.~~,~to 514

"~"'. UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

l t> WASIDNGTON, D.C. 20460 o e ~ ~ ... · ~ "'~ .._,o 

"'( PRD1tC 

MEMORANDUM 

OFFICE OF CHEMICAL 

SAFETY AND 

POLLUTION PREVENTION 

Date: April I, 2014 

SUBJECT: Tricyclazole: Human Health Risk Assessment for the Establishment of 

Tolerances with No U.S. Registration for the New Fungicide inion Imported Rice. 

PC Code: 12020 1 

Decision No.: 471295 

Petition No.: 2E8ll4 

DP Barcode: D407046 

Registration No.: NA 

Risk Assessment Type: Single Chemical Aggregate 

TXRNo.: NA 

Regulatory Action: Tolerance Petition 

CaseNo.: NA 

CAS No.: 41814-78-2 

MRIDNo.: NA 40 CFR: 180-:XXXX 

FROM: 

THROUGH: 

TO: 

Margarita Collantes, Biologist /}Jk~k--< ~~.17tfc,,z.____­ Karlyn Middleton, Toxicologist / ~ ; · 

Michael Doherty, Ph.D., Chemist ;;;t_/a~ / _ L 

Risk 

Health 

Assessment 

Effects Division 

Branch 

(HED) 

II (RABII) 

(7509P) 

• ~ 

Christina Swartz, Branch Chief~ RABII, HED (7509P) 

and -~ 

Donald Wilbur, Chemist ~ Lve{)_ ~ Elissa Reaves, Ph.D., Branch Chief Q _ . 

Risk Assessment Review Committe?~ 

Erik Kraft/ Kable Davis (RM25) 

Fungicide Branch 

Registration Division (7505P) 

This document provides the Health Effects Division's (HED's) risk assessment conducted for the 

request for tolerances with no U.S. registration for the new fungicide tricyclazole inion rice 

imported from all major rice growing countries throughout the world. 

Page 1 of57 
Table of Contents 

1.0 Executive Summary ............................................................................................................. 4 

2.0 HED Recommendations ....................................................................................................... 6 

2.1 Data Deficiencies ................................................ .. : ..... ....... ............................. ................. 6 

2.2 Tolerance Considerations ......................... ........................................................................ 6 

2.2.1 Enforcement Analytical Method ............................................................................... 6 

2.2.2 Analytical Reference Standard ................................................................................. 6 

2 .2.3 Recommended Tolerances ........................................................................................ 7 

2.2.4 Revisions to Petitioned-For Tolerances ....................................... ............................. 7 

2.2.5 International Harmonization ..................................... ................................................ 7 

2.3 Label Recommendations ..... ............................................................ ......................... ........ 8 

3.0 Introduction .......................................................................................................................... 8 

3.1 Chemical Identity ........ ........................................ ....................................... ...................... 8 

3.2 Physical/Chemical Characteristics .................. .... ............... .............................................. 8 

3.3 Pesticide Use Pattern ...... ........... ...................... ............................................................ ..... 8 

3.4 Anticipated Exposure Pathways ....................................................................................... 9 

3.5 Consideration of Environmental Justice .......................................................................... 9 

4.0 Hazard Characterization and Dose-Response Assessment.. .............................................. 10 

4.1 Toxicology Studies Available for Analysis ................................................................... 10 

4.2 Absorption, Distribution, Metabolism, Excretion (ADME) .......................................... 10 

4.2.1 Dermal Absorption ................... .... ... .... .................................................................... 11 

4.3 Toxicological Effects .................. .................. .................................. ............................... 11 

4.4 Safety Factor for Infants and Children (FQPA Safety Factor) .................................. ..... 12 

4.4.1 Completeness of the Toxicology Database ............................................................. 12 

4.4.2 Evidence ofNeurotoxicity ...................................................................................... 12 

4.4.3 Evidence of Sensitivity/Susceptibility in the Developing or Young Animal ......... 13 

4.4.4 Residual Uncertainty in the Exposure Database ..................................................... 13 

4.5 Toxicity Endpoint and Point of Departure Selections ............... ..................................... 13 

4.5.1 Dose-Response Assessment ............ ........ ................................................................ 13 

4.5.2 Recommendation for Combining Routes ofExposures for Risk Assessment.. ...... 14 

4. 5.3 Classification of Carcinogenic Potential.. ............................................................... 14 

4.5.4 Summary of Toxicological Doses and Endpoints for Tricyclazole for Use in 

Human Health Risk Assessments .................... ..................................................................... 15 

5.0 Dietary Exposure and Risk Assessment ............................................................................ 15 

5.1 Metabolite/Degradate Residue Profile .................... ........ ............................................... 15 

5 .1.1 Summary of Plant Metabolism Studies ........................................................ ........... 16 

5 .1.2 Summary of Livestock Metabolism Studies ........................................................... 16 

5.1.3 Comparison of Metabolic Pathways .................................................................... ... 16 

Page 2 of 57 
Page 3 of 57

5.14 Residues of Concern Summary and Rationale........................................................ 17

5.2 Food Residue Profile...................................................................................................... 17

5.3 Dietary Risk Assessment................................................................................................ 18

5.3.1 Description of Residue Data Used in Dietary Assessment..................................... 18

5.3.2 Percent Crop Treated Used in Dietary Assessment ................................................ 18

5.3.3 Acute Dietary Exposure and Risk........................................................................... 18

5.3.4 Chronic Dietary Risk Assessment .......................................................................... 19

5.3.5 Summary Table....................................................................................................... 19

6.0 Residential (Non-Occupational) Exposure/Risk Characterization .................................... 19

7.0 Aggregate Exposure/Risk Characterization....................................................................... 19

8.0 Cumulative Exposure/Risk Characterization..................................................................... 20

9.0 Occupational Exposure/Risk Characterization .................................................................. 20

10.0 References.......................................................................................................................... 20

Appendix....................................................................................................................................... 31
Page 4 of 57

1.0 Executive Summary

The new active ingredient (ai), tricyclazole, is a member of the azole chemistry class of 

fungicides that provides control against rice blast at all stages (leaf blast, stem blast and panicle 

blast). Tricyclazole inhibits the polyhydroxynapthaline reductase enzyme, thus inhibiting 

melanin formation in fungi. 

Dow AgroScience (DAS) (hereafter referred to as DAS or the petitioner) has petitioned to 

establish a permanent tolerance with no U.S. registration (i.e., an “import tolerance”) for residues 

of tricyclazole (5-methyl-1,2,4-triazolo[3,4-b]benzothiazole) in/on rice at 3.0 ppm.

The toxicology database is considered complete. Neurotoxicity (acute, subchronic, and 

developmental) and immunotoxicity studies are not available for tricyclazole. However, the 

Hazard and Science Policy Council (HASPOC), using a weight of the evidence (WOE)

approach, concluded that these studies are not required (TXR#0056854). Dermal toxicity and 

dermal penetration studies are not available for tricyclazole. However, these studies are not 

required to support import tolerances.

In oral rat and dog studies, decreased body weight was the primary effect observed in the 

database; in oral mouse studies, effects were mainly seen in the liver. In rats, decreased body 

weight was the only treatment related effect seen in adult animals in the subchronic and chronic 

studies, with body weight decreases occurring at a lower dose after chronic exposure. Decreased 

body weight was also seen in adult rats in developmental and reproduction studies. Other effects 

observed in rats included clinical signs (weakness, cold body, piloerection) in the developmental 

toxicity study at a dose similar to that in the subchronic study where decreased body weight was 

noted. Brain weight changes were also observed in rats in the chronic study; however, due to 

inconsistency in the data the effects were not considered treatment related. In dogs, decreased 

body weight was the only treatment related effect observed after chronic exposure. In mice,

mortality was seen after 90 days, as well as hematological changes (increased WBC, decreased 

lymphocyte count, increased neutrophil) and liver effects (increased weights, enzymes, and 

histopathology). However, it is noteworthy to mention that the 90 day subchronic study was 

considered unacceptable due to numerous deficiencies. Increased mortality was not observed in 

other toxicity studies in mice. After 10 months, only liver effects (increased weights, 

microsomal activity, and histopathology) were observed in mice and no treatment related effects 

were observed after 1 year. However, chronic exposure in mice (cancer mouse study), resulted 

in liver effects including, increased liver weights and liver histopathology (acidophilic 

degeneration and fatty change) at doses lower than those producing liver effects in the shorter 

term mouse studies. 

Delayed ossification was observed in fetuses in the rabbit developmental study while decreased 

body weight was observed in fetuses in the rat developmental study. The effects were seen in the 

absence of maternal toxicity indicating quantitative susceptibility. In the rat reproduction study, 

offspring effects included pup death [post-natal day (PND 1-4], decreases in pup body weight, 

and an increase in the number of small pups in the presence of less severe maternal toxicity 
Page 5 of 57

(decreased body weight) indicating qualitative susceptibility. Although susceptibility was 

observed in the developmental/reproduction studies, the doses and endpoints selected for risk 

assessment are protective and the degree of concern for the susceptibility observed in the studies 

is low.

Tricyclazole is classified as “not likely to be carcinogenic to humans.” There were no treatmentrelated increases in tumors observed in the submitted carcinogenicity studies in rats and/or mice. 

Since the proposed uses of tricyclazole are all non-domestic, there is no potential for drinking 

water, occupational, and/or residential exposure, and the only anticipated exposure to 

tricyclazole is dietary exposure through consumption of imported rice. Therefore, endpoints and 

points of departure (PODs) were only selected for acute and chronic dietary risk assessment. For 

acute dietary risk assessment (all populations including females 13-49), the no observed adverse 

effect level (NOAEL) of 7 mg/kg/day was selected from a reproduction study in rats. An 

increased incidence of pup death was seen at the lowest observed adverse effect level (LOAEL) 

of 26.7 mg/kg/day. Decreased pup weight and small pups were also observed at the LOAEL but 

were not considered to be single dose effects. For chronic dietary exposure, a NOAEL of 6.67 

mg/kg/day was selected from a cancer study in mice based on liver effects observed at the 

LOAEL of 21.8 mg/kg/day. For acute and chronic dietary risk assessments, a 100X uncertainty 

factor was applied (interspecies factor of 10X and intraspecies factor of 10X). The 10X Food 

Quality Protection Act (FQPA) Safety Factor (SF) was reduced to 1X.

The residue chemistry database is complete for purposes of setting tricyclazole residue tolerances 

in/on imported rice. The nature of the residue in rice is adequately understood, field trials were 

conducted to reflect worst-case residues, and the residue data are supported by acceptable 

analytical methods and storage stability data. However, an analytical reference standard has not 

been provided to the National Pesticide Standard Repository. A standard will need to be 

provided prior to the establishment of tolerances. 

Conservative screening level acute and chronic dietary (food only) exposure assessments were 

conducted for tricyclazole residues in/on rice. Acute and chronic dietary risk estimates are 

below the level of concern (LOC) for all population subgroups. At the 95th percentile, the acute 

exposure results in an estimated dietary (food only) risk equivalent to 5.7% of the acute 

population adjusted dose (aPAD) for all populations. The estimated chronic dietary (food only) 

risk is equivalent to 1.1% of the chronic population adjusted dose (cPAD) for all populations. 

The most highly exposed subpopulation is all infants for acute (19% aPAD) and chronic (4.5%

cPAD) estimated dietary risks. As indicated previously, dietary exposure is the only exposure 

pathway relevant to tricyclazole at this time; therefore, aggregate exposure and risk estimates are 

equivalent to the dietary estimates and are below HED’s LOC.

Potential areas of environmental justice concerns, to the extent possible, were considered in this 

human health risk assessment (see Section 3.5).

This risk assessment is not based on data from studies in which adult human subjects were 
Page 6 of 57

intentionally exposed to a pesticide or other chemical.

2.0 HED Recommendations

Provided a reference standard is provided to the National Pesticide Standard Repository and the 

petitioner submits a revised Section F, HED recommends in favor of the establishment of 

permanent tolerance, without U.S. registration, for residues of tricyclazole in/on rice, grain at 3.0 

ppm.

2.1 Data Deficiencies

None.

2.2 Tolerance Considerations

2.2.1 Enforcement Analytical Method

Three methods have undergone successful independent laboratory validation for use as 

enforcement analytical methods. The first method is suitable for the analysis of both parent 

tricyclazole and the alcohol metabolite. It involves acid hydrolysis, extraction of residues into 

dichloromethane, clean-up by strong cation exchange (SCX) solid-phase extraction, silylation of 

the alcohol metabolite, and analysis by GC-MS. The validated limit of quantitation (LOQ) is 

0.02 ppm for rice grain, polished rice, and rice husks, and 0.05 ppm for rice forage, straw, and 

processed byproducts. The remaining two methods are multi-residue methods (DFG S19 and 

modified QuEChERS). DFG S19 uses acetone extraction, clean-up by partitioning and gelpermeation chromatography (GPC), and analysis by GC-MS. The validated LOQ is 0.02 ppm 

(parent only). The modified QuEChERS method uses acetonitrile/water extraction, clean-up by 

solid-phase partitioning, and analysis by LC-MS/MS. The validated LOQ is 0.01 ppm each for 

the parent compound and the alcohol metabolite.

2.2.2 Analytical Reference Standard

As of 16 January 2014, a reference standard for tricyclazole had not been submitted to the EPA 

National Pesticide Standards Repository. The petitioner is required to submit a 5-gram sample of 

reference standard and supporting documentation (see OCSPP Guideline 860.1650) to the 

repository at the address below, to the attention of either Theresa Cole or Thuy Nguyen. Note 

that the full 9-digit ZIP code is mandatory or the mail will be returned to the sender. 

USEPA

National Pesticide Standards Repository/Analytical Chemistry Branch/OPP

701 Mapes Road

Fort George G. Meade, MD 20755-5350
Page 7 of 57

2 .2.3 Recommended Tolerances

HED recommends for the establishment of a permanent tolerance for residues of tricyclazole in 

rice and polished rice at 3.0 ppm. The recommended tolerance expression is as follows:

“Tolerances are established for residues of the fungicide tricyclazole, including its 

metabolites and degradates, in or on the commodities in the table below. Compliance with 

the tolerance levels specified below is to be determined by measuring only tricyclazole (5-

methyl-1,2,4-triazolo[3,4-b]benzothiazole).”

Note: The Registration Division should ensure that the entry in the Code of Federal Regulations 

(CFR) is footnoted to specify that there is no U.S. registration.

Table 2.2.2. Tolerance Summary for Tricyclazole.

Commodity Proposed Tolerance 

(ppm)

HED-Recommended 

Tolerance (ppm)

Comments 

(correct commodity definition)

Rice 3.0 3.0 Rice, grain

2.2.4 Revisions to Petitioned-For Tolerances

The recommended tolerance level of 3.0 ppm is the same as what was proposed; however, the 

petitioner should submit a revised Section F with the commodity definition specified in Table 

2.2.2. 

2.2.5 International Harmonization

According to the petitioner, Maximum Residue Limits (MRLs) have been established in other 

countries as follows:

Brazil..........................0.3 ppm,

China..........................2 ppm,

India ...........................0.02 ppm,

Italy ............................1 ppm,

Japan ..........................3 ppm,

Spain ..........................1 ppm,

Thailand .....................3 ppm,

Vietnam......................0.1 ppm.

The tolerance level and residue definition recommended by HED is harmonized with the 

maximum established MRL (3 ppm, Japan/Thailand). As summarized in Appendix C, no 

Canadian or Codex MRLs for tricyclazole exist at this time. Although the U.S. is a net exporter 

of rice, there is no use of tricyclazole in the U.S. and a 3-ppm tricyclazole tolerance is not 

expected to result in trade-irritant issues.
Page 8 of 57

2.3 Label Recommendations

None

3.0 Introduction

Tricyclazole is a systemic fungicide with translocation through the plant. The compound is part 

of the azole chemistry class that provides control against rice blast at all stages (leaf blast, stem 

blast and panicle blast). The fungicidal mode of action is inhibition of the 

polyhydroxynapthaline reductase enzyme, thus preventing melanin formation in fungi. In the 

absence of melanin, fungal appressoria fail to produce penetrating hypha or the penetrating 

hypha fails to penetrate into host tissue. Currently, tricyclazole is registered in 23 countries 

across the world, with the major rice producing and exporting countries for the US being Brazil, 

China, India, Italy, Japan, Spain, Thailand and Vietnam.

3.1 Chemical Identity

The chemical structure and nomenclature of tricyclazole is presented in Table 3.1. 

Table 3.1. Tricyclazole Nomenclature.

Compound Chemical Structure

Common name Tricyclazole

Company experimental name X59741

IUPAC name 5-methyl-1,2,4-triazolo[3,4-b][1,3]benzothiazole

CAS name 5-methyl-1,2,4-triazolo[3,4-b]benzothiazole

CAS # 41814-78-2

End-use product/EP BEAM® 75 WP, BEAM 12, BIM® 75 WP, BIM 750 BR, TROOPER®

3.2 Physical/Chemical Characteristics

Tricyclazole is a solid at environmental temperatures, with slight volatility. The technical 

compound is soluble in water and very soluble in methanol, acetone, n-octanol, xylene, ethyl 

acetate and 1,2-dichloroethane; however, it is relatively insoluble in n-heptane. Based on the 

Log KOW, tricyclazole is unlikely to partition into fat or to bioaccumulate. 

3.3 Pesticide Use Pattern

Tricyclazole is used on rice in many major rice growing countries throughout the world. 

Table 3.3 provides a summary of the registered use patterns. There are no existing/proposed 
Page 9 of 57

uses of tricyclazole in the U.S. 

Translated labels from countries with registered uses of tricyclazole were not submitted; 

however, comparison of the petition with native-language labels confirmed the use pattern 

summaries that were submitted. The information is adequate for purposes of evaluating the 

residue data for tolerance-setting purposes.

Table 3.3. Summary of Directions for Use of Tricyclazole in Major Rice-Exporting Countries.

Country MRL, 

ppm

Application Application rate per

treatment

Method Growth Stage Number

minmax

Retreatmen

t interval 

(days)

Water

(L/ha)

kg ai/ha PHI (days)

Brazil 0.3 Foliar At complete

tillering

1-2 10 0.150-0.225 30

China 2 Foliar Summer/autumn 1-2 0.225-0.300 21

India 0.02 Foliar BBCH 71-85 2-3 10 500 0.225-0.300 30

Italy 1 Foliar BBCH 47-51 1 - 400-600 0.450 Determined by

crop stage at 

application

Japan 3 Foliar 1-3 7-10 1500 0.300-0.400 7

Spain 1 Foliar BBCH 47-51 1-2 15 200-400 0.225 30

Thailand 3 Foliar Panicle stage 1-2 10 500 0.1875 30*

Vietnam 0.1 Foliar Panicle stage 1-2 10 320-400 0.200-0.300 30*

*The label rate is 14 days, but the standard practice is 30 days

3.4 Anticipated Exposure Pathways

The Registration Division has requested an assessment of human health risk to support the 

proposed new tolerance for tricyclazole in/on rice, with no U.S. registration. Since the uses of 

tricyclazole are all non-domestic, there is no domestic potential for drinking water, occupational, 

and/or residential exposures. The only potential exposure to U.S. Citizens associated with the 

petition is to tricyclazole residues of concern in food.

3.5 Consideration of Environmental Justice

Potential areas of environmental justice concerns, to the extent possible, were considered in this 

human health risk assessment, in accordance with U.S. Executive Order 12898, "Federal Actions 

to Address Environmental Justice in Minority Populations and Low-Income Populations," 

(http://www.eh.doe.gov/oepa/guidance/justice/eo12898.pdf. As a part of every pesticide risk 

assessment, OPP considers a large variety of consumer subgroups according to well-established 

procedures. In line with OPP policy, HED estimates risks to population subgroups from 

pesticide exposures that are based on patterns of that subgroup’s food and water consumption, 

and activities in and around the home that involve pesticide use in a residential setting. 

Extensive data on food consumption patterns are compiled by the United States Department of 

Agriculture (USDA) under the National Health and Nutrition Examination Survey, What We Eat 

in America (NHANES/WWEIA). These food consumption patterns are used in pesticide risk 
Page 10 of 57

assessments for all registered food uses of a pesticide. These data are analyzed and categorized 

by subgroups based on age and ethnic group. Additionally, OPP is able to assess dietary 

exposure to smaller, specialized subgroups and exposure assessments are performed when 

conditions or circumstances warrant. Whenever appropriate, non-dietary exposures based on 

home use of pesticide products and associated risks for adult applicators and for toddlers, youths, 

and adults entering or playing on treated areas post-application are evaluated. Further 

considerations are currently in development as OPP has committed resources and expertise to the 

development of specialized software and models that consider exposure to bystanders and farm 

workers as well as lifestyle and traditional dietary patterns among specific subgroups.

4.0 Hazard Characterization and Dose-Response Assessment

4.1 Toxicology Studies Available for Analysis

The toxicology database for established tolerances for residues of tricyclazole in/on imported 

rice is considered complete. Neurotoxicity (acute, subchronic, and developmental) and

immunotoxicity studies are not available for tricyclazole. However, the Hazard and Science 

Policy Council (HASPOC), using a weight of the evidence (WOE) approach, concluded that 

these studies are not required for tricyclazole at this time (TXR#0056854). The available 

toxicological studies for tricyclazole include: 1) subchronic oral toxicity studies in rats and mice, 

2) chronic/carcinogenicity studies in rats and mice, 3) a chronic oral toxicity study in dogs, 4) 

developmental studies in rats and rabbits, 5) reproduction studies in rats and mice, 6) metabolism 

studies in rats, 7) a mutagenicity battery, and 8) an acute oral lethal (LD50) study. Subchronic 

dermal and inhalation toxicity studies are not available; however these studies are not required to 

support import tolerances. The available subchronic (90 day) mouse and rat studies submitted 

for tricyclazole are considered unacceptable due to a number of deficiencies. Additionally, a 

subchronic dog study is not available; however, new 90 day studies are not needed since 

potential toxicity can be adequately characterized by the available 10 month and 1 year mouse, 

cancer mouse, chronic/cancer rat, and chronic dog studies. 

4.2 Absorption, Distribution, Metabolism, Excretion (ADME)

In oral rat metabolism studies, six treatment groups were tested with groups 1-5 receiving low 

and high (Group 2 only) oral doses and group 6 receiving an intravenous (IV) dose. Tricyclazole 

was rapidly absorbed with total absorption estimated at 89% and 96% in males and females, 

respectively. Because of extensive biliary excretion, total absorption could only be estimated for 

one orally dosed group. Tissue accumulation was low after 168 hours of treatment and was <

2.1% of the administered dose for both oral (low and high dose) and IV groups. The orally 

absorbed dose was rapidly excreted in urine (31-61% of administered dose) with slightly higher 

recoveries in the female. Most urinary excretion (66-86%) occurred within 24 hours postdosing. The IV dose was also rapidly excreted in urine (35-47% of administered dose) with the 

majority (68-84%) occurring in the first 24 hours. The percentage of the oral dose eliminated in 

feces was 39-65%. Total fecal elimination of the IV dose was 58-59%, indicating substantial

biliary excretion. 
Page 11 of 57

In orally dosed rats, elimination of the radioactivity from plasma was biphasic with an initial 

rapid elimination phase (t1⁄2α ≤0.08 hours), followed by a slower elimination phase (t1⁄2β = 34 - 51 

hours). The plasma area under the curve (AUC) at the high dose was 29- to 37-fold greater than 

the low dose. Clearance was faster at the low dose relative to the high dose group.

Extensive metabolism of the radiolabeled test material was found. A total of 29 metabolites 

were identified in the urine and fecal samples. The majority of metabolites were common 

between dose groups and gender, with some quantitative differences between sexes. Based on 

the metabolite data, the test material was conjugated with glutathione followed by subsequent βlyase cleavage to form the corresponding thiol. This was further followed by conjugation with 

glucuronide or methylation prior to excretion. Benzyl oxidation was also observed. 

4.2.1 Dermal Absorption

Dermal toxicity and dermal penetration studies are not available for tricyclazole. However, these 

studies are not required for import tolerances. 

4.3 Toxicological Effects

In oral rat and dog studies, decreased body weight was the primary effect observed in the 

database; in oral mouse studies, effects were mainly seen in the liver. In rats, decreased body 

weight was the only treatment related effect seen in adult animals in the subchronic and chronic 

studies, with body weight decreases occurring at a lower dose after chronic exposure. Decreased 

body weight was also seen in adult rats in developmental and reproduction studies. Other effects 

observed in rats included clinical signs (weakness, cold body, piloerection) in the developmental 

toxicity study at a dose similar to that in the subchronic study where decreased body weight was 

noted. Brain weight changes were also observed in rats in the chronic study; however, due to 

inconsistency in the data the effects were not considered treatment related. In dogs, decreased 

body weight was the only treatment related effect observed after chronic exposure. In mice,

mortality was seen after 90 days, as well as hematological changes (increased white blood cell 

(WBC), decreased lymphocyte count, increased neutrophil) and liver effects (increased weights, 

enzymes, and histopathology). However, it is noteworthy to mention that the 90 day subchronic 

study was considered unacceptable due to numerous deficiencies. Increased mortality was not 

observed in other toxicity studies in mice. After 10 months, only liver effects (increased 

weights, microsomal activity, and histopathology) were observed in mice and no treatment 

related effects were observed after 1 year. However, chronic exposure in mice (cancer mouse 

study), resulted in liver effects including, increased liver weights and liver histopathology 

(acidophilic degeneration and fatty change) at doses lower than those producing liver effects in 

the shorter term mouse studies. 

Delayed ossification was observed in fetuses in the rabbit developmental study while decreased 

body weight was observed in fetuses in the rat developmental study. The effects were seen in the 

absence of maternal toxicity indicating quantitative susceptibility. In the rat reproduction study, 

offspring effects included pup death (post natal day (PND) 1-4), decreases in pup body weight,
Page 12 of 57

and an increase in the number of small pups in the presence of less severe maternal toxicity 

(decreased body weight) indicating qualitative susceptibility. 

Tricyclazole is classified as “not likely to be carcinogenic to humans.” No treatment-related 

increase in tumors was observed in carcinogenicity studies in rats and/or mice. 

Since the proposed tolerances for tricyclazole are for import tolerances on rice, the only study 

required to evaluate acute lethality is the acute oral study. In an acute lethality study, acute oral 

exposure to tricyclazole resulted in moderate toxicity. 

4.4 Safety Factor for Infants and Children (FQPA Safety Factor)

The proposed use of tricyclazole on rice may result in dietary exposure to infants and children. 

Thus, FQPA hazard considerations were addressed in HED’s evaluation of the toxicological 

database. HED recommends that the 10X FQPA SF (for the protection of infants and children) 

be reduced to 1X. This recommendation is supported by the following: 1) the toxicological 

database for this assessment is complete with regard to pre-and postnatal toxicity, 2) the 

endpoints and doses selected for risk assessment are protective of the increased qualitative 

susceptibility observed in the reproduction study in rats and the increased quantitative 

susceptibility seen in the developmental rat and rabbit studies; therefore the degree of concern 

for the susceptibility is low, 3) the endpoints and doses selected for risk assessment are also 

protective of the observed clinical signs, 4) neurotoxicity and immunotoxicity studies are not 

required, 5) the dietary exposure assessments are based on tolerance level residue levels, and 6) 

there are no registered nor proposed residential uses associated with tricyclazole. 

4.4.1 Completeness of the Toxicology Database 

Based on the use pattern, the available studies are adequate for assessing and characterizing 

potential pre- and/or post-natal risk for infants and children. 

4.4.2 Evidence of Neurotoxicity

Neurotoxicity studies are not available for tricyclazole. However, only limited clinical signs 

were observed in rat and mouse repeat dose studies as well as in an acute rat oral study. The 

degree of concern for neurotoxicity is low since the clinical signs were seen at doses 18x higher 

than the current points of departure (PODs). The need for the missing neurotoxicity studies was 

evaluated by the Hazard Science Policy Council (HASPOC) and it was concluded that 

neurotoxicity studies are not required for the proposed tricyclazole import use based on the 

following considerations: 1) the absence of neurotoxicity in the toxicological database; 2) the 

liver is the target organ for tricyclazole; 3) the acute neurotoxicity (ACN), subchronic 

neurotoxicity (SCN) and developmental neurotoxicity (DNT) studies are unlikely to provide a 

lower POD or a more sensitive endpoint than those currently used for risk assessment; 4) 

although there is evidence of increased susceptibility in the developmental and reproduction 

studies, the effects seen in the young did not involve the nervous system; and 5) there are clear 
Page 13 of 57

NOAELs/LOAELs for the effects seen, 6) the endpoints and PODs are based on the most 

sensitive endpoints, and 7) the PODs selected for risk assessment are protective of potential 

developmental effects (TXR 0056854). The need for neurotoxicity studies may be re-evaluated 

for future uses. 

4.4.3 Evidence of Sensitivity/Susceptibility in the Developing or Young Animal 

Although there was evidence of quantitative susceptibility in developmental rat and rabbit 

toxicity studies and qualitative susceptibility in the reproduction study, the degree of concern for 

the effects observed in the studies is low. There are clear NOAELs/LOAELs for the fetal/pup 

effects seen and the effects observed in the developmental (3-10x) and reproduction (4x) studies 

are significantly higher than the current PODs selected for risk assessment. Therefore, the acute 

and chronic dietary risk assessments are protective of potential fetal/offspring effects. 

4.4.4 Residual Uncertainty in the Exposure Database

There are no residual uncertainties with regard to dietary (food only) exposure. The food

exposure assessments are based on high-end tolerance residue levels (that account for the parent 

compound). Actual risks from exposure to tricyclazole will likely be lower than those presented 

in this risk assessment. Furthermore, there are no anticipated residential uses.

4.5 Toxicity Endpoint and Point of Departure Selections

4.5.1 Dose-Response Assessment

The detailed descriptions of the toxicity studies used to select toxicity endpoints and points of 

departure for various exposure scenarios are presented in Appendix A.3 of this assessment.

For acute dietary exposure (all populations), the offspring NOAEL of 7 mg/kg/day from the 

reproduction study in rats was selected based on increased pup death (PND 1-4) at the LOAEL 

of 26.7 mg/kg/day. In the study, the pup deaths were reported as occurring on PND 1 or 4 and 

the exact day of death was not specified in the study report. In the absence of data outlining the 

exact day of death, it is assumed that the deaths could have resulted from a single dose exposure 

anytime between day PND 1 and 4. Therefore, the endpoint is considered appropriate to assess 

acute dietary exposure. While the pup deaths observed in the reproduction study do not appear 

to be an in utero effect, it is possibly an early post-natal effect due to maternal care or nursing 

(test article being expressed in the milk). In order to be protective of the developmental stages 

where the effect could occur, the endpoint was used to protect women of reproductive age who 

are nursing, as well as all other populations including infants and children. Therefore, the acute 

dietary exposure scenario was expanded to include all populations including infants, children and 

females 13-49 years old. The dose and endpoint are also protective of effects observed in the rat 

and rabbit developmental studies. 

For chronic dietary exposure (general population), the carcinogenicity study in mice was selected 

for risk assessment, based on liver effects (increased weight and histopathology) observed at the 
Page 14 of 57

LOAEL of 21.8 mg/kg/day; the NOAEL is 6.67 mg/kg/day. The duration of this study and the 

route of exposure are appropriate for assessing chronic dietary exposure. This endpoint and 

POD are protective of adverse effects observed at higher doses in other studies. The combined 

chronic/carcinogenicity study is considered supportive since treatment related effects were seen 

at similar doses and a comparable NOAEL was determined for the study. In the chronic rat 

study, the NOAEL is 8.1 mg/kg/day, based on decreased body weight observed at the LOAEL of 

20 mg/kg/day. 

Based on the proposed use pattern for tricyclazole (import tolerance), only dietary exposure is 

anticipated. Therefore, POD for occupational and residential dermal, inhalation and incidental 

oral exposures were not selected. For acute and chronic dietary risk assessments, a 100X 

uncertainty factor was applied (interspecies factor of 10X and intraspecies factor of 10X, FQPA 

factor of 1X). 

4.5.2 Recommendation for Combining Routes of Exposures for Risk Assessment

In the case of import tolerances with no U.S. registration, there is no potential for domestic

residential or occupational exposure. Therefore, an assessment for combined routes of exposures 

is not required.

4.5.3 Classification of Carcinogenic Potential

Tricyclazole is classified as “not likely to be carcinogenic to humans.” No treatment-related 

increase in tumors was observed in carcinogenicity studies in rats and/or mice. Mutagenicity 

studies are available for tricyclazole where positive results were observed in in vitro mouse 

lymphoma gene mutation assays. However, the assays included a colony size analysis where the 

majority of colonies seen were small, which suggests chromosome damage rather than point 

mutations (large colonies). Furthermore, negative results were observed in another in vitro gene 

mutation assay (CHO cells) and in an in vivo gene mutation assay (Ames test). Tricyclazole was 

also negative in in vivo unscheduled DNS synthesis (UDS) and mouse bone marrow assays. 

However, the in vivo mouse bone marrow assay is considered unacceptable since there is a 

question regarding the dose adequacy in the study since dose levels did not reach the maximum 

tolerated dose (MTD). Although a repeat study would better characterize the mutagenic 

potential of tricyclazole, it is not required since tricyclazole did not induce tumors in the 

carcinogenicity studies or effects in the reproduction study that would suggest a possible genetic 

component was operable. 
Page 15 of 57

4.5.4 Summary of Toxicological Doses and Endpoints for Tricyclazole for Use in Human 

Health Risk Assessments 

A summary of the toxicological doses and endpoints for tricyclazole are provided in Table 4.5.4.

Table 4.5.4 Toxicological Doses and Endpoints for Tricyclazole for Use in Dietary and Human Health Risk 

Assessments

Exposure/ 

Scenario Point of Departure Uncertainty/FQPA Safety Factors

RfD, PAD, Level of 

Concern for Risk 

Assessment

Study and Toxicological 

Effects

Acute Dietary

(All Populations 

including 

Females 13-49 

years of age)

Reproduction 

Offspring NOAEL 

= 7 mg/kg/day

Total UF: 100X

UFA= 10x

UFH=10x

FQPA SF=1x

Acute RfD = aPAD = 

0.07 mg/kg/day

Reproduction Toxicity study

(MRID48918221) in the rat 

*LOAEL = 26.7 mg/kg/day 

based on increased pup death 

(PND 1-4)

Chronic Dietary 

(All 

Populations)

NOAEL= 6.67 

mg/kg/day

Total UF: 100X

UFA= 10x

UFH=10x

FQPA SF=1x

Chronic RfD = cPAD = 

0.067 mg/kg/day

Carcinogenicity Study in mice

(MRID 48918213) Supportive: 

Carcinogenicity Study (MRID 

48918226) in the rat 

LOAEL = 21.8 mg/kg/day 

based on liver effects

(increased weights and 

histopathology).

Supportive: Carcinogenicity 

study in rats LOAEL = 20 

mg/kg/day based on decreased 

body weight. NOAEL = 8.1 

mg/kg/day

Cancer (oral, 

dermal, 

inhalation)

Classification: “Not likely to be carcinogenic to humans.”

Point of Departure (POD) = A data point or an estimated point that is derived from observed dose-response data and 

used to mark the beginning of extrapolation to determine risk associated with lower environmentally relevant human 

exposures. NOAEL = no observed adverse effect level. LOAEL = lowest observed adverse effect level. UF = 

uncertainty factor. UFA = extrapolation from animal to human (interspecies). UFH = potential variation in 

sensitivity among members of the human population (intraspecies). PAD = population adjusted dose (a = acute, c = 

chronic). RfD = reference dose. * decreased pup body weight and increased number of small pups were also seen at 

the offspring LOAEL; however, the effects were not considered attributable to a single dose exposure. 

5.0 Dietary Exposure and Risk Assessment

Since this action only assesses residues in/on imported rice, there are no drinking water or 

rotational crop considerations pertinent to this petition. Therefore, these exposure pathways are

not discussed. 

5.1 Metabolite/Degradate Residue Profile
Page 16 of 57

5.1.1 Summary of Plant Metabolism Studies

In a metabolism study, 14C-labeled tricyclazole was applied post-emergence to rice at ca. 2.35X 

the maximum worst-case label rate (i.e., the critical Good Agricultural Practice (cGAP) rate). 

Forage was harvested at various time points after application; grain, hulls, and straw were 

harvested at maturity. Samples were analyzed for total radioactivity by combustion and for 

residue characterization/identification by solvent extraction and acid digestion (selected 

samples), with analysis by high-performance liquid chromatography (HPLC) and/or thin-layer 

chromatography (TLC). For some forage, hull, and straw samples, the post-extraction solids 

(PES) were subjected to acid-detergent fiber isolation to further characterize bound residues.

Solvent extraction (acetone/water or acetonitrile) accounted for ≥90% of the radioactivity at the 

0 days after treatment (DAT) sampling point. For later sampling points, solvent extraction and 

acid digestion accounted for ca. 70- 92% of the radioactivity. Of the extracted radioactivity, 

tricyclazole was the only major residue, except for one sample of rice straw in which the alcohol 

metabolite accounted for 17% total radio-active residue (TRR) and one sample of rice grain in 

which 14C-starch was ca. 80% of the residue. Analysis of PES from forage, hull, and straw 

samples showed that the majority of the unextracted radioactivity was associated with lignin and 

cellulose.

5.1.2 Summary of Livestock Metabolism Studies

Livestock metabolism studies were not provided with the petition; however, summaries of cattle 

and poultry metabolism studies were reviewed in the European Commission’s Draft Assessment 

Report (DAR) for tricyclazole1. For both ruminants and poultry, the studies demonstrate that 

radiolabeled residues are at least an order of magnitude greater in liver and kidney than in other 

matrices and that quantifiable residues are not expected in those other matrices (milk, eggs, meat, 

and fat). Tricyclazole and the alcohol metabolite were the only residues identified in liver,

which was the only matrix where residue identification was conducted.

Based on the DAR summaries, HED would likely find deficiencies in the conduct and reporting 

of the metabolism studies. However, in combination with other studies (e.g., rat metabolism and 

livestock feeding studies), the lack of international MRLs for residues of tricyclazole in livestock 

commodities, and that there are no U.S. registrations associated with the petition, the data are 

sufficient to conclude that tolerances for tricyclazole residues in livestock commodities are not 

necessary at this time. Therefore, there is no livestock metabolism data gap associated with the 

requested tolerance. HED notes that livestock metabolism data may be required to support 

domestic registrations or other tolerances for residues in/on imported commodities.

5.1.3 Comparison of Metabolic Pathways

The submitted data are sufficient to provide an understanding of tricyclazole metabolism by rice 

plants. These data indicate that once metabolism of tricyclazole begins, the parent is extensively 

 

1 Draft Assessment Report for Tricyclazole, European Commission, October 2007
Page 17 of 57

broken down, with the components ultimately being incorporated into natural plant components 

(e.g., lignin, glucose/starch/cellulose). The only identified rice metabolite was the alcohol 

metabolite.

In rats, tricyclazole may also by oxidized to the alcohol metabolite, with additional oxidation of 

the alcohol moiety to a carboxylic acid. Both the parent compound and the oxidized metabolites 

then undergo further metabolism via glutathione conjugation, which does not appear to occur in 

rice.

While tricyclazole contains the 1,2,4-triazole moiety, the free triazole and the triazole conjugate 

metabolites common to triazole-derivative fungicides are not likely to form during breakdown of 

tricyclazole and, indeed, were not observed in the metabolism study.

5.14 Residues of Concern Summary and Rationale

HED has determined that the parent compound, tricyclazole, is a suitable residue definition for 

purposes of both tolerance enforcement and risk assessment (Table 5.1.4). This determination is 

based on tricyclazole being the only major residue in rice grain and the observation that in 

samples from field trials with quantifiable levels of the alcohol metabolite, the metabolite is 

reduced to <LOQ upon husking. It is noted that this decision applies only to the import tolerance 

use on rice. Should the use site expand in the future, this decision may need to be revisited. 

Table 5.1.4. Summary of Metabolites and Degradates to be included in the Risk Assessment and Tolerance 

Expression.

Matrix Residue Definition

Risk Assessment Tolerance Expression

Plants Primary Crop Tricyclazole Tricyclazole

Rotational Crop Not Applicable Not Applicable

Livestock Ruminant Not Determined Not Determined

Poultry Not Determined Not Determined

Drinking Water Not Applicable Not Applicable

5.2 Food Residue Profile

Field trials for the use of tricyclazole on rice are available from multiple use patterns. The data 

consistently show: (1) that residues dissipate over time, with an initial reduction the first week 

after treatment, a plateau for approximately the next two weeks, and a further reduction at longer 

time periods; (2) that husking the rice removes the majority of the residue; and (3) residues are 

further reduced on processing brown rice into polished rice. Although the number of trials at 

cGAP (9 trials) is not in strict accord with HED’s guidance (EPA 2005) for tolerances on 

imported commodities (12 trials), it is considered adequate for purposes of this petition. The atcGAP trials give relatively consistent results, all of which are well below the recommended, 

harmonized tolerance (3.0 ppm). Furthermore, proportionality, while not strictly appropriate for 

these data, supports the conclusion that residues from cGAP application are unlikely to exceed 

the recommended tolerance.

Processing studies indicate that residues of tricyclazole are associated primarily with rice hulls 
Page 18 of 57

and that processing of rough rice into brown rice, bran, and polished rice results in lower 

residues in these processed products. 

Although rice commodities may be significant livestock feedstuffs in countries with registered 

uses of tricyclazole, data from metabolism studies and livestock feeding studies indicate that 

quantifiable residues of tricyclazole would be expected only in meat byproducts. Given the 

residue profile, that no U.S. registration is being sought, and the lack of tricyclazole MRLs for 

livestock commodities in countries with registered uses of tricyclazole, HED is not 

recommending that tolerances for tricyclazole residues be established for livestock commodities.

5.3 Dietary Risk Assessment

HED conducted screening-level acute and chronic dietary risk assessments using the Dietary 

Exposure Evaluation Model DEEM-FCID, Version 3.16 which uses food consumption data from 

the U.S. Department of Agriculture’s National Health and Nutrition Examination Survey, What 

We Eat in America, (NHANES/WWEIA). Dietary risk assessment incorporates both exposure 

and toxicity of a given pesticide. For acute and chronic dietary assessments, the risk is expressed 

as a percentage of a maximum acceptable dose (i.e., the dose which HED has concluded will 

result in no unreasonable adverse health effects). This dose is referred to as the population 

adjusted dose (PAD). The PAD is equivalent to the reference dose (RfD) divided by the 

combined uncertainty factors retained due to missing data or concerns for susceptibility. For 

acute and chronic exposures, HED is concerned when estimated dietary risk exceeds 100% of the 

PAD. 

5.3.1 Description of Residue Data Used in Dietary Assessment

For the acute and chronic dietary analyses, tolerance-level residues were used for all rice 

commodities. No processing factors were used in this dietary assessment.

5.3.2 Percent Crop Treated Used in Dietary Assessment

The dietary assessment assumes 100% of all rice commodities consumed in the U.S. have been 

treated with tricyclazole.

5.3.3 Acute Dietary Exposure and Risk

A screening level acute dietary (food only) exposure analysis was performed for the general U.S. 

population and various population subgroups. Acute dietary risk estimates are not of concern for 

the general U.S. population or other population subgroups. At the 95th percentile, the acute 

exposure results in an estimated dietary (food only) risk equivalent to 5.7% of the acute 

population adjusted dose (aPAD) for the general U.S. population. The most highly exposed 

subpopulation is all infants, with an estimated risk of 19% of the aPAD. A summary of the acute 

dietary risk estimates is provided in Table 5.3.5.
Page 19 of 57

5.3.4 Chronic Dietary Risk Assessment

A screening level chronic dietary (food only) exposure analysis was performed for the general 

U.S. population and various population subgroups. Chronic dietary risk estimates are not of 

concern for the general U.S. population or other population subgroups. The estimated noncancer chronic dietary (food only) risk is equivalent to 1.1% of the chronic population adjusted 

dose (cPAD) for the general U.S. population. The most highly exposed subpopulation is all 

infants, with an estimated risk level of 4.5% of the cPAD. A summary of the chronic dietary risk 

estimates is provided in Table 5.3.5. 

5.3.5 Summary Table

Table 5.3.5 provides a summary of the dietary exposure estimates for tricyclazole.

Table 5.3.5. Summary of Dietary (Food Only) Exposure and Risk for Tricyclazole

Population Subgroup

Acute Dietary

95th Percentile Chronic Dietary

Dietary Exposure 

(mg/kg/day) % aPAD Dietary Exposure(mg/kg/day) % cPAD

General U.S. Population 0.003960 5.7 0.000763 1.1

All Infants (< 1 year old) 0.013234 19 0.002986 4.5

Children 1-2 years old 0.009106 13 0.001737 2.6

Children 3-5 years old 0.007922 11 0.001383 2.1

Children 6-12 years old 0.004887 7.0 0.000828 1.2

Youth 13-19 years old 0.003026 4.3 0.000497 < 1

Adults 20-49 years old 0.003915 5.6 0.000822 1.2

Adults 50-99 years old 0.002528 3.6 0.000467 < 1

Females 13-49 years old 0.003189 4.5 0.000589 < 1

6.0 Residential (Non-Occupational) Exposure/Risk Characterization

There are currently no proposed or registered residential uses of tricyclazole in the U.S.

7.0 Aggregate Exposure/Risk Characterization

In accordance with the FQPA, HED must consider and aggregate (add) pesticide exposures and 

risks from three major sources: food, drinking water, and residential exposures. In an aggregate 

assessment, exposures from relevant sources are added together and compared to quantitative 

estimates of hazard (e.g., a NOAEL or PAD), or the risks themselves can be aggregated. When 

aggregating exposures and risks from various sources, HED considers both the route and 

duration of exposure. Since uses of tricyclazole are all non-domestic, there is no potential for 

drinking water and/or residential exposures for the U.S. population. Therefore, aggregate 

exposure and risk estimates for tricyclazole are equivalent to the dietary estimates discussed in 

Section 5.
Page 20 of 57

8.0 Cumulative Exposure/Risk Characterization

Unlike other pesticides for which EPA has followed a cumulative risk approach based on a 

common mechanism of toxicity, EPA has not made a common mechanism of toxicity finding as 

to tricyclazole and any other substances and tricyclazole does not appear to produce a toxic 

metabolite produced by other substances, including the metabolites common to the trizole 

fungicides. For the purposes of this tolerance action, therefore, EPA has not assumed that 

tricyclazole has a common mechanism of toxicity with other substances. For information 

regarding EPA’s efforts to determine which chemicals have a common mechanism of toxicity 

and to evaluate the cumulative effects of such chemicals, see the policy statements released by 

EPA’s Office of Pesticide Programs concerning common mechanism determinations and 

procedures for cumulating effects from substances found to have a common mechanism on 

EPA’s website at http://www.epa.gov/pesticides/cumulative/.

9.0 Occupational Exposure/Risk Characterization

Since uses of tricyclazole are all non-domestic, there is no potential for occupational exposures

for U.S. handlers and workers. An occupational exposure assessment is not relevant.

10.0 References

1. Tricyclazole: Petition for the Establishment of a Permanent Tolerance for Residues 

in/on Rice, Without a U.S. Registration. Summary of Analytical Chemistry and 

Residue Data; M. Doherty, D408369; February 6, 2014

2. Tricyclazole: Acute and Chronic (Food Only) Exposure and Risk Assessments for 

Proposed Tolerances with No US Registration in/on Imported Rice; M.Collantes; 

D417241; February 7, 2014

3. Tricyclazole: Report of Residues of Concern Knowledge Subcommittee; E. Holman; 

D417768; Draft February 2014

4. NAFTA Guidance Document on Data Requirements for Tolerances on Imported 

Commodities in the United States and Canada; December 2005; US Environmental 

Protection Agency Office of Pesticide Programs and Health Canada Pest 

Management Regulatory Agency.
Page 21 of 57

Appendix A: Toxicology Profile and Executive Summaries

Appendix A.1. Toxicology Data Requirements

Toxicology Requirements for Tricyclazole

Guideline Number and Toxicity Study 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

no

no

no

no

no

yes

-

-

-

-

-

870.3100 Oral Sub-chronic (Rodent)............................................

870.3150 Oral Sub-chronic (Non-Rodent)....................................

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

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

870.3465 90/28-Day Inhalation ....................................................

yes

yes

no

no

no

Yes*

Yes*

-

-

-

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

870.3700 Developmental Toxicity (Non-rodent)..........................

870.3800 Reproduction.................................................................

yes

yes

yes

yes

yes

yes

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

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

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

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

870.4300 Chronic/Oncogenicity (Rat)..........................................

yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

870.5100 Mutagenicity: Gene Mutation - bacterial ......................

870.5300 Mutagenicity: Gene Mutation - mammalian .................

870.5395 Mutagenicity: In Vivo Cytogenetics..............................

870.5915 Mutagenicity: Sister Chromatid Exchange....................

870.5550 Mutagenicity: Unscheduled DNA Synthesis.................

yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

870.6100 Acute Delayed Neurotoxicity (Hen) .............................

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

870.6200 Acute Neurotoxicity Screening Battery (Rat) ...............

870.6200 90 Day Neuro. Screening Battery (Rat) ........................

870.6300 Developmental Neurotoxicity .......................................

no

no

yes

yes

yes

-

-

yes1

yes1

yes1

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

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

870.7800 Immunotoxicity.............................................................

yes

no

yes

yes

-

yes1

*Requirement satisfied by the available 10 month and 1 year mouse, cancer mouse, chronic/cancer rat, and 

chronic dog studies. - Not Applicable. 1 study waived by the HED HASPOC.
Page 22 of 57

Appendix A2. Toxicity Profile

Table A.2.1 Acute Toxicity Profile-Tricyclazole Technical 

Guideline 

Number

Study Type

Classification

MRID

Number

Results Toxicity Category

870.1100 Acute-oral 48918206 F: LD50 = M: LD50 = 

237mg/kg

II

870.1200 Acute-dermal Not applicable for proposed use pattern (Import tolerance)

870.1300 Acute-inhalation Not applicable for proposed use pattern (Import tolerance)

870.2400 Acute-eye 

irritation-rabbit

Not applicable for proposed use pattern (Import tolerance)

870.2500 Acute-dermal 

irritation-rabbit

Not applicable for proposed use pattern (Import tolerance)

870.2600 Skin sensitization -

guinea pig

Not applicable for proposed use pattern (Import tolerance)

Table A.2.2 Tricyclazole Subchronic, Chronic, and Other Toxicity

Guideline No. Study Type MRID No. (year)/ Classification /Doses Results

870.3100 90-Day oral toxicity –

Wistar rat: 

MRID 48918239 (1978)

ppm = 0, 282, 635, 1640

mg/kg/day = M: 0, 20.5, 46.7, 135.3; 

F: 0, , 22.5, 54.2, 186.8

Unacceptable/Guideline (numerous 

deficiencies- some of the recommended 

organ weights, clinical chemistry, 

hematology, and histopathology 

parameters not examined)

Note: Study requirement satisfied by 

combined chronic/carcinogenicity study in 

the rat)

NOAEL = 20.5 mg/kg/day

LOAEL = 46.7 mg/kg/day, based on 

decreased body weight (19%), food 

consumption. And food efficiency.

At 135.3 mg/kg/day, decreased body 

weight was seen in males (up to 31%) 

and females (up to 28%), and 

decreased food consumption and food 

efficiency seen in males and females 

(week 1 only). 

870.3100 90-Day oral toxicity –

ICR mouse 

MRID 48918238 (1978)

ppm = 0, 400, 1000, 2500, 3600

mg/kg/day = 0, 52, 130, 325, 468

Unacceptable/Guideline (numerous 

deficiencies- some recommended organ 

weights, clinical chemistry, hematology, 

and histopathology parameters not 

examined; animals not properly 

acclimated, food consumption not 

measured accurately, etc.)

Note: Study requirement satisfied by the 10 

month and 1 year studies in the mouse. 

NOAEL=52 mg/kg/day

LOAEL = 130 mg/kg/day, based on 

clinical signs (thin body, piloerection) 

and mortality.

At 325 mg/kg/day, clinical signs and 

mortality.

At 468 mg/kg/day, clinical signs,

hematological changes (increased 

WBC, decreased lymphocyte count, 

and increased neutrophil count), 

mortality, and liver effects (increased 

organs weights and ALP and bile duct 

proliferation). 
Page 23 of 57

Table A.2.2 Tricyclazole Subchronic, Chronic, and Other Toxicity

Guideline No. Study Type MRID No. (year)/ Classification /Doses Results

Non-Guideline 10-month liver toxicityICR miceMRID 48918224 (1979) 

ppm = 0, 400, 1000, 2500, 3600

mg/kg/day = 0, 52, 130, 325, 468, 

Acceptable/Nonguideline 

NOAEL = not determined

LOAEL = 52 mg/kg/day, based on 

liver histopathology (periportal 

lipocytes, and bile duct proliferation). 

At ≥ 130 mg/kg/day, increased liver 

microsomal activity (p-nitroanisole Odemethylase), increased liver weight, 

increased periportal lipocytes, and bile 

duct proliferation.

Non-Guideline One year toxicity- ICR 

mice

MRID 48918225 (1979) 

ppm = 0, 50, 140, 400, 620

mg/kg/day = 0, 6.5, 18.2, 52, 80.6

Acceptable/Nonguideline 

NOAEL = 80.6

LOAEL = not determined 

870.3200 21-Day dermal toxicity Not Required for an import tolerance on rice

870.3465 21-Day inhalation toxicity Not Required for an import tolerance on rice

870.3700b Prenatal developmental in 

rats

MRID 48918218 (2004)

mg/kg/day = 0, 5, 20, 50

Acceptable/Guideline

Maternal NOAEL = 20 mg/kg/day

Maternal LOAEL = 50 mg/kg/day, 

clinical signs (weakness, cold body, 

piloerection) and decreased body 

weight (up to 11%)

Developmental NOAEL = 5 

mg/kg/day

Developmental LOAEL = 20 

mg/kg/day, based on delayed bone 

ossification (nasal, interparietal and 

front distal phalanges). At 50 

mg/kg/day, decreased fetal body 

weight, delayed ossification of skull 

bones, cervical and caudel vertebral 

bodies, metacarpels and front distal 

phalanges, metatarsels, and hind 

proximal and distal phalanges were 

observed. High incidence of accessory 

lumbar ribs was also seen. 

870.3700b Prenatal developmental in 

rabbits

MRID 48918207 (2009)

mg/kg/day = 0, 7.5, 25, 75

Acceptable/Guideline

Maternal NOAEL = 75 mg/kg/day

Maternal LOAEL = not determined

Developmental NOAEL = 25 

mg/kg/day

Developmental LOAEL = 75 

mg/kg/day, based on decreased fetal 

body weight (15-16%) 

870.3800 Main Study-Reproduction 

in Rats

MRID 48918221 (1997)

mg/kg/diet (ppm)= 0, 30, 100, 400

mg/kg/day=

M: 0, 2.1, 7.0, and 26.7 

F: 0, 2.3, 8.0, and 30.7 

Parental/Systemic NOAEL = 7.0 

mg/kg/day

Parental/Systemic LOAEL = 26.7 

mg/kg/day, based on decreases in 

body weight (up to 9% in F0 and up to 
Page 24 of 57

Table A.2.2 Tricyclazole Subchronic, Chronic, and Other Toxicity

Guideline No. Study Type MRID No. (year)/ Classification /Doses Results

Acceptable/Guideline

15% in F1 )and food consumption in F0 

and F1 adults. 

Reproductive NOAEL = 7.0

mg/kg/day 

Reproductive LOAEL = 26.7 

mg/kg/day, based on delay in sexual 

maturation (vaginal opening).

Offspring NOAEL = 7.0 mg/kg/day

Offspring LOAEL = 26.7 mg/kg/day, 

based on pup death (PND 1-4), 

decreases in pup body weight (up to 

20%), and increase in the number of 

small pups

870.3800 RangefindingReproduction in RatsMRID 48918221 (1997)

mg/kg/diet (ppm)= 0, 200, 625, 1250, 1600 

[2 highest doses returned to control diet 

due to clinical signs (decreased body 

weight/food consumption, piloerection), no 

results reported]

mg/kg/day=

M: 0, 13.2, 38.7, 60.7, 66 

F: 0, 13.5, 35.6, 74.2, 75.6 

Acceptable/Guideline

Parental/Systemic NOAEL = 13.2 

mg/kg/day

Parental/Systemic LOAEL = 38.7 

mg/kg/day, based on decreases in body 

weight.

Reproductive NOAEL = 13.2 

mg/kg/day; Reproductive LOAEL= 

38.7 mg/kg/day, based on increased 

precoital interval.

Offspring NOAEL = 13.2 mg/kg/day

Offspring LOAEL = 38.7 mg/kg/day, 

based on decreases in pup body 

weight, decrease in the number of 

pups/litter and increase in number of 

cold/small pups. 

Non-Guideline Reproduction in Mice MRIDs 489182209 and 48918216

mg/kg/diet (ppm)= 0, 50, 375 ppm

mg/kg/day=0, 4-7, and 17-38 mg/kg/day

Unacceptable/Guideline

No effects seen in adult or offspring 

animals up to 38 mg/kg/day (HDT). 

870.4100b Chronic toxicity dogs 

(capsule)

MRID 48918222

1986)

mg/kg/day = 0, 5, 15, 45

Acceptable/Guideline 

NOAEL = 15 mg/kg/day mg/kg/day 

LOAEL = 45 mg/kg/day, based on 

decreased body weight in females 

(14%).

870.4200a Combined 

Chronic/Carcinogenicity 

Wistar rats

MRID 48918226 (1977)

ppm = 0, 100, 275, 620 and 1600ppm (90 

days then diet)

mg/kg/day = M: 0, 2.7, 8.1, 20, and 106

F: 0, 3.4, 12, 30, and 168 

Acceptable/Guideline

NOAEL = 8.1mg/kg/day

LOAEL = 20 mg/kg/day based on 

decreases in body wt (7-18%).

throughout the study. At 106 

mg/kg/day, decreases body weight was 

also seen.

No evidence of carcinogenicity
Page 25 of 57

Table A.2.2 Tricyclazole Subchronic, Chronic, and Other Toxicity

Guideline No. Study Type MRID No. (year)/ Classification /Doses Results

870.4200b Carcinogenicity ICRmiceMRID 48918213 (1985)

ppm = 0, 25, 75, 250, 1000

M: 0, 2.59, 7.98, 24.9, 101

F: 0, 2.2, 6.67, 21.8, 91

Acceptable/Guideline

NOAEL = 6.67 mg/kg/day

LOAEL = 21.8 mg/kg/day, based on 

increased liver weights, and liver 

histopathology (acidophilic 

degeneration and fatty change). At 91 

mg/kg/day, similar effects observed 

but more pronounced.

No evidence of carcinogenicity

870.4200b Carcinogenicity ICRmiceMRID 48918230 (1985)

ppm = 0, 50, 140, 400 ppm

M :0, 4.9, 13.8. 39.7

F: 0, 5.1, 14.7, 42.0

Unacceptable/Guideline

NOAEL = 39.7 mg/kg/day

LOAEL = not determined

No evidence of carcinogenicity

870.5100 Salmonella /Escherichiae

reverse gene mutation 

assay

MRID 48918208

(2011)

0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 

625, 1250, 2500, and 5000 μg/plate, 

without and with S9-mix

Acceptable/Guideline

Negative; there was no evidence of 

induced mutant colonies over 

background.

870.5300 In vitro cell gene mutation 

in CHO cells 

MRID 48918212

(2004) preliminary toxicity assay; 0, 7, 14, 

28, 56.3, 112.5, 225, 450, and 900 μg/mL 

without and with activation . first mutation 

assay; 0, 50, 100, 300, 500, 700, and 900 

μg/mL without activation and doses of 0, 

2.5, 5, 10, 20, 30, 40, 50, 80, and 100 

μg/mL with activation. 

Confirmatory (2nd mutation assay) 0, 100, 

300, 500, 700, and 900 μg/mL without 

activation and doses of 0, 2.5, 5, 10, 20, 30, 

40, 50, 80, and 100 μg/mL with activation, 

were used. 

Acceptable/Guideline

Negative; there was no evidence of 

induced mutant colonies over 

background.

870.5300 In Vitro Mammalian Cells 

in Culture Gene Mutation 

assay in Mouse 

Lymphoma

MRID 48918236; 2003

first experiment; 24 hours at 0, 2.1, 4.2, 

8.3, 17, 33, 67, 95, 136, 194, 280, 400, 570,

or 810 μg/mL in the absence of metabolic 

activation and for 4 hours at concentrations 

of 0, 2.1, 4.2, 8.3, 17, 33, 67, 95, 136, 194, 

280, 400, 570, or 810 μg/mL in the 

presence of mammalian metabolic 

activation.

Confirmatory experiment; 24 hours at 0, 

6.4, 10, 15, 23, 36, 55, 85, 131, 164, 205, 

256, 320, or 400 μg/mL in the absence of 

Evidence of a concentration related 

positive response of induced mutant 

colonies over background.
Page 26 of 57

Table A.2.2 Tricyclazole Subchronic, Chronic, and Other Toxicity

Guideline No. Study Type MRID No. (year)/ Classification /Doses Results

metabolic activation and for 4 hours at 

concentrations of 0, 0.15, 0.29, 0.59, 1.2, 

2.3, 4.7, 9.4, 19, 38, 75, 150, 300, or 600 

μg/mL in the presence of mammalian 

metabolic activation.

Acceptable/Guideline

870.5300 In Vitro Mammalian Cells 

in Culture Gene Mutation 

assay in in Mouse 

Lymphoma L5178Y cells

MRID 48918237

24 hours at concentrations of 0, 6.4, 10, 15, 

23, 36, 55, 85, 131, 164, 205, 256, 320, or 

400 μg/mL in the absence of metabolic 

activation and for 4 hours at concentrations 

of 0, 0.15, 0.29, 0.59, 1.2, 2.3, 4.7, 9.4, 19, 

38, 75, 150, 300, or 600 μg/mL in the 

presence of mammalian metabolic 

activation. Mammalian metabolic 

activation was achieved with S9 derived 

from livers of Aroclor 1254 induced male 

Wistar rats. No second (confirmatory) 

assay was needed because the results were 

so similar to those of an earlier study that 

used tricyclazole technical (95.2% a.i.) as 

the test article, as reviewed in MRID 

48918236

Acceptable/Guideline

No evidence of a concentration 

related, positive response of induced 

mutant colonies over background.

870.5395 In Vivo Mammalian

Micronucleus 

MRID 48918211

0,100, 200, 300 mg/kg

Unacceptable/Guideline

No biology significant increase in 

frequency of micronucleated 

polychromatic erythrocytes in bone 

marrow at doses tested. However, 

there is a question regarding dose 

adequacy of the HDT. No overt 

toxicity was seen in the test animals 

and no cytotoxicity toward the 

target (bone marrow cells) up to 50% 

of the LD 50.

870.5915 In Vivo Sister Chromatid 

Exchange assay

MRID 48918233, 

0, 21.25, 42.5, 85, or 170 mg/kg

Unacceptable/Guideline

No significant increase in sister 

chromatid exchanges in bone marrow 

at any dose.

870.5550 Unscheduled DNA MRID 48918214

0, 100, 250, 500, and 1000 mg/kg

Acceptable/Guideline

No evidence that unscheduled DNA 

synthesis, as determined by radioactive 

tracer procedures [nuclear silver grain 

counts], was induced.

Non-guideline DNA Repair Synthesis MRID 48918231

1981

0.5, 1, 5, 10, 50, 100, 500 and 1000 

nmol/mL, for 20 hours in the presence of 

3H-TdR.

No evidence that unscheduled DNA 

synthesis, as determined by radioactive 

tracer procedures [nuclear silver grain 

counts], was induced at the doses 

tested. However, highest test dose is 

10-fold less than the maximum dose 
Page 27 of 57

Table A.2.2 Tricyclazole Subchronic, Chronic, and Other Toxicity

Guideline No. Study Type MRID No. (year)/ Classification /Doses Results

Unacceptable/Guideline recommended.

Non-guideline Cytotoxicity study MRID 48918229

2004

400 and 800 μg/mL

Unacceptable/Non-Guideline

tricyc1azole did not induce selective 

cytotoxicity in L5178Y cells

870.6200a Acute neurotoxicity Waived

870.6200b Subchronic neurotoxicity Waived

870.6300. Developmental 

neurotoxicity 

Waived

870.7485 Metabolism in Rats MRID 48918209 

Groups 1 – 5 (oral) and Group 6 (IV) dose 

of 2 mg/kg radiolabeled test material; 

Group 2 rcvd 40 mg/kg radiolabeled test 

material. Group 5 received daily doses of 

unlabeled test material before a 2 mg/kg 

radiolabeled dose on Day 15.

Acceptable/Guideline 

radiolabeled tricyclazole was rapidly 

absorbed with total absorption 

estimated at 89% and 96% in males 

and females, respectively. Because of 

extensive biliary excretion, total 

absorption could only be estimated for 

group 1. Tissue accumulation was low 

after168 hours of treatment and was 

<2.1% of the administered dose for 

Groups 1, 2, 5, and 6. The orally 

absorbed dose was rapidly excreted in 

urine (31-61% of administered dose) 

with slightly higher recoveries in the 

female. Most urinary excretion (66-

86%) occurred within 24 hours postdosing. The IV administered test 

material was also rapidly excreted in 

urine (35-47% of administered dose) 

with the majority (68-84%) occurring 

in the first 24 hours. A higher 

percentage (39-65%) of the oral dose 

was eliminated in feces. 

In orally dosed rats, most of the 

elimination of the radioactivity from 

plasma occurred during the rapid (α) 

phase (t1⁄2α ≤0.08 hours), with a slower 

(β) elimination phase (t1⁄2β = 34 - 51 

hours). The plasma AUC at the high 

dose was 244-253 μg hours/g, which 

was 29- to 37-fold greater than the low 

dose. Clearance was faster at the low 

dose relative to the high dose-group.

Extensive metabolism of the 

radiolabeled test material was found. 

Based on the metabolite data, the test 

material was conjugated with 
Page 28 of 57

Table A.2.2 Tricyclazole Subchronic, Chronic, and Other Toxicity

Guideline No. Study Type MRID No. (year)/ Classification /Doses Results

glutathione followed by subsequent βlyase cleavage to form the corresponding thiol. This was further 

followed by conjugation with 

glucuronide or methylation prior to 

excretion. Benzyl oxidation was also 

observed. 

870.7800 Immunotoxicity Waived
Page 29 of 57

Appendix A.3 Hazard Identification and Endpoint Selection

A.3.1 Acute Dietary (All Populations including infants and children and Females 13 - 49)

Study Selected: Reproduction Toxicity Study in Rats

MRID Number: 48918221

Dose and Endpoint for Risk Assessment: Offspring NOAEL of 7 mg/kg/day, based on increased 

pup death (PND 1-4) at the LOAEL of 26.7 mg/kg/day.Decreased body weight, and increased 

number of small pups were also seen at the LOAEL; however, the effect were not considered 

attributable to a single dose exposure, at the LOAEL of 26.7 mg/kg/day. 

Uncertainty Factor(s): 100X (10X for interspecies variability, 10X for intraspecies variability, 

1X for FQPA) 

Comments about Study/Endpoint/Uncertainty Factor: For acute dietary exposure (all 

populations), the offspring NOAEL of 7 mg/kg/day from the reproduction study in rats was 

selected based on increased pup death (PND 1-4) at the LOAEL of 26.7 mg/kg/day. In the study, 

the pup deaths were reported as occurring on PND 1 or 4 and the exact day of death was not 

specified in the study report. In the absence of data outlining the exact day of death, it is assumed 

that the deaths could have resulted from a single dose exposure anytime between day PND 1 and 

4. Therefore, the endpoint is considered appropriate to assess acute dietary exposure. While the 

pup deaths observed in the reproduction study do not appear to be an in utero effect, it is 

possibly an early post-natal effect due to maternal care or nursing (test article being expressed in 

the milk). In order to be protective of the developmental stages where the effect could occur, the 

endpoint was used to protect women of reproductive age who are nursing, as well as all other 

populations including infants and children. Therefore, the acute dietary exposure scenario was 

expanded to include all populations including infants, children and females 13-49 years old. The 

dose and endpoint are also protective of effects observed in the rat and rabbit developmental 

studies. 

aRfD = aPAD = 7 mg/kg/day = 0.07 mg/kg/day

100

Chronic Dietary (All Populations)

Study Selected: Carcinogenicity study in Mice

MRID Number: 48918213

Dose and Endpoint for Risk Assessment: 6.67 mg/kg/day, based on liver effects (increased 

weights and histopathology) at the LOAEL of 21.8 mg/kg/day. This is supported by the 

carcinogenicity study in rats where the NOAEL is 8.1 mg/kg/day, based on decreased body 

weight at the LOAEL of 20 mg/kg/day. 

Uncertainty Factor(s): 100X (10X for interspecies variability, 10X for intraspecies variability, 

1X for FQPA) 
Page 30 of 57

Comments about Study/Endpoint/Uncertainty Factor: This endpoint is appropriate for the 

general population. The duration of this study and the route of exposure through feed is also 

appropriate. This endpoint is protective of adverse effects observed at higher doses in other 

studies and the observation of effects at a similar dose in the carcinogenicity study in rats, 

strengthens our confidence in the database. 

cRfD = cPAD = 6.67 mg/kg/day = 0.067 mg/kg/day

100
Page 31 of 57

Appendix A.4 EXECUTIVE SUMMARIES

Oral Subchronic Toxicity Study-Rat; OPPTS 870.3100

EXECUTIVE SUMMARY: In a 90-day oral toxicity study (MRID 48918239), tricyclazole 

(99.4%, Lot no. C53-C21-147) was administered to 15 Wistar rats/sex/dose in the diet at dietary 

concentrations of 0, 282, 635 and 1640 ppm equivalent to 0, 20.5, 46.7 or 135.3 mg/kg/day, 

respectively, in males and 0, 22.5, 54.2 or 186.8 mg/kg/day, respectively, in females. These 

dosages are time-weighted averages calculated from reported weekly averages.

There were no treatment related effects on mortality, hematology, clinical chemistry and gross 

pathology. Neurological and urine parameters were not assessed and eyes were not examined. 

Additionally, numerous parameters in hematology, clinical chemistry, organ weights and 

histopathology were not assessed.

Treatment-related decreased body weight, body weight gain, food consumption and food 

efficiency were observed at the mid- and high-dose levels in both sexes; decreased body weight 

gain was also seen in low-dose males. Impaired growth in mid- and high-dose animals resulted 

in decreased absolute and/or increased relative weights for several organs including kidney, 

heart, spleen, prostate, testes, ovaries, uterus, thyroid and adrenals. Absolute liver weights were 

not decreased in exposed groups; the average weights were slightly higher than controls in these 

groups for both sexes. This resulted in statistically significant increases in average relative liver 

weights in all of these groups. This was probably an adaptive response to compound-related 

increased mixed function oxidase activity since there was a concomitant dose-related increase in 

p-Nitroanisole metabolism. Slight centrilobular hypertrophy was seen in liver of 6/15 high-dose 

males; immature testes and small prostate were found in 3/15 and 2/15 high-dose males, 

respectively. 

The LOAEL is 46.7 mg/kg/day, based on decreased body weight, food consumption and 

food efficiency. The NOAEL is 20.5 mg/kg/day.

This 90-day oral toxicity study in the rat is Unacceptable/Guideline and does not satisfy the 

guideline requirement for a 90-day oral toxicity study (OCSPP 870.3100; OECD 408) in rats and 

is not upgradable because of numerous study deficiencies.

Oral Subchronic Toxicity Study-Mouse; OPPTS 870.3100

EXECUTIVE SUMMARY: In a 90-day oral toxicity study (MRID 48918238) Tricyclazole 

(99.4%, Lot no. C53-C48-147) was given to 15 ICR mice/sex/dose at dietary concentrations of 0, 

400, 1000, 2500 or 3600 ppm. Definitive doses of test material were not determined in this study 

due to inadequate food consumption data; reported equivalent dose ranges were 41-87, 98-225, 

255-577 and 371-819 mg/kg bw/day, respectively, in males and 41-71, 105-202, 262-522 and 

384-728 mg/kg bw/day, respectively in females based on food consumption data from a separate 
Page 32 of 57

study (M-9347). Based on a dietary conversion factor (1 ppm in the diet = 0.13 mg/kg bw/day), 

these dietary levels were equivalent to doses of 0, 52, 130, 325 and 468 mg/kg bw/day, 

respectively. The doses based on the conversion factor were used in this DER. 

There were no toxicologically significant compound-related effects on body weights, water 

consumption or gross pathology. Neurological and urine parameters were not assessed and eyes 

were not examined. Additionally, numerous parameters in hematology, clinical chemistry, organ 

weights and histopathology were not assessed.

The following findings were compound-related: clinical signs and mortalities ≥ 130 and 325 

mg/kg/day in males and females, respectively, increased platelet counts ≥ 325 mg/kg/day in 

males, increased alanine aminotransferase (SGPT) activity at 468 mg/kg/day in males and 

females, increased absolute and relative liver weights ≥ 52 and 325 mg/kg/day in females and 

males, respectively, and proliferation of small bile ducts in liver at 468 mg/kg/day in both sexes. 

It appears that the dose-related increases in absolute and relative liver weights in both sexes may 

represent an adaptive response to Tricyclazole-induced increased mixed function oxidase activity 

based on evidence in a pilot mouse study, used for dose-selection in this study, and a 13-week rat 

dietary study (R-473).

Increased absolute and relative ovary weights ≥ 130 mg/kg/day were not dose-related; therefore, 

the relevance of this finding is uncertain.

The LOAEL is 130 mg/kg/day (1000 ppm) based on clinical signs and mortality. The 

NOAEL is 52 mg/kg/day.

This 90-day oral toxicity study in the mouse is Unacceptable/Guideline and does not satisfy the 

guideline requirement for a 90-day oral toxicity study (OCSPP 870.3100; OECD 408) in mice 

and is not upgradable because of numerous study deficiencies.

EXECUTIVE SUMMARY: In a 10 month toxicity study evaluating effects on the liver (MRID 

48918224), groups of 40 male and 40 female ICR mice were administered tricyclazole technical 

(99.4% a.i., Lot # C53-C58-147) in the diet at concentrations of 0%, 0.04%,0.1%, 0.25%, or 

0.36% (equivalent to 0, 400, 1000, 2500, and 3600 ppm, respectively) for 6 months. Based on a 

food factor of 0.l3, the estimated weight-normalized doses were 0, 52, 130, 325, 468 mg/kg/day. 

After 6 months, 20 animals/sex/dose were killed, and the remaining animals were maintained on 

basal diet. Ten animals/sex/dose were killed after 3 months on basal diet, and the remaining 

animals were killed after 4 months on basal diet. The animals were observed twice a day for 

mortality and moribundity; body weight measurements and detailed clinical observations were 

conducted once a week. Daily cageside clinical observations were not conducted in this study. 

All animals including those that died early or killed at termination were subjected to a detailed 

gross examination. The livers were excised, weighed and samples of livers were processed for in 

vitro measurement of microsomal protein and p-nitroanisole O-demethylase activity. Liver 

weight relative to body weight was calculated.
Page 33 of 57

No treatment-related effects were observed on mortality, clinical signs, body weight, or weight 

gain. Absolute and relative liver weights were significantly increased at 1000, 2500, and 3600 

ppm in male and female mice after treatment with the test material for 6 months. Liver weight 

was increased in a dose related manner: 12% to 27% for absolute weight and 9% to 25% for 

relative weight in males; 15% to 30% for absolute weight and 12% to 34% for relative weight in 

females. Absolute and relative liver weights were similar to those of controls after the return to 

basal diet for 3 and 4 months. The microsomal enzyme activity, p-nitroanisole O-demethylase, 

was significantly increased in male rats by 34%, 65%, ad 87% after treatment with 1000, 2500, 

and 3600 ppm, respectively, for 6 months. Enzyme activity was significantly increased by 24% 

in female mice treated with the 3600-ppm diet for 6 months. Enzyme activity was similar to that 

of controls after the return to basal diet for 3 months. No statistically significant effect was 

observed on microsomal liver protein after treatment with the test material. Treatment-related 

microscopic lesions observed in the liver included proliferation of small bile ducts and periportal 

lipocytes; bile duct proliferation was slight to moderate in males and slight in females. 

Proliferation of small bile ducts was observed in 0/20, 0/20, 7/20, 16/20, and 20/20 males and 

1/20, 0/20, 3/19, 5/20, and 15/19 females after treatment with the 0-, 400-, 1000-, 2500-, and 

3600-ppm diets, respectively, for 6 months. The incidence after returning to the basal diet for 3 

months was 0/10, 0/10, 1/10, 0/10, and 5/10, respectively, in males. No treatment-related effect 

was observed on bile duct proliferation in females on basal diet for 3 or 4 months or in males on 

basal diet for 4 months. Periportal lipocytes were observed in 1/20, 3/20, 7/20, 13/20, and 9/20 

males and in 0/20, 2/20, 7/19, 10/20, and 15/20 females, respectively, after treatment with the 

test material for 6 months. After return to the basal diet, the incidence of periportal lipocytes 

was 0/10, 1/10, 3/10, 4/10, and 5/10, respectively, for males and 0/10, 4/10, 6/10, 7/10, and 8/10 

in females, respectively. No treatment-related effect was observed after 4 months on the basal 

diet. 

The lowest-observed-adverse-effect level (LOAEL) for is 400 ppm (0.04%, 52 mg/kg/day) 

based on microscopic lesions in the liver (periportal lipocytes and bile duct proliferation). 

A no-observed-adverse-effect level (NOAEL) was not established in this study.

This study in mice is Acceptable/Non-guideline and satisfies the requirement for a special study 

to evaluate liver toxicity in mice. The concentration of test material showed considerable 

variation and ranged from 13% to 22% below that of the target averaged over the three batches 

of diet. Therefore, the concentrations reported may not be valid. However, the study showed 

that treatment with the test material had effects on the liver, which were reversible after the 

return to basal diet.

Developmental Toxicity Study-Rat; OPPTS 870.3700a

EXECUTIVE SUMMARY: In a developmental toxicity study (MRID 48918218), tricyclazole 

(95.2% a.i.; Batch #2002090020) was administered to 28 mated outbred Wistar [Crl:(WI)WU 

BR] rats/dose in 1% carboxymethylcellulose by gavage at dose levels of 0, 5, 20, or 50 

mg/kg/day (5-mL/kg bw dosing volume) on gestation days (GDs) 0 through 20. On GD 21, 

dams were sacrificed and necropsied. Gravid and empty uterine weights, ovarian weights, 
Page 34 of 57

corpora lutea counts, and the numbers and positions of implantations, live and dead fetuses, and 

early and late resorptions were recorded. All fetuses were weighed, sexed, and examined for 

external anomalies. Approximately one-half of the fetuses from the first 20 litters of each group 

were subjected to skeletal examination, and the remaining one-half of the fetuses from the 

designated control and high-dose litters were subjected to visceral examination. All placentas 

were also weighed and examined grossly. 

The death of one high-dose female on GD 5 with no preceding abnormal clinical signs was not 

considered treatment-related. High-dose females exhibited treatment-related clinical signs 

between GDs 0 and 4, including weakness (15 animals), cold body condition (12 animals), and 

piloerection (11 animals), with a total of 15 high-dose animals affected. High-dose dams had a 

mean body weight loss over the GD 0-3 measuring interval (-12.28 g vs. +11.54 g for controls; 

p<0.01) and biologically and statistically significant decreases in absolute body weight at all 

measuring intervals between GDs 3 and 17 (9-11% less than controls; p<0.01). Mid-dose dams 

had significantly decreased body weight gains over GDs 0-3 and 3-7 (-28% and -43%, 

respectively), but absolute body weight was not affected by treatment. Mid- and high-dose dams 

both had compensatory increases in body weight gain over the GD 17-21 interval (+39% and 

+53%, respectively; p<0.01) resulting in an overall weight gain similar to that of controls at the 

mid dose and 20% less than that of control at the high dose. High-dose dams had decreased food 

consumption between GDs 0 and 17 (67%, 29-36%, and 17-21% less than controls during GDs 

0-3, 3-10, and 10-17, respectively), while mid-dose dams had decreased food consumption 

between GDs 0 and 10 (21% and 14-16% less than controls during GDs 0-3 and 3-10, 

respectively). There were no treatment-related effects on gross pathology, ovarian or empty 

uterine weight, or adjusted (for gravid uterine weight) terminal body weight or net body weight 

gain during gestation. The maternal lowest-observed-adverse-effect level (LOAEL) is 50 

mg/kg/day, based on decreased body weight and food consumption, and clinical signs

(weakness, cold body, and piloerection). The no-observed-adverse-effect level (NOAEL) is 

20 mg/kg/day. 

In the high-dose group, there were significant (p<0.01) treatment-related decreases in mean 

gravid uterine weight (-20%) and mean fetal weights for males, females, and the combined sexes 

(-13%, -15%, and -14%, respectively). In the high-dose group, there was also an increased litter 

incidence of “small fetuses” (10/22 litters vs. 1/23 control litters) with evidence of delayed 

ossification of bones of the skull (nasal, interparietal, supraoccipital, palatine, and presphenoid 

bones), cervical and caudal vertebral bodies, metacarpals and front distal phalanges, metatarsals 

and hind proximal and distal phalanges. The high-dose group also had a higher incidence of 

accessory lumbar ribs (noted in 6 litters/6 fetuses vs. no controls; p<0.05). In the mid-dose 

group, delayed ossification of the nasal and interparietal bones were seen in the absence of 

effects on fetal body weight. There were no total litter losses or dead fetuses. The mean preand postimplantation losses and numbers of corpora lutea, implantations, viable fetuses, total 

resorptions and early resorptions of the treated dams were similar to those of controls. There 

were no treatment-related effects on fecundity index, gestation index, fetal sex ratios, or the 

incidences of fetal developmental malformations or anomalies. The developmental LOAEL is 

20 mg/kg/day based on delayed ossification of the nasal and interparietal bones and front 
Page 35 of 57

distal phalanges. The NOAEL is 5 mg/kg/day.

This developmental toxicity study in the rat is classified Acceptable/Guideline and satisfies the 

guideline requirement for a developmental toxicity study (OCSPP 870.3700; OECD 414) in the 

rat.

Developmental Toxicity Study-Rabbit; OPPTS 870.3700b

EXECUTIVE SUMMARY: In a developmental toxicity study (MRID 48918207), Tricyclazole 

(99.3% a.i., Lot #2003030017; Batch #TSN104307) was administered by gavage in 0.5% 

aqueous methylcellulose to 25 time-mated, presumed pregnant New Zealand White 

[Hra:(NZW)SPF] rabbits/dose at dose levels of 0, 7.5, 25, or 75 mg/kg/day on gestation days 

(GDs) 7 through 28, inclusive. On GD 29, the does were sacrificed and necropsied. Gravid 

uterine weights, corpora lutea counts, and the numbers and positions of live and dead fetuses, 

and early and late resorptions were recorded, and all fetuses were weighed and examined for 

external anomalies. Live fetuses were sexed internally and subjected to visceral examination by 

fresh dissection (with heads examined by mid-coronal slice) then processed for and subjected to 

skeletal examination. 

High-dose dams had decreased mean maternal body weight gains over the GD 13-21 and GD 7-

29 intervals (-57% and -34% less than controls, respectively; p<0.01), which resulted in small 

but statistically significant decreases in this group’s mean absolute body weight between GDs 23 

and 26 (-5%; p<0.05). These differences were correlated with decreased mean daily food 

consumption during GDs 12-20 (18-41% less than controls; p<0.01), which resulted in decreased 

mean values over the GD 13-21 and GD 7-29 intervals (-33% and -18%, respectively; p<0.01). 

Although statistically significant, the minor decreases in body weight and body weight gain were 

not considered adverse. There was also decreased defecation in high-dose females (14/25 

animals affected between GDs 15 and 27). There were no treatment-related deaths, abortions, or 

early deliveries, or abnormal gross pathology. The maternal no-observed-adverse-effect-level 

(NOAEL) is 75 mg/kg /day, the maternal lowest-observed-adverse-effect-level (LOAEL) is 

not determined.

Total litter loss (10 late resorptions) with one dead fetus was noted in the high-dose group. 

However, the effect was only noted in 1 female and considered incidental. At the high dose, 

there were also treatment-related decreases in mean fetal weights for males, females, and the 

combined sexes (-14%, -16%, and -15%, respectively; p<0.01); the fetal weights were also 

outside of the WIL historical control data for male, female, and combined fetal weight. The 

mean pre- and postimplantation losses and numbers of corpora lutea, implantations, viable 

fetuses of the treated dams were similar to those of controls, and there were no treatment-related 

effects on fetal sex ratio. Maternal treatment did not result in an increased incidence of any fetal 

developmental malformation or skeletal variation. 
Page 36 of 57

The developmental NOAEL is 25 mg/kg/day and the developmental LOAEL is 75 

mg/kg/day based on significant decreases in fetal body weight.

The developmental toxicity study in the rabbit is classified Acceptable/Guideline and satisfies 

the guideline requirement for a developmental toxicity study (OCSPP 870.3700; OECD 414) in 

the rabbit.

Reproduction and Fertility Effects-Mouse; OPPTS 870.3800

EXECUTIVE SUMMARY: In a multi-generation study (MRID 48918220), groups of 50 male 

and 50 female ICR weanling mice per group were continuously fed diets containing tricyclazole, 

technical grade (Batch # C-53-C48-147, 99.4% a.i.; #B07-C48-20, 98.4% a.i.) at concentrations 

of 0%, 0.005%, or 0.0275%, equivalent to 0, 50, or 275 ppm. Based on estimated food 

consumption of 3 g/day, the premating doses were equivalent to 0, 4-7, 17-38 mg/kg bw/day, 

respectively for males and females. The F0 parents were fed their respective diets for 91 days, 

the F1 generation for 56 days, and the F2 generation for 63 days during the growth (premating), 

gestation, and lactation periods for three generations. Groups of 20 males and 20 females in each 

parental generation and the same dose group were paired (avoiding brother sister mating), 

observed for the presence of a copulatory plug (gestation day 0), and killed on gestation day 20 

for a separate developmental toxicity study. The remaining 30 males and 30 females in each 

generation were paired within their respective dose groups for 16 days (avoiding brother sister 

mating), the females were separated from the males, individually housed, and allowed to deliver 

their offspring naturally. The parental animals were observed daily and body weight data were 

recorded weekly. Food consumption was not measured. Litter data (number, weight, and 

survival) were recorded during lactation. Pups were weighed as litters on days 1, 7, and 14 and 

individually on day 21. Offspring were selected at weaning to parent the next generation, two 

controls and one treated pup/sex from each litter were necropsied at weaning, and the remaining 

pups were killed and discarded without further examination. All F3 weanlings were subjected to 

gross examination and tissues from one F3 weanling/sex were examined microscopically.

A total of 13 animals in the three parental generations died during the study: eight controls, two 

in the 50-ppm group, and three in the 275-ppm group. The cause of death was unknown for 

most, but was not considered treatment related. No treatment-related effects were observed on 

body weight or weight gain during the growth phase (premating) of the study. 

The parental systemic toxicity LOAEL could not be determined. The parental systemic 

toxicity NOAEL is 17-38 mg/kg/day. 

No treatment-related effects were observed on the fertility index (proportion of females 

pregnant), gestation index (proportion of pups born alive), gestation length, or the number of live 

or stillborn pups.

The reproductive toxicity LOAEL could not be determined. The reproductive toxicity 

NOAEL is 17-38 mg/kg/day.
Page 37 of 57

No treatment-related effects were observed on the 1-, 7-, 14-, or 21-day F1, F2, or F3 offspring 

survival indices. Body weights of F1, F2, or F3 pups were not affected during lactation, and no 

treatment-related effect was observed on the sex ratio at weaning. A total of 382 controls and 

697 weanlings in the treated groups were subjected to gross examination; no treatment-related 

abnormalities were reported. Microscopic examination of tissues in 265 F3 pups revealed no 

treatment-related effects. 

The LOAEL for offspring toxicity could not be determined. The NOAEL for offspring

toxicity is 17-38 mg/kg/day.

This reproduction and fertility study in mice is Acceptable/Non-guideline. Dosing was not 

considered adequate because no toxicity in parents or offspring was observed in this study; the 

highest dose tested was below the limit dose of 1000 mg/kg/day or 20,000 ppm of the test 

substance in the diet. This study was conducted prior to Subdivision F and OCSPP guidelines. 

EXECUTIVE SUMMARY: In a prenatal developmental toxicity study (MRID 48918216), 

groups of 50 male and 50 female weanling Harlan ICR mice per group were fed diets containing 

tricyclazole, technical grade (Batch # C-53-C48-147, 99.4% a.i.; #B07-C48-20, 98.4% a.i.) at 

concentrations of 0%, 0.005%, or 0.0275%, equivalent to 0, 50, or 275 ppm for three 

generations. The F0 parents were fed their respective diets for 91 days, the F1 generation for 56 

days, and the F2 generation for 63 days during the growth period (premating) before mating. The 

developmental toxicity phase of the study consisted of 20 females mated with 20 males in the 

same dose group and (avoiding brother sister mating) in each generation. The day a copulatory 

plug was found was designated gestation day (GD) 0. The maternal animals were killed on 

gestation day 20 for evaluation of maternal developmental parameters. The F0 maternal animals 

were fed their respective diets during the premating and gestation phase, the F1 maternal animals 

were fed their respective diets during premating, gestation, and lactation, and the F2 maternal 

animals were fed their respective diets during premating and gestation. The doses based on an 

estimated daily food consumption of 3 g/day were 0, 4-7, and 17-38 mg/kg bw/day, respectively. 

At termination of each generation, the reproductive status was determined for each maternal 

animal, the number of corpora lutea on each ovary was counted, and the number and distribution 

of fetuses and resorptions in each uterus was recorded. Resorptions were classified as early or 

late. The fetuses were examined for external abnormalities and viability. The sex of each fetus 

was determined and the fetuses were weighed by litter. Approximately one-third of the fetuses 

were fixed in Bouin’s solution for visceral examination by a modified Wilson’s technique, and 

the remaining fetuses were processed for skeletal examination after clearing with benzyl alcohol 

and staining with alizarin. The fertility, gestational survival, and resorption indices were 

calculated.

All animals assigned to the developmental phase of the study survived to termination. No 

treatment-related effect was observed on maternal body weight, fertility index, or number of 

implantations/mouse. 
Page 38 of 57

The maternal toxicity LOAEL could not be determined. The maternal toxicity NOAEL is 

17-38 mg/kg /day). 

No treatment-related effects were observed on developmental toxicity parameters in any 

generation including the number of resorption, resorption index, litter size, gestation index 

(proportional of live fetuses), sex ratio, fetal body weight, or external, visceral, or skeletal 

abnormalities. Common findings included 14th rib, exencephaly, misaligned sternebrae, and 

missing vertebrae. 

The developmental toxicity LOAEL could not be determined. The developmental toxicity 

NOAEL is 17-38 mg/kg /day).

This reproduction and fertility study in mice is Acceptable/Non-guideline. Dosing was not 

considered adequate because no maternal or developmental toxicity was observed in this study; 

the highest dose tested was below the limit dose of 1000 mg/kg/day or 20,000 ppm of test 

substance in the diet. This study was conducted prior to Subdivision F and OCSPP guidelines.

Reproduction and Fertility Effects-Rat; OPPTS 870.3800

EXECUTIVE SUMMARY: In a two-generation reproduction study (MRID 48918221), 

tricyclazole technical (95.2% a.i., Batch No. 2002090020) was administered in the diet to groups 

of 28 F0 and F1 male and 28 F0 and F1 female Wistar outbred (Crl:(WI)WU BR) rats at 

concentrations of 0, 30, 100, or 400 ppm (equivalent to premating doses of 0, 2.1, 7.0, and 26.7 

mg/kg/day for males and 00, 2.3, 8.0, and 30.7 mg/kg/day for females) for 70 days before and 

during mating. The male rats were terminated after mating; whereas, the female rats were 

maintained on their respective diets during gestation and lactation. The females were terminated 

after weaning their litters. Each parental generation produced only one litter. Parental systemic 

toxicity, reproductive function and performance, and offspring viability, growth, and sexual 

maturation were evaluated in this study.

No treatment-related clinical signs, mortality, or ocular abnormalities were observed in parental 

animals and no treatment-related systemic toxicity was observed at the low- or mid-dose levels. 

Body weight decreases were observed in adult F0 (up to 9%)and F1 (up to 15%) male rats during 

premating at the high-dose level compared with that of controls. The F0 and F1 males also had 

decreased body weight gains of up to 40% and 25%, respectively. Decreased body weight was 

also observed in adult F0 (up to 6.3%) and F1 females (up to 11.1%) during premating. Body 

weight gain was also decreased in F0 and F1 females (up to 10.1%) during the premating period. 

Food consumption showed a corresponding decrease in both male and female rats during 

premating. Body weight, weight gain, and food consumption were only slightly affected in F0

females during gestation and lactation. For adult F0 females, body weight decreases remained 

relatively minor (< 10%) during gestation and lactation. Body weight gain was only decreased 

by 4% during gestation but increased by 23% during lactation; food consumption was decreased 

during postnatal days (PNDs) 7-14. In adult F1 females, body weight decreases approached 9% 

during gestation and up to 10% during lactation. Body weight gain was decreased during 
Page 39 of 57

gestation (16.7%) and lactation (16.8%) and food consumption was significantly decreased 

during the early phase of gestation and during lactation (PNDs 4-14). Postmortem examination 

showed no treatment-related gross or microscopic lesions in male or female rats and the 

statistically significant changes in absolute and relative organ weights were not accompanied by 

microscopic lesions, were too small to be of toxicological concern, or were due to decreases in 

terminal body weight. 

 

The parental systemic toxicity LOAEL is 400 ppm (26.7 mg/kg/day), based on decreases in 

body weight and food consumption in F0 and F1 adult males and females. The parental 

systemic toxicity NOAEL is 100 ppm (7.0mg/kg/day). 

No treatment-related effects were observed on fertility or reproductive performance in male or 

female rats in either generation. Estrous cycle length, sperm measures, reproductive indices, precoital and gestation length, litter size, and the number of still and live births were not affected by 

treatment with the test material. No treatment-related macroscopic or microscopic lesions were 

observed in reproductive organs. Sexual maturation in F1 offspring was delayed in females at the 

high-dose level. Vaginal opening in females was attained 5.4 days later than in controls. 

Preputial separation in males was also delayed; however, the delay (1.98 days) was within 

biological variability and not considered treatment-related.

The reproductive toxicity LOAEL is 400 ppm (26.7 mg/kg/day), based on delayed sexual 

maturation (vaginal opening). The reproductive toxicity NOAEL is 100 ppm 

(7.0mg/kg/day). 

No adverse effects were observed on litter size from birth to weaning, live birth index, viability 

indices, sex ratio, anogenital distance, external abnormalities, organ weights or gross lesions. 

However, treatment-related effects were seen on pup survival/mortality. Increased incidence of 

pup death was observed in F1 and F2 pups during PND 1-4. Although not statistically 

significant, the deaths were considered biologically significant. Pup weight and weight gain 

were significantly decreased at the high-dose level in male and female pups in both generations, 

with the effect being more severe in the F2 generation than in the F1 generation. F1 male and 

female pups at the high-dose demonstrated decreased body weight starting at PND 14 (-8.7% 

to -10.6%) and decreased body weight gain after PND 7 with overall weight gain decreased by 

11.9% to 12.3% compared with that of controls. Male and female F2 pups at the high-dose level 

demonstrated decreased body weight at PND 7 and that continued to the end of lactation (-6.7% 

to -20.4%). Overall, F2 pups gained 15.6% to 16.0% less weight than controls over the 21-day 

lactation period. The number of small F2 pups was also significantly increased in the high-dose 

group compared with that of controls. 

The offspring LOAEL is 400 ppm (26.7 mg/kg/day) based on decreases in pup body weight,

increased number of small pups, and increased pup death (PND1-4). The offspring 

NOAEL is 100 ppm (7.0 mg/kg/day).
Page 40 of 57

This study is Acceptable/Guideline and satisfies the guideline requirement for a 2-generation 

reproductive toxicity study (OCSPP 870.3800; OECD 416) in rats.

Oral Chronic Toxicity study -Dog; OPPTS 870.4100

EXECUTIVE SUMMARY: In a chronic toxicity study (MRID48918222), tricyclazole (97.0% 

a.i., lot #P68931) was administered to 4 beagle dogs/sex/dose by capsule at dose levels of 0, 5, 

15, or 45 mg/kg /day for one year. There were no compound related effects on mortality, clinical 

signs, hematology, recommended clinical chemistry parameters, or gross or histological findings. 

For female dogs in the 45 mg/kg /day group, mean final body weight was 86% of the mean 

control value and mean body weight gain was 70% of the control value. Statistically significant 

increases in absolute and relative liver and kidney weight were seen in male dogs in the 45 

mg/kg/day group. Statistically significant increases in absolute and relative liver weight and 

relative kidney weight were seen in female dogs in the 45 mg/kg/day group. Hepatic pnitroanisole O-demethylase metabolism and CYP450 content were reduced in male dogs in the 

15 and 45 mg/kg/day groups. The reduced activity of p-nitroanisole O-demethylase and reduced 

CYP450 content in male dogs was considered toxicologically nonsignificant as the antipyrine 

probe taken pretreatment and at 1, 3, 6, and 12 months during the study showed that the 

oxidative drug metabolism system was unaffected in male dogs in all dose groups. The NOAEL 

is 15 mg/kg/day and the LOAEL is 45 mg/kg /day, based on decreased body weight (14%) 

in female dogs. 

This chronic study in the dog is acceptable/guideline and satisfies the guideline requirement for 

a chronic oral study [OCSPP 870.4100b, OECD 452] in dogs. 

Oral Chronic/Carcinogenecity Toxicity study -Rat; OPPTS 870.4100 and OPPTS 870.4300

EXECUTIVE SUMMARY: In a chronic/carcinogenicity study (MRID 48918226), tricyclazole

[99.5% a.i. (lot # C53-C48-147) 98.4% a.i. (lot # B07-C98-20] was administered in the diet to 

groups of 40 male and 40 female Wistar rats at concentrations of 100, 275, or 620 ppm for 2 

years. The dietary concentrations were equivalent to a dose range of 2.7-5.4, 8.1-14, and 20-34 

mg/kg/day, respectively, for males, and 3.4-7.5, 12-24, and 30-57 mg/kg/day, respectively, for 

females. An additional group of 40 male and 40 female rats were administered a dietary 

concentration of 1600 ppm (equivalent dose range = 106-112 mg/kg/day for males; 168-177 

mg/kg/day for females) for 90 days then given basal diet for 21 months. All groups administered 

the test material had one control group comprised of 60 male and 60 female Wistar rats.

There were no test-substance-related clinical findings observed during the study. The test

substance did not significantly alter survival at any time point during the two-year study. 

Survival percentages at study termination were 30, 31, 32, 25, and 32% for males and 32, 32, 38, 

30, and 34% for females in the 0, 100, 275, 620, and 1600 ppm dose groups, respectively. 

Statistically lower (p≤0.05) body weight by 8% to 18% throughout most of the experimental 

period was observed in males and females administered 620 ppm. Males in the 1600 ppm 

treatment group began the study on day 0 with statistically lower body weight by about 8% 
Page 41 of 57

relative to controls, but both males and females had statistically lower body weights by week 12 

(-26% and -23% for males and females, respectively). This statistical difference persisted for 

males until week 78 and for females until week 52. Statistically lower (p≤0.05) body weight 

gains were also observed in males and females at 620 ppm. Statistically lower food consumption 

was observed in both males and females (p≤0.05) at the 1600 ppm dietary concentration 

throughout most of the 90 days of test substance administration. The animals in the 620 ppm 

group also had statistically lower (p≤0.05) food consumption regularly throughout the 2-year 

experimental period. Statistically significant increased (p≤0.05) glucose was observed at the end 

of the two-year experimental period in females from all dietary concentration groups (75 to 87 

mg/100 mL, respectively, with a control value of 57 mg/100 mL). The glucose values were

described as being within the historical control range, but the data were not provided. There were 

no statistically significant or treatment-related differences between treated and control rats in the 

hematology or urinalysis data in either sex. There were no treatment-related findings during the 

gross examination of rats. Statistically significant increased (p≤0.01) relative liver weights were 

observed in males and females administered the test material at 620 ppm for two years. Absolute 

liver weight at this dietary concentration tended to be higher, but did not reach a level of 

significance. Statistically higher brain weight was observed in males at 620 ppm, but this finding 

was not observed in females. Treated males and females at 1600 ppm had statistically lower 

(p≤0.05) absolute brain weights at necropsy, but not relative to body weight. Due to 

inconsistency of the data, thefindings were not considered treatment-related. The microscopic 

non-neoplastic lesions observed were common to this strain of rats during the aging process and 

the incidences were no different between treated and control groups. Hepatocellular adenoma 

occurred in a few treated rats and hepatocellular carcinoma was observed in one treated rat. 

However, the incidences were low, not dose related, and within historical control range. 

The lowest-observed-adverse-effect level (LOAEL) in rats is 620 ppm (20 mg/kg/day), 

based on decreases in body weight (up to 18%) and food consumption in both sexes. The 

no-observed-adverse-effect level (NOAEL) is 275 ppm (8.1 mg/kg/day). 

At the doses tested, no treatment-related increases in the incidences of neoplastic lesions were 

observed in male or female rats when compared with those of controls. Dosing was considered 

adequate based on the appearance of microscopic liver lesions in both sexes.

This chronic/carcinogenicity study is Acceptable/Guideline and satisfies the guideline 

requirement for a chronic/carcinogenicity study [(OCSPP 870.4300); OECD 453] in the rat. 

Oral Chronic/Carcinogenecity Toxicity study -Mouse; OPPTS 870.4100 and OPPTS 

870.4200

EXECUTIVE SUMMARY: In a chronic toxicity/carcinogenicity study (MRID 48918213), 

tricyclazole technical (98.85% a.i., Lot # 133AS1) was administered in the diet to groups of 64 

male and 64 female ICR-JCL mice at concentrations of 0, 25, 75, 250, or 1000 ppm (equivalent 

to 0. 2.59, 7.98, 24.9, and 101 mg/kg/day for males and 0, 2.20, 6.67, 21.8, and 91 mg/kg/day for 

females). Ten mice/sex/dose were sacrificed at 52 weeks for interim evaluations; the remaining 
Page 42 of 57

animals were administered their respective diets for up to 95 weeks. The study was terminated at 

95 weeks because of the excessive number of deaths in all groups including the controls.

No treatment-related effects were observed on the following parameters: mortality, clinical 

signs, body weight, weight gain, food consumption, food efficiency, and urinalysis and clinical 

chemistry parameters. No treatment-related effects were observed in female mice on 

hematologic parameters. At 250 and 1000 ppm, statistically significant transient decreases were 

observed in erythrocyte count, hematocrit, and hemoglobin levels (8%-11% and 13% to 14%, 

respectively, compared with those of controls) in male mice sacrificed at 52 weeks; no 

significant effects were observed in male mice sacrificed at 95 weeks. Postmortem examination 

of mice sacrificed at 52 weeks showed significant increases in absolute and relative (to body and 

brain weight) liver weights in male and female mice in the 1000-ppm group and in male mice in 

the 250-ppm group; the liver weights were increased by 17% to 21% and 22% to 30% in male 

mice at 250 and 1000 ppm, respectively, and by 20% to 26% in female mice at 1000 ppm 

compared with those of controls. No treatment-related gross findings were observed in this 

study. Treatment-related microscopic findings in the main study consisted of fatty changes and 

acidophilic degeneration in periportal hepatocytes in male and/or female mice at 250 and 1000 

ppm. Both lesions were first observed in 1000-ppm group male and female mice sacrificed after 

treatment with the test material for 52 weeks. 

The lowest-observed-adverse-effect level (LOAEL) is 250 ppm (24.9 mg/kg/day for males 

and 21.8 mg/kg/day for females) based on increases in absolute and relative liver weights in 

males and microscopic lesions in the liver (fatty changes and/or acidophilic degeneration in

periportal hepatocytes) in both sexes. The no-observed-adverse-effect level (NOAEL) is 75 

ppm (7.98 mg/kg/day for males and 6.67 mg/kg/day for females).

At the doses tested, no treatment-related increases in the incidences of neoplastic lesions were 

observed in male or female mice when compared with those of controls. Dosing was considered 

adequate based on the appearance of microscopic liver lesions in both sexes.

This carcinogenicity study in mice is Acceptable/Guideline and satisfies the guideline 

requirement for a carcinogenicity study [OSCPP 870.4200; OECD 451] in mice. There were no 

deficiencies that would alter the interpretation of the results of this study.

Carcinogenecity Toxicity study -Mouse; OPPTS 870.4200

EXECUTIVE SUMMARY: In a 2-year chronic toxicity/carcinogenicity study (MRID 

48918230), tricyclazole technical (99.4% a.i., Lot # C53-C48-147; 98.4% a.i., Lot # B07-C98-

21) was administered in the diet to groups of 80 ICR mice/sex/dose at concentrations of 0, 

0.005%, 0.014%, or 0.04% (equivalent to 0, 50, 140, or 400 ppm) for 2 years. The controls 

consisting of 120 males and 120 females group were administered the basal diet only. Food 

consumption was not measured in this study; the investigator used data from a previous 90-day 

and 2-year study to estimate food consumption. The estimated food consumption data, average 

body weight, and percent tricyclazole in the diet was used by the investigators to calculate 
Page 43 of 57

tricyclazole intake for various intervals in the study. The time-weighted average daily doses 

estimated by the reviewer were 0, 4.9, 13.8, and 39.7 mg/kg/day, respectively, for males and 0, 

5.1, 14.7, and 42.0 mg/kg/day for females). The mice were observed two times a day for dead 

and moribund animals. All animals were weighed weekly for the first 5 months and at 2-3 week 

intervals thereafter; a detailed clinical observation was conducted on all animals at the time of 

weighing. Water consumption was measured at various intervals on three mice/sex in five 

groups at each dose level. At study termination, blood samples were collected from all surviving 

animals for evaluation of hematologic and clinical chemistry parameters. Blood was collected 

by cardiac puncture while the animals were under ether anesthesia. All animals that died early or 

were killed in extremis or at study termination by carbon dioxide asphyxiation were subjected to 

a detailed gross examination. Selected tissues were fixed in 10% buffered formalin and 

processed for microscopic examination. The heart, brain, liver, kidneys + adrenals, spleen, 

testes, and ovaries + uterus were weighed.

No treatment-related effects were observed on mortality; 28%, 31%, 34%, and 30% of males and 

28%, 28%, 29%, and 22% of females were alive at study termination. No treatment-related 

clinical signs were observed; alopecia and skin ulcers were common observations in all groups 

including controls. No treatment-related effects were observed on body weight, weight gain, or 

water consumption. Food consumption was not measured. No treatment-related effect was 

observed on hematologic or clinical chemistry parameters evaluated in this study. Postmortem 

examination showed no treatment-related effects on absolute or relative (to body weight) organ 

weights in male or female mice. No treatment-related effect was observed on gross or nonneoplastic findings in male or female mice.

The lowest-observed-adverse-effect level (LOAEL) could not be determined; the noobserved-adverse-effect level (NOAEL) is 400 ppm (39.7 and 42.0 mg/kg/day for male and 

female mice, respectively).

At the doses tested, no treatment-related increases in the incidences of neoplastic lesions were 

observed in male or female mice when compared with those of controls. Dosing was not 

considered adequate because no toxicity was observed at any dose in either sex.

This study in mice is Unacceptable/Guideline and does not satisfy the requirement for a 2-year 

carcinogenicity study [OSCPP 870.4200; OECD 451] in mice. Dosing was not considered 

adequate because no toxicity was observed at the highest dose tested, which was below the limit 

dose of 20,000 ppm of test substance in the diet or 1000 mg/kg/day. In addition, food 

consumption was not measured in this study; the intake of the test material was based on food 

consumption measured in a 90-day study and another unknown 2-year study.

Oral Chronic Toxicity study -Mouse; OPPTS 870.4100

EXECUTIVE SUMMARY: In a 1-year chronic toxicity study (MRID 48918225), tricyclazole 

technical (99.4% a.i., Lot # C53-C48-147; 98.4% a.i., Lot # B07-C98-21) was administered in 

the diet to groups of 15 ICR mice/sex/dose at concentrations of 0, 0.005%, 0.014%, 0.04%, or 
Page 44 of 57

0.062% (equivalent to 0, 50, 140, 400, or 620 ppm) for 1 year. Based on a food factor of 0.13 for 

mice, the weight-normalized doses were 0, 6.5, 18.2, 52, and 80.6 mg/kg/day, respectively, for 

male and female mice. The animals were observed twice a day for mortality and moribundity; 

clinical observations were conducted weekly. Body weights were recorded once a week for the 

first 5 months and at 2-3 week intervals thereafter. At study termination, blood samples were 

collected from all surviving animals for hematologic evaluations (erythrocyte and leukocyte 

counts, hemoglobin, hematocrit, leukocyte differential count, erythrocyte morphology, and 

platelet count) and clinical chemistry (glucose, BUN, creatinine, total bilirubin, alkaline 

phosphatase, and glutamic pyruvic transaminase (alanine aminotransferase). Blood was 

collected by cardiac puncture while the animals were under ether anesthesia. All animals killed 

by carbon dioxide asphyxiation at study termination or in extremis and those that died early were 

subjected to a detailed gross examination. Selected tissues were fixed in 10% buffered formalin 

and processed for microscopic examination. The heart, brain, liver, kidneys+adrenals, spleen, 

testes, and ovaries+uterus were weighed.

No treatment-related effects were observed on mortality, clinical signs, body weight, weight 

gain, hematologic parameters, clinical chemistry parameters, or organ weights. BUN was 

significantly decreased in male mice at 140, 400, and 620 ppm by 46%, 38%, and 41%, 

respectively; the lack of a clear dose response relationship indicate that the changes are not 

treatment related and the decrease in BUN is not associated with an adverse effect on renal 

function. Total bilirubin was significantly decreased (-23%) in female mice at 620 ppm and 

decreased (14% to 18%), but not significantly, in males at 140-620 ppm. A decrease in total 

bilirubin is not associated with an adverse effect on the liver, but may be an indication of 

microsomal enzyme induction. The small decrease (-13%) in heart weight in 620-ppm group 

males is not considered treatment related or adverse because of the lack of a microscopic 

correlate. The decrease in ovary+uterus weight was due to unusually high values in one control 

with hydrometra and one with cystic uterine hyperplasia. No treatment-related gross or 

microscopic lesions were observed in male or female mice. A high incidence of fatty 

metamorphosis in hepatocytes was observed in male and female mice in the control and the 

lower dose groups but not in the 620-ppm groups. 

The lowest-observed-adverse-effect level (LOAEL) could not be determined; the noobserved-adverse-effect level (NOAEL) is 620 ppm (80.6 mg/kg/day).

This study in mice is Acceptable/Non-guideline.

Bacterial Reverse Mutation Assay; OPPTS 870.5100

EXECUTIVE SUMMARY: In a reverse gene mutation assay in bacteria (MRID 48918208), S. 

typhimurium strains TA98, TA100, TA102, TA1535, and TA1537 were exposed to tricyclazole 

technical (98.8% purity, lot number 2003030017) dissolved in DMSO in two independent assays 

using triplicate plating. 

A preliminary cytotoxicity study, using the plate incorporation method, tested dose levels of 0, 
Page 45 of 57

9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500, and 5000 μg/plate, without and with 

S9-mix. Partial inhibition of the bacterial lawn and reduction in the number of colonies were 

observed at dose levels of 1250 and 2500 μg/plate (TA100), both in absence and presence of 

metabolic activation. Based on these results, the first mutagenicity assay (Trial I) used the plate 

incorporation method at dose levels of 0, 39.06, 78.13, 156.25, 312.5, 625, and 1250 μg/plate, 

without and with S9 (5% v/v S9-mix). A second mutagenicity assay (Trial II) was carried out, 

based on the results of Trial I. The plate incorporation method was used again, this time at dose 

levels of 0, 12.8, 32, 80, 200, 500, and 1250 μg/plate, without and with activation (10% v/v S9-

mix). The S9 fraction was obtained from Aroclor 1254-induced rat liver. There was no evidence 

that the assays were affected by cytotoxicity or insolubility. The number of revertants per plate 

was not increased over the concurrent solvent control value at any test article dose level, without 

or with S9-mix, in any tester strain, in either Trial. The solvent and positive controls induced the 

appropriate responses in the corresponding strains. There was no evidence of induced mutant 

colonies over background.

This study is classified as Acceptable/Guideline and satisfies the guideline requirement for the 

requirement for Test Guideline OCSPP 870.5100; OECD 471 for in vitro mutagenicity (bacterial 

reverse gene mutation) data.

In Vitro Mammalian Cell Gene Mutation Test; 870.5300

EXECUTIVE SUMMARY: In an in vitro mammalian cell gene mutation assay (MRID 

48918212), Chinese hamster ovary (CHO) cell cultures were exposed for 4 hours to tricyclazole 

(99.3% a.i., Lot 2003030017, Reference No. TSN104307), at concentrations of 0, 7, 14, 28, 56.3, 

112.5, 225, 450, and 900 μg/mL without and with activation in a preliminary toxicity assay. 

Based on the results of that assay, the first mutation assay used doses of 0, 50, 100, 300, 500, 

700, and 900 μg/mL without activation and doses of 0, 2.5, 5, 10, 20, 30, 40, 50, 80, and 100 

μg/mL with activation. In the confirmatory (2nd mutation assay) doses of 0, 100, 300, 500, 700, 

and 900 μg/mL without activation and doses of 0, 2.5, 5, 10, 20, 30, 40, 50, 80, and 100 μg/mL 

with activation, were used. Activation was carried out using S-9 from male Sprague-Dawley rat 

liver induced with Aroclor-1254.

Tricyclazole was tested without activation up to its limit of solubility (900 μg/mL) and with 

activation it was tested up to toxic dose levels (100 μg/mL). Either without or with activation, 

tricyclazole did not induce a mutagenic response in CHO cells at any dose level. All of the 

positive controls gave appropriate responses, without and with activation, and the concurrent 

negative controls were in agreement with historical laboratory background values. There was no 

evidence of induced mutant colonies over background.

This study is classified as Acceptable/Guideline, and satisfies the guideline requirement for Test 

Guideline OCSPP 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene 

mutation) data.
Page 46 of 57

EXECUTIVE SUMMARY: In a mammalian cell gene mutation assay at the thymidine kinase 

(TK) locus (MRID 48918237), mouse lymphoma L5178Y cells cultured in vitro were exposed to 

tricyclazole (99.3% a.i., batch #2003/03/0017) dissolved in dimethyl sulfoxide for 24 hours at 

concentrations of 0, 6.4, 10, 15, 23, 36, 55, 85, 131, 164, 205, 256, 320, or 400 μg/mL in the 

absence of metabolic activation and for 4 hours at concentrations of 0, 0.15, 0.29, 0.59, 1.2, 2.3, 

4.7, 9.4, 19, 38, 75, 150, 300, or 600 μg/mL in the presence of mammalian metabolic activation. 

Mammalian metabolic activation was achieved with S9 derived from livers of Aroclor 1254 

induced male Wistar rats. No second (confirmatory) assay was needed because the results were 

so similar to those of an earlier study that used tricyclazole technical (95.2% a.i.) as the test 

article, as reviewed in MRID 48918236.

Tricyclazole was tested up to cytotoxic conditions. At the highest concentration tested, the 

maximum mutant frequency per 106clonable cells was 1303 (versus from 102-166 for the 

solvent control) in the absence of S9-mix and 1345 (versus 120-164 for the solvent control) in 

the presence of S9-mix. There was a positive dose response both without and with S9-mix. The 

positive controls did induce the appropriate response. There was evidence of a concentration 

related, positive response of induced mutant colonies over background.

This study is classified as Acceptable/Guideline and satisfies the guideline requirement for Test 

Guideline OCSPP 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene 

mutation) data.

EXECUTIVE SUMMARY: In a mammalian cell gene mutation assay at the thymidine kinase 

(TK) locus (MRID 48918236), mouse lymphoma L5178Y cells cultured in vitro were exposed to 

tricyclazole technical (95.2% a.i., batch #2002090020) dissolved in dimethyl sulfoxide, in the 

first experiment, for 24 hours at concentrations of 0, 2.1, 4.2, 8.3, 17, 33, 67, 95, 136, 194, 280, 

400, 570, or 810 μg/mL in the absence of metabolic activation and for 4 hours at concentrations 

of 0, 2.1, 4.2, 8.3, 17, 33, 67, 95, 136, 194, 280, 400, 570, or 810 μg/mL in the presence of 

mammalian metabolic activation. Exposures in the second (confirmatory) experiment were for 

24 hours at concentrations of 0, 6.4, 10, 15, 23, 36, 55, 85, 131, 164, 205, 256, 320, or 400 

μg/mL in the absence of metabolic activation and for 4 hours at concentrations of 0, 0.15, 0.29, 

0.59, 1.2, 2.3, 4.7, 9.4, 19, 38, 75, 150, 300, or 600 μg/mL in the presence of mammalian 

metabolic activation. Mammalian metabolic activation was achieved with S9 derived from livers 

of Aroclor 1254 induced male Wistar rats.

Tricyclazole technical was tested up to cytotoxic conditions. At the highest concentration tested, 

the maximum mutant frequency per 106clonable cells was 1255 (versus 99-121 for the solvent 

control) in the absence of S9-mix and 2335 (versus 53-114 for the solvent control) in the 

presence of S9-mix. There was a positive dose response both with and without S9-mix. The 

positive controls did induce the appropriate response. There was evidence of a concentration 

related positive response of induced mutant colonies over background.

This study is classified as Acceptable (Guideline) and satisfies the guideline requirement for 
Page 47 of 57

Test Guideline OCSPP 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward 

gene mutation) data.

Micronucleus Assay in Rats; OPPTS 870.5395

EXECUTIVE SUMMARY: In an ICR CD-1 mouse bone marrow micronucleus assay (MRID 

48918211), 5 mice/sex/dose/harvest time were given a single oral administration of the test 

article, tricyclazole (96.24% purity, Batch No. P-68931) at doses of 0, 100, 200, or 300 mg/kg. 

The high dose was chosen as approximately 50% of the median lethal dose of 594.1 mg/kg in 

males, with toxicity being rather similar in both sexes. Bone marrow cells were harvested at 24, 

48 and 72 hours post-treatment. The vehicle was 10% acacia in water.

The incidence of micronucleated polychromatic erythrocytes (MPCEs) in male and female CD-l 

mice receiving tricyclazole was not significantly greater than that obtained from the 

corresponding vehicle controls at any harvest time or at any dose level examined. Furthermore, 

tricyclazole did not affect the PCE/NCE ratio of either sex at any of the harvest times or dose 

levels. A trend analysis showed no significant increase in the MPCEs with dose. In contrast, the 

positive control, cyclophosphamide, produced a significant increase in MPCE incidence in both 

males and females at the 24 h harvest time, indicating that the assay was working properly. 

There was no biologically significant increase in the frequency of micronucleated 

polychromatic erythrocytes in bone marrow after any dose, treatment interval or in either 

sex.

This study is classified as Unacceptable/Guideline and does not satisfy the guideline 

requirement for Test Guideline OCSPP 870.5395; OECD 474 for in vivo cytogenetic 

mutagenicity data. The doses in the study could have been higher since there was no evidence of 

systemic toxicity and no reduction in PCE:NCE ratio to indicate bone marrow cytotoxicity.

Unscheduled DNA Synthesis; OPPTS 870.5550

EXECUTIVE SUMMARY: In an unscheduled DNA synthesis (UDS) assay (MRID 

48918214), male Fischer 344 rats were treated, by gavage, with tricyclazole (99.3 % a.i., Lot # 

2003030017), dissolved in 0.5% methylcellulose, at doses of 0, 100, and 200 mg/kg for 2 to 4 

hours, and for 14 to 16 hours. Hepatocytes were recovered after treatment and evaluated for the 

occurrence of unscheduled DNA synthesis (UDS).

A range-finding study was performed using a single gavage treatment with the test article at 

doses of 0, 100, 250, 500, and 1000 mg/kg of the tricyclazole in 0.5% methylcellulose, using 

both male and female rats. Doses of 500 and 1000 mg/kg resulted in complete lethality in both 

sexes. Clinical signs of toxicity were seen at 250 mg/kg along with some lethality. No 

differences in toxicity were seen between the sexes. Based on this range-finding study, doses of 

0, 100, and 200 mg/kg were chosen for the UDS study, using males only. Two time points (times 

at which hepatocytes were recovered after treatment) were studied, a 2- to 4-hour time point, and 

a 14- to 16-hour time point. Neither of the test article-treated groups at either time point met the 

criteria for a positive response for an increase in mean net nuclear grain counts or in the percent 

of nuclei with five or more net grains. The vehicle control net nuclear grain results were within 
Page 48 of 57

the historical control and assay acceptance criteria ranges, and the positive control, 

dimethylnitrosoamine (DMN), treatments induced increases in nuclear labeling that met all 

criteria used to indicate UDS. The test article, tricyclazole, was therefore evaluated as negative in 

the in vivo/in vitro assay for unscheduled DNA synthesis in rat primary hepatocyte cultures at 

two time points. There was no evidence that unscheduled DNA synthesis, as determined by 

radioactive tracer procedures [nuclear silver grain counts], was induced.

This study is classified as Acceptable/Guideline and satisfies the guideline requirement for Test 

Guideline OCSPP 870.5550; OECD 482/486 for other genotoxic mutagenicity data.

COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality 

statements were provided.

EXECUTIVE SUMMARY: In a unscheduled DNA synthesis assay (MRID 48918231), 

primary rat hepatocyte cultures were exposed to tricyclazole (96.58 % a.i., Lot # P68931), 

dissolved in DMSO, at doses of 0.5, 1, 5, 10, 50, 100, 500 and 1000 nmol/mL, for 20 hours in 

the presence of 3H-TdR.

Tricyclazole was tested to determine if it could induce unscheduled DNA synthesis (UDS) in 

hepatocytes taken from a male Fischer 344 rat. No rationale was given for the selection of the 

doses used. There was no report of precipitation of the test article or of any toxic effects up to the 

highest dose that was used (1000 nmol/mL = 1mM). Hepatocytes were recovered after treatment 

and evaluated for the occurrence of unscheduled DNA synthesis (UDS). Positive control 

chemicals and vehicle controls gave appropriate responses. There was no evidence that 

unscheduled DNA synthesis, as determined by radioactive tracer procedures [nuclear silver 

grain counts], was induced. 

This study is classified as Unacceptable/Guideline and does not satisfy the guideline 

requirement for Test Guideline OCSPP 870.5550; OECD 482/486 for other genotoxic 

mutagenicity data. The highest dose used was only 1000 nmol/mL, equal to 1 mM, which 

showed no evidence of toxicity. While the Guideline for Unscheduled DNA Synthesis in 

Mammalian Cells in Culture (OCSPP 870.5550) does not specify an upper limit on the dose to 

be used where solubility is not a problem, the maximum dose of 1 mM, is still 10-fold less than 

the maximum dose recommended in other Guidelines, such as OCSPP 870.5375 In Vitro

Mammalian Chromosome Aberration Test. This study should have been carried out with higher 

doses of the test article. Also, 20 cells/concentration were evaluated rather than the required 50 

or more cells/culture.

In Vivo Sister Chromatid Exchange; OPPTS 870.5915

EXECUTIVE SUMMARY: In a Chinese hamster bone marrow in vivo sister chromatid 

exchange (SCE) assay (MRID 48918233), 3 females/dose were treated by i.p. injection with 

tricyclazole (96.58% a.i., lot # P68931) at doses of 21.25, 42.5, 85, or 170 mg/kg bw. Two 
Page 49 of 57

females were treated at a dose of 0 mg/kg bw. A pellet of bromodeoxyuridine (BUdR) was 

implanted subcutaneously into each hamster 5 hours before the injection of the test chemical, 

vehicle, or positive control chemical. Bone marrow cells were harvested at 21 hours posttreatment. Two hours before that harvest, the animals were injected with Velban® (a spindle fiber 

inhibitor). All treatments were single treatments administered by i.p. injection.

There were no signs of toxicity during the study. However, severe cytotoxicity was observed in 

the bone marrow cells of one of the three females tested at the highest dose. Tricyclazole was 

tested at an adequate dose based on cytotoxicity. The positive control induced the appropriate 

response. There was not a significant increase in sister chromatid exchanges in bone 

marrow at any dose.

This study is, however, classified as Unacceptable/Guideline and does not satisfy the guideline 

requirement for Test Guideline OCSPP 870.5915 for in vivo sister chromatid exchange data

because males were not tested, too few females were tested per dose, the number of SCEs per 

chromosome for each metaphase were not presented, and there was no indication that slides were 

coded before analysis. The study could not be upgraded to acceptable; however, it does 

demonstrate that, at least in females, the test article does not appear to have any effect on the 

frequency of SCEs.

In Vitro Cytotoxicity; Non-Guideline

EXECUTIVE SUMMARY: This cytotoxicity study was conducted because tricyclazole had 

given a positive result in the mouse lymphoma forward gene mutation assay both without and 

with S9 activation, while it has been negative in all other assays evaluating genetic damage, both 

in vitro and in vivo. It was hypothesized that the mechanism by which tricyclazole was causing a 

positive (mutagenic) response in the mouse lymphoma assay was through the selection of TK-/-

cells as a result of selective cytotoxicity to TK+/-cells. This study was conducted to determine if 

there was differential cytotoxicity of tricyclazole to TK-/-and TK+/-cells.

In this in vitro non-guideline study to assess cytotoxicity, mouse lymphoma L5178Y TK+/-and 

TK-/-cell cultures were exposed for 4 hours to tricyclazole (99.3% a.i., Lot 2003030017, 

TSN104307), at concentrations of 0, 50, 100, 200, 400, and 800 μg/mL in the absence of 

mammalian metabolic activation. (Concentration units were reported as mg/mL in Table 2A and 

2B in MRID 48918229, but in μg/mL elsewhere in the study.) 

Tricyclazole was evaluated up to toxic concentration levels of 400 and 800

μg/mL. The average relative suspension growth RSG (%) at 2 days in TK+/-cells was 61.0 and 

3.2%, for the 400 and 800 μg/mL concentrations, respectively. In TK-/-cells, cytotoxicity was 

also apparent at concentrations of 400 and 800 μg /mL, with the average RSG (%) values on day 

2 being 44.3 and 0.42 %, respectively. 

This study is classified as Acceptable/Non-guideline, it provides supplemental information to be 

considered when evaluating the results of the mouse lymphoma forward mutation assay.
Page 50 of 57

Metabolism Study-Rat; OPPTS 870.7485

EXECUTIVE SUMMARY: In a metabolism study (MRID 48918209), 14C-tricyclazole (purity 

radiolabeled = 99.6%, Lot # 027144-0001, labeled in the 3-triazole position; non-radiolabeled 

purity = 99.3%, Lot # 2003030017, TSN104307) was administered to groups of 4-8 male and 4 

female Fisher 344 rats/group. Six treatment groups were used in the study, with Groups 1 – 5 

receiving oral doses, and Group 6 receiving an IV dose of the radiolabeled test material. All 

groups received 2 mg/kg radiolabeled test material, with the exception of Group 2 which received 40 mg/kg radiolabeled test material. Rats in Group 5 received daily doses of unlabeled test 

material before a 2 mg/kg radiolabeled dose on Day 15. Rats in Groups 1, 2, and 6 were jugular 

cannulated to allow collection of whole blood 0.08, 0.17, 0.25, 0.5, 1, 2, 3, 6, 12, 24, and every 

24 hours thereafter for 168 hours. Rats in Group 3 were sacrificed at plasma Cmax, 10 and 5 

minutes after treatment for males and females, respectively, while Group 4 rats were sacrificed at 

1⁄2 plasma Cmax 6 hours after treatment. Urine and feces were collected from rats in Groups 1, 2, 

5, and 6 at 12-24 hour intervals during the 168 hour study. Expired air was collected from rats in 

Groups 1, 2, and 6. 

Recovery of radiolabel was excellent, ranging from 88% – 105% of the administered dose for all 

treatment groups. The radiolabeled test material was rapidly absorbed by all treatment groups. 

Because of extensive biliary excretion, total absorption could only be estimated in Group 1 rats, 

and was 89% and 96% in males and females, respectively. Tissue accumulation was low 

after168 hours of treatment and was <2.1% of the administered dose for Groups 1, 2, 5, and 6. 

The orally absorbed dose was rapidly excreted in urine (31-61% of administered dose) with 

slightly higher recoveries in the female. Most urinary excretion (66-86%) occurred within 24 

hours post-dosing. The IV administered test material was also rapidly excreted in urine (35-47% 

of administered dose) with the majority (68-84%) occurring in the first 24 hours. A higher 

percentage (39-65%) of the oral dose was eliminated in feces. Total fecal elimination of the IV 

dose was 58-59%, indicating substantial biliary excretion. In Groups 1, 2, 5, and 6, residual 

concentrations in the blood at 168 hours post-dosing (<0.5% of administered dose) was likely 

due to binding of tricyclazole to RBCs. In orally dosed rats, elimination of the radioactivity from 

plasma was biphasic with most of the elimination occurring during the rapid (α) phase (t1⁄2α ≤0.08 

hours), with a slower (β) elimination phase (t1⁄2β = 34 - 51 hours). There was an indication of 

enterohepatic circulation of biliary eliminated radioactivity between 3-6 hours after treatment in 

low-dose rats and was more pronounced in high-dose rats 1 – 3 hours after treatment. The 

plasma AUC at the high dose was 244-253 μg hours/g, which was 29- to 37-fold greater than the 

low dose. Clearance was faster at the low dose relative to the high dose-group.

Extensive metabolism of the radiolabeled test material was found. A total of 29 metabolites 

were identified in the urine and fecal samples from rats in Group 1, 2, 5, and 6. The majority of 

metabolites were common between dose groups and gender, with some quantitative differences 

between sexes. Nine of the metabolites were found in quantities >5% of the administered dose. 

Tentative identification was made on seven of the nine. In addition, five of twenty metabolites 

comprising <5% of the administered dose were tentatively or conclusively identified, with the 

parent compound representing one of these minor components at 1.0-4.3% of the administered 
Page 51 of 57

dose. Based on the metabolite data, the test material was conjugated with glutathione followed 

by subsequent β-lyase cleavage to form the corresponding thiol. This was further followed by 

conjugation with glucuronide or methylation prior to excretion. Benzyl oxidation was also 

observed. 

This metabolism study in the rat is classified Acceptable / Guideline and satisfies the guideline 

requirement for a metabolism study [OCSPP 870.7485, OECD 417] in the rat. 
Page 52 of 57

Appendix B. Metabolism

Figure B1: Tabular Summary of Metabolites

Table A1. Tabular Summary of Metabolites and Degradates

Chemical Name (other 

names in parenthesis) 

and Structure

Matrix Percent TRR (PPM)1

Matrices - Major 

Residue (>10%TRR)

Matrices - Minor 

Residue (<10%TRR)

Parent

*

N

N

N

S

CH3

Rice Up to 11% in grain, up to 

79% in forage at 14 DAT

--

Rat -- < 5%

Alcohol Metabolite

N

N

N

S

CH2

HO

Rice -- Up to 4%

Rat -- < 5%

Natural Plant 

Constituents (Lignin, 

Starch, etc.)

Rice Up to 60% --
Page 53 of 57

Figure B2. Metabolic Pathways

Proposed metabolic pathway for tricyclazole by rice.

.
Page 54 of 57

Proposed metabolic pathway for tricyclozole by rats
Page 55 of 57

Appendix C. Physical/Chemical Characteristics

Table 3.2. Physicochemical Properties of Tricyclazole (Technical Grade)

Parameter Value Reference

Molecular weight (g/mole) 189.24 MRID 48918201

Melting point/range (°C) 184.6

pH Not available (study in progress)

Density (g/cm3) 1.40

Water solubility ( mg/L at 20°C) 596

Solvent solubility (mg/L at 20°C) Acetone...........................13,800

1,2-Dichloroethane .........42,900

Ethyl acetate .....................4,922

n-Heptane...............................18.6 

Methanol.........................26,500

n-Octanol ..........................7,490

Xylene...............................4,490

Vapor pressure at 25°C (Pa) 1.44x10-6

Dissociation constant (pKa) Does not dissociate in water

Octanol/water partition coefficient Log(KOW) 1.41
Page 56 of 57

Appendix D: Review of Human Research

This risk assessment did not rely on data from studies in which adult human subjects were 

intentionally exposed to a pesticide or other chemical. 
Page 57 of 57

Appendix E. International Residue Limits Table

Tricyclazole (PC Code: 120201; Date of Request: 9 January 2014)

Summary of US and International Tolerances and Maximum Residue Limits 

Residue Definition:

US Canada Mexico2 Codex

40 CFR 180.New:

Plant: Tricyclazole

Livestock: None

None None

Commodity1 Tolerance (ppm) /Maximum Residue Limit (mg/kg)

US Canada Mexico2 Codex

Rice, grain 3.0

Completed: M. Negussie; 1/14/2014

1

Includes only commodities of interest for this action. Tolerance values should be the HED recommendations and not 

those proposed by the applicant.

2 Mexico adopts US tolerances and/or Codex MRLs for its export purposes.