### UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION



### MEMORANDUM

DATE: December 6, 2013

SUBJECT: Ethoprop: Review of 4

- Week Inhalation Rat Study.

DP Barcode: D401438 Registration No.: NA PC Code: 041101 Decision No.: 464348 Petition No.: NA Risk Assessment Type: Single Chemical TXR No.: 0056347 Regulatory Action: Registration Review CaseNo.: NA

MRID No.: 48779601 CAS No.: 13194

- 48

- 4 40 CFR: NA

FROM:

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TO: John Liccione, Ph.D.,

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Toxicologist Risk Assessment Branch V Health Effects Division (7509P)

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Monica Wait, Chemical Risk Manager RMIB3 Pesticide Reevaluation Division (7508P)

I. CONCLUSIONS

A 4-week inhalation rat study with ethoprop has been reviewed and is attached. The study will be included in the hazard characterization and hazard identification for risk assessment.

II. ACTION REQUES TE D Review the submitted studies for the requested registration action and risk assessment.

III. MRID Summary Table

MRID Comments 48779601 N e wD E RDATA EVALUATION RECORD

ETHOPROP (Ethoprophos)

STUDY TYPE: 4-WEEK INHALATION TOXICITY- RAT (OCSPP 870.3465)

Primary Reviewer: MRID 48779601

Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency One Potomac Yard 2777 South Crystal Drive

Arlington, VA 22202

Prepared by Summitec Corporation 9724 Kingston Pike, Suite 602

Knoxville, Tennessee 37922

Task Order No. 6-24

Thomas C. Marshall, Ph.D., DABT Signature: Date: Secondary Reviewers: H.T. Borges, Ph.D., MT(ASCP), DABT Signature: Date:

Robert Ross, M.S., Program Manager Signature: Date: Quality Assurance: Jennifer Goldberg, B.S. Signature: Date:

Disclaimer

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This review may have been altered subsequent to the contractor's signatures above.

Summitec Corp. for the U.S. Environmental Protection Agency under Contract No.EP-W-11-014ETHOPROP TECHNICAL / 041101

EPA Reviewer: Kit Farwell Risk Assessment Branch VII, Health Effects Division (7509P) EPA Secondary Reviewer :**---"-J -"'-** **'"-" hn** **""" .=.c** **cc """'** **"" o.!..On** **.=.c e**  _ __ ____ _ Health Effects Division (7509P)

TXR#: 005634 7

DATA EVALUATION RECORD

STUDY TYPE: 4

- Week Inhalation Toxicity

- Rat OCSPP 870.3465.

PC CODE: 041101 DP BARCODE: D40 1 438

TEST MATERIA L (PURITY): E thoprop Technical (96.6%)

SYNONYMS: Ethoprophos Technical

CITATION: Phillips, L. (2012)

. Ethoprop Technical: Toxicity study b y inhalation administration to rats for 4 weeks

. Huntingdon Life Sciences, Ltd., En gland

. Project ID # BDG0059, March 22
, 2012. MRID 48779601. U npublished

.

SPONSOR: AMV AC Chemical Corporation

.

EXECUTIVE SUMMARY:

In a nose

- only inhal ati on exposure exposure toxicity study (MRID 48779601) Etho prop Technical (Lot No

. PEA4000032; 96.6% purity) aerosol was administered to male and female Sprague

- Dawley rats (1 0 / sex / concentration) for 6 hours / day
, 5 days /w eek for 4 weeks at exposure concentrations of 0, 0.5, 2, or 6 !lg / L. Actual exposure concentrations as measured b y HPLC were 0.498, 2.05, and 6.36 !lg /L.

A ll animals survi ved until scheduled sacrifice. There were no treatment-related clinical findings. There were no treatment

- related effects on body weight, body weight gain
, or in any of the organ weight data in any exposure group. There were no treatment

- related effects on hematolog y or clinical chemistry parameters

. There were no test substance

- related macroscopic changes observed in any tissues. Eth oprop exposure resulted in port

- of

- entry effects at all exposure levels in the larynx consistin g of squamous cell metaplasia and epithelial h yperplasia

. Among observations in the testes (seminiferous tubular atroph y, tubular d egenerat iv e germ cells / vacuolation and in the epididymides, degenerative spermatogenic cells in the duct at 2 and 6 !lg /L), the tubular atrophy observed at 6 !lg / L could not be ruled out as a substance

- related effect since its incidence was above the historical background range and similar effects at a lower incidence were observed at 2 !lg / L. E rythrocyte cholinesterase activity was decreased

re l ative to controls at all exposure concentrations by 9% to 41% (statistically significant only in males at the highest concentration). Brain cholinest e rase activity was stat i st ic a lly d ecre ased by 13% to 37% at the two highest exposure concentrations. Brain cholinesterase activity inhibit ion
at 10% or above is generally considered toxicologically relavant.4-Week Inhalation Toxicity Study (rat) (2012) / Page 3 of 11 ETHOPROP TECHNICAL/041101 OCSPP 870.3465



### Based on the effects seen in this study, th e NOAEL in male and female Sprague-Dawley rats was 0.5 μ g/L. The LOAEL in male and female rats was 2 μ g/L based on brain cholinesterase activity inhibition.

This inhalation toxicity study is classified as Acceptable (Guideline) and satisfies the guideline requirement for 4-week inhalation toxicity study in rats (870.3465).

COMPLIANCE : Signed and dated GLP, Quality Assurance, and Data Confidentiality statements were provided.4-Week Inhalation Toxicity Study (rat) (2012) / Page 4 of 11 ETHOPROP TECHNICAL/041101 OCSPP 870.3465



### I. MATERIALS AND METHODS:

A. MATERIALS :

1. Test material : Ethoprop Technical
Description: Oily yellow liquid
Lot/batch #: PEA4000032 Purity: 96.6% a.i.
CAS # of TGAI: 13194-48-4
Structure:



### 2. Vehicle : Filtered air

3. Test animals : Species: Rat Strain: Crl:CD(SD) Age/weight at study initiation: 47-53 days/ Males 230-298 g; Females 181-239 g Source: Charles River Ltd., UK Housing: 5 of one sex/polycarbonate cage with ster ilized wood based bedding changed weekly Diet: Certified Rodent LabDiet® 5002, PM I Nutrition International, LLC, ad libitum except during exposure. Water: Tap water, ad libitum Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 19-23 0 C 40-70% Not reported 12 hours light/dark

Acclimation period: 11 days



### B. STUDY DESIGN :

1. In life dates: Start: October 31, 2011; End: November 30, 2011

2. Animal assignment and treatment: Rats were assigned to the experimental groups presented in Table 1, but the method of assignment was not specified. The exposure concentrations were selected based on the results of a one-week pilot study in which erythrocyte and brain acetylch olinesterase activity inhibiti on (AChEI) were observed at exposure concentrations of 5.42 to 66.6 μ g/L, but not at 2.17 μ g/L.4-Week Inhalation Toxicity Study (rat) (2012) / Page 5 of 11 ETHOPROP TECHNICAL/041101 OCSPP 870.3465

TABLE 1. Study Design
Experimental parameter (test relative to dosing) Ethoprop Concentrations ( μ g/L) a
0 0.5 2 6 Number of Rats Total no. of animals assigned 20 (10/se x) 20 (10/sex) 20 (10/sex) 20 (10/sex)

Blood AChEI 10/sex 10/ sex 10/sex 10/sex Brain AChEI 10/sex 10/sex 10/sex 10/sex Gross and microscopic pathol ogy 10/sex 10/sex 10/sex 10/sex

a Actual exposure levels were 0.498, 2.05, and 6.36 μ g/L. Data obtained from pages 14-22 (MRID 48779601)


### 3. Generation of the test atmosphere / chamber description : Chamber atmospheres were generated by stainless steel concentric jet atom izers coupled with a distribution system that delivered a controlled amount of vapor/aerosol to the inlet of each aluminum alloy nose-only exposure chamber. A syringe pump (Precidor) was used to deliver the test substance to the generator and combined with filtered compressed air, both of which were metered to deliver the appropriate atmosphere conc entration to the chamber. Nose -only exposure chambers (one for each exposure level) were fitted with air fl ow meters, pressure gauges, and other devices to measure temperature, humidity, and test article concentration.

4. Test atmosphere concentration : The exposure concentrations were measured three times per day by drawing a known volume of the test atmosphere at a rate of 2L/minute through a glass sintered impinger trap containing acetonitrile/water/antifoam (80/20/0.02 v/v/v). Samples were taken from the breathing zone of the animals. Analyses were conducted using HPLC with UV detection. Results are in Table 2 and show that the achieved levels were 100, 103, and106% of target values. Time to equilibrium was not reported.

Table 2. Mean Exposure Concentrations during the 4-Week Exposure Regimen.

Data obtained from page 210 (MRID 48779601)



### 5. Particle size determination : Particle size for the highest exposure concentration was determined using a Marple 290 Series cascade impactor with stainless steel substrates. The mass median aerodynamic diameter and geometri c standard deviation were determined (Table 3). Sampling intervals and the number of samples was not provided. No particle size samples were taken for the two lower concentrat ions because preliminary trials showed that the atmospheres were predominately vapor with a negligible proporti on of droplets. These preliminary data were not provided.4-Week Inhalation Toxicity Study (rat) (2012) / Page 6 of 11 ETHOPROP TECHNICAL/041101 OCSPP 870.3465

Table 3. Mean Particle Size dur ing the 4-Week Exposure Regimen.

Data obtained from page 211 (MRID 48779601)



### 6. Statistics : All analyses were conducted comparing each test substance-exposed group to the control group by sex. The overall level of si gnificance for intergroup differences was p ≤ 0.05. Body weight, body weight change, organ weight, clinical pat hology, erythrocyte AChEI, and brain AChEI activity were subjected to a series of tests to determin e intergroup differences. Bartlett’s test for variance homogeneity, if not significant (p<0.01), was followed by the F 1 

### approximate test. If not significant the William’s test for monotonic trend was applied. If the F 1 

### was significant, then Dunnett's test was us ed to compare the test substance-exposed groups to the control group. If Bartlett’s test was significan t, then the nonparametric H 1 

### test was applied, followed by either Shirley’s test for monotonic trend or St eel’s test. Clinical pathology, erythrocyte AChEI, and brain AChEI activity were further analyzed by Fisher’s Exact test if 75% of the data had the same c value. Analysis of covariance (ANCOVA) was used to analyze the effect of body weight on organ weight.

The Reviewer considers the statistical analyses used to be appropriate.

C. METHODS / OBSERVATIONS :
1. Mortality and clinical observations : Animals were observed twice daily for clinical signs, abnormal behavior, morbidity, and mortality. Detailed clinical observations were recorded weekly.

2. Body weight : Animals were weighed weekly.

3. Food consumption : Food consumption was estimated for each animal by recording weekly food usage by cage (5 rats) less spillage then calculating mean daily diet consumption as g food/rat/week.

4. Hematology and clinical chemistry : Blood was collected from all surviving animals at the end of the treatment period for hematology and cl inical chemistry; the animals were fasted overnight prior to blood collection. Blood was coll ected from the sublingual vein while the rats were under light isoflurane anesthesia . The CHECKED (X) parameters were examined.4-Week Inhalation Toxicity Study (rat) (2012) / Page 7 of 11 ETHOPROP TECHNICAL/041101 OCSPP 870.3465



### a. Hematology:

X Hematocrit (HCT)* X Leukocyte differential count* X Hemoglobin (HGB)* X Mean corpuscular HGB (MCH)* X Leukocyte count (WBC)* X Mean corpusc. HGB conc.(MCHC) X Erythrocyte count (RBC)* X Mean corpusc. volume (MCV) X Platelet count* X Reticulocyte count Blood clotting measurements* X (Thromboplastin time) (Clotting time) X (Prothrombin time) * Recommended for 28-day oral rodent studies based on Guideline 870.3050


### b. Clinical chemistry :

X ELECTROLYTES X OTHER
X Calcium X Albumin* X Chloride X Creatinine* Magnesium X Urea nitrogen* X Phosphorus X Total Cholesterol* X Potassium* Globulins X Sodium* X Glucose*
ENZYMES (more than 2 hepatic enzymes eg., *) X Total bilirubin X Alkaline phosphatase (ALK)* X Total protein (TP)* Cholinesterase (ChE) X Triglycerides Creatine phosphokinase Se rum protein electrophoresis Lactic acid dehydrogenase (LDH) X Albumin/Globulin ratio X Alanine aminotransferase (ALT/also SGPT)* X Aspartate aminotransfera se (AST/also SGOT)* Sorbitol dehydrogenase* X Gamma glutamyl transferase (GGT)* Glutamate dehydrogenase * Recommended for 28-day oral rodent studies based on Guideline 870.3050



### c. Cholinesterase determination : Blood samples were centrifuged and the plasma frozen immediately. The brain was separated at th e midline after weighi ng during necropsy and the left side snap frozen for AChE determ ination. Erythrocyte and brain cholinesterase activities were determined usi ng a modified Ellman assay.

6. Urinalysis : Urine was not examined.

7. Ophthalmoscopic examination : Eyes were examined by an ophthalmoscope prior to the first day of exposure and during week 4.

8. Sacrifice and pathology : Rats were killed by i.p. injection of sodium pentobarbital followed by exsanguination before necropsy and collectio n of tissues for microscopic examination. Any gross lesions were recorded. Tissues from animals in the control and high dose groups were processed for microscopic evaluation. The larynx, epididymides, and testes were examined in animals from all exposure groups. The CHECKED (X) parameters were collected and examined. The (XX) organs were weighed.4-Week Inhalation Toxicity Study (rat) (2012) / Page 8 of 11 ETHOPROP TECHNICAL/041101 OCSPP 870.3465

X DIGESTIVE SYSTEM X CARDIOVASC./HEMAT. X NEUROLOGIC Tongue Aorta XX Brain Salivary glands XX Heart Peripheral nerve Esophagus Bone marrow Spinal cord (3 levels) Stomach Lymph nodes Pituitary Duodenum XX Spleen Eyes (optic nerve ) Jejunum XX Thymus X GLANDULAR Ileum XX Adrenal gland
Cecum X UROGENITAL Lacrimal gland Colon XX Kidneys X Parathyroid Rectum Urinary bladder X Thyroid
XX Liver XX Testes X OTHER

Gall bladder (not rat) X Epididymid es Bone (sternum and/or femur) Bile duct (rat) Prostate Skeletal muscle Pancreas Seminal vesicles Skin
X RESPIRATORY X Ovaries X All gross lesions and masses
X Trachea X Uterus XX Lungs Mammary gland X Nasal cavity X Cervix Pharynx X Larynx



### II. RESULTS:

A. OBSERVATIONS :
1. Clinical signs and Mortality : All animals survived until sche duled sacrifice. There were no treatment-related clinical findings. There were no treatment-related effects on body weight, body weight gain, or in any of the organ wei ght data in any exposur e group. There were no treatment-related effects on hema tology or clinical chemistry pa rameters. There were no test substance-related macroscopic changes obser ved in any tissues. Ethoprop exposure resulted in minimal to mild portal-of-ent ry effects in the larynx at all exposure levels consisting of squamous cell metaplasia and epithelial hyperplasia. These were not significant toxicologically. Among observations in the te stes (seminiferous tubular atrophy, tubular degenerative germ cells/vacuolation and in th e epididymides, degenerative spermatogenic cells in the duct at 2 and 6 μ g/L), the tubular atrophy observed at 6 μ g/L could not be ruled out as a substance-related effect. Its inciden ce was above the historical background range and similar effects at a lower incidence were observed at 2 μ g/L. Erythrocyte cholinesterase activity was decreased relative to controls at all exposure concentr ations by 9% to 41% (statistically significant only in males at the highest concentration). Brain cholinesterase activity was statistically decreased by 13% to 37% at the two highest exposure concentrations. Brain cholines terase activity inhibition at 10% or above is generally considered toxicologically relevant.

B. BODY WEIGHT AND BODY WEIGHT GAIN : There were no treatment-related effects on body weight or body weight gain in any exposure group (Table 4).4-Week Inhalation Toxicity Study (rat) (2012) / Page 9 of 11 ETHOPROP TECHNICAL/041101 OCSPP 870.3465

Data obtained from pages 37-38 (MRID 48779601)



### C. FOOD CONSUMPTION : There were no treatment-relate d effects on food consumption.

D. OPHTHALMOSCOPIC RESULTS : There were no treatment-related findings during the ophthalmoscopic examinations.

E. BLOOD ANALYSES :
1. Hematology : There were no treatment-related effects on hematology parameters.

2. Clinical chemistry : There were no treatment-related effects on clinical chemistry parameters. TABLE 4.4-Week Inhalation Toxicity Study (rat) (2012) / Page 10 of 11 ETHOPROP TECHNICAL/041101 OCSPP 870.3465



### 3. Cholinesterase activity : Erythrocyte cholinesterase activity was decreased relative to controls at all exposure concentr ations by 9% to 41% (statistic ally significant only in males at the highest concentration). Brain cholineste rase activity was statis tically decreased by 13% to 37% at the two highest expos ure concentrations (Table 5).

Data obtained from page 30 (MRID 48779601) ** p ≤ 0.01



### E. SACRIFICE AND PATHOLOGY :
1. Gross pathology : There were no test substance-rela ted macroscopic changes observed in any tissues evaluated on the da y of scheduled sacrifice.

2. Organ weight : There were no test substance-related effects observed in any of the organ weight data.

3. Microscopic pathology : Microscopic alterations associat ed with 4-weeks of exposure included laryngeal ventrolateral squamous metapl asia in both males and females at all three ethoprop exposure concentrations (Table 6). La ryngeal arytenoid epit helial hyperplasia was also observed, but to a lesser ex tent than the squamous metapl asia. The squamous metaplasia and epithelial hyperplasia in the larynx were considered adaptive responses and not toxicologically significant. In the testes, seminiferous tubular atrophy and tubular degenerative germ cells/vacuolation and in th e epididymides, degenerative spermatogenic cells in the duct were seen in males exposed to 2.05 and 6.36 μ g/L at a higher incidence than controls. The findings were w ithin historical b ackground ranges except the tubular atrophy observed at 6.36 μ g/L. T ABLE 5.4-Week Inhalation Toxicity Study (rat) (2012) / Page 11 of 11 ETHOPROP TECHNICAL/041101 OCSPP 870.3465



### III. DISCUSSION AND CONCLUSIONS:

A. INVESTIGATORS’ CONCLUSIONS : The nose-only inhalation exposure of rats to ethoprop for 4 weeks caused reductions in erythr ocyte and brain cholinesterase activities in both sexes at the two highest exposure levels (2.05 and 6.36 μ g/L). There was a local irritant response of the larynx at all e xposure levels. The no-observed- adverse-effect level (NOAEL) was considered to be 0.498 μ g/L.

B. REVIEWER COMMENTS :
The reviewer generally agrees with the investig ators’ conclusions. Inhalation exposure of rats to ethoprop at concentr ations of 0, 0.5, 2, or 6 μ g/L for 6 hours/day, 5 days/week for 4 weeks resulted in port-of-entry eff ects in the larynx consisting of squamous cell metaplasia and epithelial hyperplasia in the mid and high exposure groups. Among observations in the testes, the tubular atrophy observed at 6 μ g/L could not be ruled out as a substance-related effect, since its incidence was above th e historical background range a nd similar effects at a lower incidence were observed at 2 μ g/L. Erythrocyte cholinesterase activity was decreased relative to controls at all exposure concentrations by 9% to 41% (statistica lly significant only in males at the highest concentration). Brain cholinesterase activity was statistically decreased by 13% to 37% at the two highest exposure c oncentrations. Brain cholinesterase activity inhibition at 10% or above is consid ered toxicologically significant.

Based on the effects seen in this study, th e NOAEL in male and female Sprague-Dawley rats was 0.5 μ g/L. The LOAEL in male and female rats was 2 μ g/L based on brain cholinesterase activity inhibition.

This inhalation toxicity study is classified as Acceptable (Guideline) and satisfies the guideline requirement for 4-week inhala tion toxicity study in rats (870.3465).

C. STUDY DEFICIENCIES : A minor deficiency was the time to ethoprop equilibrium in the exposure chamber was not reported. TABLE 6.