG Axis

Ovary Estrous cyclicity disruption

26 weeks 1.8/3.65 mg/kg/day Morseth (1996c)

‡ NOEL not availablePage 30 of 263 Figure 3: PROPOSED MOA SCHEMATIC

↓ LH
↓ prolactin Pituitary Steroidogenesis

↓ GnRH pulses

(Catecholamines Neuropeptides) CRH/AVP

(Catecholamines Neuropeptides) Adrenal ♀ and ♂

↑ Progesterone

↑ Corticosterone

Gonad ♀ Δ E strogen

Δ Progesterone ♂ Δ Testosterone ? CNS Atrazine

1

5 6

4 2

ACTH ↑

Altered Reproductive

Function 7

3

Figure 3: Schematic depicting the proposed mode of action of the chlorotriazines on reproductive function. The key events in this MOA are noted by the numbers. (1) Following a single exposure, atrazine has a direct effect on the neuronal pathway s regulating (2) pituitary ACTH release and (3) increasing adrenal steroid hormone secr etion. The feedback of the adrenal steroids (corticosterone and perhaps progesterone) on the brain (4) dampen the puls atile release of GnRH lowering the release of LH (5) and (6) altering gonadal function. (7) There is also evidence that exposure to atrazine and the two monodiaklyated metabolites (DIA and DEA) may directly increase steroid hormone both in vivo and in

vitro

.

3.3 Reproductive/Developmental Toxicity

Exposure to atrazine and/or its metabo lites has been linked to a number of effects on reproductive function and development including delays in puberty onset in both male and female rats and early reproductive senescence in females. These

effects are consistent with disruption of the HPG axis. This section will briefly summarize the toxicological data pertaining to atrazine-induced effects on reproductive performance and development and thei r association with the MOA.

3.3.1 Female Reproductive/Developmental Toxicity

Over the last seven years, studies have been conducted to evaluate the impact of atrazine and/or its by-p roducts on the development a nd reproductive health ofPage 31 of 263 females. 3 In 2003 Laws et al., evaluated the effects of DACT and hydroxyatrazine (a plant and bacterial metabolite of atrazine) on the pubertal development of female Wistar rats. This study is a follow-up to the res earchers’ previous fi ndings that 20 days of exposure (PND 22-41) to the parent compound – atrazine – at doses ≥ 50 mg/kg/day induced a delay in female sexual maturation manifested as a delay in vaginal opening (VO) (Laws et al., 2000). DACT exposure at high doses ( ≥ 34 mg/kg/day) from postnatal day PND 22-41 resulted in significant delays in VO (3-7 day delay) and in some instances animals failed to attain VO by t he time of their scheduled sacrifice on PND 41. Interestingly, these delays in VO were not accompanied by lower body weights thus they do not appear to be related to generaliz ed growth delays. Moreover, the NOAEL identified for DACT is the same as equimo lar equivalent NOAEL for atrazine. In contrast, hydroxyatrazine exposure at doses up to 183 mg/kg/day did not result in delays in VO. Given that atrazine is qui ckly metabolized to DACT, these results strongly suggest that DACT is the toxicologically active mo iety causing the delays in sexual maturation induced by atrazine.

Potential effects of atrazine on ovarian function were evaluated over a wide dose range (3-300 mg/kg/day) by Shi bayama and colleagues (Shibayama et al., 2009). In a companion study these researchers attempted to correlate atrazine-induced changes in ovarian function to fertility impairments. Unfo rtunately, different dose levels were used for the ovarian function and fertility studies thus precluding an appropriate comparison

between these two sets of ex periments. In the ovarian function set of experiments, atrazine exposure for 2 or 4 weeks at a high dose (300 mg/kg/day) resulted in irregular estrous cycles (typically longer cycles) due pr imarily to a lengthened diestrus. This effect was accompanied by decreased numbers of corpora lutea, follicular atresia, uterine atrophy, as well as decreased ovaria n and uterine weights. Noticeably, the duration of atrazine exposure (2 vs. 4 weeks) had no effect on the nature, severity, or dose level causing the estrous cycle disrupti on or the histopathology changes. In the fertility study, atrazine exposu res at levels up to 100 mg/kg/day for a period of time encompassing 2 weeks prior to mating up to ges tation day (GD) 7 did not result in any signs of impaired fertility. None of the si gns typically associated with impaired fertility ( e.g., number of implantation, co rpora lutea, pre- or pos t-implantation loss) were

affected.

The studies by Laws et al., and Shibayama et al., indicate that exposure to

atrazine or DACT results in effects rela ted to the development and function of the reproductive system. The effects described by both groups are consistent with the postulated MOA of perturbations of the neuroendocrine system. Sexual maturation is the culmination of a complex cascade of sex dev elopmental effects that ultimately leads

to the attainment of reproductive capacity

. Activation of the HPG axis resulting in the pulsatile secretion of GnRH and LH is critical to puberty onset. Researchers have found that disruption of GnRH release can lead to del ays in v aginal opening. Thus, it is not

3 Studies on the potential effect of atrazine ex posure on mammary gland development will be evaluated during the Fall meeting of the SAP.Page 32 of 263 unexpected that exposure to atrazine and DA CT – atrazine’s metabolite – leads to a delay in sexual maturation given atrazine’s effect on the HPG axis. Similarly, the lengthening of the diestrus stage after atrazine exposure can be readily explained by the disruptions to the HPG axis caused by atrazine. Even through the relatively short period of time evaluated by Shibayama et al., it is clear that atrazine treatment leads to a state of reproductive quiescence which – if sustained – would eventually result in the reproductive senescence characteristic of re peated atrazine exposure. It is noteworthy that the NOAEL for the effects reported after ≈ 3-4 weeks of exposure by Laws et al. (2000)

, and Shibayama et al., (25 and 30 mg/kg/day, respectively in atrazine molar equivalents) were higher than the NOAEL (1.8 mg/kg/day) for attenuation of the LH surge identified after a 6-month exposure and that is currently used as the point of departure in the Agency’s risk assessment.

3.3.2 Male Reproductive/Developmental Toxicity

Since the IRED was finalized in 2003, several studies have been conducted assessing the effect of atrazine exposur e on male reproductive function and development. Research has focused on a wi de array of possible adverse reproductive effects including delayed puberty onset, prostatitis and changes in hormonal and andrology parameters.

3.3.2.1 Delayed Puberty Onset

There have been a number of studies demon strating that atrazine delays male puberty in different strains of rats fo llowing both peripubertal and perinatal exposure (Stoker et al. , 2000; Friedmann, 2002 ;Trentacoste et al., 2001; Rayner et al. , 2006 and Rosenberg et al., 2008).

The primary data collected following the male peripubertal exposures and examination of puberty outcomes (Stoker et al. , 2001; Friedmann et al. , 2002,

Trentacoste et al. , Pogrimic et al. , 2009) is consistent with t he effect of atrazine on the central nervous system ( i.e. , suppression of GnRH/decreased pituitary secretion of LH) and subsequent alterations in the hormonal control of pubertal development (Cooper et

al. , 2000). Decreased LH secretion would lead to insufficient stimulation of the gonads during puberty to produce the necessary steroids for androgen-dependent maturation of the sex accessory tissues in the male. Therefore, the delayed growth of the reproductive tract in the male supports t he reported alteration of the hypothalamic control of pubertal progression.

A reduction in testosterone levels fo llowing atrazine exposure has been reported in a number studies in mammals, as well as ot her species revealing a consistency in the effects of atrazine on androgens. It is well k nown that the development of the size of the penis and cornification of the epithelium of the prepuc e and preputial separation in immature rats are regulated by androgens (Ma rshall, 1966). A decrease in testosterone secretion during the juvenile period can delay preputial separation (Lyons et al. , 1942)Page 33 of 263 and reduce the size of the androgen-dependent tissues, such as the ventral prostate and seminal vesicles. Normally, testosterone levels rise gradually from PND 20 to 40, and abruptly double by PND 50 (Matsumoto et al. , 1986; Monosson et al. , 1999).

In the male rat, atrazi ne exposure resulted in delays in the onset of puberty, as determined by assessment of preputial separation (PPS) (Table 4). Stoker et al. (2001) found that peripubertal exposure to atrazine delayed puberty for 1.5 to 2.5 days following exposure to 12.5 - 200 mg/kg atrazine. The NOAEL for the effect on puberty in the male Wistar rat was 6.25 mg/kg/day and the LOAEL was 12.5 mg/kg/day. They also observed significant decreases in the gr owth of the reproductive tract, namely the seminal vesicles and prostates, of the affected male rats at slightly higher doses than the doses required to delay puberty (NOAEL = 25 mg/kg). At PN D 45, intratesticular testosterone was decreased by 200 mg/kg. In the studies examining the Sprague Dawley (SD) rat (Trentacoste et al. , 2001), pubertal onset was delayed by 3 days and 4 days following exposure to 100 and 200 mg/kg at razine, respectively, with decreases in ventral prostate and seminal vesicles at the same doses (NOAEL = 50 mg/kg). Friedmann (2002) found a decrease in intratesticular and se rum testosterone at 50 mg/kg/day and 200 mg/kg/day following exposure to the peripubertal male SD rat. Thus, all three studies found decreases in eit her serum or intratesticular testosterone

and Trentacoste et al. also found a decrease in LH at the 200 mg/kg/day dose.

Ventral prostate, seminal vesicles, la teral prostates and epididymal weights are significantly reduced at doses just above th e doses which delay preputial separation (Stoker et al. , 2001). These effects provide further evidence of a decrease in androgen stimulation. Decreases in androgen horm one concentrations also correspond with the effects observed on pubertal endpoint s. For example, Stoker et al. , 2001 demonstrated that intratesticular testosterone was signifi cantly decreased following atrazine exposure peripubertally. This has corresponded to the work of others showing that serum testosterone is decreased by doses of 50, 1 00 and 200 mg/kg in a different strain of rat (SD) when dosed during a similar peri od of PND 22 to 47 (Trentacoste et al. , 2001; Friedmann, 2002) as shown in Table 4.Page 34 of 263 Table 4: Strain and Dose Comparison in Male Rats dosed peripubertally with

atrazine

EXPOSURE STRAIN EFFECT NOAEL/LOAEL REFERENCE

PND 23 to 53 Wistar Delayed PPS 6.25/12.5 Stoker et al.

, 2001

PND 22 to 47 SD Delayed PPS 50 lowest

dose/100 Trentacoste et

al. , 2001

PND 23 to 53 Wistar Decreased VP

and SV 25/50 Stoker et al.

, 2001

PND 22 to 47 SD Decreased VP

and SV 50 lowest

dose/100 Trentacoste et

al. , 2001

PND 23 to 53 SD Decreased intratesticular T on

PND 45 150/200 Stoker et al.

, 2001

PND 22 to 47 SD Decreased T on

PND 47 ND/50 Trentacoste et

al. , 2001

PND 22 to 47 SD Decreased test.

and serum T on

PND 47 ND/50 Friedmann, 2002

PND 23 to 50 Wistar Decreased

T/DHT on PND

50 50/200 Pogrimic et al.

, 2009

The study by Stoker et al. , 2002 also examined the ef fect of the atrazine metabolites, DACT, DIA and DEA on pubertal devel opment in the rat. In this study, equimolar doses of the metabolite were compar ed to the effect seen with atrazine in the earlier study (Stoker et al. , 2000). PPS was significantly delayed by DEA and DIA at

doses ≥ 25 mg/kg/day and by DACT at doses ≥ 12.5 mg/kg/day. When the males were sacrificed on PND 53, treat ments with DEA at atrazi ne equivalent dose (AED) ≥ 100 mg/kg/day, DIA at an AED ≥ 50 mg/kg/day, and DACT at an AED of 200 mg/kg/day caused a significant reduction in ventral prostate weight, while only the highest doses of DIA and DEA resulted in a significant decr ease in lateral prostate weight. Seminal vesicle weight was reduced by DEA at AEDs ≥ 25 mg/kg/day, and by DIA and DACT at

AEDs ≥ 100 mg/kg/day AED. Epididymal weight s were reduced in the DEA, DIA, and DACT at atrazine equivalent doses of 200, 200, and ≥ 100 mg/kg/day, respectively. Serum testosterone was reduced only in the males receiving the 2 highest doses of DIA. This study confirmed t hat the chlorinated metabolites of atrazine also result in similar effects on pubertal development.

It should be mentioned that other factors, in addition to those changes within the HPG, can affect the timing of puberty in th e male rat including nutritional status (leptin/body weight) and environ mental (pineal gland/secr etion of indole amines) influences. Nutritional status and body we ight are known to have effects on puberty, which are suggested to reflect the metabolic si gnals in the brain that serve as indices of

the metabolic state. These metabolic al terations associated with weight loss or decrease in growth rate are inhibitory to the reproductive system a nd may be related toPage 35 of 263 substances in the body that can alter the re lease of GnRH such as insulin, amino acids necessary for precursors of neurotransmitter synthesis and essential fatty acids (Ojeda and Urbanski, 1994).

The peripubertal exposure male study (Stoker et al. , 2001) showed decrease body weight at the 200 mg/kg dose, but a pair-fed group was included to discern the effects of changes in growth. There were changes in puberty at doses in which no body weight decreases were observed ( ≥ 50 100 mg/kg/day in females and ≥ 12.5 mg/kg/day males). However, in the study with SD rats Trentacoste et al. , found that the food restriction group (receiving ap proximately 10% less food wh ich was equivalent to the 100 mg/kg atrazine treatment food intake) showed the same effects on LH, testosterone and reproductive tract development. A re cent paper examined the effects of food restriction on the endpoints in the puberta l male exposure period and found that reductions in body weight up to 10% did not influence pubertal onset or the growth of androgen dependent tissues in the rat (Laws et al. , 2007).

Perinatally a single 100 mg/kg atrazine dose to the dam resulted in an effect on pup growth that may involve changes in mi lk quality, as the offspring showed a decreased weight of 7% on PND 4 (Rayner et al. , 2006). Work by Rosenberg and coworkers (2008) showed that prenatal (GD14-parturition) exposure to atrazine at doses

≥ 10 mg/kg/day led to a significant delay in preputial separation (1-3 days) while anogenital distance on PND 21 was reduced at 75 and 100 mg/kg/day. Measuring

testosterone levels in both the serum and intr atesticular fluid of PND 60 rats revealed

that serum testosterone leve ls were decreased at doses ≥ 75 mg/kg/day while

intratesticular fluid testosterone was reduced at doses ≥ 50 mg/kg/day. It should be

noted that all of the effect s on PPS and AGD occurred in conjunction with nonsignifcant decreases in pup body weights (Rosenberg et al., 2008). These alterations of early postnatal body weight could play a role in the delay in puberty in these male offspring.

In summary, the MOA of altered pubertal progression in the rat and decreased androgen production by the testes is plausible in humans, as GnRH and the regulation of pituitary hormones stimulat e the gonadal secretion of steroi ds and is critical for the

initiation of the set of events and time course of the HPG regulation of puberty in the human. Thus, it is essential to take into consideration that all the effects on sexual maturation and altered androgen status reported after ≈ 30 days of exposure occur at dose levels substantially higher ( ≥ 12.5 mg/kg/day) than those leading to the LH surge disruption which serves as the basis of the Agency’s current risk assessment.

3.3.2.2 Prostatitis

Prostate effects including prostatitis are oftentimes attributed to atrazine exposure. The increased incidence of pr ostatitis after atrazine exposure has been the subject of study for at least the last decade. Since 2003 , there have been two in vivo studies to further explore this potential adverse health outcome.Page 36 of 263 First reported in 1999, an increase in prostatitis has been linked to the suppression of suckling-induced prolactin rel ease. After lactating dams are exposed to

atrazine on PND1-4 at doses ≥ 12.5 mg/kg/day, the incidenc e of prostatitis in their offspring increased. At doses ≥ 25 mg/kg/day both the incidence and severity of the findings were increased. Notably, at 12.5 and 25 mg/kg/day the suckling induced release of prolactin was decreased in so me dams while the 50 mg/kg/day dose level elicited a prolactin decrease in all females. Prolactin plays a crucial role in neonatal brain development particularly in the tuberoinfundibular dopami nergic system (TIDA). In the adult male, these neurons are responsible for the down-regulation of prolactin release from the pituitary. As a consequenc e of the decreased levels of maternal prolactin during the early lactational period, the offspring of t hese dams do not receive the perinatal amounts of prolactin necessary to activate the TIDA neurons. Therefore, TIDA’s regulation of prolactin in the adult male is impaired resulting in elevated prolactin levels which in turn lead to hyperprolactinemia. It is this elevated level of circulating prolactin in the adult males t hat has been linked to an increased incidence of prostatitis. Thus, an increased incidence of prostatitis in the offspring of dams exposed to atrazine during the critical time for TIDA neurons acti vation (first postnatal week) may attributed to decreases in circulating prolactin levels in the dam.

Using a cross-fostering study design, Rayner et al. (2007) investigated if gestational exposure to atra zine could also lead to prostatitis in animals exposed in

utero. Dams were exposed to 100 mg/kg/day at razine from GD 15-19 and after birth the pups were cross-fostered resu lting in the following grouping:

• ATR-C: pups exposed to atrazine in utero only (born to atrazine-treated dams but nursed by control females)

• C-ATR: pups exposed to atrazine lactationally only ( i.e., born to control dams but nursing from atrazine-treated dams)

• ATR-ATR: pups exposed to atrazine both in utero and through the milk

• C-C: pups born to and nursing from control dams

Consistent with the findi ngs reported by Stoker et al. , both of the groups exposed postnatally ( i.e., C-ATR and ATR-ATR groups) exhibited in dications of lateral prostate inflammation on (PND 120). No prostate in flammation was observed in the ATR-C or C-C groups suggesting that t he early postnatal period enco mpasses the critical window of susceptibility to atrazine-induced prostati tis. It should be noted, however, that these effects occur at doses higher than those eliciting disruption of the LH surge.

Based on the observation that atrazi ne exposure led to a decrease in testosterone production in rats, it has been suggested that this decrease would in turn ameliorate prostate carcinoma developm ent. Kandori and coworkers (2005), however, hypothesized that rather than being a direct hormone-mediated effect, the decrease in prostate carcinogenesis is the secondary effect of a reduction in caloric intake. To test this hypothesis these researchers used a transgenic adenocarcinoma rat model typically used to study prostate carcinoge nesis. Rats received either 33 or 61.6Page 37 of 263 mg/kg/day of atrazine for a period of 13 weeks while another group was placed on a

calorie-restricted diet. Atra zine treatment at the highest dose level (61 mg/kg/day) and dietary restriction elicited a slight suppressi on of prostate carcinogenesis. Unlike other researchers, testosterone levels, however, we re not reduced by atrazine treatment in the study conducted by Kandori et al. (2005). The authors t heorized that, since testosterone levels were unaffected by at razine treatment, the decrease in prostate carcinogenesis observed after atrazine expo sure was due to reduced caloric intake (resulting from atrazine exposure) rather than hormonal disruption. It is important to know, however, that it has been demonstrated by other researchers that exposure to atrazine at the doses used in this study leads to decreases in testosterone. It is not clear if the discrepancy between this study and other studies that have demonstrated

atrazine-induced alterations of te stosterone levels is due to di fferences in administration of atrazine (dietary vs. oral gavage). In addition, testos terone levels were measured only at the end of the study and were highly variable. Also, there was a potential problem with the homogeneity of the diets (concent rations of atrazine were 60 – 91% of target values). While this study suggests the possibility that prostate carcinogenesis may be a secondary effect to a reduction in ca loric intake, a test system in which

testosterone levels are altered would provide more definitive confirmation.

In summary, the data indicate that at razine induces prostatitis at doses ≥ 12.5 mg/kg/day and that early postnatal exposure is a critical window of susceptibility to this

effect

3.3.2.3 Effects on Aromatase and steroidogenesis

Aromatase is the enzyme that catal yzes the conversion of androgens to estrogens. It is expressed in the gonadal and extragonadal tissues and is critical for

maintenance of homeostasis between the sex steroids in males and females which is necessary for reproductive function. Al though aromatase is a product of a single gene (CYP19) in mammals, its expression is quite complex as it is regulated in a speciesand tissue- dependent manner by differential us es of multiple promoters (Simpson et

al. , 2002). It is well documented that environmental contaminants can inhibit the catalytic activity of this enzyme (Vinggaar d et al., 2000; Santen et al., 2009). However, studies reporting an increase in the activity of aromatase are not as numerous.

Approximately ten years ago, Sanders on and his colleagues reported that atrazine induces estrone production and aromatase (CYP19) mRNA in H295R adrenal cortical carcinoma cells (Sanderson et al . 2000; 2001). These authors reported that elevated aromatase activity, as measured usi ng the tritiated water release assay, was observed in cells exposed to atrazine (low-effect level, LOEL, 0.3 μ M) for at least 24 hours. Since these original reports, several studies using a variety of cell lines have confirmed the atrazine-dependent increases in estrogen production in H295R cells (Heneweer et al., 2004; Higley et al., 2010) and human placental carcinoma (JEG-3) cells (Sanderson et al., 2001; Laville et al., 2006). Finally, Holloway et al. (2008) also reported that aromatase activity in hum an granulosa-lutein primary cell culturesPage 38 of 263 following treatment with atrazine (1.0 μM) was 2-fold higher than that observed for the controls. A commonality among these studies is that the effects on estrogen production are only observed in the various cell types when exposed to atrazine for at least 24 hours. This finding supports t he hypothesis that the effect s are not mediated through a direct alteration of the catalytic activity of aromatase, but rather likely involve gene transcription of the enzyme.

To date, the cellular mechanisms respons ible for the incr eased production of estrogens by aromatase have not been fully c haracterized. Sanderson et al (2000, 2002) reported that the time-re sponse profile for the inducti on of aromatase activity, CYP19 mRNA, and cyclic adenos ine monophosphate (cAMP) in H295R cells following exposure to atrazine was sim ilar to that of 8-bromo-cAMP , a known stimulant of the protein kinase-A pathway that mediates the induction of aromatase. These authors suggested that the atrazine-de pendent induction of estrogen in H295R cells results from increased CYP19 mRNA synthesis, which may be stimulated by elevated intracellular cAMP. In contrast, Fan et al., (2007a, b) reported t hat atrazine binds directly to steroidogenic factor 1 (SF-1), an orphan receptor that serves as a transcription factor for the expression of CYP19 mRNA through its interaction with aromatase promoter II. While these studies focused primarily on the up-regulation of aromatase gene expression, additional studies de monstrated that ot her cellular target sites exist for

atrazine and that thes e target sites can impact the steroidogenic process. Suzawa and Ingraham (2008) reported that atrazine does not bind directly to the SF-1, but instead stimulates three signaling pathways known to activate several of the NR5A receptors [SF-1,hLRH-1 (human liver rec eptor homolog), zff1d]. These authors also reported an upregulation of the expressi on of a group of im portant steroidogenic genes to include StAR, inhibin- α , P450scc, and11 β HSD in JEG3 cells following exposure to atrazine. Thus, these findings demonstrate that at razine can exert a mu ch broader effect on steroidogenesis. Finally Albanito et al. (2008) reported a series of in vitro experiments designed to evaluate the mechanism of action for atrazine in BG-1 cells (human ovarian cells). Their data confirm that atrazine does not bind directly to the nuclear ER α as previously reported by Cooper et al. (2007). However, atrazi ne did not alter aromatase activity or gene expression in the BG-1 cult ures following a 40-hr exposure, a finding

that contrasts with Sanderson et al. (2000) for the H295R cell line and may be a reflection of a constitutively high aromatase ac tivity that prevents fu rther amplification by exposure to atrazine. A novel finding of this study was the stimulation of extracelluar signal-regulated kinase (ERK) phosphorylation and c-fos in the BG-1 cells following exposure to atrazine (1 μ M). Using specific signaling inhibitors and gene silenc ing, these authors report that this effect is mediated through the G-protein-coupled receptor 30 (GPR30), a membrane bound orphan receptor that mediates r apid estrogen signals

The range of the LOELs (0.3 – 1 μ M) for the observed effects on cellular mechanisms provides a common factor among the in vitro studies. Recent studies in rats and mice have measured plasma and tissue concentrations of atrazine and metabolites following in vivo exposures (Ross et al. , 2007; Laws et al. 2009, Fraites etPage 39 of 263 al. , 2009). Although a complete profile of se rum and tissue atrazine concentrations for the different dosing durations tested in vivo is not available, the data reported by Fraites

et al. , for the serum concent rations of the active atrazine metabolite DIA show that it

reaches the 2-4 μ M range within 15 minutes after four daily doses of 75 mg/kg/day via gavage. Again, although more data would be needed to accurately assess the internal

dose in animals treated for different durat ions, this data does indicate that the

concentrations used in the in vitro studies discussed above are not excessive. However,

these in vivo doses are still substantially higher than those selected for points of departure in the 2003 risk assessment.

In summary, the in vitro studies demonstrate that at razine can alter aromatase activity and gene transcription. The more recent studies suggest that the cellular responses following atrazine exposure involv e a complex array of signaling events, Importantly, the fact that atrazine may exert a broad infl uence on steroidogenesis is in

accord with the in vivo studies reporting increased adrenal (Fraites et al., 2009; Laws et al., 2009) and testicular (Modic, 2004) ster oidogenesis following acute chlorotriazine dosing. It is interesting to note that alt hough changes in the production of serum steroid

hormones have been identified in vivo following atrazine exposure including estrogen, albeit at high doses (Stoker et al., 2000), alterations in aromatase gene expression or activity have not been observed in the rat testes (Modic 2004) or mouse reproductive tract (Rivest and Sanderson, 2010). Thus, suggesting that while steroidogenesis may be increased as a result of atrazine exposu re, this event is likely not mediated by

aromatase.

3.3.2.4 Andrology

Three in vitro studies have been conducted over the last seven years to evaluate the potential effect of atrazi ne exposure on sperm. Given t he effects of atrazine on a number of hormone-dependent events including triggering early reproductive senescence in female rats and causing dela ys in puberty onset, it is hypothesized that atrazine may also affect sperm parameters ( e.g., motility, viability). Two of the studies used porcine sperm while the remaining study used human sperm to explore the potential impact of atrazine exposure on spe rm. The researchers hypothesized that

effects seen after in vitro exposure to atrazine could be correlated with changes in fertility or reproductive success after in vivo exposure.

In a study designed to evaluate the effect of atrazine exposure on sperm motility, semen collected from Duroc boars was subj ected to a number of evaluations after exposure to 0, 50, 100, or 500 μ M atrazine (Betancourt et al., 2006). Exposure to

concentrations ≥ 100 μ M atrazine elicited a concentra tion-dependent and statistically signific ant decrease in sperm viability ( ↓ 8-21%) and progressive motility ( ↓ 23-75%). The authors speculated that the sperm motility effects seen after in vitro exposure would lead to an adverse effect on the sperm’ s “fertilizing ability” and hence male reproduction. This statement is not support ed by empirical data but rather conjecture based on the changes in sperm motility observed after in vitro exposures. This is butPage 40 of 263 one of the numerous lim itations of this study. The authors did not take into consideration the plau sibility that atrazine would come into direct contact with sperm

after in vivo exposure. Moreover, this study does not take into account that atrazine is quickly metabolized in the body and therefore the substance r eaching the testes is most likely an atrazine metabolite assuming it could reach the testes. Finally, there is no attempt to correlate the in vitro concentrations tested to any in vivo concentrations that live sperm may encounter during maturation in the testis, epididymal transit, in seminal fluid, or in the female oviduct where fertilizat ion takes place. In conclusion, this study evaluates mature sperm in vitro and attempts to extrapolate t hose findings to potential effects on male reproductive performance. In fact the relatively hi gh concentrations of atrazine suggests that it may have toxic proper ties at the level of the sperm membrane

or mitochondria in vitro but shows no clear relevance to in vivo exposures or a potential

MOA.

A second study conducted by the same re search group evaluated the effect of atrazine on capacitation and acro somal reaction (Maravilla-Galvan et al., 2009). Capacitation is a time-dependent physiological process that is necessary for sperm to undergo before they become capable of fertilizatio n. It usually takes place in the uterus and oviduct, but can occur under appropriate culture conditions in vitro . Capacitated sperm can bind the zona pellucida around t he oocyte, penetrate it and fuse with (fertilize) the oocyte. Unc apacitated sperm cannot. Acrosome reaction (AR) consists of an ordered, calcium dependent vesi culation and shedding of t he acrosomal vesicle that covers much of the sperm head. It can be vi sualized using chlortetracycline staining (as was done in this study). Only acrosome reacted sperm can fuse with the egg

membrane. Therefore AR is used as an i ndicator of capacitation. Progesterone may directly induce acrosome reac tion at the sperm membrane level in vitro . Once AR occurs, sperm tend to slow down and die (run out of energy). T herefore, in a mixed

culture there will be continuous ly changing populations of ac tive capacitating sperm and dying post capacitation/AR s perm. Changes in sperm mo tion called “hyperactivation” accompany capacitation and are also necessary for sperm to penetrate the zona pellucida. This study suffers from not reporting hyperactivat ed motility. The latter would help distinguish between physiologically AR sperm and dead sperm ( both of which stain similarly with CTC because they lack an acrosome).

In 2009 Maravilla-Galvan and coworkers evaluated the impact of atrazine exposure at concentrations of 0 (control), 8, 20, and 40 μM. In general, the percentage of uncapacitated acrosomes ( i.e., with intact acrosomes) and percentage of motile sperm declined in a dose-dependent manner wh ile the percentage of acrosome reacted sperm increased. There could be two interp retations for this finding. The authors’

conclusion was that atrazine stimulat ed or accelerated acrosome reaction in vitro leading to a decrease in motility. Conversely , it could be theorized that atrazine results in decreases in sperm viability and that th e dead sperm shed their acrosome after death thus appearing to be acrosome reacted. In the absenc e of data on the proportion of hyperactivated sperm, it is impossible to distinguish between these two possibilities. Regardless, these data do not provide any in formation that correla tes the findings toPage 41 of 263 adverse reproductive performance nor does it provide any rationale for hypothesizing that atrazine selectively ta rgets sperm. Consequently, the relevance of this study for risk assessment purposes is unclear.

Lastly, a study by Hase et al., examined the effect of atrazine on human sperm motility in vitro and tested the hypothesis that decr eases in sperm motility might be related to decreases in ATP availability needed for motility. S perm from consenting males was treated with 0, 0.03, 0.1, 0.3, or 1 mM atrazine. Oligomycin (0.05 mM) – a sperm metabolic poison which uncouples AT P production in mitochondria – was used as a positive control. As expected, oligomycin decreased sperm motility by approximately 20%. Atrazine also caused a decrease in sperm motility but only at considerably higher concentrations (0.3 and 1 mM) than oligomycin. In support of the authors’ original hypothesis, sperm ATP levels were also decreased at the concentration where progressive motility was impaired. Based on these findings, the authors postulate that the decreases in sperm motility observed in vitro may be the outcome of decreases in ATP availability needed for sperm flagella to move. They also point out that “the effect of ATZ [atraz ine] on sperm motility was seen at > 100 μ M, exposure levels that probably do not result from normal use of the herbicide. It is

therefore uncertain wh ether the observed phenomena are ecologically relevant.” Thus, though this is an interesting hypothesis and fi nding, its relevance to risk assessment is questionable.

In summary, three studies evaluati ng andrology parameter s have been published since the 2003 IRED. Using either porcine or human sperm samples, they evaluated atrazine’s potential effect on sperm parameters (motility, viability, capacitation, and acrosomal reaction). Although the in vitro data suggest an effect of atrazine on sperm motility and/or viability, there is no evidence of a correlation between these findings and impaired reproductive performance. Furthermo re, these studies assume that atrazine

per se reaches the testes; an assumption that has not been verified. Finally, the concentrations affecting sperm parame ters in these studies are high ( ≥ 100 μ M) and, as

Hase et al . noted, an exposure level not likely to be reached as a result from use of the

herbicide under the current rest rictions. Thus, the data in andrology measures, though interesting, provide no information that would suggest that in vivo exposure to atrazine would result in a sperm-mediated def icit in reproductive performance.

3.4 Neurotoxicity

The preponderance of the data available to date indicate s that atrazine exerts many of its toxicologically relevant effe cts through perturbations of the neuroendocrine system. Many of the reproductive effects s een after atrazine exposu re appear to be the sequelae of perturbations to the HPG and/ or HPA axes. Consequently, there is significant interest in the potential effect s of atrazine exposure on the nervous system

per se.Page 42 of 263 Over the last seven years, severa l studies have been conducted evaluating potential atrazine-induced e ffects on the dopaminergic and somatostatinergic systems as well as GnRH releasing neurons. Studies on the dopaminergic system by Coban and Filipov (2007) and Rodriguez et al. (2005) corroborate previous fi ndings on the effect of atrazine on the dopaminergic system. The work by Rodriguez et al. showed that 9- month old male rats exposed for 6 months to 10 mg/kg/day atrazine in the diet exhibited increased locomotor activity, decreased st riatal dopamine, reduc ed norepinephrine in the prefrontal cortex, decreased hypothalamic serotonin, and loss of tyrosine hydroxylase positive (TH+) and tyrosine hydrox ylase negative (TH-) cells in the ventral tegmental area and the substant ia nigra pars compacta. It should be noted that the changes in neurotransmitter levels were slig ht. In 2007, Coban and Filipov studied the effect of atrazine on male mice after a 14 day exposure to dose levels ≥ 5 mg/kg/day. The researchers reported a statistically signi ficant reduction in the number of TH+ neurons in the ventral tegment al area (day 64 of the exper iment) as well as a decrease

in the number of TH + neurons in the substantia nigra pars compacta was reported at 25 mg/kg/day at days 22 and 64. Noticeably lacki ng was a dose-response for either one of these findings. Coban and Filipov (2007) also concluded that there was a dosedependent decrease in the levels of dopamine and its metabolites in the striatum. Based on these findings, both research gr oups suggested that exposure to atrazine may be related to the etiology of Parkinso n’s disease. Both of these studies have limitations that confound the interpretation of the findings. These limitations are fully described in the study summaries found in Appen dix A. The implicat ion that these data suggest a link between atrazine and Parkins on’s disease is not supported by the data given the non-specificity of the atrazine-induced neuronal loss and the lack of histological and behavioral features char acteristic of Parkinson’s Disease.

Parkinson’s disease is a neurodegenerative disease characterized by a progressive loss of dopaminergic neurons in t he substantia nigra. The data reported by Rodriguez et al. (2005) shows loss of non-dopam inergic neurons and dopaminergic neurons following atrazine treatment in t he rat. The subpopulation of non-dopaminergic neurons includes GABA-ergic, calbindin, and cholecystok inin-like immunoreactive neurons. Other potential cell types may in clude glial cells. The data presented by Coban and Filipov (2007) reveal a statistically significant loss of dopaminergic neurons in the ventral tegmental area in the mouse at 5 mg/kg/day on day 64 of the experiment, while the loss in the substantia nigra pars compacta was significant at 25 mg/kg/day but not at 5 mg/kg/day. The loss of dopaminer gic neurons at the high dose at day 64 was comparable between the two brain regions. The ventral tegmental area contains dopaminergic cell bodies that comprise the me socorticolimbic system that is involved with functions such as cognition. The pr imary and major loss of dopaminergic neurons in Parkinson’s disease involves the substantia nigra. The nonspecific neurotoxicity of

atrazine contrasts with selective striat al dopaminergic neurotoxins such as 6- hydroxydopamine and MPTP.

In addition to the non-specificity of t he atrazine-induced neuronal loss, another

characteristic not seen after atrazine ex posure is the suite of neurobehavioral andPage 43 of 263 histological features associ ated with Parkinsons’s disease. Histologically, Parkinson’s disease is characterized by the presence of intracytoplasmic inclusions, Lewy bodies, formed mainly by α -synuclein and ubiquitin. None of the available studies have demonstrated any aggregation of proteins that form Le wy bodies after atrazine

treatment. Behavioral abnormalities noted in Parkinson’s disease include tremor, bradykinesia, hypokinesia, balance and gait disturbances. No overt behavioral signs were reported in the available atrazine st udies. This contrast s with rotenone, which causes a Parkinson-like syndrome, and resu lts in reduced mobility, flexed posture, rigidity and even catalepsy in the rat. R odriguez (2005) provided some evidence for increased locomotor activity following d-amphet amine sulfate injectio n of rats treated with atrazine. However, the authors’ asserti on that the increased locomotor activity is associated with striatal dopami ne deficiency from atrazine tr eatment should be further explored and supported with a study design that includes L-dopa and dopamine agonist and antagonist administration.

Several in vitro studies addressed the putativ e mechanism of dopaminergic toxicity of atrazine, primarily in striatal slices and striatal synaptosomes and vesicles (Hossain and Filipov 2008; Filipov et al. 2007). The results rev ealed that atrazine, in

vitro , alters striatal dopamine levels possibly by interfering with the vesicular storage and/or cellular uptake of dopam ine. Tyrosine hydroxylase per se is not a target of

atrazine. The relevance of these in vitro data to dopaminergic mechanisms in vivo is not clear given the high concentrations of atrazine examined in vitro and the complex

interactions between various neurotransmitter and endocrine systems in t he intact brain. Furthermore, as mentioned above, the availabl e data indicate that the neurotoxicity of atrazine is not specific to dopaminergic system s. The effects of atrazine on aromatic amino acid decarboxylase, monoamine oxidase and neuronal mitochondrial function are

unknown.

The somatostatinegic system in mouse br ain was used as a model by Giusi et al. (2006) to study the impact of atrazine exposur e on this system. The rationale for this study was that somatostatin plays a role in growth hormone (GH) secretion and the gender-based differences in GH secretion in rodents. The authors theorized that the differential in GH secretion may be linked to gender-related differences in the transduction of somatostatinergic mechanisms in brain and/or pituitary targets. As a result, the researchers concluded that t he GH- and SST-dependent relationships “might provide further insights into the types of neurotoxicological mechanisms involved in the delay of the estrous cycle or the alte ration of the hypothalamic-hypophyseal-gonadal axis in animals treated with atrazine (Wetzel et al. 1994).” Though the authors reported sexually dimorphic changes in the hypot halamus of females and changes in the regulation of somatostatin receptors in the hypothalamus of both males and females, the reliability of this study is uncertain given the numerous deficiencies (including poor description of the methods and poor quality of micrographs used to substantiate conclusions) identified in the study design and described in the summary review in Appendix A.Page 44 of 263 A neurobehavioral study in mice is also avai lable for consideration. In this study Belloni and coworkers (2007) report that atrazine exposure from GD14-PND21 at low doses (1 or 100 μ g/kg/day) led to behavioral effects incl uding delays in righting reflex, decreased temperature, changes in pup voca lizations and an acceleration of the ontogeny of the forepaw grasp response. It should be noted that this study also had a

number of deficiencies such as inappropriate statistical analyses and unexplained discrepancies in the sampling of pups from litters which are described in the summary review in Appendix A that call the va lidity of the results into question.

More closely related to the postulat ed MOA for atrazine is the research conducted by Cooper et al ., 2007 and Foradori et al. (2009) on the pulsatile release of GnRH neuronal activation after atrazine exposu re. The research by Foradori and coworkers show that a brief (4 da y) exposure to atrazine at doses ≥ 50 mg/kg/day resulted in a decrease in GnRH activation as indicated by a decrease in cFos activity concomitant to a significant reduction in the LH and FSH surges. Thus it appears that atrazine can induce changes in these hypothal amic neurons. Thes e data, therefore,

illustrate the close correlation between e ffects on the central nervous system ( e.g., effects on GnRH activation) and effects on the reproductive system. Whether or not this downregulation of the GnRH neurons are the resu lt of a direct action of atrazine or

atrazine metabolites on the GnRH neuron orthe result of increased hypothalamicpituitary-adrenal axis are under investigation. Indeed, recent data show that atrazine exposure leads to a rapid increase in corticosterone and progeste rone (adrenal steroid hormone) secretions which decrease GnRH secr etion (Fraites et al.,2009; Laws et al., 2009).

In summary, there are a num ber of studies indicating that atrazine and or atrazine metabolites may impact different neuronal pathways of the brain. The reliability and validity of findings on the brain soma tostatinergic system and on neurobehavior in

the mouse are uncertain due to serious deficiencies of the studies ( i.e. , Guisi et. al. 2006 and Belloni et al. 2007). The recent studi es examining changes in the rat brain dopaminergic systems and associated behavioral changes were also found to have significant deficiencies that also limit t heir utility for risk assessment purposes. In addition, results of in vivo animal toxicity tests do not support an effect of atrazine exposure on neurological func tion including Parkinson’s Dis ease. The observations that the chlorotriazines may alter GnRH neuronal activity are consistent with the proposed

neuroendocrine MOA of atrazine. Previous studies have demonstrated that atrazine can alter dopamine in hypothalamic regions that are important in t he regulation of GnRH secretion, consistent with the neuroendocrine MOA of atrazine.

3.5 Immunotoxicity

In the Agency’s 2003 evaluation of atrazi ne, the immune system was identified as one of the target systems for atrazine toxicity. Sinc e then, numerous studies both in

vivo and in vitro have been conducted to further investigate atrazine’s potential to cause immunotoxicity. The in vivo studies reported various potentia l immunological effects ofPage 45 of 263 atrazine on cellular and humoral immune systems . These included nat ural killer (NK) cell function, delayed-type hypersensitivity responses to bovine serum albumin (BSA), macrophage phagocytic activity, antibody responses to sheep erythrocytes or keyhole limpet hemocyanin, T cell prol iferation and cytolytic activity, spleen and thymic cell phenotypes, humoral response afte r immunization with heat-killed Streptococcus pneumoniae , lymphocyte populations in peri pheral blood mononuclear cells, and expression of MHC Class II proteins on sple nocytes. One study design included a host resistance test, which assesses t he function of t he immune system in toto (Karrow et al. 2005).

All of the adult in vivo immunotoxicity studies focused primarily on cellular and humoral systems. Several st udies evaluated limited systemic effects such as spleen and thymic weights and body weights. N one of the studies provided detailed and thorough histological examinations of lymphoid organs(e.g., spleen, thymus, bone marrow, lymph nodes) that would be useful in understanding the potential mechanisms of immunotoxicity. Consistent effects (e.g ., decreased splenic and thymic weights and alterations in splenic and thymic cellulari ty and phenotypes) were noted across the adult

immune studies that examined these endpoints. In many instances, the changes were slight (albeit statistically significant), and given the functional reserve capacity of the immune system, it is difficult to extrapolate the rodent findings to human health risk assessment. However, it is interesting to note that the results of t he host resistance test to validate the biological significance of changes in cellular and humoral immune function revealed significant decreases in resistance to the B16F10 tumor challenge at doses of 250 and 500 mg/kg/day atrazine, but not at 25 mg/kg/day. Host resistance assays are often considered to be the most re levant test for validating other methods and for extrapolating the potentia l of environmental agents to alter host susceptibility in the human population.

It is well known that alterations in cort icosterone levels may result in impaired immune function through the activati on of the HPA axis. Pruett et al. (2003; 2009) and

Schwab et al. (2005) exposed rats intraperitoneally to atrazine at high doses ranging from 100-300 mg/kg in an attempt to deter mine if corticosterone area under the curve could be used to predict immunotoxicity. T he effect of HPA axis perturbations on immunological parameters c an be quantified by relating immunosuppression to change in corticosterone levels represented by the area under the corticosterone vs. time curve (AUC) (Schwab et al. 2005). Pruett et al. (2003, 2009) and Schwab et al. (2005)

evaluated atrazine in the corticosterone re sponse model including acute and 28-day exposures to atrazine. In this model, atrazine treatment resu lted in a number of immune system effects (e.g., suppressed wh ite blood cell counts, increased neutrophils, decreased lymphocytes), cons istent with effects on corticosterone concentrations (Pruett 2009). However, the atrazine-in duced decrease in natural killer cell activity was not mediated by changes in the HPA axis. The latter observation suggests other

mechanisms besides HPA activation in atrazine-induced decreases in natural killer cell activity. Pruett et al. (2009) provided some supporting evidence that 28-day atrazinePage 46 of 263 exposure did not cause habituation or tole rance to the HPA axis perturbations, but immune function (e.g., antibody production) was not evaluated in this study.

It is important to note that the cort icosterone response was stimulated by intraperitoneal injection of atrazine. Pruett et al. (2003) demonstrated that a single high oral dose of 500 mg/kg atrazine also increa sed endogenous corticosterone levels. This study was intended to provide insight into the predictive value of corticosterone production over time for impairments of the immune system not to assess the immunotoxicity of atrazine per se

.

While the available studies suggest a potent ial effect of atrazine on the immune system as noted in studies prior to 2003, c onflicting results were reported in the two in

vivo developmental immunotoxicity studies available. Rooney et al. (2003) reported immunosuppression of cellular and humoral immune function in male Sprague-Dawley rats, whereas Rowe et al. (2006) reported immunopotentiat ion of immune function in

male Balb/c mice. The reasons for the cont radictory results are not known but may be related to differences in the timing and route of maternal exposure, species differences and different methodologies used to assess immune response. Rooney et al. examined Sprague-Dawley rats following oral exposure – a relevant route of human exposure – from GD 10 until PND 23 to 35 mg/kg/day at razine. Delayed type hypersensitivity response and the IgM antibody respons e to sheep red blood cell antigen were measured. In comparison, Rowe et al. used Balb/c mice which were exposed subcutaneously to atrazine for 21 days via time release pellets beginning between GD 10 and 12 through PND 10. The starting dos e was 35 mg/kg/day beginning at GD10 and then decreased to approximately 23 mg/kg/ day when the mice gave birth, due to a

constant rate of atrazine rel ease from the implant that was not adjustable for increasing maternal body weight. T cell function (proliferation and cytolytic activity) after in vitro allogeneic stimulation wa s measured. Humoral response was assessed after

immunization with heat killed streptococcus pneumoniae . Finally, it should be noted

that the results reported by Rooney et al. have not been reproduced by the same laboratory that conducted the first experiments.

It is not known if the developmental imm unological effects of atrazine reported by

Rowe et al. and Rooney et al. represent HPA axis alterati ons similar to those seen in adults at much higher doses. Rooney et al. (2003) provided some evidence that immune changes associated with atrazine during early development are not mediated through the suppression of prolactin or thyroid hormones. The precise underlying mechanism of an atrazine-mediated sex-sp ecific effect, however, is not fully

understood.

In vitro studies intended to provide mechanistic data that would further clarify atrazine’s effect on the immune system, includi ng intracellular signal transduction, have also been conducted. The underlying mechanis ms of atrazine-induced inflammatory effects such as nociception and plasma extr avasation, may be mediated through mast cell degranulation and activation of the Gq/ 11-protein coupled neurosteroid receptor,Page 47 of 263 phospholipase C and intracellular Ca 2+ (Mizota and Ueda 2006). However, the inflammatory responses were examined followin g intraplantar administration of atrazine, a route of exposure that is not lik ely to occur in humans. Pinchuk et al. (2007) provided

in vitro evidence suggesting that atrazine dire ctly targets dendritic cell maturation and decreases major histocompatibility comple x 1 (MHC-1) molecule expression on the

dendritic cell surface.

Atrazine-induced inhibition of in vitro human peripheral blood lymphocyte NK cellspecific lysis of K562 human leukemia cells may be the result of inhibition of lytic granule release, without affecti ng the ability of the natural ki ller cell to form stable conjugates with tar get cells (Rowe et al. 2007). Using human peripheral blood mononuclear cells, the in vitro study of Devos et al. (2003) suggests that atrazineinduced inhibition of human peripheral blood mononuclear cell prolif eration and cytokine production is most likely not mediated th rough nuclear receptors such as the glucocorticoid receptor, androgen receptor, me mbers of the peroxi some proliferatoractivated receptor family, and the retinoid-related orphan receptor α

.

Although in vitro studies provided some insight into potential mechanisms of

atrazine-mediated immunotoxic ity, the extrapolation of these mechanisms to the interpretation of immunotoxicity observed in vivo is difficult. Unlike the in vitro situation,

in vivo the immune system’s response to atrazine exposure involves complicated feedback communication pathways with the c entral nervous system operative (for example, potential modulation of immune cell function by endocrine hormones such as corticosterone).

In summary, the recent studies suggest a potential for atrazine to affect the immune system. However, t he underlying modes of action of atrazine-mediated immunotoxicity and its relevance to potential adverse health effects in humans are still not thoroughly understood. In adult rodents, t he indications of immunotoxicity reported in the atrazine database may be due to direct interactions between atrazine and/or its metabolites and immune system cells. Alternat ively, the immunotoxicity response could be related to endocrine hormones acting on immune cells. Consequently, no conclusions may be drawn at this time on the relationship between the immunotoxic effects observed after atrazine exposure and the activation of the HPA axis with the subsequent disruption of the HPG axis. Furthermore, data regarding the developmental immunotoxicity potential of atrazine is inconclusive since the two available developmental immunotoxicity studies yielded conflicting results. Nonetheless, the

available data do not indicate atrazine-in duced immunotoxicity is a more sensitive endpoint than the atrazine-induce d effects on neuroendocrine f unction, e.g., attenuation of LH surge in female Sprague -Dawley rats wh ich is used for the basis of the points of departure for the intermediate and ch ronic exposure scenarios.Page 48 of 263 3.6 Genotoxicity and Carcinogenesis

During the initial review of atrazine in 1986, the Agency designated this pesticide as a Group C carcinogen ( i.e., possible human carcinogen) due to the increased incidence of mammary tumors seen in Sprag ue-Dawley rats after chronic exposure and the lack of mechanistic data suggesting a MOA that could be evaluated for human

relevance. It should be noted that no other tumor sites were found in rats and there

were no tumors induced in mice.

Since the 1986 assessment, an ext ensive mechanistic database has been generated by laboratories across the world. The Agency presented its assessment of

the available mode of action information to the FIFRA SAP in 2000 (FIFRA SAP, 2000).

At that time it was concl uded, that the data show that the operative MOA for mammary gland tumorigenesis involves the perturbati on of the hormonal milieu through the HPG axis and not a genotoxic or mutagenic MOA. While the disruption of the LH surge that

is central to atrazine’s MOA is a rele vant endpoint to assess potential adverse reproductive effects, it is not relevant for mammary tumor devel opment in humans. In rats, the mammary tumors arise as a result of constant stimul ation of the mammary gland by elevated levels of circulati ng estrogen and prolacti n during the early reproductive senescence caused by the disrupt ion of the LH surge. In contrast, humans’ reproductive senescence (menopause) is not the consequence of disruptions at the hypothalamic/pituitary level, but rat her a depletion of follicles and a decrease in estrogen. That is to say, that while t he hallmark of atrazine-induced reproductive senescence in rats is elevated estrogen, in humans menopause is marked by a decrease in both estrogen and prolactin. Cons equently, in the absence of constantly elevated estrogen and prolactin levels mammary gland tumors would not develop in humans as a result of atrazine exposure.

Several genotoxicity and carcinogeni city studies using transgenic and chemically-induced rodent carcinogenesis models have been published since the 2000

SAP review and the 2003 IRED. Researchers have studied atrazine’s potential effect on tumor progression in a number of glandular ti ssues (pituitary, thyroid, ovaries, and mammary gland). It s hould be noted that none of thes e experiments study the tumorinitating capabilities of atrazine per se but rather its ability to accelerate the progression of already existing tumors. In general, these data show no evidence that atrazine exposure contributes to tumor progression or that it makes the tumors already present in the system more aggressive. In vitro studies – for the most part – corroborate the findings in the in vivo studies and show that atrazine is cytotoxic for most of the celllines tested.

Work by Fujimoto and coworkers in 2003 us ing transplanted MtT/E-2 pituitary rat cells (a cell line highly-sensitive to es trogen-induced proliferati on), showed that in ovariectomized F344 rats exposed through the di et to atrazine at dose levels ranging from 5-500 mg/kg/day atrazine exposure did not accelerate tumor progression. In contrast, tumors were observed within 9 days in animals treated with 0.5 mgPage 49 of 263 subcutaneous pellet of estradiol. In this test system, serum prolactin levels, cell proliferation, and es trogen gene activation were also u naffected by exposure to up to 500 mg/kg/day of atrazine. Similarly, work by Son et al. (2003) using a DHPN-induced thyroid carcinogenesis model demonstrated that atrazine exposure did not enhance thyroid tumor progression nor did it affect thyroid hormone levels (T 4 , T 3 , and TSH). DMBA-induced ovarian carcinogenesis was yet another model used to study atrazine’s potential to affect tumor progression. Using this model, Tanaka et al. hypothesized that the operative mode of action leading to ovarian adenocarcinomas involves hormonal disruption (particularly estrogen) . Therefore, they postulate that exposure to atrazine (which has been associated with hormone di sruption) may also affect ovarian carcinogenesis. As expected, a single expos ure to 0.5% DMBA induced ovarian tumor formation. Subsequent atrazi ne exposure through the diet at doses ranging between 5- 500 ppm led to a slight, non-statistically sign ificant decrease in the incidence of ovarian tumors seen after DMBA exposure alone. When considered collectively, the data reported by Fujimoto, Son, Tanaka and co-wor kers demonstrate that atrazine exposure did not accelerate tumor progression.

Two in vivo studies are available. Fukamachi et al. in 2004 reported that in transgenic rats carrying a human c-Ha-ras pr oto-oncogene atrazine exposure enhanced DMBA-induced mammary tumor development. Ho wever, a dose-response could not be established for this finding. Likewise, the incidence of mammary adenocarcinomas appeared to be increased but a dose-response relationship was not observed since the incidence at the low- and mid-dose was high er than in the high dose. Non-transgenic rats were also used in this study, however, the data for these animals was not included in the report. Further more, atrazine exposure in vitro did not cause MCF-7 cell proliferation – an estrogen receptor-posit ive human mammary cancer cell line. Consequently, it is difficult to ascertain if the changes seen after atrazine exposure are truly related to an inherent carcinogenic property of the compound or if it is due to the genetic background resulting from the presence of a human proto-oncogene in the cells.

Ueda et al. conducted a similar set of experiments in 2005 but instead of using a transgenic rat model they used a DMBA-induced mammary carcinogenesis model. In this study the effect of atrazine expos ure on the progressi on of DMBA-induced mammary tumors in ovariectomized Sprague Dawley rats was investigated. When DMBA-induced rats were examined, no clearly discernible correlation between atrazine exposure and the number of tumor-bear ing rats at the end of the study ( i.e., atrazine exposure did not increase the incidence of rats bearing tumors) was observed nor did atrazine exposure affect the r egression of palpable tumors observed after ovariectomy. Some statistically significant changes were reported by the authors but that is mainly due to the statistical analysis used. Exposur e to 500 ppm atrazine by itself did not induce any mammary tumors.

Agency scientists identified a number of deficiencies in the conduct, interpretation, and reporting in both t he Fukamachi and Ueda studies which are described in Appendix A. Nonetheless, thes e two sets of data suggest that atrazinePage 50 of 263 does not induce mammary tumors in transgenic rats or ovariectomized rats treated with a mammary tumor inducer thus highlighting the importance of the ovarian hormones in the development of tumors in the mammary gland.

In vitro data evaluating the cytotoxicity and cell pr oliferative propert ies in a variety

of cell lines indicate that atra zine is cytotoxic or causes decr eases in cell proliferation. In general, these data – like the in vivo data – suggest that atrazine is not genotoxic or mutagenic.

Chinese Hamster ovary (CHO) and human fibroblast cell proliferation was decreased at relatively low levels of atrazine ex posure (Kmetic et al. 2008 and Manske

et al. 2004). However, data from Yi et al. (2010) suggests that atrazine and its metabolites are not cytotoxic to two steroi dogenic cell lines (H295R and JEG-3) as well as two non-steroidogenic cell lines (HT-22 and MCF-7).

Comet assay – a sensitive and rapid met hod for DNA strand break detection in individual cells – was used to study atrazine’ s capability of inducing genotoxicity. Using this assay, Garaj-Vrhovac et al. and Zeljezic (2004) conc luded that Gesaprim (an atrazine formulation) induced ge nome damage. The limitation of these studies is that a

formulation of atrazine was used and no in formation is provi ded to determine the percentage of atrazine present in the test material. Thus, it is entirely possible that the DNA breaks reported after Gesapr im exposure may be related to other chemicals in the

formulation rather than atrazine per se . Sister chromatid ex change (SCE) analysis – a sensitive method used to study DNA instability or a deficiency in DNA repair – has also been used to study the genotoxic potential of atrazine in human peripheral lymphocytes arrested at the G2 phase of the cell cycle. Using this method, Malik et al. demonstrated

that over a concentration r ange of 5- 200 μg/L atrazine did not increase the frequency of SCE induction in G2-prematu rely condensed chromosomes.

Finally, two studies intended to evaluate if vitamin E treatment following atrazine exposure would ameliorate atrazine-induced effects are available. After atrazine exposure at a high dose of 300 mg/kg/day, Singh et al. (2008) report a marked increase in lipid peroxidation and decrease in acetyl cholinesterase activity and cell membrane lipid content suggesting an increase in oxi dative stress. Conc urrent exposure to atrazine and vitamin E dampened all of these e ffects. A subsequent study by the same research group showed that at 300 mg/kg/da y of atrazine induced DNA instability as measured by the Comet and micronucleus assa ys. Again, vitamin E exposure at 100 mg/kg/day decreased micronuclei formation and DNA breaks. The results of this study are questionable given that t he atrazine doses used to induce these genotoxic effects are orders of magnitude higher t han what is currently used as the point of departure for

human health risk assessment.Page 51 of 263 3.7 Insulin Resistance and Mitochondrial Function Impairment

In a recent publication in the el ectronic journal PLoS ONE, Lim et al. (2009) suggest that atrazine exposure leads to in sulin resistance, fat accumulation, and increased weight gain by increasing bl ood glucose levels and lowering energy expenditure. The authors pos tulate that atrazine disrupts mitochondrial function by inhibiting mitochondrial complexes I, II and III, which interact with ubiquinone for electron transport. This hypothesis is bas ed on atrazine’s known pesticidal mode of action by disrupting electron transport via plas toquinone which is similar in structure to ubiquinone in vertebrates. Mitochondria are involved in fat oxidation and insulin secretion pathways, so their disruption coul d cause steatosis or insulin pathologies. However, mitochondrial inhibition should cause decreased insulin secretion, not increased as the authors propose. In fact, a recent publicati on in the Proceedings of the Academy of Sciences (PNAS) by Hoehn et al. (2009) demonstrated that – contrary to

the Lim et al. hypothesis – mitochondrial inhibition reverses insulin resistance ( i.e. excess mitochondrial electron flow signals the over-fed state to produce insulin resistance).

The authors state that atra zine exposure led to an increase in body weight and fat deposition regardless of the caloric intake. However, nei ther the body weight nor the fat deposition increases ( ↑ 5-10%) reported in the study show a dose-response. Moreover, the blood glucose levels report ed by the authors after atrazine exposure though elevated relative to control are well within the normal range. Numerous

deficiencies have been identified in this study and are descri bed in detail in Appendix A. Amongst the most noteworthy deficiencies is the lack of several pieces of critical information in the material and methods, as we ll as inappropriate statistical analysis.

The authors concluded that exposure to at razine at dose levels as low as 30 μg/kg/day resulted in significant mitochondr ial dysfunction. Considering that NOAELs have been established in guideline in vivo toxicology studies at doses that are ≈ 60 to 300-fold higher, this seems to be unlikely. Overall, the quality of this study was poor

with numerous inconsistencies and deficiencie s in the reporting thus rendering it of limited utility in understanding the toxi cological profile of atrazine.

In summary, Lim et al. are the only researchers that have proposed a relationship between atrazine exposure and mitochondrial d ysfunction and obesity. The study used to support this hypothesis is critically flawed. Moreover, t he preponderance of the

evidence indicates that atrazine exposure leads to decreases – not increases – in body weight. Consequently, the valid ity of these data is uncerta in and renders it of little relevance for risk assessment purposes.

4. Summary of Mammalian Toxicity

Atrazine exposure has been reported to lead to a number of toxic effects including effects on the reproductive heal th of the animals, immunotoxicity andPage 52 of 263 neurotoxicity. Rather than c onsidering these toxicities as unrelated entities, it is important to examine them in the context of what is known about the PK and the MOA of this pesticide. Currently, the MOA for atrazine’s effect on the reproductive system and rat mammary carcinogenesis has been descri bed as a perturbation of the HPG axis resulting in attenuation of t he LH surge which in turn initiates a cascade of events leading to early reproductive senescence in the rat and to effects on reproduction and development. Recent data, however, suggest t hat an even earlier event in this process is the perturbation of the HPA axis manife sted as increases in corticosterone levels. Changes in corticosterone levels are postula ted to be a point of commonality for most – if not all the toxic effects – elicited by at razine. Corticosterone levels can change significantly within 15 minutes of atrazi ne exposure but quickly returns to background level after a few hours. Although, it is well-understood that a continuous 3-4 day elevation of corticosterone leads to pertur bations of the HPG ax is, the toxicological relevance of this rapid incr ease is currently unclear ( i.e. , at what duration and at what doses does this perturbation lead to an adver se outcome). Time-to-effect relationship and the correlation between changes in cortic osterone and attenuation of the LH surge suggest that HPA axis perturbation is the initial step in the MOA driving the most sensitive and biologically plaus ible toxic effect seen afte r atrazine expo sure (i.e., suppression of LH).

According to Pruett and coworkers, at high doses ( ≥ 100 mg/kg/day), atrazine and its metabolites DIA and DEA (but not DACT) have a clear effect on the secretion of pituitary adrenocorticotropin (ACTH) and adrenal steroid hormone secretion ( i.e., corticosterone and progesterone) (Pruett et al., 2003). In the rat, this response is rapid ( i.e. , blood concentrations are significantly increased within 15 minutes after oral gavage). Furthermore, this group of re searchers has correlated atrazine-induced corticosterone changes with a number of immune effects including suppressed white blood cell counts, increased neutrophils, and decreased lymphocytes. These data, however, must be interpreted cautiously since in this series of experiments atrazine exposure was through intraperitoneal injection – a route not relevant to human exposure and used high doses.

Increases in corticosterone have also been shown to affect the activity of the neurotransmitters and neuropeptides of GnRH controlling neurons (Kirby et al. , 2009), including increases in gonadotropin inhibiti ng factor, CRH and norepinephrine all of which are known to decrease GnRH pulses. Besides the neuroendocrine component, there is evidence that atrazine may affect the dopaminergic system . Given that the dopamine transporter (DAT) present in most – but not all dopaminergic neurons – has some affinity for norepinephrine, it is not unreasonable to consider that changes in corticosterone levels resulting in norepine phrine increases may af fect the dopaminergic system. In fact, in humans, a persistent incr ease in cortisol levels has been associated with changes in dopamine neuronal activity (Molcrani et al., 1997). However, no data are currently available directly linking the at razine-induced changes in corticosterone to effects in the dopam inergic system.Page 53 of 263 Interestingly, unlike other dopaminergi c neurons, pharmacological data suggest that a functional dopamine tr ansporter is not present in tuberoinfundibular (TIDA) neurons. Thus, the increased incidence in pros tatitis resulting from the dampening of the suckling-induced prolactin su rge in atrazine-treated dams is likely not related to toxic effects characteristic of the dopaminergic system. Nonetheless, the prostate effects elicited by atrazine exposure occur at higher dose levels than those suppressing the LH

surge.

In summary, the toxic effects seen afte r atrazine exposure are the result of multilevel interactions of a variety of systems, particularly the neuroendocrine, reproductive, nervous, and immune systems. Activation of the HPA axis and the subsequent disruption of the HPG axis app ear to be the corner stone for the most

sensitive effects attributed to atrazine ex posure – estrous cyclicity disruption. The extent of HPA activation (measured as increas es in corticosterone) needed to elicit the decrease in GnRH and subsequent disruption of estrous cyclicity is not clear at this time. Neither the duration of exposure nor the dose that would le ad to this adverse health outcome has been clearly described. It has been demonstrated by Fraites et al. that a 4 day exposure to atra zine at 12.5 mg/kg/day leads to an increase in both ACTH

and corticosterone. In this same time fr ame, exposures to 25 and 6.25 mg/kg/day of atrazine lead to decreases in GnRH and LH, res pectively. With the exception of the LH attenuation, a no observed effect level (NOEL) has not been identified for any of these effects. As part of the WOE evaluation to be conducted for the SAP in the Fall, the Agency is planning to conduct a benchmark dose (BMD) analysis to better characterize the dose-response curve for these effects. This analysis along with additional ongoing research investigating the putative HPA-HPG axes inte raction will help inform the Agency’s determination on the temporal and dose-response concordance of these events and how they reflect the rela tionship between the HPA and HPG axes.

5. Approaches to Evaluating Water Sampling Strategies and Frequency of Monitoring

5.1. Introduction

As a condition of re-registration, the atrazi ne registrants were required to develop a program to monitor levels of atrazi ne and its chlorinated degradates in both raw (untreated) and finished (treat ed) drinking water in areas of potential vulnerability throughout the U.S. where atrazine is used. The Atrazine Monitoring Program (AMP) conducted by the registr ant, Syngenta, began during 2003 with the collection of monitoring data from public utility Community Water Systems (CWS) located primarily in the corn and sorghum growing use areas ofthe Midwestern US. The current reevaluation of atrazine entails a re view of the most recent toxico logical data available. In conjunction with the toxicologi cal review, the Agency has init iated a re-evaluation of the sampling frequency that is necessary for dete rmining, with confidence, concentrations of the pesticide in water that sources drinking water.Page 54 of 263 Based on the best available information at the time of the 2003 IRED, the Agency

determined that the most s ensitive toxicological endpoint was associated with exposure to atrazine over a prolonged period (90-day average time frame). Syngenta employed a monitoring strategy – weekly sampling duri ng the use/high runoff season and bi-weekly sampling during other parts of the year – in tended to characterize exposure over a 90- day time frame. In implementing a monito ring program, the frequ ency of sampling is closely linked to the health effe cts of concern. If the health effects of concern relate to long-term chronic exposures, less frequent sa mpling may provide sufficient data for EPA to be reasonably confident that the result s are representative of a longer duration average concentration. However, for effect s that can occur a fter shorter exposure durations, more frequent sampling may be needed in order to be confident that the sampling has not missed a high-end or peak expo sure that could significantly affect the

estimate of short term exposure. OPP is evaluating the sampling frequency and number of samples that ma y be needed to parallel water m onitoring durations with the

durations of concern for different toxic effect s. These durations may vary from a single day peak concentration to a 3- or 7-day average concentration or even longer ( e.g., 90- day rolling average). Durations evaluated in this issue paper were selected as examples. The Agency has not yet made any decisions with respect to revising the duration of exposure concern for risk a ssessment and for drinking water monitoring

purposes.

While considering the question “How many water monitoring samples are necessary to adequately characterize varyin g exposure duration periods?”, the Agency is also interested in determining how to ma ke the best use of existing monitoring data sets with known sampling intervals. To w hat extent can pesticide concentrations be estimated between known sampling events and/or how can the uncertainty of estimated

concentrations of different averaging periods be quantified?

This section provides an overview of the nature of pesticide occurrence in surface waters and the implications both in terms of monitoring pesticides in water and interpreting monitoring studies of varyi ng sampling frequencies. The Agency discussed an approach for monitoring surface water st reams with the FIFR A SAP in December, 2007 (USEPA, 2007c). In this issue paper, the Agency presents results from two possible approaches which are similar in co ncept to the 2007 SAP analysis. Following that background, an approach is presented that models underlying distributions of data to examine the performance of different sa mpling schemes for estimating durations of

interest.

5.2 The Nature of Pesticide Occurrence in Surface Water

A unique pattern of occurrence in surface water can be i dentified for each pesticide (and, in some instances, its degrad ates) resulting from a combination of pesticide properties, use patterns, applicatio n methods and timing, weather, watershed characteristics (e.g., soil characteristics, slope, land cover type), and hydrology (GilliomPage 55 of 263 et al., 2006). While the interactions of t hese factors result in complex occurrence patterns that are difficult to adequately char acterize both geographically and temporally with monitoring (USEPA, 2000; Stone and Gi lliom, 2009), an understanding of the factors affecting pesticide distributions in water can be used to anticipate pesticide occurrence in place and time. Although such patterns also occur in differing degrees in ground water, the focus of this paper is on pesticide occurrence in surface waters of the

United States.

On a broad regional or national scale, pesticide occurrence in surface water can be largely anticipated based on the geographic distribution of pesticide use (Gilliom et al., 2006; Stone and Gilliom, 2009). Within areas of similar pesticide use, watershed characteristics such as soil susceptibility to runoff and/or erosion, weather patterns, land management practices, and hydrology can be important factors dr iving the level of pesticide concentrations in surface wa ters (USEPA, 2009a and 2009b; FIFRA SAP, 2009). Complex geographic patterns of pe sticide concentrations in water can be

characterized from the domi nant factors affecting the s patial variability (see, for example, the U.S. Geological Survey’s Wa tershed Regression on Pesticides, or WARP model, developed by Larson et al., 2004, and updated by Stone and Gilliom, 2009). Areas of the country that are particularly vulnerable to pesticide exposures in water

bodies can be identified based on a comb ination of pesticide use, weather, and watershed characteristics (see, for example, t he US EPA’s evaluation of the results of a monitoring program to assess the effects of atrazine on aquatic communities, described in USEPA, 2009a and 2009b).

While the USEPA (2009a and 2009b) wate rshed-scale vulnerability assessment for atrazine focused on small streams and aquat ic ecological community impacts, it may have potential applications for drinking wa ter exposure assessments. The Agency is currently evaluating the recommendations of the FIFRA SAP (2009 ) to improve the watershed-scale vulnerability assessments. Broader, regional-scale vulnerability approaches were also used to identify subpopulations who receive their drinking water

from vulnerable surface water sources for the cumulative assessments for both the organophosphate and the N-methyl carbam ate pesticides (USEPA, 2002, 2007b). For the cumulative risk assessments, the vulnerability of drinking water sources depended on both spatial (use, watershed characteristics, location of drinking water sources) and temporal (timing of applications for multiple pes ticides) factors. This spatial variability in pesticide concentrations in water is being addressed in other efforts; this paper will focus on addressing the temporal patterns of pesticide occurrence in surface water

bodies.

Pesticide concentrations in surface water bodies vary throughout the year, generally occurring at low to no detectable levels for most of the year, with short-term seasonal pulses of higher conc entrations that are related to the timing of pesticide use and rainfall pattern (Gilliom et al., 2006). Such short-term pul ses, or spikes, in pesticide concentrations may range from less than a day to weeks or months, depending on the fate and transport properties of the pesticide and its use, as well as the interaction ofPage 56 of 263 other driving factors (e.g., use, watershed c haracteristics, weather, land use practices) contributing to the pesticide occurrence in surface waters.

Some pesticides such as atrazine, wit h relatively widespread use and properties favorable to pesticide transport (mobility an d persistence), are often detected yearround with periods of high concentrations rangi ng from days to weeks. Other pesticides with more localized use patterns or properti es less prone to transport (less mobile and/or less persistent) may occur infrequently in water. Figure 4, ta ken from Gilliom et al. (2006), contrasts the seasonal patterns of atrazine and chlorpyr ifos in the White River in Indiana over multiple years using the same sampling frequency. A monitoring strategy that may be adequate for characteri zing the regular, periodic occurrence of atrazine may not be sufficient for a less frequently occurring pesticide such as chlorpyrifos.

Figure 4: Concentrations of atrazine a nd chlorpyrifos over time in the White River, Indiana. Source: Gilliom et al, 2006.

In any given location, the magnitude and duration of pesticide concentrations in water may vary from year to year, depending on such factors as t he magnitude, timing, and frequency of pesticide application, the timi ng and intensity of rainfall events postapplication, soil moisture conditions at the ti me of application and rainfall, and the type, timing, and extent of land management practices in place (Gilliom et al., 2006; also illustrated in Figure 4). Atrazine occurrence in most bodies of water follows a seasonal pattern that corresponds to seasonal usage and w eather patterns, with larger ‘spikes’ in concentration related to pre-emergent spring applications, particularly when followed by periods of runoff-producing rainfall.Page 57 of 263 The magnitude, duration, ti ming, and frequency of seasonal spikes in pesticide concentrations will also vary with the nat ure of the surface water body (flowing waters/streams vs. impounded waters such as reservoirs) and the size of the drainage basin. All other factors being equal, smalle r streams may have higher, more frequent spikes than larger streams wit h a larger area contributing to drainage to the water body. A different temporal exposure profile would be expected from reservoirs, which integrate pesticide loading from their watersheds and hav e slower flow-through, resulting in a greater residence time. These impounded wa ters may be expected to show smaller temporal variations than flowing wate r bodies (Blomquist et al., 2001).

In additional to seasonal and year-to-year patterns, pesticide concentrations in water show high levels of autocorrelation that are not accounted for with traditional statistical methods (Crawfor d, 2004). Pesticides such as atrazine show a serial correlation in which the probability of detecting the pesticide in water on a particular day is greater when the pesticide was detected on the previous day. Sampling designs and methods of interpreting monitoring data need to account for the temporal codependence of pesticide concentrations in wa ter: the exposures measured on one day are related to the exposures measur ed on preceding and subsequent days.

5.3 Sampling Frequency in Monitoring: Implications for Pesticide Expo sure Assessments

Depending on its quality (including locati on, timing, and frequency of sampling) and the particular exposure durations of conc ern, surface water monitoring data can be used to approximate dietary exposure to hum ans from drinking wate r sources (see, for example, the drinking water assessment for Atrazine and the cumulative assessment for

the Triazines 4 ). An important consideration in ev aluating the suitability of monitoring data for use in a dietary exposure assessm ent is how well the data represent the pesticide use both temporally and spatially. Generally, for monitoring data to be considered suitable for exposure assessments it must be targeted to the use pattern being assessed. Targeted monitoring data s hould have a study design that focuses on both where and when the pesticide is used, and should have a sample frequency designed to capture the exposure period of concern (e.g., one-day peak vs. annual average concentrations).

5.3.1 General Monitoring Strategies

A “monitoring strategy” is a combi nat ion of (a) a plan for obtaining new contaminant occurrence data and (b) a decision rule, in which the occurrence data are used to make a binary decision, such as to decide whether exposure levels are above

or below some health- or ecol ogical-based level of concern.

4 Links to both assessments can be found on the USEPA Office of Pesticide Programs atrazine web site at http ://www.epa.gov/oppsrrd1/rer egistration/atrazine/Page 58 of 263 Crafting a suitable water monitoring strategy in volves many considerations. In the case of human health concerns, these include:

• The cost of obtaining and testing sample s of water (source or drinking water)

• The spatial and temporal variability of the contaminant, particularly when occurring at or near levels of concern.

• The concentrations and durations that are associated with different health endpoints

• The severity of the adverse health outcomes

• The utility of monitoring data for informing treatment decisions

• Consequences of failing to detec t and address high occurrence (false negative)

• Consequences of false alarm (false positive)

When the above considerations lead to quantitative performanc e criteria, those

criteria become the benchmarks for evaluati ng different monitoring strategies. When available strategies fail to satisfy the criter ia, for example, becaus e they too often raise false alarm or too often fail to detect high occurrence, planners may need to reconsider the performance criteria.

5.3.2 Data Quality Objectives

Executive order 5360.1 requires the use of “a systemati c planning process to develop acceptance or performance criteria ” for environmental monitoring projects. 5
Over the past 25 years, EPA’s Quality A ssurance Staff has developed and promoted a systematic planning process for just this pur pose, called “Data Quality Objectives” or
DQOs. 6

The DQO process applies systematic plannin g and statistical hypothesis testing methodology to facilitate the design and se lection of environmental monitoring strategies. The seven steps of the process are:

1. State the Problem 2. Identify the goal of the study 3. Identify information inputs 4. Define the boundaries of the study 5. Develop the analytic approach 6. Specify performance or acceptance criteria 7. Develop the plan for obtaining data

5 USEPA Executive Order 5360.1, Policy and Program Requirements for the Mandatory Agency-Wide Quality System, Chg 1, 7/16/1998. http://www.dpnr.gov.vi/dep/5360-1c1.pdf

6 USEPA, 2006, Guidance on Systematic Planning Using the Data Quality Objectives Process (EPA QA/G4). EPA/240/B-06/001. http://www.epa.gov/quality/qs-docs/g4-final.pdfPage 59 of 263 The process becomes quantitative in Step 6, where two kinds of errors and their consequences are expressed and considered. The outputs of Step 6 are quantitative limits for the probabilities of false positives and false negativ es. A statistician can then evaluate how well selected monitoring strategi es satisfy these criteria, given different occurrence scenarios or patterns. In general, waters with high variability are the most challenging. Water having a low occurrence le vel, but high variability, can experience a false positive whenever a sample is taken during a short-lived occurrence spike. Similarly, water with a high occurrence leve l with high variability may experience a false negative and show lower levels because infr equent monitoring failed to catch a large occurrence spike. Several exam ple profiles are presented below.

5.3.3 Monitoring Strategies Related to Pesticides

The Agenc y and other researchers have st udied the issue of sampling frequency to characterize pesticide expo sure for drinking water (see, for example, ILSI, 1999; USEPA, 2000; and Crawford, 2004). Generally, longer dur ations of concern (e.g. annual average concentration for a long-term chronic effect) require less frequent sampling to approximate longer term exposures . However, as the duration of the exposure of concern shortens, the fr equency and timing of sampling become more important in determining the confidence in how well the sample data capture these short-duration exposures. This is most critical when the Agency is concerned about acute effects resulting from peak single day exposures or from short-duration exposures. For such short- term exposures of concern, the likelihood of adequately characterizing the high end of exposure fo r decreases as the time between sampling

events increases.

While the time frame of interest in relati on to the health effect (s) of concern is a key consideration in determining the samplin g frequency, logistics and cost also remain a major factor in the design of any sampli ng program (ILSI, 1999; US EPA, 2000). Often the Agency is left to determine how best to evaluate monitoring studies with samples that are taken less frequently than desirable for an effect ’s period of interest.

Few pesticide monitoring studies have cons istently sampled pesticides in water at daily time intervals. An exception is the intensive pesticide sampling efforts conducted by Heidelberg University’s Nati onal Center for Water Quality Research (NCWQR; formerly known as the Heidelber g College Water Quality Laboratory) on selected streams draining into Lake Erie 7 . Since the 1980s, the NCWQR has intensively sampled for a number of pesticides in select ed Ohio streams of various sizes. This dataset has proven valuable in evaluating varying sampling frequencies (see Crawford, 2004). The USGS National Water Quality Assessment (NAWQA), one of the more extensive national water monitoring program s that includes pesticides, generally

7 More information on the NCWQR program, its publications, and water quality data retrieval can be found

at http://www.heidelberg.edu/WQL

.Page 60 of 263 sampled weekly to twice monthly during t he time of anticipated highest pesticide use and runoff (Gilliom et al, 2006). On the other end of the sampling frequency spectrum, USEPA’s compliance monitoring regulati ons under the Safe Drinking Water Act generally rely on quarterly sampling from state water programs to determine

concentrations of contaminants in water. A number of studies of varying sampling frequencies have been conducted by pesticide regi strants, such as the Acetochlor Registration Partnership, ARP (monitoring program description and water quality data are available through USEPA, 2007) and Syngenta’s Atrazine Monitoring Program (AMP) for Community Water Systems (C WS) and Atrazine Ecological Exposure Monitoring Program (AEEMP) to assess impacts to aquatic communities (more information on these programs is availabl e through USEPA’s atra zine web site at http://www.epa.gov/pesticides/rer egistration/atrazine/in dex.htm ).

After the passage of the Food Quality Protection Act (FQPA) in 1996, the International Life Sciences Institute (I LSI) convened a panel whose charges included providing guidance on monitoring and m odeling needs for generating probability distributions of pesticide concentrations in drinking water exposure assessments. Based on their best professional judgment ( defined by ILSI, 1999, as a “statistical probability sampling... modified at ti mes to reflect the real cost or practical and logistical constraints”), the ILSI panel recomm ended a minimum of 100 samples per year, targeting the likely pesticide use seasons, for a high level of prediction accuracy for maximum peak exposures at a single water supply (ILSI, 1999). For regional and national exposures, ILSI (1999) recomm ended fewer samples at individual supply

facilities as the number of monitored water s upplies increased, reflecting the “practical cost” of sampling.

How well monitoring data capture pestici de concentrations over time depends on both the frequency and timing of sampling events relative to t he “true” exposure profile. Crawford (2004) found that, for sampling pr ograms with a fixed number of samples, seasonally weighted sampling strategies provided better estimates of time-weighted mean and percentile concentrations of pesticides (in terms of precision and accuracy) compared to fixed interval sampling frequenc ies spread throughout the year. More frequent sampling is needed to achieve the same level of precision and accuracy for small streams compared to la rger streams which integrat e pesticide loadings over a larger watershed (Crawford, 2004).

The number and frequency of sampling events will also depend on the relative

time frame of concern as it relates to t he effect endpoint and the dur ation of effects. The shorter the time averaging period requi red for a short-term effect, the more frequently the samples need to be taken to achieve the same level of precision and accuracy as seen for a longer averaging peri od (Crawford, 2004). Similarly, for a distribution of concentrations in a year, t he level of precision for a given sampling frequency decreases as the concentration per centile in the distribution increases (Crawford, 2004).Page 61 of 263 5.4 Approaches to Analyzing and Interp reting Monitoring Data Collected at Different Sampling Frequencies

In most cases, the Agency must dete rmine how to interpret monitoring data collected at different sampling frequencies. This involves determining how well the monitoring data capture the act ual pesticide concentrations in water over time for the particular toxicity endpoint duration of c oncern and providing conf idence bounds on the exposure estimate from t he monitoring data. The less frequent the sampling interval, the less information is available to characterize the “true” exposure time series of pesticide concentrations in surface waters. The Agency has evaluated or will be evaluating various methods to interpret ava ilable monitoring data in the absence of a full time series profile for the pesticide conc entrations. The applicability of the methods considered depends on both t he quality of the monitoring data and the time frame of

interest for the health effect (acute, short- or intermedi ate-term, chronic effects). Approaches to provide a confidence bound on exposure estimates follow a brief

discussion of those methods below.

5.4.1 Characterizing Exposure Ti me Series From Less Frequent

Monitorin g Data

At the most basic end of the spectrum, for risk assessment purposes the Agency

could use only the measured pesticide concentrations as the exposure

distribution for a drinking water assessment. This approach does not explicitly address what exposures occur on non-sampled days, but assumes that mean, standard deviation, and distribution of measured samp les are representativ e of the “true” exposure time series in the water body. This approach also assu mes that the maximum exposures of the true distri bution are represented in the sample. The fewer the samples taken and the greater the interval between sampling events, the less likely the sampling dataset is to represent either maxi mum exposures or the ac tual distribution of pesticide concentrations in the water body.

Another commonly used approach to interpret monitoring data is to linearly interpolate between the sampling points . This approach assumes a linear distribution between sampling dates (illustrated with the red li nes in Figure 5), which is less likely to hold the farther apart the samp les occur, and assumes no concentrations greater than what was captured in samples. As shown in Figure 5, the timing of the 14- and 30-day grab samples 8 captured exposures on eit her side of the actual concentration peak in late April but miss ed the “true” maximu m concentration. The extent to which a linear interpolation bet ween sampling points adequately characterizes the “true” exposure time series depends not only on the timing of sampling and sampling interval, but on the shape of the act ual concentration time series. Such an

8 The term “grab sample” can refer both to samples dr awn from the water that do not integrate across the water body itself and to samples taken at a particular point in time that do not integrate across any time frame. In this document, the term “grab sample ” refers solely to the temporal aspects.Page 62 of 263 approach will likely underestimate short-term or peak exposu res but may be useful to characterize pesticide concentration prof iles that have less pronounced temporal variations, such as those that may occu r in reservoirs or large rivers.

0 10 20 30 40 50 60

3/1 3/15 3/29 4/12 4/26 5/10 5/24 6/7 6/21 7/5 7/19 8/2 8/16 8/30 9/13 9/27

DateAtrazine (TCT), ug/L Daily Time Series

14-day Grab (linear)

30-day Grab (linear)

14-day Grab (stairstep)

30-day Grab (stairstep)

Figure 5: Comparison of daily time series for pesticide concentrations (dark blue line) to linear (red lines) and stair-step (light blue lines) extrapolations for samples taken at 14- or 30-day intervals.

An alternative to the linear approach is a stair-step imputation between sample points . In this method, the pesticide concentration measured on a particular sampling date is assumed to apply to subsequent days until the next sampling date (illustrated with the light blue lines in Figure 5). As with the other methods described so far, the stair-step imputation assumes no concentrations great er than what was captured in samples. The probability of mi ssing maximum concentrations increases as the interval between samples increases . The stair-step approach has a greater likelihood of overestimating longer-term, time-averaged concentrations than does the linear interpolation approach. As illustrated in Figure 5, the longer the time interval between samples, the less likely the stair-s tep imputation will resemble the “true” exposure time series.

These approaches, and similar variations, make simplified assumptions about the nature of pesticide conc entrations in the water body and how well the sample pointsPage 63 of 263 reflect the distribution of the entire time se ries. These assumptions are less likely to hold as the sampling interval increases. In addition, the approaches fail to account for the temporal autocorrelation that characte rizes pesticide concentrations in surface waters. Unless the monitoring dataset spans multiple years, it is also unlikely to account for the year-to-year variability that results from changes in land use, pesticide use patterns, and weather.

More rigorous methods of interpreting monitoring data may include statistical

methods such as bootstrapping, neural networks, kriging, random function models, regression-based models that predict pesticide concentrations from measured variables, and deterministic models based on known relationships between pesticide concentrations and physical processes in the environment. The Agency is in the process of evaluating the utility of such methods and only makes brief mention of a few selected approaches here. In the section t hat follows, the Agency presents preliminary evaluations of the simulation approach taken by Crawford (2004) as well as an alternate method that examines simulations produc ing distributions of maximum running averages. USEPA anticipates feedback and recommendations from the FIFRA SAP on these preliminary approaches, as well as other methods including those mentioned here

and others not considered.

USEPA’s Office of Water (OW) has ex plored a modeling approach using the application of neural networks that will be presented in the fo llowing section, as well.

While artificial neural networks are statisti cal modeling tools that can be used to model complex relationships or to find patterns in data, as with any of the above approaches, results are only as good as the sampling data av ailable. This approach is illustrated in more detail in Section 5.6 and Appendix C.

While kriging and random function model approaches have generally been applied to geostatistical analyses (Isaaks and Srivastava, 1989; Deutsch and Journel, 1998), their assumptions of co-linearity can be applied to the temporal autocorrelation inherent to pesticide concentration time seri es in surface waters. A nearest neighbor approach accounts for temporal autocorrelation by measuring the variance at different lag times and then linearly interpolating bet ween points with an appropriate weighting from nearest neighbors (Figure 6). Based on the nature of occurrence patterns represented by a sequence of observations, the approach estimates missing values with non-subjective linear inte rpolation techniques. A stochastic simulation adds an uncertainty analysis and provides a probabilisti c assessment of daily concentrations. Kriging may not be viable, however, for pesti cide distributions that lack significant correlation (such as the chlorpyrifos dist ributions shown in Figure 4). The data smoothing techniques illustr ated here will underestimate high occurrence events.Page 64 of 263 Figure 6: Conceptual illustration of th e nearest neighbor approach for estimating daily pesticide concentrations at l ess frequent sampling intervals.

Multiple regression models may be useful for estimating pesticide concentrations, provided the relationships ar e known and the param eters are available to populate the model. Larson et al (2004) developed the Watershed Regression on Pesticides (WARP) model for atrazine based on nationally-available geospatial data and NAWQA monitoring data for atrazine. Th e intent of the model was to provide predictions of pesticide concentrations in unmonitored or inadequately monitored streams. Because of the sampling frequen cy of the atrazine monitoring data used to develop the model (weekly to bi-weekly during the pesticide use and likely runoff season), WARP originally did not provide pesticide exposure estimates greater than the

95 th percentile of yearly distributions. At given locations, pesticide concentrations estimated by WARP were within an order of magnitude of measured concentrations in water. While predictions of percentile atrazine concentrations tended to be low in comparison to actual measur ements for most validation si tes with the original model, predictions with the updat ed model were “not substantially biased” (Stone and Gilliom, 2009). However, because the sampling frequencies of the NAWQA model-development sites were not sufficient to reliably charac terize the highest concentrations during a year, estimates of the annual maximum co ncentration from the updated WARP model are expected to underpredict the actual annual maximums (Stone and GIlliom, 2009). The authors noted that while WARP expos ure estimates “cannot replace direct measurements when reliability r equirements are high,” they provide screening estimates that can be used to target more intensiv e monitoring (Stone and GI lliom, 2009).

The 2009 FIFRA SAP reviewing the Agency’s assessment of the ecological impacts of atrazine concentrations in st reams in corn- and sorghum-growing areas recommended that the Agency consider a Cor nbelt WARP model that utilized the data and information gleaned from that monitori ng effort (FIFRA SAP, 2009). USEPA is continuing to analyze the results of that in tensive watershed monitoring effort conducted by Syngenta Crop Sciences and will be looki ng into ways the WARP model could provide improved spatial and temporal estimate s of atrazine exposure in streams.

The Agency has used deterministic models such as PRZM (Pesticide Root Zone Model) and EXAMS (Exposure Analysis M odeling System) to estimate multi-year daily pesticide exposure series for use in probabilistic drinking water exposurePage 65 of 263 assessments 9 . These deterministic models coul d be used to estimate exposures between monitoring samples provided that local soil, crop, and weather data are available. Estimated exposures generated by PRZM-EXAMS could be calibrated to the measured concentrations and provide a comple te time series between sampling dates. USEPA (2007) reviewed Syngenta Crop Science’s use of PRZM with site-specific soil, cropping, and weather data to estimate atrazine concentrations between 4-day monitoring samples as a part of the ecological exposure monitoring program. Even with the relatively frequent sampling intervals, time-averaged exposures estimated with the PRZM augmentations increased fo r the majority of the monito ring site years (66 out of 80) in comparison to stair-step imputations of the 4-day samp les (USEPA, 2007).

5.4.2 Determining Confidence Bounds on Exposure Estimates From

Monitorin g Data

At a given location, the degree to which a monitoring dataset characterizes the “true” pesticide exposure profile and the level of uncertainty in the exposure estimates from that data set depend not only on t he sampling frequency and the duration of sampling but also on the time frame of intere st for the health effect (acute, short- or intermediate-term, chronic e ffects). While the capabilitie s of the methods and models for interpreting monitoring data of various sampling frequencies can be evaluated using goodness-of-fit and residual error tools (see Stone and Gilliom, 2009, for examples), the Agency is also concerned with how accurately the available monitoring data set captures the true pesticide concentration expos ure of concern, whether it is a maximum peak or exposure over a multi-day time period.

The nature of the temporal patterns of pesticide occurrence in surface waters presents challenges in uncertainty analysis. These analyses need to account for the temporal relationships in pes ticide concentrations, the serial correlations from day to day, the periodicity in elevated concentration s within seasons and from year to year, the abundance of censored data (non-detecti ons and pesticide concentrations below analytical limits of detection), and uncertainty in the shape of the pesticide distributions in surface waters. The Agency is solicitin g feedback from the FIFR A SAP on statistical approaches that may addr ess these issues.

In the preliminary analyses below, the Agency is evaluating approaches for characterizing the level of uncertainty in predicting true pestici de concentrations over varying averaging times based on varying sampling frequencies and strategies. The resulting uncertainty can be reflected in a couple of basic approaches. In one approach, the Agency can determi ne a prediction interval with specified confidence for the predicted new exposures from monitoring data, similar to the approach described by Stone and GIlliom (2009). Or, for example, for a particular monitoring data set, the USEPA can describe the range in values with in which a given percent, e.g. 99 percent,

9 See USEPA OPP’s Water Models web page at http://www.epa.gov/oppefed1/models/water/ for more

information.Page 66 of 263 of monitoring samples are expected to occu r (tolerance interval). An upper bound of this interval can be compared to the toxici ty endpoint of concern to determine whether the true exposures could exceed the to xicity thresholds of concern.

Another approach to account for the uncerta inty in monitoring data would be to place intervals around the threshold concentrati ons of concern rather than the exposure estimate from the monitoring dataset. The lower bound on the threshold would be constructed for a given confidence bas ed on the timing and frequency of sampling events for the monitoring dataset. For exam ple, suppose the exposure threshold of concern for a particular health effect is a 3-day average concent ration of 15 ug/L. A lower threshold value of 10 ug/L might be se t for monitoring data with weekly sampling intervals during the pesticide use season. In this example, the lower threshold value reflects the maximum 3-day average concentr ation from a weekly monitoring dataset that would be expected to exceed the actual threshold concentration of concern with, for example, 95 percent confidence. ILSI (1999) provided guidance on estimating

confidence and tolerance intervals and calculat ing prediction intervals to determine the appropriate sample size for a monitoring st udy. The same or similar approaches might be adapted to evaluate the results of monito ring data against health effects thresholds

of concern.

5.5 Examining the Performance of Va rio us Sampling Strategies with Data from the Atrazine Monitoring Program (AMP)

5.5.1 Profiles of Community Water Systems in the AMP

The AMP Monitoring of Community Wate r Systems (CWS) for surface water serving 10,000 or more residents began duri ng 2003. The program focused on systems with higher expected concentrations, based on compliance monitoring, that was carried out on a weekly basis during the growing se ason (April through July or August in corn areas) and biweekly during the remainder of the year. Any CW S that exceeds an annual mean screening level conc entration of 1.6 ppb atra zine from quarterly Safe Drinking Water Act (SDWA) data is includ ed in the AMP. In the seven years of monitoring, the program has gr own from an original 130 CWS to a little over 150 CWS per year in a given year. Under the above monitoring strategy, between 30 and 35 samples each of raw and finished water are typically collected at each system annually. While both raw and treated water samples ha ve been collected, the present paper looks exclusively at raw water for summarizing information on results before any treatment

has occurred.

The numbers of raw water samples each y ear have ranged from just under 4,000 to over 4,800 samples for the 5 full y ears 2004 through 2008. Chemograph profiles of peak results and concentrations exceeding several arbitrary comparison concentrations are presented in Table 5 below.Page 67 of 263 Table 5: Summary of Atrazine Monitori ng Program (AMP) Data for Atrazine in

Raw Water*

Year Total samples Percentage

measurements. <1.0 ppb Percentage

meas. < 3.0ppb #Peaks> 12.0ppb Percentage peaks >12.0ppb 99 th

percentile

Atz.Conc. (ppb) 99.9 th

percentile

Atz.

Conc. (ppb) Maximum

Atrazine

Conc. in

Year (ppb)

2004 4429 54.4 95.6 19 0.4% 7.9 24.8 47.5

2005 4779 64.2 91.9 71 1.5% 15.4 46.0 84.8

2006 4817 77.6 93.7 30 0.6% 9.3 28.4 227

2007 4521 77.7 94.5 10 0.2% 7.1 15.9 25.8

2008 3927 72.8 92.2 49 1.3% 13.3 42.9 73.0

* Source: Raw data submitted by Syngenta annually to EPA

In general, the number of individual sample s that are below the concentration of 3 ppb is high, ranging from approximately 92- 96% of the 4,000+ sa mples each year. The number of individual samples below 1.0 ppb ranges from about 54% in 2004 to

about 78% in 2007.

Maximum concentrations are given for each year, and range fr om 25.8 ppb in 2007 to 227 ppb in 2006. Maximum peaks for t he other years vary each year as well, going from 47.5 ppb in 2004 to 84.8 ppb in 2005, to 73.0 ppb in 2008. The table also

lists the 99 th %ile and the 99.9 th %ile values for all sample s each year, with the former ranging from about 7 ppb to just over 15ppb, and the latter r anging from about 16 ppb to 46 ppb.

Since there is interest in capturing individual peak concentrations in the monitoring, the number of individual peaks exceeding several arbitrary concentration levels is also presented in Table 5 above as well as in Table 6 below. It is important to reiterate that these “peaks” are based on sampling that was carried out once a week during the season of use and biw eekly during the remainder of the year. As such, it is likely to underestimate the “true” peak c oncentrations at these CWS. In the methodology outlined below, however, the signifi cance of the relative shapes of the chemographs will become clear when they are used to create reference profiles for comparison of sampling strategies.

Looking at individual samples that equal or exceed 12 ppb in the monitoring, the number of peaks varies each year and ranges fr om 10 samples in 2007 to 71 (1.5%) in the year 2005. When 20 ppb is used as the cut-off, the num ber of peaks exceeding that level falls to 1 sample in 2007 up to 22 samples in the year 2005. At 30 ppb thePage 68 of 263 numbers decline even further, ranging from none of the 4, 521 samples reaching that level in 2007 up to 10 samples (0.3%) exceeding 30 ppb in 2008. 10

Table 6: Atrazine Monitoring Program Data for Atrazine in Raw Water*

Year Total samples #peaks>
20ppb # peaks>
30ppb #CWS w. a peak
>12ppb #CWS w. a peak >
20 ppb #CWS w. a peak
>30ppb

2004 4429 6 2 15 / 141 5 2

2005 4779 22 7 27 / 142 12 6

2006 4817 11 2 20 / 151 10 1

2007 4521 1 0 7 / 137 1 0

2008 3927 19 10 23 / 134 14 9

* Source: Raw data submitted by Syngenta annually to EPA

Generally over the course of the 5 years of moni toring presented, the peaks discussed above occurred in a fairly cons istent set of CWS each year. Peaks exceeding 12 ppb occurred in up to 20-30 CWS ov er the years, while peaks exceeding 20 ppb occurred in a subset of 5-15 of thos e systems. The few higher peaks exceeding 30 ppb occurred in 9 or fewer [ per year] of the 130-150 CWS.

Clearly, both the number and magnitude of individual atrazine peaks has varied each year within the AMP data. Across the years, however, a fairly consistent set of CWS appears to show the highest levels.

5.5.2. Approaches from the 2007 and 2009 SAP Meetings on

Monitorin g in Surface Water St reams in the Corn and Sorghum Growing Region of the US

In December 2007, the Agency summarized its preliminary review and interpretation of the initial results of the Atrazine Ecological Exposure Monitoring Program (AEEMP) before the FI FRA SAP. The Panel provided feedback related to the interpretation of the results and the extent to which the methods used by the Agency could be implemented or adapted in any fu ture aquatic assessments or monitoring

efforts to determine the extent to which water bodies exceed atrazine thresholds of

concern.

One aspect of that SAP M eeting looked at adapting Crawford’s 2004 “Sampling strategies for estimating acute and chronic exposures of pesticides in streams” to

10 The numbers presented refer to individual peak samples and do not represent averages of any

duration. For the current level of concern there were no 90-day average concentrations equal to or exceeding the 37.5 ppb concentrationPage 69 of 263 evaluate sampling frequencies. Crawford (20 04) used data from 10 years of monitoring by the Heidelberg College Wate r Quality Laborator y from four rivers and streams in northwestern Ohio. The sampling data were augmented, where necessary, using interpolation between samples to create 365-day profiles which served as the “true” chemographs for the exercise. Alternative sampling strat egies, ranging from quarterly sampling to sampling 10 times per month, were tested by sampling from the daily profiles 1,000 times at each site for each year. The performance of the various sampling strategies relative to the “tr ue” profiles was evaluated by examining differences from the daily profile for the mean and upper percentiles of each alternative strategy.

A similar approach was first recommended by Crawford and coauthors in the International Life Sciences Institute (ILS I) 1999 report, “A Framework for Estimating Pesticide Concentrations in Drinking Water for Aggregate Exposure Assessments.” The current SAP paper looks at methods similar to these approaches to present preliminary results for example chemographs from a group of ten diffe rent CWS in the Atrazine Monitoring Program.

5.5.3 Current Methods

5.5.3.1 Comparing Upper Percentile Values from Different Sampling Strategies w ith those of the “True” Profile

The first method employed in the current paper generally follows the Crawford (2004) and ILSI (1999) methods. The exam ples below used the AMP monitoring with sampling weekly during the mont hs of April through July or August, and biweekly before and after this season. For each chemograph consisting of 30-35 samples, a 365-day profile was created by linear in terpolation between any successive values that were not taken daily. For purposes of this prelimi nary exercise, these profiles served as the “true” chemographs for each site, against which those deriving from alternative sampling strategies were compared. 11 For each of the “true” chemographs, the mean and upper percentiles (90 th , 95 th , 99 th , 99.7 th , and 99.9 th ) were recorded.

From the true chemographs a number of different sampling strategies were set up to sample from t he augmented data sets on the following schedules:

Seasonal daily (D1s)—daily from April through August; biweekly before and after Every other day (2d) Every third day (3d) Every 4 th day (4d) Weekly seasonal (W1s)—weekly from April through August; biweekly before and after

11 The Agency notes that using derived chemographs that smooth out the day-to-day variations is less desirable than chemographs such as some of the He idelberg dataset which have more frequent sampling that better reflects actual day-to-day variations in exposure.Page 70 of 263 Weekly annual (W1a) Every ten days (10d) Biweekly (M2a) Monthly (M) Every 45 and 60 days Quarterly (Q)

Chemographs were resampled with r eplacement on the above schedules following a random start, wit h over 4,500 simulations ru n for each schedule. Each resample was augmented to 365 days by linear interpolati on and the mean and upper percentile values were recorded for each. The simulations were aggregated for each CWS and mean values for each percentile c oncentration were presented by sampling strategy and compared with t hose for the “true.” Resu lts for the daily peaks are presented below for several example chemographs and a subset of the sampling strategies.

Table 7 shows that for yearly profiles wit h 365 daily values it was possible to obtain estimates up to the 99.7 th %ile for each sampling strategy. For CWS #22, sampling every 4 days returned estimates of the 99.7 th %ile single day values that averaged around -12% of true, while those for the 99 th %ile underestimated the true by about 7%. It was possible to estimate the 95 th %ile of the single day peaks with good accuracy (+1.08%), as well as the 90 th and 50 th percentiles and the mean (0.31%). Estimates for the weekly and biweekly sampling followed a similar pattern, but with accuracy for the given percentiles that ranged fr om -21% to +8% for weekly and -42% to +35% for biweekly sampling. Similar to the ILSI findings, the accu racy of the percentile estimates fluctuated somewhat within each sampling set. The precision of the estimates followed an expected pattern, how ever, with good precision at the mean and wider intervals for the highest estimates.

Table 7 presents estimates for two other CWS whose results show increasing accuracy that is likely related to their shape, as discussed in the next section below. Results for CWS #8 ranged from -5-6% to +8 % for the given percentiles for sampling every 4 days, and estimated both mean and medi an with virtually no error for both 4-day and weekly sampling. For CWS #24, accuracy was good at both the upper percentiles and the mean. Figures 7 (a-c) show the chemograph profiles.

Crawford (2004) reported disparity in comparisons between true values and estimates derived from sampling at the 99 th %ile, although for larger rivers he could achieve estimates within approximately 50% of the “true.” Performance for the smaller streams was not good at that level, but was generally acceptable for the 90 th and 95 th
percentiles.Page 71 of 263 Table 7: Upper Percentile Values Derived from Different Sampling Strategies for 3 Example CWS

Mean median 90th %ile 95th %ile 99th %ile 99.7th

%ile Peak/

maximum



### CWS #22

True Chemograph (daily)

Concentration, ppb 2.718 0.306 8.293 12.368 46.806 57.34 57.98

Sampling every 4 days

(mean) Estimate 2.73 0.32 8.51 12.50 43.64 50.47

(% error) 0.31 3.03 2.67 1.08 ‐ 6.77 ‐ 11.97

(95%CI) (2.61

‐ 2.83) (0.316

‐ 0.316) (8.20

‐ 8.90) (11.56

‐ 13.43) (37.81

‐ 46.85) (41.91

‐ 57.35)

Sampling every 7 days

(mean) Estimate 2.72 0.32 8.95 12.48 38.95 45.05

(% error) 0.21 3.92 7.98 0.92 ‐ 16.78 ‐ 21.42

(95%CI) (2.49

‐ 2.93) (0.31

‐ 0.33) (8.06

‐ 9.92) (11.07

‐ 14.12) (29.85

‐ 47.73) (32.38

‐ 57.42)

Sampling every 2 weeks

(mean) Estimate 2.72 0.34 9.00 16.71 30.52 33.35

(% error) 0.19 10.29 8.49 35.10 ‐ 34.79 ‐ 41.84

(95%CI) (1.96

‐ 3.51) (0.31

‐ 0.37) (6.67

‐ 11.92) (11.60

‐ 24.21) (12.70

‐ 51.90) (12.79

‐ 57.63)



### CWS #8

True Chemograph (daily)

Concentration, ppb 3.315 0.660 6.622 25.541 40.755 44.76 44.92

Sampling every 4 days

(mean) Estimate 3.31 0.66 7.14 25.45 39.37 42.27

(% error)
‐ 0.01 0.00 7.79 ‐ 0.36 ‐ 3.40 ‐ 5.57

(95%CI) (3.245

‐ 3.38) (0.66

‐ 0.66) (6.595

‐ 8.458) (24.77

‐ 25.54) (37.35

‐ 40.75) (38.57

‐ 44.76)

Sampling every 7 days

(mean) Estimate 3.31 0.66 7.94 25.15 37.74 40.41

(% error)
‐ 0.02 0.00 19.83 ‐ 1.51 ‐ 7.39 ‐ 9.72

(95%CI) (3.18

‐ 3.44) (0.66

‐ 0.66) (6.25

‐ 9.81) (23.66

‐ 25.96) (33.71

‐ 40.91) (35.45

‐ 44.77)

Sampling every 2 weeks

(mean) Estimate 3.31 0.69 9.60 23.99 34.25 36.33

(% error)
‐ 0.08 3.98 44.96 ‐ 6.06 ‐ 15.97 ‐ 18.83

(95%CI) (2.79

‐ 3.83) (0.66

‐ 0.74) (5.05

‐ 13.05) (19.56

‐ 28.47) (28.04

‐ 41.91) (28.53

‐ 44.79)Page 72 of 263 Mean median 90th %ile 95th %ile 99th %ile 99.7th

%ile Peak/

maximum



### CWS #24

True Chemograph (daily)

Concentration, ppb 1.613 0.864 3.769 5.279 6.169 6.34 6.35

Sampling every 4 days

(mean) Estimate 1.62 0.93 3.77 5.05 6.11 6.22

(% error) 0.28 7.20 0.08 ‐ 4.28 ‐ 1.01 ‐ 1.84

(95%CI) (1.60

‐ 1.63) (0.88

‐ 1.04) (3.58

‐ 3.95) (4.71

‐ 5.29) (6.00

‐ 6.17) (6.06

‐ 6.34)

Sampling every 7 days

(mean) Estimate 1.62 0.99 3.80 4.88 6.00 6.13

(% error): 0.26 14.21 0.88 ‐ 7.61 ‐ 2.73 ‐ 3.29

(95%CI) (1.59

‐ 1.65) (0.85

‐ 1.08) (3.37

‐ 4.13) (4.57

‐ 5.29) (5.84

‐ 6.17) (5.88

‐ 6.34)

Sampling every 2 weeks

(mean) Estimate 1.62 1.04 3.80 4.70 5.70 5.93

(% error): 0.29 20.19 0.94 ‐ 10.92 ‐ 7.54 ‐ 6.54

(95%CI) (1.45

‐ 1.79) (0.88

‐ 1.13) (2.92

‐ 4.81) (4.16

‐ 5.37) (5.33

‐ 5.99) (5.36

‐ 6.33)Page 73 of 263 Figure 7: (a-c) Profiles fo r Chemographs #22, #8, and #24

(a) Chemograph for CWS #22

CWS #22 Illinois, 2005

0 10 20 30 40 50 60 70

1/1 1/31 3/2 4/1 5/1 5/31 6/30 7/30 8/29 9/28 10/28 11/27 12/27

DateAtrazine (TCT), ug/L

(b) Chemograph for CWS #8

CWS #8 Illinois, 2008

0 5 10 15 20 25 30 35 40 45 50

1/1 1/31 3/1 3/31 4/30 5/30 6/29 7/29 8/28 9/27 10/27 11/26 12/26

DateAtrazine (TCT), ug/LPage 74 of 263 (c) Chemograph for CWS #24

CWS #24 Illinois, 2005

0 1 2 3 4 5 6 7

1/1 1/31 3/2 4/1 5/1 5/31 6/30 7/30 8/29 9/28 10/28 11/27 12/27

DateAtrazine (TCT), ug/L

5.5.3.2. Examining the Ranges of Maximum Running Averages

of Differen t Durations for Various Sampling Strategies

In the process of determining exposure estimates from drinking water for the purpose of assessing risk to human populations, USEPA derives the maximum

measured concentration from monitored data to estimate potential “maximum” exposure for different durations of interest (e .g., single day peak, maximum 3-day average, maximum 7-day average, maximum 90-day aver age) for a given system by year. With the above method, it was not possible to esti mate the very highest percentile values in the year of a given system with great accuracy and precision, if at all. Data presented above for the AMP program show a large difference among values in the upper percentile range for systems with the highest peaks, even between 99.5 th %ile and

maximum values. Because of these limitatio ns in estimating highest percentile values and because the sample maximum value is t he concentration typically derived for each

duration of interest and used in OPP ri sk assessments, an alte rnative method was employed to assess the performance of diffe rent sampling schemes in capturing the “true” chemograph maximum.

.

The alternative method that follows begi ns similarly to the method discussed above. In the examples below, for eac h chemograph consisting of roughly 30-35 samples, a 365-day profile again was creat ed by linear interpolation between any successive values that were not taken daily. These profiles served as the “true” chemographs for each site, against which t hose deriving from alternative samplingPage 75 of 263 strategies were compared. For each of t he “true” chemographs, th e following ‘running’ averages were derived, and the maximum of each of those aver ages was recorded:

Single day peak 2-day average 3-day average 4-day average 7-day average 14-day average 21-day average 28-day, 60-day, 90-day, 180-day, and Annual average

At the same time a number of different sampling strategies described below were

simulated from the raw data se ts on the following schedules:

Seasonal daily (D1s—daily from April through August; biweekly before and after) Every other day (2d) Every third day (3d) Every 4 th day (4d) Weekly seasonal (W1s)—weekly from April through August; biweekly before and after Weekly annual (W1a) Every ten days (10d) Biweekly (M2a) Monthly (M) Every 45 and 60 days Quarterly (Q)

Sampling according to the above schedules was carried out with replacement after a random start, and each resample wa s augmented to 365 daily values by linear interpolation. A total of over 4,500 simulations were run for each sampling scheme, and the maximum single day peak, maximum 2-day average, maximum 3-day average, etc.

were recorded for each run.

For every sampling strategy the range and summary percentiles of the distributions of maximum averages for each duration period were compiled. For example, for sampling every 4 days, the di stribution of maximum 3-day averages, the distribution of maximum 7-day averages, and the distribution of maximum 14-day, etc., averages were compiled. In addition, the percentile of the “true” maximum average for each sampling scheme and duration was recor ded within these results. The Tables

below illustrate.Page 76 of 263 5.5.3.3 Results

As illustrated in the tables that follow, sampling according to a schedule that is less than daily makes it possible both to over estimate as well as to underestimate the true average of interest that is derived from daily sampling. As would be expected, with more frequent sampling and linear interpolat ion between sampling points, both the percentage of simulations underestimating the “t rue” average of interest as well as the magnitude of the underestimati on is less than for strategies employing less frequent sampling. Additionally, for averages of longer duration, the amount of underestimation is generally less than for those of shorter duration, so tha t, for example, the maximum 7- day average concentration is underestimated less often and by a lower amount than the maximum 3-day average or the maximum single day peak.

For longer term averages, in fact, the amount by which the maximum average of interest may be overestimated increased as the average and the sampling interval began to increase. Since the overestimation was not great, however, in this exercise

attention was directed to the lo wer range of the rea lizations to characterize how often a given maximum average might be underestimated and by how much.

For the CWS that were run using th is methodology and the 10 CWS profiles presented in this paper, some general patte rns began to emerge, where performance in capturing peaks of interest appeared to correspond to the general shapes of the chemographs assessed.

Chemograph examples: Single narrow peaks

CWS #4 in 2008 is typical of a group of chemographs that show one high and narrow peak that is detected in a given week and appears to decline substantially by the following week. The maximum daily peak of 72.99 is preceded by a concentration of 0.38 ppb measured the week before, and fo llowed by a concentration of 10.70 ppb measured 7 days later.Page 77 of 263 CWS #4 Ohio, 2008

0 10 20 30 40 50 60 70 80

1/15 2/15 3/15 4/15 5/15 6/15 7/15 8/15 9/15 10/15 11/15

DateAtrazine (TCT), ug/L

Figure 8: Illustration of a CWS chemograph with a single, relatively short duration (narrow) peak

Single Daily Peaks

When the chemograph shown in Figure 8 was sampled every 4 days, the maximum daily value of 72.99 ppb was captur ed about 26% of the time. The mean of the simulations gave a maximum value of 63.21, which underestimated the “true” maximum by a little over 13%. The lowest 1% of the resamples was 37% lower than the true maximum concentration (Table 8).

Weekly sampling was less accurate, capt uring the true maximum over 14% of the time, and underestimating it by about 23% on average and over 56% at the low end of the range. Biweekly sampling captur ed the true max about 7% of the time and underestimated the daily peak by about 46% on average and about 86% on the low

end.Page 78 of 263 Table 8: Percentiles of Maximum Concentr ations for CWS #4 Sampled at 4-, 7-, or 14-day Intervals

CWS True

Value mean 1 st % 5th% 10th% 50th% 90th% 99.9 th % % of sample

max avgs >

true Sampling every 4 Days

4 72.99 63.21 46.33 52.24 52.24 62.62 72.99 72.99 25.58

% error -13.40 -36.52 -28.42 -28.42 -14.21 0.00 0.00
Sampling every 7 Days

4 72.99 56.04 31.50 37.45 41.87 55.22 72.99 72.99 14.50

% error -23.22 -56.85 -48.70 -42.63 -24.35 0.00 0.00
Sampling every 2 Weeks

4 72.99 39.63 10.38 10.75 10.79 37.45 64.10 72.99 7.19

% error -45.70 -85.79 -85.27 -85.22 -48.70 -12.17 0.00

As may be apparent, the right hand column representing the percentage of time that the sample max is captured is close to expected for the th ree different sampling schemes, which would be about 25% of the time for sampling every 4 days, 14% for sampling every 7 days, and 7% for sampling every 14 days. For average durations of greater than 1 day, however, the numeric solu tion is more complicated. Presenting the range of outcomes from the simulations in this manner provides a straightforward

illustration of results as obtained from a large number of sampling realizations performed according to the specified strategies for the different durati ons of interest. In general, for averages approximating acute expos ures, the sampling results were more accurate (with less underestimation) as the average duration of interest increased. For durations of longer term, however, it was incr easingly possible to overestimate the true maximum when less than daily sa mpling was employed.

3-Day Averages

For 4-day sampling intervals, the ma ximum 3-day average was captured (or exceeded) about 26% of the time. The true maximum was underestimated by about 11% half the time, and about 32% for the lowest realizations. With weekly sampling the 3-day maximum average was captured or exceeded over 14% of the time and underestimated by about 22%, on average, and over 53% for the lowest runs. Sampling every 2 weeks captured or excee ded the maximum average about 7% of the time, underestimating it by about 46% in half the runs and about 86% for the lowest runs (Table 9).Page 79 of 263 Table 9: Percentiles of Maximum 3- Day Average Concentrations for CWS #4 Sampled at 4-, 7-, or 14-day Intervals

CWS True

Value mean 1st% 5th% 10th% 50th% 90th% 99.9 th % % of sample

max avgs >

true Sampling every 4 Days

4 66.57 59.12 45.59 51.06 51.06 58.97 66.57 66.57 25.58

% error -11.19 -31.52 -23.30 -23.30 -11.42 0.0 0.0

Sampling every 7 Days

4 66.57 52.32 31.17 36.70 40.21 51.44 66.57 67.36 14.50

% error -21.41 -53.17 -44.87 -39.60 -22.73 0.0 1.18

Sampling every 2 Weeks

4 66.57 37.96 10.08 10.48 10.70 35.96 61.26 69.93 7.19

% error -42.98 -84.85 -84.26 -83.93 -45.99 -7.98 5.05Page 80 of 263 7-Day Averages

For 7-day averages, sampling every 4 days returned a maximum average that captured or exceeded the tr ue max nearly 10% of the ti me (Table 10). The true maximum was underestimated by about 7% in half the runs, while the lowest maximum 7-day averages underestimated the true by about 22%. Sampling every 7 days and every 14 days returned maximum 7-day aver ages that were about 18% lower than the true and 38% lower than the true, on average, respectively, and underestimated in the lowest runs by 83% for biweekly sampling. The highest runs overestimated the true 7- day maximum by small amounts for samplin g every 4 days and every 7 days, but by over 15% for sampling every 2 weeks.

Table 10: Percentiles of Maximum 7-Day Average C oncentrations for CWS #4 Sampled at 4-, 7-, or 14-day Intervals

CWS True

Value Mean 1st% 5 th % 10th% 50th% 90th% 99.9 th % % of sample

max

avgs >

true Sampling every 4 Days

4 56.48 52.33 44.10 45.76 48.23 52.36 56.48 56.73 9.53

% error -7.35 -21.92 -18.98 -14.61 -7.31 0.0 0.44
Sampling every 7 Days

4 56.48 46.61 30.52 35.28 37.46 46.15 56.48 58.54 11.03

% error -17.47 -45.97 -37.55 -33.67 -18.29 0.0 3.64
Sampling every 2 Weeks

4 56.48 35.37 9.67 10.05 10.59 33.61 56.78 65.13 12.79

% error -37.38 -82.87 -82.21 -81.24 -40.49 0.52 15.31

In sum, for this chemograph with shor t-duration peaks, sample runs could underestimate true values by as much as 80-90% in the lowest runs for sampling every 2 weeks, but could be just 7-14% lower than true, on average, for sampling every 4 days, depending on the durati on of interest.Page 81 of 263 Moderate width peaks

Results for a peak of slightly wider s hape showed accuracy in the estimates that was higher and an amount of underestimation t hat was less than that for the narrower peak above, regardless of peak height. CWS #21 is a system that is typical of a class with one slightly wide peak that builds up and then declines over the course of several weeks (Figure 9).

CWS #21 Ohio, 2005

0 5 10 15 20 25 30 35

1/5 2/5 3/5 4/5 5/5 6/5 7/5 8/5 9/5 10/5 11/5 12/5

DateAtrazine (TCT), ug/L

Figure 9: Illustration of a CWS chemograph with a moderate duration peak and

extended tailPage 82 of 263 Single Day Peaks

For single daily peaks, 4-day sampling intervals returned a maximum value for CWS #21 that was just over 4% less than th e true, on average, while the lowest runs underestimated by just over 11% (Table 11) . For weekly sampling the maximum was underestimated by about 8% on average, and ju st over 17% for the lowest runs. Sampling every 2 weeks returned single day ma ximums that were less than the true by about 14-15%, for the average an d median of the runs, and 27% , at the low end.

Table 11: Percentiles of Maximum Daily Concentrations for CWS #21 Sampled at 4-, 7-, or 14-day Intervals

CWS True

Value mean 1st% 5th% 10th% 50th% 90th% 99.9 th % % of sample

max avgs >

true Sampling every 4 Days

21 31.25 29.88 27.68 28.54 28.54 29.46 31.25 31.25 25.43

% error -4.40 -11.43 -8.68 -8.68 -5.71 0.00 0.00
Sampling every 7 Days

21 31.25 28.89 25.82 25.89 26.79 28.57 31.25 31.25 14.09

% error -7.56 -17.37 -17.14 -14.29 -8.57 0.00 0.00
Sampling every 2 Weeks

21 31.25 26.70 22.69 23.11 23.26 26.79 30.36 31.25 7.49

% error -14.55 -27.41 -26.05 -25.55 -14.29 -2.86 0.00Page 83 of 263 3-Day Averages

Looking at the range of ma ximum 3-day averages from all resamples for CWS #21, the maximum average returned by the m ean and the median of the runs is 3-4% less than the true maximum for sampling ever y 4 days, while the lowest 1% of the

simulations underestimate the “true” ma ximum by just under 10% (Table 12).

For weekly sampling, the true 3-day maximum average was underestimated by 6-7%, on average, and about 14% for biweekly sampling. The maximum 3-day average was captured (or exceeded) at least 25% of the time with sampling every 4 days, about 14% of the time with weekly sampling, and n early 7% for sampling every 2 weeks. The amount by which the true was over estimated in all of these r uns is less than 1%.

Table 12: Percentiles of Maximum 3-Day Average C oncentrations for CWS #21 Sampled at 4-, 7-, or 14-day Intervals

CWS True

Value Mean 1st% 5th% 10th% 50th% 90th% 99.9 th % % of sample

max avgs >

true Sampling every 4 Days

21 30.36 29.24 27.37 28.09 28.24 29.15 30.36 30.36 25.43

% error -3.67 -9.84 -7.47 -6.96 -3.96 0.0 0.0
Sampling every 7 Days

21 30.36 28.29 25.58 25.58 26.41 28.16 30.36 30.43 14.09

% error -6.80 -15.72 -14.73 -12.99 -7.24 0.0 0.23
Sampling every 2 Weeks

21 30.36 26.05 22.37 22.89 23.06 25.84 29.50 30.51 6.87

% error -14.18 -26.30 -24.59 -24.04 -14.87 -2.82 0.52

7-Day Averages

For 7-day averages, sampling every 4 da ys returned maximum averages that approximated the true maximum fair ly closely a large percentage of the time (Table 13). That value was equaled or exce eded over 15% of the time, with runs that ranged from about 7% less than true to less than 1% higher in the highest runs.

Weekly and biweekly sampling underes timated the true maximum by about 5% and 13-14%, respectively, on average, and ranged from -13% and -25% to about 1.5% higher for both.Page 84 of 263 Table 13: Percentiles of Maximum 7-Day Average C oncentrations for CWS #21 Sampled at 4-, 7-, or 14-day Intervals

CWS True

Value Mean 1st% 5th% 10th% 50th% 90th% 99.9 th % % of sample

max avgs >

true Sampling every 4 Days

21 28.95 28.24 26.89 27.40 27.54 28.23 28.95 28.99 15.50

% error -2.43 -7.12 -5.43 -4.86 -2.48 0.0 0.16
Sampling every 7 Days

21 28.95 27.38 25.18 25.84 25.87 27.40 28.95 29.10 11.44

% error -5.41 -13.01 -10.75 -10.65 -5.34 0.0 0.52
Sampling every 2 Weeks

21 28.95 25.06 21.79 22.24 22.65 24.91 28.30 29.40 6.87

% error -13.42 -24.74 -23.19 -21.76 -13.96 -2.25 1.57

In sum, for this profile, a ccuracy for sampling every 4 days ranged from -7% to -11% for the lowest runs (-2.4% to -4.4% on average) and approximated the ma x almost perfectly for the highest runs. Sampling every 7 days ranged from -13% to -17% at the low end to +0.52% at the high end. Biweekly sampli ng gave estimates of the “true” that ranged from -27% among the lo west to +1.6%, with middle runs underestimating the true by
about 13-15%.Page 85 of 263 Broad or extended peaks

A third category of chemographs contai ning one broad or several continuing peaks is represented by CWS #30 (Figure 10).

CWS #30 Missouri, 2005

0 5 10 15 20 25

1/20 2/20 3/20 4/20 5/20 6/20 7/20 8/20 9/20 10/20 11/20 12/20

DateAtrazine (TCT), ug/L

Figure 10: Illustration of a Chemograph with a Broad or Extended Series of Peaks

Results for this CWS showed the most accuracy of the groups for estimating maximum averages, starting with values for si ngle daily peaks that averaged from just under 3% below true for sampling every 4 days to just over 8% less than true for sampling every 2 weeks. At the lowest range the true sample max was underestimated by 7-8% for sampling every 4 days to just over 16% for sampling every 2 weeks. The highest runs captured the true max with no error and did so 25% of the time.Page 86 of 263 Single Day Peaks

Table 14: Percentiles of Maximum Daily Concentrations for CWS #30 Sampled at 4-, 7-, or 14-day Intervals

CWS True

Value mean 1st% 5th% 10th% 50th% 90th% 99.9 th % % of sample

max avgs >

true Sampling every 4 Days

30 23.01 22.39 21.25 21.64 21.72 22.36 23.01 23.01 25.23

% error -2.71 -7.65 -5.95 -5.62 -2.81 0.00 0.00
Sampling every 7 Days

30 23.01 21.93 20.47 20.86 21.07 22.03 23.01 23.01 14.59

% error -4.71 -11.04 -9.34 -8.43 -4.26 0.00 0.00
Sampling every 2 Weeks

30 23.01 21.12 19.22 19.69 19.78 21.07 22.42 23.01 6.96

% error -8.22 -16.46 -14.43 -14.05 -8.43 -2.56 0.00

3-Day Averages

3-day averages showed a tighter range, with % differences from true values ranging from -2% to 0% error for 3-day averages sampled every 4 days. Weekly sampling gave returns that, on the low end, underestimated the max 3-day average by a little over 9%, and biweekly sampling by ju st under 15%, but averaged -4% to -6.5%. The highest runs exceeded the max by just over 2% for sampling every 2 weeks.Page 87 of 263 Table 15: Percentiles of Maximum 3-Day Average C oncentrations for CWS #30 Sampled at 4-, 7-, or 14-day Intervals

CWS True

Value mean 1st% 5 th % 10th% 50th% 90th% 99.9 th % % of sample

max avgs >

true Sampling every 4 Days

30 22.49 22.02 21.17 21.45 21.59 22.02 22.49 22.49 25.23

% error -2.09 -5.89 -4.64 -4.03 -2.11 0.0 0.0

Sampling every 7 Days

30 22.49 21.57 20.40 20.73 20.84 21.60 22.47 22.62 6.94

% error -4.10 -9.28 -7.83 -7.32 -3.96 -0.11 0.57

Sampling every 2 Weeks

30 22.49 21.04 19.14 19.61 19.73 20.92 22.40 22.97 6.96

% error -6.47 -14.90 -12.80 -12.26 -6.99 -0.43 2.12

7-Day Averages

For 7-day averages the maximum was underestimated less often and by even smaller amounts, but overesti mated more frequently and by a slightly higher amount.

The lowest runs were less than the true 7-da y max by 3% for sampling every 4 days to nearly 12% for sampling every 2 weeks. On average the simu lations ran from -1% to - 3.7% for the different sampling periods, whil e the highest runs ov erestimated the true max average by up to 5.5%.Page 88 of 263 Table 16: Percentiles of Maximum 7-Day Average C oncentrations for CWS #30 Sampled at 4-, 7-, or 14-day Intervals

CWS True

Value mean 1st% 5th% 10th% 50th% 90th% 99.9 th % % of sample

max

avgs >

true Sampling every 4 Days

30 21.7 21.44 21.01 21.16 21.22 21.47 21.70 21.72 10.53

% error -1.17 -3.16 -2.46 -2.18 -1.03 0.0 0.10
Sampling every 7 Days

30 21.7 21.06 20.27 20.42 20.54 21.07 21.61 22.01 8.19

% error -2.93 -6.56 -5.88 -5.34 -2.89 -0.39 1.43
Sampling every 2 Weeks

30 21.7 20.89 19.13 19.49 19.64 20.73 22.35 22.88 24.14

% error -3.70 -11.84 -10.18 -9.46 -4.45 3.00 5.47

Summary Tables

Results for the above systems were run wit h those from others that fell into a similar chemograph grouping that included 10 CWS in total and three categories of chemograph shape. The selection of chemographs was made from among those that included many with the highest peaks, in additi on to several that appeared to represent typical chemographs containing consistently lower values for the year. Summaries

were made of the % differences from true va lues and are presented in the tables that

follow.

The results show some consistencies among the groups in this small set of

CWS. For more defined results a much la rger number of chemographs would need to be run, and results for that group might still be better characte rized as being descriptive

of that set rather t han definitively predictive of monitori ng profiles to come. In general, however, some broad patterns seen over sev en years of monitoring may be helpful in deciding on ways to proceed for groups of sy stems that have shown some consistency in monitoring values over y ears in the program.

Table 17 below shows summary values and average % differences from “true” values for maximum 3-day averages derived from sampling every 4 days for the 10

CWS. The CWS are summarized into thr ee groups corresponding to the chemograph shapes described above.Page 89 of 263 Table 17: Maximum 3-Day Average Concentrations for All CWS Sampled at 4-day

Intervals

CWS True Value mean 1 st % 5th% 10th% 50th% 90th% 99.9th% % of sample

maximum

running
averages >
true value

4 66.57 59.12 45.59 51.06 51. 06 58.97 66.57 66.57 25.58

% error -11.19 -31.52 -23. 30 -23.30 -11.42 0.00 0.00

14 5.28 4.93 4.33 4.44 4. 44 4.95 5.28 5.28 26.40

% error -6.75 -18.02 -16.03 -16.03 -6.32 0.00 0.00

17 209.50 190.50 159.60 165.60 165.60 192.20 209.50 209.50 0.00*

% error -9.05 -23.81 -20.93 -20.93 -8.24 0.00* 0.00*

22 53.20 47.75 38.05 41.42 41. 42 47.78 53.20 53.20 9.03

% error -10.25 -28.48 -22. 14 -22.14 -10.20 0.00 0.00

average % error -9.31 -25.46 -20.60 -20.60 -9.04 0.00 0.00

8 43.36 41.13 37.79 38.65 38. 65 40.91 43.36 43.36 18.65

% error -5.15 -12.84 -10.87 -10.87 -5.64 0.00 0.00

20 28.69 27.98 26.75 27.16 27. 16 27.99 28.69 28.69 18.25

% error -2.45 -6.75 -5.31 -5.31 -2.41 0.00 0.00

21 30.36 29.24 27.37 28.09 28. 24 29.15 30.36 30.36 25.43

% error -3.67 -9.84 -7.47 -6.96 -3.96 0.00 0.00

average % error -3.76 -9.81 -7.88 -7.71 -4.00 0.00 0.00

13 5.36 5.32 5.25 5.28 5. 28 5.32 5.36 5.36 24.88

% error -0.72 -2.14 -1.59 -1.59 -0.74 0.00 0.00

24 6.26 6.18 6.01 6.06 6. 06 6.18 6.26 6.26 26.03

% error -1.41 -4.00 -3.29 -3.29 -1.28 0.00 0.00

30 22.49 22.02 21.17 21.45 21. 59 22.02 22.49 22.49 25.23

% error -2.09 -5.89 -4.64 -4.03 -2.11 0.00 0.00

average % error -1.40 -4.01 -3.17 -2.97 -1.37 0.00 0.00

* Realizations fail to equal true max by <0.001%; disparities due to rounding

The table shows that best results were achieved for the third group containing broad peak(s) so that, on average, sample runs for the CWS in this group approximated their true [max.] 3-day averages to within a 1-2% difference. Lowest runs averaged a little over 4% less than the true and highes t runs all captured the maximum average

with no error.

The first group of CWS was characterized by single narrow peaks and included CWS #17, which had the highest peak conc entration (227 ppb) across systems and years of the program. This group al so contained CWS #14, whose maximum concentration was 5.6 ppb, which rapidly dec lined by the following week. Accuracy forPage 90 of 263 every CWS in this group of similarly shaped chemographs appeared to be comparable

to that of the others in the group, regardless of peak height. The means of the resamples in this group gave 3-day maximu ms that averaged approximately 9% lower

than their true values. Lowest runs underesti mated true values by an average of about 25%, while highest runs captured t he true with essentially no error.

Summary results are also presented bel ow for single day peak concentrations derived from sampling every 7 days.

Table 18: Single Day Con centrations for All CWS Sa mpled at 7-day Intervals

CWS True Value mean 1st% 5th% 10th% 50th% 90th% 99.9th% % of sample

maximum [peaks] > true

value

4 72.99 56.04 31.50 37.45 41. 87 55.22 72.99 72.99 14.50

% error -23.22 -56.85 -48. 70 -42.63 -24.35 0.00 0.00

14 5.62 4.78 3.67 3.77 4. 06 4.84 5.62 5.62 14.50

% error -14.99 -34.70 -32. 92 -27.76 -13.88 0.00 0.00

17 227 183.10 126.10 129.90 146. 30 186.60 227.00 227.00 14.46

% error -19.32 -44.44 -42. 77 -35.55 -17.78 0.00 0.00

22 57.98 45.48 26.16 32.46 34. 11 45.22 57.98 57.98 14.46

% error -21.56 -54.89 -44. 02 -41.16 -22.01 0.00 0.00

average % error -19.77 -47.72 -42.10 -36.78 -19.50 0.00 0.00

8 44.92 40.54 34.00 35.56 37. 12 40.24 44.92 44.92 14.49

% error -9.75 -24.31 -20. 84 -17.36 -10.42 0.00 0.00

20 29.3 27.71 26.04 26.04 26. 21 27.67 29.30 29.30 14.70

% error -5.43 -11.13 -11.13 -10.55 -5.56 0.00 0.00

21 31.25 28.89 25.82 25.89 26. 79 28.57 31.25 31.25 14.09

% error -7.56 -17.37 -17.14 -14.29 -8.57 0.00 0.00

average % error -7.58 -17.60 -16.37 -14.07 -8.18 0.00 0.00

13 5.4 5.31 5.18 5.21 5. 24 5.32 5.40 5.40 14.03

% error -1.68 -4.17 -3.47 -3.02 -1.51 0.00 0.00

24 6.35 6.14 5.86 5.88 5. 97 6.16 6.35 6.35 14.09

% error -3.34 -7.76 -7.42 -5.94 -2.97 0.00 0.00

30 23.01 21.93 20.47 20.86 21. 07 22.03 23.01 23.01 14.59

% error -4.71 -11.04 -9.34 -8.43 -4.26 0.00 0.00

average % error -3.24 -7.66 -6.75 -5.80 -2.91 0.00 0.00

Results for single day maximum concentra tions again show a consistency among groups of similarly shaped chemographs, with most accuracy once again being demonstrated for the group of broad, extended peak(s). In mean runs this group returned sample maximums that averaged just over 3% less than true values and werePage 91 of 263 7-8% less for the lowest runs. The tr ue chemograph max was captured with no error
and in over 14% of the simulations.

Group 1 showed less accuracy for capturi ng single day maximum values with weekly sampling. Once again, the chem ograph max was captured in over 14% of resamples, with the lowest runs averagi ng 48% less than the true max and the mean and median runs averaging about 20% less.

Demonstrating the efficiencies gained by targeted sampling (in time), Table 19 shows results for maximum 3-day averages derived from sampling taken every day during the season of use and biweekly for the remainder of the year.

Table 19: Maximum 3-Day Averages for All CWS Sampli ng Every Day (April thru August); Every 14 Days (balance of year)

CWS True Value mean 1 st % 5th% 10th% 50th% 90th% 99.9th% % of sample

maximum

running
averages >
true value

4 66.57 64.81 60.15 60.15 60. 15 66.57 66.57 66.57 60.30

% error -2.64 -9.64 -9.64 -9.64 0.00 0.00 0.00

14 5.28 5.20 4.95 4.95 5. 01 5.28 5.28 5.28 60.31

% error -1.65 -6.35 -6.35 -5.26 0.00 0.00 0.00

17 209.50 204.90 192.00 192.00 195. 10 209.50 209.50 209.50 32.06

% error -2.17 -8.36 -8.36 -6.84 0.00 0.00 0.00

22 53.20 51.90 48.42 48.42 48. 42 53.20 53.20 53.20 60.25

% error -2.45 -8.98 -8.98 -8.98 0.00 0.00 0.00

average % error -2.23 -8.33 -8.33 -7.68 0.00 0.00 0.00

8 43.36 42.74 40.91 40.91 41. 80 43.36 43.36 43.36 61.40

% error -1.42 -5.64 -5.64 -3.60 0.00 0.00 0.00

20 28.69 28.51 28.07 28.07 28. 08 28.69 28.69 28.69 59.09

% error -0.60 -2.14 -2.14 -2.13 0.00 0.00 0.00

21 30.36 30.06 29.15 29.15 29. 56 30.36 30.36 30.36 44.05

% error -0.97 -3.96 -3.96 -2.64 0.00 0.00 0.00

average % error -1.00 -3.92 -3.92 -2.79 0.00 0.00 0.00

13 5.36 5.35 5.32 5.32 5. 34 5.36 5.36 5.36 62.01

% error -0.18 -0.74 -0.74 -0.49 0.00 0.00 0.00

24 6.26 6.24 6.18 6.18 6. 20 6.26 6.26 6.26 60.31

% error -0.35 -1.38 -1.38 -1.05 0.00 0.00 0.00

30 22.49 22.35 21.97 22.02 22. 03 22.49 22.49 22.49 60.13

% error -0.61 -2.31 -2.11 -2.05 0.00 0.00 0.00

average % error -0.38 -1.47 -1.41 -1.20 0.00 0.00 0.00Page 92 of 263 As might be expected, results for this sampling design show the most accuracy across all groups, with mean runs ranging from -2% of true to less than 1% below true. Lowest runs are only a little over 8% below true values in the group of narrow peaks and only 1.5% below true for the “broad” group. At least 50% of the simulations showed

no error for these CWS.

Finally, summary results are presented for the group of systems for 90-day averages derived from sampling that was tak en weekly during the ‘season’ of use and biweekly during the remainder of the year, a strategy that is similar to the one currently employed by the AMP.

Table 20: 90-Day Averages for All CWS Sampling Every 7 Days (April thru August); Every 14 Days (balance of year)

CWS True Value mean 1st% 5th% 10th% 50th% 90th% 99.9th% % of sample

maximum

running
averages >
true value

4 9.35 9.34 7.89 8.20 8. 56 9.35 10.12 10.84 50.30

% error -0.10 -15.67 -12.37 -8.47 0.02 8.26 15.90

14 1.49 1.49 1.41 1.43 1. 44 1.49 1.53 1.58 41.39

% error -0.53 -5.86 -4.34 -3.51 -0.48 2.32 5.75

17 25.99 25.98 22.28 23.03 23. 87 26.05 28.06 29.77 52.33

% error -0.05 -14.28 -11.39 -8.14 0.24 7.97 14.53

22 9.93 9.90 8.85 9.08 9. 29 9.92 10.50 11.06 49.39

% error -0.25 -10.90 -8.52 -6.39 -0.08 5.78 11.35

average % error -0.23 -11.68 -9.16 -6.63 -0.07 6.08 11.88

8 11.53 11.45 10.86 10.99 11. 11 11.45 11.79 12.09 39.22

% error -0.71 -5.76 -4.63 -3.64 -0.72 2.24 4.86

20 12.66 12.65 12.39 12.46 12. 50 12.65 12.80 12.99 47.33

% error -0.07 -2.14 -1.56 -1.24 -0.06 1.09 2.59

21 7.93 7.92 7.62 7.70 7. 75 7.92 8.08 8.26 44.74

% error -0.24 -4.01 -2.96 -2.38 -0.20 1.80 4.13

average % error -0.34 -3.97 -3.05 -2.42 -0.33 1.71 3.86

13 2.55 2.53 2.46 2.48 2. 49 2.53 2.56 2.61 18.77

% error -1.00 -3.53 -2.84 -2.46 -0.98 0.43 2.19

24 3.72 3.69 3.55 3.59 3. 62 3.70 3.77 3.87 37.29

% error -0.56 -4.31 -3.24 -2.64 -0.52 1.49 4.22

30 17.41 17.37 17.08 17.16 17. 20 17.37 17.54 17.75 38.84

% error -0.21 -1.88 -1.42 -1.17 -0.21 0.76 1.97

average % error -0.59 -3.24 -2.50 -2.09 -0.57 0.89 2.79Page 93 of 263 Results for the 10 CWS summarized show estimates whose averages ranged from roughly -12% to +12% of “true” for the group with t he most narrow peaks, and from

-3-4% to +3-4% for the other two groups. An examination of the 5 th %ile summaries for all groups indicates that 95% of the resa mples by group gave estimates that were no more than 10% lower than true values (wit h better performance for most CWS), while

50% were no more than 1% lower.

Finally, there appeared to be more overestimation of true values among resamples for these longer term 90-day averages . In addition, preliminary simulations using a stair-step type of im putation procedure (not pres ented here) gave indications that that method may provi de slightly higher estimates (more overestimation and less underestimation) than the linear inter polation method for these and for a ll averages.

Summary tables are available for other sampling schemes and average durations of interest, but those above demonstrate patterns for several common sampling strategies as well as for durations that may be typical approximations of both acute and chronic exposure.



### 5.5.4. Summary of Initial Analyses

The above methods examined raw water by CWS annually, and give ranges and summaries that are applicable to the system s used in the exercise. Among this small set, however, some general patterns do emerge. Results of this ex ercise demonstrate that chemographs can be categorized accordi ng to shape and that sampling strategies perform differently according to those s hape categories. Moreover, the exercise indicates that, to the extent that they can be predicted in advance, the width of the chemograph peaks, representing the period of their durati on, may be more important than their height in finding a sampling scheme to capture maximum values.

Results for this subset of 10 CWS also in dicate that the curr ent sampling strategy of weekly intervals during the period of hi ghest use and anticipated runoff appears to be adequate for capturing maximum 90-day average concentrations with reasonable to good accuracy. More frequent sampling may be necessary, however, for averages of shorter duration and for single day concentra tions, particularly for chemical profiles characterized by narrow, fast moving ( i.e. , short duration) peaks.

The methods used above employed systematic sampling (as is often done in the field) from random start dates . The Agency did not combi ne years to create larger sample sizes, as OPP risk assessments cr eate exposure profiles on a year-to-year basis. As a result, the dataset is necessarily more limited in range and in the inferences that can be made. Additionally, as ment ioned before, the “true” profiles for each chemograph were created with interpolation procedures employed on samples that were originally collected on a weekly or biw eekly basis, so that the actual pattern of intervening peaks is not known. It is likel y that these interpol ation procedures, whichPage 94 of 263 are used in common practice, may not alwa ys adequately mirror genuine variability in a true concentration profile that ex ists throughout the year.

An additional outcome that may become app arent in a larger sample is that the amount of underestimation can sometimes be gr eater for a missed peak in the range of, say, 5 ppb, than for one at higher concentra tions because the 5 ppb peak may drop to a

level that is 5-fold lower in the week that follows. This disparity generally is less common for peaks in the higher concentrations, and/or for those that extend over the

course of several weeks.

General information from the AMP presented above showed that measurements for 92-96% of the samples each year were le ss than 3 ppb. No large scale analysis has been performed to examine chemograph shapes among the CWS represented by this large majority of program samples. Bec ause the consequences of sampling that could miss peaks are greater when yearly profile s consist of samples in the higher concentration range, however, an examination of shape patterns in the CWS that have demonstrated higher concentrations in past m onitoring may be beneficial. The subset of CWS showing peak concentration values >30 ppb, >20 ppb, and >12 ppb was 9, 14, and 27 CWS, respectively, in the highest year s, with nearly complete nesting of the same CWS within these categories. The above analysis shows that it may be possible to arrange for more rigorous sampling schemes that can be applied to these subsets of CWS, if that is determined necessary.

While the analysis above has merit for presenting some general patterns that appear to hold as sampling intervals and concent ration durations increase or decrease, there is uncertainty associated both with t he estimates and with gen eralizing from the particular set(s) of chemographs analyzed to those that might be expected in future years for the same and for other systems. In the first method above, nonparametric

confidence intervals were calculated for the percentile estimates to show some measure of their precision. Because the samples we re not taken strictly at random and because

of the obvious serial correlation in the sa mples, however, there is likely to be some

amount of bias in these estimates.

In the second method the Agency present ed ranges of the lower, middle, and upper simulations which, in many cases, gav e a fairly narrow interval surrounding true values. These may also serve as the basis for discussing uncertain ty bounds for future

datasets. EPA seeks the Panel’s f eedback regarding recommended approaches, feasibility and the utility of constructing interv als for future observations in combination with larger datasets and methods similar to those above. In addition, guidance is sought in general on characterizing uncertain ty as well as on the utility of these approaches for designing future sampling plans for other potential endpoints of interest.

In the following section of the paper, USEPA presents a modeling approach for characterizing atrazine occurrence and for examining the issue of sampling frequency.Page 95 of 263 

### 5.6. Artificial Neural Network (ANN) Modeling of Atrazine

Occurrence Patterns

This section of the paper describes one potential approach for occurrence modeling

that USEPA’s Office of Water used to simu late the pattern of short-lived, high concentration peaks or “spikes,” and shows how these models can be used to assess how well candidate monitoring strategies sati sfy quantitative performance criteria. This

section illustrates one alte rnative approach for how the A gency might estimate exposure concentrations from monitoring data of va rying sampling intervals As discussed in Section 5.7.1, USEPA plans a systematic evaluation of this and other methods

described in Section 5.4.1.

5.6.1 Time Series Pl ots Using Frequent Monitoring Data

The Agenc y used monitoring data from t he Heidelberg University’s National Center for Water Quality Research (NCWQR) file for large rivers for a preliminary evaluation of ANN modeling.Page 96 of 263 Figure 11, displaying 2001 data from the Maumee River, illustrates that the ANN approach may not work well where concentra tions change rapidly between events or where short-lived peaks occur outsi de of anticipated patterns.

Figure 11: Data for Maumee River 2001Page 97 of 263 Figure 12 is a plot of Maumee 1998 ( one of the data sets used for ANN modeling) data, showing larger short-term day-to-day variabilit y or “noise” (in log units). Based on this and other plots, it appears reasonable to treat 0.25 μ g/L as a censoring limit. The natural log of 0.25 is about -1.4 . This figure clearly shows two occurrence peaks.

Figure 12: Data for Maumee River 1998Page 98 of 263 5.6.2 Artificial Neur al Netw ork Modeling

Artificial Neural Networks (ANNs) are o ften seen as magical “black boxes” that learn from data, and end up predicting how one or mo re “dependent variables” change in response to “independent variables.” Lee (2004) opens up the ANN black box, providing mathematical forms of ANN models and some real-world interpretations of the model parameters. The book goes on to des cribe parameter estimation in a Bayesian framework for multivariate regression, cla ssification, and other applications. A detailed explanation of the ANN approac h, including the equations that describe the modeling parameters, is beyond the scope of this paper, but more detail can be found in Appendix C.

Figure 13 illustrates an attempt to fit t he 1998 Maumee River data, plotted in log 10 concentrations, using a single node. A second node with a decreasing effect will be needed to pull the ANN curve down after day 130 and a third node will be needed to explain the occurrence peak around day 166.

Figure 13: Maumee 1998 and A NN Model with One Node

0 100 200 300 400 5

− 0

5 10

The “best” ANN model for any one-year data set will be the model with fewest hidden nodes that does an adequat e job of explaining the o ccurrence data. “Adequate” will be expressed in terms of log-likelihood.

The ANN model describes the expected log-concentration, but clearly, the concentration on any given day will not be exac tly that predicted by the model. Some amount of noise can be seen in all plots of occurrence data. We assume the difference between a day’s measured log-concentration and that given by the underlying model will be normal with mean zero and some variance that we can estimate using the model

and the data. We assume the variance is constant. A day’s log-concentration is regarded as a normal random va riable with mean equal to the model prediction and, forPage 99 of 263 measured values above a censoring limit, the likelihood is the normal probability density. For nondetects and measured values below a censoring limit, the likelihood is

the value of the cumulative normal distri bution function (the probability of a log concentration below the censoring limit).

Rather than a full Bayesian treatment, where prior probabilities and data combine to reduce uncertainty about the model par ameters, we seek maximum likelihood models. This simplifies the evaluation of monitoring strategies, where the true model and its parameters are fixed and data are random (simul ated) based on the fixed

occurrence model.

Once a best-fitting ANN model is identifie d, the autoregressive (AR) structure can be explored and modeled. AR structure expl ains how the deviat ion of one day’s logconcentration from the model is related to t he deviation of previous days. It turns out that days with log-concentration greater (or less) than expected tend also to be followed by days with log-concentration greater (or less) than expected. This is explained by positive coefficients in the AR model.

The time-series plot for the Maumee River in 1998 reveals two occurrence peaks. Additional ANN model developmen t, detailed in Appendi x C, found a three hidden node model with AR(2) behavior to provi de a preferable fit to the existing time series (shown in Figure 14).Page 100 of 263 Figure 14: Maumee 1998 and Max Likelihood Three-Node ANN Model

5.6.3. Predictive Analytics

This discus sion describes how the ANN oc currence models can be used to evaluate source water monitoring strategies, where the concern is for detecting situations like the following:Page 101 of 263 a) Distributions of daily concentrations where the X th percentile exceeds Y ug/L. (In this situation we don’t want to have more than a specified number of days per year above Y ug/L and the high-occurrence days do not need to be consecutive to do harm.) b) Distributions with mean concentrations gr eater than Y ug/L. (Concern is for longterm exposures, such as associated with some cancer risk.) c) Distributions with an average of 1 or more times during the year when concentrations exceed Y ug/L for Z or mo re consecutive days. (Concern is for sustained exposure over consecutive days.)

Under these models, a particular day’s concent ration will vary from year-to-year as a lognormal random variable, so each day’s dist ribution function is known. The values shown in Table 21 were calculated directly . Other percentiles co uld be derived in the

same fashion.

Table 21: 75th Percentiles and Means for the Three Node Model

Occurrence

Model a) 75 th

Percentile b) 90 th

Percentile c) Mean

Maumee 1998

Three-Node AR(2) 0.0920 μ g/L 3.02 μ g/L 1.428 μ g/L

Estimating the expected number of episodes per year having at least Z consecutive days’ concentrations above a level of interest is more involved, as it requires simulating many years of occurrence and keeping track of episodes

encountered. Simulations of this sort have not yet been performed, but are entirely feasible, should the data be assembled.

Given maximum likelihood parameter values, the Maumee 1998 occurrence levels may be considerably different than situati ons of interest. For example, neither of those occurrence levels is directly re levant if the concern is having a 90 th percentile at or

above 10 μ g/L.

Fortunately, the models can be scaled easily by adjusting the intercept term, b0. Having homogeneous variance on the log-scale ens ures that relative error (ratio of day’s standard deviation to the day’s ex pected concentration) is constant.Page 102 of 263 5.6.4. Simulating Daily Levels

Simulation is required to assess the ability of a monitoring stra tegy to “detect”

occurrence situations of interest. Simulati ng a year of daily concentrations requires adding some “error” to the ANN-predicted log- concentration for each day of the year. These daily “errors” have two components. One component is lik e “white noise,” in that it is independently and identically distributed (i .i.d.) across days. On the log-scale, the

white noise distribution is normal with a standard deviation of 0. 243 for Maumee 1998.

The other component of error is due to the AR behavior. In the case of Maumee 1998, the model is AR(2) and the error on day D includes 0.381 times the “white noise error” of the previous day (D-1) and 0.195 times the “white noise error” of day D-2.

For example, to scale Maumee 1998 so that the resulting occurrence pattern has

10 μ g/L as its 90 th percentile, parameter b0 is increased to 0.613. One year of simulated data is shown in Figure 15. In this particular simulated year, 10 μ g/L (shown in the plot as a horizontal red line) is exceeded on 34 days, so 10 falls just above the 90th percentile. Over many simulated y ears, the percentile associated with 10 μ g/L will vary. This particular simulated year just happened to have 9.3% of the values fall above10

μ g/L.Page 103 of 263 Figure 15: Simulation Based on the Maumee 1998 Model

5.6.5 Evaluating a M onitoring Strategy

Having a means of simulating daily occu rrence levels for an occurrence level and pattern of concern, one can select daily observations in accordance with a monitoring strategy and then process the selected obser vations to make the binary decision: to decide whether there is a problem or not. For example, if the strategy is to collect one sample per month (one sample every 30 days), and determine whether the maximum value exceeds Y, one could do the following:Page 104 of 263 1. Select an occurrence pattern and level of concern. This is a source water

occurrence scenario where there is conc ern about either a false positive or false negative determination. The simulati on exercise is cr afted to determine whether a strategy adequately limits dec ision error for the selected scenario. 2. Select a random start day in the first month. St artDay <- sample(1:31,1) 3. Add 30, 60, ... 330 to mark the other sampling days for the year or, for random dates, select one day at r andom from every calendar month. 4. Observe the concentrations simulated for those days in a one-year simulation 5. Identify the maximum concentration and compare to Y, recording the result 6. Repeat the steps 2 through 5, above until many years of monitoring have

been simulated 7. Across all simulated years, determine the fraction of times t hat the result was positive (because the year’s sample maximum exceeded Y)

Example: Determine the probability that monthly sampling will produce a positive result, where a positive is defi ned as having a maximum greater than 10

μ g/L when the occurrence pattern is Maumee 1998 having a 75 th percentile of 10

μ g/L. For this exercise, this level is considered “high” and a positive rate of at

least 95% is desired.

• The occurrence pattern is Maumee 1998 and coefficient b0 is adjusted so the long-term 75 th percentile is 10 μ g/L.

• Steps 2 through 5 are repeated 10,000 times

Results

• Under systematic sampling, a random sampling date is selected for January and other sampling dates follow at exac tly 30-day intervals. Among the 10,000 simulated sample maxima, only 180 exceeded 10 μ g/L. A positive

result was obtained 98.2% of the time. The goal of at least 95% positives

has been met.

• With random sampling during each of 12 30-day “months,” positive results were obtained in only 82.1% of the simu lations. The high-occurrence peak that was almost always detected with systematic sampling was often missed with random sampling. The goal of at least 95% positives has not been met.Page 105 of 263 5.6.7. Summary – Advantages a nd Disad vantages of Using the ANN

Models

There are several important advantages and disadvantages to using ANN models for the purpose of evaluating monitori ng strategies for the Atrazine Monitoring Program. First, ANN Models are flexible and can potentially represent realistic occurrence patterns. The models account fo r some autocorrelation, so they can be

used to understand the distinction between c onsecutive days above a level of concern and percentiles of the occurrenc e distribution. As with other approaches, the models can be used to assess how a monitoring strategy’s performance is related not only to the sample size (number of samples collec ted each monitoring period) but also to the sample timing with respect to occurrence pea ks. And finally, the models are scalable, so that different magnitudes of occurrence are easily simulated using the same basic occurrence pattern and relative error.

On the downside, the mathematical fo rm of an ANN model is complex, so individual model parameter s are often difficult to interpre t. Additionally, we are not yet able to produce a hierarchical model that incl udes year-to-year variability in timings, numbers, and magnitudes of spikes. Expan ding these models to account for year-toyear variability in parameters is feasible, but would require m any years of data for each location modeled. Computing and inte rpreting occurrence endpoints may be challenging.

The illustration here uses robust monito ring data, with near-daily sampling during most of the year. The Agency has not ye t evaluated the capability of the model to accurately simulate true exposure pa tterns with less frequent monitoring data.

5.7 Summary of Water Monitoring Frequency Methods

The methods discussed above illustrate tw o different approaches to examine the issue of monitoring frequency fo r assessing exposure duration s of interest in human health risk assessments. The first (section 5. 5) exercises use interpolation procedures and looks at upper percentiles of single day estimates and maximum running averages of several days’ duration. They com pare the performance of different sampling strategies for deriving estimates of the “true” concentrations for seve ral periods of time. The second (section 5.6) offers a modelin g approach that can be applied to pesticide

occurrence data and combined with different sampling strategies to evaluate their performance. A variety of data quality objec tives are addressed using specific example questions and quantitative performance criter ia. Additionally, the Agency plans to evaluate other options discussed in secti on 5.4.1 but not presented in detail here.

The relative merits of each type of approach depend on both the goals and specific questions of interest, as well as on the ability to use each approach with bothPage 106 of 263 current data and for anticipated monitoring. While the first approaches that use example profiles demonstrate the accuracy of different sampling strategies in a fairly straightforward manner, there are questions as to how well results from the small sample set can be generalized to the larger body of data, in addition to the best way to use them for designing programs for evaluating future monitoring resu lts. And although peak width may be more important than peak height for evaluating the performance of a given sampling strategy, it may be no less di fficult to determine with less than daily sampling. It may also be somewhat more variable among the same CWS from year to year, while relative peak height has demons trated some consistency in certain systems across the years of monitoring.

5.7.1. Planned Approach to Eval uate the Methods and Models for Estimating Pesticide Concentrati ons From Less Frequent Monitoring

Data

For existing monitoring data with vary ing sampling frequencies, the Agency is interested in both (1) putting confidence bounds on the exposure estimates similar to those presented in section 5.5 and (2) usi ng models to estimate exposures between measurements, as described in section 5.4.1. Such inter polation methods would als o have uncertainty bounds associated with their es timates. The Agency anticipates that some methods may be better suited for expos ures of longer durations (e.g., 90-day averaging periods) than for exposu res of short duration.

The Agency plans to use the following approach to evaluate the different interpolation methods refe renced in section 5.4.1:

• Use the robust Heidelberg NCWQR moni toring data sets and, as applicable, more frequently-sampled headwater str eams from the Atrazine Ecological Exposure Monitoring Program data to r epresent the three general exposure shapes mentioned in section 5.5, as well as capturing multiple peaks.

• Sample these data sets at different in tervals (e.g., 7-, 14, 30- day sampling intervals).

• Use the various approaches described in Section 5.4.1 (lin ear, stair step, kriging/nearest neighbor, ANN, etc) to estimate the exposures between sampling points for each of the sampled intervals.

• Generate estimated exposures for varying rolling average periods (1, 3, 7, 14, 30, 60, 90 days) and compare those es timates against the actual values.Page 107 of 263 6. Implications of MOA & Mammalian Toxicity Findings on Sampling Frequency

The Agency has conducted an extensive review of the toxicological and in vitro data published since the atrazine IRED was finaliz ed in 2003. In addition, it has initiated a re-evaluation of the sampling frequency n eeded to determine pesticide concentrations in water. The objectives of this effort are: (i) to describe the evolut ion of the state-of-the science and how these developments may be incorporated into the atrazine human health risk assessment; and (ii) evaluate the water sampling frequency that most closely corresponds to the temporal relationship bet ween atrazine exposure and toxicological

effects of concern.

The toxicological and in vitro data generated in the last seven years support the

neuroendocrine MOA described in the 2003 IRED. Moreover, these data enhance our understanding of the MOA by identifying another disrupt ed pathway in the MOA:

activation of the HPA axis manifested as incr eases in corticosterone. Atrazine-induced HPA activation leads to an increase in pituitar y ACTH release, resulting in increases in corticosterone and progesterone release from t he adrenal gland. In turn, corticosterone

increases lead to a decrease in the releas e of GnRH from the hypothalamus. The toxicological significance of the rapid HP A axis activation (15 minutes after atrazine exposure) remains unclear ( i.e., at what doses and after w hat duration of exposure do these changes lead to an adverse health outcome). Data from Cooper et al. (2007) indicate that after 4 days of atrazine exposure GnRH levels are decreased at 25 mg/kg/day (lowest dose tested) while in a s eparate set of experiment s the LH surge is attenuated at 6.25 mg/kg/day. Considered in its totality the data reported by Cooper et

al. , Laws et al., Fraites, et al., and Pruett et al., suggest a relationship between

corticosterone increases and GnRH decreases. At this time, it is unknown what level of

corticosterone increase and for what durat ion would suffice to induce the reported

decrease in GnRH. Research is current ly underway to further characterize and describe this putative dose-response and te mporal concordance between the HPA axis activation and HPG disruption.

The neurotoxic effects reported in the recent data do not support a relationship between atrazine exposure and Parkinson’s di sease. Moreover, the results of the neurotoxic studies (Rodriguez et al. , 2005) have some limitations which reduce their utility. The dopaminergic responses appear to be, generally, consistent among studies. However, these studies lack a consistent hy pothesis for the MOA t hat may be operative on the dopaminergic system. Moreover, the increases in corticosterone may be associated with these changes. The relationsh ip between corticosterone increases and dopamine changes, however, have not been invest igated. Nonetheless, the Agency believes that points of departure based on n euroendocrine effects will be protective of the changes in the dopaminergic system.

The new data indicate that atrazine-i nduced immunotoxic effects occur at higher

doses than those used to establish the NO AELs used in the risk assessment. ThePage 108 of 263 potential impact of atrazine on developmental immunotoxicity (DIT) remains unclear

since the available DIT studies offer conflic ting results or could not be replicated.

In the case of aromatase, the data continue to show that although aromatase activity is increased in vitro the same is not seen after in vivo exposure. Multiple studies

have demonstrated that in the intact anima ls aromatase changes are not seen. While informative for mechanistic studies, cellula r systems do not repres ent the full scope of pharmacodynamic and pharmacokinetic responses that are express ed in the intact animal. Consequently, with respect to the aromatase studies, the Agency puts more weight on the outcomes of in vivo mammalian studies to predict potential risks to human

health.

One of the main objectives of the re-evaluation is to evaluate the drinking water sampling frequency that most closely corres ponds to the temporal profile of the toxicological effects of concern. For instance, if the health effects of concern relate to long-term chronic exposures less frequent samp ling may be sufficient. For effects seen after shorter exposures, however, the fr equency and timing of sampling may need to increase in order to capture t hese short-duration exposures. This is most critical when the Agency is concerned about acute effect s resulting from peak, or single day, exposures. Depending on the aquatic resource being sampled, the likelihood increases that a peak exposure may be miss ed by less frequent sampling.

In the 2003 IRED, a six month LH endpoint was identified and a 90-day period was chosen as the corresponding exposure durat ion of concern. It was this 90-day average that guided the selection of the current sampling frequen cy. In preparation for the September 2010 SAP, the Agency plans to perform a more complete MOA analysis; this analysis will include a thorough evaluation of the temporal pattern of toxicity along with consideration of temporal concordance of the key ev ents in the MOA. It does appear that new toxicity data available since 2003 suggests that toxicologically relevant effects may occur after durat ions shorter than six months. For example, Table 3 provides data on several hormonal changes which occur at durations ranging from 15 minutes to four days. T he degree to which these rapid hormonal changes can be linked

to adverse health outcomes is unclear at th is time. The Agency is aware of on-going studies to consider such linkage which are expected to become avai lable in 2010. In anticipation of the availability of these data, the Agency is considering methods for evaluating the performance of different sampli ng strategies that may be necessary to assess drinking water exposures of shorte r duration. Several methods have been proposed and two are discussed in greater detail in this issue paper. The Agency is soliciting comments from the Panel on these proposed approaches. While no conclusions have been drawn at this time, pr eliminary indications suggest that if a shorter duration of toxicological concern is selected then a strategy of more frequent sampling may be considered in the future. Finally, the science that informs atrazine’ s risk assessment is complex and multifaceted. The 2003 risk assessment represent ed the state of the science at the time and was one of the most extensively peer-revi ewed assessments done by the Agency.Page 109 of 263 Since that time, a vast amount of data have been generated which enhances our understanding. At the pr esent time, the Agency is in the midst of its analysis to evaluate

all these new data. It’ s important to not e that additional data on a variety of topics such as mammary gland development, HPA-HPG axes interaction, and epidemiological data from the AHS are expected to become available within t he next few months. No firm conclusions may be drawn at this time pendi ng analysis of these data. The Agency is looking forward to the Panel’s feedback and the public’s comments on this issue paper in preparation for the dra ft WOE issue paper that will presented in the fall SAP.Page 110 of 263 

# Appendix A

EVALUATION OF ARTICLES ON ATRAZINE AND

ATRAZINE METABOLITES RELATED TO MAMMALIAN

TOXICITY STUDIES SUGGESTING POTENTIAL

ADVERSE HUMAN HEALTH OUTCOMESPage 111 of 263 A.1 Content of this Appendix

An evaluation of peer-reviewed articles related to atrazine and/or its metabolites published since 2003 was conducted. The scope of this evaluation was restricted to articles reporting on mammalian toxicity studies or in vitro studies that provided data on

atrazine and atrazine metabolites mode of action (MOA). The purpose of this evaluation was to determine the scientific qu ality of the research conducted since the atrazine Re-registration Eligibi lity Decision (RED) was finaliz ed in 2003. Ultimately, the objective of this evaluation is to ascert ain if these new data impact the Agency’s conclusions regarding points of departure, MOA and overall impact of atrazine exposure

on human health. To the extent that these data provide additional information on these areas they will be used to update the atrazine human health risk assessment.Page 112 of 263 A.2 Effects on Females

A.2.1 Dooley, G.P., Reardon, K.F., Prenni, J.E., Tjalkens, R.B., Legare, M.E., Foradori, C.D., Tessari, J.E., Hanneman, W.H. (2008). Proteomic analysis of diaminochlor otriazine adducts in Wistar rat pituitary glands and L β T2 rat pituitary cells. Chem Res Toxicol

21:844-851.

In a protein adduct study using immunohist ochemistry and Western blot techniques, ovariectomized Wistar female rats (4/group) were administered oral gavage doses of 0 or 200 mg/kg atrazine (97.9%) in carboxymethylcellulose and sacrificed on postexposure day 5. Pituitary glands were harve sted, and either froz en in liquid nitrogen

or fixed with formalin. Forma lin-fixed tissues were embedded in paraffin, sectioned, processed for immunohistochemistry and in cubated with a 1:100, 000 dilution of commercial DACT adduct antibody overnight at 4° C. Prepared slides were visualized with a Vectastain Universal ABC kit, count erstained with hematoxylin and examined for DACT-protein adducts. For the two-dimensi onal electrophoresis, pituitary glands were

frozen until use.

A similar approach was taken to prepare the L β T2 gonadotrope cells for DACT-protein adducts analysis and for the two-dimensional el ectrophoretic analysis. Cell cultures of

L β T2, grown in culture medium to 80% confluency were plated at a density of 400,000 cells on to cover slides and allowed a 48-hour attachment period. Attached cells were exposed to 300 μ M DACT, culture medium or the so lvent (dimethyl sulfoxide, DMSO)

for 24 hours. Treated cells on slides were ri nsed, fixed in methano l, mixed with Triton X, blocked with phosphate buffer, incubat ed with the DACT adduct antibody, washed and reincubated with commercial goat antirabbi t antibody labeled with the fluorescent dye, Alexa Fluor 488, and examined for DACT -protein adducts. Under similar culture conditions, L β T2 cells were grown to 80% confluency, exposed to 300 μ M DACT, culture medium or DMSO for 24 hours and frozen in liquid nitrogen until use.

For the two-dimensional electrophoresis, either the frozen pituitary glands or L β T2 cells were thawed, pooled, sonicated, and cent rifuged. Supernatants were removed; proteins were precipitated with a commercia l kit, and dissolved by sonication in 400 μ L of a rehydration buffer. Sample s of the tissue or cellular prot eins were plated onto trays containing gel and subjected to electrophoresis. At the end of the procedure, the two dimensional gels were either stained with Sypro Ruby (f or protein detection) or subjected to Western blotting for detection of the DACT-adducted proteins. Protein spots on the Western blots we re matched to spots on the Sypro Ruby-stained gels. Matched spots from pituitary tissue samples we re excised from the Sypro Ruby-stained gels, digested, rinsed and proteins were identified through MALDI-TOF/TOF MS. Matched spots from the cell cultures were al so excised from the Sypro Ruby-stained gels, digested, rinsed and processed for MS . Proteins detected in DACT-exposed L β T2 cells were identified using Sequest search of the MS/MS data. For both sample types,Page 113 of 263 MS spectra-identified prot eins were searched against the National Center for Bioinformation (NCBInr) protein dat abases using the taxonomy filter Rattus

.

DACT formed 19 spots and covalent adducts wit h 8 unique proteins in the rat pituitary and 21 spots and covalent adducts with 19 unique proteins in the L β T2 cells. The investigators reported that the proteins found in both sample types were: proteosome activator complex subunit 1 (involved in pr oteosomal degradation but effect on protein stability or function is not known); ubiquit in carboxyl-terminal hydrolase isozyme L1 (involved in ubiquitin-tagged pr otein proteosomal system degradation which is critical to cell growth, differentiation, development and transcription); tropomyosin (this protein binds to actin, one of the major components of the cytoskeleton , and acts as a mediator of internal cell motility); and ERp57 (an oxidoreductase found in the endoplasmic reticulum of cells; exact function is not completely understood). In addition, RNAbinding proteins containing active-site or solvent-exposed cysteine residues were also detected in both sample types.

Based on the results of this study in comb ination of the other Dooley studies (in PK section below) along with other studies, the authors speculate that “Because DACT is capable of forming covalent protein adducts and brain tissue is exposed to large quantities of DACT following at razine exposure, specific modi fications of proteins within the pituitary could cause aberrant function, leading to the observed suppression of the LH surge.”

Reviewer’s Comments: Adduct formation may be important in the regulation of pituitary function since adduct formation can be seen in the pituitary glands of rats exposed to

either atrazine or DACT as well as in L β T2 Rat Pituitary Cells. I n vivo exposure to 200 mg/kg atrazine resulted in 8 DACT -protein adducts. Similarly, L β T2 rat pituitary cells exposed in vitro to 300 μM DACT led to adduct formation between DACT and 19 proteins. This study was not designed to st udy a potential dose-response in terms of adduct formation nor does it prov ide any data pertaining to the functional outcomes of these adducts. Nevertheless, it is noteworthy that t hese data entail exposure to extremely high levels of atrazine and/or metabolites that far exceed the potential environmental exposures.

A.2.2 Fraites, M.J.P., Cooper, R. L., Buckalew, A., Jayaraman, S., Mills, L., and Laws , S.C. (2009). Charact erization of the Hypothalamic-Pituitary-Adrenal Ax is Response to Atrazine and

Metabolites in the Female Ra t. Tox. Sci. 112(1):88-99.

In this article the authors r eported the results of 3 studies. The objective was to investigate if atrazine and/or two of its metabolites – desethylatrazine (DEA) and diamino-s-chlorotriazine (DACT) – affected the hypothalamic-pituitary adrenal (HPA) axis in females by monitoring the levels of circulating adrenocorticotropic hormone (ACTH), corticosterone, and progesterone. Pr eviously, it had been demonstrated that

these hormones were affected in males afte r a single exposure to atrazine at a dosePage 114 of 263 level of 100 mg/kg/day. In addition, studies were also conducted to determine if the

effects seen in these hormones were similar to those elicited by restraint stress or if they were secondary to gastrointestinal discomfort.

Study 1: Oral Chloroatrazine Exposure and Restraint Stress

Single exposure

Four to nine regularly cycling Long-Evans female rats were treated by gavage with a single dose of either1% methyl cellulose (veh icle), 75 mg/kg atrazine (purity 97%), 60.2 mg/kg DIA, or 50.6 mg/kg DACT (atrazi ne equimolar doses) on the morning of proestrus. Precisely 15 minutes after treatm ent, the animals were sacrificed and their trunk blood collected to assay for ACTH, corticosterone, and progesterone. At necropsy, the pituitary and uter i were collected and weighed. In addition to examining the impact of chloroatrazine exposure on hormone levels, the investigators also evaluated the impact of restraining non-tr eated animals for 5 minutes on stress hormone levels as well as the pituitary and uterine weights.

With the exception of a statistically signifi cant 33% uterine weight decrease after DACT exposure, neither the 5 minute restraint nor treatment with a singl e dose of atrazine or DIA affected body weight, pituitary or uterin e weights. In contra st, a single dose of

atrazine or DIA resulted in a st atistically significant increase in ACTH, corticosterone, and progesterone within 15 minutes of exposure. Interestingl y, DACT though the most prevalent of the metabolites pr esent in the plasma after atrazine exposure, had little or no impact on any of the stress hormone levels.

Restraining the animals for 5 minutes and sa crificing them precisely 15 minutes after resulted in a statistically significant increas e in ACTH, progesterone, and corticosterone levels. While the magnitude of the pr ogesterone and corticosterone response was similar to that elicited by atrazine or DI A treatment, ACTH increases after restraint stress were markedly lower than the re sponses following compound exposure.

Repeated exposure

Nine to 14 regularly cycling females were treated by gavage with vehicle, 12.5 or 75 mg/kg/day atrazine, 10 or 60.2 mg/kg/day DIA, or 8.4 or 50.6 mg/kg/day DACT {atrazine equimolar doses] for 4 consecutive days starti ng on the morning of vaginal estrous and ending on the morning of proestrous. As was done during the single exposure study, animals were sacrificed precisely 15 minutes after the last administration of the compound and their trunk blood collected to assay for ACTH, corticosterone, and progesterone. At necropsy, the pituit ary gland and the uteri were collected and weighed. In addition to exam ining the impact of chlor oatrazine exposure on hormone levels, the investigators also evaluated the impact of restraining non-treated animals for 5 minutes on four consecutive days.Page 115 of 263 Body weight and uterine and pituitar y weights were unaffected by compound administration though a statistically signi ficant 20% increase in both absolute and relative uterine weights were observed after the animals were restrained for 5 minutes.

Consistent with what was observed after a single exposure, repeated atrazine and DIA exposures led to increases in ACTH, cortic osterone, and progesterone. All of these increases were statistically significant except for the ACTH increase seen after treatment with 12.5 mg/kg/day of atrazine. Again, DACT appeared to have little or no impact on stress hormone levels. Interestingly, though statistically significant, the ACTH response after repeated exposure was marked ly attenuated from the response after an acute exposure while the magnitude of the response for both corticosterone and progesterone were similar after acute or repeated exposures.

Atrazine, DEA, DIA, and DACT plasma conc entrations were obtained 15 minutes after the last treatment under both exposure par adigms. After 15 minutes, the plasma

concentrations were ranked such that atra zine<DEA<<DIA<DACT. After 4 consecutive exposures to 75 mg/kg/day atrazine, DACT plasma concentrations were ≈ 400-fold higher than the parent compound and 7-fold higher than DIA.

Study 2: Vagotomized

The objective of this study was to dete rmine if the increase in stress hormones observed after atrazine, atrazine-metabolites tr eatment were related to irritation of the gastrointestinal tract rather than a direct effect on the HPA axis.

Ten vagotomized rats and 10 intact rats were us ed in this experiment. Four to 5 of the vagotomized or intact rats received ei ther 100 mg/kg atrazine or vehicle (1% methylcellulose) by gavage. The animals we re sacrificed precisely 15 minutes after treatment and the trunk blood collected for hormone analysis. Atrazine treatment resulted in an increase in ACTH, corticos terone, and progesterone levels in both vagotomized and intact animals. For ACTH and progesterone, the response in vagotomized animals was substantially more robust than in intact animals. Corticosterone levels in both intact and vagotomized animals were also increased after

atrazine treatment but the di fference was not as marked. Nonetheless, these findings suggest that the stress hormone release not ed after atrazine or atrazine-metabolite

administration is not the out come of GI discomfort.

Study 3: Intravenous DIA Exposure

This study was also conducted to evaluat e if the stress hormone release observed shortly after atrazine or atrazine-metabolites treatment was due to GI discomfort. In this study, rats with an indwelling jugular vein cat heter were treated with either vehicle or 1 ml of 7.8 mM DIA (thus bypassing the GI tract). Blood was drawn 15 minutes after treatment and assayed for ACTH and cort icosterone levels. Both ACTH and corticosterone levels were statistically significantly increased 15 minutes after DIA administration relative to animals exposed to vehicle; suggesting that the release ofPage 116 of 263 stress hormones was not due to GI discomfort but rather a direct effect of atrazine or its

metabolites on the HPA axis.

The authors of these studies concluded that, in addition to the hypothalamic-pituitarygonadal (HPG) axis, atrazine trea tment also affects the HPA axis as evidenced by the increase in levels of circulating ACTH, co rticosterone, and progesterone. Given that

activation of the HPA axis has been shown to impact reproductive outcomes (likely through interactions of corticotropin-re leasing factors (CRF) and gonadotropin release hormone (GnRH) neurons), atrazine’s effect on the HPA axis may represent a precursor

event to the attenuation of the LH sur ge observed after atrazine exposure.

Reviewer’s Comment: The findings in these series of studies is consistent with

observations in female mice where atrazine exposure to 500 mg/kd led to an increase in

corticosterone levels. Similar to what was r eported in this study, male rats exposed to a single oral dose to atrazine, DIA, or DEA experienced an increase in ACTH, corticosterone and progesterone. Interestin gly, DACT appeared to have little or no impact on the HPA axis although it is the most prevalent of the chloroatrazine metabolites. Thus, it appears that atrazine and/or the intermediate metabolites (DEA or DIA) are critical in the activation of the HPA axis. ACTH release, however, is attenuated after repeated exposures to atrazine or DIA ye t corticosterone levels remained markedly elevated and were comparable after a single or multiple exposures.

This study is relevant for the risk assessm ent and supports the MOA for atrazine. These observations strongly suggest that the atrazi ne-induced endocrine response is a direct, chemical-induced change in the hypothalamic-pituitary-adrenal tissue and not the result of a non-specific stress response.

A.2.3 Gojmerac, T., Pleadin, J., Zur ic, M., Rajkovic-Janje, R., and Korsic, M., 2004. Serum Luteinizing Hormone Response to Administration of Gonadotropin- Releasing Hormone to AtrazineTreated Gilts. Vet Human Toxicol. 46(5): 245-247.

This study looked at porcine serum luteinizing hormone levels in response to administration of gonadotropin-releasing hormone to atrazine-treated pigs.

From the beginning of estrous (days 0 and 21) , 10 female pigs (ages 7-8 months) were given 1 mg atrazine/kg bw (99% purity) in the feed daily for 20 days of the estrous cycle. There were 5 female control pigs. One hour following the last treatment on day 20 (when the first signs of estrous were obser ved), blood samples were collected (min 0)

from control and atrazine-treat ed groups. Five atrazine-treat ed pigs were immediately given an i.v. dose of 100 μ g GnRH. Blood sample for porcine LH (pLH) determination were collected from the atrazine-expos ed and the GnRH-inject ed atrazine-treated groups at 20, 30, 40 and 90 mi nutes. Serum concentrations of pLH were determined by radioimmunoassay.Page 117 of 263 The experiment was preceded by another wh ich measured pLH concentration in peripheral blood samples from 3 intact pigs during the estrous cycle. Serum concentrations of pLH were determined in sa mples collected from the onset of estrous (days 0 and 21) between 8 and 9 am, and then twic e daily at 3-hour intervals, and on days 1,2, 4, 6, 8,10, 12, 14,16, 18,19. and 20 of the estrous cycle.

The pLH concentration from 3 normally cycling (intact) mature female pigs measured at 3 daily 3-hr intervals during the estrous cycl e was increased from 2.6 to 3.9 ng/ml on days 0 and 21, which indicated the ons et of estrous. The mean serum pLH concentration in control animals on day 20 of the estrous cycle correlated with the levels in the 3 normally cycling animal on the same day. Repeated exposure of female pigs to atrazine resulted in significant reduction (p<0.001) of serum pLH on day 20 of the estrous cycle (0.57 ±0.05 ng/ml) compared to control animals (2.24±0.20 ng/ml), which

is consistent with other studies showin g significantly reduced estrogen-induced LH response following atrazine administration in female rats. The single iv GnRH administration to atrazine-treated female pigs resulted in significant increase in pLH 20 and 30 minutes after GnRH administration, indi cating that attenuation of GnRH release in atrazine-treated animals was responsible fo r the suppression of serum pLH secretion.

Reviewer’s Comments: This study is relevant to risk assessment in that it supports the

neuroendocrine MOA identifie d for atrazine. By demonstrat ing that suppression of the LH secretion can be abrogated by providing an exogenous source of GnRH, the study authors have clearly demonstrated the re lationship between disruption of GnRH's pulsatile release and attenuat ion of the LH surge.

A.2.4 Juliani, C., Silva-Zacarin, E. , Santos, D. and Boer, P. 2008.

Effects of Atrazine on Female Wist ar Rats: Morphological Alterations in Ovarian Follicles and Immunocytochemical Labeling of 90 kDa

Heat Shock Protein. Micron. 39:607-616.

The purpose of this study was to characterize morphologically the effect of low and

sublethal doses of atrazine on ovaries of adul t female Wistar rats to determine the possible intrafollicular target site through wh ich atrazine acts. In addition, they analyzed the expression of the 90 kDa heat shock protein in both ovarian follicular cells and ooctye by immunochemical labeling, to evaluat e the role played by this protein on the ovary under stressed conditions, induced by atrazine exposure.

Female Wistar rats (n=5/dose group) were dosed via oral gavage with atrazine (97.1% purity) at doses of 0 (vehicle), 0.75 and 400 mg/kg/day. The sublethal dose of 400 mg/kg/day was chosen since it is two-fold th e atrazine dose responsible for disruption of the estrous cyclicity in female rats (Laws et al 2000). The dose of 0.75 mg/kg/day was

chosen since it was the lowest dose of atrazine that had been tested on female rats which had not induced estrous cyclicity al terations (Cooper et al. 1996; Laws et al. 2000). The study was conducted in two blo cks. In the subacute treatment block, females received 0 or 400 mg/kg/day atrazine for 14 days. In the subchronic treatmentPage 118 of 263 block, the females received 0 or 0.75 mg/kg/day atrazine for 30 days. Body weights were recorded daily. At the end of the treatment s, the animals were euthanized and ovaries were removed and prepared for hist ology and immunohistochemical analysis. Primary, preantral, and antral folli cles per ovary were analyzed.

In the subacute group, there was a decrease in food consumption and animals lost ~19% of their initial body weight after 14 days, while the control group had a 17.2% increase in body weight. Clinical signs included fatigue and weakness in the treated animals, slight trembling in hind legs and presenc e of strong odor in urine. It should be

noted that none of these data were provided in the arti cle. In the subchronic group, there was no significant decrease in food consumption in the 0.75 mg/kg/day dose group. There was an increased body weight (8 .5%) in the dosed an imals after 30 days. This was lower than the weight gain in t he control group (18%). Clinical signs included fatigue and weakness.

In the subchronic treatment group (0.75 mg/k g/day for 30 days, rare multioocytic follicles were observed in the treated gr oup. In the subacute treatment group (400 mg/kg/day for 14 days), atrazine induced oocyte toxicity as evidenced by the presence of preantral follicles with disorganized granul osa cells and/or a degenerating oocyte with discontinuous zona pellucida, which were not observed in the contro l group. Ovotoxicity was also observed in antral follicles in varied degrees. In some atretic antral follicles, where high intensity of apoptosis was observed in the granulosa cells, the oocyte showed nucleolus fragmentation and irr egularities of the Zona Pellucida.

Low doses of atrazine induced an increased ex pression of HSP90 in follicular granulosa cells in comparison to the control group. However, the positive reaction product, indicative of the HSP90 expression, was not f ound in follicular granulosa cells of ovaries collected from rats exposed to subacute treatments.

Reviewer’s Comments: It should be noted that in this study no quantification of findings was attempted. Most results were presented in the form of photomicro graphs, thus it is

difficult to assess the relevance of the fi ndings since most of the assessments were subjective (e.g. visual inspection of color change in cells.) This study, therefore, is not very useful for risk assessment.Page 119 of 263 A.2.5 Laws, S., Farrell, J., Stoker, T. and Cooper, R. (2003) Pubertal Development in Female Wistar Rats following Exposure to Propazineand Atrazine Biotransformation By-Products, Diamino-SChlorotriazine and Hydroxyatrazine. Toxicological Sciences 76:190-

200.

The purpose of this study was to evaluate the effects of two at razine by-products, diamino-s-chlorotriazine (DACT) and hydro xyatrazine (OH-ATR), and a structurally similar chlorotriazine, propazine (PRO) on female pubertal development. Previous studies by Laws et al. have shown that atrazine delays the onset of pubertal development in female rats.

Female Wistar rats (n=15/treatment group) were gavaged from postnatal days (PNDs) 22 through PND 41 with DACT (96.8% purity) (16.7, 33.8, 67.5, 135 mg/kg), OH-ATR (97.1% purity) (22.8, 45.7, 91.5, 183 mg/kg) or PRO (99. 8% purity) (13, 26.7, 53, 106.7, 213 mg/kg). The dose range for each chemical was selected as the molar equivalent of atrazine (12.5, 25, 50, 100, 200 mg/kg). The females were monitored daily for vaginal opening (VO) and killed on PND 41. The age at complete vaginal opening was recorded. Body weights were recorded daily . Tissue weights for each animal were recorded for liver, kidney, adrenals, ovaries, uterus, and pituitar y. Histological evaluations were performed on the thyroid, uter us, and ovaries. [Note: The Protocol for the Assessment of Pubertal Development and Thyroid Function in the Female Rat (Goldman et al. 200) was used for these studies . Since this protocol is undergoing validation by US EPA, thyroid hormones were evaluated in these studies even though no previous data suggested that chlorotria zines would affect thyroid function.]

DACT (33.8, 67.5, and 135 mg/kg) significa ntly (p<0.05) delay ed the age at vaginal opening by 3.2, 4.8, and 7.6 da ys, respectively, compared to controls (33.1± 0.4 (SE) days of age). Four of 15 animals in the high est DACT dose group did not attain vaginal opening by necropsy at PNDs 41-42. Body weight at the age of VO was also significantly increased in the three highest dose groups of DACT (13-19%). At necropsy the body weight in the high dose DACT animals re mained significantly lower (10.4%) than controls. PRO (106.7 and 213 mg/k g) also significantly delayed the age of VO by 3.6-4 days. A significant increase in body weight at VO was seen in females receiving the highest dose of PRO. Doses of OH-ATR (up to 200 mg/kg) did not cause a significant delay in the age of VO.

Significant reductions in kidney, pituitary, and adrenal weights were observed following exposure to the highest dose of DACT. No si gnificant differences in tissue weights were seen in any of the OH-ATR animals. Pituitar y weights in the two highest dose groups of PRO (106.7 and 213 mg/kg) were significantly decreased compared to controls.

No significant dose related effects were s een for serum T3 or TSH following exposure to any of the test chemicals. An absence of corpora lutea was noted in 4 animals in thePage 120 of 263 highest DACT treatment group that did not atta in VO prior to necropsy. No treatmentrelated microscopic changes were seen in any animals in the PRO or OH-ATR groups.

This study concluded that oral exposure to DACT and PRO from PNDs 22-41 delays the age of vaginal opening in Wistar rats in a dose-dependent manner. The no effect level (NOEL) for DACT (16.7 mg/kg) was identical to the equimolar NOEL for atrazine (25 mg/kg). The LOEL for delay in v aginal opening for DACT (33.8 mg/kg) was equimolar to that reported for atrazine (50 mg /kg). The LOEL for PRO was 2-fold higher that that for atrazine (100 mg/kg vs 50 mg /kg). OH-ATR appeared to be less potent and

did not alter the onset of puberty at doses equimolar to 200 mg/kg ATR.

Reviewer’s Comments: It appears that post-natal exposure to DACT results in a delay in sexual maturation. These delays do not appear to be secondary to low body weight since body weight at time of VO was si gnificantly higher for the delayed pups. Given that atrazine is quickly metabolized in the body, it appears that DACT is driving the delays in puberty onset. The DACT NOAEL for sexual matura tion reported by Laws et

al. (25 mg/kg/day in atrazine molar equiva lents) was higher t han the NOAEL (1.8 mg/kg/day) for attenuation of t he LH surge currently used as the point of departure in the Agency’s risk assessment.

A.2.6 McMullin, T.S., Andersen, M. E., Nagahara, A. Lund, T.D., Pak, T., Handa, R.J., Hanneman, W.H. ( 2004). Evidence that atrazine and

diaminochlorotriazine inhibit th e estrogen/progesterone induced surge of luteinizing hormone in fe male Sprague Dawley rats without changing estrogen receptor action. Tox. Sci. 79:278-286.

High oral doses of atrazine (ATRA) suppre ss the estrogen-primed luteinizing hormone (LH) surge in ovariectomized female rats. Th e mechanism by which ATRA mediates this action is unknown, but may be related to it s anti-estrogenic activity since LH release from the pituitary glan d is largely regulated by estrogen bi nding to its receptor in the hypothalamus. Since atrazine is rapidly met abolized to diaminochlorotriazine (DACT) ( i.e. , AUC for DACT is 166-fold greater than that of ATRA at 24 hours after dosing rats with 300 mg/kg bw atrazine), it could also be the actions of this metabolite on the estrogen receptor (ER). The current st udy addresses this issue by dosing ovariectomized Sprague Dawley rats by oral gavage with ATRA or DACT to investigate if the effects of atrazine and/or its major me tabolite diaminochlorotriazine (DACT) on the estradiol benzoate (EB)/pr ogesterone-induced LH surge are due to impaired ER function. As expected, ATRA suppressed the EB and progesterone-induced LH surge in a dose dependent manner. A daily dose of 300 mg ATRA/kg bw for 5 days nearly abolished the LH surge. In contrast, a dose of 77 mg DACT/kg bw (which produces the same AUC for DACT as dosing with 300 mg/kg bw ATRA) only partially attenuated the LH surge (~ 60%). The effect of DACT on pituitary function was also examined in response to exogenously added gonadotropin-releasing hormone (GNRH). DACT attenuated the LH surge by 47% as compar ed to controls, indicating a decrease in pituitary function.Page 121 of 263 Binding studies with uterine cytosolic containing ER and in vitro -transcribed ER indicate that ATRA, but not DACT at the same molar equivalent concentration, decreases estradiol binding regardless of incubation time. Based on the dose dependent inhibition of estradiol-binding, an inhibi tion binding constant (Ki) of 0.02 mM was estimated for ATRA while DACT exhibited a 10-fold greater value.

In an effort to investigate whether ATRA alters estrogen binding to hypothalamic ER, unoccupied ER levels were evaluated in two estrogen-responsive hypothalamic nuclei. Treatment with ATRA along with estradiol benzoate did not alter the levels of unoccupied ER levels as compared to estr adiol benzoate treatm ent alone. Moreover, ATRA failed to block estrogen-induced progesterone mRNA expression as compared to estrogen treatment alone.

Reviewer’s Comments: In summa ry, the results of this study indicate that (1) although

ATRA binds ER in vitro , the suppression of the LH surge in vivo may not be due to alteration of ER function in the hypothalamus by ATRA itself. This finding is not consistent with the findings of several other researchers that sh ow that atrazine does not bind directly to the ER (Cooper et al. 2007, Albanito et al. 2008, Judson et al. (2010 ) ; (2) the difference in potency between AT RA and DACT in suppressing the LH surge may be due to pharmacokinetic differences, that is, a greater internal exposure to DACT may result from dosing with ATRA than DACT itself due faster metabolism/excretion of this species; (3) the effects of DACT in suppressing the LH surge involves at least in par t decreased pituitary function.

A.2.7 Shibayama, H. Kotera, T., Sh inoda, Y., Hanada, T. Kajihara, T., Ueda, M., Tamura, H., Is hibashi, S., Ymashita, Y. , and Ochi, S. 2009.

Collaborative Work on Evaluation ofOvarian Toxicity Two- or Fourweek Repeated Dose Studies and Fertility Study of Atrazine in

Female Rats.

Atrazine was administered via oral gavage to female (n=10/group) Sprague-Dawley rats (>97% purity) for two or four weeks at doses of 0, 3, 30, or 300 mg/kg/day for the repeated dose toxicity study and at doses of 0, 3, 30, or 100 mg/kg/day for the female fertility study from two weeks befor e mating to Day 7 of gestation.

Repeated Dose Toxicity Studies: Dose levels of 0, 3, 30 or 300 mg/kg/day were

selected for these studies based on results of previous studies that showed that

treatment of female SD rats with atrazine for 21 days induced disruption of the estrous cycle at 75 mg/kg/day and inhibited follicle and corpus luteum development at 300 mg/kg/day (Cooper et al. 1996). Treatment of ovariectomized female rats with atrazine for five days suppressed the peak level of t he LH surge at 30 mg/k g/day (McMullin et al. 2004).Page 122 of 263 Five week old female SD rats were treated fo r either two or four weeks. Clinical signs and mortality were observed twice daily. Body weight/gain and food consumption were measured twice a week. Vaginal smears were taken every day and the mean estrous cycle was calculated during the dosing period. Animals were sacrificed after 2 or 4 weeks, blood samples were taken for hem atology and blood chemistry. The ovaries, uterus, and pituitary gland were weighed. These organs, as well as the vagina and mammary gland, were exami ned histopathologically.

Female Fertility Study: Dose levels of 3, 30 or 100 mg/kg/day were selected as

described above. Ten week old male and fema le SD rats were used for the fertility study. Females were treated for two weeks as the premating period. Males were not treated and were only used for mating purposes. After the premating period, females were caged with untreated male s on a 1:1 basis overnight for up to two weeks. Daily

administration of atrazine or vehicle was continued throughout the mating period and until Day 7 of gestation, or until the last day of the mating period if the animal did not copulate. Clinical signs and mortality were observed twice daily during the administration period and onc e daily during the other per iods. Body weights were measured twice a week during the pre-mating and mating periods and on Days 0-8, Day 11 and Day 14 of gestation period. Food consumption was measured twice a week during the premating period and on Days 1, 4, 8, 10, and 13 of gestation. The mean estrous cycle length during the pre-mating period was calculated. Vaginal smears and vaginal plugs were checked for the presence of sperm during the mating period. On Day 14, animals were exsanguinated and the pres ence or absence of implantations was determined after macroscopic examination of the main organs. The number of corpora lutea, implantations, live embryos, and dead embryos were counted and the copulation rate, and pregnancy rate were calculated. Animals that did not copulate were necropsied 14 days after the mating period a nd the thoracic and intraperitoneal organs were examined macroscopically.

Results for Repeated Dose Toxicity Studies : No mortality occurred in the 2- or 4-week study. In the two week study, clinical signs included lacrimation seen at 30 and 300 mg/kg/day and salivation and so iled perineal region at 300 mg/kg/day. However, no incidence for these findings was provided. In the four week study, clinical signs included lacrimation, salivat ion, and soiled perineal regi on at 30 and 300 mg/kg/day and decreased stool observed at 300 mg/kg/day. Decreased body weight/body weight gain was observed in the 300 mg/kd/ay dose groups in both the two week (11%, p<0.05/39%, p<0.01) and four week ( 17%/ 42%, both at p<0.01) studies.

Prolonged mean estrous cycle was observed at the high dose in the two week study (9.3 days vs 4.2 days, control, p<0.01) and in the 4-week study at 300 mg/kg/day (8.9 days vs 4.2 days control, p<0.01). The number of animals with irregular estrous cycles was increased (p<0.01) at 300 mg/kg/day in both the 2-week and 4-week studies (100% and 80% incidence vs 0-20% incidence in control).Page 123 of 263 Decreased absolute ovarian weights were obs erved at 300 mg/kg/day in the 2-week study (19%, p<0.01) and the 4-week study (17%, p<0.05). The uterus weight was decreased at 300 mg/kg/day (25%) in the 4-w eek study. In the two week study at the high dose, histopathological findi ngs occurred in the ovary (loss of currently formed corpora lutea, decrease in numbers of previously formed corpora lutea, increased in large-sized atretic follicles, swelling of pr eviously formed luteal cells), the uterus (atrophy), vagina (mucification and increased single cell necrosis in the vaginal epithelium) and mammary gland (lobular hyperpl asia). No weight or histological changes were observed in the pituitary gland. In the 4-week study at the high dose, histopathological findings occurred in the ovary (increase in large-sized atretic follicles (slight), swelling of previously formed lut eal cells (slight)), ut erus (atrophy), and mammary gland (lobular hyperplasia and increas ed lactation). The four week treatment group showed lower incidences of abnormal ovarian and vaginal histopathological findings then the two week treatment group. The authors noted that these results may be explained by spontaneous recovery of some animals from a disturbed to a normal estrous cycle.

Results for Female Fertility Study: No mortality occurred. Clinical signs included salivation at 30 and 100 mg/kg/day and lacr imation and decreased stools at 100 mg/kg/day. However, no incidence of these findings was provided. Atrazine did not show significant effects on the fertility study in rats. The copulation index, fertility index, number of corpora lutea, number of implantation site s, numbers of live and dead embryos, and percentage of preimplantation and postimp lantation loss were not affected by atrazine treatmen t. It should be noted that the high standard deviations for these observations call into question the quality of the data. Copulation failure caused by prolongation of diestrus was seen in 1 animal in the 100 mg/kg/day group, which could be due to the anovulatory effect of at razine. No histopathological findings were

seen in the ovaries.

The authors concluded that the effect of atrazine on female fertility in rats could be assessed by detailed histopathological exam ination of the ovaries in a two-week repeated dose toxicity study.

Reviewer’s Comments: In summary, atrazine caused irregular estrous cycles accompanied by adverse changes in ovarian histopathology at doses of 300 mg/kg. Extended dosing for 4-weeks did not further exacerbate the ef fects. In contrast, no effect on fertility was observed at a dose of 100 mg/kg. Wh ile one of the objectives of this study was to compare ovarian histology with fertility, the lack of identical doses in the repeated dose toxicity studies and the fertility studies prevented this comparison. It is noteworthy that the NOAEL for the effects reported by Shibayama et al., (30 mg/kg/day in atrazine molar equivalents) were higher than the NOAEL (1.8 mg/kg/day) for attenuation of the LH surge currently us ed as the point of departure in the Agency’s

risk assessment.Page 124 of 263 A.3 Reproductive Effects in Males

A.3.1 Betancourt, M., Reséndiz, A. , Casas, E. and Fierro, R. (2006) Effect of two insecticides and two herbicides on the porcine sperm motility patterns using computer- assisted semen analysis (CASA) in

vitro . Repro. Tox. 22:508-512.

In this study the authors investigated the effect s of four pesticides – malathion, diazinon, fenoxaprop ethyl, and atrazine – sperm viabi lity, morphology, and motility pattern in porcine semen. An in vitro viability staining method was us ed to assess viability while a computer-assisted-analyzer (CASA) was used to study sperm mobility. 12

Semen samples were collected from 3 Duro c boars of proven fertility and the sperm checked for anomalies in motility or morphol ogy. Only samples with sperm motility ≥ 85% and sperm abnormalities < 15% were used in the experiment. Sperm at a

concentration of 25 x 10 6 sperm/ml, were placed in 4 well plate samples at a final concentration of 0, 50, 100, or 500 μ M in a volume of 500 μ l tris-buffered medium [approximately 0, 5.4, 11, or 54 μ g] and incubated for 1 hour at 37°C. For the viability analysis, 10 μ l of the semen sample were pl aced on a slide and stained with 10 μ l eosin-nigrosin staining. Sperm that were vi able did not incorporate the stain while dead spem were stained purple. Fo r the motility evaluations, 8 μ l of sperm sample were placed on a slide and observed using a CASA anal yzer. Parameters measured by this device include mean velocity, linear velocity , curvilinear velocity, mean lateral head displacement, and frequency of head displacement.

Exposure to 11 or 54 μ g of atrazine resulted in an 8% and 21% statistically significant (p<0.05) decrease in sperm viability, respec tively. With respect to sperm motility, atrazine exposure at 11 or 54 μ g led to a statistically significant decrease in progressive motility of 23 and 75%, respectively. At razine exposure appeared to affect the mean velocity and linear velocity of sperm. These parameters are indicators of sperm progression. In contrast, other parameter s examined by the CASA that were not

affected by atrazine ( e.g., mean lateral head displacement and frequency of head displacement) are indicators of sperm vigor.

The authors of this study c onclude that atrazine has an effect on sperm motility and, therefore, affect fertility. It should be noted, however, t hat no data are available from this study to corroborate this hypothesis. Inte restingly, other researchers have reported

that the outcomes of in vitro fertilization (IVF) using sperm treated with up to 10 μ M

atrazine were not affected. The authors also speculate that the effects in sperm motility may be a sequela of mitochondrial function disruption – cellular re spiration, in particular.

Reviewer’s Comments: Over all, the data suggests an ef fect of atrazine on sperm motility but not morphology or viability. The study, however, entails direct exposure of

12 In this evaluation only the resu lts for atrazine will be discussedPage 125 of 263 germ cells to atrazine without taking into a ccount physiological conditions that may preclude the pesticide from eliciting this effect in vivo . Given that atrazine is rapidly metabolized, it is likely that the met abolites and not the parent compound are the moieties reaching the sperm and testis. This consideration, however, was not accounted for by the study authors. Moreover, no attempt was made by the researchers to investigate the consequences of these changes in sperm motility on reproductive health ( e.g., decrease in fertility). In the absence of these considerations, the data presented have little relevance to human health risk assessment

A.3.2 Friedmann, AS. 2002. Atrazi ne inhibition of testosterone production in rat males follow ing peripubertal exposure. Reproductive Toxicology 16: 275-279.

Juvenile Sprague-Dawley male rats (n=4-6 /dose group) were administered 0 or 50 mg/kg/day atrazine (purity not stated) via gavage for either a 3-day exposure (PND 46- 48) or a 27-day exposure (PND 22-48). Fo r both exposure scenarios, animals were

killed on PND 49 between 9 and 11 in the morn ing by decapitation. Testosterone (T) levels in trunk blood, interstitial fluid of testes and cell culture media were measured. [Note: The 50 mg/kg/day dose was based on a p ilot study in which rats were dosed with 50 and 200 mg/kg for PND 22-48 and killed on PND 49. Both doses lowered body weight, reduced serum T and inte rstitial fluid T.] For the in vitro treatment looking at testosterone production, Leydig cells isolated from rats on PND 49 were co-cultured

with 50 μ g/ml or 232 μ M atrazine in the presence of a maximally stimulating concentration of luteinizing hormone.

In the 27-day in vivo exposure to 50 mg/kg/day, mean testosterone concentrations were significantly reduced by 38% in serum and 53% in intratesticular testosterone. Decreased body weight (7%) was seen in the 50 mg/kg/day group (215 g) compared to controls (230 g). In t he 3-day exposure to 50 mg/kg/day atrazine reduced both serum (57%) and intratesticular (58%) levels of testosterone. There was no significant decrease in body weight of the atrazine group compared to control in the 3-day exposure treatment.

In vitro treatment of Leydig cell s for 3 hours with atrazine cu lture medium resulted in significantly reduced testosterone production ( 35%) compared to cont rols. Atrazine also reduced testosterone levels in Leydig cells stimulated with db-cAMP by 34%. However, atrazine did not reduce testosterone production when cells were stimulated with hydroxycholesterol.

The author concluded that atra zine inhibits steroidogenesis in vitro and in vivo . The

mode of action is via inhibiti on at the testicular level.

Friedmann argued against the earlier paper by Trentacoste (he was a co-author) that concluded the decrease in T was only the result of underfeeding. He did this by citing

the drastic reductions in food intake needed to lower serum T. This argument wasPage 126 of 263 supported in the Stoker et al., paper m entioned above in which she measured serum T and interstitial T and found no difference in the males underfed to the 200 mg/kg group. Finally, the fact that the s hort-term treated rats also had lower T values and these animals had not different BWs also argues against the suggestion that all the hormone changes observed in these studies is a function of decreased weight.

In discussing his in vitro study Friedmann concluded that T production by the Leydig cell was decreased when exposed to atrazine. T he mechanism for this, if repeatable, is unclear. Friedman did suggest that as the synt hesis of testosterone in the presence of hydroxycholesterol was not impaired, the her bicide interferes with cAMP’s role in stimulating Leydig cells Furthermore, it s hould be noted that unpub lished data from the EDSP validation studies conducted at NHEERL, used much lower doses of atrazine in culture and measured T pr oduction in H295R cells and compared the results with similar treatment to primary Leydig cells. In both cases, T production was increased (see Higley et al., paper for data on H295 R cells using doses ranging from 1-100 μ M).

Reviewer’s Comments: In summary, a limit ed number or no meas ures of key androgen dependent tissues were examined in the in vivo study presented in this paper, especially in the long-term dosed rats. The in vitro data needs repeating with a range of

doses.

A.3.3 Hase Y, Tatsuno M, Nishi T, Ka taoka K, Kabe Y, Yamaguchi Y, Ozawa N, Natori M, Handa H and Wa tanabe H. (2008) Atrazine binds

to F 1 F 0 -ATP synthase and inhibits mi crochondrial function in sperm. Biochem Biophy Research Comm 366: 66-72.

This paper demonstrates the ability of ATR to alter human sperm motility in vitro , and tests hypotheses on the mechanism through whic h this might occur, specifically by decreasing ATP availability for sperm flagellar motion. In a series of biochemical tests, the authors present evidence that ATR can bind to F 1 F 0 -ATP synthase and thereby inhibit mitochondrial function (decreasi ng ATP available for sperm motion).

Note: The title is misleading because the aut hors did not actually determine effects on sperm mitochondria – rather they used liver mitochondria. Sperm energetics are quite different from somatic cell energetics and sperm mitochondria differ from somatic mitochondria in specific ways that should be discussed.

Approach: Ejaculated human semen samples were washed to recover motile sperm and the sperm were then cultured with atrazi ne (ATZ) and ATZ-NH2, a modified version of ARZ needed for subsequent bi ochemical studies). Oli gomycin, a sperm metabolic poison which uncouples ATP production in mitrochondria, was used as a positive control. Oligomycin inhibi ted % progressive sperm at 0.05 mM; ATZ inhibited at 0.3 and 1.0 mM but not 0.1 ( i.e. was less effective than the positive control). Sperm ATP levels were also measured and shown to be significantly decreased at the same concentrations of test compounds that inhibi ted % progressive motility. MotilityPage 127 of 263 assessments were made using establishe d computer aided sperm analysis (CASA)

methods.

Data rule out several early steps in ATP syn thesis and make a solid case for ATR ability

to bind the ATPase.

The authors conclude that this likely explains earlier reports that ATR decreases porcine sperm motility in vitro (as discussed in the preceding two papers). Importantly, they also conclude that “This study was motivated by the idea that ATZ applied to fields may cause a reduction of sperm count and motility to humans and other animals. However, the effect of ATZ on sperm motility was s een at >100 μM, exposure levels that probably

do not result from normal use of the herbicide. It is, ther efore, uncertain whether the observed phenomena are ecologi cally relevant.”

Reviewer’s Comments: The relevance of these findings to risk assessment is questionable given that the concentration levels required to affect sperm motility were high and exceed likely exposure levels result ing from atrazine use in the environment. Moreover, the relevance of these ex vivo findings to the in vivo physiologically relevant

conditions is also uncertain.

A.3.4 Kandori, H, Suzuki S, Asamoto M, et al. 2005. Influence of

atrazine administration and reducti on of calorie intake on prostate carcinogenesis in probasin/SV40 T anti gen transgenic rats. Cancer

Sci: 96: 221 – 226.

The effects of a 13- week dietary admin istration of 500 and 1000 ppm atrazine on the development of androgen-dependent prostate cancer were investigated using male probasin/SV40 T antigen transgeni c (TG) rats (Sprague-Dawle y genetic background, 6 weeks of age). Based on food consumption data, the corresponding doses in mg/kg/day were 33.0 mg/kg/day for the 500 ppm group and 61.6 mg/kg/day for the 1000 ppm group. Each treatment group contained 10 ani mals. A control group was included.

The rationale for using TG rats was based on previous findings that atrazine inhibited testosterone production in the Leydig cells dire ctly of male rats. This would allow for an examination of atrazine treatment on the development of androgen dependent prostate

carcinomas. The authors noted that the TG rats have been established in their laboratory, and have developed well-differentiated adenocarcinomas in the prostate at

100% incidence before 15 week s of age. Since the tumors are considered to be completely androgen dependent, the hypothesis wa s that exposure of atrazine to the

TG rats would cause inhibition of testos terone production, resulting in reduced carcinoma development. Since atrazine has been reported to decrease body weights, a dietary-restricted TG rat group was also incl uded to assess the influence of reduction of calorie intake on the developmen t of prostate cancer.Page 128 of 263 The authors reported that mean food consumption in the dietary-restricted group was about 70% of that in the control group. At the end of 13 week tr eatment period, the following organs were subjected to histologic al examination: testes and accessory sex organs, ventral prostate, the dorsolateral pros tate including the ur ethra, and the anterior prostate/seminal vesicle complex. Serum testosterone levels were measured for every animal in each group using radioimmunoassay. Serum levels of atrazine and atrazine

metabolites were measured for 3 animals per group using gas chromatography-mass spectrometry. Immunohistochemistry fo r SV40 T antigens and androgen receptors were performed using avidin-biotin-peroxidase complex method. The ratios of prostate epithelial areas were evaluated morphometrically.

The authors reported that no deaths were obser ved. Body weights in the atrazine treated groups were significantly lower than in the control group. Bodyweights of the dietary-restricted and 1000 ppm atrazine groups were similar, and significantly lower than control values throughout the treatment period. Food consumption was lower in the atrazine treatment groups compared with control group. In the dietary-restricted group, mean relative ventral prostate weight was significantly decreased (25%) relative

to controls. No other treat ment-related changes in relative organ weights in any treatment group were reported. Serum test osterone levels were not significantly

different between the atrazi ne and dietary restricted gr oups compared with control group. Atrazine and its metabolites diamin ochlorotriazine, desethylatrazine, and desisopropylatrazine were detected in the seru m of the atrazine treated animals, but not in the control or dietary restricted groups.

Histopathological examinat ion of the ventral, lateral, an d anterior lobes of the prostate revealed lesions in all groups including cont rols. The authors described the lesions as marked epithelial proliferation with formation of irregular glands and luminal bridging to give a cribriform pattern in focal populations. The nuclei demonstrated enlargement and severe atypia. According to the authors, the lesions appeared compatible with welldifferentiated adenocarcinomas in human ca ses and were therefore diagnosed as

adenocarcinomas. These were observed in the v entral, lateral, and anterior lobes of the prostate in almost all rats. In the dorsa l lobe, adenocarcinomas were observed in two of three rats of each group. The incidences of adenocarcinomas in any treatment group

were not different from thos e of the control group. Fo ci within glands with less proliferation were also observ ed. These exhibited crowding of stratified epithelial cells with irregular spacing and occa sional luminal bridging. Nu clear atypia were described as severe, however, basic structures of t he prostate glands were mostly maintained, comparable with prostatic in traepithelial neoplasias (P IN) of human cases.

Because almost the entire lobes of the pros tate were occupied with tumor lesions at week 13, morphometric assessment ratios of the prostate epithelia l area to the whole prostate tissue area were evaluated. Morp hometric examinati on of the prostate epithelium revealed significant decreases in t he ratios of the epithelial areas to whole prostate tissue areas in the ventral, dorsal, and lateral lobes in the dietary-restricted group, and in the lateral lobe of the 1000 ppm atrazine group. The values in the dorsalPage 129 of 263 lobe in the atrazine groups showed a tendency to decrease (not statistically significant) in a dose-dependent manner. No significant ef fect was observed in the anterior lobe in any treatment groups.

SV40 Tag and androgen receptor expression we re detected immunohistochemically in almost all cells of the prostate adenocar cinomas and PIN. No treatment-related changes in the testes, epididymides, and seminal vesicles were observed.

The authors concluded that dietary administration of atrazine for 13 weeks showed slight suppressive effects on the development of prostate cancer in the probasin-SV40 T antigen transgenic rats, even though atra zine treatment did not affect serum

testosterone levels. Based on the observation that there wa s equal or greater suppression of prostate neoplastic lesion deve lopment in the dietary-restricted group,

the authors also concluded that the observed atrazine-related suppression of prostate cancer was probably caused by the decrease in calorie intake, ra ther than endocrine

effects.

Revewer’s Comments: While this study suggests the possibility that prostate carcinogenesis may be a secondary effect to a reduction in caloric intake, a test system

in which testosterone levels are altered w ould provide a more definitive confirmation.

A.3.5 Laws, S.C., Hotchkiss, M., Ferre ll, J., Jayaraman, S., Mills, L., Modic, W., Tinfo, N., Fraites, M., Stoker, T. , and Cooper, R. (2009).

Chlorotriazine Herbicides and Metabolites Activate an ACTHdependent Release of Corticosterone in Male Wistar Rats. Tox. Sci. 112(1):78-87

This study is a follow-up to a previous set of experiments study in which these

researchers demonstrated that atrazine ex posure results in an increase in steroidogenesis leading to increases in cort icosterone, progesterone, and estrogens. In this study the investigators sought to characterize the temporal changes in adrenocorticosteroid (ACTH), progesterone, and corticosterone in males receiving a single dose of atrazine, simazine, propazine or the chlorinated metabolites of these triazines (DIA, DEA,and DACT). 13
Male Wistar rats ( ≈ 50-60 days old) received 0, 5, 50, 100, or 200 mg/kg atrazine while animals exposed to DIA received 4, 10, 40, 80, or 160 mg/kg and DEA-exposed animals received 173 mg/kg. DACT-exp osed animals received 3.37, 33.7, 67.5, or 135 mg/kg.

The authors state that the maximum dose for each metabolite was the molar equivalent of atrazine at 200 mg/kg. Animals were sacrificed precisely 5, 15, 30, 60 or 180 minutes after exposure. ACTH, corticos terone, progesterone, and prolactin plasma levels were measured using commercia lly available radioimmmmunoassay kits. Furthermore atrazine, DIA, DEA, and DACT plasma levels were also measured. Statistically significant increases in ACTH levels were first detected 5 minutes after atrazine exposure at doses ≥ 100 mg/kg. Maximal increases in this hormone ( ↑ 5.6-fold)

13 This review will only discuss data for atrazine, DIA, DEA, and DACTPage 130 of 263 were observed 15 minutes after exposure to 200 mg/kg atrazine. The time-to-effect profile for corticosterone and progesterone increases were slightly slower with first statistical significant changes noted15 minutes after exposure at 50 mg/kg and above. For both of these hormones, maximum plas ma levels were seen 30 minutes after exposure ( ↑ 7.3- and 4.5 fold for corticosterone and progesterone, respectively). Exposure to DIA at doses ≥ 40 mg/kg led to increases in ACTH, corticosterone, and progesterone within 15 minutes of exposure ( ↑ 6.2, 6.3, 12.6-fold, respectively at 161 mg/kg DIA). Similarly, DEA exposures at 173 mg/kg led to a marked increase in ACTH ( ↑ 11.8-fold), corticosterone ( ↑ 6.4-fold), and progesterone ( 13.6-fold) within 15 minutes of exposure. In contrast, exposure to DA CT at doses as high as 137 mg/kg led to a minimal increase in these hormones ( ≤ 1.3-fold).

Plasma levels of atrazine, DIA, DEA, and DACT were detected within 5 minutes of exposure. The levels of the metabolites increased with time. Five minutes after exposure to atrazine DIA was the most pr evalent metabolite but by 180 minutes DACT was the predominant metabolite seen in plasma.

The authors conclude that the r apid increase in ACTH after exposure to atrazine or its

chlorinated metabolites induces adrenal steroi dogenesis indicative of initial effects at the hypothalamus and/or pituitary level.

Reviewers comments: This is a well-conduct ed study that carefully evaluates not only the dose-response but also the temporal-re sponse relationship between atrazine, DIA, or DEA exposure and changes in hormones in the HPA axis. Elevated ACTH, corticosterone, and progesterone levels can be seen 15 minutes after a single exposure

to atrazine at doses ≥ 50 mg/kg. Although t he authors did not establish a NOEL for this study, the no effect level may be establishe d at 5 mg/kg – the lowest atrazine dose tested. Interestingly, t hough DACT is the final and most prevalent atrazine metabolite, it has little impact on corticosterone, progeste rone, or ACTH levels. The relationship between these hormonal changes and the adv erse reproductive health outcomes observed after atrazine exposure, however, rema ins unclear. To what extent or for how long do these hormones must remain elevated before adverse effects are seen is not

known at this time and cannot be determined from these data.

A.3.6 Laws, SC, Stoker, TE, Ferrell , JM, Hotchkiss, MG, and Cooper

RL. 2007. Effects of Altere d Food Intake During Pubertal Development in Male and Female Wi star Rats. Toxicol. Sci. 100(1):

194-202.

Wistar rats (n=13) were underfed 10%, 20%, 30%, or 40% less than the ad libitum food consumed by controls from postnatal days (PND) 22-42 (females) or PND 23-53 (males). This time period encompasses the age of the onset of pube rty in both sexes. For any given food restriction (FR) treatment group, each animal within that group was paired with a control rat of similar initial bo dy weight. Body weight was recorded daily. The day of preputial separation (PPS) in ma les and vaginal opening (VO) in femalesPage 131 of 263 was recorded. Females were killed by decapitation on PND 41-42 and liver, kidney, adrenal ovary, uterus and pitu itary weights were recorded. Males were killed by decapitation on PND 53-54, and liver, kidney, adrenal, pituitary, left testis, ventral prostate, left epididymis, se minal vesicles with coagulating gland (with fluid) were removed and weights recorded. Thyroid and reproductive tissues were examined histologically. Serum T3, T4 and TSH, gluc ose, leptin and pituitary (males only) prolactin and LH were measured.

The terminal body weights of FR females (P ND 42) and males (PND 53) were 2, 4, 12, 19% and 2, 6, 9, 19% lower than controls for the 10, 20, 30 or 40% FR groups, respectively, being statistically significant at the top two doses. For both females and males, the age of pubertal onset was not significantly different from controls for all FR

groups.

In females, the thyroid hormone endpoint s were not affected by any FR group

associated with 2-19% reduction in body weight. In males, there was a significant

decrease in T3 and T4 at the 30% and 40% FR groups associated with 9% and 19% body weight decrease, respectively. TSH was significantly decreased in the 40% restricted group. No differences in serum test osterone, LH or prolactin were noted in any of the males. Serum glucose concentra tions in males were not different from

controls. Female rats in the 30% and 40% FR groups had significantly decreased serum glucose when compared to controls. Mean seru m leptin concentrations were decreased in the 30% and 40% FR males, but were only reduced in the 40% FR females.

The absolute weights, but not the relative weights, for pi tuitary, adrenals, liver and kidney were significantly lower in the fema les with a 12% reduction in body weight (30% FR) and significantly lower in males with a 6 or 9% reduction in body weight (20% and 30% FR, respectively). The exception was a significant change in relative liver weight in the animals with a 6, 9, and19% body weight reduction. No histopathological changes were observed in any organs.

In females, the absolute weights, but not the relative weights, for ovaries were decreased in the 30% and 40% FR groups. The mean uterine weights were not different from control for any FR group. In males, the absolute, but not re lative weights, for ventral prostate and seminal vesicles were significantly lower than the controls in the

40% FR.

Reviewer’s Comments: This study clarifies the relationship between altered growth and reproductive development in the male and fema le rat and can be useful in interpreting effects of reproductive toxicants and endocrine disrupting chemicals. Previous studies used must greater weight loss paradigms during the post natal period which usually resulted in over 50% weight loss. This st udy uses milder food restriction limits which show that a 2 to 19% difference in weight do not interfere with the endpoints measured in the male and pubertal protocols. These observations support the concept that a 10% reduction in body weight is a reasonable ba sis for setting the maximum dose for thePage 132 of 263 female pubertal protocol in conjuncti on with any other information concerning the toxicity of the test compound (e.g., clinical signs, other toxicity).

A.3.7 Maravilla-Galvan, R., Fierro, R., Gonzáles-Marquez, H. GómezArroyo, S., Jiménez, Ir and Betanc ourt, M. 2009. Eff ects of Atrazine and Fenoxaprop-Ethyl on Capacita tion and the Acrosomal Reaction in Boar Sperm. International J ournal of Toxicology. Volume 2, Number 1. pp. 24-32.

The purpose of this study was to evaluate the in vitro effects of atrazine and fenoxaprop-ethyl on capacitation, s pontaneous acrosome reaction (SAR) and progesterone-induced acrosome reaction (PIAR) in boar sperm. 14

Sperm samples were obtained from the s perm-rich ejaculated fraction of 3 healthy fertile boars. Sperm viability was evaluated by eosin-nigrosin staining. Only ejaculates showing more than 85% progressive motility, at least 90% viability, and less than 10% abnormality were used. Sperm capacitati on was done in TALP-HEPES media for 4 hours. Capacitation and SAR were evaluated immediately; PIAR was evaluated 30 minutes later. The acrosome reaction (AR) was induced by adding progesterone at a

final concentration of 10 μ g/mL and incubating for 30 minutes. The spontaneous acrosomal reaction without progesterone indu ction is called SAR. Sperm membrane status was assessed by chlortetracyc line staining and analyzed with a UV epifluorescence microscope. Sperm with a un iformly distributed fluorescence over the

whole head and an intact acrosome were considered noncapacitated; sperm showing bright fluorescence in the acrosomal regi on beside a fluorescence-free band in the postacrosomal region with an intact acrosome were considered capacitated; sperm with a dull fluorescence over the whole head except for a thin, bright ban in the equatorial segment were considered acrosome-reacted. At least 200 sperm were analyzed per

slide.

Cytotoxicity was determined by incubating sperm using concentrations of atrazine (technical grade, purity not r eported) of 0 (control), 20, 40, 60 and 100 μM. Treatment with atrazine resulted in a dose-related decrease in sperm viability. For 0, 20, 40, 60 and 100 μM atrazine the percent viability was 80, 60, 37, 27, and 6, respectively. The

median lethal concentrations of atrazine were calculated. Final concentrations of 8, 20, and 40 μM atrazine were used in the experi ments. These concentrations corresponded

to the LC 50/5 , LC 50/2 , and LC 50 , respectively. The concentrations used were sublethal for

sperm in vitro , using the LC50 as a maximum. For the capacitation and PIAR studies,

the herbicide was added at the beginning of the 4 hour inc ubation. The control was the same solvent with the herbicide (0 μM).

Sperm Capacitation: In the presence of atrazine during capacitation and PIAR, the viability of capacitated sperm decreased in a linear fashion for the 8, 20 and 40 μM dose

14 In this evaluation, only the result s for atrazine will be discussed.Page 133 of 263 groups. Capacitation decreased significantly only at the high dose (40 μM), to 79% of controls (p<0.05). SAR increased 1.9 fold for the 8 and 20 μM and 2.2 fold for the 40 μM dose groups, all at p<0.05. The nonc apacitated population was reduced at all concentrations, compared to controls (p<0.05).

PIAR: SAR observed in control sperm immediat ely after capacitation was 17%. After 30 minutes of progesterone induction of acroso me reaction, PIAR increased to 30% in controls. This increase shows that progester one acts as an AR inducer. All atrazine concentrations produced a significant dec rease (p<0.05) in noncapacitated and capacitated sperm, compared to controls. PIAR was statistically increased at 8 μM (1.6 fold), 20 μM (1.8 fold), and 40 μM (1 .8 fold), compared to controls.

Similar to a previous study by this group (Betancourt et al, 2006), the authors review

literature on atrazine effects and use, but fail to provide a sound rationale for examining

direct in vitro effects on sperm. There is no literatu re to imply that atrazine selectively targets mature sperm and impairs their fertiliz ing ability after in vivo exposure. They state that “it is necessary to carry out in vitro studies to clarify the mechanisms of action of toxic compounds,” but do not provide justification for this statement. Rather they

state that because atrazine and FE are used in Mexico and other countries, the aim of the study is to evaluate in vitro effects....on boar sperm capacitation and the progesterone-induced acrosome reaction. These two statements are unrelated and do not provide a sound rationale for the study. T here is also no evidence that atrazine reaches maturing sperm in the testis, epidi dymis or female tract at concentrations resembling those selected for testing in vitro

.

Reviewer’s Comments: In summary, these data do not appear relevant to assessment of risk of atrazine after in vivo exposu res nor provide biologi cal plausibility for suspecting that atrazine may selectively tar get sperm and thereby impact fertility or sperm function. The authors did note that th e atrazine concentrations used in this study are not in the relevant range to produce in vivo effects.

A.3.8 Mu, H. , Shang, P. and Xu, J. (2008). Testicular Toxicity and

Mechanisms of Chlorotoluron Com pounds in the Mouse. Toxicology

Mechanisms and Methods. 18: 399-403.

This study looked at the effects of chloro toluron and atrazine ingestion on male mouse testes during the peripubertal period. 15

Groups of 20 male mice (Chinese Kunming stra in) were given either chlorotoluron alone (625, 1250, 2500, 5000 mg/kg), atrazine alone (218.75, 437.5, 875, or 1750 mg/kg), or a combination of these chemicals (combinat ion 1: atrazine/chloro toluron: 218.75/625, 218.75/1250, 218.75/2500, or 218.75/5000 mg/kg; combination 2: atrazine/chlorotoluron: 437.5/625, 437.5/1250, 437.5/2500, 437.5/50 00 mg/kg.) There

15 In this evaluation, only the result s for atrazine will be discussed.Page 134 of 263 was one vehicle control group. The animals were dosed by daily gavage from day 27 to day 52. On day 52, 24 hours after the last gavage, body weights were measured and recorded and blood samples were taken. Fo llowing sacrifice test is weights were

measured and recorded. Testes tissue was processed for examination by light microscope and transmission electron micr oscope. Superoxide dismutase (SOD) activity was measured. SOD is an antio xidant (removes superoxide radicals).

There was a dose related decrease in relative testes weights at all dose levels for atrazine (4.6-13%) and combined atrazine/ chlorotoluene (6.9-14%), although the decreases were not statistically significan t. The SOD enzyme activities increased in a

dose-related manner in animals treated with atrazine and combined, with the effects of

treatment with the combinati on of herbicides being greater than the single chemical. Control values were not reported.

Histologic changes in the testes that occu rred in treated animals included shedding of spermatogenic cells, increased intercellula r space, appearance of mild interstitial edema, and modest congestion of tissue. No tables were provided and it wasn’t clear whether these findings were for atrazine or chlortoluron, or both . Findings

for mice treated with a comb ination of chlorotoluon and atrazine included seminiferous tubules that were shrunken and had indentations and increased shedding of spermatogenic cells and Sertoli cells. Other effects seen by electron microscopy included decreased smooth endoplas mic reticulum and Golgi (at combined dose of 625 mg/kg chlorotoluron plus 218.75 mg/kg atrazi ne) and heterochromatin in the mural cells which appeared in a blocked state, decreased nuclear matrix, spermatogenic cells with swollen appearance, segmental degeneration of cell membranes and tight junctions which were partially damaged (at combi ned dose of 1250 mg/kg chlorotoluron plus 437.5 mg/kg atrazine).

Comments: This study was poorly written such that it was unclear whether the findings

were for atrazine or chlorotoluron. Cons equently, this study was not useful for risk

assessment.

A.3.9 Pogrmic K, Fa S, Dakic V, Kaisarevic S, and Kovacevic R. 2009. Atrazine Oral Exposure of Peripubertal Male Rats Downregulates Steroidogenesis Gene Expression in Leydig Cells. Toxicological Sciences 111(1):189-197.

The purpose of this study was to attempt to identify the possible mechanism of inhibition of Leydig cell steroiodogenesis in per ipubertal male Wistar rats after in vivo exposure to atrazine. The study was based on earlier pu blished papers (described above) that

identified a decreased testosterone fo llowing atrazine exposure when dosing began shortly after weaning until after sexual maturation (defined as PPS).

Male Wistar rats were treated by gavage wi th 0, 50 or 200 mg/kg/day atrazine (98% purity) from PND 23-50. Animals were sacrif iced 24 hours after last dose of atrazine.Page 135 of 263 They reported that oral admin istration of atrazine (200 mg /kg/ay) produced significant reduction in serum T/DHT(assay they used was sensitive to both). At 200 mg/kg/day, body weight was also reduced significantly as were the weig hts of andr ogen-dependent organs (testes and seminal vesicles at both, dorsal and ventral prostate at high dose only). But LH was not altered at 50 and not significantly reduced in the 200 mg group. 50 mg/kg atrazine did not alter serum hormo ne levels nor the weight of androgen dependent tissues. Adrenal and Testes weight s were affected in both dose groups. They pointed out that Trentacoste et al. (2001) suggested that testosterone-reducing ability of atrazine is se condary to weight loss.

Their in vitro dosing is of interest as both doses caused significant decrease in basal

and hCG stimulated testosterone releas e. There was no dose response and no variance presented in all the cell-stimulation. However, the variance was presented for

the dose of 10 μ M hCG in a bar graph for hCG 10 μ M. In addition to a decrease in T production, there was also a dose dependent dec rease in the cAMP concentrations. When the cells were incubated in the pres ence of hydroxycholesterol, pregnolone, progesterone and androsteinedione, in every case , the amount of T produced from the Leydig cells obtained from t he 50 and 200 mg/kg/ atazine-tr eated animals was reduced compared to controls. Importantly, there was no dose response. Finally the authors reported reduced levels of transcripts fo r CYP17A1 and 17bHSD, which seem to indicate an effect upstream and downstream of androgen synthesis. LH did appear reduced at 200 mg/kg but was not significant. Th is is in contrast to the observation of Friedmann, 2002 who reported that in vitro treatment with atrazi ne suppressed T from Leydig cells of untreated animals, but t he suppression could be overcome by coincubating with hydroxycholestero l. If these observations ar e both correct then there is

a difference in the ex vivo vs. the in vitro exposed Leydig cell.

This group also measured the transcription of several genes involved in steroidogenesis (including the LH receptor) after p.o. adm inistration. They report a clear dosedependent decrease in gene expression of LHR, SR-B1, StAR, TSPO, SF-1, PDE4B, 3bHSD, CYP17A1, 17bHSD in Leydig cells. There was no effect on CYP11A1 and ßHSD. They conclude that the inhibition of all these genes following atrazine may be

related to decreased c-AMP stimul ation. However, there are many inconsistencies with the literature ( i.e. , others found SF1 increased after atrazine exposure in vitro ) and within their own data ( i.e. , cAMP is lowered, as is PDE4B....the enzym e that degrades cAMP).

Reviewer’s Comments: The quality of this manuscript is hard to judge. There is a lot of data, much of which appears solid, however, the lack of a dose response in the Leydig

cell incubation studies and the decrease in t he expression of so many genes in the RTPCR studies demands confirmation. Finally, as to whether or not these data support the HPG MOA or indicate a direct action of Atrazine on the Leydig cell can not be determined. In addition, it should be noted t hat the doses used in this study werePage 136 of 263 relatively high compared to the doses e liciting the neuroendocrine effects and used for regulatory purposes.

A.3.10 Rayner J L., Enoch R R, et al. 2007. Atrazine-induced reproductive tract alterations after transplacental and/or lactational exposure in male Long-Evans rats. Tox Appl Pharmacol: 218: 238 –

248.

The authors note that pr evious studies have shown that early postnatal exposure to atrazine delayed preputial separation (PPS) and increased incidence of prostate

inflammation in adult Wistar rats. The aut hors stated that a cro ss-fostering paradigm was used in their study to determine if gestati onal exposure to atrazine would also result in altered puberty and reproductive tissue effe cts in the male rat. Timed-pregnant LongEvans rats were dosed by gavage on gestati onal days (GD) 15 – 19 with 100 mg/kg/day atrazine or 1% methylcellulose (controls). On postnatal day (PND) 1, half litters were cross-fostered to create the following four treatment groups: birt h dam or gestational exposure only (ATR-C); milk source or lactational expos ure only (C-ATR), exposure both pre-and postnatal (ATR-ATR), or neit her (C-C). Beginning on PND 37, male offspring (n>18males/group) were evaluated for puberty ( i.e. , PPS). Necropsy was performed on either PND120 (n = 10/group) or 220 (n = 9-10/group); organs included pituitary gland, testes, lateral and ventral prostates, and seminal vesicles. Serum prolactin, testosterone, androstenedione and estrone levels were measured by radioimmunoassay. The left lateral pros tates were analyzed for myeloperoxidase activity. Histological exam ination of the right latera l and ventral prostates were conducted. The distribution scoring and severi ty classification were provided in the study. Body weights were monitored throughout the study.

Mean body weight of the ATR dams on GD19 was reduced 13% when compared to control dams. The male offspring in t he ATR-ATR group were 6.5% smaller when compared to offspring in the C-C and C-ATR groups on PND4. A significant 2 1⁄2 day pubertal delay was seen only in males in the AT R-ATR group. Body we ights at the time

of PPS were also decreased in the ATR-ATR animals.

At PND120, the lateral prostates of the AT R-ATR offspring group we ighed significantly more than those in any other group, even when corrected for body weight. The average ATR-ATR lateral prostate weight was 49% higher than control on PND120. At PND220, lateral prostate weights were significantly increased (45 – 110%) in animals within all ATR exposure groups compared to control animals. When adjusted for body weight,

animals in ATR-C and ATR-AT R groups had significantly in creased lateral prostate weights.

At PND120, necropsy evaluati on of offspring indicated ma croscopic abnormalities of the left and right lateral prostate in the ATR-ATR group; the abnormalities were characterized by nodular pale white foci pres ent on the outer surfac e. At PND 220, all atrazine groups had the abnormalities, most ly on the right lateral prostate.Page 137 of 263 Histological examination of lateral and ventral prostates revealed an increased

incidence and distribution of inflammation in the lateral prostates of C-ATR males at PND 120. Only one out of ten ATR-ATR male s also exhibited inflammation; there was no inflammation in the C-C and ATR-C groups. The C-ATR and ATR-ATR lateral prostates had neutrophils as the predominant in flammatory cells. In regard to ventral prostates at PND 120, inflammation was evi dent in all groups (C-C, ATR-C, C-ATR and ATR-ATR) indicating no effect of treatment. The authors stated that it is difficult to assess inflammation in the ventral prostate because of a high background present. The authors also noted that although necropsy re vealed macroscopic abnormalities on the surface of the lateral prostate of males in the ATR-ATR group, these abnormalities “may not have penetrated deep within the gland.” By PND220, lateral prostate weights were significantly increased for ATR-C and ATR- ATR groups, but there were no significant changes in inflammation in either the lateral or ventral prostate.

The results of the myeloperoxidase assay demonstrated no significant effects; the authors noted the discrepancy between the assa y and the pathological findings. The authors explained the discrepancy as possibly due to not having both lateral prostates for assay (one used for the assay, the other fo r pathology). No statistically significant

differences in serum hormone concentrations of total testosterone, androstenedione, estrone, and prolactin were observed among the different treatment groups.

The authors concluded t hat the results of the study s uggest that the dev eloping lateral prostate gland in the Long-Evans rat is sens itive to atrazine-in duced changes that are transplacental or via nursing. In addition, la ctational exposure to these atrazine-induced changes increases lateral prostate inflammation.

Reviewer’s Comments: This study only examines one dose, so there are no dose response evaluations. The authors do not menti on the time of day wh ich they killed the males at 120 or 200 days of age or how long t he necropsy lasted, as this would be very important when evaluating effects on the endocrine system. The increased lateral prostate weights are reflective of an increase in lateral prostate inflammation, which has been found previously following early postnatal exposure of atrazine to the lactating dam (Stoker et al., 1999). There is also a delay of PPS in the ATR-ATR dose group only, indicating that it is the combinati on of gestational exposure to the dam and the postnatal milk from those mo thers responsible for the effect. Rosenberg et al, 2008 also observed a delay in PPS at 100 mg/kg with an oral perinatal exposure only, a condition that would be similar to the ATR-CON group in whom Rayner found no effect

on PPS. It should be noted that these effects in the Rayner et al. study occur at doses higher than those eliciting di sruption of the LH surge.Page 138 of 263 A.3.11 Rosenberg, B.G., Chen, H., Folmer, J., Liu, J., Papadopoulos, V., and Zirkin, B.R. (2008). Gestational Exposure to

Atrazine: Effects on the Postnatal D evelopment of Male Offspring. J. Andrology 29(3):304-311.

In this study, the authors investigated t he impact of atrazine exposure during gestation on the development of Sprague-Dawley (SD) ra ts male offspring. Fourteen to 16 pregnant dams were exposed via gavage from gestation day (GD) 14 - 21 to 0, 1, 10, 50, 75, or 100 mg/kg/day of at razine (purity 98.5%). The male offspring of 7-8 dams per dose group were sacrificed on post-natal day (PND) 0. The testes from these males

were collected and used to measure testicular te stosterone levels in the newborn pups. The litters of the remaining 7-8 dams were cu lled on PND2 to 8 pups (4/sex/litter). Body weights and food consumption of pregnant dams were recor ded daily from GD 14-21. On PND21 the body weights and anogenital distance (AGD) were measured for each pup. The 4 male pups from each litter were also examined for preputial separation beginning on PND35. On PND 60 the pups we re sacrificed by decapitation and their blood (serum), testes, ventral prostate, and seminal vesicles collected. The testes, ventral prostate, and seminal vesicles were weighed. Testosterone levels were

measured in both the serum and intratesticular fluid.

Maternal body weight was statistica lly significantly reduced at doses ≥ 75 mg/kg/day starting on GD18. This decrease coin cided with a 33-47% decrease in food consumption for the 50, 75 and 100 mg/kg/day dose groups. Litter size was unaffected by atrazine treatment. However, pup death between PND 0 and 2 was increased by approximately 10-25% at doses ≥ 10 mg/kg/day, with statistical significance attained at the 75 and 100 mg/kg/day doses (~20-25%). Pup weight decreases of 10-17% were reported for pups whose dams were exposed to doses 50 mg/kg/day or higher. None of these pup weight decrements, however, were statistically significant.

Intratesticular testosterone levels m easured from neonates did not reveal any statistically significant effects at any dose level. Although a 60% increase in

intratesticular testosterone was noted at the 10 mg/kg/day dose level, there was no statistical significance or dose response for this effect ( i.e., testosterone levels at ≥ 50 mg/kg/day were comparable to control). It is noteworthy, that at this dose level a nonstatistically significant increase in AGD was observed in males on PND 21. while AGD was significantly reduced at 100 mg /kg/dose levels on PND 21.

A statistically significant delay in PPS rangi ng from 1-3 days was noted at 50 mg/kg/day

and above. (Note: Graph and text differ: graph indicates ss only at 100 mg/kg/day (p<0.004) while text says ss at ≥ 50 mg/kg/day (p<0.05, p<0.01)).

On PND 60, the animals used to assess PPS and AGD were sacrificed and their testes, seminal vesicles, and ventral prostate weig hts measured. In addition, testosterone

levels in both the serum and intratesticula r fluids were measured. No changes in terminal body weight or organ weights were noted. Serum testosterone levels,Page 139 of 263 however, were decreased at ≥ 50 mg/kg/day while intrat esticular testosterone was reduced at doses of 75 and 100 mg/kg/day. [Note: graph and text r eport of T differ; they are reversed.]

The authors of this st udy concluded that in utero exposure resulted in changes that manifested in the neonatal, per ipubertal, and adult life stages . The authors, however, did point out that these effects were observed at concentrations that “are unlikely to be experienced under any but ex perimental conditions.”

Reviewer’s Comments : The highest two doses 75 and 100 mg/kg/day resulted in significant reductions in the dam’s BW gai n. There was a dose-dependent loss of pups -- ~20-25% pup death at 75 and 100 mg/kg from PND 0-2. There were no differences in neonatal T at PND 0, but there did appear to be effect s on androgen dependent measures later in life, including reduced AGD at PND 21, a delay in PPS in the 100 mg/kg group and a decrease in serum T at 60 days of age in the 50, 75 and 100 mg/kg group. Oddly, none of the androgen-dependent tissue weights, including the seminal vesicles or ventral prostate were affected even at the highest dose. It should be noted that the effects on maturation and altered androgen status occurred in this study at dose levels substantially higher t han those leading to LH surge disruption, which serves as the basis of the Agency’s current risk assessment.

A.3.12 Stoker TE, Guidici DL, Laws SC, Cooper RL. 2002. The

Effects of Atrazine Metabolites on Puberty and Thyroid Function in

the Male Wistar Rat. Toxicol ogical Sciences 67: 198-206.

Male Wistar rats were gavaged (betw een 8 and 9 am) from PND 23 –PND 53 with atrazine metabolites (97-98% purity), deethylatrazine (DEA), deisopropylatrazine (DIA), and diaminochlorotriazine (DACT) at doses equivalent to the atrazine equimolar doses (AED) of 0, 6.25, 12.5, 25, 50, 100, or 200 mg/kg/day.. The day of preputial separation (PPS) was measured. Males were killed by decapitation 2 hours following the last dose on PND 53. The pituitary, adrenal, thyroid, te stes, epididymides, seminal vesicles, and ventral and lateral prostates were wei ghed and examined histol ogically. Blood and serum were assayed for hormones (testosterone, estrogen, LH, prolactin, TSH, T4, T3).

There was a significant decrease in body weight seen with all the metabolites at the two highest dose groups. For all metabolites at 200 mg/kg/day, there was a greater than 10% decrease in body weight at PND 43 and 53. For all metabolites at 100 mg/kg/day, body weight decreases ranged from 9-12% at PND 43 to 7-13% at PND 53, with DEA being the metabolite exceeding 10% body we ight decrease at both time points.

The day of PPS was delayed by all 3 met abolites. For DIA, PPS was significantly delayed at 25, 100, and 200 mg/kg/day AED by 1.2, 1.2 and 3.4 days, respectively, resulting in a NOAEL for delayed PPS of 12.5 mg/kg/day. For DEA, PPS was significantly delayed at 25, 100, and 200 mg/kg/day AED by 1, 2 and 4 days, respectively, with a NOAEL of 12.5 mg/k g/day. For DACT, PPS was significantlyPage 140 of 263 delayed at 12.5, 25, 100, and 200 mg/kg/da y AED by 1, 2.3, 1, 4, and 4.2 days, respectively with a NOAEL of 6.25 mg/kg/day.

Testicular weights were similar to contro ls in all treatment groups for all three

metabolites. When males were killed on PND 53, DEA (100 and 200), DIA (50-200 AED), and DACT (200 AED) treatments caused significant reduction in ventral prostate weight, while the highest doses of DIA and DEA resulted in significant decrease in lateral prostate weight. Seminal vesicle weight was reduced at 100 and 200 AED by all three metabolites. Epididymal weights were decreased in the DEA (200 AED), DIA (200 AED), and DACT (100 and 200 AED) treatment groups.

Serum testosterone was significantly decr eased only in DIA exposed males at 100 and 200 AED. Serum estrone was significantly in creased only by DACT at 100 and 200

AED.

The mean anterior pituitary weight was si gnificantly decreased on PND 53 at 100 and 200 AED by DEA and DACT and the highest dos e of DIA. On PND 53, serum LH showed a significant trend for a dose-dependent decrease from control to high dose in the DIA group. Pituitary LH was significantly decreased in the 100 AEd of DIA and the 200 AED of DEA compared to controls. Pitu itary prolactin content was significantly

decreased at 100 and 200 AEd of DEA and at 200 AED of DACT.

TSH, T3 and T4 were unaffected by any trea tment group. No effects were observed on thyroid histology of high dose or controls.

Minimal hypospermia (defined as rats in whic h the overall density of spermatozoa within the epididymal ducts appeared to be decreased compared to control samples() was seen in 25% of the high dose group of DIA, 40% of the hi gh dose group of DACT, and 12.5% of the high dose group of DEA. The author reported that this observation is probably a result of the length of the delay in puberty in t he high dose groups of atrazine metabolites and the age of sacrifice.

Reviewer’s Comments: This study shows t he effects of the three primary atrazine metabolites on pubertal onset and growth of androgen dependent tissues. Interestingly, many of the same effects were obser ved including delayed PPS and decreased reproductive tract development. DIA was the only metabolite which resulted in

decreased serum testosterone and DACT increas ed serum estrone. The doses appear

to be similar to those that were effective with atrazine for the same endpoints, including PPS. DACT delayed puberty at the same doses (12.5 to 200 AED). It is noteworthy

that the lowest NOAEL for t he effects reported by Stoker et al. 2002 (6.25 mg/kg/day in atrazine molar equivalents) was higher than t he NOAEL (1.8 mg/kg/day) for attenuation of the LH surge currently used as the point of departure in the Agency’s risk

assessment.Page 141 of 263 A.3.13 Stoker, T.E., Laws, S.C ., Guidici, D., and Cooper, R.L.

2000. The effects of atrazine on puberty in male wistar rats: An evaluation of a protocol for the assessment of pubertal development and thyroid function. Toxicological Sciences. 58: 50-59.

Intact male Wistar rats (n=6 -20) were treated with atrazine ( 97.1% purity) via gavage at doses of 0, 12.5, 25, 50, 100, 150, or 200 mg/k g/day from postnatal day (PND) 23 to 53. An additional pair-fed group was added to co mpare effects of any decreased food consumption in the high dose group. Daily body weight and preputial separation were measured. Males were killed by decapitation on either PND 45, 53, (or 54) or 120. The pituitary, thyroid, testes, epididymides, seminal vesicles, and ventral and lateral prostates were weighed and examined hist opathologically. Blood and serum were assayed for hormones (testosterone, es trogen, LH, prolactin, TSH, T4, T3).

There was a significant reduction in body we ight in the high dose (200 mg/kg/day) on PND 43 (14%) and PND 50 (17%). The pair fed group of 200 mg/kg/day had a significant reduction in body weight on PPS day (13%), PND 43 (20%) and PND 53 (15%).

Preputial separation was sign ificantly delayed in the 12.5, 50, 100, 150, and 200 mg/kg/day dose groups by 2.3, 1.8, 1.7, 1.6 and 3 days as compared to controls. PPS was also delayed by 2 days in the pair-fed animals compared to controls. The delay in PPS observed in the pair-fed animals was not as severe as that observed in the atrazine 200 mg/kg/day group.

This study showed that atrazine suppre ssed the growth of androgen dependent reproductive tissues at 50 mg/kg and up (ventral prostate). Ventral prostate weight was also decreased in the pair-fed group. Seminal vesicle and epididymal weights on PND 53 were decreased in the 200 mg/kg/day at razine group and pair-fed animals. By PND 120, all reproductive tract organ weights retu rned to control levels in all treatment groups, except for the ventral prostrate which was still signifi cantly decreased in the 200 mg/kg/day atrazine group (784.8 ± 46.1, controls vs 638.13 ± 25.3, 250 mg/kg/day). Lateral prostate and testicular weights were not different from control in any of the atrazine or pair-fed rats killed on PND 53.

There was no effect on serum testosterone at 53 days of age, but intra-testicular

testosterone was decreased on PND 45 which might explain the delay in pubertal development. There was no significant e ffect on serum LH on PND 53, but serum estrogens were increased at 200 mg/kg/day. These effects did not appear to be due solely to decreased growth, as there were effects at doses which did not affect growth (both PPS and tissue weights).

Reviewer’s Comments: Peripubertal exposure to atrazine resulted in delayed puberty onset and delayed growth of the reproductive tract of male rats. These data support the reported alteration of the hypothalamic control of pubertal progression.Page 142 of 263 A.3.14 Trentacoste, S V.. Friedmann, AS, Youker, RT, Breckenridge, CB, and Zirkin, BR. 2001. Atrazine Effects on Testosterone Levels and Androgen-Dependent Reproductive Organs in Peripubertal Male Rats. Journal of Andrology, Vol. 22, No. 1

, January/February 2001 ; pp. 142-148.

Male Sprague-Dawley rats received atra zine (96.1%) from PND 22-47 by gavage in methyl cellulose at doses of 0, 1, 2.5, 5, 10, 25, 50, 100 or 200 mg/kg/day (n = 9-12 rats/dose) and killed by decapitation on PN D 48 (24 hours following last dose; doesn’t state time of day killed). A food restriction study was conduct ed with rats pair fed to the 100 mg/kg/day atrazine treated group. In life meas ures included daily (? ) body weights. The day of preputial separation (PPS) wa s measured in controls and only the 100 and 200 mg/kg/day dose groups. Testosterone (serum and interstitial fluid) and serum LH

concentrations were measured.

Body weight (9%, 100 mg/kg/day/21%, 200 mg /kg/day), seminal vesicle (32%/55%) and ventral prostate (26%/48%) weights, seru m T (34%/32%) and intratesticular fluid T (41%, 45%), LH (17%, not s.s./20%) and PPS (3 days/4 days) were significantly reduced in the 100 and 200 mg/kg/day groups. PPS was only evaluated in the 100 and 200 dose groups. As body weight was affe cted by the atrazine 100 and 200 mg/kg/day, a group of males were pair f ed to the 100 mg/kg/day group. The underfed only animals

had a decrease in T and LH on PND 47 that wa s similar to the 100 mg/kg/dose group.

The authors concluded t hat these results indicate that atrazine exposure, up to 50 mg/kg/day, had no effect on the hy pothalamic-pituitary-testicular axis, and that at levels greater than 50 mg/kg per day , the effects observed in response to atrazine could not be distinguished from the effe cts observed after reduced food consumption. The author’s emphasized that caution must be exercised before c oncluding that atrazine (or any potentially toxic chemical) has direct, de trimental effects when the toxicant also produces significant changes in body weight. They further emphasize that body weight

must be both monitored and accounted for in t he assessment of potential toxic effects.

Reviewer’s Comments: The study author’s contention that the changes in the male reproductive tissue weight and PPS changes obs erved was the result of decrease body weight is not supported by an earlier study by Stoker et al., 2000. In that study, Stoker

examined similar measures in the male Wistar rat. This group reported that 100 mg/kg/day reduced BW approximat ely 7% (vs. approximately 10% in the present study with Sprague-Dawley males). However, Stoker also found a decrease in ventral prostate weights at 50 mg/kg/day males (whi ch had BW reduction of 3%) and delays in preputial separation following 12.5 mg/kg/day and above. Stoker also noted lower

serum testosterone values in males tr eated with 25 mg/kg/day and above, which was very similar to that observed by Rosenberg et al., 2008, following gestational dosing). Stoker et al., also pair fed males during the experimental period base on the food consumed by the 200 mg/kg/day animals. The body weight in the pair fed animals was 84% of control body weights (reduced 16%). Se rum T was not reduced in Stoker et alPage 143 of 263 study, however they did have smaller ventral pr ostates, seminal vesicles and a delay in PPS (albeit significantly less than that observ ed in the 200 mg/kg/day rats). Importantly, the Trentacoste et al., paper did not look at the onset of preputial separation in animals dosed with less than 100 and 200 mg /kg/day, the very endpoint that set the LOAEL an NOAEL in the Stoker paper. No explanation is given for this omission. Freidman (2002) reported a significant decrease in both serum testoste rone and interstitial testosterone in Sprague-Dawley rats tr eated during the same PND period, and body weight was reduced 7% (230 = control 215 = atrazine 50 mg/kg/day from 22-48.

A.3.15 Victor-Costa, AB, Bandeir a SMC, Oliveira AG, Mahecha GAB, Oliveira, CA. 2010. Chang es in Testicular Morphology and Steroidogenesis in Adult Rats Ex posed to Atrazine. Reproductive Toxicology. oi10.1016/j.reprotox.2009.12.006.

This study evaluated the effects of atrazine on the steroidogenic pathway in adult male rats by analyzing the 3 β -HSD expression, plasma and testicular estrogen and testosterone levels, expression of andr ogen receptor, and morphophysiological changes

in the testes.

Adult male Wistar rats (4-7/group) (120 days old) were given daily doses of atrazine (purity not provided) via gavage for 7 days (300 mg/kg/day), 15 days (50 and 200 mg/kg/day) or 40 days (200 mg/kg/day). A commercialized mix of atrazine called Gesaprim ® was used. This formulation consisted of 500 g/l atrazine and 500 g/l of inert ingredients. Control animals were treated for similar periods with the vehicle, corn oil.

After treatment, rats were weighed and sacrificed. Testes and adrenal glands were weighed. Testes were exam ined histopathologically . Changes in the expression of 3 β

hydroxysteroid dehydrogenase (3 β -HSD) enzyme and androgen receptor (AR) in the testes were studied by immunohistochemistry. 3 β -HSD is the enzyme responsible for various stages of the steroi dogenic pathway, including synthesis of adrostenedione, the testosterone precursor. Adrenal gl ands were also evaluated for the 3 β -HSD by immunodetection for comparison between ster oidogenic tissues. Immunostaining was quantified by computer-assi sted image analysis. AR st aining was quantified in 30 interstitial Leydig cells as well as 30 Serto li cells lining seminiferous epithelium stages VII or VIII, per animal, ex cept for those treated with at razine 200 mg/kg/day/40 days in which the testes were atrophic and the st ages of the seminiferous epithelium cycle could not be identified. In this case, the Sert oli cell positivity was measured in randomly

observed tubules. Plasma testosterone and estradiol were measured by both radioimmunoassay (RIA) and enzyme linked i mmuno sorbent assay (ELISA). Western blot assays were performed to determine t he specificity of t he antibodies and the changes in the expression of 3 β -HSD enzyme in testis and adrenal and androgen receptor in testes of treated groups.Page 144 of 263 There were significant body weight decreases (p<0.05) in the rats treated with atrazine 200 mg/kg/day (15 and 40 days) (35%) and 300 mg/kg/day (7 days) (38%). A significant increase (p<0.05) in the relative and absolute testes weights were seen in rats treated with atrazine 200 mg/kg/day/ 15 days (80% and 16%) and 300 mg/kg/day/7 days (83% and 14%). However, exposure to atrazine 200 mg /kg/day/40 days resulted in

a 41% and 60% reduction in the relative and abs olute testis weight, respectively. There was a significant increase (p<0.05) in the relative adrenal weight of 64%, 104%, and

41% for the rats treated with atrazine 200 mg/kg/day/15 days, 200 mg/kg/day/40 days, and 300 mg/kg/day/7 days, respectively. Ther e was no effect on body weight, testis or adrenal weights in animals treated wit h atrazine 50 mg/kg/day for 15 days.

Plasma testosterone levels, as measured in RIA assays, were statistically decreased (p<0.05) compared to controls in all tr eated animals (65% for 50 mg/kg/day/15 days; 85% for 200 mg/kg/day/15 days; 86% fo r 200 mg/kg/day/40 days; 87% for 300 mg/kg/day/7 days). ELISA assay confirm ed the reduction in te stosterone (data not provided). Testicular levels of testoster one were also significantly (p<0.05) reduced (32%) after treatment with atrazine 200 mg/k g/day/15 days. However, plasma and testicular estradiol levels were signific antly increased by 33% and 38%, respectively,

after treatment with atrazine 200 mg/kg/day/15 days. At a ll other dose levels, the plasma and testicular estradiol le vels were similar to controls.

Atrazine exposure caused changes in testis morphology. Atrazine exposure caused dilation of seminipherous tubules at doses of 200 mg/kg/day/15 days and 300 mg/kg/day/7 days. Pleomorphic Leydig cells with irregular nuclei were observed in all atrazine treated animals. Atrazine 200 mg/kg/ day/40 days resulted in testis atrophy and

reduction in the seminiferous tubules lumen. In this group giant multinucleated bodies and a large number of apoptotic cells were frequent within the seminiferous tubules.

Immunopositivity for AR was detected in Leydi g cells, Sertoli cells, and Myoid cells of both treated (200 mg/kg/day/15 and 40 days; 300 mg/kg/day/7days) and control rats. There was no difference in AR protein leve ls when animals treated with atrazine 200 mg/kg/day/15 days and controls were compared by Western blotting assay. Quantification of the AR immunop ositivity in specific cell ty pes after exposure to atrazine at the same dose (200 mg/kg/ day/15 days) showed equal AR intensity in Sertoli cells and Leydig cells compared to controls, which confirmed the Western blotting results. Treatment with higher doses of atrazine (300 mg/kg/day/7 days) resulted in decreased AR intensity in both Sertoli and Leydig cells. Longer treatment with atrazine (200 mg/kg/day/40 days) resulted in decreased AR st aining in Sertoli cells but increased AR staining in Leydig cells. It was noted that the rats tr eated for 40 days had atrophied testis and most of the tubules presented AR- positive Sertoli cells, with reduced staining intensity.

In control animals, 3 β -HSD positivity in the testis was found in the Leydig cells cytoplasm. After atrazine exposure, there was a great reduction in Leydig cells 3 β -HSDPage 145 of 263 immunoreactions (either weakly stained or negative). These data were confirmed by Western blotting, in which rats treated with atrazine 200 mg/kg/day/15 days showed significant reduction (40%) in the intensity of 3 β -HSD positive bands in the testis. The expression of 3 β -HSD immunoreaction was found in ce lls of the three zones of the adrenal cortex. The pattern of dist ribution and intensity of the 3 β -HSD immunoreaction

in the adrenal cortex was similar in both control and treated animals. Western blotting was in agreement with these re sults, showing that the 3 β -HSD protein was not altered

in adrenal cortex of treated ani mals compared to controls.

Reviewer’s Comments: This study used some unusual treatment regi mens. There is no dose-response examined, as there is only on e dose per treatment period except in the 15 day exposure. For exampl e, in the 15 day exposure, they include a 50 and 200 mg/kg dose, but do not show any effect s for the 50 mg/kg except for decreased testosterone. It is difficult to interpret the results with this experimental design and the high doses. They do include some measures that other studies in this area did not examine, including the measurement of testic ular and adrenal 3B-HSD and testicular androgen receptors. 3B-HSD is a key enzyme in the androgen biosynthetic pathway, but changes in the expression or protein levels could be a result of the suppression of luteinizing hormone. The fact that they found a decrease in B-HSD in the testes is different than the Pogrmic paper wherein this was one of only two (of the many examined) that did NOT c hange, but the current paper l ooks at the protein using immunocytochemistry and the Pogrmic paper looked at message. The changes in testicular weight are interesting and haven’ t been shown before. For example, testis increased weight with transi ent exposures (200 for 15 days or 300 for 7 days), but showed atrophy with exposed for 40 days at 200 mg/kg. The larger weights correlated

with increased lumens of the seminiferous tubules. The increased lumen is likely due to increased fluid accumulation (backflow). Over a period of time, this can lead to atrophy, which may be why it is observed in the 40 day exposure. The high doses and the fact that 50% of this was made from “other-ingr edients” in the commercial mixture make the

results difficult to inte rpret. This paper adds little to what we know.

A.4 Aromatase

A.4.1 Albanito L, Lappano R, Made o A, Chimento A, Prossnitz ER, Cappello AR, Dolce V, Abonante S, Pezzi V, Maggiolini M. (2008). Gprotein-coupled receptor 30 and estrogen receptor- α are involved in the proliferative effects induced by atrazine in ovarian cancer cells. Environ Health Perspect. 116(12):1648-55.

This study used in vitro methods to further evaluate the effects of atrazine in multiple cell lines. The hypothesis for the study is based upon previously reported endocrine disrupting effects of atra zine for androgen- and estrogen-medi ated processes following

in vivo and in vitro exposures. Using a series of molecular approaches, the study evaluates a number of potent ial cellular mechanisms to include potential effects onPage 146 of 263 estrogen receptor expression and other estr ogen target genes, extr acellular signalrelated kinase (ERK) phosphorylation, and cell proliferation.

Studies were conducted using human BG-1, MCF-7 (breast cancer), Ishikawa (human endometrial), and SkBr3 (breast cancer) cell li nes. BG-1, MCF-7, Is hikawa cells were (transiently) transfected with ER lucife rase reporter plasmids, and SkBr3 was cotransfected with ER α expression plasmid and ER-luc. All transfected cell line were maintained for 24 hrs., and then exposed to solv ent, estradiol (100 nM), atrazine (1 – 10

μ M)m, ICI ER antagonist (10 μ M), or combinations of ICI + E2, ATR + E2 for an additional 24 hrs. Figure 2 shows that atrazine did not stim ulate luciferase expression or block E2 induction in any of the cell lines at concentrations up to 10 μ M. A study to

determine whether or not atrazine regulated ER α expression was conducted using BG1 cells, and results demonstrated that atrazine (1 μ M) did not alter mRNA or protein concentrations of the receptor (Fig. 3) as compared to the down -regulation of these measures in those cells exposed to estradi ol. In addition, the study confirms that atrazine does not bind directly to the ER as previously reported by Cooper et al. 2007. Together, these data demonstrate that at razine does not bind or activate ER α

.

In experiments using the triated-water releas e assay, atrazine did not alter aromatase activity or gene expression in the BG-1 cells following a 40-hr exposure to 1 μ M. This finding is in contrast to that reported by Sanders on et al. (2000) for the H295R cell line. However, the current study r eports only aromatase activity as % of controls. Without

absolute values for aromatase acti vity of the controls, it is difficult to assess whether or not the BG-1 cells exhibit constitutively hi gh aromatase activity as observed in the R2C Leydig cell line (Heneweer et al., 2004) that would prevent further amplification by exposure to atrazine. In addition, the current study does not provide a positive control for either aromatase activity or gene expression.

To test the hypothesis that atrazine may generate rapid signaling via second messenger systems such as Ca2+, cAMP, nitric oxide, and G-proteins, which in turn can lead to activation of downstream kinase, an experiment was conducted to evaluate the ability of atrazine to produce effects on extracellula r signal-related kinase (ERK) phosphorylation in the BG-1 cells. Figure 5A-B and 6 show that atrazine (1 μ M) stimulated ERK phosphorylation within 20 min., albeit a high er concentration and prolonged time was required as compared with effect s of estradiol. A similar effect was observed in 2008 ovarian cancer cells. The ER agonist, ICI, prevented the atrazine and E2 mediated effects on ERK phosphoryl ation (Fig. 6).

Given the results on ERK phosphorylation, t he authors further evaluated the ability of atrazine to up-regulate the expression of c-fo s, an early gene that responds to a variety of extracellular stimuli, including estrogens . An 1-hr exposure to atrazine (1uM) significantly enhanced c-fos and cyclin A mRNA levels (200-269% increase as compared with control), and longer exposure (24- hr) resulted with significant elevations in progesterone receptor, pS2k and cyclin A. The authors conclude “Atrazine is able toPage 147 of 263 stimulate the expression of diverse estrogen target genes without an apparent activation

of ER α . Additional experiments demonstrat ed that the atra zine dependent upregulation of c-fos was abolished after 2- hr incubation with ER antagonist ICI, the EGFR inhibitor AG, or the ERK inhibitor, PD. Thus, the authors concluded “in ovarian
cancer cells, atrazine involves ER α and the EGFR/MAPK pathway to trigger c-fos protein increase.”

The study further evaluated the effects of at razine on the G-protein- coupled receptor 30 (GPR30), a seven-transmembrane receptor which is struct urally unrelated to the nuclear ER, but has recently been shown to mediate rapid actions of estrogens. The authors have also recently shown that c-fo s stimulation of E2 occurs through GPR30 and requires ER α and EGFR-mediated signaling in cancer cells (Albanito et al 2007, Cancer Res. 67:1859-1866). In the cu rrent study, both at razine-dependent ERK phosphorylation and c-fos induction were abo lished in BG-1, 2008 ovarian, and SkBr3 cells after silencing of GPR30 with sma ll interfering RNA (siRNA), Fig. 10.

Reviewer’s Comments: This study shows no evidence that ATR binds directly or

activate ER- α in cancer cells. It does offer intr iguing evidence that atrazine may bind to a membrane receptor that modifies a si gnaling pathway associat ed with c-AMP, ERK,

and P13k activation. The concentrations used to indicate this effect are well within, or below the range of concentrations known to m odify the steroidogenesis. The fact that the authors identify GPR30 as the target site is also interesting in that although there is clear evidence that this membrane receptor mediates very rapid (non-genomic) cellular responses, the finding that the ligand typically used in these experiments is estradiol. This manuscript, in combination with t he Suzawa and Ingraham and Higley et al., papers argues that atrazine influences signal transduction and provides an alternative to phosphodiesterase as the membrane associated target molecule for the herbicide.

A.4.2 Benachour N, Moslemi S, Si pahutar H, Seralini GE. (2007). Cytotoxic effects of aromatase i nhibition by xenobiotic endocrine disrupters alone and in combinat ion. Toxicology and Applied Pharmacology. 222:129-140.

The purpose of this study was to evaluate the in vitro effects of known endocrine disrupting chemicals on aromatase and cytochr ome P450 reductase activities, the later being the second moiety of t he aromatase enzyme complex t hat serves as the electron donor during the conversion of androgens to estrogens. Chemicals (aroclor 1254, atrazine, o,p’-DDT, vinclozolin, p,p’DDE, bisphenol A, chlordecone, nonylphenol, tributylin oxide, and diethylstilbestrol) were tested in both human placental microsomal and purified equine testicular ar omatase preparations, as well as in human embryonic 293 cells (transiently) transfect ed with human aromatase cDNA.

Human embryonic 293 cells (50,000/well) were grown in 24-well plates to 80% confluency and transfected with human aromatase cDNA 48 hrs prior to exposure toPage 148 of 263 test chemicals. An initial evaluation of ce ll viability following exposure to each chemical (up to 500 μ M) was determined using the MMT method. Following exposure to the test chemicals, media was removed, cells were washed with serum-free media, and tested for aromatase activity using the tritiated-wa ter release assay. Specifically, the cells

were incubated for 45 min. with 3H-androstenedi one, a substrate for aromatase. 3Hwater, released during the conversion of each molecule of androgen to estrogen is a direct measure of estrogen production and en zyme activity. Additional assays were conducted to determine the inhibitory effects of the test chemicals on aromatase activity in human placenta microsomes and equine test icular preparations containing the full aromatase (reductase) enzyme co mplex. Direct effects on t he reductase moiety of the aromatase complex was test ed by measuring the absor bance corresponding to the reduced form of cytochrome c in the presence of NADPH + . For this assay, reductase (isolated from equine testes) was preincubated wit h the test chemical for 15 min. prior to

initiation of the reaction.

In general, results from the study are difficult to interpret. Initial experiments to detect inhibitory effects on aromatase activity in human placental microsomes were conducted for each test chemical at a single high dose of 500 uM . Atrazine had no inhibitory

effect at 500 uM. Chemical s that reduced aromatase acti vity by 50% were further

tested at concentrations r anging from 10 – 1000uM. BPA inhibited reductase activity, albeit the IC50 was exceptiona lly high, 1250 uM. The authors report “a slight increase in reductase activity in the presence of atrazine” but the actual concentration responsible for the effect is unclear. Viab ility of the embryonic cells was affected after

24 hrs in culture at chemical concentrations ≥ 50 uM. Thus, maximal concentrations used for the aromatase assays in the cultures was 20 uM. Binary mixtures were tested for some of the chemicals, but again, data are difficult to interpret. Fig. 7 shows that atrazine significantly inhibited aromat ase activity in the embryonic cells.

Reviewer’s Comments: In conclusions, the data reported by Benachour et al. are not

useful for the current assessment of atra zine due to an incomplete documentation of methods, doses, and results. Atrazine is fair ly insolubility at concentrations higher than 50 uM, especially in media. Sections of the discussion are poorly written and cite data from other studies incorrectly ( e.g. , Sanderson, 2000; Crain 1997).

A.4.3 Fan W, Yanase T, Morinaga H, Gonda S, Okabe T, Nomura M, Hayes T, Takayanagi R, Nawata H. (2007a). Herbicide atrazine activates SF-1 by direct affinity and concomitant co-activators

recruitments to induce aromatase expression via promoter II. Biochemical and Biophysical Research Communications. 355:1012-

1018.

The authors were interested in confirming pr evious findings that atrazine induces aromatase expression via promoter II (ArPII) in a SF-1 (s teroidogenic factor 1) manner. They were also interested in studying bindi ng of atrazine to t he receptor, and whether affinity is independent of the ligand binding domain. The in vitro assays utilized H295RPage 149 of 263 cells (human adrenocortical cancer cells), HIH-3T3 fibroblasts cells, and KGN tumor cell line (human ovarian granulose-like tumor cell line). A luciferase gene reporter system

was examined. Information on the constr uction of the human CYP19 promoter II luciferase reporter and human SF -1 reporter was provided.

The in vitro experiments with H295R cells and luciferase gene reporter system showed that the ArPII reporter responded (dose-dependently) to atrazine (10 -7 to 10 -5 M) without exogenous SF-1 co-expression. To confi rm that SF-1 is essential for atrazine stimulation, endogenous SF-1 in H295R ce lls were knocked down by siRNA. The results indicated a complete loss of respons iveness of ArPII to either atrazine (10 -5 M) or simazine (10 -5 M). In the presence of ex ogenous SF-1, atrazine and simazine induced ArPII in the normally non-responsiv e KGN cells. The authors also noted that both atrazine and simazine were not cytotoxic and didn’t affect SF-1 gene expression.

Data were presented to demonstrate that protein kinase a (PKA) and liver cell homolog1 (LRH-1) are not essential fo r atrazine stimulation. To assess if the affinity between SF-1 and atrazine is followed by co-activat or recruitment, mammalian two hybrid-assays for SF1-TIF2 interaction were performed. Results suggested that atrazine enhanced

the interaction between SF-1 and its co-activ ator TIF2. The aut hors concluded that atrazine-SF-1 affinity is coupled with enhanc ed receptor-co-activator interactions. To

examine the functional domain of SF-1 in mediating atra zine stimulation over ArPII,

mutations were introduced to the DNA bind ing domain (DBD) and the ligand binding domain (LBD). Results revealed that mutations in DBD, but not LBD of SF-1 alter

atrazine stimulation. The authors concluded that atrazine binds to and activates SF-1

to induce ArPII.

Reviewer’s Comments: Fan et al 2007a, b showed that atrazine binds directly to SF-1 and induces aromatase expression in vitro . It should be noted, however, that after aromatase activity is not affected after in vivo exposure in mammalian species.

A.4.4 Fan W, Yanase T, Morinaga H, Gondo S, Okabe T, Nomura M, Komatsu T, Morohashi K, Hayes TB, Takayanagi R, Nawata H. (2007b). G Atrazine-induced aromatase expression is SF-1 dependent: implications for endocri ne disruption in wildlife and reproductive cancers in humans. Environ Health Perspect. 115(5):720-7.

This in vitro study focused on the role of steroi dogenic factor 1 (SF-1) in atrazineinduced aromatase expression. A lucife rase reporter driven by the SF-1 dependent aromatase promoter (ArPII) was used to examine activation of this promoter by atrazine and simazine. Atrazine-responsive adrenal ca rcinoma cells (H295R) and atrazine nonresponsive cells, ( i.e. , HIH-3T3 fibroblasts cells, and KGN tumor cell line (human ovarian granulose-like tumor cell line)) we re examined. A luciferase gene reporter system was used for the transfection assays.Page 150 of 263 Atrazine-responsive H295R cells expr essed 54 times more SF-1 than the nonresponsive KGN cells. Exogenous SF-1 conferred aromatase responsiveness to

atrazine and simazine in KGN cells. The re sults of the chromatin-immunoprecipitation (ChIP) assay indicated that atrazine and simazi ne increased binding of SF-1 to ArPII. Reducing the aromatase promot er II to 516 base pairs did not affect atrazine-induced aromatase response. However, a mutation in the SF-1 bind ing site on promoter II (AFFTCA to ATTTCA) eliminated the res ponsiveness to atrazine and simazine

treatment.

The authors concluded that atrazine binds direct ly to SF-1, showing that atrazine is a ligand for this “orphan” receptor. The resu lts of this study ar e supported by another study conducted by these investigators (Fan et al. 2007b).

Reviewer’s Comments: Fan et al 2007a, b showed that atrazine binds directly to SF-1 and induces aromatase expression in vitro . It should be noted, however, that after aromatase activity is not affected after in vivo exposure in mammalian species

A.4.5 Heneweer M, van den Berg M, Sanderson JT. (2004). A comparison of human H295R and rat R2C cell lines as in vitro screening tolls for effects on arom atase. Toxicol. Lett. 149(2):183-

194.

The purpose of this study was to compar e two cell lines, rat Leydig cell carcinoma (R2C) and human adrenocorticocarcinoma (H295 R) cells, for their suitability as a screening method to detect effe cts on aromatase expression and catalytic activity following exposure to selected xenobiotics. Fo r each cell line, aromatase activity using the tritiated-water release assay and promot er-specific mRNA levels were measured following exposure to prochloraz, fadrozole, epoxyconazole, atrazine, 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), prost aglandin E2, phorbol 12-myristate 13- acetate, and dexamethasone. This study is unique in that it is one of the first efforts to evaluate changes in aromatase acti vity of the test chemicals as well as the indentify the specific promoter regions of the CYP19 gene that is affected during exposure.

For all experiments, cells were cultured in 24-well plates, and exposed to each test chemical (1 – 10 uM,) for 24 hr prior to assa y for aromatase. These cells were used to evaluate effects on aromatase activity that required mRNA synth esis of the CYP19 gene and/or other transcription fa ctors. In contra st, another group of untreated cells were exposed to the test chemicals only during the 3 –hr inc ubation period of the aromatase assay to evaluate effects related to direct (rapid) inhibition of the enzyme through competitive or non-comp etitive binding of the test chemicals to the enzyme binding sites, binding to allosteric sites of the enzyme causing conformational changes and loss of enzymatic activity, or alterati on of other posttransla tional events that generally occur rapidly. Appropriate controls were used to demonstrate the specificity of the aromatase assay and measurement of pr omoter-specific mRNA levels; solvent controls, 4-OH-androstenedione (known inhibitor of catalytic activity of aromatase), andPage 151 of 263 8-BR-cAMP (inducer of aromatase activity). Cell viability was evaluated using the MTT reduction assay.

Results from positive and negative controls c onfirm assay specificity and demonstrate the precision of the methods to detect significant differences in aromatase activity and gene expression in both cell lines. At razine (0.3, 3 and 30 uM) caused a dosedependent increase in aromatase activity (LOEL 10 uM) in the H295R cells only when exposed for 24 hrs. (Fig.5) indicating t hat gene transcription and/ or other longer-term cellular event was required. While an increase in the amplification response of I.3 and pII-containing aromatase transcr ipts, the change as shown in Fi g. 6 was not significantly different from control or 8-Br-cAMP (100 uM ). The authors suggest that the change in these particular promoters may be related to an increase in cAMP, as this transcription factor is crucial for stimulation of this par ticular promoter. In contrast, no effects on aromatase activity was observed in the R2 C Leydig cells followi ng 3 or 24-hrs of exposure to atrazine or 8-Br-cAMP. This cell line, believed to be under the primary regulation of promoter pII, is also simulated by cAMP . The authors report that basal aromatase activity in the R2C cell line is very high as compared to that of the H295R cells, and that it is possible that one or mo re aromatase regions are fully activated, leading to constitutively high aromatase expression in the R2C cells with no further room for induction by chemicals.

Reviewer’s Comments: In conclusion, thes e data support earlier findings of atrazinedependent increases in aromatase activity in the H295R cell lin e, and provide some insight on the possible cellular regulation of aromatase during the 24-hr exposure period. It is clear that atrazine does not alter enzymatic activity through a rapid (competitive or non-competitive inhibition, allosteric modi fication ) alteration of the enzyme. However, failure to evaluate ot her promoters within t he H295R cells is a limitation of this study , and stops short of providing firm (significant) correlation between increased transcripts with I.3 and pII with elevated aromatase activity. In addition, the study demonstrates that the R2C Leydig cell line is not suitable for screening chemicals for identification of induction of aromatase gene expression.

Aromatase activity (AA) : H295R cells displayed dose dependant increase in AA (0.3 , 10 and 30 uM) with 3-fold induction as compared to control at highest dose. LOEL 10 uM, NOEL 0.3 uM) R2C cells = no sign. effect on AA or cell viability (at 30uM), NOEL 30 uM (Authors suggest lack of effect was likely due to constitutively high aromatase expression as compared with that observed for the H295R cells.

Promoter-specific mRNA Levels : H295R cells – Following 24 hrs exposure, an increase in I.3 and pII transcripts was observed in cells treated with 30 uM, but was not sign. different than control or 8-Br-cAMP. (Thus, it is still unclear whether or not cAMP and/or activation of these promoters are totally responsibl e for elevated aromatase activity).Page 152 of 263 A.4.6 Higley EB, Newsted JL, Zhang X, Giesy JP, Hecker M. (2010). Assessment of chemicals effects on aromatase activity using the

H295R cell line. Environ Sci Pollu t Res Int. Jan 20. (Epub ahead of print).

This study compared the effects of sev en model chemicals (atrazine, forskolin, prometon, ketoconazole, ami noglutethimide, letrozole, and prochloraz) on aromatase activity and steroid production (estr ogen, testosterone) in H295R human adenocarcinoma cells. The suite of chemicals selected for the study were selected because they were chemicals known to indu ce aromatase activity through up-regulation of gene expression and/or transcription fact ors. Using a tritiated-water release aromatase assay to quantify t he conversion of radiolabeled androstenedione to estrone, the authors tested an approach to determine whether or not a given chemical affected aromatase directly (e.g., imm ediate competitive inhibition of enzyme), indirectly (e.g., altered activity requiring a longer exposure time), or a combination of both direct and

indirect effects. Measur ement of estrogen and testoster one allowed an evaluation of the predictability of these endpoints for effe cts on aromatase activity in the cultured

cells.

For each experiment, H295R cells were cultur ed in 24-well plates, 300,000 cell/ml, and allowed to attach for 24 hrs prior to tr eatment. Media was changed and cells were exposed to test chemicals for an additional 48 hrs. DMSO ( ≤ 0.1% v/v) was used as solvent, and six concentrations of the each c hemical (0.01 to 10 uM) along with solvent controls were tested in duplicate or trip licate. MMT assay used for detection of cytotoxicity. At the end of each experimen t, culture media was collected for hormone assay (ELISA), and the cells were further s ubjected to the tritiated-water release aromatase assay for 1.5 hrs. To differentiate between direc t, indirect or a combination

of direct and indirect effects on aromatase acti vity, the exposure of the cells to the test chemical was varied in one of 3 ways: (1) indirect effect- no fu rther exposure of the cells during the 1.5 hr. incubation, (2) combi nation of direct and indirect effects- cells exposed for 48 hrs in culture and then again during the 1.5 hr a ssay, and (3) untreated cells during the 48 hrs culture were exposed to the test chemical only during the 1.5 hr assay period.

Reviewer’s Comments: Overall, the results of this study indicate that the increase in

estradiol observed in the H295R cell assa y was not due to a direct increase in aromatase activity. Atrazine, like forsko lin (which activates the enzyme adenylyl cyclase and increases the intracellular le vels of cAMP) enhanced steroidogenesis as evidence by increased T and E. In contrast , when atrazine and forskilin were tested in the “direct” tritiated water assay, ther e was no apparent change in aromatase activity (even at concentrations gr eater than the concentrations that changed estradiol production. These observations are important because they demonstrate that atrazine does not interact directly with the enzyme aromatase (as indicat ed earlier by the paper of Sanderson using the same cell line) and ca sts further doubt on the hypotheses that any adverse effects of the chemical are m ediated through activation of aromatase.Page 153 of 263 In addition, significant elevations of (indire ct) aromatase activity were only observed following a longer exposure fo r both of these chemicals. The authors conclude, “The hormone and aromatase activity profiles for forskolin and atrazine were comparable, and suggest similar mechanisms of activity even though exposure to forskolin resulted in greater concentrations of estrogen and testosterone, and greater aromatase activity

at lower concentrations than observed for atra zine.” In contrast, known inhibitors of aromatase (prochloraz, letr ozole, aminoglutethimide, ke toconazole) caused decreases in estrogen, testosterone and (direct) aromat ase activity measured following the 1.5 hrs exposure. The novel finding of increased (indi rect) aromatase activity following longer exposures to these inhibitors suggests that a compensatory feedback regulation of aromatase gene expression may also occu r in response to the low estrogen concentrations. Thus, the authors conclude that multiple measures of aromatase activity to evaluate immediate (direct) e ffects on the enzyme as well as longer term (indirect) effects should be measured in using in vitro culture systems such as the

H295R cell line.

Figure 16: Effect of Atrazine Exposure on Aromatase Activity

and Hormone Levels

(A1, A2) Atrazine: 48-hr s. exposure, 0.01 – 100 uM, E2, 7.2X increase (100uM), LOEL (1 uM), NOEL (0.1 uM) T, 1.4X increase (100 uM), LOEL (10 uM), NOEL (1.0 uM) Direct AA: no effect (100uM)

Indirect AA: increase LOEL (1 uM), NOEL (0.1 uM)Page 154 of 263 Limitations of study:

‐ Data for all endpoints reported as fold-change, with no actual values for

means ± SEM for any observations for ster oid concentrations or aromatase activity. Without control data, it is difficult to gauge how consistent data are among experiments within the stud y or compare with data reported

elsewhere.

‐ The large discrepancy between the fold changes in AA and E2 (ATR (100 uM) AA was 2.25X versus 7.2X for E2 as compared to respective controls) in Fig 17. is believed to be a reflection of different collection times, e.g , AA is that change which occurs in tritiated water release over a 1.5 hr period, versus E2 production over t he 48 hrs in culture.

‐ Although positive controls used to demonstrate specificity of aromatase assay (e.g., radio inert androstenedione and fadrozole), no concentrations for androstenedione reported and vague informa tion on that used for fadrozole.

‐ Solubility of ATR (100uM) is questionable.

A.4.7 Holloway, A. Anger, D., Cran kshaw, D., Wu, M, and Foster, W. 2008. Atrazine-induced Changes in Aromatase Activity in Estrogen Sensitive Target Tissues. Journal of Applied Toxicology. 28:260-270.

The purpose of this study was to determi ne the effects of atrazine treatment on aromatase activity in cells from estrogen sensitive target tissues in the ovary and

endometrium.

The effect of atrazine on ar omatase activity was measured in human granulosa-lutein cell cultures, cells that abundantly expre ss aromatase, and endomet rial stromal cell (ESC) cultures, cells that do not express ar omatase. Aromatase activity was quantified by the tritiated water met hod and the specificity of the assay was confirmed by coincubation with 4-hydr oxyandrostenedione, an irreversible inhibitor of the catalytic activity of aromatase, to blo ck the formation of tritiated water. The cells were treated for 24 hours with increasing concentrations (1 nM to 1 μ M) of atrazine. Prostaglandin E 2 (PGE 2 , 1 μ M) and vomitoxin (a selective cyclooxy genase-II inducer, 250 mg/ml) were used as positive controls.

In the granulosa-lutein cell cultures, atrazine treatment i nduced a dose-dependent increase in aromatase activity (p=0.007). Ar omatase activity in atrazine treated (1 nM0.1 mM) granulosa-lutein cells (GC) was increased more than 2-fold compared with control cultures, with significant ar omatase activities at 0.1 and 1.0 μ M. Atrazine

treatment had no effect on ESC aromatase ac tivity at any concentration tested. Atrazine treatment had no effect on GC or ESC protein concentrations, which indicatedPage 155 of 263 no change in cell number, suggesting that the atrazine induced changes in aromatase activity were not due to cell prolifer ation. The positive controls PGE 2 (1.0 μ M) and vomitoxin (250 ng/ml) induced a significant increase in aromatase activity (132 ± 10 and 149± 13% of control, respectively, p<0.05) . There was also no effect of atrazine

treatment on human recombinant aromatase activity in ce ll free test system. It was

concluded that μ M concentrations of atrazine can increase aromatase activity of human granulosa cells but not ESC and this effect is not elicited at the enzyme level.

Reviewer’s Comments: The authors concluded that atrazine treatment induced an increase in aromatase activity in human luteinized granulosa cells, however, the

concentrations needed to induce the observ ed change is considered to be outside the range likely to be achieved in human tissues and, therefore, is unlikely to pose a health threat through this mechanism.

A.4.8 Laville N, Balaguer P, Brion F, Hinfray N, Casellas C, Porcher JM, Ait-Aissa S. (2006). Modulati on of aromatase activity and mRNA by various selected pesticides in the human choriocarcinoma JEG-3 cell line. Toxicology. 228:98-108.

This study evaluates 30 pesticides for their ab ility to modulate aromatase activity in the

human choriocarcinoma JEG-3 cell line. Pest icides were selected based upon chemical

structure and environmental re levance, but also included a number of known aromatase

inhibitors and inducers. Atrazine was am ong one of the reference pesticides used in this study. By using the tr itiated water release assay a fter both short (2 hr) and long (24hr) exposures to each test chemical, the aut hors were able to identify either inhibition or induction (requiring longer term exposur e) of aromatase enzymatic activity. Pesticides that were newly found to induce ar omatase activity were further evaluated for their action at the transcriptional level wit h measurement of CYP 19 mRNA and through the retinoic acid receptor signaling pathway.

JEG-3 cells were seeded in 24-well plates, density of 50,000 cells/w ell, and allowed to attach for 24 hrs. before changing media and ex posing the cells to the test chemicals (1, 3, 10 uM) for an additional 24 hrs. Media was collected at the end of the exposure period, cells were washed once with media, and further elevated for ar omatase activity. The tritiated water release assay was used to quantify aromatase activity in cells exposed to test chemicals for 24 hrs (to detect possible effects of chemicals on aromatase gene expression, or changes at transcription or ( post) transcription levels), and in untreated cells exposed to the test chemical for only 2 hrs (e.g., direct competitive effects on the aromatase enzymatic comple x). OH-Androstenedione, an

irreversible inhibitor of aromatase acti vity was used to demonstrate time- and concentration-dependent inhibition. Forskolin and TTNPB were used as positive controls for up-regulation of aromatase expression. Cell viability was determined using the methyl-thiazol-tetra zolium (MMT) assay.Page 156 of 263 Reviewer’s Comments: While neither atrazine nor forskolin had any effect on aromatase activity following a 2 hr expos ure, both of these chemicals increased aromatase activity after 24 hrs of exposure. Approximately a 2-fold increase in aromatase activity was

observed in cells treated with 10 uM atra zine, but no change in CYP19 mRNA was

observed.

Atrazine: JEG3 cells, doses 1, 3, and 10 uM (24 hrs)

LOEL: 10 uM NOEL: 3 uM

Strengths of study : Use of positive controls to demonstrate specific ity of aromatase assay and CYP19 mRNA measurements. This study supports other

in vitro studies demonstrating that atrazi ne does not alter aromatase enzyme competitive inhibition, but rather requires longer expos ure time such that enzyme stability, synthesis and/or post-transcriptional events can be altered.

A.4.9 Rayner, J., Wood, C. , and Fenton, S. (2003) Exposure Parameters Necessary for Delayed Puberty and Mammary Gland Development in Long-Evans Rats Ex posed in Utero to Atrazine. Toxicology and Applied Pharmacology 195:23-34.

This study looked at the effects on del ayed puberty and mammary gland development in rats exposed in utero to atrazine. 16 Groups of 20 timed- pregnant Long-Evans rats were gavaged on gestational days (GD) 15-19 wit h atrazine (purity 97.1%) at doses of 100 mg/kg bw or vehicle control (C). On P ND 1, half of each litte r was cross-fostered, creating four treatment groups: C-C, ATR- C, C-ATR, and ATR-ATR (dam-milk source, respectively). Litters we re weighed and equalized to 10 pups on PND4. On PND 22 (weaning), animals were weighed and separat ed into 2-4 females/cage. Some females were sacrificed on PND 4, 22, 33, 40 and 58. The data from a tr eated-dam’s offspring were evaluated with the dam (t wo pups/dam or litter) as t he experimental unit, with the exception of vaginal smears.

On PND 29 females (n>29/group) were eval uated for vaginal opening (VO). The PND of complete VO and body weight on that day were recorded. Daily vaginal smears were collected beginning the day a fter VO for each animal until PND 58 to determine cyclicity patterns. Female pups (n=14-16) were sa crificed on PND58. Blood was collected and the pituitary gland, ovaries, and uterus we re weighed and prepared for histopathology. Serum luteinizing hormone (LH), prolactin (PRL), and thyroid stimulating hormone (TSH) were analyzed.

Pups in the ATR-ATR group were statistically significantly smaller than those in the control group on PND4 (p<0.01) (8.2±0.1 g ATR-ATR vs 8.7±0.1g, C-C), however, their mean weight was reduced 5.7% from contro l animals, which was within normal range.

16 This review will concentrate on the assessment of delayed puberty onset. The discussion of the mammary gland development will be defer red until the September SAP meeting.Page 157 of 263 There was no difference in body weight among groups at PND 22, 33, 40 or 58. A significant delay (p<0.02) in VO was seen only in animals nursing from ATR-treated dams (C-ATR 35.4±0.6 days, and ATR-ATR 37. 1±0.5 days vs C-C 33±0.4 days). Mean body weight on day of VO was significantly (p<0.002) increased in the ATR-ATR group (~10%, estimated from graph), but not in t he C-ATR group, com pared to C-C group. There were no significant differences in the number of consecutive normal estrous cycles between VO and PND 58 or in the nu mber of animals demonstrating persistent estrous (PE) or persistent diestrous (PD). However, while not statistically significant, there appeared to be a lower num ber of animals with irregular cycles in the ATR-ATR group (3%), compared to controls (23%) and the number of animals with normal cycles (2-5 days) appears to be increased in the AT R-ATR animals, compared to controls.

No significant treatment-rela ted changes were seen in circ ulating hormones (serum LH, PRL, and TSH) in any treatment group at PND 33, 40 or 58.

At PND 58, all atrazine ex posure groups had increased pitu itary glands; however only the pituitary glands of the animals in the ATR-ATR group were significantly increased, compared to controls. There we re no statistically significant differences in reproductive organ weights am ong the groups.

Reviewer’s Comments: The researchers in this articl e demonstrate that atrazine in utero and perinatal exposure to atrazine c an lead to a delay in vaginal opening in female rats. These findings are c onsistent with those reported by Laws et al. in 2000 and 2003. It should be noted, however, that t hese effects are seen at substantially higher dose levels than those used as point s of departure in the Agency’s human health

risk assessment.

A.4.10 Suzawa, M and Ingraham , HA. 2008. The herbicide atrazine activates endocrine gene networks via non-steroidal NR5A nuclear receptors in fish and ma mmalian cells. P LoS ONE 3(5): e2117. doi:10.1371/journal.pone.0002117.

* In this evaluation, only the results per taining to mammalian cells are discussed. The purpose of this in vitro study was to examine the potent ial effects of atrazine on NR5A receptors (steroidogenic fact or 1 or SF-1). The spec ific interest was on whether Cyp19A1 (aromatase), a known target of NR5A receptors would be selectively activated by atrazine. The focus on SF-1 was a result of the two paper s by Fan et al., who argued that this NR5A receptor was the target molecule for atrazine evoking changes in aromatase activity and potentia lly the molecular mechanism for many assumed adverse outcomes following exposure to this herbicide in vivo. Cellular reporter assays, utilizing JEG3 human placental cells, were performed. JEG3 cells contain modest amounts of SF-1. In one experiment, the effects of in vitro atrazine on luciferase activity after transfection of human placental JEG3 cells with aromatase-luciferase reporter and the parent reporter (pGL3-luciferase) with mS F-1 (mouse SF-1) and hLRH-1 (human liver receptor homolog) expression vectors were examined. The data revealed a dose-Page 158 of 263 dependent increase in luciferase activity whic h the authors interpreted as activation of aromatase promoters. The effects were significant at atrazine concentrations of ≥ 1 μM. No activation was reported for the par ent reporter or with mu tant SF-1 binding

sites. Atrazine also activated mSF-1 in He pG2 (human liver) cells. The authors also provided data demonstrating activation of otherSF-1 reporters, such as MIS-Luc, StARLuc, and HSD3B-Luc. The authors conclude d that atrazine effects depend on DNA binding and receptor o ccupancy of the promoter.

In a knock-down experimen t, the authors provided result s showing knocking-down hLRH-1 in human HepG2 liver cells attenuated atrazine- induced activation of the aromatase reporter, which the authors interpreted as demonstrating a dependence on this NR5A receptor. In addition, they conc lude that all NR5A subfamily receptors will be responsive to atrazine.

Atrazine treatment was wit hout effects on the estrogen responsive zcyp19a2 (ERE-Luc reporter). No significant activation of NR5A was observed with other estrogenic compounds tested.

Several additional experiments were per formed to determine the underlying cellular mechanisms of activation of NR5A receptors. These included mitogen-activated protein protein kinase (MAPK), phosphatidyl 3-kinas e (Pl3K), G proteins, phospholipase C (PLC) in JEG3 cells. Pharmacological inhibi tors of these systems were examined. The

results indicated that inhibitors of both the MAPK and Pl3K signaling decreased activation by atrazine, whereas the inhibitors did not affect G protein or PLC signaling. The finding of phosphorylation of AKT/protein kinase B provided further support for effects on MAPK and Pl3K signaling. Atrazine ( ≥ 1 μM) significantly increased cAMP

levels in JEG3 cells.

The authors also assessed whether or not atra zine binds directly to SF-1 to either increase DNA binding or act as a ligand. At razine had no effect on DNA binding activity to a high affinity SF-1 bindi ng site (the mMIS bindi ng site was used). Atrazine also did not activate a Gal4-SF-1 ligand binding domain fusion construct. The authors concluded that these results suggest that atrazine may not directly interact with NR5A receptors, but instead activates three si gnaling pathways known to activate NR5A receptors, including phosphor ylation of SF-1, generation of SF-1 ligands, and increased production of cAMP. The author s also reported that atrazine can increase a cluster of genes involved in endocrine signaling in spec ific cell types; however, very limited data were presented to corroborate these findings.

Reviewer’s Comments: Overall, this study provides some preliminary research on the underlying cellular mechanism of arom atase expression following atrazine in vitro treatment. The study has lim itations including lack of details on methodology of luciferase activity and transfection of cells. Only a few poor qu ality results of PCR analyses and western blots were provided to corroborate the findings. The authors

made several references to “data not show n” to support their conclusions. ThePage 159 of 263 activation of important second messenger pa thways, and lack of evidence for direct binding to SF-1 does not support the reports of Fan et al ., who conclude that atrazine

has a direct SF-1 mediated activation of ar omatase and subsequently increase estradiol production. The results indicating an indirect activation of aromatase are also in agreement with the observations of Higley et al reviewed above. The experiment examining the effect of atrazine on zebra fi sh was previously reviewed by the Agency

and numerous deficiencies were identified.

A.4.11 Yi D K, Kim P J, et al. 2010. Dose- and time-dependent

effect of atrazine or its metabolites on aromatase mRNA in vitro

. Only a preprint of article available.

The purpose of this study (available only as a preprint) was to examine the potential effects of dose and duration of expos ure to atrazine and its metabolites (diaminochlorotriazine, DACT; deethyl at razine, DEA; deisopropyl atrazine, DIA) in vitro on the expression of aromatase m RNA in two steroidogenic lines, i.e. , human adrenocortical cancer cells (H295R), and hum an placental carcinoma cells (JEG3).

There were no details in the methods se ction of the articl e pertaining to the

concentrations used and the duration of treatm ent. Based on information in the abstract and figures, atrazine and metabolites (all we re reported to be 97% purity and dissolved in DMSO) were administered in vitro at equimolar concentrations of 0, 100 nM, 1 μM or 10 μM for 15 minutes to 72 hours. There were descriptions of cell cultures, RNA extraction and cDNA synthesis, pr imer design, reverse transcription PCR, and statistical analyses.

Exposure to 10 μM atrazine increased arom atase mRNA expression in H295R cells approximately 2.2 fold after 2 hours and approx imately 3-fold after 4 to 24 hours. Aromatase mRNA expression was increased about 2.5 fold at 48 hours and 2.2 fold after 72 hours. At 1 μM atrazine, there wa s about a 2-fold increas e in aromatase mRNA expression at 24 hours but not at other time points. No ex pression was observed at the

low concentration of atrazine.

Exposure of H295R cells to 10 μM DEA in creased aromatase mRNA approximately 1.5- fold after 2 to 24 hour time period. Ex posure of H295R cells to 10 μM DIA increased aromatase mRNA approximately 1. 8-fold after 2 to 48 hour ti me period. There were no apparent treatment-related effects with hydroxyatrazine and DACT.

Reviewer’s Comments: The authors concl uded that exposure to 10 μM atrazine in vitro resulted in an increased expressi on of aromatase mRNA. This in vitro response is dose- and time-dependent with peak expres sion occurring after 4 to 24 hours of exposure. Atrazine metabolites, DACT and hydroxyatrazine, failed to show a consistent response.Page 160 of 263 A.4.12 Yi, KD, Perez, EJ, Br eckenridge, CB, and Simpkins, JW (2010 ?) Ex vivo effects of atrazines or metabolites on phosphodiesterase activity.

This study concentrated on phosphodiesterase (P DE) activity and whether atrazine and its metabolites inhibit this process, while also noting effects on aromatase in vivo . It provides several theories on the possible MOA regarding the incidence of mammary tumors, delayed onset of puberty, and immune suppression.

The premise that there is an alternative MOA for mammary tumors in female SpragueDawley rats is that mRNA expression and aromatase activity was increased in H295R adrenocortical carcinoma cells when exposed to a minimum of 10μM atrazine. Increased cyclic AMP (cAMP) levels were correlated with the induction of aromatase activity by atrazine; increases in cAMP ma y be mediated either via increases in adenylyl cyclase which catalyzes the conversion of AT P to cAMP, or via the inhibition of PDE which catalyzes the breakdown of cAMP to AMP. Therefore, high concentrations of

atrazine in vitro may be mediated by increases in intracellular cAMP, activating adenylyl cyclase, or by inhibiting PDE.

Atrazine, deethyl atrazine (DEA), deisopropyl atrazine (DIA), diaminochlorotriazine (DACT), ammeline (AM), and hydroxyatrazine (hydroxyl-ATZ) used were approximately 97% pure. IBMX, 3-isobutyl-1-methylxanthine was used as the positive control. The IMAP phosphodiesterase assay kit was used along with bovine PDE at varying concentrations (0.000025 to 0.5 PDE U/ml in 96-well plates). To determine the effects of addition of atrazine after completion of the PDE reaction, an hour long incubation period was allowed and then to assess the effects of atrazine and its metabolites on PDE activity, the potential inhibitors were added at concentrations ranging from 5pM to 5 nM in 10 μl of assay buffer enzyme wit h additional 90 minute substrate/incubation period. Competitive inhibi tion studies were similarly conducted with the addition of varying concentrations of DACT with atrazine. Determining IC 50 values was done using a 4-parameter log curve and creati ng sigmoidal standard curves.

The results from the optimized assay fo r PDE inhibition showed a concentrationdependent increase in the relative fluorescenc e output of the assa y at both 30 minutes and 2 hours after the addition of the binding reagent. Maximal out put, of 0.025 PDE U/ml was used for all subsequent assays. A ddition of atrazine after completion of the enzymatic reaction didn’t have any effect on the fluorescence output of the assay. Regarding the effects of atrazi ne and to a much lesser extent its metabolites inhibited PDE in a cell-free system. The IC 50 for atrazine (39.52 μM) wh ile for IBMX it was 1.749 μM. The IC 50 for DIA was 1371 μM and DEA at 7156 μM were 35 and 181-fold less potent inhibitors of PDE than was atrazine; a ll other metabolites were inactive. DACT, however dose-dependently caused a rightward shift in the dose-response curve of the atrazine mediated inhibition of PDE activity suggesting to the authors that although DACT does not inhibit PDE out right, it perhaps modifies the effect of atrazine on PDE.Page 161 of 263 Also at both 5 and 50 μM conc entrations of DACT, the IC 50 of atrazine was several folds higher, suggesting competitive i nhibitory effects of DACT.

The authors concluded that atrazine at all concentrations which exceed its aqueous solubility limits, is a weak inhibitor of PD E and that its metabolites have extremely weak, or no effect on PDE activity. The primary metabolite of atrazine, DACT was shown to act as a competitive inhibitor of atrazine-induced inhibition of PDE activity. Although, not supported, the authors elude to the possibility that the i nhibition by atrazine of PDE

in ex vivo condition, a cell-free system with a complex mixture of different PDE isozymes and substrate, could be due to a non- specific effect of the precipitate on the

kinetics of the reaction. The IC 50 for atrazine-mediated inhibiti on of PDE activity in this study (40 μM) was vastly different, 22-fold higher, form the concentration reported by Roberge et al (2004). No true rationale was given for this discrepancy – however, they went on to describe the many similarities in results from the two l aboratories. Even at high concentrations of atrazine there have been no shown increases in aromatase mRNA or enzyme activity in vivo with the exception of a study on juvenile zebrafish. Therefore the study concludes that a general or local increase in the expression of this

enzyme in vivo , would not account for the observed effects of high doses of atrazine on estrous cycles, mammary tumors or the age of onset of puberty.

Reviewer’s Comments: In mammals, atrazi ne is NOT acutely toxic (oral LD= 3090 mg/kg) and is only moderately toxic followin g repeat dosing to rats, mice, rabbits, or dogs. Atrazine-mediated inhibition of PDE ex vivo is unlikely to account for the tissue, sex, strain and species-specific effects of chronic high-dose exposure to atrazine. The bottom-line is that it is unlikely that an at razine-mediated inhibition PDE activity would occur at environmentally relevant concent rations. The potential human exposure to atrazine is low and equally unlikely to result in PDE inhibition in humans. The study goes on to endorse the NOEL (1.8 mg/kg/da y) which is currently used to regulate chronic exposure to atrazine and its chloro metabolites and appears to be protective of any potential effects in humans.

A.5. Neurotoxicity

A.5.1 Belloni V, Alleva E, et al. 2007. E ffects of low doses of

atrazine on the neurobehavioural development of mice. Ethology Ecology & Evoluti on 19: 309 – 322.

The authors stated that the aim of their work was to study possible neurodevelopmental effects of perinatal exposure to low, envir onmentally relevant doses of atrazine in mouse pups. Specifically, the focus was on somatic parameters and a wide range of

reflexes to assess sensor imotor maturation.

The authors noted that mice of an outbred Swi ss-derive strain (CD-1) were used in the study; no details on age or body weights were provided. Breeding pairs were formed,Page 162 of 263 and on GD 12, males were removed and females housed singly. GD 14 females were assigned to one of three treatment groups: control (received corn oil); low-dose atrazine (1 μg/kg/day) and high-dose atrazine (100 μg/kg/day). Atrazine (98.4% purity) was dissolved in corn oil and administered daily to female mice by self administration drinking from a modified syringe. The aut hors stated that the procedure was not stressful since the animals were previously trained to drink from the apparatus. The treatment period began GD 14 until PND 21. The pups were not treated directly and were only exposed by transplacental transfer or via lactation. On PND 2, litters were culled to 8 per litter, 4 males and 4 females.

For behavioral testing, two male and two female pups from 32 different litters were used for postnatal assessment of somatic and neur obehavioral development. A total of 128 mice were subjected to behavioral testi ng between 2 and 15 days of age. Assessment of somatic growth and sensorimotor devel opment included the following parameters: righting reflex, cliff aversion, forepaw grasp, and auditory startle. Only brief descriptions of these tests were provided. Scoring was as follows: 0, no response; 1, uncertain; 2, incomplete; and 3, full. On PND 2, 4, 9, and 12, pups were weighed.

Evaluation of pup vocalizations (ultrasonic calls) was also performed on PND 4, 9, and 12. The authors provided adequate details on this evaluation. A spectrographic analysis of ultrasonic calls was also perfo rmed on PND 9 (which the authors mention is the peak day in the ontogenetic profile of ultr asonic vocalization). In addition, body temperature was recorded on PND 4, 9, and 12.

The authors reported that there were no diffe rences in dam body weight gain during gestation. A pup body weight decrease of appr oximately 8% was noted in both atrazine treatment groups compared with the control group on PND 2; no effects were noted at PND 4, 9 and 12. Righting reflex (expressed as “day of full response”) was delayed in the high-dose atrazine group (mean of 6.60 vs. control m ean of 4.60). There was an acceleration of forepaw gras p (“day of full response”) in the low-dose atrazine group (mean of 5.18) and high-dose atrazine group (mean of 5.25) in comparison with the control group (mean of 7.50).

On PND 9, mean body weight te mperatures were significant ly lower in the males and females of the low-dose atrazine group (5 % decrease in males; 4% decrease in females) when compared with controls. Th ere were no significant changes on PND 4 and 12 and no significant changes in the high-dose group.

Reviewer’s Comments: The authors indica ted that were was a tendency towards a

decrease in the mean number of ultrasonic calls in atrazine-treated pups when compared to controls on PND 9. However, the data (in contrast to controls) show a decrease (not statistically significant) in atrazine-treated females (both low and high dose groups) and only the low-dose group for at razine-treated males. Moreover, the

decreases in mean number of ultrasonic calls in atrazine-treated females on PND 9 did not display a clear dose-response relationshi p. Based on spectrographic analyses ofPage 163 of 263 the ultrasonic calls, the authors claim that there was a tendency towards a longer ultrasonic call mean interpulse (seconds) in atrazine-treated animals compared to control groups. However, the data indicate that this tendency was only apparent in the low-dose atrazine group of females or when data from males and females were

combined. No differences were noted in the high-dose atrazine groups, whether data are presented as sex specific or combi ned. Peak frequency was slightly (not statistically significant) increased in atrazi ne-treated males (low and high dose groups)

and in atrazine-treated females (just the high dose group).

The authors concluded that exposure to lo w doses of atrazine during the fetal and suckling period interferes with some aspects of somatic growth of p ups in a reversible manner, and more clearly with their neurobehavio ral development. However, this study

has serious deficiencies and flaws. The primary problem entails the inappropriate sampling of pups from litters and the statistical evaluation of the data. The authors fail to account for the potential effects of litter, and the key results re ported by the authors rely on only individual pup measurements which inflates sample size. The failure to

account for litter effects can result in in creased type 1 errors and overestimate any effects, thus reducing the validity of the findings of the study.

In addition to inappropriate statistical analys es, there were unexplained discrepancies in the sampling of pups from litters. For inst ance, a lower number of pups were evaluated for somatic growth and sensorimotor devel opment, body temperature, and ultrasonic call emittance compared to the actual number of pups availabl e. For the spectrographic analyses of ultrasonic vocalization, not only we re significantly less animals evaluated in

contrast with the assessment of mean ultrasonic calls, but the reported total numbers of pups evaluated did not match the total repor ted for males and females separately. The number of animals evaluated for spectrographic analyses of the ultrasonic calls should

match the numbers evaluated for the mean number of ultrasonic calls.

Other deficiencies of the study include a la ck of details on the procedures used for assessment of somatic growth and sensorimot or development, a lack of inclusion of positive controls that would support the reliability of the methodology, the lack of objective and validated behavioral tests, lack of neuropathology, and the lack of actual spectrograms to corroborate t he spectrographic results. T he serious deficiencies of these studies raise questions about the validit y and reliability of the data and its use for risk assessment purposes.

A.5.2 Coban A, and Filipov, N M. 2007. Dopaminergic toxicity associated with oral exposure to th e herbicide atrazine in juvenile

male C57BL/6 mice. Journal of Neurochemistry 100: 1177-1187.

The objectives of this study were to determine the following: (1) t he effects of atrazine exposure on striatal neurochemistry and striat al expression of tyro sine hydroxylase; (2)

whether atrazine effects on striatal neuroc hemistry are associ ated with dopaminergicPage 164 of 263 neuron loss; and (3) whether the neuronal lo ss is permanent and specific to the substantia nigra pars compacta.

One-month-old male C57BL/6 mice were administered atrazine (5, 25, 125, and 250 mg/kg/day) by daily gavage for 14 days. Several indices of neurotoxicity were evaluated on days 15, 22 or 64. D opamine and its met abolites, 3, 4- hydroxyphenylacetic acid (DOPAC), and hom ovanillic acid (HVA) were measured by high performance liquid chromatography. Stri atal tyrosine hydroxylase levels were determined by western-blot analysis. Fixed half-brains were used to analyze neurons in the substantia nigra pars compac ta and ventral tegmental area by immunohistochemistry/unbiased stereology.

No effects of atrazine treat ment on body weights were r eported (data not shown). Atrazine at doses of 125 and 250 mg/kg decreased (dose dependently) striatal dopamine (approximately 80 ng/mg protein at 250 mg/kg vs. 100 ng/mg protein in controls), DOPAC (approximately 6 ng/mg pr otein at 250 mg/kg vs. 5 ng/mg protein in controls) and HVA (approximatel y 6 ng/mg protein at 250 mg/k g vs. 8 ng/mg protein in controls) levels on day 15. There was no difference between control and atrazinetreated animals in striatal dopamine levels at day 22. Slight decreases in DOPAC and HVA were present at day 22. At day 64, there were no statistically significant effects on

DOPAC and HVA. There were no effects of at razine treatment on striatal levels of TH as determined by western blot analysis.

On day 15, there was a decrease in the num ber of TH-positive neurons in both the substantia nigra pars compacta and ventral tegmental area. The loss of neurons was statistically significant at 250 mg/kg/day (the high dose). At day 22, the loss of neurons in the substantia nigra pars compacta was si gnificant at 25 mg/kg/day dose. In regard to the ventral tegmental area, there was a significant de crease in the number of THpositive neurons at days 14 and 22. The dec rease in the ventral tegmental area was reported to be 19% in contra st to a 32% decrease in t he substantia nigra pars compacta. On day 64, atrazine exposure decreased the number of TH-positive neurons in both the substantia nigra pars compacta and ventral tegmental ar ea, with the overall effect being significant at all dose levels in the ventral tegmental area. The authors reported that at 64 days, t he effect on the neurons in the substantia nigra pars compacta at the high dose was only margina lly greater than the effect in the ventral tegmental area (33% cell loss in substantia nigra pars compacta vs. 28% cell loss in the ventral tegmental area).

Reviewer’s Comments: There are se veral limitations with this study:

• The reason for a lack of a dose-respons e relationship with respect to the number of TH+ neurons is not clear. The study di d not include a positive control such as 6-hydroxydopamine which produces dos e dependent alterations in dopamine and dopamine metabolites and lo ss of tyrosine hydroxylase-immunoreactive cells in the substantia nigra.Page 165 of 263 • The results reported for the atrazine- induced TH+ neuronal cell numbers (only bar graphs presented) cannot be corroborated since there was no presentation of photomicrographs of brain sections to corr elate with the findings presented in the bar graphs. Also, there was no information on the calibration of the stereological test system. The only stat ement on the precision of t he estimate of neuronal

counts was the calculation of a Gundersen coefficient of error.

• No histological examination of the s ubstantia nigra and ventral tegmental area was performed; this would provide insight into any morphologic al alterations to support their cell number data.

• The dose-response relationships with regard to dopamine and dopamine metabolite levels were flat. Also, the decreases were slight even at the two highest doses tested ( i.e. , 125 and 250 mg/kg/day). A positive control, for example, 6-hyroxydopamine (discussed abov e) was not included in the study.

• There were no functional assessment s of the dopaminergic alterations, for example, assessment of behavior and moto r activity to correlate with the neurochemical findings.

• Neurochemical analyses were not per formed for the ventral tegmental area which would have provided some support fo r the observed findings of the loss of dopaminergic neurons in this region.

• Coban and Filipov also presented dat a for days 22 and 64 of the experiment revealing only slight changes in the dopamine metabolites DOPAC and HVA at day 22 (flat dose response) and no alterati ons in dopamine and its metabolites at day 64. However, these data ( i.e. , Figure 3 on page 1182 of the study) are contradicted by the description of the dat a in which the aut hors state that the

“some of the animals received MPTP on da y 15” (no further details provided).

MPTP has been associated with a Parkin sonian-like syndrome, and it is not certain if the word MPTP was a typographi cal error, if MPTP was meant as a possible positive control (not discussed in methods or any other section) or if the wrong results were presented. If MPTP wa s included as a positive control it is not clear why it was not administered to mi ce at the same treat ment start time as

atrazine.

A.5.3 Filipov N M, Stewart M A et al . 2007. Dopaminergic toxicity of

the herbicide atrazine in rat striatal slices. Toxicology 232: 68 – 78.

This in vitro study examined the effects of atrazi ne treatment of various aspects of dopamine metabolism in rat striatal slices. Striatal tissues from adult male SpragueDawley rats were incubated with up to 500 μM atrazine for 4 hours. The following were evaluated: tyrosine hydroxylase activity using L-DOPA accumulation as an indicator, high potassium-stimulated dopamine release, media dopamine levels in the presence of the dopamine uptake blocker nomifensine, and striatal dopamine metabolism. In addition, the effects of in vitro exposure to atrazine on LDH levels was performed to determine if any potential dopaminergic effects ar e caused by non-specif ic cytotoxicity.Page 166 of 263 Dopamine and metabolites, 3,4- hydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were measured.

Atrazine treatment at concentrations of 100 μM or greater result ed in a dose-dependent decrease in tissue levels of dopamine. At 50 μM or greater concentration of atrazine, DOPAC + HVA/DA ratio was dose-dependently increased. Tyrosine hydroxylase protein levels and activity were not affect ed by atrazine treatment. However, atrazine decreased high potassium-induced dopamine re lease; the increase in media dopamine in the presence of nomifensine was increa sed even further by atrazine in a dosedependent manner. These effects were observed at levels that were not toxic to the

tissue. The authors concluded that their resu lts suggest that atrazine decreases tissue dopamine levels not by affecting tyrosine hydroxylase activity, but possibly by interfering with the vesicular storage and/or cellular uptake of dopamine.

Reviewer’s Comments: The results revealed that atrazine, in vitro , alters striatal dopamine levels possibly by interfering with the vesicular storage and/or cellular uptake of dopamine. Tyrosine hydroxylase per se is not a target of atrazine. The relevance of

these in vitro data to dopaminergic mechanisms in vivo is not clear given the high

concentrations of atrazine examined in vitro and the complex interactions between

various neurotransmitter and endocrine systems in the intact brain. The effects of

atrazine on aromatic amino acid deca rboxylase, monoamine oxidase and neuronal

mitochondrial function are unknown.

A.5.4 Foradori, C D, Hinds, L R, et al. 2009. Atrazine inhibits pulsatile luteinizing hormone re lease without altering pituitary sensitivity to a gonadotropin-releasi ng hormone receptor agonist in female wistar rats. Biology of Reproduction. 81: 40 – 45.

The objectives of this study were to exam ine the effects of atrazine on normal tonic release of luteinizing hormone (LH) and to eluc idate the site of action of atrazine in the hypothalamic-pituitary-gonadal axis. To assess potential effects of atrazine on pulsatile LH release, ovariectomized adult female Wist ar rats were administered atrazine (50, 100, or 200 mg/kg/day by gavage) or vehicle (1% carboxymethylcellulose sodium salt) control for 4 days. At termination, bl ood samples were obtained using an indwelling right atrial cannula (implanted on second day of treatment). Samples were examined for LH levels. There was a significant reduction in LH pulse frequency and a concomitant increase in pulse amplitude in the 200 mg/kg/day group.

The second experiment involved validation of a protocol for GnRH immunoneutralization and pituitary response to GnRH agonist. Ut ilizing the same experimental procedure above, animals were administered GnRH antiserum via cannula to block the actions of endogenous GnRH. The anti-GnRH serum was prepared against keyhole limpet hemocyanin-conjugated GnRH. Following blood sampling, animals were treated with

one of four doses of Gn RH receptor agonist, D-Ala 6 -des-Gly-NH 2 10 -GnRH-ethylamide (0.01, 0.1, 1.0, or 10 ng; 4- 6 animals per treatment group). LH levels were measured.Page 167 of 263 The results showed that LH levels were at or below the detection level of the assay in

animals administered the GnRH antiserum, indicating that the effect of endogenous GnRH on the pituitary release of LH was su ppressed. Administration of GnRH agonist resulted in dose-dependent increases in mean plasma LH levels.

The third experiment entailed an examination of the effect s of atrazine on in vivo pituitary response to GnRH agonist. The ob jective was to determine whether atrazine reduces pituitary sensitivity to a GnRH agonist. Ovariectom ized rats were administered atrazine (0, 50, 100, or 200 mg/kg/day by gav age) for 4 days. On the second day of treatment, intra-atrial cannul ae were implanted, and rats were treated with anti-GnRH serum via cannula. On the last day of treatment, sampling of blood was performed and

D-Ala 6 -des-Gly-NH 2 10 -GnRH-ethylamide (0.1 or 1.0 ng) was administered via catheter. Sampling was continued for an additional 90 mi nutes. Atrazine-treated animals that had

been immunoneutralized with the anti-GnRH serum showed an LH response comparable to that observed in the c ontrol group. Both doses of D-Ala 6 -des-Gly-NH 2 10

- GnRH-ethylamide caused an increase in the circ ulating concentration of LH. However, there was no significant change in the levels of LH related to atrazine treatment.

The authors concluded that al though LH pulse period and amplitude were increased in atrazine-treated animals, the anterior pituitary function does not seem to be altered by atrazine, as LH levels were not reduced in atrazine-treated animals in response to a GnRH agonist after GnRH immunoneutralization. Additionally, the inhibition of LH pulses and the disruption of cyclicity in female rats treated with atrazine are likely to be mediated by interference with central mec hanisms controlling GnRH release from the hypothalamus. The brain appear s to be the primary target mediating the effect of

atrazine on LH release in the rat.

Reviewer’s Comments: The Foradori study provides added suppor t for the hypothesis that atrazine targets the br ain to modify the neuronal cont rol of pituitary function. Evaluating the secretion of gonadotropin releasing hormone (G nRH) in rats revealed

that atrazine alters the release of this neur opeptide from the brain. GnRH is essential for LH release from the pituitary.

A.5.5 Foradori, C D, Hinds, L R, et al. 2009. Effects of atrazine on

GnRH neuroendocrine function afte r its withdrawal in the adult female wistar rat. Biology of Reproduction 81(6): 1099 – 1105.

The authors previously reported that atrazine treatment decreased LH pulse frequency and increased pulse amplitude in ovariect omized rats without changing pituitary sensitivity to a GnRH receptor agonist. The authors interpreted these results to suggest a neural site of atrazine’s action. In order to explore this possibility, the authors

examined the effect of atrazine on the es trogen and progesterone-induced LH surge in freely moving, ovariectomized rats to determine if atrazine’s inhibition of LH secretion

coincides with a reduction in GnRH neuronal activation. The immediate early gene product FOS (cFos) was colocalized in GnRH neur ons as a measure of cellular activity.Page 168 of 263 In addition, withdrawal studies were perfo rmed to determine whether LH levels and

measures of GnRH neuronal activation would re turn to control levels following the

termination of atrazine treatment.

To examine the potential effects of atrazine on GnRH neuronal activation, ovariectomized young adult female Wistar rats were treated (by gavage in 1% carboxymethylcellulose sodium salt) once daily with atrazine (0, 50, 100, or 200 mg/kg) for 4 consecutive days, and were prim ed with 17beta-estradiol-3-benzoate and progesterone (EB + P) to induce LH surge. Near the peak of the predicted LH surge, animals (n=4-6) were anesthetized and perfus ed, and brains were removed. Brains were processed for GnRH and cFOS using i mmunohistochemistry. To determine if atrazine alone would have a stimulatory effe ct on GnRH neuronal activation, a second cohort of animals was treated similarly exc ept that no hormones were given to induce GnRH and LH surges. For this part of t he experiment, rats were treated with 50 mg/kg

atrazine or vehicle for 4 da ys. To examine the effect s of atrazine withdrawal on hormone-induced LH surge and GnRH activation, ovariectomized animals were given a 4-day regimen of atrazine (200 mg/kg) by gavage. Control animals received vehicle only. An LH surge was induced in these ani mals either during the atrazine treatment period or 2 days or 4 days after the ce ssation of atrazine treatment.

Atrazine treatment inhibited t he EB+P LH surge at all doses. Atrazine also reduced the area under the curve (AUC) for the LH surge at all doses examined. Atrazine treatment did not affect peak FSH levels; however, at razine treatment at 200 mg/kg reduced the AUC for FSH levels in this group. Tr eatment with 100 mg/kg and 200 mg/kg atrazine reduced the percentage of GnRH neurons immunoreactive for cFOS compared with the vehicle-treated and 50 mg/kg atrazine groups . There was no difference between the 100 and 200 mg/kg atrazine groups. Treatm ent with 100 mg/kg and 200 mg/kg atrazine

also reduced the mean number of GnRH neurons immunoreactive for FOS; there was no difference between these groups. The resu lts also indicated that atrazine alone does not induce FOS immunoreactivity in GnRH neurons.

Within 4 days (but not 2 days) after cessati on of atrazine treatment, the reductions in GnRH activation and hormone-induced LH surge returned to control levels.

The authors concluded that t heir data indicate that at razine affects neuroendocrine function in the female rat by actions at t he level of the GnRH neuron and that the acute effects of high doses of atrazine can be reversed within 4 days after withdrawal of

treatment. The authors ackno wledge that “it remains uncl ear whether atrazine acts directly on GnRH neurons or on one of many neurotransmitter containing neurons in the

brain that are known to have a role in c ontrolling the preovulatory surge of GnRH.”

Reviewer’s Comments: This study provides further support to thei r previous findings (Foradori et al. 2009).Page 169 of 263 A.5.6 Giusi G, Facciolo RM, et al. 2006. The endocrine disruptor atrazine accounts for a dimorphic somatostatinergic neuronal expression pattern in mice. Toxico logical Sciences 89(1): 257 – 264.

The intent of this study was to examine the potential effects of atrazine on various somatostatin receptor s ubtypes (in particular, SST 2, 3, 5 ) including possible regional specificity in some hypothalamic and ex tra-hypothalamic estrogen-enriched brain regions. The authors provided a rationale for t he selection of the somatostatin receptor subtypes. The authors noted that the widely distributed SST 2, 3, 5 appear relatively early during development but also remain stable du ring adult life and exert a key role in the organization of the central nervous system . The authors also noted the role of

somatostatin in the control of the secretion of growth hormone (GH) and the striking

differences in GH release between male and female rodents. T he authors stated that differentiated GH secretion may be due to gende r-related differences in the transduction of somatostatinergic mechanisms in brain and/or pituitary targets. Finally, the authors concluded that the GH- and SST-dependent relationships “might provide further insights into the types of neurotoxicological mechani sms involved in the delay of the estrous cycle or the alteration of the hypothalamic -hypophyseal-gonadal axis in animals treated with atrazine (Wetzel et al. 1994).” The intere st in regional specificity was based on the observations that altered SST levels have been reported for hypothalamic nuclei.

Outbred Swiss-derived strain (CD-1) mice were used in t he study. Gestation day (GD) 14 females were divided into 2 groups: one group (low-dose) received 1ug/kg day atrazine via drinking water from a syringe, while the second group (high-dose) received 100 ug/kg day atrazine. The authors noted that the procedure was monitored to make sure that pups did not attemp t to drink the solution from the syringe. Treatment period was from GD14 to postnatal day (PND) 21. On PND 21, mothers were removed and F1 mice of both sexes (n = 6 for both sexes) continued with the treatm ent protocol (control group was included). At PND 60 – 65, the animals were sacr ificed and brains removed. Brain sections were prepared. Amino cupric silver staining was used for examining potential degenerative changes. Reverse transcr iption polymerase chain reaction was applied for the detection of SST 2,3,5 . In situ hybridization was utilized for mRNA.

The authors reported a hi gh-dose atrazine-dependent di morphic neurodegeneration pattern based on the amino cupric silver st aining results. The authors described the neurodegeneration as “a consistently evident argyrophilic reaction of axonal processes and perikarya in some extra-hypothalamic areas, specifically the external pyramidal neuron layer of the female hippo campal area, but not those of the male.” The authors

also indicated that examinati on of another hippocampal field ( i.e. , the dentate gyrus), the neurons showed a rather significant overall reduction in the total number of neuronal cells accompanied by significant perikarya l swelling plus well-developed astrocytic

formations in the female. T he dimorphic pattern was also evident in the hypothalamus, as demonstrated by significantly darker ax onal processes in the female arcuate hypothalamic nucleus. The dimorphic patte rn was not observed in extra-hypothalamic

areas such as the cortex or striatum.Page 170 of 263 In the high-dose female group, the author s reported a very strong (p < 0.001) upregulation of sst2 expressing neurons in the female hypot halamic areas, specifically the suprachiasmatic nucleus (+110%) and vent romedial hypothalamic nucleus (+65%), whereas a similar downregulat ory trend was noted in the so me extrahypothalamic areas such as the striatum (- 96%) and central amy gdaloid nucleus (-45%). In the high-dose male group, the authors repor ted very strong upregulator y and downregulatory actions for sst3 expressing neurons, with upregulati on in the arcuate hypothalamic nucleus (+120%) and basolateral amygdaloid nucle us (+78%), and downregulation in the cortical areas ( - 88%) and dendate gyrus (- 105%).

The authors concluded that the observed neurotoxicological effects of atrazine are “very likely linked to dimorphic expression pattern s of specific somatostain subtypes in discrete but key hypothalamic and extrahy pothalamic areas” of the mouse.

Reviewer’s Comments:

• There was a lack of details on the age at which mice were bred, the source and purity of atrazine, and the body weights of the animals, and systemic toxicity measurements were not performed. It is not clear if atrazine was dissolved in

corn oil which was administered to the contro ls. It is also not clear if the treated animals received the same amount of solution ( i.e. , 0.1 ml) as was administered during a pretreatment period to train the mice to drink from a syringe.

• Dams were treated with atrazine duri ng gestational and lactational phases, however, there was no mention of standardi zing litter sizes. Standardization of litter size would reduce between-litter variability.

• Also, several of the reference citati ons did not match or support statements presented elsewhere in the study. For exam ple, the reference to Surrales (1995) pertains to alachlor, mito cyn, and vinblastine. Gould et al. (1997) refers to research on polychlorinated biphenyl mixtures, not atrazine.

• The few photomicrographs of the amino cupric silver staining results utilized to

demonstrate critical neuronal degeneration were of ve ry poor quality and not presented in color to portray the reac tive staining, and were based on a small sample size, and only representative sections were provided. A “blind” evaluation was not performed. There was no presentation of count data (all stained reactive cells within an area) with statistical analyses to quantify the results of the reactive staining. Furthermore, no cell number data (with appropriate statistical analyses) were pr ovided to support the claim that there was “a rather significant overall reduction in the total number of neuronal cells...”.

Their claim cannot be confirm ed without these data.

• Only one representative photo on reve rse-transcriptase polymerase chain amplification results was provided, and there was no mention if there was a control for possible genomic contam ination in RNA preparations.Page 171 of 263 • No rational basis to explain the purpor ted differential neuronal expression and dimorphic sex effects was offered. For example, striatal neuronal degeneration was reported in both males and female s (although the photomicrographs were too poor in quality to verify thes e findings), yet downregulation of somatostatinergic receptor subtype 2 (sst 2) was noted only in the females. In

other brain areas of the female there was upregulation of sst2. There were similar difficulties in correlating neuronal degeneration with diffe rential expression of sst subtypes and dimorphic sex distri bution in other brai n regions, and no plausible biological mechanism to explai n the pattern of effects was discussed. Moreover, the relevancy of these findings is not clear.

• The study did not address the pot ential confounding by corticosterone.

Based on the number of serious limitations and no other corroborative support in the literature, the preliminar y results of this study are inconclusive.

A.5.7 Hossain M M and Filipov N M. 2008. Alteration of dopamine uptake into rat striatal vesicles and synaptosomes caused by an in

vitro exposure to atrazine and some of its metabolites. Toxicology

248: 52 – 58.

To better clarify the potential mechanisms of atrazine-mediated effects on striatal dopamine, this study examined the effe cts of atrazine on the vesicular and synaptosomal uptake of striatal dopamine, which may result in disrupted vesicular storage and/or cellular uptak e of dopamine. The study design involved in vitro atrazine exposure using isolated rat striatal synaptosomes and synaptic vesicles. Besides atrazine, effects of atrazine metabolites, i.e. , didealkly atrazine (DACT), desethyl atrazine (DE) and desiopropyl atrazine (DIP) we re also investigated. Limited details were provided on the various methods such as preparation of synaptic vesicles and striatal synaptosomes, vesicular uptake of dopamine, and synaptosomal uptake of dopamine.

Atrazine (1 – 250 μM) inhibited dopamine uptake into synaptic vesicles, and the inhibition was dose dependent. DACT did not affect vesicular dopamine uptake, while DE and DIP significantly decreased vesicu lar dopamine uptake. Kinetic analysis (nonlinear isotherm regression) of the ve sicular dopamine uptake showed that atrazine

decreased the V max parameter but not K
m .

Atrazine and DE increased (6 – 13%) sy naptosomal dopamine uptake; DACT and DIP were without effect. Atrazine, DE and DIP increased the ratio of synaptosomal/vesicular uptake of dopamine. The authors concluded t hat atrazine and two of its metabolites (DE and DIP) decrease striatal dopamine levels, at least in part, by increasing cytosolic dopamine, which is prone to ox idative breakdown. The author s note that the effects of

atrazine on aromatic amino acid deca rboxylase, monoamine oxidase and neuronal

mitochondrial function remain unknown. Prev iously, the authors (Filipov et al. 2007)

showed that atrazine does not alter the func tion of tyrosine hydroxylase substantially.Page 172 of 263 Reviewer’s Comments: The re sults revealed that atrazine, in vitro , alters striatal dopamine levels possibly by interfering with the vesicular storage and/or cellular uptake of dopamine. Tyrosine hydroxylase per se is not a target of atrazine. The relevance of

these in vitro data to dopaminergic mechanisms in vivo is not clear given the high

concentrations of atrazine examined in vitro and the complex interactions between

various neurotransmitter and endocrine systems in the intact brain. The effects of

atrazine on aromatic amino acid deca rboxylase, monoamine oxidase and neuronal

mitochondrial function are unknown.

A.5.8 Rodriguez V M, Thiruchelvam M, Cory-Slechta D A. 2005. Sustained Exposure to the widely used herbicide atrazine: altered

function and loss of neurons in brain monoamine systems. Environmental Health Perspectives. 113(6): 708-715.

The purpose of this study was to examine the hypothesis that atrazine adversely affects brain dopamine systems, in particular, the nigr ostriatal and mesocorticolimbic pathways. In addition, the underlying mechanisms of any potential changes in dopamine function in these pathways were explored by examini ng the acute effects of atrazine on striatal dopamine release.

In the chronic phase of the study, 30 male Long-Evans rats (9 months of age) were exposed via the diet to 0, 5 or 10 mg/kg/day atrazine for 6 months. Body weights and food consumption were monitored throughout the study. Locomotor activity was recorded at 2, 3, and 6 months of atrazi ne treatment and 2 weeks after cessation of

treatment. At the 2-mont h time point, locomotor activity was measured on 3 consecutive days, in which the rats were injected saline intr aperitoneally 5 minutes before the session during the first 2 days, an d an injection of 1 mg/kg d-amphetamine sulfate on day 3. Only a single locomotor activity session was performed at the 3- and 6-month time points and at 2 weeks after termi nation of treatment. Locomotor activity was recorded during the light phase for 7 hour s. The hypothalamus, prefrontal cortex, nucleus accumbens, and the striatum were removed from sacrificed animals 2 weeks

after termination of the treatment. Monoamine and monoamine metabolites were measured by HPLC with electrochemical detection. Immunohistochemical and unbiased stereologic analyses of tyrosine hydrox ylase from the substantia nigra pars compacta and ventral tegmental area were performed. Microdialysis was used to evaluate changes in striatal dopamine release after acute i.p. exposures to 100 or 200 mg/kg atrazine in sessions lasting 7 hours. Animals were subjected to stereotaxic surgery and cannulation. Brain histology was performed on the ra ts at the completion of micordialysis sampling.

No treatment-related changes in body weights or food consumption were reported (data were not shown). Rats treated for 2 m onths with 10 mg/kg atrazine exhibited an enhanced horizontal locomotor activity respons e (70% relative to the 0 and 5 mg/kg groups) to d-amphetamine. T he increased locomotor activity persisted at the 3- and 6-Page 173 of 263 month time periods, as well as 2 weeks afte r treatment, time periods carried out in the absence of d-amphetamine adminis tration. At 2 weeks a fter treatment with atrazine, locomotor activity was 40% above control val ues. A statistically significant decrease (approximately 20%) in striat al dopamine levels at 10 mg/k g/day was noted. Dopamine levels were increased in the prefrontal co rtex in an inverse U-shaped fashion; the increase at 5 mg/kg/day was 30 – 40%, while le vels declined towards control levels at 10 mg/kg/day dose. Significant decreases (10 – 15%) in hypothalamic serotonin level were observed at the 5- and 10-mg/kg/ day doses. The 10 mg/kg/day dose was associated with a significant decrease (15 – 20%) in norephineprine levels in the prefrontal cortex. The author s reported no changes in the me tabolites of serotonin or dopamine or dopamine turnover in any region.

At 2 weeks after the 6-month treatment per iod, the number of tyrosine hydroxylase positive (TH+) and tyrosine hydroxylase negat ive (TH-) cells were reduced in the substantia nigra pars compacta and ventral t egmental areas. This effect was primarily due to the 10 mg/kg/day dose level. Mean reduc tions in cell numbers ranged from 9 to 13% in the 10 mg/kg/day group, whereas those in the 5 mg/kg/day group ranged from 0

to 3%.

Following an acute i.p. administration of 100 or 200 mg/kg atrazine, a significant decrease in basal dopamine release was no ted as measured at 90, 120, 150, and 180 minutes postadminstration. By 150 minut es, the decreases averaged approximately 40% and were seen in both dose groups. The authors also noted a dose-dependent decrease in potassium-stimulated dopamine release. No changes in the dopamine metabolites (DOPAC or HVA) were observe d in the microdialysis component of the study.

The authors concluded that t here findings “demonstrate that atrazine may have broad

effects on brain monoamine systems and thereby influence a wide range of behavioral

functions.” The authors ackno wledge that “additional studies are needed to unravel the targets of atrazine and the mechanisms of it s effects, as well as the ultimate human health consequences of such exposure s for behavioral and/or neurological dysfunctions.”

Reviewer’s Comments: This study has a number of limitations, including:

• No photomicrographs were provided to support and corroborate the findings of stereological examination of TH+ and TH - cell loss (only cell number data were provided), and no histological examination to describe actual morphological changes was discussed. There was no in formation on the calibration of the stereological test system and no informati on on control for potential sources of variability in the stereological esti mations of cell numbers. There was no assessment of the precision of the estimations of neuronal counts.Page 174 of 263 • Behavioral assessment was limited to d-amphetamine-induced locomotor activity; inclusion of dopamine agonists and antagonists and L-Dopa in the experimental design would help support the findings.

• The study did not include a positive cont rol such as 6-hydroxydopamine which produces dose dependent alterations in dopamine and dopam ine metabolites and loss of tyrosine hydroxylase-immunoreacti ve cells in the substantia nigra.

A.6 Immunotoxicity

A.6.1 Cimino-Reale, G. , Ferrario, D., Casati, B., Brustio, R. Diodovich, C., Collotta, A., Vahter, M. , Gribaldo, L. (2008). Combined

in utero and juvenile exposure of mice an d atrazine in drinking water modulates gene expression and clonogenicity of myeloid progenitors. Toxicol. Letters 180:59-66.

In this study, groups of four adult female CD-1 mice were exposed to 1mg/L sodium arsenate (NaHAsO 4 ) in drinking water for 10 days before mating and during gestation. Other groups of NaHAsO 4 -exposed male and female (4 /sex/group) offspring were treated for 4 months with 1 mg /L atrazine (unreported purity ), an additional dose of 1mg/L NaHAsO 4, or a combination of atrazine + NaHAsO 4 in drinking water. At the end of treatment, animals were sacrificed and bone marrow cells were collected from one femur, frozen and used for the colony forming unit-granulocyte/macrophage (CFU-GM) assay. Cells from the second femur were harvested, flushed with 1 mL of commercial

RNA-Later to stabilize the RNA for later isol ation, and held frozen until use in the gene expression phase of testing.

For the CFU-GM assay, bone marrow cells we re thawed, centrifuged, and resuspended in culture medium specific for murine hem atopoietic cells containing 30% fetal bovine serum, bovine pancreatic insulin, human tran sferrin iron-saturated, 2-mercaptoethanol, interleukin-3 and-6, and stem cell growth fa ctor. Cell viability was determined; cells were adjusted to 6.0 x 10 5 viable cells/mL, seeded to yield a final density of 4.0 x

10 4 viable cells/plate and incubated. Triplicat e plates were prepared per group and five replicated experiments were conducted. After 7 days, colonies were counted to determine cytotoxicity.

For the gene expression phase of testing, total RNA was extracted from bone marrow cells, and [ 33 P]-labeled cDNA was obtained by converting 1 μ g of total RNA into cDNA using reverse transcriptase in the presence of Mouse Cancer CDS primer mix. The cDNA was purified, hybridized for 16 hour s on microarray membranes, washed, and exposed on a phosphor-image screen for 21-24 hour s. Transcripts were considered to be differentially expressed if the p value was ≤ 0.05.Page 175 of 263 Bone marrow cells were also investigat ed for modulation of gene expression of estrogen receptors alpha and beta using real-tim e PCR. For this approach, 500 ng of total RNA, extracted from the bone marro w of treated males and females, was converted to cDNA through reverse transcr iption using random hex amer primers HighCapacity cDNA Archive Kit and Moloney murine leukemia virus reverse transcriptase.

No gross pathological changes were seen in the treated mice. A significant (p<0.05) ~19% (males) and ~23% (females) decreas e in the percentage of CFU-GM was see after exposure to 1 mg/L atrazine alone. NaHAsO 4 had no cytotoxic effect on the bone marrow. However, cell growth increased si gnificantly (p<0.05) by 12% (138 vs. 123 colonies) after the combined atrazine/ NaHAsO 4 treatment of females, compared to control. Results from the microarray anal ysis showed that there was no significant change in gene expression in bone marrow cells following exposure to either atrazine

alone or NaHAsO 4 . However, the combined ex posure to atrazine/NaHAsO 4 demonstrated the overexpression of 20 differentially expressed genes (DEGs). For the analysis of changes in gene expression of estrogen receptors alpha and beta For the analysis of changes in gene expression of estrogen receptors alpha and beta (ER α and β ), quantitative RT-PCR of exposed bone marrow cells revealed that the ER α was not modified in either sex after dosing with t he three treatment groups. However, ER β gene expression was significantly increased in both sexes and all treatment groups. Nevertheless, the high variability among indivi dual females in expression of either ER α

or β genes limits the interpretati on of these data. CFU-GM assay, quantitative RT-PCR of exposed bone marrow cells revealed that the ER α was not modified in either sex after dosing with the three treat ment groups. However, ER β gene expression was significantly increased in both sexes and all treatment groups.

Reviewer’s Comments: With the exception of the CFU- GM assay results showing inhibition of myeloid cells, suggestive of an adverse effect on the immune system, no

definitive conclusion can be reached that would shed light on the mechanism(s) responsible for the bone marrow immunotoxicit y. This is because of the lack of an effect on gene expression in the treated bone marrow and the wide variation in the gene expression of ER β among individual females.

A.6.2 Devos S, Bosscher K De, et al. 2003. Inhibi tion of cytokine production by the herbicide atrazine – Search for nuclear receptor targets. Biochemical Pharmacology 65: 303 – 308.

The purpose of this study was to test the hypothesis that atrazine affects leukocyte function via nuclear receptors such as t he glucocorticoid rec eptor (GR), the androgen receptor (AR), members of the peroxisome proliferato r-activated receptor (PPAR) family, and the retinoid-related orphan receptor (ROR) α . The authors noted previous work that showed weak binding of atrazine to the AR. The GR was investigated because of the similarity of effects induced by atrazine and the synthetic glucocorticoid

DEX. Glucocorticoids have anti-inflamma tory and immunosuppressive properties and

interfere with nuclear factor – κ B (NF- κ B) and AP-1, and in this way repress thePage 176 of 263 expression of inflammation-related genes such as interleukin (IL )-6, IL-8, ICAM, and Eselectin, that contain NF- κ B and AP-1 response elements. Activation of PPARs has been reported to have effects on leukocytes. ROR α can also affect leukocytes function.

In this study, adult (2- or 7 -month-old) CF1 mice were inje cted i.p. on days 1 and 4 with atrazine (100 mg/kg). Control mice received the vehicle only (carboxymethylcellulose containing 0.5% Tween 80). Mice were sa crificed on day 7. Liver and kidneys were removed. Brief descriptions of cells (murine fibrosacrcoma L929sA and rabbit kidney RK13) and culture procedures, plasmids (p1168hu.IL6P-luc+, p98.IL8p-luc+, p133.IL8pluc+, pELAmP-luc+), transfection and repor ter gene analysis, ELISA, AR assay, and peroxisome analysis were provided.

Inhibition of human peripheral blood m ononuclear cell (PBMC) proliferation and cytotokine production was observed in cultures with atrazine (3 x 10-7 M to 1.5 X 10-5 M). This effect was not prevented by the gluc ocorticoid inhibitor RU4 86. In contrast to DEX, atrazine did not reduce IL-8 production. The authors concl uded that these data suggest that the effects of atrazine on PBMC are not mediated through the GR. Atrazine did not affect TNF-induced IL-8 promoter activity, in contrast to DEX. This was demonstrated in stable cell pools transfected wit h an IL-8 promoter-driven reporter gene construct, p98.IL8p-luc+, and then induced wit h TNF and treated with DEX and atrazine (3, 0.3, and 0.03 μM) alone or in combinatio n. TNF is an inflammatory stimulus activating the reporter genes. Testing a longer variant of the IL-8 promoter, p133.IL8pluc+, yielded the same overall results (dat a were not shown). To test whether the results with the IL-8 promoter were promoter-specific, the aut hors also tested the effect of atrazine on other proinflammatory gene cons truct, containing the E-selectin promoter (with NF- κ B elements) or the IL-6 promoter (with NF- κ B and AP-1 elements), that are both inducible by TNF. The authors indicated that there were no s ubstantial effects with atrazine. They concluded that atrazine does not exhibit glucocorticoid-like properties

and does not interact with the GR.

No peroxisome proliferation was observed in the liver or kidneys of mice treated with atrazine. Moreover, no PPA R-mediated induction of promoter activity was seen on targets of PPAR α , PPAR γ , or PPAR δ . No binding of atrazine to the AR was observed.

The authors concluded that their study shows that AR, GR, PPARs, or ROR α are not likely targets of atrazine.

Reviewer’s Comments: Using human per ipheral blood mononuclear cells, the in vitro study of Devos et al. (2003) suggests that atrazi ne-induced inhibition of human peripheral blood mononuclear ce ll proliferation and cytokine pr oduction is most likely not mediated through nuclear receptors such as the glucocorticoid receptor, androgen receptor, members of the per oxisome proliferator-activat ed receptor family, and the retinoid-related orphan receptor α . The relevance of this study to human healt h risk assessment is limited since it entails intraperi toneal injection of atrazine to the animals prior to in vitro experiments.Page 177 of 263 A.6.3 Devos S, Van Den Heuvel R, et al. 2004. Li mited effect of selected organic pollutants on cyto kine production by peripheral blood leukocytes. Eur. Cytoki ne Netw: 15: 145 – 151.

This study examined t he potential effects of in vitro atrazine treatment on cytokine production by peripheral blood mononuclear ce lls (PBMC) from control and allergic donors. Cells were cultured for six days in the presence of atrazi ne (3 μM) or vehicle with phytohemagglutinin (PHA) or Dermatophagoides pteronyssinus (Dpt) extract, then for one day in the presence of PHA + phorbol 12-myristate 13-acetate. Information on the donors (selection, ethical approval, allergic disease description) and methods (PBMC cultures, cytokine assays, IgE plasma level determination, radioallergosorbent

or RAST test to confirm Dpt allergy) was provided.

Atrazine treatment significantly reduc ed cytokine production IL-5 and IFN- γ in the donors. This effect was apparent in PHA-stimul ated cultures, but not Dpt stimulation.

Reviewer’s Comments: The authors concluded that since the effects on cells from allergic and non-allergic donors were similar, and the effects on cytokines were limited, the impact of atrazine on allerg ic responses is unlikely.

A.6.4 Filipov, N M, Pinchuk, L M, et. al. 2005. Immunotoxic effects of short-term atrazine exposure in young male C57BL/6 mice. Toxicological Sciences: 86(2): 324-332.

The objective of this study was to assess t he effects of atrazine exposure on selected immune parameters in young mice. Parameters included: thymus and spleen weights, cellularity, lymphocyte subpopulations in the spleen, thymus, and peripheral blood

mononuclear cells. To assess short- vs. long-term effects, t he parameters were measured at one day, one week, and seven wee ks after administration of the last

atrazine dose. Atrazine was administered to one-month-old male C57BL/6 mice for 14 days by oral gavage at doses of 5, 25, 125, and 250 mg/kg bw.

Spleen weight was decreased at the 250 mg/kg dose level on day 1, while thymus weights were decreased at the 125 and 250 mg/kg/day dose levels. Decreased thymus weights persisted one week after the last dose of atrazine, but resolved by seven weeks. Spleen and thymus cellu larity was dose-dependently decreased by atrazine treatment (statistica lly significant at 125 and 250 mg /kg doses) at day 1. The decreased cellularity persisted one week a fter cessation of atra zine treatment. However, decreased thymic cellularity was resolved by seven weeks, whereas decreased splenic cellularity was significantly decreased at all doses levels of atrazine. Thymic T-cell populations were significantly and dose-dependently decr eased at day 1. The authors reported that one week after cessa tion of atrazine trea tment, the decreases in most T-cell populations were still present at the higher doses of atrazine, whereas at the lower doses a non-significant rebounding effect was observed (data not shown). The authors also reported that by seven weeks after the last atrazine dose, thePage 178 of 263 decreases in the thymic subpopulations were numerically present, but were no longer significant (data not shown).

In regard to splenic cell phenotypes, both MHC II+ and CD19+ cells were dosedependently decreased, whereas only the high dose of atrazine increased the percentage of CD3+ cells. In addition, atrazine treatment decreased the number of splenic naïve T helper and T cytotoxic cell s, whereas it increased the percentage of highly activated cytotoxic/memory T cells. The proportion of mature splenic dendritic cells (DC; CDIIc high ) was also decreased, which persi sted for at least one week. No effects on splenic cell phenotypes were seen at seven weeks post treatment time point.

With respect to peripheral blood mononuc lear cells, a dose-dependent decrease in circulating CD4+ cells was observed one day a fter the last atrazine dose. The decrease in CD4+ cells was still present at the 250 mg /kg dose (high dose) at seven days after the last dose. In addition, at seven days, the MHC-II+ cells were also decreased at the high dose. The authors stated that mo st of the changes in peripheral blood mononuclear cells were no longer apparent at se ven weeks after the last atrazine dose, although data were not shown. The authors concluded that “atrazine exposure appears to be detrimental to the immune system of ju venile mice by decreasing cellularity and affecting lymphocyte distribution, with certai n effects persisting long after exposure has

been terminated.”

Reviewer’s Comments: Although T cell subp opulations were decreased at all dose levels on Day 1 the fact that they quickly recover diminishes the toxicological significance of this finding ( i.e., its ability to predict impaired function). A similar conclusion was reached by The Joint FA O/WHO Meeting on Pesticides Monograph on Atrazine (JMPR 2007).

A.6.5 Karrow, N A, McCay, J A, et. al. 2005. Oral exposure to

atrazine modulates cell-mediated immune function and decreases

host resistance to the B16F10 tumor model in female B6C3F1 mice. Toxicology: 15-28.

The purpose of this study was to examine the potential immunomodul atory properties of atrazine and was designed to assess changes in cellular and antibody immune function

in the female B6C3F1 mouse model. Th is model also included pathogenic B16F10 tumor and Listeria monocytogen es challenge models to assess potential biological significance of any observed changes in i mmune function. In this study, female B6C3F1 were administered atrazine by gavage (0, 25, 250, and 500 mg/kg/day) for 14 days then evaluated for immune responsive ness. Immune assays and toxicity parameters included: body weights, organ weights (i ncluding spleen), hematology, differential cell counts, serum IgM titers, spleni c natural killer (NK) cell activity, splenic mixed lymphocyte response (MLR), splenic IgM antibody-forming cell (AFC) response, reticulocyte counts, splenic lymphocyte pro liferation, splenic B and T cell enumeration, splenic cytotoxic T lymphocyte respons e (CTLR) to P815 mastocytoma cells,Page 179 of 263 mononuclear phagocytic system (MPS) eval uation, L. mono cytogenes challenge and B16F10 tumor challenge.

A decrease in body weights was observed in the atrazine treated groups. A significant dose-dependent decrease in spleen and thymus weights was also observed. Kidney weights were slightly but significantly reduced at the high dose. Hematological alterations included reductions in erythro cyte counts (all dose levels), decreased hematocrit (high dose), and increased mean co rpuscular hemoglobin (all doses). At the high dose, the percentage of CD3 + and CD4 - CD8 + T cells was significantly increased by 10 and 18%, respectively. Atrazine treatment did not significantly alter the spleen cell proliferative respons e to mitogens Con A or LPS. There was no effect of treatment on splenic primary AFC response and serum IgM titers to sRBCs. The spleen cell MLR to DBA/2 murine spleen cells was stimulated (30%) in the high-dose mice. The splenic CTLR was significantly stimulated in mice that were treated with both 25 and 250 mg/kg atrazine. The NK cell activity in the spleen was only inhibited in the 250 mg/kg/day group and the authors di d not consider this effect to be of biological significance. A dose-related decreasing trend in MPS function across the treatment groups was noted.

Host resistance to B16F10 tumor challenge was reduced in atrazine treated mice at both challenge levels. At the 3 x 10 5 cells/mouse challenge le vel, a significant dosedependent increase in the number of B16F10 nodules was observed in the lungs of treated mice. The greatest increase in t he number of nodules (122%) was observed at the 500 mg/kg treatment level. Although ther e were no statistically significant changes in host resistance to L. monocytogenes, t he authors stated that the data suggest that atrazine may alter host resistance to this pathogen. The percent mortalities for the various treatment groups at the challenge leve l of 6.3 x 10 4 CFU/mouse were 67, 92, 58, and 33 for the control, 25, 250, and 500 mg/kg atrazine treatment groups.

Reviewer’s Comments: This study design included a host resistance test, which

assessed the function of the immune system in toto.

A.6.6 Mizota K and Ueda H. 2006. Endocrine disrupting chemical atrazine causes degranulation through G q/11 protein-coupled neurosteroid receptor in mast cells . Toxicological Sciences 90(2):

362 – 368.

The authors examined the in vitro effects of several putative endocrine disrupting chemicals including atrazine on β -hexosaminidase release fr om mast cells and their putative neurosteroid receptor involvement. Drawing from their previous research, the authors proposed that NS2-type neurosteroid re ceptors exist on mast cells and mediate histamine release, which, in turn, stimulates nociceptor endings. They previously demonstrated that neurosteroids cause extravasation as well as hyperalgesia, which were abolished by diphenhydramine, a repr esentative H1 receptor antagonist.

Neurosteroid-induced extravasation has been a ttributed to degranulation of mast cells.Page 180 of 263 Degranulation of mast cells was determi ned by measuring the release of granuleassociated β -hexosaminidase from RBL-2H3 cells . Degranulation was also evaluated by Alexa 488-annexin V binding to the phosphat idylserine, which is externalized during degranulation. Antagonism of atrazine-induced Alexa 488- annexin V binding in RBL2H3 cells by BSA-conjugated progesterone (PROG-BSA) was examined. β

Hexosaminidase release was characterized by various inhibitors including antisenseoligodeoxynucleotide for G α q/11, pertussis toxin, phosph olipase C inhibitor U-73122, inositol 1,4,5-triphosphte receptor inhibitor xestospongi n C and Ca2+ channel blocker

lanthanum chloride.

Based on the authors previous findings that intraplantarly administered neurosteroids cause pain-like responses, such as biting and licking behaviors in a diphenhydraminereversible manner, these responses were also evaluated in male ddY mice. Atrazine (10 fmol) was administered intraplantarly (i. pl.) into the hind paw. The total amount of time showing pain responses, such as biting and licking, was measur ed for 10 min. after i.pl. injection.

Atrazine (10 nM – 1 μM) caused a conc entration-dependent degranul ation in RBL-2H3 cells and peritoneal mast cells as measured by β -hexosaminidase release. This release was blocked by PROG-BSA, a membrane impermeable neurosteroid receptor antagonist. PROG-BSA also blocked atrazi ne-mediated increase in Alexa 488-annexin V binding (which labels phosphatidylserine ex ternalization in the plasma membrane representing degranulati on) in RBL-2H3 cells. The authors indicated that the

observation of atrazine-induced increase in Alexa 488-annexin V bind ing shows that the atrazine-induced degranulation is un likely due to cell damage.

Antisense-oligodeoxynucl eotide pre-treatment r educed atrazine-induced β

hexosaminidase release. Atrazine- induced degranulation was abolished by phospholipase C inhibitor U-73122. Lanthanum chloride, which inhibits Ca2+ influx in cells, had no effect. The aut hors interpreted the findings to reveal that the signal transduction involved in the degranulation is mediated though G q/11 , PLC and mobilization of intracellular Ca2+, c haracteristics of neurosteroids.

The results also demonstrated atrazi ne-induced pain response that occurred immediately and ceased within 5 mi nutes after i.pl. administration. The responses were dose-dependent between 1 and 10 fmol (i.pl.). The effects were abolished by PROG – BSA and DPH, which is a histamine receptor antagonist. This was interpreted by the authors to indicate that the responses we re caused by neurosteroid receptor-mediated histamine release from mast cells, which was supported by atrazine-induced plasma extravasation assay using Evans blue. Ov erall, the authors c oncluded that atrazine induces some inflammatory effects, including nociception and plasma extravasation, and the underlying mechanisms are mediat ed through mast cell degranulation and

activation of the G q/11 -protein coupled neurosteroid receptor, phospholipase C and

intracellular Ca2+ mobilization.Page 181 of 263 Reviewer’s Comments: This study conc luded that the underly ing mechanisms of atrazine-induced inflammatory effects such as nociception and plasma extravasation, may be mediated through mast cell degranulat ion and activation of the Gq/11-protein coupled neurosteroid receptor, phospholipase C and intracellular Ca 2+ . However, the inflammatory responses were examined followin g intraplantar administration of atrazine, a route of exposure that is not likely to occur in humans.

A.6.7 Pinchuk L M, Lee S-R, and Filipov N M. 2007. In vitro atrazine exposure affects the phenotypic and functional maturation of dendritic cells. Toxicology and A pplied Pharmacology 223: 206 –

217.

Based on prior results revealing that atrazi ne may affect dendritic cell (DC) function (Rowe et al. 2006; Filipov et al. 2005), the obje ctives of this study was to investigate the

effects of in vitro atrazine exposure on the phenotypic and functional maturation of a growth factor-dependent, bone-marrow-derived immature murine DC cell line JAWSII. For comparative studies, limited studies we re performed with primary thymic DC. Details were provided on the following mate rials and methods used for the analyses: dendritic cell line, primary thymic DC preparation, anti bodies and flow cytometry, endocytosis assay, apoptosis assay, PCR analysis, and real-time PCR analysis.

JAWSII cells were induced to mature in t he presence of growth factors and selected

maturational stimuli in vitro . The DC line was exposed to atrazine (1 – 300 μM) and the effects on phenotypic and functional maturati on of DC were examined. Phenotypic changes caused by atrazine included a dos e-dependent removal of surface MHC-1 with a significant decrease at 1 μM concentration . In addition, atrazine treatment decreased the expression of the costim ulatory molecule CD86 and it downregulated the expression of the CD11b and CD11c accessory molecule s and the myeloid developmental marker CD14. The phenotypic changes in JAWSII DC maturation were associated with

functional inhibition of matura tion at higher concentrations of atrazine where receptormediated antigen uptake was increased. The aut hors stated that thei r data suggest that atrazine directly targets DC maturation and removes MHC-1 molecules from the DC

surface.

Reviewer’s Comments: Pinchuk et al. (2007) provided in vitro evidence suggesting that atrazine directly targets dendritic cell matu ration and decreases major histocompatibility complex 1 (MHC-1) molecule expression on the dendritic cell surface.Page 182 of 263 A.6.8 Pruett, S B, Fan, R, et al. 2003. Modeling and predicting immunological effects of chemical stressors: characterization of a quantitative biomarker for imm unological changes caused by

atrazine and ethanol. Toxicol ogical Sciences: 75: 343-354.

The authors stated that the study was conducted to determine if the “stress-induced immunological effects of atra zine and ethanol could be predicted, and if it is feasible to use one immunological parameter as a biomar ker of stress to predict the quantity of changes expected in other immunological par ameters.” Consequen tly, doses were

selected in order to elicit an increase in cort icosterone levels as a predictor of immune function; this study was not intended as a hazard identification study of atrazine. Female C57BL/6 X C3H F1 (B6C3F1) mice (8 – 12 weeks of age following a two-week accommodation period) were used in t he study. Atrazine was administered intraperitoneally (only once) at dose levels of 100-, 200-, or 300-mg/kg bw in corn oil (group size was 5 mice). A naïve control group was included

. The authors selected corticosterone as a mediator that might predict the quantity of immunosuppression caused by stress responses. Therefore, t he corticosterone area under the curve (AUC)

values for atrazine-treated mice were dete rmined. Immunological parameters included: thymus and spleen cellularity, lymphocyte s ubpopulations in the thymus and spleen, expression of MHC class II protein on sp lenocytes, antibody responses to keyhole limpet hemocyanin, and natural killer cell activity. Model s were derived describing the relationship between corticosterone AUC and potential immunological changes.

The peak corticosterone response (approximat ely 1200 ng/ml) to atrazine occurred at 6 hours, and was dose-dependent. In order to assess if th e observed corticosterone response was unique to the intraperitoneal rout e of exposure, the authors included an additional experiment in which five mice were treated by oral gavage (corn oil) to 500 mg/kg atrazine, while five mice were admin istered the vehicle. The 500 mg/kg dose was selected in order to match the high dose used in the National Toxicology Program’s study on atrazine. Three hours after dosi ng, serum corticosterone levels were

measured. The authors reported the following corticostero ne values: controls: 164 ± 88; atrazine-treatment: 676 ± 79.

Linear regression models of corticoster one AUC vs. thymus- and spleen-cell numbers and subpopulations revealed that atrazi ne treatment was asso ciated with decreased thymus cell number and percentage of thymic CD4+CD8+, and decreased spleen total cell number and splenic B220+ cells. Linear regression models of corticosterone AUC vs. expression of MHC Class II protein on splenocytes indica ted a dose-dependent decrease in the expression of MHC Class II protein on splenocytes in atrazine-treated

animals. The results of linear models al so indicated dose-dependent decreases in splenic NK cell activity and IgG1 and IgG2a responses to KLH.

The authors also assessed the potential correlations between observed effects of atrazine and effects predicted using models derived from mice tr eated with exogenousPage 183 of 263 corticosterone or restraint stress with linear m odeling. The author s stated that the modeling results indicated that the atrazine effects were sim ilar to results obtained from mice subjected to restraint stress; however, some of the atrazine effects were greater than predicted on the basis of the stress response alone.

Reviewer’s Comments: Pruett et al. (2003) demonstrated that a single high oral dose of 500 mg/kg atrazine also increased endogenous co rticosterone levels. This study was intended to provide insight into the predictiv e value of corticosterone production over time for impairments of the immune system not to assess the immunotoxicity of atrazine

per se

.

A.6.9 Pruett, S B, Fan, R, et al . 2009. Patterns of immunotoxicity associated with chronic as compared with acute exposure to chemical or physical stressors and their relevance with regard to the role of stress and with regard to immunotoxicity testing. Toxicological Sciences: 109(2): 265-275.

This study is an extension of an earlier study (Pruett et al. 2003) that entailed single dose exposure to atrazine. The purpose was to determine if mice can become tolerant or habituated with regard to the stress re sponse and accompanying immunological effects.” Female C57BL/6 X C3H F1 (B6C 3F1) mice (8 – 12 weeks of age following a two-week accommodation period) were used in the study. Mice were dosed daily (intraperitoneally in corn oil) for 28 days with atrazine (up to 150 mg/kg/day) or were subjected to restraint, which can also in duce neuroendocrine stress responses and alter immunological parameters. T he exact doses of atrazine were not specified except for the high dose of 150 mg/kg/day . At the end of t he study, the followi ng parameters were

measured: white blood cell count and differential blood count, lymphocyte phenotypes in the blood and spleen, natur al killer (NK) cell activity in splenocytes, and corticosterone concentrations (at various time s after dosing or restraint). The results were compared with those from the acute study (P ruett et al. 2003).

The authors provided results for the corticos terone response in mice treated with 150 mg/kg/day atrazine with that observed in t he acute study (at 200 mg/kg dose level). The corticosterone response was very similar after the 28-day treatment as observed after a single treatment. T he authors concluded that the 28-day atrazine treatment did

not cause tolerance or habituation.

Restraint had little effect on white blood cell c ounts following one treatm ent or after a 28 day treatment period. Atrazine suppressed white blood cell counts; the suppression was greater after 28 day treatment compared with the single dose tr eatment. Atrazine caused essentially the same increase in neut rophils and decrease in lymphocytes after 28 days of treatment as it did after 1 dose administration. C hanges in the percentage of MHC class II expression by B lym phocytes and MHC class II expression on B lymphocytes in the spleen were comparabl e between the single dose and 28-day daily dose regimens. Atrazine caused a similar decrease in B cell percentage in the bloodPage 184 of 263 and spleen in the 28-day and single day studies. Restraint did not affect the percentage of B lymphocytes following ac ute or 28-day treatment. A dose-dependent decrease in CD4+ and CD8+ T lymphocytes in the blood wa s noted after single dose administration of atrazine; however, 28-day tr eatment increased this param eter. Restraint for 28-days decreased the T cell percentage in the blood, whereas acute restraint caused a smaller decrease. Decreases in NK cell activity of splenic lymphocytes were noted with both acute and 28-day atrazine treatments; however , decreases were greater with the latter

treatment.

Reviewer’s Comments: Pruett et al. (2009) provided some s upporting evidence that 28- day atrazine exposure did not cause habitu ation or tolerance to the HPA axis perturbations, but immune function (e.g., antibody production) was not evaluated in this study.

A.6.10 Rooney, A A, Matulka R, and Luebke, R. 2003. Developmental atrazine exposure suppresses immune function in male, but not female Sprague-Dawle y rats. Toxicological Sciences

76: 366-375.

This study was designed to test the hypothe sis that atrazine may cause developmental immunotoxicity through its disr uption of prolactin (PRL) or thyroid hormones during a critical early period of dev elopment. Pregnant Sprague-Dawle y (SD) rats were exposed via oral gavage to 35-mg atrazine/kg/day fr om gestational days (GD) 10 (to avoid the possible interference of atrazine with embryo implantation) through postnatal day (PND)

23. The authors indicated t hat the dose of 35 mg/kg/day was set “just above the NOEL for atrazine-induced developmental endocrine dysfunction to examine the potential for atrazine to indirectly induce immunotoxicity.” The authors st ated that this dose is likely to induce endocrine effects in both sexes. Separate groups were exposed to bromocryptine (BCR) at 0. 2 mg/kg/2x/day to induce hypoprolactinemia or to propylthiouracil (PTU) at 2 mg/kg/day to i nduce hypothyroidism. At 7 weeks old, the following immune functions in the offspring we re evaluated: natural killer (NK) cell function, delayed-type hypersensitivity (D TH) responses to bovine serum albumin (BSA), phagocytic activity of peritoneal macrophages, and antibody response to sheep erythrocytes (SRBC).

There was an increase in offspring mortality in atrazine treated animals during PND 2 – 14. The average % offspring mortalit y/litter in atrazine-treated dams was 13.2 compared to 2.3% for contro l dams. The authors noted that increased mortality was “unexpected, as this has not been reported for th is dose of atrazine.” They further state

that “it is not known whether the weight loss or death of pups was due to an effect on the physiology or behavior of the dams or the pups themselv es.” The authors stated that male offspring body weight of the atra zine group was decreased relative to controls (mean control body weight was 20.1 g comp ared to a mean atrazi ne group body weight of 19.0 g).Page 185 of 263 The primary (IgM) antibody response to SRBC was suppressed in atrazine-treated males (p = 0.0149), but not female, offspr ing at 8 weeks of age. There was no difference among treatment groups in the Ig G antibody response to SRBC. There was no difference between the offspring of ATR- exposed dams and the control offspring in the IgM antibody response to SRBC in 6-m onth-old-animals (data were not shown).

The latter results indicate that that imm une function returned to normal by 6 months. The DTH response of male, but not female , offspring of atrazine treated dams was reduced relative to the control animals (p = 0.0308) when the animals were sensitized to BSA at 8 weeks of age and measured at 9 weeks of age. The decrease in DTH response of male offspring of the atrazi ne treatment group persisted until at least 12 weeks of age. There was no difference in the DTH response betw een the offspring of

atrazine-treated dams and the c ontrol offspring in 6-month- old animals (data were not shown). There was no effect of atrazine treatment on NK cell activity. There was no effect of atrazine treatment on phago cytosis by peritoneal macrophages.

The authors concluded that atrazi ne treatment causes gender-dependent immunosuppression of cellular and humoral immune function, specifically suppression of primary antibody production and DTH responses. The authors also concluded that the immune changes associated with atra zine are not mediated through the suppression of PRL or THs. In addition, the authors concl uded that “the current data do not support nor completely refute a rela tionship between atrazine’s immunotoxic potential and its endocrine disrupting properties.”

Reviewer’s Comments: Rooney et al. (2003) provided some evidence that immune changes associated with atrazine during early development are not mediated through the suppression of prolactin or thyroid ho rmones. The precise underlying mechanism of an atrazine-mediated sex-specific effect, however, is not fully understood.

A.6.11 Rowe, A M, Brundage, K M, Sc hafer, R, and Barnett J B. 2006. Immunomodulatory effects of maternal atrazine exposure on male Balb/c mice. Toxicology a nd applied Pharmacology 21: 69 –

77.

The aim of this study was to test the hypot hesis that prenatal/lacta tional exposure to atrazine would affect the development of t he immune system and alter immune function in the adult offspring in the Balb/c mous e model. Pregnant Balb/c dams were exposed subcutaneously for 21 days via time releas e pellets to 700 ug atrazine/day beginning between days 10 and 12 of pregnancy. The im planted pellet was designed to release the same amount of atrazine every minute of every hour of every day until the end of the treatment period. The rate of exposure at the beginni ng of treatment was reported to be approximately 35 mg/kg/day, however, the daily dose dropped to approximately 23 mg/kg/day by the time the mice gave birth. The authors reported that the prenatal/lactational exposure “caused no overt physical malformations in the offspring

and had no effect on the number of litters ca rried to term or t he litter size.”Page 186 of 263 Immune status was evaluated at approximatel y 3 months of age. Body weight and spleen weights (absolute and relative) were monitored. T cell function was assessed by measuring proliferation and cytolytic activity after in vitro allogeneic stimulation. Allogeneic stimulation was assessed using two different assays. In one assay, splenocytes were isolated from unimmunized prenatally/lactationally exposed Balb/c mice and stimulated using irradiated spl enocytes from female C57B1/6 mice.

Proliferation of alloreactive ly stimulated lymphocytes wa s assessed by measuring the incorporation of 3 H-thymidine into newly synthesiz ed DNA following co-culture with

alloreactive stimulator cells and incubation with 3 H-thymidine. In another assay, the ability of the T cells to lyse alloreactive target cells ( i.e. , cytotoxic T lymphocyte response), was assessed following alloreactive stimulation. EL-4 lymphoma cells were used as targets. The humoral immune res ponse was assessed after immunization with heat killed Streptococcus pneumoniae (HKSP). The number of HKSP-specific B cells was quantified by the ELISpot assay. Spleen cell phenotype (specifically, CD4 + , CD8 +

, and B220 + ), was determined by flow cytometry.

There were no changes in body weights or sp leen weights. No changes were observed

in the number of CD4 + T cell, CD8 + T cell, and B220 + B cell subpopulations in the spleen. Male mice (3-month old) exhibited an increase in both T cell proliferation (atrazine offspring: 67 ± 5 x 10 3 cpm; control offspring: 46 ± 4 x 10 3 cpm) and cytolytic activity (approximately 66% increase). A si gnificant increase in the number of HKSPspecific IgM secreting B cells in the spl een of treated male mice was noted. Male atrazine-exposed offspring had 78.7 ± 4.5 H KSP-specific IgM antibody secreting cells (ASC) per 10 6 B220 + cells compared to 58.6 ± 3.0 ASC per 10 6 B220 + cells for male control offspring. Three-month-old female offspring showed no significant difference in immune response from controls. The authors concluded that the results indicate “a male-specific immunopotentiation of the imm une response following prenatal/lactational atrazine exposure.”

In a review article (Rowe et al. 2008; Basi c & Clinical Pharmacology & Toxicology, 102, 139-145), the study authors r eported on the results of similar assays performed on available littermates at 6 months of age (def ined as “long-term”). Limited data were presented. The authors noted t hat too few male offspring were available to perform replicate assessments. Howeve r, they stated that a si ngle iteration of the mixed lymphocyte response assay in the males show ed a significant decrease (this contrasts

with observations at 3 months of age). Female offspring were immunized with the

HKSP vaccine at 6 months and sera collected at 7 and 14 days after immunization. The authors reported that six-m onth-old atrazine-treated fe males had a significant and reproducible reduction in the number of phosphorylcholine (PC)-specific responding splenocytes following HKSP vaccination (PC- specific IgM-producing B cells in the spleen of HKSP-vaccinated atrazi ne-treated females were 69% of control). In addition, spleens from the atrazine-treated groups pr oduced a mean of 72% of control IgG PCspecific B cells per million splenocytes. The MLR response was also assessed in female offspring. Results indicated t hat atrazine-treated females had a significantPage 187 of 263 decrease in the MLR (spleen cell proliferat ion level was 72% of control and was statistically significant).

The authors also stated that in the 6-month-ol d females, the total number of splenocytes and the lymphocyte subpopulation phenotypes we re also unaffected. The mean spleen weight and total spleen cell number were s lightly higher in the treated groups. The percentage of spleen cell subtypes (CD4, CD8 , and B220) was not significantly different between the two groups. Based on the findings of the 3- and 6-month evaluations, the study authors concluded t hat the findings “demonstr ate a sex- and age-dependent effect of prenatal exposure to atrazine on the immune system of the adult offspring.”

Reviewer’s Comments: Rowe et al. (2006) reported imm unopotentiation of immune function in male Balb/c mice. However, these results conflicted with the results by Rooney et al. (2003) who reported immunosuppression of cellular and humoral immune function in male Sprague-Dawley rats. The r easons for the contradictory results are not known but may be related to differences in the timing and route of maternal exposure, species differences and different met hodologies used to assess immune response.

A.6.12. Rowe A M, Brundage K M and Barnett J B. 2007. In

vitro atrazine-exposure inhibits human natural killer cell lytic granule release. Toxicology and Applied Pharmacology 221: 179 – 188.

Earlier studies have revealed effects of atra zine on natural killer (NK) cells. In the

current in vitro study, Rowe et al. were interested in testing the hypothesis that atrazine exposure would decrease tar get cell adhesion and subsequent granule release in order to clarify precise changes in NK cell lytic function. Peripheral blood lymphocytes were isolated from healthy human male donors and expo sed to atrazine (0 , 10 or 30 μM) for 24 hours. The ability of NK cells to bind target cells (K562 human leukemia cells that are sensitive to lysis by human NK cells) as well as the ability of the NK cells to subsequently release lytic granule was assessed. Details on the methods used were provided in the study including: cell cu lture, peripheral bl ood lymphocyte harvest, cytotoxic lymphocyte assay, enrichment of NK cells, flow cytometry, NK cell adhesion, perforin ELISA and western blots.

Results showed that atrazine decreased NK cell function of PBLs under the in vitro experimental conditions, confirming observations by other investigator s, e.g., Whalen et al. 2003. The authors also reported that at razine did not affect viability or NK cell subpopulation distributions (dat a were not shown). For this experiment, PBLs were assessed for viability and subpopulation dist ribution via flow cyt ometry. Subpopulation

distribution was determined us ing four lymphocyte mark ers (CD56, CD16, CD3 and CD8), and viability was determined using 7-ADD exclusion.

The basal level of lytic proteins ( i.e. , perforin, granzyme A and B levels) was not changed following a 24-hour exposure to atrazine. Atrazine also did not affect the posttranslational modification of per forin. In addition, atrazine treatment did not affect thePage 188 of 263 ability of NK cells to bind target cells. Ho wever, atrazine treatment decreased the ability of NK cells to release granule proteins.

Reviewer’s Comments: Rowe et al. 2007 s howed that atrazine-i nduced inhibition of in

vitro human peripheral blood lymphocyte NK cell-s pecific lysis of K562 human leukemia cells may be the result of inhibition of lytic granule release, without affecting the ability of

the natural killer cell to form stabl e conjugates with target cells.

A.6.13 Schwab, C L, Fan R, et al. 2005. Modeling and predicting stress-induced imm unosuppression in mice using blood parameters. Toxicological Sciences 83: 101 – 113.

This study is an extension of the Pruett et al. (2003) study described above except that the blood rather than the spleen was of intere st. Utilizing the same approach as Pruett et al. (2003), Schwab et al. (2005) showed t hat the atrazine-induced decreases in CD4+

and CD8+ T cells in blood were simila r to exogenous corticosterone.

Reviewer’s Comments: This study provides further suppor t for the predictive value of

corticosterone for immune markers.

A.6.14 Whalen M M, Logana than B G, et al. 2003.

Immunomodulation of human natural killer cell cytotoxic function by triazine and carbamate pesticides. Chemico-Biological Interactions

145: 311 – 319.

The effects of in vitro atrazine treatment on the ability of human natural killer (NK) cells to lyse tumor cells were investigated. At razine was tested in both purified NK cells as well as a cell preparation that contained both T and NK lymphocytes (T/NK cells). Lymphocytes were exposed to atrazine (10 μM) for periods of time ranging from 1 hour to 6 days. Information on t he methods used (isolation of NK cells, cell treatments and cell viability, and chromium release assay for NK cytotoxicity) was presented in the study.

Exposure of highly purified NK cells to atra zine inhibited K562 tumor cell lysis by 63 ± 25 % after 24-hour exposure, and by 83 ± 21 % after a 6-day exposur e. Atrazine also caused significant decreases in lytic function when T/NK cells were exposed for 24- hours (61 ± 13%) and after 6-days (54 ± 12%).

Reviewer’s Comments: This in vitro study provides some evidence that atrazine may affect NK cell cytotoxic function; however, t he relevancy of these findings to in vivo exposure remains unclear.Page 189 of 263 A.7 Carcinogenesis/Genotoxicity

A.7.1 Fujimoto, N and Honda, H. (2003). Effects of Environmental Estrogenic Compounds on Growth of a Transplanted Estrogen Responsive Pituitary Tumor Cell Li ne in Rats. Food Chem. Toxicol.

41:1711-1717. The objective of this study was to evaluate the role genistein, p-nony lphenol, or atrazine as tumorigenic promoters for estrogen-sensitive tumors. A rat pituitary cell-line – MtT/E2 – that is responsive to estrogen exposure was transplanted into ovariectomized rats. The rats were subsequently exposed to estr adiol, genistein, p-nonylphenol, or atrazine and examined twice a week for the development of tumors. 17 In addition, MtT/E-2 cell cultures were transfected with an estrogen-re sponse-element-luciferase reporter gene and evaluated for activation of the reporte r gene. These cells were also grown in vitro and evaluated for cell proliferat ion after exposure to the c hemicals under evaluation. Finally serum collected at the time of sacrifice was used to evaluate prolactin levels.

Six week old ovariectomized F3 44 rats (sample size not r eported) were inoculated with MtT/E-2 tumors cells and subsequently expos ed through the diet to 0, 5, 50, or 500 mg/kg atrazine (purity not reported) for 33 days. Animals were examined for tumor development twice per week and sacrificed when the tumor size reached 1-2 cm in diameter. At necropsy, t he pituitary gland and uterus were collected and weighed and blood serum obtained to measure for prol actin levels. For cell proliferation assessments, MtT/E-2 cell cultur es were exposed to 0 or10 -7 -10 -4 M atrazine for 7 days and cell proliferation measur ed using the WST-1 assay (reduction WST-1 into formazan dye by viable cells). Activation of the ERE-luc reporter gene was evaluated by measuring luciferase activity in MtT/E-2 ce lls transfected with the ERE-luc reporter gene and subsequently exposed to 0 or 10 -7 -10 -4 M atrazine for 24 hours.

Atrazine exposure did not result in MtT/E-2 cell pr oliferation according to the WST-1 assay nor did it have an estrogenic response as evidenced by a lack of response in the ERE reporter gene assay. After 33 days of exposure, atrazine did not cause any changes in body, uterine, or pituitary weight s. Serum prolactin levels were also unaffected by exposure to up to 500 mg/kg atra zine. In contrast, exposure to a 0.5 mg subcutaneous pellet of estradiol resulted in a marked decrease in body weight, and an increase in uterine and pi tuitary weights along with a marked increase in serum prolactin ( ↑ 7-fold, p<0.01). Tumor development wa s also unaffected by treatment with

atrazine with evidence of tumor devel opment first appearing on day 24 of the experiment for animals in the control or atrazine treated groups as opposed to 9 days

for animals treated with estradiol.

Reviewer’s Comments: The current study had a number of deficiencies including the lack of data on the purity of the atrazine us ed in the study or the sample size. Nonetheless, some general conclusions may be drawn from this study. In particular,

17 In this evaluation only the resu lts for atrazine will be discussedPage 190 of 263 that atrazine exposure up to 500 mg/kg did not enhance the appearance of estrogendependent pituitary tumors nor does it resu lt in increased cell proliferation. When considered collectively, t he data reported by Fujimoto, Son, Tanaka and co-workers demonstrate that atrazine exposure did not accelerate tumor progression.

A.7.2 Fukamachi, K., Han, B.S., Kim, C.K., Takasuka, N., Matsuoka, Y., Matsuda, E., Yamasaki, T., and Tsuda, H. (2004). Possible enhancing effects of atrazi ne and nonylphenol on 7,2- dimethylbenz[a]anthracene-induced mammary tumor development in human c-Ha-ras proto-oncogene transgenic rats. Cancer Sci. 95(5):404-410.

This study evaluates the potential of atra zine or nonylphenol to promote growth of mammary tumors induced by 7,2-dimethylbenz [a]anthracene (DMBA) in transgenic (Tg) rats carrying the human c-HA-ra s proto-oncogene. These rats are exquisitely sensitive to DMBA-induced mammary tumors. 18 Two-hundred seventy six female and male Tg and non-Tg rats were administered a single 25 mg/kg dose of DMBA via gavage at an age of 50 days. Beginning on the following day, animals were exposed in the diet to 0, 5, 50, or 500 ppm atrazine (purity unknown) [ ≈ 0, 0.55, 6.2, or 36.4 mg/kg/day ( ♀ ) and 0, 0.26, 2.75, or 24.23 mg/kg/day ( ♂ )]. The Tg females were exposed to atrazine for 8 weeks while the non-Tg females and all males were exposed to atrazine for 20 weeks. At the end of the exposure period, animals were sacrificed by exsanguination, the numbers of grossly observed tumors were recorded and subsequently measured and subjected to histopathological examination. In addition, body, liver, kidney, ovarian, uterine, and testis weights were recorded. Levels of serum atrazine were measured after necropsy by liquid chromatography and mass spectrometry (LC/MS). The potential impact of atrazine on cell proliferat ion was also measured by exposing the human mammary cancer cell line MCF-7 to atra zine for 3 days. Ce ll proliferation was then measured using the WST-1 assay (measures viable cells capability to reduce WST-1 into formazan dye).

Tg females exposed to 500 ppm atrazine exhibi ted a statistically significant decrease in body weight ( ↓ 14%, p<0.01) with a concomitant decrease in food consumption ( ↓ 10%, p< 0.05). A statistically si gnificant increase in the in cidence of mammary adenomas ( ↑ 67%, p<0.01 vs 17% control) was noted following exposu re of Tg female rats to 5 ppm atrazine. However, no increase was obs erved after exposure to 50 or 500 ppm atrazine. The incidence of mammary adenoc arcinomas at 5 and 50 ppm atrazine was 100% (control: 50%) although the in cidence at 500 ppm was lower ( ≈ 86%). In contrast to the apparent increase in mammary tumors in Tg females, Tg males exposed to 500 ppm atrazine had a decrease in the incidence of skin tumors (40% vs. 67% control) although a non-statistically significant increase in the incidence of mammary adenocarcinomas (100% vs 56% control) was also reported at this dose level. No data were provided on non-Tg rats (males or females).

18 In this evaluation only the resu lts for atrazine will be discussedPage 191 of 263 The study authors concluded that atra zine appeared to enhance mammary tumors in female Tg rats but without any dose relations hip while decreasing the incidence of skin tumors in Tg males. Furthermore, the author s concluded that atrazine did not increase cell proliferation of MCF-7 cells . Several limitations were i dentified in this study that hinders a meaningful interpretation of the resu lts. As an example, Tg female rats received atrazine for 8 weeks, the remainder of the animals in the study received atrazine for a considerably longer period ( 20 weeks). In addition , the purity of the atrazine used in the study was not reported, no exposure concentrations were provided for the MCF-7 experiment, and no data were pr esented for the non-Tg rats or for the MCF-7 cell proliferation assay.

Reviewer’s Comments: The lack of data on non-Tg rats is a critical deficiency in this study. In experiments conducted by Ueda et al. using non-Tg rats treated with DMBA to induce mammary tumors, a relationship betw een atrazine exposure and increases in the incidence of tumor-bearing rats cannot be drawn. Thus, in t he current study the

different treatment and lack of data on the non-Tg rats precludes reviewers from drawing a conclusion on whether the in creases seen in mammary adenocarcinomas were related to atrazine exposure. The lack of dose-response on the incidence of mammary adenomas and adenocarcinomas further confounds the interpretation of

these data.

A.7.3 Garaj-Vrhovac, V. and Zeije zic, D. (2004) Genotoxicity evaluation of pesticide formulations containing alachlor and atrazine in multiple mouse tissues (blood, kidney, liver, bone marrow, spleen) by comet assay. Ne oplasma 51.3:198-203.

In this study the authors evaluated possibl e genome damage caused by two pesticide formulations (Bravo® and Gesaprim®) cont aining alachlor and atrazine as active ingredients by applying the comet assay on mouse cells from blood, kidney, liver, bone marrow, and spleen. 1

The focus was intended to uncover the possi ble genotoxicity of pesticide formulations and not only their active ingredients. Work ers who handle these products during their production and application stages are exposed to the complex mixture contained in the formulation versus just the isolated active ingredients – which in turn could give results that don’t correspond to the actual conditions.

Gesaprim®, containing 500 mg/ml of atrazine as the active ingredient were given at the doses of 1.08 ml/kg (540 mg/kg a.i.) and 0.07 μl/kg (3.5 X 10 -2 mg/kg a.i.) to five male CBA mice assigned to 4 treatment groups and a control group injected intraperitoneally once. The lower dose represents the or al chronic RfD and the higher dose administered is compatible with that dose which shows survival in mice in vivo . Methyl methanesulfonate (MMS) at 70 mg/kg was used as a positive control. The mice were sacrificed 24 hours after treatment; blood (t aken into heparinized vacutainers) andPage 192 of 263 selected organs were weighed, minced, and suspended in a chilled homogenization

buffer.

The alkaline comet assay of blood samp les was performed according to standard protocol, while organ samples were subjected to a modified assay (S asaki, YF, 1997). Each organ and pesticide concentration, 100 cells were analyzed by the Comet assay II automatic digital analysis system to det ermine tail length and tail moment. Any differences of assay endpoints between control blood and organs and those from

treated mice were evaluated us ing the Mann-Whitney U-test.

Results showed no death, morbidity, or distinctive clinical signs observed after any treatment (including during nec ropsy). Mean values of t he comet assay endpoints were found to be statistically significant (p<0.01) regarding an increase of tail length for all 5 tissues examined in mice in low (0.07 μl/k g) and high (1.08 ml/kg) treatment groups compared to control. The DNA of the ki dney and liver, for both concentrations given, showed the largest migration. There was also a significant increase for all tissues in the higher concentration treated gr oup when compared to the lowe r concentration group. For mice given the lower concentration, the tail length values of the kidney and liver were significantly higher than those of blood, bone marrow, and spleen from the same group but non significant difference was found between the liver and kidney; while in the higher dose group the kidney and liver tail le ngths were significantly higher but no

statistical difference was found. Tail lengths in the kidney DNA from mice treated with 1.08 ml/kg Gesaprim® were up to 99 μm versus 84 μm for mice in the 0.07 μl/kg group (data not shown). For the liver, the tail l engths at the higher dose were 97 μm whereas at the lower dose it was 39 μm, much lower when compared to the kidney.

The authors have concluded that regardless of the dose, genome dam age in all tissues

examined was detectable with the comet assa y. The most affected organs were the kidney and liver. This paper also cites severa l studies which show contradictory results

both in vitro and in vivo . This study also gave descriptions of studies on human lymphocytes showing no appearance of genotox icity and provided a rationale as to why the toxic activity maybe slightly increased at higher doses when co mpared to the RfD, could be due to the removal of apoptotic cells (data not shown). Other apoptotic detection methods should be employed befo re presenting those results.

Reviewer’s Comments: Significant (p<0.01) increases in the tail length and tail moment

were recorded for all tissues at both dos es of atrazine. However, there are uncertainties regarding the atrazine findings. For example, the authors stated, “genome damage for both pesticides was found to be higher for the higher dose given”. It was noted, however, that the “dose response” is very flat considering that more than two orders of magnitude separate t he atrazine doses that were tested. Nevertheless, the relevance of the DNA damage data for atra zine is uncertain because neither the species (rat) nor the target ti ssue for tumors (mammary tumors ) were examined. Overall, the use of the alka line comet assay on different tissue samples could provide evidence of genotoxic effects of pesticides in vivo . Garaj-Vrhovac et al. and ZeljezicPage 193 of 263 and Garaj-Vrhovac (2004) concluded that Ges aprim (an atrazine formulation) induced genome damage. The limitation of these studies is that a formulation of atrazine was used and no information is provided to deter mine the percentage of atrazine present in the test material. Thus, it is entirely possible that the DNA breaks reported after Gesaprim exposure may be related to other chemicals in the formulation rather than

atrazine per se

.

A.7.4 Kmetic, I., Srcek, G., Slivac, I., Simi c, B., Knle wald, Z., Kniewald, J. 2008. Atrazine Expos ure Decreases Cell Proliferation in Chinese Hamster Ovary (CHO-K1) Ce ll Line. Bull Environ Contam

Toxicol. 81: 205-209.

The purpose of this study was to determine th e toxic effect of atrazine at the ovarian

cellular level. The Chinese Hamster Ovary (CHO-K1) cell line was used as the test system for evaluation of in vitro atrazine cytotoxicity and the morphological changes were followed during cell death.

CHO-K1 cells were treated with atrazine at concentrations ranging from 10-160 μ g atrazine/mL of cell suspension (46-742 μ M) for 24, 48 or 72 hours. The cell viability was determined by four different bioassays: Tr ypan blue exclusion method, Kenacid Blue, Neutral red and MTT assays. Trypan blue excl usion method was used to estimate the proportion of viable cells. The Kenacid bl ue assay determines the total cell protein content. The Neutral red uptake assay dete rmines the accumulation of the neutral red dye in the lysosomes of viable uninjured ce lls. The MTT assay determines the viability

of cells.

All four methods showed clear dose-dependent cytotoxic effe cts. Concentrations of atrazine 20-80 ug/mL follow ed by Trypan blue method sh owed significantly decreased proliferation of cells in CHO-K1 cells li ne at all dose levels, with the toxicity progressively increased with high atrazi ne concentrations. After 72 hours incubation period, the 20 ug/mL atrazine concentration si gnificantly decreased cell proliferation by 19% in CHO-K1. Cell viability was decreas ed 49% at 60 ug atrazine/mL and 55% at 80 ug atrazine/mL, both at p<0.001.

IC 20 and IC 50 values were determined after 24, 48 and 72 hours for the four methods. A comparison of the IC 20 values after 48 hours or IC 20 and IC 50 after 72 hours of incubation showed that Neutral red, Kenac id blue and MTT assays showed a similar rate of toxicity. The Trypan blue assay was more sensitive. The IC 50 value for trypan

blue after 72 hours was two times lower than the IC 50 s for the other 3 assays. Morphologically, the level of apoptosis in atrazine treated cells was low.

Reviewer’s Comments: Cell proliferation was decreased in Chinese Hamster ovary (CHO) cell lines at relatively lo w levels of atrazine exposure.Page 194 of 263 A.7.5 Malik, S. I., Terzoudi, G.I., a nd Pantelias, G.E. (2004). SCE analysis in G2 lymphocytes prematurely condensed chromosomes after exposure to atrazine: the non-dose-dependent increase in homologous recombinational events does not support its genotoxic mode of action. Cytogenet Genome Res 104: 315-319.

Human peripheral lymphocytes, collected from seven healthy volunteers, were grown, stimulated with phytohemagglutinin and expos ed to atrazine ( unreported purity) concentrations ranging from 5 to 220 μ g/mL atrazine, the solvent (dimethyl sulfoxide ) or the positive control (mitomycin C) for 24 hours. Three separate experiments were

conducted. Prior to the end of treatmen t, calyculin-A was added to the appropriate cultures for the last 1 hour of incubation (to arrest cells at G2 ) or colcemid for the last 3 hours [to arrest cells at metaphase (M-phase)]. The rationale for using cell arrest at G2

rather than the conventional approach of colcemid treatment to collect cells at metaphase was to determine whether atra zine would increase the frequency of homologous recombination, a DNA repair proc ess associated with SCE induction. Cells were collected, stained by the fluorescent -plus-Giemsa technique and 30-50 cells were scored for each culture; triplicate cult ures were prepared per treatment group.

Results from the first experim ent showed that concentrations ≥ 220 μ g/mL atrazine could be scored for SCE induction in G2-phase cells while no metaphases were found at 120

and 220 μ g/mL in the colcemid-tr eated cultures. Thus, SCEs could be scored even at

levels that were lethal in the conventiona l assay and there was no significant increase in

SCE induction at 60-220 μ g/mL. Results for the second experiment were in good agreement with the first trial and indicated that atrazine over a concentration range of 5

to 200 μ g/mL did not increase the frequency of SCE induction in G2-prematurely condensed chromosomes (PCCs). Using cells treated with 20 μ g/mL and arrested with either colcemid or calyculin-A, trial three results indicated that the variation in SCE frequencies in lymphocyte cultures is signifi cantly lower in G2-PCCs than in SCEs scored in the metaphase cells.

Reviewer’s Comment: The assay was well- conducted and indicate s that there was convincing evidence that atrazine is not genotoxic in this test system.

A.7.6 Manske, M., Beltz, L, a nd Dhanwada, K. 2004. Low-Level Atrazine Exposure Decreases Ce ll Proliferation in Human

Fibroblasts. Arch. Environ. Contam. Toxicol. 46:438-444.

This purpose of this study was to examine t he effect of low-level, short-term atrazine exposures on human fibroblast cells in cult ure by assessing cell proliferation and apoptosis.

Normal human fibroblasts, DET 551, were us ed in the saturation density analysis to measure cell proliferation. In this a ssay, DET 551 cells were plated at 2x10 5 cells/100

mm dish in MEM with 0.5% F BS (low serum medium). Cells were incubated at 37oCPage 195 of 263 with 5% CO 2 for 48 hours in the low serum medium to synchronize cells. After 48 hour the medium was removed and replaced with complete medium with 10% FBS containing increasing concentrations of atrazi ne or DMSO (0, 3, 10, 50, 100, 200, and

300 μ g/L). Atrazine dilutions were made from an atrazine stock concentration of 32 mg/L dissolved in DMSO. Cells were grown for 5 days at 37oC and 5% CO 2. After harvesting, individual cells we re counted by the trypan blue exclusion method. Viability of cells in all treatment groups and controls was over 99%. The actual number of cells treated with increasing concentrations of atra zine or solvent control was compared to

the number of control cells. Cells were c ounted using a hemocytometer. All experiments were run in triplicate.

An MTT assay was run to confirm the results for cell proliferation. This method does not rely on individual cells being counted. It is a calorimetric assay. DET 551 cells were plated in flat-bottom 96-well tissue culture trea ted plates at 2000 cells/well in low serum

medium and incubated for 48 hours. After 48 hours, the medium was removed and replaced with complete medium with 10% FBS and increasing concentrations of atrazine or DMSO (0, 3, 10, 50, 100 and 200 μ g/L) for an additional 48 or 72 hours . The

atrazine stock concentration was the same as before, 32 mg/L in DMSO. For MTT analysis, 20 μ l of 5 mg/ml MTT in PBS was added to cells and plates were incubated for 4 hours at 37oC. HCl in isopropanol was ad ded to each well, and optical density was read on a UV/VIS spectrophotometer. A similar procedure as followed for human peripheral blood mononuclear cells ( PBMCs), composed of lymphocytes and monocytes. One million PBMCs were plated in 24 well tissue culture plates in PRMI with 5% FMS containing increasing concentrati ons of atrazine or DM SO (40, 100 or 200

μ g/ml). Cells were incubated for 96 hours at 37oC and 5% CO 2. Then 150 μ l of MTT

solution was added and cells were incubated for an additional 4 hours at 37oC. HCl in isopropanol was added to each well, and optic al density was read at 570 nm. The increase in OD had a positive correlation with viable cell number and indicated enhanced cell growth compared to control cells. All experiments were run in triplicate.

To determine if the decrease in cell number was due to apoptosis of the treated cells, two assays were done—a qualitative immuno histochemical analysis and a quantitative enzymatic analysis (caspase assay). In t he qualitative immunohist ochemical analysis, apoptotic cells are detected with a biotin-streptavidin system complexed with horseradish peroxidase and a diaminobenzid ine-based stain. DET 551 cells were exposed to atrazine or DMSO at concentrations of 0, 3, 50 or 200 μ g/L for 48 hours before the assay was perform ed. Cells treated with TACS nuclease was the positive control. The Caspase assay was used to measure apoptosis enzymatically. Caspase-3 activity in cell extracts was measured us ing the CaspASE colorimetric assay system protocol. The CaspASE assay is a colorimetric assay that measure activity of caspase3. Cytosolic thiol proteases called caspases are key enzymes involved in apoptosis. DET 551 cells were exposed to increasing concentrations of atrazine or DMSO (0, 3, 100, 200, and 500 μ g/L) for 48 hours before analysis.Page 196 of 263 Cell Proliferation Results : The saturation density analysis showed that DET 551 fibroblasts exposed to increasing concentrati ons of atrazine showed a decrease in cell growth compared to control cell s. Cells exposed to 50, 200 and 300 μ g/L atrazine had a statistically significant decrease (1.2-1.5 fo ld) in cell number compared to control cells (p<0.05). The authors also noted a decrease (not statistically significant) in cell growth in the highest concentration of DMSO (300 μ g/L) compared to the other DMSO control

values. This could indicate that this conc entration of DMSO may be slightly toxic by itself since the percentage of DMSO in these cells is higher than in the other samples and these cells were exposed to the chemical for 5 days, 2 days longer than in the MTT analysis.

In the MTT analysis, a 48 hour exposure to at razine showed a statistically significant decrease (p<0.05) in DET 551 cell pro liferation. Cells exposed to 10-200 μ g/L had a dose related significantly decreased absorbanc e (1.1-1.7 fold) compared to controls, indicating a decrease in cell proliferation. There was no statistically significant decrease in proliferation in solvent-c ontrol-treated cells. There was no effect on cell proliferation by MTT analysis when human PBMCs were tr eated with increasing concentrations of

atrazine for 96 hours.

Apoptosis Results : In the immunohistochemical analysis, DET 551 cells were exposed to 0, 3, 50, or 200 μ g/L atrazine or DMSO for 48 hours before the assay was performed. The atrazine and DMSO-treated samples we re compared qualitatively to a TACs nuclease-positive control. When the cells treated with 200 μ g/L atrazine or DMSO were compared to control cells, there was only a sight increase in nuclear staining of atrazine-treated cells, however, the level of apoptosis in treated–treated cells was low compared to the positive cont rol. The caspase assay was then done to determine if the small increase in the apoptotic cells was significant. The results showed that all atrazine-treated samples (3-500 μ g/L) had caspase activity (7.31-9.53 pmol/ μ l) similar to the negative control (7.31 pmol/ μ l). No significant differences were detected between the control treatments (DMSO) and atrazine-treated cells.

Conclusion: In vitro exposure to low levels of atrazine (as low as10 μ g/L) for a short exposure (48 hours) causes a decrease in cell proliferation in hum an fibroblasts but not in PBMCs. This study showed that the decrease in cell proliferation of human fibroblasts was not attributed to an increase in apoptosis.

Reviewer’s Comments: In vitro data evaluating the cell pro liferative properties in a variety of cell lines suggest indicate that atrazi ne causes decreases in cell proliferation. In general, these data suggest that atrazine is not genotoxic or mutagenic. Manske et al. 2004 showed that human fibroblast cell prolif eration was decreased at relatively low levels of atrazine exposure.Page 197 of 263 A.7.7 Mastrangelo, S., Tomassetti, M, Carratu, M.R., Evandri, M.G., and Bolle, P. (2006). Quercetin reduces chromosome aberrations induced by atrazine in the Al lium cepa test. Environ and Mol Mutagen 47:254-259.

In this study, groups of six peel ed, white commercial bulbs of Allium cepa, also known as the “garden onion”, were placed in gl ass tubes containing mineral water and the flavonoid, quercetin (Q), which is thought to have antioxidant proper ties, was tested at

0.1 to 20 μ g/mL Q. Bulbs were also exposed to atrazine (unreported purity) at 0, 2.5,

5.0 or 7.5 μ g/ml and varying combinations of Q and atrazine (0.5 or 5 μ g/mL Q with 2.5,

5.0 or 7.5 μ g/mL atrazine), and incubated in the dark for 5 days. Test solutions were renewed daily and “topped-up” on days 3, 4, and 5. Ten replicate bulbs per each treatment group were prepared. Bulbs were observed da ily throughout the 5-day exposure period for root length and form and the presence of “crochet hooks” and

tumors. At the end of treatm ent, the meristematic cell ( analogous to stem cells in mammalian tissue) region from the root tips was harvested and processed for cytogenetic evaluation. One t housand cells were counted to determine the mitotic index (MI) and a total of 400 metaphases and 400 ana-/telophases (40 metaphases /slide and 40 ana-/telophases/slide) were scored for each treatment group.

No effects were observed on root morphology afte r exposure to Q or atrazine. Similarly, Q alone did not affect the MI at any concent ration. By contrast, atrazine alone caused an 18% reduction in the MIs at the highest level tested (7.5 μ g/mL); no cytotoxic effects

were noted at lower doses. Combined with either 0.5 or 5 μ g/mL Q, 7.5 μ g/mL atrazine affected a slightly lower reduction in the MI (~23% ↓ for both Q concentrations) then when the pesticide was tested alone. Neither Q nor 2.5 or 5.0 μ g/mL atrazine were clastogenic; however, 7.5 μ g/mL atrazine induced a signif icant (p<0.001) increase in

structural chromosome aberration. T he most prominent type of aberration was fragments. When the plant ce lls were assayed with 0.5 μ g/mL Q in combination with

7.5 μ g/mL atrazine, the frequency of chromosome aberrations was listed as significantly (p<0.05) lower (~43% ↓ ) compared to 7.5 μ g/mL atrazine alone. However, no increased protection was afforded by the higher concentration of Q.

Reviewer’s Comments: The study was well- conducted and these data provide evidence of a clastogenic response induced by atrazine at 7.5 μ g/mL atrazine, a concentration resulting in adequate cytotoxicity (~ 20% ↓ in the MI). The data also suggest that the clastogenic activity is likely to be c onfined to a narrow range of doses because

concentrations >7.5 μ g/mL atrazi ne would likely exceed the recommended level of cytotoxic and no cytotoxicity or clastogenicity was noted at ≤ 5.0 μ g/mL.Page 198 of 263 A.7.8 Singh, M., Sandhir, R., and Kiran, R. 2008. Atrazine-Induced Alterations in Rat Erythrocyte Me mbranes: Ameliorating Effect of

Vitamin E. J. Biochem. Molecular Toxicology. Vol 22, No. 5: pgs. 363-

369.

This study evaluated the possible beneficia l effects of vitamin E against atrazineinduced oxidative stress in erythrocytes. Erythrocytes are susc eptible to oxidative damage and a good model to understand oxi dative damage induced by xenobiotics.

Vitamin E is an antioxidant.

Male Wistar rats (n=18 ani mals/group) were administer ed atrazine (300 mg/kg/day, 97.83% purity), Vitamin E (100 mg/k g/day), and combined atrazine (300 mg/kg/day)/Vitamin E (100 mg/kg/day) for a period of 7, 14, and 21 days. A vehicle control group was similarly treated. Blood sa mples were collected at the end of the treatment periods. Erythrocyte memb ranes were prepared and analyzed for acetylcholinesterase (AChE) activity, lipid peroxidation (LPO), and lipid composition (total lipid content, phospholipid phosphorus, cholesterol). Protein content was also determined. Scanning electron microscopy ( SEM) was used to examine morphological alterations of erythrocytes.

Atrazine treatment resulted in significant (p <0.05) decreases in AChE activity in erythrocytes at 7 days (22. 32%), 14 days (25.58%), and 21 days (26.96%), compared to

controls. Atrazine/Vitamin E treatment resu lted in significant increases (12-14%) in AChE activity at 7, 14, and 21 days compared to atrazine alone treated animals.

Atrazine treatment resulted in a significant increase (p<0.05) in lipid peroxidation (nmol MDA/mg protein) compared to controls, wit h the effects being mo re pronounced with increasing duration. After 21 days treatm ent there was a 218.29% increase in MDA levels. The increase in LPO suggests that oxidative stress is induced by atrazine. Vitamin E treatment alone significantly (p <0.05) decreased MDA levels compared to controls. Atrazine/Vitamin E treatment signific antly (p<0.05) decreased lipid peroxidation when compared to animals tr eated with atrazine alone.

Atrazine treatment resulted in a significant (p<0.05) decrease (24%) in the total lipid content of erythrocyte membr anes only after 21 days, compared to controls. Vitamin E along with atrazine treatment significantly in creased total lipid content to normal levels after 21 days, as compared to the at razine treated group. Atrazine treatment significantly (p<0.05) decreased cholestero l content after 14 days (20.12%) and 21 days (32.94%). Atrazine/Vitamin E treatment significantly increased cholesterol content after 14 days (33.86%) and 21 days (57.02%), compar ed to the atrazine treated group. Atrazine treatment resulted in a significant decrease in phospholip id content after 14 days (16.76%) and 21 days (37.20%), compared to controls. Atrazine/Vitamin E treatment significantly in creased phospholipid content a fter 14 days (22.86%) and 21 days (57.92%), compared to atrazine treated group.Page 199 of 263 Erythrocytes of rats treat ed with atrazine showed significant morphological changes as compared to the control group. These changes included mild to moderate distortion in shape, significant ruptured me mbranes, and echinocyte formation, central or peripheral protuberances. SEM of erythrocytes of atrazine/vitamin E group shows improved erythrocyte topography as compared to atrazi ne-treated group. Extent of protuberance significantly decreased as co mpared to the control group.

Reviewer’s Comments: The results of this study are questionable give n that the atrazine doses used to induce these genotoxic effects are orders of magnitude higher than what is currently used as the point of depar ture for human health risk assessment.

A.7.9 Singh, M., Kaur, P., Sandhir, R., Kiran, R. (2008). Protective effect of vitamin E against atrazi ne-induced genotoxicity in rats.

Mutat Res 654:145-149.

The protective effect of an antioxidant, vita min E, against atrazine-induced genotoxicity was investigated in young (~4 -5 weeks old, based on the weight range of 100-120 g) 19a

Wistar male rats dosed with vitamin E alon e, atrazine (97.83%) al one or a combination

of atrazine and vitamin E in the comet and micr onucleus assays. Accordingly, groups of six male rats were administered oral gavage doses of corn oil, 100 mg/kg/day vitamin E, 300 mg/kg/day atrazine or a combination of comparable doses of vi tamin E and atrazine for 7, 14 or 21 days. At the end of treatm ent, peripheral blood and liver were collected simultaneously from each test animal, processed and assayed for micronucleus induction and/or DNA damage (gel elec trophoresis and Comet assay).

Results from the gel electrophoresis on DNA is olated from the liver show that atrazine alone induced DNA damage as indicated by “shearing of the DNA” at 7, 14 and 21 days; the maximum effect was observed at 14 days. The invest igators stated that

atrazine did not induce the classic DNA ladd er pattern, which is associated with apoptosis but shearing of DNA suggested DNA damage to the atrazi ne-treated livers. Similarly, significant (p<0.001) increases in the DNA tail length and tail moment were obtained from the comet assay performed on both blood and liver sampled at all harvest times. The response was generally ti me-related and in good agreement with the electrophoresis data showing a marked incr ease in DNA damage at 14 and 21 days. By contrast, vitamin E alone was not genotoxic in either tissue. However, significantly (p<0.001) reduced (23-56% ↓ ) DNA tail lengths liver and blood samples were obtained for the tissues treated with atrazine plus vita min E, compared to atrazine treatment alone. In agreement with thes e data, the results of the liver cell micronucleus assay demonstrated that atrazine alone increased (p<0 .001) the frequency of micronuclei at all sample times and the response was time-related with the largest increase occurring at 21 days. The treatment of the rats with atrazine (300 mg/kg/day) plus vitamin E (100

19 According to personal communications with Charles Parady, Senior Specialist, Charles River, male Wistar rats in the weight range of 100-120 gm would be approximately 4-5 weeks old.Page 200 of 263 mg/kg) caused significant (p<0.001) reduc tions in the frequency of micronuclei at 7 (23% ↓ ) and 21 (14% ↓ ) days as compared to at razine-treated rats.

Reviewer’s Comments: The results of this study are questionable gi ven that the atrazine doses used to induce these genotoxic effect s are orders of magnit ude higher than what is currently used as the point of depar ture for human health risk assessment.

A.7.10 Son, H-Y., Nishikawa, A., Okazaki, K., Lee, K-Y., Imazawa, T., and Hirose, M. (2003) . Lack of Modifying Effects of

Atrazine and/or Tamoxifen on Th yroid Carcinogenesis in Rats Pretreated with N-bis(2-hydroxyp ropyl)nitrosamine (DHNP). Food

Chem. Toxicol. 41:1811-1816.

In this study the researchers investigated the effect of atrazine and/ or tamoxifen on the progression of DHPN-induced thyroid carcinogenesis. 20 The goal of the study was to assess the impact of atrazine (with or without concomitant tamoxifen exposure) on thyroid hormone changes and thyroid lesions induced by DHPN.

Twenty male F344 rats per group received a subcutaneous injection of 2800 mg/kg DHPN or saline. Starting one we ek later, the animals were exposed to 0, 5, 50, or 500 ppm atrazine (purity unknown) through the diet for 24 weeks. Rats receiving the saline injections were exposed to 0 or 500 ppm at razine. At the end of the exposure, animals were sacrificed and the thyroid, pituitar y, liver, lungs, kidney, pancreas, testis, epididymis, and brain were collected, wei ghed and subjected to histopathological examination. In addition, se rum from 5 rats/group (except the 5 and 50 ppm atrazine groups) was collected and used to measure thyroid hormones (T 3 , T 4 , and TSH).

Exposure to 500 ppm atrazine resulted in a decrease in body weight ( ↓ 12-14%, p<0.05) regardless of DHPN exposure. The relative brain, liver, testicular, and epididymal weights from rats receiv ing both DHPN and 500 ppm atra zine were increased by ≈ 9- 13% (p<0.05) when compared to animals rece iving DHPN only. Moreover, when these rats were compared to the control group ( no DHPN or atrazine exposure), the changes in body weight and relative organ weights were similar. Comparison of rats exposed to 500 ppm only (no DHPN injection) revealed that these animal s experienced a 14% decrease in body weight (p<0.05) along wit h an 11-12% increase in relative brain, testicular, an epididymal weight. However, unlike pre-exposure to DHPN followed by 500 ppm atrazine, exposure to 500 ppm atrazi ne alone did not elicit an increase in the relative liver weight. Histopathology asse ssments did not reveal any compound-related changes though the data were not provided in the study report. Hormone analysis revealed that while there is a statistically significant decrease in T 4 levels ( ↓ 16%, p<0.05) after exposure to DHPN + 500 ppm at razine relative to exposure to DHPN alone and no changes in T 3 or TSH. When compared to the control group, the

decrease in T 4 is ≈ 10% and non-statistically significan t. Furthermore, exposure to 500

20 In this evaluation only the resu lts for atrazine will be discussedPage 201 of 263 ppm atrazine alone did not elicit changes in any of the thyroid hormones while exposure to DHPN by itself resulted in a 7% increase in T 4 . This suggests that the apparent decrease in the hormone after exposure to DHPN + atrazine may be related to the increase above background caused by DHPN and not indicative of an effect of atrazine on thyroid hormone homeostasis.

Reviewer’s Comments: The results of this st udy indicate that atrazine does not have an impact on thyroid carcinogenesis or thyroid hormone homeostasis. With the exception of a decrease in body weight, atrazine di d not appear to have any effects on the parameters evaluated in this study. No hi stopathological changes were reported after exposure to 500 ppm atrazine and the increases in relative organ weights observed are most likely related to this decrease in body weight rather than a direct effect on the organs. Similarly, thyroid hormone assessm ents did not suggest a direct effect of atrazine on thyroid hormone homeostasis. It should be noted, however, that this report does not include information on the purity of t he atrazine used in the study and that the histopathological data are not provided.

A.7.11 Tanaka, T., Kohno, H., Suzu ki, R., Sugie, S. (2004) Lack of modifying effects of an est rogenic compound atrazine on 7,12- dimethylbenz(a)anthracene-induced ovarian carcinogenesis in rats.

Cancer Lett. 210:129-137.

The effect of atrazine on DM BA-induced ovarian carcinogenesis was investigated in this study. The authors hypothesis is that the op erative mode of action leading to ovarian adenocarcinomas involves hormonal disruption (particularly estrogen). Therefore, exposure to atrazine (which has been associ ated with hormone disruption) may affect ovarian carcinogenesis.

In this series of experiments, the res earchers exposed 12-20 female Sprague-Dawley rats to 0.5% DMBA (7,12-dimethylbenz(a)ant hracene) injected directly into the left ovaries. One week after exposure to DMBA, ani mals were fed diets containing 0, 5, 50, or 500 ppm atrazine (purity unknown) for 50 weeks. Another group did not receive a DMBA injection but were exposed to 0 or 500 ppm atrazine through the diet. On week 51, the animals were sacrificed, necropsie d and tissues with gross lesions, as well as, the liver, kidney, mammary gland, lung, and heart subjected to histopathological evaluation. In addition, immunohistochemistry evaluations for prolif erating cell nuclear antigen (PCNA), expression of t he androgen or estrogen receptors ( α and β ) and the BRCA-1 gene were conducted.

Neither DMBA, nor atrazine or DMBA + at razine exposure led to any changes in ovarian, uterine, or body weight. DMBA expo sure followed by dietary exposure to 500 ppm atrazine exposure led to a 10% statisti cally significant increase in absolute liver weight, consistent with what has been report ed by other investigat ors. DMBA exposure
led to a 45% incidence in ovarian adenocarcinomas vs 0% in control. Atrazine appeared to slightly modify the effect of DMBA since exposure to 5, 50, or 500 ppmPage 202 of 263 atrazine subsequent to DMBA exposure resulted in a slight non-statistically significant

decrease in the incidence of ovarian tumors (22, 28, or 26%, respectively) compared to

45% incidence after DMBA treatment-alone. There were no compound-related effects that could be unequivocally attributed to ei ther DMBA, atrazine, or DMBA + atrazine exposure in the other tumor types seen in his study (malignant fibrous histiocytomas, hemangiomas, mammary tumors ). PCNA-labeling after treatment with DMBA alone was markedly increased ( ↑ 1644%) over unexposed animals. Atrazine exposure did not affect PCNA labeling index. Similarly, immunohistochemistry assessments did not reveal an impact of atrazine exposure on the expression of the BRCA-1 protein or the estrogen or androgen receptors after DMBA treatment.

Reviewer’s Comments: The study authors concl uded that atrazine did not result in any modifying effects on DMBA-induced rat ovar ian carcinogenesis. It should be noted, however, that though not statistically signific ant the incidence of ovarian tumors due to DMBA exposure was reduced by half when DM BA treatment was followed by atrazine exposure at dietary conc entrations of 5-500 ppm (0. 09-8.5 mg/rat/day). The toxicological significance of this finding, however, is unclear given the lack of doseresponse over a two-order of magnitude difference in atra zine exposures. However, when considered collectively, the data reported by Fuji moto, Son, and Tanaka and coworkers demonstrate that atra zine exposure did not accelerate tumor progression.

A.7.12 Ueda, M., Imai, T., Taki zawa, T., Onodera, H., Mitsumori, K., Mtsui, T. and Hirose, M. (2005) Possible enhancing effects of atrazine on growth of 7,12-di methylbenz(a)anthracene-induced mammary tumors in ovariectomiz ed Sprague-Dawley rats. Cancer Sci. 96(1):19-25.

The objective of this study was to evaluat e the effect of atrazine exposure on late promotion/progression of mammary tumors in ovariectomized rats after treatment with 7,12-dimethylbenz(a)anthracene (DMBA).

Two hundred and six female Sprague-Dawley rats were used in the study. One hundred eighty six received a single gavage dose of DMBA (50 mg/kg) while 20 control

rats received vehicle. Nineteen weeks a fter DMBA treatment (when palpable tumor

incidence was ≈ 50%), the animals were ovariectomiz ed. The 89 rats bearing tumors at this time were designated as DMBA-tumors (+) [Group A] while the 90 rats without tumors by week 19 were designated as DMBA- tumors (-) [Group B]. These rats were distributed among 4 subgroups (22-23 rats/group) that received 0, 5, 50, or 500 ppm atrazine (purity unknown) [appr oximately 0, 0.25, 2.5, and 25 mg/kg/day] through the

diet for 34 weeks. A third group (Group C) consisting of animals not exposed to DMBA but exposed to atrazine at 0 or 500 ppm (10 rats/dose) for 34 weeks was also examined. Body weights, food consumption, the number of rats bearing tumors, number of tumors/rat, size of tumors, and location of palpable tumors were recorded once per week during atrazine-treatment. At the terminal sacrif ice, whole skin with mammary glands and tumo rs were collected. In additi on, the liver, kidneys, uterus,Page 203 of 263 pituitary glands, and adrenal glands were removed and weighed. Histopathology evaluations were conducted on mammary tumors , as well as, the liver, kidneys, spleen, lungs, heart, Zymbal glands, uterus, vagina, pituitary, adrenals, thyroids, mammary glands (not having gross indications of tu mors), and scars of the ovariectomized regions. If indications that traces of ovarian tissue remained after the ovariectomy, the animals were removed from the study. Thus the number of Group A animals treated

with the various concentrations of atrazine was between 18-21/group and Group B animals treated with atrazine ranged betwee n12-19 rats/group. Immunohistochemistry analysis was used to detect potential increase s in proliferating- cell nuclear antigen (PCNA), single strand DNA ( ssDNA), and estrogen receptor α (ER α ) in mammary tumors (except for fibromas). Finally, estradiol, progester one, prolactin, and luteinizing hormone levels were measured for Group A animals.

No effects on body weights or organ weights were reported for any treatment group. Mortality was higher in Group A (rats with early tumors) than Group B (rats with late tumors) but there was no apparent corre lation to atrazine exposure.

Following ovariectomy, the num ber of Group A tumor-bearing rats and total number of tumors palpated decreased by 6-29% and 50-68%, respectively, regardless of atrazine exposure. Similarly, the types of tumors ( i.e., benign vs . carcinoma) were not affected by atrazine treatment and t hough there was an apparent dose -related increase in tumor

size at doses ≥ 50 ppm ( ↑ 43 -261%) there was no statisti cal significance due to the large variability in the Gr oup A. Immunohistochemistry ev aluations showed that Group A rats treated with 500 ppm atrazine s howed an increase in the number of ER α -positive tumor cells relative to control (3/3 vs. 2/5) as well as in the number of PCNA-positive cells ( ↑ 92%, p<0.05) in malignant mammary tu mors. The authors report that no changes in hormone levels that could be attri buted to atrazine exposure were observed. However, no data were provided to corroborate this latter statement.

Interestingly, at the end of the study Group B rats ( i.e., DMBA-treated rats with late tumors) had a statistically significant (p<0.0 5) increase in the num ber of tumor-bearing rats exposed to 500 ppm atrazine relative to the Group B control animals (58% vs. 26% in control) yet a comparison with the Group A control animals revealed a lower incidence of tumor-bearing rats in the atrazi ne-treated animals than in the control. A

≈ 1300% increase in the size of tumors was al so noted for the Group B rats treated with

≥ 50 ppm atrazine; again comparison to the Group A control animals revealed tha the tumors in the atrazine group were smaller than in the Group A controls. There was no

evidenc e of a clear correlation between atrazine exposure and the incidence of malignancies for either Group A or Group B rats.Page 204 of 263 Figure 17: Treatment Groups

The authors concluded that atrazine has t he potential to facilitate mammary tumors during the promotion/progression stage. These conclusions are based on increases in the Group B incidence of tumor-bearing rats , number of carcinomas, tumor size at

doses ≥ 50 ppm, and in the number of cells pr oliferating (PCNA-positive cells) and expressing ER α after exposure to 500 ppm atrazine.

The preponderance of the evidence, however , does not support t he statement that

atrazine contributes to mamma ry tumor development. For ex ample, for Group A rats (tumors present at week 19) , there was no correlation between atrazine exposure and the number of tumor-bearing ra ts, number of new tumors ( appearing after ovariectomy),

number of tumors/rat or the incidence of carcinomas. Moreover, the regression of palpable tumors observed after ovariectomy was similar for rats in the control and atrazine-treated Group A rats. Although for Gr oup B a statistically significantly higher incidence of tumor-bearing rats is seen after exposure to 500 ppm atrazine relative to the Group B control, it shoul d be noted that the incidence in the Group B control is substantially lower than in the Group A cont rol (Control B: 26% vs Control A: 72%). When the Group B 500 ppm atrazine-treated rats are compared to the Group A controls, the incidence of tumor-bearing rats is lowe r after 500 ppm treatment than in the Group

A controls. It should be noted that the inci dence of tumor-bearing rats in the Group B control was substantially lower than in the Group A control. When both control groups are combined (Group A + Group B) , the incidence of tumor-bearing rats at the Group B500 ppm atrazine dose level is increased by 9% and is not statis tically significant. 21 In fact, if Group B rats treat ed with 500 ppm atrazine are com pared to the Group A control rats, the tumor incidence afte r atrazine exposure is lower than in the Group A controls.

21 Calculated by reviewer and analyzed using Fisher’s Exact test A

B

C

Table excerpted from Ueda et al. , (2005) p. 22Page 205 of 263 For rats treated with 500 ppm at razine in Group B (no tumors at week 19), the incidence of tumor-bearing rats was lower than the num ber of tumor-bearing rats in the Group A control (58% vs 72%). Similarly, the number of carcinomas present in atrazine-treated Group B rats did not show a dose respons e and at 500 ppm atrazine the carcinoma incidence was lower than for the Group A control rats.

With regards to the immunohistochemical evaluat ions, a statistically significant increase ( ↑ 92%, p< 0.05) in the number of PCNA-positive cell carcinomas was noted for Group A rats treated with 500 ppm atrazine. This is not unexpected given that carcinomas are quickly proliferating lesions. Interestingly, at razine exposure in and of itself does not appear to contribute to the increase in PCNA-pos itive cells as evidenced by the fact that for late developing carcinomas (Group B rats ) no statistically significant increase was noted at the 500 ppm atrazine dose level. An increase in the incidence of ER α -positive carcinomas was reported at the 500 pm atrazine level in Group A rats (100% vs 40% control). It should be noted, how ever, that a 100% incidence of ER α -positive carcinomas was also reported for the cont rol and 5 ppm atrazine Group B rats (latedeveloping tumors) while the high dose atra zine rats in Group B had an incidence of

75%.

Reviewer’s Comments: Based on the multiple lines of evidence presented in this study, a relationship between atrazine exposure and increases in the incidence of tumor rats cannot be drawn. In many instances it appear s that the incidence reported for the 500 ppm atrazine Group B rats was comparable or at times lower than the incidence reported for the control Group A rats. Since control rats in Groups A and B were treated the same (only difference is detection of pal pable tumors at week 19), a comparison of the treated groups with either cont rol group or a combination of both is valid. While it is true that increases are occasionally seen in the 500 ppm atrazine-treated rats that developed tumors after week 19 of the study when they were compared to the Group A control rats or when the Group A and Group B control rats (pre- and post-week 19 tumor development) were combined, the increase was no longer evident.

While there are a number of deficiencies in the conduct, interpretation, and reporting in the Ueda study, these data s uggest that atrazine in an of itself does not induce mammary tumors in ovariectomized rats treated with a mammary tumor inducer thus demonstrating the importance of the ovarian hormones in the development of tumors in the mammary gland.Page 206 of 263 A.7.13 Yi, Kun Don, Breckenridge, Charles B, and Simpkins, James W. 2010. Cytotoxicity of Atrazine and its Metabolites in vitro

. Unpublished data. Syngenta Crop Protection Inc.

In this study, the cytotoxi city of atrazine and its met abolites (diaminochlorotrizine [DACT], deisopropyl atrazine [DIA], ammeli ne [AM], hydroxyatrazine [OH-ATR]) was evaluated in four cell lines. The purity of the compounds was 97%. The four cell lines used were HT-22 cells (immortalized mu rine hippocampal neurons), MCF-7 cells (human breast adenocarcinomas), H295R cells (human adrenocortical carcinoma), and JEG03 cells (human placental choriocarcinoma). The cells were cultured in the appropriate media and the H 295R, JEG-3, and MCF-7 cells were maintained at 100% confluency, while HT-22 cells were maintained at 50% confluency.

For the cell viability assay, cells were seeded 24 hours before initiation at varying densities depending on the cell type in 96-well plates: H295R cells – 25,000 cells/well, JEG-3 cells – 3500 cells/well, MCF-7 cells – 15,000 cells/well, and HT-22 cells- 3500 cells/well. Cells were exposed to atrazine or its metabolites at concentrations ranging

from 0.001 μ M to 300 μ M in physiologically buffered sali ne (PBS) or in sterile water with dimethyl sulfoxide (DMSO) for up to 72 hours. After exposure , cell viability was measured using the membrane-permeant calcei n-AM dye. Calcein-AM is a fluorogenic esterase substrate that is hydrolyzed to a fluorescent product in cells having esterase activity and intact membranes. Fluorescence was determined using an Infinity M120 microplate reader with an excitation/emission of 485/530 nm. The results, obtained in relative fluorescent units (R FU), were expressed as the percentage of untreated or

vehicle treated control values. For each a ssay, six independent experiments were run with triplicate wells for eac h treatment per experiment.

The effects of atrazine on mitochondrial activi ty was measured in H295R cells. H295R cells were seeded in 96-well plates at 25,000 cells/well 24 hours befor e the initiation of the assay. Varying concentra tions of atrazine were dissolved in DMSO (0.001-300 μ M) or PBS (0.0003-300 μ M) for 24 hours. Mitochondrial acti vity was assessed using a MTT

assay.

Cytosolic reactive oxygen species (ROS) wa s estimated by measuring the amount of ROS by the fluorescent dye 2  ́ ,7  ́ -dichlorofluorescin diacetate (DCFH-DA), which measures hydrogen peroxide (H 2 O 2 ). Cells were plated in 96-well plates at a density of 25,000 cells/well 24 hours before the initiation of the assay. Cells were exposed to atrazine (10-300 μ M) dissolved in DMSO or PBS. RO S production was monitored for up to 72 hours. DCF-2,7-dichlorofluore scin fluorescence was determined at an

excitation of 485 nm and an emission of 538 nm using an Infinity M120 microplatereader.

The effects of atrazine on the cytotoxic e ffects of other known toxins was measured using H295R cells seeded in 96-well plates at 25,000 cells/well 24 hours before thePage 207 of 263 initiation of the assay. Vary ing concentrations of atrazine, DACT, DEA, DIA, ammeline, and OH-ATR dissolved in DMSO were added. Cell viability was measured 24 hours

after treatment. H295R cells were co-treat ed with varying concentrations of atrazine and other know toxins (glutamate – 15 mM; 3-NPA – 15 mM; H 2 O 2 – 250 μ M) for 24

hours.

The results of this study showed that atrazi ne and its metabolites were not cytotoxic in four cell lines, irrespective of being solu bilized in DMSO or PBS. Atrazine and its metabolites did not produce cytotoxicity as i ndicated by an absence of any effect on cell viability in two steroidogenic (H295R and JEG-3) and two non-ster oidogenic (HT-22 and MCF-7) cell lines. Atrazine had no effect on mitochondrial activity in H295R cells regardless of whether it wa s dissolved in DMSO or PBS. Atrazine did not induce oxidative stress in H295R cells, as measured by cellular production of hydrogen peroxide (H 2 O 2 ).

The potential of atrazine or its metabolites to exacerbate the effects of other compounds known to induce cytotoxicity was evaluated. H295R cells were trea ted concurrently with atrazine and glutamate, or 3-nitropropionic acid (3-NPA), or H 2 O 2 24 hours. Atrazine and its metabolites did not affect the cyt otoxicity of glutamate, 3-NPA, or H 2 O 2.

Reviewer’s Comments: The methods sect ion of the paper fail ed to include the evaluation of mitochondrial activity or m easuring the effects of atrazine or its metabolites on cell viability when treated in conjunction with known cytotoxicants. Nevertheless, data from Yi et al. (2010) suggest that atrazine and its metabolites are not cytotoxic to two steroidogenic cell lines (H295R and JEG-3) as well as two nonsteroidogenic cell line s (HT-22 and MCF-7).

A.8 Insulin/Mitochondria Function

A.8.1 Lim, S., Ahn, S.Y., Song, I.C ., Chung, M.H., Jang, H.C., Park, K.S., Lee, K-U, Pak, Y. K., Lee, H.K. (2009). Chronic Exposure to the Herbicide, Atrazine, Causes Mito chondrial Dysfunction and Insulin Resistance. PLoS ONE 4(4): e5186, p.1-11.

The authors have performed a series of in vivo and in vitro experiments to assess the

extent to which low levels of atrazine in water effect mitochondrial function and insulin resistance. The authors have treated Sprague- Dawley rats with 0, 30 or 300 μg/kg/day atrazine in drinking water for 5 months. [These levels of at razine are significantly lower than the dose levels used in most atrazine studies.] Rats were given either a standard diet or a high fat diet. The authors stated that there were no differences in water consumption during the study. The manuscript does not describe the vehicle or how often the water was prepared. In additi on, the manuscript does not provide any

information on the concentration of atrazine in t he water or estimates of the water intakePage 208 of 263 during the course of the study. In parti cular it is important to understand if the

concentration of atrazine in the water or t he vehicle changed the wa ter consumption.

The following measurements were made as part of the in vivo experiments: body weight, visceral fat, motor ac tivity, intramuscular and intrahepatic lipid content, plasma glucose, serum insulin, total cholesterol and triglycerides, intravenous glucose tolerance test, high insulin challenge (h yperinsulinemic-euglycemic st udy), energy expenditure, electron microscopy of mitochondria, and pr otein expression levels of mitochondrial OXPHOS complex. In vitro experiments were conducted to evaluate endogenous cellular oxygen consumption and mitochondrial respiratory chain activities. In general, the methods section lacks some important in formation and references to the methods used. For example, the met hods state that motor activity was measured 4hrs/month. However, it is unclear whether this refe rs to one 4 hr session or one hr/week. The authors performed student’s t-tests and ANO VA. However, a 2-way ANOVA with ATZand diet as the independent variables may be more appropriate. Based on limited reanalysis conducted, further scrutiny of the statistical approaches may be useful in the

future.

Generally, the results section of this paper is hard to follow. Figure labels and legends are difficult to interpret.

Body weight and fat determinations. Based on the results sectio n, atrazine at these doses increased body weights in both normal and high fat diet, although, there is a flat dose response relationship. The lowest dose has an equivalent effect of the highest water concentration. Similarly, ther e is poor dose dependent effect on the fat measures, either the low dose is equivalent to the high dose or the low dose is more affected than the high dose (see intrahepatic lipid/water graph; Fig 1F). These results are in direct conflict with many other atrazi ne studies which suggest that atrazine results in decreased body weight, not increased.

ATZ and insulin and HOMA-IR: The authors report a dose related increase in insulin

and HOMA-IR levels.

ATZ insulin resistance: Fasting plasma glucose levels of atrazine treated rats on the regular and high fat diets were higher than thos e in controls. The atrazine treated rats showed a reduced response to insulin loading , although the dose-response curves are

flat.

In vitro studies: The authors observe minor effects on mitochondrial function of atrazine

in their in vitro model at concentrations up to 460 uM. Rats given 150 mg/kg have peak

blood concentrations of 7.5 uM. There s eems to be a real disconnect between the in

vitro concentrations needed to see effects on t he mitochondria and the doses that produce effects on mitochondrial function in vivo . In order to achieve similar

concentrations in vivo compared to the in vitro studies, exposures to atrazine would have to be well over 150 mg/kg.Page 209 of 263 Reviewer’s Comments: To believe their dat a, one would have to believe that atrazine has significant and near maximal effects on mitochondrial function at doses of 30 ug/kg/d (a LOEL) in animals on a normal die t. This seems unlikely given that most

studies have not observed effects of at razine until exposures are approximately 100- 1000 times higher. Most notably, the results of this study are based on the statistical procedures used (which may be inappropriate ). Preliminary analyses by Agency

scientists do not confirm the resu lts reported in the study. Th is study is not sufficiently robust for use in a regulatory context and s hould be repeated to confirm the findings.

A.9 Pharmacokinetics: Internal Dosi metry, Metabolism, & Time Course

A.9.1 Dooley, G.P, Prenni, J.E., Pren tiss, P.L., Cranmer, B.K., Anderson, M.E., Tessari, J.D. ( 2006). Identification of novel hemoglobin adduct in Sprague Dawley rats exposed to atrazine.

Chem Res Toxicol 19:692-700.

The potential of atrazine to form coval ent adducts with hemoglobin was studied in groups of two female Sprague Dawley rats re ceiving oral gavage doses of 0, 10, 30, 100 or 300 mg/kg atrazine (97.1%) for 3 days. Baseline blood samples were taken the morning before treatment and at 24, 48 and 72 hours post-initiation; subsequent samples were collected at 10 days and 1 and 2 months after treatment had ceased. Samples were centrifuged, and the plasma was withdrawn. Packed erythrocytes were rinsed and processed to collect purify globin. Globin samples were spotted on MALDI plates and subjected to MALDI-TOF/TO F MS (Matrix-Assisted Laser Desorption Ionization- Time of Flight/Time of Flight Ma ss Spectrometry). Globi n samples were also analyzed by HPLC; fractions collected via HPLC and peptides were extracted via electrophoresis and tryptic digestion, purif ied and prepared for mass spectrometry analysis. Similar procedures were used to prepare samples of rat hemoglobin, harvested from unexposed bone marro w cells, which were treated in vitro with 90 ppm diaminochlorotriazine (DACT, a reactive electrophilic metabolite of atrazine).

The investigators repor ted that an additional β globin peak was seen in exposed animals during the HPLC and MALDI-TO F/TOF MS analysis with a mass 110 Da greater than the normal β subunits. Subsequent tryptic digestion of the β peak revealed a peptide of 1449.9 m/z ; this corresponds to a modified peptide of amino acids 121-132. Sequencing of this pepti de yielded a 110 Da addition to Cys-125 of the major β globin chain. These findings were confirmed in vitro in bone marrow cells reacted with DACT, resulting in a peptide of 1449.9 m/z with a similar sequence of this modified peptide.

Reviewer’s Comments: Ov erall, the study was we ll-conducted and support the investigators’ conclusion that atrazine exposur e resulted in the formation of a covalent adduct with cysteine residues of hemoglobin in Sprague Dawley rat bone marrow cells

both in vivo and in vitro . Given that, unlike rat hem oglobin, human hemoglobin doesPage 210 of 263 not have a reduced cysteine residue at positi on 125, the relevance of rat hemoglobin

adduct formation to human ri sk assessment is limited.

A.9.2 Dooley, G.P., Hanneman, W.H., Carbone, D.L., Legare, M.E., Anderson, M.E., Tessari, J.D. , (2007). Development of an immunochemistry detection method for atrazine-induced albumin

adducts. Chem Res Toxicol 20:1061-1066.

These study authors investigated atrazine in teractions with cellular macromolecules using rat and human albumins as the targets. For the in vivo phase, ovariectomized Wistar female rats (3/group) were adminis tered oral gavage doses of 0, 5, 10 or 20 mg/kg atrazine (97.1%) and sacrificed on poste xposure day 4. A second grouping of animals was exposed as describe to higher doses of atrazine (0, 50 or 200 mg/kg). Blood was collected and the plasma albumin wa s purified; proteins were purified by gel electrophoresis, isolated and digested with thermolysin. Recovered peptides were spotted on a MALDI plate and analyzed by MALDI-TOF/TOF MS.

For the in vitro phase, commercially prepared rat and human albumins were reacted with 90 μg/mL DACT for 5 days. Loaded onto polyacryamide gel and electrophoreses

for 1 hour. Purified albumin was transferred to a polyvinylidene fluride (PVDF) Western blotting membrane, rinsed, and probed wit h commercially prepared anti-DACT adduct antibody. The DACT adduct antibody comp lex was examined by chemiluminescence using Western Blot analysis. DACT adducts were confirmed if a band was detected by

chemiluminescence.

The investigators indicated that using ma ss spectrometry, a covalent bond with a mass of 110 DA was located on Cys-34 of the al bumin from rats dosed with 20- 200 mg/kg atrazine. This same adduct was also detected in rat and human albumin exposed to 90 μg/mL DACT. Using immunochemical analysi s, a dose-related increase in adduct

formation was detected in the purified albumin samples colle cted from rats treated with a wide range of atrazine doses (5-200 mg/kg). DACT adducts were also detected in rat and human albumin preparati ons exposed to 90 μg/mL.

Reviewer’s Comments: The findings of this well-conduced study provide evidence that DACT can form protein adducts. Additionally, the investigators proposed this methodology as a potential biomarker screen for the detection of atrazine in exposed

humans.Page 211 of 263 A.9.3 Kim D. and Breckenridge C. (Syngenta). Derivation of the short-term inhalation NOAEL using a physiologically based pharmacokinetic model. January 29, 2010.

Physiologically based pharmacokinetic (PBPK) models can be used for extrapolation of internal dose across different species and routes of exposure. In this study, a previously published PBPK model (McMullin 2007) for orally-administe red atrazine (ATRA) was

modified to include inhalation ex posure for the extrapolation of a rat oral NOAEL to the equivalent rat and human inhalation NOAELs. The rationale for using a PBPK model is based on a request by US EPA for a 28-day i nhalation study aimed at examining the effects of ATRA on the estrous cycle and luteinizing hormone (LH) surge. Such inhalation studies can be reportedly difficult to carry out experimentally since inhalation confinements by themselves without food and water would likely alter the estrous cycle. Therefore, a well-par ameterized PBPK model offers an alternative approach for estimating inhalation exposures of ATRA asso ciated with a given internal dose metric. The rat oral NOAEL of 6.25 mg/kg/day used in this study was based on a delay in preputial separation from a study aimed at examining the e ffects of ATRA on puberty in

male rats.

The PBPK model for orally-administered ATRA consisting of gut, liver, and body/plasma (McMullin 2007) was modified to allow inhal ation uptake based on species-specific pulmonary ventilation rates and amount of ATRA in air, and assuming that ATRA is not absorbed in the respiratory trac t nor eliminated by exhalation. The internal dose metric selected for predicting the equivalent ra t and human inhalation NOAELs was the area under the 24-hr plasma concentration-time cu rve (AUC). Thus, the model was first used to estimate the AUC resulting from the ora lly-administered ATRA at the NOAEL of 6.25 mg/kg/day. Inhalation pharmacokinetics (6-h ex posures, while setting oral dose to zero) were then simulated to result in the equiva lent AUC for rats and humans (using a body weight of 0.3 and 70 kg for rats and humans , respectively). The inhalation exposures that resulted in the same AUC (reported as 0.58 mmol/L/day) as the rat oral NOAEL of 6.25 mg/kg were 4.5 and 5.1 mg/m 3 for rats and humans, respectively.

Since a PBPK model accounts of interspecie s differences in pharmacokinetics, the uncertainty factor of 10 ( i.e. , 10 1⁄2 for pharmacokinetics and 10 1⁄2 for pharmacodynamics) used for interspecies extrapolation can be reduced to 10 1⁄2 ( i.e. , 3.2) to account only for pharmacodynamic differences. Thus, the composite UF of 100 ( i.e. , including UF of 10 for intraspecies extrapolation) can be reduced to 32 with the use of a PBPK model. Margin of exposure (MOE) estimates were derived based on the PBPK model-derived inhalation NOAELs and report ed measurements of ATRA i nhalation exposures. The reported MOE estimates are greater than the PBPK model-refined level of 32 as well as

the default benchmark level of 100.

Reviewer’s Comments: There are two main deficiencies associated with this study. First, the PBPK model for orally-administere d ATRA (McMullin 2007) does a relativelyPage 212 of 263 poor job of simulating ATRA plasma levels following oral dosing (See Figure 7A of McMullin 2007). Second, ATRA dealkylated ch lorinated metabolites were not included in the estimation of AUC following oral dosin g at the rat NOAEL of 6.25 mg/kg. This is particularly important since chlorinat ed ATRA metabolites are known to be toxicologically-active with respect to t he estrous cycle and LH surge, and ATRA is rapidly metabolized (particularl y following oral administrati on). Inclusion of chlorinated ATRA metabolites would result in a great er AUC for a given oral ATRA dose and correspondingly lower estimates for inhalation NOAELs.

A.9.4 McMullin T.S. (2003) Pharm acokinetic modeling of disposition and time-course studies with [ 14 C]-atrazine. J. Toxicol. Environ. Health, 66(A):941-964.

This manuscript describes the development of a physiologically based pharmacokinetic model in an attempt to estima te total plasma chlorotriazines (atrazine and its chlorinated metabolites: deisopropylatrazine, deethylatr azine, and diaminochlorotriazine) time courses. The development of the model is based on two previous studies: a single dose (30 mg/kg bw) study whereby individual metabolites (c hlorinated and non-chlorinated) were identified and quantified; and a repeat ed dose (1, 3, 7, 10, 50, and 100 mg/kg bw/day) study where, in addition to plasma, ur ine, and feces, reactivity of ATRA with red blood cells (RBCs) was examin ed. The compartments in the model are as follows: liver (the primary site of metabolism, brain (postulated site of action), a plasma compartment (containing RBCs), and a rest of body com partment. Individual metabolites are not tracked in the model. Instead, two pools of compounds are described: chlorinated

triazines and metabolites derived from glutathione conjugation. In the model, chlorotriazines can bind plasma proteins, react with RBCs, conjugate with glutathione, or be eliminated in urine. In contrast, glutathione conjugat es are considered toxicologically-inactive and can be eliminat ed in the model through feces and urine.

Parameterization of the model was carried out as follows: (1) since the single oral dose study quantified individual metabolites, the ra tes of glutathione conjugation, chlorinated triazine elimination, and glut athione conjugate elimination were fitted to the reported urinary percentages of 73% chlorinated triazi nes and 27% glutathio ne conjugates. This parameterization reasonably described the cumulative urinary elimination of presumed radiolabeled equivalents (chlorinated tria zine and glutathione conjugates) reported in the single dose study as well as at all doses in the repeated dose study. The accumulation of ATRA-derived reactivity in RBCs was described in the model by assuming a reaction with hemoglobin. A second order rate constant was estimated by fitting the radioactivity in RBCs reported at the different administered doses in the repeated dosing study. After incorporating RBC reactivity, it was noticed that the model consistently underestimated the single dose plas ma data at the longer time points. In an effort to better describe this dataset, plasma protein binding/reactivity was also included. A binding rate constant was estimated based on albumin, the major plasma binding protein of known concentration and turnover, by fitting the plasma dat a for retention of radioactivity in the single dose study and t hen evaluating the abilit y of the model toPage 213 of 263 describe the repeated-dosing study plasma data. Overall the model reasonably predicted plasma levels from the repeated-dosing study at t he early (first 50 hours postdose) and late time points (>200 hours), whil e systematically underpredicting levels at the mid-time points.

The study authors also reported on a time-course study whereby a single dose of 90 mg ATRA/kg BW was administered by oral gav age to rats and individual chlorinated triazines were monitored in plasma. It is unc lear to this reviewer why this new dataset was not used to evaluate the performance of the model. Instead, a l og-likelihood best fit to a one-compartment model is described to presumably show that a slower absorption rate constant describes the higher dose pl asma data, and that a similar terminal elimination phase exist across studies ( i.e. , consistent with slow absorption of ATRA and the rapid metabolism of ATRA to DACT).

Reviewer’s Comments: The major deficiency of this study is that the model developed was not reported as having been ev aluated using an independent dataset.

A.9.5 McMullin, T.S., Hanneman, W. H., Cranmer, B.K., Tessari, J.D., and Andersen, M.E. (2007). Oral ab sorption and oxidative metabolism

of atrazine in rats evaluate d by physiologically modeling approaches. Toxicology 240:1-14.

This study reports on physiologically-based m odeling approaches to evaluate the role of absorption and oxidative metabolism on t he pharmacokinetics of atrazine and its chlorinated metabolites. High oral doses of atrazine and its di-dealkylated metabolite diaminochlorotriazine (DACT), but not its non-chlorinated metabolites, have been associated with alterations in the hypothalam ic pituitary axis, leading to neuroendocrine responses such as suppression of the st eroid-induced luteinizing hormone surge in

adult ovariectomized female rats. Based on t hese findings, a net tissue exposure to chlorinated triazines may be necessary to conduct human health risk assessment for

atrazine.

Atrazine undergoes metabolism by P450 enzym es to the mono-dealkylated chlorinated species deisopropylatrazine (ETHYL) and deethylatrazine (ISO). Further P450- mediated dealkylation yields DA CT. Atrazine and all three of its chlorinated metabolites can undergo glutathione-S-trans ferase-mediated dechlorination to yield the respective non-chlorinated species.

A physiologically-based pharmacoki netic (PBPK) model based on in vivo pharmacokinetic studies with 14 C-atrazine was previously dev eloped to estimate total chlorinated triazines in plasma after si ngle and multiple or al gavage exposures. However, the predictive capabi lity of the model was limited by the lack of specific information on the rates of saturable, oxi dative metabolic clearance of individual chlorinated species. This study addresses this deficiency by developing a set of PBPK

models based on in vitro- derived metabolic parameters and in vivo pharmacokineticPage 214 of 263 data of individual chlorinated triazines. The resulting individual m odel parameters were integrated into a final composite model in an attempt to describe the kinetics of all four chlorinated triazine species after high dose oral administrat ion of atrazine.

Individual chlorinated tr iazine model structures

The model structures for all chlori nated triazines consisted of a GI subcompartment to evaluat e the potential for pre-systemic metabolism and absorption processes producing multiple peaks in plasma profiles; a liver as the primary site of metabolism; and a lumped rest of body compartment to account for the remaining tissues.

In vivo time-course pharmacokinetic data for individual chlorinated

triazines Female Sprague Dawley rats were dosed by oral gavage with 150 mg/kg bw atrazine or an equimolar amount of one of its chlorinated metabolites: ETHYL, ISO, or DACT. Plasma samples were analyz ed for all four chlorinated species.

Sequential Parameter estimation fo r individual model structures The following model parameters were esti mated sequentially by fitting the model to the respective time-c ourse pharmacokinetic data: (1). DACT: volume of distribution and DACT elimination rate constant (2). ETHYL: Vmax for metabolism of ETHYL to DACT and ETHYL elimination

rate constant (3). ISO: Vmax for metabolism of ISO to DACT and IS O elimination rate constant

Model evaluation for individua l chlorinated triazines DACT: plasma time-course data was simu lated using first order absorption and

elimination. ETHYL and ISO: First order absorpti on could not describe a double peak phenomenon; peak plasma concentrations occu rred at 0.5 h, followed by a rapid decline over 2 h. A second, smaller peak occurred at 2 h and extended over 24 h. The model was adjusted so that t he multipeak behavior was simulated by describing different rates of uptake from the gut; a rapid firs t order uptake and a slower zero order uptake of the remaining dose. Absorp tion rate constants for the different uptake processes were estimat ed by fitting to the time-course data.

Composite model evaluation for atrazine The uptake of atrazine could not be simula ted as a single first order absorption process due to a double peak phenomenon. As with ETHYL and ISO, this phenomenon was described by a combinatio n of first order and zero order descriptions. The authors hypothesiz ed that the double-peak phenomenon observed for ETHYL, ISO, and atrazine is reflective of limited compound solubility in the gut at the high dose s used in the study. The continuing absorption of more slowly dissolving co mpound along the GI tract contributes to the complex time-course behavior. A ddition of pre-systemic intestinalPage 215 of 263 metabolism of atrazine to mono-alkylated metabolites was also necessary to adequately describe the bioavailability of atrazine without adjusting the in vitro

derived metabolic parameters.

Reviewer’s Comments: The current study underscores some of the parameters that may contribute to the complex pharmacoki netics of atrazine observed following high dose oral administration. The utility of the co mposite model for atrazine is questionable. First, there is no direct evi dence cited in support of multiple absorption processes and

GI intestinal metabolism used to fit the mode l prediction to the plasma concentrationtime profiles. Second, the final composite model significantly underpredicts plasma

levels of chlorinated metabolites after 8 hours while underpredicting atrazine levels at all time points.

A.9.6 Ross, M.K., Jones, T.L., and Filipov, M.N. (2009). Disposition of the herbicide 2-chloro-4-(ethylami no)-6-(isopropylamino)-s-triazine (atrazine) and its major metabolites in mice: a liquid chromatography/mass spectrometry an alysis of urine, plasma, and tissue levels. Drug Metab. Dispos. 37(4): 776-786.

Mice are increasing being used to assess the potential adverse effects following atrazine (ATRA) exposure. However, disposit ion studies of ATRA in this particular species are lacking. This study was aimed at examining the metabol ism and disposition of ATRA in mice and assessing how tissue levels may correlate with levels of ATRA and its metabolites found in plasma and urine, the most accessible biological fluids. Thus, following an overnight fast, mice (5-8/dose/time) were admini stered a single dose of 0, 5, 25, 125, and 250 mg/kg bw ATRA in corn oil by oral gavage. Plasma and tissue

levels were measured as soon as 0.5 hours following dosing while urine and feces were

collected at 24-h intervals.

Plasma: Levels of ATRA exhibited a biphasic behavior; maximum levels were seen at 0.5 hours post dose, followed by a rapid decline before a sec ond peak occurred at 12 h. The biphasic phenomenon was attributed to discont inuous absorption fr om the GI tract.

ATRA levels were below the limit of detection by 24 hrs. In contras t, levels of the didealkylated metabolite diaminochlorotriazine (D ACT), the major metabolite identified (~ 10-fold higher levels than mono-dealkylated metabolites), concomitantly increased and peaked at 2 hrs at all dose levels. The clearance of DACT was dose-dependent and relatively slow during the first 24 hours (hal flives of 7.5 and 42 hrs for 5 and 250 mg/kg bw ATRA, respectively). When plasma area under the curves (AUCs) for DACT were plotted versus ATRA dose, a signifi cant linear correlation was observed (r 2 = 0.987).

Urine: DACT levels were 2 orders of m agnitude higher (as high as 5400 μM) than ATRA or its mono-dealkylated metabolites in the 0-24 hr time period.

Feces: Analysis of fecal extracts did not re veal significant amounts of metabolites.

Detected amounts of ATRA most likely reflected unabsorbed compound.Page 216 of 263 Liver and kidney: ATRA and all of its metabolites including glutathione conjugates

were detectable at 4 hrs. DACT was t he most abundant species followed by ATRA. Lower levels of each compound were found in the kidneys as compared to the liver.

Brain: Significant levels of DACT (as much as 50 μM at the highest dose) along with low levels of ATRA ( ~1 μM at the highes t dose) were found at 4 hrs. ATRA was still detectable in the brain at all dose levels at 24 hrs, while DACT levels remained high at

24 hrs.

Spleen and thymus: At the low doses of 5 and 25 mg/kg bw, maximum levels of ATRA (~2-3 μM) were detected at 1 hr, whereas at the two highest doses (125 and 250 mg/kg bw ATRA), maximum ATRA levels were seen at 4 hrs. Peak levels of DACT, the major species detected, were seen at 4 hrs at the two highest doses of ATRA.

Reviewer’s Comments: Much of the information reported in this study for mice seems to be strikingly similar to that reported in rats (see McMullin reviews). In particular, the biphasic absorption phenomenon of ATRA is consistent across these two rodent species. Plasma kinetics (maximum leve ls, time to reach maximum levels, and clearance) of ATRA and DACT are also ve ry similar for a given administered ATRA dose. This disposition information reported in this study may be important in the use of pharmacokinetic models that can link target ti ssue dose to toxicological effects of ATRA

and in vivo metabolites.Page 217 of 263 

# Appendix B

Atrazine immunotoxicity dataPage 218 of 263 UNITED STATES ENVIRONMENTAL PROTECTION AGENCY National Health and Environmental Effects Research Laboratory Cardiopulmonary and Immunotoxicology Branch

Environmental Public Health Divis ion, Research Triangle Park, NC 27711

OFFICE OF

RESEARCH AND DEVELOPMENT

DATE: January 28, 2010

SUBJECT: Atrazine immunotoxicity data

TO: Tina Levine, Ph.D. Director, Health Effects Division Office of Office of Pesticide Programs

US EPA

THROUGH: MaryJane Selgrade, Ph.D. /s/ Chief, Cardiopulmonary and Immunotoxicology Branch

Environmental Public Health Division

US EPA

FROM: Robert Luebke, Ph.D. /s/ Cardiopulmonary and Immunotoxicology Branch

Environmental Public Health Division

US EPA

Dear Dr. Levine,

In 2003, my laboratory published a paper reporting the effects of maternal atrazine exposure on immune function in male and female offspring (attached). Timed-pregnant Sprague Dawley rats were exposed to 35 mg atrazine/kg body weight/d from gestational day (GD)10 through postnatal day (PND) 23. The dose of atrazine was chosen to be just above the NOAEL for delayed onset of puberty in female offspring (25 mg/kg/d), as reported by (Laws et al., Toxicol. Sci. 58, 366–376, 2000). Maternal atrazine exposure has also been reported to disrupt prolactin production ( Stoker et al., Reprod. Toxicol. 13, 463–472, 1999) and thyroid hormones (Kornilovskaya et al., Biomed. Environ. Sci. 9, 60–66,1996). Alterations in either of these hormones has been reported to affect immune function. To test our hypothesis that disruption of prolactin or thyroid hormone homeostasis was causally related to defects in immune function, separate groups of rats were exposed to bromocryptine (0.2 mg/kg, given twice/d LOAEL for prostatitis development; Stoker et al., 1999) or propylthiouracil (2 mg/kg/d, a dose that would have minimal overtly toxic effects on the dam or offspring; Kevin Crofton, US EPA, personal communication). Only one dose level of atrazine was included in the study so that all members of exposure cohorts could be examined on a single day. Both the antibody response to immunization and the cell mediated response to a foreign protein were suppressed in male, but not in female, offspring of dams exposed to atrazine. Bromocryptine and propylthiouracilPage 219 of 263 exposure did not affect immune function. Pup mortality was greater in the atrazine offspring on postnatal day 7 (13% vs. 2% in controls) as was the number of litters in which at least one pup died (19 vs 4 in controls). Each experiment was done twice, over approximately 18-24 months. Similar results were obtained in both replicates. These studies were completed prior to our move to the new EPA facility in RTP.

After moving to a new laboratory facility, additional studies were done to derive a dose response for atrazine developmental immunotoxicity. Two replicate studies were conducted in timed-pregnant Sprague Dawley rats from the same supplier at doses of 0, 3.5, 17.5 and 35 mg atrazine/kg/day, from GD10 through PND 23, as in the original studies. Litters were standardized for number and gender of pups, following the same procedures described in the published studies. Antibody and delayed hypersens itivity responses were evaluated, following the same operating procedures as before. However, only male pups were evaluated immunologically, based on our previous results and the need to reduce the experiment size to accommodate the workload in my lab. The second study also included a group of adult rats that were injected as adults with the known immunotoxicant, cyclophospham ide, as a positive control for suppression of the antibody assay. Maternal atrazine exposure did not affect immune function in the offspring. Cyclophosphamide significantly (p<0.001) suppressed antibody production, indicating that the assay was able to detect immunosuppression. To assess possible strain effects, a third study was done in which timed pregnant Sprague Dawley, Long Evans and Wistar rats, all from the same supplier, were exposed to 35 mg atrazine/kg/d, following the same exposure and evaluation protocols used in previous studies; only male offspring were evaluated. As in the first two studies, immune function was not affected in the offspring.

I have complete confidence in the technical soundness and results of our published studies and in our attempts to generate a dose response. The same personnel were involved in both sets of studies, the same operating procedures were followed for exposure and assessment of immune function and reagents and supplies were all obtained from the same vendors as before. Three different lots of atrazine were used in these studies, all from the same vendor. Two different lots of atrazine were used in the initial and replicate studies reported by Rooney et al. and the outcome of both studies was the same. It is possible that the lot used for the dose response series was compromised in some way, but that seems unlikely. Animal dosing was completed

between 7 and 10 AM for all studies. Animal health was monitored in both the old and new facility; sentinel animals were housed in the same rooms as experimental animals and routinely evaluated for selected pathogenic viral, bacterial and parasitic infections and the results were consistently negative.

Based on these facts, I believe that both sets of studies are “correct” and reflect all conditions under which the experiments were done. I believe that the disparate outcomes are the result of unknown environmental factors, which are unlikely to be identified, and that the lack of effects in the second set of studies do not negate our published conclusions that exposure to ATR during immune system development and maturation may m odulate immune function. I will be happy to discuss our data with you and with members of the SAP.Page 220 of 263 

# Appendix C

Artificial Neural Network Modeling of Atrazine

Occurrence PatternsPage 221 of 263 

### Artificial Neural Network Modelin g of Atrazine Occurrence Patterns 22

by Michael J. Messner, Ph.D.

USEPA Office of Ground Wa ter and Drinking Water March 3, 2010



### Contents

ABSTRACT........................................................................................... 222

BACKGRO UND

.................................................................................... 223

M ONITORING S TRATEGIES

....................................................................................................................... 223

D ATA Q UALITY O BJECTIVES

..................................................................................................................... 223

A TRAZINE O CCURRENCE P ATTERNS

........................................................................................................ 224

OCCURRENCE DATA.......................................................................... 226

F REQUENT M ONITORING D ATA

. ................................................................................................................. 95

T IME S ERIES P LOTS

.................................................................................................................................. 95

D ATASETS S UITABLE FOR M ODELING

....................................................................................................... 231

ARTIFICIAL NEURAL NETWORK MO DELING..................................... 98

ANN MODELS

. .......................................................................................................................................... 98

ANN MODELING FOR THE M AUMEE R IVER

, 1998. ..................................................................................... 235

ANN MODELING FOR THE S ANDUSKY R IVER

, 1995. .................................................................................. 239

PREDICTIVE AN ALYTICS ................................................................... 100

D IRECT D ERIVATION OF THE E XPOSURE

................................................................................................... 242

S CALING THE O CCURRENCE P ATTERNS

................................................................................................... 243

S IMULATING D AILY L EVELS

...................................................................................................................... 243

E VALUATING A M ONITORING S TRATEGY

................................................................................................... 103

SUMMARY – ADVANTAGES AND DISADV ANTAGES OF USING THE ANN

MODELS ............................................................................................... 105

Disclaimer : Views and opinions expressed in th is paper are those of the author and do not necessarily state or reflect those of t he Office of Ground Wate r and Drinking Water, the Environmental Protection Ag ency of the US Government.

22 File is ANN_Occurrence 3_3_2010.docxPage 222 of 263 C.1 Abstract

This paper assumes that EPA/OPP has dev eloped or will develop some quantitative performance criteria for their Atrazine Monitoring Program (AMP) and that these criteria will include some that relate to the detection of short-lived, high-concentration periods, called “spikes” or “peaks.” This paper describes one form of model (Artificial Neural Network) that can be used to simulate this kind of occurrence pattern and shows how these models can be used to assess how well candidate monitoring strategies satisfy quantitative performance criteria.Page 223 of 263 C.2 Background

C.2.1 Monitoring Strategies

A “monitoring strategy” is a combi nation of (a) a plan for obtaining new contaminant occurrence data and (b) a decision rule, in wh ich the occurrence data are used to make a binary decision, such as to decide whether exposure levels are above or below some

health-based level of concern.

Crafting a suitable water monitoring strategy involves many considerations. These include the following:

• The cost of obtaining and testing samples of drinking water (or source water)

• The spatial and temporal variability of the contaminant, particularly when occurring at

levels of concern.

• The occurrence levels and durations that are associated with different health endpoints

• The severity of the adverse health outcomes

• The utility of monitoring data for informing treatment decisions

• Consequences of failing to detect and address high occurrence (false negative)

• Consequences of false alarm (false positive)

When the above considerations lead to quantitative performance criteria, those criteria become the benchmarks for evaluating different monitoring strategies. When available strategies fail to satisfy the criteria, for example, because they too often raise false alarm or too often fail to detect high occurrence, planners may need to reconsider the performance criteria.

This paper assumes that EPA/OPP has dev eloped or will develop some quantitative performance criteria for their Atrazine Monitoring Program (AMP) and that these criteria will include some that relate to the detection of short-lived, high-concentration periods, called “spikes” or “peaks.” This paper describes a form of occurrence modeling that can be used to simulate this kind of occurrence pattern and shows how these models can be used to assess how well candidate monitoring strategies satisfy quantitative performance criteria.

C.2.2 Data Quality Objectives

Executive order 5360.1 requires the use of “a systematic planning process to develop acceptance or performance criteria” fo r environmental monitoring projects. 23 Over the past 25 years, EPA’s QA Staff has devel oped and promoted a systematic planning process for just this purpose, called “Data Quality Objectives” or DQOs. 24

23 USEPA Executive Order 5360.1, Policy and Program Requirements for the Mandatory Agency-Wide Quality System, Chg 1, 7/16/1998. http://www.dpnr.gov.vi/dep/5360-1c1.pdf

24 USEPA, 2006, Guidance on Systematic Planning Using the Data Quality Objectives Process (EPA QA/G4). EPA/240/B-06/001. http://www.epa.gov/quality/qs-docs/g4-final.pdfPage 224 of 263 The DQO process applies systematic plann ing and statistical hypothesis testing methodology to facilitate the design and se lection of environmental monitoring strategies. The seven steps of the process are:

1. State the Problem 2. Identify the goal of the study 3. Identify information inputs 4. Define the boundaries of the study 5. Develop the analytic approach 6. Specify performance or acceptance criteria 7. Develop the plan for obtaining data

The process really gets quantitative in Step 6, where two kinds of errors and their consequences are expressed and considered. The outputs of Step 6 are quantitative limits for the probabilities of false positives and false negativ es. A statistician can then evaluate how well selected monitoring strategi es satisfy these criteria, given different occurrence scenarios or patterns. In general, waters with high variability are the most challenging. Water having a low occurrence le vel, but high variability, can experience a false positive whenever a sample is taken during a short-lived occurrence spike. Similarly, water with a high occurrence leve l with high variability can “get lucky,” and experience a false negative because infrequ ent monitoring failed to catch a large occurrence spike.

C.2.3 Atrazine Occurrence Patterns

Atrazine occurrence is known to be greatest following rainfall events in the spring and summer and lowest prior to the pes ticide’s first use in the spring. 25 Occurrence is more variable in flowing streams than in reserv oirs and lakes, and variability tends to be inversely related to flowing stream size, wit h greatest variability in headwater streams in regions where the pesticide is used. 26

Monitoring data provided by Missouri American Water Company and displayed in Figure 18 reveals a strong seasonal occurrence pattern with high variability. 27

25 USGS. Herbicides in Rainfall Across the Midw estern and Northeastern United States , USGS Fact Sheet 181-97, June 1998, h ttp://ks.water.usgs.gov/pubs /fact-sheets/fs.181-97.html

26 Graziano, N., et.al, 2004 National Atrazine Occurrence Monitoring Program Using Abraxis ELISA Method, ES&T, 40, 1163-1171, 2006.

27 Data provided by Dr. Mark LeChevallier, American Water Works Service Company.Page 225 of 263 Figure 18 Atrazine Occurrence at Missouri-American Water Intake

Drinking water sources monitored under t he Atrazine Monitoring Program (AMP) include some with variability like that shown above, but also include many waters with less variability like those shown in Figures 19 and 20.Page 226 of 263 Figure 19: Source Water with Low Variability

Figure 20: Source Water wi th Moderate Variability

In the Figures above, the actual monitoring data are displayed as black dots. The red

lines are Artificial Neural Network cu rves that have been fit to the data.

C.3 Occurrence Data

C.3.1 Frequent Monitoring Data.

The data used for the modeling described be low was collected by the Heidelberg University’s National Center for Water Qualit y Research (NCWQR) file for large rivers.

Other data from Headwater Streams and CWS were plotted and evaluated, but were not used in the modeling exercise.Page 227 of 263 C.3.2 Time Series Plots.

Figure 21 displays 2001 data from the Maum ee River, one of the Large Rivers . The Figure has been marked to highlight some features of the data, including:

• Data that are not consistent with their near-time neighbors due to rapid changes in the

occurrence level or measurement error.

• Single measurements that appear to be repeated on following days. It turns out that days without data were filled in with the most recent previous measurement result.

• A short-lived occurrence peak appears near day 200, but another, smaller peak may have occurred around day 120. With a single meas urement, it is difficult to say whether that point is an actual occurrence spike or a measurement error.Page 228 of 263 Figure 21: Data for Maumee River 2001Page 229 of 263 Figure 22 is a plot of Maum ee 1998 (one of the data sets us ed for ANN modeling) data, showing larger short-term day-to-day vari ability or “noise” (in log units) when

concentrations are below e -1.5 than when above e -1.5 . Based on this and other plots, it appears reasonable to treat 0.25 g/L as a cens oring limit. The natural log of 0.25 is about -1.4. This figure clearly shows two occurrence peaks.

Figure 22: Data for Maumee River 1998Page 230 of 263 Figure 23 displays data from headwater stre am NE 07 2005 revealing several sharp occurrence peaks (marked by red arrows in the plot below). This number of peaks would be very difficult to model wit h such a small amount of data.

Figure 23: Data for a Headwater StreamPage 231 of 263 C.3.3 Datasets Suitable for Modeling

Two datas ets are used to illustrate Artificial Neural Network modeling. Both Maumee 1998 and Sandusky 1995 exhibit high variability, with at most two occurrence peaks. The data seem to be randomly dispersed, wit h no obvious outliers – at least, at first glance. Both sets have mostly-daily monitoring in high-occurrence periods.

Original data were processed in R to (a) replace the repeated values with “NA” and (b) replace concentrations below 0.25 with 0. Figure 24 is a plot of Sandusky 1995 original data (black) overlaid with data that were retained as measured values (r ed). Red points that are follo wed by horizontal strings of black dots represent single measured values followed by days wit hout measurements. The horizontal red line represents our censoring limit (log(0.25) = -1.4). Below that line, horizontal strings of black dots are days t hat followed measured values less than our censoring limit. Red points for those meas urements don’t show on the plot because they were replaced by zeros before log tr ansformation, but they do appear in the “rug,” where a fiber is shown for each day having a measured value, whether above or below our censoring limit.Page 232 of 263 Figure 24: Data for Sandusky River 1995Page 233 of 263 C.4 Artificial Neur al Network Modeling

C.4.1 ANN models.

Artificial Neural Networ ks (ANNs) are o ften seen as magical “black boxes” that magically learn from data, and end up pr edicting how one or more “dependent variables” change in response to “independent va riables.” A recent book by Herbert K. H. Lee opens up the ANN black box, providin g mathematical forms of ANN models and some real-world interpretations of the model parameters. 28 The book goes on to describe parameter estimation in a Bayesian framework for multivariate regression, classification, and other applications.

The equation below describes one form of ANN used to model Atrazine occurrence. Subscript day is the day of a calendar year (0:364) and lnC is the natural logarithm of the concentration, g/L.

lnC day b0 b1

1 e b5

− b6 day

⋅− + + b2

1 e b7

− b8 day

⋅− + + b3

1 e b9

− b10 day

⋅− + + b4

1 e b11

− b12 day

⋅− + + :=

The first parameter (b 0) is an intercept term in the s ense that, as the denominators of the other terms get large, the contributions of the other terms become negligible. Parameters b1 through b4 determine the potential magnitudes of the increases and

decreases of concentration due to those four terms, called “hidden nodes.” Parameters in the denominators determine how quickly the nodes’ contributions increase or decrease with time and the location or day on which the incremental increase or decrease is greatest. The first fraction’s c ontribution per unit time is greatest around day –b5/b6 and parameter b6 determines the magnitude of the increase on neighboring days. On days far removed from day = -b5/ b6, the first node’s contribution approaches either zero (when the denomi nator is huge) or b1 (when t he denominator is nearly 1).

Figure 25 shows the sum of b0 and a single hidden node toget her with the observed log

concentrations for the Maumee River in 1998 . A second node with a decreasing effect will be needed to pull the ANN curve dow n after day 130 and a third node will be needed to explain the occurrence peak around day 166.

28 Lee, Herbert H.K., Bayesian Nonparametrics via Neural Networks , ASA-SIAM, 2004.Page 234 of 263 Figure 25: Maumee 1998 and A NN Model with One Node

0 100 200 300 400 5

− 0

5 10

The “best” ANN model for any one-year data set will be the model with fewest hidden nodes that does an adequate job of explaining the occurrence data. “Adequate” will be expressed in terms of log-likelihood.

The ANN model describes the expected log-c oncentration, but clearly, the concentration on any given day will not be exac tly that predicted by the model. Some amount of noise can be seen in all plots of occurrence data. We assume the difference between a day’s measured log-concentration and that given by the underlying model will be normal with

mean zero and some variance that we can esti mate using the model and the data. We assume the variance is constant. A day’s log-concentration is regarded as a normal random variable with mean equal to the model prediction and, for measured values above a censoring limit, the lik elihood is the normal probabilit y density. For nondetects and measured values below a censoring lim it, the likelihood is the value of the cumulative normal distribution function (the probability of a log concentration below the censoring limit).

Rather than a full Bayesian treatment, where prior probabilities and data combine to reduce uncertainty about the model parameters, we seek maximum likelihood models. This simplifies the evaluation of monitoring strategies, where the true model and its parameters are fixed and data are random (simulated) based on the fixed occurrence

model.

Once a best-fitting ANN model is identified, the autoregressive (AR) structure can be explored and modeled. AR structure expl ains how the deviation of one day’s logconcentration from the model is related to t he deviation of previous days. It turns out that days with log-concentration greater (or less) than expected tend to also be followedPage 235 of 263 by days with log-concentration greater (or less) than expected. This is explained by positive coefficients in the AR model.

The following sections describe ANN model dev elopment for two river-years of data; Maumee 1998 and Sandusky 1995. The prefe rred model for Maumee 1998 has three hidden nodes and AR(2) behavior, while t he preferred model for Sandusky 1995 has two hidden nodes and AR(1) behavior.

C.4.2 ANN modeling for the Maumee River, 1998.

Modeling begins with simple plot s of log- concentration vs. time. All plotting and analyses are done using R version 2.10.0. 29 R-code for this can be found at https://kestrel.rtpnc.epa.gov/OGWDW_ Strategies/index. php/ModelingMaumee1998

The time-series plot reveals two occurrenc e peaks. ANN modeling begins with a node to generate the increase of concentration to the first peak and a node to begin reducing log-concentration to where the second spik e begins. A third node is added to generate the increase to the second peak and a fourth node is added to bring the level back down following the second peak.

lnC day b0 b1

1 e b5

− b6 day

⋅− + + b2

1 e b7

− b8 day

⋅− + + b3

1 e b9

− b10 day

⋅− + + b4

1 e b11

− b12 day

⋅− + + :=

Initial values for the model parameters were selected using profe ssional judgment. The process for the first node went something like this:

• b0 was set to ln(0.1) to establish an “intercept” of ln(0.1 g/L), as that appeared to be a

reasonable “floor” for Atrazine occurrence

• b1 was set to ln(8/0.1) = 4.382 so that this second fraction could max out at ln(8 g/L), which is a bit greater than the highest value of the first occurrence peak. This allows the second fraction to begin having an effect some time before the peak.

• The day of steepest ascent was set at 120 and this was set equal to the ratio of –b5/b6, where b5 and b6 are in the node’s denominator (exp(-b5-b6*day)).

• Various levels of b6 were selected, and b5 found to fit the day of steepest ascent, and the resulting model was plotted against the data. Parameter b6 was then adjusted up or

down to better fit the rate of increase seen in the data.

Once good initial values were found for all four nodes, a mean deviance was calculated and a log likelihood function was cons tructed. Given fixed data, this is a function of the parameters (the 13 model coeffi cients and the precision, 1/va riance). Log likelihood is a sum of the log likelihood cont ribution of each observation. For observations that fall above ln(0.25), a censoring limit, the cont ribution is natural log of the following probability density (dnorm):

29 The R Foundation for Statistical Computing, http://www.r-project.org/Page 236 of 263 dnorm Z j 2

, b0 b1

1 e b5

− b6 Z j 0

, ⋅− + + b2

1 e b7

− b8 Z j 0

, ⋅− + 

# ( ) + b3

1 e b9

− b10 Z j 0

, ⋅− + 

# ( ) + b4

1 e b11

− b12 Z j 0

, ⋅− + 

# ( ) +

, b13 0.5

−

, ⎡ ⎢ ⎣ ⎤ ⎥ ⎦

For observations that fall below the censoring limit, the contri bution is the natural log of the distribution function (pnorm):

pnorm DL b0 b1

1 e b5

− b6 Z j 0

, ⋅− + + b2

1 e b7

− b8 Z j 0

, ⋅− + + b3

1 e b9

− b10 Z j 0

, ⋅− + + b4

1 e b11

− b12 Z j 0

, ⋅− + +

, b13 0.5

−

, ⎛ ⎜ ⎝ ⎞ ⎟ ⎠

The precision (b13) also contributes a value to the log likelihood, ln(b13), which gently penalizes small precisions. In a Bayesian framework, this woul d be consistent with having a vague, flat pr ior on the precision.

Maximum likelihood values were identified by repeated application of a maximization function. A solution was found at b = c(-3.35, 10.664, -5.008, 3.618, -7.379, -15.524, 0.125, -36.097, 0.273, -214.029, 1.311, -7.213, 0.04, 29. 062). Figure 26 shows that, while the model does a nice job of fitti ng the data, there is an odd-looking bump between the two peaks. The fourth node, wh ich was supposed to explain the second decline, appears to have an impact mid-way th rough the first decline. This suggests that perhaps only one node is needed to explain both declines in log- concentration.Page 237 of 263 Figure 26: Maumee 1998 and Max Likelihood Four-Node ANN ModelPage 238 of 263 The three-node equation looks like the form abov e, but without the final term. As before, initial values were identified usi ng professional judgment. Maximum likelihood parameters were found to be c(-1.81,3. 68,-7.194,3.844,-50.084, 0.423,-7.222,0.04,- 202.475,1.241,24.58). The resulting curve and the data are shown in Figure 27.

Figure 27: Maumee 1998 and Max Likelihood Three-Node ANN Model

The maximum log likelihood under the f our-node model was 18.109 and the error standard deviation (log units) was 0.185. T he log likelihood of the three-node model shown above was 11.855, with error standard devia tion 0.202. Given that the four-nodePage 239 of 263 model has three more paramet ers than the three-node model and that the difference in log likelihood is slightly great er than 6, the two m odels are nearly equal in quality-of-fit.

The three-node model is selected for its simplicity and better “appearance.”

Residuals for the three-node model we re calculated for each non-censored measurement. The resulting autocorrelati on function, shown in Figure 28, shows a wave pattern that is commonly associated with AR(2) models. 30

Figure 28: Residuals for Maumee 1998 Three-Node Model

R function “resid.ar” confirmed that t he best autoregressive model is AR(2) and provided maximum likelihood coefficients 0.381 and 0.195. This means that, when simulating occurrence under the model, eac h day’s “error” will have two components, one of which is random and independent of other days’ errors, while the other is a function of the two previous days’ errors.

C.4.3 ANN modeling for the Sandusky River, 1995.

As before, modeling begins with simple time se ries plots of log-conc entration, Figure 7. The plot reveals a single occurrence peak, so a two-node ANN model is needed.

30 Cowpertwait, Paul S.P. and Metcalfe, Andrew V., Introductory Time Series with R , Springer, 2009.Page 240 of 263 Initial parameter values were set using professional judgment. The process was similar

to that described above for Maumee 1998. Maximum likelihood estimates were found, and the resulting curve looked reasonable, but the log likelihood was very small (-110). Log likelihoods for individual observations rev ealed six that contributed large amounts of the negative likelihood. The six observati ons are marked in Figure 29 and include:

• two points prior to the peak, at significantly greater concentrations than their neighbors (suggesting that these may have actually been two short-lived peaks)

• one high concentration near the peak that, immediately preceded and followed by nondetects (suggesting large measurement error), and

• one nondetect around day 200 that also appears to be a measurement error.

When these six spurious observations were removed, the maximum likelihood two-node model’s log likelihood improved to -50.75 and the error standard deviation reduced from

0.516 to 0.331.Page 241 of 263 Figure 29: Data Contributing to Small Likelihood for Two-Node Model

Maximum likelihood parameters were c(-1.189 , 4.197, -4.138, -37. 513, 0.286, -6.436, 0.034, 9.434) and the resulting curve (not sh own) was similar to that shown above.

Residuals were plotted and evaluated us ing R function “resid.ar.” The best autoregressive model was found to be AR(1), with parameter 0.479.Page 242 of 263 C.5 Predictive Analytics

The two occurrence patterns explored abov e (Maumee 1998 and Sandusky 1995) are among the most variable or “spiky” of all drinking water sources evaluated to date. Headwater stream data exhibits even greater variability, with several occurrence spikes per year. Modeling headwater stream occurrence may be useful if the monitoring objectives concern contamination of headwater streams and the contaminant’s impact on stream ecology.

This discussion describes how the ANN oc currence models can be used to evaluate source water monitoring strategies, where the concern is for detecting situations like the following:

a) Distributions of daily concentrations where the 75 th percentile exceeds X g/L. (Wedon’t want to have more than about 90 days per year above X g/L and the highoccurrence days do not need to be consecutive to do harm.) b) Distributions with mean concentrations greater than Y g/L. (Concern is for long-term exposures, such as associated with some cancer risk.) c) Distributions with an average of 1 or more times during the year when concentrations exceed Z g/L for W or more consecutive days. (Concern is for sustained exposure over consecutive days.)

C.5.1 Direct Derivat ion of the Exposure

Under these models, a particular day’s concent ration will vary from year-to-year as a lognormal random variable, so each day’s dist ribution function is known. The values shown in Tabl3 23 were calculated directly.

Table 22: 75th Percentiles a nd Means for Selected Models

Occurrence Model d) 75 th

Percentile e) 90 th

Percentile f) Mean

Maumee 1998 Three-Node AR(2) 0.0920 g/L 3.02 g/L 1.428 g/L

Sandusky 1995 Two-Node AR(1) 0.0958 g/L 4.50 g/L 1.375 g/L

Estimating the expected number of episodes per year having at least W consecutive days’ concentrations above a level of interest is more involved, as it requires simulating many years of occurrence and keeping track of episodes encountered. Simulations of this sort have not yet been performed, but are entirely feasible, should concern be expressed for these kinds of exposures.Page 243 of 263 C.5.2 Scaling the Occurrence Patterns

Given maximum likelihood parameter va lues, the Maumee 1998 and Sandusky 1995 occurrence levels may be considerably differ ent than situations of interest. For example, neither of those occurrence levels is directly relevant if the concern is having a

90 th percentile at or above 10 g/L.

Fortunately, the two models can be scaled eas ily by adjusting the intercept term, b0. Having homogeneous variance on the log-scale ens ures that relative error (ratio of day’s standard deviation to the day’s ex pected concentration) is constant.

Example 1: Scaling Maumee 1998 so that the resulting occurrence pattern has a

90 th percentile equal to 10 g/L.

The original model for Maumee 1998 has a 10 th percentile of 3.02 g/L, which is smaller than 10 by a factor of 3.31. Pa rameter b0 must increas e from -1.81 to - 0.613, which is -1.81+ln(3.31). Example 2: Scaling Sandusky 1995 so that the resulting long-term average concentration is 10 g/L.

The original model for Sandusky 1995 has a long-term average concentration of 1.375 g/L, which is smaller than 10 by a factor of 7.27. Parameter b0 must be increased from -1.189 to 0.795, which is -1.189+ln(7.27).

C.5.3 Simulating Daily Levels

Simulation is required to assess the ability of a monitoring stra tegy to “detect”

occurrence situations of interest. Simulati ng a year of daily concentrations requires adding some “error” to the ANN-predicted log- concent ration for each day of the year. These daily “errors” have two components. One component is lik e “white noise,” in that it is independently and identically distributed (i .i.d.) across days. On the log-scale, the

white noise distributions are normal with standard deviations 0.243 and 0.265 for Maumee 1998 and Sandusky 1995, respectively.

The other component of error is due to the AR behavior. In the case of Maumee 1998, the model is AR(2) and the error on day D incl udes 0.381 times the “white noise error” of the previous day (D-1) and 0.195 times the “white noise error” of day D-2. In the case of Sandusky 1995, the model is AR(1 ), and the error on day D includes 0.265

times the “white noise erro r” of the previous day.Page 244 of 263 Example 1: Maumee 1998 is scaled so that the resulting occurrence pattern has 10 g/L as its 90 th percentile. Parameter b0 has been increased to 0.613. One year of simulated data is shown in Figure 30 . In this particular simulated year, 10 g/L (shown in the plot as a horizontal red line) is exceeded on 34 days, so 10 falls just above the 90th per centile. Over many simu lated years, the percentile associated with 10 g/L will vary. This particular simulated year just happened to have 9.3% of the values fall above 10 g/L.

Figure 30: Simulation Based on the Maumee 1998 ModelPage 245 of 263 Example 2: Sandusky 1995 is scaled so that the resulting long-term average concentration is 10 g/L. Parameter b0 has been increased to 0.795. One year of simulated data is shown in Figure 31. In this particular simulated year, the average concentration is 9.68 g/L. There happen to be 79 days on which the concentration exceeds 10 g/L. As the number of simulated years increases, the average concentration will converge to 10 g/L.

Figure 31: Simulation Based on the Sandusky 1995 ModelPage 246 of 263 C.5.4 Evaluating a Monitoring Strategy

Having a means of simulating daily occu rrence levels for an occurrence level and pattern of concern, one can select daily obs ervations in accordance with a monitoring strategy and then process the selected obser vations to make the binary decision; to decide whether or not there is a problem. For example, if the strategy is to collect one sample per month (one sample every 30 days), and determine whether the maximum value exceeds X, one could do the following:

1. Select an occurrence pattern and level of concern. This is a source water

occurrence scenario where there is concern about either a false positive or false negative determination. The simulation exer cise is crafted to determine whether a strategy adequately limits decision error for the selected scenario. 2. Select a random start day in the first month. St artDay <- sample(1:31,1) 3. Add 30, 60, ... 330 to mark the other sampling days for the year or, for random dates, select one day at random from every calendar month.

4. Observe the concentrations simulated for those days in a one-year simulation 5. Identify the maximum concentration and compare to X, recording the result 6. Repeat the steps 2 through 5, above unt il many years of monitoring have been

simulated 7. Across all simulated years, determine the fraction of times that the result was positive (because the year’s sample maximum exceeded X)

Example: Determine the probability that mont hly sampling will produce a positive result, where a positive is defined as having a maximum greater than 10 g/L when the occurrence pattern is Maumee 1998 having a 75 th percentile of 10 g/L. For this exercise, this level is considered “high” and a positive rate of at least 95% is desired.

• The occurrence pattern is Maumee 1998 and coefficient b0 is adjusted so the long-term 75 th percentile is 10 g/L. Figure 14 di splays a year of simulated data for this pattern and level.

• Steps 2 through 5 are repeated 10,000 times

Results

• Under systematic sampling, a random sa mpling date is selected for January and other sampling dates follow at exactl y 30-day intervals. Among the 10,000 simulated sample maxima, only 180 exceeded 10 g/L. A positive result was

obtained 98.2% of the time. Our goal of at least 95% positives has been met.

• With random sampling during each of 12 30-day “months,” positive results were obtained in only 82.1% of the simulations . The high-occurrence peak that was almost always detected with systematic sampling was often missed with random sampling. Our goal of at least 95% positives has not been met.Page 247 of 263 C.6 Summary – Advantages and Disad vantages of Using the ANN Models

Figure 31 lists some important advantages and disadvantages of using ANN models for the purpose of evaluating monitoring strategi es for the Atrazine Monitoring Program. (Space is provided for additional remarks.)

Figure 32 Utility of ANN Modeling

Advantage Disadvantage Models are flexible and can potentially represent realistic occurrence patterns. The mathematical form of an ANN model is complex, so individual model parameters are difficult to interpret.

The models account for some autocorrelation, so can be used to

understand the distinction between consecutive days above a level of concern and percentiles of the occurrence

distribution. We are not yet able to produce ahierarchical model that includes year-toyear variability in timings, numbers, and magnitudes of spikes. Our sense is that the benefit of developing such models is less than the cost / effort required.

The models can be used to assess how a monitoring strategy’s performance is related not only to the sample size (number of samples collected each monitoring period) but also to the sample timing with respect to occurrence peaks. Expanding these models to account for year-to-year variability in parameters is feasible, but would require many years of

data for each location modeled. Computing and interpreting occurrence endpoints may be challenging.
The models are scalable, so that relative error (departure from the model’s baseline curve) is maintained on the measurement scale (g/L). Different magnitudes of occurrence are easily simulated using the same basic occurrence pattern.Page 248 of 263 Bibliography

Post 2003 References

1. Albanito, L., Lappano, R., Madeo, A., Ch imento, A., Prossnitz, E.R., Cappello, A.R., Dolce, V., Abonante, S., Pezzi, V ., & Maggiolini, M. (2008). G-proteincoupled receptor 30 and estrogen receptor-alpha are involved in the proliferative effects induced by atrazine in ovarian c ancer cells. Environ Health Perspect, 116(12): 1648-55.

2. Albanito, L., Madeo, A., Lappano, R., Vivacq ua, A., Rago, V., Carpino, A., Oprea, T., Prossnitz, E., Musti, A.M., Andò, S., & Maggiolini, M. (2007). G Protein– Coupled Receptor 30 (GPR30) Mediates Gene Expression Changes and Growth Response to 17ß-Estradiol and Selective GPR30 Ligand G-1 in Ovarian Cancer Cells. Cancer Res, Feb 15; 67(4): 1859-66.

3. Bakke B., De Roos A.J., Barr D.B., Stewart P.A., Bl air A., Freeman L.B., Lynch C.F., Allen R.H., Alavanja M.C., Vermeulen R. (2009). Exposure to atrazine and selected non-persistent pesticides among corn farmers during the growing season. J Expo Sci Environ Ep idemiol., Sep; 19(6): 544-54.

4. Belloni V, Alleva E, et al. (2007). Effects of low doses of atrazine on the neurobehavioural development of mice. Et hology Ecology & Evolution, 19: 309-

22.

5. Benachour, N., Moslemi, S., Sipahutar, H., & Seralin i G.E. (2007). Cytotoxic effects and aromatase inhibition by xenob iotic endocrine disrupters alone and in combination, Toxicol Appl Ph armacol, Jul 15; 222(2): 129-40.

6. Betancourt, M., Resendiz, A., & Fierro, E. C. (2006). Effects of two insecticides and two herbicides on the porcine spe rm motility patterns using computerassisted semen analysis (CASA) in vitro . Reproductive Toxicology, Oct, 22(3):

508-12.

7. Boobis, A.R., J.E. Doe, B. Heinrich- Hirsch et al. 2008. IPCS framework for analyzing the relevance of a noncancer m ode of action for humans. Crit Rev Toxicol, 38(2):87-96.

8. Breckenridge, C.B., Yi, K. D., & Simpkins, J.W. (2010a). Cytotoxicity of atrazine

and its metabolites in vitro . Unpublished data from Syngenta Crop Protection,

Inc.

9. Breckenridge, C.B., Yi, K.D., Perez, E.J., & Simpki ns, J.W. (2010b). Dose and time dependent effects of atrazine and metabolites on aromatase mRNA in multiple cell types in vitro . Unpublished data from SyngentaPage 249 of 263 10. Breckenridge, C.B., Yi, K. D., Perez, E.J., & Simpkins , J.W. (2010c). Ex vivo

assessment of the effects of atrazine or its metabolites on phosphodiesterase activity. Unpublis hed data from Syngenta

11. Cimino-Reale, G., Ferrario, D., Casati, B., Brustio, R., Diodovich, C., Collotta, A., Vahter, M., & Gribaldo, L. (2008). Comb ined in utero and juvenile exposure of

mice to arsenate and atrazine in drin king water modulates gene expression and clonogenicity of myeloid progenitors. Toxicol Lett, Jul 30; 180(1): 59-66.

12. Coban, A., & Filipov, N.M. (2007). Dopam inergic toxicity associated with oral exposure to the herbicide atrazine in juvenile male C57BL/6 mice. J. Neurochem, Mar, 100(5): 1177-87.

13. Coder, Pragati Sawhney (Syngenta) ( 2010). Atrazine-An oral developmental toxicity study of atrazine in the rat, including cross-foster ing and pair-feeding. Unpublished data from Syngenta, 3000 pgs

14. Cooper R.L., Laws S., Das P.C., Narotsky M.G., Go ldman J.M., Tyrey E.L., Stoker T.E. (2007). Atra zine and reproductive function: mode and mechanism of

action studies. Birth Defects Res B Dev Reprod Toxicol, Apr; 80(2): 98-112.

15. Crawford, C.G. 2004. Sampling Strat egies for Estimating Acute and Chronic Exposures of Pesticides in Streams. Journal of the American Water Resources Association (JAWRA) 40(2):485-502.

16. Devos, S., Hooghe, R., Hooghe-Peters , E.L., De Bosscher, K., Vanden Berghe, W., Haegeman, G., Staels, B., Bauer, E., Roels, F., & Hooghe, R. (2003). Inhibition of cytokine production by the herbicide atrazine: Search for nuclear receptor targets. Biochem P harmacol, Jan 15; 65(2): 303-8.

17. Devos S., Van Den Heuvel R., et al. (2004). Limited effect of selected organic pollutants on cytokine production by peripheral blood leukocytes. Euro Cytokine Netw, Apr-Jun; 15(2): 145-51.

18. Dooley, G., Hanneman, W., Carbone, D., Legare, M., Anderson, M., & Tessari, J. (2007). Development of an immunochem istry detection method for atrazineinduced albumin adducts. Chem Res Toxicol, Jul; 20(7): 1061-6.

19. Dooley, G., Prenni, J., Pr entiss, P., Cranmer, B., A nderson, M., & Tessari, J. (2006). Identification of a novel hemoglobin adduct in Sprague-Dawly rats exposed to atrazine. Chem Re s Toxicol, May; 19(5): 692-700.

20. Dooley, G.P., Reardon, K.F., Prenni, J. E., Tjalkens, R.B., Legar e, M.E., Foradori, C.D., Tessari, J.E., & Hanneman, W. H. (2008). Proteomic analysis ofPage 250 of 263 diaminocholorotriazine adducts in Wistar rat pituitary glands and LbetaT2 rat pituitary cells. Chem Res Toxicol, Apr, 21(4): 844-51.

21. Enoch, R.R., Stanko, J.P., Greiner, S.N., Youngblood, G.L. , Rayner, J.L., & Fenton, S.E. (2007). Mamma ry gland development as a se nsitive end point after acute prenatal exposure to an atrazine metabolite mixture in female Long-Evans

rats. Environ Health Pe rspect, Apr, 115(4): 541-7.

22. Fan, W., Yanase, T., Morinaga, H., G onda, S., Okabe, T., No mura, M., Hayes, T., Takayanagi, R., & Nawata, H. (2007a). Herbicide atrazine activates SF-1 by direct affinity and concomitant co-activ ators recruitments to induce aromatase expression via promoter II. Bioc hemical and Biophysical Research Communications, 355(4): 1012-8.

23. Fan, W., Yanase, T., Morinaga, H ., Gondo, S., Okabe, T., Normura, M., Komatsu, T., Moroashi, K., Hayes, T.B., Takayanagi, R., & Nawata, H. (2007b). Atrazine-induced aromatase expression is SF-1 dependent: implications for endocrine disruption in wildlife and reproduc tive cancers in humans. Environ Health Perspect, May, 115(5): 720-7.

24. FIFRA Scientific Advisory Panel (SAP). (2003). “Characterization of Epidemiology Data Relating to Prosta te Cancer and Exposure to Atrazine.” Report from the FIFRA Scientific Advisory Panel Meeting of July, 2003. FIFRA Scientific Advisory Panel, Office of Sci ence Coordination and Policy, Office of Prevention, Pesticides and Toxic Substances, U.S. Environmental Protection Agency. Washington, DC. Available: http://www.epa.gov/scipoly/sap/t ools/atozind ex/atrazine.htm

25. Filipov, N.M., Pinchuk, L.M., Boyd, B.L., & Crittenden P.L. ( 2005). Immunotoxic effects of short-term atrazine exposure in young male C57BL/6 mice. Toxicol Sci, Aug; 86(2): 324-32.

26. Filipov, N.M., Stewart, M. A., Carr, R.L., & Sistrunk, S.C. (2007). Dopaminergic toxicity of the herbicide atrazine in rat striatal slices. Toxicology, Mar 22; 232(1- 2): 68-78.

27. Foradori, C., Hinds, L., Hanneman, W., & Handa, R. (2009a). Effects of atrazine and its withdrawal on gonadotropin-releas ing hormone neuroendocrine function

in the adult female Wistar rat. Biol Reprod, Dec, 81(6): 1099-105.

28. Foradori, C., Hinds, L., Hanneman, W., Legare, M., Clay, C., & Handa, R. (2009b). Atrazine inhibits pulsatile lute inizing hormone release without altering pituitary sensitivity to a gonadotropin-re leasing hormone receptor agonist in

female Wistar rats. Biol Reprod, Jul; 81(1): 40-5.Page 251 of 263 29. Fraites, M.J.P., Cooper, R.L., Buckalew, A., Jayaraman, S., Mills, L., and Laws, S.C. (2009). Characterization of the Hypothalamic-Pituitary-Adrenal Axis Response to Atrazine and Metabolites in the Female Rat. Tox. Sci. 112(1):88-

99.

30. Fujimoto, N. and Honda, H. (2003). Effects of environmental estrogenic compounds on growth of a transplanted estrogen responsiv e pituitary tumor cell

line in rats. Food Chem To xicol, Dec, 41(12): 1711-7.

31. Fukamachi, K., Han, B.S., Kim, C.K ., Takasuka, N., Matsuoka, Y., Matsuda, E., Yamasaki, T., and Tsuda, H. (2004). Possible enhancing effects of atrazine and nonylphenol on 7,2-dimethylbenz[a] anthracene-induced mammary tumor development in human c-Ha-ras proto- oncogene transgenic rats. Cancer Sci., May; 95(5): 404-10.

32. Gilliom R.J., Barbash, J.E., Crawford, C.G., Ham ilton, P.A., Martin, J.D., Nakagaki, N., Nowell, L.H., Scott, J.C., St ackelberg, P.E., Thelin, G.P. & Wolock, D.M. (2006). The quality of our Nation’s waters—Pesticides in the Nation’s streams and ground water, 1992-2001. U.S. Geological Survey Circular 1291, 172 p. Available at http://pubs.usgs.gov/circ/2005/1291/pdf/circ1291.pdf

33. Giusi, G., Facciolo, R.M., Canoaco, M., Alleva, E., Santucci, D., Belloni, V., & Dessi-Fulgheri, F. (2006). The endocrine disruptor atrazine accounts for a dimorphic somatostatinergic neuronal expre ssion pattern in mice. Toxicol Sci, Jan; 89(1): 257-64.

34. Gojmerac, T., Pleadin, J., Zuric, M., Rajkovic-Janje, R., & Korsic, M. (2004). Serum luteinizing hormone response to administration of gonadotropin-releasing hormone to atrazine-treated gilts. Ve t Hum Toxicol, Oct, 6(5): 245-7.

35. Hase, Y., Tatsuno, M., Nishi, T., Kabe, Y., Yamaguchi, Y., Hanada, H., Kataoka, K., Ozawa, N., Natori, M., Handa, H., & Wantabe, H. (2 008). Atrazine binds to F1F0-ATP synthase and inhibits mitoc hondrial function in sperm. Biochem Biophys Res Commun, Feb 1; 366(1): 66-72.

36. Heneweer, M., Van den Berg, M., & Sander son, J.T. (2004). A comparison of

human H295R and rat R2C cell lines as in vitro screening tools for effects on

aromatase. Toxicol Le tt, Jan 15; 146(2): 183-94.

37. Higley, E.B., Newsted, J.L., Zhang, X., Giesy, J.P., & Hecker, M. (2010).

Assessment of chemicals effects on aromatas e activity using the H295R cell line. Environ Sci Pollut Res Int., Jan 20, Epub ahead of print

38. Hocking, B. (2009). Mela mine-contaminated powdered fo rmula and urolithiasis. N Engl J Med., June 18; 360(25): 2676-7.Page 252 of 263 39. Hoehn K., Salmon A., Hohnen-Behrens C., Turner N., Ho y A., Maghzal G., Socker R., Van Remmen H., Kraegen E., Cooney G., Richardson A., James D. (2009). Insulin resistance is a cellular antioxidant defense mechanism. Proc Natl Acad Sci U S A, Oct 20 106(42):17787-92.

40. Holloway, A.C., Anger, D.A., Crankshaw , D.J., Wu, M., & Fo ster, W.G. (2008). Atrazine-induced changes in aromatase ac tivity in estrogen sensitive target tissues. J. Appl. Toxico l., Apr, 28(3): 260-70.

41. Hossain, M. M. & Filipov, N. M.(2008). Alteration of dopamine uptake into rat striatal vesicles and synaptosomes caused by an in vitro exposure to atrazine

and some of its metabolites. To xicology, Jun 3; 248(1): 52-8.

42. Judson R.S., Houck K.A., Kavlock R.J., K nudsen T.B., Martin M.T ., et al. (2009).

In vitro Screening of Environmental Chemical s for Targeted Testing Prioritization - The Tox Cast Project. Envir on Health Perspect, doi:10.1289/ehp.0901392

43. Juliani, C.C., Silva-Zacari n, E.C., Santos, D.C., & B oer, P.A.(2008). Effects of atrazine on female Wistar rats: morpholog ical alterations in ovarian follicles and immunocytochemical labeling of 90 kDa heat shock protein. Micron, Jul, 39(5):

607-16.

44. Kandori, H., Suzuki, S., Asamoto, M. , et al. (2005). Influence of atrazine

administration and reduction of calorie in take on prostate carcinogenesis in probasin/SV40 T antigen transgenic rats. Cancer Sci, Apr; 96(4): 221-6.

45. Karrow, N.A., McCay, J.A., Brown, R. D., Musgrove, D.L., Guo, T.L., Germolec, D.R., & White, K.L. Jr ( 2005). Oral exposure to at razine modulates cell-mediated

immune function and decreases host resist ance to the B16F10 tumor model in

female B6C3F1 mice. Toxi cology, Apr 1; 209(1): 15-28.

46. Kim, D., Breckenridge, C. (Syngenta) ( 2010). Atrazine - Derivation of the shortterm inhalation NOAEL using a physiologi cally-based pharmacokinetic model. Unpublished data from Syngenta

47. Kirby E.D., Geraghty A.C., Ubuka T., B entley G.E., Kaufer D. (2009). Stress increases putative gonadotropin inhibito ry hormone and decreases luteinizing

hormone in male rats. Proc Natl Acad Sci USA, 106( 27): 11324-9.

48. Kmetic, I., Gaurina Srcek, V., Silvac, I., Simic, B., Kniewald, Z., & Kniewald, J. (2008). Atrazine exposure decreases cell proliferation in Chinese Hamster Ovary (CHO-K1) cell line. Bull Environ C ontam Toxicol., Aug, 81(2): 205-9.

49. Kniewals, J., Kmeti č , I., Gaurina Sr č ek, V., Šimi ċ ,B., & Kniewald, Z. (2003). Cytotoxicity of Herbicide Atrazine On Chinese Hamster ovary cell Line In Monolayer. Toxicol Lett, S ept; 144 (Suppl 1):S111-S112.Page 253 of 263 50. Larson, S.J., C.G. Crawford, and R. J. Gilliom. (2004). Development and application of Watershed Regressions fo r Pesticides (WARP) for estimating

atrazine concentration distributions in st reams. U.S. Geolog ical Survey WaterResources Investigations Repor t 03–4047, 68 p. Available at http://pubs.usgs.gov/wri/wri034047/

51. Laville, N., Balaguer, P., Br ion, F., Hinfray, N., Casella s, C., Porcher, J.M., & AitAissa, S. (2006). Modulation of arom atase activity and mRNA by various selected pesticides in the human chorioca rcinoma JEG-3 cell line. Toxicology, Nov 10; 228(1): 98-108.

52. Laws, S.C., Ferrell, J.M., Stoker, T.E., & Cooper, R.L. (2003). Pubertal development in female Wistar rats following exposure to propazine and atrazine biotransformation by-products, diamino-S- chlorotriazine and hydroxyatrazine. Toxicol Sci, Nov; 76(1): 190-200.

53. Laws, S.C., Hotchkiss, M., Ferrell, J., Jayaraman, S., Mills, L., Modic, W., Tinfo, N., Fraites, M., Stoker, T., & Cooper, R. (2009). Chlorotriazine Herbicides and Metabolites Activate an ACTH-Dependent Release of Corticosterone in Male Wistar Rats. Tox. Sci., 112(1): 78-87.

54. Laws, S.C., Stoker, T.E., Ferrell, J.M. , Hotchkiss, M.G., & Cooper R.L. (2007). Effects of Altered Food Intake During Pubertal Development in Male and Female Wistar Rats. Toxicol. Sci., Nov; 100(1): 194-202.

55. Lee, H.K. (2004). Baye sian Nonparametrics vi a Neural Networks. Philadelphia:

ASA/SIAM.

56. Lim, S., Ahn, S.Y., Song, I.C., Chung, M.H., Jang, H.C. , Park, K.S., Lee, K., Pak, Y.K., & Lee, H.K. (2009). Chronic exposure to the her bicide, atrazine, causes mitochondrial dysfunction and insulin resi stance. PLOS One, 4(4): 1-11.

57. Malik, S.I., Terzoudi, G.I ., & Pantelias, G.E. (2004). SCE analysis in G2 lymphocyte prematurely condensed chromoso mes after exposure to atrazine: the non-dose-dependent increase in homologous recombinational events does not support its genotoxic mode of action. Cytogenet and Genome Research, 104(1- 4): 315-9.

58. Manske, M., Beltz, L, and Dhanwada, K. (2004). Low-Level Atrazine Exposure

Decreases Cell Proliferation in Human Fibroblasts. Arch. Environ. Contam. Toxicol., 46: 438-44.

59. Maravilla-Galvan, R., Fierro, R., Gonzalez-Marquez, H., Gomez-Arroyo, S., Jimenez, I., & Betancourt, M. (2009). Effe cts of atrazine and fe noxaprop-ethyl onPage 254 of 263 capacitation and the acrosomal reaction in boar sperm. Int. J. Toxicol., Jan-Feb, 28(1): 24-32.

60. McMullin T.S. (2003). Pharmacokinetic modeling of disposition and time-course studies with [ 14 C]-atrazine. J. Toxicol. Environ. Health, May 23;66(10):941-64.

61. McMullin, T.S., Andersen, M.E., H anneman, W.H., Nagahara, A., Lund, T.D., Pak, T., & Handa, R.J. (2004). Evidence that atrazine and diaminochlorotriazine inhibit the estrogen/progesterone induced sur ge of luteinizing hormone in female Sprague-Dawley rats without changing estro gen receptor action. Toxicol Sci, Jun; 79(2): 278-86.

62. McMullin, T.S., Hanneman, W.H., Cranmer , B.K., Tessari, J.D., and Andersen, M.E. (2007). Oral absorpt ion and oxidative metabolism of atrazine in rats evaluated by physiologically modeling approaches. Toxicol, Oct 30; 240(1-2): 1-

14.

63. Meek, M.E., J.R. Bucher, S.M. Cohen et al. (2003). A framework for human relevance analysis of information on carc inogenic modes of action. Crit Rev Toxicol, 33(6): 591-653.

64. Mizota, K. & Ueda, H. (2006). Endocrine disrupting chemical atrazine causes degranulation through Gq/11 prot ein-coupled neurosteroid receptor in mast cells. Toxicol Sci., Apr, 90(2): 362-8.

65. Modic, MW. (2004). The role of testic ular aromatase in t he atrazine mediated changes in estrone and estradiol in the ma le wistar rat. North Carolina State University.

66. Mu, H., Zhang, P., & Xu, J. (2008). Testicular toxicity and mechanisms of chlorotoluron compounds in the mouse. Toxicology Mechanisms and Methods, 18(5): 399-403.

67. Pinchuk, L.M., Lee, S.R ., & Filipov, N.M. (2007). In vitro atrazine exposure affects the phenotypic and functional matura tion of dendritic cells. Toxicology and Applied Pharmacology, Sept, 223(3): 206-17.

68. Pogrmic, K., Fa, S., Dakic, V., Kaisarev ic, S. & Kovacevic, R. (2009). Atrazine oral exposure of peripubertal male rats downregulates steroidogenesis gene expression in Leydig cells. Toxicol. Sci., 111(1): 189-97.

69. Pruett, S.B., Fan, R., Zheng, Q., Myers, L.P., & Hebert, P. (2003). Modeling and predicting immunological effe cts of chemical stressors : characterization of a quantitative biomarker for immunologica l changes caused by atrazine and ethanol. Toxicol Sci., Oct, 75(2): 343-54.Page 255 of 263 70. Pruett, S.B., Fan, R., Zheng, Q., & Schwab, C. (2009). Patterns of immunotoxicity associated with chronic as compared with acute exposures to chemical or physical stressors and their relevance with regard to the role of stress and with regard to immunotoxicity te sting. Toxicological Sciences, June, 109(2): 265-75.

71. Rayburn, A.L., Moody, D. D., and Freeman, J.L. (2005). Cytotoxicity of technical grade versus formulations of atrazine and acetochlor using mammalian cells. Bull. Environ. Contam. Toxicol., 75: 691-8.

72. Rayner, J.L., Wolf, D.C., Rayner, J.L., Enoch, J.L., Enoch, R.R., & Fenton, S.E. (2007). Atrazine-induced reproductive trac t alterations after transplacental and/or lacational exposure in male Long- Evans rats. Toxicology and Applied Pharmacology, Feb, 218(3): 238-48.

73. Rayner, J. L., Wood, C., Fenton, S. E. (2004). Exposure parameters necessary for delayed puberty and mammary gland development in Long–Evans rats exposed in utero to atrazine. Toxicol Appl Pharmacol, Feb 15; 195(1): 23-34.

74. Rivest, P. & Sanderson, J. (2010). Effects of pesticides on aromatase expression in bioluminescent mice and GSK 3BETA/BETA-signalling in LNCAp human prostate cancer cells. The Toxicologist 114, 405.

75. Rodriguez, V.M., Thiruchelvam, M., & Cory-Slechta, D.A. (2005). Sustained exposure to the widely used herbicide atrazine: Altered function and loss of

neurons in brain monoamine systems. Environmental Health Perspectives, Jun, 113(6): 708-15.

76. Rooney, A.A., Matulka, R.A., & Luebke, R.W. (2003). Developmental atrazine exposure suppresses immune function in male, but not female Spragus-Dawley rats. Toxicol Sci., Dec, 76(2): 366-75.

77. Rosenberg, B.G., Chen, H., Folmer, J., Liu, J., Papadopoulos, V., & Zirkin, B.R. (2008). Gestational exposure to atrazine: effects on the postnatal development of male offspring. J A ndrol., May-June; 29(3): 304-11.

78. Ross, M.K., Jones, T.L., and Filipov, M.N. (2009). Disposition of the herbicide 2- chloro-4-(ethylamino)-6-(isopropylamino)-s -triazine (atrazine) and its major metabolites in mice: a liquid chromat ography/mass spectrometry analysis of urine, plasma, and tissue levels. Dr ug Metab. Dispos., Apr; 37(4): 776-86.

79. Rowe, A.M., Brundage, K.M., & Bar nett, J.B. (2008). Developmental immunotoxicity of atrazine in rodents. Basic Clin Pharmacol Toxicol, Feb; 102(2): 139-45.Page 256 of 263 80. Rowe, A.M., Brundage, K.M ., & Barnett, J.B. (2007). In vitro atrazine exposure inhibits human natural killer cell lytic gr anule release. Toxicol Appl Pharmacol, Jun 1; 221(2): 179-88.

81. Rowe, A.M., Brundage, K.M., Schafer , R., & Barnett, J.B. (2006). Immunomodulatory effects of maternal atrazine exposure on male Balb/c mice. Toxicol Appl Pharmacol, July 1; 214(1): 69-77

82. Rusiecki, J.A., De Roos, A., Lee, W.J. , Dosemci, M., Lubin J.H., Hoppin, J.A., Blair, A., & Alavanja, M. C. (2004). Cancer incidenc e among pesticide applicators exposed to atrazine in the Agricultural Heal th Study. J Natl C ancer Inst, Sept 15; 96(18): 1375-82.

83. Sanderson, J.T., Boerma, J., Gideon, W.A., Lansbergen, G.W. & van den Berg, M. (2002). Induction and in hibition of aromatase (CYP 19) activity by various classes of pesticides in H295R human adrenocortical carcinoma cells. Toxicol Appl Pharmacol. Jul 1; 182: 44-54.

84. Santen R.J., Brodie H., Simpson E.R., S iiteri P.K., Brodie A.. (2009). History of Aromatase: saga of an impor tant biological m ediator and ther apeutic target. Endocr Rev, Jun; 30(4): 343-75.

85. Schwab, C. L., Fan R., Zheng, Q., Myer s, L.P., Hébert,P., & Pruet, S.B. (2005). Modeling and predicting stress-induced immunosuppression in mice using blood parameters. Toxicol Sc i, Jan; 83(1): 101-13.

86. Seed, J., E.W. Carney, R.A. Corley et al. (2005). Overvi ew: Using mode of action and lifestage information to evaluate the human relevance of animal toxicity data. Crit. Rev. To xicol., Oct-Nov; 35(8-9):664-72.

87. Shibayama, H., Kotera, T., Shinoda, Y., Hanada, T., Kajhara, T., Ueda, M., Tamura, H., Ishibashi, S., Yamashita, Y ., & Ochi, S. (2009). Collaborative work on evaluation of ovarian toxicity. 14) Tw o- or four-week repeated-dose studies and fertility study of atrazine in female rats. J toxicol Sci., 34: 147-55.

88. Singh, M., Kaur, P., Sandhir, R., & Kiran, R. (2008). Pr otective effects of vitamin E against atrazine-induced genotoxicity in ra ts. Mutat Res, Jul 31; 654(2): 145-9.

89. Singh, M., Sandhir, R., & Kiran, R.(2008). Atrazine- induced alterations in rat erthrocyte membranes: ameliorating efect of vitamin E. J Biochem Mol Toxicol, Sept, 225: 363-9.

90. Son, H.Y., Nishikawa, A., Okazaki, K., Lee, K., Imazawa, T., & Hirose, M. (2003). Lack of modifying effects of atrazine and/ or tamoxifen on thyroid carcinogenesis in rat pretreated with N-bis(2-hydroxyp ropyl)nitrosamine (DHPN). Food Chem Toxicol, Dec; 41(12): 1811-6.Page 257 of 263 91. Stoker T.E & Cooper R.L. (2007). Distribution of 14C-atrazine following an acute lactational exposure in the Wistar ra t. Reprod Toxicol, Jun; 23(4):607-10.

92. Stone, W.W. & Gilliom, R.J. (2009). Update of watershed regressions for pesticides (WARP) for predicting atrazi ne concentration in streams. U.S. Geological Survey Open-File Re port 2009–1122, 22 p. Available at http://pubs.usgs.gov/of/2009/1122/

93. Stone, W.W., Gilliom, R. J. & Crawford, C.G. (2008). Watershed Regressions for Pesticides (WARP) for Predicting Annual Maximum and Maximum MovingAverage Concentrations of Atrazine in St reams. U.S. Geological Survey OpenFile Report 08-1186, 19 p. Available at http://pubs.usgs.gov/of/2008/1186/

94. Suzawa, M. & Ingraham, H.A. (2008). The herbicide atrazine activates endocrine gene networks via non-steroidal NR5A nucl ear receptors in fish and mammalian cells. PloS ONE, 3(5): 1-11.

95. Tanaka, T., Kohno, H., Suzuki, R., & Sugi e, S. (2004). Lack of modifying effects of an estrogenic compound atrazine on 7, 12-dimethylbenz(a)anthracene-induced ovarian carcinogenesis in rats. Cancer Lett, Jul 16; 210(2): 129-37.

96. Tinfo, N., & Laws, S.C. (2009). Understanding the Effects of Atrazine on Steroidogenesis in Wistar rats. Soci ety of Toxicology, Itinerary Planner, Baltimore, MD.

97. Ueda, M., Imai, T., Takizawa, T., O nodera, H., Mitsumori, K., Matsul, T., & Hirose, M. (2005). Possibl e enhancing effects of atrazine on growth of 7,12- dimethylbenze(a) anthracene-induced mammary tumors in ovariectomized Sprague-Dawley rats. Cancer Sci., Jan, 96(1): 19-25.

98. U.S. Environmental Protection Agency. (2010). DRAFT Framework for Incorporating Human Epidemio logic & Incident Data in Health Risk Assessment (Draft dated January 7, 2010). Presented to the FIFRA Scientific Advisory Panel, February 2-4, 2010. http://www.r egulations.gov (Docket EPA-HQ-OPP-2009- 0851).

99. U.S. Environmental Protection Agency (USEPA). (2009a). The Ecological Significance of Atrazine Effects on Prim ary Producers in Surface Water Streams in the Corn and Sorghum Growing Region of the United States (Part II). Prepared for the May 12 - 15, 2009 FIFRA Scientific Advisory Panel. Document available

at http://www.epa.gov/scipoly/sap/ meetings/2009/051209meeting. html

100. U.S. Environmental Protection Agency (USEPA). (2009b). The Ecological Significance of Atrazine Effects on Prim ary Producers in Surface Water Streams in the Corn and Sorghum Growing Region of the United States (Part II).Page 258 of 263 Addendum to Section VI: Additional Analysis of the 40 AEEMP Sampling Sites to Identify Additional Areas That May Exce ed the LOC. Prepared for the May 12 - 15, 2009 FIFRA Scientific Advisory Panel. Document available at http://www.epa.gov/scipoly/sap/m eetings/20 09/051209meeting.html

101. U.S. Environmental Protection Agency (USEPA). (2007a). Acetochlor Registration Agreement and Monitoring Program. Docum ents are available through the Federal docket. Docket ID : EPA-HQ-OPP-2007-0725. Available at http://www.regulations.gov/search/Regs /ho me.html#docketDetail?R=EPA-HQOPP-2007-0725

102. U.S. Environmental Protection Agency (USEPA). (2007b). N-methyl

Carbamate Revised Cumulative Risk A ssessment. Available through the USEPA Office of Pesticide Programs web site at http://www.epa.gov/oppsrrd1/REDs/nmc_revised_cra.pdf

103. U.S. Environmental Protection Ag ency (USEPA). (2007c). Preliminary Interpretation of the Ecological Si gnificance of Atrazine Stream-Water Concentrations Using a Statistically-Desi gned Monitoring Program. Prepared for the December 4 - 7, 2007 FI FRA Scientific Advisory Panel. Document available

at http://www.epa.gov/scipoly/sa p/meetings/2007/120407_mtg.htm

104. US EPA. (2006). Decision Docum ents for Atrazine. Atrazine IREDs (January and October 31, 2003) and Atra zine RED (April 6, 2006). EPA-HQOPP-2003-0367.

105. US EPA. (2005). Guidelines fo r Carcinogen Risk Assessment. U.S. Environmental Protection Agency, Risk Assessment Forum, Washington, DC.

EPA/630/P-03/001F. Federal Register 70(66):17765-1781 7. Available at http://www.epa.gov/raf

106. US EPA. (2003). Interim Re-regist ration Eligibility Decision (IRED)

107. Victor-Costa, A.B., Carozzi Band eira, S.M., Olivei ra, A.G., Bohorquiz Mahecha, G.A. & Oliveira, C.A. (2010). Changes in testicular morphology and steroiodogenesis in adult rats exposed to at razine. Reprod Toxicol, Jan 4. [Epub ahead of print] Doi:10.1016/j. reprotox.2009.12.006

108. Whalen M.M., Loganathan B.G., Yamash ita, N., Saito, T. (2003). Immunomodulation of human natural killer cell cytot oxic function by triazine and carbamate pesticides. Chemico-Biological Interactions, Jun 15; 145(3): 311-19.

109. Zeljeciz D, & Garaj-Vrhovac, V. (2004a). Atrazine genotoxicity evaluation in different mouse organs by comet assa y. Periodicum Bi ologorum, 106(2): 155-

9.Page 259 of 263 110. Zeljezic, D. & Garaj-Vrhovac, V. (2004b). Genotoxicity evaluation of pesticide formulations containing alachlor and atrazine in multiple mouse tissues (blood, kidney, liver, bone marrow, spleen) by comet assay. Neoplasma, 51(3):

198-203.

111. Zeljezic, D., Garaj-Vrhovac, V., & Perkovic, P. (2006). Evaluation of DNA damage induced by atrazine and atrazine- based herbicide in human lymphocytes

in vitro using a comet and DNA diffusion assay. Toxicol In vitro , Sept; 20(6):

923-35.

112. Zorrilla, L.M., Gibson, E.K., Stoker (2009) Effect of Chlorotriazine Pesticides on Gonadotrophi n Releasing Hormone in the Neuronal GT1-7 Cell Line and Hypothalamic Explants

. Annual Meeting of the Society for Study of Reproduction, Pittsburgh, PA. Biol Reprod, 81: 638.

Pre-2003 References

1. Blomquist, J.D., Denis, J. M., Cowles, J.L., Hetrick, J. A., Jones, R.D. & Birchfield N.B. (2001). Pesticides in Selected Water-Supply Reservoirs and Finished Drinking Water, 1999-2000: Summary of Results from a Pilot Monitoring Program. U.S. Geological Survey O pen-File Report 01-456. Published in cooperation with the U.S. Envi ronmental Protection Agency

2. Cooper, R.L., Stoker, T.E., Goldman, J. M., Parrish, M.B, & Tyrey, L. (1996).

Effect of atrazine on ovarian function in the rat. Reprod Toxicol, Jul-Aug; 10(4):

257-64.

3. Cooper, R.L., Stoker, T.E., Tyrey, L., Goldman, J.M., & McEl roy, W.K. (2000). Atrazine disrupts the hypothalam ic control of pituitary-ov arian function. Toxicol Sci., Feb; 53(2): 297-307

4. Das, P.C., McElroy, W. K. & Cooper, R.L. (2001). Al teration of Catecholamines in Pheochromocytoma (PC12) Cells in vitro by the Metabolites of Chlorotriazine

Herbicide. Toxicol Sc i, Jan; 59(1): 127-37.

5. Deutsch, C.V. & Journel, A.G. (1998). GSLIB: Geostatistical Software Library

and User’s Guide. Second Edition. Oxford University Press (N ew York, Oxford).

6. Eldridge, J.C., Wetzel, L.T., & Tyre y, L. (1999). Estrous cycle patterns of Sprague-Dawley rats during acute and chroni c atrazine administration. Reprod Toxicol, Nov-Dec; 13(6): 491-9.Page 260 of 263 7. FIFRA Scientific Advisory Panel (SAP) . (2000a). “Atrazine: Hazard and DoseResponse Assessment and Characterization.” Report from the FIFRA Scientific Advisory Panel Meeting of June 27-29, 2000. FIFRA Scientific Advisory Panel, Office of Science Coordination and Policy, Office of Prevention, Pesticides and Toxic Substances, U.S. Environmental Protection Agency. Washington, DC.

Available: http://www.epa.gov/scipoly/sap/t ools/atozind ex/atrazine.htm

8. FIFRA SAP. (2000b). "Atrazine :Hazard and Dose-Response Assessment and Characterization." Report fr om the FIFRA Scientific Ad visory Panel Meeting of June 27-29, 2000. FIFRA Scie ntific Advisory Panel, Office of Science Coordination and Policy, Offi ce of Prevention, Pestic ides and Toxic Substances, U.S. Environmental Protection Agency. Washington, DC. SAP Report 2000-05.

Available: http://www.epa.gov/scipoly/sap/meet ings/20 00/june27/finalatrazine.pdf

9. FIFRA Scientific Advisory Panel ( SAP). (1988). “A set of issues being considered by the Agency in connection with the peer review classification of atrazine a class C carcinogen.” Report from the FIFRA Scientific Advisory Panel Meeting of September 14, 1988. FIFRA Scientific Advisory Panel, Office of Science Coordination and Policy, Office of Prevention, Pe sticides and Toxic Substances, U.S. Environmental Pr otection Agency. Washington, DC.

Available: http://www.epa.gov/scipoly/sap/1988 *

10. Friedmann, A.S. (2002). Atrazine inhibition of testosterone production in rat males following peripubertal exposure. R eprod. Toxicol, May-Jun; 16: 275-9.

11. Gao H.B., Shan L.X., Monder C., Hardy M.P. (1996). Suppression of endogenous corticosterone levels in vivo in creases the steroidogenic capacity of purified rat Leydig cells in vitro . Endocrinology, May; 137(5):1714-8.

12. ILSI (International Life Sciences Inst itute), (1999). A Framew ork for Estimating

Pesticide Concentrations in Drin king Water for Aggregate Exposure

Assessments. International Life Sciences Institute Risk Science Institute Working Group Report, Washington, D. C., 61 pp. Available at http://www.ilsi.org/Research Foun dation/Publications/Estimating%20Pesticide%2 0Concentrations.pdf

13. Isaaks, E.H., and R.M. Srivastava. 1989. Applied Geostati stics. Oxford University Press (New York, Oxford).

14. Kalra, S.P., & Kalra, P.S.(1983). Neural regulati on of luteinizing hormone

secretion in the rat. E ndocr Rev., Fall; 4(4): 311-51.

15. Laws, S. C., Ferrell, J. M. , Stoker, T. E., Schmid, J. & Cooper, R. L. (2000). The

Effects of Atrazine on Female Wistar Ra ts: An Evaluation of the Protocol for Assessing Pubertal Development and Thyroid Function. Toxicol Sci, 58: 366-76.Page 261 of 263 16. Lyons, W.R., Berlin, I., and Friendland er, S. (1942) Corn ification of balanopreputial epithelium in normal rats and ca strated rats treated with testosterone proprionate. Endocrinology, 31(6): 659-63.

17. Marshall, F. (1966) Marshall’s Physi ology of Reproduction. Longmans, Green

and Co. LTD

18. Matsumoto AM, Karpas AE, Southwor th MB, Dorsa DM, Br emner WJ. (1986). Evidence for activation of the central nervous system-pituitary mechanism for gonadotropin secretion at the ti me of puberty in the male rat. Endocrinology, Jul; 119(1): 362-9.

19. Molcrani M.C., Duval F., Crocq M.A., Bailey P., Macher J.P. (1997). HPA axis dysfunction in depression: Correlati on with monoamine system abnormalities. Psychoneuroendocrinology, 22, Suppl 1: S63-8

20. Monosson E, Kelce WR, Lambr ight C, Ostby J, Gray LE Jr. (1999). Peripubertal exposure to the antiandrogenic fungicide, vi nclozolin, delays puberty, inhibits the development of androgen-dependent tissues, and alters androgen receptor

function in the male rat. Toxi col Ind Health, 15(1-2): 65-79.

21. Morseth S. (1996) Evaluat ion of the Luteinizing Hormone (LH) Surge in AtrazineExposed Female Sprague-Dawley Rats--(Final) 6-Month Interim Report: Lab Project Number: CHV 2386-111: 2386-111: 6791E. Unpublished study prepared by Corning Hazleton In c. 727 p. MRID 44152102

22. Ojeda, S.R., and Urbanski, H.F. (1994). P uberty in the rat. In E. Knobil and J.D. Neill (Eds.): The Physiology of Reproduction, 2 nd ed. New York, Raven Press.

23. Rivest, .S & Rivier, C. (1995). The ro le of corticotropin-releasing factor and interleukin-1 in the regulation of neurons controlling reproductive functions. Endocr Rev, 16(2): 177-99.

24. Rivier, C. & Rivest, S. (1991) . Effect of stress on the activity of the hypothalamicpituitary-gondal axis: peripheral and cent ral mechanisms. Biol Reprod., October 1; 45 (4): 523-532

25. Rivier, C., Rivier, J., & Vale, W. (1986). Stress-induced inhibition of reproductive functions: role of endogenous corticotr opin-releasing factor. Science, 231(4738): 607-9.

26. Sanderson J.T., Boerma J., Lansber gen G.W., van den Berg M. (2002). Induction and inhibition of aromatase (CYP1 9) activity by various classes of pesticides in H295R human adrenocortical carcinoma cells. Toxicol Appl Pharmacol, Jul 1; 182(1): 44-54.Page 262 of 263 27. Sanderson J.T., Let cher R.J., Heneweer M., Giesy J.P., van den Berg M. (2001).

Effects of chloro-s-triazine herbicides and metabolites on aromatase activity in various human cell lines and on vitellogenin production in male carp hepatocytes. Environ health Perspe ct, Ovt; 109(10): 1027-31.

28. Sanderson J.T., Seinen W., Giesy J.P., van den Berg M. (2000). 2-Chloro-striazine herbicides induce aromatase (CYP19) activity in H295R human

adrenocortical carcinoma cells: a novel mechanisms for estrogenicity? Toxicol Sci, Mar; 54(1): 121-27.

29. Selye, H. (1939). Effect to adaptati on to various damaging agents on the female sex organs in the rat. Endocrinology, 25(4): 615-24.

30. Simpson, E.R., Clyne, C.D., Rubin, G ., Boon, W.C., Robertson, K., Britt, K., Speed, C., & Jones, M.E. ( 2002). Aromatase: a brief review. Annu Rev Physiol,

64: 93-127.

31. Simpson, E.R., & Davis, S.R. (2001). Minireview: aromatase and the regulation of estrogen biosynthesis – some new perspectives. Endocrinology, Nov; 142(11): 4589-94.

32. Sonich-Mullin, C., R. Fielde r, J. Wiltse et al. (2001). IPCS conceptual framework for evaluating a mode of action for chem ical carcinogenesis. Regul Toxicol Pharmacol, Oct; 34(2):146-52.

33. Stoker, T.E., Guidici, D.L., Laws, S.C. & Cooper, R.L. (2002). The Effects of Atrazine Metabolites on Puberty and Thyroid Function in the Male Wistar Rat: An

Evaluation in the Male P ubertal Protocol. Toxicol. Sci., 67(2): 198-206.

34. Stoker, T.E., Laws, S.C., Guidici, D ., & Cooper, R.L. (2000) . The effects of atrazine on puberty and thryroid function in t he male wistar rat: An evaluation of a protocol for the assessment of puberta l development and thyroid function. Toxicol. Sci., 58: 50-9.

35. Stoker T.E., Robinette, C.L. & Cooper , R.L. (1999). Maternal exposure to atrazine during lactation suppresses su ckling- induced prolactin release and results in prostatitis in the adult offspring. Toxicol Sci, 52: 68-79

36. Trentacoste, S.V., Friedm ann, A.S., Youker, R.T., Bre ckenridge, C.B. & Zirkin, B.R. (2001). Atrazine effects on te stosterone levels and androgen-dependent reproductive organs in peripubertal male rats . J. Androl, Jan-Feb; 22(1): 142-8.

37. US EPA. (1999). Guidelines for carcinogen risk assessment. Risk Assessment Forum. SAB review draft. Washington, DC: U.S. Enviro nmental Protection Agency. www.epa.gov/ncea/raf/crasab.htm . *Page 263 of 263 38. Wetzel L.T., Luempert L.G., III Breck enridge C.B., Tisdel M.O., Stevens J.T. (1994). Chronic effects of atrazine on estrus and mammary tu mor formation in female Sprague-Dawley and Fisher 344 rats. J Toxicol. Environ. Health, Oct; 43(2):169-82.