2

applied through various ground methods, such as ground booms and soil incorporation 

equipment.” None of the end use products containing isoxaflutole are manufactured as 

emulsifiable concentrates. The end use products are suspension concentrates or dry flowables.

Also on page 4 of the Summary Document (and as noted in the Ecological Problem Formulation 

Document) the Agency notes that the most recent isoxaflutole ecological risk assessment was 

conducted in April 2010 for an Experimental Use Permit (EUP) for use on soybeans. An 

assessment conducted in 1997 was the next most current assessment.

EFED Response: The comment is noted.

#2 Bayer Comment:

BCS would like to remind the Agency of the extensive additional ecotoxicology studies, 

environmental fate studies, and environmental monitoring studies that were conducted after the 

1997 assessment as summarized by Allen et al. (2004, MRID 46330201). Examples would 

include a comment made on page 9 of the Ecological Problem Formulation Document 

wherein EPA states: Isoxaflutole’s terminal metabolite, RPA 203328 is expected to persist and 

accumulate. The results of the field dissipation, ground water, tile drain, and surface water 

studies as summarized by Allen et al. (2004, MRID 46330201) do not support the statement that 

RPA 203328 persists and accumulates in the environment.

EFED Response: EFED will re-examine existing data and information on the physicalchemical and fate properties of the RPA 203328 degradate. Any changes in EPA‟s 

characterization of that degradate will then be reflected in the forthcoming drinking water and 

ecological risk assessments in support of registration review.

Environmental fate data gaps

GLN 835.4100. - Aerobic soil Metabolism

#3 Bayer Comment:

As described in Allen et al. (2004, MRID 46330201), three aerobic soil metabolism studies have 

been conducted on a total of six different soils (Ferreira et al., 1994, MRID 43588006; Ferreira 

et al., 1996, MRID 45658805; Burr, 1996a; MRID 45658804). Kinetics evaluations conducted 

by Hardy et al. (2002, MRID 45658807) for all six soils are also summarized in Allen et al.

(2004, MRID 46330201). This information should be sufficient to meet the aerobic soil 

metabolism study requirements. EPA may not have considered Ferreira et al., 1996, MRID 

45658805, such that only the data from three soils were considered for aerobic soil metabolism 

results.

EFED Response: As noted in Table 9 of the Isoxaflutole Summary Document MRID‟s 

43588006 and 45658804 are categorized as „supplemental‟. As such they may contain useful 

information that may be used in the Agency‟s assessment of parent isoxaflutole but do not fully 

meet the requirements of Guideline No. 835.4100. More specifically, the individual study results

are not based on the analyses of four soil samples. Collectively, however, since the total number 

of studies account for four or more soils then no additional data is needed. 
3

GLN 835.4300. - Aerobic Aquatic metabolism

#4 Bayer Comment:

As summarized by Allen et al. (2004, MRID 46330201), an aerobic aquatic metabolism study on 

two water sediment systems was conducted by Ayliffe and Newby (1995, MRID 44291502). 

Isoxaflutole was rapidly degraded in both systems and no longer detected after 7 days. This 

study should satisfy the requirements for an aerobic aquatic metabolism study.

EFED Response: The Agency completed a review of an aerobic aquatic metabolism study by 

Ayliffe and Newby (MRID) 44291502) in February 1998 and found it to be acceptable with one 

notable exception. The analysis only satisfied the data requirements for the parent and the 

metabolite RPA 202248; it did not include the metabolite RPA 203328. As a result, the analysis 

was considered incomplete. Subsequent to that review the Agency has determined that the RPA 

203328 metabolite is not a chemical of toxicological concern. Therefore, no additional data are 

needed.

For degradate RPA 202248

#5 Bayer Comment:

In the Ecological Problem Formulation Document, EPA stated that “There have been no 

registrant submitted environmental fate studies for the compound‟s primary degradate.” 

Numerous studies for RPA 202248 have been submitted for review by the Agency. For 

example, three of the adsorption/desorption studies cited in Table 9 of the formulation document 

used RPA 202248 degradate as the test material. In many of the degradation studies, the 

behavior of the RPA 202248 degradate can be determined from the studies starting with parent 

isoxaflutole.

EFED Response: EPA will change the above language to note that data have been submitted. 

Anticipated data requirements for degradate RPA 202248

Guideline No. 835.1230 - Adsorption/desorption (batch equilibrium)

#6 Bayer Comment:

Three of the studies listed in Table 9 of the formulation document (Burr, 1995, MRID 43940301; 

Burr, 1996b, MRID 44065801; and Lowden et al., MRID 48108103) were conducted with RPA 

202248. These studies have been classified as acceptable in Table 9. 

EFED Response: No additional adsorption/desorption study data (GLN 835.1230) are 

requested for the RPA 202248 degradate.
4

Guideline No. 835.1240 - Leaching Studies 

#7 Bayer Comment:

The study cited for parent isoxaflutole (MRID 43588008) should satisfy the requirement for 

RPA 202248 since the parent isoxaflutole had degraded to RPA 202248 before the start of the 

leaching phase of the study. 

EFED Response: No additional leaching study data (GLN 835.1240) are requested for the RPA 

202248 degradate.

Guideline No. 835.4100 - Aerobic Soil Metabolism 

#8 Bayer Comment:

As described in Allen et al. (2004, MRID 46330201), three aerobic soil metabolism studies have 

been conducted on a total of six different soils (Ferreira et al., 1994, MRID 43588006; Ferreira 

et al., 1996, MRID 45658805; Burr, 1996a, MRID 45658804) starting with parent isoxaflutole. 

Kinetics evaluations conducted by Hardy et al. (2002, MRID 45658807) for all six soils are also 

summarized in Allen et al. (2004, MRID 46330201). In these studies the degradation rate for 

RPA 202248 has been determined, so these studies should be sufficient to meet the aerobic soil 

metabolism study requirements for the RPA 202248 degradate.

EFED Response: See response to BCS comment #3. No additional aerobic soil metabolism data 

are needed.

Guideline No. 835.4200 - Anaerobic Soil Metabolism 

#9 Bayer Comment:

The study starting with parent isoxaflutole is not being required since “additional data are not 

expected to change risk conclusions.” Given the relatively rapid degradation of RPA 202248, the 

same reasoning applies to RPA 202248 and therefore an anaerobic soil metabolism with RPA 

202248 should not be required.

EFED Response: No additional anaerobic soil metabolism study data (GLN 835.4200) are 

needed to assess the RPA 202248 degradate.

Guideline No. 835.4300 - Aerobic Aquatic Metabolism 

#10 Bayer Comment:

As summarized by Allen et al. (2004, MRID 46330201), an aerobic aquatic metabolism study on 

two water sediment systems was conducted by Ayliffe and Newby (1995, MRID 44291502). 

Since isoxaflutole was rapidly degraded in both systems to RPA 202248, this study should 

satisfy the requirements for an aerobic aquatic metabolism study with RPA 202248. Note that in 
5

response to the slow degradation rate encountered in the laboratory study, an outdoor mesocosm 

study was also performed (Lui et al., 2003, MRID 46081105; also Rupprecht et al., 2004). This 

study should also be considered as fulfilling this requirement.

EFED Response: See response to BCS comment #4. No additional aerobic aquatic metabolism 

study data (GLN 835.4300) are needed to assess the RPA 202248 degradate.

Guideline No. 835.4400 - Anaerobic Aquatic Metabolism

#11 Bayer Comment:

As summarized by Allen et al. (2004, MRID 46330201), an anaerobic aquatic metabolism study 

on one water sediment system was conducted by Burr et al. (1995, MRID 43588007). This 

study was found to be acceptable to satisfy the requirement for parent and so should also satisfy 

the requirement for RPA 202248 since isoxaflutole was rapidly degraded to RPA 202248, which 

remained stable throughout the remainder of the study.

EFED Response: The comment is noted. No additional anaerobic aquatic metabolism study 

data (GLN 835.4400) are needed to assess the RPA 202248 degradate.

Guideline No. 835.6200 - Environmental Chemistry Analytical Methods and ILV (Water) 

#12 Bayer Comment:

The methods and associated independent laboratory validations that apparently were acceptable 

for parent isoxaflutole (since there was no data requirement in this area) also include RPA 

202248. Therefore, no additional work is needed to meet this guideline for RPA 202248. 

There are three methods that include parent and RPA 202248 that have undergone independent 

laboratory validations. The first was an enforcement method (Hunt and Lopes, 1997, MRID 

44399901) with three associated independent laboratory validations (Wickremesinhe, 1997, 

MRID 44399902; Davis, 1997, MRID 44399903; Hughes, 1997, MRID 44399904). The second 

method was the method based on internal standards that was used in the monitoring work 

conducted by the registrant during the period of the conditional registration (Smitley, 1999, 

MRID 44830501). The independent validation of the second method was conducted by Zheng 

(1999, MRID 44830502). A later revision of this method (Smitley et al., 2000) does not seem to 

be listed independently in the list of MRID values, but was included in its entirely in the reports 

of several monitoring studies (for example pages 320 to 360 in Lee and Nandihalli, 2002, MRID 

45696003). The third method was an immunoassay method required by EPA as a condition of 

registration and internal (Nandihalli, 2006, MRID 47171602) and independent (Perez, 2006, 

MRID 47171603) laboratory validations have been submitted to the Agency. The instructions for 

applying the immunoassay methods are included in the validation reports.

EFED Response: No additional environmental chemistry methods and independent laboratory 

validation documentation (GLN 835.6200) are needed for the RPA 202248 degradate.
6

For degradate RPA 203328, General comment on EFED’s recommendations 

to request chronic toxicity data

#13 Bayer Comment: “All available data indicate that RPA 203328 is not of toxicological 

significance as indicated in the recent assessment for soybean uses (USEPA, 2010). No or 

limited toxicity has been observed with all species (plants, fish, Daphnia, algae, Lemna, birds, 

soil microgranisms, earthworms and rats) tested to date with RPA 203328. In the few cases 

where a toxicological response has been observed, it has been at high dose levels. Requiring 

extensive chronic ecotoxicity testing of a metabolite solely based on the potential for trace levels 

in the environment, when data exists to demonstrate the lack of toxicity, appears to a major 

policy shift by the Agency. Based on how the Agency has historically handled metabolites, in 

general and specifically in this case, and the available Agency guidance on this topic, Bayer 

believes that additional testing is not warranted with RPA 203328. 

“As summarized in Allen et al., 2004 and Allen et al., 2010, an extensive set of monitoring data 

are available for RPA 203328, RPA 202248 and parent isoxaflutole. In the vast majority of 

samples, RPA 203328 concentrations were below 0.3 ppb. A comparative example from these 

data indicate that the lowest EC50 value (for algae) was estimated by EPA to be 5900 ppm. Even 

the EECs estimated by the Agency using PRZM-EXAMS in 1997 (EPA, 1997) are 1000x lower 

than the lowest toxicity value. Based on the depth and breadth of available information, Bayer 

requests that the Agency reconsider their request for additional ecotoxicity testing with RPA 

203328.”

EFED Response: In order to evaluate the utility of degradate toxicity data, EFED uses both 

exposure potential and available toxicity data to determine if additional data would be useful to 

the risk assessment process. In the case of isoxaflutole, the two major degradates are converted 

at fairly high rates, and they are expected to be persistent and mobile. Therefore, evaluating 

potential risks from these degradates is considered an important aspect of the risk assessment. 

In light of comments received from the registrant, EFED has re-evaluated the utility of obtaining 

additional data on the two degradates as explained in the following sections. EFED notes that 

monitoring data rarely captures high-end exposure values from vulnerable areas. 

For degradate RPA 202248, Guidelines 850.4100 and 850.4150 - Terrestrial 

plant toxicity, Tier II (seeding emergence and vegetative vigor) 

#14 Bayer Comment: “The agency has requested tier II seeding emergence and vegetative 

vigor studies with RPA 202248. With respect to the vegetative vigor study request, it is difficult 

for BCS to come up with a scenario where plants would be directly oversprayed with RPA 

202248, which makes such a study irrelevant to the risk assessment. If additional data is 

necessary, then it would seem that exposure via the soil (seedling emergence study design) 

would be the more relevant route of exposure for any additional NTTP testing with RPA 202248.

“As stated by EPA, a non-target terrestrial plant (NTTP) screening study with RPA 202248 

exists, and Bayer has acknowledged that RPA 202248 is herbicidally active at a level similar to 

parent. We believe the toxicity of RPA 202248 is covered in studies with parent, since it is so 
7

rapidly formed in plants and soils after application of the parent isoxaflutole. A large number of 

NTTP studies, lab and field, are available with isoxaflutole, its formulations and to a lesser 

extent metabolites. Additional standard NTTP studies are unlikely to change the characterization 

of isoxaflutole’s risk to terrestrial plants. With the exception of more recent studies, this work 

has been summarized in Cappy, et al., 2002 and Dobbs et al., 2002.”

EFED Response: EFED agrees with the comment that vegetative vigor studies conducted with 

parent isoxaflutole would also include exposures of plants to degredate 202248. Since toxicity 

endpoints from vegetative vigor studies are expressed in terms of the amount of parent applied to 

the plant and they are intended to be compared to exposures of non-target plants to pesticide 

exposures through spray drift, existing studies with parent isoxaflutole are sufficient to 

characterize the effects of spray drift transport of isoxaflutole and its herbicidally active

degradate, RPA 202248. 

EFED recommends that PRD request seedling emergence data for RPA 202248 because 1) RPA 

202248 may form on the treated field for days before runoff events occur, leading to an exposure 

of non-target plants to RPA 202248 through runoff transport and 2) the available seedling 

emergence studies with isoxaflutole may have a smaller proportion of RPA 202248 than could be 

found on non-target areas exposed to this degredate through runoff. Therefore, it is important to 

have an understanding of the effects of RPA 202248 to the seedling emergence of non-target 

plants.

For degradates RPA 202248 & RPA 203328, Guideline No. 850.2300 - Avian 

reproduction 

#15 Bayer Comment: “The agency has requested 3 avian reproduction studies that include a 

mallard duck and bobwhite quail study with RPA 203328, and a mallard study with RPA 202248 

(a quail study already exists). While BCS believes that the available information is adequate to 

assess the risk to birds, BCS agrees to perform a mallard reproduction study with RPA 202248 as 

requested. However, as previously mentioned, BCS strongly disagrees that additional testing of 

RPA 203328 is necessary.”

EFED Response: Given that the available reproduction test with the bobwhite quail exposed to 

degradate 202248 did not show reproductive effects at 500 mg/kg-food, which is an order of 

magnitude below the expected environmental concentration (EEC) of isoxaflutole on potential 

food items of birds (if 100% conversion of isoxaflutole to degradate 202248 is assumed, the most 

conservative EEC is 34 mg/kg-food), EFED recommends that PRD waive the avian reproduction 

study with the mallard duck exposed to RPA 202248. 

In regards to degradate RPA 203328, EFED no longer considers this a degradate of 

toxicological concern for avian reproductive effects. This conclusion is based on three lines of 

evidence. First, this degradate would need to be at least an order of magnitude more toxic that 

degradate 202248 to pose a concern for birds on a chronic exposure basis. Second, a

developmental toxicity study in rats conducted with RPA 203328 has been submitted to the 

Agency (MRID 45655906). The results of the study show that in the presence of material toxicity 

at the highest doses tested (750 mg/kg/day), there was no teratogenicity or developmental 
8

toxicity. Third, the Health Effects Division (HED) has determined that degradate RPA 203328 is 

not of toxicological concern for humans. Therefore, EFED recommends that PRD does not 

require avian reproduction studies with this degradate.

For degradates RPA 202248 & RPA 203328, Guideline No. 850.1300 -

Freshwater invertebrate life cycle 

#16 Bayer Comment: “The Agency expressed concern about the lack of chronic invertebrate 

studies with isoxaflutole or its metabolites. Three studies are available to address this concern 

and will be submitted for review by the Agency. These studies include two Daphnia magna

studies that examined the impact of isoxaflutole around the practical limit of solubility and a 

limit test with Chironomous riparius at much lower concentration.

“The first Daphnia magna study was a flow-through study (McElligott, 1995; M-213123-01-1) 

where undissolved test material, that likely confounded the study results, was present at the two 

high test concentrations (0.75 and 1.5 mg/L nominal). The rapid addition of a concentrated stock 

to water with insufficient mixing time likely led to precipitate formation and continual delivery 

to the test chambers. Survival was impacted (65 and 73%) at the two high treatment levels 

where undissolved test substance was observed, but not at any of the treatment levels (0.09, 0.9, 

and 0.38 mg/L) where the test compound appeared to be fully in solution. No treatment related 

effects on reproduction were observed.

“A second Daphnia chronic study (Putt, 1998; M-210464-01-1) was conducted with isoxaflutole, 

using a static renewal study design. Nominal test levels is this study were 10, 3.1, 0.98, 0.31, 

0.095, and 0.030 mg/L. Undissolved test material was observed at the highest treatment of 10 

mg/L after mixing of new test solution, but prior to distribution to individual replicates, 

consistent with the reported solubility limit for isoxaflutole of 6.2 mg/L. No treatment related 

effects were observed at any test level, resulting in a NOEC of 5.7 mg/L based on measured 

concentrations.

“A water-sediment study (Odin-Feurtet, M., 1997; (M-211474-01-1) with Chironomous riparius

is available. There was a single treatment level of 100 ug/L of isoxaflutole spiked to the water. 

The isoxaflutole did degrade to predominately RPA 202248 during the 28 study. No negative 

effects were observed during the study.

“The 1997 studies showed no negative effect to aquatic invertebrates due to chronic exposure of 

solubilized isoxaflutole. Acute exposure testing of parent isoxaflutole, and the two primary 

metabolites, indicated no toxicity to Daphnia. While BCS believes there is sufficient information 

available to conclude that RPA 202248 and RPA 202328 pose minimal risk to aquatic 

invertebrates, BCS is willing to conduct a Daphnia magna chronic study with RPA 202248 to 

increase the Agency confidence in the risk assessment.

EFED Response: When the chronic invertebrate studies conducted with isoxaflutole that were 

referenced by Bayer are submitted, EFED will review them and determine whether or not they 

are sufficient for risk assessment under the guideline requirement. EFED agrees that submission 

of chronic toxicity data for RPA 202248 would increase the Agency‟s confidence in the risk 
9

assessment for isoxaflutole. If these studies are acceptable, EFED will consider using the 

submitted chronic toxicity studies involving exposures of D. magna to isoxaflutole to derive 

ACRs. These ACRs may be applied to the acute toxicity data for RPA 202248 and RPA 203328 

to estimate the chronic toxicities of these degradates to freshwater invertebrates. 

For degradates RPA 202248 & RPA 203328, Guideline No. 850.1350 -

Saltwater invertebrate life cycle 

#17 Bayer Comment: “The Agency has requested chronic mysid studies with RPA 202248 and 

RPA 203328. A mysid chronic is available for isoxaflutole, along with acute mysid studies with 

parent and both metabolites. As noted by the Agency in the problem formulation document, the 

metabolites are over two and close to four orders of magnitude less toxic to shrimp than parent. 

The 96-hr mysid LC50 values are 0.0178, 3.6. and 145 mg/L for isoxaflutole. The acute values 

for the two metabolites are above the 1 mg/L criteria as listed in the 40 CFR Part 158 guidance 

and the expected EECs for the metabolites are well below the LC50 values. The statement by 

the Agency that the chronic mysid study with parent suggests the potential for reproductive 

effects is difficult to understand. Survival was the most sensitive endpoint in mysid chronic with 

parent and there were not significant effects on reproduction. Even at the two highest test 

levels,(where survival was 13 and 48 percent, respectively), reproduction on an 

offspring/female/reproductive day was comparable to the control.

“BCS requests the Agency to reconsider their request for mysid chronic studies with two 

metabolites, or possibly provide a more detail explanation as to why these studies are triggered 

based on existing guidance.”

EFED Response: Available acute toxicity data for isoxaflutole and RPA 202248 indicate that 

this degradates is less toxic (on an acute exposure basis) than isoxaflutole by two orders of 

magnitude. If the available ACR for available toxicity data for the mysid shrimp exposed to 

isoxaflutole (17.8) is applied to the acute toxicity endpoint for RPA 202248 (3600 ppb), the 

estimated chronic toxicity endpoint for mysid shrimp exposed to this degradate would be 200 

ppb. In the most recent ecological risk assessment of isoxaflutole (DP Barcode 370959), EFED 

derived a 21-d EEC of 7.6 ppb for RPA 202248. If this EEC and the estimated chronic toxicity 

endpoint were used to determine risk to estuarine/marine invertebrates, this degradate would not 

be of concern. In order to pose a risk to estuarine/marine invertebrates, RPA 202248 would have 

to be 2 orders of magnitude more toxic on a chronic exposure basis to be of concern. 

Available acute toxicity data for isoxaflutole and RPA 203328 indicate that this degradates is

less toxic than isoxaflutole by four orders of magnitude. Using the ACR for isoxaflutole (17.8) 

and the acute toxicity endpoint for RPA 203328, the estimated chronic toxicity endpoint for this 

degradate is 8300 ppb. Given that the EEC for isoxaflutole‟s other degradate, i.e., RPA 202248 

is orders of magnitude below this level, it is not expected that degradate RPA 203328 will be 

present at levels sufficient to pose a risk. 

EFED believes that available acute and chronic toxicity data for isoxaflutole are sufficient to 

derive ACRs that may be used to estimate the chronic toxicities of degradates 202248 and 
10

203328 to estuarine/marine invertebrates. EFED does not believe that it is necessary to require 

chronic toxicity studies involving exposures of mysid shrimp to degradates 202248 and 203328.

For degradates RPA 202248 & RPA 203328, Guideline No. 850.1400 -

Freshwater and saltwater fish early life stage 

#18 Bayer Comment: “BCS will conduct a freshwater fish ELS study with RPA 202248 as 

requested by Agency, but requests that an estuarine fish ELS study be held in reserve contingent 

on the outcome of the freshwater ELS. As discussed, based on the structure and activity 

information available for RPA 203328, BCS does not believe additional testing of RPA 203328 

is necessary or warranted.”

EFED Response: If the registrant conducts an acceptable ELS study with a freshwater fish (of 

most interest would be a test species for which acute toxicity data are available) exposed to RPA 

202248, EFED would be willing to consider applying an ACR based on RPA 202248 to estimate 

the chronic toxicity of RPA 203328 to freshwater fish. This same ACR could also be applied to 

estuarine/marine fish. Therefore, it would only be necessary for the registrant to submit one fish 

ELS study.

For degradates RPA 202248 & RPA 203328, Guideline No. 850.5400 - Aquatic 

non-vascular plant growth Tier II 

#19 Bayer Comment: “The Agency appears to have requested a total of six (6) additional algal 

studies with RPA 202248 and RPA 203328 that include Navicula pelliculosa, Anabaena flosaquae, and Skeletonema costatum for each metabolite. BCS targets metabolite testing to 

determine the relative toxicity of the metabolite to parent. In the case of isoxaflutole, RPA 

202248 and RPA 203328, and this specific data request, both metabolites are orders magnitude 

less toxic to Selenastrum capricornutum. In fact, for all aquatic taxa tested isoxaflutole 

metabolites are at least an order less toxic than parent. BCS believes that the additional algal 

testing with the metabolites is excessive and highly unlikely that and requests that the Agency 

reconsider the need for these additional studies.”

EFED Response: EFED agrees that the available green algae (S. capricornutum) toxicity data 

are sufficient to allow for characterization of the relative toxicities of isoxaflutole, RPA 202248 

and 203328 to algae. EFED no longer recommends that PRD request additional toxicity data 

involving exposures of the other 3 species of algae to RPA 202248 and 203328.

References:

Allen, R., P. Cain, P. Freesman, I. Hardy, R. Jones, I. Kelly, R. Lee, B. Narasimhan, T. S. 

Ramanarayanan, G. Sabbagh, L. Somerville, and R. Srinivasan. 2004. The Environmental Fate 

of Isoxaflutole Following Use on Corn in the U.S. Bayer CropScience Report No. B004653. 

Bayer Document No. M-251278-01-1. 374 pages. MRID No. 46330201
11

Allen, R., M. Dobbs, R. Jones, G. Sabbagh. 2010. Environmental Fate and Ecological Risk 

Assessment for Isoxaflutole Use in Corn and Soybean. Bayer CropScience Report No. US0061, 

Document No. M-372827-01-1, 28 p.

USEPA, 2010. Ecological Risk Assessment Addressing the “Crop Destruct” Experimental Use 

Permit (EUP) for Isoxaflutole and its End-product Balance® PRO Herbicide for use on Select 

Soybean varieties. Memorandum from M. Davy and W. Shaughnessy (OPP/EFED) to Kathryn 

Montague and James Stone (RD). DP Barcode 370959. April 16, 2010