Site visit: Crystal Lake Automotive, Inc. 16055 Buck Hill Road Lakeville, MN 55044 Participants: Jim Siegfried Crystal Lake Marv Hays Crystal Lake Teena Wooten EPA OSW Jim O'Leary EPA OSW Nancy Ellefson MN Pollution Control Agency Date: May 10, 2006 Jim O'Leary and Teena Wooten with the Environmental Protection Agency's Office of Solid Waste and Nancy Ellefson with the Minnesota Pollution Control Agency visited the Crystal Lake Automotive facility in Lakeville, MN on May 10, 2006. Mr. Siegfried escorted the group to the painting area where he introduced Mr. Hays. The group watched a technician remove sealer from a car. He first sprayed a small amount of solvent on the vehicle and then used a disposable towel to remove the solvent and sealer. A petroleum based solvent is used for the initial cleaning. Before painting, the technician will use a water soluble solvent. After use, the wipe, which is considered an industrial solid waste, is thrown into the trash container.

Crystal Lake uses disposal wipes for its degreasing operations. The facility changed to disposal wipes approximately six years ago because of the lint from reusable wipes. Crystal Lake also changed from applying the solvent to the wipe to using a sprayer. This reduced the quantity of solvent needed and the number of wipes used. The facility is a very small quantity user under the MPCA criteria. Crystal Lake uses a solvent containing vat to clean the spray guns. Once used the solvent is reclaimed on site. By reclaiming the solvent, the facility has reduced its waste from 12 drums of sludge to one drum.


4	Dermal/Inhalation Dose = Unit Exposure (μg/lb ai) x Conversion Factor (0.001 mg/μg) x Application Rate (lb ai/acre or gal) x Area Treated or Amount  Handled Daily (A or gal/day) / BW (80 kg).
5	Dermal/Inhalation MOE = NOAEL (ST/IT = 1.25 mg/kg/day) / Dose (mg/kg/day).
6   Field Crop Typical = Vegetable Transplants (Brassica, Bulb Vegetables, Cucurbit Vegetables, Fruiting Vegetables
7   Combined MOEs = NOAEL (ST/IT = 1.25 mg/kg/day) / Dermal Dose (mg/kg /day) + Inhalation Dose (mg/kg/day).
9.3	Post-Application Exposure

HED uses the term post-application to describe exposures that occur when individuals are present in an environment that has been previously treated with a pesticide (also referred to as re-entry exposure).  Such exposures may occur when workers enter previously treated areas to perform job functions, including activities related to crop production, such as scouting for pests or harvesting.  Post-application exposure levels vary over time and depend on such things as the type of activity, the nature of the crop or target that was treated, the type of pesticide application, and the chemical's degradation properties.  In addition, the timing of pesticide applications, relative to harvest activities, can greatly reduce the potential for post-application exposure.

Dermal Exposure
Post-application dermal exposure and risk estimates for the proposed new uses on legume subgroup 6C and bushberry subgroup 13-07B; post-harvest applications on pome fruit 11-10; and on ornamental plants and vegetable transplants grown in both indoor and outdoor production facilities are all expected to result in comparable/identical exposure scenarios to those assessed in previous occupational exposure assessments for this chemical (D398608, I. Nieves, 05/30/2012; D412811, I. Nieves, 11/13/2013).   The application rates proposed for these new uses are identical to the ones previously assessed (0.11 lb ai/A for legumes and bushberries; 1.35x10[-6] lb ai/ lb fruit for Pome Fruits; and 0.11 for vegetable transplants). No risk estimates of concern were identified for any of the previous uses.  Based on the premise that no new Dislodgeable Foliar Residue studies have been recently submitted, there have been no revisions to the toxicological database/endpoints and/or to the occupational post-application SOPs since the date of the oldest Occupational/Residential Exposure Risk Assessment (2012), all post-applications activities related to the proposed new uses are considered to be not of concern to the Agency.

The post-application potential for dermal exposure of difenoconazole from the proposed use on ornamentals have been re-visited to reflect recent updates (2012) on HEDs Occupational SOPs and body weight assumptions.  The following post-application activities have been identified from the proposed use on ornamentals: hand harvesting; hand set irrigation; hand pruning; scouting; container moving; hand weeding; propagating; hand pruning; transplanting; pinching; and tying/training.  Hand set irrigation was identified with the highest exposure potential.  Table 9.3.1 provides a summary of the anticipated post-application activities and associated transfer coefficients for the proposed crops/use sites.

9.3.1. Anticipated Post-Application Activities and Dermal Transfer Coefficients.
                                Proposed Crops
                                  Crop Height
                                Foliage Density
                      Transfer Coefficients[*] (cm[2]/hr)
                                  Activities
                Nursery Crop (Ornamentals, Non-bearing Plants)
                                   High/Low
                                   Full/Min
                                     1,900
Hand Set Irrigation

                                      230
Hand harvesting; hand pruning; scouting; container moving; hand weeding; propagating; hand pruning; transplanting; pinching; tying/training.
*It is the policy of HED to use the best available data to assess post-application exposure.  Sources of generic post-application data, used as surrogate data in the absence of chemical-specific data, are derived from ARTF exposure monitoring studies, and, as proprietary data, are subject to the data protection provisions of FIFRA.  The standard values recommended for use in predicting post-application exposure that are used in this assessment, known as "transfer coefficients", are presented in the ExpoSAC Policy 3" which, along with additional information about the ARTF data, can be found at the Agency website.

No risk estimates of concern were identified for any of the post-application scenarios assuming treated area re-entry immediately after application. Table 9.3.2 presents results for the highest exposure potential scenario (hand set irrigation) at the maximum proposed application rate (0.13 lb ai/A).

Table 9.3.2.  Occupational Post-application Non-Cancer Exposure and Risk Estimates for Difenoconazole.
                                   Crop/Site
                                  Activities
                        Transfer Coefficient (cm[2]/hr)
                                    DFR[1]
                                  Dermal Dose
                                (mg/kg/day)[2]
                                    MOE[3]
                         Short- and Intermediate- term
                Nursery Crop (Ornamentals, Non-bearing Plants)
                              Hand Set Irrigation
                                     1,900
                                     0.36
                                    0.0042
                                      300
1	DFR = Application Rate x F x (1-D)t x 4.54E8 ug/lb x 2.47E-8 acre/cm[2]; where F = 0.25 and D = 0.10 per day
2	Daily Dermal Dose = [DFR (ug/cm[2]) x Transfer Coefficient x 0.001 mg/ug x 8 hrs/day x dermal absorption (%)]  BW (kg).
3	MOE = POD (mg/kg/day) / Daily Dermal Dose.

Inhalation Exposure
Based on the Agency's current practices, a quantitative post-application inhalation exposure assessment was not performed for difenoconazole at this time primarily because of the low acute inhalation toxicity (Toxicity Category III and IV), low vapor pressure (2.5 x 10[-10] mm Hg at 25 ºC), and low proposed use rates (highest proposed rate = 0.13 lb ai/A).  However, there are multiple potential sources of post-application inhalation exposure to individuals performing post-application activities in previously treated fields.  These potential sources include volatilization of pesticides and resuspension of dusts and/or particulates that contain pesticides.  The Agency sought expert advice and input on issues related to volatilization of pesticides from its Federal Insecticide, Fungicide, and Rodenticide Act Scientific Advisory Panel (SAP) in December 2009, and received the SAP's final report on March 2, 2010. The Agency is in the process of evaluating the SAP report as well as available post-application inhalation exposure data generated by the ARTF and may, as appropriate, develop policies and procedures, to identify the need for and, subsequently, the way to incorporate occupational post-application inhalation exposure into the Agency's risk assessments.  If new policies or procedures are put into place, the Agency may revisit the need for a quantitative occupational post-application inhalation exposure assessment for difenoconazole.

Although a quantitative occupational post-application inhalation exposure assessment was not performed, an inhalation exposure assessment was performed for occupational/commercial handlers.  Handler exposure resulting from application of pesticides outdoors is likely to result in higher exposure than post-application exposure.  Therefore, it is expected that these handler inhalation exposure estimates would be protective of most occupational post-application inhalation exposure scenarios.

	9.3.1 	Restricted Entry Interval

The REI specified on the proposed label is based on the acute toxicity of difenoconazole.  Difenoconazole is classified as Toxicity Category III for  acute dermal toxicity and eye irriitation, and Toxicity Category IV for skin irritation potential.  It is not a skin sensitizer.  Short- and intermediate-term post-application risk estimates were not a concern on day 0 (12 hours following application) for all post-application activities.  Under 40 CFR 156.208 (c) (2) (iii), active ingredients classified as Acute III or IV for acute dermal, eye irritation and primary skin irritation are assigned a 12-hour REI.  Therefore, the [156 subpart K] Worker Protection Statement interim REI of 12 hours on the proposed labels is adequate to protect agricultural workers from post-application exposures to difenoconazole.

10.	REFERENCES

Difenoconazole.  New Foliar Uses on Pea and Bean, Dried Shelled, Except Soybean, Subgroup 6C and Bushberry Subgroup 13-07B.  Summary of Analytical Chemistry and Residue Data. - B. Cropp-Kohlligian, D418479 and D421194, 10/15/2014

Difenoconazole.  Request to Register New Multiple Active Ingredient (MAI) End-Use Product (Academy(TM) Fungicide) for New Post-Harvest Uses on Pome Fruit Group 11-10.  Summary of Analytical Chemistry and Residue Data. - B. Cropp-Kohlligian, D417611, 10/17/2014

Difenoconazole.  Acute and Chronic Aggregate Dietary Exposure and Risk Assessments for the Petition for Use of Difenoconazole on Pea and Bean, Dried Shelled, Except Soybean, Subgroup 6C, Bushberry Subgroup 13-07B and an amended use on Pome Fruit to add Post-Harvest Use of Difenoconazole. - T. Morton, D421430, 2/24/15

Drinking Water Exposure Assessment in support of the new use registration of multiple difenoconazole formulated products  on Artichoke, Berry Bushberry Subgroup 13-07B, Ginseng,  and Bean and Pea, Dried Shelled (except Soybean) Subgroup 6C as well as Conversion of Stone Fruit Crop Group 12-12 and  Tree Nuts Crop Group 14-12..   -  F. Khan, D421092, 11/13/14

Difenoconazole.  Occupational and Residential Exposure Assessment for a Proposed Use on Canola and Oilseed Subgroup 20A. - I. Nieves, D412811, 09/13/2013

Difenoconazole.  Occupational and Residential Exposure Assessment to Evaluate the Risk from Proposed Uses on Post-Harvest Potatoes, and the Expansion of Various Crop Groups Including: Citrus Fruit Group 10-10; Fruiting Vegetable Subgroups 8-10; Low Growing Berry Subgroup 13-07G, except cranberry; Pome Fruit, Crop Group 11-10 and Fruiting Vegetable, Crop Group 8-10). - I. Nieves, D398608, 5/30/2012

Difenoconazole. Occupational and Residential Exposure Assessment for the Proposed New Use of Difenoconazole on Strawberry, Carrot, Chickpeas, Soybean, Stone Fruit: Group 12 and Golf Course Turfgrass. - Rebecca Daiss; D371037; February 24, 2011.

Difenoconazole. Occupational and Residential Exposure/Risk Assessment for the Proposed New Uses of Difenoconazole on Fruiting Vegetables, Tuberous and Corm Vegetables, Pome Fruit, Sugar Beets and Ornamentals - Mark I. Dow; D340044; July 27, 2007.

APPENDICES

A 	TOXICOLOGY DATA SUMMARY

	A.1 	Guideline Data Requirements - Difenoconazole

                                 Guideline No.
                                                 Study Type
                                   Technical
                                   MRID No.

                                   Required
                                   Submitted

                  870.3100

                  870.3150
                  870.3200

                  870.3250
                  870.3465
                  Subchronic (Oral) Toxicity - Rodent

                  Subchronic (Oral) Toxicity - Non-Rodent
                  21/28-Day Dermal Toxicity

                  90-Day Dermal Toxicity
                  90-Day Inhalation Toxicity
                                       Y

                                       Y
                                       N

                                       N
                                      N*
                                       Y

                                       Y
                                       Y

                                       N
                                       N
                                   42090022
                                   42090021
                                   42090013
                                   42090013
                                   46950310
870.3700a

870.3700b

870.3800
                  Prenatal Developmental Toxicity - Rodent

                  Prenatal Developmental Toxicity - Non-Rodent

                  Reproduction and Fertility Effects
                                       Y

                                       Y

                                       Y
                                       Y

                                       Y

                                       Y
                                   42090016
                                   42710008
                                   42090017
                                   42710008
                                   42090018
870.4100a

870.4100b

870.4200a

870.4200b

870.4300
                  Chronic (Oral) Toxicity - Rodent

                  Chronic (Oral) Toxicity - Non-Rodent (Dog)

                  Carcinogenicity - Rat

                  Carcinogenicity - Mouse

                  Combined Chronic Toxicity /Carcinogenicity
                                       Y

                                       Y

                                       Y

                                       Y

                                       Y
                                       Y

                                       Y

                                       Y

                                       Y

                                       Y
                                   42090015
                                   42710006
                                   42090012
                                   42710005
                                   42090019
                                   42710010
                                   42090015
                                   42710006
                              42090019, 42710010
870.6100a
870.6100b
870.6200a
870.6200b
870.6300
870.7485
870.7600

870.7800
                  Neurotoxicity - Acute Delayed Neurotox.- Hen
                  Neurotoxicity  - Subchronic - Hen
                  Neurotoxicity - Acute - Rat
                  Neurotoxicity -Subchronic - Rat
                  Developmental Neurotoxicity..................
                  General Metabolism
                  Dermal Penetration

                  Immunotoxicity.............................
                                       N
                                       N
                                       Y
                                       Y
                                       N
                                       Y
                                       Y

                                       Y
                                       N
                                       N
                                       Y
                                       Y
                                       N
                                       Y
                                       Y

                                       Y
                                      ---
                                      ---
                               46950327 46950329
                                      --
                                   42090028
                                   47453201
                                   46950333
                                   47453202
                                   47453203
                                   48696701
* The Hazard and Science Policy Council (HASPOC) concluded that a 28-day inhalation toxicity study is not required at this time (TXR 0054074). A.2	Toxicity Profiles

Table A.1.	Acute Toxicity Profile  -  Difenoconazole
Guideline No.
Study Type
MRID No.
Results
Toxicity Category
870.1100
Acute oral
42090006
                              LD50  = 1450 mg/kg
                                      III
870.1200
Acute dermal
42090007
                             LD50 > 2010 mg/kg
                                      III
870.1300
Acute inhalation
42090008
                              LC50 > 3.3 mg/L
                                      III
870.2400
Eye irritation
42090009
                     Mild irritation reversible in 7 days
                                      III
870.2500
Dermal irritation
40789807
                               Slight irritation
                                      IV
870.2600
Skin sensitization
42090011, 42710004
                                   Negative
                                      N/A

Table A.2.	Subchronic, Chronic and Other Toxicity Profile of Difenoconazole
Guideline No.
Study Type
MRID No. (year)/ Classification /Doses
Results
870.3100

90-Day oral toxicity (rat)
42090022 (1987)
Acceptable/guideline
0, 20, 200, 750, 1500 or 3000 ppm
0, 1, 10, 37.5, 75 and 150 mg/kg/d
NOAEL = 20 ppm (1 mg/kg/day)
LOAEL = 200 ppm (10 mg/kg/day) based on the 10% decrease in body weight in the 200 ppm females (as well as a negative trend in feed consumption) and Increases in absolute liver weights in both sexes

870.3100

90-Day oral toxicity (mouse)
42090021 (1987)
 Acceptable/guideline
0, 20, 200, 2500, 7500 or 15,000 ppm
M: 0, 2.9, 30.8, 383.6, 1125 and 2250 mg/kg/d
F: 0, 4.1, 41.5, 558.9, 1125 and 2250 mg/kg/d
NOAEL = 20 ppm (2.9 mg/kg/day)
LOAEL = 200 ppm (30.8 mg/kg/day) based on body weight changes & liver histopathology.
870.3150
26-Week oral toxicity
42090012 (1987)
Acceptable / guideline
0, 100, 1000, 3000 or 6000 ppm
M: 0, 3.6, 31.3, 96.6 and 157.8 mg/kg/d
F: 0, 3.4, 34.8, 110.6 and 203.7 mg/kg/d
NOAEL = 3000 ppm (31.3 mg/kg/day in males/34.8 mg/kg/day in females)
LOAEL = 6000 ppm (96.6 mg/kg/day in males/110.6 mg/kg/day in females), based primarily on microscopic examination of CGA 169374-related lenticular cataracts.
870.3200

21/28-Day dermal toxicity (rat)
42090013 (1987)
Acceptable / guideline
0, 10, 100 and 1000 mg/kg/d
NOAEL = 10 mg/kg/day
LOAEL = 100 mg/kg/day based on statistically significant decrements in body weight, body weight gain, and food consumption.
870.3200

21/28-Day dermal toxicity (rat)
46950310 (2000)
Acceptable/ guideline
0, 10, 100 and 1000 mg/kg/d
NOAEL (systemic) = 1000 mg/kg/day
LOAEL (systemic) was not determined.
NOAEL (dermal) = 100 mg/kg/day
LOAEL (dermal) = 1000 mg/kg/day based on hyperkeratosis at the skin application site.
870.3700a

Prenatal developmental in (rat)
42090016, 42710007 (1987)
Acceptable / guideline
0, 2, 20, 100 or 200 mg/kg/d from GD 6-15 (nominal doses  differed widely from theoretical, this required altering NOAEL/LOAEL values)
Maternal NOAEL = 16 mg/kg/day
LOAEL = 85 mg/kg/day based on decreased body weight gain and food consumption.
Developmental NOAEL = 85 mg/kg/day
LOAEL = 171 mg/kg/day based on alterations in fetal ossification.
870.3700b

Prenatal developmental in (rabbit)
42090017, 42710008 (1987)
Acceptable / guideline
0, 1, 25 or 75 mg/kg/d from GD 7-19

Maternal NOAEL = 25 mg/kg/day
LOAEL = 75 mg/kg/day based on decreased body weight gain and food consumption.
Developmental NOAEL = 25 mg/kg/day
LOAEL = 75 mg/kg/day based on nonsignificant increases in postimplantation loss and resorptions/doe and a significant decrease in fetal weight.
870.3800

Reproduction and fertility effects
(rat)
42090018 (1988)
Acceptable / guideline
0, 25, 250 or 2500 ppm
0, 1.25, 12.5 and 125 mg/kg/d

Parental/Systemic NOAEL = 25 ppm (1.25 mg/kg/day)
LOAEL = 250 ppm (12.5 mg/kg/day) based on reductions (statistically nonsignificant) in body weight gain which appear to be part of a dose-related trend days 70-77 prior to mating, days 0-7 of gestation, and days 7-14 of lactation
Offspring NOAEL = 25 ppm (1.25 mg/kg/day)
LOAEL = 250 ppm (12.5 mg/kg/day) based on a significant reduction in the body weight of F1 male pups at day 21 in the 250 ppm group.
870.4100b

Chronic toxicity
(dog)
42090012, 42710005 (1988)
Acceptable / guideline
0, 20, 100, 500 or 1500 ppm
M: 0, 0.71, 3.4, 16.4 and 51.2 mg/kg/d
F: 0, 0.63, 3.7, 19.4 and 44.3 mg/kg/d
NOAEL = 100 ppm (3.4 mg/kg/day in males/3.7 mg/kg/day in females)
LOAEL = 500 ppm (16.4 mg/kg/day in males/19.4 mg/kg/day in females), based on significant inhibition of body weight gain in females.

870.4200

Carcinogenicity
(rat)
42090019, 42710010 (1989)
Acceptable / guideline
0, 10, 20, 500 or 2500 ppm
M: o, 0.48, 0.96, 24.12 and 123.7 mg/kg/d
F: 0, 0.64, 1.27, 32.79 and 169.6 mg/kg/d
NOAEL = 20 ppm (0.96 mg/kg/day in males/1.27 mg/kg/day in females)
LOAEL = 500 ppm (24.1 mg/kg/day in males/ 32.8  mg/kg/day in females) based on reductions in cumulative body weight gains in the 500 and 2500 ppm groups.

No evidence of carcinogenicity
870.4300

Carcinogenicity
(mouse)
42090015, 42710006 (1989)
Acceptable / guideline
0, 10, 30, 300, 2500 or 3000 ppm
M: 0, 1.51, 4.65, 46.29, 423.1 and 818.9 mg/kg/d
F: 0, 1.9, 5.63, 57.79 and 512.6 mg/kg/d
NOAEL = 30 ppm (4.7 mg/kg/day in males/5.6 mg/kg/day in females)
LOAEL = 300 ppm (46.3 mg/kg/day in males/57.8 mg/kg/day in females) based on reductions in the cumulative body weight gains and hepatocellular hypertrophy, liver necrosis, fatty changes in the liver and bile stasis in the 300, 2500 & 4500 ppm groups.

Evidence of carcinogenicity (liver adenoma/carcinoma in both sexes)
870.5100
In vitro bacterial gene mutation (Salmonella typhimurium/ E. coli)/ mammalian activation gene mutation assay
42090019, 42710010 (1989)
Acceptable / guideline
340 - 5447 ug/plate;
85 - 1362 ug/plate (repeat assay with TA1537 and TA98)

There were sufficient and valid data to conclude that CGA 169374 technical was negative in the microbial gene mutation assay.
870.5300
in vitro mammalian cell gene mutation assay in mouse lymphoma cells

42090024 (1986)
Unacceptable/ guideline
No conclusion can be reached from the three nonactivated and two S9 activated mouse lymphoma forward mutation assays conducted with difenoconazole technical. The study was seriously compromised.
870.5375
In vitro Mammalian Cytogenetics (chromosomal aberrations) assay in Chinese hamster CHO cells
46950319 (2001)
Acceptable/ guideline
0, 21.99, 27.49, or 34.36 ug/mL (-S9)
0, 34.36, 53.69 or 67.11 ug/mL (+S9)
There was evidence of a weak induction of structural chromosomal aberrations over background in the presence of S9-mix.

870.5375
In vitro Mammalian Cytogenetics (chromosomal aberrations) assay in Chinese hamster CHO cells
46950321 (2001)
Acceptable/ guideline
0, 26.3, 39.5 or 59.3 ug/mL (-S9)
0, 11.7 or 17.6 ug/mL (+S9)
There was evidence of a weak induction of structural chromosomal aberrations over background.

870.5375
In vitro Mammalian Cytogenetics (chromosomal aberrations) assay in human lymphocytes
46950323 (2001)
Acceptable/ guideline
0, 5, 30 or 75 ug/mL (-S9)
0, 5, 30 or 62 ug/mL (+S9)
There was no evidence of structural chromosomal aberrations induced over background.

870.5385
In vivo mammalian chromosomal aberration test Assay in Mice
42090023 (1986)
Unacceptable/guideline
250, 500 or 1000 mg/kg
There was no evidence of a cytotoxic effect on the target organ or significant increase in the frequency of nuclear anomalies (micronuclei). However, the study was compromised.
870.5395
In vivo mammalian cytogenetics - erythrocyte micronucleus assay in mice
41710011 (1992)
Acceptable/guideline
Doses up to 1600 mg/kg
Mice bone marrow - No increase in micronucleated polychromatic erythrocytes occurred with CGA-1 69374 (91.2% a.i).
870.5550
Unscheduled DNA Synthesis in Mammalian Cells in Culture

4210012 (1992)
Acceptable/ guideline
Doses up to 50 ug/mL

CGA-i69374 tech. (92.2% a.i.) was considered to be negative in the unscheduled DNA synthesis assay in rat primary hepatocytes as measured by an autoradiographic method at concentrations up to 50.0 ug/mL.
870.5550
Unscheduled DNA Synthesis in Mammalian Cells in Culture

42090027 (1985)
Unacceptable/ guideline
0.25-31.25 ug/mL

No conclusion can be reached from the unscheduled DNA synthesis (UDS) primary rat hepatocyte assay conducted with difenoconazole technical at concentrations ranging from 0.25 to 31.25 ug /mL. The sensitivity of the study was severely compromised.
870.5550
Unscheduled DNA Synthesis in Mammalian Cells in Culture
42090026 (1985)
Unacceptable/ guideline
0.08-10 ug/mL
No conclusion can be reached from the unscheduled DNA synthesis (UDS) human fibroblast assay conducted with difenoconazole tech. at conc. ranging from 0.08 to 10 ug /mL.
870.6200a

Acute neurotoxicity screening battery
46950327 (2006)
Acceptable/ guideline
0, 25, 200 or 2000 mg/kg/d
NOAEL (M) = 25 mg/kg/day
LOAEL (M) = 200 mg/kg/day based on reduced fore-limb grip strength in males on day 1 and increased motor activity on Day 1.
NOAEL (F) = 200 mg/kg/day
LOAEL (F) = 2000 mg/kg/day based on decreased body weight, the following clinical signs: upward curvature of the spine, tip-toe gait, decreased activity, piloerection and sides pinched in and decreased motor activity.
870.6200b

Subchronic neurotoxicity screening battery
46950329 (2006)
Acceptable/ guideline
0, 40, 250, or 1500 ppm  M; 0, 2.8, 17.3 or 107.0 mg/kg/d
F: 0, 3.2, 19.5, or 120.2 mg/kg/d
NOAEL (M) = 40 ppm (2.8 mg/kg/day)
LOAEL (M) = 250 ppm (17.3 mg/kg/day) based on decreased hind limb strength.
NOAEL (F) = 250 ppm (19.5 mg/kg/day)
LOAEL (F) = 1500 (120.2 mg/kg/day) based on decreased body weight, body weight gain and food efficiency.
870.7800
Immunotoxicity [dietary] - Mouse
48696701 (2011)
Acceptable/ guideline
0, 20, 200, 1000, or 1500 pm (0, 3, 35, 177, or 247  mg/kg/day) for 28 days.
Systemic toxicity NOAEL = 200 ppm (35 mg/kg/day)
Systemic toxicity LOAEL = 1000 ppm (177 mg/kg/day) based on decreased body weight gains and liver toxicity

Immunotoxicity NOAEL = 200 ppm (35 mg/kg/day)
Immunotoxicity LOAEL = 1000 ppm (177 mg/kg/day)
based on decreased mean anti-SRBC IgM levels.
870.7600    Dermal Penetration

In vivo Dermal Penetration in the Rat,
In vitro
47453201 (2007)
See TXR 0056473
870.7600    Dermal Penetration

In vivo Dermal Penetration in the Rat,
46950333 (2003)
See TXR 0056473
870.7600    Dermal Penetration

In vitro Absorption through Human Epidermis;
47453202 (2007)
See TXR 0056473
870.7600    Dermal Penetration

In vitro Absorption through Rat Epidermis;
47453203 (2007)
See TXR 0056473
870.7485

Metabolism and pharmacokinetics
(rat)
42090028 (1990)
Acceptable/ guideline
14 daily doses of  0.5 or 300 mg/kg
Male and female Sprague-Dawley rats. Animals were administered a single oral gavage dose of 0.5 or 300 mg/kg [[14]C]CGA- 169374, or 0.5 mg/kg unlabeled GGA-169374 by gavage for 14 days followed by a single gavage dose of 0.5 mg/kg [[14]C)CGA-169374 on day 15. The test compound was labeled with C[14] at either the phenyl or triazole ring.
870.7485
Metabolism and pharmacokinetics
(rat)
42090028 (1990)
42090029 (1987)
42090030 (1987)
42090031 (1988)
Acceptable/ guideline
Single oral dose 0.5 or 300 mg/kg
14 daily doses of  0.5 or 300 mg/kg
Male and female Sprague-Dawley rats. Animals were administered a single oral gavage dose of 0.5 or 300 mg/kg [[14]C]CGA- 169374, or 0.5 mg/kg unlabeled GGA-169374 by gavage for 14 days followed by a single gavage dose of 0.5 mg/kg [[14]C)CGA-169374 on day 15. The test compound was labeled with C[14] at either the phenyl or triazole ring.

[[14]C] CCA 169374  was rapidly and extensively distributed, metabolized, and excreted in rats for all dosing regimens. The metabolism of difenoconazole appears to be extensive because the metabolites accounted for most of the recovered radioactivity in the excrete. Three major metabolites were identified in the feces (i.e. metabolites A, B, and C).  Two of the metabolites were separated into isomers (i.e., Al, A2, B1, and B2).  Metabolite C was detected only in the high-dose groups, indicating that metabolism of difenoconazole is dose-related and involves saturation of the metabolic pathway. Free triazole metabolite was detected in the urine of triazole-labeled groups and its byproduct was detected in the liver of phenyl labeled groups only. Other urinary metabolites were not characterized.
870.7485
Metabolism and pharmacokinetics
(rat)
42090028 (1990)
42090029 (1987)
42090030 (1987)
42090031 (1988)
Acceptable/ guideline in conjunction with MRIDs 420710013, 42710014  listed below
Single oral dose 0.5 or 300 mg/kg
14 daily doses of  0.5 or 300 mg/kg
The absorption, distribution, metabolism, and excretion of CGA 169374 were studied in groups of male and female Sprague-Dawley rats. Animals were administered a single oral gavage dose of 0.5 or 300 mg/kg [[14]C]CGA- 169374, or 0.5 mg/kg unlabeled GGA-169374 by gavage for 14 days followed by a single gavage dose of 0.5 mg/kg [[14]C)CGA-169374 on day 15. The test compound was labeled with C[14] at either the phenyl or triazole ring.

[[14]C] CCA 169374  was rapidly and extensively distributed, metabolized, and excreted in rats for all dosing regimens. The extent of absorption is undetermined pending determination of the extent of biliary excretion. The 4-day recoveries were 97.94-107.75% of the administered dose for all dosing groups. The elimination of radioactivity in the feces (78.06-94.61% of administered dose) and urine (8.48-21.86%) were almost comparable for all oral dose groups, with slightly higher radioactivity found in the feces of the high-dose group than the low-dose groups. This was probably due to biliary excretion, poor absorption or saturation of the metabolic pathway. The radioactivity in the blood peaked at about 24-48 hours.  Half-lives of elimination appear to be approximately 20 hours for the low-dose groups and 33-48 hours for the high-dose group. The study results also indicate that difenoconazole and/or its metabolites do not bioaccumulate to an appreciable extent following oral exposure since all the tissues contained negligible levels (< 1%) of radioactivity 7 days post exposure.

The metabolism of difenoconazole appears to be extensive because the metabolites accounted for most of the recovered radioactivity in the excrete. Three major metabolites were identified in the feces (i.e. metabolites A, B, and C).  Two of the metabolites were separated into isomers (i.e., Al, A2, B1, and B2).  Metabolite C was detected only in the high-dose groups, indicating that metabolism of difenoconazole is dose-related and involves saturation of the metabolic pathway. Free triazole metabolite was detected in the urine of triazole-labeled groups and its byproduct was detected in the liver of phenyl labeled groups only. Other urinary metabolites were not characterized.

These studies indicate that distribution, metabolism, and elimination of CGA-169374 were not sex related. There was a slight dose difference in the metabolism and elimination of CGA-169374. In phenyl and triazole labeling studies, fecal excretion of radioactivity was higher in the high dose animals compared to the low dose animals, and an additional metabolite was found in the feces of the high dose animals compared to the low dose animals. There was no major difference in the distribution and excretion of radioactivity with labeling at the phenyl and triazole ring positions, however, there were some different metabolites identified. The studies also showed that administration of 0.5 and 300 mg/kg CGA- 169314 did not induce any treatment related clinical effects.

A.3	Toxicological Endpoints

A.3.1	Acute Population Adjusted Doses (aPAD)  -  All Populations

Selected Study: Acute Neurotoxicity Study in Rats
MRID 46950327
Dose and Endpoint for Establishing an aPAD:  NOAEL is 25 mg/kg/day.  LOAEL is 200 mg/kg/day based on reduced fore-limb grip strength in males on day 1.
Uncertainty Factor (UF): 100 This includes 10x for interspecies extrapolation and 10x for intraspecies variation, 1X FQPA SF.
Comments about Study/Endpoint:  The selected endpoint is considered appropriate for acute dietary exposure because effects were seen after a single dose.  The endpoint is protective of the general population and all subpopulations for effects seen in the acute neurotoxicity study in rats.  It is also protective of developmental and maternal effects observed in the rabbit developmental toxicity study at the LOAEL of 75 mg/kg/day and NOAEL of 25 mg/kg/day.
      General Population aPAD =        (NOAEL) 25 mg/kg    =  0.25 mg/kg
                                                              (UF) 100
General Population aPAD =        (NOAEL) 25 mg/kg    =  0.25 mg/kg
                                                              (UF) 100

A.3.2	Chronic Population Adjusted Dose (cPAD)  -  All Populations

Selected Study: Chronic/Oncogenicity Study in Rats
MRID 42090019/20
Dose and Endpoint for Establishing an cPAD:  The NOAEL is 0.96 mg/kg/day.  The LOAEL is 24.12 mg/kg/day based on cumulative decreases in body weight gains at 24.12 mg/kg/day in males.

General Population  cPAD  =         (NOAEL) 0.96 mg/kg/day    = 0.01 mg/kg/day
		                                          (UF) 100
General Population  cPAD  =         (NOAEL) 0.96 mg/kg/day    = 0.01 mg/kg/day
		                                          (UF) 100

Uncertainty Factor (UF): 100:  This includes 10X for interspecies extrapolation,10x for intraspecies variation, 1X FQPA SF.

A.3.3	Incidental Oral Exposure (Short-Term)

Selected Study:  Two Generation Reproduction Study in Rats
MRID 42090018
Dose and Endpoint for Establishing POD: The NOAEL is 1.25 mg/kg/day based on decreased pup weight in males at 12.5 mg/kg/day (LOAEL) on day 21, and reductions in body weight gain in F0 females.
Uncertainty Factor (UF): An MOE 100 is required for the short- and intermediate-term scenarios for dermal exposure is based on the conventional uncertainty factor of 100.  This includes 10x for interspecies extrapolation and 10x for intraspecies variation.

Comments about Study/Endpoint:  There are no residential uses for difenoconazole that would result in incidental oral exposure to children.  However, a short term oral exposure endpoint is required for aggregate risk assessment.

A.3.4	Dermal Absorption

	A dermal absorption factor (DAF) is applied when dermal exposure endpoints are selected from oral toxicity studies.  The dermal factor converts the oral dose to an equivalent dermal dose for the risk assessment.  A DAF of 6% was selected for use in risk assessment based on available in vivo dermal absorption studies in rat and in vitro dermal absorption studies conducted with rat and human skin (TXR 0056473).

A.3.5	Dermal Exposure (Short and Intermediate-Term)

      Selected Study:  Two Generation Reproduction Study in Rats (MRID 42090018)

      See Section A.4.3

      Dose and Endpoint for Establishing POD: The NOAEL is 1.25 mg/kg/day based on decreased pup weight in males at 12.5 mg/kg/day (LOAEL) on day 21 and reductions in body weight gain in F0 females..  Dermal absorption is 6%.

      Uncertainty Factor (UF): An MOE 100 is required for the short- and intermediate-term scenarios for dermal exposure is based on the conventional uncertainty factor of 100.  This includes 10x for interspecies extrapolation and 10x for intraspecies variation.

      Comments about Study/Endpoint:  Although dermal toxicity studies are available, a POD from an oral study was selected because effects in young animals (decreased pup weight) the primary effect of concern for short, intermediate and long term exposure is not specifically evaluated in the available dermal toxicity studies that only assess adult animals.   The selected endpoint is protective of offspring  effects from dermal exposure.  A DAF of 6% is applied to the POD for dermal exposure.

A.3.6 	Inhalation Exposure (Short- and Intermediate-Term)

      Selected Study:  Two Generation Reproduction Study in Rats (MRID 42090018)

      See Section A.4.3

A.4		EXECUTIVE SUMMARIES FOR SUPPORTING TOXICITY STUDIES

A.4.1	Subchronic Toxicity

	870.3100	90-Day Oral Toxicity  -  Rat MRID 42090022

CGA-169374 Technical was administered orally in feed admixtures to six groups of rats of both sexes at 0 ppm, 20 ppm, 200 ppm, 750 ppm, 1500 ppm, and 3000 ppm for 13 weeks. The results of this dietary subchronic evaluation of the toxicity of the test article were generally unremarkable. There was a significant trend for decreased body weights in both sexes, and the 200 ppm female rats showed an approximate 10% decrease in body weight relative to their controls concomitant with decreased food consumption. There was one dose -- related effect of the chemical discovered during the histopathology examination, that identified modest diffuse hepatocellular enlargement, vis a vis. increased liver weights, in rats of both sexes at the two highest doses tested. Additionally, although not statistically significant, compared to the other groups there was an increase in the frequency and quantity of ketones in the urine of group 6 males. The presence of elevated ketone levels may be due to gluconeogenesis driven by decreased protein intake from the diet as a result of decreased food intake. The somewhat compromised nutritional status of the rats could possibly and indirectly have promoted the hepatocellular enlargement as well.
It is possible to conclude from this study, that based on approximately 10% decrease in body weight in the 200 ppm females (concomitant with a negative trend for food consumption) and increases in absolute liver weights in both sexes appearing at 750 ppm, the LOAEL is 200 ppm. The NOAEL was 20 ppm.
Core Classification: Minimum

	870.3100	90-Day Oral Toxicity  -  Mouse MRID 42090021

CGA 169374 was offered in feed admixtures to five groups of mice composed of 15 animals/group/sex and 20 mice per sex for controls in dietary concentrations of 20 ppm, 200 ppm, 2500 ppm, 7500 ppm, or 15000 ppm for 13 weeks. Most of the mice fed 7500 ppm or 15,000 ppm test article, groups 5 and 6 respectively, died during the first week on study. There were some CGA 169374-related effects. The statistical analysis of total food consumption and body weight changes over the course of the study showed significantly reduced body weight gain for paired group 4 (2500 ppm) females and a significant negative trend. Compound -- related effects from histologic examination were confined to the liver. Hepatotoxicity in mice that DOS was evidenced by hepatocellular enlargement and necrosis of individual hepatocytes. Those mice that survived to the end of the study showed hepatotoxicity that included hepatocellular enlargement in group 4 animals and group 3 males and hepatocytic vacuolization in group 4 animals. Furthermore, coagulative necrosis was observed in the livers of 4/9 group 4 females. This finding, however, was not considered treatment related, because the foci were frequently small and random. The animals in groups 5 and 6, which represent the unscheduled deaths, had a high incidence of changes consistent with stress. The changes included lymphoid depletion or necrosis of the spleen, lymph nodes, and thymus, hypocellularity of the femoral marrow, mucosal erosion/ulceration of the glandular stomach, and in the female mice necrosis of individual cells in the adrenal cortex, specifically in the zona reticularis. Hyperkeratosis of the nonglandular stomach was observed in males especially from group 6. The study director suggests the "stress" effects may be related to inappetence and a failure to eat as opposed to a direct effect of the test article. On the strength of the available data as they relate to the dose levels tested and to the parameters observed, the body weight changes and the liver histopathology form the basis for setting the NOAEL at 20 ppm, and the LOAEL at 200 ppm. The mortality data indicate the MTD was exceeded and is likely S 7500 ppm.

      870.3150 26 Week Oral Feeding study  - dog OPPTS MRID 42090012

CGA 169374 was offered in feed admixtures to five groups of beagle dogs composed of three animals/group/sex in dietary concentrations of 0 ppm, 100 ppm, 1000 ppm, 3000 ppm, or 6000 ppm for a minimum of 28 weeks. None of the dogs DOS. Compound --  related effects, developed essentially at the 3000 ppm and 6000 ppm dose levels. The singularly most striking compound effect was bilateral lenticular cataracts ophthalmoscopically-observed in all dogs at 6000 ppm and in one female beagle at 3000 ppm. Additionally, iridic changes (irregular pupillary margins, miosis), secondary to lens induced uveitis, were also present in the affected animals. There were also reductions in mean body weight in females and males at 6000 ppm test compound throughout the study; weight loss was observed during the first three weeks on study. Body weight loss was precipitated by moderate to severe reductions in mean food consumption in females and males at 6000 ppm during the study with slight reductions observed in males at 3000 ppm and 1000 ppm and in one female at 3000 ppm. Furthermore, there were slight reductions in values for red blood cell count, hemoglobin, and hematocrit in females and males at 6000 ppm. There were also decrements in some serum clinical chemistry measurements including calcium and total protein in females at 6000 ppm and moderate increases in serum alkaline phosphatase in one or both sexes at 3000 ppm. There were modest alterations in several absolute and/or relative organ weight measurements to include the heart, prostate gland, salivary gland, uterus, kidney, liver, and brain at the highest dose tested (HOT). Nevertheless, liver weight measurements were also increased in Group 4 females. There were no other test article -- related changes in any other parameter examined. On the strength of the available data as they relate to the dose levels tested and the parameters observed, the LOAEL and the NOAEL for the test article in female and male beagle dogs were 3000 ppm and 1000 ppm, respectively, based primarily on microscopic examination of CGA 169374-related lenticular cataracts. Core Classification: Minimum

A.4.2	Prenatal Developmental Toxicity

	870.3700a Prenatal Developmental Toxicity Study  -  Rat MRID 42090016

CGA 169347 technical was administered by gavage on days 6-15 of gestation to presumed pregnant rats at 0, 2, 20, 100, or 20a mg/kg. Significant decreases in maternal body weight gain and feed consumption were observed during the dosing period for the feed consumption were observed during the dosing period for the 100 and 200 mg/kg groups. These animals also exhibited a significant increase in the incidence of excess salivation. There was a non-significant decrease in the mean number of fetuses per dam, and non-significant increases in the mean number of resorptions per dam and % postimplantation loss in the 200 mg/kg group. There was a slight (non-significant) decrease in mean fetal body weight at the 200 mg/kg group. The following represents the significant alterations in the development of fetuses in the 200 mg/kg group. The incidence of bifid or unilateral ossification of the thoracic vertebrae was significantly increased on the fetal basis. There were also significant increases in the average number of ossified hyoid and decreases in the average number of sternal centers of ossification (per fetus per litter). The average number of ribs was significantly increased (with accompanying increases in the number of thoracic vertebrae), and decreases in the number of lumbar vertebrae in this group. These findings may be related to maternal toxicity. This study may be upgraded after satisfactory review of the response to the noted deficiencies.
core classification: supplementary.  NOTE: Due to the relatively high percent deviation of the actual doses tested from the theoretical concentration the effect levels have been modified accordingly. This modification may be subject to change as the purity is currently unknown.
Maternal NOAEL = 16 mg/kg; Maternal LOEL = 85 mg/kg; Developmental Toxicity NOAEL = 85 mg/kg; Developmental Toxicity LOAEL = 171 mg/kg

	870.3700b Prenatal Developmental Toxicity Study  -  Rabbit  MRID 42090017

CGA 169347 technical was administered by gavage on days 7 -- 19 of gestation to presumed pregnant rabbits at 0, 1, 25, or 73 mg/kg. Maternal toxicity was observed in this study as the death of one doe and abortions observed in two other high dose does. In addition, significant reductions in body weight gain of high dose does, were present days 7-10, 10 -- 14, 7-20, and 0 -- 29. These reductions correspond with reduced feed consumption during these intervals (significant reductions in feed consumption in the HDT were only observed during the treatment period, not after treatment). Slight non-significant increases in postimplantation loss and resorptions/doe were observed in the HDT. The significant decrease in fetal weight at the HDT may have been due to treatment. The significant differences in fetal weight observed at the low and mid dose were apparently not due to treatment.
Core Classification: supplementary
Maternal NOAEL = 25 mg/kg; Maternal LOEL = 75 mg/kg
Developmental Toxicity NOAEL 25 mg/kg; Developmental Toxicity LOEL = 75 mg/kg

A.4.3	Reproductive Toxicity

	870.3800 Reproduction and Fertility Effects  -  Rat MRID 42090018

In a two generation reproduction study, difenoconazole was administered in the diet to male and female rats at 0, 25, 250, or 2500 ppm [0, 1.25, 12.5, or 125 mg/kg/day, respectively]. Statistically significant reductions in body weight gains of F0 and F1 males were observed at 2500 ppm during Days 70-77 and during the course of the study [terminal body weight minus Day 0 body weight]. Significant reductions in body weight gains of F0 and F1 females were seen during the pre-mating, gestation, and lactation periods. A dose-related, but non-statistically significant decreases in body weight gain was seen in F0 females at 250 ppm during Days 70-77 prior to mating, Days 0-7 of gestation, and Days 7-14 of lactation:
At 2500 ppm, significant reductions in pup body weight were detected on Days 0, 4 [pre- and post culling], 7, 14, and 21 for males and females of both generations. There was a significant reduction in the body weight of F1 male pups on Day 21 in the 250 ppm group. The percentage of male pups in the F1 generation surviving Days 0-4 was significantly reduced in the 2500 ppm group: For parental toxicity, the LOAEL of 250 ppm [12.5 mg/kg/day is based on the decreased maternal body weight gain; the NOAEL is 25 ppm [1.25 mg/kg/day. For offspring toxicity, the LOAEL of 250 ppm [12.5 mg/kg/day] is based on decreased pup weights at Day 21; the NOAEL is 25 ppm [1.25 mg/kg/day].

A.4.4	Chronic Toxicity

        870.4100a (870.4300) Combined Chronic Toxicity/Carcinogenicity  -  Rat MRIDs 42090019/ -20

CGA 169374 was administered in the diet to male and female rats [80/sex/dose] for 104 weeks at 0; 10; 20; 500; and 2500 ppm. There were reductions in cumulative body weight gains in the 500 and the 2500 ppm groups. Mean liver weight was increased at week 53 and t termination in the 2500 ppm group. Hepatocellular hypertrophy was observed in the 500 and the 2500 ppm animals at termination. Additional findings in the clinical chemistry data also indicated that liver was the primary target organ for toxicity. No treatment related increased incidences of neoplastic findings were observed in this study. The NOAEL for the study was 20 ppm which was equal to 0.96 and 127 mg/kg/d for males and females respectively. The LOAEL was 500 ppm equal to 24.12 and 32.79mg/kg/day for males and females respectively based on cumulative decreases in body weight gains.  Discussion of Tumor Data No treatment related increased incidences of neoplastic findings were observed in this study. Adequacy of the Dose Levels Tested The dose levels tested were considered adequate by the Cancer Peer Review Committee. (memorandum of July 27,1994 from B. Rinde of the Health Effects Division)

      870.4100b Chronic Toxicity - Dog  MRID 42090012

CGA 169347 was administered in the diet to male and female dogs at 0, 20, 100, 500, or 1500 ppm. The NOAEL was 100 ppm and the LOAEL was
500 ppm based on the following. Females receiving 1500 ppm in the diet had a significant reduction in body weight gain on day 7. Females in the 500 and 1500 ppm groups, although not statistically significant, had inhibited body weight gain throughout the study. These animals also had significant reductions in food consumption on days 7, 35, 70, and 357. The reduction in mean percent reticulocytes at the highest dose tested on day 359 may have been related to treatment, Significant increases (treatment related at day 85; dose -- related at days 175 and 359) were observed in alkaline phosphatase in males receiving 1500 ppm. This study may be upgraded upon satisfactory review of the registrants response to the deficiencies (submission of the purity and raw daily observation data).
Classification: core -- supplementary

A.4.5	Carcinogenicity

	870.4200a Carcinogenicity/Chronic Study  -  Mice MRIDs 42090015 and 42710006

CD-I mice were fed diets containing difenoconazole at 0; 10; 30; 300; 2500or 4500 [males only] for 78 weeks. The NOAEL was 30 ppm equal to 4.65 mg/kg/d in males and 5.63mg/kg/d in females respectively. The LOAEL was 300 ppm equal to 46.29 mg/kg/d in males and 57.79mg/kg/d in females based on reductions in the cumulative body weight gains at the higher dose levels.

Discussion of Tumor Data: Difenoconazole was reviewed by the HED-CPRC on May 18, 1994 (memorandum of July 27, 1994 from E. Rinde of the HED CPRC to C. Giles-Parker of RD) and classified as a Category C carcinogen without a q-star. The margin-of-exposure (MOE) approach was selected because there was only very weak (limited) evidence of carcinogenic potential at dose levels not considered to be excessive with significant changes observed only at excessive doses. There was no evidence for genotoxicity. There was a statistically significant increase in liver adenomas, carcinomas, and combined liver adenomas and carcinomas in both sexes at doses of 2500 and 4500 ppm. These doses were considered to be excessively high for cancer testing. Liver necrosis and liver adenomas were also noted in males at 300 ppm. There were no statistically significant increases in liver tumors at 10 or 30 ppm.  Adequacy of the Dose Levels Tested: The Health Effects Division Cancer Peer Review Committee considered the doses adequate and the study acceptable.

	870.4200b Carcinogenicity (feeding)  -  Rat MRIDs 42090019/ -20

CGA 169374 was administered in the diet to male and female rats [80/sex/dose] for 104 weeks at 0; 10; 20; 500; and 2500 ppm. There were reductions in cumulative body weight gains in the 500 and the 2500 ppm groups. Mean liver weight was increased at week 53 and t termination in the 2500 ppm group . Hepatocellular hypertrophy was observed in the 500 and the 2500 ppm animals at termination. Additional findings in the clinical chemistry data also indicated that liver was the primary target organ for toxicity. No treatment related increased incidences of neoplastic findings were observed in this study. The NOAEL for the study was 20 ppm which was equal to 0.96 and 127 mg/kg/d for males and females respectively. The LOAEL was 500 ppm equal to 24.12 and 32.79 mg/kg/day for males and females respectively based on cumulative decreases in body weight gains.  Discussion of Tumor Data No treatment related increased incidences of neoplastic findings were observed in this study. Adequacy of the Dose Levels Tested The dose levels tested were considered adequate by the Cancer Peer Review Committee. (memorandum of July 27,1994 from B. Rinde of the Health Effects Division)

A.4.6	Mutagenicity

	Gene Mutation
Guideline # 870.5100 Bacterial assay
42090019, 42710010 Minimum/ guideline
Guideline #870.5300, In vitro mammalian cell gene mutation test
MRID 42090024
Unacceptable Guideline
Not mutagenic

No conclusion can be reached from the three non-activated and two S9 activated mouse lymphoma forward mutation assays conducted with difenoconazole technical. The study was seriously compromised.

Cytogenetics
Guideline # 870.5375,
Clastogenicity in mammalian cells
MRID 46950319, 46950321
Acceptable Guideline
MRID 46950323

Guideline #870.5395
Micronucleus test in bone marrow
MRID 41710011
Acceptable Guideline

Guideline #870.5550
Unscheduled DNA Synthesis in Mammalian Cells in Culture
4210012 (1992)
Acceptable/ guideline
There was evidence of a weak induction of structural chromosomal aberrations over background in the presence of S9-mix.

There was no evidence of structural chromosomal aberrations induced over background. Mice bone marrow - No increase in micronucleated polychromatic erythrocytes occurred with CGA-169374 (91.2% a.i).

CGA-169374 tech. (92.2% a.i.) was considered to be negative in the unscheduled DNA synthesis assay in rat primary hepatocytes as measured by an autoradiographic method at concentrations up to 50.0 ug/mL.

A.4.7	Neurotoxicity

	870.6100 Delayed Neurotoxicity Study  -  Hen - NA

	870.6200 Acute Neurotoxicity Screening Battery  -  Rat MRID 46950327

In an acute neurotoxicity study (MRID 46950327), groups of fasted Alpk:APfSD Wistar-derived rats (10/sex/dose), at least 42 days old, were given a single oral dose of difenoconazole technical (CGA169374) (94.3% w/w, batch/lot # WM806228) in 1% w/v aqueous carboxymethylcellulose (CMC) at doses of 0, 25, 200, or 2000 mg/kg bw and observed for 14 days. Dose levels selected for this study were based on the results of preliminary acute neurotoxicity study (MRID 46950325). Neurobehavioral assessment (functional observational battery and motor activity testing) was performed on 10 animals/sex/group on days -7, 1, 8, and 15. Body weight and food consumption were measured weekly throughout the study. At study termination, 5 animals/sex/group were euthanized and perfused in situ for neuropathological examination; brain weight was recorded from these animals. Of the perfused animals, 5 animals/sex from the control and high dose groups were subjected to histopathological evaluation of brain and peripheral nervous system tissues.

There were no unscheduled deaths at any dose level.  Weight change on the day of dosing by the control, low-, mid-, and high-dose groups was -2.1, - 1.0, -7.8, and -18.3 g, respectively, for males and 0.0, 2.1, -3.8, and -13.0 g, respectively, for females. Body weight for females had recovered to control levels by day 8. Food consumption for males given 2000 mg/kg was approximately 20% less than control during week 1 only (p<0.01). Food consumption for these animals recovered to control levels during week 2. There were no differences from control for females at any dose level or for males at the lower dose levels. These effects on body weight and food consumption were not toxicologically significant.

At 2000 mg/kg, a number of adverse clinical signs were observed on day I (at the time of peak effect), including: upward curvature of the spine (8 males, 9 females); tip-toe gait (3, 8); decreased activity (6, 7); piloerection (3, 5); sides pinched in (3, 7); and subdued (1, 0). Females were affected more than males. All treatment-related clinical signs observed on day 1 showed complete recovery by day 5 (males) or day 7 (females).
Significant decreases in fore-limb grip strength were seen in mid- (23%) and high-dose (26%) males on day 1. Females dosed with 2000 mg/kg had lower motor activities on day 1 (37%), at the time of peak effect, and on day 8 (31%). Males dosed with 200 or 2000 mg/kg had higher motor activities than the controls on day 1, 50% and 55%, respectively, at the time of peak effect. There were no effects on brain weight at any dose level. Neuropathological examination of the central and peripheral nervous system showed no effects of treatment at doses of 2000 mg/kg in both sexes. The LOAEL for acute neurotoxicity of difenoconazole technical (CGA169374) in male rats is 200 mg/kg bw based on reduced fore-limb grip strength in males on day 1. The NOAEL is 25 mg/kg bw. The LOAEL for acute neurotoxicity of difenoconazole technical (CGA169374) in female rats is 2000 mg/kg.  Based on decreased body weight, the following clinical signs: upward curvature of the spine, tip-toe gait, decreased activity, piloerection and sides pinched in, and decreased motor activity. The NOAEL is 200 mg/kg bw.

	870.6200 Subchronic Neurotoxicity Screening Battery

In a subchronic neurotoxicity study (MRID 46950329) difenoconazole technical (94.5% w/w, batch no. WM806228) was administered to groups of 12 male and 12 female Alpk:APfSD (Wistar-derived) rats at concentrations of 0, 40, 250, or 1500 ppm in the diet for 90 days.  Respective dose levels corresponded to 0, 2.8, 17.3 or 107.0 mg/kg bw/day for males and 0, 3.2, 19.5, or 120.2 mg/kg bw/day for females.  Neurobehavioral assessment (functional observational battery and motor activity testing) was performed in 12 animals/sex/group pretest and during weeks 2, 5, 9, and 14.  Cholinesterase activity was not determined.  At study termination, 5 animals/sex/group were euthanized and perfused in situ for neuropathological examination.  Of the perfused animals, 5/sex from the control group and 5/sex from the 1500 ppm group were subjected to histopathological evaluation of brain and peripheral nervous system tissues. Treatment with difenoconazole at concentrations up to 1500 ppm in the diet had no effect on mortality or clinical signs.  Relative to respective control weight, final body weight of males and females in the 1500 ppm group was reduced by 9% and 7%.  Body weight gain was reduced by 22% in males and 23% in females.  Food consumption was reduced in this group (statistically significant only in females [7%]), and food efficiency was significantly reduced in males by 21% (p<=0.05) and in females by 21% (ns).  Lower dose groups were unaffected.  Absolute liver weight in males and females in the 1500 ppm group was increased over respective control weight by 38% and 45%.  Liver was not weighed in lower dose groups.  The increase in liver weight was considered a normal response to chemical treatment.

During weeks 2, 9 and 14, hind-limb grip strength in males in the 1500 ppm group was reduced by 18 to 27% relative to the control values.  At week 14, hind-limb grip strength in males in the 250 ppm group was significantly (p<=0.05) reduced by 20% relative to the control values.  FOB observations in females were unaffected by treatment.  Motor activity was unaffected in both sexes at all observation times.  Brain weight was unaffected by treatment and there were no treatment-related neuropathological lesions.

The LOAEL in male rats is 250 ppm in the diet (17.3 mg/kg bw/day), based on decreased hind limb strength.  The NOAEL is 40 ppm (2.8 mg/kg bw/day). The LOAEL in female rats is 1500 ppm in the diet (120.2 mg/kg bw/day), based on decreased body weight, body weight gain and food efficiency.  The NOAEL is 250 ppm  (19.5 mg/kg bw/day).  The study is classified as Acceptable/Guideline

A.4.8	Metabolism

      870.7485	Metabolism  -  Rat

Study 1
The absorption, distribution, metabolism, and excretion of difenoconazole were studied in groups of male and, female Sprague-Dawley rats.  Animals were administered a single oral gavage dose of 0.5 or 300 mg/kg [[14]C] difenoconazole or 0.5 mg/kg unlabeled difenoconazole by gavage for 14 days followed by a single gavage dose of 0.5 mg/kg [[14]C] difenoconazole on day 15. The test compound was labeled with [[14]C] at either the phenyl or triazole ring.

[[14]C] CCA 169374  was rapidly and extensively distributed, metabolized, and excreted in rats for all dosing regimens. the extent of absorption is undetermined pending determination of the extent of biliary excretion. The 4-day recoveries were 97.94-107.75% of the administered dose for all dosing groups. The elimination of radioactivity in the feces (78.06-94.61% of administered dose) and urine (8.48-21.86%) were almost comparable for all oral dose groups, with slightly higher radioactivity found in the feces of the high-dose group than the low-dose groups. This was probably due to biliary excretion, poor absorption or saturation of the metabolic pathway. The radioactivity in the blood peaked at about 24-48 hours for an dosing group.  Half-lives of elimination appear to be approximately 20 hours for the low-dose groups and 33-48 hours for the high-dose group. The study results also indicate that difenoconazole and/or its metabolites do not bioaccumulate to an appreciable extent following oral exposure since all the tissues contained negligible levels (< 1%) of radioactivity 7 days post exposure.

The metabolism of difenoconazole appears to be extensive because the metabolites accounted for most of the recovered radioactivity in the excreta. Three major metabolites were identified in the feces (i.e. metabolites A, B, and C).  Two of the metabolites were separated into isomers (i.e., Al, A2, B1, and B2).  Metabolite C was detected only in the high-dose groups, indicating that metabolism of difenoconazole is dose-related and involves saturation of the metabolic pathway. Free triazole metabolite was detected in the urine of triazole-labeled groups and its byproduct was detected in the liver of phenyl labeled groups only. Other urinary metabolites were not characterized.

These study results indicate that distribution, metabolism, and elimination of difenoconazole were not sex related.  There was a slight dose-related difference in the metabolism and elimination difenoconazole. In phenyl- and triazole-labeling studies, fecal excretion of radioactivity was higher in the high-dose animals compared to the low-dose animals, and an additional metabolite was found in the feces of the high-dose animals compared to the low-dose animals. There were no major differences in the distribution and excretion of radioactivity with labeling at the phenyl and triazole ring positions, however, there were some different metabolites identified. The studies also showed that administration of 0.5 and 100 mg/kg difenoconazole did not induce any apparent treatment-related clinical effects.

The study is classified as acceptable guideline when considered together with data provided in additional rat metabolism studies (MRIDs 42710014, 42710013) submitted as supplemental to this study. This study may be upgraded if the following additional information is provided and is judged to be acceptable:

Study 2
These studies (MRIDs 42710014, 42710013) were submitted because EPA requested additional information not provided in the Sponsor's previously submitted metabolism studies (MRID Nos. 420900-28/29/30/31). The present studies describe the absorption, distribution, and excretion, as well as pharmacokinetics, of [[14]C] difenoconazole after a single oral gavage dose of 0.5 or 300 mg/kg in rats (Report 1) and isolated and identified urinary metabolites in three females after a single oral gavage dose of 300 mg/kg (Report 2).

Following oral administration of 0.5 or 300 mg/kg [14]C-CCA 169374 in rats, the test compound was adequately absorbed and mainly eliminated via the bile; no evidence of bioaccumulation in any tissue was noted. After 48 hours, total recovery (independent of dose and sex) was ≈ 96% of the administered dose. Biliary excretion constituted the main route of elimination with some dose- and sex-dependency (≈ 75% at the low dose for both sexes; 56% for males and 39% for females at the high dose). Urinary and fecal eliminations exhibited a dose-related pattern at 48 hours. In the urine, 9-l4% was eliminated at the low dose versus 1% in the high-dose rats. In the feces,  2-4% was eliminated at the low dose versus 17-22% at the high dose. In cannulated males after 48 hours, ≈ 80% was eliminated via the bile, while ≈ 4% and ≈ 14% were eliminated via urine and feces, respectively. Therefore, this study indicates that most of the dose following oral administration is absorbed as indicated by the biliary excretion data. The dose-related difference in elimination suggests that saturation is reached at the higher dose level resulting in an increase
of unabsorbed test material.

Maximum concentration in blood was reached within 2 hours at the low dose and 4 hours at the high dose. By 24 hours, <0.05 ppm equivalent was detected in the blood. Total recovery ranged from 95% to 97% after 48 hours, irrespective of dose and sex. During the first 12 hours, slight differences were evident between males and females with regard to Tmax, Cmax, and rate of elimination. The concentration in females was approximately half of that in males and was eliminated faster than in males. Mean half-lives in males and females from Tmax to 12 hours, were 6.2 and 4.4 hours, respectively; from 24 to 168 hours, they were 2.8 and 3.7 days, respectively.

Following administration of 300 mg/kg of ([14]C-phenyl) CGA 169374, 3 major urinary metabolites were identified: sulfate conjugates (and their isomers) of HO-CGA 205375, isomers of HO-CGA 205375, and the hydroxyacetic metabolite of H0-CGA 205373. The major urinary metabolites of CGA 169374 have been identified and no single unknown metabolite accounted for >1.1% of the dose.

 These studies alone do not meet the minimum requirements for Guidelines 85-1. However, these studies combined with previously submitted studies (MRID Nos. 420900-28/29/30/31) are considered to be acceptable,

A.4.9	Immunotoxicity

	870.7800	Immunotoxicity  -  Rat

In an immunotoxicity study (MRID 48696701), difenoconazole (97.4% a.i., Batch # SMO4H493) was administered to female Crl:CD-1 (ICR) mice (10/dose) in the diet at concentrations of 0, 20, 200, 1000, or 1500 pm (equivalent to doses of 0, 3, 35, 177, or 247  mg/kg body weight (bw)/day, respectively) for 28 days.  Animals in the positive control group received cyclophosphamide at a dose of 10 mg/kg bw/day by oral gavage for 28 consecutive days.  On Day 25, animals in all groups were immunized with a suspension of sheep red blood cells (SRBC) by intravenous injection (2x10[8] SRBC/animal, 0.25 mL/animal dose volume).  On Day 29 the animals were sacrificed and blood was collected.  All animals were evaluated for mortality, clinical signs, body weight changes, and food and water consumption. Gross pathology and spleen, thymus, and liver weights were evaluated at necropsy.  Histopathology was performed on the liver and spleen of the vehicle control and treatment groups.  Immunotoxicity was assessed for all animals by an enzyme-linked immunosorbent assay (ELISA) that measured the concentrations of serum anti-SRBC IgM.

There were no treatment-related effects on mortality, clinical signs, food and water consumption, or spleen and thymus weights.  Decreased body weight gains over the course of the study were observed at 1000 and 1500 ppm (-25% and  - 36%, respectively); the differences did not reach statistical significance.  Statistically significant increases in mean absolute and adjusted liver weights were seen at 1000 ppm (+39% and +43%, respectively) and 1500 ppm (+54% for both).  Hepatocyte vacuolation, centrilobular hepatocyte hypertrophy, and increased incidences of pale-colored liver and prominent lobulation of the liver were noted in the 1000 and 1500 ppm groups

The systemic toxicity LOAEL for difenoconazole in female mice is 1000 ppm (equivalent to 177 mg/kg bw/day) based on decreased body weight gains and liver toxicity.  The NOAEL for systemic toxicity is 200 ppm (equivalent to 35 mg/kg bw/day).

For immunotoxicity, decreased anti-SRBC IgM levels were found at 1000 and 1500 ppm (-36% and -51%, respectively) as measured by an ELISA, reaching statistical significance at 1500 ppm.  There were no treatment-related effects on thymus and spleen weights and macropathology or on spleen histopathology.  High inter-individual variability in anti-SRBC antibody levels was noted in all the treatment groups as well as in the control group.  However, evaluation of individual animal showed that 40% of the animals in the 1000 ppm group and 50% of the animals in the 1500 ppm group had values that were below the range of the control group.  The positive control group showed a statistically significant reduction in the anti-SRBC IgM response, confirming the validity of the immunotoxicity assay.

A natural killer (NK) cell activity assay was not performed in this study.  The HED guidance stated that if the test substance produces dose-related suppression of the TDAR (anti-SRBC response), then the test substance is considered as immunotoxic and no further study is required. A NK cell activity assay is not required at this time.

Under the conditions of this study, the LOAEL for immunotoxicity is 1000 ppm (equivalent to 177 mg/kg bw/day) based on decreased mean anti-SRBC IgM levels.  The NOAEL for immunotoxicity is 200 ppm (equivalent to 35 mg/kg bw/day).

This immunotoxicity study is classified acceptable/guideline and satisfies the guideline requirement for an immunotoxicity study (OPPTS 870.7800) in the mouse.

APPENDIX B.  Chemical Names And Structures Of Metabolites

B.1 	Chemical Names And Structures
 Difenoconazole Nomenclature.
Chemical structure

Common name
Difenoconazole
Company experimental name
CGA-169374
IUPAC name
1-({2-[2-chloro-4-(4-chlorophenoxy)phenyl]-4-methyl-1,3-dioxolan-2-yl}methyl)-1H-1,2,4-triazole
CAS name
1-[[2-[2-chloro-4-(4-chlorophenoxy)phenyl]-4-methyl-1,3-dioxolan-2-yl]methyl]- 1H-1,2,4-triazole
CAS registry number
119446-68-3
Chemical structure of
CGA-205375 livestock metabolite

Chemical structure of
1,2,4-Triazole (1,2,4-T)

Chemical structure of Triazolylalanine (TA)

Chemical structure of
Triazolylacetic acid (TAA)

APPENDIX C. Physical/Chemical Properties

Physicochemical Properties of Difenoconazole.
Parameter
Value
Reference
Melting point
78.6 ºC
DP#s 172067 and 178394, 10/26/92, R. Lascola
pH
6-8 at 20 ºC (saturated solution)

Density
1.37 g/cm[3] at 20 ºC

Water solubility
3.3 ppm at 20 ºC

Solvent solubility
	g/100 mL at 25 ºC:
n-hexane:	0.5
1-octanol:	35
toluene:	77
acetone:	88
ethanol:	89

Vapor pressure
2.5 x 10[-10] mm Hg at 25 ºC

Dissociation constant, pKa
pure grade (99.3% +- 0.3%) difenoconazole in water (with 4% methanol) at 20ºC is 1.1
DP# 375159, 5/26/10, B. Cropp-Kohlligian
Octanol/water partition coefficient, Log(KOW)
4.2 at 25 ºC
DP#s 172067 and 178394, 10/26/92, R. Lascola
UV/visible absorption spectrum
max at about 200 and 238 nm (in methanol at 26 ºC)
PMRA Proposed Regulatory Decision Document on Difenoconazole, 4/14/99 (PRDD99-01)

APPENDIX D.  Studies Reviewed for Ethical Conduct

This risk assessment relies in part on data from studies in which adult human subjects were intentionally exposed to a pesticide or other chemical.  These studies were determined to require a review of their ethical conduct, have received that review and have been determined to be ethical.

      The PHED Task Force, 1995.  The Pesticide Handlers Exposure Database, Version 1.1.  Task Force members Health Canada, U.S. Environmental Protection Agency, and the National Agricultural Chemicals Association, released February, 1995.

      The Agricultural Handler Exposure Task Force (AHETF), 2011. The Occupational Handler Unit Exposure Surrogate Reference Table.  U.S. Environmental Protection Agency.  Released June 21, 2011.

Klonne, D. (1999) Integrated Report for Evaluation of Potential Exposures to Homeowners and Professional Lawn Care Operators Mixing, Loading, and Applying Granular and Liquid Pesticides to Residential Lawns:  Lab Project Number:  OMA005: OMA001: OMA002.  Unpublished study prepared by Riceerca, Inc., and Morse Laboratories.  2213 p. (MRID 44972201).

The PHED Task Force, 1995.  The Pesticide Handlers Exposure Database, Version 1.1.  Task Force members Health Canada, U.S. Environmental Protection Agency, and the National Agricultural Chemicals Association, released February, 199