Oiethofencarb Human Health Risk Assessment

UNITED ST A TES ENVIRONMENT AL PROT ECTION AGENCY WASHINGTON, D.C. 20460 0418227

OFFICE OF C llEMI CAL SAFE TY AND PO L LUTION PREVENT I ON

MEMORANDUM

Date: 27-AUG-2015

SUBJECT: Di e th ofe n carb. I Ium an H ea l th Ri sk Assessme nt fo r the Proposed To ler ances without a U.S. Registration for Residues of Diethofencarb inion Ban a na s.

PC Code: 112102 Deci s i on No.: 486694 Pet iti on No.: 4 E 8232 DP Barcode: 0418227 Registrati o n No.: NA Regu l atory Actio n : Tolerances Without U.S. Registration Ri s k Assessment Type: Sing l e Chemical/Agg r egate TXR No.: NA Case No.: NA CAS No.: 87130-20-9 40 CFR: § 180.TBD MRIO No.: NA

FROM: Cass i L. Walls, Ph .D.

, Senior Biologist

## ~ 2.

## ~ Risk Assessment Branch I (RAB l) 

### -....Jt2-"'-**~** Myron Ottley

, Toxicologist 

## /'hr)d} f /...,-**~** William Wassell, C hemis t Risk Assessment Branch 3 (RAB3) Health Effects Di vision (HE D ; 7509P)

THROUGH: Christine O li nger , Branch Chief /} 

## ;f 

# /.JC------

RAB3, HED (7509P) 

# if G 

# r

Charles Smith

, Designated Reviewer

### ~ AND

## ~~ Donald Wilbur, Designated R eviewer

### ~(..,.£, Risk Assessment Review Committee (RARC / HED)

TO: Rachel Holloman/Driss Benmhend Fungicide-Herbicide Branch Registration Division ( RD ) (7505P)

The Health Effects Division ( H ED) of the Office of Pesticide Programs (OP P ) is charged with estimating the risk to human health from exposure to p est icid es. RD of OPP has reque sted that HED evaluate hazard and exposure data and conduc t a dietary exposure assessment as needed, to estimate the risk to human health that wi ll result from the proposed use of the new active ingredient

, die th ofencarb

, 1-methylethyl N-(3,4-diethoxyphe n y l )carbamate.

A summary of the findings and an assessmen t of human risk resulting from the proposed uses are provided in thi s document. The risk assessment and dietary risk assessment were provided by Cassi Walls (RAB I )

, the hazard characterization was prov id ed by Myron Ottley (RAB3) and the residue chemis tr y review was provided by William Wassell (RAB I ).

Page I of 49Diethofencarb Hum a n Health Ri sk Assessment 0418227
Table of Contents 1.0 Executive Sumn1ary

................ .. ........... . ... ......... ... .. .......... .. .. ............................ ... ... . ........... 3 2.0 HED R ecommendations

.................................................................................................... 4 2.1 Data D efic i enc i es

..................................................................................................... .. .. ... 4 2.2 Tolerance Cons id erat i o n s

.......... .. .. ... .............. .. .......... .. ..... .. .. ........ ... . ............. . ...... . .. ..... 4 2

. 2.1 Enforcement Ana l ytical Method

.. .. .. .. ...... ... .. . ................................... . ....................... 4 2.2.2 Recommended To l e r ances

.......... ........................... ... .... .. .. . ........................................ 4 2.2.3 Revision s to Pet it ioned-For Tolerances

.................................................................... 5 2.2.4 International H a rmonization

.......... ... .......... .. .. .. .. .......... . ..... .. ... ........... ... ... .............. . . 5 3.0 Intr oductio n

.... .. ... .... . .......... . .......... . ... .. ...... .. ... .. ...... ...... ................. ........ ... .. . ...... . ................ 5 3.1 Chemica l Identity

... . ...... . ... . ................... .. ...... .. ....... .. ......... ... .... .. .. ....................... . .......... 5 3 .2 P h ysical/Chem ic a l Properties

.................................................................... ........ .... . ....... 5 3.3 Use Pattern

. .. .......................................................... .. .. .. ..... ... . ... .... . ... .. ............... . ......... . ... 5 3.4 Ant icip ated Exposure Pathways

. .. ........................... .. ... ... ... ........................... .. ......... . ... 6 4.0 Hazard C haract eriza ti on and Dose-Response Assessment

...................... .. ..... ............... 6 4.1 Tox i co l ogy Stud i es Availab l e for Ana l ys i s

................................. . ......... .. ...................... 6 4.2 Absorption, Di st ribution, Metabolism

, & E limin at i on (ADME)

............................... 6 4.3 Tox i co l ogical E ffects

.......................................................................................... .. . ..... .. .. 7 4.4 Safety Factor for In fants and Ch ildr e n (FQPA Safety Factor)

.... . .. ...... .. . ................. 7 4.4.1 Comp let eness of the Toxicology Database

.. .. .......... . ............................................. 8 4.4.2 Evidence of Neurotoxic i ty

...................................................................................... 8 4.4.3 Evidence of Sensit i vity / Susceptibility in the Developing or Young Anima l

...... 8 4.4.4 Re s idual Uncerta in ty in the Exposure Database

.. ... ................ ........ .... ........ .. ...... . 8 4.5 Toxicity Endpoint and Point of Departure Se l ections

.... .............. .. ............................ 8 4.5.1 Do se -Respon se Assessment

.. ...... .. .. .. .............................................................. ........ 8 4.5

. 2 Cancer C l ass ifi cation and Risk Assessment Recommendation

.......................... 9 4.5.3 S umm ary of Points of Departure and Toxicity Endpo int s Used in Human Risk Assessn1ent

.......................................................................................... .. ......... .. .... ... ............... 9 5.0 Dietary Expos ure and Risk Assessme nt..

........................................ .. .. ........................... 10 5.1 Metabo lit e/Degradate Residue Profile

....................... . ......... ......... .. ..... . ..................... 1 0 5.1.1 Summary of Plant and Animal Metabolism Studies

................................ . ........ 10 5.1.2 Summary of Environme nt al Degradation

.......................................................... 1 0 5.1.3 Comparison of Metabol ic Pathways

.................................................................... 10 5.1.4 Residues of Concern Summary and Rationale

.......... ......................................... 11 5.2 Food Residue Profile

...................................................................... .. ............................ 11 5 .3 Drinking Water Residue Profile

................................................................................. 1 2 5.4 Dietary (Food Onl y) Risk Assessment

........................................................................ 1 2 6.0 Re s idential (Non-Occupationa l ) Expos ure/Ri sk Characterization

............................. 12 7.0 Aggregate Expos ur e/Risk Character i zat ion

... .............. ................................................ . 12 8.0 Cumu lati ve Exposure ! Risk Characterization

............................................................... 1 3 9.0 Occupational Exposure/Risk Characterization

... .. ................... .... ................................ 1 3 Appendix A. Toxico l ogy Profile and Executive S umm aries

................................... .. ............ . 14 A.1 Toxico l ogy Data Requirements

.. .. .. ... ..... ... .. .. .......... ..... .. .. .. ......... .... .. .. ... ..................... 14 A .2 Toxicity Profile s

.............................. ................... . ..... ... ..... .. .. .... ... .. ............................ ... . 15 Appendix B. Metabolism Considerations

................................................................................ 46 B .1

. Metabo li sm Summary Table

................................................................................... .. .. 46 B.2. Metabo li c Path\vays

.. ...... . .. .. ...... . ........... ... .. .. ............... . .......... . ...... . ...... . ... .. ..... . .. ...... .... ...... 48

Pa ge 2 of 49Diethofencarb Hum an Health Risk Assessment D418227

Page 3 of 49 1.0 Executive Summary

The new active ingredient (ai) diethofencarb ( 1

- methylethyl N

- (3,4

- diethoxyphenyl)carbamate ) is a n N

- phenyl, O

- alkylcarbamate fungicide

. It is a broad spectrum fo liar fungicide for use on a variety of fruits and vegetables

. Diethofencarb is currently registered in the Europe an Union and Japan

. The registrant (Bryant Christie Inc. on behalf of Sumitomo Chemical Co.) submitted a petition for establishment of a toleranc e for residues of diethofencarb in/on bananas . There are no proposed or established domestic uses . The proposed tolerance is expressed in terms of the parent only.

Exposure Profile: Based on the proposed uses of diethofencarb , dietary exposure (food only) is the only anticipated exposure pathway. As there are no domestic agricultural or residential uses register ed or proposed for diethofencarb , there is no potential for drinking water, residential, or occupational exposure.

Hazard Assessment: The risk assessment for diethofencarb is based on a well

- characterized and complete toxicology database. In repeated dose animal studies, the liver was a target organ in the rat, mouse and dog. The most common effects were increased liver weights, hyper trophy and increase cholesterol levels which were not considered adverse. However, increased liver pigmentation observed histologically in the dog, and foci of necrosis and hepatocellular hyperplasia in the mouse, were considered adverse and evidence of t oxicity . Other target organs identified were the kidney (proteinaceous cast and regenerative epithelium), urinary bladder (submucosal lymphoid hyperplasia) and thyroid (follicular cell adenomas and carcinomas) in the mouse, and the nervous system (changes in functional observational battery parameters, decreased motor activity and decreased pupillary reflex) in the rat. Decreased body weight and food consumption and increased salivation were observed in the dog, and increased abortions were observed in th e rabbit. Decreased body weight was also seen in F2 pups during lactation in the rat in the absence of parental toxicity, raising a concern for increased susceptibility in offspring. However, appropriate endpoints and points of departure were used to add ress the susceptibility issue and there are no residual pre

- and /or post

- natal uncertainties, so the Food Quality Protection Act (FQPA) Safety factor has been reduced to 1X. The level of concern (LOC) for all durations and routes of exposure is 100. Mutag enicity tests were negative and data were submitted suggesting a non

- genotoxic mode of action for the thyroid tumors observed in male rats. Diethofencarb is classified as “suggestive evidence of carcinogenicity” with no quantification required

. There was no evidence of immunotoxcity in the immunotoxcity study or any other studies in the database.

Dietary Risk Estimates (Food Only ): A chronic dietary (food only) exposure and risk assessment was conducted for the general U.S. population and various population subgroups using the Dietary Exposure Evaluation Model software with the Food Commodity Intake D atabase (DEEM

- FCID) Version 3.16 . This software uses 2003

- 2008 food consumption data from the U.S. Depart ment of Agriculture’s (USDA’s) National Health and Nutrition Examination Survey, What We Eat in America, (NHANES/WWEIA). The unrefined chronic

asses sment assumed that diethofencarb residues are present in all commodities at tolerance levels and that 100% of all crops are treated. DEEM Version 7.81 default processing factors were used to estimate re sidues in processed commodities . Drinking water estimates were not incorporated into the chronic dietary assessment, as there are no domestic uses proposed or established. Using the DEEM

- FCID softwar e, d ietary risk is estimated at <1 % of the cPAD for the general U.S. population and children 1 to 2 years old , the population subgroup with the highest estimatedDiethofencarb Hum an Health Risk Assessment D418227

Page 4 of 49 chronic d ietary exposure to diethofencarb . The chron ic dietary risk assessment shows that for all included commodities, the chronic dietary risk estimates are below HED’s level of concern (i.e., <100% cPAD). An endpoint of concern attributable to a single dose was not identified; therefore, an acute dietar y assessment was not performed.

Aggregate Exposure Assessment: Because this action is for a tolerance w ithout U.S.

r egistration and there are no proposed o r existing uses of diethofencarb that would result in drinking water or residential exposures

, th e chronic dietary (food only) exposure estimates represent the aggregate exposure assessment

.

2.0 HED Recommendations

T here are no data gaps that would preclude establishing the recommended toleranc es for residues of diethofencarb in/on the requested crop . The sp ecific tolerance recommendation is discussed in Section 2.2.2.

2.1 Data Deficiencies

T here are no data deficiencies at this time ; however, a revised Section F is required to amend the proposed tolerance level. HED notes that additional plant metabolism, livestock metabolism, confined rotational crop, and livestock feeding studies may be necessary to support any future use proposed for the U. S.

2.2 Tolerance Considerations

2.2.1 Enforcement Analytical Method

A high

- performance liquid chromatography method with tandem mass spectrometry detection (HPLC/MS/MS), PTRL West Method No. 2348W is proposed for the determination of residues of diethofencarb in banana pulp and banana peel. The method is adequate for the determi nation of diethofencarb in banana peel and pulp with a validated LOQ of 0.01 ppm. The method was adequately validated using samples of banana peel and banana pulp fortified with diethofencarb at 0.01 ppm and 0.10 ppm. An adequate independent laboratory v alidation (ILV) was conducted.

QuEChERS

- method DIN 15662:2009

- 02 and DFG

- S19 ha ve been successfully tested with diethofencarb. HED concludes adequate enforcement methodology are available to enforce the proposed tolerance.

2.2.2 Recommended Tolerances

HED has reviewed the available residue data and has determined the appropriate tolerance leve ls for residues of diethofencarb (Table 2.2.2.1). The recommended tolerance expression is as follows:

(a) General. (1) Tolerances are established for residues of the fungicide diethofencarb

, including its metabolites and degradates, in or on the commodities in the table below. Compliance with the tolerance levels specified below is to be determined by measuring only (1

- methylethyl N

- (3,4

- diethoxyphenyl)carbama te).Diethofencarb Hum an Health Risk Assessment D418227

Page 5 of 49 Table 2.2.2.1 . Tolerance Summary for Diethofencarb

. Commodity Proposed Tolerance (ppm) HED

- Recommended

Tolerance (ppm) Comments

( correct commodity definition ) Banana 1 0.09 0.10

1 No U.S. registration as of [publication date of the tolerances]

.

2.2. 3 Revisions to Petitioned

- For Tolerances

As per the Organization for Economic Co

- operation and Development Maximum Residue Limit ( OECD MRL ) methodologies , the submitted residue data support a tolerance level slightly higher than what was proposed. A revised Section F is required.

2.2. 4 International Harmonization

There are no Codex, Canadian, and Mexican MRLs established for diethofencarb in/on bananas. There are no harmonization issues.

3.0 In troduction

3.1 Chemical Identity

The chemical structure and nomenclature of diethofencarb is presented in Table 3.1.1.

Table 3.1.1. Diethofencarb Nomenclature. Compound: ai Chemical Structure

Common name Diethofencarb Company experimental name None IUPAC name i sopropyl 3,4

- diethoxycarbanilate CAS name 1

- methylethyl N

- (3,4

- diethoxyphenyl)carbamate CAS # 87130

- 20

- 9 End

- use product/EP Powmyl 25 SC (250 g/kg diethofencarb, suspension concentrate). Not registered in the US.

3.2 Physical/Chemical Properties

Technical

- grade diethofencarb is a solid at room temperature and all environmentally relevant temperatures. The compound is not volatile (i.e., vapor pressure is 9.44 x 10

- 6 Pa at 20° C) . The technical

- grade material has a log K OW of 2.89 , indicating tha t the compound is fat

- soluble. Appendix C presents the physical/chemical parameters for diethofencarb.

3.3 Use Pattern

The petitioner provided copies and English tra nslations of the proposed POWMYL ® 25 SC label

. POWMYL ® is a SC formulation containing 25% diethofencarb for ground or aerial applicationsDiethofencarb Hum an Health Risk Assessment D418227

Page 6 of 49 to bananas . The proposed use directions , as identified by the petitioner, are detailed in Table 3.3.1.

Table 3.3. 1 Summary of Directions for Use of Diethofencarb on Bananas 1

. Applic. Timing, Type, and Equip. Formulation Applic. Rate Max. No.

Applic. per Season Max. Seasonal

Applic. Rate PHI

(days) Use Directions and

Limitations

Banana

Foliar 250 g/kg SC 0.15

- 0.20 kg ai/ha ; ( 0.13

- 0.18 lb ai/A) 6 1.2 kg ai/ha ( 1.1 lb ai/A) 0 Application interval is 5

– 8 days.

1 No domestic registrations (uses) are requested. The proposed label is for Guatemala, Belize, El Salvador, Honduras, Nicaragua, Costa Rice, Panama, and Dominican Republic and does not contain use information. The information entered is taken from the European Import Tolerance Request Tier II, Document M (MRID #49267481), prepared by the applicant.

3.4 Anticipated Exposure Pathways

Based on the proposed diethofencarb application scenarios, dietar y exposure (food only) is the only anticipated exposure pathway. As there are no domestic agricultural or residential uses register ed or proposed for diethofencarb , there is no potential for drinking water, residential, or occupational exposure.

4.0 Haza rd Characterization and Dose

- Response Assessment

Diethofencarb

, a FRAC 10 N

- phenyl carbamate , is a new active ingredient (a

. i

. ) fungicide with broad spectrum activity in fruits and vegetables. It is a systemic fungicide with protective and curative action. It is readily absorbed though the leaves and roots and translocated throughout the plant. It inhibits mitosis in grey mold germ tubes, and has secondary activity against powdery mildews. This mode of action has not been established in mammals.

4. 1 Toxicology Studies Available for Analysis

All of the required toxicity studies have been submitted to support the proposed food

- use tolerance for diethofencarb and the potential exposure scenarios associated with this action. The available toxicology d atabase is sufficient to characterize the hazard of diethofencarb for tolerance in the absence of a U.S. registration . Available repeated dose studies include oral subchronic and chronic studies in rat s, mice and dog s , oral carcinogenicity studies in the rats and mice, oral pre

- natal developmental studies in rats and rabbits, a multi

- generation reproduction study in rats, a mutagenicity battery, a neurotoxicity battery in rats, an immunotoxicity study in mice, and a dermal subchronic study in rats.

4.2 Ab sorption, Distribution, Metabolism, & Elimination (ADME)

Data from the ADME tests indicate that there is quick and extensive absorption of diethofencarb, limited distribution in tissues, extensive metabolism into sulfate and glucuronide metabolites, and q uick and extensive excretion with no bioaccumulation. In a standard series of ADME tests with single and multiple gavage dosing at various dose levels, metabolism of diethofencarb in rats was found to be independent of sex, dose level and pre

- treatment. Oral absorption is quickDiethofencarb Hum an Health Risk Assessment D418227

Page 7 of 49 and extensive ( > 87% of administered dose or AD by day 2). Organ/tissue radioactivity decreased rapidly and was mainly found in liver, adrenals, blood/blood cells, kidney and skin. T max was 0.5 to 1.0 hr in blood and tissue. A plasma T 1/2 value was not included in the report. Diethofencarb was extensively biotransformed into s everal metabolites, mainly sulf ate and glucuronide conjugates. Excretion was extensive and quick ( > 93% of AD by day 2) and occurred in urine ( > 61% of AD ), expired air ( > 21% of AD) and feces ( > 13% of AD). This resulted in organ/tissue depuration t 1/2 values of < 35 hours based on dat a up to 72 hr post

- dose and a low retained fraction ( < 2 % of AD on day 7), and the deduction that there is no significant

en tero

- hepatic recirculation. The quick and extensive excretion, as well as the rapid organ/tissue decrease in radioactivity suggested there is no bioaccumulation.

4.3 Toxicological Effects

In repeated dose animal studies, the liver was a target organ in the rat, mouse and dog. The most common effects were increased cholesterol levels, increased liver weights, and hypertrophy ; f oci of necrosis and hepatocellular hyperplasia were observed in the mouse. Other target organs identified in the database were the kidney (proteinaceous cast and regenerative epithelium), urinary bladder (submucosal lymphoid hyperplasia) and thyroid (follicular cell adenomas and carcinomas) in the mouse

. Decreased bo dy weight and food consumption and increased sali vation and vomiting were observed in the dog

. In developmental toxicity studies, no adverse effects were seen in rat dams or fetuses up to the limit dose. In rabbits, an increase in late

- term abortions o ccurred only at dose levels approaching the limit dose. In the multi

- generation reproduction study in rats, no parental or reproductive effects were observed at any dose level tested; however, d ecreased body weight was seen in pups of the F2 generation dur ing lactation in the absence of parental toxicity, suggesting increased quantitative susceptibility in offspring

. There was no evidence of immunotoxicity in the database, including the immunotoxicity study. Mutagenicity tests were negative , but as ment ioned above, thyroid follicular cell tumors wer e seen in the rat and HED has determined a cancer classification for diethofencarb as “suggestive evidence of carcinogencity to humans” with no quantification required based on evidence of adenomas and carcinomas in one species (rat). There was no evidence of tumors in the mouse.

In acute studies, diethofencarb is categorized as Toxicity Category IV following oral and dermal exposure, and Toxicity Category III following inhalation exposure; it cause d slight irritation to the eye (Toxicity Category III), no irritation to the skin (Toxicity Category IV) and is not a dermal sensitizer.

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

Based on the currently available toxicity and exposure data, the risk assessment team recommends that the FQPA Safety Factor be reduce d to 1X

. The toxicity database for diethofencarb is considered adequate for risk assessment for the following reasons: 1) the dietary exposure assessment is based on reliable data and will not underestimate exposure; 2) there is evidence of reproductive su sceptibility as shown in the multi

- generation reproduction study, but the effect is well

- characterized; and 3) there is a clear No Observed Adverse Effects Level ( NOAEL ) for this effect and there are no residual pre

- or post

- natal uncertainties.Diethofencarb Hum an Health Risk Assessment D418227

Page 8 of 49 4.4.1 Completeness of the Toxicology Database

The diethofencarb toxicology database for FQPA assessment is considered adequate for purposes of setting a tolerance in the absence of a U.S. registration

. It includes developmental toxicity studies in rats and rabbits, a multi

- generation reproduction study, an acute and subchronic neurotoxicity battery

, and an immunotoxicity study

.

4.4.2 Evidence of Neurotoxicity

The available database did not show diethofencarb to be neurotoxic. In the available acute

neuro toxicity study (MRID 49267464), transient changes (observed on day 0 only) in decreased forelimb grip strength

, reduced body temperature , increased landing foot

- splay, decreased muscle tone and pupillary reflex, and reductions in motor activity were observ ed at the LOAEL of 2000 mg/kg in males and/or females

. The NOAEL was 600 mg/kg

. In the subchronic neurotoxicity study (MRID 49267463), decreased pupillary reflex seen in males at 571 mg/kg/day was not considered biologically relevant because the degree o f decrease was rated as “slight” in all affected animals, with no other changes in neurobehavioral or neuropathological parameters noted. No adverse effects were seen in females in this study. Therefore, the NOAELs for the study were 571 and 654 mg/kg bw /day, in males and females, respectively , the highest dose level tested

.

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

I n the rat developmental stud y (MRID 49267450 ), t here were no indications of toxicity in the dam s or fetus es up to the limit dose. An acceptable (non

- guideline) rabbit developmental toxicity study showed late

- term abortions (considered evidence of both maternal and fetal toxicity) at dose levels near the limit dose (800 mg/kg/day and above)

. In the rat reproduction study, offspring effect s (decreased pup body weight in F 2 males and females) were noted below the parental NOAEL, indicating increased quantitative susceptibility in offspring. However,

c lear NOAELs and LOAELs are available for all parent al and offspring effects and endpoints and PODs are based on the effects in the offspring.

4.4.4 Residual Uncertainty in the Exposure Database

There are no residual uncertainties in the exposure database. The exposure databases are sufficient
to determine the nature/magnitude of the residue in food and dietary analyses are unlikely to underestimate ris k of exposure from diethofencarb

. In addition, there are no currently proposed residential uses.

4.5 Toxicity Endpoint and Point of Departure Selections

The toxicity e ndpoints and PODs are summariz ed in Table 4.5.3

.

4.5.1 Dose

- Response Assessment

For acute dietary exposure ( all populations including Females 13+ )

, an endpoint attributable to a single dose has not been identified in the databas e; therefore, there is no risk assessment for acute dietary exposure . The acute neurotoxicity study produced effects only at the limit dose ofDiethofencarb Hum an Health Risk Assessment D418227

Page 9 of 49 2000 mg/kg which is not relevant for risk assessment purposes, and the developmental and other available studies produced no effects that could be attributed to a single dose.

The chronic dietary endpoint was chosen from the chronic dog study ( MRID 4926745 2 )

. The NOAEL was 50 mg/kg/day and the LOAEL was 250 mg/kg/day in males and females, based on decreased body weights and increased emesis

. This endpoint is considered to be robust and appropriate for the duration and route of exposure. It is considered protective of effects seen in the database following chronic oral exposur es in mice, rats

, and dogs. Although the 2

- generation reproduction study had a higher NOAEL and a similar LOAEL to the chronic dog study, it was not selected because the effects in the reproduction study were seen only the F 2 pups and it is uncertain what duration of exposure is needed to elicit the pup effects, whereas, the duration to cause adverse effects in the chronic dog study is clear.

Application of a 100

- fold uncertainty factor has been determined to be appropriate (10X inter

species extrapolatio n and 10X intra

- species variation). The FQPA SF is 1X, the cRfD is equivalent to the cPAD.

cRfD = c PAD = (50 mg/kg/day) = 0.50 mg/kg/day

100

Additional oral endpoints, as well as dermal and inhalation endpoints, were not evaluated or selected because they are not required for this import tolerance registration action.

4.5.2 Cancer Classification and Risk Assessment Recommendation

Diethofencarb did not produce a significant increase in tumors in a 78

- week mouse oncogenicity study at dosages up to 2108 mg/kg/day, twice the limit dose. In rats, thyroid follicular cell tumors (adenomas in females and carcinomas in males) were observed at 220.3 /288.19 mg/kg/day, respectively. Special studies are available which suggest that the mode of action for thes e thyroid tumors is not quantitatively relevant to humans and there is no risk to human s , but the CARC examined these data and found they were inadequate to establish a mode of action. Consequently the CARC has assigned the classification “suggestive evide nce of carcinogenicity to humans” based on the rat thyroid tumors, but quantification is not required. In both the mouse and rat studies, dosing was considered adequate to assess carcinogenic potential

.

4.5.3 Summary of Points of Departure and Toxicity En dpoints Used in Human Risk Assessment

Table 4.5.3 Summary of Toxicological Do ses and Endpoints for Diethofencarb for Use in Non

Occupational /Residential Human Health Risk Assessments a

Exposure/ Scenario Point of

Departure Uncertainty/

FQPA Safety Factors RfD, PAD, Level of

Concern for Risk Assessment Study and Toxicological Effects

Acute Dietary (All Populations including Females 13+ ) An endpoint attributable to a single dose was not identified in the data base.Diethofencarb Hum an Health Risk Assessment D418227

Page 10 of 49 Table 4.5.3 Summary of Toxicological Do ses and Endpoints for Diethofencarb for Use in Non

Occupational /Residential Human Health Risk Assessments a

Exposure/ Scenario Point of

Departure Uncertainty/

FQPA Safety Factors RfD, PAD, Level of

Concern for Risk Assessment Study and Toxicological Effects

Chronic Dietary (All Populations) NOAEL = 50

mg/kg/day UF A = 10X

UF H = 10X FQPA SF = 1 X Chronic RfD = 0.50 mg/kg/day

cPAD = 0.50 mg/kg/day Chronic Toxicity, Dog. LOAEL = 250 mg/kg/day based on decreased body weights and emesis. Cancer (oral, dermal, inhalation) Classification: “suggestive evidence of carcinogenicity to humans” based on the rat thyroid

follicular cell tumors; quantification is not required.

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

- response data and used to mark the beginning of extrapolation to determine risk associated with lower environmentally relevant human exposures. NOAEL = no observed adverse effect level. LOAEL = lowest observed adverse effect level. UF = uncertainty factor. UF A = extrapolatio n from animal to human (interspecies). UF H = potential variation in sensitivity among members of the human population (intraspecies). FQPA SF = FQPA Safety F actor. RfD = Reference Dose. cPAD = chronic Population Adjusted Dose.

5.0 Dietary Exposure and Risk Assessment

D421677 , W. Wassell

, xx

- AUG

- 2015 (residue chemistry chapter for the current action) D428591 , C. Walls

, 18

- AUG

- 2015 (dietary exposure analysis for the current action) D428492, E. Holman, xx

- AUG

- 2015 (ROCKS memo for the current action)

5.1 Metabolite/Degradate Residue Profile

5.1.1 Summary of Plant and Animal Metabolism Studies

The metabolism of radiolabeled diethofencarb was studied in grapes, tomatoes, and lettuce. Studies were performed with [ 14 C

- phenyl]

- and [ 14 C

- isopropyl]

- diethofenc arb. No study was conducted with banana. The result s showed that only parent was found as a surface residue. I n the three plant metabolism studies, d iethofencarb is a major portion of the residue (>20%)

, however, the parent concentration decreases with increasing pre

- harvest interval ( PHI )

. Residues in plants undergo hydroxylation and conjugation while maintaining the diethofencarb backbone.

Livestock metabolism data is not required as there are no feed items associated with bananas.

5.1.2 Summary of Environmental Degradation

As there are no domestic agricultural or residential uses register ed or proposed for diethofencarb

, environmental degradation is not relevant for this petition.

5.1.3 Comparison of Metabolic Pathways

Plant metabolism studies indicate that unchanged parent is a major portion of the residue. The total amount of parent declined with increasing PHI. Parent was 70.6% and 48.0% Total Radioactive Residues ( TRR ) in tomato, and lettuce, respectively at short PHIs (7

– 10

- days). At the longer PHI of 35 days for grapes, parent declined to 21.5% TRR. Identified metabolites represent hydrolysis and subsequent conjugation of the parent. All metabolites have theDiethofencarb Hum an Health Risk Assessment D418227

Page 11 of 49 backbone structure of diethofencarb. Significant amounts of the TRRs were unidentified and attributed to many minor metabolites of relatively low concentration (<10% TRR).

Rat metabolism studies have shown that there is quick and extensive absorption of diethofencarb, limited distribution in tissues, extensive metabolism into s ulfate and glucuronide metabolites, and quick and extensive excretion with no bioaccumulation. Appendix B presents the chemical structures of parent and metabolites of diethofencarb.

No confined rotational crop studies were submitted. Additionally, no livestock metabolism studies were provided. These studies are not relevant to a tolerance without U.S. registration on a crop with no livestock feed components. Additionally, residues in water are not a concern for a tolerance without U.S. registration.

5.1.4 Residues of Concern Summary and Rationale

Upon examination of the available data, the HED Residues of Concern Knowledgebase Subcommittee (ROCKS) determined that th e parent compound, diethofencarb , is a suitable residue definition for purposes of both tolerance enforcement and risk assessment in plants

.

Table 5.1.4. 1. Summary of Metabolites and Degradates to be included in the Risk Assessment and Tolerance Expression for Bananas

Matrix Residues included in Risk Assessment Residues included in Tolerance Expression

Plants Primary Crop Diethofencarb Diethofencarb

Rotational Crop Not applicable 1 Not applicable 1

Livestock Ruminant Not applicable 1 Not applicable 1

Poultry Not applicable 1 Not applicable 1 Drinking Water Not applicable 1 Not Applicable

1 Request is for a tolerance without U.S. registration on banana only.

5.2 Food Residue Profile

Ten independent banana field trials were conducted during the 2012 growing season in Costa Rica (3 trials), Ecuador (2 trials), Guatemala (3 trials), and Honduras (2 trials). The number and distribution of trials are adequate to support a tolerance for diethofencarb without U.S. registration on banana. Guatemala, Ecuador, Costa Rica, Co lombia, and Honduras account for about 94% of U.S. banana imports (University of Florida, IFAS Extension, 2012). The trials reflect the proposed use directions, and the analytical method is adequate for the analysis of banana samples. Samples were stored frozen for a period of demonstrated residue stability.

P eel and pulp samples were analyzed separately for parent only. Residues in the peel ranged from non

- detectable to 0.15 ppm; residues in the pulp were non

- detectable. Calculated residue levels in w hole bananas (unbagged) ranged for <0.01 to 0.071 ppm in samples collected at PHI of 0

- day. Decline studies were conducted in two trials and residues of parent were non

detectable within five days of the final application

. In summary, the HED has conclud ed that due to the very short pre

- harvest interval any exposure will be to the parent only, but exposure to diethofencarb will likely be minimal in the edible commodity

.Diethofencarb Hum an Health Risk Assessment D418227

Page 12 of 49 5.3 Drinking Water Residue Profile

As there are no domestic agricultural or reside ntial uses register ed or proposed for diethofencarb

, a drinking water assessment is not relevant for this petition.

5.4 Dietary (Food Only ) Risk Assessment

A chronic (food only) analysis was performed using DEEM

- FCID, ver. 3.16, which incorporates consumption data from the USDA NHANES/WWEIA; 2003

- 2008, to estimate the dietary exposure of the U.S. population and various population subgroups. The unrefined chronic

asses sment assumed that diethofencarb residues are present in all commodities at recomm ended tolerance levels and that 100% of all crops are treated. DEEM Version 7.81 default processing factors were used to estimate re sidues in processed commodities . Drinking water estimates were not incorporated into the chronic dietary assessment, as th ere are no domestic uses proposed or established. The results are summarized in Table 5.4.1. The resulting chronic food estimates are not of concern to HED (<100% cPAD) for U.S. general population a nd all population subgroups (≤ 1 % cPAD); the most highly exposed population subgroup is children 1

- 2 years old. The chronic dietary exposure and risk estimates are highly conservative since they assume that all subject crops consumed in this country have been treated (but use is not registered not in the U.S.) and that residues are present at tolerance levels. Dietary exposure estimates could be refined using anticipated residues and percent crop imported data. However, since estimated exposures and risks using conservative assumptions are below HED’s level o f concern, such refinement is not warranted.

Table 5.4.1. Summary of Dietary (Food Only) Exp osure and Risk for Diethofencarb .*

Population Subgroup Chronic Dietary Dietary Exposure (mg/kg/day) % cPAD*
General U.S. Population 0.000038 < 1 All Infants (<1 year old) 0.000185 < 1 Children 1

- 2 years old 0.00021 3 < 1 Children 3

- 5 years old 0.00010 4 < 1 Children 6

- 12 years old 0.000040 < 1 Youth 13

- 19 years old 0.00001 6 < 1 Adults 20

- 49 years old 0.00002 2 < 1 Adults 50

- 99 years old 0.00003 5 < 1 Females 13

- 49 years old 0.00002 2 < 1 *Population subgroup with the highest exposure and risk is in bold type.

6.0 Residential (Non

- Occupational) Exposure/Risk Characterization

As there are no domestic agricultural or residential uses register ed or proposed for diethofencarb

, a residential exposure assessment was not performed.

7.0 Aggregate Exposure/Risk Characterization

In accordance with the FQPA, HED must consider and aggregate (add) pesticide exposures and risks from three major sources: foo d, drinking water, and residential exposures. In an aggregate assessment, exposures from relevant sources are added together and compared to quantitativeDiethofencarb Hum an Health Risk Assessment D418227

Page 13 of 49 estimates of hazard (e.g., a NOAEL or PAD), or the risks themselves can be aggregated. When aggregat ing exposures and risks from various sources, HED considers both the route and duration of exposure. Because this action is for tolerance s without a U.S. registration and there are no proposed o r existing uses of diethofencarb which would result in drinki ng water or residential exposures

, the dietary (food only) exposure estimates provided in Table 5.4.1 represent the aggregate exposure assessment

.

8.0 Cumulative Exposure/Risk Characterization

Unlike other pesticides for which EPA has followed a cumulative r isk approach based on a common mechanism of toxicity, EPA has not made a common mechanism of toxicity finding as
to diethofencarb and any other substances and diethofencarb does not appear to produce a toxic metabolite produced by other substances. For th e purposes of this tolerance action, therefore, EPA ha s not assumed that diethofencarb has a common mechanism of toxicity with other substances. For information regarding EPA’s efforts to determine which chemicals have a common mechanism of toxicity and t o evaluate the cumulative effects of such chemicals, see the policy statements released by EPA’s Office of Pesticide Programs concerning com mon mechanism determinations and procedures for cumulating effects from substances found to have a common mechanism on EPA’s website at http://www.epa.gov/pesticides/cumulative/.

9.0 Occupational Exposure/Risk Characterization

Since the proposed use is for a tolerance without an U.S. registration, an occupational exposure assessment is unnecessary.

Appendix A. Toxicology Profile and Executive Summaries Appendix B. Metabolism Considerations Appendix C. Physical/Chemical Properties of DiethofencarbDiethofencarb Hum an Health Risk Assessment D418227

Page 14 of 49 A ppendix A

. Toxicology Profile and Executive Summaries

A.1 Toxicology Data Requirements

The requ irements (40 CFR 158.340) for a food use for diethofencarb are in Table A 1. Use of the new guideline numbers does not imply that the new (1998) guideline protocols were used.
Table A1. Summary of Toxicology Data Requirements

Guideline No. / Study Type Technical MRID No. Required Submitted 870.3100 Subchronic (Oral) Toxicity

- Rodent

................................ ........... 870.3150 Subchronic (Oral) Toxicity

- Non

- Rodent

................................ ... 870.3200 21/28

- Day Dermal Toxicity

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

- Day Dermal Toxicity

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

- Day Inhalation Toxicity

................................ ......................... Y Y Y N N a Y

Y Y N N 49267444

49267445 49267447

--

-- 870.3700a Prenatal Developmental Toxicity

- Rodent

................................ .. 870.3700b Prenatal Developmental Toxicity

- Non

- Rodent

......................... 870.3800 Reproduction and Fertility Effects

................................ .............. Y Y Y Y

Y Y 49267448

49267449 49267451 870.4100a Chronic (Oral) Toxicity

- Rodent

................................ ................ 870.4100b Chronic (Oral) Toxicity

- Non

- Rodent

................................ ........ 870.4200a Carcinogenicity

- Rat

................................ ................................ .... 870.4200b Carcinogenicity

- Mouse

................................ ............................. 870.4300 Combined Chronic Toxicity /Carcinogenicity

............................. N Y N Y Y Y

Y Y Y Y 49267454 b

49267452 49267454 b 49267453 49267454 b 870.6100a Neurotoxicity

- Acute Delayed Neurotox.

- Hen

.......................... 870.6100b Neurotoxicity

- Subchronic

- Hen

................................ .............. 870.6200a Neurotoxicity Screening Battery

- Acute

- Rat

............................ 870.6200b Neurotoxicity Screening Battery

- Subchronic

- Rat

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

................................ .................... N N Y Y N N

N Y Y N --

-- 49 267464 49 267463

-- 870.5100 Bacterial Reverse Mutation Test

................................ .................

870.5300 In Vitro Mammalian Gene Mutation Test

– Mouse Lymphoma Cells

................................ ................... 870.5375 In Vitro Mammalian Chromosome Aberration Test

.................... 870.5395 In Vivo Erythrocyte Micronucleus Test

- Mice

........................... 870. 5550 Unscheduled DNA Synthesis

................................ ...................... 870.5900 In Vitro Sister Chromatid Exchange

................................ ........... Y

Y N Y N N Y

Y Y Y Y Y 49267456

49267457

49267458 49267459 49267460 49267461 49267475 870.7485 Metabolism and Pharmacokinetics

................................ ..............

870.7200 Companion Animal Safety

................................ .......................... 870 .7600 Dermal Penetration

- Rat

................................ ............................. 870.7 600 Dermal Penetration

- Human

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

- Rat

................................ ................................ .. Y

N CR a CR a Y Y

N N N Y 49 267467

49 267468 49 267469 49 267470 49 267471

--

--

-- 49267472

a This study is not required for a tolerance on imported foodstuffs

. b The requirements for the chronic oral (rat) and carcinogenicity (rat) studies are satisfied by the combined chronic/oncogenic ity (rat) study

.Diethofencarb Hum an Health Risk Assessment D418227

Page 15 of 49 A.2 Toxicity Profiles

Table A.2.1 Acute Toxicity Profile for Diethofencarb Guideline No. Study Type MRID(s) Results Toxicity Category 870.1100 Acute oral (rat) 49267431 LD 50 > 5000 mg/kg IV 870.1100 Acute oral (mouse) 49267432 LD 50 > 5000 mg/kg IV 870.1100 Acute oral (mouse) 49267433 LD 50 > 5000 mg/kg IV 870.1200 Acute dermal (rat) 49267434 LD 50 > 5000 mg/kg IV 870.1200 Acute dermal (mouse) 49267435 LD 50 > 5000 mg/kg IV 870.1300 Acute inhalation (rat) 49267436 LC 50 > 1.05 mg/L III 870.2400 Acute eye irritation (rabbit) 49267437 Slight irritation III 870.2500 Acute dermal irritation (rabbit) 49267437 Non

- irritating IV 870.2600 Skin sensitization (mouse) 49267438 Not a dermal sensitizer N/A

Table A.2.2 Repeated

- Dose Toxicity Profile for Diethofencarb Guideline Study, Species MRID (Year), Dose Levels, Classification Results

870.3050 28

- day oral dietary, rat 49267440 (1986) 0, 100, 1000, 3000, and 10,000 ppm M: 0, 9.02, 89.1, 276, 878 mg/kg bw/day F: 0, 9.94, 98.9, 300, 1009 mg/kg bw/day

Acceptable/Guideline NOAEL = 878 / 1009 mg/kg bw/day (M/F) LOAEL = not established. Increased blood cholesterol, increased liver weight and swelling of centrilobular hepatocytes considered adaptive and not adverse.

870.3050 28

- day oral dietary,

mouse 49267442 (1986) 0, 100, 3000, 10,000, 30,000 ppm 0, 18.7, 563, 1852, 6169 mg/kg bw/day (M) 0, 4.4, 720, 2337, 8450 mg/kg bw/day (F)

Acceptable / non

guideline NOAEL = 563/720 mg/kg bw/day (M/F) LOAEL = 1852/2337 mg/kg bw/day (M/F) based on higher platelet counts (increases of 10% and 17% in males and females, respectively), higher total cholesterol concentrations in blood (increases of 26% in both sexes), higher absolute liver weights (increases of 14% and 23% in males and females, respectively), higher relative liver weights (increases o f 17% and 20% in males and females, respectively), as well as foci of necrosis in livers . Males only had submucosal lymphoid hyperplasia in urinary bladders and hepatocellular hypertrophy

No analysis of dietary admixtures performed to investigate the homogeneity, stability, and achieved concentrations of the test substance. Range

finding 28

- day oral capsule, dog 49267443 (1986) 0, 10, 50, 250, 1000 mg/kg/day

Acceptable / non

guideline NOAEL and LOAEL are not determined because the inadequate number of animals used makes data interpretation difficult. S alivation and emesis, and decreases in food consumption, body weight and fecal output were observed in males and females, with the female being the more sensitive

sex. Increased liver weight and hyper trophy considered adaptive.

Range

- finding studyDiethofencarb Hum an Health Risk Assessment D418227

Page 16 of 49 Table A.2.2 Repeated

- Dose Toxicity Profile for Diethofencarb Guideline Study, Species MRID (Year), Dose Levels, Classification Results

870.3100 13

- week oral dietary, rat 49267444 (1986) 0, 300, 1000, 3000, 10,000 ppm 0, 21.3, 78.2, 232 752 mg/kg/day (M) 0, 27.2, 90.8, 275 824 mg/kg/day (F)

Acceptable / Non

Guideline NOAEL = 275 mg/kg bw/day (F) LOAEL = 824 mg/kg bw/day (F) based on decreased body weight (

- 15%, p < 0.01) and food consumption (

- 25%, p < 0.01) in females. LOAEL not established in males

Study did not meet the GLP standards. Although the stability of the test com pound was confirmed in a 28

- day rat study (MRID 49267440), no dietary analyses for the homogeneity and concentration analysis were provided 870.3150 13

- week oral capsule,

dog 49267445 (1986) 0, 10, 30, 100 300 mg/kg bw/day

Acceptable/Guideline NOAEL = 300 mg/kg bw/day LOAEL = not established.

Note: emesis was observed at 100 and 300 mg/kg bw/day in males and females, with both the number of animals affected and the frequency of emesis increasing with dose. However, there were no changes in body weight or food consumption to suggest animal is compromised. 870.3200 28

- day dermal, rat 49267447 (2002) 0, 100, 300, 1000 mg/kg bw/day

Acceptable/Guideline NOAEL=1000 mg/kg bw/day LOAEL not established

No local or systemic effects were seen at any dose level.

870.3465 90/28

- day inhalation, rat Study Not Available Study Not Available

870.3700a Developmental Toxicity,

rat 49267450 (1987) 0, 100, 300, or 1000 mg/kg bw/day

Acceptable/Guideline Maternal NOAEL = 1000 mg/kg bw/day Maternal LOAEL = not established

Developmental NOAEL = 1000 mg/kg bw/day Developmental LOAEL = not established 870.3700b Developmental Toxicity,

rabbit 49267447 (1986) 0, 50, 125, 300, or 750 mg/kg bw/day

Unacceptable/non

guideline NOAEL = not identified LOAEL = not identified

The non

- treatment

- related maternal wastage (deaths, abortions/early deliveries) and total litter resorptions resulted in fewer than twelve litters per group at caesarean in four of five groups, which decreased the study’s sensitivity. In addition, the tot al numbers of deaths, abortions/early deliveries, and total litter resorptions call into question whether there might have been a problem with laboratory technique, the environmental conditions, or animal health.Diethofencarb Hum an Health Risk Assessment D418227

Page 17 of 49 Table A.2.2 Repeated

- Dose Toxicity Profile for Diethofencarb Guideline Study, Species MRID (Year), Dose Levels, Classification Results

870.3700b Developmental Toxicity,

rabbit 4 9267448 (1987) 0, 125, 300, or 750 mg/kg bw/day

Unacceptable/non

guideline NOAEL = not identified LOAEL = not identified

20% frequency of implantation loss in controls suggests unknown factors at play that make data interpretation difficult. Low number of dams for evaluation in the high dose (just 9) is too much at variance with guideline minimums. 870.3700b Developmental Toxicity,

rabbit 49267449 (1992) 0, 800, or 900 mg/kg bw/day

Acceptable/non

guideline Maternal NOAEL < 800 mg/kg/day, not established LOAEL = 800 mg/kg/day based on late

term abortions

Developmental NOAEL < 800 mg/kg/day, not established LOAEL = 800 mg/kg/day based on late

term abortions 870.3800 Multi

- generation

reproduction, rat 49267451 (1988) 0, 200, 1000, or 5000
ppm. Pre

- mating doses: 0, 13.1, 65.5, 328 mg/kg bw/day F0 (M); 0, 16.0, 88.0, 427 mg/kg bw/day F0 (F); 0, 16.6, 84.8, 420 mg/kg bw/day F1 (M) 0, 19.4, 105, 510 mg/kg bw/day in F1 (F)

. Acceptable/Guideline Parental NOAEL = 328/347 mg/kg bw/day M/F Parental LOAEL = not established

Reproductive NOAEL =328/347 mg/kg bw/day M/F Reproductive LOAEL = not established

Offspring NOAEL =65.5/88.0 mg/kg bw/day M/F Offspring LOAEL = 328/347 mg/kg bw/day M/F based on decreased absolute body weights (

- 11% to

- 15%, p < 0.05) in F2 offspring of both sexes during lactation (PNDs 4, 14, 21)

. 870.4100 Chronic Toxicity, dog 49267452 (1988) 10, 50, 250, and 1000 mg/kg/day

Acceptable/Guideline NOAEL (M) = 50 mg/kg/day LOAEL (F) = 250 mg/kg/day based on decreased body weight and increased emesis. NOAEL (F) = 250 mg/kg/day LOAEL (F) = 1000 mg/kg/day based on decreased absolute and relative uterine weights.Diethofencarb Hum an Health Risk Assessment D418227

Page 18 of 49 Table A.2.2 Repeated

- Dose Toxicity Profile for Diethofencarb Guideline Study, Species MRID (Year), Dose Levels, Classification Results

870.4200 Oncogenicity, mouse 49267453 (1988) 0, 10, 100, 1000, 10,000 ppm 0, 1.63, 16.47, 163.8, 1682 mg/kg/day (M) 0, 2. 03, 20.53, 202.9, 2108 mg/kg/day (F)

Acceptable/Guideline NOAEL = 163.8 / 202.9 mg/kg/day (M/F) LOAEL = 1682 / 2108 mg/kg/day (M/F) based on a dose

- related increased incidence of hepatocellular hyperplasia in males (18%, p<0.01), and decreased survival rates at 78 weeks ( p<0.05; 32% vs. 66% in controls in females.

At 1682 mg/kg/day a 27% increase (p < 0.05 in testes weight was observed in males but there were no histopathological changes.

No increase in tumor incidence.

Dosing was considered adequate to assess carcinogenic potential

. 870.4300 Chronic/ Oncogenicity

Study, rat 49267454 (1989) 0, 40, 200, 1000, 5000
ppm 0, 1.69, 8.42, 42.70, 220.30 mg/kg/day (M) 0, 2.18, 10.90, 54.50, 288.19 mg/kg/day (F)

Acceptable/Guideline NOAEL = 220.3 / 288.2 (M/F) LOAEL not established because no non

neoplastic effects seen at any dose level.

Dosing was considered adequate based on a treatment

- related increase in thyroid follicular cell tumor incidence (carcinomas in males, adenomas in females)

870.5100 In vitro Bacterial Gene

Mutation 49267456 (1984) 0, 5, 10, 50, 100, 500, and 1000 μg/plate, with and without S9

- mix

Acceptable/Guideline Negative. There was no evidence of induced mutant colonies over background.

870.5100 In vitro Bacterial Gene

Mutation 492674 57 (1988) of 0, 50, 100, 200, 500, 1000 and 2000 μg/plate, with and without S9

- mix

Acceptable/Guideline Negative. There was no evidence of induced mutant colonies over background.

870.5300 In Vitro Mammalian

Cells in Culture Gene Mutation Assay 49267458 (1989) 0, 20, 50, 100, or 200 μg/mL in three independent experiments in the absence of metabolic activation and in two independent experiments in the presence of metabolic activation

Acceptable/Guideline Negative. There was no evidence of induce d mutant colonies over background.Diethofencarb Hum an Health Risk Assessment D418227

Page 19 of 49 Table A.2.2 Repeated

- Dose Toxicity Profile for Diethofencarb Guideline Study, Species MRID (Year), Dose Levels, Classification Results

870.5375 In vitro Mammalian

Cytogenetics Chromosome Aberration Assay 49267459 (1989) 0, 13.37, 26.73, 53.46 and 133.66 μg/mL without S9; 0, 26.73, 53.46, 133.66 and 267.32 μg/mL in the presence of S9 activation

Acceptab le/Guideline Negative. There was no evidence of chromosome aberrations induced over background.

870.5395 In vivo Mammalian

Cytogenetics

- Erythrocyte Micronucleus assay in the mouse 49267460 (1987)

1250, 2500 or 5000 mg/kg

Acceptable/non

guideline Negative. T here were no significant increases in the frequencies of micronucleated polychromatic erythrocytes in bone marrow after any treatment time or dose level.

Test conduct ed in males only, females excluded without justification.

870.5550 Unscheduled DNA

Synthesis in Primary Rat Hepatocytes/Mammalian Cell Cultures 49267461 (1989)

25.3, 202 μg/mL

Una cceptable/Guideline Negative. There was no evidence that unscheduled DNA synthesis, as determined by radioactive tracer procedures [nuclear silver grain counts], was induced.

The results of the two assays were radically different, with an approximately 8

- fold difference in cytotoxicity levels. This apparent discrepancy brings into question the validity of the procedure and results. 870.5900 In Vitro Sister

Chromatid Exchange Assay in CHO Cells 49267475 (1986) First assay: 0, 0.27, 0.81, 2.67, 8.01, 26.73, 80.19, 267.32 μg/mL, without and with activation Second Assay: 0, 26.73, 53.46, 133.65 and 267.32 μg/mL, without and with activation

Accep table/Guideline Negative

There was no evidence of an increase in sister chromatid exchanges in CHO

- K1 cells induced over background

870.6200a Acute Neurotoxicity, rat 49267464 (2013) 0, 200, 600, or 2000 mg/kg bw

Acceptable/Guideline NOAEL = 600 mg/kg bw (M/F) LOAEL = 2000 mg/kg bw ( M/F) based on decreased forelimb grip strength, reduced body temperature and decreased alertness (M) and decreased muscle tone and pupillary reflex, reduced body temperature and increased landing footsplay (F)Diethofencarb Hum an Health Risk Assessment D418227

Page 20 of 49 Table A.2.2 Repeated

- Dose Toxicity Profile for Diethofencarb Guideline Study, Species MRID (Year), Dose Levels, Classification Results

870.6200b Subchronic

Neurotoxicity, rat 49267463 (2013) 0, 1000, 3000, or 10,000 ppm 0, 56.7, 172, and 571 mg/kg bw/day (M) 0, 65.4, 194, 654 mg/kg bw/day (F)

Acceptable/Guideline NOAEL = 571 / 654 mg/kg bw/day (M/F) LOAEL Not established

870.7485 Absorption Distribution

Metabolism Excretion 49267467, 49267468, 49267469, 49267470, 49267471

10 or 300 mg/kg bw as a single or two

- day dose. In an addition, male rats were treated with a 10 mg/kg bw/day unlabeled test material for 14 days followed by a single radiolabeled dose of the test material.

Acceptable/Guideline Metabolism of diethofencarb in rats was found to be independent of sex, dose level and pre

- treatment. Oral absorption is quick and extensive ( > 87% of AD by day 2). Organ/tissue radioactivity decreases rapidly and is mainly found in lever, adrenals, blood/blood cells, kidney and skin. Diethofencarb has no bioaccumulation potential. It is extensively biotransformed into s everal metabolites, mainly sulf ate and glucuronide conjugate. Excretion is extensive and quick extensive ( > 93% of AD by day 2) and occurs in urine ( > 61% of AD), expired air ( > 21% of AD) and feces ( > 13% of AD). This results in organ/tissue depuration t 1/2 values of < 35 hours based on data up to 72 hr pose

- dose and a low retained fraction ( <2 % of AD on day 7), and the deduction that there is no sig nificant entero

- hepatic recirculation. 870.7800 Immunotoxicity, rat

(female only) 49267472 (2012) 0, 1000, 3000, 10,000
ppm 0, 79.6, 236, 764 mg/kg bw/day (F)

Acceptable/Guideline Systemic NOAEL = 764 mg/kg bw/day Systemic LOAEL = not established

Immuno toxicity NOAEL = 764 mg/kg bw/day Immunotoxicity LOAEL = not established Non

guideline 14

- day thyroid

mechanistic study, rat 49267473 (1989) 0, 5000, 20,000 ppm 0, 430, 1720 mg/kg bw/day for 7

- wk olds; 0, 345, 1380 mg/kg bw/day for 37

- and 66

- wk olds

Acc e ptable/non

guideline;

N umerous procedural deficiencies found. However, data corroborated and found useful in conjunction with MRID 49267474 Treatment with 20,000 ppm for up to two weeks decreased serum T4 concentrations and induced slight increases in TSH
concentra tions with no effect on T3 concentrations. In the bioaccumulation study, the kidney had the highest 14 C

concen trations followed by the liver, skin and hair following both 7

- and 14

- days of
treat ment. No indication of bioaccumulation of radiolabel was found in the thyroid. The data also suggested that increased T4 excretion in the bile arose through metabolic processes related to hepatic UDP

- GT induction.Diethofencarb Hum an Health Risk Assessment D418227

Page 21 of 49 Table A.2.2 Repeated

- Dose Toxicity Profile for Diethofencarb Guideline Study, Species MRID (Year), Dose Levels, Classification Results

Non

guideline 3

- month thyroid

mechanistic study, rat 49267474 (1989) 0, 5000, 20,000 ppm 0, 430, 1720 mg/kg bw/day

Acceptable/non

guideline Decreased body weight and food consumption were observed at 5000 ppm and higher. Examinations at termination demonstrated a decrease in serum free T 4 level and an increase in serum TSH level in the 20000 ppm group. However, no compound related effects were observed in the thyroid weight, the thyroid uptake of

125 I, autopsy and histopathological examination of the thyroid. Liver weight increase at and above 5000 ppm, and an increase of UDP

- GT (UDP

glucuronyltransferase) activity at 20000 ppm, were observed.Diethofencarb Hum an Health Risk Assessment D418227

Page 22 of 49 

### EXECUTIVE SUMMARIES

Subchronic Toxicity

870.3050 28

- Day Oral Toxicity

— Mouse

Repeated dose oral toxicity 28

- day feeding study in rats (MRID 49267442)

. This 28

- day oral toxicity study in the rat is Acceptable/ Non

- Guideline and does not satisf y the guideline requirement for a 4

- week toxicity study in rodents ( OCSPP 870.3050; OECD 408)

. Groups of CD

- 1 (ICR) BR mice (10/sex/group) were administered diethofencarb (97.2% a.i., Batch PKG 84093) in the diet at the dietary concentrations of 0, 100, 3 000, 10,000 or 30,000 ppm for 28 days. No analyses of dietary admixtures were performed to investigate the homogeneity, stability, and achieved concentrations of the test substance. Mortality, clinical signs, body weight, and food consumption were recorded . Samples of blood and were collected for clinical pathology evaluation at the end of treatment . All surviving animals in all groups were then sacrificed and subjected to gross necropsy. Selected organs were weighed, and, in the control and 30,000 ppm grou ps, a standard range of organs and tissues was removed, fixed, and examined microscopically. Livers, kidneys, and urinary bladders of mice in all groups were examined microscopically. The 100, 3000, 10,000 and 30,000 ppm dietary levels corresponded to nomi nal dose

- levels of 18.7, 563, 1852, and 6169 mg/kg bw/day in males and to 24.4, 720, 2337, and 8450 mg/kg bw/day in females for the 28 days of feeding.

No mortality occurred. Both sexes at the 30,000 ppm diet had higher platelet counts (increases of 16% and 24% in males and females, respectively), higher albumin concentrations (increases of 13% and 20% in males and females, respectively), higher protein concentrations (increases of 15% and 17% in males and females, respectively), higher total cholesterol concentrations in blood (increases of 74% and 66% in males and females, respectively), higher alanine aminotransferase (ALT) activities (increases of 88% and 79% in males and females, respectively), higher absolute liver weights (increases of 34% and 53% i n males and females, respectively), higher relative liver weights (increases of 63% and 61% in males and females, respectively), dark livers in 6/10 males and in all 10 females, as well as foci of necrosis in livers, hepatocellular hypertrophy, proteinaceo us casts and regenerative epithelium in kidneys, and submucosal lymphoid hyperplasia in urinary bladders in both sexes. Males only at the 30,000 ppm diet showed a reduction in body weight gain, which amounted to 49%. The LOAEL in CD

- 1 (ICR) BR mice of both sexes was the 10,000 ppm diet (corresponding to nominal dose levels of 1852 and 2337 mg/kg bw/day in males and females, respectively) based on higher platelet counts (increases of 10% and 17% in males and females, respectively), higher total cholesterol c oncentrations in blood (increases of 26% in both sexes), higher absolute liver weights (increases of 14% and 23% in males and females, respectively), higher relative liver weights (increases of 17% and 20% in males and females, respectively), as well as fo ci of necrosis in livers. Males only at the 10,000 ppm dietary level had submucosal lymphoid hyperplasia in urinary bladders and hepatocellular hypertrophy.

No treatment

- related adverse effects were observed at the 100 or 3000 ppm dietary levels in eithe r sex. The NOAEL in both sexes was the 3000 ppm diet (corresponding to nominal doses of 563 and 720 mg/ kg bw/day in males and females, respectively). The target organs were identified as the liver, kidneys, and urinary bladder.Diethofencarb Hum an Health Risk Assessment D418227

Page 23 of 49 This repeated dose 28

- da y oral toxicity study in mice is Acceptable / non

- g uideline and does not satisfy the requirements of Guideline OCSPP 870.3050 (OECD 407) because of analytical deficiencies (see Deficiencies section). However, it does provide data useful in characterizing the toxicity of diethofencarb in the mouse and serves as a range

- finding study.

This Data Evaluation Record is a modified version of the Ministere de l’Agriculture et de la Peche (France) Draft Assessment Report (DAR), Diethofencarb Vol. 3, Annex B6, pp. 248

- 251 (2007). Portions of this document were altered by the EPA reviewer to reflect Agency policies and conclusions

.

870.3050 28

- Day Oral Toxicity

— Rat

Repeated dose oral toxicity 28

- day feeding study in rats (MRID 49267440)

. This 28

- day oral toxici ty study in the rat is Acceptable/Guideline and satisf ies the guideline requirement for a 4

- week toxicity study in rodents ( OCSPP 870.3050; OECD 408)

.

In a 28

- day feeding study (MRID 49267440), g roups of Sprague Dawley rats (10/sex/group) were administered diethofencarb (97.4% a.i., Batch PK 84042) in the diet at concentrations of 0, 100, 1000, 3000, or 10,000 ppm for 4 weeks. Dietary admixtures were analyzed to investigate the homogeneity, stabil ity (over 7 days), and achieved concentrations of the test substance. Mortality, clinical signs, body weight, and food consumption were recorded. Samples of blood and urine were collected for clinical pathology evaluation at the end of treatment . At study termination, all surviving animals from the two highest dose groups and the controls were examined ophthalmologically. All surviving animals in all groups were then sacrificed and subjected to gross necropsy. Selected organs were weighed, and, in the contr ol and 10,000 ppm dose group, a standard range of organs and tissues were removed, fixed, and examined microscopically. Livers of rats in all groups were examined microscopically. The 100, 1000, 3000, and 10,000 ppm concentrations corresponded to dose

- leve ls of 9.02, 89.1, 276, and 878 mg/kg bw/day in males and to 9.94, 98.9, 300, and 1009 mg/kg bw/day in females for the 28 days of feeding.

No mortality occurred. Both sexes at the 10,000 ppm dose level had higher blood cholesterol concentrations (increase s of 43% and 22% in males and females, respectively), and swelling of centrilobular hepatocytes in all animals. Males only at 10,000 ppm dose level had lower food intake (a reduction of 11%), higher absolute liver weight (increase of 14%), and higher relat ive liver weight (increase of 25%). These increases in cholesterol and liver weights are considered adaptive responses that are not adverse. No treatment

- related adverse effects were observed at 10 or 1000 ppm dose levels in either sex. The NOAEL was 10, 0 00 ppm (878 / 1009 mg/kg bw/day , respectively ) in males and females and the LOAEL was not established.

This repeated dose 28

- day oral toxicity study in rats is a cceptable/ g uideline and satisfies the requirements of G uideline OCSPP 870.3050 (OECD 407)

.

This Data Evaluation Record is a modified version of the Ministere de l’Agriculture et de la Peche (France) Draft Assessment Report (DAR), Diethofencarb Vol. 3, Annex B6, pp. 246

- 248 (2007). Portions of this document were altered by the EPA reviewer to r eflect EPA policies and conclusions.Diethofencarb Hum an Health Risk Assessment D418227

Page 24 of 49 Non

- guideline 28

- day Oral Toxicity

– Dog

28

- Day feeding study the dog. MRID 49 267443

. This 28

- day range

- finding oral toxicity study in the dog is Acceptable/Non

- guideline and does not satisfy a guideline requirement.

In a four

- week toxicity study, g roups of Beagle dogs (2/sex/group) were administered diethofencarb (97.2% a.i., Batch PKG 84093) in gelatin capsules at 0, 10, 50, 250, or 1000 mg/kg bw/day for 28 days. Mortality, clinical signs, body weight, and food c onsumption were recorded. Samples of blood were collected for hematological and blood biochemistry investigations prior to treatment (at week

- 1) and at the end of the treatment . Urinalyses were performed at the same times. Plasma and red blood cell cholin esterase concentrations were investigated prior to treatment and at 1 and 3 hours after dosing on treatment weeks 2 and 4. Brain cholinesterase concentration was determined at the time of necropsy. Ophthalmological examinations were performed on all dogs p rior to treatment and at study termination, at which time all surviving animals were sacrificed and subjected to gross necropsy. Selected organs were weighed, and the following organs and tissues were removed, fixed, and examined microscopically for all do gs: hearts, lungs, pancreases, livers, kidneys, testes with epididymides, ovaries, spleens, urinary bladders, and any gross lesions.

No mortality occurred. Males and females at the 1000 mg/kg bw/day dose had few or no feces, emesis, salivation, weight lo ss over the time of study (

- 15 % to

- 18% over both sexes), lower food intake (of 30% through 61% in males and of 65% through 81% in females), increased absolute liver weights (37% and 76%, respectively, in males and females), and increased liver weights (6 4% and 113%, respectively, in males and females). At this same dose, one of two males had tremors and compound

- colored stools, and one of two females showed thinness, dehydration, and ataxia. Since this study used just two animals/sex/dose group, the findi ngs are difficult to interpret, and the NOAEL and LOAEL are not determined

. This repeated dose 28

- day oral toxicity study in mice is a cceptable/ non - g uideline

. The number of animals used/group was not sufficient. Nonetheless, i t provides useful information as a
sup portive range

- finding study.

This Data Evaluation Record is a modified version of the Ministere de l’Agriculture et de la Peche (France) Draft Assessment Report (DAR), Diethofencarb Vol. 3, Annex B6, pp. 2 52

- 25 4 (2007). Portions of thi s document were altered by the EPA reviewer to reflect Agency policies and conclusions

.

870.3100 90

- Day Oral Toxicity

— Rat

90

- day feeding study in rats. MRID 49267444 This study is classified as A cce ptable / Non

- Guideline and satisfies the guideline requirements for an oral 90

- day subchronic Toxicity Study in the rat (OCSPP 870.3100; OECD 408).

In a 13

- week study (MRID 49267444), groups of 15 male and 15 female Sprague

- Dawley rats

were fed S

- 32165 (Diethofencarb, 97.4% a.i., Batch PK 84042) at diet ary concentrations of 0, 300, 1000, 3000 or 10,000 ppm, for a period of 90 days (corresponding to 0, 21.3, 78.2, 232 orDiethofencarb Hum an Health Risk Assessment D418227

Page 25 of 49 752 mg/kg/day in males, and 0, 27.2, 90.8, 275 or 824 mg/kg/day in females). Animals were observed for mortality and clinical signs dai ly and body weight, food and water consumption were recorded weekly. Opthalmoscopic examinations were performed. Blood samples were taken during Week 13 for hematology and blood biochemistry investigations

. Cholinesterase activity in plasma, erythrocyte s and brain was investigated. Following completion of the scheduled treatment period, a detailed necropsy was performed on all animals and various organs were weighed. A full set of tissues and organs were prepared and subjected to histopahtology.

Oral administration of S

- 32165 caused statistically significant (p < 0.05, p < 0.01) decreases in body weights in both sexes, which were marginal ( < 10% in males) ; and lower food consumption and water intake in females at 10,000 ppm. Increases in absolute and relative liver weights accompanied by centrilobular hepatocellular hypertrophy as well as hypertrophy of endoplasmic reticulum in males at 3000 ppm and both sexes at 10,000 ppm were considered as adaptive responses to S

- 32165

- in duced toxicity.

The LOAEL is 824 mg/kg bw/day based on decreased body weight in females. The female NOAEL was 275 mg/kg bw/day . The LOAEL in males was is not established, and the NOAEL is 752 mg/kg bw/day.

This study is classified as A cce ptable / Non

- Guideline and satisfies the guideline requirements for an oral 90

- day subchronic Toxicity Study in the rat (OCSPP 870.3100; OECD 408). The study did not meet the GLP standards. The stability of the test compound was not provided, but was confirmed in a 28

- day rat study ( MRID 49267440); no dietary analyses for the homogeneity and concentration analysis were provided.

This Data Evaluation Record is a modified version of the Draft Assessment Report (DAR) of the Initial Risk Assessment by the Reporter Memb er State: France (2007), Diethofencarb Vol.3. Annex B, part 2/A, B.6., pages 254

- 257. Portions of this document were altered by the EPA reviewer to reflect Agency policies and conclusions.

870.3150 90

- Day Oral Toxicity

— Dog

90

- Day oral toxicity study in dogs. MRID 49267445. This study can be classified as A cceptable / Guideline and satisfies the guideline requirement for a 90

- day subchronic toxicity study in non

- rodents [ OCSPP 870.3150; OECD 409 ]

. In a subchronic toxicity study (MRID 49267445), Dieth ofencarb ( 98.1%; Batch No. PEG 85045) was administered orally in gelatin capsules to groups of 4 Beagle dogs/sex at dose levels of 0, 10, 30, 100 or 300 mg/kg bw/day for 13 weeks. The control group received empty gelatin capsules. The dogs were observed twice daily for mortality and once daily for clinical signs. Body weights and food consumption were recorded weekly. Blood samples were taken for hematology and blood biochemistry investigations and urine samples were collected

. Ophthalmological examin ations were performed. Following completion of the scheduled treatment period, a detailed necropsy was performed on all animals and various organs were weighed. A full set of tissues and organs were collected, processed and examined microscopically.Diethofencarb Hum an Health Risk Assessment D418227

Page 26 of 49 The only treatment

- related effect in male and female dogs was emesis seen at 100 and 300 mg/kg bw/day. However, this effect was not sufficiently severe to cause significant decreases in food consumption or body weight, and is not considered adverse in this s tudy

. A LOAEL was not established. The NOAEL was 300 mg/kg bw/day.

This study can be classified as A cceptable / Guideline and satisfies the guideline requirement for a 90

- day subchronic toxicity study in non

- rodents [ OCSPP 870.3150; OECD 409 ]

. This Data Evaluation Record is a modified version of the Draft Assessment Report (DAR) of the Initial Risk Assessment by the Reporter Member State: France (2007), Diethofencarb Vol.3. Annex B, part 2/A, B.6., pages 257

- 259

. Portions of this document were altered by the EPA reviewer to reflect Agency policies and conclusions.

870.3200 Subchronic Dermal Toxicity

— Rat

28

- Day dermal toxicity study in rat. MRID 49267446

. This 28

- day dermal toxicity study in the Sprague Dawley rat is Acceptable / Guideline and satisfies the guideline requirement for a 28

- day dermal toxicity study (OCSPP 870.3200; OECD 410) in the rat.

Diethofencarb was applied moistened to the moistened clipped dorsal skin to groups of 10 male and 10 female Sprague Dawley rats at dose

- levels of 0, 100, 300, or 1000 mg/kg bw/day for a period of 28 days. Dose

- levels were selected based on a 7

- day repeat ed dose dermal toxicity study in rats. Observation for clinical signs and skin reactions were carried out daily. The body weight and food consumption were recorded once a week. Opthal mos copic and veterinary examinations were done and blood and urine samples were collected for clinical investigations at weekly intervals during the study. Following completion of the sche dul ed t reatment period, a detailed necropsy was done on all animals and various organs were weighed. A full set of tissues and organs were prepared and examined for histopathology.

Following dermal application of diethofencarb for 28 days there were no deaths a nd no
treatment

- related clinical signs, skin reactions or ophthalmologic lesions. There were no significant differences in body weight, weight gain, food intake, hematological and blood biochemistry parameters as well as organs weights, between treated an d control groups. There
were no treatment

- related gross and microscopic findings. There were no local or systemic effects seen at any dose level.

The NOAEL for Sprague Dawley rats was 1000 mg/kg bw /day (Limit dose). A LOAEL was not identified. This st udy is classified A cceptable / Guideline.

This Data Evaluation Record is a modified version of the Draft Assessment Report of the Initial Risk Assessment by the Reporter Member State: France (2007), Diethofencarb Vol.3. Annex B, part 2/A, B.6., pages 262

- 263. The EPA reviewer may have altered portions of this document to reflect Agency policies and conclusions.

870.3700 Prenatal Developmental Toxicity

Developmental toxicity study in rats. MRID 49267450.Diethofencarb Hum an Health Risk Assessment D418227

Page 27 of 49 The developmental toxicity study in the rat is classified acceptable / guideline and satisfies the guideline requirement for a developmental toxicity study [OCSPP 870.3700; OECD 414] in the
rat.

In a developmental toxicity study (MRID 49267450 ) S

- 32165 (Diethofencarb; 98.2% a.i.; Lot no. PSG 86018) was administered to 25 mated female Sprague

- Dawley rats/dose by gavage in 0.5% aqueous methylcellulose at dose levels of 0, 100, 300, or 1000 mg/kg bw/day on gestation days (GDs) 6

- 15

. On GD 21, dams were sacrificed and subjected to necropsy and caesarea n section. Gravid uterine weights and numbers of corpora lutea, live and dead fetuses, early and late resorptions

, and implantation sites were recorded . Fetuses were examined grossly for external malformations/variations, w eighed

, and sexed. One half were fixed in Bouin’s fluid and the remaining half in alcohol, to evaluate visceral and skeletal anomalies , respectively.

There were no deaths or treatment

- related clinical signs . During GDs 6

- 8, the 1000 mg/kg bw/day dams had decr eased food consumption (

- 17% ; p<0.05 ) , which was not considered
treatment

- related or adverse because there was not a corresponding decrease in absolute body weight. There were no treatment

- related effects on maternal intrauterine parameters. The maternal N OAEL is 1000 mg/kg bw/day. The maternal L OAEL was not established

.

No treatment

- related effects were observed on mean implantation efficiency, fetal viability, fetal body weight , or fetal sex ratio. At 1000 mg/kg bw/day, m oderately dilated ureters we re observed in 5.8% of fetuses (and 25% of litters) compared to 2.3% (and 13%) of control fetuses (and litters). However, since these fetus and litter values are within historical control ranges, they are not considered biologically relevant

. The develop mental LOAEL is not identified . The developmental NOAEL is greater than or equal to 1000 mg/kg bw/day.

The developmental toxicity study in the rat is classified Acceptable/Guideline and satisfies the guideline requirement for a developmental toxicity study (OCSPP 870.3700; OECD 414) in the
rat.

This Data Evaluation Record is a modified version of the Rapporteu r M ember States (RMS) Summary, Evaluation a nd Asse ssment o f t he Data a nd Information

, Minist è re de l’ Agriculture et de l a P ê che

, Direction G éné rale de l’ Alimentation , France (2007) Draft Assessment Report (DAR)

, public version , Diethofencarb, Volume 3

– Annex B.6, pp. 307

- 309. Portions of this document were altered by the EPA reviewer to reflect Agency policies and conclusions

. Developmental toxicity study in rabbits. MRID 49267447

. This study is classified as U nacceptable/non

- guideline) and does not satisfy the guideline requirement for a developmental toxicity study in non

- rodents ( OPPTS 870.3700; OECD 414).

In a developmental toxicity study (MRID 49267447 ) S

- 32165 (Diethofencarb; 98.1% a.i.; batch No.: PEG 85045 ) was administered to 18 presumed pregnant New Zealand white rabbits/dose by
gavage in 0.5% aqueous methylcellulose solution, at dose le v els of 0 , 50, 125, 300

, or 750 mg/kg bw /d a y (dosing volume 3 m L/ kg bw) on gestation days (GDs) 7 through 19, inclusive. On GD 29

, does were sacrificed and subjected to caesarean section. Gravid uterine weights and numbers of corpora lut e a, l ive and dead f etuses, early and l ate resorptions

, and implantation sites were recorded. All f

etuses were given a gross external examination, weighed

, sexed , and subjected to visceral and skeletal examinations. Heads of one ha l f of the live fetuses were cut for

ex a mination of eyes, ears, palate, nasal s eptum , and brain

.Diethofencarb Hum an Health Risk Assessment D418227

Page 28 of 49 Deaths of one dam from each of the control and 125

- mg/kg groups and abortions/early deliveries by 1 , 2, 3, 0

, and 3 animals from the 0

- , 50

- , 1 25

- , 300

- , a nd 750

- mg /kg groups , respectively

, were not considered treatment

- related

. Relati ve to controls, dams treated at 750 mg/kg bw/day had st atistically significant decreases in body weights (

- 7% to

- 10%) during treatment , and this group also had a mean weight loss of 50 g (vs. a weight gain of 140 g for controls ) during the treatment inter val. In addition, the food consumption of the 750

- mg/kg dams was 38% less than that of controls during treatment.

In the 0

- , 50

- , 125

- , 300

- , and 750

- mg /kg groups, there were respectively 15, 16, 12, 10

, and 14 pregnant females (out of 18 initially) , i.e. pregnancy was achieved by 56

- 89% of the animals in each group or ~74% overall. Due to maternal deaths, abortions/early deliveries, and total litter resorptions of three control litters and one 50

- mg/kg litter, at c aesarean , there were respectively 10, 13, 8, 10, and 11 litters with live fetuses in the 0

- , 50

- , 125

- , 300

- , and 750

- mg /kg groups

. Mean numbers of corpora lutea and implantations and the mean percentages implantation efficiency of the treate d groups were similar to those of controls. There were no evident
treatment

- related effects on postimplantation loss, numbers of early/late resorptions or dead fetuses, live litter sizes, fetal weight, or fetal sex ratio. Neither the pattern nor frequenc y of fetal malformations or variations indicated a treatment

- related effect

. In four of the five groups there were twelve or more pregnant rabbits, which was the minimum required by the guidelines before January 2000. However, the non

- treatment

- related maternal wastage (deaths, abortions/early deliveries) and total litter resorptions resulted in fewer than twelve litters per group at caesarean in four of five groups, which decreased the study’s sensitivity. In addition, the total numbers of deaths, abo rtions/early deliveries, and total litter resorptions call into question whether there might have been a problem with laboratory technique, the environmental conditions, or animal health.

Under the conditions of this study, the maternal NOAEL and LOAEL co uld not be determined, and t he developmental NOAEL and LOAEL cannot be identified

. The developmental toxicity study in the rabbit is classified Unacceptable/Non

- guideline and does not satisfy the guideline requirement for a developmental toxicity study (O CSPP 870.3700; OECD 414) in the rabbit.

This DER is a modified version of the Rapporteu r M ember States (RMS) Summary, Evaluation

a nd Asse ssment o f t he Data a nd Information

, Minist è re de l’ Agriculture et de l a P ê che

, Direction G éné rale de l’ Alimentatio n , France (2007) Draft Assessment Report (DAR)

, public version

, Diethofencarb, Volume 3

– Annex B.6, pp. 307

- 309. Portions of this document were altered by the EPA reviewer to reflect Agency policies and conclusions

.

Developmental toxicity study in rabbits. MRID 4926744 8

. This study is classified as U nacceptable/non

- guideline and does not satisfy the guideline requirement for a developmental toxicity study in non

- rodents ( OPPTS 870.3700; OECD 414).

In a developmental toxicity study (MRID 49267448 )

, S

- 32165 (Diethofencarb; 98.2% a.i.; Batch no. PSG 86018) was administered to 16 presumed pregnant New Zealand white rabbits/dose byDiethofencarb Hum an Health Risk Assessment D418227

Page 29 of 49 gavage in 0.5% aqueous methylcellulose solution, at dose le v els of 0 , 125, 300

, or 750 mg/kg bw /d a y (dosing volume 3 m L/ kg bw) on gestation days (GDs) 7 through 19, inclusive. On GD 29

, does were sacrificed and subjected to caesarean section . Gravid uterine weights and numbers of corpora lut e a, l ive and dead f etuses, early and l ate resorptions

, and implantation sites were

re corded. All f

etuses were given a gross external examination, weighed

, sexed , and subjected to visceral and skeletal examinations. Heads of one ha l f of the live fetuses were cut (mid

- coronal slice) for ex a mination of eyes, ears, palate, nasal septum

, and brain . This study was a repeat of earlier study ( MRID 49267447 ) in which the pregnancy rate was low. The two studies were discussed and interpreted together by the notifier ( Laboratory report no. HHT

- 0112, 2001).

T here were no treatment

- related effects on clinical signs, body weight, or food consumption. Single deaths in each of the low

- and high

- dose groups were not clearly attributable to exposure to the test material or to gavage error. A treatment

- related increased incidence of total litter resorpt ions in the high

- dose group is considered both a maternal effect and a developmental effect; at caesarean section, total litter resorptions were seen in 1/14, 1/12, 2/14, and 6/15 of the pregnant does in the control, low

- , mid

- , and high

- dose groups, respe ctively.

No dead fetuses were observed, and there were no treatment

- related effects on fetal weight, fetal sex ratios, or live litter sizes in the litters that were not totally resorbed. The total litter resorptions of 6/15 high

- dose litters resulted in increased mean post

- implantation loss for this group (45% vs. 20% for controls). This reflected greater numbers of resorptions per dam in the dams with total litter resorptions: the six affected high

- dose dams had an average of 3.3 resorptions (range: 1

- 8 ) compared to 1, 2, and 1.5 in the control, low

- , and high

- dose groups, respectively. In the affected animals, all of the resorptions were early resorptions, and in one dam from each of the mid

- and high

- dose groups, the corpora lutea appeared to have com pletely regressed. A total of 82 (13), 56 (11), 77 (12), and 60 (9) fetuses (and litters) were evaluated in the control, low

- , mid

- , and high

- dose groups, respectively. Fused frontal bones (an uncommon malformation) were observed in 0 (0), 0 (0), 1 (1), and 3 (3) fetuses (and litters) in these same respective groups. Because this finding is considered uncommon, it is considered treatment

related and adverse despite the low incidence. The overall incidence of external, visceral, and/or skeletal malformat ions and/or variations were not affected by treatment.

There was a 20% frequency in post

- implantation loss in the controls, which is unacceptably high, suggesting other unknown factors are in play which make data evaluation and interpretation difficult. In addition, the number of litters drops to nine in the high

- dose group, which is unacceptably low even for the guidelines extant in 1987 (when the study was conducted) which required a minimum of 12 animals.

Therefore, under the conditions of this study

, t he maternal NOAEL and LOAEL are not determined, and t he developmental LOAEL and NOAEL are not determined

. This study is classified as Unacceptable/Non

- guideline and does not satisfy the guideline requirement for a pre

- natal development toxicity study O CSPP 870.3700b [§83

- 3b]; OECD 414} in rabbits.

This Data Evaluation Record is a modified version of the Rapporteu r M ember States (RMS) Summary, Evaluation a nd Asse ssment o f t he Data a nd Information

, Minist è re de l’ Agriculture et de l a P ê che

, Direction G éné rale de l’ Alimentation , France (2007) Draft Assessment ReportDiethofencarb Hum an Health Risk Assessment D418227

Page 30 of 49 (DAR)

, public version , Diethofencarb, Volume 3

– Annex B.6, pp. 309

- 312. The EPA reviewer may have altered portions of this document to reflect Agency policies and conclusions.

Developmental toxicity study in rabbits. MRID 4926744 9

. This study is classified as A cceptable/non

- guideline and satisf ies the guideline requirement for a developmental toxicity study in non

- rodents ( OPPTS 870.3700; OECD 414).

In a developmental toxicity study (MRID 49267449 ) , S

- 32165 (98.7% a.i., Lot # S

- 7509

- TG) was administered to 25

- 26 New Zealand White rabbits/dose by gavage at dose levels of 0, 800

, or 900 mg/kg bw/day from days 7 through 28 of gestation. On gestation day (GD) 29, the females were sacrificed and fetuses removed by cesarean section. The evaluated parameters included clinical signs, mortality, body weight, body weight gain, food consumption, embryo

fetal death, fetal growth, and fetal malf ormations.

One animal at 900 mg/kg bw/day died on GD 22 and another delivered a litter before GD 29. At 800 and 900 mg/kg bw/day, two and seven females, respectively, aborted during GD 22

- 27. Decreased feces and/or urine were observed at a higher frequ ency in treated animals. Body weights in both treatment groups were significantly decreased by 3

- 8% (p<0.05, p<0.01), and food consumption decreased by 10

- 53% (p<0.05, p<0.01). No differences were observed in cesarean parameters. No gross lesions were o bserved in animals that survived to scheduled necropsy. While the decreases in body weight were minimal and are not normally considered adverse, the decreased food consumption is considered to have led to decreased body weights and malnutrition, in turn l eading to an increased incidence of aborted litters. The maternal LOAEL was 800 mg/kg bw/day, increased late

- term abortions . The maternal NOAEL was not established

.

There were no treatment

- related effects in developmental parameters such as growth, devel opmental variations or malformations . Late

- term abortions were increased at 900 mg/kg bw/day . However, the two dose levels are close and considered NOT to be biologically different

. The developmental LOAEL was 800 mg/kg bw/day based on increased late

- ter m abortions . The developmental NOAEL not established

. The developmental toxicity study in the rabbit is classified Acceptable/non

- guideline and nevertheless satisfies the guideline requirement for a developmental toxicity study (OCSPP 870.3700; OECD 414 ) in rabbits.

This Data Evaluation Record is a modified version of the Rapporteu r M ember States (RMS) Summary, Evaluation a nd Asse ssment o f t he Data a nd Information

, Minist è re de l’ Agriculture et de l a P ê che

, Direction G éné rale de l’ Alimentation , France (2007) Draft Assessment Report (DAR)

, public version , Diethofencarb, Volume 3

– Annex B.6. Portions of this document were altered by the EPA reviewer to reflect Agency policies and conclusions

.

870.3800 Reproduction and Fertility Effects

2

- Generation reproduction study in rats. MRID 49267451. This study is classified as acceptable/guideline and satisfied the guideline requirements (O PPTS 870.3800; OECD N0. 416) for a multi

- generation reproduction and fertility study.Diethofencarb Hum an Health Risk Assessment D418227

Page 31 of 49 In a two

- generation reproduction study (MRID 49267451) S

- 32165 (Diethofencarb; batch No.: PKG 84093, purity: 97.2%; batch No.: PEG 85045, purity: 98.1%; and batch No.: PSG 86018, purity: 98.2%) was administered to groups of 26 CD rats/sex/dose in the diet at concentrations of 0, 200, 1000, or 5000 ppm. Pre

- mating doses were respectively as follows: 0, 13.1, 65.5, or 328 mg/kg bw /day in F0 males ; 0, 16.0, 88.0, or 427 mg/kg bw /day in F0 females; 0, 16.6, 84.8, or 42 0 mg/kg bw /day in F1 males ; and 0, 19.4, 105, or 510 mg/kg bw /day in F1 females

. F0 and F1 parental animals were administered the treated or control diets for at least 14 weeks prior to mating, during mating, and either throughout gestation, and lactation , and until necropsy after weaning of litters (females) or until pups were born (males). One litter was produced by each generation.

F0 males treated at 5000 ppm had a small but statistically significant decrease in absolute body weight (

- 7% at week 10). In the 5000

- ppm males and females of the F1 generation, statistically significant decreases in body weight were also seen during pre

- mating weeks 0

- 10 and/or 0

- 14 but were of biologically insignificant magnitude (males:

- 7%; females:

- 4% to

- 7%). At 500 0
ppm, relative and/or absolute liver weights were significantly increased in both sexes and generations, as follows (for absolute/relative weights): F0 males, +19%/+26%; F0 females, +7%/+13%; F1 males, +12%/+18%; and F1 females, +12%/+16%. Ho wever there were no histopathological lesions in the liver. Therefore, t he parental systemic N OAEL is 5000 ppm ( 328 mg/kg bw/day in males and 427 mg/kg bw/day in females )

. The parental systemic L OAEL was not established

. Statistically significant (p < 0.05) lower body w eights were recorded in F2 pups at 5000 ppm; absolute body weights were 1 0 % / 15% lower in males / females at day 4 , respectively, 11% lower in males / females at day 14, and 1 5 % / 13% lower in males / females at day 21, respectively, compared with controls. Although this effect was only present in one generation, it was considere d to be biologically relevant. The offspring LOAEL is 5000 ppm ( 328 mg/kg bw/day in males and 427 mg/kg bw/day in females ), based on decreased absolute body weights in F2 offspring of both sexes during lactation . The offspring NOAEL is 1000 ppm ( 65.5 mg/kg bw/day in males and 88.0 mg/kg bw/day in females ).

No evidence of reproductive toxicity was observed

. The reproductive LOAEL is therefore unidentified, and the reproductive NOAEL is greater than or equal to 5000 ppm ( 328 mg/kg bw/day in males and 427 mg/kg bw/day in females).

This study is Acceptable/Guideline and satisfies the guideline requirement for a two

- generation reproductive study (O CSPP 870.3800 ; OEC D 416 ) in the rat.

This Data Evaluation Record is a modified version of the Rapporteu r M ember States (RMS) Summary, Evaluation a nd Asse ssment o f t he Data a nd Information

, Minist è re de l’ Agriculture et de l a P ê che

, Direction G éné rale de l’ Alimentation , France (2007) Draft Assessment Report (DAR)

, public version , Diethofencarb, Volume 3

– Annex B.6, pp. 309

- 312. Portions of this document were altered by the EPA reviewer to reflect Agency policies and conclusions

. 870.4100b Chronic Toxicity

— Dog

One

- year oral toxicity study in dogs. MRID 49267452.Diethofencarb Hum an Health Risk Assessment D418227

Page 32 of 49 This chronic oral toxicity study in the dog is Acceptable/Guideline and satisfies the guideline requirement for a chronic oral toxicity study (OCSPP 870.4100b; OECD 452) in a non

- rodent species

.

In a chron ic study (MRID 49267452 ), g roups of Beagle dogs (6/sex/group) were administered Diethofencarb daily via capsule at dose levels of 0, 10, 50, 250, and 1000 mg/kg/day for 52 weeks. Mortality, clinical signs, body weight, and food consumption were recorded. Samples of blood and urine were collected for clinical pathology evaluation prior to study initiation and on weeks 13, 26, 39, and 52. At study termination, all animals were sacrificed and subjected to gross necropsy. Selected organs were weighed and a standard range of organs and tissues were removed, fixed, and examined microscopically.

Both sexes at 1000 mg/kg/day and 250 mg/kg/day had increased frequency of emesis, increased absolute and relative liver weights, dark livers (increased liver pigment observed histologically), and hepatocellular enlargement. In males, body weights were decreased >10% (up to 23%) beginning at week 13 in the 250 and 1000 mg/kg/day groups. While these decreases reached statistical significance only at 1000 mg/kg/day (p<0.05, on week 13), the decreases were considered treatment related and adverse. Increased salivation and emesi s w ere observed in both sexes at 1000 mg/kg/day. A statistically significant decrease (p<0.05) in absolute and relative uterine weights was observed at 1000 mg/kg/day. While a clear dose

- response relationship could not be established at lower dose levels , this finding is considered biologically relevant because it is more severe in magnitude than at any other dose level, and is statistically significant. No
treatment

- related biological effects were observed at 10 mg/kg/day or 50 mg/kg/day. The increased incidence of emesis in the presence of decreased body weights and food consumption in males was considered adverse.

Therefore, the LOAEL in males for this study is 250 mg/kg/day based on increased emesis and decreased body weights; the NOAEL in males is 50 mg/kg/day. In females the LOAEL is 1000 mg/kg/day based on decreased absolute and relative uterine weights and the NOAEL is 250 mg/kg/day.

This chronic toxicity study in dog s is A cceptable/Guideline and satisfies the requirements of G uideline OCSPP 870.4100 ( OECD 452 )

. Disclaimer

This Data Evaluation Record is a modified version of the Ministere de l’Agriculture et de la Pe che (France) Draft Assessment Report (DAR), Diethofencarb Vol. 3, Annex B6, p. 259 (2007). Portions of this document were altered by the EPA reviewer to reflect EPA policies and conclusions.

870.4200b Carcinogenicity (feeding)

— Mouse

Mouse carcinogenicity study. MRID 49267453 This study is classified as Acceptable/guideline , and satisfies the guideline requirements (OSCPP 870.4200b; OECD 451) for a carcinogenicity study in the mouse

.
In a mouse carcinogenicity study ( MRID 49267453 ) Five g roups of 75 Crl:CD

- 1(ICR)BR mice/sex/group were administered Diethofencarb (98.1% a.i., Lot Numbers PEG 85045 and PEG 86018) in the diet at 0, 10, 100, 1000, or 10,000 ppm (equivalent to 0, 1.63, 16.47, 163.8, or 1682 mg/kg/day in males; 0, 2.03, 20.53, 20 2.9, or 2108 mg/kg/day in females) for 78 weeksDiethofencarb Hum an Health Risk Assessment D418227

Page 33 of 49 with interim sacrifices of 10/sex/group at 13 weeks and 15/sex/group at 52 weeks. Mortality, clinical signs, body weight, and food and water consumption were recorded. Differential leukocyte counts and cell morphology were conducted on blood from all surviving animals after 52 and 78 weeks of treatment. Gross necropsies, organ weights, and microscopic examination of tissues were performed on decedents and on those sacrificed on schedule.

Females at 10,000 ppm had a significantly lower survival rate relative to controls at 78 weeks (

- 33%, p < 0.05). The incidence of hepatocellular hyperplasia in high dose males was significantly higher (+18%, p < 0.05) relative to controls.

Under the conditions of this stud y, the systemic LOAEL for Diethofencarb in CD

- 1 mice was 10000 ppm in the diet in males (1682 mg/kg/day) based on increased incidence of hepatocellular hyperplasia, and 10000 ppm in females (2108 mg/kg/day) based on reduced survival. The NOAEL was 1000 pp m in males and females (163.8 and 202.9 mg/kg/day, respectively).

At the doses tested, there was not a treatment

- related increase in tumor incidence when compared to controls. Dosing was considered adequate to assess carcinogenicity based on hepatocellular hyperplasia observed in males at 1682 mg/kg/day) , and reduced survival in females at 2108 mg/kg/day. Dosing was also considered adequate because administered dose levels exceeded the limit dose.

This carcinogenicity study in mice is A cceptable/ G uideline and satisfies the guideline requirements for a cancer study ( O CSPP 870.4200b [§83

- 2b]; OECD 451 ) in the mouse.

This Data Evaluation Record is a modified version of the Ministere de l’Agriculture et de la Peche (France) Draft Asses sment Report (DAR), Diethofencarb Vol. 3, Annex B6, p. 288 (2007). Portions of this document were altered by the EPA reviewer to reflect EPA policies and conclusions.

870.4 3 00 Combined Chronic Toxicity/Onco genicity (feeding)

— Rat

Rat chronic/oncogenicit y study. MRID 4926745 4 This study is classified as Acceptable/guideline , and satisfies the guideline requirements (OSCPP 870. 4300 ; OECD 45 3 ) for a chronic/oncogenicity study in the rat

. In a rat chronic/oncogenicity study (MRID 49267454) f ive groups of 80 Crl:CD ® (SD)BR rats/sex/group were administered Diethofencarb (Batch Nos. PKG 84093, PEG 85045, PSG 86018) in the diet at 0, 40, 200, 1000, or 5000 ppm (equivalent to 0, 1.69, 8.42, 42.70, or 220.30 mg/kg/day in males; 0, 2.18, 10.90, 54.50, or 288.19 mg /kg/day in females) for 104 weeks with interim sacrifices of 10/sex/group at 52 weeks and 20/sex/group at 104 weeks. Mortality, clinical signs, body weight, and food and water consumption were recorded. Samples of blood and urine were collected for clinic al pathology evaluation, including cholinesterase activities in plasma and red blood cells, at weeks 13, 26, 52, 78, and 104. Brain tissue for cholinesterase determination was collected at weeks 52 and 104. Gross necropsies, organ weights, and microscopi c examination of tissues were performed on decedents and on those sacrificed on schedule.

Food conversion efficiency was lower in both sexes at the high dose and water consumption was also lower for females. Platelet counts and cholesterol levels were elevated in high dose females.Diethofencarb Hum an Health Risk Assessment D418227

Page 34 of 49 However, the elevated platelet counts were within the range of historical control and are not considered adverse; the elevated cholesterol was not considered adverse in the absence of corroborating effects such as changes in liver weight or liver histopathology.

T here was a significantly higher incidence of thyroid follicular tumors in both sexes at 5000 ppm relative to controls. (220.3 mg/kg/day and 288.19 mg/kg/day in males and females, respectively, as evidenced by a treat ment

- related increase in thyroid follicular cell tumor incidence (carcinomas in males, adenomas in females). The NOAEL for this study was 5000ppm (220 mg/kg/day in males, 288 mg/kg/day in females) , the highest dose level test. The LOAEL was not establish ed because there were insufficient indicators of non

- neoplastic toxicity at the dose levels tested.

Dosing was considered adequate to assess carcinogenic potential based on the presence of treatment

- related thyroid tumors at 5000ppm (220 mg/kg/day in males, 288 mg/kg/day in females)

. This study in rats is classified as A cceptable/ G uideline and meets the guideline requirements for

a c ombined c hronic Toxicity / Oncogenicity study in the rat [ O CSPP 870. 4300 ; OECD 45 3]

.

This Data Evaluation Record is a modified version of the Ministere de l’Agriculture et de la Peche (France) Draft Assessment Report (DAR), Diethofencarb Vol. 3, Annex B6, p. 284 (2007). Portions of this document were altered by the EPA reviewer to reflect EPA policies and conclusions.



### 870.5100 Bacterial reverse mutation test

In vitro Bacterial Gene Mutation (MRID 49067456) This mutagenicity study is classified as a cceptable / g uideline and satisfies the requirement for Test Guideline OCSPP 870.5100 1 ; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

In a reverse gene mutation assay in bacteria (MRID 49 267456 ), S. typhimurium strains TA98, TA100, TA1535, and TA1537, TA1538 and E. coli strain WP2 uvr A were exposed to diethofe ncarb (9 7

. 4 % a.i.

, batch number PK

- 84042 ) dissolved in dimethylsulfoxide ( DMSO ) in two independent assays using the pre

- incubation method and duplicate plating. These mutagenesis assays were preceded by a range

- finding assay performed at concentrations ranging from 10 through 5000 μg/plate. Both mutagenesis assays tested concentrations of 0, 5 , 10, 50, 100, 500, and 1 000 μg/plate, with and without S9

- mix. The S9 fraction was obtained from polychlorinated biphenyl (PCB)

- induced male Sprague

- Dawley rat liv er.

The test article, diethofencarb , was tested up to, but not exceeding, the limit at which the presence of precipitate was expected to seriously interfere with the counting of revertant colonies, both in the absence and presence of S9 mix, based on find ings in the range

- finding test. At higher concentrations the precipitate precluded the counting of revertant colonies. No cytotoxicity was seen in the range

- finding assay. Also, no cytotoxicity was observed in the two mutagenesis assays. There was no menti on of a precipitate in the mutagenesis assays. The number of revertants per plate was not increased over the concurrent solvent control value at any test material concentration, with or without S9

- mix, in any tester strain. The solvent and positiveDiethofencarb Hum an Health Risk Assessment D418227

Page 35 of 49 control s induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as a cceptable / g uideline and satisfies the requirement for Test Guideline OCSPP 870.5100 1 ; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

This Data Evaluation Record is a modified version of the Ministere de l’Agriculture et de la Peche (France) Draft Assessment Report (DAR), Diethofencarb Vol. 3, Annex B6, pp. 267

- 269 (2007 ). Portions of this document were altered by the EPA reviewer to reflect Agency policies and conclusions

.

In vitro Bacterial Gene Mutation (MRID 49067457) This mutagenicity study is classified as a cceptable / g uideline and satisfies the requirement for Test Guideline OCSPP 870.5100 1 ; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

In a reverse gene mutation assay in bacteria (MRID 49 267457 ), S. typhimurium strains TA98, TA100, TA1535, and TA1537, TA1538 and E. coli strain WP2 uvr A were exposed to diethofencarb (9 8

. 2 % a.i.

, batch number PSG

- 86018 ) dissolved in dimethylsulfoxide ( DMSO ) in two independent assays using the pre

- incubation method and duplicate pl ating. These mutagenesis assays were preceded by a range

- finding assay performed at concentrations ranging from 15 through 5000 μg/plate. Both mutagenesis assays tested concentrations of 0, 50, 100, 200, 500, 1000 and 2 000 μg/plate, with and without S9

- mix . The S9 fraction was obtained from polychlorinated biphenyl (PCB)

- induced male Sprague

- Dawley rat liver.

Based on findings in the range

- finding test, t he test article, diethofencarb , was tested up to and, in some cases, beyond the limit at which the presence of precipitate was expected to seriously interfere with the counting of revertant colonies, both in the absence and presence of S9 mix. At higher concentrations, the precipitate precluded the counting of revertant colonies. No cytotoxicity was seen in the range

- finding assay. Also, no cytotoxicity was observed in the two mutagenesis assays. In the mutagenesis assays, a precipitate was observed at 1000 and 2000 μg/plate both in the presence and absence of S9

- mix. The precipitate was so heavy at the concentration of 2000 μg/plate with S9

- mix that it was impossible to count the colonies. The number of revertants per plate was not increased over the concurrent solvent control value at any
test mate rial concentration, with or without S9

- mix, in any tester strain. The solvent and positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as a cceptable / g uideline and satisfies the requirement for Test Guideline OCSPP 870.5100 1 ; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

This Data Evaluation Record is a modified version of the Ministere de l’Agriculture et de la Peche (France) Draft Assessment Report (DAR), Diethofencarb Vol. 3, Annex B6, pp. 269

- 271 (2007). Portions of this document were altered by the EPA reviewer to reflect Agency policies and conclusions

. 

### 870.5300 In vitro mammalian cell gene mutation testDiethofencarb Hum an Health Risk Assessment D418227

Page 36 of 49 In Vitro Mammalian Cells in Culture Gene Mutation Assay in Chinese Hamster V79 Lung Cells (MRID 49067458) This study is classified as acceptable/g uideline and satisfies the guideline requirement for Test Guideline O CSPP 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data

. In a mammalian cell gene mutation assay at the hypoxanthine

- guanine phosphoribosyl transferase (HGPRT) locus (MRID 49267458), Chinese hamster lung V79 cells cultured in vitro were exposed to technical diethofencarb (98.2% a.i., batch PSG

- 86018) dissolved in dimethyl sulfoxide (DMSO) for 5 hours at concentrations of 0, 20, 50, 100, or 200 μg/mL in three independent experiments in the absence of metabolic activation and in two independent experiments in t he presence of metabolic activation. These concentrations were chosen based on the results of a range

- finding assay that tested concentrations of the test article of 0, 5, 10, 20, 50, 100, 200, and 500 μg/mL in the absence and presence of metabolic activat ion in exposures that lasted 5 hours. Mammalian metabolic activation was achieved using the S9 fraction obtained from polychlorinated biphenyl (PCB)

- induced male Sprague

- Dawley rat liver.

In the range

- finding assay, the test article induced only slight cy totoxicity, with relative survival ranging from 71 through 96% of that of the solvent control at the four highest concentrations tested without and with metabolic activation. A precipitate was observed at concentrations of 200 and 500 μg/mL in the absence and presence of metabolic activation. In all mutagenesis assays, the test substance was tested to above the limit of solubility. The positive controls induced the appropriate responses. Although the first mutagenesis assay in the absence of S9

- mix suggeste d that mutations were induced at the maximum concentration tested, two repeat experiments in the absence of S9

- mix provided no indication of mutation induction. Neither mutagenesis assay in the presence of S9

- mix provided any suggestion of mutation inducti on. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable/g uideline and satisfies the guideline requirement for Test Guideline O CSPP 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene

mu tation) data.

This Data Evaluation Record is a modified version of the Ministere de l’Agriculture et de la Peche (France) Draft Assessment Report (DAR), Diethofencarb Vol. 3, Annex B6, pp. 271

- 273 (2007). Portions of this document were altered by the EPA reviewer to reflect Agency policies and conclusions

.



### 870.5375 In vitro mammalian chromosome aberration test

In vitro Mammalian Cytogenetics: Chromosome Aberration Assay Using Chinese Hamster Ovary Cells (MRID 49267459) This study is classified as Acceptable/ guideline

, and satisf ies the guideline requirement for Test Guideline OCSPP 870.5375; OECD 473 for in vitro cytogenetic mutagenicity data

.

In an in vitro mammalian cell chromosome aberration assay (MRID 49267459 ), Chinese hamster ovary cells were exposed to Diethofencarb, ( 97.4% a.i., Lot No. PK

- 84042 ) in a range

- finding test, for 48 h without S9 activation and for 6 h with activation, at concentrations ranging fromDiethofencarb Hum an Health Risk Assessment D418227

Page 37 of 49 10

- 6 to 10

- 2 M (0.27

- 2673.20 μg/mL). Based on the range

- finding test, in the definitive test, without S9, the concentrations were 5x10

- 5 , 10

- 4 , 2x10

- 4 , and 5x10

- 4 M, i.e. respectively, 13.37, 26.73, 53.46 or 133.66 μg/mL. The exposures were for 24 and 48 h with no recovery period. In the presence of S 9 activation, the concentrations were 10

- 4 , 2x10

- 4 , 5x10

- 4 , and 10

- 3 M, i.e. respectively, 26.73, 53.46, 133.66 or 267.32 μg/mL. The exposures were for 6 h with an 18 h recovery period. S9 came from male Sprague

- Dawley rats induced with polychlorinated biphenyls (PCB).

Diethofencarb was tested up to a maximum concentration of 1 3 3 .66 μg/ mL (limit of
cy totoxicity) in the absence of S 9 mix and up to 267.32 μg/ mL (limit of solubility) in the presence of S9 mix. No increase in the frequencies of polyploid me taphases was found after treatment with the test article, without or with activation. All of the positive controls gave appropriate responses, without and with activation, as did the concurrent negative contro ls. No historical laboratory positive or negati ve control data were reported

. There was no evidence of chromosome aberrations induced over background.

This study is classified as Acceptable /Guideline, and satisfies the guideline requirement for Test Guideline OCSPP 870.5375; OECD 473 for in vitro cytogenetic mutagenicity data . O nly 100 metaphases were scored inst e a d of the required 200 , but acceptable positive control data were available and the concentrations of test substance reached the limits of cytotoxicity and solubility, so the results of t he tests are not considered to be compromised.

This Data Evaluation Record is a modified version of the Ministere de l’Agriculture et de la Peche (France) Draft Assessment Report (DAR), Diethofencarb Vol. 3, Annex B, part B.6 Toxicology and metabolism, pp . 273

- 276 (2007). Portions of this document were altered by the EPA reviewer to reflect Agency policies and conclusions

. 

### 870.5395 Mammalian erythrocyte micronucleus test

In vivo Mammalian Cytogenetics

– Mouse Erythrocyte Micronucleus assay ( MRID 49267460 ) This study is classified as acceptable/non

- guideline and does not satisf y the guideline requirement for Test Guideline O CSPP 870.5395; OECD 474 for in vivo cytogenetic mutagenicity data

.

In an ICR mouse bone marrow micronucleus assay (MRID 4 9267460 ), 6 male mice/ group were each given a single intraper itoneal (i.p.) injection of diethofencarb ( test article S

- 32165, purity 97.4 %, Lot No. PK84042) in corn oil. Two independent studies were conducted: a time

- course study and a dose

- response study. In the tim e

- course study, three groups of mice received a dose of 5000 mg/kg (guideline limit dose is 2000 mg/kg). Six animals were sacrificed at 24, 48 and 72 h after treatment. Two additional groups of 6 mice apiece were treated with the vehicle only (20 mL/kg) as a negative control, or with cyclophosphamide as a positive control (80 mg/kg bw). In the dose

- response study, three groups of mice were treated with diethofencarb at dose

- levels of 1250, 2500 or 5000 mg/kg. Two additional groups of 6 mice apiece were trea ted with the vehicle only (20 mL/kg) as a negative control, or with 9,10

- dimethyl

- 1,2

- benzanthracene (DMBA) at a dose of 50 mg/kg bw. All animals were sacrificed 48 h after treatment.

In the dose

- response study, the test article produced small, but statis tically significant, decreases in the polychromatic erythrocytes ( PCE s), which were also dose

- related. T he time

- course stud ies at 48 and 72 h also showed reductions in the PCE s . This was an indication that the se treatmentsDiethofencarb Hum an Health Risk Assessment D418227

Page 38 of 49 caused moderate bone marrow cell toxicity. Administration of the positive control chemicals in both studies resulted in significantly higher numbers of micronucleated

- PCEs compared with the corresponding vehicle controls, demonstrating the validity of the assay. At the doses tested, t here were no significant increases in the frequencies of micronucleated polychromatic erythrocytes in bone marrow after any treatment time or dose level

. Note: T he guideline calls for testing both male and female animals unless there is data that show no sex d ifferences exist in toxicity to the test article. No data was presented in this study to justify the exclusion of female mice.

This study is classified as acceptable/non

- guideline and does not satisf y the guideline requirement for Test Guideline O CSPP 870 .5395; OECD 474 for in vivo cytogenetic mutagenicity data , because no data were presented to justify the exclusion of female mice. T he study can be upgraded to guideline if data can be presented that show that no sex differences exist in the toxicity of diethofencarb in mice.

This Data Evaluation Record is a modified version of the Ministere de l’Agriculture et de la Peche (France) Draft Assessment Report (DAR), Diethofencarb Vol. 3, Annex B, part B.6 Toxicology and metabolism, pp. 280

- 282 (2007). Portio ns of this document were altered by the EPA reviewer to reflect Agency policies and conclusions

. 

### 870.5550 Unscheduled DNA synthesis in mammalian cells in culture

Unscheduled DNA Synthesis in Primary Rat Hepatocytes/Mammalian Cell Cultures (MRID 4926746 1) This study is classified as una cceptable/guideline and does not satisf y the guideline requirement for Test Guideline OCSPP 870.5550; OECD 482/486 for other genotoxic mutagenicity data

. In an unscheduled DNA synthesis (UDS) assay (MRID 49267461 ), primary hepatocyte cultures from male Fischer 344 rats were exposed in vitro to diethofencarb (9 8.2 % a.i

. , Lot # PSG

86018 ), dissolved in DMSO, at concentrations of 0, 0.507, 1.01, 2.53, 5.07, 10.1, or 25.3 μ g/mL for 21.3 h in assay 1 and at concentrations of 0, 6.32, 12.6, 25.3, 50.6, 101, or 202 μ g/mL for 19.0 h in assay 2.

Diethofencarb was tested up to cytotoxic concentrations (64.5% survival at 25.3 μ g/mL and 67.7% survival at 202 μ g/mL) in assays 1 and 2, respectively. In assay 2, the cytotoxicity wa s much weaker: at 50.6 μg/mL, the relative survival was unaffected (97%) while the same concentration was found to be highly cytotoxic in assay 1. In a similar manner, the cytotoxicity at 25.3 μg/mL was about 35% less in assay 2 (relative survival of 99.5% ) than in assay 1 (relative survival of 64.5 %). Overall, there was an approximately 8

- fold difference in sensitivity to cytotoxicity between the cells used in assays 1 and 2, with the same relative survival being observed at 25.3 μg/mL in assay 1 and at 2 02 μg/mL in assay 2. None of the treatments with diethofencarb caused nuclear labeling to differ significantly from the solvent control. However, both assays showed a non

- statistically

- significant increase in the mean net nuclear grain counts at the highes t concentration level and, for both assays, the average percent of nuclei having five or more net grains is two times higher at the highest concentration level than at the next lower concentration level (for assay 1, these concentrations were 25.3 μg/mL an d 10.1 μg/mL, respectively, and for assay 2, they were 202 μg/mL and 101 μg/mL, respectively). These results are not considered reliable

, and the suggested negative result must be confirmed by another study (preferably done in vivo ). The positive control elicited the expected nuclear labeling. There wasDiethofencarb Hum an Health Risk Assessment D418227

Page 39 of 49 no evidence that unscheduled DNA synthesis, as determined by radioactive tracer procedures [nuclear silver grain counts] , was induced.

This study is classified as una cceptable/guideline and does not satisf y the guideline requirement for Test Guideline OCSPP 870.5550; OECD 482/486 for other genotoxic mutagenicity data

. Because of strong differences in the cytotoxicity seen in the two assays, because both assays showed non

- statistically significant increases in the mean net nuclear grain counts at the highest concentrations tested, and because the average percentage of nuclei having five or more net grains is two times higher at the highest concentration level than the next lower concentration level in both as says, the suggested negative result s must be confirmed by another study.

This Data Evaluation Record is a modified version of the Ministere de l’Agriculture et de la Peche (France) Draft Assessment Report (DAR), Diethofencarb Vol. 3, Annex B, part B.6 Tox icology and metabolism, pp. 278

- 280 (2007). Portions of this document were altered by the EPA reviewer to reflect Agency policies and conclusions

.


### 870.5 900 In Vitro Sister Chromatid Exchange Assay 

### in mammalian cells in culture

In Vitro Sister Chromatid Exchange Assay in Chinese Hamster Ovary Cells (MRID 49267475) In a Chinese hamster in vitro sister chromatid exchange (SCE) assay (MRID 49267475) , CHO

Kl cells were exposed to diethofencarb (purity 97.4 %, Lot No. PK

- 84042) dissolved in D MSO and culture medium at concentrations of 0, 0.27, 0.81, 2.67, 8.01, 26.73, 80.19 and 267.32 μg/mL, without and with activation in the first assay and at concentrations of 0, 26.73, 53.46, 133.65 and 267.32 μg/mL, without and with activation in the secon d assay. Exposure was for 2 hours followed by a 28

- hour recovery period. The S9 came from PCB

- treated male Sprague

Dawley rats.

Diethofencarb was tested up to cytotoxic and precipitating concentration s. The positive controls induced the appropriate response. There was no evidence of an increase in sister chromatid exchanges in CHO

- K1 cells induced over background

. This study is classified as acceptable/guideline and satisfies the guideline requirement for Test Guideline OCSPP 870.5900; OECD 479 for in vi tro cytogenetic mutagenicity data.

This Data Evaluation Record is a modified version of the Ministere de l’Agriculture et de la Peche (France) Draft Assessment Re port (DAR), Diethofencarb Vol. 3, Annex B, part B.6 Toxicology and metabolism, pp. 276

- 278 (2007). Portions of this document were altered by the EPA reviewer to reflect Agency policies and conclusions

.

870. 62 00 Neurotoxicity Battery

Acute Neurotoxicity study

– Rat (MRID 49267464 ) This acute neurotoxicity study is classified acceptable/guideline and satisfies the guideline requirement for an immunotoxicity study (OCSPP 870. 6200a ) in the rat

. In an acute neurotoxicity study ( MRID 49267464) , groups of 10 non

- fasted , ~ 6

- week

- old Crl:CD(SD) rats/ sex/dose were given a single oral dose of Diethofencarb (99.5% a.i , b atch/lot # S

- 7509

- TG) in 0.5% sodium methylcellulose at doses of 0, 200

, 600, or 2000 mg/kg bw and observed for 14 days. Neurobehavioral asses sment (functional observational battery and motorDiethofencarb Hum an Health Risk Assessment D418227

Page 40 of 49 a ctivity testing) was performed o n 10 animals/sex/group prior to initiation of treatment (

- 1 week) and at 6 hours (Day 0), 7 days, and 14 days post dosing. Cholinesterase activity was not determined. At s tudy termination, 5 a nimals/sex/group were euthanized and perfused in situ for neuropathological examination. Of the perfused animals, rats from the control and high

- dose
groups were subjected to histopathological evaluation of brain and peripheral nervou s system tissues.

Treatment with up to 2000 mg/kg bw of D iethofencarb did not produce any changes in clinical signs, mortality, body weight or body weight gain

, or gross and neuro

- histological findings.

Treatment with 600 and/or 2000 mg/kg bw produced neurological effects 6 hours post dosing on Day 0. In males at 600 and 2000 mg/kg bw, changes included decreased forelimb grip strength (474 and 449 g, respectively, vs. 527 g for controls) and reduced body temperature (37.4 and 37.2°C, respectively, vs. 37.8°C for controls), with males at 2000 mg/kg bw additionally exhibiting decreased alertness (9/10 high

- dose males affected vs. 1/10 controls). In females at 2000 mg/kg bw, changes included a slight decrea se in muscle tone (8/10 high

- dose females affected vs. 0/10 for controls) and pupillary reflex (7/10 high

- dose females affected vs. 0/10 for controls), reduced body temperature ( 35.8°C compared to 37.9°C for controls) , and increased landing footsplay (101 mm in high

- dose females vs. 86 mm for controls). Although not attaining statistical significance, a decrease in alertness was noted during handling in high dose females (4/10) compared to controls (1/10). Reductions in motor activity accompanied the cha nges in FOB parameters observed after dosing on Day 0. Overall mean motor activity was significantly decreased in males at 600 and 2000 mg/kg bw (532 and 492 counts, respectively, vs. 1190 for controls). When viewing the intersession data, males at 600 m g/kg bw had significantly reduced activity only over the first interval, while males at 2000 mg/kg bw had significantly decreases in motor activity over the first two intervals. In females, a decrease in mean motor activity was noted at 2000 mg/kg bw, but the decrease did not attain statistical significance. Examination of intersession data revealed a significant decrease in activity only over the first interval.

Observed in this study is a suite of functional observational battery (FOB) effects. These include d decreased forelimb grip strength, reduced body temperature

, and decreased alertness in males and decrease in muscle tone and pupillary reflex , reduced body temperature , and increased landing footsplay in females. While the FOB effects are sl ight and individually would not have been considered adverse, the suite of effects as a whole are considered adverse and are consistent with the effects indicative of a sympathetic nervous system response. This is considered to be a conservative call.

Th e LOAEL in this study is 2000 mg/kg in males and females based on decreased forelimb grip strength, reduced body temperature and decreased alertness (M) and decreased muscle tone and pupillary reflex, reduced body temperature and increased landing footspla y (F). The NOAEL was in males and females is 600 mg/kg bw

. This neurotoxicity study is classified as A cceptable / Guideline and satisfies the guideline requirement for an acute neurotoxicity study in rats ( OCSPP 870.6200; OECD 424).

Subchronic Neurotoxicity study

– Rat (MRID 4926746 3 ) This subchronic neurotoxicity study is classified acceptable/guideline and satisfies the guideline requirement for an immunotoxicity study (OCSPP 870. 6200b ) in the rat

.Diethofencarb Hum an Health Risk Assessment D418227

Page 41 of 49 In a subchronic neurotoxicity study (MRID 4 9267463 ) , Diethofencarb (99.5% a.i, b atch/lot # S

7509

- TG) was administered daily to 10 Crl:CD(SD) rats/ sex/dose in the diet at doses of 0, 10 00, 30 00, or 10, 000 ppm (equivalent to 0, 56.7, 172, or 571 mg/kg bw/day in males, respectively, and 0, 65.4, 194 , or 654 mg/kg bw/day in females, respectively) for 13 weeks

. Neurobehavioral assessment (functional observational battery [FOB] and motor activity testing) was performed o n 10 animals/sex/group during pre

- exposure and during Weeks 2, 4, 8, and 13 . Choli nesterase activity was not measured. At study termination, five animals/sex/group were euthanized and perfused in situ for neuropathological examination. Of the perfused animals, rats from the control and high

- dose groups were subjected to histopathological evaluation of brain and peripheral nervous system tissues. The rats not selected for neuropathological examination were sacrificed and discarded.

Treatment of rats with 1000 or 3000 ppm of Diethofencarb did not produce any changes in clinical signs, mortality, body weight or body weight gain, food consumption, FOB parameters, motor activity, or gross or neuro

- histological findings. The only death occurred in one male from the 3000 ppm group before the commencement of d osing and therefore was not related to
treatment.

Treatment with 10,000 ppm Diethofencarb produced minimal changes. Females had reductions in body weight gain over the intervals assessed by the reviewer (Weeks 0

- 1 and 0

- 13; 83 and 84% of controls, resp ectively) but absolute body weight remained comparable to controls. No significant changes in absolute body weight or body weight gain were noted in males. Food consumption was unaffected in males and females. No other changes in FOB parameters were obs erved in males or females at any other time point. No changes in motor activity or gross or neuropathological findings were noted.

The NOAEL in males and females was 10,000ppm (571 and 654 mg/kg bw/day, respectively), and the LOAEL was not established.

This neurotoxicity study is classified as A cceptable / Guideline and satisfies the guideline requirement for a subchronic neurotoxicity study in rats (OCSPP 870.6200; OECD 424).

870.7485 Metabolism

— Rat

Absorption, distribution, metabolism, and elimination study in rats. MRID s 49267467

– 49267471

. This metabolism study in the rat is classified Acceptable / Guideline and satisfies the guideline requirement for a metabolism study [OCSPP 870.7485, OECD 417] in the rat

.

In a series of metabolism stud ies (MRID s 49267467

– 49267471), [Phenyl

14 C] Diethofencarb ( Unlabeled Lot No. PK

- 84042, purity ~98%, Radiolabeled: [Phenyl

14 C]Diethofencarb, Batch No. C

- 84

- 127, radiochemical purity >99%, specific activity 30.1 mCi/mmol used in MRIDs 49267467and 4926 7468). (Unlabeled Batch No. S0518G, purity 98.2%, Radiolabeled: [Phenyl

14 C]Diethofencarb, Batch No. CFQ12311, radiochemical purity >99.2%, specific activity 9.6MBq/mg used in MRIDs 49267469, 49267470 ) was administered to groups of male or female
rats at doses of 10 or 300 mg/kg bw as a single or two

- day dose. In an addition, male rats were treated with a 10 mg/kg bw/day unlabeled test material for 14 days followed by a singleDiethofencarb Hum an Health Risk Assessment D418227

Page 42 of 49 radiolabeled dose of the test material. Absorption, tissue distribution, meta bolism and excretion of the test material were monitored.

The studies indicate that oral absorption was rapid and extensive; with 87

– 95% being eliminated in the urine and feces, and absorption was not saturated at doses up to 300 mg/kg bw/day or by mu ltiple low dose exposures. No sex

- related effects on absorption were found. The blood and tissue T max was ~0.5 hours and was independent of sex or dose in one study and was ≤ 1 hour in another.

Organ/tissue radioactivity was very low 7 days after trea tment; the highest total radioactivity residue after a 10 mg/kg bw dose was in the liver (0.05

- 0.08 μg equivalent /g), a drenals (0.02

- 0.04 μg equivalent

/g), whole blood, blood cells and kidney ( each 0.01

- 0.02 μg equivalent /g ) and skin ( 0.01 μg equivalent

/g). Based on these results and the rapidly decreasing fat/blood radioactivity ratio (1.07 at 1 hour down to 0.36 at 24 hours ), Diethofencarb was not bioaccumu

lated.

Diethofencarb was extensively bio

- transformed into many metabolites , irrespective o f dose or pretreatment, recovered in the urine. The primary metabolites were identified as: ( 4

- OH

- DFC sulf ate (

~ 49% administered dose); 4

- O H

- DFC glucuronide (6

- 16% administered dose ); 3

- OEt

- 4

- OH

- AA sulf

ate (10

- 17 % administered dose ); 3

- O Et

- 4

- O H

- AA glucur onide (2

- 3 % administered dose ); sulf

ate and/or glucuronide conjugates of 3

- OEt

- 4

- O ACA

- AA (3 % administered dose ), and an unknown metabolite that comprised 3% of the administered dose . Numerous minor metabo

lites were also present with each contributing <1 % of the administered dose. In the feces, numerous metabolites were identified with the unconjugated metabolite 4

- O H

- DFC comprising 2

- 4% of the administered dose being in the highest concentration.

Based on these results , the major metabolic routes inv olved: 1) de

- ethylation of the 4

- ethoxy group; 2) cleavage of the carbamate linkage ; 3) acetylation of the resultant amino group; and 4) conjugation of the resulta n t phenols with sulf uric acid and/or glucuronic acid. Additional minor metabolic routes were : 1) oxidation o f the isopropyl group; 2) cyclization of the oxidized isopropyl carbamate group; 3) oxidation of the 4

- ethoxy group ; 4) conjugation of th e phenols with glutathione; 5) γ

- glutamyltranspeptidation and depeptidation of glutathione to form the cysteine conjugate ( 4

- O H

- 5

- Cys

- DFC was not d etec ted); 6) N

- acetylation of the cysteine; 7) cleavage of C

- S linkage of the cysteine followed by methylation; and 8) oxidation of the S

methyl group. A minor metabolite, 3,4

- OEt

- 6

- O H

- AA, was detected and ma y be produced by cleavage of the carbamate linkage followed by oxidation of C

- 6 position of the phenyl ring.

Excretion was rapid; being 93

- 100% of the administered dose by day 2 and was i ndependent of sex, dose

, and pre

- treatment . Most of the radiolabel was eliminated in the urine (≥80%) with the remainder eliminated in the feces. The short tissue distribution times and very short retained fraction s on day 7 (<0.2 % of the administered dose) do not suggest the metabolites undergo extensive entero hepatic recirculation

. This metabolism study in the rat is classified A cceptable/Guideline and satisfies the guideline requirement s for a metabolism study [ OCSPP 870.7485, OECD 417] in the rat. Biliary or IV studies should have been con ducted to determine the contribution of liver or intestinal microflora metabolites recovered in the feces, as well as whether cleavage of the isopropyl linkage of the test material described in a report, but data not provided to the Reviewer, was the resul t of an isotopic effect or metabolism.Diethofencarb Hum an Health Risk Assessment D418227

Page 43 of 49 Portions of this Data Evaluation Record is a modified version of the Ministere de l’Agriculture et de la Peche (France) Draft Assessment Report (DAR), Diethofencarb Vol. 3, Annex B6, (2007). Portions of this docume nt were altered by the EPA reviewer to reflect Agency policies and conclusions

.

870.7800 Immunotoxicity

28

- Day Immunotoxicity study

– mouse (MRID 49267472 ) This immunotoxicity study is classified acceptable/guideline and satisfies the guideline requirement for an immunotoxicity study (OCSPP 870.7800) in mice.

In an immunotoxicity study (MRID 49267472), Diethofencarb (99.5% a.i., Lot No. S

- 7509

- TG) was administered to groups of 10 female Sprague Dawley rats in the d iet at concentra tions of 0, 1000, 3000, or 10,000 ppm ( equivalent to 0, 79.6, 236, or 764 mg/kg bw/day) for 28 days. A concurrent positive control group of 8 female rats was fed the basal diet for 28 days, and receiv

ed Cyclophosphamide (CPS), 10 mg/kg b w/day, on Days 23

– 27 by oral gavage. Six days before scheduled sacrifice, all rats were immunized with a single 0.5 mL IV injection of 2 × 10 8 sheep red blood cells (SRBC) into the tail vein.

One rat in the low

- dose group was found moribund unrelated to treatment and euthanized in extremis on study day 14 . No clinical signs of toxicity were found from the remaining rats and all other rats survived to scheduled sacrifice. The body weight of high

- dose rats was statistically decreas ed ~8% on study days 21

– 28, which was not sufficient to be to be considered biologically significant. In addition, food consumption was ≥ 1 g/rat/day lower for high

- dose rats and was statistically decreased during Weeks 1 and 3. No decreases in body weight, or f ood consumption were found in lower dose rats or rats receiving the positive control. No effects on absolute or relative to body thymus and spleen weights were found in Diethofencarb

- treated rats, however the absolute and relative to body thymus and splee n weights of CPS positive control rats was statistically decreased ~65% and 27%, respectively. T he systemic Diethofen carb LOAEL for female Sprague Dawley rats not established. The corresponding NOAEL was 10,000 ppm (equivalent to 764 mg/kg bw/day).

The anti

- SRBC titer of Diethofencarb

- treated rats was not decreased by treatment while the anti

SRBC titer of CPS

- treated rats was decreased 97%, indicating the validity of the study. The immunotoxicity LOAEL for Diethofencarb was not found. The NOAEL ≥ 10,000 ppm (≥ 764 mg/kg bw/day).

This immunotoxicity study is classified Acceptable / Guideline and satisfies the guideline requirement for an immunotoxicity study ( OCSPP 870.7800) in the rat.

This Data Evaluation Record is a modified version of the Mi nistere de l’Agriculture et de la Peche (France) Draft Assessment Report (DAR), Diethofencarb Vol. 3, Annex B6 ( 2007). Portions of this document were altered by the EPA reviewer to reflect Agency policies and conclusions

.

Non

- Guideline: Thyroid Mechanist ic Studies

14

- day Special Thyroid Mechanistic Study MRID 49267473.Diethofencarb Hum an Health Risk Assessment D418227

Page 44 of 49 This special study is Acceptable / Non

- guideline and does not satisfy a guideline requirement

. The study has numerous deficiencies but when combined with the companion study (MRID 4926747 4) provides data that can be considered reliable.

In a series of non

- guideline studies to determine effects on the thyroid gland (MRID 49267473), Diethofencarb (purity 97.8% and 98.2% and Lot numbers PK

- 84042 and PSG

- 86018M, respec

tively , and/or radiochemical purity >98% uniformly labeled on phenyl ring) was administered to groups of 10 male Sprague Dawley rats 7

- , 37

- , and 66

- weeks of age at concentrations of 0, 5000 or 20,000 ppm for 2 weeks to determine effects on thyroid hormone concentration; to groups of ten 31

- week male Sprague Dawley rats at concentrations of 20,000 ppm for 7 and 14 days to determine tissue distribution; and to groups of two to four 7

- and 37

- week male Sprague Dawley
rats at concen trations of 0 or 20,000 ppm for 14 days fo r biliary excretion and metabolism studies. The study report does not make clear whether non

- radiolabeled compound was administered to rats during the two

- week and biliary excretion and metabolism studies. Rats in the biliary excretion/metabo lism study received an IV injection of 2 μg/kg bw 125 I

- thyroxin in 0.5 mL saline at the end of feeding period to determine radiolabeled excretion. Since individual animal body weights were not provided in the study reports, the estimated doses to animals in the
two

- week feeding study were approximately 0, 430, or 1720 mg/kg bw/day for 7

- week animals, and 0, 345, or 1380 mg/kg bw/day for 37

- and 66

- week animals, respectively. These were calculated by the Reviewer using food conversion factors of 0.086 for 7 week and 0.069 for 37

- and 66

- week animals, respectively. For the tissue distribution study, the rats received an estimated dose of approximately 1380 mg/kg bw/day (based on a food conversion factor of 0.069 and assuming food consumption was similar to the 2

- week study). Finally, the estimated doses for rats treated with the test material were approximately 430 and 1380 mg/kg bw/day for 7

- week and 37

- week rats treated with 20,000 ppm, respectively (again assuming food consumption was similar to the 2

- week study

. Since body weights were not reported except terminal body weights in the 2

- week study, these were not included in the assumptions for bioaccumulation and biliary excretion studies. )

. Summary data from these studies indicate treatment with 20,000 ppm Die thofencarb for up to two weeks decreased serum T4 concentrations and induced slight increases in TSH concentra

tions with no effect on T3 concentrations. In the bioaccumulation study, t he kidney had the highest 14 C concen trations followed by the liver

, s kin and hair following both 7

- and 14

- days of
treat ment . No indication of bioaccumulation of radiolabel was found in the thyroid. The data also suggested that increased T4 excretion in the bile arose through metabolic processes related to hepatic UDP

- GT induction

. This study is classified as A cceptable /non

- guideline , and does not satisfy a guideline requirement. It has numerous deficiencies; however, but when evaluated in conjunction with MRID 49267474, which is a similar and better conducted study, and which produced similar, corroborating results, this study is considered useful in determining the effects of diethofencarb on thyroid tissue and function.

This Data Evaluation Record is a modified version of the Ministere de l’Agriculture et de la Pe che (France) Draft Assessment Report (DAR), Diethofencarb Vol. 3, Annex B6 (2007). Portions of this document were altered by the EPA reviewer to reflect Agency policies and conclusions

.

90

- day Special Thyroid Mechanistic Study MRID 4926747 4

.Diethofencarb Hum an Health Risk Assessment D418227

Page 45 of 49 This special study is Acceptable / Non

- guideline and does not fulfill a guideline requirement.

In a 90

- day Non

- guideline oral toxicity study to determine effects on thyroid function (MRID 49267474), Diethofencarb (98.2% a.i., Lot No. PSG

- 86018

- M ) was administered to 2 0 male SD rats/sex/ dose in the diet at levels of 0, 5000, or 20,000 ppm (equivalent to 0, 307.9, or 1277.9 mg/kg bw/day). Twenty

- four hours before scheduled sacrifice, half the rats in each group were given a single IP injection of 125 I to determine uptak e. Histological examinations were limited to the thyroid of rats not treated with 125 I.

Treatment of male rats with 20,000 ppm Diethofencarb for 90 days induced alopecia on 7

- 8/20 rats during the first 8 days of treatment. Thereafter, alopecia was not increased through the remainder of the study. The body weight of 20,000 ppm male rats was statistically decreased from Day 5 through the remainder of the study and total weight gain was decreased 16%. Body weight of 5000 ppm rats was decreased 4

– 6% fr om Day 12 through study end, with total weight gain decreased 8%. Food consumption was slightly decreased (<10%) through week 10 of the study for both 5000 ppm and 20,000 ppm rats. Slight dose

- related statistical decreases in circulating free T 4 and incr eases in TSH (thyroid stimulating hormone) were found in 20,000 ppm rats at study end; however, total T 4 was not statistically different from control rats, though decreased 16%. No significant effects were found on total or free T 3 in rats treated with 20,000
ppm. There was a 16% increase in relative to body thyroid weight of 20,000 male rats, but no effects were found on absolute thyroid weight or 125 I thyroid uptake, and no microscopic effects were found. Statis tically signific ant increases in liver UDP

- GT (UDP

- glucuronyl transferase) activit ies were found in 20,000 ppm rats with activity/kg bw (3.7 fold), activity/g liver weight (2.6 fold), a nd activity/mg protein (1.9 fold). Slight increases were also found in 5000 ppm rats f or the same parameters, but were not statistically increased (1.3 fold for activity/kg bw, 1.4 fold for activity/g liver weight, and 1.4 fold for activity/mg protein). The absolute liver weight of 20,000 ppm rats was statistically increased 25%, while th e liver relative to body weight was increased 42%. The liver weight relative to body weight was statistically increased 13% in 5000 ppm rats, while no significant increase in absolute liver weight was found.

These results suggest that Diethofencarb had n o direct effect on the thyroid, but the effects were due to accelerated excretion of thyroid hormones from the liver as evidenced by the increase in liver UDP

- GT activity. This resulted in a decrease in serum free T 4 concentrations that likely triggered t he feedback mechanism of the pituitary gland, and promotion of TSH release.

The LOAEL for Diethofencarb in male SD rats was 20,000 ppm (equivalent to 1277.9 mg/kg bw /day ) based on decreased body weight and body weight gain, decreased serum free T 4 , increased TSH, and increased liver UDP

- GT activities. The NOAEL was 5000 ppm (307.9 mg/kg bw /day )

.

This 90

- day oral toxicity supplemental study in the male rat is Acceptable / Non

- guideline and does not satisfy a guideline requirement.

This Data Evalu ation Record is a modified version of the Ministere de l’Agriculture et de la Peche (France) Draft Assessment Report (DAR), Diethofencarb Vol. 3, Annex B6 (2007). Portions of this document were altered by the EPA reviewer to reflect Agency policies and

con clusions

.Diethofencarb Hum an Health Risk Assessment D418227

Page 46 of 49 Appendix B. Metabolism Considerations

B.1. Metabolism Summary Table

Summary of Metabolites and Metabolism Studies 1

Name; Abbreviation; Structural formula Crop Major Residue ; %TRR Minor

Residue; % TRR Diethofencarb 1

- methylethyl(3,4

- diethoxyphenyl)carbamate Tomato 69; 72.9

---

Lettuce 57.3; 52.0

---

Grape 19.9; 23.1

---

DFC

- COOH 2

- [(3,4

- diethoxyphenylcarbamoyl)oxy]propionic acid Lettuce

--- ---

Tomato

--- 2.6; 2.4 2

Grape

--- ---

Rat Feces, 2

- 4% AD

4

- OH

- DFC

Isopropyl 3

- ethoxy

- 4

- hydroxycarbanilate Tomato

--- 2.6; 2.4 2

Lettuce

--- 0.5; 0.3
Grape

--- 0.4; 0.9

Rat Multiple conjugates were found

in urine, individually up to 49%

DPO 3

- (3,4

- diethoxyphenyl)

- 5

- methyloxazolidine

- 2,4

- dione Lettuce

--- ---

Tomato

--- 0.9; 1.2

Grape

--- ---

4

- OH

- 5

- TLA DFC ; (OTD)

3

- {3

- ethoxy

- 2

- hydroxy

- 5

- [(isopropoxycarbonyl)amino] phenylthio}

- 2

- hydroxypropionic acid Lettuce

--- 0.6; 0.6

Tomato

--- ---

Grape

--- 3.2; 2.0Diethofencarb Hum an Health Risk Assessment D418227

Page 47 of 49 Summary of Metabolites and Metabolism Studies 1

Name; Abbreviation; Structural formula Crop Major Residue ; %TRR Minor

Residue; % TRR 4

- Glc

- 5

- TLA DFC ; (GTD) 3

- {3

- ethoxy

- 2

- 

- glucopyranosyloxy

- 5

- [(isopropoxycarbonyl)amino] phenylthio

- 2

- hydroxypropionic acid Lettuce

--- 0.9; 1.9 3

Tomato

--- 2.6; ND 3

Grape

--- 1.6; 1.9

4

- Glc

- DFC ; (GD) Isopropyl 3

- ethoxy

- 4

- 

- glucopyranosyloxycarbanilate Lettuce

--- 0.9; 1.9 3

Tomato

--- 2.6; ND 3

Grape 15.5; 14.6

---

4

- OH

- 5

- (OGlc

- TLA) DFC 3

- {3

- ethoxy

- 2

- hydroxy

- 5

- [(isopropoxycarbonyl)amino] phenylthio}

- 2

- 

- glucopyranosyloxypropionic acid Tomato

--- 1.7; 2.4

Lettuce

--- ---

Grape 21.5; 20.7

---

Unknown 1 4 Tomato 11.8 (isopropyl) 6.9 (phenyl)

Tomato

- [Phenyl

- 14C] ; two applications; PHI = 3 days; TRR = 0.116 ppm; 2 x 375 g a.i ./ha/application Tomato

- [Isopropyl

14 C] ; two applications; PHI = 3 days; TRR = 0.085 ppm; 2 x 375 g a.i ./ha/application Lettuce

- [Phenyl

- 14C] ; five applications; PHI = 7 days; TRR = 2.02 ppm; 5 x 38 g ai./ha/application. Lettuce

- [Isopropyl

14 C] ; five applications; PHI 7 Days ; TRR = 1.76 ppm; 5 x 38 g ai./ha/application. Grape

- [Phenyl

- 14C] ; one application; PHI = 35 days; TRR = 2.83 ppm; 1 x 500 g a.i ./ha/application Grape

- [Isopropyl

14 C] ; one application; PHI 35 Days ; TRR = 5.46 ppm; 1 x 500 g a.i ./ha/application (Note that the current labels for bananas allow up to 6 applications at 150 g ai/ha with a 0 day PHI.)

1 If two values are listed, results from phenyl label study listed first. Summary at bottom of table. Three crop studies were conducted at different PHIs and are listed in increasing PHIs.

2 4

- Glc

- 5

- TLA DFC could not be distinguished from 4

- Glc DFC (GTD or GD) using method employed so both analytes are reported in this table.

3 4

- OH DFC could not be distinguished from DFC

- COOH using method employed so both analytes are reported in this table.

4 Although this unknown was >10% of the residue in tomatoes (isopropyl l abel only) , the level was low, 0.01 ppm. It did not co

- chromatograph with any reference standard and could not be identified.Diethofencarb Hum an Health Risk Assessment D418227

Page 48 of 49 B

. 2. Metabolic Pathways

Proposed Metabolic Pathway in Plants (Copied directly from EFSA Draft Assessment Report for Diethofencarb, Vol 3

– Annex B7)

.

a T he metabolites 4

- Glc

- DFC and 4

- Glc

- 5

- TLA DFC could not be separated; the percentage of TRR given corresponds to the sum of the amount of both metabolites

.

Tomato: 73 %, Grape 23%

, Lettuce 55% TRR Tomato: 2.6 % TRR Tomato: 1.2 % TRR

Tomato: 2.6 % , Grape 0.9%

, Lettuce 0.5% TRR

Grape: 3.2 % Lettuce 0.6% TRR T Tomato: 2.6 % Grape 1.9% lettuce 1.5% TRR a Tomato: 2.4 % Grape 21%TRR Tomato: 2.6 %

, Grape 15%

, Lettuce 1.5% TRR aDiethofencarb Hum an Health Risk Assessment D418227

Page 49 of 49 Appendix C

. Physical/Chemical Properties

Physicochemical Properties of the Nominal 99% Diethofencarb Parameter Value Reference Molecular weight (g/mole) 267.32 g/mol MRID 49267477. EFSA Journal 2010:8(9):1721, Appendix A Melting point/range (°C) 98.3

- 100.9 MRID 49267477. EFSA Journal 2010:8(9):1721, Appendix A pH Does not dissociate MRID 49267477. EFSA Journal 2010:8(9):1721, Appendix A Density (g/cm 3 ) 1.206 at 25 o C EC Draft Assessment Report, Vol3, Annex B, B.2.1.2 Water solubility ( mg/L at 20°C) 27.64 MRID 49267477. EFSA Journal 2010:8(9):1721, Appendix A Solvent solubility (g/L at 25°C) Methanol 106.1 Acetone 207.4

n

- Octanol >28.9 Xylene 29.9 Ethyl acetate 140.4 Dichloro

methane 413.2

n

- Heptane 1.23 Vapor pressure at 25°C (Pa) 9.44 X 10

- 6 Dissociation constant (pK a ) Does not dissociate between pH 2 and pH 12 Octanol/water partition coefficient Log(K OW ) 2.89 at 20 o C