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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 Protection Agency

Office of Pesticide Programs

Health Effects Division

and

Environmental Fate and Effects Division

in collaboration with the

Office of Research and Development

Presented On:

September 14-17, 2010
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Page 2 of 677

TABLE OF CONTENTS

1. INTRODUCTION ............................................................................. 16

2. MODE OF ACTION (MOA) & EXPERIMENTAL TOXICOLOGY..... 21

2.1 Historical Background..........................................................................................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 Spontaneous 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 EPIDEMIOLOGY & EXPERIMENTAL 

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 Daily 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 Triazine 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 CONSIDERATIONS IN POTENTIAL SENSITIVITY OF 

INFANTS & CHILDREN ......................................................................... 82
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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 EVALUATING WATER SAMPLING 

STRATEGIES AND FREQUENCY OF MONITORING ........................... 93

7.1. Introduction...........................................................................................................93

7.2 General Strategy For Designing a Monitoring Study 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 

Frequencies...............................................................................................................119

7.4.1 Filling in Values Between Samples With Linear or Stair-step Interpolation 120

7.4.2 Artificial Neural Network .............................................................................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. IMPLICATIONS OF MOA & TOXICITY PROFILE ON WATER 

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 THIS APPENDIX.................................................. 157

A.2 EFFECTS ON FEMALES.............................................................. 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.............................................................................................................................161
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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..............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 Report. 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., 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. 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 pituitary 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). Characterization 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 Juliani, C., Silva-Zacarin, E., Santos, D. and Boer, P. 2008. Effects of Atrazine 

on Female Wistar Rats: Morphological Alterations in Ovarian Follicles and 

Immunocytochemical Labeling of 90 kDa Heat Shock Protein. Micron. 39:607-616.179

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 Hydroxyatrazine. 

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 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..........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 Following 

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 period of mammary gland growth. Tox. Sci. 

87(1):255-266............................................................................................................188

A.3 REPRODUCTIVE EFFECTS 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 computerassisted semen analysis (CASA) in vitro. Repro. Tox. 22:508-512. ..........................197
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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 F1F0-ATP synthase and 

inhibits microchondrial 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., 

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.....................................................................................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 

Expression 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 lactational 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). Gestational 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 Atrazine 

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 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..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 2001 ; 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 Atrazine. Reproductive Toxicology. oi10.1016/j.reprotox.2009.12.006.................217

A.4 AROMATASE ............................................................................... 219

Summary: Effects on Aromatase and Steroidogenesis, 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 estrogen receptor-α are involved in the proliferative effects induced by atrazine in 

ovarian cancer cells. Environ Health Perspect. 116(12):1648-55..............................224
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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 Atrazine-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 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.................................................................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 Atrazine. 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 receptors 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 metabolites 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 metabolites 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.............................................................................250
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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. Toxicology 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 monoamine 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 juvenile 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 (Gavage) 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 Devos S, Bosscher K De, et al. 2003. Inhibition of cytokine production by the 

herbicide atrazine – Search for nuclear receptor targets. Biochemical Pharmacology 

65: 303 – 308............................................................................................................265

A.6.4 Devos S, Van Den Heuvel R, et al. 2004. Limited effect of selected organic 

pollutants on cytokine production by peripheral blood leukocytes. Eur. Cytokine Netw: 

15: 145 – 151............................................................................................................266

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. 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 Gq/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. Toxicological 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 stressors 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 Johansson, O. (2010). Combined Exposure of 

Peripubertal Male Rats to the Endocrine-Disrupting Compound Atrazine and PowerFrequency Electromagnetic Fields Causes Degranulation of Cutaneous Mast Cells: A 

New Toxic Environmental Hazard? Arch. Environ. Contam. Toxicol. DOI 

10:1007/s00244-010-9477-6 .....................................................................................273
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A.6.12 Rooney, A A, Matulka R, and Luebke, R. 2003. Developmental atrazine 

exposure suppresses immune function in male, but not female Sprague-Dawley rats. 

Toxicological Sciences 76: 366-375. ........................................................................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 Balb/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 granule 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 parameters. 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 carbamate pesticides. ChemicoBiological Interactions 145: 311 – 319......................................................................278

A.7 CARCINOGENESIS/GENOTOXICITY.......................................... 279

Summary: Carcinogenesis/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 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.......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 Expression 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 after exposure to atrazine: the 

non-dose-dependent increase in homologous recombinational events does not support 

its genotoxic mode of action. Cytogenet Genome Res 104: 315-319.......................289

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

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 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....................................................................296
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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-hydroxypropyl)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 SpragueDawley rats. Cancer Sci. 96(1):19-25........................................................................300

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

A.8 INSULIN/MITOCHONDRIA 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 DOSIMETRY, 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 of 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). Derivation 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 [14C]-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 absorption 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 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................................................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 ..................................................................................................318
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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 LITERATURE REVIEW 

METHODOLOGY ................................................................................. 322

APPENDIX B.2: REVIEW OF EPIDEMIOLOGIC INVESTIGATIONS OF 

NON-CANCER HEALTH EFFECTS OF ATRAZINE EXPOSURE...... 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 rates 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 

system 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 Retardation 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 Ochoa- 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. ―Pesticide 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 Birth 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 (1997)]..................................................................................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-forgestational-age,‖ [Environmental Health Perspectives, 62(6): 400-405 (2005)].........364
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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: SUMMARIES OF BMD ANALYSES OF STUDIES ON LH 

SURGE................................................................................................. 372

C.1 NHEERL STUDY .......................................................................... 372

C.2 MORSETH ONE MONTH LH STUDY ........................................... 381

APPENDIX D.1: CHARACTERIZATION OF ATRAZINE MONITORING 

PROGRAM (AMP) CWS CHEMOGRAPH SHAPES AND MONITORING 

DATA FOR CWS WITH THE GREATEST TCT CONCENTRATIONS.. 440

APPENDIX D.2 SAMPLING FREQUENCY CHEMOGRAPHS............. 442

APPENDIX E. UPDATE FOR SAP....................................................... 582

APPENDIX F. TOXICOLOGY CHAPTER OF THE REREGISTRATION 

ELIGIBILITY DECISION....................................................................... 585

F. 1.0 HAZARD CHARACTERIZATION.............................................. 587

F.2.0 REQUIREMENTS........................................................................ 588

F.3.0 DATA GAPS............................................................................... 590

F.4.0 HAZARD ASSESSMENT ............................................................ 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 ....................................................................................................602

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 Estrous 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-amino striazine; 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- hydroxy - 4(ethylamino)- 6(isopropylamino) - striazine...................................................................................................................643

F.5.0 TOXICITY ENDPOINT SELECTION........................................... 647
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F.5.3 Classification of Carcinogenic Potential............................................................647

F.5.3.1 Conclusions...............................................................................................648

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

F.6.0 FQPA CONSIDERATIONS......................................................... 649

F.6.1 Special Sensitivity to Infants and Children.......................................................649

F.6.2 Recommendation for a Developmental Neurotoxicity Study............................650

F.7.0 REFERENCES (LISTED IN ORDER OF ASCENDING MRID 

NUMBER)............................................................................................. 651

F.8.0 APPENDICES ............................................................................. 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..............................................................................................................676
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LIST OF FIGURES

Figure 5.1: Metabolic Profile of Atrazine......................................................................62

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)64

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)...................................65

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...................................................66

Figure 5.5: Plasma concentration 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 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...............................................................................................69

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 thick 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.................................................................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) and 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 Levels231
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Page 14 of 677

Figure A- 2: Treatment 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-day 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 chemograph (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 chemographs (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 (red 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...............................574
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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: Summary of BMD analyses .......................................................................76

Table 5-3: Conversion of AUC estimates to administered dose ..................................77

Table 6-1: Summary of available atrazine 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: Summary 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 Offspring............................................................................................................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 Chemograph 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), Maximum 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 Averages 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........................................................................................................590
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1. INTRODUCTION

Atrazine – a chlorotriazine herbicide – is currently one of the most widely used 

agricultural herbicides in the United States, with approximately 70 million pounds of 

active ingredient applied domestically per year. The Interim Reregistration Eligibility 

Document (IRED) for atrazine was finalized 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 evaluating 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 collaboration with the Office of Research and 

Development (ORD) and the Office of Water is re-evaluating atrazine and its 

metabolites‘ [deethyl-atrazine (DEA), deisopropyl-atrazine (DIA), and diamino-schlorotriazine (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, 2010. 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 (USEPA, 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 and 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 exposure2. This collaborative project continues; 

an update on the current status is provided in Appendix E to this document3. 

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) analysis 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 infers its chlorinated metabolites (DEA, DIA, and 

DACT) unless otherwise specified.

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

found at http://www.regulations.gov (Docket EPA-HQ-OPP-2009-0851).

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 meeting. The AHS-EPA collaborative project will be discussed in more detail in the upcoming 

2011 SAP meeting on cancer epidemiology studies.
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Section 1 - Page 17 of 677

biological organization. The draft framework relies heavily on existing Agency and OPP 

guidance documents, particularly the Mode of Action and Human Relevance 

Frameworks (U.S. EPA, 1999, 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 Hill criteria like that in the MOA Framework as a tool to 

organize, review, and interpret data (U.S. EPA, 1999, 2005; Boobis et al., 2008; SonichMullin et al ., 2001; Meek et al ., 2003; Seed et al., 2005). 

Briefly, the concept of MOA involves the identification of a biologically plausible 

sequence of key events that are obligatory and quantifiable leading to an adverse 

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

temporal concordance, coherence, and consistency. In this analysis, data from humans 

and experimental in vitro and in vivo studies can be evaluated together for biological 

plausibility, to identify areas of confidence and characterize areas of uncertainty. The 

SAP‘s report from the February meeting became available 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 process. 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 integrative 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 the 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 duration along with key gaps in data or scientific 

information. Specifically 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 Agency with an opportunity to get peer review on a subset of noncancer epidemiology studies; the SAP‘s feedback was useful as the Agency evaluated 

the remaining non-cancer 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 the
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Section 1 - Page 18 of 677

Agency with a peer review of 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. The Agency is 

proposing an approach for performing dose-response assessment and extrapolation

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 analysis 

phase of the atrazine exposure characterization. 

The current document provides a review of new experimental toxicology studies 

available between January 30 and July 15, 20104. Twenty new experimental toxicology 

studies became available during 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 atrazine 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 current 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 20 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 cycles, 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 assessment. 

 

4

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

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

January 30 and July 15, 2010, respectively. 
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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 studies 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 benchmark 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 knowledge 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 health 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 endpoint 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 concern 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 missed by less 

frequent sampling. This is most critical when the Agency is concerned about very shortterm 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 within 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 conservative pairing of exposure 

duration with LH effects as seen in the animal 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 state 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 drinking water monitoring frequency and suggests 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. 
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Section 1 - Page 20 of 677

Section 7 (Approaches for Evaluating 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 for 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 conducted 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 proposes 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-science 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 appropriate 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 frequency may be needed to accurately assess atrazine concentrations.
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Section 2 - Page 21 of 677

 

2. MODE OF ACTION (MOA) & EXPERIMENTAL TOXICOLOGY

2.1 Historical Background

The 2003 human health risk assessment for atrazine is complex and 

incorporated significant data development, 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 rat mammary gland tumor response 

seen in adult cancer studies in 1988 (FIFRA SAP, 1988). At that time, the SAP noted 

that a ―hormonal influence‖ might be an important consideration in the development of 

these mammary gland tumors in adult rats. The Agency 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 its 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-pituitary-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. The Agency concluded, and the April 2010 SAP concurred, that new

research conducted in the last seven years supports the neuroendocrine MOA identified 

in the 2003 IRED and the conclusions regarding the human relevance of the mode of 

action for rat mammary gland tumors. As described in detail in this draft issue paper, 

the 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, the SAP evaluated the available epidemiology studies

suggesting an association between atrazine and prostate cancer in workers at an 

atrazine manufacturing 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 could likely be explained by the increase in Prostate Specific 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 before 
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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 

epidemiologic 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 toxicity in both males and females, neurotoxicity, 

immunotoxicity, mammary gland development, mixtures effects, andrology, aromatase 

and insulin resistance and mitochondrial function impairment. 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 the new literature are provided on 

each of the scientific areas in Appendix A. 

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

system leading to reproductive toxicity as the most biologically plausible and sensitive 

effects attributable to atrazine exposure. These adverse outcomes include 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 presented 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 

conclusively 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. Since 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 effects 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 laboratory (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/degradates (DEA, DIA, DACT, hydroxy-atrazine) on mammary 

gland development in female pups. In the Rayner 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 levels resulted in a delay in 

mammary gland development. In 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. conclude that the mixture study suggests 
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Section 2 - Page 23 of 677

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 research has become 

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

hypothalamic-pituitary-adrenal (HPA) axis manifested as increases 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 hypothesis but that there was insufficient data to make this linkage and 

that the available information was not sufficiently robust for incorporation into 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, 1999, 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 frameworks 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, consistency, 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 have 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 implications 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 (measurable 

parameters) that are thought to underlie the MOA.
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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 association. 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 presentation 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 the 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 likelihood 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 inconsistent 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 preparation 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 detailed information, please 

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

2.3.2 Key Events – Hypothalamic-Pituitary-Gonadal (HPG) Axis

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) described in the 2003 IRED are:

Hypothalamic effects resulting in changes in catecholamine function and 

regulation of the pulsatile release of gonadotropin releasing hormone (GnRH).

Attenuation of the LH surge and disruption of ovarian cycles
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Section 2 - Page 25 of 677

Cessation of ovulation 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

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

., 1996b

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 

(1996b)

‡ NOEL not available

Although 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 hypothalamicpituitary 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 points 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, sexual maturation is the culmination of a 
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Section 2 - Page 26 of 677

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. For instance, decreased LH during puberty 

would lead to insufficient stimulation of the gonads to reduce the circulating hormone 

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 (VO) and preputial 

separation (PPS). 

In addition to identifying the key events in the MOA, a dose-response and 

temporal concordance analysis was also part of the 2003 evaluation. The disruption of 

ovarian cycles has been attributed by multiple investigators to an atrazine-induced 

interruption 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 

observed 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 after 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 the current considerations, the LH surge attenuation occurs at doses ≤ to 

those where these effects were observed (Table 2-2). With respect to temporal 

concordance, attenuation of LH surge precedes changes in estrous cyclicity. One of the 

recent advances in the MOA understanding is that the timing of LH suppression in the 

rat appears 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 endpoints from 

developing rats

Life Stage LH Hormone 

(NOAELa/LOAELb)

Apical Endpoint

NOAEL/LOAEL

Pre-Natal 

(Fetus)

10/70 mg/kg/day; Delays 

in ossification 

10/50 mg/kg/day; Delayed 

PPS

Perinatal 6.25/12.5 mg/kg/day; 

increased prostatitis

Peripubertal

6.25/12.5 mg/kg/day; 

increased prostatitis, 

delayed PPS

25/50 mg/kg/day; delays 
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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 studies have further characterized the cellular and 

neuroendocrine changes responsible for how atrazine interferes with the regulation 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 the 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 activity of GnRH neurons (Kalra and Kalra, 

1983). Research from two laboratories has shown that atrazine interferes with the 

pattern and amplitude of GnRH release by slowing the frequency of the pulses and 

lowering the overall amount of GnRH released into the portal system (Cooper et al ., 

2007; Foradori et al ., 2009). In contrast, the 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; Foradori et al ., 2009b). Further evidence 

supporting this observation comes from the work done on pigs demonstrating that a 

single intravenous injection of GnRH (100 μg) was able to partially reverse or counteract 

atrazine-induced suppression of the LH surge (Gojmerac et al ., 2004). There have 

been some reports indicating that atrazine, or its metabolites, may interfere directly with 

GnRH activity (e.g., Zorrilla et al ., 2010) as well as the activity of other neurons (Das et 

al ., 2001). 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 neuroendocrine 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

effects as the most relevant and sensitive health outcome reported in animals. Because 

human reproduction and development are controlled by the neuroendocrine system, 
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there is the potential that atrazine exposure could lead to adverse health consequences.

Thus, 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 

inform 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 

differences in humans and animals. 
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3. EVALUATION OF NON-CANCER EPIDEMIOLOGY LITERATURE

3.1 Overview

This chapter describes the epidemiology studies that evaluated the association 

between atrazine exposure and adverse non-cancer health effects. To identify articles 

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 

review 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 articles 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 association 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 using 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 epidemiologic analysis 

in performing this review. Among these 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 size 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 consider 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 exposure 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 relevance of the biomarker to 

actual environmental exposures. A detailed review addressing these points, among 

others, for each article can be found in Appendix B-2. Table 3-1 delineates key study 

characteristics. 
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Section 3 - Page 30 of 677

3.2 Literature Review

3.2.1 Reproductive System Effects

Sex steroid hormones regulate the reproductive cycle. As summarized in Section 

2, there is evidence that atrazine may disrupt steroid hormone synthesis and signaling. 

Therefore, epidemiologic researchers have 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 including 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 pesticide 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 missed 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 maneb/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  ̳hormonally 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 compared 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 intermenstrual 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 atrazinespecific risk estimates in this 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) in 
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association 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 

tetrachlorine, 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 

authors 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 diabetogenic, 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 use 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 crops 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, agricultural 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 depiction 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 affect 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 with 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 this 
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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 of menopause, Farr et al . (2006) conducted a welldesigned 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 endpoint. 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 association 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 noncausal 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 understand 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 without 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 National Institute of Environmental Health Science 

(NIEHS). 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)). This study included use of biomarkers of both 

semen quality and pesticide 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 exposure 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, disallowing meaningful exposure-response 

assessment. Researchers indicated that the proportion of men reporting occupational 

pesticide use, recent pesticide use, and residential proximity to treated field were 

comparable between men with and without poor semen parameters. However, 

researchers were not able to fully adjust for the co-exposure to other environmental 
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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 poor semen 

parameters. Larger prospective studies with the ability to account for multiple 

environmental exposures are needed to clarify the potential association between 

atrazine use and semen parameters. 

3.2.2 Fetal and Perinatal Outcomes

As summarized in Section 2, published reports indicate that atrazine 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 20

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 available 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 and 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 further 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 risk 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. 
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Participants reported using several different pesticides within the time period of 

susceptibility. 

In a different study, Savitz et al. (1997) estimated the association between preconception male pesticide exposure and 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 preconception period. A major limitation of the study is 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 exposure misclassification (likely nondifferential 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 literature 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 assessed 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 hypothesize 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). Gastroschisis and omphalocele, 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 (Canfield 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 factors including smoking, alcohol use, 

and medication use have also been epidemiologically associated 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 analysis 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 greater 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 in 
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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 comparability. This limitation may have resulted 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 exposure. 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 different study designs.

Hornemann et al . (2008) investigated the prevalence of omphalocele in 

association with atrazine exposure in a part of northern 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 prevalence 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. Therefore, 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 association 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. Overall the study provides 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 exposure 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) 

looked 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. 

natality 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 date of conception 

during 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), muscular 
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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-statistically 

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 rates 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 defects 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 observed 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 an 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 association.

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 builds 

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 

ascertain 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 measure. 

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. Although the study design, population-based case-control 

study, is generally considered more targeted than ecologic study designs, the method of 

exposure assessment Waller et al . was pseudo-ecologic. Therefore, uncertainty 

remains as to whether mothers of neonates diagnosed with gastroschisis were exposed 
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to atrazine (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, presents uncertainties in the interpretation of these data. 

The five studies investigating atrazine exposure in association with birth defects, 

including abdominal wall defects, 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 which 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 utilized 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 ascertained 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 states (Watkins et al ., 1996). For studies in which 

individual-level confounder assessment was possible (Waller, Hornemann, and OchoaAcuna), researchers were able to measure most relevant demographic characteristics;

however, no study was able to investigate 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 environmental 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 (<10th percentile), preterm delivery (<37 

weeks), low birth weight (<2500 grams), and intra-uterine growth retardation, as well as 

another population birth characteristic, male to female sex ratio. Given the suspected 

effect atrazine has on the hormonal milieu, authors hypothesize that these birth 

outcomes could potentially relate to maternal or paternal 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 exposure for certain adverse effects, and then 
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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 investigated 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 concentration 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 enhancement 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 the 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 notable. 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) and 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 (MayOctober in this study [France]), 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 on the farm on which both male and female partners 

live and work (study based upon sample in Poland). 
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A recent publication using AHS data reported no association between maternal 

pesticide activity (indirect agricultural exposures, pesticide use in the home, 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 pregnancy) (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 same 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, smallfor-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 of crop- or yardpesticides and atrazine exposure by male partners during the three months prior to 

conception; however, 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 yardherbicides likely 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. 

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

the case-control and cohort studies, authors assessed exposure through either selfreport questionnaire (Savitz et al ., 1997 and Dabrowski et al . , 2003) or drinking water 

exposure measurement (Villanueva et al ., 2005 and Ochoa-Acuna et al., 2009). 

Uncertainties 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 

exposure in relation to the period of susceptibility 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 relatively low 

statistical power. Birth outcomes were ascertained through either self-report or birth 
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records, which are generally considered to be accurate with respect to birth weight, and 

moderately valid and consistent reporting of gestational age. None of the 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 with 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 within the AHS; however, atrazine or triazine exposure was not 

considered among the a priori hypotheses posited by these authors. Participants selfreported 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 significant 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 association; 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 association (Hoppin et al., 2009). Additional studies are 

needed to better understand and characterize the potential respiratory effects of 

atrazine use.

An additional study investigated the potential effect atrazine or triazines could 

have upon respiratory health, however this study includes several limitations the 

greatest of which is the inability to isolate the effect of atrazine in relation to respiratory 

health. Kossman et al . (1997) evaluated several respiratory parameters including 

pulmonary ventilation 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 manufacture 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 plant 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 plant 
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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, effect atrazine or triazines may have on this outcome. 

Among a similar occupational cohort as 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 assessment 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 population 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 respiratory 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., possibly 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, include 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 

able to make a firm cause to effect linkage 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 exist 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 observed 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 without 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 been found to be reliably selfreported in epidemiologic analysis, several studies identify birth defects and measure 
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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 

studies 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 

exposure; 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 exists as to the extent the authors were able to 

correctly 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 

overlaying 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 OchoaAcuna et al., (2009) measured atrazine concentration in drinking water in association 

with maternal residence 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 is 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 using 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 authors. Swan et al. (2003) utilized urinary concentration of atrazine 

mercapture (AM), a biomarker of atrazine exposure. Biomarkers are generally 
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Section 3 - Page 43 of 677

considered superior measures of exposure. However, recent publication indicates use 

of only AM as a biomarker of atrazine 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 understanding 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 as 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 performed 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. While 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 specifically, 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 excluded 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 of 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 

of 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 and 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 these 
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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 exposure 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 statistical 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 reported direct, agricultural exposure during 

the first trimester of pregnancy. Two studies report an increased probability of small-forgestational age and the authors posit atrazine later in pregnancy, during the third 

trimester, may be an important exposure window to consider in future research.

Consistent 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 studies. 

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 essentially 

at the aggregate level. However, this study warrants additional follow-up. Mattix (2007) 

reports rates of this outcome are higher in Indiana, a high atrazine use state, than the 

rest of the U.S. However, given the relatively weak design of these studies, 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, wellconducted hypothesis generating studies, and warrant additional follow-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 quantitative 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 design, 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. 
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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/Spontaneous Abortion

Arbuckle 2001

Nested CaseControl 

(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 

exposure

Birth Defects

Waller 2010

Population 

based casecontrol 

(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 

defects [AWD] 

(U.S., state birth 

registry)

Atrazine 

concentration in 

surface water 

(USGS)

n/a

AWD rate 

U.S.: 2.75/105 

v. AWD rate 

IN: 3.75/105; 

correlation 

Hypothesis 

generating 

study; 

supports 

additional 

Limits of study 

design; underreporting of 

outcome 

possible
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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/Spontaneous Abortion

monthly 

atrazine and 

AWD, 0.69 

(IN), 

p=0.0125

research

Hornemann 2008

Populationbased casecontrol [N. 

Germany] 

(N=66/28,935)

Sonographic 

diagnosis 

omphalocele 

(medical records)

Season of 

conception Unadjusted

No 

association 

season and 

omphalocele; 

no 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

Atrazine 

concentration in 

surface water 

(USGS)

Maternal age, 

alcohol, 

smoking, 

diabetes, year of 

birth

Higher rates 

of 11/22 birth 

defects if 

Spring 

conception

Hypothesisgenerating 

study; 

additional 

research 

necessary

Limits of study 

design; underreporting of 

outcome likely
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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/Spontaneous 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) per 10 

hectacres 

from field

Hypothesisgenerating, 

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 2009

Retrospective 

cohort 

(n=several 

hundred 

cases/24,154 

live births)

Small-forgestational age 

(SGA), pre-term 

delivery (State 

birth registry)

Drinking water 

concentration 

atrazine, four 

data sources, 

time series 

analysis

Maternal health 

behavior and 

demographic 

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 

unknown
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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/Spontaneous Abortion

Villanueva 2005

Retrospective 

Cohort 

(n=3,510)

Preterm delivery, 

SGA, low birth 

weight (LBW) 

(regional birth 

registry)

Drinking water 

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, 

preterm 

delivery OR 

1.36 (95% CI 

0.95-1.95))

Drinking 

water 

atrazine 

concentratio

n measured; 

subanalyses 

informative 

to possible 

critical 

window

Exposure 

measurement 

error possible; 

narrow 

exposure range

Munger 1997

Ecologic 

(N=several 

hundred 

cases/6,500 live 

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)

Atrazine, 

cyanazine 

concentration 

correlated 

IUGR, 

associated 

IUGR (multivariable 

model)

Hypothesisgenerating 

study; 

additional 

research 

necessary

Limits study 

design
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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/Spontaneous Abortion

Sathyanaraya

na

2010 Cross-sectional, 

AHS (N=2,246 )

Birth weight 

(maternal selfreport)

Questionnaire 

categorized 

(None, indirect 

(e.g., planting), 

residential, 

agricultural); 

Lifetime everuse atrazine

Pregnancy 

duration, 

maternal prepregnancy body 

mass index 

(BMI), maternal 

height, parity, 

state, and 

maternal 

smoking

No 

association; 

nonsignificant 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

Populationbased casecontrol 

(N=117/377)

LBW, preterm 

delivery (medical 

records)

Pesticide 

exposure 

(questionnaire; 

validated)

Infant sex, 

maternal prepregnancy 

weight, height, 

maternal 

smoking, year of 

birth, 

involvement in 

field work

Triazine 

observed/exp

ected (O/E), 

LBW: -259g 

(p=0.43); O/E 

pregnancy 

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 Reproductive
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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/Spontaneous Abortion

Saldena 2007 Cross-sectional 

(n=11,273)

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 exposure 

referent) 2.2 

(95% CI 1.5, 

3.3); 

increased 

odds GDM 

with lifetime 

ever-use 

atrazine 

among direct 

users 

(graphical 

depiction 

only)

Hypothesisgenerating 

study; 

additional 

research 

necessary

Lacks atrazine 

exposure 

measure during 

pregnancy; 

multiple 

statistical 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]

Hypothesistesting, 

hormonally 

active 

pesticides; 

large sample 

size; 

analysis 

appropriate 

to study 

Design

Outcome 

misclassificatio

n possible; lack 

exposure 

measure at 

relevant 

lifestages
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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/Spontaneous Abortion

Farr 2004

Cross-sectional 

analysis of AHS 

data (N=8,038)

Menstrual cycle 

length (>36 days, 

<24 days, 

normal) irregular 

cycle, missed 

period, intermenstrual 

bleeding 

(Questionnaire)

Atrazine or 

lindane use; 

lifetime days of 

use of any 

pesticide (0, 1-9 

days/yr, 10+ 

days/yr)

Age, smoking, 

BMI

Atrazine 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)

Hypothesistesting; large 

sample size; 

analysis 

appropriate 

to study 

design

No atrazinespecific 

estimate; 

potential 

residual 

confounding 

physical 

activity; 

information comorbid 

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; 

analysis 

appropriate 

to study 

design

Small sample; 

atrazine 

biomarker 

under-estimates 

total atrazine; 

generalizable to 

healthy, white 

men only
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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/Spontaneous Abortion

Savitz 1997

Case-control 

analysis 

[Ontario Farm 

Family study] 

(N=3,984)

Preterm delivery, 

SGA, 

miscarriage, 

secondary 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 offfarm job, 

father's off-farm 

job, maternal 

smoking, 

maternal 

alcohol, time 

period 

conception to 

interview 

interval

OR 

miscarriage: 

crop-/yardherbicides 

1.2-1.5 (n.s.); 

OR preterm 

delivery: crop-

/yardherbicides 

2.4-4.9*(s.s.); 

OR SGA: 0.5 

(n.s.); OR sex 

ratio: crop 

herbicide 0.8 

(n.s.)

Response 

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); 

exposureresponse by 

category of 

annual days 

of use 

Hypothesisgenerating; 

additional 

research 

necessary

Multiple 

statistical tests 

performed; no 

biologically 

plausible 

mechanism 

proposed
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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/Spontaneous 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 

employment, 

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 

database

Inadequate 

atrazine 

exposure 

measurement

Kossman 1997

Populationbased 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

Hypothesisgenerating 

study; 

additional 

research 

necessary

Inadequate 

atrazine 

exposure 

measurement
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Section 3 - Page 54 of 677
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Section 4 - Page 55 of 677

4. INTEGRATION OF EPIDEMIOLOGY & EXPERIMENTAL TOXICOLOGY 

Although typically the Agency‘s human health risk assessments 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 mode 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 atrazine, 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 laboratories 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 availability 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 either 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, regardless 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 differences 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 disrupts the 

regulation 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., 1996 & 

2000, Morseth, 1996b, 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 al., 2004; 

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, Farr 
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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 ratio 

(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., 2003; 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 with lindane toxicity; 

this may or may not explain this finding.

In contrast to the findings on estrous cyclicity disruption where rodents and 

humans 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 aging and dampening of the 

GnRH pulse. Given these differences 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) among women that ever used (i) any of the pesticides in the AHS 

study, (ii) hormonally active pesticides5in 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 menopause delay (i.e., Were the women exposed 

consistently over their lifetimes or only once or twice?, How much atrazine were these 

women potentially exposed to over their lifetime?, Were the women only exposed years 

prior to any changes in menopause?). In 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.
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Section 4 - Page 57 of 677

the menstrual cycle disruptions reported by Farr et. al are associated with atrazine 

exposure. 

Changes in andrology parameters (sperm parameters and male reproductive 

performance) have also been investigated in regards to atrazine exposure (See 

Appendix A). The dataset for these types of studies is sparse relative to the studies on 

the female reproductive system. Experimental data on these parameters come 

primarily from in vitro studies using either porcine or human sperm. Two studies used 

porcine sperm to evaluate sperm viability, motility, 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 effect 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 consideration. 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 researchers 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 pesticide 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 effects 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 humans) 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; Villanueva 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 outcome or exposure is possible using 

these methods. Authors reported a 20-50% increase in the prevalence of SGA (Ochoa-
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Section 4 - Page 58 of 677

Acuna et al., 2009; Villanueva et al.,2005); while Savitz et al. (1997) observed a 50% 

decreased risk. 

For low birth weights, the evidence of a correlation with atrazine exposure is less 

compelling in that results are either based upon sub-analyses including small numbers 

(Villanueva et al.,2005), 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 significant 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 epidemiology database include abdominal wall 

defects (omphalocele 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 conducted 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 atrazine. 

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 female 

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. Therefore, 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. However, 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 and 
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Section 4 - Page 59 of 677

developmental outcomes, as noted above, 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 instances the inability 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 epidemiology 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 important 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-fold 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. 
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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 exposures, short- and intermediate 

incidental oral exposures, and short-, intermediate-, and long-term dermal and 

inhalation exposures. Effects observed in developmental toxicity studies (i.e., delayed 

ossification of certain cranial bones in fetuses, and decreased body weight gain in 

adults that are not likely due to atrazine‘s MOA but rather to general toxicity) were 

selected as the endpoints 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 of 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 

benchmark 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 

chemical species (e.g., parent chemical, metabolites, or both) represents an important 

step for linking exposure to response. In the case of atrazine, perturbations of the 

neuroendocrine system resulting in attenuation of the hypothalamus-controlled preovulatory LH surge (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 

disrupt 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). 
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Section 5 - Page 61 of 677

5.2.2 Data Supporting the Use of Internal Dose in Dose Response 

Assessment

 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 direct 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, atrazine 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 chlorinated 

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.

Glutathione conjugation

Deisopropylatrazine (DIA) Deethylatrazine (DEA)

Atrazine

Diaminochlorotriazine (DACT)

CYP450

CYP450

Dealkylation Dealkylation

Dealkylation Dealkylation

Glutathione conjugation

Deisopropylatrazine (DIA) Deethylatrazine (DEA)

Atrazine

Diaminochlorotriazine (DACT)

CYP450

CYP450

Dealkylation Dealkylation

Dealkylation Dealkylation
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Section 5 - Page 62 of 677

Figure 5.1: Metabolic Profile of Atrazine

 

Studies examining more directly the contribution of individual triazine species 

(atrazine and its metabolites) in attenuating the LH surge also suggest 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 monodealkylated 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 Modeling 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 pharmacokinetic 

(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 . (2007) 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 

compartment (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 (McMullin et al ., 2003). It should be noted that although red blood cells 

were modeled as a potential site for bioaccumulation, no toxicity related to red blood cell 

dysfunction has been reported to date. Although a substantial amount of data was 

used for model 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).
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Section 5 - Page 63 of 677

Because there is no robust pharmacokinetic model available and a measure of 

internal dose is warranted to account 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 Area 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) attenuated 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, 200, 

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 risk 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 concentration-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. 
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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)

Time (h)

Dose (mg/kg)

Time (h)

Dose (mg/kg-d)
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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 State AUC for Triazine Equivalents 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 oscillate 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 dosed 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 elimination half-life of the 

radiolabel (Thede, 1987). Thus, following the rough approximation that 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 of 

Vehicle 1.56 3.12 6.25 12.5 25

0

5

10

15

20

25

Atrazine (mg/kg-d)

 Leutinizing Hormone

Serum (ng/ml)
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Section 5 - Page 66 of 677

daily dosing. In the case of the high doses (50 and 100 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 the 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 linear 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 postdosing), 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.

0 50 100 150 200 250 300 350 400 450 500 550 600

0

200

400

600 Pseudo Steady State

Hypothetical

Elimination Phase

{

time (hr)

Serum Levels
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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).

ATRA dose (mg/kg/day) Elimination rate constant (hr-1)

1 0.010

3 0.015

7 0.013

10 0.014

50 0.376

100 0.308

Table 5-1: Estimated elimination rate 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 plasma levels by 24. 

If steady state is a condition under which the LH surge is attenuated by triazine 

0 24 48 72 96 120 144 168 192 216 240 264 288

0

2

4

6

20

40

60

1 mg/kg bw ATRA

3 mg/kg bw ATRA

7 mg/kg bw ATRA

10 mg/kg bw ATRA

50 mg/kg bw ATRA

100 mg/kg bw ATRA

pseudo-steady state Daily dosing stopped

time (hrs)

Plasma radiolabeled equivalents

 (mg/L)
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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 between 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 doses 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 beginning 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 doseresponse 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.

0 20 40 60 80 100

0

2500

5000

7500

10000

12500 y= 116.4x - 1.67

R

2

 = 0.999

ATRA dose (mg/kg/day)

AUC (mg/L*h)
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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.

0 20 40 60 80 100

0

10

20

30

40

50

60 y= 0.541x - 0.357

R

2

 = 0.994

ATRA dose (mg/kg/day)

pseudo-steady state

serum levels (mg/L)

0 20 40 60 80 100

0

250

500

750

1000

1250

1500

y= 13.0x - 8.57

R

2

 = 0.994

ATRA dose (mg/kg/day)

pseudo-daily

steady state AUC

(mg/L-h)
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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 Equivalents 

The implications of reaching a pseudo steady state are significant from the 

standpoint 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 

constant 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 achieved. 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 regardless 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 established 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-day 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 LOAELs 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 of the LH surge seems to be reached at end of the 4-d doseresponse curve. 
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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 thick 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.

 In 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 and 

0 5 10 15 20 25 30

0

25

50

75

100

125

NOAELs

LOAELs

Morseth 1996 - 6 month study (Sprague Dawley)

Cooper 2010 - 4-day study (Long Evans)

McMullin 2004 - 5-day study (Sprague Dawley)

Minnema 2001 - 1 month study (Sprague Dawley)

{

{

ATRA dose (mg/kg/day)

LH Surge Attenuation

(% control)
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Section 5 - Page 72 of 677

thus were omitted. 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 atrazine 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 equivalents, 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 Minnema (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 studies. Thus, although 

repeated daily dosing continued for much longer in the 1- and 6-month studies, the LH 

surge attenuation did not increase and remained fairly constant.

The remarkably similar effects seen across studies from different laboratories 

involving different durations (4-days, 5-days, 1-month, and 6-months), modes 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 exposure

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 primarily 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 oral administration, steady state 
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Section 5 - Page 73 of 677

appears to be an 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 points 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 PoD. 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, the only BMD 

analyses provided here and in Appendix C relate to LH attenuation. Although, it would 

be preferred to model additional endpoints, as shown in Table 5-2, the other outcomes 

reported following atrazine exposure are less sensitive than 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 (the 

BMDL corresponds to the 95% lower bound 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, 
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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/concurrent controls) and statistical 

considerations (sample size, variability, etc). However, in 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 and 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 including but not limited to the 

life-stage and functional outcomes under consideration (e.g., estrous 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 surge 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 reproductive 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 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. 

Based 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 surge 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 clearly 

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 metric for atrazine including administered 

dose and average daily steady state AUC. 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 pre-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 the 
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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 Critical Studies 

Several oral toxicity studies in the rat provided LH data that were considered for 

dose-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 deviations). 

A one month oral study by Morseth et al . (1996a) 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 . 1996b) 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 calculated 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 evaluate 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 exponential, Hill, 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-
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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 Month 

(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 internal 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), and 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. 
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Section 5 - Page 77 of 677

The estimates 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. The 

Hill model provided the best fits for both AUC internal dose metrics; these results are 

presented in Figure 5.10 for ―area under the triazine equivalent plasma-concentration 

time curve‖, and Figure 5.11for ―daily steady state AUC for triazine equivalents.‖ The 

following table summarizes BMD and BMDL estimates 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. Applying 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 is comparable to the 

value (i.e., 1.96 mg/kg/day) estimated with administered dose as the dose metric.
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Section 5 - Page 78 of 677

 5

 10

 15

 20

 25

 0 10 20 30 40 50 60 70

Mean Response

dose

Exponential Model 4 with 0.95 Confidence Level

14:07 07/20 2010

BMDL BMD

 

Exponential

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. 
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Section 5 - Page 79 of 677

 5

 10

 15

 20

 25

 0 2 4 6 8 10 12

Mean Response

dose

Hill Model with 0.95 Confidence Level

12:15 07/30 2010

BMDL BMD

 

Hill

Figure 5.10: EPA BMDS Hill Model (Constant Variance; BMR = 1 SD from 

control mean) Results for ―Area Under the Triazine Equivalent PlasmaConcentration Time Curve‖ as the Dose Metric - NHEERL Data 

(Attenuation of LH Surge in Female Rats Administered Atrazine by 

Gavage). 
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Section 5 - Page 80 of 677

 5

 10

 15

 20

 25

 0 50 100 150 200 250 300

Mean Response

dose

Hill Model with 0.95 Confidence Level

12:45 07/30 2010

BMDL BMD

 

Hill

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). 

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 total triazine equivalents 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 such, 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 of 
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Section 5 - Page 81 of 677

1.86 mg/kg/day (based on AUC dose metrics) is proposed as a point of departure (PoD) 

for atrazine risk assessment for repeated exposure scenarios. Possible approaches for 

extrapolating this animal PoD to humans is discussed in Section 8 below.
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Section 6 - Page 82 of 677

6. SCIENTIFIC CONSIDERATIONS IN POTENTIAL SENSITIVITY OF INFANTS & 

CHILDREN

6.1 Background 

The FFDCA, as amended by the FQPA (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 and 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 (including 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 from 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 assessing 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 throughout 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, 
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Section 6 - Page 83 of 677

several new studies in developing rats have been conducted. Generally, 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 during 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 considered 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 dosimetry 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 tissues 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/day. In contrast, Rayner et al. (2007), 

observed no change in the timing of male puberty, but did report a higher incidence in 

prostatitis at 100 mg/kg/day. 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 Prostatitis
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Section 6 - Page 84 of 677

(2007)

Table 6-2 a &b: Extracted from Stoker et al. (2010) 
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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 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. A 100 mg/kg atrazine group was used as a positive control. The 

authors 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 preclude 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 plays 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 studies 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 concentrations 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 higher fat content of milk at PND3 

compared to PND10, different analytical techniques, across laboratory variability, or 

other factors.
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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 14CAtrazine 0.658 ± 0.37

4 mg 14CAtrazine 1.172 ± 0.65

Brain

2 mg 14CAtrazine 0.013 ± 0.0002

4 mg 14CAtrazine 0.020 ± 0.0002

Table 6-4: a & b. Extracted from Stoker et al. (2010)
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Section 6 - Page 88 of 677

Table 6-5: Summary of available atrazine experimental toxicology studies 

from the lactational periods

AUTHOR (YR) EXPOSURE NOAEL/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 crossfostered pups on PND 1 to/from atrazine or vehicle exposed dams to create four groups 

(control-control, atrazine-control, control-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 exposure
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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 pups 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 offspring. 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 reported. 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/day 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 number 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 important 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 sensitivity, 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 peripubertal 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 PPS
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Stoker et al ., 2001 PND 23 to 

53 25/50 Decreased 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 in the literature are pertinent to the 

consideration of the FQPA Safety Factor. Epidemiologic 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 estrous 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 everusing atrazine or lindane in their lifetime (Farr et al., 2004). In a similar study 

population, researchers 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 validated exposure 

information (ever-use in a lifetime), relatively large sample sizes, and adjustment for 

potentially confounding variables including physical activity. However, given the data 

available to these researchers, neither of these studies was able to definitively 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) among 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 necessary.

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 

drinking 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., 2009; 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 other 
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birth outcomes is generally weak at this time, and study results are inconsistent 

(Munger et al., 1997; Sathyanarayana et al., 2010; Dabrowski et al., 2003; 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 al.,

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., 2010). Uncertainty 

exists as to whether these estimates reflect true increased risk or are the result 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 association 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 atrazine 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 lactation, 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 findings. 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 in goats and cows which show that 2% and 2.7% 

respectively of the atrazine 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 1.86 mg/kg/day derived from LH data 

collected after four days 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 et 
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al., 2007). This mixture study suffers 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 

potential 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 

combination 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; however, 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 design, 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 not 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 incomplete 

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. 
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7. APPROACHES TO EVALUATING WATER SAMPLING STRATEGIES AND 

FREQUENCY OF MONITORING

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, 2010), 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). The 90-day 

rolling average corresponded to: (i) the period within the year during which most peak 

concentrations are likely to occur; and (ii) the most sensitive effect (LH surge 

attenuation) occurring 4-5 months after daily dosing in a 6 month toxicity study. The 

registrant, Syngenta, employed a monitoring 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 atrazine 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 shorter 

duration of exposure for the toxicological endpoint of concern. If the re-evaluation 

determines that a shorter duration of exposure is more appropriate for atrazine effects 

on humans, the USEPA must consider how well the existing AMP monitoring design 

characterizes shorter durations of exposure and whether the monitoring program needs

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, how frequently 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, artificial neural networks, kriging/random function models, 

regression-based models and deterministic models. While the background document 
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Section 7 - Page 94 of 677

presented some exploratory analyses using the weekly chemographs from the AMP 

program, the USEPA proposed to continue exploring different exposure estimation 

methods using 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 methods 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, smoothing out the variability and providing a 

biased 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 for 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 appropriateness 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 that the minimum 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 design must also consider the tradeoffs between the 

cost of improved reliability and accepting probability-based regulatory actions based on 

estimates that may have lower precision. For new monitoring studies, the SAP 

recommended considering autosamplers that 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 

peak concentrations over shorter exposure durations are important, the USEPA needs

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 recommended exploring the possibility of combining regressionbased models, such as WARP, with kriging or random-function models. The panel also 

suggested exploring extreme value theory, especially for rolling averages. 

This section outlines 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 analyze 
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Section 7 - Page 95 of 677

varying sampling strategies and methods for estimating exposures from less frequent 

monitoring data. The chapter 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 exposure. 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 monitoring study takes 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. An acute or short-term exposure duration will require more frequent sampling

to reliably characterize exposures than a longer-duration exposure period. Because it is 

not possible to measure daily concentrations of pesticides at all places across multiple

years to capture the full extent of pesticide occurrence, the monitoring study must 

balance the frequency 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 periods 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 Monitoring 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 2nd 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. 
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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 

highest 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 sorghum 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:
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Section 7 - Page 97 of 677

(1) Target Monitoring to the Most Vulnerable Areas: The SAP recommended 

using a model like WARP to identify the most vulnerable areas at a watershed 

scale that is appropriate for water bodies 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 representative 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 Intensive Sampling to the High Occurrence Period: 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 beginning 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 runoff. The duration of this period will depend on 

application patterns and the persistence and mobility of the pesticide. 

(3) Base Sampling Frequency on the Toxicological Exposure Duration of 

Concern: As noted in the April 2010 SAP, a time-averaged concentration 

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 uncertainty 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 Exposure Periods of Interest: 

Consider using autosamplers as a supplement or an alternative to grab samples, 

particularly 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 period 
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Section 7 - Page 98 of 677

of concern, potentially reducing costs 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 adequately 

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 

samples 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 frequently-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 the 

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 

USEPA 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 from 

rivers or streams, and 4 from combined (reservoirs and rivers) sources. In 2 CWS (OH14 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 

analysis, 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, shorter-duration peaks 

between sampling events may have been missed. Table 7-1 summarizes 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 peaks 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). 
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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 date and magnitude of peak concentration for each of the CWS years. The 

patterns, illustrated in the CWS graphs in Appendix D.1, show that where multiple TCT 

peaks were detected in a given year, the majority of peak profiles (rise, peak, and 

decline) overlapped. 

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 for the 44 CWS with TCT detects of 15 ug/L or greater 

(see Appendix D.1). Little 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 TCT 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 sampling, 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:
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Section 7 - Page 100 of 677

(1) No treatment effect is evident: 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 and 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, IN06, IN-12 (2004-05), KS-07, KS-19, KS-20, KS-22, KS-27, LA-01, OH-06, OH-09 

(except 2009), OH-12. 

IL-24, 2004-05

0

5

10

15

20

25

30

35

40

45

50

J-04 M-04 M-04 J-04 S-04 N-04 J-05 M-05 M-05 J-05 S-05 N-05

Date

TCT, ug/L (ppb)

TCT Source

TCT Treated

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 concentrations 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 levels 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 OH21.
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Section 7 - Page 101 of 677

IA-07, 2004-05

0

10

20

30

40

50

60

J-04 M-04 M-04 J-04 S-04 N-04 J-05 M-05 M-05 J-05 S-05 N-05

Date

TCT, ug/L (ppb)

TCT Source

TCT Treated

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 treatment: These could be CWS that use activated carbon, but not 

enough to be fully effective, and/or blend water from multiple sources. The 

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. 
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Section 7 - Page 102 of 677

IN-13, 2004-05

0

5

10

15

20

25

30

J-04 M-04 M-04 J-04 S-04 N-04 J-05 M-05 M-05 J-05 S-05 N-05

Date

TCT, ug/L (ppb)

TCT Source

TCT Treated

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 

address the effects of drinking water treatment separately on a case-by-case basis in 

the risk assessment process. To evaluate monitoring sampling 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 drinking 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 sampled water 

monitoring data for analyses. 

7.3.3 Intensively Sampled Datasets To Evaluate Sampling Strategies and 

Exposure Estimation Methods
Do 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 reflective 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 CWS 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 monitoring 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 analysis 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 Chemograph Shapes: 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 published 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 Heidelberg 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 Model/Exposure Analysis 

Modeling System8) to Generate Chemographs for Static Water Bodies: 

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 daily 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#aquatic
Do 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 1980s9. 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, including 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 across 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 over 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 found 

at http://www.heidelberg.edu/WQL .
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Section 7 - Page 105 of 677

(a) Actual Chemograph (red arrows point to spikes)

Maumee River, 2008

0

10

20

30

40

50

60

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill), avg

4-day avg

(b) Sampled at 7-day fixed intervals, beginning on Day 2

Maumee River, 2008: 7-Day Sample Interval #2

0

10

20

30

40

50

60

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

Measured 4-day avg

7-da interval 2

linear 7-da interval 2

4-day avg, linear 7-da int 2
Do not cite

Section 7 - Page 106 of 677

(c) Sampled at 7-day fixed intervals, beginning on Day 6

Maumee River, 2008: 7-Day Sample Interval #6

0

10

20

30

40

50

60

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

Measured 4-day avg

7-da interval 6

linear 7-da interval 6

4-day avg, linear 7-da int 6
Do 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 effectively 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 the 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

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

Maumee 

River, 

2005

Actual 15 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 

River, 

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 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 

River, 

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 0
Do not cite

Section 7 - Page 108 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 7 5 9.6 1 8.4 0 0

Maumee 

River, 

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 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

Sandusky 

River, 

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 

River, 

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

Sandusky 

River, 

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 

River, 

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
Do not cite

Section 7 - Page 109 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 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 

River, 

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 sampling beginning on the 1st through 7th 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 2nd to 5th 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 subset 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, and 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 available 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 extremes 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 actual 
Do not cite

Section 7 - Page 110 of 677

peaks in a chemograph 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 chemographs (red) 

sampled at 7-day fixed intervals, MO-01, 2007 (Data Source: AEEMP)

(a) Actual Chemograph (red arrows point to spikes)

MO-01, 2007

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

4-day avg
Do not cite

Section 7 - Page 112 of 677

(b) Sampled at 7-day fixed intervals, beginning on Day 4

MO-01, 2007: 7-Day Sample Interval #4

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

Measured 4-day avg

7-da interval 4

linear 7-da interval 4

4-day avg, linear 7-da int 4
Do not cite

Section 7 - Page 113 of 677

(c) Sampled at 7-day fixed intervals, beginning on Day 6

MO-01, 2007: 7-Day Sample Interval #6

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

Measured 4-day avg

7-da interval 6

linear 7-da interval 6

4-day avg, linear 7-da int 6

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)
Do not cite

Section 7 - Page 114 of 677

(a) Actual Chemograph (red arrows point to spikes)

MO-02, 2006 adjusted for flow

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Flow Adjusted

4-day avg
Do not cite

Section 7 - Page 115 of 677

(b) Sampled at 7-day fixed intervals, beginning on Day 1

MO-02, 2006: 7-Day Sample Interval #1

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Flow Adjusted

7-da interval 1

linear 7-da interval 1

4-day avg, linear 7-da int 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 sites 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: 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

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 14 106.0 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 9
Do 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 da, 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 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

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 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

MO-02, 

2006

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

Actual 11 16.2 1 10.8 0 0

7 da, beg day 1 4 7.0 1 6.9 0 0
Do 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 1st through 7th day of the week. 

While the Heidelberg NCWQR and AEEMP datasets provide a pool of intensivelysampled monitoring data for atrazine concentrations in flowing water bodies, 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 predominant 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 concentrations 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 concentrations 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 streams 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 currently 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 sorghum 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 

across 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 atrazine 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 year. 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 rivers and streams, more frequent flow 

parameters will be needed for the reservoir simulations.
Do not cite

Section 7 - Page 119 of 677

OH Reservoir - PRZM/EXAMS Simulation (89)

0

1 0

2 0

3 0

4 0

5 0

6 0

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

Date

Atrazine, ug/L

Simulated Daily Atrazine (P/E)

Figure 7.8: A single year of daily atrazine concentrations simulated for an 

index reservoir in OH using PRZM and 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 samples. In 

this case, estimated 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 used PRZM with site-specific soil, cropping, and 

weather 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 

degree of reliability for a toxicity endpoint of concern with a 90-day duration but is much 
Do 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 in 

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 reviews 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 predicted 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 linear 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 shortduration exposures, especially 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 value 

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. 
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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 continued 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 useful 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 estimated 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 that 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 this 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 concentrations 

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 atrazine 

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. While most of the literature addresses methods for 

use with independent samples, the Panel cited a recent paper that deals with modeling 
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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-Term Modeling Approaches Combining Random Function 

and Regression Models

In April 2010 the USEPA sought feedback from the Panel on several different longerterm modeling 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 covariates 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 variables 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 herbicides 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 authors 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 specific 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 different 

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 

atrazine 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 

where 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 the 
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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 have 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 the 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 for 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 term 

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. 
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8. IMPLICATIONS OF MOA & TOXICITY PROFILE ON 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 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. 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 epidemiology 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 precision 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. (1996b) at doses ≥ 3.65 mg/kg/day 

(NOAEL/LOAEL = 1.8/3.65 mg/kg/day). Based on the current review, LH attenuation 

continues 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).
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LH plays a critical role in a variety of reproductive and developmental processes 

in rats and humans (Goldman et al. ̧2000; Stoker et al., 2000; Grumbach, 2002; Ebling, 

2005). Multiple lines of evidence demonstrate that decreases in circulating LH levels 

lead to disruptions in estrous cyclicity in rats (Cooper et al., 1996; Morseth et 

al.,1996a&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 result 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 experimental 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 feedback 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 observed 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 in 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 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 in rats, the offspring of these dams do not receive the perinatal 

levels of prolactin necessary 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 males 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 

decreases 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 

resulting 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 the 
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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 

developmental 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 

cyclicity, testosterone decreases, puberty onset in both sexes, and prostatitis) since 

decreases in LH occur at doses lower than those eliciting these outcomes. The doseresponse relationships of these health outcomes are described more fully in earlier 

sections 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 exposures. 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 levels 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 LHsurge. 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 similar 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. However, 

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. Data on 

the time course of atrazine and/or its metabolites in the brain are not available at this 
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time. However, at steady state plasma levels, brain and plasma will be at equilibrium 

making plasma levels a good surrogate for brain levels. 

The extrapolation 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 equivalent 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 

just 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 

shortcomings 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 daily 

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 

exceed 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 was 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 high atrazine 

exposures. As a rough approximation to gain insight into human relevance, the 

elimination rate constants were allometrically scaled for an average human female BW 

of 60 kg. It should be noted that BW3/4 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., t1/2 = 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 provide valuable perspective on the extrapolation of 

rat findings to humans. For example, 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 allometrically scaled human equivalent time estimates are 
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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, the 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 

ATRA dose (mg/kg/day) rat kel (hr-1) Allometrically scaled human female kel (hr-1) 1t1/2 (days) 2

time to steady state (days) 3

1 0.010 0.00343 8.42 25.3 - 42.1

3 0.015 0.00527 5.48 16.4 - 27.4

7 0.013 0.00471 6.14 18.4 - 30.7

10 0.014 0.00495 5.84 17.5 - 29.2

1 Scaled to BW-1/4 based on a female BW of 60 kg

2 Assuming t1/2 = ln2/kel

3 Range obtained by assuming a time of 3 -5 half-lives 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 power 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 possible 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 considered. 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 the 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 question arises as to whether a brief 

exposure (e.g., a few days) in humans could lead to an attenuation of the LH surge. 
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Evidence of chemically-induced decreases in GnRH or LH secretion is sparse in 

humans and non-human primates relative to rodents. The 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 of 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 corroborated 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 health 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 qualitatively 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 reproductive/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 vulnerability. 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 Interval

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 

average 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 cycle
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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 of 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 frequency 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 effects 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 methods 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, more 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 Summary 

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 

within 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 

precision 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 

potential 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 

or additional scientific factors to consider in establishing the critical duration of interest. 
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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.
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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-coupled 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., Oprea, T., 

Prossnitz, E., Musti, A.M., Andò, S., & Maggiolini, M. (2007). G Protein–Coupled 

Receptor 30 (GPR30) Mediates Gene Expression Changes and Growth Response to 

17ß-Estradiol and Selective GPR30 Ligand G-1 in Ovarian Cancer Cells. Cancer Res, 

Feb 15; 67(4): 1859-66.

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; PubMed 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 

herbicides 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.
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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.

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Appendix A - Page 156 of 677

APPENDIX A

EVALUATION OF ARTICLES ON ATRAZINE AND 

ATRAZINE METABOLITES RELATED TO MAMMALIAN 

TOXICITY STUDIES SUGGESTING POTENTIAL 

ADVERSE HUMAN HEALTH OUTCOMES
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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 studies 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 atrazine 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 objective 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 malathion, 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 the 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 Rats.

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 Preovulatory 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 prenatal 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 Fields 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 Protein 

Expression of MCF-7 Human Cells.

A.7

Garaj-Vrhovac, V. and Zeijezic, D. (2004) Genotoxicity evaluation of pesticide formulations A.7
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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 attenuation 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 Long-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 

Hormone Receptor and Affects Growth Hormone Gene Expression

A.11
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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 the 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 effects 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 ovarian function set of experiments, atrazine 

exposure for 2 or 4 weeks at a high dose (300 mg/kg/day) resulted in irregular estrous 

cycles (typically longer cycles) due primarily to a lengthened diestrus. This effect was 

accompanied by decreased numbers of corpora lutea, follicular atresia, uterine atrophy, 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 fertility 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 fertility (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 development will be evaluated 

during the Fall meeting of the SAP.
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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 reproductive 

system. The effects described by both groups are consistent with the postulated MOA of 

perturbations of the neuroendocrine system. Sexual maturation is the culmination of a 

complex cascade of sex 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 explained by the disruptions to the HPG axis caused by 

atrazine. Even through the relatively short period of time evaluated by Shibayama et al., it 

is clear that atrazine treatment leads to a state of reproductive quiescence which – if 

sustained – would eventually result in the reproductive senescence characteristic of 

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 Development.

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 (Daniel 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 (Hennighausen 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 back 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 unreasonable 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., 2004; 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) on 
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Appendix A - Page 161 of 677

mammary gland development in female pups. Briefly, the authors concluded that atrazine 

delays mammary gland development using a subjective scoring scale to evaluate 

mammary gland development.The Agency‘s reviews of these studies are summarized in 

this appendix. The 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 critical 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 μM concentrations. 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 maturing. 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 concentration required to produce death in 50% of the oocytes (lethal 

concentration 50, LC50) and the concentration needed to inhibit the maturation in 50% of 

the oocytes (inhibition of maturation 50, IM50) were calculated by a non-linear regression. 

For atrazine, 664 oocytes (total) were analyzed in five assays at 0, 50, 100 and 500 μM 

atrazine concentrations. For viability, the Spearman correlation was not significant, 
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Appendix A - Page 162 of 677

therefore, it was not possible to calculate the LC50. For maturation, the Spearman 

correlation was significant and the estimated IM50 was 143 μM. Exposure to 100 and 500 

μM 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 affected 

at any dose level. The percentage of mature oocytes (MII) decreased at 100 μM (33%) and 

500 μM (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 vesicle 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 cross-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 the 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 F1 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 4th mammary gland. Two hours prior 

to sacrifice the dams were administered BrDU to assess for cell proliferation in the 

mammary tissue. Another F1 female pup/litter was mated with untreated males to produce 

an F2 generation and allowed to deliver and rear their offspring until PND 21. The F2 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 assessments
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Appendix A - Page 163 of 677

were sexed and subjected to clinical observations, body weight and food consumption 

determinations.

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 level (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 F1 pups. Once they reached adulthood, no effects 

were reported in F1 animals. Similarly, F2 pups were unaffected.14

Mammary gland assessments were conducted on the 4th 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 number 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 4th 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 effects 

were noted on PND 33. However, on the day of VO an increase in the number of end buds 

 

14 F1 and F2 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 F1 generation
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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 development 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 effects. 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 weight 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.

Reviewer 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 be 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 exposure 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 length 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 in 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 rats were assigned to receive 0 or 100 

mg/kg/day of atrazine (purity 98.8% a.i.) for 4 consecutive days after ovariectomy and 
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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), animals 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 sampling, 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 statistically 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 corticosterone plasma levels were measured 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 (GraphPad 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 corticosterone 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 readily quantified
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the corticosterone peak for the atrazine treated group was slightly 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 increase 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 

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. Study completion date: July 14, 

2010. Report Number: WIL-639060.

The purpose of this study was to assess the effects of atrazine on sexual maturation (as 

indicated by the occurrence of vaginal opening), the estrous cycle, and the estrogeninduced luteinizing hormone (LH) surge in female Sprague-Dawley [Crl:CD(SD)] rats 

following oral gavage dosing 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 F0 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).
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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) and 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 (beginning 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 naïve 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 attainment 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-treatment 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 Sprague-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 light:dark schedule (lights on at 0500 hours: lights out at 1900 hours). 

Following 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 lactation 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, 
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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 shown 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 effective 
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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 was 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 females/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 ovariectomy, 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 corticosterone) 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 turned 

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. 
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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 body 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 decreased 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 mean 

F1 body weights (6.5-25%) compared to controls were seen throughout the post-weaning 

period, 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%). 

Food 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 of 
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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 food 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 number of pups born, liver litter size, percentage of 

males at birth, postnatal survival, and offspring weights for the F1 litters of the untreated F0 

dams in Cohort II were similar across dose groups. No remarkable clinical findings were 

noted in F1 pups.

F1 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 II (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 results 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 of 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.
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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 greater 

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 dose 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/day 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 suppressed LH secretion at 6.25 mg/kg/day in LongEvans female rats (Cooper et al. 2007). Using a ovariectomized, 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 the 
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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., Narotsky, 

M., Stoker, T. 2010. Evaluating the Effect of the Chlorotriazine Herbicide 

Atrazine on the Amplitude of the Pre-ovulatory LH surge in the LongEvans 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 

mg/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 

measure 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 significantly 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; 1800 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 surge 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 are amenable to benchmark dose analysis to more clearly 

describe the dose-response curve for the attenuation of the LH surge.
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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. (2008). 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 female rats (4/group) were administered oral gavage doses of 0 or 

200 mg/kg atrazine (97.9%) in carboxymethylcellulose and sacrificed on postexposure day 

5. Pituitary glands were harvested, and either frozen in liquid nitrogen or fixed with formalin. 

Formalin-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, counterstained 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 and 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 μM DACT, culture medium or the solvent (dimethyl sulfoxide, DMSO) for 24 hours. 

Treated cells on slides were rinsed, fixed in methanol, mixed with Triton 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 μM DACT, culture medium or DMSO for 24 

hours and frozen in liquid nitrogen until use. 

For the two-dimensional electrophoresis, either the frozen pituitary glands or LβT2 cells 

were thawed, pooled, sonicated, and centrifuged. Supernatants were removed; proteins 

were precipitated with a commercial kit, and dissolved by sonication in 400 μL of a 

rehydration buffer. Samples of the tissue or cellular proteins were plated onto trays 

containing gel and subjected to electrophoresis. At the end of the procedure, the two 

dimensional gels were either stained with Sypro Ruby (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. 

 
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DACT formed 19 spots and covalent adducts with 8 unique proteins in the rat pituitary and 

21 spots 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 binds to actin, one of the major 

components of the cytoskeleton and acts as a mediator of internal cell motility); and ERp57 

(an oxidoreductase found in the endoplasmic reticulum of cells; exact function is not 

completely understood). In addition, RNA-binding proteins containing active-site or solventexposed 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 speculate 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 LβT2 Rat Pituitary Cells. In vivo exposure to 200 mg/kg 

atrazine resulted in 8 DACT-protein adducts. Similarly, LβT2 rat pituitary cells exposed in 

vitro to 300 μM DACT led to adduct formation between DACT and 19 proteins. This study 

was not designed to study 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 

metabolites 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-PituitaryAdrenal Axis Response to Atrazine and Metabolites 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-schlorotriazine (DACT) – affected 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 after 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 gastrointestinal 

discomfort.
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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 necropsy, 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 nontreated animals for 5 minutes on stress hormone levels 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, pituitary 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 metabolites 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 ACTH, 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 following compound exposure.

Repeated exposure

Nine to 14 regularly cycling females were treated by gavage with vehicle, 12.5 or 75 

mg/kg/day atrazine, 10 or 60.2 mg/kg/day DIA, or 8.4 or 50.6 mg/kg/day DACT {atrazine 

equimolar doses] for 4 consecutive days starting 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 weights 
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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 statistically 

significant except for the ACTH increase seen after treatment with 12.5 mg/kg/day of 

atrazine. Again, DACT appeared to have little or no impact on stress hormone levels. 

Interestingly, though statistically significant, the ACTH response after repeated exposure 

was 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 concentrations 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 concentrations were ≈400-fold higher 

than the parent compound and 7-fold higher than DIA.

Study 2: Vagotomized

The objective of this study was to determine if the increase in stress hormones observed 

after atrazine, atrazine-metabolites treatment were related to irritation of the gastrointestinal 

tract rather than a direct effect on the HPA axis.

Ten vagotomized rats and 10 intact rats were used in this experiment. Four to 5 of the 

vagotomized or intact rats received either 100 mg/kg atrazine or vehicle (1% 

methylcellulose) 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 difference 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 to 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 

exposed 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.
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Appendix A - Page 178 of 677

The authors of these studies concluded that, in addition to the hypothalamic-pituitarygonadal (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 through 

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 oral 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 appears 

that atrazine and/or the intermediate metabolites (DEA or DIA) are critical in the activation 

of the HPA axis. ACTH release, however, is attenuated after repeated exposures to 

atrazine or DIA yet corticosterone levels remained markedly elevated and were 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 of estrous (days 0 and 21), 10 female pigs (ages 7-8 months) were 

given 1 mg atrazine/kg bw (99% purity) in the feed daily for 20 days of the estrous cycle. 

There were 5 female control pigs. One hour following the last treatment on day 20 (when 

the first signs of estrous were 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 

from 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.
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Appendix A - Page 179 of 677

The experiment was preceded by another which measured pLH concentration in peripheral 

blood 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, 12, 

14,16,18,19. and 20 of the estrous cycle.

The pLH concentration from 3 normally cycling (intact) mature female pigs measured at 3 

daily 3-hr intervals during the estrous cycle was increased from 2.6 to 3.9 ng/ml on days 0 

and 21, which indicated the onset 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 female rats. The single iv GnRH administration to atrazine-treated female 

pigs resulted in significant increase in pLH 20 and 30 minutes after GnRH administration, 

indicating that attenuation of GnRH release in atrazine-treated animals was responsible for 

the 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 

Atrazine 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). The 
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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 article. 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 (18%). 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 induced 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 follicles in varied degrees. In some atretic antral follicles, where high 

intensity of apoptosis was observed in the granulosa cells, the oocyte showed nucleolus 

fragmentation and irregularities of the Zona Pellucida.

Low doses of atrazine induced an increased 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 subacute treatments.

Reviewer‘s Comments: It should be noted that in this study no quantification of findings was 

attempted. Most results were presented in the form of 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.
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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 atrazine by-products, diaminos-chlorotriazine (DACT) and hydroxyatrazine (OH-ATR), and a structurally similar 

chlorotriazine, propazine (PRO) on female pubertal development. Previous studies by 

Laws et al. have shown that atrazine delays the onset of pubertal development in female 

rats. 

Female Wistar rats (n=15/treatment group) were gavaged from postnatal days (PNDs) 22 

through PND 41 with DACT (96.8% purity) (16.7, 33.8, 67.5, 135 mg/kg), OH-ATR (97.1% 

purity) (22.8, 45.7, 91.5, 183 mg/kg) or PRO (99.8% purity) (13, 26.7, 53, 106.7, 213 

mg/kg). The dose range for each chemical was selected as the molar equivalent of 

atrazine (12.5, 25, 50, 100, 200 mg/kg). The females were monitored daily for vaginal 

opening (VO) and killed on PND 41. The age at complete vaginal opening was recorded. 

Body weights were recorded daily. Tissue weights for each animal were recorded for liver, 

kidney, adrenals, ovaries, uterus, and pituitary. Histological evaluations were performed on 

the thyroid, uterus, and ovaries. [Note: The Protocol for the Assessment of Pubertal 

Development and Thyroid Function in the Female Rat (Goldman et al. 200) was used for 

these studies. Since this protocol is undergoing validation by US EPA, thyroid hormones 

were evaluated in these studies even though no previous data suggested that 

chlorotriazines would affect thyroid function.]

DACT (33.8, 67.5, and 135 mg/kg) significantly (p<0.05) delayed the age at vaginal 

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. Body weight at the age of VO was also significantly increased 

in the three highest dose groups of DACT (13-19%). At necropsy the body weight in the 

high dose DACT animals 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 of OH-ATR (up to 200 mg/kg) did not cause a significant delay in the age of VO. 

Significant reductions in kidney, pituitary, and adrenal weights were observed following 

exposure to the highest dose of DACT. No significant differences in tissue weights were 

seen in any of the OH-ATR animals. Pituitary weights in the two highest dose groups of 

PRO (106.7 and 213 mg/kg) were significantly decreased compared to controls. 

No significant dose related effects were seen for serum T3 or TSH following exposure to 

any of the test chemicals. An absence of corpora lutea was noted in 4 animals in the 
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Appendix A - Page 182 of 677

highest DACT treatment group that did not attain VO prior to necropsy. No treatmentrelated microscopic changes were seen in any animals in the PRO or OH-ATR groups. 

This study concluded that oral exposure to DACT and PRO from PNDs 22-41 delays the 

age of vaginal opening in Wistar rats in a dose-dependent manner. The no effect level 

(NOEL) for DACT (16.7 mg/kg) was identical to the equimolar NOEL for atrazine (25 

mg/kg). The LOEL for delay in 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: It appears that post-natal exposure to DACT results in a delay in 

sexual maturation. These delays do not appear to be secondary to low body weight since 

body weight at time of VO was significantly higher for the delayed pups. Given that atrazine 

is quickly metabolized in the body, it appears that DACT is driving the delays in puberty 

onset. The DACT NOAEL for sexual maturation reported by Laws et al. (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 assessment.

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 

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 atrazine (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 gland 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)/progesteroneinduced LH surge are due to impaired ER function. As expected, ATRA suppressed the EB 

and progesterone-induced LH surge in a dose dependent manner. A daily dose of 300 mg 

ATRA/kg bw for 5 days nearly abolished the LH surge. In contrast, a dose of 77 mg 

DACT/kg bw (which produces the same AUC for DACT as dosing with 300 mg/kg bw 

ATRA) only partially attenuated the LH surge (~ 60%). The effect of DACT on pituitary 

function was also examined in response to exogenously added gonadotropin-releasing 

hormone (GNRH). DACT attenuated the LH surge by 47% as compared to controls, 

indicating a decrease in pituitary function. 
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Appendix A - Page 183 of 677

Binding studies with uterine cytosolic containing ER and in vitro-transcribed ER indicate 

that ATRA, but not DACT at the same molar equivalent concentration, decreases estradiol 

binding regardless of incubation time. Based on the dose dependent inhibition of estradiolbinding, an inhibition binding constant (Ki) of 0.02 mM was estimated for ATRA while DACT 

exhibited a 10-fold greater value.

In an effort to investigate whether ATRA alters estrogen binding to hypothalamic ER, 

unoccupied ER levels were evaluated in two estrogen-responsive hypothalamic nuclei.

Treatment with ATRA along with estradiol benzoate did not alter the levels of unoccupied 

ER levels as compared to estradiol benzoate treatment alone. Moreover, ATRA failed to 

block estrogen-induced progesterone mRNA expression as compared to estrogen 

treatment 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 finding 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 involves 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 repeated 

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 studies based on results of previous studies that showed that treatment of female 

SD rats with atrazine for 21 days induced disruption of the estrous cycle at 75 mg/kg/day 

and inhibited follicle and corpus luteum development at 300 mg/kg/day (Cooper et al. 

1996). Treatment of ovariectomized female rats with atrazine for five days suppressed the 

peak level of the LH surge at 30 mg/kg/day (McMullin et al. 2004). 
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Appendix A - Page 184 of 677

Five week old female SD rats were treated for either two or four weeks. Clinical signs and 

mortality were observed twice daily. Body weight/gain and food consumption were 

measured twice a week. Vaginal smears were taken every day and the mean estrous cycle 

was calculated during the dosing period. Animals were sacrificed after 2 or 4 weeks, blood 

samples were taken for 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 were 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, females 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 animal 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 and Day 14 of gestation period. Food consumption was 

measured twice a week during the premating period and on Days 1, 4, 8, 10, and 13 of 

gestation. The mean estrous cycle length during the pre-mating period was calculated. 

Vaginal smears and vaginal plugs were checked for the presence of sperm during the 

mating period. On Day 14, animals were exsanguinated and the 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 macroscopically. 

Results for Repeated Dose Toxicity Studies: No mortality occurred in the 2- or 4-week 

study. In the two week study, clinical signs included lacrimation seen at 30 and 300 

mg/kg/day and salivation and soiled perineal region at 300 mg/kg/day. However, no 

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% incidence in control).
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Appendix A - Page 185 of 677

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 at the high dose, 

histopathological findings occurred in the ovary (loss of currently formed corpora lutea, 

decrease in numbers of previously formed corpora lutea, increased in large-sized atretic 

follicles, swelling of 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, histopathological 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 treatment group showed lower incidences of abnormal 

ovarian and vaginal histopathological findings then the two week treatment group. The 

authors noted that these results may be explained by spontaneous recovery of some 

animals from a disturbed to a normal estrous cycle.

Results for Female Fertility Study: No mortality occurred. Clinical signs included salivation 

at 30 and 100 mg/kg/day and lacrimation and decreased stools at 100 mg/kg/day. 

However, no incidence of these findings was provided. Atrazine did not show significant 

effects on the fertility study in rats. The copulation index, fertility index, number of corpora 

lutea, number of implantation sites, numbers of live and dead embryos, and percentage of 

preimplantation and postimplantation loss were not 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 group, 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 examination of the ovaries in a two-week repeated 

dose toxicity study.

Reviewer‘s Comments: In summary, atrazine caused irregular estrous cycles accompanied 

by adverse changes in ovarian histopathology at doses of 300 mg/kg. Extended dosing for 

4-weeks did not further exacerbate the 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 

studies and the fertility studies prevented this comparison. It is noteworthy that the NOAEL 

for the effects reported by Shibayama et al., (30 mg/kg/day in atrazine molar equivalents) 

were higher than the NOAEL (1.8 mg/kg/day) for attenuation of the LH surge currently used 

as the point of departure in the Agency‘s risk assessment.
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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/Lactational 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. Two 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 after 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 the 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 detected 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 accounted 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, respectively. 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 quantitation (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, DIA 
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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 content 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

Atrazine DACT DIA

Dams Pups Dams Pups Dams Pups

Plasma 

(ng/ml)

0 LOQaLOQ 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 LOQ

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-conducted 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 most common metabolite found in dams, fetus, 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 tested. 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. 
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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 concentrations 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 particular, 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-Evans (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 days (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 females/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 F2 generation. On 

PND 4, the F2 were standardized to10 pups/litter. Subsequently, 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.
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Appendix A - Page 189 of 677

collected as well as the pituitary and mammary glands. The pituitary was 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 decapitation; 

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 

weighed. 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, estrone, 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 after 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 test 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 authors stated that atrazine-exposure 

delayed the development 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 examination 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 pups 
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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 

uterus) 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 lactational 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 dams 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 and 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 gavage 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 weight 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 size (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).
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Appendix A - Page 191 of 677

Conclusions

Differences seen in gestational 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 were 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 

Summary: 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 focused 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 strains of rats following both peripubertal and perinatal exposure (Stoker et al., 

2000; Friedmann, 2002 ;Trentacoste et al., 2001; Rayner et al., 2006 and Rosenberg et 

al., 2008).

 

The primary data collected following the male peripubertal exposures and 

examination of puberty outcomes (Stoker et al., 2001; Friedmann et al., 2002, Trentacoste 

et al., Pogrimic et al., 2009) is consistent with the effect of atrazine on the central nervous 

system (i.e., suppression of GnRH/decreased pituitary secretion of LH) and subsequent 

alterations in the hormonal control of pubertal development (Cooper et al., 2000). 

Decreased LH secretion would lead to insufficient stimulation of the gonads during puberty 

to produce the necessary steroids for androgen-dependent maturation of the sex accessory 

tissues in the male. Therefore, the delayed growth of the reproductive tract in the male 

supports the reported alteration of the hypothalamic control of pubertal progression. 

A reduction in testosterone levels following atrazine exposure has been reported in a 

number studies in mammals, as well as other species revealing a consistency in the effects 

of atrazine on androgens. It is well known that the development of the size of the penis and 

cornification of the epithelium of the 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-dependent tissues, such as the ventral prostate and seminal vesicles. 

Normally, testosterone levels rise gradually from PND 20 to 40, and abruptly double by 

PND 50 (Matsumoto et al., 1986; Monosson et al., 1999). 
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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 Sprague Dawley (SD) rat 

(Trentacoste et al., 2001), pubertal onset was delayed by 3 days and 4 days following 

exposure to 100 and 200 mg/kg 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 and serum testosterone at 50 mg/kg/day and 200 mg/kg/day

following exposure to the peripubertal male SD rat. Thus, all three studies found 

decreases in either serum or intratesticular testosterone and Trentacoste et al. also found a 

decrease in LH at the 200 mg/kg/day dose.

Ventral prostate, seminal vesicles, lateral prostates and epididymal weights 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 intratesticular testosterone was significantly decreased following atrazine exposure 

peripubertally. This has corresponded to the work of others showing 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 (Trentacoste 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 23 to 53 SD Decreased intratesticular T on PND 

45

150/200 Stoker et al., 2001

PND 22 to 47 SD Decreased T on 

PND 47

ND/50 Trentacoste et al., 

2001

PND 22 to 47 SD Decreased test. ND/50 Friedmann, 2002
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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 prostate weight. Seminal vesicle weight was reduced by 

DEA at AEDs ≥25 mg/kg/day, and by DIA and DACT at AEDs ≥100 mg/kg/day AED.

Epididymal weights were reduced in the DEA, DIA, and DACT at atrazine equivalent doses 

of 200, 200, and ≥100 mg/kg/day, respectively. Serum testosterone was reduced only in 

the males receiving the 2 highest doses of DIA. This study confirmed 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 weight 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 

to 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 peripubertal exposure male study (Stoker et al., 2001) showed decrease body 

weight at the 200 mg/kg dose, but a pair-fed group was included to discern the effects of 

changes in growth. There were changes in puberty at doses in which no body weight 

decreases were observed (≥50 100 mg/kg/day in females and ≥12.5 mg/kg/day males). 

However, in the study with SD rats Trentacoste et al., found that the food restriction group 

(receiving approximately 10% less food which was equivalent to the 100 mg/kg atrazine 

treatment food intake) 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 influence pubertal onset or the growth of androgen dependent tissues in the rat (Laws 

et al., 2007). 

Perinatally a single 100 mg/kg atrazine dose to the dam resulted in an effect on pup 

growth that may involve changes in milk quality, as the offspring showed a decreased 
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Appendix A - Page 194 of 677

weight of 7% on PND 4 (Rayner 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 anogenital 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 decreases in pup body weights (Rosenberg et al., 2008). 

These alterations of early postnatal body weight could play a role in the delay in puberty in 

these male offspring. 

In summary, the MOA of altered pubertal progression in the rat and decreased androgen 

production by the testes is plausible in humans, as GnRH and the regulation of pituitary 

hormones stimulate the gonadal secretion of steroids and is critical for the initiation of the 

set of events and time course of the HPG regulation of puberty in the human. Thus, it is 

essential to take into consideration that all the effects on sexual maturation and altered 

androgen status reported after ≈ 30 days of exposure occur at dose levels substantially 

higher (≥ 12.5 mg/kg/day) than those leading to the LH surge disruption which serves as 

the basis of the Agency‘s current risk assessment.

Prostate effects 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 further 

explore this potential adverse health outcome. 

First reported in 1999, an increase in prostatitis has been linked to 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 neonatal 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 elevated 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 offspring 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.
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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 to atrazine in utero only (born to atrazine-treated dams 

but nursed by control females)

C-ATR: pups exposed to atrazine lactationally only (i.e., born to control dams 

but nursing from atrazine-treated dams)

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

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

Consistent with the 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 development. Kandori and coworkers (2005), however, hypothesized 

that rather than being a direct hormone-mediated effect, the decrease in prostate 

carcinogenesis is the secondary effect of a reduction in caloric intake. To test this 

hypothesis these researchers used a transgenic adenocarcinoma rat model typically used 

to study prostate 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 

treatment 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) rather than hormonal 

disruption. It is important to know, however, that it has been demonstrated by other 

researchers that exposure to atrazine at the doses used in this study leads to decreases in 

testosterone. It is not clear if the discrepancy between this study and other studies that 

have demonstrated atrazine-induced alterations of 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 and 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 may 
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Appendix A - Page 196 of 677

be a secondary effect to a reduction in caloric intake, a test system in which testosterone 

levels are altered would provide 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, H2O2-mediated oxidation of Fe2+ to 

Fe3+, superoxide dismutase (SOD), γ-glutamyl transferase ( -GT), glutathione-Stransferase (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 statistically 

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..
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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 points.

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 atrazine 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 increased 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 changes 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 epididymal 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) in 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 porcine 
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Appendix A - Page 198 of 677

semen. An in vitro viability staining method was used to assess viability while a computerassisted-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 samples with sperm motility ≥ 85% 

and sperm abnormalities < 15% were used in the experiment. Sperm at a concentration of 

25 x 106sperm/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-buffered 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 

exposure 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 eliciting 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. Moreover, no attempt was made by the researchers to investigate the 

consequences of these changes in sperm motility on reproductive health (e.g., decrease in 

fertility). In the absence of these considerations, the data presented have little relevance to 

human health risk assessment

 

17 In this evaluation only the results for atrazine will be discussed
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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 μM atrazine in the presence of a maximally stimulating concentration of luteinizing 

hormone. 

In the 27-day in vivo exposure to 50 mg/kg/day, mean testosterone concentrations were 

significantly reduced by 38% in serum and 53% in intratesticular testosterone. Decreased 

body weight (7%) was seen in the 50 mg/kg/day group (215 g) compared to controls (230 

g). In 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 control 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 cells 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 inhibition at the testicular level.

Friedmann argued against the earlier paper by Trentacoste (he was a co-author) that 

concluded the decrease in T was only the result of underfeeding. He did this by citing the 

drastic reductions in food intake needed to lower serum T. This argument 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 production by the Leydig cell 

was decreased when exposed to atrazine. The mechanism for this, if repeatable, is 
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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 interferes 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 compared the results with similar 

treatment to primary Leydig cells. In both cases, T production was increased (see Higley et 

al., paper for data on 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 of doses.

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 F1F0-ATP 

synthase and inhibits microchondrial function in sperm. Biochem 

Biophy Research Comm 366: 66-72.

This paper demonstrates the ability of ATR to alter human sperm motility in vitro, and tests 

hypotheses on the mechanism through 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 F1F0-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 mitochondria – rather they used liver mitochondria. Sperm energetics are quite 

different from somatic cell energetics and sperm mitochondria differ from somatic 

mitochondria in specific ways that should be discussed. 

Approach: Ejaculated human semen samples were washed to recover motile sperm and 

the sperm were then cultured with 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 mitrochondria, 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 significantly 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 make a solid case for ATR ability to 

bind the ATPase.

The authors conclude that this likely explains earlier reports that ATR decreases porcine 

sperm motility in vitro (as discussed in the preceding two papers). Importantly, they also 
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Appendix A - Page 201 of 677

conclude that ―This study was motivated by the idea that ATZ applied to fields may cause a 

reduction of sperm count and motility to humans and other animals. However, the effect of 

ATZ on sperm motility was 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 relevant.‖ 

Reviewer‘s Comments: The relevance of these findings to risk assessment is questionable 

given that the concentration levels required to affect sperm motility were high and exceed 

likely exposure levels 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, 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.

The effects of a 13- week dietary administration of 500 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 corresponding doses in mg/kg/day 

were 33.0 mg/kg/day for the 500 ppm group and 61.6 mg/kg/day for the 1000 ppm group. 

Each treatment group contained 10 animals. A control group was included. 

The rationale for using TG rats was based on previous findings that atrazine inhibited 

testosterone production in the Leydig cells 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 established 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 reduction 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 subjected 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-mass 
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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 group. Bodyweights of the dietaryrestricted 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 were 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 treated 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 epithelial proliferation with formation of irregular glands and luminal bridging to give 

a cribriform pattern in focal populations. The nuclei demonstrated enlargement and severe 

atypia. According to the authors, the lesions appeared compatible with well-differentiated 

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. 

Because 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 revealed 

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 lobe 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 immunohistochemically in 

almost all cells of the prostate adenocarcinomas and PIN. No treatment-related changes in 

the testes, epididymides, and seminal vesicles were observed. 
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The authors concluded that dietary administration of atrazine for 13 weeks showed slight 

suppressive effects on the development of prostate cancer in the probasin-SV40 T antigen 

transgenic rats, even though atrazine treatment did not affect serum testosterone levels. 

Based on the observation that there was equal or greater suppression 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 effects. 

Revewer‘s Comments: While this study suggests the possibility that prostate 

carcinogenesis may be a secondary effect to a reduction in caloric intake, a test system in 

which testosterone levels are altered 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 using 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 changes 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 DACT
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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 minutes 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 increase 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 hormones 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 Development 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 period 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 

preputial 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.
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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 associated 

with 2-19% reduction in body weight. In males, there was a significant decrease in T3 and 

T4 at the 30% and 40% FR groups associated with 9% and 19% body weight decrease, 

respectively. TSH was significantly decreased in the 40% restricted group. No differences 

in serum 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, and19% body weight reduction. No histopathological changes were observed in 

any organs.

In females, the absolute weights, but not the relative weights, for ovaries were decreased in 

the 30% and 40% FR groups. The mean uterine weights were not different from control for 

any FR group. In males, the absolute, but not 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 growth 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 which 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 concept 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 Boar 
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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 fenoxapropethyl 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 

were 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 

μg/mL and 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 distributed 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 capacitated; sperm with a dull fluorescence over the whole head except for a 

thin, bright ban in the equatorial segment were considered acrosome-reacted. At least 200 

sperm were analyzed per slide.

Cytotoxicity was determined by incubating sperm using concentrations of atrazine 

(technical grade, purity not 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, 27, and 6, respectively. The median lethal 

concentrations of atrazine were calculated. Final concentrations of 8, 20, and 40 μM 

atrazine were used in the experiments. These concentrations corresponded to the LC50/5, 

LC50/2, and LC50, respectively. The concentrations used were sublethal for sperm in vitro, 

using the LC50 as a maximum. For the capacitation and PIAR studies, the herbicide was 

added at the beginning of the 4 hour incubation. The control was the same solvent with the 

herbicide (0 μM).

Sperm Capacitation: In the presence of atrazine during capacitation and PIAR, the viability 

of capacitated sperm decreased in a linear fashion for the 8, 20 and 40 μM dose groups. 

Capacitation decreased significantly only at the high dose (40 μM), to 79% of controls 

(p<0.05). SAR increased 1.9 fold for the 8 and 20 μM and 2.2 fold for the 40 μM dose 

groups, all at p<0.05. The noncapacitated population was reduced at all concentrations, 

compared to controls (p<0.05).

 

19 In this evaluation, only the results for atrazine will be discussed.
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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<0.05) in noncapacitated and capacitated 

sperm, compared to controls. PIAR was statistically increased at 8 μM (1.6 fold), 20 μM 

(1.8 fold), and 40 μM (1.8 fold), compared to controls. 

Similar to a previous study by this group (Betancourt et al, 2006), the authors review 

literature on atrazine effects and use, but fail to provide a sound rationale for examining 

direct in vitro effects on sperm. There is no literature to imply that atrazine selectively 

targets mature sperm and impairs their fertilizing ability after in vivo exposure. They state 

that ―it is necessary to carry out in vitro studies to clarify the mechanisms of action of toxic 

compounds,‖ but do not provide justification for this statement. Rather they state that 

because atrazine and FE are used in Mexico and other countries, the aim of the study is to 

evaluate in vitro effects....on boar sperm capacitation and the progesterone-induced 

acrosome reaction. These two statements are unrelated and do not provide a sound 

rationale for the study. There 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 effects 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 1750 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 recorded. 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.
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Superoxide dismutase (SOD) activity was measured. SOD is an antioxidant (removes 

superoxide radicals).

There was a dose related decrease in relative testes weights at all dose levels for atrazine 

(4.6-13%) and combined atrazine/chlorotoluene (6.9-14%), although the decreases were 

not statistically significant. The SOD enzyme activities increased in a dose-related manner 

in animals treated with atrazine 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 

spermatogenic 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 endoplasmic 

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 

degeneration 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 

Steroidogenesis Gene Expression in Leydig Cells. Toxicological 

Sciences 111(1):189-197.

The purpose of this study was to attempt to identify the possible mechanism of inhibition of 

Leydig cell steroiodogenesis in peripubertal male Wistar rats after in vivo exposure to 

atrazine. The study was based 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 sensitive to both). At 200 mg/kg/day, body 
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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 significantly 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) suggested 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 was 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, pregnolone, 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 authors 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 Friedmann, 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 difference in the ex vivo vs. the in vitro

exposed Leydig cell. 

This group also measured the transcription of several genes involved in steroidogenesis 

(including the LH receptor) after p.o. administration. They report a clear dose-dependent 

decrease in gene expression of LHR, SR-B1, StAR, TSPO, SF-1, PDE4B, 3bHSD, 

CYP17A1, 17bHSD in Leydig cells. There was no effect on CYP11A1 and 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 quality of this manuscript is hard to judge. There is a lot of 

data, much of which appears solid, however, the lack of a dose response in the Leydig cell 

incubation studies and the decrease in the expression of so many genes in the RT-PCR 

studies demands confirmation. Finally, as to whether or not these data support the HPG 

MOA or indicate a direct action of Atrazine on the Leydig cell can not be determined. In 

addition, it should be noted that the doses used in this study were relatively high compared 

to the doses eliciting the neuroendocrine effects and used for regulatory purposes.
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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 Appl 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 LongEvans rats were dosed by gavage on gestational days (GD) 15 – 19 with 100 mg/kg/day 

atrazine or 1% methylcellulose (controls). On postnatal day (PND) 1, half litters were 

cross-fostered to create the following four treatment groups: birth dam or gestational 

exposure only (ATR-C); milk source or lactational 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 myeloperoxidase 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 weight 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 males 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 weight. The average 

ATR-ATR lateral prostate weight was 49% higher than control on PND120. At PND220, 

lateral prostate weights were significantly increased (45 – 110%) in animals within all ATR 

exposure groups compared to control animals. When adjusted for body weight, animals in 

ATR-C and ATR-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 abnormalities 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 increased incidence 

and distribution of inflammation in the lateral prostates of C-ATR males at PND 120. Only 
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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 prostates 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 assess 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 ―may not have penetrated 

deep within the gland.‖ By PND220, lateral prostate weights were significantly increased 

for ATR-C and ATR-ATR groups, but there were no significant changes in inflammation in 

either the lateral or ventral prostate.

The results of the myeloperoxidase assay demonstrated no significant effects; the authors 

noted the discrepancy between the assay and the pathological findings. The authors 

explained the discrepancy as possibly due to not having both lateral prostates for assay 

(one used for the assay, the other 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 increases 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 postnatal exposure of atrazine to the lactating dam (Stoker et al., 

1999). There is also a delay of PPS in the ATR-ATR dose group only, indicating that it is 

the combination of gestational exposure to the dam and the postnatal milk from those 

mothers responsible for the effect. Rosenberg et al, 2008 also observed a delay in PPS at 

100 mg/kg with an oral perinatal exposure only, a condition that would be similar to the 

ATR-CON group in whom Rayner found no effect on PPS. It should be noted that these 

effects in the Rayner et al. study occur at doses higher than those eliciting 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 

Postnatal Development of Male Offspring. J. Andrology 29(3):304-311.
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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 exposed 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 

weights and anogenital distance (AGD) were measured for each pup. The 4 male pups 

from each litter were also examined for preputial separation beginning on PND35. On PND 

60 the pups were sacrificed by decapitation and their blood (serum), testes, ventral 

prostate, and seminal vesicles collected. The testes, ventral prostate, and seminal vesicles 

were weighed. Testosterone levels were measured in both the serum and intratesticular 

fluid. 

Maternal body weight was 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 approximately 10-

25% at doses ≥ 10 mg/kg/day, with statistical significance attained at the 75 and 100 

mg/kg/day doses (~20-25%). Pup weight decreases of 10-17% were reported for pups 

whose dams were exposed to doses 50 mg/kg/day or higher. None of these pup weight 

decrements, however, were statistically significant.

Intratesticular testosterone levels measured from neonates did not reveal any statistically 

significant effects at any dose level. Although a 60% increase in intratesticular testosterone 

was noted at the 10 mg/kg/day dose level, there was no statistical significance or dose 

response for this effect (i.e., testosterone levels at ≥ 50 mg/kg/day were comparable to 

control). It is noteworthy, that at this dose level a 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 PND 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 body 

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.]
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Appendix A - Page 213 of 677

The 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 to 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 mg/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 days of age in the 50, 75 and 100 mg/kg group. Oddly, none of the 

androgen-dependent tissue weights, including the seminal vesicles or ventral prostate were 

affected even at the highest dose. It should be noted that the effects on maturation and 

altered androgen status occurred in this study at dose levels substantially higher 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), deisopropylatrazine (DIA), and 

diaminochlorotriazine (DACT) at doses equivalent to the atrazine equimolar doses (AED) of 

0, 6.25, 12.5, 25, 50, 100, or 200 mg/kg/day.. The day of preputial separation (PPS) was 

measured. Males were killed by decapitation 2 hours following the last dose on PND 53. 

The pituitary, adrenal, thyroid, 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 significantly delayed 

at 25, 100, and 200 mg/kg/day AED by 1.2, 1.2 and 3.4 days, respectively, resulting in a 

NOAEL for delayed PPS of 12.5 mg/kg/day. For DEA, PPS was significantly delayed at 25, 

100, and 200 mg/kg/day AED by 1, 2 and 4 days, respectively, with a NOAEL of 12.5 

mg/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.
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Appendix A - Page 214 of 677

Testicular weights were similar to controls in all treatment groups for all three metabolites. 

When males were killed on PND 53, DEA (100 and 200), DIA (50-200 AED), and DACT 

(200 AED) treatments caused significant reduction in ventral prostate weight, while the 

highest doses of DIA and DEA resulted in significant decrease in lateral prostate weight. 

Seminal vesicle weight was reduced at 100 and 200 AED by all three metabolites. 

Epididymal weights were decreased in the DEA (200 AED), DIA (200 AED), and DACT 

(100 and 200 AED) treatment groups. 

Serum testosterone was significantly 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 group 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 primary 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 decreased 

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 assessment.
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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 decreased food 

consumption in the high dose group. Daily body weight and preputial separation were 

measured. Males were killed by decapitation on either PND 45, 53, (or 54) or 120. The 

pituitary, thyroid, testes, epididymides, seminal vesicles, and ventral and lateral prostates 

were weighed and examined 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 groups by 2.3, 1.8, 1.7, 1.6 and 3 days as compared to controls. PPS was also 

delayed by 2 days in the pair-fed animals compared to controls. The delay in PPS observed 

in the pair-fed animals was not as severe as that observed in the atrazine 200 mg/kg/day 

group. 

This study showed that atrazine 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 53 

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 testosterone at 53 days of age, but intra-testicular 

testosterone was decreased on PND 45 which might explain the delay in pubertal 

development. There was no significant effect on serum LH on PND 53, but serum 

estrogens were increased at 200 mg/kg/day. These effects did not appear to be due solely 

to decreased growth, as there were effects at doses which did not affect growth (both PPS 

and tissue weights).
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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 support 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 Testosterone Levels and 

Androgen-Dependent Reproductive Organs in Peripubertal Male Rats. 

Journal of Andrology, Vol. 22, No. 1, January/February 2001 ; pp. 142-

148.

Male Sprague-Dawley rats received atrazine (96.1%) from PND 22-47 by gavage in methyl 

cellulose at doses of 0, 1, 2.5, 5, 10, 25, 50, 100 or 200 mg/kg/day (n = 9-12 rats/dose) and 

killed by decapitation on 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 concentrations were 

measured.

Body weight (9%, 100 mg/kg/day/21%, 200 mg/kg/day), seminal vesicle (32%/55%) and 

ventral prostate (26%/48%) weights, 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 potential 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 that 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 SpragueDawley 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 in 
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Appendix A - Page 217 of 677

males treated with 25 mg/kg/day and above, which was very similar to that observed by 

Rosenberg et al., 2008, following gestational dosing). Stoker et al., also pair fed males 

during the experimental period base on the food consumed by the 200 mg/kg/day animals. 

The body weight in the pair fed animals was 84% of control body weights (reduced 16%). 

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. Freidman (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 Victor-Costa, AB, Bandeira SMC, Oliveira AG, Mahecha GAB, Oliveira, 

CA. 2010. Changes in Testicular Morphology and Steroidogenesis in 

Adult Rats Exposed to Atrazine. Reproductive Toxicology. 

oi10.1016/j.reprotox.2009.12.006.

This study evaluated the effects 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 rats (4-7/group) (120 days old) were given daily doses of atrazine (purity 

not provided) via gavage for 7 days (300 mg/kg/day), 15 days (50 and 200 mg/kg/day) or 

40 days (200 mg/kg/day). A commercialized mix of atrazine called Gesaprim® was used. 

This formulation consisted of 500 g/l atrazine and 500 g/l of inert ingredients. Control 

animals were treated for similar periods with the vehicle, corn oil. 

After treatment, rats were weighed and sacrificed. Testes and adrenal glands were 

weighed. Testes were examined 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 pathway, 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 stages 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 determine 
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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 body weight decreases (p<0.05) in the rats treated with atrazine 200 

mg/kg/day (15 and 40 days) (35%) and 300 mg/kg/day (7 days) (38%). A significant 

increase (p<0.05) in the relative and absolute testes weights were seen in rats treated with 

atrazine 200 mg/kg/day/15 days (80% and 16%) and 300 mg/kg/day/7 days (83% and 

14%). However, exposure to atrazine 200 mg/kg/day/40 days resulted in a 41% and 60% 

reduction in the relative and 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 

treated 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 treated animals. 

Atrazine 200 mg/kg/day/40 days resulted in testis atrophy and reduction in the seminiferous 

tubules lumen. In this group giant multinucleated bodies and a large number of apoptotic 

cells were frequent within the seminiferous tubules. 

Immunopositivity for AR was detected in 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 cells compared 

to controls, which confirmed the Western blotting results. Treatment with higher doses of 

atrazine (300 mg/kg/day/7 days) resulted in decreased AR intensity in both Sertoli and 

Leydig cells. Longer treatment with atrazine (200 mg/kg/day/40 days) resulted in decreased 

AR 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 intensity.
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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 by 

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 adrenal 

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 altered 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 example, 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. 3BHSD is a key enzyme in the androgen biosynthetic pathway, but changes in the expression 

or protein levels could be a result of the suppression of luteinizing hormone. The fact that 

they found a decrease in B-HSD in the testes is different than the Pogrmic paper wherein 

this was one of only two (of the many examined) that did NOT change, but the current 

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 period of time, this can lead to 

atrophy, which may be why it is observed in the 40 day exposure. The high doses and the 

fact that 50% of this was made from ―other-ingredients‖ in the commercial mixture make the 

results difficult to interpret. This paper adds 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 critical 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 species- and tissuedependent manner by differential uses of multiple promoters (Simpson et al., 2002). It is 
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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 his colleagues reported that atrazine 

induces estrone production and aromatase (CYP19) mRNA in H295R adrenal cortical 

carcinoma cells (Sanderson et al. 2000; 2001). These authors reported that elevated 

aromatase activity, as measured 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 atrazinedependent increases in estrogen production in H295R cells (Heneweer et al., 2004; Higley 

et al., 2010) and human placental carcinoma (JEG-3) cells (Sanderson et al., 2001; Laville 

et al., 2006). Finally, Holloway et al. (2008) also reported that aromatase activity in human 

granulosa-lutein primary cell cultures following treatment with atrazine (1.0 μM) was 2-fold 

higher than that observed for the controls. A commonality among these studies is that the 

effects on estrogen production are only observed in the various cell types when exposed to 

atrazine for at least 24 hours. This finding supports 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 characterized. 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 known 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 aromatase promoter II. While these studies focused primarily on 

the up-regulation 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 reported 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 effect on steroidogenesis. Finally Albanito et al. (2008) reported a series of 

in vitro experiments designed to evaluate the mechanism of action for atrazine in BG-1 

cells (human ovarian cells). Their data confirm that atrazine does not bind directly to the 

nuclear ER as previously reported by Cooper et al. (2007). However, atrazine did not 
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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 LOELs (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 concentrations of the active atrazine metabolite DIA show that it reaches the 2-4 μM 

range within 15 minutes after four daily doses of 75 mg/kg/day via gavage. Again, although 

more data would be needed to accurately assess the internal dose in animals treated for 

different durations, this data does indicate that the concentrations used in the in vitro

studies discussed above are not excessive. However, these in vivo doses are still 

substantially higher than those selected for points of departure in the 2003 risk 

assessment.

In summary, the in vitro studies demonstrate that atrazine can alter aromatase activity and 

gene transcription. The more recent studies suggest that the cellular responses following 

atrazine exposure involve a complex array of signaling events, Importantly, the fact that 

atrazine may exert a broad influence 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 changes in the production of serum steroid hormones have been identified in vivo 

following atrazine exposure including estrogen, albeit at high doses (Stoker et al., 2000), 

alterations in aromatase gene expression or activity have not been observed in the rat 

testes (Modic 2004) or mouse reproductive tract (Rivest and Sanderson, 2010). Thus, 

suggesting that while steroidogenesis may be increased as a result of atrazine exposure, 

this event is likely not mediated by aromatase.

Three in vitro studies have been 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 delays in puberty onset, it is hypothesized that atrazine may also affect 

sperm parameters (e.g., motility, viability). Two of the studies used porcine sperm while the 

remaining study used human sperm to explore the potential impact of atrazine exposure on 
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sperm. 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 statistically 

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 male 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 consideration 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 is most likely an atrazine metabolite assuming 

it could reach the testes. Finally, there is no attempt to correlate the in vitro concentrations 

tested to any in vivo concentrations that live sperm may encounter during maturation in the 

testis, epididymal transit, in seminal fluid, or in the female oviduct where 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 relatively 

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 research 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 reacted 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 to penetrate the zona pellucida. This study suffers from not reporting 

hyperactivated motility. The latter would help distinguish between physiologically AR 
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sperm and dead sperm (both of which stain similarly with CTC because they lack an 

acrosome). 

In 2009 Maravilla-Galvan and coworkers evaluated the impact of atrazine exposure 

at concentrations of 0 (control), 8, 20, and 40 μM. In general, the percentage of 

uncapacitated acrosomes (i.e., with intact acrosomes) and percentage of motile sperm 

declined in a dose-dependent manner 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 decrease 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 hyperactivated 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 atrazine 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 in 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 mitochondria – was used as a positive control. 

As expected, oligomycin decreased sperm motility by approximately 20%. Atrazine also 

caused a decrease in sperm motility but only at considerably higher concentrations (0.3 

and 1 mM) than oligomycin. In support of the authors‘ original hypothesis, sperm ATP 

levels were also decreased at the concentration where progressive motility was impaired. 

Based on these findings, the authors postulate that the decreases in sperm motility 

observed in vitro may be the outcome of decreases in ATP availability needed for sperm 

flagella to move. They also point out that ―the effect of ATZ [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 

relevant.‖ 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 samples, they evaluated 

atrazine‘s potential effect on sperm parameters (motility, viability, capacitation, and 

acrosomal reaction). Although the in vitro data suggest an effect of atrazine on sperm 

motility and/or viability, there is no evidence of a correlation between these findings and 

impaired reproductive performance. 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 studies are high (≥ 100 μM) and, as 

Hase et al. noted, an exposure level not likely to be reached as a result from use of the 
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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-proteincoupled 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 cotransfected with ER expression plasmid and ER-luc. All transfected cell line were 

maintained for 24 hrs., and then exposed to solvent, estradiol (100 nM), atrazine (1 – 10 

μM)m, ICI ER antagonist (10 μM), 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 μM. A 

study to determine whether or not atrazine regulated ER expression was conducted using 

BG-1 cells, and results demonstrated that atrazine (1 μM) did not alter mRNA or protein 

concentrations of the receptor (Fig. 3) as compared to the down-regulation of these 

measures in those cells exposed to estradiol. In addition, the study confirms that atrazine 

does not bind directly to the ER as previously reported by Cooper et al. 2007. Together, 

these data demonstrate that 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 following a 40-hr exposure to 1 μM. 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 difficult 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 does not provide a positive control for either 

aromatase activity or gene expression. 
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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 μM) 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, including estrogens. An 1-hr exposure to atrazine (1uM) significantly 

enhanced c-fos and cyclin A mRNA levels (200-269% increase as compared with control), 

and longer exposure (24-hr) resulted with significant elevations in progesterone receptor, 

pS2k and cyclin A. The authors conclude ―Atrazine is able 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 abolished 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 no 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 below 

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) cellular 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 alternative to phosphodiesterase as the 

membrane associated target molecule for the herbicide. 
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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 aromatase 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-well 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 exposure to each chemical (up to 500 μM) was 

determined using the MMT method. Following exposure to the test chemicals, media was 

removed, cells were washed with serum-free media, and tested for aromatase activity using 

the tritiated-water release assay. Specifically, the cells were incubated for 45 min. with 3Handrostenedione, a substrate for 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 corresponding 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 difficult to interpret. Initial experiments to detect 

inhibitory effects on aromatase activity in human placental microsomes were conducted for 

each test chemical at a single high dose of 500 uM. Atrazine had no inhibitory effect at 

500 uM. Chemicals that 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 presence 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 in 

the cultures was 20 uM. Binary mixtures were tested for some of the chemicals, but again, 

data are difficult to interpret. Fig. 7 shows that atrazine significantly inhibited aromatase 

activity in the embryonic cells.
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Reviewer‘s Comments: In conclusions, 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. Sections of the discussion are poorly written and cite data from other 

studies incorrectly (e.g., Sanderson, 2000; Crain 1997).

A.4.3 Fan W, Yanase T, Morinaga H, Gonda S, Okabe T, Nomura M, Hayes T, 

Takayanagi R, Nawata H. (2007a). Herbicide atrazine activates SF-1 by 

direct affinity and concomitant co-activators recruitments to induce 

aromatase expression via promoter II. Biochemical and Biophysical 

Research Communications. 355:1012-1018.

The authors were interested in confirming 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 in 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 (10-7to 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 simazine (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.
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Reviewer‘s Comments: Fan et al 2007a, b showed that atrazine binds directly to SF-1 and 

induces aromatase expression in vitro. It should be noted, however, that after aromatase 

activity is not affected after in vivo exposure in mammalian species.

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

T, Morohashi K, Hayes TB, Takayanagi R, Nawata H. (2007b). G Atrazineinduced aromatase expression 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 expression. 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., HIH-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 the 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. Reducing 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: Fan et al 2007a, b showed that atrazine binds directly to SF-1 and 

induces aromatase expression in vitro. It should be noted, however, that after aromatase 

activity is not affected after in vivo exposure in mammalian species

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

human H295R and rat R2C cell lines as in vitro screening tolls for 

effects on 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 tritiated-water 

release assay and promoter-specific mRNA levels were measured following exposure to 
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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 dexamethasone. 

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 experiments, 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 other 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 

competitive 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 occur 

rapidly. Appropriate controls were used to demonstrate the specificity of the aromatase 

assay and measurement of promoter-specific mRNA levels; solvent controls, 4-OHandrostenedione (known inhibitor of catalytic activity of aromatase), and 8-BR-cAMP 

(inducer 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 activity 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 that the change in these particular 

promoters may be related to an increase in cAMP, as this transcription factor is crucial for 

stimulation of this particular promoter. In contrast, no effects on aromatase activity was 

observed in the R2C Leydig cells following 3 or 24-hrs of exposure to atrazine or 8-BrcAMP. This cell line, believed to be under the primary regulation of promoter pII, is also 

simulated by cAMP. The authors report that basal aromatase activity in the R2C cell line is 

very high as compared to that of the H295R cells, and that it is possible that one or 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 conclusion, these data support earlier findings of atrazinedependent 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 atrazine does not alter enzymatic activity through a rapid (competitive or noncompetitive inhibition, allosteric modification ) alteration of the enzyme. However, failure to 

evaluate other promoters within the H295R cells is a limitation of this study, and stops short 
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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 identification of induction of aromatase gene 

expression. 

Aromatase activity (AA) : H295R cells displayed dose dependant increase in AA 

(0.3 , 10 and 30 uM) with 3-fold induction as compared to control at highest dose.

 LOEL 10 uM, NOEL 0.3 uM)

R2C cells = no sign. effect on AA or cell viability (at 30uM), NOEL 30 uM (Authors 

suggest lack of effect was likely due to constitutively high aromatase expression as 

compared with that observed for the H295R cells. 

Promoter-specific mRNA Levels: 

H295R cells – Following 24 hrs exposure, an increase in I.3 and pII transcripts was 

observed in cells treated with 30 uM, but was not sign. different than control or 8-BrcAMP. (Thus, it is still unclear whether or not cAMP and/or activation of these 

promoters are totally 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 print).

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 adenocarcinoma cells. 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 quantify 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 requiring 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 each 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 (ELISA), 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, 
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Appendix A - Page 231 of 677

the exposure of the cells to the test chemical 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) untreated cells during the 48 hrs culture were exposed to the test 

chemical only during the 1.5 hr assay period. 

Reviewer‘s Comments: Overall, the results of this study indicate that the increase in 

estradiol observed in the H295R cell assay was not due to a direct increase in aromatase 

activity. Atrazine, like forskolin (which activates the enzyme adenylyl cyclase and 

increases the intracellular levels of cAMP) enhanced steroidogenesis as evidence by 

increased T and E. In contrast, when atrazine and forskilin were tested in the ―direct‖ 

tritiated water assay, 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 demonstrate 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 activation 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 atrazine were comparable, and 

suggest similar mechanisms of activity even though exposure to forskolin resulted in 

greater concentrations of estrogen and testosterone, and greater aromatase activity at 

lower concentrations than observed for atrazine.‖ In contrast, 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) aromatase 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 aromatase 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) 
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Appendix A - Page 232 of 677
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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 difficult 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 controls) in Fig 17. 

is believed to be a reflection of different collection times, e.g, AA is that change 

which occurs in tritiated water release over a 1.5 hr period, versus E2 production 

over the 48 hrs in culture. 

- Although positive controls used to demonstrate specificity of aromatase assay 

(e.g., radio inert androstenedione and fadrozole), no concentrations for 

androstenedione reported and vague 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 stromal 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 the 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 E2 (PGE2, 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) was 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 protein concentrations, which indicated no change in cell number, suggesting 

that the atrazine induced changes in aromatase activity were not due to cell proliferation. 
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Appendix A - Page 234 of 677

The positive controls PGE2 (1.0 μM) and vomitoxin (250 ng/ml) induced a significant 

increase in aromatase activity (132 ± 10 and 149± 13% of control, respectively, p<0.05). 

There was also no effect of atrazine treatment on human recombinant aromatase activity in 

cell free test system. It was concluded that μM concentrations of atrazine can increase 

aromatase activity of human granulosa cells but not ESC and this effect is not elicited at the 

enzyme level.

Reviewer‘s Comments: The authors concluded that atrazine treatment induced an increase 

in aromatase activity in human luteinized granulosa cells, however, the concentrations 

needed to induce the 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, AitAissa 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 among 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 longer 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 signaling 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 the 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 effects of chemicals on aromatase gene 

expression, or changes at transcription or (post) transcription levels), and in untreated cells 

exposed to the test chemical for only 2 hrs (e.g., direct competitive effects on the 

aromatase enzymatic complex). OH-Androstenedione, 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 using the methyl-thiazol-tetrazolium (MMT) 

assay. 
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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. Approximately 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 longer 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 the 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 histopathology. Serum 

luteinizing hormone (LH), prolactin (PRL), and thyroid stimulating hormone (TSH) were 

analyzed. 

Pups in the ATR-ATR group were statistically significantly smaller than those in the control 

group on PND4 (p<0.01) (8.2±0.1 g ATR-ATR vs 8.7±0.1g, C-C), however, their mean 

weight was reduced 5.7% from 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 until the September SAP meeting.
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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 significant 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 animals 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 atrazine 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 substantially 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., who 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, utilizing JEG3 human placental 

cells, were performed. JEG3 cells contain modest amounts of SF-1. In one experiment, 

the effects of in vitro atrazine on luciferase activity after transfection of human placental 

JEG3 cells with aromatase-luciferase reporter and the parent reporter (pGL3-luciferase) 

with mSF-1 (mouse SF-1) and hLRH-1 (human liver receptor homolog) expression vectors 
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Appendix A - Page 237 of 677

were examined. The data revealed a dose-dependent increase in luciferase activity which 

the authors interpreted as activation of 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 also 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. 

Several 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 

JEG3 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 increased 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: Overall, 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 transfection of cells. Only a few poor quality results of PCR analyses and 

western blots were provided to corroborate the findings. The authors made several 
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Appendix A - Page 238 of 677

references to ―data not shown‖ to support their conclusions. The activation of important 

second messenger 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 an indirect activation of aromatase are also in agreement with the observations of 

Higley et al reviewed above. The experiment examining the effect of atrazine on zebra 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 potential 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 synthesis, 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 

concentration 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 doseand time-dependent with peak expression occurring after 4 to 24 hours of exposure. 

Atrazine metabolites, DACT and hydroxyatrazine, failed to show a consistent response. 
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Appendix A - Page 239 of 677

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

effects 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 

several theories on the possible MOA regarding the incidence of mammary tumors, 

delayed onset of puberty, and immune suppression.

The premise that there is an alternative MOA for mammary tumors in female SpragueDawley rats is that mRNA expression and aromatase activity was increased in H295R 

adrenocortical carcinoma cells when exposed to a minimum of 10μM atrazine. Increased 

cyclic AMP (cAMP) levels were correlated with the induction of aromatase activity by 

atrazine; increases in cAMP 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, deethyl atrazine (DEA), deisopropyl atrazine (DIA), diaminochlorotriazine (DACT), 

ammeline (AM), and hydroxyatrazine (hydroxyl-ATZ) used were approximately 97% pure. 

IBMX, 3-isobutyl-1-methylxanthine was used as the positive control. The IMAP 

phosphodiesterase assay kit was used along with bovine PDE at varying concentrations 

(0.000025 to 0.5 PDE U/ml in 96-well plates). To determine the effects of addition of 

atrazine after completion of the PDE reaction, an hour long incubation period was allowed 

and then to assess the effects of atrazine and its metabolites on PDE activity, the potential 

inhibitors were added at concentrations ranging from 5pM to 5 nM in 10 μl of assay buffer 

enzyme with additional 90 minute substrate/incubation period. Competitive inhibition 

studies were similarly conducted with the addition of varying concentrations of DACT with 

atrazine. Determining IC50 values was done using a 4-parameter log curve and creating 

sigmoidal 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. Maximal 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 IC50 for atrazine (39.52 μM) while for IBMX it was 1.749 μM. The IC50 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 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 concentrations 
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Appendix A - Page 240 of 677

of DACT, the IC50 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 IC50 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 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. 

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 potential human exposure to atrazine is low 

and equally unlikely to result in PDE inhibition in humans. The study goes on to endorse 

the NOEL (1.8 mg/kg/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.

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 neuroendocrine 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 nervous 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 Coban and Filipov 

(2007) and Rodriguez et al. (2005) corroborate previous findings on the effect of atrazine 
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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 diet 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 ventral 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 reported 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 striatum. 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 fully 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 histological 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 substantia 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 selective 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 

histological 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. 
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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 

available 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 activity following damphetamine 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 supported 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 vesicles (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 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. 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 mitochondrial 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 hormone (GH) secretion and the gender-based 

differences in GH secretion in rodents. The authors theorized that the differential in GH 

secretion may be linked to gender-related differences in the transduction of 

somatostatinergic mechanisms in brain and/or pituitary targets. As a result, the 

researchers concluded that 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-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 both males and females, the reliability of this study is 

uncertain given the numerous deficiencies (including poor description of the methods and 

poor quality of micrographs used to substantiate conclusions) identified in the study design 

and described in the summary review in Appendix A.

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 including 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 of 
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Appendix A - Page 243 of 677

deficiencies such as inappropriate statistical analyses and unexplained discrepancies in the 

sampling of pups from litters which are described in the summary review in Appendix A that 

call the validity of the results into question. 

More closely related to the postulated MOA for atrazine is the research conducted 

by Cooper et al., 2007 and Foradori et al. (2009) on the pulsatile release of GnRH neuronal 

activation after atrazine exposure. The research by Foradori and coworkers show that a 

brief (4 day) exposure to atrazine at doses ≥ 50 mg/kg/day resulted in a decrease in GnRH 

activation as indicated by a decrease in cFos activity concomitant to a significant reduction 

in the LH and FSH surges. Thus it appears that atrazine can induce changes in these 

hypothalamic neurons. These data, therefore, illustrate 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 atrazine 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 hormone) secretions which decrease GnRH secretion 

(Fraites et al.,2009; Laws et al., 2009).

In summary, there 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 atrazine 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 atrazine 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. Specifically, the focus was on somatic parameters and a wide range of reflexes to 

assess sensorimotor maturation. 
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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). Atrazine (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 growth 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, incomplete; 

and 3, full. On PND 2, 4, 9, and 12, pups were weighed. 

Evaluation of pup vocalizations (ultrasonic calls) was also performed on PND 4, 9, and 12. 

The authors provided adequate details on this evaluation. A spectrographic analysis of 

ultrasonic calls was also 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 highdose 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 pups when compared 

to controls on PND 9. However, the data (in contrast to controls) show a decrease (not 
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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. Moreover, 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 longer ultrasonic call mean interpulse 

(seconds) in atrazine-treated animals compared to control groups. However, the data 

indicate that this tendency was only apparent in the low-dose atrazine group of females or 

when data from males and females were combined. No differences were noted in the highdose 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 atrazine-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 their 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 reducing 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 sensorimotor 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 assessment 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 numbers 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 support the reliability of the methodology, the lack of objective 

and validated behavioral tests, lack of neuropathology, and the lack of actual spectrograms 

to corroborate the spectrographic results. The serious deficiencies of these studies raise 

questions about the validity and reliability of the data and its use for risk assessment 

purposes. 
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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 Neurochemistry 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 neurochemistry 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 gavage 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 tyrosine hydroxylase levels were determined by western-blot analysis. Fixed halfbrains were used to analyze neurons in the substantia nigra pars compacta and ventral 

tegmental area by immunohistochemistry/unbiased stereology. 

No effects of atrazine treatment 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 protein 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 day 22. Slight decreases in DOPAC and HVA were present at day 22. 

At day 64, there were no statistically significant effects on DOPAC and HVA. There were 

no effects of 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 tegmental area (33% cell loss in substantia 

nigra pars compacta vs. 28% cell loss in the ventral tegmental area). 
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Appendix A - Page 247 of 677

Reviewer‘s Comments: There are several limitations with this study:

The reason for a lack of a dose-response relationship with respect to the number 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 

graphs. 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 substantia 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 dopamine and its metabolites at day 64. 

However, these data (i.e., Figure 3 on page 1182 of the study) are contradicted by 

the description of the data in which the authors state that the ―some of the animals 

received MPTP on day 15‖ (no further details provided). MPTP has been associated 

with a Parkinsonian-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-
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Appendix A - Page 248 of 677

Dawley rats were incubated with up to 500 μM atrazine for 4 hours. The following were 

evaluated: tyrosine hydroxylase activity using L-DOPA accumulation as an indicator, high 

potassium-stimulated dopamine release, media dopamine levels in the presence of the 

dopamine uptake blocker nomifensine, and striatal dopamine metabolism. In addition, the 

effects of in vitro exposure to atrazine on LDH levels was performed to determine if any 

potential dopaminergic effects are caused by non-specific cytotoxicity. Dopamine and 

metabolites, 3,4-hydroxyphenylacetic 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 nomifensine 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 affecting tyrosine hydroxylase activity, but possibly by interfering with the vesicular 

storage and/or cellular uptake of dopamine.

Reviewer‘s Comments: The results revealed that atrazine, in vitro, alters striatal dopamine 

levels possibly by interfering with the vesicular storage and/or cellular uptake of dopamine. 

Tyrosine hydroxylase per se is not a target of atrazine. The relevance of these in vitro data 

to dopaminergic mechanisms in vivo is not clear given the high concentrations of atrazine 

examined in vitro and the complex interactions between various neurotransmitter and 

endocrine systems in the intact brain. The effects of atrazine on aromatic amino acid 

decarboxylase, monoamine oxidase and neuronal mitochondrial function are unknown. 

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

luteinizing hormone release without altering pituitary sensitivity to a 

gonadotropin-releasing hormone receptor agonist in female wistar rats. 

Biology of Reproduction. 81: 40 – 45.

The objectives 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 pulsatile 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 indwelling right atrial cannula 

(implanted on second day of treatment). Samples were examined for LH levels. There 

was a significant reduction in LH pulse frequency and a concomitant increase in pulse 

amplitude in the 200 mg/kg/day group. 
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Appendix A - Page 249 of 677

The second experiment 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 anti-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-Ala6-des-Gly-NH2

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 suppressed. Administration of GnRH agonist resulted in dosedependent 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 determine 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, intraatrial 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-Ala6-des-Gly-NH2

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-Ala6-des-Gly-NH2

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 are 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 study provides added support for the hypothesis that 

atrazine targets the brain to modify the neuronal control of pituitary function. Evaluating the 

secretion of gonadotropin 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. 
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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 Reproduction 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 interpreted these results to suggest a neural site of 

atrazine‘s action. In order to explore this possibility, the authors examined the effect of 

atrazine on the estrogen and progesterone-induced LH surge in freely moving, 

ovariectomized rats to determine if atrazine‘s inhibition of LH secretion coincides with a 

reduction in GnRH neuronal activation. The immediate early gene product FOS (cFos) was 

colocalized in GnRH neurons as a measure of cellular activity. In addition, withdrawal 

studies were performed to determine 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 neuronal 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 

atrazine withdrawal on hormone-induced LH surge and GnRH activation, ovariectomized 

animals were given a 4-day regimen of atrazine (200 mg/kg) by gavage. Control animals 

received vehicle only. An LH surge was induced in these animals either during the atrazine 

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 treatment 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 difference 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. 
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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 controlling 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 somatostatinergic 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, SST2, 3, 5) including possible 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 SST2, 3, 5 appear 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) and 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 GHand 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-gonadal 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 the study. Gestation day (GD) 14 

females were divided into 2 groups: one group (low-dose) received 1ug/kg day atrazine via 

drinking water from a syringe, while the second group (high-dose) received 100 ug/kg day 

atrazine. The authors noted that the procedure was monitored to make sure that pups did 

not 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 the 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 polymerase chain reaction was applied for the detection of 

SST2,3,5. In situ hybridization was utilized for mRNA. 
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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 swelling 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 observed 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 authors 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 dendate 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 did 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.
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The 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 differential 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 somatostatinergic 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 and 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 corticosterone.

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 striatal vesicles and synaptosomes caused by an in vitro exposure 

to atrazine and some of its metabolites. Toxicology 248: 52 – 58.

To better clarify the potential mechanisms of atrazine-mediated effects on striatal 

dopamine, this study examined the effects 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 preparation of synaptic vesicles and striatal synaptosomes, vesicular 

uptake of dopamine, and synaptosomal uptake of dopamine.
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Atrazine (1 – 250 μM) inhibited dopamine uptake into synaptic vesicles, and the inhibition 

was dose dependent. DACT did not affect vesicular dopamine uptake, while DE and DIP 

significantly decreased vesicular dopamine uptake. Kinetic analysis (nonlinear isotherm 

regression) of the vesicular dopamine uptake showed that atrazine decreased the Vmax

parameter but not Km. 

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) decrease 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 function 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 possibly by interfering with the vesicular storage and/or cellular uptake of dopamine. 

Tyrosine hydroxylase per se is not a target of atrazine. The relevance of these in vitro data 

to dopaminergic mechanisms in vivo is not clear given the high concentrations of atrazine 

examined in vitro and the complex interactions between various neurotransmitter and 

endocrine systems in the intact brain. The effects of atrazine on aromatic amino acid 

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 measured 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 7 
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Appendix A - Page 255 of 677

hours. The hypothalamus, prefrontal cortex, nucleus accumbens, and the striatum were 

removed from sacrificed animals 2 weeks after termination of the treatment. Monoamine 

and monoamine metabolites were measured by HPLC with electrochemical detection. 

Immunohistochemical and unbiased stereologic analyses of tyrosine hydroxylase from the 

substantia nigra pars compacta and ventral tegmental area were performed. Microdialysis 

was used to evaluate changes in striatal dopamine release after acute i.p. exposures to 

100 or 200 mg/kg atrazine in sessions lasting 7 hours. Animals were subjected to 

stereotaxic surgery and cannulation. Brain histology was performed on the 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 exhibited an enhanced 

horizontal locomotor activity response (70% relative to the 0 and 5 mg/kg groups) to damphetamine. 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 damphetamine 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 serotonin level were observed at the 5- and 10-

mg/kg/day doses. The 10 mg/kg/day dose was associated with a significant decrease (15 

– 20%) in norephineprine levels in the prefrontal cortex. The authors reported no changes 

in the metabolites of serotonin or dopamine 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 tegmental 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 authors 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.‖
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Reviewer‘s Comments: This study has a number of limitations, including:

 

No photomicrographs were provided to support and corroborate the findings of 

stereological examination of TH+ and TH- cell loss (only cell number data were 

provided), and no histological examination to describe actual morphological changes 

was discussed. There was no information on the calibration of the stereological 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-amphetamine-induced locomotor activity; 

inclusion of dopamine agonists and antagonists and L-Dopa in the experimental 

design would help support the findings.

 

The study did not include a positive 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.

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.

In an in vitro study using neuronal differentiation as an indicator of developmental toxicity, 

Solari et al. (2010) exposed cultured mouse P16 embryonic carcinoma cells to atrazine 

along with NaNO3 and NaNO2. 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 metabolite of vitamin A) metabolism and cause 

developmental toxicity since RA is an essential micronutrient 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 treatment, 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) using 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. 
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Appendix A - Page 257 of 677

Reviewer Comments: 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 limited 

(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 evaluation 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. The 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 phagocytic 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, lymphocyte 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 adult 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 examinations of lymphoid organs(e.g., spleen, thymus, bone marrow, lymph 

nodes) that would be useful in understanding the potential mechanisms of immunotoxicity. 

Consistent effects (e.g., decreased splenic and thymic weights and alterations in splenic 

and thymic 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 revealed significant decreases in 

resistance to the B16F10 tumor challenge at doses of 250 and 500 mg/kg/day atrazine, but 

not at 25 mg/kg/day. Host resistance assays are often considered to be the most relevant 

test for validating other methods and for extrapolating the potential of environmental agents 

to alter host susceptibility in the human population. 
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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 perturbations 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 neutrophils, 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 perturbations, 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 

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 time for impairments of the immune system not to assess the immunotoxicity 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 SpragueDawley 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 subcutaneously 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. 
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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 effects 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 development 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 mediated through mast cell 

degranulation and activation of the Gq/11-protein coupled neurosteroid receptor, 

phospholipase C and intracellular Ca2+ (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 expression on the 

dendritic cell surface. 

Atrazine-induced inhibition of in vitro human peripheral blood lymphocyte NK cellspecific lysis of K562 human leukemia cells may be the result of inhibition of lytic granule 

release, without 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 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-related orphan 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 vitro 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 are still not thoroughly 
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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 the subsequent disruption of the HPG 

axis. Furthermore, data regarding the developmental immunotoxicity potential of atrazine is 

inconclusive since the two available developmental immunotoxicity studies yielded 

conflicting results. Nonetheless, the available data do not indicate atrazine-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 departure 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 (NaHAsO4) in drinking water for 10 days before mating and during gestation. 

Other groups of NaHAsO4-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 

NaHAsO4, or a combination of atrazine + NaHAsO4 in drinking water. At the end of 

treatment, animals were sacrificed and bone marrow cells were collected from one femur, 

frozen and used for the colony forming unit-granulocyte/macrophage (CFU-GM) assay. 

Cells from the second femur were harvested, flushed with 1 mL of commercial RNA-Later 

to stabilize the RNA for later 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 hematopoietic cells containing 30% fetal bovine serum, 

bovine pancreatic insulin, human transferrin iron-saturated, 2-mercaptoethanol, interleukin3 and-6, and stem cell growth factor. Cell viability was determined; cells were adjusted to 

6.0 x 105viable cells/mL, seeded to yield a final density of 4.0 x 104viable 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 [33P]-labeled cDNA was obtained by converting 1 μg of total RNA into cDNA using 

reverse transcriptase in the presence of Mouse Cancer CDS primer mix. The cDNA was 
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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 primers 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 see after 

exposure to 1 mg/L atrazine alone. NaHAsO4 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/ NaHAsO4 treatment of females, compared to control. 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 NaHAsO4. However, the 

combined exposure to atrazine/NaHAsO4 demonstrated the overexpression of 20 

differentially expressed genes (DEGs). For the analysis of changes in gene expression of 

estrogen receptors alpha and beta For the analysis of changes in gene expression of 

estrogen receptors alpha and beta (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 variability 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 variation 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, Ashland, 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) 
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Appendix A - Page 262 of 677

(98.8%) in a 1% methylcellulose deionized water vehicle 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 – cyclophosphamide 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, hematological 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), progesterone, 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 tissue 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 functional 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 formulation 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 dose administration. There were no other test substancerelated 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 100 mg/kg/day dose groups 

when compared to the untreated vehicle control group. The decrease found in body weight 
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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 be  ̳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 subsets 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 consumption 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 control 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-days 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 hematocrit, 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 cytological 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 appeared 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 treatment 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 found 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 corticosterone 
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levels were unaffected by ATR treatment 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 effects 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 number 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 control 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 animals 

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 male 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 absolutely 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 atrazine 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., 
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decreased thymus 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 activity 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. 

Biochemical Pharmacology 65: 303 – 308.

The purpose of this study was to test the hypothesis that atrazine affects leukocyte function 

via nuclear receptors such as the glucocorticoid receptor (GR), the androgen receptor (AR), 

members of the peroxisome proliferator-activated receptor (PPAR) family, and the retinoidrelated 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 antiinflammatory and immunosuppressive properties and interfere with nuclear factor – κB (NFκB) 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 and 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). Control 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 procedures, plasmids (p1168hu.IL6P-luc+, p98.IL8p-luc+, p133.IL8pluc+, 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 TNFinduced IL-8 promoter activity, in contrast to DEX. This was demonstrated in stable cell 

pools transfected with an IL-8 promoter-driven reporter 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 a 
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Appendix A - Page 266 of 677

longer variant of the IL-8 promoter, p133.IL8p-luc+, yielded the same overall results (data 

were not shown). To test whether the results with the IL-8 promoter were promoterspecific, the authors also tested the effect of atrazine on other proinflammatory gene 

construct, containing the E-selectin 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 glucocorticoid-like properties and does not interact with the GR. 

No peroxisome proliferation was observed in the liver or kidneys of mice treated with 

atrazine. Moreover, no PPAR-mediated induction of promoter activity was seen on targets 

of PPARα, PPARγ, or PPARδ. 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 study 

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-related 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 effect 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 mononuclear 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 + phorbol 12-myristate 13-acetate. Information on the donors 

(selection, ethical approval, allergic disease description) and methods (PBMC cultures, 

cytokine assays, IgE plasma level determination, radioallergosorbent or RAST test to 

confirm Dpt allergy) was provided.

Atrazine treatment significantly 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 cytokines were limited, the impact 

of atrazine on allergic responses is unlikely.
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A.6.5 Filipov, N M, Pinchuk, L M, et. al. 2005. Immunotoxic effects of shortterm 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 

mononuclear 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 

gavage at doses of 5, 25, 125, and 250 mg/kg bw. 

Spleen weight was decreased at the 250 mg/kg dose level on day 1, while thymus weights 

were decreased at the 125 and 250 mg/kg/day dose levels. Decreased thymus weights 

persisted one week after the last dose of atrazine, but resolved by seven weeks. Spleen 

and thymus 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 cessation 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-dependently 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 phenotypes, both MHC II+ and CD19+ cells were dosedependently decreased, whereas only the high dose of atrazine increased the percentage 

of CD3+ cells. In addition, atrazine treatment decreased the number of splenic naïve T 

helper and T cytotoxic cells, whereas it increased the percentage of highly activated 

cytotoxic/memory T cells. The proportion of mature splenic dendritic cells (DC; CDIIchigh) 

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 250 mg/kg dose (high dose) at seven days after the last 

dose. In addition, at seven days, the MHC-II+ cells were also decreased at the high dose. 

The authors stated that 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 immune 
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Appendix A - Page 268 of 677

system of juvenile mice by decreasing cellularity and affecting lymphocyte distribution, with 

certain 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-mediated immune function and decreases host 

resistance to the B16F10 tumor model in female B6C3F1 mice. 

Toxicology: 15-28.

The purpose of this study was to examine the potential immunomodulatory properties of 

atrazine and was designed to assess changes in cellular 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), hematology, 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 treated groups. A significant 

dose-dependent decrease in spleen and thymus weights was also observed. Kidney 

weights were slightly but significantly reduced at the high dose. Hematological alterations 

included reductions in 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 alter 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 mice. The splenic CTLR was 

significantly stimulated in mice that were treated with both 25 and 250 mg/kg atrazine. The 

NK cell activity in the spleen was only inhibited in the 250 mg/kg/day group and the authors 

did not consider this effect to be of biological significance. A dose-related decreasing trend 

in MPS function across the treatment groups was noted. 
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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 105cells/mouse challenge level, 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 statistically 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 104 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 Ueda H. 2006. Endocrine disrupting chemical atrazine 

causes degranulation through Gq/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 and mediate histamine 

release, which, in turn, stimulates nociceptor endings. They previously demonstrated that 

neurosteroids cause extravasation as well as hyperalgesia, which were abolished by 

diphenhydramine, a representative H1 receptor antagonist. Neurosteroid-induced 

extravasation has been attributed to degranulation of mast cells. 

Degranulation of mast cells was determined by measuring the release of granuleassociated 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 binding 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, inositol 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 diphenhydraminereversible manner, these responses were also evaluated in male ddY mice. Atrazine (10 

fmol) was administered intraplantarly (i. pl.) into the hind paw. The total amount of time 

showing pain responses, such as biting and licking, was measured for 10 min. after i.pl. 

injection. 
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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 impermeable 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 indicated that the observation of atrazineinduced 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 U73122. Lanthanum chloride, which inhibits Ca2+ influx in cells, had no effect. The authors 

interpreted the findings to reveal that the signal transduction involved in the degranulation 

is mediated though Gq/11, PLC and mobilization of intracellular Ca2+, characteristics of 

neurosteroids. 

The results also demonstrated atrazine-induced pain response that occurred immediately 

and ceased within 5 minutes after i.pl. administration. The responses were dosedependent 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 Gq/11-

protein coupled neurosteroid receptor, phospholipase C and intracellular Ca2+ mobilization. 

Reviewer‘s Comments: This study concluded that the underlying mechanisms of atrazineinduced inflammatory effects such as nociception and plasma extravasation, may be 

mediated through mast cell degranulation and activation of the Gq/11-protein coupled 

neurosteroid receptor, phospholipase C and intracellular Ca2+ . However, the inflammatory 

responses were examined following intraplantar administration of atrazine, a route of 

exposure that is not likely to occur 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 that atrazine may affect dendritic cell (DC) function (Rowe 

et al. 2006; 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 factordependent, bone-marrow-derived immature murine DC cell line JAWSII. For comparative 

studies, limited studies were performed with primary thymic DC. Details were provided on 
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Appendix A - Page 271 of 677

the following materials and methods used for the analyses: dendritic cell line, primary 

thymic 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 concentration. 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 removes MHC-1 molecules from the DC surface. 

Reviewer‘s Comments: Pinchuk et al. (2007) provided in vitro evidence suggesting that 

atrazine directly targets dendritic cell maturation and decreases major histocompatibility 

complex 1 (MHC-1) molecule expression 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. 

Toxicological 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 biomarker 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 hazard identification study of atrazine. Female C57BL/6 X C3H F1 

(B6C3F1) mice (8 – 12 weeks of age following a two-week accommodation period) were 

used in the study. Atrazine was administered intraperitoneally (only once) at dose levels of 

100-, 200-, or 300-mg/kg bw in corn oil (group size was 5 mice). A naïve control group 

was included. The authors selected corticosterone as a mediator that might predict the 

quantity of immunosuppression caused by stress responses. Therefore, 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 responses to keyhole limpet hemocyanin, and natural killer cell 

activity. Models were derived describing the relationship between corticosterone AUC and 

potential immunological changes. 
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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 (corn 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 levels were measured. The 

authors reported the following corticosterone values: controls: 164 ± 88; atrazinetreatment: 676 ± 79. 

Linear regression models of corticosterone AUC vs. thymus- and spleen-cell numbers and 

subpopulations revealed that atrazine 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 splenocytes 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 responses 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 stated 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. 

Reviewer‘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 time 

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 atrazine. 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) were used in the study. Mice were dosed daily (intraperitoneally in 

corn oil) for 28 days with atrazine (up to 150 mg/kg/day) or were subjected to restraint, 
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Appendix A - Page 273 of 677

which can also induce neuroendocrine stress responses and alter immunological 

parameters. The exact 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, natural 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 corticosterone 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 percentage 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 spleen 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 both 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 HPA 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 treatments of atrazine (98% 

purity) and power-frequency electromagnetic fields (EMFs) on cutaneous mast cells in 
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juvenile/peripubertal male Wistar rats. The investigators chose to study the effects of 

atrazine and EMFs because they thought it likely that humans 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/day to EMFs (50 Hz, 

100-300 μT, 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 

treatments was previously described in Rajkovic et al. 2005a22. Dose levels were selected 

based on previous studies on atrazine effects on prepubertal rats (Stoker et al. 2000b23). 

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-μm-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 stereologically 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 

intact 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 SpragueDawley 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 development. 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 indicated 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):197-252
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induced developmental endocrine dysfunction to examine the potential for atrazine to 

indirectly induce immunotoxicity.‖ The authors stated that this dose is likely to induce 

endocrine effects in both sexes. Separate groups were exposed to bromocryptine (BCR) at 

0.2 mg/kg/2x/day to induce hypoprolactinemia or to propylthiouracil (PTU) at 2 mg/kg/day 

to induce hypothyroidism. At 7 weeks old, the following immune functions in the offspring 

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 increase 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 has 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 offspring 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 treatment 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 the 
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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 exposure 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 

the 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 

same amount of atrazine every minute of every hour of every day until the end of the 

treatment period. The rate of exposure at the 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/lactational 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 

stimulation was assessed using two different assays. In one assay, splenocytes were 

isolated from unimmunized prenatally/lactationally exposed Balb/c mice and stimulated 

using irradiated splenocytes from female C57B1/6 mice. Proliferation of alloreactively 

stimulated lymphocytes was assessed by measuring the incorporation of 3H-thymidine into 

newly synthesized DNA following co-culture with alloreactive stimulator cells and incubation 

with 3H-thymidine. In another assay, the ability of the T cells to lyse alloreactive target cells 

(i.e., cytotoxic T lymphocyte response), was assessed following alloreactive stimulation. 

EL-4 lymphoma cells were used as targets. The humoral immune 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 were observed in 

the number of CD4+T cell, CD8+ T cell, and B220+ B cell subpopulations in the spleen. 

Male mice (3-month old) exhibited an increase in both T cell proliferation (atrazine 

offspring: 67 ± 5 x 103cpm; control offspring: 46 ± 4 x 103cpm) 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 secreting cells (ASC) per 106 B220+ 

cells compared to 58.6 ± 3.0 ASC per 106 B220+ cells for male control offspring. Threemonth-old female offspring showed no significant difference in immune response from 
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controls. The authors concluded that the results indicate ―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 results 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-producing B cells in the spleen of HKSPvaccinated 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 also 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-old 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, CD8, and B220) was not significantly different 

between the two groups. Based on the findings of the 3- and 6-month evaluations, the 

study authors concluded that the findings ―demonstrate a sex- and age-dependent effect of 

prenatal exposure to atrazine on the immune system of the adult offspring.‖

Reviewer‘s Comments: Rowe et al. (2006) reported 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 used to assess immune response.

A.6.14 Rowe A M, Brundage K M and Barnett J B. 2007. In vitro atrazineexposure 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 clarify 
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precise changes 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 lymphocyte assay, enrichment 

of NK cells, flow cytometry, NK cell adhesion, perforin ELISA and western blots.

Results showed that atrazine decreased NK cell function of PBLs under the in vitro

experimental conditions, confirming observations by other investigators, 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 cytometry. 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 

following 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 inhibition 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 stressinduced immunosuppression in mice using blood parameters. 

Toxicological Sciences 83: 101 – 113.

This study is an extension of the Pruett et al. (2003) study described above except that the 

blood rather than the spleen was of 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.
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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 

Summary: 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 after chronic exposure and the lack of 

mechanistic data suggesting a MOA that could be evaluated for human relevance. It 

should be noted that no other tumor sites were found in rats and there were no tumors 

induced in mice. 

Since the 1986 assessment, an 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 show 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 endpoint 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 prolactin 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 follicles 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 the 
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Appendix A - Page 280 of 677

absence of constantly elevated estrogen and prolactin levels mammary gland tumors would 

not develop in humans as a result of atrazine exposure.

Several genotoxicity and carcinogenicity studies using transgenic and chemicallyinduced 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 these 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 that it makes the tumors already present in the system 

more aggressive. In vitro studies – for the most part – corroborate the findings in the in 

vivo studies and show that atrazine is cytotoxic for most of the cell-lines tested.

Work by Fujimoto and coworkers 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 exposure 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 were 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 thyroid hormone levels (T4, T3, and TSH). DMBA-induced ovarian carcinogenesis 

was yet another model used to study atrazine‘s potential to affect tumor progression. Using 

this model, Tanaka et al. hypothesized that the operative mode of action leading to ovarian 

adenocarcinomas involves hormonal disruption (particularly estrogen). Therefore, they 

postulate that exposure to atrazine (which has been associated with hormone 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 considered 

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 carrying 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 dose-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, atrazine exposure in vitro did not cause MCF-7 cell proliferation – an 
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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 

carcinogenic property of the compound or if it is due to the genetic background resulting 

from the presence of a human proto-oncogene in the cells. 

Ueda et al. conducted a similar set of experiments in 2005 but instead of using a 

transgenic rat model they used a DMBA-induced mammary carcinogenesis model. In this 

study the effect of atrazine exposure on the progression of DMBA-induced mammary 

tumors in ovariectomized Sprague Dawley rats was investigated. When DMBA-induced rats 

were examined, no clearly discernible correlation between atrazine exposure and the 

number of tumor-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 cell 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 nonsteroidogenic 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 inducing 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 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 related to other chemicals in the formulation 

rather than atrazine per se. Sister chromatid exchange (SCE) analysis – a sensitive 
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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 following atrazine 

exposure would ameliorate atrazine-induced effects are available. After atrazine exposure 

at a high dose of 300 mg/kg/day, Singh et al. (2008) report a marked increase in lipid 

peroxidation and decrease in acetylcholinesterase activity and cell 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 instability 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 effects 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 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.

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 evaluation. 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 weighed 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 discussed
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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, uterine, 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 

and 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 control or 

atrazine treated groups as opposed to 9 days for animals treated with estradiol. 

Reviewer‘s Comments: The current study had a number of deficiencies including the lack of 

data on the purity of the atrazine used in the study or the sample size. Nonetheless, 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 considered 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 Tsuda, H. (2004). Possible enhancing 

effects of atrazine and nonylphenol on 7,2-dimethylbenz[a]anthraceneinduced mammary tumor development in human c-Ha-ras protooncogene transgenic rats. Cancer Sci. 95(5):404-410.

This study evaluates the potential of atrazine 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-hundred seventy six female and male Tg and 

non-Tg rats were administered a single 25 mg/kg dose of DMBA via gavage at an age of 

50 days. Beginning on the following day, animals were exposed in the diet to 0, 5, 50, or 

500 ppm atrazine (purity unknown) [≈ 0, 0.55, 6.2, or 36.4 mg/kg/day (♀) and 0, 0.26, 2.75, 

or 24.23 mg/kg/day (♂)]. The Tg females were exposed to atrazine for 8 weeks while the 

non-Tg females and all males were exposed to atrazine for 20 weeks. At the end of the 

exposure period, animals were sacrificed by exsanguination, the numbers of grossly 

observed tumors were recorded and subsequently measured and subjected to 

histopathological examination. In addition, body, liver, kidney, ovarian, uterine, and testis 

weights were recorded. Levels of serum atrazine were measured after necropsy by liquid 

chromatography and mass spectrometry (LC/MS). The potential impact of atrazine on cell 

 

25 In this evaluation only the results for atrazine will be discussed
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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% (control: 

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-statistically 

significant increase in the incidence of mammary adenocarcinomas (100% vs 56% control) 

was also reported at this dose level. No data were provided on non-Tg rats (males or 

females). 

The study authors concluded that atrazine appeared to enhance 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 addition, the purity of the atrazine used in the study was not 

reported, no exposure concentrations were provided for the MCF-7 experiment, and no 

data were presented for the non-Tg rats or for the MCF-7 cell proliferation assay.

Reviewer‘s Comments: The lack of data on non-Tg rats is a critical deficiency in this study. 

In experiments conducted by Ueda et al. using non-Tg rats treated with DMBA to induce 

mammary tumors, a relationship between atrazine exposure and increases in the incidence 

of tumor-bearing 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 lack of dose-response on the incidence of mammary adenomas and 

adenocarcinomas further confounds the interpretation of these data. 

A.7.3 Garaj-Vrhovac, V. and 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.

In this study the authors evaluated possible genome damage caused by two pesticide 

formulations (Bravo® and Gesaprim®) containing alachlor and atrazine as active 
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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 ingredients. 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 the actual conditions.

Gesaprim®, containing 500 mg/ml of atrazine as the active ingredient were given at the 

doses of 1.08 ml/kg (540 mg/kg a.i.) and 0.07 μl/kg (3.5 X 10-2 mg/kg a.i.) to five male CBA 

mice assigned to 4 treatment groups and a control group injected intraperitoneally once. 

The lower dose represents the 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 moment. 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 during 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 control. 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 higher 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 kidney 
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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 methods 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 atrazine findings. For example, the authors stated, ―genome damage for both 

pesticides was found to be higher for the higher dose given‖. It was noted, however, that 

the ―dose response‖ is very flat considering that more than two orders of magnitude 

separate the 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 comet 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 

related 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 cytotoxicity and the morphological changes were followed 

during cell death.

CHO-K1 cells were treated with atrazine at concentrations ranging 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 dose levels, with the toxicity progressively 
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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 60 ug atrazine/mL and 55% at 80 ug atrazine/mL, both at 

p<0.001.

IC20 and IC50 values were determined after 24, 48 and 72 hours for the four methods. A 

comparison of the IC20 values after 48 hours or IC20 and IC50 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 IC50 value for trypan blue after 72 hours 

was two times lower than the IC50s for the other 3 assays. Morphologically, the level of 

apoptosis in atrazine treated cells was low.

Reviewer‘s Comments: Cell proliferation was decreased in Chinese Hamster ovary (CHO) 

cell lines at relatively low levels of atrazine exposure.

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 Expression of MCF-7 Human Cells. 

Journal of Proteome Research.

The present study aims to investigate the proteomic effects of endocrine disrupting 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. Proteins 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, digested 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 were 

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 databases using GPS Explorer 

Software v3.6. The results 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 tryptophan (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 

significant homology were considered. The acceptance criteria allowed for ion scores greater than 

26 or 43 indicate homology and identity, respectively.
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The results for morphology and adhesion of MCF-7 cells showed different observations between 

the four conditions 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 atrazine, 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 different proteins. Out of 

these, 22 different proteins were identified by at least two peptides, with a sufficiently high ion 

score. Some proteins (far upstream element binding protein, histone H1b, actin, heat shock 70 kDa 

protein 8) were identified in more than one spot, suggesting the presence of isoforms and/or posttranslational modifications that affect the pI and/or the molecular mass of the protein. 

Identified proteins were classified with respect to their subcellular localization and biological 

processes, 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 effects of atrazine and 

PCBs on the Proteome of MCF-7 Human Cells showed that among the 22 differentially expressed 

proteins, 10 were specifically 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 treatment 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 proteins, 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 

nor 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 also 

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 expression 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 future studies should target 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 is 
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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 exposure to 

atrazine: the non-dose-dependent increase in homologous 

recombinational events does not support its genotoxic mode of action. 

Cytogenet Genome Res 104: 315-319.

Human peripheral lymphocytes, collected from seven healthy volunteers, were grown, 

stimulated with phytohemagglutinin and exposed to atrazine (unreported purity) 

concentrations ranging from 5 to 220 μg/mL atrazine, the solvent (dimethyl sulfoxide ) or 

the positive control (mitomycin C) for 24 hours. Three separate experiments were 

conducted. Prior to the end of 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 at 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 collected, stained by the 

fluorescent-plus-Giemsa technique and 30-50 cells were scored for each culture; triplicate 

cultures were prepared per treatment group.

Results from the first experiment showed that concentrations ≥220 μg/mL atrazine could be 

scored for SCE induction in G2-phase cells while no metaphases were found at 120 and 

220 μg/mL in the colcemid-treated cultures. Thus, SCEs could be scored even at levels 

that were lethal in the conventional assay and there was no significant increase in SCE 

induction at 60-220 μg/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 μg/mL 

did not increase the frequency of SCE induction in G2-prematurely condensed 

chromosomes (PCCs). Using cells treated with 20 μg/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, short-term atrazine 

exposures on human fibroblast cells in culture by assessing cell proliferation and apoptosis.
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Normal human fibroblasts, DET 551, were used in the saturation density analysis to 

measure cell proliferation. In this assay, DET 551 cells were plated at 2x105cells/100 mm 

dish in MEM with 0.5% FBS (low serum medium). Cells were incubated at 37oC with 5% 

CO2 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% CO2. After harvesting, 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 compared 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 calorimetric 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 increasing 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 plates 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 and 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% CO2. Then 150 μl of MTT solution was added and 

cells were incubated for an additional 4 hours at 37oC. HCl in isopropanol was added to 

each well, and 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 DMSO 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 

measured using the CaspASE colorimetric assay system protocol. The CaspASE assay is 

a colorimetric assay that measure activity of caspase-3. Cytosolic thiol proteases called 
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Appendix A - Page 291 of 677

caspases are key enzymes involved in apoptosis. DET 551 cells were exposed to 

increasing concentrations of atrazine or DMSO (0, 3, 100, 200, and 500 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 exposed 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 to 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 significantly 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 proliferation 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 nucleasepositive 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 low levels of atrazine (as low as10 μg/L) for a short 

exposure (48 hours) causes a decrease in cell 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 properties 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. 
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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 Mutagen 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 properties, was tested at 0.1 to 20 

μg/mL Q. Bulbs were also exposed to atrazine (unreported purity) at 0, 2.5, 5.0 or 7.5 

μg/ml and varying combinations of Q and atrazine (0.5 or 5 μg/mL Q with 2.5, 5.0 or 7.5 

μg/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 ―crochet 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 index (MI) and a total of 400 metaphases and 400 ana-

/telophases (40 metaphases /slide and 40 ana-/telophases/slide) were scored for each 

treatment group. 

No effects were observed on root morphology after exposure to Q or atrazine. Similarly, Q 

alone did 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 μg/mL); no cytotoxic effects were 

noted at lower doses. Combined with either 0.5 or 5 μg/mL Q, 7.5 μg/mL atrazine affected 

a slightly lower reduction in the MI (~23% ↓ for both Q concentrations) then when the 

pesticide was tested alone. Neither Q nor 2.5 or 5.0 μg/mL atrazine were clastogenic; 

however, 7.5 μg/mL atrazine induced a significant (p<0.001) increase in structural 

chromosome aberration. The most prominent type of aberration was fragments. When the 

plant cells were assayed with 0.5 μg/mL Q in combination with 7.5 μg/mL atrazine, the 

frequency of chromosome aberrations was listed as significantly (p<0.05) lower (~43% ↓) 

compared to 7.5 μg/mL atrazine alone. However, no increased protection was afforded by 

the higher concentration of Q. 

Reviewer‘s Comments: The study was well-conducted and these data provide evidence of 

a clastogenic response induced by atrazine at 7.5 μg/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

μg/mL atrazine would likely exceed the recommended level of cytotoxic and no cytotoxicity 

or clastogenicity was noted at ≤ 5.0 μg/mL.
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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

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 endpoints 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 20 % 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 doserelated effects on AP activity, the investigators concluded that the inhibition of intestinal 

cells maturation could 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 

integrity 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 250 
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μM atrazine had a ―strong effect‖ on the ―intestinal cell monolayer development 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 largely 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, harvested 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 support 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 

cytotoxicity (~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 

treated 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 cytotoxicity 

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 oxidative 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 + vitamin 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 (SOD), 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. 
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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 and 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 included 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. 
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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.

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 acetylcholinesterase (AChE) activity, lipid peroxidation 

(LPO), and lipid composition (total lipid content, phospholipid phosphorus, cholesterol). 

Protein content was also determined. Scanning electron microscopy (SEM) was used to 

examine morphological alterations of erythrocytes. 

Atrazine treatment resulted in significant (p<0.05) decreases in AChE activity in 

erythrocytes at 7 days (22.32%), 14 days (25.58%), and 21 days (26.96%), compared to 

controls. Atrazine/Vitamin E treatment 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 with 

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 controls. 

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 membranes 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) 

decreased 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 

atrazine treated group.
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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 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.

The protective effect of an antioxidant, vitamin E, against atrazine-induced genotoxicity was 

investigated in young (~4 -5 weeks old, based on the weight range of 100-120 g)26a 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 rats 

were administered oral gavage doses of corn oil, 100 mg/kg/day vitamin E, 300 mg/kg/day 

atrazine or a combination of comparable doses of vitamin E and atrazine for 7, 14 or 21 

days. At the end of treatment, peripheral blood and liver were collected simultaneously 

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 time-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 samples 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. The 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 would be approximately 4-5 weeks old.
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mg/kg/day) plus vitamin E (100 mg/kg) caused significant (p<0.001) reductions in the 

frequency 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 than 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 Carcinogenesis 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 The goal of the study was to 

assess the impact of atrazine (with or without concomitant tamoxifen exposure) on thyroid 

hormone changes and thyroid lesions induced by DHPN.

Twenty male F344 rats per group received a subcutaneous injection of 2800 mg/kg DHPN 

or saline. Starting one 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 groups) was collected 

and used to measure thyroid hormones (T3, T4, 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 

only (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, exposure 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 there is a statistically 

significant decrease in T4 levels (↓16%, p<0.05) after exposure to DHPN + 500 ppm 

atrazine relative to exposure to DHPN alone and no changes in T3 or TSH. When 

compared to the control group, the decrease in T4 is ≈10% and non-statistically significant. 

 

27 In this evaluation only the results for atrazine will be discussed
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Furthermore, exposure to 500 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 T4. This 

suggests that the apparent decrease in the hormone after exposure to DHPN + atrazine 

may be related to the increase above background caused by DHPN and not indicative of an 

effect of atrazine on thyroid hormone homeostasis.

Reviewer‘s Comments: The results of this 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 relative organ weights observed are most likely related 

to this decrease in body weight rather than a direct effect on the organs. Similarly, thyroid 

hormone assessments did not suggest a direct effect of atrazine on thyroid hormone 

homeostasis. It should be noted, however, that this report does not include information on 

the purity of the atrazine used in the study and that the histopathological data are not 

provided.

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.

The effect of atrazine on DMBA-induced ovarian carcinogenesis was investigated in this 

study. The authors hypothesis is that 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 series 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 

atrazine (purity unknown) for 50 weeks. Another group did not receive a DMBA injection 

but were exposed to 0 or 500 ppm atrazine through the diet. On week 51, the animals 

were sacrificed, necropsied and tissues with gross lesions, as well as, the liver, kidney, 

mammary gland, lung, and heart subjected to histopathological evaluation. In addition, 

immunohistochemistry evaluations for proliferating cell nuclear antigen (PCNA), expression 

of the androgen or estrogen 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 slightly 
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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-alone. 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-labeling after treatment with DMBA alone was markedly increased (↑1644%) over 

unexposed animals. Atrazine exposure did not affect PCNA labeling index. Similarly, 

immunohistochemistry assessments did not reveal an impact of atrazine exposure on the 

expression of the BRCA-1 protein or the estrogen or androgen receptors after DMBA 

treatment. 

Reviewer‘s Comments: The study authors 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 incidence 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 twoorder 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 progression. 

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 

subgroups (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 terminal 
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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 tissue remained after the ovariectomy, the 

animals were removed from the study. Thus the number of Group A animals treated with 

the various concentrations of atrazine was between 18-21/group and Group B animals 

treated with atrazine ranged between12-19 rats/group. 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, prolactin, and luteinizing hormone levels were 

measured for Group A animals.

No effects on body weights or organ weights were reported for any treatment group. 

Mortality was higher in Group A (rats with early tumors) than Group B (rats with late 

tumors) but there was no apparent 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 types 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 Group 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.
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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 not 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 (appearing after ovariectomy), number of 

tumors/rat or the incidence of carcinomas. Moreover, the regression of palpable tumors 

observed after ovariectomy was similar for rats in the control and atrazine-treated Group A 

rats. Although for Group B a statistically significantly higher incidence of tumor-bearing rats 

is seen after exposure to 500 ppm atrazine relative to the Group B control, it 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%). When 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 tumor-bearing rats in the Group B control was substantially lower than in the Group A 

control. When both control groups are combined (Group A + Group B), the incidence of 

tumor-bearing rats at the Group 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. 22
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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 response 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 significant 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- and post-week 19 tumor development) were combined, the 

increase was no longer evident.

While there are a number of deficiencies in the conduct, interpretation, and reporting in the 

Ueda study, these data suggest that atrazine in an of itself does not induce mammary 

tumors in ovariectomized rats treated with a mammary tumor inducer thus demonstrating 

the importance of the ovarian hormones in the development of tumors in the mammary 

gland. 
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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], hydroxyatrazine [OH-ATR]) was evaluated in 

four cell lines. The purity of the compounds was 97%. The four cell lines used were HT-22 

cells (immortalized 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 viability assay, cells were seeded 24 hours before initiation at varying densities 

depending on the cell type in 96-well plates: H295R cells – 25,000 cells/well, JEG-3 cells –

3500 cells/well, MCF-7 cells – 15,000 cells/well, and HT-22 cells- 3500 cells/well. Cells 

were exposed to atrazine or its metabolites at concentrations ranging from 0.001 μM to 300

μM in physiologically buffered saline (PBS) or in sterile water with dimethyl sulfoxide 

(DMSO) for up to 72 hours. After exposure, cell viability was measured 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 triplicate 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 μM) or PBS 

(0.0003-300 μM) 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 measures

hydrogen peroxide (H2O2). 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 μM) dissolved in DMSO 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 of 
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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; H2O2 – 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 and 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 by cellular production of hydrogen peroxide (H2O2).

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 H2O2 24 hours. Atrazine and 

its metabolites did not affect the cytotoxicity of glutamate, 3-NPA, or H2O2. 

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/MITOCHONDRIA FUNCTION

Summary: Insulin/Mitochondria Function

In a recent publication in the electronic journal PLoS ONE, Lim et al. (2009) suggest 

that atrazine exposure leads to insulin resistance, fat accumulation, and increased weight 

gain by increasing blood glucose levels and lowering energy expenditure. The authors 

postulate that atrazine disrupts mitochondrial function by inhibiting mitochondrial 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 ubiquinone 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 mitochondrial electron flow signals the over-fed state to produce insulin 

resistance).
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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 authors 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. Moreover, 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, KU, 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 have performed a series of in vivo and in vitro experiments to assess the 

extent to which low levels of atrazine in water effect mitochondrial function and insulin 

resistance. The authors have treated Sprague-Dawley rats with 0, 30 or 300 μg/kg/day 

atrazine in drinking water for 5 months. [These levels of 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 stated that there were no differences in water consumption 

during the study. The manuscript does not describe the vehicle or how often the water was 

prepared. In addition, the manuscript does not provide any information on the 

concentration of atrazine in the water or estimates of the water intake during 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, 
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Appendix A - Page 307 of 677

serum insulin, total cholesterol and triglycerides, intravenous glucose tolerance test, high 

insulin challenge (hyperinsulinemic-euglycemic study), energy expenditure, 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, the methods section 

lacks some important information and references to the methods used. For example, the 

methods state that motor activity 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. Based on the results section, atrazine at these doses 

increased body weights in both normal and high fat diet, although, there is a flat dose 

response relationship. The lowest dose has an equivalent effect of the highest water 

concentration. Similarly, 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: Fasting plasma glucose levels of atrazine treated rats on the 

regular and high fat diets 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 near maximal effects on mitochondrial function at 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. 
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Appendix A - Page 308 of 677

Most notably, the results of this study are based on the statistical procedures used (which 

may be inappropriate). Preliminary analyses by Agency scientists do not confirm the 

results reported in the study. This study is not sufficiently robust for use in a regulatory 

context and should be repeated to confirm the findings.

A.9 PHARMACOKINETICS: INTERNAL DOSIMETRY, METABOLISM, & TIME 

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 MALDITOF/TOF MS (Matrix-Assisted Laser Desorption Ionization- Time 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 procedures 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 an 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.
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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 authors 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 polyacryamide 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 by 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 collected 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. 
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Appendix A - Page 310 of 677

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

inhalation NOAEL using a physiologically based pharmacokinetic 

model. January 29, 2010.

Physiologically based pharmacokinetic (PBPK) models can be used for extrapolation of 

internal dose across different species and routes of exposure. In this study, a previously

published PBPK model (McMullin 2007) for orally-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 

based 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 confinements 

by themselves without food and water would likely alter the estrous cycle. Therefore, a wellparameterized PBPK model offers an alternative approach for estimating inhalation 

exposures of ATRA associated with a given internal dose metric. The rat oral NOAEL of 

6.25 mg/kg/day used in this study was based on a delay in preputial separation from a 

study aimed at examining the 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 exposures that 

resulted in the same AUC (reported as 0.58 mmol/L/day) as the rat oral NOAEL of 6.25 

mg/kg were 4.5 and 5.1 mg/m3for rats and humans, respectively.

Since a PBPK model accounts of interspecies differences in pharmacokinetics, the 

uncertainty factor of 10 (i.e., 101⁄2for pharmacokinetics and 101⁄2for pharmacodynamics) 

used for interspecies extrapolation can be reduced to 101⁄2(i.e., 3.2) to account only for 

pharmacodynamic differences. Thus, the composite UF of 100 (i.e., including UF of 10 for 

intraspecies extrapolation) can be reduced to 32 with the use of a PBPK model. Margin of 

exposure (MOE) estimates were derived based on the PBPK model-derived inhalation 

NOAELs and 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.
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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 relatively 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 timecourse studies with [14C]-atrazine. J. Toxicol. Environ. Health, 66(A):941-

964.

This manuscript describes the development of a physiologically based pharmacokinetic 

model in an attempt to estimate total plasma chlorotriazines (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 (postulated 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 conjugation. 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 through 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 elimination 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 glutathione conjugates) reported in the 

single dose study as well as at all doses in the repeated dose study. The accumulation of 

ATRA-derived reactivity in RBCs was described in the model by assuming a reaction with 

hemoglobin. A second order rate constant was estimated by fitting the radioactivity in RBCs 

reported at the different administered doses in the repeated dosing study. After 

incorporating RBC reactivity, it was noticed that the model consistently underestimated the 

single dose plasma data at the longer time points. In an effort to better describe this 

dataset, plasma protein binding/reactivity was also included. A binding rate constant was 
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Appendix A - Page 312 of 677

estimated based on albumin, the major plasma binding protein of known concentration and 

turnover, by fitting the 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 

the 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 onecompartment model is described 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. (2007). 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 metabolism 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 hypothalamic 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 necessary to conduct human health 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. Atrazine 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 with 14C-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 saturable, 
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Appendix A - Page 313 of 677

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 triazines. 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 model 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 

metabolism; and a lumped rest of body compartment to account for the remaining 

tissues.

In vivo time-course pharmacokinetic data for individual chlorinated triazines

Female Sprague Dawley rats were dosed by oral gavage with 150 mg/kg bw 

atrazine or an equimolar amount of one of its chlorinated metabolites: ETHYL, ISO, 

or DACT. Plasma samples were analyzed for all four chlorinated species. 

Sequential Parameter estimation for individual model structures

The following model parameters were estimated sequentially 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 metabolism 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 describe 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 evaluation 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. The authors hypothesized that the double-peak phenomenon observed 
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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 the GI tract contributes to the complex time-course 

behavior. Addition of pre-systemic intestinal metabolism of atrazine to monoalkylated metabolites was also necessary to adequately describe the bioavailability 

of atrazine without adjusting the in vitro-derived metabolic parameters.

Reviewer‘s Comments: The current study underscores some of the parameters that may 

contribute to the complex 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 significantly underpredicts plasma levels of chlorinated 

metabolites after 8 hours while underpredicting atrazine levels at all time points.

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

herbicide 2-chloro-4-(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.

Mice are increasing being used to assess the potential adverse effects 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 levels of ATRA and its 

metabolites found in plasma and urine, the most accessible biological fluids. Thus, 

following an overnight fast, mice (5-8/dose/time) were administered a single dose of 0, 5, 

25, 125, and 250 mg/kg bw ATRA in corn oil by oral gavage. Plasma and tissue levels were 

measured as soon as 0.5 hours following dosing while urine and feces were collected at 

24-h intervals. 

Plasma: Levels of ATRA exhibited a biphasic behavior; maximum levels were seen at 0.5 

hours post dose, followed by a rapid decline before a 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 diaminochlorotriazine (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 hours (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 (r2= 0.987).

Urine: DACT levels were 2 orders of magnitude higher (as high as 5400 μM) than ATRA or 

its mono-dealkylated metabolites in the 0-24 hr time period.
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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 compound.

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 biphasic 

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 

information 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 investigators 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 were measured but the focus of this study is on development of the 

mammary gland following in utero exposure. 
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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 4th and/or 5th inguinal 

mammary gland were whole mounted and examined them subjectively using a 1-4 scoring 

system (1 = poor development/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 patterns (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 

controls. 

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 authors 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 require 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 responseadditivity 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 evaluations. 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. 
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Appendix A - Page 317 of 677

In the specific case 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 mixture 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 and 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 potentially 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 

data 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 or 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 scoring 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 above, 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 characteristics (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 currently available information, the mixture study, as currently 

reported, is not sufficiently robust for use in setting regulatory endpoints. 

A.11 OTHER
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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 

confounding 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 thoroughly 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 exclusion 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) and 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 treated 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 based 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, and 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 pituitary cells were assayed after 0.5, 1.0, 2.0, and 
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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. Contrary 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 decreases 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 remarkable 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 antibodies 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. They 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 LH 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 formation 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 that 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 of
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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 increase 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 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 

several theories on the possible MOA regarding the incidence of mammary tumors, 

delayed onset of puberty, and immune suppression.

The premise that there is an alternative MOA for mammary tumors in female SpragueDawley rats is that mRNA expression and aromatase activity was increased in H295R 

adrenocortical carcinoma cells when exposed to a minimum of 10μM atrazine. Increased 

cyclic AMP (cAMP) levels were correlated with the induction of aromatase activity by 

atrazine; increases in cAMP 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, deethyl atrazine (DEA), deisopropyl atrazine (DIA), diaminochlorotriazine (DACT), 

ammeline (AM), and hydroxyatrazine (hydroxyl-ATZ) used were approximately 97% pure. 

IBMX, 3-isobutyl-1-methylxanthine was used as the positive control. The IMAP 

phosphodiesterase assay kit was used along with bovine PDE at varying concentrations 

(0.000025 to 0.5 PDE U/ml in 96-well plates). To determine the effects of addition of 

atrazine after completion of the PDE reaction, an hour long incubation period was allowed 

and then to assess the effects of atrazine and its metabolites on PDE activity, the potential 

inhibitors were added at concentrations ranging from 5pM to 5 nM in 10 μl of assay buffer 

enzyme with additional 90 minute substrate/incubation period. Competitive inhibition 

studies were similarly conducted with the addition of varying concentrations of DACT with 

atrazine. Determining IC50 values was done using a 4-parameter log curve and creating 

sigmoidal 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. Maximal output, of 0.025 PDE U/ml was used for 

all subsequent assays. Addition of atrazine after completion of the enzymatic reaction 
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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 IC50 for atrazine (39.52 μM) while for IBMX it was 1.749 μM. The IC50 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 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 concentrations 

of DACT, the IC50 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 IC50 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 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 atrazine. 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 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 appears to be protective of any potential effects in humans.
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Appendix B1 - Page 322 of 677

APPENDIX B-1: EPIDEMIOLOGY LITERATURE 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 researchers 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/Medline 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 below:

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 aggregate stud*[tw])

Web of Science: atrazine OR (triazine* AND (pesticide* OR herbicide*))) AND 

(epidemiolog* OR cohort* OR (case-control OR case-control's OR casecontroled OR case-controlled OR case-controlling OR case-controls) OR 

cluster* OR "environmental exposure" 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 search 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 eliminated 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 published in the open 

literature. Therefore, there were 67 epidemiological investigations of the cancer and noncancer 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 included 
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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 epidemiology 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 not 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-cancer 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. 
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Appendix B1 - Page 324 of 677

Figure B- 1:
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Appendix B2 - Page 325 of 677

APPENDIX B.2: REVIEW OF EPIDEMIOLOGIC INVESTIGATIONS OF 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 these 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 strengths 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-conception 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 included 

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 pregnancy 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 medically confirmed. Authors evaluated 
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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 occurred 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 pesticide 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 calculated 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 among 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 potential 

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 

pesticide  ̳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 preconception (3 months prior to conception, and month of conception) exposure to several 

different pesticide groupings including glyphosate, carbaryl, phenoxy acetic acids, triazines 
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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 later (12-19 weeks) in pregnancy. There was no association between postconception exposure to atrazine and spontaneous abortion. For those instances of earlyterm 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 based upon initial exploratory analyses of these statistical 

interactions.

Author‘s Conclusions: Researchers state that their work provides evidence that preconception exposure to certain pesticides including atrazine, in addition to glyphosate, 

carbaryl, and 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 

increase 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 

association 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 exploratory 

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 are 
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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 based 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 adjust 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 age 

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. Given 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 investigate 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 atrazine, 

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 clarify 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)].
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Appendix B2 - Page 329 of 677

Background: Authors 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 recruitment 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 that 

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 who 

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 and 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 information 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 and 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 labor 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 low 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 pregnancy 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), and 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 pesticide 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) may 
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Appendix B2 - Page 330 of 677

also be observed. This suggests a small-for-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-based 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 women 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 reported 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 atrazine 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 relatively 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 outcomes, 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 

Agricultural Health Study (AHS),‖ [Am J Epidemiol,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 included 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 pesticide 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. 
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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 hormonal 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 enrollment, 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 

outcome 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 less 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 the 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 midpoint 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 between 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 pesticide, 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) resulted 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 

menopause), 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.
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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 atrazine, 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 relatively 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 incorporation 

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 menopause 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 most 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 the 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 state 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 research, 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 include more frequent ascertainment of 

menopausal status during the course of follow-up.

With regard to the exposure assessment method, researchers utilized self-reported everuse 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 atrazine/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 and 
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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 role 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 purposes 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 timing 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, including 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 the 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 12 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 concerning 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 evaluating 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 

cycle characteristics and pesticide use was also collected via self-reported questionnaire. 
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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). 

Identification 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. However, 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 characteristics), 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 measurement 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. Across 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 atrazine+cyanazine in this study), authors observed a non-significant decreased odds of 

short cycle (<24 days), increased odds missed period, and no association with intermenstrual bleeding (OR 1.0, 95% CI 0.37, 2.9). No prevalence estimates for the 

association 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 

hormonally 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; missed 
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Appendix B2 - Page 335 of 677

period, OR 1.7, 95% CI 1.3-2.2), and the association with irregular period also persisted 

(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 missed 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, OR 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 intermenstrual bleeding 1.7 (95% CI 1.1, 2.7) 

The results of sensitivity 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 association 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 menstrual 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. Therefore, 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 assessing 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 months, 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 characteristics, 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 researchers 
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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 cycle 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 diaries 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 were 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 point 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 with 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 estimates 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 preceded 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.
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Appendix B2 - Page 337 of 677

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].

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 Study (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 metric 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 questionnaire 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 wheeze 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 pesticides evaluated in association with wheeze, 11 were significantly 

associated with the outcome. Of these 11, researchers observed a significant exposureresponse relation with 10, including atrazine. Researchers observed a 20% increase in the 

prevalence of wheeze 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 

categories 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). Neither 
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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 wheeze 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 exposures 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 information 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 hypotheses 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 (Blair 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 relatively 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, and 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 enhances 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 North Carolina (the AHS). The study is absent large sources of 
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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 and 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) 

condition 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 omphalocele 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 diagnosed 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 omphalocele 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 conception 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 difference 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 change 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 in 

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.
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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 cited. 

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, or 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 screening 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 inherent 

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 information 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 study, 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 Monit; 7(6): 1270-1274 

(2001)].
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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 represent 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 measured 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, results 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 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 exposure assessment, this evaluation offers limited additional 

information to consider with regard to atrazine toxicity, although as research into the 

association between atrazine exposure and respiratory health effects progresses, use of 

these immune system biomarkers may prove useful to future epidemiologic analyses. 
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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 the 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 community 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 

participants 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 spirometry 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 types, 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 exposure 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, including atrazine, and respiratory health. Given the lack of specificity 
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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 and 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: Mattix, 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 Natality 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 Nebraska.

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, the 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 correlation 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 Midwest 

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, authors reported that 

the annual rate of AWD in Indiana was significantly higher than the rate in the entire 

Midwest in 1998.
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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 visual 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 descriptive 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 rates 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. 

Specifically, 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 comparison 

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 water 

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 Midwest 

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. 
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Appendix B2 - Page 345 of 677

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 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 exposure 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 lesser 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 work, 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 other 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 water 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 community-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 retardation (<10th

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, 
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Appendix B2 - Page 346 of 677

information regarding demographic characteristics including potentially 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 the 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 between 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 various 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 communities, 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 communities), 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 cyanazine (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 

variability in the model (R20.19 and 0.16, respectively). Final models indicate the rate of 

age-adjusted IUGR changes 25-30% for each unit increase in atrazine or cyanazine 
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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 causal 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 addition, 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. 

Statistical 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 

drinking 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-morbid 

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 Critique:

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 the 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 outcome at the individual, 

rather than the group, level are needed. 
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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 criteria 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 estimate 

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 period 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 Indiana and prevalence of small-forgestational-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 exposure 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 to reconstruct 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 outcome 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 birth 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 varied 
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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 atrazine 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 concentrations 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 averaged 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, 

last 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 infant 

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 exposure 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, 1.24).

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 monitoring data that allowed the 

investigators to perform a robust statistical analysis. Additionally, the study was able to 
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Appendix B2 - Page 350 of 677

adjust for individual-level potential confounders, and considered seasonal trends in adverse 

birth outcomes. 

Although the study used 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 

statistical 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 

concentrations 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 

investigators 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-construct 

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 summarized.

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. 407: 

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 the 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 population.

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 pregnancy 
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Appendix B2 - Page 351 of 677

using maternal home proximity to land with corn and soy fields as an 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 Agriculture. 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 the 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 

variables, 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 care, 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 generally 

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 defects 

(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; the 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% increase 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 agricultural 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 defects, although other birth defects were also elevated among women 

who resided in close proximity to treated fields.

EPA Critique:
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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 

agrichemical. 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 alternative 

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, authors 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 to 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 

association 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 controlling 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 been 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 report 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 were 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 also obtained through self-report questionnaires 
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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) pregnancy. 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 pesticide 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 between 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 pregnancy. 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, race 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, malathion, 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 prevalence 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 the 
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Appendix B2 - Page 354 of 677

first trimester of the index pregnancy and prevalent 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 application 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-analysis, 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: Researchers 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 well-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; therefore, 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 

there 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 reported 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 the 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 association only among women who report agricultural use 

of pesticides, and not all pesticide users, support that reporting bias is not likely, i.e., if 
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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.2.14 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 series 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 occurred 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 such 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 were 

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 for 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 prepregnancy body mass index (BMI), maternal 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 exposure 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 reported no pesticide exposure was the greatest among the pesticide 

categories, a reduction of 0.72 grams (95% confidence interval -272, 79). Examining ever 
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Appendix B2 - Page 356 of 677

use of several pesticides during the lifetime and infant birth weight, authors observed a 

statistically significant 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 birthweight.

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 assessment 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 were 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 the 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 birth 

weight among women who used this pesticide during the lifetime. Overall, this is a welldesigned exploratory study of this question and adds to our knowledge of this association. 

However, pesticide exposure measurement during the first trimester, or other 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 activities, 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 ratio.

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 families 

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 less (child-bearing 
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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 performed 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 defined 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 include 

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 exposure 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 reported 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 

(95% 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 variables. 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.
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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 exposure 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 specifically 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 sex 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 with 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 dated 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 acknowledged 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. However, 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 pesticide 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 to 
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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 bias 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 were 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. Information 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 causally 

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 control 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 who 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).
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Appendix B2 - Page 360 of 677

Methods: Authors performed a case-control analysis using cross-sectional 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 analysis 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, white 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 met 

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 assigns a  ̳normal‘ rating to samples with absolutely no abnormalities. Authors did not 

provide any measure of reliability or validity of measures; however, cases and controls 
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Appendix B2 - Page 361 of 677

were similar with respect to abstinence time and other risk factors for poor semen quality. 

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 simple 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 sperm 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-parameterizing 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 reported 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 reflected 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 (of 
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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 atrazine 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 researchers 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, this 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 occupational 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 literature 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 

male 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 written 

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 provided 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.
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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 

compared 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 association 

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 

exposure (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 semen 

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 exposurestatus, 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 concurs 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 

strategy 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-estimation of exposure due to 
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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. Strengths 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 given 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 studies 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,‖ [Environmental 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 SGA.

Methods: The study 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 by 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 that 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 sampling period. Based on distribution of their exposure index, 

the investigators then created low, medium, and high exposure groups using tertiles as cutoffs for both raw (≤ 0.05, > 0.0.05– –

0.036, > 0.036
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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 statistical 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 category, 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 

pregnancy 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 

trimester 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 preterm 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-response 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 significant) 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% 

increase 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 atrazine 

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 significant (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 comparison 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 the 
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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, controlling 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 ability 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 and 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-October in this region), authors report elevated odds of LBW and small-forgestational 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 

borderline). 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 provide 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 exposure 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 Washington 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 abdominal 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 inclusion in this study. Maternal 

residence as reported in birth records was linked to USGS surface water data to determine 
Do 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 and 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 present, 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 of 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 

seasonal concentration of each agrichemical of interest were averaged to generate an 

annual average, and the annual averages between 2001-2006 were averaged. This sitespecific annual average was compared to U.S. EPA acceptable contaminant levels to 

determine areas 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; 

however, 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‖ agrichemical 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 variable 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

parity, 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 3616 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 exposure 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, 

nitrate and nitrite, were not associated with the risk of gastroschisis. As a result of multivariable 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 surface 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 many 

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 been informative, 

given the reported influence of young maternal age on the incidence of gastroschisis 

(Williams, 2005). 
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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 records 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 appropriate 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 result 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 

speculate 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 ―high‖ 

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 appropriate 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 

interpretation 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, recreation 

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: Winchester, 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 estimated using data collected through USGS‘s National WaterQuality Assessment Program (NAWQA), which has collected water quality data from 

several major U.S. river basins and aquifers since 1991. Based on these health outcome 
Do not cite

Appendix B2 - Page 370 of 677

and drinking water monitoring sources, 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 the 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, several 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 agrichemicals. 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 seasonal 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 awareness 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 utilize 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 studies 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-level 
Do not cite

Appendix B2 - Page 371 of 677

may not 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 surface 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 little 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 individual-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 common 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 response for exposure = dose;

 sign = +1 for increasing trend in data;

 sign = -1 for decreasing trend.
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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.

 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 = 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 Parameter 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 0.0449039 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 0
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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

 Dose 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 11.81 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 Scaled 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 0.6071
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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)

 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 -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 the
Do 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.

 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 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 appropriate to model the data.
Do not cite

Appendix C - Page 377 of 677

 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 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. 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
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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 1.962

 5

 10

 15

 20

 25

 0 10 20 30 40 50 60 70

Mean Response

dose

Exponential Model 2 with 0.95 Confidence Level

14:07 07/20 2010

BMDL BMD

 

Exponential
Do not cite

Appendix C - Page 379 of 677

 5

 10

 15

 20

 25

 0 10 20 30 40 50 60 70

Mean Response

dose

Exponential Model 3 with 0.95 Confidence Level

14:07 07/20 2010

BMDL BMD

 

Exponential
Do not cite

Appendix C - Page 380 of 677

 5

 10

 15

 20

 25

 0 10 20 30 40 50 60 70

Mean Response

dose

Exponential Model 4 with 0.95 Confidence Level

14:07 07/20 2010

BMDL BMD

 

Exponential
Do not cite

Appendix C - Page 381 of 677

 5

 10

 15

 20

 25

 0 10 20 30 40 50 60 70

Mean Response

dose

Exponential Model 5 with 0.95 Confidence Level

14:07 07/20 2010

BMDL BMD

 

Exponential

________________________________________________________________

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

aNonhomogeneous Variance recommended by BMDS output
Do 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 3 523.7 174.7 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/BMDS212/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 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 = 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 5
Do 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 -0.2357
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Appendix C - Page 385 of 677

 2.5 2798 2060 0.3324

 5 2780 2060 -0.09725

 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)

 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 -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 Tests
Do 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)

 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 43.94 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 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
Do 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 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 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 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 BMDL

 ------- ------------ ----------

 2 152.443 25.6454

 3 152.443 25.6454
Do not cite

Appendix C - Page 388 of 677

 4 129.57 11.8371

 5 0 0 Not computed

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Exponential Model 2 with 0.95 Confidence Level

13:45 07/22 2010

BMDL BMD

 

Exponential
Do not cite

Appendix C - Page 389 of 677

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Exponential Model 3 with 0.95 Confidence Level

13:45 07/22 2010

BMDL BMD

 

Exponential
Do not cite

Appendix C - Page 390 of 677

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Exponential Model 4 with 0.95 Confidence Level

13:45 07/22 2010

BMDL BMD

 

Exponential
Do not cite

Appendix C - Page 391 of 677

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Exponential Model 5

13:45 07/22 2010

BMD

Exponential

________________________________________________________________

Morseth One Month LH Study: BMD OUTPUT for Exponential Model Runs 

(Nonhomogeneous Variance)

==================================================================== 

 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 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]))

 The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)

 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 -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 -- 1
Do 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 0.035215 0.0290775

 c -- -- 0.273458 0.291831

 d -- 1 -- 2.06427

 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 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

 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 0.05275
Do 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 constant 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 43.94 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-homogeneous
Do 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 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 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 Model

 Model BMD BMDL
Do 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 computed

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Mean Response

dose

Exponential Model 2 with 0.95 Confidence Level

09:35 08/06 2010

BMDL BMD

 

Exponential
Do not cite

Appendix C - Page 398 of 677

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dose

Exponential Model 3 with 0.95 Confidence Level

09:35 08/06 2010

BMDL BMD

 

Exponential
Do not cite

Appendix C - Page 399 of 677

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Exponential Model 4

09:35 08/06 2010

BMD

Exponential
Do not cite

Appendix C - Page 400 of 677

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Exponential Model 5

09:35 08/06 2010

BMD

Exponential

________________________________________________________________

3. Morseth Six Month LH Study 

Model 

(Constant 

Variance)

BMD BMDL p-value AIC

Exponential 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 0.61 661.9
Do 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 

completion in Optimization routine.

==================================================================== 

 Exponential Model. (Version: 1.7; Date: 12/10/2009) 

 Input Data File: C:/Usepa/BMDS212/Data/exp_Actualruns_Setting.(d) 

 Gnuplot Plotting 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 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.

 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 0.0471466 0.14585 0.14585

 c -- -- 0.21433 0.21433

 d -- 1 -- 1

 (S) = Specified

 Parameter Estimates by Model
Do not cite

Appendix C - Page 403 of 677

 Variable Model 2 Model 3 Model 4 Model 5

 -------- ------- ------- ------- -------

 lnalpha 15.3924 15.3924 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 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 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-007
Do 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-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 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 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 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 .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. 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 seems
Do 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 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 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:

 Specified 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 3.66097
Do not cite

Appendix C - Page 407 of 677

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 5000

 6000

 0 5 10 15 20 25 30

Mean Response

dose

Exponential Model 2 with 0.95 Confidence Level

13:57 07/29 2010

BMDL BMD

 

Exponential
Do not cite

Appendix C - Page 408 of 677

 1000

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Mean Response

dose

Exponential Model 3 with 0.95 Confidence Level

13:57 07/29 2010

BMDL BMD

 

Exponential
Do not cite

Appendix C - Page 409 of 677

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Mean Response

dose

Exponential Model 4 with 0.95 Confidence Level

13:57 07/29 2010

BMDL BMD

 

Exponential
Do not cite

Appendix C - Page 410 of 677

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Mean Response

dose

Exponential Model 5 with 0.95 Confidence Level

13:57 07/29 2010

BMDL BMD

 

Exponential

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) 

 Gnuplot Plotting File: 

Thu Jul 29 14:00:34 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}]
Do not cite

Appendix C - Page 411 of 677

 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]))

 The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)

 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 -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 5
Do 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 0.0471113 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

 29.4 857.9 394.8 0.001045
Do 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 8 642.6425

 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. 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.

 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:

 Specified Effect = 1.000000

 Risk Type = Estimated standard deviations from control

 Confidence Level = 0.950000

 BMD and BMDL by Model

 Model BMD BMDL

 ------- ------------ ----------

 2 36.1811 17.0918

 3 36.1811 17.0918

 4 0 0 Not computed

 5 0 0 Not computed
Do not cite

Appendix C - Page 416 of 677

 1000

 2000

 3000

 4000

 5000

 6000

 0 5 10 15 20 25 30 35

Mean Response

dose

Exponential Model 2 with 0.95 Confidence Level

14:00 07/29 2010

BMDL BMD

 

Exponential
Do not cite

Appendix C - Page 417 of 677

 1000

 2000

 3000

 4000

 5000

 6000

 0 5 10 15 20 25 30 35

Mean Response

dose

Exponential Model 3 with 0.95 Confidence Level

14:00 07/29 2010

BMDL BMD

 

Exponential
Do not cite

Appendix C - Page 418 of 677

 1000

 2000

 3000

 4000

 5000

 6000

 0 5 10 15 20 25 30

Mean Response

dose

Exponential Model 4

14:00 07/29 2010

BMD

Exponential
Do not cite

Appendix C - Page 419 of 677

 1000

 2000

 3000

 4000

 5000

 6000

 0 5 10 15 20 25 30

Mean Response

dose

Exponential Model 5

14:00 07/29 2010

BMD

Exponential

________________________________________________________________

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 0.38 274.7
Do not cite

Appendix C - Page 420 of 677

Minnema One Month 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

 -------- ------- ------- ------- -------

 lnalpha 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 Residual

 ------- ------ ---------- --------- ----------------

 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

 40 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

 Model A2: Yij = Mu(i) + e(ij)

 Var{e(ij)} = Sigma(i)^2
Do 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 -134.3092 3 274.6183

 3 -134.3092 3 274.6183

 4 -132.9359 4 273.8719

 5 -132.9359 4 273.8719

 Additive 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: 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)
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 curve 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 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 Test 6b is greater than .05. Model 4 does
Do not cite

Appendix C - Page 425 of 677

 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.

 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 computed
Do not cite

Appendix C - Page 426 of 677

 1

 1.5

 2

 2.5

 3

 3.5

 4

 4.5

 5

 5.5

 0 100 200 300 400 500 600

Mean Response

dose

Exponential Model 2 with 0.95 Confidence Level

08:35 07/23 2010

BMDL BMD

 

Exponential
Do not cite

Appendix C - Page 427 of 677

 1

 1.5

 2

 2.5

 3

 3.5

 4

 4.5

 5

 5.5

 0 100 200 300 400 500 600

Mean Response

dose

Exponential Model 3 with 0.95 Confidence Level

08:35 07/23 2010

BMDL BMD

 

Exponential
Do not cite

Appendix C - Page 428 of 677

 1.5

 2

 2.5

 3

 3.5

 4

 4.5

 5

 5.5

 0 50 100 150 200

Mean Response

dose

Exponential Model 4

08:35 07/23 2010

BMD

Exponential
Do not cite

Appendix C - Page 429 of 677

 1.5

 2

 2.5

 3

 3.5

 4

 4.5

 5

 5.5

 0 50 100 150 200

Mean Response

dose

Exponential Model 5

08:35 07/23 2010

BMD

Exponential

BMD Output Summaries – AUC

1. AUC for Steady State Serum Levels of Total Triazine Equivalents

Model 

(Constant 

Variance)

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 0.10 410.1
Do 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 File: 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

 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 = 1.1704

 k = 1.67918
Do 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 specified 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 -0.0933
Do 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 the 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 = 0.64721
Do not cite

Appendix C - Page 434 of 677

 5

 10

 15

 20

 25

 0 2 4 6 8 10 12

Mean Response

dose

Hill Model with 0.95 Confidence Level

12:15 07/30 2010

BMDL BMD

 

Hill

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 0.10 410.1
Do 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

 Independent 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 Estimates
Do 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 intercept 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 Conf. Limit

 alpha 63.7164 10.2688 43.5899 83.8429

 intercept 21.8193 1.88737 18.1201 25.5185

 v -14.7416 3.24394 -21.0996 -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 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 0.0144

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 -0.358
Do 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 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 -198.446021 4 404.892043

 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)

(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 = 15.5588
Do not cite

Appendix C - Page 439 of 677

 5

 10

 15

 20

 25

 0 50 100 150 200 250 300

Mean Response

dose

Hill Model with 0.95 Confidence Level

12:45 07/30 2010

BMDL BMD

 

Hill
Do not cite

Appendix D1 - Page 440 of 677

APPENDIX D.1: CHARACTERIZATION OF ATRAZINE MONITORING PROGRAM (AMP) CWS CHEMOGRAPH 

SHAPES AND MONITORING DATA FOR CWS WITH THE GREATEST TCT CONCENTRATIONS

Worksheet Table / 

Parameter Description

Max TCT Concentration 

by Year

D.1.a Table summarizing the maximum concentrations of atrazine (Atz) and total 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 tables 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 target 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 concentration, 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 either 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 Graphs 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

Appendix D1 - Page 441 of 677

Worksheet Table / 

Parameter Description

Atz Source Atrazine concentration, in ug/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) concentration, 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 sample

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 water 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, simazine, DDA, DIA, DEA), in ug/L, in 

the treated water sample
Do not cite

Appendix D2 - Page 442 of 677

APPENDIX D.2 SAMPLING FREQUENCY CHEMOGRAPHS

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

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 D2.1)

Actual 14 106.0 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 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 day 7 2 43.1 1 40.4 16 2

MO-01, 

2007

(Figure D2.2)

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 D2.3)

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-01, 

2009

(Figure D2.4)

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 1st through 7th 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)

MO-01, 2006

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

4-day avg
Do not cite

Appendix D2 - Page 444 of 677

(b) Sampled at 7-day fixed intervals, beginning on Day 1

MO-01, 2006: 7-Day Sample Interval #1

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 1

linear 7-da interval 1

4-day avg, linear 7-da int 1
Do not cite

Appendix D2 - Page 445 of 677

(c) Sampled at 7-day fixed intervals, beginning on Day 2

MO-01, 2006: 7-Day Sample Interval #2

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval2

linear 7-da interval 2

4-day avg, linear 7-da int 2
Do not cite

Appendix D2 - Page 446 of 677

(d) Sampled at 7-day fixed intervals, beginning on Day 3

MO-01, 2006: 7-Day Sample Interval #3

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 3

linear 7-da interval 3

4-day avg, linear 7-da int 3
Do not cite

Appendix D2 - Page 447 of 677

(e) Sampled at 7-day fixed intervals, beginning on Day 4

MO-01, 2006: 7-Day Sample Interval #4

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 4

linear 7-da interval 4

4-day avg, linear 7-da int 4
Do not cite

Appendix D2 - Page 448 of 677

(f) Sampled at 7-day fixed intervals, beginning on Day 5

MO-01, 2006: 7-Day Sample Interval #5

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 5

linear 7-da interval 5

4-day avg, linear 7-da int 5
Do not cite

Appendix D2 - Page 449 of 677

(g) Sampled at 7-day fixed intervals, beginning on Day 6

MO-01, 2006: 7-Day Sample Interval #6

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 6

linear 7-da interval 6

4-day avg, linear 7-da int 6
Do not cite

Appendix D2 - Page 450 of 677

(h) Sampled at 7-day fixed intervals, beginning on Day 7

MO-01, 2006: 7-Day Sample Interval #7

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 7

linear 7-da interval 7

4-day avg, linear 7-da int 7
Do 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)

MO-01, 2007

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

4-day avg
Do not cite

Appendix D2 - Page 452 of 677

(b) Sampled at 7-day fixed intervals, beginning on Day 1

MO-01, 2007: 7-Day Sample Interval #1

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 1

linear 7-da interval 1

4-day avg, linear 7-da int 1
Do not cite

Appendix D2 - Page 453 of 677

(c) Sampled at 7-day fixed intervals, beginning on Day 2

MO-01, 2007: 7-Day Sample Interval #2

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval2

linear 7-da interval 2

4-day avg, linear 7-da int 2
Do not cite

Appendix D2 - Page 454 of 677

(d) Sampled at 7-day fixed intervals, beginning on Day 3

MO-01, 2007: 7-Day Sample Interval #3

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 3

linear 7-da interval 3

4-day avg, linear 7-da int 3
Do not cite

Appendix D2 - Page 455 of 677

(e) Sampled at 7-day fixed intervals, beginning on Day 4

MO-01, 2007: 7-Day Sample Interval #4

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 4

linear 7-da interval 4

4-day avg, linear 7-da int 4
Do not cite

Appendix D2 - Page 456 of 677

(f) Sampled at 7-day fixed intervals, beginning on Day 5

MO-01, 2007: 7-Day Sample Interval #5

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 5

linear 7-da interval 5

4-day avg, linear 7-da int 5
Do not cite

Appendix D2 - Page 457 of 677

(g) Sampled at 7-day fixed intervals, beginning on Day 6

MO-01, 2007: 7-Day Sample Interval #6

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 6

linear 7-da interval 6

4-day avg, linear 7-da int 6
Do not cite

Appendix D2 - Page 458 of 677

(h) Sampled at 7-day fixed intervals, beginning on Day 7

MO-01, 2007: 7-Day Sample Interval #7

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 7

linear 7-da interval 7

4-day avg, linear 7-da int 7
Do 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, 2008

(a) Actual Chemograph (red arrows point to spikes)

MO-01, 2008

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

4-day avg
Do not cite

Appendix D2 - Page 460 of 677

(b) Sampled at 7-day fixed intervals, beginning on Day 1

MO-01, 2008: 7-Day Sample Interval #1

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 1

linear 7-da interval 1

4-day avg, linear 7-da int 1
Do not cite

Appendix D2 - Page 461 of 677

(c) Sampled at 7-day fixed intervals, beginning on Day 2

MO-01, 2008: 7-Day Sample Interval #2

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval2

linear 7-da interval 2

4-day avg, linear 7-da int 2
Do not cite

Appendix D2 - Page 462 of 677

(d) Sampled at 7-day fixed intervals, beginning on Day 3

MO-01, 2008: 7-Day Sample Interval #3

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 3

linear 7-da interval 3

4-day avg, linear 7-da int 3
Do not cite

Appendix D2 - Page 463 of 677

(e) Sampled at 7-day fixed intervals, beginning on Day 4

MO-01, 2008: 7-Day Sample Interval #4

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 4

linear 7-da interval 4

4-day avg, linear 7-da int 4
Do not cite

Appendix D2 - Page 464 of 677

(f) Sampled at 7-day fixed intervals, beginning on Day 5

MO-01, 2008: 7-Day Sample Interval #5

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 5

linear 7-da interval 5

4-day avg, linear 7-da int 5
Do not cite

Appendix D2 - Page 465 of 677

(g) Sampled at 7-day fixed intervals, beginning on Day 6

MO-01, 2008: 7-Day Sample Interval #6

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 6

linear 7-da interval 6

4-day avg, linear 7-da int 6
Do not cite

Appendix D2 - Page 466 of 677

(h) Sampled at 7-day fixed intervals, beginning on Day 7

MO-01, 2008: 7-Day Sample Interval #7

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 7

linear 7-da interval 7

4-day avg, linear 7-da int 7
Do not cite

Appendix D2 - Page 467 of 677

Figure D-2.4: Actual chemograph (blue) and resulting chemographs (dark red) 

sampled at 7-day fixed intervals (red dots) for MO-01, 2009

(a) Actual Chemograph (red arrows point to spikes)

MO-01, 2009

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

4-day avg
Do not cite

Appendix D2 - Page 468 of 677

(b) Sampled at 7-day fixed intervals, beginning on Day 1

MO-01, 2009: 7-Day Sample Interval #1

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 1

linear 7-da interval 1

4-day avg, linear 7-da int 1
Do not cite

Appendix D2 - Page 469 of 677

(c) Sampled at 7-day fixed intervals, beginning on Day 2

MO-01, 2009: 7-Day Sample Interval #2

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval2

linear 7-da interval 2

4-day avg, linear 7-da int 2
Do not cite

Appendix D2 - Page 470 of 677

(d) Sampled at 7-day fixed intervals, beginning on Day 3

MO-01, 2009: 7-Day Sample Interval #3

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 3

linear 7-da interval 3

4-day avg, linear 7-da int 3
Do not cite

Appendix D2 - Page 471 of 677

(e) Sampled at 7-day fixed intervals, beginning on Day 4

MO-01, 2009: 7-Day Sample Interval #4

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 4

linear 7-da interval 4

4-day avg, linear 7-da int 4
Do not cite

Appendix D2 - Page 472 of 677

(f) Sampled at 7-day fixed intervals, beginning on Day 5

MO-01, 2009: 7-Day Sample Interval #5

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 5

linear 7-da interval 5

4-day avg, linear 7-da int 5
Do not cite

Appendix D2 - Page 473 of 677

(g) Sampled at 7-day fixed intervals, beginning on Day 6

MO-01, 2009: 7-Day Sample Interval #6

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 6

linear 7-da interval 6

4-day avg, linear 7-da int 6
Do not cite

Appendix D2 - Page 474 of 677

(h) Sampled at 7-day fixed intervals, beginning on Day 7

MO-01, 2009: 7-Day Sample Interval #7

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-01 Measured

7-da interval 7

linear 7-da interval 7

4-day avg, linear 7-da int 7
Do 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 D2.5)

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 D2.6)

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 D2.7)

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 D2.8)

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 1st through 7th 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 fixed intervals (red dots) for MO-02, 2006

(a) Actual Chemograph (red arrows point to spikes)

MO-02, 2006 adjusted for flow

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Flow Adjusted

4-day avg

on Day 1
Do not cite

Appendix D2 - Page 477 of 677

MO-02, 2006: 7-Day Sample Interval #1

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Flow Adjusted

7-da interval 1

linear 7-da interval 1

4-day avg, linear 7-da int 1
Do not cite

Appendix D2 - Page 478 of 677

(c) Sampled at 7-day fixed intervals, beginning on Day 2

MO-02, 2006: 7-Day Sample Interval #2

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Flow Adjusted

7-da interval2

linear 7-da interval 2

4-day avg, linear 7-da int 2
Do not cite

Appendix D2 - Page 479 of 677

(d) Sampled at 7-day fixed intervals, beginning on Day 3

MO-02, 2006: 7-Day Sample Interval #3

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Flow Adjusted

7-da interval 3

linear 7-da interval 3

4-day avg, linear 7-da int 3
Do not cite

Appendix D2 - Page 480 of 677

(e) Sampled at 7-day fixed intervals, beginning on Day 4

MO-02, 2006: 7-Day Sample Interval #4

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Flow Adjusted

7-da interval 4

linear 7-da interval 4

4-day avg, linear 7-da int 4
Do not cite

Appendix D2 - Page 481 of 677

(f) Sampled at 7-day fixed intervals, beginning on Day 5

MO-02, 2006: 7-Day Sample Interval #5

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Flow Adjusted

7-da interval 5

linear 7-da interval 5

4-day avg, linear 7-da int 5
Do not cite

Appendix D2 - Page 482 of 677

(g) Sampled at 7-day fixed intervals, beginning on Day 6

MO-02, 2006: 7-Day Sample Interval #6

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Flow Adjusted

7-da interval 6

linear 7-da interval 6

4-day avg, linear 7-da int 6
Do not cite

Appendix D2 - Page 483 of 677

(h) Sampled at 7-day fixed intervals, beginning on Day 7

MO-02, 2006: 7-Day Sample Interval #7

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Flow Adjusted

7-da interval 7

linear 7-da interval 7

4-day avg, linear 7-da int 7
Do 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-02, 2007

(a) Actual Chemograph (red arrows point to spikes)

MO-02, 2007

0

10

20

30

40

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

4-day avg
Do not cite

Appendix D2 - Page 485 of 677

(b) Sampled at 7-day fixed intervals, beginning on Day 1

MO-02, 2007: 7-Day Sample Interval #1

0

10

20

30

40

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

7-da interval 1

linear 7-da interval 1

4-day avg, linear 7-da int 1
Do not cite

Appendix D2 - Page 486 of 677

(c) Sampled at 7-day fixed intervals, beginning on Day 2

MO-02, 2007: 7-Day Sample Interval #2

0

10

20

30

40

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

7-da interval2

linear 7-da interval 2

4-day avg, linear 7-da int 2
Do not cite

Appendix D2 - Page 487 of 677

(d) Sampled at 7-day fixed intervals, beginning on Day 3

MO-02, 2007: 7-Day Sample Interval #3

0

10

20

30

40

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

7-da interval 3

linear 7-da interval 3

4-day avg, linear 7-da int 3
Do not cite

Appendix D2 - Page 488 of 677

(e) Sampled at 7-day fixed intervals, beginning on Day 4

MO-02, 2007: 7-Day Sample Interval #4

0

10

20

30

40

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

7-da interval 4

linear 7-da interval 4

4-day avg, linear 7-da int 4
Do not cite

Appendix D2 - Page 489 of 677

(f) Sampled at 7-day fixed intervals, beginning on Day 5

MO-02, 2007: 7-Day Sample Interval #5

0

10

20

30

40

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

7-da interval 5

linear 7-da interval 5

4-day avg, linear 7-da int 5
Do not cite

Appendix D2 - Page 490 of 677

(g) Sampled at 7-day fixed intervals, beginning on Day 6

MO-02, 2007: 7-Day Sample Interval #6

0

10

20

30

40

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

7-da interval 6

linear 7-da interval 6

4-day avg, linear 7-da int 6
Do not cite

Appendix D2 - Page 491 of 677

(h) Sampled at 7-day fixed intervals, beginning on Day 7

MO-02, 2007: 7-Day Sample Interval #7

0

10

20

30

40

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

7-da interval 7

linear 7-da interval 7

4-day avg, linear 7-da int 7
Do 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-02, 2008

(a) Actual Chemograph (red arrows point to spikes)

MO-02, 2008

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

4-day avg
Do not cite

Appendix D2 - Page 493 of 677

(b) Sampled at 7-day fixed intervals, beginning on Day 1

MO-02, 2008: 7-Day Sample Interval #1

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

7-da interval 1

linear 7-da interval 1

4-day avg, linear 7-da int 1
Do not cite

Appendix D2 - Page 494 of 677

(c) Sampled at 7-day fixed intervals, beginning on Day 2

MO-02, 2008: 7-Day Sample Interval #2

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

7-da interval2

linear 7-da interval 2

4-day avg, linear 7-da int 2
Do not cite

Appendix D2 - Page 495 of 677

(d) Sampled at 7-day fixed intervals, beginning on Day 3

MO-02, 2008: 7-Day Sample Interval #3

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

7-da interval 3

linear 7-da interval 3

4-day avg, linear 7-da int 3
Do not cite

Appendix D2 - Page 496 of 677

(e) Sampled at 7-day fixed intervals, beginning on Day 4

MO-02, 2008: 7-Day Sample Interval #4

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

7-da interval 4

linear 7-da interval 4

4-day avg, linear 7-da int 4
Do not cite

Appendix D2 - Page 497 of 677

(f) Sampled at 7-day fixed intervals, beginning on Day 5

MO-02, 2008: 7-Day Sample Interval #5

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

7-da interval 5

linear 7-da interval 5

4-day avg, linear 7-da int 5
Do not cite

Appendix D2 - Page 498 of 677

(g) Sampled at 7-day fixed intervals, beginning on Day 6

MO-02, 2008: 7-Day Sample Interval #6

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

7-da interval 6

linear 7-da interval 6

4-day avg, linear 7-da int 6
Do not cite

Appendix D2 - Page 499 of 677

(h) Sampled at 7-day fixed intervals, beginning on Day 7

MO-02, 2008: 7-Day Sample Interval #7

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

7-da interval 7

linear 7-da interval 7

4-day avg, linear 7-da int 7
Do 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-02, 2009

(a) Actual Chemograph (red arrows point to spikes)

MO-02, 2009

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

4-day avg

109 (max) 155 (max)
Do not cite

Appendix D2 - Page 501 of 677

(b) Sampled at 7-day fixed intervals, beginning on Day 1

MO-02, 2009: 7-Day Sample Interval #1

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

7-da interval 1

linear 7-da interval 1

4-day avg, linear 7-da int 1

109 (max) 155 (max)
Do not cite

Appendix D2 - Page 502 of 677

(c) Sampled at 7-day fixed intervals, beginning on Day 2

MO-02, 2009: 7-Day Sample Interval #2

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

7-da interval2

linear 7-da interval 2

4-day avg, linear 7-da int 2

109 (max) 155 (max)
Do not cite

Appendix D2 - Page 503 of 677

(d) Sampled at 7-day fixed intervals, beginning on Day 3

MO-02, 2009: 7-Day Sample Interval #3

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

7-da interval 3

linear 7-da interval 3

4-day avg, linear 7-da int 3

109 (max) 155 (max)
Do not cite

Appendix D2 - Page 504 of 677

(e) Sampled at 7-day fixed intervals, beginning on Day 4

MO-02, 2009: 7-Day Sample Interval #4

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

7-da interval 4

linear 7-da interval 4

4-day avg, linear 7-da int 4

109 (max) 155 (max)
Do not cite

Appendix D2 - Page 505 of 677

(f) Sampled at 7-day fixed intervals, beginning on Day 5

MO-02, 2009: 7-Day Sample Interval #5

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

7-da interval 5

linear 7-da interval 5

4-day avg, linear 7-da int 5

109 (max) 155 (max)
Do not cite

Appendix D2 - Page 506 of 677

(g) Sampled at 7-day fixed intervals, beginning on Day 6

MO-02, 2009: 7-Day Sample Interval #6

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

7-da interval 6

linear 7-da interval 6

4-day avg, linear 7-da int 6

109 (max) 155 (max)
Do not cite

Appendix D2 - Page 507 of 677

(h) Sampled at 7-day fixed intervals, beginning on Day 7

MO-02, 2009: 7-Day Sample Interval #7

0

20

40

60

80

100

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date

Atrazine, ug/L

MO-02 Measured

7-da interval 7

linear 7-da interval 7

4-day avg, linear 7-da int 7

109 (max) 155 (max)
Do 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 

average

Days 4-

da avg 

>20 ug/L

Days 4-

da avg 

>40 ug/L

Maumee 

R, 2005

(Figure 

D-2.9)

Actual 15 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

(Figure 

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 1st through 7th 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 fixed intervals (red dots) for Maumee River (Heidelberg), 

2005

(a) Actual Chemograph (red arrows point to spikes)

Maumee River, 2005

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill), max

Measured (w/ infill), avg

4-day avg
Do not cite

Appendix D2 - Page 510 of 677

(b) Sampled at 7-day fixed intervals, beginning on Day 1

Maumee River, 2005: 7-Day Sample Interval #1

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 1

linear 7-da interval 1

4-day avg, linear 7-da int 1
Do not cite

Appendix D2 - Page 511 of 677

(c) Sampled at 7-day fixed intervals, beginning on Day 2

Maumee River, 2005: 7-Day Sample Interval #2

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 2

linear 7-da interval 2

4-day avg, linear 7-da int 2
Do not cite

Appendix D2 - Page 512 of 677

(d) Sampled at 7-day fixed intervals, beginning on Day 3

Maumee River, 2005: 7-Day Sample Interval #3

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 3

linear 7-da interval 3

4-day avg, linear 7-da int 3
Do not cite

Appendix D2 - Page 513 of 677

(e) Sampled at 7-day fixed intervals, beginning on Day 4

Maumee River, 2005: 7-Day Sample Interval #4

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 4

linear 7-da interval 4

4-day avg, linear 7-da int 4
Do not cite

Appendix D2 - Page 514 of 677

(f) Sampled at 7-day fixed intervals, beginning on Day 5

Maumee River, 2005: 7-Day Sample Interval #5

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 5

linear 7-da interval 5

4-day avg, linear 7-da int 5
Do not cite

Appendix D2 - Page 515 of 677

(g) Sampled at 7-day fixed intervals, beginning on Day 6

Maumee River, 2005: 7-Day Sample Interval #6

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 6

linear 7-da interval 6

4-day avg, linear 7-da int 6
Do not cite

Appendix D2 - Page 516 of 677

(h) Sampled at 7-day fixed intervals, beginning on Day 7

Maumee River, 2005: 7-Day Sample Interval #7

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 7

linear 7-da interval 7

4-day avg, linear 7-da int 7
Do not cite

Appendix D2 - Page 517 of 677

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

(a) Actual Chemograph (red arrows point to spikes)

Maumee River, 2006

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill), max

Measured (w/ infill), avg

4-day avg
Do not cite

Appendix D2 - Page 518 of 677

(b) Sampled at 7-day fixed intervals, beginning on Day 1

Maumee River, 2006: 7-Day Sample Interval #1

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 1

linear 7-da interval 1

4-day avg, linear 7-da int 1
Do not cite

Appendix D2 - Page 519 of 677

(c) Sampled at 7-day fixed intervals, beginning on Day 2

Maumee River, 2006: 7-Day Sample Interval #2

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 2

linear 7-da interval 2

4-day avg, linear 7-da int 2
Do not cite

Appendix D2 - Page 520 of 677

(d) Sampled at 7-day fixed intervals, beginning on Day 3

Maumee River, 2006: 7-Day Sample Interval #3

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 3

linear 7-da interval 3

4-day avg, linear 7-da int 3
Do not cite

Appendix D2 - Page 521 of 677

(e) Sampled at 7-day fixed intervals, beginning on Day 4

Maumee River, 2006: 7-Day Sample Interval #4

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 4

linear 7-da interval 4

4-day avg, linear 7-da int 4
Do not cite

Appendix D2 - Page 522 of 677

(f) Sampled at 7-day fixed intervals, beginning on Day 5

Maumee River, 2006: 7-Day Sample Interval #5

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 5

linear 7-da interval 5

4-day avg, linear 7-da int 5
Do not cite

Appendix D2 - Page 523 of 677

(g) Sampled at 7-day fixed intervals, beginning on Day 6

Maumee River, 2006: 7-Day Sample Interval #6

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 6

linear 7-da interval 6

4-day avg, linear 7-da int 6
Do not cite

Appendix D2 - Page 524 of 677

(h) Sampled at 7-day fixed intervals, beginning on Day 7

Maumee River, 2006: 7-Day Sample Interval #7

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 7

linear 7-da interval 7

4-day avg, linear 7-da int 7
Do not cite

Appendix D2 - Page 525 of 677

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

(a) Actual Chemograph (red arrows point to spikes)

Maumee River, 2007

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill), max

Measured (w/ infill), avg

4-day avg
Do not cite

Appendix D2 - Page 526 of 677

(b) Sampled at 7-day fixed intervals, beginning on Day 1

Maumee River, 2007: 7-Day Sample Interval #1

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 1

linear 7-da interval 1

4-day avg, linear 7-da int 1
Do not cite

Appendix D2 - Page 527 of 677

(c) Sampled at 7-day fixed intervals, beginning on Day 2

Maumee River, 2007: 7-Day Sample Interval #2

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 2

linear 7-da interval 2

4-day avg, linear 7-da int 2
Do not cite

Appendix D2 - Page 528 of 677

(d) Sampled at 7-day fixed intervals, beginning on Day 3

Maumee River, 2007: 7-Day Sample Interval #3

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 3

linear 7-da interval 3

4-day avg, linear 7-da int 3
Do not cite

Appendix D2 - Page 529 of 677

(e) Sampled at 7-day fixed intervals, beginning on Day 4

Maumee River, 2007: 7-Day Sample Interval #4

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 4

linear 7-da interval 4

4-day avg, linear 7-da int 4
Do not cite

Appendix D2 - Page 530 of 677

(f) Sampled at 7-day fixed intervals, beginning on Day 5

Maumee River, 2007: 7-Day Sample Interval #5

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 5

linear 7-da interval 5

4-day avg, linear 7-da int 5
Do not cite

Appendix D2 - Page 531 of 677

(g) Sampled at 7-day fixed intervals, beginning on Day 6

Maumee River, 2007: 7-Day Sample Interval #6

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 6

linear 7-da interval 6

4-day avg, linear 7-da int 6
Do not cite

Appendix D2 - Page 532 of 677

(h) Sampled at 7-day fixed intervals, beginning on Day 7

Maumee River, 2007: 7-Day Sample Interval #7

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 7

linear 7-da interval 7

4-day avg, linear 7-da int 7
Do 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), 

2008

(a) Actual Chemograph (red arrows point to spikes)

Maumee River, 2008

0

10

20

30

40

50

60

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill), max

Measured (w/ infill), avg

4-day avg
Do not cite

Appendix D2 - Page 534 of 677

(b) Sampled at 7-day fixed intervals, beginning on Day 1

Maumee River, 2008: 7-Day Sample Interval #1

0

10

20

30

40

50

60

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 1

linear 7-da interval 1

4-day avg, linear 7-da int 1
Do not cite

Appendix D2 - Page 535 of 677

(c) Sampled at 7-day fixed intervals, beginning on Day 2

Maumee River, 2008: 7-Day Sample Interval #2

0

10

20

30

40

50

60

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 2

linear 7-da interval 2

4-day avg, linear 7-da int 2
Do not cite

Appendix D2 - Page 536 of 677

(d) Sampled at 7-day fixed intervals, beginning on Day 3

Maumee River, 2008: 7-Day Sample Interval #3

0

10

20

30

40

50

60

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 3

linear 7-da interval 3

4-day avg, linear 7-da int 3
Do not cite

Appendix D2 - Page 537 of 677

(e) Sampled at 7-day fixed intervals, beginning on Day 4

Maumee River, 2008: 7-Day Sample Interval #4

0

10

20

30

40

50

60

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 4

linear 7-da interval 4

4-day avg, linear 7-da int 4
Do not cite

Appendix D2 - Page 538 of 677

(f) Sampled at 7-day fixed intervals, beginning on Day 5

Maumee River, 2008: 7-Day Sample Interval #5

0

10

20

30

40

50

60

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 5

linear 7-da interval 5

4-day avg, linear 7-da int 5
Do not cite

Appendix D2 - Page 539 of 677

(g) Sampled at 7-day fixed intervals, beginning on Day 6

Maumee River, 2008: 7-Day Sample Interval #6

0

10

20

30

40

50

60

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 6

linear 7-da interval 6

4-day avg, linear 7-da int 6
Do not cite

Appendix D2 - Page 540 of 677

(h) Sampled at 7-day fixed intervals, beginning on Day 7

Maumee River, 2008: 7-Day Sample Interval #7

0

10

20

30

40

50

60

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 7

linear 7-da interval 7

4-day avg, linear 7-da int 7
Do not cite

Appendix D2 - Page 541 of 677

Table D-2.4: 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 

average

Days 4-

da avg 

>20 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 da, beg day 2 3 36.6 2 32.8 8 0
Do 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 resulting chemographs (dark 

red) sampled at 7-day fixed intervals (red dots) for Sandusky River 

(Heidelberg), 2004

(a) Actual Chemograph (red arrows point to spikes)

Sandusky River, 2004

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill), max

Measured (w/ infill), avg

4-day avg
Do not cite

Appendix D2 - Page 543 of 677

(b) Sampled at 7-day fixed intervals, beginning on Day 1

Sandusky River, 2004: 7-Day Sample Interval #1

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 1

linear 7-da interval 1

4-day avg, linear 7-da int 1
Do not cite

Appendix D2 - Page 544 of 677

(c) Sampled at 7-day fixed intervals, beginning on Day 2

Sandusky River, 2004: 7-Day Sample Interval #2

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 2

linear 7-da interval 2

4-day avg, linear 7-da int 2
Do not cite

Appendix D2 - Page 545 of 677

(d) Sampled at 7-day fixed intervals, beginning on Day 3

Sandusky River, 2004: 7-Day Sample Interval #3

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 3

linear 7-da interval 3

4-day avg, linear 7-da int 3
Do not cite

Appendix D2 - Page 546 of 677

(e) Sampled at 7-day fixed intervals, beginning on Day 4

Sandusky River, 2004: 7-Day Sample Interval #4

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 4

linear 7-da interval 4

4-day avg, linear 7-da int 4
Do not cite

Appendix D2 - Page 547 of 677

(f) Sampled at 7-day fixed intervals, beginning on Day 5

Sandusky River, 2004: 7-Day Sample Interval #5

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 5

linear 7-da interval 5

4-day avg, linear 7-da int 5
Do not cite

Appendix D2 - Page 548 of 677

(g) Sampled at 7-day fixed intervals, beginning on Day 6

Sandusky River, 2004: 7-Day Sample Interval #6

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 6

linear 7-da interval 6

4-day avg, linear 7-da int 6
Do not cite

Appendix D2 - Page 549 of 677

(h) Sampled at 7-day fixed intervals, beginning on Day 7

Sandusky River, 2004: 7-Day Sample Interval #7

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 7

linear 7-da interval 7

4-day avg, linear 7-da int 7
Do 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 

(Heidelberg), 2005

(a) Actual Chemograph (red arrows point to spikes)

Sandusky River, 2005

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill), max

Measured (w/ infill), avg

4-day avg
Do not cite

Appendix D2 - Page 551 of 677

(b) Sampled at 7-day fixed intervals, beginning on Day 1

Sandusky River, 2005: 7-Day Sample Interval #1

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 1

linear 7-da interval 1

4-day avg, linear 7-da int 1
Do not cite

Appendix D2 - Page 552 of 677

(c) Sampled at 7-day fixed intervals, beginning on Day 2

Sandusky River, 2005: 7-Day Sample Interval #2

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 2

linear 7-da interval 2

4-day avg, linear 7-da int 2
Do not cite

Appendix D2 - Page 553 of 677

(d) Sampled at 7-day fixed intervals, beginning on Day 3

Sandusky River, 2005: 7-Day Sample Interval #3

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 3

linear 7-da interval 3

4-day avg, linear 7-da int 3
Do not cite

Appendix D2 - Page 554 of 677

(e) Sampled at 7-day fixed intervals, beginning on Day 4

Sandusky River, 2005: 7-Day Sample Interval #4

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 4

linear 7-da interval 4

4-day avg, linear 7-da int 4
Do not cite

Appendix D2 - Page 555 of 677

(f) Sampled at 7-day fixed intervals, beginning on Day 5

Sandusky River, 2005: 7-Day Sample Interval #5

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 5

linear 7-da interval 5

4-day avg, linear 7-da int 5
Do not cite

Appendix D2 - Page 556 of 677

(g) Sampled at 7-day fixed intervals, beginning on Day 6

Sandusky River, 2005: 7-Day Sample Interval #6

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 6

linear 7-da interval 6

4-day avg, linear 7-da int 6
Do not cite

Appendix D2 - Page 557 of 677

(h) Sampled at 7-day fixed intervals, beginning on Day 7

Sandusky River, 2005: 7-Day Sample Interval #7

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 7

linear 7-da interval 7

4-day avg, linear 7-da int 7
Do 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), 2006

(a) Actual Chemograph (red arrows point to spikes)

Sandusky River, 2006

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill), max

Measured (w/ infill), avg

4-day avg
Do not cite

Appendix D2 - Page 559 of 677

(b) Sampled at 7-day fixed intervals, beginning on Day 1

Sandusky River, 2006: 7-Day Sample Interval #1

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 1

linear 7-da interval 1

4-day avg, linear 7-da int 1
Do not cite

Appendix D2 - Page 560 of 677

(c) Sampled at 7-day fixed intervals, beginning on Day 2

Sandusky River, 2006: 7-Day Sample Interval #2

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 2

linear 7-da interval 2

4-day avg, linear 7-da int 2
Do not cite

Appendix D2 - Page 561 of 677

(d) Sampled at 7-day fixed intervals, beginning on Day 3

Sandusky River, 2006: 7-Day Sample Interval #3

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 3

linear 7-da interval 3

4-day avg, linear 7-da int 3
Do not cite

Appendix D2 - Page 562 of 677

(e) Sampled at 7-day fixed intervals, beginning on Day 4

Sandusky River, 2006: 7-Day Sample Interval #4

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 4

linear 7-da interval 4

4-day avg, linear 7-da int 4
Do not cite

Appendix D2 - Page 563 of 677

(f) Sampled at 7-day fixed intervals, beginning on Day 5

Sandusky River, 2006: 7-Day Sample Interval #5

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 5

linear 7-da interval 5

4-day avg, linear 7-da int 5
Do not cite

Appendix D2 - Page 564 of 677

(g) Sampled at 7-day fixed intervals, beginning on Day 6

Sandusky River, 2006: 7-Day Sample Interval #6

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 6

linear 7-da interval 6

4-day avg, linear 7-da int 6
Do not cite

Appendix D2 - Page 565 of 677

(h) Sampled at 7-day fixed intervals, beginning on Day 7

Sandusky River, 2006: 7-Day Sample Interval #7

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 7

linear 7-da interval 7

4-day avg, linear 7-da int 7
Do not cite

Appendix D2 - Page 566 of 677

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

(a) Actual Chemograph (red arrows point to spikes)

Sandusky River, 2007

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill), max

Measured (w/ infill), avg

4-day avg
Do not cite

Appendix D2 - Page 567 of 677

(b) Sampled at 7-day fixed intervals, beginning on Day 1

Sandusky River, 2007: 7-Day Sample Interval #1

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 1

linear 7-da interval 1

4-day avg, linear 7-da int 1
Do not cite

Appendix D2 - Page 568 of 677

(c) Sampled at 7-day fixed intervals, beginning on Day 2

Sandusky River, 2007: 7-Day Sample Interval #2

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 2

linear 7-da interval 2

4-day avg, linear 7-da int 2
Do not cite

Appendix D2 - Page 569 of 677

(d) Sampled at 7-day fixed intervals, beginning on Day 3

Sandusky River, 2007: 7-Day Sample Interval #3

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 3

linear 7-da interval 3

4-day avg, linear 7-da int 3
Do not cite

Appendix D2 - Page 570 of 677

(e) Sampled at 7-day fixed intervals, beginning on Day 4

Sandusky River, 2007: 7-Day Sample Interval #4

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 4

linear 7-da interval 4

4-day avg, linear 7-da int 4
Do not cite

Appendix D2 - Page 571 of 677

(f) Sampled at 7-day fixed intervals, beginning on Day 5

Sandusky River, 2007: 7-Day Sample Interval #5

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 5

linear 7-da interval 5

4-day avg, linear 7-da int 5
Do not cite

Appendix D2 - Page 572 of 677

(g) Sampled at 7-day fixed intervals, beginning on Day 6

Sandusky River, 2007: 7-Day Sample Interval #6

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 6

linear 7-da interval 6

4-day avg, linear 7-da int 6
Do not cite

Appendix D2 - Page 573 of 677

(h) Sampled at 7-day fixed intervals, beginning on Day 7

Sandusky River, 2007: 7-Day Sample Interval #7

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 7

linear 7-da interval 7

4-day avg, linear 7-da int 7
Do 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), 2008

(a) Actual Chemograph (red arrows point to spikes)

Sandusky River, 2008

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill), max

Measured (w/ infill), avg

4-day avg
Do not cite

Appendix D2 - Page 575 of 677

(b) Sampled at 7-day fixed intervals, beginning on Day 1

Sandusky River, 2008: 7-Day Sample Interval #1

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 1

linear 7-da interval 1

4-day avg, linear 7-da int 1
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Appendix D2 - Page 576 of 677

(c) Sampled at 7-day fixed intervals, beginning on Day 2

Sandusky River, 2008: 7-Day Sample Interval #2

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 2

linear 7-da interval 2

4-day avg, linear 7-da int 2
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Appendix D2 - Page 577 of 677

(d) Sampled at 7-day fixed intervals, beginning on Day 3

Sandusky River, 2008: 7-Day Sample Interval #3

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 3

linear 7-da interval 3

4-day avg, linear 7-da int 3
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Appendix D2 - Page 578 of 677

(e) Sampled at 7-day fixed intervals, beginning on Day 4

Sandusky River, 2008: 7-Day Sample Interval #4

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 4

linear 7-da interval 4

4-day avg, linear 7-da int 4
Do not cite

Appendix D2 - Page 579 of 677

(f) Sampled at 7-day fixed intervals, beginning on Day 5

Sandusky River, 2008: 7-Day Sample Interval #5

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 5

linear 7-da interval 5

4-day avg, linear 7-da int 5
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Appendix D2 - Page 580 of 677

(g) Sampled at 7-day fixed intervals, beginning on Day 6

Sandusky River, 2008: 7-Day Sample Interval #6

0

5

10

15

20

25

30

35

40

45

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 6

linear 7-da interval 6

4-day avg, linear 7-da int 6
Do not cite

Appendix D2 - Page 581 of 677

(h) Sampled at 7-day fixed intervals, beginning on Day 7

Sandusky River, 2008: 7-Day Sample Interval #7

0

5

10

15

20

25

30

35

40

45

50

4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19

Date Atrazine, ug/L

Measured (w/ infill)

7-da interval 7

linear 7-da interval 7

4-day avg, linear 7-da int 7
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Appendix E - Page 582 of 677

APPENDIX E. UPDATE FOR SAP

EPA is convening 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 installment 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 Study 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://www.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 case 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 Agricultural 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 focuses 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 peerreview. The methodology the Agency uses to consider occupational exposure differs from 

the approaches used in the AHS, and in epidemiological 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., 200229). Generically, exposure assessment in observational 

epidemiological investigations relies on self-reporting on an individual level to define and 

measure exposure. At times, this 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 resulting 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, Guittard 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.
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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 exposure 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 exposure 

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 cohort. 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 out 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 Health (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 especially 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 whether available historical pesticide usage 

would help refine the AHS and OPP collaborative efforts, or better characterize the results
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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.
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Appendix F - Page 585 of 677

APPENDIX F. TOXICOLOGY CHAPTER OF 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 number 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 Scientist

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 revised Atrazine Toxicology Chapter of the Reregistration Eligibility 

Decision.
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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-2000
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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 studies 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 hypothalamicpituitary-ovarian axis. The neuroendocrine disruptions 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 pulse 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 prolonged exposure to estrogen and 

prolactin. Although, there are certain similarities in the control of the hypothalamicpituitary- ovarian axis between humans and rats in that the hypothalamus can play a 

key regulatory role in primates, there are fundamental differences. 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/developmental 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 demonstrated 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 qualitative 
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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 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]. 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 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 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 via 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 recommends, 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 protocols 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.
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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

870.1200.............................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)

870.3150............... 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

yesa

yes

-

yesb

870.3700a Developmental Toxicity (Rodent) .....

870.3700b Developmental Toxicity (Nonrodent).............................................

870.3800.................... 2-Generation 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

870.5375Mutagenicity—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 Neurotoxicity 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 Requirement 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 study
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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 described 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 MRID 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 Gaps 

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 Reproduction 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 oral 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 offspring 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 special studies examining the 

CNS alterations following atrazine exposure be performed.
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Appendix F - Page 591 of 677

F.4.0 HAZARD ASSESSMENT

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 Toxicity 

Category

870.1000 Oral LD50 - rat LD50 > 1,869 mg/kg (M&F 

combined)

 III

870.1200 Dermal LD50 - rat LD50 > 2,000 mg/kg (M&F 

combined)

 III

870.1300 Inhalation LC50 -

rat

LC50 > 5.8 mg/L (M&F combined) IV

870.1400 Eye Irritation -

rabbit

PISa= 0.0/110 IV

870.1500 Dermal Irritation -

rabbit

PISa= 0.2/8.0 IV

870.1600 Dermal 

Sensitization -

guinea pig

Non-sensitizing ---

870.6100 Acute Delayed 

Neurotoxicity

Not Required ---

a PIS=Primary Irritation Score

870.1100. Acute oral toxicity - rats

A pair of acute LD50 studies in the rat using technical grade atrazine have 

shown the LD50 to be: 1,869 mg/kg (Accession No. 230303; MRID 00024706) and 2,850 

mg/kg (MRID 00027097). Numbers in all these studies 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 LD50 studies using the rat resulted in LD50s of >2,000 mg/kg and > 

3,100 mg/kg for both sexes combined (Accession Nos. 00027097 and 230303; MRID 

00024708). A study using rabbits, the preferred species in the OPPTS Series 870 
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Appendix F - Page 592 of 677

guidelines, resulted in an LD50 of 7,550 mg/kg for both sexes combined (Accession No. 

231466). Atrazine is at least toxicity category III for dermal exposure. 

870.1300. Acute inhalation toxicity - rats

The inhalation LC50 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 negative 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 studies 

(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

Adequacy 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), Atrazine Technical (purity 

97.1 %) was administered in the diet to Tif: RAIf (SPF), RII/1 x RII/2 hybrids, (SpragueDawley 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 sacrificed (S2).

One female in the control group died during the study. No treatment-related clinical 

effects were noted. Treatment with atrazine technical resulted in significant (p<0.01) 
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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 chemistry parameters showed no significant treatment related effects. 

Decreased liver and kidney weights in high dose males occurred as a result 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 guideline 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 42089902) 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% 

relative 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 (compared 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 reductions in 

food consumption, mean body weight, body weight gain and increases in spleen weight.
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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 

reduced 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 Developmental 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 

period, 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 observation 

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 dose 

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 clearly 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 various 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 ossification 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 atrazine under the conditions described in this study seemed to have few 

embryo/fetotoxic effects.
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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 AcceptableGuideline 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 was 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 

abdomens; 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 csection was comparable to controls (mean of 12.7 per litter for controls vs 13.4 for the 

high dose). 

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 of 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.

The 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 

satisfies the OPPTS 870.3700 guideline requirement for a developmental toxicity study 

in rats.
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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%) was 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 significantly 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 effects 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 

elements 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 study is classified Acceptable-Guideline and satisfies the guideline 

requirement for a developmental toxicity study (OPPTS 870.3700) in rabbits.
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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-generation 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 little variation in test article consumption between generations; the F0 

and F1 males had similar test article consumption during the 70-day premating period as 

did the F0 and F1 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 F0 and F1 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 F0 high-dose 

males and females at 70 days into the study were decreased by 12% and 15%, 

respectively while F1 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 

NOAEL 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 toxicity 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 Carcinogenicity 
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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 (SpragueDawley)

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 and 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. Food 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 in 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. Interestingly, 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 glucose. 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 weight 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 the 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 findings 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 < 
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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 weight 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 food 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/finalparta_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 

levels 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 significantly 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 or 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.
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Appendix F - Page 600 of 677

At the doses tested, there was no treatment-related increase in tumor incidence when 

compared to controls. Dosing was considered adequate based on decreased body 

weight gain.

This chronic toxicity/carcinogenicity study in the Fischer 344 rat is AcceptableGuideline and satisfies the guideline requirement for a chronic toxicity/carcinogenicity 

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 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 F-344 rats (see 

http://www.epa.gov/scipoly/sap/2000/june27/finalparta_atz.pdf ).

870.4100b Chronic Toxicity - Dog

In a chronic 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 as 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 highdose males was this increase statistically significant compared to the 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%. 

The 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 highdose group, on the other hand, displayed moderate to severe atrial dilation in 4/5 males 
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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 both 

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 

determined 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 

requirement (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 40431302), 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 mean 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 both 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, 
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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 groups. 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) 

has 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 

dimethylsufoxide, 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 

requirement (OPPTS 870.5100) for in vitro mutagenicity (bacterial reverse gene 

mutation) data.
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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 consisted 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 

requirement (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 activity. 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 Acceptable- Guideline, and it satisfies the guideline 

requirement (OPPTS 870.5450) for rodent dominant lethal data.

870.5550 UDS Assay 
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Appendix F - Page 604 of 677

In an unscheduled DNA synthesis assay (MRID 00161790/40722301) primary 

rat hepatocytes from adult male Tif:RAIf rats that 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-Guideline, 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 dimethyl 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 unscheduled 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. Atrazine 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 (specifically, 

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 

OPPTS 870.7485 guideline requirements. These studies are summarized below.
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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 labeled] 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 group 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 by 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 tissues 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 of 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 requirement 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 all 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 requirement 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.
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Appendix F - Page 606 of 677

In a metabolism study (MRID 40431306) atrazine (purity of unlabeled 

compound not reported but radiolabeled purity >97%), 14C-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-Nonguideline 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 

fecal 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 [U14C] 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, 
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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 deaths. 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, absorption was relatively rapid. The time to maximum blood 

concentration (tcmax) 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 100% 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 the 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 

(tcmax) at 2 hours and 24 hours for the low-dose and high-dose groups, respectively. 

Time to half maximum tissue concentrations (1⁄2 tcmax) for these groups 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, and 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 relatively 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 minimal 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 of which 

represented more than 2.42% of the total administered radioactivity.
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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) and 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 870.7485). 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 given a single oral low dose 

(1 mg/kg) or high dose (100 mg/kg) of [U14C] 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/cm2of 14C-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 exposed 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 kept 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 the 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 group. 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.
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Appendix F - Page 609 of 677

870.7600 Dermal Absorption - Human

In a dermal absorption study (MRID 44152114), 10 human volunteers were 

exposed to a single topical dose of [triazine ring-U14C] atrazine (94.3-96.3% a.i., 98.0-

98.4% radiochemical purity) at 6.7 (4 volunteers) or 79 μg/cm2(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%, respectively. 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 only 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 analysis 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 

methods. 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 Triazines

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 weight, 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 exposure 
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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 positive 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, exposure 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 in 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 radioactive 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 simazine 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 exhibit 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, diaminochlorotriazine (DACT, a mammalian metabolite of both atrazine and 

simazine) and simazine (>96 % a.i. for all three compounds) to the estrogen receptor 
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Appendix F - Page 611 of 677

(ER), several different experiments were performed utilizing extracted cytosolic ER from 

adult female Sprague-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-dependant 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 

10-9to 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 (IC50) for 

unlabeled estradiol was approximately 10-9 M whereas the IC50's for the triazine were 

20 μM for 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) relative 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 estradiol 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 atrazine 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 without 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 centrifugation. 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 tracer. 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 tracer. 
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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 competitive 

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 assays 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 progesterone 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 simazine. 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 anything 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: 10 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. 
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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 receptor 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. Polyacrylamide 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 

sequence 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 complex 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 indirect 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 luciferase 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 

in 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 displayed 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 using atrazine or simazine plus estradiol were performed 
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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 results 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 antiestrogenic 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: proliferation 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 system. 

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 PRPRE 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 estradiol 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 indirectly 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 measured 

luciferase activity as a surrogate measure of estrogen receptor activity. Doses used 

were 10-13M 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 

and increased exponentially up to 10-8 M where light production plateaued. Atrazine 
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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 atrazine 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 exhibits 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 validity of a proposed protocol for 

testing the effect of atrazine exposure on the proestrus afternoon luteinizing hormone 

(LH) surge, estradiol (E2) was administered to 70 ovariectomized female Sprague 

Dawley rats through a surgically implanted silastic capsule. Serum LH, E2, 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 appeared 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 

administered 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 body 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). 
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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 blocks 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 weight 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 rats 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 the 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 week 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 273%). 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 animals 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 clinical 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.
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Appendix F - Page 617 of 677
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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 rats 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. Hormone 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 for 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, gross 

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 GehanBreslow test there was a statistically significant negative trend for survival (survival 

decreased as the dose increased). Another statistical 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. Food 
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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, decreased 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 increased 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 compared 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 the 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 three 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 group 
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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 with 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 animals 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 histomorphology 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 

42085001. 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 

implies 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 time point. By three months the number of 

animals which did not have old corpora lutea present increased in a slight, but dosedependant 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 lutea, 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 grade 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 anovulation. These differences between control 

and treated groups became more extreme at the nine month timepoint. Seven of ten 
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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 present 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 the 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 months 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 and 

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 parameters 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 index 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 prolactin.

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 time 

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 though 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 high dose. 

Acinar/Lobular Development - An early onset of this parameter is evident. The oneand 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 increased 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. 
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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 

compared 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 parameters 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) SpragueDawley 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 following 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 

confirmed histologically indicated an early onset of mammary tumors. No statisticallysignificant 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 body 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 be 
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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 tested. 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 exposed 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 assessed 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 statisticallysignificantly 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 finding 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-neoplastic 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 finding 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 group. 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 the intact animals. No OVX animals 
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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 found 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 carcinogenicity.

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 incidence of 

pituitary adenomas compared to OVX animals.
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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 mammary 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 also 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 number 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 animals 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 aspects 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 direct 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 controls. 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 requirement 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 libitum 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, 

15, and 18 months exposure to the test article. 
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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 animals 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 tumors 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 

statistically-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 

corticosterone 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 were 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 and 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. 
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Appendix F - Page 627 of 677

An increase in prolactin levels might be expected 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 above) 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 decreases 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 consequence 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 that 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 

cycling 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 evidence 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 aging 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, 2000 document that was reviewed by the 

June, 2000 SAP [see http://www.epa.gov/scipoly/sap/2000/june27/finalparta_atz.pdf]. 
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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 chronological 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 herbicide, 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 tested (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 employed 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 profile 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 MRID 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 lactational 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., hyperprolactinemia) 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 day (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 been 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 the initiation of 
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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 atrazine-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 incidence 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 with 

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 herbicide, 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 

chlorotriazine 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 36Clflux. Diazepam (10 microM) and pentobarbital (100 microM) potentiated 

GABA-stimulated 36Cl- flux to 126 and 166% of control, respectively. At concentrations 
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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. 

(2000) 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 surge 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 suppressed 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 suppressed 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 (GnRH) 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 conclusion, 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. These 
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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., McElroy, 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 pointed 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 effect 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 NE 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 cyanazine 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 agonist, 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 and 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 inhibition 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.

Cummings, 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 reproductive effects including 

alterations in levels of pituitary hormones such as prolactin and luteinizing hormone (LH) in 
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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 secretion 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 periods 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 four 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 but 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 postimplantation 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 decreased 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 susceptible 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., 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.

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 resorption 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.
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Appendix F - Page 633 of 677

The effects of atrazine, a chlorotriazine herbicide, on the onset of puberty were 

evaluated in Wistar rats. Female 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 opening 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 the 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 females 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 weight 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 lateral 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 weights 
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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 

decreased 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 mean 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 difference 

in LH receptor number at PND 45 or 53. Serum estrone and estradiol showed doserelated 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 

screen 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 dehalogenation 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 capable 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 hydroxyatrazine 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. Hydroxyatrazine 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 appropriate 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 

chlorometabolites 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, desisopropyl atrazine , and desethyl atrazine. A limited 

toxicology database is available for these three metabolites and these studies are 

summarized below in sections F4.10.1, F4.10.2, and F4.10.3. A limited toxicology 

database is also available for hydroxyatrazine and is shown below under section 

F4.10.4.
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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 metabolite 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 the 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-related 

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 consumption 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, there 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 effects 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 dogs
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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, the 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. Moribundity 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, thickened 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, centrilobular 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 abdominal 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 spleen, 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 reversible 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 3.43 

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 rats
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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 SpragueDawley 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 in 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 bodyweight 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 affected 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 increase 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 doserelated increase in incomplete ossification of several bones 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, interparietals, 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 over 
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Appendix F - Page 638 of 677

controls in revertant colony counts) 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 activation 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 

Desisopropyl 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 or 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, 90, 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 treatment 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), decreased 

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 

significance and may not be treatment-related. Increased relative liver weights (+13%) 
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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 slight 

hypertrophy of the thyroid follicular epithelium in males at 500 ppm suggest treatmentrelated effects. Additionally, extramedullary hematopoiesis of the spleen in females at 

500 ppm and hypertrophy of pituitary cells in males at 500 ppm may suggest treatment 

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 subchronic 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-ppm 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 

groups. 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 

treatment-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 

myocardial 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 

weight 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 

considered 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 exposed 

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. 

. 
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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. 

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 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). Corrected 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/day, 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/kg/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 tested up to the limit dose of 

5000 μg/plate. This study is classified Acceptable-Guideline (MRID 43093101).

870.5385 Mammalian Bone Marrow Chromosome Aberration Test
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Appendix F - Page 641 of 677

The atrazine metabolite G-28279 (97.4%) was reported negative for the 

induction of micronuclei 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 atrazine 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) striazine; 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 of 

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 treatmentrelated 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 LOAEL 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. Treatment 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 renal tubular hyperplasia/basophilia in 3/4 male and 2/4 
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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) heart weights, normocytic/normochromic anemia, 

paroxysmal 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 satisfies 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% of 

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 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/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 posterior 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 

ossification 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.
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Appendix F - Page 643 of 677

870.5100 Bacterial Reverse 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, 5000 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 mice treated once orally up to clinically (but not 

cytologically) toxic doses (MTD = 480 mg/kg). This study is classified AcceptableGuideline (MRID 43093104).

870.5550 UDS Assay 

The atrazine metabolite G-30033 (99.3%) was negative for inducing 

unscheduled 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 reactions 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), hydroxyatrazine (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 exception 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% reduction 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 selected timepoints. Hematocrit and erythrocyte counts were 

significantly depressed in both sexes at the high dose only (-7.2% and 7.6% in males 
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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 (+38% 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 was 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 body 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 males 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 

guideline 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 26/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 seen 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 enlarged 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/litter 
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Appendix F - Page 645 of 677

incidence of 20%/56% vs 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 fetal 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:CD (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 toxicity, 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 

parameters (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 including 
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Appendix F - Page 646 of 677

deposition 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 resulting 

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. At 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, in 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 histopathological effects in the 

kidneys. The NOAEL 1.0 mg/kg/day.
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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 Reverse 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. This 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 hepatocytes up to and beyond 

precipitating concentrations (> 500 μg/mL). This study is classified Acceptableguideline (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 ENDPOINT 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 maximum 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 Potential
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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 carcinogenicity 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 to 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 27th to June 29th, 2000. Both the CARC 

and SAP agreed that atrazine is associated with mammary 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 tumors 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 accelerating 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- ovarian 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 conditions 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 development. 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 etiology 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 conducive 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. Additionally, 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 Carcinogenic To Humans‖ [HED Document 014431; December 13, 2000 meeting], 

based on the overall weight of evidence that:
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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 rat 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 lead 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 epidemiological 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 CONSIDERATIONS

F.6.1 Special Sensitivity 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 susceptibility 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 bodyweight gain and clinical signs in the maternal animal]. There were other 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 tumor 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 adverse 

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 

alterations. 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 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 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 

alterations 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 dietary [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 for 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 NUMBER)

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 Dermal 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 Sensitization 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. Autoradiographic DNA repair test on rat hepatocytes. CibaGeigy. 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/Mammalian--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

40431301O'Connor, D.; McCormick, G.; Green, J. (1987) Chronic Toxicity Study in Dogs: 

Atrazine Technical: Laboratory Study No. 852008. Unpublished study prepared 

by Ciba-Geigy Corp. 1405 p.

40431302Hazelette, 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.; Youreneff, 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.

40431304Orr, G. (1987) Disposition of Atrazine in Rat: (General Metabolism): Atrazine: 

Laboratory Study No.: ABR-87048. Unpublished study prepared by Ciba-Geigy 

Corp. 62 p

40431305Thede, 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.

40431306Miles, B. (1987) Characterization and Identification of Atrazine Metabolites from Rat 

Urine: (General Metabolism): Laboratory Study No.: ABR-87115. Unpublished 

study prepared by Ciba-Geigy Corp. 35 p.

40437501Orr, 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 Supplement 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. Unpublished 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, Inc. 316 p.
Do not cite

Appendix F - Page 653 of 677

40722301Ceresa, 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 Test: 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 Fibroblasts: 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 Corp. 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.

41065201Giknis, 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 prepared by CibaGeigy Corp. 362 p.

41293501Rudzki, 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., Pharmaceuticals Div. 408 

p.

41392401Thompson, 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.

41479401Ceresa, C. (1988) Hydroxyatrazine: Structural Chromosomal Aberration Test: Lab 

Project Number: 871373. Unpublished study prepared by Ciba-Geigy, Ltd. 32 p.

42085001Thakur, 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.

42165503Thede, B. (1991) Response to Data Request in EPA Review of Atrazine Registration 

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. Unpublished 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.

42547105Hertner, 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) Determination 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 Technical: 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.

43013203Thompson, 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. Ciba-Geigy Corp. 429 

p

43013205Schneider, 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.

43013206Gerspach, 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.

43013207Pettersen, J.; Richter, 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.

43013208Marty, J. (1992) G-28273 Technical: Teratology Study in Rats: Developmental Toxicity 

(Teratogenicity) Study in Rats with G-28279 Technical (Oral Administration): 

Lab Project Number: 901262. Unpublished study prepared by Ciba-Geigy Ltd. 

298 p.

43013209Marty, J. (1992) G-30033 Technical: Teratology Study in Rats: Developmental Toxicity 

(Teratogenicity) Study in Rats with G-30033 Technical (Oral Administration): Lab 

Project Number: 901265. Unpublished study prepared by Ciba-Geigy Ltd. 296 

p.

43016502Sova, J. (1993) Atrazine Technical: Response to the EPA Review of the Acute 

Inhalation Toxicity Study. Unpublished study prepared by Ciba Plant Protection. 

5 p.

43093101Deparade, E. (1990) Salmonella and Escherichia/Liver-Microsome Test: G-28279 

Technical: Gene Mutations Test: Lab Project Number: 891243. Unpublished 

study prepared by Ciba-Geigy Limited. 39 p.
Do not cite

Appendix F - Page 656 of 677

43093102Deparade, 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.

43093103Ogorek, 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 Test: 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.

43093106Geleick, D. (1991) Tests for Other Genotoxic Effects: Autoradiographic DNA Repair 

Test on Rat Hepatocytes: G-30033 Technical: Lab Project Number: 901310. 

Unpublished study prepared by Ciba-Geigy Limited. 94 p.

43314302Chengelis, 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.

43532001Chow, 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.

43598617Tennant, 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.

43598618Tennant, 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 43:197-211
Do not cite

Appendix F - Page 657 of 677

43598619Safe, S.; Chen, I.; Liu, H.; et al. (1995) Failure of Atrazine and Simazine to Induce 

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, and 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 compounds 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 

2386-109: 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.

44152102Morseth, S. (1996) Evaluation of the Luteinizing Hormone (LH) Surge in AtrazineExposed Female Sprague-Dawley Rats--(Final) 6-Month Interim Report: Lab 

Project Number: CHV 2386-111: 2386-111: 6791E. Unpublished study prepared 

by Corning Hazleton 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 Laboratories 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 Protection. 

297 p.

44713802 Paul, H. J.; Dunsire, J.P.; Hedley, D. (1993). The Absorption, Distribution, 

Degradation and Excretion of [U14C]-Triazine G 30027 in the Rat. 

Inveresk Research International, Tranent, EH33 2NE, Scotland. IRI Report 

No. 9523. December 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. Unpublished 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 chlorotriazine 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 evaluation 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 Dec 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. Toxicol. 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, Vienna 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 LD50 - rat LD50 > 1,869 mg/kg (M&F 

combined)

 III

870.1200 Dermal LD50 -

rat

LD50 > 2,000 mg/kg (M&F 

combined)

 III

870.1300 Inhalation LC50 -

rat

LC50 > 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 weight
Do 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 

chronic 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, and 

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

870.3700a

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 

consumption.

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

Maternal 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, increased 

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

See 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, Fischer-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 tumor 
Do not cite

Appendix F - Page 662 of 677

Guideline No./ 

Study Type

MRID No. (year) 

/Doses

Results

response in this strain following atrazine 

exposure.

870.4200

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, SpragueDawley)

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, 

1250, 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, 150 

μg/mL

Negative in primary rat hepatocytes up to 

150 μg/mL
Do 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-dependent 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 the 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 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.

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 than 

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 42165503 

(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 concentration 

(tcmax) 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 by 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 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. 
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/cm2

The percent absorbed 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 maximum 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 was 

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 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 performed a trio of assays: 

uterotrophic response assay; progesterone 

receptor competitive binding assay; 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 some 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 above 

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 receptor; 

and, luciferase reporter gene assay in MCF7 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 -

Estrous 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 hormone (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 attenuation
Do 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 study1-

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 Ovarian 

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 anovulation as indicated by ovarian 

histomorphology and early onset of 

indicators of prolonged/increased hormone 

exposure in the mammary gland

Special study2-

Two-year 

bioassay in F344 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 tumors.

Special study2- 42743902 (1993) NOAEL = at least 33.4 mg/kg/day
Do 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 confirm 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 study2-

Mammary Gland 

and Ovarian 

Histomorphology 

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 histomorphology 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-year 

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 

animal
Do 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 atrazinetreated animals compared to controls.

Special studyDirect 

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 days 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 contains 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 42146101 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.
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. (year) /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/day 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)

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.

870.3700a

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 

activation
Do not cite

Appendix F - Page 671 of 677

Guideline 

No./ Study 

Type

MRID No. (year) /Doses Results

870.5550

UDS assay

40722303 (1987)

0, 5.56, 16.67, 50, 150, 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

LD50 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 weight 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 rodents

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 fetal 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 800 
Do 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

Results

870.100

Acute oral 

toxicity

43013202 (1991)

500, 2000, 3500, 

5050 mg/kg

LD50 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 

hyperplasia/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

870.5375

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 concentrations of 1000 
Do not cite

Appendix F - Page 675 of 677

Guideline No./ 

Study Type

MRID No. (year) 

/Doses

Results

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 based 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 decreased 

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 males 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 μg/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 was 

found up to the limits of solubility (increasing 

precipitation from 500 μg/mL) and at 

concentrations approaching toxicity (1500 μg/mL) in 
Do 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 μg/mL) and severe 

cytotoxicity (1500 μg/mL) in human fibroblast cells.

HIARC Report [TXR NO. 0050592]