Page 1 of 210

APPENDIX A

EVALUATION OF ARTICLES ON ATRAZINE AND ATRAZINE METABOLITES 

RELATED TO MAMMALIAN TOXICITY STUDIES SUGGESTING POTENTIAL 

ADVERSE HUMAN HEALTH OUTCOMES
Page 2 of 210

A.1 CONTENT OF THIS APPENDIX ..........................................................................................................................8

A.2 EFFECTS ON FEMALES ................................................................................................................................... 10

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

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 UNPUBLISHED SYNGENTA STUDY................................................................................................................13

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...................................................................................15

A.2.4 CODER P. (2011E). 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 REPORT. REPLACES MRID 48180103) UNPUBLISHED 

DATA FROM SYNGENTA CROP PROTECTION, INC. STUDY COMPLETION DATE: FEBRUARY 4, 2011. REPORT NUMBER: WIL-639060.

MRID 48381001 ......................................................................................................................................................17

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

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

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

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

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. ...............................................................................................................................................28

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

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. ...........31

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

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. ............................34

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.........................................................................36

A.2.15 DAVIS LK, MURR AS, BEST DS, FRAITES MJP, ZORRILLA LM, MAROTSKY MG, STOKER TE, GOLDMAN JM, COOPER

RL. 2011. THE EFFECTS OF PRENATAL EXPOSURE TO ATRAZINE ON PUBERTAL AND POSTNATAL REPRODUCTIVE INDICES IN THE FEMALE 

RAT. REPROD. TOXICOL., DOI:10.1016/J.REPROTOX.2011.04.004 ...................................................................................39

A.2.16 GOLDMAN J, DAVIS L, MURR A, COOPER R. (2011). THE INFLUENCE OF 1, 2, AND 4 DAYS OF ATRAZINE TREATMENTS ON 

THE LH SURGE IN OVARIECTOMIZED/ESTRADIOL-PRIMED RATS: PRELIMINARY REPORT TO THE OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION. INTERNAL REPORT USEPA.........................................................................................................42

A.2.17 CODER, P. S. (2011) AN ORAL (GAVAGE AND DIETARY) STUDY OF THE EFFECTS OF ATRAZINE ON THE SPONTANEOUS 

LUTEINIZING HORMONE SURGE IN FEMALE SPRAGUE-DAWLEY RATS, WIL RESEARCH LABORATORIES, LLC, 1407 GEORGE ROAD,

ASHLAND, OH. UNPUBLISHED. STUDY NUMBER: WIL-639080...........................................................................................43
Page 3 of 210

A.2.18 CODER, P. S. (2011) AN STUDY OF THE EFFECTS OF SHORT-TERM ATRAZINE EXPOSURE ON THE ESTROGEN-INDUCED 

LUTEINIZING HORMONE (LH) SURGE IN OVARIECTOMIZED YOUNG-ADULT SPRAGUE DAWLEY FEMALE RATS, WIL RESEARCH 

LABORATORIES, LLC, 1407 GEORGE ROAD, ASHLAND, OH. UNPUBLISHED. STUDY NUMBER: WIL-639091 ...............................47

A.2.19 CODER, P. (2011D) ATRAZINE: AN ORAL (GAVAGE AND DIETARY) STUDY OF THE EFFECTS OF ATRAZINE ON FERTILITY AND 

REPRODUCTIVE PERFORMANCE IN FEMALE LONG EVANS AND SPRAGUE-DAWLEY RATS: FINAL REPORT. REPORT NUMBER:

WIL/639090, TASK NO. TK0052068. UNPUBLISHED STUDY. PERFORMING LABORATORY: WIL RESEARCH LABORATORIES, INC. 966

P. COMPLETION DATE: APRIL 6, 2011. MRID 48470001. ................................................................................................49

A.3 REPRODUCTIVE EFFECTS IN MALES ............................................................................................................... 51

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. ..........................................................................................56

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.

................................................................................................................................................................................58

A.3.3 FRIEDMANN, AS. 2002. ATRAZINE INHIBITION OF TESTOSTERONE PRODUCTION IN RAT MALES FOLLOWING PERIPUBERTAL 

EXPOSURE. REPRODUCTIVE TOXICOLOGY 16: 275-279. .....................................................................................................59

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.....................................................................................................................................................60

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. .61

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

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. ................................64

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. .....................................................................65

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

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. ........68

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

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

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......................................................................73

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. .................................................................................................................................................74

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

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

A.3.17 FRAITES MJP, NAROTSKY MG, BEST DS, STOKER TE, DAVIS LK, GOLDMAN JM, HOTCHKISS MG, KLINEFELTER GR, KAMEL 

A, QIAN Y, PODHORNIAK L, COOPER RL. (2011). GESTATIONAL ATRAZINE EXPOSURE EFFECTS ON MALE REPRODUCTIVE 

DEVELOPMENT AND METABOLITE DISTRIBUTION IN THE DAM, FETUS, AND NEONATE. REPROD. TOXICOL.,

DOI:10.1016/J.REPROTOX.2011.04.003.......................................................................................................................79
Page 4 of 210

A.4 AROMATASE................................................................................................................................................. 80

Summary: Effects on Aromatase and Steroidogenesis, Andrology..................................................................80

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

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. .86

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

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. ........88

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

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). ....................................90

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

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

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. .......................................................................................................................................94

A.4.10 SUZAWA, M AND INGRAHAM, HA. 2008. THE HERBICIDE ATRAZINE ACTIVATES ENDOCRINE GENE NETWORKS VIA NONSTEROIDAL NR5A NUCLEAR RECEPTORS IN FISH AND MAMMALIAN CELLS. PLOS ONE 3(5): E2117. 

DOI:10.1371/JOURNAL.PONE.0002117. .......................................................................................................................96

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. ................................................................................................97

A.4.12 YI, KD, PEREZ, EJ, BRECKENRIDGE, CB, AND SIMPKINS, JW (2010?) EX VIVO EFFECTS OF ATRAZINES OR METABOLITES 

ON PHOSPHODIESTERASE ACTIVITY...................................................................................................................................98

A.4.13 POGRMIC-MAJKIC K, FA S., DAKIC V, KAISAREVIC S., KOVACEVIC R. 2010. UPREGULATION OF PERIPUBERTAL LEYDIG CELL 

STEROIDOGENESIS FOLLOWING 24 H IN VITRO AND IN VICO EXPOSURE TO ATRAZINE. TOXICOL. SCI. 118(1): 52-60.....................99

A.5 NEUROTOXICITY ......................................................................................................................................... 101

Summary: Neurotoxicity..................................................................................................................................102

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

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

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. .......................................................................................................................................108

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. ...............................................................................................................................109

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

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

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. ....114
Page 5 of 210

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

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...........117

A.5.10 BELLONI V, DESSI-FULGHERI, F, ET AL. 2010. EARLY EXPOSURE TO LOW DOSES OF ATRAZINE AFFECTS BEHAVIOR IN JUVENILE 

AND ADULT CD1 MICE. TOXICOLOGY (2010), DOI:10.1016/J.TOX.2010.07.002...............................................................117

A.6 IMMUNOTOXICITY...................................................................................................................................... 121

Summary: Immunotoxicity..............................................................................................................................121

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

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)

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

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

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

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

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

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

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

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

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

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............................................................................................................................................................137

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

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

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. ................................................141

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.......................................................................................141

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

A.7 CARCINOGENESIS/GENOTOXICITY .............................................................................................................. 142

Summary: Carcinogenesis/Genotoxicity.........................................................................................................142

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. ............145
Page 6 of 210

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. ....................146

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

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

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. ...............................................................................................................................................151

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. .....152

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..........................................................................153

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.......155

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

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 .......................................158

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

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

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

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. .162

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

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

A.8 INSULIN/MITOCHONDRIA FUNCTION ......................................................................................................... 168

Summary: Insulin/Mitochondria Function......................................................................................................168

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

A.9.1. PHARMACOKINETIC CALCULATIONS ........................................................................................................ 170

A.9.2. PHARMACOKINETIC STUDIES- MINIREVIEWS .............................................................................................199

A Physiologically Based Pharmacokinetic (PBPK) Model for Atrazine and its Main Metabolites in the Adult 

Male C57BL/6 Mouse Toxicology and Applied Pharmacology 251 (2011) 16-31.............................................199

In Vitro Metabolism of Atrazine in Rat and Human Hepatocytes....................................................................201

PBPK Model for Atrazine and its Chlorinated Metabolites...............................................................................201

A. 10 MIXTURES................................................................................................................................................. 202
Page 7 of 210

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 AFTER ACUTE PRENATAL EXPOSURE TO AN ATRAZINE METABOLITE MIXTURE IN FEMALE 

LONG-EVANS RATS. ENVIRONMENTAL HEALTH PERSPECTIVES. 115(4): 541-547. ..............................................................202

A.11 OTHER....................................................................................................................................................... 207

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

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 ........................................................................................................................209
Page 8 of 210

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

Coder P. (2011e). 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 Report. Replaces MRID 48180103) Unpublished data from Syngenta Crop 

Protection, Inc. Study completion date: February 4, 2011. Report Number: WIL-639060. 

MRID 48381001

A.2.4

Davis LK, Murr AS, Best DS, Fraites MJP, Zorrilla LM, Marotsky MG, Stoker TE, 

Goldman JM, Cooper RL. 2011. The Effects of Prenatal Exposure to Atrazine on 

Pubertal and Postnatal Reproductive Indices in the Female Rat. Reprod. Toxicol., 

doi:10.1016/j.reprotox.2011.04.004

A.2.15

Goldman J, Davis L, Murr A, Cooper R. (2011). The Influence of 1, 2, and 4 Days of 

Atrazine Treatments on the LH Surge in Ovariectomized/Estradiol-Primed Rats: 

Preliminary Report to the Office of Chemical Safety and Pollution Prevention. Internal 

Report USEPA

A.2.16

Coder, P. S. (2011) An Oral (Gavage and Dietary) Study of the Effects of Atrazine on the 

Spontaneous Luteinizing Hormone Surge in Female Sprague-Dawley Rats, WIL 

Research Laboratories, LLC, 1407 George Road, Ashland, OH. Unpublished. Study 

Number: WIL-639080

A.2.17

Coder, P. S. (2011) An Study of the Effects of Short-Term Atrazine Exposure on the 

Estrogen-Induced Luteinizing Hormone (LH) Surge in Ovariectomized Young-Adult 

Sprague Dawley Female Rats, WIL Research Laboratories, LLC, 1407 George Road, 

Ashland, OH. Unpublished. Study Number: WIL-639091

A.2.18

Coder, P. (2011d) Atrazine: An Oral (Gavage and Dietary) Study of the Effects of 

Atrazine on Fertility and Reproductive Performance in Female Long Evans and 

Sprague-Dawley Rats: Final Report. Report Number: WIL/639090, Task No. TK0052068. 

Unpublished study. Performing Laboratory: WIL Research Laboratories, Inc. 966 p. 

Completion Date: April 6, 2011. MRID 48470001.

A.2.19

Fraites MJP, Narotsky MG, Best DS, Stoker TE, Davis LK, Goldman JM, Hotchkiss MG, 

Klinefelter GR, Kamel A, Qian Y, Podhorniak L, Cooper RL. (2011). Gestational Atrazine 

Exposure Effects on Male Reproductive Development and Metabolite Distribution in the 

Dam, Fetus, and Neonate. Reprod. Toxicol., doi:10.1016/j.reprotox.2011.04.003

A.3.17

Pogrmic-Majkic K, Fa S., Dakic V, Kaisarevic S., Kovacevic R. 2010. Upregulation of 

Peripubertal Leydig Cell Steroidogenesis Following 24 h In Vitro and In Vico Exposure 

A.4.13
Page 9 of 210

to Atrazine. Toxicol. Sci. 118(1): 52-60

Belloni V, dessi-Fulgheri, F, et al. 2010. Early exposure to low doses of atrazine affects 

behavior in juvenile and adult CD1 mice. Toxicology (2010), 

doi:10.1016/j.tox.2010.07.002

A.5.10

Enoch, R.R., Stanko, J.P., Greiner, S.N., Youngblood, G.L. Rayner, J.L., Fenton, S.E., 

2007. Mammary Gland Development as a Sensitive End Point after Acute Prenatal 

Exposure to an Atrazine Metabolite Mixture in Female Long-Evans Rats. 

Environmental Health Perspectives. 115(4): 541-547.

Stanko, J., Enoch, R., Rayner, J., Davis, C., Wolf, D., Malarkey, D., and Fenton S. 2010. 

Effects of Prenatal Exposure to a Low Dose Atrazine Metabolite Mixture on Pubertal 

Timing and Prostate Development of Male Long Evans Rats. Reproductive Toxicology. 

30:540-549.

A.10.1
Page 10 of 210

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.1

 

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.

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 

 1 Studies on the potential effect of atrazine exposure on mammary gland development will be evaluated 

during the Fall meeting of the SAP.
Page 11 of 210

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 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 
Page 12 of 210

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, 

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 
Page 13 of 210

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 lowand 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 2.2

 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).3 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 were sexed and 

subjected to clinical observations, body weight and food consumption determinations.

For P dams

 2 This aspect of the study will not be discussed further in this review.

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 midand high-dose remained lower relative to control (↓4-11%) probably due to the initial 

3 Only litters comprised of ≥ 6 female pups and at least 10 pups total were used for litter standardization 

and subsequent assessments
Page 14 of 210

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 ≥ 50mg/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.4

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 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 

 4 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
Page 15 of 210

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 

implantation of an estradiol benzoate containing capsule (4 mg/ml)5

 5 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

. 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) 
Page 16 of 210

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 postoperative 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, 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.
Page 17 of 210

A.2.4 Coder P. (2011e). 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 Report. Replaces MRID 

48180103) Unpublished data from Syngenta Crop Protection, Inc. 

Study completion date: February 4, 2011. Report Number: WIL639060. MRID 48381001

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 F1 female Sprague-Dawley [Crl:CD(SD)] rats 

(approximately 13-14 weeks of age on gestation day 0) following oral gavage dosing to 

two separate Cohorts (I and II) of animals at atrazine doses of 0, 6.5, 25 or 50 

mg/kg/day. In Cohort I, F1 females were exposed to atrazine from conception through 

sexual maturation (vaginal opening) or through PND 133. In Cohort II, naïve (untreated) 

F1 females were exposed to atrazine beginning at weaning (PND 21) through sexual 

maturation or for 14 days in adulthood (PND 121-133).

Study Design: Atrazine was administered daily via oral gavage to Crl:CD(SD) rats (F0 

females and/or their F1 female offspring) at dose levels of 0, 6.5, 25 or 50 mg/kg/day. 

The study was divided into 2 cohorts (I and II) each with 4 dose 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. Each subset consisted of 15-25 F1

females/group. The study design is shown in the following table and figure (taken from 

pages 23-24 of Syngenta study report).
Page 18 of 210

GD = Gestation Day LD = Lactation Day PND = Postnatal Day

VO = Day of vaginal opening

OVX = Day of ovariectomy

B/N = Day of blood collection and necropsy

E = Beginning of 3-week estrous evaluation period

Cohort I: Mated female rats (25/group) (F0 generation) were administered atrazine (0, 

6.5, 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 F1 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, which was the day euthanasia for 

Subset B. Subset C had a 3-week post-treatment recovery period. 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, 6.5, 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. 

Animals
Page 19 of 210

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, F1 offspring were housed individually in suspended wire-mesh cages until 

euthanasia. LLC Certified Rodent LabDiet®5002 and water were provided ad libitum.

Dosing: All animals were dosed at approximately the same time each day. F1 females in 

Subsets B and C, which received a total of 113 doses, were given daily doses at 

approximately 6-9 hours post-lights (1100 hours to 1400 hours). All other animals on 

study (F0 females in Cohort I and F1 females in Subsets A, D,E,F) which received 14-43 

doses of atrazine, were given daily doses at approximately 7-8:45 hours post-lights on 

(1200 hours to 1345 hours).

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.

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 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 6.5 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 
Page 20 of 210

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. 

Results: 

Cohort I: 

Gestational and lactational exposure to atrazine in F0 mated females resulted in 

reduced F1 postnatal survival, and lower pre-weaning pup body weight and body weight 

gains at 50 mg/kg/day. Continued administration of atrazine to F1 females following 

weaning resulted in lower post-weaning body weights and body weight gains at 50 

mg/kg/day (Subsets A, B and C). 

A 1.4- to 2.2-day delay in the age of attainment of vaginal patency was noted in the 50 

mg/kg/day group F1 females in Subsets A, B, C. When these data were combined there 

was a 1.6-day delay (p<0.01) in the age of attainment of vaginal patency noted at 50 

mg/kg/day. The mean body weight on the day of the attainment in the 50 mg/kg/day 

was similar to the controls. At 6.5 and 25 mg/kg/day there were no effects on the ages 

of attainment of vaginal patency or body weights at attainment for F1 females in 

Subsets A, B or C of Cohort 1. The combined data are presented below.

There was also no dose-related effect on the mean estrous cycle length for Subsets B 

and C.

Cohort II

Administration of atrazine from PND 120-133 to F1 females in Subsets E and F resulted 

in lower mean body weight gain and/or losses during week 1 of treatment at 25 

mg/kg/day and throughout the 2-week treatment at 50 mg/kg/day.

Atrazine administration at 6.5, 25 and 50 mg/kg/day had no effect on the attainment of 

vaginal patency for F1 females in Subsets D, E, and F or on the mean estrous cycle 

length for F1 females in Subsets E and F. In Subsets D and E, vaginal patency was 

attained prior to the beginning of atrazine treatment on PND 120.

Plasma Hormone Analysis: An LH surge was detected in all treatment groups (0, 6.5, 

25 and 50 mg/kg/day) for Cohort I (Subsets A, B, C) and Cohort II (subsets D, E, F). 
Page 21 of 210

Also for both cohorts and corresponding subsets there were no significant differences in 

either the peak height of the LH surge or the area under the curve for the LH surge in 

any of the treatment groups. 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.

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 and their 

subsets), compared to controls. The study authors, however, stated that the timing of 

blood collection was not ideal for elucidating an effect on corticosterone. [Three days 

following ovariectomy, blood samples for corticosterone were collected from all 

surviving females 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).]

Concentrations of atrazine and metabolites in Rat plasma: Plasma concentrations of

atrazine and its chlorometabolites 10.5 to 12 hours after dose administration indicated 

that little or no atrazine remained in the plasma. The chlorometabolites (DIA, DEA and 

DACT) were detectable at all dose levels. The concentrations of DACT were greater 

than DEA and DIA. 

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. No effect on LH surge was seen at 6.5, 

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 Long-Evans 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, however the study 

authors stated that their timing of blood collection was not ideal for elucidating an effect 

on CORT. 

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.
Page 22 of 210

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.

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 
Page 23 of 210

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. 

 

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, RNAbinding proteins containing active-site or solvent-exposed cysteine residues were also 

detected in both sample types. 
Page 24 of 210

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-Pituitary-Adrenal 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-s-chlorotriazine (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.

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 non-treated animals for 5 minutes on stress 

hormone levels as well as the pituitary and uterine weights.
Page 25 of 210

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 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
Page 26 of 210

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.

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 
Page 27 of 210

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 GonadotropinReleasing 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.

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 
Page 28 of 210

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 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, 
Page 29 of 210

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.
Page 30 of 210

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, 

diamino-s-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 

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. 
Page 31 of 210

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)/progesterone-induced LH surge are due to impaired ER 

function. As expected, ATRA suppressed the EB and progesterone-induced LH surge in 

a dose dependent manner. A daily dose 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. 

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 
Page 32 of 210

of estradiol-binding, 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). 

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. 
Page 33 of 210

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).

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, 
Page 34 of 210

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.

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 
Page 35 of 210

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 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- 1: Atrazine and Predominant Metabolites Tissue Levels in Dams Exposed GD14-PND10 and 

their Offspring
Page 36 of 210

Atrazine 

dose 

(mg/kg/day)

Tissue Content

Atrazine DACT DIA

Dams Pups Dams Pups Dams Pups

Plasma 

(ng/ml)

0 LOQa LOQ LOQ LOQ LOQ LOQ

5 0.9 ± 0.4 LOQ 960.5 ± 36.2 96.8 ± 4.1

(10%) b 38.3 ± 6.8 LOQ

25 9.8 ± 8.3 LOQ 2477 ± 

863.6

330.7 ± 79.2

(13%)

173.8 ± 111.4 1.8 ± 0.32

(1%)

Adrenal 

(ng/g)

0 LOQ LOQ LOQ LOQ LOQ LOQ

5 10,3 ± 4.2 2.9 ± 2 332.9 ± 151 LOQ 10.6 ± 6.8 LOQ

25 58.7 ±45.6 LOQ 424.9 ± 

173.9

86 ± 50.8

(20%)

44.5 ± 28.6 LOQ

Brain

(ng/g)

0 LOQ LOQ LOQ LOQ LOQ LOQ

5 1.2 ± 0.2 5.6 ± 5.4

(367%)

710.4 ± 42.5 100.1 ± 9.9

(14%)

11 ± 2.1 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. 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 
Page 37 of 210

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).6

 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 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 prenatal 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

 6 Male pups were used for another study unrelated to these investigations.
Page 38 of 210

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 (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.
Page 39 of 210

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).

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.2.15 Davis LK, Murr AS, Best DS, Fraites MJP, Zorrilla LM, Marotsky MG, Stoker 

TE, Goldman JM, Cooper RL. 2011. The Effects of Prenatal Exposure to Atrazine 

on Pubertal and Postnatal Reproductive Indices in the Female Rat. Reprod. 

Toxicol., doi:10.1016/j.reprotox.2011.04.004

In this study the authors investigated the potential impact of in utero exposure on female 

reproductive development as measured by puberty onset (day of vaginal opening), 

mammary gland (MG) development, and estrous cyclicity. This study is the companion 

study to Fraites et al., (2011) in which effects of gestational exposure on male 

reproductive development were described. In addition, the investigators studied the 

transfer of atrazine and its metabolites into the fetus after maternal exposure during 

gestation (GD 18-20 or GD 14-20).7

Time-pregnant Sprague-Dawley rats (12 dams/dose) were exposed to 0, 1, 5, 20, and 

100 mg/kg/day atrazine (97% a.i. purity) by gavage once daily (s.i.d) or divided into 2 

daily doses (b.i.d) during GD 14-21. On PND 7 pups were weighed and AGD 

measured. Beginning on PND 28 all female offspring were evaluated daily for vaginal 

opening and the body weights were recorded on each day. Mammary gland 

development was assessed using whole mounts of the 4th abdominal mammary gland 

of PND 45 old pups. This day was selected on the basis of a recommendation from a 

 7 The results of the atrazine distribution experiments were described in review A.2.13 in Appendix A of 

this document and will not be discussed further in this review.
Page 40 of 210

workshop on MG development held in 2009.8

No effects on pup viability, pup weight, or attainment of vaginal opening (VO) are 

reported at doses ≤ 20 mg/kg/day. Early postnatal pup mortality (PND0-4) was 

increased at the 100 mg/kg/day dose level in both the s.i.d. (9.7% mortality vs. 1.4 in 

control) and b.i.d. dosing regimen (33.6% mortality vs 0.9% control). Other effects 

reported at 100 mg/kg/day included decreases in pup weight after s.id. (↓15-17%) or 

b.i.d. ((↓12-15%) exposures and delays in VO (<2 day delay). A statistically significant 

decrease in pup weight (↓10%, p<0.05) at the 5 mg/kg/day dose level (s.i.d. exposure) 

was not considered to be toxicologically significant since it showed no dose-response, 

had resolved by PND4, and was not observed in the b.i.d. exposure regimen. No effects 

on estrous cyclicity or MG development were noted at any dose level regardless of the 

dosing regimen. 

 MG development was evaluated using 

both a morphometric approach and subjective scoring in accordance to the 

methodology used by Enoch et al. (2004). In the morphometric methodology, 

photographs of each mammary gland were evaluated using ImagePro® Software linked 

to a camera mounted on a microscope. Measures were taken of various regions and 

coefficient of variation determined to assess the representativeness of the area. Branch 

points, terminal end buds, and the distance between the lymph nodes and the ductal 

field were measured using this approach. Using the procedures described by Enoch, 

the mammary glands were subjectively scored for their stage of development where a 

score of 1 represented poor MG development while a score of 4 represented normal 

development for a PND45 rat. Finally, after all females attained vaginal patency, 

estrous cycles were monitored at 14 day intervals to assess cyclicity status.

Reviewer Comments: This is a well conducted study that evaluates the impact of in 

utero atrazine exposure on female reproductive indices including VO attainment, 

extrous cyclicity, and MG development. This study confirms previous findings by 

Rayner et al. (2004 and 2005). Toxicologically relevant effects including decreases in 

pup weight, increased early postnatal mortality, and delays in VO were noted at the 100 

mg/kg/day dose only. Most notably, however, is the failure to affect MG development at 

any dose level whether it was evaluated using a morphometric analysis or the 

subjective scoring described by Enoch et al. (2004).

Figure 1. Quantitative analysis of mammary gland morphology in female rats prenatally 

exposed to atrazine on GD 14-21 either once per day (s.i.d.) or a half dose twice per 

day (b.i.d.). Slides were quantified by at least 2 individuals blinded to the experimental 

 8 Mammary gland whole mount round robin summary. 2009; http://www.silentspring.org/pdf/our_research/

MammaryGlandWholeMountRoundRobinSummary.pdf
Page 41 of 210

treatment. Numbers in parentheses above the columns indicate group size.

0 1 5 20 100 0

5

10

15

20

25 A

(10) (10) (10)

(9)

(9)

ATR treatment (mg/kg)

Branches / 9 mm2

0 1 5 20 100 0

5

10

15

20

25 D

(11) (6)

(10) (10) (9)

ATR treatment (mg/kg)

Branches / 9 mm2

0 1 5 20 100 0

10

20

30

40

50 B(8)

(9) (7)

(9)

(6)

ATR treatment (mg/kg)

Number of TEBs

0 1 5 20 100 0

10

20

30

40

50

60 E

(11) (6)

(9)

(8)

(9)

ATR treatment (mg/kg)

Number of TEBs

0 1 5 20 100 0

5

10

15

C (9) (9)

(8)

(10) (9)

ATR treatment (mg/kg)

Distance (mm)

0 1 5 20 100 0

5

10

15

F (11) (9) (10) (10) (6)

ATR treatment (mg/kg)

Distance (mm)

s.i.d. b.i.d.
Page 42 of 210

Figure 2. Subjective scoring of the control and high dose mammary gland whole 

mounts analyzed in Figure 1. Slides were scored by two individuals based on the 

system described by Enoch et al. (2007).

s.i.d. b.i.d.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5 control

100 mg/kg

Dosing regimen

Subjective score

A.2.16 Goldman J, Davis L, Murr A, Cooper R. (2011). The Influence of 1, 2, 

and 4 Days of Atrazine Treatments on the LH Surge in 

Ovariectomized/Estradiol-Primed Rats: Preliminary Report to the 

Office of Chemical Safety and Pollution Prevention. Internal Report 

USEPA

The purpose of this study was to investigate the impact of 1, 2, or 4 days of atrazine 

exposure on the LH surge and circulating progesterone (one of the hormones involved 

in the regulation of the LH surge) levels. While a brief increase in progesterone levels 

on the day of proestrous acts in conjunction with estradiol to trigger the LH surge, a 

prolonged period of elevated progesterone results in an attenuation of the LH surge. 

This is in fact, one of the basic principles used in the production of oral contraceptives.

Nine to eleven regularly cycling Sprague Dawley rats were bilaterally ovariectomized 

and implanted with an estradiol benzoate capsule (4 mg/ml in sesame oil). Three days 

after surgery, blood was collected at 1400, 1600, and 1800 hours from the lateral tail 

vein. At 2000 hours, the animals were sacrificed and trunk blood collected. Prior to 

blood collection, these animals had been exposed to 0 or 100 mg/kg/day atrazine (97% 

a.i. purity) by gavage once daily (at 1300 hours) for 1, 2 or 4 days. The group that 

received only 1 day of atrazine exposure was dosed at 1300 hours on the day of 

sacrifice; the group exposed for 2 days was dosed for the last two days before sacrifice, 

while the group exposed for 4 days received atrazine every day beginning the day 

before the ovariectomy. Measurements of circulating LH level were conducted using a 

radioimmunoassay with materials from the National Hormone and Peptide Program. 

Estradiol and progesterone levels were measured with a Siemens Healthcare 

Diagnostic antibody-coated tube kit.
Page 43 of 210

For animals receiving a single dose of atrazine on the day of sacrifice a statistically 

significant (p<0.01) increase in progesterone levels were observed one hour after 

dosing. However, progesterone levels returned to background (i.e., comparable to 

control) 3 hours after exposure. An LH surge is observed (manifested as an increase in 

both peak amplitude and area under the curve) in the animals receiving a single dose of 

atrazine. In contrast, 4 days of atrazine exposure did not change progesterone levels 

from background. Without the physiological increase in progesterone, the LH surge 

was attenuated in these animals both in terms of peak amplitude and area under the 

curve. 

Reviewers Comments: This is a well conducted study that begins to investigate the 

potential impact of single exposures to atrazine on the organism’s hormonal milieu. The 

data show that a single day of exposure elicits a marked increase in progesterone 

resulting in an LH surge similar to what is observed in intact cycling female rats. Given 

that the rats in this study had been ovariectomized prior to the atrazine exposure, the 

increase in progesterone seen is due to a rise of its synthesis in the adrenal glands. 

The observation that the elevated progesterone levels are observed one hour after a 

single exposure to atrazine and resolved by 3 hours suggests that atrazine’s effect on 

the adrenal glands is rapid and transient. The toxicological relevance and relationship 

between these changes in progesterone and LH and potential adverse health outcomes 

is unclear. In addition,100 mg/kg/day is not an environmentally relevant dose thus a 

dose-response for these findings would be informative in the future.

A.2.17 Coder, P. S. (2011) An Oral (Gavage and Dietary) Study of the Effects 

of Atrazine on the Spontaneous Luteinizing Hormone Surge in 

Female Sprague-Dawley Rats, WIL Research Laboratories, LLC, 1407 

George Road, Ashland, OH. Unpublished. Study Number: WIL-639080 

In an oral gavage and dietary study the purpose was to evaluate the effects of atrazine 

exposure on the spontaneous luteinizing hormone (LH) surge in intact female Sprague 

Dawley rats following a distributed dose (diet) versus bolus (gavage) exposure over a 4-

day estrous cycle. In addition, the potential immunotoxic effects of atrazine were 

evaluated following distributed dose (diet) versus bolus (gavage) exposure after a 

minimum of 28 days of exposure. 

NOTE* The previously submitted study by Coder, P. S. (2010) An Oral (Gavage) 

Immunotoxicity and Hormone Evaluation Study for the Effects of Atrazine on Male Rats 

concluded that 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. This current 

study assessed other components of the immune system for their function and has 

concluded that no functional deficits in humoral and innate immunity occurred. These 

studies in combination have shown equivocal evidence of toxicity to the immune system 

and further the toxicology database for atrazine is complete.
Page 44 of 210

STUDY DESIGN:

For immunotoxicity assessments in Phase II, 2 positive control groups were also 

assigned to study. Animals in positive control group 1 (Group 8; Antibody-Forming Cell 

[AFC] assay) were administered cyclophosphamide (50 mg/kg/day) once daily by 

intraperitoneal injection for 4 consecutive days prior to the day of scheduled euthanasia. 

Animals in positive control group 2 (Group 9; Natural Kill Cell [NKC] activity assay) were 

administered approximately 1.0 mL of diluted Anti Asialo GM1 by intravenous injection 

at approximately 24 hours prior to the scheduled euthanasia. The positive control 

groups were composed of 10 females each.

 Atrazine, was administered to female Crl:CD(SD) rats for comparison 

between distributed dose (dietary) versus bolus (oral gavage) exposure to atrazine. This 

study was divided into 2 phases (Phase I and Phase II), and each phase consisted of 2 

cohorts (Cohort A and Cohort B). Animals were administered the test substance over 

one 4-day (or 5-day) estrous cycle in Phase I for luteinizing hormone (LH) surge 

assessments, and selected animals from Phase I continued to be administered the test 

substance for a minimum of 28 days (up to 36 days of exposure) in Phase II for 

immunotoxicity assessments. Within each phase, animals in Cohort A were exposed to 

the test substance in the diet and animals in Cohort B were administered the test 

substance via oral gavage. In Phase I, female rats received the test substance on a 

continuous basis in the diet (Cohort A) or orally by gavage once daily (Cohort B) over 

one 4-day (or 5-day) estrous cycle. In Phase II, selected females from designated 

groups in Phase I continued to receive the test substance on a continuous basis in the 

diet (Cohort A) or orally by gavage (Cohort B) following completion of Phase I. 

RESULTS: The results revealed no treatment related deaths. No test substancerelated clinical findings were noted at any dose level during Phase I in Cohorts A or B. 

In Cohort A (dietary exposure), the average quantities of test substance consumed 

during Phase I (study days -1 to 3) in the 0, 30, 100, and 500 ppm groups were 0, 3, 9, 

and 40 mg/kg/day, respectively. Lower mean body weight gains were noted in the 500 

ppm group during the first 3 days of exposure, resulting in a lower mean body weight 

change for the overall Phase I treatment period (study days -1 to 4). In addition, mean 

body weights in the 500 ppm group were 4.1% to 5.1% lower than the control group 

during study days 1-4. Mean daily food consumption in the 500 ppm group was lower 

than the control group throughout the Phase I treatment period, primarily as the result of 

decreased food consumption during the dark photoperiods. Mean body weights, body 

weight gains, and food consumption in the 30 and 100 ppm groups were unaffected by 

test substance exposure during Phase I.

In Cohort B (oral gavage administration), lower mean body weight gains were noted in 

the 50 mg/kg/day group during study days 0-2, resulting in a lower mean body weight 

change for the overall Phase I treatment period (study days 0 to 4). These lower mean 

body weight gains did not have a remarkable effect on mean body weights at 50 

mg/kg/day. Mean food consumption in the 50 mg/kg/day group was slightly lower than 

the control group during study days 0-3 and for the overall Phase I treatment period 

(study days 0-4). Mean body weights, body weight gains, and food consumption in the 

0.75, 1.5, 3, 6, and 10 mg/kg/day groups were unaffected by atrazine administration 

during Phase I. Bolus (gavage) administration of atrazine for 1 complete estrous cycle 
Page 45 of 210

resulted in prolongation of the estrous cycle at 50 mg/kg/day. Five of the 21 females in 

the 50 mg/kg/day group exhibited a 5-day estrous cycle during the Phase I treatment 

period. This prolongation of the estrous cycle from 4 to 5 days in the 50 mg/kg/day 

group was considered to be a result of atrazine administration. In addition, distributed 

dose (diet) exposure to atrazine at 30, 100, and 500 ppm had no effects on the length of 

the estrous cycle.

Atrazine, when administered via oral gavage at a dose of 50 mg/kg/day for 4 

consecutive days was sufficient to inhibit the spontaneous LH surge in intact female 

rats, as indicated by decreased values for peak LH and area under the curve (AUC). 

An equivalent distributed dose of atrazine delivered in the diet (500 ppm) had no effect 

on the spontaneous LH surge, suggesting that inhibition of the LH surge is a 

consequence of achieving peak levels of atrazine above a currently undefined 

threshold.

During Phase II, in Cohort A, the average quantities of test substance consumed in the 

0, 30, and 500 ppm groups were 0, 3, and 51 mg/kg/day, respectively, for the AFC 

animals (study days 5-28) and the NKC animals (study days 5-30). No test substancerelated clinical findings were noted at any dose level during Phase II in Cohort A. Lower 

mean body weight gains/losses were noted intermittently throughout the treatment 

period which resulted in a statistically significantly lower mean body weight gain - noted 

in the 500 ppm group during the overall Phase II treatment period (study days 5-28 for 

AFC animals and study days 5-30 for NKC animals) evaluation. The lower mean body 

weights noted at 500 ppm during Phase I generally continued during Phase II (up to 

7.4% and 10.2% lower for the AFC and NKC assay animals, respectively). The 

reductions in mean food consumption noted in the 500 ppm group during Phase I were 

not sustained during Phase II. Mean body weights, body weight gains, and food 

consumption in the 30 ppm group were unaffected by test substance exposure during 

Phase II.

There were no test substance-related, toxicologically significant effects on hematology 

parameters at any dose level or exposure route (dietary or oral gavage) in the NKC 

animals. Treatment with atrazine via dietary or oral gavage exposure for a minimum of 

28 days did not produce biologically relevant effects on the humoral or innate immune 

components of the immune response, when evaluated using the AFC assay and the 

NKC activity assay. There were no effects in absolute or relative spleen or thymus 

weights or spleen cell numbers (non-Atrazine treated groups) from non-immunized 

animals (NKC animals) treated with atrazine as compared to the respective (dietary or 

oral gavage) vehicle control group. 

Levels of urinary corticosterone were elevated in all groups during the second 6-hour 

collection interval (9-15 hours) compared to the first 6-hour collection interval (0-6 

hours), but the elevation during the 9-15 hour interval was statistically significantly less 

in the 50 mg/kg/day group compared to the control group. There was no effect on the 

diurnal rise in urinary corticosterone in the 3 or 6 mg/kg/day groups. Atrazine did not 

alter circulating levels of prolactin, progesterone, corticosterone,
Page 46 of 210

androstenedione, aldosterone, estrone or estradiol when administered via the diet 

(Cohort A) or by oral gavage (Cohort B) for 28 days.

CONCULSIONS: Lower body weight gains (or body weight losses) and/or reduced food 

consumption were observed following distributed dose (diet) exposure to atrazine at 500 

ppm and bolus (gavage) exposure to atrazine at 50 mg/kg/day. In addition, 

administration of atrazine via bolus (gavage) exposure resulted in prolongation of the 

estrous cycle at 50 mg/kg/day, but there was no effect on estrous cycle length at a 

distributed dose (diet) of 500 ppm. Treatment with atrazine via dietary or via oral 

gavage exposure for a minimum of 28 days did not produce biologically relevant effects 

on the humoral or innate immune components of the immune response, when evaluated 

using the AFC assay and the NKC activity assay. Based on the results of this study, the 

no-observed-effect level (NOEL) for suppression of the immune system in female 

Crl:CD(SD) rats administered atrazine for 28 days via the diet or orally by gavage was 

500 ppm and 50 mg/kg/day, respectively.

Bolus (gavage) exposure to atrazine for 4 consecutive days at a dose of 50 mg/kg/day 

was sufficient to impair the spontaneous LH surge in intact female rats. An equivalent 

distributed dose (dietary) was ineffective in blocking the surge of LH. When these 

animals were maintained on the same treatment paradigm for 28 days, atrazine did not 

alter circulating levels of prolactin, progesterone, corticosterone, androstenedione, 

aldosterone, estrone or estradiol, but there was a decrease in the diurinal rise (9-15 

hours) of urinary corticosterone at a dose of 50 mg/kg/day. The NOELs in this study are 

based on the design using distributed (diet) or bolus (gavage) doses of atrazine up to 

500 ppm (diet) or 50 mg/kg/day for 4 or 28 consecutive days.

Endpoint (ppm) (mg/kg/day)

Body weight/food consumption 100 10

Immunotoxicity 

(humoral,innate)

500 50

Estrous cycle 500 10

LH surge attenuation 500 10

Plasma progesterone, 

prolactin, corticosterone, 

androstenedione, Aldosterone, 

estrone, estradiol

NA 50

Urinary corticosterone NA 6

EPA Reviewer Comments: Results seem vastly similar to what was presented at 

previous SAPs and therefore conclusions to that of the registrant are also similar. 

Dietary dosing having no effect is not new knowledge. A statement regarding “rebleeding" the animals if they were pro the first time they tried raises concerns. Since 

this issue could be problematic, further review is warranted to see how many rats were 

treated this way. The immunotoxicity component to this study provided little to know 

new knowledge on the lack of an effect on the humoral or innate components of the rats 

exposed.
Page 47 of 210

A.2.18 Coder, P. S. (2011) An Study of the Effects of Short-Term Atrazine 

Exposure on the Estrogen-Induced Luteinizing Hormone (LH) Surge 

in Ovariectomized Young-Adult Sprague Dawley Female Rats, WIL 

Research Laboratories, LLC, 1407 George Road, Ashland, OH. 

Unpublished. Study Number: WIL-639091

In an oral gavage study the purpose was to assess the effects of short-term atrazine 

exposure on the estrogen-induced luteinizing hormone (LH) surge in ovariectomized 

young adult female Sprague-Dawley rats following oral administration over 4, 8, or 14 

days.

STUDY DESIGN: Atrazine was administered in 1% methylcellulose in deionized water 

by gavage to ovariectomized female Crl:CD(SD) rats. This study was divided into 3 

cohorts (A, B, and C), and each cohort was composed of 4 groups (dose levels) of 25 

ovariectomized females per dose. Dose levels were 0 (vehicle only), 6.5, 50, and 100 

mg/kg/day administered at a volume of 5 mL/kg. The test substance or vehicle was 

administered for 4, 8, or 14 days to Cohorts A, B, and C, respectively, prior to collection 

of blood samples for LH evaluation; all animals received an additional dose the following 

day for possible assessment of atrazine plasma levels. The females were approximately 

12 weeks of age at the beginning of treatment. Ovariectomies were performed on all 

females 3 days prior to the scheduled day of blood collection for LH assessment (study 

days 0, 4, and 10 for Cohorts A, B, and C, respectively). Blood samples for plasma 

atrazine analysis were collected from up to 12 females/group/time point at 30 minutes or 

approximately 4 hours after dose administration on the day following LH blood collection 

(study days 4, 8, and 14 for Cohorts A, B, and C, respectively).

RESULTS: The results revealed no treatment related deaths. Clinical findings related 

to treatment consisted of red material around the nose and yellow material on the 

urogenital area in the 6.5, 50, and/or 100 mg/kg/day groups (Cohorts A and C), and 

salivation in the 100 mg/kg/day group of Cohort C. Mean body weight losses and lower 

mean body weights and food consumption were noted in the 50 and 100 mg/kg/day 

groups during the treatment period for all cohorts (study days 0-4, 0-8, and 0-14 for 

Cohorts A, B, and C, respectively). Females generally exhibited 4-5 day estrous cycles 

during pretest (all cohorts). Vaginal lavages were discontinued beginning on the day of 

ovariectomy. In Cohort C, 4 and 5 females in the 50 and 100 mg/kg/day groups, 

respectively, exhibited 6-7 day estrous cycles, compared with no females in the control 

group. Estrous cycle length could not be evaluated in Cohort A (ovariectomy occurred 

on the first day of treatment) or in Cohort B (due to the limited number of days [4] prior 

to ovariectomy). There were no treatment-related macroscopic findings at the 

scheduled euthanasia, and mean organ weights in all cohorts were generally similar 

across all groups, including the control group. Statistically significantly lower LH levels 

(peak concentration and AUC values) were noted in the 50 and 100 mg/kg/day groups 

with respect to the control group following 4 days of atrazine administration (Cohort A). 

Although LH levels in the 6.5 mg/kg/day group females in Cohort A were significantly 

lower at 13 hours post lights-on, there was no effect on peak concentration or AUC and 

hence the effect noted at this single time point was not considered adverse. Following 8 
Page 48 of 210

and 14 days of atrazine administration, the 100 mg/kg/day group females (Cohorts B 

and C respectively) had significantly lower LH levels (peak concentration and AUC 

values) as compared to the control group. Although mean LH levels in the 50 mg/kg/day 

group females in Cohort C were statistically significantly lower at 11 and 13 hours post 

lights-on, there was no effect on peak LH level or AUC and hence the effect noted at 50 

mg/kg/day following 8 and 14 days of exposure was not considered adverse - Although 

the study indicates that a 1-way ANOVA for peak LH measures revealed a 

significant effect of treatment (p<0.001), and a 1-way ANOVA of LH AUC also 

revealed a significant effect of treatment (p=0.00311).

CONCULSIONS: Treatment-related clinical findings were noted at all dose levels in 

Cohorts A and C but not in Cohort B. The incidence of these clinical observations in the 

100 mg/kg/day groups of Cohorts and C were higher than the other treatment groups. 

Treatment-related decreases for body weight and food consumption parameters were 

noted for the 50 and 100 mg/kg/day groups during the treatment period for all cohorts. 

A treatment-related effect in the number of animals with an increased cycle length (6-7 

days versus 4 days in pretest) was noted during the 10-day treatment period prior to 

ovariectomy in the 50 and 100 mg/kg/day groups (Cohort C). Significantly lower LH 

levels (peak concentration and AUC values) were noted in the 50 and 100 mg/kg/day 

groups following 4 days of treatment (Cohort A), but only in the 100 mg/kg/day groups 

following 8 or 14 days of treatment (Cohorts B and C, respectively). The registrant 

determined no-observed-effect levels (NOELs) in this study are based on the dosing 

design in which 6.5, 50, and 100 mg/kg/day atrazine was administered for 4, 8, or 14 

days to Cohorts A, B, and C, respectively (found in Table I).

Table I.

Endpoint Cohort A 

(mg/kg/day)

Cohort B 

(mg/kg/day)

Cohort C 

(mg/kg/day)

Clinical observations* 50 100** 50

Body weight/food 

consumption

6.5 6.5 6.5

Estrous cyclicity ND ND 6.5

LH surge* 6.5 50 50

Macroscopic exam 100 100 100

*No-observed-adverse-effect level (NOAEL).

**No test substance-related clinical findings noted in Cohort B.

ND - Not determined due to insufficient number of days of assessment prior to ovariectomy.

EPA Reviewer Comments: The dosing formulations were visually inspected by the 

Study Director and therefore, stability and homogeneity analyses were not conducted. 

In Cohort A, one rat per dosing group (control, 6.5, 50, and 100 mg/kg/day) died on the 

day of surgery (study day 0), this seems questionable as well as the fact that of those 

animals that died, one from each the control and 50 mg/kg/day group were found to 

have both ovaries present. Section 3.4.3 Euthanasia of Unselected Females indicates 

that there were 24 females/group versus the before mentioned 25/group. In Cohort B, 
Page 49 of 210

one female in the 50 and 100 mg/kg/day groups were found dead about 4 hours after 

surgery while none were reported to have died in Cohort C. This is inconclusive since it 

doesn’t indicate whether the older the females were they either could withstand the 

surgery more so, or did the handlers get better with their technique over time? In 

Cohorts A and C clinical signs were found to be treatment related and were shown as 

increased incidences of red material around the nose, (eyes, and mouth); yellow 

material on or around the urogenital area within 2 hours of dose administration. While 

in Cohort B these same clinical findings appeared but because they occurred in ‘single’ 

animals and in ‘similarity’ to the control group were NOT considered dose related. Also 

found was hair loss, scabbing on forelimbs – all effects considered not dose-related 

since they mimicked what was seen in Cohort B. The analysis of LH levels showed that 

Cohorts A, B, and C with all doses there was a decrease in the estradiol induced LH 

surge in the afternoon of the 4th, 8th, and 14th day of treatment, compared to the control 

group, while decreased peak LH and AUC were noted in the 50 and 100 mg/kg/day 

groups but not the 6.5 mg/kg/day group for Cohort A and only noted in group 100 

mg/kg/day for Cohorts B and C.

Also noted in Cohort C was a treatment related increase in the cycle length (6-7 days 

versus 4 days in the pretest) during the 10-day treatment prior to the ovariectomy in the 

two high doses tested. This effect was not further elaborated upon, but could have 

reasonable significance related to the lengthening of the estrous cycle in females 

exposed to atrazine repeatedly at those dose levels (50 and 100 mg/kg/day) for this 

particular duration (>7 days). Ultimately it brings up the question as to whether in acute 

situations is the LH suppression truly adverse?

The NOAEL for LH surge suppression is found to be 6.5 mg/kg/day (chosen as a no 

affect level) for this study during 4 days of exposure. The LOAEL of 50 mg/kg/day was 

true for clinical observations at this duration. 

A.2.19 Coder, P. (2011d) Atrazine: An Oral (Gavage and Dietary) Study of 

the Effects of Atrazine on Fertility and Reproductive Performance in 

Female Long Evans and Sprague-Dawley Rats: Final Report. Report 

Number: WIL/639090, Task No. TK0052068. Unpublished study. 

Performing Laboratory: WIL Research Laboratories, Inc. 966 p. 

Completion Date: April 6, 2011. MRID 48470001.

Summary:

This study compared the effects of atrazine exposure on ovulation, fertility, and 

reproductive performance in Sprague-Dawley (SD) and Long Evans (LE) intact female 

rats following diet versus gavage exposure over a 4-day estrous cycle period. 

Study Design: Twelve-week old female rats (25/dose level) with repeated 4-day estrous 

cycles during the pretest period were exposed to atrazine (97.5%) for 4 days (or 5 days) 

via oral gavage up to 100 mg/kg/day (Cohort A: SD rats, doses 0, 12, 50, 100 

mg/kg/day and Cohort B: LE rats, doses 0, 1.5, 3, 6, 12, 50, 100 mg/kg/day) or via the 
Page 50 of 210

diet up to 1460 ppm (Cohort C: LE rats, doses 160, 660, 1460 ppm (0, 11, 35, 51 

mg/kg/day). For Cohorts A and B, the vehicle and atrazine were administered orally via 

gavage once daily, beginning lights-ON (0500 hours) on the first day of the estrous 

cycle (day of estrous; study day 0) and continuing over 1 complete 4- or 5-day estrous 

cycle. Females in Cohort C were given the control and test diets ad libitum beginning at 

lights-OFF (1900 hours) on the 4th day of the previous estrous cycle (study day -1) and 

continuing over the next complete 4- or 5-day estrous cycle. 

Vaginal lavages were performed daily during the pre-treatment and treatment periods to 

determine the estrous cycle. Only females that exhibited 4-day estrous cycles 

repeatedly throughout the pretest period were assigned to the study. On the last day of 

treatment, females were paired with untreated males. Animals were paired for breeding 

prior to 1200 hours as the females were expected to be in proestrus that afternoon and 

in estrus the following morning. If evidence of mating was noted, the day was 

designated as day 0 of gestation. If evidence of mating was absent, the stage of estrous 

was recorded. If the female was in estrus, the female was separated from the male 

without further opportunity for mating. If the female was in diestrus the morning following 

the initial pairing, the female was re-paired with an untreated male rat that successfully 

mated. In the absence of mating on the second day, the stage of estrous was noted and 

the female was separated from the male without further opportunity for mating. 

Clinical observations were recorded. Body weight and food consumption were recorded 

daily during treatment, on GDs 0, 3, 6, 9, 12, 15, 18 and 20 for females with evidence of 

mating. A laparohysterectomy was performed on GD 20 for each female with evidence 

of mating. 

Functional endpoints (ovulation, fertility, and reproductive performance) were evaluated.

Results: 

There were no survival issues noted in the study for Cohorts A, B or C. No treatmentrelated clinical observations were noted for Cohorts A or B. Decreased defecation and 

small feces were noted in the 1460 ppm dose group (Cohort C) during the exposure 

period (study days -1 to 3). These findings were considered treatment-related and 

correlated with a low mean food consumption observed during the treatment period.

During the treatment period, treatment related body weight losses and/or reduced body 

weight gains with corresponding reductions in mean food consumption were noted at 

the two highest doses of atrazine administered (50 and 100 mg/kg/day in SD (Cohort A) 

and LE rats (Cohort B) and 660 and 1460 ppm in LE rats (Cohort C)). These effects did 

not affect mean maternal weights during gestation in any of the cohorts, which were 

comparable to controls by GD 20. 

For Cohorts A, B and C, atrazine treatment did not affect the rat estrous cycle in either 

LE or SD rats.
Page 51 of 210

Female reproductive performance (mating, fertility, or conception indices), the number 

of corporal lutea or implantation sites, intrauterine growth and survival or external fetal 

morphology in intact female SD or LE rats were unaffected by gavage doses up to 100 

mg/kg/day in LE or SD rats or by dietary doses up to 1460 ppm (51 mg/kg/ay) in LE 

rats. 

Reviewer’s comments: The underlying assumption in this experiment was that the 

animals would be dosed with an LH suppressing dose of atrazine. The animals were 

then bred to see if there were any affects on ovulation, fertility, and reproductive 

performance. This was done using two strains of rats via gavage and dietary exposures. 

The study authors concluded that “"Female reproductive performance (mating, fertility, 

or conception indices) was unaffected by bolus doses up to 100 mg/kg/day in LE or SD 

rats or by distributed doses up to 1460 ppm (51 mg/kg/day) in LE rats. Furthermore, 

mean numbers of corpora lutea, mean litter proportion of pre-implantation loss, and the 

mean number of implantation sites were unaffected by treatment. Intrauterine growth 

and survival and external fetal morphology were unaffected by atrazine administration. 

These results indicate that the magnitude of the atrazine-induced preovulatory surge 

suppression shown in previous studies was not sufficient to influence ovulation or 

reproductive performance of SD or LE female rats as evaluated in this study as the 

doses tested". There are some elements that confound the conclusions reached by the 

researchers. While it is not unreasonable to think that the atrazine induced LH 

suppression occurred at some of these doses, the LH plasma levels were not 

measured. A primary concern of this study was that the design did not really provide 

an opportunity to test the potential adverse effects of atrazine on the endpoints 

examined. The assumption was that if atrazine decreased LH then this decrease would 

lead to some identifiable adverse outcome, at least in the high-dose, gavaged animals. 

However, the design of this study did not allow for a true test of the assumption. For 

example, it is well-known that if the spontaneous LH surge is compromised by 

pharmacological treatment (e.g., phenobarbitol, the usual positive control in such 

studies) and the female is mated later on the day of vaginal proestrus (as was done in 

the current study), mating itself will not be altered (because you have not interfered with 

the steroid hormones regulating sexual behavior) and that the act of mating (i.e., coital 

stimulation of the vaginal cervix) induces an LH surge (they have become reflex 

ovulators) and normal pregnancies will ensue (Tyler and Gorski 1980 Tyler, J and 

Gorski R. 1980. Temporal Limits for Copulation-Induced Ovulation in the PentobarbitalBlocked Proestrous Rat. Endocrinology. Vol. 106, No. 6: 1815-1819; Cooper et al. 1994 

Cooper, R. Barrett, M, et al. 1994. Pregnancy Alterations Following Xenobiotic-Induced 

Delays in Ovulation in the Female Rat. Fundamentals and Applied Toxicology. 22, 474-

480.). Thus, it should be noted that even if the atrazine induced LH suppression had 

occurred, the mating itself may have produced a surge and this alone may be 

responsible for the lack of any adverse effects on pregnancy in this study.

A.3 REPRODUCTIVE EFFECTS IN MALES 

Summary: Reproductive/Developmental Toxicity, Delayed Puberty and Prostatitis
Page 52 of 210

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). 

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. 
Page 53 of 210

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- 2: Strain and Dose Comparison in Male Rats dosed peripubertally with 

atrazine

EXPOSURE STRAIN EFFECT NOAEL/LOAEL REFERENCE

PND 23 to 53 Wistar Delayed PPS 6.25/12.5 Stoker et al., 2001

PND 22 to 47 SD Delayed PPS 50 lowest 

dose/100 

Trentacoste et al., 

2001

PND 23 to 53 Wistar Decreased VP and 

SV

25/50 Stoker et al., 2001

PND 22 to 47 SD Decreased VP and 

SV

50 lowest 

dose/100

Trentacoste et al., 

2001

PND 23 to 53 SD Decreased intratesticular T on 

PND 45

150/200 Stoker et al., 2001

PND 22 to 47 SD Decreased T on 

PND 47

ND/50 Trentacoste et al., 

2001

PND 22 to 47 SD Decreased test. 

and serum T on 

PND 47

ND/50 Friedmann, 2002

PND 23 to 50 Wistar Decreased T/DHT 

on PND 50

50/200 Pogrimic et al., 

2009

The study by Stoker et al., 2002 also examined the 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 
Page 54 of 210

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 

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
Page 55 of 210

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.

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.
Page 56 of 210

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 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 (γ-GT), 

glutathione-S-transferase (GST) activity, glutathione (GSH), lipid peroxidation (LPO), 

which was quantified as malondialdehyde MDA), ascorbate (AA) and protein content. 
Page 57 of 210

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..

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. γ-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.
Page 58 of 210

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 semen. An in vitro viability staining method was used to assess viability while a 

computer-assisted-analyzer (CASA) was used to study sperm mobility. 9

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 106 sperm/ml, were placed in 4 well plate samples at a final 

concentration of 0, 50, 100, or 500 μM in a volume of 500 μl tris-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 

 9 In this evaluation only the results for atrazine will be discussed
Page 59 of 210

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

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.
Page 60 of 210

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 

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 
Page 61 of 210

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 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. 
Page 62 of 210

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 

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 

dietary-restricted and 1000 ppm atrazine groups were similar, and significantly lower 

than control values throughout the treatment period. Food consumption was lower in 

the atrazine treatment groups compared with control group. In the dietary-restricted 

group, mean relative ventral prostate weight was significantly decreased (25%) relative 

to controls. No other treatment-related changes in relative organ weights in any 

treatment group were reported. Serum testosterone levels 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 welldifferentiated 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 
Page 63 of 210

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. 

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 ACTHdependent Release of Corticosterone in Male Wistar Rats. Tox. Sci. 

112(1):78-87.

This study is a follow-up to a previous set of experiments study in which these 

researchers demonstrated that atrazine 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).10

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 

 10 This review will only discuss data for atrazine, DIA, DEA, and DACT
Page 64 of 210

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 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 
Page 65 of 210

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.

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 
Page 66 of 210

Fenoxaprop-Ethyl on Capacitation and the Acrosomal Reaction in 

Boar Sperm. International Journal of Toxicology. Volume 2, Number 

1. pp. 24-32.

The purpose of this study was to evaluate the in vitro effects of atrazine and 

fenoxaprop-ethyl on capacitation, spontaneous acrosome reaction (SAR) and 

progesterone-induced acrosome reaction (PIAR) in boar sperm.11

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).

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 

 11 In this evaluation, only the results for atrazine will be discussed.
Page 67 of 210

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. 12

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. 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 

 12 In this evaluation, only the results for atrazine will be discussed.
Page 68 of 210

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 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. 
Page 69 of 210

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 coincubating 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 dosedependent decrease in gene expression of LHR, SR-B1, StAR, TSPO, SF-1, PDE4B, 

3bHSD, CYP17A1, 17bHSD in Leydig cells. There was no effect on CYP11A1 and ßHSD. They conclude that the inhibition of all these genes following atrazine may be 

related to decreased c-AMP 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 RTPCR studies demands confirmation. Finally, as to whether or not these data support 

the HPG MOA or indicate a direct action of Atrazine on the Leydig cell can not be 

determined. In addition, it should be noted that the doses used in this study were 

relatively high compared to the doses eliciting the neuroendocrine effects and used for 

regulatory purposes.

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 Long-
Page 70 of 210

Evans 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 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.
Page 71 of 210

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.

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. 
Page 72 of 210

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 nonstatistically significant increase in AGD was observed in males on PND 21. while AGD 

was significantly reduced at 100 mg/kg/dose levels on PND 21. 

A statistically significant delay in PPS 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.]

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.
Page 73 of 210

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.

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.
Page 74 of 210

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.

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 
Page 75 of 210

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).

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. 
Page 76 of 210

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 Sprague-Dawley males). However, Stoker also found a decrease in ventral 

prostate weights at 50 mg/kg/day males (which had BW reduction of 3%) and delays in 

preputial separation following 12.5 mg/kg/day and above. Stoker also noted lower 

serum testosterone values in 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 3β-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. 
Page 77 of 210

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

weighed. Testes were examined histopathologically. Changes in the expression of 3βhydroxysteroid dehydrogenase (3β-HSD) enzyme and androgen receptor (AR) in the 

testes were studied by immunohistochemistry. 3β-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 3β-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 the specificity of the antibodies and the 

changes in the expression of 3β-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. 
Page 78 of 210

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.

In control animals, 3β-HSD positivity in the testis was found in the Leydig cells 

cytoplasm. After atrazine exposure, there was a great reduction in Leydig cells 3β-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 3β-HSD positive bands in the testis. The 

expression of 3β-HSD immunoreaction was found in cells of the three zones of the 

adrenal cortex. The pattern of distribution and intensity of the 3β-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 3β-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. 3B-HSD is a key enzyme in the androgen biosynthetic pathway, 

but changes in the expression or protein levels could be a result of the suppression of 

luteinizing hormone. The fact that they found a decrease in B-HSD in the testes is 

different than the Pogrmic paper wherein this was one of only two (of the many 

examined) that did NOT 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 
Page 79 of 210

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.3.17 Fraites MJP, Narotsky MG, Best DS, Stoker TE, Davis LK, Goldman 

JM, Hotchkiss MG, Klinefelter GR, Kamel A, Qian Y, Podhorniak L, 

Cooper RL. (2011). Gestational Atrazine Exposure Effects on Male 

Reproductive Development and Metabolite Distribution in the Dam, 

Fetus, and Neonate. Reprod. Toxicol., 

doi:10.1016/j.reprotox.2011.04.003

In this study the authors investigated the potential impact of in utero exposure on male 

reproductive development. Various androgen-dependent endpoints were evaluated 

including: (i) testosterone production at birth and on PND 59, (ii) rough and tumble play 

behavior, (iii) AGD and PPS, and (iv) androgen-dependent organ weights. In addition, 

the investigators studied the transfer of atrazine and its metabolites into the fetus after 

maternal exposure during gestation (GD 18-20 or GD 14-20).13

Time-pregnant Sprague-Dawley rats (12 dams/dose) were exposed to 0, 1, 5, 20, and 

100 mg/kg/day atrazine (97% a.i. purity) by gavage once daily (s.i.d) or divided into 2 

daily doses (b.i.d) during GD 14-21. At birth, 2 male pups/litter were selected for testis 

histology or unstimulated or LH-stimulated testosterone production by the testis. AGD 

measures were taken on PND 7 and beginning on PND 38 pups were examined daily 

for PPS – an indicator of puberty onset. On PND 30-33, male pairs were assessed for 

rough and tumble play behavior (pinning, chase, on the back posture, wrestling, or 

boxing). At the time of necropsy, 2 males/litter were decapitated within 15 seconds of 

removing them from the cage and their trunk blood was collected. Serum testosterone 

and LH levels were measured. In addition, the pituitary, testes, left epididymis, seminal 

vesicles with coagulating gland (with fluid), and ventral prostate were removed and 

weighed. 

Exposure to atrazine did not affect PPS, AGD, rough and tumble play behavior, serum 

or interstitial testosterone, serum LH, unstimulated and LH-stimulated testosterone 

production by the testes (either at birth or on PND 59), or androgen-dependent organ 

weights. The only atrazine-related effects observed in the study occurred at the 100 

mg/kg/day dose level and included decrease in maternal body weights, pup body 

weights (↓6-12%), and pup viability between PND 1-4 after s.i.d administration (9.7% 

pup loss vs 1.4% in control) or b.i.d. administration (33.6% pup loss vs 0.9% control). A 

statistically significant decrease in absolute pituitary weight ((↓12%) was also observed 

at the 100 mg/kg/day dose level on PND 59 after s.i.d administration (but not b.i.d) but 

this was likely related to the decreases in pup weight since relative pituitary weight was 

unaffected.

Reviewers comments: This is a well conducted study that indicates that at the doses 

tested (1-100 mg/kg/day) late-gestational exposure to atrazine does not affect male 

 13 The results of the atrazine distribution experiments were described in review A.2.13 in Appendix A of 

this document and will not be discussed further in this review.
Page 80 of 210

reproductive development or the androgen-dependent endpoints measured in this 

study.

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 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 

atrazine-dependent increases in estrogen production in H295R cells (Heneweer et al., 

2004; Higley et al., 2010) and human placental carcinoma (JEG-3) cells (Sanderson et 

al., 2001; Laville et al., 2006). Finally, Holloway et al. (2008) also reported that 

aromatase activity in 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 atrazinedependent 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 
Page 81 of 210

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 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 signalregulated kinase (ERK) phosphorylation and c-fos in the BG-1 cells following exposure 

to atrazine (1M). Using specific signalinginhibitors 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 
Page 82 of 210

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 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 
Page 83 of 210

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 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 
Page 84 of 210

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 

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). Gprotein-coupled receptor 30 and estrogen receptor-α are involved in 

the proliferative effects induced by atrazine in ovarian cancer cells. 

Environ Health Perspect. 116(12):1648-55.

This study used in vitro methods to further evaluate the effects of atrazine in multiple 

cell lines. The hypothesis for the study is based upon previously reported endocrine 

disrupting effects of 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 signalrelated kinase (ERK) phosphorylation, and cell proliferation. 

Studies were conducted using human BG-1, MCF-7 (breast cancer), Ishikawa (human 

endometrial), and SkBr3 (breast cancer) cell lines. BG-1, MCF-7, Ishikawa cells were 

(transiently) transfected with ER luciferase reporter plasmids, and SkBr3 was cotransfected with ERα 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 ERα 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 downregulation 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 

ERα.

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 
Page 85 of 210

exposure to atrazine. In addition, the current study does not provide a positive control 

for either aromatase activity or gene expression. 

To test the hypothesis that atrazine may generate rapid signaling via second messenger 

systems such as Ca2+, cAMP, nitric oxide, and G-proteins, which in turn can lead to 

activation of downstream kinase, an experiment was conducted to evaluate the ability of 

atrazine to produce effects on 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 ERα. Additional experiments demonstrated that the atrazine dependent upregulation of c-fos was abolished after 2-hr incubation with ER antagonist ICI, the EGFR 

inhibitor AG, or the ERK inhibitor, PD. Thus, the authors concluded “in ovarian cancer 

cells, atrazine involves ERα 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 ERα 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. 
Page 86 of 210

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 3H-androstenedione, a substrate for aromatase. 3Hwater, 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.
Page 87 of 210

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 (10-7 to 10-5 M) without 

exogenous SF-1 co-expression. To confirm that SF-1 is essential for atrazine 

stimulation, endogenous SF-1 in H295R cells were knocked down by siRNA. The 

results indicated a complete loss of responsiveness of ArPII to either atrazine (10-5 M) 

or 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 homolog1 (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.

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.
Page 88 of 210

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.

This in vitro study focused on the role of steroidogenic factor 1 (SF-1) in atrazineinduced 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 nonresponsive 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 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. 
Page 89 of 210

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-OH-androstenedione (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 dosedependent 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-Br-cAMP. This cell line, believed to be under the primary 

regulation of promoter pII, is also simulated by cAMP. The authors report that basal 

aromatase activity in the R2C cell line is very high as compared to that of the H295R 

cells, and that it is possible that one or 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 non-competitive inhibition, allosteric modification ) alteration of the 

enzyme. However, failure to evaluate other promoters within the H295R cells is a 

limitation of this study, and stops short of providing firm (significant) correlation between 

increased transcripts with I.3 and pII with elevated aromatase activity. In addition, the 

study demonstrates that the R2C Leydig cell line is not suitable for screening chemicals 

for identification of induction of aromatase gene expression. 
Page 90 of 210

Aromatase activity (AA)

 LOEL 10 uM, NOEL 0.3 uM)

: 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.

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, 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. 
Page 91 of 210

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.
Page 92 of 210

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) 

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. 
Page 93 of 210

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 coincubation 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 nM0.1 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. 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 
Page 94 of 210

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. 

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

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.

: Use of positive controls to demonstrate specificity of aromatase 

assay and CYP19 mRNA measurements. This study supports other

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.14

 14 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.

 Groups of 20 timed-pregnant Long-Evans rats 
Page 95 of 210

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 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.
Page 96 of 210

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 were examined. The data revealed a dosedependent 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, StARLuc, 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.
Page 97 of 210

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 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 
Page 98 of 210

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 

dose- and time-dependent with peak expression occurring after 4 to 24 hours of 

exposure. Atrazine metabolites, DACT and hydroxyatrazine, failed to show a 

consistent response. 

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. 
Page 99 of 210

The results from the optimized assay for PDE inhibition showed a concentrationdependent 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 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.4.13 Pogrmic-Majkic K, Fa S., Dakic V, Kaisarevic S., Kovacevic R. 2010. 

Upregulation of Peripubertal Leydig Cell Steroidogenesis Following 
Page 100 of 210

24 h In Vitro and In Vico Exposure to Atrazine. Toxicol. Sci. 118(1): 

52-60

In this study the authors investigated the impact of direct exposure of Leydig cells to 

atrazine on steroidogenesis by studying changes in the levels of cAMP, androgen, and 

progesterone synthesis by Leydig cells. They pursued this line of investigation using 

both in vitro and ex-vivo approaches. For the in vitro studies, Leydig cells obtained 

from peripubertal (PND 51) male Wistar rats were exposed to atrazine (98% purity) at 

concentrations of 0, 0.001, 1, 10, 20, and 50 μM for 24 hours. At the end of the 

incubation period the culture medium was collected and cells were stimulated with 

different concentrations of human chorion gonadotropin hormone (hCG), progesterone 

or ∆4

-androstenedione for 2 hrs. For the ex-vivo studies male Wistar rats were 

exposed to 200 mg/kg/day atrazine (via gavage) for 6 days (PND45-50), 3 days 

(PND48-50) and 1 day (PND50). Twenty four hours after the last exposure the animals 

were sacrificed and Leydig cells collected to measure levels of cAMP and testosterone 

after the cells were incubated for 2 hrs with hCG.

In vitro studies:

In addition to the assessments on the role of cAMP and PKA in androgen biosynthesis, 

an evaluation of the mRNA transcripts for genes involved in androgenesis including 

Luteinizing Hormone Receptor (LHR), Steroidogenic acute regulatory protein (StAR, 

involved in cholesterol transport), Steroidogenic factor -1 (SF-1), CYP17A1, 17βhydroxysteroid dehydrogenase (17β-HSD), scavenger receptor B-1 (SR-B1), and 

translocator protein (TSPO) was conducted to ascertain what genes – if any – were 

activated after exposure to 1 or 20 μM atrazine. While SF-1 was statistically 

significantly (p<0.05) increased after exposure to either of the atrazine concentrations 

(↑141 and 191%, respectively), StAR, CYP17A1 and 17βHSD were only increased at 

 A 24 hr exposure to either 1 or 20 μM atrazine resulted in an increase 

in basal cAMP levels of Leydig cells. Interestingly, in hCG-stimulated Leydig cells the 

increase in cAMP was only observed after exposure to 20 μM atrazine. Since cAMP 

has been implicated in the regulation of Leydig cell steroidogenesis, the impact of 

atrazine exposure on basal progesterone and androgen production was also evaluated. 

Increases in androgen production (↑31-98%) measured as testosterone + 

dihydrotestosterone levels were statistically significant and dose-dependent at 

concentrations ≥ 1μM. In contrast, progesterone levels exhibited an inverted doseresponse pattern with increases in progesterone levels after exposure to 0.001 or 1 μM 

atrazine (↑24 and 41%, respectively) and decreases at concentrations of 20 or 50 μM 

atrazine (↓13 and 22%, respectively). Together these findings lend credence to the 

hypothesis that androgen synthesis is dependent on cAMP-protein kinase A (PKA). To 

further test this hypothesis, hCG-stimulated or unstimulated Leydig cells exposed to 0 or 

20 μM atrazine for 24 hrs were subsequently treated with PKA-inhibitor H89 (10 μM). 

While the atrazine induced increase in cAMP was not affected, androgen production 

was markedly reduced (≈60%) after H89 treatment. Thus, under the conditions of this 

study, the pathway leading to androgenesis is blocked at the PKA step suggesting this 

kinase plays a critical role in in vitro androgen synthesis. 
Page 101 of 210

the 20 μM concentration (↑162, 247, and 153%, respectively). In contrast, neither LHR, 

SR-B1, or TSPO were affected by atrazine exposure (data not shown). Given that 17βHSD and CYP17A1 transcript levels were increased after exposure to 20 μM atrazine, 

the investigators evaluated whether this translates into an increase in the protein levels 

of these enzymes by measuring the conversion of progesterone to testosterone as well 

as the conversion of ∆4

-androstenedione to testosterone. After a 24 hours exposure to 

0, 0.001, 1, 20 or 50 μM atrazine, Leydig cells were treated with either progesterone or 

∆4

-androstenedione at concentrations of 0.25, 0.5, or 10 μM for 2 hrs. Exposure to 

atrazine at ≥ 20 μM atrazine resulted in an increased conversion of progesterone to 

testosterone (≈227%, p<0.05). For cells receiving 0.25 μM ∆4 -androstenedione, 

atrazine exposure at concentrations ≥ 1 μM resulted in a statistically significant increase 

in testosterone although the response is not as robust as for the progesterone to 

testosterone conversion. It is noteworthy that for cells receiving 0.5 or 10 μM of the 

steroid substrate (∆4 –androstenedione) an increase in testosterone levels was only 

noted at the 20 μM atrazine level.

In the ex vivo experiments, peripubertal male Wistar rats were exposed by gavage to 0 

or 200 mg/kg/day atrazine for 1(PND 50), 3 (PND48-50), or 6 days (PND 45-50). 

Twenty four hours after the last exposure Leydig cells were isolated and cAMP and 

testosterone levels measured. After a single exposure, cAMP and testosterone levels 

were increased (↑40%) regardless of whether the cells were hCG-stimulated or 

unstimulated. Peak increases in both cAMP and testosterone were reached after 3 

days of atrazine exposure in unstimulated cells (≈↑150-180%) and hCG-stimulated cells 

(≈↑100%). After 6 days, however, cAMP levels were comparable between the control 

and atrazine exposed groups. In the case of testosterone, the levels in the atrazine 

treated group continued to be elevated relative to control but decreased to the levels 

observed after a single exposure.

Ex vivo experiments

Reviewer comments: In general, this appears to be a well-conducted study. However, 

details on the study design and results are lacking. As an example, the number of rats 

treated during the ex vivo experiments is not provided and findings at the 50 μM 

atrazine concentration in the in vitro experiments are only reported for the progesterone 

or androstenedione-stimulated testosterone synthesis and the basal androgen and 

progesterone synthesis assessments. Given that a bidirectional dose-response was 

observed during the basal progesterone synthesis assessment, inclusion of the entire 

dose-response curve for these experiments is considered to be crucial. In conclusion, 

this set of experiments suggests that cAMP signaling and subsequent PKA activation 

are critical in androgenesis by catalyzing the upregulation of genes involved in 

steroidogenesis such as SF-1, StAR, CYP17A1, and 17βHSD. Regardless, the ex vivo 

data effects reported in this study occurred at doses that are approximately 2 orders of 

magnitude higher than the point of departure currently being used for regulatory 

purposes. Thus, the utility of these data for risk assessment purposes is limited. 

A.5 NEUROTOXICITY 
Page 102 of 210

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 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 dosedependent 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 
Page 103 of 210

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. 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 d-amphetamine sulfate injection of rats treated 

with atrazine. However, the authors’ assertion that the increased locomotor activity is 

associated with striatal dopamine deficiency from atrazine treatment should be further 

explored and 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 genderbased 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 
Page 104 of 210

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 

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.
Page 105 of 210

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. 

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 high-dose atrazine group (mean of 6.60 vs. control mean of 4.60). There was an 

acceleration of forepaw grasp (“day of full response”) in the low-dose atrazine group 

(mean of 5.18) and high-dose atrazine group (mean of 5.25) in comparison with the 

control group (mean of 7.50). 

On PND 9, mean body weight temperatures were significantly lower in the males and 

females of the low-dose atrazine group (5% decrease in males; 4% decrease in 
Page 106 of 210

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 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 high-dose atrazine groups, whether data 

are presented as sex specific or combined. Peak frequency was slightly (not 

statistically significant) increased in atrazine-treated males (low and high dose groups) 

and in 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. 
Page 107 of 210

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 half-brains were used to analyze neurons in 

the substantia nigra pars compacta and ventral tegmental area by 

immunohistochemistry/unbiased stereology. 

No effects of atrazine 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 atrazinetreated animals in striatal dopamine levels at day 22. Slight decreases in DOPAC and 

HVA were present at day 22. At day 64, there were no statistically significant effects on 

DOPAC and HVA. There were no effects of 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 THpositive 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). 
Page 108 of 210

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 Parkinsonianlike 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 SpragueDawley rats were incubated with up to 500 μM atrazine for 4 hours. The following were 

evaluated: tyrosine hydroxylase activity using L-DOPA accumulation as an indicator, 

high potassium-stimulated dopamine release, media dopamine levels in the presence of 
Page 109 of 210

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 dosedependent 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. 

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 
Page 110 of 210

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 dose-dependent increases in mean plasma LH levels. 

The third experiment entailed an examination of the effects of atrazine on in vivo 

pituitary response to GnRH agonist. The objective was to 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, intra-atrial cannulae were implanted, and rats were treated with anti-GnRH 

serum via cannula. On the last day of treatment, sampling of blood was performed and 

D-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. 

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 
Page 111 of 210

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. 

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).
Page 112 of 210

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 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).” 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. 

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. 
Page 113 of 210

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.

• 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 
Page 114 of 210

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.

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 
Page 115 of 210

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 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 d-amphetamine. The increased locomotor activity persisted at the 3- and 6-

month time periods, as well as 2 weeks after treatment, time periods carried out in the 

absence of d-amphetamine administration. At 2 weeks after treatment with atrazine, 

locomotor activity was 40% above control values. A statistically significant decrease 

(approximately 20%) in striatal dopamine levels at 10 mg/kg/day was noted. Dopamine 

levels were increased in the prefrontal cortex in an inverse U-shaped fashion; the 

increase at 5 mg/kg/day was 30 – 40%, while levels declined towards control levels at 
Page 116 of 210

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.”

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.
Page 117 of 210

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 (atRA). 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 antigen1 (SSEA1, a cell-surface glycoconjugate marker of the undifferentiated state) and βIIItubin (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. 

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.5.10 Belloni V, dessi-Fulgheri, F, et al. 2010. Early exposure to low doses 

of atrazine affects behavior in juvenile and adult CD1 mice. 

Toxicology (2010), doi:10.1016/j.tox.2010.07.002

The authors stated that the aim of the study “was to evaluate effects of perinatal 

exposure to environmentally relevant subtoxic doses of atrazine (ATZ), on 

neurobehavioral development in mice and investigate possible alterations in steroid 

hormone metabolism.” 

Female and male mice delivered from CD1 dams were exposed daily from Gestational 

Day 14 (GD14) until Postnatal Day 21 (PND21) to 1 or 100 μg/kg bw ATZ. Locomotor 

and exploratory activity, social interactions and cognitive performance were evaluated at 

PND 16, 31 and 60, respectively. On PND 60-65, 10 F1 males (1/litter in each 

treatment and control group), were randomly selected and sacrificed by cervical 
Page 118 of 210

dislocation. Liver and testes from control and treated groups were removed and 

weighed. Testicular parameters (sperm head number and concentration on right 

testes, histological evaluation of left testes), rate of testosterone metabolism (hepatic 

microsomal testosterone hydroxylase) and aromatase activity in F1 male liver were 

analyzed. 

The authors reported that there were no clinical signs of toxicity in dams and pups. No 

significant changes in absolute and relative testicular weights were noted in F1 male 

mice at PND 60-65. A reduction in testicular sperm count and concentration was 

observed in treated animals. The authors indicated that there were no significant 

morphological changes of the germinal epithelium in treated animals, although in 1/8 in 

both ATZ-treated groups the epithelium organization was altered in some tubules. 

In regard to the open-field test, the authors concluded that duration of exploratory 

behavior was increased when compared to controls. For the novel object test, the 

authors indicated an increase in duration of sniffing and a tendency to perform higher 

number of contacts with the object in both ATZ-treated groups compared to control (no 

differences between sexes). 

With respect to social interactions, the authors reported a “significant main effect of 

treatment” on investigative-affilative and explorative behaviors in males, in particular, for 

the low-dose ATR group there was a higher frequency of elements grouped in “factor 1” 

(squire, genital sniff, following, and allogrooming) compared to controls. A less 

pronounced and nonsignificant similar trend was noted for the high-dose ATR group. 

The authors also stated that non-social exploratory component of “factor 2” (explore, 

wall rearing, sniff) was affected by the high-dose ATR treatment, in which exposed 

animals exhibited a clear cut reduction in the habituation profile when compared to 

controls.

The results of the passive avoidance test revealed improved performance in the 

learning session for the high-dose ATR group compared with the low-dose ATR and 

control groups. The authors mentioned that “females showed higher latency than males 

to enter the escape compartment.” Also, no effect of treatment was observed in the 

retest session, whereas, sex affected the passive avoidance response (females taking 

longer in stepping-through than males). 

The authors reported effects of atrazine on hepatic testosterone hydroxylase and 

aromatase activities in the liver. They stated that treatment with both ATZ doses 

produced slight changes in the rate of selected testosterone hydroxylases. The affected 

isoforms were CYP 2C and 2B, as reflected by the differences in the formation of 16αand 16β-hydroxyl-T in the livers from treated and control animals. The authors also 

reported a slight decrease in hepatic aromatase activity (statistically not significant) in 

the treated animals compared with controls. The authors concluded that 

“developmental exposure to non-toxic, environmentally relevant doses of ATZ can 

produce subtle functional alterations” in juvenile and adult mice. 

Reviewer’s Comments: This published study was not designed as a GLP compliant 

study to meet Agency toxicology test guidelines for data requirement, but rather to 
Page 119 of 210

explore potential effects of atrazine on behavior in juvenile and adult mice. A strong 

point of the study is that the “litter” was considered in the design and analyses of the 

results. The behavioral methods are typical of the methods utilized with measuring 

social and non-social behaviors in mice. However, there are several notable limitations 

of the study:

Dosing - Dosing of the dams was done via licking the dosing solution from a syringe 

which is not a common dosing procedure. This procedure assumes that the total dose 

obtained from the syringe was swallowed. There was also no analytical confirmation of 

atrazine concentrations of the dosing solution. 

Only 2 dose levels were examined. The authors acknowledge the “controversy” of 

nonmonotonic dose- response relationships associated with “endocrine disruptors.” 

Examination of more than 2 dose levels would assist in interpretation of the findings. 

In-house breeding - Mice were bred in-house which means that births were likely spread 

across multiple days. If this is the case, the authors should have presented a table 

indicating the number of animals from each group born on each day. The implication is 

that for testing at very early ages one may not have representative pups from all dose 

groups.

 

Blind observations – There was no mention if the observer was “blind” to treatment 

which would ensure objectivity. This is critical for behavioral studies of this nature.

Positive Controls - The study did not include positive controls which would be helpful in 

interpretation of behavioral findings, and ensure that the investigators can actually 

detect chemicals that interfere with behavior. As an example, the biological significance 

of exploratory behavior (figure 1), in which changes were of small magnitude and nondose-response related is dubious. The same problem is also noted for the reported 

effects of perinatal atrazine administration on frequency and duration of sniff object 

behavior in novelty test on PND16 (figure 2). There was large variability in the data and 

the findings are of doubtful toxicological significance. 

This is also apparent with the results of the passive avoidance test in which the reported 

effect is small and not dose related. In addition, the high-dose ATZ treated animals 

exhibited improved performance compared with the low-dose ATZ and control groups, 

implying that high dose ATZ animals had better memory and learning performance than 

the control group. The figures did not distinguish between male and female results 

although the authors noted significant differences between the sexes in regard to 

passive avoidance. There were also no statistical analyses to compare the retest 

(retention) results from the pre-multitrial (learning) session. In regard to their findings on 

passive avoidance, the authors claim that their results are consistent with the findings of 

rats perinatally treated with bisphenol A (Negishi et al. 2004). However, Negishi et al. 

(2004) expressed reservation of the effects of bisphenol A on passive avoidance and 

stated “...such changes were statistically ambiguous because of the large individual 

differences in the experimental conditions used in this study.” Furthermore, Belloni et 

al. acknowledge several potential confounders which further complicate the 

interpretation of the passive avoidance results. Belloni et al. state that “possible effects 

of development ATZ exposure on shock sensitivity thresholds, that could mask 
Page 120 of 210

associative learning alterations in treated mice, cannot be dismissed on the basis of our 

results, since estrogens and estrogenic substances can influence nociceptive sensory 

processing in rodents (Aloisi et al., 2002).” Also, they mentioned that “we cannot 

exclude that ATZ may induce alterations in dams, resulting in changes in maternal 

cares, and, consequently, in offspring’s altered developmental behavioral trajectories.” 

Problems with figure labeling – The labels associated with a number of endpoints are 

incorrect. For example, the authors reported an effect of atrazine treatment on 

“duration” of exploring behavior in the open-field test, however, the relevant figure 

(figure 1) indicates “frequency.” Figure 1 also depicts “duration” to describe “crossing”, 

and “rearing”; however, “frequency” is the correct descriptor. 

Additionally, the figures did not distinguish between males and females, although the 

authors often made conclusions based on individual sex.

Testosterone metabolism and aromatase activity – The reported effects of atrazine on 

testosterone metabolism and aromatase activity are of doubtful biological significance 

given the slight and non-dose-related changes. In addition, one of the references (i.e., 

Traina et al. 2003) relating to methodology for examining testicular parameters was not 

presented in the reference section.

Principal Component Analysis - The application of principal component analysis (PCA) 

as an exploratory data analysis of social interaction data is an acceptable technique 

since behavioral variables may be correlated. However, there are limitations with the 

use of PCA. Most notably, the authors applied a linear PCA method. Since 

neurobehavioral data may exhibit nonlinearity, a nonlinear PCA approach should have 

been explored. Other significant problems with PCA are that it can fail if the data are 

non-Gaussian or time-dependent. The authors did not address these potential and 

likely issues. The results of the linear PCA should be interpreted with caution and may 

be misleading. 

The authors provided little description of how the PCA was performed, e.g., the software 

used, how transformation was performed (orthogonal or other approach), eiganvalue 

decomposition approach, and the matrix (correlation or covariance) examined. The 

authors also reported a total variance of 75% following varimax rotation. However, the 

table of individual factors adds up to 66% and it is not clear if the 66% is a typo, the 

correct value following rotation, or if it represents an unrotated value. Reporting of both 

unrotated and rotated values would be helpful. It is also unclear if the loading factors 

presented in Table 3 represent values before varimax rotation or after varimax rotation. 

Once again, it would be helpful to see the results of both which can aid in interpretation 

of the results. It is interesting to note that even with varimax rotation, the PCA analyses 

cannot explain all of the information, which raises questions about the applicability of 

linear PCA methodology. Finally, it would have been useful if the authors had applied a 

statistical analyses of the data separate from the PCA analyses for comparative 

purposes given that the assumptions of the PCA may be flawed. 
Page 121 of 210

In summary, this study has a number of significant limitations that limit its use for 

quantitative risk assessment purposes. There are no other studies that have validated 

the findings presented by Belloni et al.

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. 

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 
Page 122 of 210

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 

Sprague-Dawley rats following oral exposure – a relevant route of human exposure –

from GD 10 until PND 23 to 35 mg/kg/day atrazine. Delayed type hypersensitivity 

response and the IgM antibody response to sheep red blood cell antigen were 

measured. In comparison, Rowe et al. used Balb/c mice which were exposed 

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. 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 
Page 123 of 210

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 atrazineinduced 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 proliferatoractivated receptor family, and the retinoid-related orphan receptor α. 

Although in vitro studies provided some insight into potential mechanisms of atrazinemediated 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 

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 atrazineinduced immunotoxicity is a more sensitive endpoint than the atrazine-induced effects 
Page 124 of 210

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, 

interleukin-3 and-6, and stem cell growth factor. Cell viability was determined; cells 

were adjusted to 6.0 x 105 viable cells/mL, seeded to yield a final density of 4.0 x 

104

viable cells/plate and incubated. Triplicate plates were prepared per group and five 

replicated experiments were conducted. After 7 days, colonies were counted to 

determine cytotoxicity. 

For the gene expression phase of testing, total RNA was extracted from bone marrow 

cells, and [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 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 HighCapacity cDNA Archive Kit and Moloney murine leukemia virus reverse transcriptase. 

No gross pathological changes were seen in the treated mice. A significant (p<0.05) 

~19% (males) and ~23% (females) decrease in the percentage of CFU-GM was see 
Page 125 of 210

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) 

(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 
Page 126 of 210

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 substance-related clinical findings for animals at any dose level within 2 hours of 

dosing, or during daily examinations. The positive control group II showed an increase 

of red, brown, and yellow fur staining, but no other remarkable clinical findings. No 

clinical signs were noted in doses at 25 or 6 mg/kg/day.

Treatment resulted in dose-dependent, statistically significant decreases in body weight 

gain and food consumption for ATR-treated rats in the 25 and 100 mg/kg/day dose 

groups when compared to the untreated vehicle control group. The decrease found in 

body weight 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 
Page 127 of 210

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 dosedependent 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 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.
Page 128 of 210

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 doseresponse 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., 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.

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 retinoid-related orphan receptor (ROR) α. The authors noted previous 

work that showed weak binding of atrazine to the AR. The GR was investigated 

because of the similarity of effects induced by atrazine and the synthetic glucocorticoid 
Page 129 of 210

DEX. Glucocorticoids have anti-inflammatory and immunosuppressive properties and 

interfere with nuclear factor – κB (NF- κB) and AP-1, and in this way repress the 

expression of inflammation-related genes such as interleukin (IL)-6, IL-8, ICAM, and Eselectin, 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 TNF-induced 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 longer variant of the IL-8 promoter, p133.IL8pluc+, yielded the same overall results (data were not shown). To test whether the 

results with the IL-8 promoter were promoter-specific, the authors also tested the effect 

of atrazine on other proinflammatory gene construct, containing the E-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.
Page 130 of 210

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.

A.6.5 Filipov, N M, Pinchuk, L M, et. al. 2005. Immunotoxic effects of 

short-term atrazine exposure in young male C57BL/6 mice. 

Toxicological Sciences: 86(2): 324-332.

The objective of this study was to assess the effects of atrazine exposure on selected 

immune parameters in young mice. Parameters included: thymus and spleen weights, 

cellularity, lymphocyte subpopulations in the spleen, thymus, and peripheral blood 

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 
Page 131 of 210

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 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, 
Page 132 of 210

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. 

Host resistance to B16F10 tumor challenge was reduced in atrazine treated mice at 

both challenge levels. At the 3 x 105 cells/mouse challenge level, a significant dosedependent 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. 
Page 133 of 210

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 RBL2H3 cells by BSA-conjugated progesterone (PROG-BSA) was examined. βHexosaminidase release was characterized by various inhibitors including antisenseoligodeoxynucleotide 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. 

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 atrazine-induced increase in Alexa 488-annexin V binding shows that the 

atrazine-induced degranulation is unlikely due to cell damage. 

Antisense-oligodeoxynucleotide pre-treatment reduced atrazine-induced βhexosaminidase release. Atrazine-induced degranulation was abolished by 

phospholipase C inhibitor U-73122. 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 

dose-dependent between 1 and 10 fmol (i.pl.). The effects were abolished by PROG –

BSA and DPH, which is a histamine receptor antagonist. This was interpreted by the 

authors to indicate that the responses were caused by neurosteroid receptor-mediated 

histamine release from mast cells, which was supported by atrazine-induced plasma 

extravasation assay using Evans blue. Overall, the authors concluded that atrazine 

induces some inflammatory effects, including nociception and plasma extravasation, 

and the underlying mechanisms are mediated through mast cell degranulation and 

activation of the Gq/11-protein coupled neurosteroid receptor, phospholipase C and 

intracellular Ca2+ mobilization. 
Page 134 of 210

Reviewer’s Comments: This study concluded that the underlying mechanisms of 

atrazine-induced inflammatory effects such as nociception and plasma extravasation, 

may be mediated through mast cell 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 factor-dependent, bone-marrow-derived immature murine DC cell line JAWSII. 

For comparative studies, limited studies were performed with primary thymic DC. 

Details were provided on 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 receptormediated 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 
Page 135 of 210

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. 

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; atrazine-treatment: 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. 
Page 136 of 210

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, 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 
Page 137 of 210

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 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. 2005a15. Dose 

levels were selected based on previous studies on atrazine effects on prepubertal rats 

(Stoker et al. 2000b16). 

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. 

 15 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.

16 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
Page 138 of 210

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.

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-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. 
Page 139 of 210

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 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 3

H-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. 
Page 140 of 210

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 103 cpm; control offspring: 46 ± 4 x 103 cpm) and cytolytic 

activity (approximately 66% increase). A significant increase in the number of HKSPspecific 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. Three-month-old female offspring showed no significant difference in 

immune response from controls. The authors concluded that the results indicate “a 

male-specific immunopotentiation of the 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 HKSP-vaccinated atrazine-treated females were 69% of control). In addition, 

spleens from the atrazine-treated groups produced a mean of 72% of control IgG PCspecific 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.
Page 141 of 210

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 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 posttranslational 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.
Page 142 of 210

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.

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 
Page 143 of 210

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 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 celllines 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 DMBAinduced 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 
Page 144 of 210

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 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 method used to study DNA instability or a deficiency in DNA repair – has also 
Page 145 of 210

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/E2 – 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.17 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 transfected with the ERE-luc reporter gene 

and subsequently exposed to 0 or 10-7-10-4 M atrazine for 24 hours.

 17 In this evaluation only the results for atrazine will be discussed
Page 146 of 210

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 estrogendependent 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]anthracene-induced mammary tumor development in 

human c-Ha-ras proto-oncogene transgenic rats. Cancer Sci. 

95(5):404-410.

This study evaluates the potential of 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.18

 18 In this evaluation only the results for atrazine will be discussed

 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 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).
Page 147 of 210

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 

ingredients by applying the comet assay on mouse cells from blood, kidney, liver, bone 

marrow, and spleen. 1 
Page 148 of 210

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 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, 
Page 149 of 210

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 increased with high atrazine concentrations. After 72 hours incubation 

period, the 20 ug/mL atrazine concentration significantly decreased cell proliferation by 
Page 150 of 210

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.
Page 151 of 210

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.

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, 
Page 152 of 210

suggesting the presence of isoforms and/or post-translational 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 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 
Page 153 of 210

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.

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 2x105 cells/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 
Page 154 of 210

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 caspase3. Cytosolic thiol proteases called caspases are key enzymes involved in apoptosis. 

DET 551 cells were exposed to increasing concentrations of atrazine or DMSO (0, 3, 

100, 200, and 500 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 
Page 155 of 210

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 

nuclease-positive control. When the cells treated with 200 μg/L atrazine or DMSO were 

compared to control cells, there was only a sight increase in nuclear staining of 

atrazine-treated cells, however, the level of apoptosis in treated–treated cells was low 

compared to the positive control. The caspase assay was then done to determine if the 

small increase in the apoptotic cells was significant. The results showed that all 

atrazine-treated samples (3-500 μg/L) had caspase activity (7.31-9.53 pmol/μl) similar 

to the negative control (7.31 pmol/μl). No significant differences were detected between 

the control treatments (DMSO) and atrazine-treated cells. 

Conclusion: In vitro exposure to 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. 

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, 
Page 156 of 210

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.

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. 
Page 157 of 210

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 reduc ed 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 dose-related 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 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, 
Page 158 of 210

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), glutathiones-transferase (GST) and glucose-6-transferase (GTS). Body and liver weights were 

also recorded at each sacrifice interval. 

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 
Page 159 of 210

“antioxidant status of the liver”. They further conclude that the administration of vitamin 

E along with atrazine ameliorates the toxic effects of atrazine. 

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 atrazineinduced 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 
Page 160 of 210

treatment significantly increased phospholipid content after 14 days (22.86%) and 21 

days (57.92%), compared to atrazine treated group.

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)19a

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 mg/kg/day) plus vitamin E (100 

 19 According to personal communications with Charles Parady, Senior Specialist, Charles River, male 

Wistar rats in the weight range of 100-120 gm would be approximately 4-5 weeks old.
Page 161 of 210

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 Nbis(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.20 The goal of the study was to 

assess the impact of atrazine (with or without concomitant tamoxifen exposure) on 

thyroid hormone changes and thyroid lesions induced by DHPN.

Twenty male F344 rats per group received a subcutaneous injection of 2800 mg/kg 

DHPN or saline. Starting one 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. 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 

 20 In this evaluation only the results for atrazine will be discussed
Page 162 of 210

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 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 
Page 163 of 210

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 doseresponse over a two-order of magnitude difference in atrazine exposures. However, 

when considered collectively, the data reported by Fujimoto, Son, and Tanaka and coworkers 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 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 
Page 164 of 210

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.

Figure A- 1: Treatment Groups
Page 165 of 210

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 B500 ppm atrazine dose level is increased by 9% and is not statistically significant.21 In 

fact, if Group B rats treated with 500 ppm atrazine are 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 

 21 Calculated by reviewer and analyzed using Fisher’s Exact test

B

C

A

Table excerpted from Ueda et al., (2005) p. 22
Page 166 of 210

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 (latedeveloping 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. 
Page 167 of 210

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 microplatereader. 

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 the assay. Varying concentrations of atrazine, DACT, DEA, DIA, ammeline, 

and OH-ATR dissolved in DMSO were added. Cell viability was measured 24 hours 
Page 168 of 210

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 nonsteroidogenic 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).

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 
Page 169 of 210

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, K-U, Pak, Y.K., Lee, H.K. (2009). Chronic Exposure to the 

Herbicide, Atrazine, Causes Mitochondrial Dysfunction and Insulin 

Resistance. PLoS ONE 4(4): e5186, p.1-11.

The authors 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, 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 
Page 170 of 210

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 reanalysis conducted, further scrutiny of the statistical approaches may be useful in the 

future. 

Generally, the results section of this paper is hard to follow. Figure labels and legends 

are difficult to interpret. 

Body weight and fat determinations. Based on the results 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. 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.9.1. PHARMACOKINETIC CALCULATIONS

(a). Oral Gavage Dosing with Atrazine: Estimation of Plasma Half-lives
Page 171 of 210

Since atrazine was barely detected if at all by 24 hours post-dosing, each treatment day 

was analyzed separately for elimination rate constant (kel) half-life at dose levels of 3 

and 10 mg/kg bw/day. At the high dose of 50 mg/kg bw/day, analysis was carried out on 

the last treatment day (TD4) to include the washout period. In all cases, a significant 

linear behavior was observed. Figure 1a shows the plasma levels of atrazine plotted 

versus time in a semi-logarithmic scale following each treatment day (TD) with 3 mg/kg 

bw/day atrazine.

Atrazine Analysis:

Figure 1a: Plasma elimination phase of atrazine resulting from oral gavage dosing with 

3 mg/kg bw/day atrazine plotted versus time for treatment days 1- 4 (TD1-4) on a semilogarithmic scale. The dashed line represents the 95% confidence interval of the linear 

regression.

Table 1a. Linear regression analysis for Figure 1a (3 mg/kg bw/day atrazine). 

Table 1b: Summary of kel and half-life estimates for atrazine at 3 mg/kg bw/day from 

Table 1a.

ATRAZINE: 3 mg/kgbw/day: Elimination Phase Analysis

20 40 60 80 100

-8

-6

-4

-2

0

2

4

TD1 TD2 TD3 TD4

time (hr)

Ln (plasma atrazine) (

μmoles/L)

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

95% Confidence Intervals

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

Goodness of Fit

R square

Sy.x

Is slope significantly non-zero?

F

DFn, DFd

P value

Deviation from zero?

Data

Number of X values

Maximum number of Y replicates

Total number of values

Number of missing values

Plasma ATRA (TD1, 3 mg/kg bw)

-0.9065 ± 0.06656

1.651 ± 0.1103

1.821

-1.103

-1.041 to -0.7724

1.429 to 1.873

1.623 to 2.051

0.8013

0.5696

185.5

1.000, 46.00

< 0.0001

Significant

48

1

48

102

Plasma ATRA (TD2, 3 mg/kg bw)

-1.175 ± 0.07359

30.23 ± 1.865

25.74

-0.8514

-1.325 to -1.024

26.41 to 34.05

25.56 to 25.93

0.9010

0.5534

254.7

1.000, 28.00

< 0.0001

Significant

30

1

30

120

Plasma ATRA (TD3, 3 mg/kg bw)

-0.8740 ± 0.04943

43.41 ± 2.476

49.67

-1.144

-0.9788 to -0.7692

38.16 to 48.66

49.42 to 49.92

0.9513

0.4293

312.7

1.000, 16.00

< 0.0001

Significant

18

1

18

132

Plasma ATRA(TD4, 3 mg/kg bw)

-0.8065 ± 0.08785

59.34 ± 6.517

73.57

-1.240

-0.9919 to -0.6212

45.59 to 73.09

73.04 to 74.04

0.8322

0.7809

84.30

1.000, 17.00

< 0.0001

Significant

19

1

19

131
Page 172 of 210

Figure 2a: Plasma elimination phase of atrazine resulting from oral gavage dosing with 10 mg/kg 

bw/day atrazine plotted versus time for treatment days 1- 4 (TD1-4) on a semi-logarithmic scale. 

The dashed line represents the 95% confidence interval of the linear regression.

logarithmic scale. The dashed line represents the 95% confidence interval of the linear 

regression.

kel (hr-1) Error

TD1 0.907 0.067

TD2 1.175 0.074

TD3 0.874 0.049

TD4 0.807 0.088

Mean = 0.941 0.069

t1/2 (hr)= 0.737 hr

t1/2 (min)= 44.22 min

 ATRAZINE:10 mg/kg bw/day elimination phase analysis

24 48 72 96

-8

-6

-4

-2

0

2

4

6

TD1 TD2 TD3 TD4

time (hr)

Ln (plasma atrazine) (

μg/L)
Page 173 of 210

Table 2a. Linear regression analysis for Figure 2a (10 mg/kg bw/day atrazine). 

Table 2b: Summary of kel and half-life estimates for atrazine at 10 mg/kg bw/day from 

Table 2a.

Figure 3a: Plasma elimination phase of atrazine resulting from oral gavage dosing with 50 mg/kg 

bw/day atrazine plotted versus time for treatment day 4 (TD4) on a semi-logarithmic scale. The 

dashed line represents the 95% confidence interval of the linear regression.

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

95% Confidence Intervals

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

Goodness of Fit

R square

Sy.x

Is slope significantly non-zero?

F

DFn, DFd

P value

Deviation from zero?

Data

Number of X values

Maximum number of Y replicates

Total number of values

Number of missing values

TD1

-0.5541 ± 0.03521

2.506 ± 0.1434

4.523

-1.805

-0.6246 to -0.4837

2.220 to 2.793

4.079 to 5.032

0.8077

0.8678

247.8

1.000, 59.00

< 0.0001

Significant

61

1

61

110

TD2

-0.6182 ± 0.04295

17.69 ± 1.142

28.61

-1.618

-0.7057 to -0.5306

15.36 to 20.01

28.12 to 29.19

0.8662

0.7523

207.2

1.000, 32.00

< 0.0001

Significant

34

1

34

137

TD3

-0.6041 ± 0.06079

31.66 ± 3.136

52.41

-1.655

-0.7305 to -0.4776

25.13 to 38.18

51.78 to 53.12

0.8246

0.8714

98.75

1.000, 21.00

< 0.0001

Significant

23

1

23

148

TD4

-0.5000 ± 0.04798

38.22 ± 3.645

76.44

-2.000

-0.5993 to -0.4007

30.68 to 45.76

75.68 to 77.25

0.8252

0.9193

108.6

1.000, 23.00

< 0.0001

Significant

25

1

25

146

10 mg/kg bw atrazine

kel (hr-1) Error

TD1 0.554 0.035

TD2 0.618 0.043

TD3 0.604 0.061

TD4 0.500 0.048

Mean = 0.569 0.047

t1/2 (hr)= 1.22

ATRAZINE: 50 mg/kg bw/day TD4 elimination phase analysis

80 90 100 110 120

-10

-8

-6

-4

-2

0

2

4

 time (hr)

Ln (plasma atrazine,

μg/L)
Page 174 of 210

Table 3a: Linear regression analysis for Figure 3a (50 mg/kg bw/day atrazine).

Table 3b: Summary of kel and half-life estimates for atrazine at 50 mg/kg bw/day from 

Table 3a.

50 mg/kg bw atrazine

kel (hr-1) Error

TD4 0.238 0.013

t1/2 (hr)= 2.91

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

95% Confidence Intervals

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

Goodness of Fit

R square

Sy.x

Is slope significantly non-zero?

F

DFn, DFd

P value

Deviation from zero?

Data

Number of X values

Maximum number of Y replicates

Total number of values

Number of missing values

Plasma ATRA (50 mg/kg)

-0.2378 ± 0.01295

20.52 ± 1.041

86.31

-4.206

-0.2642 to -0.2114

18.40 to 22.65

85.20 to 87.59

0.9158

0.6354

337.1

1.000, 31.00

< 0.0001

Significant

33

1

33

27
Page 175 of 210

DEA Analysis:

Figure 4a: Plasma elimination phase of DEA resulting from oral gavage dosing with 3 mg/kg 

bw/day atrazine plotted versus time for treatment day 4 (TD4) on a semi-logarithmic scale. The 

dashed line represents the 95% confidence interval of the linear regression.

Table 4a: Linear regression analysis for DEA (Figure 4a, 3 mg/kg bw/day atrazine).

 DEA: 3 mg/kg bw/day atrazine-elimination phase analysis

75 80 85 90 95 100

-2

0

2

4

6

Ln (plasma DEA) (

time (hr)

μg/L)

DEA: 10 mg/kg bw atrazine- elimination phase analysis

80 100 120 140 160 180 200

-10

-5

0

5

10

time (min)

Ln (plasma DEA) (μg/L)

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

95% Confidence Intervals

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

Goodness of Fit

R square

Sy.x

Is slope significantly non-zero?

F

DFn, DFd

P value

Deviation from zero?

Data

Number of X values

Maximum number of Y replicates

Total number of values

Number of missing values

DEA (μg/L, 3mg/kg ATRA)

-0.2040 ± 0.009172

18.23 ± 0.7376

89.34

-4.902

-0.2227 to -0.1853

16.72 to 19.73

88.29 to 90.55

0.9392

0.4239

494.6

1.000, 32.00

< 0.0001

Significant

34

1

34

2
Page 176 of 210

Figure 5a: Plasma elimination phase of DEA resulting from oral gavage dosing with 10 mg/kg 

bw/day atrazine plotted versus time for treatment day 4 (TD4) on a semi-logarithmic scale. The 

dashed line represents the 95% confidence interval of the linear regression.

Table 5a: Linear regression analysis for DEA (Figure 4a, 10 mg/kg bw/day atrazine).

Figure 6a: Plasma elimination phase of DEA resulting from oral gavage dosing with 50 mg/kg 

bw/day atrazine plotted versus time for treatment day 4 (TD4) on a semi-logarithmic scale. The 

dashed line represents the 95% confidence interval of the linear regression.

Table 6a: Linear regression analysis for DEA (Figure 6a, 50 mg/kg bw/day atrazine).

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

95% Confidence Intervals

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

Goodness of Fit

R square

Sy.x

Is slope significantly non-zero?

F

DFn, DFd

P value

Deviation from zero?

Data

Number of X values

Maximum number of Y replicates

Total number of values

Number of missing values

DEA

-0.09281 ± 0.003397

10.37 ± 0.3795

111.8

-10.78

-0.09960 to -0.08602

9.613 to 11.13

109.4 to 114.2

0.9222

0.8949

746.5

1.000, 63.00

< 0.0001

Significant

65

1

65

11

DEA: 50 mg/kg bw atrazine- elimination phase analysis

80 100 120 140 160 180 200

-4

-2

0

2

4

6

8

time (hr)

Ln (plasma, μg/L)
Page 177 of 210

DIA Analysis

A small, secondary (β) phase of elimination was sometimes observed for this metabolite 

at the later time points, but given its magnitude compared to the much greater α-phase, 

it was neglected.

Figure 7a: Plasma elimination phase of DIA resulting from oral gavage dosing with 3 mg/kg 

bw/day atrazine plotted versus time for treatment day 4 (TD4) on a semi-logarithmic scale. The 

dashed line represents the 95% confidence interval of the linear regression.

Table 7a: Linear regression analysis for DIA (Figure 7a, 3 mg/kg bw/day atrazine).

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

95% Confidence Intervals

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

Goodness of Fit

R square

Sy.x

Is slope significantly non-zero?

F

DFn, DFd

P value

Deviation from zero?

Data

Number of X values

Maximum number of Y replicates

Total number of values

Number of missing values

DEA 

-0.06783 ± 0.003481

9.720 ± 0.4158

143.3

-14.74

-0.07477 to -0.06088

8.891 to 10.55

138.8 to 148.5

0.8369

1.103

379.8

1.000, 74.00

< 0.0001

Significant

76

1

76

0

DIA: 3 mg/kg bw atrazine - analysis of elimination phase

(from 72 - 80 hrs)

72 74 76 78 80

0

2

4

6

8

time (hr)

Ln (plasma DIA) (μg/L)
Page 178 of 210

Figure 8a: Plasma elimination phase of DIA resulting from oral gavage dosing with 10 mg/kg 

bw/day atrazine plotted versus time for treatment day 4 (TD4) on a semi-logarithmic scale. The 

dashed line represents the 95% confidence interval of the linear regression.

Table 8a: Linear regression analysis for DIA (Figure 8a, 10 mg/kg bw/day atrazine).

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

95% Confidence Intervals

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

Goodness of Fit

R square

Sy.x

Is slope significantly non-zero?

F

DFn, DFd

P value

Deviation from zero?

Data

Number of X values

Maximum number of Y replicates

Total number of values

Number of missing values

DIA (3mg/kg)

-0.5636 ± 0.03036

45.99 ± 2.290

81.60

-1.774

-0.6266 to -0.5006

41.24 to 50.74

80.90 to 82.46

0.9400

0.4442

344.7

1.000, 22.00

< 0.0001

Significant

24

1

24

0

DIA: 10 mg/kg bw atrazine- elimination phase analysis

80 90 100 110 120

-4

-2

0

2

4

6

8

10

time (hr)

Ln (plasma DIA) (μg/L)

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

95% Confidence Intervals

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

Goodness of Fit

R square

Sy.x

Is slope significantly non-zero?

F

DFn, DFd

P value

Deviation from zero?

Data

DIA, 10 mg/kg bw ATRA

-0.2425 ± 0.01456

22.99 ± 1.219

94.83

-4.124

-0.2720 to -0.2129

20.52 to 25.47

92.98 to 97.04

0.8852

1.023

277.5

1.000, 36.00

< 0.0001

Significant
Page 179 of 210

Figure 9a: Plasma elimination phase of DIA resulting from oral gavage dosing with 50 mg/kg 

bw/day atrazine plotted versus time for treatment day 4 (TD4) on a semi-logarithmic scale. The 

dashed line represents the 95% confidence interval of the linear regression.

Table 9a: Linear regression analysis for DIA (Figure 9a, 50 mg/kg bw/day atrazine).

DIA: 50 mg/kg bw atrazine- elimination phase analysis

80 90 100 110 120 130 140

-4

-2

0

2

4

6

8

10

time (hr)

Ln (plasma DIA) (

μg/L)

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

95% Confidence Intervals

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

Goodness of Fit

R square

Sy.x

Is slope significantly non-zero?

F

DFn, DFd

P value

Deviation from zero?

Data

Number of X values

Maximum number of Y replicates

Total number of values

Number of missing values

DIA 

-0.1757 ± 0.006532

19.72 ± 0.5857

112.2

-5.692

-0.1889 to -0.1625

18.53 to 20.90

110.2 to 114.5

0.9439

0.7099

723.6

1.000, 43.00

< 0.0001

Significant

45

1

45

3
Page 180 of 210

DACT

Figure 10a: Plasma elimination phase of DACT resulting from oral gavage dosing with 3 mg/kg 

bw/day atrazine plotted versus time for treatment day 4 (TD4) on a semi-logarithmic scale. The 

dashed line represents the 95% confidence interval of the linear regression.

Table 10a: Linear regression analysis for DACT (Figure 10a, 3 mg/kg bw/day atrazine).

DACT: 3 mg/kg bw atrazine- elimination phase analysis

80 100 120 140 160 180 200

-8

-4

0

4

8

time (hr)

Ln(Plasma,

μg/L)

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

95% Confidence Intervals

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

Goodness of Fit

R square

Sy.x

Is slope significantly non-zero?

F

DFn, DFd

P value

Deviation from zero?

Data

Number of X values

Maximum number of Y replicates

Total number of values

Number of missing values

DACT (3 mg/kg bw ATRA)

-0.08730 ± 0.006230

12.80 ± 0.7536

146.7

-11.45

-0.09974 to -0.07486

11.30 to 14.31

140.0 to 154.7

0.7400

2.064

196.3

1.000, 69.00

< 0.0001

Significant

71

1

71

11
Page 181 of 210

Figure 11a: Plasma elimination phase of DACT resulting from oral gavage dosing with 10 mg/kg 

bw/day atrazine plotted versus time for treatment day 4 (TD4) on a semi-logarithmic scale. The 

dashed line represents the 95% confidence interval of the linear regression.

Table 11a: Linear regression analysis for DACT (Figure 10a, 10mg/kg bw/day 

atrazine).

DACT: 10 mg/kg bw atrazine- elimination phase analysis

72 96 120 144 168 192

-10

-5

0

5

10

time (hr)

Ln (plasma DACT,

μg/L)

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

95% Confidence Intervals

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

Goodness of Fit

R square

Sy.x

Is slope significantly non-zero?

F

DFn, DFd

P value

Deviation from zero?

Data

Number of X values

Maximum number of Y replicates

Total number of values

Number of missing values

DACT (ug/L)

-0.08449 ± 0.006908

14.05 ± 0.9067

166.3

-11.84

-0.09830 to -0.07068

12.24 to 15.87

158.2 to 176.8

0.7070

2.100

149.6

1.000, 62.00

< 0.0001

Significant

64

1

64

6
Page 182 of 210

Figure 12a: Plasma elimination phase of DACT resulting from oral gavage dosing with 50 mg/kg 

bw/day atrazine plotted versus time for treatment day 4 (TD4) on a semi-logarithmic scale. The 

dashed line represents the 95% confidence interval of the linear regression.

Table 11a: Linear regression analysis for DACT (Figure 10a, 50 mg/kg bw/day 

atrazine).

(b). Oral Gavage Dosing with Atrazine: Estimation of Plasma AUC

Plasma AUC was estimated based on the trapezoid rule as implemented by Graphpad 

Prism (version 5). In the case of atrazine, DEA, and DIA, plasma levels at the last time 

point of 24 hours were very minimal, and thus no correction was made for estimating 

the area from the last time point to infinity. Only in the case of DACT, a correction was 

made by estimating the area from the last time point to infinity by dividing the last 

plasma time point by the estimated elimination rate constant.

DACT: 50 mg/kg bw atrazine- elimination phase analysis

84 96 108 120 132 144 156

0

2

4

6

8

10

time (hr)

Ln (plasma DACT,

μg/L)

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

95% Confidence Intervals

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

Goodness of Fit

R square

Sy.x

Is slope significantly non-zero?

F

DFn, DFd

P value

Deviation from zero?

Data

Number of X values

Maximum number of Y replicates

Total number of values

Number of missing values

-0.07522 ± 0.01020

14.69 ± 1.186

195.3

-13.29

-0.09596 to -0.05448

12.28 to 17.11

177.3 to 226.7

0.6154

1.196

54.41

1.000, 34.00

< 0.0001

Significant

36

1

36

0
Page 183 of 210

Figure 13a: 24-h plasma profile for atrazine following oral gavage dosing with 3, 10, and 50 mg/kg 

bw atrazine.

Table 13a: AUC estimates for plasma levels of atrazine from Figure 13a.

0 1 2 3 4 5 6 7 8

0

10

20

30

40

10 12 14 16 18 20 22 24

3 mg/kg bw/day ATRA

10 mg/kg bw/day ATRA

50 mg/kg bw ATRA

time (hr)

Plasma atrazine (μg/L)

Baseline

Total Area

Total Peak Area

Number of Peaks

Peak 1

First X=

Last X=

Peak X=

Peak Y=

Area=

%Area=

3 mg/kg bw/day ATRA

0.0

6.411

6.361

1.000

0.0

6.000

0.3333

6.432

6.361

100.0

10 mg/kg bw/day ATRA

0.0

24.87

24.72

1.000

0.0

18.00

0.3333

18.04

24.72

100.0

50 mg/kg bw ATRA

0.0

109.6

109.6

1.000

0.0

24.00

0.1667

31.07

109.6

100.0

3 mg/kg bw atrazine

0 1 2 3 4 5 6

0

50

100

150

200

250

300

8 10 12 14 16 18 20 22 24

DEA

DIA

time (hr)

Plasma Levels (

μmoles/L)
Page 184 of 210

Figure 14a: 24-h plasma profile for DEA and DIA following oral gavage dosing with 3 mg/kg bw 

atrazine.

Table 14a: AUC estimates for plasma levels of DEA and DIA from Figure 14a.

Baseline

Total Area

Total Peak Area

Number of Peaks

Peak 1

First X=

Last X=

Peak X=

Peak Y=

Area=

%Area=

DEA (ug/L)

0.0

153.5

153.5

1.000

0.0

24.00

0.3333

58.17

153.5

100.0

DIA (ug/L)

0.0

370.3

370.3

1.000

0.0

24.00

0.3333

220.7

370.3

100.0
Page 185 of 210

Figure 15a: 24-h plasma profile for DACT following oral gavage dosing with 3 mg/kg bw atrazine

Table 15a: AUC estimates for plasma levels of DACT from Figure 15a.

DACT AUC estimate - 3 mg/kg bw atrazine

0 2 4 6 8 10 12 14 16 18 20 22 24

0

200

400

600

800

1000

time (hr)

Plasma Levels (

μg/L)

Baseline

Total Area

Total Peak Area

Number of Peaks

Peak 1

First X=

Last X=

Peak X=

Peak Y=

Area=

%Area=

DACT

0.0

7947

7947

1.000

0.0

24.00

2.000

682.0

7947

100.0

∞

AUC

0 last

AUC

0 ∞

AUC

last

= +

= 7947 + Cplast

kel

= 7947 + 84 mg/L

0.0873/hr

= 7947 + 962.2

= 8909.2 (mg/L)*hr
Page 186 of 210

Figure 16a: 24-h plasma profile for DEA, DIA, and DACT following oral gavage dosing with 10 

mg/kg bw atrazine

Table 16a: AUC estimates for plasma levels of DEA, DIA, and DACT from Figure 16a.

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

0

500

1000

1500

2000

2500

DEA

DIA

DACT

time (hr)

Plasma Levels (

μg/L)

Baseline

Total Area

Total Peak Area

Number of Peaks

Peak 1

First X=

Last X=

Peak X=

Peak Y=

Area=

%Area=

DEA

0.0

465.3

465.3

1.000

0.0

24.00

1.000

141.0

465.3

100.0

DIA

0.0

1336

1336

1.000

0.0

24.00

1.000

614.2

1336

100.0

DACT

0.0

25139

25139

1.000

0.0

24.00

6.000

2125

25139

100.0

∞

AUC

0 last

AUC

0 ∞

AUC

last

= +

= 25139 + Cplast

kel

= 25139 + 367.83 mg/L

0.0845/hr

= 25139 +

= 29492.02 (mg/L)*hr

DACT:

 4353.02
Page 187 of 210

Figure 17a: 24-h plasma profile for DEA, DIA, and DACT following oral gavage dosing with 10 

mg/kg bw atrazine

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

0

2000

4000

6000

8000

10000

DEA

DIA

DACT

time (hr)

Plasma Levels (

μg/L)
Page 188 of 210

Table 17a: AUC estimates for plasma levels of DEA, DIA, and DACT from Figure 17a.

Baseline

Total Area

Total Peak Area

Number of Peaks

Peak 1

First X=

Last X=

Peak X=

Peak Y=

Area=

%Area=

DEA

0.0

2277

2277

1.000

0.08333

24.00

0.7500

219.7

2277

100.0

DIA

0.0

7255

7255

1.000

0.08333

24.00

2.000

1066

7255

100.0

DACT

0.0

121819

121819

1.000

0.08333

24.00

8.000

8614

121819

100.0

∞

AUC

0 last

AUC

0 ∞

AUC

last

= +

= 121819 + Cplast

kel

= 121819 + 2053.4 mg/L

0.0752/hr

= 121819 +

= 149124.9 (mg/L)*hr

DACT:

 27305.9
Page 189 of 210

(b). Dietary Dosing with Atrazine: Estimation of Plasma Half-lives

Figure 18a: Elimination phase analysis of DEA following dosing with atrazine through the diet at 

the nominal dose level of 3 mg/kg bw/day atrazine.

DEA: 3 mg/kg/day atrazine: elimination phase analysis

96 120

-6

-4

-2

0

2

4

time (hr)

Ln (plasma DEA) (

μg/L)

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

95% Confidence Intervals

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

Goodness of Fit

R square

Sy.x

Is slope significantly non-zero?

F

DFn, DFd

P value

Deviation from zero?

Data

Number of X values

Maximum number of Y replicates

Total number of values

Number of missing values

DEA (3 mg/kg bw atrazine)

-0.1871 ± 0.01258

19.90 ± 1.365

106.4

-5.344

-0.2126 to -0.1616

17.14 to 22.67

105.2 to 107.4

0.8534

0.6294

221.2

1.000, 38.00

< 0.0001

Significant

40

1

40

1
Page 190 of 210

Figure 19a: Elimination phase analysis of DEA following dosing with atrazine through the diet at 

the nominal dose level of 10 mg/kg bw/day (actual dose level = 9 mg/kg bw/day).

DEA: 9 mg/kg/day atrazine - elimination phase analysis

96 120 144 168 192

-6

-4

-2

0

2

4

time (hr)

Ln (plasma DEA) (

μg/L)

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

95% Confidence Intervals

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

Goodness of Fit

R square

Sy.x

Is slope significantly non-zero?

F

DFn, DFd

P value

Deviation from zero?

Data

Number of X values

Maximum number of Y replicates

Total number of values

Number of missing values

DEA (10 mg/kg atrazine)

-0.07614 ± 0.003619

9.377 ± 0.4506

123.2

-13.13

-0.08338 to -0.06890

8.476 to 10.28

120.9 to 125.4

0.8824

0.6570

442.6

1.000, 59.00

< 0.0001

Significant

61

1

61

17
Page 191 of 210

Figure 20a: Elimination phase analysis of DEA following dosing with atrazine through the diet at 

the nominal dose level of 50 mg/kg bw/day (actual dose level = 43 mg/kg bw/day).

DEA: 43 mg/kg/day atrazine - elimination phase analysis

96 120 144

-2

0

2

4

6

Ln (plasma DEA) (

time (hr)

μg/L)

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

95% Confidence Intervals

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

Goodness of Fit

R square

Sy.x

Is slope significantly non-zero?

F

DFn, DFd

P value

Deviation from zero?

Data

Number of X values

Maximum number of Y replicates

Total number of values

Number of missing values

DEA (43 mg/kg bw/day atrazine)

-0.1042 ± 0.004897

14.15 ± 0.5488

135.8

-9.598

-0.1141 to -0.09432

13.04 to 15.26

133.5 to 138.6

0.9078

0.3849

452.7

1.000, 46.00

< 0.0001

Significant

48

1

48

0
Page 192 of 210

Figure 21a: Elimination phase analysis of DIA following dosing with atrazine through the diet at 

the nominal dose level of 3 mg/kg bw/day.

DIA: 3 mg/kg bw/day atrazine - elimination phase analysis

96 120

-4

-2

0

2

4

time (hr)

ln (plasma levels,

μg/L)

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

95% Confidence Intervals

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

Goodness of Fit

R square

Sy.x

Is slope significantly non-zero?

F

DFn, DFd

P value

Deviation from zero?

Data

Number of X values

Maximum number of Y replicates

Total number of values

Number of missing values

DIA (μg/L)

-0.2309 ± 0.01586

24.41 ± 1.693

105.7

-4.331

-0.2632 to -0.1986

20.96 to 27.86

104.7 to 106.7

0.8688

0.6730

211.9

1.000, 32.00

< 0.0001

Significant

34

1

34

7
Page 193 of 210

Figure 22a: Elimination phase analysis of DIA following dosing with atrazine through the diet at 

the nominal dose level of 10 mg/kg bw/day (actual dose level = 9 mg/kg bw/day).

 DIA: 9 mg/kg bw/day - elimination phase analysis

100 120

-4

-2

0

2

4

6

 time (hr) ln (plasma levels,

μg/L)

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

95% Confidence Intervals

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

Goodness of Fit

R square

Sy.x

Is slope significantly non-zero?

F

DFn, DFd

P value

Deviation from zero?

Data

Number of X values

Maximum number of Y replicates

Total number of values

Number of missing values

-0.2485 ± 0.01497

27.08 ± 1.612

109.0

-4.025

-0.2788 to -0.2181

23.82 to 30.35

108.1 to 110.0

0.8816

0.7126

275.4

1.000, 37.00

< 0.0001

Significant

39

1

39

3
Page 194 of 210

Figure 23a: Elimination phase analysis of DIA following dosing with atrazine through the diet at 

the nominal dose level of 50 mg/kg bw/day (actual dose level = 43 mg/kg bw/day).

DIA: 43 mg/kg bw/day - elimination phase analysis

96 108 120 132

-2

0

2

4

6

8

time (hr)

Ln (Plasma) (

μg/L)

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

95% Confidence Intervals

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

Goodness of Fit

R square

Sy.x

Is slope significantly non-zero?

F

DFn, DFd

P value

Deviation from zero?

Data

Number of X values

Maximum number of Y replicates

Total number of values

Number of missing values

DIA (μg/L)

-0.2428 ± 0.01319

27.99 ± 1.433

115.3

-4.119

-0.2694 to -0.2161

25.09 to 30.88

114.2 to 116.5

0.8943

0.6867

338.6

1.000, 40.00

< 0.0001

Significant

42

1

42

0
Page 195 of 210

Figure 24a: Elimination phase analysis of DACT following dosing with atrazine through the diet at 

the nominal dose level of 3 mg/kg bw/day.

DACT: 3 mg/kg bw atrazine- elimination phase analysis

96 120 144 168 192

-4

-2

0

2

4

6

8

Plasma DACT (ug/L)

time (hr)

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

95% Confidence Intervals

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

Goodness of Fit

R square

Sy.x

Is slope significantly non-zero?

F

DFn, DFd

P value

Deviation from zero?

Data

Number of X values

Maximum number of Y replicates

Total number of values

Number of missing values

Plasma DACT (3 mg/kg bw atraz)

-0.08977 ± 0.001865

14.68 ± 0.2404

163.5

-11.14

-0.09350 to -0.08605

14.20 to 15.16

161.7 to 165.5

0.9735

0.4019

2316

1.000, 63.00

< 0.0001

Significant

65

1

65

12
Page 196 of 210

Figure 25a: Elimination phase analysis of DACT following dosing with atrazine through the diet at 

the nominal dose level of 10 mg/kg bw/day (actual dose level = 9 mg/kg bw/day).

DACT: 9 mg/kg bw/day atrazine- elimination phase analysis

96 120 144 168 192 216 240

-2

0

2

4

6

8

time (hr)

Plasma Levels (

μg/L)

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

95% Confidence Intervals

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

Goodness of Fit

R square

Sy.x

Is slope significantly non-zero?

F

DFn, DFd

P value

Deviation from zero?

Data

Number of X values

Maximum number of Y replicates

Total number of values

Number of missing values

DACT (9 mg/kg bw atrazine)

-0.07287 ± 0.002136

14.02 ± 0.2814

192.4

-13.72

-0.07714 to -0.06860

13.46 to 14.58

188.6 to 196.7

0.9440

0.5050

1164

1.000, 69.00

< 0.0001

Significant

71

1

71

7
Page 197 of 210

Figure 26a: Elimination phase analysis of DACT following dosing with atrazine through the diet at 

the nominal dose level of 50 mg/kg bw/day (actual dose level = 43 mg/kg bw/day).

DACT: 43 mg/kg bw/day atrazine - elimination phase analysis

96 120 144 168 192

0

2

4

6

8

10

time (hr)

Ln (plasma levels,

μg/L)

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

95% Confidence Intervals

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

Goodness of Fit

R square

Sy.x

Is slope significantly non-zero?

F

DFn, DFd

P value

Deviation from zero?

Data

Number of X values

Maximum number of Y replicates

Total number of values

Number of missing values

DACT (43 mg/kg bw atrazine)

-0.07298 ± 0.001597

15.74 ± 0.2190

215.7

-13.70

-0.07617 to -0.06979

15.31 to 16.18

212.1 to 219.7

0.9649

0.4387

2088

1.000, 76.00

< 0.0001

Significant

78

1

78

0
Page 198 of 210

(III). Estimating the volume of distribution at steady state for plasma triazines

Figure 27a: First moment-time curve following dosing of rats with 100 mg/kg bw 

atrazine

100 mg/kg Atrazine: Area under the first moment curve (AUMC) analysis

0 100 200 300 400

0

100

200

300

400

500

Paul et al. 1993

time (hr)

Cp*t (mg*hr/L)

Baseline

Total Area

Total Peak Area

Number of Peaks

Peak 1

First X=

Last X=

Peak X=

Peak Y=

Area=

%Area=

AUMC

0.0

57445

57445

1.000

0.0

336.0

24.00

386.9

57445

100.0
Page 199 of 210

Figure 28a. Area under the first moment curve analysis for estimating Vdss.

A.9.2. PHARMACOKINETIC STUDIES- MINIREVIEWS

A Physiologically Based Pharmacokinetic (PBPK) Model for Atrazine and 

its Main Metabolites in the Adult Male C57BL/6 Mouse Toxicology and 

Applied Pharmacology 251 (2011) 16-31

This manuscript describes the development of a PBPK model in the adult male C57BL/6 

mouse based on a previously published disposition study of atrazine (ATRA) and its 

chlorinated metabolites deethylatrazine (DEA), deisopropylatrazine (DIA), and 

diaminochlorotriazine (DACT) following a single oral gavage dose of 5, 25, 125, and 250 

mg/kg bw of ATRA (Ross et al. 2009). The PBPK model consists of four submodels 

describing the pharmacokinetics of each species (ATRA, DEA, DIA, and DACT) linked 

via systemic circulation into an integrated model. In the case of ATRA and its main 

0 ∞

Vdss =

0 ∞

Dose *

Where AUCM is the area under the first moment of the plasma concentration-time curve and AUC is

the area under the concentration-time curve

 AUCM (mg*hr2

/L)

AUC2 (mg2

*hr2

/L2

)

For 100 mg/kg atrazine, AUCM to last = 57445 mg*hr2

/L

0 ∞

AUCM = 57445 + Cplast*tlast

kel

+

 Cplast

kel2

= 57445 + 50.3 + 0.15

0.013 hr-1 (0.013 hr-1)

2

= 62201.83 mg*hr2/L

For 100 mg/kg atrazine, AUC to last = 963 (mg/L)*hr

0 ∞

AUC = 963 + 0.148/0.014 = 974.4 (mg/L)*hr

Vdss = 100 mg/kg bw *

(974.4mg*hr/L)2

= 6.55 L/kg bw 62201.83 mg*hr2/L
Page 200 of 210

metabolite DACT, the compartments include blood, brain (potential target organ), liver 

(primary site of metabolism), kidney (primary site of elimination), rapidly- and slowlyperfused compartments. The submodels for DEA and DIA consist of five compartments, 

namely blood, brain, liver, slowly- and rapidly-perfused compartments. The uptake of 

atrazine from the gut was described using a 2-compartment model used previously for 

lipophilic chemicals consisting of an initial rapid absorption phase from the stomach 

lumen, followed by gastric emptying into the intestinal lumen where a slow, sustained 

uptake takes place into the systemic circulation. All three rate constants were estimated 

via visually fitting plasma concentration of ATRA and its chlorinated metabolites. The 

following assumptions were used in modeling of ATRA and its metabolites: flow-limited 

distribution, saturable metabolism, and first-order urinary elimination. Binding to plasma 

proteins and red blood cells was included in the model via second order equations 

assuming that 26% of ATRA and DACT are bound to plasma proteins and 1.5% of total 

chlorinated triazines are bound to RBCs based on information from previous studies. 

The model was calibrated with dosimetry data from the highest dose tested of 250 

mg/kg bw (Ross et al. 2009). Partition coefficients (PCs) were also estimated from the 

highest dose tested based on the area under the curve (AUC) ratio of the particular 

tissue over that of plasma. Due to the lack of appropriate information, liver PCs for DEA 

and DIA were estimated as the 4-h time point concentration in the liver over that of 

plasma. In vivo hepatic metabolism of ATRA to DEA and DIA was estimated by 

manually adjusting in vitro metabolic rates in the mouse for better fit to the plasma data. 

Optimal fitting was obtained when in vitro metabolic rates were increased by 2-fold. 

Metabolism of DEA and DIA to DACT was based on the rat metabolic rate used in a 

previous rat model (McMullin 2007)

This modeling effort in the mouse involved significant parameter-fitting to the measured 

data of mice dosed with 250 mg/kg bw ATRA. Significant parameter adjustments were 

made to oral uptake, metabolism, and urinary excretion. Being that dose dependent 

changes were observed in the oral uptake of ATRA as well as PCs, questions remain 

regarding whether the data for the highest dose of 250 mg/kg is appropriate for model 

calibration. While plasma profile of DACT can be reasonably described by the model at 

the high dose levels of 250, 125, and 25 mg/kg ATRA, plasma levels of ATRA, DEA, 

and DIA were not, particularly at the later time point of 24 hours. Brain levels of ATRA 

and DACT were much better simulated than liver levels were at the same high dose 

levels. Simulation of brain levels of ATRA and DACT improved but still were not 

adequately described when PCs in the model were replaced with those based on the 

low dose level of 5 mg/kg ATRA, indicating potentially dose-dependent changes in PCs. 

The model should have been calibrated with data from a lower dose of ATRA to be 

more relevant to risk assessment applications. When the mouse PBPK model was 

scaled to rats, the plasma profile of ATRA was significantly over-predicted while doing a 

better job simulating plasma levels of DACT. This species extrapolation exercise 

underscores the limited potential of this current model for use in cross-species 

dosimetry extrapolation, though it represents a good effort in identifying data needs for a 

more reliable model.
Page 201 of 210

In Vitro Metabolism of Atrazine in Rat and Human Hepatocytes

Jennifer A. Thomas, Ph.D., WIL Research Laboratories, LLC (Report # WIL-639084), 

Sponsored by Syngenta Crop Protection 

 The in vitro metabolism of atrazine (ATZ) to its chlorometabolites, 

deisopropylatrazine (DIA), deethylatrazine (DEA), and diaminochlorotriazine (DACT) 

was investigated using cryopreserved hepatocytes from rat and human donors during a 

4-hour period. The initial concentrations of atrazine were 1.7, 1.0, or 0.5 μM. Samples 

were collected after 0, 5, 10, 20, 30, 45, 90, 120, 180, and 240 minutes of incubation at 

which time they were snap-frozen and sent for analysis of ATZ, DIA, DEA, and DACT.

 The hepatocyte suspension cultures were deemed acceptable for investigating the in 

vitro metabolism of atrazine based on confirmation of enzymatic activity and cell 

viability. The de-ethylation of 7-ethoxycoumarin was used as confirmation of enzymatic 

activity. The starting hepatocyte viability was > 70% for both rats and humans as 

required by the protocol but decreased to final average cell viability of 29% in rats and 

49% in humans. The rates of metabolism, however, were not affected by decreases in 

viability and/or hepatocyte enzyme activity.

 Atrazine was metabolized within 30 to 60 minutes concurrent with the appearance of

DEA and DIA. After approximately 30 to 45 minutes of incubation, the concentrations of 

DEA and DIA began declining concomitantly with the appearance of DACT 

progressively over the entire 4 hour incubation period. Rat hepatocytes metabolized 

atrazine preferentially to DIA, whereas human hepatocytes preferentially metabolized 

ATZ to DEA. Atrazine fell to non-detectable levels after 30 to 60 minutes, depending 

upon the initial concentration. The time to maximum levels (tmax) for DEA, DIA, and 

DACT was 20, 30, and 240 minutes in rat hepatocytes exposed to 0.5 μM atrazine, 

whereas with human hepatocytes, the corresponding times were 30, 30, and 180 

minutes, respectively. The experimental maximum levels (Cmax) for DEA, DIA, and 

DACT were 14, 36, and 27 ng/mL for rat hepatocytes, and the corresponding Cmax 

values for human hepatocytes were 50, 11, and 27 ng/mL, respectively. When adjusted 

for decreases in cell viability, the corresponding Cmax were 21, 58, and 127 ng/mL for 

rat hepatocytes and 80, 18, and 63 ng/mL, respectively, for human hepatocytes. The 

metabolism rates obtained from this in vitro study can be used to parameterize a PBPK 

model for atrazine and its chlorinated metabolites DEA, DIA, and DACT.

PBPK Model for Atrazine and its Chlorinated Metabolites

Jerry Campbell, Melvin Andersen, and Harvey Clewell, the Hamner Institutes for Health 

Sciences, Center for Human Health Assessment, Sponsored by Syngenta Crop 

Protection 

 Although the Agency has not performed a thorough review and evaluation of the 

model, a preliminary review indicates that the original McMullin 2007 model (McMullin et 

al. 2007) has been expanded to include compartments of suspected target tissues 

including mammary gland, hypothalamus, pituitary, testes/ovaries, and adrenals. The 

oral uptake of atrazine has also been refined by removing a saturable uptake 

description and replacing it with separate compartments for insoluble/bound and free 

fractions of atrazine as well as describing the potential slow release of bound atrazine. 
Page 202 of 210

New empirically determined tissue:blood partition coefficients and hepatic in vitro

metabolic rates were also incorporated. Most importantly, however, was the model 

calibration performed with the new in vivo time-intensive rat pharmacokinetic study 

described above where the chlorinated metabolites DEA, DIA, and DACT were 

individually monitored in female SD rats following dosing with atrazine via the oral route 

of administration (Coder 2011). The Agency will consider using the newly improved 

PBPK in the future when it has undergone a thorough review and evaluation process.

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 after Acute Prenatal Exposure to an Atrazine 

Metabolite Mixture in Female Long-Evans Rats. Environmental 

Health Perspectives. 115(4): 541-547.

Stanko, J., Enoch, R., Rayner, J., Davis, C., Wolf, D., Malarkey, D., 

and Fenton S. 2010. Effects of Prenatal Exposure to a Low Dose 

Atrazine Metabolite Mixture on Pubertal Timing and Prostate 

Development of Male Long Evans Rats. Reproductive Toxicology. 

30:540-549.

Two articles, Enoch et al. 2007 and Stanko et al. 2010, were produced from one study. 

The Enoch et al article focused on mammary gland development and Stanko et al. 2010 

focused on reproductive development of male rats following prenatal exposure to an 

atrazine metabolite mixture (AMM). 

The study was conducted in 2 replicate blocks with 5 treatment groups/block. Based on 

the results from these blocks, a third block was conducted to include a 180-day 

necropsy. In this study, the investigators dosed pregnant female Long-Evans rats (n≥6 

treatment/block) with atrazine or a mixture of atrazine and its metabolites from 

gestational day (GD) 15-19. Exposure was conducted on GD 15-19 because this 

dosing window is a critical period of development for both the mammary gland and 

prostate. 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 (positive control). 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 the Enoch et al. 2007 article is on 

development of the mammary gland following in utero exposure. The animals in the 

Stanko et al. 2010 article were the siblings of those in Enoch et al. 2007 and focused on 

pubertal timing and prostate development following in utero exposure. Reproductive 

organs were weighed and hormone measurements were taken. 

Enoch et al. 2007: 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 subjectively 
Page 203 of 210

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 affected; 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, increasing the AMM dose by 100-fold 

changes the response very little.

Stanko et al. 2010: Two male pups/dam from ≥5 dams were necropsied on PND 120 

in blocks one and two (n≥10) and on PND 120 and 180 in block three. For prostate 

evaluations, the ventral and lateral lobes of the prostate were processed by routine 

methods and embedded in paraffin block. The lobes were graded as a whole because 

the inflammation involved both the ventral prostate (VP) and lateral prostate (LP). 

Severity of inflammation was scored as follows: “1 - minimal multifocal interstitial 

infiltration of inflammatory cells involving less than 10% of the gland, 2 – multifocal to 

coalescing moderate interstitial inflammation affecting 10-25% of the gland, with or 

without focally marked accumulations of perivascular collections of inflammatory cells or 

mild fibrosis, 3 – multifocal, marked interstitial inflammation involving 25-75% of the 

gland, with or without focally marked accumulations of perivascular collections of 

inflammatory cells, moderate fibrosis, or sloughing and necrosis of the epithelial lining 

with intraluminal accumulations of neutrophils (micro-abscesses), or 4- diffuse and 

extensive interstitial inflammation involving > 75% of the gland often with interstitial 

fibrosis and occasional microabscesses.”
Page 204 of 210

No effect was seen on pup body weight of the AMM or ATZ treated groups compared to 

controls on PND 4 or 21. A slight statistically significant decrease in pup body weight 

was seen in the 0.09 mg AMM group (5.7%) on PND 120, but this is not considered to 

be biologically relevant. An 8-9% increase in pup body weight compared to controls 

was seen in the 0.87 and 8.73 AMM dose groups, respectively, on PND 180. AMM 

treated dam weights were not affected, however, there was a 61% decrease in maternal 

body weight gain in the 100 mg/kg/day atrazine dams during the dosing period. 

Preputial separation (PPS) was significantly delayed in male offspring by 0.8 days in the 

0.87 mg AMM (42.3 d) and 8.73 mg AMM (42.3 d) dose groups, and by 1.6 days in the 

atrazine group, (43.1 d) compared to controls (41.5 d), however, these marginal delays 

were not considered to be adverse since they were within ±2 day variation. PPS had not 

occurred by PND44 (a day randomly chosen based on the general window for PPS 

timing) in 4.5% of control males, ≥13% in the AMM treated groups, and 28% in the ATR 

treated group. All animals achieved PPS by PND51. Prolactin measures were not 

affected at PND 120 or 180 (no statistical significance). Serum testosterone levels were 

increased 60% in the 100 mg ATR group at PND 120 but decreased 60% in the 8.73

mg AMM (60%) at PND 180. Serum estradiol was decreased 33-41% at all dosed AMM 

groups and 40% in the 100 mg ATR, compared to controls. In 100 mg ATR, serum 

estrone was increased 46% at PND 120 but decreased 47% at PND 180. Despite the 

different hormone changes seen, all mean hormone concentrations were within the 

normal reported ranges.

At PND 120, anterior pituitary weight was significantly greater in 0.87 AMM (16%) and 

100 mg ATR (17%) males compared to controls. The mean ventral prostate weights of 

8.73 mg AMM were statistically decreased (13%) compared to controls at PND 120. No 

other reproductive tissue weights were affected at PND 120 or PND 180.

The study authors reported a significant increase in the percentage of males exhibiting 

lipomatous epididymal fat masses collected at necropsy at PND 120 and 180 at all dose 

levels. There was also a significant increase in the incidence of pale prostatic foci of 

discoloration noted in the males at necropsy at both 120 and 180 days at all dose 

levels.

There was a significant increase in the incidence of prostatitis and in the severity of 

prostate inflammation in all AMM dose groups (67-80%) and the atrazine dose group 

(81%) on PND 120 compared to controls (48%) (Table 3). The severity scores of 

affected males on PND 120 were greater in all AMM treatment groups than in the ATR 

exposed group. The incidence of prostatitis and severity scores decreased over time in 

all AMM dose groups and 100 mg ATR dose group and was diminished by PND 180 

(Table 4).
Page 205 of 210

There are a number issues with this study which require further discussion.

1. Study design (applicable to both Enoch and Stanko): When conducting a 

mixture study, one of the critical components is the ability to distinguish the effects of an 

individual chemical component to the effects of the mixture. Most often, investigators 

begin with a hypothesis of dose-additivity or response-additivity and subsequently test 

their hypothesis. No such hypothesis testing was performed here. Furthermore, the 

investigators failed to evaluate each of the individual components in separate doseresponse 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. 
Page 206 of 210

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, hydroxyatrazine (HA), in 

the AMM. HA is not a mammalian metabolite, but is instead an environmental 

degradate. 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 of the Enoch et al study, 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. 

Similarly, in the Stanko et al study, unless otherwise indicated, treated dams were 

utilized as the unit of measure for endpoints measured prior to PPS. Although not 

specifically stated, it follows that after PPS, statistics were done on the pups. It was 

difficult to track the number of pups in this study. For example, the data presented in 

Figure 4 for Day of PPS shows a potential pup loss in two groups when looking at the 

n’s in each group (pups n = 67, 47, 64, 66 and 40, for 0, 0.09 mg/kg AMM, 0.87 mg/kg 

AMM, 8.73 mg/kg AMM, and 100 mg/kg atrazine respectively). The study author did not 

report any sort of litter difference or pup loss to explain this decrease. 

3. Subjective 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. 
Page 207 of 210

Conclusion: The mixture study, as currently reported, is not sufficiently robust for use 

in setting regulatory endpoints. Overall, in both studies, either the dose-response 

relationships are weak and/or there are critical concerns with study design and data 

reporting/analysis, which are discussed above, which preclude meaningful conclusions. 

A.11 OTHER

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 
Page 208 of 210

another approach to block adrenocorticotrophic (ACTH) and pituitary adenylate cyclaseactivating 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 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 
Page 209 of 210

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 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 concentrationdependent 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. 
Page 210 of 210

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. Atrazinemediated 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.