UNITED STATES ENVIRONM E NTAL PROT E CTION AGENCY

WASHINGTON, D.C. 20460

MEMORANDUM

Date: October 31, 2013 OFFI CE OF
C HEMICAL SA F ETY AND
POLLUTION PREVENTION

SU B JECT : Pr oq uin azid: Human Health Risk Assessment for the To leran ce on Imp orted

G r apes

PC Co de : 044502
Decision N o.: 459391 Petition No.: 1 E7972

Risk Assess ment Type : S in gle Chem ical
TXRNo

. : NA
MRIDNo

. : NA DP Barcode: 399842 Registration No.: None Regulatory Action : Tolerance, No U.S.
Re gistrat ion
Case No.: NA
CAS No.: 189278-12-4

4 0 CFR: 180.TBD

FROM: Jessica Kidw e ll, Risk Assessor Marquea King, Ph D

,**Toxicolo~·~~7J.** Thurston Morton, Chemist

Susan H ummel, Chemist

RAB IV, H ea l th Effects Di v i si on (HED) (7509P)

THROUG H: E l issa Reave s, Branch Chief

RAB IV, HED (750 9P)

Ray Kent, Chemist 

# c--:? /\

I mmediate Office, HED (7509 P)

## ·-**~** \

Chris t ine Olin ger, Branch Chief

# ~ /

# ~**~** RAB III, HE D (7509P)

# v~r--<-

TO: Tony Kish and Rosemary Kearns, PM22 Cynt h ia Gi les- P arker, Branch Chief Fungicide Branch, Re gistration Divi sion (75 05P)Proquinazid Human Health Ri s k Assessment DP No

. 399842

1.0 Executive Summary

.......... . . ............ .... ... ....................................................... ... . ................. 4

2.0 HED Recommendations

.................................................................................................... 5

2.1

2.2

2 .2. 1
2.2.2

2.2.3 Data Deficiencies

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

.......... . . ...... . ....................................................... .. .................. 6

E nforcement Analytical Method

...... ... .... ...... ............................... .. .. ...... .. ................. 6
Recommended Tolerances

............. .. .. .. . .............. . .............. .. ..... ...... .. .......... .. ...... ....... 6
Revisions to Petitioned-For Tolerances

.......................................... . ......................... 7

2.2.4 International Harmonization

......... . .. ......... . ... .. ......... .. .. ................... . ........ .... .......... ... 7
2.3 Label Recommendations

................... . ................ .. .. .......................... .. . ........................... 7

3

. 0 Introduction

.......................... ...... ........................................................................................ 7

3.1 C hemical Identity

. ............................... ...................................................... .. . .......... ..... ... 7
3.2 Physical/Chemical Characteristics

.............. ... .......... ..................... ............................... 8

3.3 Pesticide Use Pattern

....................... ...................................... .. .. .. .. ...... . ..................... ..... 8
3.4 Anticipated Exposure Pathways

..................... ................................. ............................. 9

3.5 Co nsideration of Environmental Justice

...................................................................... 9
4.0 Hazard C haracterization and Dose-Response Assessment

........................... ................. 9
4. 1 Toxicology Studies Available for Analysis

.. ......... .. . ........................ ............................. 9

4.2 Absorption, Distribution, Metabolism, & Elimination (ADME)

.................. ....... .... 1 0
4.2.1 Dermal Absorption

.............................. ................ . ... ...... . ... . . ... .. ..... ...... .. ............. ...... 10
4.3 Toxicological Effects

............... ............................................................ .. .................. ..... 1 1

4.4 FQPA Safety Factor for Infants and Children

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

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

4.4.2 Evidence of N eurotoxicity

. . ...................................................................................... 12
4.4.3 Evidence of Sensitivity/Susceptibility in the Developing or Young Animal..

...... 1 3

4.4.4 Residual U ncertainty in the Exposure Database

.......................................... ......... 13

4.5 Toxicity Endpoint and Point of Departure (POD) Selections

.................................. 13

4.5.1 Dose-Response Assessment

...................... ................... ......... .. . .. ... . ........................... 13
4.5.2 Recommendation for Combining Routes of Exposures for Risk Assessment

..... 14

4.5.3 Cancer Classification and Risk Assessment Recommendation

....................... ..... 14

4.5.4 Summary of Points of Departure and Toxicity Endpoints Used in Human Risk
Assessment

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

5.0 Dietary E xposure and Risk Assessment..

.............................................. ......................... 17
5. 1 Metabolite/Degradate Residue Profile

....................................................................... 17
5.1.1 Summary of Plant and Animal Metabolism Studies

..................................... .. ...... 17

5.1.2 Summary of Environmental Degradation

........................................... ................... 17
5.1.3 Comparison of Metabolic Pathways

.......................................................... . .. .. . ...... . 17

5.1.4 Residues of Concern Summary and Rationale

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

....................... . ...... ..... .. ...... .. .. . ........................... . ... . ... ................ . 18

5.3 Water Residue Profile

.................................................................................................. 19

5.4 Dietary Risk Assessment

............................................................................ . ................. 1 9
5.4.1 Description of Residue Data Used in Dietary Assessment..

.................................. 19
5.4.2 Percent Cr op Treated Used in Dietary Assessment

................................... ....... .... 1 9

5.4.3 Acute Dietary Risk Assessment..

...................................... .. .. ................................... 19
5.4.4 Chronic Dietary Risk Assessment

........................................................................... 20

5.4.5 Ca ncer Dietary Risk Assessment..

.......................................................................... . 20

5.4 .6 Summary Table

................................ . .. ........ . ... . .......... .. . ............................................ 20

Page 2 of49Proquinazid Human Health Risk Assessment DP No. 399842

Page 3 of 49 6.0 Residential (Non

- Occupational) Exposure/Risk Characterization

............................. 20
7.0 Aggregate Exposure/Risk Characterization

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

................................ ............................... 21
9.0 Occupational Exposure/Risk Characterization

................................ ............................ 21
10.0 References

................................ ................................ ................................ ......................... 21 Appendix A. Toxicology Profile and Executive Summaries

................................ .................. 22
A.1 Toxicology Data Requirements

................................ ................................ ................... 22
A.2 Toxicity Profiles

................................ ................................ ................................ ............ 23
A.3 Hazard Identification and Endpoint Selection

................................ .......................... 36
A.4 Executive Summaries

................................ ................................ ................................ ... 37 Appendix B. Metabolism Summary Table

................................ ................................ ............... 48 Appendix C. Physicochemical Properties Table

................................ ................................ ...... 49Proquinazid Human Health Risk Assessment DP No. 399842

Page 4 of 49 1.0 Executive Summary

Proquinazid is a new fungicide used to c ontrol powdery mildew on grapes. It acts primarily by preventing infection through disruption of secondary appressorial formation (parasitic fungi penetration into host tissues). T he formulated product is referred to as Proquinazid 200 g/L EC

, produced by DuPont Agricultural Products

. This is the first food use for this chemical.
Tolerances of 0.5 ppm for proquinazid residues on grapes and 1.0 ppm for proquinazid residues
on raisins are proposed, but no registration is being proposed in the U nited States (U. S )

. Since the request is for an i mport tolerance only and there are no U.S. registrations being proposed, only dietary (food only) exposures are assessed. Occupational and residential risk assessments are not required. The present action is being conducted as a joint review with Health Ca nada’s Pesticide Management Regulatory Authority (PMRA). This risk assessment was reviewed by the Risk Assessment Review Committee (RARC) on July 10, 2013.

The database of experimental toxicology studies available for proquinazid provides a robust
charact erization of the hazard potential of this pesticide. T here are no outstanding studies according to Part 158 data requirements for the current request for an import tolerance on grapes. However, HED has determined that a comparative thyroid assay in rats i s required to assess the potential impact of proquinazid exposure on thyroid function in the young. The agency is requiring these data given the pivotal role of thyroid hormones in brain development.

The main target tissues for p roquinazid are the thyroid and liver, with effects seen in several species. The lowest effect levels following exposure to proquinazid are based primarily on thyroid and liver effects in rats, liver toxicity in mice, and body weight decrements in dogs. Proquinazid was classified as “Suggestive Evidence of Carcinogenic Potential” . This
determination was b ased on the treatment

- related liver tumors observed in female rats. The thyroid follicular cell tumors observed in male rats and the cholangiocarcinomas observed in
female rats wer e supported by mode of action data. No treatment

- related tumors were observed
in male or female mice. There is no mutagenicity concern from the in vivo or in vitro genetic toxicity assays

. Proquinazid has no significant acute toxicity via the oral, dermal, or inhalation routes of exposure. It is not an eye or skin irritant and does not cause skin sensitization. Based on

the results of a 28

- day dermal study in rats (as well as the dermal LD

50 study), proquinazid is
poorly absorbed through the skin. No eviden ce of increased quantitative or qualitative susceptibility was seen in the proquinazid hazard database following in utero exposure with rats or rabbits in the prenatal developmental studies or in young rats in the 2

– generation reproduction study. Decreased motor activity in both sexes was seen following acute oral exposure and no neurotoxic effects were seen in the subchronic study

.

Based on the current petition for an import tolerance on grapes, exposure is expected only from food through the oral route of exposure. Therefore, endpoints for acute and chronic dietary risk

assessment s only were selected at this ti me. The endpoint chosen for acute dietary exposure for the general population was from the acute neurotoxicity study in rats and was based on d ecreased motor activity. An acute dietary endpoint was not selected which was specific for Females 13

- 49 years old since no developmental effects that could be attributed to a single exposure were identified in the proquinazid database. The endpoint chosen for chronic dietary exposure was from the chronic toxicity/carcinogenicity study in the rat and was based on a dose

- relatedProquinazid Human Health Risk Assessment DP No. 399842

Page 5 of 49 increase in non

- neoplastic liver lesions and changes in the thyroid hormones and thyroid pathology

. Quantification of risk using a non

- linear approach (i.e., reference dose (RfD)) will adequately protect for all chronic toxicity, including carcinogenicity, that could result from exposure to proquinazid.

The FQPA Safety Factor (SF) was retained at 10X as a database uncertainty factor due to lack of a comparative thyroid assay. The objective of this study is to evaluate the potential for a chemical to alter thyroid homeostasis via reduction of circulating thyroid hormones because it is known that adverse effects on the thyroid affect d evelopment, especially of the nervous system. Effects on the thyroid (manifested as changes in hormones, weight and histopathology) following proquinazid exposure were consistently observed in adult animals (rats) following subchronic and chronic exposure s. There is, however, no data regarding the potential effects of proquina z id on thyroid homeostasis in infants and children. This lack of characterization in the young creates uncertainty with regards to potential life stage sensitivities due to exposure t o proquinazid, and raises the A gency’s level of concern.

The acute reference dose (RfD) is derived from a Point of Departure (POD) of 50 mg/kg/day based on decreased motor activity at 100 mg/kg/day and an uncertainty factor of 1000

, which
includes 10x fo r interspecies extrapolation, 10x for intraspecies variability, and 10x database uncertainty factor. The chronic RfD is derived from a POD of 1.2 mg/kg/day based on liver and thyroid toxicity and an uncertainty factor of 300, which includes 3X for interspe cies extrapolation, 10x intraspecies variation, and 10X database uncertainty factor. Since the POD for deriving the chronic RfD is based on thyroid toxicity, the 3X toxicodynamics part of the 10X interspecies extrapolation factor can be removed because of several important quantitative dynamic differences between rats and humans with respect to thyroid function. A reduction in the 10X interspecies extrapolation cannot be made for the a cute dietary risk since the endpoint of concern is neurotoxicity.

A cute and chronic dietary analyses were performed using conservative assumptions ( i.e ., use of
tolerance level residues and 100% crop treated ). T here is no drinking water exposure associated with the establishment of an import tolerance ; therefore

, the acute and chronic dietary exposure and risk assessments include food residues only

. The acute dietary exposure estimates were less than HED’s level of concern ( ≤ 100% acute Population Adjusted Dose ( PAD ) ) for the general U.S. population (3.8% aPAD) an d all population sub

- groups. The most highly exposed population subgroup was Children 1

- 2 years old with an estimated risk of 18% aPAD at the 95 th
percentile. The resulting chronic exposure estimates were less than HED’s level of concern ( ≤ 100% c hro nic PAD) for general U.S. population ( 8.1 % cPAD ) and all population sub

- group s

. Th e most highly exposed population subgroup was Children 1

- 2 year s old with an estimated risk
of 47 % cPAD. As noted above, quantification of risk using a non

- linear approach (i .e., RfD) will adequately account for all chronic toxicity, including carcinogenicity, that could result from exposure to proquinazid.

2.0 HED Recommendations

HED concludes that there a re no risk issues that would preclude establishing toler a nces for the residues of proquinazid

, including its metabolites and degradates, in or on grapes and raisins.Proquinazid Human Health Risk Assessment DP No. 399842

Page 6 of 49 Additional data are needed as outlined in section 2.1 below. Specific tolerance recommendations
are discussed in section 2.2.

2.1 Data Deficiencies

2.1.1 Toxicity Studies

T here are no outstanding studies according to Part 158 data requirements for the current request for an import tolerance on grapes; however, HED has determined that a comparative thyroid assay in rats is required to generate specific data on the thyroid hormone levels to protect the developing nervous system from thyroid hormone disrupting chemicals. In the absenc e of this data, t he 10X FQPA database uncertainty factor has been retained in this risk assessment for the uncertainty associate d with the potential for enhanced sensitivity of the developing organism to prenatal or postnatal thyroid toxicity. This uncertainty may be addressed by submission of a comparative thyroid assay o r a proposal describing an alternative approach that adequat ely addresses the issue of disruption of thyroid hormone homeostasis during development.

2.1.2. Residue Chemistry

There are no residue chemistry issues which would preclude setting the recommended
tolerances.

2.2 Tolerance Considerations

2.2.1 Enforcement Analytical Method

An adequate analytical method is available to enforce the proposed tolerances for residues of proquinazid on grape commodities. The gas chromatography (GC) with electron capture detection (ECD) and GC with mass spectrometry ( MS ) for confirmation method AMR 4943

- 98 has been adequately validated and has undergone a successful independent laboratory validation (ILV) . It is considered adequately radiovalidated and has been reviewed by the Agency for appropriateness as an enforceme nt method.

The limit of quantitation ( LOQ ) was reported as 0.01 ppm in grapes

. The LOD was estimated to be ≤0.001 ppm ( g rape ).

Proquinazid is completely recovered using the FDA PAM I multiresidue protocol E using GC/Electron Capture Detection (ECD), no t recovered using protocol D, without Florisil cleanup, and variably recovered by protocol F.

2.2. 2 Recommended Tolerances

Currently there are no tolerances established for residues of proquinazid. Based on the HED Interim Guidance on Tolerance Expr essions ( Knizner, S.

, 05/27/2009), the following tolerance expression is recommended.Proquinazid Human Health Risk Assessment DP No. 399842

Page 7 of 49 Tolerances are established for residues of the fungicide, proquinazid, 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 proquinazid [ 6

- i odo

- 2

- propoxy

- 3

- propyl

- 4(3H)

- quinazolinone ] in or on the commodity.

Ta ble 2.2.2

. Tolerance Summary for Proquinazid. § 180.xxx

Commodity Current/Proposed Tolerance (ppm) Recommended

Tolerance (ppm) Comments; Correct Commodity

Definition

Grape 0.5 0.50

Raisin 1.0 1.0 Grape, raisin

2.2. 3 Revisions to Petitioned

- For Tolerances

The Agency is recommending that tolerances be set for the commodities requested by the petitioner at the levels requested, with the change in the commodity definition for raisins as specified in Table 2.2.2 above. The change in the commodity de finition is recommended to be consistent with Agency naming

- conventions for commodities and crop groups. No changes are recommended for the proposed tolerance levels, but the grape tolerance is changed to 0.50 to correct the number of significant figures.

2.2. 4 International Harmonization

CODEX has not established any maximum residue limits (MRLs) for proquinazid. Canada has
not established MRLs for proquinazid, but is concurrently planning to establish an MRL on grape at 0.5 0 ppm, and raisin at 1.0 ppm. T herefore, there are no issues related to international harmonization associated with this petition.

2.3 Label Recommendations

Since this action is for tolerances without a U.S. registration, a discussion of labels is not pertinent to this risk assessme nt.

3.0 In troduction

3.1 Chemical Identity

The structure and nomenclature for proquinazid are provided below in Table 3.1.Proquinazid Human Health Risk Assessment DP No. 399842

Page 8 of 49 Table 3.1. Proquinazid Nomenclature.
Compound

Common name Proquinazid
Company experimental name DPX

- KQ926

IUPAC name 6

- Iodo

- 2

- propoxy

- 3

- propyl

- 3 H

- quinazolin

- 4

- one

CAS name 6

- Iodo

- 2

- propoxy

- 3

- propyl

- 4(3H)

- quinazolinone
CAS registry number 189278

- 12

- 4
Molecular weight 372.21 g/mole

End

- use product (EP) TALENDO and TALIUS

.

3.2 Physical/Chemical Characteristics

The physical and chemical properties for proquin azid are detailed in Appendix C

.

Proquinazid has a low vapor pressure (10 - 5 Pa ) and is, therefore, not a volatile pesticide. Proquinazid has fairly high water solubility and does not dissociate . It is very soluble in most organic solvents, both polar and nonpolar. E nvironmental fate p roperties are not available since no use is proposed in the U.S.

3.3 Pesticide Use Pattern

No U. S. r egistration is proposed at the present time for proquinazid. The registrant has provided a summary of the use pattern for the Agency’s consideration. T he formulated product is referred to as Proquinazid 200 g/L EC , produced by DuPont Agricultural Products

. Registrations have been proposed in the E uropean U nion and Australia

.

Proquinazid 200 g/L EC is diluted in water and applied using broadcast air

- assisted sprayers, variable geometry sprayers, ducted air

- assisted sprayers and hand

- held sprayers. The appli cation use pattern is summarized in the table below.

Table 3 .3

. Summary of Proposed Directions for Use Proquinazid 200 g/L EC

Formulation [EPA Reg. No.],
a.i. Applic. Timing, Type, and Equip. Applic. Rate (g ai/ha) Max.

Seasonal Applic. Rate ( g ai/A) PHI

(days) Use Directions and Limitations

Grapes

200 g/L EC Foliar ground broadcast

application in 100 L

- 500 L

water. 75 300 60 14 day interval between applications.Proquinazid Human Health Risk Assessment DP No. 399842

Page 9 of 49 3.4 Anticipated Exposure Pathways

The Registration Division has requested an assessment of human health risk to support the proposed new use of proquinazid on imported grapes. This request is for an import tolerance, therefore, humans may be exposed to pro q u inazid in food only. Since there is no U.S. use on

grapes, there is no potential for residues to end up in drinking water or potential for domestic worker exp osure to residue s of proquinazid during the application to grapes. Lastly, there are no registered residential uses; therefore, there is no potential for domestic residential exposure to proquinazid

.

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 subgr oup’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 USDA under the National Health and Nutrition Survey/What We Eat in America (NHANES/WWEIA) and are used in pesticide risk 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 small er, 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, yout hs, and adults entering or playing on treated areas postapplication 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 database of experimental toxicolog y studies available for proquinazid provides a robust characterization of the hazard potential of this pesticide. T here are no outstanding studies according to Part 158 data requirements for the current request for an import tolerance on grapes. However, HED has determined that a comparative thyroid assay in rats is required to assess the potential impact of proquinazid exposure on thyroid function in the young (Dellarco et al. 2005)

. The agency is requiring these data given the pivotal role of thyroid hor mones in brain development. In the absence of these data, the 10X FQPA database uncertainty factor has been
retained.

The data from the following studies were used to evaluate the hazard potential of proquinazid:Proquinazid Human Health Risk Assessment DP No. 399842

Page 10 of 49 - Developmental Rat Study

- Developmenta l Rabbit Study

- Reproduction Rat Study

- Cancer Mouse Study

- Chronic/Cancer Rat Study

- 28 Day Oral Mouse Study

- 90 Day Rat Study

- 90 Day and 1 year Dog Study

- Acute Neurotoxicity (ACN) Rat Study

- Subchronic Neurotoxicity (SCN) Rat Study

- 28

- day Dermal Rat Study

- Genotoxicity Studies

- Immunotoxicity Study

- Metabolism and Pharmacokinetics

Although most of the acute studies submitted were conducted with proquinazid manufactured by the current production process, most of the other toxicology studies submi tted were conducted with proquinazid manufactured by the old production process. As a result of these differences, bridging studies were conducted to compare the toxicity of material s produced by the two
methods. These studies demonstrated that studies conducted with the current production process exhibited equivalent or lesser toxicity than the studies conducted with the old production process (see section A.4. 10 ).

4.2 Absorption, Distribution, Metabolism, & Elimination (ADME)

Proquinazid is extensively absorbed (86

- 89%) after a single oral administration in rats within 48 hours. Peak plasma concentration was reached after 4

- 8h (1 mg/kg bw) or 6

- 10h (20 mg/kg bw). Proquinazid and metabolites are widely distributed in the body (highest tissue levels in adrenals, liver, kidneys, fat). Excretion is rapid and extensive, equally important via urine and feces, and with biliary excretion accounting for nearly all of the fecal excretion. There is no significant
accumulation of [phenyl

14 C] proquinazid residues in tissues, as radioactive tissue concentrations after 7 consecutive doses are similar to those generated after a single dose. In addition, major metabolites in urine and feces are the same following single or multiple doses. There are no signifi cant gender differences in the disposition or elimination of radioactive residues. The
elimination half

- life is 8

- 12 hours. Proquinazid is extensively metabolized (>98 % of dose), mainly through phenyl ring hydroxylation and hydroxylation at the propyl and propoxy side chains, as well as some hydrolysis of side chains.

4.2.1 Dermal Absorption

Dermal exposure is not expected with an import tolerance.Proquinazid Human Health Risk Assessment DP No. 399842

Page 11 of 49 4.3 Toxicological Effects

Proquinazid is a new fungicide with a novel pesticidal mode of action (MOA) . It is highly active against powdery mildew diseases and acts by preventing secondary appressorial formation (parasitic fungi penetration into host tissues) , which is essential for successful infection by powdery mildew fungi.

The liver and thyroid are the primary target organs for proquinazid . In rodents, body weight/body weight gain reductions, increased liver and thyroid organ weights , hypertrophy /hyperplasia , liver enzyme induction, and thyroid hormone changes were seen across varying dur ations and routes of exposure in rodents but not dogs. Effects seen in the toxicity studies where proquinazid was administered by the oral and dermal routes of exposures are qualitatively similar but the effects occur at much higher dose levels by the derm al route compared to the oral route, indicating relatively poor dermal absorption of the technical material.

In the 90

- day oral rat stud y, the low dose effects of proquinazid are characterized primarily by altered thyroid hormone s and associated follicula r cell hypertrophy in the thyroid. Decrements in body weight and nutritional parameters, as well as histopathological changes in the liver (including hypertrophy) were observed at higher doses. In a 28

- day oral rat study, hypertrophy of the thyroid and liv er was completely reversible after a 6 week recovery period. In chronic rodent
studies , non

- neoplastic effects in both mice and rats included thyroid follicular hyperplasia and hypertrophy, with associated thyroid hormone changes (only investigated in rats ), and some marked hepatic lesions, i.e., necrosis and hyperplasia (including oval cell hyperplasia in rats). In addition chronic exposure in rats led to increases in the incidence of l iver and thyroid tumors

.

Mechanistic investigations revealed that cyto chrome P

- 450 content in rats and mice and peroxisome proliferation in mice were increased in the liver after 1 week, with lesser increases after exposure for 6 or 12 months. The mode of action ( MOA) for the thyroid tumors in rats was based on early changes in liver enzyme regulation that lead to dysregulation of thyroid hormone
homeostasis (↑ thyroid stimulating hormone ( TSH ) , ↓ triiodothyronine ( T3 )

, ↓ thyroxine ( T4 ))

, thyroid follicular hypertrophy/hyperplasia, and thyroid follicular adenoma formation. Proquinazid was classified as “Suggestive Evidence of Carcinogenic Potential”. This

determination was based on the treatment

- related liver tumors observed in female rats. The thyroid follicular cell tumors observed in male rats and the cholangiocarcinomas observed in female rats were supported by mode of action data. The hypothesized mode of action (i.e, non

genotoxic) for each tumor type (i.e., the thyroid and cholangiocarcinoma ) was supported by adequate studies that clearly identified the sequence of key events, dose

- response concordance

, and temporal relationship to the tumor types. No treatment

- related tumors were observed in male or female mice. There is no mutagenicity concern from the in vivo or in vitro genetic toxicity

assays.

Proquinazid was not found to be immunotoxic.

No evidence of increased quantitative or qualitative susceptibility was seen following in utero exposure to proquinazid with rats or rabbits in the prenatal developmental studies or in youngProquinazid Human Health Risk Assessment DP No. 399842

Page 12 of 49 rats in the 2

- generation reproduction study. The 2

- generation rat reproduction s tudy resulted in no effects on reproduction or fertility. The offspring effects (decreases in F 1 pup weight during lactation) occurred at the same dose which caused parental effects (thyroid hypertrophy, reduced
bo dy weight gain and food consumption). Evidence of developmental delays were observed in developmental toxicity studies in rabbits and rats and were characterized by reduced fetal weight and an increased incidence of retarded ossification and patent ductus arteriosus , respectively. These developmental effects occurred in the presence of maternal toxicity and w ere considered of equal toxicity

.

D ecreased motor activity in both sexes was seen following acute oral exposure and no neurotoxic
effects were seen in the subchronic study

.

4.4 FQPA Safety F actor for Infants and Children

No evidence of increased quantitative or qualitative susceptibility was seen following in utero exposure to proquinazid with rats or rabbits in the prenatal developmental studies o r in young
rats in the 2

- generation reproduction study. There is only limited evidence for neurotoxicity following oral exposures to proquinazid. Thyroid toxicity was seen following subchronic and chronic exposures to adult rats in multiple studies. There is, however, no data regarding the potential effects of proquinazid on thyroid homeostasis in the young animals. This lack of characterization creates uncertainty with regards to potential life stage sensitivities due to exposure to proquinazid, and raises the Agency’s level of concern. Therefore, the Agency is requiring a comparative thyroid assay in rats with proquinazid. This study will better address the concern for potential thyroid toxicity in the young. There are no residual uncertainties in the

proq uinazid residue database with regards to dietary exposure. Therefore, the FQPA Safety Factor is retained at 10X in the form of a database uncertainty factor (UF DB )

.

4.4 .1 Completeness o f the Toxicology Database

T here are no outstanding studies according to Part 158 data requirements for the current request for an import tolerance on grapes; however, HED has determined that a comparative thyroid assay in rats is required as discussed above. The uncertainty for the lack of this study is
address ed by retaining the FQPA Safety Factor of 10X as the database UF DB.

4.4.2 Evidence of N eurotoxicity

There is limited evidence for neurotoxicity following oral exposures to proquinazid. Following a single exposure, evidence for neurotoxicity at the LOAEL was limited to decreased motor activity in both sexes with no behavioral or neuropathology changes

. A t doses above the study LOAEL other effects including decreased grip strength and foot splay were observed. Following repeated (dietary) exposures, there were no treatment

- related clinical signs of neurotoxicity, behavioral changes or neuropathology. Typically, if there is a concern for neurotoxicity, the Agency would require a developmental neurotoxicity study. However, in the case of proquinazid, since the evidence for neurotoxicity was limited and the thyroid was shown to be the target organ in adult rats raising the concern for thyroid toxicity in the young, the Agency is requiring a comparative thyroid assay

. The comparative thyroid assay will better address this concern than a developmental neurotoxicity study.Proquinazid Human Health Risk Assessment DP No. 399842

Page 13 of 49 4.4.3 Evidence o f Sensitivity/Su sceptibility in the Developing o r Young Animal

No evidence of increased quantitative or qualitative susceptibility was seen following in utero exposure to proquinazid with rats or rabbits in the prenatal developmental studies or in young
rats in the 2

- generation reproduction study. The 2

- generation rat reproduction study resulted in no effects on reproduction or fertil ity. The offspring effects (decreases in F 1 pup weight during lactation) occurred at the same dose which caused parental effects (thyroid hypertrophy, reduced body weight gain and food consumption). Evidence of developmental delays were observed in develop mental toxicity studies in rabbits and rats and were characterized by reduced fetal weight and an increased incidence of retarded ossification and patent ductus arteriosus, respectively. These developmental effects occurred in the presence of maternal toxi city. For the rats, the developmental effects were seen in the presence of clear maternal toxicity

, including a marked reduction in body weight gain after adjustment for uterine contents and were considered to be of equal severity

. As stated above in the F QPA section, t here is, however, no data regarding the potential effects of proquinazid on thyroid homeostasis in the young animals. This lack of characterization creates uncertainty with regards to potential life stage sensitivities due to
exposure to proq uinazid, and raises the Agency’s level of concern. Therefore, the Agency is requiring a comparative thyroid assay in rats with proquinazid.

4.4.4 Residual Uncertainty in the Exposure Datab ase

There are no residual uncertainties in the proquinazid residue database with regards to dietary

exposure

. HED used conservative assumptions in the dietary exposure assessment, inc l uding tolerance level residues and 100% crop treated. No water, residential, or worker assessments are required for this action.

4.5 Toxicity Endpoint and Point of D eparture (POD) Selections

4.5.1 Dose

- Response Assessment

The specific toxicity endpoints and PODs are presented in Table 4.5.4.1. Based on the current petition for an import tolerance on grapes, exposure is expected only from food through the oral route of exposure. Therefore, endpoints for acute and chronic dietary risk assessment s were only
selected at this time.

The endpoint chosen for acute dietary exposure for the general population was from the acute

neurotox icity study in rats (MRID 48696359). D ecreased motor activity at a LOAEL of 100 mg/kg/day (NOAEL=50 mg/kg/day) in females in the acute neurotoxicity study was considered a

treatment

- related effect and appropriate for endpoint selection. This was a single d ose effect seen on day 1. While the dose

- response for the decreased motor activity was fairly flat, the percentage decreases were statistically significant and >40% and were considered treatment

- related and adverse for all doses ≥100 mg/kg/day. Furthermore , selection of an observation time of 1

- 3 hours post dosing on day one in the ACN was too early and not ideal since the ADME study showed a plasma t
max of 6

- 10 hours following a single dose of 20 mg/kg/day. If the endpoint was evaluated
at the time of peak plasma concentration , a more robust response may have been seen. For this risk assessment the total uncertainty factor is 1000 (10 X interspecies factor, 10 X intraspeciesProquinazid Human Health Risk Assessment DP No. 399842

Page 14 of 49 variability, and 10 X database uncertainty factor). The FQPA SF was retained at 10 X as a database uncertainty factor for lack of a comparative thyroid study. A n acute dietary endpoint
was not selected specifically for Females 13

- 49 years old since no developmental effects that could be attributed to a single exposure were identified in t he proquinazid database. The acute dietary risk assessment is a screening

- level assessment. Children <6 years old and females 13

- 49 years old are the population subgroups that require the 10X FQPA safety factor due to the potential for thyroid effects; ho wever, the 10X FQPA safety factor has been applied to all population subgroups as a conservative assumption. In the future, if refinements are required for the acute dietary assessment, the subpopulations can be assessed separately; however, since the

asse ssment is a screening

- level and health

- protective, this level of refinement is not required at
this time.

The endpoint chosen for chronic dietary exposure was from the chronic toxicity/carcinogenicity study in the rat (MRID 48696348). This was based on a dose

- related increase in non

- neoplastic liver lesions, changes in the thyroid hormones and thyroid pathology in females at a LOAEL of 12 mg/kg/day (NOAEL = 1.2 mg/kg/day). Since the POD for the chronic dietary risk assessment is based on thyroid toxicity in adult rats, the interspecies uncertainty factor was reduced to 3X to account for the toxicodynamics differences between rats and humans. These differences stem from markedly longer half

- life for T4 in humans than in rats (5

- 9 days vs 12 hrs) due to the presence of thyroxin binding globulin in the humans resulting in slower metabolic degradation and clearance of thyroid hormones. Therefore, TSH levels in rats are ≈ 25

- fold higher than in humans reflecting the increased activity of the thyroid

- pituitary a xis in rats to maintain thyroid
hormone homeostasis. Thus, on a t oxico dynamic basis, adult rats are more sensitive than adult
humans. The combined total uncertainty factor for the chronic dietary risk assessment is, therefore, 300 (3X for interspecies extr apolation, 10x for intraspecies variation, and 10X database uncertainty factor ).

4.5.2 Recommendation for Combining Routes of Exposures for Risk Assessment

Since only oral exposure is anticipated for proquinazid, it is not relevant to this action to discuss combining routes of exposure at this time.

4.5.3 Cancer Classification and Risk Assessment Recommendation

I n accordance with the EPA's Final Guideline s for Carcinogen Risk Assessment (March, 2005), the CARC classified proquinazid as having “Suggestive Evidence of Carcinogenic Potential” (CARC Report, Rowland, J and Middleton, K, 4/24/2013, TXR No. 0056645) . This
determination was b ased on the following : (i) t reatment

- related liver tumors were observed in
female rats at doses that were considered adequate to assess carcinogenicity; (ii) t reatment

related thyroid follicular cell tumors were observed in male rats; (i i i) t reatment

- related cholangiocarcinom as were observed in female rats; (i v ) n o treatment

- related tumors were observed in male or female mice at doses that were considered adequate to assess carcinogenicity; (v) t here is no mutagenicity concern from the in vivo or in vitro genetic toxicity

assa ys ; and(vi) t he hypothesized mode of action (i.e, non

- genotoxic) for each tumor type (i.e.,
the thyroid and cholangiocarcinoma ) was supported by adequate studies that clearly identifiedProquinazid Human Health Risk Assessment DP No. 399842

Page 15 of 49 the sequence of key events, dose

- response concordance

, and temporal re lationship to the tumor types. The mode of action met the criteria established by the Agency.

The overall weight

- of

- evidence was considered sufficient to demonstrate that proquinazid thyroid follicular tumors are the result of an antithyroidal mode of action and r ats are substantially more sensitive than humans to the development of thyroid follicular cell tumors in response to thyroid hormone imbalance.

P roquinazid induced cholangiocarcinomas in fe m a le rats only at doses that produced marked
liver tox icity and oval cell hyperplasia m i cro s copically. In contrast, in both m ale and f e m ale rats, doses that produced less severe or no hepatotoxicity or oval cell proliferation did not prod u ce cholangiocarcino m as . Therefore, at high enough doses, proquinazid ca n cause these biochemical and histopathological effects in livers of rodents but is unlikely to be carcinogenic at doses below those causing these changes.

Quantification of risk using a non

- linear approach (i.e., reference dose (RfD)) will adequately

pr otect for all chronic toxicity, including carcinogenicity, that could result from exposure to proquinazid.Proquinazid Human Health Risk Assessment DP No. 399842

Page 16 of 49 4. 5

. 4 Summary of Points of Departure and Toxicity Endpoints Used in Human Risk
Assessment

Table 4 .5. 4. 1 Summary of Toxicol ogical Doses and Endpoints for Proquinazid for Use in Dietary 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 (General Population, including
Infants and Children) NOAEL= 50

mg/kg/bw UF A = 10x

UF H = 10x FQPA UF DB = 10x Acute RfD =

aPAD = 0. 0 50 mg/kg/bw Acute Neurotoxicity Study

- Rat

(48696359) LOAEL = 100 mg/kg/bw based on decreased motor activity seen in females on day 1

Chronic Dietary (All Populations) NOAEL= 1.2

mg/kg/day UF A = 3 x

UF H = 10 x FQPA UF DB = 10x Chronic R fD =

cPAD = 0.004 mg/kg/day Chronic Toxicity/ Carcinogenicity Study

-

Rat (48696348)

LOAEL = 12 mg/kg/day based on
increases in non

- neoplastic liver lesions and changes in thyroid hormones and thyroid pathology

Cancer (oral, dermal, inhalation) Classification: “ Suggestive Evidence of Carcinogenic Potential ”

.

A non

- linear approach (i.e., reference dose (RfD) will ad equately protect for all chronic toxicity, including carcinogenicity, that could result from exposure to proquinazid.

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 ob served adverse effect level. LOAEL = lowest observed adverse effect level. UF = uncertainty factor. UF A = extrapolation from animal to human (interspecies). UF H = potential variation in sensitivity among members of the human population (intraspecies). UF DB = to account for the absence of key data (i.e., lack of a critical study). FQPA SF = FQPA Safety Factor. PAD = population adjusted dose (a = acute, c = chronic). RfD = reference dose.Proquinazid Human Health Risk Assessment DP No. 399842

Page 17 of 49 5.0 Dietary Exposure and Risk Assessment

5.1 Metabolite/Degradate Residue Profile

5.1.1 Summary of Plant and Animal Metabolism Studies

The grape metabolism study is adequate to support a tolerance for proquinazid residues on

grapes

. The primary residue on grapes was the parent compound, proquinazid , present at 35

- 40% parent. No other metabolites were found at >10% of the residue. Approximately 60% of the total radioactive residues ( TRR ) was identified or characterized. Parent r esidues of proquinazid were
shown to decline from 75% to 53% of the resid ue on grape foliage; however, no metabolites on grape leaves were reported. The proposed metabolic pathway for proquinazid in grapes primarily
involves the loss of the iodine substituent to form IN

- MM671

, followed by O

- dealkylation of the propoxy side chain to form IN

- MM991 (see Appendix B for structures). These metabolites were further metabolized and incorporated into unextractable naturally occurring compounds associated with the lignin fraction of the plant.

Livestock metabolism data were not revi ewed by the Agency and are not required to support the requested grape tolerance without U.S. registration.

5.1.2 Summary of Environmental Degradation

Since there is no U.S. registration proposed for grapes, a discussion of the environmental fate parameters for proquinazid is not required at this time.

5.1.3 Comparison of Metabolic Pathways

Only one plant metabolis m study is available for proquinazid and no additional data are required at this time; theref

ore it is not possible to comment on comparative metabolic profiles in plants. Similarly, no livestock metabolism data are required; therefore it is not possible to draw conclusions with respect to similarities between ruminant, poultry and rat metabolic profiles. Of note, however, is the fact that the two metabolites identified in the grape metabolism study were not identified in the rat metabolism study. The metabolism pathways in rats and grapes appear to
be different.

5.1.4 Residues of Concern Summary and Rationale

The residue of concern for proquinazid on grapes is proquinazid, per se (Table 5.1.4)

. For future tolerances and registrations of proquinazid, the registrant must continue to monitor for the
metabolite IN

- MM671 in field trials. Limited tox icity data indicate that IN

- MM671 may have equal toxicity compared to the parent, although additional toxicity data for the metabolite could preclude this concern. However, based on the lack of IN

- MM671 residues in exaggerated rate field trials in grapes a t the 28

- day pre

- harvest interval ( PHI ) , there is not likely to be any significant exposure and

, therefore

, this metabolite is not being included in the residue definition.

The two minor plant metabolites in grapes were not found as rat metabolites.Proquinazid Human Health Risk Assessment DP No. 399842

Page 18 of 49 Tabl e 5.1.4. Summary of Metabolites and Degradates to be included in th e Risk
Assessment and Tolerance Expression

Matrix Residues Included In Risk

Assessment Residues Included In Tolerance Expression

Plants Primary Crop

- grapes Proquinazid Proquinazid

Rotational Crop *

-- --

Livestock Ruminant

-- --

Poultry

-- --

Drinking Water

-- Not Applicable since this is an import use * Since no registration is being requested in the US at the present time, and grapes are not included in plant rotations, no rotational crop studies have been submitted nor are required for the use on grapes.

5 .2 Food Residue Profile

DuPont Inc. has submitted residue data for proquinazid (KQ926) from grape field trials. Twenty

eight trials were conducted in Europe during 1997 a nd 2001

- 2002 growing seasons.

In the European trials, the treated plots in each field received 4 to 6 foliar broadcast spray applications of proquinazid at a rate of 75 g a.i./ha, with a 14 day re

- treatment interval (RTI) for
a total rate of 375

- 450 g a.i. /ha, amounting to Good Agricultural Practices ( GAP ) to 1.5x the maximum seasonal rate for GAP. In the Australian trials, four applications of proquinazid were made to grapes at a nominal rate of either 60 g a.i./ha or 179 g a.i./ha, with a 14 day RTI, for a total rate of either 240 g a.i./ha or 716 g a.i./ha. The actual total application rates in the
Australian trials were 191

- 1648 g a.i./ha. The Australian trials differed so much from GAP that they were not further considered.

Field trials were conducted at the proposed foliar use rate (GAP) and higher seasonal applications rates, except in Australia. Samples were analyzed by a validated analytical method and were found to be below the recommended tolerance, but quantifiable. Prior to analysis, the samples were stored in a manner in which residues of proquinazid and IN

- MM671 were shown
to be stable. In decline studies, residues were reported to decline with increasing PHI from 0 to 28 days. No quantifiable residues were found of the metabolite, IN

- MM671 i n any of the field
trials. In future field trials, the petitioner must continue to analyze for this metabolite.Proquinazid Human Health Risk Assessment DP No. 399842

Page 19 of 49 Grape Field Trial Data are summarized in Table 5.2 below. The Australian trials are excluded from the summary because the use patterns in the Australian trials were not close to the registered single or seasonal application rate. It should be noted that residues in the Australian trials are much lower than the residues in the European trials.

TABLE 5.2 . Summary of Residue Data from Grape Trials with Proquinazid.

Commodity Total Rate of Proquinazid (g a i/ha) PHI

(days) Residue Levels (ppm) *

N Min. Max. Mean Median St. Dev.
Proquina zid

Grapes 300

(4 applications at 75 g ai/ha ) 28 6 <0.01 0.25 0.14 0.13 0.08

375 (5 applications at 75 g ai/ha ) 25

- 29 6 0.02 0.35 0.20 0.19 0.14

450 ( 6 applications at 75 g ai/ha ) 28 16 0.02 0.37 0.13 0.12 0.09

* Based on average field trial residues.

The processing data indicated that residues of proquinazid may concentrate in raisins ( 2.6x ), wet pomace (3.9x) and dry pomace (12.5x), but do not concentrate in juice (0.4x)

, wine (0.3x) , or must or must deposit/lees (<1x).

5.3 Water Residue Profile

An assessment of residues in drinking water is not required for this assessment because there is no drinking water exposure in the U.S. associated with the establishment of an import tolerance.

5.4 Dietary Risk Assessment

5.4.1 Description of Re sidue Data Used in Dietary Assessment

For the acute and chronic dietary analys e s, tolerance level residues were used

. Default processing factors were used for grape juice. The Agency considers these to be highly
conservative assessments.

5.4.2 Percent Crop Treated Used in Dietary Assessment

For the acute and chronic dietary analys e s, a 100% crop treated assumption was used

.

5.4.3 Acute Dietary Risk Assessment

The results of the acute dietary exposure analyses are reported in Table 5.4.6

. The acute risk estimates were less than HED’s level of concern ( ≤ 100% aPAD) for the general U.S. population ( 3.8 % aPAD) and all population sub

- groups . T he most highly exposed population subgroup was
Children 1

- 2 year s old with an estimated risk of 18 % aPAD at the 95 th percentile.Proquinazid Human Health Risk Assessment DP No. 399842

Page 20 of 49 5.4.4 Chronic Dietary Risk Assessment

The results of the chronic dietary exposure analyses are reported in Table 5.4.6. The resulting
chronic risk estimates were less than HED’s level of concern ( ≤ 100% cPAD) for the general U.S. population ( 8.1 % cPAD ) and all population sub

- group. Th e most highly exposed population subgroup was Children 1

- 2 year s old with an estimated risk of 47 % cPAD.

5.4.5 Cancer Dietary Risk Assessment

T he Agency has determined that quantification of ris k using a non

- linear approach (i.e., reference dose ( RfD )) will adequately protect for all chronic toxicity, including carcinogenicity
that could result from exposure to proquinazid. As noted in Section 5.4.4, there are no chronic risks of concern for proquinazid.

5.4.6 Summary Table

Table 5.4.6. Summary of Dietary (Food Only) Exposure and Risk for Proquinazid

Population Subgroup Acute Dietary
95th Percentile Chronic Dietary Cancer

Dietary Exposure (mg/kg/day) %

aPAD Dietary

Exposure (mg/kg/day) %

cPAD Dietary

Exposure (mg/kg/day) Risk

General U.S. Population 0.001890 3.8 0.000324 8.1

NA All Infants (< 1 year old) 0.004074 8.2 0.000672 17

Children 1

- 2 years old 0.009079 18 0.001881 47

Children 3

- 5 years old 0.005852 12 0.001138 28

Children 6

- 12 years old 0.002913 5.8 0.000447 11

Youth 13

- 19 years old 0.000793 1.6 0.000147 3.7

Adults 20

- 49 years old 0.001333 2.7 0.000205 6.1

Adults 50

- 99 years old 0.001426 2.9 0.000237 5.9

Females 13

- 49 years old 0.001370 2.7 0.000214 5.4

6.0 Residential (Non

- Occupational) Exposure/Risk Characterization

There are no residential uses for proquinazid and the food use is for imported grapes. As a result, a residential exposure risks assessment in not required

.

7.0 Aggregate Exposure/Risk Characterization

There are no proposed registrations for proquinazid in the U.S. Therefore, the only human

expo sure to proquinazid in the U.S. is from imported grapes and grape processed commoditi es treated with proquinazid. D ietary exposure is from food only. There is no potential drinking

water or residential exposur e resulting from use on grapes. Therefore, the only component of the aggregate risk assessment is food, and those risks are not of c oncern and have been detailed in Section 5.4.3 and 5.4.4, above.Proquinazid Human Health Risk Assessment DP No. 399842

Page 21 of 49 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 mecha nism of toxicity finding as to proquinazid and any other substances and proquinazid does not appear to produce a toxic metabolite produced by other substances. For the purposes of this tolerance action, there fore, EPA has not assumed that proqu inazid 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 re leased 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 Expo sure/Risk Characterization

This action is for imported grape and no U.S. registration is requested. An occupati onal exposure
risk assessment is not required.

10.0 References

Dellarco, V. et al. 11/01/ 2005. Interim Guidance: Thyroid Disrupting Pesticides : Use of Rat Thyroid Data and Application of Uncertainty Factors for RfD Derivation. Prepared and Reviewed by the Hazard Science Policy Council.

OPP Guidance. 10/24/2005. Guidance for Thyroid Assays in Pregnant Animals, Fetuses and Postnatal Animals, and Adult Animals. Office of Pesticide Programs, Health Effects Division, Washington, DC. Cited in Dellarco, V. et al 2005

Hummel , S. 6/20/ 20 13. D410206. Proquinazid

- Petition for Establishment of Permanent Tolerances on Imported Grapes (1E7972). Summary of Chemistry and Residue Data.

Knizner, S. 05/27/2009. “ Interim Guidance on Tolerance Expressions,” EPA Memorandum

.

Morton, T. 10 / 3 0/ 20 13. D410207. Pro quinazid

– Acute and Chronic Dietary (Food Only) Exposure and Risk Assessments for the Use on Imported Grapes

.

Rowland, J and Middleton, K. 4/24/ 2013. TXR No. 0056645. Proquinazid: Report of the Cancer
Assessment Review Committee.

Scollon, E. 5/29/2 013. Email from E d Scollon to Sue Hummel. Proquinazid ROCKS decision.

Van Alstine, J. 9/03/13. D399842. RARC Report: Summary of the RARC Meeting to Discuss the Proquinazid Human Health Risk Assessment for a Tolerance on Imported Grapes.Proquinazid Human Health Risk Assessment DP No. 399842

Page 22 of 49 A ppendix A

. Toxicology Profile and Executive Summaries

A.1 Toxicology Data Requirements

The requirements ( 40 CFR 158.500 ) for the Food Use of Proquinazid are in Table A.1 . Use of the new guideline numbers does not imply that the new (1998) guideline protocols were used.

Table A.1. Toxicology Data Requirements

Study Technical

Required Satisfied

870.1100 Acute Oral Toxicity

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

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

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

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

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

................................ ...................... yes

yes

yes

yes

yes

yes yes

yes

yes

yes

yes

yes

870.3050 28

- day oral (rodent).........................................

870.3100 Oral Subchronic (rodent)

................................ ............... 870.3150 Oral Subchronic (nonrodent)

................................ ......... 870.3200 21

- Day Dermal

................................ .............................. 870.3250 90

- Day Dermal

................................ .............................. 870.3465 90

- Day Inhalation

................................ .......................... yes

yes

yes

yes

cr

cr yes

yes

yes

yes

no

no

870.3700a Developmental Toxicity (rodent)

................................ ... 870.3700b Developmental Toxicity (nonrodent)

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

................................ ................................ . yes

yes

yes yes

yes

yes

870.4100a Chronic Toxicity (rodent)

................................ .............. 870.4100b Chronic Toxicity (nonrodent)

................................ ........ 870.4200a Oncogenicity (rat)

................................ .......................... 870.4200b Oncogenicity (mouse)

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

................................ .................... n/a

yes n/a

yes

yes -

yes

- yes

yes

870.5100 Mutagenicity

— Gene Mutation

- bacterial

..................... 870.5300 Mutagenicity

— Gene Mutation

- mammalian

................ 870.5375 Mutagenicity

— Structural Chromosomal Aberrations

... 870.5395 Mutagenicity

— Other Genotoxic Effects

........................ yes

yes

yes

yes yes

yes yes

yes

870.6100a Acute Delayed Neurotox. (hen)

................................ ..... 870.6100b 90

- Day Neurotoxicity (hen)

................................ ........... 870.6200a Acute Neurotox. Screening Battery (rat)

....................... 870.6200b 90

- Day Neuro. Screening Battery (rat)

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

................................ .............................. no

no

yes

yes

cr -

-
yes yes n/a

870.7485 General Metabolism

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

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

................................ ............................. yes

cr

yes yes

yes

yesProquinazid Human Health Risk Assessment DP No. 399842

Page 23 of 49 Table A.1. Toxicology Data Requirements

Study Technical

Required Satisfied

Special Studies for Ocular Effects

Acute Oral (rat)

................................ ........................... Subchronic Oral (rat)

................................ .................. Six

- month Oral (dog)

................................ .................. Other Special Studies

Comparative Thyroid Assa y (rat)...................... no

no

no

yes -

-

-

no n/a=not applicable

A.2 Toxicity Profile s

Table A.2.1 Table A.2.1 Acute Toxicity Profile

– Proquinazid, TGAI

Guideline No. Study Type MRID(s) Results Toxicity Category

870.1100 Acute oral, rat 48696329 LD 50 = 4846 mg/kg females

LD 50 > 5000
mg/kg males III

870.1100 Acute oral, rat (IN

- MM671,

metabolite of DPX

- KQ926) 48696330 LD 50 = 2052 mg/kg females

LD 50 = 2890
mg/kg males III

870.1200 Acute dermal, rat 48696331 LD 50 > 5000 mg/kg IV

870.1300 Acute inhalation, rat 48696332 LC 50 > 5.2 mg/L IV

870.2400 Acute eye irritation, rabbit 48696333 irritation at 1 hr cleared by 24 hrs;
redness through 72 hrs cleared by day 7;
discharge at 1 hr cleared by 24 hrs III

870.2500 Acute dermal irritation, rabbit 48696334 very slight erythema through

48 hrs cleared by
72 hrs IV

870.2600 Skin sensitization, guinea pig 48696335 Negative

--Proquinazid Human Health Risk Assessment DP No. 399842

Page 24 of 49 Table A.2.2 Subchronic, Chronic and Other Toxicity Profile

Guideline

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

8 70.3050 28

- Day oral

toxicity ( rat ) 48696336 (2002)

Acceptable/Guideline

0, 10,30, 300, ppm 0, 0.6, 1.9, 19, mg/kg/day NOAEL = 30 ppm (1.9 mg/kg bw/day) in
males

LOAEL = 300 ppm 19 mg/kg bw/day)
based on increased serum TSH and rT 3 , decreased serum T 3 and T 4 , increased UDP

glucuronyltransferase activity, decreased 5’

- deiodinase activity, and thyroid follicular cell hypertrophy; substance

- related changes in thyroid hormones and UDP

glucuronyltransferase activity at 30 ppm are considered to be adaptive, but not adverse

fin dings . T he reversibility of the increase in liver weight and hepatocellular hypertrophy, and absence of cell proliferation in the liver, is consistent with an adaptive response in the
liver.

3000ppm was tested but all rats were killed on day 18 due to exc essive bw decreases

870.3100 90

- Day oral

toxicity (rat)

Proquinazid

Note: bridging study with proquinazid from

current manufacturing

process 48696337 (2002)

Acceptable/Guideline

M: 0, 30, 100, 300, or 2000 ppm

F: 0, 30, 100, 300, or 600

ppm

M: 0, 2, 6, 19, and 127 mg/kg bw/day
F: 0, 2, 8, 24 and 50 mg/kg bw/day NOAEL= 30 ppm (2 mg/kg bw/day) in males

NOAEL= 100 ppm (8 mg/kg bw/day) in
females.

LOAEL =100 ppm (6 mg/kg/bw/day) for males based on thyroid hypertrophy and hormonal changes associated with the thyroid

LOAEL= 300 ppm (24 mg/kg bw/day) for
females based on decreases in white blood cell

counts.

870.3100 90

- Day oral

toxicity (mouse) N/A No study submitted

. The applicant states that reports of 28

- day and 90

- day feeding studies
with mice were not submitted because they were conducted on an early laboratory sample that is not representative of the current production process and add no additional
information.Proquinazid Human Health Risk Assessment DP No. 399842

Page 25 of 49 Table A.2.2 Subchronic, Chronic and Other Toxicity Profile

Guideline

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

870.3150 90

- Day oral

toxicity (dog) 48696339 (1997)

48696340 (1998) Supplement No. 1

Acceptable/Guideline

0, 500, 2000, or 4000/3000 ppm

M: 0, 17, 62, and 87, mg/kg/day

F: 0, 18, 56, and 95 mg/kg/day NOAEL = 2000 ppm

LOAEL = 4000/3000 ppm
Substance

- related toxicity (ocular discharge, red ears and increased liver weight) was seen at all dose levels, i.e. ≥ at 500 ppm (> 17

- 18 mg/kg bw/day). Although proquinazid is not classified as an eye irritant based on acute exposure of rabbits, the ext ent to which ocular discharge in this dog study was due to a local irritation rather than to a systemic effect is unclear. The p resence of red ears at all doses and conjunctivitis in one top dose animal is consistent with an irritant response. However

in creased ocular discharge was also seen in the

1

- year dog study with dosing by capsule.

870.3200 21/28

- Day dermal

toxicity (rat) 48696341 ( 2002 )

Acceptable/Guideline

M/F: 100, 500, 1000, or 2000 mg/kg bw/day The NOAEL for systemic effects = 500 mg/kg bw/day for males and 100 mg/kg bw/day for
females.

In males, the LOAEL= 1000 mg/kg bw/day based on thyroid hypertrophy and increased liver weig ht and hepatocyte hypertrophy.

In females, the LOAEL=500 mg/kg bw/day
was based on based on decreme nts in body weight and nutritional parameters as well as increased liver weight and hepatic hypertrophy at 500 mg/kg bw/day and above.

The NOAEL for local dermal effects was 500 mg/kg bw/day in male and female rats based on desquamation and/or erythema at the application site at 1000 mg/kg bw/day and
above.

[ It is notable that at the NOAEL of 100 mg/kg bw/day, the amount of proquinazid applied per cm2 can be calculated to be about 1.2 mg. ]Proquinazid Human Health Risk Assessment DP No. 399842

Page 26 of 49 Table A.2.2 Subchronic, Chronic and Other Toxicity Profile

Guideline

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

870.3700a Prenatal

developmental in (rat)

Dosed from Gestation Days 7

- 21 48696342 (1997)

Acceptable/ G uideline

0, 5, 10, 30, or 60 mg/kg bw/day Maternal and developmental NOAEL in rats = 30 mg / kg /day.

M aternal L OAEL = 60 mg/ kg /day based on reduced body weight gain and food

consu mption, and clinical signs.

Developmental L OAEL = 60 mg/kg /day based on reduced fetal weight and increased incidence
of two structural variations (retarded sternal ossification and patent ductus arteriosus)

.

F etal effects were seen in the presence of markedly reduced maternal body w eight gain.

870.3700b Prenatal

developmental in (rabbit)

Dosed from Gestation Days 7

- 28. 48696343 (1998)

Acceptable/Guideline

0, 1, 2.5, 5, or 10 mg/kg bw/day. The NOAEL in rabbits is 2.5 mg/kg bw/day for both maternal and developmental effects.

Maternal LOAEL = 5 mg/kg/day based on decreased body weight gain and food consumption.

Developmental L OAEL = 5 mg/kg/day based on decreased mean fetal weightProquinazid Human Health Risk Assessment DP No. 399842

Page 27 of 49 Table A.2.2 Subchronic, Chronic and Other Toxicity Profile

Guideline

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

870.3800 Reproduction and

fertility effects (rat) 48696344 ( 2002 )

The acceptability of the study has not been
assessed due to a more recent study.

0, 150, 300, 600 ppm M 0 : 0, 19, 17, 35 mg/kg/day

F 0 : 0, 11, 21, 44 mg/kg/day

premating Parental/Systemic NOAEL = not established LOAEL = 150 ppm based on thyroid gland
effects Reproductive NOAEL = 300 ppm,

LOAEL= 600 ppm based on reductions in implantation site number s and implantation efficiency

Offspring NOAEL = 150 ppm

LOAEL = 300 ppm based on reductions in pup weight. Reductions in pup viability were
present at 600 ppm

The applicant states that the in

- life phase of this study predates the January, 2001 update of OECD 416. During the conduct of this study, more detailed protocols for 2

- generation reproduction studies were promulgated. Some endpoints included in these new guidelines

w ere added to the study protocol for the F 1 generation. However, not all additional endpoints could be included and sperm parameters, though included, were not considered valid due to methodological concerns. Therefore, the 2

- generation study was repeated in accordance with more recent regulatory guidelines, see B.6.6.1.a.

870.3800 Reproduction and

fertility effects (rat) 48696345 ( 2003 )

Acceptable/Guideline

0, 10, 30, 150, or 600 ppm M 0 : 0, 0.6, 1.8, 9, 35 mg/kg/day

F 0 : 0, 0.7, 2.3, 11, 44 mg/kg/day

Premating Parental/Systemic NOAEL = 30 ppm (2 mg/kg bw/day)
LOAEL = 150 ppm (9

- 11 mg/kg bw/day) based on thyroid hypertrophy in P 1 and

F 1 males and females fed.

Reproductive NOAEL = 600 ppm (35

- 44 mg/kg bw/day), the highest concentration tested, based on absence of effects on reproductive parameters and reproductive

organs. LOAEL = Not established

Offspring NOAEL = 150 ppm (11 mg/kg/day)
LOAEL = 600 ppm (44 mg/kg bw/day) based on a marginal reduction in total litter weight of

F 1 pups during lactation, which was likely secondary to maternal toxicity in P 1 females.Proquinazid Human Health Risk Assessment DP No. 399842

Page 28 of 49 Table A.2.2 Subchronic, Chronic and Other Toxicity Profile

Guideline

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

870.4100b Chronic toxicity

(dog) 48696346 (2002)

Acceptable/Guideline

0 (empty capsules), 15, 60, or 180 mg/kg bw/day

ppm

. The dose level for the high dose group was 60 mg/kg/day for Study
Week 0, 120 mg/kg/day for Study Week 1, and 180 mg/kg/day for the remainder of the study. NOAEL = 15 mg/kg bw/day (males)

LOAEL = 60 mg/kg bw/day (males) based reduced body weight gain. NOAEL = 60 mg/kg bw/day (females) LOAEL =180 mg/kg bw/day (females) based on decreases in body weight and/or reductions in body weight gains

870.4200 Carcinogenicity

(mouse) 48696347 ( 2002 )

Acceptable/Guideline

0, 5, 30, 200, or 2000 ppm

Males: 0, 0. 7, 4, 27, and 282 mg/kg bw/day

Females: 0, 1.0, 6, 38, and 415 mg/kg bw/day NOAEL = 30 ppm for males and females (4 and 6 mg/kg/day, respectively).

LOAEL= 200 ppm (27 and 38 mg/kg bw/day)
based on non

- neoplastic lesions in the liver.

Non

- neoplastic lesions were also present in the thyroid gland of male and female mice fed 2000 ppm. Increased liver weight and hepatocyte hypertrophy at 2000 ppm was probably due, at least in part, to the observed induction of cytochrome P

- 450 and increase in peroxisome proliferation.

No treatment

- related tumors in male or female
mice

870.4300 Chronic Toxicity/

Carcinogenicity (rat) 48696348 ( 2002 )

Acceptable/Guideline

Males: 0, 10, 30, 300, 1000 and 2000 ppm Females: 0, 10, 30, 300, 600 and 1200 ppm

Males: 0, 0.4, 1.2, 12, 43, and 92 mg/kg bw/day.

Females: 0, 0.5, 1.4, 16, 35 and 76 mg/kg bw/day. NOAEL = 30 ppm in males and females (1.2 and 1.4 mg/kg/day, respectively) LOAEL = 300 ppm (12 and 16 mg/kg bw/day)
based on test substance

- related increases in

non

- neoplastic liver lesions and changes in thyroid hormones and pathology

.

Liver tumors and cholangiocarcinomas (with acceptable MOA data) in female rats

. Thyroid tumors (with acceptable MOA data) in
male ratsProquinazid Human Health Risk Assessment DP No. 399842

Page 29 of 49 Table A.2.2 Subchronic, Chronic and Other Toxicity Profile

Guideline

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

870.5100 Bacterial Reverse

Mutation

Proquinazid 48696349 (1998)

Acceptable/Guideline

Nominal concentrations of 10, 50, 100, 500, 1000, 2500, and 5000 μ g/plate Proquinazid (DPX

- KQ926

- 75) was negative for mutagenic activity in the non activated and S9

- activated test systems in the in vitro bacterial gene mutation assay.

870.5100 Bacterial Reverse

Mutation

Proquinazid 48696350 (1997)

Acceptable/Guideline

Nominal concentrations of 10, 50, 100, 250, 500, 1000, 2500, and 5000

μ g/plate were initially evaluated in Trial 1 using
strains TA100 and E. Coli only. Proquinazid (DPX

- KQ926

- 45) was negative for mutagenic activity in the non activated and S9

- activated test systems in the in vitro bacterial gene mutation assay.

870.5100 Bacterial Reverse

Mutation

Metabolite IN

MM671 48696351 (1999)

Acceptable/Guideline

Nominal concentrations of 10, 50, 100, 500, 1000, 2500, and 5000 μ g/plate
were evaluated in Trial 1.
Nominal concentrations of 10, 50, 100, 500, 750, 1000, and 2500 μ g/plate
were evaluated in Trial 2. Metabolite IN

- MM671 was negative for mutagenic activity in the non

- activated and S9 activated test systems in the in vitro bacterial gene mutation assay.

870.5300 In vitro

Mammalian Cell
Gene Mutation 48696352 (1997)

Acceptable/Guideline

Initial assay (nonactivated): 1 .5, 5.0, 10, 15, 20, 30, or 40

μ g/mL.

Initial assay (activated): 5, 15, 50, 60, 75, 100, or 125

g μ /mL

Independent repeat assay (nonactivat ed): 10, 15, 20, 25, 30, or 35 g μ /mL

Independent repeat assay (activated): 25, 50, 55, 60, 65, or 70 μ g/mL

Third assay (activated): 40, 50, 60, 65, 70, 75, 90,

or 100 μ g/mL. The findings of th is study were considered to
be uni nterpretable. It was recommended that a mouse lymphoma assay conducted to internationally accepted standards be provided
to address thi s endpoint. The applicant has now

s ubmitted such a study (48696353 ).Proquinazid Human Health Risk Assessment DP No. 399842

Page 30 of 49 Table A.2.2 Subchronic, Chronic and Other Toxicity Profile

Guideline

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

870.5300 In vitro Gene

Mutation

Mouse Lymphoma 48696353 (2005)

Acceptable/Guideline

Experiment 1 : L5178 TK+ mouse lymphoma cells (duplicate cultures per dose) were exposed
for 3 h at 37 o C to the test
substance at
concentrations of 20 to

180 μ g/mL in the non

activated test system. In
the S9 activated test system, cultures were exposed for 3 hours at 37

o C to concentrations of 45 to 180 μ g/mL.

Experiment 2 : C ultures were exposed for 24 h at

37 C to the test substance
at concentrations of 10 to

140 g/mL in the non

activated test system, and

for 3 h at 37 C to
concentrations of 45

to180 g/mL in the S9 activated test system. Proquinazid was negative in the non

- activated
and S9

- activated test systems in this mouse lymphoma assay.Proquinazid Human Health Risk Assessment DP No. 399842

Page 31 of 49 Table A.2.2 Subchronic, Chronic and Other Toxicity Profile

Guideline

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

870.5375 In vitro

Mammalian
Chromosome
Aberration 48696354 (1999)

Acceptable/Guideline

Doses (2 replicates/dose)
were as follows.

Non

- activated initial

assay:

79, 157, 313, 625*, 1250*, 2500*, and 5000*

μ g/mL.

S9

- activated initial assay:

79*, 157*, 313 *, 625*, 1250*, 2500, and 5000

μ g/mL.

Non

- activated independent repeat assay:

79, 157, 313, 625*,
1250 *, 2500*, and 5000*

μ g/mL.

S9

- activated independent
repeat assay:

40, 79*, 157*, 313*, 625*, 1250, 2500, and
5000 μ g/mL.

[* = doses for which a cytogenetic evaluation was made] Proquinazid (DPX

- KQ926

- 45) was negative for
structural and numerical chromosome
aberrations in the non

- activated and S9

- activated test systems in the in vitro mammalian cytogenetics test using human peripheral blood lymphocytes. However the study was not completely adequate by current standards because there was no investigation of the effect of continuous exposure in the absence of metabolic activation (which could have limited to ability to detect any test
substance induced structural and numerical aberrations). Also the study may have had limited sensitivity because the doses of

p roquinazid used had resulted in high levels of cytotoxicity and current positive control responses were relatively low. These study limitations have been confirmed by the UK Committee on Mutagenicity (see Appendix 6).

870.5395 Mammalian

Erythrocyte
Micronucleus 48696355 ( 1999)

Acceptable/Guideline

0, 360, 720, or 1400 mg/kg bw by single

- dose oral gavage Proquinazid (DPX

- KQ926

- 45) did not induce
an increase in micronuclei in bone marrow cells of mice and is considered negative in this in vivo assay. The clinical signs of toxicity seen in mice (especially ataxia and lethargy in the absence of diarrhea) are indicative of systemic exposure (and hence likely exposure of the bone marrow) to proquinazid and/or
metabolites.

In the sin gle dose ADME study with rats, with dosing of radiolabelled proquinazid by gavage (B.6.1.1a), radiolabel was found in bone marrow at similar levels to that plasma and blood. It is likely that radiolabel reaching the bone marrow is a mixture of parent and metabolites because metabolism of parent is extensive in rats, with biliary excretion being important.Proquinazid Human Health Risk Assessment DP No. 399842

Page 32 of 49 Table A.2.2 Subchronic, Chronic and Other Toxicity Profile

Guideline

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

870.5395 Mammalian

Erythrocyte
Micronucleus 48696356 (1999a)

Acceptable/Guideline

Male mice received 720, 1440, or 2000 mg/kg bw by single

- dose oral

gavage.

Doses in females were 360, 720, or 1440 mg/kg
bw. Proquinazid (DPKQ926

- 75) did not induce an
increase in micronuclei in bone marrow cells of mice and is considered negative in this in vivo assay. The rapid appearance of clinical signs of toxicity in mice is indicative of rapid absorption and likely systemic exposure (including bone marrow) to proquinazid and/or
metabolites.

In the single dose ADME study with rats, with
dosin g of radiolabelled proquinazid by gavage (B.6.1.1a), radiolabel was found in bone marrow at similar levels to that plasma and blood. It is likely that radiolabel reaching the bone marrow is a mixture of parent and metabolites because metabolism of parent is extensive in rats, with biliary excretion being important.

870.5395 Mammalian

Erythrocyte
Micronucleus

Metabolite IN

MM671 48696357 (1999b)

Acceptable/Guideline

Male and female mice received 0, 500, 1000, or 2000 mg/kg by single

dose oral gavage. IN

- MM671 did not induce an increase in micronuclei in bone marrow cells of mice, and is considered negative in this in vivo assay.

870.5550 Unscheduled

DNA Synthesis in
Mammalian
Cultured Cells 48696358 (1999)

Acceptable/Guideline

In both initial and independent repeat assays,
the test substance was tested at eight dose l evels, ranging from 3.9 to 500

μ g/mL, and fully
evaluated for UDS at five
dose levels o f 7.8, 15.6, 31.3, 63, and 125 μ g/mL. Proquinazid (DPX

- KQ926

- 45) did not cause a significant incr ease in unscheduled DNA synthesis and was concluded to be negative in this assay.Proquinazid Human Health Risk Assessment DP No. 399842

Page 33 of 49 Table A.2.2 Subchronic, Chronic and Other Toxicity Profile

Guideline

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

870.6200a Acute

neurotoxicity screening battery 48696359 (1997)

Acceptable/Guideline

M/F: 0, 100, 500, 2000 mg/kg/day

In order to establish a NOAEL for females, 12
females/dose were similarly administered a single dose by oral gavage
of: 0, 15, 30, or 50 mg/kg/day NOAEL = 100 mg/kg bw for males

LOAEL= 500 mg/kg bw for males based on decreased motor activity on the day of exposure

NOAEL = 50 mg/kg bw for females LOAEL = 100 mg/kg/day for females, based on decreased motor activity on the day of

exposure. While the dose

- response for the decreased motor activity in females was fairly flat, the percentage decreases were statistically significant and >40% and were con sidered

treatment

- related and adverse for all doses ≥100 mg/kg/day. [While there was a 23% decrease in motor activity at 50 mg/kg/day, this was not statistically significant and was considered a NOAEL.]

870.6200b Subchronic

neurotoxicity screening battery

Combined subchronic (90

- day) feeding study
and subchronic neurotoxicity study (proquinazid from previous manufacturing

process ) 48696338 (2003)

Acceptable/Guideline

M: 0, 30, 100, 300, 2000ppm 0, 2, 6, 19, 135 mg/kg/day

F: 0, 30, 100, 600ppm

0, 2, 8, 23, 50 mg/kg/day Neurotoxicity

NOAEL = 2000 ppm (135 mg/kg bw/day) in males and 600 ppm (50 mg/kg bw/day) in females, HDT
LOAEL= not observed

(systemic toxicity of proquinazid (DPX

KQ926

- 45)

NOAEL = 30 ppm (2 mg/kg bw/day) in males and 100 ppm (8 mg/kg bw/day) in females
LOAEL= 100 ppm (6 mg/kg bw/day) in males and at 300 ppm (23 mg/kg bw/day) in females based on hormonal and morphological changes (hypertrophy) in the thyroid; d ecrements in body weig ht and nutritional parameters, as well as microscopic and clinical pathological changes in the liver, were also present at higher
doses.Proquinazid Human Health Risk Assessment DP No. 399842

Page 34 of 49 Table A.2.2 Subchronic, Chronic and Other Toxicity Profile

Guideline

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

870.7485 Metabolism and

pharmacokinetics (rat) 48696360 (1999)

Acceptable/Guideline

M/F: 1 mg/kg/ 7 day s Male and female rats were dosed with approximately 1 mg/kg of [phenyl

14 C(U)] proquinazid for 7 consecutive days and sacrificed at approximately 4.4

- 6.1, 24

- 25, 48

- 49, and 169

- 170 hours after the last dose. The concentration of radioactivity in tissues
de clined rapidly following the last dose. Tissue residue concentrations were generally lower than plasma concentrations with the exception of liver, kidney, and fat. There appeared to be little potential for bioaccumulation. Extensive metabolism of proqui nazid was observed. The
most abundant metabolites in urine and f eces were the same as observed in the single dose study. A small percentage of 14 C

- proquinazid (up to 6%) was deiodinated and excreted as a ring hydroxylated product.

870.7485 Metabolism and

pharmacokinetics

(rat) 48696361 (1998)

Acceptable/Guideline

M/F: 1 and 20 mg/kg/day Proquinazid was virtually completely absorbed at the 1 and 20 mg/kg doses. There was extensive metabolism, based on very low excretion of parent compound and extensive
excretion of metabolised dose in urine and feces. Biliary excretion accounted for essentially all of the fecal excretion. The
elimination half

- life for total radioactive
residues was 8

- 12 hours, and by 7 days post dosing little potential for ac cumulation of residues was seen (<1% of the dose in the body). Major biotransformations observed
were the loss of side chains and hydroxylation/oxidation both at the phenyl ring
and side chains

870.7800 Immunotoxicity

(mouse) 48778901 (2012)

Acceptable/Guideline

0, 50, 200, 600, or 1200

ppm ( 0, 4, 14, 42, or 82 mg/kg/day ) The systemic toxicity LOAEL is 1200 ppm (82 mg/kg/day) based on decreases in terminal body weight, body weight gain, food consumption and food efficiency. The NOAEL
for systemic toxicity is 600 ppm (42 mg/kg/day). The immunotoxicity NOAEL is 1200 ppm (82 mg/kg/day), the highest dose tested. The
LOAEL was not established.Proquinazid Human Health Risk Assessment DP No. 399842

Page 35 of 49 Table A.2.2 Subchronic, Chronic and Other Toxicity Profile

Guideline

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

Non

guideline

Special
Studies Scientific

Advisory Panel
Review of Thyroid 48696364 (2003) The colloid alteration was shown to occur at doses where alterations in thyroid hormone homeostasis were minimal or absent, as well as in control rats. Furthermore, this change was not observed in mice or dogs even at high doses. Therefore, as with changes in other thyroid parameters, the rat appears to be uniquely susceptible to the colloid alteration
described in these studies. Based on these observations, the SAP considered the colloid changes in rats not to be relevant to humans.

Non

guideline

Special
Studies Scientific

Advisory Panel
Review of Liver 48696365 (2003) The precise mechanisms for the development of proliferative cholangiofibrotic lesions in the liver, as well as the precise cell of origin, have not been definitively determined. However, it is clear that significant hepatotoxicity is a prerequisite for th e eventual development of cholangiofibrotic lesions including intestinal

type cholangiocarcinomas in the liver. Such hepatotoxicity was evident in association with cholangiofibrotic lesions in female rats fed high dietary concentrations of proquinazid [R MS:
these tumor s were seen at > 600ppm i.e. at > 35 mg/kg bw/day which is a moderate, not high, dose level]Proquinazid Human Health Risk Assessment DP No. 399842

Page 36 of 49 A.3 Hazard Identification and Endpoint Selection

A.3.1 Acute Reference Dose (aRfD)

- Females age 13

- 49

An endpoint was not selected for Females 13

- 49 years old since no developmental effects that could be attributed to a single exposure were identified in the proquinazid database.

A.3.2 Acute Reference Dose (aRfD)

- General Population

Study selected: Acute neurotoxicity study (rat)

MRID No.: 48696359

Dose and Endpoint for Risk Assessment: NOAEL = 50 mg/kg/day based on decreased motor activity seen at the LOAEL of 100 mg/kg/day in females on day 1.

Comments about Study/Endpoint/Uncertainty Factors: D ecreased motor activity in the a cute neurotoxicity study was considered a treatment

- related effect and appropriate for endpoint selection. This is a single dose effect seen on day 1. While the dose

- response for the decreased motor activity was fairly flat, the percentage decreases were s tatistically significant and >40%
and were considered treatment

- related and adverse for all doses ≥100 mg/kg/day

. The selection
of an observation time of 1

- 3 hours post dosing on day one in the ACN was considered to be suboptimal since the ADME study showe d a plasma t
max of 6

- 10 hours followin g a single dose of 20 mg/kg/day. If the endpoint had been evaluated at the time of peak plasma concentration

, a more robust response may h ave been seen. The total UF is 1000 (10X for interspecies uncertainty factor, 10X for intraspecies variability, and a 10X FQPA da tabase uncertainty factor)

.

aRfD = aPAD = 50 mg/kg/day = 0.050 mg/kg/day

1000

A.3.3 Chronic Reference Dose (cRfD ) `

Stud y Selected: Chronic toxicity/carcinogenicity study (rat)

MRID No.: 48696348

Dose and Endpoint for Risk Assessment: NOAEL = 1.2 mg/kg/day (males) based on test
substance

- related increases in non

- neoplastic liver lesions and changes in thyroid hormones and pathology seen at LOAEL = 12 mg/kg/day (males)

.

Comments about Study/Endpoint/Uncertainty Factors: This study was selected since the database reveals that the rodent is the most sensitive species and effects to the liver and thyroid
are considered the most sensitive endpoint s. The duration of 2 years and route of exposure through feed is also appropria te. It is a robust endpoint that is protective of adverse effectsProquinazid Human Health Risk Assessment DP No. 399842

Page 37 of 49 observed in other studies, including cancer effects. The NOAEL (1.2 mg/kg/day) is similar to the NOAEL observed in the rat reproduction study (2 mg/kg/day). The total UF is 300 ( a n interspe cies scaling factor of 3x, lowered from 10 X for pharmaco dynamic s (rats eliminate thyroxine (a thyroid hormone) at a higher rate than humans), an intraspecies variability factor of 10X, and a 10X FQPA database uncertainty factor ).

cRfD = cPAD = 1.2 mg/ kg/day = 0.004 mg/kg/day

300

A.4 Executive Summaries

A.4.1 Subchronic Toxicity

870.3050 28

- Day Oral Toxicity

– Rat

This executive summary and classification determination was prepared by the EPA reviewer. The purpose of these studies was to investigate the toxicity of DPX

- KQ926 technical to male rats when administered via the diet for 28 consecutive days (MRID 48696336)

. Young adult male

Crl:CD (IGS)BR rats were administered diets containing 0, 10, 30, 300, or 3000 ppm proquinazid (adj usted for purity of 97.03%) for 28 days. Rats were about 10 weeks old at the start of dosing. The test substance was dissolved in acetone and then mixed in the diet. There were 100 rats per concentration, with generally different subsets of rats being use d for hormonal analysis, for cell proliferation and for biochemistry/histopathology at each concentration. Satisfactory data were provided for homogeneity, stability and achieved concentration of the active ingredient in the diet. Due to excessive body we ight depression in the 3000 ppm dose group, all rats in that group were killed on Test Day 18 and did not contribute further to the study. The mean daily intake of proquinazid was 0.6 1.9 and 19 mg/kg/day for the 10, 30, and

300 ppm dose groups, respective ly. The increase of liver weight and hepatocellular hypertrophy was considered adaptive. The NOAEL was 30 ppm (1.9 mg/kg bw/day) based on the spectrum of adverse effects (increased serum TSH and rT 3 , decreased serum T 3 and T 4 , increased UDP

glucuronyltrans ferase activity, decreased 5’

- deiodinase activity, and thyroid follicular cell hypertrophy) that was observed at the LOAEL of 300 ppm (19 mg/kg bw/day).

This 28

- day oral toxicity study in the rat is classified as acceptable/guideline

.

870.3100 90

- Day O ral Toxicity

– Rat

This executive summary and classification determination was prepared by the EPA reviewer. The purpose of these studies was to investigate the toxicity of DPX

- KQ926

- 75 technical to both
female and male rats when administered via the diet for 90 + consecutive days (MRID 48696337)

. Young adult Crl:CD (SD) IGS BR rats (10 rats/sex/concentratio n) were administered diets containing proquinazid (DPX

- KQ926

- 75) at concentrations of 0, 30, 100, 300, or 2000 ppm for males, and 0, 30, 100, 300, or 60 0 ppm for females for approximately 104 days

. Mean daily intakes were: males: 0, 2, 6, 19, and 127 mg/kg bw/day and females: 0 , 2, 8, 24 and 50 mg/kg bw/day. The follicular cell hypertrophy of the thyroid in the absence of persistentProquinazid Human Health Risk Assessment DP No. 399842

Page 38 of 49 changes in thyroid hormones is not considered to be a non

- adverse response and hence concluded that the NOAEL was 30 ppm (2 mg/kg bw/day) in males and 100 ppm (8 mg/kg bw/day) in females. The L OAELs were based on thyroid hypertrophy coupled with hormonal changes associated with the thyroid at 100 ppm (6 mg/kg/ bw/day) and above for males and on decreases in white blood cell counts at 300 ppm (24 mg/kg bw/day) and above for females. Decrements in body weight and nutritional parameters were also present at higher doses, toge ther with hepatocyte hypertrophy (but no clear evidence of hepatocellular damage)

. At 30 ppm, increased severity of altered colloid in the thyroid of males is not considered an adverse effect.

This 90

- day oral toxicity study in the rat is classified as acceptable/guideline

.

870.3100 90

- Day Oral Toxicity

- Mouse

No study was submitted . The applicant states that reports of 28

- day and 90

- day feeding studies with mice were not submitted because they were conducted on an early laboratory sample that is

not representative of the current production process and add no additional information.

870.3150 90

- Day Oral Toxicity

– Dog

This executive summary and classification determination was prepared by the EPA reviewer. The purpose of these studies was to invest igate the toxicity of DPX

- KQ926 technical to through dietary administration to dogs for 90

- days (MRIDs 48696339 and48696340)

. Proquinazid was offered to beagle dogs (4/sex/concentration) in 400g of food for 1

- 2h/day at concentrations of 0, 500, 2000, or 40 00/3000 ppm; these concentrations were corrected for purity of 97%. Due to decreased food consumption and body weight loss, the 4000 ppm groups received basal diet during Study Week 5 and resumed test substance administration during Study Week 6 at 3000

pp m. Originally, ‘dry’ proquinazid was mixed with the diet but because of poor homogeneity from week 3 proquinazid was added to acetone before being mixed in the diet; satisfactory homogeneity data were then achieved

. Satisfactory data were provided for stability and achieved
concentration of the a. i . in the diet. The mean daily intakes were: m ales: 0, 17, 62, and 87 mg/kg bw/day and f

emales: 0, 18, 56, and 95 mg/kg bw/day. The clinical findings included: decreases in body weight, body weight gain at di etary concentrations of 3000/40 00 ppm (LOAEL) and
above. Other findings, including eye discharge and decreased food consumption may have been due to local effects (ocular irritation) or decreased palatability rather than systemic toxicity, respectively, t he refore, the NOAEL for systemic toxicity was found to be 2000 ppm

.

This 90

- day oral toxicity study in the dog is classified as acceptable/guideline and is considered supplemental.

870.3200 21/28

- Day Dermal Toxicity

– Rat

This executive summary and classification determination was prepared by the EPA reviewer. The purpose of th is stud y was to investigate the toxicity of DPX

- KQ926 technical to male rats when applied via the dermal route for 28 consecutive days (MRID 48696341)

. In a 28

- day dermal study , proquinazid was moistened with deionised water to form a paste and applied to the shaved, intact dorsal skin of male and female Crl: CD (SD)BR rats (10/sex/dose) for 28 dailyProquinazid Human Health Risk Assessment DP No. 399842

Page 39 of 49 (consecutive) applications. The rats were exposed to the test substance, applie d under gauze and
self

- adhesive bandages, for 6 hours per day. Exposure doses we re 100, 500, 1000, or 2000 mg/kg bw/day (adjusted for a purity of 96.4%). There were also control animals (10/sex). The main finding in this 28

- day dermal study in rats, hyper trophy of thyroid and liver, and reduced body weight gain. The NOAEL for systemic effects was 500 mg/kg bw/day for males and 100 mg/kg bw/day for females. In males, a LOAEL of 1000 mg/kg bw/day based on thyroid hypertrophy and increased liver weight and h epatocyte hypertrophy. In females, the LOAEL was 500mg/kg bw/day was based on body weight decrements, nutritional parameters, increased liver weight, and hepatic hypertrophy at the LOAEL and doses greater than that. Male and female rats both showed desqu amation and/or erythema at the application site at 1000mg/kg bw/day and above with a NOAEL of 500 mg/kg bw/day for local dermal effects.

This 28

- day dermal toxicity study in the rat is classified as acceptable/ guideline

.

A.4.2 Prenatal Developmental Toxicity

870.3700a Prenatal Developmental Toxicity Study

– Rat

This executive summary and classification determination was prepared by the EPA reviewer. Proquinazid was administered by oral gavage to mated Crl:CD (SD)BR female rats (25/dose group) on Gestation Days 7

- 21 (MRID 48696342). Gavage doses in 0.5% methyl cellulose vehicle were 0, 5, 10, 30, or 60 mg/kg bw/day

. The maternal and developmental NOAEL was 30 mg/kg bw/day based on the evidence of delayed fetal development (reduced weight and an
incr eased incidence of retarded ossification and patent ductus arteriosus) seen in the presence of clear maternal toxicity (including a marked reduction in body weight gain after adjustment for
uterine contents , reduced food consumption and clinical signs ) wit h a LOAEL of 60 mg/kg bw/day.

This prenatal developmental tox icity study in the rat is classified as acceptable/guideline

.

870.3700b Prenatal Developmental Toxicity Study

– Rabbit

This executive summary and classification determination was prepared by the EPA reviewer. Proquinazid was administered by oral gavage to time

- mated Hra:(NZW)SPF female rabbits (22/dose group) on Gestation Days 7

- 28 (MRID 48696343). Gavage doses in 0.5% met hyl cellulose vehicle were 0, 1, 2.5, 5, or 10 mg/kg bw/day

. In rabbits, decreased fetal weight was seen in the presence of maternal toxicity (reduction in body weight gain after adjustment for uterine contents). The NOAEL was 2.5 mg/kg bw/day for both ma ternal and developmental endpoints, while the LOAELs for both were 5 mg/kg bw/day with maternal effects being decreased body weight gains and food consumption and the developmental effects were decreased mean fetal weight.

This prenatal developmental to x icity study in the r abbits is classified as acceptable/guideline

.Proquinazid Human Health Risk Assessment DP No. 399842

Page 40 of 49 A.4.3 Reproductive Toxicity

870.3800 Reproduction and Fertility Effects

- Rat

This executive summary and classification determination was prepared by the EPA reviewer. During the conduct of the 2002 study, a more detailed protocol for 2

- generation reproduction
studies was promulgated. Therefore, the 2

- generation study (2003) was repeated in accordance with more recent regulatory guidelines (MRID 48696345)

. A two

- generation reprodu ction study was conducted with proquinazid ( DPX

- KQ926

- 85) involving the production of one set of litters in each generation. Throughout this study, Crl:CD (SD)IGS BR rats (30/sex/concentration) were fed diets containing 0, 10, 30, 150, or 600 ppm proquinaz id . Proquinazid was dissolved in acetone before being mixed with diet. Following at least 70 days of diet administration (premating), the P 1 and F 1 generation males and females were co

- housed within their respective treatment groups, to produce F 1 and F 2 litters, respectively. Dams were allowed to deliver and rear their offspring until weaning (Postpartum Day 21). At weaning, 30 F 1 rats/sex/group were randomly selected as the F 1 parental generation and were given the same concentration level as
their res pective P 1 generation sires and dams. F 1 and F 2 litters were culled to 4 pups/sex/litter (litter size permitting) on Postnatal Day 4; all remaining pups were discarded without further
evaluation. No deaths and no test substance

- related clinical signs were observed. Thyroid hypertrophy in P 1 and F 1 males and females occurred at the lowest dose tested (9

- 11 mg/kg bw/day).

. Increased incidence/severity of altered colloid in thyroid at 10 and 30 ppm is not considered to be an adverse finding. There were no effects on reproductive parameters and reproductive organs. Pup growth and development was effected with a marginal reduction in total litter weight of F 1 pups during lactation at 44 mg/kg bw/day.

The p arental/s ystemic NOAEL was 30 ppm (2 mg/kg bw/day). The parental/systemic
LOAEL was 150 ppm (9

- 11 mg/kg bw/day) based on thyroid hypertrophy in P 1 and F 1 males
and females fed.

The reproductive NOAEL was 600 ppm (35

- 44 mg/kg bw/day), the highest concent ration tested, based on absence of effects on reproductive parameters and reproductive organs. The reproductive LOAEL was n ot established.

The o ffspring NOAEL was 150 ppm (11 mg/kg/day) . The offspring LOAEL was 600 ppm (44 mg/kg bw/day) based on a marginal reduction in total litter weight of F 1 pups during lactation, which was likely secondary to maternal toxicity in P 1 females.

The repeated 2

- generation reproduction study in the rats is classified as acceptable/guideline

.

A.4.4 Chronic Toxicity

870.4100a (870.4300) Chronic Toxicity

– Rat

See 870.4300.Proquinazid Human Health Risk Assessment DP No. 399842

Page 41 of 49 870.4100b Chronic Toxicity

– Dog

This executive summary and classification determination was prepared by the EPA reviewer. The purpose of th is stud y was to investigate the toxicity of DPX

- KQ926 technical to beagle dogs administered via the diet for 1 year (MRID 48696346)

. Proquinazid was administered to beagle dogs (5/sex/concentration) at doses of 0 (empty capsules), 15, 60, or 180 mg/kg bw/day for
5 4 consecutive weeks. The dose level for the high dose group was 60 mg/kg/day for Study
Week 0, 120 mg/kg/day for Study Week 1, and 180 mg/kg/day for the remainder of the study. Concentrations were corrected for purity of 97%. A NOAEL of 15 mg/kg bw/day is proposed for males based on reduced body weight gain seen at a LOAEL of 60 mg/kg bw/day

. A NOAEL
of 60 mg/kg/day for females was based on decreases in body weight and/or reductions in body weight gains at a LOAEL of 180 mg/kg/day

.

This 1

- year chronic toxicity study in the dog is classified as acceptable/ guideline

A.4.5 Carcinogenicity

870.4200a Carcinogenicity Study

– rat

See 870.4300.

870.4200b Carcinogenicity (feeding)

– Mouse

In a carcinogenicity study (MRID 48696347 ), proquinazid (94.1

- 98.4% a.i.; Batch No. KQ926

- 45) was administered in the diet to Crl:CD

- 1 (ICR) BR mice ( 70/sex/group ) for up to 80 weeks

at concentrations of 0, 5, 30, 200, or 2000 ppm (equivalent to 0/0, 0.7 / 1.0

, 4 / 6

, 27/38, and

282 / 415 mg/kg bw/day in males/female s). The NOAEL in mice was 30 ppm for males and females (4 and 6 mg/kg/day, respectively). This LOAEL was based on non

- neoplastic lesions in the liver at 200 ppm (27 and 38 mg/kg bw/day). Non

- neoplastic lesions were also present in the thyroid gland of m ale and female mice fed 2000 ppm. Increased liver weight and hepatocyte hypertrophy at 2000 ppm was probably due, at least in part, to the observed induction of cytochrome P

- 450 and increase in peroxisome proliferation. No treatment

- related tumors were

o bserved in male or female mice.

This study is classified as acceptable/guideline and satisfies the guideline requirements

.

870.4300 Chronic Toxicity/Carcinogenicity (feeding)

- Rat

In a 2 year c ombined chronic toxicity/ carcino genicity feeding study in rats (MRID 48696348 ), proquinazid ( 93.1

- 96.5% a.i.; Batch No. KQ926

- 45) was administered in the diet to

Crl:CD (SD)BR rats ( 8 0/sex/group ) at concentrations of males/females 0 /0

, 10/10

, 30/ 30, 300/300, 1000/600 , or 2000 /1200 ppm (equivalent to 0/0, 0.4 / 0 .5

, 1.2 / 1.4

, 12/16, 43 / 35, and
92/76 mg/kg bw/day in males/females). The NOAEL in rats was 30 ppm in males and females (1.2 and 1.4 mg/kg/day, respectively) based on test substance

- related increases in non

- neoplastic liver lesions and changes in thyroid h ormones and pathology at a LOAEL of 300 ppm (12 and 16 mg/kg bw/day).Proquinazid Human Health Risk Assessment DP No. 399842

Page 42 of 49 Proquinazid was oncogenic, causing thyroid tumors (follicular cell adenomas in males at 1000 ppm and above) and liver tumors (hepatocellular adenomas in females at 600 ppm and above). The thyroid tumors had acceptable mode of action (MOMA) data. In females at 600 ppm and
above there was treatment related incidence and acceptable MOA data for cholangiocarcinomas

.

This study is classified as acceptable/guideline and satisfies the guid eline requirements.

A.4.6 Mutagenicity

870.5100 Bacterial Reverse Mutation

The purpose of these studies was to investigate the in vitro toxicity of DPX

- KQ926

- 45,

- 75, and
metabolite IN

- MM671. IN

- MM671 was evaluated for mutagenicity in Salmonella typhimurium strains TA97a, TA98, TA100, TA1535, and in Escherichia coli strain WP2 uvr A (pKM101) with and without an exogenous metabolic activation system (10% Aroclor

- induced rat liver S9 (v/v), 4.0 mg/mL protein)

. Nominal concentrations of 10, 50, 100, 500, 1000, 2500, and 5000 g/plate
were evaluated in Trial 1

. Nominal concentrations of 10, 50, 100, 500, 750, 1000, and 2500 g/plate were evaluated in Trial 2

. The two 2 independent trials used standard plate incorporation methods

. The highest dose level in Trial 1 was set based on the limit dose for this test system, and in Trial 2 based on cytotoxicity observed in Trial 1

. The test substance was administered to the test system as a solution in dimethyl sulfoxide (DMSO). Proquinazid ( DPX

KQ926

- 45) ; ( DPX

- KQ926

- 75); and Metabolite IN

- MM671 were all negative for mutagenic activity in the non

- activated and S9

- activated test systems in the in vitro bacterial gene mutation

assay ( MRIDs 48696349, 48696350, 48696351)

These bacterial reverse mutation studi es ( in vitro ) are classified as acceptable/ guideline

.

870. 53 00 Gene Mutation

Proquinazid was tested in the CHO/HPRT mutation assay with and without an exogenous metabolic activation system (Aroclor1254

- induced rat liver S9)

. An initial, independent repeat and third mutagenesis assay were conducted

. The third assay (S9 activated test system only) was conducted due to the lack of toxicity observed in the independent repeat assay

. The test
substance was dissolved in acetone at a maximum concentration of 500 mg/mL. The purpose of these studies was to investigate the in vitro toxicity of DPX

- KQ926

- 45 and L5178 mouse lymphoma cells. T he findings of the initial study (MRID 48696352) were considered by the UK Committee on Mutagenicity to be not interpreta ble

. Upon recommendation a mouse lymphoma assay conducted to internationally accepted standards and provided to address this endpoint. Proquinazid was negative in the non

- activated and S9

- activated test systems in this mouse lymphoma assay (MRID 48696353)

.

This gene mammalian mutation study ( in vitro ) is classified as acceptable/ guideline

.Proquinazid Human Health Risk Assessment DP No. 399842

Page 43 of 49 870. 5375 Chromosome Aberration

Proquinazid (DPX

- KQ926

- 45) was tested in the in vitro mammalian cytogenetic test using human peripheral blood lymphocytes (from 3 adult males and 3 adult females) both with and without an exogenous metabolic activation system (Aroclor

- induced rat li ver S9). The purpose of these studies was to investigate t he in vitro toxicity of Proquinazid (DPX

- KQ926

- 45) and was found to be negative for structural and numerical chromosome aberrations in the non

- activated
and S9

- activated test systems in the in vitro mammalian cytogenetics test using human peripheral
blood lymphocytes.

This mammalian chromosomal aberration study ( in vitro ) is classified as acceptable/guideline

.

870. 5395 Mammalian Erythrocyte Micronucleus

Proquinazid was evaluated for its ability to induce micronucleated polychromatic erythrocytes (MNP CEs) in the bone marrow of Cr1: CD

- 1 (ICR)BR mice (5/sex/dose/sample time, with 5/sex extra at the top dose). Male and female mice received 0, 360, 720, or 1400 mg/kg bw by single

- dose oral gavage. Proquinazid ( DPX

- KQ926

- 45) did not induce an increase in micronuclei in bone marrow cells of mice and is considered negative in this in vivo assay. The clinical signs of toxicity seen in mice (especially ataxia and let hargy in the absence of diarrhe a) are indicative of systemic expo sure (and hence likely exposure of the bone marrow) to proquinazid and/or metabolites (MRID 48696355)

.

Proquinazid was evaluated for its ability to induce micronucleated polychromatic erythrocytes (MNPCEs) in the bone marrow of Cr1:CD

- 1 (ICR)BR mice (5/ sex/dose/sampling time, with an extra 5/sex at the top dose). Male mice received 720, 1440, or 2000 mg/kg bw by single

- dose oral gavage. Doses in females were 360, 720, or 1440 mg/kg bw. The highest dose level was set based on a previous mouse micronucl eus study conducted with the technical material DPX

KQ926

- 45 and a range finding study conducted with the current batch of technical material. The test substance was suspended in corn oil at a top concentration of 200 mg/mL. Proquinazid ( DPKQ926

- 75) did not induce an increase in micronuclei in bone marrow cells of mice and is considered negative in this in vivo assay. The rapid appearance of clinical signs of toxicity in mice is indicative of rapid absorption and likely systemic exposure (including bone marrow) to proquinazid and/or metabolites (MRID 48696356).

IN

- MM671 was evaluated for its ability to induce micronucleated polychromatic erythrocytes (MNPCEs) in the bone marrow of Cr1:CD

- 1 (ICR)BR mice (5/sex/dose/sampling time, with an
extra 5/sex at th e top dose )

. Male and female mice received 0, 500, 1000, or 2000 mg/kg by single

- dose oral gavage

. The highest dose level was set based on the limit dose for this test
system

. The test substance was dissolved in corn oil at concentrations of 51.0, 103. 3, and 205.0 mg/mL after adjusting for purity

. Cyclophosphamide (40 mg/kg) was used as the positive
control

. IN

- MM671 did not induce an increase in micronuclei in bone marrow cells of mice, and is considered negative in this in vivo assay (MRID 4869635 7).Proquinazid Human Health Risk Assessment DP No. 399842

Page 44 of 49 These mammalian erythrocytes micronucleus studies ( in viv o ) are classified as acceptable/ guideline

.

870. 5550 Unscheduled DNA Synthesis

The purpose of these studies was to investigate the in vitro toxicity of Proquinazid (DPX

KQ926

- 45) and was found to not cause a significant increase in unscheduled DNA synthesis and was concluded to be negative in this assay. DPX

- KQ926

- 45 was tested in t he unscheduled DNA synthesis (UDS) assay using primary cultures of rat hepatocytes. In both initial and independent repeat assays, the test substance was tested at eight dose levels, ranging from 3.9 to 500 g/mL, and fully evaluated for UDS at five dose levels of 7.8, 15.6, 31.3, 63, and 125 g/mL. The highest dose level was set based on the limit dose for this test system, and cytotoxicity data from a range finding experiment. For both the initial and repeat assays, the test substance was
administered to the test system as a solution in acetone at a maximum concentration of 500 mg/mL. Proquinazid (DPX

- KQ926

- 45) did not cause a significant increase in unscheduled DNA synthesis and was conclud ed to be negative in this assay (MRID 4869635 8).

This unsched uled DNA synthesis in mammalian cultured cell study ( in vitro ) is classified as acceptable/ guideline

.

A.4.7 Neurotoxicity

870.6200 Acute Neurotoxicity Screening Battery

This executive summary and classification determination was prepared by the EPA reviewer. The purpose of these studies was to investigate the toxicity of DPX

- KQ926 technical to rats in an acute neurotoxicity screening battery (MRID 48696359)

. Proquinazid was administered to

Crl:CD BR rats (12 rats/sex/dose) by single

- dose gavage as a suspension in 0.5% methyl
cellulose in water. Dose volumes were 10 mL/kg bw. Dose levels (adjusted for purity) in both

sexes were: 0, 100, 500, or 2000 mg/kg bw . In order to establish a NOAEL for females, 12 females/dose were similarly administered a si ngle dose by oral gavage of: 0, 15, 30, or 50 mg/kg
bw.

The NOAEL for neurotoxicity was 100 mg/kg bw for males and 50 mg/kg bw for females based on decreased motor activity on the day of exposure at the LOAEL of 500 mg/kg bw and above in
males and at 100 mg/kg bw and above in females. While the dose

- response fo r the decreased motor activity in females was fairly flat, the percentage decreases were statistically significant
and >40% and were considered treatment

- related and adverse for all doses ≥100 mg/kg/day

. While there was a 23% decrease in motor activity in females at 50 mg/kg/day, this finding was not toxicologically relevant and considered a NOAEL because it was (i) not statistically significant ( i.e., within the standard deviation) and (ii) within the normal variability historically
observed for motor acti vity in adult rats (typically the variability is 20

- 53%) reported in studies from labs worldwide (Moser et al ., 2007). Females in the 2000 mg/kg group had increased incidences of hunched posture and staining of the skin in various locations. On Test Day 8 , hind limb grip strength and foot splay were significantly decreased in 2000 mg/kg bw females, andProquinazid Human Health Risk Assessment DP No. 399842

Page 45 of 49 foot splay was significantly decreased in 2000 mg/kg bw males and females. Affected rats appeared to completely recover by Test Day 15

.

This acute neurotoxicity screening battery in the rat is classified as acceptable/ guideline

.

870.6200 Subchronic Neurotoxicity Screening Battery

This executive summary and classification determination was prepared by the EPA reviewer. The purpose of these studies was to investigate the toxicity of DPX

- KQ926

- 45 and

- 75 technical to rats when administered via the diet for 90 consecutive days as well as the subchronic
neurotoxic effects (MRID 48696338)

. In a combined subchronic toxicity study and subchronic

neurotoxi city study, Crl:CD BR rats (22 rats/sex/concentration) were administered diets containing 0, 30, 100, 300, or 2000 ppm proquinazid (DPX

- KQ926

- 45) for males or 0, 30, 100, 300, or 600 ppm for females for approximately 90 days. The mean daily intakes were : males: 0, 2, 6, 19, and 135 mg/kg bw/day and females: 0, 2, 8, 23, and 50 mg/kg bw/day. Within each dose group, 10 rats were designated as the toxicity subgroup and 12 rats were designated as the neurotoxicity subgroup.

The NOAEL for the systemic toxi city of proquinazid (DPX

- KQ926

- 45 ) was 30 ppm (2 mg/kg bw/day) in males and 100 ppm (8 mg/kg bw/day) in females based on hormonal and morphological changes (hypertrophy) in the thyroid at a LOAEL of 100 ppm (6 mg/kg bw/day) in males and at 300 ppm (23 mg/k g bw/day) in females. Decrements in body weight and nutritional parameters, as well as microscopic and clinical pathological changes in the liver, were also present at higher doses. The NOAEL for neurotoxicity was 2000 ppm (135 mg/kg/day) in
males and 60 0 ppm (49.7 mg/kg/day) in females, the highest dietary concentrations tested.

Under the conditions of the later study with a newer manufacturing process , the NOAEL values
in the 90

- day feeding study with proquinazid (DPX

- KQ926

- 75 ) was the same with a NOAEL of 30 ppm (2 mg/kg bw/day) in males and 100 ppm (8 mg/kg bw/day) in females based on thyroid hypertrophy and hormonal changes associated with the thyroid at a LOAEL of 100 ppm (6 mg/kg/bw/day) and above for males and on decreases in w hite blood cell counts at 300 ppm (24 mg/kg bw/day) and above for females. Decrements in body weight and nutritional parameters were also present at higher doses, together with hepatocyte hypertrophy (but no clear evidence of hepatocellular damage)

.

This combined 90

- day oral toxicity study and subchronic neurotoxicity screening battery in the

rat are classified as acceptable/ guideline

.

870.6300 Developmental Neurotoxicity Study

A.4.8 Metabolism

870.7485 Metabolism

– Rat

This executive summary and classification determination was prepared by the EPA reviewer. The purpose of these studies was to investigate the toxicity of DPX

- KQ926 technical to rats inProquinazid Human Health Risk Assessment DP No. 399842

Page 46 of 49 both a single dose and a repeat dose study of 7 consecutive days (MRIDs 48696360 and 48696361). Experiments were conducted in male and female Sprague

- Dawley CVF+ rats using the radiolabel [phenyl

14 C(U)]proquinazid (DPX

- KQ926) at gavage doses of 1 mg/kg and
20 mg/kg, and a dose volume of 4 ml/kg. Single dose: Proquinazid w as virtually completely absorbed at the 1 and 20 mg/kg doses. There was extensive metabolism, based on very low excretion of parent compound and extensive excretion of metabolise d dose in urine and f eces. Biliary excretion account ed for essentially all o f the f ecal excretion. The elimination half

- life for
total radioactive residues was 8

- 12 hours, and by 7 days post dosing little potential for accumulation of residues was seen (<1% of the dose in the body). Major biotransformations
observed were the los s of side chains and hydroxylation/oxidation both at the phenyl ring and
side chains. Repeat dose: Male and female rats were dosed with approximately 1 mg/kg of [phenyl

14 C(U)]proquinazid for 7 consecutive days and sacrificed at approximately 4.4

- 6.1, 24

- 25, 48

- 49, and 169

- 170 hours after the last dose. The concentration of radioactivity in tissues declined rapidly following the last dose. Tissue residue concentrations were generally lower than plasma concentrations with the exception of liver, kidney, and fat. There appeared to be little potential for bioaccumulation. Extensive metabolism of proquinazid was observed. The
most abundant metabolites in urine and f eces were the same as observed in the single dose study. A small percentage of 14 C

- proquin azid (up to 6%) was deiodinated and excreted as a ring hydroxylated product.

This metabolism and pharmacokinetics studies in the rat are classified as acceptable/guideline

.

A.4.9 Immunotoxicity

870.7800 Immunotoxicity

In an immunotoxicity study (MRID No. 48 778901), Proquinazid (95%, Lot No. 30041) was administered in the diet to female Crl:CD(SD) rats (10/group) at concentrations of 0, 50, 200, 600, or 1200 ppm (equivalent to 0, 4, 14, 42, or 82 mg/kg/day, respectively) for 28 days. A group
of 10 fema les in the positive control group received intraperitoneal injection of Cyclophosphamide at a dose of 25 mg/kg/day during Days 23

- 27. On Day 23, animals in all groups were immunized with a suspension of sheep red blood cells (SRBC) by intravenous injection (4 x10 8 SRBC/animal, 0.5 mL/animal dose volume). Animals were sacrificed on Day 28. All animals were evaluated for mortality, clinical signs, body weight changes, food consumption, water consumption, organ weights and gross pathology. Immunotoxicity was assessed for all animals by using an enzyme

- linked immunosorbent assay (ELISA); brain, spleen and thymus weights were determined at necropsy. There were no effects of test substance treatment on mortality, clinical signs, food consumption, water consumpti on, spleen or thymus weights, or gross pathology. At the highest dose 1200 ppm (82 mg/kg/day), final body weight (test day 28) and overall body weight gains, food consumption, and food efficiency (test days 0

- 28) were reduced compared to the control. The systemic toxicity LOAEL is 1200 ppm (82 mg/kg/day) based on decreases in terminal body weight, body weight gain, food consumption and food efficiency. The NOAEL for systemic toxicity is 600 ppm (42 mg/kg/day).

This immunotoxicity study is classified accep table /guideline and satisfy the guideline requirements

.Proquinazid Human Health Risk Assessment DP No. 399842

Page 47 of 49 A.4. 10 Additional info rmation on batch variability

The toxicity tests summarized in this document were conducted with one of three specific
batches of the technical material ( DPX

- KQ926

- 45

, DPX

- KQ926

- 75

, and DPX

- KQ926

- 85). A complete battery of mammalian toxicity studies was originally conducted with DPX

- KQ926

- 45. During the course of the development of proquinazid the manufacturing process was modified and improved. Batches DPX

- KQ926

- 75 and DPX

- KQ926

- 85 are equivalent with respect to purity and impurity profile (see the Product Chemistry section, for full technical composition), and are representative of the commercial product produced by the current, improved manufacturing process. These tec hnical materials are 98.3% and 97.7% (w/w) proquinazid, respectively. As there were differences in the purity and impurity profiles of technical material produced by the old and current methods, selected bridging studies were conducted to
demonstrate the e quivalent or lesser toxicity of material produced using the current method to that produced under the original process. These bridging studies (conducted with DPX

- KQ926

- 75) were a 90

- day feeding study in rats, a bacterial reverse mutation test and an in vi vo mouse micronucleus assay. These studies demonstrated that DPX

- KQ926

- 75 exhibited equivalent or lesser toxicity to DPX

- KQ927

- 45 and t hat like DPX

- KQ927

- 45, is not likely to be genotoxic. Based on the results of these bridging studies, the remaining genot oxicity studies, as well as the chronic studies, the 90

- day dog study, and the developmental and neurotoxicity studies
conducted with DPX

- KQ926

- 45 were not repeated using the current process material, thus greatly diminishing the use of animals. However, a complete battery of acute toxicity studies and

a 2

- generation reproductive study were conducted with the current process material (either DPX

KQ926

- 75 or

- 85). The original two

- generation reproduction study in rats was conducted with material produced by the previous process, DPX

- KQ926

- 45, and was initiated prior to promulgation of the most recent (22 January, 2001) version of the OECD 416 guidelines for two

generation reproduction toxicity studies. However, during the conduct of the study, some end points included in the 2001 update were included for the F
1 (but not the P
1 ) generation. The

repeat two

- generation reproduction study was conducted with test material representative of the current manufacturing process. In addition, this study was necessary in o rder to evaluate specific developmental and reproductive endpoints required in the updated OECD 416 guidelines that were not covered by the original study. Metabolism studies were conducted using a batch of pure proquinazid (DPX

- KQ926

- 21, 99.2% w/w proqui nazid).

Comparison of the results of 90

- day studies in rats with proquinazid produced by the old method (batch KQ926

- 45) and the current method (batch KQ926

- 75) shows that the toxicity of the materials is mostly equivalent with respect to the thyroid eff ects, which generally form the basis
of the NOAELs in rats

. Also although liver weights were affected to a similar extent, other effects on the liver (clinical pathology and microscopic findings both sexes) were more pronounced with batch KQ926

- 45. Hence there are indications for the lesser toxic potential of proquinazid manufactured by the current process. Although a reduction in white blood cells in females (due primarily to reduced numbers of lymphocytes) was seen only with proquinazid manufactured by the new process, the toxicological significance of this finding is uncertain as there were no correlative effects on the histology of lymphoid organs (spleen, lymph node or thymus) or bone marrow. Taken together, these 90

- day studies indicate similar tar get organs for the test materials produced by the former and current processes

.Proquinazid Human Health Risk Assessment DP No. 399842

Page 48 of 49 A ppendix B. Metabolism Summary Table

Table B.1. Tabular Summary of Proquinazid 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 Grapes 35

- 39% (0.08

- 0.09 ppm)
Grape Vines 52

- 75%

IN

- MM671 Grapes 3

- 8% (00.01 ppm)

Grape Vines

--

IN

- MM991 Grapes 2

- 3% (<0.01 ppm)
Grape Vines

--

The final row of the table should have a concise summary of relevant parameters. Crop 1; MRID No.48696316; Application Rate : 3 applications at 200 g ai/ha; Level of exaggeration compared to label rate (4x single application rate; 3x seasonal application rate); timing 30 day intervals (14 days on label); pre

harvest interval: samples c ollected 0, 14 and 29 days after the last application , compared to 60 day PHI on label.Proquinazid Human Health Risk Assessment DP No. 399842

Page 49 of 49 A ppendix C

. Physicochemical Properties Table

Table 3.2. Physicochemical Properties of the Technical Grade Proquinazid.

Parameter Value Reference

Melting point/range 61.5 °C

– 62 °C 48696308

pH 6.48 48696308
Density 1.57 g/m L 48696308
Water solubility (25°C) 0.97 mg/ L 486963 13
Solvent solubility (g/100 mL at 20°C to 25°C) Acetone >250 g/kg
Acetonitril e 154 g/L Dichloromethane >250 g/kg Dimethylformamide >250 g/kg Ethyl acetate >250 g/kg

n

- hexane >250 g/kg methanol 136 g/ L

1

- octanol >250 g/kg

o

- xylene >250 g/kg 486963 13

Vapor pressure (25°C) 9 x 10

- 5 Pa 486963 15
Dissociation constant, pK

a No dissociation between pH 2.4 and 11.6 486963 13
Octanol/water partition coefficient, log

P OW (25°C) 5.5 486963 14

UV/visible absorption spectrum No absorbance maxima ( λ

max ) above 325 nm at pH <2, 7, and >10 buffer at
concentrations of 12 and 19 μg/mL 486963 12