Do not cite

Re

- Evaluation of Human Health Effects of Atrazine:

Review of Non

- Cancer Epidemiology, Experimental Animal and

In vitro Studies and Drinking Water Monitoring Frequency

Presented Jointly To The FIFRA Scientific Advisory Panel By:

U.S. Environmental Prot ection Agency Office of Pesticide Programs

Health Effects Division

and

Environmental Fate and Effects Division

i n collaboration with the

Office of Research and Development

Presented On:

September 14

- 17 , 2010Do not cite

Page 2 of 677 TABLE OF CONTENTS

1. INTRODUCTION

................................ ................................ ............. 16

2. MODE OF ACTION (MOA) & EXPERIMENTAL TOXIC OLOGY

..... 21

2.1 Historical Bac kground

................................ ................................ .......................... 21
2.2 2010 Scientific Re

- Evaluation

................................ ................................ .............. 22 2.3 Mode of Action (MOA)

................................ ................................ ......................... 23 2.3.1 Background

................................ ................................ ................................ .. 23 2.3.2 Key Events

– Hypothalamic

- Pituitary

- Gonadal (HPG) Axis

.......................... 24 2.4 Summary

................................ ................................ ................................ ............... 27

3. EVALUATION OF NON

- CANCER EPIDEMIOLOGY LITERATURE

29

3.1 Overview

................................ ................................ ................................ ................ 29

3. 2 Literature Review

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

3.2.1 Reproductive System Effects

................................ ................................ ...... 30 3.2.1.1 Female Reproductive System

................................ ................................ ........... 30 3.2.1.2 Male Reproductive System

................................ ................................ ............... 32 3.2.2 Fetal and Perinatal Outcomes

................................ ................................ ....... 33 3.2.2.1 Spont aneous Abortion

................................ ................................ ...................... 33

3.2.2.2 Birth Defects

................................ ................................ ................................ ..... 34
3.2.2.3 Adverse Birth Outcomes

................................ ................................ ................... 37 3.2.3 Respiratory Effects

................................ ................................ ........................ 40 3.3 Synthesis of Epidemiological Data

................................ ................................ ........ 41
3.4 Conclusions

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

4. INTEGRATION OF EPIDE MIOLOGY & EXPERIMENT AL
TOXICOLOGY

................................ ................................ ........................ 55

5. PROPOSED UPDATES TO THE DOSE

- RESPONSE ASSESSMENT

60

5.1 Overview

................................ ................................ ................................ ............... 60 5.2 Dosimetry: Internal vs. External

................................ ................................ ........... 60 5.2.1 Background

................................ ................................ ................................ .. 60 5.2.2 Data Supporting the Use of Internal Dose in Dose Response Assessment

. 61 5.2.3 Temporal Aspects and Pharmacokinetic Modeling Efforts

........................... 62 5.2.4 The Area under the Triazine Equivalent Plasma

- Concentration Time Curve (AUC) as Internal Dose Metric

................................ ................................ ................. 63

5.2.5 The D aily Steady State AUC for Triazine Equivalents as Internal Dose Metric

................................ ................................ ................................ ................................ 65 5.2.6 Comparison of Different LH

- Surge Attenuation Studies that Likely Achieved Steady State Plasma Levels of Triazi ne Equivalents

................................ .............. 70 5.2.7 Dosimetry Summary

................................ ................................ ..................... 72 5.3 Benchmark Dose (BMD) Analysis

................................ ................................ ......... 73
5.3.1 Identification of Critical Studies

................................ ................................ .... 75

5.3.2 Methods & Results

................................ ................................ ....................... 75 5.4 Proposed Point of Departure

................................ ................................ ................ 80

6. SCIENTIFIC CONSIDERA TIONS IN POTENTIAL S ENSITIVITY OF

INFANTS & CHILDREN

................................ ................................ ......... 82Do not cite

Page 3 of 677 6.1 Background

................................ ................................ ................................ ........... 82 6.2 Experimental Toxicology

................................ ................................ ....................... 83 6.2.1 Gestational Exposure (Dosimetry and Effects)

................................ ............. 83

6. 2.2 Lactational Exposure (Dosimetry and Effects)

................................ .............. 86
6.2.3 Cross

- fostering Studies

................................ ................................ ................ 88

6.2.4 Peri

- pubertal Exposure

................................ ................................ ................. 89 6.3 Epidemiology

................................ ................................ ................................ ....... 90 6.4 Summary

................................ ................................ ................................ .............. 91

7. APPROACHES TO EVALUA TING WAT ER SAMPLING
STRATEGIES AND FREQU ENCY OF MONITORING

........................... 93

7.1. Introduction

................................ ................................ ................................ ........... 93 7.2 General Strategy For Designing a Monitoring Stud y to Characterize Drinking Water Exposures

................................ ................................ ................................ ......... 95 7.2 A Set of Intensively Sampled Datasets To Evaluate Sampling Strategies and Exposure Estimation Methods

................................ ................................ ..................... 98 7.3.1 Analysis of CWS Chemograph Shapes

................................ ......................... 98 7.3.2 Consideration of Drinking Water Treatment Effects

................................ ....... 99 7.3.3 Intensively Sampled Datasets To Evaluate Sampling Strategies and Exposure Estimation Methods

................................ ................................ ............... 102 7.4 Analyzing and Interpreting Monitoring Data Collected at Different Sampling Freq uencies

................................ ................................ ................................ ............... 119 7.4.1 Filling in Values Between Samples With Linear or Stair

- step Interpolation 120
7.4.2 Artificial Neural Netw ork

................................ ................................ ............. 120 7.4.3 Kriging

................................ ................................ ................................ ........ 121 7.4.4 Extreme Value Theory

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

7.4 .5 Longer

- Term Modeling Approaches Combining Random Function and Regression Models

................................ ................................ ................................ 122

7.4.6 Directions Forward

................................ ................................ ..................... 122

8. IMPLI CATIONS OF MOA & TOX ICITY PROFILE ON WAT ER

MONITORING

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

8.1 Toxicological & MOA Considerations

................................ ................................ .. 124
8.1.1 LH as the Critical Endpoint for Risk Assessment

................................ ........ 124
8.1.2 Pharmacokinetic Considerations

................................ ................................ 126 8.1.3 Pharmacodynamic Considerations

................................ ............................. 128 8.2 Drinking Water Considerations & the Monitoring Sampling Interval

.................... 129

9. NEXT STEPS

................................ ................................ ................ 131

10. REFERENCES

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

Post 2003 References

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

Pre

- 2003 References

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

APPENDIX A

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

A .1 CONTENT OF THI S APPENDIX

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

A.2 EFFECTS ON FEMA LES

................................ .............................. 159

A.2.1 Casas, E., Bonilla, E., Ducolomb Y, Betancourt M. 2010. Differential effects of herbicides atrazine and fenoxaprop

- ethyl, and insecticides diazinon and malathion, on viability and maturation of porcine oocytes in vitro . Toxicology in vitro 24 (2010); 224

- 230.

................................ ................................ ................................ ............................ 161Do not cite

Page 4 of 677 A.2.2 Coder, P.S. 2010. An Oral Developmental Toxicity Study of Atrazine in the Rat, Including Cross

- Fostering and Pair

- Feeding. Unpubli shed Syngenta study

.............. 162 A.2.3 Coder, P.S. 2010. An Oral (Gavage) Study to Assess the Potential for Detecting the Estrogen

- Induced Luteinizing Hormone Surge in Female Rats. Syngenta Report.

................................ ................................ ................................ ....... 164 A.2.4 Coder P. (2010). Atrazine: An Oral (Gavage) Study of the Effects of Atrazine on the Estrous Cycle and the Estrogen

- Induced Luteinizing Hormone Surge in Female

Rats. Final Interim R eport. Unpublished data from Syngenta Crop Protection, Inc. Study completion date: July 14, 2010. Report Number: WIL

- 639060.

....................... 166 A.2.5 Cooper, R., Buckalew, A., Fraites, M., Goldman, J., La ws, S., Narotsky, M., Stoker, T. 2010. Evaluating the Effect of the Chlorotriazine Herbicide Atrazine on the Amplitude of the Pre

- ovulatory LH surge in the Long

- Evans Rat. EPA Internal Report.

................................ ................................ ................................ ................................ ... 173

A.2.6 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 pi tuitary glands and LβT2 rat pituitary cells.
Chem Res Toxicol 21:844

- 851.

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

A.2.7 Fraites, M.J.P., Cooper, R.L., Buckalew, A., Jayaraman, S., Mills, L., and Laws, S.C. (2009). Characterizat ion of the Hypothalamic

- Pituitary

- Adrenal Axis Response to Atrazine and Metabolites in the Female Rat. Tox. Sci. 112(1):88

- 99.

.. 175

A.2.8 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.

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

A.2.9 Julian i, 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. 179

A.2.10 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 Hy droxyatrazine. Toxicological Sciences 76:190

- 200.

................................ ................................ .......... 181 A.2.11 McMullin, T.S., Andersen, M.E., Nagahara, A. Lund, T.D., Pak, T., Handa, R.J., Hanneman, W.H. (2004). Evidence that atrazine and d iaminochlorotriazine inhibit the estrogen/progesterone induced surge of luteinizing hormone in female Sprague Dawley rats without changing estrogen receptor action. Tox. Sci. 79:278

- 286.

......... 182

A .2.12 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.

................................ ................................ ............ 183 A.2.13 Stoker, T.E., Zorrilla, L., Strader, L., Gibson, E., Kamel, A., Qian, Y., Podhorniak, L. 2010. The Distribution of Atrazine in the Wistar Rat Followi ng Gestational/Lactational Exposures. EPA Internal Report.

................................ ......... 186 A.2.14 Rayner, J.L., Enoch, R.R., and Fenton, S.E. (2005) Adverse effects of prenatal exposure to atrazine during a critical pe riod of mammary gland growth. Tox. Sci. 87(1):255

- 266.

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

A.3 REPRODUCTIVE EF FECTS IN MALES

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

A.3.1 Abarikwu S., Adeslyan A., Oyeloja, T., Farombi, E. 2010. Changes in Sperm Characteristics and Induction of Oxidative Stress in the Testis and Epididymis of Experimental Rats by a Herbicide, Atrazine. Arch. Environ Contam Toxicol 58:874

- 882. DOI 10.1007/s00244

- 009

- 9371

- 2.

................................ ................................ ..... 196 A.3.2 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.

.......................... 197Do not cite

Page 5 of 677 A.3.3 Friedmann, AS. 2002. Atrazine inhibition of testosterone production in rat males following peripubertal exposure. Reproductive Toxicology 16 : 275

- 279.

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

A.3.4 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 microchondr ial function in sperm. Biochem Biophy Research Comm 366: 66

- 72. 200

A.3.5 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.

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

A.3.6 Laws, S.C., Hotchkiss, M., Ferrell, J., Jayaraman, S., Mills, L., Modic, W.,

Tinf o, N., Fraites, M., Stoker, T., and Cooper, R. (2009). Chlorotriazine Herbicides and

Metabolites Activate an ACTH

- dependent Release of Corticosterone in Male Wistar

Rats. Tox. Sci. 112(1):78

- 87.

................................ ................................ .................... 203

A.3.7 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.

................................ ............................... 204

A.3.8 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.

................................ ............................. 205

A.3.9 Mu, H. , Shang, P. and Xu, J. (2008). Testicular Toxicity and Mechanisms of Chlorotoluron Compounds in the Mouse. Toxicology Mechanisms and Methods. 18:

399

- 403.

................................ ................................ ................................ .................... 207 A.3.10 Pogrmic K, Fa S, Dakic V, Kaisarevic S, and Kovacevic R. 2009. Atrazine Oral Exposure of Peripubertal Male Rats Downregulates Steroidogenesis Gene Expr ession in Leydig Cells. Toxicological Sciences 111(1):189

- 197.

........................ 208

A.3. 11 Rayner J L., Enoch R R, et al. 2007. Atrazine

- induced reproductive tract alterations after transplacental and/or lac tational exposure in male Long

- Evans rats. Tox Appl Pharmacol: 218: 238

– 248.

................................ ................................ ...... 210 A.3.12 Rosenberg, B.G., Chen, H., Folmer, J., Liu, J., Papadopoulos, V., and Zirkin, B.R. (2008). Gestat ional Exposure to Atrazine: Effects on the Postnatal Development of Male Offspring. J. Andrology 29(3):304

- 311.

................................ ........................ 211 A.3.13 Stoker TE, Guidici DL, Laws SC, Cooper RL. 2002. The Effects of Atr azine Metabolites on Puberty and Thyroid Function in the Male Wistar Rat. Toxicological
Sciences 67: 198

- 206.

................................ ................................ ............................... 213 A.3.14 Stoker, T.E., Laws, S.C., Guidici, D., and Cooper, R.L. 2000. The eff ects 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.

. 215

A.3.15 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 20 01 ; pp. 142

- 148.

................................ ................................ ....... 216

A.3.16 Victor

- Costa, AB, Bandeira SMC, Oliveira AG, Mahecha GAB, Oliveira, CA. 2010. Changes in Testicular Morphology and Steroidogenesis in Adult Rats Exposed

to Atraz ine. Reproductive Toxicology. oi10.1016/j.reprotox.2009.12.006.

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

A.4 AROMATASE

................................ ................................ ............... 219

Summary: Effects on Aromatase and Steroi dogenesis, Andrology

............... 219

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 estr ogen receptor

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

- 55.

............................. 224Do not cite

Page 6 of 677 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.

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

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. 227

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 Atr azine

- induced aromatase expression is SF

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

- 7.

228

A.4.5 Hen eweer 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.

................................ ................................ 228

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).

................................ ................................ ...... 230

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.

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

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.

..................... 234

A.4.9 Rayner, J. , Wood, C., and Fenton, S. (2004 ) Exposure Parameters Necessary for Delayed Puberty and Mammary Gland Development in Long

- Evans Rats Exposed

in Utero to Atraz ine. Toxicology and Applied Pharmacology 195:23

- 34.

................... 235 A.4.10 Suzawa, M and Ingraham, HA. 2008. The herbicide atrazine activates endocrine gene networks via non

- steroidal NR5A nuclear rece ptors in fish and mammalian cells. PLoS ONE 3(5): e2117. doi:10.1371/journal.pone.0002117.

..... 236

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

- and time

- dependent effect of atrazine

or its met abolites on aromatase mRNA in vitro . Only a preprint of article available. 238

A.4.12 Yi, KD, Perez, EJ, Breckenridge, CB, and Simpkins, JW (2010?) Ex vivo

effects of atrazines or metaboli tes on phosphodiesterase activity.

............................ 239

A.5 NEUROTOXICITY

................................ ................................ ......... 240

Summary: Neurotoxicity

................................ ................................ ..................... 240

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.

243

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.

................................ ................................ .............. 246

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.

................................ ............. 247

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. 248

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.

................................ ................................ ............ 250Do not cite

Page 7 of 677 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.

................................ ................................ ....................... 251 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. Toxicolo gy 248: 52

– 58.

................................ ................... 253

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 mon oamine systems. Environmental Health Perspectives. 113(6): 708

- 715.

254 A 5.9 Solari, M, Paquin, J, Ducharme, P, Boily, M (2010). P19 neuronal differentiation

and retinoic acid metabolism as criteria to investigate atrazine, nitrite, and nitrate developmental toxicity. Toxicol Sci 113 (1):116

- 126.

................................ ................ 256

A.6 IMMUNOTOXICITY

................................ ................................ ....... 257

Summary: Immunotoxicity

................................ ................................ ................. 257

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

- 66.

................................ ...................... 260 A.6.2 Coder, P. S. (2010) An Oral (Ga vage) Immunotoxicity and Hormone Evaluation Study for the Effects of Atrazine on Male Rats, WIL Research Laboratories, LLC, 1407 George Road, Ashland, OH. Unpublished. (TK0023175)

................................ .......... 261

A.6.3 De vos 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.

................................ ................................ ................................ ........... 265

A.6.4 Devo s 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.

................................ ................................ ................................ ........... 266

A.6.5 Fil ipov, 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. 267

A.6.6 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.

................................ .................. 268 A.6.7 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.

................................ ................................ . 269

A.6.8 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.

................................ ................................ .... 270

A.6.9 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. Toxi cological Sciences: 75:

343

- 354.

................................ ................................ ................................ .................... 271 A.6.10 Pruett, S B, Fan, R, et al. 2009. Patterns of immunotoxicity associated with chronic as compared with acute exposure to chemical or physical s tressors and their relevance with regard to the role of stress and with regard to immunotoxicity testing. Toxicological Sciences: 109(2): 265

- 275.

................................ ................................ 272 A.6.11 Rajkovic, V, Matavulj, M and J ohansson, O. (2010). Combined Exposure of Peripubertal Male Rats to the Endocrine

- Disrupting Compound Atrazine and Power

Frequency Electromagnetic Fields Causes Degranulation of Cutaneous Mast Cells: A

New Toxic Environmental Hazard? Arch. Environ. Conta m. Toxicol. DOI

10:1007/s00244

- 010

- 9477

- 6

................................ ................................ ..................... 273Do not cite

Page 8 of 677 A.6.12 Rooney, A A, Matulka R, and Luebke, R. 2003. Developmental atrazine exposure suppresses immune function in male, but not female Sprague

- D awley rats. Toxicological Sciences 76: 366

- 375.

................................ ................................ ........ 274 A.6.13 Rowe, A M, Brundage, K M, Schafer, R, and Barnett J B. 2006. Immunomodulatory effects of maternal atrazine exposure on male Bal b/c mice. Toxicology and applied Pharmacology 21: 69

– 77.

................................ ................. 276 A.6.14 Rowe A M, Brundage K M and Barnett J B. 2007. In vitro atrazine

- exposure inhibits human natural killer cell lytic gra nule release. Toxicology and Applied Pharmacology 221: 179

– 188.

................................ ................................ ................. 277 A.6.15 Schwab, C L, Fan R, et al. 2005. Modeling and predicting stress

- induced immunosuppression in mice using blood p arameters. Toxicological Sciences 83: 101

– 113.

................................ ................................ ................................ ......................... 278 A.6.16 Whalen M M, Loganathan B G, et al. 2003. Immunomodulation of human natural killer cell cytotoxic function by triazine and carb amate pesticides. Chemico

Biological Interactions 145: 311

– 319.

................................ ................................ ..... 278

A.7 CARCINOGENESIS/ GENOTOXICITY

................................ .......... 279

Summary: Carcinogen esis/Genotoxicity

................................ ........................... 279

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.

................................ .................... 282

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 at razine 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.

...... 283

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.

................... 284

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.

................................ ............... 286 A.7.5 Lasserre, JP, Fack, F, Revets, D, Planchon S, Renaut J, Hoffmann L, Gutleb, Muller CP, Bohn T, (2009) Effects of the Endocrine Disruptors Atrazine and PCB 153 on the Protein Expressio n of MCF

- 7 Human Cells. Journal of Proteome Research

. 287

A.7.6 Malik, S. I., Terzoudi, G.I., and Pantelias, G.E. (2004). SCE analysis in G2 lymphocytes prematurely condensed chromosomes aft er 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.

...................... 289

A.7.7 Mans ke, M., Beltz, L, and Dhanwada, K. 2004. Low

- Level Atrazine Exposure
Decreases Cell Proliferation in Human Fibroblasts. Arch. Environ. Contam. Toxicol.

46:438

- 444.

................................ ................................ ................................ ............... 289

A.7.8 Mastrangelo, S., T omassetti, 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.

................................ .................... 292 A.7.9 Olejnik, Am, Marecik, R, Bialas, W, Cyplik, P. (2010). In vitro studies on atrazine

effects on human intestinal cells. Water Air Soil Pollut. DOI 10.1007/s11270

- 010

- 0394

- 0; published inline: April 16, 2010

................................ ................................ .... 293 A.7.10 Singh, M, Sandhir, R, Kiran, Ravi. (2010). Effects on antioxidant status of liver following atrazine exposure and its attenuation by vitamin E. Exp and Toxicol Pathol doi:10.1016/jetp.2010.01.005

................................ ................................ .................... 294 A.7.11 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.

................................ ................................ ... 296Do not cite

Page 9 of 677 A.7.12 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.

................ 297 A.7.13 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

- hydrox ypropyl)nitrosamine (DHNP).

Food Chem. Toxicol. 41:1811

- 1816.

................................ ................................ .......... 298 A.7.14 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.

................................ ....... 299 A.7.15 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.

................................ ................................ ....... 300

A.7.1 6 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.

................................ ................................ ................................ ............ 304

A.8 INSULIN/M ITOCHONDRIA FUNCTION

................................ ........ 305

Summary: Insulin/Mitochondria Function

................................ ......................... 305

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.

. 306

A .9 PHARMACOKINETICS : INTERNAL DOSIMETR Y, METABOLISM,

& TIME COURSE

................................ ................................ ................. 308

A.9.1 Dooley, G.P, Prenni, J.E., Prentiss, P.L., Cranmer, B.K., Anderson, M.E., Tessari, J.D. (2006). Identification o f novel hemoglobin adduct in Sprague Dawley rats exposed to atrazine. Chem Res Toxicol 19:692

- 700.

................................ ............... 308

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.

..................... 309

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

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

... 310

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.

..................... 311

A.9.5 McMullin, T.S., Hanneman, W.H., Cranmer, B.K., Tessari, J.D., and Andersen, M.E. (2007). Oral absorpti on and oxidative metabolism of atrazine in rats evaluated by physiologically modeling approaches. Toxicology 240:1

- 14.

................................ ..... 312

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.

............................... 314

A. 10 MIXTURES

................................ ................................ .................. 315

A 10.1 Enoch, R.R., Stanko, J.P., Greiner, S.N., Youngblood, G.L,. Rayner, J.L., Fenton, S.E., 2007. Mammary gland de velopment as a sensitive end point acute prenatal exposure to an atrazine metabolite mixture in female Long

- Evans rats. Environmental Health Prospective. 115(4): 541

- 547

................................ ................ 315

A.11 OTHER

................................ ................................ ........................ 317

A.11.1 Fakhouri, WD, Nunez, JL, Trail, F. (2010?) Atrazine Binds to the Growth Hormone Releasing Hormone Receptor and Affects Growth Hormone Gene Expression . In press

................................ ................................ ................................ .. 318Do not cite

Page 10 of 677 A.11.2 Yi, KD, Perez, EJ, Breckenridge, CB, and Simpkins, JW (2010) Ex vivo effects of atrazines or metabolites on phosphodiesterase activity. In press

.......................... 320

APPENDIX B

- 1: EPIDEMIOLOGY LITE RATURE REVIEW

METHODOLOGY

................................ ................................ ................. 322

APPENDIX B.2: REVIE W OF EPIDEMIOLOGIC I NVESTIGATIONS OF

NON

- CANCER HEALTH EFFE CTS OF ATRAZINE EXPO SURE

...... 325

B.2.1 Arbuckle et al. ―An Exploratory Analysis of the Effect of Pesticide Exposure on the Risk of Spontaneous Abortion in an Ontario Farm Population,‖ [ Environ Health Perspect 109:851

– 857 (2001).]

................................ ................................ ................. 325 B.2.2 Dabrowski, S. et al. ―Pesticide exposure and birthweight: an epidemiologiucal study in central poland,‖[International Journal of Occupational Medicine and Environmental Health; 16(1): 31

— 39. (2003)].

................................ .......................... 328 B.2.3 Farr et al. ―Pesticide Exposure and the Timing of Menopause in the Agricultural Health Study (AHS),‖ [Am J Epidemiol,163:731

– 742 (2006)].

................................ ... 330 B. 2.4 Farr et al. ―Pesticide Use and Menstrual Cycle Characteristics among Premenopausal Women in the Agricultural Health Study,‖ [Am J Epidemiol
2004;160:1194

– 1204 ((2004)].

................................ ................................ .................. 333 B.2.5 Hoppin et al. 2002. ―Chemical Predictors of Wheeze among Farmer Pesticide Applicators in the Agricultural Health Study,‖ [Am J Respir Crit Care Med Vol 165. pp

683

– 689].

................................ ................................ ................................ ................... 337 B.2.6 Hornemann et al. ―Seasonality of omphalocele in Northern Germany,‖ [Ultraschall in Med. 29:591

- 595 (2008)].

................................ ................................ .... 339 B.2.7 Klucinski et al. ―Humoral and cellular immunity rat es in chemical plant workers producing dust pesticides,‖ [Med Sci Monit; 7(6): 1270

- 1274 (2001)].

....................... 340 B.2.8 Kossman et al. ―Role of respiratory muscles in the impairment of the respiratory

sy stem function in the workers of a chemical plant division producing pesticides,‖ [Przeglas lekarski, 54(10): 702

- 6 (1997)].

................................ ................................ .. 342 B.2.9 Mattix K.D., Winchester P.D., and Scherer L.R. ―Incidence of abdominal wall defects is related to surface water atrazine and nitrate levels,‖ [ Journal of Pediatric Surgery. 42, 947

- 949 (2007)].

................................ ................................ .................... 343 B.2.10 Munger et al. ―Intrauterine Growth Retarda tion in Iowa Communities with

Herbicide

- contaminated Drinking Water Supplies,‖ [Env Health Perspec. 105: 308

- 314 (1997)].

................................ ................................ ................................ ...................... 345
B.2.11 Ochoa

- Acuña H., Frankenberger J., Hahn L., and Carbajo C. ―Drinking water herbicide exposure in Indiana and prevalence of small

- for

- gestational

- age and preterm delivery,‖ [Environmental Health Perspectives. 117(10): 1619

- 1624 (2009)].

............ 348
B.2.12 Ocho a

- Acuña H., Carbajo C. ―Risk of limb birth defects and mother‘s home proximity to cornfields,‖ [ Science of the Total Environment . 407: 4447

- 4451 (2009)].

................................ ................................ ................................ ................................ ... 350
B. 2.13 Saldena et al. ―Pesticid e Exposure and Self

- Reported Gestational Diabetes Mellitus in the Agricultural Health Study,‖ [Diabetes Care 30:529

– 534 (2007)].

........ 352 B.2.14 Sathyanarayana et al. ―Maternal Pesticide Use and B irth Weight in the Agricultural Health Study,‖ [J. Agromed, 15: 2, 127

- 136. (2010)].

.............................. 355 B.2.15 Savitz et al. ―Male Pesticide Exposure and Pregnancy Outcome,‖ [ Am J Epidemiol ;146:1025

- 36 (199 7)].

................................ ................................ ................. 356 B.2.16 Swan et al. ―Semen Quality in relation to biomarkers of pesticide exposure,‖ [Environ Health Perspect 111:1478

– 1484 (2003)].

................................ .................... 359 B.2.17 Villanueva C., Durand G., Coutte M., Chevrier C., and Cordier S. ―Atrazine in municipal drinking water and risk of low birth weight, preterm delivery, and small

- for

gestational

- age,‖ [ Environmental Health Perspectives, 62(6): 400

- 4 05 (2005)].

........ 364Do not cite

Page 11 of 677 B.2.18 Waller et al. ―Agricultural

- related chemical exposures, season of conception, and risk of gastroschisis in Washington State,‖ [Am J Obstet Gynecol;202:241.e1

- 6. (2010)]

. ................................ ................................ ................................ ...................... 366 B.2.19 Winchester P.D., Huskins J., and Ting J. ―Agrichemicals in surface water and birth defects in the United States,‖ [ Acta Paediatrica . 98(4): 664

- 669 (2009)].

.......... 369

APPENDIX C: SUMMARI ES OF BMD ANALYSES O F STUDIES ON LH

SURGE

................................ ................................ ................................ . 372

C.1 NHEERL STUDY

................................ ................................ .......... 372

C.2 MORSETH ONE MON TH LH STUDY

................................ ........... 381

APPENDIX D.1: CHARAC TERIZATION OF ATRAZI NE MONITORING PROGRAM (AMP) CWS CH EMOGRAPH SHAPES AND MONITORING
DATA FOR CWS WITH TH E GREATEST TCT CONCE NTR ATIONS

.. 440

APPENDIX D.2 SAMPLIN G FREQUENCY CHEMOGRA PHS

............. 442

APPENDIX E. UPDATE F OR SAP

................................ ....................... 582

APPENDIX F. TOXICOL OGY CHAPTER OF THE R EREGISTRATION

ELIGIBILITY DECISION

. ................................ ................................ ...... 585

F. 1.0 HAZARD CHARA CTERIZATION

................................ .............. 587

F.2.0 REQUIREMENTS

................................ ................................ ........ 588

F.3.0 DATA GAPS

................................ ................................ ............... 590

F.4.0 HAZARD ASSESSM ENT

................................ ............................ 591

F.4.1 Acute Toxicity

................................ ................................ ................................ ... 591 F.4.2 Subchronic Toxicity

................................ ................................ ......................... 592 F.4.3 Prenatal Developmental Toxicity

................................ ................................ ..... 594 F.4.4 Reproductive Toxicity

................................ ................................ ...................... 597 F.4.5 Chronic Toxicity and Carcinogenicity

................................ ............................... 597 F.4.6 Mutagenicity

................................ ................................ ................................ .... 6 02 F.4.7 Neurotoxicity

................................ ................................ ................................ .... 604

F.4.8 Metabolism

................................ ................................ ................................ ...... 604 F.4.9 Special/Other Studies

................................ ................................ ...................... 609 F.4.9.1 Assays of Direct Estrogenic Activity of Triazines

................................ ...... 609

F.4.9.2 Estrou s Cycle Alterations and LH Surge Attenuation

............................... 615
F.4.9.3 Other Two

- Year Bioassays

................................ ................................ ...... 618

F.4.9.4 NHEERL Publications

................................ ................................ .............. 627 F.4.10 Toxicology data for major metabolites of atrazine

................................ ......... 634 F.4.10.1 Diaminochlorotriazine metabolite (DACT)

- 2

- chloro

- 4

- amino

- 6

- ami no s

triazine; didealkyl atrazine; G

- 28273

................................ ................................ ..... 635
F.4.10.2 G

- 28279 metabolite

- 2

- chloro

- 4

- (ethylamino)

- 6

- (amino) s

- triazine; ethylatrazine; deisopropyl atrazine

................................ ................................ ........ 638
F.4.10.3 G

- 30033 metabolite

- 2

- chloro

- 4

- amino

- 6

- (isopropylamino) s

- triazine; deethyl atrazine; isopropyl atrazine.

................................ ................................ ...... 641 F.4.10.4 Hydroxyatrazine

- 2

- hydro xy

- 4(ethylamino)

- 6(isopropylamino)

- s

triazine

................................ ................................ ................................ ................... 643

F.5.0 TOXICITY ENDP OINT SELECTION

................................ ........... 647Do not cite

Page 12 of 677 F.5.3 Classification of Carcinoge nic Potential

................................ ............................ 647
F.5.3.1 Conclusions

................................ ................................ ............................... 648 F.5.3.2 Classification Carcinogenic Potential

................................ ........................ 648

F.6.0 FQPA CONSIDER ATIONS

................................ ......................... 649

F.6.1 Special Sensitivity to Infants and Children

................................ ....................... 649

F.6.2 Recomme ndation for a Developmental Neurotoxicity Study

............................ 650

F.7.0 REFERENCES (LI STED IN ORDER OF ASC ENDING MRID NUMBER)

................................ ................................ ............................. 651

F.8.0 APPEND ICES

................................ ................................ ............. 659

F.8.1 Toxicity Profile Summary Tables

................................ ................................ ...... 659 F.8.1.1 Acute Toxicity Table for Atrazine

................................ .............................. 659 F.8.1.2 Subchronic, Chronic and Other Toxicity Table for Atrazine

...................... 659 F.8.1.3 Toxicity Table for DACT . All available acceptable toxicity studies are

shown.

................................ ................................ ................................ ................... 670 F.8.1.4 Toxicity Table for G

- 28279. All available acceptable toxicity studies are

shown.

................................ ................................ ................................ ................... 672 F.8.1.5 Toxicity Table for G

- 30033. All available acceptable toxicity studies are

shown.

................................ ................................ ................................ ................... 674 F.8.1.6 Toxicity Table for hydroxyatrazine . All available acceptable toxicity studies

are shown.

................................ ................................ ................................ ............. 676Do not cite

Page 13 of 677 

### LIST OF FIGURES

Figure 5.1: Metabolic Profile of Atrazine

................................ ................................ ...... 62 Figure 5.2: Attenuation of the leutenizing Hormone (LH) surge i n Long Evans

- Hooded rats following a single versus 3 oral gavage daily doses of atrazine (Figure is from Cooper et al . 2000) 64 Figure 5.3: Attenuation of the leutinizing hormone (LH) surge f ollowing repeating daily dosing for four days via oral gavage (Unpublished data from the Cooper Lab).

................................ .. 65 Figure 5.4: Hypothetical depiction of pseudo steady state plasma levels reached upon re peatedly daily
dosing. The dashed lines indicate the mean value with lower and upper bounds, respectively. The elimination phase is indicative of when dosing stops.

................................ .................. 66 Figure 5.5: Plasma conce ntration profiles of total radiolabeled triazine equivalents corresponding to different administered doses of radiolabeled atrazine (Data from Thede 1987).

......... 67 Figure 5.6: Relationship between administered dose of atrazine and AUC for total radiolabeled triazine
equivalents. The dashed line indicates the 95% confidence interval.

.......................... 68 Figure 5.7: Relationship between pseudo

- steady stat e plasma levels and steady state AUC for total
radiolabeled triazine equivalents versus administered dose of atrazine. The dashed line indicates the

95% confidence interval.

................................ ................................ .............................. 69 Figure 5.8: Att enuation of the pre

- ovulatory LH surge following atrazine exposure across different studies and rat strains where daily dosing was performed for at least 4 days. The thick line represents

the 4

- day unpublished study from the Cooper Lab. The other studies a re either shown as single data
points or dashed lines to aid in comparison.

................................ ................................ 71 Figure 5.9: EPA BMDS Exponential Model (Constant Variance; BMR = 1 SD from control mean)

Results for Attenuation of LH Surge in Female Rats Administered Atrazine by Gavage

- NHEERL

Data.

................................ ................................ ................................ ............................ 78 Figure 5.10: EPA BMDS Hill Model (Constant Variance; BMR = 1 SD from control mean) Results for

―Area Under the Triazine Equivalent Plasma

- Concentration Time Curve‖ as the Dose Metric

- NHEERL Data (Attenuation of LH Surge in Female Rats Administered Atrazine by Gavage). 79 Figure 5.11: EPA BMDS Hill Model (Constant Variance; BMR = 1 SD from control mean) Results for ―Daily Steady State AUC for Triazine Equivalents‖ as the Dose Metric

- NHEERL Data (Attenuation of LH Surge in Female Rats Administered Atrazine by Gavage).

................................ .... 80 Figure 7.1: Location of CWS Monitored in the AMP in relation to vulnerable watersheds identified by
WARP. The WARP vulnerability ranking is based on atrazine use on corn and sorghum only. 96 Figure 7.2: Example of a CWS in which TCT concentrations are similar in pattern and magnitude for
both source (blue) and treated (pink) water samples.

................................ ................ 100 Figure 7.3: Example of a CWS in which TCT concentrations in treated (pink) water samples drop to
near the limits of detection in comparison to source water concentrations (blue).

..... 101 Figure 7. 4: Example of a CWS where TCT concentrations in the treated (pink) water samples are
reduced in relation to source (blue) water samples, but remain above the limit of detection. 102 Figure 7.5 : Actual chemograph (blue) and 2 resulting chemographs (red) sampled at 7

- day fixed intervals, Maumee River, 2008 (Data Source: Heidelberg U. NCWQR)

.................... 107 Figure 7.6 Actual chemograph (blue) an d 2 resulting chemographs (red) sampled at 7

- day fixed
intervals, MO

- 01, 2007 (Data Source: AEEMP)

................................ ......................... 111 Figure 7.7: Actual chemograph (blue) and 1 of the resulting chemographs (red) sampled at 7

- day fixed
intervals, MO

- 02, 2006 (Data Source: AEEMP)

................................ ......................... 113 Figure 7.8: A single year of daily atrazine concentrations simulated for an index reservoir in OH using

PRZM and EXAMS.

................................ ................................ ................................ ... 119 Figure A

- 1: Effect of Atrazine Exposure on Aromatase Activity and Hormone Levels 231Do not cite

Page 14 of 677 Figure A

- 2: Treatm ent Groups

................................ ................................ ................. 302 Figure B

- 1:

................................ ................................ ................................ ................ 324 Figure D

- 2.1: Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for MO

- 01, 2006

................................ ................................ .......... 443 Figure D

- 2.2: Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- d ay fixed intervals (red dots) for MO

- 01, 2007

................................ ................................ .......... 451 Figure D

- 2.3: Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for MO

- 01, 2008

................................ ................................ .......... 459 Figure D

- 2.4: Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for MO

- 01, 2009

................................ ................................ .......... 467 Figure D

- 2.5: Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for MO

- 02, 2006

................................ ................................ .......... 476 Figure D

- 2.6: Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for MO

- 02, 2007

................................ ................................ .......... 484 Figure D

- 2.7: Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for MO

- 02, 2008

................................ ................................ .......... 492 Figure D

- 2.8: Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for MO

- 02, 2009

................................ ................................ .......... 500 Figure D

- 2.9: Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for Maumee River (Heidelberg), 2005

................................ ......... 509 Figure D

- 2.10: Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for Maumee River (Heidelberg), 2006

................................ 517 Figure D

- 2.11: Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for Maumee River (Heidelberg), 2007

................................ 525 Figure D

- 2.12: Actual chemo graph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for Maumee River (Heidelberg), 2008

................................ 533 Figure D

- 2.13: Actual chemograph (blue) and resulting che mographs (dark red) sampled at 7

- day fixed intervals (red dots) for Sandusky River (Heidelberg), 2004

............................... 542 Figure D

- 2.14: Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for Sandusky River (Heidelberg), 2005

............................... 550 Figure D

- 2.15: Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (re d dots) for Sandusky River (Heidelberg), 2006

............................... 558 Figure D

- 2.16: Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for Sandusky River (Heidelberg), 2007

............................... 566 Figure D

- 2.17: Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for Sandusky River (Heidelberg), 2008

............................... 574Do not cite

Page 15 of 677 

### LIST OF TABLES

Table 2

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

....................... 25

Table 2

- 2: Comparison of LH data from adult rats to apical endpoints from developing rats 26 Table 3. 1: Summary of the Non

- Cancer Epidemiology Studies included in Literature Review 45

Table 5

- 1: Estimated elimination rate constants assuming linear kinetics for the different dose groups
in Figure 5.4.

................................ ................................ ................................ ................ 67

Table 5

- 2: S ummary of BMD analyses

................................ ................................ ....... 76

Table 5

- 3: Conversion of AUC estimates to administered dose

................................ .. 77

Table 6

- 1: Summary of available atrazi ne experimental toxicology studies from the gestational period

................................ ................................ ................................ ................................ .... 83

Table 6

- 2 a &b: Extracted from Stoker et al. ( 2010)

................................ .................... 84

Table 6

- 3: Total reactivity measured in PND 3 pups following lactational exposure to dams exposed to
atrazine [ Extracted from Stoker et al. (2007)].

................................ ............................. 87

Table 6

- 4: a & b. Extracted from Stoker et al

. ................................ ............................. 87

Table 6

- 5 : Summary of available atrazine experimental toxicology studies from the lactational periods

................................ ................................ ................................ ................................ .... 88

Table 6

- 6 : Summa ry of available atrazine experimental toxicology studies from the peripubertal period

................................ ................................ ................................ ................................ .... 89

Table 7

- 1: Summary of Annual Frequency of Peak Occurrences by Source Water Type 99

Table 7

- 2: Analysis of Chemograph Peaks (Spikes), Maximum Detects, and 4

- Day Averages based

on 7

- Day Fixed Sampling Intervals for 2 OH Rivers (Source for original data: Heidelberg U. NCWQR)

................................ ................................ ................................ ................................ .. 107

Table 7

- 3: Analysis of Chemograph Peaks (Spikes), Maximum Detects, and 4

- Day Averages based on

7

- Day Fixed Sampling Intervals for 2 AEEMP Monitoring Sites

................................ . 115

Table 8

- 1: Estimates of human half

- lives for triazine equivalents and the predicted time to reach steady

state

................................ ................................ ................................ ........................... 128

Table A

- 1: Plasma concentrations of atrazine and its chlorometabolites 10.5 to 11.5 hours after dose

administration

................................ ................................ ................................ ............ 172

Table A

- 2: Atrazine and Predominant Metabolites Tissue Levels in Dams Exposed GD14

- PND10 and

their Off spring

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

Table A

- 3: Strain and Dose Comparison in Male Rats dosed peripubertally with atrazine 192

Table D

- 2.1: Analysis of Chemograph Peaks (Spikes), Maximum Detects, and 4

- Day Averages based

on 7

- Day Fixed Sampling Intervals for 2 AEEMP Monitoring Sites

............................ 442

Table D

- 2.2: Analysis of C hemograph Peaks (Spikes), Maximum Detects, and 4

- Day Averages based

on 7

- Day Fixed Sampling Intervals for 2 AEEMP Monitoring Sites

............................ 475

Table D

- 2.3: Analysis of Chemograph Peaks (Spikes), Maxi mum Detects, and 4

- Day Averages based

on 7

- Day Fixed Sampling Intervals for 2 Heidelberg Monitoring Sites

....................... 508

Table D

- 2.4: Analysis of Chemograph Peaks (Spikes), Maximum Detects, and 4

- Day Av erages based

on 7

- Day Fixed Sampling Intervals for 2 Heidelberg Monitoring Sites

....................... 541

Table F

- 1: The requirements (CFR 158.340) for food use of atrazine

...................... 589

Table F

- 2: Data Gaps Listed in 1988 RED Document and Studies That Have Been Submitted to Fill these Data Gaps.

................................ ................................ ................................ ....... 590Do not cite

Section 1

- Page 16 of 677 1. INTRODUCTION

Atrazine

– a chlorotriazine herbicide

– is currently one of the most widely used agricultural herbicides in the United States, with ap proximately 7 0 million pounds of active ingredient applied domestically per year. The Interim Reregistration Eligibility Document (IRED) for atrazine was final ized in January 2003 with an addendum added in October 2003 (US EPA 2003). The Agency completed the reregistration eligibility document (RED) for atrazine in April 2006. In recent years, numerous experimental toxicology and epidemiologic studies evaluati ng the toxicity profile and/or mode of action (MOA) of atrazine have become available. To consider the extent to which these new studies may impact the Agency‘s human health risk characterization for atrazine, the Office of Pesticide Programs (OPP) in col laboration with the Office of Research and Development (ORD) and the Office of Water is re

- evaluatin g atrazine and its metabolites‘ [deethyl

- atrazine (DEA), deisopropyl

- atrazine (DIA), and diamino

- s

chlorotriazine (DACT)]. 1

As part of the scientific re

- evaluation of the health effects of atrazine, three meetings of the FIFRA Scientific Advisory Panel (SAP) are scheduled in 2010 with an additional meeting focused on cancer epidemiology studies in 2011. The first of these meetings was held February 2

- 4, 2 010. The February, 2010 SAP meeting focused on a number of broad scientific issues related to the use of epidemiology and human incident data in risk assessment. As part of the February meeting, the Agency presented two case studies involving atrazine (U SEPA, 2010) . The first case study considered selected epidemiology studies associating atrazine exposure with birth outcomes. The studies included in the February case study have been evaluated in combination with

other non

- cancer epidemiology studies an d integrated with the animal toxicology data in this draft issue paper. The second case study from the February SAP described a collaborative effort between the Agency and investigators at the Agricultural Health Study (AHS) ( http://aghealth.nci.nih.gov ) to compare exposure assessment approaches used to evaluate pesticide applicator exposure 2 . This collaborative project continues; an update on the current status is provided in Appendix E to this document 3

.

At the February SAP, the Agency also presented a draft framework for incorporating epidemiology and human incident data into risk assessment (USEPA, 2010). This draft framework described a proposed approach for developing a weight of the evidence (WOE) a nalysis using data from multiple sources (e.g., in vitro and in vivo experimental studies along with epidemiology studies) and from multiple levels of

1 Henceforth mention of atrazine in this document also in fers 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.regulati ons.gov (Docket EPA

- HQ

- OPP

- 2009

- 0851 )

. 3 Note, the update on the AHS

- EPA collaborative exposure assessment project is provided as a courtesy for purposes of transparency. The Agency will not solicit feedback from the Panel on this work at the September m eeting. The AHS

- EPA collaborative project will be discussed in more detail in the upcoming 2011 SAP meeting on cancer epidemiology studies.Do not cite

Section 1

- Page 17 of 677 biological organization. The draft framework relies heavily on existing Agency and OPP guidance document s, particularly the Mode of Action and Huma n Relevance

Frameworks ( U.S. EPA, 1 999, 2005 ; Boobis et al . , 2008; Sonich

- Mullin et al .

. , 2001 ; Meek et al .

, 2003 ; Seed et al. , 2005 )

. Specifically, the draft framework describes the

use of modified Bradford H ill criteria like that in the MOA Framework as a tool to organize, review, and interpret data ( U.S. EPA, 1 999, 2005 ; Boobis et al. , 2008; Sonich

Mullin et al .

, 2001 ; Meek et al .

, 2003 ; Seed et al. , 2005 )

.

Briefly, t he concept of MOA i nvolve s the ident ification of a biologically plausible sequence of key events that are obligatory and quantifiable leading to an adverse

outcome . The MOA analysis takes into account factors such as dose

- response and temporal concordance, coherence

, and consistency. In th is analysis, data from humans and experimental in vitro and in vivo studies can be evaluated together for biological plausibility, to identify areas of confidence and characterize areas of uncertainty . The SAP‘s report from the February meeting became av ailable in April (FIFRA SAP, 2010a);

the Agency is currently reviewing the suggested revisions in preparation to improve the

earlier draft.

The Agency‘s draft Framework describes conducting problem formulation at the beginning of the risk assessment pro cess. Because of the distinct strengths and limitations of experimental toxicology and epidemiology studies, the SAP highlighted the importance of problem formulation as a strong starting point for developing a scientifically

- based and transparent integra tive analysis

. Generally, problem formulation

starts with a planning dialogue between risk assessors and risk managers to identify goals for the analysis and possible risk management strategies. This initial dialogue provides the regulatory context for t he scientific analysis and helps define the scope of such an analysis. The problem formulation stage also involves consideration of the available information regarding the pesticide use/usage

, toxicological effects of concern

and exposure pathways and dur ation along with key gaps in data or scientific information. Specifi c ally for atrazine, Agency scientists and risk managers have determined that, consistent with the RED, exposure in drinking water continues to be the major exposure pathway of concern for atrazine. In addition, it has been determined that the two key goals for the current re

- evaluation are: 1) to determine the extent to which new science indicates a need for the Agency 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.

The problem formulation phase of the atrazine non

- cancer re

- evaluation is being completed in a step

- wise manner. The atrazine case studies from the February meeting provided the Age ncy with an opportunity to get peer review on a subs et of non

cancer epidemiology studies; the SAP‘s feedback was useful as the Agency evaluated the remaining non

- cance r epidemiology studies. In Section 3 and Appendix B

- 1 , the Agency summarizes all of the identified epidemiology studies and describes their potential role in informing the risk assessment. The April SAP meeting provided theDo not cite

Section 1

- Page 18 of 677 Agency with a peer review o f the state of the science with respect to new information on experimental toxicology studies and MOA and possible statistical approaches for determining drinking water monitoring frequency strategies. The September SAP meeting will bring the topics from the two previous SAP meetings together in an integrated evaluation; specifically, epidemiology, experimental toxicology, and drinking water exposure will all be considered . As discussed in Section 4 , this draft issue paper (and associated appendices) basically completes the problem formulation stage for the hazard assessment component of the atrazine risk assessment

. T he Agency is proposing an approach for performing dose

- response assessment and ext rapolation using the best available information on the pharmacokinetic (PK), pharmacodynamic (PD), and temporal characteristics of atrazine in drinking water sources . With respect to the drinking water exposure assessment, since the April SAP, the Agency has continued to explore statistical approaches for evaluating drinking water monitoring strategies. Pending feedback from Panel in September, the Agency expects to be in a position to select one or more statistical methods for use in the drinking water a nalysis phase of the atrazine exposure characterization

.

The current document provides a review of new experimental toxicology studies available between January 30 and July 15, 2010 4

. Twenty new experimental toxicology

studies became available durin g this time period; these include key studies from EPA‘s Office of Research and Development (ORD) on the leutenizing hormone ( LH ) effects of atrazine following four days of exposure as well as studies from ORD and Syngenta characterizing the effects of atr azine from in utero or post

- natal exposure. The mammary gland development literature was not considered in the April SAP meeting. A review of the literature on the potential effects of atrazine on mammary gland development has also been included in the c urrent issue paper (See Appendix A). As such, any studies on the effects of atrazine exposure on mammary gland development

available since the 2003 risk assessment are included.

The Agency has reviewed approximately 2 0 non

- cancer epidemiology studies on a variety of topics such as birth defects, birth outcomes, male fertility, and female reproductive effects. In short, most of the non

- cancer epidemiology studies are either initial hypothesis generating studies or involve weak exposure assessment

. As such, they lack quantitative information for using directly in a human health risk assessment or for establishing a causal link between reported outcomes and exposure to atrazine

and/or its metabolites. However, epidemiology studies reporting findings on small for gestational age (SGA)

, lengthening of menstrual cycle s , and lengthening time to menopause provide qualitative information on the human relevance of the experimental toxicology findings and thus increase the overall confidence in the hazard asses sment.

4 To allow the Agency time to evaluate new information and provide draft issue papers for SAP reviews,

cut

- off date s were established for the April and September SAP meetings. These cut

- off dates were January 30 and July 15, 2010, respectively.Do not cite

Section 1

- Page 19 of 677 The epidemiology chapter is followed by an integrative narrative which discusses the relative strengths and limitations of the epidemiology and experimental toxicology studies along with preliminary conclusions about the degree to which these st udies can inform a human health risk assessment. This integrative narrative provides the

foundation for Sections 5 and 6 on estimating internal dose of atrazine and its metabolites, calculating ben chmark doses, and considering potential susceptibility of infants and children as required under the Food Quality Protection Act (FQPA, 1996)

. The proposed updates to the dose

- response assessment and extrapolation for atrazine

are made in the context of k nowledge about the hypothalamic

- pituitary

- gonadal (HPG) neuroendocrine MOA. Like the 2003 human health risk assessment, attenuation of the preovulatory LH surge data from female rats continues to provide the most sensitive endpoint for assessing human hea lth risk.

It is prudent for the Agency to evaluate the hazard potential of atrazine and the drinking water monitoring strategy together. Namely, the frequency of drinking water monitoring is related to the temporal pattern of the toxicological endpoi nt of concern

used for the risk assessment. Generally, longer durations of toxicological concern (e.g., a long

- term chronic effect) require a less frequent drinking water sampling design to approximate longer term exposures. However, as the duration of c oncern shortens, the frequency and timing of sampling become more important in determining how well the sample data capture short

- duration exposures. Depending on the aquatic resource being sampled, the likelihood increases that a peak exposure may be mis sed by less frequent sampling. This is most critical when the Agency is concerned about very short

term effects resulting from only a few days of exposure. As such, in 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.

In the 2003 risk assessment, a 90

- day rolling average drinking water

concentration was selected to correspond to: (i) the period with in the year during which most peak atrazine water concentrations are likely to occur; and (ii) the most sensitive effect (LH surge attenuation) occurring 4

- 5 months after daily dosing in a 6 month toxicity study. This combination represented the most cons ervative pairing of exposure

duration with LH effects as seen in the a nimal studies available at that time . The current drinking water monitoring frequency being conducted by Syngenta and required by EPA, involves weekly sampling during the growing season and bi

- weekly ( i.e., every other week) sampling for the remainder of the year. Section 5 of this draft issue paper describes the available PK and PD (particularly LH data) which indicate that atrazine

reaches pseudo steady st ate in the adult rat after a few days of dosing. The Agency is proposing that the timing of pseudo steady state is an important factor in evaluating appropriate drinki ng water monitoring frequency and suggest s that a critical temporal window of < 90 days appears appropriate at this time. Section 7 describes strengths and limitations of possible options for selecting a temporal window(s) for drinking water monitoring.Do not cite

Section 1

- Page 20 of 677 Section 7 (Approaches for E valuating Water Sampling Strategies & Frequency of Monitoring) addresses both sampling frequency considerations in monitoring study designs and methods of estimating drinking water concentrations between sampling points. The section presents a framework f or designing a monitoring study to adequately characterize drinking water exposures based on recommendations from the April 2010 SAP and lessons learned from both the drinking water monitoring program and a separate ecological exposure monitoring program c onducted by Syngenta for atrazine. In response to the April 2010 SAP concerns that the weekly CWS sampling smoothes out variability by missing short

- duration peaks and, thus, may provide a biased representation of actual concentration profiles, the USEPA p roposes a set of more intensively sampled monitoring data to evaluate sampling designs and methods for estimating exposures between sampling points. Finally, the Agency presents options for estimating exposures between existing sampling points for either a short (e.g., 4

- day) or a longer term duration of toxicological concern.

The previous atrazine human health risk assessment was based on a strong science foundation, supported by significant external peer review, and represented the

state

- of

- the

- scien ce at the time. The 90

- day rolling average was determined to be a health protective duration of exposure given the state

- of

- the

- science for atrazine prior to 2006. As such, the drinking water monitoring frequency required by the Agency in the RED was app ropriate for protecting health given that knowledge. Given that the Agency is proposing a significant shortening of the critical duration of exposure based on new

data , pending peer review by the SAP, more frequent monitoring than the current sampling fre quency may be needed to accurately assess atrazine concentrations.Do not cite

Section 2

- Page 21 of 677 2. MODE OF ACTION (MOA) & EXPERIMENTAL TOXICOL OGY

2.1 Historical Background

The 2003 human health risk assessment for atrazine is complex and incorporated si gnificant data developmen t, regulatory evaluation, and FIFRA SAP

review. Prior to the 2010 re

- evaluation, the Agency sought advice from 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 r at mammary gland tumor response

seen in adult cancer studies in 1988 ( FIFRA SAP, 1988 ). At that time, t he SAP noted that a ―hormonal influence‖ might be an important consideration in the development of these mammary gland tumors in adult rats

. T he A gency returned 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 relevance of these findings.

The SAP agreed with the Agency ‘s proposal for atrazine‘s neuroendocrine MOA, and they further concluded that it is unlikely that the mechanism by which atrazine induces mammary tumors in adult female Sprague Dawley rats could be operational in

humans. Based on the 2000 SAP guidance, EPA changed i ts position on atrazine and reclassified it from a ―possible carcinogen‖ to ―not likely to be carcinogenic in humans‖. Nevertheless, the SAP further concluded that it is not unreasonable to expect that atrazine might cause adverse effects on hypothalamic

- p ituitary

- gonadal function in humans if exposures were sufficiently high and that the MOA was relevant for developmental and reproductive effects (FIFRA SAP, 2000). It is these developmental and reproductive effects of atrazine which have been the focus of significant research in the last few years. T h e Agency concluded , and the April 2010 SAP concurred, that new

research conducted in the last seven years support s the neuroendocrine MOA identified

in the 2003 IRED and the conclusions regarding the human re levance of the mode of action for rat mammary gland tumors

. As described in detail in this draft issue paper,

t he critical and most sensitive adverse effects continue to be related to the reproductive /developmental consequences of perturbations to the HPG axis.

In 2003

, the Agency presented its evaluation on prostate cancer to the FIFRA SAP. At that meeting, t he SAP evaluated the available epidemiology studies suggesting an association between atrazine and prostate cancer in workers at an

atrazine manu facturing plant in St. Gabriel

, 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 increase in incidence of prostate cancer in workers at the plant co uld likely be explained by the increase in Prostate S pecific Antigen (PSA) screening at the plant (FIFRA SAP, 2003)

. The SAP also stated that a more thorough and systematic review of the biologic and epidemiologic literature on the topic of the potential or observed carcinogenic effects of atrazine exposure was required beforeDo not cite

Section 2

- Page 22 of 677 concluding that atrazine exposure is an unlikely explanation for at least part of the excess of prostate cancer in the St. Gabriel manufacturing plant. Review of the cancer epidemi ologic studies with atrazine is on

- going and will be considered as part of the
SAP meeting in spring 2011.

2.2 2010 Scientific Re

- Evaluation

In preparation for the April and September SAP meetings, the Agency has reviewed more than 100 new experimental studies on a variety of topics including reproductive/developmental toxic ity in both males and females, neurotoxicity, immunotoxicity, mammary gland development, mixtures effects, andrology, aromatase and insulin resistance and mitochondrial function impa irment

. The Agency‘s literature review is current as of July 15, 2010. In addition, the Agency has evaluated numerous
studies on atrazine‘s mode of action . The Agency‘s reviews on all these topics are provided in Appendix A. In addition summaries of t he new literature are provided on each of the scientific areas in Appendix A.

In the 2003 IRED, the Agency identified perturbations of the neuroendocrine

s ystem leading to reproductive toxicity as the most biologically plausible and sensitive

effects at tributable to atrazine exposure. These adverse outcomes include disruption of estrous cyclicity and delays in puberty onset (males and females) occurring as a consequence of disruptions to the HPG axis in rats

. During the April SAP meeting, the Agency p resented its review of the state of the science on the potential of atrazine to elicit other toxic effects ( e.g., immunotoxicity or neurotoxicity). The SAP concluded and the Agency concurs that

– at this time

- there are no atrazine

- specific data to

concl usively establish linkages between effects on either the immune or nervous system and atrazine‘s neuroendocrine MOA. Effects on the nervous and immune systems, however, occur at doses higher than those eliciting the reproductive/developmental

effects . Sin ce the April SAP, few new studies on these topics have become available. None of these new studies alter the overall weight of evidence and would not impact the Agency‘s risk assessment conclusions. As a result, the potential neurotoxic and

immunotoxic e ffects attributed to atrazine will not be discussed in this draft issue paper. However, a synopsis of the Agency‘s evaluation of the neurotoxicity and immunotoxicity data is available in Appendix A of this document.

Several papers from the same laborat ory ( Rayner et al. , 2004 ; Rayner et al.

, 2005; Enoch et al.

, 2007 ) describe the effects of either atrazine or a mixture of atrazine and several metabolites/degr adates (DEA, DIA, DACT, hydroxy

- atrazine) on mammary gland development in female pups. In the R ayner et al. publications, the doses used to assess the impact of atrazine exposure were high (≥ 50 mg/kg/day) relative to the doses eliciting other effects ( e.g., LH surge attenuation and prostatitis). The authors concluded that atrazine exposure at these high dose l evels resulted in a delay in mammary gland development. I n the Enoch et al. , (2007) paper

, dams were exposed orally to atrazine or a mixture of atrazine and metabolites for five consecutive days

. As described in detail in Appendix A, Enoch et al. conclud e that the mixture stud y suggest sDo not cite

Section 2

- Page 23 of 6 77 effects may occur at doses lower than those currently being used by EPA to assess the risk of atrazine and its metabolites. However, in all three mammary gland development

studies , the dose

- response relationships are weak and there are concerns with study design and data reporting / analysis that preclude definitive conclusions.

The current evaluation of the post

- 2003 data, supports the neuroendocrine MOA and key events identified in the 2003 IRED . In addition, new resea rch has become

available on another potentially disrupted pathway in the MOA: activation of the hypothalamic

- pituitary

- adrenal (HPA) axis manifested as incr eases in corticosterone levels leading to perturbations of the HPG axis. At the April SAP meeting, the Panel
evaluated the connection between the HPA and HPG axes and determined that this was a plausible hypoth e sis but that there was insufficient data to make this linkage and

that the available information was not sufficiently robust for incorporati o n i nto the Agency‘s risk assessment. As such, the remainder of this draft issue paper focuses on the key events related to disruption of the HPG axis and the implications for this

neuroendocrine MOA in the human health risk assessment for atrazine.

2

. 3 Mode of Action (MOA)

2

. 3 .1 Background

In describing and analyzing a MOA for any chemical, the Agency applies the
MOA framework for organizing and analyzing the available data ( U.S. EPA, 1 999, 2005 ; Boobis et al. , 2008; Sonich

- Mullin et al.

, 2001 ; Meek et al.

, 2003 ; Seed et al. , 2005 )

. Recently, OPP proposed extending this MOA framework and related Human Relevance Framework to the integration of epidemiology and experimental toxicology data into a WOE analysis (USEPA, 2010). The MOA and WOE framework s rely heavily on the

Bradford

- Hill Criteria, often used in epidemiology for establishing causality. These steps are provided below (extracted from Seed et al. , 2005)

. In brief the first step in this approach is to ascertain 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, con sistency, and specificity of the relationship between the key events and the toxic response. Finally, the data are analyzed to determine if the key events are biologically plausible in humans both on a qualitative and quantitative basis. Once the data ha ve been subjected to this evaluation, other potential MOAs considered and shown to be unlikely, the analysis culminates in a statement of confidence, analysis, and implications that clearly articulates the uncertainties, strengths, weaknesses, and implicat ions of the analysis.

o Postulated MOA . A brief description of the sequence of measured events, starting with chemical administration, to the formation of the toxic effect at a given site.

o Key events . Clear descriptions of each of the key events (measurab le parameters) that are thought to underlie the MOA.Do not cite

Section 2

- Page 24 of 677 o Dose

- response relationships . Dose

- response relationships identified for each key event, and comparisons presented of dose

- response relationships among key events and with the toxic effect.

o Temporal assoc iation . Sequence of key events over time that lead to the toxic effect. This includes any MOA differences specific to different life stages.

o Strength, consistency, and specificity of association of key events and the

toxic effect . Complete assessment and p resentation of the relationships among key events, precursor lesions, and the toxic effect. portrayal of the consistency of observations across studies of different designs.

o Biological plausibility and coherence . Determination of whether key

events and t he sequence of events are consistent with current biological thinking, regarding both the specific toxic effect and the specific chemical

under review.

o Other MOAs . Alternative MOAs that may be applicable for the chemical under review. Comparison of their l ikelihood vis

- à

- vis the proposed MOA.

o Conclusion about the MOA . Overall indication of the level of confidence in the postulated MOA.

o Uncertainties, inconsistencies, and data gaps. Identification of information deficiencies in the case; description of incon sistent findings in the data at large; evaluation of uncertainties; proposal of pointed research that could significantly inform the case.

In the specific case of atrazine, a MOA framework analysis using these steps was conducted by EPA (USEPA, 2000) in p reparation for the 2003 risk assessment

, supported by the SAP (2000) and confirmed by WHO (2005 ). Given the extensive peer review received on the HPG MOA/ key events and since the April SAP draft issue paper

described the most recent science to inform the MOA, a complete MOA framework analysis is not repeated here

. Instead, the important information regarding the key events leading to the neuroendocrine MOA needed for considering updated points of departure and extrapolation are provided here. For more de tailed information, please

refer to USEPA ( 2000 ); and WHO (2005)

.

2

. 3 .2 Key Events

– H ypothalamic

- P ituitary

- G onadal (HPG) A xis

Initially postulated to elucidate the physiological events and endocrine changes leading 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 development. The key events ( Table 2

- 1 ) de scribed in the 2003 IRED are:

Hypothalamic effects resulting in changes in catecholamine function and regulation of the pulsatile release of gona d otropin releasing hormone (GnRH)

. Attenuation of the LH surge and disruption of ovarian cyclesDo not cite

Section 2

- Page 25 of 677 Cessation of o vulation with the ensuing persistent release of estrogen Increased prolactin release by the pituitary as a secondary consequence resulting from the elevated estrogen levels Prolactin and estrogen

- induced proliferative processes in the mammary gland leading to tumorigenesis.

Table 2

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

Concordance

Axis Key Event Time Dose ( NOEL/ LOEL) Reference

HPG Axis Hypothalamus

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

2007

Pituitary

Attenuation of LH surge 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

26we eks 1.8/ 3.65 mg/kg/day Morseth et al

. , 1996 b

Ovary Estrous cyclicity disruption 1

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

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

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

‡ NOEL not available

Alt hough the MOA as it relates to the development of mammary gland tumors is not operative in humans, it is not unreasonable to assume that the same endocrine perturbations that induce tumors in rats may play a role in at least some developmental effects (not associated with reproductive aging) that may be relevant to hypothalamic

pituitary function in humans. As such, the Agency used an early key event (i.e., attenuation of preovulatory LH surge) from atrazine‘s toxicity pathway as the basis for setting the point s of departure for the intermediate and chronic assessments . Similarly, the effect of atrazine on the neuroendocrine control of reproduction was considered a key step in the atrazine

- induced delay in pubertal development in both sexes ( Stoker et
al . , 2000; Laws et al . , 2000 ) and the disruption of prostate function in the male offspring when the dam is exposed immediately following birth. The perturbation of the LH surge is the cornerstone of the cascade of events leading to the adverse reproductive outcomes (e.g., disruption of ovarian cycling and sexual maturation) attributed to atrazine exposure. For example, s exual maturation is the culmination of aDo not cite

Section 2

- Page 26 of 677 complex cascade of sex developmental effects that ultimately leads to the attainment of reproduct ive capacity

. Activation of the HPG axis resulting in the pulsatile secretion of GnRH and LH is critical to puberty onset. For instance, decreased LH during puberty would lead to insufficient stimulation of the gonads to reduce the circulating hormone

le vels needed for development of sex accessory tissues in males and females. Moreover, researchers have found that disruption of GnRH release and the ensuing dampening of the LH surge can lead to delays in vaginal opening (VO) and preputial separation (PPS )

.

In addition to identifying the key events in t he MOA, a dose

- response and temporal concordance analysis was also part of the 2003 evaluation. Th e disruption of ovarian cycles has been attributed by multiple investigators to an atrazine

- induced

inte rruption of the ovulatory surge of 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 )

. The attenuation of the LH surge was

observ ed at a dose of 3.65 mg/kg/day after 26 weeks of exposure (Morseth et al .

, 1996b ) . The dose

- response profile for the LH surge attenuation closely parallel the profile for estrous cycle disruption and ultimately mammary tumor formation which

occurred afte r two years of atrazine exposure at a dose level of 3.1 mg/kg/day implying the effect occurred sometime during the first 26 weeks . Therefore, in general there was good dose

- concordance between the key events in the MOA and mammary tumors

. Important for t he current considerations, the LH surge attenuation occur s at doses ≤ to
those where these effects were observed ( Table 2

- 2 )

. With respect to temporal concordance, attenuation of LH surge pr ece d e s changes in estrous cyclicity . O ne of the recent advances in the MOA understanding is that the timing of LH suppression in the

rat appear s to occur much earlier (4 days of exposure) than previously observed (Cooper et al. , 2010)

.

Table 2

- 2 : Comparison of LH data from adult rats to apical endpoi nts from developing rats

Life Stage LH Hormone (NOAEL a

/LOAEL b ) Apical Endpoint
NOAEL/LOAEL

Pre

- Natal (Fetus) 10/70 mg/kg/day; Delays

in ossification 10/50 mg/kg/day; Delayed
PPS

Perinatal 6.2 5/12.5 mg/kg/day; increased prostatitis

Peripubertal 6.25/12.5 mg/kg/day; increased prostatitis, delayed PPS 25/50 mg/kg/day; delaysDo not cite

Section 2

- Page 27 of 677 in vaginal opening 50/100 mg/kg/day; Delays in vaginal opening,

decreased testosterone

Adult 1.56/3.12 mg/kg/day (4 day exposure) 1.8/3.65 mg/kg/day; disrupted cyclicity 50/100 mg/kg/day; disrupted cyclicity 1.8/3.65 mg/kg/day (26 week

exposure)

a. NOAEL is the no

- observed

- adverse

- effect

- level
b. LOAEL is the lowest

- observed

- adverse

- effect

- level

A number of recent st udies have further characterize d the cellular and neuroendocrine changes responsible for how atrazine interferes with the regulat i on of LH secretion. The preponderance of evidence provides added support for the hypothesis that atrazine targets the brain to modify the neuronal control of pituitary

function. Importantly, the HPG axis in humans and rodents is maintained by the activity of GnRH neurons, whose cells are distributed throughout the preoptic region including the medial preoptic area (mPOA) of th e anterior hypothalamus ( Rivest and Rivier, 1995). The nerve terminals of these neurons project to the median eminence, where

GnRH is released into the hypophysial portal circulation. The pulsatile secretion and the ovulatory surge of LH depend on the act ivity of GnRH neurons ( Kalra and Kalra,

1983 ). Research from two laboratories has shown that atrazine interferes with the pattern an d 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 ability 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; Fora dori et al . , 2009b ). Further evidence supporting this ob servation comes from the work done on pigs demonstrating that a single intravenous injection of GnRH (100 μg) was able to partially reverse or counteract

atrazine

- induced suppression of the LH surge (Gojmerac et al .

, 2004). There have been some reports i ndicating that atrazine, or its metabolites, may inter fere directly with
GnRH ac tiv it y ( e.g.

, Zorrilla et al . , 2010 ) as well as the activity of other neurons ( Das et
al . , 2001 ). While these new data enhance our understanding of the processes involved

in the HPG axis

- mediated attenuation of the LH surge and subsequent toxic effects, they do not

– at this time

– suggest a lower effect level than the one used in the 2003

IRED.

2. 4 Summary

The Agency has used the MOA Framework to evaluate the neuroendocrin e MOA leading to disruption of the HPG axis (USEPA, 2000; WHO, 2005); this analysis has been recently confirmed and updated using new LH time course and mechanistic data on the pulsatile release of GnRH. The experimental toxicology data support the LH

eff ects as the most relevant and sensitive health outcome reported in animals. Because human reproduction and development are controlled by the neuroendocrine system,Do not cite

Section 2

- Page 28 of 677 there is the potential that atrazine exposure could lead to adverse health consequences.

Th us , attenuation of LH provides a strong foundation for assessing the human health effects of atrazine. In the following sections, the Agency describes the review of the

non

- cancer epidemiology literature and discusses how these epidemiology studies can

in form the human health risk assessment. Consistent with OPP‘s draft WOE framework for evaluating epidemiology and experimental toxicology data, the integrated analysis uses the MOA as the starting point for considering qualitative and quantitative

differen ces in humans and animals.Do not cite

Section 3

- Page 29 of 677 3. EVALUATION OF NON

- CANCER EPIDEMIOLOGY LITERATURE

3 .1 Overview

This chapter describes the epidemiology studies that evaluat ed the association between atrazine exposure and adverse n on

- cancer health effects. To identify article s relevant to this question, EPA searched major biomedical databases, performed citation mapping, and hand

- searched selected reference lists of pertinent articles. A full description of the literature review methodology is presented in Appendix B

- 1 . This

r eview includes studies which utilized a cohort, case

- control, cross

- sectional or ecologic study design, as well as some hybrid study designs

. This information will be integrated with the experimental animal toxicology database presented in Section 4

.

The nineteen ar ticles included in this review estimated statistical associations between use of the herbicide and several major types of systemic health effects. The majority of articles reviewed investigated atrazine exposure in a ssociation with either male or female reproductive effects, or fetal or peri

- natal effects , including major birth defects. EPA evaluated the quality of these studies and considered the usefulness of

this database to inform the atrazine risk assessment usin g a modified Bradford

- Hill criteria as presented to the FIFRA SAP in February 2010. Recommendations received from the February FIFRA SAP report greatly aided this review (FIFRA SAP, 2010).

EPA considered several factors indicative of good quality epidemi ologic analysis in performing this review. Among the se factors were the ability to accurately measure the health effect (the outcome) as well as atrazine exposure; the identification and accurate measurement of potential confounding factors; the sample siz e and statistical power to measure the association; the appropriateness of the statistical analysis to the study design; and, the interpretation of the overall study results. Environmental epidemiologic research includes a set of additional factors to cons i der when assessing the validity and reliability of study results. Key among these is the method of exposure assessment. Specifically, whether authors were able to perform qualitative or quantitative exposure assessment; the ability to measure low level ex posure and how researchers dealt with observations below the limit of detection (LOD), when applicable; the ability to measure atrazine exposure during relevant time period for the health effect; the range of the exposure captured in the study; and, the re levance of the biomarker to

actual environmental exposure s . A detailed review addressing these points, among others, for each ar ticle can be found in Appendix B

- 2

. Table 3

- 1 delineates key study

characteristics.Do not cite

Section 3

- Page 30 of 677 3.2 Literature Review

3.2.1 Reproduct ive System Effects

Sex steroid hormones regulate the reproductive cycle . As summarized in S ection

2

, there is evidence that atrazine may disrupt steroid hormone synthesis and signaling. Therefore, epidemiologic researchers ha ve hypothesized male and female reproductive effects may result from atrazine exposure. Several epidemiology studies were identified that investigate the potential association between atrazine exposure and specific reproductive health endpoints/outcomes in cluding timing of menopause, menstrual cycle characteristics, gestational diabetes

, and, among men, semen quality. These epidemiological studies will be considered in combination with experimental toxicology

studies in Section 4

.

3. 2.1.1 Female Reproductive System

Farr et al. (2004 and 2006) investigated the role of atrazine use on both menstrual cycle characteristics, and timing of menopause, respectively, among female pesticide applicators and female spouses of male pes ticide applicators enrolled in the Agricultural Health Study (AHS) [www.aghealth.org]. In Farr et al . (2004), researchers compared the percentage of women reporting abnormal cycle length (<24 days, >36 days, irregular), inter

- menstrual bleeding, and misse d periods among those who reported using certain pesticides, including atrazine, during their lifetime and those who did not. Based upon the authors‘ review of the available literature, the authors posited a priori that atrazine, as well as lindane and man eb/mancozeb, would be associated with menstrual cycle abnormalities due to the pesticides‘ suspected interference with the estrous cycle

. The odds of a long cycle and missed period were elevated for women using any pesticides and author

- defined  ̳hormonall y active‘ pesticides (60

- 100% greater than for ever use of any pesticide). The odds of these menstrual cycle characteristics

were more than 2

- fold greater among women who reported using atrazine or lindane (or both), excluding users of maneb/mancozeb, as c ompared to never users of pesticides. Specifically, in comparison with never users of pesticides, the odds of long or missed cycle among ever users of atrazine or lindane over the lifetime were 2.7 (95% CI 1.3

- 5.5), and 2.0 (95 % CI 1.3

- 3.3), respectively, controlling for age, body mass index and smoking. Results did not differ when adjusting for occupational physical activity; leisure time physical activity information was not available. A borderline significant association with irregular cycle (OR 0.45, 95 % CI 0.18, 1.1) and a significant association with inter

menstrual bleeding 1.7 (95% CI 1.1, 2.7) was also observed for women who reported

ever

- using atrazine or lindane. Unfortunately, authors were not able to present atrazine

specific risk estimates in t his paper

.

To study the potential association between atrazine use and timing of menopause, Farr et al . (2006) studied a similar group of women enrolled in AHS. In a survival analysis, the authors modeled time to menopause (ovarian failure) inDo not cite

Section 3

- Page 31 of 677 associat ion with ever

- use of any pesticide, functional group ( i.e ., herbicide) and by pesticide class ( e.g.

, triazine), as well as with those pesticides designated by the authors as ―hormonally active‖ or ovotoxic . These included atrazine , carbaryl, carbon

tetrach lorine, DDT, lindane, and maneb/mancozeb. As compared to those who never used pesticides, the authors observed a reduced hazard (increased time to menopause) among women who used any AHS pesticide ( hazard ratio (HR) 0.87 (95% CI 0.78

- 0.97,

a 3

- month delay) , and among women who used any hormonally active or ovotoxic pesticide (v. never users) (HR 0.77, 95% CI 0.65

- 0.92). With regard to atrazine, the strength of the association was slightly greater than that for ever

- users of any hormonally active pesticide: HR 0.79 (95% CI 0.63, 0.99). Inclusion of variables reflecting occupational or leisure

- time physical activity had little effect on the estimated

risk . Authors adjusted for smoking and oral contraceptive use in their final model

. The

a uthors were not able to assess the timing of pesticide exposure or exposure

- response relation using the data available at this time of this study.

Because it has been suggested that pesticide exposure and other environmental contaminants are possibly dia beto genic, Saldena et al . (2007) studied the association between pesticide use and gestational diabetes among female spouses in the AHS. Gestational diabetes occurs in women who did not have diabetes before pregnancy. In this study, pesticide exposure was measured based upon activities reported by women during the first trimester of their last pregnancy. The average time between last pregnancy and questionnaire completion was 11.4 years among the participants in this study. Exposure was assessed considering broad pesticide us e categories as follows: no exposure (referent), indirect exposure (planning, pruning, weeding, picking or harvesting), residential direct use (applying pesticides to garden or inside house), or agricultural direct use (mixing or applying pesticides to cro ps or repairing pesticide application equipment). Among women who reported direct agricultural use of pesticides during the first trimester of the last (index) pregnancy, the authors compared

lifetime ever

- and never

- users of 50 pesticides included in the AHS ( www.aghealth.org ) including atrazine, in association with self

- reported gestational diabetes. Researchers

observed a two

- fold increased risk (odds) of gestational diabetes among women who reported direct, agric ultural use of pesticides as compared to never users of any pesticide. Among women who reported direct agricultural exposure during the first trimester, a statistically significant association with ever

- use (lifetime) of atrazine was observed (graphical de piction only, no exact point estimate presented in study).

The limited epidemiologic database related to female reproductive effects and atrazine use generally support the hypothesis that atrazine may influence the hormonal milieu in such a way as to aff ect female reproductive outcomes such as menstrual cycle characteristics and menopause, however

, significant uncertainties exist. In the study of menstrual cycle characteristics, authors were not able to measure atrazine use specifically in association wit h any of the menstrual cycle characteristics; and, the lack of a refined estimate of the timing of pesticide exposure in relation to change in menstrual cycle characteristics limit the use of these data in risk assessment. The clinical significance of a 5

- month delay in time to menopause is not understood at thisDo not cite

Section 3

- Page 32 of 677 time. Strengths are the relatively large sample of women among the most highly exposed in the general population, and ability to control for potentially confounding factors. With regard to timing o f menopause, Farr et al . (2006) conducted a well

designed investigation of the use of potentially hormonally active pesticides among a large sample of women who are among the most highly exposed to pesticides in the

U.S. population in relation to this end point. As acknowledged by authors, more frequent assessment of menopausal status among the women in the cohort, enhanced pesticide exposure assessment allowing exposure

- response assessment, and improved confounding factor analysis, specifically with regard to physical activity and possibly other aspects of a healthy population, would be enhancements to the current, initial investigation. Although authors connect the results of their two investigations in the following manner: they postulate that the associa tion between pesticide use  ̳hormonally active‘ pesticides and delay in timing of menopause may be explained by the observation of increased odds of long

- or missed menstrual cycles in association with

use of hormonally active pesticides. However, there is not enough epidemiological evidence to date to suggest with certainty that atrazine exposure is related to either a delay in the timing of menopause or with long

- or missed menstrual cycles ; other non

causal explanations cannot be eliminated at this time. Additional epidemiologic

evaluations of these associations are needed to reduce the current uncertainties in the

database. The hypothesis

- generating research into the association between pesticides

and gestational diabetes requires replication to better un derstand any possible

association

.

3.2 .1.2 Male Reproductive System

Swan et al . (2003) performed a case

- control analysis comparing concentration of atrazine metabolite (atrazine mercapturate), in addition to other pesticides, between

men with and wit hout poor semen quality (sperm concentration below the study population median). Participants were derived from a larger study cohort, the Study for Future Families, sponsored by N ational Institute of Environmental Health Science (N IEHS ) . Authors reported statistically significant elevated odds of poor semen quality (sperm concentration, morphology and motility) in association with atrazine exposure (odds ratio 11.3 (95% CI 1.3, 98.9)). T his study include d use of biomarkers of both semen quality and pestici de exposure, and use of appropriate analytic methods which maximize the amount of information gained from the study. Limitations include the relatively small sample size (n<10 for certain exposure

- outcome groups), and some limitations with respect to expos ure assessment. Authors measured atrazine mercapturate (AM) only, a method which has since been shown to under

- estimate total atrazine exposure ( Barr et al. 2007)

. As a result, a significant proportion of observations were below the limit of detection, dis allowing meaningful exposure

- response
assessment. R esearchers indicated that the proportion of men reporting occupational pesticide use, recent pesticide us e, and residential proximity to treated field were comparable between men with and without poor seme n parameters. However, researchers were not able to fully adjust for the co

- exposure to other environmentalDo not cite

Section 3

- Page 33 of 677 exposures which the authors acknowledge as a potential limitation of the current work

. In sensitivity analyses, authors devised an ad hoc score test accounting for multiple pesticide exposures in association with poor semen parameters and observed that exposure to more than one pesticide was more frequent among men with poo r semen parameters. L arger prospective studies with the ability to account for multiple

environmental exposure s are needed to clarify the potential association between atrazine use and semen parameters.

3.2.2 Fetal and Perinatal Outcomes

As summarized in S ection 2 , p ublished reports indicate that atr azine exposure during gestation may affect rat pup weight. As a result, several authors have hypothesized that either male or female atrazine exposure, or that pre

- conception, early

- or late

- fetal atrazine exposure may be related to adverse birth outcomes

.

3. 2.2.1 Spontaneous Abortion

Spontaneous abortion (also known as miscarriage) is defined as the loss of a

fetus before the 2 0 th week of gestation. Most spontaneous abortions occur within the first trimester. A variety of factors may contribute to the occurrence of a spontaneous abortion ranging from chromosomal abnormalities (the most common cause during the first trimester) to maternal uterine malformations, polycystic ovary syndrome, uncontrolled gestational diabetes, and environmental factors. Pesticide exposure is among the environmental factors that have been hypothesized as potentially associated with spontaneous abortions (Weselak et al

., 2008 ). Prior to the study published by

Arbuckle et al . (2001) little epidemiologic information was ava ilable to evaluate whether and which pesticides may be related to this outcome and what were the potential critical windows of exposure.

In the Ontario Farm Family study, authors estimated the association of pre

- and post

- conception (3 months prior to an d the estimated month of conception, or first trimester, respectively) agricultural and residential pesticide use reported at the farm level, including atrazine, with spontaneous abortion (defined in this study as prior to 20 weeks gestation). Authors furt her stratified the outcome variable as early

- (<12 weeks gestation) and late

- fetal age (12

- 19 weeks gestation) spontaneous abortion. Researchers reported that particularly early gestational age spontaneous abortion triazines, glyphosate, carbaryl, phenoxy herbicides, and thiocarbamates were associated with spontaneous abortion, particularly early gestational age spontaneous abortion. Specifically, with respect to atrazine and triazines, pre

- conception exposure to atrazine resulted in a slightly increased r isk of spontaneous abortion, OR (95% CI) was 1.2 (0.9

– 1.7), risks were increased for the instances of spontaneous abortion that occurred early (<12 weeks), but not later (12

- 19 weeks) in pregnancy. There was no association between post

- conception exposure to atrazine and spontaneous abortion.Do not cite

Section 3

- Page 34 of 677 Participants reported using several different pesticides within the time period of susceptibility.

In a different study, Savitz et al

. (1997) estimated the association between pre

conception male pesticide exposure a nd adverse birth outcomes

. Non

- significant elevated risks were observed for miscarriage among pregnancies in which the male partner reported application of crop herbicides and atrazine use during the pre

conception period . A major limitation of the study i s the quality of exposure assessment: authors were not able to measure atrazine use directly in association with application of

crop

- and yard

- herbicides , authors utilized a probabilistic method of exposure

assessment . This likely resulted in a degree of e xposure misclassification ( likely non

differential exposure misclassification )

. Using this method of exposure assessment, authors were not able to eliminate the role of maternal pesticide exposure in this

association.

While these studies add to the litera ture by providing results that inform the possible susceptible window of exposure and potential target sites of toxicity (embryo or fetus), the method of exposure assessment limits the utility of the results. In both studies, pesticide exposure was assesse d at the farm

- level as a surrogate for both maternal and paternal exposure. This approach, as acknowledged by the authors, likely resulted in some exposure misclassification.

3. 2 .2.2 Birth Defects

In this section, several studies in which authors hypo thesize pesticide exposures including atrazine exposure may be related to birth defects are presented . Three of the

five studies reviewed herein estimated the link between atrazine exposure and

abdominal wall birth defects (AWD) . Gastroschi s is and omphaloc ele, are different types of abdominal wall defects, are congenital malformations in which abdominal organs protrude from the body through a defect in the fetal abdominal wall. The incidence of gastroschisis and omphalocele is approximately 0.04% each (Can field et al .

, 2006) with

a 6

- fold higher incidence in infants born to very young mothers (<20 years of age) (Williams et al. 2005). Several environmental factor s including smoking, alcohol use,

and medication use have also been epidemiologically associat ed with increased incidences of gastroschisis ( Siega

- Riz et al . 2009)

. Five epidemiological investigations as to the relation between atrazine exposure and birth defects are presented in this

section.

Mattix et al . (2007) performed an ecologic analysi s using cases of abdominal wall defects reported to the national U.S. natality data set, and USGS collected atrazine surface water concentration. Authors reported that the rate of this birth defect in the state of Indiana (3.75 per 10,000 births) was great er than the U.S. (2.75 per 10,000 births in the U.S.), and greater than the Midwest region (2.91 per 10,000). The authors reported a statistically significant correlation between monthly atrazine concentration inDo not cite

Section 3

- Page 35 of 677 surface waters in the Midwest and rates of omphalocele and gastroschisis in Indiana (p=0.0125). Several limitations were noted. Specifically, not all natality data are comparable across states, especially with respect to birth defects reporting, although efforts have been made to enhance comparabil ity. This limitation may have result ed in

measurement errors in the study. In addition, the measurement of atrazine in surface water using USGS collected data likely leads to some exposure measurement error when used as a surrogate for drinking water expos ure. However, the major limitation of this study is that it is not known whether women whose infants were born with this birth defect were actually exposed to atrazine (whether in drinking water or via another relevant exposure pathway ) given the ecologic study design. While other non

- causal explanations for the correlation observed between atrazine concentration in surface water and month of conception of fetus with AWD cannot be excluded based upon these data, this study has spurred other work using diffe rent study designs.

Hornemann et al . (2008) investigated the prevalence of omphalocele in association with atrazine exposure in a part of n orthern Germany. Authors identified cases of omphalocele through a prenatal clinic in the area, and utilized season of conception (spring/summer v. fall/winter) as an indicator of potential seasonal exposure to atrazine. Researchers observed no distinct pattern in the rates of omphalocele over the time period of the study (1993

- 2002), and reported no differences in pre valence of this birth defect between pregnancies conceived in the spring/summer as opposed to the fall/winter. However, as stated by the researchers, the study was performed during a time period in which use of atrazine in agriculture was prohibited. There fore, if atrazine was still present in the environment after the registration was revoked, it is likely that atrazine use would not differ greatly between seasons of the year or over the time period of this study, possibly explaining the lack of associatio n reported herein. Authors hypothesized that residual environmental concentration would have still been great enough to observe an effect in the population, if an association between atrazine exposure and this birth outcome exists. O verall the study provid es little additional evidence as to the developmental toxicity of atrazine.

Subsequent to the Mattix et al . (2007) and Hornemann et al . (2008) studies, two additional epidemiologic investigations evaluated the potential association between atrazine exp osure and birth defects. In contrast to Mattix and Hornemann, these studies did not posit a specific hypothesis regarding atrazine‘s potential developmental toxicity,

e.g. , abdominal wall defects. Winchester et al . (2009) and Ochoa

- Acuna et al . (2009)

look ed at atrazine exposure in association with several types of birth defects using national and state birth records, respectively. Winchester et al . (2009) published a second ecologic study comparing rates of several birth defects reported to the U.S.

natal ity dataset and USGS collected atrazine concentration in surface water for the entire U.S. for the time period 1996

- 2002. Authors reported increased prevalence of 11 of 22 birth defects investigated among pregnancies with estimated dat e of conception

durin g the Spring months in comparison to all other months. Birth defects rates were higher in the Spring versus all other months of the year for the following broad types of birth defects: spinal cord, circulatory system, cleft lip, limb defects (adactyly), mu scularDo not cite

Section 3

- Page 36 of 677 problems, Down‘s syndrome, other congenital birth defects, tracheo

- larengyl, gastrointestinal (omphalocele higher but not statistically significant), urogenital and clubfoot. It should also be noted that the rates of omphalocele was non

- statisticall y different between pregnancies conceived in the Spring in comparison to other seasons (28.89/100,000 births in Spring v. 28.10/100,000 births other seasons).

In a retrospective cohort study (Ochoa

- Acuna et al .

, 2009), authors compared

birth defect rate s reported to the Indiana birth registry in association with geographic proximity of maternal residence to agricultural area (corn and soybean production) in the state of Indiana. In this study, authors observed increased odds of prevalent limb

birth defec ts including adactyly among pregnancies among women who lived in closer proximity to corn fields; authors reported an approximate 20% increase in limb defects

for each 10 ha increment of distance a maternal residence was from a corn field.

Authors also obs erved a non

- significant increased odds of abdominal cavity birth defects in association with closer (<3.4 ha) versus farther (>3.4 ha) distance between maternal residence and corn field or soybean field (OR 1.50 (95% CI 0.72, 3.10). This study represents a n improvement over the ecologic study (Winchester 2009), however proximity to corn field as a measure of atrazine exposure likely results in some exposure misclassification. Although preliminary in nature, it is notable that both investigations identified limb birth defects and abdominal cavity defects in association with modeled atrazine exposure. Given the potential biases likely in the design and conduct of each of these studies, additional research is necessary to determine the true

nature of the associ ation.

Waller et al . (2010) recently performed a population

- based case

- control study of the association between atrazine concentrations in surface water samples (USGS) and the prevalence of gastroschisis in Washington state. This study

, in large

, part bu ilds upon the evidence provided by Mattix et al . (2007), Winchester et al . (2009), and

Ochoa

- Acuna (2009) using an improved study design and appropriate confounder analysis. Waller et al . (2010) utilized birth records and hospital discharge records to

a scertain cases of gastroschisis, and randomly selected a control series (1:4) from all births reported to the state in the study time period, frequency matching on year of birth. Medical record review was performed to enhance the validity of the outcome me asure. To measure atrazine exposure, the authors measured the distance between the zip code in which the mother resided at the time of delivery, and the nearest ―high‖ atrazine

surface water monitoring site (―high‖ was defined as >3 ug/L, the EPA limit at the time of this study). Authors reported a 40

- 60% increased odds of prevalent gastroschisis among mothers who resided less than 50 km from a ―high‖ atrazine surface water site [25

- 50 km: odds ratio 1.4 (95% CI, 1.2

- 1.7); <25 km: OR 1.6 (95% CI 1.1

- 2.3), p trend 0.049). Strengths of this study include the relatively large sample size, and hospital record review to properly categorize cases of gastroschisis, omphalocele, and

abdominal wall defects. A lthough the study design, population

- based case

- control

st udy, is generally considered more targeted than ecologic study designs, the method of exposure assessment Waller et al . was pseudo

- ecologic. Therefore, u ncertainty remains as to whether mothers of neonates diagnosed with gastroschisis were exposedDo not cite

Section 3

- Page 37 of 677 to atra zine (in the drinking water, through recreational use, or other environmental exposure) during the relevant exposure window, i.e., the period of pregnancy . Other potential biases including relatively high prevalence of maternal relocation during the period of pregnancy, are well described by the authors. The strength of the associations presented as well as the apparent positive, monotonic exposure

- response relation between distance to atrazine ―high‖ USGS site are interesting aspects of this study ; however

, potential exposure measurement error

, given the method of exposure

assessment

, present s uncertainties in the interpretation of these data

.

The five studies investigating atrazine exposure in association with birth defects, including abdominal wall def ects, utilize several different methods of exposure assessment. Assessment methods include state, regional or national average atrazine concentration of atrazine in surface water using the USGS dataset; distance between

maternal residence and a USGS site w hich on average reported atrazine concentrations greater than the regulatory limit; season of conception; and maternal proximity to corn and soybean fields in a state in which atrazine is predominately used on corn. In each instance, exposure assessment ut ilized an aggregate measure

. Similarly, cases of birth defects were identified using U.S. or state level birth records which included report of the birth defect. There is no national birth defect registry in the U.S. The Hornemann et
al . (2008) study asce rtained cases of omphalocele using sonographic records. With respect to birth record data, while attempts have been made to enhance the consistency and completeness in reporting, under

- reporting of birth defects is generally

considered to occur in most sta tes (Watkins et al . , 1996) . For studies in which

individual

- level confounder assessment was possible (Waller, Hornemann, and Ochoa

Acuna), researchers were able to measure most relevant demographic charac t eristics ; however

, no study was able to investigat e possible confounding with other pesticides or environmental exposures which may also coincide with atrazine use and may be related

to birth defects. However, to have a significant influence on the measure of association, the potentially confounding envir onmental agent would have to be strongly related to

both atrazine use and birth defects. Overall, the database for the association between early gestation exposure to atrazine and abdominal wall defects

, including gastroschisis and omphalocele

, is weak

.

3.2.2.3 Adverse Birth Outcomes

Several studies evaluated how atrazine use or exposure relates to adverse birth outcomes including small

- for

- gestational age (<10 th percentile), preterm delivery (<37 weeks), low birth weight (<2500 grams), and intra

- uter ine growth retardation, as well as another population birth characteristic, male to female sex ratio. Given the suspected

e ffect atrazine has on the hormonal milieu, authors hypothesize that these birth outcomes could potentially relate to maternal or pate rnal pre

- conception exposure, or in

utero exposure to the fetus during different periods of gestation (i.e., trimester). It should be noted, however, that a challenge to conducting reproductive epidemiology research is determining the critical window of ex posure for certain adverse effects, and thenDo not cite

Section 3

- Page 38 of 677 measuring exposure well during this period (Pryor et al . , 2000) . The studies included in this review varied in this respect. However, looking across studies, some new knowledge can be gleaned

.

Three studies in vestigated small

- for

- gestation age (SGA) in relation to atrazine exposure. Ochoa

- Acuna et al . (2009) performed a retrospective cohort study which included several hundred cases of SGA reported to the Indiana state birth registry in

association with concent ration of atrazine in drinking water. In this study, authors used

time

- series analysis tools to model weekly and monthly atrazine concentrations in drinking water combining data from four different water monitoring programs in the state, which is an enhanc ement over other aggregate exposure measurement methods included in this review. Authors reported a 20% increase in the prevalence of SGA among pregnancies exposed in the third trimester, a period of rapid (non

- linear) growth of the fetus, suggesting late pregnancy exposure may be a susceptible window of exposure for SGA. Additionally, Villanueva et al. (2005), used the same study design to examine this question in an area in France noted for high agricultural production and herbicide use including atrazine . These authors used birth outcomes reported to regional birth registries and concentration of atrazine in drinking water, matching community drinking water levels to individual residences. Authors reported no
association between atrazine and SGA overall ; however

, they included a very narrow exposure range in this study, possibly too limited in range to discern a true difference between groups given the sample size. In sub

- analyses restricting the sample pregnancies in which the third trimester overlapped t he period of higher atrazine use (May

- October in this region), researchers observed a statistically significant 50% increased odds of SGA. Although limitations are present, the consistency between

Villanueva et al . and Ochoa

- Acuna et al . (2009b) is notab le. An ecologic study published more than 10 years ago reported a correlation between residence in an area with high atrazine concentration in surface water in southern Iowa and intra

- uterine growth retardation (Munger 1997).

Ochoa

- Acuna et al . (2009) a nd Villanueva et al

, (2005) also both measured the association between atrazine exposure in drinking water and preterm delivery, defined as delivery before 37 completed weeks gestation. Ochoa

- Acuna et al . (2009) reported no association, using time

- series analysis of monthly atrazine concentration in drinking

water. Villanueva et al . (2005) also reported no association overall ; however

, among pregnancies in which the first trimester overlapped the period of high atrazine use (May

October in this study [Fra nce]), odds of preterm delivery increased more than 30%, although the result was not statistically significant. These authors also report an increased odds of low birth weight in association with pregnancies in which the third trimester overlapped with the season of high atrazine use in the region. Measuring atrazine exposure on the farm through questionnaire response, a study by Dabrowski et
al . (2003) [Poland] reported an average 2 week decrease in gestation length in

association with use of a triazine o n the farm on which both male and female partners live and work (study based upon sample in Poland).Do not cite

Section 3

- Page 39 of 677 A recent publication using AHS data reported no association between maternal pesticide activity (indirect agricultural exposures, pesticide use in the h ome, and direct pesticide application) during the first trimester of pregnancy and a significant change in birthweight among births which occurred within 5 years of enrollment into the cohort (enhancing the probability that pesticide use preceded the pregn ancy) (Sathuanarayana

et al .

, 2010). However, maternal engagement in direct pesticide application during the first trimester of pregnancy resulted in the greatest change in birthweight, a 72 gram reduction, which was not statistically significant. In the s ame study, comparing ever

- use of several pesticides over the lifetime in association with pregnancies which occurred within 5 years of enrollment into the cohort, authors observed a non

- statistically significant decrease in birthweight (

- 39 grams, 95% CI

- 171, 92 grams) among women who reported ever

- use of atrazine in comparison to never

- users of atrazine. Overall, Sathuanarayana et al . (2010) offer little additional evidence that maternal atrazine exposure is associated with birthweight.

Savitz et al

. (1997) estimated the association between pesticide exposure

, including atrazine

, among male partners in a pregnancy during the three months prior to conception and adverse birth outcomes including miscarriage, preterm delivery, small

for

- gestational age, and secondary sex ratio. Savitz et al . (1997) reported a 2

- to 4

- fold increased risk of preterm delivery in association with application o f crop

- or yard

pesticides and atrazine exposure by male partners during the three months prior to conception ; howe ver

, estimates were based upon a small number of exposed cases and were therefore imprecise (large confidence intervals). A major limitation of the study is the quality of exposure assessment: authors were not able to measure atrazine use with application of crop

- and yard

- herbicides directly. This likely resulted in a degree of exposure misclassification, however likely non

- differential exposure misclassification. The study provides some qualitative evidence that application of crop

- and yard

herbicides l ikely involving the application of atrazine may be associated with preterm delivery.

Overall, this body of evidence provides some suggestion that atrazine exposure may relate to adverse birth outcomes, particularly small

- for

- gestational age. Epidemiologi cal evidence for a link with both preterm delivery (<37 weeks) and low birth weight (<2500 grams) is less compelling at this time. The studies included in this section

of the literature review reflect case

- control and cohort studies, measuring individual

l evel variables; however, Munger et al . (1997) performed an ecologic analysis. Among

the case

- control and cohort studies, authors assessed exposure through either self

report questionnaire (Savitz et al .

, 1997 and Dabrowski et al .

, 2003) or drinking wat er exposure measurement (Villanueva et al .

, 2005 and Ochoa

- Acuna et al., 2009).

U ncertainties remain in the measurement of individual

- level atrazine exposure in each study with both the measurement of actual atrazine exposure and the time period of

expos ure in relation to the period of susceptib ility for these birth outcomes. Villanueva et
al . (2005) attempted to refine the period of susceptibility for SGA, preterm delivery and (low birth weight) LBW. These results are based upon sub

- analyses with relati vely low statistical power. Birth outcomes were ascertained through either self

- report or birthDo not cite

Section 3

- Page 40 of 677 records, which are generally considered to be accurate with respect to birth weight , and moderately valid and consistent reporting of gestational age . None of t he studies were able to investigate co

- exposure to other pesticides or environmental chemicals that may be related to both atrazine seasonality or exposure and birth outcomes. In general, there is a suggestion that an association may exist, particularly wi th SGA and possibly with preterm delivery

.

3.2.3 Respiratory Effects

Among the studies selected for this literature review, one hypothesis generating study examined the relation between atrazine use, among other pesticides, and prevalence of wheeze w ithin the AHS; however, atrazine or triazine exposure was not considered among the a priori hypotheses posited by these authors. Participants self

reported instances of wheeze; those who indicated they experienced one or more episode of wheeze in the last year were included as  ̳cases‘ of wheeze. Pesticide usage was evaluated for the year prior to questionnaire completion. A strength of this study was that both exposure and outcome measures reflect a similar time period. Researchers observed a statistically significant increased prevalence of wheeze among those who reported using atrazine in the index year (OR 1.20, 95% CI 1.07, 1.34). In addition, when researchers examined exposure

- response pattern by days of year of use of atrazine, they observed a signific ant trend (p trend<0.01). In all, this represents a high quality hypothesis generating study within the AHS. A particular strength of the study is the ability to control for co

- exposure to grain dusts as a potential confounder in the

atrazine

- wheeze associ ation; atrazine is an herbicide used primarily on corn and wheat in Iowa, one of the 2 states included in the AHS. In subsequent analysis of the

association between atrazine use and adult

- onset asthma within the AHS cohort,

researchers did not observe an a ssociation (Hoppin et al

. , 2009). A dditional studies are

needed to better understand and characterize the potential respiratory effects of

atrazine use.

An additional study investigated the potential e ffect atrazine or triazines could have upon respirato ry health, however this study includes several limitations the greatest of which is the inability to isolate the e ffect of atrazine in relation to respiratory

health. Kossman et al . (1997) evaluated several respiratory parameters including pulmonary venti lation rate and muscular force among a group of pesticide manufacturing employees in Poland. Using an occupational cohort design, authors selected 37 male and 17 female employees of the plant, the female employees were not involved in direct pesticide manu facture but in packaging of the products; employment at the plant was considered a surrogate for pesticide exposure including, but not limited to, triazine exposure. The unexposed series was identified from the surrounding population. Employees in the plan t were responsible for manufacturing and packing several types of pesticides including triazines, carbamates, 2,4

- D, captan, carbofuran and other solvents. Authors performed simple statistical comparisons (t

- test and percent of values below the population median), and concluded that respiratory parameters, specifically forced maximum inspiratory and expiratory volume, were lower among plantDo not cite

Section 3

- Page 41 of 677 employees than residents of the surrounding population. This study provides some evidence that pesticide manufacture may be associated with suppressed lung function, however lack of specificity in the exposure assessment methodology makes it impossible to discern what, if any, e ffect atrazine or triazines may have on this outcome.

Among a similar occupational cohort a s Kossman et al . (1997), Kluchinski et al . (2001) investigated certain immune parameters hypothesized to be precursor biomarkers for the impaired respiratory functions observed in the two studies by Kossman. However, similar limitations in exposure asses sment exist in this study as were present in the Kossman research, i.e., lack of specificity in exposure measurement. Therefore, while Klunchinski also observed aberrant levels of immune system cell counts among those employed in pesticide manufacturing as compared to population residents (non

- exposed), it is not possible to discern what role, if any, is played by atrazine. In addition to the significant limitations presented by the method of exposure assessment, the selection of non

- occupational, general p opulation controls is likely flawed; residents of the surrounding area may also be exposed (do not represent an unexposed series) due to potential environmental contamination. Overall, the

database for an association between atrazine or triazines and respi ratory effects weak

at this time.

3.3 Synthesis of Epidemiological Data

The epidemiologic evaluations of the non

- cancer health effects potentially related to atrazine exposure are largely consistent with the experimental toxicology database, i.e., pos sibly influence of atrazine exposure on male and female reproductive system, and possibly small

- for

- gestation age birth outcomes. Additional associations are suggested in the epidemiology literature based upon high

- quality hypothesis generating studies, in clude respiratory effects (wheeze), and gestation diabetes. At this time, the epidemiologic database concerning the non

- cancer health effects of atrazine exposure

is not sufficiently robust or mature enough to support causal inference, ( i.e ., EPA is not

ab le to make a firm cause to effect link age between atrazine exposure and any of the

non

- cancer health effects examined in these studies )

. Additional research is needed to clarify the signals in these data. While limitations in the epidemiology database exis t at this time, the information can be used to support hazard identification and characterization steps of risk assessment.

Several methodological limitations in the design, conduct or analysis of the studies prohibit inferring causal associations were o bserved across these studies, and interfere with use of these data in quantitative risk assessment. Accurate outcome identification is of key importance in epidemiologic research. Across this database, outcome is ascertained either through self

- report with out medical record review, or through birth registry databases which are inconsistent . The one exception is the study by Swan et al . (2003) in which they measured biomarkers of semen quality. While some birth outcome information like birth weight have bee n found to be reliably self

reported in epidemiologic analysis, several studies identify birth defects and measureDo not cite

Section 3

- Page 42 of 677 birth outcomes using birth record reporting databases. Adverse birth outcomes and birth defects are likely under

- reported to these registries . In addition, several studies include a relatively small sample size, particularly with reference to sub

- analyses evaluating potential effect modification or specific exposure windows. Therefore, replication is necessary. Sufficient confounder analysis is also lacking in several of these studies, including the evaluation of physical activity in the association of atrazine use with both menstrual cycle characteristics and also menopause, and co

- exposure to other pesticides or environmental chemicals.

The quality of the atrazine exposure measurement across several of the studies included in this review is a major limitation to concluding a possible causal association

exists at this time , or using these data in quantitative risk assessment. Several of the

st udies utilize a surrogate for atrazine exposure including concentration of atrazine in USGS surface water monitoring programs as a surrogate for atrazine in the drinking water; season of conception (spring v. other) as a surrogate for maternal atrazine

exp osure; or, geographic location or proximity to atrazine use site, e.g. , agricultural field, as a surrogate for maternal or in utero atrazine exposure. Using these types of surrogate measures, uncertainty exist s as to the extent the authors were able to

cor rectly capture atrazine exposure among those with certain non

- cancer health effects under evaluation. In studies of adverse birth outcomes, researchers with the Ontario Farm Family study measured maternal, paternal and in utero atrazine exposure by overlay ing typical months of the year of atrazine use in an average farming year (considering the most recent 15 years of farming experience) with the maternal reported period of pregnancy. Therefore, while a reasonable surrogate measure at the time of this study , there is uncertainty as to the assessment of maternal, paternal or in utero exposure, and the period of development in which the exposure likely occurred.

For the non

- cancer health outcomes evaluated, specifically female reproductive

effects and birth outcomes, the exposure window is of key importance. Most studies lacked the ability to refine the exposure window. Villanueva et al ., (2005) and Ochoa

Acuna et al

. , (2009) measured atrazine concentration in drinking water in association

with maternal resi dence at the time of birth to estimate maternal atrazine exposure. While measurement in drinking water, as opposed to surface water, is an improvement over other investigations included herein, others have measured that maternal mobility during pregnancy i s relatively high in some investigations

, up to 30 % (Canfield et al .

, 2006). Therefore, uncertainty remains as to whether the method of atrazine exposure assessment correctly measures maternal atrazine exposure through drinking water.
Authors with the AHS investigated pesticide exposure, including atrazine, in association with female reproductive outcomes including menstrual cycle characteristics, timing of menopause and gestational diabetes. However, in each investigation atrazine exposure

was assessed us ing an ever

- and never

- exposure metric over the course of the lifetime. While sensitivity analyses in these investigations indicate this exposure measure may be reasonably accurate, more research with enhanced exposure assessment is suggested by the author s. Swan et al. ( 2003 ) utilized urinary concentration of atrazine
mercaptur e (AM) , a biomarker of atrazine exposure. Biomarkers are generallyDo not cite

Section 3

- Page 43 of 677 considered superior measures of exposure. However, recent publication indicates use of only AM as a biomarker of at razine exposure likely under

- estimates total atrazine

exposure

, and that measurement of other metabolites may be more appropriate for epidemiologic analyses (Barr et al

. , 2007). Lastly, most studies lack the ability to perform exposure

- response assessment, i.e. , typically an ever

- use , or two

- level

measure was evaluated.

Although limitations exist, some new insights into the potential health

consequences of atrazine may also be gleaned looking across the observational epidemiology database. Given our unders tanding of atrazine‘s mode of action and its toxicity in experimental animals, the findings in the observational studies identified in the published literature are plausible. The majority of the studies investigated male and female reproductive endpoints a s well as adverse birth outcomes. Regarding female reproductive health, three studies provide some additional evidence to consider in conjunction with the experimental toxicology database. In 2006, Farr et al . published results of a cohort analysis perfor med within the AHS that concluded atrazine use may be associated with delay in the timing of menopause; researchers estimated a 5 month delay in menopause in relation to ever

- use of atrazine and with users of certain other  ̳hormonally active‘ pesticides. W hile limitations exist in the performance of this study, authors present these result along with the published results of Farr et al

. ( 2004 ) in which use of  ̳hormonally active‘ pesticide, a group that included atrazine, and atrazine or lindane users specif ically, was associated with increased probability of long

- or missed menstrual cycles. This suggests a plausible explanation may be that a delay in menopause is due to reduced ovarian function/number of cycles ; however

, other explanations cannot be exclude d at this time . Atrazine exposure assessment in both of these analyses reflects some uncertainty, but given what is known about atrazine‘s effect on the HPG axis these results are biologically plausible, warranting additional

follow

- up epidemiology studies . Therefore, at this time, the data do not support the conclusion that atrazine exposure is linked to either a delay in the timing of menopause or with long

- or missed menstrual cycles, or both. Additional research, specifically designed to overcome some o f the limitations articulated by the respective study

authors as well as this review are needed.

As stated above , studies suggest pesticide exposure may influence male reproductive health. Swan et al

. ( 2003 ) suggests that among men who reside in an area

o f high use of agricultural chemicals, men who have higher levels of pesticide biomarkers in the urine, including atrazine mercapturate as a measure of exposure to atrazine, have lower semen parameters including sperm concentration, motility and morphology. While based upon a small sample, this study measures both exposure and disease using biomarkers. Both Savitz et al

. (1997) and later in Arbuckle et al

. (2001), researchers report atrazine use among male partners may be associated with preterm delivery an d possible risk of miscarriage (non

- statistically significant result), and atrazine use (measured at the level of the farm site) may also be associated with early term spontaneous abortion. While additional research is necessary to elucidate the degree if any , to which atrazine exposure, may partially or completely explain theseDo not cite

Section 3

- Page 44 of 677 results, the database indicates a potential role for male pesticide exposure, possibly including atrazine, in male reproductive health.

There is some evidence that atrazine exposur e may be associated with adverse birth outcomes including pre

- term delivery, low birth weight, and SGA ; however

, the database is weak at this time. Some research indicates a potential increased risk for preterm delivery ; however

, not all estimates reach st atistical significance, and one study observed no association between atrazine use and this birth outcome. Low birth weight is also associated with atrazine use in some studies; however, a recent AHS publication reported no association among women who repo rted direct, agricultural exposure during the first trimester of pregnancy. Two studies report an increased probability of small

- for

gestational age and the authors posit atrazine later in pregnancy, during the third trimester, may be an important exposure window to consider in future research.

Consisten t results between two of the superior studies investigating these outcomes (Ochoa

- Acuna et al

. , 2009; Villanueva et al

. , 2005) are notable; however, there are

inconsistencies in results across all of these s tudies.

There were five studies that investigated atrazine exposure in association with birth defects; three evaluated abdominal wall defects

, specifically. Waller et al. (2010) report a 50% increased odds of the outcome among women who resided less than 25 km from a USGS surface water monitoring site which registered atrazine concentrations above the EPA limit (3 ug/L). While a notable finding, significant potential biases may exist with the study including the method of exposure assessment which was ess entially at the aggregate level. However, this study warrants additional follow

- up. Mattix (2007) reports rates of this outcome are higher in I ndiana , a high atrazine use state, than the rest of the U.S. However, given the relatively weak design of these s tudies, they offer little additional supportive evidence to the Waller et al . (2010) finding. The study of both gestational diabetes (Saldena 2007) and wheeze (Hoppin 2002) are novel, well

conducted hypothesis generating studies, and warrant additional fo llow

- up.

3.4 Conclusions

At this time, the epidemiologic database concerning the non

- cancer health effects potentially related to atrazine does not support the ability to make causal inference, nor are the data robust enough for inclusion in quantitat ive risk assessment. There are several limitations present in the current database, and the quality of atrazine exposure assessment is paramount among the limitations. Additional epidemiologic research addressing some of the current weaknesses in study des ign, conduct or analysis will enhance our understanding of the potential association between atrazine exposure and non

- cancer health effects. There are several suggested associations, however, that cannot be discounted at this time.Do not cite

Section 3

- Page 45 of 677 Table 3

- 1 : Summary of the Non

- Cancer Epidemiology Studies included in Literature Review

First Author Year Study Design (sample size) Outcome

Ascertainment Exposure

Assessment Confounders Measure of

Association Strengths Weaknesses

Fetal Death/Spo ntaneous Abortion

Arbuckle 2001 Nested Case

Control (N=3936) Spontaneous

Abortion (SA) (self

- reported) Months of year

of pesticide use during estimated

critical window Unadjusted Odds ratio (OR) and

95%
confidence interval (95% CI) 1.2 (0.9

– 1.7),

association stronger SA

<12 weeks Efficient

design; SA medically confirmed;

refinement of possible

critical

window Exposure

measurement error possible,

no direct pesticide

exposure

measure (probabilistic); no adjustment other pesticide

e xposure Birth Defects

Waller 2010 Population

based case

control (N=806/3616) Gastroschisis

(state birth registry) Proximity (km) to

"high" atrazine
USGS site Maternal age,

smoking, parity <25km: OR

1.6 (95% CI

1.1

- 2.3), 25

- 50 km: OR 1.4 (95% CI

1.2

- 1.7),

>50km (referent) (trend=0.049) Hypothesis

testing study; relatively large sample

size Exposure

measurement error possible;

under

- reporting

of outcome using birth

records possible

Mattix 2007 Ecologic (9,871

cases/~35 million births) Abdominal wall

defect s [AWD] (U.S., state birth registry) Atrazine

concentration in

surface water (USGS) n/a AWD rate

U.S.: 2.75/10 5

v. AWD rate
IN: 3.75/10 5 ; correlation Hypothesis

generating study; supports
additional L imits of study

design; under

reporting of

outcome possibleDo not cite

Section 3

- Page 46 of 677 First Author Year Study Design (sample size) Outcome

Ascertainment Exposure

Assessment Confounders Measure of

Association Strengths Weaknesses

Fetal Death/Spo ntaneous Abortion
monthly

atrazine and AWD, 0.69 (IN), p=0.0125 research

Hornemann 2008 Population

based case

control [N. Germany] (N=66/28,935) Sonographic

diagnosis omphalocele (medical records) Season of conception Unadjusted No

association

season and omphalocele;

n o trend over

time Sample size;

method of

outcome
ascertainme

nt (sonography ) Atrazine

exposure low (regulatory

cessation of use); exposure

measurement error possible

Winchester 2009 Ecologic (30

million live births) U.S. natality

dataset, birth

defects Atr azine

concentration in

surface water (USGS) Maternal age,

alcohol, smoking, diabetes, year of

birth Higher rates

of 11/22 birth

defects if Spring conception Hypothesis

generating study;

additional

research

necessary Limits of study

design; under

reporting of outcome likelyDo not cite

Section 3

- Page 47 of 677 First Author Year Study Design (sample size) Outcome

Ascertainment Exposure

Assessment Confounders Measure of

Association Strengths Weaknesses

Fetal Death/Spo ntaneous Abortion

Ochoa

- Acuna 2009 Retrospective

cohort (n=48,216 births) State birth

registry Maternal

proximity to corn, soybean field maternal and

infant demographics,

behavioral risk factors,

seasonal factors Odds limb

birth defects 1.22 (1.01

- 1.47) p er 10

hectacres

from field Hypothesis

generating,

measured

several

different

birth defects Exposure

measurement error likely;

under

- reporting

in birth record data; small sample size for

some birth

defects investigated

Adverse Birth Outcomes

Ochoa

- Acuna 2 009 Retrospective

cohort (n=several

hundred cases/24,154 live births) Small

- for

gestational age (SGA), pre

- term delivery (State birth registry) Drinking water

concentration atrazine, four data sources,

time series analysis Maternal health

behavior and

dem ographic characteristics, reproductive history SGA

Prevalence

rate ratio (PRR) 1.14 (95% CI 1.03, 1.24); 3rd

trimester exposure 17

- 19%

increased prevalence of SGA; no

association

pre

- term delivery Drinking

water
atrazine

concentratio n measured;

time

- series analysis Exposure

measurement error possible; comparability of four monitoring programs used

in time

- series analysis

unknownDo not cite

Section 3

- Page 48 of 677 First Author Year Study Design (sample size) Outcome

Ascertainment Exposure

Assessment Confounders Measure of

Association Strengths Weaknesses

Fetal Death/Spo ntaneous Abortion

Villanueva 2005 Retrospective
Cohort (n=3,510) Preterm delivery,

SGA, low birth weight (LBW) (regional birth registry) Drinking w ater

concentration

atrazine Maternal age,

infant gender, percent of sample below

LOD No

association with any

outcome

overall ; 3rd

trimester

atrazine

exposure: SGA: OR 1.54 (95% CI

1.11

- 2.13);

LBW: OR 1.24 (0.88

- 1.74); 1st

trimester

atrazine

exposure, prete rm delivery OR 1.36 (95% CI

0.95

- 1.95)) Drinking

water
atrazine

concentratio n measured;

sub

analyses

informative to possible

critical

window Exposure

measurement error possible;

narrow

exposure range

Munger 1997 Ecologic (N=several

hundred cases/6,500 liv e births) Intra

- uterine

growth

retardation (IUGR), LBW, preterm delivery (state birth registry) Residence in

county with high triazine (RRWA) Percent women

in labor force,

Kessner index prenatal care, median income,

maternal

education (group level) Atrazin e,

cyanazine

concentration

correlated IUGR,

associated IUGR (multi

variable model) Hypothesis

generating study;

additional

research

necessary Limits study

designDo not cite

Section 3

- Page 49 of 677 First Author Year Study Design (sample size) Outcome

Ascertainment Exposure

Assessment Confounders Measure of

Association Strengths Weaknesses

Fetal Death/Spo ntaneous Abortion

Sathyanaraya

na 2010 Cross

- sectional,

AHS (N=2,246 ) Birth weight

(maternal self

report) Quest ionnaire

categorized (None, indirect (e.g., planting), residential, agricultural);

Lifetime ever

use atrazine Pregnancy

duration, maternal pre

pregnancy body

mass index (BMI), maternal height, parity, state, and

maternal smoking No

association;

non

signifi cant 39 gram reduced birth weight

with ever

- use Accuracy

ever

- use pesticide

exposure (AHS);

outcome report reliable; large sample

size Lack of

precision of pesticide exposure during

pregnancy

Dabrowski 2003 Population

based case

control (N=117/377) LBW, p reterm

delivery (medical records) Pesticide

exposure (questionnaire; validated) Infant sex,

maternal pre

pregnancy weight, height,

maternal smoking, year of birth,

involvement in

field work Triazine

observed/exp ected (O/E),

LBW:

- 259g (p=0.43); O/E

pregnan cy duration:

- 2weeks (p=0.18) Outcome

ascertained

medical

record review;

validated

exposure estimate; potential

recall bias

reduced No atrazine

measure (triazine); small

number of

triazine exposed

Female ReproductiveDo not cite

Section 3

- Page 50 of 677 First Author Year Study Design (sample size) Outcome

Ascertainment Exposure

Assessment Confounders Measure of

Association Strengths Weaknesses

Fetal Death/Spo ntaneous Abortion

Saldena 2007 Cross

- sectional (n=11,27 3) Gestational

diabetes [GDM] (Questionnaire) Questionnaire

categorized (None, indirect (e.g., planting), residential, agricultural);

lifetime ever

- use atrazine BMI, maternal

age, parity,
race, state, 5

most common pesticides (AHS) OR

agricultural (no expo sure referent) 2.2 (95% CI 1.5, 3.3);

increased
odds GDM

with lifetime

ever

- use atrazine among direct

users (graphical depiction only) Hypothesis

generating study;

additional

research

necessary Lacks atrazine

exposure measure during

pregnancy; multiple

sta tistical tests

Farr 2006 Cohort analysis (N=3,103) [AHS] Menopause

(Questionnaire) Author identified

"hormonally active" pesticide and atrazine,

lifetime ever

- use Smoking, oral

contraceptive

use Hazard ratio

(HR) atrazine 0.79 (95% CI 0.63, 0.99) [5

month delay

in time to menopause] Hypothesis

testing, hormonally

active pesticides; large sample size; analysis appropriate to study Design Outcome

misclassificatio n possible; lack

exposure
measure at

relevant lifestagesDo not cite

Section 3

- Page 51 of 677 First Author Year Study Design (sample size) Outcome

Ascertainment Exposure

Assessment Confounders Measure of

Association Strengths Weaknesses

Fetal Death/Spo ntaneous Abortion

Farr 2004 Cross

- sectional analysis of A HS data (N=8,038) Menstrual cycle

length (>36 days, <24 days, normal) irregular cycle, missed period, inter

menstrual bleeding (Questionnaire) Atrazine or

lindane use; lifetime days of use of any pesticide (0, 1

- 9 days/yr, 10+ days/yr) Age, smoking,

BMI At razine or

lindane use, OR >36 days: 2.7 (95% CI

1.3

- 5.5), missed cycle, OR 2.0 (05%

CI 1.3

- 3.3) (referent: never users) Hypothesis

testing; large sample size; analysis appropriate to study design No atrazine

specific estimate; potential

residual

confoundin g physical activity;

information co

morbid

conditions

needed

Male Reproductive

Swan 2006 Cross

- sectional analysis (N=86) Semen

parameters (concentration, motility, morphology) Urinary

concentration of

atrazine mercapturate (AM) Non

- parametric,

parametric tests performed; in

hierarchical model,

confounder score, study site, and pesticidexcenter interaction term;

others unadjusted OR atrazine

(above/below LOD): 11.3 (95% CI 1.3

- 98.9) Use of

biomarkers

outcome
and

exposure; potential

observer

bias reduced;

a nalysis appropriate to study design Small sample;

atrazine

biomarker

under

- estimates total atrazine; generalizable to healthy, white men onlyDo not cite

Section 3

- Page 52 of 677 First Author Year Study Design (sample size) Outcome

Ascertainment Exposure

Assessment Confounders Measure of

Association Strengths Weaknesses

Fetal Death/Spo ntaneous Abortion

Savitz 1997 Case

- control analysis [Ontario Farm Family study] (N=3,984) Preterm delivery,

SGA, miscarriage,

secon dary sex ratio (self

- report questionnaire) Pesticide

activity, pesticide use, months of year of use;

matched

pregnancy period Maternal age,

parity, maternal education, paternal education, per capita income,

mother's off

farm job,

father's off

- farm job, mat ernal smoking,

maternal alcohol, time period conception to

interview

interval OR

miscarriage:

crop

- /yard

herbicides

1.2

- 1.5 (n.s.); OR preterm delivery: crop

- /yard

herbicides

2.4

- 4.9*(s.s.);

OR SGA: 0.5 (n.s.); OR sex ratio: crop

herbicide 0.8 (n.s.) Resp onse

rate good; possible

recall bias

reduced Exposure

measurement error possible (probabilistic); inability discern maternal/patern al exposure

Respiratory Health

Hoppin 2002 Cross

- sectional [AHS] (N=20,468) Wheeze

(Questionnaire) Pesticide lifetime

ever

- use, atrazine; number of days of year

of use in index

year Age, state,

smoking, asthma or atopy OR ever use

atrazine: 1.20 (95% CI 1.07

- 1.34);

exposure

response by category of annual days

of use Hypothesis

gen erating;

additional

research

necessary Multiple

statistical tests performed; no biologically plausible

mechanism proposedDo not cite

Section 3

- Page 53 of 677 First Author Year Study Design (sample size) Outcome

Ascertainment Exposure

Assessment Confounders Measure of

Association Strengths Weaknesses

Fetal Death/Spo ntaneous Abortion
positive,

monotonic (OR: 1.1

- 1.5, ss) (p<0.01)

Klucinski 2001 Population

based cohort study (N=156) Biomarkers of

immune system function (IgA, CD3,4,8,1 and, CIC) Occupational

empl oyment, community unexposed

series n/a Evidence of

immune system impairment based upon

several

biomarkers

measured Hypothesize

d biologically plausible explanation

of respiratory findings (Kossman 1997); occupational

, manufacturi ng setting unique in

this

da tabase Inadequate

atrazine

exposure
measurement

Kossman 1997 Population

based cohort study (N=91) Respiratory

health, spirometry

function tests Occupational

series based upon location of work in plant, or community

identified unexposed

series n/a Evidence of reduced lung

function

among exposed Hypothesis

generating study;

additional

research

necessary Inadequate

atrazine

exposure
measurementDo not cite

Section 3

- Page 54 of 677Do not cite

Section 4

- Page 55 of 677 4. INTEGRATION OF EPIDEMIOLOGY & EX PERIMENTAL T OXICOLOGY

Although typically the Agency‘s human health risk assess ments rely heavily on experimental toxicity data obtained from test species (rodents, non

- rodents, and canines), the ultimate goal of the risk assessment is to evaluate the potential impact of a toxicant exposure on the health of the human population. A m ode of action understanding of the critical effects found in experimental animals provides a basis for evaluating human relevance. Moreover, it is important to consider the data collected in epidemiology studies, whenever available. In the case of atrazi ne, an experimental toxicology database , a toxicological mode of action understanding and an epidemiology

data are available . The experimental database is comprehensive with hundreds of studies conducted in academic, industrial, and government laboratorie s around the world. In contrast, the epidemiology database concerning the potential non

- cancer health effects of atrazine exposure consists of 19 studies identified in the published

literature . Though not as extensive as the toxicity database, the availa bility of 19 epidemiology studies represents a larger epidemiology database than what is typically available for pesticides

. However , as previously described in Section 3 of this document, these epidemiology studies are eithe r hypothesis

- generating in nature or reflect use of exposure surrogates, sometimes at the aggregate (group) level. While the observational data, to date, are not sufficiently robust to allow investigators to make conclusions regarding the cause

- and

- effect relationship between atrazine exposure and the adverse health outcomes reported, there are some suggestive signals in the data

that warrant consideration, especially in light of what is understood from the experimental toxicology database. Therefore, regar dless of the discrepancy in the quantity and quality of data available from the two disciplines, it is important to consider to what degree these datasets do or do not complement each other. This section of the

document will discuss the similarities and di fferences in the findings reported in toxicology and epidemiology studies evaluating potential adverse non

- cancer health outcomes associated with atrazine exposure.

Multiple lines of evidence have demonstrated that atrazine exposure disrupt s the regulatio n of pituitary hormone secretion and as a result alter a number of reproductive

functions in rats. Therefore, it is not surprising that one of the hallmarks of atrazine toxicity is effects on the reproductive system and the developing reproductive system

. Both toxicity and epidemiology studies have explored this aspect of atrazine toxicity. In the rat, repeated exposure to atrazine at doses as low as 3.65 mg/kg/day leads to estrous cyclicity disruption and early reproductive senescence (Cooper et al.

, 19 96 & 2000, Morseth, 1996 b , Laws et al .

, 2000, Shibayama et al . 2009) as a result of the LH surge attenuation. In humans and primates reproductive function/ovarian cycling is also influenced by the hypothalamic GnRH (Goldfien and Monroe, 1994 ; Plant et a l

., 200 4; Terasawa and Nyberg, 1997). Therefore, a potential consequence in humans is disrupted or irregular menstrual cycles which can lead to gynecological problems such as diminished fertility, prolonged menses or excessive bleeding. In 2004 and 2006

, FarrDo not cite

Section 4

- Page 56 of 677 and co

- workers evaluated the potential impact of atrazine exposure on the menstrual cycle and menopause onset of female pesticide applicators and female spouses of male pesticide applicators enrolled in the Agricultural Health Study (AHS). The odds r atio (OR) for a long cycle or a missed period was elevated among women who reported ever using atrazine or lindane during their lifetime [OR 2.7]. This group also exhibited a slight association with irregular cycle and a significant association with inter

- menstrual bleeding. This study included a large sample size, was performed among the women likely most highly exposed to pesticides in the general population, and reflected a pesticide exposure metric with high validity and reliability (Blair et al

. , 200 3; Hoppin et
al

. , 2002). However, given the data available, the authors were not able to estimate the association between atrazine use with menstrual cycle functioning independent of lindane. Therefore, it is not possible to ascribe atrazine use to any of the menstrual cycle characteristics evaluated, or estimate the timing of pesticide exposure in relation to changes in the menstrual cycle. Moreover, the direction of the association for the

atrazine or lindane sub

- group examined is somewhat consistent wi th lindane toxicity; this may or may not explain this finding.

In contrast to the findings on estrous cyclicity disruption where rodent s and

human s appear to have a similar suite of effects (prolonged/missed cycles), menopause onset in women appears to be delayed while rats enter an early reproductive senescence after atrazine exposure. It is noteworthy that in humans menopause is due to ovarian atresia, whereas in the rat it is the result of the a ging and dampening of the
GnRH pulse. Given these differe nces in the reproductive aging physiology between rats and humans it is not entirely unexpected that they would show a different pattern of

effects. In a 2006 epidemiology study , Farr et al . reported a reduced hazard ratio ( i.e., delayed menopause) amon g women that ever used (i) any of the pesticides in the AHS study, (ii) hormonally active pesticides 5 in the AHS study, or (iii) atrazine. There are, however, limitations of this study that preclude concluding that atrazine exposure is causally linked to this delay in menopause onset. Some of the limitations acknowledged by the study authors include: (i) limited frequency in assessing menopausal status of the women in the cohort, (ii) pesticide exposure assessment do not allow exposure

- response assessment , (iii) analysis of confounding factors that needs to be enhanced, including the measure of physical activity. In addition, based on the exposure metric in this study it is not possible to correlate the timing and/or extent of atrazine exposure with the m enopause delay ( i.e., W ere the women exposed consistently over their lifetimes or only once or twice ? , H ow much atrazine were these women potentially exposed to over their lifetime ? , W ere the women only exposed years prior to any changes in menopause?). I n conclusion, while a similar pattern of effects on menstrual cyclicity parameters is emerging from the experimental toxicity and epidemiology data, there are insufficient epidemiology data to definitely conclude that

5 Classification of a chemical as ―hormonally active‖ is based on the investigators interpretation of the

literature. The authors considered atrazine as one of the hormonally active compounds.Do not cite

Section 4

- Page 57 of 677 the menstrual cycle disruptions report ed by Farr et. al are associated with atrazine

exposure.

Changes in andrology parameters (sperm parameters and male reproductive performance) ha ve also been investigated in regards to atrazine exposure (See Appendix A) . The dataset for these types of s tudies is sparse relative to the studies on the female reproductive system. Experimental d ata on these parameters come primarily from in vitro studies using either porcine or human sperm . Two studies used porcine sperm to evaluate sperm viability, motili ty, and capacitation (a process undergone by sperm in order to be capable of fertilization) after exposure to atrazine in

vitro . A third study evaluated the impact of an in vitro atrazine exposure on human sperm motility. All these studies suggest an eff ect of atrazine on sperm motility and/or viability; however, there is no evidence of a correlation between these findings and impaired reproductive performance. Only one epidemiology study on male reproductive health is currently available for considerati on. In 2003, Swan et al . compared atrazine

and other non

- persistent pesticide exposure between men with and without poor semen quality parameters. The measure of atrazine exposure was the concentration of atrazine mercapturate

– an atrazine metabolite

– in the urine. Although the authors reported an increase in the odds of poor semen quality in association with atrazine exposure, given the relatively small sample size the precision of this estimate was low (odds ration 11.3, 95% CI 1.3, 98.9). While res earchers reported that the proportion of men reporting occupational pesticide use, recent pesticide use

, or residential proximity to treated field was comparable between men with and without poor semen parameters,

sub

- analyses indicate men with multiple pe sticide exposure were more likely to have poor semen quality parameters. Therefore, multiple pesticide exposures, or other environmental exposure, may play a role in the observed. Considering both the in vitro

and human data , there is a suggestion that e ffects on sperm parameters may be indicative of atrazine toxicity. However, the data from the mode of action and epidemiology literature do not provide sufficient information to draw any causal

inferences while the in vitro data (both in swine and human s) fail to provide a functional correlate to the purported andrology effects reported in the studies.

Another adverse health outcome suggested by the epidemiology data is the potential for low birth weight or small for gestational age (SGA). A comparable finding is observed in the animal data with decreases in fetal/offspring weight observed in toxicity

studies in rats but at high experimental doses only (≥ 50 mg/kg/day) which is likely due to general toxicity and not associated with atrazine neuroendocrine mode of action . The prevalence of SGA in these epidemiology studies was obtained from birth registries (Ochoa

- Acuna et al

. , 2009; Villan ueva et al

. , 2006), or maternal self

- report of birthweight and gestational age (Savitz et al

. , 1997), and the exposure assessment was based on atrazine drinking water concentrations or self

- reported atrazine use at the

farm level . Therefore, misclassification of the o utcome or exposure is possible using

these methods. Authors reported a 20

- 50% increase in the prevalence of SGA (Ochoa

-Do not cite

Section 4

- Page 58 of 677 Acuna et al

. , 2 009; Villanueva et al

. , 2005) ; while Savitz et al

. (1997) observed a 50%

decreased risk.

For low birth weights, the evid ence of a correlation with atrazine exposure is less compelling in that results are either based upon sub

- analyses including small numbers (Villanueva et al

. , 2 005), measure overall triazine use, as opposed to atrazine use (Dabrowski 2003), or were not able to measure atrazine exposure during pregnancy (Sathuanarayana et al .

, 2010). A recent study within the AHS, indicate that among women who reported ever using atrazine (use of atrazine not necessarily during pregnancy) there was a non

- statistically signi ficant decrease in birth weight (Sathuanarayana et al .

, 2010). Even for women reporting pesticide use during the first trimester of the pregnancy, the decreases in birth weight were not statistically significant.

Other effects reported in the epidemiolo gy database include a bdominal wall defects (omphaloc ele and gastroschisis), gestational diabetes, pre

- term delivery, and wheeze. However, overall the epidemiology data on these effects is not robust. With the exception of gestational diabetes, one would expect atrazine toxicity testing would have identified these types of effects, and no effects of this nature were reported in the experimental toxicology database. With regards to gestational diabetes, clinical chemistry measures are not routinely conduct ed on pregnant animals in order to minimize stress. Consequently, there are no available data to address this issue. Nonetheless, it is noteworthy that no indication of glucose imbalance is observed in any of the adult toxicity studies available for atra zine.

For the purposes of quantitative risk assessment, the experimental toxicology data (particularly in vivo data from rat) will continue to be utilized for endpoint selection

and dose

- response assessment. However, the epidemiologic and toxicological databases can be used together to inform both hazard identification and also hazard

characterization. The non

- cancer epidemiology database provides important information that adds to the human relevance of the animal findings, particularly related to femal e and male reproductive effects and small for gestational age birth outcome.

With the review of the experimental toxicology and non

- cancer epidemiology data, the problem formulation stage of hazard assessment is fundamentally complete

and is articulated as follows. Because of the similarity between rats and humans of the influence of hypothalamic GnRH on the reproductive system, the reproductive effects seen in rats should be assumed to be relevant to humans, in the absence of contradictory evidence. The refore, we consider that several of the non

- cancer health outcomes examined in relation to atrazine exposure in epidemiologic studies may plausibly be explained by atrazine‘s influence on hypothalamic GnRH signaling including estrous cycle disruption. Howe ver, other modes of action may also play a role in the human population, e.g. , direct testicular toxicity in relation to semen quality. There are suggestions in the epidemiological data base of adverse reproductive andDo not cite

Section 4

- Page 59 of 677 developmental outcomes, as noted abov e, although at this time we cannot exclude

other non

- causal explanations for the apparent relation. Use of aggregate level exposure assessment and quality of exposure assessment across studies, small sample size and limited statistical power, and in some i nstances the in ability to control for potentially confounding factors that may influence reported point estimates, e.g., other environmental agents

, may explain the associations observed. Among the key

studies in the epidemiology database, studies in which the potential for other non

- causal explanations is comparatively lower, evidence indicates a possible role for atrazine exposure (Farr et al

. , 2004; Farr et al

. , 2006; Swan et al

. , 2003; Ochoa

- Acuna et al

. , 2009; and, Villanueva et al

. , 2005). Additional research in both disciplines would aid our understanding of the nature of the association between atrazine exposure and reproductive and developmental effects.

In conclusion, given the important uncertainties regarding exposure assessment in the epidemio logy studies (e.g., lack of quantitative measures) the experimental toxicology data (particularly in vivo data from rat) provides the strongest data for performing quantitative risk assessment. The non

- cancer epidemiology data do, however, provide importa nt information that adds to the human relevance of the animal findings. With the review of the experimental toxicology and non

- cancer epidemiology data, the problem formulation for hazard assessment is fundamentally complete. As
discussed in Section 2 above, attenuation of LH is a critical event leading to reproductive/developmental outcomes and is the most sensitive endpoint observed. As

shown in Table 2

- 2 , LOAELs for changes in LH are at least 5

- fo ld more sensitive LOAELs for reproductive endpoints. In the previous risk assessment, attenuation of LH was used to assess intermediate and chronic scenarios; as described in Section 5 below, attenuation of LH remains the most sensitive and human relevant data for extrapolating risk.Do not cite

Section 5

- Page 60 of 677 5. PROPOSED UPDATES TO THE DOSE

- RESPONSE ASSESSMENT

5.1 Overview

The previous EPA human health risk assessment identified non

- cancer effects of atrazine following acute and chronic dietary e xposures, 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 s of concern for acute dietary exposure (NOAEL/LOAEL of 10/70 mg/kg/day). Delayed preputial separation in male rats (which is associated with atrazine‘s neuroendocrine MOA) was the basis for assessing the short

- term oral, dermal and inhalation exposures (NOAEL/LOAEL of 6.25/12.5 mg/kg/day). Effects indicative of disruption of hypothalamic function ( i.e. , attenuation o f pre

- ovulatory luteinizing hormone surge) were determined to be appropriate toxicity endpoints of concern for chronic dietary, intermediate incidental oral, and intermediate

- and long

- term dermal and inhalation exposures (NOAEL/LOAEL of 1.8/3.65 mg/kg/day ).

As part of the atrazine re

- evaluation, the Agency has considered key improvements to the points of departure for atrazine and its metabolites; namely using an internal dose metric instead of the typical external dose metric and application of the

be nchmark dose (BMD) technique over the NOAEL/LOAEL approach. These improvements are discussed below.

5 .2 Dosimetry : Internal vs. External

5

. 2 .1 Background

Background

Understanding the pharmacokinetics and internal dosimetry of the relevant

c hemical species (e.g., parent chemical, metabolites, or both) represents an important step for linking exposure to response. In the case of atrazine, p erturbations of the neuroendocrine system resulting in attenuation of the hypothalamus

- controlled pre

ovu latory LH s urge (i.e., the HPG axis) and the ensuing reproductive toxicity have

been identified as the most sensitive response attributable to exposure (IRED 2003)

. The adverse outcomes are the result of alterations in pituitary hormone secretions that

dis rupt ovarian function including estrous cyclicity and pregnancy (Cummings et al .

, 2000; Morseth, 1996a, 1996b; Narotsky et al . , 2001) ; suppress the suckling

- induced prolactin release during lactation (Cooper et al . , 2000) ; and delay puberty onset in

male and female rats (Cooper et al . , 2000; Laws et al . , 2000)

.Do not cite

Section 5

- Page 61 of 677 5 .2 .2 Data Supporting the Use of Internal Dose in Dose Response

As sessmen t

Data obtained from metabolism studies support the use of an internal dose over the traditional administered dose metric as a way to refine the linkage between attenuation of the pre

- ovulatory LH surge and atrazine exposure. Upon entering the body via the oral route, atrazine is quickly and extensively metabolized to dealkylated chlorinated metabolites, namely DEA, DIA, and DACT, and non

- chlorinated glutathione conjugates as illustrated in Figure 5

. 1 . Although no dire ct plasma half

- life measurements are available for the parent chemical atrazine, several indications from pharmacokinetic/metabolism studies suggest a very short in vivo half

- life. For instance, following an oral gavage dose of 90 mg/kg atrazine to rats, a trazine accounted for only about 4% of total chlorotriazines in plasma at 30 minutes post

- dose while the contribution of DACT was far greater at more than 50%. By 24 hours, atrazine was barely detectable while DACT accounted for more than 98% of total chlo rinated triazines in plasma (McMullin et al .

, 2003). Such profile of circulating plasma levels is

indicative of minimal and transient in vivo atrazine levels and suggestive of an important contribution of metabolites in the toxicity profile.Do not cite

Section 5

- Page 62 of 677 Figure 5

. 1 : Metabolic Profile of Atrazine

S tudies examining more directly the contribution of individual triazine species (atrazine and its metabolites) in attenuating the LH surge also sugges t an important contribution of metabolites in exerting this effect. In independent studies by Minnema (2001) and McMullin et al. (2004), the direct activity of DACT in attenuating the LH

surge was demonstrated at equimolar levels as atrazine (McMullin et al. , 2004; Minnema, 2001) . No specific studies have been carried out to date on the mono

dealkylated metabolites (DEA and DIA); however, it is presumed

, based on their intact chlorinated nature, that they may also be active in attenuating the LH surge . On the other hand, the role of glutathione conjugates remains largely unknown with respect to the LH surge. It has been postulated, based on urine radioactivity percentages, that glutathione conjugation may represent as much as 30% of atrazine metabolism, thus raising concerns about their potential contribution (Timchalk et al . , 1990) . Therefore, based on the exhibited direct activity by DACT, the presumed activity of DEA and DIA

, and the uncertainty surrounding glutathione conjugates, the use of an internal dose

metric for total triazines is warranted to account for the contribution of all the relevant triazine species in the attenuation of the LH surge . The use of total triazine equivalents has the added convenience that data from conventional disposition studies that monitor core radiolabel can be readily used without having to distinguish between different pools

of metabolites.

5

. 2 .3 Temporal Aspects and Pharmacokinetic Mode ling Efforts

Characterization of the pharmacokinetics of atrazine is critical to understand the temporal relationship between exposure and observed effects. Efforts to develop pharmacokinetic models, particularly physiologically based pharmacok inetic (PBPK) models that can simulate the pharmacokinetics of atrazine have been hampered by a complex behavior at high administered doses and the inability to distinguish between parent chemical and metabolites in animal toxicity studies. One of

the best efforts to date is a published composite PBPK model developed by McMullin et
al . ( 200 7 ) that focused on the gut as the site for absorption and potential site for presystemic metabolism, liver as the primary site of metabolism, and a rest of body compartm ent (McMullin et al . , 2007)

. Other modeling efforts also put emphasis on the

brain as a potential target organ for toxicity, and red blood cells as a potential site for

bioaccumulation (McMulli n et al . , 2003)

. It should be noted that although red blood cells were modeled as a potential site for bio accumulation, no toxicity related to red blood cell dysfunction has been reported to date. Although a substantial amount of data was

used for mode l parameterization, the final physiologically based model had several shortcomings including the inability to simulate plasma levels of atrazine and its chlorinated metabolites. The result was significant underprediction of plasma levels of

atrazine at all time points and those for its chlorinated metabolites at the later time points (McMullin et al . , 2007)

.Do not cite

Section 5

- Page 63 of 677 Because there is no robust pharmacokinetic model available and a measure of

internal dose is warranted to ac count for the contribution of all relevant triazine species,

non

- compartmental analysis of the existing pharmacokinetic data represents the best method to estimate an internal dose metric without exceeding the limits of the available

data.

5

. 2 .4 The Are a under the Triazine Equivalent Plasma

- Concentration Time

Curve (AUC) as Internal Dose Metric

By definition, dose response assessment is an evaluation of the conditions under which effects may be observed. In the case of atrazine, it has been noted that in addition to dose, duration of atrazine exposure is an important parameter that must be considered in evaluating the relationship between dose and attenuation of the LH surge

. The primary support for this is the observation that a single high oral gavage atrazine dose may have little or no effect on the LH surge, while a much lower dose administered repeatedly can markedly attenuate the LH surge. In a study by Cooper et
al . (2000), a single oral gavage dose as high as 200 mg/kg administered on the day of proestrous failed to elicit any attenuation of the LH surge in Long Evans rats at 1, 3, or 6 hours post

- dose ( Figure 5

. 2 ). In contrast, a 4

- fold lower dose of 50 mg/kg administered repeatedly (daily for 3 days) attenua ted the LH surge by more than 75% at the 3 h timepoint which was more than the mild attenuation observed post

- dose from a single dose of 300 mg/kg (Cooper et al . , 2000) . Similarly, the other higher doses of 100, 20 0, and 300 mg/kg markedly suppressed the LH surge only when administered repeatedly (Figure 5.2 ). A recent unpublished study from the Cooper lab sets the LOAEL as low as 3.12 mg/kg/day (NOAEL = 1.56 mg/kg/day) for attenuation of the LH

- effect on the day of proestrous when rats were dosed daily for 4 days ( Figure 5

. 3 ). In combination, the data

summarized in Figures 5. 1

- 5.3 strongly suggest that attenuation of the LH surge is not governed by peak levels (i.e., Cmax) of atrazine and that duration of exposure is a critical parameter for selecting an appropriate internal dose metric. Based on the postulated activity of metabolites that support the use of internal dose and the need to incorporate duration of exposure in the atrazine ris k assessment (not just plasma levels

of atrazine or triazine equivalents ), the most appropriate internal dose metric for refining the dose response assessment of atrazine associated with the attenuation of the LH surge is the area under the plasma concentr ation

- time curve (AUC) for total triazine equivalents. The selection of this dose metric is conservative, since not all metabolites may be active, and incorporates the latest dose

- response information available for

atrazine.Do not cite

Section 5

- Page 64 of 677 Figure 5

. 2 : Attenuation of the leutenizing Hormone (LH) surge in Long

Evans

- Hooded rats following a single versus 3 oral gavage daily doses of atrazine (Figure is from Cooper et al . 2000)Do not cite

Section 5

- Page 65 of 677 Figure 5

. 3 : Attenuation of the leutinizing hormone (LH) surge following repeating daily dosing for four days via oral gavage (Unpublished data from the Cooper Lab).

5

. 2 .5 The Daily Steady Stat e AUC for Triazine Equ ivalents as Internal

Dose Metric

In a continued evaluation of the conditions under which the LH surge is attenuated following atrazine exposure, it was noted that in one of the most thorough pharmacokinetic datasets available for atrazine that the plasma levels of total radiolabeled triazine equivalents achieve (or nearly achieve) pseudo

- steady state by the fourth day of repeated daily dosing. At pseudo

- steady state, plasma levels of triazine equivalents can be expected to oscilla te about a mean value but remain fairly constant upon continuous ( e.g., daily) dosing, as indicated hypothetically in Figure 5

. 4 . An elimination phase would be observed once dosing stops. In the study by Thede ( 1987 )

, rats were do sed daily with 1, 3, 7, 10, 50, and 100 mg/kg radiolabeled atrazine for 10 consecutive days. Blood samples were taken at 24, 48, 72, 96, 144, 192, 219, 240, and 264 hours after the initial dose. One rat from each dose group was sacrificed at 3 and

72 hours after the tenth dose to assess the elimination phase of the radiolabel. The whole plasma profile of the radiolabel is depicted in Figure 5

. 5 . In the study report, a plasma half

- life of 38.6 hours was estimated based on the elimin ation half

- life of the

radiolabel (Thede, 1987) . Thus, following the rough approximation th at it would take 3

- 5 half

- lives to reach steady state, the result of 4.825 days (3x 38.6 hours) is consistent with plasma levels being at (or nearly at) pseudo steady state levels by the fourth day ofDo not cite

Section 5

- Page 66 of 677 daily dosing. In the case of the high doses (50 and 1 00 mg/kg/day), plasma levels did not change significantly after three daily doses indicating that pseudo steady state may be reached (or nearly reached) at least 24 hours earlier as compared to the lower doses

of 1

- 10 mg/kg/day. Another indication that t he pharmacokinetic behavior of triazine equivalents may be different at the high doses came from analyzing the elimination phase of each dose group. As indicated in Table 5

- 1 , when each elimination phase was analyzed assuming line ar behavior (i.e., plotting natural log of plasma levels versus time), a different elimination rate constant was estimated for the high dose groups. While a comparable rate constant of 0.010 to 0.015 hr - 1 was estimated for the low dose groups (i.e., 1

- 10 mg/kg/day), a more than 30

- fold higher value of 0.308 to 0.376 was estimated for the 50 and 100 mg/kg/day dose groups. At steady state, a faster clearance is indicative of faster uptake of administered atrazine into the plasma (assuming no change postdosi ng), and thus this observation is consistent with a shorter

time to reach pseudo

- steady state for the high dose groups.

The information provided in Figure 5

. 2 indicating the lack of effect on the LH

surge following a single high administered dose of atrazine and the contrasting marked LH surge attenuation following daily administration of a much lower dose for 4 days ( Figure 5

. 3 ) , when combined with the pharmacokinetic information in Figure 5

. 5 is

consistent with the hypothesis that pseudo steady state is strongly associated with

attenuation of the LH surge

.

Figure 5

. 4 : Hypothetical depiction of pseudo steady state plasma levels reached upon repeatedly daily dosing. The dashed lines indicate the mean value with lower and upper bounds, respectively. The elimination phase is indicative of when dosing stops.Do not cite

Section 5

- Page 67 of 677 Figure 5

. 5 : Plasma concentration profiles of total radiolabeled triazine equivalents corresponding to different administered doses of radiolabeled atrazine (Data from Thede 1987).

Table 5

- 1 : Estimated elimination rat e constants assuming linear kinetics for the different dose groups in Figure 5

. 4

.

One of the advantages of being at steady state is that a daily steady state AUC (AUCss) may be estimated by simply multiplying steady state pl asma levels by 24. If steady state is a condition under which the LH surge is attenuated by triazineDo not cite

Section 5

- Page 68 of 677 equivalents, then a daily AUCss dose metric for total triazine equivalents may be an appropriate internal dose metric to better characterize the link betwe en atrazine exposure and effect. In an effort to investigate how this dose metric may change as a function of administered dose of atrazine, the AUC for each plasma

- concentration profile in Figure 5

. 5 was first estimated using the traditional trapezoidal method and plotted versus administered dose of atrazine. As shown in Figure 5

. 6 , a linear relationship exists between AUC for total plasma triazine equivalents and administered

dose of atrazine for all dos es tested (1

- 100 mg/kg/day), suggestive of no apparent

dose

- dependent changes in absorption, distribution, metabolism and elimination (ADME) processes within the given dose range. When pseudo

- steady state plasma levels (estimated at the average value beg inning at 96 hours and ending at 219 hrs) and corresponding daily AUCss for total radiolabeled triazine equivalents were plotted versus administered dose of atrazine, a linear relationship was also observed ( Figure

5

. 7 ). Therefore , there are no apparent dose

- dependent changes in ADME processes that may preclude the use of a daily AUCss for total triazine equivalents in the dose

response assessment of atrazine at administered atrazine doses as high as 100 mg/kg/day

.

Figure 5

. 6 : Relationship between administered dose of atrazine and AUC for total radiolabeled triazine equivalents. The dashed line indicates the

95% confidence interval.Do not cite

Section 5

- Page 69 of 677 Figure 5

. 7 : Relationship between pseudo

- steady state plasma levels and steady state AUC for total radiolabeled triazine equivalents versus

administered dose of atrazine. The dashed line indicates the 95%

confidence interval.Do not cite

Section 5

- Page 70 of 677 5

. 2 .6 Comparison of Different LH

- Surge Attenuation Studies that Likely Achieved Steady State Plasma Levels of Triazine Equi valents

The implications of reaching a pseudo steady state are significant from the standpoi nt of establishing a more refined link between time, exposure, and effect (i.e.,

the time

- dose response relationship). For one thing, steady state plasma levels of triazine equivalents can be expected to oscillate about a mean value but remain fairly

const ant upon continuous dosing (See hypothetical depiction in Figure 5

. 4 ). In essence, plasma levels are only impacted by the time it takes to reach pseudo steady state, but not by how long dosing continues once such condition is achi eved. If the attenuation of

the LH surge is driven by steady state plasma levels of triazine equivalents, then studies

that achieve pseudo

- steady state would elicit the same level of LH surge attenuating

effect once pseudo steady state is reached regardle ss of how long repeated dosing continues. In effort to investigate this idea further, all the available studies where attenuation of the LH surge following atrazine exposure was examined after at least 4 days of daily dosing were compiled and analyzed for comparison. It should be noted that data was expressed as percent control to account for differences in rat strains and interlaboratory variability. One of the critical studies, the 6

- month LH surge special study in Sprague Dawley rats used to establish a chronic reference dose in the last risk assessment was included (IRED 2003). In this study, atrazine was administered in the diet to female Sprague Dawley rats at levels corresponding to 0, 1.8, 3.65, or 29.4 mg/kg/day. A NOAEL of 1.8 mg/kg/day was establi shed based on a statistically significant attenuation of the LH surge at 1800 h on the day of proestrous at the LOAEL of 3.65 mg/kg/day.

This dataset was compared to the 4

- d ay Cooper et al. (2010) study carried out in Long Evans rats via oral gavage at atrazine doses of 1.5, 3.12, 6.25, 12.5, and 25 mg/kg/day. Although more doses were included in the 4

- d versus 6 month study, the dose range covered in the two studies is very comparable. As shown in Figure 5

. 8 , the
NOAELs and LOA ELs for the two studies are remarkably indistinguishable. Moreover, the result obtained at 29.4 mg/kg/day in the 6

- month study is consistent with that obtained at 25 mg/kg/day in the 4

- day study in that a plateau indicative of nearly complete attenuation o f the LH surge seems to be reached at end of the 4

- d dose

response curve.Do not cite

Section 5

- Page 71 of 677 Figure 5

. 8 : Attenuation of the pre

- ovulatory LH surge following atrazine exposure across different studies and rat strains where daily dosing was performed for at least 4 days. The thi c k line represents the 4

- day unpublished study from the Cooper Lab. The other studies are either shown as single data points or dashed lines to aid in comparison.

I n continuing to gather evidence supporting the condition of steady state for attenuation of the LH surge by triazine equivalents, a study by McMullin et al . (2004) was identified where a dose of 30 mg/kg/day was evaluated in Sprague Dawley rats following 5 days of repeated daily dosing. Although doses of 100 and 300 mg/kg/day were also evaluated in that study, these doses are outside the dose range covered by the other two LH studies (i.e., 4

- day and 6

- month) being compared in this analysis andDo not cite

Section 5

- Page 72 of 677 thus were o mitted. Moreover, the doses of 100 and 300 mg/kg/day did not seem to result in any more statistically significant attenuation as compared to the 30 mg/kg/day dose group (data not shown). The attenuation of the LH surge resulting from an

administered atrazi ne dose of 30 mg/kg/day was selected for comparison on the basis that it is comparable to the high dose of 25 and 29.4 mg/kg/day from the 4

- day and 6

- month study, respectively. If steady state is related to attenuation of the LH surge by triazine equivalen ts, then the effect following dosing for 6 months at 29.4 mg/kg/day should be fairly similar to that resulting from 5 days of daily dosing at 30 mg/kg/day since only 4 daily doses seem to be required to reach pseudo steady state ( Figure 5

. 5 ).

As can be seen from Figure 5

. 8 , the effect seen after 5 daily doses of 30 mg/kg/day is remarkably similar to that seen in the 6

- month study at 29.4 mg/kg/day. Further

evidence was obtained from a one

- month study by Mi nnema (2001) where Sprague Dawley rats were dosed by oral gavage at dose levels of 0, 2.5, 5, 40, and 200 mg/kg/day atrazine (Minnema, 2001) . Only the doses of 2.5 and 5.0 mg/kg/day were selected for comparison to the other studies for the same reasons cited above. It should also be noted these doses were the NOAEL and LOAEL, respectively, selected in the study. As indicated in Figure 5

. 8 , the NOAEL and LOAEL from the one

- month study are remarkably comparable to those from the 6

- month and 4

- day stud ies . Thus, although repeated daily dosing continued for much longer in the 1

- and 6

- month studies, the LH

surge attenuation did not increase and remain ed fairly constant.

The remarkabl y similar effects seen across studies from different laboratories involving different durations (4

- days, 5

- days, 1

- month, and 6

- months), m odes of oral exposure (oral gavage versus dietary), and rat strains (Sprague Dawley and Long Evans) strongly supports the hypothesis that pseudo

- steady state is one of the

conditions associated with the attenuation of the LH surge following atrazine exposu re in rats . This finding provides a critical piece of information for refining the linkage between atrazine exposure and attenuation of the LH surge

.

5

. 2 .7 Dosimetry Summary

The results of this analysis indicate that the link between atrazine exposure and attenuation of the LH surge can be better characterized through the use of an internal dose metric that serves as an umbrella for all relevant chemical species (parent and metabolites). The short in vivo half

- life of the parent chemical due pr imarily to metabolism and the intrinsic activity of metabolites form the basis for an internal dose response assessment over the traditional administered dose analysis. The observation that repeated dosing has a more pronounced attenuating effect on the LH surge than an equivalent or even a much higher single administered dose supports the use of AUC on the basis that it accounts for levels as well as duration of exposure. Based on the pharmacokinetic profile exhibited by repeated daily dosing with atrazine coupled with LH

surge attenuation studies carried out in different laboratories at different administered doses of atrazine, under different repeated daily dosing regimens (but at least for four days), with different rat strains, and different modes of or al administration, steady stateDo not cite

Section 5

- Page 73 of 677 appears to be a n important condition under which the LH surge attenuating effect occurs following atrazine exposure, suggesting that a daily steady state AUC for triazine equivalents may be the most appropriate internal dose metric.

Section 8 describes possible approaches for extrapolating these rat results to humans for establishing an appropriate frequency of water monitoring for triazine species.

5

. 3 Benchmark Dose (BMD) Analysis

Detailed information about the BMD analysis summarized below can be found in Appendix C.

Numerous scientific peer review panels over the last decade have supported the Agency‘s application of the BMD approach as a scientifically supportable method for deriving poi nts of departures ( PoDs ) in human health risk assessment, and as an improvement over the historically applied approach of using no

- observed

- adverse

- effect levels (NOAELs) or lowest

- observed

- adverse

- effect

- levels (LOAELs). The NOAEL/LOAEL approach does not account for the variability and uncertainty in the experimental results, which are due to characteristics of the study design, such as dose selection, dose spacing, and sample size. With the BMD approach, all the dose response data are used to derive a P oD. Moreover, the response level used for setting regulatory limits can vary based on the chemical and/or type of toxic effect and for a given chemical, all available studies can be evaluated using a common response level

which aids interpretation ( USEPA, 2000 )

.

In recent months, the Agency pursued performing BMD analysis on a variety of endpoints (attenuation of LH, prostatitis , delayed PPS, delayed VO). However, after an initial scoping exercise , it became clear that the LH data were the only datasets with

sufficient dose

- response data to support empirical modeling. For the other endpoints

, toxicological effects have only been observed at 1) only at very high doses; 2) only at single dose levels; or 3) exhibit flat dose

- response curves. As such, th e only BMD analyses provide d here and in Appendix C relate to LH attenuation. Although, it would be preferred to model additiona l endpoints, as shown in Table 5

- 2 , the other outcomes reported following atrazine exposure are less sensitive tha n the LH data. As such, the Agency is focusing on the data which are most sensitive and thus most protective of

human health.

Generally, EPA calculates both the BMD (central estimate) and the BMDL ( t he BMDL corresponds to the 95% lower bou nd on dose ) . As a matter of science policy, EPA uses the BMDL as the point of departure. A key consideration when performing BMD modeling is determining the response level

— known as the benchmark response (BMR). In the case of continuous endpoints, like LH attenuation, the BMR most often represents an X% change from background levels (or untreated controls). Typically,Do not cite

Section 5

- Page 74 of 677 the BMR is selected on the basis of a combination of biological (mode of action, quantitative link between key events, historical/concur rent controls) and statistical

considerations (sample size, variability, etc). However, i n the absence of information concerning the level of response (or % change) associated with an adverse effect, the Agency‘s draft BMD guidance suggests that the BMD a nd BMDL corresponding to a change in the mean response equal to one standard deviation from the control mean be

used as the BMR . In the case of atrazine, the level of attenuation of the LH surge

considered to be adverse is a function of several factors in cluding but not limited to the

life

- stage and functional outcomes under consideration ( e.g. , estr o us cyclicity disruptions in rats ). Moreover, the differences in reproductive cycles/aging between rodents and humans add an additional level of complexity to establishing a specific BMR value. These differences notwithstanding, in general, the reproductive physiology among mammals is remarkably similar making the use of the LH surge as a critical effect of concern appropriate. This perturbation of the LH sur ge is the cornerstone of the cascade of events leading to the adverse reproductive outcomes attributed to atrazine exposure. Sexual maturation is the culmination of a complex cascade of developmental effects that ultimately leads to the attainment of repr oductive capacity

. Activation of the HPG axis resulting in the pulsatile secretion of GnRH and LH is critical to puberty onset. For instance, decreased LH during puberty would lead to insufficient stimulation of the gonads to reduce the circulating hormo ne levels needed for development of sex accessory tissues in males and females. Moreover, researchers have found that disruption of GnRH release and the ensuing dampening of the LH surge can lead to delays in vaginal opening and preputial separation.

B ased on the available data, attenuation of the LH surge continues to be the most sensitive endpoint in the developmental/reproductive toxicity profile for atrazine

. As shown in Table 5

- 2 of this document, attenuation of the LH su rge occurs at doses that are either comparable to or ≈ 4

- to 30

- fold lower than those eliciting other developmental/reproductive effects ( e.g. , delayed ossification, estrous cyclicity disruptions, delays in sexual maturation, and prostatitis). Taking into account these challenges in establishing a response level

, a BMR of one standard deviation from the control mean was chosen as the suggested choice by the EPA BMDS 2000 guidance whenever the biologically significant degree of change in a response is not cl early defined. The Agency will solicit comments from the SAP on selection of an appropriate

BMR.

As discussed in Section 5 .2 Dosimetry : Internal vs. External , there are various options for selection of the appropriate dose m etric for atrazine including administered

dose and average daily steady state A UC

. The average daily AUC steady state dose metric may be an appropriate internal dose metric to better characterize the link between atrazine exposure and attenuation of the p re

- ovulatory LH surge since it considers the contributions of all the relevant triazine species in the attenuation of the LH surge . BMD modeling was conducted using the administered dose for each of theDo not cite

Section 5

- Page 75 of 677 studies identified below and using the AUC metric for the study selected as the most robust for deriving a point of departure (4

- day exposure data from Cooper et al. , 2010).

5.3. 1 Identification of C ritical S tudies

Several oral toxicity studies in the rat provided LH data that were considered for

do se

- response modeling. These include Morseth et. al. ( 1996a & b ), Minnema (2001; 2002) and Cooper et al. (2010). Recent data from the NHEERL provided the most robust LH data in terms of dose selection ( number of dose levels

- particularly low dose

range

- and the spacing between dose levels ) and variability of the data. The study design addressed the low

- dose region of the dose

- response curve and exhibited less data variability (i.e., smaller standard deviation s ).

A one month oral study by Morseth et al . (1996 a ) established a LOAEL of 40 mg/kg/day and a NOAEL of 5 mg/kg/day for an effect on LH surge. A limitation of the study is the large variability of the data (as reflected by standard deviations). A subsequent 6 month study (Morseth et al . 1996 b ) established a LOAEL of 3.65 mg/kg/day for attenuation of LH surge; the NOAEL was 1.8 mg/kg/day. The data from this study also indicated large variability. The NOAEL from this study was the basis for the point of departure used to establish the chronic reference dose (cRfD) in the EPA‘s

2003 human health risk assessment for atrazine.

Minnema (2001) established a LOAEL of 40 mg/kg/day for attenuation of LH surge in rats treated with atrazine for 1 month. The NOAEL was 5 mg/kg/day. These

data also displayed variability. In a follow

- up 6 month dietary study (Minnema 2002), atrazine (1.8, 3.4, 4.9, and 29.1 mg/kg/day) was without effect on LH surge in

ovariectomized female SD rats. Confidence in this study is low ; BMD /BMDL values

were not calcula ted for Minnena (2002) . Based on historical control data, the induction of LH surge in control animals seems to be suboptimal. Rats exhibited an infection that may have impacted LH surge response. In addition, estradiol data were not submitted

to evalu ate LH induction response.

5

. 3 .2 Methods & Results

EPA ‘s Benchmark Dose Software (BMDS) version 2.1.2 was utilized to estimate BMD estimates from the available LH studies. All models pertinent for continuous data

were evaluated including exponent ial, H ill, power, polynomial and linear. Criteria used to assess the best fit included statistical (goodness

- of

- fit) values, model criteria (Akaike Information Criteria; AIC), BMD/BMDL ratios, and visual inspection.

The most reliable model fits and BMD and BMDL estimates were obtained from

the 4

- day data from Cooper et al. (2010)

. In contrast to the NHEERL dataset, less reliable model fits and failure of some models to compute BMD/BMDL values were

encountered with the other available studies that had more data variability and less well

-Do not cite

Section 5

- Page 76 of 677 defined dose selection. There is uncertainty in the BMD/BMDL estimates from these

other studies. Table 5

- 2 below summarizes the results of BMD analyses of the available

LH studies and the level of confidence in the estimates

. Detailed results are provided in Appendix C.

Table 5

- 2 : Summary of Administered Dose BMD analyses

LH Studies BMD BMDL Confidence

NHEERL 4 day (oral gavage) 4.23 1.96 High

– robust dataset (less data variability; well

- defined

dose selection ) ; describes LH response very well.

Minnema 1 Month (oral gavage) 653.3 245.1 Low

– data variability; both

BMD and BMDL estimates outside experimental range; large BMD/BMDL ratio; failure of some exponential models to compute estimates.

Morseth 1 Month (oral gavage) 523.7 174.7 Low

– data variability; BMD estimate outside experimental range; large BMD/BMDL ratio; failure of some exponential models to compute estimates.

Morseth 6 M onth (dietary) 36.2 17.1 Low

– data variability;BMD estimate outside experimental range; failure of some exponential models to compute estimates.

For the Cooper et al. (2010) 4

- day data, EPA calculated the BMD/BMDL for the

administered dose and interna l dose metrics. When administered dose was used as the dose metric, the exponential model (subset model 4) provided the best fit to the data, and based on statistical (goodness

- of

- fit values), modeling criteria (Akaike Information Criteria; AIC values), an d visual inspection, BMD and BMDL estimates were 4.23 and 1.96 mg/kg/day, respectively (see Figure 5

. 9 ; BMD output and summaries provided in Appendix C ). The BMD and BMDL estimates described the LH surge data very well.Do not cite

Section 5

- Page 77 of 677 The e stimates for the 4

- day Cooper et al. (2010) data using administered dose

as the dose metric were supported by BMD analyses of internal AUC dose metric . T he Hill model provided the best fits for both AUC internal dose metrics; these results are presented i n Figure 5

. 10 for ―area under the triazine equivalent plasma

- concentration time curve‖, and Figure 5

. 11 for ―daily steady state AUC for triazine equivalents.‖ The following table summarizes BMD and BMDL estimate s corresponding to administered
dose and AUC as the dose metrics.

Table 5

- 3 : Conversion of AUC estimates to administered dose

BMD and BMDL estimates (BMR =

1SD from control mean) SS levels (mg/L ) of

Total Triazine Equivalents Daily AUCss (mg/L

- hr)

BMD 2.07966 49.9819

BMDL 0.64721 15.5588

BMDL values of 0.64721 mg/L and 15.5588 (mg/L

- hr) were estimated for steady state levels of Total Triazine Equivalents and Daily AUCss, respectively. Apply ing the appropriate equations (Section 6 ) for the relationship between AUC dose metrics and administered dose, the BMDL values of 0.64721 mg/L and 15.5588 (mg/L

- hr) correspond to an administered dose of 1.86 mg/kg/day, which i s comparable to the value (i.e., 1.96 mg/kg/day) estimated with administered dose as the dose metric.Do not cite

Section 5

- Page 78 of 677 Figure 5

. 9 : EPA BMDS Exponential Model (Constant Variance; BMR = 1 SD from control mean) Results fo r Attenuation of LH Surge in Female Rats Administered Atrazine by Gavage

- NHEERL Data.Do not cite

Section 5

- Page 79 of 677 Figure 5

. 10 : EPA BMDS Hill Model (Constant Variance; BMR = 1 SD from control mean) Results for ―Area Under t he Triazine Equivalent Plasma

Concentration Time Curve‖ a s the Dose Metric

- NHEERL Data (Attenuation of LH Surge in Female Rats Administered Atrazine by Gavage).Do not cite

Section 5

- Page 80 of 677 Figure 5

. 11 : EPA BMDS Hill Model (Co nstant Variance; BMR = 1 SD from control mean) Results for ―Daily Steady State AUC for Triazine Equivalents‖ as the Dose Metric

- NHEERL Data (Attenuation of LH Surge in Female Rats Administered Atrazine by Gavage).

5.4 Proposed Point of Departure

As shown in Figure 5

. 8 , atrazine and its metabolites appear to reach pseudo PK steady state at or near four days of exposure in the adult rat. Specifically, after four days of exposure, the plasma levels of t otal triazine equiva lents plateau during periods of repeated dosing. Similarly, data from multiple laboratories ranging in duration from four days up to six months of exposure show that attenuation of LH is fairly constant at a given dose and suggest PD steady state. As suc h, given both PK and PD steady state, it is prudent to select the most robust study from those studies where steady state has been reached (i

. e., any study four days or longer). As described in Sections 5.3, the new data from Cooper et al. (2010) contain multiple doses across a broad range

and provide comparatively low variability . As such, the data from Cooper et al. (2010) are proposed as the most appropriate for deriving a PoD. Specifically, the BMDL ofDo not cite

Section 5

- Page 81 of 677 1.86 mg/kg/day (based on AUC dose metrics) is p roposed as a point of departure (PoD)

for atrazine risk assessment for repeated exposure scenarios. Possible approaches for extrapolati n g this animal PoD to humans is discussed in Section 8 below.Do not cite

Section 6

- Page 82 of 677 6. SCIENTIFIC CONSIDERA TIONS IN POTENTIAL S ENSITIVITY OF I NFANTS &

CHILDREN

6

. 1 Background

The F FDCA, as amended by the F QPA (1996)

, requires the Agency to give special attention to the potential risk to infants and children. Specifically, FQPA instructs EPA, in making its ―reasonable certainty of no harm‖ finding, that in ―the case of threshold effects, an additional tenfold margin of safety for the pesticide chemical residue and other sources of exposure shall be applied for infants and children to take into account potential pre

- and post

- natal toxicity a nd completeness of data with respect to exposure and toxicity to infants and children .‖ Section 408 (b)(2)(C) further states that ―the Administrator may use a different margin of safety for the pesticide chemical residue only if, on the basis of reliable data, such margin will be safe for infants and

children.‖ This additional margin of safety is referred to as the ―FQPA Safety Factor.‖

In this section, the Agency describes the state of the science with respect to the potential for pre

- and post

- natal toxicity and the completeness of data with respect to toxicity to infants and children. This discussion focuses on experimental toxicology and epidemiology data relevant for considering pre

- and post

- natal toxicity (in cluding the peripubertal period ). Although exposure considerations are a component of the FQPA Safety Factor analysis, this section of the draft issue paper does not include drinking water exposure. Drinking water exposure and statistical approaches for assessing monitoring frequency are covered in Section 7

.

The Agency is aware of key experimental toxicology studies important for the FQPA analysis which are still on

- going. These include studies evaluating 1) the potential hormonal changes and outcomes fro m gestational exposure to male and female rats; 2) behavioral changes in male rats (e.g., rough and tumble play) following gestational exposure (GD 14

- 21) and 3) other possible latent effects manifested in adults from gestational and lactational exposure. Because the Agency is still evaluating potential approaches for assessing drinking water monitoring frequency and important experimental toxicology data are still being collected, at this time the scientific analysis related to determining potential for lifestage sensitivity is incomplete for both hazard and exposure. As such, this draft issue paper does not propose a new FQPA Safety Factor. Instead, the Agency will solicit comment from the SAP on 1) the current state of the science with respect to asse ssing potential sensitivity to the young and 2) the overall approach for determining the FQPA Safety Factor.

In documents supporting the previous risk assessment, the Agency noted datagaps
related to ― information on Atrazine concerning exposure througho ut all critical developmental periods (i.e., gestation through puberty in both sexes), in particular, early in development ....(FQPA memo , 2002).‖ Since the last risk assessment for atrazine,Do not cite

Section 6

- Page 83 of 677 several new studies in developing rats have been conducted. Gener ally, among the new studies, there are far more experimental toxicology studies evaluating the potential health effects of atrazine during the peripubertal period than gestational or lactational periods. There are new tissue dosimetry data collected durin g both the gestational and lactational periods which aid in considering the dose that reaches the fetus/pup, respectively. Epidemiology studies on reported associations between presumed atrazine exposure and birth defects and birth outcomes are also consi dered in this

section.

6.2 Experimental Toxicology

6.2. 1 Gestational Exposure (Dosimetry and Effects)

Recently

, EPA‘s ORD and OPP‘s Biological and Economics Analysis Division (BEAD) worked collaboratively to collect and analyze tissue dosimetr y data from dams and fetuses exposed to atrazine from GD 18

- 20 or 14

- 20 (Stoker et al. , 2010; Kamel et
al. , 2010). As shown in Table 6

- 2 , the levels of atrazine and its metabolites in the fetuses are almost equal to that measured in the blood of dams suggesting that the placenta offers little barrier for these chemicals. It is important to note that atrazine and metabolites were only measured in whole fetuses and therefore , the concentration in specific tissue s is not known. In the dam, the levels of atrazine and its metabolites are remarkably similar across the GD 18

- 20 (3 days) and GD 14

- 20 (7 days) durations and support the concept that a pseudo

- steady state is reached upon repeated daily dosing

as described in Section 5

.

With respect to toxicity outcomes following gestational exposure, Rosenberg, et
al. (2008) reported delays in PPS at 50 mg/kg/d ay . In contrast, Rayner et al. (2007), observed no change in the timing of male puberty, but did repo rt a higher incidence in prostatitis at 100 mg/kg/d ay . Delays in mammary gland development of female offspring following high doses have also been reported (Rayner et al. , 2005) ( Table

6

- 1 ).

Table 6

- 1 : Summary of available atrazine experimental toxicology studies from the gestational period

AUTHOR (YR) EXPOSURE NOAEL/LOAEL mg/kg/day EFFECT

Gestation Rosenberg (2008) GD14

-

parturition 10/50 Delayed PPS

Rayner et al. (2005) GD 13

- 19 NA/100 Delay in mammary gland development Rayner et al. (2005) GD 17

- 19 NA/100 Delay in mammary gland development Rayner et al. GD 15

- 19 NA/100 ProstatitisDo not cite

Section 6

- Page 84 of 677 (2007)

Table 6

- 2 a &b: Extracted from Sto ker et al. (2010)Do not cite

Section 6

- Page 85 of 677Do not cite

Section 6

- Page 86 of 677 Enoch et al. , ( 2007 ) describe the effects of a mixture of atrazine and several metabolites/degradates on mammary gland development in female pups. In this study, dams were exposed orally to an atrazine metabolite mixture from GD 1 5

- 19. The atrazine metabolite mixture (AMM) was made up of atrazine, DACT, DEA, DIA, and hydroxyatrazine (HA) at 0, 0.09 mg/kg AMM, 0.87 mg/kg AMM, 8.73 mg/kg AMM, and 100 mg/kg atrazine. A 100 mg/kg atrazine group was used as a positive control. The

au thors conclude that the findings suggest adverse findings in both mammary gland development at all AMM doses. However, the dose

- response relationships are equivocal and there are critical concerns with study design and data reporting/analysis which preclu de meaningful conclusions. For example, the authors did not evaluate the dose response relationships for the individual metabolites and one cannot determine if the findings are related to a single metabolite or an effect of the mixture. In addition, the methods and results appear inconsistent with each other and the tables and figures are hard to interpret . Thus, Enoch et al. (2007) does not provide sufficient scientific basis for use in risk assessment and in setting regulatory limits. Moreover, given the concerns over study design and data reporting, this mixture study play s a minor role in overall weight of the evidence for assessing potential sensitivity to the young.

6.2.2 Lactational Exposure (Dosimetry and Effects)

Two tissue dosimetry stu dies have been conducted by EPA laboratories to evaluate lactational transfer of atrazine and its metabolites to lactating pups (Stoker and Cooper , 2007; Stoker et al. , 2010; Kamel et al. , 2010; Table 6

- 3 & Table 6

- 4 ). In general, these studies show a decrease in the concentration of atrazine and its metabolites as the chemicals move from the dam‘s mammary gland → milk in the pup

stomach → pup plasma and pup brain, such that the concentrations in the pup plasma and brain are approximately 10

- fold (or more) less than that in the dam plasma. DACT is the major metabolite in milk collected from pup stomachs with only small amounts of atrazine, DIA, and DEA detected in the milk.

In the Stoker/Kamel et al. (2010) study, different tissues from dams and pups were collected and analyzed following repeated oral gavage dosing of dams with atrazine from GD 14 to PND 10 ( Table 6

- 4 ). In Stoker and Cooper (2007), tissue dosimetry data for dams and pups was obtained following exposure of lactating dams to a single oral gavage dose of atrazine on PND 3 ( Table 6

- 3 ). The concentr ations of atrazine and its metabolites in the milk collected from the pup stomachs at PND3 were approximately 3

- fold higher in the single dose study (Stoker and Cooper, 2007) at the 4 mg/kg/day dose than in the 5 mg/kg/day dose group of the repeated dosing study (Stoker et al. , 2010, Kamel et al. , 2010). The source of this difference is not known but may be explained by a number of factors including a h igher fat content of milk at PND3 compared to PND10, different analytical techniques, across laboratory v ariability, or

other factors.Do not cite

Section 6

- Page 87 of 677 Table 6

- 3 : Total reactivity measured in PND 3 pups following lactational exposure to dams exposed to atrazine [Extracted from Stoker and Cooper

. (2007)].

Atrazine

Dose Atrazine Dose Concentration of 14C

- CITRI residues, based on percentage of dose

Stomach (Milk) 2 mg 14 C

Atrazine 0.658 ± 0.37

4 mg 14 C

Atrazine 1.172 ± 0.65

Brain 2 mg 14 C

Atrazine 0.013 ± 0.0002

4 mg 14 C

Atrazine 0.020 ± 0.0002

Table 6

- 4 : a & b. Extracted from Stoker et al. (2010)Do not cite

Section 6

- Page 88 of 677 Table 6

- 5 : Summary of available atrazine experimental toxicology studies

from the lac t ational periods

AUTHOR (YR) EXPOSURE NOAE L/LOAEL mg/kg/day EFFECT

Lactation

Stoker 1999 PND1

- 4 12.5/25 Prostatitis

PND 6

- 9 NA/25 Non

- stat sig prostatitis

6.2.3 Cross

- fostering Studies

Two cross

- fostering studies are available which involved gestational exposure to

atrazine followed by cross

- fostering with presumed lactational transfer.

Rayner et al . (2007) exposed dams at 100 mg/kg from GD 15

- 19 and cross

fostered pups on PND 1 to/from atrazine or vehicle exposed dams to create four groups (control

- control, atrazine

- control, con trol

- atrazine, atrazine

- atrazine). 6 As such, some pups were nursed by dams exposed to atrazine during gestation. The authors assume
that sufficient atrazine and/or its metabolites remain for lactational transfer. Litters receiving milk from dams exposed to 100 mg/kg/day during gestation showed a delay in

6 Dam exposure

– offspring exposureDo not cite

Section 6

- Page 89 of 677 VO and in mammary gland development. Rayner et al. (2007) also measured LH, prolactin, and TSH on PND 33, 40, and 58 in the cross

- fostered pups and found no biologically meaningful hormonal changes in p ups exposed during lactation.

A study (Coder, 2010) recently submitted to the Agency evaluated the impact of gestational exposure (GD 13

- 19; the presumptive critical period for mammary gland development) on the development of mammary gland in the offsprin g. With the exception of a statistically significant elongation of mammary ductal length and a decrease in the proliferation of epithelial cells in the mammary gland at doses ≥ 50 mg/kg/day on PND1 only , no effects in mammary gland development were report ed. It is noteworthy that no effects on mammary gland development were noted at any of the other time point evaluated (PND 21, 33, day of VO, or first day of diestrous after PND 58). The author also reported an increase in pup mortality at the 100 mg/kg/d ay dose level. This effect, however, was attributed to poor maternal care evidenced by poor nesting behavior, aggression towards pups, and diminished nursing behavior.

6.2.4 Peri

- pubertal Exposure

As shown in Table 6

- 6 , a n umber of new studies have evaluated the effects of atrazine in male and female pups during the peri

- pubertal period. Overall, there is qualitative consistency among these studies as they show delays in the onset of puberty in both sexes, although the dose

- response relationships differ somewhat among
studies. The Stoker et al. (2001) study provides the most sensitive NOAEL/LOAEL at 6.25/12.5 mg/kg/day atrazine. With respect to hormone measurements, changes in testosterone have been shown at high doses ( ≥ 50 mg/kg). Given the inherent variability on testosterone levels during the peripubertal period, it is not unexpected that significant changes in testosterone were only reported after atrazine exposure at relatively high dose levels. It is also importa nt to consider that although LH stimulates testosterone secretion from the Leydig cells, this modulation is the result of increased sensitivity of Leydig cells to the LH stimuli rather than an increase in circulating LH. As

a result of this increased sens itivity, substantial decreases in LH are needed before changes in testosterone are observed.

Table 6

- 6 : Summary of available atrazine experimental toxicology studies from the gestational, lactational , and pe ripubertal periods

AUTHOR (YR) EXPOSURE NOAEL/LOAEL mg/kg/day EFFECT

Peripubertal

Stoker et al . , 2001 PND 23 to

53 6.25/12.5 Delayed PPS

Trentacoste et al .

, 2001 PND 22 to

47 50 lowest dose/100 Delayed PPSDo not cite

Section 6

- Page 90 of 677 Stoker et al . , 2001 PND 23 to

53 25/50 Dec reased VP and SV

Trentacoste et al .

, 2001 PND 22 to

47 50 lowest dose/100 Decreased VP and SV

Stoker et al . , 2001 PND 23 to

53 ND/200 ‡ Decreased intra

- testicular T on PND

45

Trentacoste et al .

, 2001 PND 22 to

47 ND/50 Decreased T on PND 47

Friedmann , 2002 PND 22 to

47 ND/50 Decreased test. and serum T on

PND 47

Pogrimic et al . , 2009 PND 23 to

50 50/200 Decreased T/DHT on PND 50

Laws (2000) PND 22

- 41 25/50 Delayed VO

‡ Lowest dose tested

6.3 Epidemiology

Several research articles identified i n the literature are pertinent to the consideration of the FQPA Safety Factor. E pidemiologic data when evaluated in the
context of the MOA for atrazine are supportive of the current atrazine toxicity and exposure profile with respect to effects on the estr ous cycle and possibly sperm

characteristics

. One study observed increased odds of menstrual cycles greater than 36 days and increased odds of missed menstrual cycles among women who reported ever

using atrazine or lindane in their lifetime (Farr et al

. , 2 004). In a similar study population, r esearchers have also observed that exposure to hormonally active pesticides, including atrazine, may delay menopause by several months (Farr et al

. , 2006). Both studies were conducted within the AHS, include validate d exposure information (ever

- use in a lifetime), relative ly large sample size s , and adjustment for potentially confounding variables including physical activity. However, given the data available to these researchers, neither of these studies was able to d efinitively link atrazine exposure with these female reproductive effects . Regarding male reproductive health effects, Swan et al

. (2003) reported increased prevalence of poor semen quality (sperm concentration, motility and morphological abnormalities) amo ng men with urinary concentration of atrazine mercapturate, a metabolite of atrazine, above the limit

of detection . While these results are suggestive, they are based upon the relatively small sample size. Extension and replication of these findings is nec essary

.

In addition, two studies observed a slightly elevated risk of SGA, particularly if atrazine exposure occurred in the third trimester of pregnancy. Both of these investigations measured atrazine in drinking water, as opposed to a surrogate for

drin king water exposure, however, the association between SGA and atrazine exposure in the late stages of pregnancy are based upon exploratory sub

- analyses (Ochoa

- Acuna

et al

. , 2 009; Villanueva et al

. , 2005). The relationship, if any, between the MOA and

these types of adverse health outcomes is not known. Nonetheless, reduced pup weight is also seen in the toxicology database. The strength of the association for otherDo not cite

Section 6

- Page 91 of 677 birth outcomes is generally weak at this time, and study results are inconsistent (Munger e t al

. , 1997; Sathyanarayana et al

. , 2010; Dabrowski et al

. , 2 003; Savitz et al

. , 1997; Arbuckle et al

. , 2001). Hypothesis

- generating studies suggested a possible role for atrazine in the etiology of major birth defects (Mattix et al

. , 2007; Winchester et a l

. , 2009; Hornemann et al

. , 2008). However, subsequent evaluations have not presented strong supporting evidence mainly due to limitations in exposure measurement and the ability to correlate atrazine exposure and the timing of pregnancy. Studies reported marginally statistically significant 20

- 60% increased odds of limb abnormalities and gastroschisis, respectively (Ochoa

- Acuna et al

. , 2009; Waller et al

. , 2 010). Uncertainty

exists as to whether these estimates reflect true increased risk or are the resul t of exposure or outcome measurement error.

EPA has concluded that, at this time, the epidemiologic database concerning the

non

- cancer health effects of atrazine exposure is not robust or mature enough to support causal inference, i.e ., EPA is not able to make a firm cause to effect link between atrazine exposure and any of the non

- cancer health effects examined in these studies including health effects to infants, children and the developing fetus. However, EPA is also not able to discount a possible asso ciation between atrazine and several health outcomes related to women of childbearing years and the developing fetus based upon these data.

6.4 Summary

With respect to the experimental toxicology database, new tissue dosimetry

studies show that atrazi ne and/or its metabolites crosses the placenta barrier and reach the fetus at levels similar to that in the dam‘s blood during gestation. Health outcome measurements do not show any evidence of increased sensitivity by the developing fetus. During lactat ion, the levels of atrazine or its metabolites in the pups are much lower than those in the dam. As such, it is not surprising that none of the studies evaluating pups during lactation indicate that they are more sensitive than the adult female LH finding s. The levels of atrazine found in the milk of these more recent rat studies show that the % of administered dose is very small, which is consistent with previous test guideline studies i n goats and cows which show that 2% and 2.7% respectively of the a trazine dose is transferred to milk. With respect to peri

- pubertal studies, a number of studies consistently show delays in puberty in male and female

rats.

EPA is proposing to use a BMDL of 1.8 6 mg/kg/day derived from LH data collected after four day s of exposure to adult females as the PoD for human health risk assessment. None of the available studies with atrazine evaluating the rats exposed during gestation, lactation or the per

- pubertal periods have shown effects at doses lower than the four day LH adult study. The only exception is a mixture study of atrazine, DACT, DIA, DEA, and hydroxyatrazine where the author claims a LOAEL of 0.09 mg/kg/day in females based on delays in mammary gland development (Enoch etDo not cite

Section 6

- Page 92 of 677 al.

, 2007) . This mixture study su ffers from significant limitations in study design and

dose

- response relationships that preclude definitive conclusions. Thus, of the quality

studies on the developing rat, Stoker et al. (2001) provides the most sensitive endpoint, namely delayed PPS at a NOAEL/LOAEL of 6.25/12.5 mg/kg/day. The NOAEL from
Stoker et al. (2001) is approximately three

- fold higher than the four day LH data, thus adding confidence that the proposed PoD would be protective for this effect as well as others occurring at higher doses.

In the previous risk assessment, the Agency identified data gaps in critical periods of development (gestation through puberty). Significant research has been conducted in this area since the 2003 risk assessment and key studies are on

- going. For example, on

- going studies at EPA are evaluating potential effects of atrazine on behavior as well as on hormonal changes in female and male pups from gestational exposure. In addition, Syngenta is conducting a multi

- lifestage study evaluating the pote ntial latent effects from early life atrazine exposure. This study is still on

- going; although completed components were recently submitted to EPA in preparation for the September SAP (Coder et al. , 2010b). The Agency anticipates that these studies, in

c ombination with other recent studies, will fill gaps in knowledge identified in the previous risk assessment,

Epidemiologic studies suggest possible alteration in menstrual cycle characteristics (e.g., length) among women exposed to atrazine or lindane, and a change in reproductive senescence in women exposed to atrazine as well as a potential link between semen quality in atrazine exposed men. The health effects are plausible considering our knowledge of atrazine‘s ability to affect the HPG axis; howeve r, other explanations may exist. There are weakly suggestive data regarding birth outcomes, particularly small

- for

- gestation age in relation to atrazine exposure late in pregnancy (third trimester). However, several methodological limitations in the desi gn, conduct or analysis of the studies prohibit making clear causal connections, and discerning clear evidence for additional susceptibility to infants, children or the developing fetus. EPA is

not able to make a firm cause to effect link between atrazine exposure and any of the

non

- cancer health effects examined in these studies including health effects to infants, children and the developing fetus. However, given the qualitative consistency of the epidemiologic findings with the animal MOA, EPA is also no t able to discount a possible

association between atrazine and several health outcomes related to women of childbearing years and the developing fetus based upon these data.

The analysis to evaluate potential sensitivity to infants and children is incomp lete since key studies are still ongoing. In addition, the approach to evaluate the drinking water monitoring interval is still being developed. At this time, the Agency is not proposing an updated FQPA Safety Factor based on the new information. As new data become available, the Agency plans to consider this new information and evaluate the

extent to which it is or is not consistent with the existing database of studies and with existing knowledge about the MOA for atrazine.Do not cite

Section 7

- Page 93 of 677 7. APPROACHES TO EVALUA TING WATER SAMPLING STRAT EGIES AND
FREQUENCY OF MONITOR ING

7 .1. Introduction

As a part of its April 2010 consultation with the SAP on a Review of Experimental Animal and In vitro Studies and Drinking Water Monitoring Frequency related to atrazine (USEPA, 201 0), the USEPA presented a preliminary review of approaches for evaluating water monitoring strategies and estimating pesticide exposure concentrations in drinking water sources for use in human health risk assessments. Using the best available information at the time of the 2003 IRED (USEPA, 2003) , the
USEPA determined that the most sensitive toxicological endpoint was associated with exposure to atrazine over a prolonged period (90

- day average time frame). T he 90

- day rolling average corresponded to: (i) t he 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 dosing in a 6 month toxicity study. The registrant, Syngenta

, employed a monitor ing strategy

– weekly sampling during the use/high runoff season and bi

- weekly sampling during other parts of the year

– to characterize exposure over a 90

- day time frame for both source and treated waters at vulnerable community water systems (CWS) in the atrazine use area

. Any CWS that exceeded annual mean concentrations of 1.6 ug/L (ppb) atrazine based on quarterly Safe Drinking Water Act (SDWA) sampling was included in this more intensively sampled Atrazine Monitoring Program (AMP). The AMP sampled atr azine and other

chloro

- triazines for 130 to 150 CWS per year over the past seven years.

The current re

- evaluation of the human health effects of atrazine based on new data related to the hazard potential and dose

- response for atrazine may result in a sho rter duration of exposure for the toxicological endpoint of concern. If the re

- evaluation

determines that a shorter dura tion of exposure is more appropriate for atrazine effects on humans, the USEPA must consider h ow well the existing AMP monitoring desig n characterize s shorter durations of exposure and whether the monitoring program need s to include more frequent sampling or other means to augment existing data. In the April 2010 SAP, the USEPA explored two general questions to drinking water monitoring:

(1) For new monitoring studies, h ow frequent ly does sampling need to be in order to adequately characterize various exposure magnitudes and durations? (2) For existing monitoring studies, how much exposure information can we derive from sampling that may be less frequent than the exposure duration of concern?

The April 2010 background document for the SAP (USEPA, 2010) listed several approaches for analyzing and interpreting monitoring data, including linear or stair

- step interpolation, bootstrapping, artifici al neural networks, kriging/random function models, regression

- based models and deterministic models. While the background documentDo not cite

Section 7

- Page 94 of 677 presented some exploratory analyses using the weekly chemographs from the AMP program, t he USEPA proposed to continue expl oring different exposure estimation

methods u s ing more intensively sampled monitoring data sets.

In its comments, the SAP emphasized that simulations of candidate monitoring strategies and evaluations of the utility of different exposure estimation method s need to

be benchmarked against intensive empirical data for an adequately representative range of sites (FIFRA SAP, 2010). The SAP expressed concern that the weekly sampling may miss short

- duration peaks, smooth ing out the variability and provid ing a bi ased representation of actual concentration profiles

.

The SAP noted that the toxicological exposure timeframe of interest will define the importance of peaks and determine the most useful approach f o r designing a monitoring study and for evaluating the utility of existing monitoring studies. Thus, the exposure duration of toxicological interest will define the importance of peak events and their duration and the importance of clusters of peaks, their duration, and the intra

- cluster

maxima . The a ppropri ateness of combining inference, modeling, and monitoring to meet risk assessment needs must be evaluated specifically for the toxicological level and duration of concern and for the accuracy and precision needed for the assessment.

The SAP recommended th at the m inimum sampling interval should be more frequent than the toxicological exposure duration of interest , with at least 2 points within the timeframe of interest necessary to provide an average concentration for that timeframe. However, the study des ign must also c onsider the tradeoffs between the cost of improved reliability and accepting probability

- based regulatory actions based on estimates that may have low er precision . For new monitoring studies, the SAP

recommended considering autosamplers tha t produce composite samples over biologically relevant time frames. A predictive model , such as USGS‘s Watershed Regression on Pesticides ( WARP ), can be used to target sites that merit the most detailed monitoring and target the time periods when atrazine is most likely to be present (FIFRA SAP, 2010). This is similar to the approach used for the separate atrazine ecological exposure monitoring program (see USEPA, 2007).

To estimate exposures from monitoring data sampled at intervals, particularly where

p eak concentrations over shorter exposure durations are important, the USEPA n eed s methods that can predict values that may be greater than those sampled. This is a

limitation of linear or stair

- step interpolations and boot

- strapping techniques (FIFRA SAP, 2010 ) . The SAP r ecommended exploring the possibility of combining regression

based models, such as WARP, with kr iging or random

- function models. The panel also suggested exploring e xtreme value theory, especially f o r rolling averages

.

This section ou tlines a basic approach for designing a monitoring study to adequately characterize drinking water exposures for human health assessments. USEPA then describes a set of intensive monitoring data it proposes to use to analyzeDo not cite

Section 7

- Page 95 of 677 varying sampling strategies an d methods for estimating exposures from less frequent monitoring data. The cha pter closes with a brief review of the 2010 SAP recommendations on approaches for estimating exposures from monitoring data. Although the USEPA has not yet defined the critical exposure magnitude and timeframe of toxicological interest for atrazine to determine the appropriate sampling design or methods/ models for estimating exposures, this chapter will explore options for both a short (e.g., 4

- day) or longer duration of exposu re. While the approaches described here focus on atrazine, they may also be appropriate to use for other pesticides as well.

7 .2 General Strategy For Designing a Monitoring Study to Characterize Drinking Water Exposures

A well

- designed surface water m onitoring study take s into account both spatial and temporal patterns of exposure , focusing sampling on when and where the pesticide is

used . Sampling frequency depends not only on the nature of the pesticide (e.g., fate and transport properties), its use pattern, the nature of the water body, and the relative vulnerability of the use area to runoff, but also on the exposure duration of toxicological

concern . A n acute or short

- term exposure duration will require more frequent sampling to reliably characte rize exposures than a longer

- duration exposure period. Because i t is not possible to measure daily concentrations of pesticides at all places across multiple years to capt ure the full extent of pesticide occurrence, the monitoring study must

balance the f requency and location of sampling in the context of the level of certainty specified for making regulatory decisions.

The SAP recommended using a model such as WARP to target sampling in those

areas identified as most vulnerable based on model estimates (FIFRA SAP, 2010). Such a targeted monitoring approach has been used for the Atrazine Ecological Exposure Monitoring Program (AEEMP) 7 , where WARP was used to identify a candidate pool of vulnerable watersheds for monitoring (USEPA, 2007 and 2009).

While the CWS included in the AMP drinking water study are based on SDWA compliance monitoring data, they coincide with the most vulnerable watersheds identified by WARP ( Figure 7

. 1 ).

The SAP also recommended targeting critical perio ds for monitoring, sampling more frequently during the spring and summer months when atrazine levels could be high. Both the AMP and the AEEMP sampled more intensively in the spring and summer months, beginning shortly before the likely planting date for corn/sorghum and

7 As a condition of re

- registration in 2003, the USEPA required Syngenta to conduct two separate water monitoring programs. One, the Atrazine Monitor ing Program (AMP), focused on drinking water exposures, sampling from vulnerable CWS. The second, the Atrazine Ecological Exposure Monitoring Program (AEEMP), focused on exposures to aquatic plant communities in headwater watersheds (generally 2 nd to

5 th order streams). While the focus of this paper is on drinking water exposure, lessons learned from both studies have been helpful in determining approaches for designing monitoring studies and interpreting the

results of these studies.Do not cite

Section 7

- Page 96 of 677 continuing through the summer. The AMP sampled source and treated water samples

for atrazine and other chloro

- triazines weekly during the time when atrazine was most likely to occur in water, typically beginning in early April and running through July or early August, and less intensively

– bi

- weekly or longer

– during the remaining portion of the year. The AEEMP analyzed samples for atrazine at 4

- day intervals beginning in April and running through the end of August or early September. For a subset of AEEMP sites, Syngenta also used autosamplers to provide more frequent, often daily, samples. Such focused sampling characterized atrazine exposure profiles (chemographs) during the periods when atrazine was most likely to be found in the

h ighest concentrations.

Figure 7

. 1 : Location of CWS Monitored in the AMP in relation to vulnerable watersheds identified by WARP. The WARP vulnerability ranking is based on atrazine use on corn and sorghu m only.

Based on the recommendations of the SAPs for both human health (FIFRA SAP, 2010) and ecological concerns (FIFRA SAP, 2009) and on the lessons gained from both the AMP and AEEMP studies for atrazine, the USEPA recommends the following

framework for designing a targeted monitoring study for atrazine or other pesticides:Do not cite

Section 7

- Page 97 of 677 (1) Target M onitoring to the M ost V ulnerable A reas : The SAP recommended using a model like WARP to identify the most vulnerable areas at a watershed scale that is appropriate for water b odies of interest. A spatially balanced design, such as the generalized random tessellation stratified (GRTS) design used for the AEEMP (see USEPA, 2007, for a description of the approach) can facilitate selecting monitoring sites that are spatially repre sentative of the vulnerable water bodies. While the WARP model was developed for atrazine, a multi

- chemical version of WARP could be used for pesticides other than atrazine. Alternatively, GIS tools and data layers can be used to identify potential high exposure areas based on an overlap of high

- runoff potential soil/hydrologic conditions, use area (actual use or potential use based on crop acreage), and precipitation.

(2) Target I ntensive S ampling to the High Occurrence P eriod : Determining the period when the pesticide is most likely to appear in water will depend on the usage pattern, pest occurrence, method of application, pesticide persistence and mobility, and vulnerability of the area to runoff. The intensive monitoring period should cover the beginnin g of pesticide applications in order to capture potential early season runoff events, and extend beyond the application season for a sufficient period to capture late runoff events that may occur while the pesticide is still present and available for runof f. The duration of this period will depend on application patterns and the persistence and mobility of the pesticide.

(3) Base Sampling F requency on the T oxicological E xposure D uration of
C oncern : As noted in the April 2010 SAP, a time

- averaged concentrati on requires sampling to occur more frequently than the toxicological exposure period of concern in order to provide an average concentration for the time interval. Characterization of the peak concentration is critical, particularly for

short

- durations of concern. Because of the variable nature of pesticide occurrence in water, the probability of capturing the peak concentration decreases as the interval between samples increases. For risk assessments, the
USEPA must take into consideration the level of u ncertainty in exposure assessments related to sampling frequency. Thus, as acknowledged by the SAP, the sampling design must balance the trade

- off between increased sampling to improve reliability versus probability

- based risk assessments that may reflect lower

- precision estimates (i.e., the level of certainty necessary for making regulatory decisions).

(4) Use Autosamplers to Collect Data for E xposure Periods of Interest:

C onsider using autosamplers as a supplement or an alternative to grab samples

, particul arly for collecting monitoring data over short exposure periods

. Autosamplers are being employed more frequently in water monitoring studies

and can integrate exposure measurements over the biologically relevant periodDo not cite

Section 7

- Page 98 of 677 of concern , potentially reducing cost s related to collection of grab samples and number of samples analyzed.

7.2 A Set of Intensively Sampled Datasets To Evaluate Sampling Strategies and Exposure Estimation Methods

The USEPA needs a set of intensively sampled monitoring data that adequat ely represent a range of sites reflective of the pesticide use area in order to evaluate sampling strategies and modeling approaches to estimate exposures from less frequent sampling (FIFRA SAP, 2010). The SAP expressed concern that using the weekly CWS

sa mples is not appropriate because it provides a ―biased representation‖ of actual concentration profiles (FIFRA SAP, 2010). In its issue paper for the SAP, the USEPA proposed using the robust Heidelberg NCWQR monitoring data and, as applicable, the

more fre quently

- sampled headwater streams from the AEEMP for such evaluations (USEPA, 2010). This section evaluates these monitoring datasets in context of the chemographs for the CWS in the AMP and outlines an approach for developing a set of intensively sampled chemographs representing both rivers/streams and reservoirs.

7 .3.1 Analysis of CWS Chemograph Shapes

Appendix D .1 characterizes the chemograph shapes (maximum measured concentration, number of peaks, duration of rise and decline, shape of decline) for t he CWS in the AMP, focusing on the 44 CWS that had at least one detection of total

chloro

- triazines ( TCT ) of 15 ug/L or more in either the source or treated water samples. Although both source and treated water samples are included in the datasets, the
USE PA focused on the source water chemographs for reasons discussed below. The CWS are concentrated in the Midwestern US, from OH to KS ( Figure 7

. 1 ).

Of these 44 CWS chemographs

, 16 draw source water from lakes or reservoirs

, 22 fr om rivers or streams , and 4 from combined (reservoirs and rivers) sources. In 2 CWS ( OH

14 and OH

- 17), both the reservoir and the river sources were analyzed . Only those years in which TCT concentrations equaled or exceeded 15 ug/L are included in the

an alysis , totaling 106 chemograph years

.

For each year, USEPA counted the number of upward spikes, or peaks, of at least 1 ug/L. Because the CWS were sampled at weekly intervals , s horter

- duration peaks between sampling events may have been missed. Table 7

- 1 summar izes the frequency of occurrence of upward spikes per sampling year by source water type where TCT concentrations of 15 ug/L or greater were detected . Approximately 80 percent of the 106 chemograph years had 2 to 4 pe aks detected in a given year. Less than 4% of the chemograph years had only a single TCT peak. The river/ stream chemographs had a higher percentage of years with 5 or more peak occurrences

– 18%

vs. 12% for reservoirs and 11% for mixed sources ( Table 7

- 2 ).Do not cite

Section 7

- Page 99 of 677 Table 7

- 1 : Summary of Annual Frequency of Peak Occurrences by Source Water Type

Lake/

Reservoir River/ Stream Mixed
Sources Total of All

Sources

Total Pct Total Pct Total Pct Total Pct

No. CWS 18* 24* 4 46*

No Years TCT >15 33 64 9 106 No. of Peaks Detected Per Sampling Year

Frequency of

Peak
Occurrences Per Sample

Year: 1 1 3% 2 3% 1 11% 4 4%

2 7 21% 19 30% 2 22% 28 26%

3 11 33% 17 27% 4 44% 32 30%

4 10 30% 14 22% 1 11% 25 24%

5 3 9% 9 14% 1 11% 13 12%

> 6 1 3% 3 4% 0 0% 4 4%
* The total include 2 CWS where both reservoir and river source waters were analyzed.

Table D .1.d in Appendix D .1 breaks down the dates and duration of rise and decline

and dat e and magnitude of peak concentration for each of the CWS years. The patterns, illustrated in the CWS graphs in Appendix D .1, show that w here multiple TCT peaks were detected in a given year, the majority of peak profiles (rise, peak, and decline) overlap ped.

7 .3.2 Consideration of Drinking Water Treatment Effects

To determine the extent to which drinking water treatment may have affected TCT concentrations, USEPA compared chemographs for source water and treated water samples taken on the same day f or the 44 CWS with TCT detects of 15 ug/L or greater (see Appendix D .1). L ittle difference was found in concentrations between source water and treated water samples for 16 of the 44 CWS . Only 7 of the 44 CWS showed

evidence of consistent removal of most T CT residues between source and treated

waters. In the remaining CWS, TCT concentrations were reduced but not completely

removed . At one CWS (IN

- 12), TCT concentrations in treated water were similar to source water samples during the first 2 years of sampl ing , suggesting no treatment effect, but were reduced in subsequent years, suggesting a possible change in treatment processes.

The observed CWS chemographs can be grouped into three categories based on likely

treatment effects:Do not cite

Section 7

- Page 100 of 677 (1) No treatment effect is e vident : This likely occurs in CWS that use conventional
treatment processes

– flocculation, sedimentation, and disinfection

– which do not remove atrazine from water (USEPA, 2000, 2001) and do not blend drinking water

sources . For the most part, source a nd treated water concentrations are similar and follow the same pattern , as illustrated in Figure 7

. 2

. The following CWS, based on dominant trends, fall into this category: IL

- 02, IL

- 03, IL

- 05, IL

- 24, IN

- 02, IN

- 04, IN

06, IN

- 12 ( 2004

- 05), KS

- 07, KS

- 19, KS

- 20, KS

- 22, KS

- 27, LA

- 01, OH

- 06, OH

- 09 (except 2009), OH

- 12.

Figure 7

. 2 : Example of a CWS in which TCT concentrations are similar in pattern and magnitude for both source (blue ) and treated (pink) water samples.

(2) Most of atrazine/TCT is removed by treatment : The CWS in this group likely use the best available treatment technology for atrazine

– granular activated carbon ( GAC )

– in sufficient amounts to knock down the TCT conce ntrations on a

consistent basis (USEPA, 2000, 2001)

. Figure 7

. 3 illustrates a CWS where the TCT concentrations in the treated samples do not follow the patterns seen in the source water samples. In this group, the elevated TCT l evels in source waters are not making it through the treatment process. The following CWS, based on dominant trends, fall into this category: IA

- 07, IL

- 29, IL

- 30, MO

- 17, OH

- 04, OH

- 14, and OH

21

.Do not cite

Section 7

- Page 101 of 677 Figure 7

. 3 : Example of a CWS in which TCT concentrations in treated (pink) water samples drop to near the limits of detection in comparison to source water concentrations (blue)

.

(3) Atrazine/TCT concentrations are reduced/dampened but not completely removed by treatm ent : These could be CWS that use activated carbon, but not enough to be fully effective

, and/or blend water from multiple sources. T he

maximum values for the treated water would likely be less than that in the source
water, although the relative dampening effect may vary from year

- to

- year or even day

- to

- day depending on CWS

- specific practices. GAC is often used to treat odor or taste problems rather than pesticides and may not necessarily be used at the time atrazine is present in the water. Figure 7

. 4 illustrates a CWS where TCT concentrations for the treated water still track the same patterns seen in the source water, but at a dampened (reduced) level. The remaining chemographs fall into this category.Do not cite

Section 7

- Page 102 of 677 Figure 7

. 4 : Example of a CWS where TCT concentrations in the treated (pink) water samples are reduced in relation to source (blue) water samples, but remain above the limit of detection.

In keeping with the approach vetted at a September 2000 SAP (USEPA , 2000 ) and documented in a subsequent science policy paper (USEPA, 2001) the USEPA will

a ddress the effects of drinking water treatment separately on a case

- by

- case basis in the risk assessment process. To evaluate monitoring sa mpling frequencies and exposure estimation approaches, the USEPA will focus on the source water samples to eliminate the uncertainty in drinking water treatment effects and because of the importance in protecting source water. For those CWS where no drinki ng water treatment effect is evident, the magnitudes, patterns, and variability in chemographs are expected to be similar for both source and treated waters. In the cases where drinking water treatment results in partial or complete removal of TCT residues

, a reduction not only in concentrations but in variability would be expected. An analysis of sampling variability using source water chemographs is not only reasonably protective for all CWS, but provides an opportunity to incorporate more intensively sam pled water monitoring data for analyses.

7 .3.3 Intensively Sampled Datasets To Evaluate Sampling Strategies and Exposure Estimation MethodsDo not cite

Section 7

- Page 103 of 677 The SAP noted the need for intensively sampled monitoring data that adequately represent a range of sites ref lective of the pesticide use area in order to evaluate sampling strategies and modeling approaches to estimate pesticide exposures (FIFRA SAP, 2010). While the AMP covers the geographic range of atrazine use, with particular

focus on the most vulnerable CW S within that range, the weekly sampling interval is not sufficient because it provides a ―biased representation‖ of actual concentration profiles (FIFRA SAP, 2010). In April 2010 issue paper, the USEPA proposed using the more intensively sampled monitorin g data from Heidelberg University‘s National Center for Water Quality Research (NCWQR) and, as applicable, the more frequently

- sampled

headwater streams from the AEEMP for such evaluations (USEPA, 2010).

Based on feedback from the April SAP and further an alysis of the CWS chemographs (Appendix D .1) and available atrazine monitoring studies, the USEPA proposes the following approach to develop a set of intensively sampled datasets that represent both a geographic and temporal range in atrazine use for both flowing (rivers and streams) and static (lakes and reservoirs) water bodies:

(1) Group the CWS by Water Source and Classify C hemograph S hape s: An initial analysis of the CWS chemographs in Appendix D .1 suggests a grouping by

source water source (river/stream, lake/reservoir) may be warranted. Until further information becomes available on watershed area and land use (under development but not yet available), additional factors influencing chemograph shape have not been evaluated. Although USEPA found no publis hed standard classification for chemographs, a general grouping could be accomplished based

on the number

, duration , and magnitude of peaks . The chemograph classification would be used to match CWS with more intensively monitored datasets.

(2) Use The Heidel berg NCWQR Monitoring Data For Streams And Rivers And Intensively

- Sampled Monitoring Data From The AEEMP To Provide Intensive Monitoring Data For Flowing Water Bodies : The two datasets are described below. Although they both represent ambient streams and not intakes for CWS, the sites could be used to represent different chemograph shapes. If necessary the measured peaks could be scaled to match magnitudes found in
the CWS or to focus on concentrations of concern

.

(3) Use PRZM/EXAMS (Pesticide Root Zone Mode l/Exposure Analysis Modeling System 8 ) to G enerate C hemographs for S tatic W ater B odies : USEPA uses PRZM and EXAMS to estimate pesticide exposures in water (reservoirs) as a part of its routine risk assessments. The linked models can generate 30 years of da ily concentrations based on local weather data, application rates and timing, and local soil, hydrologic, and management

8 More information on PRZM/EXAMS is available on the USEPA Office of Pesticide Program‘s Aquatic Models page at http://www.epa.gov/pesticides/science/models_pg.htm#aquaticDo not cite

Section 7

- Page 104 of 677 conditions. The model could be used to generate chemographs of daily concentrations for static water bodies where intensively

- sampled monitoring data

are not available.

Heidelberg University‘s NCWQR has intensively sampled pesticides in selected streams and rivers draining into Lake Erie since the 1980s 9 . While the Heidelberg dataset is geographically limited to the eastern portion of the atrazine use area for corn and sorghum and does not sample directly from CWS intakes, it provides many years of intensive sampling

– including daily and multi

- daily samples during the atrazine runoff

season

– for a range of rivers and streams, includin g those that are representative of flowing water sources of drinking water for Ohio.

As a preliminary scoping exercise, the USEPA generated samples from the actual chemographs for the Maumee and Sandusky Rivers at 7

- day fixed sample intervals, varying a cross each of the 7 possible days. Figure 7

. 5 illustrates two of the resulting 7

- day sampling intervals overlaid on the actual monitored chemograph. The entire set of sampled chemographs for both the Maumee and Sandusky Rivers ov er multiple years can be found in Appendix D .2. The figures and data summary assume a fixed 7

- day sample interval. Because actual sampling intervals for the AMP may not be fixed within the week, bootstrapping methods will be used to reflect the variations in sampling dates within a weekly window.

An examination of the chemographs shows the degree of data smoothing that occurs as a result of less frequent sampling intervals. The chemographs sampled at weekly intervals had fewer spikes, or peaks, than the actual chemographs (Appendix D .2 and Table 3). This illustrates the data smoothing concerns raised by the SAP in April (FIFRA SAP, 2010). It also suggests that attempts to match chemograph shapes based on number and duration of peaks will need to account for the additional variability from more frequent monitoring.

9 More information on the NCWQR program, its publications, and water quality data retrieval can be foun d

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

.Do not cite

Section 7

- Page 105 of 677 (a) Actual Chemograph (red arrows point to spikes)

(b) Sampled at 7

- day fixed intervals, beginning on Day 2Do not cite

Section 7

- Page 106 of 677 (c) Sampled at 7

- day fixed intervals, beginning on Day 6Do not cite

Section 7

- Page 107 of 677 Figure 7

. 5 : Actual chemograph (blue) and 2 resulting chemographs (red) sampled at 7

- day fixed intervals, Maumee River, 2008 (Data Source: Heidelberg U. NCWQR)

Table 7.2 illustrates a preliminary step in assessing how ef fectively different sampling strategies capture peak or short

- duration exposures, comparing the sampled peak and

4

- day average concentrations of the fixed 7

- day sample intervals to the actual chemographs. The chemographs could also be compared based on th e number of days an average of concern (for this illustration, a 4

- day average was used) exceeds threshold values (20 and 40 ug/L were used as examples).

Table 7

- 2 : Analysis of Chemograph Peaks (Spikes), Maximum Detects, and

4

- Day Averages based on 7

- Day Fixed Sampling Intervals for 2 OH Rivers (Source for original data: Heidelberg U. NCWQR)

Site/Year Sampling

Interval N umber

of Spikes Max

Detect Spike #

w/ Max
Conc Max 4

- da

average Days 4

-

da avg >20 ug/L Days 4

-

da avg >40 ug/L

Maumee

R iver

, 2005 Actual 1 5 22.7 1 14.7 0 0

7 da, beg day 1 3 9.5 1 8.6 0 0

7 da, beg day 2 2 8.3 1 7.6 0 0

7 da, beg day 3 2 7.9 1 7.2 0 0

7 da, beg day 4 2 8.5 1 8.1 0 0

7 da, beg day 5 3 22.7 1 20.3 1 0

7 da, beg d ay 6 2 15.5 1 13.9 0 0

7 da, beg day 7 3 11.2 1 10.0 0 0

Maumee

R iver

, 2006 Actual 12 32.9 3 28.8 9 0

7 da, beg day 1 1 22.8 1 22.3 11 0

7 da, beg day 2 1 19.3 1 17.9 0 0

7 da, beg day 3 1 32.9 1 30.7 11 0

7 da, beg day 4 1 24.1 1 22.7 6 0

7 d a, beg day 5 1 24.3 1 22.6 4 0

7 da, beg day 6 1 28.8 1 26.9 8 0

7 da, beg day 7 1 26.0 1 25.0 10 0

Maumee

R iver

, 2007 Actual 11 12.1 1 9.4 0 0

7 da, beg day 1 4 8.6 1 7.5 0 0

7 da, beg day 2 3 12.1 1 10.5 0 0

7 da, beg day 3 3 8.9 1 7.8 0 0

7 da, beg day 4 3 8.1 1 7.1 0 0

7 da, beg day 5 4 5.2 1 4.5 0 0

7 da, beg day 6 4 3.6 2 3.5 0 0Do not cite

Section 7

- Page 108 of 677 Site/Year Sampling

Interval N umber

of Spikes Max

Detect Spike #

w/ Max
Conc Max 4

- da

average Days 4

-

da avg >20 ug/L Days 4

-

da avg >40 ug/L

7 da, beg day 7 5 9.6 1 8.4 0 0

Maumee

R iver

, 2008 Actual 9 52.2 4 49.7 6 2

7 da, beg day 1 2 49.9 2 44.8 12 3

7 da, beg day 2 2 19.2 2 17.6 0 0

7 d a, beg day 3 2 19.2 2 17.6 0 0

7 da, beg day 4 2 9.5 2 9.1 0 0

7 da, beg day 5 3 46.5 2 41.8 10 3

7 da, beg day 6 3 52.2 2 46.6 11 4

7 da, beg day 7 3 49.9 2 44.8 12 3

Sandusky

R iver

, 2004 Actual 6 24.7 4 18.2 0 0

7 da, beg day 1 3 18.6 1 17.5 0 0

7 da, beg day 2 3 18.2 1 16.8 0 0

7 da, beg day 3 3 15.5 1 14.2 0 0

7 da, beg day 4 3 11.9 1 10.9 0 0

7 da, beg day 5 2 10.1 2 9.6 0 0

7 da, beg day 6 2 24.7 2 22.2 4 0

7 da, beg day 7 2 18.5 2 16.8 0 0

Sandusky

R iver

, 2005 Actual 7 11.3 1 10.3 0 0

7 da, beg day 1 4 10.4 1 9.3 0 0

7 da, beg day 2 3 11.2 1 9.9 0 0

7 da, beg day 3 3 7.5 1 7.3 0 0

7 da, beg day 4 3 9.6 1 8.9 0 0

7 da, beg day 5 3 11.3 1 10.2 0 0

7 da, beg day 6 5 10.7 1 9.5 0 0

7 da, beg day 7 4 8.9 1 7.9 0 0

San dusky

R iver

, 2006 Actual 12 37.7 3 34.5 6 0

7 da, beg day 1 2 37.7 1 33.9 9 0

7 da, beg day 2 2 34.2 1 30.7 8 0

7 da, beg day 3 2 13.5 1 12.8 0 0

7 da, beg day 4 1 20.5 1 20.0 0 0

7 da, beg day 5 3 19.8 1 19.1 0 0

7 da, beg day 6 1 34.2 1 31.6 12 0

7 da, beg day 7 1 31.7 1 28.4 7 0

Sandusky

R iver

, 2007 Actual 5 17.5 2 14.5 0 0

7 da, beg day 1 2 14.5 2 13.0 0 0

7 da, beg day 2 2 9.5 2 8.9 0 0Do not cite

Section 7

- Page 109 of 677 Site/Year Sampling

Interval N umber

of Spikes Max

Detect Spike #

w/ Max
Conc Max 4

- da

average Days 4

-

da avg >20 ug/L Days 4

-

da avg >40 ug/L

7 da, beg day 3 2 12.5 2 11.4 0 0

7 da, beg day 4 2 8.4 2 8.1 0 0

7 da, beg day 5 2 5.4 1 5. 0 0 0

7 da, beg day 6 2 16.5 2 14.7 0 0

7 da, beg day 7 2 17.5 2 15.5 0 0

Sandusky

R iver

, 2008 Actual 8 41.2 4 38.8 6 0

7 da, beg day 1 3 36.6 2 32.9 8 0

7 da, beg day 2 3 36.6 2 32.8 8 0

7 da, beg day 3 3 40.9 2 37.9 14 0

7 da, beg day 4 3 41 .2 2 37.9 11 0

7 da, beg day 5 3 35.3 2 32.6 10 0

7 da, beg day 6 3 15.0 2 14.6 0 0

7 da, beg day 7 3 21.9 2 20.4 2 0

1 Actual = values generated based on the entire chemograph (daily monitoring). The next 7 rows represent 7

- day fixed interval samp ling beginning on the 1 st through 7 th day of the week.

While the Heidelberg NCWQR data are robust in time, they are limited in area. Syngenta‘s AEEMP , focusing on impacts to aquatic communities

, covers the same geographic area and time frame as the AMP for CWS (USEPA, 2007, 2009). The AEEMP monitored headwater streams (generally 2 nd to 5 th order) that typically have a smaller watershed than all but the smallest CWS. However, the AEEMP sites were sampled more frequently (4

- day fixed intervals) and a sub set of those sites included autosamplers that captured additional exposures between the grab samples. For sampling frequency analyses, the USEPA is only looking at the AEEMP sites that augmented grab samples with autosamplers.

For a preliminary scoping analysis, the USEPA evaluated the chemographs for MO

- 01, which generally had a few (2

- 5) spikes per year, and MO

- 02, which tended to have more spikes (7

- 14) per year. Additional sites with autosample

- augmented monitoring data can be found in NE, IL, IN, a nd OH. In addition, Syngenta has commenced a new phase of monitoring using autosamplers that will span from OH west to NE and KS and south to TX. These sites provide an additional geographic range to the pool of intensively sampled monitoring datasets av ailable for atrazine.

Figure 7

. 6 is representative of the shape of a number of CWS with 2 peaks in a year; the actual magnitude may differ; for now the focus is on the shape and number of peaks. This figure contrasts the extre mes in monitoring that can occur with a simple chemograph shape sampled at 7

- day intervals when the peaks are of relatively short duration. In contrast, Figure 7

. 7 shows that, while weekly samples may miss the actualDo not cite

Section 7

- Page 110 of 677 peaks in a ch emograph with a broad period of elevated exposures, the general chemograph shape is evident no matter which day the fixed sampling interval begins.Do not cite

Section 7

- Page 111 of 677 Figure 7

. 6 Actual chemograph (blue) and 2 resulting chem ographs (red) sampled at 7

- day fixed intervals

, MO

- 01, 2007 (Data Source: AEEMP)

(a) Actual Chemograph (red arrows point to spikes)Do not cite

Section 7

- Page 112 of 677 (b) Sampled at 7

- day fixed intervals, beginning on Day 4Do not cite

Section 7

- Page 113 of 677 (c) Sampled at 7

- day fixed intervals, beginning on Day 6

Figure 7

. 7 : Actual chemograph (blue) and 1 of the resulting chemographs (red) sampled at 7

- day fixe d intervals, MO

- 02 , 200 6 (Data Source: AEEMP)Do not cite

Section 7

- Page 114 of 677 (a) Actual Chemograph (red arrows point to spikes)Do not cite

Section 7

- Page 115 of 677 (b) Sampled at 7

- day fixed intervals, beginning on Day 1

Table 7

- 3 summarizes the preliminary analysis for 7

- day fixed sample intervals for the 2 MO sites with intensive sampling intervals from the AEEMP. The entire set of sampled chemographs for these sit es can be found in Appendix D .2. These analyses illustrate the importance of the chemograph shape, particularly duration of the peaks, in sampling frequency. Further analysis is planned with bootstrapping methods for variable dates within weekly sampling windows. These can be used to evaluate the potential variability in exposure estimates from different sampling designs compared with actual measured

concentrations for the entire set of data.

Table 7

- 3 : Anal ysis of Chemograph Peaks (Spikes), Maximum Detects, and

4

- Day Averages based on 7

- Day Fixed Sampling Intervals for 2 AEEMP Monitoring Sites

Site/Year Sampling

Interval Number

of Spikes Max

Detect Spike #

w/ Max

Conc Max 4

- da

average Days 4

-

da avg >20 ug/L Days 4

-

da avg >40 ug/L

MO

- 01,

2006 Actual 1 4 106.0 2 71.9 23 8

7 da, beg day 1 2 36.8 1 35.0 16 0

7 da, beg day 2 2 36.8 1 34.9 15 0

7 da, beg day 3 3 82.8 1 73.6 26 9Do not cite

Section 7

- Page 116 of 677 Site/Year Sampling

Interval Number

of Spikes Max

Detect Spike #

w/ Max
Conc Max 4

- da

average Days 4

-

da avg >20 ug/L Days 4

-

da avg >40 ug/L

7 da, beg day 4 2 82.8 1 75.6 26 14

7 da, beg day 5 2 43.1 1 40.1 19 1

7 d a, beg day 6 2 106.0 1 95.5 21 12

7 da, beg day 7 2 43.1 1 40.4 16 2

MO

- 01,

2007 Actual 2 91.6 2 59.7 13 (6,7) 4

7 da, beg day 1 2 42.0 2 37.3 11 (2,9) 0

7 da, beg day 2 2 33.3 2 29.6 9 (2,7) 0

7 da, beg day 3 2 26.2 2 23.3 6 (2,4) 0

7 da, beg d ay 4 2 29.7 1 28.3 10 0

7 da, beg day 5 2 23.0 1 21.5 4 0

7 da, beg day 6 2 91.6 2 80.4 15 (2,13) 9

7 da, beg day 7 2 71.8 2 62.9 14 (3,11) 7

MO

- 01,

2008 Actual 5 78.2 2 63. 0 8 4

7 da, beg day 1 1 70.1 1 61.3 11 7

7 da, beg day 2 1 78.2 1 68.3 11 7

7 da, beg day 3 1 51.7 1 44.6 8 4

7 da, beg day 4 2 51.7 1 44.8 10 4

7 da, beg day 5 2 51.7 1 44.8 10 4

7 da, beg day 6 2 12.5 1 11.4 0 0

7 da, beg day 7 1 8.1 1 7.3 0 0

MO

- 01,

2009 Actual 3 71.0 2 64.7 9 7

7 da, beg day 1 2 62.4 2 54.8 1 1 6

7 da, beg day 2 2 57.8 2 53.4 16 8

7 da, beg day 3 2 34.9 2 33.0 13 0

7 da, beg day 4 2 71.0 2 63.3 14 8

7 da, beg day 5 2 63.1 2 55.6 11 6

7 da, beg day 6 2 63.2 2 55.7 11 6

7 da, beg day 7 2 61.6 2 54.0 11 5

MO

- 02,

2006 Actual 7 43.9 1 4 3.2 25 3

7 da, beg day 1 2 32.7 1 31.6 20 0

7 da, beg day 2 2 34.9 1 33.3 21 0

7 da, beg day 3 2 43.2 1 41.6 28 3

7 da, beg day 4 2 39.5 1 36.9 29 0

7 da, beg day 5 2 43.9 1 40.5 28 2

7 da, beg day 6 2 26.6 1 26.3 18 0

7 da, beg day 7 2 37.3 1 35.0 20 0

MO

- 02,

2007 Actual 11 16.2 1 10.8 0 0

7 da, beg day 1 4 7.0 1 6.9 0 0Do not cite

Section 7

- Page 117 of 677 Site/Year Sampling

Interval Number

of Spikes Max

Detect Spike #

w/ Max
Conc Max 4

- da

average Days 4

-

da avg >20 ug/L Days 4

-

da avg >40 ug/L

7 da, beg day 2 4 10.9 3 9.9 0 0

7 da, beg day 3 3 9.2 1 8.6 0 0

7 da, beg day 4 3 16.2 1 14.4 0 0

7 da, beg day 5 4 10.9 1 9.6 0 0

7 da, beg day 6 3 8.2 1 8.0 0 0

7 da, beg day 7 3 9.1 1 8.5 0 0

MO

- 02,

2008 Actual 9 56.6 2 35.5 7 (4,3) 0

7 da, beg day 1 4 56.6 1 49.1 10 4

7 da, beg day 2 2 38.2 2 33.5 11 (4,7) 0

7 da, beg day 3 4 21.6 2 19.0 0 0

7 da, beg day 4 3 33.6 2 28.8 6 0

7 da, beg day 5 3 47. 9 3 41.2 8 2

7 da, beg day 6 2 13.5 1 13.0 0 0

7 da, beg day 7 3 36.4 1 31.5 6 0

MO

- 02,

2009 Actual 14 155.2 11 68.9 21 (16,5) 8 (4,4)

7 da, beg day 1 4 108.9 2 96.2 27 (15,12) 19 (11,8)

7 da, beg day 2 4 155.2 3 133.5 18 (6,12) 10

7 da, beg day 3 5 66.0 2 58.2 20 (12,8) 7

7 da, beg day 4 3 49.0 2 43.6 11 3

7 da, beg day 5 3 45.4 2 42.0 14 3

7 da, beg day 6 2 48.6 2 44.5 13 4

7 da, beg day 7 3 17.8 2 17.2 0 0

1 Actual = values generated based on the entire chemograph (daily monitoring). The next 7 rows represent 7

- day fixed interval sampling beginning on the 1 st through 7 th day of the week.

While the Heidelberg NCWQR and AEEMP datasets provide a pool of intensively

sampled monitoring data for atrazine concentrations in flowing water bo dies, a similar set of intensive monitoring does not exist for reservoirs, lakes, and other static water bodies. The USGS sampled 12 CWS reservoirs in 1999

- 2000, collecting source

- and

treated

- water samples at weekly or bi

- weekly intervals following predo minant pesticide application periods and at quarterly intervals during the remainder of the year (Blomquist et al. , 2001). This is similar in intensity to the AMP monitoring. While we would expect smaller temporal variability in peak pesticide concentrat ions in reservoirs which integrate pesticide loading from their watersheds and have a longer residence time (slower flow

- through), we do not have monitoring data more intensive than weekly samples to evaluate day

- to

- day variability in pesticide concentrati ons in these water

bodies.Do not cite

Section 7

- Page 118 of 677 One approach for evaluating temporal variability in reservoirs is to assume that pesticide concentrations in reservoirs will be less variable in time than in flowing waters and let the uncertainty assessment for river and strea ms apply to reservoirs and lakes as well. A second approach would be to use a deterministic model, such as the linked PRZM and EXAMS models used by the USEPA in its risk assessments, to generate artificial chemographs of daily concentrations. The USEPA c urrently uses the model with a standard set of scenarios representing vulnerable runoff conditions for a variety of crops at localized areas and with meteorological data measured over a 30

- year period for the specific scenario location. For corn and sorgh um use, standard scenarios exist for corn in IL, OH, MS, and PA and for sorghum in KS. Additional corn scenarios have been developed for IA, IN, KS, MN, and NE.

Using atrazine fate and transport properties and varying application rates and timing

acros s the likely application window, the USEPA can generate a potential distribution of 30 years of daily chemographs based on weather conditions. Figure 7

. 8 illustrates a single chemograph year out of a 30

- year simulation for atrazi ne applied at 4 different times across the historic application window using the OH corn scenario. The number of peaks varied each year in the simulations depending on the magnitude and timing of rainfall events in relation to the application in a given ye ar. The concave decline curve after the last peak, characteristic of all of the years of simulation, is likely an artifact of using average monthly flow in the reservoir simulation. While flow in reservoirs is expected to be less variable than flow in ri vers and streams, more frequent flow parameters will be needed for the reservoir simulations.Do not cite

Section 7

- Page 119 of 677 Figure 7

. 8 : A single year of daily atrazine concentrations simulated for an index reservoir in OH using PRZM an d EXAMS.

PRZM and EXAMS can also be used to simulate daily pesticide concentrations at monitored sites using site

- specific application, soil, hydrology, and weather information. Such simulations could be used to estimate exposures between monitoring samp les. In this case, e stimated exposures generated by PRZM and EXAMS could be calibrated to the measured concentrations and provide a complete time series between sampling

dates. As an example, Syngenta use d PRZM with site

- specific soil, cropping, and

weath er data to estimate atrazine concentrations between 4

- day monitoring samples in the AEEMP (see USEPA

, 2007 , for more information ). Such an approach may identify

short

- duration peaks that occurred as a result of runoff events between sampling

intervals.

7.4 Analyzing and Interpreting Monitoring Data Collected at Different Sampling Frequencies

At the April 2010 SAP, the USEPA noted a need to be able to analyze existing monitoring data collected at different sample intervals to determine its reliability in estimating drinking water exposures in relation to a particular toxicity endpoint and duration of concern. For example, the existing AMP study with weekly sampling intervals during the period around and following atrazine applications provides a high

d egree of reliability for a toxicity endpoint of concern with a 90

- day duration but is muchDo not cite

Section 7

- Page 120 of 677 less certain if the duration of concern is for a much shorter period (e.g. 4 days ) . The USEPA presented a number of potential approaches analyzing monitoring data i n context to the endpoint and duration of concern and solicited additional

recommendations from the SAP.

In its comments, the April 2010 SAP emphasized the importance of defining the exposure magnitude and timeframe of interest in order to determine both the appropriate sampling design for monitoring studies and the appropriate methods/models for estimating exposures from existing monitoring studies. While the exposure level and duration of toxicological concern has not been finalized, this section revie ws the SAP recommendations and identifies likely future directions.

7 .4.1 Filling in Values Between Samples With Linear or Stair

- step Interpolation

The April 2010 SAP noted that, because neither stair

- step imputation nor linear interpolation give predi cted values greater than those sampled, they have the potential to underestimate the maximum if residues occurred on a day that was not sampled. This underestimation could be substantial. Furthermore, if the average of interest is of short duration (e.g. , 4 days), a [missed] maximum value will have a more significant effect on that average than on a longer term average (i.e., 90 days).

Both methods, on the other hand, will tend to overestimate exposure during a  ̳concave‘

decline in the curve. While l inear interpolation may overestimate these declining values by a smaller amount, the potential for missing the maximum value is most important, especially for shorter exposure durations. For longer durations, this potential for missing maximum values may be balanced to some unknown extent by the overestimation of those values that follow, although this is not regarded as a compensating occurrence.

While these commonly used interpolation methods are likely to underestimate short

duration exposures, espec ially when the sampling interval is similar to or broader than the toxicological duration of concern, they may be useful for estimating exposures for longer

- term durations of concern when multiple samples are taken within the duration of

concern. Even in these instances, the estimations should include a characterization of the level of uncertainty resulting from the sampling intervals.

7 .4.2 Artificial Neural Network

The Panel noted that the artificial neural network (ANN) approach can predict a va lue that is larger than those observed in the data. The SAP emphasized the importance of finding the right number of nodes for getting good estimates, stressing that too many or too few will result in a curve that is not a good fit, with the potential to miss the shape of the peak and the maximum.Do not cite

Section 7

- Page 121 of 6 77 The Panel commented that the USEPA‘s use of autoregressive errors in the modeling may be too complicated for general practical use and recommended eliminating it, if possible. The Agency is considering the co ntinued pursuit of this approach, along with others mentioned below, but has not done further development at this point.

7 .4.3 Kriging

In the April 2010 background paper, the USEPA noted that kriging and random function model approaches may be usefu l in evaluating monitoring data because their assumptions of co

- linearity can be applied to the temporal autocorrelation inherent to pesticide concentration time series in surface waters (USEPA, 2010). The SAP noted that kriging methods can produce smooth curves that can estimate concentrations greater than the actual sampled points and may better approximate a missed maximum

value. A stochastic simulation process, generating multiple realizations of temporal chemographs, would be necessary to provide estim ated concentrations greater than those measured (see Deutsch and Journel, 1998). The Panel cautioned that standard stationary models may not be adequate to characterize the curves and mentioned the possibility of analyses involving non

- stationary models th at account for changing correlation. Because these are very complicated to implement on a system

- by

- system basis, the SAP recommended estimating correlation structures across a number of pooled (and variable) systems. The USEPA is further considering thi s approach in combination with regression modeling, as recommended by the SAP and discussed

below . An additional option, which may also tie in with the efforts discussed in the longer

- term modeling approaches is to consider co

- kriging measured concentrati ons with rainfall events or with daily streamflow measurements.

7 .4.4 Extreme Value Theory

Several members of the April Panel suggested exploring extreme value theory in modeling and analyzing monitoring data. This has been used to model extreme and high end events in climatology and hydrology over the years, especially in data rich environments where the record consists of daily measurements for multiple decades.

Huang and Batterman (2003) used a deterministic deposition

- leaching model to predict daily concentrations of soluble pollutants in soil with 50 years of precipitation data. A stochastic model was then used to simulate 1,000 years of data for analysis of extreme concentrations. This method may have parallel applications for the current a trazine monitoring data as supplemented with outputs from other deterministic models employed by the Agency, such as PRZM/EXAMS.

In using these methods, the existence of seasonality and serial correlation of values would greatly affect the outcomes. Wh ile most of the literature addresses methods for use with independent samples, the Panel cited a recent paper that deals with modelingDo not cite

Section 7

- Page 122 of 677 extreme values from a correlated process (Padoan, et al . , 2010). The Agency will be investigating the recent literature to discover any new work that might have applications

in this area.

7 .4.5 Longer

- T erm M odeling A pproaches Combining Random Function and Regression Models

In April 2010 the USEPA sought feedback from the Panel on several different longer

term modelin g approaches that have been used by various researchers for assessing pesticide concentrations, including the use of random function models and regression

models. The Panel confirmed their usefulness, noting the utility of known shape patterns and covaria tes such as weather and use patterns when the approaches are combined. This was done in several papers they cited in the recent literature.

The USGS has developed regression and time series models for predicting pesticide concentrations from existing v ariables that may be available on a regional or local level

. Vecchia and Crawford (2006) developed a time series model to simulate daily pesticide concentrations that reproduced the distributions, seasonal variability, and serial

correlation in data for h erbicides and insecticides from 112 monitoring stations in the National Water Quality Assessment (NAWQA) and National Stream Quality Accounting Network (NASQAN) programs. Monthly precipitation and temperature data for the watersheds were used to reproduce seasonal patterns and generate the correlated time series of daily pesticide concentrations. Repeated realizations of the daily time series were generated for each pesticide

- station for the ten years of data, and percentile

values were examined. The aut hors noted , however, that the model was designed ―to simulate daily pesticide concentrations for which the long

- term traces could be used to assess long

- term pesticide

- exposure risks

, ‖ rather than for the purpose of determining specific maximum values on s pecific days.

In more recent work, Vecchia et al . (2008 and 2009 ) examined seasonal variability

, trend, and introduced a stream flow term to develop a SEAWAVE

- Q model that appeared to be robust for approximately monthly sampling frequencies and for diff erent amounts of censoring in the data. Further work would be necessary to develop time series for USEPA pesticide assessments, but in personal communications with Agency staff, the authors were optimistic that a useful application could be developed for

a trazine assessments using existing monitoring data.

7 .4.6 Directions Forward

The April Panel emphasized the utility of regression, random function, and deterministic models for aiding in the assessment process. The literature demonstrates examples

wh ere the above types of models have been used to augment less frequent sampling and develop daily time series for assessment purposes. Thus, there is potential for
models like the SEAWAVE

- Q and PRZM/EXAMS to be extended for use with theDo not cite

Section 7

- Page 123 of 677 current chemographs to create longer

- term ―traces‖ from which probability statements may be made with regard to exceeding levels of concern.

As both the Agency and the Panel ha ve noted , however, specific approaches forward do depend on the level and the duration of concern that will be determined. Future approaches that employ more frequent sampling may become more important for levels of concern where durations are relatively short term. For these, targeted and supplemental monitoring methods may be more important than t he different modeling methods in carrying out the assessment. The Panel noted that ―for public water systems for which short duration LOCs are likely to be exceeded, and thus, where reliability requirements are high, there probably will be no substitute f or intensive monitoring.‖ The WARP model and knowledge from current chemographs can be used to determine CWS/areas with potentially high concentrations, where more frequent sampling would lead to improved accuracy and precision in estimates of shorter ter m concentrations. For longer term durations the current data along with modeling efforts may be sufficient. In the present paper the Agency has discussed a number of approaches that can be used in a flexible strategy once levels of concern are set.Do not cite

Section 8

- Page 124 of 677 8. IMPLICATIONS OF MOA & TOXICITY PROFILE O N WATER MONITORING

As described in the Introduction to this document (Section 1), the goals of the

current atrazine re

- evaluation are 1) to determine the extent to which new science indicates a need for the Agency t o 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. Two important issues related to achieving these goals are determining the point of departure and identifying the critical associated duration of exposure. Using this information, EPA will determine an appropriate water monitoring frequency. The proposed point of departure, discussed at length in Section 5 , is based on attenuation of the LH surge in female rats, a key event in the neuroendocrine MOA for atrazine. The purpose of this section is to describe the available science for determining the duration of exposure necessary to produce such an effect interest. This section provides an integration of information on MOA, PK, human relevance, and species extrapolation in context with drinking water exposure and monitoring frequency.

Observation epidemio logy studies reviewed in Section 3 suggest possible links between atrazine exposure and reproductive and developmental outcomes but these epidemiology studies suffer from limitations which prevent strong conclusions. Although some of these studies do provide qualitative support the human relevance of the endpoints identified through the experimental toxicological database, they provide little to no information on the critical duration of exposure. In addition, the MOA and PK database are also lacking in human specific information on the effects of atrazine which could be used to quantitatively extrapolate between species. As such, the information available to evaluate the critical duration of exposure is lacks quantitative pr ecision but is instead derived by inferring knowledge from a variety of scientific disciplines.

8.1 Toxicological & MOA Considerations

8.1.1 LH as the Critical Endpoint for Risk Assessment

In the 2003 human health risk assessment for atrazine, the PoD for the intermediate and chronic exposure risk assessments was based on the attenuation of the LH surge reported by Morseth et al. (1996 b ) at doses ≥ 3.65 mg/kg/day (NOAEL/LOAEL = 1.8/3.65 mg/kg/day). Based on the current review, LH attenuation

c ontinues to be the most sensitive endpoint identified to date in the database. The Agency is proposing a BMDL of 1.86 mg/kg/day based on a 4

- day study involving repeated doses once per day from Cooper et al (2010).Do not cite

Section 8

- Page 125 of 677 LH plays a critical role in a variety of reproductive and developmental processes in rats and humans (Goldman et al . ̧2000 ; St oker et al., 2000; Grumbach, 2002; Ebling, 2005). Multiple lines of evidence demonstrate that decreases in circulating LH levels lead to disruptions in estrous cyclicit y in rats ( Cooper et al., 1996; Morseth et al., 1996 a&b, Cooper et al., 2000; Laws et al. 2000; Shibayama, et al., 2009). Furthermore, in males, LH stimulates testosterone secretion from the Leydig cells in the
testes. Since LH levels are decreased as a r esult of atrazine exposure, the decreased testosterone synthesis reported in several studies (Friedmann, 2002; Stoker, et al

. , 2000 and 2001; Trentacoste, 2001; Progrimic 2009) can be linked to decreases in LH, albeit these findings were induced at high ex perimental doses (50

- 200 mg/kg). LH attenuation is implicated in delays in sexual maturation

. In rats, atrazine clearly causes a delay in the onset of puberty in both males and females. The role of androgens, in general, and testosterone, in particular , in preputial separation is well understood (Marshall, 1966 and Lyons et al., 1942). A decrease in testosterone

– such as would be caused by decreased circulating LH

– during the peripubertal period can lead to delays in preputial separation as well as reduced weights of androgen

- dependent tissues. With respect to pubertal development in females, LH levels modulate the synthesis of estradiol in the ovaries. As estradiol concentration steadily increases during the peripubertal period, there is positive f eedback on the mechanisms controlling the pulsatile release of GnRH. It is this mechanism that allows for the generation of the LH surge necessary for ovulation. Again, the fact that atrazine impairs LH release was implicated in the delay in puberty obser ved in the female rat (Laws et al ., 2000).

Another effect attributed to atrazine exposure is the increased incidence of prostatitis in the male offspring of female rats treated during the peri

- natal period. This

effect is associated with a decrease i n the suckling

- induced release of prolactin. Prolactin plays a crucial role in neonatal brain development particularly in the tuberoinfundibular dopaminergic system (TIDA) in rats (Shyr, et al., 1986; Stoker et al

., 1999). These neurons are responsible f or the down

- regulation of prolactin release from the pituitary. As a consequence of the decreased levels of maternal prolactin during the early lactational period in rats, the offspring of these dams do not receive the perinatal

levels of prolactin necessa ry to activate the TIDA neurons. Therefore, TIDA‘s regulation of prolactin in the adult male rat is impaired resulting in elevated prolactin levels which in turn lead to hyperprolactinemia. It is this elevated level of circulating prolactin in the

adult m ales that 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 activation (first postnatal week) may be attributed to

de creases in circulating prolactin levels in the dam. Although a direct interaction between the mechanisms regulating the sequence of events leading to prostatitis and

LH attenuation has not been described LH attenuation occurs at doses lower than those

res ulting in prostatitis. Therefore, the Agency determined that regulating for decreases in LH would also prevent the decreases in prolactin leading to prostatitis. To date, there are no data available to address the potential impact of atrazine exposure on theDo not cite

Section 8

- Page 126 of 677 potential of humans to develop prostatitis. Nonetheless, the finding of prostatitis in rats is consistent with a primary action of atrazine on the HPG axis.

The Agency has concluded that a potential exists in humans for reproductive and develop mental disruption to occur as a consequence of HPG disturbance , and selection of LH attenuation is a sensitive and critical effect for establishing a point of departure that would be protective of potential health outcomes described here (e.g., estrous

cyc licity, testosterone decreases, puberty onset in both sexes, and prostatitis) since decreases in LH occur at doses lower than those eliciting these outcomes. The dose

response relationships of these health outcomes are described more fully in earlier

sect ions of this paper . Moreover, LH attenuation is a key event in disrupting estrous cyclicity and sexual maturation.

The Agency performs risk assessments on a variety of durations of exposure ranging from a single day exposure to chronic or lifetime expo sures. In the most recent risk assessment, LH attenuation was used for extrapolating risk from durations of one month and longer, typically called intermediate

- term (1

- 6 months) and chronic (6 months or longer). Given that pseudo

- steady state plasma leve ls of triazine equivalents are reached at approximately four days of once

- per

- day exposures to atrazine and such condition appears to be strongly associated with the LH attenuating effect to occur (Section 5 ), exposure periods of shorter duration than one month may be appropriate for use in risk assessments. However, extrapolating the once

- per

- day for four day exposure effect measured in the rat to humans is a difficult issue. The science available

to inform the rat to human extrapolation with respect to plausible durations of interest is

discussed below.

8.1.2 Pharmacokinetic Considerations

Characterization of the pharmacokinetics of atrazine is critical to understand

the time

- dose response relationship associated with attenuation of the pre

- ovulatory LH

surge. The observation that plasma levels of total triazine equivalents in the rat achieve (or nearly achieve) pseudo steady state upon repeated once per day daily dosing for 4 days combined with a remarkably simil ar LH

- surge attenuating effect across different rat studies of different durations that likely achieve such state of plasma levels suggest that a daily steady state AUC for total triazine equivalents may be an appropriate internal

dose metric to refine the time

- dose response relationship (discussed in Section 5 ). Given that the neuroendocrine MOA for atrazine involves the pulsatile release of GnRH from the brain, it would be preferred to have tissue dosimetry in the brain. Howe ver, use of plasma as a surrogate for brain is supportable based on the results of Stoker et al (2010) and Kamel et al (2010) who have shown that in pregnant and lactating dams that brain and plasma concentrations of atrazine and DACT are very similar. D ata on
the time course of atrazine and/or its metabolites in the brain are not available at thisDo not cite

Section 8

- Page 127 of 677 time. However, at steady state plasma levels , brain and plasma will be at equilibr ium making plasma levels a good surrogate for brain levels

.

The extrapola tion of these rat findings to humans, however, is not a simple task. The preferred approach for interspecies extrapolation involves a properly calibrated and evaluated PBPK model that can be used in a ―reverse dosimetry‖ approach to estimate a human equiva lent atrazine exposure that would likely result in an equivalent AUC as a rat point of departure. Ideally, the PBPK model would also be amenable to addressing variations in daily dose and inherent interindividual variability so that a distribution, not jus t point estimates, of AUC outputs can be predicted for a population of interest. The best PBPK effort to date is a published rat composite model for atrazine and its metabolites by McMullin et al (2007). The McMullin et al (2007) model has several

shortco mings including the inability to reasonably simulate rat plasma levels of atrazine and its chlorinated metabolites in the rat following oral dosing with atrazine (See reviews in Appendix A). Because there is currently no robust PBPK model available, the d aily steady state AUC estimates described in this issue paper relied entirely on a noncompartmental analysis approach. It should be noted that such approach contrasts with the use of a PBPK model in that it is empirical in nature and by definition cannot

e xceed the limits of the available data.

Some inference on human relevance may still be derived from the results of the noncompartmental analysis of the rat plasma data. When the plasma elimination phase of each dose group in the Thede (1987) study wa s analyzed for an elimination rate constant (kel) assuming linear behavior (i.e., plotting the natural log of plasma levels versus time), a very comparable range of values of 0.010

– 0.015 hr - 1 was obtained for the dose range of 1

- 10 mg/kg/day (See Table 7.1 below). The high dose groups of 50 and 100 mg/kg/day were omitted here since they exhibit a much higher elimination rate, and humans are also less likely to encounter such h igh atrazine

exposures . As a rough approximation to gain insight into human re levance, the elimination rate constants were allometrically scaled for an average human female BW of 60 kg. It should be noted that BW 3/4 allometric scaling as a means to derive toxicologically equivalent doses across species is an accepted risk assessment practice in the absence of the preferred approach involving a PBPK model (Rhomberg and Lewandowski, 2006; USEPA, 2006 ) . When the corresponding BW - 1/4 allometrically

scaled elimination rate constants were converted to half

- lives, again assuming linear behavior (i.e., t 1/2 = ln2/kel), a range of 5.48

– 8.42 days was obtained. Assuming that it would take between 3 and 5 plasma half

- lives to reach steady state, a time range of

16.4

– 30.7 days was estimated as the time it would take to reach steady state plasma levels of triazine equivalents in a typical human female of BW of 60 kg. It should be noted that given the surrounding uncertainties, these are rough estimates but that in the absence of any other information prov ide valuable perspective on the extrapolation of rat findings to humans. For examp le, all the estimates in Table 8 .1 are between 2.5 and

4 weeks and are thus all shorter than the 90

- day rolling average used in the previous

risk assessment. These allometr ically scaled human equivalent time estimates areDo not cite

Section 8

- Page 128 of 677 based on rat studies involving once per day repeated oral gavage dosing with the same dose of atrazine for 4 days. It should be noted as an additional uncertainty that human consumption of drinking water, t he primary mode of human exposure to atrazine, is unlikely to be once per day, but spread in intervals throughout the day. The concentration of atrazine and its metabolites in water is also unlikely to be constant from day to day. The implications of these differences in daily dose and input rate on achieving steady state are unknown.

Table 8

- 1 : Estimates of human half

- lives for triazine equivalents and the predicted time to reach steady state

Allometric scaling can also be used to estimate a human equivalent dose (HED) based on the rat BMDL of 1.86 mg/kg/day. In US EPA‘s allometric scaling document, a dosimetric adjustment factor (DAF) based on the ratio of animal to human body weight raised to the 1⁄4 powe r is recommended (USEPA, 2006) . Using an average rat BW of 0.250 kg and an average human female BW of 60 kg results in a DAF of 0.254. Application of this DAF to the rat BMDL of 1.86 mg/kg/day results in a HED estimate of 0.473 mg/kg/day. Again, these calculations are considered rough estimates given the absence of more appropriate information.

8.1.3 Pharmacodynamic Considerations

As noted above, little is known about the direct effects of atrazine in humans and available epidemiology studies are not sufficiently robust for making strong conclusions. However, one can infer information about possib le windows of susceptibility from what is known about human physiology and from the pharmaceutical literature. In considering the relevance and temporal concordance of a toxic effect in a test species to human health, a variety of factors need to be consi dered. Among them is the potential window of susceptibility to the toxic effect. For atrazine, the experimental data available indicate that, in rats, a 4

- day exposure at doses as low as 3.12 mg/kg/day (BMDL = 1.86 mg/kg/day) is sufficient to attenuate t he LH surge

– the most sensitive endpoint in the atrazine risk assessment. Since the estrous cycle in rats is 4 days long, this window of susceptibility is not unexpected. However, in humans, the menstrual cycle lasts

– on average

– 28 days. Thus, the q uestion arises as to whether a brief exposure ( e.g., a few days) in humans could lead to an attenuation of the LH surge.Do not cite

Section 8

- Page 129 of 677 Evidence of chemically

- induced decreases in GnRH or LH secretion is sparse in

humans and non

- human primates relative to rodents. Th e available evidence in humans comes primarily from the pharmaceutical arena. Nal

- Glu, Cetrorelix®, and Ganirelix are three GnRH antagonists used to block the LH surge and ovulation in women prior to in vitro fertilization (IVF) procedures. In a series o f experiments, regularly ovulating women received two 5 mg injections of Nal

- Glu on days 8 and 11 of the follicular phase of the natural cycle (Frydman et al. 1992). This treatment resulted in a block of the spontaneous LH surge. This work was further cor roborated by Olivennes

et al. who demonstrated that a single 3 mg administration of the GnRH antagonist Cetrorelix® on day 8 of the follicular phase was sufficient to block the LH surge. Ganirelix exposure during the late follicular phase of the menstrual cycle has also been demonstrated to inhibit the LH surge and ovulation by competing with the endogenous GnRH for receptor binding (Fauser et al., 2002). One must consider these studies with caution with respect to how they relate to atrazine human healt h risk since the potency and pharmacokinetics of these pharmaceuticals relative to atrazine is unknown. Moreover, data in rats have shown that one dose of atrazine (up to 200 mg/kg administered in the morning of the expected LH surge) is not sufficient to block the LH surge (Cooper et al., 2000). As such, given the current database of atrazine studies, the Agency does not believe that one or two exposures of atrazine is sufficient to block the LH surge in humans. However, these studies do help qualitativ ely inform a potential window of vulnerability to chemicals disrupting the HPG axis in women. Specifically, all of these pharmaceutical agents are administered during the late follicular phase of the menstrual cycle (days 8

- 12 of the follicular phase) 10

. Thus, one can infer that the follicular phase (lasting ≈ 12 days) and possibly the late follicular phase (days 8

- 12 of the follicular phase) of the menstrual cycle may be a possible window of susceptibility in

humans.

With regard to the other reproductiv e/developmental effects elicited by atrazine exposure ( e.g., puberty onset delays and prostatitis), the complexity and inherent variability in the human population for these developmental processes makes it difficult to identify a critical window of vulner ability. Notwithstanding this complexity, in humans it is unlikely that a single exposure would affect these processes given that he emergence of the LH surge occurs over several years.

8.2 Drinking Water Considerations & the Monitoring Sampling Inter val

When assessing exposure via the drinking water pathway, the USEPA may use either models or monitoring data to predict potential levels of a chemical throughout the year under various scenarios. When monitoring data are used, the Agency derives an

ave rage for the duration period of concern. In the case of atrazine and the 90

- day duration of concern, the Agency, computed consecutive [―rolling‖] 90

- day averages throughout the year by interpolating between the weekly monitoring points. This

10 In humans, the follicular phase lasts approximately 12 days, assuming a 28

- day menstrual cycleDo not cite

Section 8

- Page 130 of 677 distribution of averages was then compared against the level of concern for the 90

- day period to see if computed rolling average values exceeded the LOC and, if so, how

often.

The method used to estimate concentrations from monitoring data depends on

the duration o f concern and how critical it is to estimate peak concentrations. For the

90

- day duration of toxicological concern identified in the IRED, the effect of missing peak concentrations was less of a concern than for a shorter duration and the weekly sampling f requency provided adequate exposure estimates with a given level of confidence (see USEPA, 2010, Crawford, 2004, and ILSI, 1999, for analyses of the level of confidence of different sampling intervals).

The current scientific re

- evaluation of the health e ffects of atrazine suggests a shorter critical duration of exposure than used in the previous risk assessment. The

outcome of this re

- evaluation will determine the extent to which the current monitoring study can be used in estimating exposures and what me thods may be necessary to augment the monitoring data. When the duration of concern is shorter than the sampling interval, then either modeling methods must be used to estimate concentrations between sampling points or more frequent sampling may be needed. Simple interpolations that don‘t account for the potential of concentrations greater than what was measured can lose a large amount of information about the levels and variability of the underlying distribution of concentrations. In these instances, mor e complex estimation methods that allow for the possibility of predicting concentrations greater than what was measured in the monitoring study or other means of bounding the level of confidence in the exposure estimates would be needed.

8 .3 Summar y

The Agency has used MOA as the foundation for considering PK and PD characteristics of atrazine and other HPG active chemicals in the context of determining the critical duration of interest. Evidence of the sequalae of the key events identified

withi n the mode of action, i.e., LH surge attenuation, is suggested in the observational epidemiology literature. However, the epidemiology data are not sufficiently robust to draw firm conclusions. Given the current state of the science, there is a lack of

pr ecision in establishing the duration of interest. As such, the Agency has considered biologically plausible ranges. In summary, according to the Agency‘s analysis of the pharmaceutical data and allometric scaling of the rodent pharmacokinetics data the

p otential durations of interest lie between a few days of exposure to approximately 4 weeks of exposure. Given the importance of this issue in determining monitoring frequency, the Agency will solicit comment from the SAP on this analysis and alternative

o r additional scientific factors to consider in establishing the critical duration of interest.Do not cite

Section 9

- Page 131 of 677 9. NEXT STEPS

The Agency will be soliciting comment from the FIFRA SAP on many important and challenging scientific issues at the September meeting. Following that meeting, the Agency expects to be in a position to begin the analysis phase

— namely quantitatively evaluating the monitoring frequency for atrazine. Moreover, the Agency is planning to hold another meeting of the SAP in spring 2011 to discuss issues related to cancer epidemiology findings for atrazine.Do not cite

Section 10

- Page 132 of 677 10. REFERENCES

Post 2003 References

Albanito, L., Lappano, R., Madeo, A., Chimento, A., Prossnitz, E.R., Cappello, A.R., Dolce, V., Abonante, S., Pezzi, V., & Maggiolini, M. (2008). G

- protein

- cou pled receptor 30 and estrogen receptor

- alpha are involved in the proliferative effects induced by atrazine in ovarian cancer cells. Environ Health Perspect, 116(12): 1648

- 55.

Albanito, L., Madeo, A., Lappano, R., Vivacqua, A., Rago, V., Carpino, A., Opre a, 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; 6 7(4): 1859

- 66.

Bakke B., De Roos A.J., Barr D.B., Stewart P.A., Blair 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 Epidemiol., Sep; 19(6): 544

- 54.

Barr DB, Panuwet P, Nguyen JV, Udunka S, Needham LL. Assessing exposure to atrazine and its metabolites using biomonitoring. Environ Health Perspect. 2007 Oct;115(10):1474

- 8. PubMed PMID: 17938738; PubMe d Central PMCID: PMC2022667.

Belloni V, Alleva E, et al . (2007). Effects of low doses of atrazine on the neurobehavioural development of mice. Ethology Ecology & Evolution, 19: 309

- 22.

Benachour, N., Moslemi, S., Sipahutar, H., & Seralini G.E. (2007 ). Cytotoxic effects and aromatase inhibition by xenobiotic endocrine disrupters alone and in combination, Toxicol Appl Pharmacol, Jul 15; 222(2): 129

- 40.

Betancourt, M., Resendiz, A., & Fierro, E.C. (2006). Effects of two insecticides and two

herbicide s on the porcine sperm motility patterns using computer

- assisted semen analysis (CASA) in vitro

. Reproductive Toxicology, Oct, 22(3): 508

- 12.

Blair A, Tarone R, Sandler D, Lynch CF, Rowland A, Wintersteen W, Steen WC, Samanic C, Dosemeci M, Alavanja MC. (2002) Reliability of reporting on life

- style and agricultural factors by a sample of participants in the Agricultural Health Study from

Iowa. Epidemiology., Jan;13(1):94

- 9. PubMed PMID: 11805592.Do not cite

Section 10

- Page 133 of 677 Boobis, A.R., J.E. Doe, B. Heinrich

- Hirsch et al . 2008. IPCS framework for analyzing

the relevance of a noncancer mode of action for humans. Crit Rev Toxicol, 38(2):87

- 96.

Breckenridge, C.B., Yi, K.D., & Simpkins, J.W. (2010a). Cytotoxicity of atrazine and its

metabolites in vitro . Unpublished data from Sy ngenta Crop Protection, Inc.

Breckenridge, C.B., Yi, K.D., Perez, E.J., & Simpkins, J.W. (2010b). Dose and time dependent effects of atrazine and metabolites on aromatase mRNA in multiple cell types in vitro . Unpublished data from Syngenta

Breckenrid ge, 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. Unpublished data from Syngenta

Canfield MA, Ramadhani TA, Langlois PH, Waller DK. Residential mob ility patterns and exposure misclassification in epidemiologic studies of birth defects. J Expo Sci Environ Epidemiol. 2006 Nov;16(6):538

- 43. Epub 2006 May 31.

PubMed PMID: 16736057.

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 to arsenate and atrazine in drinking water modulates gene expression and clonogenicity of myeloid progenitors. Toxicol Lett, Jul 30; 180(1): 59

- 66.

Coban, A ., & Filipov, N.M. (2007). Dopaminergic toxicity associated with oral exposure to the herbicide atrazine in juvenile male C57BL/6 mice. J. Neurochem, Mar, 100(5):

1177

- 87.

Coder, P. (2010 ). Atrazine: An Oral Developmental Toxicity Study of Atrazine in t he Rat, Including Cross

- Fostering and Pair

- Feeding. Final Report. January 28, 2010. Project Number: WIL/639025, T007794/08, 140/107. Unpublished study prepared by WIL Research Laboratories, Inc. 2827 p. MRID 47972801.

Coder, P. (2010 ). Atrazine: An Oral ( Gavage) Study to Assess the Potential for Detecting the Estrogen

- Induced Luteinizing Hormone Surge in Female Rats. Final Report. Unpublished data from Syngenta Crop Protection, Inc. Report Number WIL

639062. Study Completion Date July 2, 2010.

Coder, P. ( 2010 ). Atrazine: An Oral (Gavage) Immunotoxicity and Hormone Evaluation Study for the Effects of Atrazine on Male Rats. Final Report. Unpublished data from Syngenta Crop Protection, Inc. Report Number WIL

- 639072. Study Completion Date July 13, 2010.Do not cite

Section 10

- Page 134 of 677 Coder , P. (2010 ). Atrazine: An Oral (Gavage) Study of the Effects of Atrazine on the Estrous Cycle and the Estrogen

- Induced Luteinizing Hormone Surge in Female Rats. Final Interim Report. Unpublished data from Syngenta Crop Protection, Inc. Report

Number WIL

- 63 9062. Study Completion Date July 14, 2010.

Cooper, R, Buckalew, A. Fraites, M., et al. (2010). Internal Report: Evaluation of Effects of the Chlorotriazine Herbicide Atrazine on the Amplitude of the Pre

- ovulatory LH Surge in the Long Evans Rat. Memorandum dated July 8, 2010 from Ralph Cooper, NHEERL/ORD to Elizabeth Mendez, HED.

Cooper, R, Buckalew, A. Fraites, M., et al. (2010). Internal Report: Evaluation of Effects of the Chlorotriazine Herbicide Atrazine on the Amplitude of the Pre

- ovulatory LH Surge in the Long Evans Rat. Memorandum dated July 8, 2010 from Ralph Cooper, NHEERL/ORD to Elizabeth Mendez, HED. Corrected Report.

Cooper R.L., Laws S., Das P.C., Narotsky M.G., Goldman J.M., Tyrey E.L., Stoker T.E. (2007). Atrazine and reproductive functio n: mode and mechanism of action studies. Birth Defects Res B Dev Reprod Toxicol, Apr; 80(2): 98

- 112.

Crawford, C.G. 2004. Sampling Strategies for Estimating Acute and Chronic Exposures

of Pesticides in Streams. Journal of the American Water Resources As sociation (JAWRA) 40(2):485

- 502.

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 Pharmacol, Jan 15; 65(2): 303

- 8.

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): 1 45

- 51.

Dooley, G., Hanneman, W., Carbone, D., Legare, M., Anderson, M., & Tessari, J. (2007). Development of an immunochemistry detection method for atrazine

- induced albumin adducts. Chem Res Toxicol, Jul; 20(7): 1061

- 6.

Dooley, G., Prenni, J., Prentis s, P., Cranmer, B., Anderson, M., & Tessari, J. (2006). Identification of a novel hemoglobin adduct in Sprague

- Dawly rats exposed to atrazine. Chem Res Toxicol, May; 19(5): 692

- 700.

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 diaminocholorotriazineDo not cite

Section 10

- Page 135 of 677 adducts in Wistar rat pituitary glands and LbetaT2 rat pituitary cells. Chem Res Toxicol, Apr, 21(4): 844

- 51.

Ebling, F.J.P. (2005) The Neuroendocrine Timing of Puberty. Reproduction 129: 675

- 683

Enoch, R.R., Stanko, J.P., Greiner, S.N., Youngblood, G.L., Rayner, J.L., & Fenton, S.E. (2007). Mammary gland development as a sensitive end point after acute prenatal exposure to an atrazine metabolite mixt ure in female Long

- Evans rats. Environ Health Perspect, Apr, 115(4): 541

- 7.

Falagas ME, Pitsouni EI, Malietzis GA, Pappas G. (2008) Comparison of PubMed, Scopus, Web of Science, and Google Scholar: strengths and weaknesses. FASEB J., Feb;22(2):338

- 42. E pub 2007 Sep 20. PubMed PMID: 17884971.

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(4): 1012

- 8.

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 reproductive cancers in humans. Environ Health Perspect, May, 115(5):

720

- 7.

Farr, S., Cai, J., Savitz, D., Sandler, D., Hoppin, J ., & Cooper, G. (2006). Pesticide exposure and timing of menopause: the Agricultural Health Study. Am J Epidemiol,

163 (8), 731

- 742. doi: kwj099 [pii] 10.1093/aje/kwj099.

Farr, S., Cooper, G., Cai, J., Savitz, D., & Sandler, D. (2004). Pesticide use and

me nstrual cycle characteristics among premenopausal women in the Agricultural Health Study. Am J Epidemiol, 160 (12), 1194

- 1204. doi: 160/12/1194 [pii] 10.1093/aje/.

Feldkamp ML, Carey JC, Sadler TW. Development of gastroschisis: review of hypotheses, a nove l hypothesis, and implications for research. (2007) Am J Med Genet A., Apr 1;143(7):639

- 52. Review. PubMed PMID: 17230493.

FIFRA Scientific Advisory Panel (SAP). (2003). ―Characterization of Epidemiology Data Relating to Prostate Cancer and Exposure to A trazine.‖ Report from the FIFRA Scientific Advisory Panel Meeting of July, 2003. FIFRA Scientific Advisory Panel, Office of Science Coordination and Policy, Office of Prevention, Pesticides and ToxicDo not cite

Section 10

- Page 136 of 677 Substances, U.S. Environmental Protection Agency. W ashington, DC. Available: http://www.epa.gov/scipoly/sap/tools/atozindex/atrazine.htm

FIFRA Scientific Advisory Panel (FIFRA SAP). 2009. A Set of Scientific Issues Being

Conside red by the Environmental Protection Agency Regarding: The Ecological Significance of Atrazine Effects on Primary Producers in Surface Water Streams in the Corn and Sorghum Growing Region of the United States (Part II). SAP Minutes No.

2010

- 04. FIFRA Scient ific Advisory Panel, Office of Science Coordination and Policy, Office of Prevention, Pesticides and Toxic Substances, U.S. Environmental Protection Agency. Washington, DC. Available at http://www.epa.gov/scipoly/sap/meetings/2009/051209meeting.html

FIFRA Scientific Advisory Panel (FIFRA SAP). 2010. A Set of Scientific Issues Being Considered by the Environmental Protection Agency Regarding: Re

- Evaluation of

Human Heal th Effects of Atrazine: Review of Experimental Animal and In Vitro Studies and Drinking Water Monitoring Frequency. SAP Minutes No. 2010

- 04. FIFRA Scientific Advisory Panel, Office of Science Coordination and Policy, Office of Prevention,

Pesticides and To xic Substances, U.S. Environmental Protection Agency. Washington,

DC. Available: http://www.epa.gov/scipoly/sap/meetings/2010/042610meeting.html

FIFRA Scientific Advisory Panel (FIFRA SAP). 2010. Transmittal of Meeting of the FIFRA Scientific Advisory Panel Meeting on the Draft Framework and Case Studies on Atrazine, Human Incidents, and the Agricultural Health Study: Incorporation of Epidemiology and Human Incident Data in Human Health Risk Assessment. SAP

Minutes No. 2010

- 03 . 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/meetings/2010/index.html

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.

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.

Foradori, C., Hinds, L., Hanneman, W., & Handa, R. (2009a). Effects of atrazine and

its withdrawal on gonadotropin

- releasing hormone neuroendocrine function in the adult female Wistar rat. Biol Reprod, Dec, 81(6): 1099

- 105.

Foradori, C., Hinds, L., Hanneman, W., Legare, M., Clay, C., & Handa, R. (2009b). Atrazine inhibits pulsatile luteini zing hormone release without altering pituitary sensitivityDo not cite

Section 10

- Page 137 of 677 to a gonadotropin

- releasing hormone receptor agonist in female Wistar rats. Biol Reprod, Jul; 81(1): 40

- 5.

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.

Fujimoto, N. and Honda, H. (2003). Effects of environmental estrogenic compounds on growth of a transplant ed estrogen responsive pituitary tumor cell line in rats. Food Chem Toxicol, Dec, 41(12): 1711

- 7.

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 an d 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.

Gilliom R.J., Barbash, J.E., Crawford, C.G., Hamilton, P.A., Martin, J.D., Nakagaki, N.,

N owell, L.H., Scott, J.C., Stackelberg, 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

Giusi, G., Facciolo, R.M., Canoaco, M., Alleva, E., Santucci, D., Belloni, V., & Dessi

Fulgheri, F. (2006). The endocrine disruptor atrazine account s for a dimorphic somatostatinergic neuronal expression pattern in mice. Toxicol Sci, Jan; 89(1): 257

- 64.

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. Vet Hum Toxicol, Oct, 6(5): 245

- 7.

Goldfein A, Monroe SE. (1994) Ovaries. In: Greenspan FS, Baxter JD, editors.Basic and clinical endocrinology 4th ed. Norwalk, CT: Appleton and Lange, . pp. 419

- 70.

Goldman, J.M., Laws, S.C., Balchak, S.K., Cooper, R.L. and Kavlock, R.J. Endocrine disrupting chemicals: Prepubertal exposures and effects on sexual maturation and thyroid activity in the female rat. A review of the EDSTAC recommendations. Critical

Reviews in To xicology . 30: 135

- 196, 20

Hase, Y., Tatsuno, M., Nishi, T., Kabe, Y., Yamaguchi, Y., Hanada, H., Kataoka, K., Ozawa, N., Natori, M., Handa, H., & Wantabe, H. (2008). Atrazine binds to F1F0

- ATP synthase and inhibits mitochondrial function in sperm. Bioc hem Biophys Res Commun, Feb 1; 366(1): 66

- 72.Do not cite

Section 10

- Page 138 of 677 Heneweer, M., Van den Berg, M., & Sanderson, J.T. (2004). A comparison of human
H295R and rat R2C cell lines as in vitro screening tools for effects on aromatase. Toxicol Lett, Jan 15; 146(2): 183

- 94.

Higle y, E.B., Newsted, J.L., Zhang, X., Giesy, J.P., & 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

Hocking, B. (2009). Melamine

- contaminated p owdered formula and urolithiasis. N Engl J Med., June 18; 360(25): 2676

- 7.

Hoehn K., Salmon A., Hohnen

- Behrens C., Turner N., Hoy A., Maghzal G., Socker R., Van Remmen H., Kraegen E., Cooney G., Richardson A., James D. (2009). Insulin

resistance is a ce llular antioxidant defense mechanism. Proc Natl Acad Sci U S A, Oct 20 106(42):17787

- 92.

Holloway, A.C., Anger, D.A., Crankshaw, D.J., Wu, M., & Foster, W.G. (2008).

Atrazine

- induced changes in aromatase activity in estrogen sensitive target tissues. J. Appl. Toxicol., Apr, 28(3): 260

- 70.

Hoppin JA, Umbach DM, London SJ, Henneberger PK, Kullman GJ, Coble J, Alavanja MC, Beane Freeman LE, Sandler DP. (2009) Pesticide use and adult

- onset asthma among male farmers in the Agricultural Health Study. Eur Resp ir J., Dec;34(6):1296

- 303. Epub 2009 Jun 18. PubMed PMID: 19541724; PubMed Central PMCID:

PMC2822385.

Hornemann et al . ―Seasonality of omphalocele in Northern Germany,‖ (2008) [Ultraschall in Med. 29:591

- 595. ].

Hornemann, A., Weichert, J., Thill, M., A ltgassen, C., Luedders, D., & Bohlmann, M. K. (2009). Seasonality of Omphalocele in Northern Germany. Ultraschall in Der Medizin,

30 (6), 591

- 595. doi: 10.1055/s

- 2008

- 1027896. [German]

Huang, Y., Batterman, S. 2003. An Extreme Value Analysis of Pollutant Concentrations in Surface Soils Due to Atmospheric Deposition. Human and Ecological Risk Assessment, 9:1729

- 1746.

Hossain, M. M. & Filipov, N. M.(2008). Alteration of dopamine uptake into rat striatal vesicles and synaptosomes caused by an in vitro ex posure to atrazine and some of its metabolites. Toxicology, Jun 3; 248(1): 52

- 8.Do not cite

Section 10

- Page 139 of 677 Judson R.S., Houck K.A., Kavlock R.J., Knudsen T.B., Martin M.T., et al . (2009). In

vitro Screening of Environmental Chemicals for Targeted Testing Prioritization

- The

To x Cast Project. Environ Health Perspect, doi:10.1289/ehp.0901392

Juliani, C.C., Silva

- Zacarin, E.C., Santos, D.C., & Boer, P.A.(2008). Effects of atrazine on female Wistar rats: morphological alterations in ovarian follicles and immunocytochemical label ing of 90 kDa heat shock protein. Micron, Jul, 39(5): 607

- 16.

Kamel, A., Qian, Y., Podhorniak, L. (2010). Final Report on ―Determination of Atrazine and Its Metabolites in Rat Plasma, Milk, and Tissue.‖ ACB Project B10

- 15. Memorandum dated July 13, 201 0. Analytical Chemistry Branch/BEAD/OPP to Ralph Cooper, NHEERL/ORD

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 transgeni c rats. Cancer Sci, Apr; 96(4): 221

- 6.

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 atrazine modulates cell

- mediated immune

function and decreases host resistance to the B16 F10 tumor model in female B6C3F1 mice. Toxicology, Apr 1; 209(1): 15

- 28.

Kim, D., Breckenridge, C. (Syngenta) (2010). Atrazine

- Derivation of the short

- term inhalation NOAEL using a physiologically

- based pharmacokinetic model. Unpublished data from Sy ngenta

Kirby E.D., Geraghty A.C., Ubuka T., Bentley G.E., Kaufer D. (2009). Stress increases putative gonadotropin inhibitory hormone and decreases luteinizing hormone in male

rats. Proc Natl Acad Sci USA , 106(27): 11324

- 9.

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 Contam Toxicol., Aug, 81(2): 205

- 9.

Kniewals, J., Kmetič, I., Gaurina Srček, V., Ši miċ,B., & Kniewald, Z. (2003). Cytotoxicity of Herbicide Atrazine On Chinese Hamster ovary cell Line In Monolayer. Toxicol Lett, Sept; 144 (Suppl 1):S111

- S112.

Larson, S.J., C.G. Crawford, and R.J. Gilliom. (2004). Development and application of

Waters hed Regressions for Pesticides (WARP) for estimating atrazine concentration distributions in streams. U.S. Geological Survey Water

- Resources Investigations Report

03

– 4047, 68 p. Available at http://pubs. usgs.gov/wri/wri034047/Do not cite

Section 10

- Page 140 of 677 Laville, N., Balaguer, P., Brion, F., Hinfray, N., Casellas, C., Porcher, J.M., & Ait

- Aissa, S. (2006). Modulation of aromatase activity and mRNA by various selected pesticides
in the human choriocarcinoma JEG

- 3 cell line. Toxic ology, Nov 10; 228(1): 98

- 108.

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 hydroxyatr azine. Toxicol Sci, Nov; 76(1): 190

- 200.

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 o f Corticosterone in Male Wistar Rats. Tox. Sci., 112(1): 78

- 87.

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.

Lee, H.K. (2004). Bayesian Nonparametrics via Neural Networks. Philadelphia:

ASA/SIAM.

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 herbicide, a trazine, causes mitochondrial dysfunction and insulin resistance. PLOS One, 4(4): 1

- 11.

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

- dose

depen dent increase in homologous recombinational events does not support its genotoxic mode of action. Cytogenet and Genome Research, 104(1

- 4): 315

- 9.

Manske, M., Beltz, L, and Dhanwada, K. (2004). Low

- Level Atrazine Exposure
Decreases Cell Proliferation in H uman Fibroblasts. Arch. Environ. Contam. Toxicol., 46:

438

- 44.

Maravilla

- Galvan, R., Fierro, R., Gonzalez

- Marquez, H., Gomez

- Arroyo, S., Jimenez, I., & Betancourt, M. (2009). Effects of atrazine and fenoxaprop

- ethyl on capacitation and

the acrosomal reac tion in boar sperm. Int. J. Toxicol., Jan

- Feb, 28(1): 24

- 32.

Mattix, K., Winchester, P., & Scherer, L. (2007). Incidence of abdominal wall defects is

related to surface water atrazine and nitrate levels. J Pediatr Surg, 42 (6), 947

- 949. doi:

S0022

- 3468(07 )00056

- 5 [pii] 10.1016/j.jpedsurg.2007.01.027.Do not cite

Section 10

- Page 141 of 677 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.

McMullin, T.S., Andersen, M.E., Hanneman, W.H., Nagahara, A., Lund, T.D., Pak, T., & Handa, R.J. (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. Toxi col Sci, Jun; 79(2): 278

- 86.

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. Toxicol, Oct 30; 240(1

- 2 ): 1

- 14.

Meek, M.E., J.R. Bucher, S.M. Cohen et al . (2003). A framework for human relevance analysis of information on carcinogenic modes of action. Crit Rev Toxicol, 33(6): 591

- 653.

Mizota, K. & Ueda, H. (2006). Endocrine disrupting chemical atrazi ne causes degranulation through Gq/11 protein

- coupled neurosteroid receptor in mast cells. Toxicol Sci., Apr, 90(2): 362

- 8.

Modic, MW. (2004). The role of testicular aromatase in the atrazine mediated changes

in estrone and estradiol in the male wistar rat. North Carolina State University.

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.

Ochoa

- Acuña, H., & Carbajo, C. (2009). Risk of lim b birth defects and mother's home proximity to cornfields. Sci Total Environ, 407 (15), 4447

- 4451. doi: S0048

- 9697(09)00373

- 8 [pii] 10.1016/j.scitotenv.2009.04.028.

Ochoa

- Acuña, H., Frankenberger, J., Hahn, L., & Carbajo, C. (2009). Drinking

- water
herbicid e exposure in Indiana and prevalence of small

- for

- gestational

- age and preterm delivery. Environ Health Perspect, 117 (10), 1619

- 1624. doi: 10.1289/ehp.0900784.

Padoan, S., Ribatet, M., Sisson, S. 2010. Likelihood

- Based Inference for Max

- Stable

Processes. J ournal of the American Statistical Association 105 (489): 263

- 277.

Plant, T.M.; Barker

- Gibb, L.M. (2004). Neurobiological mechanisms of puberty in higher primates . Human Reproduction Upda t e 10(1): 67

- 77

Pinchuk, L.M., Lee, S.R., & Filipov, N.M. (2 007). In vitro atrazine exposure affects the phenotypic and functional maturation of dendritic cells. Toxicology and Applied Pharmacology, Sept, 223(3): 206

- 17.Do not cite

Section 10

- Page 142 of 677 Pogrmic, K., Fa, S., Dakic, V., Kaisarevic, S. & Kovacevic, R. (2009). Atrazine oral

exposu re of peripubertal male rats downregulates steroidogenesis gene expression in Leydig cells. Toxicol. Sci., 111(1): 189

- 97.

Pryor JL, Hughes C, Foster W, Hales BF, Robaire B. (2000) Critical windows of exposure for children's health: the reproductive syst em in animals and humans. Environ Health Perspect., Jun;108 Suppl 3:491

- 503. Review. PubMed PMID: 10852849; PubMed Central PMCID: PMC1637840.

Pruett, S.B., Fan, R., Zheng, Q., Myers, L.P., & Hebert, P.(2003). Modeling and predicting immunological effects of chemical stressors: characterization of a quantitative biomarker for immunological changes caused by atrazine and ethanol. Toxicol Sci., Oct, 75(2): 343

- 54.

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 testing. Toxicological Sciences, June, 109(2): 265

- 75.

Rayburn, A.L., Moody, D.D., an d Freeman, J.L. (2005). Cytotoxicity of technical grade versus formulations of atrazine and acetochlor using mammalian cells. Bull. Environ. Contam. Toxicol., 75: 691

- 8.

Rayner, J.L., Wolf, D.C., Rayner, J.L., Enoch, J.L., Enoch, R.R., & Fenton, S.E. (2 007).

Atrazine

- induced reproductive tract alterations after transplacental and/or lacational exposure in male Long

- Evans rats. Toxicology and Applied Pharmacology, Feb, 218(3): 238

- 48.

Rayner JL, Enoch RR, Fenton SE. (2005) Adverse effects of prenatal exposure to atrazine during a critical period of mammary gland growth. Toxicol Sci. 2005 Sep;87(1):255

- 66..

Rayner, J. L., Wood, C., Fenton, S. E. (2004). Exposure parameters necessary for delaye d puberty and mammary gland development in Long

– Evans rats exposed in utero to atrazine. Toxicol Appl Pharmacol, Feb 15; 195(1): 23

- 34.

Rhomberg, L.R., and Lewandowski, T.A. (2006). Methods for identifying a default cross

species scaling factor. Hum Ecol Risk Assess 12 , 1094

- 1127.

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.Do not cite

Section 10

- Page 143 of 677 Rodriguez, V.M., Thiruche lvam, 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.

Rooney, A.A., Matulka, R.A., & Lueb ke, R.W. (2003). Developmental atrazine exposure suppresses immune function in male, but not female Spragus

- Dawley rats. Toxicol Sci., Dec, 76(2): 366

- 75.

Rosenberg, B.G., Chen, H., Folmer, J., Liu, J., Papadopoulos, V., & Zirkin, B.R. (2008).

Gestatio nal exposure to atrazine: effects on the postnatal development of male offspring. J Androl., May

- June; 29(3): 304

- 11.

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., Apr; 37(4): 776

- 86.

Rowe, A.M., Brundage, K.M., & Barnett, J.B. (2008). Developmental immunotoxicit y of atrazine in rodents. Basic Clin Pharmacol Toxicol, Feb; 102(2): 139

- 45.

Rowe, A.M., Brundage, K.M., & Barnett, J.B. (2007). In vitro atrazine exposure inhibits human natural killer cell lytic granule release. Toxicol Appl Pharmacol, Jun 1; 221(2):

179

- 88.

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

Rusiecki, J.A., De Roos, A., Lee, W.J., Dosemci, M., Lub in J.H., Hoppin, J.A., Blair, A., & Alavanja, M.C. (2004). Cancer incidence among pesticide applicators exposed to atrazine in the Agricultural Health Study. J Natl Cancer Inst, Sept 15; 96(18): 1375

- 82.

Saldana, T. M., Basso, O., Hoppin, J. A., Baird, D. D., Knott, C., Blair, A., et al . (2007). Pesticide exposure and self

- reported gestational diabetes mellitus in the agricultural health study. Diabetes Care, 30 (3), 529

- 534. doi: 10.2337/dc06

- 1832.

Santen R.J., Brodie H., Simpson E.R., Siiteri P.K., Br odie A

. (2009). History of Aromatase: saga of an important biological mediator and therapeutic target. Endocr Rev, Jun; 30(4): 343

- 75.Do not cite

Section 10

- Page 144 of 677 Sathyanarayana S, Basso O, Karr CJ, Lozano P, Alavanja M, Sandler DP, Hoppin JA. 2010. Maternal pesticide use and bir th weight in the agricultural health study. J Agromedicine. 2010 Apr;15(2):127

- 36.

Schwab, C. L., Fan R., Zheng, Q., Myers, L.P., Hébert,P., & Pruet, S.B. (2005). Modeling and predicting stress

- induced immunosuppression in mice using blood parameters. T oxicol Sci, Jan; 83(1): 101

- 13.

Seed, J., E.W. Carney, R.A. Corley et al . (2005). Overview: Using mode of action and lifestage information to evaluate the human relevance of animal toxicity data. Crit. Rev. Toxicol., Oct

- Nov; 35(8

- 9):664

- 72.

Shibaya ma, 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) Two

- or four

- week repeated

- dose studies and fertility study of

atraz ine in female rats. J toxicol Sci., 34: 147

- 55.

Siega

- Riz AM, Herring AH, Olshan AF, Smith J, Moore C; National Birth Defects Prevention Study. (2009) The joint effects of maternal prepregnancy body mass index and age on the risk of gastroschisis. Paedi atr Perinat Epidemiol., Jan;23(1):51

- 7.

Review. PubMed PMID: 19228314.

Singh, M., Kaur, P., Sandhir, R., & Kiran, R. (2008). Protective effects of vitamin E against atrazine

- induced genotoxicity in rats. Mutat Res, Jul 31; 654(2): 145

- 9.

Singh, M., San dhir, R., & Kiran, R.(2008). Atrazine

- induced alterations in rat erthrocyte membranes: ameliorating efect of vitamin E. J Biochem Mol Toxicol, Sept, 225: 363

- 9.

Son, H.Y., Nishikawa, A., Okazaki, K., Lee, K., Imazawa, T., & Hirose, M. (2003). Lack

of m odifying effects of atrazine and/or tamoxifen on thyroid carcinogenesis in rat pretreated with N

- bis(2

- hydroxypropyl)nitrosamine (DHPN). Food Chem Toxicol, Dec; 41(12): 1811

- 6.

Stoker, T., Zorilla, L., Strader, L. Et al. 2010. Internal Report:The Distr ibution of Atrazine in the Wistar Rat Following Gestational/Lactational Exposures. Memorandum dated July 14, 2010 from Tammy Stoker, NHEERL/ORD to Elizabeth Mendez,

HED/OPP.

Stoker T.E & Cooper R.L. (2007). Distribution of 14C

- atrazine following an acute lactational exposure in the Wistar rat. Reprod Toxicol, Jun; 23(4):607

- 10.Do not cite

Section 10

- Page 145 of 677 Stone, W.W. & Gilliom, R.J. (2009). Update of watershed regressions for pesticides (WARP) for predicting atrazine concentration in streams. U.S. Geological Survey Open

File Repor t 2009

– 1122, 22 p. Available at http://pubs.usgs.gov/of/2009/1122/

Stone, W.W., Gilliom, R.J. & Crawford, C.G. (2008). Watershed Regressions for Pesticides (WARP) for Predicting Annual Maximum and Maximu m Moving

- Average Concentrations of Atrazine in Streams. U.S. Geological Survey Open

- File Report 08

- 1186, 19 p. Available at http://pubs.usgs.gov/of/2008/1186/

Suzawa, M. & Ingraham, H.A. (2008). The her bicide atrazine activates endocrine gene

networks via non

- steroidal NR5A nuclear receptors in fish and mammalian cells. PloS ONE, 3(5): 1

- 11.

Swan, S. H., Kruse, R. L., Liu, F., Barr, D. B., Drobnis, E. Z., Redmon, J. B., et al . (2003). Semen quality in relation to biomarkers of pesticide exposure. Environmental Health Perspectives, 111 (12), 1478

- 1484. doi: 10.1289/ehp.6417.

Swan SH. Semen quality in fertile US men in relation to geographical area and pesticide exposure. Int J Androl. 2006 Feb;29(1):62

- 8; discussion 105

- 8. PubMed

PMID: 16466525.

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, Jul 1 6; 210(2): 129

- 37.

Tinfo, N., & Laws, S.C. (2009). Understanding the Effects of Atrazine on Steroidogenesis in Wistar rats. Society of Toxicology, Itinerary Planner, Baltimore, MD.

Ueda, M., Imai, T., Takizawa, T., Onodera, H., Mitsumori, K., Matsul, T ., & Hirose, M. (2005). Possible 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.

US EPA. (2003). Interim Re

- registration Eligibilit y Decision (IRED)

US EPA. (2005). Guidelines for Carcinogen Risk Assessment. U.S. Environmental Protection Agency, Risk Assessment Forum, Washington, DC. EPA/630/P

- 03/001F. Federal Register 70(66):17765

- 17817. Available at http://www.epa.gov/raf

US EPA. (2006). Decision Documents for Atrazine. Atrazine IREDs (January and October 31, 2003) and Atrazine RED (April 6, 2006). EPA

- HQ

- OPP

- 2003

- 0367.Do not cite

Section 10

- Page 146 of 677 USEPA (2006). Harmonization in interspecies extrapolation: use of body weight 3/4 as the default method in derivation of the oral reference dose. External Review Draft.
Office of the Science Advisor.

U.S. Environmental Protection Agency (USEPA). 2007. Preliminary Interpretation of the Ecological Significance of Atrazine Stream

- Water Concentrations Using a Statistically

Designed Monitoring Program. Prepared for the December 4

- 7, 2007 FIFRA Scientific Advisory Panel. Document available at http:/ /www.epa.gov/scipoly/sap/meetings/2007/120407_mtg.htm

U.S. Environmental Protection Agency (USEPA). (2007a). Acetochlor Registration Agreement and Monitoring Program. Documents are available through the Federal

docket. Docket ID: EPA

- HQ

- OPP

- 2007

- 0725. Available at http://www.regulations.gov/search/Regs/home.html#docketDetail?R=EPA

- HQ

- OPP

2007

- 0725

U.S. Environmental Protection Agency (USEPA). (2007 b). N

- methyl Carbamate Revised Cumulative Risk Assessment. Available through the USEPA Office of Pesticide Programs web site at http://www.epa.gov/oppsrrd1/REDs/nmc_revised_cra.pdf

U.S. Environmental Protection Agency (USEPA). (2007c). Preliminary Interpretation of the Ecological Significance of Atrazine Stream

- Water Concentrations Using a Statistically

- Designed Monitoring Program. Prepared for the December 4

- 7, 2007
FIFRA Scie ntific Advisory Panel. Document available at http://www.epa.gov/scipoly/sap/meetings/2007/120407_mtg.htm

U.S. Environmental Protection Agency (USEPA). 2009. The Ecological Signif icance of Atrazine Effects on Primary 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

U.S. Environmental Protection Agency (USEPA). (2009a). The Ecological Significance of Atrazine Effects on Primary Producers in S urface 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

U.S. Environmental Protection Agency (USEPA). (2009b). The Ecological Significance of Atrazine Effects on Primary Producers in Surface Water Streams in the Corn and Sorghum Growing Region of the United States (Part II). Addendum to Section VI: Additional Analysis of the 40 AEEMP Sampling Sites to Identify Additional Areas ThatDo not cite

Section 10

- Page 147 of 677 May Exceed the LOC. Prepared for the May 12

- 15, 2009 FIFRA Scientific Advisory

Panel. Document available at http://www.epa.gov/scipoly/sap/meetings/2009/051209meeting.html

U.S. Environmental Protection Agency (USEPA). 2010. Re

- Evaluation of Human

Health Effects of Atrazine: Rev iew of Experimental Animal and In Vitro Studies and Drinking Water Monitoring Frequency. Prepared for the April 26

- 29, 2010 FIFRA Scientific Advisory Panel. Document available at http://www.epa.gov/scipoly/sap/meetings/2010/042610meeting.html

U.S. Environmental Protection Agency. (2010). DRAFT Framework for Incorporating Human Epidemiologic & Incident Data in Health Risk Assessment (Draft dated January 7, 2010). Presented t o the FIFRA Scientific Advisory Panel, February 2

- 4, 2010. http://www.regulations.gov (Docket EPA

- HQ

- OPP

- 2009

- 0851).

Vecchia A.V., Crawford, C.G. 2006. Simulation of Daily Pesticide Concentrations from Watershed Characteristics and Monthly Climatic Data : U.S. Geological Survey Scientific Investigations Report 2006

- 5181, 60 p.

Vecchia, A.V., Martin, J.D., Gilliom, R.J. 2008. Modeling Variability and Trends in

Pesticide Concentrations in Streams. Journal of the American Water Resources Association (JAWRA ) 44(5): 1308

- 1324.

Vecchia, A.V., Gilliom, R.J., Sullivan, D.J., Lorenz, D.L., Martin, J.D. 2009. Trends in Concentrations and Use of Agricultural Herbicides for Corn Belt Rivers, 1996

- 2006. Environ. Sci. Technol. 43, 9096

- 9102.

Victor

- Costa, A.B., Caro zzi Bandeira, S.M., Oliveira, A.G., Bohorquiz Mahecha, G.A. & Oliveira, C.A. (2010). Changes in testicular morphology and steroiodogenesis in adult rats exposed to atrazine. Reprod Toxicol, Jan 4. [Epub ahead of print] Doi:10.1016/j.reprotox.2009.12.006

Villanueva, C., Durand, G., Coutté, M., Chevrier, C., & Cordier, S. (2005). Atrazine in municipal drinking water and risk of low birth weight, preterm delivery, and small

- for

gestational

- age status. Occup Environ Med, 62 (6), 400

- 405. doi: 62/6/400 [pii]

10.1136/oem.2004.016469.

Waller, S. A., Paul, K., Peterson, S. E., & Hitti, J. E. (2010). Agricultural

- related chemical exposures, season of conception, and risk of gastroschisis in Washington
State. American Journal of Obstetrics and Gynecology, 202 (3). doi: 10.1016/j.ajog.2010.01.023.

Weselak M, Arbuckle TE, Walker MC, Krewski D. The influence of the environmentDo not cite

Section 10

- Page 148 of 677 and other exogenous agents on spontaneous abortion risk. (2008) J Toxic ol Environ Health B Crit Rev., Mar;11(3

- 4):221

- 41. Review. PubMed PMID: 18368554

Whalen M.M., Loganathan B.G., Yamashita, N., Saito, T. (2003). Immunomodulation of human natural killer cell cytotoxic function by triazine and carbamate pesticides.
Chemico

- Biological Interactions, Jun 15; 145(3): 311

- 19.

Williams LJ, Kuci k JE,AlversonCH, OlneyRS, CorreaA.Epidemiology of gastroschisis in metropolitan Atlanta, 1968 through 2000. Birth Defects Res Part A Clin Mol Teratol
2005;73:177

- 83.

Winchester, P., Huskins, J., & Ying, J. (2009). Agrichemicals in surface water and birth
defects in the United States. Acta Paediatr, 98 (4), 664

- 669. doi: APA1207 [pii] 10.1111/j.1651

- 2227.2008.01207.x.

Zeljeciz D, & Garaj

- Vrhovac, V. (2004a). Atrazine genotoxicity evaluation in different mouse organs by comet assay. Periodicum Biologorum, 106(2): 155

- 9.

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.

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.

Zorrilla, L.M. , Gibson, E.K., Stoker (2009) Effect of Chlorotriazine Pesticides on Gonadotrophin 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

Arbuckle, T. E., Lin, Z. Q., & Mery, L. S. (2001). An exploratory analysis of the effect of pesticide exposure on the risk of spontaneous abortion in an Ontario farm population. Environmental Health Perspectives, 109 (8), 851

- 857.

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

Su rvey Open

- File Report 01

- 456. Published in cooperation with the U.S. Environmental Protection AgencyDo not cite

Section 10

- Page 149 of 677 Canfield MA, Honein MA, Yuskiv N, Xing J, Mai CT, Collins JS, et al . (2006) National

estimates and race/ethnic

- specific variation of selected birth defe cts in theUnited States,

1999

- 2001. BirthDefects Res A Clin Mol Teratol, 76:747

- 56.

Canfield MA, Ramadhani TA, Langlois PH, Waller DK. (2006) Residential mobility patterns and exposure misclassification in epidemiologic studies of birth defects. J Expo
Sc i Environ Epidemiol., Nov;16(6):538

- 43. Epub 2006 May 31. PubMed PMID:

16736057.

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.

Coo per, R.L., Stoker, T.E., Tyrey, L., Goldman, J.M., & McElroy, W.K. (2000). Atrazine disrupts the hypothalamic control of pituitary

- ovarian function. Toxicol Sci., Feb; 53(2):

297

- 307

Cummings, A. M., B. E. Rhodes, et al . (2000). "Effect of atrazine on implantation and early pregnancy in 4 strains of rats." Toxicol Sci 58 (1): 135

- 143.

Dabrowski, S., Hanke, W., Polańska, K., Makowiec

- Dabrowska, T., & Sobala, W. (2003). Pesticide exposure and birthweight: an epidemiological study in Central Poland.

Int J Occup Med Environ Health, 16 (1), 31

- 39.

Das, P.C., McElroy, W.K. & Cooper, R.L. (2001). Alteration of Catecholamines in Pheochromocytoma (PC12) Cells in vitro by the Metabolites of Chlorotriazine Herbicide. Toxicol Sci, Jan; 59(1): 127

- 37.

Deutsch, C .V. & Journel, A.G. (1998). GSLIB: Geostatistical Software Library and User‘s Guide. Second Edition. Oxford University Press (New York, Oxford).

Eldridge, J.C., Wetzel, L.T., & Tyrey, L. (1999). Estrous cycle patterns of Sprague

Dawley rats during acut e and chronic atrazine administration. Reprod Toxicol, Nov

Dec; 13(6): 491

- 9.

Fauser, B.C., de Jong, D., Olivennes, F. et al . (2002). Endocrine Profiles after Triggering of Final Oocyte Maturation with GnRH Agonist after Cotreatment with the GnRH Antag onist Ganirelix during Ovarian Hyperstimulation for in Vitro Fertilization.
J Clin Endocrinol Metab. 87 (2):709

- 715.

FIFRA Scientific Advisory Panel (SAP). (2000a). ―Atrazine: Hazard and Dose

Response Assessment and Characterization.‖ Report from the F IFRA Scientific Advisory Panel Meeting of June 27

- 29, 2000. FIFRA Scientific Advisory Panel, Office of Science Coordination and Policy, Office of Prevention, Pesticides and ToxicDo not cite

Section 10

- Page 150 of 677 Substances, U.S. Environmental Protection Agency. Washington, DC. Availa ble: http://www.epa.gov/scipoly/sap/tools/atozindex/atrazine.htm

FIFRA SAP. (2000b). "Atrazine :Hazard and Dose

- Response Assessment and Characterization." Report from the FIFR A 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. SAP Report 200 0

- 05. Available: http://www.epa.gov/scipoly/sap/meetings/2000/june27/finalatrazine.pdf

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, Pesticides and Toxic Substances, U.S. Environmental Protection Agency. Washington, DC. Available: http://www.epa.gov/scipoly/sap/1988 *

Friedmann, A.S. (2002). Atrazine inhib ition of testosterone production in rat males following peripubertal exposure. Reprod. Toxicol, May

- Jun; 16: 275

- 9.

Frydman, R., Cornel, C., de Ziegler D. et al . (1992) Spontaneous luteininzing hormone surges can be reliably prevented by the timely adm inistration of donadotrophin releasing hormone antagonist (Nal0Glu) during the late follicular phase. Human Reproduction

7 (7):930

- 933

Gao H.B., Shan L.X., Monder C., Hardy M.P. (1996). Suppression of endogenous

corticosterone levels in vivo increases th e steroidogenic capacity of purified rat Leydig

cells in vitro . Endocrinology, May; 137(5):1714

- 8.

Goldman, J.M., Laws, S.C., Balchak, S.K., Cooper, R.L. and Kavlock, R.J. (2000) Endocrine disrupting chemicals: Prepubertal exposures and effects on sexual maturation and thyroid activity in the female rat. A review of the EDSTAC

recommendations. Critical Reviews in Toxicology . 30: 135

- 196, 20 Grumbach, M.M. (2002) The Neuroendocrinology of Human Puberty Revisited. Horm.

Res. 57(supp 2): 2

- 14

Henderson KD , Bernstein L, Henderson B, Kolonel L, Pike MC. (2008) Predictors of the timing of natural menopause in the Multiethnic Cohort Study. Am J Epidemiol. 2008 Jun 1;167(11):1287

- 94. Epub Mar 21. PubMed PMID: 18359953.

Hoppin JA, Umbach DM, London SJ, Hennebe rger PK, Kullman GJ, Coble J, Alavanja MC, Beane Freeman LE, Sandler DP. (2009) Pesticide use and adult

- onset asthma among maleDo not cite

Section 10

- Page 151 of 677 farmers in the Agricultural Health Study. Eur Respir J., Dec;34(6):1296

- 303. Epub 2009 Jun 18. PubMed PMID: 19541724; PubMed Ce ntral PMCID: PMC2822385.

Hoppin, J. A., Umbach, D. M., London, S. J., Alavanja, M. C. R., & Sandler, D. P. (2002). Chemical predictors of wheeze among farmer pesticide applicators in the agricultural health study. American Journal of Respiratory and Criti cal Care Medicine,

165 (5), 683

- 689. doi: 10.1164/rccm.2106074.

Hoppin JA, Yucel F, Dosemeci M, Sandler DP. (2002) Accuracy of self

- reported pesticide use duration information from licensed pesticide applicators in the Agricultural Health Study. J Expo Ana l Environ Epidemiol., Sep;12(5):313

- 8. PubMed PMID:

12198579.

ILSI (International Life Sciences Institute), (1999). A Framework for Estimating Pesticide Concentrations in Drinking Water for Aggregate Exposure Assessments. International

Life Sciences Insti tute Risk Science Institute Working Group Report, Washington, D.C., 61 pp. Available at http://www.ilsi.org/ResearchFoundation/Publications/Esti mating%20Pesticide%20Conce ntrations.pdf

Isaaks, E.H., and R.M. Srivastava. 1989. Applied Geostatistics. Oxford University Press (New York, Oxford).

Kalra, S.P., & Kalra, P.S.(1983). Neural regulation of luteinizing hormone secretion in

the rat. Endo cr Rev., Fall; 4(4): 311

- 51.

Kossmann, S., Konieczny, B., & Hoffmann, A. (1997). The role of respiratory muscles in the impairment of the respiratory system function in the workers of a chemical plant division producing pesticides. Przegl Lek, 54 (10), 702

- 706.

Kłuciński, P., Kossmann, S., Tustanowski, J., Friedek, D., & Kamińska

- Kołodziej, B. (2001). Humoral and cellular immunity rates in chemical plant workers producing dust pesticides. Med Sci Monit, 7 (6), 1270

- 1274. doi: 1270 [pii].

Laws, S. C., Ferr ell, J. M., Stoker, T. E., Schmid, J. & Cooper, R. L. (2000). The Effects of Atrazine on Female Wistar Rats: An Evaluation of the Protocol for Assessing Pubertal Development and Thyroid Function. Toxicol Sci, 58: 366

- 76.

Lyons, W.R., Berlin, I., and Fri endlander, S. (1942) Cornification of balano

- preputial epithelium in normal rats and castrated rats treated with testosterone proprionate. Endocrinology, 31(6): 659

- 63.Do not cite

Section 10

- Page 152 of 677 Marshall, F. (1966) Marshall‘s Physiology of Reproduction. Longmans, Green and Co.

LTD

Matsumoto AM, Karpas AE, Southworth MB, Dorsa DM, Bremner WJ. (1986). Evidence for activation of the central nervous system

- pituitary mechanism for gonadotropin secretion at the time of puberty in the male rat. Endocrinology , Jul; 119(1): 362

- 9.

M innema, D. (2001). "Special Study (non

- guideline). Comparison of the LH surge in female rats administered atrazine, simazine, or diaminochlorotriazine (DACT) via gavage for one month." Covance Laboratories Inc. Vienna, VA Laboratory report

number 6117

- 398 ( MRID # 45471002).

Minnema, D.J. (2002). 52

- Week toxicity study of simazine, atrazine, and DACT administered in the diet to female rats. Covance Laboratories Inc., 9200 Leesburg Pike, Vienna, VA. Laboratory Identification Number 6117

- 399. February 21, 2 002. MRID 45622309. Unpublished

Molcrani M.C., Duval F., Crocq M.A., Bailey P., Macher J.P. (1997). HPA axis dysfunction in depression: Correlation with monoamine system abnormalities. Psychoneuroendocrinology, 22, Suppl 1: S63

- 8

Monosson E, Kelce WR , Lambright C, Ostby J, Gray LE Jr. (1999). Peripubertal exposure to the antiandrogenic fungicide, vinclozolin, delays puberty, inhibits the development of androgen

- dependent tissues, and alters androgen receptor function in

the male rat. Toxicol Ind Hea lth , 15(1

- 2): 65

- 79.

Morseth, S. (1996a). "Evaluation of the lutenizing hormone (LH) surge in atrazine expose female Sprague Dawley rats." Covance Laboratories Inc. Vienna, VA Laboratory report # CHV 2386

- 111 (MRID # 43934406).

Morseth, S. (1996b). "Eva luation of the lutenizing hormone (LH) surge in atrazine exposed female Sprague Dawley rats

- 6 month report." Covance Laboratories Inc. Vienna, VA Laboratory study no. CHV 2386

- 108 (MRID # 44152102).

Munger, R., Isacson, P., Hu, S., Burns, T., Hanson , J., Lynch, C. F., et al . (1997). Intrauterine growth retardation in Iowa communities with herbicide

- contaminated drinking water supplies. Environmental Health Perspectives, 105 (3), 308

- 314.

Narotsky, M. G., D. S. Best, et al . (2001). "Strain comparisons o f atrazine

- induced pregnancy loss in the rat." Reprod Toxicol 15 (1): 61

- 69.Do not cite

Section 10

- Page 153 of 677 Olivennes, F., Alvarez, S., Bouchard, P. et al . (1998). The use of GnRH antagonist (Cetrorelix®) in a single dose protocol in IVF

- embryo transfer: a dose finding study of 3

versu s 2 mg. Human Reproduction 13 (9):2411

- 2413

Ojeda, S.R., and Urbanski, H.F. (1994). Puberty in the rat. In E. Knobil and J.D. Neill (Eds.): The Physiology of Reproduction, 2 nd ed. New York, Raven Press.

Rivest, .S & Rivier, C. (1995). The role of co rticotropin

- releasing factor and interleukin

- 1 in the regulation of neurons controlling reproductive functions. Endocr Rev, 16(2): 177

- 99.

Rivier, C. & Rivest, S. (1991). Effect of stress on the activity of the hypothalamic

pituitary

- gondal axis: periph eral and central mechanisms. Biol Reprod., October 1; 45 (4): 523

- 532

Rivier, C., Rivier, J., & Vale, W. (1986). Stress

- induced inhibition of reproductive functions: role of endogenous corticotropin

- releasing factor. Science, 231(4738): 607

- 9.

Sanders on, J.T., Boerma, J., Gideon, W.A., Lansbergen, G.W. & van den Berg, M. (2002). Induction and inhibition of aromatase (CYP19) activity by various classes of pesticides in H295R human adrenocortical carcinoma cells. Toxicol Appl Pharmacol. Jul 1; 182: 44

- 54.

Sanderson J.T., Letcher 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 Perspect, Ovt; 109(10): 1027

- 31.

Sanderson J.T., Seinen W., Giesy J.P., van den Berg M. (2000). 2

- Chloro

- s

- triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: a novel mechanisms for estrogenicity? Toxicol Sci, Mar; 54(1): 121

- 27.

Savitz, D., Arbuckle, T., Kaczor, D., & Curtis, K. (1997). Male pesticide exposure and pregnancy outcome. Am J Epidemiol, 146 (12), 1025

- 1036.

Selye, H. (1939). Effect to adaptation to various damaging agents on the fema le sex organs in the rat. Endocrinology, 25(4): 615

- 24.

Shyr, S.W., Crowley, W.R., and Grosvenor, C.E. (1986) Effect of Neonatal Prolactin Deficiency on Prepubertal tuberoinfundibular and tuberohypophyseal dopaminergic neuronal activity. Endocrinology 119: 1217

- 1221Do not cite

Section 10

- Page 154 of 677 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.

Simpson, E.R., & Davis, S.R. (2001). Minireview: aromatase and the re gulation of estrogen biosynthesis

– some new perspectives. Endocrinology, Nov; 142(11): 4589

- 94.

Sliwiński, Z., Hermanowicz, A., Kossmann, S., & Hrycek, A. (1991). Neutrophil function in chemical plant workers employed in the production of dust pesticide s. Pol J Occup Med Environ Health, 4 (3), 241

- 247.

Sonich

- Mullin, C., R. Fielder, J. Wiltse et al . (2001). IPCS conceptual framework for evaluating a mode of action for chemical carcinogenesis. Regul Toxicol Pharmacol, Oct; 34(2):146

- 52.

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 Pubertal Protocol. Toxicol. Sci., 67(2): 198

- 206.

Stoker, T.E., Laws, S.C., Guidici, D., & Cooper, R.L. (2000). The effects of atrazine on puberty and thryroid function in the male wistar rat: An evaluation of a protocol for the assessment of pubertal development and thyroid function. Toxicol. Sci., 58: 50

- 9.

Stoker T.E., Robinette, C.L . & Cooper, R.L. (1999). Maternal exposure to atrazine during lactation suppresses suckling

- induced prolactin release and results in prostatitis in the adult offspring. Toxicol Sci, 52: 68

- 79

Stoker, T.E., Parks, L.G., Gray, L.E., and Cooper, R.L. Eff ects of endocrine disrupting chemicals on puberty in the male rat: A review of the EDSTAC

recommendations. Critical Reviews in Toxicology 30: 197

- 252, 2000.

Terasawa, E., and C.L. Nyberg. 1997. LHRH pulse generation in the monkey: in vivo

and in vitro st udies. In: Neural control of reproduction: physiology and behavior, K. Maeda, H. Tsukamura, and E. Yokoyama, eds., pp. 57

- 70. JSS Press, Tokyo.

Thede, "Study of 14C

- atrazine dose/response relationship in the rat (general metabolism)"

. MRID # 40431305 , 19 87.

Timchalk, C., M. D. Dryzga, et al . (1990). "Determination of the effect of tridiphane on the pharmacokinetics of [14C]

- atrazine following oral administration to male Fischer 344

rats." Toxicology 61 (1): 27

- 40.Do not cite

Section 10

- Page 155 of 677 Trentacoste, S.V., Friedmann, A.S., You ker, R.T., Breckenridge, C.B. & Zirkin, B.R. (2001). Atrazine effects on testosterone levels and androgen

- dependent reproductive organs in peripubertal male rats. J. Androl, Jan

- Feb; 22(1): 142

- 8.

US EPA. (1999). Guidelines for carcinogen risk assessmen t. Risk Assessment Forum. SAB review draft. Washington, DC: U.S. Environmental Protection Agency. www.epa.gov/ncea/raf/crasab.htm . *

U.S. Environmental Protection Agency (USEPA). 2000. Atrazine: Hazard and Dose

Response A ssessment and Characterization.

U.S. Environmental Protection Agency (USEPA). 2000. Progress Report on Estimating Pesticide Concentrations in Drinking Water and Assessing Water Treatment Effects on

Pesticide Removal and Transfo rmation: A Consultation. Prepared for Session VI of the September 26

- 29, 2000 FIFRA Scientific Advisory Panel.

USEPA. (2000). ―Benchmark Dose Technical Guidance Document.‖ Draft report. Risk Assessment Forum, Office of Research and Development, U.S. Envi ronmental Protection Agency. Washington, DC. EPA/630/R

- 00/001.

U.S. Environmental Protection Agency (USEPA). 2001. Water Treatment Effects on Pesticide Removal and Transformation. Federal Register Docket Number: OPP

- 00731.

Available at http://www.epa.gov/pesticides/trac/science/water_treatment.pdf

Watkins ML, Edmonds L, McClearn A, Mullins L, Mulinare J, Khoury M. (1996) The

surveillance of birth defects: the usefulness of t he revised US standard birth certificate. Am J Public Health., May;86(5):731

- 4. PubMed PMID: 8629729; PubMed Central

PMCID: PMC1380486.

Wetzel L.T., Luempert L.G., III Breckenridge C.B., Tisdel M.O., Stevens J.T. (1994).

Chronic effects of atrazine on e strus and mammary tumor formation in female Sprague

Dawley and Fisher 344 rats. J Toxicol. Environ. Health, Oct; 43(2):169

- 82.

Williams LJ,Kucik JE,AlversonCH, OlneyRS, CorreaA. (2005) Epidemiology of gastroschisis in metropolitan Atlanta, 1968 through 2 000. Birth Defects Res Part A Clin Mol Teratol, 73:177

- 83.Do not cite

Appendix A

- Page 156 of 677 

## APPENDIX A

EVALUAT ION OF ARTICLES ON ATRAZINE AND

ATRAZINE METABOLITES RELATED TO MAMMALIAN

TOXICITY STUDIES SUGGESTING POTENTIAL

ADVERSE HUMAN HEALTH OUTCOMESDo not cite

Appendix A

- Page 157 of 677 A .1 CONTENT OF THIS APPENDIX

An evaluation of peer

- reviewed articles related to atrazine and/or its metabolites published since 2003 was conducted in preparation for the April 2010 SAP and updated prior to the September meeting. The following table provides a listing of the new stu dies available between January 30 and July 15, 2010 (deadlines for the April and September meetings, respectively). The scope of this evaluation was restricted to articles reporting on mammalian toxicity studies or in vitro studies that provided data on a trazine and atrazine metabolites mode of action (MOA). The purpose of this evaluation was to determine the scientific quality of the research conducted since the atrazine Re

- registration Eligibility Decision (RED) was finalized in 2003. Ultimately, the o bjective of this evaluation is to ascertain 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.

Author/Title Section Casas, E., Bonilla, E., Ducolomb Y, Betancourt M. 2010. Differential effects of herbicides atrazine and fenoxaprop

- ethyl, and insecticides diazinon and ma lathion, on viability and maturation of porcine oocytes in vitro. A.2

Coder, P.S. 2010d. An Oral Developmental Toxicity Study of Atrazine in the Rat, Including

Cross

- Fostering and Pair

- Feeding. A.2

Coder, P.S. 2010a. An Oral (Gavage) Study to Assess t he Potential for Detecting the Estrogen

- Induced Luteinizing Hormone Surge in Female Rats. A.2

Coder P. (2010c). Atrazine: An Oral (Gavage) Study of the Effects of Atrazine on the Estrous Cycle and the Estrogen

- Induced Luteinizing Hormone Surge in Female R ats. A.2

Cooper, R., Buckalew, A., Fraites, M., Goldman, J., Laws, S., Narotsky, M., Stoker, T. 2010. Evaluating the Effect of the Chlorotriazine Herbicide Atrazine on the Amplitude of the Pre

ovulatory LH surge in the Long

- Evans Rat. A.2

Stoker, T.E., Zorrilla, L., Strader, L., Gibson, E., Kamel, A., Qian, Y., Podhorniak, L. 2010. The Distribution of Atrazine in the Wistar Rat Following Gestational/Lactational Exposures. A.2

Rayner, J.L., Enoch, R.R., and Fenton, S.E. (2005). Adverse effects of prena tal exposure to atrazine during a critical period of mammary gland growth A.2

Rayner, J. , Wood, C., and Fenton, S. (2004 ) Exposure Parameters Necessary for Delayed Puberty and Mammary Gland Development in Long

- Evans Rats Exposed in Utero to Atrazine. A.4

Solari, M, Paquin, J, Ducharme, P, Boily, M (2010). P19 neuronal differentiation and retinoic acid metabolism as criteria to investigate atrazine, nitrite, and nitrate developmental toxicity. A.5

Coder, P. S. (2010b) An Oral (Gavage) Immunotoxicity and Hormone Evaluation Study for the Effects of Atrazine on Male Rats, A.5

Rajkovic, V, Matavulj, M and Johansson, O. (2010). Combined Exposure of Peripubertal

Male Rats to the Endocrine

- Disrupting Compound Atrazine and Power

- Frequency Electromagnetic Fiel ds Causes Degranulation of Cutaneous Mast Cells: A New Toxic

Environmental Hazard? A.5

Lasserre, JP, Fack, F, Revets, D, Planchon S, Renaut J, Hoffmann L, Gutleb, Muller CP, Bohn T, (2009) Effects of the Endocrine Disruptors Atrazine and PCB 153 on the Pr otein Expression of MCF

- 7 Human Cells. A.7

Garaj

- Vrhovac, V. and Zeijezic, D. (2004) Genotoxicity evaluation of pesticide formulations A .7Do not cite

Appendix A

- Page 158 of 677 containing alachlor and atrazine in multiple mouse tissues (blood, kidney, liver, bone marrow, spleen) by comet assay. Olejnik, Am, Marecik, R, Bialas, W, Cyplik, P. (2010). In vitro studies on atrazine effects on

human intestinal cells. A.7

Singh, M, Sandhir, R, Kiran, Ravi. (2010). Effects on antioxidant status of liver following atrazine exposure and its attenuati on by vitamin E. Exp and Toxicol A.7

Enoch, R.R., Stanko, J.P., Greiner, S.N., Youngblood, G.L,. Rayner, J.L., Fenton, S.E., 2007. Mammary gland development as a sensitive end point acute prenatal exposure to an atrazine

metabolite mixture in female Lon g

- Evans rats A.10

Yi, KD, Perez, EJ, Breckenridge, CB, and Simpkins, JW (2010) Ex vivo effects of atrazines or metabolites on phosphodiesterase activity. A.11

Fakhouri, WD, Nunez, JL, Trail, F. (2010?) Atrazine Binds to the Growth Hormone Releasing

Hormo ne Receptor and Affects Growth Hormone Gene Expression A.11Do not cite

Appendix A

- Page 159 of 677 A.2 EFFECTS ON FEMALES

Summary: Vaginal Opening, Ovarian Function

.

Over the last seven years, studies have been conducted to evaluate the impact of atrazine and/or its by

- products on the development and reproductive health of females. 11

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 th e researchers‘ previous findings 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 post

- natal day PND 22

- 41 resulted in significant delays in VO (3

- 7 day delay) and in some instances animals failed to attain VO by the 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 generalized growth delays. Moreover, the NOAEL identified for DACT is the same as equimolar 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 quickly metabolized to DACT, these results strongly suggest that DACT is the toxicologically active moiety causing the delays in sexual maturation induced by atrazine.

Potential effe cts of atrazine on ovarian function were evaluated over a wide dose range (3

- 300 mg/kg/day) by Shibayama and colleagues (Shibayama et al.

, 2009). In a companion study these researchers attempted to correlate atrazine

- induced changes in

ovarian function to fertility impairments. Unfortunately, different dose levels were used for the ovarian function and fertility studies thus precluding an appropriate comparison between these two sets of experiments. In the ovar ian function set of experiments, atrazine

exp osure for 2 or 4 weeks at a high dose (300 mg/kg/day) resulted in irregular estrous cycles (typically longer cycles) due primarily to a lengthened diestrus. This effect was accompanied by decreased numbers of corpora lutea, follicular atresia, uterine atr ophy, as well as decreased ovarian 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 disruption or the histopathology changes. In the fertilit y study, atrazine exposures at levels up to 100 mg/kg/day for a period of time encompassing 2 weeks prior to mating up to gestation day (GD) 7 did not result in any signs of impaired fertility. None of the signs typically associated with impaired fertilit y ( e.g.

, number of implantation, corpora lutea, pre

- or post

- implantation loss) were affected.

11 Studies on the potential effect of atrazine exposure on mammary gland develo pment will be evaluated during the Fall meeting of the SAP.Do not cite

Appendix A

- Page 160 of 677 The studies by Laws et al.

, and Shibayama et al.

, indicate that exposure to atrazine or DACT results in effects related to the development and function of the r eproductive 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 developmental 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 delays in vaginal opening. Thus, it is not unexpected that exposure to
atrazine and DACT

– 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 explaine d 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 eventuall y result in the reproductive senescence characteristic of repeated 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.

Summary: Mammary Gland Devel opment

.

Mammary gland development is a complex biological process controlled by multiple hormonal interactions. The interplay of pituitary, ovarian, and adrenal hormones regulate the development, differentiation, and proliferation in mammary glands (Dani el et al ., 1987; Imagawa et al., 1990) . Over the last century, extensive research has been conducted to understand the underlying biological processes involved in mammary gland development and the hormonal signals and pathways that modulate it (Hennighau sen and Robinson, 2001). In a series of seminal experiments in 1900, Haban first described the role ovaries played in mammary gland development by demonstrating that in ovariectomized animals mammary gland tissue regressed and that transplanting ovaries b ack into these animals prevented this atrophy. In the 1920s several investigators showed that the pituitary gland was also involved in mammary gland development. Thus, began the era of mammary gland development research.

Development of mammary tissue occurs in defined stages linked to sexual maturation and reproduction including embryonic, pre

- and peripubertal periods, as well as pregnancy and lactation. Given the effects atrazine has on the hormonal environment and reproductive systems, it is not un reasonable to hypothesize that it may also affect mammary gland development. A number of studies have been conducted to investigate this possibility.
Several studies from the same research group (Rayner et al., 200 4 ; Rayner et al. , 2005 ; and Enoch et al. , 2007 [see mixtures section below]) describe effects of either atrazine or a mixture of atrazine metabolites/degradates (DIA, DEA, DACT, hydroxy

- atrazine) onDo not cite

Appendix A

- Page 161 of 677 mammary gland development in female pups. Briefly, the authors concluded that atrazine delays ma mmary gland development using a subjective scoring scale to evaluate mammary gland development. The Agency‘s reviews of these studies are summarized in this appendix. T he Agency concluded that there are shortcomings in the study design, data reporting and analysis that compromise the interpretation of the studies.

A fourth study recently submitted to the Agency also assessed the impact of atrazine exposure on mammary gland development (Coder, 2010). Animals were exposed in utero during the presumptive c ritical period for mammary gland development (GD 13

- 19) to atrazine at doses ranging from 6.5

- 100 mg/kg/day. In contrast to the Rayner et al . and

Enoch et al. studies that used a subjective scoring scale to assess mammary gland development, Coder employed an objective measure (morphometry) to evaluate mammary gland development. Unlike the previous studies, the Coder data suggest that atrazine does not affect mammary gland development. The Agency‘s review of this study is also included in this appendix.

A.2.1 Casas, E., Bonilla, E., Ducolomb Y, Betancourt M. 2010. Differential effects of herbicides atrazine and fenoxaprop

- ethyl , and insecticides diazinon and malathion, on viability and maturation of porcine oocytes

in vitro

. Toxicology in vitro 24 (2010); 224

- 230.

The purpose of this study was to evaluate the effect of two commercially

- formulated insecticides (malathion and diazinon) and two technical grade herbicides (atrazine and fenoxaprop

- ethyl) on viability and in vitro maturation of porcine oocytes. Only the results of atrazine will be discussed in this summary.

Ovaries were collected from slaughtered 6

- month old gilts and oocytes were matured in increasing concentrations of atrazine for 44 hours. Atrazine was added in 0, 50, 100 and

500 μ M concent rations. Viability and in vitro maturation were evaluated simultaneously in the same oocyte. After in vitro maturation, the oocytes were stained with methylthiazolydiphenyl

- tetrazolium bromide (MTT) for 2 hours to assess viability. To assess maturation, oocytes were treated with 10 ug/ml bisbenzimide. Purple stained oocytes were considered alive when evaluated under a light microscope. Oocytes having a germinal vesicle (GV) were considered immature. Oocytes in first metaphase (MI) were judged to be matu ring. Those showing a second metaphase (MII) and a polar body were classified as matured. The correlation among pesticide concentration and its effect on ooctye viability and maturation was assessed using Spearman‘s rank correlation

coefficient. The conc entration required to produce death in 50% of the oocytes (lethal concentration 50, LC 50 ) and the concentration needed to inhibit the maturation in 50% of the oocytes (inhibition of maturation 50, IM 50 ) were calculated by a non

- linear regression.

For at razine, 664 oocytes (total) were analyzed in five assays at 0, 50, 100 and 500 μ M atrazine concentrations. For viability, the Spearman correlation was not significant,Do not cite

Appendix A

- Page 162 of 677 therefore, it was not possible to calculate the LC 50 . For maturation, the Spearman

cor relation was significant and the estimated IM 50 was 143 μ M. Exposure to 100 and 500

μ M atrazine significantly increased (p<0.05) the percentage of oocytes in the GV stage to 40% and 79%, respectively, compared to controls (16%). The MI values were not af fected at any dose level. The percentage of mature oocytes (MII) decreased at 100 μ M (33%) and

500 μ M (2%), compared to controls (61%).

Reviewer‘s Comments: Atrazine had an effect on maturation but not on viability, due to a blockage at the germinal ves icle stage.

A.2.2 Coder, P.S. 2010. An Oral Developmental Toxicity Study of Atrazine in the Rat, Including Cross

- Fostering and Pair

- Feeding. Unpublished Syngenta study

The purpose of this study was to assess the impact of atrazine on: (i) mammary gland development; (ii) in utero only or lactational only exposure; as well as the effect of maternal food consumption on the offspring.

Long

- Evans dams received atrazine (97.1% a.i.) from GD 13

- 19 [the presumptive critical period for mammary gland development ] at doses of 0, 6.5, 50, and 100 mg/kg/day. The control and high

- dose groups contained 42 dams each while the low

- and mid

- dose contained 18 dams). The larger number in the control and high dose group was to

accommodate the need for rats used in the cro ss

- fostering portion of the study. Initially, 15 dams (and their litters) from the control and high dose groups were intended to be assigned

to the cross

- fostering module of this study. However, due to maternal aggression and cannibalization of pups this portion of the study was terminated after only 8 litters were assigned before this aspect of the study was terminated on PND 2. 12 An additional group of 18 dams was used for the pair

- feeding component of the study and their feed was

restricted to match th e food consumption of the 100 mg/kg/day rats. Litters were standardized on PND 4 to 10 pups/litter (with as many females retained as possible). 13 In addition to the common assessments ( clinical observations, body weight and food consumption measures), all female pups were weighed on the day of vaginal opening (VO), and vaginal lavages performed daily to check for estrous cyclicity beginning on PND 50

- 72.
One F 1 female pup/litter was sacrificed on PND 1, 21, 333, day of VO, first day of diestrous

after PND 58 to conduct mammary gland evaluations. Mammary gland evaluations consisted of morphometric analysis of whole mounted 4 th mammary gland. Two hours prior to sacrifice the dams were administered BrDU to assess for cell proliferation in the mammary tissue . Another F 1 female pup/litter was mated with untreated males to produce

an F 2 generation and allowed to deliver and rear their offspring until PND 21. The F 2 pups

12 This aspect of the study will not be discussed further in this review.

13 Only litters comprised of ≥ 6 female pups and at least 10 pups total were used for litter standardization and subsequent assessmentsDo not cite

Appendix A

- Page 163 of 677 were sexed and subjected to clinical observations, body weight and food consumption

determi nations.

For P dams a slight but statistically significant decrease in body weight was noted at the 50 and 100 mg/kg/day dose levels ( ↓4

- 13%) during gestation. Body weight gains were also statistically significantly (p<0.01) reduced during this period at 50 and 100 mg/kg/day (↓30 and 59%, respectively). Although overall body weight gain during lactation was increased at the high dose ( ↑ 26%, p<0.05), the body weight at the mid

- and high

- dose remained lower relative to control (↓4

- 11%) probably due to the initial lower body weight because of the gestational body weight decrements. In general the body weight of the pair

- fed group

animals was statistically significantly lower than control (↓33%) and the highest dose tested (↓47%). During gestation, decreases in food consumption and food efficiency were noted at doses ≥ 50 mg/kg/day (↓22

- 37% and 11

- 39%, respectively). Food consumption and food efficiency were either unaffected or increased during lactation. No significant clinical signs or necropsy findings were noted at any dose level. Also unaffected was the gestation length.

Total litter loss was increased at the 100 mg/kg/day dose le vel (21% vs. 9% control). For most dams this was attributed to diminished nursing behavior (no milk band on pups), poor nesting behavior and/or aggression towards pups. In addition, exposure to 100 mg/kg/day

atrazine resulted in decreases in litter size (↓11%), live litter size (↓15%), pup survival

between PND 0

- 1 (76% survival vs. 91% control), litter weight (↓5

- 14%), and pup weight gain from PND 10

- 14 (↓16

- 19%, p<0.05). Sixty eight pups were sacrificed in extremis at the high dose (21 of these pups had no milk in their stomachs) vs. 36 in control. No other findings were reported in the surviving F 1 pups. Once they reached adulthood, no effects were reported in F 1 animals. Similarly, F 2 pups were unaffected. 14

Mammary gland assessments were conducted on the 4 th mammary gland collected on PND 1, 21, 33, day of VO, and first diestrous after PND 58. The left mammary gland was used for whole mount and subjected to morphometric measurements of the ductal length, ductal network area, epithelial area, and nu mber of end buds. Three hundred seventy six specimens were collected but 38 could not be used (the unusable specimens were equally distributed among dose groups).The 4 th right mammary gland was used for cell proliferation analysis using a BrDU immunoassay

.

No end buds were present on PND1 animals and a few of the PND21 animals. On PND1

, a statistically significant (p<0.01) 20% increase on mammary ductal length was reported at

doses ≥ 50 mg/kg/day along with a decrease in the proliferation of epithelial cells at 100 mg/kg/day (↓31%, p< 0.01). Though not statistically significant, an increase in the number of end buds (↑27%) was noted at the 100 mg/kg/day dose level on PND21

. No effe cts
were noted on PND 33 . However, on the day of VO an increase in the number of end buds

14 F 1 and F 2 pups were never directly exposed to atrazine. Thus any effects seen in either of these generations would have been the result of in utero exposure of the F 1 generationDo not cite

Appendix A

- Page 164 of 677 was noted for the 50 and 100 mg/kg/day dose group as well as the offspring of the pair

- fed (↑32, 17, and 113%, respectively). No effects on mammary gland developmen t were
observed in animals sacrificed after PND 58.

Except for a decrease in pup survival from PND 4

- 21 (83% survival vs 98% in control), and an increase in the number of end buds on the day of VO, the offspring of pair

- fed animals

exhibited little effect s. The pair

- fed dams themselves had a decrease in body weight relative to control ( ↓7

- 16%, p<0.01) during GD 13

- 19. Body weight gain during the treatment period was also decreased relative to both control and the high

- dose group (↓78 and 45%, p<0.01 respectively) as well as food efficiency (↓63%, p<0.01). During lactation, the body wei ght of pair fed dams was 16

- 25% lower than control and 5

- 19% lower than the high

- dose. The body weight gain during this period was 33% lower than control and 45% lower than the high

- dose group while the food efficiency was 28% lower than control.

Reviewe r Comments: In general, this is a well

- conducted study. One of the initial goals of this study was to evaluate the effect of atrazine exposure in utero only or during lactation only in the development of the offspring. This portion of the study had to b e terminated on PND 2 due to maternal aggression and cannibalism. The other major purpose of the study was to conduct an objective and quantitative evaluation of mammary gland development after atrazine exposure during GD 13

- 19. With the exception of an increase in the mammary ductal length reported on PND 21 offspring, there were no effects on mammary gland development that could be attributed to in utero atrazine exposure. Although there was an increase in the number of end buds observed on the day of VO in the 50 and 100 mg/kg/day dose levels relative to control, the increase in the number of end buds in the pair

- fed group was even larger suggesting that this effect may be more related to the decrease in body weight/body weight gain than to atrazine ex posure per se . The NOAEL for both dams and their offspring is 6.5 mg/kg/day and the LOAEL is 50 mg/kg/day based on decreases in body weight, body weight gain, food consumption, and food efficiency (maternal animals) and an increase in mammary ductal lengt h and decrease in epithelial cell proliferation on PND1 (offspring). It should be noted, however, that these putative effects on mammary gland development perinatally did not result in any sustained effects that could be observed at any other time point i n development. Thus their toxicological significance is equivocal.

A.2.3 Coder , P.S. 2010. An Oral (Gavage) Study to Assess the Potential for Detecting the Estrogen

- Induced Luteinizing Hormone Surge in Female Rats. Syngenta Report.

In this study the author attempted to replicate results previously reported by Cooper et al. regarding the impact of a brief (4

- day) exposure to atrazine on the estrogen

- induced LH surge in ovariectomized (OVXD) females.

Initially, 20 adult nulliparous Sprague Dawley ra ts were assigned to receive 0 or 100 mg/kg/day of atrazine (purity 98.8% a.i.) for 4 consecutive days after ovariectomy andDo not cite

Appendix A

- Page 165 of 677 implantation of an estradiol benzoate containing capsule (4 mg/ml) 15 . On study day 0 (first day of study), all females were OVXD and implanted with both an estradiol benzoate capsule and a femoral vein catheter (to allow rapid collection of multiple blood samples). Due to mortalities during or shortly after the surgical procedure (typically before the first atrazine treatment), animal s were reassigned to ensure that at least 16 rats/group were available for hormone assessments. Thirty minutes after surgery animals were gavaged with 0 or 100 mg/kg/day of atrazine. Treatment was done each subsequent day at approximately the same time of day (1200 to 1549 hours). On the fourth day of treatment (study day 3), blood samples were collected at 1300, 1600, 1800, 2000, 2300, and 0100 (study day 4) hours to analyze for luteinizing hormone (LH) and corticosterone blood levels.

After the last s ampling, animals were sacrificed as quickly as possible and a terminal blood sample was collected for the possible evaluation of plasma levels of estradiol, estriol, and

estrone.

Five and 2 rats died prior to the first administration of atrazine in the 0 and 100 mg/kg/day group, respectively. One female in the 100 mg/kg/day group died after the first atrazine administration but necropsy indicated that this death was also due to post

- operative complications.

The incidence of wet yellow material in the urogenital area and right hindlimb was increased in the treatment group (13/16 and 10/16, respectively vs 1

- 2/16 control). Also increased in the 100 mg/kg/day group was the incidence of dried red material around the mouth (10/16 vs 2/16 control). A stat istically significant (p< 0.01) decrease (↓6%) in body weight was reported at the end of the study for the treatment group. This is consistent with a marked decrease in food consumption (↓54

- 250%, p<0.01).

LH and corticoster o ne plasma levels were measur ed using radioimmunoassay techniques. Peak amplitudes of LH were measured by determining the peak hormone concentration regardless of timing. The area under the curve (AUC) for the hormone was plotted using all time points and using Prism1 software (Grap hPad Software). Atrazine exposure resulted in a statistically significant decrease in the LH surge at the 1600 and 1800 hour time points ( ↓62% {p<0.001} and 57% {p<0.01}, respectively). Non

- statistically significant but substantial decreases in LH circulating levels were also noted at the 1300 and 2000 hours (↓43 and 51%, respectively). The LH peak was reduced by 51% (p<0.001) while the AUC was decreased by 53% (p<0.01) after a 4

- day exposure to atrazine at 100 mg/kg/day. Statistically significant (p<0.001) increases in corticosterone levels were only noted at the 1300 hour (↑102%). A non

- statistically significant increase in corticosteron e was also noted at the 2000 time point (↑22%). At all other time points, corticosterone levels in the treatment group were lower than in control (↓7

- 36%). Though not statistically significant,

15 The estradiol benzoate implants produced a daily afternoon LH surge that yielded a synchronized cohort of animals that yielded a surge where the timing and amplitude of each surge could be readil y quantifiedDo not cite

Appendix A

- Page 166 of 677 the corticosterone peak for the atrazine treated group was s lightly higher than for control (↑16%) while the corticosterone AUC was comparable between both groups.

The study author concluded that atrazine exposure at a dose level of 100 mg/kg/day for 4 consecutive days resulted in an attenuated LH surge and an i ncrease in corticosterone levels. Clinical signs observed after this treatment were not severe (urogenital, hindlinmb, and mouth staining) and the body weight decreases were not substantial (≈6%) and of questionable toxicological significance.

Reviewer‘s comments: This is a well

- conducted study that partially corroborates previous findings reported by Cooper et al. (2007). The LH surge

– though not completely abrogated by atrazine treatment at 100 mg/kg/day

– was significantly dampened. However,

unlike previous reports, this study suggests that corticosterone levels do not remain elevated after atrazine exposure but rather return to baseline (or even lower) shortly after.

A. 2.4 Coder P. (2010). Atrazine: An Oral (Gavage) Study of the Effects of

Atrazin e on the Estrous Cycle and the Estrogen

- Induced Luteinizing Hormone Surge in Female Rats. Final Interim Report. Unpublished data from Syngenta Crop Protection, Inc . Study completion date: July 14, 2010. Report Number: WIL

- 639060.

The purpose of this stu dy was to assess the effects of atrazine on sexual maturation (as indicated by the occurrence of vaginal opening), the estrous cycle, and the estrogen

induced luteinizing hormone (LH) surge in female Sprague

- Dawley [Crl:CD(SD)] rats following oral gavage d osing to two separate Cohorts of animals at atrazine doses of 0, 6.25, 25 or 50 mg/kg/day.

Study Design : Atrazine was administered daily via oral gavage to Crl:CD(SD) rats (F1 females and/or their female offspring) at dose levels of 0, 6.25, 25 or 50 mg /kg/day. The study was divided into 2 cohorts (I and II) each with 4 groups of 25 bred F 0 female rats (approximately 13

- 14 weeks of age on GD 0). In each cohort, the F1 female offspring were further divided into 3 subsets. The study design is shown in the following table (taken from page 18 of Syngenta study report).Do not cite

Appendix A

- Page 167 of 677 Cohort I : Mated female rats (25/group) (F0 generation) were administered atrazine (0, 6.25, 25 or 50 mg/kg/day) by oral gavage from gestation day 0 (GD 0) through lactation day 20 (LD 20) a nd continued administration of the offspring (F1 generation) from weaning (PND 21) until 5 days after vaginal opening for a total of 15

- 26 doses (Subset A). The F1 females in Subsets B and C of cohort I were administered atrazine following weaning (beginni ng on PND 21) until PND 133 (day of Subset B euthanasia or initiation of a 3

- week post

- treatment recovery period [Subset C]). Subsets B and C received a total of 113 doses.

Cohort II: Naïve (untreated) F0 females were utilized exclusively to generate n aïve F1 offspring. Naïve F1 females were assigned to subsets D, E, or F. The F1 females in Subset D were administered atrazine (0, 6.25, 25 or 50 mg/kg/day) following weaning (beginning on PND 21) and continuing until euthanasia (5 days following attainme nt of vaginal patency) for a total of 15

- 26 doses. The F1 females in Subsets E and F of cohort II were

administered atrazine from PND 120

- 133 (for 2 weeks prior to the scheduled LH surge evaluation and euthanasia (Subset E) or initiation of a 3

- week post

- t reatment recovery period (Subset F). Subsets E and F received a total of 14 doses.

[Note: The post

- weaning data from F1 offspring in subsets B, C, E, and F were not presented in the interim report because data collection is ongoing.]

Animals

Female Sp rague

- Dawley [Crl:CD(SD)] rats (70 days old) were received from Charles River Laboratories (Raleigh, NC) and housed singly in a room maintained at 71 5oF (22 3oC) with a 14:10 h ligh t :dark schedule (lights on at 0500 hours: lights out at 1900 hours).

Fol lowing acclimation, the rats were paired for mating in the home cage of the male. Following positive evidence of mating, females were individually housed in plastic maternity cages with nesting material. The F0 females were housed in these cages until lact ation day 21, the scheduled day of euthanasia. Following weaning, F1 offspring were housed together by litter in plastic maternity cages with nesting material until PND 28. At PND 28,Do not cite

Appendix A

- Page 168 of 677 F1 offspring were housed individually in suspended wire

- mesh cages until euthanasia. LLC
Certified Rodent LabDiet ® 5002 and water were provided ad libitum

.

Dosing Solutions and Procedures

Atrazine (purity 98.8%) was received from Syngenta Crop Protection, Inc (Greensboro, NC). All dosing was administered by oral gavage in a suspension of 1% methylcellulose/deionized water in a volume of 5.0 mL dosing solution/kg body weight. Control animals received 1% methylcellulose vehicle (5.0 mL/kg) only.

Experimental Procedures

Details of the study design were presented above. The F0 animals were assigned to study groups as follows:

The F1 females were assigned to subsets (A

- F) as follows:

Dose levels were selected based on the results from previous studies. The highest dose level of 50 mg/kg/day was selected because it was sho wn by Foradori et al 2009 to be a dose that had an effect on the LH surge in female Wistar rats and higher than the effectiveDo not cite

Appendix A

- Page 169 of 677 dose of 30 mg/kg/day seen in female SD rats fed atrazine for 6 months in a study by Morseth 1986. The low dose of 6.25 mg/kg/day w as selected to be slightly higher than the previously identified NOAEL for atrazine on LH

- dependent effects in male rats (Stoker et al. 2000).

To eliminate endogenous estradiol, ovariectomies were performed on 1

- 2 F1 female pups/ litter (maximum of 20 fem ales/group/subset) at the following time points: the second day following the day of attainment of vaginal patency (Subsets A and D), PND 130 (Subsets B and E) and following a 3

- week post

- treatment period (PND 151; Subset C and F). At the

time of ovariecto my, a femoral vein catheter attached to an SAI Quick Connect and a subcutaneous silastic capsule containing 4 mg/mL of estradiol benzoate in sesame oil were implanted in each female. Three days following ovariectomy, blood samples for hormone (LH and cort icosterone) evaluated by radioimmunoassay were collected from all surviving females (that underwent surgery) via the surgically implanted femoral vein access port at 6, 9, 11, 13, 15 and 18 hours post

- lights on (lights were turned on at 0500 hours and turn ed off at 0900 daily). Following the 18

- hour post

- lights on blood sample collection, females were euthanized and terminal blood samples for plasma analysis of atrazine (and metabolites) were collected from the vena cava. Each female was subjected to gross necropsy and examined for the absence of ovaries, the presence of the estradiol capsule and the presence and placement of the femoral vein catheter.Do not cite

Appendix A

- Page 170 of 677 Results:

F0 Generation (Cohort I ): All F0 maternal animals in all dose groups survived until

scheduled necropsies. Clinical findings included salivation and evidence of clear material around mouth seen at 25 and 50 mg/kg/day at the time of dosing or 2 hours after dosing during late gestation (Day 15

- 20) or throughout lacatation (Day 0

- 20).

No changes in b ody weight or body weight gains were seen in any dosed group compared to controls during gestation or lactation.

No changes in food consumption were seen in any dose group during gestation. During lactation, at 50 mg/kg/day mean food consumption was de creased compared to control on lactation days 4

- 7 (8%, not ss), 7

- 10 (7%, not ss), 10

- 14 (8%, not ss), 14

- 17 (13%, p<0.05)

and 17

- 21 (7%, not ss) and during the overall lacation period (lactation day 1

- 21) (8%, not ss). Mean food consumption was unaffected in the other dosed groups compared to

controls.

No treatment

- related effects were noted on gestation length, parturition or macroscopic findings.

F1 Litter Data (Cohort I ): In the 50 mg/kg/day dose group, postnatal survival through PND

4 was decreased 4

- 19% compared to control. At the high dose, offspring pup body weights

were decreased from PND 1

- 21 in females (4

- 11.5%) with statistically significant decreases seen on PNDs 1 (6.3%) and 4 (11.5%). In males, pup weights were decreased (not s.s.) on

PNDs 4 (9.8%) and 7 (6%). Mean pup body weight gains in the 50 mg/kg/day dose groups were statistically decreased on PND 1

- 4 (21%, male; 28% female) and slightly lower than controls throughout the rest of the study for both sexes (2

- 6%). No effects on survival , pup body weight/gain were seen in the lower dose groups compared to control.

F1 Generation (Cohort I , Subset A ): In the 50 mg/kg/day dose group, decreases in m ean F1 body weights (6.5

- 25%) compared to controls were seen throughout the post

- weaning perio d, with the decreases being statistically significant except on PND 21 and 46. Decreases in body weight gains in females were observed in the 50 mg/kg/day dose group during PND 21

- 35 (5

- 29%), with significant differences seen during PND 21

- 25 (29%).

Foo d consumption was reduced in the 50 mg/kg/day dose group during PND 28

- 32 (13%)

and PND 35

- 39 (15%). No effects on F1 body weight/gain or food consumption were seen in the lower dose groups compared to control.

A delay in the age of attainment of vaginal patency (1.4 days, p<0.05) was noted in the 50 mg/kg/day dose group compared to controls. This was considered to be treatment

- related. Although not statistically significant, mean body weight on the day of attainment of vaginal patency at 50 mg/kg/day was 4.5% lower than the controls. Mean ages of attainment ofDo not cite

Appendix A

- Page 171 of 677 vaginal patency and mean body weights at the age of attainment were unaffected in the lower dose groups. No treatment

- related macroscopic findings were noted.

F0 Generation (Cohort II ): All Cohort 2 F0 maternal animals in all dose groups survived until scheduled necropsies. Clinical findings of hair loss on various body surfaces and red material around the nose and/or mouth were noted in all groups. No changes in body weight

, body weight gains or f ood consumption were seen in any dosed group compared to controls during gestation or lactation. No treatment

- related effects were noted in any dose

groups on gestation length, parturition or macroscopic findings.

F1 Litter Data (Cohort II ): The mean num ber of pups born, liver litter size, percentage of males at birth, postnatal survival, and offspring weights for th e F1 litters of the untreated F0

dams in C ohort II were similar across dose groups. No remarkable clinical findings were noted in F1 pups.

F 1 Generation (Cohort II, Subset D) : There were no effects on post

- weaning survival, clinical or microscopic findings, post

- weaning body weight, body weight gains, food consumption, the age of attainment of vaginal patency, the mean body weight at the age of vaginal patency, or macroscopic findings for F1 females in Subset D in any dose group compared to the controls.

Hormone Analysis : An LH surge was detected in all treatment groups (0, 6.25, 25 and 50 mg/kg/day) of both Cohort I (subset A) and Cohort I I (subset D). In Cohort 1, subset A at 50 mg/kg/day, atrazine exposure resulted in non

- statistically significant but substantial decreases in the LH surge at the 6 and 9 hour time points (↓62% and ↓47%, respectively). The LH peak was reduced 12% and the AUC was reduced 20%, but both these decreases were not statistically significant. The results at 50 mg/kg/day suggest there was no effect on the suppression of LH surge. These resu lts are unusual in that they contradict the results of other studies by Foradori et al, Morseth et al. and Cooper et al. showing that doses of 50 mg/kg/day and lower result in a statistically significant decrease in LH surge.

An assessment of the effect o f atrazine on LH surge in female SD rats 5 days after becoming sexually mature (as indicated by the occurrence of vaginal opening) indicated that atrazine at dose levels of 6.25 and 25 mg/kg/day did not reduce peak LH or the LH area under the curve (AUC), irrespective of whether treatment began in utero (Cohort I) or post weaning (Cohort II) or at 50 mg/kg/day (Cohort II), compared to controls.

Corticosterone: There were no significant differences in plasma CORT levels, peak CORT levels, or AUC for CORT in any treatment group (cohorts I and II) compared to controls, however, since the time of dosing was not reported, it is difficult to interpret the CORT

results.Do not cite

Appendix A

- Page 172 of 677 Concentrations of atrazine and metabolites in Rat plasma: Plasma concentrations of

atrazine and its chlorometabolites 10.5 to 11.5 hours after dose administration indicated that little or no atrazine remained in the plasma. For Subset D at 50 mg/kg/day, plasma concentrations of DIA, DEA and DACT were approximately 30

- , 60

- , and 6000

- fold greate r than atrazine concentrations. For Subset A at 50 mg/kg/day, plasma concentrations of DIA,

DEA and DACT were 23

- . 30

- , and 2500

- fold greater than atrazine concentrations. See table below (data taken from p. 54 of study report).

.

Table A

- 1 : Plasma concentrations of atrazine and its chlorometabolites 10.5

to 11.5 hours after dose administration

Reviewer’s Comments: The impetus of this study was to see whether or not pre

- or perinatal exposure to atrazine would shift the dose

- response curve to the left. The results seem to indicate that this is not the case. Gestational and lactational exposure does not seem to be affecting cycling. The results showed a delay in the age of attainment of vaginal patency in the 50 mg/kg/day dos e group.

Technically, this was a well conducted study however, the time of day the animals were dosed was not reported. This is very important in interpreting both the LH and CORT data. No effect on LH surge was seen at 6.25, 25 mg/kg/day, or 50 mg/kg/ day. The lack of a specific effect on LH surge at ≤50 mg/kg/day in OVX

- estrogen primed animals in this study contradicts the results of other studies by Foradori et al.2009, Cooper et al 200, and Morseth et al 1996. Five daily doses of 50 or 100 mg/kg/da y atrazine administered to ~13 week old Wistar rats caused a significant suppression of the LH surge (Foradori et al. 2009). Four daily doses of atrazine suppr essed LH secretion at 6.25 mg/kg / d ay in Long

Evans female rats (Cooper et al. 2007). Using a ov ariectomized, estrogen primed model, Morseth et al. 1996 reported a significantly lower dose (3.65 mg/kg/day) of atrazine was required to decrease the LH surge in females treated for a period of 6 months prior to measuring the hormone. No changes in CORT levels were noted at any dose. Since theDo not cite

Appendix A

- Page 173 of 677 study is still ongoing and the results of all the cohorts are not reported, it is difficult to make any definite conclusions at this time.

A.2.5 Cooper, R., Buckalew, A., Fraites, M., Goldman, J., Laws, S., Na rotsky, M., Stoker, T. 2010. Evaluating the Effect of the Chlorotriazine Herbicide Atrazine on the Amplitude of the Pre

- ovulatory LH surge in the Long

Evans Rat. EPA Internal Report.

In this study, the authors continued the work reported in Cooper et al

. 2007 trying to identify a NOAEL for attenuation of the LH surge in the LE rats after 4 days of exposure to atrazine. Previous research indicated that a dose as low as 6.25 mg/kg/day for 4 consecutive days resulted in attenuation of the LH surge. Thus, the work reported herein includes lower doses to try to establish a NOAEL.

Regularly cycling females were exposed to atrazine (purity: 97.1% a.i.) starting on the day of vaginal estrous until the day after proestrous (4 consecutive days) to 0, 25, or 75

m g/kg/day [Block 1]; 0, 6.25, 12.5, 25 mg/kg/day [Block 2] or 0, 1.56, 3.12, 6.25 mg/kg/day [Block 3]. The number of animals used in the analysis varied because in order to be included they had to meet 3 criteria at the time of the terminal sacrifice: (i) have a proestrous vaginal smear, (ii) increased uterine weight, (iii) increased progesterone concentration. To assess for the spontaneous ovulatory LH surge animals were decapitated at 1200, 1400, 1600, 1800, or 2000 hrs. Radioimmunoassays were used to

m easure serum LH and progesterone levels.

Uterine weights, progesterone levels, and the onset of the LH surge ( i.e., timing) were unaffected by atrazine treatment in females reaching proestrous. The magnitude of the LH surge, however, was statistically si gnificantly decreased at 3.12 mg/kg/day (analysis of the data for the entire day) or 6.25 mg/kg/day (analysis for the time of peak LH surge; 18 00 hr) (↓ ≈27% and ≈46%% , respectively).

Reviewer Comments: This is a well

- conducted study that provides valuable information for both hazard characterization and risk assessment purposes. Serum progesterone levels, uterine weight, and timing of LH surg e were unaffected for animals reaching proestrous but the magnitude of the LH surge was dampened at doses ≥ 3.12 mg/kg/day. A NOAEL of 1.56 mg/kg/day has been identified for the attenuation of the LH surge. The reviewer

considers selection of this lower dose as a NOAEL appropriate since it takes into consideration the totality of the data rather than concentrating in one specific time point (1800 hrs the time of the afternoon LH surge) which would have resulted in a NOAEL of 3.12 mg/kg/day. These data ar e amenable to benchmark dose analysis to more clearly

describe the dose

- response curve for the attenuation of the LH surge.Do not cite

Appendix A

- Page 174 of 677 A.2. 6 Dooley, G.P., Reardon, K.F., Prenni, J.E., Tjalkens, R.B., Legare, M.E., Foradori, C.D., Tessari, J.E., Hanneman, W.H. (200 8). Proteomic analysis of diaminochlorotriazine adducts in Wistar rat pituitary glands and LβT2 rat pituitary cells. Chem Res Toxicol 21:844

- 851

.

In a protein adduct study using immunohistochemistry and Western blot techniques,

ovariectomized Wistar fem ale 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 harvested, and either frozen in liquid nitrogen or fixed with formalin.

Formali n

- fixed tissues were embedded in paraffin, sectioned, processed for immunohistochemistry and incubated 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, co unterstained with hematoxylin and examined for DACT

- protein

adducts. For the two

- dimensional electrophoresis, pituitary glands were frozen until use.

A similar approach was taken to prepare the LβT2 gonadotrope cells for DACT

- protein adducts analysis an d for the two

- dimensional electrophoretic analysis. Cell cultures of LβT2, 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 solvent (dimethyl sulfoxide, DMSO) for 24 hours. Treated cells on slides were rinsed, fix ed in methanol, mixed with Trit on X, blocked with phosphate buffer, incubated with the DACT adduct antibody, washed and reincubated with commercial goat antirabbit antibody labeled with the fluorescent dye, Alexa Fluor 488, and
examined for DACT

- protein adducts. Under similar culture conditions, LβT2 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 o r LβT2 cells were thawed, pooled, sonicated, and centrifuged. Supernatants were removed; proteins were precipitated with a commercial kit, and dissolved by sonication in 400 μ L of a rehydration buffer. Samples of the tissue or cellular proteins were plat ed onto trays containing gel and subjected to electrophoresis. At the end of the procedure, the two dimensional gels were either stained with Sypro Ruby (for protein detection) or subjected to Western blotting for detection of the DACT

- adducted proteins. Protein spots on the Western blots were matched to spots on the Sypro Ruby

- stained gels. Matched spots from pituitary tissue samples were 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 also excised from the Sypro Ruby

- stained gels, digested, rinsed and processed for MS. Proteins detected in DACT

- exposed LβT2 cells were identified using Sequest search of the MS/MS data. For both sample types, MS spectra

- identified proteins were searched against the National Center for Bioinformation (NCBInr) protein databases using the taxonomy filter Rattus

.Do not cite

Appendix A

- Page 175 of 677 DACT formed 19 spots and covalent adducts with 8 unique proteins in the rat pituitary and 21 sp ots and covalent adducts with 19 unique proteins in the LβT2 cells. The investigators reported that the proteins found in both sample types were: proteosome activator complex subunit 1 (involved in proteosomal degradation but effect on protein stability or function is not known); ubiquitin carboxyl

- terminal hydrolase isozyme L1 (involved in ubiquitin

- tagged protein proteosomal system degradation which is critical to cell growth, differentiation, development and transcription); tropomyosin (this protein bi nds 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, RNA

- bindi ng proteins containing active

- site or solvent

exposed cysteine residues were also detected in both sample types.

Based on the results of this study in combination of the other Dooley studies (in PK section below) along with other studies, the authors sp eculate that ―Because DACT is capable of forming covalent protein adducts and brain tissue is exposed to large quantities of DACT following atrazine exposure, specific modifications 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 c ells exposed in

vitro to 300 μM DACT led to adduct formation between DACT and 19 proteins. This study was not designed to s tudy a potential dose

- response in terms of adduct formation nor does it provide any data pertaining to the functional outcomes of these adducts. Nevertheless, it is noteworthy that these data entail exposure to extremely high levels of atrazine and/or

met abolites that far exceed the potential environmental exposures.

A.2.7 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 Metabo lites in the Female Rat. Tox. Sci. 112(1):88

- 99.

In this article the authors reported 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)

– aff ected the hypothalamic

- pituitary adrenal (HPA) axis in females by monitoring the levels of circulating adrenocorticotropic hormone (ACTH), corticosterone, and progesterone. Previously, it had been demonstrated that these hormones were

affected in males af ter a single exposure to atrazine at a dose 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 gastrointestina l

discomfort.Do not cite

Appendix A

- Page 176 of 677 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 (vehicle), 75 mg/kg atrazine (purity 97%), 60.2 mg/kg DIA, or 50.6 mg/kg DACT (atrazine equimolar doses) on the morning of proestrus. Precisely 15 minutes after treatment, the animals were sacrificed and their trunk blood collected to assay for ACTH, corticosterone, and progesterone. At nec ropsy, the pituitary and uteri 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

treated animals for 5 minutes on stress hormone lev els as well as the pituitary and uterine weights.

With the exception of a statistically significant 33% uterine weight decrease after DACT exposure, neither the 5 minute restraint nor treatment with a single dose of atrazine or DIA affected body weight, p ituitary or uterine weights. In contrast, a single dose of atrazine or DIA resulted in a statistically significant increase in ACTH, corticosterone, and progesterone within 15 minutes of exposure. Interestingly, DACT though the most prevalent of the meta bolites present 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 sacrificing them precisely 15 minutes after resulted in a statistically significant increase in A CTH, progesterone, and corticosterone levels. While the magnitude of the progesterone and corticosterone response was similar to that elicited by atrazine or DIA treatment, ACTH increases after restraint stress were markedly lower than the responses follo wing 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 s tarting 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 pituitary gland and the uteri 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

- treated animals for 5 minutes on four consecutive days.

Body weight and uterine and pituitary weights were unaffected by compound administration though a statistically significant 20% increase in both absolute and relative uterine weight sDo not cite

Appendix A

- Page 177 of 677 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, corticosterone, and progesterone. All of these increases were statis tically 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 rep eated exposure was markedly 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 concentration s were obtained 15 minutes after the last treatment under both exposure paradigms. After 15 minutes, the plasma

concentrations were ranked such that atrazine<DEA<<DIA<DACT. After 4 consecutive exposures to 75 mg/kg/day atrazine, DACT plasma concentration s were ≈400

- fold higher than the parent compound and 7

- fold higher than DIA.

Study 2: Vagotomized

The objective of this study was to determine if the increase in stress hormones observed after atrazine, atrazine

- metabolites treatment were related to ir ritation of the gastrointestinal

tract rather than a direct effect on the HPA axis.

Ten vagotomized rats and 10 intact rats were used in this experiment. Four to 5 of the vagotomized or intact rats received either 100 mg/kg atrazine or vehicle (1% methyl cellulose) by gavage. The animals were sacrificed precisely 15 minutes after treatment and the trunk blood collected for hormone analysis. Atrazine treatment resulted in an increase in ACTH, corticosterone, 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 differ ence was not as marked. Nonetheless, these findings suggest that the stress hormone release noted

after atrazine or atrazine

- metabolite administration is not the outcome of GI discomfort.

Study 3: Intravenous DIA Exposure

This study was also conducted t o evaluate 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 catheter 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 corticosterone levels. Both ACTH and corticosterone levels were statistically significantly increased 15 minutes after DIA administration relative to animals

exp osed to vehicle; suggesting that the release of stress hormones was not due to GI

discomfort but rather a direct effect of atrazine or its metabolites on the HPA axis.Do not cite

Appendix A

- Page 178 of 677 The authors of these studies concluded that, in addition to the hypothalamic

- pituitary

gonadal (HPG) axis, atrazine treatment also affects the HPA axis as evidenced by the increase in levels of circulating ACTH, corticosterone, and progesterone. Given that activation of the HPA axis has been shown to impact reproductive outcomes (likely thr ough interactions of corticotropin

- releasing 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 surge 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 reported in this study, male rats exposed to a single o ral dose to atrazine, DIA, or DEA experienced an increase in ACTH, corticosterone and progesterone. Interestingly, DACT appeared to have little or no impact on the HPA axis although it is the most prevalent of the chloroatrazine metabolites. Thus, it app ears 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 yet corticosterone levels remained markedly elevated and w ere comparable after a single or multiple exposures.

This study is relevant for the risk assessment and supports the MOA for atrazine. These observations strongly suggest that the atrazine

- 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.2.8 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.

This study looked at porcine serum luteinizing hormone levels in response to administration of gonadotropin

- releasing hormone to atrazine

- treated pigs.

From the beginning o f 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 si gns of estrous were observed), blood samples were collected (min 0) from control

and atrazine

- treated groups. Five atrazine

- treated pigs were immediately given an i.v. dose of 100 μg GnRH. Blood sample for porcine LH (pLH) determination were collected

f rom the atrazine

- exposed and the GnRH

- injected atrazine

- treated groups at 20, 30, 40 and 90 minutes. Serum concentrations of pLH were determined by radioimmunoassay.Do not cite

Appendix A

- Page 179 of 677 The experiment was preceded by another which measured pLH concentration in peripheral

bl ood samples from 3 intact pigs during the estrous cycle. Serum concentrations of pLH were determined in samples collected from the onset of estrous (days 0 and 21) between 8 and 9 am, and then twice daily at 3

- hour intervals, and on days 1,2, 4, 6, 8,10, 1 2, 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 cycle was increased from 2.6 to 3.9 ng/ml on days 0 and 21, which indicated the on set 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 showing significantly reduced estrogen

- induced LH response following atrazine

administration in f emale rats. The single iv GnRH administration to atrazine

- treated female pigs resulted in significant increase in pLH 20 and 30 minutes after GnRH administration, indicating that attenuation of GnRH release in atrazine

- treated animals was responsible for

t he suppression of serum pLH secretion.

Reviewer‘s Comments: This study is relevant to risk assessment in that it supports the

neuroendocrine MOA identified for atrazine . By demonstrating that suppression of the LH secretion can be abrogated by providing an exogenous source of GnRH, the study authors have clearly demonstrated the relationship between disruption of GnRH's pulsatile release and attenuation of the LH surge.

A.2. 9 Juliani, C., Silva

- Zacarin, E., Santos, D. and Boer, P. 2008. Effects of

Atrazi ne on Female Wistar 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 adult female Wistar rats to determine the possible intrafollicular target site through which 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 evaluate 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 the 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 alterations (Cooper et al. 1996; Laws et al. 2000). TheDo not cite

Appendix A

- Page 180 of 677 study was conducted in two blocks. In the subacute treatment block, females received 0 or 400 mg/kg /day atrazine for 14 days. In the subchronic treatment 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 treatments, the animals were euthanized and ovaries were removed and prepared for histology and immunohistochemical analysis. Primary, preantral, and antral follicles 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 presence 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 animals after 30 days. This was lower than the weight gain in the control group (1 8%). Clinical signs included fatigue and weakness.

In the subchronic treatment group (0.75 mg/kg/day for 30 days, rare multioocytic follicles were observed in the treated group. In the subacute treatment group (400 mg/kg/day for 14 days), atrazine induce d oocyte toxicity as evidenced by the presence of preantral follicles with disorganized granulosa cells and/or a degenerating oocyte with discontinuous zona pellucida, which were not observed in the control group. Ovotoxicity was also

observed in antral f ollicles 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 irregularities of the Zona Pellucida.

Low doses of atrazine induced an incre ased expression of HSP90 in follicular granulosa cells in comparison to the control group. However, the positive reaction product, indicative of the HSP90 expression, was not found in follicular granulosa cells of ovaries collected from rats exposed to sub acute 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 photomicrographs

, thus it is difficult to assess the relevance of the findings 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.Do not cite

Appendix A

- Page 181 of 677 A.2.10 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.

The purpose of this study was to evaluate the effects of two a trazine by

- products, diamino

s

- chlorotriazine (DACT) and hydroxyatrazine (OH

- ATR), and a struct ur ally similar

chlorotr iazine, propazine (PRO) on female pubertal development. Previous studies by

Laws et al. h ave shown that atrazine delays the onse t of pubertal development in female

rat s.

Female Wistar rats (n=15/treatment group) were gavaged fro m 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 record ed. Body weights were recorded daily. Tissue weights for each animal were recorded for liver, kidney, adrenals, ovaries, uterus, and pituitary. Histological evaluations were performed on the thyroid, uterus, and ovaries. [Note: The Protocol for the Asses sment 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 sug gested that chlorotriazines would affect thyroid function.]

DACT (33.8, 67.5, and 135 mg/kg) significantly (p<0.05) delayed the age at va ginal opening by 3.2, 4.8, and 7.6 days, respectively, compared to controls (33.1± 0.4 (SE) days of age). Four of 15 animals in the highest DACT dose group did not attain vaginal opening by necropsy at PNDs 41

- 42. B ody 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 dos e DACT animals remained significantly lower (10.4%) than controls. PRO (106.7 and 213 mg/kg) 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 o f 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 significant differe nces in tissue weights wer e seen in any of the OH

- ATR animals. Pituitary weight s 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 seen for serum T3 or TSH f ollowing exposure to any of the test chemicals. An absence of corpo ra lutea was noted in 4 animals in theDo not cite

Appendix A

- Page 182 of 677 highest DACT treatment group that did not attain VO prior to necropsy. No treatment

related microscopic changes were seen in any animals in the PRO or OH

- ATR groups.

Th is study concluded that oral exposure to DACT and PRO from PNDs 22

- 41 delays the age of v aginal opening in Wistar rats in a dose

- dependent manner. The no effect level (NOEL) for DACT (16.7 mg/kg) was ident ical to the e quimol ar NOEL fo r atrazin e (25 mg/kg). T he LOEL for delay in vaginal 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: I t appears that post

- natal exposure to D ACT 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 significantly higher for the delayed pups. Given that atrazine is quickly metabolized in the body, it appears that DAC T is driving the delays in puberty
onset. The DACT NOAEL for sexual maturation reported by Laws et al. (25 mg/kg/day in

atrazine mola r equivalents) was higher than the 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.

A.2.11 McMullin, T.S., Andersen, M.E., Nagahara, A. Lund, T.D., Pak, T., Handa, R.J., Hannem an, 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.

High oral doses of atr azine (ATRA) suppress the estrogen

- primed luteinizing hormone (LH) surge in ovariectomized female rats. The mechanism by which ATRA mediates this action is unknown, but may be related to its anti

- estrogenic activity since LH release from the pituitary glan d is largely regulated by estrogen binding to its receptor in the hypothalamus. Since atrazine is rapidly metabolized 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 study 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 metabolite diaminochlorotriazine (DACT) on the estradiol benzoate (EB)/progesterone

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 o f 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

fu nction was also examined in response to exogenously added gonadotropin

- releasing hormone (GNRH). DACT attenuated the LH surge by 47% as compared to controls, indicating a decrease in pituitary function.Do not cite

Appendix A

- Page 183 of 677 Binding studies with uterine cytosolic containing E R 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 inhibition binding constan t (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 estradiol benzoate treatment alone. Moreover, ATRA failed to block estrogen

- induced progesterone mRNA expression as compared to estrogen

trea tment alone.

Reviewer‘s Comments: In summary, 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 findin g is not consistent with the findings of several other researchers that show 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 ATRA 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 invo lves at least in part decreased pituitary

function.

A.2.12 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.

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 rep eated dose toxicity study and at doses of 0, 3, 30, or 100 mg/kg/day for the female fertility study from two weeks before 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 studie s 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 the LH surge at 30 mg/kg/day (McMullin et al. 2004).Do not cite

Appendix A

- Page 184 of 677 Five week old female SD rats were treated for either two or four weeks. Clinical signs and mortality w ere 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 we re taken for hematology and blood chemistry. The ovaries, uterus, and pituitary gland were weighed. These organs, as well as the vagina and mammary gland, were examined histopathologically.

Female Fertility Study: Dose levels of 3, 30 or 100 mg/kg/day we re selected as described above. Ten week old male and female 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, f emales were caged with untreated males 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 anim al did not copulate. Clinical signs and mortality were observed twice daily during the administration period and once daily during the other periods. Body weights were measured twice a week during the pre

- mating and mating periods and on Days 0

- 8, Day 11 a nd 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 presence 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 and the thoracic and intraperitoneal organs were examined macroscopicall y.

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 soiled perineal region at 300 mg/kg/day. However, n o incidence for these findings was provided. In the four week study, clinical signs included lacrimation, salivation, and soiled perineal region 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% inc idence in control).Do not cite

Appendix A

- Page 185 of 6 77 Decreased absolute ovarian weights were observed 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

- week study. In the two week study a t the high dose, histopathological findings occurred in the ovary (loss of currently formed co r pora lutea, decrease in numbers of previously formed corpora lutea, increased in large

- sized atretic follicles, swelling of previously formed luteal cells), the uterus (atrophy), vagina (mucification and increased single cell necrosis in the vaginal epithelium) and mammary gland (lobular hyperplasia). No weight or histological changes were observed in the pituitary gland. In the 4

- week study at the high dose, h istopathological findings occurred in the ovary (increase in large

- sized atretic follicles (slight), swelling of previously formed luteal cells (slight)), uterus (atrophy), and mammary gland (lobular hyperplasia and increased lactation). The four week tre atment 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 es trous cycle.

Results for Female Fertility Study: No mortality occurred. Clinical signs included salivation at 30 and 100 mg/kg/day and lacrimation and decreased stools at 100 mg/kg/day. However, no incidence of these findings was provided. Atrazine did n ot show significant effects on the fertility study in rats. The copulation index, fertility index, number of corpora lutea, number of implantation sites, numbers of live and dead embryos, and percentage of preimplantation and postimplantation loss were n ot affected by atrazine treatment. 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 grou p, which could be due to the anovulatory effect of atrazine. 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 examinatio n 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 fur ther exacerbate the effects. In contrast, no effect on fertility was observed at a dose of 100 mg/kg. While one of the objectives of this study was to compare ovarian histology with fertility, the lack of identical doses in the repeated dose toxicity

st udies 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 surg e currently used as the point of departure in the Agency‘s risk assessment.Do not cite

Appendix A

- Page 186 of 677 A.2.13 Stoker, T.E., Zorrilla, L., Strader, L., Gibson, E., Kamel, A., Qian, Y., Podhorniak, L. 2010. The Distribution of Atrazine in the Wistar Rat Following Gestational/Lactat ional Exposures. EPA Internal Report.

In this study the authors studied the transfer of atrazine and its metabolites into the fetus or the pups after maternal exposure during gestation (GD 18

- 20 or GD 14

- 20) or during gestation and lactation (GD14

– PND 10). Also evaluated was the potential of atrazine

and/or its metabolites to cross the blood

- brain

- barrier (BBB) by measuring the amounts of compounds present in the brains of both maternal animals and neonates.

Time

- pregnant animals were exposed to 0, 5, or 25 mg/kg/day atrazine (97% a.i. purity) by gavage during GD 18

- 20, 14

- 20, or GD 14

- PND10. Dams sacrificed on GD20 received the compound 2 hours prior to sacrifice and the blood, brain, adrenal and mammary glands were collected and tissues weighed. Tw o fetuses/sex/dam were removed from the uterus and examined for atrazine and/or metabolites content (whole body). Dams sacrificed on PND 10 were separated from their pups 3 hours before the time of sacrifice, treated with compound or vehicle one hour afte r separation from the pups, and allowed to nurse their pups for 30 minutes just before sacrifice. Dams‘ tissues collected included blood, brain, adrenal, and mammary glands and solid tissues weighed. One male and one female pup per dam was perfused and t he plasma, milk from stomach, adrenal glands, gonads, and brains were collected and analyzed for atrazine and/or metabolite content.

In general, the major metabolite detected in dams, fetuses, and pups was DACT although atrazine, DEA, and DIA were also de tected albeit at substantially lower levels. In dams exposed solely during gestation ( i.e., GD18

- 20 or 14

- 20) , DACT accounted for ≈ 77% of the total chlorotriazines (TCT) detected in plasma, 85% of TCT in brain, 65% of TCT in mammary glands, and 60% of TCT in adrenal glands. After DACT, DIA was the most common metabolite detected with levels ranging between 11

- 20 % of the TCT detected in

tissues. Atrazine per se accounted for <1% of TCTs in plasma and brain and 6 and 11% of the TCTs in mammary and adrenal glands, respectively. In fetuses, DACT accounted for ≈
78% of the TCTs detected in the whole fetus and DIA ac counted for ≈ 18%, atrazine per se accounted for <1%).

Similarly, for dams exposed from GD14

- PND10 DACT was the most prominent metabolite. DACT accounted for ≈ 90, 95, 75, and 90% of TCTs detected in plasma, brain, adrenal, and mammary glands, respective ly. DIA, the second most common metabolite in all tissues (except the adrenals), accounted for 3

- 8% of the TCTs detected. In the adrenal glands, the parent compound (atrazine) accounted for ≈ 11% of the TCTs while in other

tissues it accounted for <1%. In pups, DACT was by far the predominant metabolite detected accounting for 99, 91, 100, and 96% of TCTs in pup plasma, stomach content, adrenal glands, and brain, respectively. DIA was either not detected or detected at levels below the level of quantita tion (LOQ) in the adrenals and detected barely above the LOQ in plasma and brain at the 25 mg/kg/day dose level. In the stomach contents, however, DIADo not cite

Appendix A

- Page 187 of 677 accounted for ≈6% of the TCT. Due to the small size of the gonads in the neonate, the tissues (ovaries or testes) were only analyzed for the presence of DACT. DACT was only detected in the ovaries of the offspring of dams treated at the 25 mg/kg/day dose while it

was detected in the testes at both doses.

It is noteworthy that for all tissues

– except the brain

– the content of atrazine and/or its metabolites was markedly higher in dams than in their pups (≈80

- 97% higher). In the brain, however, the content of atrazine in the pups was comparable or ≈5

- fold higher than in the
dams while the DACT and DIA con tent in the pup brain was 3

- 19% of that detected in the

dams‘ brain.

Table A

- 2 : Atrazine and Predominant Metabolites Tissue Levels in Dams Exposed GD14

- PND10 and their Offspring

Atrazine

dose (mg/kg/day) Tissue Content

At razine DACT DIA

Dams Pups Dams Pups Dams Pups

Plasma (ng/ml) 0 LOQ a LOQ LOQ LOQ LOQ LOQ

5 0.9 ± 0.4 LOQ 960.5 ± 36.2 96.8 ± 4.1 (10%) b 38.3 ± 6.8 LOQ

25 9.8 ± 8.3 LOQ 2477 ±

863.6 330.7 ± 79.2

(13%) 173.8 ± 111.4 1.8 ± 0.32 (1%)

Adrenal (ng/g) 0 LOQ LOQ LOQ LOQ LOQ LOQ

5 10,3 ± 4.2 2.9 ± 2 332.9 ± 151 LOQ 10.6 ± 6.8 LOQ

25 58.7 ±45.6 LOQ 424.9 ±

173.9 86 ± 50.8

(20%) 44.5 ± 28.6 LOQ

Brain (ng/g) 0 LOQ LOQ LOQ LOQ LOQ LOQ

5 1.2 ± 0.2 5.6 ± 5.4 (367%) 710.4 ± 42.5 100.1 ± 9.9 (14%) 11 ± 2.1 L OQ

25 10.6 ± 8.8 9.7 ± 10.1 (92%) 1961 ±675.8 382 ± 102.6 (19%) 55.5 ± 37.1 1.5 ± 0.36 (3%)

a Undetectable or below the level of quantitation (LOQ)

b Numbers in parenthesis represent percentage of maternal content

Reviewer Comments: This is a well

- con ducted study that provides valuable information on the distribution of atrazine and its metabolites into the fetus or neonates following gestational and lactational exposure to the dams. As expected given atrazine‘s fast metabolic profile, DACT was the mo st common metabolite found in dams, fetu s, and pups in all tissues examined. In dams, DACT represented 60

- 95% of the TCT detected in the tissues tested, in fetus DACT was ≈ 78% of the TCT detected in the whole fetus while in PND10 pups DACT accounted for 91

- 100% of the TCTs in the tissues teste d. Overall TCT content in the fetus is at least 40% lower than in the dams (given that only selected maternal tissues were analyzed for TCT content vs whole fetus TCT content, the whole maternal TCT content is most likely higher than what was measured in this experiment). In pups, the overall TCT content for tissues examined is ≈ 80% lower than in the dams.Do not cite

Appendix A

- Page 188 of 677 These data demonstrate that although transfer of atrazine and/or its metabolites between the dam and her offspring occurs, the overall concentratio ns of these compounds reaching the tissues in fetuses and pups is appreciably lower than in the dams. Also noted is that atrazine and its metabolites can cross the BBB both in adults and in pups.

A.2.14 Rayner, J.L., Enoch, R.R., and Fenton, S.E. (2005 ) Adverse effects of prenatal exposure to atrazine during a critical period of mammary gland growth. Tox. Sci. 87(1):255

- 266.

In this study, the authors investigated the effects of prenatal atrazine exposure on mammary gland (MG) development. In particu lar, the objective of this study was to identify critical periods of susceptibility during which atrazine exposure may result in delays in MG development and whether the delays previously reported were associated/secondary to other effects ( e.g., decreases in pup weight).

Two major experiments were conducted as part of this study: (i) critical period experiment

– designed to clarify the critical periods of susceptibility during gestation that resulted in delays in MG development after atrazine exposure, and (ii) fetal weight experiment

– designed to study if decrements in maternal body weight/weight gain affected the number and weight of fetuses at the end of gestation.

Critical Period Experiment

Study Design

Two blocks consisting of 40 pregnant Long

- Eva ns (LE) dams per block were exposed to 0 or 50 mg/kg (2x daily for a total daily exposure of 100 mg/kg/day) atrazine. Eight dams/exposure period/ treatment were exposed for different periods during gestation. Control animals were exposed from gestation d ays (GD) 13

- 19

while atrazine

- treated dams were exposed during GD 13

- 15, GD 15

- 17, GD 17

- 19,
or GD 13

- 19. On PND 4, the litters were culled to 10 pups/litter (6 females and 4 males, when possible). 16 Pups were weaned on PND 22 or 25, weighed and caged

2 f emales/cage. Female pups (2/dam, 4 dams/group/block) were sacrificed on PND4 [from culled animals], PND 22, 25, 33, 46, and 67. It should be noted that females in block 2 were retained until PND67 due to persistent effects on MG development noted in PND 46 females in the first block.

In the lactational challenge portion of the experiment, regularly cycling female pups surviving after PND 67 were bred to control males to produce an F 2 generation. On PND 4, the F 2 were standardized to10 pups/litter. Su bsequently, on PND 11 these pups were separated from their dams for two hours the returned to their dams and allowed to suckle for 20 minutes before being decapitated. Trunk blood was

16 Male pups were used for another study unrelated to these investigations.Do not cite

Appendix A

- Page 189 of 677 collected as well as the pituitary and mammary glands. The pituitary w as weighed while the MGs were prepared for whole mount analysis. The dams were also sacrificed and their uterus examined for enumeration of implantation sites.

Twenty

- four female pups/group were examined for vaginal opening (VO) beginning

on PND 29. The day after VO, the animals were weighed. For females in Block 2, vaginal smears were collected from PND 37

- 67 to determine the impact, if any, of atrazine pre

- natal exposure on cyclicity.

Two or more female pups were terminated on PND 33, 46, or 67 by de capitation; trunk blood was collected on PND 33 and 67. On PND 33, MGs were removed and prepared for whole mount from female pups that had undergone VO. On PND 67, in addition to blood, the pituitary gland, ovaries, and wet uterus were removed and weighe d. The MGs were also collected and prepared for whole mount.

The blood collected during the sacrifices was used to obtain serum that could be analyzed via immunoassay for levels of total testosterone, androstenedione, corticosterone, progesterone, estr one, estradiol, and prolactin.

Results

In general, exposure to 100 mg/kg/day atrazine resulted in substantial and at times statistically significant decreases (↓26

- 100%) in maternal body weight gain regardless of the period of exposure. Pup mortality a fter in utero exposure during

GD 13

- 15, 15

- 17, or 17

- 19 was increased to 33, 41, and 30%, respectively relative to control (25% mortality). Interestingly, pup weight was unaffected by treatment with atrazine until PND 67 when animals exposed in utero from GD15

- 17 or GD 13

- 19 exhibited a statistically significant (p<0.01) decrease in weight. Data not provided.

Onset of vaginal opening was slightly but statistically significantly (p<0.0004) delayed (≈ 2 days) for pups exposed in utero during GD13

- 19 in the absence of body weight decreases. Estrous cyclicity appeared to also be unaffected by

treatment with the te st compound. On PNDs 4, 22, and 25 the MG area was decreased (↓32

- 60%) by treatment with 100 mg/kg/day atrazine during various gestational periods; however, length of the epithelium was not affected. When MD development was scored subjectively , the auth ors stated that atrazine

- exposure delayed the d e velopmen t of the gland based on displays of ―abundant terminal end

buds in 2

- 3 sides of epithelial tree, limited lobules, and sparse branching.‖ These differences, however, were no longer noted during the ex amination of PND 33 animals though subjective scoring continued to consider MG development for these pups to be delayed. During the examination of PND 46 females, MG development was markedly different from control in that the MG of atrazine

- treated pupsDo not cite

Appendix A

- Page 190 of 677 ( particularly GD17

- 19 and GD 13

- 19) exhibited less dense epithelial branching and contained many terminal end buds.

Serum hormone measurements were obtained to assess the impact

– if any

– of atrazine exposure on serum hormone levels. Hormone measures on PND 33 revealed a statistically significant increase (↑269%, p<0.009) in corticosterone level accompanied by a 206% increase in testosterone levels (p< 0.04). In contrast, on PND 67 none of the hormones measured or the reproductive organs (ovaries and

ute rus) weighed appeared to be altered by atrazine exposure.

Female pups exposed to atrazine in utero were mated to control males in an attempt to elucidate if the delays in MG development led to detrimental effects in the dams‘ ability to care for their young. With the exception of one female in the GD 17

- 19 exposure group, all females mated were fertile. Body weights and pituitary gland weights were unaffected by atrazine exposure. On PND4, the litters were standardized to 10 pups and on PND 11 the la ctational challenge took place as previously described. In general, on PND 4 and 11 the offspring of dams exposed during GD 17

- 19 or 13

- 19 were smaller and weighed less (↓13

- 25%) than control. The MGs of female pups sacrificed on PND4 that were born to d ams exposed on
GD17

- 19 or 13

- 19, were taken and assessed for development. Unlike the MGs taken from control female pups, the MGs of females born to dams exposed in utero
on GD 17

- 19 or 13

- 19 had few ductal buds and were undersized both during the

PND 4 an d PND 11 evaluations. However, when the pup body weight is used as a covariate there were no statistical difference between the MGs of these treated pups

and control.

Fetal Weight Experiment

Study Design

Eight pregnant LE dams/dose were exposed by gav age to 0, 25, 50, or 100 mg/kg/day atrazine during GD 15

- 19. On GD20, the dams were weighed, sacrificed and their litters removed. The fetuses were sexed and weighed. In addition, prolactin

levels in maternal blood were measured.

Results Maternal body we ight were statistically significantly reduced (↓42

- 63%) at doses ≥ 50 mg/kg/day. Neither fetal weights nor percentage of resorbed fetuses were affected by atrazine exposure suggesting that the decreases in maternal body weight were not related to litter s ize (weight or number of pups). Though no statistically significant differences in the levels of prolactin were noted at any dose level, prolactin levels were decreased by 49% at the highest dose tested (100 mg/kg/day).Do not cite

Appendix A

- Page 191 of 677 Conclusions

Differences seen in g estational durations were most notable from PND 15

- 17, however, at such high doses it is difficult to determine the exact critical period because the mode of action of Atrazine is unknown and also there were no developmental delays in the rat MG. There wer e no changes in puberty or hormones that could be associated with delayed MG development in this study and presumably because traditional endpoints were measured only, they showed no association with
MG effects

.

A.3 REPRODUCTIVE EFFECTS IN MALES

Summa ry: Reproductive/Developmental Toxicity, Delayed Puberty and Prostatitis

Since the IRED was finalized in 2003, several studies have been conducted assessing the effect of atrazine exposure on male reproductive function and development.

Research has focus ed on a wide array of possible adverse reproductive effects including delayed puberty onset, prostatitis and changes in hormonal and andrology parameters.

There have been a number of studies demonstrating that atrazine delays male puberty in

different st rains of rats following both peripubertal and perinatal exposure (Stoker et al.

, 2000; Friedmann, 2002 ;Trentacoste et al.

, 2001; Rayne r et al. , 2006 and Rosenberg et
al.

, 2008)

.

The primary data collected f ollowing the male peripubertal exposures an d examination of puberty outcomes (Stoker et al. , 2001; Friedmann et al. , 2002, Trentacoste

et al.

, Pogrimic et al. , 2009) is consistent with the effect of atrazine on the central nervous system ( i.e. , suppression of GnRH /decreased pituitary secretion of L H ) 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 matura tion of the sex accessory tissues in the male. Therefore, the delayed growth of the reproductive tract in the male supports the reported alteration of the hypothalamic control of pubertal progression.

A reduction in testosterone levels following atrazi ne exposure has been reported in a

number studies in mammals, as well as other species revealing a consistency in the effects

of atrazine on andr ogens. It is well known that the development of the size of the penis and cornification of the epithelium of t he prepuce and preputial separation in immature rats are regulated by androgens (Marshall, 1966). A decrease in testosterone secretion during the juvenile period can delay preputial separation (Lyons et al. , 1942) and reduce the size of the androgen

- depen dent 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).Do not cite

Appendix A

- Page 192 of 677 In the male rat, atrazine 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 growth 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 PND 45, intratesticular testosterone was decreased by 200 mg/kg. In the studies examining the S prague D awley (SD) rat (Trentacoste et al. , 2001), pubert al onset was delayed by 3 days and 4 days following exposure to 100 and 200 mg/kg atrazine, respectively, with decreases in ventral prostate and seminal vesicles at the same doses (NOAEL = 50 mg/kg). Friedmann (2002) found a

decrease in intratesticular a nd serum testosterone at 50 mg/k g /day and 200 mg/ kg/day following exposure to the peripubertal male SD rat. Thus, a ll three studies found

decreases in either serum or intratesticular testosterone and Trentacoste et al. also found a

decrease in LH at the 200 mg/kg /day dose.

Ventral prostate, s eminal v esicles , lateral prostates and epididym al weight s are significantly reduced at doses just above the doses which delay preputial separation (Stoker et al. , 2001). These effects provide further evidence of a decrease in androgen stimulation. Decreases in androgen hormone concentrations also correspond with the effects observed on pubertal endpoints. For example, Stoker et al. , 2001 demonstrated

that intra testicular testosterone was significantly decreased f ollo wing atrazine exposure peri pubertally. This has corresponded to the work of others show i n g that serum testosterone is decreased by doses of 50, 100 and 200 mg/kg in a different strain of rat (SD) when dosed during a similar period of PND 22 to 47 (Tre ntacoste et al. , 2001; Friedmann, 2002) as shown in Table 4

.

Table A

- 3 : 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 2 3 to 53 SD Decreased intra

testicular 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. ND/50 Friedmann, 2002Do not cite

Appendix A

- Page 193 of 677 and serum T on

PND 47

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 effect of the atrazine metabolites, DACT, DIA and DEA on pubertal development in the rat. In this study, equimolar doses of the metabolite were compared 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

, treatments with DEA at atrazine 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 decrease in lateral p rostate 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 weights were reduced in the DEA , DIA, and DACT at atrazine equivalent doses of 200, 200 , and ≥ 100 mg/kg/day, respe ctively . Serum testosterone was reduced only in the males receiving the 2 highest doses of DIA. This study confirmed that 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 the male rat including nutritional status (leptin/body weight) and environmental (pineal gland/secretion of indole amines) influences. Nutritional status and body w eight are known to have effects on puberty, which are suggested to reflect the metabolic signals in the brain that serve as indices of the metabolic state. These metabolic alterations associated with weight loss or decrease in growth rate are inhibitory

t o the reproductive system and may be related to substances in the body that can alter the release of GnRH such as insulin, amino acids necessary for precursors of neurotransmitter synthesis and essential fatty acids (Ojeda and Urbanski, 1994).

The peri pubertal 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 w ere observed ( ≥ 50 100 mg/kg /day in female s and ≥ 12.5 mg/kg /day male s). However, in the study with SD rats Trentacoste et al. , found that the food restriction group (receiving approximately 10% less food which was equivalent to the 100 mg/kg atrazine

treatment food inta ke) showed the same effects on LH, testosterone and reproductive tract development. A recent paper examined the effects of food restriction on the endpoints in the pubertal male exposure period and found that reductions in body weight up to 10% did

not in fluence 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 milk quality, as the offspring sho wed a decreasedDo not cite

Appendix A

- Page 194 of 677 weight of 7% on PND 4 (Rayne r et al. , 2006). Work by Rosenberg and co

- workers (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 a nogenital distance on PND21 was reduced at 75 and 100 mg/kg/day. Measuring testosterone levels in both the serum

and intratesticular fluid of PND 60 rats revealed that serum testosterone levels 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 effects on PPS and AGD occurred in conjunction with nonsignifcant decrease s 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, t he 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

ho rmones stimulate the gonadal secretion of steroids and is critical for the initiation of the set of events and time course of the HPG reg ulation of puberty in the human

. Thus, it is

essential to take into consideration that all the effects on sexual matur ation 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.

Prostate e ffects including prostatitis are oftentimes attributed to atrazine exposure. The increased incidence of prostatitis 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 fur ther explore this potential adverse health outcome.

First reported in 1999, an increase in prostatitis has been linked t o the suppression of suckling

- induced prolactin release. After lactating dams are exposed to atrazine on

PND1

- 4 at doses ≥ 12.5 mg/kg/day, the incidence 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 some dams while the 50 mg/kg/day dose level elicited a prolactin decrease in all females. Prolactin plays a crucial role in neona tal brain development particularly in the tuberoinfundibular dopaminergic system (TIDA). In the adult male, these neurons are responsible for the down

- regulation of prolactin release from the pituitary. As a consequence of the decreased levels of maternal prolactin during the early lactational period, the offspring of these 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 elev ated prolactin levels which in turn lead to hyperprolactinemia. It is this elevated level of circulating prolactin in the adult males that has been linked to an increased incidence of prostatitis. Thus, an increased incidence of prostatitis in the offspri ng of dams exposed to atrazine during the critical time for TIDA neurons activation (first postnatal week) may attributed to decreases in circulating prolactin

levels in the dam.Do not cite

Appendix A

- Page 195 of 677 Using a cross

- fostering study design, Rayner et al. (2007) investigated if gestational exposure to atrazine could also lead to prostatitis in animals exposed in utero. Dams were exposed to 100 mg/kg /day atrazine from GD 15

- 19 and after birth the pups were cross

- fostered resulting in the following grouping:

ATR

- C: pups exposed t o 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 an d through the milk

C

- C: pups born to and nursing from control dams

Consistent with the findings reported by Stoker et al. , both of the groups exposed postnatally ( i.e.

, C

- ATR and ATR

- ATR groups) exhibited indications of lateral prostate inflammation on ( PND 120). No prostate inflammation was observed in the ATR

- C or C

- C groups suggesting that the early postnatal period encompasses the critical window of susceptibility to atrazine

- induced prostatitis. 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 atrazine exposure led to a decrease in testosterone production in rats, it has been suggested that this decrease would in turn ameliorate prostate carcinoma de velopment. 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 resear chers used a transgenic adenocarcinoma rat model typically used to study prostate carcinogenesis. Rats received either 33 or 61.6 mg/kg/day of atrazine for a period of 13 weeks while another group was placed on a calorie

- restricted diet. Atrazine

treatme nt at the highest dose level (61 mg/kg/day) and dietary restriction elicited a slight suppression of prostate carcinogenesis. Unlike other researchers, testosterone levels, however, were not reduced by atrazine treatment in the study conducted by Kandori et al. (2005). The authors theorized that, since testosterone levels were unaffected by atrazine treatment, the decrease in prostate carcinogenesis observed after atrazine exposure was due to reduced caloric intake (resulting from atrazine exposure) rathe r 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 testosterone levels is due to differences in administration of atrazine (dietary vs. oral gavage). In addition, testosterone levels were measured only at the end of the study a nd were highly variable. Also, there was a potential problem with the homogeneity of the diets (concentrations of atrazine were 60

– 91% of target values ). While this study suggests the possibility that prostate carcinogenesis mayDo not cite

Appendix A

- Page 196 of 677 be a secondary effect t o a reduction in caloric intake, a test system in which testosterone levels are altered would provi de more definitive confirmation.

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

A.3.1 Abarikwu S., Adeslyan A., Oyeloja, T., Farombi, E. 2010. Changes in Sperm Characteristics and Induction of Oxidative Stress in the Testis and Epididymis of Experimental Rats by a Herbicide, Atrazine. Arch.
Environ Contam Toxicol 58:874

- 882. DOI 10.1007/s00244

- 009

- 9371

- 2.

In this study, the authors investigated the effects of atrazine on oxidative stress in rat reproductive organs.

Adult male Wistar rats (10/group) were exposed to 0, 120, or 200 mg/kg/day atrazine (commercial product cotrazine 80WP, an 80% wettable powder) orally (dissolved in soya oil) for 7 or 16 days. Body weight, food intake, and reproductive organ weights were recorded. The following biochemical assays to measure oxidative stress were conducted on the testes and epididymides: catalase (CAT) activity, H 2 O 2 - mediated oxidation of Fe 2+ to

Fe 3+

, superoxid e dismutase (SOD), γ

- glutamyl transferase (

- GT), glutathione

- S

transferase (GST) activity, glutathione (GSH), lipid peroxidation (LPO), which was quantified as malondialdehyde MDA), ascorbate (AA) and protein content. Testicular and epididymal sperm number and progressive sperm motility were measured. Morphological and viability of spermatozoa were examined. Daily sperm production (DSP) and DSP/gram

testis were calculated.

At 200 mg/kg/day body weight was significantly decreased (p<0.05) compared to controls at 7 days (↓21 %) and 16 days (↓19%). No decrease in body weight was seen at 120 mg/kg/day at either 7 or 16 days. Food intake was significantly decreased at 200 mg/kg/day at both 7 and 16 days.

Animals exposed to the high dose of atrazine (200 mg/kg/day) showed stati stically significant (p<0.05) reductions in reproductive organ weights, including epididymis (↓25% absolute/↓12% relative at 7days; ↓29% absolute/↓17% relative at 16 days), seminal vesicle (↓41% absolute at 7 days; ↓28% absolute at 16 days) and prostate (↓ 38% absolute/↓48% relative at 7 days; ↓50% absolute/↓40% relative at 16 days). No changes in these organ weights compared to controls were observed at the low dose of 120 mg/kg/day. The absolute testicular weight remained unaffected at both dose levels, but the relative testicular weights were significantly decreased by 23

- 29% at both 7 and 16 days at both dose levels, however, this was not considered to be adverse since there was no change in absolute testicular weight..Do not cite

Appendix A

- Page 197 of 677 Sperm motility, testicular sperm number, epididymal sperm number and daily sperm production were significantly (p<0.05) decreased at both dose levels and both time points. Significant increases (p<0.05) were seen in dead sperms and abnormal sperm rate at both dose levels and both time p oints.

There was a significant dose related increase in testicular GST and GSH activities during the 7 day study while SOD and CAT activities were similar to controls. The GSH activity was considered to be an adaptive response to oxidative stress. When a trazine treatment was increased to 16 days, CAT activity decreased in both dose groups in epididymis but remained unchanged in the testes, while SOD activity decreased in both dose groups in both the testes and epididymis.

- GT was significantly increase d in the testes at both doses and timepoints, while AA remained unchanged. Epididymal LPO was significantly increased at both dose levels after 16 days. MDA levels in the testes were significantly increased at both dose levels at 16 days but not at 7 days . Hydrogen peroxide generation in the testes remained unchanged in both dose levels after exposure for 7 or 16 days. According to the study author, the seminiferous tubules were normal except for some tubules with mild degeneration. No morphological change s were seen in the epididymis (data not shown),

Significant positive correlations were seen with the following: 1) testis and epididymal weights and testicular sperm number (TSN) and epididymal sperm number (ESN), respectively; 2) testicular and epididyma l MDA levels and abnormal sperm rate; 3) MDA levels with testis SOD activity. Significant negative correlations were seen with the following: 1) testicular and epididymal MDA levels and sperm numbers and 2) epididymal

MDA levels with CAT and SOD activities

.

Comments : The results suggest that the observed biochemical and spermatogenic aberrations may be due to oxidative stress in the rat testis and epididymis, however, the influence of secondary factors such as decreased body weight and food intake cannot be ruled out. There were only mild changes in the seminiferous tubules. The measures of oxidative stress were for the most part modest with emphasis on the high dose effects for a few measures. The overall functional significance of these changes remains unclear given the degree of change in the responses.

A

. 3.2 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) i n vitro

. Repro. Tox. 22:508

- 512.

In this study the authors investigated the effects of four pesticides

– malathion, diazinon, fenoxaprop ethyl, and atrazine

– sperm viability, morphology, and motility pattern in porcineDo not cite

Appendix A

- Page 198 of 677 semen. An in vitro viability stain ing method was used to assess viability while a computer

assisted

- analyzer (CASA) was used to study sperm mobility. 17

Semen samples were collected from 3 Duroc boars of proven fertility and the sperm checked for anomalies in motility or morphology. Only s amples 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 μM in a volume of 500 μl tris

- bu ffered medium [approximately 0, 5.4, 11, or 54 μg] and incubated for 1 hour at 37°C. For the viability analysis, 10 μl of the semen sample were placed on a slide and stained with 10 μl eosin

- nigrosin staining. Sperm that were viable did not incorporate the stain while dead spem were stained purple. For the motility evaluations, 8 μl of sperm sample were placed on a slide and observed using a CASA analyzer. 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 μg of atrazine resulted in an 8% and 21% statistically significant (p<0.05) decrease in sperm viability, respectively. With respect to sperm motility, atrazine

expos ure at 11 or 54 μg led to a statistically significant decrease in progressive motility of 23 and 75%, respectively. Atrazine exposure appeared to affect the mean velocity and linear velocity of sperm. These parameters are indicators of sperm progression. In contrast, other parameters 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 conclude that atrazine has an effect on sperm motility and, therefore, affect fertility. It should be noted, however, that no data are available from this study to corroborate this hypothesis. Interestingly, other researchers have reported that the

outcomes of in vitro fertilization (IVF) using sperm treated with up to 10 μM atrazine were

not affected. The authors also speculate that the effects in sperm motility may be a sequela of mitochondrial function disruption

– cellular respiration, in particular.

Reviewer‘s Comments: Overall, the data suggests an effect of atrazine on sperm motility but not morphology or viability. The study, however, entails direct exposure of germ cells to atrazine without taking into account physiological conditions that may preclude the pesticide from eliciti ng this effect in vivo . Given that atrazine is rapidly metabolized, it is likely that the metabolites and not the parent compound are the moieties reaching the sperm and testis. This consideration, however, was not accounted for by the study

authors. Mo reover, 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

hum an health risk assessment

17 In this evaluation only the results for atrazine will be discussedDo not cite

Appendix A

- Page 199 of 677 A.3. 3 Friedmann , AS. 2002. Atrazine inhibition of testosterone production in rat males following 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). For both exposure scenarios, animals were killed on PND 49 between 9 and 11 in the morning 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 pilot 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 interstitial fluid T.] For the in vitro treatment looking at testosterone production, Leydig cells isolated from rats on PND 49 were co

- cultured with 50 μg/ml or

232 μ M atrazine in the presence of a maximally stimulating concen tration 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 the 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 cont rol in the 3

- day exposure treatment.

In vitro treatment of Leydig cells for 3 hours with atrazine culture medium resulted in significantly reduced testosterone production (35%) compared to controls. Atrazine also reduced testosterone levels in Leydig cell s stimulated with db

- cAMP by 34%. However, atrazine did not reduce testosterone production when cells were stimulated with hydroxycholesterol.

The author concluded that atrazine inhibits steroidogenesis in vitro and in vivo . The mode

of action is via inh ibition 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 lo wer serum T. This argument was supported in the Stoker et al., paper mentioned 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 short

- 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 pr oduction by the Leydig cell was decreased when exposed to atrazine. The mechanism for this, if repeatable, isDo not cite

Appendix A

- Page 200 of 677 unclear. Friedman did suggest that as the synthesis of testosterone in the presence of hydroxycholesterol was not impaired, the herbicide interf eres with cAMP‘s role in stimulating Leydig cells Furthermore, it should be noted that unpublished data from the EDSP validation studies conducted at NHEERL, used much lower doses of atrazine in culture and measured T production in H295R cells and compare d the results with similar treatment to primary Leydig cells. In both cases, T production was increased (see Higley et al., paper for data on H295R cells using doses ranging from 1

- 100 μM).

Reviewer‘s Comments: In summary, a limited number or no measures 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 o f doses.

A.3. 4 Hase Y, T atsuno 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. Bioch e m Biophy Research Comm 366: 66

- 72

.

Thi s paper demonstrates the ability of ATR to alter human sperm motility in vitro , and tests hypotheses on the mechanism through which 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 (decreasing ATP available for sperm motion).

Note: The title is misleading because the authors did not actually determine effects on

sperm m itochondria

– 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 sam ples were washed to recover motile sperm and the sperm were then cultured with atrazine (ATZ) and ATZ

- NH2, a modified version of ARZ needed for subsequent biochemical studies). Oligomycin, a sperm metabolic poison which uncouples ATP production in mitroch ondria, was used as a positive control. Oligomycin inhibited % 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 significan tly decreased at the same concentrations of test compounds that inhibited % progressive motility. Motility assessments were made using established computer aided sperm analysis (CASA) methods.

Data rule out several early steps in ATP synthesis and ma ke 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 alsoDo not cite

Appendix A

- Page 201 of 677 conclude that ―This s tudy 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 seen at >100 μM, exposure levels that probably do not result

from nor mal use of the herbicide. It is, therefore, uncertain whether the observed phenomena are ecologically relevant.‖

Reviewer‘s Comments: The relevance of these findings to risk assessment is questionable given that the concentration levels required to aff ect sperm motility were high and exceed likely exposure levels resulting 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.3.5 Kandori, H, Suzuk i 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

.

The effects of a 13

- week dietary administration of 50 0 and 1000 ppm atrazine on the development of androgen

- dependent prostate cancer were investigated using male probasin/SV40 T antigen transgenic (TG) rats (Sprague

- Dawley genetic background, 6 weeks of age). Based on food consumption data, the correspond ing 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 animals. A control group was included.

The rationale for using TG rats was based on previous findings that at razine inhibited testosterone production in the Leydig cells directly 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 esta blished in their laboratory, and have developed well

- differentiated adenocarcinomas in the prostate at 100% incidence before 15 weeks of age. Since the tumors are considered to be completely androgen dependent, the hypothesis was that exposure of atrazine to the TG rats would cause inhibition of testosterone production, resulting in reduced carcinoma development. Since atrazine has been reported to decrease body weights, a dietary

- restricted TG rat group was

also included to assess the influence of reduct ion of calorie intake on the development of
prostate cancer.

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 treatment period, the following

organs were s ubjected to histological examination: testes and accessory sex organs, ventral prostate, the dorsolateral prostate including the urethra, 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

- massDo not cite

Appendix A

- Page 202 of 677 spectrometry. Immunohistochemistry for 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 observed. Body weights in the atrazine treated groups were significantly lower than in the control g roup. 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 treatment

- related changes in relative organ weights in any treatment group were reported. Serum testosterone levels w ere not significantly different between the atrazine and dietary restricted groups compared with control group. Atrazine and its metabolites diaminochlorotriazine, desethylatrazine, and desisopropylatrazine were detected in the

serum of the atrazine treat ed animals, but not in the control or dietary restricted groups.

Histopathological examination of the ventral, lateral, and anterior lobes of the prostate revealed lesions in all groups including controls. The authors described the lesions as marked ep ithelial 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 well

- d ifferentiated adenocarcinomas in human cases and were therefore diagnosed as adenocarcinomas. These were observed in the ventral, lateral, and anterior lobes of the prostate in almost all rats. In the dorsal lobe, adenocarcinomas were observed in two of three rats of each group. The incidences of adenocarcinomas in any treatment group were not different from those of the control group. Foci within glands with less proliferation were also observed. These exhibited crowding of stratified epithelial cells with irregular spacing and occasional luminal bridging. Nuclear atypia were described as severe, however, basic structures of the prostate glands were mostly maintained, comparable with prostatic intraepithelial neoplasias (PIN) of human cases.

Becaus e almost the entire lobes of the prostate were occupied with tumor lesions at week 13, morphometric assessment ratios of the prostate epithelial area to the whole prostate tissue area were evaluated. Morphometric examination of the prostate epithelium rev ealed significant decreases in the 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 dorsal lo be in the atrazine groups showed a tendency to decrease (not statistically significant) in a dose

- dependent manner. No significant effect was observed in the anterior lobe in any treatment groups.

SV40 Tag and androgen receptor expression were detected i mmunohistochemically in almost all cells of the prostate adenocarcinomas and PIN. No treatment

- related changes in the testes, epididymides, and seminal vesicles were observed.Do not cite

Appendix A

- Page 203 of 677 The authors concluded that dietary administration of atrazine for 13 weeks sh owed slight suppressive effects on the development of prostate cancer in the probasin

- SV40 T antigen transgenic rats, even though atrazine treatment did not affect serum testosterone levels. Based on the observation that there was equal or greater suppres sion of prostate neoplastic lesion development 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, rather than endocrine effect s.

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 would provide a more definitive confirmation

.

A.3.6 Laws, S.C., Hotchkiss, M., Ferrell, J., Jayaraman, S., Mills, L., Modic, W., Tinfo, N., Fraites, M., Stoker, T., and Cooper, R. (2009). Chlorotriazine

Herbicides and Metabolites Activate an ACTH

- dependent 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 exposure results in an increase in steroidogenesis leading to increases in corticosterone, 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). 18
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

- exposed 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, corticosterone, progesterone, and prolactin plasma levels were

measured usin g commercially 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) 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 cha nges noted15 minutes after exposure at 50 mg/kg and above. For both of these hormones, maximum plasma 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

18 This review will only discuss data for atrazine, DIA, DEA, and DACTDo not cite

Appendix A

- Page 204 of 677 progesterone (13.6

- fold) within 15 minutes of exposure. In contrast, exposure to DACT 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 minute s of exposure. The levels of the metabolites increased with time. Five minutes after exposure to atrazine DIA was the most prevalent metabolite but by 180 minutes DACT was the predominant metabolite seen in plasma.

The authors conclude that the rapid in crease in ACTH after exposure to atrazine or its chlorinated metabolites induces adrenal steroidogenesis indicative of initial effects at the hypothalamus and/or pituitary level.

Reviewers comments: This is a well

- conducted study that carefully evaluates not only the

dose

- response but also the temporal

- response 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 the authors did not establish a NOEL for this study, the no effect level may be established at 5 mg/kg

– the lowest atrazine dose tested. Interestingly, though DACT is the final and most prevalent atrazine metabolite, it has little impact on corticosterone, progesterone, or ACTH levels. The relationship between these hormonal changes and the adverse reproductive health outcomes observed after atrazine exposure, however, remains unclear. To what extent or for how long do these hormo nes must remain

elevated before adverse effects are seen is not known at this time and cannot be

determined from these data.

A.3.7 Laws, SC, Stoker, TE, Ferrell, JM, Hotchkiss, MG, and Cooper RL. 2007. Effects of Altered Food Intake During Pubertal Deve lopment in Male and

Female Wistar 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 per iod encompasses the age of the onset of puberty 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 body weight. Body weight was recorded daily. The day of

prepu tial separation (PPS) in males and vaginal opening (VO) in females was recorded. Females were killed by decapitation on PND 41

- 42 and liver, kidney, adrenal ovary, uterus and pituitary weights were recorded. Males were killed by decapitation on PND 53

- 54, and liver, kidney, adrenal, pituitary, left testis, ventral prostate, left epididymis, seminal vesicles with coagulating gland (with fluid) were removed and weights recorded. Thyroid and reproductive tissues were examined histologically. Serum T3, T4 and TSH, glucose, leptin and pituitary (males only) prolactin and LH were measured.Do not cite

Appendix A

- Page 205 of 677 The terminal body weights of FR females (PND 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 endpoints were not affected by any FR group asso ciated

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 testosterone, LH or prolactin were noted in any of the males. Serum glucose

concentrations 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 serum 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 pituitary, adrenals, liver and kidney were significantly lower in the females 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, an d19% 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 relative 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 g rowth and reproductive development in the male and female 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 postnatal period whic h usually resulted in over 50% weight loss. This study 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 con cept that a 10% reduction in body weight is a reasonable basis for setting the maximum dose for the female pubertal protocol in conjunction with any other information concerning the toxicity of the test compound (e.g., clinical signs, other toxicity).

A.3 .8 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 BoarDo not cite

Appendix A

- Page 206 of 677 Sperm. International Journal 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, spontaneous acrosome reaction (SAR) and progesterone

- induced acrosome reaction (PIAR) in boar sperm. 19

Sperm samples were obtained from the sperm

- 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

we re used. Sperm capacitation 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 a nd incubating for 30 minutes. The spontaneous acrosomal reaction without progesterone induction is called SAR. Sperm membrane status was assessed by chlortetracycline staining and analyzed with a UV epifluorescence microscope. Sperm with a uniformly distri buted fluorescence over the whole head and an intact acrosome were considered noncapacitated; sperm showing bright fluorescence in the acrosomal region

beside a fluorescence

- free band in the postacrosomal region with an intact acrosome were considered capa citated; 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 concen trations of atrazine (technical grade, purity not reported) 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, 2 7, and 6, respectively. The median lethal concentrations of atrazine were calculated. Final concentrations of 8, 20, and 40 μM atrazine were used in the experiments. These concentrations corresponded to the LC 50/5 , LC 50/2 , and LC 50 , respectively. The conce ntrations 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 incubation. The control was the same solvent with the herbicide (0 μM).

Sperm Capa citation: 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 groups. Capacitation decreased significantly only at the high dose (40 μM), to 79% of control s (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 noncapacitated population was reduced at all concentrations, compared to controls (p<0.05).

19 In this evaluation, only th e results for atrazine will be discussed.Do not cite

Appendix A

- Page 207 of 677 PIAR: SAR observed in control sperm immediately after capacitation was 17%. After 30 minutes of progesterone induction of acrosome reaction, PIAR increased to 30% in controls. This increase shows that progesterone acts as an AR inducer. All atrazine concentrations produced a significant decrease (p<0.0 5) 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 literature to imply that atrazine selectively targets mature sperm and impairs their fertilizing abili ty 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 use d 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. Ther e is also no evidence that atrazine reaches maturing sperm in the testis, epididymis 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 exposures nor provide biological plausibility for suspecting that atrazine may selectively target sperm and thereby impact fertility or sperm function. The

authors did note that the atrazine concentrations used in this study are not in the relevant range to produce in vivo effects.

A.3. 9 Mu, H. , Shang, P. and Xu, J. (2008). Testicular Toxicity and Mechanisms of Chlorotoluron Compounds in the Mouse. Toxicology Mechanisms

and Methods. 18: 399

- 403.

This study looked at the effect s of chlorotoluron and atrazine ingestion on male mouse testes during the peripubertal period. 20

Groups of 20 male mice (Chinese Kunming strain) were given either chlorotoluron alone (625, 1250, 2500, 5000 mg/kg), atrazine alone (218.75, 437.5, 875, or 1 750 mg/kg), or a combination of these chemicals (combination 1: atrazine/chlorotoluron: 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, 437.5/5000 mg/kg.) There 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. Following sacrifice testis weights were measured and reco rded. Testes tissue was processed for examination by light microscope and transmission electron microscope.

20 In this evaluation, only the results for atrazine will be discussed.Do not cite

Appendix A

- Page 208 of 677 Superoxide dismutase (SOD) activity was measured. SOD is an antioxidant (removes superoxide radicals).

There was a dose related decrease in relativ e testes weights at all dose levels for atrazine (4.6

- 13%) and combined atrazine/chlorotoluene (6.9

- 14%), although the decreases were not statistically significant. The SOD enzyme activities increased in a dose

- related manner

in animals treated with atrazi ne and combined, with the effects of treatment with the combination of herbicides being greater than the single chemical. Control values were not reported.

Histologic changes in the testes that occurred in treated animals included shedding of spermatogeni c cells, increased intercellular 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 combination 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 endoplasmi c reticulum and Golgi (at combined dose of 625 mg/kg chlorotoluron plus 218.75 mg/kg atrazine) and heterochromatin in the mural cells which appeared in a blocked state, decreased nuclear matrix, spermatogenic cells with swollen appearance, segmental degene ration of cell membranes and tight junctions which were partially damaged (at combined 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. Consequently, this study was not useful for risk

assessment.

A.3.10 Pogrmic K, Fa S, Dakic V, Kaisarevic S, and Kovacevic R. 2009. Atrazine Oral Exposure of Peripubertal Male Rats Downregulates
Steroi d ogenesis 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 peripubertal male Wistar rats after in vivo exposure to atrazine. The study was base d on earlier published papers (described above) that identified a decreased testosterone following atrazine exposure when dosing began shortly after weaning until after sexual maturation (defined as PPS).

Male Wistar rats were treated by gavage with 0, 50 or 200 mg/kg/day atrazine (98% purity)

from PND 23

- 50. Animals were sacrificed 24 hours after last dose of atrazine.

They reported that oral administration of atrazine (200 mg/kg/ay) produced significant reduction in serum T/DHT(assay they used was sen sitive to both). At 200 mg/kg/day, bodyDo not cite

Appendix A

- Page 209 of 677 weight was also reduced significantly as were the weights of androgen

- 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 signi ficantly reduced in the 200 mg group. 50 mg/kg atrazine did not alter serum hormone levels nor the weight of androgen dependent tissues. Adrenal and Testes weights were affected in both dose groups. They pointed out that Trentacoste et al. (2001) sugge sted that testosterone

- reducing ability of atrazine is secondary to weight

loss.

Their in vitro dosing is of interest as both doses caused significant decrease in basal and hCG stimulated testosterone release. There was no dose response and no variance presented in all the cell

- stimulation. However, the variance wa s presented for the dose of 10 μM hCG in a bar graph for hCG 10 μM. In addition to a decrease in T production, there was also a dose dependent decrease in the cAMP concentrations. When the cells were incubated in the presence of hydroxycholesterol, pregn olone, progesterone and androsteinedione, in every case, the amount of T produced from the Leydig cells obtained from the 50 and 200 mg/kg/ atrazine

- treated animals was reduced compared to controls. Importantly, there was no dose response. Finally the au thors reported reduced levels of transcripts for 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. This is in contrast to the observation of Fri edmann, 2002 who reported that in

vitro treatment with atrazine suppressed T from Leydig cells of untreated animals, but the suppression could be overcome by co

- incubating with hydroxycholesterol. If these

observations are both correct then there is a dif ference 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. administration. They report a clear dose

- dependent decrease in ge ne expression of LHR, SR

- B1, StAR, TSPO, SF

- 1, PDE4B, 3bHSD, CYP17A1, 17bHSD in Leydig cells. There was no effect on CYP11A1 and ß

- HSD. They conclude that the inhibition of all these genes following atrazine may be related to

decreased c

- AMP stimulation. 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 enzyme that degrades cAMP).

Reviewer‘s Comments: The qua lity 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 the expression of so many genes in the RT

- PCR

studies demands con firmation. 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 that the doses used in this study were relatively high compared

to t he doses eliciting the neuroendocrine effects and used for regulatory purposes.Do not cite

Appendix A

- Page 210 of 677 A. 3. 11 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 Ap pl Pharmacol: 218: 238

– 248

.

The authors note that previous studies have shown that early postnatal exposure to atrazine delayed preputial separation (PPS) and increased incidence of prostate

inflammation in adult Wistar rats. The authors stated that a cross

- fostering paradigm was used in their study to determine if gestational exposure to atrazine would also result in altered puberty and reproductive tissue effects in the male rat. Timed

- pregnant Long

Evans rats were dosed by gavage on gestational da ys (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: birth dam or gestational exposure only (ATR

- C); milk source or lactati onal exposure only (C

- ATR), exposure both

pre

- and postnatal (ATR

- ATR), or neither (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 prostates were analyzed for myelo peroxidase activity. Histological examination of the right lateral and ventral prostates were conducted. The distribution scoring and severity classification were provided in the study. Body weights were monitored throughout the study.

Mean body weigh t of the ATR dams on GD19 was reduced 13% when compared to control dams. The male offspring in the 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 male s in the ATR

- ATR group. Body weights at the time of PPS were also

decreased in the ATR

- ATR animals.

At PND120, the lateral prostates of the ATR

- ATR offspring group weighed significantly more than those in any other group, even when corrected for body w eight. 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 f or body weight, animals in

ATR

- C and ATR

- ATR groups had significantly increased lateral prostate weights.

At PND120, necropsy evaluation of offspring indicated macroscopic abnormalities of the left and right lateral prostate in the ATR

- ATR group; the ab normalities were characterized by nodular pale white foci present on the outer surface. At PND 220, all atrazine groups had the abnormalities, mostly on the right lateral prostate.

Histological examination of lateral and ventral prostates revealed an inc reased incidence and distribution of inflammation in the lateral prostates of C

- ATR males at PND 120. OnlyDo not cite

Appendix A

- Page 211 of 677 one out of ten ATR

- ATR males also exhibited inflammation; there was no inflammation in
the C

- C and ATR

- C groups. The C

- ATR and ATR

- ATR lateral pros tates had neutrophils as the predominant inflammatory cells. In regard to ventral prostates at PND 120, inflammation was evident 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 a ssess inflammation in the ventral prostate because of a high background present. The authors also noted that although necropsy revealed macroscopic abnormalities on the surface of the lateral prostate of males in the ATR

- ATR group, these abnormalities ―ma y 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 assay and the pathological findings. The authors explained the discrepancy as possibly due to not having both lateral prostates for assay (one use d for the assay, the other for 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 that the results of the study suggest that the developing lateral prostate gland in the Long

- Evans rat is sensitive to atrazine

- induced changes that are transplacental or via nursing. In addition, lactational exposure to these atrazine

- induced changes in creases lateral prostate inflammation.

Reviewer‘s Comments: This study only examines one dose, so there are no dose response evaluations. The authors do not mention the time of day which they killed the males at 120 or 200 days of age or how long the 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 postna tal 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 combination of gestational exposure to the dam and the postnatal milk from those mothers responsibl e 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 disruption of the LH surge.

A

. 3.12 Rosenberg, B.G., Chen, H., Folmer, J., Liu, J., Papadopoulos, V., and Zirkin, B.R. (2008). Gestational Exposure to Atrazine: Effects on the

Postnat al Development of Male Offspring. J. Andrology 29(3):304

- 311.Do not cite

Appendix A

- Page 212 of 677 In this study, the authors investigated the impact of atrazine exposure during gestation on the development of Sprague

- Dawley (SD) rats male offspring. Fourteen to 16 pregnant dams were expos ed via gavage from gestation day (GD) 14

- 21 to 0, 1, 10, 50, 75, or 100 mg/kg/day of atrazine (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 testosterone levels in the newborn pups. The litters of the remaining 7

- 8 dams were culled on PND2 to 8 pups (4/sex/litter). Body weights and food consumption of pregnant dams were recorded daily from GD 14

- 21. On PND21 the body weight s 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 were sacrificed by decapitation and their blood (serum), testes, ventral
prosta te, 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 statistically significantly reduced at doses ≥ 75 mg/kg/day starting on GD18. This decrease coincided 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 app roximately 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 pu p weight decrements, however, were statistically significant.

Intratesticular testosterone levels measured from neonates did not reveal any statistically significant effects at any dose level. Although a 60% increase in intratesticular testosterone

was n oted 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 non

- statistically 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 ranging 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 PN D 60, the animals used to assess PPS and AGD were sacrificed and their testes, seminal vesicles, and ventral prostate weights measured. In addition, testosterone levels in both the serum and intratesticular fluids were measured. No changes in terminal bo dy weight or organ weights were noted. Serum testosterone levels, however, were decreased at ≥ 50 mg/kg/day while intratesticular testosterone was reduced at doses of 75 and 100 mg/kg/day. [Note: graph and text report of T differ; they are reversed.]Do not cite

Appendix A

- Page 213 of 677 Th e authors of this study concluded that in utero exposure resulted in changes that manifested in the neonatal, peripubertal, and adult life stages. The authors, however, did point out that these effects were observed at concentrations that ―are unlikely t o be experienced under any but experimental conditions.‖

Reviewer‘s Comments : The highest two doses 75 and 100 mg/kg/day resulted in significant reductions in the dam‘s BW gain. There was a dose

- dependent loss of pups

-- ~20

- 25% pup death at 75 and 100 m g/kg from PND 0

- 2. There were no differences in neonatal T at PND 0, but there did appear to be effects 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 da ys 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 an drogen status occurred in this study at dose levels substantially higher than those leading to LH surge disruption, which serves as the basis of the Agency‘s current risk

assessment.

A.3.13 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.

Male Wistar rats were gavaged (between 8 and 9 am) from PND 23

– PND 53 with atrazine metabolites (97

- 98% purity), deethylatrazine (DEA), deiso propylatrazine (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 hour s following the last dose on PND 53. The pituitary, adrenal, thyroid, testes, epididymides, seminal vesicles, and ventral and lateral prostates were weighed and examined histologically. 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 weight decrease at both time points.

The day of PPS was delayed by all 3 metabolites. For DIA, PPS was si gnificantly 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/kg/day. For DACT, PPS was significantly delayed at 12.5, 25, 100, and 200 mg/kg/day AED by 1, 2.3, 1, 4, and 4.2 days, respectively with a NOAEL of 6.25 mg/kg/day.Do not cite

Appendix A

- Page 214 of 677 Testicular weights were similar to controls in all treatment gro ups 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 decr ease 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 s ignificantly decreased only in DIA exposed males at 100 and 200 AED. Serum estrone was significantly increased only by DACT at 100 and 200 AED.

The mean anterior pituitary weight was significantly decreased on PND 53 at 100 and 200 AED by DEA and DACT and the highest dose 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. Pituitary 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 treatment group. No effects were observed on thyroid histology of high dose or controls.

Minimal hypospermia (defined as rats in which 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 high dose group of DACT, and 12.5% of the high dose gr oup of DEA. The author reported that this observation is probably a result of the length of the delay in puberty in the high dose groups of atrazine metabolites and the age of sacrifice.

Reviewer‘s Comments: This study shows the effects of the three prima ry atrazine metabolites on pubertal onset and growth of androgen dependent tissues. Interestingly, many of the same effects were observed including delayed PPS and decreased reproductive tract development. DIA was the only metabolite which resulted in de creased serum testosterone and DACT increased 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 the effects reported by Stoker et al. 2002 (6.25 mg/kg/day in atrazine molar equivalents) was higher than the NOAEL (1.8 mg/kg/day) for attenuation of the LH surge currently used as the point of departure in the Agency‘s risk assessme nt.Do not cite

Appendix A

- Page 215 of 677 A.3.14 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/kg/day from postnatal day (PND) 23 to 53. An additional pair

- fed group was added to compare effects of any decre ased 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 an d lateral prostates were weighed and examined histopathologically. Blood and serum were assayed for hormones (testosterone, estrogen, LH, prolactin, TSH, T4, T3).

There was a significant reduction in body weight 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 significantly delayed in the 12.5, 50, 100, 150, and 200 mg/kg/day dose group s 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

gro up.

This study showed that atrazine suppressed 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 5 3 were decreased in the 200 mg/kg/day atrazine group and pair

- fed animals. By PND 120, all reproductive tract organ weights returned to control levels in all treatment groups, except for the ventral prostrate which was still significantly 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 testosteron e 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 effect on serum LH on PND 53, but serum estrogens were increased at 200 mg/kg/day. These effect s 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).Do not cite

Appendix A

- Page 216 of 677 Reviewer‘s Comments: Peripubertal exposure to atrazine resulted in delayed puberty onset and delayed growth of the reproductive tract of male rats. These data su pport the reported alteration of the hypothalamic control of pubertal progression.

A.3.15 Trentacoste, S V.. Friedmann, AS, Youker, RT, Breckenridge, CB, and Zirkin, BR. 2001. Atrazine Effects on Tes tosterone 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 atrazine (96.1%) from PND 22

- 47 by gavage in methyl

cellulo se 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 PND 48 (24 hours following last dose; doesn‘t state time of day killed). A food restriction study was conducted with rats pair fed to the 100 mg/kg/day atrazine treated group. In life measures included daily (?) body weights. The day of preputial separation (PPS) was measured in controls and only the 100 and 200 mg/kg/day dose groups. Testosterone (serum and interstitial fluid) and serum LH con centrations were

measured.

Body weight (9%, 100 mg/kg/day/21%, 200 mg/kg/day), seminal vesicle (32%/55%) and ventral prostate (26%/48%) weights, serum 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 affected by the atrazine 100 and 200 mg/kg/day, a group of males were pair fed to the 100 mg/kg/day group. The underfed only animals had a decrease in T and LH on PND 47 that was similar to the 100 mg/kg/dose group. The authors concluded that these results indicate that atrazine exposure, up to 50 mg/kg/day, had no effect on the hypothalamic

- 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 effects observed after reduced food consumption. The author‘s emphasized that caution must be exercised before concluding that atrazine (or any potentially toxic chemical) has direct, detrimental 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 the assessment of potentia l toxic effects.

Reviewer‘s Comments: The study author‘s contention that the changes in the male reproductive tissue weight and PPS changes observed was the result of decrease body weight is not supported by an earlier study by Stoker et al., 2000. In t hat study, Stoker examined similar measures in the male Wistar rat. This group reported that 100 mg/kg/day reduced BW approximately 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 (which 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 inDo not cite

Appendix A

- Page 217 of 677 males treated with 25 mg/kg/day and above, which was very si milar 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%). Serum T was not reduced in Stoker et al study, however they did have smaller ventral prostates, seminal vesicles and a delay in PPS (albeit significantly less than that observed 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. Freidma n (2002) reported a significant decrease in both serum testosterone and interstitial testosterone in Sprague

- Dawley rats treated during the same PND period, and body weight was reduced 7% (230 = control 215 = atrazine 50 mg/kg/day

from 22

- 48.

A.3. 16 Vic tor

- 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

.

This study evaluated the effect s of atrazine on the steroidogenic pathway in adult male rats by analyzing the 3

- HSD expression, plasma and testicular estrogen and testosterone levels, expression of androgen receptor, and morphophysiological changes in the testes.

Adult male Wistar rat s (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 formula tion 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 exami ned histopathologically. Changes in the expression of 3

- hydroxysteroid dehydrogenase (3

- HSD) enzyme and androgen receptor (AR) in the testes were studied by immunohistochemistry. 3

- HSD is the enzyme responsible for various stages of the steroidogenic p athway, including synthesis of adrostenedione, the testosterone precursor. Adrenal glands were also evaluated for the 3

- HSD by immunodetection for comparison between steroidogenic tissues. Immunostaining was quantified by computer

- assisted image analysis. AR staining was quantified in 30 interstitial Leydig cells as well as 30 Sertoli cells lining seminiferous epithelium stages VII or VIII, per animal, except for those treated with atrazine 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 Sertoli cell positivity was measured in randomly observed tubules. Plasma testosterone and estradiol were measured by both radioimmunoassay (RIA) and enzyme

linked immuno sorbent assay (ELISA). Western blot assays were performed to determineDo not cite

Appendix A

- Page 218 of 677 the specificity of the antibodies and the changes in the expression of 3

- HSD enzyme in testis and adrenal and androgen receptor in testes of treated groups.

There were significant bod y 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 absolute 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. There was no effect on body weight, testis or adrenal weights in animals

treate d with 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 treated animals (65% for 50 mg/kg/day/15 days; 85% for 200 mg/kg/day/15 days; 86% for 200 mg /kg/day/40 days; 87% for 300 mg/kg/day/7 days). ELISA assay confirmed the reduction in testosterone (data not provided). Testicular levels of testosterone were also significantly (p<0.05) reduced (32%) after treatment with atrazine 200 mg/kg/day/15 days. However, plasma and testicular estradiol levels were significantly increased by 33% and 38%, respectively, after treatment with atrazine 200 mg/kg/day/15 days. At all other dose levels, the plasma and testicular estradiol levels 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 tr eated 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.

Immu nopositivity for AR was detected in Leydig 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 levels when animals treated with atrazine 200 mg/kg /day/15 days and controls were compared by Western blotting assay. Quantification of the AR immunopositivity in specific cell types after exposure to atrazine at the same dose (200 mg/kg/day/15 days) showed equal AR intensity in Sertoli cells and Leydig ce lls 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 da ys) resulted in decreased AR staining in Sertoli cells but increased AR staining in Leydig cells. It was noted that the rats treated for 40 days had atrophied testis and most of the tubules presented AR

- positive Sertoli cells, with reduced staining intensi ty.Do not cite

Appendix A

- Page 219 of 677 In control animals, 3

- HSD positivity in the testis was found in the Leydig cells cytoplasm. After atrazine exposure, there was a great reduction in Leydig cells 3

- HSD immunoreactions (either weakly stained or negative). These data were confirmed b y Western blotting, in which rats treated with atrazine 200 mg/kg/day/15 days showed significant reduction (40%) in the intensity of 3

- HSD positive bands in the testis. The expression of 3

- HSD immunoreaction was found in cells of the three zones of the a drenal cortex. The pattern of distribution and intensity of the 3

- HSD immunoreaction in the adrenal cortex was similar in both control and treated animals. Western blotting was in agreement with these results, showing that the 3

- HSD protein was not alter ed in adrenal cortex of treated animals compared to controls.

Reviewer‘s Comments: This study used some unusual treatment regimens. There is no

dose

- response examined, as there is only one dose per treatment period except in the 15 day exposure. For exa mple, in the 15 day exposure, they include a 50 and 200 mg/kg dose, but do not show any effects 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 testicular 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 pro tein 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 change, but the cu rrent paper looks 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 transient 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 peri od 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

- ingredients‖ in the commercial mixture make the results difficult to interpret. This paper add s little to what we know.

A.4 AROMATASE

Summary: Effects on Aromatase and Steroidogenesis, Andrology

Aromatase is the enzyme that catalyzes the conversion of androgens to estrogens

. It is expressed in the gonadal and extragonadal tissues and is crit ical for maintenance of homeostasis between the sex steroids in males and females which is necessary for reproductive function. Although aromatase is a product of a single gene (CYP19) in mammals, its expression is quite complex as it is regulated in a sp ecies

- and tissue

dependent manner by differential uses of multiple promoters (Simpson et al. , 2002). I t isDo not cite

Appendix A

- Page 220 of 677 well documented that environmental contaminants can inhibit the catalytic activity of this enzyme (Vinggaard 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, Sanderson and hi s colleagues reported that atrazine induces estrone production and aromatase (CYP19) mRNA in H295R adrenal cortical

c arcinoma cells (Sanderson et al. 2000; 2001). These authors reported that elevated aromatase activity, as measured using the tritiated water release assay, was observed in cells exposed to atrazine (low

- effect level, LOEL, 0.3 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 ( Sand erson et al. , 2001; Laville et al., 2006). Finally, Holloway et al. (2008) also reported that aromatase activity in human granulosa

- lutein primary cell cultures following treatment with atrazine (1.0 μM) was 2

- fold higher than that observed for the contro ls. 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 the hypothesis that the effects 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 responsible for the increased production of estrogens by aromatase have not been fully characterize d. Sanderson et al (2000, 2002) reported that the time

- response profile for the induction of aromatase activity, CYP19 mRNA, and cyclic adenosine monophosphate (cAMP) in H295R cells following exposure to

atrazine was similar to that of 8

- bromo

- cAMP, a kno wn stimulant of the protein kinase

- A pathway that mediates the induction of aromatase. These authors suggested that the

atrazine

- dependent 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 that 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 aromata se promoter II. While these studies focused primarily on the up

- regula tion of aromatase gene expression, additional studies demonstrated that other cellular target sites exist for atrazine and that these 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 receptor homolog), zff1d] . These

authors also r eported an upregulation of the expression of a group of important steroidogenic genes to include StAR, inhibin

- α, P450scc, and11βHSD in JEG3 cells following exposure to atrazine. Thus, these findings demonstrate that atrazine can exert a

much broader effe ct 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 as previously reported by Cooper et al. (2007). However, atrazine did notDo not cite

Appendix A

- Page 221 of 677 alter aromatase activity or gene expression in the BG

- 1 cultures 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 activity that prevents further 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 M). Using specific signaling inhibitors and gene silencing, these authors report that this effect is mediated through the G

- protein

- coupled receptor 30 (GPR30), a membrane bound orphan receptor that mediates rapid estrogen signals

The range of the LOEL s (0.3

– 1 μM) 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 et al. , 2009). Although a complete profile of serum 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 concentr ations of the active atrazine metabolite DIA show that it reach es the 2

- 4 μM range within 15 minu tes 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 durations, this data does indicate that the concentrat ions 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 involve a complex array of signal i ng events, Importantly, the fact that atrazine may exert a broad influen ce 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) steroidogenesis following acute chlorotriazine dosing. It is interesting to note that although c hanges 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 r at testes (Modic 2004) or mouse reproductive tract (Rivest and Sanderson, 2010) . Thus, suggesting that while steroidogenesis may be increased as a result of atrazine exposure, this event is likely not mediated by aromatase.

Three in vitro studies have be en conducted over the last seven years to evaluate the potential effect of atrazine exposure on sperm. Given the 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 onDo not cite

Appendix A

- Page 222 of 677 sp erm. 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 subjected to a number of evaluations after exposure to 0, 50, 100, or 500 μM atrazine (Betancourt et al.

, 2006). Exposure to concentrations ≥ 100 μM atrazine elicited a concentration

- dependent and statistica lly significant 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 mal e reproduction. This statement is not supported by empirical data but rather conjecture based on the changes in sperm motility observed after in vitro exposures. This is but one of the numerous limitations of this study. The authors did not take into co nsideration the plausibility 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 reaching the testes i s 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 tr ansit, in seminal fluid, or in the female oviduct where fertilization takes place. In conclusion, this study evaluates mature sperm in vitro and attempts to extrapolate those findings to potential effects on male reproductive performance. In fact the rel atively high concentrations of atrazine suggests that it may have toxic properties 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 rese arch group evaluated the effect of atrazine on capacitation and acrosomal 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 fertilization. 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 the oocyte, penetrate it and fuse with (fertilize) the oocyte. Uncapacitated sperm cannot . Acrosome reaction (AR) consists of an ordered, calcium dependent vesiculation and shedding of the acrosomal vesicle that covers much of the sperm head. It can be visualized using chlortetracycline staining (as was done in this study). Only acrosome re acted sperm can fuse with the egg membrane. Therefore AR is used as an indicator of capacitation. Progesterone may directly induce acrosome reaction at the sperm membrane level in vitro . Once AR occurs, sperm tend to slow down and die (run out of energy ). Therefore, in a mixed culture there will be continuously changing populations of active capacitating sperm and dying post capacitation/AR sperm. Changes in sperm motion called ―hyperactivation‖ accompany capacitation and are also necessary for sperm t o penetrate the zona pellucida. This study suffers from not reporting hyperactivated motility. The latter would help distinguish between physiologically ARDo not cite

Appendix A

- Page 223 of 677 sperm and dead sperm (both of which stain similarly with CTC because they lack an acrosome).

I n 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 while the percentage of acrosome reacted sperm increased. There could be two interpretations for this finding. The authors‘ conclusion was

that atrazine stimulated or accelerated acrosome reaction in vitro leading to a decreas e in motility. Conversely, it could be theorized that atrazine results in decreases in sperm viability and that the dead sperm shed their acrosome after death thus appearing to be acrosome reacted. In the absence of data on the proportion of hyperactivat ed sperm, it is impossible to distinguish between these two possibilities. Regardless, these data do not provide any information that correlates the findings to adverse reproductive performance nor does it provide any rationale for hypothesizing that atra zine selectively targets 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 decreases i n sperm motility might be related to decreases in ATP availability needed for motility. Sperm 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 ATP production in mi tochondria

– 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 suppo rt 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 out come of decreases in ATP availability needed for sperm flagella to move . They also point out that ―the effect of ATZ [atrazine] on sperm motility was seen at > 100 μM, exposure levels that probably do not result from normal use of the herbicide. It is therefore uncertain whether the observed phenomena are ecologically

relev ant.‖ Thus, though this is an interesting hypothesis and finding, its relevance to risk assessment is questionable.

In summary, three studies evaluating andrology parameters have been published since the 2003 IRED. Using either porcine or human sperm s amples, 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 corr elation between these findings and impaired reproductive performance. Furthermore, these studies assume that atrazine per

se reaches the testes; an assumption that has not been verified. Finally, the concentrations affecting sperm parameters in these stu dies are high ( ≥ 100 μM) and, as

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

Appendix A

- Page 224 of 677 herbicide under the current restrictions. 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 deficit in reproductive performance.

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

.

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 atrazine for androgen

- and estrogen

- mediated processes following in vivo and in

vitro exposures. Using a series of molecular approaches, the study evaluates a number of potential cellular mechanisms to include potential effects on estrogen receptor expression and other estrogen target genes, extracellular signal

- related 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 lines. BG

- 1, MCF

- 7, Ishikawa cells were (transiently) transfected with ER luciferase reporter plasmids, and SkBr3 was co

transfected with ER expression plasmid and ER

- luc. All transfected cell line were maintained for 24 hrs., and then exposed to solvent, estrad iol (100 nM), atrazine (1

– 10 μM)m, ICI ER antagonist (10 μ M), or combinations of ICI + E2, ATR + E2 for an additional

24 hrs. Figure A

- 2 : Treatment Groups shows that atrazine did not stimulate 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 expression was conducted using

BG

- 1 cells, and results demonstrated that atrazine (1 μ M) did not alter mRNA or protein

concent rations of the receptor (Fig. 3) as compared to the down

- regulation of these measures in those cells exposed to estradiol. In addition, the study confirms that atrazine does not bind directly to the ER as previously reported by Cooper et al. 2007. Togeth er,
these data demonstrate that atrazine does not bind or activate ER

.

In experiments using the triated

- water release assay, atrazine did not alter aromatase activity or gene expression in the BG

- 1 cells f ollowing a 40

- hr exposure to 1 μ M. This finding is in contrast to that reported by Sanderson et al. (2000) for the H295R cell line. However, the current study reports only aromatase activity as % of controls. Without absolute values for aromatase activity of the controls, it is diffi cult to assess whether or not
the BG

- 1 cells exhibit constitutively high 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 do es not pro vide a positive control for eith er aromatase activity or gene expression.Do not cite

Appendix A

- Page 225 of 677 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 extracellular 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 higher concentration and prolonged time was required as compared with effects 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 phosphorylation ( Fig. 6).

Given the results on ERK phosphorylation, the authors further evaluated the ability of atrazine to up

- regulate the expression of c

- fos, an early gene that responds to a variety of extracellular stimuli, includ ing 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 cycl in A. The authors conclude ―Atrazine is able to stimulate the expression of diverse estrogen target genes without an apparent activation of ER . Additional experiments demonstrated that the atrazine dependent up

- regulation 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 and the EGFR/MAPK pathway to trigger c

- fos protein increase.‖

The study further evaluated the effects of atrazine on the G

- protein

- coupled receptor 30 (GPR30), a seven

- transmembrane receptor which is structurally unrelated to the nuclear ER, but has recently been shown to mediate rapid actions of estrogens. The authors have also recently shown that c

- fos stimulation of E2 occurs through GPR30 and requires ER

and EGFR

- mediated signaling in cancer cells (Albanito et al 2007, Cancer Res. 67:1859

- 1866). In the current study, both atrazine

- dependent ERK phosphorylation and c

- fos

induction were abo lished in BG

- 1, 2008 ovarian, and SkBr3 cells after silencing of GPR30 with small interfering RNA (siRNA), Fig. 10.

Reviewer‘s Comments: This study shows n o evidence that ATR binds directly or activate

ER

- α in cancer cells . It does offer intriguing evidence that atrazine may bind to a membrane receptor that modifies a signaling pathway associated with c

- AMP, ERK, and P13k activation. The concentrations used to indicate this effect are well within, or belo w the range of concentrations known to modify 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) cellu lar responses, the finding that the ligand typically used in these experiments is estradiol. This manuscript, in combination with the Suzawa and Ingraham and Higley et al., papers argues that atrazine influences signal transduction and provides an alternat ive to phosphodiesterase as the membrane associated target molecule for the herbicide.Do not cite

Appendix A

- Page 226 of 677 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

.

The purpose of this study was to evaluate the in vitro effects of known endocrine disrupting chemicals on aromatase and cytochrome P450 reductase activities, the later being the second moiety of the a romatase enzyme complex that 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 aromatase preparations, as well as in human embryonic 293 cells (transiently)

transfected with human aromatase cDNA.

Human embryonic 293 cells (50,000/well) were grown in 24

- we ll plates to 80% confluency and transfected with human aromatase cDNA 48 hrs prior to exposure to test chemicals. An initial evaluation of cell viability following exposu re to each chemical (up to 500 μ M) was determined using the MMT method. Following ex posure to the test chemicals, media was removed, cells were washed with serum

- free media, and tested for aromatase activity using

the tritiated

- water release assay. Specifically, the cells were incubated for 45 min. with 3H

androstenedione, a substrate fo r aromatase. 3H

- water, released during the conversion of each molecule of androgen to estrogen is a direct measure of estrogen production and enzyme activity. Additional assays were conducted to determine the inhibitory effects of

the test chemicals on aromatase activity in human placenta microsomes and equine testicular preparations containing the full aromatase (reductase) enzyme complex. Direct effects on the reductase moiety of the aromatase complex was tested by measuring the absorbance correspondi ng to the reduced form of cytochrome c in the presence of

NADPH + . For this assay, reductase (isolated from equine testes) was preincubated with the test chemical for 15 min. prior to initiation of the reaction.

In general, results from the study are di fficult 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. Chemicals tha t reduced aromatase activity by 50% were further tested at concentrations ranging from 10

– 1000uM. BPA inhibited reductase activity, albeit the IC50 was exceptionally high, 1250 uM. The authors report ―a slight increase in reductase activity in the pre sence of atrazine‖ but the actual concentration responsible for the effect is unclear. Viability of the embryonic cells was affected after 24 hrs in culture at chemical concentrations ≥ 50 uM. Thus, maximal concentrations used for the aromatase assays i n 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 aromatase activity in the embryonic cells.Do not cite

Appendix A

- Page 227 of 677 Reviewer‘s Comments: In conclusio ns, the data reported by Benachour et al. are not useful for the current assessment of atrazine due to an incomplete documentation of methods, doses, and results. Atrazine is fairly insolubility at concentrations higher than 50 uM, especially in media. S ections 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 acti vates 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 previous findings that atrazine induces aromatase expression via promoter II (ArPII) in a SF

- 1 (steroidogenic factor 1) manner. They were also interested in studying binding of atrazine to the receptor, and whether affinity is independent of the ligand binding domain. The i n vitro assays utilized H295R 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 construction 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 confirm that SF

- 1 is essential for atrazine stimulation, endogenous SF

- 1 in H295R cells were knocked down by siRNA. The results indicated a complete loss of responsiveness of ArPII to either atrazine (10 - 5 M) or s imazine (10 - 5 M). In the presence of exogenous SF

- 1, atrazine and simazine induced ArPII in the normally

non

- responsive 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 homolog

- 1 (LRH

- 1) are not essential for atrazine stimulation. To assess if the affinity between SF

- 1 and atrazine is followed by co

- activator recruitment, mammalian two hybrid

- assays for SF1

- TIF2 interaction were performed. Results suggested that atrazine enhanced the interaction

between SF

- 1 and its co

- activator TIF2. The authors concluded that atrazine

- SF

- 1 affinity is coupled with enhanced receptor

- co

- activator interactions. To examine the functional
domain of SF

- 1 in mediating atrazine stimulation over ArPII, mutations were introduced to the DNA binding 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.Do not cite

Appendix A

- Page 228 of 677 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, tha t 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 . (2007 b ). G Atrazine

induced aromatase expressi on is SF

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

- 7

.

This in vitro study focused on the role of steroidogenic factor 1 (SF

- 1) in atrazine

- induced
aromatase expre ssion. A luciferase 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 carcinoma cells (H295R) and atrazine non

- responsive cells, ( i.e. , H IH

- 3T3 fibroblasts cells, and KGN tumor cell line (human ovarian granulose

- like tumor cell line)) were examined. A luciferase gene reporter system was used for the transfection assays.

Atrazine

- responsive H295R cells expressed 54 times more SF

- 1 than t he nonresponsive KGN cells. Exogenous SF

- 1 conferred aromatase responsiveness to atrazine and
simazine in KGN cells. The results of the chromatin

- immunoprecipitation (ChIP) assay indicated that atrazine and simazine increased binding of SF

- 1 to ArPII. R educing the aromatase promoter II to 516 base pairs did not affect atrazine

- induced aromatase response. However, a mutation in the SF

- 1 binding site on promoter II (AFFTCA to ATTTCA) eliminated the responsiveness to atrazine and simazine treatment.

The authors concluded that atrazine binds directly to SF

- 1, showing that atrazine is a ligand for this ―orphan‖ receptor. The results of this study are supported by another study conducted by these investigators (Fan et al. 2007b).

Reviewer‘s Comments: Fa n 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 aromatase. Toxicol. Lett. 149(2):183

- 194

.

The purpose of this study was to compare two cell lines, rat Leydig cell carcinoma (R2C)

and human adrenocorticocarcinoma (H295R) cells, for their suitability as a screening method to detect effects on aromatase expression and catalytic activity following exposure to selected xenobiotics. For each cell line, aromatase activity using the tritiate d

- water release assay and promoter

- specific mRNA levels were measured following exposure toDo not cite

Appendix A

- Page 229 of 677 prochloraz, fadrozole, epoxyconazole, atrazine, 8

- bromo

- cyclic adenosine monophosphate (8

- Br

- cAMP), prostaglandin E2, phorbol 12

- myristate 13

- acetate, and dexam e th asone. This study is unique in that it is one of the first efforts to evaluate changes in aromatase activity of the test chemicals as well as the indentify the specific promoter regions of the CYP19 gene that is affected during exposure.

For all experim ents, cells were cultured in 24

- well plates, and exposed to each test chemical (1

– 10 uM,) for 24 hr prior to assay for aromatase. These cells were used to evaluate effects on aromatase activity that required mRNA synthesis of the CYP19 gene

and/or othe r transcription factors. In contrast, another group of untreated cells were exposed to the test chemicals only during the 3

– hr incubation period of the aromatase assay to evaluate effects related to direct (rapid) inhibition of the enzyme through competi tive or non

- competitive 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 alteration of other posttranslational events that generally occu r rapidly. Appropriate controls were used to demonstrate the specificity of the aromatase assay and measurement of promoter

- specific mRNA levels; solvent controls, 4

- OH

androstenedione (known inhibitor of catalytic activity of aromatase), and 8

- BR

- cAMP (in ducer of aromatase activity). Cell viability was evaluated using the MTT reduction assay.

Results from positive and negative controls confirm assay specificity and demonstrate the precision of the methods to detect significant differences in aromatase ac tivity and gene expression in both cell lines. Atrazine (0.3, 3 and 30 uM) caused a dose

- dependent increase in aromatase activity (LOEL 10 uM) in the H295R cells only when exposed for 24 hrs. (Fig.5) indicating that 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 transcripts, the change as shown in Fig. 6 was not significantly different from

control or 8

- Br

- cAMP (100 uM). The authors suggest tha t the change in these particular promoters may be related to an increase in cAMP, as this transcription factor is crucial for stimulation of this particular promoter. In contrast, no effects on aromatase activity was observed in the R2C Leydig cells foll owing 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 compare d to that of the H295R cells, and that it is possible that one or more 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 co nclusion, these data support earlier findings of atrazine

dependent increases in aromatase activity in the H295R cell line, and provide some insight on the possible cellular regulation of aromatase during the 24

- hr exposure period. It is

clear that atrazi ne does not alter enzymatic activity through a rapid (competitive or non

competitive inhibition, allosteric modification ) alteration of the enzyme. However, failure to evaluate other promoters within the H295R cells is a limitation of this study, and sto ps shortDo not cite

Appendix A

- Page 230 of 677 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 identificat ion 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 s ign. 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

– F ollowing 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 tota lly responsible for elevated aromatase activity).

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 prin t)

.

This study compared the effects of seven model chemicals (atrazine, forskolin, prometon, ketoconazole, aminoglutethimide, letrozole, and prochloraz) on aromatase activity and steroid production (estrogen, testosterone) in H295R human ad enocarcinoma ce lls. The suite of chemicals selected for the study were selected because they were chemicals known to induce aromatase activity through up

- regulation of gene expression and/or transcription factors. Using a tritiated

- water release aromatase assay to quant ify the conversion of radiolabeled androstenedione to estrone, the authors tested an approach to determine whether or not a given chemical affected aromatase directly (e.g., immediate competitive inhibition of enzyme), indirectly (e.g., altered activity re quiring a longer exposure time), or a combination of both direct and indirect effects. Measurement of estrogen and testosterone allowed an evaluation of the predictability of these endpoints for effects on aromatase activity in the cultured cells.

For ea ch experiment, H295R cells were cultured in 24

- well plates, 300,000 cell/ml, and allowed to attach for 24 hrs prior to treatment. 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 chemical (0.01 to 10 uM) along with solvent controls were tested in duplicate or triplicate. MMT assay used for detection of cytotoxicity. At the end of each experiment, culture media was collected for hormone assay (EL ISA), and the cells were further subjected to the tritiated

- water release aromatase assay for 1.5 hrs. To differentiate between direct, indirect or a combination of direct and indirect effects on aromatase activity,Do not cite

Appendix A

- Page 231 of 677 the exposure of the cells to the test c hemical was varied in one of 3 ways: (1) indirect

effect

- no further exposure of the cells during the 1.5 hr. incubation, (2) combination of

direct and indirect effects

- cells exposed for 48 hrs in culture and then again during the 1.5 hr assay, and (3) u ntreated 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 assay was not due t o a direct increase in aromatase activity. Atrazine, like forskolin (which activates the enzyme adenylyl cyclase and

increas es the intracellular levels of cAMP ) enhanced steroidogenesis as evidence by increased T and E. In contrast, when atrazine and for skilin were tested in the ―direct‖ tritiated water assay, there was no apparent change in aromatase activity (even at concentrations greater than the concentrations that changed estradiol production. These observations are important because they demonstra te that atrazine does not interact directly with the enzyme aromatase (as indicated earlier by the paper of Sanderson using the same cell line) and casts further doubt on the hypotheses that any adverse effects of the chemical are mediated through activati on of aromatase.

In addition, significant elevations of (indirect) aromatase activity were only observed following a longer exposure for both of these chemicals. The authors conclude, ―The hormone and aromatase activity profiles for forskolin and atrazin e 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 atrazine.‖ In contras t, known inhibitors of aromatase (prochloraz, letrozole, aminoglutethimide, ketoconazole) caused decreases in estrogen, testosterone and (direct) aromatase activity measured following the 1.5 hrs exposure. The novel finding of increased (indirect) aromata se activity following longer exposures to these inhibitors suggests that a compensatory feedback regulation of aromatase gene expression may also occur in response to the low estrogen concentrations. Thus, the authors conclude that multiple measures of a romatase activity to evaluate immediate (direct) effects 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 A

- 1 : Effect of Atrazine Exposure on Aromatase Activity and Hormone

Levels

(A1, A2) Atrazine: 48

- hrs. 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)Do not cite

Appendix A

- Page 232 of 677Do not cite

Appendix A

- Page 233 of 677 Limitations of study:

- Data for all endpoints reported as fold

- change, with no actual values for means SEM for any observations for steroid concentrations or aromatase activity. Without control data, it is di fficult to gauge how consistent data are among experiments within the study 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 contro ls) in Fig 1 7

. 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 the 48 hrs in culture.

- Although positive controls used to demonstr ate specificity of aromatase assay (e.g., radio inert androstenedione and fadrozole), no concentrations for androstenedione reported and vague information on that used for fadrozole.

- Solubility of ATR (100uM) is questionable.

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.

The purpose of this study was to determine 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 aromatase activity was measured in human granulosa

- lutein cell cultures, cells that abundantly express aromatase, and endometrial s tromal cell (ESC) cultures, cells that do not express aromatase. Aromatase activity was quantified by the tritiated water method and the specificity of the assay was confirmed by co

- incubation with

4

- hydroxyandrostenedione, an irreversible inhibitor of th e catalytic activity of aromatase, to block the formation of tritiated water. The cells were treated for 24 hours with increasing concentrations (1 nM to 1μM) of atrazine. Prostaglandin E 2 (PGE 2 , 1 μM) and vomitoxin (a selective cyclooxygenase

- II inducer, 250 mg/ml) were used as positive controls.

In the granulosa

- lutein cell cultures, atrazine treatment induced a dose

- dependent increase in aromatase activity (p=0.007). Aromatase activity in atrazine treated (1 nM

- 0.1 mM) granulosa

- lutein cells (GC) wa s increased more than 2

- fold compared with control cultures, with significant aromatase activities at 0.1 and 1.0 μM. Atrazine treatment had no effect on ESC aromatase activity at any concentration tested. Atrazine treatment had no effect on GC or ESC pro tein concentrations, which indicated no change in cell number, suggesting that the atrazine induced changes in aromatase activity were not due to cell proliferation.Do not cite

Appendix A

- Page 234 of 677 The positive controls PGE 2 (1.0 μM) and vomitoxin (250 ng/ml) induced a significant

inc rease 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 cell free test system. It was concluded that μM concentrations of atrazine can i ncrease 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 observed 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). Modulation 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 ability to modulate aromatase activity in the
human choriocarcinoma JEG

- 3 cell line. Pesticides were selected based upon chemical structure and environmental relevance, but also included a number of known aromatase

inhibitors and inducers. Atrazine was a mong one of the reference pesticides used in this study. By using the tritiated water release assay after both short (2 hr) and long (24hr) exposures to each test chemical, the authors were able to identify either inhibition or induction (requiring longe r term exposure) of aromatase enzymatic activity. Pesticides that were newly found to induce aromatase activity were further evaluated for their action at the transcriptional level with measurement of CYP19 mRNA and through the retinoic acid receptor sign aling pathway.

JEG

- 3 cells were seeded in 24

- well plates, density of 50,000 cells/well, and allowed to attach for 24 hrs. before changing media and exposing the cells to the test chemicals (1, 3, 10 uM) for an additional 24 hrs. Media was collected at th e end of the exposure period, cells were washed once with media, and further elevated for aromatase activity. The tritiated water release assay was used to quantify aromatase activity in cells exposed to test chemicals for 24 hrs (to detect possible effe cts 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 complex). OH

- Andros tenedione, an irreversible inhibitor of aromatase activity 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 u sing the methyl

- thiazol

- tetrazolium (MMT)

assay.Do not cite

Appendix A

- Page 235 of 677 Reviewer‘s Comments: While neither atrazine nor forskolin had any effect on aromatase activity following a 2 hr exposure, both of these chemicals increased aromatase activity after 24 hrs of exposure. App roximately a 2

- fold increase in aromatase activity was observed in cells treated with 10 uM atrazine, 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 specificity of aromatase assay
and CYP19 mRNA measurements. This study supports other

in vitro studies demonstrating that atrazine does not alter aromatase enzyme competitive inhibition, but rather requires longe r exposure time such that enzyme stability, synthesis and/or post

- transcriptional events can be altered.

A

. 4.9 Rayner, J. , Wood, C., and Fenton, S. (2004 ) 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

.

This study looked at the effects on delayed puberty and mammary gland development in rats exposed in utero to atrazine. 21 Groups of 20 timed

- pregnant Long

- Evans rats were gavaged on gestational days (GD) 15

- 19 with atrazine (purity 97.1%) at doses of 100 mg/kg bw or vehicle control (C). On PND 1, half of each litter was cross

- fostered, creating four treatment groups: C

- C, ATR

- C, C

- ATR, and ATR

- ATR (dam

- milk source, respectively). Litters were weighed and equalized to 10 pups on PND4. On PND 22 (weaning), animals were weighed and separated into 2

- 4 females/cage. Some females were sacrificed on PND 4, 22, 33, 40 and 58. The data from a treated

- dam‘s offspring were evaluated with th e dam (two pups/dam or litter) as the experimental unit, with the exception of vaginal smears.

On PND 29 females (n>29/group) were evaluated 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 after VO for each animal until PND 58 to determine cyclicity patterns. Female pups (n=14

- 16) were sacrificed on PND58. Blood was collected and the pituitary gland, ovaries, and uterus were weighed and prepared for histopat hology. 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 control animals, which was within normal range. There was

21 This review will concentrate on the assessment of delayed puberty onset. The discussion of the mammary gland development will be deferred un til the September SAP meeting.Do not cite

Appendix A

- Page 236 of 677 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 the C

- ATR group, compared 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 number of animals demonstrating persistent estrous (PE) or persistent diestrous (PD). However, while not statistically significant, there appeared to be a lower number 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 ATR

- ATR animals, compared to controls.

No sig nificant treatment

- related changes were seen in circulating hormones (serum LH, PRL, and TSH) in any treatment group at PND 33, 40 or 58.

At PND 58, all atrazine exposure groups had increased pituitary glands; however only the pituitary glands of the ani mals in the ATR

- ATR group were significantly increased, compared to controls. There were no statistically significant differences in reproductive organ weights among the groups.

Reviewer‘s Comments: The researchers in this article demonstrate that atraz ine in utero and perinatal exposure to atrazine can lead to a delay in vaginal opening in female rats. These findings are consistent with those reported by Laws et al. in 2000 and 2003. It should be noted, however, that these effects are seen at substant ially higher dose levels than those used as points 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 mammalian cells. PLoS ONE 3(5): e2117. doi:10.1371/journal.pone.0002117

.

* In this evaluation, only the results pertaining to mammalian cells are discussed. The purpose of this in vitro study was to examine the potential effects of atrazine on NR5A receptors (steroidogenic factor 1 or SF

- 1). The specific 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 papers by Fan et al., wh o argued that this NR5A receptor was the target molecule for atrazine evoking changes in aromatase activity and potentially the molecular mechanism for many assumed adverse outcomes following exposure to this herbicide in vivo. Cellular reporter assays, ut ilizing 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 mSF

- 1 (mouse SF

- 1) and hLRH

- 1 (human liver receptor homolog) expression vectorsDo not cite

Appendix A

- Page 237 of 677 were examined. The data revealed a dose

- dependent increase in luciferase activity which the authors interpreted as activation o f aromatase promoters. The effects were significant at atrazine concentrations of ≥ 1 μM. No activation was reported for the parent reporter or

with mutant SF

- 1 binding sites. Atrazine also activated mSF

- 1 in HepG2 (human liver)

cells. The authors al so provided data demonstrating activation of other SF

- 1 reporters,
such as MIS

- Luc, StAR

- Luc, and HSD3B

- Luc. The authors concluded that atrazine effects depend on DNA binding and receptor occupancy of the promoter.

In a knock

- down experiment, the authors provided results 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 conclude that all NR5A subfamily receptors will be responsive to

atrazine.

Atrazine treatment was without effects on the estrogen responsive zcyp19a2 (ERE

- Luc reporter). No significant activation of NR5A was observed with other estrogenic compounds tested.

Severa l additional experiments were performed to determine the underlying cellular mechanisms of activation of NR5A receptors. These included mitogen

- activated protein protein kinase (MAPK), phosphatidyl 3

- kinase (Pl3K), G proteins, phospholipase C (PLC) in

JEG 3 cells. Pharmacological inhibitors 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 atrazine binds directly to SF

- 1 to either increase DNA binding or act as a ligand. Atrazine had no effect on DNA binding activity to a high affinity SF

- 1 binding site (the mMIS binding 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 signaling pathways known to activate NR5A receptors, including phosphorylation of SF

- 1, generation of SF

- 1 ligands, and i ncreased production of cAMP. The authors also reported that atrazine can increase a cluster of genes involved in endocrine signaling in specific cell types; however, very limited data were presented to corroborate these findings.

Reviewer‘s Comments: Ov erall, this study provides some preliminary research on the underlying cellular mechanism of aromatase expression following atrazine in vitro treatment. The study has limitations including lack of details on methodology of luciferase activity and transfec tion of cells. Only a few poor quality results of PCR analyses and western blots were provided to corroborate the findings. The authors made severalDo not cite

Appendix A

- Page 238 of 677 references to ―data not shown‖ to support their conclusions. The activation of important

second messen ger pathways, 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 aromatase and subsequently increase estradiol production. The results indicating a n 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 fish 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 potent ial effects of dose and duration of exposure to atrazine and its metabolites (diaminochlorotriazine, DACT; deethyl atrazine, DEA; deisopropyl atrazine, DIA) in vitro on the expression of aromatase mRNA in two steroidogenic lines, i.e. , human adrenocortical cancer cells (H295R), and human placental carcinoma cells (JEG3). There were no details in the methods section of the article pertaining to the concentrations used and the duration of treatment. Based on information in the abstract and figures, atrazine and metabolites (all were 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 sy nthesis, primer design, reverse transcription PCR, and statistical analyses.

Exposure to 10 μM atrazine increased aromatase mRNA expression in H295R cells approximately 2.2 fold after 2 hours and approximately 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 was about a 2

- fold increase in aromatase mRNA expression at 24 hours but not at other time points. No expression was observed at the low

concentratio n of atrazine.

Exposure of H295R cells to 10 μM DEA increased aromatase mRNA approximately 1.5

- fold after 2 to 24 hour time period. Exposure of H295R cells to 10 μM DIA increased aromatase mRNA approximately 1.8

- fold after 2 to 48 hour time period. There were no apparent treatment

- related effects with hydroxyatrazine and DACT.

Reviewer‘s Comments: The authors concluded that exposure to 10 μM atrazine in vitro resulted in an increased expression of aromatase mRNA. This in vitro response is dose

and time

- dependent with peak expression occurring after 4 to 24 hours of exposure. Atrazine metabolites, DACT and hydroxyatrazine, failed to show a consistent response.Do not cite

Appendix A

- Page 239 of 677 A.4.12 Yi, KD, Perez, EJ, Breckenridge, CB, and Simpkins, JW ( 2010 ?) Ex vivo

e ffects of atrazines or metabolites on phosphodiesterase activity.

This study concentrated on phosphodiesterase (PDE) activity and whether atrazine and its metabolites inhibit this process, while also noting effects on aromatase in vivo . It provides

sever al 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 Sprague

Dawley rats is that mRNA expression and aromatas e 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 may be mediated either via increases in adenylyl cyclase which catalyzes the conversion of ATP 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 c AMP, 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

- m ethylxanthine 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 w ith additional 90 minute substrate/incubation period. Competitive inhibition 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 creating

s igmoidal standard curves.

The results from the optimized assay for PDE inhibition showed a concentration

- dependent increase in the relative fluorescence output of the assay at both 30 minutes and 2 hours after the addition of the binding reagent. Maxim al output, of 0.025 PDE U/ml was used for all subsequent assays. Addition of atrazine after completion of the enzymatic reaction didn‘t have any effect on the fluorescence output of the assay. Regarding the effects of

atrazine and to a much lesser extent its metabolites inhibited PDE in a cell

- free system.
The IC 50 for atrazine (39.52 μM) while 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; all other metabol ites 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. Also at both 5 and 50 μM concentrationsDo not cite

Appendix A

- Page 240 of 677 of DACT, the IC 50 of atrazine was several folds higher, suggesting competitive inhibitory
effects of DACT.

The authors concluded that atrazine at all concentrations which exceed its aque ous solubility limits, is a weak inhibitor of PDE 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 activi ty. Although, not supported, the authors elude to the possibility that the inhibition 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 inhibition 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 th is discrepancy

– however, they went on to describe the many similarities in results from the two laboratories. 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 ons et of puberty.

Reviewer‘s Comments: In mammals, atrazine is NOT acutely toxic (oral LD= 3090 mg/kg) and is only moderately toxic following 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 atrazine

- mediated inhibition PDE activity would occur at environmentally relevant concentrations. The pote ntial 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/day) which is currently used to regulate chronic exposure to atrazine and its chlorometabolites and appear s to be protective of any potential effects in humans.

A.5 NEUROTOXICITY

Summary: Neurotoxicity

The preponderance of the data available to date indicates that atrazine exerts many of its toxicologically relevant effects through perturbations of the ne uroendocrine system. Many of the reproductive effects seen after atrazine exposure appear to be the sequelae of perturbations to the HPG and/or HPA axes. Consequently, there is significant interest in the potential effects of atrazine exposure on the ner vous system per se.

Over the last seven years, several studies have been conducted evaluating potential

atrazine

- induced effects on the dopaminergic and somatostatinergic systems as well as GnRH releasing neurons. Studies on the dopaminergic system by C oban and Filipov (2007) and Rodriguez et al. (2005) corroborate previous findings on the effect of atrazineDo not cite

Appendix A

- Page 241 of 677 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 die t exhibited increased locomotor activity, decreased striatal dopamine, reduced norepinephrine in the prefrontal cortex, decreased hypothalamic serotonin, and loss of tyrosine hydroxylase positive (TH+) and tyrosine hydroxylase negative (TH

- ) cells in the v entral tegmental area and the substantia nigra pars compacta . It should be noted that the changes in neurotransmitter levels were slight. 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 significant reduction in the number of TH+ neurons in the ventral tegmental area (day 64 of the experiment) as well as a decrease in the number of TH + neurons in the substantia nigra pars compacta was repo rted at 25 mg/kg/day at days 22 and 64. Noticeably lacking was a dose

- response for either one of these findings. Coban and Filipov (2007) also concluded that there was a dose

- dependent decrease in the levels of dopamine

and its metabolites in the striatu m. Based on these findings, both research groups suggested that exposure to atrazine may be related to the etiology of Parkinson‘s disease. Both of these studies have limitations that confound the interpretation of the findings.

These limitations are ful ly described in the study summaries found in Appendix A. The implication that these data suggest a link between atrazine and Parkinson‘s disease is not supported by the data given the non

- specificity of the atrazine

- induced neuronal loss and

the lack of h istological and behavioral features characteristic of Parkinson‘s Disease

.

Parkinson‘s disease is a neurodegenerative disease characterized by a progressive loss of dopaminergic neurons in the substantia nigra. The data reported by Rodriguez et al. (2005) shows loss of non

- dopaminergic neurons and dopaminergic neurons following atrazine treatment in the rat. The subpopulation of non

- dopaminergic neurons includes
GABA

- ergic , calbindin, and cholecystokinin

- like immunoreactive neurons. Other potential

cell types may include 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 subs tantia nigra pars compacta was significant at 25 mg/kg/day but not at 5 mg/kg/day. The loss of dopaminergic 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 mesocorticolimbic system that is involved with functions such as cognition. The primary and major loss of dopaminergic neurons in Parkinson‘s disease involves the substantia nigra. The nonspecific neurotoxicity of atrazine contrasts with sele ctive striatal dopaminergic neurotoxins such as 6

- hydroxydopamine and MPTP.

In addition to the non

- specificity of the atrazine

- induced neuronal loss, another characteristic not seen after atrazine exposure is the suite of neurobehavioral and histologica l features associated 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 Lewy bodies after atrazine treatment.Do not cite

Appendix A

- Page 242 of 677 Behavioral abnormalities noted in Parkinson‘s disease include tremor, bradykinesia, hypokinesia, balance and gait disturbances. No overt behavioral signs were reported in the

av ailable atrazine studies. This contrasts with rotenone, which causes a Parkinson

- like syndrome, and results in reduced mobility, flexed posture, rigidity and even catalepsy in the rat. Rodriguez (2005) provided some evidence for increased locomotor activi ty following d

amphetamine sulfate injection of rats treated with atrazine. However, the authors‘ assertion that the increased locomotor activity is associated with striatal dopamine deficiency from atrazine treatment should be further explored and support ed with a study design that

includes L

- dopa and dopamine agonist and antagonist administration.

Several in vitro studies addressed the putative mechanism of dopaminergic toxicity of atrazine, primarily in striatal slices and striatal synaptosomes and ves icles (Hossain and Filipov 2008; Filipov et al. 2007). 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 relevanc e 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. Furthermore, as mentioned above, the available data indicate that the neurotoxicity of atrazine is not specific to dopaminergic systems. The effects of atrazine on aromatic amino acid decarboxylase,

monoamine oxidase and neuronal mitoc hondrial function are unknown.

The somatostatinegic system in mouse brain was used as a model by Giusi et al. (2006) to study the impact of atrazine exposure on this system. The rationale for this study was that somatostatin plays a role in growth horm one (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 pit uitary targets. As a result, the
researchers concluded that the 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 alteration of the hypotha lamic

- hypophyseal

- gonadal axis in animals treated with atrazine (Wetzel et al. 1994).‖ Though the authors reported sexually dimorphic changes in the hypothalamus of females and changes in the regulation of somatostatin receptors in the hypothalamus of bot h 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.

A neurobehavioral study in mice is also available for consideration. In this study Belloni and coworkers (2007) report that atrazine exposure from GD14

- PND21 at low doses (1 or 100 μg/kg/day) led to behavioral effects inclu ding delays in righting reflex, decreased temperature, changes in pup vocalizations and an acceleration of the ontogeny of the forepaw grasp response. It should be noted that this study also had a number ofDo not cite

Appendix A

- Page 243 of 677 deficiencies such as inappropriate statistical a nalyses and unexplained discrepancies in the sampling of pups from litters which are described in the summary review in Appendix A that call the validity of the results into question.

More closely related to the postulated MOA for atrazine is the researc h conducted by Cooper et al

., 2007 and Foradori et al. (2009) on the pulsatile release of GnRH neuronal activation after atrazine exposure. The research by Foradori and coworkers show that a brief (4 day) exposure to atrazine at doses ≥ 50 mg/kg/day resul ted 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 hypothalamic neurons. These data, therefore, illus trate the close correlation between effects 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

result of a direct action of atrazine or at razine metabolites on the GnRH neuron or the result of increased hypothalamic

- pituitary

- adrenal axis are under investigation. Indeed, recent data show that atrazine exposure leads to a rapid increase in corticosterone and progesterone (adrenal steroid hor mone) secretions which decrease GnRH secretion (Fraites et al.,2009; Laws et al., 2009).

In summary, the re are a number 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 somatostatinergic 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 studies examining changes in the rat brain dopaminergic systems and associated behavioral changes were also found to have significant deficiencies that also limit their utility for risk assessment purposes. In addition, results of in vivo animal toxicity tests do not support an effect of atra zine exposure on neurological function including Parkinson‘s Disease. The observations that the chlorotriazines may alter GnRH neuronal activity are consistent with the proposed neuroendocrine MOA of atrazine. Previous

studies have demonstrated that atra zine can alter dopamine in hypothalamic regions that are important in the regulation of GnRH secretion, consistent with the neuroendocrine MOA

of atrazine.

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

.

The authors stated that the aim of their work was to study possible neurodevelopmental effects of perinatal exposure to low, environmentally relevant doses of atrazine in mouse pups. Spec ifically, the focus was on somatic parameters and a wide range of reflexes to

assess sensorimotor maturation.Do not cite

Appendix A

- Page 244 of 677 The authors noted that mice of an outbred Swiss

- derive strain (CD

- 1) were used in the study; no details on age or body weights were provided. Breeding pairs were formed, 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). A trazine (98.4% purity) was dissolved in corn oil and administered daily to female mice by self administration drinking from a modified syringe. The authors 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 neurobehavioral development. A total of 128 mice were subjected to behavioral testing between 2 and 15 days of age. Assessment of somatic gr owth and sensorimotor development 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, in complete; 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

ultraso nic calls was also performed on PND 9 (which the authors mention is the peak day in the ontogenetic profile of ultrasonic vocalization). In addition, body temperature was recorded on PND 4, 9, and 12.

The authors reported that there were no differences in dam body weight gain during gestation. A pup body weight decrease of approximately 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 mean of 4.60). There was an acceleration of forepaw grasp (―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 temperatures were significantly lower in the males and

females of the low

- dose atrazine group (5% decrease in males; 4% decrease in females) when compared with controls . There were no significant changes on PND 4 and 12 and no significant changes in the high

- dose group.

Reviewer‘s Comments: The authors indicated that were was a tendency towards a

decrease in the mean number of ultrasonic calls in atrazine

- treated pup s when compared to controls on PND 9. However, the data (in contrast to controls) show a decrease (notDo not cite

Appendix A

- Page 245 of 677 statistically significant) in atrazine

- treated females (both low and high dose groups) and only the low

- dose group for atrazine

- treated males. Moreove r, the decreases in mean

number of ultrasonic calls in atrazine

- treated females on PND 9 did not display a clear

dose

- response relationship. Based on spectrographic analyses of the ultrasonic calls, the authors claim that there was a tendency towards a lo nger 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 combi ned. No differences were noted in the high

dose atrazine groups, whether data are presented as sex specific or combined. Peak frequency was slightly (not statistically significant) increased in atrazine

- treated males (low and high dose groups) and in atr azine

- treated females (just the high dose group).

The authors concluded that exposure to low doses of atrazine during the fetal and suckling period interferes with some aspects of somatic growth of pups in a reversible manner, and more clearly with thei r neurobehavioral 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 reported by the authors rely on only individual pup measurements which inflates sample size. The failure to account for litter effects can result in increased type 1 errors and overestimate any effects, thus reducin g the validity of the findings of the study.

In addition to inappropriate statistical analyses, there were unexplained discrepancies in the sampling of pups from litters. For instance, a lower number of pups were evaluated for somatic growth and senso rimotor development, body temperature, and ultrasonic call emittance compared to the actual number of pups available. For the spectrographic analyses of ultrasonic vocalization, not only were significantly less animals evaluated in

contrast with the asses sment of mean ultrasonic calls, but the reported total numbers of pups evaluated did not match the total reported for males and females separately. The number of animals evaluated for spectrographic analyses of the ultrasonic calls should

match the number s evaluated for the mean number of ultrasonic calls

.

Other deficiencies of the study include a lack of details on the procedures used for assessment of somatic growth and sensorimotor development, a lack of inclusion of positive controls that would suppo rt the reliability of the methodology, the lack of objective and validated behavioral tests, lack of neuropathology, and the lack of actual spectrograms to corroborate the spectrographic results. The serious deficiencies of these studies raise questions a bout the validity and reliability of the data and its use for risk assessment

purposes.Do not cite

Appendix A

- Page 246 of 677 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 Ne urochemistry 100: 1177

- 1187.

The objectives of this study were to determine the following: (1) the effects of atrazine exposure on striatal neurochemistry and striatal expression of tyrosine hydroxylase; (2)

whether atrazine effects on striatal neuroche mistry are associated with dopaminergic neuron loss; and (3) whether the neuronal loss 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 g avage for 14 days. Several indices of neurotoxicity were evaluated on days 15, 22 or 64. Dopamine and its metabolites, 3, 4

- hydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were measured by high performance liquid chromatography.

Striatal t yrosine hydroxylase levels were determined by western

- blot analysis. Fixed half

brains were used to analyze neurons in the substantia nigra pars compacta and ventral tegmental area by immunohistochemistry/unbiased stereology.

No effects of atrazine tre atment on body weights were reported (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 prot ein at 250 mg/kg vs. 5 ng/mg protein in controls) and HVA (approximately 6 ng/mg protein at 250 mg/kg vs. 8 ng/mg protein in controls) levels on day

15. There was no difference between control and atrazine

- treated animals in striatal dopamine levels at da y 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 atrazine treatment on striatal levels of TH as determined by western blot analysis.

On day 15, there was a decrease in the number 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 significant at 25 mg/kg/day dose. In regard to the ventral tegmental area, there was a significant decrease in the number of TH

- positive neurons at days 14 and 22. The decrease in the ventral tegmental area was reported to be 19% in contrast to a 32% decrease in the 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 area, with the overall effect being significant at all dose levels in the ventral tegmental area. The authors reported that at 64 days, the effect on the neurons in the substantia nigra pars compacta at the high dose was only marginally greater than the effect in the ventral tegmenta l area (33% cell loss in substantia nigra pars compacta vs. 28% cell loss in the ventral tegmental area).Do not cite

Appendix A

- Page 247 of 677 Reviewer‘s Comments: There are several limitations with th is study:

The reason for a lack of a dose

- response relationship with respect to the num ber of TH+ neurons is not clear. The study did not include a positive control such as 6

- hydroxydopamine which produces dose dependent alterations in dopamine and dopamine metabolites and loss of tyrosine hydroxylase

- immunoreactive cells in the

substantia nigra. 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 correlate with the findings presented in the bar

grap hs. Also, there was no information on the calibration of the stereological test system. The only statement on the precision of the 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 morphological 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 above) was not included in the study. There were no functional assessments of the dopaminergic alterations, for example, assessment of behavior and motor activity to correlate with the neurochemical findings. Neurochemical analyses were not performed for the ventral tegmental area which would have provided some support for the observed findings of the loss of dopaminergic neurons in this region. Coban and Filipov also presented data 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 alterations in dopami ne 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 data in which the authors state that the ―some of the animals received MPTP on day 15‖ (no further details provided ). M PTP has been associated

with a P arkinsonian

- like syndrome, and it is not certain if the word MPTP was a typographical 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 was included as a positive control it is not clear why it was not administered to mice

at the same treatment 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 atrazine treatment of various aspects of dopamine metabolism in rat striatal slices. Striatal tissues from adult male Sprague

-Do not cite

Appendix A

- Page 248 of 677 Dawley rats were incubated with up to 5 00 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 nomifensin e, 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 are caused by non

- specific cytotoxicity. Dopamine and metabolites, 3,4

- hydroxy phenylacetic acid (DOPAC) and homovanillic acid (HVA) were

measured.

Atrazine treatment at concentrations of 100 μM or greater resulted 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 affected by atrazine treatment. However, atrazine decreased high potassium

- induced dopamine release; the increase in media dopamine in the presence of nom ifensine was increased even further by atrazine in a dose

- dependent

manner. These effects were observed at levels that were not toxic to the tissue. The authors concluded that their results suggest that atrazine decreases tissue dopamine levels not by af fecting 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 wi th 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 decarboxylase, monoamine oxidase and neuronal mitochondrial function are unknown.

A.5.4 For adori, 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

.

The o bjectives of this study were to examine the effects of atrazine on normal tonic release of luteinizing hormone (LH) and to elucidate the site of action of atrazine in the hypothalamic

- pituitary

- gonadal axis. To assess potential effects of atrazine on puls atile LH release, ovariectomized adult female Wistar 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, blood samples were obtained using an indwelli ng 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.Do not cite

Appendix A

- Page 249 of 6 77 The second experimen t involved validation of a protocol for GnRH immunoneutralization and pituitary response to GnRH agonist. Utilizing the same experimental procedure above,

animals were administered GnRH antiserum via cannula to block the actions of endogenous GnRH. The a nti

- GnRH serum was prepared against keyhole limpet hemocyanin

- conjugated GnRH. Following blood sampling, animals were treated with one of four doses of GnRH 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. 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 effects of atrazine on in vivo pituitary response to GnRH agonist. The objective was to determ ine whether atrazine reduces pituitary sensitivity to a GnRH agonist. Ovariectomized rats were administered atrazine (0, 50, 100, or 200 mg/kg/day by gavage) for 4 days. On the second day of treatment, intra

atrial cannulae 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 minutes. Atrazine

- treated animals that had been immunoneutralized

with the anti

- GnRH serum showed an LH response comparable to that observed in the control group. Both doses of D

- Ala 6

- des

- Gly

- NH 2 10

- GnRH

- ethylamide caused an increase in the circulating concentration of LH. However, there was no significant change in the

levels of LH related to atrazine treatment.

The authors concluded that although 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 a re likely to be mediated by interference with central mechanisms controlling GnRH release from the hypothalamus. The brain appears to be the primary target mediating the effect of atrazine on LH release in

the rat.

Reviewer‘s Comments: The Foradori st udy provides added support for the hypothesis that atrazine targets the brain to modify the neuronal control of pituitary function . Evaluating the secretion of gona d otropin releasing hormone (GnRH) in rats revealed that atrazine alters

the release of this neuropeptide from the brain. GnRH is essential for LH release from the pituitary.Do not cite

Appendix A

- Page 250 of 677 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 Reprodu ction 81(6): 1099

– 1105

.

The authors previously reported that atrazine treatment decreased LH pulse frequency and increased pulse amplitude in ovariectomized rats without changing pituitary sensitivity to a GnRH receptor agonist. The authors interpret ed 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 estrogen and progesterone

- induced LH surge in freely moving,

ovariectomized rats to determine if atraz ine‘s inhibition of LH secretion coincides with a reduction in GnRH neuronal activation. The immediate early gene product FOS (cFos) was colocalized in GnRH neurons as a measure of cellular activity. In addition, withdrawal studies were performed to dete rmine whether LH levels and measures of GnRH neuronal activation would return 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 primed 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 perfused, and brains were removed. Brains were processed for GnRH and cFOS using immunohistochemistry. To determine if atrazine alone would have a stimulatory effect on
GnRH neuron al activation, a second cohort of animals was treated similarly except that no hormones were given to induce GnRH and LH surges. For this part of the experiment, rats were treated with 50 mg/kg atrazine or vehicle for 4 days. To examine the effects of

at razine 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 animals either during the atra zine treatment period or 2 days or 4 days after the cessation of atrazine treatment.

Atrazine treatment inhibited the 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 tr eatment did not affect peak FSH levels; however, atrazine treatment at 200 mg/kg reduced the AUC for FSH levels in this group. Treatment 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. Treatment with 100 mg/kg and 200 mg/kg atrazine also reduced the mean number of GnRH neurons immunoreactive for FOS; there was no diffe rence between these groups. The results also indicated that atrazine alone does not induce FOS immunoreactivity in GnRH neurons.

Within 4 days (but not 2 days) after cessation of atrazine treatment, the reductions in GnRH

activation and hormone

- induced LH surge returned to control levels.Do not cite

Appendix A

- Page 251 of 677 The authors concluded that their data indicate that atrazine affects neuroendocrine function in the female rat by actions at the 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 acknowledge that ―it remains unclear 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 contr olling the preovulatory surge of GnRH.‖

Reviewer‘s Comments : This study provides further support to their previous findings (Foradori et al. 2009).

A.5.6 Giusi G, Facciolo RM, et al. 2006. The endocrine disruptor atrazine accounts for a dimorphic som atostatinergic neuronal expression pattern in mice. Toxicological Sciences 89(1): 257

– 264

.

The intent of this study was to examine the potential effects of atrazine on various somatostatin receptor subtypes (in particular, SST 2, 3, 5 ) including possib le regional specificity in some hypothalamic and extra

- hypothalamic estrogen

- enriched brain regions. The authors provided a rationale for the selection of the somatostatin receptor subtypes. The authors noted that the widely distributed SST 2, 3, 5 appea r relatively early during development but also remain stable during 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) a nd the striking differences in GH release

between male and female rodents. The authors stated that differentiated GH secretion may be due to gender

- 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 mechanisms involved in the delay of the estrous cycle or the alteration of the hypothalamic

- hypophyseal

- gonada l axis in animals treated with atrazine (Wetzel et al. 1994).‖ The interest 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 proce dure was monitored to make sure that pups did not attempt 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 th e treatment protocol (control group was included). At

PND 60

– 65, the animals were sacrificed and brains removed. Brain sections were prepared. Amino cupric silver staining was used for examining potential degenerative changes. Reverse transcription po lymerase chain reaction was applied for the detection of

SST 2,3,5 . In situ hybridization was utilized for mRNA.Do not cite

Appendix A

- Page 252 of 677 The authors reported a high

- dose atrazine

- dependent dimorphic neurodegeneration pattern based on the amino cupric silver staining 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 hippocampal area, but not those of the male.‖ The authors also indicated that examination 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 perikaryal swelli ng plus well

- developed astrocytic formations in the female. The dimorphic pattern was also evident in the hypothalamus, as demonstrated by significantly darker axonal processes in the female arcuate hypothalamic nucleus. The dimorphic pattern was not ob served in extra

- hypothalamic areas such as the cortex or

striatum.

In the high

- dose female group, the authors reported a very strong (p < 0.001) upregulation of sst2 expressing neurons in the female hypothalamic areas, specifically the suprachiasmatic nucleus (+110%) and ventromedial hypothalamic nucleus (+65%), whereas a similar downregulatory trend was noted in the some extrahypothalamic areas such as the striatum (

- 96%) and central amygdaloid nucleus (

- 45%). In the high

- dose male group, the author s reported very strong upregulatory and downregulatory actions for sst3 expressing neurons, with upregulation in the arcuate hypothalamic nucleus (+120%) and basolateral amygdaloid nucleus (+78%), and downregulation in the cortical areas (

- 88%) and denda te gyrus (

- 105%).

The authors concluded that the observed neurotoxicological effects of atrazine are ―very likely linked to dimorphic expression patterns of specific somatostain subtypes in discrete but key hypothalamic and extrahypothalamic 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 controls. 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 during gestational and lactational phases, however, there was no mention of standardizing litter sizes. Standardization of litter size would

reduce between

- litter variability.
Also, several of the reference citations di d not match or support statements presented elsewhere in the study. For example, the reference to Surrales (1995) pertains to alachlor, mitocyn, and vinblastine. Gould et al. (1997) refers to research on polychlorinated biphenyl mixtures, not atrazine.Do not cite

Appendix A

- Page 253 of 677 Th e few photomicrographs of the amino cupric silver staining results utilized to demonstrate critical neuronal degeneration were of very poor quality and not presented in color to portray the reactive 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 provided to support the claim that there was ―a rather significant overall

reduction in the total number of neuronal cells...‖. Their claim cannot be confirmed

without these data. Only one representative photo on reverse

- transcriptase polymerase chain amplification results was provided, and there was no mention if there was a control for possible genomic contamination in RNA preparations. No rational basis to explain the purported differen tial neuronal expression and dimorphic sex effects was offered. For example, striatal neuronal degeneration was reported in both males and females (although the photomicrographs were too poor in quality to verify these findings), yet downregulation of som atostatinergic receptor subtype 2 (sst2) 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 differential expression of sst subtypes a nd dimorphic sex distribution in other brain regions, and no plausible biological mechanism to explain the pattern of effects was discussed. Moreover, the relevancy of these findings is

not clear. The study did not address the potential confounding by cor ticosterone

.

Based on the number of serious limitations and no other corroborative support in the literature, the preliminary results of this study are inconclusive.

A

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

rat str iatal 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 effect s of atrazine on the vesicular and synaptosomal uptake of striatal dopamine, which may result in disrupted vesicular storage and/or cellular uptake 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) were also investigated. Limited details were provided on the various methods such as prepara tion of synaptic vesicles and striatal synaptosomes, vesicular uptake of dopamine, and synaptosomal uptake of dopamine.Do not cite

Appendix A

- Page 254 of 677 Atrazine (1

– 250 μM) inhibited dopamine uptake into synaptic vesicles, and the inhibition was dose dependent. DACT did not affect ve sicular dopamine uptake, while DE and DIP significantly decreased vesicular dopamine uptake. Kinetic analysis (nonlinear isotherm regression) of the vesicular dopamine uptake showed that atrazine decreased the V max parameter but not K m .

Atrazine and DE increased (6

– 13%) synaptosomal dopamine uptake; DACT and DIP were without effect. Atrazine, DE and DIP increased the ratio of synaptosomal/vesicular uptake of dopamine. The authors concluded that atrazine and two of its metabolites (DE and DIP) decrea se striatal dopamine levels, at least in part, by increasing cytosolic dopamine, which is prone to oxidative breakdown. The authors note that the effects of atrazine on aromatic amino acid decarboxylase, monoamine oxidase and neuronal

mitochondrial functi on remain unknown. Previously, the authors (Filipov et al. 2007) showed that atrazine does not alter the function of tyrosine hydroxylase substantially.

Reviewer‘s Comments: The results revealed that atrazine, in vitro , alters striatal dopamine levels p ossibly 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 concentrati ons 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 decarboxylase, 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 nigrostriatal and mesocorticolimbic pathways. In addition, the underlying mechanisms of any potential changes in dopamine function in these pathways were explored by examining 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 atrazine treatment and 2 weeks after cessation of treatment. At the 2

- month time point, locomotor activity was mea sured on 3 consecutive days, in which the rats were injected saline intraperitoneally 5 minutes before the session during the first 2 days, and 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 termination of treatment. Locomotor activity was recorded during the light phase for 7Do not cite

Appendix A

- Page 255 of 677 hours. The hypothalamus, prefrontal cortex, nucleus accumbens, and the striatum were

removed from sacrificed ani mals 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 hydroxylase from the substantia nigra pars compac ta 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 c annulation. Brain histology was performed on the rats 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 months with 10 mg/kg atrazine exh ibited an enhanced horizontal locomotor activity response (70% relative to the 0 and 5 mg/kg groups) to d

amphetamine. The increased locomotor activity persisted at the 3

- and 6

- month time periods, as well as 2 weeks after treatment, time periods carried out in the absence of d

amphetamine administration. At 2 weeks after treatment with atrazine, locomotor activity was 40% above control values. A statistically significant decrease (approximately 20%) in striatal dopamine levels at 10 mg/kg/day was noted . Dopamine levels were increased in the prefrontal cortex in an inverse U

- shaped fashion; the increase at 5 mg/kg/day was 30

– 40%, while levels declined towards control levels at 10 mg/kg/day dose. Significant decreases (10

– 15%) in hypothalamic seroto nin 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 authors reported no changes

in the metabolites of serotonin or do pamine or dopamine turnover in any region.

At 2 weeks after the 6

- month treatment period, the number of tyrosine hydroxylase positive (TH+) and tyrosine hydroxylase negative (TH

- ) cells were reduced in the substantia nigra pars compacta and ventral tegm ental areas. This effect was primarily due to the 10 mg/kg/day dose level. Mean reductions 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 noted as measured at 90, 120, 150, and 180 minutes postadminstration. By 150 minutes, the decreases averaged approximately 40% and were seen in both dose groups. The aut hors also noted a dose

- dependent decrease in potassium

- stimulated dopamine release. No changes in the dopamine metabolites (DOPAC or HVA) were observed in the microdialysis component of the study.

The authors concluded that there findings ―demonstrate that atrazine may have broad effects on brain monoamine systems and thereby influence a wide range of behavioral functions.‖ The authors acknowledge that ―additional studies are needed to unravel the targets of atrazine and the mechanisms of its effects, as well as the ultimate human health consequences of such exposures for behavioral and/or neurological dysfunctions.‖Do not cite

Appendix A

- Page 256 of 677 Reviewer‘s Comments: This study has a number of limitations, including:

No photomicrographs were provided to support and corroborate t he 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 information on the calibration of the stereologic al test system and no information on control for potential sources of variability in the stereological estimations of cell numbers. There was no assessment of the precision

of the estimations of neuronal counts.

Behavioral assessment was limited to d

- am phetamine

- 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 control such as 6

- hydroxydopamine which produces dose depend ent alterations in dopamine and dopamine metabolites and loss of tyrosine hydroxylase

- immunoreactive cells in the substantia nigra.

A 5.9 Solari, M, Paquin, J, Ducharme, P, Boily, M (2010). P19 neuronal

differentiation and retinoic acid metabolism as cri teria to investigate atrazine, nitrite, and nitrate developmental toxicity. Toxicol Sci 113 (1):116

- 126.

In an in vitro study using neuronal differentiation as an indicator of developmental toxicity, Solari et al. (2010) exposed cultured mouse P16 embry onic carcinoma cells to atrazine along with NaNO 3 and NaNO 2. The basis of the assay is that P16 cells, which can differentiate into neurons in response to retinoic acid (RA), are sensitive to agents that can influence all trans RA (atRA, the active metabo lite of vitamin A) metabolism and cause developmental toxicity since R A is an essential micronutri ent for growth and embryonic development. Consequently, undifferentiated P16 cells, grown in differentiation culture medium, were treated with 0, 500, 1000 or 2000 μg/L of atrazine, prepared in dimethyl sulfoxide in the presence and absence of 0.05 μM all

- trans RA (at

- RA). Cells were allowed to aggregate for 4

- 5 days and differential medium, containing atrazine, was replenished at days 2.and 4. After treatmen t, cells were dissociated with trypsin, processed and analyzed for the expression of stage

- specific embryonic antigen

- 1 (SSEA1, a cell

- surface glycoconjugate marker of the undifferentiated state) and βIII

- tubin (a neuron

- specific cytoskeletal protein) usin g immunostaining and flow cytometry. Extracts, prepared .from lysed cells, were subjected to gel electrophoresis for confirmation of gene expression using Western blot analysis. Cell viability and proliferation were also determined as well as

retinoid and protein content. The limited data that were available for atrazine suggest that no adverse effects on cell viability, cell proliferation or RA metabolism resulted from exposure to atrazine concentrations observed in rivers in Quebec.Do not cite

Appendix A

- Page 257 of 677 Reviewer Commen ts: In general, the study represents the development of a new in vitro assay to detect potential developmental toxicants; thus the major emphasis in the article is on techniques that validate the test system. Since the extent of data on atrazine are limit ed (generally presented as bar graphs) and the highest concentration tested (2 μg/mL) was not cytotoxic, the relevance of these findings to the atrazine risk assessment is questionable.

A.6 IMMUNOTOXICITY

Summary: Immunotoxicity

In the Agency‘s 2003 ev aluation of atrazine, the immune system was identified as one of the target systems for atrazine toxicity. Since then, numerous studies both in vivo

and in vitro have been conducted to further investigate atrazine‘s potential to cause immunotoxicity. Th e in vivo studies reported various potential immunological effects of atrazine on cellular and humoral immune systems. These included natural killer (NK) cell function, delayed

- type hypersensitivity responses to bovine serum albumin (BSA), macrophage phag ocytic activity, antibody responses to sheep erythrocytes or keyhole limpet hemocyanin, T cell proliferation and cytolytic activity, spleen and thymic cell phenotypes, humoral response after immunization with heat

- killed Streptococcus pneumoniae , lymphocyt e populations in peripheral blood mononuclear cells, and expression of MHC Class II proteins on splenocytes. One study design included a host resistance test, which assesses the function of the immune system in toto (Karrow et al. 2005).

All of the adul t in vivo immunotoxicity studies focused primarily on cellular and humoral systems. Several studies evaluated limited systemic effects such as spleen and thymic weights and body weights. None of the studies provided detailed and thorough histological exa minations 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 th ymic cellularity 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 the host resistance test to validate the biological significance of changes in cellular and humoral immune function reveal ed 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 relevant test for validating other methods and for extrap olating the potential of environmental agents to alter host susceptibility in the human population.Do not cite

Appendix A

- Page 258 of 677 It is well known that alterations in corticosterone levels may result in impaired immune function through the activation 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 determine if corticosterone area under the curve could be used to predict immunotoxicity. The effect of HPA axis pe rturbations on immunological parameters can 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 response model including acute and 28

- day exposures to atrazine. In this model, atrazine treatment resulted in a number of immune system effects (e.g., suppressed white blood cell counts, increased neut rophils, decreased lymphocytes), consistent with effects on corticosterone concentrations (Pruett 2009). However, the atrazine

- induced 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 atrazine exposure did not cause habituation or tolerance to the HPA axis perturbatio ns, but immune function (e.g., antibody production) was not evaluated in this study.

It is important to note that the corticosterone response was stimulated by intraperitoneal injection of atrazine. Pruett et al. (2003) demonstrated that a single high

o ral dose of 500 mg/kg atrazine also increased 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 immunotoxici ty of

atrazine per se

.

While the available studies suggest a potential effect of atrazine on the immune system as noted in studies prior to 2003

, conflicting 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 immunopotentiation of immune function in male

Balb/c mice. The reasons 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 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 atrazine. Delayed type hypersensitivity response and the IgM antibody response to sheep red blood cell antigen were measured. In comparison, Rowe et al. used Balb/c mice which were exposed subcuta neously to atrazine for 21 days

via time release pellets beginning between GD 10 and 12 through PND 10 . The starting dose 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 release from the implant that was not adjustable for increasing maternal body weight . T cell function (proliferation and cytolytic activity) after in vitro allogeneic stimulation was measured.Do not cite

Appendix A

- Page 259 of 677 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 immunological eff ects of atrazine reported by

Rowe et al. and Rooney et al. represent HPA axis alterations 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 developme nt are not mediated through the suppression of prolactin or thyroid hormones. The precise underlying mechanism of an

atrazine

- mediated sex

- specific 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, including intracellular signal transduction, have also been conducted. The underlying mechanisms of atrazine

- induced inflammatory effects such as nociception and plasma extravasation, may be mediate d through mast cell degranulation and activation of the Gq/11

- protein coupled neurosteroid receptor, phospholipase C and intracellular Ca 2+ (Mizota and Ueda 2006). However, the inflammatory responses were examined following intraplantar administration of atrazine, a route of exposure that is not likely to occur in humans. Pinchuk et al. (2007) provided in

vitro evidence suggesting that atrazine directly targets dendritic cell maturation and decreases major histocompatibility complex 1 (MHC

- 1) molecule exp ression on the

dendritic cell surface.

Atrazine

- induced inhibition of in vitro human peripheral blood lymphocyte NK cell

specific 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 stable conjugates with target cells (Rowe et al. 2007). Using human peripheral blood mononuclear cells, the in

vitro study of Devos et al. (2003) suggests that atrazine

- induced inhibition of human peripheral blood mono nuclear cell proliferation and cytokine production is most likely not mediated through nuclear receptors such as the glucocorticoid receptor, androgen receptor, members of the peroxisome proliferator

- activated receptor family, and the

retinoid

- related orp han receptor α.

Although in vitro studies provided some insight into potential mechanisms of

atrazine

- mediated immunotoxicity, the extrapolation of these mechanisms to the interpretation of immunotoxicity observed in vivo is difficult. Unlike the in vi tro situation, in

vivo the immune system‘s response to atrazine exposure involves complicated feedback communication pathways with the central 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, the underlying modes of action of atrazine

- mediated immunotoxicity and its relevance to potential adverse health effects in humans a re still not thoroughlyDo not cite

Appendix A

- Page 260 of 677 understood. In adult rodents, the indications of immunotoxicity reported in the atrazine database may be due to direct interactions between atrazine and/or its metabolites and immune system cells. Alternatively, 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 th e 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 availab le data do not indicate atrazine

- induced immunotoxicity is a more sensitive endpoint than the atrazine

- induced effects on neuroendocrine function, e.g., attenuation of LH surge in female Sprague

- Dawley rats which is used for the basis of the points of dep arture for the intermediate and chronic exposure scenarios.

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

.

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 matin g 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 wat er. 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 isolation, and held frozen until use in the gene expression phase of testing.

For the CFU

- GM assay, bone marrow cells were thawed, centrifuged, and resuspended in culture medium specific for murine hem atopoietic cells containing 30% fetal bovine serum, bovine pancreatic insulin, human transferrin iron

- saturated, 2

- mercaptoethanol, interleukin

3 and

- 6, and stem cell growth factor. Cell viability was determined; cells were adjusted to

6.0 x 10 5 viable ce lls/mL, seeded to yield a final density of 4.0 x 10 4 viable cells/plate and incubated. Triplicate 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 wasDo not cite

Appendix A

- Page 261 of 677 purified, hybridized for 16 hours on microarray membranes, washed, and exposed on a phosphor

- image screen for 21

- 24 hours. Transcripts were considered to be differentially expressed if the p value was ≤0.05.

Bone marrow cells were also investigated for modulation of gene expression of estrogen receptors alpha and beta using real

- time PCR. For this approach, 500 ng of total RNA, extracted from the bone marrow of treated males and females, was converted to cDNA through reverse transcription using random hexamer pri mers High

- Capacity 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) decrease in the percentage of CFU

- GM was se e after exposure to 1 mg/L atrazine alone. NaHAsO 4 had no cytotoxic effect on the bone marrow. However, cell growth increased significantly (p<0.05) by 12% (138 vs. 123 colonies) after

the combined atrazine/ NaHAsO 4 treatment of females, compared to cont rol. Results from the microarray analysis 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 exposure to atrazine/NaHAsO 4 demonstrated the over expression 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 (ERα and β), quantitative RT

- PCR of exposed bone marrow cells revealed that the ERα was not modified in either sex after dosing with the three treatment groups. However, ERβ gene expression was significantly increased in both sexes and all treatment groups. Nevertheless, the high varia bility among individual females in expression of either ERα or β genes limits the interpretation of these data. CFU

- GM assay, quantitative RT

- PCR of exposed bone marrow cells revealed that the ERα was not modified in either sex after dosing with the three treatment groups. However, ERβ 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 immunotoxicity. This is because of the lack of an effect on gene expression

in the treated bone marrow and the wide va riation in the gene expression of ERβ among

individual females.

A.6.2 Coder, P. S. (2010) An Oral (Gavage) Immunotoxicity and Hormone Evaluation Study for the Effects of Atrazine on Male Rats, WIL Research Laboratories, LLC, 1407 George Road, Ashlan d, OH. Unpublished. (TK0023175)

In an oral gavage, 28

- day immunoxicity and hormone evaluation study, 71

- day old male Sprague

- Dawley [Crl:CD(SD)] rats (10/subset/group) were administered Atrazine (ATR)Do not cite

Appendix A

- Page 262 of 677 (98.8%) in a 1% methylcellulose deionized water vehicl e by oral gavage at 0, 6.5, 25, 100, and 0 pair

- fed control mg/kg. These doses were equivalent to 0, 1.3, 5, 20, and 0 mg/mL. Positive control animals were given either of the following: substance I

– Anti Asialo GM1 (rabbit) or substance II

– cyclophosp hamide monohydrate and also used a pair

- fed control group (table attached, pg 29

- WIL 639072 study). The vehicle for the test and control substances was the same. Animals were examined twice daily for signs of mortality and morbidity. Individual clinical observations of toxicity were recorded 2 hours following dose administration. Food consumption and body weight were recorded daily. Urine examinations were collected in certain subsets only to assess urinary corticosterone levels. Clinical, hematologica l and hormonal parameters were measured at termination. All animals were euthanized following dose administration of atrazine (within 30 minutes) by decapitation. Hormonal analysis was performed on corticosterone, adrenocorticotrophic hormone (ACTH), pro gesterone, estradiol, estrone, estriol, and prolactin. Bioanalysis was done on androstenedione, testosterone, and dihydrotestosterone (DHT). Tissues were collected following blood sampling and organ weights were recorded for adrenal glands,

intestinal ti ssue with Peyer‘s patches, liver, spleen, thymus, lymph nodes (axillary, mandibular, mesenteric). All tissues were fixed from subsets A

- D and tissues from subsets C & D were evaluated histopathologically. The spleen from animals assigned to subset D

were used to perform the NKC Function assay. Those animals in subset E were used for the splenic AFC assay following immunization with sRBC on study day 24 (table attached, pg 29

- WIL 639072 study). On day 28, blood samples were taken and the harvested spleen tissues were used to determine the specific IgM antibody

- forming cells.

Data acquisition and reporting was done by WIL Laboratories, as was the statistical testing with the exception of daily body weight and food consumption data. The AFC and

NKC func tional assay data and organ (spleen/thymus) weight data were analyzed by ImmunoTox®, Inc.

Concentration analysis of group 2 (1.3 mg/mL.) describes the relative standard deviation (RSD%) created on March 22 (2.5%) as nearly two times that of the formulat ion created on April 12 (1.3%).

No treatment related mortality and morbidity were observed. Few atrazine

- related clinical signs of toxicity were noted, with only the 100 mg/kg/day dose group displaying evidence of salivation after 2 hours following dos e administration. There were no other test substance

related clinical findings for animals at any dose level within 2 hours of dosing, or during daily examinations. The positive control group II showed an increase of red, brown, and yellow fur staining, but no other remarkable clinical findings. No clinical signs were noted in doses at 25 or 6 mg/kg/day.

Treatment resulted in dose

- dependent, statistically significant decreases in body weight gain and food consumption for ATR

- treated rats in the 25 and 1 00 mg/kg/day dose groups when compared to the untreated vehicle control group. The decrease found in body weightDo not cite

Appendix A

- Page 263 of 677 and food consumption persisted throughout the treatment period. Mean body weights for rats treated in the 6.5 mg/kg/day group were found to b e  ̳marginally‘ statistically significantly lower than the vehicle control group during study days 10

- 24, only when those data from subsets D & E were combined. The analysis for subsets D and E were combined from day 1 to 24, because animals in these subse ts were not necessarily comparable beyond day 24 (pg. 415

- WIL 639072 study). Food consumption for rats in this same dose group did not show a consistent effect of treatment on subsets, while individually there was

also no effect of treatment on food consu mption in the 6.5 mg/kg/day group.

Hematology data was assessed in only subsets C & D. Rats treated for 14 days with ATR at any dose showed no test substance related alterations. In subset C, statistically significant variations from the vehicle contr ol group were higher in mean corpuscular hemoglobin concentrations (MCHC) in the 25 and 100 mg/kg/day groups, higher mean percent neutrophil count in the 100 mg/kg/day group, and a lower percent eosinophil count

as well. These results were considered not toxicologically relevant since absolute counts are more relevant for interpretative purposes, hence these values did not show statistically significant or pathologically meaningful differences. Subset D, however, showed that

treatment with ATR for 28

- day s displayed multiple test substance related and statistically significant alterations in red blood cell parameters in the 100 mg/kg/day group when compared to the vehicle control and pair

- fed control group. These include low hemoglobin

concentration and h ematocrit, higher MCHC, reticulocyte counts (absolute and percentage), red cell distribution width, and hemoglobin distribution width values. No other variations in hematology were attributed to test substance administration.

Subset C data displayed no c ytological alterations or differences in bone marrow smear cytology between the control and the atrazine

- treated groups following 14 days of atrazine treatment. Overall ATR treatment at doses 25 and 100 mg/kg/day were associated with a rapid reduction in absolute thymus weights within 30 minutes following the first dose, although not found to be statistically significant. However statistical significance was found in 100 mg/kg/day group on days 0, 6, 13, and 27 when compared to the control group.

There a ppeared to be no adverse toxicologically relevant effects of ATR treatment on thymus weights at any dose and there were no ATR related effects revealed on any other organ weight.

Microscopic evaluation showed that animals euthanized after 14 days of tre atment showed a 50% increase in periportal glycogen content in the liver and an increased absolute and relative liver weight in this group. There were no treatment effects evident in the 25 or 6.5 mg/kg/day groups. There appeared to be a trend towards a dose

- dependent increase in Adrenocorticotrophic Hormone (ACTH) within 30 minutes following ATR administration at all dose groups. However, statistical significance was not found due to a high degree of

inter

- animal variability. No effect on ACTH was foun d after 7, 14, or 28 days of treatment.

Dose

- related, statistical significance was found in plasma corticosterone levels in all ATR treatment groups on study day 0 but not on study day 6, 13, or 27. Urinary corticosteroneDo not cite

Appendix A

- Page 264 of 677 levels were unaffected by ATR tr eatment on days 9 and 27. Plasma progesterone levels were statistically significantly elevated after the first dose on days 7, 14, and 28 at doses of 25 and 100 mg/kg/day in ATR treated groups. Prolactin plasma levels were highly variable

and no noted ef fects of ATR treatment in any dose at any time interval. In plasma corticosterone and prolactin, the pair

- fed control group, on day 27 displayed an increase when compared to the vehicle control group.

Atrazine had no effect on male (androgens) or female (estrogens) sex hormones at any level at any interval. Aldosterone levels in ATR treated groups on study day 0 were higher than control groups on the same day, while statistical significance was found in the 100 mg/kg/day treated group. Any other changes found in other dose groups were considered not related to the ATR administration. This was based on changes in control levels with time, variability in the magnitude and the direction of the changes observed over time, and

the absence of a dose

- response in the ATR treated groups.

Immunotoxicity assessments showed that ATR treatment at any dose did not suppress the IgM AFC response when compared to controls, even at doses which caused statistically significant decreases on food intake and body weight gain over 28 days. Total spleen activity showed an enhanced AFC response in the 25 mg/kg/day dose group only, although it was not considered biologically significant since no dose

- response was observed and the

increase was further attributed to a limited numb er of high responding animals. The positive control group I displayed a substantial and significant reduction in AFC activity.

Exposure to ATR at any dose didn‘t decrease the ability of Natural Killer (NK) cells to kill target tumor cells. The 100 mg/kg /day dosed animals displayed an enhanced ability to kill target tumor cells compared to vehicle control group, with a percent kill at effector to target ratio 200 to 1 and 100 to 1. Significantly suppressed NK cell activity was observed in the positive con trol group at all effector (NK cell) to target (Tumor cell) ratio. The pair

- fed control group had higher responses than the vehicle controls, but none of the increases reached a level of statistical significance. There was no evidence that the pair

- fed a nimals showed any reduction in the cell mediated immunity AFC response or in NK cell activity although a slight, but not statistically significant increase in NK cell activity was observed in the pair

- fed control group.

In conclusion, treatment of the mal e rats with ATR orally for 28 days did not ultimately produce adverse biologically relevant effects on the AFC and NK assay, while other components of the immune system were not assessed for their function

– it is premature to definitively conclude that ab solutely no functional deficits in humoral and innate immunity occurred. The study has shown equivocal evidence of toxicity to the immune system; further the immune system has not been proven to be a primary target organ. The toxicology database for atraz ine does not indicate that the immune system is the primary target organ. The observed effects on the immune system have been well characterized in the adult rat and these effects were seen at doses that produce overt systemic toxicity, i.e.,Do not cite

Appendix A

- Page 265 of 677 decreased th ymus weight at the highest does tested without histopathological findings. The NOEL for immune suppression is found to be 25 mg/kg/day for this study. The LOEL was the HDT, or 100 mg/kg/day. This is based upon a decrease in thymus weight and NK cell act ivity at effector : target site.

CLASSIFICATION

This study is classified as acceptable/guideline study.

A.6.3 Devos S, Bosscher K De, et al. 2003. Inhibition of cytokine production by the herbicide atrazine

– Search for nuclear receptor targets.

Bio chemical Pharmacology 65: 303

– 308

.

The purpose of this study was to test the hypothesis that atrazine affects leukocyte function via nuclear receptors such as the glucocorticoid receptor (GR), the androgen receptor (AR), members of the peroxisome proli ferator

- activated receptor (PPAR) family, and the retinoid

r elated 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

inflammatory and immunosuppressive properties and interfere with nuclear factor

– κB (NF

κB) and AP

- 1, and in this way repress the expression of inflammation

- related genes such as interleukin (IL)

- 6, IL

- 8, ICAM, and E

- selectin, that contain NF

- κB a nd AP

- 1 response elements. Activation of PPARs has been reported to have effects on leukocytes. RORα can also affect leukocytes function.

In this study, adult (2

- or 7

- month

- old) CF1 mice were injected i.p. on days 1 and 4 with atrazine (100 mg/kg). Co ntrol mice received the vehicle only (carboxymethylcellulose containing 0.5% Tween 80). Mice were sacrificed on day 7. Liver and kidneys were removed. Brief descriptions of cells (murine fibrosacrcoma L929sA and rabbit kidney RK13) and culture procedure s, plasmids (p1168hu.IL6P

- luc+, p98.IL8p

- luc+, p133.IL8p

luc+, pELAmP

- luc+), transfection and reporter gene analysis, ELISA, AR assay, and peroxisome analysis were provided.

Inhibition of human peripheral blood mononuclear 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 glucocorticoid inhibitor RU486. In contrast to DEX, atrazine did

not reduce IL

- 8 production. The authors concluded 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 with an IL

- 8 promoter

- driven report er gene construct, p98.IL8p

- luc+, and then induced with TNF and treated with DEX and atrazine (3, 0.3, and 0.03 μM) alone or in combination. TNF is an inflammatory stimulus activating the reporter genes. Testing aDo not cite

Appendix A

- Page 266 of 677 longer variant of the IL

- 8 promoter, p13 3.IL8p

- luc+, yielded the same overall results (data were not shown). To test whether the results with the IL

- 8 promoter were promoter

specific, the authors also tested the effect of atrazine on other proinflammatory gene construct, containing the E

- select in promoter (with NF

- κB elements) or the IL

- 6 promoter (with NF

- κB and AP

- 1 elements), that are both inducible by TNF. The authors indicated that there were no substantial effects with atrazine. They concluded that atrazine does not exhibit glucocortic oid

- 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 PPAR

- mediated induction of promoter activity was seen on targets of PPARα, PPARγ, or PPA Rδ. No binding of atrazine to the AR was observed. The authors concluded that their study shows that AR, GR, PPARs, or RORα are not likely targets of atrazine.

Reviewer‘s Comments: Using human peripheral blood mononuclear cells, the in vitro stud y of Devos et al. (2003) suggests that atrazine

- induced inhibition of human peripheral blood mononuclear cell proliferation and cytokine production is most likely not mediated through nuclear receptors such as the glucocorticoid receptor, androgen receptor , members of the peroxisome proliferator

- activated receptor family, and the retinoid

- rel ated orphan receptor α. The relevance of this study to human health risk assessment is limited since it entails intraperitoneal injection of atrazine to the animals prior to in vitro experiments.

A.6.4 Devos S, Van Den Heuvel R, et al. 2004. Limited ef fect of selected organic pollutants on cytokine production by peripheral blood leukocytes. Eur. Cytokine Netw: 15: 145

– 151.

This study examined the potential effects of in vitro atrazine treatment on cytokine production by peripheral blood mononucl ear cells (PBMC) from control and allergic donors. Cells were cultured for six days in the presence of atrazine (3 μM) or vehicle with phytohemagglutinin (PHA) or Dermatophagoides pteronyssinus (Dpt) extract, then for one day in the presence of PHA + phor bol 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 p rovided.

Atrazine treatment significantly reduced cytokine production IL

- 5 and IFN

- γ in the donors. This effect was apparent in PHA

- stimulated 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 c ytokines were limited, the impact of atrazine on allergic responses is unlikely.Do not cite

Appendix A

- Page 267 of 677 A.6.5 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 the 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

mon onuclear cells. To assess short

- vs. long

- term effects, the parameters were measured at one day, one week, and seven weeks after administration of the last atrazine dose.

Atrazine was administered to one

- month

- old male C57BL/6 mice for 14 days by oral

ga vage 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 cellularity was dose

- dependently decreased by atrazine treatment (statistically significant at 125 and 250 mg/kg doses) at day 1. The decreased cellularity persisted one

week after cessati on of atrazine 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

- depende ntly decreased at day 1. The authors reported that one week after cessation of atrazine treatment, 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, the decreases in the thymic subpopulations were numerically present, but were no longer significant (data not shown ).

In regard to splenic cell phe notypes, both MHC II+ and CD19+ cells were dose

dependently decreased, whereas only the high dose of atrazine increased the percentage of CD3+ cells. In addition, atrazine treatment decreased the number of sp l enic naïve T helper and T cytotoxic cells, wh ereas 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 persisted 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 mononuclear cells, a dose

- dependent decrease in circulating CD4+ cells was observed one day after the last atrazine dose. The decrease in CD4+ cells was still present at the 2 50 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 most of the changes in peripheral blood mononuclear cells were no longer apparent at seven weeks after the last atrazine dose, although data were not shown. The authors concluded that ―atrazine exposure appears to be detrimental to the immuneDo not cite

Appendix A

- Page 268 of 677 system of juvenile mice by decreasing cellularity and affecting lymphocyte distribution, with

cer tain effects persisting long after exposure has been terminated.‖

Reviewer‘s Comments: Although T cell subpopulations 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 FAO/WHO Meeting on Pesticides Monograph on Atrazine (JMPR 2007 )

.

A.6

. 6 Karrow, N A, McCay, J A, et. al. 2005. Oral exposure to atrazine

modulates cell

- media ted 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 immunomodulatory properties of atrazine and was designed to assess changes in ce llular and antibody immune function in the female B6C3F1 mouse model. This model also included pathogenic B16F10 tumor and Listeria monocytogenes challenge models to assess potential biological significance of any observed changes in immune 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 responsiveness. Immune assays and toxicity parameters included: body weights, organ weights (including spleen), hematolog y, differential cell counts, serum IgM titers, splenic natural killer (NK) cell activity, splenic mixed lymphocyte response (MLR), splenic IgM antibody

- forming cell (AFC) response, reticulocyte counts, splenic lymphocyte proliferation, splenic B and T cell enumeration, splenic cytotoxic T lymphocyte response (CTLR) to P815 mastocytoma cells, mononuclear phagocytic system (MPS) evaluation, L. monocytogenes challenge and B16F10 tumor challenge.

A decrease in body weights was observed in the atrazine treate d 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 erythrocyte counts (all dose levels ), decreased hematocrit (high dose), and increased mean corpuscular 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 a lter the spleen cell proliferative response 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 m ice. 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

did not consider this effect to be of biol ogical significance. A dose

- related decreasing trend in MPS function across the treatment groups was noted.Do not cite

Appendix A

- Page 269 of 677 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 l evel, a significant dose

- dependent increase in the number of B16F10 nodules was observed in the lungs of treated mice. The greatest increase in the number of nodules (122%) was observed at the 500 mg/kg treatment level. Although there were no statistical ly significant changes in host resistance to L. monocytogenes, the 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 level 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

. 7 Mizota K and Ue da 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 from mast cells and their putative neurosteroid receptor involvement. Drawing from their previous research, the authors proposed that NS2

- type neurosteroid receptors exist on mast cells a nd 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 representative H1 receptor antagonist. Ne urosteroid

- induced extravasation has been attributed to degranulation of mast cells.

Degranulation of mast cells was determined by measuring the rel ease of granule

associated β

- hexosaminidase from RBL

- 2H3 cells. Degranulation was also evaluated by

Alexa 488

- annexin V binding to the phosphatidylserine, which is externalized during degranulation. Antagonism of atrazine

- induced Alexa 488

- annexin V bind ing in RBL

- 2H3 cells by BSA

- conjugated progesterone (PROG

- BSA) was examined. β

- Hexosaminidase release was characterized by various inhibitors including antisense

- oligodeoxynucleotide for Gαq/11, pertussis toxin, phospholipase C inhibitor U

- 73122, inosito l 1,4,5

- triphosphte receptor inhibitor xestospongin 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 diphenhydramine

reversible 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 meas ured for 10 min. after i.pl. injection.Do not cite

Appendix A

- Page 270 of 677 Atrazine (10 nM

– 1 μM) caused a concentration

- dependent degranulation in RBL

- 2H3 cells and peritoneal mast cells as measured by β

- hexosaminidase release. This release was blocked by PROG

- BSA, a membrane impermea ble neurosteroid receptor antagonist.
PROG

- BSA also blocked atrazine

- mediated increase in Alexa 488

- annexin V binding (which labels phosphatidylserine externalization in the plasma membrane representing degranulation) in RBL

- 2H3 cells. The authors indic ated that the observation of atrazine

induced increase in Alexa 488

- annexin V binding shows that the atrazine

- induced degranulation is unlikely due to cell damage.

Antisense

- oligodeoxynucleotide pre

- treatment reduced 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. T he authors 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+, characteristics of

neurosteroids.

The results also demonstrated atrazine

- induc ed pain response that occurred immediately and ceased within 5 minutes 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 were caused by neurosteroid receptor

- mediated histamine release from mast cells, which was supported by atrazine

- induced plasma extravasation assay using Evans blue. Overall, the authors concluded that atrazine induces some inflammatory effects, including nociception and plasma extravasation, and the underlying mechanisms are mediated through mast cell degranulation and activation of the G q/11 - protein coupled neurosteroid receptor, phosph olipase C and intracellular Ca2+ mobilization.

Reviewer‘s Comments: This study concluded that the underlying mechanisms of atrazine

induced inflammatory effects such as nociception and plasma extravasation, may be mediated through mast cell degranulati on and activation of the Gq/11

- protein coupled neurosteroid receptor, phospholipase C and intracellular Ca 2+ . However, the inflammatory responses were examined following intraplantar administration of atrazine, a route of exposure that is not likely to o ccur in humans.

A.6

. 8 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

.

Based on prior results revealing th at atrazine may affect dendritic cell (DC) function (Rowe

et al. 20 0 6; Filipov et al. 2005), the objectives 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 were performed with primary thymic DC. Details were provided onDo not cite

Appendix A

- Page 271 of 677 the following materials and methods used for the analyses: dendritic cell line, primary

t hymic DC preparation, antibodies and flow cytometry, endocytosis assay, apoptosis assay, PCR analysis, and real

- time PCR analysis.

JAWSII cells were induced to mature in the 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 maturation of DC were examined. Phenotypic changes caused by atrazine included a dose

- dependent removal of surface MHC

- 1 with a significant decrease at 1 μM concentrati on. In addition, atrazine treatment decreased the expression of the costimulatory molecule CD86 and it downregulated the expression of the CD11b and CD11c accessory molecules and the myeloid developmental marker CD14. The phenotypic changes in JAWSII DC maturation were associated with functional inhibition of maturation at higher concentrations of atrazine where receptor

- mediated antigen uptake was increased. The authors stated that their data suggest that atrazine directly targets DC maturation and remo ves 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 maturation and decreases major histocompatibility complex 1 (MHC

- 1) molecule expressi on on the dendritic cell surface.

A.6.9 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.

To xicological Sciences: 75: 343

- 354

.

The authors stated that the study was conducted to determine if the ―stress

- induced immunological effects of atrazine 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 parameters.‖ Consequently, doses were selected in order to elicit an increase in corticosterone levels as a predictor of immune function; this study

was not intended as a haz ard 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 the study. Atrazine was administered intraperitoneally (only once) at dose levels of

100

- , 200

- , or 3 00

- 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, the corticosterone

area under the curve (AUC) values for atrazine

- treated mice were determined. Immunological parameters included: thymus and spleen cellularity, lymphocyte subpopulations in the thymus and spleen, expression of MHC class II protein on splenocytes, antibody resp onses to keyhole limpet hemocyanin, and natural killer cell activity. Models were derived describing the relationship between corticosterone AUC and potential immunological changes.Do not cite

Appendix A

- Page 272 of 677 The peak corticosterone response (approximately 1200 ng/ml) to atrazine occurred at 6 hours, and was dose

- dependent. In order to assess if the observed corticosterone response was unique to the intraperitoneal route of exposure, the authors included an additional experiment in which five mice were treated by oral gavage (cor n oil) to 500 mg/kg atrazine, while five mice were administered 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 dosing, serum corticosterone leve ls were measured. The authors reported the following corticosterone values: controls: 164 ± 88; atrazine

treatment: 676 ± 79.

Linear regression models of corticosterone AUC vs. thymus

- and spleen

- cell numbers and subpopulations revealed that atra zine treatment was associated 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 s plenocytes indicated a dose

- dependent decrease in the expression of MHC Class II protein on splenocytes in atrazine

- treated animals. The results

of linear models also indicated dose

- dependent decreases in splenic NK cell activity and IgG1 and IgG2a respon ses to KLH.

The authors also assessed the potential correlations between observed effects of atrazine and effects predicted using models derived from mice treated with exogenous corticosterone or restraint stress with linear modeling. The authors state d that the modeling results indicated that the atrazine effects were similar 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.

Review er‘s Comments: Pruett et al. (2003) demonstrated that a single high oral dose of 500 mg/kg atrazine also increased endogenous corticosterone levels. This study was intended to provide insight into the predictive value of corticosterone production over tim e for impairments of the immune system not to assess the immunotoxicity of atrazine per se

.

A.6.10 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 atrazi ne. The purpose was to determine if mice can become tolerant or habituated with regard to the stress response and accompanying immunological effects.‖ Female C57BL/6 X C3H F1 (B6C3F1) mice (8

– 12 weeks of age following a two

- week accommodation period) w ere 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,Do not cite

Appendix A

- Page 273 of 677 which can also induce neuroendocrine stress responses and alter immunological parameters. The exa ct doses of atrazine were not specified except for the high dose of 150 mg/kg/day. At the end of the study, the following parameters were measured: white blood cell count and differential blood count, lymphocyte phenotypes in the blood and spleen, natura l killer (NK) cell activity in splenocytes, and corticosterone concentrations (at various times after dosing or restraint). The results were compared with those from the acute study (Pruett et al. 2003).

The authors provided results for the corticoster one response in mice treated with 150 mg/kg/day atrazine with that observed in the 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. The authors concluded that the 28

- day atrazine treatment did not cause

tolerance or habituation.

Restraint had little effect on white blood cell counts following one treatment 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 treatment. Atrazine caused essentially the same increase in neutrophils and decrease in lymphocytes after 28 days of treatment as it did after 1 dose administration. Changes in the percen tage of MHC class II expression by B lymphocytes and MHC class II expression on B lymphocytes in the spleen were comparable between the single dose and 28

- day daily dose regimens. Atrazine caused a similar decrease in B cell percentage in the blood and s pleen in the 28

- day and single day studies. Restraint did not affect the percentage of B lymphocytes following

acute or 28

- day treatment. A dose

- dependent decrease in CD4+ and CD8+ T lymphocytes in the blood was noted after single dose administration of atrazine; however,

28

- day treatment increased this parameter. 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 bot h acute and 28

- day atrazine treatments; however, decreases were greater with the latter treatment.

Reviewer‘s Comments: Pruett et al. (2009) provided some supporting evidence that 28

- day atrazine exposure did not cause habituation or tolerance to the HP A axis perturbations, but immune function (e.g., antibody production) was not evaluated in this study.

A

. 6

. 11 Rajkovic, V, Matavulj, M and Johansson, O. (2010). Combined Exposure of Peripubertal Male Rats to the Endocrine

- Disrupting Compound Atrazine and Power

- Frequency Electromagnetic Fields Causes Degranulation of Cutaneous Mast Cells: A New Toxic

Environmental Hazard? Arch. Environ. Contam. Toxicol. DOI

10:1007/s00244

- 010

- 9477

- 6

This study evaluated the in vitro effects of single and combined trea tments of atrazine (98% purity) and power

- frequency electromagnetic fields (EMFs) on cutaneous mast cells inDo not cite

Appendix A

- Page 274 of 677 juvenile/peripubertal male Wistar rats. The investigators chose to study the effects of atrazine and EMFs because they thought it likely that huma ns could be exposed simultaneously to various combinations of chemical and physical environmental agents, which raises a concern for possible health effects. Atrazine was chosen since it is represents a chemical found in drinking water and EMFs generated by power lines and electric appliances represent an unavoidable physical factor in the environment.

Male Wistar rats were treated from postnatal day (PND) 23 to PND 53. Animals were divided into 6 groups (n=10 animals/group): 1) control ; 2) 4 hours/da y to EMFs (50 Hz,

100

- 300 μ T, 54

- 160 V/m); 3) 20 mg/kg/day atrazine ( per os , dissolved in olive oil); 4) 200 mg/kg/day atrazine ( per os , dissolved in olive oil); 5) combined EMF with 20 mg/kg/day atrazine; 6) combined EMF with 200 mg/kg/day atrazine. The exposure system for EMF

tr eatments was previously described in Rajkovic et al. 2005a 22 . Dose levels were selected based on previous studies on atrazine effects on prepubertal rats (Stoker et al. 2000b 23 ).

Light Microscopy: Animals were decapitated and skin samples (10/group) were taken from the interscapular region, fixed in 4% paraformaldehyde, embedded in paraffin, and

sectioned into 5

- μ m

- thick sections. The histochemical staining method with toluidine blue was used for the histological analysis and quantitative estimation of mast cells.

Stereological Analysis: Skin sections from 10 rats in each experimental group were analyzed stereolo gically using a multipurpose stereological grid M

- 42 placed in the ocular of a Reichert light microscope under a total magnification of x400. The numerical (Nvm) and volume (Vvm) densities of the total mast cell population of degranulated cells and of

int act cells in the skin dermis were determined. Estimations were made by the same

observer on blindcoded sections.

A.6

. 12 Rooney, A A, Matulka R, and Luebke, R. 2003. Developmental atrazine exposure suppresses immune function in male, but not female Spr ague

Dawley rats. Toxicological Sciences 76: 366

- 375.

This study was designed to test the hypothesis that atrazine may cause developmental immunotoxicity through its disruption of prolactin (PRL) or thyroid hormones during a critical early period of dev elopment. Pregnant Sprague

- Dawley (SD) rats were exposed via oral gavage to 35

- mg atrazine/kg/day from gestational days (GD) 10 (to avoid the possible interference of atrazine with embryo implantation) through postnatal day (PND) 23. The

authors indicate d that the dose of 35 mg/kg/day was set ―just above the NOEL for atrazine

-

22 Rajkovic, V et al. 2005a. The effect of extremely low

- frequency electromagnetic fields on skin and thyroid

amine

- and peptide

- containing cells in rats. An immunohistochemical and morphometrical study. Environ Res 99(3):369

- 377.

23 Stoker TE et al. 2000b. Endocrine

- disrupting chemicals: prepubertal exposures and effects on sexual maturation and thyroid function in the male rat. A focus on the EDSTAC recommendations. Crit Rev Toxicol. 30(2):197

- 252Do not cite

Appendix A

- Page 275 of 677 induced developmental endocrine dysfunction to examine the potential for atrazine to indirectly induce immunotoxicity.‖ The authors stated that this dose is likely to induce

endocri ne effects in both sexes. Separate groups were exposed to bromocry ptine (BCR) at 0.2 mg/kg/2x/day to induce hypoprolactinemia or to propylthiouracil (PTU) at 2 mg/kg/day to induce hypothyroidism. At 7 weeks old, the following immune functions in the offs pring were evaluated: natural killer (NK) cell function, delayed

- type hypersensitivity (DTH) responses to bovine serum albumin (BSA), phagocytic activity of peritoneal macrophages, and antibody response to sheep erythrocytes (SRBC).

There was an increa se in offspring mortality in atrazine treated animals during PND 2

– 14. The average % offspring mortality/litter in atrazine

- treated dams was 13.2 compared to 2.3% for control dams. The authors noted that increased mortality was ―unexpected, as

this ha s not been reported for this 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 themselves.‖ The authors stated that male offspri ng body weight of the atrazine group was decreased relative to controls (mean control body weight was 20.1 g compared to a mean atrazine group body weight of 19.0 g).

The primary (IgM) antibody response to SRBC was suppressed in atrazine

- treated males ( p = 0.0149), but not female, offspring at 8 weeks of age. There was no difference among treatment groups in the IgG 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

- month

- old

- animals (data were not shown). The latter results indicate that that immune 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 atrazine treatment group persisted until at least 12 weeks of age. There was no difference in the DTH response between the offspring of atrazine

- treated dams and the control 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 treatmen t on phagocytosis by peritoneal macrophages.

The authors concluded that atrazine 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 atrazine are not mediated through the suppression of PRL or THs. In addition, the authors concluded that ―the current data do not support nor completely refute a relationship 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 theDo not cite

Appendix A

- Page 276 of 677 suppression of prolactin or thyroid hormones. The precise underlying mechanism of an

atrazine

- mediated sex

- specific effect, however, is not fully understood.

A.6

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

– 77.

The aim of this study was to test the hypothesis that prenatal/lactational exposure to atrazine would affect the development of the immune system and alter immune function in

t he adult offspring in the Balb/c mouse model. Pregnant Balb/c dams were exposed subcutaneously for 21 days via time release pellets to 700 ug atrazine/day beginning between days 10 and 12 of pregnancy. The implanted pellet was designed to release the

sam e amount of atrazine every minute of every hour of every day until the end of the treatment period. The rate of exposure at the beginning 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/lac ta tional exposure ―caused no overt physical malformations in the offspring and

had no effect on the number of litters carried to term or the litter size.‖

Immune status was evaluated at approximately 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

stimula tion was assessed using two different assays. In one assay, splenocytes were isolated from unimmunized prenatally/lactationally exposed Balb/c mice and stimulated using irradiated splenocytes from female C57B1/6 mice. Proliferation of alloreactively

stim ulated lymphocytes was assessed by measuring the incorporation of 3 H

- thymidine into newly synthesized 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 allor eactive target cells ( i.e. , cytotoxic T lymphocyte response), was assessed following alloreactive stimulation.

EL

- 4 lymphoma cells were used as targets. The humoral immune response 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 spleen weights. No changes wer e 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 significant increase in the number of HKSP

- specific IgM secreting B cells in the spleen of treated male mice was noted. Male atrazine

- exposed offspring had 78.7 ± 4.5 HKSP

- specific IgM antibody secr eting 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 fromDo not cite

Appendix A

- Page 277 of 677 controls. The authors concluded that the results in dicate ―a male

- specific immunopotentiation of the immune response following prenatal/lactational atrazine exposure.‖

In a review article (Rowe et al. 2008; Basic & Clinical Pharmacology & Toxicology, 102,

139

- 145), the study authors reported on the re sults of similar assays performed on available littermates at 6 months of age (defined as ―long

- term‖). Limited data were presented. The authors noted that too few male offspring were available to perform replicate assessments. However, they stated that a single iteration of the mixed lymphocyte response assay in the males showed 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

- month

- old atrazine

- treated females had a significant and reproducible reduction in the number of phosphorylcholine (PC)

- specific responding splenocytes following HKSP vaccination (PC

- specific IgM

- prod ucing B cells in the spleen of HKSP

vaccinated atrazine

- treated females were 69% of control). In addition, spleens from the

atrazine

- treated groups produced a mean of 72% of control IgG PC

- specific B cells per million splenocytes. The MLR response was al so assessed in female offspring. Results

indicated that atrazine

- treated females had a significant decrease in the MLR (spleen cell proliferation level was 72% of control and was statistically significant).

The authors also stated that in the 6

- month

- o ld females, the total number of splenocytes and the lymphocyte subpopulation phenotypes were also unaffected. The mean spleen weight and total spleen cell number were slightly higher in the treated groups. The percentage of spleen cell subtypes (CD4, CD 8, 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 that the findings ―demonstrate a sex

- and age

- dependent effect of prenatal exposure to atrazine on th e immune system of the adult offspring.‖

Reviewer‘s Comments: Rowe et al. (2006) reported immunopotentiation 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 reasons 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 methodologies us ed to assess immune response

.

A. 6

. 14 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 atrazine 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 target cell adhesion and subsequent granule release in order to clarifyDo not cite

Appendix A

- Page 278 of 677 precise c hanges in NK cell lytic function. Peripheral blood lymphocytes were isolated from healthy human male donors and exposed 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 culture, peripheral blood lymphocyte harvest, cytotoxic lymphocy te 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 atrazine did not affect viability or NK cell subpopulation distributions (data were not shown). For this experiment, PBLs were assessed for viability and subpopulation distribution via flow cyto metry. Subpopulation distribution was determined using four lymphocyte markers (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

fo llowing a 24

- hour exposure to atrazine. Atrazine also did not affect the post

- translational modification of perforin. In addition, atrazine treatment did not affect the ability of NK cells to bind target cells. However, atrazine treatment decreased the ability of NK cells to release granule proteins.

Reviewer‘s Comments: Rowe et al. 2007 showed that atrazine

- induced inhibition of in vitro human peripheral blood lymphocyte NK cell

- specific lysis of K562 human leukemia cells may be the result of inhibit ion of lytic granule release, without affecting the ability of the natural killer cell to form stable conjugates with target cells.

A.6. 15 Schwab, C L, Fan R, et al. 2005. Modeling and predicting stress

induced immunosuppression in mice using blood p arameters. 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 interest. Utilizing the same approach as Pruett et al. (2003), Schwab et al. (2005) showed that the atrazine

- induced decreases in CD4+ and CD8+ T cells in blood were similar to exogenous corticosterone.

Reviewer‘s Comments: This study provides further support for the predictive value of

corticosterone for immune markers.

A.6.16 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

.Do not cite

Appendix A

- Page 279 of 677 The effects of in vitro atrazine treatment on the ability of human natural killer (NK) cells to lyse tumor cells were investigated. Atrazine 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 the 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 atrazine inhibited K562 tumor cell lysis by 63 ± 25 %

after 24

- hour exposure, and by 83 ± 21 % after a 6

- day exposure. 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, the relevancy of these findings to in vivo exposure

remains unclear.

A.7 CARCINOGENESIS/ GENOTOXICITY

Su mmary: Carcinogenesis/Genotoxicity

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 Sprague

- Dawley rats a fter 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 extensive 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 concluded, that the data sh ow that the operative MOA for mammary gland tumorigenesis involves the perturbation 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 relevant endp oint to assess potential adverse reproductive effects, it is not relevant for mammary tumor development in humans. In rats, the mammary tumors arise as a result of constant stimulation of the mammary gland by elevated levels of circulating estrogen and pro lactin during the early reproductive senescence caused by the disruption of the LH surge. In contrast, humans‘ reproductive senescence (menopause) is not the consequence of disruptions at the hypothalamic/pituitary level, but rather a depletion of follicl es and a decrease in estrogen. That is to say, that while the hallmark of

atrazine

- induced reproductive senescence in rats is elevated estrogen, in humans menopause is marked by a decrease in both estrogen and prolactin. Consequently, in theDo not cite

Appendix A

- Page 280 of 677 absence of c onstantly elevated estrogen and prolactin levels mammary gland tumors would not develop in humans as a result of atrazine exposure.

Several genotoxicity and carcinogenicity 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 tissues (pituitary, thyroid, ovaries, and mammary gland). It should be noted that none of thes e experiments study the tumor

- initating 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 tha t 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 cell

- lines tested.

Work by Fujimoto and cowork ers in 2003 using transplanted MtT/E

- 2 pituitary rat cells (a cell line highly

- sensitive to estrogen

- induced proliferation), showed that in ovariectomized F344 rats exposed through the diet to atrazine at dose levels ranging from

5

- 500 mg/kg/day atrazine e xposure did not accelerate tumor progression. In contrast, tumors were observed within 9 days in animals treated with 0.5 mg subcutaneous pellet of estradiol. In this test system, serum prolactin levels, cell proliferation, and estrogen gene

activation w ere also unaffected 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 thyr oid 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 ovar ian adenocarcinomas involves hormonal disruption (particularly estrogen). Therefore, they postulate that exposure to atrazine (which has been associated with hormone disruption) may also affect ovarian carcinogenesis. As expected, a single exposure to 0. 5% DMBA induced ovarian tumor formation. Subsequent atrazine exposure through the diet at doses ranging between 5

- 500 ppm led to a slight, non

- statistically significant decrease in the incidence of ovarian tumors seen after DMBA exposure alone. When cons idered collectively, the data reported by Fujimoto, Son, Tanaka and co

- workers 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 carryi ng a human c

- Ha

- ras proto

- oncogene atrazine exposure enhanced

DMBA

- induced mammary tumor development. However, a dose

- response could not be established for this finding. Likewise, the incidence of mammary adenocarcinomas appeared to be increased but a do se

- response relationship was not observed since the

incidence at the low

- and mid

- dose was higher 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. Furthermore, at razine exposure in vitro did not cause MCF

- 7 cell proliferation

– anDo not cite

Appendix A

- Page 281 of 677 estrogen receptor

- positive human mammary cancer cell line. Consequently, it is difficult to ascertain if the changes seen after atrazine exposure are truly related to an inherent

carcino genic 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 exposure on the progression of DMBA

- induced mammary tumors in ovariectomized Sprague Dawley rats was investigated. When DMBA

- induced rats were examined, no clearly discerni ble correlation between atrazine exposure and the

number of tumor

- bearing 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 regression 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. Exposure 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 the Fukamachi and Ueda studies which are described in Appendix A. Nonetheless, these two sets of data suggest that atrazine 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 cel l proliferative properties in a variety of cell lines indicate that atrazine is cytotoxic or causes decreases 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 exposure (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 steroidogenic 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 method for DNA strand break detection in

individual cells

– was used to study atrazine‘s capability of in ducing genotoxicity. Using this assay, Garaj

- Vrhovac et al. and Zeljezic (2004) concluded that Gesaprim (an atrazine formulation) induced genome damage. The limitation of these studies is that a formulation of atrazine was used and no information is prov ided to determine 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

. Sister chromat id exchange (SCE) analysis

– a s ensitiveDo not cite

Appendix A

- Page 282 of 677 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 range of 5

- 200 μg/L atrazine did not increase the frequency of SCE

induction in G2

- prematurely condensed chromosomes.

Finally, two studies intended to evaluate if vitamin E treatment follo wing 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 acetylcholinesterase activity and c ell membrane lipid content suggesting an increase in oxidative stress. Concurrent exposure to atrazine and vitamin E dampened all of these effects. A subsequent study by the same research group showed that at 300 mg/kg/day of atrazine induced DNA instabi lity as measured by the Comet and micronucleus assays. Again, vitamin E exposure at 100 mg/kg/day decreased micronuclei formation and DNA breaks. The results of this study are questionable given that the atrazine doses used to induce these genotoxic effe cts are orders of magnitude higher than what is currently used as the point of departure for human health risk assessment.

A.7.1 Fujimoto , N an d Honda , H. (2003). Effects of Environmental Estrogenic Compounds on Growth of a Transplanted Estrogen Respon sive Pituitary Tumor Cell Line in Rats. Food Chem. Toxicol. 41:1711

- 1717

. The objective of this study was to evaluate the role genistein, p

- nonylphenol, or atrazine as tumorigenic promoters for estrogen

- sensitive tumors. A rat pituitary cell

- line

– MtT/E

- 2

– that is responsive to estrogen exposure was transplanted into ovariectomized rats. The rats were subsequently exposed to estradiol, genistein, p

- nonylphenol, or atrazine and examined twice a week for the development of tumors. 24 In addition, MtT/E

- 2 cell cultures were transfected with an estrogen

- response

- element

- luciferase reporter gene and evaluated for activation of the reporter gene. These cells were also grown in vitro and evaluated for cell proliferation after exposure to the chemicals under ev aluation. Finally serum collected at the time of sacrifice was used to evaluate prolactin levels.

Six week old ovariectomized F344 rats (sample size not reported) were inoculated with

MtT/E

- 2 tumors cells and subsequently exposed 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, the pituitary gland and uterus were collected and weighe d and blood serum obtained to measure for prolactin levels. For cell proliferation assessments, MtT/E

- 2 cell cultures were exposed to 0 or10 - 7

- 10 - 4 M atrazine for 7 days and cell proliferation measured 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 cells

24 In this evaluation only the results for atrazine will be discussedDo not cite

Appendix A

- Page 283 of 677 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 proliferation 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, ute rine, or pituitary weights. Serum prolactin levels were also unaffected by exposure to up to 500 mg/kg atrazine. 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

a nd pituitary weights along with a marked increase in serum prolactin (↑7

- fold, p<0.01). Tumor development was also unaffected by treatment with atrazine with evidence of tumor development first appearing on day 24 of the experiment for animals in the cont rol 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 used in the study or the sample size. N onetheless, some general conclusions may be drawn from this study. In particular, that atrazine exposure up to 500 mg/kg did not enhance the appearance of estrogen

- dependent pituitary tumors nor does it result in increased cell proliferation. When conside red collectively, the 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 T suda, 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

.

This study evaluates the potential of a trazine 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

- ras proto

- oncogene. These rats are exquisitely sensitive to

DMBA

- induced mammary tumors. 25 Two

- hundr ed 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 expose d 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 mea sured 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

25 In this evaluation only the results for atrazine will be discussedDo not cite

Appendix A

- Page 284 of 677 proliferation was also measured by exposing the human mammary cancer cell line MCF

- 7 to atrazine for 3 days. Cell 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 exhibited a statistically significant decrease in body weight (↓14%, p<0.01) with a concomitant decrease in food consumption (↓10%, p< 0.05). A statistically significant increase in the incidence of mammary adenomas (↑67%, p<0.01 vs 17% control) was noted following exposure of Tg female rats to 5 ppm atrazine. However, no increase was observed after exposure to 50 or 500 ppm atrazine. The incidence of mammary adenocarcinomas at 5 and 50 ppm atrazine was 100% (contro l: 50%) although the incidence 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

- stat istically 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).

The study authors concluded that atrazine appeared to enhanc e mammary tumors in

female Tg rats but without any dose relationship while decreasing the incidence of skin tumors in Tg males . Furthermore, the authors concluded that atrazine did not increase cell proliferation of MCF

- 7 cells. Several limitations were identified in this study that hinders a meaningful interpretation of the results. 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 add ition, 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 presented for the non

- Tg rats or for the MCF

- 7 cell proliferation assay.

Reviewer‘s Comments: The la ck 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 between atrazine exposure and incr eases in the incidence

of tumor

- bearin g rats cannot be drawn . Thus, in the current study the different treatment

and lack of data on the non

- Tg rats precludes reviewers from drawing a conclusion on whether the increases seen in mammary adenocarcinomas were related to atrazine exposure. The l ack 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 Zeijezic, D. (2004) Genotoxicity evaluation of pesticide formulations containing alachlor and at razine in multiple mouse tissues (blood, kidney, liver, bone marrow, spleen) by comet assay. Neoplasma 51.3:198

- 203

.

In this study the authors evaluated possible genome damage caused by two pesticide formulations (Bravo® and Gesaprim®) containing alachlor and atrazine as activeDo not cite

Appendix A

- Page 285 of 677 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 possible genotoxicity of pesticide formulations and not only their active ingredien ts. Workers 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 th e 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 grou p injected intraperitoneally once. The lower dose represents the oral 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 (taken into heparinized vacutainers) and selected organs were weighed, minced, and suspended in a chilled homogenization buffer.

The alkaline comet assay of blood samples was performed according to standard protocol, while organ samples were subjected to a modified assay (Sasaki, YF, 1997). Each organ and pesticide concentration, 100 cells were analyzed by the Comet assay II automatic digital analysis system to determine tail length and tail mom ent. Any differences of assay endpoints between control blood and organs and those from treated mice were evaluated using the Mann

- Whitney U

- test.

Results showed no death, morbidity, or distinctive clinical signs observed after any treatment (including d uring necropsy). Mean values of the 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/kg) and high (1.08 ml/kg) treatment groups compared to co ntrol. The DNA of the kidney and liver, for both concentrations given, showed the largest migration. There was also a significant increase for all tissues in the higher concentration treated group when compared to the lower 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 hi gher dose group the kidney and liver tail lengths 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 lengths 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 damage in all tissues

examined was detectable with the comet assay. The most affected organs were the kidneyDo not cite

Appendix A

- Page 286 of 677 and liver. This paper also cites several 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 genotoxicity and provided a rationale as to why the toxic activity maybe slightly increased at higher doses when compared to the RfD, could be due to the removal of apoptotic cells (data not shown). Other apoptotic detection metho ds should be employed before 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 doses of atrazine. However, there are uncertainties regarding the atrazi ne 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 atrazine is uncertain because neither the species (rat) nor the target tissue for tumors (mammary tumors) were examined. Overall, the use of the alkaline com et assay on different tissue samples could provide evidence of genotoxic effects of pesticides in vivo

. Garaj

- Vrhovac et al. and Zeljezic and Garaj

- Vrhovac (2004) concluded that Gesaprim (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 determine the percentage of atrazine present in the test material. Thus, it is entirely possible that the DNA breaks reported after Gesaprim exposure may be

relate d to other chemicals in the formulation rather than atrazine per se

.

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

.

The purpose of this study was to determine the 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 cytoto xicity and the morphological changes were followed during cell death.

CHO

- K1 cells were treated with atrazine at concentrations ra nging from 10

- 160 μg atrazine/mL of cell suspension (46

- 742 μM) for 24, 48 or 72 hours. The cell viability was determined by four different bioassays: Trypan blue exclusion method, Kenacid Blue, Neutral red and MTT assays. Trypan blue exclusion method was used to estimate the proportion of viable cells. The Kenacid blue assay determines the total cell protein content. The Neutral red uptake assay determines the accumulation of the neutral red dye in the lysosomes of viable uninjured cells. The MTT assay determines the viability of cells.

All four methods showed clear dose

- dependent cytotoxic effects. Concentrations of

atrazine 20

- 80 ug/mL followed by Trypan blue method showed significantly decreased proliferation of cells in CHO

- K1 cells line at all dos e levels, with the toxicity progressivelyDo not cite

Appendix A

- Page 287 of 677 increased with high atrazine concentrations. After 72 hours incubation period, the 20 ug/mL atrazine concentration significantly decreased cell proliferation by 19% in CHO

- K1. Cell viability was decreased 49% at 6 0 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, Kenacid 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

a poptosis in atrazine treated cells was low.

Reviewer‘s Comments: Cell proliferation was decreased in Chinese Hamster ovary (CHO) cell lines at relatively low levels of atrazine exposure.

A.7.5 Lasserre, JP, Fack, F, Revets, D, Planchon S, Renaut J, Hof fmann L, Gutleb, Muller CP, Bohn T, ( 2009 ) Effects of the Endocrine Disruptors Atrazine and PCB 153 on the Protein Expression of MCF

- 7 Human Cells.

Journal of Proteome Research

.

The present study aims to investigate the proteomic effects of endocrine dis rupting compounds using MCF

- 7 cells as a model. MCF

- 7 cells were chosen as these cells are derived from a human breast cancer and their growth is being stimulated by 17

- β estradiol. To identify changes of protein expression in MCF

- 7 human cells, they were treated with either 100 ppb atrazine or PCB153 during 24 h. A negative control containing DMSO was included as well as a positive control containing 17

- β estradiol. Pro teins from the two groups (Atrazine, PCB153) and two controls (positive and negative controls) were separated by isoelectric focusing (IEF) followed by SDS

- PAGE. After separation and analysis, the differentially expressed protein spots were excised, digest ed by trypsin, analyzed by MALDIToF and identified by comparison with databank searches.

The chemicals and materials used consisted of Atrazine (98% purity), PCB153 (100% DMSO with 2mg/mL stock solution), 17

- β estradiol (DMSO 10uM solution), cell cultures were done in monolayers (MCF

- 7bus cells), protein extraction and analysis were performed and used to obtain sufficient biological material. Protein labeling was done with a pooled ―internal standard‖ and wer e labeled prior to mixing and electrophoresis with CyDyes. Bidimensional electrophoresis was also performed. Labeled protein images were then captured and analyzed by excitation of gels. Proteins were then identified by searching against the NCBInr data bases using GPS Explorer

Software v3 .6. The re sults were displayed in showing a total of 5,828,094 sequences of which 272,706 were for humans. The search parameters allowed fixed modifications for cysteine and methionine and variable modifications of try ptophan (kynurenin, oxidation and double oxidation) as well as peptide N

- terminal E or Q as pyroGlu. All identifications were manually verified and validated. Only the proteins having a protein score superior to MASCOT protein score threshold for signific ant homology were considered. The acceptance criteria allowed for ion scores greater than 26 or 43 indicate homology and identity, respectively.Do not cite

Appendix A

- Page 288 of 677 The results for m orphology and adhesion of MCF

- 7 cells showed differ ent observations between

the four conditi ons indicated that the different treatments had an effect on morphology and adhesion of cells. Cells exposed to 17

- β estradiol were spread out, whereas the cells were more rounded and comparatively smaller for the atraz ine, PCB153 and

DMSO treatment. The analysis of the different treatments revealed that overall and within all pairwise comparisons, 66 spots were differentially regulated in their expression ( P < 0.05, 2

- sided).

From these 66 protein spots, 49 were tentatively identified, belonging to 29 d ifferent proteins. Out of these, 22 different proteins were identified by at least two peptides, with a suffi ciently high ion

score . Some proteins (far upstream element binding protein, histone H1b, actin, heat shock 70 kDa protein 8) were identified in mo re than one spot, suggesting the presence of isoforms and/or post

translational modifications that affect the p I and/or the molecular mass of the protein.

Identified proteins were classified with respect to their subcellular localization and biological

p rocesses , according to information collected on Swiss

- Prot (http://www.expasy.ch/sprot/). The majority of the identified proteins were intracellular proteins (88.6 %), mostly distributed in the nucleus (34.3%) and cytosol (34.3%). Concerning the function of the proteins of interest, results showed that the proteins identified were involved in various processes. The e ffects of a trazine and
PCBs on the Proteome of MCF

- 7 Human Cells showed that a mong the 22 differentially expressed proteins, 10 were specifical ly differentially expressed upon atrazine treatment, 4 were specifically differentially expressed following the PCB treatment and 8 were both differentially expressed after
atrazine and PCB treatment . Among 18 differentially expressed proteins during the atrazine treatment, 88% were underexpressed, and among 12 differentially expressed proteins during the PCB treatment, 75% were overexpressed. These results indicate that atrazine treatment seems to decrease the activity of the cells, whereas the PCB153 tre atment stimulated the cells

.

The authors concluded that this is the first report on the effects of atrazine and PCB153 at the proteomic level on MCF

- 7 human cells, a cancer cell line. They revealed a total of 22 differentially expressed identified protei ns, predominantly involved in transcription processes, stress regulation and structural components. They claim that their findings indicate that

atrazine treatment seemed to decrease the activity of cells, whereas the

PCB153 treatment stimulated the cells . The present findings indicate that the differentially expressed proteins were not located in a single compartment of the cell. Furthermore, different functions of the cell were affected, indicating that atrazine targeted neither a unique compartment

no r a single process in the cell. The causes are purely speculatory.

The observed expression levels of these proteins following atrazine and PCB153 exposure may

have contributed not only to DNA damage as previously described for PCB153 or atrazine but al so to changes of the expression level of certain genes. The present study aimed to understand the

mechanism of action of atrazine and PCB153 on MCF

- 7 human cells in particular. Here, it was ultimately found that atrazine and PCB153 have effects on the ex pression of several proteins located in different cell compartments and are involved in various processes such as oxidative stress, DNA damages, shape of the cell, gene expression regulation or spermatogenesis. It is suggested that f uture studies should t arget cellular subfractions such as membrane, cytosol and

nucleus. Also performing studies which are designed to go into further detail with various

concentrations of additional individual EDC compounds would be useful

. Since the environment isDo not cite

Appendix A

- Page 289 of 677 most often polluted by several pollutants that could act in synergy it would be prudent to study the

effects of mixtures.

A.7.6 Malik, S. I., Terzoudi, G.I., and Pantelias, G.E. (2004). SCE analysis in G2 lymphocytes prematurely condensed chromosomes after exposu re 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,

sti mulated with phytohemagglutinin and exposed 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

conducte d. Prior to the end of treatment, 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 a t G2 rather than the conventional approach of colcemid treatment to collect cells at metaphase was to determine whether atrazine would increase the frequency of homologous recombination, a DNA repair process associated with SCE induction. Cells were colle cted, stained by the

fluorescent

- plus

- Giemsa technique and 30

- 50 cells were scored for each culture; triplicate cultures were prepare d per treatment group.

Results from the first experiment 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

- treated cultures. Thus, SCEs could be scored even at l evels that were lethal in the conventional 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 significantly lower in G2

- PCCs than in SCEs scored in the metaphase cells.

Reviewer‘s Comment: The assay was well

- conducted and indicates that there was convincing evidence that atrazine is not genotoxic in this test system.

A

. 7.7 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

.

This purpose of this study was to examine the effect of low

- level, sh ort

- term atrazine exposures on human fibroblast cells in culture by assessing cell proliferation and apoptosis.Do not cite

Appendix A

- Page 290 of 677 Normal human fibroblasts, DET 551, were used in the saturation density analysis to measure cell proliferation. In this assay, DET 551 cells we re plated at 2x10 5 cells/100 mm dish in MEM with 0.5% FBS (low serum medium). Cells were incubated at 37oC 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 atrazine or DMSO (0, 3, 10, 50, 100, 200, and 300 μg/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 ha rvesting, individual cells were 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 atrazine or solvent control was compare d to the number of control cells. Cells were counted 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 ca lorimetric assay. DET 551 cells were plated

in flat

- bottom 96

- well tissue culture treated 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 in c reasing concentrations of atrazine or DMSO (0, 3, 10, 50, 100 and 200 μg/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 μl of 5 mg/ml MTT in PBS was added to cells and pl ates were incubated for 4 hours at 37oC. HCl in isopropanol was added 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 an d monocytes. One million PBMCs were plated in 24 well tissue culture plates in PRMI with 5% FMS containing increasing concentrations of atrazine or DMSO (40, 100 or 200 μg/ml). Cells were

incubated for 96 hours at 37oC and 5% CO 2. Then 150 μl of MTT solu tion was added and cells were incubated for an additional 4 hours at 37oC. HCl in isopropanol was added to each well, and optical 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 immunohistochemical analysis and a quantitative enzymatic analysis (caspase assay). In the qualitative immunohistochemical analysis, apoptotic cells are detected with a biotin

- streptavidin system complexed with horseradish peroxidase and a diaminobenzidine

- based stain. DET 551 cells were exposed to atrazine

or D MSO at concentrations of 0, 3, 50 or 200 μg/L for 48 hours before the assay was performed. 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

m easured using the CaspASE colorimetric assay system protocol. The CaspASE assay is a colorimetric assay that measure activity of caspase

- 3. Cytosolic thiol proteases calledDo not cite

Appendix A

- Page 291 of 677 caspases are key enzymes involved in apoptosis. DET 551 cells were exposed to

incr easing concentrations of atrazine or DMSO (0, 3, 100, 200, and 500 μg/L) for 48 hours before analysis.

Cell Proliferation Results : The saturation density analysis showed that DET 551 fibroblasts exposed to increasing concentrations of atrazine showed a decrease in cell growth compared to control cells. Cells expo sed to 50, 200 and 300 μg/L atrazine had a statistically significant decrease (1.2

- 1.5 fold) 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 μg/L) compared to the other DMSO control values. This could indicate that this concentration 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 t o the chemical for 5 days, 2 days longer than in the MTT analysis.

In the MTT analysis, a 48 hour exposure to atrazine showed a statistically significant decrease (p<0.05) in DET 551 cell proliferation. Cells exposed to 10

- 200 μg/L had a dose

related si gnificantly decreased absorbance (1.1

- 1.7 fold) compared to controls, indicating a decrease in cell proliferation. There was no statistically significant decrease in proliferation

in solvent

- control

- treated cells. There was no effect on cell proliferatio n by MTT analysis when human PBMCs were treated 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 μg/L atrazine or DMSO for 48 hours before the assay was performed. The

atrazine and DMSO

- treated samples were compared qualitatively to a TACs nuclease

positive control . When the cells treated with 200 μg/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 control. 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 μg/L) had caspase activity (7.31

- 9.53 pmol/μl) similar to the negative control (7.31 pmol/μl). No significant differences were detected between the control treatments (DMSO) and

atrazine

- treated cells.

Conclusion: In vitro exposure to lo w levels of atrazine (as low as 10 μg/L) for a short exposure (48 hours) causes a decrease in ce ll proliferation in human 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 proliferative pro perties in a variety of cell lines suggest indicate that atrazine 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 proliferation was decreased at relatively low levels of atrazine exposure.Do not cite

Appendix A

- Page 292 of 677 A.7.8 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 Mu tagen 47:254

- 259

.

In this study, groups of six peeled, white commercial bulbs of Allium cepa, also known as the ―garden onion‖, were placed in glass tubes containing mineral water and the flavonoid, quercetin (Q), which is thought to have antioxidant prop erties, 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 daily throughout the 5

- day exposure period for root length and form and the presence of ―croche t hooks‖ and tumors. At the end of treatment, the meristematic cell (analogous to stem cells in mammalian tissue) region from the root tips was harvested and processed for cytogenetic evaluation. One thousand cells were

counted to determine the mitotic i ndex (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 after exposure to Q or atrazine. Similarly, Q

alone di d not affect the MI at any concentration. 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 af f ected 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 significant (p<0.001) increase in struc tural chromosome aberration. The most prominent type of aberration was fragments. When the plant cells 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) lowe r (~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 atra zine 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 confined to a narrow range of doses because concentrations >7.5

μ g/mL atrazine would lik ely exceed the recommended level of cytotoxic and no cytotoxicity or clastogenicity was noted at ≤ 5.0 μ g/mL.Do not cite

Appendix A

- Page 293 of 677 A.7.9 Olejnik, Am, Marecik, R, Bialas, W, Cyplik, P. (2010). In vitro studies on

atrazine effects on human intestinal cells. Water Air Soil Po llut. DOI
10.1007/s11270

- 010

- 0394

- 0; published inline: April 16, 2010

In an in vitro study, cultured Caco

- 2 cells from the established Caco

- 2 cells line, which is a continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells, were exposed to atrazine (purity not specified) concentrations of 0, 1, 10, 50, and 250 μM, prepared in dimethyl sulfoxide (DMSO). A series of experiments were carried out to simulate long

- term exposure (cells treated with atrazine levels ranging from 1

- 250 μM for 7 days or continuously treated with 10 μM for 4 weeks) and short

- term exposures (cells cultured for 7, 14 or 21 days were harvested and exposed to 50 and 250 μM atrazine for 3 hours). Six replicate cultures were prepared for the negative control ( culture media alone), the solvent control (0.1% DMSO), and the treatment groups. After exposure, treated cells were monitored for viability, proliferation, differentiation, monolayer integrity and DNA damage.

Results were as follows for the key endpoi nts that were measured ( i.e., cytotoxicity and genotoxicity):

Cytotoxicity

For the long

- term exposure, cell viability and cell growth (indicated by generation time) were unaffected by treatment with concentrations as high as 250 μM for 4 days. After 5 days of treatment with concentrations ≥50 μM, however, cell survival was reduced 17 and 40 % and cell generation time was increased from a background level of 46 hours to 52 or 64 hours for cultures dosed with 50 or 250 μM, respectively. In support of the above observations, decreased cell viability and cell density were clearly time

- related as indicated by the marked and significant reduction in the cell viability and density of cultures treated chronically with 10 μM atrazine (decreases of ~20, 40 and 80% after 14, 21 or 28 days, respectively). Mitochondrial activity was also assessed as an indicator of viability, and the investigators found that metabolic activity was significantly altered in cultures treated with 250 μM for 14 and 21 days (~30 and 2 0 % reduced, respectively, compared to control). In addition, alkaline phosphatase (AP) activity was measured to determine enterocytic cell differentiation (an indicator of the activity of microvilli within the entrocytic surface). These data show that at 10, 50 or 250 μM, AP activity was 2.5, 4.4, or

10.2

- fold lower than control, respectively. Based on the evidence of significant and dose

related effects on AP activity, the investigators concluded that the inhibition of intestinal

cells maturation cou ld be the main consequence of the long

- term exposure to atrazine. The final test conducted to assess cytotoxicity was the transepithelial electrical resistance (TEER) test; this procedure is designed to determine the effects of a test substance on the

inte grity of the monolayer as an indicator of the integrity of the intestinal epithelial barrier. In agreement with the data from the other assays, the investigators found that 50 and 250Do not cite

Appendix A

- Page 294 of 677 μM atrazine had a ―strong effect‖ on the ―intestinal cell monolayer dev elopment and
confluence‖.

For the acute exposure, the low atrazine concentration (50 μM) had no adverse effects on cell viability in cultures grown for up to 21 days prior to the 3

- hour exposure. At 250 μM, 7

- day old cultures were also large ly unaffected. However, as the cultures differentiated and aged, viability diminished by ~5

- 10% in 14 and 21

- day old cultures.

Genotoxicity

Caco

- 2 cell cultures, grown for 7, 14 or 21 days were treated with 0, 50 and 250 μM atrazine for 24 hours, h arvested and subjected to single cell gel electrophoresis (Comet test). Significant DNA damage was observed at all time intervals but only at the highest

concentration tested.

Reviewer Comment: In general the study was well

- performed and the data suppor t the study authors‘ conclusions. Overall the data indicate that in this in vitro study, acute exposure to atrazine concentrations up to 250 μM was not cytotoxic and only minimal cytotoxicit y (~5

- 10%↓ in survival) was seen in older cultures. By contrast, cytotoxicity, manifested as reduced cell viability and density, delayed growth generation, reduced metabolic activity, and reduced monolayer integrity occurred in 14

- and 21

- day cultures

tr eated with 50 or 250 μM (equivalent to 10 or 54 μg/mL). Significant damage to DNA in cells grown for 7, 14 or 21 days was only observed at the highest concentration; no DNA damage was induced at 50 μM. The investigators claimed, however, that the cytotox icity

and DNA

- damaging activity of atrazine occurred at concentrations that would not be encountered in the environment. Hence, the relevance of these findings to the risk

assessment is not clear.

A.7.10 Singh, M, Sandhir, R, Kiran, Ravi. (2010). Effects on antioxidant status of liver following atrazine exposure and its attenuation by vitamin E. Exp and Toxicol Pathol doi:10.1016/jetp.2010.01.005

Singh et al. (2010) investigated the potential of the non

- enzymatic antioxidant, vitamin E to

inhibit oxidat ive stress induced by atrazine. Accordingly, male Wistar rats (6/group/sacrifice time) received single oral administrations of the vehicle (1 mL corn oil), atrazine (97.83%) alone (300 mg/kg in corn oil), vitamin E (100 mg/kg in corn oil) or atrazine + vi tamin E ( 300 mg/kg atrazine + 100 mg/kg vitamin E). Animals were sacrificed 7, 14 and 21 days post treatment; livers were harvested, homogenized and samples were used to measure lipid peroxidation (LP), reduced glutathione (GSH), superoxide dismutase (S OD), catalase, glutathione peroxidase (GSH

- Px), glutathione

- s

- transferase (GST) and glucose

- 6

- transferase (GTS). Body and liver weights were also recorded at each sacrifice interval.Do not cite

Appendix A

- Page 295 of 677 Body weight of male rats receiving atrazine alone was significantly lower that control at all sample times; percent weight decreases ranged from

- 15% at 7 days to

- 26% at 21 days. By contrast, animals dosed with atrazine and vitamin E (A+VE) showed a weight loss at 7 days that was ~50% less then the atrazine only group. By 14 days, a significant (17%) increase in body weight was recorded for the A+VE group, which continued until the

terminal 21

- day sacrifice. Liver weights were consistently lower than control for the atrazine group and showed a 17% reduction at 14 days a nd a significant reduction at 21 days. Liver weights of the A+VE group were largely unaffected by treatment with atrazine. These finding suggest that vitamin E diminishes the toxic effects of atrazine on body and organ weight.

Similar evidence of the protective capacity of vitamin E toward atrazine

- induced liver damage was found in the data showing that the significant increases in LP and significant
decreases in GSH and G

- 6

- P D, which were generally evident by day 7, were lessened

when vitamin E was i ncluded in the dosing regime. The adverse effects of atrazine on the antioxidant defense system of the intact animal, as indicated by the increased SOD, catalase, GPx, and G6PD activity in the livers of atrazine

- treated rats, which were clearly

diminished in the presence of vitamin E, supports this assessment.

Reviewer Comment : In general the study was well

- performed and the data support the study authors‘ conclusions that atrazine causes oxidative damage and alters the

―antioxidant status of the liver ‖. They further conclude that the administration of vitamin E along with atrazine ameliorates the toxic effects of atrazine.Do not cite

Appendix A

- Page 296 of 677 A

. 7.11 Singh, M., Sandhir, R., and Kiran, R. 2008. Atrazine

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

- 369

.

This study evaluated the possible beneficial effects of vitamin E against atrazine

- induced oxidative stress in erythrocytes. Erythrocytes are susceptible to oxidative damage and a good model to understand oxidative damage induced by xenobiotics. Vitamin E is an

antioxidant.

Male Wistar rats (n=18 animals/group) were administered atrazine (300 mg/kg/day, 97.83% purity), Vitamin E (100 mg/kg/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 samples were collected at the end of the treatment periods. Erythrocyte membranes were prepared and analyzed for acetylcholinesteras e (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 erythro cytes.

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 resulted 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, with the effects being more pronounced w ith increasing duration. After 21 days treatment 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 contro ls. Atrazine/Vitamin E treatment significantly (p<0.05) decreased lipid peroxidation when compared to animals treated 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 increased total lipid content to normal levels after 21 days, as compared to the atrazine treated group. Atrazine treatment significantly (p<0.05)

decreas ed cholesterol 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%), compared to the atrazine treated group. Atrazine

treatment resulted in a significant decrease in phospholipid content after 14 days (16.76%) and 21 days (37.20%), compared to controls. Atrazine/Vitamin E treatment significantly increased phospholipid content after 14 days (22.86%) and 21 days (57.92%), compared to

atrazin e treated group.Do not cite

Appendix A

- Page 297 of 677 Erythrocytes of rats treated with atrazine showed significant morphological changes as compared to the control group. These changes included mild to moderate distortion in shape, significant ruptured membranes, and echinocyte formation, central or peripheral protuberances. SEM of erythrocytes of atrazine/vitamin E group shows improved erythrocyte topography as compared to atrazine

- treated group. Extent of protuberance significantly decreased as compared to the control group.

Reviewer‘s Comments: The results of this study are questionable given that the atrazine doses used to induce these genotoxic effects are orders of magnitude higher than what is currently used as the point of departure for human health risk assessment.

A.7.12 Sing h, M., Kaur, P., Sandhir, R., Kiran, R. (2008). Protective effect of vitamin E against atrazine

- induced genotoxicity in rats. Mutat Res

654:145

- 149

.

The protective effect of an antioxidant, vitamin E

, against atrazine

- induced genotoxicity was investigat ed in young (~4

- 5 weeks old, based on the weight range of 100

- 120 g) 26 a Wistar male rats dosed with vitamin E alone, atrazine (97.83%) alone or a combination of atrazine and vitamin E in the comet and micronucleus assays. Accordingly, groups of six male r ats 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 vitamin E and atrazine for 7, 14 or 21 days. At the end of treatment, peripheral blood and liver were collected si multaneously from each test animal, processed and assayed for micronucleus induction and/or DNA damage (gel electrophoresis and Comet assay).

Results from the gel electrophoresis on DNA isolated 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 investigators stated that atrazine did not induce the classic DNA ladder pattern, which is associated with apoptosis but shearing of DNA suggested DNA damage to the atrazine

- 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 tim e

- related and in good agreement with the electrophoresis data showing a marked increase 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 sampl es were obtained for the tissues treated with atrazine plus vitamin E, compared to atrazine treatment alone. In agreement with these 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. T he treatment of the rats with atrazine (300

26 According to personal communications with Charles Parady, Senior Specialist, Charles River, male Wistar rats in the weight range of 100

- 120 gm wo uld be approximately 4

- 5 weeks old.Do not cite

Appendix A

- Page 298 of 677 mg/kg/day) plus vitamin E (100 mg/kg) caused significant (p<0.001) reductions in the frequen cy of micronuclei at 7 (23%↓) and 21 (14%↓) days as compared to atrazine

- treated

rats.

Reviewer‘s Comments: The results of this study are questionable given that the atrazine doses used to induce these genotoxic effects are orders of magnitude higher tha n what is currently used as the point of departure for human health risk assessment.

A

. 7.13 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 Carcino genesis in Rats Pretreated with N

- bis(2

- hydroxypropyl)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. 27 Th e 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 week 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 atrazine. At the end of the exposure, animals were sacrificed and the thyroid, pituitary, liver, lungs, kidney, pancreas, testis, epididymis, and brain were collected, weighed and subjected to histopathological examination. In addition, serum from 5 rats/group (except the 5 and 50 ppm atrazine group s) 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 receiving both DHPN and 500 ppm atrazine were increased by ≈9

- 13% (p< 0.05) when compared to animals receiving 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

onl y (no DHPN injection) revealed that these animals experienced a 14% decrease in body weight (p<0.05) along with an 11

- 12% increase in relative brain, testicular, an epididymal weight. However, unlike pre

- exposure to DHPN followed by 500 ppm atrazine, expo sure to 500 ppm atrazine alone did not elicit an increase in the relative liver weight. Histopathology assessments did not reveal any compound

- related changes though the data were not provided in the study report. Hormone analysis revealed that while the re is a statistically significant decrease in T 4 levels (↓16%, p<0.05) after exposure to DHPN + 500 ppm atrazine 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 significant.

27 In this evaluation only the results for atrazine will be discussedDo not cite

Appendix A

- Page 299 of 677 Furthermore, exposure to 5 00 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 i ncrease above background caused by DHPN and not indicative of an effect of atrazine on thyroid hormone homeostasis.

Reviewer‘s Comments: The results of this study 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 did not appear to have any effects on the parameters evaluated in this study. No histopathological changes were reported after exposure to 500 ppm atrazine and the increases in relati ve organ weights observed are most likely related to this decrease in body weight rather than a direct effect on the organs. Similarly, thyroid hormone assessments did not suggest a direct effect of atrazine on thyroid hormone

homeostasis. It should be n oted, however, that this report does not include information on the purity of the atrazine used in the study and that the histopathological data are not provided.

A

. 7 .1 4 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

.

The effect of atrazine on DMBA

- induced ovarian carcinogenesis was investigated in this study. The authors hypothesis is tha t the operative mode of action leading to ovarian adenocarcinomas involves hormonal disruption (particularly estrogen). Therefore, exposure to atrazine (which has been associated with hormone disruption) may affect ovarian carcinogenesis.

In this serie s of experiments, the researchers exposed 12

- 20 female Sprague

- Dawley rats to 0.5% DMBA ( 7,12

- dimethylbenz(a)anthracene ) injected directly into the left ovaries. One week after exposure to DMBA, animals were fed diets containing 0, 5, 50, or 500 ppm

atraz ine (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, necropsied and tissues with gross lesions, as well as, the liver, kidne y, mammary gland, lung, and heart subjected to histopathological evaluation. In addition, immunohistochemistry evaluations for proliferating cell nuclear antigen (PCNA), expression of the androgen or estroge n receptors (α and β) and the BRCA

- 1 gene were conducted.

Neither DMBA, nor atrazine or DMBA + atrazine exposure led to any changes in ovarian, uterine, or body weight. DMBA exposure followed by dietary exposure to 500 ppm atrazine exposure led to a 10% statistically significant increase in absolute liver weight, consistent with what has been reported by other investigators. DMBA exposure led to a 45%

incidence in ovarian adenocarcinomas vs 0% in control. Atrazine appeared to slightlyDo not cite

Appendix A

- Page 300 of 677 modify the effect of DMBA since exposure to 5, 50, or 500 ppm 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

- al one. There were no compound

- related effects that could be unequivocally attributed to either DMBA, atrazine, or DMBA + atrazine exposure in the other tumor types seen in his study (malignant fibrous histiocytomas, hemangiomas, mammary tumors).

PCNA

- label ing 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 concluded that atrazine did not result in any modifying effects on DMBA

- induced rat ovarian carcinogenesis. It should be noted, however, that though not statistically significant the incidenc e of ovarian tumors due to DMBA exposure was reduced by half when DMBA treatment was followed by atrazine exposure at dietary concentrations of 5

- 500 ppm (0.09

- 8.5 mg/rat/day). The toxicological significance of this finding, however, is unclear given the lack of dose

- response over a two

order of magnitude difference in atrazine exposures. However, when considered collectively, the data reported by Fujimoto, Son, and Tanaka and co

- workers demonstrate that atrazine exposure did not accelerate tumor progressi on.

A

. 7.15 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

.

The objective of this study was to evaluate 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 after DMBA treatment (when palpable tumor incidence

was ≈ 50%), the animals were ovariectomized. 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

su bgroups (22

- 23 rats/group) that received 0, 5, 50, or 500 ppm atrazine (purity unknown) [approximately 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 terminalDo not cite

Appendix A

- Page 301 of 677 sacrifice, whole skin with mammary glands and tumors were collected. In addition, the liver, kidneys, uterus, 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 tumors), and scars of the ovariectomized regions. If indications that traces of ovarian tissu e 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 between12

- 19 rats/gr oup. Immunohistochemistry analysis was used to detect potential increases in proliferating

- cell nuclear antigen (PCNA), single strand DNA (ssDNA), and estrogen receptor α (ER α) in mammary tumors (except for fibromas). Finally, estradiol, progesterone, p rolactin, 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) b ut there was no apparent correlation to atrazine exposure.

Following ovariectomy, the number 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 t ypes of tumors ( i.e.

, benign vs . carcinoma) were not affected by atrazine treatment and though there was an apparent dose

- related increase in tumor size at doses ≥ 50 ppm (↑43

- 261%) there was no statistical significance due to the large variability in the Group A. Immunohistochemistry evaluations showed that Group A rats treated with 500 ppm atrazine showed an increase in the number of ERα

- 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 tumors. The authors report that no changes in hormone levels that could be attributed to atrazine exposure were observed. However, no data were provided to corroborate this latter statement.

Interestingly, at the end of the study G roup B rats ( i.e.

, DMBA

- treated rats with late tumors) had a statistically significant (p<0.05) increase in the number 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 atrazine

- treated animals than in the control. A ≈1300% increase in the size of tumors was also 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 evidence of a clear correlation between atrazine exposure and the incidence of malignancies for either Group A or Group

B rats.Do not cite

Appendix A

- Page 302 of 677 Figure A

- 2 : Treatment Groups

The authors concluded that atrazine has the 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 proliferating (PCNA

- positive cells) and expressing ERα after exposure to 500 ppm atrazine.

The preponderance of the evidence, however, does n ot support the statement that atrazine contributes to mammary tumor development. For example, for Group A rats (tumors present at week 19), there was no correlation between atrazine exposure and the number

of tumor

- bearing rats, number of new tumors (appe aring 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 Group B a statistica lly significantly higher incidence of tumor

- bearing rats is seen after exposure to 500 ppm atrazine relative to the Group B control, it should be noted that the incidence in the Group B control is substantially lower than in the Group A control (Control B: 26% vs Control A: 72%). W hen the Group B 500 ppm atrazine

- treated rats are compared to the Group A controls, the incidence of tumor

- bearing rats is lower after 500 ppm treatment than in the Group A controls. It should be noted that the incidence

of tumo r

- 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 B

- 500 ppm atrazine dose level is increased by 9% and is not statistically significant. 28 In fact, if Group B rats treated with 500 ppm atrazine are

28 Calculated by reviewer and analyzed using Fisher‘s Exact test A

B

C

Table excerpted from Ueda et al. , (2005) p. 22Do not cite

Appendix A

- Page 303 of 677 compared to the Group A control rats, the tumor incidence after atrazine exposure is lower than in the Group A controls.

For rats treated with 500 ppm atrazine in Group B (no tumors at week 19), the incidence of

tumor

- bearing rats was lower than the number 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 respo nse and at 500 ppm atrazine the carcinoma incidence was lower than for the Group A control rats.

With regards to the immunohistochemical evaluations, 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, atrazine exposure in and of itself does not appear to contribute to the increase in PCNA

- positive cells as evidenced by the fact that for late developing carcinomas (Group B rats) no statistically signifi cant increase was noted at the 500 ppm atrazine dose level. An increase in the incidence of ERα

- positive carcinomas was reported at the 500 pm atrazine level in Group A rats (100% vs 40% control). It should be noted, however, that a 100% incidence of ERα

- positive carcinomas was also reported for the control and 5 ppm atrazine Group B rats (late

- developing tumors) while the high dose atrazine 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 appears 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 palpable tumors at week 19), a comparison of the treated groups with either control 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

- a nd 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 suggest that atrazine in an of itself does not indu ce 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.Do not cite

Appendix A

- Page 304 of 677 A

. 7.16 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 cytotoxicity of atrazine and its metabolites (diaminochlorotrizine [DACT], deisopropyl atrazine [DIA], ammeline [AM], hydr oxyatrazine [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 murine 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 H295R, JEG

- 3, and MCF

- 7 cells were maintained at 100% confluency, while HT

- 22 cells were maintained at 50% confluency.

For the cell v iability 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/wel l. Cells were exposed to atrazine or its metabolites at concentrations ranging from 0.001 μ M to 300

μ M in physiologically buffered saline (PBS) or in sterile water with dimethyl sulfoxide (DMSO) for up to 72 hours. After exposure, cell viability was measur ed using the

membrane

- permeant calcein

- 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 (RFU), were expressed as the percentage of untreated or vehicle treated control values. For each assay, six independent experiments were run with triplic ate wells for each treatment per experiment.

The effects of atrazine on mitochondrial activity was measured in H295R cells. H295R

cells were seeded in 96

- well plates at 25,000 cells/well 24 hours before the initiation of the assay. Varying concentrations of atrazine were dissolved in DMSO (0.001

- 300 μ M) or PBS (0.0003

- 300 μ M) for 24 hours. Mitochondrial activity was assessed using a MTT assay.

Cytosolic reactive oxygen species (ROS) was estimated by measuring the amount of ROS by the fluorescent dye 2 ́,7  ́

- dichlorofluorescin diacetate (DCFH

- DA), which measure s 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 D MSO or PBS. ROS production was monitored for up to 72 hours.

DCF

- 2,7

- dichlorofluorescin fluorescence was determined at an excitation of 485 nm and an emission of 538 nm using an Infinity M120 microplate

- reader.

The effects of atrazine on the cytotoxic effects of other known toxins was measured using

H295R cells seeded in 96

- well plates at 25,000 cells/well 24 hours before the initiation ofDo not cite

Appendix A

- Page 305 of 677 the assay. Varying 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

- treated with varying concentrations of atrazine and other know toxins (glutamate

– 15 mM; 3

- NPA

– 15 mM; H 2 O 2 – 250 μM) for 24 hours.

The results of this study showed that atrazine and its metabolites were not cytotoxic in four cell lines, irrespective of being solubilized in DMSO or PBS. Atrazine and its metabolites did not produce cytotoxicity as indicated by an absence of any effect on cell viability in two steroidogenic (H295R a nd JEG

- 3) and two non

- steroidogenic (HT

- 22 and MCF

- 7) cell lines. Atrazine had no effect on mitochondrial activity in H295R cells regardless of whether it was dissolved in DMSO or PBS. Atrazine did not induce oxidative stress in H295R cells, as

measured b y 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 treated 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 cytotoxicity of glutamate, 3

- NPA, or H 2 O 2

.

Reviewer‘s Comments: The methods section of the paper failed to include the evaluation of mitochondrial activity or measuring 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 non

- steroidogenic cell lines (HT

- 22 and MCF

- 7).

.

A.8 INSULIN/MITOCHO NDRIA FUNCTION

Summary: Insulin/Mitochondria Function

In a recent publication in the electronic journal PLoS ONE, Lim et al. (2009) su ggest that atrazine exposure leads to insulin resistance, fat accumulation, and increased weight gain by increasing blood glucose levels and lower ing energy expenditure . The authors postulate that atrazine disrupts mitochondrial function by inhibiting mit ochondrial complexes I, II and III, which interact with ubiquinone for electron transport. This hypothesis is based on atrazine‘s known pesticidal mode of action by disrupting electron transport via plastoquinone which is similar in structure to ubiquinon e in vertebrates. Mitochondria are involved in fat oxidation and insulin secretion pathways, so their disruption could cause steatosis or insulin pathologies. However, mitochondrial inhibition should cause decreased insulin secretion, not increased as the authors propose. In fact, a recent publication 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 mitoc hondrial electron flow signals the over

- fed state to produce insulin resistance).Do not cite

Appendix A

- Page 306 of 677 The authors state that atrazine exposure led to an increase in body weight and fat deposition regardless of the caloric intake. However, neither the body weight nor the fat deposition increases (↑5

- 10%) reported in the study show a dose

- response. Moreover, the blood glucose levels reported 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 described 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 well as inappropriate statistical analysis.

The autho rs concluded that exposure to atrazine at dose levels as low as 30 μg/kg/day resulted in significant mitochondrial 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 deficiencies in the reporting thus rendering it of limited utility in understanding the toxicological profile of atrazine.

In summary, Lim et al. are the only researchers that have proposed a relationship between atrazine exposure and mitochondrial dysfunction and obesity. The study used to support this hypothesis is critically flawed. M oreover, the preponderance of the evidence indicates that atrazine exposure leads to decreases

– not increases

– in body weight. Consequently, the validity of these data is uncertain and renders it of little relevance for risk
assessment purposes.

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

.

The authors ha ve 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 atrazine 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 state d 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 addition, the manuscript does not provide any information on the

concentration of atrazine in the water or estimates of the water intake d uring the course of the study. In particular it is important to understand if the concentration of atrazine in the water or the vehicle changed the water consumption.

The following measurements were made as part of the in vivo experiments: body weight

, visceral fat, motor activity, intramuscular and intrahepatic lipid content, plasma glucose,Do not cite

Appendix A

- Page 307 of 677 serum insulin, total cholesterol and triglycerides, intravenous glucose tolerance test, high insulin challenge (hyperinsulinemic

- euglycemic study), energy expendi ture, electron microscopy of mitochondria, and protein expression levels of mitochondrial OXPHOS complex. In vitro experiments were conducted to evaluate endogenous cellular oxygen consumption and mitochondrial respiratory chain activities. In general, t he methods section lacks some important information and references to the me thods used. For example, t he

methods state that motor a ctivity was measured 4hrs/month

. However, it is unclear whether
this refers to one 4 hr session or one hr/week. The authors performed student‘s t

- tests and ANOVA. However, a 2

- way ANOVA with ATZ and diet as the independent variables may be more appropriate. Based on limited re

- analysis 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. B ased on the results section, atrazine at these doses increased body weights in both normal and h igh fat diet, although, there is a flat dose response relationship. The lowest dose has an eq uivalent effect of the highest water

concentration . Similarly, there 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 atrazine 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: Fast ing plasma glucose levels of atrazine treated rats on the regular and high fat diet s were higher than those 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 seems to be a real disconnect between the in vitro concentrations needed to see effects on the 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.

Reviewer‘s Comments: To believe their data, one would have to believe that atrazine has significant and n ear maximal effects on mitochondrial function a t doses of 30 ug/kg/d (a LOEL) in animals on a normal diet. This seems unlikely given that most studies have not observed effects of atrazine until exposures are approximately 100

- 1000 times higher.Do not cite

Appendix A

- Page 308 of 677 Most not ably, 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 results reported in the study. This study is not sufficiently robust for use in a regu latory context and should be repeated to confirm the findings.

A.9 PHARMACOKINETIC S: INTERNAL DOSIMET RY, METABOLISM, & TI ME
COURSE

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

.

The potential of atrazine to form covalent adducts with hemoglobin was studied in groups of two female Sprague Dawley rats receiving 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/TOF MS (Matrix

- Assisted Laser Desorption Ionization

- Tim e of Flight/Time of Flight Mass Spectrometry). Globin samples were also analyzed by HPLC; fractions collected via HPLC and peptides were extracted via electrophoresis and tryptic digestion, purified and prepared for mass spectrometry analysis. Similar pr ocedures were used to prepare samples of rat hemoglobin, harvested from unexposed bone marrow cells, which were

treated in vitro with 90 ppm diaminochlorotriazine (DACT, a reactive electrophilic metabolite of atrazine).

The investigators reported that a n additional β globin peak was seen in exposed animals during the HPLC and MALDI

- TOF/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 peptide 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: Overall, the study was well

- conducted and support the investigators‘ conclusion that atrazine exposure 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 hemoglobin, human hemoglobin does not have a reduced cysteine residue at position 125, the relevance of rat hemoglobin adduct formation

to human risk assessment is limited.Do not cite

Appendix A

- Page 309 of 677 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 author s investigated atrazine interactions with cellular macromolecules using rat and human albumins as the targets. For the in vivo phase, ovariectomized Wistar female rats (3/group) were administered oral gavage doses of 0, 5, 10 or 20 mg/kg atrazine (97.1%) and sacrificed on postexposure 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 was 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 polyacryami de gel and electrophoreses for 1 hour. Purified albumin was transferred to a polyvinylidene fluride (PVDF) Western blotting membrane, rinsed, and probed with commercially prepared anti

- DACT adduct antibody. The DACT adduct antibody complex was examined b y chemiluminescence using Western Blot analysis. DACT adducts were confirmed if a band was detected by

chemiluminescence.

The investigators indicated that using mass spectrometry, a covalent bond with a mass of 110 DA was located on Cys

- 34 of the albumin 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 analysis, a dose

- related increase in adduct formation was detected in the purified albumin samples co llected from rats treated with a wide range of atrazine doses (5

- 200 mg/kg). DACT adducts were also detected in rat and human albumin preparations 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.Do not cite

Appendix A

- Page 310 of 677 A.9.3 Kim D. and Breckenridge C. (Syngenta). Derivation of the short

- term
inhalati on 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

- administered atrazine (ATRA) was modified to include inhalation exposure for the extrapolation of a rat oral NOAEL to the equivalent rat and human inhalation NOAELs. The rationale for using a PBPK model is

b ased on a request by US EPA for a 28

- day inhalation 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 confi nements by themselves without food and water would likely alter the estrous cycle. Therefore, a well

parameterized PBPK model offers an alternative approach for estimating inhalation exposures of ATRA associated with a given internal dose metric. The rat o ral 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 effects 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 inhalation uptake based on species

- specific pulmonary ventilation rates and amount of ATRA in air, and assuming that ATRA is not absorbed in the respiratory tract nor eliminated by exhalation. The internal dose metric selected for predicting the equivalent rat and human inhalation NOAELs was the area

under the 24

- hr plasma concentration

- time curve (AUC). Thus, the model was first used to estimate the AUC resulting from the orally

- administered ATRA at the NOAEL of 6.25 mg/kg/day. Inhalation pharmacokinetics (6

- h exposures, while setting oral dose to zero) were then simulated to result in the equivalent AUC for rats and humans (using a body weight of 0.3 and 70 kg for rats and humans, respectively). The inhalation exposur es 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 interspecies differences in pharmacokinetics, the uncertainty facto r 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 intrasp ecies 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 reported measurements of ATRA inhalation exposures. The reported MOE

estimates are greater than the PBPK model

- refined level of 32 as well as the default

benchmark level of 100.Do not cite

Appendix A

- Page 311 of 677 Reviewer‘s Comments: There are two main deficiencies associated with this study. First, the PBPK model for orally

- administered ATRA (McMullin 2007) does a rela tively poor job of simulating ATRA plasma levels following oral dosing (See Figure 7A of McMullin 2007). Second, ATRA dealkylated chlorinated metabolites were not included in the estimation of AUC following oral dosing at the rat NOAEL of 6.25 mg/kg. This is particularly important since chlorinated ATRA metabolites are known to be toxicologically

- active with respect to the estrous cycle and LH surge, and ATRA is rapidly metabolized (particularly following oral administration). Inclusion of chlorinated ATRA metabolites would result in a greater AUC for a given oral ATRA dose and correspondingly lower estimates for inhalation NOAELs.

A

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

course studies with [ 14 C]

- atrazine. J. Toxicol. Env iron. Health, 66(A):941

- 964

.

This manuscript describes the development of a physiologically based pharmacokinetic

model in an attempt to estimate total plasma chlorotriazine s (atrazine and its chlorinated metabolites: deisopropylatrazine, deethylatrazine, 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 (chlorinated and non

- chlorinated) were identified and quantified; and a repeated dose (1, 3, 7, 10, 50, and 100 mg/kg bw/day) study where, in addition to plasma, urine, and feces, reactivity of ATRA with red blood cells (RBCs) was examined. The compartments in the model are as follows: liver (the primary site of metabolism, brain (postulate d site of action), a plasma compartment (containing RBCs), and a rest of body compartment. Individual metabolites are not tracked in the model. Instead, two pools of compounds are described: chlorinated triazines and metabolites derived from glutathione co njugation. In the model, chlorotriazines can bind plasma proteins, react with RBCs, conjugate with glutathione, or be eliminated in urine. In contrast, glutathione conjugates are considered toxicologically

- inactive and can be eliminated in the model throug h feces and urine.

Parameterization of the model was carried out as follows: (1) since the single oral dose study quantified individual metabolites, the rates of glutathione conjugation, chlorinated triazine elimination, and glutathione conjugate eliminat ion were fitted to the reported urinary percentages of 73% chlorinated triazines and 27% glutathione conjugates. This parameterization reasonably described the cumulative urinary elimination of presumed radiolabeled equivalents (chlorinated triazine and gl utathione 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 plasma data at the l onger time points. In an effort to better describe this dataset, plasma protein binding/reactivity was also included. A binding rate constant wasDo not cite

Appendix A

- Page 312 of 677 estimated based on albumin, the major plasma binding protein of known concentration and turnover, by fitting t he plasma data for retention of radioactivity in the single dose study and then evaluating the ability of the model to describe the repeated

- dosing study plasma data. Overall the model reasonably predicted plasma levels from the repeated

- dosing study at

th e early (first 50 hours post

- dose) and late time points (>200 hours), while 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 gavage to rats and individual chlorinated triazines were monitored in plasma. It is unclear to this reviewer why this new dataset was not used to evaluate the performance of the model. Instead, a log

- likelihood best fit to a one

compartment model is d escribed to presumably show that a slower absorption rate constant describes the higher dose plasma 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 evaluated using an independent dataset.

A

. 9

. 5 McMullin, T.S., Hanneman, W.H., Cranmer, B.K., Tessari, J.D., and Andersen, M.E. (2 007). Oral absorption and oxidative metabolism of atrazine in rats evaluated by physiologically modeling approaches. Toxicology 240:1

- 14

.

This study reports on physiologically

- based modeling approaches to evaluate the role of absorption and oxidative meta bolism on the 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 hy pothalamic pituitary axis, leading to neuroendocrine responses such as suppression of the steroid

- induced luteinizing hormone surge in adult ovariectomized female rats. Based on these findings, a net tissue exposure to chlorinated triazines may be necessar y to conduct human heal th risk assessment for atrazine.

Atrazine undergoes metabolism by P450 enzymes to the mono

- dealkylated chlorinated species deisopropylatrazine (ETHYL) and deethylatrazine (ISO). Further P450

- mediated dealkylation yields DACT . Atrazi ne and all three of its chlorinated metabolites can undergo glutathione

- S

- transferase

- mediated dechlorination to yield the respective non

- chlorinated species.

A physiologically

- based pharmacokinetic (PBPK) model based on in vivo pharmacokinetic

studies wi th 14 C

- atrazine was previously developed to estimate total chlorinated triazines in plasma after single and multiple oral gavage exposures. However, the predictive capability of the model was limited by the lack of specific information on the rates of satu rable,Do not cite

Appendix A

- Page 313 of 6 77 oxidative 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 pharmacokinetic data of individual chlorinated triazi nes. The resulting individual model 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

administration of atrazine.

Individual chlorinated triazine m o del structures

The model structures for all chlorinated triazines consisted of a GI subcompartment to evaluate the potential for pre

- systemic metabolism and absorption processes producing multiple peaks in plasma profiles; a liver as the primary site of

me tabolism; 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 equi molar amount of one of its chlorinated metabolites: ETHYL, ISO,

or DACT. Plasma samples were analyzed for all four chlorinated species.

Sequential Parameter estimation for individual model structures The following model parameters were estimated sequenti ally by fitting the model to the respective time

- course 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 metab olism of ISO to DACT and ISO elimination rate constant

Model evaluation for individual chlorinated triazines DACT: plasma time

- course data was simulated using first order absorption and

elimination. ETHYL and ISO: First order absorption could not describ e a double peak phenomenon; peak plasma concentrations occurred 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 the multipeak behavior was simulated by describing different rates of uptake from the gut; a rapid first order uptake and a slower zero order uptake of the remaining dose. Absorption rate constants for the different uptake processes were estimated by fitting to the time

- course data.

Composite model evalu ation for atrazine The uptake of atrazine could not be simulated as a single first order absorption process due to a double peak phenomenon. As with ETHYL and ISO, this phenomenon was described by a combination of first order and zero order descriptions. T he authors hypothesized that the double

- peak phenomenon observedDo not cite

Appendix A

- Page 314 of 677 for ETHYL, ISO, and atrazine is reflective of limited compound solubility in the gut at the high doses used in the study. The continuing absorption of more slowly dissolving compound along th e GI tract contributes to the complex time

- course behavior. Addition of pre

- systemic intestinal metabolism of atrazine to mono

alkylated metabolites was also necessary to adequately describe the bioavailability of atrazine without adjusting the in vitro

- d erived metabolic parameters.

Reviewer‘s Comments: The current study underscores some of the parameters that may contribute to the complex pharmacokinetics of atrazine observed following high dose oral administration. The utility of the composite model for atrazine is questionable. First, there is no direct evidence cited in support of multiple absorption processes and GI intestinal metabolism used to fit the model prediction to the plasma concentration

- time profiles. Second, the final composite model signi ficantly 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

- (ethylamino)

- 6

- (isopro pylamino)

- 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

.

Mice are increasing being used to assess the potential adverse e ffects following atrazine (ATRA) exposure. However, disposition studies of ATRA in this particular species are lacking. This study was aimed at examining the metabolism and disposition of ATRA in mice and assessing how tissue levels may correlate with leve ls 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 administered a single dose of 0, 5, 25, 125, and 250 mg/kg bw ATRA in corn oil by oral gavage. Pla sma 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 d ecline before a second peak occurred at 12 h. The biphasic phenomenon was attributed to discontinuous absorption from the GI tract. ATRA levels were below the limit of detection by 24 hrs. In contrast, levels of the di

- dealkylated

metabolite diaminochlorot riazine (DACT), 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 ho urs (halflives 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 significant linear correlation was observed (r 2 = 0.987).

Urine: DACT levels were 2 orders of ma gnitude higher (as high as 5400 μM) than ATRA or

its mono

- dealkylated metabolites in the 0

- 24 hr time period.Do not cite

Appendix A

- Page 315 of 677 Feces: Analysis of fecal extracts did not reveal significant amounts of metabolites. Detected amounts of ATRA most likely reflected unabsorbed c ompound.

Liver and kidney: ATRA and all of its metabolites including glutathione conjugates were detectable at 4 hrs. DACT was the 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 highest 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 biphasi c absorption phenomenon of ATRA is consistent across these two rodent species. Plasma kinetics (maximum levels, time to reach maximum levels, and clearance) of ATRA and DACT are also very similar for a given administered ATRA dose. This disposition

informa tion reported in this study may be important in the use of pharmacokinetic models that can link target tissue dose to toxicological effects of ATRA and in vivo metabolites.

A. 10 MIXTURES

A 10.1 Enoch, R.R., Stanko, J.P., Greiner, S.N., Youngblood, G.L, . Rayner, J.L., Fenton, S.E., 2007. Mammary gland development as a sensitive end point acute prenatal exposure to an atrazine metabolite mixture in female Long

- Evans rats. Environmental Health Prospective. 115(4):

541

- 547

In this study, the investiga tors dosed pregnant female rats with atrazine or a mixture of atrazine and its metabolites from gestational day (GD) 15

- 19. The atrazine metabolite mixture (AMM) was made up of atrazine, DACT, DEA, DIA, and hydroxyatrazine (HA) at 0, 0.09 mg/kg AMM, 0.87 mg/kg AMM, 8.73 mg/kg AMM, and 100 mg/kg atrazine. The proportions of metabolites in the AMM are provided in Table 1 of the paper. The 100 mg/kg atrazine group was used as a positive control for effects on mammary gland development. Several endpoints wer e measured but the focus of this study is on development of the mammary gland following in utero exposure.Do not cite

Appendix A

- Page 316 of 677 Two female pups/dam (for ≥ 5 dams) were necropsied on post

- natal days (PND) 4, 25, 33, 40, and 60. For mammary gland developmental evaluations, the 4 th and/or 5 th inguinal mammary gland were whole mounted and examined them subjectively using a 1

- 4 scoring system (1 = poor develop ment/structure; 4 = normal development/structure). The scoring criteria varied depending on the age of the pups. The scoring system is intended to evaluate: primary, secondary, lateral branching; longitudinal growth toward the lymph node; budding patter ns (or lack thereof); and the presence of terminal end buds or ducts.

Pup body weights were increased compared to controls in the three AMM treated groups. Dam weights were not effected; the two lower doses of AMM showed 7

- 12% increases in body weight compared to controls. The day of vaginal opening was the same in treated groups compared to controls. Similarly, the estrous cycles in treated animals were similar to controls. The AMM treated animals exhibited increased pituitary weights compared to

co ntrols.

With respect to the mammary gland development findings, the investigators evaluated pups at PND 4, at weaning, PND 33, PND 40, and PND 60. The results are provided below. Representative images are provided in Figure 5 of the paper. The author s argue that adverse findings are observed as low as 0.09 mg/kg AMM. There is, however, a weak

dose

- response curve. In fact, an increasing the AMM dose by 100

- fold changes the response very little.

There are a number issues with this study which re quire further discussion.

1. Study design: When conducting a mixture study, one of the critical components is the ability to distinguish the effects of an individual chemical component to the effects of the mixture. Most often, investigators begin with a hypothesis of dose

- additivity or response

additivity and subsequently test their hypothesis. No such hypothesis testing was performed here. Furthermore, the investigators failed to evaluate each of the individual components in separate dose

- response e valuations. There are known differences in potency for atrazine, DACT, DIA, and DEA for some endpoints. As such, with the exception of atrazine, one can not rule out that the findings reported are simply caused by a single chemical.Do not cite

Appendix A

- Page 317 of 677 In the specific ca se of atrazine, pharmacokinetic studies show that the parent compound is rapidly metabolized to DACT, DEA, and DIA. As such, atrazine exposure results in a mixture of metabolites in the body. It would be helpful to have internal dosimetry data for

the mi xture study to compare the atrazine exposed animals to the AMM exposed animals. The authors have indicated to the Agency that such tissue dose data may be available in

the future.

Qualitatively the major difference between the atrazine exposed animals a nd the AMM exposed animals is the inclusion of the hydroxy metabolite in the AMM. HA is not a major mammalian metabolite. HA is believed to have a different target organ (kidney) than atrazine, DACT, DEA, and DIA. As such, the investigators have potenti ally developed a mixture using chemicals with different modes of action, further highlighting the importance of the individual component dose

- response curves.

2. Reporting of the findings: In the methods section, the authors state they analyzed all

da ta by dam and block. As such, with n ≥ 6/treatment/block the n for the statistical analysis should be n ≥ 12. The analyses in the figures have n values that often exclude 12. As such, it is difficult to ascertain if they looked at every endpoint/block o r just used the two blocks for different endpoints. In addition, they report that they started with n dams ≥ 6

then sacrificed n ≥5 (2 pups/dam) at PND 4, 25, 33, 40, and 60 . It is unclear what happened to the missing dam and respective pups. Lastly, the text below some tables/figures (e.g., Table 2, Figure 4) suggests that statistics were actually done on the pups, not the dams.

3. Subject scale used to evaluate the mammary gland whole mounts: The investigators use a subjective scale

--- a 1

- 4 scor ing system (1 = poor development/structure; 4 = normal development/structure). The scoring criteria varied depending on the age of the pups. This score attempts to capture a number of different characteristics in a single value. As shown in the table ab ove, the reported mean values for all groups, including controls, lie between 1.70 and 3.83 with control means as low as 2.50. As such, the 1

- 4 scale has very little resolution. Furthermore, these values provide no information around the specific charact eristics (e.g., branching, budding patterns, etc)

which have been effected. It is also of concern that the control means have been scored as low as 2.50 when a score of 4 is expected to represent normal development/structure.

Conclusion: Based on the c urrently available information, the mixture study, as currently reported, is not sufficiently robust for use in setting regulatory endpoints.

A.11 OTHERDo not cite

Appendix A

- Page 318 of 677 A.11.1 Fakhouri, WD, Nunez, JL, Trail, F. ( 2010 ?) Atrazine Binds to the Growth Hormone Releasing Hormone Receptor and Affects Growth Hormone Gene Expression . In press

This study concentrated on identifying a ligand binding receptor for atrazine in pituitary cells that may explain the mechanism of action at the gene expression level.

The effect of atrazine (ATR) on animals has been difficult to elucidate. Studies on whole animals have correlated low environmental concentrations (0.1

- 1.0 ppb) with alterations in sexual development in Xenopus laevis along with mammary tumors in rats. Other

confo unding studies have shown that  ̳environmentally relevant‘ doses of ATR had little effect on these same subjects. This debate prompted research to identify possible mechanism of endocrine interference. Specifically, examination of the binding specificity of ATR to the growth hormone releasing hormone receptor (GHRHR) and the effects on the expression of its target gene, growth hormone (GH), and the expression of other pituitary hormonal balance genes.

The pituitary cell culture and treatments were thoroug hly explained and described, use of PND 7 male rats from breeder females were approved and followed NIH guidelines. Pituitary glands were dissected and cells were dissociated by trituration, with cell number and viability determined by trypan blue exclusi on methods. Cultured cells were treated with one of four exposure compounds: ATR, dexamethasone (Dex), RU38486 and/or rat GHRF to determine the competitive inhibition of each compound on the gene expression. All treatments were initiated on day in vitro (DIV) 0. For the combinations of treatments the first agent was administered 30 minutes prior to the application of the second agent. All chemicals were dissolved in DMSO (final DMSO concentration < 0.5% v/v). For real time quantitative PCR (RTqPCR) an d competitive inhibition assays, cells were harvested and rinsed with 0.1 M PBS, then covered with RIPA buffer. The trypan blue exclusion method was used to determine cell number and cells were counted with a hemocytometer. Total

RNA was extracted from t reated and untreated pituitary cells. For each experiment, results from the treatment replicates were averaged and the amount of mRNA of each gene was normalized to the mRNA of the histone H3 gene. The standard error of each treatment was

calculated base d on the variation among the replicates of each tested gene, normalized to the standard error of histone H3 in those samples. Each biological experiment was repeated at least two times.

Protein levels in atrazine

- treated pituitary cell (GH, LH, PRL, an d CHRH receptor) cultures

were determined in AtT

- 20 mouse pituitary tumor cell line (ATCC, Val). In another approach to block adrenocorticotrophic (ACTH) and pituitary adenylate cyclase

- activating protein (PACAP) receptors, protein levels of GH in the rat GH3 pituitary tumor cell line were quantified after treatment with 5 and 10 μl ACTHR antibodies at a concentration of 0.2 mg/ml, 15 minutes prior to treatment with 1ppm ATR. This experiment was repeated twice. Competitive binding assays of ATR to pituita ry cells were assayed after 0.5, 1.0, 2.0, andDo not cite

Appendix A

- Page 319 of 677 3.0 hours at 37 degrees Celsius. The radioactivity of each vial was measured using a scintillation counter. Saturation was measured over a 3 hour time course. No significant increase in binding was observed after 0.5 hours of incubation. Testing the ability of GHRF to competitively inhibit the binding of radio labeled ATR was also performed. Actin and tubulin were immunofluorescently labeled and cells were visualized and imaging was performed.

The results presented describe a remarkable reduction in the mRNA levels of genes encoding GH (growth hormone) and LH (luteinizing hormone) were found after a 1.0ppm ATR exposure to pituitary cells, while there was an increase mRNA level of PRL (prolactin)

in treated cells compared to controls. Expression of GH was reduced in ATR treated cells compared to controls; however, GH expression did not appear to be inhibited in cells

treated with ATR and GHRF

– suggesting that GHRF may displace ATR binding to the

GHRHR. C ontrary results showed that GH expression was also lower following 1ppm GHRF treatment versus 0.5ppm GHRF. Further, the expression was also slightly reduced in cells treated with ATR and 2ppm GHRF compared to cells treated with ATR and 1ppm GHRF. Slight d ecreases and increases were found in GH, LH and PRL proteins, respectively. Competitive binding was found with ATR only and ATR and GHRF increasing mixtures. The author reported Dex+ATR were comparable to ATR alone and that there

didn‘t seem to be a rema rkable difference when compared to RU38486 alone due to variation within technical replicates. Cells were considered to have been functioning physiologically when observing histone H3 expression after ATR treatment. Blocking ACTH receptor with their anti bodies did not interfere with the antagonistic effect of ATR on GH. Moderate alleviation of the antagonistic effect of ATR on GH protein levels was

observed.

The authors concluded that the ligand

- binding receptor, AGHRHR, was responsive to

atrazine. Th ey further conclude that their hypothesis of the pituitary gland being the target of ATR is supported by a Stoker and Cooper, 2007 study which round ―a small concentration of ATR present in the anterior pituitary). They observed reductions in the GH

and L H expression in cultured rat pituitary cells following ATR exposure. They further insist that LH expression may have been reduced due to a reduction in GH expression, resulting from ATR mediated effects on GHRHR. They also attribute mammary tumor formati on to an increase in PRL expression in ATR treated pituitary cells. They expand to say that there is an increased somatostain receptor expression which may be secondary to an antagonistic effect of ATR on GHRHR. Their results did lead them to believe tha t the relatively moderate effect on GH expression in response to ATR on the PACAP receptor shows that ATR is not a primary ligand for the PACAPR.

The authors conclude that ATR targets the GHRHR of pituitary cells and that inhibition of GH production in these cells results from competition between ATR and GHRF for GHRHR binding. They make claims that this is the first time a receptor for ATR has been identified. Ultimately, the authors would like us to believe that identification of GHRHR as a target ofDo not cite

Appendix A

- Page 320 of 677 ATR may facilitate future studies on the effects of ATR and will inform efforts to develop guidelines for safe levels of ATR in water systems.

The Agency reviewer concludes that the results shown are speculatory at best and the slight or very slight incr ease and/or decreases seen in competitive binding and protein expression are not definitive.

A.11.2 Yi, KD, Perez, EJ, Breckenridge, CB, and Simpkins, JW (2010) Ex vivo effects of atrazines or metabolites on phosphodiesterase activity. In

press

This stu dy concentrated on phosphodiesterase (PDE) 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 Sprague

Dawley rats is that mRNA expression and aromatase activity was increased in H295R adrenocortical carcinoma cells when expos ed to a minimum of 10μM atrazine. Increased cyclic AMP (cAMP) levels were correlated with the induction of aromatase activity by atrazine; increases in cAMP may be mediated either via increases in adenylyl cyclase which catalyzes the conversion of ATP 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, deethy l 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 with additional 90 minute substrate/incubation period. Competitive inhibiti on 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 creating sigmoidal standard curves.

The results from the optimized assay for PDE i nhibition showed a concentration

- dependent increase in the relative fluorescence output of the assay at both 30 minutes and 2 hours after the addition of the binding reagent. Maximal output, of 0.025 PDE U/ml was used for all subsequent assays. Addition of atrazine after completion of the enzymatic reactionDo not cite

Appendix A

- Page 321 of 677 didn‘t have any effect on the fluorescence output of the assay. Regarding the effects of

atrazine and to a much lesser extent its metabolites inhibited PDE in a cell

- free system.
The IC 50 for atrazin e (39.52 μM) while 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; all other metabolites were inactive. DACT, however dose

- dependently caused a rightward shif t 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. Also at both 5 and 50 μM concentrations
of DAC T, the IC 50 of atrazine was several folds higher, suggesting competitive inhibitory
effects of DACT.

The authors concluded that atrazine at all concentrations which exceed its aqueous solubility limits, is a weak inhibitor of PDE 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 inhibition 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

- mediate d inhibition 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 laboratories. 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.

In mammals, atrazine is NOT acutely toxic (oral LD= 3090 mg/kg) and is only moderately toxic following 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 atraz ine. The bottom

- line is that it is unlikely that an

atrazine

- mediated inhibition PDE activity would occur at environmentally relevant concentrations. The potential human exposure to atrazine is low and equally unlikely to

result in PDE inhibition in huma ns. The study goes on to endorse the NOEL (1.8 mg/kg/day) which is currently used to regulate chronic exposure to atrazine and its chlorometabolites and appears to be protective of any potential effects in humans.Do not cite

Appendix B1

- Page 322 of 677 APPENDIX B

- 1: EPIDEMIOLOGY LITE RATURE REVIEW METHODOLOGY

To identify the epidemiological investigations of the association between atrazine exposure and adverse health effects (both cancer and non

- cancer), we queried PubMed/Medline and

the Institute of Scientific Information‘s Web of Science. We also performed limited searches using Google.Scholar. Querying these three search engines is considered a comprehensive way to identify relevant research articles (Falagas, 2008). PubMed is the most commonly used biomedical search engine used by resear chers today, however Web of Science offers similar journal coverage in addition to citation mapping capabilities. We performed citation mapping using Web of Science, examining other articles which referenced the articles included in the literature review, to identify additional relevant

research articles. We generated the following search strings. Emphasis was placed upon identification of all possible epidemiological studies available, and the ability to use the identical search string in both PubMed/Med line and Web of Science. Regarding Google.Scholar, we attempted use of similar search strings as well as the advanced search capabilities

available at: http://scholar.google.com/advanced_scholar_search?hl=en&as_sdt=20000].

The search strings are found b elow:

PubMed : (atrazine[tw] OR (triazine*[tw] AND (pesticide*[tw] OR herbicide*[tw]))) AND (epidemiolog*[tw] OR cohort*[tw] OR case

- control*[tw] OR cluster*[tw] OR environmental exposure*[tw] OR occupational exposure*[tw] OR ecologic stud*[tw] OR aggrega te stud*[tw])

Web of Science : atrazine OR (triazine* AND (pesticide* OR herbicide*))) AND (epidemiolog* OR cohort* OR (case

- control OR case

- control's OR case

controled OR case

- controlled OR case

- controlling OR case

- controls) OR

cluster* OR "environmental e xposure" OR "environmental exposures" OR "occupational exposure" OR "occupational exposures" OR "ecologic study" OR "ecologic studies" OR "aggregate study" OR "aggregate studies")

After elimination of duplicates with identical citations between the 2 sea rch engines, we initially identified 386 articles of potential interest. Upon review of the titles, we determined that 135 were investigations of human health effects, in contrast to environmental science or biology research papers. Of these 135, we elimin ated an additional 46 studies that reported on exposure assessment methodology only. We then performed an abstract review of the remaining 89 papers and eliminated an additional 22

that were review articles , editorials, or other opinion narratives publishe d in the open literature. Therefore, there were 67 epidemiological investigations of the cancer and non

cancer health effects of atrazine. In the review reflected herein, we are only considering the

non

- cancer related epidemiology studies. EPA will repeat the literature search using similar methodology at a later date to assure that all relevant atrazine cancer studies are includedDo not cite

Appendix B1

- Page 323 of 677 in the upcoming cancer evaluation. There were 29 non

- cancer epidemiology studies

included in our full

- text review of the epidem iology literature.

Upon review of the full

- text articles, we additionally determined that three were case reports of acute adverse health effects only, two were only measured atrazine exposure (not in association with a non

- cancer outcome), and two did n ot measure atrazine or triazines independently. In addition, one of the references included in this group based upon abstract review has not yet been published as a full

- text article. Therefore, there were 21 epidemiological investigations of the non

- cance r health effects of atrazine exposure included in the literature review, two of which were published in languages other than English, and are not included in this review as we were unable to obtain Polish

- English translation. Our process of identifying and selecting articles for this literature review including the criteria used is depicted in Figure B

- 1 : below

.Do not cite

Appendix B1

- Page 324 of 677 Figure B

- 1 :Do not cite

Appendix B2

- Page 325 of 677 APPENDIX B. 2: REVIEW OF EPIDEMIOLO GIC INVESTIGATIONS O F NON

- CANCER

HEALTH EFFECTS OF ATRAZINE EXPOSURE

A detailed review of the strengths and weaknesses of the nineteen studies included in the EPA literature search evaluating the non

- cancer health effects of atrazine exposure are provided in this appendix. In performing thes e reviews, EPA considered the comments and suggestions received from the FIFRA SAP panel convened February 2

- 4, 2010 regarding incorporation of epidemiologic data into the Office of Pesticide Program‘s risk assessment process. Considerations include streng ths and weaknesses of the study designs, ascertainment of the outcome, measure of atrazine exposure, assessment and analysis of relevant confounding factors, appropriateness of the statistical analyses performed, presentation of the data, interpretation of the results in the context of the literature at the time, and statistical power to estimate the association with precision, among other factors noted on a study specific basis. These studies are considered individually within the context

of these critical reviews, but are considered in conjunction with the toxicological data in
Section 4

.

B.2.1 Arbuckle et al. ―An Exploratory Analysis of the Effect of Pesticide Exposure on the Risk of Spontaneous Abortion in an Ontario Farm Population,‖ [ Environ Health Perspect 109:851

– 857 (2001).]

Background : Researchers investigated the ways in which pre

- and post

- conception pesticide exposure is related to risk of early

- (<12 weeks) and later

- (12

- 19 weeks) in gestational age spontaneous abortion. In hypothesis generating interaction analyses, the authors examined the potential effect modifying role of various known risk factors for spontaneous abortion, such as age, smoking, alcohol consumption in the association between pre

- and post

- co nception pesticide exposure and spontaneous abortion (<20 weeks).

Methods : Authors performed this study within the Ontario Farm Family study (FFS) which included male and female partners who lived and worked on a farm. To be eligible for the FFS, couples must live year round on the farm, include a female partner less than 44 years of age, and include at least one member of the couple that works of the farm at the time of the study. Data were collected using three self

- administered questionnaires and inclu ded information about the demographic characteristics, reproductive health and history, and pesticide exposure.

Women were asked to recall each pregnancy which has occurred at the time of enrollment. For each pregnancy, gestational age at the end of preg nancy and month of conception was calculated using date of last menstrual period (LMP). The outcome was ascertained through self

- reported instances of spontaneous abortion (gestational age <20 weeks); all but 5 of the cases included in this study were medi cally confirmed. Authors evaluatedDo not cite

Appendix B2

- Page 326 of 677 association between pesticide use and spontaneous abortions which occurred less than 12

weeks and 12

- 19 weeks to determine critical windows of exposure as well as possible target sites of toxic effect. Pregnancies which o ccurred when women were not residing on the farm and pregnancies for which the current male partner living on the farm was not the father were excluded from this study.

In written questionnaires regarding pesticide exposure, the farm operator (either the male or female partner in the couple, or the farm operator) provided information about ever

- use of certain pesticide products (queried using both  ̳drop

- down‘ menus and blank  ̳write

- in‘ opportunities), and, during an average year of use, the months of the year in which the product was used. Authors matched pesticide products with active ingredients using a database of Canadian registered pesticide products. In this study, 17 pesticide  ̳units‘ were assessed which included 4 pesticide types, e.g., herbicide; 4 pesticide classes, e.g., triazine; and, 9 specific pesticides. Authors pooled pesticide exposure information reported by the farm operator, and male or female partner in the farm family, and the critical windows of embryo and fetal exposure to develop an historical pesticide use exposure record for each farm site. In this study, pesticide use aggregated to the farm level was used as a surrogate for individual pesticide exposure. Authors stated that they were able to measure both direct and indirect pestic ide use using this method; however, they could not discern exposures which occurred to the male or female partner. Specifically, authors were interested in pesticide exposure during the time periods 3 months prior to the month of conception and including the month of conception (pre

- conception), and the first trimester. Authors compared exposure during these two time windows with total number of spontaneous abortion, and then early

- (<12 weeks), and late

- (12

- 19 weeks) spontaneous

abortion.

Authors calcu lated odds ratios and 95% confidence intervals (95% CI) for the association between pesticide use in the pre

- and post

- conception period and spontaneous abortion (<20 weeks), and early and later in gestational age spontaneous abortion. Researchers

stated a mong the potential risk factors evaluated, none were strong confounding factors in the assessment of this association, therefore unadjusted estimates were provided. In a hypothesis generating sub

- analysis, authors were also interested in exploring potentia l effect modification by pesticide use in the association between known risk factors for spontaneous abortion and spontaneous abortion (<20 weeks) using CART analysis (Classification and Regression Tree methods). This resulted in 367 comparisons (17 pestic ide  ̳units‘ and 21 potential risk factors).

Results : There were over 2,000 couples included in the study and 3936 pregnancies, 395 of which ended in spontaneous abortion. Overall, increased risk of spontaneous abortion, and particularly early (<12 weeks) , spontaneous abortion was associated with the pre

conception (3 months prior to conception, and month of conception) exposure to several different pesticide groupings including glyphosate, carbaryl, phenoxy acetic acids, triazinesDo not cite

Appendix B2

- Page 327 of 677 and thiocarbamates. With respect to atrazine, pre

- conception exposure to atrazine resulted in a slightly increased risk of spontaneous abortion, OR and 95% CI 1.2 (0.9

– 1.7), risks were increased for the instances of spontaneous abortion that occurred early (<12 weeks),

but not la ter (12

- 19 weeks) in pregnancy. There was no association between post

conception exposure to atrazine and spontaneous abortion. For those instances of early

term spontaneous abortion, the odds of pre

- conception atrazine use was elevated as compared to post

- conception exposure, OR 1.7 (95% CI 0.8, 3.8). The risk of late term spontaneous abortion was also elevated for pre

- conception atrazine exposure as compared to post

- conception exposure OR 1.4 (95% CI 0.6, 3.5).

In the analysis of potential interactions between pesticide use and risk factors for spontaneous abortion, authors observed that maternal age was the strongest risk factor for the outcome. In addition, they report that women 35 years of age or older and who reported use of triazines during the pre

- conception period had an approximately 3

- fold increased risk of spontaneous abortion (OR 2.7, 95% CI 1.1, 6.9). Older women (35 years or older) who reported use of both triazines and thiocarbamates had an 8

- fold increased risk in spontaneous abortion base d upon initial exploratory analyses of these statistical

interactions.

Author‘s Conclusions : Researchers state that their work provides evidence that pre

conception exposure to certain pesticides including atrazine, in addition to glyphosate, carbaryl, an d 2,4

- D, may result in 20

- 40% increased risk of spontaneous abortion; no associations between post

- conception pesticide exposure and the outcome were observed. From this work, authors suggest that pesticide exposure possibly including atrazine, may

increas e risk of early

- but not late term spontaneous abortion. Authors posit that sperm damage is a possible biological mechanism for this observation.

EPA Critique

The authors performed a well

- designed and conducted hypothesis

- generating study of the

associa tion between pre

- and post

- conception pesticide use and spontaneous abortion (<20 weeks gestation). This study adds to our knowledge of potential critical windows of effect and target sites for pesticide toxicity, including atrazine. In addition, in explor atory work, authors have shown that maternal age and pesticide exposure, including atrazine, may both contribute to the risk of spontaneous abortion, particularly exposures which occurred pre

- conception. There is some evidence that pre

- conception exposure to atrazine is associated with spontaneous abortion, however several other pesticides and pesticide

classes are as well.

The suggestion that maternal age and pesticide exposures including atrazine may result in an additional risk of spontaneous abortion is qualitatively informative, as it may suggest cumulative exposure to pesticides is an important consideration. Additional studies areDo not cite

Appendix B2

- Page 328 of 677 needed to test this hypothesis; caution should be used in interpreting the results of these exploratory analyses.

This study was performed in the same cohort, the Ontario Farm Family study, as the study by Savitz et al. (1997) which investigated adverse birth outcomes including pre

- term delivery, small

- for

- gestational age, and secondary sex ratio. As stated in the summary of Savitz et al., the exposure characterization methods used in this study are a limiting factor to use of these results in overall causal inference. Reconstruction of historical pesticide exposure was performed through matching self

- report information on year and months of year of use of specific pesticide products on the farm (ascertained based on the 5 years prior to study enrollment and extrapolated back to the initiation of the farm employment) and the critical windows of embryo and fetal exposure base d upon maternal self

- report of gestational age at birth. Therefore, no direct personal pesticide exposure was made in this study. As acknowledged by the authors, this approach likely resulted in exposure misclassification. In addition, authors did not adju st for highly correlated, multiple pesticide exposure in their analyses, if any.

Authors achieved an adequate response rate from the sampled population. In addition, misclassification of the critical window of exposure using LMP to estimate gestational ag e and month of conception is possible; however, medical confirmation of most all the cases is a strength. Authors state that there analysis indicated there were no strong confounding factors in the analysis of pesticide exposure and spontaneous abortion. G iven the role of maternal age in understanding adverse pregnancy outcomes, an age

- adjusted estimate may have been informative. Researchers acknowledge recall bias through differential recall of pesticide use is unlikely, but not impossible, given typically men reported pesticide use and female partners reported spontaneous abortion. Reverse causation, a case in which persons would reduce pesticide exposure based upon a previous spontaneous abortion, is possible but authors state they were not able to invest igate given the low numbers of previous spontaneous abortion. The use of CART is an innovative approach and appropriate to the hypothesis generating nature of the sub

- analysis.

Overall, this work provides some evidence that pesticide exposure, including a trazine, during the pre

- conception period (3 months prior to conception and the month of conception) may be associated with spontaneous abortion, particularly early (<12 weeks gestation) events. However, additional epidemiological research is needed to cla rify these suggested signals.

B.2.2 Dabrowski, S. et al. ―Pesticide exposure and birthweight: an epidemiologiucal study in central poland,‖[International Journal of Occupational Medicine and Environmental Health; 16(1): 31

— 39. (2003)].Do not cite

Appendix B2

- Page 329 of 677 Background: Autho rs hypothesized that maternal pesticide exposure during pregnancy was related to low birth weight (<2500 grams) and preterm delivery (<37 weeks).

Methods : Authors initially attempted to identify risk factors for low birth weight infants, but

within the re cruitment process decided to focus the study on pesticide exposure and low birth weight and preterm delivery. Women were randomly selected from hospitals from several cities in Poland to participate in the study; in two geographic locations, all births tha t took place between January and June 2001 were considered for inclusion in the study (non

- random selection). From this population

- based sample, authors identified low birth weight infants and pregnancies that ended preterm. Authors only considered women w ho reported at least 7 days of pesticide use during pregnancy as potentially exposed, and authors excluded women with other health conditions, such as diabetes, which may have

resulted in adverse birth outcomes.

Researchers collected pesticide exposure an d relevant demographic information through physician administered questionnaire. Pesticide use as well as engagement in heavy physical labor was assessed for each trimester of pregnancy. Occupational health professionals validated the pesticide exposure in formation reported by mothers with pesticide applicators on the farm, typically the husband/father. Authors state that the method of exposure assessment captured both agricultural and indirect residential pesticide exposure.

Authors utilized both linear a nd logistic regression to model the association between pesticide use and low birth weight infants and preterm delivery, adjusting for infant sex, maternal age, pre

- pregnancy weight, height, maternal smoking, year of birth, and involvement in physical labo r in the field. For pesticide specific analysis, researchers compared observed and expected birth weight and pregnancy duration using the rates for the region as the standard.

Results : Authors report ever

- use of any pesticide was associated with odds of l ow birth weight (adjusted odds ratio (OR) 1.29, 95% confidence interval (95% CI) 0.63, 2.65). In linear regression analyses, ever use of any pesticide was associated with a reduction in infant birth weight of 189 grams (p=0.01), and a reduction in pregnanc y duration of half of a week (p=0.05). Infants of mothers who reported triazine exposure during the first or second trimesters of pregnancy were observed to have a non

- significantly lower birth weight than expected (259 gram less than expected, p=0.43), an d pregnancy duration among triazine exposed mothers was also less than expected (2 weeks less than expected, p=0.18). No atrazine specific estimates were provided, based upon the data available.

Author‘s conclusions : Researchers conclude that maternal pes ticide exposure may result in a small, but significant reduction in infant birth weight, and that analyses illustrate that a reduction in the pace of fetal growth (birth weight adjusted for pregnancy duration) mayDo not cite

Appendix B2

- Page 330 of 677 also be observed. This suggests a small

- fo r

- gestational age effect, according to the authors. With respect to specific types of pesticides, authors report preliminary results that triazines and organophosphates may be linked to low birth weight.

EPA Critique :

Authors performed a population

- b ased case

- control study of the association between maternal pesticide exposure and infant birth weight; however, information regarding the specific relation between maternal atrazine exposure and infant birth weight was limited.

The random selection of wom en into the study (with the exception of 2 sampling locations) enhances comparability of the case and control series. Strengths of the study include ascertainment of birth weight from medical records, validation of pesticide exposure among women who report ed pesticide exposure, inclusion of numerous potential occupational hazards in the questionnaire likely reducing the chance of recall bias, and sufficient confounder analysis. A limitation of this study, for the purpose of this review, is the lack of

an at razine specific exposure estimate. The analysis of observed and expected infant birth weight and pregnancy duration by pesticide class suggests a potential role for triazines and organophosphates, but it is based upon an unadjusted analysis and a relativel y small sample size. Authors report seven women in this study sample who listed triazine use during the pregnancy. This study offers some limited additional evidence to consider as to the possible role of maternal triazine exposure and adverse birth outcom es, but small sample size and lack of adjusted estimates of the association between triazine exposure and low birth weight are limitations.

B.2.3 Farr et al. ―Pesticide Exposure and the Timing of Menopause in the Agri cultural Health Study (AHS),‖ [ Am J E pidemiol,163:731

– 742 (2006)].

Background: Authors investigated whether pesticide use, and use of pesticides designated as hormonally active or ovotoxic specifically including atrazine, is associated with timing of

menopause.

Methods: Researchers include d female pesticide applicators or female spouses of pesticide applicators enrolled in the Agricultural Health Study (AHS). Authors included 8,038 women in the study who were premenopausal at enrollment, ages 35

- 55, and who reported information regarding pe sticide exposure, menopausal status, and relevant co

- variables ( i.e. , these data were not missing). Menopausal status was ascertained through self

- report questionnaire. At enrollment, women were asked  ̳ ̳Have you gone through menopause (the change of life) or had surgery that caused you to completely stop having menstrual periods?‘‘ Approximately 5 years later, during the follow

- up interview, women were again questioned regarding menopausal status.Do not cite

Appendix B2

- Page 331 of 677 Women reported information regarding their ever

- use of 50 different pesticides during their lifetime in the AHS questionnaires (Female and Family Health questionnaire for spouses,

and enrollment and take

- home questionnaires for female applicators). Researchers included only the 40 of the 50 pesticides which were available for use since the enrollment period (early 1990‘s) because authors wanted to ensure pesticide use could be temporally linked to the timing of menopause. Authors identified six of the 40 pesticides for which there was evidence of plausible hormona l activity or ovotoxicity. These pesticides were of a priori interest to the researchers. Information regarding demographic characteristics, reproductive health, and co

- morbid conditions was collected through self

- report questionnaire at the time

of enroll ment, and through follow

- up (periodic re

- questioning of cohort participants).

Researchers utilized Cox proportional hazards regression methods to measure survival of ovarian function, i.e. , time to menopause, using age at last menstrual period as the

outc ome variable in the analysis. In this survival analysis, the number of events ( i.e., woman who experienced menopause) is compared among pesticide users and non

- users at the time of each event ( i.e ., within each risk set). In this analysis, hazard ratios le ss than 1.0 indicate longer time to menopause. Although authors collected pesticide use and reproductive history ( e.g., age at menarche) since birth (information collected at enrollment into AHS reflected lifetime exposure history), because women entered t he sample at different ages ( i.e., left truncated data), authors considered time at risk for menopause to begin after the age at enrollment. Participants were censored ( i.e ., follow

- up time ended) at either age at natural or surgical menopause (using the m idpoint of the year of age at menopause) or age at follow

- up interview. Analyses were adjusted for smoking and oral contraceptive use. The validity of the proportional hazards model assumptions were tested using Kaplan

- Meier curves and interaction terms be tween each variable and time (since enrollment).

Results: Authors reported that ever use of any pesticide (hazard ratio (HR) (95% CI ) 0.87 (0.78

- 0.97)), ever use of any herbicide (HR (95% CI) 0.88 (0.74

- 1.05)) or ever use of any hormonally or ovotoxic p esticide, including atrazine, (HR (95% CI ) 0.86 ( 0.77

- 0.97)) resulted in an approximate 3 month longer time to menopause among users as compared

to non

- users of these groups of pesticides. Ever use of any triazine (atrazine, cyanazine, metribuzin) resul ted in a reduced hazard of ovarian failure (increased time to menopause) (HR (95% CI ) 0.63 (0.36

- 1.12)). For atrazine specifically, ever use of the chemical resulted in a statistically significant decreased risk of ovarian failure (increase in time to

men opause), a 5 month increase in the time to menopause (HR (95% CI ) 0.79 (0.63, 0.99)).

Several sensitivity analyses were performed to assess the influence of potential biases in the estimates. Authors reported no significant changes to the point estimates in the main analysis as a result of these sub

- analyses.Do not cite

Appendix B2

- Page 332 of 677 EPA Critique:

The authors conducted a well

- designed research study to determine the association between use of pesticides, and author

- defined hormonally

- active and ovotoxic pesticides, including a trazine, and timing of menopause. This hypothesis is based upon limited published experimental and observational literature. The sample size is large and the prevalence of use of pesticides overall, and proportion of women who were menopausal

was relativel y high allowing precise estimation of risk. However, atrazine use in this cohort among female applicators and female spouses of applicators is more limited, approximately 8%. Although a well conducted study, some limitations exist that hinder full incorpor ation into a quantitative risk assessment, most of which are also acknowledged by the authors. EPA may use this study to qualitatively inform the atrazine risk assessment process.

In this study, outcome was determined through self

- reported date of menopau se assessed

at follow

- up approximately 5

- years after initial enrollment into the AHS. Although whether or not a woman has gone through menopause is likely to be self

- reported with relative accuracy, timing of menopause is more difficult to determine. For m ost women, menopause is not an instantaneous event; women may be in a perimenopausal status for several years. In this study, researchers did not use a definition of time since last menstrual period as a measure of menopause, they measured whether or not t he event occurred, and if it occurred, the date of LMP. Therefore, authors did not measure the perimenopausal status directly. It is likely many women who responded ―do not know‖ were indeed perimenopausal. Authors classified women in the perimenopausal st ate as menopausal. Pesticide exposure may also affect the perimenopausal state. It is not known how pesticide exposure may affect the timing or duration of the perimenopausal state. Given the authors utilized time to menopause as the outcome for this resea rch, misclassification of timing of menopause due to the measurement error around definition of menopause may affect the results. Authors did not report any methods to validate this measure, e.g., medical record

review. Authors recommend future studies inc lude more frequent ascertainment of menopausal status during the course of follow

- up.

With regard to the exposure assessment method, researchers utilized self

- reported ever

use of pesticides as well as number of days per year of use of any pesticide (not specific pesticides like atrazine). As a result, no information regarding an exposure

- response gradient for atrazine can be derived from the study. In addition, this study is not able to address the question as to when in the reproductive life span does at razine/pesticide exposure affect timing of menopause. The ever

- never metric does not allow evaluation of cyclic use of pesticides or periods of cessation of use.

Authors identified and measured several potentially confounding variables in the

association between pesticides and timing of menopause including both leisure time andDo not cite

Appendix B2

- Page 333 of 677 occupational physical activity. Consideration of a family history of early (or late) menopause may also have been informative, if available. It is likely there may be a genetic rol e for timing of menopause (Henderson 2008). In addition, if the woman‘s mother also resided on a farm, which is common in farming communities, a family history of timing of menopause may also be environmental, possibly including pesticide exposure.

For pu rposes of risk assessment, EPA is not able to conclude that atrazine influences timing of menopause with certainty based upon these data. The data suggest that some pesticides, possibly those with evidence of hormonal ovotoxic activity as suggested by the authors, may affect timing of menopause, but additional research is needed to reduce the uncertainties present. Specifically, as noted by the authors, more frequent assessment of menopausal status through prospective study designs, better measure of the ti ming and dose of specific pesticides, and enhanced confounder assessment particularly with respect to physical activity would aid the understanding of this potential association.

B. 2.4 Farr et al. ―Pesticide Use and Menstrual Cycle Characteristics among Premenopausal Women in the Agricultural Health Study,‖ [Am J Epidemiol 2004;160:1194

– 1204 ((2004)].

Background : The authors studied the association between use of hormonally active or directly ovotoxic pesticides and menstrual cycle characteristics, incl uding length of cycle (>36 days, 24

- 36 days, <24 days, or irregular), missed periods, or inter

- menstrual bleeding.

Methods : Authors studied this question in a sample of female pesticide applicators or female spouses of pesticide applicators enrolled in th e Agricultural Health Study (AHS). Authors identified five menstrual cycle characteristics to measure in association with pesticide use. Information concerning the prevalence of these menstrual cycle characteristics within the sample was obtained via self

- report questionnaire. Menstrual cycle characteristics evaluated included: long cycle length (>35 days), short cycle length (<24 days), irregular cycles based upon the question ―what is your average cycle length,‖ as well as missed periods within the past 1 2 months, and inter

- menstrual bleeding within

the last 12 months.

Female pesticide applicators and female spouses of male pesticide applicators completed the ―Female and Family Health Questionnaire‖ (FFHQ) within which self

- reported

information concernin g lifetime ever

- use of 50 different farm pesticides was ascertained. In addition, the FFHQ asked ―how many days per year, on average, they mixed or applied pesticides.‖ Therefore, based upon the pesticide exposure data available, analyses were performed e valuating lifetime ever

- use of specific pesticides as well as days of use of any pesticide per year (no exposure, 1

- 9 days per year, and more than 10 days per year). Information concerning factors which could possibly be associated with both menstrual cycl e characteristics and pesticide use was also collected via self

- reported questionnaire.Do not cite

Appendix B2

- Page 334 of 677 In this analysis, prevalence estimates were adjusted for the effects of age (continuous variable), smoking status (ever/never) and body mass index (BMI).

Identificat

ion of “Probably or Possible Hormonally

- Active Pesticides”: Authors conducted a literature review to determine which of the 50 pesticides included in the AHS ―showed evidence of ovarian effects, disruption of estrous cycles in animal models, or evidence of endocrine disruptions in in vivo or in vitro tests.‖ Authors identified several of the pesticides included in the AHS with active registrations in 1993 (the year of enrollment) that were, according to the authors, ―possible‖ or ―probable‖ with respect to evidence published in the open literature as to the hormonal activity of the pesticides. Atrazine is considered by the authors as probable hormonally active pesticide with respect to endocrine disruption, ovarian effects or estrous cycle disruption. Howev er, given the data available authors were not able to present atrazine

- specific prevalence estimates.

Because the researchers were simultaneously evaluating the association between pesticide use and 5 correlated health outcomes (the 5 menstrual cycle char acteristics), they used a type of analysis that would account for the correlated nature of the data: marginal models and generalized estimating equations. Several sensitivity analyses were performed

to assess the influence of uncertainties in the measureme nt of certain study parameters to determine if biases exist in the prevalence estimates presented.

Results : Authors observed that ever

- use of any pesticide was associated with increased odds of a longer cycle and missed period, and a decreased odds of an irregular period. Similar results were observed for the pesticide groupings carbamate, crop insecticides, and herbicides, however use of herbicides and fumigants were also negatively and positively associated with a short cycle (<24 days), respectively. A cross the groupings inclusive of the 40 pesticides examined, no statistically significant association with inter

- menstrual bleeding was observed. It should be noted, prevalence estimates for short

- , missed periods and

inter

- menstrual bleeding for women who reported use of triazine herbicides, but not

atrazine alone.

As mentioned above, no estimates were provided for atrazine specifically in relation to the menstrual cycle characteristics examined. In association with ever

- use of triazines (defined

as atra zine+cyanazine in this study), authors observed a non

- significant decreased odds of short cycle (<24 days), increased odds missed period, and no association with inter

menstrual bleeding (OR 1.0, 95% CI 0.37, 2.9). No prevalence estimates for the

associati on between triazine use and long

- or missed periods were provided, most likely due to low numbers of triazine exposed women who reported these characteristics.

When authors restricted the analysis to only those women who reported using a probable

hormona lly active or ovotoxic pesticide, the associations long cycles and missed periods persisted (long cycle: odds ratio (OR) 1.6, 95% confidence interval (CI) 1.0

- 2.5; missedDo not cite

Appendix B2

- Page 335 of 677 period, OR 1.7, 95% CI 1.3

- 2.2), and the association with irregular period also persi sted (OR 0.52 (95% CI 0.36

- 0.77). Authors also observed a borderline statistically significant

association with inter

- menstrual bleeding (OR 1.2, 95% CI 0.97

- 1.6). The trend for number of days of any pesticide use per year were significant for long

- and mi ssed periods. Among women who reported using lindane or atrazine or both (excluding users of maneb/mancozeb) as compared to never users of any pesticides, authors observed decreased odds of short cycle, increased odds of long or missed cycle (long cycle, O R 2.7,
95% CI 1.3

- 5.5; missed period OR 2.0, 95% CI 1.3

- 3.3), a borderline significant association with irregular cycle (OR 0.45, 95% CI 0.18, 1.1), and a significant association with inter

menstrual bleeding 1.7 (95% CI 1.1, 2.7)

The results of sensitiv ity analyses indicate that residual confounding due to differences

between ever

- and never

- users of certain pesticides, exposure misclassification as a result of the measurement of lifetime pesticide use, and confounding due to physical activity in

the ass ociation between pesticide use and menstrual cycle characteristics do not likely explain these results in total.

EPA Critique :

The authors conducted a well designed research study to measure the association between ever use of pesticides and certain m enstrual cycle characteristics among female pesticide applicators and female spouses of pesticide applicators enrolled in the Agricultural Health Study (AHS). This research question is based upon a priori evidence in the toxicological literature, and tests the hypothesis that pesticides with suggested hormonal effects may alter menstrual cycle characteristics. However, with respect to atrazine, no specific estimates are provided most likely due to a low number of atrazine exposed women in the sample. Theref ore, given the lack of atrazine

- specific estimates, EPA cannot draw many firm conclusions from this study regarding the way in which atrazine exposure may affect the menstrual cycle.

Understanding the limitations of this study to EPA‘s purpose of assessi ng atrazine toxicity, EPA notes several points in brief. In this study authors compared ―average‖ menstrual cycle length over the course of a lifetime (21

- 40 years) as well as the prevalence of missed periods or inter

- menstrual bleeding in the last 12 mont hs, with lifetime pesticide use. Therefore, the timing of the measurement of pesticide exposure in association with menstrual cycle characteristics is not precise; ideally one would measure concurrent pesticide use and menstrual cycle characteristics. This approach assumes lifetime pesticide use and menstrual cycle characteristics are relatively consistent, and it is possible both exposure and outcome change over time. However, authors argue that women are likely accurately reporting lifetime characteristic s, and illustrate that excluding female applicators and spouses of male applicators who reported no use of hormonally active pesticides within the prior 12 months, the effect estimate are comparable. The researchersDo not cite

Appendix B2

- Page 336 of 677 also discuss that the effect of exposure measurement error of this type (timing of exposure), if present, would likely attenuate the estimated associations.

Menstrual cycle characteristics were ascertained via self

- report questionnaire. Although the timeframe for two of the five menstrual cycl e characteristics were restricted to more recent time period (last 12 months), in general there is likely some misclassification in menstrual cycle characteristics as a result of this method of outcome ascertainment. Authors recognize that the use of diari es would have been preferable way to collect this information. However, it is unlikely that pesticide use would be associated with differential likelihood of reporting menstrual cycle characteristics in this study because menstrual cycle

characteristics we re not of a priori interest at the time of enrollment.

The AHS collects a wide range of information regarding potentially confounding variables. Authors state that while several potentially confounding variables were evaluated as to the effect on the poi nt estimate (±10%), only age (continuous), body mass index (BMI) (normal, overweight, obese), and smoking (ever/never) were included in final models. Physical activity is known to be associated with menstrual cycle functioning and also likely

associated wi th pesticide use. Authors reported that analysis which adjusted for occupational physical activity (―how many days per year did you work in the fields?‖), prevalence estimates were slightly, but not significantly, affected. Ideally, leisure time as

well as occupational physical activity would have been available.

Regarding co

- morbid conditions, authors did not have information regarding polycystic ovarian syndrome, endometriosis, anorexia, or other common disorders which may also

interfere with menstrual function. A sensitivity analysis excluding these women may have informed the nature of the pesticide

- menstrual cycle characteristic association. Authors were however able to evaluate the effect of thyroid disorders and reported no differences in

the estima tes presented.

Authors performed an analysis of cross

- sectional data and studied whether the prevalence of these menstrual cycle characteristics differed by ever use of certain pesticides. Therefore, one cannot say with certainty whether pesticide use p receded the disruption of the hormone cycle. However, given the average age of the women included in this study is 35 years and they average number of years lived on a farm is 21, it is likely most of these

women lived and/or worked on a farm most of their reproductive life.

Overall, authors performed a high

- quality investigation of a novel research hypothesis. However, lack of atrazine

- specific effect estimates limits use in risk assessment.Do not cite

Appendix B2

- Page 337 of 677 B.2.5 Hoppin et al. 2002. ―Chemical Predictors of Wheeze amon g Farmer Pesticide Applicators in the Agricultural Health Study,‖ [Am J Respir Crit Care Med Vol 165. pp 683

– 689].

Background : Authors hypothesize that chemical use over the previous year is associated

with self

- reported episodes of wheeze. Specifically, authors hypothesize that use of OP, carbamates or thiocarbamates may be associated with wheeze, based upon previous

literature.

Methods: Authors studied this question in a cross

- sectional analysis among pesticide applicators in the Agricultural Health S tudy (AHS). Upon enrollment, pesticide applicators in the AHS responded to written questionnaires regarding pesticide use and non

- cancer health outcomes. Participants who self

- reported one or more instance of wheeze over the year prior to completion of the questionnaire were considered wheeze sufferers for the purpose of the study (cases of wheeze).

Researchers measured pesticide use with both an ever

- never exposure metric and, for those pesticides positively associated with wheeze using this exposure me tric or for which authors proposed a priori hypotheses, days of use over the past year were categorized. Because recent exposure was of interest to the researchers, only the pesticides available for use in the reference year (the year prior to the question naire completion) were included in the study (40 of 50 available pesticides). Neither atrazine nor triazine use was of a priori

interest to the researchers.

Authors compared use of certain pesticides between those who did and did not report episodes of wh eeze in the previous year. Models were adjusted for age, state, smoking and presence of asthma or atopy. Authors defined atopy as physician diagnosis of eczema or hay fever. To determine whether the relation between pesticide use and wheeze differed as

to whether participants reported physician diagnosis of asthma and atropy, authors used interaction terms in the models. In each model, the reference group was comprised of those who reported never

- use of the specific pesticide.

Results: Of the 40 pesticide s evaluated in association with wheeze, 11 were significantly associated with the outcome. Of these 11, researchers observed a significant exposure

response relation with 10, including atrazine. Researchers observed a 20% increase in the prevalence of whee ze among those who report ever using atrazine over the year preceding the questionnaire completion as compared to those who report never using atrazine over the past year [odds ratio (OR) 1.20, 95% confidence interval (95% CI) 1.07

- 1.34]. Across 5 categori es of days of use of atrazine over the past year, authors observed a significant

exposure

- response association (p for trend <0.01). Odds ratios (95% CI) for the five categories are as follows: never users 1.0 (referent); 5 days, 1.12 (0.96, 1.29); 5

– 9 days

, 1.17 (1.01, 1.36); 10

– 19 days, 1.27 (1.08, 1.51); and, 20 days, 1.53 (1.21, 1.95). NeitherDo not cite

Appendix B2

- Page 338 of 677 adjustment for correlated pesticides nor for corn or grain dust exposure changed the

observed results. No difference in the association between atrazine use and wh eeze was observed between those who did and did not report diagnosis of asthma or allergy.

Author‘s Conclusions: Researchers state that this study illustrates pesticides may be associated with increased odds of wheeze, controlling for other farm

- related e xposures that may confound the association. Authors acknowledge that the association between atrazine and wheeze was not hypothesized. In this study, authors were able to investigate the association between atrazine and wheeze, independent of the affect of corn and grain dust, which are important confounding factors to consider in this association. Authors acknowledge that additional information concerning product formulation and use site would have enhanced the exposure characterization, but the informatio n was not available.

EPA Critique :

The authors conducted a well

- designed cross

- sectional analysis of the odds of wheeze in association with pesticide use both measured over the year prior to questionnaire completion. Authors did not present a priori hyp otheses regarding use of triazines/atrazine and wheeze; however, they noted previous findings with regard to farming and pesticide use in general. In other research, AHS private and commercial pesticide applicators have been found to supply reliable (Blai r 2002) and valid (Hoppin 2002) pesticide use estimates. The outcome was ascertained through self

- report questionnaire responses administered via computer assisted telephone interview (CATI).

As noted by the authors, strengths of this study include the r elatively large sample size (nearly 20,000 pesticide applicators, and approximately 3300 wheeze cases) and the ability to control for many known and suspected confounding variables in the association measured. Regarding the association between atrazine use and wheeze, it is particularly notable that authors were able to simultaneously control for exposure to corn and grain dust. Atrazine is primarily used on these types of crops, and these exposures could also be

related to wheeze. The measured association between atrazine and wheeze independent of corn and grain dust is a contribution to our knowledge of atrazine‘s respiratory effects.

Given the relatively high percentage of wheeze sufferers in the sample, 19%, which is comparable to other populations, an d the high prevalence of atrazine use in the sample, the sample size is sufficient to detect a difference between wheeze cases and controls with regard to atrazine use. The parsimonious models used in the multi

- variate analysis of the

association also enha nces statistical power to detect associations.

Authors performed a well

- conducted hypothesis

- generating, cross

- sectional analysis of the association between pesticide use including atrazine and wheeze in a sample of pesticide applicators in Iowa and Nort h Carolina (the AHS). The study is absent large sources ofDo not cite

Appendix B2

- Page 339 of 677 systematic error which may result in an inaccurate measure of the association. Sample size is adequate to detect an association, if one truly exists. However, the association between

atrazine use a nd wheeze is not previously hypothesized and, at this time, there is not strong biologically plausible explanation proposed as to the mechanism of action for this effect. Additional research including information concerning co

- morbid (respiratory)

conditio n and alternative study designs will aid the understanding of this association. Additional mechanistic and experimental work may aid our understanding of the mechanism of action to explain the proposed association.

B.2.6 Hornemann et al. ―Seasonality of omphalocele in Northern Germany,‖ [Ultraschall in Med. 29:591

- 595 (2008)].

Based upon German

- English translation provided by Dina Schreinemachers, USEPA, 5

- 17

- 2010.

Background : Authors compared season of conception (summer v. winter) and prevalence

of o mphalocele between the years 1993

- 2007 to determine whether there were seasonal differences that may be attributed to atrazine exposure.

Methods : Authors performed a population

- based case

- control analysis. Researchers identified all cases of omphalocele d iagnosed at a clinic serving Schleswig

- Holstein and western Mecklenburg

- Vorpommeren area, an area of high agricultural production, between

1993

- 2007. The clinic provided genetic counseling and routine screening of pregnancies early in gestation. Cases of o mphalocele were further screened to determine whether chromosomal aberrations were also present ( e.g. , trisomy 18). Month of conception was

determined at the time of ultra

- sonic evaluation. Based upon this information, authors classified season of concepti on as summer (April

- September) or winter (October

- March). Cases with other abdominal birth defects including gastroschisis were excluded.

Statistically comparisons were made using the non

- parametric Mann

- Whitney test.

Results : Authors observed no differ ence in season of conception between omphalocele

cases and non

- cases. Authors did not observe any difference between rural and urban populations with regard to season of conception or prevalence of omphalocele. Authors did not identify a clear pattern of c hange in prevalence of this birth defect over the time period of this study.

Authors‘ Conclusions: The authors concluded that there was no discernible trend in omphalocele rates of the time period of this study, nor were there identifiable differences i n the rate of omphalocele between pregnancies conceived in the spring/summer as compared to the fall/winter, that may have been possibly related to atrazine, or other seasonal environmental exposure.Do not cite

Appendix B2

- Page 340 of 677 EPA critique:

Authors investigated the seasonality of omphalocele prevalence in order to identify environmental factors, especially atrazine. In addition, authors evaluated trend in omplalocele prevalence over time. However, this study took place after a ban was placed upon the use of atrazine in agriculture. Therefore, the magnitude and range of environmental atrazine exposure during the time period of this study is likely low (not explicitly reported by authors), and the differences in environmental atrazine exposure between spring/summer and fall/winter is also likely low or non

- significant, given the ban. However, as stated by the authors, although a ban in atrazine use took place in the early 1990‘s, the herbicide was still detectable in the environment; environmental sampling data were not provided or cit ed.

Authors identified cases of omphalocele through case registries of women who attended the clinic. Presumably, women attended the clinic either routine screening for genetic anomalies in the pregnancy, due to family history of genetic abnormalities, o r prior pregnancy history. Therefore, it is possible a selection bias may have occurred with this

method of case ascertainment if attendance atthe clinic is also related to a factor related to atrazine exposure, i.e. , one must have had access to genetic sc reening via ultra

- sound to be identified as a case in this study. In this study, the authors identified no significant differences in maternal age between cases and controls.

Use of season of conception as a measure of atrazine exposure includes many in herent assumptions including, but not limited to, the degree to which season correlates with atrazine exposure, whether the individual women would be exposed to atrazine in this season (source of drinking water, recreational water use during summer months in this region of northern Germany). Information bias due to exposure misclassification is possible, however use of broad categories of exposure (summer v. winter) likely lessens the magnitude of the bias.

Overall, this study offers limited additional inf ormation with regard to the association between atrazine exposure and prevalence of omphalocele, given a ban in use of the chemical was in place during the time period of the study. Given a ban to atrazine use was in place during the time period of this st udy, the magnitude and range of atrazine environmental concentration was likely limited among the participants of this study.

B.2.7 Klucinski et al. ―Humoral and cellular immunity rates in chemical plant workers producing dust pesticides,‖ [Med Sci Moni t; 7(6): 1270

- 1274 (2001)].Do not cite

Appendix B2

- Page 341 of 677 Background : Authors investigated whether impaired respiratory health was due to a change in hummoral and cellular immunity as well as a change in lymphocyte composition. The study investigated a possible biological mechanism to explain the findings reported by Kossman (1997).

Methods : Authors compared biomarkers of immune system functioning between individuals exposed to pesticides and those who were not. Authors selected a sample of pesticide manufacturing employees to represe nt the exposed series, and compared presence of biomarkers of immune response among individuals from the surrounding community. Participants reported smoking and chronic bronchitis. The study was performed in a community in Poland.

At the manufacturing plant from which the exposed series was selected, pesticide manufactured included: triazines, carbamate, cabendazim, captan, dodine, 2,4

- D, among others. Exposure was measured using ―air dust‖ concentrations.

Biomarkers of immune system functioning measur ed in this study included IgA, CD3,4,8,1 and, CIC.

Statistical comparisons were made using simple t

- tests.

Results: Authors reported several differences in immune parameters between the exposed and unexposed series sampled in this study. Generally, resu lts indicated immune functioning was compromised among those in the exposed series for some, but not all,

biomarkers measured.

EPA Critique:

There are several limitations to this study as presented. For the purposes of this literature review, the most no table is the lack of the ability to isolate exposure to atrazine, or triazine, among the several pesticides that were manufactured at this plant. The use of population based unexposed series may or may not have reflected a truly unexposed series, if

enviro nmental contamination was a possibility.

Given the limitations of the exposure assessment, this evaluation offers limited additional information to consider with regard to atrazine toxicity, although as research into the association between atrazine expos ure and respiratory health effects progresses, use of these immune system biomarkers may prove useful to future epidemiologic analyses.Do not cite

Appendix B2

- Page 342 of 677 B.2.8 Kossman et al. ―Role of respiratory muscles in the impairment of the respiratory system function in the workers of a chemical plant division producing pesticides,‖ [Przeglas lekarski, 54(10): 702

- 6 (1997)].

Background: Authors evaluated pulmonary ventilation and muscular force among chemical plant workers engaged in producing ―dust and liquid pesticides‖ including triazines, carbendazim, 2,4

- D, captan, carboxin, and solvents.

Methods : Authors selected male and female employees of the chemical plant to compare respiratory function. In general, the male participants were considered more exposed to pesticides than t he female participants. The female participants were largely employed in pesticide packaging and not pesticide manufacture. Researchers compared measures of respiratory health between male and female chemical plant workers and a selected sample

of communit y residents to serve as ―healthy controls.‖ Authors used the t

- test and a comparison of median level of the respiratory markers to evaluate whether an association between pesticide manufacture and respiratory impairment is probable. Thirty

- seven male parti cipants and 17 female participants from the chemical plant, and 22 male and 15 female residents of the surrounding community were selected into this study.

Results : Exposed (male) and unexposed (female) participants were comparable with
respect to most sp irometry function tests, with the exception of peak expiratory volume (PEV) and forced expiratory volume (FEV). Maximum inspiratory respiratory volume was significantly lower than the community

- based controls. Researchers report that chemical plant workers more frequently exhibited respiratory function measures beyond the normal

range.

Author conclusion : Authors conclude that this study provides some evidence that employment in a chemical plant producing pesticides, including triazines among many other typ es, may result in impaired respiratory function.

EPA Critique :

There are several limitations to this study as presented. For the purposes of this literature review, the most notable is the lack of the ability to isolate exposure to atrazine, or triazine, among the several pesticides that were manufactured at this plant. The use of population based unexposed series may or may not have reflected a truly unexposed series, if environmental contamination was a possibility.

Given the limitations of the exposur e assessment, this evaluation offers limited additional information to consider with regard to atrazine toxicity, although on a limited basis, this information can be considered in context of other epidemiologic research concerning pesticide exposure, incl uding atrazine, and respiratory health. Given the lack of specificityDo not cite

Appendix B2

- Page 343 of 677 in the pesticide exposure assessment, use of community unexposed series, and lack of control for confounding factors including smoking, these results offer limited additional

evidence an d should be interpreted with caution.

B.2.9 Mattix K.D., Winchester P.D., and Scherer L.R. ―Incidence of abdominal wall defects is related to surface water atrazine and nitrate levels,‖ [ Journal of Pediatric Surgery. 42, 947

- 949 (2007)].

Background: M attix, Winchester, and Scherer (2007) conducted an ecologic study to describe and compare the rates of congenital abdominal wall defects (AWD) in the U.S., the state of Indiana and the Midwest (defined as Indiana, Illinois, Iowa, Ohio and Nebraska), and to estimate the correlation between the AWD rates in the state of IN and

surface water atrazine and nitrate levels in the Midwest.

Methods: The national rate of AWD was determined using the Centers for Disease Control and Prevention‘s (CDC) National Natalit y Dataset for the years 1990 and 1995

- 2002; US CDC natality datasets were not available for the years 1991

- 1994, according to the authors. Comparison of AWD rates across geographic regions were made using the same time frames. Similarly, the incidence of omphalocele and gastroschisis in Indiana was determined using birth registry data collected by the Indiana State Department of Health

from 1990

- 2002. The investigators then characterized potential population

- level exposure to atrazine and nitrates during 1990

- 2002 using surface water monitoring data from U.S. Geologic Service (USGS). The USGS surface water data were not only collected from Indiana but also represented the Midwest region of the U.S., which was defined as Indiana, Illinois, Iowa, and Nebras ka.

The investigators performed a descriptive analysis comparing the rate of AWD in Indiana, the Midwest, and the U.S. A chi

- squared test was then used to statistically compare the annual rate of AWD in Indiana with the Midwest and U.S. rates. Lastly, t he investigators evaluated the correlation between the monthly rate of AWD in IN using state birth registry data with monthly mean estimates of surface water levels of atrazine and nitrate in the Midwest. A descriptive analysis was performed and Pearson c orrelation coefficients were calculated to evaluate the linear association between the monthly AWD rate in IN and mean monthly atrazine and nitrate levels in the midwest.

Results: The annual rate of AWD in Indiana was generally higher than the entire Midw est and U.S., using CDC natality records for identifying birth defects for the years 1990 and

1995

- 2002 combined. For the years 1996, 1998, 2001, the annual rate of AWD in Indiana was reported to be significantly higher than the U.S. rate. Similarly, auth ors reported that the annual rate of AWD in Indiana was significantly higher than the rate in the entire

Midwest in 1998.Do not cite

Appendix B2

- Page 344 of 677 Based on their descriptive analysis, the monthly AWD rate in IN based upon estimated date of conception (using LMP or other measure) in Indiana and surface levels of atrazine and nitrate in the Midwest were reported to follow similar seasonal patterns and exhibited peak values during the month of June. While both atrazine and nitrate surface water levels

followed somewhat similar visua l patterns, only the associations between the monthly AWD rate and atrazine levels was reported to be statistically significant. This correlation was observed when using both the CDC national natality data (0.60 Pearson correlation coefficient, p

- value = 0.0392) and Indiana state registry data (0.69 Pearson correlation coefficient, p

- value = 0.0125).

EPA Critique:

Mattix, Winchester, and Scherer (2007) performed descriptive analyses of ADW trends in both Indiana and the U.S. These descriptive analyses provide information on the burden of AWD in Indiana relative to the Midwest and to the entire U.S., as well as evidence that both

ADW rates and atrazine levels in Midwest surface waters follow the same seasonal trend. While the study provides useful descr iptive information and has been used to identify hypotheses to test in other studies, given the design the study holds limited utility in risk

assessment.

Researchers used the CDC natality dataset which included reports of birth defects to compare AWD ra tes between the U.S., the Midwest region and the state of IN for the years

1990 and 1995

- 2002. It is appropriate to compare across similar datasets and similar time periods, however, authors did not comment as to the quality of US natality data. Specifical ly, there may be differences between states in the quality of birth record reporting, and those differences may also relate to potential risk factors for birth defects leading to potential information and selection bias. In addition, as evidenced by the co mparison between the number of cases reported to the Indiana neonatal tertiary care facility and the IN state birth records, under

- reporting of AWD exists in these data.

Authors measured atrazine and nitrate concentration in surface waters using USGS wat er monitoring data which likely leads to some exposure measurement error in the measurement of drinking water atrazine concentration. In addition, authors compared AWD monthly rates in IN using state birth registry data with average atrazine concentration in surface water for the entire Midwest region. A more relevant comparison which would more closely measure the association of interest would have been to use state of IN USGS atrazine concentration data; authors provided no explanation as to why total Mid west USGS data records were used instead. Authors were not able to investigate individual level confounders (e.g. demographic and behavioral risk factors) in this analysis given the limitations of the study design.Do not cite

Appendix B2

- Page 345 of 677 The major limitation of this study is t hat it is not known whether women whose infants were born with this birth defect were actually exposed to atrazine in drinking water or via another relevant exposure pathway. While other non

- causal explanations for the correlation

observed between atrazine concentration in surface with and month of conception of fetus with AWD cannot be excluded based upon these data, this study has spurred other work using different study designs to inform the true nature of the potential association between atrazine expos ure and AWD.

B.2.10 Munger et al. ―Intrauterine Growth Retardation in Iowa Communities

with Herbicide

- contaminated Drinking Water Supplies,‖ [Env Health Perspec. 105: 308

- 314 (1997)].

Background : Authors investigated the hypothesis that neonates born to mothers residing in areas with high pesticide contaminant levels measured in finished drinking water have greater probability of intra

- uterine growth retardation (IUGR), low birth weight and prematurity than neonates born to mothers residing in areas of l esser pesticide contaminant levels in finished drinking water.

Methods: Using an ecologic study design, authors compared rates of IUGR, low birth weight and prematurity between areas of southern IA with different sources of drinking water. In previous wor k, finished drinking water samples in the Rathbun regional water authority (RRWA) were measure to have high concentration of triazine herbicides. Therefore, authors compared rates between RRWA and other communities in southern Iowa. Comparison groups were defined using both political boundaries and primary source of drinking water for each community in the region and included: the RRWA; all communities in Rathbun County not served by the RRWA; communities served by surface water drinking water supplies othe r than those in Rathbun county; communities served primarily by groundwater drinking water sources from alluvial and glacial drift; and communities served by primarily groundwater from bedrock aquifers drinking water sources. Authors assigned drinking wate r source according to the type of source delivered to the majority (>75%) of households in the community served, therefore comparison groups were mutually exclusive. Concentration of 35 different compounds including some pesticides were measured in each of the 856 municipal water supplies in the state. Researchers applied knowledge of drinking water finishing practices such as chlorination from records of the USGS. Concentration of pesticides and other contaminants in drinking

water measured at the communit y

- level were assigned to each female in the study according to her residence at the time of birth.

Authors studied certain birth characteristics including low birth weight (<2500 grams), premature birth (<37 weeks at delivery), and intrauterine growth ret ardation (<10 th
percentile for gestational age using the state of CA birth records for non

- Hispanic whites). Some births were included in more than one type of outcome. In this ecologic study,Do not cite

Appendix B2

- Page 346 of 677 information regarding demographic characteristics including pot entially confounding factors, was obtained through both the 1980 U.S. Census and available birth records. Authors calculated the percentage of mothers within each comparison group with certain characteristics such as high school degree, percent who report smoking during pregnancy and included these aggregate level variables in statistical models. Age

- adjustment was accomplished by including the age distribution of mothers recorded at birth in the state in 5

- year age groups. Authors attempted to control for differences in demographic characteristics between mothers who reside in northern versus southern Iowa by restricting the sample to communities in the southern part of the state.

Authors included non

- Hispanic white neonates only in this study because th e number of

non

- White neonates was too small for a robust analysis. Researchers excluded multiple births as there are well understood reasons for low birth weight among multiples. Births for which gestational age was not recorded in the birth records were also excluded.

Authors calculated rate of birth characteristics of interest by comparing the number of affected births in the community/comparison group to the total number of live births in the community/comparison group. To measure the association betw een pesticide contamination of drinking water and the same birth characteristics, authors utilized multiple linear regression of the rate of IUGR, low birth weight and premature birth, age

- adjusted, on drinking water contaminant levels, controlling for var ious potentially confounding factors measured at the community

- level.

Results : Regarding pesticide contamination of drinking water, the communities served within the RRWA had the greatest levels of atrazine and cyanazine as compared to the other community groups in the study. Mean (and median) atrazine levels were approximately 2 mg/L, and mean (and median) cyanazine levels were 1.4 mg/L. All communities sampled in the statewide survey reported measurable/detectable levels of

both herbicides. In the RRWA c ommunities, the percentage of live births in which IUGR was identified was 11.2, while authors reported that the other comparison communities‘ percentage of IUGR was less than 7%. Differences in the percentage of low birth weight and premature birth across drinking water comparison communities were not notable. To estimate the association between specific drinking water contaminants including atrazine, and IUGR (the only birth characteristic observed to be different between RRWA and other groups of communit ies), authors using linear regression modeling to estimate the change in the rate of IUGR by certain predictors including atrazine and cyanazine. Significant predictors were determined through stepwise regression methods. Models including atrazine and cyan azine (separately) as well as percentage of birth records missing date of LMP (as a measure of accuracy of birth record reporting) and percentage of maternal smoking reported among births in the community explained the greatest proportion of the

variabilit y in the model (R 2 0.19 and 0.16, respectively). Final models indicate the rate of

age

- adjusted IUGR changes 25

- 30% for each unit increase in atrazine or cyanazineDo not cite

Appendix B2

- Page 347 of 677 concentration (mg/L) in the finished drinking water (p

- value 0.001 and 0.009), respectively, adjusting for the percent of records in the communities with date of LMP and for percent of mothers who reported smoking during pregnancy.

Author‘s Conclusions : Authors state that their work illustrates that atrazine (and other triazines) are greater in the RRWA than other communities in southern Iowa, and the percentage of IUGR reported in RRWA is greater than the other communities in the region. Linear regression results observe indicate atrazine and cyanazine are among the most significant predictors of IUGR in this study, in addition to reporting of date of LMP and maternal smoking during pregnancy. LMP reporting was associated with a greater change in the regression coefficient than cyanazine in that model.

Researcher state that strengths of their study include use of comprehensive, stratified sampling of statewide drinking water sources in which pesticide, and other contaminant, levels were measured with accuracy (at the time). Of course, the use of an ecologic study design precludes any type of c ausal inference based upon this study alone; it is not known with certainty whether women who gave birth to neonates with measured IUGR had the higher levels of atrazine or cyanazine in their drinking water. Therefore, authors encourage replication. In ad dition, although authors attempted to handle potential confounding through both restriction and adjustment using group level variables, unmeasured or residual confounding cannot be ruled out as an explanation for the linear regression model results.
Statis tical adjustment for confounding variables at the group level is not always advisable (CITATAION: ROTHMAN TEXT). In addition, use of maternal residence reported by mothers at birth as a surrogate for in utero exposure, inability to include all sources of

d rinking water in the exposure measure, and potentially inaccurate methods of measuring the outcome IUGR (head circumference is a superior method), are all limitations to the study. Authors also state they did not have information concerning other co

- morbi d conditions which may affect both drinking water consumption and IUGR, the ability to examine the correlated nature of many drinking water contaminants in their analysis, or the ability to assess the influence of different water treatment methods.

EPA C ritique:

Authors performed an ecologic study to determine whether concentration of triazine herbicides in drinking water were correlated with proportion of certain birth characteristics including IUGR in a sample of communities in southern Iowa. Given th e limitations of this study design, authors were able to determine there is an apparent correlation between this exposure and outcome, however, as acknowledged by the authors, additional research using alternative study designs which measure exposure and o utcome at the individual, rather than the group, level are needed.Do not cite

Appendix B2

- Page 348 of 677 Authors discuss the limitations of the study design, as well as the exposure assessment and outcome ascertainment methods, and EPA concurs with their assessment. Conclusions from ecologic studies can be useful for additional study hypotheses, however results cannot be used to determine whether triazine/atrazine exposure is (causally) associated with birth characteristics such as IUGR. In addition, the specific inclusion and

exclusion crite ria applied to the births selected for this study resulted in a sample that

reflected non

- Hispanic White residents who lived in areas served by municipal water supply (v. rural well water). . Based upon the results of Table 1, the concentration of triazine herbicides appears different in RRWA in comparison to the other groups included in the study, enhancing the statistical power to detect statistically significant changes. Also, the use of drinking water data collected in a survey conducted 1986

- 87 to est imate concentration of contaminants in the drinking water of mothers who gave birth 1984

- 1990, the period of this study, relies upon the assumption that the relative rankings of the communities based upon the 1986

- 87 survey is the same throughout the perio d of the study.

The study does illustrate that group with the greatest atrazine concentration in drinking water also experiences the greatest proportion of IUGR births, and atrazine and cyanazine were significant predictors of the rate of IUGR in linear regression analyses. Results of this study must be considered in context of the study design, and therefore has limited utility in

risk assessment.

B.2.11 Ochoa

- Acuña H., Frankenberger J., Hahn L., and Carbajo C. ―Drinking water herbicide exposure in Ind iana and prevalence of small

- for

gestational

- age and preterm delivery,‖ [Environmental Health Perspectives. 117(10): 1619

- 1624 (2009)].

Background: Ochoa

- Acuña et al. (2009) conducted a retrospective study to assess the association between atrazine exposu re and the prevalence of preterm delivery and SGA births among women in Indiana.

Methods: The investigators restricted their study area to community water systems in urban areas that had well defined service boundaries and sufficient drinking water data t o re

construct atrazine concentrations over the exposure periods of interest. Based on these restrictions, the investigators identified 19 community water systems with sufficient atrazine monitoring data. Infant and maternal characteristics and health ou tcome information was then identified using the Indiana Birth Record Database. Mother‘s residence at the time of delivery was used to match each mother to a specific community water system within the state. A spatial analysis was then performed to link bi rth records to the specific community water systems that supplied water to the maternal address. Based on this approach, a total of 25,154 births were linked to one of the 19 community water systems with atrazine

concentration data. The number of births within each community water system variedDo not cite

Appendix B2

- Page 349 of 677 considerably based on the populations served and number of years of atrazine data, although roughly 70% of the birth records came from the Ft. Wayne community water system.

The investigators then classified atrazi ne exposure using water monitoring data. In Indiana, drinking water monitoring data were available through four data sources: (1) Safe Drinking Water Act IDEM system quarterly measurements; (2) Acetochlor Registration Partnership (Hackett et al. 2005); (3 ) Novartis Atrazine Public Water System Voluntary Monitoring (Tierney et al. 1999); and (4) Atrazine and Simazine Re

- registration Program (U.S. EPA, 2006). Using these data sources, the investigators re

- constructed average (monthly) atrazine concentration s in finished drinking water by interpolating atrazine

concentrations between the 7

- 14 day sampling intervals in the summer months, and 1

- 6 month sampling intervals in the winter months. These re

- constructed atrazine concentration profiles were then averag ed over the exposure period of interest to estimate exposure (i.e., first month of pregnancy, last month of pregnancy, third trimester, or entire pregnancy).

The prevalence of preterm delivery and SGA were compared between mothers who resided in community water systems with low, medium or high concentration of atrazine in drinking water; categories were defined using the <25th, 25

- 75th, >75th percentiles of the continuous distribution of atrazine concentration distribution during either the first month,

la st month, last trimester, or entire pregnancy period, depending on the research question. The investigators then used a log

- binomial model to calculate prevalence ratios after adjusting for several potential confounders, including various maternal and infa nt demographics characteristics, behavioral risk factors, and potential seasonal patterns in pregnancy outcomes.

Results: The investigators reported that there was an association between atrazine concentration in drinking water and the prevalence of small

- for

- gestation age infants, but not for preterm delivery. When the third trimester was used as the period of exposure, the reported prevalence of SGA in the medium and high exposure groups were 19% and 17% higher than the prevalence reported for the low e xposure group, respectively. Similarly, when entire pregnancy period was used as the exposure period, the reported prevalence of SGA in the high exposure group was 14% higher than the low exposure group (Adjusted prevalence ratio = 1.14, 95% CI: 1.03, 1.2 4).

EPA Critique:

The investigators conducted a well

- designed epidemiologic investigation on atrazine and the birth outcomes preterm delivery and SGA. The study shares many similarities with Villanueva et al., 2005 (see below), and utilized atrazine mon itoring data that allowed the investigators to perform a robust statistical analysis. Additionally, the study was able toDo not cite

Appendix B2

- Page 350 of 677 adjust for individual

- level potential confounders, and considered seasonal trends in adverse

birth outcomes.

Although the study use d a more refined measure of exposure than the previous studies, additional information regarding the method of interpolation of the atrazine drinking water concentrations could be helpful to fully understand the study‘s results. For example, the

statistic al error involved in interpolation of average monthly concentrations of atrazine in drinking water using measurements 7

- 14 days apart in the spring and summer months is likely different than that involved in attempting to interpolate monthly atrazine

conce ntrations in drinking water between measurements taken 1

- 6 months apart in the winter months. While the possible bias due to misclassification due to interpolation may be low, especially in the assignment of low, medium, high exposure categories, the

inve stigators did not comment as to the impact of the varying data quality between spring/summer and fall/winter. In addition, investigators did not comment on the quality and comparability of the four drinking water monitoring programs utilized to re

- constru ct atrazine drinking water concentration over the study period. Ideally, a comparison of detection methods, percent coefficient of variability between and within the separate programs, limit of detection, and other measures of quality control would be sum marized.

In this study, authors were able to adjust for individual level confounding variables including season of year. However, controlling for season may have also inadvertently controlled for the main exposure variable. Additional sensitivity analysis and report of the change in the

effect estimate with and without this variable would have been illustrative.

B.2.12 Ochoa

- Acuña H., Carbajo C. ―Risk of limb birth defects and mother’s home proximity to cornfields,‖ [ Science of the Total Environment . 4 07:

4447

- 4451 (2009)].

Background : Ochoa

- Acuña and Carbajo (2009) conducted a retrospective cohort study to assess the relation between birth defects and maternal home proximity to corn and soy

fields.

Methods: The study population was identified using t he Indiana Birth Records Database, which includes data on maternal demographics, behavioral risk factors, and health outcomes of infants delivered in Indiana. Using the Indiana Birth Records Database, the investigators defined their study population as (1 ) mothers living in rural geographic boundaries based on the 2000 U.S. Census who had (2) singleton births conceived between May 1 and August 31 of the years 2000 through 2004. Based on these selection criteria, 48,216 births were included in the study po pulation.

Births defect cases were then identified using standardized birth anomaly disease codes as reported to the IN birth registry, and exposure levels were assigned to each pregnancyDo not cite

Appendix B2

- Page 351 of 677 using maternal home proximity to land with corn and soy fields as a n indicator of agrichemical exposure including atrazine. To estimate maternal home proximity to corn and soy fields, the investigators performed a spatial analysis of maternal address data and land crop cover data collected by the U.S. Department of Agric ulture. Using this spatial analysis, the investigators classified the exposure of each birth record in their study population two ways. First, they created dichotomous variables in which births were defined as ―exposed‖ to a given crop (corn or soy) if t he planted crop area near a maternal address exceeded the median planted crop area for the entire study population. Second, they created continuous exposure variables by utilizing their spatial analysis to develop estimates of exposure expressed in terms of areas of corn and soy that were within 500

meters of each maternal address.

The investigators performed two separate statistical analyses to assess whether there were associations between birth defects and their dichotomous and continuous exposure

vari ables, respectively. In both statistical analyses, multivariate logistic regression models were used to calculate birth defect odds ratios, after controlling for potential demographic and maternal risk factors, including age, race, education, prenatal car e, smoking, use of alcohol during pregnancy, and health status during pregnancy.

Results: Based on the analysis that utilized dichotomous exposure variables for corn and soy fields, the authors reported that a higher number of birth defect cases were gene rally observed in the ―exposed‖ groups for both corn and soybeans. However, the only statistically significant association was between limb birth defects and exposure to corn fields (Odds Ratio = 1.76, 95% CI: 1.12, 2.78). Elevated odds of neural tube de fects (NTD), abdominal cavity and craniofacial defects were also observed, however several of these estimates were based upon a small number of cases. In association with proximity to soy fields, elevated odds of craniofacial and NTD were also observed; th e estimate for NTD was based upon only 9 cases. When maternal home proximity to corn and soy fields were treated as continuous exposure variables, a similar pattern of associations was also identified. In particular, it was reported that there was a 22% i ncrease in the risk of limb birth defects per every 10 ha increase in planted area with corn (Odds Ratio = 1.22, 95% CI: 1.01, 1.47). Borderline statistically significant association was also observed for NTD in association with percent of area agricultura l in proximity to maternal residence (OR 1.72,

95% CI 0.98

- .032).

Author‘s Conclusion : Researchers conclude that their study provides evidence that proximity to corn and soybean fields may be associated with adverse birth outcomes, specifically limb defec ts, although other birth defects were also elevated among women who resided in close proximity to treated fields.

EPA Critique:Do not cite

Appendix B2

- Page 352 of 677 The ability to estimate individual

- level exposure was a strength of the study; however, the use of a proximity to corn fields is only a surrogate of potential chemical exposure and was not validated in this study. Without actual exposure information, it is difficult to link the potential increase in limb birth defects or other birth defects to atrazine or any other specific

agr ichemical. The investigators address this issue in their discussion and suggest that exposure to agrichemicals applied preferentially to corn, including higher quantities of the fertilizer nutrients nitrogen, phosphorous, potassium and atrazine, may be al ternative explanations of their findings. Therefore, it would be beneficial to collect environmental or human exposure data in follow

- up research to validate that proximity to corn fields is associated with increased chemical exposure.

In this study, aut hors focused upon the statistically significant association between proximity to cornfields and limb defects, however other potentially biologically significant links were observed; additional follow

- up studies including mechanistic studies may be

needed t o replicate these findings. Specifically, non

- significant but elevated odds of abdominal cavity defects were observed in association with proximity to cornfields. In addition, 25

- 50% increased odds of craniofacial defects and NTD were also observed in

asso ciation with proximity to both corn and soybean fields. Results should be interpreted

with caution as there were a small number of cases in several of these birth defect categories, and several comparisons were made. In addition, the heterogeneity of birth defects reflected within the groups analyzed herein may be significant, indicating different etiologies, if a true association exists. With regard to adjustment for ―other farm exposures,‖ authors state this decision was predicated on an interest in contr olling for residual confounding, however it may also be true that analysts inadvertently adjusted for their main exposure variable in the analysis, thus attenuating the risk (odds) estimated presented. Stratification by presence of farm exposure may have b een illustrative. Overall, given the relatively small number of cases in some of these birth defect categories and the number of statistical tests performed, chance cannot be excluded for some or all of these findings.

B. 2.13 Saldena et al. ―Pesticide Exposure and Self

- Reported Gestational Diabetes Mellitus in the Agricultural Health Study,‖ [Diabetes Care

30:529

– 534 (2007)].

Background: Women with greater probability of pesticide exposure during the first trimester of pregnancy are more likely to repo rt gestational diabetes mellitus (GDM) than women with no reported pesticide exposure during the first trimester of pregnancy.

Methods: Using a cross

- sectional study design, authors collected information regarding pesticide use and gestational diabetes we re ascertained at enrollment into the AHS (1993

- 1997). Authors identified cases of GDM among eligible pregnancies during the index pregnancy, the most recent pregnancy at the time of enrollment. Information regarding potentially confounding variables was a lso obtained through self

- report questionnairesDo not cite

Appendix B2

- Page 353 of 677 administered at enrollment. Authors applied certain inclusion and exclusion including pregnancies which would require extreme periods of recall among participants (more than 25 years in the past), pregnancies among very young or older participants which may be at high risk for other reasons, and prevalent diabetes (non

- GDM). In all, there were 11,273 pregnancies included in the study. Authors used self

- reported cases GDM provided by female spouses of pesticide applicators in the AHS to define cases of GDM.

To measure pesticide exposure, authors defined four categories of pesticide use based upon participant responses to questions regarding pesticide use during the first trimester of the most recent (index) pre gnancy. The four categories included: 1) no exposure, 2) indirect exposure (pruning, planting activities in which some pesticide exposure may be possible), 3) residential application of pesticides, and, 4) mixing or loading of pesticides or repair of pesti cide related equipment in an agricultural setting. Authors assumed that spouses who reported mixing and loading pesticides during pregnancy had the greatest potential for pesticide exposure. Authors state if a woman reported activities related to

more than one pesticide exposure category during the first trimester of pregnancy, she was categorized according to the most common pesticide use activity she reported; therefore, pesticide exposure categories were mutually exclusive.

To evaluate associations bet ween GDM and specific pesticide use, author utilized information regarding ever

- use of 50 specific pesticides during the lifetime among women who reported directly engaging in agricultural practice, i.e. , not necessarily during the first trimester of pregn ancy. However, authors only reported pesticide

- specific results for those

substances for a priori hypotheses were proposed or for which there were at least 5 pesticide exposed cases of GDM in each exposure category.

Authors modeled the association between GDM and pesticide use (none, indirect, residential direct, and agricultural direct) during the first trimester of pregnancy, as well as

ever

- use of specific pesticides reported during a woman‘s lifetime among women who reported direct use of agricultural pesticides during the first trimester of the index pregnancy using unconditional logistic regression. Statistical models were adjusted for body mass index (BMI) reported at enrollment (and not at the time of pregnancy), maternal age at delivery, parity, ra ce and state (IA or NC). In the analysis of ever

- use of specific pesticides during the lifetime with GDM, authors also adjusted for the five most common pesticides reported by women in the AHS who reported applying pesticides: glyphosate, carbaryl, malathi on, 2,4

- D, and diazinon.

Results: Authors reported that compared to women who reported no pesticide use during the first trimester of pregnancy, women who reported engaging directly in agricultural practice were observed to have a 2

- fold increase prevalen ce of GDM (odds ratio (OR) 2.2, 95% confidence interval (95% CI) 1.5, 3.3, p<0.0001). Authors observed no association between women who reported either indirect or direct residential pesticide use during theDo not cite

Appendix B2

- Page 354 of 677 first trimester of the index pregnancy and preva lent GDM. When authors restricted the analysis to pregnancies within 12 years of enrollment, no difference in the estimated

association was observed. Ever

- use of any pesticide during the lifetime among women directly engaged in agricultural pesticide appli cation was not associated with GDM.

Authors investigated ever

- use of 15 specific pesticides for which there was a specific a priori hypothesis regarding GDM or for which there were at least 15 exposed GDM cases available for analysis. In this sub

- analysi s, ever

- use of atrazine during a women‘s lifetime was statistically significantly associated with GDM [actual odds ratio and confidence interval not specifically reported]; other pesticide

- GDM associations were also reported.

Author‘s Conclusion: Researc hers conclude there is evidence that direct, agricultural pesticide exposure during the first trimester of pregnancy may be associated with GD, however authors acknowledge limitation of their ability to measure specific pesticide exposure, such as atrazine , during this period as a limitation to the current study. Atrazine use was not included among the a priori hypotheses posited, however the observation of a significant association is notable, and warrants replication.

EPA Critique:

Authors performed a w ell

- conducted hypothesis

- generating study regarding the potential association between pesticide use during the first trimester of pregnancy and prevalent GDM. EPA notes neither atrazine nor triazine use was previously hypothesized to be related to GDM; the refore, the observed association, while not statistically significant and based upon small number of cases, is nevertheless notable. However, the role of chance cannot be eliminated. Regarding atrazine specifically, the study offers limited evidence that

t here may be an association. This validity of this measurement is limited by the inability to match the time period of atrazine use (authors measured ever

- use over the lifetime) and the first trimester of pregnancy, and the small number of women who reporte d applying atrazine. Clearly, additional research is needed to replicate and expand upon these findings.

As acknowledged by the authors, the period of recall between questionnaire completion and the last pregnancy is lengthy, e.g. , up to 25 years for some participants. However, when restricting the analysis to more recent pregnancies (within 12 years of enrollment; 50% reduction in period of recall), authors state the results did not change. Authors state that reporting/recall bias is not likely because t he outcome, GDM, was not the focus of the questionnaire, therefore respondents with GDM would not likely to be likely to respond differently to questions regarding pesticide use than respondents with no reported GDM. In addition, the observation of an asso ciation only among women who report agricultural use of pesticides, and not all pesticide users, support that reporting bias is not likely, i.e., ifDo not cite

Appendix B2

- Page 355 of 677 reporting bias were likely observed associations across pesticide exposure categories would be expected.

Authors have contributed to the scientific literature with regard to the possible role pesticide exposure may play in the etiology of GDM. However, additional research is necessary to better understand the possible role of atrazine in this relation.

B.2.1 4 Sathyanarayana et al. ―Maternal Pesticide Use and Birth Weight in the Agricultural Health Study,‖ [J. Agromed, 15: 2, 127

- 136. (2010)].

Background : Authors investigated the relation between maternal pesticide exposure and infant birth weight among a se ries of female participants in the Agricultural Health Study (AHS).

Methods: Among 9,202 women who reported a pregnancy that ended in a live birth between 1975 and enrollment into the AHS, the authors restricted their analysis to pregnancies which occurre d within 5 years of enrollment to increase the probability that the pesticide exposure occurred prior to that pregnancy. Other exclusion criteria were applied including women who had other co

- morbid conditions that may have affected infant birth weight suc h as diabetes. Women self

- reported infant birth weight. Information concerning pesticide exposure during pregnancy, overall lifetime pesticide use, and other potentially confounding factors was ascertained through self

- administered questionnaire. There wer e 2,246 women included in the study.

Using the same method as Saldena et al. (2007), authors classified first trimester pesticide use in the following manner: no exposure, indirect agricultural exposure ( e.g. , picking or pruning), direct residential use, or direct agricultural application. No information regarding use of specific pesticides during the first trimester, or any other period, was available. Authors compared lifetime ever

- use of specific pesticides and infant birth weight for those pesticides f or which at least 20 women reported use.

Authors used multiple linear regression to estimate the change in birth weight in association with pesticide exposure category, controlling for pregnancy duration, maternal pre

pregnancy body mass index (BMI), mat ernal height, parity, state, and maternal smoking.

Results : Among this sample, seven percent of women reported preterm delivery and three percent report low birth weight infant (<2500 grams). Researchers observed no association between any pesticide expo sure category and change in birth weight, including direct, agricultural exposure. However, the beta

- coefficient for the change in infant birth weight comparing women who reported direct agricultural exposure during the first trimester to those who reporte d no pesticide exposure was the greatest among the pesticide categories, a reduction of 0.72 grams (95% confidence interval

- 272, 79). Examining everDo not cite

Appendix B2

- Page 356 of 677 use of several pesticides during the lifetime and infant birth weight, authors observed a statistically si gnificant change in infant birth weight in association with ever use of carbaryl. In association with ever use of atrazine, researchers observed a 39 gram reduction (95%

CI:

- 171, 92) in infant birth weight.

Author‘s conclusion : Researchers summarize that carbaryl use may be associated with a reduction in infant birth weight, but caution additional epidemiologic analyses are needed to confirm these initial findings. Authors did not observe an association between ever

- use of atrazine and change in birthweig ht.

EPA Critique:

Authors offer additional evidence regarding the potential association between pesticide use and infant birth weight. Strengths of this study include the reliability of maternal self

- report of infant birth weight, pesticide exposure asse ssment methodology which has been shown to provide reliable and valid measures of pesticide use, availability of relevant information concerning potentially confounding variables, and the relatively large sample size to

estimate the association. Authors we re not, however, able to measure specific pesticide usage during the first trimester of pregnancy, or any other relevant exposure window. Results suggest there is no association between pesticide use, including direct, agricultural pesticide use, during th e first trimester of pregnancy and infant birth weight. Among specific pesticides evaluated, use of carbaryl during the lifetime may be associated with infant birth weight. Regarding atrazine use, authors did not observe a significant change in infant birt h weight among women who used this pesticide during the lifetime. Overall, this is a well

designed exploratory study of this question and adds to our knowledge of this association. However, pesticide exposure measurement during the first trimester, or othe r relevant exposure window, would enhance utility of these data for risk assessment purposes.

B.2.15 Savitz et al. ―Male Pesticide Exposure and Pregnancy Outcome,‖ [ Am J Epidemiol ;146:1025

- 36 (1997)].

Background : Authors hypothesized that farm related a ctivities, including pesticide exposure, three months prior to conception among male partners enrolled in this study are associated with adverse birth outcomes including miscarriage, preterm delivery, small for gestational age, as well as secondary sex rat io.

Methods: Authors performed a nested case

- control analyses within the Ontario Farm Family Study. Pregnancies were identified for inclusion in the current study from the larger Ontario Farm Family Study, a study of reproductive outcomes among farm famil ies sampled from the 1986 Canadian census of Agriculture. Farm families were eligible for inclusion into the Ontario Farm Family study if families (male/female partners) were living on the farm year round, and included female partners 44 years of age of le ss (child

- bearingDo not cite

Appendix B2

- Page 357 of 677 age). Pregnancies were included in this study if the time period of the pregnancy could be determined; the time period of the pregnancy occurred while female living on the farm; and, the husband, or partner currently living on the farm at the time of the survey, was the male partner in the pregnancy. Researchers recorded information for preterm delivery, small for gestational age, miscarriage, and male:female sex ratio based on maternal self

- report.

Information regarding farm activities including pesticide usage was ascertained by paternal

self

- report. Upon enrollment, male partners were asked to report the common farm activities in which he engaged over the 5

- year period prior to enrollment, and the months of the year in which he perform ed the activity. Researchers assumed farm activities reported over this period accurately reflected farm activities during the entire period of living and working on the farm, including the time window 3 months prior to the index pregnancy. Exposure was de fined as having engaged in a potential pesticide related activity for at least 1 month in the three months prior to estimated conception. Authors also ascertainment whether participants used personal protective equipment (PPE) (date of initiation of use), and whether certain farm pesticides were used on the farm (as reported by the farm operatory who may or may not have been the paternal partner in the couple) during the 5 years prior to enrollment. Authors determined which farm activities would likely incl ude pesticide usage and categorized participants who reported these activities as potentially chemically exposed.

Therefore, to measure pesticide exposure, authors combined information regarding farm activities that would likely include pesticide exposur e such as ―mixing or applying crop herbicides‖ with reported pesticide usage on the farm during the same time period such as triazine or atrazine. However, given this method of exposure assessment, it is not known with certainty whether the farm activity r eported by the participant actually involved usage of that farm chemical. The exposure assessment methodology is probabilistic. Authors acknowledge that women living and working on the farm could have been exposed to these chemicals directly or indirectly, i.e., take home exposure. Authors cannot exclude the possibility that female pesticide exposure may account for the observed associations reported in the study.

Authors used logistic regression to estimate odds ratios (OR) and 95% confidence intervals (9 5% CI) for performance of certain pesticide related farm activities as well as use of specific pesticides in the performance of certain activities deemed by the authors to likely include use of pesticides, adjusting for certain relevant confounding variabl es. Authors did not additionally adjust or utilize statistical methods that are able to reflect intra

- person correlations to reflect the inclusion of more than one pregnancy per female study participant

because the affect of these correlations of the risk estimate was minimal according to the

authors.Do not cite

Appendix B2

- Page 358 of 677 Results: Sixty

- four percent of the eligible farms were included in the Ontario Farm Family study and 68% of pregnancies were included in this study. The period of recall with respect to both pregnancy and exp osure information was 5

- 15 years.

Authors observed that pesticide use was associated with miscarriage and preterm delivery, but not associated with small for gestation age or male:female sex ratio. The risk of miscarriage in association with reported use of crop herbicides, triazine and atrazine specifically was 1.4 (95% CI 1.0, 2.0), 1.4 (95% CI 0.9, 2.2), and 1.5 (95% CI 0.9, 2.4), respectively. The risk of preterm delivery in association with crop herbicide use, triazine use and atrazine use specifical ly was 1.4 (95% CI 0.6, 2.0), 1.5 (95% CI 0.6, 3.8), and 2.4 (95% CI 0.8, 7.0), respectively. Reported use of other pesticides in association with farm activities that likely include pesticide exposure were also statistically significantly

associated with both miscarriage (thiocarbamates and carbaryl) and preterm delivery 2,4

- DB.

Authors Conclusions: The authors conclude that their study illustrates possible association between male pesticide use three months prior to conception and adverse birth outcomes preterm delivery and miscarriage, although less evidence than preterm delivery. Authors suggest there is little evidence from this study that male pesticide use immediately prior to conception is associated with small

- for

- gestational age and male:female s ex ratio. Although estimates are not measured with precision, the researchers state that for the risk of preterm delivery appears greatest among male partners who reported triazine/atrazine use, and the risk of miscarriage appears greatest in association w ith thiocarbamate/carbaryl use.

EPA Critique :

Given the time period of this study, the early 1990‘s, and the advancements in pesticide exposure assessment within epidemiologic investigations which have taken place since that time, the current study is d ated and somewhat weak in terms of exposure assessment. The inability to directly link farm activities such as applying crop herbicides and use of specific herbicide such as atrazine within each participant is the major weakness of this study, as acknowled ged by the authors. This probabilistic exposure assessment likely resulted in accurate exposure assessment for some of the participants in the study, but the potential exposure misclassification due to this method of exposure assessment is significant. How ever, the misclassification would likely be non

- differential given that the male partners are reporting pesticide exposure and the female partners are reported pregnancy outcomes, most likely having attenuated the risk estimates presented. Female partner p esticide exposure was not measured in this study, and may also have contributed to the risk of adverse birth outcomes, as stated by the authors. Given that it is not known with certainty whether atrazine exposure did occur among men engaged in certain farm activities during the three months prior to conception, this study can be considered toDo not cite

Appendix B2

- Page 359 of 677 provide slightly suggestive qualitative evidence of an association between pre

- term delivery

and atrazine use..

Authors adequately addressed potential sources of bia s in their risk estimates, in addition to the information bias mentioned above. Participation rate was sufficient to discount a high probability of selection bias into the study (64%). Recall bias is unlikely, however not impossible, given male partners we re reporting pesticide exposure and female partners were reporting pregnancy outcomes. Temporal bias is not likely as pesticide exposure, was ascertained for the time period prior to conception and, therefore, prior to the adverse birth

outcome. Informatio n bias due to outcome misclassification is likely given the period of recall is significant (5

- 15 years), and estimating gestational age (to determine pre

- term and

small

- for

- gestational age outcomes) is challenging . However, this factor likely results in

non

- differential outcome misclassification and an attenuation of the risk estimate. Researchers identified relevant confounding factors and were conservative in the inclusion of these factors in the final statistical models, erring on the side of parsimony in their model development, which enhances statistical power. Evaluation of co

- exposure to other pesticides was not performed in this study and may be a source of unmeasured confounding if pesticide usage is correlated and more than one pesticide may be c ausally related to these birth outcomes, possibly via different biological mechanisms, e.g., direct testicular effect or interference with lutenizing hormore or follicle stimulating hormone. As a

nested case

- control study, authors the likelihood that the c ontrol series was derived from that same source population as the cases was enhanced.

Savitz et al encourage future research, since the time of publication [1997], regarding male pesticide exposure and sperm parameters. The study included in this review by Swan et al. (2003) in large part responds to this suggestion. While this study provides an important foundation for future work, in and of itself, the study provides only slightly suggestive, qualitative evidence that atrazine use may be associated with pre

- term delivery, and possibly miscarriage.

B.2.16 Swan et al. ―Semen Quality in relation to biomarkers of pesticide exposure,‖ [Environ Health Perspect 111:1478

– 1484 (2003)].

Background : Researchers hypothesized that men from the general population w ho reside in Missouri (MO), an area of high use of non

- persistent agricultural chemical use such as atrazine, have reduced semen quality than men from the general population who reside in Minneapolis, Minnesota (MN), an area of lower use of non

- persistent agricultural pesticides. Authors utilized two biomarkers to estimate this association: urinary concentration of pesticide metabolites and semen quality (concentration, motility, morphology).Do not cite

Appendix B2

- Page 360 of 677 Methods: Authors performed a case

- control analysis using cross

- s ectional data from the Study on Future Families cohort for 2 of the 4 study sites included in the cohort study, Columbia, MO and Minneapolis, MN. The SFF is a population

- based case

- control study including male, female and fetus trios recruited at four pre

- natal clinics around the country (NYC, LA, Columbia, and Minneapolis). For this study male partners recruited at the Columbia, MO and Minneapolis, MN study sites were compared with respect to semen parameters. Upon agreement to participate in the SFF, male partners of pre

- natal clinic patients completed a written questionnaire and provide blood, semen, and urine sample. The authors state that data collection methods were identical across the four study sites.

For purposes of the nested case control analys is planned in this study, authors defined a case as a man with sperm concentration below the total SFF population median, and a control as a man with sperm concentration above the population median. Using this definition, motility and morphology cases and controls likewise fell below and above the population medians.

Authors sought to exclude men from the study with risk factors for poor semen parameters, unrelated to the hypothesized role of pesticide use. Therefore, inclusion criteria were ages

21

- 40, w hite race, non

- smoker, no history of sexually transmitted infections, and no report of fever within 3 months of semen donation. Because too few men met these criteria, researchers elected to enrich the sample with additional participants who may not have m et these criteria. Authors provided characteristics of all participants with respect to risk factors for poor semen quality, and authors support that any bias introduced through the altering

the inclusion criteria would be non

- differential (bias toward the null). Authors enhanced statistical power of the study using this approach.

Pesticide Selection and Analysis: Initially researchers selected 15 non

- persistent pesticides with reported high use in MO and low use in MN for inclusion in this study; however , only 8 were included in the final analyses. Reasons for pesticide exclusion were greater use in MN than MO which was counter to the hypothesis, and no detectable analytes in participants. Urine samples were measured with good laboratory techniques ((CDC/ NCEH

- national center environmental health). Technicians were blinded to case status when making the pesticide measurement.

Semen Collection and Analysis: Male participants provided 2 semen samples, typically 24 days apart. Semen samples were analyzed within 45 minutes of collection. Authors adjusted for abstinence time in the analysis of semen parameters. Sperm concentration and motility were measured using standard practices. Determination of sperm morphology was made utilizing a WHO method

which assi gns a  ̳normal‘ rating to samples with absolutely no abnormalities. Authors did not provide any measure of reliability or validity of measures; however, cases and controlsDo not cite

Appendix B2

- Page 361 of 677 were similar with respect to abstinence time and other risk factors for poor semen qu ality. Authors analyzed both semen samples provided using a mixed, hierarchical model which accounts for multiple measures of the outcome within participants.

Statistical Analysis: Authors state they used a series of statistical analyses ranging from simp le non

- parametric tests to multiple variable adjusted mixed effect models in order to maximize use of the data collected to inform the study question. Analyses were appropriate to the research question, and model assumptions were met. In the analysis of sp erm parameters and pesticide concentration as continuous variables, authors state the distributions were not normally distributed even after log

- transformation. Due to this fact, authors consider this analysis exploratory only.

Authors handled potential confounding factors through both study design and statistical analysis. Authors excluded men with risk factors for poor semen parameters, with the exception noted above. In addition, due to the relatively small sample size and the risk of

over

- parameterizi ng their statistical model, authors utilized a confounder score to simultaneously adjust for the predicted value of all confounder

- exposure associations in the risk estimate. With respect to other pesticide exposure, authors compared percent of men who rep orted occupational use of pesticides and who reported living within one half

- mile of an agricultural field. In addition, to account for multiple pesticide exposure in association with semen parameters, authors devised an ad hoc high pesticide score which r eflected the number of pesticides for which a participant was in the ―high‖ category. Comparison of semen quality by score was made.

Results : Participants were predominantly white, non

- smokers, with few instances of sexually transmitted infections (STI) or a history of infertility, based upon the inclusion criteria. Approximately 25% of cases and controls from MO reported a recent pesticide exposure; however the proportions were comparable between cases and controls. Cases

and controls were similar with respect to abstinence time and time to analysis of sample, but differed significantly with respect to semen parameters. The concentration of the atrazine metabolite atrazine mercapturate is higher in this sample than reported in the NHANES Human Exposures report, as noted by the authors.

Authors reported that concentration of alachlor, atrazine and diazinon differed between cases and controls in MO, but not in MN based upon the non

- parametric Wilcoxan rank sum test. No significant differences in pesticide metabolite level was observed for metolachlor and 2,4

- D by study site. Samples provided in the Spring/Summer were more likely to be poor semen quality, i.e., to be a case. Men who reported recent pesticide use (n=16) were more likely to be a case and more likely to have more than one pesticide analyte in the  ̳high‘ category, i.e., a high score. For atrazine specifically, authors report that those with urinary metabolite levels above the LOD, the odds of poor semen parameters (ofDo not cite

Appendix B2

- Page 362 of 677 being a case) was 11.3 (95% CI 1.3, 98.9) in MO; estimates could not be calculated for the MN study site as there were no cases observed above the LOD. In an exploratory analysis modeling both atrazine mercapturate and semen parameters as continuous variables, authors reported atrazi ne exposure resulted in a greater change (beta

- estimate, slope of regression line) in sperm motility than sperm concentration or morphology, but a negative slope was observed in association with each measured semen parameter.

Author‘s Conclusion : The rese archers conclude that there is some evidence that exposure

to non

- persistent pesticide commonly used in agriculture in MO, but not in MN, including atrazine, alachlor and diazinon may be associated with poor semen parameters. With respect the atrazine, thi s association is elevated and statistically significant, although the confidence bounds are large, and in exploratory analyses, sperm motility is most affected by atrazine exposure. Authors support that exposures are likely environmental, as opposed

to occ upational or residential use, and suggest drinking water exposure as the likely source pathway. Researchers acknowledge that although the half

- life of these non

- persistent pesticides in the body is short (days), use of the chemicals in the Midwest is high and relatively consistent over the time period of this study, and conclude that both semen samples reflect influence of the same exposures.

EPA Critique:

Authors presented a well

- designed and conducted study with a hypothesis supported by previous lite rature with respect to persistent pesticides; this is the first evaluation of the question with respect to non

- persistent pesticides and represented novel research. The exploration of the association between environmental exposures such as pesticides and

m ale reproductive health is needed and fulfills a gap in the epidemiologic literature on this point.

Authors utilize biomarkers to measure both the outcome semen parameters and the exposure to pesticides. Biomarkers are generally considered superior to wri tten questionnaire or other forms of measurement, particularly when evaluating a short

- term effect. Measurement of the respective biomarkers was performed using standard practices, however no report as to the accuracy or precision of the estimates were pro vided in this study, e.g., coefficient of variation. Since this time of this study, it has been determined that atrazine mercapturate only reflects a small proportion of total atrazine exposure (Barr 2007), and other atrazine biomarkers have been proposed for epidemiologic analysis. In addition, when using biomarkers of exposure, the question as to how well the biomarker reflects actual dose is of key importance. Authors did not comment on this point. In general, use of biomarkers is a strength of the study and likely adds precision to the exposure and

outcome measurements.Do not cite

Appendix B2

- Page 363 of 677 Authors attempted to control for confounding through both design (restriction) and analysis (adjustment and stratification). With respect to confounding by other pesticides, authors

comp ared proportion who reported recent pesticide use and proximity to agricultural field between cases and controls and reported results were comparable. They also used a  ̳score‘ test to characterize the influence of multiple pesticide exposure on the associa tion and reported there was evidence that multiple chemical exposure alters the association (those with greater score has lower semen parameters). Only three men reported occupational pesticide exposure and only 16 (of the 84) reported recent pesticide use

. Exclusion of these men from the analysis (in a sensitivity analysis) did not alter the inference. However, as stated by the authors (pp.1483), unmeasured or residual confounding due to pesticides or other environmental contaminants cannot be ruled out.

With regard to the temporality of pesticide exposure and semen parameter alteration, authors support that because pesticide use is common and consistent in the Midwest, semen parameters measured could reflect the influence of the exogenous pesticide

expos ure (semen parameters reflect exposure over previous three months) which occurred close in time to the sample collection. However, as a cross

- sectional study, authors cannot fully rule out temporal bias in the association between pesticide exposure and sem en parameters, i.e. , it is not known with certainty from these data whether or not pesticide exposure preceded semen parameter abnormality.

In general, authors successfully address the sources of potential bias in their study and discuss the likely affect these sources of bias, if present, would have on the inference (risk estimates presented). Researchers state the low participation rate (27%) while comparable for studies of semen parameters, would not likely result in selection bias as men would

have to know both their pesticide exposure level and semen parameters to make a decision to participate on these factors; this is not likely the case. In addition, in the measurement of semen and pesticide biomarkers, technicians were blinded as to case

- and expos ure

status, likely reducing the chance of observer bias. While a balance must be struck between cost (sample size) and the ability to increase and diversify the sample collection, one must acknowledge the limits this places upon generalizability.

EPA conc urs with the authors‘ approach to utilize several analytic methods from simple

non

- parametric tests to multi

- variable adjusted regression estimates to maximize the information gained from the collection of these data. This is an appropriate and efficient

s trategy given the cost of collecting these data. Overall, authors report a trend of lower quality semen parameters with higher pesticide exposure, including atrazine exposure. Whereas these data provide some qualitative evidence of an association, the low sample size, particularly with respect to atrazine exposed controls in MO (n=1) and low sample size in MN to see an effect; potential for residual or unmeasured confounding (co

- exposure to other pesticides or another environmental agent); and under

- estimat ion of exposure due toDo not cite

Appendix B2

- Page 364 of 677 use of the atrazine mercapturate biomarker reduce the quality of these data for risk

assessment.

Overall, this is a well

- designed and executed study evaluating the association between pesticide exposure and semen parameters. Strengt hs include the analysis of biomarkers for both exposure and outcome, quality of the lab techniques and consistency achieved across study sites (data not shown), and authors‘ ability to address potential sources of bias. Limits include use of AM biomarker g iven this likely under

- estimates exposure, potential temporal bias, generalizabiity of results, and low sample size. Additional prospective studies, utilizing alternative biomarkers of atrazine exposure would be informative. Although based upon exploratory analyses, the indication that atrazine may be associated with adverse sperm morphology as well as reduced motility may be informative to the purported association with adverse birth outcomes as a result of male pesticide exposure. However, additional stud ies are needed to fully elucidate this connection, if any.

B.2.17 Villanueva C., Durand G., Coutte M., Chevrier C., and Cordier S. ―Atrazine in municipal drinking water and risk of low birth weight, preterm delivery, and small

- for

- gestational

- age,‖ [ Envi ronmental Health Perspectives, 62(6): 400

- 405 (2005)]

.

Background: Villanueva et al. (2005) conducted a retrospective cohort study to evaluate the association between atrazine levels in municipal drinking water and the risk of preterm delivery, LBW, and S GA.

Methods: T he stu dy focused on Finisteŕe, a region of France that is highly agricultural and uses a large amount of pesticides in corn production. Birth record data were identified for 9,721 live births in Finisteŕe during October 1, 1997 to September 30, 1998. All birth records data were obtained before infants were discharged from hospitals and included infant health information, maternal demographic characteristics, and municipality of maternal residence prior to birth.

Preterm delivery, LBW, and SGA were determined b y comparing infant health information with a standard definition of preterm delivery and French population growth curves. Atrazine exposure levels for each birth were then classified using drinking water monitoring data from 2,661 atrazine measurements th at were sampled from 112 water distribution units during 1990 to 1998. Using this atrazine monitoring data, an exposure index was created by calculating the geometric mean atrazine levels for each water distribution unit over the entire eight

- year samplin g period. Based on distribution of their exposure index, the investigators then created low, medium, and high exposure groups using tertiles as cut

offs for both raw (≤ 0.05, > 0.0.05

– – 0.036, > 0.036Do not cite

Appendix B2

- Page 365 of 677 were linked to the exposure index data by determining the water distribution unit that serves the municipality of each maternal residence.

The investigators performed two statist ical analyses to evaluate the association between their measure of maternal atrazine exposure and risk of preterm delivery, LBW, and SGA. First, logistic regression was used to calculate odds ratios for each health outcome relative to the low exposure cate gory, adjusting for several potential confounders, including infant sex, maternal age, and percent of samples below the limit of detection (1 μg/l). Second, the investigators considered the timing of potential exposure relative to each trimester of

pregna ncy because their descriptive analysis of the atrazine monitoring data showed a distinct peak during the late spring through summer (i.e. May to September). Logistic regression was used to calculate odds ratios of preterm delivery, LBW, and SGA for each

t rimester of pregnancy by comparing trimester periods that overlapped with any part of May

- September or with October

- April.

Results: Based on the investigators first analysis, no statistically significant associations were reported between atrazine and pre term delivery, LBW, or SGA. There was a slight indication that atrazine concentration in treated drinking water during high use months of May

- Sept. was associated with preterm delivery (OR 1.93, 95% CI 0.85

- 4.35), with a monotonic, positive exposure

- respo nse trend observed (p trend 0.126).

When the investigators evaluated timing of potential exposure relative to each trimester of exposure, a significant association was identified between SGA (statistically significant) and LBW (non

- statistically signific ant) with third trimester overlap with the May

- Sept. high use period; and, preterm delivery with first trimester overlap with this period (n.s.). In particular, the investigators reported that births with third trimesters in May

- September had a 37%

increa se in the risk of SGA when compared with births that had their third trimester in

October

- April (Odd Ratio = 1.37, 95% CI: 1.04, 1.81). When authors estimated risk of SGA by number of months (0,1,2,3) of the third trimester which overlapped the high use at razine period, they observed a statistically significant, positive exposure

- response trend (p trend 0.019); the odds of SGA among those pregnancies for which the third trimester occurred entirely during the high use May

- Sept. period was statistically signi ficant (OR 1.48, 95% CI

1.04

- 2.09).

EPA Critique:

Villanueva et al. (2005) utilized atrazine measurements from drinking water sampling to estimate exposure. Authors‘ use of atrazine concentration in raw and treated drinking

water is an enhancement in co mparison to other studies investigating this question included in this review (Munger 1997; Dabrowski 2003). Researchers compared monthly geometric mean atrazine concentration in raw and finished drinking water averaged over

1990

- 1998, and performed ANOVA analyses to determine factors contributing to theDo not cite

Appendix B2

- Page 366 of 677 amount of variation in the data. After controlling for season, and region, only random variation in atrazine concentration was observed. Therefore, authors used monthly average concentration of atrazine, co ntrolling for these factors, over this extended time period to estimate atrazine concentration for the period 1997

- 1998.. However, uncertainty in the ability to match atrazine in drinking water with actual maternal exposure remains. As acknowledged by the authors, the number of observations below the limit of detection for atrazine (parent) and metabolites DIA and DEA were significant; 33

- 50% for the parent, and up to 99% for the metabolites. As a result, the exposure range was quite narrow, reducing the ab ility to observe a significant difference in exposure groups given the sample size. To the extent that this exposure reflected in this study differs from other populations, results may or may not be generalizable. In addition, differences in atrazine use a nd use profile, drinking water consumption patterns among pregnant women including use of bottled water, may also affect external validity.

While authors report no statistically significant association between atrazine concentration in drinking water in this area of France and either preterm delivery, low birth weight, or

small

- for

- gestational age infants, sub

- analysis indicate some suggestive findings. For example, among pregnancies in which the third trimester overlaps a high atrazine use period (May

- Oc tober in this region), authors report elevated odds of LBW and small

- for

gestational age births. Similarly, for pregnancies in which the first trimester overlaps this high use period, elevated odds of preterm delivery were observed (non

- significant. but

bo rderline). Regarding SGA and atrazine exposure during the third trimester, the more months of the trimester that overlapped this period of high use the greater the odds of SGA (p trend statistically significant). While based upon sub

- analyses, these provi de initial suggestions that potential exposure during different periods of development may elicit different adverse birth outcomes. However, the authors acknowledge that other non

- causal explanations cannot be excluded at the time of the study, including e xposure to other highly correlated environmental chemicals that may have a similar temporal and geographic distribution in the population.

B.2.18 Waller et al. ―Agricultural

- related chemical exposures, season of conception, and risk of gastroschisis in W ashington State,‖ [Am J Obstet Gynecol;202:241.e1

- 6. (2010)].

Background: Authors hypothesize that in utero exposure to agrichemicals including atrazine, 2,4

- D, nitrate and nitrite, is related to prevalence of gastroschisis, a birth defect of

the abdomina l wall.

Methods : Authors performed a population

- based case

- control study using birth records and hospital discharge records in WA state to identify eligible cases and controls. All singleton,

live births in the state between 1987

- 2006 were eligible for in clusion in this study. Maternal residence as reported in birth records was linked to USGS surface water data to determineDo not cite

Appendix B2

- Page 367 of 677 relative maternal exposure to atrazine, 2,4

- D, nitrate and nitrite measured a distance between maternal residence reported at birth an d a ―high‖ chemical surface water monitoring site.

All cases of gastroschisis reported in the state between 1987 and 2006 were eligible for selection into this study. Cases of gastroschisis were excluded from this study if chromosomal abnormalities were p resent, if there were other known genetic syndromes identified in the birth records, if the pregnancy resulted in fetal death, or if the zip code of maternal residence (at time of birth) was not reported in the birth records. Control subjects were randomly selected from all live births reported in WA state between 1987 and 2006 in a ratio of 4:1, controls to cases. Controls were frequency matched to cases by year of birth. Authors measured month of conception and gestational age using last menstrual period (LMP).

Demographic characteristics of the participants and maternal residence were ascertained from birth certificate records database. Information concerning age, race, smoking status, county of residence at time of birth, longitude/latitude coordinate o f maternal residence during pregnancy (assumed from residence at birth), education, and occupation were collected from the hospital discharge records.

Surface water concentration of contaminants was determined using USGS surface water records reported to the state. For each USGS monitoring site in the state (n=390), authors collected season pesticide concentration data for the years 2001

- 06, the only years within the study period (1987

- 2006) for which USGS surface water data were available. Average

season al concentration of each agrichemical of interest were averaged to generate an annual average, and the annual averages between 2001

- 2006 were averaged. This site

specific annual average was compared to U.S. EPA acceptable contaminant levels to

determine ar eas of ―high‖ pesticide concentration in surface water (drinking water), i.e., those with annual average greater than the US EPA acceptable limit were identified as ―high.‖ Authors assumed a ―high‖ exposure could have occurred during the in utero period;

h owever, this is not measured directly.

To measure maternal pesticide exposure authors measured the distance between the center of the zip code of the reported maternal residence to the nearest USGS monitoring site which was determined to have a ―high‖ agr ichemical concentration (using US EPA criteria). Authors also investigated the association between season of conception and prevalence of gastroschisis.

Authors compared characteristics of cases and controls using standard tests (t

- test for

continuous var iable and chi

- squared test for categorical). Both logistic and linear regression was used to model the outcomes (case/control status) and distance from treated field, respectively. Several potentially confounding factors were investigated including age,Do not cite

Appendix B2

- Page 368 of 677 p arity, maternal smoking, season of conception, distance from high exposure measurement point (USGS measurement station; however, only maternal age, smoking and parity were

included in the final statistical models.

Results: Authors included 806 cases and 3 616 controls in their study. Cases and controls were statistically different with respect to maternal age, percent married, race, education, parity, smoking, income, paternal age, gestational age, birthweight. Authors report that among women who resided ( at the time of birth) in an area less than 25 km from a ―high‖ atrazine exposure area the prevalence of gastroschisis is increased 60% (odds ratio, OR, 1.6, 95% confidence interval (95% CI) 1.1, 2.3, p=0.014). Authors also observed that women residing 25

- 50 km from a ―high‖ atrazine exposure area are also observed to have a statistically significant increase in risk of gastroschisis in their children (OR 1.4, 95% CI 1.2, 1.7). When authors measured distance between maternal residence and ―high‖ atrazine ex posure area as a continuous variable (as opposed to three categories of distance from ―high‖ atrazine), the measure of association was statistically significant, although borderline, p=0.049. Other environmental agents investigated in this study, 2,4

- D,

ni trate and nitrite, were not associated with the risk of gastroschisis. As a result of multi

variable models, authors report that conception in the Spring season, in addition to nulliparity and smoking, was a significant predictor of gastroschisis (OR 1.2, 95% CI 1.1, 1.5).

Authors conclusion: The researchers conclude that their work suggests environmental exposures possibly including atrazine may be associated with the prevalence of gastroschisis, a major form of abdominal wall birth defects. EPA Critique:

Researchers investigated a novel hypothesis with regard to the association between atrazine concentration in surface water and prevalence of gastroschisis. The approach taken by Waller et al. is reasonable, i.e., use of state birth records and USGS surfa ce water monitoring data, given the large sample size needed to observe an association between exposure and this rare birth defect. However, limitations were present.

A review of Table 1 indicates that cases and controls were different with respect to man y potentially confounding variables including maternal age, paternal age, birth characteristics, smoking, education and other measures of socio

- economic status; however, only maternal age, smoking and parity were included in their final statistical model. The method of confounder selection was not clearly described by the authors. Additional sub

- analyses including stratification of the association between gastroschisis and surface water concentration of atrazine by maternal age (+/

- 18 years) would have bee n informative, given the reported influence of young maternal age on the incidence of gastroschisis (Williams, 2005).Do not cite

Appendix B2

- Page 369 of 677 There are several sources of bias which could affect the interpretation of the prevalence estimates presented herein. The use of birth r ecords and hospital discharge records to identify cases of birth defects have been well characterized elsewhere (Watkins 1996). In general, under

- reporting of birth defects in records of this type are a concern. Exposure measurement in this study is approp riate to the goal of performing the analysis on a large enough sample to achieve statistical power, however significant misclassification could have occurred. Maternal relocation during pregnancy or immediately after delivery is a possibility which would r esult in incorrect measurement of atrazine exposure (Canfield, 2006). It is not certain whether the surface water monitoring site is the source of drinking water for the zip code in which the mother lived at the time of delivery, and authors do not specula te as to the probability of atrazine exposure through the drinking water based upon a 25km distance from a surface water monitoring site. If the surface water source measured serves as recreational site as well as a drinking water site, under

- estimation of exposure could have occurred. The authors did not comment as to why they used a different set of exposure categories for atrazine as compared to the other 4 agrichemicals measures; however, it is possible that there were too few cases within 10km of a ―hi gh‖ atrazine monitoring site to perform a robust statistical analysis, so authors collapsed exposure categories. However, authors did not comment on this point.

Overall, authors evaluated a novel hypothesis with outcome and exposure measurement methods ap propriate to the goal of achieving the large sample size needed to study this question. However, potential misclassification due to exposure and outcome measurement error was possible . Elaboration as to their confounder selection process would aid interpr etation of the results. Given the limitations present, these data require replication with improved exposure assessment methodology to better understand the nature of the association between atrazine exposure in the environment, via drinking water, recreat ion water, or other environmental exposure, and this type of birth defect.

B.2.19 Winchester P.D., Huskins J., and Ting J. ―Agrichemicals in surface water and birth defects in the United States,‖ [ Acta Paediatrica . 98(4):

664

- 669 (2009)].

Background: W inchester, Huskins, and Ying (2009) conducted an ecologic study to investigate if the rate of birth defects is greater in months with the highest concentrations of several surface water agrichemicals, including atrazine.

Methods: National pregnancy and birth outcome data from 1996

- 2002 were obtained through the national natality database maintained by the Centers for Disease Control and Prevention (CDC). Monthly surface water concentrations of atrazine, nitrates, and ―other pesticides‖ were then estima ted using data collected through USGS‘s National Water

Quality Assessment Program (NAWQA), which has collected water quality data from several major U.S. river basins and aquifers since 1991. Based on these health outcomeDo not cite

Appendix B2

- Page 370 of 677 and drinking water monitoring so urces, the investigators presented average monthly birth defect rates and geometric mean atrazine concentrations by aggregating monthly data for the year 1996

- 2002

The investigators performed two analyses to explore the relationship between birth defects and agrichemical surface water concentrations. First, they compared the risk of 22 different birth defect types during April through July

– the months with the highest levels of atrazine

– with the rest of the year using a logistic regression model that adjusted for potential confounders, including race/ethnicity, metropolitan residence, alcohol use, tobacco use, maternal diabetes, and year. Second, they assessed the relationship between birth defects and atrazine, nitrates, and ―other pesticides‖ over t he entire calendar year using a logistic regression model that adjusted for the same potential confounders.

Results: The rates of 11 different birth defect types were significantly higher in April

- July when compared with all other months. Similarly, seve ral positive associations were observed to be statistically significant in their second analysis. In particular, the odds ratios for 9 of 11 birth defect types were associated with atrazine when their model treated atrazine separately from the other agric hemicals. Similarly, when they considered all three groups of agrichemicals in their logistic regression, 5 of 6 birth defects were positively

associated with atrazine.

EPA Critique:

Overall, the study provides useful descriptive information on the seas onal patterns of both birth defects and the levels of several agrichemicals, including atrazine, in surface water. Specifically, the study demonstrated that monthly U.S. birth defect rates generally follow a seasonal pattern that peaks during late spring to early summer. Similarly, it also highlighted that the use of pesticides and fertilizers can lead to higher levels of residues in surface waters, some of which may subsequently be sources of public drinking water supplies. These findings help raise awa reness in the public health community, help generate research hypotheses, and inform future birth defect research on atrazine and other risk factors that exhibit similar seasonal patterns. However, there are significant limitations as to our ability to uti lize results in risk assessment.

Although the study reported positive associations between several types of birth defects and atrazine levels in surface water, it has limitations to consider when evaluating the

results in the context of risk assessment. The most important limitations of the study relate to its ecologic design and use of USGS surface water monitoring data as an exposure surrogate. In ecologic designs, the unit of analysis is at a group level, rather than individual level. Ecologic stud ies are frequently used to generate hypotheses in epidemiologic research, since they utilize existing data sources and are not resource intensive in terms of time and cost. A limitation of ecologic studies, however, is that findings at a group

- levelDo not cite

Appendix B2

- Page 371 of 677 may n ot translate to individuals. For example, Winchester, Huskins, and Ying (2009) characterized exposure levels using monthly geometric mean estimates of the concentration of atrazine in U.S. surface water. As this measure of exposure is based on regional s urface water data, rather than drinking water data, it cannot provide direct evidence that mothers who had children with birth defects were actually exposed to elevated levels of atrazine. Overall, effect sizes presented are small and may represent very l ittle variation in the population. Given the study design, authors were not able to control for other environmental exposures in their analysis. Due to this limitation, the validity of the study must be confirmed through additional research that uses indiv idual

- level exposure data. The investigators do emphasize the limitations of their ecologic study design and indicate, "While a causal link between agrichemicals and birth defects cannot be proven from this study an association might provide clues to comm on factors shared by

both variables."Do not cite

Appendix C

- Page 372 of 677 APPENDIX C: SUMMARIES OF BMD ANALYSES OF STUDIES ON LH SURGE

C.1 NHEERL STUDY

Model (Constant Variance) BMD BMDL p

- value AIC

Exponential 2 58.5 29.6 0.004 515.8 Exponential 3 58.5 29.6 0.004 515.8 Exponential 4 4.23 1.96 0.94 501.3 Exponential 5 4.31 1.96 0.86 503.3

Hill 4.16 1.68 0.87 503.3

Linear 78.4 53.1 0.002 518.1 Polynomial 78.4 53.1 0.002 518.1

Power 78.4 53.1 0.002 518.1

NHEERL Study: BMD OUTPUT for Exponential Model Runs (Constant Variance; Best Model Fit)

==================================================================== Exponential Model. (Version: 1.7; Date: 12/10/2009) Input Data File: C:/Usepa/BMDS212/Data/exp_Actualruns_Setting.(d) Gnuplot Plotting File:

Tue Jul 20 14:07:40 2010

====================================================================

BMDS Model Run

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

The form of the response function by Model:

Model 2: Y [dose] = a * exp{sign * b * dose} Model 3: Y[dose] = a * exp{sign * (b * dose)^d} Model 4: Y[dose] = a * [c

- (c

- 1) * exp{

- b * dose}] Model 5: Y[dose] = a * [c

- (c

- 1) * exp{

- (b * dose)^d}]

Note: Y[dose] is the median respons e for exposure = dose; sign = +1 for increasing trend in data; sign =

- 1 for decreasing trend.Do not cite

Appendix C

- Page 373 of 677 Model 2 is nested within Models 3 and 4.

Model 3 is nested within Model 5.

Model 4 is nested within Model 5.

Dependen t variable = Mean Independent variable = Dose Data are assumed to be distributed: normally Variance Model: exp(lnalpha +rho *ln(Y[dose]))

rho is set to 0.

A constant variance model is fit.

Total number of dose groups = 7

Total number of records with missing values = 0

Maximum number of iterations = 250 Relative Function Convergence has been set to: 1e

- 008 Parameter Convergence has been set to: 1e

- 008

MLE solution provided: Exact

Initial P arameter Values

Variable Model 2 Model 3 Model 4 Model 5

-------- ------- ------- ------- ------- lnalpha 4.07816 4.07816 4.07816 4.07816 rho(S) 0 0 0 0

a 11.0009 11.0009 22.9404 22.9404

b 0.00794702 0.00794702 0.044 9039 0.0449039

c

-- -- 0.352881 0.352881

d

-- 1

-- 1

(S) = Specified

Parameter Estimates by Model

Variable Model 2 Model 3 Model 4 Model 5

-------- ------- ------- ------- ------- lnalpha 4.25603 4.25603 4.08595 4.08581

rho 0 0 0 0Do not cite

Appendix C

- Page 374 of 677 a 16.8823 16.8823 21.9754 21.9037

b 0.0117718 0.0117718 0.219622 0.22213

c

-- -- 0.422997 0.427096

d

-- 1

-- 1.07687

Table of Stats From Input Data

D ose N Obs Mean Obs Std Dev

----- --- ---------- ------------- 0 16 21.85 7

1.56 11 18.57 10.8

3.12 12 16.05 9.3

6.25 17 11.8 1 7

12.5 7 11.88 9

25 14 8.5 7.6

75 20 9.6 6.7

Estimated Values of Interest

Model Dose Est Mean Est Std Sc aled Residual

------- ------ ---------- --------- ---------------- 2 0 16.88 8.398 2.365

1.56 16.58 8.398 0.7878

3.12 16.27 8.398

- 0.09425

6.25 15.68 8.398

- 1.902

12.5 14.57 8.398

- 0.8482

25 12.58 8.398

- 1.817

75 6. 982 8.398 1.394

3 0 16.88 8.398 2.365

1.56 16.58 8.398 0.7878

3.12 16.27 8.398

- 0.09425

6.25 15.68 8.398

- 1.902

12.5 14.57 8.398

- 0.8482

25 12.58 8.398

- 1.817

75 6.982 8.398 1.394

4 0 21.98 7.714

- 0.06604

1.56 18.3 7.714 0.1173

3.12 15.69 7.714 0.1613

6.25 12.51 7.714

- 0.3737

12.5 10.11 7.714 0.6071Do not cite

Appendix C

- Page 375 of 677 25 9.348 7.714

- 0.4113

75 9.296 7.714 0.1765

5 0 21.9 7.713

- 0.02886

1.56 18.47 7.713 0.04191

3.12 15.75 7.713 0.1316

6.25 12.38 7.713

- 0.303

12.5 9.978 7.713 0.6526

25 9.377 7.713

- 0.4255

75 9.355 7.713 0.1421

Other models for which likelihoods are calculated:

Model A1: Yij = Mu(i) + e(ij)

V ar{e(ij)} = Sigma^2

Model A2: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma(i)^2

Model A3: Yij = Mu(i) + e(ij) Var{e(ij)} = exp(lalpha + log(mean(i)) * rho)

Model R: Yij = Mu + e(i) Var{e(ij)} = Sigma^2

Likelihoods of Interest

Model Log(likelihood) DF AIC

------- ----------------- ---- ------------ A1

- 246.2909 8 508.5818

A2

- 243.8179 14 515.6358

A3

- 246.2909 8 508.5818

R

- 261.9739 2 527.9478

2

- 254.9177 3 515.8354

3

- 254.9177 3 515.8354

4

- 246.6687 4 501.3374

5

- 246.6617 5 503.3234

Additive constant for all log

- likelihoods =

- 89.14. This constant added to theDo not cite

Appendix C

- Page 376 of 677 above values gives the log

- likelihood including the term that does not depend on the model parameters.

Explana tion of Tests

Test 1: Does response and/or variances differ among Dose levels? (A2 vs. R) Test 2: Are Variances Homogeneous? (A2 vs. A1) Test 3: Are variances adequately modeled? (A2 vs. A3) Test 4: Does Model 2 fit the data? (A3 vs. 2)

Test 5a: Does Model 3 fit the data? (A3 vs 3) Test 5b: Is Model 3 better than Model 2? (3 vs. 2)

Test 6a: Does Model 4 fit the data? (A3 vs 4) Test 6b: Is Model 4 better than Model 2? (4 vs. 2)

Test 7a: Does Model 5 fit the data? (A3 vs 5) Test 7b: Is Model 5 better than Model 3? (5 vs. 3) Test 7c: Is Model 5 better than Model 4? (5 vs. 4)

Tests of Interest

Test

- 2*log(Likelihood Ratio) D. F. p

- value

-------- -------- ---------------- ------ -------------- Test 1 36.31 12 0.0002886

Test 2 4.946 6 0.5508

Test 3 4.946 6 0.5508

Test 4 17.25 5 0.004043

Test 5a 17.25 5 0.004043

Test 5b

- 7.39e

- 013 0 N/A

Test 6a 0.7556 4 0.9443

Test 6b 16.5 1 < 0.0001

Test 7a 0.7416 3 0.8634

Test 7b 16.51 2 0.0002597

Test 7c 0.01397 1 0.9059

The p

- value for Test 1 is less than .05. There appears to be a difference between response and/or variances among the dose levels, it seems appr opriate to model the data.Do not cite

Appendix C

- Page 377 of 6 77 The p

- value for Test 2 is greater than .1. A homogeneous variance model appears to be appropriate here.

The p

- value for Test 3 is greater than .1. The modeled variance appears to be appropriate here.

The p

- value for Test 4 is less than .1. Model 2 may not adequately describe the data; you may want to consider another model.

The p

- value for Test 5a is less than .1. Model 3 may not adequately describe the data; you may want to consider another model.

Degrees of freedom for Test 5b are less than or equal to 0.
The Chi

- Square test for fit is not valid.

The p

- value for Test 6a is greater than .1. Model 4 seems to adequately describe the data.

The p

- value for Te st 6b is less than .05. Model 4 appears

to fit the data better than Model 2.

The p

- value for Test 7a is greater than .1. Model 5 seems to adequately describe the data.

The p

- value for Test 7b is less than .05. Model 5 appears

to fit the data better than Model 3.

The p

- value for Test 7c is greater than .05. Model 5 does

not seem to fit the data better than Model 4.

Benchmark Dose Computations:

Specified Effect = 1.000000

Risk Type = Estimated standard deviations from control

Confidence Level = 0.950000

BMD and BMDL by ModelDo not cite

Appendix C

- Page 378 of 677 Model BMD BMDL

------- ------------ ---------- 2 58.451 29.553

3 58.451 29.553

4 4.26795 1.9583

5 4.30751 1.962Do not cite

Appendix C

- Page 379 of 677Do not cite

Appendix C

- Page 380 of 677Do not cite

Appendix C

- Page 381 of 677 ________________________________________ ________________________

C. 2 MORSETH ONE MONTH LH STUDY

Model (Constant Variance) a BMD BMDL p

- value AIC

Exponential 2 152.4 25.6 0.78 819.9 Exponential 3 152.4 25.6 0.78 819.9 Exponential 4 129.6 11.8 0.85 821.2 Exponential 5 Not Computed Not Computed

-- -- Hill Not Computed Not Computed

-- -- Linear 226.5 126.7 0.54 821.0 Polynomial 215.9 128.8 0.59 820.8

Power 215.9 128.8 0.59 820.8

a Nonhomogeneous Variance recommended by BMDS outputDo not cite

Appendix C

- Page 382 of 677 Model (Nonhomogeneous Variance) BMD BMDL p

- value AIC

Exponential 2 523.7 174.7 0.017 795.3 Exponential 3 52 3.7 174.7 0.017 795.3 Exponential 4 Not

Computed Not

Computed -- --

Exponential 5 Not

Computed Not

Computed -- --

Hill Not

Computed Not

Computed -- --

Linear 291.3 197.3 0.006 797.5 Polynomial 291.3 197.3 0.006 797.5

Power 291.3 197.3 0.006 797.5

Morseth One Month LH Study: BMD OUTPUT for Exponential Model Runs (Constant Variance)

==================================================================== Exponential Model. (Version: 1.7; Date: 12/10/2009) Input Data File: C:/Usepa/BMDS2 12/Data/exp_Actualruns_Setting.(d) Gnuplot Plotting File:

Thu Jul 22 13:45:53 2010

====================================================================

BMDS Model Run

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~

The form of the response function by Model: Model 2: Y[dose] = a * exp{sign * b * dose} Model 3: Y[dose] = a * exp{sign * (b * dose)^d} Model 4: Y[dose] = a * [c

- (c

- 1) * exp{

- b * dose}] Model 5: Y[dose] = a * [c

- (c

- 1) * exp{

- (b * dose)^d}]

Note: Y[dose] is the median response for exposure = dose;Do not cite

Appendix C

- Page 383 of 677 sign = +1 for increasing trend in data; sign =

- 1 for decreasing trend.

Model 2 is nested within Models 3 and 4.

Model 3 is nes ted within Model 5.

Model 4 is nested within Model 5.

Dependent variable = Mean Independent variable = Dose Data are assumed to be distributed: normally Variance Model: exp(lnalpha +rho *ln(Y[dose]))

rho is set to 0.

A constant va riance model is fit.

Total number of dose groups = 5 Total number of records with missing values = 0

Maximum number of iterations = 250 Relative Function Convergence has been set to: 1e

- 008 Parameter Convergence has been set to: 1e

- 008

MLE solution provided: Exact

Initial Parameter Values

Variable Model 2 Model 3 Model 4 Model 5

-------- ------- ------- ------- ---- --- lnalpha 15.2574 15.2574 15.2574 15.2574 rho(S) 0 0 0 0

a 1413.18 1413.18 3165.75 3165.75

b 0.00610828 0.00610828 0.0210595 0.0210595

c

-- -- 0.244281 0.244281

d

-- 1

-- 1

(S) = Specified

Parameter Estimates by Model

Variable Model 2 Model 3 Model 4 Model 5Do not cite

Appendix C

- Page 384 of 677 -------- ------- ------- ------- ------- lnalpha 15.2794 15.2794 15.2638 15.2609

rho 0 0 0 0

a 2764.3 2764.3 2913.32 2803.53

b 0.00915046 0.00915045 0.0267425 0.0265255

c

-- -- 0.269019 0.28969

d

-- 1

-- 2.20144

NC = No Convergence

Table of Stats From Input Data

Dose N Obs Mean Obs Std Dev

----- --- ---------- ------------- 0 10 2650 2389

2.5 10 3015 3220

5 10 2717 2542

40 10 1450 857

200 10 812 470

Estimated Values of Interest

Model Dose Est Mean Est Std Scaled Residual

------- ------ ---------- --------- ---------------- 2 0 2764 2079

- 0.1738

2.5 2702 2079 0.4764

5 2641 2079 0.1161

40 1917 2079

- 0.7103

200 443.4 2079 0.5606

3 0 2764 2079

- 0.1738

2.5 2702 2079 0.4764

5 2641 2079 0.1161

40 1917 2079

- 0.7103

200 443.4 2079 0.5606

4 0 2913 2063

- 0.4036

2.5 2776 2063 0.367

5 2647 2063 0.1076

40 1514 2063

- 0.09875

200 793.9 2063 0.0278

5 0 2804 2060

- 0.2357Do not cite

Appendix C

- Page 385 of 677 2.5 2798 2060 0.3324

5 2780 2060

- 0.097 25

40 1449 2060 0.0007715

200 812.2 2060

- 0.0002382

Other models for which likelihoods are calculated:

Model A1: Yij = Mu(i) + e(ij) Var{e( ij)} = Sigma^2

Model A2: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma(i)^2

Model A3: Yij = Mu(i) + e(ij) Var{e(ij)} = exp(lalpha + log(mean(i)) * rho)

Model R: Yij = Mu + e(i)

V ar{e(ij)} = Sigma^2

Likelihoods of Interest

Model Log(likelihood) DF AIC

------- ----------------- ---- ------------ A1

- 406 .4352 6 824.8704

A2

- 388.3924 10 796.7849

A3

- 406.4352 6 824.8704

R

- 410.3636 2 824.7272

2

- 406.986 3 819.9719

3

- 406.986 3 819.9719

4

- 406.5955 4 821.1911

5

- 406.5231 5 823.0462

Additive constant for all log

- likelihoods =

- 45.95. This constant added to the above values gives the log

- likelihood including the term that does not depend on the model parameters.

Explanation of TestsDo not cite

Appendix C

- Page 386 of 677 Test 1: Does response and/or variances differ among Dose levels? (A2 vs. R) Test 2: Are Variances Homogeneous? (A2 vs. A1) Test 3: Are variances adequately modeled? (A2 vs. A3) Test 4: Does Model 2 fit the data? (A3 vs. 2)

Tes t 5a: Does Model 3 fit the data? (A3 vs 3) Test 5b: Is Model 3 better than Model 2? (3 vs. 2)

Test 6a: Does Model 4 fit the data? (A3 vs 4) Test 6b: Is Model 4 better than Model 2? (4 vs. 2)

Test 7a: Does Model 5 fit the data? (A3 vs 5)

Te st 7b: Is Model 5 better than Model 3? (5 vs. 3) Test 7c: Is Model 5 better than Model 4? (5 vs. 4)

Tests of Interest

Test

- 2*log(Likelihood Ratio) D. F. p

- value

-------- ------------- ----------- ------ -------------- Test 1 43.94 8 < 0.0001

Test 2 36.09 4 < 0.0001

Test 3 36.09 4 < 0.0001

Test 4 1.102 3 0.7767

Test 5a 1.102 3 0.7767

Test 5b

- 1.819e

- 011 0 N/A

Test 6a 0.3207 2 0.8518

Test 6b 0.7808 1 0.3769

Test 7a 0.1758 1 0.675

Test 7b 0.9257 2 0.6295

Test 7c 0.1449 1 0.7034

The p

- value for Test 1 is less than .05. There appears to be a difference between response and/or variances among the dose levels, it seems appropria te to model the data.

The p

- value for Test 2 is less than .1. Consider running

a non

- homogeneous variance model.

The p

- value for Test 3 is less than .1. You may want toDo not cite

Appendix C

- Page 387 of 677 consider a different variance model.

The p

- value for Test 4 is greater than .1. Model 2 seems to adequately describe the data.

The p

- value for Test 5a is greater than .1. Model 3 seems to adequately describe the data.

Degrees of freedom for Test 5b are less than or equal to 0.
The Ch i

- Square test for fit is not valid.

The p

- value for Test 6a is greater than .1. Model 4 seems to adequately describe the data.

The p

- value for Test 6b is greater than .05. Model 4 does

not seem to fit the data better than Model 2.

The p

- value for Test 7a is greater than .1. Model 5 seems to adequately describe the data.

The p

- value for Test 7b is greater than .05. Model 5 does

not seem to fit the data better than Model 3.

The p

- value for Test 7c is great er than .05. Model 5 does

not seem to fit the data better than Model 4.

Benchmark Dose Computations:

Specified Effect = 1.000000

Risk Type = Estimated standard deviations from control

Confidence Level = 0.950000

BMD and BMDL by Model

Model BMD BMDL

------- ------------ ---------- 2 152.443 25.6454

3 152.443 25.6454Do not cite

Appendix C

- Page 388 of 677 4 129 .57 11.8371 5 0 0 Not computedDo not cite

Appendix C

- Page 389 of 677Do not cite

Appendix C

- Page 390 of 677Do not cite

Appendix C

- Page 391 of 677 ________________________________________________________________

Morseth One Month LH Study: BMD OUTPUT for Exponential Model Runs (Nonhomogeneous Vari ance)

==================================================================== Exponential Model. (Version: 1.7; Date: 12/10/2009) Input Data File: C:/Usepa/BMDS212/Data/exp_Actualruns_Setting.(d) Gnuplot Plotting File: Fri Aug 06 09:35:01 2010

====================================================================

BMDS Model Run

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

The form of the response function by Model:Do not cite

Appendix C

- Page 392 of 677 Model 2: Y[dose] = a * exp{sign * b * dose} Model 3: Y[dose] = a * exp{sign * (b * dose)^d} Model 4: Y[dose] = a * [c

- (c

- 1) * exp{

- b * dose}] Model 5: Y[dose] = a * [c

- (c

- 1) * exp{

- (b * dose)^d}]

Note: Y[dose] is the median response for exp osure = dose; sign = +1 for increasing trend in data; sign =

- 1 for decreasing trend.

Model 2 is nested within Models 3 and 4.

Model 3 is nested within Model 5.

Model 4 is nested within Model 5.

Dependent variabl e = Mean Independent variable = Dose Data are assumed to be distributed: normally Variance Model: exp(lnalpha +rho *ln(Y[dose])) The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)

Total number of dose groups = 5

T otal number of records with missing values = 0

Maximum number of iterations = 250 Relative Function Convergence has been set to: 1e

- 008 Parameter Convergence has been set to: 1e

- 008

MLE solution provided: Exact

Initial Parameter Values

Variable Model 2 Model 3 Model 4 Model 5

-------- ------- ------- ------- ------- lnalpha

- 7.57613

- 7.57613

- 7.57613

- 7.57613

rho 2.94035 2.94035 2.94035 2.94035

a 1413.18 1413.18 3165.75 3165.75

b 0.00610828 0.00610828 0.0210595 0.0210595

c

-- -- 0.244281 0.244281

d

-- 1

-- 1Do not cite

Appendix C

- Page 393 of 677 Parameter Estimates by Model

Variable Model 2 Model 3 Model 4 Model 5

-------- ------- ------- ------- ------- lnalpha

- 7.94834

- 7.94834

- 7.47498

- 7.51913

rho 2.99971 2.99971 2.91611 2.92104

a 2583.98 2583.98 3033.27 2862.72

b 0.00589281 0.00589281 0.03 5215 0.0290775

c

-- -- 0.273458 0.291831

d

-- 1

-- 2.06427

NC = No Convergence

Table of Stats From In put Data

Dose N Obs Mean Obs Std Dev

----- --- ---------- ------------- 0 10 2650 2389

2.5 10 3015 3220

5 10 2717 2542

40 10 1450 857

200 10 812 470

Estimated Values of Interest

Model Dose Est Mean Est Std Scaled Residual

------- ------ ---------- --------- ----- ----------- 2 0 2584 2466 0.08467

2.5 2546 2412 0.6146

5 2509 2359 0.2788

40 2041 17 32

- 1.08

200 795.1 421 0.1266

3 0 2584 2466 0.08467

2.5 2546 2412 0.6146

5 2509 2359 0.2788

40 2041 1732

- 1.08

200 795.1 421 0.1266

4 0 3033 2842

- 0.4264

2.5 2848 2592 0.2043

5 2677 2369 0.05275Do not cite

Appendix C

- Page 394 of 677 40 1368 890.3 0.2903

200 831.4 430.6

- 0.1425

5 0 2863 2606

- 0.2581

2.5 2854 2594 0.1967

5 2825 2556

- 0.1339

40 1353 871.8 0.3532

200 835.4 431.3

- 0.1718

Other models for which likelihoods are calculated:

Model A1: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma^2

Model A2: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma(i)^2

Model A3: Yij = Mu(i) + e(ij) Var{e(ij)} = exp(lalpha + log(mean(i)) * rho)

Model R: Yij = Mu + e(i) Var{e(ij)} = Sigma^2

Likelihoods of Interest

Model Log(likelihood) DF AIC

------- ----------------- ---- ------------ A1

- 406.4352 6 824.8704

A2

- 388.3924 10 796.7849

A3

- 388.5412 7 791.0824

R

- 410.3636 2 824.7272

2

- 393.6414 4 795.2827

3

- 393.6414 4 795.2827

4

- 389.3963 5 788.7925

5

- 389.0987 6 790.1974

Additive constant for all log

- likelihoods =

- 45.95. This const ant added to the above values gives the log

- likelihood including the term that does not depend on the model parameters.Do not cite

Appendix C

- Page 395 of 677 Explanation of Tests

Test 1: Does response and/or variances differ among Dose levels? (A2 vs. R) Test 2: Are Variances Homogeneous? (A2 vs. A1) Test 3: Are variances adequately modeled? (A2 vs. A3) Test 4: Does Model 2 fit the data? (A3 vs. 2)

Test 5a: Does Model 3 fit the data? (A3 vs 3)

Test 5b: Is Model 3 better than Model 2? (3 vs. 2)

Test 6a: Does Model 4 fit the data? (A3 vs 4) Test 6b: Is Model 4 better than Model 2? (4 vs. 2)

Test 7a: Does Model 5 fit the data? (A3 vs 5) Test 7b: Is Model 5 better than Model 3? (5 vs. 3)

Test 7c: Is Model 5 better than Model 4? (5 vs. 4)

Tests of Interest

Test

- 2*log(Likelihood Ratio) D. F. p

- value

-------- ------------------------ ------ -------------- Test 1 43.9 4 8 < 0.0001

Test 2 36.09 4 < 0.0001

Test 3 0.2976 3 0.9605

Test 4 10.2 3 0.01694

Test 5a 10.2 3 0.01694
Test 5b 0 0 N/A

Test 6a 1.71 2 0.4253

Test 6b 8.49 1 0.003571

Test 7a 1.115 1 0.291

Test 7b 9.085 2 0.01065

Test 7c 0.5951 1 0 .4405

The p

- value for Test 1 is less than .05. There appears to be a difference between response and/or variances among the dose levels, it seems appropriate to model the data.

The p

- value for Test 2 is less than .1. A non

- homogene ousDo not cite

Appendix C

- Page 396 of 677 variance model appears to be appropriate.

The p

- value for Test 3 is greater than .1. The modeled variance appears to be appropriate here.

The p

- value for Test 4 is less than .1. Model 2 may not adequately describe the data; you may want to consider another model.

The p

- value for Test 5a is less than .1. Model 3 may not adequately describe the data; you may want to consider another model.

Degrees of freedom for Test 5b are less than or equal to 0.

The C hi

- Square test for fit is not valid.

The p

- value for Test 6a is greater than .1. Model 4 seems to adequately describe the data.

The p

- value for Test 6b is less than .05. Model 4 appears

to fit the data better than Model 2.

The p

- value for Test 7a is greater than .1. Model 5 seems to adequately describe the data.

The p

- value for Test 7b is less than .05. Model 5 appears

to fit the data better than Model 3.

The p

- value for Test 7c is greater than .05. Mod el 5 does

not seem to fit the data better than Model 4.

Benchmark Dose Computations:

Specified Effect = 1.000000

Risk Type = Estimated standard deviations from control

Confidence Level = 0.950000

BMD and BMDL by Model

Model BMD BMDLDo not cite

Appendix C

- Page 397 of 677 ------- ------------ ---------- 2 523.696 174.68

3 523.696 174.68

4 0 0 Not computed 5 0 0 Not computedDo not cite

Appendix C

- Page 398 of 677Do not cite

Appendix C

- Page 399 of 677Do not cite

Appendix C

- Page 400 of 677 ________________________________________ ________________________

3. Morseth Six Month LH Study

Model (Constant Variance) BMD BMDL p

- value AIC

Expo nential 2 20.3 4.6 0.67 661.7 Exponential 3 20.3 Not Computed 0.67 661.7 Exponential 4 16.7 3.4 0.38 663.7 Exponential 5 3.9 3.7 N/A 664.5

Hill 4.2 3.3 0.58 663.2

Linear 26.2 16.3 0.61 661.9 Polynomial 20.6 12.9 0.05 666.9

Power 25.9 16.3 0.61 661.9Do not cite

Appendix C

- Page 401 of 677 Model (Nonhomogeneous Variance) BMD BMDL p

- value AIC

Exponential 2 36.2 17.1 0.52 636.1 Exponential 3 36.2 17.1 0.52 636.1 Exponential 4 Not

Computed Not

Computed -- --

Exponential 5 Not

Computed Not

Computed -- --

Hill Not

Computed Not

Computed -- --

Linear 32.6 22.9 0.36 636.9 Polynomial 32.6 22.9 0.36 636.9

Power 32.6 22.9 0.36 636.9

Morseth 6 Month LH Study

- Exponential (Constant Variance) Model Results

Warning: optimum may not have been found form model 2 (BMDL). Bad process compl etion in Optimization routine.

==================================================================== Exponential Model. (Version: 1.7; Date: 12/10/2009) Input Data File: C:/Usepa/BMDS212/Data/exp_Actualruns_Setting.(d) Gnuplot Plotti ng File:

Thu Jul 29 13:57:08 2010

====================================================================

BMDS Model Run

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

The form of the response function by Model: Model 2: Y[dose] = a * exp{sign * b * dose} Model 3: Y[dose] = a * exp{sign * (b * dose)^d} Model 4: Y[dose] = a * [c

- (c

- 1) * exp{

- b * dose}] Model 5: Y[dose] = a * [c

- (c

- 1) * exp{

- (b * dose)^d}]Do not cite

Appendix C

- Page 402 of 677 Note: Y[dose] is t he median response for exposure = dose; sign = +1 for increasing trend in data; sign =

- 1 for decreasing trend.

Model 2 is nested within Models 3 and 4.

Model 3 is nested within Model 5.

Model 4 is nested within Model 5.

Dependent variable = Mean Independent variable = Dose Data are assumed to be distributed: normally Variance Model: exp(lnalpha +rho *ln(Y[dose]))

rho is set to 0.

A constant variance model is fit.

Total number of dose groups = 4 Total number of records with missing values = 0

Maximum number of iterations = 250 Relative Function Convergence has been set to: 1e

- 008 Parameter Convergence has been set to: 1e

- 008

MLE solution provided: Exact

Initial Parameter Values

Variable Model 2 Model 3 Model 4 Model 5

-------- ------- ------- ------- ------- lnalpha 15.3723 15.3723 15.3723 15.3723 rho(S) 0 0 0 0

a 1497.19 1497.19 3812.55 3812.55

b 0.0471466 0.047146 6 0.14585 0.14585

c

-- -- 0.21433 0.21433

d

-- 1

-- 1

(S) = Specified

Parameter Estimates by ModelDo not cite

Appendix C

- Page 403 of 677 Variable Model 2 Model 3 Model 4 Model 5

-------- ------- ------- ------- ------- lnalpha 15.3924 1 5.3924 15.3913 15.3746

rho 0 0 0 0

a 3458.46 3458.46 3544.45 3483.5

b 0.0498021 0.0498021 0.0805078 0.262698

c

-- -- 0.16111 0.246304

d

-- 1

-- 18

Table of Stats From I nput Data

Dose N Obs Mean Obs Std Dev

----- --- ---------- ------------- 0 10 3336 3138

1.8 10 3631 2732

3.65 10 2500 1897

29.4 10 858 416

Estimated Values of Interest

Model Dose Est Mean Est Std Scaled Residual

------- ------ ---------- --------- ---------------- 2 0 3458 2200

- 0.176

1.8 3162 2200 0.6743

3.65 2884 2200

- 0.5514

29.4 799.8 2200 0.08362

3 0 3458 2200

- 0.176

1.8 3162 2200 0.6743

3.65 2884 2200

- 0.5514

29.4 799.8 2200 0.08362

4 0 3544 2199

- 0.2998

1.8 3143 2199 0.7014

3.65 2787 2199

- 0.4133

29.4 849.9 2199 0.01171

5 0 3484 2180

- 0.2139

1.8 3483 2180 0.2139

3.65 2500 2180

- 9.656e

- 007

29.4 858 2180 2.459e

- 007Do not cite

Appendix C

- Page 404 of 677 Other models for which likelihoods are calculated:

Model A1: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma^2

Model A2: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma(i)^2

Model A3: Yij = Mu(i) + e(ij) Var{e(ij)} = exp(lalpha + log(mean(i)) * rho)

Model R: Yij = Mu + e(i) Var{e(ij)} = Sigma^2

Likelihoods of Interest

Model Log(likelihood) DF AIC

------- ----------------- ---- ------------ A1

- 327.446 5 664.892

A2

- 313.3212 8 642.6425

A3

- 327.446 5 664.892

R

- 331.828 2 667.6559

2

- 327.8484 3 661.6967

3

- 327.8484 3 661.6967

4

- 327.826 4 663.652

5

- 327.4918 5 664.9836

Additive constant for all log

- likelihoods =

- 36.76. This constant added to the above values gives the log

- like lihood including the term that does not depend on the model parameters.

Explanation of Tests

Test 1: Does response and/or variances differ among Dose levels? (A2 vs. R) Test 2: Are Variances Homogeneous? (A2 v s. A1) Test 3: Are variances adequately modeled? (A2 vs. A3) Test 4: Does Model 2 fit the data? (A3 vs. 2)Do not cite

Appendix C

- Page 405 of 677 Test 5a: Does Model 3 fit the data? (A3 vs 3) Test 5b: Is Model 3 better than Model 2? (3 vs. 2)

Test 6a: Does Model 4 fit the dat a? (A3 vs 4) Test 6b: Is Model 4 better than Model 2? (4 vs. 2)

Test 7a: Does Model 5 fit the data? (A3 vs 5) Test 7b: Is Model 5 better than Model 3? (5 vs. 3) Test 7c: Is Model 5 better than Model 4? (5 vs. 4)

T ests of Interest

Test

- 2*log(Likelihood Ratio) D. F. p

- value

-------- ------------------------ ------ -------------- Test 1 37.01 6 < 0.0001

Test 2 28.25 3 < 0.0001

Test 3 28.25 3 < 0.0001

Test 4 0.8047 2 0.6687

Test 5a 0.8047 2 0.6687

Test 5b

- 1.137e

- 013 0 N/A

Test 6a 0.7599 1 0.3833

Test 6b 0.04476 1 0.8325

Test 7a 0.09163 0 N/A

Test 7b 0.7131 2 0.7001

Test 7c 0.6683 1 0.4136

The p

- value for Test 1 is less than .0 5. There appears to be a difference between response and/or variances among the dose levels, it seems appropriate to model the data.

The p

- value for Test 2 is less than .1. Consider running

a non

- homogeneous variance model.

The p

- value for Test 3 is less than .1. You may want to

consider a different variance model.

The p

- value for Test 4 is greater than .1. Model 2 seems to adequately describe the data.

The p

- value for Test 5a is greater than .1. Model 3 seemsDo not cite

Appendix C

- Page 406 of 677 to adequately describe the data.

Degrees of freedom for Test 5b are less than or equal to 0.

The Chi

- Square test for fit is not valid.

The p

- value for Test 6a is greater than .1. Model 4 seems to adequately describe the d ata.

The p

- value for Test 6b is greater than .05. Model 4 does

not seem to fit the data better than Model 2.

Degrees of freedom for Test 7a are less than or equal to 0.

The Chi

- Square test for fit is not valid.

The p

- value for Test 7b is greater than .05. Model 5 does

not seem to fit the data better than Model 3.

The p

- value for Test 7c is greater than .05. Model 5 does

not seem to fit the data better than Model 4.

Benchmark Dose Computations:

Specif ied Effect = 1.000000

Risk Type = Estimated standard deviations from control

Confidence Level = 0.950000

BMD and BMDL by Model

Model BMD BMDL

------- ------------ --- ------- 2 20.2989 4.57536 3 20.2989 Bad completion

4 16.7073 3.43496

5 3.92994 3.66097Do not cite

Appendix C

- Page 407 of 677Do not cite

Appendix C

- Page 408 of 677Do not cite

Appendix C

- Page 409 of 677Do not cite

Appendix C

- Page 410 of 677 Morseth 6 Month LH Study

- Exponential (Nonhomogeneous Variance) Model

Results

==================================================================== Exponential Model. (Version: 1.7; Date: 12/10/2009) Input Data File: C:/Usepa/BMDS212/Data/exp_Actualruns_Setting.(d) Gnupl ot Plotting File:

Thu Jul 29 14:00:34 2010

====================================================================

BMDS Model Run

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

The form of the response function by Mo del: Model 2: Y[dose] = a * exp{sign * b * dose} Model 3: Y[dose] = a * exp{sign * (b * dose)^d} Model 4: Y[dose] = a * [c

- (c

- 1) * exp{

- b * dose}]Do not cite

Appendix C

- Page 411 of 677 Model 5: Y[dose] = a * [c

- (c

- 1) * exp{

- (b * dose)^d}]

Note: Y[d ose] is the median response for exposure = dose; sign = +1 for increasing trend in data; sign =

- 1 for decreasing trend.

Model 2 is nested within Models 3 and 4.

Model 3 is nested within Model 5.

Model 4 is nested wit hin Model 5.

Dependent variable = Mean Independent variable = Dose Data are assumed to be distributed: normally Variance Model: exp(lnalpha +rho *ln(Y[dose])) The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)

T otal number of dose groups = 4 Total number of records with missing values = 0

Maximum number of iterations = 250 Relative Function Convergence has been set to: 1e

- 008 Parameter Convergence has been set to: 1e

- 008

MLE solution provided: Exa ct

Initial Parameter Values

Variable Model 2 Model 3 Model 4 Model 5

-------- ------- ------- ------- ------- lnalpha

- 6.65625

- 6.65625

- 6.65625

- 6.65625

rho 2.77485 2.77485 2.77485 2.77485

a 1497.19 1497.19 3812.55 3812.55

b 0.0471466 0.0471466 0.14585 0.14585

c

-- -- 0.21433 0.21433

d

-- 1

-- 1

Parameter Estimates by Model

Variable Model 2 Model 3 Model 4 Model 5Do not cite

Appendix C

- Page 412 of 677 -------- ------- ------- ------- ------- lnalpha

- 7.30123

- 7.30123

- 7.07367

- 6.96831

rho 2.84795 2.84795 2.81591 2.80183

a 3418.7 3418.7 3725.66 3553.63

b 0.047111 3 0.0471113 0.124728 0.213467

c

-- -- 0.210352 0.241407

d

-- 1

-- 2.9234

NC = No Convergence

Table of Stats From Input Data

Dose N Obs Mean Obs Std Dev

----- --- ---------- ------------- 0 10 3336 3138

1.8 10 3631 2732

3.65 10 2500 1897

29.4 10 858 416

Estimated Values of Interest

Model Dose Est Mean Est Std Scaled Residual

------- ------ ---------- --------- -------------- -- 2 0 3419 2797

- 0.0935

1.8 3141 2479 0.6254

3.65 2879 2190

- 0.5468

29.4 855.7 389.2 0.01852

3 0 3419 2797

- 0.0935

1.8 3141 2479 0.6254

3.65 2879 2190

- 0.5468

29.4 855.7 389. 2 0.01852

4 0 3726 3105

- 0.3968

1.8 3134 2434 0.6456

3.65 2650 1922

- 0.2464

29.4 858.9 393.4

- 0.007039

5 0 3554 2891

- 0.2381

1.8 3394 2711 0.2765

3.65 2522 1789

- 0.03959

29.4 8 57.9 394.8 0.001045Do not cite

Appendix C

- Page 413 of 677 Other models for which likelihoods are calculated:

Model A1: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma^2

Model A2: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma(i)^2

Model A3: Yij = Mu(i) + e(ij) Var{e(ij)} = exp(lalpha + log(mean(i)) * rho)

Model R: Yij = Mu + e(i) Var{e(ij)} = Sigma^2

Likelihoods of Interest

Model Log(likelihood) DF AIC

------- ----------------- ---- ------------ A1

- 327.446 5 664.892

A2

- 313.3212 8 642.64 25

A3

- 313.4172 6 638.8345

R

- 331.828 2 667.6559

2

- 314.0727 4 636.1453

3

- 314.0727 4 636.1453

4

- 313.7385 5 637.477

5

- 313.4172 6 638.8345

Additive constant for all log

- likelihoods =

- 36.76. This constant added to the above values gives the log

- likelihood including the term that does not depend on the model parameters.

Explanation of Tests

Test 1: Does response and/or variances differ among Dose levels? (A2 vs. R)

Test 2: Are Variances Homogeneous? (A2 vs. A1) Test 3: Are variances adequately modeled? (A2 vs. A3) Test 4: Does Model 2 fit the data? (A3 vs. 2)Do not cite

Appendix C

- Page 414 of 677 Test 5a: Does Model 3 fit the data? (A3 vs 3) Test 5b: Is Model 3 better than Model 2? (3 vs. 2)

Test 6a: Does Model 4 fit the data? (A3 vs 4) Test 6b: Is Model 4 better than Model 2? (4 vs. 2)

Test 7a: Does Model 5 fit the data? (A3 vs 5) Test 7b: Is Model 5 better than Model 3? (5 vs. 3) Test 7c: Is Model 5 better than Model 4? (5 vs. 4)

Tests of Interest

Test

- 2*log(Likelihood Ratio) D. F. p

- value

-------- ------------------------ ------ -------------- Test 1 37.01 6 < 0.0001

Test 2 28.25 3 < 0.0001

Test 3 0.192 2 0.9084

Test 4 1.311 2 0.5192

Test 5a 1.311 2 0.5192

Test 5b

- 1.137e

- 013 0 N/A

Test 6a 0.6425 1 0.4228

Test 6b 0.6683 1 0.4136

Test 7a

- 1.137e

- 013 0 N/A

Test 7b 1.311 2 0.5192

Test 7c 0.6425 1 0.4228

The p

- value for Test 1 is less than .05. There appears to be a difference between response and/or variances among the dose levels, it seems appropriate to model the data.

The p

- value for Test 2 is less than .1. A non

- homogeneous

variance model appears to be appropriate.

The p

- value for Test 3 is greater than .1. The modeled variance appears to be appropriate here.

The p

- value for Test 4 is greater than .1. Model 2 seems to adequately describe the data.Do not cite

Appendix C

- Page 415 of 677 The p

- value for Test 5a is greater than .1. Model 3 seems to adequately describe the data.

Degrees of freedom for Test 5b are less than or equal to 0.

The Chi

- Square test for fit is not valid.

The p

- value for Test 6a is greater than .1. M odel 4 seems to adequately describe the data.

The p

- value for Test 6b is greater than .05. Model 4 does

not seem to fit the data better than Model 2.

Degrees of freedom for Test 7a are less than or equal to 0.
The Chi

- Square tes t for fit is not valid.

The p

- value for Test 7b is greater than .05. Model 5 does

not seem to fit the data better than Model 3.

The p

- value for Test 7c is greater than .05. Model 5 does

not seem to fit the data better than Model 4.

Benchmark Dose Computations:

Specified Effect = 1.000000

Risk Type = Estimated standard deviations from control

Confidence Level = 0.950000

BMD and BMDL by Model

Model BMD BM DL

------- ------------ ---------- 2 36.1811 17.0918

3 36.1811 17.0918 4 0 0 Not computed

5 0 0 Not computedDo not cite

Appendix C

- Page 416 of 677Do not cite

Appendix C

- Page 417 of 677Do not cite

Appendix C

- Page 418 of 677Do not cite

Appendix C

- Page 419 of 677 ________________________________________ ________________________

4. Minnema One Month LH Study

Model (Constant Variance) BMD BMDL p

- value AIC

Exponential 2 653.3 245.1 0.40 274.6 Exponential 3 653.3 245.1 0. 40 274.6 Exponential 4 Not Computed Not Computed

-- -- Exponential 5 Not Computed Not Computed

-- -- Hill Not Computed Not Computed

-- -- Linear 500.8 240.7 0.38 274.7 Polynomial 500.8 239.9 0.38 274.7

Power 500.8 240.7 0.38 274.7Do not cite

Appendix C

- Page 420 of 677 Minnema One M onth LH Study

- Exponential (Constant Variance) Model Results

==================================================================== Exponential Model. (Version: 1.7; Date: 12/10/2009) Input Data File: C:/Usepa/BMDS212/Data/exp_Actualruns_ Setting.(d) Gnuplot Plotting File:

Fri Jul 23 08:35:00 2010

====================================================================

BMDS Model Run

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

The form of the response function by Model: Model 2: Y[dose] = a * exp{sign * b * dose} Model 3: Y[dose] = a * exp{sign * (b * dose)^d} Model 4: Y[dose] = a * [c

- (c

- 1) * exp{

- b * dose}] Model 5: Y[dose] = a * [c

- (c

- 1) * exp{

- (b * dose)^d}]

Note: Y[dose] is the median response for exposure = dose; sign = +1 for increasing trend in data; sign =

- 1 for decreasing trend.

Model 2 is nested within Models 3 and 4.

Model 3 is nested within Model 5.

Model 4 is nested within Model 5.

Dependent variable = Mean Independent variable = Dose Data are assumed to be distributed: normally Variance Model: exp(lnalpha +rho *ln(Y[dose]))

rho is set to 0.

A constant variance model is fit.Do not cite

Appendix C

- Page 421 of 677 Total number of dose groups = 5 Total number of records with missing values = 0

Maximum number of iterations = 250 Relative Function Convergence has been set to: 1e

- 008 Parameter Convergence has been set to: 1e

- 008

MLE solution provided: Exact

Initial Parameter Values

Variable Model 2 Model 3 Model 4 Model 5

-------- ------- ------- ------- ------- lnalpha 1.65681 1.65681 1.65681 1.65681 rho(S) 0 0 0 0

a 3.0427 3.0427 4.305 4.305

b 0.00143069 0.00143069 0.0127345 0.0127345

c

-- -- 0.588463 0.588463

d

-- 1

-- 1

(S) = Specified

Parameter Estimates by Model

Variable Model 2 Model 3 Model 4 Model 5

-------- ------- ------- ------- ------- l nalpha 1.68618 1.68618 1.65872 1.65872

rho 0 0 0 0

a 3.55899 3.55899 4.04195 4.04195

b 0.00161956 0.00161956 0.128035 0.128035

c

-- -- 0.669638 0.669638

d

-- 1

-- 1

NC = No Convergence

Table of Stats From Input Data

Dose N Obs Mean Obs Std Dev

----- --- ---------- -------------Do not cite

Appendix C

- Page 422 of 677 0 20 4.1 2.52

2.5 20 3.51 2. 41

5 20 3.53 2.57

40 20 2.66 1.71

200 20 2.75 2.43

Estimated Values of Interest

Model Dose Est Mean Est Std Scaled Residua l

------- ------ ---------- --------- ---------------- 2 0 3.559 2.324 1.041

2.5 3.545 2.324

- 0.06662

5 3.53 2.324

- 0.0005584

40 3.336 2.324

- 1.301

200 2.574 2.324 0.3382

3 0 3.559 2.324 1.041

2.5 3.545 2 .324

- 0.06662

5 3.53 2.324

- 0.0005584

40 3.336 2.324

- 1.301

200 2.574 2.324 0.3382

4 0 4.042 2.292 0.1133

2.5 3.676 2.292

- 0.3243

5 3.411 2.292 0.233

40 2.715 2.292

- 0.1066

200 2.707 2.292 0.08461

5 0 4.042 2.292 0.1133

2.5 3.676 2.292

- 0.3243

5 3.411 2.292 0.233

4 0 2.715 2.292

- 0.1066

200 2.707 2.292 0.08461

Other models for which likelihoods are calculated:

Model A1: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma^2

Mo del A2: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma(i)^2Do not cite

Appendix C

- Page 423 of 677 Model A3: Yij = Mu(i) + e(ij) Var{e(ij)} = exp(lalpha + log(mean(i)) * rho)

Model R: Yij = Mu + e(i) Var{e(ij)} = Sigma^2

Likelihoods of Interest

Model Log(likelihood) DF AIC

------- ----------------- ---- ------------ A1

- 132.8405 6 277.6809

A2

- 130.8789 10 281.7577

A3

- 132.8405 6 277.6809

R

- 135.53 2 275.0599

2

- 13 4.3092 3 274.6183

3

- 134.3092 3 274.6183

4

- 132.9359 4 273.8719

5

- 132.9359 4 273.8719

Additiv e constant for all log

- likelihoods =

- 91.89. This constant added to the above values gives the log

- likelihood including the term that does not depend on the model parameters.

Explanation of Tests

Test 1: D oes response and/or variances differ among Dose levels? (A2 vs. R) Test 2: Are Variances Homogeneous? (A2 vs. A1) Test 3: Are variances adequately modeled? (A2 vs. A3) Test 4: Does Model 2 fit the data? (A3 vs. 2)

Test 5a: Does Model 3 fit the data? (A3 vs 3) Test 5b: Is Model 3 better than Model 2? (3 vs. 2)

Test 6a: Does Model 4 fit the data? (A3 vs 4) Test 6b: Is Model 4 better than Model 2? (4 vs. 2)

Test 7a: Does Model 5 fit the data? (A3 vs 5)

Test 7b: Is Model 5 bette r than Model 3? (5 vs. 3) Test 7c: Is Model 5 better than Model 4? (5 vs. 4)Do not cite

Appendix C

- Page 424 of 677 Tests of Interest

Test

- 2*log(Likelihood Ratio) D. F. p

- value

-------- ------------------------ ------ -------------- Test 1 9.302 8 0.3174

Test 2 3.923 4 0.4165

Test 3 3.923 4 0.4165

Test 4 2.937 3 0.4014

Test 5a 2.937 3 0.4014
Test 5b 0 0 N/A

Test 6a 0.191 2 0.9089

Test 6b 2.746 1 0.09747

Test 7a 0.191 2 0.9089

Test 7b 2.746 1 0.09747

Test 7c 2.274e

- 013 0 N/A

The p

- value for Test 1 is greater than .05. There may not be a diffence between responses and/or variances among the dose levels Modelling the data with a dose/response cur ve may not be appropriate.

The p

- value for Test 2 is greater than .1. A homogeneous variance model appears to be appropriate here.

The p

- value for Test 3 is greater than .1. The modeled variance appears to be appropriate here.

The p

- value for Test 4 is greater than .1. Model 2 seems to adequately describe the data.

The p

- value for Test 5a is greater than .1. Model 3 seems to adequately describe the data.

Degrees of freedom for Test 5b are less than or eq ual to 0.
The Chi

- Square test for fit is not valid.

The p

- value for Test 6a is greater than .1. Model 4 seems to adequately describe the data.

The p

- value for Test 6b is greater than .05. Model 4 doesDo not cite

Appendix C

- Page 425 of 677 not seem to fit the data b etter than Model 2.

The p

- value for Test 7a is greater than .1. Model 5 seems to adequately describe the data.

The p

- value for Test 7b is greater than .05. Model 5 does

not seem to fit the data better than Model 3.

Degrees of freedom for Test 7c are less than or equal to 0.
The Chi

- Square test for fit is not valid.

Benchmark Dose Computations:

Specified Effect = 1.000000

Risk Type = Estimated standard deviations from control

Confidence Level = 0.950000

BMD and BMDL by Model

Model BMD BMDL

------- ------------ ---------- 2 653.287 245.122

3 653.287 245.122 4 0 0 Not computed 5 0 0 Not computedDo not cite

Appendix C

- Page 426 of 677Do not cite

Appendix C

- Page 427 of 677Do not cite

Appendix C

- Page 428 of 677Do not cite

Appendix C

- Page 429 of 677 BMD Output Summaries

– AUC

1. AUC for Steady State Serum Levels of Total Triazine Equivalents

Model (Constant

Va riance) BMD BMDL p

- value AIC

Exponential 2 6.73 3.66 0.25 407.8 Exponential 3 6.73 3.66 0.25 407.8 Exponential 4 2.19 0.82 0.89 405.1 Exponential 5 2.19 0.82 0.89 405.1

Hill 2.08 0.65 0.92 404.9

Linear 9.91 7.00 0.10 410.1 Polynomial 9.91 7.00 0.10 410.1Do not cite

Appendix C

- Page 430 of 677 Power 9.91 7.00 0.10 410.1

==================================================================== Hill Model. (Version: 2.15; Date: 10/28/2009) Input Data File: C:/Usepa/BMDS212/Data/hil_Actualruns_Opt.(d) Gnuplot Plotting F ile: C:/Usepa/BMDS212/Data/hil_Actualruns_Opt.plt

Fri Jul 30 12:15:38 2010

====================================================================

BMDS Model Run

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

The form of the response function is:

Y[dose] = intercept + v*dose^n/(k^n + dose^n)

Dependent variable = Mean Independent variable = Dose

rho is set to 0 Power parameter restricted to be greater than 1

A constant variance model is fit

Tot al number of dose groups = 6 Total number of records with missing values = 0

Maximum number of iterations = 250 Relative Function Convergence has been set to: 1e

- 008 Parameter Convergence has been set to: 1e

- 008

Default Ini tial Parameter Values alpha = 68.6432 rho = 0 Specified intercept = 21.848

v =

- 13.348

n = 1.1704

k = 1.67918Do not cite

Appendix C

- Page 431 of 677 Asymptotic Correlation Matrix of Parameter Estimates

( *** The model parameter(s)

- rho

- n have been estimated at a boundary point, or have been s pecified by the user, and do not appear in the correlation matrix )

alpha intercept v k

alpha 1 2.4e

- 009 1.1e

- 008

- 2.1e

- 008

intercept 2.4e

- 009 1

- 0.37

- 0.52

v 1.1e

- 008

- 0.37 1

- 0.48

k

- 2.1e

- 008

- 0.52

- 0.48 1

Parameter Estimates

95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit alpha 63.7164 10.2688 43.5899 83.843 intercept 21.819 1.88733 18.1199 25.5181

v

- 14.7403 3.24361

- 21.0977

- 8.38296

n 1 NA

k 1.76071 1.38154

- 0 .947057 4.46847

NA

- Indicates that this parameter has hit a bound implied by some inequality constraint and thus

has no standard error.

Table of Data and Estimated Values of Interest

Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res.

------ --- -------- -------- ----------- ----------- ----------

0 16 21.8 21.8 7 7.98 0.0145

0.4545 11 18.6 18.8 10.8 7 .98

- 0.0933Do not cite

Appendix C

- Page 432 of 677 1.331 12 16 15.5 9.3 7.98 0.248

3.024 17 11.8 12.5 7 7.98

- 0.358

6.406 7 11.9 10.3 9 7.98 0.538

13.17 14 8.5 8.82 7.6 7.98

- 0.149

Model Descriptions for likelihoods calculated

Model A1: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma^2

Model A2: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma(i)^2

Model A3: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma^2 Model A3 uses any fixed variance parameters that were specified by the user

Model R: Yi = Mu + e(i) Var{e(i)} = Sigma^2

Likelihoods of Interest

Model Log(likelihood) # Param's AIC

A1

- 198.190188 7 410.380377

A2

- 196.288688 12 416.577376

A3

- 198.190188 7 410.380377

fitted

- 198.446043 4 404.892085

R

- 209.919649 2 423.839298

Explanation of Tests

Test 1: Do responses and/or variances differ among Dose levels? (A2 vs. R) Test 2: Are Variances Homogeneous? (A1 vs A2) Test 3: Are variances adequately modeled? (A2 vs. A3) Test 4: Does the Model for the Mean Fit? (A3 vs. fitted)Do not cite

Appendix C

- Page 433 of 677 (Note: When rho=0 the results of Test 3 and Test 2 will be th e same.)

Tests of Interest

Test

- 2*log(Likelihood Ratio) Test df p

- value

Test 1 27.2619 10 0.002367

Test 2 3.803 5 0.5781

Test 3 3.803 5 0.5781

Test 4 0.511709 3 0.9163

The p

- value for Test 1 is less than .05. There appears to be a difference between response and/or variances among the dose levels It seems appropriate to model the data

The p

- value for Test 2 is greater than .1. A homogeneous variance model appears to be appropriate here

The p

- value for Test 3 is greater than .1. The modeled variance appears to be appropriate here

The p

- value for Test 4 is greater than .1. The model chosen seems to adequately describe the data

Benchmark Dose Computation

Specified effect = 1

Risk Type = Estimated standard deviations from the control mean

Confidence level = 0.95

BMD = 2.07966

BMDL = 0.64721Do not cite

Appendix C

- Page 434 of 677 2. Predicted Daily Steady State Serum AUC

Model (Constant Variance) BMD BMDL p

- value AIC

Exponential 2 161.7 88.0 0.25 407.8 Exponential 3 161.7 88.0 0.25 407.8 Exponential 4 52.5 19. 8 0.87 405.1 Exponential 5 52.5 19.8 0.87 405.1

Hill 49.9 15.6 0.92 404.9

Linear 238.2 168.2 0.10 410.1 Polynomial 238.2 168.2 0.10 410.1Do not cite

Appendix C

- Page 435 of 677 Power 238.2 168.2 0.10 410.1

==================================================================== Hill Model. (Version: 2.15; Date: 10/28/2009) Input Data File: C:/Usepa/BMDS212/Data/hil_Actualruns_Opt.(d) Gnuplot Plotting File: C:/Usepa/BMDS212/Data/hil_Actualruns_Opt.plt

Fri Jul 30 12:45:13 2010

=================================== =================================

BMDS Model Run

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

The form of the response function is:

Y[dose] = intercept + v*dose^n/(k^n + dose^n)

Dependent variable = Mean Indep endent variable = Dose

rho is set to 0 Power parameter restricted to be greater than 1

A constant variance model is fit

Total number of dose groups = 6 Total number of records with missing values = 0

Maximum number of iterations = 250 Relative Function Convergence has been set to: 1e

- 008 Parameter Convergence has been set to: 1e

- 008

Default Initial Parameter Values alpha = 68.6432

rho = 0 Specified intercept = 21.848

v =

- 13.348

n = 1.17066

k = 40.3586

Asymptotic Correlation Matrix of Parameter Es timatesDo not cite

Appendix C

- Page 436 of 677 ( *** The model parameter(s)

- rho

- n have been estimated at a boundary point, or have been specified by the user, and do not appear in the correlation matrix )

alpha interce pt v k

alpha 1 8.8e

- 010 9.7e

- 009

- 1.9e

- 008

intercept 8.8e

- 010 1

- 0.37

- 0.52

v 9.7e

- 009

- 0.37 1

- 0.48

k

- 1.9e

- 008

- 0. 52

- 0.48 1

Parameter Estimates

95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Co nf. Limit alpha 63.7164 10.2688 43.5899 83.8429 intercept 21.8193 1.88737 18.1201 25.5185

v

- 14.7416 3.24394

- 21.0 996

- 8.38356

n 1 NA

k 42.3243 33.205

- 22.7563 107.405

NA

- Indicates that this parameter has hit a bound implied by some inequality constrai nt and thus

has no standard error.

Table of Data and Estimated Values of Interest

Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res.

------ --- -------- -------- ----------- ----------- ----------

0 16 21.8 21.8 7 7.98 0.0144

10.93 11 18.6 18.8 10.8 7.98

- 0.093

31.99 12 16 15.5 9.3 7.98 0.248

72.68 17 11.8 12.5 7 7.98

- 0.358Do not cite

Appendix C

- Page 437 of 677 153.9 7 11.9 10.3 9 7.98 0.538

316.4 14 8.5 8.82 7.6 7.98

- 0.149

Model Descriptions for likelihoods calculated

Model A1: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma^2

Model A2: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma(i)^2

Model A3: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma^2 Model A3 uses any fixed variance p arameters that were specified by the user

Model R: Yi = Mu + e(i) Var{e(i)} = Sigma^2

Likelihoods of Interest

Model Log(likelihood) # Param's AIC

A1

- 198.190 188 7 410.380377

A2

- 196.288688 12 416.577376

A3

- 198.190188 7 410.380377

fitted

- 198.446021 4 404.892043

R

- 209.919 649 2 423.839298

Explanation of Tests

Test 1: Do responses and/or variances differ among Dose levels? (A2 vs. R) Test 2: Are Variances Homogeneous? (A1 vs A2) Test 3: Are variances adequately modeled? (A2 vs. A3) Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) (Note: When rho=0 the results of Test 3 and Test 2 will be the same.)Do not cite

Appendix C

- Page 438 of 677 Tests of Interest

Test

- 2*log(Likelihood Ratio) Test df p

- value

Test 1 27.2619 10 0.002367

Test 2 3.803 5 0.5781

Test 3 3.803 5 0.5781

Test 4 0.511666 3 0.9163

The p

- value for Test 1 is less than .05. There appears to be a difference between response and/or variances among the dose levels It seems appropriate to model the data

The p

- value for Test 2 is greater than .1. A homogeneous variance model appears to be appropriate here

The p

- value for Test 3 is greater than .1. The modeled variance appears to be appropriate here

The p

- value for Test 4 is greater than .1. The model chosen seems to adequately describe the data

Benchmark Dose Computation

Specified effect = 1

Risk Type = Estimated standard deviations from the control mean

Confidence level = 0.95

BMD = 49.9819

BMDL = 15.5588Do not cite

Appendix C

- Page 439 of 677Do not cite

Ap pendix D1

- Page 440 of 677 APPENDIX D.1: CHARAC TERIZATION OF ATRAZI NE MONITORIN G PROGRAM (AMP) CWS CHEMOGRAPH

SHAPES AND MONITORIN G DATA FOR CWS WITH THE GREATEST TCT CON CENTRATIONS

Worksheet Table /

Parameter Description

Max TCT Concentration by Year D.1.a Table summarizing the maximum concentrations of atrazine (Atz) and tota l chloro

- triazines (TCT) in both source and treated water samples by year for all of the CWS in the AMP.

Days Exceeding TCT
Conc This worksheet applies to the subset of 44 CWS that had a detect of > 15 ug/L TCT in one or more years. The summary table s are for all of the years (2004

- 09) for those CWS and not just the years with the 15 ug/L or greater detect.

D.1.b Summary of the total number of samples taken by month and year as well as the total number of

source water samples with TCT concentrations > 5, 10, or 20 ug/L (ppb).

D.1.c Summary of Days Exceeding TCT concentrations of 5, 10, or 20 ug/L, assuming sampled

concentration extends to next sampling event (stairstep approach)

NOTE: This may result in an estimate of more days exceeding the tar get concentration than days in the month where the sample was taken near the end of the month and the period until the next sample extends into the next month. Peak Analysis D.1.d Analysis of the number of peaks, including date, duration of rise, peak con centration, duration of decline, and shape of decline for each of the CWS years that had detects >15 ug/L. Only the source water chemographs were analyzed, even if concentrations > 15 ug/L may have occurred in treated water samples. (that cutoff is for ei ther source or treated, although the peak analysis is for the source water)

D.1.e Summary of Annual Frequency of Peak Occurrences by CWS Grouped by Source Water Type

D.1.f Summary of Annual Frequency of Peak Occurrences by Source Water Type

CWS Gr aphs Chemographs for each CWS sample year that had detects of > 15 ug/L TCT.

Remaining worksheets Monitoring data for TCT components for each of the 44 CWs that had detects of >15 ug/L TCT in 1

or more years.Do not cite

Ap pendix D1

- Page 441 of 6 77 Worksheet Table /

Parameter Description

Atz Source Atrazine concentration, in u g/L, in the source water sample

G28273
Source Didealkylatrazine (DDA) concentration, in ug/L, in the source water sample

G28279
Source Deisopropylatrazine (DIA) concentration, in ug/L, in the source water sample

G30033
Source Deethylatrazine (DEA) co ncentration, in ug/L, in the source water sample

Sim
Source Simazine concentration, in ug/L, in the source water sample

TCT
Source Summation of total chloro

- triazine concentrations (atrazine, simazine, DDA, DIA, DEA), in ug/L, in

the source water sampl e

Atz

Treated Atrazine concentration, in ug/L, in the treated water sample

G28273

Treated Didealkylatrazine (DDA) concentration, in ug/L, in the treated water sample

G28279

Treated Deisopropylatrazine (DIA) concentration, in ug/L, in the treated wate r sample

G30033

Treated Deethylatrazine (DEA) concentration, in ug/L, in the treated water sample

Sim

Treated Simazine concentration, in ug/L, in the treated water sample

TCT

Treated Summation of total chloro

- triazine concentrations (atrazine, simazi ne, DDA, DIA, DEA), in ug/L, in

the treated water sampleDo not cite

Appendix D2

- Page 442 of 677 APPENDIX D.2 SAMPLIN G FREQUENCY CHEMOGRA PHS

Table D

- 2. 1 : Analysis of Chemograph Peaks (Spikes), Maximum Detects, and

4

- Day Averages based on 7

- Day Fixed Sampling Inte rvals for 2 AEEMP Monitoring Sites

Site/Year Sampling

Interval No

Upward Spikes Max

Detect Spike #

w/ Max
Conc Max 4

- da

average Days 4

-

da avg >20 ug/L Days 4

-

da avg >40 ug/L

MO

- 01,

2006 ( Figure D

2. 1 ) Actual 1 4 1 06.0 2 71.9 23 8

7 da, beg day 1 2 36.8 1 35.0 16 0
7 da, beg day 2 2 36.8 1 34.9 15 0 7 da, beg day 3 3 82.8 1 73.6 26 9
7 da, beg day 4 2 82.8 1 75.6 26 14 7 da, beg day 5 2 43.1 1 40.1 19 1
7 da, beg day 6 2 106.0 1 95.5 21 12 7 da, beg da y 7 2 43.1 1 40.4 16 2

MO

- 01,

2007 ( Figure D

2. 2 ) Actual 2 91.6 2 59.7 13 (6,7) 4

7 da, beg day 1 2 42.0 2 37.3 11 (2,9) 0
7 da, beg day 2 2 33.3 2 29.6 9 (2,7) 0 7 da, beg day 3 2 26.2 2 23.3 6 (2,4) 0

7 da, beg day 4 2 29.7 1 28.3 10 0 7 da, beg day 5 2 23.0 1 21.5 4 0
7 da, beg day 6 2 91.6 2 80.4 15 (2,13) 9 7 da, beg day 7 2 71.8 2 62.9 14 (3,11) 7

MO

- 01,

2008 ( Figure D

2. 3 ) Actual 5 78.2 2 63. 0 8 4

7 da, beg day 1 1 70.1 1 61.3 11 7 7 da, beg day 2 1 78.2 1 68.3 11 7 7 da, beg day 3 1 51.7 1 44.6 8 4
7 da, beg day 4 2 51.7 1 44.8 10 4 7 da, beg day 5 2 51.7 1 44.8 10 4
7 da, beg day 6 2 12.5 1 11.4 0 0 7 da, beg day 7 1 8.1 1 7.3 0 0

MO

- 0 1,

2009 ( Figure D

2. 4 ) Actual 3 71.0 2 64.7 9 7

7 da, beg day 1 2 62.4 2 54.8 11 6
7 da, beg day 2 2 57.8 2 53.4 16 8 7 da, beg day 3 2 34.9 2 33.0 13 0
7 da, beg day 4 2 71.0 2 63.3 14 8 7 da, beg day 5 2 63.1 2 55.6 11 6
7 da, beg day 6 2 63.2 2 55.7 11 6 7 da, beg day 7 2 61.6 2 54.0 11 5

1 Actual = values generated based on the entire chemograph (daily monitoring). The next 7 rows represent 7

- day fixed interval sampling beginning on the 1 st throu gh 7 th day of the week.Do not cite

Appendix D2

- Page 443 of 677 Figure D

- 2. 1 : Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for MO

- 01, 2006

(a) Actual Chemograph (red arrows point to spikes)Do not cite

Appendix D2

- Page 444 of 677 (b) Sample d at 7

- day fixed intervals, beginning on Day 1Do not cite

Appendix D2

- Page 445 of 677 (c) Sampled at 7

- day fixed intervals, beginning on Day 2Do not cite

Appendix D2

- Page 446 of 677 (d) Sampled at 7

- day fixed intervals, beginning on Day 3Do not cite

Appendix D2

- Page 447 of 677 (e) Sampled at 7

- day fixed intervals, beginning on Day 4Do not cite

Appendix D2

- Page 448 of 677 (f) Sampled at 7

- day f ixed intervals, beginning on Day 5Do not cite

Appendix D2

- Page 449 of 677 (g) Sampled at 7

- day fixed intervals, beginning on Day 6Do not cite

Appendix D2

- Page 450 of 677 (h) Sampled at 7

- day fixed intervals, beginning on Day 7Do not cite

Appendix D2

- Page 451 of 677 Figure D

- 2. 2 : Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for MO

- 01, 2007

(a) Actual Chemograph (red arrows point to spikes)Do not cite

Appendix D2

- Page 452 of 677 (b) Sampled at 7

- day fixed intervals, beginning on Day 1Do not cite

Appendix D2

- Page 453 of 677 (c) Sampled at 7

- day fixed intervals, beginning on Day 2Do not cite

Appendix D2

- Page 454 of 677 (d) Samp led at 7

- day fixed intervals, beginning on Day 3Do not cite

Appendix D2

- Page 455 of 677 (e) Sampled at 7

- day fixed intervals, beginning on Day 4Do not cite

Appendix D2

- Page 456 of 677 (f) Sampled at 7

- day fixed intervals, beginning on Day 5Do not cite

Appendix D2

- Page 457 of 677 (g) Sampled at 7

- day fixed intervals, beginning on Day 6Do not cite

Appendix D2

- Page 458 of 677 (h) Sampled at 7

- day fixed intervals, beginning on Day 7Do not cite

Appendix D2

- Page 459 of 677 Figure D

- 2. 3 : Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for MO

- 01, 200 8

(a) Actual Chemograph (red arrows point to spikes )Do not cite

Appendix D2

- Page 460 of 677 (b) Sampled at 7

- day fixed intervals, beginning on Day 1Do not cite

Appendix D2

- Page 461 of 677 (c) Sampled at 7

- day fixed intervals, beginning on Day 2Do not cite

Appendix D2

- Page 462 of 677 (d) Sampled at 7

- day fixed intervals, beginning on Day 3Do not cite

Appendix D2

- Page 463 of 677 (e) Sampled at 7

- day fixed intervals, beginning on Day 4Do not cite

Appendix D2

- Page 464 of 677 (f) Sa mpled at 7

- day fixed intervals, beginning on Day 5Do not cite

Appendix D2

- Page 465 of 677 (g) Sampled at 7

- day fixed intervals, beginning on Day 6Do not cite

Appendix D2

- Page 466 of 677 (h) Sampled at 7

- day fixed intervals, beginning on Day 7Do not cite

Appendix D2

- Page 467 of 677 Figure D

- 2. 4 : Actual chemograph (blue) and resul ting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for MO

- 01, 200 9

(a) Actual Chemograph (red arrows point to spikes)Do not cite

Appendix D2

- Page 468 of 677 (b) Sampled at 7

- day fixed intervals, beginning on Day 1Do not cite

Appendix D2

- Page 469 of 677 (c) Sampled at 7

- day fixed intervals, beginning on D ay 2Do not cite

Appendix D2

- Page 470 of 677 (d) Sampled at 7

- day fixed intervals, beginning on Day 3Do not cite

Appendix D2

- Page 471 of 677 (e) Sampled at 7

- day fixed intervals, beginning on Day 4Do not cite

Appendix D2

- Page 472 of 677 (f) Sampled at 7

- day fixed intervals, beginning on Day 5Do not cite

Appendix D2

- Page 473 of 677 (g) Sampled at 7

- day fixed intervals, beginning on Day 6Do not cite

Appendix D2

- Page 474 of 677 (h) Sampled at 7

- day fixed intervals, beginning on Day 7Do not cite

Appendix D2

- Page 475 of 677 Table D

- 2. 2 : Analysis of Chemograph Peaks (Spikes), Maximum Detects, and

4

- Day Averages based on 7

- Day Fixed Sampling Intervals for 2 AEEMP Monitoring Sites

Site/Year Sampling

Interval No

Upward Spikes Max

Detect Spike #

w/ Max
Conc Max 4

- da

average Days 4

-

da avg >20 ug/L Days 4

-

da avg >40 ug/L

MO

- 02,

2006 ( Figure D

2. 5 ) Actual 7 43.9 1 43.2 25 3

7 da, beg day 1 2 32.7 1 31 .6 20 0
7 da, beg day 2 2 34.9 1 33.3 21 0 7 da, beg day 3 2 43.2 1 41.6 28 3
7 da, beg day 4 2 39.5 1 36.9 29 0 7 da, beg day 5 2 43.9 1 40.5 28 2
7 da, beg day 6 2 26.6 1 26.3 18 0 7 da, beg day 7 2 37.3 1 35.0 20 0

MO

- 02,

2007 ( Figure D

2. 6 ) Actual 11 16.2 1 10.8 0 0

7 da, beg day 1 4 7.0 1 6.9 0 0
7 da, beg day 2 4 10.9 3 9.9 0 0 7 da, beg day 3 3 9.2 1 8.6 0 0
7 da, beg day 4 3 16.2 1 14.4 0 0 7 da, beg day 5 4 10.9 1 9.6 0 0
7 da, beg day 6 3 8.2 1 8.0 0 0 7 da, beg day 7 3 9.1 1 8.5 0 0

MO

- 02,

2008 ( Figure D

2. 7 ) Actual 9 56.6 2 35.5 7 (4,3) 0

7 da, beg day 1 4 56.6 1 49.1 10 4
7 da, beg day 2 2 38.2 2 33.5 11 (4,7) 0 7 da, beg day 3 4 21.6 2 19.0 0 0
7 da, beg day 4 3 33.6 2 28.8 6 0 7 da, beg day 5 3 47.9 3 41.2 8 2
7 da, beg day 6 2 13.5 1 13.0 0 0 7 da, beg day 7 3 36.4 1 31.5 6 0

MO

- 02,

2009 ( Figure D

2. 8 ) Actual 14 155.2 11 68.9 2 1 (16,5) 8 (4,4)

7 da, beg day 1 4 108.9 2 96.2 27 (15,12) 19 (11,8)

7 da, beg day 2 4 155.2 3 133.5 18 (6,12) 10 7 da, beg day 3 5 66.0 2 58.2 20 (12,8) 7 7 da, beg day 4 3 49.0 2 43.6 11 3 7 da, beg day 5 3 45.4 2 42.0 14 3
7 da, beg day 6 2 48.6 2 44.5 13 4 7 da, beg day 7 3 17.8 2 17.2 0 0

1 Actual = values generated based on the entire chemograph (daily monitoring). The next 7 rows represent 7

- day fixed interval sampling beginning on the 1 st through 7 th day of the week.Do not cite

Appendix D2

- Page 476 of 677 Figure D

- 2. 5 : Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixe d intervals (red dots) for MO

- 02 , 200 6

(a) Actual Chemograph (red arrows point to spikes)

on Day 1Do not cite

Appendix D2

- Page 477 of 677Do not cite

Appendix D2

- Page 478 of 677 (c) Sampled at 7

- day fixed intervals , beginning on Day 2Do not cite

Appendix D2

- Page 479 of 677 (d) Sampled at 7

- day fixed intervals, beginning on Day 3Do not cite

Appendix D2

- Page 480 of 677 (e) Sampled at 7

- day fixed intervals, beginning on Day 4Do not cite

Appendix D2

- Page 481 of 677 (f) Sampled at 7

- day fixed intervals, beginning on Day 5Do not cite

Appendix D2

- Page 482 of 677 (g) Sampled at 7

- day fixed intervals, beginning on Day 6Do not cite

Appendix D2

- Page 483 of 677 (h) Sampled at 7

- day fixed intervals, beginning on Day 7Do not cite

Appendix D2

- Page 484 of 677 Figure D

- 2. 6 : Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for MO

- 0 2 , 200 7

(a) Actual Chem ograph (red arrows point to spikes)Do not cite

Appendix D2

- Page 485 of 677 (b) Sampled at 7

- day fixed intervals, beginning on Day 1Do not cite

Appendix D2

- Page 486 of 677 (c) Sampled at 7

- day fixed intervals, beginning on Day 2Do not cite

Appendix D2

- Page 487 of 677 (d) Sampled at 7

- day fixed intervals, beginning on Day 3Do not cite

Appendix D2

- Page 488 of 677 (e) Sampled at 7

- day fixed interv als, beginning on Day 4Do not cite

Appendix D2

- Page 489 of 677 (f) Sampled at 7

- day fixed intervals, beginning on Day 5Do not cite

Appendix D2

- Page 490 of 677 (g) Sampled at 7

- day fixed intervals, beginning on Day 6Do not cite

Appendix D2

- Page 491 of 677 (h) Sampled at 7

- day fixed intervals, beginning on Day 7Do not cite

Appendix D2

- Page 492 of 677 Figure D

- 2. 7 : Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for MO

- 0 2 , 200 8

(a) Actual Chemograph (red arrows point to spikes)Do not cite

Appendix D2

- Page 493 of 677 (b) Sampled at 7

- day fixed intervals, beginning on Day 1Do not cite

Appendix D2

- Page 494 of 677 (c) Sampled at 7

- d ay fixed intervals, beginning on Day 2Do not cite

Appendix D2

- Page 495 of 677 (d) Sampled at 7

- day fixed intervals, beginning on Day 3Do not cite

Appendix D2

- Page 496 of 677 (e) Sampled at 7

- day fixed intervals, beginning on Day 4Do not cite

Appendix D2

- Page 497 of 677 (f) Sampled at 7

- day fixed intervals, beginning on Day 5Do not cite

Appendix D2

- Page 498 of 677 (g) Sampled at 7

- day fixed int ervals, beginning on Day 6Do not cite

Appendix D2

- Page 499 of 677 (h) Sampled at 7

- day fixed intervals, beginning on Day 7Do not cite

Appendix D2

- Page 500 of 677 Figure D

- 2. 8 : Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for MO

- 0 2 , 200 9

(a) Actual Chemograph (red arrows point to spikes)Do not cite

Appendix D2

- Page 501 of 677 (b) Sampled at 7

- day fixed intervals, beginning on Day 1Do not cite

Appendix D2

- Page 502 of 677 (c) Sampled at 7

- day fixed intervals, beginning on Day 2Do not cite

Appendix D2

- Page 503 of 677 (d) Sampled at 7

- day fixed intervals, beginning on Day 3Do not cite

Appendix D2

- Page 504 of 677 (e) Sampled at 7

- day fixed intervals, beginning on Day 4Do not cite

Appendix D2

- Page 505 of 6 77 (f) Sampled at 7

- day fixed intervals, beginning on Day 5Do not cite

Appendix D2

- Page 506 of 677 (g) Sampled at 7

- day fixed intervals, beginning on Day 6Do not cite

Appendix D2

- Page 507 of 677 (h) Sampled at 7

- day fixed intervals, beginning on Day 7Do not cite

Appendix D2

- Page 508 of 677 Table D

- 2. 3 : Analysis of Chemograph Peaks (Spikes), Maximum Detects, and

4

- Day Averages based on 7

- Day Fixed Sampling Intervals for 2 Heidelberg Monitoring Sites

Site/Year Sampling

Interval No

Upward Spikes Max

Detect Spike #

w/ Max
Conc Max 4

- da

averag e Days 4

-

da avg >20 ug/L Days 4

-

da avg >40 ug/L

Maumee R, 2005 ( Figure

D

- 2. 9 ) Actual 1 5 22.7 1 14.7 0 0

7 da, beg day 1 3 9.5 1 8.6 0 0
7 da, beg day 2 2 8.3 1 7.6 0 0
7 da, beg day 3 2 7.9 1 7.2 0 0

7 da , beg day 4 2 8.5 1 8.1 0 0
7 da, beg day 5 3 22.7 1 20.3 1 0
7 da, beg day 6 2 15.5 1 13.9 0 0
7 da, beg day 7 3 11.2 1 10.0 0 0

Maumee R, 2006 ( Figur e D

- 2. 10 ) Actual 12 32.9 3 28.8 9 0

7 da, beg day 1 1 22.8 1 22.3 11 0
7 da, beg day 2 1 19.3 1 17.9 0 0
7 da, beg day 3 1 32.9 1 30.7 11 0
7 da, beg day 4 1 24.1 1 22.7 6 0
7 da, beg day 5 1 24.3 1 22.6 4 0
7 da, beg day 6 1 28.8 1 26.9 8 0
7 da, beg day 7 1 26.0 1 25.0 10 0

Maumee R, 2007 ( Figure

D

- 2. 11 ) Actual 11 12.1 1 9.4 0 0

7 da, beg day 1 4 8.6 1 7.5 0 0
7 da, beg day 2 3 12.1 1 10.5 0 0
7 da, beg day 3 3 8.9 1 7.8 0 0
7 da, beg day 4 3 8.1 1 7.1 0 0
7 da, beg day 5 4 5.2 1 4.5 0 0

7 da , beg day 6 4 3.6 2 3.5 0 0
7 da, beg day 7 5 9.6 1 8.4 0 0

Maumee R, 2008 ( Figure

D

- 2. 12 ) Actual 9 52.2 4 49.7 6 2

7 da, beg day 1 2 49.9 2 44.8 12 3
7 da, beg day 2 2 19.2 2 17.6 0 0
7 da, beg day 3 2 19 .2 2 17.6 0 0
7 da, beg day 4 2 9.5 2 9.1 0 0
7 da, beg day 5 3 46.5 2 41.8 10 3
7 da, beg day 6 3 52.2 2 46.6 11 4
7 da, beg day 7 3 49.9 2 44.8 12 3

1 Actual = values generated based on the entire chemograph (daily monitoring). The next 7 rows represent 7

- day fixed interval sampling beginning on the 1 st through 7 th day of the week.Do not cite

Appendix D2

- Page 509 of 677 Figure D

- 2. 9 : Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day f ixed intervals (red dots) for Maumee Riv er (Heidelberg)

, 200 5

(a) Actual Chemograph (red arrows point to spikes)Do not cite

Appendix D2

- Page 510 of 677 (b) Sampled at 7

- day fixed intervals, beginning on Day 1Do not cite

Appendix D2

- Page 511 of 677 (c) Sampled at 7

- day fixed intervals, beginning on Day 2Do not cite

Appendix D2

- Page 512 of 677 (d) Sampled at 7

- day fixed intervals, beginning on Day 3Do not cite

Appendix D2

- Page 513 of 677 (e) Sampled at 7

- day fixed intervals, beginning on Day 4Do not cite

Appendix D2

- Page 514 of 677 (f) Sampled at 7

- day fixed intervals, beginning on Day 5Do not cite

Appendix D2

- Page 515 of 677 (g) Sampled at 7

- day fixed intervals, beginning on Day 6Do not cite

Appendix D2

- Page 516 of 677 (h) Sampled at 7

- day fixed intervals, beginning on Day 7Do not cite

Appendix D2

- Page 517 of 677 Figur e D

- 2. 10 : Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixe d intervals (red dots) for Maumee River (Heidelberg)

, 200 6

(a) Actual Chemograph (red arrows point to spikes)Do not cite

Appendix D2

- Page 518 of 677 (b) Sampled at 7

- day fixed intervals, beginning on Day 1Do not cite

Appendix D2

- Page 519 of 677 (c) Sampled at 7

- day fixed intervals, beginning on Day 2Do not cite

Appendix D2

- Page 520 of 677 (d) Sampled at 7

- day fixed intervals, beginning on Day 3Do not cite

Appendix D2

- Page 521 of 677 (e) Sampled at 7

- day fixed intervals, beginning on Day 4Do not cite

Appendix D2

- Page 522 of 677 (f) Sampled at 7

- day fixed inter vals, beginning on Day 5Do not cite

Appendix D2

- Page 523 of 677 (g) Sampled at 7

- day fixed intervals, beginning on Day 6Do not cite

Appendix D2

- Page 524 of 677 (h) Sampled at 7

- day fixed intervals, beginning on Day 7Do not cite

Appendix D2

- Page 525 of 677 Figure D

- 2. 11 : Actual chemograph (blue) and resulting chemographs (dark

re d) sampled at 7

- day fixed intervals (red dots) for Maumee River (Heidelberg)

, 200 7

(a) Actual Chemograph (red arrows point to spikes)Do not cite

Appendix D2

- Page 526 of 677 (b) Sampled at 7

- day fixed intervals, beginning on Day 1Do not cite

Appendix D2

- Page 527 of 677 (c) Sampled at 7

- day fixed intervals, beginning on Day 2Do not cite

Appendix D2

- Page 528 of 677 (d) Sampled at 7

- day fixed intervals, beginning on Day 3Do not cite

Appendix D2

- Page 529 of 677 (e) Sampled at 7

- day fixed intervals, beginning on Day 4Do not cite

Appendix D2

- Page 530 of 677 (f) Sampled at 7

- day fixed intervals, beginning on Day 5Do not cite

Appendix D2

- Page 531 of 677 (g) Sampled at 7

- day fixed intervals, beginning on Day 6Do not cite

Appendix D2

- Page 532 of 677 (h) Samp led at 7

- day fixed intervals, beginning on Day 7Do not cite

Appendix D2

- Page 533 of 677 Figure D

- 2. 12 : Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for Maumee River (Heidelberg)

, 200 8

(a) Actual Chem ograph (red arrows point to spikes)Do not cite

Appendix D2

- Page 534 of 677 (b) Sampled at 7

- day fixed intervals, beginning on Day 1Do not cite

Appendix D2

- Page 535 of 677 (c) Sampled at 7

- day fixed intervals, beginning on Day 2Do not cite

Appendix D2

- Page 536 of 677 (d) Sampled at 7

- day fixed intervals, beginning on Day 3Do not cite

Appendix D2

- Page 537 of 677 (e) Sampled at 7

- day fixed interv als, beginning on Day 4Do not cite

Appendix D2

- Page 538 of 677 (f) Sampled at 7

- day fixed intervals, beginning on Day 5Do not cite

Appendix D2

- Page 539 of 677 (g) Sampled at 7

- day fixed intervals, beginning on Day 6Do not cite

Appendix D2

- Page 540 of 677 (h) Sampled at 7

- day fixed intervals, beginning on Day 7Do not cite

Appendix D2

- Page 541 of 677 Table D

- 2. 4 : An alysis of Chemograph Peaks (Spikes), Maximum Detects, and

4

- Day Averages based on 7

- Day Fixed Sampling Intervals for 2 Heidelberg Monitoring Sites

Site/Year Sampling

Interval No

Upward Spikes Max

Detect Spike #

w/ Max
Conc Max 4

- da

average Days 4

-

da avg

>2 0 ug/L Days 4

-

da avg >40 ug/L

Sandusk y R, 2004 Actual 6 24.7 4 18.2 0 0

7 da, beg day 1 3 18.6 1 17.5 0 0
7 da, beg day 2 3 18.2 1 16.8 0 0
7 da, beg day 3 3 15.5 1 14.2 0 0
7 da, beg day 4 3 11.9 1 10.9 0 0
7 da, beg day 5 2 10.1 2 9.6 0 0

7 da, beg day 6 2 24.7 2 22.2 4 0
7 da, beg day 7 2 18.5 2 16.8 0 0

Sandusk y R, 2005 Actual 7 11.3 1 10.3 0 0

7 da, beg day 1 4 10.4 1 9.3 0 0
7 da, beg day 2 3 11.2 1 9.9 0 0
7 da, beg day 3 3 7.5 1 7.3 0 0
7 da, beg day 4 3 9.6 1 8.9 0 0

7 da, beg day 5 3 11.3 1 10.2 0 0
7 da, beg day 6 5 10.7 1 9.5 0 0
7 da, beg day 7 4 8.9 1 7.9 0 0

Sandusk y R, 2006 Actual 12 37.7 3 34.5 6 0

7 da, beg day 1 2 37.7 1 33.9 9 0
7 da, beg day 2 2 34.2 1 30.7 8 0
7 da, beg day 3 2 13.5 1 12.8 0 0

7 da , beg day 4 1 20.5 1 20.0 0 0
7 da, beg day 5 3 19.8 1 19.1 0 0
7 da, beg day 6 1 34.2 1 31.6 12 0
7 da, beg day 7 1 31.7 1 28.4 7 0

Sandusk y R, 2007 Actual 5 17.5 2 14.5 0 0

7 da, beg day 1 2 14.5 2 13.0 0 0
7 da, beg day 2 2 9.5 2 8.9 0 0

7 da, beg day 3 2 12.5 2 11.4 0 0
7 da, beg day 4 2 8.4 2 8.1 0 0
7 da, beg day 5 2 5.4 1 5.0 0 0
7 da, beg day 6 2 16.5 2 14.7 0 0
7 da, beg day 7 2 17.5 2 15.5 0 0

Sandusk y R, 2008 Actual 8 41.2 4 38.8 6 0

7 da, beg day 1 3 36.6 2 32.9 8 0

7 d a, beg day 2 3 36.6 2 32.8 8 0Do not cite

Appendix D2

- Page 542 of 677 7 da, beg day 3 3 40.9 2 37.9 14 0
7 da, beg day 4 3 41.2 2 37.9 11 0
7 da, beg day 5 3 35.3 2 32.6 10 0
7 da, beg day 6 3 15.0 2 14.6 0 0

Figure D

- 2. 13 : Actual chemograph (blue) and res ulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for Sandusky River (Heidelberg), 2004

(a) Actual Chemograph (red arrows point to spikes)Do not cite

Appendix D2

- Page 543 of 677 (b) Sampled at 7

- day fixed intervals, beginning on Day 1Do not cite

Appendix D2

- Page 544 of 677 (c) Sampled at 7

- day fixed i ntervals, beginning on Day 2Do not cite

Appendix D2

- Page 545 of 677 (d) Sampled at 7

- day fixed intervals, beginning on Day 3Do not cite

Appendix D2

- Page 546 of 677 (e) Sampled at 7

- day fixed intervals, beginning on Day 4Do not cite

Appendix D2

- Page 547 of 677 (f) Sampled at 7

- day fixed intervals, beginning on Day 5Do not cite

Appendix D2

- Page 548 of 677 (g) Sampled at 7

- day fixed intervals, be ginning on Day 6Do not cite

Appendix D2

- Page 549 of 677 (h) Sampled at 7

- day fixed intervals, beginning on Day 7Do not cite

Appendix D2

- Page 550 of 677 Figure D

- 2. 14 : Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for Sandusky River (He idelberg), 200 5

(a) Actual Chemograph (red arrows point to spikes)Do not cite

Appendix D2

- Page 551 of 677 (b) Sampled at 7

- day fixed intervals, beginning on Day 1Do not cite

Appendix D2

- Page 552 of 677 (c) Sampled at 7

- day fixed intervals, beginning on Day 2Do not cite

Appendix D2

- Page 553 of 677 (d) Sampled at 7

- day fixed intervals, beginning on Day 3Do not cite

Appendix D2

- Page 554 of 677 (e ) Sampled at 7

- day fixed intervals, beginning on Day 4Do not cite

Appendix D2

- Page 555 of 677 (f) Sampled at 7

- day fixed intervals, beginning on Day 5Do not cite

Appendix D2

- Page 556 of 677 (g) Sampled at 7

- day fixed intervals, beginning on Day 6Do not cite

Appendix D2

- Page 557 of 677 (h) Sampled at 7

- day fixed intervals, beginning on Day 7Do not cite

Appendix D2

- Page 558 of 677 Figure D

- 2. 15 : Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for Sandusky River (Heidelberg), 200 6

(a) Actual Chemograph (red arrows point to spikes)Do not cite

Appendix D2

- Page 559 of 677 (b) Sampled at 7

- day fixe d intervals, beginning on Day 1Do not cite

Appendix D2

- Page 560 of 677 (c) Sampled at 7

- day fixed intervals, beginning on Day 2Do not cite

Appendix D2

- Page 561 of 677 (d) Sampled at 7

- day fixed intervals, beginning on Day 3Do not cite

Appendix D2

- Page 562 of 677 (e) Sampled at 7

- day fixed intervals, beginning on Day 4Do not cite

Appendix D2

- Page 563 of 677 (f) Sampled at 7

- day fixed intervals, beginning on Day 5Do not cite

Appendix D2

- Page 564 of 677 (g) Sampled at 7

- day fixed intervals, beginning on Day 6Do not cite

Appendix D2

- Page 565 of 677 (h) Sampled at 7

- day fixed intervals, beginning on Day 7Do not cite

Appendix D2

- Page 566 of 677 Figure D

- 2. 16 : Actual chemograph (blue) and resulting chemographs (dark red) s ampled at 7

- day fixed intervals (red dots) for Sandusky River (Heidelberg), 200 7

(a) Actual Chemograph (red arrows point to spikes)Do not cite

Appendix D2

- Page 567 of 677 (b) Sampled at 7

- day fixed intervals, beginning on Day 1Do not cite

Appendix D2

- Page 568 of 677 (c) Sampled at 7

- day fixed intervals, beginning on Day 2Do not cite

Appendix D2

- Page 569 of 6 77 (d) Sampled at 7

- day fixed intervals, beginning on Day 3Do not cite

Appendix D2

- Page 570 of 677 (e) Sampled at 7

- day fixed intervals, beginning on Day 4Do not cite

Appendix D2

- Page 571 of 677 (f) Sampled at 7

- day fixed intervals, beginning on Day 5Do not cite

Appendix D2

- Page 572 of 677 (g) Sampled at 7

- day fixed intervals, beginning on Day 6Do not cite

Appendix D2

- Page 573 of 677 (h) Sample d at 7

- day fixed intervals, beginning on Day 7Do not cite

Appendix D2

- Page 574 of 677 Figure D

- 2. 17 : Actual chemograph (blue) and resulting chemographs (dark red) sampled at 7

- day fixed intervals (red dots) for Sandusky River (Heidelberg), 200 8

(a) Actual Chem ograph (red arrows point to spikes)Do not cite

Appendix D2

- Page 575 of 677 (b) Sampled at 7

- day fixed intervals, beginning on Day 1Do not cite

Appendix D2

- Page 576 of 677 (c) Sampled at 7

- day fixed intervals, beginning on Day 2Do not cite

Appendix D2

- Page 577 of 677 (d) Sampled at 7

- day fixed intervals, beginning on Day 3Do not cite

Appendix D2

- Page 578 of 677 (e) Sampled at 7

- day fixed interv als, beginning on Day 4Do not cite

Appendix D2

- Page 579 of 677 (f) Sampled at 7

- day fixed intervals, beginning on Day 5Do not cite

Appendix D2

- Page 580 of 677 (g) Sampled at 7

- day fixed intervals, beginning on Day 6Do not cite

Appendix D2

- Page 581 of 677 (h) Sampled at 7

- day fixed intervals, beginning on Day 7Do not cite

Appendix E

- Page 582 of 677 APPENDIX E. UPDATE FOR SAP

EPA is convenin g a meeting of the FIFRA Scientific Advisory Panel (SAP) in September, 2010 to provide peer review on the Re

- evaluation of Human Health Effects of Atrazine:

Review of Non

- Cancer Effects and Drinking Water Monitoring Frequency. This meeting is

the third ins tallment on the atrazine scientific reevaluation efforts set forth by the US Environmental Protection Agency (hereafter referred to as the Agency) and the Office of Pesticide Programs (OPP). This document contains an update and progress report for

Case St udy B, Office of Pesticide Programs & The Agricultural Health Study Comparison of Exposure Assessment Approaches, presented as part of the February, 2010 FIFRA SAP (Available: http://ww w.regulations.gov/search/Regs/home.html#home

- Docket Number

EPA

- HQ

- OPP

- 2009

- 0851).

The original February 2010 submission provided details on the project plan for the incorporation of epidemiology studies within risk assessment. The intent of the cas e study is to develop an understanding of how to better incorporate results from high quality epidemiologic studies to inform human health risk assessments from problem formulation to risk characterization. The case study is based on information from the A gricultural Health Study (AHS) ( http://aghealth.nci.nih.gov/ ). The AHS is an extensive, high quality prospective epidemiologic study which collected information on the patterns of pesticide exposures of its cohort.

The specific case study analysis focuse s on individuals who mix, load, or apply pesticides (commonly referred to as handlers). The Agency has well

- developed, transparent, and peer reviewed methods for estimating occupational handler exposures (http://www.epa.gov/scipoly/sap/meetings/2007/010907 _mtg.htm). Similarly, the AHS approach for exposure classification is well

- developed and has undergone significant peer

review. The methodology the Agency uses to consider occupational exposure differs from the approaches used in the AHS, and in epidemiolo gical analyses in general, because the goals of the two types of analyses are decidedly different. The general differences in scope and purpose between exposure assessment in observational epidemiologic investigations and quantitative risk assessment have been well articulated in the scientific literature (van

den Brandt et al

., 2002 29 ). Generically, exposure assessment in observational epidemiological investigations relies on self

- reporting on an individual level to define and measure exposure. At times, th is information can be coupled with measured exposure data to create a more robust dataset. The exposure assessment methodology in the Agency‘s human health risk assessment tends to focus on designed exposure studies that represent a population. While the r esulting information captured in the two approaches may lead to a

29 van den Brandt P, Voorrips L, Hertz

- Picciotto I, Shuker D, Boeing H, Speijers G, Guit tard C, Kleiner J, Knowles M, Wolk A, Goldbohm A. 2002 The contribution of epidemiology Food Chem Toxicol. 2002 Feb

- Mar;40(2

- 3):387

- 424.Do not cite

Appendix E

- Page 583 of 677 similar conclusion of exposure, the context of the exposure assessment are different. These differences are reflected in the inputs and algorithms used in the two occupational

handler expos ure assessment approaches.

In order to assess the two approaches, the Agency presented a three tier process for the analysis during the February 2010 FIFRA SAP meeting : Step 1: Complete an evaluation of the exposure determinants used in the AHS and Agency approaches for pesticide applicator exposure assessment to illustrate potential differences. Step 2: Building on the findings from Step 1, use exposure and biomonitoring data applicable to a subset of pesticide applicators in the AHS cohort to compare ex posure metrics for those individuals calculated using the Agency and AHS approaches. Step 3: This step involves scaling up the process in Step 2 to a wider comparative analysis using information pertaining to the atrazine and alachlor users in the AHS coho rt. Prior to the conduct of this step, a feasibility analysis will be evaluated by the Agency and investigators from the AHS.

Since the February 2010 meeting, the Agency and the AHS exposure team have met to design a work plan to achieve the tasks laid ou t in the Case Study B submission paper.

The team includes individuals from National Institute of Environmental Health Sciences (NIEHS), National Cancer Institute (NCI), National Institutes of Health (NIH), National Institute for Occupational Safety and He alth (NIOSH), Centers for Disease Control (CDC), and the Agency. In order to maintain communication and progress on this project, the Agency‘s working group engages in monthly teleconferences with the AHS. The purpose of the teleconference is not only to update all the members about the progression of the project, but also to discuss any issues in the data analysis or facilitate any data sharing as

necessary.

The anticipated timeline for the project involves completing the data analysis by mid

- fall

2010 with intent of presenting the findings during the atrazine FIFRA SAP scheduled for spring, 2011.

The Agency is also proposing a second data request to the AHS for a related but separate analysis. The AHS occurred in the midst of significant changes in US agriculture (and pesticide usage), and it is important for the Agency to have a thorough understanding of how the AHS measured pesticide exposures and accounted for uncertainties, confounders and biases in the analysis of the data. The Agency is especi ally interested in the parameters that make up the lifetime cumulative exposure score, co

- occurring use patterns of pesticides leading to concurrent exposures, and in the generalizability of the results. Additionally, the Agency plans to investigate whethe r available historical pesticide usage would help refine the AHS and OPP collaborative efforts, or better characterize the resultsDo not cite

Appendix E

- Page 584 of 677 The second data request is to be submitted and reviewed in the summer of 2010. This analysis will not be presented as part of the Spring 2011 FIFRA SAP meeting, but will rather be part of a longer term assessment for future application.Do not cite

Appendix F

- Page 585 of 677 APPENDIX F

. TOXICOLOGY CHAPTER O F THE REREGISTRATION ELIGIBILITY
DECISION.

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

OFFICE OF PREVENTION, PESTICIDES AND
TOXIC SUBSTANCES

TXR NO. 0050644

MEMORANDUM Date: April 11, 2002

SUBJECT: Atrazine : Toxicology Chapter of the Reregistration Eligibility Decision.

SECOND REVISION

Chemical numbe r 080803.

DP Barcode D272007

FROM: Linda L. Taylor, Ph.D.

Reregistration Branch I Health Effects Division (7509C)

THROUGH: Steve Knizner, Branch Chief

Reregistration Branch III Health Effects Division (7509C)

Whang Phang, Ph.D., Branch Senior Scienti st Reregistration Branch I Health Effects Division (7509C)

TO: Cathy Eiden, Risk Assessor

Reregistration Branch III Health Effects Division (7509C)

Pam Noyes, Chemical Review Manager Special Review and Reregistration Division (7508C)

Attached is the re vised Atrazine Toxicology Chapter of the Reregistration Eligibility

Decision.Do not cite

Appendix F

- Page 586 of 677

Atrazine

PC Code: 080803

Toxicology Disciplinary Chapter for the Reregistration Eligibility Decision

Document

Date completed: November 15, 2000; REVISED: January 18, 2001 and April 11, 2002

Prepared : Roger Hawks [Revised: Linda Taylor]

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency

form: FINAL 21 MAR

- 2000Do not cite

Appendix F

- Page 587 of 677 F. 1.0 HAZARD CHARACTERIZATION

Atrazine has low acute toxicity. Toxicity categories are III or IV by all routes of exposures (oral, dermal, inhalation, ocular) and atrazine is not a skin sensitizer.

Atrazine has a low toxicity by the dermal route as indicated by the acute dermal toxicity study and by a 21

- day dermal toxicity study using New Zealand white rabbits. This 21 day study found a LOAEL at the limit dose of 1,000 mg/kg/day and had a NOAEL at 100 mg/kg/day.

Numerous mutagenicity studies, both submitted guideline st udies and open literature studies, are available. The weight of evidence does not support a mutagenic potential

or concern for Atrazine.

The primary effects seen in rats following chronic exposure to atrazine are neuroendocrine perturbations and mammary and pituitary gland tumors. The primary underlying events that lead to mammary and pituitary tumor formation following atrazine exposure of Sprague

- Dawley female rats involve disruption of the hypothalamic

pituitary

- ovarian axis. The neuroendocrine disrupt ions include alterations in hypothalamic neurotransmitter and neuropeptide levels. Decreases in serum levels of pituitary luteinizing hormone (LH) and prolactinare presumably a consequence of these hypothalamic alterations. Hypothalamic neuronal

- GnRH pu lse modulation of pituitary releases of LH is a central driver of ovulation in the SD female rat, and atrazine is essentially accelerating the aging process of the CNS control of ovulation. This leads to a constant state of estrus (anovulation) and prolong ed exposure to estrogen and prolactin. Although, there are certain similarities in the control of the hypothalamic

pituitary

- ovarian axis between humans and rats in that the hypothalamus can play a key regulatory role in primates, there are fundamental di fferences. Although atrazine might cause adverse effects on hypothalamic

- pituitary function in humans, the hormonal environment conducive to tumor development (i.e., elevated or prolonged exposure to estrogen or prolactin) that is found in SD female rats can not be established in humans. The Health Effects Division Cancer Assessment Review Committee [CARC] concluded

that the neuroendocrine alterations in the SD rat constitute the mode of action for

atrazine

- associated carcinogenicity. CARC has classified atrazine as a "Not Likely To Be Carcinogenic To Humans‖.

These same neuroendocrine alterations, which constitute the mode of action [MOA] for cancer, could be expected to be associated with reproductive or developmental effects. Reproductive/developmen tal effects, including prostate inflammation in male rats whose mothers‘ prolactin was suppressed due to treatment with atrazine while lactating, as well as delay of puberty in both male and female rats exposed prior to pubertal onset,

have been demonstrat ed following atrazine exposure.

Though special studies conducted at EPA labs indicate developmental/reproductive toxicity, guideline studies, for the most part, have not indicated developmental/reproductive toxicity. There was no increased quantitative or qualitativeDo not cite

Appendix F

- Page 588 of 677 susceptibility in any of the guideline studies on atrazine in the rat [two developmental toxicity studies and a 2

- generation reproduction study], and there was no increased quantitative susceptibility in the rabbit developmental toxicity stu dy. However, there was increased qualitative susceptibility in the rabbit study [increased resorptions (deaths) at a dose level that resulted in decreased body

- weight gain and clinical signs in the maternal animal]. Studies in the open literature indicate increased qualitative susceptibility. Dosing of dams immediately following parturition [postnatal days 1

- 4] resulted in prostatitis in male offspring, and dosing of the young following weaning resulted in delayed puberty in both sexes. The mode of action f or these two effects (prostate inflammation and delayed puberty) is believed to be similar to the mode of

action described for atrazine

- associated cancer and involves a central nervous system (CNS) toxicity

- specifically, neurotransmitter and neuropeptide alterations at the level of the hypothalamus.

Atrazine is not readily absorbed through dermal exposure. Based on both a rat and a human dermal absorption study, HIARC recommended a dermal absorption factor of

6%.

There is one datagap. A 28

- day study v ia the inhalation route of exposure is required to assess the estrous cycle/LH surge in females for comparison with the 28

- day oral exposure study. There are no other data gaps for atrazine according to the Subdivision F Guideline requirements. HIARC recom mends, but does not require , that special studies examining atrazine's associations with delayed puberty and prostatitis in offspring of dams exposed shortly after parturition, be conducted. Should such studies be conducted, it is recommended that study p rotocols be approved by HED prior to commencement of any such study. In addition, HIARC recommends, but does not require , that special studies examining the CNS alterations following atrazine exposure be performed.

F.2.0 REQUIREMENTS

The requirements (CFR 158.340) for food use of atrazine are in Table 1.Do not cite

Appendix F

- Page 589 of 677 Table F

- 1 : The requirements (CFR 158.340) for food use of atrazine

Test Technical

Required Satisfied

870.1100

................................ . Acute Oral Toxicity
870.12 00

............................. Acute Dermal Toxicity
870.1300

......................... Acute Inhalation Toxicity
870.2400

.............................. Primary Eye Irritation
870.2500

......................... Primary Dermal Irritation
870.2600

............................... Dermal Sensitization yes yes yes yes yes yes yes

yes yes yes yes yes

870.3100

....................... Oral Subchronic (Rodent)
870.315 0

............... Oral Subchronic (Non

- Rodent)
870.3200

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

- Day Dermal
870.3250

................................ ....... 90

- Day Dermal
870.3465

................................ ... 90

- Day Inhalation yes yes yes

no

no yes

yes a

yes

- yes b

870.3700a Developmental Toxicity (Rodent)

..... 870.3700b Developmental Toxicity (Non

rodent)

................................ ............. 870.3800

.................... 2

- Gener ation Reproduction yes yes yes yes

yes yes

870.4100a Chronic Toxicity (Rodent)

................ 870.4100b Chronic Toxicity (Non

- rodent)

.......... 870.4200a Oncogenicity (Rat)

........................... 870.4200b Oncogenicity (Mouse)

...................... 870.4300

............................. Chronic/Oncogenicity yes yes yes yes yes yes

yes yes yes yes

870.5100 Mutagenicity

— Gene Mutation

- bacterial
870.5375 Mutagenicity

— Structural Chromosomal Aberrations
870.5550

.. Mutagenicity

— Other Genotoxic Effects yes

yes yes yes

yes yes

870.6100a Acute Delayed Neurotox. (Hen)

....... 870.6100b 90

- Day Neuro toxicity Hen)

............... 870.6200a Acute Neurotox. Screening Battery

(Rat)

................................ ................ 870.6200b 90 Day Neuro. Screening Battery

(Rat)

................................ ................ 870.6300

................................ ...... Develop. Neuro no

no

no

no

no -

-

no

no

no

870.7485

............................... General Metabolism
870.7600

................................ Dermal Penetration yes yes yes

yes
a Req uirement waived because a chronic study in the non

- rodent ( i.e

. - dog) is available

b 28

- day inhalation study in female SD rats to assess the estrous cycle/LH surge for comparison with the 28

- day oral exposure studyDo not cite

Appendix F

- Page 590 of 677 F. 3.0 DATA GAPS

Status of previous datagaps

The previous Toxicology RED chapter for atrazine (TXR 006937) completed in 1988, identified several datagaps. Table two displays those data gaps and the studies that have been submitted to fill these datagaps. All datagaps d escribed in the previous Toxicology RED chapter have been satisfied.

Table F

- 2 : Data Gaps Listed in 1988 RED Document and Studies That Have Been Submitted to Fill these Data Gaps.

1988 Data Gap Results of Submitted Study MRI D No(s).

81

- 3, Acute Inhalation Study Acceptable

- Data Gap

Filled 42089901,

43016502

82

- 2, Subchronic Dermal Study Acceptable

- Data Gap

Filled 42089902

84

- 2, Mutagenicity

- a. Dominant Lethal Assay b. Direct DNA Damage Studies Acceptable

- Data Gap s Filled 426370003

and

42547105

Current datagaps

Previously, there were no datagaps for atrazine or the atrazine metabolites listed in this document, with the exception of DACT. The HIARC previously required that a OPPTS Series 870.3800 Reproduc tion and Fertility Effects study be performed with the atrazine metabolite diaminochlorotriazine (DACT)

- a mammalian metabolite of both atrazine and simazine. Subsequently, a re

- evaluation of the data by the HIARC determined that the OPPTS 870.3800 study was not required for DACT [TXR No. 0050592].

Additionally, it was determined by the HIARC that a 28

- day study via the inhalation route of exposure is required for atrazine to assess the estrous cycle/LH surge in females for comparison with the 28

- day or al exposure study. There are no other data gaps for atrazine according to the Subdivision F Guideline requirements. HIARC recommends, but does not require , that special studies examining atrazine's associations with delayed puberty and prostatitis in offsp ring of dams exposed shortly after parturition, be conducted. Should such studies be conducted, it is recommended that study protocols be approved by HED prior to commencement of any such study. In addition, HIARC recommends, but does not require , that sp ecial studies examining the CNS alterations following atrazine exposure be performed.Do not cite

Appendix F

- Page 591 of 677 F. 4.0 HAZARD ASSESSMEN T

F.4.1 Acute Toxicity

Adequacy of data base for acute toxicity : The data base for acute toxicity is considered complete. No additional studies are required at this time.

The acute toxicity data on atrazine technical is summarized below in Table 3.

Table 3 : Acute Toxicity Data of Technical Atrazine

Guideline

No. Test Results Toxi city Category

870.1000 Oral LD 50 - rat LD 50 > 1,869 mg/kg (M&F combined) III

870.1200 Dermal LD 50 - rat LD 50 > 2,000 mg/kg (M&F combined) III

870.1300 Inhalation LC 50 -

rat LC 50 > 5.8 mg/L (M&F combined) IV

870.1400 Eye Irrita tion

-

rabbit PIS a = 0.0/110 IV

870.1500 Dermal Irritation

-

rabbit PIS a = 0.2/8.0 IV

870.1600 Dermal

Sensitization

- guinea pig Non

- sensitizing

---

870.6100 Acute Delayed

Neurotoxicity Not Required

---

a PIS=Primary Irri tation Score

870.1100. Acute oral toxicity

- rats

A pair of acute LD 50 studies in the rat using technical grade atrazine have

shown the LD 50 to be: 1,869 mg/kg (Accession No. 230303; MRID 00024706) and 2,850 mg/kg (MRID 00027097). Numbers in all these st udies are for both sexes combined. The toxicity category for acute oral exposure is at least III.

870.1200. Acute dermal toxicity

- rat and rabbit

Two dermal LD 50 studies using the rat resulted in LD 50 s of >2,000 mg/kg and > 3,100 mg/kg for both sexes c ombined (Accession Nos. 00027097 and 230303; MRID

00024708 ). A study using rabbits, the preferred species in the OPPTS Series 870Do not cite

Appendix F

- Page 592 of 677 guidelines, resulted in an LD 50 of 7,550 mg/kg for both sexes combined (Accession No. 231466). Atrazine is at least toxicity c ategory III for dermal exposure.

870.1300. Acute inhalation toxicity

- rats

The inhalation LC 50 was found to be 5.82 mg/L (MRIDs 42089901 and 43016502). This exceeds the limit dose of 5.0 mg/L and places atrazine in toxicity category IV.

870.2400. Primary ocular irritation

- rabbits

Atrazine tested negative for primary eye irritation and has been placed in toxicity category IV for this endpoint (Accession No. 230303; MRID 00024709).

870.2500. Primary dermal irritation

- rats

Atrazine tested nega tive for primary skin irritation, and has been placed in toxicity category IV for this endpoint (Accession No. 230303; MRID 00024710).

870.2600. Dermal sensitization

- guinea pigs

Atrazine has been shown to be non

- sensitizing in dermal sensitization stud ies (MRID 00105131 ).

870.6100. Acute and delayed type neurotoxicity of organophosphorus substances

Acute neurotoxicity studies have not been performed on atrazine, and none are required.

F. 4.2 Subchronic Toxicity

Ad equacy of data base for subchronic toxicity : The data base for subchronic toxicity is considered complete. No additional studies are required at this time.

870.3100 90

- Day Oral Toxicity

- Rat

In a subchronic oral toxicity study (MRID 44723701), Atraz ine Technical (purity 97.1 %) was administered in the diet to Tif: RAIf (SPF), RII/1 x RII/2 hybrids, (Sprague

Dawley derived) rats 10/sex/dose at dose levels of 0, 10, 50, or 500 ppm for 92 days. These doses were equivalent to 0, 0.6, 3.3, and 34.0 mg/ kg/day for males, and 0, 0.659, 3.35, and 35.3 mg/kg/day for females. Rats were treated for 13 weeks, then sacrificed (S1). Additional recovery groups of 10 rats/sex/dose were fed 0 or 500 ppm for 13 weeks, remained untreated for 4 weeks, and were then s acrificed (S2).

One female in the control group died during the study. No treatment

- related clinical

effects were noted. Treat ment with atrazine technical resulted in significant (p<0.01)Do not cite

Appendix F

- Page 593 of 677 decreases in body weight in high dose males and females compared to controls. Body weight losses were partially regained during the recovery period. Hematological and

clinical chem istry parameters showed no significant treatment related effects. Decreased liver and kidney weights in high dose males occurred as a resu lt of the marked body weight loss. There were no treatment

- related effects on gross pathology or ophthalmology. Hemosiderin pigment was found in the spleen at an increased incidence and severity in high dose animals of both sexes, at the end of treatment and recovery periods.

As tested in this study, the LOAEL for Atrazine is 34.5 mg/kg/day, based on decreased body weight. The NOAEL is 3.3 mg/kg/day.

This subchronic oral toxicity study is classified as Acceptable /Guideline and satisfies the guidelin e requirement for a subchronic oral study (OPPTS 870.3100) in rats.

870.3150 90

- Day Oral Toxicity

- Dog

Study has been waived because an acceptable chronic study is available.

870.3200 21

- Day Dermal Toxicity

- Rabbit

A 21 day dermal study (MRID 4208 9902) in New Zealand White rabbits was conducted. Rabbits, 5 rabbits/sex/dose, were administered atrazine (97.6%) 6 hrs per day for 25 days at concentrations of 0, 10, 100 or 1000 mg/kg/day.

Systemic toxicity was observed at the highest dose tested and consisted of reductions in food consumption (decreased 28% for males and 31% for females at day 21 vs controls) and mean body weight (12% at day 25 vs controls for males and 19% for females). Percent body weight gain was decreased (

- 4.4% vs +9.5 at day 21 for males

and

- 11% vs +8% for females). Compared to controls, relative and absolute spleen weights in both sexes were increased (males had an 81% absolute increase and 75% increase relative to body weight; females had an 86% increase absolutely and 94%

rel ative to body weight).

Additional findings were reduced red blood cell and hematocrits counts (each decreased by 12% vs controls), and increased cholesterol and triglyceride levels (increased by 101 and 55% vs controls) in the highest dose tested females and reduced total protein and chloride levels (decreased by 7 and 5% vs controls) in the high

- dose males. Dermal findings were: limited

- to

- slight acanthosis for both sexes at all dose levels; minimal

- to

- moderate acanthosis in 3 high

- dose females (compa red to zero control females having this finding); and 3 cases of focal subacute inflammation in the treated skin of high

- dose females (compared to zero cases of this in the controls). The NOAEL is 100 mg/kg/day and the LOAEL at 1000 mg/kg/day based on red uctions in food consumption, mean body weight, body weight gain and increases in spleen weight.Do not cite

Appendix F

- Page 594 of 677 The LOAEL is 1000 mg/kg/day, based on reduced food consumption, mean body weight and body weight gain, increased relative and absolute spleen weights, and

redu ced red blood cell counts and hematocrits. The NOAEL is 100 mg/kg/day

.

This study is classified Acceptable

- Guideline and satisfies the OPPTS 870.3200 guidelines for a 21 day dermal toxicity study in the rabbit.

F. 4.3 Prenatal Developmental Toxicity

Adequacy of data base for Prenatal Developmental Toxicity : The data base for prenatal developmental toxicity is considered complete. No additional studies are required at this time.

870.3700 Prenatal Develop mental Toxicity Study

- Rat (1989 study)

In a developmental toxicity study (MRID 41065201), atrazine (97.6%) was administered by gavage to 104 mated female Sprague

- Dawley rats, 26/dose, at dose levels of 0, 5, 25, 100 mg/kg/day from days 6 through 15 of gestation.

Maternal toxicity findings were almost exclusively confined to the high dose group. Compared to controls, high dose dams displayed: reduced food consumption (decreased 13%, p < 0.5); reduced total body weight gain (reduced 18% during dosing

pe riod, p < 0.5); reduced corrected (minus uterine weight) weight gain (reduced 20% for entire gestation, p < 0.5); and increased alopecia 1/26 controls vs 5/26 high dose). One high dose animal died on gestation day 20 and salivation was noted as an observat ion in 18/26 high dose animals. The only observations seen outside the high dose group

were: an abortion from one of the mid

- dose animals on gestation day 19; a fluid

- filled hollow right kidney in a mid

- dose animal; and hollow discolored kidneys in a low d ose animal.

The maternal LOAEL is 100 mg/kg/day, based on reduced body weight gain and food consumption. The maternal NOAEL is 25 mg/kg/day.

The few malformations seen upon external examination of the fetuses were seen only in the control groups and clea rly could not be compound related. Likewise, there was no increased incidence of visceral malformation in dosed groups vs control groups. There

were no skeletal malformations observed but there was an increased incidence of incomplete ossification of vario us bones in the highest dose tested. Hyoids (control fetal

incidence of 11% vs 21.7% at high dose), occipitals (7.7% vs 21.1%) and parietals (

2.2% vs 8.4%) showed incomplete ossification. There was also an increased incidence (p < 0.05) of incomplete ossi fication of the interparietals in all dose groups compared to

controls.

Fetal body weight, number of resorptions and implantations, and live fetuses/litter were not significantly affected by atrazine treatment. Exposure of gravid Sprague

- Dawley rats

to a trazine under the conditions described in this study seemed to have few embryo/fetotoxic effects.Do not cite

Appendix F

- Page 595 of 677 The developmental LOAEL is 100 mg/kg/day, based on increased incidence of delayed ossification of skull bones. The developmental NOAEL is 25 mg/kg/day.

The developmental toxicity study (MRID 41065201) in the rat is classified Acceptable

Guideline and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700) in the rat.

870.3700 Prenatal Developmental toxicity

- Rat (1984 study)

In a developmental toxicity study (MRID 00143008 and 40566302), atrazine (96.7%) was administered to 104 Charles River CD rats 27/dose by gastric intubation at dose levels of 0, 10, 70, or 700 mg/kg/day from days 6 through 15 of gestation.

Mortality wa s very high for the 700 mg/kg/day animals in this study. All but 6 of the 27 females in this group died during the gestation period. Other statistically significant findings in this group included: salivation; oral and nasal discharge; ptosis; swollen

abdo mens; blood on the vulva; enlarged stomachs and adrenal; and discolored lungs. Body weight gains were statistically significantly reduced throughout most of the gestation period. Significant reductions in food consumption were observed. Although mortality was quite high in the high dose group, pregnancy rates were comparable to controls (85.2% for controls vs 96.3% high dose) and the numbers of live fetuses at c

section was comparable to controls (mean of 12.7 per litter for controls vs 13.4 for the high do se).

There were few findings in either the low or mid dose animals. Alopecia was observed in mid dose animals, but was not considered to be biologically significant. Body weight gain in the mid dose group was significantly reduced for the first 5 days o f compound exposure (gestation days 6

- 10). Food consumption in the intermediate group was reduced, but only at day 17 of gestation. Low dose body weight gain was increased on days 6

- 10 of gestation and food consumption was increased on gestation day 9.

Th e maternal LOAEL is 70 mg/kg/day, based on reduced body weight gain. The maternal NOAEL is 10 mg/kg/day.

Fetal weights were statistically significantly reduced in the high dose group. Skeletal examinations were not conducted in the high dose group due to the extremely low fetal weights. Visceral and external examinations were conducted, though, and no group, including the high dose group, displayed any findings significantly different from control values. Skeletal anomalies, however, were observed in the mid dose group.

The developmental LOAEL was found to be 70 mg/kg/day, based on delayed or no ossification at several sites. The developmental NOAEL is 10 mg/kg/day.

The developmental toxicity study in the rat is classified Acceptable

- Guideline and

satisf ies the OPPTS 870.3700 guideline requirement for a developmental toxicity study

in rats.Do not cite

Appendix F

- Page 596 of 677 870.3700 Prenatal Developmental Toxicity Study

- Rabbit

In a developmental toxicity study (Accession No. 254979 and MRIDs
00143006, 40566301), atrazine (96.3%) wa s administered by gavage to 76 mated female New Zealand White rabbits, 19/dose, at dose levels of 0, 1, 5, or 75 mg/kg/day, from days 7 through 19 of gestation.

Clinical signs seen in 75 mg/kg/day (highest dose tested) animals that were considered

to be related to compound treatment were stool changes (none, little or soft stool; 9/19

controls vs 19/19 at high dose), and the appearance of blood in the cage or on the vulva (0/19 controls vs 4/19 at high dose). Body weight gain was reduced in high dose dams and, at many time points, body weight was below day zero values. At gestation days 14, 19, 21 and 25, mean maternal body weights were 12%, 19%, 18%, and 10% below control values (p < 0.01 for all four of these time points).

High dose animals displayed si gnificantly reduced food consumption during treatment. During gestation days 12 to 17, the average feed consumption at the high dose was only 1

- 6 grams of feed per animal per day compared to 175

- 182 grams for the controls.

The mid and low dose groups had no alterations that could be attributed to atrazine

exposure.

The maternal toxicity LOAEL is 75 mg/kg/day, based on decreased body weight, food consumption and increased incidence of clinical signs. The maternal toxicity NOAEL is 5 mg/kg/day.

Fetal eff ects were: increased resorptions

- mean of 1.3/dam in controls vs 4.8/dam at the high dose (p < 0.01), reduced live fetuses per litter

- mean of 8.8/dam in controls vs 5.9/dam at the high dose (p < 0.05), and increased delayed ossification of appendicular

elem ents were observed in the high dose group.

The low and intermediate groups had no fetal findings that could be attributed to compound exposure.

The findings in the high dose group were determined to be secondary to maternal toxicity and thus the LOAEL and NOAEL for embryo/fetotoxicity match the maternal

LOAEL and NOAEL.

The developmental toxicity LOAEL is 75 mg/kg/day, based on reduced litter size, increased resorptions and delayed ossification. The developmental toxicity NOAEL is 5 mg/kg/day

The stud y is classified Acceptable

- Guideline and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700) in rabbits.Do not cite

Appendix F

- Page 597 of 677 F. 4.4 Reproductive Toxicity

Adequacy of data base for Reproductive Toxicity : The database for reproductive toxicity is considered complete. There is an acceptable multi

- generation reproduction study available for atrazine. No additional studies are required at this time.

870.3800 Reproduction and Fertility Effects

- Rat

In a 2

- g eneration reproduction study (MRID 40431303), atrazine (purity not specified but said to be technical grade) was administered to 240 Charles River (CRCD, VAF/PLUS) rats 30/sex/dose in the diet at dose levels of 0, 10, 50, and 500 ppm.

There was very litt le variation in test article consumption between generations; the F 0 and F 1 males had similar test article consumption during the 70

- day premating period as

did the F 0 and F 1 females. The average values for the two generations are 0, 0.75, 3.78, 39.0 mg/kg /day for males and 0, 0.86, 3.70, 42.8 mg/kg/day for females. Test article consumption for the F 0 and F 1 generation females during their gestation period did not vary greatly between generation. Mean compound consumption for both generations
were 0, 0.66, 3.33 and 35.43 mg/kg/day.

Parental body weights, body weight gain, and food consumption were statistically significantly reduced at the 500 ppm dose (highest dose tested) in both sexes and both generations throughout the study. Compared to controls, body weights for F 0 high

- dose males and females at 70 days into the study were decreased by 12% and 15%, respectively while F 1 body weight for the same time period was decreased by 15% and 13% for males and females, respectively. The only other parental effect that may have been treatment related was a slight, but statistically significant, increase in relative testes weight, which occurred in both generations at the high dose. There did not appear to be any reproductive effects from compound exposure. Measured reproductive parameters from both generations did not appear to be altered in a dose

- related

manner.

The LOAEL is 500 ppm (39 mg/kg/day in males, 42.8 mg/kg/day in females) based on decreased body weights, body weight gains and food consumption. The

NOA EL is 50 ppm(3.78 mg/kg/day in males, 3.7 mg/kg/day in females)

The developmental toxicity LOAEL is 39 mg/kg/day in males, 42.8 mg/kg/day in females, based on decreased body weights in both generations of males at postnatal day 21. The developmental toxic ity NOAEL is 3.78 mg/kg/day in males and 3.7 mg/kg/day in females.

This study is classified Acceptable

- Guideline and satisfies the guideline requirement (OPPTS 870.3800) for a 2

- generation reproductive study in the rat.

F. 4.5 Chronic Toxicity and Carci nogenicityDo not cite

Appendix F

- Page 598 of 677 Adequacy of data base for chronic toxicity : The data base for chronic toxicity is considered complete. No additional studies are required at this time.

870.4100 (870.4300) Combined Chronic Toxicity/Carcinogenicity

– Rat (Sprague

Dawley)

In a combined 2

- year feeding/oncogenicity study (MRID 40629302), atrazine (95.9% +/

- 2.3%) was administered to Sprague

- Dawley rats, 20/sex/dose, in the diet, at dose levels of 0, 10, 70, 500, and 1000 ppm, ( 0, 0.5, 3.5 25, 50 mg/kg/day) for 2 years.

Toxicologic alteration appeared to be confined to the 500 and 1000 ppm dose groups.

25 mg/kg/day: Both male and female rats showed reductions in body weight gain, and food consumption. Body weight for males an d females at 104 weeks was 8% and 19% below 104 week control values. Food consumption at 104 weeks was not significantly below control, but at weeks 1, 13 and 26 for males and week 1 for females, food consumption was statistically

- significantly reduced. F ood consumption was reduced 11% at week 1, 11% at week 13 and 9% at week 26 in males and 8% at week 1 in females. Males displayed no unusual histopathology findings in this group, but females had statistically

- significant increases of myeloid hyperplasia i n the bone marrow of the femur (25 control incidences vs 38) and sternum (21 vs 33), and splenic extra medullary hematopoiesis (12 vs 22).

50 mg/kg/day: Both sexes showed reductions in body weight gain (19% vs controls at 104 weeks for males, 27% at 104 weeks for females)and food consumption (reduced vs controls 20%, 16%, 14% and 11% at weeks 1, 13, 26 and 52 for males and 18%, 8%,and 7% at weeks 1, 13, and 26 for females). Female survival was 50% at 104 weeks in controls and 26% in 1000 ppm animals. Inte restingly, survival was increased in 1000 ppm males

- 44% control survival vs 67% 1000 ppm at 104 weeks. Altered hematology and clinical chemistry findings were noted in females and included: decreased hemoglobin concentration; hematocrit; RBC; and serum g lucose. Males did not display these alterations but did display decreased serum triglyceride levels throughout the course of the study. Organ

- to

- body weight ratios were decreased in high dose animals which may have been the result of decreases in body weig ht gain. Histopathology findings in females dosed 1000 ppm consisted of statistically

- significant increases in incidences of (% increase vs controls in parenthesis): retinal degeneration (83%); centrilobular necrosis in the liver (300%); degeneration of t he rectus

- femoris muscle (160%); transitional epithelial hyperplasia in the bladder (150%) and the kidney (82%); splenic extra medullary hematopoiesis (133%); and myeloid hyperplasia in both the femur (108%) and sternum bone marrow (119%). Histopathology f indings in males dosed 1000 ppm consisted of statistically

- significant increases in incidences of: degeneration rectus femoris muscle (366%); prostate epithelial hyperplasia (141%); calculi in the kidney pelvis(106%); and acinar hyperplasia of the mammary gland (200%).

A carcinogenicity study run concurrently with the toxicity study determined that there

was a dose

- related increase in mammary adenocarcinomas (p value for the trend <Do not cite

Appendix F

- Page 599 of 677 0.00005) in 70, 500 and 1000 ppm females. Adenocarcinomas incidences were : controls

- 15/66; 70 ppm

- 26/68; 500 ppm

- 27/65; 1000 ppm

- 35/64.

The dose response curve in this study appeared to be adequate as the low doses (10 and 70 ppm) showed few toxic effects; the mid

- dose level showed some toxic effects (reduced body weigh t gain and food consumption); while the high dose showed the same effects as the mid dose plus hematology/clinical chemistry and histopathology findings.

The LOAEL for non

- cancer effects is 500 ppm (25 mg/kg/day), based on reduced body weight gain and fo od consumption. The NOAEL is 70 ppm (3.5 mg/kg/day). Mammary tumors [adenocarcinomas] were observed at dose levels of 70 ppm (3.5 mg/kg/day) and above.

This chronic toxicity/carcinogenicity study in the rat is classified Acceptable

- Guideline

and satisfies the guideline requirement (870.4300) for a combined chronic oral/carcinogenicity study in the rat.

NOTE: Please refer to Hazard Assessment and Review of Available Studies [Part B] of the May 22, 2000 PRELIMINARY DRAFT Hazard and Dose

- Response

Assessment and Characterization

- ATRAZINE, presented to the FIFRA Scientific Advisory Panel [SAP] on June 27

- 29, 2000 for a summary and details of this study as well as the other long

- tern studies in SD rats (see http://www.epa.gov/scipoly/sap/2000/june27/finalpart a_atz.pdf )

.

870.4100 (870.4300) Combined Chronic Toxicity/Carcinogenicity

– Rat (F

- 344)

In a chronic toxicity/carcinogenicity toxicity study (MRID 42227001), atrazine [97.0%) was administered to 600 Fischer

- 344 rats, 60/sex/dose in the diet at dose

le vels of 0, 10, 70, 200, 400 ppm (0, 0.49, 3.43, 9.87 and 20.17 mg/kg/day for males and 0, 0.61, 4.35, 12.71, and 26.18 mg/kg/day for females) for 104 weeks.

Administration of atrazine did not affect animal survival nor, with the exception of

thinness in the high

- dose group, were any clinical signs apparent. Body weight and body weight gain were significantly reduced in 200 and 400 ppm exposed animals of both sexes throughout most of the study. Food consumption in the high dose group males was significant ly reduced throughout the study, and significantly reduced in high dose females for the first 13 weeks of exposure. No findings were seen at necropsy which could be attributed to compound exposure. There were no significant increases in either neoplastic o r non

- neoplastic findings which could be attributed to compound

exposure.

The LOAEL is 3.9 mg/kg/day based on decreased body weight gain. The NOAEL is 0.55 mg/kg/day.Do not cite

Appendix F

- Page 600 of 677 At the doses tested, there was no treatment

- related increase in tumor incidence whe n compared to controls. Dosing was considered adequate based on decreased body weight gain.

This chronic toxicity/carcinogenicity study in the Fischer 344 rat is Acceptable

Guideline and satisfies the guideline requirement for a chronic toxicity/carcinoge nicity study (OPPTS 870.4300) in the rat.

NOTE: Please refer to Hazard Assessment and Review of Available Studies [Part B] of the May 22, 2000 PRELIMINARY DRAFT Hazard and Dose

- Response
Assessment and Characterization

- ATRAZINE, presented to the FIFRA Sc ientific Advisory Panel [SAP] on June 27

- 29, 2000 for a summary and details of this study as well as the other long

- tern studies in F

- 344 rats (see http://www.epa.gov/scipoly/sap/2000/june27/finalparta_atz.pdf )

.

870.4100b Chronic Toxicity

- Dog

In a c hronic toxicity study (MRID 40431301), atrazine (97% a.i.) was administered to 40 Beagle dogs, 4/sex in the low and intermediate dose groups and 6/sex in the control and high dose groups, in the diet at dose levels of 0, 15, 150 and 1000 ppm for 52 weeks. The mean daily dose was 0, 0.48, 4.97, 33.65 mg/kg for males. Female doses were the same for the low and mid dose and only slightly different [33.80 mg/kg/day] in the high dose group.

Percent increases in body weight during the course of the study were a s follows: male: 46.6% in control; 52.5% [low dose]; 36.8% [mid dose] and 29.5% [high dose]; female: controls; 44.4% [low dose]; 35.9% [mid dose]; and 21.5% [high dose]. Only for the high

dose males was this increase statistically significant compared to t he control increase.

Although only statistically

- significant for the first quarter, high

- dose males and females showed reduced food consumption throughout the course of the study. By studies‘ end, mean food consumption was decreased 22% in males and 20% in females.

High dose animals recorded statistically

- significant changes in organ weights for both sexes compared to controls. Absolute heart weight was decreased in females by 14% and male liver weight relative to body weight was increased by 47%.

Th e major treatment

- related findings in this study were cardiac findings. EKG alterations such as tachycardia and increased P wave amplitude (P

- II), were observed at many timepoints during the study in high

- dose animals. Mean heart rate was increased 59% (p < 0.05) and P

- II was decreased 78% (p < 0.01) on day 361 in high

- dose males compared to controls. Female heart rates at the same time point were increased 46% which was not statistically significant, but P

- II was decreased 71% which was significant at p < 0.01 . Gross pathology findings were also seen in the heart and were mostly concentrated in the high

- dose group. Minimal atrial dilation seen in 3/6 males and 2/6 females was the only cardiac finding seen at necropsy in the control animals. The high

dose group, on the other hand, displayed moderate to severe atrial dilation in 4/5 malesDo not cite

Appendix F

- Page 601 of 677 and 5/5 females who survived until scheduled sacrifice. Two of 5 high

- dose males who survived until scheduled sacrifice also displayed enlarged, soft hearts and 1/5 had fluid

in the pericardium.

Histopathology findings were also seen in the heart. Three of 6 high

- dose males and 6/6 high

- dose females were found to have cardiac myolysis while no control animals of either sex had myolysis. Focal atrophy of the heart was seen in 2 /6 high

- dose males and 5/6 high

- dose females while no controls of either sex displayed this finding.

Three animals, two high

- dose [male on day 250; female on day 113] and one mid

- dose male [day 75], were sacrificed in moribund condition during the course of the study. The two high dose sacrifices may have been due to compound

- related cardiac dysfunction.

The LOAEL for both sexes is 1000 ppm (33.65 mg/kg/day for males and 33.8 for females), based on cardiac effects. The NOAEL is 150 (4.97 mg/kg/day) for bo th

sexes.

The major finding of this study was cardiopathology. A broad array of cardiac dysfunctions seen in the high dose group such as: electrocardiography alterations; the appearance of both microscopic and macroscopic cardiac abnormalities, were

deter mined to be due to atrazine exposure. The wide variety of effects seen, as well as possible compound

- related animal deaths, indicate the severity of the cardiac effects.

This chronic toxicity study is Acceptable

- Guideline and satisfies the guideline

requ irement (OPPTS 870.4100) for a chronic oral toxicity study in dogs.

870.4200 Carcinogenicity Study

- rat

See above under "870.4100 Chronic Toxicity

– rat" for Executive Summary.

870.4200 Carcinogenicity

- mouse

In an carcinogenicity study (MRID 40431 302), atrazine (purity not given) was

administered to CD

- 1 mice, 59

- 60/sex/dose, in the diet at dose levels of 0, 10, 300, 1500 and 3000 ppm (male/female mean daily dose 0/0, 1.4/1.6, 38.4/47.9, 194.0/246.9, 385.7/482.7 mg/kg/day) for 91 weeks. Female mice in the 300, 1500 and 3000 ppm groups received a daily atrazine dose about 25% higher than their counterpart males.

No dose

- related increases in neoplasms were observed. The dose response curve seemed adequate since toxic effects, such as a decrease in m ean body weight of both sexes and an increase in cardiac thrombi in the females, are seen at both 1500 and 3000 ppm, while no dose

- related toxic effects are seen at 10 and 300 ppm. In addition to the toxic effects just mentioned, the 3000 ppm animals of bo th sexes also displayed decreases in food consumption and decreases in RBC, hematocrit, and hemoglobin concentration. Female mice, but not males, at 3000 ppm showed decreased mean group brain and kidney weights and decreased percentages of neutrophils and lymphocytes. There was also an increase in mortality (p < 0.05) in 3000 ppm females,Do not cite

Appendix F

- Page 602 of 677 but not males, with only 25% of the females surviving vs 39

- 43% of the females surviving in the other female dose groups.

The cardiac thrombi found at both 1500 and 3000 ppm may have contributed to unscheduled female deaths during the course of the study. The incidence of unscheduled death in mice with cardiac thrombi is statistically significantly different from the incidence of unscheduled death in mice from control grou ps. The occurrence of

cardiac thrombi is considered a severe effect.

The LOAEL is 222.0 mg/kg/day, based on decreased body weight gain in both sexes and increased cardiac thrombi in the females. The NOAEL is 43 mg/kg/day

.

At the doses tested, there was not a treatment

- related increase in tumor incidence when compared to controls. Dosing was considered adequate due to the occurrence of decreased body weight gain and cardiac thrombi.

This carcinogenicity study in the mouse is Acceptable

- Guideline, and it satisfies the guideline requirement for a carcinogenicity study in the mouse (OPPTS 870. 4200).

F. 4.6 Mutagenicity

Adequacy of data base for Mutagenicity : The data base for Mutagenicity is considered adequate based on current and pre

- 1991 mutagenicity guidelines. No additional studies are required at this time.

Numerous open literature mutagenicity studies are available for evaluation. The entire mutagenicity database for atrazine (including these open literature studies)

ha s been reviewed by CARC (October 13, 1999; April 12, 2000; May 22, 2000 {http://www.epa.gov/scipoly/sap/ #june27}, October 19, 2000) and by the Science Advisory Panel (June, 28

- 30, 2000) and both panels stated that the available

evidence did not indicate a mutagenic effect of atrazine exposure

.

870.5100 Bacterial reverse mutation test

In a reverse gene mutation assay in bacteria (MRID 40246601), strains TA 98, 100, 1535 and 1537 of S. typhimurium were exposed to atrazine (98.2% a.i.), in dimethylsufoxid e, at concentrations of 0, 20, 78, 313, 1250, and 5000 μg/plate. Test were conducted in the presence and absence of mammalian metabolic activation S9

fraction of Tif:RAIf rats treated with Aroclor 1254.

Atrazine was tested up to the limit concentration, 5000 μg/plate. The positive controls did induce the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as Acceptable

- Guideline . It does satisfy the guideline

req uirement (OPPTS 870.5100) for in vitro mutagenicity (bacterial reverse gene mutation) data.Do not cite

Appendix F

- Page 603 of 677 870.5385 Mammalian Bone Marrow Chromosome Aberration Test

A mouse bone marrow micronucleus test was conducted using Tif:MAGF mice (MRID 40722301). The test co nsisted of two parts. The first portion consisted of 24 male and 24 female mice being dosed with 2250 mg/kg atrazine (98.2% a.i.). Eight animals of each sex were then sacrificed at 16, 24 or 48 hours following treatment. The second portion of the study 24 mice, 8/sex/dose, were treated with atrazine (98.2% a.i.) at doses of 562.5, 1175, 2250 mg/kg. Bone marrow cells were harvested at 24 hours
post

- treatment. The vehicle in both portions of the study was carboxymethyl cellulose. Exposure in both portions of the study was accomplished by a single gastric intubation.

There were no signs of toxicity in either portion of the study. Atrazine was tested at an adequate doses being that these were doses that induced death in mice. The positive

control induced the appropriate response. There was not a significant increase in the frequency of micro nucleated polychromatic erythrocytes in bone marrow after any treatment time or dose.

This study is classified as Acceptable

- Guideline, and it satisfies the guideline

re quirement (OPPTS 870.5375) for in vivo cytogenetic mutagenicity data.

870.5450 Dominant Lethal Assay

In a mouse dominant lethal assay (MRID 42637003), groups of 30 male Tif: MAGf (SPF) mice were treated orally by gavage with Atrazine technical (97.1% a .i., batch #SG8029BA10) at doses of 0, 500, 1000, 2000, or 2400 mg/kg body weight in a volume of 10 mL/kg. The vehicle was corn oil. Starting immediately after dosing, each male was mated with 2 untreated females per interval for days 1

- 4, days 4

- 8, and days

8

- 12. Each male was then mated with 2 untreated females per week for weeks three through eight.

Atrazine technical was tested at an adequate dose. There were signs of toxicity after dosing as evidenced by piloerection and decreased locomotor activ ity. The females were sacrificed on gestation day 13

- 15 and the uteri examined for the number of alive, early, and late dead embryos and resorptions. Cyclophosphamide served as the positive control.

There was no significant difference between the control group and treated groups with respect to post

- implantation mortality of embryos. Under the conditions of this study atrazine technical did not induce dominant lethal mutations in male mice at doses as high as 2400 mg/kg.

This study is classified as Accep table

- Guideline , and it satisfies the guideline requirement (OPPTS 870.5450) for rodent dominant lethal data.

870.5550 UDS AssayDo not cite

Appendix F

- Page 604 of 677 In an unscheduled DNA synthesis assay (MRID 00161790/40722301) primary rat hepatocytes from adult male Tif:RAIf rats tha t were exposed to concentrations of atrazine of 0, 1.2, 6, 30, 150 μg/mL for five hours. No concentration induced UDS, including the high dose of 150 μg/mL, where precipitation of the test article

occurred.

This study is classified as Acceptable

- Guid eline , and it satisfies the requirement (OPPTS 870.5550) for other genotoxic mutagenicity data.

870.5550 UDS Assay

In an unscheduled DNA synthesis assay (MRID 42547105), primary rat hepatocyte cultures were exposed to atrazine, (97.1% a.i.), in dim ethyl sulfoxide at concentrations of 15, 46, 139, 417, 835, and 1670 μg/mL for 16

- 18 hours.

Atrazine was tested up to precipitating concentrations, 139 μg/mL. The positive controls did induce the appropriate response. There was no evidence that unschedu led DNA synthesis, as determined by nuclear silver grain counts, was induced. This study is classified as Acceptable

- Guideline , and it satisfies the guideline requirement (OPPTS 870.5550) for other genotoxic mutagenicity data.

F. 4.7 Neurotoxicity

Acute and subchronic neurotoxicity studies (OPPTS 870.6200) are not available for atrazine. Special studies submitted by the registrant (MRIDs 44152102 and 43934406) and published in the open literature (Cooper, et al. 2000. Atrazin e disrupts the hypothalamic control of pituitary

- ovarian function. Tox. Sci . 53: 297

- 307 [MRID 45166902]) provide evidence of atrazine

- associated central nervous system (CNS) toxicity. The neurotoxicity seen in these studies was a CNS toxicity (specifica lly, neuroendocrine alterations at the hypothalamus).

F. 4.8 Metabolism

Adequacy of data base for metabolism: The data base for metabolism is considered to be complete. No additional studies are required at this time.

870.7485 Metabolism

- Rat

Several metabolism studies have been submitted to fulfill the OPPTS 870.7485 guideline requirements. MRIDs 40431304; 40431305; 40431306; and 42165503 together satisfy the metabolism guidelines. MRID 44713802, by itself, satisfies the

OP PTS 870.7485 guideline requirements. These studies are summarized below.Do not cite

Appendix F

- Page 605 of 677 In a metabolism study (MRID 40431304) [atrazine, chemical purity of unlabeled compound not given, radio

- purity of labeled compound > 98%, all carbons on the triazine ring were labe led] was administered to 34 Sprague Dawley CD rats in four dose groups, 5/sex/dose, except controls which were 2 sex/dose. Three groups were dosed acutely at dose levels of 0, 1, and 100 mg/kg for a single dose given through oral gavage. A fourth dose gro up was dosed subchronically at 1.0 mg/kg/day for 15 days by oral gavage.

Distribution, accumulation

Distribution was dose

- dependent and independent of sex. In the 100 mg/kg group, the highest distribution of radiolabel was in red blood cells followed b y the heart, spleen, lung and liver. In the group given 1 mg/kg for 15 days distribution was highest in the red blood cell, followed by the liver, spleen, kidney and lung. Distribution appeared to follow

first

- order kinetics and the half

- life in the tissue s was 31.3 hours. This indicates that

atrazine does not bioaccumulate.

Excretion Approximately 95% of the atrazine was excreted within 7 days of dosing. The urinary

route accounted for about 75% of the excretion while feces accounted for 20%. The

route o f excretion did not seem to vary among sexes or with dose.

This metabolism study in the rat is classified Acceptable

- Nonguideline and, when considered in conjunction with studies: MRID Nos.: 404313

- 05;

- 06; and 421655

- 03

, satisfies the guideline require ment for a metabolism study (OPPTS 870.7485) in the rat. Only in conjunction with these four studies does MRID 404313

- 04 satisfy the metabolism guideline requirement.

In a metabolism study (MRID 40431305) Atrazine, 98.8% a.i., was labeled with

14 C on al l carbons of the triazine moiety, radiopurity 97.9%, and was administered to 14 female Sprague

- Dawley rats 2/dose. The animals were dosed daily through a stomach tube with dose levels of 0, 1, 3, 7, 10, 50 or 100 mg/kg/day. One animal from each

group was sacrificed 3 hours and the other was sacrificed 72 hours following the last

dose.

Distribution, accumulation

The location of 14 C label did not vary with dose but the amount distributed did vary with dose. Distribution was highest in the red blood cell, followed by the liver, ovary and kidney. When the dose increased the amount distributed in the tissues increased. The distribution appeared to follow first

- order kinetics and the tissue half

- life was 38.6 hours. This indicates that atrazine, with possible exception of the red blood cell, does not

bioaccumulate.

This metabolism study in the rat is classified Acceptable

- Nonguideline and, when considered in conjunction with studies: MRID Nos.: 404313

- 04;

- 06; and 421655

- 03

, satisfies the guideline require ment for a metabolism study (OPPTS 870.7485) in the rat. Only in conjunction with these four studies does MRID 404313

- 04 satisfy the metabolism guideline requirement.Do not cite

Appendix F

- Page 606 of 677 In a metabolism study (MRID 40431306) atrazine (purity of unlabeled compound not reporte d but radiolabeled purity >97%), 14 C

- labeled on all carbon of the triazine moiety, was administered to 13 female Sprague

- Dawley rats. The test article was given through the stomach tube in a single oral dose. Five animals were given 100 mg/kg and urine and fecal samples were collected 24, 48 and 72 hours later, after which

the animals were sacrificed and 5 mL of blood and the liver were obtained. The remaining 8 animals were given between 16.18 and 19.64 mg/kg and urine was collected over a 24 hr period. The urine was analyzed for metabolites using anion/cation exchange chromatography or reverse phase HPLC followed by infrared and mass spectrometry.

Excretion In the rats given 100 mg/kg, 100% of the administered radioactivity was accounted for

within 3 days of dosing. Urine was found to contain 47.3% of the radioactivity and the

feces 49.3%. The tissues contained 5.75% and 1.4% was found in the blood.

Metabolism Metabolites detected from the 8 rats given 16.18

- 10.64 mg/kg indicated that

dechlorination of the triazine ring and N

- dealkylation are the major metabolic pathways for atrazine in rats. Oxidation of the alky substituents of atrazine appears to be of minor metabolic importance.

This metabolism study in the rat is classified Acceptable

- Nonguide line and, when considered in conjunction with studies: MRID Nos.: 404313

- 04;

- 05; and 421655

- 03

, satisfies the guideline requirement for a metabolism study (OPPTS 870.7485) in the rat.

In a metabolism study (MRID 42165503) fecal and urinary samples from rats exposed to 1 or 100 mg/kg radiolabeled atrazine in a separate metabolism study (MRID 40431304) were obtained and analyzed by thin layer chromatography to determine metabolism profiles. No sex differences in metabolic profiles were evident. The major

f ecal metabolite was DACT which accounted for 40% of the total fecal radioactivity. The major urinary metabolite was also DACT.

This metabolism study in the rat is classified Acceptable

- Nonguideline and, when considered in conjunction with studies: MRID Nos.: 404313

- 04;

- 05; and

- 06 , satisfies the guideline requirement for a metabolism study (OPPTS 870.7485) in the rat

In a metabolism study (MRID 44713802), groups of male and female rats (3

- 12 per group depending on the experiment) were each given [U

1 4 C] Triazine G 30027 (batch no. GAN

- XVIII

- 77

- 5; purity>95%) and non

- radiolabeled Triazine (batch no. AMS 126/105; purity 99%) to provide a total single oral dose of 1 or 100 mg/kg. Urinary and fecal excretion were monitored up to 168 hours, tissue burdens were assessed up to 336 hours post dosing, and major metabolites quantified and, where possible,Do not cite

Appendix F

- Page 607 of 677 identified. Biliary samples were taken from four male rats up to 48 hours post dosing and metabolite analysis performed.

There were no treatment

- related de aths. Recovery of administered radioactivity was 95.84% and 98.17%, respectively, for the administered high (168 hours postdosing) and low dose (48 hours postdosing). Based upon urinary and biliary excretion data, and tissue/carcass burden data, absorptio n was relatively rapid. The time to maximum blood concentration (t cmax ) was 2 hours and 24 hours for the low and high dose groups, respectively. Under the conditions of this study, absorption and elimination did not appear to be saturated. The major route of excretion was via the urine, accounting for

64.72% of the total administered low dose over a 48

- hour period and 66.16% of the total administered high dose over a 168

- hour period. Time

- course studies revealed that within 48 hours urinary excretion was 1 00% and 94% complete for the low

- dose and high

- dose group, respectively. Fecal elimination accounted for 10.80% and 19.69% of the total dose for the low and high dose groups, respectively. Over a 48

- hour period, biliary secretions accounted for 7.35% of t he administered dose (68% of total amount excreted via the feces) in the low

- dose group. Total fecal elimination of radioactivity accounted for 10.80% and 19.69% of the total administered dose, a portion of which was due to biliary secretion resulting from absorbed test material.

Tissue distribution appeared to be extensive and reached maximum concentrations (t cmax ) at 2 hours and 24 hours for the low

- dose and high

- dose groups, respectively. Time to half maximum tissue concentrations (1⁄2 t cmax ) for these g roups were 48 hours and 168 hours, respectively. Although test article associated radioactivity was widely distributed, concentrations were greatest in red blood cells and highly perfused tissues and organs. With exception of red blood cells, whole blood , and skeletal muscle, tissue burden for any specific tissue or organ represented less than 1% of the administered dose by 14 days post dosing. Whole

- body autoradiography confirmed the extensive distribution of the test material and/or its metabolites, an d the rapid removal of radioactivity from the gastrointestinal tract. The pattern of tissue distribution did not appear to be significantly affected at the doses tested. Removal of radioactivity from tissues was a biphasic process characterized by a relat ively rapid phase at 48 hours followed by a slower phase (48

- 336 hours). Depuration of radioactivity from red blood cells, however, exhibited a slower monophasic pattern over 336 hours.

Urinary, biliary and fecal metabolites were detected and revealed m inimal dose

- related qualitative differences in metabolite profile. Quantitative differences were not inconsistent with that expected for a 100

- fold difference in dose. Twenty six urinary metabolites were detected although only two accounted for more than 5 % of the administered dose. Chromatographic analysis using known reference standards allowed for identification of four of the urinary metabolites. Nine biliary metabolites were detected, although none of the biliary metabolites accounted for more than 1.6 % of the total administered dose. Three biliary metabolites were also identified as urinary and fecal metabolites. Twelve metabolites were identified in the feces of high

- dose rats and

nine metabolites were identified in the feces of low

- dose rats, none o f which represented more than 2.42% of the total administered radioactivity.Do not cite

Appendix F

- Page 608 of 677 This study adequately described the metabolism and disposition of Triazine in rats administered single oral doses of 1 or 100 mg/kg. Inventory of administered radioactivity was acceptable and metabolite profiles indicate that, at the doses tested, Triazine G 30027 is nearly completely metabolized. The conclusion of the study authors that the major metabolic pathway appears to be dealkylation to the major metabolite (G 28273) an d that dechlorination to CGA 10582 is a minor pathway is consistent with the data presented in the study report.

This metabolism study in rats is Acceptable/Guideline and satisfies the requirements for a Metabolism and Pharmacokinetics Study (OPPTS 870.74 85). This study (MRID 44713802) was properly designed and conducted, and provided data regarding the absorption, distribution, and elimination of the test material, and quantitation and identification of urinary, biliary, and fecal metabolites in rats giv en a single oral low dose (1 mg/kg) or high dose (100 mg/kg) of [U

14 C] Triazine G 30027.

870.7600 Dermal Absorption

- Rat

In a dermal absorption study (MRID 43314302), male Charles River CD rats received either 0.01, 0.1 or 1 mg/cm 2 of 14 C

- atrazine( two vials used: radiochemical purity: 98.7 and 98.9%, specific activity: 1.9 uCi/mg and 19.1 uCi/mg). Animals were exposed in three groups: animals in group one were exposed to a single dose of the 3 above listed concentrations for 0.5, 1, 2, 4, 10, or 24 hours and were sacrificed immediately following dosing; group two animals were exposed to a single dose of the above listed concentrations for 10 hours and were sacrificed 34, 58 or 82 hours following the start of exposure; and group three animals were ex posed to a single dose of the above listed concentrations for 24 hours and then sacrificed 48, 72 or 96 hours following the start of exposure. The exposure sites were washed with liquid Dove and water at the end of the exposure period. The animals were kep t in metabolism cages during exposure and after exposure until sacrifice.

The percent absorbed increased with time and decreased with dose. Regardless of the dose or exposure time, the majority (65

- 95%) of the radio labeled atrazine was

recovered in th e washes or was found associated with the skin at the site of exposure. The maximum percent absorbed was in the animals exposed to the low dose for 24 hours and sacrificed 96 hours following the start of exposure. Absorption was approximately 30% in this g roup. This indicates that much of what remains on the skin is subsequently absorbed.

The percent absorbed increased with time and decreased with dose. A significant portion of compound remaining on washed skin is subsequently absorbed given enough

time.

This dermal absorption study in the rat is classified Acceptable

- Guideline , and satisfies the guideline requirement for a dermal absorption study (870.7600) in the rat.Do not cite

Appendix F

- Page 609 of 677 870.7600 Dermal Absorption

- Human

In a dermal absorption study (MRID 44152114), 1 0 human volunteers were exposed to a single topical dose of [triazine ring

- U

14 C] atrazine (94.3

- 96.3% a.i., 98.0

- 98.4% radiochemical pur ity) at 6.7 (4 volunteers) or 79 μg/cm 2 (6 volunteers) for 24 hours; equivalent to 0.1667 and 1.9751 mg of [14C] atrazine for the low and high doses, respectively.

Overall recoveries of radioactivity from the low

- and high

- dose groups were 101 and 92%, r espectively. The majority (91.1

- 95.5%) of the dose remained unabsorbed and was detected in skin wash samples taken 24 hours after dosing. After 168 hours, only

5.6% of the dose was absorbed and excreted in the urine and feces of the low

- dose group and on ly 1.2% in the high

- dose group. The renal excretion half

- life was 19.6

- 29

hours for the low

- dose group and 25.9

- 31 hours for the high

- dose group. In both dose groups, peak urinary elimination occurred at 24

- 48 hours and peak fecal elimination

occurred at 48

- 72 hours.

Total chlorotriazine residues detected by TLC in a high

- dose 0

- 24 hour composite urine sample accounted for 9.16% of the TRR and included deethyl atrazine (3.88% TRR) and didealkyl atrazine (5.28% TRR). No atrazine was detected. GC/MSD ana lysis of urine samples also did not detect atrazine or its chlorotriazine metabolites.

Enzyme immunoassays indicated that levels of atrazine mercapturate, an expected glutathione pathway metabolite was near the limit of detection/quantitation for the

meth ods. Mercapturic acid conjugates of atrazine and its chlorotriazine metabolites were not detected by LC/MS/MS in urine samples.

Some similarities in HPLC profiles of urine from i.v. dosed monkeys (MRID 44152112)

and dermally treated humans were observed

.

This dermal absorption study in humans is Acceptable

- Nonguideline . This study was not meant to satisfy an OPPTS 870.7600 guideline.

F. 4.9 Special/Other Studies

F. 4.9.1 Assays of Direct Estrogenic Activity of Triaz ines

Study 1:

This study (MRID 43598617) combines three in vivo assays to investigate the estrogenic effects of atrazine, simazine, and diaminochlorotriazine [DACT, (a.i. > 96%)] on body w eight, body weight gain, uterine weight, progesterone receptor binding capacity, and thymidine incorporation on female Sprague

- Dawley rats.

For body weight and uterine weight measurements, ovariectomized rats were dosed by oral gavage daily for 3 days at 20, 100, and 300 mg/kg/day in single exposureDo not cite

Appendix F

- Page 610 of 677 experiment and in co

- exposure experiments with 2 μg estradiol on day 2 and 3. Body weight gains were decreased for rats exposed to the vehicle control, 0.5% carboxymethylcellulose, (

- 3.5%) and to the positi ve control, 2 μg estradiol, (

- 6.9%). Larger decreases in weight gain were observed in rats exposed to triazines. At 20 mg/kg/day weight losses ranged from 6

- 9%. At 100 mg/kg/day, weight losses ranged

from 8

- 13.5%. At 300 mg/kg/day, weight losses ranged from 11

- 17%.

Chemicals with estrogenic activity cause proliferation and thickening of the uterine wall. The uterine weights of rats exposed to triazines were similar to vehicle control uterine weights. In co

- exposure experiments with estradiol, exposur e to atrazine and DACT at 100 and 300 mg/kg/day resulted in a dose

- dependant and statistically significant decrease in uterine weight relative to the positive control. In co

- exposure experiments with estradiol, uteri of rats exposed to simazine at 100 and 300 mg/kg/day were significantly decreased relative to the positive control.

Thymidine incorporation was measured to assess the effect triazines on DNA synthesis in the uterus of exposed rats. Dose groups included: exposure to 300 mg/kg/day of

triazine alone administered by oral gavage for 2 days; and exposure to 1,10, 20, 50, 100, and 300 mg/kg/day of triazine administered by oral gavage for 2 days plus an injection of 0.15 μg estradiol on day 2. Exposure to triazines alone at 300 mg/kg/day

resulted i n significant decrease relative to the vehicle control. Exposure to the positive control, 0.15 μg estradiol, resulted in approximately 2.5 fold increase (p < 0.05) in thymidine incorporation relative to the vehicle control. Exposure to 50, 100, and 300 mg/kg/day in co

- exposure with 0.15 μg estradiol resulted in a significant reduction in thymidine incorporation relative to the positive control.

Levels of inducible progesterone receptor (PR) were indirectly measured by the PR binding capacity to a radio active ligand. The PR was isolated from uteri of rats exposed by oral gavage to atrazine, DACT, and simazine alone at 300 mg/kg/day for 2 days and to atrazine, DACT, and simazine at 50 and 300 mg/kg/day in co

- exposure with 1 μg/day estradiol for 2 days. The PR binding capacity in rat uteri exposed to 300 mg/kg/day triazine alone was statistically decreased relative to vehicle control. PR binding capacity decreased significantly relative to the positive control with exposure to atrazine, DACT, and simazi ne at 300 mg/kg/day in co

- exposure experiments with 1 μg estradiol.

According to the effects on uterine weight, progesterone binding capacity, and thymidine incorporation at the concentrations tested in this study, atrazine, DACT, and simazine do not exhi bit estrogenic activity.

This study is classified as Acceptable

- nonguideline as a special study on in vivo and

in vitro estrogenic effects.

Study 2:

In a special study (MRID 43598618) on in vitro and in vivo competitive binding of atrazine, diamin ochlorotriazine (DACT, a mammalian metabolite of both atrazine and simazine) and simazine (>96 % a.i. for all three compounds) to the estrogen receptorDo not cite

Appendix F

- Page 611 of 677 (ER), several different experiments were performed utilizing extracted cytosolic ER from adult female Sp rague

- Dawley rats. Experiments observed the displacement of radiolabeled estradiol by atrazine, simazine, and DACT under both equilibrium (simultaneous exposure to triazine and radiolabeled estradiol at 4 C ) and at disequilibrium conditions (pre

- exposure to triazine at 25 C for 30 min. prior to exposure to radiolabeled estradiol). Results of equilibrium experiments indicated that atrazine, simazine, and DACT did not displace any radiolabeled estradiol whereas the positive

control exhibited a dose

- dependa nt displacement of the radiolabeled estradiol. Under disequilibrium conditions in a time

- course experiment, uterine cytosolic extracts were first incubated with triazine at 100 μM for 30 min. prior to exposure to radiolabeled

estradiol for 5

- 150 minutes . With time radiolabeled estradiol displaced triazine that had bound during the initial exposure. In a dose

- response experiment under disequilibrium conditions, uterine cytosolic extracts were first incubated with triazine at concentrations

10 - 9 to 10 - 3 M or unlabeled estradiol at 10 - 11 to 10 - 7 M prior to exposure to radiolabeled estradiol. The concentration at which 50% inhibition of binding occurred (IC 50 ) for
unlabeled estradiol was approximately 10 - 9 M whereas the IC 50 's for the triazine were 20 μM f or atrazine and 100 μM for simazine and DACT.

An additional experiment under disequilibrium conditions was performed to mimic a Scatchard type analysis using constant molar excess of triazine (100X estradiol, 10,000X atrazine, or 10,000X simazine) relativ e to the radiolabeled estradiol (0.2 nM, 0.5 nM, 1.5 nM, and 5.0 nM). Results indicated the triazines competed with the radioligand better at lower concentrations of tracer. For example, there was 60% displacement of 0.5 nM radiolabeled estradiol when co

- exposed with 5 μM atrazine whereas there was 11% displacement of 5 nM radiolabeled estradiol when co

- exposed with 50 μM atrazine. In the Scatchard

- type plot, dissociation constants were produced; 0.5 nM in the presence of 100

- fold molar excess of estr adiol and 1 nM in the presence of 10,000 fold molar excess of atrazine and simazine. The x

- intercepts of the plot were approximately equal (210 fmol/mg) in the presence of estradiol and simazine indicating competitive binding. The x

- intercept for the atr azine plot was slightly less (158 fmol/mg) indicating the potential for competitive binding but also some noncompetitive binding under the disequilibrium conditions of this study.

Experiments using uteri extracted with KCl to extract the total ER and with out KCl to extract the ER not bound or only loosely bound to chromatin were performed using cytosol fractions incubated with 50 μM triazine or 500 nM estradiol under disequilibrium conditions followed by separation of the sample by sucrose density centrifu gation. One hundred twenty five mL fractions were removed and counted for radioactivity. The authors generated plots representing the fraction number versus the radioactivity in the given fraction in order to observe which fraction exhibited the binding. In the extractions without KCl, the receptor binding peaks were detected in the 7

- 8S fraction for both estradiol and atrazine. Atrazine displaced an average of 10.7% of the radiolabeled estradiol whereas estradiol displaced all of the radiolabeled trace r. In the conditions favoring the extraction of total ER, activated and unactivated, binding was

detected in the 4

- 5S fraction. Atrazine displaced an average of 29.6% of the radiolabeled estradiol whereas estradiol displaced all of the radiolabeled trace r.Do not cite

Appendix F

- Page 612 of 677 Making the assumption that living rats contain the activated form of the ER, in order to test whether triazines competed more effectively against the transformed ER, ovariectomized rats were dosed with triazines prior to uterine dissection and compe titive binding experiments. The binding of radiolabeled estradiol was reduced by an average of 33%, 39%, and 24% for atrazine, simazine, and DACT, respectively (p < 0.05 vs. vehicle controls) in the 300 mg/kg/day group. In the 50 mg/kg/day group, binding of radiolabeled estradiol was reduced (not statistically significant) by 18, 21, and 13% .

Overall the results indicate that atrazine, triazine, and DACT do exhibit some competitive binding with estradiol but only under conditions which favor triazine binding.

This special study on in vitro and in vivo competitive binding of atrazine, DACT and simazine binding to the estrogen receptor in the rat is Acceptable

- nonguideline.

Study 3:

Another study (MRID 43598619) combines the following in vitro assa ys to investigate the estrogenic and antiestrogenic activity of atrazine and simazine: binding to hepatocyte Ah receptor; proliferation of MCF

- 7 (human breast cancer cell line) cells; gel electrophoresis mobility shift assay to measure levels of progestero ne receptor; and
MCF

- 7 transfection using a luciferase reporter gene.

Competitive binding to the aryl hydrocarbon (Ah) receptor was measured using 2,3,7,8

- tetrachlorodibenzo

- p

- dioxin (TCDD) as the competitor. This assay is performed because some dioxins, which are strong Ah receptor agonists, have also been shown to exhibit antiestrogenic activity. Cytosol from male rat hepatocytes containing Ah receptor was isolated and incubated with the positive control, radiolabeled TCDD, plus

either atrazine or si mazine. With addition of hydroxylapatite to this mixture, high molecular weight compounds, such as test compound bound to the Ah receptor, will precipitate out of solution. Subsequent centrifugation of this solution will result in the Ah receptor, and an ything bound to it, being spun out into a pellet. By counting the radioactivity in the pellet, it is possible to measure the amount of radiolabeled TCDD displaced indicating competitive inhibition with the triazine. Competitive binding was

measured as the decrease in radioactivity versus the positive control. At concentrations up to 10,000 nM, atrazine and simazine did not displace TCDD from the Ah receptor.

A cell proliferation assay using the MCF

- 7 cell line was performed using the following

doses: 1 0 μM, 1.0 μM, 0.1 μM, and 0.01 μM for atrazine and simazine and 1 nM for estradiol. Experiments using individual chemicals plus triazine were performed. Results indicated that the positive control increased the density of MCF

- 7 cells two fold

relative to the negative control after 11 days. Exposure to atrazine and simazine alone neither increased nor decreased cell number at day 11 relative to the negative control.

Results of co

- exposure experiments indicate that cell number did not change relative to

the positive control.Do not cite

Appendix F

- Page 613 of 677 Estradiol has been shown to increase the level of progesterone receptor in mammalian cells. This assay measures the effect of estradiol and/or triazine on the binding capacity of a high affinity ligand (R5020) to the progesterone rec eptor which is an indirect measure of progesterone level. The gel electrophoresis mobility shift assay utilized indirectly measures progesterone receptor (PR) levels in MCF

- 7 cells following exposure to atrazine, simazine, or estradiol for 3 days. Polyacr ylamide gel electrophoresis can separate molecules, such as DNA, or complexes of molecules, such as protein

- DNA complexes, based on size. The DNA used in this study is a radioactive oligonucleotide of a specific sequence which binds to the PR. This

seq uence is called a progesterone response element (PRE). PR isolated from MCF

- 7
cells is first incubated with R5020. PR

- R5020 complex was then incubated with

radiolabeled PRE. The R5020

- PR

- PRE complex was then run on a polyacrylamide gel.

The unbound PRE was separated from the bound complex based on differences in mobility through the gel. Doses used were 1 μM for atrazine and simazine and 10 nM for estradiol. Exposure to estradiol resulted in a 3.5

- fold increase in reactivity of the

R5020

- PR

- PRE comple x relative to the negative control. The radioactivity in the

R5020

- PR

- PRE band following exposure to atrazine and simazine was similar to negative control. The radioactivity in the band representing the R5020

- PR

- PRE complex was measured to get an indirec t measure of PR levels in MCF

- 7 cells. Results indirectly indicate that the levels of PR did not increase or decrease with exposure to atrazine or simazine.

Transfection experiments were performed where the human estrogen receptor along

with the lucif erase expression system were inserted into MCF

- 7 . The MCF

- 7 cell assay measured luciferase activity ( i.e ., light production) and is quantitative in nature ( i.e

., concentration of estrogenic chemical is directly related to light production). Doses used

i n the luciferase experiments were 10 - 13 M to 10 - 8 M estradiol and 10 - 9 M to 10 - 5 M atrazine and simazine. Atrazine and simazine did not induce luciferase activity above background at concentrations as high as 10 - 5 M (concentrations above 10 - 5 M are toxic
to MCF

- 7 cells). Exposure to the positive control, estradiol, resulted in significant luciferase activity beginning at 10 - 12 M. Light production increased exponentially up to

10 - 8 M where light production plateaued.

Neither atrazine nor simazine displ ayed estrogenic activity or interacted with the Ah receptor in the set of experiments described in this paper.

This study is classified as Acceptable

- nonguideline as a special study on in vitro estrogenic effects. Study 4:

Another study (MRID 43934403 ) , combines three in vivo and four in vitro assays to investigate the estrogenic and antiestrogenic activity of atrazine and

simazine. In vivo experiments, 4 or 5 female Sprague

- Dawley rats/dose were dosed orally or by intraperitoneal injection. In order to test for estrogenic activity, rats were exposed to atrazine (> 97% a.i.) or simazine (> 97% a.i.) at 50, 150, and 300 mg/kg/day or estradiol (positive control) at 10 μg/kg/day. In order to test for antiestrogenic activity,

co

- exposure experiments usin g atrazine or simazine plus estradiol were performedDo not cite

Appendix F

- Page 614 of 677 using the same concentrations. Following a three day exposure, the following in vivo endpoints were measured: uterine weight, progesterone receptor levels and uterine peroxidase.

Exposure to estradiol r esults in statistically significant increased uterine weights, increased levels of the progesterone receptor, and increased activity of uterine peroxidase. Exposure to atrazine and simazine individually resulted in negative effects indicating antiestrogen ic activity are shown in slightly reduced uterine weights, statistically significantly reduced levels of the progesterone receptor, and statistically significantly reduced activity of uterine peroxidase.

The following in vitro assays were performed: proli feration of MCF

- 7 (human breast cancer cell line) cells; gel electrophoresis mobility shift assay to measure levels of progesterone receptor; MCF

- 7 transfection using the luciferase reporter gene; and yeast transfection using a selective media expression s ystem.

Doses for the cell proliferation assay when tested individually and in combination were 10 μM, 1.0 μM, 0.1 μM, and 0.01 μM for atrazine and simazine and 1 nM for estradiol. Results indicated that the positive control significantly increased the density of MCF

- 7 cells three fold relative to the negative control after 11 days . Exposure to atrazine and simazine alone neither increased nor decreased cell number at day 11 relative to the negative control. Results of co

- exposure experiments indicate that cell number did not change relative to the positive control .

The gel electrophoresis mobility shift assay used in this study indirectly measures progesterone receptor (PR) levels in MCF

- 7 cells following exposure for 3 days. The oligonucleotide used contains progesterone response element (PRE) which binds to the

PR. PR from MCF

- 7 cells was isolated and incubated with radiolabeled PRE. The PR

PRE complex was then run on a polyacrylamide gel to separate bound and unbound

PRE based on differences in mobility through the gel. The radioactivity in the band representing the PR

- DNA complex was measured as an indirect measure of PR levels. Doses used were 1 μM for atrazine and simazine and 10 nM for estradiol. Results indicated that exposure to est radiol resulted in a 3.5

- fold increase in reactivity of the

DNA

- PR complex relative to the negative control. The radioactivity in the DNA

- PR band following exposure to atrazine and simazine was similar to negative control. These

results indicate indirect ly that the levels of PR did not increase or decrease with exposure to atrazine or simazine.

Two transfection experiments were performed where the human estrogen receptor was inserted into the genome of MCF

- 7 and yeast cells. The MCF

- 7 cell assay measur ed luciferase activity as a surrogate measure of estrogen receptor activity. Doses used

were 10 - 13 M to 10 - 8 M estradiol and 10 - 9 M to 10 - 5 M atrazine and simazine. Atrazine and simazine did not induce luciferase activity above background at concentratio ns as high as 10 - 5 M (concentrations above 10 - 5 M are toxic to MCF

- 7 cells). Exposure to the positive control, estradiol, resulted in significant luciferase activity beginning at 10 - 12 M and increased exponentially up to 10 - 8 M where light production plat eaued. AtrazineDo not cite

Appendix F

- Page 615 of 677 and simazine did not exhibit in estrogenic or antiestrogenic activity and did not interact directly with the estrogen receptor. In the yeast transfection assay, the human estrogen receptor linked to a necessary

amino acid was inserted into the yeast. Growth on selective media is a qualitative measure of activity of estrogen receptor. Yeast were dosed with 1 nM estradiol or 10 μM atrazine or simazine. Yeast proliferated when exposed to estradiol but did not proliferate when exposed to atr azine or simazine. These results indicate that atrazine and simazine did not exhibit estrogenic activity and did not directly interact with the estrogen receptor.

In conclusion, the results of these experiments indicate that in vivo atrazine and

simazine exhibit s some antiestrogenic activity, but no estrogenic activity. Based

on the in vitro results, this antiestrogenic activity is not the result of direct interaction with the estrogen receptor.

This study is classified as Acceptable

- nonguideline as a special study on in vivo and

in vitro estrogenic effects.

F. 4.9.2 Estrous Cycle Alterations and LH Surge Attenuation

1. Pilot study

In a study (MRID 43934404) to determine the valid ity of a proposed protocol for testing the effect of atrazine exposure on the proestrus afternoon luteinizing hormone (LH) surge, estradiol (E 2 ) was administered to 70 ovariectomized female Sprague Dawley rats through a surgically implanted silastic capsul e. Serum LH, E 2, and prolactin levels were measured 3 days later to determine if the LH and prolactin surges could be measured and to evaluate success of the estradiol implantation procedure.

The ovariectomy followed by estradiol implantation procedure ap peared to be effective in inducing LH and prolactin surges, as both these surges were clearly evident in rats undergoing this procedure.

This special study in the rat is Acceptable

- nonguideline . This study does not satisfy any guideline requirements and was not submitted with the intention of satisfying a guideline requirement.

2. 28 day study

In a study to evaluate the effect of atrazine exposure on the proestrus afternoon luteinizing hormone (LH) surge (MRID 43934406) atrazine, 97.1% a.i., was

admini stered to 450 female Sprague Dawley rats in the diet. Dose levels were 0 (vehicle control), 2.5, 5, 40 and 200 mg/kg/day for 28 days.

Mortality, clinical signs, gross pathology and pituitary weights were not affected in this study. Food consumption and b ody weights are decreased at the 40 and 200 mg/kg/day doses (body weight decreased 13% and 47% at 40 and 200 mg/kg/day respectively).Do not cite

Appendix F

- Page 616 of 677 The number of animals with diestrus blocks were increased at 40 and 200 mg/kg/day.

The number of animals with estrus block s were increased at 200 mg/kg/day. The prolactin surge was attenuated at 200 mg/kg/day. The LH surge was attenuated at 40 and 200 mg/kg/day [non

- repeat bleed rats].

The LOAEL is 40 mg/kg/day, based on decreases in food consumption, body weight, body weigh t gain, estrous cycle alterations and LH surge attenuation. The NOAEL is 5 mg/kg/day.

This special study in the rat is Acceptable

- nonguideline . This study does not satisfy any guideline requirements and was not submitted with the intention of satisfying a guideline requirement.

3. Six month study

In a study to evaluation the effect of long

- term atrazine exposure on the proestrus afternoon luteinizing hormone (LH) surge (MRID 44152102) atrazine, 97.1% a.i., was administered to 360 female Sprague Dawley ra ts in the diet. Dose levels were 0 (negative control), 25, 50, and 400 ppm (0, 1.80, 3.65, 29.44 mg/kg/day) for 26 weeks (approximately six months).

Body weight, body weight gain and food consumption were significantly (p 0.05)

decreased in animals at th e highest dose tested compared to controls (body weight decreased 8.5% at the end of the study and food consumption decreased 3.75% for the entire study). The percentage of days in estrus were significantly increased (p 0.01) during the 21

- 22 and 25

- 26 we ek time periods at the high

- dose level. Percent days in estrus were also increased during the 21

- 22 and 25

- 26 week time periods at the mid dose, but the increase was only significant (p 0.05) for the 21

- 22 week time period. The proestrus afternoon LH surge was severely attenuated at the high dose (LH levels at most sampling time points were actually decreased compared to baseline) and less so at the mid dose (maximum increase over baseline was 157% compared to maximum

increase over baseline in controls of 2 73%). Pituitary weights were increased at the high dose (absolute weight increased 22% and weight relative to body weight was increased 28%). Pituitary weights at the other two doses were not affected. At the high dose, there was a slight increase in anima ls displaying enlarged pituitaries (0% in controls compared to 3.4% at 29.44 mg/kg/day) and thickened mammary glands (0% in controls compared to 6.7% at 29.44 mg/kg/day). There were no other gross necropsy findings in the high dose that could be attributed to compound exposure and there were no compound

- related gross pathology findings at the mid

- or low

- dose. Selected tissues were saved for histopathology but those results have not been reported.

There were no compound

- related effects in mortality or clin ical signs. The proestrus afternoon prolactin surge was not affected by compound exposure at any dose. The low dose had no effects on the estrous cycle, LH or prolactin surges.

The LOAEL is 3.65 mg/kg/day, based on estrous cycle alterations and LH surge attenuation. The NOAEL is 1.8 mg/kg/day.Do not cite

Appendix F

- Page 617 of 677Do not cite

Appendix F

- Page 618 of 677 This special study in the rat is Acceptable

- nonguideline . this study does not satisfy any guideline requirements and was not submitted with the intention of satisfying a guideline requirement.

F. 4.9.3 Other Two

- Year Bioassays

Three other two

- year bioassays have been submitted to the Agency. These are: 1. a serial sacrifice study using Sprague

- Dawley female rats (Study 1, below); 2. a study using Sprague

- Dawley female r ats with two dose groups and a control (Study 4, below); 3. a serial sacrifice study using F

- 344 female rats (Study 5, below).

Certain data collected in the two serial sacrifice studies were submitted separately and have their own MRID numbers. Hormon e measurements and estrous cycle data from both SD and F

- 344 bioassays were submitted under the MRID number 42743902. Histomorphology data from these two bioassays was submitted under the MRID number 43598622. The table below displays the MRID numbers f or each

submission.

SD Serial Sacrifice Bioassay F

- 344 Serial Sacrifice Bioassay

Measurements MRID # Measurements MRID #

Body wt., food cons., clinical observations, gross pathology 42085001 Body wt., food cons., clinical observations, gro ss pathology 42146101

Hormone measurements and Estrous cycle

measurements 42743902 Hormone measurements and Estrous cycle

measurements 42743902

Histomorphology 43598622 Histomorphology 43598622

Study 1 (SD serial sacrifice bioassay):

In a 2

- year bioassay (MRID 42085001) seventy SD female rats (no males were used) were exposed, through the diet, to doses of Atrazine (97%) at 0, 70 and 400 ppm ( 0, 4.23 and 26.23 mg/kg/day) for 2 years. Ten females per dose were sacrificed at 1,
3, 9, 12, 15, 18 and 24 months. Vaginal smears were taken for two weeks prior to each sacrifice date and serum was collected for measurements of estradiol, progesterone, prolactin and corticosterone, on the day of sacrifice.

Mortality was increased in a dose

- dependent manner. There were five unscheduled deaths in the control group, 6 in the 70 ppm group and 8 at 400 ppm. Using the Gehan

Breslow test there was a statistically significant negative trend for survival (survival decreased as the dose increased). Another st atistical test

- the Cox

- Tarone test

- did not indicate a significant trend in either direction. Body weights at several timepoints in the 400 ppm group were statistically significantly reduced compared to controls. FoodDo not cite

Appendix F

- Page 619 of 677 consumption was also statistically significantly reduced at several timepoints in the 400 ppm group compared to controls. Body weights and food consumption did not appear to be altered in the 70 ppm group compared to controls.

Atrazine treatment at 26.2 mg/kg/day increased mortality, de creased body weights and decreased food consumption.

Study 2 (hormone measurement and estrous cycle evaluations in SD rats):

In a special study (MRID 42743902) female SD rats from the two year bioassay with serial sacrifices described above under "Study 1" (MRID 42085001) had their estrous cycles measured and serum levels of estradiol, progesterone, prolactin and corticosterone measured prior to or at each serial sacrifice. The results of these estrous cycle evaluation and serum hormone measurements are summarized below.

Estrous cycle evaluations (vaginal smears)

The percent days spent in estrus in control animals increases as the animals age at the expense of days spent in diestrus. The percent days of the cycle spent in estrus was dramatically incre ased in dosed animals compared to controls at nine, 12 and 18 months. Females in the 4.23 and 26.23 mg/kg/day groups at nine months spent an average of 34.3 and 44.8% of their days in estrus, respectively (p<0.05 at 70 and p<0.01 at 400 ppm). This is com pared to 24% spent in estrus at the controls. The

dose

- related trend, as determined using a Terpstra

- Jonckbeere Trend Test, showed a significant increase in % days spent in estrus (p<0.01). At 12 months there was also a

dose

- related trend (p<0.05) but th e increases in dosed groups compared to controls as determined by ANOVA were not significant at either dose.

Atrazine

- treated animals showed an early onset of increased days in the estrus phase of the estrous cycle. This effect occurred at both doses in this study.

Hormone measurements

Serum progesterone and corticosterone levels did not show any significant

dose

- related alterations compared to controls. Serum estradiol levels in the control rats in this study showed a positive trend (levels increased as time increased) over months one through nine. Exposure to atrazine did not alter this trend. The 4.23 and 26.23 mg/kg/day dose groups also showed a significantly positive trend from months one through nine. These trends are expected as constant estrus would be expected to begin to set in as these animals approach nine months of age. Examination of the pairwise comparisons at three months indicates that treated animals had an early onset of increased serum estradiol levels compared to controls. At thr ee months control estradiol levels were 3.5 ng/mL, 70 ppm levels were 11.2, and 400 ppm levels were 16.2 ng/mL. The increase at 70 ppm was significant at p<0.05, the increase at 400 ppm was significant at p<0.01 and the trend, determined using a Terpstra

- Jonckbeere Trend Test, was positive at p<0.05. At nine months control and 4.23 mg/kg/day groupDo not cite

Appendix F

- Page 620 of 677 estradiol levels were similar, but the 26.23 mg/kg/day group compared to controls was increased 44%. At nine months estradiol levels were elevated compared to control, but not significantly so.

An early onset of increased serum estradiol levels is seen in both doses of

atrazine

- treated females

.

Study 3 (histomorphology in SD):

In a special study (MRID 43598622) female SD rats from the two year bioassay wi th serial sacrifices described above under "Study 1" (MRID 42085001) had certain tissues, including their ovarian and mammary gland tissues, examined histopathologically. An evaluation was made of parameters in the ovarian tissues

which indicated the an imals current and recent estrous cycle states. The mammary gland was examined by histopathology for indications of exposure to steroid hormones such as estrogens, progesterone and prolactin. The examination of tissues, through histology, for morphologic alterations which provide information about certain aspects of an animals' physiologic state (such as the animal's estrous cycling ability or their exposure to hormones) is termed histomorphology. The results of the ovarian and mammary gland histomorpholo gy from evaluations from this study are summarized

below.

Ovarian Gland Histomorphology :

Selected tissues (including both ovaries and mammary glands) were removed and preserved at the serial sacrifice intervals described above under study 1, MRID

4208500 1. Several ovarian parameters were evaluated including the number and type of corpora lutea and the number of antral follicles. The presence of many "old" corpora lutea indicates that an animal went through normal estrous cycles in the past. A complete absence of corpora lutea indicates that an animal is not cycling and has not gone through an estrous cycle in some time. Antral follicles are mature follicles that are close to ovulation. An increased number of these means that they are accumulating and

i mplies that the signal for ovulation (an LH surge) may not be available or of sufficient magnitude to stimulate ovulation.

There were no differences in number of old corpora lutea or antral follicles between controls and dose groups at the one month ti me point. By three months the number of animals which did not have old corpora lutea present increased in a slight, but dose

dependant manner, from zero in controls to one at 4.23 mg/kg/day and two at 26.23 mg/kg/day. Of the animals that had corpora lute a, there was an increased incidence in the dose groups of animals with reduced numbers of corpora lutea. Twenty percent of the controls were found to have reduced numbers of corpora lutea compared to 33% at 4.23 and 37.5% at 26.23 mg/kg/day. The mean gra de per animal for antral follicles ( i.e

., the sum total of all grades for all animals in the group divided by ten) was 2.1 and 2.2 in the 4.23 and 26.23 mg/kg/day groups, respectively, compared to 1.2 for controls. A higher mean antral grade indicates an ovulation. These differences between control and treated groups became more extreme at the nine month timepoint. Seven of tenDo not cite

Appendix F

- Page 621 of 677 and ten of ten animals at nine months in the 4.23 and 26.23 mg/kg/day dose groups, respectively, had no corpora lutea at all pr esent in their ovaries. This is compared to six of ten animals in the controls which did not have corpora lutea present. The mean antral follicle grade was 3.1 and 3.8 at the 4.23 and 26.23 mg/kg/day dose groups, respectively. This is compared to 2.6 in t he controls. Data from the 12 month timepoint indicated that control animals had equalized with the treated animals in terms of parameters representing anovulation. Ten of ten controls and ten of ten high dose animals displayed no corpora lutea at 12 mon ths while eight of ten low dose animals displayed no corpora lutea. The mean antral follicle scores were 3.3, 2.9 and 3.1 for the control, low dose, and high dose, respectively.

Compared to controls there was an early onset in dosed groups, at three an d nine months, of parameters such as absence of old corpora lutea, absence of any corpora lutea, corpora lutea reduced in number, and increased numbers of antral follicles. These parameters indicate lack of ovulation and the appearance of these parameter s in greater numbers in dose groups than in controls, at three and nine months, provides evidence that atrazine exposure is associated with and early onset of anovulation in the this assay.

Mammary Gland Histomorphology :

Mammary gland histomorphology for several parameters is discussed below. Index weighted scores are used to describe the results of the histomorphic evaluations. An index weighted score assigns a numerical value to the severity of the finding assigned by the pathologist. The higher the i ndex weighted score, the more severe was the finding in that group. These results show an early onset of increased index weighted scores for several histomorphic parameters which indicate exposure of the mammary tissue to estrogens, progesterone and prol actin.

Acinar Development

- An early onset of increased exposure to estrogen is indicated by acinar development histomorphology index scores indicate and early onset of this finding in atrazine

- exposed female SD rats. The index scores at the one

- month t ime point are slightly higher in the dose groups than in the control, but a dose

- response relationship was not seen. At the three

- month time point the index scores are again increased over control. The increase is at this time point is dose

- related thou gh with the high dose being more severe than the low dose. At both nine and 12 months the index score again indicate more severe acinar development in the dose groups compared to the controls with the increase in severity being especially obvious at the h igh dose.

Acinar/Lobular Development

- An early onset of this parameter is evident. The one

and three

- month timepoints have index scores in the dose groups that are similar to the control index scores. The dose group index scores are clearly increas ed compared to controls at nine and 12 month timepoints though. This indicates that the

atrazine

- treated animals were exposed to elevated levels of prolactin at an earlier time point than the control animals.Do not cite

Appendix F

- Page 622 of 677 Secretory Activity

- The index scores for secretory activity also demonstrate an early onset of increased prolactin exposure in the dose groups compared to the controls. Index scores at one and three months in the dose groups were similar to control values, while index scores in both dose groups were clearly elevated compared to controls at

the nine

- and 12

- month timepoints.

Dilated Ducts with Secretion

- Index scores at one and three months in the dose groups were similar to control values. The index scores for the 4.23 mg/kg/day group

compar ed to controls were only slightly elevated. The index scores for the 26.23 mg/kg/day group were greatly elevated compared to controls

Galactocele Incidence and Severity

- No galactoceles were observed in any group at

the one

- and three

- month timepoints. At nine and 12 months galactoceles in the dose groups were increased in both number and severity. By 15 months galactocele incidence and severity were similar between control and dose groups.

An early onset of several mammary gland histomorphology para meters in atrazine exposed SD rats of both doses in this study indicates an early onset of exposure to increased levels of steroid hormones

- particularly estrogens and prolactin

.

Study 4 (two dose two

- year bioassay):

Atrazine technical was given in the diet to groups of 60 (female only) Sprague

Dawley rats for 24 months (MRID 42204401) at concentrations of 0, 70, or 400 ppm (approximating 0, 3.79, 23.01 mg/kg/day respectively). Water and food were available

ad libitum . Test doses were selected followin g a 2

- year study using 0, 10, 70, 500 or 1000 ppm of atrazine in the diet (MRID 00158930).

A slight reduction (a negative trend) in survival found amongst the dosed groups was considered to be equivocal because the data were statistically significant (p< 0.05) by the Gehan Breslow test but not by the Cox

- Tarone test. No treatment

- related increases in clinical signs were noted in the study. Body weight gains were statistically significantly reduced relative to controls (approx. 12 to 13%) only at 400 ppm during

weeks 0

- 76. Food consumption was only minimally reduced at the highest dose. Slight alteration in red blood cell shapes and incidence of nucleated RBCs were transient in occurrence. Spleen weights were slightly increased but the increase was not statistically significant. Other organ weight and organ:body weight values from the 400 ppm group were not significantly different from controls. nonneoplastic lesion findings were comparable in controls and treatment groups. Palpation times for tumors

co nfirmed histologically indicated an early onset of mammary tumors. No statistically

significant increase in mammary tumors was observed.

The NOAEL for systemic toxicity is 70 ppm (calculated by the reviewer to be approximately 3.79 mg/kg/day) based on bod y weight gains of this group being

12

- 13% less than controls as well as statistically significant decreases in body weights in the 0

- 76 week period. Also, a reduction in survival considered to beDo not cite

Appendix F

- Page 623 of 677 equivocal is reported at 400 ppm. An MTD and effect level was determined in a previous chronic feeding study (MRID 00158930).

The study is classified Acceptable

- nonguideline. This study alone does not fulfill Guideline requirements 870.4300 for carcinogenicity evaluation because only 2 dose

levels were teste d. Other studies have completed this Guideline requirement.

Study 5 (Intact vs OVX females and long

- term estrous cycle evaluations):

In a carcinogenicity study intended to provide information about the mode of action for oncogenicity in atrazine expos ed Sprague

- Dawley rats,(MRID 44544701), atrazine, [97.1% a.i.] was administered to 800 female Sprague

- Dawley rats. The rats were divided into 2 groups of 400 each. One group was ovariectomized (OVX) while the

other was left intact. Atrazine was mixed with the diet at dose levels of 0 (control) 25, 50, 70 and 400 ppm (0, 1.5, 3.1, 4.2, 24.4 mg/kg/day for intact animals and 0, 1.2, 2.5, 3.5, and 20.9 mg/kg/day for ovariectomized animals) for 2 years.

Hematology, clinical chemistry, and urinalysis were not a ssessed in this study. Food consumption in the dose groups compared to the controls was not altered by compound exposure. The trend for survival was statistically

- significantly decreased in the dosed groups compared to the controls. Survival was as follows : 43.3% in controls; 31.7%

- 25 ppm; 28.8%

- 50 ppm; 31.6%

- 70 ppm; 21.7% 400 ppm. Body weight was statistically

significantly reduced in the first half of the study in the 400 ppm group (other groups were not significantly altered), but by the end of the study body weights were similar to control values. Organ weights for pituitary, uterus and the ovaries were taken in this study. No organ weights in either the intact or ovariectomized group were altered by compound exposure.

The only gross necropsy find ing which was altered by compound exposure was the occurrence of mammary masses in the intact dosed animals. Dosed animals showed a higher incidence of mammary masses (many of which were confirmed by histopathology to be tumors).

There were no non

- neoplas tic findings at histology that were increased in dosed OVX animals compared to controls. OVX animals in all groups displayed very high incidences of juvenile uterus and castration cells in the pituitary which would be expected in an OVX animal. The only fi nding in dosed intact animals which was increased in incidence over controls were ovarian cysts which were slightly increased at 70 and 400 ppm compared to controls. There were though, many findings which were prevalent in the intact animals, yet were not seen (or were seen at lower levels) in the OVX animals. Mammary gland galactoceles were seen in anywhere from 65 to 78% of the intact animals, depending on the dose group, but the highest percentage of galactoceles in the OVX group was 24% in the 50 ppm gr oup. Mammary gland secretory activity was seen in from 34 to 46% of the intact animals but was not seen in any OVX animals.

Uterine dilation was seen in from 8

- 18% of the intact animals and uterine cystic endometrial hyperplasia was seen in about 50% of th e intact animals. No OVX animalsDo not cite

Appendix F

- Page 624 of 677 displayed uterine dilation and only about 20% displayed cystic endometrial hyperplasia. Intact animals also had increased incidence of pituitary findings compared to OVX

animals. From 65 to 73% of the intact animals were fo und to have sinusoid ectasia/angiectasis but the range in OVX animals was 31 to 45%. The differences in mammary gland, uterine and pituitary findings between OVX and intact animals may provide information about the mode of action of atrazine‘s carcinogenic ity.

Neoplastic histopathology findings were mostly limited to the pituitary and the mammary gland. Neither OVX nor intact animals showed an increase in pituitary tumors compared to their respective controls, but intact animals did show a 20

- 30% greater i ncidence of pituitary adenomas compared to OVX animals.Do not cite

Appendix F

- Page 625 of 677 There were few mammary tumors in the interim sacrifice animals, which is not surprising given that these animals were sacrificed after only one

- year. Excluding the interim sacrifice and looking only at those animals which were sacrificed at 24 months and those which died prematurely, there was an increase in mammary tumor incidence at all intact dose groups compared to controls. In ascending order of dose the percentage of animals with any type of ma mmary tumors was: 38.3% in controls; 53.3%; 71.2%; 56.6% and 68.3% at 400 ppm. Looking at carcinomas alone values are: 18.3%; 36.7%; 33.9%; 20%; and 41.7%. Fibroadenomas alone were: 26.6%; 40%; 52.5%; 45%; and 40%.

A decrease in the time

- to

- tumor is als o evident from exposure to atrazine. In the control group 50% of the tumors occurred in the last 6 months of the study. The percentage of tumors which appeared in the last 6 months of the study in the dose groups were: 35.8%; 37.5% 36.5% and 33.4%. The num ber of tumors which occurred in the first year of the study was slightly increased at 25, 50 and 760 ppm and greatly increased at 400 ppm: 9% in controls; 10.7%; 10%; 12% and 17.9% in the 400 ppm group.

An increased incidence of mammary tumor in intact an imals was observed at doses of 50 ppm (3.1 mg/kg/day) and above.

The purpose of this study was to examine mammary tumor carcinogenesis in female Sprague

- Dawley rats. Thus, a large part of this review focuses on carcinogenicity.

One of the more striking a spects of the study was the complete lack of mammary tumors in OVX animals. Not a single mammary tumor of any sort was seen in any OVX animal. The lack of mammary tumors in OVX animals provides evidence that the mode

of action of atrazine is neither a dire ct genotoxic nor estrogenic effect on the mammary gland. Rather, an indirect hormonally

- mediated effect involving the ovary is implied.

At the 50 ppm and above doses, there was a treatment related increase in mammary tumor incidence when compared to contr ols. Dosing was considered adequate based on decreases in body weight. Additionally, there was a decreased time

- to

- tumor at doses of 25 ppm and above.

This carcinogenicity study in the rat is Acceptable

- nonguideline , and does not satisfy the guideline r equirement for a carcinogenicity study (870.4300) in the rat nor was it submitted with the intention of satisfying a guideline.

Study 6 (F

- 344 serial sacrifice bioassay):

In MRID 42146101 , seventy F

- 344 rats (females only) per dose were exposed

ad libi tum to diet that had been mixed with atrazine (97.1%) to the appropriate doses of 0 (negative control), 10, 70, 200 and 400 ppm (0, 0.68, 4.82, 14.05, 34.33 mg/kg/day). Ten animals per dose group were sacrificed after approximately one, three, nine, 12,

1 5, and 18 months exposure to the test article.Do not cite

Appendix F

- Page 626 of 677 There was not an increase in mortality due to compound exposure, and there was no increased incidence of clinical signs in dosed animals compared to controls. The doses tested appeared to be sufficiently high because there was a decreased absolute body weight and body weight gain in the 400 ppm group compared to the controls. Group mean absolute body weight in the 400 ppm group compared to controls was also significantly decreased compared to controls at several time points though the final mean body weight was not significantly decreased compared to controls. The final group mean body weight for the 400 ppm group was 6.6% less than the mean control value. During the course of the study the 400 ppm anima ls gained an average of 116.7 grams compared to the weight gain in the control group of 133.3 grams (14% less than controls). This difference in body weight gain was statistically

- significant at a p value of

0.05.

There was not an increase in mammary t umors or any other type of tumor at any dose group in either sex.

Studies 7 and 8, described below, are studies which performed hormone measurements, estrous cycle evaluations, and histomorphological evaluations on ovarian and mammary tissues in animals from this study.

Study 7 (hormone measurement and estrous cycle evaluations in F

- 344):

The hormone measurements data and estrous cycle evaluation results (vaginal smears)

from the animals in MRID 42146101 are shown in MRID 42743902. HED believes that the estrous cycle evaluation data reported in MRID 42743902 are unreliable. The histomorphology data from MRID 43598622 are instead used to determine stage of the estrous cycle.

The results of the hormone measurements did not reveal any consistently

sta tistically

- significant alterations in serum hormone levels compared to controls for any of the hormones tested. There were occasional significant alterations such as significantly decreased (p< 0.05) progesterone levels in the 4.82 mg/kg/day dose and

cort icosterone levels in the 34.33 mg/kg/day group at the 12

- month time point compared to controls; significant negative trends in estradiol levels at the 12

- month time point, progesterone levels at the 12

- and 18

- month timepoints, and corticosterone levels at the

12

- and 15

- month timepoints, and prolactin levels displayed a significant positive trend

at the three

- month time point only. Careful consideration of these alterations indicated that they did not appear to be related to atrazine exposure.

There wer e alterations in serum hormone levels that were seen in control as well as treated rats. These were: decreases in estradiol levels in the later half of the study; significantly increasing progesterone levels from one to 18 months. Decreased

estradiol an d increased progesterone are expected in rats undergoing a reproductive aging process involving pseudopregnancy. Exposure to atrazine did not alter the

age

- related changes in estradiol or progesterone levels.Do not cite

Appendix F

- Page 627 of 677 An increase in prolactin levels might be exp ected in an aging rat undergoing pseudopregnancy. However, consistent increases in serum prolactin levels were not

seen.

Study 8 (histomorphology in F

- 344):

The histomorphologic evaluation of tissue from animals in MRID 42146101 (study 6,

described abov e) is presented as MRID 43598622.

Ovarian Histomorpology : The great majority of F

- 344 rats in all groups, both control and dose groups, maintained corpora lutea throughout most of the study. Only at the final,

24

- month, time point were there dramatic d ecreases in corpora lutea numbers. The

atrazine

- treated animals at this time point did not show decreases in corpora lutea numbers any more severe than the control animals. The reduction in corpora lutea numbers at this late time point appears to be a co nsequence of a natural progression of the animals from persistent diestrus into acyclicity. All animals in all dose groups maintained moderate numbers of secondary, antral and atretic follicles throughout the study, including the 24

- month time point.

The ovarian histomorpohology seen in this study is what would be expected in a normally aging female F

- 344 rat undergoing a normal aging process towards a state of repetitive pseudopregnancy. Repetitive pseudopregnancy is associated with vaginal

smears t hat are primarily leukocytic and is, thus, sometimes referred to as "persistent

diestrous".

The ovarian histomorphology data indicates atrazine treatment did not alter the number of animals in repetitive pseudopregnancy/persistent diestrous. Estrous cycl ing in atrazine treated F

- 344 females was not altered.

Mammary Gland Histomorphology : Animals in all dose groups displayed mammary gland histomorphologic alterations that would be expected in a normally aging F

- 344
females rats i.e., there was some evid ence of lobular/acinar development with secretory activity and occasional galactoceles in all dose groups at the 15, 18 and 24 month timepoints.

The mammary gland histomorphologic alterations that are seen are those that would be expected in a normally a ging F

- 344 female rat. Atrazine exposure did not increase the severity of any mammary gland histomorphology finding or

decrease their time of onset.

F. 4.9.4 NHEERL Publications

Substantial research has been conducted on the toxicologic effects of atrazine exposure at the Reproductive Toxicology Division of EPA's National Heath and Environmental Effects Research Laboratories (NHEERL) at Research Triangle Park, N.C. . These studies have been evaluated in the May 22, 2 000 document that was reviewed by the June, 2000 SAP [see http://www.epa.gov/scipoly/sap/2000/june27/finalparta_atz.pdf].Do not cite

Appendix F

- Page 628 of 677 This research includes studies investigating the neuroendocrine basis of the mode of

action for atrazine

- associated carcinogenesis, as well as studies investigating developmental and reproductive effects associated with atrazine exposure. Much of these data have been published in the open literature. The abstracts from these open literature publications are shown below in chronologica l order of publication.

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. 1996 Jul

- Aug; 10(4):257

- 64. MRID not yet assigned.

The effect of the chlorotriazine h erbicide, atrazine, on ovarian function was studied in Long

- Evans hooded (LE

- hooded) and Sprague

- Dawley (SD) rats. Atrazine was administered by gavage for 21 d to females displaying regular 4

- d estrous cycles. In both strains, 75 mg/kg/d disrupted the 4

- d ovarian cycle; however, no distinct alteration ( i.e. , irregular cycles but not persistent estrus or diestrus) was apparent at this dose. At 150 mg/kg/d, atrazine induced repetitive pseudopregnancies in females of both strains. The highest dose tes ted (300 mg/kg/d) also induced repetitive pseudopregnancies in the SD females, while the ovaries of the LE

- hooded female appeared regressed and the smear cytology was indicative of the anestrous condition. Although a NOAEL was not established, the doses em ployed in this experiment were in excess of those used in chronic feeding studies in which an early onset of mammary gland tumors was noted. These data demonstrate that atrazine can disrupt ovarian function and bring about major changes in the endocrine pr ofile of the female.

Stoker, T.E., Robinette, C.L., Cooper, R.L. (1999) Maternal exposure to atrazine during lactation suppresses suckling

- induced prolactin release and results in prostatitis in the adult offspring. Toxicol Sci 1999 Nov; 52(1):68

- 79 MR ID 45166902

The availability of prolactin to the neonatal brain is known to affect the development of the tuberoinfundibular (TIDA) neurons and, as a consequence, lead to alterations in subsequent prolactin regulation. Without early lactati onal exposure to prolactin (derived from the dam's milk), TIDA neuronal growth is impaired and elevated prolactin levels are present in the prepubertal male. These observations, combined with the finding that alterations in prolactin secretion ( i.e ., hype rprolactinemia) in the adult male rat have been implicated in the development of prostatitis, led us to hypothesize that early lactational exposure to agents that suppress suckling

- induced prolactin release would lead to a disruption in TIDA development , altered prolactin regulation, and subsequent prostatitis in the male offspring. To test this hypothesis, suckling

- induced prolactin release was measured in Wistar dams treated twice daily with the herbicide atrazine (atrazine, by gavage, on postnatal da y (PND) 1

- 4 at 0, 6.25, 12.5, 25, and 50 mg/kg body weight), or twice daily with the dopamine receptor agonist bromocriptine (BROM, sc, at 0.052, 0.104, 0.208, and 0.417 mg/kg); BROM is known to suppress prolactin release. Similarly, atrazine has also be en reported to suppress prolactin in adult females. Serum prolactin was measured on PND 3 using a serial sampling technique and indwelling cardiac catheters. A significant rise in serum prolactin release was noted in all control females within 10 min of t he initiation ofDo not cite

Appendix F

- Page 629 of 677 suckling. Fifty

- mg/kg atrazine inhibited suckling

- induced prolactin release in all females, whereas 25 and 12.5 mg/kg atrazine inhibited this measure in some dams and had no discernible effect in others. The 6.25 mg/kg dose of atrazine was without effect. BROM, used here as a positive control, also inhibited suckling

- induced prolactin release at doses of 0.104 to 0.417 mg/kg, with no effect at 0.052 mg/kg. To examine the effect of postnatal atrazine and BROM on the incidence and severity of inflammation of the lateral prostate of the offspring, adult males were examined at 90 and 120 days. While no effect was noted at 90 days of age, at 120 days, both the incidence and severity of prostate inflammation was increased in those offspring of atr azine

- treated dams (25 and 50 mg/kg). The 12.5 mg/kg atrazine and the two highest doses of BROM increased the incidence, but not the severity, of prostatitis. Combined treatment of ovine prolactin and 25 or 50 mg/kg atrazine on PND 1

- 4 reduced the incidenc e of inflammation observed at 120 days, indicating that this increase in inflammation, seen after atrazine alone, resulted from the suppression of prolactin in the dam.

To determine whether there is a critical period for these effects, dams were dosed wit h 25 and 50 mg/kg on PND 6

- 9 and PND 11

- 14. Inflammation was increased in those offspring from dams treated on PND 6

- 9, but this increase was not significant. Dosing

on PND 11

- 14 was without effect. These data demonstrate that atrazine suppresses suckling

- induced prolactin release and that this suppression results in lateral prostate inflammation in the offspring. The critical period for this effect is PND 1

- 9.

Shafer, T.J., Ward, T.R., Meacham, C.A., Cooper, R.L. (1999) Effects

of the chlorotriazine herb icide, cyanazine, on GABA(A) receptors in cortical tissue from rat brain. Toxicology 1999 Dec 20;142(1):57

- 68. MRID not yet assigned.

Chlorotriazine herbicides disrupt luteinizing hormone (LH) release in female rats following in vivo exposure . Although the mechanism of action is unknown, significant evidence suggests that inhibition of LH release by chlorotriazines may be mediated by effects in the central nervous system. GABA(A) receptors are important for neuronal regulation of gonadotropin releasing hormone and LH release. The ability of chlorotriazine herbicides to interact with GABA(A) receptors was examined by measuring their effects on [3H]muscimol, [3H]Ro15

- 4513 and [35S]tert

- butylbicyclophosphorothionate (TBPS) binding to rat cortical membranes. Cyanazine (1

- 400 microM) inhibited [3H]Ro15

- 4513 binding with an IC50 of approximately 105 microM (n=4). Atrazine (1

- 400 microM) also inhibited [3H]Ro15

- 4513 binding, but was less potent than cyanazine (IC50 = 305 microM). However, the

chlorotr iazine metabolites diaminochlorotriazine,

2

- amino

- 4

- chloro

- 6

- ethylamino

- s

- triazine and

2

- amino

- 4

- chloro

- 6

- isopropylamino

- s

- triazine were without significant effect on [3H]Ro15

- 4513 binding. Cyanazine and the other chlorotriazines were without effect on [3H]muscimol or [35S]TBPS binding. To examine whether cyanazine altered GABA(A) receptor function, GABA

- stimulated 36Cl

- flux into synaptoneurosomes was examined. Cyanazine (50

- 100 microM) alone did not significantly decrease GABA

- stimulated 36Cl

flux. D iazepam (10 microM) and pentobarbital (100 microM) potentiated

GABA

- stimulated 36Cl

- flux to 126 and 166% of control, respectively. At concentrationsDo not cite

Appendix F

- Page 630 of 677 of 50 and 100 microM, cyanazine decreased potentiation by diazepam to 112 and 97% of control, respectively , and decreased potentiation by pentobarbital to 158 and 137% of control (n = 6). Interestingly, at lower concentrations (5 microM), cyanazine shifted the

EC50 for GABA

- stimulated 36Cl

- flux into synaptoneurosomes from 28.9 to 19.4 microM, respectively (n = 5). These results suggest that cyanazine modulates benzodiazepine, but not the muscimol (GABA receptor site) or TBPS (Cl

- channel), binding sites on GABA(A) receptors. Furthermore, at low concentrations, cyanazine may slightly enhance function of GABA(A) receptors, but at higher concentrations, cyanazine antagonizes GABA(A) receptor function and in particular antagonizes the positive modulatory effects of diazepam and pentobarbital.

Cooper, R.L., Stoker, T.E., Tyrey, L., Goldman, J.M., McElroy, W.K. (20 00) Atrazine disrupts the hypothalamic control of pituitary

- ovarian function. Toxicol Sci 2000 Feb; 53(2):297

- 307. MRID 45166901

The chloro

- S

- triazine herbicides ( i.e ., atrazine, simazine, cyanazine) constitute the largest group of herbicides sold in the United States. Despite their extensive usage, relatively little is known about the possible human

- health effects and mechanism(s) of action of these compounds. Previous studies in our laboratory have shown that the chlorotriazines disrupt the hormonal control of ovarian cycles. Results from these studies led us to hypothesize that these herbicides disrupt endocrine function primarily through their action on the central nervous system. To evaluate this hypothesis, we examined the estrogen

- induced surges of luteinizing hormone (LH) and prolactin in ovariectomized Sprague

- Dawley (SD) and Long

- Evans hooded (LE) rats treated with atrazine (50

- 300 mg/kg/day, by gavage) for 1, 3, or 21 days. One dose of atrazine (300 mg/kg) suppressed the LH and prolactin sur ge in ovariectomized LE, but not SD female rats. Atrazine (300 mg/kg) administered to intact LE females on the day of vaginal proestrus was without effect on ovulation but did induce a pseudopregnancy in 7 of 9 females. Three daily doses of atrazine suppre ssed the estrogen

- induced LH and prolactin surges in ovariectomized LE females in a dose

- dependent manner, but this same treatment was without effect on serum LH and prolactin in SD females. The estrogen

- induced surges of both pituitary hormones were supp ressed by atrazine (75

- 300 mg/kg/day) in a dose

- dependent manner in females of both strains evaluated after 21 days of treatment. Three experiments were then performed to determine whether the brain, pituitary, or both organs were the target sites for the chlorotriazines. These included examination of the ability of (1) the pituitary lactotrophs to secrete prolactin, using hypophysectomized females bearing pituitary autotransplants (ectopic pituitaries); (2) the synthetic gonadotropin

- releasing hormone (GnR H) to induce LH secretion in females treated with high concentrations of atrazine for 3 days; and (3) atrazine (administered in vivo or in vitro) to suppress LH and prolactin secretion from pituitaries, using a flow

- through perfusion procedure. In conclusi on, the results of these studies demonstrate that atrazine alters LH and prolactin serum levels in the LE and SD female rats by altering the hypothalamic control of these hormones. In this regard, the LE female appeared to be more sensitive to the hormone suppressive effects of atrazine, as indicated by the decreases observed on treatment

- day 3. TheseDo not cite

Appendix F

- Page 631 of 677 experiments support the hypothesis that the effect of atrazine on LH and prolactin secretion is mediated via a hypothalamic site of action.

Das, P.C., McElro y, W.K., Cooper, R.L. (2000) Differential modulation of catecholamines by chlorotriazine herbicides in pheochromocytoma (PC12) cells in vitro. Tox. Sci. Aug; 56(2): 324

- 31. MRID not yet assigned.

Epidemiological, wildlife, and laboratory studies have po inted to the possible adverse health effects of chlorotriazine herbicide ( i.e. , atrazine, simazine, and cyanazine) exposure. However, the cellular mechanism(s) of action of these compounds remains unknown. Recently, it was reported by Cooper et al. (2000, Toxicol. Sci. 53, 297

- 307) that atrazine disrupts ovarian function by altering hypothalamic catecholamine concentrations and subsequently the regulation of luteinizing hormone (LH) and prolactin secretion by the pituitary. In this study, we

examined the e ffect of three chlorotriazines on catecholamine metabolism in vitro using PC12 cells. Intracellular norepinephrine (NE) and dopamine (DA) concentrations and spontaneous NE release were measured following treatment with different

concentrations of atrazine , simazine (0, 12. 5, 25, 50, 100, and 200 microM) and cyanazine (0, 25, 50, 100, and 400 microM) for 6, 12, 18, 24, and 48 h. Atrazine and simazine significantly decreased intracellular DA concentration in a

concentration

- dependent manner. Intracellular N E concentration was also significantly decreased by 100 and 200 microM atrazine and 200 microM simazine. Similarly, there

was a dose

- dependent inhibition of NE release with 100 and 200 microM concentrations of both compounds. Although 100 and 400 microM cy anazine increased intracellular NE concentration, 50, 100, and 400 microM cyanazine significantly increased NE release at 24 and 36 h. In contrast, intracellular DA concentration was decreased by cyanazine, but only at 400 microM. The GABA(A)

- receptor ag onist, muscimol (0, 0.01, 0.1, and 1.0 microM) had no effect on either the release or on intracellular catecholamine concentrations from 6 through 24 h of treatment. Cell viability was somewhat lower in the groups exposed to 100 and 200 microM atrazine an d simazine. However, the reduction in viability was significant only in the highest dose of atrazine used (200 microM) at 24 h. Cyanazine did not have an effect on the viability at any of the doses tested, and the cells were functional, even up to 48 h of exposure. These data indicate that both atrazine and simazine inhibit the cellular synthesis of DA mediated by the tyrosine hydroxylase (TH), and NE mediated by dopamine beta

- hydroxylase (DbetaH), and, as a result, there is a partial or significant inhibit ion of NE release. Cyanazine, on the other hand, stimulated the synthesis of intracellular NE, and not DA. Thus, chlorotriazine compounds presumably act at the enzymatic steps or sites of DA biosynthesis to modulate monoaminergic activity in PC12 cells.

C ummings, A. M., Rhodes, B.E., and Cooper, R.L. (2000). Effect of atrazine on implantation and early pregnancy in four strains of rats.
Tox. Sci. Nov. 58: 135

- 143. MRID not yet assigned.

Atrazine is an herbicide that has been shown to have adverse repr oductive effects including alterations in levels of pituitary hormones such as prolactin and luteinizing hormone (LH) inDo not cite

Appendix F

- Page 632 of 677 female LE rats when administered at doses of 200 mg/kg/day for 1 and 3 days. Since prolactin’s action to promote progesterone secretio n is essential for the initiation of pregnancy in rats, this study was designed to examine the effect of exposure to atrazine during early pregnancy on implantation and short

- term pregnancy maintenance. Rats were divided into two groups representing perio ds of dosing with atrazine prior to the diurnal or nocturnal surges of prolactin. Within each group, four groups consisting of four strains of rats (Holtzman, HLZ; Sprague Dawley, SD; Long Evans, LE; Fisher 344, F344) were each further subdivided into fou r atrazine dosages. Rats were dosed by gavage with 0, 50, 100, or 200 mg/kg/day atrazine on days 1

- 8 of pregnancy (day 0 = sperm +). All animals were necropsied on day 8 or 9 of pregnancy. The 200 mg/kg dose of atrazine reduced body weight gain in all b ut one group. Two groups of animals dosed at 100 and 200 mg/kg/day in the nocturnal dosing period showed an increase in percent preimplantation loss, and both of these were F344 rats. Holtzman rats were the only strain to show a significant level of post implantation loss and a decrease in serum progesterone at 200 mg/kg/day both following diurnal and nocturnal dosing. Doses of 100 mg/kg/day also produced postimplantation loss following diurnal and nocturnal dosing, but progesterone levels were only decre ased after nocturnal dosing. Alterations in serum LH were seen in several groups. Serum estradiol was significantly increased only in Sprague Dawley rats dosed at the diurnal interval with 200 mg/kg atrazine. We conclude that F344 rats are most suscepti ble to preimplantation effects of atrazine and that HLZ rats appear most sensitive to the postimplantation effects of the chemical. LE and SD rats were least sensitive to effects of atrazine during very early pregnancy.

Narotsky M. G., Best, D.S., Gui dici, D. L., and Cooper, R.L. (2000). Strain comparisons of atrazine

- induced pregnancy loss in the rat. Repro Toxicol. In Press. MRID not yet assigned.

Atrazine was administered by gavage, in 1% methylcellulose, to F344 Sprague

- Dawley (SD), and Long Evans (LE) rats at 0, 25, 50, 100, or 200 mg/kg/d on gestation days 6 through 10. The dams were allowed to deliver and litters were examined postnatally. The F344 strain was the most sensitive to atrazine‘s effects on pregnancy, showing full

- litter resorp tion at $50 mg/kg. In surviving F344 litters, prenatal loss was increased at 200 mg/kg. In SD and LE rats, full

- litter resorption occurred only at 200 mg/kg. Delayed parturition was seen at $100 mg/kg in F344 and SD rats. Regarding maternal toxicity, the SD dams were the most sensitive, with weight loss at $ 25 mg/kg. When 200 mg/kg was administered to F344 rats on days 11 through 15 (after the LH

- dependent period of pregnancy), no full

- litter resorption was seen. These findings suggest that

atrazine

- induced full

- litter resorption is maternally mediated, and consistent with loss of LH support of the corpora lutea.

Laws, S.C., Ferrell, J.M., Stoker, T.E., Schmid, J., and Cooper, R.L. (2000). The effect of atrazine on puberty in female Wistar rats: an evaluation in the protocol for the assessment of pubertal development and thyroid function. Tox. Sci . 58 (2): 366

- 76. MRID not yet assigned.Do not cite

Appendix F

- Page 633 of 6 77 The effects of atrazine, a chlorotriazine herbicide, on the onset of puberty were

evaluated in Wistar rats. Fema le rats were dosed by oral gavage from postnatal day (PND) 22 through PND 41 with 0, 12.5, 25, 50, 100 or 200 mg atrazine /kg. Vaginal opening was significantly delayed 3.4, 4.5 or greater than 6.8 days by 50, 100 and 200 mg/kg, respectively. Vaginal ope ning did not occur in 4 of 15 females in the 200 mg/kg group by the time of necropsy (PND 41). Body weight at necropsy was reduced in the 200 mg/kg group by 11.6%, but was not different from the control (0) in the 50 and 100 mg/kg groups. To examine th e influence of reduced body weight on pubertal development, a group of pair

- fed controls was included whose daily food intake was dependent upon the amount consumed by their counterpart in the 200 mg/kg group. Although necropsy body weight was reduced to the same extent as the atrazine females, vaginal opening in the pair

- fed controls was not significantly delayed. Adrenal, kidney, pituitary, ovary and uterine weights were reduced by 200 mg/kg atrazine. Serum T3 [tri

- iodothyronine], T4 [thyroxine], and TSH [thyroid stimulating hormone] were unaltered by atrazine which were consistent with no histopathologic/morphologic changes in the thyroid. Estrous cyclicity was monitored in a second group of females from vaginal opening

- PND 149. The number of fem ales displaying regular 4 or 5

- day estrous cycles during the first 15

- day interval after vaginal opening was lower in the 100 and 200 mg/kg atrazine and pair

- fed controls. Irregular cycles were characterized by extended periods of diestrus. By the end of the second 15

- day interval (PND 57

- 71), no effects on estrous cyclicity were observed. These data show that atrazine can delay the onset of puberty and alter estrous cyclicity in the female Wistar rat ( NOAEL of 25 mg/kg). Reduced food consumption and body weight did not account for the delay in vaginal opening because this effect was not observed in the pair

- fed controls. In addition, the effect on estrous cyclicity was observed in the 100 mg/kg atrazine group where no significant reduction in body w eight was observed.

Stoker, T.E., Laws, S.C., Guidici, D. and Cooper, R.L. (2000) The effect of atrazine on puberty in male Wistar rats: An evaluation in the protocol for the assessment of pubertal development and thyroid

function. Toxicol. Sci. Nov. 58: 50

- 59.

Since atrazine, a chlorotriazine herbicide, has been shown previously to alter the secretion of luteinizing hormone (LH) and prolactin through a direct effect on the CNS, we hypothesized that exposure to atrazine in the EDSTAC male pubertal protocol (juvenile to peripubertal) would alter the development of the male rat reproductive system. We dosed intact male Wistar rats from postnatal day (PND) 23 to 53 and examined several reproductive endpoints. Atrazine (0, 12.5, 25, 50, 100, 150 or 200 mg/kg) was administered by gavage and an additional pair

- fed group was added to compare the effects of any decreased food consumption in the high dose group. Preputial separation was significantly delayed in the 12.5, 50, 100, 150 and 200 mg/kg

atrazine dose groups. Preputial separation was also delayed in the pair

- fed group, although significantly less than in the high dose atrazine group. The males were killed on PND 53 or 54 and pituitary, thyroid, testes, epididymides, seminal vesicles, ventral

and la teral prostates were removed. Atrazine (50 to 200 mg/kg) treatment resulted in a significant reduction in ventral prostate weights, as did the pair

- fed group. Testes weights were unaffected by atrazine treatment. Seminal vesicle and epididymal weightsDo not cite

Appendix F

- Page 634 of 677 were decreased in the high dose atrazine group and the control pair

- fed group. However, the difference in epididymal weights was no longer significantly different when body weight was entered as a covariable. Intratesticular testosterone was significantly

de creased in the high dose atrazine group on PND 45, but apparent decreases in serum testosterone were not statistically significantly on PND 53, There was a trend for a decrease in luteinizing hormone as the dose of atrazine increased, however, dose

group m ean LH were not different from controls. Due to the variability of serum prolactin concentrations on PND 53, no significant difference was identified. Although prolactin is involved in the maintenance of LH receptors prior to puberty, we observed no diffe rence in LH receptor number at PND 45 or 53. Serum estrone and estradiol showed dose

related increases that were significant only in the 200 mg/kg atrazine group. No differences were observed in thyroid stimulating hormone (TSH) and thyroxine (T4)

between the atrazine groups and the control, however tri

- iodothyronine (T3) was elevated in the high dose atrazine group. No differences in hormone levels were observed in the pair

- fed animals. These results indicate that atrazine delays puberty in

the male rat and its mode of action appears to be altering the secretion of steroids and subsequent effects on the development of the reproductive tract, which appear to be due to atrazine‘s effects on the CNS. Thus, atrazine tested positive in the pubertal male

scree n that EDSTAC is considering as an optional screen for endocrine disrupters.

F. 4.10 Toxicology data for major metabolites of atrazine

Metabolism of atrazine can occur through hydrolytic de halogenation at the 2 position of the s

- triazine ring resulting in the formation of hydroxyatrazine. Dealkylation reactions at the 4 and 6 positions

- either in the absence or presence of dehalogenation

- may also occur. Both plants and animals are capab le of performing the dealkylation reactions. Hydroxyatrazine is the major metabolite in plants, but is only a minor metabolite in animals where the varying forms of the delkylated chlorometabolites dominate instead. The metabolism of atrazine to hydroxyatr azine in plants is a detoxification reaction as the phytotoxicity of hydroxyatrazine is greatly reduced compared to the parent compound. Bacteria are believed to be capable of the dealkylation reactions that form the chlorometabolites of atrazine. Hydrox yatrazine and the chlorometabolites can be found in both drinking water and the diet.

The HED Metabolism Committee (October 17, 2000) has determined that for chronic dietary risk, the residues of concern are parent and chlorometabolites compared
to appro priate atrazine endpoints. A separate chronic dietary risk assessment will be conducted for hydroxyatrazine metabolites compared to the appropriate hydroxyatrazine endpoint. For acute dietary risk assessment the residues of concern are the parent

chloromet abolites compared to appropriate acute atrazine endpoints. A separate acute assessment will not be conducted for hydroxyatrazine as HIARC did not select an acute RfD for hydroxyatrazine. The three chlorometabolites of concern are diaminochlorotriazine, des isopropyl atrazine , and desethyl atrazine. A limited toxicology database is available for these three metabolites and these studies are

summarized below in sections F 4.10.1, F 4.10.2, and F 4.10.3. A limited toxicology database is also available for hydro xyatrazine and is shown below under section

F 4.10.4.Do not cite

Appendix F

- Page 635 of 677 F. 4.10.1 Diaminochlorotriazine metabolite (DACT)

- 2

- chloro

- 4

- amino

- 6

- amino s

- triazine; didealkyl atrazine; G

- 28273

The DACT metabolite is the terminal mammalian metabolite of both atrazine and the closely related herbicide simazine. DACT is primarily a mammalian metabolite but can also be produced in plants and bacteria. DACT is produced through dealkylation of the isopropyl group at position 2 of the triazine ring in atrazine (dealkylation of the ethyl group at the 2 position of the triazine ring in simazine) and dealkylation of the ethyl group at the 4 position of the triazine ring in either atrazine or simazine. DACT is the primary metaboli te produced in rats following atrazine treatment. It is the major fecal metabolite in rats accounting for 40% of the radioactivity found in feces following exposure by oral gavage. DACT is also the major urinary metabolite in rats accounting for 26% of th e total administered dose following exposure by oral

gavage.

870.3100

- Subchronic oral toxicity in rats

Groups of 15 male and 15 female CD Sprague

- Dawley rats were fed diets containing diaminochlorotriazine (G

- 28273) (purity 98.2%) at concentrations of 0, 10, 100, 250, or 500 ppm for 13 weeks (MRID 43013207). The average consumption of test material was 0.7, 6.7, 16.7, or 34.1 mg/kg/day (males) and 0.7, 7.6, 19.7, or 40.2 mg/kg/day (females). All animals survived to study termination. No treatment

- rela ted clinical signs of toxicity including ocular lesions were seen at any dose level. At 500 ppm, mean body weights of male rats were lower than controls during most of the study period, decreasing to 87% of controls at week 12. Body weight gain at week 12 was 82% of controls for males receiving 500 ppm, and 85% and 83% of controls for females receiving 250 and 500 ppm, respectively. No treatment

- related effects on body weight or body weight gain were seen at the lower doses in either sex. Food consumptio n was not affected by administration of the test material. There were no biologically significant effects on hematology, clinical chemistry, urinalysis, and gross or histopathology at any dose level. Although several organ weight changes were observed, th ere were no histologic or functional correlates. Estrous cycle data indicated a treatment

- related

effect at doses of 100 ppm. The effects, generally more pronounced on days 70

- 85 than on days 14

- 28 and 42

- 56, included lengthening of the estrus cycle and/ or an increased incidence of rats exhibiting cycles with persistent estrus and/or diestrus. There were no apparent effects on serum levels of estradiol, progesterone, prolactin,

and corticosterone.

The LOAEL is 7.6 mg/kg/day, based on estrous cycle effe cts in female rats. The NOAEL is 0.7 mg/kg/day.

This study is classified as Acceptable

- Guideline and satisfies the guideline requirements for a subchronic dietary toxicity study (OPPTS 870.3100) in rats.

870.3150 and 870.4100

- Subchronic and chronic oral toxicity in dogsDo not cite

Appendix F

- Page 636 of 677 Diaminochlorotriazine was fed to male and female dogs at dietary levels of 0, 5, 100, or 1500 ppm for 13 or 52 weeks (MRID 41392401). Because of severe toxicity at the highest dose, which was evident after 6 weeks of treatment, th e high

- dose dogs were fed a diet containing 750 ppm. Females tolerated this dose level and received 750 ppm until termination at 13 or 52 weeks, or through 13 weeks followed by a 39

- week recovery period. Since males continued to exhibit signs of toxicity at 750 ppm, they were fed untreated diet for 9 weeks through 13. Four male dogs were then placed again on a diet containing 750 ppm until termination at 52 weeks.

The mean daily doses for male dogs receiving dietary levels of 5, 100, and 1500/750 ppm for 52 weeks were 0.187, 3.61, and 24.1 mg/kg/day, and the doses were 0.195, 3.43, and 32.7 mg/kg/day for females receiving the same dietary levels. Among the high

- dose dogs, five males and two females were sacrificed moribund during the treatment period. Mor ibundity was attributed to impairment of heart function, the primary

treatment

- related effect of Diaminochlorotriazine, which was accompanied by several clinical and pathological changes. Pathological cardiac findings included enlargement, softness, thick ened valves, lesions, distension, red/dark color, thrombosis, chronic myocarditis, necrosis, inflammation, hemorrhage, and hemosiderosis. Secondary

treatment

- related changes in the high

- dose animals were seen in the liver (enlargement, congestion, centril obular fibrosis/atrophy, bile stasis, necrosis, hemosiderosis, red/dark color, lesions, adhesions, mottling, and rough texture); testes (hypospermatogenesis and hypospermia); thymus (atrophy); bone marrow (hyperplasia); and pericardium, thoracic, and abdom inal cavities (fluid accumulation). Recovery females did not exhibit any clinical ophthalmological signs of cardiac impairment. Other effects at the high dose included decreased body weight gains in males and females dosed for 6 weeks; increased mean spl een, liver, and kidney weights; anemia with accompanying reticulocytosis (a reversible effect); decreases in albumin, calcium and total cholesterol levels; nonsignificant increase in lactic acid dehydrogenase activity ; and elevations of platelet levels. High

- dose males continued to lose weight when the dose was lowered to 750 ppm. Severe anemia with reticulocytosis was noted in only one of the two recovery females. The effects of diaminochlorotriazine on cholesterol levels and erythroid parameters were r eversible as were noted in almost all animals of both sexes and beginning at week 6 and extending until week 14. No adverse effects were observed at dietary levels of 5 or 100 ppm. Although there was appreciable mortality at the highest dose of 1500 ppm, a sufficient number of animal were at risk to evaluate histopathology. Administration of dietary levels of 750 ppm to dogs is associated with symptomatology of cardiac impairment.

The LOAEL is 24.1 mg/kg/day, based on cardiac effects. The NOAEL is 3.4 3 mg/kg/day.

This study is classified as Acceptable

- Guideline and satisfies the guideline requirements for a subchronic dietary toxicity study (OPPTS 870.3150) and also a chronic dietary toxicity study (OPPTS 870.4100) in dogs.

870.3700

- Developmental toxicity in ratsDo not cite

Appendix F

- Page 637 of 677 In a developmental toxicity study (MRID 41392402), diaminochloro

- triazine [DACT] (98.2% a.i., batch # FL 871423) was administered to 26 female Sprague

Dawley rats/dose via gavage at dose levels of 0, 2.5, 25, 75, and 150 mg/kg bw/day

from days 6 through 16 of gestation.

There were no treatment

- related effects on mortality or clinical signs. Maternal body weight

- gain was reduced significantly [33.2%] during the 20

- day gestation interval at the high

- dose level , and there was a decrease i n body

- weight gain during the dosing interval [days 6

- 16] at the two

- highest dose levels. During the initial dosing interval [days 6

- 8], dams at the 25 mg/kg/day dose level displayed a 32% decrease in body

weight gain, and the dams at the 75 and 150 mg/kg/ day dose levels displayed negative body

- weight gains. Decreased food consumption was observed at the highest dose level [days 6

- 16; 66% of control value] during the dosing period. Only a slight decrease in food consumption was observed at the 25 and 75 mg/ kg/day dose levels during the dosing period.

The maternal LOAEL is 25 mg/kg bw/day, based on decreased body

- weight gain during the initial three days of the dosing period.. The maternal NOAEL is 2.5mg/kg bw/day.

Pregnancy rate was not adversely affecte d by treatment, and there were no abortions. Comparable numbers of corpora lutea/dam and implantations/dam were observed among the groups, and there was no dose

- related effect on pre

- implantation loss. At the high

- dose level [150 mg/kg/day], there was an i ncrease in resorptions [both early and late], and post

- implantation loss was significantly increased [18.9% vs 5.6% in the control] at this dose level. There were fewer live fetuses at 150 mg/kg/day [86% of control]. There were no dead fetuses, and the sex ratio was comparable among the groups. Gravid uterine weight was decreased significantly compared to the control [77% of control] at the 150 mg/kg/day dose level, and fetal body weights were lower at 75 mg/kg/day [91%

- 92% of control] and 150 mg/kg/day [81 % of controls] in both sexes. There was an increased incidence of absent renal papilla [22% vs 3.3%] and pitted kidneys [4.8% vs 0%] at the high

- dose level compared to the control. There was a dose

related increase in incomplete ossification of several bon es at the 75 and 150 mg/kg/day dose levels and in three bones at 25 mg/kg/day [ interparietal, parietals, and hyoids (unossified)].

The developmental LOAEL is 25 mg/kg bw/day, based on increases in the incidences of incompletely ossified parietals, inter parietals, and unossified hyoids. The developmental NOAEL is 2.5 mg/kg bw/day.

The developmental toxicity study in the rat is classified Acceptable/Guideline , and it satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700) in the rat.

870.5100 Bacterial Reverse Mutation

In replicate (separate) trials, the atrazine metabolite diaminochlorotriazine (DACT, 97.0%) failed to induce reverse mutation (as evidenced by no increase overDo not cite

Appendix F

- Page 638 of 677 controls in revertant colony c ounts) in Ames testing at concentrations up to 5000 μg/0.1 mL, at which dose precipitation of compound occurred. This study is classified Acceptable/Guideline (MRID 40722302).

870.5550 UDS Assay

The atrazine metabolite diaminochlorotriazine (DACT, 97 .0%) did not induce unscheduled DNA synthesis in cells of the human fibroblast cell CRL 1521 exposed to concentrations resulting in compound precipitation (400 to 600 μg/mL), but only in the absence of a metabolic activation system (assay without activati on was not performed). This study is classified Acceptable/ nonguideline (MRID 40722303).

F. 4.10.2 G

- 28279 metabolite

- 2

- chloro

- 4

- (ethylamino)

- 6

- (amino) s

- triazine; ethylatrazine; deisopropyl atrazine

Desisopropy l atrazine is a metabolite of both atrazine and simazine. Desisopropyl atrazine is primarily a mammalian metabolite but can also be produced in plants and bacteria. Dealkylation of the isopropyl group from the 2 position of the triazine ring in atrazine o r simazine yields desisopropyl atrazine/simazine.

870.3100

- Subchronic oral toxicity in rats

In a 90

- day oral toxicity study (MRID 43013205), groups of 10 male and 10 female RAIf(SPF) rats were fed diets containing G

- 28279 technical (purity 96.7%; Lot

#FL

- 901747) at concentrations of 0, 10, 50, or 500 ppm for 13 weeks. The average consumption of test material was 0.602, 3.20, or 34.9 mg/kg/day (males) and 0.641, 3.34, or 37.5 mg/kg (females).

All animals survived to study termination. No treatment

- related clinical signs or ocular lesions were seen at any dose level. At 10 ppm, there were no effects on body weight, clinical chemistry parameters, hematologic values, or organ weights, nor were macroscopic or histopathologic changes observed. Slightly decreased body weights and body

- weight gains were seen in males administered 50 or 500 ppm of the test material. At week 13, the body weights of males were 93% and 88% of control values at 50 and 500 ppm, respectively, and the body

- weight gains were 96, 9 0, or 84% of control values at 10, 50, or 500 ppm, respectively. For female rats exposed to 500 ppm, the body weights were 9

- 12% below those of controls from treatment week 2 to 13, and the mean body

- weight gain was 80% of controls at the end of the treat ment period. Food consumption was not significantly affected by treatment with G

- 28279 technical. Organ weight changes (absolute and relative to body weight) in males at 500 ppm included increased relative kidney weights (14% above control values), decr eased absolute heart weights (

- 10%), increased relative testes weights (+21%), and increased relative brain weights (+18%). In the absence of histopathologic lesions in the corresponding organs, these organ weight changes are of uncertain toxicological

si gnificance and may not be treatment

- related. Increased relative liver weights (+13%)Do not cite

Appendix F

- Page 639 of 677 and extramedullary hematopoiesis of the liver seen in female rats at 500 ppm indicate slight hepatotoxicity. Minimal to moderate fatty changes of adrenal cortex and sli ght hypertrophy of the thyroid follicular epithelium in males at 500 ppm suggest treatment

related effects. Additionally, extramedullary hematopoiesis of the spleen in females at 500 ppm and hypertrophy of pituitary cells in males at 500 ppm may suggest t reatment
related effects.

The LOAEL is 3.3 mg/kg/day, based on decreased body weights and body weight gains in both sexes. The NOAEL is 0.6 mg/kg/day.

This study is classified as Acceptable/Guideline and satisfies the guideline requirements for a subch ronic dietary toxicity study (OPPTS 870.3100) in rats.

870.3150

- Subchronic oral toxicity in dogs

In a subchronic oral toxicity study (MRID 43013203), male and female beagle dogs(four/sex/group) were fed diets containing 0,15,100, 500, or 1000 ppm G

- 28279 technical for at least 14 weeks (equivalent to compound intakes of 0, 0.6, 3.8, 18.9, and 33.4 mg/kg/day for male dogs, and 0.6, 3.8, 18.0, and 33.3 mg/kg/day for female dogs).

Male dogs in the 1000

- ppm and female dogs in the 500

- ppm and 1000

- pp m groups exhibited significant decreases in food consumption and decreased body weight gain relative to controls and to respective baseline values. Mean food efficiency was negative in male dogs of the 1000

- ppm and female dogs of the 500

- ppm and 1000

- ppm

g roups. Clinical chemistry, hematology, and urinalysis data showed alterations that were of sporadic or intermittent occurrence, were not dose

- related, and were not sustained for any period of time. Gross and histopathological examinations revealed no

tre atment

- related findings. A statistically significant decrease in mean absolute and mean relative (to brain) heart weight were observed for the 500

- ppm and 1000

- ppm male dogs but there were no histopathologic or functional (EKG) correlates indicating

myoca rdial toxicity. Although the study authors attributed the alterations in heart weight to be secondary to inanition, the significant decrease (17% in the 500

- ppm group, 0.01<p<0.05; 25% in the 1000

- ppm group, p 0.01) in mean relative (to brain) heart weigh t may be indicative of a treatment

- related effect. Similar findings were noted for

organ

- to

- brain weight values for testes and prostate glands in the 500

- ppm and 1000

- ppm dogs. Two dogs (litter mates) were found to be anemic but the condition was not

con sidered to be related to test article exposure. Although most of the observed effects may be secondary to inanition, the decreased organ

- to brain weight ratios for the heart, testes, and prostate gland, and the negative food efficiency observed in dogs ex posed to dietary G

- 28279 at concentrations of 500 or 1000 ppm are indicative of marginally

toxic effects.

The LOAEL is 18.9 mg/kg/day, based on decreased food consumption, body weight, and heart weight (relative to brain weight). The NOAEL is 3.8 mg/ kg/day.

.Do not cite

Appendix F

- Page 640 of 677 This study is classified as Acceptable / Guideline and satisfies the guideline requirements for a subchronic feeding study (OPPTS 870.3150) in dogs.

870.3700

- Developmental toxicity in rats

In a developmental toxicity study (MRID 43013208 ), 24 Tif: RAI rats per group

received G

- 28279 technical by gavage on gestational days (GDs) 6

- 15, inclusive, at dose levels of 0, 5, 25, or 100 mg/kg/day. Aqueous corn starch (3%) served as the

control substance and vehicle for the test mixture.

Matern al toxicity was observed at 25 and 100 mg/kg/day in a dose

- related manner, as evidenced by effects on body weight/weight gain and food consumption. Body weights were significantly decreased at 25 mg/kg/day on GDs 9 and 13 (96% of control) and at 100 mg/kg/ day on GD 7 through GD 18 (93% of control). Weight gains were decreased

on GDs 6

- 11 at 25 mg/kg/day (73% of control, nonsignificant) and at 100 mg/kg/day (30% of control, significant) and on GDs 11

- 16 at 100 mg/kg/day (89% of control, nonsignificant). Cor rected weight gain was significantly decreased at 100 mg/kg/day (55% of control). Food consumption was significantly decreased on GDs 6

- 11 at 25 mg/kg/day (91% of control) and at 100 mg/kg/day (70% of control).

The maternal toxicity LOAEL is 25 mg/kg/d ay, based on decreased body weight and food consumption. The NOAEL is 5 mg/kg/day.

Developmental toxicity was observed at 25 and 100 mg/kg/day, as evidenced by effects on skeletal development. Fetal and litter incidences of fused sternebrae 1 and 2 were significantly increased at both these dose levels, while fetal incidences of poor ossification in sternebrae 2 and absent ossification in several digits were also increased significantly, but only at 100 mg/kg/day.

The developmental toxicity is 25 mg/k g/day, based on increased fetal and litter incidences of fused sternebrae. The NOAEL is 5 mg/kg/day.

This study is classified as Acceptable

- Guideline and satisfies the guideline requirements for a developmental toxicity study (OPPTS 870.3700) in rats. 870.5100 Bacterial Reverse Mutation Assay

The atrazine metabolite G

- 28279 (97.4%) was negative for inducing genetic reversions at the histidine locus in the Ames battery of Salmonella typhimurium or tryptophan prototrophy in Escheria coli WP2uvrA, when t ested up to the limit dose of 5000 μg/plate. This study is classified Acceptable

- Guideline (MRID 43093101).

870.5385 Mammalian Bone Marrow Chromosome Aberration TestDo not cite

Appendix F

- Page 641 of 677 The atrazine metabolite G

- 28279 (97.4%) was reported negative for the

induction of mic ronuclei in polychromatic erythrocytes of Tif:MAGF mice treated orally at single doses up to clinically (but not cytologically) toxic levels (MTD = 480 mg/kg). This study is classified Acceptable

- Guideline (MRID 43093103).

870.5550 UDS Assay

The atraz ine metabolite G

- 28279 (97.4%) was negative for inducing unscheduled DNA synthesis in primary rat hepatocyte cultures up to cytotoxic concentrations (800 μg/mL). This study is classified Acceptable

- Guideline (MRID 43093105).

F. 4.10.3 G

- 30033 metabolite

- 2

- chloro

- 4

- amino

- 6

- (isopropylamino) s

triazine; deethyl atrazine; isopropyl atrazine.

Deethyl atrazine is a metabolite of atrazine, but not simazine . Deethyl atrazine is primarily a mammalian metabolite but can also be produced in plants and bacteria. Dealkylation of the ethyl group from the 4 position of the triazine ring yields deethyl atrazine.

870.3100

- Subchronic oral toxicity in rats

In a subchronic study (MRID 43013206) G

- 30033 Technical, a metabolite o f atrazine was given in the diet to four groups of 10 male and 10 female RAIf (SPF) rats at a concentration of 0, 10, 50, or 500 ppm for 13

- weeks. The doses were equivalent to an average dose of 0.68, 3.2, and 35.1 mg/kg body weight/day or 0.72, 3.3, and 38.1 mg/kg body weight/day for male and female rats, respectively. The only treatment

related effects reported include: a decrease in the body weight of high dose female rats and a decrease in the food efficiency of high

- dose male and female rats.

The LO AEL is 35.1 mg/kg/day, based on decreased body weight and food efficiency. The NOAEL is 3.2 mg/kg/day.

This study is classified as Acceptable

- Guideline and satisfies the guideline (OPPTS 870.3100) requirements for a subchronic oral toxicity study in rats.

870.3150

- Subchronic oral toxicity in dogs

In a subchronic oral toxicity study (MRID 43013203), Beagle dogs were divided into four groups, each containing four males and four females and given diets containing 0, 15, 100, or 1000 ppm G

- 30033 (equivalent to 0, 0.56, 3.71, or 28.85 mg/kg/day for males and 0, 0.51, 3.88, and 32.18 mg/kg/day for females) for 13 weeks. Dietary exposure did not induce mortality, clinical signs of toxicity, ocular toxicity, or

effects detectable in the urine. Treat ment significantly decreased the weight gain of male and female dogs in the 1000 ppm group; however, the effect was attributed to inanition as a result of food palatability rather than an effect induced by the test material.

The test material induced rena l tubular hyper plasia/basophilia in 3/4 male and 2/4Do not cite

Appendix F

- Page 642 of 677 female dogs in the 1000 ppm treatment group. Marginal threshold effects found in male and female dogs fed diets containing 1000 ppm test material include: decreased absolute and relative (to brain) he art weights, normocytic/normochromic anemia,

paroxys mal atrial fibrillation, and right atrial wall hemorrhagic inflammation with angiomatous hyperplasia. The marginal threshold effects may be related to G

- 30033 treatment; however, without the use of pair

- fed control dogs, they cannot be separated from effects induced by inanition.

The LOAEL for male and female dogs is 30 mg/kg/day, based on renal tubular hyperplasia

. The NOAEL is 3.8 mg/kg/day.

This study is classified as Acceptable

- Guideline and sati sfies the guideline requirements for a subchronic feeding study (OPPTS 870.3150) in beagle dogs.

870.3700

- Developmental toxicity in rats

In a developmental toxicity study (MRID 43013209), 24 Tif: RAI rats per group

received G

- 30033 technical by gavage on gestational days (GDs) 6

- 15, inclusive, at dose levels of 0, 5, 25, or 100 mg/kg/day. Aqueous corn starch (3%) served as the

control substance and vehicle for the test mixture.

Maternal toxicity was observed at 25 and 100 mg/kg/day in a dose

- related manner as evidenced by effects on body weight/weight gain and food consumption. Body weights were significantly decreased at 100 mg/kg/day (94% of control) on GD 7 through GD 20. Weight gains were significantly decreased on GDs 6

- 11 at 25 mg/kg/day (83% o f control) and at 100 mg/kg/day (41% of control) and on GDs 11

- 16 at 100 mg/kg/day (87% of control). Corrected weight gains were non

- significantly decreased at 25 mg/kg/day (73% of control) and at 100 mg/kg/day (80% of control). Food consumption was signif icantly decreased on GDs 6

- 11 at 25 mg/kg/day (91% of control) and at 100 mg/kg/day (70% of control).

The maternal toxicity LOAEL is 25 mg/kg/day, based on decreased body

- weight gain and food consumption. The NOAEL is 5 mg/kg/day.

Developmental toxicity was observed at 100 mg/kg/day as evidenced by effects on skeletal development. Fetal and litter incidences of fused sternebrae 1 and 2 were significantly increased at 100 mg/kg/day and the fetal incidence of poor ossification of the proximal phalanx of po sterior digit 5 (a skeletal variation) at 100 mg/kg/day was also increased significantly.

The LOAEL for developmental toxicity is 100 mg/kg/day, based on increased fetal and litter incidences of fused sternebrae and increased fetal incidence of poor

ossif ication of the proximal phalanx of posterior digit 5. The NOAEL is 25 mg/kg/day.

This study is classified as Acceptable

- Guideline , and it satisfies the guideline requirements for a developmental study (OPPTS 870.3700) in rats.Do not cite

Appendix F

- Page 643 of 677 870.5100 Bacterial Rever se Mutation Assay

The atrazine metabolite G

- 30033 (99.3%) was negative for inducing reverse gene mutation at the histidine locus of Salmonella typimurium and for the tryptophan

locus in Escheria coli when tested to the limit dose, 5 000 μg/plate. This study is

classified Acceptable

- Guideline (MRID 43093102).

870.5385 Mammalian Bone Marrow Chromosome Aberration Test

The atrazine metabolite G

- 30033 (99.3%) was reportedly negative for induction

of micronuclei in the PCE of Tif:MAGF mi ce treated once orally up to clinically (but not cytologically) toxic doses (MTD = 480 mg/kg). This study is classified Acceptable

Guideline (MRID 43093104).

870.5550 UDS Assay

The atrazine metabolite G

- 30033 (99.3%) was negative for inducing

unsched uled DNA synthesis in primary rat hepatocyte cultures exposed up to cytotoxic concentrations (1000 μg/mL). This study is classified Acceptable

- Guideline (MRID 43093106).

F. 4.10.4 Hydroxyatrazine

- 2

- hydroxy

- 4(ethylamino)

- 6(isopropylamino)

- s

- triazine

Hydroxyatrazine is a metabolite of both atrazine and simazine. Hydroxyatrazine is primarily a plant metabolite but can also be produced in mammals and bacteria. Hydroxyatrazine is produced by dehalogenation of the chlorine from the triazine ring of either atrazine or simazine. Dealkylation rea ctions preceding or following the dehalogenation allow for the production of diamino, desisopropyl, and deethyl hydroxyatrazine or hydroxysimazine.

870.3100

- Subchronic oral toxicity in rats

In a subchronic toxicity study (MRID 41293501), hydroxyatrazi ne (97.1% ) was administered to 150 Sprague

- Dawley rats, 15/sex/dose in the diet at dose levels of 0, 10, 100, 300 and 600 ppm (0, 0.64, 6.3, 18.89 and 37.47 mg/k/g/day for males and 0, 0.75, 7.35, 22.75 and 45.64 mg/kg/day for females).

With the excep tion of kidney alterations, most of the adverse effects of hydroxyatrazine exposure were limited to the high dose group. Body weights were non

- significantly reduced in both sexes at the high dose only (7% reduction compared to controls in

males and 5% redu ction in females). Percent body weight gain was significantly decreased in both sexes at the high dose only (14.4% decrease compared to controls in males and 13.6% in females). Food consumption was significantly decreased in males only and only at a few se lected timepoints. Hematocrit and erythrocyte counts were significantly depressed in both sexes at the high dose only (

- 7.2% and 7.6% in malesDo not cite

Appendix F

- Page 644 of 677 and females respectively for hematocrit and

- 5.4% and 4.4% for males and females respectively for red blood cell counts). Hemoglobin was also significantly decreased in high dose males only (

- 6.4% compared to controls). Serum blood urea nitrogen was significantly increased in both sexes at the high dose only (+76% in males, +79.6% in females) as was creatinine (+3 8% in both sexes) sodium (+1.4% in males, +0.7% in females) and chloride (+2% in males, + 2.9% in females). Urine volume was significantly increased in both sexes at the high dose (+227% in males and +147% in females compared to controls). Urine volume wa s also significantly increased in males in the 300 ppm group (+38% compared to controls). Kidney weights

- absolute and relative to both body and brain weight

- were significantly increased in both sexes at the high dose only. Kidney weights relative to bo dy weight were increased 44% in males and 45% in females compared to controls.

Kidney alterations at both gross necropsy and at histopatholgy were seen in both the 300 and 600 ppm dose groups. Toxic nephrosis classified as "minimal" was seen in 7 of

15 m ales and 11 of 15 females at 300 ppm. Toxic nephrosis classified as severe was see in all animals of both sexes at the high dose. Tubule crystals were seen in 10 of 15 males and 11 of 15 females at the high dose. There were no compound

- related effects on mortality, clinical signs, or ophthalmology in either sex at any dose.

The LOAEL is 21 mg/kg/day, based on kidney alterations . The NOAEL is 6.8 mg/kg/day.

This subchronic toxicity study is classified Acceptable

- Guideline , and it satisfies the guide line requirement for a subchronic oral toxicity study (OPPTS 870.3100) in the rat.

870.3700

- Developmental toxicity in rats

In a developmental toxicity study (MRID 41065202), hydroxyatrazine, 97.6% a.i., was administered to 104 Sprague

- Dawley females 2 6/dose by gavage at dose levels of 0, 5, 25, or 125 mg/kg/day from days 6 through 15 of gestation.

There were no maternal deaths in this study. Two of the 22 pregnant females in the high dose group had enlarged, mottled kidneys, findings which were not s een in any other animal and are considered compound

- related. Food consumption was significantly decreased (8.7%; p<0.05) at the high dose level only, during the 6

- 16 day dosing period. Body weight and body weight gain were unaltered by atrazine treatment. There were no clinical signs attributable to compound exposure. There were no alterations in cesarean section parameters in dose groups vs controls.

The maternal LOAEL is 125 mg/kg/day, based on decreased food consumption during the dosing period and enla rged and mottled kidneys. The maternal NOAEL is 25 mg/kg/day.

Fetal death and resorptions were not altered by compound exposure. The high dose group only displayed increases in partially ossified interparietals (control fetal/litterDo not cite

Appendix F

- Page 645 of 677 incidence of 20%/56% v s high dose fetal/litter of 44%/91%); partially ossified hyoid (control fetal/litter 6%/20%, vs 17%/55%). Mean male and female fetal body weight at the high dose was significantly (p<0.05) reduced compared to controls (Control male/female body weight 3.6/3 .4 grams vs male/female high dose 3.5/3.3 grams. There were no increases in any visceral findings in dosed fetuses.

The developmental LOAEL is 125 mg/kg/day, based on increased incidence of partially ossified interparietal and hyoid bones and decreased fe tal body weight. The developmental NOAEL is 25 mg/kg/day.

The developmental toxicity study in the rat is classified Acceptable

- Guideline , and it satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700; §83

- 3a) in the rat.

870.4100 (870.4300) Chronic Toxicity

– Rat

In a 2

- year combined chronic feeding/carcinogenicity study (MRID 43532001),

G

- 34048 technical (hydroxyatrazine) of 97.1% purity, was administered in the diet to groups of 70 or 80 male and 70 or 80 female Crl:C D (SD) BR strain rats at dose levels of 0 (control), 10, 25, 200 or 400 ppm (equivalent to 0, 0.388, 0.962, 7.75, or 17.4 mg/kg/day in males and to 0, 0.475, 1,17, 9.35, or 22.3 mg/kg/day in females). Ten or 20 rats/sex/group were sacrificed at 12 months; the remaining 60 animals in each group were scheduled to be sacrificed at 24 months. Due to high mortality in the 400 ppm group, however, the surviving males and females in the 400 ppm group were sacrificed at 18 months. Mortality, clinical signs of tox icity, body weights, food consumption and water consumption were monitored. Ophthalmologic examinations were performed. Hematological examinations, clinical chemistries and urinalysis were also performed. Necropsy examinations were conducted on all animals and organ weight determinations were made on all animals sacrificed at 12, 18, 24 months. Histopathological examinations were made on a complete set of organs/tissues from all animals in the control, 200 and 400 ppm groups. Histopathological examinations were also performed on a limited set of organs/tissues from the 10 and 25 ppm groups.

At 400 ppm, an excessive, treatment

- related, mortality was observed for both males and females, and this dose level was terminated at 18 months. Severe renal failure was the predominant cause of death for these animals. Prior to death or sacrifice, these animals exhibited emaciation, dehydrated bodies, pallor and other clinical signs of toxicity expected in animals with severe renal failure. Greatly decreased body weights , body weight gains, and food consumption were observed in these animals throughout the study (to 18 months). Water consumption was increased during the first year of the study. Changes in hematology parameters (including anemia), in clinical chemistries

p arameters (indicating renal disturbances), and in urinalysis parameters (including crystalline material in urine samples) were observed in 400 ppm males and females. Gross necropsies, organ weights, and histopatholgy indicated that kidney and lower urinary tract were the primary target organs in both males ands females at 400 ppm. Kidney effects included discoloration, calculi and rough pitted surfaces seen at gross necropsy; increased kidney weights; and severe histopathological changes includingDo not cite

Appendix F

- Page 646 of 677 depositio n of crystalline material within collecting ducts and renal pelvises, calculi, other morphological changes, and accelerated chronic progressive nephropathy. In addition, secondary effects in extra renal tissues reflected the severe renal damage and result ing

renal failure in these animals.

At 200 ppm, similar but less severe gross and histopathological effects on the kidneys were observed in both males and females. Secondary effects in extra renal tissues were generally not observed at this dose level. A t 25 ppm, no treatment

- related effects were

observed in either male or female rats. An accumulation of interstitial matrix in the papilla of the kidneys of female rats was observed at this dose level, but the toxicologic significance of this observation, i n the absence of any other signs of renal damage or impaired renal function, was highly questionable.

A treatment

- related increased incidence of tumors of any type was not observed in the treated male or female animals in this study. In particular, there was no increase above control levels in the incidence of mammary gland tumors in either males or females. In addition, onset times for mammary gland tumors in female rats were not decreased in this study.

The LOAEL is 8.7 mg/kg/day, based on gross and h istopathological effects in the kidneys. The NOAEL 1.0 mg/kg/day.Do not cite

Appendix F

- Page 647 of 677 This study is classified as Acceptable

- Guideline , and it satisfies the OPPTS 870.4300 guideline requirement for a combined chronic/carcinogenicity toxicity study.

870.5100 Bacterial Reve rse Mutation Assay

The atrazine metabolite hydroxyatrazine (99% purity) failed to induce reverse mutation (as evidenced by no increase over controls in revertant colony counts) in Ames testing at concentrations up to the limit dose of 5000 μg/0.1 mL. T his study is

classified Acceptable

- Guideline (MRID 40722304).

870.5385 Mammalian Bone Marrow Chromosome Aberration Test

The atrazine metabolite hydroxyatrazine (99% purity) did not induce an increase in micronuclei in mice fed concentrations up to the limit dose of 5000 mg/mL. This study is classified Acceptable

- Guideline (MRID 41479401).

870.5550 Other genetic effects

The atrazine metabolite hydroxyatrazine (96

- 99% purity) was negative for inducing unscheduled DNA synthesis in primary rat hepatocyt es up to and beyond precipitating concentrations (> 500 μg/mL). This study is classified Acceptable

guideline (MRID 40422305).

870.5550 Other genetic effects

The atrazine metabolite hydroxyatrazine (96

- 99% purity) was negative for inducing unscheduled DNA synthesis in human fibroblasts (CRL 1521) up to and beyond precipitating concentrations (> 500 μg/mL). This study is classified Acceptable

- guideline (MRID 40888101).

F. 5.0 TOXICITY ENDPOI NT SELECTION

F. 5.1 See Section F. 8.2 for endpoint selection table [TXR NO. 0050592]

F. 5.2 Dermal Absorption

The maximum percentage of atrazine absorbed in the rat study after a 10 hr (representative of a typical workday) exposure was 21.6% (rounded up to 22%). The maximum percent absorbed after any duration of exposure in the human dermal penetration study described above under section 4.8, was 5.6% (rounded up to 6%).

Because the ma ximum percent absorbed is being used and because an ample amount of time (168 hours) was allowed for absorption to occur, 6% is deemed to be a protective estimate of dermal penetration in the human.

F. 5.3 Classification of Carcinogenic PotentialDo not cite

Appendix F

- Page 648 of 677 F. 5.3.1 Conclusions

Numerous studies, both guideline and non

- guideline studies submitted by the registrant, as well as studies performed by EPA's NHEERL [described in this document], examine the carcinogen icity and mode of action of atrazine. In a series of meetings [October 13, 1999 , April 12, 2000, and October 19, 2000] held subsequent to the Fifth HED Cancer Peer Review Committee (CPRC) of July, 1996, the Cancer Assessment Review Committee (CARC) met t o examine these studies. The data pertaining to atrazine's carcinogenicity and mode of action were also examined by the FIFRA Science Advisory Panel (SAP) on June 27 th to June 29 th , 2000. Both the CARC and SAP agreed that atrazine is associated with mamm ary and pituitary tumor formation in the female SD rat, but not in male SD rats or CD

- 1 mice and F

- 344 rats of either sex. CARC and SAP also agreed that there are sufficient data available to support a proposed mode of action (MOA) to account for the tumor s seen in SD female rats and that this MOA would likely not result in carcinogenicity in humans. Hypothalamic

neuronal

- GnRH pulse modulation of pituitary releases of LH is a central driver of ovulation in the SD female rat, and atrazine is essentially ac celerating the aging process of the CNS control of ovulation, which leads to a constant state of estrus (anovulation) and prolong exposure to estrogen and prolactin. Although there are certain similarities in the control of the hypothalamic

- pituitary

- ovar ian axis between humans and rats in that the hypothalamus can play a key regulatory role in primates, there are fundamental differences. For example, unlike the SD rat, CNS modulation is not the driving factor on

human GnRH and LH releases. Human conditi ons of anovulation are associated with aberrant GnRH and LH pulsatile releases. Even if atrazine induced anovulation in humans like in the SD rat, there is no evidence for the potential of an unopposed estrogen condition that would lead to tumor developme nt. It appears that when LH is low, such as in hypothalamic amenorrhea (HA), a state of low serum estrogen is found, not elevated or prolong estrogen exposure. It should be noted that there is no known cancer risk associated with HA patients, albeit they are at risk to a number of other clinical conditions (e.g., osteoporosis, heart disease, infertility). Another condition of anovulation, polycystic ovarian syndrome (PCOS), is also not a good model for atrazine

cancer mode of action in SD rats. The etiol ogy of PCOS is multifactorial, and LH secretion is elevated due to increased synthesis of androgen and their conversion to estrogens. Although atrazine might cause adverse effects on hypothalamic

- pituitary function in humans, the hormonal environment condu cive to tumor development (i.e., elevated or prolonged exposure to estrogen or prolactin) that is found in SD rats cannot be established in humans. Therefore, it is unlikely that atrazine‘s cancer mode of action in SD rats is operative in humans. Additiona lly, mutagenic and estrogenic activity do not appear to play a significant role in atrazine

- associated carcinogenicity.

F. 5.3.2 Classification Carcinogenic Potential

The CARC concluded that atrazine should be classified as a " Not Likely To Be Carcinogen ic To Humans ‖ [HED Document 014431; December 13, 2000 meeting], based on the overall weight of evidence that:Do not cite

Appendix F

- Page 649 of 677 1. The mode of carcinogenic activity in the female SD rat is supported by the

data.

2. The mode of carcinogenic activity in the female SD ra t essentially involves an acceleration of the reproductive aging process.

3. The mode of action for the carcinogenicity of atrazine is unlikely to be expressed in humans; no human conditions can be established that support a potential for atrazine to lea d to carcinogenicity in humans.

4. Other modes of action are not supported by the available data and, in particular, mutagenic and estrogenic activity do not appear to significantly contribute to atrazine‘s carcinogenic potential.

5. Although a few ep idemiological studies suggest a possible association between atrazine (or triazine) exposure and NHL and ovarian cancer, these cancers do not appear to be plausible based on atrazine‘s mode of action. Therefore, the human studies by themselves do not make a strong case for an

association.

5.3.3 Quantification of Carcinogenic Potential

Not applicable as atrazine is classified as a "Not Likely To Be Carcinogenic To

Humans".

F. 6.0 FQPA CONSIDERAT IONS

F. 6.1 Special Sensit ivity to Infants and Children

There was no increased quantitative or qualitative susceptibility in any of the guideline studies on atrazine in the rat, and there was no increased quantitative susc eptibility in the rabbit study. However, there was increased qualitative susceptibility in the rabbit study [increased resorptions (deaths) at a dose level that resulted in decreased body

weight gain and clinical signs in the maternal animal]. There were o ther non

- guideline studies on atrazine that show evidence of endocrine disruption [prostatitis study, delayed puberty study, and data on LH surge attenuation, and estrous cycle alterations]. The primary underlying events that led to mammary and pituitary t umor formation following atrazine exposure of Sprague

- Dawley female rats involved disruption of the hypothalamic

- pituitary

- ovarian axis. Since aspects related to this axis are involved in reproductive and developmental competency, there is a concern for ad verse reproductive and developmental effects in maternal animals and their offspring. Several special studies showed that treatment of pregnant rats with atrazine can lead to reproductive and developmental effects that may be associated with endocrine

alte rations. Additionally, the neurotoxicity seen in the non

- guideline studies with atrazine is a central nervous system (CNS) toxicity

- specifically, neurotransmitter and neuropeptide alterations at the level of the hypothalamus.Do not cite

Appendix F

- Page 650 of 677 Studies in the open litera ture indicate increased qualitative susceptibility. Dosing of dams immediately following parturition [postnatal days 1

- 4] resulted in prostatitis in male offspring, and dosing of the young following weaning resulted in delayed puberty in both

sexes. The mo de of action for these two effects (prostate inflammation and delayed puberty) is believed to be similar to the mode of action described for atrazine

- associated cancer and involves the CNS neuroendocrine alterations, specifically, neuroendocrine

alteration s at the hypothalamus.

Following a Degree of Concern Analysis [TXR No. 0050592; see 8.2 below], the HIARC concluded that an additional Special FQPA Safety Factor of 3X would be adequate to

account for the hazard

- based residual uncertainties for chronic d ietary [cRfD] and residential (incidental oral, dermal, inhalation) exposures. Additionally, it was concluded that an additional Special FQPA Safety Factor would not be required for acute dietary exposure [aRfD]. Refer to the Risk Assessment on Atrazine fo r a discussion of the FQPA Safety Factor used for the various exposure scenarios [D272009].

F. 6.2 Recommendation for a Developmental Neurotoxicity Study

A developmental toxicity study is not available and HIARC did not recommend that one be performed.Do not cite

Appendix F

- Page 651 of 677 F. 7.0 REFERENCES (LISTED IN ORDER OF ASCENDING MRID NUMBE R)

00024706 Sachsse, K.; Bathe, R. (1975) Acute Oral LD450^ of Technical Atrazine (G

30027) in the Rat: Project No. Siss 4569. (Unpublished study received Jun 2,

1977 under 100

- 529; prepared by Ciba

- Geigy, Ltd., submitted by Ciba

- Geigy Corp., Greensboro, N.C.; CDL:230303

- E)

00024709 Sachsse, K.; Ullmann, L. (1976) Eye Irritation in the Rabbit of G 30027: Project

No. Siss 5663. (Unpublished study received Jun 2, 1977 under 100

- 529; prepared by Ciba

- Geigy, Ltd., submitted by Ciba

- Geigy Corp., Greensboro, N.C.;

CDL:230303

- H)

00024710 Sachsse, K.; Ullmann, L. (1976) Skin Irritation in the Rabbit after Single

Application of G 30027: Project No. Siss 5663. (Unpublished study received Jun 2, 1977 under 100

- 529; prepared by Ciba

- Geigy, Ltd., submitted by Ciba

- Geigy Corp., Greensboro, N.C.; CDL:230303

- I)

00027097 Consultox Laboratories, Limited (1974) Atrazine: Acute Oral and D ermal Toxicity

Evaluation. (Unpublished study received April 22, 1976 under 33660

- 1; submitted by Industria Prodotti Chemici, S.p.a., Novate Milanese, Italy;

CDL:225976

- A)

00105131 Burlingham, M.; Young, S.; Adamik, E. (1979) Comparative Skin Sensitizati on of

Technical Atrazine: Project No. WIL

- 1214

- 78. (Unpublished study received May 12, 1982 under 201

- 411; prepared by WIL Research Laboratories, Inc., submitted by Shell Chemical Co., Washington, DC; CDL:247726

- A)

00150623 Puri, E. 1984. Autoradiographi c DNA repair test on rat hepatocytes. Ciba

Geigy. Basle, Switzerland. Study No. 831371. May 16, 1984.

00143006 Arthur, A. (1984) Segment II Teratology Study in Rabbits: Toxicology/Pathology

Report No. 68

- 84. Unpublished study prepared by Ciba

- Geigy Corp . 220 p.

00143008 Infurna, R. (1984) A Teratology Study of Atrazine Technical in Charles River

Rats: Toxicology/Pathology Report No. 60

- 84. Unpublished study prepared by

Ciba

- Geigy Corp. 269 p.

40246601 Deparade, E. (1986) Atrazine: Salmonella/Mammali an

-- Microsome Mutagenicity

Test: Laboratory Study No. 861172. Unpublished study prepared by Ciba

- Geigy Ltd. 23 p.Do not cite

Appendix F

- Page 652 of 677 40431301 O'Connor, D.; McCormick, G.; Green, J. (1987) Chronic Toxicity Study in Dogs:

Atrazine Technical: Laboratory Study No. 852008. Unp ublished study prepared by Ciba

- Geigy Corp. 1405 p.

40431302 Hazelette, J.; Green, J. (1987) Oncogenicity Study in Mice: Atrazine Technical:

Laboratory Study No. 842120. Unpublished study prepared by Ciba

- Geigy Corp. 3160 p.

40431303 Mainiero, J.; Yo ureneff, M.; Giknis, M.; et al. (1987) Two

- generation

Reproduction Study in Rats: Atrazine Technical: Laboratory Study No. 852063. Unpublished study prepared by Ciba

- Geigy Corp. 1395 p.

40431304 Orr, G. (1987) Disposition of Atrazine in Rat: (General Met abolism): Atrazine:

Laboratory Study No.: ABR

- 87048. Unpublished study prepared by Ciba

- Geigy Corp. 62 p

40431305 Thede, B. (1987) Study of ?Carbon 14F

- Atrazine Dose/Response Relationship in the

Rat: (General Metabolism): Laboratory Study No. ABR

- 87087. Unpublished study prepared by Ciba

- Geigy Corp. 53p.

40431306 Miles, B. (1987) Characterization and Identification of Atrazine Metabolites from Rat

Urine: (General Metabolism): Laboratory Study No.: ABR

- 87115. Unpublished study prepared by Ciba

- Geigy C orp. 35 p.

40437501 Orr, G. (1987) A Summary of the Disposition, Kinetics and Metabolism of Atrazine in

the Rat (General Metabolism): Laboratory/Study No. ABR

- 87116. Unpublished study prepared by Ciba

- Geigy Corp. 44 p.

40566301 Arthur, A. (1984) A Sup plement to a Teratology Study of Atrazine Technical in

New Zealand White Rabbits. Unpublished study prepared by Ciba

- Geigy Corporation. 4 p.

40566302 Infurna, R. (1984) A Supplement to a Teratology Study of Atrazine Technical in

Charles River Rats. Unp ublished study prepared by Ciba

- Geigy Corporation. 6

p.

40629302 Hardisty, J. (1987) Supplement to Two

- year Chronic Feeding/Oncogenicity Study

in Rats Administered Atrazine: 410

- 1102. Unpublished study prepared by Experimental Pathology Laboratories, In c. 316 p.Do not cite

Appendix F

- Page 653 of 677 40722301 Ceresa, C. (1988) Atrazine: Structural Chromosomal Aberration Test Micronucleus

Test, Mouse: Study No. 871546. Unpublished study prepared by Ciba

- Geigy Corp. 29 p.

40722302 Deparade, E. (1987) Diaminochlorotriazine: Gene Mutations T est: Salmonella/

Mammalian Mutagenicity Test: Study No. 871372. Unpublished study prepared by Ciba

- Geigy Corp. 26 p.

40722303 Meyer, A. (1987) Diaminochlorotriazine: Tests for Other Genetoxic Effects:

Autoradiographic DNA Repair Test on Human Fibroblas ts: Study No. 871371. Unpublished study prepared by Ciba

- Geigy Corp. 81 p.

40722304 Deparade, E. (1988) Hydroxyatrazine: Gene Mutations Test: Salmonella/

Mammalian

- Microsome Mutagenicity Test: Study No. 871376. Unpublished study prepared by Ciba

- Geigy C orp. 27 p.

40722305 Hertner, T. (1988). Tests for other genotoxic effects

- auto

- radiographic

DNA repair test on rat hepatocytes. Ciba

- Geigy, Basle, Switzerland. Laboratory report number: 871374. January 22, 1988. Unpublished.

40888101 Meyer, A. (1988) Hydroxyatrazine: Tests for other genotoxic effects,

autoradiographic DNA repair tests on human fibroblasts. Ciba

- Geigy, Basle, Switzerland . Study No. 871375. January 11, 1988. Unpublished.

41065201 Giknis, M. (1989) A Teratology (Segment II) Study in Rats: Atrazine Technical:

Laboratory Study No. 882049. Unpublished study prepared by Ciba

- Geigy Corp. 357 p

41065202 Giknis, M. (1989) A Teratology (Segment II) Study in Rats: Hydroxyatrazine

Technical: Laboratory Study No. 872202. Unpublished study pr epared by Ciba

Geigy Corp. 362 p.

41293501 Rudzki, M.; McCormick, G.; Arthur, A. (1989) 90

- Day Oral Toxicity Study in Rats:

Hydroxyatrazine: Lab Study Number 882146; MIN 882146; 89015. Unpublished study prepared by Ciba

- Geigy Corp., Research Dept., Phar maceuticals Div. 408

p.

41392401 Thompson, S.; Batastini, G.; Arthur, A. (1990) 13/52

- Week Oral Toxicity Study in

Dogs: Lab Project Number: 872151. Unpublished study prepared by Ciba

- Geigy Corp. 784 p.Do not cite

Appendix F

- Page 654 of 677 41392402 Hummel, H.; Youreneff, M.; Giknis; et al . (1989) Diaminochlorotriazine: A

Teratology (Segment II) Study in Rats: Lab Project Number: 872177. Unpublished study prepared by Ciba

- Geigy Corp. 428 p.

41479401 Ceresa, C. (1988) Hydroxyatrazine: Structural Chromosomal Aberration Test: Lab

Project Num ber: 871373. Unpublished study prepared by Ciba

- Geigy, Ltd. 32 p.

42085001 Thakur, A. (1991) Determination of Hormone Levels in Sprague

- Dawley Rats Treated

with Atrazine Technical: Final Report: Lab Project Number: 483

- 278. Unpublished study prepared by Hazleton Washington, Inc. 1132 p.

42089901 Holbert, M. (1991) Acute Inhalation Toxicity Study in Rats: Atrazine Technical:

Lab Project Number: 8079

- 91. Unpublished study prepared by Stillmeadow Inc. 23 p.

42146101 Thakur, A. (1991) Determination of Hormone Levels in Fischer

- 344 Rats

Treated with Atrazine Technical: Final Report: Lab Project Number: 483

- 279. Unpublished study prepared by Hazleton Washington, Inc. 1534 p.

42165503 Thede, B. (1991) Response to Data Request in EPA Review of Atrazine R egistration

Standard

-- Toxicity Studies: Lab Project Number: ABR

- 91076. Unpublished study prepared by Ciba

- Geigy Corp. 301 p.

42204401 Thakur, A. (1992) Oncogenicity Study in Sprague

- Dawley Rats with Atrazine

Technical: Lab Project Number: 483

- 275. Unpu blished study prepared by Hazleton Washington, Inc. 1092 p.

42227001 Thakur, A. (1992) Oncogenicity Study in Fischer 344 Rats with Atrazine

Technical: Lab Project Number: 483

- 277. Unpublished study prepared by Hazleton Washington, Inc. 2999 p.

42547105 Hertner, T. (1992) Autoradiographic DNA Repair Test on Rat Hepatocytes Tests for

Other Genotoxic Effects: Lab Project Number: 911246. Unpublished study prepared by Ciba

- Geigy Limited. 98 p.

42743902 Eldridge, J., Wetzel, L., Tisdel, M., and Luempert, L .G. 1993a.

Determination of Hormone Levels in Sprague

- Dawley Rats Treated with Atrazine Technical: Revised Supplement to Final Report. Hazleton Washington, Inc. Lab Project Number: 483

- 278.

42743903 Eldridge, J.; Wetzel, L.; Tisdel, M. et al. (1993) De termination of Hormone

Levels in Fischer

- 344 Rats Treated with Atrazine Technical: Revised Supplement to Final Report: Lab Project Number: 483

- 279. Unpublished study prepared by Bowman Gray School of Medicine. 166 p.Do not cite

Appendix F

- Page 655 of 677 43013201 Kuhn, J. (1991) G

- 28279 Tec hnical: Acute Oral Toxicity Study in Rats: Lab Project

Number: 7803/91. Unpublished study prepared by Stillmeadow, Inc. 27 p.

43013202 Kuhn, J. (1991) G

- 30033 Technical: Acute Oral Toxicity Study in Rats: Lab

Project Number: 7802/91. Unpublished study prepared by Stillmeadow, Inc. 28 p.

43013203 Thompson, S.; Batastini, G.; Arthur, A. (1992) G

- 28279 Technical: 90

- Day Oral

Toxicity Study in Dogs: 13

- Week Feeding Study in Dogs: Lab Project Number: 912021. Unpublished study prepared by Research Dept. Ci ba

- Geigy Corp. 429

p

43013205 Schneider, M. (1992) G

- 28279 Technical: 90

- Day Oral Toxicity Study in Rats: 3

-

Month Oral Toxicity Study in Rats (Administration in Food): Lab Project Number: 901261. Unpublished study prepared by Ciba

- Geigy Ltd. 263 p.

430 13206 Gerspach, R. (1991) G

- 30033 Technical: 90

- Day Oral Toxicity Study in Rats: 3

- Month

Oral Toxicity Study in Rats (Administration in Food): Lab Project Number: 901264. Unpublished study prepared by Ciba

- Geigy Ltd. 254 p.

43013207 Pettersen, J.; Richt er, A.; Gilles, P. (1991) Diaminochlorotriazine (G

- 28273): 90

- Day

Oral Toxicity Study in Rats: Lab Project Number: F

- 00006. Unpublished study prepared by Ciba

- Geigy Corp. 450 p.

43013208 Marty, J. (1992) G

- 28273 Technical: Teratology Study in Rats: Deve lopmental Toxicity

(Teratogenicity) Study in Rats with G

- 28279 Technical (Oral Administration): Lab Project Number: 901262. Unpublished study prepared by Ciba

- Geigy Ltd. 298 p.

43013209 Marty, J. (1992) G

- 30033 Technical: Teratology Study in Rats: Devel opmental Toxicity

(Teratogenicity) Study in Rats with G

- 30033 Technical (Oral Administration): Lab Project Number: 901265. Unpublished study prepared by Ciba

- Geigy Ltd. 296

p.

43016502 Sova, J. (1993) Atrazine Technical: Response to the EPA Review of th e Acute

Inhalation Toxicity Study. Unpublished study prepared by Ciba Plant Protection. 5 p.

43093101 Deparade, E. (1990) Salmonella and Escherichia/Liver

- Microsome Test: G

- 28279

Technical: Gene Mutations Test: Lab Project Number: 891243. Unpublished

s tudy prepared by Ciba

- Geigy Limited. 39 p.Do not cite

Appendix F

- Page 656 of 677 43093102 Deparade, E. (1989) Salmonella and Escherichia/Liver

- Microsome Test: G

- 30033

Technical: Gene Mutations Test: Lab Project Number: 891236. Unpublished study prepared by Ciba

- Geigy Limited. 37 p.

430931 03 Ogorek, B. (1991) Structural Chromosomal Aberration Test: Micronucleus Test,

Mouse: G

- 28279 Technical: Lab Project Number: 901307. Unpublished study prepared by Ciba

- Geigy Limited. 37 p.

43093104 Ogorek, B. (1991) Structural Chromosomal Aberration T est: Micronucleus Test,

Mouse: G

- 30033 Technical: Lab Project Number: 901309. Unpublished study prepared by Ciba

- Geigy Limited. 40 p.

43093105 Geleick, D. (1991) Tests for Other Genotoxic Effects: Autoradiographic DNA

Repair Test on Rat Hepatocytes: G

- 28279 Technical: Lab Project Number: 901308. Unpublished study prepared by Ciba

- Geigy Limited. 94 p.

43093106 Geleick, D. (1991) Tests for Other Genotoxic Effects: Autoradiographic DNA Repair

Test on Rat Hepatocytes: G

- 30033 Technical: Lab Project Numb er: 901310. Unpublished study prepared by Ciba

- Geigy Limited. 94 p.

43314302 Chengelis, C. (1994) A Dermal Radiotracer Absorption Study in Rats with (carbon 14)

Atrazine: Final Report: Lab Project Number: 82048: 89/90/B. Unpublished study prepared by WIL Research Laboratories, Inc. 510 p.

43532001 Chow, E.; Hart, S. (1995) 2

- Year Dietary Toxicity/Oncogenicity Study with G

- 34048

(Hydroxyatrazine) in Rats: Final Report: Lab Project Number: F

- 00125. Unpublished study prepared by Ciba

- Geigy Corp. 2599 p.

43598617 Tennant, M.; Hill, D.; Eldridge, J.; et al. (1994) Possible antiestrogenic properties of

chloro

- s

- triazines in rat uterus. Journal of Toxicology and Environmental Health

43:183

- 196.

43598618 Tennant, M.; Hill, D.; Eldridge, J.; et al. (1994 ) Chloro

- s

- triazine antagonism of

estrogen action: Limited interaction with estrogen receptor binding. Journal of Toxicology and Environmental Health 43:197

- 211Do not cite

Appendix F

- Page 657 of 677 43598619 Safe, S.; Chen, I.; Liu, H.; et al. (1995) Failure of Atrazine and Simazine to Indu ce

Estrogenic Responses in MCF

- 7 Human Breast Cancer Cells. Unpublished study prepared by Texas A&M Univ.; and Univ. of Western Ontario. 25 p.

43598622 McConnell, R. (1995) A Histomorphologic Reevaluation of the Ovaries, Uterus,

Vagina, Mammary Gland, a nd Pituitary Gland From Sprague

- Dawley and

Fischer

- 344 Female Rats Treated With Atrazine: Lab Project Numbers: 483

- 278:

483

- 279. Unpublished study prepared by Ciba

- Geigy Corp. 107 p.

43934403 Safe, S. (1995) Failure of chloro

- s

- triazine derived compound s to induce

estrogenic responses in vivo and in vitro . in Fundamental Applied Toxicology (in Press).

43934404 Morseth, S. (1996) Evaluation of the Luteinizing Hormone (LH) in Female

Sprague

- Dawley Rats

-- Pilot Study: Final Report: Lab Project Number: CHV

2 386

- 109: 6791. Unpublished study prepared by Corning Hazleton Inc. (CHV). 33 p.

43934406 Morseth, S. (1996) Evaluation of the Luteinizing Hormone (LH) Surge in

Atrazine

- Exposed Female Sprague

- Dawley Rats: Interim Report: Lab Project
Number: CHV 2386

- 111 : 6791E: 2386

- 111. Unpublished study prepared by Corning Hazleton Inc. (CHV). 624 p.

44152102 Morseth, S. (1996) Evaluation of the Luteinizing Hormone (LH) Surge in Atrazine

Exposed Female Sprague

- Dawley Rats

-- (Final) 6

- Month Interim Report: Lab Project Number: CHV 2386

- 111: 2386

- 111: 6791E. Unpublished study prepared by Corning Hazleton Inc. 727 p.

44544701 Morseth, S. (1998) Chronic (12

- 24 Month) Study in Rats with Atrazine Technical.

Study No. 2386

- 108. Unpublished study prepared by Covance Labora tories Inc. 2251 p.

44152114 Hui, X.; Gilman, S.; Simoneaux, B.; et al. (1996) In vivo Percutaneous Absorption of

Atrazine in Man: Lab Project Number: ABR

- 96067: BDH

- 081

- 2: H832

- 11835

- 01. Unpublished study prepared by UCSF; UC Davis; and Ciba Crop Protec tion. 297 p.

44713802 Paul, H. J.; Dunsire, J.P.; Hedley, D. (1993). The Absorption, Distribution,

Degradation and Excretion of [U

14 C]

- Triazine G 30027 in the Rat. Inveresk Research International, Tranent, EH33 2NE, Scotland. IRI Report

No. 9523. Decemb er 10,Do not cite

Appendix F

- Page 658 of 677 45058701 Minnema, D.J. (2000) Comparison of the LH surge in female rats

administered atrazine, simazine or DACT via oral gavage for one month.

Covance # 6117

- 398 Novartis # 1198

- 98. Covance Labs, Vienna, VA. Feb. 29, 2000. Unpublish ed study.

45166901 Cooper, R.L., Stoker, T.E., Tyrey, L., Goldman, J.M., and McElroy, W.K.

2000. Atrazine disrupts the hypothalamic control of pituitary

- ovarian

function. Tox. Sci . 53: 297

- 307.

45166902 Stoker, T.E., Robinette, C.L., and Cooper, R.L. ( 1999) Maternal exposure

to atrazine during lactation suppresses suckling

- induced prolactin release and results in prostatitis in the adult male offspring. Tox. Sci . 52;68

- 79.

Studies which do not have an MRID number:

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 1996 Jul

- Aug;10(4):257

- 64. MRID not yet assigned.

Das, P.C., McElroy, W.K., Cooper, R.L. (2000) Differential modulation of catecholamines by chloro triazine herbicides in pheochromocytoma (PC12) cells in vitro. Tox. Sci. Aug; 56(2): 324

- 31. MRID not yet assigned.

Laws, S.C., Ferrell, J.M., Stoker, T.E., Schmid, J., and Cooper, R.L. (2000).The effect of atrazine on puberty in female wistar rats: an e valuation in the protocol for the assessment of pubuertal development and thyroid function. Tox. Sci . In Press. MRID not yet assigned.

Narotsky M. G., Best, D.S., Guidici, D. L., and Cooper, R.L. (2000). Strain comparisons of atrazine

- induced pregnancy loss in the rat. Repro Toxicol. In Press. MRID not yet assigned.

Shafer, T.J., Ward, T.R., Meacham, C.A., Cooper, R.L. (1999) Effects of the chlorotriazine herbicide, cyanazine, on GABA(A) receptors in cortical tissue from rat brain. Toxicology 1999 De c 20;142(1):57

- 68. MRID not yet assigned.

Stoker, TE, Laws, SC, Guidici, D and Cooper, RL. (2000) The effect of atrazine on puberty in male Wistar rats: An evaluation in the protocol for the assessment of pubertal development and thyroid function. To xicol. Sci. 58: 50

- 59.

Thakur, A.K. (1999). Addendum 1 to study no. 2386

- 108: Chronic (12/24 month) study in rats with atrazine technical (EPA MRID No. 445701). Unaudited draft interim report of the estrous cycle data through week 46. Covance Lab, V ienna VA. Covance Lab

No. 2386

- 108. Novartis Nexus # 810

- 95. March 12, 1999.Do not cite

Appendix F

- Page 659 of 677 F. 8.0 APPENDICES

F. 8.1 Toxicity Profile Summary Tables

F. 8.1.1 Acute Toxicity Table for Atrazine

Acute Toxicity Data of Technical Atrazine

Guideline

No. Test Results Tox. Cat.

870.1000 Oral LD 50 - rat LD 50 > 1,869 mg/kg (M&F combined) III

870.1200 Dermal LD 50 -

rat LD 50 > 2,000 mg/kg (M&F

combined) III

870.1300 Inhalation LC 50 -

rat LC 50 > 5.8 mg/L (M&F combined) IV

870.1400 Eye Irritation

-

rabbit PIS = 0.0/110 IV

870.1500 Dermal Irritation

- rabbit PIS = 0.2/8.0 IV

870.1600 Dermal

Sensitization

- guinea pigs Non

- sensitizing

---

870.6100 Acute Delayed

Neurotoxicity Not Required

---

PIS=Primary Irritation Score

F. 8.1.2 Subchronic, Chronic and Other Toxicity Table for Atrazine

Subchronic, Chronic and Other Toxicity Table. Only studies considered acceptable by

HED are shown.

Guideline No./ Study Type MRID No. (year)

/Doses Results

870.3100

90

- Day oral toxicity rodents 44723701 (1994)

0, 10, 50, or 500 ppm 0, 0.6 , 3.3, 34.0 mg/kg/day

- males NOAEL = 3.30 mg/kg/day LOAEL = 34.5 mg/kg/day based on decreased body weightDo not cite

Appendix F

- Page 660 of 677 Guideline No./ Study Type MRID No. (year)

/Doses Results

0, 0.659, 3.35, 35.3 mg/kg/day

- females.

870.3150

90

- Day oral toxicity in

nonrodents (dogs) --- Study waived because an acceptable

chroni c oral study in the dog is available.

870.3200
21/28

- Day dermal toxicity 42089902 (1989)

0, 10, 100 or 1000 mg/kg/day NOAEL = 100 mg/kg/day LOAEL = 1000 mg/kg/day based on statistically significant reductions in food consumption, mean body weight, an d percent weight gain in both sexes, statistically significantly increased absolute and relative spleen weights in both sexes

870.3250

90

- Day dermal toxicity --- Study not required

870.3465

90

- Day inhalation toxicity --- Study not required

87 0.3700a

Prenatal developmental in

rodents 40566302 (1984)

0, 10, 70, or 700 mg/kg/day Maternal NOAEL = 10 mg/kg/day. Maternal LOAEL = 70 mg/kg/day, based on reduced body weight gain

Developmental NOAEL = 10 mg/kg/day Developmental LOAEL = 70 mg/kg/day based on delayed or no ossification at

several sites

870.3700a

Prenatal developmental in

rodents 41065201 (1989)

0, 5, 25, 100 mg/kg/day Maternal NOAEL = 25 mg/kg/day. Maternal LOAEL = 100 mg/kg/day based on reduced body weight gain and food

consum ption.

Developmental NOAEL = 25 mg/kg/day. Developmental LOAEL = 100 mg/kg/day based on increased incidence of delayed

ossification of skull bones.Do not cite

Appendix F

- Page 661 of 677 Guideline No./ Study Type MRID No. (year)

/Doses Results

870.3700b

Prenatal developmental in

nonrodents 00143006, 40566301

(1984)
0, 1, 5, or 75 mg/kg/day Mat ernal NOAEL = 5 mg/kg/day Maternal LOAEL = 75 mg/kg/day based on decreased body weight, food consumption and increased incidence of clinical signs

Developmental NOAEL = 5 mg/kg/day Developmental LOAEL = 75 mg/kg/day based on reduced litter size, incre ased resorptions and delayed ossification.

870.3800 Reproduction and fertility effects 40431303 (1987)

0, 10, 50,and 500

ppm 0, 0.75, 3.78, 39.0 mg/kg/day

- males 0, 0.86, 3.70, 42.8 mg/kg/day

- females. NOAEL = 3.78 mg/kg/day LOAEL = 39 mg/kg/day in males based on decreased body weights, body weight gains and food consumption

Offspring NOAEL = 3.78 mg/kg/day Offspring LOAEL = 39 mg/kg/day based on decreased body weights in both generations

of males at PND 21.

870.4100a Chronic toxicity

rodents 40629302 (1986)

0, 10, 70, 500, 1000

ppm, 0, 0.5, 3.5 25, 50 mg/kg/day This guideline satisfied by 870.4300 Combined chronic toxicity/carcinogenicity NOAEL = 3.5 mg/kg/day LOAEL = 25 mg/kg/day, based on reduced body

- weight gain and food consumption
Se e below under 870.4300 for details

870.4100b Chronic toxicity

nonrodents (dogs) 40431301 (1987)

0, 15, 150, 1000

ppm 0, 0.5, 5.0 33.7 mg/kg/day NOAEL = 5.0 mg/kg/day LOAEL = 33.7 mg/kg/day based on cardiac

effects.

870.4200 Carcinogenicity (rat, Fi scher

- 344) 42227001 (1992)

0, 10, 70, 200, 400

ppm 0, 0.5, 3.4, 9.9, 20.2 mg/kg/day

- males 0, 0.6, 4.4, 12.7, 26.2 mg/kg/day

- females NOAEL = 3.4 mg/kg/day

- males; 4.4 mg/kg/day

- females LOAEL = 9.9 mg/kg/day

- males; 12.7 mg/kg/day

- females based on decreased body weight gain

There was no treatment

- related increase in tumor incidence when compared to controls. This study used Fischer

- 344 rats. The purpose was to demonstrate a lack of tumorDo not cite

Appendix F

- Page 662 of 677 Guideline No./ Study Type MRID No. (year)

/Doses Results

response in this strain following atrazine

exposure.

87 0.4200 Carcinogenicity (mice) 40431302 (1987)

0,10,300,1500, 3000

ppm 0, 1.4, 38.4, 194, 385.7 mg/kg/day

- males 0, 1.6, 47.6, 246.9, 482.7 mg/kg/day

- females NOAEL = 43 mg/kg/day LOAEL = 222.0 mg/kg/day based on decreased body weight gain in both sexes

and increased cardiac thrombi in the

females.

No evidence of carcinogenicity was seen.

870.4300
Combined chronic toxicity/ carcinogenicity (rat, Sprague

Dawley) 40629302 (1986)

0, 10, 70, 500, 1000

ppm, 0, 0.5, 3.5 25, 50 mg/kg/day NOAEL = 3.5 mg/ kg/day LOAEL = 25 mg/kg/day ,based on reduced body weight gain and food consumption. Mammary tumors increased at 3.5 mg/kg/day and above. This study used the Sprague Dawley strain

of rat.

870.5100

Bacterial reverse mutation assay 00060642 (1977)

0, 50, 100, 500, 1000, 5000 μg/plate Negative in strains TA 98, 100, 1535 and
1537 of S. typhimurium up to the limit concentration of 5000 μg/plate both with and

without activation

870.5100

Bacterial reverse mutation assay 40246601 (1986)

0, 20, 78, 313,

1 250, and 5000 μg/plate Negative in strains TA 98, 100, 1535, 1537
and 1538 of S. typhimurium up to the limit concentration of 5000 μg/plate both with and

without activation

870.5385

Micronucleus

assay 40722301 (1988)

562.5, 1175, 2250 mg/kg Negative in doses which induced death in
Tif:MAGF mice.

870.5450

Rodent dominant lethal assay 42637003 (1993)

0, 500, 1000, 2000, or 2400 mg/kg Negative in Tif: MAGf mice at doses 2400 mg/kg.

870.5550 UDS assay 00161790/40722301

(1984)
0, 1.2, 6, 30, 15 0 μg/mL Negative in primary rat hepatocytes up to 150 μg/mLDo not cite

Appendix F

- Page 663 of 677 Guideline No./ Study Type MRID No. (year)

/Doses Results

870.5550 UDS assay 42547105 (1992)

0, 15, 46, 139, 417, 835, 1670 μg/mL Negative in primary rat hepatocyte cultures up to and beyond a dose which caused precipitation (139 μg/mL)

870.6300 Developmental neurotoxicity

870.7485

Metabolism and

Pharmacokinetics 40431304 (1987)

0, 1, and 100 mg/kg for a single dose given through oral gavage. 1.0 mg/kg/day for 15 days by oral gavage. Distribution, accumulation

Distribution was dose

- de pendent and independent of sex. Distribution appeared to

follow first

- order kinetics and the half

- life in

the tissues was 31.3 hours.

Excretion Approximately 95% of the atrazine excreted within 7 days of dosing. Urinary route

accounted for about 75% of t he excretion

feces accounted for 20%. Route of excretion did not seem to vary among sexes or with

dose.

870.7485

Metabolism and pharmacokinetics MRID 40431305

(1987)

The animals were dosed daily for 10 days through a

stomach tube with

dose levels of 0 , 1, 3, 7, 10, 50 or 100 mg/kg/day. Distribution, accumulation Distribution was highest in the red blood cell, followed by the liver, ovary and kidney.

When the dose increased the amount

distributed in the tissues increased. The distribution appeared to f ollow first

- order

kinetics and the tissue half

- life was 38.6 hours. This indicates that atrazine, with possible exception of the red blood cell,

does not bioaccumulate.

870.7485

Metabolism and pharmacokinetics MRID 40431306

(1987) Rats were given test 100 mg/kg article was given through

the stomach tube in a single oral dose.
Other rats were given 16.18 and Excretion In the rats given 100 mg/kg greater th an 100% of the administered radioactivity was recovered within 3 days of dosing. Urine

was found to contain 47.3% of the radioactivity and the feces 49.3%. The

tissues contained 5.75% and 1.4% was

found in the blood.Do not cite

Appendix F

- Page 664 of 677 Guideline No./ Study Type MRID No. (year)

/Doses Results

19.64 mg/kg and

urine was collected over a 24 hr period.

The urine was analyzed for

metabolites. Metabolism

Metabolites indicate that dechlorination of the triazine ring and N

- dealkylation are the major metabolic pathways for atrazine in rats. Oxidation of the alky substituents of atrazine appears to be of minor metabolic importance.

870.7485

Metabolism and pharmacokinetics MRID 421 65503

(1993) Fecal and urinary samples from rats exposed in a separate metabolism study (MRID 40431304) were

obtained and analyzed to

determine metabolism profiles. Metabolic profile

No sex differences in metabolic profiles were evident. The major fecal metabolite
was DACT which accounted for 40% of the total fecal radioactivity.

870.7485

Metabolism and pharmacokinetics MRID 44713802

(1993) single oral dose of 1 or 100 mg/kg through oral gavage Distribution, accumulation

Time to maximum blood concent ration (t cmax ) was 2 hours and 24 hours for the low and high dose groups, respectively. With exception of red blood cells, whole blood, and skeletal muscle, tissue burden for any specific tissue or organ represented less

than 1% of the administered dose b y 14 days post dosing

Excretion Urinary excretion was 64.72% of the total

administered low dose over a 48

- hour period and 66.16% of the total administered high dose over a 168

- hour period. Within 48 hours urinary excretion was 100% and 94% complete for th e low

- dose and high

- dose group, respectively.

Fecal elimination accounted for 10.80% and

19.69% of the total dose for the low and high dose groups, respectively.Do not cite

Appendix F

- Page 665 of 677 Guideline No./ Study Type MRID No. (year)

/Doses Results

870.7600

Dermal penetration

- rat 43314302 (1994)

0.01, 0.1 or 1 mg/cm 2 The percent a bsorbed increased with exposure time and decreased with dose. Regardless of the dose or exposure time, the majority (65

- 95%) of the radio labeled

atrazine was recovered in the washes or

was found associated with the skin at the site of exposure. The maxi mum percentage of atrazine absorbed in the rat study after a 10 hr (representative of a typical workday) exposure

was 21.6 %.

870.7600

Dermal penetration

- human 44152114 (1996)

0.17 and 2.0 mg of [14C] atrazine The majority (91.1

- 95.5%) of the dose wa s not absorbed. After 168 hours, only 5.6% of

the dose was absorbed and excreted in the

urine and feces of the low

- dose group and only 1.2% in the high

- dose group.

Special study

-

Assays of direct estrogenic activity 43598617 (1994)

This MRID numbe r describes more than one assay. The

doses varied depending on the assay. See above

under section 4.9.1 for specific details on

doses used. This study performed a trio of assays: uterotrophic response assay; progesterone receptor competitive binding assa y; and, a uterine thymidine incorporation assay. All doses tested displayed a lack of clear effects on uterine weight, progesterone binding capacity, and thymidine incorporation. This indicates that atrazine (and DACT and simazine, which were also tested) do not exhibit direct estrogenic activity.

Special study

-

Assays of direct estrogenic activity 43598618 (1994)

This MRID number describes one type of assay conducted under a variety of experimental

conditions. The
doses varied. See

above under section 4.9.1 for specific

details on doses

used. This study describes a series of estrogen receptor competitive binding assays, both in

vivo and in vitro

. Overall the results indicate that atrazine (and DACT and simazine, which were also tested) do exhibit so me competitive binding with estradiol but only under conditions which favor binding.Do not cite

Appendix F

- Page 666 of 677 Guideline No./ Study Type MRID No. (year)

/Doses Results

Special study

- Assays of direct estrogenic activity 43598619 (1995)

This MRID number

describes more than one assay. The

doses varied depending on the assay. See abo ve under section 4.9.1 for specific details on

doses used. This study describes four separate assays: competitive binding assay with the hepatocyte Ah receptor; MCF

- 7 cell proliferation; gel electrophoresis mobility shift assay using the progesterone rec eptor; and, luciferase reporter gene assay in MCF

7 cells. Neither atrazine (nor simazine, which was also tested) displayed estrogenic activity or interacted with the Ah receptor in the set of experiments described in this paper.

Special study

-

Assays of direct estrogenic activity 43934403 (1995)

This MRID number

describes more than one assay. The

doses varied depending on the assay. See above

under section 4.9.1 for specific details on

doses used. This study described several assays, both in

vivo and in vitro

. In vivo assays: uterine weight, progesterone receptor levels and uterine peroxidase. In vitro assays: MCF

- 7 cell proliferation; gel electrophoresis mobility shift assay using the progesterone receptor; luciferase reporter gene assay in MCF

- 7 cells; and, a selective medium growth assay in yeast cells.

The results of these experiments indicate

that in vivo atrazine and simazine may exhibit some antiestrogenic activity but no estrogenic activity either in vivo or in vitro

.
Special study

-

Es trous Cycle and LH Surge

Measurements (Pilot study) 43934404 (1996)

atrazine not given This study was a pilot study in which the validity of a proposed protocol for testing the effect of atrazine exposure on the proestrus afternoon luteinizing hormon e (LH) was evaluated. This study demonstrated that the proposed protocol adequately tested the parameters to examined.

Special study

- Estrous Cycle and LH Surge

Measurements (28 day exposure) 43934406 (1996)

0, 2.5, 5, 40 and 200 mg/kg/day NOAEL = 5 mg/kg/day. LOAEL = 40 mg/kg/day based on decreases in food consumption, body weight, body weight gain, estrous cycle alterations and LH surge attenuationDo not cite

Appendix F

- Page 667 of 677 Guideline No./ Study Type MRID No. (year)

/Doses Results

Special study

- Estrous Cycle and LH Surge

Measurements (6 month exposure) 44152102 (1996)

0, 25, 50, and 400

ppm 0, 1.8, 3.7, 29.4 mg/kg/day NOAEL = 1.8 mg/kg/day LOAEL = 3.7 mg/kg/day based on estrous cycle alterations and LH surge attenuation

Special study 1

-

Hormone and estrous cycle

measurements in
SD rats 42085001,

42743902 (1991, 1993)

See also 43598622
0, 70, 400 ppm 0, 4.2, 26.2 mg/kg/day Ten females per

dose were sacrificed
at 1, 3, 9, 12, 15, 18,

24 mo. NOAEL = none found LOAEL = 4.2 mg/kg/day based on estrous cycle alterations

Special Study 1

-

Mammary Gland

and Ova rian Histomorphology
in SD rats 43598622 (1995)

See also 42085001,

42743902 0, 70, 400 ppm 0, 4.2, 26.2 mg/kg/day Ten females per

dose were sacrificed at 1, 3, 9, 12, 15, 18, 24 mo. NOAEL = none found LOAEL = 4.2 mg/kg/day based early onset

of anovu lation as indicated by ovarian histomorphology and early onset of indicators of prolonged/increased hormone exposure in the mammary gland

Special study 2

-

Two

- year bioassay in F

344 rats 42146101 (1991)

See also 4274392

and 43598622

0, 0.7, 4.8, 14, 33.4 mg/kg/day Ten females per

dose were sacrificed
at 1, 3, 9, 12, 15, 18,

24 mo. NOAEL = 14 mg/kg/day LOAEL = 33.4 mg/kg/day based on decreased body weight gain.

There was not an increased incidence of any tumor type, nor an early onset of mammary tu mors.

Special study 2

- 42743902 (1993) NOAEL = at least 33.4 mg/ kg/dayDo not cite

Appendix F

- Page 668 of 677 Guideline No./ Study Type MRID No. (year)

/Doses Results

Hormone and estrous cycle

measurements in

F

- 344 rats See also 42146101

and 43598622

0, 0.7, 4.8, 14, 33.4 mg/kg/day

Ten females per

dose were sacrificed
at 1, 3, 9, 12, 15, 18,

24 mo. LOAEL = none found

This study examined hormones and estrous cycles in the animals used in 42146101

. The estrous cycle evaluations from this study are deemed unreliable by HED. The ovarian histomorphology data from MRID

43598622 is used to confir m a lack of effect of atrazine treatment on estrous cycles.

The animals exhibited hormone levels indicative of normally aging females of the strain. Exposure to atrazine had no effect on

hormone levels.

Special study 2

-

Mammary Gland

and Ovarian

Histomo rphology

in F

- 344 rats 43598622 (1995)

See also 42146101

and 42743902

0, 0.7, 4.8, 14, 33.4 mg/kg/day

Ten females per

dose were sacrificed
at 1, 3, 9, 12, 15, 18,

24 mo. NOAEL = at least 33.4 mg/kg/day

LOAEL = none found

This study examined histomor phology in the

animals used in 42146101

. The animals exhibited ovarian and mammary gland histomorphology indicative of normally aging females of the strain. Exposure to atrazine had no effect on these histomorphologic parameters.

Special study

-

Two

- yea r bioassay with the
SD strain of rat 42204401 (1992)

0, 70, 400 ppm

0, 3.8, 23.0 mg/kg/day NOAEL = 3.8 mg/kg/day LOAEL = 23 mg/kg/day based on decreased body weight gains as well as statistically significant decreases in body weights in the

0

- 76 week period A statistically significant increase in mammary tumors was seen at the high dose. An early onset of mammary

carcinomas was seen.

Special study

-

Tumor incidence

in ovariectomized (OVX) vs intact

animals 44544701 (1998)

0, 25, 50, 70, 400

ppm 0, 1.5, 3.1, 4.2, 24.4 mg/kg/day

- intact 0, 1.2, 2.5, 3.5, 20.9 Intact animal Mammary tumors

No mammary tumors seen in any OVX

animalDo not cite

Appendix F

- Page 669 of 677 Guideline No./ Study Type MRID No. (year)

/Doses Results

mg/kg/day

- OVX

Special study

-

Long

- term estrous cycle

measurements No MRID assigned

(1999)
0, 25, 50, 70, 400

ppm 0, 1.5, 3.1, 4.2, 24.4 mg/kg/day This is the unaudited draft report of the interim estrous cycle data from MRID

44544701

These data demonstrate an early onset of increased % days in estrus in atrazine

treated animals compared to controls.

Speci al study

Direct comparison of LH surge attenuation of atrazine, simazine, DACT No MRID assigned

(2000)

0, 2.5, 5, 40, 200 mg/kg/day This is the unaudited draft report of a study in which SD females were exposed to atrazine, simazine or DACT for 28 da ys and the ability of these chemicals to attenuate the LH surge was measured.

These data showed that simazine and DACT are able to attenuate the LH surge at

doses similar to those at which atrazine attenuates the surge.

1 This study in SD rats contain s three parts submitted under three separate MRID

numbers:

MRID 42085001 contains the results of the animal bioassay part of this study (the clinical observations, body weights, food consumption, gross pathology,

etc. );

MRID 42743902 contains the results of the vaginal smears/estrous cycle determinations and serum hormone measurements;

MRID 43598622 contains the results of the histomorphologic analysis.

2 This study in F

- 344 rats contains three parts submitted under three separate MRID

numbers:

MRID 421 46101 contains the results of the animal bioassay part of this study (the clinical observations, body weights, food consumption, gross pathology,

etc. );

MRID 42743902 contains the results of the vaginal smears/estrous cycle determinations

and serum hormo ne measurements;
MRID 43598622 contains the results of the histomorphologic analysis.Do not cite

Appendix F

- Page 670 of 677 F. 8.1.3 Toxicity Table for DACT

. All available acceptable toxicity studies

are shown.

Guideline No./ Study Type MRID No. (ye ar) /Doses Results

870.3100

90

- Day oral toxicity rodents 43013207 (1991)

0, 0, 10, 100, 250, or 500

ppm 0, 0.7, 6.7, 16.7, or 34.1 mg/kg/day

- males 0, 0.7, 7.6, 19.7, or 40.2 mg/kg/day

- females. Male NOAEL= 16.7 mg/kg/day LOAEL= 34.1 mg/kg/da y based on decreased body weight and body weight gain

Female NOAEL= 0.7 mg/kg/day LOAEL=7.6 mg/kg/day based on estrous cycle alterations

870.3100

90

- Day oral toxicity in

nonrodents (dog) 41392401 (1990)

0, 5, 100, and 1500/750 ppm (high dose reduced after 6 wks) 0, 0.187, 3.61, and 24.1mg/kg/day

- males 0, 0.195, 3.43, and 32.7 mg/kg/day

- females NOAEL = 3.4 mg/kg/day LOAEL = 24.1 mg/kg/day based on

cardiac effects and tremors in the males.

870.4100

Chronic toxicity

in nonrodent (dog) 41392 401 (1990)

0, 5, 100, and 1500/750 ppm (high dose reduced after 6 wks) 0, 0.187, 3.61, and 24.1mg/kg/day

- males 0, 0.195, 3.43, and 32.7 mg/kg/day

- females NOAEL = 3.4 mg/kg/day LOAEL = 24.1 mg/kg/day based on

cardiac effects and tremors in the males.

870.3700a

Prenatal developmental toxicity in

rodents 41392402 (1989)

0, 2.5 , 25, 75 or 150 mg/kg/day Maternal NOAEL = 2.5 mg/kg/day Maternal LOAEL = 25 mg/kg/day, based on decreased body weight gain [

- 32%] during the initial dosing period [GD 6

- 8]. Developmental NOAEL is 2.5 mg/kg/day. Developmental LOAEL = 25 mg/kg/day,

based on increases in incidences of incompletely ossified parietals, interparietals and unossified hyoids.

870.5100

Bacterial

reverse mutation assay 40722302 (1987)

0, 20 , 78 , 313, 1250, 5000 μg/plate Negative in the Salmonella /Ames assay up to limit concentration of 5000 μg per plate in strains TA 98, TA100, TA1535,

and TA1537 with and without metabolic

activationDo not cite

Appendix F

- Page 671 of 677 Guideline No./ Study Type MRID No. (ye ar) /Doses Results

870.5550 UDS assay 40722303 (1987)

0, 5.56, 16.67, 50, 1 50, 300, 600 μg/mL Negative up to 600 μg/mL (which exceeded the solubility limit of 400 μg/mL) in UDS assay using isolated human fibroblasts without activation only (not performed with activation)Do not cite

Appendix F

- Page 672 of 677 F. 8.1.4 Toxicity Table for G

- 28279. All available acceptable toxicity

studies are shown.

Guideline No./ Study Type MRID No. (year)

/Doses Results

870.100

Acute oral toxicity 43013201 (1991)

500, 2000, 3500, 5050 mg/kg LD 50 for both sexes combined= 1240 mg/kg

870.3100

90

- Day oral toxicity rodents MRID 43013205

(1992) 0, 10, 50, or 500 ppm 0, 0.602, 3.20, 34.9 mg/kg/day

- males
0, 0.641, 3.34, 37.5 mg/kg

- females NOAEL=3.2 mg/kg/day

LOAEL=34.9 based on based on decreased body weights and body weigh t gains in both

sexes. and possible histopathologic effects in the liver, adrenal cortex, and thyroid.

870.3100

90

- Day oral toxicity in

nonrodents (dog) MRID 43013203

(1992)
0,15,100, 500, or 1000

ppm 0, 0.6, 3.8, 18.9, 33.4 mg/kg/day

- male 0, 0.6, 3.8, 18.0, 33.3 mg/kg/day

- female NOAEL=3.8 mg/kg/day

LOAEL=18.9 based on decreased food consumption and body weight gain and decreased organ

- to brain weight ratios for the heart, testes, and prostate gland

870.3700a

Prenatal developmental

in roden ts 43013208 (1992)

0, 5, 25, or 100 mg/kg/day. Maternal NOAEL= 5 mg/kg/day

Maternal LOAEL= 25 mg/kg/day based on decreased body weight gain and food consumption

Developmental NOAEL= 5 mg/kg/day Developmental LOAEL 25 mg/kg/day based on .

increased fe tal and litter incidences of fused

sternebrae 1 and 2

870.5100

Bacterial

reverse mutation

assay 43093101 (1990)

0, 313, 625, 1250, 2500, 5000 μg/mL Negative in the Salmonella /Ames and E. Coli WP2uvrA assays up to limit concentration of 5000 μg per plate. Tested in S. Typhimurium in strains TA 98, TA100, TA1535, and TA1537

with and without metabolic activation

870.5375

micronucleus

assay 43093103 (1991)

0, 120, 240, 480 mg/kg Negative in Tif:MAGF mice treated up to the maximum tolerated dose of 480 mg/kg

870.5550 43093105 (1991) Negative up to cytotoxic concentrations of 800Do not cite

Appendix F

- Page 673 of 677 Guideline No./ Study Type MRID No. (year)

/Doses Results

UDS assay
0, 7.4, 22.2, 66.6, 200,

400, 800 μg/mL μg/mL in primary rat hepatocyte cultures.Do not cite

Appendix F

- Page 674 of 677 F. 8.1.5 Toxicity Table for G

- 30033. All available acceptable toxicity

studies are shown.

Guideline No./ Study Type MRID No. (year)
/Doses Re sults

870.100

Acute oral toxicity 43013202 (1991)

500, 2000, 3500, 5050 mg/kg LD 50 for both sexes combined= 1111 mg/kg

870.3100

90

- Day oral toxicity rodents 43013206 (1991)

0, 10, 50, or 500 ppm 0, 0.68, 3.2, and 35.1 mg/kg

- male 0, 0.72, 3.3, and 38.1 mg/kg /day

- female NOAEL = 3.2 mg/kg/day LOAEL = 35.1 mg/kg/day based on decreased body weight of high dose female rats and decreased food efficiency of high

- dose male and
female rats.

870.3100

90

- Day oral toxicity in

nonrodents (dog) 43013203 (1992)

0, 15, 100, or 1000

ppm 0, 0.56, 3.71, 28.85 mg/kg/day

- males
0, 0.51, 3.88,32.18 mg/kg/day

- females NOAEL = 3.7 mg/kg/day LOAEL= 28.9 mg/kg/day based on renal tubular hyper plasia/basophilia in 3/4 male and 2/4 female dogs

870.3700a

Prenatal developmental

in rodents 43013209 (1992)

0, 5, 25, or 100 mg/kg/day Maternal NOAEL = 5 mg/kg/day

Maternal LOAEL = 25 mg/kg/day based decreased food consumption, body weight/weight gain and food consumption

Developmental NOAEL = 25 mg/kg/ day Developmental LOAEL = 100 mg/kg/day based

on increased fetal and litter incidences of fused

sternebrae 1 and 2 and increased fetal incidence of poor ossification of the proximal phalanx of posterior digit 5 (a skeletal variation)

870.5100

Bacterial

reverse mutation

assay 43093102 (1991)

0, 313, 625, 1250, 2500, 5000 μg/ml Negative in the Salmonella /Ames and E. Coli WP2uvrA assays up to limit concentration of 5000 μg per plate. Tested in S. Typhimurium in strains TA 98, TA100, TA1535, and TA1537 with and

without metabolic activation

87 0.5375

Micronucleus

assay 43093104 (1991)

0, 120, 240, 480 mg/kg Negative in Tif:MAGF mice treated up to the maximum tolerated dose of 480 mg/kg

870.5550 43093106 (1991) Negative up to cytoxic con centrations of 1000Do not cite

Appendix F

- Page 675 of 677 Guideline No./ Study Type MRID No. (year)
/Doses Re sults

UDS assay 0, 9.25, 27.7, 83.3,

250, 500, 1000 μg/ml μg/mL in primary rat hepatocyte cultures.Do not cite

Appendix F

- Page 676 of 677 F. 8.1.6 Toxicity Table for hydroxyatrazine . All available acceptable toxicity

studies are shown.

Guideline No./ Study Type MRID No. (year )
/Doses Results

870.3100

90

- Day oral toxicity rodents MRID 41293501

(1989)
0, 10, 100, 300, 600

ppm 0, 0.6, 6.3, 18.9, 37.5 mg/kg/day

- males 0, 0.8, 7.4, 22.8, 45.6 mg/kg/day

- females NOAEL = 6.3 mg/kg/day in males and 7.4 mg/kg/day in females LOAEL = 18.9 mg/kg/day in males and 22.8 mg/kg/day in females based on kidney

alterations .

870.3700a

Prenatal developmental

in rodents MRID 41065202 (1989)

0, 5, 25, or 125 mg/kg/day Maternal NOAEL = 25 mg/kg/day Maternal LOAEL = 125 mg/kg/day ba sed on decreased food consumption during the dosing period and enlarged and mottled kidneys. Developmental NOAEL = 25 mg/kg/day. Developmental LOAEL = 125 mg/kg/day based on increased incidence of partially ossified interparietal and hyoid bones and decrea sed fetal body weight.

870.4100a (870.4300)

Combined Chronic Toxicity/ Oncogenicity

– Rat MRID 43532001 (1995)

0, 10, 25, 200, 400 ppm
0, 0.39, 1.0, 7.8, 17.4 mg/kg/day

- males 0, 0.5, 1.2, 9.4, 22.3 mg/kg/day

- females NOAEL = 1.0 mg/kg/day for mal es and 1.2 mg/kg/day for females LOAEL = 7.8 mg/kg/day for males and 9.5 mg/kg/day for females based on gross and histopathological effects in the kidneys

870.5100

Bacterial

reverse mutation

assay MRID 40722304 ( 1988 )

0, 20, 78, 313, 1250, 5000 μg/0.1 ml No increases in revertant colonies in TA 98, 100, 1535, and 1537 Salmonella strains exposed to precipitating concentrations (313 μ g/plate and above) both with and without activation system.

870.5375

Micronucleus

assay MRID 41479401 ( 1988)

0, 1250 , 2500, 5000 mg/ml No increase in micronuclei in mice treated with acute incubated doses up to the limit dose of 5000 mg/mL.

870.5550

UDS assay MRID 40722305 (1988 )

0, 13.9, 41.7, 125, 375, 750, 1500 μg/ml No evidence of unscheduled DNA synthesis wa s found up to the limits of solubility (increasing precipitation from 500 μ g/mL) and at concentrations approaching toxicity (1500 μ g/mL) inDo not cite

Appendix F

- Page 677 of 677 Guideline No./ Study Type MRID No. (year )
/Doses Results

primary hepatocyte cultures treated in vitro

.

870.5550

UDS assay MRID 40888101 ( 1988)

0, 13.9, 41.7, 125, 375, 750, 1500 μg/ml Negative up to the limits of solubility (increasing precipitation from 500 μ g/mL) and severe cytotoxicity (1500 μ g/mL) in human fibroblast cells.

HIARC Report [TXR NO. 0050592]