Document ID: EPA-HQ-OPP-2012-0445-0027
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2020-02-04T05:00Z

DATA EVALUATION RECORD
                                       
                                       
                                   BROMACIL
                                       
      Study Type:  OCSPP Non-Guideline; In Vitro Dermal Absorption Study
                             (Human and Rat Skin)
                                       
                 Work Assignment No. 32-23-035 (MRID 50753901)
                                       
                                       
                                 Prepared for
                            Health Effects Division
                         Office of Pesticide Programs
                     U.S. Environmental Protection Agency
                           2777 South Crystal Drive
                              Arlington, VA 22202
                                       
                                       
                                  Prepared by
                                       
                        3201 Jermantown Rd., Suite 400
                               Fairfax, VA 22030

Primary Reviewer:
                                                                     Signature:
                                       
Scott D. Studenberg, Ph.D., DABT
                                                                          Date:
                                  05/16/2019
Secondary Reviewer:
                                                                     Signature:
                                       
Michael E. Viana, Ph.D., DABT
                                                                          Date:
                                  05/21/2019
Quality Assurance:
                                                                     Signature:
                                       
Sarah E. Saucier, Ph.D.
                                                                          Date:
                                  05/23/2019
Project Manager:
                                                                     Signature:
                                       
Michael E. Viana, Ph.D., DABT
                                                                          Date:
                                  05/24/2019

                                  Disclaimer

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by CDM/CSS-Dynamac Joint Venture personnel.  Contractor's role did not include establishing Agency policy.
 
EPA Reviewer:     Anwar Y. Dunbar, Ph.D.   	Signature: 	
Risk Assessment Branch I, Health Effects Division (7509P)	Date:        09/27/19	

EPA Secondary Reviewer:    Monique Perron, D.Sc. 	Signature: 	
Risk Assessment Branch I, HED (7509P)	                                         Date:      09/27/2019	
	Template version 02/06

                            DATA EVALUATION RECORD

STUDY TYPE:	In Vitro Dermal Absorption Study (Human and Rat Skin), OCSPP Non-Guideline; OECD 428.

PC CODE:  012301	DP BARCODE:  451257
TXR #:  0057955

TEST MATERIAL (RADIOCHEMICAL PURITY):  [[14]C]-bromacil (100%)

SYNONYMS:  5-bromo-6-methyl-3-(1-methylpropyl)-2,4(1H,3H)-pyrimidinedione

CITATION:	Estigoy, L.L. and Reifenrath, W.G. (2018) Skin penetration of [[14]C]-bromacil after topical application of Bayer Hyvar X Formulation to excised human skin, and Bayer Hyvar X-L and Alligare Bromacil 80 WDG formulation to excised rat and human skin.  EAG Laboratories, Hercules, CA.  Laboratory Study No.: 3051W, November 15, 2018.  MRID 50753901.  Unpublished.

SPONSOR:	ADAMA USA, 3120 Highwoods Boulevard, Suite 100, Raleigh, NC

EXECUTIVE SUMMARY:  In an in vitro dermal absorption study (MRID 50753901), [[14]C]-bromacil (100% radiochemical purity; Lot #: 86929-1-31-1) was applied to human and rat skin membranes in flow-through diffusion cells for exposure periods of 8 h with receptor fluid sample collection through 24 h post-application.  All skin membranes were washed at 8-h post-application.  The target doses for the dilutions were 42 (low), 250 or 420 (intermediate), and 1369 or 1375 ug/cm[2] (high), respectively.

After single applications of [[14]C]-bromacil in three different formulations applied at target doses of 42 ug/cm[2], 250 or 420 ug/cm[2], and 1369 or 1375 ug/cm[2] to human and rat skin samples, mean total recoveries of 88-105% and 93-106% for human and rat skin, respectively, were obtained with no discernible differences between formulations or species.

Potentially-absorbable bromacil was reported as the sum of radioactive residues present in the receptor fluid and the dermis.  Mean percutaneous absorption of [[14]C]-bromacil in the formulations through human skin ranged from 0.4% to 3.8%, with mean absorption through rat skin ranging from 2.0-7.8%.  Radioactive materials removed by washing at 8 h, recovered from the surface (tape strips 1-2) at the end of the exposure period, and in the epidermis were considered unabsorbed.  Most of the radioactive residues were recovered in the skin washes at 8 h post-application, with mean percentage values of 85-98% and 81-98% for human and rat skin, respectively.  The unabsorbed dose remaining after the skin wash accounted for 0.2-5.0% and 0.7-14.4% for human and rat skin, respectively.

For human skin samples, there was no discernible pattern between dose level and the percentage of absorbable dose across the three formulations.  There also was no discernable difference in absorption between the three formulations.  For rat skin samples, greatest absorption was observed at the low dose for either tested formulations; percentage absorption was similar between the intermediate- and high-dose levels.  Absorption was greater from the Hyvar X-L formulation relative to the 80 Bromacil Alligare WDG formulation.  It was stated that an effect of dose (p=0.02) was noted for rat skin but no effect was observed for the percutaneous absorption values.

Under current practices, the reviewer's calculations differed slightly from the study author's.  The reviewer added both receptor fluid values in addition to both the dermis and the epidermis values.   The EPA reviewer further considered the doses were the radioactivity seemed to still be moving through the skin at the end of the exposure period.  Throughout the study, the highest amounts absorbed were in the 42 ug/cm[2] low dose groups based upon the amounts recovered in the receptor fluid.  These doses were therefore considered the most appropriate for risk assessment.  There was no in vitro data calculated for the Hyvar X formulation for the rat.  The amounts calculated are as follows:
    
Human at 42 ug/cm[2]: Hyvar X-L- 3.2%, Alligare WDG- 3.05% and Hyvar X- 2.52%
    
Rat at 42 ug/cm[2]: Hyvar X-L- 10.93% and Alligare WDG- 8.64%
    
It is also worth noting that the study director included both tape strips one and two in their dermal absorption calculation.  Under current practices, the agency does not include these two tape strips.  The reviewers did look at the amount of radioactivity present on the two tape strips, and determined that the amounts didn't change the overall absorption data significantly and thus were left in place in this instance.  For the triple pack calculations the ratios of the human to the rat values for each formulation can be used along with the appropriate in vivo rat data to generate the dermal absorption factor for bromacil. 

This study is classified acceptable / non-guideline and it demonstrates the comparative in vitro percutaneous absorption of [[14]C]-bromacil in up to three different formulations through human and rat skin samples.

COMPLIANCE:  Signed and dated Data Confidentiality, GLP Compliance, Flagging, and Quality Assurance statements were provided.  It was stated that the present study was conducted in compliance with the current US EPA FIFRA GLP Standards (40 CFR Part 160) that are compatible with the corresponding European Commission Regulations and JMAFF GLP (11 Nousan No. 6283). 
I.	MATERIALS AND METHODS

A.	MATERIALS

1.	Test material

    Radiolabeled test material:
[2-[14]C]-Bromacil

 Radiochemical purity:
100% (HPLC)

 Specific activity:
58 mCi/mol

 Lot No.:
86929-1-31-1

 Expiration / Storage:
Not reported / −20°C or below

 Structure:

* denotes position of radiolabel

 Non-radiolabeled TGAI:
Bromacil Pestanal

 Description:
Not provided

 Batch #:
SFBF139XV

 Purity:
98.5%

 CAS # of TGAI:
314-40-9

 Expiration / Storage:
Approximately five years / room temperature

 Structure:

 Formulated product 1:
80 Bromacil Alligare WDG

 Description:
Granular

 Lot #:
PM16011344

 Purity:
80%

 Expiration / Storage:
Not provided

 Formulated product 2:
Hyvar X

 Description:
Granular

 Lot #:
JUN25LE020

 Purity:
80%

 Expiration / Storage:
Not provided
 
 Formulated product 3:
Hyvar X-L
 
 Description:
Liquid

 Lot #:
APR15LE002

 Purity:
21.9% (21.4% according to statement of formula)

 Expiration / storage
Not provided

2.	Relevance of test material to proposed formulation(s):  Bromacil is a commercially available broad spectrum, non-selective herbicide used as a weed killer  The proposed formulations represent mixtures of [[14]C]-labeled bromacil combined with three non-labeled bromacil commercial formulations (granular or liquid) to yield the desired concentrations.  

3.	Test systems

 Species:
Human skin (donor sex not provided)

 Source:
Full-thickness skin samples obtained from the Cooperative Human Tissue Network (address not provided)

 Age:
Not provided

 Origin/Anatomical site:
Not provided

 

 Species:
Rat (males only)

 Strain:
Sprague-Dawley [SPF]

 Age / mean weight at euthanasia:
9 weeks / 250-300 g

 Source:
Charles River Laboratories (Hollister, CA)

B.	TEST SYSTEM AND STUDY DESIGN

1.	Dose

    Dose selection rationale:  It was stated that the dosages selected bracketed the range of the minimum skin deposition that allowed reliable determination of dermal absorption with undiluted radiolabel and the maximum deposition likely to occur on human skin.  The dosages were intended to simulate human exposure to undiluted Hyvar X-L liquid or aqueous tank mixes of Hyvar X, Hyvar X-L, and 80 Bromacil Alligare WDG.  The three formulations were each prepared at three concentrations.  The contractor observed that the study protocol stated that all intermediate dose concentrations would be 420 ug bromacil/cm[2].  In the summary and study design sections (pages 13-15 of MRID 50753901) this dose was stated as being changed to 250 ug bromacil/cm[2] for the Hyvar X-L intermediate dose to align with the dose used in a companion in vivo study.  Calculations conducted by the contractor reviewers based on the formulation preparation data and applied dpm data for each pertinent run do not support this dose level.  See Table 1 and accompanying spreadsheet calculations for apparent doses.  The Hyvar and 80 Bromacil Alligare WDG granular formulations were diluted to 1375, 420, and 42 ug bromacil/cm[2], respectively. 

    High-dose Hyvar X-L preparation:  [[14]C]-bromacil (228 μL, 5.91 x 10[8] dpm) was transferred to a 10-mL tube and evaporated to dryness under nitrogen.  A 1655.17-mg portion of the Hyvar X-L formulation was weighed and added to the radiolabeled material.  The contractor reviewers prepared calculations for all dosing formulations in an attempt to determine actual concentrations and amounts of bromacil administered for each experiment.  As the X-L formulation used was reported in mg, a conversion of 1 mg = 1 uL was estimated for the volume calculations (see the accompanying spreadsheet created by the contractor). The preparation was vortexed for approximately one min and placed on the stir plate for one h.  The gross weight of the preparation was determined and three 5-μL portions (approximately 5 mg each) were counted with a liquid scintillation counter (LSC) to determine the radioactivity count (dpm).] 

    Intermediate-dose Hyvar X-L preparation:  he contractor reviewers stated that following preparation yields the originally planned 420 ug/cm[2] dose formulation.]  [[14]C]-bromacil (228 μL, 5.91 x 10[8] dpm) was transferred to a 10-mL tube and evaporated to dryness under nitrogen.  A 503.94-mg portion of the Hyvar X-L formulation was weighed and added to the radiolabeled material, followed by three 500-μL water washes with additional water to yield a total weight of 1603.46 mg.  The preparation was vortexed for approximately one min and placed on the stir plate for one h.  The gross weight of the preparation was determined and three 5-μL portions (approximately 5 mg each) were counted by LSC.  The contractor reviewers noted that to achieve the reported target doses, the following dose preparation section was re-calculated by the Reviewers (see accompanying spreadsheet).]  [[14]C]-bromacil (228 μL, 5.91 x 10[8] dpm) was transferred to a 10-mL tube and evaporated to dryness under nitrogen.  A 302.40-mg portion of the Hyvar X-L formulation was weighed and added to the radiolabeled material, followed by three 500-μL water washes with additional water to yield a total weight of 1603.46 mg.  The preparation was vortexed for approximately one min and placed on the stir plate for one h.  The gross weight of the preparation was determined and three 5-μL portions (approximately 5 mg each) were counted by LSC.

    Low-dose Hyvar X-L preparation:  [[14]C]-bromacil (228 μL, 5.91 x 10[8] dpm) was transferred to a 10-mL tube and evaporated to dryness under nitrogen.  A 45.49-mg portion of the Hyvar X-L formulation was weighed and added to the radiolabeled material, followed by three 500-μL water washes with additional water to yield a total weight of 1647.84 mg.  The preparation was vortexed for approximately one min and placed on the stir plate for one h.  The gross weight of the preparation was determined and three 5-μL portions (approximately 5 mg each) were counted by LSC.

    High-dose 80 Bromacil Alligare WDG preparation:  80 Bromacil Alligare WDG (455.06 mg) was weighed into a tube and combined with [[14]C]-bromacil (228 μL, 5.91 x 10[8] dpm) and 200 μL acetone.  The solution was vortexed and evaporated to dryness under nitrogen.  A one-mL portion of water was added to the tube, the tube was vortexed, and the remaining water (2,320 μL) was added.  The preparation was vortexed for approximately one min and placed on the stir plate for one h.  The gross weight of the preparation was determined and three 5-μL portions (approximately 5 mg each) were counted by LSC to determine the radioactivity count (dpm).

    Intermediate-dose 80 Bromacil Alligare WDG preparation:  80 Bromacil Alligare WDG (138.02 mg) was weighed into a tube and combined with [[14]C]-bromacil (228 μL, 5.91 x 10[8] dpm) and 200 μL acetone.  The solution was vortexed and evaporated to dryness under nitrogen.  A one-mL portion of water was added to the tube, the tube was vortexed, and the remaining water (2,320 μL) was added.  The preparation was vortexed for approximately one min and placed on the stir plate for one h.  The gross weight of the preparation was determined and three 5-μL portions (approximately 5 mg each) were counted by LSC to determine the radioactivity count (dpm).

    Low-dose 80 Bromacil Alligare WDG preparation:  80 Bromacil Alligare WDG (12.50 mg) was weighed into a tube and combined with [[14]C]-bromacil (228 μL, 5.91 x 10[8][ ]dpm) and 200 μL acetone.  The solution was vortexed and evaporated to dryness under nitrogen.  A one-mL portion of water was added to the tube, the tube was vortexed, and the remaining water (2,320 μL) was added.  The preparation was vortexed for approximately one min and placed on the stir plate for one h.  The gross weight of the preparation was determined and three 5-μL portions (approximately 5 mg each) were counted by LSC to determine the radioactivity count (dpm).

    High-dose Hyvar X preparation:  Hyvar X (21.97 mg) was weighed into a vial and combined with [[14]C]-bromacil (11 μL, 2.85 x 10[7] dpm).  The solution was evaporated to dryness under nitrogen.  A 160-uL portion of water was added to the tube and the preparation was placed on the stir plate for one h.  The gross weight of the preparation was determined and three 5-μL portions (approximately 5 mg each) were counted by LSC to determine the radioactivity count (dpm).  The preparation was mixed on the stir plate for a second h due to poor homogeneity and re-counted.

    A second Hyvar X high-dose formulation was prepared by weighing 43.85 mg of Hyvar X into a vial combined with [[14]C]-bromacil (22 μL, 5.70 x 10[7] dpm) and 100 μL of acetone. The preparation was evaporated to dryness under nitrogen and approximately 300 μL of water was added to the vial and the vial was sonicated, vortexed, and stirred for one h.  The contractor noted that for the high and intermediate formulation repeat preparations, the volume is reported as approximately 300 uL.  The exact volume is unknown, but 300 uL was used in the accompanying spreadsheets to estimate the concentrations prepared.  The gross weight of the preparation was determined and three 5-μL portions (approximately 5 mg each) were counted by LSC to determine the radioactivity count (dpm).]

    Intermediate-dose Hyvar X preparation:  Hyvar X (6.70 mg) was weighed into a vial and combined with [[14]C]-bromacil (11 μL, 2.85 x 10[7] dpm).  The solution was evaporated to dryness under nitrogen.  A 160-uL portion of water was added to the tube and the preparation was placed on the stir plate for one h.  The gross weight of the preparation was determined and three 5-μL portions (approximately 5 mg each) were counted by LSC to determine the radioactivity count (dpm).  The preparation was mixed on the stir plate for a second h due to poor homogeneity and re-counted.

    A second Hyvar X intermediate-dose formulation was prepared by weighing 13.31 mg of Hyvar X into a vial combined with [[14]C]-bromacil (22 μL, 5.70 x 10[7] dpm) and 100 μL of acetone. The preparation was evaporated to dryness under nitrogen and approximately 300 μL of water was added to the vial and the vial was sonicated, vortexed, and stirred for one h.  The gross weight of the preparation was determined and three 5-μL portions (approximately 5 mg each) were counted by LSC to determine the radioactivity count (dpm).

    Low-dose Hyvar X preparation:  Hyvar X (1.23 mg) was weighed into a vial and combined with [[14]C]-bromacil (22 μL, 5.70 x 10[7] dpm).  The preparation was evaporated to dryness under nitrogen and 320 μL of water was added to the vial and the vial was stirred for one h.  The gross weight of the preparation was determined and three 5-μL portions (approximately 5 mg each) were counted by LSC to determine the radioactivity count (dpm).  The preparation was mixed on the stir plate for a second h due to poor homogeneity and re-counted.
    
    Nominal doses:  1369 ug/cm[2], 250 ug/cm[2] (see the contractor reviewer's comments above), and 42 ug/cm[2] for the Hyvar X-L formulation and 1375 ug/cm[2], 420 ug/cm[2], and 42 ug/cm[2] for the 80 Bromacil Alligare WDG and Hyvar X formulations.

    Radiochemical purity (%):  99.4-100% (Hyvar X-L); 98.7-98.9% (80 Bromacil Alligare WDG); 99.0-99.5% (Hyvar X)

    Percentage dpm recovery (%):  100.5-104.1% (Hyvar X-L); 94.6-101.0% (80 Bromacil Alligare WDG); 93.7-114.4% (Hyvar X, initial preparations); 96.8-105.3% (Hyvar X, repeat preparations)

    Homogeneity (% CV; calculated by the contractor reviewers):  Not reported

    Stability:  Not reported

    Dose volume:  4-5 uL (5-6.25 uL/cm[2]) for the Hyvar X-L formulation and 10-12 uL (12.5-15 uL/cm[2]) for the 80 Bromacil Alligare WDG and Hyvar X formulations.

    Termination periods:  24 h

    Number of skin preparations/dose group/dose duration
        Human: eight
        Rat: six

TABLE 1.	Dosing [a]
                                     Level
                             Termination time (h)
                                  Theoretical
                                    Actual
                                       
                                       
                             Dose a.i. (ug/cm[2])
                            Amount a.i. (ug-equiv)
                           Dose a.i. (ug/cm[2]) [b]
                          Amount a.i. (ug-equiv) [b]
                        Specific activity (dpm/ug) [b]
                              Bromacil Hyvar X-L
                                   High [c]
                                      24
                                     1369
                                     1095
                                     1122
                                     897.6
                                     1995
                                   High [d]
                                      24
                                     1369
                                     1095
                                     1122
                                     897.7
                                     1995
                               Intermediate [c]
                                      24
                                    420 [l]
                                      336
                                     366.4
                                     293.1
                                     3619
                               Intermediate [e]
                                      24
                                    420 [l]
                                      336
                                     422.6
                                     338.1
                                     3619
                               Intermediate [f]
                                      24
                                    420 [l]
                                      336
                                     338.1
                                     270.5
                                     4553
                               Intermediate [g]
                                      24
                                    250 [m]
                                      200
                                     221.4
                                     177.1
                                     5988
                               Intermediate [h]
                                      24
                                    250 [m]
                                      200
                                     255.4
                                     204.3
                                     5988
                               Intermediate [i]
                                      24
                                    250 [m]
                                      200
                                     204.3
                                     163.4
                                     7534
                                    Low [c]
                                      24
                                      42
                                     33.6
                                     41.1
                                     32.9
                                     54585
                                    Low [j]
                                      24
                                      42
                                     33.6
                                     41.93
                                     33.55
                                     54548
                                    Low [k]
                                      24
                                      42
                                     33.6
                                     83.87
                                     104.8
                                     34911
                           80 Bromacil Alligare WDG
                                   High [c]
                                      24
                                     1375
                                     1100
                                     1471
                                     1177
                                     1516
                                     High
                                      24
                                     1375
                                     1100
                                     1513
                                     1210
                                     1516
                               Intermediate [c]
                                      24
                                      420
                                      336
                                     457.9
                                     366.3
                                     4867
                                 Intermediate
                                      24
                                      420
                                      336
                                     462.3
                                     369.8
                                     4866
                                    Low [c]
                                      24
                                      42
                                     33.6
                                     42.51
                                     34.01
                                     52521
                                    Low [e]
                                      24
                                      42
                                     33.6
                                     42.15
                                     33.72
                                     52440
                                    Low [f]
                                      24
                                      42
                                     33.6
                                     50.58
                                     40.47
                                     43879
                                Bromacil Hyvar
                                   High [c]
                                      24
                                     1375
                                     1100
                                     1409
                                     1127
                                     1584
                                     High
                                      24
                                     1375
                                     1100
                                     1405
                                     1124
                                     1584
                               Intermediate [c]
                                      24
                                      420
                                      336
                                     442.4
                                     353.9
                                     4998
                                 Intermediate
                                      24
                                      420
                                      336
                                     444.5
                                     355.6
                                     4996
                                    Low [c]
                                      24
                                      42
                                     33.6
                                     41.93
                                     33.55
                                     52966
                                      Low
                                      24
                                      42
                                     33.6
                                     42.93
                                     34.34
                                     52794
                                 High  repeat
                                      24
                                     1375
                                     1100
                                     1496
                                     1197
                                     1488
                              Intermediate repeat
                                      24
                                      420
                                      336
                                     470.9
                                     376.7
                                     4716
 a	Data were obtained on pages 15-16, 20-22, 31-33, and 87-142 (Appendix C) of MRID 50753901.
 b	Values calculated by the Reviewers from individual data.
 c	Run 1 for human (based on reported dose preparation).
 d	Runs 2 through 8 for human and 1 through 6 rat.
 e	Runs 2 through 6 for human and 1 through 6 for rat (based on reported dose preparation).
 f	Runs 7 and 8 for human only (based on reported dose preparation).
 g	Run 1 for human (re-calculated).
 h	Runs 2 through 6 for human and 1 through 6 for rat (re-calculated).
 i	Runs 7 and 8 for human only (re-calculated).
 j	Runs 2 through 8 for human and 1 through 6 rat except for a single replicate in Run 3 (rat).
 k	Single replicate in Run 3 for rat.
 l	Although the study design reported stated that the dose of the intermediate Hyvar X-L formulation would be 250 ug/cm[2], the formulation data presented yield a dose of 420 ug/cm[2] (reported in the study protocol).
 m	Re-calculated (by the Contractor Reviewers) intermediate Hyvar X-L formulation preparation data do yield a 250 ug/cm[2] dose formulation as reported in MRID 50753901(see Reviewer comments).

2.	Receptor fluid:  The selected tissue culture medium was RPMI formula 1640 modified with L-glutamine, sodium bicarbonate, 4% bovine serum albumin, and no phenol red.  The medium was prepared and used within one week.

    Solubility of bromacil in the receptor fluid under the proposed study conditions was demonstrated as follows.  The aqueous solubility of bromacil was stated to be 815 mg/L (815 ug/mL).  At the proposed receptor fluid flow rate of approximately 2 mL/h over 24 h, the total volume of receptor fluid (rounded up to 50 mL) could solubilize 40,750 ug bromacil (815 ug/mL x 50 mL).  It was calculated that one hundred percent absorption of the highest dose administered (1375 ug/cm[2]) would yield 1100 ug of bromacil over the 24 h period which is <3% of the potential solubility capacity of the receptor fluid for bromacil.

3.	Skin preparation

    Human:  Fresh full-thickness human skin samples (within 24 h of excision) were obtained as surgical discards from the Cooperative Human Tissue Network (CHTN).  Each sample was stored on gauze containing RPMI medium and gentamycin sulfate and shipped overnight on wet ice (approximately 4°C).  Each sample was prepared and used on the date of receipt.

    Rat:  Fresh skin samples were obtained from male Sprague-Dawley rats (SPF, approximately nine weeks old and 250-300 g [Charles River Laboratories, Hollister, CA]).  Rats were euthanized with pentobarbital/phenytoin and full-thickness skin strips (approximately 4 in x 4 in) were excised from the upper dorsum of each animal after clipping.  Skin strips were folded (visceral side to visceral side) and shipped on wet ice.  After dermatoming and cutting of disks (below), the prepared disks were stored on gauze containing RPMI medium and gentamycin sulfate overnight at approximately 4°C for use the following day (within 24 h of receipt).

    Dermatome procedure:  Full-thickness skin samples were dermatomed with an electrodermatome to yield sections that were approximately 200 to 400 um thick.  Disks of skin from these sections were cut with a #15 cork borer.

4.	Study design

    Diffusion cell:  An in vitro skin penetration (with or without an evaporation apparatus) system was used to allow measurement of potential evaporation from the skin and penetration through the skin.  A diagram of the overall diffusion apparatus was included as Figure 1 of the study protocol in Appendix A on page 75 of MRID 50753901 and is included as Appendix I at the end of this DER.  A stainless steel diffusion cell (with or without an evaporation apparatus) was used; diagrams of the diffusion cell and evaporation apparatus were included as Figures 2 and 3, respectively, of the study protocol in Appendix A on pages 76-77 of MRID 50753901 and are included as Appendix II at the end of this DER.  The donor chamber had a volume of approximately 1 mL and was unoccluded (in the absence of the evaporation apparatus).  The skin disk was positioned on the penetration (receptor) cell and the donor cell was clamped to the penetration cell with the skin disk in the center and an exposed area of 0.8 cm[2].  Radiolabeled material  penetrating the skin was collected by serial fractionation during each 24-h test period.  The target receptor fluid flow rate was 2 mL/h (range 1-3 mL/h).  Water baths were used to supply water to the cell holders (target 37°C) and evaporation cells (target 17°C); actual temperatures and humidity levels were monitored (44-66%).  It was stated that actual skin temperatures were 30-32°C which was considered normal.

    Membrane integrity:  Membrane integrity was determined by tritiated water ([3]H2O) penetration.  [3]H2O (125 μL; approximately 190,000-200,000 dpm) was applied to the membrane surface and fractions were collected over 1-h intervals for a total of 3 h.  At the conclusion, [3]H2O remaining on the surface of the membrane was removed (further details regarding the test procedure were not provided).  Radioactivity was determined in the receptor fluid samples by LSC.  It was stated that if the radioactivity in the perfusate collected during the first hourly interval was >3000 dpm, the skin sample was not used.

    Dose determination:  Quality control portions (6.4 μL) of each dose formulation were collected throughout application.  The samples were diluted to a volume of 10 mL with acetonitrile and duplicate 0.25-mL portions were analyzed by LSC.  No further details were provided, but identical doses (dpm) within each dose formulation level for a given dose volume were reported as delivered across runs and both species.

    Absorption and distribution:  For the initial (pilot) experiment with human skin (Human Run 1), evaporation cells were attached to the tops of the diffusion cells to trap potential volatile gases.  The adsorbent in the vapor traps (Tenax TA) was collected and replaced with fresh adsorbent at 2, 4, 8, 22, and 24 h.  Trapped radiolabeled material in the adsorbent was determined by LSC.  Because the percentage of recovered radioactive material was <=0.6% of any applied dose, evaporation cells were not used for any further human or rat skin sample experiments.

    Including the pilot experiment, human skin samples from eight different donors were used to test 8-9 of the nine formulations.  Rat skin samples from six different animals were used to test the Hyvar X-L and 80 Bromacil Alligare WDG formulations only (Hyvar X formulations were not tested on rat skin samples).  Four- to 12-μL portions of the formulations were applied with dedicated 5-μL blunt-tipped Hamilton syringes or a micro-pipette.  No data regarding analysis of syringes or pipette tips by LSC after dosing to determine residual radioactivity were presented.

    Receptor fluid samples exiting the receptor cells were collected at four 1-h intervals (1-4 h), four 2-h intervals (6, 8, 10, and 12 h), and three 4-h intervals (16, 20, and 24 h) with a fraction collector.

    At 8 h post-application, the donor chamber was removed from each skin surface and membranes were washed successively with three cotton wipes containing liquid Ivory soap, three cotton wipes wetted with distilled water, and two dry cotton wipes.  Wipes were placed in separate vials containing scintillant for LSC analysis.  After washing, each donor chamber was replaced and reclamped for the remainder of the 24-h test period.

    At termination (24 h post-application), each skin disk was removed, placed dermis side down on a cutting board protected with plastic film, pinned, and tape-stripped (human skin samples only) twice with Highland Invisible tape (3M, Minneapolis, MN).  The tape strips were placed in vials containing tetrahydrofuran for dissolution followed by addition of scintillant and counting by LSC.  Each skin was unpinned and the epidermis was covered with a polyvinyl film (Stretch-tite plastic food wrap for microwave ovens, Sutton, MA).  A heated (65°C) brass weight (200 g) was pressed against the epidermis for 90 sec and the epidermis was separated from the dermis with forceps.  Each epidermis and dermis were placed in separate LSC vials, solubilized with approximately l mL of tissue solubilizer (Soluene 350, Packard Instruments) heated to 50-65°C for approximately 1 h or until dissolved, mixed with scintillant, and counted by LSC. The film used for heat separation and the underlying film were placed in separate vials, mixed with scintillant, and counted by LSC.  For rat skin samples, each epidermis was pinned on a cutting board protected with plastic film, peeled from the underlying dermis with forceps, and the separated epidermis, dermis, and plastic were treated as described for human skin samples.

    After collection of the 20-24 h receptor fluid sample at termination, the remaining volume of receptor fluid in each penetration cell also was collected.  All receptor fluid samples were combined with scintillant and counted by LSC.  Each donor cell was wiped with a cotton-tip applicator wetted with ethanol and a second applicator that was dry.  Each penetration cell was wiped with two successive dry cotton-tip applicators.  Each wipe was placed in separate vial containing scintillant for LSC analysis.

    Quantification:  Radioactive residues were measured by LSC.  No further details regarding sample handing prior to counting, count time, or background/limit of detection were provided.  

    An HPLC method with radiodetection was used for radiochemical purity checks.  Details of the method were provided on page 23 of MRID 50753901 and are included as Appendix III at the end of this DER.

5.	Statistical analysis:  The results of replicate measurements were presented as mean values.  Summary data was presented as mean (+- standard deviation [SD]).  It was stated that the variability of the data was expressed as the coefficient of variation (CV).  Statistical analyses were conducted with BMDP software (Bio-Medical Data Package, BMDP Statistical Software, Los Angeles, CA).  If a significant F occurred by analysis of variance (ANOVA), a multiple comparison test (Student-Newman-Keuls multiple-range test) was used to determine the combinations of variables that were significant at p = 0.05.

II.	RESULTS

A.	EPIDERMAL INTEGRITY AND SELECTION

1.	Human:  For human skin sample #1, cell H was not used because the overall [3]H-H2O recovery was low (5%) and appeared to have been mis-dosed.  For human skin sample #2, cell B was not used because the radioactivity in the perfusate collected during the first hourly interval was 73,944 dpm (>3000 dpm).  Another cell (cell F) was used but showed counts of 3942 and 3575 dpm during the first and second hourly collections, respectively (no reason was provided).  For human skin sample #3, cell H was not used because the radioactivity in the perfusate collected during the first hourly interval was 98,796 dpm.  All other human skin sample disks met the selection criterion.

2.	Rat:  For rat skin sample #5, cells B, H, and I were not used because the radioactivity in the perfusates collected during the first hourly interval were 3725 dpm, 3980 dpm, and 6544 dpm, respectively.  A fourth cell (cell G) had a count of 2546 dpm during the first interval but was not used (no reason was provided).  For rat skin sample #6, cell G was not used because no penetration was observed through the skin (0-1 dpm) during the 3-h collection period.

B.	DISTRIBUTION OF RADIOACTIVE RESIDUES

1.	Human:  Data for human skin (mass and percentage of the applied doses) were calculated by the Reviewers and are presented in Tables 2a (Hyvar X-L formulations), 2b (80 Bromacil Alligare WDG formulations), and 2c (Hyvar X formulations).  The percentage data were similar to summary results reported in Tables 5-8 on pages 35-38 of MRID 50753901; these tables are included as Appendices IV and V at the end of this DER.

a.	Hyvar X-L

i.	Low dose:  Following application of the target dose of 42 ug/cm[2] [[14]C]-bromacil (41.0 or 41.9 ug/cm[2]) to human skin, 1.8% of the applied dose was recovered in the receptor fluid over the 24-h period with an additional 0.2% remaining in the receptor fluid at termination.  Radioactive residues in the vapor traps (from Run 1 only) accounted for 0.3% of the dose.  Radioactive residues in the dermis accounted for 0.5% of the dose and the absorbable dose was considered 2.5%.  Most of the radioactive residues were recovered in the skin wash (88.7%) with 0.4% associated with the superficial stratum corneum (tape strips) and 0.3% in the epidermis.  Radioactive residues recovered in the donor and receptor chambers accounted for <0.1% each.  The total unabsorbed radioactive residues after the skin wash equaled 0.93% of the dose and the mean total recovery was 92.1% (38.4 ug-equiv/cm[2]).

ii.	Intermediate dose:  Following application of the target dose of 250 ug/cm[2] [[14]C]-bromacil (221, 255, or 204 ug/cm[2]) to human skin, 2.3% of the applied dose was recovered in the receptor fluid over the 24-h period with <0.1% remaining in the receptor fluid at termination.  Radioactive residues in the vapor traps (from Run 1 only) accounted for 0.6% of the dose.  Radioactive residues in the dermis accounted for 1.5% of the dose and the absorbable dose was considered 3.8%.  Most of the radioactive residues were recovered in the skin wash (95.9%) with 2.5% associated with the superficial stratum corneum (tape strips) and 2.1% in the epidermis.  Radioactive residues recovered in the donor and receptor chambers were 0.2% and 0.1%, respectively.  The total unabsorbed radioactive residues after the skin wash equaled 4.95% of the dose and the mean total recovery was 104.6% (247.5 ug-equiv/cm[2]).

iii.	High dose:  Following application of the target dose of 1369 ug/cm[2] [[14]C]-bromacil (1122 ug/cm[2]) to human skin, 0.8% of the applied dose was recovered in the receptor fluid over the 24-h period with <0.1% remaining in the receptor fluid at termination.  Radioactive residues in the vapor traps (from Run 1 only) accounted for 0.1% of the dose.  Radioactive residues in the dermis accounted for 0.2% of the dose and the absorbable dose was considered 0.9%.  The majority of the radioactive residues was recovered in the skin wash (88.9%) with 0.1% associated with the superficial stratum corneum (tape strips) and <0.1% in the epidermis.  Radioactive residues recovered in the donor and receptor chambers accounted for 0.1% each.  The total unabsorbed radioactive residues after the skin wash equaled 0.22% of the dose and the mean total recovery was 90.1% (1011 ug-equiv/cm[2]).

Table 2a.	Mean (+- SD) disposition of radioactive residues (mass and % applied dose) at 24 h after topical administration of [[14]C]-bromacil in the Hyvar X-L formulations to excised human skin for 8 h of exposure [a]
Termination
                       Target concentration of bromacil

                                42 ug/cm[2 b]
                               250 ug/cm[2] [c]
                              1369 ug/cm[2] [d]

                                ug-equiv/cm[2]
                                % applied dose
                                ug-equiv/cm[2]
                                % applied dose
                                ug-equiv/cm[2]
                                % applied dose
                            Receptor fluid (0-24 h)
                                     24 h
                                 0.77 +- 1.03
                                 1.84 +- 2.47
                                 5.53 +- 6.70
                                 2.30 +- 2.78
                                 8.70 +- 12.49
                                 0.78 +- 1.11
                         Receptor fluid (termination)
                                     24 h
                                 0.07 +- 0.17
                                 0.16 +- 0.42
                                 0.02 +- 0.02
                                 0.01 +- 0.01
                                 0.04 +- 0.08
                               <0.01 +- 0.01
                                   Vapor [e]
                                     24 h
                                 0.12 +- 0.14
                                 0.29 +- 0.34
                                     1.25
                                     0.57
                                     0.76
                                     0.07
                                    Dermis
                                     24 h
                                 0.20 +- 0.34
                                 0.48 +- 0.84
                                 3.34 +- 3.82
                                 1.46 +- 1.68
                                 1.68 +- 2.43
                                 0.15 +- 0.22
                               Total absorbable
                                     24 h
                                 1.04 +- 1.26
                                 2.49 +- 3.04
                                 8.89 +- 8.57
                                 3.78 +- 3.45
                                10.42 +- 14.44
                                 0.93 +- 1.29
                                Skin wash (8 h)
                                     24 h
                                 37.00 +- 1.82
                                 88.68 +- 4.30
                                226.72 +- 55.59
                                95.91 +- 16.48
                                997.58 +- 83.68
                                 88.90 +- 7.46
                             Tape strips (surface)
                                     24 h
                                 0.17 +- 0.39
                                 0.42 +- 0.95
                                 5.92 +- 7.08
                                 2.52 +- 2.83
                                 0.53 +- 0.41
                                 0.05 +- 0.04
                                     Film
                                     24 h
                                 1.84 +- 2.47
                                  0.05 +- 0.8
                                 0.20 +- 0.22
                                 0.08 +- 0.09
                                 0.18 +- 0.21
                                 0.02 +- 0.02
                               Receptor chamber
                                     24 h
                                 0.01 +- 0.02
                                 0.02 +- 0.04
                                 0.17 +- 0.21
                                 0.07 +- 0.08
                                 0.56 +- 0.88
                                 0.05 +- 0.08
                                 Donor chamber
                                     24 h
                                 0.02 +- 0.02
                                 0.04 +- 0.06
                                 0.37 +- 0.67
                                 0.17 +- 0.33
                                 0.72 +- 0.97
                                 0.06 +- 0.09
                                   Epidermis
                                     24 h
                                 0.13 +- 0.15
                                 0.32 +- 0.37
                                 5.11 +- 8.17
                                 2.06 +- 3.19
                                 0.43 +- 0.25
                                 0.04 +- 0.02
                    Total unabsorbable after skin wash [f]
                                     24 h
                                 0.38 +- 0.58
                                 0.93 +- 1.41
                                11.87 +- 13.63
                                 4.95 +- 5.32
                                 2.52 +- 2.16
                                 0.22 +- 0.19
                                Total recovered
                                     24 h
                                 38.42 +- 1.18
                                 92.10 +- 3.16
                                247.49 +- 54.34
                                104.64 +- 14.11
                               1010.53 +- 84.71
                                 90.06 +- 7.55
a	Data were obtained by Reviewer-derived calculations based on the individual raw data on pages 86-142 of MRID 50753901; reported cells from 8 donors (single or duplicate replicates; N = 8 runs for the low and high dose and 9 runs for the intermediate dose).
b	41.07 ug/cm[2] applied for Run 1 and 41.93 ug/cm[2] applied for Runs 2 and 4-8.
c	221.41 ug/cm[2] applied for Run 1, 255.36 ug/cm[2] applied for Runs 2-6, and 204.28 ug/cm[2] applied for Runs 7-8.
d	1121.97 ug/cm[2] applied for Run 1 and 1122.13 ug/cm[2] applied for Runs 2-8.
e	Vapor traps used during Run 1 only (duplicate determinations for the low-dose formulation).
f	Total recovered  -  (skin wash + total absorbable).

b.	80 Bromacil Alligare WDG

i.	Low dose:  Following application of the target dose of 42 ug/cm[2] [[14]C]-bromacil (42.2, 42.5, or 50.6 ug/cm[2]) to human skin, 2.2% of the applied dose was recovered in the receptor fluid over the 24-h period with <0.1% remaining in the receptor fluid at termination.  Radioactive residues in the vapor traps (from Run 1 only) accounted for 0.1% of the dose.  Radioactive residues in the dermis accounted for 0.2% of the dose and the absorbable dose was considered 2.4%.  Most of the radioactive residues were recovered in the skin wash (85.0%) with 0.2% associated with the superficial stratum corneum (tape strips) and 0.7% in the epidermis.  Radioactive residues recovered in the donor and receptor chambers accounted for <0.1% each.  The total unabsorbed radioactive residues after the skin wash equaled 2.37% of the dose and the mean total recovery was 88.4% (40.2 ug-equiv/cm[2]).
ii.	Intermediate dose:  Following application of the target dose of 420 ug/cm[2] [[14]C]-bromacil (458 or 462 ug/cm[2]) to human skin, 0.5% of the applied dose was recovered in the receptor fluid over the 24-h period with <0.1% remaining in the receptor fluid at termination.  Radioactive residues in the vapor traps (from Run 1 only) accounted for <0.1% of the dose.  Radioactive residues in the dermis accounted for 0.5% of the dose and the absorbable dose was considered 1.0%.  Most of the radioactive residues were recovered in the skin wash (94.1%) with 0.2% associated with the superficial stratum corneum (tape strips) and 0.2% in the epidermis.  Radioactive residues recovered in the donor and receptor chambers were 0.1% and <0.1%, respectively.  The total unabsorbed radioactive residues after the skin wash equaled 0.95% of the dose and the mean total recovery was 95.7% (441.7 ug-equiv/cm[2]).

iii.	High dose:  Following application of the target dose of 1375 ug/cm[2] [[14]C]-bromacil (1471 or 1513 ug/cm[2]) to human skin, 0.3% of the applied dose was recovered in the receptor fluid over the 24-h period with <0.1% remaining in the receptor fluid at termination.  Radioactive residues in the vapor traps (from Run 1 only) accounted for <0.1% of the dose.  Radioactive residues in the dermis accounted for 0.2% of the dose and the absorbable dose was considered 0.4%.  Most of the radioactive residues were recovered in the skin wash (92.1%) with 0.1% associated with the superficial stratum corneum (tape strips) and 0.1% in the epidermis.  Radioactive residues recovered in the donor and receptor chambers accounted for <0.1% each.  The total unabsorbed radioactive residues after the skin wash equaled 0.20% of the dose and the mean total recovery was 92.7% (1398 ug-equiv/cm[2]).

Table 2b.	Mean (+- SD) disposition of radioactive residues (mass and % applied dose) at 24 h after topical administration of [[14]C]-bromacil in the 80 Bromacil Alligare WDG formulations to excised human skin for 8 h of exposure [a]
Termination
                       Target concentration of bromacil

                               42 ug/cm[2] [b]
                               420 ug/cm[2] [c]
                              1375 ug/cm[2] [d]

                                ug-equiv/cm[2]
                                % applied dose
                                ug-equiv/cm[2]
                                % applied dose
                                ug-equiv/cm[2]
                                % applied dose
                            Receptor fluid (0-24 h)
                                     24 h
                                 1.05 +- 1.53
                                 2.18 +- 2.98
                                 2.24 +- 4.28
                                 0.48 +- 0.92
                                 3.83 +- 5.58
                                 0.26 +- 0.37
                         Receptor fluid (termination)
                                     24 h
                             <0.01 +- <0.01
                                 0.01 +- 0.01
                                 0.04 +- 0.09
                                 0.01 +- 0.02
                                 0.04 +- 0.03
                             <0.01 +- <0.01
                                   Vapor [e]
                                     24 h
                                     0.03
                                     0.08
                                     0.13
                                     0.03
                                     0.47
                                     0.03
                                    Dermis
                                     24 h
                                 0.09 +- 0.14
                                 0.19 +- 0.27
                                 2.11 +- 3.87
                                 0.46 +- 0.84
                                 2.78 +- 2.26
                                 0.18 +- 0.15
                               Total absorbable
                                     24 h
                                 1.15 +- 1.60
                                 2.38 +- 3.11
                                 4.38 +- 5.32
                                 0.95 +- 1.15
                                 6.64 +- 6.24
                                 0.44 +- 0.42
                                Skin wash (8 h)
                                     24 h
                                 38.61 +- 8.29
                                85.02 +- 14.89
                               434.56 +- 127.51
                                94.11 +- 27.61
                               1388.32 +- 188.12
                                92.07 +- 12.51
                             Tape strips (surface)
                                     24 h
                                 0.10 +- 0.07
                                 0.22 +- 0.13
                                 0.87 +- 1.12
                                 0.19 +- 0.24
                                 1.02 +- 0.90
                                 0.06 +- 0.06
                                     Film
                                     24 h
                                 0.02 +- 0.01
                                 0.05 +- 0.02
                                 0.26 +- 0.35
                                 0.06 +- 0.08
                                 0.41 +- 0.40
                                 0.03 +- 0.03
                               Receptor chamber
                                     24 h
                                 0.01 +- 0.01
                                 0.01 +- 0.03
                                 0.18 +- 0.22
                                 0.04 +- 0.05
                                 0.46 +- 0.67
                                 0.03 +- 0.04
                                 Donor chamber
                                     24 h
                                 0.01 +- 0.01
                                 0.02 +- 0.03
                                 0.34 +- 0.42
                                 0.07 +- 0.09
                                 0.42 +- 0.49
                                 0.03 +- 0.03
                                   Epidermis
                                     24 h
                                 0.30 +- 0.16
                                 0.67 +- 0.34
                                 1.09 +- 0.98
                                 0.24 +- 0.21
                                 0.87 +- 0.46
                                 0.06 +- 0.03
                    Total unabsorbable after skin wash [f]
                                     24 h
                                 0.44 +- 0.16
                                 2.37 +- 3.11
                                 2.79 +- 2.28
                                 0.95 +- 1.15
                                 2.93 +- 1.91
                                 0.20 +- 0.13
                                Total recovered
                                     24 h
                                 40.20 +- 8.68
                                88.37 +- 14.63
                               441.73 +- 125.36
                                95.66 +- 27.15
                               1397.9 +- 184.63
                                92.71 +- 12.31
a	Data were obtained by Reviewer-derived calculations based on the individual raw data on pages 86-142 of MRID 50753901; reported cells from 8 donors (single or duplicate replicates; N = 11, 10, and 9 runs for the low, intermediate, and high doses, respectively).
b	42.51 ug/cm[2] applied for Run 1, 42.15 ug/cm[2] applied for Runs 2-6, and 50.58 ug/cm[2] applied for Runs 7-8.
c	457.91 ug/cm[2] applied for Run 1 and 462.25 ug/cm[2] applied for Runs 2-8.
d	1471.40 ug/cm[2] applied for Run 1 and 1512.68 ug/cm[2] applied for Runs 2-8.
e	Vapor traps used during Run 1 only (duplicate determinations for the low-dose formulation).
f	Total recovered  -  (skin wash + total absorbable).

c.	Hyvar X

i.	Low dose:  Following application of the target dose of 42 ug/cm[2] [[14]C]-bromacil (41.9 or 42.9 ug/cm[2]) to human skin, 1.3% of the applied dose was recovered in the receptor fluid over the 24-h period with <0.1% remaining in the receptor fluid at termination.  Radioactive residues in the vapor traps (from Run 1 only) accounted for 0.1% of the dose.  Radioactive residues in the dermis accounted for 0.7% of the dose and the absorbable dose was considered 2.1%.  Most of the radioactive residues were recovered in the skin wash (93.0%) with 0.2% associated with the superficial stratum corneum (tape strips) and 0.5% in the epidermis.  Radioactive residues recovered in the donor and receptor chambers accounted for 0.1% each.  The total unabsorbed radioactive residues after the skin wash equaled 0.88% of the dose and the mean total recovery was 95.9% (41.1 ug-equiv/cm[2]).

ii.	Intermediate dose:  Following application of the target dose of 420 ug/cm[2] [[14]C]-bromacil (442 or 444 ug/cm[2]) to human skin, 0.4% of the applied dose was recovered in the receptor fluid over the 24-h period with <0.1% remaining in the receptor fluid at termination.  Radioactive residues in the vapor traps (from Run 1 only) accounted for 0.1% of the dose.  Radioactive residues in the dermis accounted for 0.3% of the dose and the absorbable dose was considered 0.7%.  Most of the radioactive residues were recovered in the skin wash (98.2%) with 0.1% associated with the superficial stratum corneum (tape strips) and 0.1% in the epidermis.  Radioactive residues recovered in the donor and receptor chambers were <0.1% and 0.1%, respectively.  The total unabsorbed radioactive residues after the skin wash equaled 0.34% of the dose and the mean total recovery was 99.3% (441.0 ug-equiv/cm[2]).

iii.	High dose:  Following application of the target dose of 1375 ug/cm[2] [[14]C]-bromacil (1405 or 1409 ug/cm[2]) to human skin, 0.2% of the applied dose was recovered in the receptor fluid over the 24-h period with <0.1% remaining in the receptor fluid at termination.  Radioactive residues in the vapor traps (from Run 1 only) accounted for <0.1% of the dose.  Radioactive residues in the dermis accounted for 1.6% of the dose and the absorbable dose was considered 1.8%.  Most of the radioactive residues were recovered in the skin wash (93.7%) with 1.8% associated with the superficial stratum corneum (tape strips) and 0.9% in the epidermis.  Radioactive residues recovered in the donor and receptor chambers accounted for 0.2% each.  The total unabsorbed radioactive residues after the skin wash equaled 3.36% of the dose and the mean total recovery was 98.8% (1390 ug-equiv/cm[2]).

Table 2c.	Mean (+- SD) disposition of radioactive residues (mass and % applied dose) at 24 h after topical administration of [[14]C]-bromacil in the Hyvar X formulations to excised human skin for 8 h of exposure [a]
Termination
                       Target concentration of bromacil

                           42 ug/cm[2] ( ug equiv)
                        420 ug/cm[2] ( ug equiv) [b]
                          1375 ug/cm[2] ( ug equiv)

                                ug-equiv/cm[2]
                                % applied dose
                                ug-equiv/cm[2]
                                % applied dose
                                ug-equiv/cm[2]
                                % applied dose
                            Receptor fluid (0-24 h)
                                     24 h
                                 0.56 +- 0.42
                                 1.32 +- 1.02
                                 1.76 +- 0.79
                                 0.40 +- 0.18
                                 2.52 +- 1.58
                                 0.18 +- 0.11
                         Receptor fluid (termination)
                                     24 h
                             <0.01 +- <0.01
                                 0.01 +- 0.01
                                 0.03 +- 0.05
                                 0.01 +- 0.01
                                 0.04 +- 0.04
                             <0.01 +- <0.01
                                   Vapor [c]
                                     24 h
                                     0.04
                                     0.09
                                     0.23
                                     0.05
                                 0.57 +- 0.03
                               0.04 +- <0.01
                                    Dermis
                                     24 h
                                 0.31 +- 0.37
                                 0.72 +- 0.86
                                 1.51 +- 3.45
                                 0.34 +- 0.78
                                22.11 +- 39.77
                                 1.57 +- 2.82
                               Total absorbable
                                     24 h
                                 0.88 +- 0.56
                                 2.05 +- 1.33
                                 3.29 +- 3.39
                                 0.74 +- 0.76
                                24.68 +- 40.52
                                 1.75 +- 2.88
                                Skin wash (8 h)
                                     24 h
                                 39.81 +- 3.17
                                 93.00 +- 7.18
                               436.22 +- 123.62
                                98.18 +- 27.78
                               1317.59 +- 198.16
                                93.70 +- 14.07
                             Tape strips (surface)
                                     24 h
                                 0.08 +- 0.07
                                 0.18 +- 0.16
                                 0.37 +- 0.28
                                 0.08 +- 0.06
                                24.98 +- 62.05
                                 1.77 +- 4.40
                                     Film
                                     24 h
                                 0.05 +- 0.06
                                 0.11 +- 0.14
                                 0.11 +- 0.16
                                 0.03 +- 0.04
                                 3.75 +- 8.40
                                 0.27 +- 0.60
                               Receptor chamber
                                     24 h
                                 0.02 +- 0.03
                                 0.05 +- 0.06
                                 0.23 +- 0.37
                                 0.05 +- 0.08
                                 2.70 +- 6.27
                                 0.19 +- 0.45
                                 Donor chamber
                                     24 h
                                 0.05 +- 0.08
                                 0.11 +- 0.18
                                 0.16 +- 0.22
                                 0.04 +- 0.05
                                 2.98 +- 4.80
                                 0.21 +- 0.34
                                   Epidermis
                                     24 h
                                 0.20 +- 0.12
                                 0.47 +- 0.27
                                 0.59 +- 0.51
                                 0.13 +- 0.11
                                12.58 +- 27.36
                                 0.89 +- 1.94
                    Total unabsorbable after skin wash [d]
                                     24 h
                                 0.78 +- 0.92
                                 0.88 +- 0.55
                                 1.51 +- 1.11
                                 0.34 +- 0.25
                                47.34 +- 102.64
                                 3.36 +- 7.28
                                Total recovered
                                     24 h
                                 41.06 +- 2.73
                                 95.93 +- 6.23
                               441.02 +- 124.48
                                99.26 +- 27.97
                               1389.61 +- 119.70
                                 98.82 +- 8.42
a	Data were obtained by Reviewer-derived calculations based on the individual raw data on pages 86-142 of MRID 50753901; reported cells from 8 donors (single or duplicate replicates; N = 8 runs for the low and high dose and 10 runs for the intermediate dose).
b	457.91 ug/cm[2] applied for Run 1 and 462.25 ug/cm[2] applied for Runs 2-8.
c	Vapor traps used during Run 1 only (duplicate determinations for the low-dose formulation).
d	Total recovered  -  (skin wash + total absorbable).

2.	Rat:  Data for rat skin (mass and percentage of the applied doses) were calculated by the Reviewers and are presented in Tables 3a (Hyvar X-L formulations) and 3b (80 Bromacil Alligare WDG formulations).  The percentage data were similar to summary results reported in Tables 9-11 on pages 39-41 of MRID 50753901; these tables are included as Appendices VI and VII at the end of this DER.

a.	Hyvar X-L

i.	Low dose:  Following application of the target dose of 42 ug/cm[2] [[14]C]-bromacil (41.9 or 83.9 ug/cm[2] [single cell]) to rat skin, 4.3% of the applied dose was recovered in the receptor fluid over the 24-h period with <0.1% remaining in the receptor fluid at termination.  Radioactive residues in the dermis accounted for 3.5% of the dose and the absorbable dose was considered 7.8%.  Most of the radioactive residues were recovered in the skin wash (81.0%) with 3.1% in the epidermis.  Radioactive residues recovered in the donor and receptor chambers accounted for 0.2% and 0.5%, respectively.  The total unabsorbed radioactive residues after the skin wash equaled 4.00% of the dose.  The mean total recovery was 92.8% (38.9 ug-equiv/cm[2] for the regular dose and 78.5 ug-equiv/cm[2] for the doubled dose).

ii.	Intermediate dose:  Following application of the target dose of 250 ug/cm[2] [[14]C]-bromacil (255 ug/cm[2]) to rat skin, 1.6% of the applied dose was recovered in the receptor fluid over the 24-h period with <0.1% remaining in the receptor fluid at termination.  Radioactive residues in the dermis accounted for 3.3% of the dose and the absorbable dose was considered 4.9%.  Most of the radioactive residues were recovered in the skin wash (86.9%) with 14.1% in the epidermis.  Radioactive residues recovered in the donor and receptor chambers were 0.1% and <0.1%, respectively.  The total unabsorbed radioactive residues after the skin wash equaled 14.4% of the dose and the mean total recovery was 106.1% (307.1 ug-equiv/cm[2]).

iii.	High dose:  Following application of the target dose of 1369 ug/cm[2] [[14]C]-bromacil (1122 ug/cm[2]) to rat skin, 3.4% of the applied dose was recovered in the receptor fluid over the 24-h period with <0.1% remaining in the receptor fluid at termination.  Radioactive residues in the dermis accounted for 1.3% of the dose and the absorbable dose was considered 4.7%.  The majority of the radioactive residues was recovered in the skin wash (86.5%) with 1.7% in the epidermis.  Radioactive residues recovered in the donor and receptor chambers were 0.1% and <0.1%, respectively.  The total unabsorbed radioactive residues after the skin wash equaled 1.81% of the dose and the mean total recovery was 93.0% (1044 ug-equiv/cm[2]).

Table 3a.	Mean (+- SD) disposition of radioactive residues (mass and % applied dose) at 24 h after topical administration of [[14]C]-bromacil in the Hyvar X-L formulations to excised rat skin for 8 h of exposure [a]
Termination
                       Target concentration of bromacil

                                42 ug/cm[2 b]
                               250 ug/cm[2] [c]
                              1369 ug/cm[2] [d]

                                ug-equiv/cm[2]
                                % applied dose
                                ug-equiv/cm[2]
                                % applied dose
                                ug-equiv/cm[2]
                                % applied dose
                            Receptor fluid (0-24 h)
                                     24 h
                                 2.06 +- 1.74
                                 4.28 +- 2.77
                                 4.38 +- 1.89
                                 1.55 +- 0.70
                                38.32 +- 65.63
                                 3.42 +- 5.85
                         Receptor fluid (termination)
                                     24 h
                                 0.01 +- 0.01
                                 0.02 +- 0.01
                                 0.05 +- 0.02
                                 0.02 +- 0.01
                                 0.09 +- 0.09
                                 0.01 +- 0.01
                                    Dermis
                                     24 h
                                 1.58 +- 1.77
                                 3.52 +- 4.19
                                 8.98 +- 14.00
                                 3.30 +- 5.53
                                14.42 +- 16.85
                                 1.28 +- 1.50
                               Total absorbable
                                     24 h
                                 3.65 +- 2.88
                                 7.83 +- 5.72
                                13.41 +- 15.00
                                 4.87 +- 5.95
                                52.83 +- 72.66
                                 4.71 +- 6.48
                                Skin wash (8 h)
                                     24 h
                                 36.62 +- 9.46
                                 80.99 +- 9.61
                                251.13 +- 76.77
                                86.91 +- 14.93
                               970.44 +- 100.43
                                 86.48 +- 8.95
                                     Film
                                     24 h
                                 0.09 +- 0.20
                                 0.14 +- 0.25
                                 0.12 +- 0.17
                                 0.04 +- 0.07
                                 0.31 +- 0.36
                                 0.03 +- 0.03
                               Receptor chamber
                                     24 h
                                 0.21 +- 0.64
                                 0.50 +- 1.52
                                 0.05 +- 0.08
                                 0.02 +- 0.03
                                 0.48 +- 1.27
                                 0.04 +- 0.11
                                 Donor chamber
                                     24 h
                                 0.07 +- 0.10
                                 0.15 +- 0.24
                                 0.38 +- 0.90
                                 0.14 +- 0.36
                                 0.78 +- 1.14
                                 0.07 +- 0.10
                                   Epidermis
                                     24 h
                                 1.51 +- 1.22
                                 3.11 +- 1.60
                                41.73 +- 31.21
                                 14.06 +- 7.58
                                18.61 +- 23.44
                                 1.66 +- 2.09
                    Total unabsorbable after skin wash [e]
                                     24 h
                                 1.92 +- 1.79
                                 4.00 +- 3.27
                                42.55 +- 30.94
                                 14.4 +- 7.47
                                20.27 +- 23.11
                                 1.81 +- 2.06
                                Total recovered
                                     24 h
                               38.89 +- 3.26 [f]
                                 92.81 +- 7.41
                                307.09 +- 80.23
                                106.14 +- 6.72
                               1043.54 +- 23.97
                                 93.00 +- 2.14
a	Data were obtained by Reviewer-derived calculations based on the individual raw data on pages 86-142 of MRID 50753901; reported cells from 6 donors (duplicate replicates; N = 6 runs for the low dose and N = 5 runs for the intermediate and high doses).
b	41.93 ug/cm[2] applied for Runs 1-6, except one replicate for Run 3 was doubled to 83.87 ug/cm[2].
c	255.36 ug/cm[2] applied for Runs 1-4 and 6.
d	1122.13 ug/cm[2] applied for Runs 1-4 and 6.
e	Total recovered  -  (skin wash + total absorbable).

b.	80 Bromacil Alligare WDG

i.	Low dose:  Following application of the target dose of 42 ug/cm[2] [[14]C]-bromacil (42.2 ug/cm[2]) to rat skin, 4.8% of the applied dose was recovered in the receptor fluid over the 24-h period with <0.1% remaining in the receptor fluid at termination.  Radioactive residues in the dermis accounted for 1.8% of the dose and the absorbable dose was considered 6.5%.  Most of the radioactive residues were recovered in the skin wash (88.6%) with 2.1% in the epidermis.  Radioactive residues recovered in the donor and receptor chambers accounted for <0.1% each.  The total unabsorbed radioactive residues after the skin wash equaled 2.21% of the dose and the mean total recovery was 97.4% (40.0 ug-equiv/cm[2]).

ii.	Intermediate dose:  Following application of the target dose of 420 ug/cm[2] [[14]C]-bromacil (462 ug/cm[2]) to rat skin, 0.8% of the applied dose was recovered in the receptor fluid over the 24-h period with <0.1% remaining in the receptor fluid at termination.  Radioactive residues in the dermis accounted for 1.3% of the dose and the absorbable dose was considered 2.1%.  Most of the radioactive residues were recovered in the skin wash (97.9%) with 1.2% in the epidermis.  Radioactive residues recovered in the donor and receptor chambers were <0.1% and 0.1%, respectively.  The total unabsorbed radioactive residues after the skin wash equaled 1.31% of the dose and the mean total recovery was 101.2% (463.0 ug-equiv/cm[2]).

iii.	High dose:  Following application of the target dose of 1375 ug/cm[2] [[14]C]-bromacil (1513 ug/cm[2]) to human skin, 0.5% of the applied dose was recovered in the receptor fluid over the 24-h period with <0.1% remaining in the receptor fluid at termination.  Radioactive residues in the dermis accounted for 1.6% of the dose and the absorbable dose was considered 2.0%.  Most of the radioactive residues were recovered in the skin wash (89.8%) with 0.6% in the epidermis.  Radioactive residues recovered in the donor and receptor chambers accounted for 0.1% each.  The total unabsorbed radioactive residues after the skin wash equaled 0.68% of the dose and the mean total recovery was 92.5% (1393 ug-equiv/cm[2]).

Table 3b.	Mean (+- SD) disposition of radioactive residues (mass and % applied dose) at 24 h after topical administration of [[14]C]-bromacil in the 80 Bromacil Alligare WDG formulations to excised rat skin for 8 h of exposure [a]
Termination
                       Target concentration of bromacil

                               42 ug/cm[2] [b]
                               420 ug/cm[2] [c]
                              1375 ug/cm[2] [d]

                                ug-equiv/cm[2]
                                % applied dose
                                ug-equiv/cm[2]
                                % applied dose
                                ug-equiv/cm[2]
                                % applied dose
                            Receptor fluid (0-24 h)
                                     24 h
                                 1.90 +- 2.54
                                 4.77 +- 6.02
                                 3.69 +- 3.46
                                 0.82 +- 0.74
                                 7.98 +- 4.32
                                 0.47 +- 0.26
                         Receptor fluid (termination)
                                     24 h
                             <0.01 +- <0.01
                                 0.01 +- 0.01
                                 0.01 +- 0.01
                             <0.01 +- <0.01
                                 0.03 +- 0.02
                             <0.01 +- <0.01
                                    Dermis
                                     24 h
                                 0.70 +- 0.52
                                 1.76 +- 1.46
                                 5.72 +- 9.69
                                 1.26 +- 2.09
                                29.87 +- 31.86
                                 1.57 +- 1.63
                               Total absorbable
                                     24 h
                                 2.60 +- 2.50
                                 6.54 +- 5.87
                                 9.43 +- 12.91
                                 2.08 +- 2.78
                                37.88 +- 35.21
                                 2.04 +- 1.80
                                Skin wash (8 h)
                                     24 h
                                36.43 +- 13.15
                                88.62 +- 30.24
                                447.07 +- 22.75
                                 97.85 +- 5.06
                               1344.52 +- 60.95
                                 89.78 +- 4.65
                                     Film
                                     24 h
                                 0.01 +- 0.02
                                 0.03 +- 0.06
                                 0.10 +- 0.14
                                 0.02 +- 0.03
                                 0.19 +- 0.14
                                 0.01 +- 0.01
                               Receptor chamber
                                     24 h
                                 0.02 +- 0.04
                                 0.04 +- 0.10
                                 0.38 +- 1.07
                                 0.08 +- 0.23
                                 0.76 +- 1.10
                                 0.05 +- 0.07
                                 Donor chamber
                                     24 h
                                 0.02 +- 0.02
                                 0.04 +- 0.05
                                 0.15 +- 0.17
                                 0.03 +- 0.04
                                 0.91 +- 0.62
                                 0.05 +- 0.04
                                   Epidermis
                                     24 h
                                 0.91 +- 0.33
                                 2.10 +- 0.71
                                 5.91 +- 4.70
                                 1.18 +- 0.99
                                 8.47 +- 8.43
                                 0.55 +- 0.56
                    Total unabsorbable after skin wash [e]
                                     24 h
                                 0.96 +- 0.33
                                 2.21 +- 0.70
                                 6.52 +- 4.64
                                 1.31 +- 0.98
                                 10.70 +- 9.92
                                 0.68 +- 0.67
                                Total recovered
                                     24 h
                                39.99 +- 13.11
                                97.37 +- 30.01
                                463.02 +- 23.68
                                101.24 +- 5.13
                               1393.09 +- 66.53
                                 92.50 +- 4.71
a	Data were obtained by Reviewer-derived calculations based on the individual raw data on pages 86-142 of MRID 50753901; reported cells from 6 donors (duplicate replicates, except for a single replicate of the intermediate dose for Run 6).
b	42.15 ug/cm[2] applied for Runs 1-6.
c	462.25 ug/cm[2] applied for Runs 1-6.
d	1512.68 ug/cm[2] applied for Runs 1-6.
e	Total recovered  -  (skin wash + total absorbable).

C.	COMPARATIVE ABSORPTION:  After single applications of [[14]C]-bromacil in three different formulations applied at target doses of 42 ug/cm[2], 250 or 420 ug/cm[2], and 1369 or 1375 ug/cm[2] to human and rat skin samples, mean total recoveries (percentage of applied dose) ranged from 88-105% for human skin and 93-106% for rat skin were obtained.  There were no discernible differences in recovery between formulations or species.
    The original study design included bromacil formulations of 42, 420, and 1369-1375 ug/cm[2].  It was stated that these doses were also selected for a companion in vivo study to compare in vitro and in vivo rat percutaneous absorption.  Because the intermediate dose for the Hyvar X-L formulation in the in vivo study was approximately 40% lower (250 ug/cm[2]), it was stated that the in vitro dose was adjusted to match.  As the reported doses prepared for the Hyvar X-L intermediate formulation did not indicate this adjustment, the formulation preparation and actual doses administered were re-calculated by the Reviewers (Table 1).  In general, only the actual doses for the low-dose groups for all three formulations (and both species) and the intermediate X-L dose (Runs 2-6) approximated the target dose (generally within approximately +-2%, except for Runs 7 and 8 [human] with the 80 Bromacil Alligare WDG formulation [↑20%] and the single cell [rat] with the Hyvar X-L formulation that received a double dose).  The intermediate and high doses were generally +- 2-18% of the target doses.

    The individual absorption time-course profiles were reported but not discussed, and absorption rate data were not presented.  As the actual amounts of radiolabel administered within each dose level and amounts of radiolabel or administration volumes varied between doses and between formulations, comparison of absorption rates may not have been appropriate.

    Potentially absorbable bromacil was reported as the sum of radioactive residues present in the receptor fluid and the dermis.  Radioactive materials removed by washing at 8 h, recovered from the surface (tape strips 1-2) at the end of the exposure period, in the epidermis or on the films used to hold the skin samples, and residual materials in the donor and receptor chambers were considered unabsorbed.  With human skin samples, there was no discernable pattern between dose level and the percentage of absorbable or unabsorbable dose across the three formulations.  There also was no discernable difference in absorption between the three formulations.  For rat skin samples, greatest absorption was observed at the low dose for either tested formulation; percentage absorption was similar between the intermediate- and high-dose levels.  Absorption appeared to be slightly greater for the Hyvar X-L formulation relative to the 80 Bromacil Alligare WDG formulation.  It was stated that an effect of dose (p=0.02) was noted for rat skin but no effect was observed for the percutaneous absorption values. 

    Finally, it was noted that the use of bromacil formulations as suspensions in water (with the inherent difficulty of maintaining homogenous suspensions on a small scale) contributed to variability in the absorption results.

III.	DISCUSSION AND CONCLUSIONS

A.	INVESTIGATORS CONCLUSIONS:  Based on its relatively high molecular mass as a measure of its molecular size and diffusability, bromacil was not expected to have high skin absorption.  The values obtained with human skin were less than 5% of applied dose regardless of chemical dose or formulation.  The rat skin absorptions were nominally higher but even for the more permeable rat skin, absorptions were less than 10% for all combinations of formulation and dose.  There was a trend in the data for higher skin absorption with lower chemical dose as might be expected, but this was not statistically significant.  There was also a trend for higher skin absorption with the Hyvar X-L liquid formulation as compared to the solid formulations (Hyvar X and Alligare WDG) but again this was not statistically significant.

    The in vitro rat data generated in this study will be used, along with in vivo rat data from a companion study, to calculate an in vivo/in vitro ratio for rat skin.  This ratio will be used to estimate a human in vivo absorption value by multiplying the in vitro human value generated in this report by the ratio (the so-called 3-pack or parallelogram approach).  A separate report will provide this calculation.

B.	REVIEWER COMMENTS:  The contractor created a spreadsheet in order to back check the registrant's calculations.  A single applications of [[14]C]-bromacil in three different formulations applied at target doses of 42 ug/cm[2], 250 or 420 ug/cm[2], and 1369 or 1375 ug/cm[2] to human and rat skin samples, mean total recoveries ranging from 88-105% for human skin and 93-106% for rat skin were obtained with no discernible differences between formulations or species.  The contractor observed that the study protocol stated that all intermediate dose concentrations would be 420 ug bromacil/cm[2].  In the summary and study design sections (pages 13-15 of MRID 50753901) this dose was stated as being changed to 250 ug bromacil/cm[2] for the Hyvar X-L intermediate dose to align with the dose used in a companion in vivo study.  Calculations conducted by the Reviewers based on the formulation preparation data and applied dpm data for each pertinent run do not support this dose level.  See Table 1 and accompanying spreadsheet calculations for apparent doses.  The Hyvar and 80 Bromacil Alligare WDG granular formulations were diluted to 1375, 420, and 42 ug bromacil/cm[2], respectively.

    Potentially absorbable bromacil was reported as the sum of radioactive residues present in the receptor fluid and the dermis.  Mean percutaneous absorption of [[14]C]-bromacil in the formulations through human skin ranged from 0.4% to 3.8%, with mean absorption through rat skin ranging from 2.0% to 7.8%.  Radioactive materials removed by washing at 8 h, recovered from the surface (tape strips 1-2) at the end of the exposure period, and in the epidermis were considered unabsorbed.  Most of the radioactive residues were recovered in the skin washes at 8 h post-application with mean percentage values of 85-98% for human skin and 81-98% for rat skin.  The unabsorbed dose remaining after the skin wash accounted for 0.2-5.0% for human skin and 0.7-14.4% for rat skin.

    With human skin samples, there was no discernable pattern between dose level and the percentage of absorbable dose across the three formulations.  There also was no discernable difference in absorption between the three formulations.  For rat skin samples, greatest absorption was observed at the low dose for either tested formulation; percentage absorption was similar between the intermediate- and high-dose levels.  Absorption appeared to be slightly greater for the Hyvar X-L formulation relative to the 80 Bromacil Alligare WDG formulation.  It was stated that an effect of dose (p=0.02) was noted for rat skin but no effect was observed for the percutaneous absorption values.
    
    Under current practices, the reviewer's calcualtions differed slightly from the study author's.  The reviewer added both receptor fluid values in addition to both the dermis and the epidermis values.   The EPA reviewer further considered the doses were the radioactivity seemed to still be moving through the skin at the end of the exposure period.  Throughout the study, the highest amounts absorbed were in the 42 ug/cm[2] low dose groups based upon the amounts recovered in the receptor fluid.  These doses were therefore considered the most appropriate for risk assessment.  There was no in vitro data calculated for the Hyvar X formulation for the rat.  The amounts calculated are as follows:
    
    Human at 42 ug/cm[2]: Hyvar X-L- 3.2%, Alligare WDG- 3.05% and Hyvar X- 2.52%
    
    Rat at 42 ug/cm[2]: Hyvar X-L- 10.93% and Alligare WDG- 8.64%
    
    It is also worth noting that the study director included both tape strips one and two in their dermal absorption calculation.  Under current practices, the agency does not include these two tape strips.  The reviewers did look at the amount of radioactivity present on the two tape strips, and determined that the amounts didn't change the overall absorption data significantly and thus were left in place in this instance.  For the triple pack calculations the ratios of the human to the rat values for each formulation can be used along with the appropriate in vivo rat data to generate the dermal absorption factor for bromacil. 
    
    This study is classified acceptable / non-guideline and it demonstrates the comparative in vitro percutaneous absorption of [[14]C]-bromacil in up to three different formulations through human and rat skin samples.

C.	STUDY DEFICIENCIES:  Minor study deficiencies were noted as follows:

     The study protocol stated that all intermediate dose concentrations would be 420 ug bromacil/cm[2].  In the summary and study design sections (pages 13-15 of MRID 50753901) this dose was stated as 250 ug bromacil/cm2 for the Hyvar X-L intermediate dose to align with the dose used in a companion in vivo study.  Although the dpm delivered for the Hyvar X-L intermediate dose were approximately 60% of the high and low doses, the calculations conducted by the Reviewers based on the formulation preparation data did not support a reduction to 250 ug bromacil/cm[2].  The formulation data were re-calculated by the Reviewers to yield the reported dose level.

     Actual doses delivered within some formulations/dose levels were changed for some runs without explanation.  Additionally, individual doses were not assessed for dose (dpm) delivered.  Identical doses (dpm) within each dose formulation level for a given dose volume were reported as delivered across runs and both species.
Appendix I.  Flow-through system set up.

                                       
                                       
                                         (copied from page 75 of MRID 50753901)

Appendix II.  Flow-through diffusion cell and evaporation cell designs.

                                       
                                       
                                       
                                       
                                     (copied from pages 76-77 of MRID 50753901)

Appendix III.  HPLC method with radiodetection.

                                                                               
                                         (copied from page 23 of MRID 50753901)

Appendix IV.  Summary of percutaneous absorption of [[14]C]-bromacil through human skin.

                                         (copied from page 35 of MRID 50753901)

Appendix V.  Disposition of the applied dose of [[14]C]-bromacil with human skin.
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                     (copied from pages 36-38 of MRID 50753901)

Appendix VI.  Summary of percutaneous absorption of [[14]C]-bromacil through rat skin.

                                         (copied from page 39 of MRID 50753901)

Appendix VII.  Disposition of the applied dose of [[14]C]-bromacil with human skin.
                                       
                                       
                                       
                                       
                                       
                                     (copied from pages 40-41 of MRID 50753901)