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APPENDIX E
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APPENDIX E. TOXICOLOGY CHAPTER OF THE REREGISTRATION ELIGIBILITY 

DECISION.

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C. 20460

OFFICE OF 

PREVENTION, PESTICIDES AND

TOXIC SUBSTANCES

TXR NO. 0050644

MEMORANDUM Date: April 11, 2002

SUBJECT: Atrazine: Toxicology Chapter of the Reregistration Eligibility Decision. 

SECOND REVISION

Chemical number 080803. 

DP Barcode D272007

FROM: Linda L. Taylor, Ph.D.

Reregistration Branch I

Health Effects Division (7509C)

THROUGH: Steve Knizner, Branch Chief

Reregistration Branch III

Health Effects Division (7509C)

Whang Phang, Ph.D., Branch Senior Scientist

Reregistration Branch I

Health Effects Division (7509C)

TO: Cathy Eiden, Risk Assessor

Reregistration Branch III

Health Effects Division (7509C)

Pam Noyes, Chemical Review Manager

Special Review and Reregistration Division (7508C)

Attached is the revised Atrazine Toxicology Chapter of the Reregistration Eligibility 

Decision.
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Atrazine

PC Code: 080803

Toxicology Disciplinary Chapter for the Reregistration Eligibility Decision 

Document

Date completed: November 15, 2000; REVISED: January 18, 2001 and April 11, 2002

Prepared : Roger Hawks [Revised: Linda Taylor]

Health Effects Division

Office of Pesticide Programs

U.S. Environmental Protection Agency

form: FINAL 21 MAR-2000
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E. 1.0 HAZARD CHARACTERIZATION

Atrazine has low acute toxicity. Toxicity categories are III or IV by all routes of 

exposures (oral, dermal, inhalation, ocular) and atrazine is not a skin sensitizer. 

Atrazine has a low toxicity by the dermal route as indicated by the acute dermal toxicity 

study and by a 21-day dermal toxicity study using New Zealand white rabbits. This 21 

day study found a LOAEL at the limit dose of 1,000 mg/kg/day and had a NOAEL at 100 

mg/kg/day.

Numerous mutagenicity studies, both submitted guideline studies and open literature 

studies, are available. The weight of evidence does not support a mutagenic potential 

or concern for Atrazine.

The primary effects seen in rats following chronic exposure to atrazine are 

neuroendocrine perturbations and mammary and pituitary gland tumors. The primary 

underlying events that lead to mammary and pituitary tumor formation following atrazine 

exposure of Sprague-Dawley female rats involve disruption of the hypothalamicpituitary-ovarian axis. The neuroendocrine disruptions include alterations in 

hypothalamic neurotransmitter and neuropeptide levels. Decreases in serum levels of 

pituitary luteinizing hormone (LH) and prolactinare presumably a consequence of these 

hypothalamic alterations. Hypothalamic neuronal - GnRH pulse modulation of pituitary 

releases of LH is a central driver of ovulation in the SD female rat, and atrazine is 

essentially accelerating the aging process of the CNS control of ovulation. This leads to 

a constant state of estrus (anovulation) and prolonged exposure to estrogen and 

prolactin. Although, there are certain similarities in the control of the hypothalamicpituitary- ovarian axis between humans and rats in that the hypothalamus can play a 

key regulatory role in primates, there are fundamental differences. Although atrazine 

might cause adverse effects on hypothalamic-pituitary function in humans, the hormonal 

environment conducive to tumor development (i.e., elevated or prolonged exposure to 

estrogen or prolactin) that is found in SD female rats can not be established in humans. 

The Health Effects Division Cancer Assessment Review Committee [CARC] concluded 

that the neuroendocrine alterations in the SD rat constitute the mode of action for 

atrazine-associated carcinogenicity. CARC has classified atrazine as a "Not Likely To 

Be Carcinogenic To Humans”. 

These same neuroendocrine alterations, which constitute the mode of action [MOA] for 

cancer, could be expected to be associated with reproductive or developmental effects. 

Reproductive/developmental effects, including prostate inflammation in male rats whose 

mothers’ prolactin was suppressed due to treatment with atrazine while lactating, as 

well as delay of puberty in both male and female rats exposed prior to pubertal onset, 

have been demonstrated following atrazine exposure. 

Though special studies conducted at EPA labs indicate developmental/reproductive 

toxicity, guideline studies, for the most part, have not indicated 

developmental/reproductive toxicity. There was no increased quantitative or qualitative 

susceptibility in any of the guideline studies on atrazine in the rat [two developmental 

toxicity studies and a 2-generation reproduction study], and there was no increased 
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quantitative susceptibility in the rabbit developmental toxicity study. However, there was 

increased qualitative susceptibility in the rabbit study [increased resorptions (deaths) at 

a dose level that resulted in decreased body-weight gain and clinical signs in the 

maternal animal]. Studies in the open literature indicate increased qualitative 

susceptibility. Dosing of dams immediately following parturition [postnatal days 1-4] 

resulted in prostatitis in male offspring, and dosing of the young following weaning 

resulted in delayed puberty in both sexes. The mode of action for these two effects 

(prostate inflammation and delayed puberty) is believed to be similar to the mode of 

action described for atrazine-associated cancer and involves a central nervous system 

(CNS) toxicity - specifically, neurotransmitter and neuropeptide alterations at the level of 

the hypothalamus.

Atrazine is not readily absorbed through dermal exposure. Based on both a rat and a 

human dermal absorption study, HIARC recommended a dermal absorption factor of 

6%.

There is one datagap. A 28-day study via the inhalation route of exposure is required to 

assess the estrous cycle/LH surge in females for comparison with the 28-day oral 

exposure study. There are no other data gaps for atrazine according to the Subdivision 

F Guideline requirements. HIARC recommends, but does not require, that special 

studies examining atrazine's associations with delayed puberty and prostatitis in 

offspring of dams exposed shortly after parturition, be conducted. Should such studies 

be conducted, it is recommended that study protocols be approved by HED prior to 

commencement of any such study. In addition, HIARC recommends, but does not 

require, that special studies examining the CNS alterations following atrazine exposure 

be performed.

E.2.0 REQUIREMENTS

The requirements (CFR 158.340) for food use of atrazine are in Table 1.
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Table F- 1: The requirements (CFR 158.340) for food use of atrazine

Test Technical

Required Satisfied

870.1100 ................................. Acute Oral Toxicity

870.1200 ............................ Acute Dermal Toxicity

870.1300 ........................ Acute Inhalation Toxicity

870.2400 ..............................Primary Eye Irritation

870.2500 ........................ Primary Dermal Irritation

870.2600 .............................. Dermal Sensitization

yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

870.3100 .......................Oral Subchronic (Rodent)

870.3150 ...............Oral Subchronic (Non-Rodent)

870.3200 ....................................... 21-Day Dermal

870.3250 ....................................... 90-Day Dermal

870.3465 ................................... 90-Day Inhalation

yes

yes

yes

no

no

yes

yesa

yes

-

yesb

870.3700a Developmental Toxicity (Rodent).....

870.3700b Developmental Toxicity (Nonrodent) .............................................

870.3800 .................... 2-Generation Reproduction

yes

yes

yes

yes

yes

yes

870.4100a Chronic Toxicity (Rodent) ................

870.4100b Chronic Toxicity (Non-rodent)..........

870.4200a Oncogenicity (Rat) ...........................

870.4200b Oncogenicity (Mouse)......................

870.4300 ............................. Chronic/Oncogenicity

yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

870.5100 Mutagenicity—Gene Mutation - bacterial

870.5375Mutagenicity—Structural Chromosomal Ab

870.5550 ..Mutagenicity—Other Genotoxic Effects

yes

yes

yes

yes

yes

yes

870.6100a Acute Delayed Neurotox. (Hen) .......

870.6100b 90-Day Neurotoxicity Hen)...............

870.6200a Acute Neurotox. Screening Battery 

(Rat).................................................

870.6200b 90 Day Neuro. Screening Battery 

(Rat).................................................

870.6300 ...................................... Develop. Neuro

no

no

no

no

no

-

-

no

no

no

870.7485 ............................... General Metabolism

870.7600 ................................ Dermal Penetration

yes

yes

yes

yes

a Requirement waived because a chronic study in the non-rodent (i.e. - dog) is available b 28-day inhalation study in female SD rats to assess the estrous cycle/LH surge for 

comparison with the 28-day oral exposure study
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E.3.0 DATA GAPS

Status of previous datagaps

The previous Toxicology RED chapter for atrazine (TXR 006937) completed in 

1988, identified several datagaps. Table two displays those data gaps and the studies 

that have been submitted to fill these datagaps. All datagaps described in the previous 

Toxicology RED chapter have been satisfied.

Table F- 2: Data Gaps Listed in 1988 RED Document and Studies That Have Been Submitted to 

Fill these Data Gaps.

1988 Data Gap Results of Submitted Study MRID No(s).

81-3, Acute Inhalation Study Acceptable-Data Gap 

Filled

42089901, 

43016502

82-2, Subchronic Dermal Study Acceptable-Data Gap 

Filled

42089902

84-2, Mutagenicity -

a. Dominant Lethal Assay

b. Direct DNA Damage

Studies Acceptable-Data Gaps 

Filled

426370003 

 and

42547105

Current datagaps

Previously, there were no datagaps for atrazine or the atrazine metabolites 

listed in this document, with the exception of DACT. The HIARC previously required that 

a OPPTS Series 870.3800 Reproduction and Fertility Effects study be performed with 

the atrazine metabolite diaminochlorotriazine (DACT) - a mammalian metabolite of both 

atrazine and simazine. Subsequently, a re-evaluation of the data by the HIARC 

determined that the OPPTS 870.3800 study was not required for DACT [TXR No. 

0050592]. 

Additionally, it was determined by the HIARC that a 28-day study via the inhalation 

route of exposure is required for atrazine to assess the estrous cycle/LH surge in 

females for comparison with the 28-day oral exposure study. There are no other data 

gaps for atrazine according to the Subdivision F Guideline requirements. HIARC 

recommends, but does not require, that special studies examining atrazine's 

associations with delayed puberty and prostatitis in offspring of dams exposed shortly 

after parturition, be conducted. Should such studies be conducted, it is recommended 

that study protocols be approved by HED prior to commencement of any such study. In 

addition, HIARC recommends, but does not require, that special studies examining the 

CNS alterations following atrazine exposure be performed.
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E.4.0 HAZARD ASSESSMENT

E.4.1 Acute Toxicity

Adequacy of data base for acute toxicity: The data base for acute toxicity is 

considered complete. No additional studies are required at this time.

The acute toxicity data on atrazine technical is summarized below in Table 3.

Table 3: Acute Toxicity Data of Technical Atrazine

Guideline 

No.

Test Results Toxicity 

Category

870.1000 Oral LD50 - rat LD50 > 1,869 mg/kg (M&F 

combined)

 III

870.1200 Dermal LD50 - rat LD50 > 2,000 mg/kg (M&F 

combined)

 III

870.1300 Inhalation LC50 -

rat

LC50 > 5.8 mg/L (M&F combined) IV

870.1400 Eye Irritation -

rabbit

PISa = 0.0/110 IV

870.1500 Dermal Irritation -

rabbit

PISa = 0.2/8.0 IV

870.1600 Dermal 

Sensitization -

guinea pig

Non-sensitizing ---

870.6100 Acute Delayed 

Neurotoxicity

Not Required ---

a PIS=Primary Irritation Score

870.1100. Acute oral toxicity - rats

A pair of acute LD50 studies in the rat using technical grade atrazine have 

shown the LD50 to be: 1,869 mg/kg (Accession No. 230303; MRID 00024706) and 2,850 

mg/kg (MRID 00027097). Numbers in all these studies are for both sexes combined. 

The toxicity category for acute oral exposure is at least III. 

870.1200. Acute dermal toxicity - rat and rabbit

Two dermal LD50 studies using the rat resulted in LD50s of >2,000 mg/kg and > 

3,100 mg/kg for both sexes combined (Accession Nos. 00027097 and 230303; MRID 

00024708). A study using rabbits, the preferred species in the OPPTS Series 870 

guidelines, resulted in an LD50 of 7,550 mg/kg for both sexes combined (Accession No. 

231466). Atrazine is at least toxicity category III for dermal exposure. 
Page 9 of 90

870.1300. Acute inhalation toxicity - rats

The inhalation LC50 was found to be 5.82 mg/L (MRIDs 42089901 and 

43016502). This exceeds the limit dose of 5.0 mg/L and places atrazine in toxicity 

category IV. 

870.2400. Primary ocular irritation - rabbits

Atrazine tested negative for primary eye irritation and has been placed in 

toxicity category IV for this endpoint (Accession No. 230303; MRID 00024709).

870.2500. Primary dermal irritation - rats

Atrazine tested negative for primary skin irritation, and has been placed in 

toxicity category IV for this endpoint (Accession No. 230303; MRID 00024710).

870.2600. Dermal sensitization - guinea pigs

Atrazine has been shown to be non-sensitizing in dermal sensitization studies 

(MRID 00105131). 

870.6100. Acute and delayed type neurotoxicity of organophosphorus substances

Acute neurotoxicity studies have not been performed on atrazine, and none are 

required. 

E.4.2 Subchronic Toxicity

Adequacy of data base for subchronic toxicity: The data base for subchronic 

toxicity is considered complete. No additional studies are required at this time. 

870.3100 90-Day Oral Toxicity - Rat

In a subchronic oral toxicity study (MRID 44723701), Atrazine Technical (purity 

97.1 %) was administered in the diet to Tif: RAIf (SPF), RII/1 x RII/2 hybrids, (SpragueDawley derived) rats 10/sex/dose at dose levels of 0, 10, 50, or 500 ppm for 92 days. 

These doses were equivalent to 0, 0.6, 3.3, and 34.0 mg/kg/day for males, and 0, 0.659, 

3.35, and 35.3 mg/kg/day for females. Rats were treated for 13 weeks, then sacrificed 

(S1). Additional recovery groups of 10 rats/sex/dose were fed 0 or 500 ppm for 13 

weeks, remained untreated for 4 weeks, and were then sacrificed (S2).

One female in the control group died during the study. No treatment-related clinical 

effects were noted. Treatment with atrazine technical resulted in significant (p<0.01) 

decreases in body weight in high dose males and females compared to controls. Body 

weight losses were partially regained during the recovery period. Hematological and 

clinical chemistry parameters showed no significant treatment related effects. 

Decreased liver and kidney weights in high dose males occurred as a result of the 
Page 10 of 90

marked body weight loss. There were no treatment-related effects on gross pathology 

or ophthalmology. Hemosiderin pigment was found in the spleen at an increased 

incidence and severity in high dose animals of both sexes, at the end of treatment and 

recovery periods. 

As tested in this study, the LOAEL for Atrazine is 34.5 mg/kg/day, based on 

decreased body weight. The NOAEL is 3.3 mg/kg/day.

This subchronic oral toxicity study is classified as Acceptable /Guideline and satisfies 

the guideline requirement for a subchronic oral study (OPPTS 870.3100) in rats.

870.3150 90-Day Oral Toxicity - Dog

Study has been waived because an acceptable chronic study is available.

870.3200 21-Day Dermal Toxicity - Rabbit

A 21 day dermal study (MRID 42089902) in New Zealand White rabbits was 

conducted. Rabbits, 5 rabbits/sex/dose, were administered atrazine (97.6%) 6 hrs per 

day for 25 days at concentrations of 0, 10, 100 or 1000 mg/kg/day. 

Systemic toxicity was observed at the highest dose tested and consisted of reductions 

in food consumption (decreased 28% for males and 31% for females at day 21 vs

controls) and mean body weight (12% at day 25 vs controls for males and 19% for 

females). Percent body weight gain was decreased (-4.4% vs +9.5 at day 21 for males 

and -11% vs +8% for females). Compared to controls, relative and absolute spleen

weights in both sexes were increased (males had an 81% absolute increase and 75% 

increase relative to body weight; females had an 86% increase absolutely and 94% 

relative to body weight). 

Additional findings were reduced red blood cell and hematocrits counts (each 

decreased by 12% vs controls), and increased cholesterol and triglyceride levels 

(increased by 101 and 55% vs controls) in the highest dose tested females and reduced 

total protein and chloride levels (decreased by 7 and 5% vs controls) in the high-dose 

males. Dermal findings were: limited- to- slight acanthosis for both sexes at all dose 

levels; minimal-to-moderate acanthosis in 3 high-dose females (compared to zero 

control females having this finding); and 3 cases of focal subacute inflammation in the 

treated skin of high-dose females (compared to zero cases of this in the controls). The 

NOAEL is 100 mg/kg/day and the LOAEL at 1000 mg/kg/day based on reductions in 

food consumption, mean body weight, body weight gain and increases in spleen weight.

The LOAEL is 1000 mg/kg/day, based on reduced food consumption, mean body 

weight and body weight gain, increased relative and absolute spleen weights, and 

reduced red blood cell counts and hematocrits. The NOAEL is 100 mg/kg/day.

This study is classified Acceptable-Guideline and satisfies the OPPTS 870.3200 

guidelines for a 21 day dermal toxicity study in the rabbit. 
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E.4.3 Prenatal Developmental Toxicity

Adequacy of data base for Prenatal Developmental Toxicity: The data base for 

prenatal developmental toxicity is considered complete. No additional studies are 

required at this time.

870.3700 Prenatal Developmental Toxicity Study - Rat (1989 study)

In a developmental toxicity study (MRID 41065201), atrazine (97.6%) was 

administered by gavage to 104 mated female Sprague-Dawley rats, 26/dose, at dose 

levels of 0, 5, 25, 100 mg/kg/day from days 6 through 15 of gestation. 

Maternal toxicity findings were almost exclusively confined to the high dose group. 

Compared to controls, high dose dams displayed: reduced food consumption 

(decreased 13%, p < 0.5); reduced total body weight gain (reduced 18% during dosing 

period, p < 0.5); reduced corrected (minus uterine weight) weight gain (reduced 20% for 

entire gestation, p < 0.5); and increased alopecia 1/26 controls vs 5/26 high dose). One 

high dose animal died on gestation day 20 and salivation was noted as an observation 

in 18/26 high dose animals. The only observations seen outside the high dose group 

were: an abortion from one of the mid-dose animals on gestation day 19; a fluid-filled 

hollow right kidney in a mid-dose animal; and hollow discolored kidneys in a low dose 

animal.

The maternal LOAEL is 100 mg/kg/day, based on reduced body weight gain and 

food consumption. The maternal NOAEL is 25 mg/kg/day.

The few malformations seen upon external examination of the fetuses were seen only in 

the control groups and clearly could not be compound related. Likewise, there was no 

increased incidence of visceral malformation in dosed groups vs control groups. There 

were no skeletal malformations observed but there was an increased incidence of 

incomplete ossification of various bones in the highest dose tested. Hyoids (control fetal 

incidence of 11% vs 21.7% at high dose), occipitals (7.7% vs 21.1%) and parietals ( 

2.2% vs 8.4%) showed incomplete ossification. There was also an increased incidence 

(p < 0.05) of incomplete ossification of the interparietals in all dose groups compared to 

controls. 

Fetal body weight, number of resorptions and implantations, and live fetuses/litter were 

not significantly affected by atrazine treatment. Exposure of gravid Sprague-Dawley rats 

to atrazine under the conditions described in this study seemed to have few 

embryo/fetotoxic effects.

The developmental LOAEL is 100 mg/kg/day, based on increased incidence of 

delayed ossification of skull bones. The developmental NOAEL is 25 mg/kg/day.

The developmental toxicity study (MRID 41065201) in the rat is classified AcceptableGuideline and satisfies the guideline requirement for a developmental toxicity study 

(OPPTS 870.3700) in the rat.

870.3700 Prenatal Developmental toxicity - Rat (1984 study)
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In a developmental toxicity study (MRID 00143008 and 40566302), atrazine 

(96.7%) was administered to 104 Charles River CD rats 27/dose by gastric intubation at 

dose levels of 0, 10, 70, or 700 mg/kg/day from days 6 through 15 of gestation. 

Mortality was very high for the 700 mg/kg/day animals in this study. All but 6 of the 27 

females in this group died during the gestation period. Other statistically significant 

findings in this group included: salivation; oral and nasal discharge; ptosis; swollen 

abdomens; blood on the vulva; enlarged stomachs and adrenal; and discolored lungs. 

Body weight gains were statistically significantly reduced throughout most of the 

gestation period. Significant reductions in food consumption were observed. Although 

mortality was quite high in the high dose group, pregnancy rates were comparable to 

controls (85.2% for controls vs 96.3% high dose) and the numbers of live fetuses at csection was comparable to controls (mean of 12.7 per litter for controls vs 13.4 for the 

high dose). 

There were few findings in either the low or mid dose animals. Alopecia was observed 

in mid dose animals, but was not considered to be biologically significant. Body weight 

gain in the mid dose group was significantly reduced for the first 5 days of compound 

exposure (gestation days 6-10). Food consumption in the intermediate group was 

reduced, but only at day 17 of gestation. Low dose body weight gain was increased on 

days 6-10 of gestation and food consumption was increased on gestation day 9.

The maternal LOAEL is 70 mg/kg/day, based on reduced body weight gain. The 

maternal NOAEL is 10 mg/kg/day.

Fetal weights were statistically significantly reduced in the high dose group. Skeletal 

examinations were not conducted in the high dose group due to the extremely low fetal 

weights. Visceral and external examinations were conducted, though, and no group, 

including the high dose group, displayed any findings significantly different from control 

values. Skeletal anomalies, however, were observed in the mid dose group.

The developmental LOAEL was found to be 70 mg/kg/day, based on delayed or no 

ossification at several sites. The developmental NOAEL is 10 mg/kg/day.

The developmental toxicity study in the rat is classified Acceptable-Guideline and 

satisfies the OPPTS 870.3700 guideline requirement for a developmental toxicity study 

in rats.

870.3700 Prenatal Developmental Toxicity Study - Rabbit

In a developmental toxicity study (Accession No. 254979 and MRIDs 

00143006, 40566301), atrazine (96.3%) was administered by gavage to 76 mated 

female New Zealand White rabbits, 19/dose, at dose levels of 0, 1, 5, or 75 mg/kg/day, 

from days 7 through 19 of gestation. 

Clinical signs seen in 75 mg/kg/day (highest dose tested) animals that were considered 

to be related to compound treatment were stool changes (none, little or soft stool; 9/19 

controls vs 19/19 at high dose), and the appearance of blood in the cage or on the vulva 
Page 13 of 90

(0/19 controls vs 4/19 at high dose). Body weight gain was reduced in high dose dams 

and, at many time points, body weight was below day zero values. At gestation days 14, 

19, 21 and 25, mean maternal body weights were 12%, 19%, 18%, and 10% below 

control values (p<0.01 for all four of these time points). 

High dose animals displayed significantly reduced food consumption during treatment. 

During gestation days 12 to 17, the average feed consumption at the high dose was 

only 1-6 grams of feed per animal per day compared to 175-182 grams for the controls. 

The mid and low dose groups had no alterations that could be attributed to atrazine 

exposure.

The maternal toxicity LOAEL is 75 mg/kg/day, based on decreased body weight, 

food consumption and increased incidence of clinical signs. The maternal toxicity 

NOAEL is 5 mg/kg/day.

Fetal effects were: increased resorptions - mean of 1.3/dam in controls vs 4.8/dam at 

the high dose (p<0.01), reduced live fetuses per litter - mean of 8.8/dam in controls vs

5.9/dam at the high dose (p<0.05), and increased delayed ossification of appendicular 

elements were observed in the high dose group. 

The low and intermediate groups had no fetal findings that could be attributed to 

compound exposure. 

The findings in the high dose group were determined to be secondary to maternal 

toxicity and thus the LOAEL and NOAEL for embryo/fetotoxicity match the maternal 

LOAEL and NOAEL.

The developmental toxicity LOAEL is 75 mg/kg/day, based on reduced litter size, 

increased resorptions and delayed ossification. The developmental toxicity 

NOAEL is 5 mg/kg/day

The study is classified Acceptable-Guideline and satisfies the guideline 

requirement for a developmental toxicity study (OPPTS 870.3700) in rabbits.

E.4.4 Reproductive Toxicity

Adequacy of data base for Reproductive Toxicity: The database for reproductive toxicity 

is considered complete. There is an acceptable multi-generation reproduction study 

available for atrazine. No additional studies are required at this time.

870.3800 Reproduction and Fertility Effects - Rat

In a 2-generation reproduction study (MRID 40431303), atrazine (purity not 

specified but said to be technical grade) was administered to 240 Charles River (CRCD, 

VAF/PLUS) rats 30/sex/dose in the diet at dose levels of 0, 10, 50, and 500 ppm. 

There was very little variation in test article consumption between generations; the F0 

and F1 males had similar test article consumption during the 70-day premating period as 
Page 14 of 90

did the F0 and F1 females. The average values for the two generations are 0, 0.75, 3.78, 

39.0 mg/kg/day for males and 0, 0.86, 3.70, 42.8 mg/kg/day for females. Test article 

consumption for the F0 and F1 generation females during their gestation period did not 

vary greatly between generation. Mean compound consumption for both generations 

were 0, 0.66, 3.33 and 35.43 mg/kg/day.

Parental body weights, body weight gain, and food consumption were statistically 

significantly reduced at the 500 ppm dose (highest dose tested) in both sexes and both 

generations throughout the study. Compared to controls, body weights for F0 high-dose 

males and females at 70 days into the study were decreased by 12% and 15%, 

respectively while F1 body weight for the same time period was decreased by 15% and 

13% for males and females, respectively. The only other parental effect that may have 

been treatment related was a slight, but statistically significant, increase in relative 

testes weight, which occurred in both generations at the high dose. There did not 

appear to be any reproductive effects from compound exposure. Measured reproductive 

parameters from both generations did not appear to be altered in a dose-related 

manner.

The LOAEL is 500 ppm (39 mg/kg/day in males, 42.8 mg/kg/day in females) based 

on decreased body weights, body weight gains and food consumption. The 

NOAEL is 50 ppm(3.78 mg/kg/day in males, 3.7 mg/kg/day in females)

The developmental toxicity LOAEL is 39 mg/kg/day in males, 42.8 mg/kg/day in 

females, based on decreased body weights in both generations of males at 

postnatal day 21. The developmental toxicity NOAEL is 3.78 mg/kg/day in males 

and 3.7 mg/kg/day in females.

This study is classified Acceptable-Guideline and satisfies the guideline requirement 

(OPPTS 870.3800) for a 2-generation reproductive study in the rat.

E.4.5 Chronic Toxicity and Carcinogenicity 

Adequacy of data base for chronic toxicity: The data base for chronic toxicity is 

considered complete. No additional studies are required at this time. 

870.4100 (870.4300) Combined Chronic Toxicity/Carcinogenicity – Rat (SpragueDawley)

In a combined 2-year feeding/oncogenicity study (MRID 40629302), atrazine 

(95.9% +/-2.3%) was administered to Sprague-Dawley rats, 20/sex/dose, in the diet, at 

dose levels of 0, 10, 70, 500, and 1000 ppm, (0, 0.5, 3.5 25, 50 mg/kg/day) for 2 years. 

Toxicologic alteration appeared to be confined to the 500 and 1000 ppm dose groups. 

25 mg/kg/day: Both male and female rats showed reductions in body weight gain, and 

food consumption. Body weight for males and females at 104 weeks was 8% and 19% 

below 104 week control values. Food consumption at 104 weeks was not significantly 

below control, but at weeks 1, 13 and 26 for males and week 1 for females, food 

consumption was statistically- significantly reduced. Food consumption was reduced 
Page 15 of 90

11% at week 1, 11% at week 13 and 9% at week 26 in males and 8% at week 1 in 

females. Males displayed no unusual histopathology findings in this group, but females 

had statistically-significant increases of myeloid hyperplasia in the bone marrow of the 

femur (25 control incidences vs 38) and sternum (21 vs 33), and splenic extra medullary 

hematopoiesis (12 vs 22).

50 mg/kg/day: Both sexes showed reductions in body weight gain (19% vs controls at 

104 weeks for males, 27% at 104 weeks for females)and food consumption (reduced vs

controls 20%, 16%, 14% and 11% at weeks 1, 13, 26 and 52 for males and 18%, 

8%,and 7% at weeks 1, 13, and 26 for females). Female survival was 50% at 104 

weeks in controls and 26% in 1000 ppm animals. Interestingly, survival was increased 

in 1000 ppm males - 44% control survival vs 67% 1000 ppm at 104 weeks. Altered 

hematology and clinical chemistry findings were noted in females and included: 

decreased hemoglobin concentration; hematocrit; RBC; and serum glucose. Males did 

not display these alterations but did display decreased serum triglyceride levels 

throughout the course of the study. Organ-to-body weight ratios were decreased in high 

dose animals which may have been the result of decreases in body weight gain. 

Histopathology findings in females dosed 1000 ppm consisted of statistically-significant 

increases in incidences of (% increase vs controls in parenthesis): retinal degeneration 

(83%); centrilobular necrosis in the liver (300%); degeneration of the rectus-femoris 

muscle (160%); transitional epithelial hyperplasia in the bladder (150%) and the kidney 

(82%); splenic extra medullary hematopoiesis (133%); and myeloid hyperplasia in both 

the femur (108%) and sternum bone marrow (119%). Histopathology findings in males 

dosed 1000 ppm consisted of statistically-significant increases in incidences of: 

degeneration rectus femoris muscle (366%); prostate epithelial hyperplasia (141%); 

calculi in the kidney pelvis(106%); and acinar hyperplasia of the mammary gland 

(200%). 

A carcinogenicity study run concurrently with the toxicity study determined that there 

was a dose-related increase in mammary adenocarcinomas (p value for the trend < 

0.00005) in 70, 500 and 1000 ppm females. Adenocarcinomas incidences were: 

controls -15/66; 70 ppm - 26/68; 500 ppm - 27/65; 1000 ppm - 35/64.

The dose response curve in this study appeared to be adequate as the low doses (10 

and 70 ppm) showed few toxic effects; the mid-dose level showed some toxic effects 

(reduced body weight gain and food consumption); while the high dose showed the 

same effects as the mid dose plus hematology/clinical chemistry and histopathology 

findings. 

The LOAEL for non-cancer effects is 500 ppm (25 mg/kg/day), based on reduced 

body weight gain and food consumption. The NOAEL is 70 ppm (3.5 mg/kg/day). 

Mammary tumors [adenocarcinomas] were observed at dose levels of 70 ppm (3.5

mg/kg/day) and above.

This chronic toxicity/carcinogenicity study in the rat is classified Acceptable-Guideline

and satisfies the guideline requirement (870.4300) for a combined chronic 

oral/carcinogenicity study in the rat. 
Page 16 of 90

NOTE: Please refer to Hazard Assessment and Review of Available Studies [Part 

B] of the May 22, 2000 PRELIMINARY DRAFT Hazard and Dose-Response 

Assessment and Characterization - ATRAZINE, presented to the FIFRA Scientific 

Advisory Panel [SAP] on June 27-29, 2000 for a summary and details of this study 

as well as the other long-tern studies in SD rats (see 

http://www.epa.gov/scipoly/sap/2000/june27/finalparta_atz.pdf ).

870.4100 (870.4300) Combined Chronic Toxicity/Carcinogenicity – Rat (F-344)

In a chronic toxicity/carcinogenicity toxicity study (MRID 42227001), atrazine 

[97.0%) was administered to 600 Fischer- 344 rats, 60/sex/dose in the diet at dose 

levels of 0, 10, 70, 200, 400 ppm (0, 0.49, 3.43, 9.87 and 20.17 mg/kg/day for males 

and 0, 0.61, 4.35, 12.71, and 26.18 mg/kg/day for females) for 104 weeks. 

Administration of atrazine did not affect animal survival nor, with the exception of 

thinness in the high-dose group, were any clinical signs apparent. Body weight and 

body weight gain were significantly reduced in 200 and 400 ppm exposed animals of 

both sexes throughout most of the study. Food consumption in the high dose group 

males was significantly reduced throughout the study, and significantly reduced in high 

dose females for the first 13 weeks of exposure. No findings were seen at necropsy 

which could be attributed to compound exposure. There were no significant increases in 

either neoplastic or non-neoplastic findings which could be attributed to compound 

exposure. 

The LOAEL is 3.9 mg/kg/day based on decreased body weight gain. The NOAEL 

is 0.55 mg/kg/day.

At the doses tested, there was no treatment-related increase in tumor incidence when 

compared to controls. Dosing was considered adequate based on decreased body 

weight gain.

This chronic toxicity/carcinogenicity study in the Fischer 344 rat is AcceptableGuideline and satisfies the guideline requirement for a chronic toxicity/carcinogenicity 

study (OPPTS 870.4300) in the rat.

NOTE: Please refer to Hazard Assessment and Review of Available Studies [Part 

B] of the May 22, 2000 PRELIMINARY DRAFT Hazard and Dose-Response 

Assessment and Characterization - ATRAZINE, presented to the FIFRA Scientific 

Advisory Panel [SAP] on June 27-29, 2000 for a summary and details of this study 

as well as the other long-tern studies in F-344 rats (see 

http://www.epa.gov/scipoly/sap/2000/june27/finalparta_atz.pdf ).

870.4100b Chronic Toxicity - Dog

In a chronic toxicity study (MRID 40431301), atrazine (97% a.i.) was 

administered to 40 Beagle dogs, 4/sex in the low and intermediate dose groups and 

6/sex in the control and high dose groups, in the diet at dose levels of 0, 15, 150 and 

1000 ppm for 52 weeks. The mean daily dose was 0, 0.48, 4.97, 33.65 mg/kg for males. 
Page 17 of 90

Female doses were the same for the low and mid dose and only slightly different [33.80 

mg/kg/day] in the high dose group. 

Percent increases in body weight during the course of the study were as follows: male: 

46.6% in control; 52.5% [low dose]; 36.8% [mid dose] and 29.5% [high dose]; female: 

controls; 44.4% [low dose]; 35.9% [mid dose]; and 21.5% [high dose]. Only for the highdose males was this increase statistically significant compared to the control increase. 

Although only statistically-significant for the first quarter, high-dose males and females 

showed reduced food consumption throughout the course of the study. By studies’ end, 

mean food consumption was decreased 22% in males and 20% in females. 

High dose animals recorded statistically-significant changes in organ weights for both 

sexes compared to controls. Absolute heart weight was decreased in females by 14% 

and male liver weight relative to body weight was increased by 47%. 

The major treatment-related findings in this study were cardiac findings. EKG alterations 

such as tachycardia and increased P wave amplitude (P-II), were observed at many 

timepoints during the study in high-dose animals. Mean heart rate was increased 59% 

(p<0.05) and P-II was decreased 78% (p<0.01) on day 361 in high-dose males 

compared to controls. Female heart rates at the same time point were increased 46% 

which was not statistically significant, but P-II was decreased 71% which was significant 

at p<0.01. Gross pathology findings were also seen in the heart and were mostly 

concentrated in the high-dose group. Minimal atrial dilation seen in 3/6 males and 2/6 

females was the only cardiac finding seen at necropsy in the control animals. The highdose group, on the other hand, displayed moderate to severe atrial dilation in 4/5 males 

and 5/5 females who survived until scheduled sacrifice. Two of 5 high-dose males who 

survived until scheduled sacrifice also displayed enlarged, soft hearts and 1/5 had fluid 

in the pericardium. 

Histopathology findings were also seen in the heart. Three of 6 high-dose males and 6/6 

high-dose females were found to have cardiac myolysis while no control animals of 

either sex had myolysis. Focal atrophy of the heart was seen in 2/6 high-dose males 

and 5/6 high-dose females while no controls of either sex displayed this finding.

Three animals, two high-dose [male on day 250; female on day 113] and one mid-dose 

male [day 75], were sacrificed in moribund condition during the course of the study. The 

two high dose sacrifices may have been due to compound-related cardiac dysfunction.

The LOAEL for both sexes is 1000 ppm (33.65 mg/kg/day for males and 33.8 for 

females), based on cardiac effects. The NOAEL is 150 (4.97 mg/kg/day) for both 

sexes.

The major finding of this study was cardiopathology. A broad array of cardiac 

dysfunctions seen in the high dose group such as: electrocardiography alterations; the 

appearance of both microscopic and macroscopic cardiac abnormalities, were 

determined to be due to atrazine exposure. The wide variety of effects seen, as well as 

possible compound-related animal deaths, indicate the severity of the cardiac effects. 
Page 18 of 90

This chronic toxicity study is Acceptable-Guideline and satisfies the guideline 

requirement (OPPTS 870.4100) for a chronic oral toxicity study in dogs.

870.4200 Carcinogenicity Study - rat

See above under "870.4100 Chronic Toxicity – rat" for Executive Summary. 

870.4200 Carcinogenicity - mouse

In an carcinogenicity study (MRID 40431302), atrazine (purity not given) was 

administered to CD-1 mice, 59-60/sex/dose, in the diet at dose levels of 0, 10, 300, 

1500 and 3000 ppm (male/female mean daily dose 0/0, 1.4/1.6, 38.4/47.9, 194.0/246.9, 

385.7/482.7 mg/kg/day) for 91 weeks. Female mice in the 300, 1500 and 3000 ppm 

groups received a daily atrazine dose about 25% higher than their counterpart males. 

No dose-related increases in neoplasms were observed. The dose response curve 

seemed adequate since toxic effects, such as a decrease in mean body weight of both 

sexes and an increase in cardiac thrombi in the females, are seen at both 1500 and 

3000 ppm, while no dose-related toxic effects are seen at 10 and 300 ppm. In addition 

to the toxic effects just mentioned, the 3000 ppm animals of both sexes also displayed 

decreases in food consumption and decreases in RBC, hematocrit, and hemoglobin 

concentration. Female mice, but not males, at 3000 ppm showed decreased mean 

group brain and kidney weights and decreased percentages of neutrophils and 

lymphocytes. There was also an increase in mortality (p < 0.05) in 3000 ppm females, 

but not males, with only 25% of the females surviving vs 39-43% of the females 

surviving in the other female dose groups.

The cardiac thrombi found at both 1500 and 3000 ppm may have contributed to 

unscheduled female deaths during the course of the study. The incidence of 

unscheduled death in mice with cardiac thrombi is statistically significantly different from 

the incidence of unscheduled death in mice from control groups. The occurrence of 

cardiac thrombi is considered a severe effect.

The LOAEL is 222.0 mg/kg/day, based on decreased body weight gain in both 

sexes and increased cardiac thrombi in the females. The NOAEL is 43 mg/kg/day.

At the doses tested, there was not a treatment-related increase in tumor incidence when 

compared to controls. Dosing was considered adequate due to the occurrence of 

decreased body weight gain and cardiac thrombi.

This carcinogenicity study in the mouse is Acceptable-Guideline, and it satisfies the 

guideline requirement for a carcinogenicity study in the mouse (OPPTS 870. 4200).

E.4.6 Mutagenicity

Adequacy of data base for Mutagenicity: The data base for Mutagenicity is considered 

adequate based on current and pre -1991 mutagenicity guidelines. No additional studies 

are required at this time.
Page 19 of 90

Numerous open literature mutagenicity studies are available for evaluation. 

The entire mutagenicity database for atrazine (including these open literature studies) 

has been reviewed by CARC (October 13, 1999; April 12, 2000; May 22, 2000 

{http://www.epa.gov/scipoly/sap/ #june27}, October 19, 2000) and by the Science 

Advisory Panel (June, 28 - 30, 2000) and both panels stated that the available 

evidence did not indicate a mutagenic effect of atrazine exposure.

870.5100 Bacterial reverse mutation test

In a reverse gene mutation assay in bacteria (MRID 40246601), strains TA 98, 

100, 1535 and 1537 of S. typhimurium were exposed to atrazine (98.2% a.i.), in 

dimethylsufoxide, at concentrations of 0, 20, 78, 313, 1250, and 5000 μg/plate. Test 

were conducted in the presence and absence of mammalian metabolic activation S9 

fraction of Tif:RAIf rats treated with Aroclor 1254. 

Atrazine was tested up to the limit concentration, 5000 μg/plate. The positive controls 

did induce the appropriate responses in the corresponding strains. There was no 

evidence of induced mutant colonies over background.

This study is classified as Acceptable-Guideline. It does satisfy the guideline 

requirement (OPPTS 870.5100) for in vitro mutagenicity (bacterial reverse gene 

mutation) data.

870.5385 Mammalian Bone Marrow Chromosome Aberration Test 

A mouse bone marrow micronucleus test was conducted using Tif:MAGF mice 

(MRID 40722301). The test consisted of two parts. The first portion consisted of 24 

male and 24 female mice being dosed with 2250 mg/kg atrazine (98.2% a.i.). Eight 

animals of each sex were then sacrificed at 16, 24 or 48 hours following treatment. The 

second portion of the study 24 mice, 8/sex/dose, were treated with atrazine (98.2% a.i.) 

at doses of 562.5, 1175, 2250 mg/kg. Bone marrow cells were harvested at 24 hours 

post-treatment. The vehicle in both portions of the study was carboxymethyl cellulose. 

Exposure in both portions of the study was accomplished by a single gastric intubation. 

There were no signs of toxicity in either portion of the study. Atrazine was tested at an 

adequate doses being that these were doses that induced death in mice. The positive 

control induced the appropriate response. There was not a significant increase in the 

frequency of micro nucleated polychromatic erythrocytes in bone marrow after 

any treatment time or dose.

This study is classified as Acceptable-Guideline, and it satisfies the guideline 

requirement (OPPTS 870.5375) for in vivo cytogenetic mutagenicity data.

870.5450 Dominant Lethal Assay 

In a mouse dominant lethal assay (MRID 42637003), groups of 30 male Tif: 

MAGf (SPF) mice were treated orally by gavage with Atrazine technical (97.1% a.i., 

batch #SG8029BA10) at doses of 0, 500, 1000, 2000, or 2400 mg/kg body weight in a 

volume of 10 mL/kg. The vehicle was corn oil. Starting immediately after dosing, each 
Page 20 of 90

male was mated with 2 untreated females per interval for days 1-4, days 4-8, and days 

8-12. Each male was then mated with 2 untreated females per week for weeks three 

through eight.

Atrazine technical was tested at an adequate dose. There were signs of toxicity after 

dosing as evidenced by piloerection and decreased locomotor activity. The females 

were sacrificed on gestation day 13-15 and the uteri examined for the number of alive, 

early, and late dead embryos and resorptions. Cyclophosphamide served as the 

positive control.

There was no significant difference between the control group and treated groups 

with respect to post-implantation mortality of embryos. Under the conditions of 

this study atrazine technical did not induce dominant lethal mutations in male 

mice at doses as high as 2400 mg/kg.

This study is classified as Acceptable- Guideline, and it satisfies the guideline 

requirement (OPPTS 870.5450) for rodent dominant lethal data.

870.5550 UDS Assay 

In an unscheduled DNA synthesis assay (MRID 00161790/40722301) primary 

rat hepatocytes from adult male Tif:RAIf rats that were exposed to concentrations of 

atrazine of 0, 1.2, 6, 30, 150 μg/mL for five hours. No concentration induced UDS, 

including the high dose of 150 μg/mL, where precipitation of the test article 

occurred. 

This study is classified as Acceptable-Guideline, and it satisfies the requirement 

(OPPTS 870.5550) for other genotoxic mutagenicity data.

870.5550 UDS Assay 

In an unscheduled DNA synthesis assay (MRID 42547105), primary rat 

hepatocyte cultures were exposed to atrazine, (97.1% a.i.), in dimethyl sulfoxide at 

concentrations of 15, 46, 139, 417, 835, and 1670 μg/mL for 16-18 hours. 

Atrazine was tested up to precipitating concentrations, 139 μg/mL. The positive controls 

did induce the appropriate response. There was no evidence that unscheduled DNA 

synthesis, as determined by nuclear silver grain counts, was induced.

This study is classified as Acceptable-Guideline, and it satisfies the guideline 

requirement (OPPTS 870.5550) for other genotoxic mutagenicity data.

E.4.7 Neurotoxicity

Acute and subchronic neurotoxicity studies (OPPTS 870.6200) are not available for 

atrazine. Special studies submitted by the registrant (MRIDs 44152102 and 43934406) 
Page 21 of 90

and published in the open literature (Cooper, et al. 2000. Atrazine disrupts the 

hypothalamic control of pituitary- ovarian function. Tox. Sci. 53: 297-307 [MRID 

45166902]) provide evidence of atrazine-associated central nervous system (CNS) 

toxicity. The neurotoxicity seen in these studies was a CNS toxicity (specifically, 

neuroendocrine alterations at the hypothalamus). 

E.4.8 Metabolism

Adequacy of data base for metabolism: The data base for metabolism is considered to 

be complete. No additional studies are required at this time.

870.7485 Metabolism - Rat 

Several metabolism studies have been submitted to fulfill the OPPTS 870.7485 

guideline requirements. MRIDs 40431304; 40431305; 40431306; and 42165503 

together satisfy the metabolism guidelines. MRID 44713802, by itself, satisfies the 

OPPTS 870.7485 guideline requirements. These studies are summarized below.

In a metabolism study (MRID 40431304) [atrazine, chemical purity of unlabeled 

compound not given, radio-purity of labeled compound > 98%, all carbons on the 

triazine ring were labeled] was administered to 34 Sprague Dawley CD rats in four dose 

groups, 5/sex/dose, except controls which were 2 sex/dose. Three groups were dosed 

acutely at dose levels of 0, 1, and 100 mg/kg for a single dose given through oral 

gavage. A fourth dose group was dosed subchronically at 1.0 mg/kg/day for 15 days by 

oral gavage. 

Distribution, accumulation

Distribution was dose-dependent and independent of sex. In the 100 mg/kg group, the 

highest distribution of radiolabel was in red blood cells followed by the heart, spleen, 

lung and liver. In the group given 1 mg/kg for 15 days distribution was highest in the red 

blood cell, followed by the liver, spleen, kidney and lung. Distribution appeared to follow 

first-order kinetics and the half-life in the tissues was 31.3 hours. This indicates that 

atrazine does not bioaccumulate. 

Excretion

Approximately 95% of the atrazine was excreted within 7 days of dosing. The urinary 

route accounted for about 75% of the excretion while feces accounted for 20%. The 

route of excretion did not seem to vary among sexes or with dose. 

This metabolism study in the rat is classified Acceptable-Nonguideline and, when 

considered in conjunction with studies: MRID Nos.: 404313-05; -06; and 421655-03, 

satisfies the guideline requirement for a metabolism study (OPPTS 870.7485) in the rat. 

Only in conjunction with these four studies does MRID 404313-04 satisfy the 

metabolism guideline requirement.

In a metabolism study (MRID 40431305) Atrazine, 98.8% a.i., was labeled with 

14 C on all carbons of the triazine moiety, radiopurity 97.9%, and was administered to 14 

female Sprague-Dawley rats 2/dose. The animals were dosed daily through a stomach 

tube with dose levels of 0, 1, 3, 7, 10, 50 or 100 mg/kg/day. One animal from each 
Page 22 of 90

group was sacrificed 3 hours and the other was sacrificed 72 hours following the last 

dose. 

Distribution, accumulation

The location of 14 C label did not vary with dose but the amount distributed did vary with 

dose. Distribution was highest in the red blood cell, followed by the liver, ovary and 

kidney. When the dose increased the amount distributed in the tissues increased. The 

distribution appeared to follow first-order kinetics and the tissue half-life was 38.6 hours. 

This indicates that atrazine, with possible exception of the red blood cell, does not 

bioaccumulate.

This metabolism study in the rat is classified Acceptable-Nonguideline and, when 

considered in conjunction with studies: MRID Nos.: 404313-04; -06; and 421655-03, 

satisfies the guideline requirement for a metabolism study (OPPTS 870.7485) in the rat. 

Only in conjunction with these four studies does MRID 404313-04 satisfy the 

metabolism guideline requirement.

In a metabolism study (MRID 40431306) atrazine (purity of unlabeled 

compound not reported but radiolabeled purity >97%), 14C-labeled on all carbon of the 

triazine moiety, was administered to 13 female Sprague-Dawley rats. The test article 

was given through the stomach tube in a single oral dose. Five animals were given 100 

mg/kg and urine and fecal samples were collected 24, 48 and 72 hours later, after which 

the animals were sacrificed and 5 mL of blood and the liver were obtained. The 

remaining 8 animals were given between 16.18 and 19.64 mg/kg and urine was 

collected over a 24 hr period. The urine was analyzed for metabolites using 

anion/cation exchange chromatography or reverse phase HPLC followed by infrared 

and mass spectrometry.

Excretion

In the rats given 100 mg/kg, 100% of the administered radioactivity was accounted for 

within 3 days of dosing. Urine was found to contain 47.3% of the radioactivity and the 

feces 49.3%. The tissues contained 5.75% and 1.4% was found in the blood. 

Metabolism

Metabolites detected from the 8 rats given 16.18-10.64 mg/kg indicated that 

dechlorination of the triazine ring and N-dealkylation are the major metabolic pathways 

for atrazine in rats. Oxidation of the alky substituents of atrazine appears to be of minor 

metabolic importance. 

This metabolism study in the rat is classified Acceptable-Nonguideline and, when 

considered in conjunction with studies: MRID Nos.: 404313-04; -05; and 421655-03, 

satisfies the guideline requirement for a metabolism study (OPPTS 870.7485) in the rat.

In a metabolism study (MRID 42165503) fecal and urinary samples from rats exposed 

to 1 or 100 mg/kg radiolabeled atrazine in a separate metabolism study (MRID 

40431304) were obtained and analyzed by thin layer chromatography to determine 

metabolism profiles. No sex differences in metabolic profiles were evident. The major 
Page 23 of 90

fecal metabolite was DACT which accounted for 40% of the total fecal radioactivity. The 

major urinary metabolite was also DACT.

This metabolism study in the rat is classified Acceptable-Nonguideline and, when 

considered in conjunction with studies: MRID Nos.: 404313-04; -05; and -06, satisfies 

the guideline requirement for a metabolism study (OPPTS 870.7485) in the rat

In a metabolism study (MRID 44713802), groups of male and female rats (3-12 

per group depending on the experiment) were each given [U14C] Triazine G 30027 

(batch no. GAN-XVIII-77-5; purity>95%) and non-radiolabeled Triazine (batch no. AMS 

126/105; purity 99%) to provide a total single oral dose of 1 or 100 mg/kg. Urinary and 

fecal excretion were monitored up to 168 hours, tissue burdens were assessed up to 

336 hours post dosing, and major metabolites quantified and, where possible, 

identified. Biliary samples were taken from four male rats up to 48 hours post dosing 

and metabolite analysis performed.

There were no treatment-related deaths. Recovery of administered radioactivity was 

95.84% and 98.17%, respectively, for the administered high (168 hours postdosing) and 

low dose (48 hours postdosing). Based upon urinary and biliary excretion data, and 

tissue/carcass burden data, absorption was relatively rapid. The time to maximum blood 

concentration (tcmax) was 2 hours and 24 hours for the low and high dose groups, 

respectively. Under the conditions of this study, absorption and elimination did not 

appear to be saturated. The major route of excretion was via the urine, accounting for 

64.72% of the total administered low dose over a 48-hour period and 66.16% of the total 

administered high dose over a 168-hour period. Time-course studies revealed that 

within 48 hours urinary excretion was 100% and 94% complete for the low-dose and 

high-dose group, respectively. Fecal elimination accounted for 10.80% and 19.69% of 

the total dose for the low and high dose groups, respectively. Over a 48-hour period, 

biliary secretions accounted for 7.35% of the administered dose (68% of total amount 

excreted via the feces) in the low-dose group. Total fecal elimination of radioactivity 

accounted for 10.80% and 19.69% of the total administered dose, a portion of which 

was due to biliary secretion resulting from absorbed test material.

Tissue distribution appeared to be extensive and reached maximum concentrations 

(tcmax) at 2 hours and 24 hours for the low-dose and high-dose groups, respectively. 

Time to half maximum tissue concentrations (1⁄2 tcmax) for these groups were 48 hours 

and 168 hours, respectively. Although test article associated radioactivity was widely 

distributed, concentrations were greatest in red blood cells and highly perfused tissues 

and organs. With exception of red blood cells, whole blood, and skeletal muscle, tissue 

burden for any specific tissue or organ represented less than 1% of the administered 

dose by 14 days post dosing. Whole-body autoradiography confirmed the extensive 

distribution of the test material and/or its metabolites, and the rapid removal of 

radioactivity from the gastrointestinal tract. The pattern of tissue distribution did not 

appear to be significantly affected at the doses tested. Removal of radioactivity from 

tissues was a biphasic process characterized by a relatively rapid phase at 48 hours 

followed by a slower phase (48-336 hours). Depuration of radioactivity from red blood 

cells, however, exhibited a slower monophasic pattern over 336 hours.
Page 24 of 90

Urinary, biliary and fecal metabolites were detected and revealed minimal dose-related 

qualitative differences in metabolite profile. Quantitative differences were not 

inconsistent with that expected for a 100-fold difference in dose. Twenty six urinary 

metabolites were detected although only two accounted for more than 5% of the 

administered dose. Chromatographic analysis using known reference standards allowed 

for identification of four of the urinary metabolites. Nine biliary metabolites were 

detected, although none of the biliary metabolites accounted for more than 1.6% of the 

total administered dose. Three biliary metabolites were also identified as urinary and 

fecal metabolites. Twelve metabolites were identified in the feces of high-dose rats and 

nine metabolites were identified in the feces of low-dose rats, none of which 

represented more than 2.42% of the total administered radioactivity.

This study adequately described the metabolism and disposition of Triazine in rats 

administered single oral doses of 1 or 100 mg/kg. Inventory of administered 

radioactivity was acceptable and metabolite profiles indicate that, at the doses tested, 

Triazine G 30027 is nearly completely metabolized. The conclusion of the study 

authors that the major metabolic pathway appears to be dealkylation to the major 

metabolite (G 28273) and that dechlorination to CGA 10582 is a minor pathway is 

consistent with the data presented in the study report.

This metabolism study in rats is Acceptable/Guideline and satisfies the requirements 

for a Metabolism and Pharmacokinetics Study (OPPTS 870.7485). This study (MRID 

44713802) was properly designed and conducted, and provided data regarding the 

absorption, distribution, and elimination of the test material, and quantitation and 

identification of urinary, biliary, and fecal metabolites in rats given a single oral low dose 

(1 mg/kg) or high dose (100 mg/kg) of [U14C] Triazine G 30027.

870.7600 Dermal Absorption - Rat

In a dermal absorption study (MRID 43314302), male Charles River CD rats 

received either 0.01, 0.1 or 1 mg/cm2 of 14C-atrazine(two vials used: radiochemical 

purity: 98.7 and 98.9%, specific activity: 1.9 uCi/mg and 19.1 uCi/mg). Animals were 

exposed in three groups: animals in group one were exposed to a single dose of the 3 

above listed concentrations for 0.5, 1, 2, 4, 10, or 24 hours and were sacrificed 

immediately following dosing; group two animals were exposed to a single dose of the 

above listed concentrations for 10 hours and were sacrificed 34, 58 or 82 hours 

following the start of exposure; and group three animals were exposed to a single dose 

of the above listed concentrations for 24 hours and then sacrificed 48, 72 or 96 hours 

following the start of exposure. The exposure sites were washed with liquid Dove and 

water at the end of the exposure period. The animals were kept in metabolism cages 

during exposure and after exposure until sacrifice. 

The percent absorbed increased with time and decreased with dose. Regardless of the 

dose or exposure time, the majority (65 - 95%) of the radio labeled atrazine was 

recovered in the washes or was found associated with the skin at the site of exposure. 

The maximum percent absorbed was in the animals exposed to the low dose for 24 

hours and sacrificed 96 hours following the start of exposure. Absorption was 
Page 25 of 90

approximately 30% in this group. This indicates that much of what remains on the skin 

is subsequently absorbed. 

The percent absorbed increased with time and decreased with dose. A significant 

portion of compound remaining on washed skin is subsequently absorbed given enough 

time.

This dermal absorption study in the rat is classified Acceptable-Guideline, and 

satisfies the guideline requirement for a dermal absorption study (870.7600) in the rat.

870.7600 Dermal Absorption - Human

In a dermal absorption study (MRID 44152114), 10 human volunteers were 

exposed to a single topical dose of [triazine ring-U14C] atrazine (94.3-96.3% a.i., 98.0-

98.4% radiochemical purity) at 6.7 (4 volunteers) or 79 μg/cm2 (6 volunteers) for 24 

hours; equivalent to 0.1667 and 1.9751 mg of [14C] atrazine for the low and high doses, 

respectively. 

Overall recoveries of radioactivity from the low- and high-dose groups were 101 and 

92%, respectively. The majority (91.1-95.5%) of the dose remained unabsorbed and 

was detected in skin wash samples taken 24 hours after dosing. After 168 hours, only 

5.6% of the dose was absorbed and excreted in the urine and feces of the low-dose 

group and only 1.2% in the high-dose group. The renal excretion half-life was 19.6-29 

hours for the low-dose group and 25.9-31 hours for the high-dose group. In both dose 

groups, peak urinary elimination occurred at 24-48 hours and peak fecal elimination 

occurred at 48-72 hours.

Total chlorotriazine residues detected by TLC in a high-dose 0-24 hour composite urine 

sample accounted for 9.16% of the TRR and included deethyl atrazine (3.88% TRR) 

and didealkyl atrazine (5.28% TRR). No atrazine was detected. GC/MSD analysis of 

urine samples also did not detect atrazine or its chlorotriazine metabolites.

Enzyme immunoassays indicated that levels of atrazine mercapturate, an expected 

glutathione pathway metabolite was near the limit of detection/quantitation for the 

methods. Mercapturic acid conjugates of atrazine and its chlorotriazine metabolites 

were not detected by LC/MS/MS in urine samples.

Some similarities in HPLC profiles of urine from i.v. dosed monkeys (MRID 44152112) 

and 

dermally treated humans were observed.

This dermal absorption study in humans is Acceptable-Nonguideline. This study was 

not meant to satisfy an OPPTS 870.7600 guideline.

E.4.9 Special/Other Studies

E.4.9.1 Assays of Direct Estrogenic Activity of Triazines

Study 1:
Page 26 of 90

This study (MRID 43598617) combines three in vivo assays to investigate the 

estrogenic effects of atrazine, simazine, and diaminochlorotriazine [DACT, (a.i. > 96%)] 

on body weight, body weight gain, uterine weight, progesterone receptor binding 

capacity, and thymidine incorporation on female Sprague-Dawley rats. 

For body weight and uterine weight measurements, ovariectomized rats were dosed by 

oral gavage daily for 3 days at 20, 100, and 300 mg/kg/day in single exposure 

experiment and in co-exposure experiments with 2 μg estradiol on day 2 and 3. Body 

weight gains were decreased for rats exposed to the vehicle control, 0.5% 

carboxymethylcellulose, (-3.5%) and to the positive control, 2 μg estradiol, (-6.9%). 

Larger decreases in weight gain were observed in rats exposed to triazines. At 20 

mg/kg/day weight losses ranged from 6-9%. At 100 mg/kg/day, weight losses ranged 

from 8-13.5%. At 300 mg/kg/day, weight losses ranged from 11-17%.

Chemicals with estrogenic activity cause proliferation and thickening of the uterine wall. 

The uterine weights of rats exposed to triazines were similar to vehicle control uterine 

weights. In co-exposure experiments with estradiol, exposure to atrazine and DACT at 

100 and 300 mg/kg/day resulted in a dose-dependant and statistically significant 

decrease in uterine weight relative to the positive control. In co-exposure experiments 

with estradiol, uteri of rats exposed to simazine at 100 and 300 mg/kg/day were 

significantly decreased relative to the positive control.

Thymidine incorporation was measured to assess the effect triazines on DNA synthesis 

in the uterus of exposed rats. Dose groups included: exposure to 300 mg/kg/day of 

triazine alone administered by oral gavage for 2 days; and exposure to 1,10, 20, 50, 

100, and 300 mg/kg/day of triazine administered by oral gavage for 2 days plus an 

injection of 0.15 μg estradiol on day 2. Exposure to triazines alone at 300 mg/kg/day 

resulted in significant decrease relative to the vehicle control. Exposure to the positive 

control, 0.15 μg estradiol, resulted in approximately 2.5 fold increase (p < 0.05) in 

thymidine incorporation relative to the vehicle control. Exposure to 50, 100, and 300 

mg/kg/day in co-exposure with 0.15 μg estradiol resulted in a significant reduction in 

thymidine incorporation relative to the positive control.

Levels of inducible progesterone receptor (PR) were indirectly measured by the PR 

binding capacity to a radioactive ligand. The PR was isolated from uteri of rats exposed 

by oral gavage to atrazine, DACT, and simazine alone at 300 mg/kg/day for 2 days and 

to atrazine, DACT, and simazine at 50 and 300 mg/kg/day in co-exposure with 1 μg/day 

estradiol for 2 days. The PR binding capacity in rat uteri exposed to 300 mg/kg/day 

triazine alone was statistically decreased relative to vehicle control. PR binding 

capacity decreased significantly relative to the positive control with exposure to atrazine, 

DACT, and simazine at 300 mg/kg/day in co-exposure experiments with 1 μg estradiol.

According to the effects on uterine weight, progesterone binding capacity, and 

thymidine incorporation at the concentrations tested in this study, atrazine, 

DACT, and simazine do not exhibit estrogenic activity. 

This study is classified as Acceptable-nonguideline as a special study on in vivo and 

in vitro estrogenic effects. 
Page 27 of 90

Study 2:

In a special study (MRID 43598618) on in vitro and in vivo competitive binding 

of atrazine, diaminochlorotriazine (DACT, a mammalian metabolite of both atrazine and 

simazine) and simazine (>96 % a.i. for all three compounds) to the estrogen receptor 

(ER), several different experiments were performed utilizing extracted cytosolic ER from 

adult female Sprague-Dawley rats. Experiments observed the displacement of 

radiolabeled estradiol by atrazine, simazine, and DACT under both equilibrium 

(simultaneous exposure to triazine and radiolabeled estradiol at 4°C ) and at 

disequilibrium conditions (pre-exposure to triazine at 25°C for 30 min. prior to exposure 

to radiolabeled estradiol). Results of equilibrium experiments indicated that atrazine, 

simazine, and DACT did not displace any radiolabeled estradiol whereas the positive 

control exhibited a dose-dependant displacement of the radiolabeled estradiol. Under 

disequilibrium conditions in a time-course experiment, uterine cytosolic extracts were 

first incubated with triazine at 100 μM for 30 min. prior to exposure to radiolabeled 

estradiol for 5-150 minutes. With time radiolabeled estradiol displaced triazine that had 

bound during the initial exposure. In a dose-response experiment under disequilibrium 

conditions, uterine cytosolic extracts were first incubated with triazine at concentrations 

10-9 to 10-3 M or unlabeled estradiol at 10-11 to 10-7 M prior to exposure to radiolabeled 

estradiol. The concentration at which 50% inhibition of binding occurred (IC50) for 

unlabeled estradiol was approximately 10-9 M whereas the IC50's for the triazine were 

20 μM for atrazine and 100 μM for simazine and DACT.

An additional experiment under disequilibrium conditions was performed to mimic a 

Scatchard type analysis using constant molar excess of triazine (100X estradiol, 

10,000X atrazine, or 10,000X simazine) relative to the radiolabeled estradiol (0.2 nM, 

0.5 nM, 1.5 nM, and 5.0 nM). Results indicated the triazines competed with the 

radioligand better at lower concentrations of tracer. For example, there was 60% 

displacement of 0.5 nM radiolabeled estradiol when co-exposed with 5 μM atrazine 

whereas there was 11% displacement of 5 nM radiolabeled estradiol when co-exposed 

with 50 μM atrazine. In the Scatchard-type plot, dissociation constants were produced; 

0.5 nM in the presence of 100-fold molar excess of estradiol and 1 nM in the presence 

of 10,000 fold molar excess of atrazine and simazine. The x-intercepts of the plot were 

approximately equal (210 fmol/mg) in the presence of estradiol and simazine indicating 

competitive binding. The x-intercept for the atrazine plot was slightly less (158 fmol/mg) 

indicating the potential for competitive binding but also some noncompetitive binding 

under the disequilibrium conditions of this study.

Experiments using uteri extracted with KCl to extract the total ER and without KCl to 

extract the ER not bound or only loosely bound to chromatin were performed using 

cytosol fractions incubated with 50 μM triazine or 500 nM estradiol under disequilibrium 

conditions followed by separation of the sample by sucrose density centrifugation. One 

hundred twenty five mL fractions were removed and counted for radioactivity. The 

authors generated plots representing the fraction number versus the radioactivity in the 

given fraction in order to observe which fraction exhibited the binding. In the 

extractions without KCl, the receptor binding peaks were detected in the 7-8S fraction 

for both estradiol and atrazine. Atrazine displaced an average of 10.7% of the 

radiolabeled estradiol whereas estradiol displaced all of the radiolabeled tracer. In the 
Page 28 of 90

conditions favoring the extraction of total ER, activated and unactivated, binding was 

detected in the 4-5S fraction. Atrazine displaced an average of 29.6% of the 

radiolabeled estradiol whereas estradiol displaced all of the radiolabeled tracer. 

Making the assumption that living rats contain the activated form of the ER, in order to 

test whether triazines competed more effectively against the transformed ER, 

ovariectomized rats were dosed with triazines prior to uterine dissection and competitive 

binding experiments. The binding of radiolabeled estradiol was reduced by an average 

of 33%, 39%, and 24% for atrazine, simazine, and DACT, respectively (p < 0.05 vs. 

vehicle controls) in the 300 mg/kg/day group. In the 50 mg/kg/day group, binding of 

radiolabeled estradiol was reduced (not statistically significant) by 18, 21, and 13% . 

Overall the results indicate that atrazine, triazine, and DACT do exhibit some 

competitive binding with estradiol but only under conditions which favor triazine 

binding.

This special study on in vitro and in vivo competitive binding of atrazine, DACT and 

simazine binding to the estrogen receptor in the rat is Acceptable-nonguideline.

Study 3:

Another study (MRID 43598619) combines the following in vitro assays to 

investigate the estrogenic and antiestrogenic activity of atrazine and simazine: binding 

to hepatocyte Ah receptor; proliferation of MCF-7 (human breast cancer cell line) cells; 

gel electrophoresis mobility shift assay to measure levels of progesterone receptor; and 

MCF-7 transfection using a luciferase reporter gene.

Competitive binding to the aryl hydrocarbon (Ah) receptor was measured using 2,3,7,8-

tetrachlorodibenzo-p-dioxin (TCDD) as the competitor. This assay is performed 

because some dioxins, which are strong Ah receptor agonists, have also been shown 

to exhibit antiestrogenic activity. Cytosol from male rat hepatocytes containing Ah 

receptor was isolated and incubated with the positive control, radiolabeled TCDD, plus 

either atrazine or simazine. With addition of hydroxylapatite to this mixture, high 

molecular weight compounds, such as test compound bound to the Ah receptor, will 

precipitate out of solution. Subsequent centrifugation of this solution will result in the Ah 

receptor, and anything bound to it, being spun out into a pellet. By counting the 

radioactivity in the pellet, it is possible to measure the amount of radiolabeled TCDD 

displaced indicating competitive inhibition with the triazine. Competitive binding was 

measured as the decrease in radioactivity versus the positive control. At concentrations 

up to 10,000 nM, atrazine and simazine did not displace TCDD from the Ah receptor. 

A cell proliferation assay using the MCF-7 cell line was performed using the following 

doses: 10 μM, 1.0 μM, 0.1 μM, and 0.01 μM for atrazine and simazine and 1 nM for 

estradiol. Experiments using individual chemicals plus triazine were performed. 

Results indicated that the positive control increased the density of MCF-7 cells two fold 

relative to the negative control after 11 days. Exposure to atrazine and simazine alone 

neither increased nor decreased cell number at day 11 relative to the negative control. 

Results of co-exposure experiments indicate that cell number did not change relative to 

the positive control. 
Page 29 of 90

Estradiol has been shown to increase the level of progesterone receptor in mammalian 

cells. This assay measures the effect of estradiol and/or triazine on the binding capacity 

of a high affinity ligand (R5020) to the progesterone receptor which is an indirect 

measure of progesterone level. The gel electrophoresis mobility shift assay utilized 

indirectly measures progesterone receptor (PR) levels in MCF-7 cells following 

exposure to atrazine, simazine, or estradiol for 3 days. Polyacrylamide gel 

electrophoresis can separate molecules, such as DNA, or complexes of molecules, 

such as protein-DNA complexes, based on size. The DNA used in this study is a 

radioactive oligonucleotide of a specific sequence which binds to the PR. This 

sequence is called a progesterone response element (PRE). PR isolated from MCF-7 

cells is first incubated with R5020. PR-R5020 complex was then incubated with 

radiolabeled PRE. The R5020-PR-PRE complex was then run on a polyacrylamide gel. 

The unbound PRE was separated from the bound complex based on differences in 

mobility through the gel. Doses used were 1 μM for atrazine and simazine and 10 nM 

for estradiol. Exposure to estradiol resulted in a 3.5-fold increase in reactivity of the 

R5020-PR-PRE complex relative to the negative control. The radioactivity in the 

R5020-PR-PRE band following exposure to atrazine and simazine was similar to 

negative control. The radioactivity in the band representing the R5020-PR-PRE 

complex was measured to get an indirect measure of PR levels in MCF-7 cells. 

Results indirectly indicate that the levels of PR did not increase or decrease with 

exposure to atrazine or simazine. 

Transfection experiments were performed where the human estrogen receptor along 

with the luciferase expression system were inserted into MCF-7 . The MCF-7 cell assay 

measured luciferase activity (i.e., light production) and is quantitative in nature (i.e., 

concentration of estrogenic chemical is directly related to light production). Doses used 

in the luciferase experiments were 10-13 M to 10-8 M estradiol and 10-9 M to 10-5 M 

atrazine and simazine. Atrazine and simazine did not induce luciferase activity above 

background at concentrations as high as 10-5 M (concentrations above 10-5 M are toxic 

to MCF-7 cells). Exposure to the positive control, estradiol, resulted in significant 

luciferase activity beginning at 10-12 M. Light production increased exponentially up to 

10-8 M where light production plateaued. 

Neither atrazine nor simazine displayed estrogenic activity or interacted with the 

Ah receptor in the set of experiments described in this paper.

This study is classified as Acceptable-nonguideline as a special study on in vitro

estrogenic effects. 

Study 4:

Another study (MRID 43934403), combines three in vivo and four in vitro

assays to investigate the estrogenic and antiestrogenic activity of atrazine and 

simazine. In vivo experiments, 4 or 5 female Sprague-Dawley rats/dose were dosed 

orally or by intraperitoneal injection. In order to test for estrogenic activity, rats were 

exposed to atrazine (> 97% a.i.) or simazine (> 97% a.i.) at 50, 150, and 300 mg/kg/day 

or estradiol (positive control) at 10 μg/kg/day. In order to test for antiestrogenic activity, 

co-exposure experiments using atrazine or simazine plus estradiol were performed 

using the same concentrations. Following a three day exposure, the following in vivo
Page 30 of 90

endpoints were measured: uterine weight, progesterone receptor levels and uterine 

peroxidase.

Exposure to estradiol results in statistically significant increased uterine weights, 

increased levels of the progesterone receptor, and increased activity of uterine 

peroxidase. Exposure to atrazine and simazine individually resulted in negative effects 

indicating antiestrogenic activity are shown in slightly reduced uterine weights, 

statistically significantly reduced levels of the progesterone receptor, and statistically 

significantly reduced activity of uterine peroxidase.

The following in vitro assays were performed: proliferation of MCF-7 (human breast 

cancer cell line) cells; gel electrophoresis mobility shift assay to measure levels of 

progesterone receptor; MCF-7 transfection using the luciferase reporter gene; and yeast 

transfection using a selective media expression system. 

Doses for the cell proliferation assay when tested individually and in combination were 

10 μM, 1.0 μM, 0.1 μM, and 0.01 μM for atrazine and simazine and 1 nM for estradiol. 

Results indicated that the positive control significantly increased the density of MCF-7 

cells three fold relative to the negative control after 11 days . Exposure to atrazine and 

simazine alone neither increased nor decreased cell number at day 11 relative to the 

negative control. Results of co-exposure experiments indicate that cell number did not 

change relative to the positive control . 

The gel electrophoresis mobility shift assay used in this study indirectly measures 

progesterone receptor (PR) levels in MCF-7 cells following exposure for 3 days. The 

oligonucleotide used contains progesterone response element (PRE) which binds to the 

PR. PR from MCF-7 cells was isolated and incubated with radiolabeled PRE. The PRPRE complex was then run on a polyacrylamide gel to separate bound and unbound 

PRE based on differences in mobility through the gel. The radioactivity in the band 

representing the PR-DNA complex was measured as an indirect measure of PR levels. 

Doses used were 1 μM for atrazine and simazine and 10 nM for estradiol. Results 

indicated that exposure to estradiol resulted in a 3.5-fold increase in reactivity of the 

DNA-PR complex relative to the negative control. The radioactivity in the DNA-PR band 

following exposure to atrazine and simazine was similar to negative control. These 

results indicate indirectly that the levels of PR did not increase or decrease with 

exposure to atrazine or simazine. 

Two transfection experiments were performed where the human estrogen receptor was 

inserted into the genome of MCF-7 and yeast cells. The MCF-7 cell assay measured 

luciferase activity as a surrogate measure of estrogen receptor activity. Doses used 

were 10-13M to 10-8 M estradiol and 10-9 M to 10-5 M atrazine and simazine. Atrazine 

and simazine did not induce luciferase activity above background at concentrations as 

high as 10-5 M (concentrations above 10-5 M are toxic to MCF-7 cells). Exposure to the 

positive control, estradiol, resulted in significant luciferase activity beginning at 10-12 M 

and increased exponentially up to 10-8 M where light production plateaued. Atrazine 

and simazine did not exhibit in estrogenic or antiestrogenic activity and did not interact 

directly with the estrogen receptor.

In the yeast transfection assay, the human estrogen receptor linked to a necessary 

amino acid was inserted into the yeast. Growth on selective media is a qualitative 
Page 31 of 90

measure of activity of estrogen receptor. Yeast were dosed with 1 nM estradiol or 10 

μM atrazine or simazine. Yeast proliferated when exposed to estradiol but did not 

proliferate when exposed to atrazine or simazine. These results indicate that atrazine 

and simazine did not exhibit estrogenic activity and did not directly interact with the 

estrogen receptor.

In conclusion, the results of these experiments indicate that in vivo atrazine and 

simazine exhibits some antiestrogenic activity, but no estrogenic activity. Based 

on the in vitro results, this antiestrogenic activity is not the result of direct

interaction with the estrogen receptor.

This study is classified as Acceptable-nonguideline as a special study on in vivo and 

in vitro estrogenic effects. 

E.4.9.2 Estrous Cycle Alterations and LH Surge Attenuation

1. Pilot study 

In a study (MRID 43934404) to determine the validity of a proposed protocol for 

testing the effect of atrazine exposure on the proestrus afternoon luteinizing hormone 

(LH) surge, estradiol (E2) was administered to 70 ovariectomized female Sprague 

Dawley rats through a surgically implanted silastic capsule. Serum LH, E2, and prolactin 

levels were measured 3 days later to determine if the LH and prolactin surges could be 

measured and to evaluate success of the estradiol implantation procedure.

The ovariectomy followed by estradiol implantation procedure appeared to be effective 

in inducing LH and prolactin surges, as both these surges were clearly evident in rats 

undergoing this procedure. 

This special study in the rat is Acceptable-nonguideline. This study does not satisfy 

any guideline requirements and was not submitted with the intention of satisfying a 

guideline requirement.

2. 28 day study

In a study to evaluate the effect of atrazine exposure on the proestrus 

afternoon luteinizing hormone (LH) surge (MRID 43934406) atrazine, 97.1% a.i., was 

administered to 450 female Sprague Dawley rats in the diet. Dose levels were 0 (vehicle 

control), 2.5, 5, 40 and 200 mg/kg/day for 28 days. 

Mortality, clinical signs, gross pathology and pituitary weights were not affected in this 

study. Food consumption and body weights are decreased at the 40 and 200 mg/kg/day 

doses (body weight decreased 13% and 47% at 40 and 200 mg/kg/day respectively). 

The number of animals with diestrus blocks were increased at 40 and 200 mg/kg/day. 

The number of animals with estrus blocks were increased at 200 mg/kg/day. The 

prolactin surge was attenuated at 200 mg/kg/day. The LH surge was attenuated at 40 

and 200 mg/kg/day [non-repeat bleed rats].
Page 32 of 90

The LOAEL is 40 mg/kg/day, based on decreases in food consumption, body 

weight, body weight gain, estrous cycle alterations and LH surge attenuation. The 

NOAEL is 5 mg/kg/day.

This special study in the rat is Acceptable-nonguideline. This study does not satisfy 

any guideline requirements and was not submitted with the intention of satisfying a 

guideline requirement.

3. Six month study

In a study to evaluation the effect of long-term atrazine exposure on the 

proestrus afternoon luteinizing hormone (LH) surge (MRID 44152102) atrazine, 97.1% 

a.i., was administered to 360 female Sprague Dawley rats in the diet. Dose levels were 

0 (negative control), 25, 50, and 400 ppm (0, 1.80, 3.65, 29.44 mg/kg/day) for 26 weeks 

(approximately six months). 

Body weight, body weight gain and food consumption were significantly (p≤0.05) 

decreased in animals at the highest dose tested compared to controls (body weight 

decreased 8.5% at the end of the study and food consumption decreased 3.75% for the 

entire study). The percentage of days in estrus were significantly increased (p≤0.01) 

during the 21-22 and 25-26 week time periods at the high-dose level. Percent days in 

estrus were also increased during the 21-22 and 25-26 week time periods at the mid 

dose, but the increase was only significant (p≤0.05) for the 21-22 week time period. The 

proestrus afternoon LH surge was severely attenuated at the high dose (LH levels at 

most sampling time points were actually decreased compared to baseline) and less so 

at the mid dose (maximum increase over baseline was 157% compared to maximum 

increase over baseline in controls of 273%). Pituitary weights were increased at the high 

dose (absolute weight increased 22% and weight relative to body weight was increased 

28%). Pituitary weights at the other two doses were not affected. At the high dose, there 

was a slight increase in animals displaying enlarged pituitaries (0% in controls 

compared to 3.4% at 29.44 mg/kg/day) and thickened mammary glands (0% in controls 

compared to 6.7% at 29.44 mg/kg/day). There were no other gross necropsy findings in 

the high dose that could be attributed to compound exposure and there were no 

compound-related gross pathology findings at the mid- or low-dose. Selected tissues 

were saved for histopathology but those results have not been reported.

There were no compound-related effects in mortality or clinical signs. The proestrus 

afternoon prolactin surge was not affected by compound exposure at any dose. The low 

dose had no effects on the estrous cycle, LH or prolactin surges.

The LOAEL is 3.65 mg/kg/day, based on estrous cycle alterations and LH surge 

attenuation. The NOAEL is 1.8 mg/kg/day.
Page 33 of 90

This special study in the rat is Acceptable-nonguideline. this study does not satisfy 

any guideline requirements and was not submitted with the intention of satisfying a 

guideline requirement.

E.4.9.3 Other Two-Year Bioassays

Three other two-year bioassays have been submitted to the Agency. These 

are: 1. a serial sacrifice study using Sprague-Dawley female rats (Study 1, below); 2. a 

study using Sprague-Dawley female rats with two dose groups and a control (Study 4, 

below); 3. a serial sacrifice study using F-344 female rats (Study 5, below). 

Certain data collected in the two serial sacrifice studies were submitted 

separately and have their own MRID numbers. Hormone measurements and estrous 

cycle data from both SD and F-344 bioassays were submitted under the MRID number 

42743902. Histomorphology data from these two bioassays was submitted under the 

MRID number 43598622. The table below displays the MRID numbers for each 

submission. 

SD Serial Sacrifice Bioassay F-344 Serial Sacrifice Bioassay

Measurements MRID # Measurements MRID #

Body wt., food cons., clinical 

observations, gross 

pathology

42085001 Body wt., food cons., clinical 

observations, gross 

pathology

42146101

Hormone measurements 

and Estrous cycle 

measurements

42743902 Hormone measurements 

and Estrous cycle 

measurements

42743902

Histomorphology 43598622 Histomorphology 43598622

Study 1 (SD serial sacrifice bioassay):

In a 2-year bioassay (MRID 42085001) seventy SD female rats (no males were 

used) were exposed, through the diet, to doses of Atrazine (97%) at 0, 70 and 400 ppm 

( 0, 4.23 and 26.23 mg/kg/day) for 2 years. Ten females per dose were sacrificed at 1, 

3, 9, 12, 15, 18 and 24 months. Vaginal smears were taken for two weeks prior to each 

sacrifice date and serum was collected for measurements of estradiol, progesterone, 

prolactin and corticosterone, on the day of sacrifice.

Mortality was increased in a dose-dependent manner. There were five unscheduled 

deaths in the control group, 6 in the 70 ppm group and 8 at 400 ppm. Using the GehanBreslow test there was a statistically significant negative trend for survival (survival 

decreased as the dose increased). Another statistical test - the Cox-Tarone test - did 

not indicate a significant trend in either direction. Body weights at several timepoints in 

the 400 ppm group were statistically significantly reduced compared to controls. Food 

consumption was also statistically significantly reduced at several timepoints in the 400 
Page 34 of 90

ppm group compared to controls. Body weights and food consumption did not appear 

to be altered in the 70 ppm group compared to controls. 

Atrazine treatment at 26.2 mg/kg/day increased mortality, decreased body 

weights and decreased food consumption.

Study 2 (hormone measurement and estrous cycle evaluations in SD 

rats):

In a special study (MRID 42743902) female SD rats from the two year 

bioassay with serial sacrifices described above under "Study 1" (MRID 42085001) had 

their estrous cycles measured and serum levels of estradiol, progesterone, prolactin 

and corticosterone measured prior to or at each serial sacrifice. The results of these 

estrous cycle evaluation and serum hormone measurements are summarized below. 

Estrous cycle evaluations (vaginal smears)

The percent days spent in estrus in control animals increases as the animals 

age at the expense of days spent in diestrus. The percent days of the cycle spent in 

estrus was dramatically increased in dosed animals compared to controls at nine, 12 

and 18 months. Females in the 4.23 and 26.23 mg/kg/day groups at nine months spent 

an average of 34.3 and 44.8% of their days in estrus, respectively (p<0.05 at 70 and 

p<0.01 at 400 ppm). This is compared to 24% spent in estrus at the controls. The 

dose-related trend, as determined using a Terpstra-Jonckbeere Trend Test, showed a 

significant increase in % days spent in estrus (p<0.01). At 12 months there was also a 

dose-related trend (p<0.05) but the increases in dosed groups compared to controls as 

determined by ANOVA were not significant at either dose.

Atrazine-treated animals showed an early onset of increased days in the estrus 

phase of the estrous cycle. This effect occurred at both doses in this study.

Hormone measurements

Serum progesterone and corticosterone levels did not show any significant 

dose-related alterations compared to controls. Serum estradiol levels in the control rats 

in this study showed a positive trend (levels increased as time increased) over months 

one through nine. Exposure to atrazine did not alter this trend. The 4.23 and 26.23 

mg/kg/day dose groups also showed a significantly positive trend from months one 

through nine. These trends are expected as constant estrus would be expected to 

begin to set in as these animals approach nine months of age. Examination of the 

pairwise comparisons at three months indicates that treated animals had an early onset 

of increased serum estradiol levels compared to controls. At three months control 

estradiol levels were 3.5 ng/mL, 70 ppm levels were 11.2, and 400 ppm levels were 

16.2 ng/mL. The increase at 70 ppm was significant at p<0.05, the increase at 400 ppm 

was significant at p<0.01 and the trend, determined using a Terpstra-Jonckbeere Trend 

Test, was positive at p<0.05. At nine months control and 4.23 mg/kg/day group 

estradiol levels were similar, but the 26.23 mg/kg/day group compared to controls was 

increased 44%. At nine months estradiol levels were elevated compared to control, but 

not significantly so.
Page 35 of 90

An early onset of increased serum estradiol levels is seen in both doses of 

atrazine-treated females.

Study 3 (histomorphology in SD): 

In a special study (MRID 43598622) female SD rats from the two year 

bioassay with serial sacrifices described above under "Study 1" (MRID 42085001) had 

certain tissues, including their ovarian and mammary gland tissues, examined 

histopathologically. An evaluation was made of parameters in the ovarian tissues 

which indicated the animals current and recent estrous cycle states. The mammary 

gland was examined by histopathology for indications of exposure to steroid hormones 

such as estrogens, progesterone and prolactin. The examination of tissues, through 

histology, for morphologic alterations which provide information about certain aspects of 

an animals' physiologic state (such as the animal's estrous cycling ability or their 

exposure to hormones) is termed histomorphology. The results of the ovarian and 

mammary gland histomorphology from evaluations from this study are summarized 

below.

Ovarian Gland Histomorphology:

Selected tissues (including both ovaries and mammary glands) were removed and 

preserved at the serial sacrifice intervals described above under study 1, MRID 

42085001. Several ovarian parameters were evaluated including the number and type 

of corpora lutea and the number of antral follicles. The presence of many "old" corpora 

lutea indicates that an animal went through normal estrous cycles in the past. A 

complete absence of corpora lutea indicates that an animal is not cycling and has not 

gone through an estrous cycle in some time. Antral follicles are mature follicles that are 

close to ovulation. An increased number of these means that they are accumulating and 

implies that the signal for ovulation (an LH surge) may not be available or of sufficient 

magnitude to stimulate ovulation. 

There were no differences in number of old corpora lutea or antral follicles between 

controls and dose groups at the one month time point. By three months the number of 

animals which did not have old corpora lutea present increased in a slight, but dosedependant manner, from zero in controls to one at 4.23 mg/kg/day and two at 26.23 

mg/kg/day. Of the animals that had corpora lutea, there was an increased incidence in 

the dose groups of animals with reduced numbers of corpora lutea. Twenty percent of 

the controls were found to have reduced numbers of corpora lutea compared to 33% at 

4.23 and 37.5% at 26.23 mg/kg/day. The mean grade per animal for antral follicles (i.e., 

the sum total of all grades for all animals in the group divided by ten) was 2.1 and 2.2 in 

the 4.23 and 26.23 mg/kg/day groups, respectively, compared to 1.2 for controls. A 

higher mean antral grade indicates anovulation. These differences between control 

and treated groups became more extreme at the nine month timepoint. Seven of ten 

and ten of ten animals at nine months in the 4.23 and 26.23 mg/kg/day dose groups, 

respectively, had no corpora lutea at all present in their ovaries. This is compared to six 

of ten animals in the controls which did not have corpora lutea present. The mean 

antral follicle grade was 3.1 and 3.8 at the 4.23 and 26.23 mg/kg/day dose groups, 

respectively. This is compared to 2.6 in the controls. Data from the 12 month timepoint 
Page 36 of 90

indicated that control animals had equalized with the treated animals in terms of 

parameters representing anovulation. Ten of ten controls and ten of ten high dose 

animals displayed no corpora lutea at 12 months while eight of ten low dose animals 

displayed no corpora lutea. The mean antral follicle scores were 3.3, 2.9 and 3.1 for the 

control, low dose, and high dose, respectively. 

Compared to controls there was an early onset in dosed groups, at three and 

nine months, of parameters such as absence of old corpora lutea, absence of 

any corpora lutea, corpora lutea reduced in number, and increased numbers of 

antral follicles. These parameters indicate lack of ovulation and the appearance 

of these parameters in greater numbers in dose groups than in controls, at three 

and nine months, provides evidence that atrazine exposure is associated with 

and early onset of anovulation in the this assay.

Mammary Gland Histomorphology:

Mammary gland histomorphology for several parameters is discussed below. Index 

weighted scores are used to describe the results of the histomorphic evaluations. An 

index weighted score assigns a numerical value to the severity of the finding assigned 

by the pathologist. The higher the index weighted score, the more severe was the 

finding in that group. These results show an early onset of increased index weighted 

scores for several histomorphic parameters which indicate exposure of the mammary 

tissue to estrogens, progesterone and prolactin.

Acinar Development - An early onset of increased exposure to estrogen is indicated by

acinar development histomorphology index scores indicate and early onset of this 

finding in atrazine-exposed female SD rats. The index scores at the one-month time 

point are slightly higher in the dose groups than in the control, but a dose-response 

relationship was not seen. At the three- month time point the index scores are again 

increased over control. The increase is at this time point is dose-related though with the 

high dose being more severe than the low dose. At both nine and 12 months the index 

score again indicate more severe acinar development in the dose groups compared to 

the controls with the increase in severity being especially obvious at the high dose. 

Acinar/Lobular Development - An early onset of this parameter is evident. The oneand three-month timepoints have index scores in the dose groups that are similar to the 

control index scores. The dose group index scores are clearly increased compared to 

controls at nine and 12 month timepoints though. This indicates that the 

atrazine-treated animals were exposed to elevated levels of prolactin at an earlier time 

point than the control animals. 

Secretory Activity - The index scores for secretory activity also demonstrate an early 

onset of increased prolactin exposure in the dose groups compared to the controls. 

Index scores at one and three months in the dose groups were similar to control values, 

while index scores in both dose groups were clearly elevated compared to controls at 

the nine- and 12-month timepoints.

Dilated Ducts with Secretion - Index scores at one and three months in the dose groups 

were similar to control values. The index scores for the 4.23 mg/kg/day group 
Page 37 of 90

compared to controls were only slightly elevated. The index scores for the 26.23 

mg/kg/day group were greatly elevated compared to controls

Galactocele Incidence and Severity - No galactoceles were observed in any group at 

the one- and three-month timepoints. At nine and 12 months galactoceles in the dose 

groups were increased in both number and severity. By 15 months galactocele 

incidence and severity were similar between control and dose groups.

An early onset of several mammary gland histomorphology parameters in 

atrazine exposed SD rats of both doses in this study indicates an early onset of 

exposure to increased levels of steroid hormones - particularly estrogens and 

prolactin.

Study 4 (two dose two-year bioassay):

Atrazine technical was given in the diet to groups of 60 (female only) SpragueDawley rats for 24 months (MRID 42204401) at concentrations of 0, 70, or 400 ppm 

(approximating 0, 3.79, 23.01 mg/kg/day respectively). Water and food were available 

ad libitum. Test doses were selected following a 2-year study using 0, 10, 70, 500 or 

1000 ppm of atrazine in the diet (MRID 00158930).

A slight reduction (a negative trend) in survival found amongst the dosed groups was 

considered to be equivocal because the data were statistically significant (p<0.05) by 

the Gehan Breslow test but not by the Cox-Tarone test. No treatment-related increases 

in clinical signs were noted in the study. Body weight gains were statistically 

significantly reduced relative to controls (approx. 12 to 13%) only at 400 ppm during 

weeks 0-76. Food consumption was only minimally reduced at the highest dose. Slight 

alteration in red blood cell shapes and incidence of nucleated RBCs were transient in 

occurrence. Spleen weights were slightly increased but the increase was not 

statistically significant. Other organ weight and organ:body weight values from the 400 

ppm group were not significantly different from controls. nonneoplastic lesion findings 

were comparable in controls and treatment groups. Palpation times for tumors 

confirmed histologically indicated an early onset of mammary tumors. No statisticallysignificant increase in mammary tumors was observed.

The NOAEL for systemic toxicity is 70 ppm (calculated by the reviewer to be 

approximately 3.79 mg/kg/day) based on body weight gains of this group being 

12-13% less than controls as well as statistically significant decreases in body 

weights in the 0-76 week period. Also, a reduction in survival considered to be 

equivocal is reported at 400 ppm. An MTD and effect level was determined in a 

previous chronic feeding study (MRID 00158930). 

The study is classified Acceptable-nonguideline. This study alone does not fulfill 

Guideline requirements 870.4300 for carcinogenicity evaluation because only 2 dose 

levels were tested. Other studies have completed this Guideline requirement.

Study 5 (Intact vs OVX females and long-term estrous cycle evaluations):
Page 38 of 90

In a carcinogenicity study intended to provide information about the mode of 

action for oncogenicity in atrazine exposed Sprague-Dawley rats,(MRID 44544701), 

atrazine, [97.1% a.i.] was administered to 800 female Sprague-Dawley rats. The rats 

were divided into 2 groups of 400 each. One group was ovariectomized (OVX) while the 

other was left intact. Atrazine was mixed with the diet at dose levels of 0 (control) 25, 

50, 70 and 400 ppm (0, 1.5, 3.1, 4.2, 24.4 mg/kg/day for intact animals and 0, 1.2, 2.5, 

3.5, and 20.9 mg/kg/day for ovariectomized animals) for 2 years. 

Hematology, clinical chemistry, and urinalysis were not assessed in this study. Food 

consumption in the dose groups compared to the controls was not altered by compound 

exposure. The trend for survival was statistically-significantly decreased in the dosed 

groups compared to the controls. Survival was as follows: 43.3% in controls; 31.7% - 25 

ppm; 28.8% - 50 ppm; 31.6% - 70 ppm; 21.7% 400 ppm. Body weight was statisticallysignificantly reduced in the first half of the study in the 400 ppm group (other groups 

were not significantly altered), but by the end of the study body weights were similar to 

control values. Organ weights for pituitary, uterus and the ovaries were taken in this 

study. No organ weights in either the intact or ovariectomized group were altered by 

compound exposure.

The only gross necropsy finding which was altered by compound exposure was the 

occurrence of mammary masses in the intact dosed animals. Dosed animals showed a 

higher incidence of mammary masses (many of which were confirmed by 

histopathology to be tumors).

There were no non-neoplastic findings at histology that were increased in dosed OVX 

animals compared to controls. OVX animals in all groups displayed very high incidences 

of juvenile uterus and castration cells in the pituitary which would be expected in an 

OVX animal. The only finding in dosed intact animals which was increased in incidence 

over controls were ovarian cysts which were slightly increased at 70 and 400 ppm 

compared to controls. There were though, many findings which were prevalent in the 

intact animals, yet were not seen (or were seen at lower levels) in the OVX animals. 

Mammary gland galactoceles were seen in anywhere from 65 to 78% of the intact 

animals, depending on the dose group, but the highest percentage of galactoceles in 

the OVX group was 24% in the 50 ppm group. Mammary gland secretory activity was 

seen in from 34 to 46% of the intact animals but was not seen in any OVX animals. 

Uterine dilation was seen in from 8-18% of the intact animals and uterine cystic 

endometrial hyperplasia was seen in about 50% of the intact animals. No OVX animals 

displayed uterine dilation and only about 20% displayed cystic endometrial hyperplasia. 

Intact animals also had increased incidence of pituitary findings compared to OVX 

animals. From 65 to 73% of the intact animals were found to have sinusoid 

ectasia/angiectasis but the range in OVX animals was 31 to 45%. The differences in 

mammary gland, uterine and pituitary findings between OVX and intact animals may 

provide information about the mode of action of atrazine’s carcinogenicity.

Neoplastic histopathology findings were mostly limited to the pituitary and the mammary 

gland. Neither OVX nor intact animals showed an increase in pituitary tumors compared 

to their respective controls, but intact animals did show a 20-30% greater incidence of 

pituitary adenomas compared to OVX animals.
Page 39 of 90

There were few mammary tumors in the interim sacrifice animals, which is not 

surprising given that these animals were sacrificed after only one-year. Excluding the 

interim sacrifice and looking only at those animals which were sacrificed at 24 months 

and those which died prematurely, there was an increase in mammary tumor incidence 

at all intact dose groups compared to controls. In ascending order of dose the 

percentage of animals with any type of mammary tumors was: 38.3% in controls; 

53.3%; 71.2%; 56.6% and 68.3% at 400 ppm. Looking at carcinomas alone values are: 

18.3%; 36.7%; 33.9%; 20%; and 41.7%. Fibroadenomas alone were: 26.6%; 40%; 

52.5%; 45%; and 40%. 

A decrease in the time-to-tumor is also evident from exposure to atrazine. In the control 

group 50% of the tumors occurred in the last 6 months of the study. The percentage of 

tumors which appeared in the last 6 months of the study in the dose groups were: 

35.8%; 37.5% 36.5% and 33.4%. The number of tumors which occurred in the first year 

of the study was slightly increased at 25, 50 and 760 ppm and greatly increased at 400 

ppm: 9% in controls; 10.7%; 10%; 12% and 17.9% in the 400 ppm group.

An increased incidence of mammary tumor in intact animals was observed at 

doses of 50 ppm (3.1 mg/kg/day) and above.

The purpose of this study was to examine mammary tumor carcinogenesis in female 

Sprague-Dawley rats. Thus, a large part of this review focuses on carcinogenicity.

One of the more striking aspects of the study was the complete lack of mammary 

tumors in OVX animals. Not a single mammary tumor of any sort was seen in any OVX 

animal. The lack of mammary tumors in OVX animals provides evidence that the mode 

of action of atrazine is neither a direct genotoxic nor estrogenic effect on the mammary 

gland. Rather, an indirect hormonally-mediated effect involving the ovary is implied.

At the 50 ppm and above doses, there was a treatment related increase in mammary 

tumor incidence when compared to controls. Dosing was considered adequate based 

on decreases in body weight. Additionally, there was a decreased time-to-tumor at

doses of 25 ppm and above.

This carcinogenicity study in the rat is Acceptable-nonguideline, and does not satisfy 

the guideline requirement for a carcinogenicity study (870.4300) in the rat nor was it 

submitted with the intention of satisfying a guideline. 

Study 6 (F-344 serial sacrifice bioassay):

In MRID 42146101 , seventy F-344 rats (females only) per dose were exposed 

ad libitum to diet that had been mixed with atrazine (97.1%) to the appropriate doses of 

0 (negative control), 10, 70, 200 and 400 ppm (0, 0.68, 4.82, 14.05, 34.33 mg/kg/day). 

Ten animals per dose group were sacrificed after approximately one, three, nine, 12, 

15, and 18 months exposure to the test article. 

There was not an increase in mortality due to compound exposure, and there was no 

increased incidence of clinical signs in dosed animals compared to controls. The doses 
Page 40 of 90

tested appeared to be sufficiently high because there was a decreased absolute body 

weight and body weight gain in the 400 ppm group compared to the controls. Group 

mean absolute body weight in the 400 ppm group compared to controls was also 

significantly decreased compared to controls at several time points though the final 

mean body weight was not significantly decreased compared to controls. The final 

group mean body weight for the 400 ppm group was 6.6% less than the mean control 

value. During the course of the study the 400 ppm animals gained an average of 116.7 

grams compared to the weight gain in the control group of 133.3 grams (14% less than 

controls). This difference in body weight gain was statistically-significant at a p value of 

0.05. 

There was not an increase in mammary tumors or any other type of tumor at any 

dose group in either sex.

Studies 7 and 8, described below, are studies which performed hormone 

measurements, estrous cycle evaluations, and histomorphological evaluations on 

ovarian and mammary tissues in animals from this study.

Study 7 (hormone measurement and estrous cycle evaluations in F-344): 

The hormone measurements data and estrous cycle evaluation results (vaginal smears) 

from the animals in MRID 42146101 are shown in MRID 42743902. HED believes that 

the estrous cycle evaluation data reported in MRID 42743902 are unreliable. The 

histomorphology data from MRID 43598622 are instead used to determine stage of the 

estrous cycle.

The results of the hormone measurements did not reveal any consistently 

statistically-significant alterations in serum hormone levels compared to controls 

for any of the hormones tested. There were occasional significant alterations such as 

significantly decreased (p< 0.05) progesterone levels in the 4.82 mg/kg/day dose and 

corticosterone levels in the 34.33 mg/kg/day group at the 12-month time point compared 

to controls; significant negative trends in estradiol levels at the 12-month time point, 

progesterone levels at the 12- and 18-month timepoints, and corticosterone levels at the 

12- and 15-month timepoints, and prolactin levels displayed a significant positive trend 

at the three-month time point only. Careful consideration of these alterations indicated 

that they did not appear to be related to atrazine exposure. 

There were alterations in serum hormone levels that were seen in control as well as 

treated rats. These were: decreases in estradiol levels in the later half of the study; 

significantly increasing progesterone levels from one to 18 months. Decreased 

estradiol and increased progesterone are expected in rats undergoing a reproductive 

aging process involving pseudopregnancy. Exposure to atrazine did not alter the 

age-related changes in estradiol or progesterone levels. 

An increase in prolactin levels might be expected in an aging rat undergoing 

pseudopregnancy. However, consistent increases in serum prolactin levels were not 

seen.

Study 8 (histomorphology in F-344):
Page 41 of 90

The histomorphologic evaluation of tissue from animals in MRID 42146101 (study 6, 

described above) is presented as MRID 43598622.

Ovarian Histomorpology: The great majority of F-344 rats in all groups, both control and 

dose groups, maintained corpora lutea throughout most of the study. Only at the final, 

24-month, time point were there dramatic decreases in corpora lutea numbers. The 

atrazine-treated animals at this time point did not show decreases in corpora lutea 

numbers any more severe than the control animals. The reduction in corpora lutea 

numbers at this late time point appears to be a consequence of a natural progression of 

the animals from persistent diestrus into acyclicity. All animals in all dose groups 

maintained moderate numbers of secondary, antral and atretic follicles throughout the 

study, including the 24-month time point. 

The ovarian histomorpohology seen in this study is what would be expected in a 

normally aging female F-344 rat undergoing a normal aging process towards a state of 

repetitive pseudopregnancy. Repetitive pseudopregnancy is associated with vaginal 

smears that are primarily leukocytic and is, thus, sometimes referred to as "persistent 

diestrous".

The ovarian histomorphology data indicates atrazine treatment did not alter the 

number of animals in repetitive pseudopregnancy/persistent diestrous. Estrous 

cycling in atrazine treated F-344 females was not altered.

Mammary Gland Histomorphology: Animals in all dose groups displayed mammary 

gland histomorphologic alterations that would be expected in a normally aging F-344 

females rats i.e., there was some evidence of lobular/acinar development with secretory 

activity and occasional galactoceles in all dose groups at the 15, 18 and 24 month 

timepoints. 

The mammary gland histomorphologic alterations that are seen are those that 

would be expected in a normally aging F-344 female rat. Atrazine exposure did

not increase the severity of any mammary gland histomorphology finding or 

decrease their time of onset.

E.4.9.4 NHEERL Publications

Substantial research has been conducted on the toxicologic effects of atrazine exposure 

at the Reproductive Toxicology Division of EPA's National Heath and Environmental 

Effects Research Laboratories (NHEERL) at Research Triangle Park, N.C. . These 

studies have been evaluated in the May 22, 2000 document that was reviewed by the 

June, 2000 SAP [see http://www.epa.gov/scipoly/sap/2000/june27/finalparta_atz.pdf]. 

This research includes studies investigating the neuroendocrine basis of the mode of 

action for atrazine-associated carcinogenesis, as well as studies investigating 

developmental and reproductive effects associated with atrazine exposure. Much of 

these data have been published in the open literature. The abstracts from these open 

literature publications are shown below in chronological order of publication.
Page 42 of 90

Cooper, R.L., Stoker, T.E., Goldman, J.M., Parrish, M.B., Tyrey, L. 

(1996) Effect of atrazine on ovarian function in the rat. Reprod. 

Toxicol. 1996 Jul-Aug; 10(4):257-64. MRID not yet assigned.

The effect of the chlorotriazine herbicide, atrazine, on ovarian function was 

studied in Long-Evans hooded (LE-hooded) and Sprague-Dawley (SD) rats. Atrazine 

was administered by gavage for 21 d to females displaying regular 4-d estrous cycles. 

In both strains, 75 mg/kg/d disrupted the 4-d ovarian cycle; however, no distinct

alteration (i.e., irregular cycles but not persistent estrus or diestrus) was apparent at this 

dose. At 150 mg/kg/d, atrazine induced repetitive pseudopregnancies in females of both 

strains. The highest dose tested (300 mg/kg/d) also induced repetitive 

pseudopregnancies in the SD females, while the ovaries of the LE-hooded female 

appeared regressed and the smear cytology was indicative of the anestrous condition. 

Although a NOAEL was not established, the doses employed in this experiment were in 

excess of those used in chronic feeding studies in which an early onset of mammary 

gland tumors was noted. These data demonstrate that atrazine can disrupt ovarian 

function and bring about major changes in the endocrine profile of the female.

Stoker, T.E., Robinette, C.L., Cooper, R.L. (1999) Maternal exposure 

to atrazine during lactation suppresses suckling-induced prolactin 

release and results in prostatitis in the adult offspring. Toxicol Sci 

1999 Nov; 52(1):68-79 MRID 45166902

 

The availability of prolactin to the neonatal brain is known to affect the 

development of the tuberoinfundibular (TIDA) neurons and, as a consequence, lead to 

alterations in subsequent prolactin regulation. Without early lactational exposure to 

prolactin (derived from the dam's milk), TIDA neuronal growth is impaired and elevated 

prolactin levels are present in the prepubertal male. These observations, combined with 

the finding that alterations in prolactin secretion (i.e., hyperprolactinemia) in the adult 

male rat have been implicated in the development of prostatitis, led us to hypothesize 

that early lactational exposure to agents that suppress suckling-induced prolactin 

release would lead to a disruption in TIDA development, altered prolactin regulation, 

and subsequent prostatitis in the male offspring. To test this hypothesis, 

suckling-induced prolactin release was measured in Wistar dams treated twice daily 

with the herbicide atrazine (atrazine, by gavage, on postnatal day (PND) 1-4 at 0, 6.25, 

12.5, 25, and 50 mg/kg body weight), or twice daily with the dopamine receptor agonist 

bromocriptine (BROM, sc, at 0.052, 0.104, 0.208, and 0.417 mg/kg); BROM is known to 

suppress prolactin release. Similarly, atrazine has also been reported to suppress 

prolactin in adult females. Serum prolactin was measured on PND 3 using a serial 

sampling technique and indwelling cardiac catheters. A significant rise in serum 

prolactin release was noted in all control females within 10 min of the initiation of 

suckling. Fifty-mg/kg atrazine inhibited suckling-induced prolactin release in all females, 

whereas 25 and 12.5 mg/kg atrazine inhibited this measure in some dams and had no 

discernible effect in others. The 6.25 mg/kg dose of atrazine was without effect. BROM, 

used here as a positive control, also inhibited suckling-induced prolactin release at 

doses of 0.104 to 0.417 mg/kg, with no effect at 0.052 mg/kg. To examine the effect of 

postnatal atrazine and BROM on the incidence and severity of inflammation of the 

lateral prostate of the offspring, adult males were examined at 90 and 120 days. While 

no effect was noted at 90 days of age, at 120 days, both the incidence and severity of 
Page 43 of 90

prostate inflammation was increased in those offspring of atrazine-treated dams (25 and 

50 mg/kg). The 12.5 mg/kg atrazine and the two highest doses of BROM increased the 

incidence, but not the severity, of prostatitis. Combined treatment of ovine prolactin and 

25 or 50 mg/kg atrazine on PND 1-4 reduced the incidence of inflammation observed at 

120 days, indicating that this increase in inflammation, seen after atrazine alone, 

resulted from the suppression of prolactin in the dam.

To determine whether there is a critical period for these effects, dams were dosed with 

25 and 50 mg/kg on PND 6-9 and PND 11-14. Inflammation was increased in those 

offspring from dams treated on PND 6-9, but this increase was not significant. Dosing 

on PND 11-14 was without effect. These data demonstrate that atrazine suppresses 

suckling-induced prolactin release and that this suppression results in lateral prostate 

inflammation in the offspring. The critical period for this effect is PND 1-9.

Shafer, T.J., Ward, T.R., Meacham, C.A., Cooper, R.L. (1999) Effects 

of the chlorotriazine herbicide, cyanazine, on GABA(A) receptors in 

cortical tissue from rat brain. Toxicology 1999 Dec 20;142(1):57-68. 

MRID not yet assigned.

 

Chlorotriazine herbicides disrupt luteinizing hormone (LH) release in female 

rats following in vivo exposure. Although the mechanism of action is unknown, 

significant evidence suggests that inhibition of LH release by chlorotriazines may be 

mediated by effects in the central nervous system. GABA(A) receptors are important for 

neuronal regulation of gonadotropin releasing hormone and LH release. The ability of 

chlorotriazine herbicides to interact with GABA(A) receptors was examined by 

measuring their effects on [3H]muscimol, [3H]Ro15-4513 and 

[35S]tert-butylbicyclophosphorothionate (TBPS) binding to rat cortical membranes. 

Cyanazine (1-400 microM) inhibited [3H]Ro15-4513 binding with an IC50 of 

approximately 105 microM (n=4). Atrazine (1-400 microM) also inhibited [3H]Ro15-4513 

binding, but was less potent than cyanazine (IC50 = 305 microM). However, the 

chlorotriazine metabolites diaminochlorotriazine, 

2-amino-4-chloro-6-ethylamino-s-triazine and 

2-amino-4-chloro-6-isopropylamino-s-triazine were without significant effect on 

[3H]Ro15-4513 binding. Cyanazine and the other chlorotriazines were without effect on 

[3H]muscimol or [35S]TBPS binding. To examine whether cyanazine altered GABA(A) 

receptor function, GABA-stimulated 36Cl- flux into synaptoneurosomes was examined. 

Cyanazine (50-100 microM) alone did not significantly decrease GABA-stimulated 36Clflux. Diazepam (10 microM) and pentobarbital (100 microM) potentiated 

GABA-stimulated 36Cl- flux to 126 and 166% of control, respectively. At concentrations 

of 50 and 100 microM, cyanazine decreased potentiation by diazepam to 112 and 97% 

of control, respectively, and decreased potentiation by pentobarbital to 158 and 137% of 

control (n = 6). Interestingly, at lower concentrations (5 microM), cyanazine shifted the 

EC50 for GABA-stimulated 36Cl- flux into synaptoneurosomes from 28.9 to 19.4 

microM, respectively (n = 5). These results suggest that cyanazine modulates 

benzodiazepine, but not the muscimol (GABA receptor site) or TBPS (Cl- channel), 

binding sites on GABA(A) receptors. Furthermore, at low concentrations, cyanazine 

may slightly enhance function of GABA(A) receptors, but at higher concentrations, 

cyanazine antagonizes GABA(A) receptor function and in particular antagonizes the 

positive modulatory effects of diazepam and pentobarbital.
Page 44 of 90

Cooper, R.L., Stoker, T.E., Tyrey, L., Goldman, J.M., McElroy, W.K. 

(2000) Atrazine disrupts the hypothalamic control of pituitary-ovarian 

function. Toxicol Sci 2000 Feb; 53(2):297-307. MRID 45166901

The chloro-S-triazine herbicides (i.e., atrazine, simazine, cyanazine) constitute 

the largest group of herbicides sold in the United States. Despite their extensive usage, 

relatively little is known about the possible human-health effects and mechanism(s) of 

action of these compounds. Previous studies in our laboratory have shown that the 

chlorotriazines disrupt the hormonal control of ovarian cycles. Results from these 

studies led us to hypothesize that these herbicides disrupt endocrine function primarily 

through their action on the central nervous system. To evaluate this hypothesis, we 

examined the estrogen-induced surges of luteinizing hormone (LH) and prolactin in 

ovariectomized Sprague-Dawley (SD) and Long-Evans hooded (LE) rats treated with 

atrazine (50-300 mg/kg/day, by gavage) for 1, 3, or 21 days. One dose of atrazine (300 

mg/kg) suppressed the LH and prolactin surge in ovariectomized LE, but not SD female 

rats. Atrazine (300 mg/kg) administered to intact LE females on the day of vaginal 

proestrus was without effect on ovulation but did induce a pseudopregnancy in 7 of 9 

females. Three daily doses of atrazine suppressed the estrogen-induced LH and 

prolactin surges in ovariectomized LE females in a dose-dependent manner, but this 

same treatment was without effect on serum LH and prolactin in SD females. The 

estrogen-induced surges of both pituitary hormones were suppressed by atrazine 

(75-300 mg/kg/day) in a dose-dependent manner in females of both strains evaluated 

after 21 days of treatment. Three experiments were then performed to determine 

whether the brain, pituitary, or both organs were the target sites for the chlorotriazines. 

These included examination of the ability of (1) the pituitary lactotrophs to secrete 

prolactin, using hypophysectomized females bearing pituitary autotransplants (ectopic 

pituitaries); (2) the synthetic gonadotropin-releasing hormone (GnRH) to induce LH 

secretion in females treated with high concentrations of atrazine for 3 days; and (3) 

atrazine (administered in vivo or in vitro) to suppress LH and prolactin secretion from 

pituitaries, using a flow-through perfusion procedure. In conclusion, the results of these 

studies demonstrate that atrazine alters LH and prolactin serum levels in the LE and SD 

female rats by altering the hypothalamic control of these hormones. In this regard, the 

LE female appeared to be more sensitive to the hormone suppressive effects of 

atrazine, as indicated by the decreases observed on treatment-day 3. These 

experiments support the hypothesis that the effect of atrazine on LH and prolactin 

secretion is mediated via a hypothalamic site of action.

Das, P.C., McElroy, W.K., Cooper, R.L. (2000) Differential modulation 

of catecholamines by chlorotriazine herbicides in pheochromocytoma 

(PC12) cells in vitro. Tox. Sci. Aug; 56(2): 324-31. MRID not yet 

assigned.

Epidemiological, wildlife, and laboratory studies have pointed to the possible 

adverse health effects of chlorotriazine herbicide (i.e. , atrazine, simazine, and 

cyanazine) exposure. However, the cellular mechanism(s) of action of these 

compounds remains unknown. Recently, it was reported by Cooper et al. (2000, 

Toxicol. Sci. 53, 297-307) that atrazine disrupts ovarian function by altering 
Page 45 of 90

hypothalamic catecholamine concentrations and subsequently the regulation of 

luteinizing hormone (LH) and prolactin secretion by the pituitary. In this study, we 

examined the effect of three chlorotriazines on catecholamine metabolism in vitro using 

PC12 cells. Intracellular norepinephrine (NE) and dopamine (DA) concentrations and 

spontaneous NE release were measured following treatment with different 

concentrations of atrazine, simazine (0, 12. 5, 25, 50, 100, and 200 microM) and 

cyanazine (0, 25, 50, 100, and 400 microM) for 6, 12, 18, 24, and 48 h. Atrazine and 

simazine significantly decreased intracellular DA concentration in a 

concentration-dependent manner. Intracellular NE concentration was also significantly 

decreased by 100 and 200 microM atrazine and 200 microM simazine. Similarly, there 

was a dose-dependent inhibition of NE release with 100 and 200 microM concentrations 

of both compounds. Although 100 and 400 microM cyanazine increased intracellular NE 

concentration, 50, 100, and 400 microM cyanazine significantly increased NE release 

at 24 and 36 h. In contrast, intracellular DA concentration was decreased by cyanazine, 

but only at 400 microM. The GABA(A)-receptor agonist, muscimol (0, 0.01, 0.1, and 1.0 

microM) had no effect on either the release or on intracellular catecholamine 

concentrations from 6 through 24 h of treatment. Cell viability was somewhat lower in 

the groups exposed to 100 and 200 microM atrazine and simazine. However, the 

reduction in viability was significant only in the highest dose of atrazine used (200 

microM) at 24 h. Cyanazine did not have an effect on the viability at any of the doses 

tested, and the cells were functional, even up to 48 h of exposure. These data indicate 

that both atrazine and simazine inhibit the cellular synthesis of DA mediated by the 

tyrosine hydroxylase (TH), and NE mediated by dopamine beta-hydroxylase (DbetaH), 

and, as a result, there is a partial or significant inhibition of NE release. Cyanazine, on 

the other hand, stimulated the synthesis of intracellular NE, and not DA. Thus, 

chlorotriazine compounds presumably act at the enzymatic steps or sites of DA 

biosynthesis to modulate monoaminergic activity in PC12 cells.

Cummings, A. M., Rhodes, B.E., and Cooper, R.L. (2000). Effect of 

atrazine on implantation and early pregnancy in four strains of rats. 

Tox. Sci. Nov. 58: 135-143. MRID not yet assigned.

Atrazine is an herbicide that has been shown to have adverse reproductive effects 

including alterations in levels of pituitary hormones such as prolactin and luteinizing 

hormone (LH) in female LE rats when administered at doses of 200 mg/kg/day for 1 and 

3 days. Since prolactin’s action to promote progesterone secretion is essential for the 

initiation of pregnancy in rats, this study was designed to examine the effect of exposure 

to atrazine during early pregnancy on implantation and short-term pregnancy 

maintenance. Rats were divided into two groups representing periods of dosing with 

atrazine prior to the diurnal or nocturnal surges of prolactin. Within each group, four 

groups consisting of four strains of rats (Holtzman, HLZ; Sprague Dawley, SD; Long 

Evans, LE; Fisher 344, F344) were each further subdivided into four atrazine dosages. 

Rats were dosed by gavage with 0, 50, 100, or 200 mg/kg/day atrazine on days 1-8 of 

pregnancy (day 0 = sperm +). All animals were necropsied on day 8 or 9 of pregnancy. 

The 200 mg/kg dose of atrazine reduced body weight gain in all but one group. Two 

groups of animals dosed at 100 and 200 mg/kg/day in the nocturnal dosing period 

showed an increase in percent preimplantation loss, and both of these were F344 rats. 

Holtzman rats were the only strain to show a significant level of postimplantation loss 

and a decrease in serum progesterone at 200 mg/kg/day both following diurnal and 
Page 46 of 90

nocturnal dosing. Doses of 100 mg/kg/day also produced postimplantation loss 

following diurnal and nocturnal dosing, but progesterone levels were only decreased 

after nocturnal dosing. Alterations in serum LH were seen in several groups. Serum 

estradiol was significantly increased only in Sprague Dawley rats dosed at the diurnal 

interval with 200 mg/kg atrazine. We conclude that F344 rats are most susceptible to 

preimplantation effects of atrazine and that HLZ rats appear most sensitive to the 

postimplantation effects of the chemical. LE and SD rats were least sensitive to effects 

of atrazine during very early pregnancy. 

Narotsky M. G., Best, D.S., Guidici, D. L., and Cooper, R.L. (2000). 

Strain comparisons of atrazine-induced pregnancy loss in the rat. 

Repro Toxicol. In Press. MRID not yet assigned.

Atrazine was administered by gavage, in 1% methylcellulose, to F344 

Sprague-Dawley (SD), and Long Evans (LE) rats at 0, 25, 50, 100, or 200 mg/kg/d on 

gestation days 6 through 10. The dams were allowed to deliver and litters were 

examined postnatally. The F344 strain was the most sensitive to atrazine’s effects on 

pregnancy, showing full-litter resorption at $50 mg/kg. In surviving F344 litters, prenatal 

loss was increased at 200 mg/kg. In SD and LE rats, full-litter resorption occurred only 

at 200 mg/kg. Delayed parturition was seen at $100 mg/kg in F344 and SD rats. 

Regarding maternal toxicity, the SD dams were the most sensitive, with weight loss at 

$25 mg/kg. When 200 mg/kg was administered to F344 rats on days 11 through 15 

(after the LH-dependent period of pregnancy), no full-litter resorption was seen. These 

findings suggest that atrazine-induced full-litter resorption is maternally mediated, and 

consistent with loss of LH support of the corpora lutea.

Laws, S.C., Ferrell, J.M., Stoker, T.E., Schmid, J., and Cooper, R.L. 

(2000). The effect of atrazine on puberty in female Wistar rats: an 

evaluation in the protocol for the assessment of pubertal development 

and thyroid function. Tox. Sci. 58 (2): 366-76. MRID not yet assigned.

The effects of atrazine, a chlorotriazine herbicide, on the onset of puberty were 

evaluated in Wistar rats. Female rats were dosed by oral gavage from postnatal day 

(PND) 22 through PND 41 with 0, 12.5, 25, 50, 100 or 200 mg atrazine /kg. Vaginal 

opening was significantly delayed 3.4, 4.5 or greater than 6.8 days by 50, 100 and 200 

mg/kg, respectively. Vaginal opening did not occur in 4 of 15 females in the 200 mg/kg 

group by the time of necropsy (PND 41). Body weight at necropsy was reduced in the 

200 mg/kg group by 11.6%, but was not different from the control (0) in the 50 and 100 

mg/kg groups. To examine the influence of reduced body weight on pubertal 

development, a group of pair-fed controls was included whose daily food intake was 

dependent upon the amount consumed by their counterpart in the 200 mg/kg group. 

Although necropsy body weight was reduced to the same extent as the atrazine 

females, vaginal opening in the pair-fed controls was not significantly delayed. Adrenal, 

kidney, pituitary, ovary and uterine weights were reduced by 200 mg/kg atrazine. 

Serum T3 [tri-iodothyronine], T4 [thyroxine], and TSH [thyroid stimulating hormone] 

were unaltered by atrazine which were consistent with no histopathologic/morphologic 

changes in the thyroid. Estrous cyclicity was monitored in a second group of females 

from vaginal opening - PND 149. The number of females displaying regular 4 or 5-day 
Page 47 of 90

estrous cycles during the first 15-day interval after vaginal opening was lower in the 100 

and 200 mg/kg atrazine and pair-fed controls. Irregular cycles were characterized by 

extended periods of diestrus. By the end of the second 15-day interval (PND 57-71), no 

effects on estrous cyclicity were observed. These data show that atrazine can delay the 

onset of puberty and alter estrous cyclicity in the female Wistar rat ( NOAEL of 25 

mg/kg). Reduced food consumption and body weight did not account for the delay in 

vaginal opening because this effect was not observed in the pair-fed controls. In 

addition, the effect on estrous cyclicity was observed in the 100 mg/kg atrazine group 

where no significant reduction in body weight was observed. 

Stoker, T.E., Laws, S.C., Guidici, D. and Cooper, R.L. (2000) The 

effect of atrazine on puberty in male Wistar rats: An evaluation in the 

protocol for the assessment of pubertal development and thyroid 

function. Toxicol. Sci. Nov. 58: 50-59. 

Since atrazine, a chlorotriazine herbicide, has been shown previously to alter 

the secretion of luteinizing hormone (LH) and prolactin through a direct effect on the 

CNS, we hypothesized that exposure to atrazine in the EDSTAC male pubertal protocol 

(juvenile to peripubertal) would alter the development of the male rat reproductive 

system. We dosed intact male Wistar rats from postnatal day (PND) 23 to 53 and 

examined several reproductive endpoints. Atrazine (0, 12.5, 25, 50, 100, 150 or 200 

mg/kg) was administered by gavage and an additional pair-fed group was added to 

compare the effects of any decreased food consumption in the high dose group. 

Preputial separation was significantly delayed in the 12.5, 50, 100, 150 and 200 mg/kg 

atrazine dose groups. Preputial separation was also delayed in the pair-fed group, 

although significantly less than in the high dose atrazine group. The males were killed 

on PND 53 or 54 and pituitary, thyroid, testes, epididymides, seminal vesicles, ventral 

and lateral prostates were removed. Atrazine (50 to 200 mg/kg) treatment resulted in a 

significant reduction in ventral prostate weights, as did the pair-fed group. Testes 

weights were unaffected by atrazine treatment. Seminal vesicle and epididymal weights 

were decreased in the high dose atrazine group and the control pair-fed group. 

However, the difference in epididymal weights was no longer significantly different when 

body weight was entered as a covariable. Intratesticular testosterone was significantly 

decreased in the high dose atrazine group on PND 45, but apparent decreases in 

serum testosterone were not statistically significantly on PND 53, There was a trend for 

a decrease in luteinizing hormone as the dose of atrazine increased, however, dose 

group mean LH were not different from controls. Due to the variability of serum prolactin 

concentrations on PND 53, no significant difference was identified. Although prolactin is 

involved in the maintenance of LH receptors prior to puberty, we observed no difference 

in LH receptor number at PND 45 or 53. Serum estrone and estradiol showed doserelated increases that were significant only in the 200 mg/kg atrazine group. No 

differences were observed in thyroid stimulating hormone (TSH) and thyroxine (T4) 

between the atrazine groups and the control, however tri-iodothyronine (T3) was 

elevated in the high dose atrazine group. No differences in hormone levels were 

observed in the pair-fed animals. These results indicate that atrazine delays puberty in 

the male rat and its mode of action appears to be altering the secretion of steroids and 

subsequent effects on the development of the reproductive tract, which appear to be 

due to atrazine’s effects on the CNS. Thus, atrazine tested positive in the pubertal male 

screen that EDSTAC is considering as an optional screen for endocrine disrupters.
Page 48 of 90

E.4.10 Toxicology data for major metabolites of atrazine

Metabolism of atrazine can occur through hydrolytic dehalogenation at the 2 

position of the s-triazine ring resulting in the formation of hydroxyatrazine. Dealkylation 

reactions at the 4 and 6 positions - either in the absence or presence of dehalogenation 

- may also occur. Both plants and animals are capable of performing the dealkylation 

reactions. Hydroxyatrazine is the major metabolite in plants, but is only a minor 

metabolite in animals where the varying forms of the delkylated chlorometabolites 

dominate instead. The metabolism of atrazine to hydroxyatrazine in plants is a 

detoxification reaction as the phytotoxicity of hydroxyatrazine is greatly reduced 

compared to the parent compound. Bacteria are believed to be capable of the 

dealkylation reactions that form the chlorometabolites of atrazine. Hydroxyatrazine and 

the chlorometabolites can be found in both drinking water and the diet.

The HED Metabolism Committee (October 17, 2000) has determined that for 

chronic dietary risk, the residues of concern are parent and chlorometabolites compared 

to appropriate atrazine endpoints. A separate chronic dietary risk assessment will be 

conducted for hydroxyatrazine metabolites compared to the appropriate hydroxyatrazine 

endpoint. For acute dietary risk assessment the residues of concern are the parent 

chlorometabolites compared to appropriate acute atrazine endpoints. A separate acute 

assessment will not be conducted for hydroxyatrazine as HIARC did not select an acute 

RfD for hydroxyatrazine. The three chlorometabolites of concern are 

diaminochlorotriazine, desisopropyl atrazine , and desethyl atrazine. A limited 

toxicology database is available for these three metabolites and these studies are 

summarized below in sections F4.10.1, F4.10.2, and F4.10.3. A limited toxicology 

database is also available for hydroxyatrazine and is shown below under section 

F4.10.4.

E.4.10.1 Diaminochlorotriazine metabolite (DACT) - 2-chloro-4-amino-6-

amino s-triazine; didealkyl atrazine; G-28273 

The DACT metabolite is the terminal mammalian metabolite of both atrazine 

and the closely related herbicide simazine. DACT is primarily a mammalian metabolite 

but can also be produced in plants and bacteria. DACT is produced through 

dealkylation of the isopropyl group at position 2 of the triazine ring in atrazine 

(dealkylation of the ethyl group at the 2 position of the triazine ring in simazine) and 

dealkylation of the ethyl group at the 4 position of the triazine ring in either atrazine or 

simazine. DACT is the primary metabolite produced in rats following atrazine treatment. 

It is the major fecal metabolite in rats accounting for 40% of the radioactivity found in 

feces following exposure by oral gavage. DACT is also the major urinary metabolite in 

rats accounting for 26% of the total administered dose following exposure by oral 

gavage.

870.3100 - Subchronic oral toxicity in rats

Groups of 15 male and 15 female CD Sprague-Dawley rats were fed diets 

containing diaminochlorotriazine (G-28273) (purity 98.2%) at concentrations of 0, 10, 

100, 250, or 500 ppm for 13 weeks (MRID 43013207). The average consumption of 
Page 49 of 90

test material was 0.7, 6.7, 16.7, or 34.1 mg/kg/day (males) and 0.7, 7.6, 19.7, or 40.2 

mg/kg/day (females). All animals survived to study termination. No treatment-related 

clinical signs of toxicity including ocular lesions were seen at any dose level. At 500 

ppm, mean body weights of male rats were lower than controls during most of the study 

period, decreasing to 87% of controls at week 12. Body weight gain at week 12 was 

82% of controls for males receiving 500 ppm, and 85% and 83% of controls for females 

receiving 250 and 500 ppm, respectively. No treatment-related effects on body weight 

or body weight gain were seen at the lower doses in either sex. Food consumption was 

not affected by administration of the test material. There were no biologically significant 

effects on hematology, clinical chemistry, urinalysis, and gross or histopathology at any 

dose level. Although several organ weight changes were observed, there were no 

histologic or functional correlates. Estrous cycle data indicated a treatment-related 

effect at doses of ≥100 ppm. The effects, generally more pronounced on days 70-85 

than on days 14-28 and 42-56, included lengthening of the estrus cycle and/or an 

increased incidence of rats exhibiting cycles with persistent estrus and/or diestrus. 

There were no apparent effects on serum levels of estradiol, progesterone, prolactin, 

and corticosterone. 

The LOAEL is 7.6 mg/kg/day, based on estrous cycle effects in female rats. The 

NOAEL is 0.7 mg/kg/day.

This study is classified as Acceptable-Guideline and satisfies the guideline 

requirements for a subchronic dietary toxicity study (OPPTS 870.3100) in rats. 

870.3150 and 870.4100 - Subchronic and chronic oral toxicity in dogs

Diaminochlorotriazine was fed to male and female dogs at dietary levels of 0, 

5, 100, or 1500 ppm for 13 or 52 weeks (MRID 41392401). Because of severe toxicity at 

the highest dose, which was evident after 6 weeks of treatment, the high-dose dogs 

were fed a diet containing 750 ppm. Females tolerated this dose level and received 750 

ppm until termination at 13 or 52 weeks, or through 13 weeks followed by a 39-week 

recovery period. Since males continued to exhibit signs of toxicity at 750 ppm, they 

were fed untreated diet for 9 weeks through 13. Four male dogs were then placed again 

on a diet containing 750 ppm until termination at 52 weeks.

The mean daily doses for male dogs receiving dietary levels of 5, 100, and 1500/750 

ppm for 52 weeks were 0.187, 3.61, and 24.1 mg/kg/day, and the doses were 0.195, 

3.43, and 32.7 mg/kg/day for females receiving the same dietary levels. Among the 

high-dose dogs, five males and two females were sacrificed moribund during the 

treatment period. Moribundity was attributed to impairment of heart function, the primary 

treatment-related effect of Diaminochlorotriazine, which was accompanied by several 

clinical and pathological changes. Pathological cardiac findings included enlargement, 

softness, thickened valves, lesions, distension, red/dark color, thrombosis, chronic 

myocarditis, necrosis, inflammation, hemorrhage, and hemosiderosis. Secondary 

treatment-related changes in the high-dose animals were seen in the liver (enlargement, 

congestion, centrilobular fibrosis/atrophy, bile stasis, necrosis, hemosiderosis, red/dark 

color, lesions, adhesions, mottling, and rough texture); testes (hypospermatogenesis 

and hypospermia); thymus (atrophy); bone marrow (hyperplasia); and pericardium, 

thoracic, and abdominal cavities (fluid accumulation). Recovery females did not exhibit 
Page 50 of 90

any clinical ophthalmological signs of cardiac impairment. Other effects at the high 

dose included decreased body weight gains in males and females dosed for 6 weeks; 

increased mean spleen, liver, and kidney weights; anemia with accompanying 

reticulocytosis (a reversible effect); decreases in albumin, calcium and total cholesterol 

levels; nonsignificant increase in lactic acid dehydrogenase activity ; and elevations of 

platelet levels. High-dose males continued to lose weight when the dose was lowered 

to 750 ppm. Severe anemia with reticulocytosis was noted in only one of the two 

recovery females. The effects of diaminochlorotriazine on cholesterol levels and 

erythroid parameters were reversible as were noted in almost all animals of both sexes 

and beginning at week 6 and extending until week 14. No adverse effects were 

observed at dietary levels of 5 or 100 ppm. Although there was appreciable mortality at 

the highest dose of 1500 ppm, a sufficient number of animal were at risk to evaluate 

histopathology. Administration of dietary levels of ≥ 750 ppm to dogs is associated with 

symptomatology of cardiac impairment. 

The LOAEL is 24.1 mg/kg/day, based on cardiac effects. The NOAEL is 3.43 

mg/kg/day.

This study is classified as Acceptable-Guideline and satisfies the guideline 

requirements for a subchronic dietary toxicity study (OPPTS 870.3150) and also a 

chronic dietary toxicity study (OPPTS 870.4100) in dogs. 

870.3700 - Developmental toxicity in rats

In a developmental toxicity study (MRID 41392402), diaminochloro-triazine 

[DACT] (98.2% a.i., batch # FL 871423) was administered to 26 female SpragueDawley rats/dose via gavage at dose levels of 0, 2.5, 25, 75, and 150 mg/kg bw/day 

from days 6 through 16 of gestation. 

There were no treatment-related effects on mortality or clinical signs. Maternal body 

weight-gain was reduced significantly [33.2%] during the 20-day gestation interval at the 

high-dose level , and there was a decrease in body-weight gain during the dosing 

interval [days 6-16] at the two-highest dose levels. During the initial dosing interval 

[days 6-8], dams at the 25 mg/kg/day dose level displayed a 32% decrease in bodyweight gain, and the dams at the 75 and 150 mg/kg/day dose levels displayed negative 

body-weight gains. Decreased food consumption was observed at the highest dose 

level [days 6-16; 66% of control value] during the dosing period. Only a slight decrease 

in food consumption was observed at the 25 and 75 mg/kg/day dose levels during the 

dosing period. 

The maternal LOAEL is 25 mg/kg bw/day, based on decreased body-weight gain 

during the initial three days of the dosing period.. The maternal NOAEL is 

2.5mg/kg bw/day.

Pregnancy rate was not adversely affected by treatment, and there were no abortions. 

Comparable numbers of corpora lutea/dam and implantations/dam were observed 

among the groups, and there was no dose-related effect on pre-implantation loss. At the 

high-dose level [150 mg/kg/day], there was an increase in resorptions [both early and 

late], and post-implantation loss was significantly increased [18.9% vs 5.6% in the 
Page 51 of 90

control] at this dose level. There were fewer live fetuses at 150 mg/kg/day [86% of 

control]. There were no dead fetuses, and the sex ratio was comparable among the 

groups. Gravid uterine weight was decreased significantly compared to the control [77% 

of control] at the 150 mg/kg/day dose level, and fetal body weights were lower at 75 

mg/kg/day [91%-92% of control] and 150 mg/kg/day [81% of controls] in both sexes. 

There was an increased incidence of absent renal papilla [22% vs 3.3%] and pitted 

kidneys [4.8% vs 0%] at the high-dose level compared to the control. There was a doserelated increase in incomplete ossification of several bones at the 75 and 150 

mg/kg/day dose levels and in three bones at 25 mg/kg/day [ interparietal, parietals, and 

hyoids (unossified)].

The developmental LOAEL is 25 mg/kg bw/day, based on increases in the 

incidences of incompletely ossified parietals, interparietals, and unossified 

hyoids. The developmental NOAEL is 2.5 mg/kg bw/day.

The developmental toxicity study in the rat is classified Acceptable/Guideline, and it 

satisfies the guideline requirement for a developmental toxicity study (OPPTS 

870.3700) in the rat. 

870.5100 Bacterial Reverse Mutation 

In replicate (separate) trials, the atrazine metabolite diaminochlorotriazine 

(DACT, 97.0%) failed to induce reverse mutation (as evidenced by no increase over 

controls in revertant colony counts) in Ames testing at concentrations up to 5000 μg/0.1 

mL, at which dose precipitation of compound occurred. This study is classified 

Acceptable/Guideline (MRID 40722302).

870.5550 UDS Assay 

The atrazine metabolite diaminochlorotriazine (DACT, 97.0%) did not induce 

unscheduled DNA synthesis in cells of the human fibroblast cell CRL 1521 exposed to 

concentrations resulting in compound precipitation (400 to 600 μg/mL), but only in the 

absence of a metabolic activation system (assay without activation was not performed). 

This study is classified Acceptable/ nonguideline (MRID 40722303).

E.4.10.2 G-28279 metabolite -2-chloro-4-(ethylamino)-6-(amino) s-triazine; 

ethylatrazine; deisopropyl atrazine 

Desisopropyl atrazine is a metabolite of both atrazine and simazine. 

Desisopropyl atrazine is primarily a mammalian metabolite but can also be produced in 

plants and bacteria. Dealkylation of the isopropyl group from the 2 position of the 

triazine ring in atrazine or simazine yields desisopropyl atrazine/simazine. 

870.3100 - Subchronic oral toxicity in rats

In a 90-day oral toxicity study (MRID 43013205), groups of 10 male and 10 

female RAIf(SPF) rats were fed diets containing G-28279 technical (purity 96.7%; Lot 

#FL-901747) at concentrations of 0, 10, 50, or 500 ppm for 13 weeks. The average 
Page 52 of 90

consumption of test material was 0.602, 3.20, or 34.9 mg/kg/day (males) and 0.641, 

3.34, or 37.5 mg/kg (females). 

All animals survived to study termination. No treatment-related clinical signs or ocular 

lesions were seen at any dose level. At 10 ppm, there were no effects on body weight, 

clinical chemistry parameters, hematologic values, or organ weights, nor were 

macroscopic or histopathologic changes observed. Slightly decreased body weights 

and body-weight gains were seen in males administered 50 or 500 ppm of the test 

material. At week 13, the body weights of males were 93% and 88% of control values 

at 50 and 500 ppm, respectively, and the body-weight gains were 96, 90, or 84% of 

control values at 10, 50, or 500 ppm, respectively. For female rats exposed to 500 ppm, 

the body weights were 9-12% below those of controls from treatment week 2 to 13, and 

the mean body-weight gain was 80% of controls at the end of the treatment period. 

Food consumption was not significantly affected by treatment with G-28279 technical. 

Organ weight changes (absolute and relative to body weight) in males at 500 ppm 

included increased relative kidney weights (14% above control values), decreased 

absolute heart weights (-10%), increased relative testes weights (+21%), and increased 

relative brain weights (+18%). In the absence of histopathologic lesions in the 

corresponding organs, these organ weight changes are of uncertain toxicological 

significance and may not be treatment-related. Increased relative liver weights (+13%) 

and extramedullary hematopoiesis of the liver seen in female rats at 500 ppm indicate 

slight hepatotoxicity. Minimal to moderate fatty changes of adrenal cortex and slight 

hypertrophy of the thyroid follicular epithelium in males at 500 ppm suggest treatmentrelated effects. Additionally, extramedullary hematopoiesis of the spleen in females at 

500 ppm and hypertrophy of pituitary cells in males at 500 ppm may suggest treatment 

related effects.

The LOAEL is 3.3 mg/kg/day, based on decreased body weights and body weight 

gains in both sexes. The NOAEL is 0.6 mg/kg/day.

This study is classified as Acceptable/Guideline and satisfies the guideline 

requirements for a subchronic dietary toxicity study (OPPTS 870.3100) in rats.

870.3150 - Subchronic oral toxicity in dogs

In a subchronic oral toxicity study (MRID 43013203), male and female beagle 

dogs(four/sex/group) were fed diets containing 0,15,100, 500, or 1000 ppm G-28279 

technical for at least 14 weeks (equivalent to compound intakes of 0, 0.6, 3.8, 18.9, and 

33.4 mg/kg/day for male dogs, and 0.6, 3.8, 18.0, and 33.3 mg/kg/day for female dogs). 

Male dogs in the 1000-ppm and female dogs in the 500-ppm and 1000-ppm groups 

exhibited significant decreases in food consumption and decreased body weight gain 

relative to controls and to respective baseline values. Mean food efficiency was 

negative in male dogs of the 1000-ppm and female dogs of the 500-ppm and 1000-ppm 

groups. Clinical chemistry, hematology, and urinalysis data showed alterations that 

were of sporadic or intermittent occurrence, were not dose-related, and were not 

sustained for any period of time. Gross and histopathological examinations revealed no 
Page 53 of 90

treatment-related findings. A statistically significant decrease in mean absolute and 

mean relative (to brain) heart weight were observed for the 500-ppm and 1000-ppm 

male dogs but there were no histopathologic or functional (EKG) correlates indicating 

myocardial toxicity. Although the study authors attributed the alterations in heart weight 

to be secondary to inanition, the significant decrease (17% in the 500-ppm group, 

0.01<p<0.05; 25% in the 1000-ppm group, p≤0.01) in mean relative (to brain) heart 

weight may be indicative of a treatment-related effect. Similar findings were noted for 

organ-to-brain weight values for testes and prostate glands in the 500-ppm and 1000-

ppm dogs. Two dogs (litter mates) were found to be anemic but the condition was not 

considered to be related to test article exposure. Although most of the observed effects 

may be secondary to inanition, the decreased organ-to brain weight ratios for the heart, 

testes, and prostate gland, and the negative food efficiency observed in dogs exposed 

to dietary G-28279 at concentrations of 500 or 1000 ppm are indicative of marginally 

toxic effects. 

The LOAEL is 18.9 mg/kg/day, based on decreased food consumption, body 

weight, and heart weight (relative to brain weight). The NOAEL is 3.8 mg/kg/day. 

. 

This study is classified as Acceptable/Guideline and satisfies the guideline 

requirements for a subchronic 

feeding study (OPPTS 870.3150) 

in dogs.

870.3700 - Developmental toxicity in rats

In a developmental toxicity study (MRID 43013208), 24 Tif: RAI rats per group 

received G-28279 technical by gavage on gestational days (GDs) 6-15, inclusive, at 

dose levels of 0, 5, 25, or 100 mg/kg/day. Aqueous corn starch (3%) served as the 

control substance and vehicle for the test mixture. 

Maternal toxicity was observed at 25 and 100 mg/kg/day in a dose-related manner, as 

evidenced by effects on body weight/weight gain and food consumption. Body weights 

were significantly decreased at 25 mg/kg/day on GDs 9 and 13 (96% of control) and at 

100 mg/kg/day on GD 7 through GD 18 (93% of control). Weight gains were decreased 

on GDs 6-11 at 25 mg/kg/day (73% of control, nonsignificant) and at 100 mg/kg/day 

(30% of control, significant) and on GDs 11-16 at 100 mg/kg/day (89% of control, 

nonsignificant). Corrected weight gain was significantly decreased at 100 mg/kg/day 

(55% of control). Food consumption was significantly decreased on GDs 6-11 at 25 

mg/kg/day (91% of control) and at 100 mg/kg/day (70% of control). 

The maternal toxicity LOAEL is 25 mg/kg/day, based on decreased body weight 

and food consumption. The NOAEL is 5 mg/kg/day.

Developmental toxicity was observed at 25 and 100 mg/kg/day, as evidenced by effects 

on skeletal development. Fetal and litter incidences of fused sternebrae 1 and 2 were 

significantly increased at both these dose levels, while fetal incidences of poor 

ossification in sternebrae 2 and absent ossification in several digits were also increased 

significantly, but only at 100 mg/kg/day. 
Page 54 of 90

The developmental toxicity is 25 mg/kg/day, based on increased fetal and litter 

incidences of fused sternebrae. The NOAEL is 5 mg/kg/day. 

This study is classified as Acceptable-Guideline and satisfies the guideline 

requirements for a developmental toxicity study (OPPTS 870.3700) in rats.

870.5100 Bacterial Reverse Mutation Assay

The atrazine metabolite G-28279 (97.4%) was negative for inducing genetic 

reversions at the histidine locus in the Ames battery of Salmonella typhimurium or 

tryptophan prototrophy in Escheria coli WP2uvrA, when tested up to the limit dose of 

5000 μg/plate. This study is classified Acceptable-Guideline (MRID 43093101).

870.5385 Mammalian Bone Marrow Chromosome Aberration Test

The atrazine metabolite G-28279 (97.4%) was reported negative for the 

induction of micronuclei in polychromatic erythrocytes of Tif:MAGF mice treated orally at 

single doses up to clinically (but not cytologically) toxic levels (MTD = 480 mg/kg). This 

study is classified Acceptable-Guideline (MRID 43093103).

870.5550 UDS Assay 

The atrazine metabolite G-28279 (97.4%) was negative for inducing 

unscheduled DNA synthesis in primary rat hepatocyte cultures up to cytotoxic 

concentrations (800 μg/mL). This study is classified Acceptable-Guideline (MRID 

43093105).

E.4.10.3 G-30033 metabolite -2-chloro-4-amino-6-(isopropylamino) striazine; deethyl atrazine; isopropyl atrazine. 

Deethyl atrazine is a metabolite of atrazine, but not simazine . Deethyl 

atrazine is primarily a mammalian metabolite but can also be produced in plants and 

bacteria. Dealkylation of the ethyl group from the 4 position of the triazine ring yields 

deethyl atrazine. 

870.3100 - Subchronic oral toxicity in rats

In a subchronic study (MRID 43013206) G-30033 Technical, a metabolite of 

atrazine was given in the diet to four groups of 10 male and 10 female RAIf (SPF) rats 

at a concentration of 0, 10, 50, or 500 ppm for 13-weeks. The doses were equivalent to 

an average dose of 0.68, 3.2, and 35.1 mg/kg body weight/day or 0.72, 3.3, and 38.1 

mg/kg body weight/day for male and female rats, respectively. The only treatmentrelated effects reported include: a decrease in the body weight of high dose female rats 

and a decrease in the food efficiency of high-dose male and female rats. 

The LOAEL is 35.1 mg/kg/day, based on decreased body weight and food 

efficiency. The NOAEL is 3.2 mg/kg/day. 
Page 55 of 90

This study is classified as Acceptable-Guideline and satisfies the guideline 

(OPPTS 870.3100) requirements for a subchronic oral toxicity study in rats. 

870.3150 - Subchronic oral toxicity in dogs

In a subchronic oral toxicity study (MRID 43013203), Beagle dogs were 

divided into four groups, each containing four males and four females and given diets 

containing 0, 15, 100, or 1000 ppm G-30033 (equivalent to 0, 0.56, 3.71, or 28.85 

mg/kg/day for males and 0, 0.51, 3.88, and 32.18 mg/kg/day for females) for 13 weeks. 

Dietary exposure did not induce mortality, clinical signs of toxicity, ocular toxicity, or 

effects detectable in the urine. Treatment significantly decreased the weight gain of 

male and female dogs in the 1000 ppm group; however, the effect was attributed to 

inanition as a result of food palatability rather than an effect induced by the test material. 

The test material induced renal tubular hyperplasia/basophilia in 3/4 male and 2/4 

female dogs in the 1000 ppm treatment group. Marginal threshold effects found in male 

and female dogs fed diets containing 1000 ppm test material include: decreased 

absolute and relative (to brain) heart weights, normocytic/normochromic anemia, 

paroxysmal atrial fibrillation, and right atrial wall hemorrhagic inflammation with 

angiomatous hyperplasia. The marginal threshold effects may be related to G-30033 

treatment; however, without the use of pair-fed control dogs, they cannot be separated 

from effects induced by inanition. 

The LOAEL for male and female dogs is 30 mg/kg/day, based on renal tubular 

hyperplasia. The NOAEL is 3.8 mg/kg/day.

This study is classified as Acceptable-Guideline and satisfies the guideline 

requirements for a subchronic feeding study (OPPTS 870.3150) in beagle dogs.

870.3700 - Developmental toxicity in rats

In a developmental toxicity study (MRID 43013209), 24 Tif: RAI rats per group 

received G-30033 technical by gavage on gestational days (GDs) 6-15, inclusive, at 

dose levels of 0, 5, 25, or 100 mg/kg/day. Aqueous corn starch (3%) served as the 

control substance and vehicle for the test mixture. 

Maternal toxicity was observed at 25 and 100 mg/kg/day in a dose-related manner as 

evidenced by effects on body weight/weight gain and food consumption. Body weights 

were significantly decreased at 100 mg/kg/day (94% of control) on GD 7 through GD 20. 

Weight gains were significantly decreased on GDs 6-11 at 25 mg/kg/day (83% of 

control) and at 100 mg/kg/day (41% of control) and on GDs 11-16 at 100 mg/kg/day 

(87% of control). Corrected weight gains were non-significantly decreased at 25 

mg/kg/day (73% of control) and at 100 mg/kg/day (80% of control). Food consumption 

was significantly decreased on GDs 6-11 at 25 mg/kg/day (91% of control) and at 100 

mg/kg/day (70% of control).

The maternal toxicity LOAEL is 25 mg/kg/day, based on decreased body-weight 

gain and food consumption. The NOAEL is 5 mg/kg/day.
Page 56 of 90

Developmental toxicity was observed at 100 mg/kg/day as evidenced by effects on 

skeletal development. Fetal and litter incidences of fused sternebrae 1 and 2 were 

significantly increased at 100 mg/kg/day and the fetal incidence of poor ossification of 

the proximal phalanx of posterior digit 5 (a skeletal variation) at 100 mg/kg/day was also 

increased significantly.

The LOAEL for developmental toxicity is 100 mg/kg/day, based on increased fetal 

and litter incidences of fused sternebrae and increased fetal incidence of poor 

ossification of the proximal phalanx of posterior digit 5. The NOAEL is 25 

mg/kg/day.

This study is classified as Acceptable-Guideline, and it satisfies the guideline 

requirements for a developmental study (OPPTS 870.3700) in rats.

870.5100 Bacterial Reverse Mutation Assay 

The atrazine metabolite G-30033 (99.3%) was negative for inducing reverse 

gene mutation at the histidine locus of Salmonella typimurium and for the tryptophan 

locus in Escheria coli when tested to the limit dose, 5000 μg/plate. This study is 

classified Acceptable-Guideline (MRID 43093102).

870.5385 Mammalian Bone Marrow Chromosome Aberration Test

The atrazine metabolite G-30033 (99.3%) was reportedly negative for induction 

of micronuclei in the PCE of Tif:MAGF mice treated once orally up to clinically (but not 

cytologically) toxic doses (MTD = 480 mg/kg). This study is classified AcceptableGuideline (MRID 43093104).

870.5550 UDS Assay 

The atrazine metabolite G-30033 (99.3%) was negative for inducing 

unscheduled DNA synthesis in primary rat hepatocyte cultures exposed up to cytotoxic 

concentrations (1000 μg/mL). This study is classified Acceptable-Guideline (MRID 

43093106).

E.4.10.4 Hydroxyatrazine - 2- hydroxy - 4(ethylamino)- 6(isopropylamino) 

- s- triazine

Hydroxyatrazine is a metabolite of both atrazine and simazine. Hydroxyatrazine 

is primarily a plant metabolite but can also be produced in mammals and bacteria. 

Hydroxyatrazine is produced by dehalogenation of the chlorine from the triazine ring of 

either atrazine or simazine. Dealkylation reactions preceding or following the 

dehalogenation allow for the production of diamino, desisopropyl, and deethyl 

hydroxyatrazine or hydroxysimazine.

870.3100 - Subchronic oral toxicity in rats

In a subchronic toxicity study (MRID 41293501), hydroxyatrazine (97.1% ) was 

administered to 150 Sprague-Dawley rats, 15/sex/dose in the diet at dose levels of 0, 
Page 57 of 90

10, 100, 300 and 600 ppm (0, 0.64, 6.3, 18.89 and 37.47 mg/k/g/day for males and 0, 

0.75, 7.35, 22.75 and 45.64 mg/kg/day for females). 

With the exception of kidney alterations, most of the adverse effects of hydroxyatrazine 

exposure were limited to the high dose group. Body weights were non-significantly 

reduced in both sexes at the high dose only (7% reduction compared to controls in 

males and 5% reduction in females). Percent body weight gain was significantly 

decreased in both sexes at the high dose only (14.4% decrease compared to controls in 

males and 13.6% in females). Food consumption was significantly decreased in males 

only and only at a few selected timepoints. Hematocrit and erythrocyte counts were 

significantly depressed in both sexes at the high dose only (-7.2% and 7.6% in males 

and females respectively for hematocrit and -5.4% and 4.4% for males and females 

respectively for red blood cell counts). Hemoglobin was also significantly decreased in 

high dose males only (-6.4% compared to controls). Serum blood urea nitrogen was 

significantly increased in both sexes at the high dose only (+76% in males, +79.6% in 

females) as was creatinine (+38% in both sexes) sodium (+1.4% in males, +0.7% in 

females) and chloride (+2% in males, + 2.9% in females). Urine volume was 

significantly increased in both sexes at the high dose (+227% in males and +147% in 

females compared to controls). Urine volume was also significantly increased in males 

in the 300 ppm group (+38% compared to controls). Kidney weights - absolute and 

relative to both body and brain weight - were significantly increased in both sexes at the 

high dose only. Kidney weights relative to body weight were increased 44% in males 

and 45% in females compared to controls. 

Kidney alterations at both gross necropsy and at histopatholgy were seen in both the 

300 and 600 ppm dose groups. Toxic nephrosis classified as "minimal" was seen in 7 of 

15 males and 11 of 15 females at 300 ppm. Toxic nephrosis classified as severe was 

see in all animals of both sexes at the high dose. Tubule crystals were seen in 10 of 15 

males and 11 of 15 females at the high dose.

There were no compound- related effects on mortality, clinical signs, or ophthalmology 

in either sex at any dose. 

The LOAEL is 21 mg/kg/day, based on kidney alterations . The NOAEL is 6.8 

mg/kg/day.

This subchronic toxicity study is classified Acceptable-Guideline, and it satisfies the 

guideline requirement for a subchronic oral toxicity study (OPPTS 870.3100) in the rat.

870.3700 - Developmental toxicity in rats

In a developmental toxicity study (MRID 41065202), hydroxyatrazine, 97.6% 

a.i., was administered to 104 Sprague-Dawley females 26/dose by gavage at dose 

levels of 0, 5, 25, or 125 mg/kg/day from days 6 through 15 of gestation. 

There were no maternal deaths in this study. Two of the 22 pregnant females in the high 

dose group had enlarged, mottled kidneys, findings which were not seen in any other 

animal and are considered compound-related. Food consumption was significantly 

decreased (8.7%; p<0.05) at the high dose level only, during the 6-16 day dosing 

period. Body weight and body weight gain were unaltered by atrazine treatment. There 
Page 58 of 90

were no clinical signs attributable to compound exposure. There were no alterations in 

cesarean section parameters in dose groups vs controls.

The maternal LOAEL is 125 mg/kg/day, based on decreased food consumption 

during the dosing period and enlarged and mottled kidneys. The maternal NOAEL 

is 25 mg/kg/day.

Fetal death and resorptions were not altered by compound exposure. The high dose 

group only displayed increases in partially ossified interparietals (control fetal/litter 

incidence of 20%/56% vs high dose fetal/litter of 44%/91%); partially ossified hyoid 

(control fetal/litter 6%/20%, vs 17%/55%). Mean male and female fetal body weight at 

the high dose was significantly (p<0.05) reduced compared to controls (Control 

male/female body weight 3.6/3.4 grams vs male/female high dose 3.5/3.3 grams. There 

were no increases in any visceral findings in dosed fetuses.

The developmental LOAEL is 125 mg/kg/day, based on increased incidence of 

partially ossified interparietal and hyoid bones and decreased fetal body weight. 

The developmental NOAEL is 25 mg/kg/day.

The developmental toxicity study in the rat is classified Acceptable-Guideline, and it 

satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700; 

§83-3a) in the rat. 

870.4100 (870.4300) Chronic Toxicity – Rat

In a 2-year combined chronic feeding/carcinogenicity study (MRID 43532001), 

G-34048 technical (hydroxyatrazine) of 97.1% purity, was administered in the diet to 

groups of 70 or 80 male and 70 or 80 female Crl:CD (SD) BR strain rats at dose levels 

of 0 (control), 10, 25, 200 or 400 ppm (equivalent to 0, 0.388, 0.962, 7.75, or 17.4 

mg/kg/day in males and to 0, 0.475, 1,17, 9.35, or 22.3 mg/kg/day in females). Ten or 

20 rats/sex/group were sacrificed at 12 months; the remaining 60 animals in each group 

were scheduled to be sacrificed at 24 months. Due to high mortality in the 400 ppm 

group, however, the surviving males and females in the 400 ppm group were sacrificed 

at 18 months. Mortality, clinical signs of toxicity, body weights, food consumption and 

water consumption were monitored. Ophthalmologic examinations were performed. 

Hematological examinations, clinical chemistries and urinalysis were also performed. 

Necropsy examinations were conducted on all animals and organ weight determinations 

were made on all animals sacrificed at 12, 18, 24 months. Histopathological 

examinations were made on a complete set of organs/tissues from all animals in the 

control, 200 and 400 ppm groups. Histopathological examinations were also performed 

on a limited set of organs/tissues from the 10 and 25 ppm groups.

At 400 ppm, an excessive, treatment-related, mortality was observed for both males and 

females, and this dose level was terminated at 18 months. Severe renal failure was the 

predominant cause of death for these animals. Prior to death or sacrifice, these animals 

exhibited emaciation, dehydrated bodies, pallor and other clinical signs of toxicity 

expected in animals with severe renal failure. Greatly decreased body weights, body 

weight gains, and food consumption were observed in these animals throughout the 

study (to 18 months). Water consumption was increased during the first year of the 
Page 59 of 90

study. Changes in hematology parameters (including anemia), in clinical chemistries 

parameters (indicating renal disturbances), and in urinalysis parameters (including 

crystalline material in urine samples) were observed in 400 ppm males and females. 

Gross necropsies, organ weights, and histopatholgy indicated that kidney and lower 

urinary tract were the primary target organs in both males ands females at 400 ppm. 

Kidney effects included discoloration, calculi and rough pitted surfaces seen at gross 

necropsy; increased kidney weights; and severe histopathological changes including 

deposition of crystalline material within collecting ducts and renal pelvises, calculi, other 

morphological changes, and accelerated chronic progressive nephropathy. In addition, 

secondary effects in extra renal tissues reflected the severe renal damage and resulting 

renal failure in these animals. 

At 200 ppm, similar but less severe gross and histopathological effects on the kidneys 

were observed in both males and females. Secondary effects in extra renal tissues were 

generally not observed at this dose level. At 25 ppm, no treatment-related effects were 

observed in either male or female rats. An accumulation of interstitial matrix in the 

papilla of the kidneys of female rats was observed at this dose level, but the toxicologic 

significance of this observation, in the absence of any other signs of renal damage or 

impaired renal function, was highly questionable.

A treatment-related increased incidence of tumors of any type was not observed in the 

treated male or female animals in this study. In particular, there was no increase above 

control levels in the incidence of mammary gland tumors in either males or females. In 

addition, onset times for mammary gland tumors in female rats were not decreased in 

this study.

The LOAEL is 8.7 mg/kg/day, based on gross and histopathological effects in the 

kidneys. The NOAEL 1.0 mg/kg/day.
Page 60 of 90

This study is classified as Acceptable-Guideline, and it satisfies the OPPTS 870.4300 

guideline requirement for a combined chronic/carcinogenicity toxicity study. 

870.5100 Bacterial Reverse Mutation Assay 

The atrazine metabolite hydroxyatrazine (99% purity) failed to induce reverse 

mutation (as evidenced by no increase over controls in revertant colony counts) in 

Ames testing at concentrations up to the limit dose of 5000 μg/0.1 mL. This study is 

classified Acceptable-Guideline (MRID 40722304).

870.5385 Mammalian Bone Marrow Chromosome Aberration Test

The atrazine metabolite hydroxyatrazine (99% purity) did not induce an 

increase in micronuclei in mice fed concentrations up to the limit dose of 5000 mg/mL. 

This study is classified Acceptable-Guideline (MRID 41479401).

870.5550 Other genetic effects 

The atrazine metabolite hydroxyatrazine (96-99% purity) was negative for 

inducing unscheduled DNA synthesis in primary rat hepatocytes up to and beyond 

precipitating concentrations (> 500 μg/mL). This study is classified Acceptableguideline (MRID 40422305).

870.5550 Other genetic effects 

The atrazine metabolite hydroxyatrazine (96-99% purity) was negative for 

inducing unscheduled DNA synthesis in human fibroblasts (CRL 1521) up to and 

beyond precipitating concentrations (> 500 μg/mL). This study is classified 

Acceptable-guideline (MRID 40888101).

E.5.0 TOXICITY ENDPOINT SELECTION

E.5.1 See Section E.8.2 for endpoint selection table [TXR NO. 0050592]

E.5.2 Dermal Absorption

The maximum percentage of atrazine absorbed in the rat study after a 10 hr 

(representative of a typical workday) exposure was 21.6% (rounded up to 22%). The 

maximum percent absorbed after any duration of exposure in the human dermal 

penetration study described above under section 4.8, was 5.6% (rounded up to 6%). 

Because the maximum percent absorbed is being used and because an ample amount 

of time (168 hours) was allowed for absorption to occur, 6% is deemed to be a 

protective estimate of dermal penetration in the human. 

E.5.3 Classification of Carcinogenic Potential

E.5.3.1 Conclusions
Page 61 of 90

Numerous studies, both guideline and non-guideline studies submitted by the 

registrant, as well as studies performed by EPA's NHEERL [described in this 

document], examine the carcinogenicity and mode of action of atrazine. In a series of 

meetings [October 13, 1999 , April 12, 2000, and October 19, 2000] held subsequent to 

the Fifth HED Cancer Peer Review Committee (CPRC) of July, 1996, the Cancer 

Assessment Review Committee (CARC) met to examine these studies. The data 

pertaining to atrazine's carcinogenicity and mode of action were also examined by the 

FIFRA Science Advisory Panel (SAP) on June 27th to June 29th, 2000. Both the CARC 

and SAP agreed that atrazine is associated with mammary and pituitary tumor formation 

in the female SD rat, but not in male SD rats or CD-1 mice and F-344 rats of either sex. 

CARC and SAP also agreed that there are sufficient data available to support a 

proposed mode of action (MOA) to account for the tumors seen in SD female rats and 

that this MOA would likely not result in carcinogenicity in humans. Hypothalamic 

neuronal - GnRH pulse modulation of pituitary releases of LH is a central driver of 

ovulation in the SD female rat, and atrazine is essentially accelerating the aging process 

of the CNS control of ovulation, which leads to a constant state of estrus (anovulation) 

and prolong exposure to estrogen and prolactin. Although there are certain similarities 

in the control of the hypothalamic-pituitary- ovarian axis between humans and rats in 

that the hypothalamus can play a key regulatory role in primates, there are fundamental 

differences. For example, unlike the SD rat, CNS modulation is not the driving factor on 

human GnRH and LH releases. Human conditions of anovulation are associated with 

aberrant GnRH and LH pulsatile releases. Even if atrazine induced anovulation in 

humans like in the SD rat, there is no evidence for the potential of an unopposed 

estrogen condition that would lead to tumor development. It appears that when LH is 

low, such as in hypothalamic amenorrhea (HA), a state of low serum estrogen is found, 

not elevated or prolong estrogen exposure. It should be noted that there is no known 

cancer risk associated with HA patients, albeit they are at risk to a number of other 

clinical conditions (e.g., osteoporosis, heart disease, infertility). Another condition of 

anovulation, polycystic ovarian syndrome (PCOS), is also not a good model for atrazine 

cancer mode of action in SD rats. The etiology of PCOS is multifactorial, and LH 

secretion is elevated due to increased synthesis of androgen and their conversion to 

estrogens. Although atrazine might cause adverse effects on hypothalamic-pituitary 

function in humans, the hormonal environment conducive to tumor development (i.e., 

elevated or prolonged exposure to estrogen or prolactin) that is found in SD rats cannot 

be established in humans. Therefore, it is unlikely that atrazine’s cancer mode of action 

in SD rats is operative in humans. Additionally, mutagenic and estrogenic activity do not 

appear to play a significant role in atrazine-associated carcinogenicity. 

E.5.3.2 Classification Carcinogenic Potential

The CARC concluded that atrazine should be classified as a "Not Likely To 

Be Carcinogenic To Humans” [HED Document 014431; December 13, 2000 meeting], 

based on the overall weight of evidence that:

1. The mode of carcinogenic activity in the female SD rat is supported by the 

data. 

2. The mode of carcinogenic activity in the female SD rat essentially involves 

an acceleration of the reproductive aging process.
Page 62 of 90

3. The mode of action for the carcinogenicity of atrazine is unlikely to be 

expressed in humans; no human conditions can be established that support a 

potential for atrazine to lead to carcinogenicity in humans.

4. Other modes of action are not supported by the available data and, in 

particular, mutagenic and estrogenic activity do not appear to significantly 

contribute to atrazine’s carcinogenic potential.

5. Although a few epidemiological studies suggest a possible association 

between atrazine (or triazine) exposure and NHL and ovarian cancer, these 

cancers do not appear to be plausible based on atrazine’s mode of action. 

Therefore, the human studies by themselves do not make a strong case for an 

association.

5.3.3 Quantification of Carcinogenic Potential

Not applicable as atrazine is classified as a "Not Likely To Be Carcinogenic To 

Humans".

E.6.0 FQPA CONSIDERATIONS

E.6.1 Special Sensitivity to Infants and Children

There was no increased quantitative or qualitative susceptibility in any of the guideline 

studies on atrazine in the rat, and there was no increased quantitative susceptibility in 

the rabbit study. However, there was increased qualitative susceptibility in the rabbit 

study [increased resorptions (deaths) at a dose level that resulted in decreased bodyweight gain and clinical signs in the maternal animal]. There were other non-guideline 

studies on atrazine that show evidence of endocrine disruption [prostatitis study, 

delayed puberty study, and data on LH surge attenuation, and estrous cycle alterations]. 

The primary underlying events that led to mammary and pituitary tumor formation 

following atrazine exposure of Sprague-Dawley female rats involved disruption of the 

hypothalamic-pituitary-ovarian axis. Since aspects related to this axis are involved in 

reproductive and developmental competency, there is a concern for adverse 

reproductive and developmental effects in maternal animals and their offspring. Several 

special studies showed that treatment of pregnant rats with atrazine can lead to 

reproductive and developmental effects that may be associated with endocrine 

alterations. Additionally, the neurotoxicity seen in the non-guideline studies with atrazine 

is a central nervous system (CNS) toxicity - specifically, neurotransmitter and 

neuropeptide alterations at the level of the hypothalamus.

Studies in the open literature indicate increased qualitative susceptibility. Dosing of 

dams immediately following parturition [postnatal days 1-4] resulted in prostatitis in male 

offspring, and dosing of the young following weaning resulted in delayed puberty in both 

sexes. The mode of action for these two effects (prostate inflammation and delayed 

puberty) is believed to be similar to the mode of action described for atrazine-associated 

cancer and involves the CNS neuroendocrine alterations, specifically, neuroendocrine 

alterations at the hypothalamus.
Page 63 of 90

Following a Degree of Concern Analysis [TXR No. 0050592; see 8.2 below], the HIARC 

concluded that an additional Special FQPA Safety Factor of 3X would be adequate to 

account for the hazard-based residual uncertainties for chronic dietary [cRfD] and 

residential (incidental oral, dermal, inhalation) exposures. Additionally, it was concluded 

that an additional Special FQPA Safety Factor would not be required for acute dietary 

exposure [aRfD]. Refer to the Risk Assessment on Atrazine for a discussion of the 

FQPA Safety Factor used for the various exposure scenarios [D272009]. 

E.6.2 Recommendation for a Developmental Neurotoxicity Study

A developmental toxicity study is not available and HIARC did not recommend that one 

be performed.
Page 64 of 90

E.7.0 REFERENCES (LISTED IN ORDER OF ASCENDING MRID NUMBER)

00024706 Sachsse, K.; Bathe, R. (1975) Acute Oral LD450^ of Technical Atrazine (G 

30027) in the Rat: Project No. Siss 4569. (Unpublished study received 

Jun 2, 1977 under 100-529; prepared by Ciba-Geigy, Ltd., submitted by 

Ciba-Geigy Corp., Greensboro, N.C.; CDL:230303-E)

00024709 Sachsse, K.; Ullmann, L. (1976) Eye Irritation in the Rabbit of G 30027: 

Project No. Siss 5663. (Unpublished study received Jun 2, 1977 under 

100-529; prepared by Ciba-Geigy, Ltd., submitted by Ciba-Geigy Corp., 

Greensboro, N.C.; CDL:230303-H)

00024710 Sachsse, K.; Ullmann, L. (1976) Skin Irritation in the Rabbit after Single 

Application of G 30027: Project No. Siss 5663. (Unpublished study 

received Jun 2, 1977 under 100-529; prepared by Ciba-Geigy, Ltd., 

submitted by Ciba-Geigy Corp., Greensboro, N.C.; CDL:230303-I)

00027097 Consultox Laboratories, Limited (1974) Atrazine: Acute Oral and Dermal 

Toxicity Evaluation. (Unpublished study received April 22, 1976 under 

33660-1; submitted by Industria Prodotti Chemici, S.p.a., Novate 

Milanese, Italy; CDL:225976-A)

00105131 Burlingham, M.; Young, S.; Adamik, E. (1979) Comparative Skin 

Sensitization of Technical Atrazine: Project No. WIL-1214-78. 

(Unpublished study received May 12, 1982 under 201-411; prepared by 

WIL Research Laboratories, Inc., submitted by Shell Chemical Co., 

Washington, DC; CDL:247726-A)

00150623 Puri, E. 1984. Autoradiographic DNA repair test on rat hepatocytes. CibaGeigy. Basle, Switzerland. Study No. 831371. May 16, 1984. 

00143006 Arthur, A. (1984) Segment II Teratology Study in Rabbits: 

Toxicology/Pathology Report No. 68-84. Unpublished study prepared by 

Ciba-Geigy Corp. 220 p.

00143008 Infurna, R. (1984) A Teratology Study of Atrazine Technical in Charles 

River Rats: Toxicology/Pathology Report No. 60-84. Unpublished study 

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40246601 Deparade, E. (1986) Atrazine: Salmonella/Mammalian--Microsome 

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Page 65 of 90

40431301 O'Connor, D.; McCormick, G.; Green, J. (1987) Chronic Toxicity Study in 

Dogs: Atrazine Technical: Laboratory Study No. 852008. Unpublished 

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40431302 Hazelette, J.; Green, J. (1987) Oncogenicity Study in Mice: Atrazine 

Technical: Laboratory Study No. 842120. Unpublished study prepared by 

Ciba-Geigy Corp. 3160 p.

40431303 Mainiero, J.; Youreneff, M.; Giknis, M.; et al. (1987) Two-generation 

Reproduction Study in Rats: Atrazine Technical: Laboratory Study No. 

852063. Unpublished study prepared by Ciba-Geigy Corp. 1395 p.

40431304 Orr, G. (1987) Disposition of Atrazine in Rat: (General Metabolism): 

Atrazine: Laboratory Study No.: ABR-87048. Unpublished study prepared 

by Ciba-Geigy Corp. 62 p

40431305 Thede, B. (1987) Study of ?Carbon 14F-Atrazine Dose/Response 

Relationship in the Rat: (General Metabolism): Laboratory Study No. ABR87087. Unpublished study prepared by Ciba-Geigy Corp. 53p.

40431306 Miles, B. (1987) Characterization and Identification of Atrazine Metabolites 

from Rat Urine: (General Metabolism): Laboratory Study No.: ABR-87115. 

Unpublished study prepared by Ciba-Geigy Corp. 35 p.

40437501 Orr, G. (1987) A Summary of the Disposition, Kinetics and Metabolism of 

Atrazine in the Rat (General Metabolism): Laboratory/Study No. ABR87116. Unpublished study prepared by Ciba-Geigy Corp. 44 p.

40566301 Arthur, A. (1984) A Supplement to a Teratology Study of Atrazine 

Technical in New Zealand White Rabbits. Unpublished study prepared by 

Ciba-Geigy Corporation. 4 p.

40566302 Infurna, R. (1984) A Supplement to a Teratology Study of Atrazine 

Technical in Charles River Rats. Unpublished study prepared by CibaGeigy Corporation. 6 p.

40629302 Hardisty, J. (1987) Supplement to Two-year Chronic 

Feeding/Oncogenicity Study in Rats Administered Atrazine: 410-1102. 

Unpublished study prepared by Experimental Pathology Laboratories, Inc. 

316 p.

40722301 Ceresa, C. (1988) Atrazine: Structural Chromosomal Aberration Test 

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Page 66 of 90

40722302 Deparade, E. (1987) Diaminochlorotriazine: Gene Mutations Test: 

Salmonella/ Mammalian Mutagenicity Test: Study No. 871372. 

Unpublished study prepared by Ciba-Geigy Corp. 26 p.

40722303 Meyer, A. (1987) Diaminochlorotriazine: Tests for Other Genetoxic Effects: 

Autoradiographic DNA Repair Test on Human Fibroblasts: Study No. 

871371. Unpublished study prepared by Ciba-Geigy Corp. 81 p.

40722304 Deparade, E. (1988) Hydroxyatrazine: Gene Mutations Test: Salmonella/ 

Mammalian-Microsome Mutagenicity Test: Study No. 871376. 

Unpublished study prepared by Ciba-Geigy Corp. 27 p.

40722305 Hertner, T. (1988). Tests for other genotoxic effects - auto-radiographic 

DNA repair test on rat hepatocytes. Ciba-Geigy, Basle, Switzerland. 

Laboratory report number: 871374. January 22, 1988. Unpublished.

40888101 Meyer, A. (1988) Hydroxyatrazine: Tests for other genotoxic effects, 

autoradiographic DNA repair tests on human fibroblasts. Ciba-Geigy, 

Basle, Switzerland . Study No. 871375. January 11, 1988. Unpublished.

41065201 Giknis, M. (1989) A Teratology (Segment II) Study in Rats: Atrazine 

Technical: Laboratory Study No. 882049. Unpublished study prepared by 

Ciba-Geigy Corp. 357 p

41065202 Giknis, M. (1989) A Teratology (Segment II) Study in Rats: 

Hydroxyatrazine Technical: Laboratory Study No. 872202. Unpublished 

study prepared by Ciba-Geigy Corp. 362 p.

41293501 Rudzki, M.; McCormick, G.; Arthur, A. (1989) 90-Day Oral Toxicity Study 

in Rats: Hydroxyatrazine: Lab Study Number 882146; MIN 882146; 

89015. Unpublished study prepared by Ciba-Geigy Corp., Research 

Dept., Pharmaceuticals Div. 408 p.

41392401 Thompson, S.; Batastini, G.; Arthur, A. (1990) 13/52-Week Oral Toxicity 

Study in Dogs: Lab Project Number: 872151. Unpublished study prepared 

by Ciba-Geigy Corp. 784 p.

41392402 Hummel, H.; Youreneff, M.; Giknis; et al. (1989) Diaminochlorotriazine: A 

Teratology (Segment II) Study in Rats: Lab Project Number: 872177. 

Unpublished study prepared by Ciba-Geigy Corp. 428 p.

41479401 Ceresa, C. (1988) Hydroxyatrazine: Structural Chromosomal Aberration 

Test: Lab Project Number: 871373. Unpublished study prepared by CibaGeigy, Ltd. 32 p.
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42085001 Thakur, A. (1991) Determination of Hormone Levels in Sprague-Dawley 

Rats Treated with Atrazine Technical: Final Report: Lab Project Number: 

483-278. Unpublished study prepared by Hazleton Washington, Inc. 

1132 p.

42089901 Holbert, M. (1991) Acute Inhalation Toxicity Study in Rats: Atrazine 

Technical: Lab Project Number: 8079-91. Unpublished study prepared by 

Stillmeadow Inc. 23 p.

42146101 Thakur, A. (1991) Determination of Hormone Levels in Fischer-344 Rats 

Treated with Atrazine Technical: Final Report: Lab Project Number: 483-

279. Unpublished study prepared by Hazleton Washington, Inc. 1534 p.

42165503 Thede, B. (1991) Response to Data Request in EPA Review of Atrazine 

Registration Standard--Toxicity Studies: Lab Project Number: ABR-91076. 

Unpublished study prepared by Ciba-Geigy Corp. 301 p.

42204401 Thakur, A. (1992) Oncogenicity Study in Sprague-Dawley Rats with 

Atrazine Technical: Lab Project Number: 483-275. Unpublished study 

prepared by Hazleton Washington, Inc. 1092 p.

42227001 Thakur, A. (1992) Oncogenicity Study in Fischer 344 Rats with Atrazine 

Technical: Lab Project Number: 483-277. Unpublished study prepared by 

Hazleton Washington, Inc. 2999 p.

42547105 Hertner, T. (1992) Autoradiographic DNA Repair Test on Rat Hepatocytes 

Tests for Other Genotoxic Effects: Lab Project Number: 911246. 

Unpublished study prepared by Ciba-Geigy Limited. 98 p.

42743902 Eldridge, J., Wetzel, L., Tisdel, M., and Luempert, L.G. 1993a. 

Determination of Hormone Levels in Sprague-Dawley Rats Treated with 

Atrazine Technical: Revised Supplement to Final Report. Hazleton 

Washington, Inc. Lab Project Number: 483-278. 

42743903 Eldridge, J.; Wetzel, L.; Tisdel, M. et al. (1993) Determination of Hormone 

Levels in Fischer-344 Rats Treated with Atrazine Technical: Revised 

Supplement to Final Report: Lab Project Number: 483-279. Unpublished 

study prepared by Bowman Gray School of Medicine. 166 p.

43013201 Kuhn, J. (1991) G-28279 Technical: Acute Oral Toxicity Study in Rats: Lab 

Project Number: 7803/91. Unpublished study prepared by Stillmeadow, 

Inc. 27 p.

43013202 Kuhn, J. (1991) G-30033 Technical: Acute Oral Toxicity Study in Rats: Lab 

Project Number: 7802/91. Unpublished study prepared by Stillmeadow, 

Inc. 28 p.
Page 68 of 90

43013203 Thompson, S.; Batastini, G.; Arthur, A. (1992) G-28279 Technical: 90-Day 

Oral Toxicity Study in Dogs: 13-Week Feeding Study in Dogs: Lab Project 

Number: 912021. Unpublished study prepared by Research Dept. CibaGeigy Corp. 429 p

43013205 Schneider, M. (1992) G-28279 Technical: 90-Day Oral Toxicity Study in 

Rats: 3-Month Oral Toxicity Study in Rats (Administration in Food): Lab 

Project Number: 901261. Unpublished study prepared by Ciba-Geigy Ltd. 

263 p.

43013206 Gerspach, R. (1991) G-30033 Technical: 90-Day Oral Toxicity Study in 

Rats: 3-Month Oral Toxicity Study in Rats (Administration in Food): Lab 

Project Number: 901264. Unpublished study prepared by Ciba-Geigy Ltd. 

254 p.

43013207 Pettersen, J.; Richter, A.; Gilles, P. (1991) Diaminochlorotriazine (G28273): 90-Day Oral Toxicity Study in Rats: Lab Project Number: F-00006. 

Unpublished study prepared by Ciba-Geigy Corp. 450 p.

43013208 Marty, J. (1992) G-28273 Technical: Teratology Study in Rats: 

Developmental Toxicity (Teratogenicity) Study in Rats with G-28279 

Technical (Oral Administration): Lab Project Number: 901262. 

Unpublished study prepared by Ciba-Geigy Ltd. 298 p.

43013209 Marty, J. (1992) G-30033 Technical: Teratology Study in Rats: 

Developmental Toxicity (Teratogenicity) Study in Rats with G-30033 

Technical (Oral Administration): Lab Project Number: 901265. 

Unpublished study prepared by Ciba-Geigy Ltd. 296 p.

43016502 Sova, J. (1993) Atrazine Technical: Response to the EPA Review of the 

Acute Inhalation Toxicity Study. Unpublished study prepared by Ciba 

Plant Protection. 5 p.

43093101 Deparade, E. (1990) Salmonella and Escherichia/Liver-Microsome Test: 

G-28279 Technical: Gene Mutations Test: Lab Project Number: 891243. 

Unpublished study prepared by Ciba-Geigy Limited. 39 p.

43093102 Deparade, E. (1989) Salmonella and Escherichia/Liver-Microsome Test: 

G-30033 Technical: Gene Mutations Test: Lab Project Number: 891236. 

Unpublished study prepared by Ciba-Geigy Limited. 37 p.

43093103 Ogorek, B. (1991) Structural Chromosomal Aberration Test: Micronucleus 

Test, Mouse: G-28279 Technical: Lab Project Number: 901307. 

Unpublished study prepared by Ciba-Geigy Limited. 37 p.
Page 69 of 90

43093104 Ogorek, B. (1991) Structural Chromosomal Aberration Test: Micronucleus 

Test, Mouse: G-30033 Technical: Lab Project Number: 901309. 

Unpublished study prepared by Ciba-Geigy Limited. 40 p.

 43093105 Geleick, D. (1991) Tests for Other Genotoxic Effects: Autoradiographic 

DNA Repair Test on Rat Hepatocytes: G-28279 Technical: Lab Project 

Number: 901308. Unpublished study prepared by Ciba-Geigy Limited. 94 

p.

43093106 Geleick, D. (1991) Tests for Other Genotoxic Effects: Autoradiographic 

DNA Repair Test on Rat Hepatocytes: G-30033 Technical: Lab Project 

Number: 901310. Unpublished study prepared by Ciba-Geigy Limited. 94 

p.

43314302 Chengelis, C. (1994) A Dermal Radiotracer Absorption Study in Rats with 

(carbon 14) Atrazine: Final Report: Lab Project Number: 82048: 89/90/B. 

Unpublished study prepared by WIL Research Laboratories, Inc. 510 p.

43532001 Chow, E.; Hart, S. (1995) 2-Year Dietary Toxicity/Oncogenicity Study with 

G-34048 (Hydroxyatrazine) in Rats: Final Report: Lab Project Number: F00125. Unpublished study prepared by Ciba-Geigy Corp. 2599 p.

43598617 Tennant, M.; Hill, D.; Eldridge, J.; et al. (1994) Possible antiestrogenic 

properties of chloro-s-triazines in rat uterus. Journal of Toxicology and 

Environmental Health 43:183-196.

43598618 Tennant, M.; Hill, D.; Eldridge, J.; et al. (1994) Chloro-s-triazine 

antagonism of estrogen action: Limited interaction with estrogen receptor 

binding. Journal of Toxicology and Environmental Health 43:197-211

43598619 Safe, S.; Chen, I.; Liu, H.; et al. (1995) Failure of Atrazine and Simazine to 

Induce Estrogenic Responses in MCF-7 Human Breast Cancer Cells. 

Unpublished study prepared by Texas A&M Univ.; and Univ. of Western 

Ontario. 25 p.

43598622 McConnell, R. (1995) A Histomorphologic Reevaluation of the Ovaries, 

Uterus, Vagina, Mammary Gland, and Pituitary Gland From SpragueDawley and Fischer-344 Female Rats Treated With Atrazine: Lab Project 

Numbers: 483-278: 483-279. Unpublished study prepared by Ciba-Geigy 

Corp. 107 p.
Page 70 of 90

43934403 Safe, S. (1995) Failure of chloro-s-triazine derived compounds to induce 

estrogenic responses in vivo and in vitro. in Fundamental Applied 

Toxicology (in Press).

43934404 Morseth, S. (1996) Evaluation of the Luteinizing Hormone (LH) in Female 

Sprague-Dawley Rats--Pilot Study: Final Report: Lab Project Number: 

CHV 2386-109: 6791. Unpublished study prepared by Corning Hazleton 

Inc. (CHV). 33 p.

43934406 Morseth, S. (1996) Evaluation of the Luteinizing Hormone (LH) Surge in 

Atrazine-Exposed Female Sprague-Dawley Rats: Interim Report: Lab 

Project Number: CHV 2386-111: 6791E: 2386-111. Unpublished study 

prepared by Corning Hazleton Inc. (CHV). 624 p.

44152102 Morseth, S. (1996) Evaluation of the Luteinizing Hormone (LH) Surge in 

Atrazine-Exposed Female Sprague-Dawley Rats--(Final) 6-Month Interim 

Report: Lab Project Number: CHV 2386-111: 2386-111: 6791E. 

Unpublished study prepared by Corning Hazleton Inc. 727 p.

44544701 Morseth, S. (1998) Chronic (12-24 Month) Study in Rats with Atrazine 

Technical. Study No. 2386-108. Unpublished study prepared by Covance 

Laboratories Inc. 2251 p.

44152114 Hui, X.; Gilman, S.; Simoneaux, B.; et al. (1996) In vivo Percutaneous 

Absorption of Atrazine in Man: Lab Project Number: ABR-96067: BDH081-2: H832-11835-01. Unpublished study prepared by UCSF; UC Davis; 

and Ciba Crop Protection. 297 p.

44713802 Paul, H. J.; Dunsire, J.P.; Hedley, D. (1993). The Absorption, Distribution, 

Degradation and Excretion of [U14C]-Triazine G 30027 in the Rat. 

Inveresk Research International, Tranent, EH33 2NE, Scotland. IRI Report 

No. 9523. December 10, 

45058701 Minnema, D.J. (2000) Comparison of the LH surge in female rats 

administered atrazine, simazine or DACT via oral gavage for one month. 

Covance # 6117-398 Novartis # 1198-98. Covance Labs, Vienna, VA. 

Feb. 29, 2000. Unpublished study.

45166901 Cooper, R.L., Stoker, T.E., Tyrey, L., Goldman, J.M., and McElroy, W.K. 

2000. Atrazine disrupts the hypothalamic control of pituitary-ovarian 

function. Tox. Sci. 53: 297-307.

45166902 Stoker, T.E., Robinette, C.L., and Cooper, R.L. (1999) Maternal exposure 

to atrazine during lactation suppresses suckling-induced prolactin release 

and results in prostatitis in the adult male offspring. Tox. Sci. 52;68-79.
Page 71 of 90

Studies which do not have an MRID number:

Cooper, R.L., Stoker, T.E., Goldman, J.M., Parrish, M.B., Tyrey, L. (1996) Effect of 

atrazine on ovarian function in the rat. Reprod Toxicol 1996 Jul-Aug;10(4):257-64. 

MRID not yet assigned.

Das, P.C., McElroy, W.K., Cooper, R.L. (2000) Differential modulation of 

catecholamines by chlorotriazine herbicides in pheochromocytoma (PC12) cells in vitro. 

Tox. Sci. Aug; 56(2): 324-31. MRID not yet assigned.

Laws, S.C., Ferrell, J.M., Stoker, T.E., Schmid, J., and Cooper, R.L. (2000).The effect 

of atrazine on puberty in female wistar rats: an evaluation in the protocol for the 

assessment of pubuertal development and thyroid function. Tox. Sci. In Press. MRID 

not yet assigned.

Narotsky M. G., Best, D.S., Guidici, D. L., and Cooper, R.L. (2000). Strain 

comparisons of atrazine-induced pregnancy loss in the rat. Repro Toxicol. In Press. 

MRID not yet assigned.

Shafer, T.J., Ward, T.R., Meacham, C.A., Cooper, R.L. (1999) Effects of the 

chlorotriazine herbicide, cyanazine, on GABA(A) receptors in cortical tissue from rat 

brain. Toxicology 1999 Dec 20;142(1):57-68. MRID not yet assigned.

Stoker, TE, Laws, SC, Guidici, D and Cooper, RL. (2000) The effect of atrazine on 

puberty in male Wistar rats: An evaluation in the protocol for the assessment of 

pubertal development and thyroid function. Toxicol. Sci. 58: 50-59. 

Thakur, A.K. (1999). Addendum 1 to study no. 2386-108: Chronic (12/24 month) study 

in rats with atrazine technical (EPA MRID No. 445701). Unaudited draft interim report 

of the estrous cycle data through week 46. Covance Lab, Vienna VA. Covance Lab 

No. 2386-108. Novartis Nexus # 810-95. March 12, 1999.
Page 72 of 90

E.8.0 APPENDICES

E.8.1 Toxicity Profile Summary Tables

E.8.1.1 Acute Toxicity Table for Atrazine

Acute Toxicity Data of Technical Atrazine

Guideline 

No.

Test Results Tox. Cat.

870.1000 Oral LD50 - rat LD50 > 1,869 mg/kg (M&F 

combined)

 III

870.1200 Dermal LD50 -

rat

LD50 > 2,000 mg/kg (M&F 

combined)

 III

870.1300 Inhalation LC50 -

rat

LC50 > 5.8 mg/L (M&F combined) IV

870.1400 Eye Irritation -

rabbit

PIS = 0.0/110 IV

870.1500 Dermal Irritation 

- rabbit

PIS = 0.2/8.0 IV

870.1600 Dermal 

Sensitization -

guinea pigs

Non-sensitizing ---

870.6100 Acute Delayed 

Neurotoxicity

Not Required ---

PIS=Primary Irritation Score

E.8.1.2 Subchronic, Chronic and Other Toxicity Table for Atrazine

Subchronic, Chronic and Other Toxicity Table. Only studies considered acceptable by 

HED are shown.

Guideline No./ 

Study Type

MRID No. (year) 

/Doses

Results

870.3100

90-Day oral 

toxicity rodents

44723701 (1994)

0, 10, 50, or 500 

ppm 0, 0.6, 3.3, 34.0 

mg/kg/day - males

0, 0.659, 3.35, 35.3 

mg/kg/day - females. 

NOAEL = 3.30 mg/kg/day

LOAEL = 34.5 mg/kg/day based on 

decreased body weight
Page 73 of 90

Guideline No./ 

Study Type

MRID No. (year) 

/Doses

Results

870.3150

90-Day oral 

toxicity in 

nonrodents 

(dogs)

---

Study waived because an acceptable 

chronic oral study in the dog is available. 

870.3200

21/28-Day 

dermal toxicity 

42089902 (1989)

0, 10, 100 or 1000 

mg/kg/day

NOAEL = 100 mg/kg/day

LOAEL = 1000 mg/kg/day based on 

statistically significant reductions in food 

consumption, mean body weight, and 

percent weight gain in both sexes, 

statistically significantly increased absolute 

and relative spleen weights in both sexes

870.3250

90-Day dermal 

toxicity

---

Study not required

870.3465

90-Day inhalation

toxicity

---

Study not required

870.3700a

Prenatal 

developmental in 

rodents

40566302 (1984)

0, 10, 70, or 700 

mg/kg/day

Maternal NOAEL = 10 mg/kg/day.

Maternal LOAEL = 70 mg/kg/day, based on 

reduced body weight gain

Developmental NOAEL = 10 mg/kg/day

Developmental LOAEL = 70 mg/kg/day 

based on delayed or no ossification at 

several sites

870.3700a

Prenatal 

developmental in 

rodents

41065201 (1989)

0, 5, 25, 100 

mg/kg/day

Maternal NOAEL = 25 mg/kg/day.

Maternal LOAEL = 100 mg/kg/day based on 

reduced body weight gain and food 

consumption.

Developmental NOAEL = 25 mg/kg/day.

Developmental LOAEL = 100 mg/kg/day 

based on increased incidence of delayed 

ossification of skull bones.

870.3700b

Prenatal 

developmental in 

00143006, 

40566301 (1984)

0, 1, 5, or 75 

Maternal NOAEL = 5 mg/kg/day

Maternal LOAEL = 75 mg/kg/day based on 

decreased body weight, food consumption 
Page 74 of 90

Guideline No./ 

Study Type

MRID No. (year) 

/Doses

Results

nonrodents mg/kg/day and increased incidence of clinical signs 

Developmental NOAEL = 5 mg/kg/day

Developmental LOAEL = 75 mg/kg/day 

based on reduced litter size, increased 

resorptions and delayed ossification. 

870.3800

Reproduction and 

fertility effects

40431303 (1987)

0, 10, 50,and 500 

ppm

0, 0.75, 3.78, 39.0 

mg/kg/day - males 

0, 0.86, 3.70, 42.8 

mg/kg/day - females.

NOAEL = 3.78 mg/kg/day

LOAEL = 39 mg/kg/day in males based on 

decreased body weights, body weight gains 

and food consumption 

Offspring NOAEL = 3.78 mg/kg/day

Offspring LOAEL = 39 mg/kg/day based on 

decreased body weights in both generations 

of males at PND 21. 

870.4100a

Chronic toxicity 

rodents

40629302 (1986)

0, 10, 70, 500, 1000 

ppm, 

0, 0.5, 3.5 25, 50 

mg/kg/day

This guideline satisfied by 870.4300

Combined chronic toxicity/carcinogenicity

NOAEL = 3.5 mg/kg/day

LOAEL = 25 mg/kg/day, based on reduced 

body-weight gain and food consumption

See below under 870.4300 for details

870.4100b

Chronic toxicity 

nonrodents 

(dogs)

40431301 (1987)

0, 15, 150, 1000 

ppm

0, 0.5, 5.0 33.7 

mg/kg/day

NOAEL = 5.0 mg/kg/day

LOAEL = 33.7 mg/kg/day based on cardiac 

effects.

870.4200

Carcinogenicity 

(rat, Fischer-344)

42227001 (1992)

0, 10, 70, 200, 400 

ppm

0, 0.5, 3.4, 9.9, 20.2 

mg/kg/day - males

0, 0.6, 4.4, 12.7, 

26.2 mg/kg/day -

females

NOAEL = 3.4 mg/kg/day -males; 4.4 

mg/kg/day - females

LOAEL = 9.9 mg/kg/day - males; 12.7 

mg/kg/day - females based on decreased 

body weight gain

There was no treatment-related increase in 

tumor incidence when compared to controls. 

This study used Fischer- 344 rats. The 

purpose was to demonstrate a lack of tumor 

response in this strain following atrazine 

exposure.

870.4200

Carcinogenicity 

40431302 (1987)

0,10,300,1500, 3000 

ppm

NOAEL = 43 mg/kg/day

LOAEL = 222.0 mg/kg/day based on 

decreased body weight gain in both sexes 
Page 75 of 90

Guideline No./ 

Study Type

MRID No. (year) 

/Doses

Results

(mice) 0, 1.4, 38.4, 194, 

385.7 mg/kg/day -

males

0, 1.6, 47.6, 246.9, 

482.7 mg/kg/day -

females

and increased cardiac thrombi in the 

females.

No evidence of carcinogenicity was seen.

870.4300

Combined 

chronic toxicity/

carcinogenicity

(rat, SpragueDawley)

40629302 (1986)

0, 10, 70, 500, 1000 

ppm, 

0, 0.5, 3.5 25, 50 

mg/kg/day

NOAEL = 3.5 mg/kg/day

LOAEL = 25 mg/kg/day ,based on reduced 

body weight gain and food consumption.

Mammary tumors increased at 3.5 

mg/kg/day and above.

This study used the Sprague Dawley strain 

of rat.

870.5100

Bacterial reverse 

mutation assay

00060642 (1977)

0, 50, 100, 500, 

1000, 5000 μg/plate

Negative in strains TA 98, 100, 1535 and 

1537 of S. typhimurium up to the limit 

concentration of 5000 μg/plate both with and 

without activation

870.5100

Bacterial reverse 

mutation assay

40246601 (1986)

0, 20, 78, 313, 

1250, and 5000 

μg/plate

Negative in strains TA 98, 100, 1535, 1537 

and 1538 of S. typhimurium up to the limit 

concentration of 5000 μg/plate both with and 

without activation

870.5385

Micronucleus 

assay 

40722301 (1988)

562.5, 1175, 2250 

mg/kg

Negative in doses which induced death in 

Tif:MAGF mice.

870.5450

Rodent dominant 

lethal assay

42637003 (1993)

0, 500, 1000, 2000, 

or 2400 mg/kg

Negative in Tif: MAGf mice at doses 2400 

mg/kg.

870.5550

UDS assay

00161790/40722301 

(1984)

0, 1.2, 6, 30, 150 

μg/mL

Negative in primary rat hepatocytes up to 

150 μg/mL

870.5550

UDS assay

42547105 (1992)

0, 15, 46, 139, 417, 

835, 1670 μg/mL

Negative in primary rat hepatocyte cultures 

up to and beyond a dose which caused 

precipitation (139 μg/mL)

870.6300

Developmental 

neurotoxicity
Page 76 of 90

Guideline No./ 

Study Type

MRID No. (year) 

/Doses

Results

870.7485

Metabolism and 

Pharmacokinetics

40431304 (1987)

0, 1, and 100 mg/kg 

for a single dose 

given through oral 

gavage. 1.0 

mg/kg/day for 15 

days by oral gavage. 

Distribution, accumulation

Distribution was dose-dependent and 

independent of sex. Distribution appeared to 

follow first-order kinetics and the half-life in 

the tissues was 31.3 hours.

Excretion

Approximately 95% of the atrazine excreted 

within 7 days of dosing. Urinary route 

accounted for about 75% of the excretion 

feces accounted for 20%. Route of excretion 

did not seem to vary among sexes or with 

dose. 

870.7485

Metabolism and 

pharmacokinetics

MRID 40431305 

(1987)

The animals were 

dosed daily for 10 

days through a 

stomach tube with 

dose levels of 0, 1, 

3, 7, 10, 50 or 100 

mg/kg/day.

Distribution, accumulation

Distribution was highest in the red blood 

cell, followed by the liver, ovary and kidney. 

When the dose increased the amount 

distributed in the tissues increased. The 

distribution appeared to follow first-order 

kinetics and the tissue half-life was 38.6 

hours. This indicates that atrazine, with 

possible exception of the red blood cell, 

does not bioaccumulate.

870.7485

Metabolism and 

pharmacokinetics

MRID 40431306 

(1987)

Rats were given test 

100 mg/kg article 

was given through 

the stomach tube in 

a single oral dose.

Other rats were 

given 16.18 and 

19.64 mg/kg and 

urine was collected 

over a 24 hr period. 

The urine was 

analyzed for 

metabolites.

Excretion

In the rats given 100 mg/kg greater than 

100% of the administered radioactivity was 

recovered within 3 days of dosing. Urine 

was found to contain 47.3% of the 

radioactivity and the feces 49.3%. The 

tissues contained 5.75% and 1.4% was 

found in the blood. 

Metabolism

Metabolites indicate that dechlorination of 

the triazine ring and N-dealkylation are the 

major metabolic pathways for atrazine in 

rats. Oxidation of the alky substituents of 

atrazine appears to be of minor metabolic 

importance. 

870.7485

Metabolism and 

MRID 42165503 

(1993)

Metabolic profile
Page 77 of 90

Guideline No./ 

Study Type

MRID No. (year) 

/Doses

Results

pharmacokinetics Fecal and urinary 

samples from rats 

exposed in a 

separate metabolism 

study (MRID 

40431304) were 

obtained and 

analyzed to 

determine 

metabolism profiles.

No sex differences in metabolic profiles 

were evident. The major fecal metabolite 

was DACT which accounted for 40% of the 

total fecal radioactivity. 

870.7485

Metabolism and 

pharmacokinetics

MRID 44713802 

(1993)

single oral dose of 1 

or 100 mg/kg 

through oral gavage

Distribution, accumulation

Time to maximum blood concentration 

(tcmax) was 2 hours and 24 hours for the low 

and high dose groups, respectively. With 

exception of red blood cells, whole blood, 

and skeletal muscle, tissue burden for any

specific tissue or organ represented less 

than 1% of the administered dose by 14 

days post dosing

Excretion

Urinary excretion was 64.72% of the total 

administered low dose over a 48-hour 

period and 66.16% of the total administered 

high dose over a 168-hour period. Within 48 

hours urinary excretion was 100% and 94% 

complete for the low-dose and high-dose 

group, respectively. 

Fecal elimination accounted for 10.80% and 

19.69% of the total dose for the low and 

high dose groups, respectively. 

870.7600

Dermal 

penetration - rat

43314302 (1994) 

0.01, 0.1 or 1 

mg/cm2

The percent absorbed increased with 

exposure time and decreased with dose. 

Regardless of the dose or exposure time, 

the majority (65 - 95%) of the radio labeled 

atrazine was recovered in the washes or 

was found associated with the skin at the 

site of exposure. The maximum percentage 

of atrazine absorbed in the rat study after a 

10 hr (representative of a typical workday) 

exposure was 21.6%.
Page 78 of 90

Guideline No./ 

Study Type

MRID No. (year) 

/Doses

Results

870.7600

Dermal 

penetration -

human

44152114 (1996)

0.17 and 2.0 mg of 

[14C] atrazine

The majority (91.1-95.5%) of the dose was 

not absorbed. After 168 hours, only 5.6% of 

the dose was absorbed and excreted in the 

urine and feces of the low-dose group and 

only 1.2% in the high-dose group. 

Special study -

Assays of direct 

estrogenic 

activity

43598617 (1994)

This MRID number 

describes more than 

one assay. The 

doses varied 

depending on the 

assay. See above 

under section 4.9.1 

for specific details on 

doses used.

This study performed a trio of assays: 

uterotrophic response assay; progesterone 

receptor competitive binding assay; and, a 

uterine thymidine incorporation assay.

All doses tested displayed a lack of clear 

effects on uterine weight, progesterone 

binding capacity, and thymidine 

incorporation. This indicates that atrazine 

(and DACT and simazine, which were also 

tested) do not exhibit direct estrogenic 

activity.

Special study -

Assays of direct 

estrogenic 

activity

43598618 (1994)

This MRID number 

describes one type 

of assay conducted 

under a variety of 

experimental 

conditions. The 

doses varied. See 

above under section 

4.9.1 for specific 

details on doses 

used.

This study describes a series of estrogen 

receptor competitive binding assays, both in 

vivo and in vitro.

Overall the results indicate that atrazine 

(and DACT and simazine, which were also 

tested) do exhibit some competitive binding 

with estradiol but only under conditions 

which favor binding.

Special study -

Assays of direct 

estrogenic 

activity

43598619 (1995)

This MRID number 

describes more than 

one assay. The 

doses varied 

depending on the 

assay. See above 

under section 4.9.1 

for specific details on 

doses used.

This study describes four separate assays: 

competitive binding assay with the 

hepatocyte Ah receptor; MCF-7 cell 

proliferation; gel electrophoresis mobility 

shift assay using the progesterone receptor; 

and, luciferase reporter gene assay in MCF7 cells.

Neither atrazine (nor simazine, which was 

also tested) displayed estrogenic activity or 

interacted with the Ah receptor in the set of 

experiments described in this paper.

Special study - 43934403 (1995) This study described several assays, both in 
Page 79 of 90

Guideline No./ 

Study Type

MRID No. (year) 

/Doses

Results

Assays of direct 

estrogenic 

activity

This MRID number 

describes more than 

one assay. The 

doses varied 

depending on the 

assay. See above 

under section 4.9.1 

for specific details on 

doses used.

vivo and in vitro. In vivo assays: uterine 

weight, progesterone receptor levels and 

uterine peroxidase. In vitro assays: MCF-7 

cell proliferation; gel electrophoresis mobility 

shift assay using the progesterone receptor; 

luciferase reporter gene assay in MCF-7 

cells; and, a selective medium growth assay 

in yeast cells.

The results of these experiments indicate 

that in vivo atrazine and simazine may 

exhibit some antiestrogenic activity but no 

estrogenic activity either in vivo or in vitro.

Special study -

Estrous Cycle 

and LH Surge 

Measurements

(Pilot study)

43934404 (1996)

atrazine not given

This study was a pilot study in which the 

validity of a proposed protocol for testing the 

effect of atrazine exposure on the proestrus 

afternoon luteinizing hormone (LH) was 

evaluated. This study demonstrated that the 

proposed protocol adequately tested the 

parameters to examined. 

Special study -

Estrous Cycle 

and LH Surge 

Measurements

(28 day 

exposure)

43934406 (1996)

0, 2.5, 5, 40 and 200 

mg/kg/day 

NOAEL = 5 mg/kg/day.

LOAEL = 40 mg/kg/day based on 

decreases in food consumption, body 

weight, body weight gain, estrous cycle 

alterations and LH surge attenuation

Special study -

Estrous Cycle 

and LH Surge 

Measurements

(6 month 

exposure)

44152102 (1996)

0, 25, 50, and 400 

ppm 

0, 1.8, 3.7, 29.4 

mg/kg/day

NOAEL = 1.8 mg/kg/day

LOAEL = 3.7 mg/kg/day based on estrous 

cycle alterations and LH surge attenuation 

Special study1-

Hormone and 

estrous cycle 

measurements in 

SD rats

42085001, 

42743902 

(1991, 1993)

See also 43598622 

0, 70, 400 ppm

0, 4.2, 26.2 

mg/kg/day

Ten females per 

NOAEL = none found

LOAEL = 4.2 mg/kg/day based on estrous 

cycle alterations
Page 80 of 90

Guideline No./ 

Study Type

MRID No. (year) 

/Doses

Results

dose were sacrificed 

at 1, 3, 9, 12, 15, 18, 

24 mo.

Special Study 1-

Mammary Gland 

and Ovarian 

Histomorphology

in SD rats

43598622 (1995) 

See also 42085001, 

42743902

0, 70, 400 ppm

0, 4.2, 26.2 

mg/kg/day

Ten females per 

dose were sacrificed 

at 1, 3, 

9, 12, 15, 18, 24 mo.

NOAEL = none found

LOAEL = 4.2 mg/kg/day based early onset 

of anovulation as indicated by ovarian 

histomorphology and early onset of 

indicators of prolonged/increased hormone 

exposure in the mammary gland

Special study2 -

Two-year 

bioassay in F344 rats

42146101 (1991)

See also 4274392 

and 43598622

0, 0.7, 4.8, 14, 33.4 

mg/kg/day

Ten females per 

dose were sacrificed 

at 1, 3, 9, 12, 15, 18, 

24 mo.

NOAEL = 14 mg/kg/day

LOAEL = 33.4 mg/kg/day based on 

decreased body weight gain.

There was not an increased incidence of 

any tumor type, nor an early onset of 

mammary tumors.

Special study2 -

Hormone and 

estrous cycle 

measurements in 

F-344 rats

42743902 (1993)

See also 42146101 

and 43598622

0, 0.7, 4.8, 14, 33.4 

mg/kg/day

Ten females per 

dose were sacrificed 

at 1, 3, 9, 12, 15, 18, 

24 mo.

NOAEL = at least 33.4 mg/kg/day

LOAEL = none found

This study examined hormones and estrous 

cycles in the animals used in 42146101. 

The estrous cycle evaluations from this 

study are deemed unreliable by HED. The 

ovarian histomorphology data from MRID 

43598622 is used to confirm a lack of effect 

of atrazine treatment on estrous cycles.

The animals exhibited hormone levels 

indicative of normally aging females of the 

strain. Exposure to atrazine had no effect on 

hormone levels.

Special study2-

Mammary Gland 

43598622 (1995)

See also 42146101 

and 42743902 

NOAEL = at least 33.4 mg/kg/day

LOAEL = none found
Page 81 of 90

Guideline No./ 

Study Type

MRID No. (year) 

/Doses

Results

and Ovarian 

Histomorphology 

in F-344 rats

0, 0.7, 4.8, 14, 33.4 

mg/kg/day

Ten females per 

dose were sacrificed 

at 1, 3, 9, 12, 15, 18, 

24 mo.

This study examined histomorphology in the 

animals used in 42146101.

The animals exhibited ovarian and 

mammary gland histomorphology indicative 

of normally aging females of the strain. 

Exposure to atrazine had no effect on these 

histomorphologic parameters.

Special study -

Two-year 

bioassay with the 

SD strain of rat

42204401 (1992)

0, 70, 400 ppm

0, 3.8, 23.0 

mg/kg/day

NOAEL = 3.8 mg/kg/day

LOAEL = 23 mg/kg/day based on decreased 

body weight gains as well as statistically 

significant decreases in body weights in the 

0-76 week period

A statistically significant increase in 

mammary tumors was seen at the high 

dose. An early onset of mammary 

carcinomas was seen.

Special study -

Tumor incidence 

in ovariectomized 

(OVX) vs intact 

animals 

44544701 (1998)

0, 25, 50, 70, 400 

ppm 

0, 1.5, 3.1, 4.2, 24.4 

mg/kg/day - intact 

0, 1.2, 2.5, 3.5, 20.9 

mg/kg/day - OVX

Intact animal Mammary tumors

No mammary tumors seen in any OVX 

animal

Special study -

Long-term 

estrous cycle 

measurements

No MRID assigned 

(1999)

0, 25, 50, 70, 400 

ppm 

0, 1.5, 3.1, 4.2, 24.4 

mg/kg/day 

This is the unaudited draft report of the 

interim estrous cycle data from MRID 

44544701

These data demonstrate an early onset of 

increased % days in estrus in atrazinetreated animals compared to controls.

Special studyDirect 

comparison of LH 

surge attenuation 

of atrazine, 

simazine, DACT 

No MRID assigned 

(2000)

0, 2.5, 5, 40, 200 

mg/kg/day

This is the unaudited draft report of a study 

in which SD females were exposed to 

atrazine, simazine or DACT for 28 days and 

the ability of these chemicals to attenuate 

the LH surge was measured.

These data showed that simazine and 

DACT are able to attenuate the LH surge at 

doses similar to those at which atrazine 
Page 82 of 90

Guideline No./ 

Study Type

MRID No. (year) 

/Doses

Results

attenuates the surge.

1 This study in SD rats contains three parts submitted under three separate MRID 

numbers:

MRID 42085001 contains the results of the animal bioassay part of this study 

(the clinical observations, body weights, food consumption, gross pathology, 

etc.);

MRID 42743902 contains the results of the vaginal smears/estrous cycle 

determinations and serum hormone measurements;

MRID 43598622 contains the results of the histomorphologic analysis.

2 This study in F-344 rats contains three parts submitted under three separate MRID 

numbers:

MRID 42146101 contains the results of the animal bioassay part of this study 

(the clinical observations, body weights, food consumption, gross pathology, 

etc.);

MRID 42743902 contains the results of the vaginal smears/estrous cycle 

determinations and serum hormone measurements;

MRID 43598622 contains the results of the histomorphologic analysis.
Page 83 of 90

E.8.1.3 Toxicity Table for DACT. All available acceptable toxicity studies 

are shown.

Guideline 

No./ Study 

Type

MRID No. (year) /Doses Results

870.3100 

90-Day oral 

toxicity rodents

43013207 (1991)

0, 0, 10, 100, 250, or 500 

ppm 

0, 0.7, 6.7, 16.7, or 34.1 

mg/kg/day - males 

0, 0.7, 7.6, 19.7, or 40.2 

mg/kg/day -females. 

Male NOAEL= 16.7 mg/kg/day

LOAEL= 34.1 mg/kg/day based on 

decreased body weight and body weight 

gain

Female NOAEL= 0.7 mg/kg/day

LOAEL=7.6 mg/kg/day based on estrous 

cycle alterations

870.3100 

90-Day oral 

toxicity in 

nonrodents 

(dog)

41392401 (1990) 

0, 5, 100, and 1500/750 ppm 

(high dose reduced after 6 

wks)

0, 0.187, 3.61, and 

24.1mg/kg/day -males

0, 0.195, 3.43, and 32.7 

mg/kg/day - females

NOAEL = 3.4 mg/kg/day

LOAEL = 24.1 mg/kg/day based on 

cardiac effects and tremors in the males. 

870.4100 

Chronic toxicity 

in nonrodent 

(dog)

41392401 (1990) 

0, 5, 100, and 1500/750 ppm 

(high dose reduced after 6 

wks)

0, 0.187, 3.61, and 

24.1mg/kg/day -males

0, 0.195, 3.43, and 32.7 

mg/kg/day - females

NOAEL = 3.4 mg/kg/day

LOAEL = 24.1 mg/kg/day based on 

cardiac effects and tremors in the males. 

870.3700a

Prenatal 

developmental 

toxicity in 

rodents

41392402 (1989)

0, 2.5 , 25, 75 or 150 

mg/kg/day

Maternal NOAEL = 2.5 mg/kg/day

Maternal LOAEL = 25 mg/kg/day, based 

on decreased body weight gain [-32%] 

during the initial dosing period [GD 6-8]. 

Developmental NOAEL is 2.5 mg/kg/day.

Developmental LOAEL = 25 mg/kg/day, 

based on increases in incidences of 

incompletely ossified parietals, 

interparietals and unossified hyoids. 

870.5100

Bacterial 

reverse 

mutation assay

40722302 (1987)

0, 20, 78, 313, 1250, 5000 

μg/plate

Negative in the Salmonella/Ames assay 

up to limit concentration of 5000 μg per 

plate in strains TA 98, TA100, TA1535, 

and TA1537 with and without metabolic 

activation

870.5550 40722303 (1987) Negative up to 600 μg/mL (which 
Page 84 of 90

Guideline 

No./ Study 

Type

MRID No. (year) /Doses Results

UDS assay

0, 5.56, 16.67, 50, 150, 300, 

600 μg/mL

exceeded the solubility limit of 400 μg/mL) 

in UDS assay using isolated human 

fibroblasts without activation only (not 

performed with activation)
Page 85 of 90

E.8.1.4 Toxicity Table for G-28279. All available acceptable toxicity 

studies are shown.

Guideline No./ 

Study Type

MRID No. (year)

/Doses

Results

870.100

Acute oral 

toxicity

43013201 (1991)

500, 2000, 3500, 5050 

mg/kg

LD50 for both sexes combined= 1240 mg/kg 

870.3100 

90-Day oral 

toxicity rodents

MRID 43013205 

(1992)

0, 10, 50, or 500 ppm 

0, 0.602, 3.20, 34.9 

mg/kg/day - males 

0, 0.641, 3.34, 37.5 

mg/kg - females

NOAEL=3.2 mg/kg/day

LOAEL=34.9 based on based on decreased 

body weights and body weight gains in both 

sexes.

and possible histopathologic effects in the liver, 

adrenal cortex, and thyroid.

870.3100 

90-Day oral 

toxicity in 

nonrodents 

(dog)

MRID 43013203 

(1992)

0,15,100, 500, or 1000 

ppm

0, 0.6, 3.8, 18.9, 33.4 

mg/kg/day -male 

0, 0.6, 3.8, 18.0, 33.3 

mg/kg/day - female 

NOAEL=3.8 mg/kg/day

LOAEL=18.9 based on decreased food 

consumption and body weight gain and 

decreased organ-to brain weight ratios for the 

heart, testes, and prostate gland

870.3700a

Prenatal 

developmental 

in rodents

43013208 (1992)

0, 5, 25, or 100 

mg/kg/day.

Maternal NOAEL= 5 mg/kg/day

Maternal LOAEL= 25 mg/kg/day based on 

decreased body weight gain and food 

consumption

Developmental NOAEL= 5 mg/kg/day

Developmental LOAEL 25 mg/kg/day based on . 

increased fetal and litter incidences of fused 

sternebrae 1 and 2

870.5100

Bacterial 

reverse mutation 

assay

43093101 (1990)

0, 313, 625, 1250, 

2500, 5000 μg/mL

Negative in the Salmonella/Ames and E. Coli

WP2uvrA assays up to limit concentration of 

5000 μg per plate. Tested in S. Typhimurium in 

strains TA 98, TA100, TA1535, and TA1537 

with and without metabolic activation

870.5375

micronucleus 

assay

43093103 (1991)

0, 120, 240, 480 mg/kg

Negative in Tif:MAGF mice treated up to the 

maximum tolerated dose of 480 mg/kg

870.5550

UDS assay

43093105 (1991) Negative up to cytotoxic concentrations of 800 

μg/mL in primary rat hepatocyte cultures.
Page 86 of 90

Guideline No./ 

Study Type

MRID No. (year)

/Doses

Results

0, 7.4, 22.2, 66.6, 200, 

400, 800 μg/mL
Page 87 of 90

E.8.1.5 Toxicity Table for G-30033. All available acceptable toxicity 

studies are shown.

Guideline No./ 

Study Type

MRID No. (year) 

/Doses

Results

870.100

Acute oral 

toxicity

43013202 (1991)

500, 2000, 3500, 

5050 mg/kg

LD50 for both sexes combined= 1111 mg/kg 

870.3100 

90-Day oral 

toxicity rodents

43013206 (1991) 

0, 10, 50, or 500 ppm 

0, 0.68, 3.2, and 35.1 

mg/kg -male

0, 0.72, 3.3, and 

38.1 mg/kg /day -

female 

NOAEL = 3.2 mg/kg/day

LOAEL = 35.1 mg/kg/day based on decreased 

body weight of high dose female rats and 

decreased food efficiency of high-dose male and 

female rats. 

870.3100 

90-Day oral 

toxicity in 

nonrodents 

(dog)

43013203 (1992)

0, 15, 100, or 1000 

ppm

0, 0.56, 3.71, 28.85 

mg/kg/day - males 

0, 0.51, 3.88,32.18 

mg/kg/day - females

NOAEL = 3.7 mg/kg/day

LOAEL= 28.9 mg/kg/day based on renal tubular 

hyperplasia/basophilia in 3/4 male and 2/4 female 

dogs

870.3700a

Prenatal 

developmental 

in rodents

43013209 (1992) 

0, 5, 25, or 100 

mg/kg/day

Maternal NOAEL = 5 mg/kg/day

Maternal LOAEL = 25 mg/kg/day based

decreased food consumption, body weight/weight 

gain and food consumption

Developmental NOAEL = 25 mg/kg/day

Developmental LOAEL = 100 mg/kg/day based 

on increased fetal and litter incidences of fused 

sternebrae 1 and 2 and increased fetal incidence 

of poor ossification of the proximal phalanx of 

posterior digit 5 (a skeletal variation)

870.5100

Bacterial 

reverse mutation 

assay

43093102 (1991)

0, 313, 625, 1250, 

2500, 5000 μg/ml

Negative in the Salmonella/Ames and E. Coli

WP2uvrA assays up to limit concentration of 5000 

μg per plate. Tested in S. Typhimurium in strains 

TA 98, TA100, TA1535, and TA1537 with and 

without metabolic activation

870.5375

Micronucleus 

assay

43093104 (1991)

0, 120, 240, 480 

mg/kg

Negative in Tif:MAGF mice treated up to the 

maximum tolerated dose of 480 mg/kg

870.5550 43093106 (1991)

0, 9.25, 27.7, 83.3, 

250, 500, 1000 μg/ml

Negative up to cytoxic concentrations of 1000 

μg/mL in primary rat hepatocyte cultures.
Page 88 of 90

Guideline No./ 

Study Type

MRID No. (year) 

/Doses

Results

UDS assay
Page 89 of 90

E.8.1.6 Toxicity Table for hydroxyatrazine. All available acceptable toxicity 

studies are shown.

Guideline No./ 

Study Type

MRID No. (year) 

/Doses

Results

870.3100

90-Day oral 

toxicity rodents

MRID 41293501 

(1989)

0, 10, 100, 300, 600 

ppm 

0, 0.6, 6.3, 18.9, 37.5 

mg/kg/day - males 

0, 0.8, 7.4, 22.8, 45.6 

mg/kg/day - females

NOAEL = 6.3 mg/kg/day in males and 7.4 

mg/kg/day in females

LOAEL = 18.9 mg/kg/day in males and 22.8 

mg/kg/day in females based on kidney 

alterations . 

870.3700a

Prenatal 

developmental 

in rodents

MRID 41065202 (1989)

0, 5, 25, or 125 

mg/kg/day

Maternal NOAEL = 25 mg/kg/day

Maternal LOAEL = 125 mg/kg/day based on 

decreased food consumption during the dosing 

period and enlarged and mottled kidneys.

Developmental NOAEL = 25 mg/kg/day.

Developmental LOAEL = 125 mg/kg/day based 

on increased incidence of partially ossified 

interparietal and hyoid bones and decreased 

fetal body weight.

870.4100a 

(870.4300) 

Combined 

Chronic Toxicity/

Oncogenicity –

Rat

MRID 43532001 (1995) 

0, 10, 25, 200, 400 ppm 

0, 0.39, 1.0, 7.8, 17.4 

mg/kg/day - males

0, 0.5, 1.2, 9.4, 22.3 

mg/kg/day - females

NOAEL = 1.0 mg/kg/day for males and 1.2 

mg/kg/day for females

LOAEL = 7.8 mg/kg/day for males and 9.5 

mg/kg/day for females based on gross and 

histopathological effects in the kidneys

870.5100

Bacterial 

reverse mutation 

assay

MRID 40722304 (1988)

0, 20, 78, 313, 1250, 

5000 μg/0.1 ml

No increases in revertant colonies in TA 98, 

100, 1535, and 1537 Salmonella strains 

exposed to precipitating concentrations (313 

μg/plate and above) both with and without 

activation system.

870.5375

Micronucleus 

assay

MRID 41479401 (1988)

0, 1250, 2500, 5000 

mg/ml

No increase in micronuclei in mice treated with 

acute incubated doses up to the limit dose of 

5000 mg/mL.

870.5550

UDS assay

MRID 40722305 (1988)

0, 13.9, 41.7, 125, 375, 

750, 1500 μg/ml

No evidence of unscheduled DNA synthesis 

was found up to the limits of solubility 

(increasing precipitation from 500 μg/mL) and 

at concentrations approaching toxicity (1500 

μg/mL) in primary hepatocyte cultures treated in 
Page 90 of 90

Guideline No./ 

Study Type

MRID No. (year) 

/Doses

Results

vitro.

870.5550

UDS assay

MRID 40888101 (1988)

0, 13.9, 41.7, 125, 375, 

750, 1500 μg/ml

Negative up to the limits of solubility (increasing 

precipitation from 500 μg/mL) and severe 

cytotoxicity (1500 μg/mL) in human fibroblast 

cells.

HIARC Report [TXR NO. 0050592]