### UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION



### MEMORANDUM

DATE: September 12

, 2014

SUBJECT: Ethoxyquin. Human Health Assessment Scoping Document to Support Registration Review.

PC Code: 055501
Decision No.: 491210 DP Barcode : 420308 Registration No.: N / A Petition No.: Not Applicab l e (N / A)
Assessment Type: N/ A Regulatory Action: Registration Review Reregistration Case No.: 0003
TXRNo.: N / A CAS No.: 91-53-2
MRIDNo.: None 40 CFR: §180.178

FROM:

THROUGH:

TO: Bonnie Cropp-Kohlligian, Risk

# Assessor-.::t:S~**~** •

# ~\~ Abdallah Khasawinah, Ph.D.,

## Toxicologi**t** /

## ~ Susan V. 

### Hummel

, Senior Chemist A.-or- 

# V**~** Kelly O'Rourke, ORE Assessor

. . . 

## 1
Risk Assessment Branch (RAB) IV '7), ()

### 2~ Health Effects Division (HED) (7509P)

.**---;;? ,..__~** 



### Elissa Reaves, Ph.D.

, Branch Chief

# ~ - RAB IV, HED (7509P)

Khue Nguyen/Thomas Moriarty RM 51
Risk Management and Implementation Branch 1
Pesticide Re-Evaluation Division (7508P)

Executive Summary

The Health Effects Division (HED) has evaluated the human health risk assessments for ethoxyquin and its toxicity

, exposure and usage databases

, as well as the most current Agency science and assessment policies to determine the scope of work necessary to support the Ethoxyquin Registration Review. The primary sources of information for this
evaluation were the 2004 Ethoxyquin Revised Human Health Risk Assessment for the Reregistration E l igibility Decision (D31 0459; hereafter referred to as the 2004 RED)

, the subsequent 2008 occupational and residential exposure (ORE) risk assessment conducted

to evaluate a thermal fogging use on pears (D342695), and updated occupational

Page 1 

### of28Ethoxyquin Registration Review Human Health Assessment Scoping Document



### expos ur e estimates reflecting current methodology generated fo r the HED Hazard and
Sc i ence Policy Council (HASPOC) evaluation (TXR# 0053945).

E thoxyquin ( 1 ,2-dihydro-6 -e t hox y

- 2,2,4-trimethylquinolene) i s an antioxidant currently

r egis ter e d for u se as a deterrent of scald in pears through po st -harv est (indoor) application v ia a drench/in

- line spray treatment, thermal fogging and/or impregnated

wraps. Ethoxyquin is also used for preservation of co l o r in the p r oduction of chili powder

, paprika

, and ground chili and as a preservative i n animal feed with food additive

t olerances established b y the Food and Drug Adm ini stra tio n (FDA) in/on the specified spices, as well as , meat

, poultry and eggs (2 1 CFR 

### § 172.140).

Based on the currently registered pesticidal uses, exposures to ethoxyquin can be expected to occur via the dietary (food only) and occupa tional (dermal and inhalation)

routes

. With adequate label restrictions regarding wash-water r ecycling practices, it is

not likel y to occur in drinking water and there are no registered resident i a l uses of ethoxyquin.

The residue chemistry and occupationa l exposure databases for ethoxyqu i n are adequate for Registration Review

. The to x i cology database fo r ethoxyquin is adequate with the

exce p tion of dermal penetration and inhalation toxicity studies. The HED HASPOC (TXR# 0053945) determined that dermal penetration and 28 -d ay inhalation stud i es are required due to updated occupational exposure est im ates refl ecting current methodology
that identified risks of concern u sing oral point of departures for assessing risk by the
dermal and inhalation routes. As part of Registration Review

, toxicological endpo i n t s, points of departure and uncertainty / safety facto r s used in the most recent human health
risk assessments may be re-evaluated according to current HED polic y. Updated

exposure and risk assessments may need to be conducted using current po li cy and procedures i f there are any s igni ficant changes to ethoxy quin endpoints

, points of departure

, uncertainty / safety factors

, and/or exposure estimates / assumptions. Th e current tol erance expression will need to be updated to reflect the HED Interim Gu i dance on To leranc e Ex pressions (5/27/09, S

. Knizner).

T he primary target organs for ethoxyquin in experimental animals were the liver and kidney. Dogs we r e the most sensitive species tested. The major effects in liver, as seen in the 90-da y dog study

, were elevated enzymes and microscopic fmdings (cytop lasmic

vac uolation and minimal hepatocellular n ec ro s i s). The major effects in kidney

, as seen i n the 90 -d ay rat study, were m ic roscopic l esions ( renal papillary necro s i s, hya lin droplets in
the cort i cal tubular epithe li al cells and nephropathy). There is no evidence of increased suscept ib i lit y following pre-natal exposure to rats. The available data does not indicate a neurotoxic concern for ethoxy quin. Ethoxyquin has been the subject of numerous studies
because of its wide use as an antioxidant and its role as a microsomal oxidase system inducer. Pub l ished s tudies in rats and dogs indicate that reproductive /offs pring toxicity

occurs at higher doses than maternal toxicity doses. In mutagenicity testing

, ethoxyquin is negative in 

### Salmonella

, negative in the micronucleus assay , negati ve for chromosomal
aberrations and positive for 

### in vitro sister chromati d exchanges in Chinese hamster ovary cells. There are no guideline studies assessing ethoxyquin carcinogenicity in animals. However, i t is structurally similar to Flectol H which showed evidence of carcinogenicity (kidney tumors) in male rats

. Ethoxyquin is also a nephrotoxin in male rats. A

Page 2 of28Ethoxyquin Registration Review Human Health Assessment Scoping Document



### Page 3 of 28 theoretical upper bound oral slope factor (Q 1 * 

### ) for ethoxyquin was calculated to be 0.04 (mg/kg/day)

- 1 

### , based on a model that correlates cancer slope factor with maximum tolerated dose.

Acute, chronic and cancer dietary risk assessments for food only were previously conducted for ethoxyquin and included exposures from its uses on pears as well as from its uses as a preservative in foods and feeds. No dietary risks of concern were identif ied. At the 95 th 

### percentile, 14% of the acute Population Adjusted Dose (aPAD) was used for females 13

- 49 (the only population subgroup identified as relevant for the acute dietary endpoint) and 14% or less of the chronic Population Adjusted Dose (cPAD) wa s used for the general U.S. population and all other population subgroups. The cancer dietary risk for ethoxyquin was estimated at 2 x 10

- 6 

### and assumed additional refinements to the exposure; the most notable being an adjustment factor for the fraction of fresh pears that were not of the Bartlett variety since ethoxyquin use on pears was limited to the Bosc and Anjou varieties. Updated dietary exposure and risk assessments may be necessary if exposure assumptions used in the previous assessments change an d/or if there are any significant changes to ethoxyquin endpoints, points of departure and uncertainty/safety factors.

Although no occupational risks of concern were identified in previous risk assessments, updated occupational exposure and risk assessmen ts are likely to be necessary given the required dermal penetration and inhalation toxicity studies and current occupational exposure assessment evaluation methodology for post

- harvest treatments.

While there are currently no registered residential uses o f ethoxyquin, residential bystander exposures resulting from off

- site transport (i.e., volatilization) may occur as a result of occupational applications. The need for a bystander risk assessment for ethoxyquin will be considered during Registration Revie w.

Introduction

Ethoxyquin ( 1,2

- dihydro

- 6

- ethoxy

- 2,2,4

- trimethylquinolene) is an antioxidant currently registered for use as a deterrent of scald in pears through post

- harvest (indoor) application via a drench/in

- line spray treatment, thermal fogging and /or impregnated wraps. Currently eight products are registered; two emulsifiable concentrates, one soluble concentrate and five impregnated materials. There are four ethoxyquin registrants: Wrap Pack Inc., Pace International, Seaman Paper Company and Bu rrows Paper Corporation. The maximum application rates vary by treatment method; 2,700 parts per million (ppm) for drench/in

- line spray treatment (i.e., 0.022 pounds active ingredient [ai] per gallon solution), 1.07 x 10

- 5 

### pounds ai/pound of fruit for thermal fogging, and 1,000 ppm per piece for impregnated paper wraps.

The primary sources of information for this evaluation were the 2004 RED ( D310459 ), the subsequent 2008 ORE risk assessment conducted to evaluate a thermal fogging use on pears ( D342695 ), and updated occupational exposure estimates reflecting current methodology generated for the HED HASPOC (TXR# 0053945) evaluation. The nomenclature and physiochemical properties of ethoxyquin are presented in Attachment 1

.Ethoxyquin Registration Review Human Health Assessment Scoping Document



### Page 4 of 28 Hazard Identification/Toxicology

The toxicology database for e thoxyquin is adequate to support existing registrations with the exception of dermal penetration and inhalation studies. The HED HASPOC (TXR# 0053945) granted waivers for the requirements fo r immunotoxicity, neurotoxicity battery and developmental neurotoxicity studies but determined that dermal penetration and 28

- day inhalation studies are required for Registration Review . The HED HASPOC decision to require additional dermal penetration and inhalation studies was based on updated occupational exposure estimates reflecting current evaluation methodology which identified risks of concern using oral points of departure (PODs) for assessing risk via dermal and inhalation routes of exposure.

T he published toxicology database on ethoxyquin is very extensive; however, guideline toxicology studies are very limited. Ethoxyquin has been the subject of numerous studies because of its wide use as an antioxidant and its role as a microsomal oxidase sy stem inducer. It has been extensively investigated along with other antioxidants for the modification of the carcinogenic properties of known carcinogens. It was nominated by the FDA in 1990 for National Toxicology Program (NTP) testing due to “uncertain ty concerning its toxicological effects stemming from reports of purported toxicity in dogs, the need for adequate toxicity data to re

- evaluate the currently approved levels in animal feed and human feed and being used as an antioxidant in animal feed”. T he current status of the NTP testing can be found at: http://ntp.niehs.nih.gov/testing/status/agents/ts

m88005.html

.
Ethoxyquin has low to moderate acute toxicity by the oral (C ategory III), dermal (Category III) and inhalation (Category III) exposure routes. It is not an eye irritant (Category IV), and produces minimal irritation to the skin (Category IV). Tests in animals show it to have a weak sensitizing potential; however, extensive human experience from the use of this chemical showed strong association with contact dermatitis that ceased upon discontinuation of working in an ethoxyquin environment.

The primary target organs for ethoxyquin in experimental animals are the liver and kidney. Dogs are more susceptible to ethoxyquin toxicity than rats with effects occurring at doses as low as 4 mg/kg/day over a 90 day feeding period. The major effects in liver, as seen in the 90

- day dog study, were elevated enzymes and micros copic findings (cytoplasmic vacuolation and minimal hepatocellular necrosis). The major effects in kidney, as seen in the 90

- day rat study, were microscopic lesions (renal papillary necrosis, hyalin droplets in the cortical tubular epithelial cells and ne phropathy). Ethoxyquin did not cause developmental effects in rats tested at doses of 350 mg/kg/day during the gestation period or in rats at doses as high as 500 mg/kg/day of a 67% ethoxyquin formulation. Published studies in rats and dogs indicate that reproductive/offspring toxicity occurs at higher doses than maternal toxicity doses for ethoxyquin. Published studies do not indicate a neurotoxic concern for ethoxyquin.

There is low concern (and no residual uncertainty) for pre

- and/or postnatal toxic ity resulting from exposure to ethoxyquin. The available data provided no indication of increased susceptibility (quantitative or qualitative) to rats or rabbits to in utero exposure.Ethoxyquin Registration Review Human Health Assessment Scoping Document



### Page 5 of 28 Therefore, the special FQPA safety factor was reduced to 1X in risk as sessments for this chemical.

The only suggestion of a potential carcinogenic effect for ethoxyquin came from Manson et al ( 1987, Ethoxyquin alone induces preneoplastic changes in rat kidney while preventing induction of such lesions in liver by aflatoxin b

- 1. Carcinogenesis (London); 8 (5):723

- 728 ) study where feeding of male Fisher 344 rats of ethoxyquin at 0.5% in the diet (5000 ppm, equivalent to 250 mg/kg/day) for 23 weeks caused severe damage to the kidneys and produced many hyperplastic and putative preneoplastic tubules. However, guideline studies in rats and mice have not been submitted. In mutagenicity testing, ethoxyquin is negative in Salmonella , negative in the micronucleus assay, negative for chromosomal aberrations and positive for in vitro sister chromatid exchanges in Chinese hamster ovary cells. Ethoxyquin is structurally similar to Flectol H (1,2

- dihydro

- 2,2,4

- trimethylquinoline) which showed evidence of carcinogenicity (kidney tumors) in male

rats. Flectol carcinogenicity studies were conducted by the dermal route in F344/N rats and B6C3F 1 

### mice in the National Toxicology Program (NTP 1997. Toxicology and Carcinogenesis Studies of 1,2

- Dihydro

- 2,2,4

- trimethylquinoline (CAS No. 147

- 47

- 7) in F344/N Rats and B6C3F 1 

### Mice and the Initiation/Promotion (Dermal Study) in Female Sencar Mice. NTP Technical Report Series No. 456). Ethoxyquin is also a nephrotoxin in male rats. Hence, based on Flectol H data, a theoretical upper bound oral slope factor (Q 1 * 

### ) for ethoxyqu in was calculated to be 0.04 (mg/kg/day)

- 1 

### for cancer risk (2 004 RED (D310459)).

The ethoxyquin risk assessment for the 2004 RED (D310459) relied on the published literature and the few available guideline studies, the limited use patterns and potential exposures in selecting the relevant toxicity endpoints for risk assessment. Subsequently an inhalation point of departure was selected for the 2008 ORE risk assessment conducted to evaluate the thermal fogging use on pears (D342695). Toxicity profile and relevant toxicological endpoints are provided in Attachments 2 and 3, respectively.

Conclusions. The toxicology database for e thoxyquin is adequate to support existing registrations with the exception of dermal penetration and inhalation studies. The H ED HASPOC (TXR# 0053945) determined that dermal penetration and 28

- day inhalation studies are required for Registration Review due to updated occupational exposure estimates reflecting current evaluation methodology that identified risks of concern using

o ral points of departure for assessing risk via the dermal and inhalation routes of

exposure. Until the inhalation study is submitted and found acceptable (or alternative information is provided to reduce the uncertainty), a 10X database uncertainty factor will be applied for the inhalation route. Once these studies have been received and reviewed, the toxicological endpoint selections and uncertainty/safety factors may be re

- evaluated.

Dietary Exposure

The qualitative nature of the residue of ethoxyqui n for post

- harvest uses on pears is adequately understood. The HED Metabolism Committee (meeting held 12/20/1994) determined that ethoxyquin is the residue of concern in/on pears for tolerance enforcement and risk assessment. There is no reasonable expec tation of finite residues of ethoxyquin in livestock commodities from the registered pesticidal uses on pears.Ethoxyquin Registration Review Human Health Assessment Scoping Document



### Page 6 of 28 Adequate analytical enforcement methods are available . No additional residue chemistry data are required.

Acute, chronic and cancer dietary ri sk assessments for food only were conducted for ethoxyquin to support the 2004 RED (D310459) using Lifeline Model Version 2.0 and Dietary Exposure Evaluation Model (DEEM

- FCID  , Version 2.03) which uses food consumption data from the USDA’s Continuing Surve ys of Food Intakes by Individuals (CSFII) from 1994

- 1996 and 1998. These assessments included food from all sources of potential exposure to ethoxyquin including its uses as a preservative in food and feeds. The conclusions of the assessments were based on Lifeline analyses.

The acute and chronic (non

- cancer) dietary exposure and risk assessments (D297740) assumed tolerance level residues and 100 % crop/feed treated. Ethoxyquin

- treated pears were assumed to be for fresh market only. Available meat/po ultry cooking study data were used to reduce estimated residues in cooked livestock tissues by 96%. At the 95 th 

### percentile, 14% of the acute Population Adjusted Dose (aPAD) was used for females 13

- 49 (the only population subgroup identified as relevant fo r the acute dietary endpoint) and 14% or less of the chronic Population Adjusted Dose (cPAD) was used for the general U.S. population and all other population subgroups.

The cancer dietary exposure and risk assessment (2004 RED; D310459) incorporated the following additional refinements: (1) assumed the highest average field trial value for fresh pears instead of the tolerance, (2) used an adjustment factor for the fraction of fresh pears that were not of the Bartlett variety (45% according to USDA Agric ultural Statistics for 2003) since ethoxyquin use on pears was limited to the Bosc and Anjou varieties, and (3) assumed that only dried spices would have incurred residues of ethoxyquin since ethoxyquin is not applied to fresh herbs and spices. The cancer dietary risk for ethoxyquin was estimated at 2 x 10

- 6



### .

Conclusions. The residue chemistry database for ethoxyquin is adequate for Registration Review. An updated dietary exposure and risk assessment may be conducted if there are significant changes to ethoxyquin endpoints, points of departure, uncertainty/safety factors, and/or exposure estimates/assumptions. The assessment, if conducted, will use the newest version of DEEM and reflect updated consumption information.

Residential Exposure and Risk Ass essment

Residential bystander exposures resulting from off

- site transport (e.g., spray drift or volatilization) may occur as a result of applications of ethoxyquin. The potential for spray drift will be quantitatively evaluated for each pesticide during the Registration Review process which ensures that all uses for that pesticide will be considered concurrently. The approach is outlined in the revised (2012) Standard Operating Procedures for Residential Risk Assessment (SOPs)

– Residential Exposure Asse ssment Standard Operating Procedures Addenda 1: Consideration of Spray Drift. This document outlines the quantification of indirect non

- occupational exposure to drift. In terms of volatilization, the Agency has developed a Volatilization Screening Tool and a subsequent Volatilization Screening Analysis ( http://www.regulations.gov/#!docketDetail;D=EPA

- HQ

- OPP

- 2014

- 0219 ).Ethoxyquin Registration Review Human Health Assessment Scoping Document



### Page 7 of 28 During Registration Review, the Agency will utili ze this analysis to determine if data (i.e., flux studies, route

- specific inhalation toxicological studies) or further analysis is required for ethoxyquin. While spray drift is not expected due to the nature of the application methods, volatilization pote ntial may be addressed with more information regarding filtering practices associated with the ventilation of thermal fogging applications.

Conclusions. There are no currently registered residential uses for ethoxyquin; therefore, a quantitative resident ial exposure and risk assessment is not necessary during Registration Review. The need for spray drift and volatilization risk assessment for ethoxyquin will be examined during Registration Review

.

Aggregate Exposure and Risk Assessment

In accordance with the Food Quality Protection Act (FQPA), HED must consider and aggregate pesticide exposures and risks from three major sources: food, drinking water, and residential exposures. The aggregate risk assessment evaluates the combined risk from dietary ex posure (food and water) and any non

- occupational (residential) uses. With adequate label restrictions regarding wash

- water recycling practices, ethoxyquin is not likely to occur in drinking water and it is not currently registered for residential uses.
T herefore, an aggregate risk assessment from these exposures to ethoxyquin is not expected under Registration Review.

Occupational Exposure

Occupational handler and post

- application exposures are expected based on the registered agricultural uses of etho xyquin. The most recent occupational and residential exposure (ORE) risk assessment was conducted in 2008 to evaluate the thermal fogging use on pears (D342695); prior to that, the ORE risk assessment for drench/in

- line spray treatments and impregnated wr aps was conducted for the 2004 RED (D310459). The level of concern (LOC) is for dermal margins of exposure (MOEs) less than 100. Until the required inhalation study is submitted and found acceptable (or alternative information is provided to reduce the u ncertainty), a 10X database uncertainty factor will be applied for the inhalation route (TXR# 0053945); hence, in the absence of inhalation data, the LOC would be for inhalation MOEs less than 1000.

Occupational Handlers There is the potential for dermal and inhalation exposure to occupational handlers from the currently registered use pattern of ethoxyquin. Because no chemical

- specific data are available, occupational handler exposure is based on surrogate unit exposures from the Pesticide Handlers Expos ure Database (PHED). Previous risk calculations were based on the maximum application rates and assumed maximum amount treated per day. Handler exposure and risk were calculated at baseline (long pants, a long

- sleeved shirt, no chemical

- resistant gloves, and no respirator) and with personal protective equipment (PPE) when necessary (e.g., chemical

- resistant gloves, respirators, and engineering controls).Ethoxyquin Registration Review Human Health Assessment Scoping Document



### Page 8 of 28 The previous exposure and risk calculations were updated for the HED HASPOC (TXR# 0053945) evaluati on to reflect current methodology. Short

- and intermediate

- term exposure durations resulted in MOEs for dermal exposure with chemical

- resistant gloves ranging from 5.6 (direct spraying with hand

- held equipment) to 590 (mixing/loading for thermal fogging). Inhalation MOEs without a respirator ranged from 1,900 (direct spraying with hand

- held equipment) to 100,000 (mixing/loading for thermal fogging). Cancer estimates range from 5.6 x 10

- 6 

### (mixing/loading for thermal fogging) to 5.9 x 10

- 4 

### (direct spraying with hand

- held equipment).

Occupational Post

- Application There are potentially significant short and intermediate

- term post

- application exposures to workers who sort, cull and pack treated fruit. Because no chemical

- specific data are available, post

- app lication exposure calculations were updated for the HED HASPOC (TXR# 0053945) based on current evaluation methodology and standard assumptions for assessing post

- harvest commodities. The current assessment methodology for this use pattern does not utilize dislodgeable foliar residue (DFR) data; therefore, the requirement for DFR data is not applicable. The dermal MOE with chemical

- resistant gloves was 56, and the inhalation MOE without a respirator was 88. The post

- application cancer estimate was 9.6 x 1 0

- 5



### .

Conclusions. The occupational exposure database is adequate to support the Registration Review process for ethoxyquin. Updated occupational handler and post

- application exposure assessments conducted for the HED HASPOC (TXR# 0053945) evaluation using current methodolo gy did identify risks of concern. Registration Review is likely to include updated occupational exposure and risk assessments which will be based on current policies and the required toxicity studies (i.e., dermal penetration and inhalation).

Public He alth and Pesticide Epidemiology Data

Both the OPP Incident Data System (IDS) and the Centers for Disease Control and Prevention/National Institute for Occupational Safety and Health (CDC/NIOSH) Sentinel Event Notification System for Occupational Risk

- Pes ticides (SENSOR) databases were consulted for pesticide incident data on the active ingredient ethoxyquin (D420779). Ethoxyquin is not included in the Agricultural Health Study (AHS). The purpose of the database sea rch was to identify potential patterns in the frequency and severity of the health effects attributed to ethoxyquin exposure.

Conclusions. The Agency has reviewed the available incident data and concludes that

t here does not appear to be a concern that would warrant further investigation, s ince t here

were no incidents involving ethoxyquin reported to either IDS or SENSOR

- Pesticides. The Agency will continue to monitor the incident information and if a concern is triggered, additional analysis will be included in the risk assessment.

Tolera nce Assessment and International Harmonization

A tolerance is established for residues of ethoxyquin from post

- harvest use in/on pears at 3 ppm under 40 CFR §180.178. There is also an exemption from the requirement of a tolerance for ethoxyquin ( 1,2

- dihy dro

- 6

- ethoxy

- 2,2,4

- trimethylquinolene) as an inert (PCEthoxyquin Registration Review Human Health Assessment Scoping Document



### Page 9 of 28 Code 855501) when used as an antioxidant in insecticides under 40 CFR §180 .910. FDA has established additional food additive tolerances under 21 CFR §172.140(a) at 100 ppm in/on chili powder, paprika and ground chili as an antioxidant for preservation of color in their production. FDA has also established food additive tolerances in livestock commodities under 21 CFR §172.140(b) at 5 ppm in/on the uncooked fat of meat from animals except poultry, 3 ppm in/on the uncooked liver and fat of poultry, 0.5 ppm in/on the uncooked muscle meat of animals and in poultry eggs in order to provide for the safe use of the additive in feed prepared in accordance with 21 CFR §573.380 and 21 CFR §573.400.

The U.S. tolerance is harmonized with the established Codex and Canadian maximum residue limits (MRLs) for residues of ethoxyquin in/on pear. Mexico adopts U.S. tolerances and/or Codex MRLs for its export purposes. The U.S. tolerance expression will need to be updated to reflect the HED Interim Guidance on Tolerance Expressions (5/27/09, S. Knizner).

Cumulative Risk Assessment

Unlike other p esticides for which EPA has followed a cumulative risk approach based on a common mechanism of toxicity, EPA has not made a common mechanism of toxicity finding as to ethoxyquin and any other substances and ethoxyquin does not appear to produce a toxic met abolite produced by other substances. For information regarding EPA’s efforts to determine which chemicals have a common mechanism of toxicity and to evaluate the cumulative effects of such chemicals, see the policy statements released by EPA’s Office of P esticide Programs concerning common 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/

.

Human Studies

The previous ethoxyquin risk assessments relied in part on data from studies in which adult human subjects were intentionally exposed to a pesticide or other chemical. These data, which include studies from Pestic ide Handler Exposure Database (PHED) 1.1; the Agricultural Handler Exposure Task Force (AHETF) database; the Agricultural Reentry Exposure Task Force (ARTF) database; and, are (1) subject to ethics review pursuant to 40 CFR 26, (2) have received that revie w, and (3) are compliant with applicable ethics requirements. For certain studies, the ethics review may have included review by the
Human Studies Review Board. Descriptions of data sources, as well as guidance on their

use, can be found at the Agency we bsite 1



### .

Data Requirements

The following additional toxicology data are required to refine the occupational risk

assessment.

 GLN 870.7600 Dermal penetration

 GLN 870.3465 28

- Day inhalation toxicity study (rat)

1 http://www.epa.gov/pesticides/science/handler

- exposure

- data.html and http://www.epa.gov/pest icides/science/post

- app

- exposure

- data.htmlEthoxyquin Registration Review Human Health Assessment Scoping Document



### Page 10 of 28 References

Table 1. Memoranda Relevant to Registration Review.

Author Barcode/TXR No. Date Title

Clock, M. D342695 4/1/2008 Ethoxyquin. Occupational Risk Assessment for Proposed Uses on Stored Pears. ORE Thermal Fogging Review.

Funk, S. None 1/26/1995 The HED Metabolism Committee Meeting Held on December 20, 1994: Ethoxyquin.

Hrdy, D., et al

. D310495 7/20/2004 Ethoxyquin. Health Effects Division (HED) Revised Risk Assessment for the Reregistration Eligibility Decision.

Hrdy, D. D297740 7/2/2004 Ethoxyquin Acute, Chronic, and Cancer Dietary Aggregate Exposure Assessments for the Reregistration Eligibility Decision.

Knizner, S. None 5/27/2009 HED Interim Guidance on Tolerance Expressions

Leshin, J. 0053945 8/21/2014 Ethoxyquin: Summary of Hazard and Science Policy Council (HASPOC) Meeting of June 5, 2014: Recommendation on Multiple Toxicology Studies.

Recore, S. D420779 7/16/2014 Ethoxyquin: Tier I Review of Human Incidents



### Attachments:

Attachment 1: Ethoxyquin Identity and Physicochemical Properties Attachment 2: Ethoxyquin Toxicity Profile Tables Attachment 3: Ethoxyquin Toxicological Doses and Endpoints for Risk Assessment Attachment 4: Ethoxyquin International Residue LimitsEthoxyquin Registration Review Human Health Assessment Scoping Document



### Page 11 of 28 Attachment 1. Ethoxyquin Identity and Physicochemical Pr operties

Table A1.1. Test Compound Nomenclature

Chemical Structure

Empirical Formula C 14 H 19 NO

Common name Ethoxyquin

IUPAC name 1,2

- dihydro

- 2,2,4

- trimethylquinolin

- 6

- yl ethyl ether

CAS name 6

- ethoxy

- 1,2

- dihydro

- 2,2,4

- trimethylquinoline

CAS Registry Number 91

- 53

- 2

Chemical Class Quinoline

Table A1.2. Physicochemical Properties

Parameter Value Reference

Molecular Weight 217.34

Melting point/range Approximately 0  C Sax and Lewis, 1987

pH No information found.

Density No information found.

Water solubility (20 C) Insoluble Monsanto, 1959

Solvent solubility (temperature not specified) Miscible with animal and vegetable fat and oils Monsanto, 1959

Vapor pressure (25 C) 1.82 x 10

- 5 mm Hg @ 0 C (purified material); 2.56 x10

- 4 mm Hg @ 25  C Monsanto, 1987

Dissociation constant, pKa No information found.

Octanol/water partition coefficient, logP OW (25 C) No information found.

UV/visible absorption spectrum 1.569

- 1.572 @ 25  C Sax and Lewis, 1987Ethoxyquin Registration Review Human Health Assessment Scoping Document



### Page 12 of 28 Attachment 2. Ethoxyquin Toxicity Profile Tables

Table A2.1. Acute Toxicity Profile: Ethoxyquin

Guideline
No. Study Type MRID #(s) Results Toxicity Category

870.1100 Acute Oral

- Rats 43885901 (1995) LD 50 = 1726 mg/kg

 III

870.1200 Acute Dermal

- Rats 43885902 (1995) LD 50 = > 2000 mg/kg III

870.1300 Acute Inhalation 43894101 (1996) LC 50 = > 1.97 mg/L III

870.2400 Primary Eye Irritation 43885903 (1995) not an eye irritant IV

870.2500 Primary Skin Irritation 43885904 (1995) slight irritant IV

870.2600 Dermal Sensitization 43885905 (1995) weak sensitizer NA

Table A2.2. Ethoxyquin Subchronic, Chronic and Other Toxicity Profile

GL # MRID Study Type Results and Classification Ethoxyquin: Chronic Studies
Non

guideline Hard & Neal,

1992 Fund. Appl.
Tox. 18 : 185

- 189 Chronic (dietary) toxicity

study at 0 or 0.5% of 90% ethoxyquin (65.5/70.6 mg/kg/day for males/females) in arachis oil. Fisher 344 rats (6

- 20/sex/dose/sacrifice at 4,
12

- 14, 24, 58 weeks or 18 months. Body weight gains in treated rats: 80%/ 90% of controls for males/females, feed consumption not affected. Interstitial degeneration of the extremity of the renal papilla was seen in male rats after 4 weeks which progressed to necrosis and pyelonephritis with loss of part of the papilla and foci of mineralization in the abscission layer by week 24. Degeneration in female rats paralleled that of males at the 4 or 12 to 14 week points; neither pyelonephritis nor urothelial hyperplasia was seen. At 18 months, all treated males had complete papillary necrosis with tissue loss and severe end stage chronic progressive nephropathy. All treated females had interstitial degeneration of the papillary tip, but the lesion had not progressed beyond the earlier stage. No evidence of recovery of the lesions was seen. Ethoxyquin is a potent inducer of renal papillary necrosis in male rats and that female rats are far less susceptible to it s effects. A proposed LOAEL is <65 mg/kg/day and a NOAEL would be much less.
Non

guideline Neal et al

,

2003 Food Chem.
Tox. 41 : 193

- 200 Chronic (diet) tox study

Fisher 344 rats ethoxyquin 90% in the diet at 0, 0.01, 0.05, 0.01, 0.25 or 0.5% (5, 25, 50, 125, or 250 mg/kg/day) for 3 or 6 months (5 males /group for 3 months feeding; 8/sex/group in the 6 month feeding except in the 0.05, 0.1, and 0.25% 5 females/group were used). Proposed LOAEL = 5000 ppm (250 mg/kg/day) based on a sharp threshold in male rats with either interstitial degeneration of the papilla or frank papillary necrosis being evident after 3 and 6 months but not at 2500 pp m or lower dose. In the female rats, 6 months of feeding produced early minimal interstitial degeneration in one individual only at the 0.5% exposure level. When C

- 14 ethoxyquin was administered ip or orally by gavage (10 mg/kg), the radiolabel was assoc iated with urinary albumin. The ratio of the excreted radioactivity in the urine to feces was 7.3

- 8.2. Autoradiographic sections of the kidneys did not show differences among the sexes inEthoxyquin Registration Review Human Health Assessment Scoping Document



### Page 13 of 28 Table A2.2. Ethoxyquin Subchronic, Chronic and Other Toxicity Profile

GL # MRID Study Type Results and Classification the distribution of the radioactivity or retention of ethoxyquin. Fecal and urinary metabolites’ profiles were similar in the two sexes. No unmetabolised ethoxyquin was detected in any sample. Treatment of the urine and fecal extracts with β

- glucuronidase failed to produce any detectable level of non

- polar labeled com

pounds.
Non

guideline Hernández et

al , 1993 Food Chem.
Tox. 31 : 363

- 367 Renal cortical slices

prepared from Wistar male rats that had consumed a diet containing ethoxyquin 94% (0.01, 0.05, 0.1, 0.2, 0.5, 1.0 or 2.0 mM equivalent to 0.3, 1.5, 3, 6, 15, 30, 60 mg/rat/day), from weanling to adult age Ethoxyquin inhibited the specific uptake of C 14

- labeled p

- aminohippurate ([14C]PAH) and tetraethylammonium ([14C]TEA), markers of organic anion and cation tubular secretion, respectively, in a concentration d ependent manner. The specific uptake of [14C]TEA was five

- fold more sensitive to ethoxyquin than [14C]PAH uptake (IC 50 = 0.33 and 1.51 mM, respectively). Ethoxyquin (1 mM) decreased Na+/K(+)

- ATPase activity from 1.58 to 1.0 mumol inorganic phosphate/mg protein/min in renal microsomes. The activity of this enzyme provides the energy for the function of both secretory systems.
Non

guideline Imazawa et

al , 1989 Bull Natl Hyg Sci 107 : 68

- 72 Male Wistar rats were

treated with 100 ppm
N

- methyl

- N'

- nitro

- N

- ni tro soguanidine (MNNG) in the drinking water for 8 weeks, followed by a dietary supplement of 0.1% ethoxyquin for 32 weeks. An additional group received the ethoxyquin treatment alone. Calcification and stones were observed from the renal papilla to the pe lvis region in all ethoxyquin treated groups. The incidence and grade of the lesion in the combined treated groups were much greater than in the group treated with ethoxyquin alone. Ultrastructurally, microvilli on the surface of the renal papilla cells a ppeared degenerated or disappeared completely in the treated groups. Chemical element analysis of the renal stones revealed P and Ca to be the primary constituents.

Non

guideline Rudra et al.,

1974
MRID 5000419 Chronic dietary, Wistar

male rats (5/group) for 500 days; 0.0, 0.5% ETX in arachis oil (estimated 250 mg/kg/day) The ETX rats ate less than controls and experienced a decreased growth rate. After 280 days, 4 animals died from severe kidney failure.

Non

guideline Hazelton

Labs, 1955; Monsanto, 1985; NTP, 1990 Chronic, capsule, dog, 0

(I), 10 (II), 50 (III) or 100 (IV) mg ETX/kg (2

- 3 dogs mixed sex/group; dosed 5 days/week except group III daily for 1 st 6 weeks then twice/day afterwards. Group IV discontinued after 6 weeks due to toxic ity, sacrificed at week 9, replaced with two dogs at 3 mg/kg (V) Abdominal tenderness, anorexia, depression, soft feces, dark brown livers, discolored urine, irritation of the large and small intestine, enlarged liver seen in some or all dogs at some or at all treatment levels. Kidney swelling with fatty accumulation was seen in groups I, III, and V. Granulation of cells in collecting tubules was also seen in groups 2 and 3. Liver degeneration and fatty metamorphosis of the liver was seen in groups 2, 3, a nd 5. Groups 3 and 4 exhibited marked retention of exogenous pigment in the liver. One dog in group 3 had massive fibrosis. These histological changes were thought to be temporary and may be reversible. The NOAEL in this study for microscopic pathological changes was 28 mg/kg/day at day 84.
Non

guideline Western URB

1955; Monsanto, 1985; Chronic, dietary in alfalfa,

rats. Trial I . (10 rats/sex/group) at 0, 0.2%, In trial I males and females weighed significantly less (p<0.05

- 0.01) than controls. In trial II, a small indication of kidney lesions was seen in the 0.1 and 0.2% ETX groups. Liver weights among females inEthoxyquin Registration Review Human Health Assessment Scoping Document



### Page 14 of 28 Table A2.2. Ethoxyquin Subchronic, Chronic and Other Toxicity Profile

GL # MRID Study Type Results and Classification

(NTP, 1990) 0.4%, ETX (0, 100 or 200

mg/kg/day) for 155 days. Trial II. (10 males/group)

at 0, 0.0062%, 0.0125%, 0.05%, 0.1%, 0.2% (3, 6, 25, 50, or 100 mg/kg/day) and (10 females/group) at 0, 0.05%, 0.1%, 0.2% ETX for 225 days. the 0.2% ETX group and males in the 0.1% group as well as liver weights in all higher dose groups were significantly greater (p<0.01) than controls. Kidneys weights were significantly greater (p<0.01) in the 0.2% (trials 1 and 2) and 0.4% (trial 1) dose groups versus controls. Kidneys of 2 male rats in the 0.4% ETX group, had stones in the renal pelvis. Microscopic examination revealed well

- developed chronic pyelone phritis among males in the high dose groups. Two kidneys had areas of calcification. The thyroid glands of male rats exposed to higher doses of ETX had evidence of hyperplasia. All male rats in the 0.2% ETX group, 3/4 in the 0.1% group, and 2/5 in the 0 .05% group had small kidney scars. Proposed LOAEL is 25 mg/kg/day based on kidney lesions, proposed NOAEL is 6 mg/kg/day.
Non

guideline Davies et al

,

1993 Carcinogenes is 14(1):47

- 52 Chronic, albino rats. ETX

in the diet (0.5%) fed to 21 days old or 6 weeks old until 9 months of age. A marked body weight gain reduction (15

- 20%) reported. The conversion of 2N nuclei to 4N and 8N nuclei as measured by flow cytometry was slowed down. Ethoxyquin is a very potent inducer of gamma

glutamyltranspeptidase (GGT), but expression depended on the age of the animals, the length of treatment time and the ploidy status of the liver.
Non

guideline Manson et al

,

1992 Arch Toxicol 66(1):51

- 56 Chronic (dietary) toxicity

study at 0 or 0.5% ETX (250 mg/kg/day) in weanling or adult rats at the beginning of the experiment for 26 weeks Female rats were much less susceptible to the toxic effects of ethoxyquin than males of the same age. In males damage to the cortex, mainly as an acceleration of the normal ageing proc ess, was similar in both age groups, but rats exposed to ethoxyquin as weanlings also suffered from extensive papillary necrosis. Male rats were more prone than females to proteinuria. Ethoxyquin: Sub

- chronic Studies 82

- 1 870.3100 44123801 &

44222501 28

- Day oral gavage

– Rat

Acceptable/Non

Guideline 5/sex at 0, 50, 250, 500, 1000 mg/kg/day in corn oil (5 mL/kg) LOAEL = 250 mg/kg/day, based on decreased hematology parameters (RBC count, hemoglobin, hematocrit), increased serum chemistry values (globulin , total protein, Ca, P, K, cholesterol), increased liver weights, and histopathological findings in the kidneys. At the 1000 mg/kg/day all animals died within 2

- 3 days. NOAEL = 50 mg/kg/day.
870.3100 44123901 90

- Day oral gavage

– Rat Acceptable/Guideline 10/sex at 0, 20, 40, 200 or 400 mg/kg/day in corn oil (5 mL/kg) LOAEL = 40 mg/kg/day based on decreased mean body weight gain (

- 14%) in males. At 200 and 400 mg/kg/day these effects were noted: decreased RBC, hematocrit and hemoglobin , increased mean reticulocyte counts, decreased mean WBC counts, increased serum albumin, and globulin, total protein, total bilirubin, GGT, cholesterol, decreased Serum T4 level and increased TSH level. Reddened and/or enlarged thyroid glands, increased liver and kidney weights. Microscopic lesions on kidneys: renal papillary necrosis, hyalin droplets in the cortical tubular epithelial cells and nephropathy. NOAEL = 20 mg/kg/day.Ethoxyquin Registration Review Human Health Assessment Scoping Document



### Page 15 of 28 Table A2.2. Ethoxyquin Subchronic, Chronic and Other Toxicity Profile

GL # MRID Study Type Results and Classification
870.3150 44149001 28

- Day oral, capsule

– Dog Acceptable/Non

Guideline 2/sex at 0, 25, 50, 100 or 200 mg/kg/day LOAEL(estimated) = 25 mg/kg/day based on decreased body weight gain (

- 17%) in females , elevated AP, ALT, AST and GGT enzyme levels and necropsy findings (dark liver, dilated vessel in the heart, GI changes, moder ate to severe endogenous pigment on the liver). The 100 and 200 mg/kg animals were sacrificed in extremis at 5

- 17 days with the following effects: hypoactivity, ataxia, hypothermia, emaciation, pale gums, dermal atonis, green ocular discharge, decreased d efecation, brown urine, severe body weight losses, and reduction in food consumption. NOAEL not established (<25 mg/kg/day).

870.3150 44148901 90

- Day oral, capsule

– Dog Acceptable/Guideline 2/sex at 0, 2, 4, 20 or 40 mg/kg/day LOAEL = 4 mg/kg based on elevated liver enzymes and microscopic findings in the liver (cytoplasmic vacuolation and minimal hepatocellular necrosis). Effects at 20 and 40 mg/kg: brown areas of skin on the urogenital and ventral abdominal areas, brown sclera, discolored ur ine (brown or amber), decreased and/or black mucoid feces and/or emesis, one 40 mg/kg female sacrificed in extremis on day 13, clinical chemistry findings, bile duct hyperplasia in 90% of the dogs. NOAEL = 2 mg/kg/day.
Non

guideline Neal et al

,

2003 Food Chem.
Tox. 41 : 193

- 200 Sub

- chronic (diet) tox

study Fisher 344 rats ethoxyquin 90% in the diet at 0, 0.01, 0.05, 0.01, 0.25 or 0.5% (5, 25, 50, 125, or 250 mg/kg/day) for 3 or 6 months (5 males /group for 3 months feeding; 8/sex/group in the 6 month feeding except in the 0.05,
0 .1, and 0.25% 5 females/group were used). Proposed LOAEL = 5000 ppm (250 mg/kg/day) based on a sharp threshold in male rats with either interstitial degeneration of the papilla or frank papillary necrosis being evident after 3 and 6 months but not at 250 0 ppm or lower dose. In the female rats, 6 months of feeding produced early minimal interstitial degeneration in one individual only at the 0.5% exposure level. When C

- 14 ethoxyquin was administered ip or orally by gavage (10 mg/kg), the radiolabel was a ssociated with urinary albumin. The ratio of the excreted radioactivity in the urine to feces
was 7.3

- 8.2. Autoradiographic sections of the kidneys did not show differences among the sexes in the distribution of the radioactivity or retention of ethoxyqu in. Fecal and urinary metabolites’ profiles
were similar in the two sexes. No un

- metabolized ethoxyquin was detected in any sample. Treatment of the urine and fecal extracts with β

- glucuronidase failed to produce any detectable level of non

- polar labele d compounds.
Non

guideline Shibata et al

,

1989 Tox Appl Pharmaco 99 : 37

- 49 0.8% ethoxyquin (640

mg/kg/day) in drinking water to Fisher rats for 4 or 8 weeks. Significant body weight and food consumption reduction. Leafy or ropy microridges and or short

m icrovilli observed on the bladder epithelial surface. Increased urinary bladder DNA synthesis.

Non

guideline Kim, 1985

J Tox Env. Health, 15: 663

- 671 0, 0.25 or 0.5% ethoxyquin

or ethoxyquin

- HCl, dietary to mice (5/dose) for 20 days. Dose

- dependent weigh t loss. Dose

- dependent hepatic hypertrophy (p<0.0001) compared to controls. Liver weights in the high dose group (0.5%) were greater in the ethoxyquin group than those in the ethoxyquin

- hydrochloride group. Tissue thiol levelsEthoxyquin Registration Review Human Health Assessment Scoping Document



### Page 16 of 28 Table A2.2. Ethoxyquin Subchronic, Chronic and Other Toxicity Profile

GL # MRID Study Type Results and Classification increased in both ethoxyqu in and ethoxyquin

- hydrochloride groups, especially in the small intestine (p=0.007). Liver tissue thiol levels also increased significantly (p=0.116). Thiol levels in the stomach of ethoxyquin treated rats were significantly higher (p=0.024) than levels observed in the ethoxyquin

- hydrochloride and control groups.
Non

guideline Ward et al

,

1989 Fund. Appld Toxicology,
12: 163

- 171 5000 ppm, dietary to male

F344/DuCrj rats for 6 weeks Induced toxic proliferative biliary lesions.

Ethoxyquin: Contact Dermatitis
Non

guideline Mehlhorn &

Beetz, 1971 Berufsdermat osen; 19 (2):84

- 91 Animal feed First case reported of occupational contact eczema attributed to ethoxyquin in an animal feeder.

Burrows, 1975
BR J DERMAT.; 92: 167

- 170 Animal feed mill,

ethox yquin added as vitamin E antioxidant Contact dermatitis in a 30

- yr old male and 48 yr old foreman.

O’Malley, 1997 Occup. Medicine:, 12:327

- 345 Apple and pear packing

processing Skin irritation in 20 workers from handling paper impregnated with ethoxyquin

Savini et al

, 1989 Contact Dermatitis, 21: 342

- 343 Poultry feed in a poultry

farm Puritic erythematous vesciualr dermatitis in a 21

- yr old female.

Nishioka et al , 1996 Contact Dermatitis; 35:241

- 245 Rice farmers wearing

rubber boots

- ethoxyquin Contact dermatitis: itch erythema, papules, scales and erosion on the legs, and erythema, vesicles, hyperkeratosis, scales and fissures on the soles, leading occasionally to generalization of eczema (autosensitization dermatitis) Zachariae,
1 978 Contact Dermatitis
4:117

- 118 MRID 5000444

Animal feed containing ethoxyquin Three cases of dermatitis reported

Alankoa, 1997 J European Acad Derm Venereology 9(S1): S167 Multivitamin powder

preparation with ethoxyquin Extensive eczemztous rash in a 43

- yr old man exposed for 17 years. Potential skin sensitizer.

Rubel and Freeman, 1998 Australas J. Dermatol. 39(2):89

- 91 Chicken feed with

ethoxyquin as vitamin antioxidant A case of allergic contact dermatitis reported

Vaneckova & Ettler, 1994 Rubber surgical gloves

confining ethoxyquin Dermatitis of hands in health care personnel.Ethoxyquin Registration Review Human Health Assessment Scoping Document



### Page 17 of 28 Table A2.2. Ethoxyquin Subchronic, Chronic and Other Toxicity Profile

GL # MRID Study Type Results and Classification

Contact Dermatitis, 31: 266

- 268 Hecke, 1977 Contact Dermatitis, 3 : 341

- 352 Animal feed farmer and

animal feed factory production workers Scaly dermatitis on face, hands, arms and legs.

Schubert et al , 1973 Dermatolozif che Monatffchrif 159:791

- 796 Animal feed mill Acute contact dermatitis in workers attributed to ethoxyquin in the feed.

Brandao, 1983 Contact Dermatitis Volume 9, Issue 3, page 240 Animal feed mill Contact dermatitis in animal feed workers.

Ethoxyquin: Developmental and Reproduction Studies 870.3700 MRID

44098901 Developmental Tox study

rat. Acceptable/Guideline 25/group at 0, 50, 150, or 350 mg/kg/day on GD 6

- 19 Maternal toxicity LOAEL = 150 mg/kg/day based on clinical signs of toxicity (wet yellow staining in the urogenital area, wet clear matting on the mout h, dried brown staining around the mouth) and reduced body weight gains and food consumption. Maternal toxicity NOAEL = 50 mg/kg/day. Developmental toxicity NOAEL =  350 mg/kg/day Developmental toxicity LOAEL was not identified.

Non

guideline Khera et al

,

1979 J Env Sci Hlth B 14: 563

- 577 Developmental tox study

in rats. 20/group t 0, 125, 250 or 500 mg/kg Santoquin (67% ethoxyquin) No maternal toxicity reported. It was concluded by the study authors that the overall effect of ethoxyquin formulation was not teratogenic to rats.

Non

guideline Telford et al,

1962
MRID 05000428 Developmental tox study

in rats. 57 mg/kg/day from
GD 1

- 22 Lower fetal absorption rate than in the control.

Non

guideline King, 1964

J NUTR 83:123

- 132 Developmental tox study

i n rats. Examined the effect of ethoxyquin on the outcome of pregnancy in vitamin E deficient rats Ethoxyquin reduced the incidence of resorptions and decreased the occurrence of gross congenital malformations.

Non

guideline DeSesso &

Goeringer, 1990 Repro Tox 4(4): 267

- 275 Developmental tox study

in rabbits. 7

- 10 / group, subcutaneous injection on GD 12 with 0, 950 mg/kg/day ethoxyquin alone or followed with 650 mg/kg hydroxyurea (HU) or 650 mg/kg of hydroxyurea injection alone (positive control) Maternal treatment with HU caused severe developmental toxicity in term fetuses: reduced mean fetal weight (p<0.01) and 100% malformations (cleft lip, cleft palate, limb and tail deformities) vs to 0% malformations in controls. Treatment with the ETX pri or to HU resulted in significant reduction of HU

induced fetal malformations (p<0.001) and an increase in the mean fetal body weight compared to the HU treatment only. Microscopic analysis of limb buds from HU

- treated embryos revealed severe mesenchymal cell necrosis in the limb buds within 4 hours of maternal injection. Pre

- treatment withEthoxyquin Registration Review Human Health Assessment Scoping Document



### Page 18 of 28 Table A2.2. Ethoxyquin Subchronic, Chronic and Other Toxicity Profile

GL # MRID Study Type Results and Classification ethoxyquin reduced the cell necrosis to less than 20% during the first 4 hours, but by 12 hours it was comparable to the HU treatment.
Non

guideline Isenstein,

1970 AM J VET RES 31:907

- 909 Developmental tox study

in rabbits. 16

- 20 group, dietary at 0, 0.0025% or 0.005% ethoxyquin 10 day prior to breeding to 2 weeks after parturition. Study repeated 4 months later at 0, 0.0025% or 0.01% ethoxyquin Results were inco nsistent, but do suggest that ethoxyquin had no effect on newborn mortality or abortions in pregnant rabbits at these dietary doses.

Non

guideline Delaney et al

,

1995 Toxicologist Mar;15(1):16 5 Reproduction study in rats.

Ethoxyquin in corn oil at 0, 75, 150, or 300 mg/kg/day (20/sex/group) using the continuous breeding protocol. Proposed LOAEL for reproductive toxicity is 300 mg/kg/day, based on decreased litters/pair (22%), live pups/ litter (22%), and increased cumulative days to litter (8%

- 17%), decreased pup weight (20%) in pups born to treated females and decreased body weights in F0 and F1 males(7

- 20%). Proposed NOAELfor reproductive toxicity is 150 mg/kg/day. Proposed parental toxicity LOAEL is 75 mg/kg/day based on increased liver and kidn ey weights (13% to 56% with increasing dose) in F0 and F1 animals. Proposed parental toxicity NOAEL is <75 mg/kg/day
Non

guideline Gilman &

Voss, 1995 in JMPR 1998 Reproduction study in

dogs 0, 100, 225 ppm of ethoxyquin (Santoquin®) dietary (0, 2.5, 6

m g/kg/day) for two generations Proposed LOAEL for reproductive toxicity is >6 mg/kg/day. Proposed NOAEL for reproductive toxicity is 6 mg/kg/day. Proposed parental toxicity LOAEL is 2.5 mg/kg/day based on increased incidences of clinical signs such as exc ess lachrymation and dehydration, clinical chemical changes and pigment deposition in the liver. Ethoxyquin: Metabolism and Absorption Studies
Non

guideline Skaare &

Nafstad, 1979
Acta Pharmacologi

ca et Toxicologica 44:303

- 307 Metabolism Study

-

Distribution & Whole body autoradiography

- Male Rat. 2 rats/sacrifice time of 0.5, 1, 2, 4, 8, 10, 12, 16, 20, 24, 48 & 144 hours given single oral dose of 14C

- ETX. 0.5 hr autoradiograms showed that radioactivity was distributed throughout most tissues, highest in GI tract, liver and kidneys. By 24 hours no radioactivity detected in the stomach but still persisted in the intestines. At 48 hours, radioactivity declin ed with exception of the GI tract, the medulla of the kidney, and the blood. At 144 hours, high radioactivity persisted in the renal cortex and moderate to weak radioactivity in the liver, the intestines, the lungs, and heart blood.
Non

guideline Skaa re &

Solheim, 1979 Xenobiotica 9 (11): 649

- 657 Metabolism Study

Metabolite Identification

male rats. 14C

- ETX, 100 mg/kg oral dose The major metabolic pathway was deethylation and hydroxylation. An average of 28 & 36% of dose of radioactivity was recove red in bile in 12 & 24 hr respectively following intragastric admin of C14

- ETX to bile duct cannulated rats. Biliary metabolites were identified. A 95% of the AD was accounted for within 6 days. Less than 1% of the AD was detected in 24 hour respired ai r samples.
Non

guideline Kim et al

,

1992 Metabolism

- Sheep and

rats. Sheep given ETX.HCl as 0.5% of diet. ETX was identified in all sheep urine samples

co llected during the first 24 h of feeding, and ETX and hydroxylated ETX were identified in all urineEthoxyquin Registration Review Human Health Assessment Scoping Document



### Page 19 of 28 Table A2.2. Ethoxyquin Subchronic, Chronic and Other Toxicity Profile

GL # MRID Study Type Results and Classification

Tox Environ Health; 37 (2): 341

- 347 Rats given ETX in corn oil

0.08 g/day/rat orally for 7 days samples collected after 12 days of feeding. In contrast, ETX, hydroxylated ETX, and dihydroxylated ETX were identified in urine from rats fed ETX for 7 day s. Non

guideline Sanders et al

,

1996 Xenobiotica; 26 (6):583

- 595 NTP metabolism study,

rats and mice. 14 C

- ETX per oral or intravenous. Excretion studies The disposition of single doses up to 25 mg/kg was similar in the rat and mouse. About 90% of AD was excreted in urine and feces within 24 h. In contrast, no more than 60% of a 250 mg/kg dose was excreted within 24 h following p.o. administration. Urinary excretion is the predominant route. Its metabolism was rapid in both the rat and mouse follow ing either p.o. or i.v. administration. Little or no parent compound was detected in cumulative 24

- h excreta.
ETX

- derived radioactivity bioaccumulated in some tissues (liver, kidney and adipose tissue) following repeated exposure to rat of either 25 or 25 0 mg/kg by

gavage. Non

guideline Burka et al

,

1996 Xenobiotica; 26 (6):597

- 611 NTP metabolism study,

rats and mice. 14 C

- ETX po or iv. Metabolite identification ETX is extensively metabolized by oxidation, deethylation and conjugation to endogenous substances prior to urinary elimination. Rat urine sulphate conjugates: 1,2

- dihydro

- 6

- hydroxy

- 2,2,4

- trimethylquinoline sulphate, and 1,2,3,4

- tetrahydro

- 3,6

- dihydroxy

- 4

- methylene

- 2,2

- di methylquinoline sulphate. Mouse urine had a sulfate conjugate and a g lucuronide conjugate. ETX

- derived radioactivity was excreted in rat bile, mainly as GSH conjugates, with little unchanged ETX present.
Non

guideline Wilson et al

,

1981 Xenobiotica 9 (6): 343

- 51 Metabolism study

- rats,

repeated feeding (0.005%
ETX in diet) for 10 days followed by a single oral dose 1.5 mg of 14 C

- ETX and sacrificed 1, 2, 7 days for males and 7, 14, 28 days for females. Liver and kidney contained the highest concentrations (0.3 ppm

- 0.4 ppm) of radioactivity after one week. The heart, skeletal muscle, and brain contained lower levels while the spleen, blood, and abdominal fat contained intermediate levels of radioactivity. The estimated ETX turnover rate in rats was 10 days. ETX is rapidly excreted within one or two days. Oxidation of ETX to carbon dioxide was negligible. Placental transfer of 14 C

- ETX or its metabolites was demonstrated.
Non

guideline Meulen et al

,

1980 Journal of Animal Physiology and Animal Nutrition) 43: 164

- 170 Metabolism study

Excretion and metabolite identification, male rats. Single oral dose of 40 mg of 14 C

- ETX and sacrificed after 6, 12, 24, 48, 72 hrs. The highest values of the AD observed in the liver, kidney, and stomach were 8.0%, 0.3%, and 56.5%,

resp ectively, after 6 hours. Levels of radioactivity found in the small intestine, large intestine, caecum and blood were 11.3%, 0.9%, 6.9% and 0.9% of the AD, respectively. 14 C

- ETX concentration in urine exceeded that in feces by two

- fold. After 72 hours,
8 4.8% of the total labeled dose was found to be eliminated in the urine and feces. Unchanged ETX, one unknown metabolite and five known metabolites were detected in urine. Ethoxyquin: Special Studies
Non

guideline Cabral &

Neal, 1983 Cancer Letters, 19: 125

- 132 Inhibitory Effects Of

Ethoxyquin On The Carcinogenic Action Of Aflatoxin B1 In Rats. Rats fed diets containing 0 or 4 Ethoxyquin produced an inhibition of liver carcinogenicity that was dose related. There was a high incidence of altered foci, neoplastic nodules, and liver cell tumors in rats fed aflatoxin only. Addition of ethoxyquin to a diet containing aflatoxinEthoxyquin Registration Review Human Health Assessment Scoping Document



### Page 20 of 28 Table A2.2. Ethoxyquin Subchronic, Chronic and Other Toxicity Profile

GL # MRID Study Type Results and Classification ppm aflatoxin or ethoxyquin 0.05 or 0.5% with ethoxyquin administered alone or after aflatoxin or before aflatoxin or together. comp letely inhibited liver tumors. When ethoxyquin was administered after aflatoxin, there was considerable reduction in the incidence of altered foci and neoplastic nodules of the liver. No liver tumors were found in rats fed ethoxyquin alone or in

untreate d controls. No metastases were found from any liver tumor.
Non

guideline Wischi, 1981

Toxicology; 21: 95

- 104 Inhibitory effects When male mice were injected i.p. with a single dose of urethanE and fed 0.75% ETX in the diet and killed 4 months after uret hane, exposure to ETX did not enhance formation of lung tumors.
Non

guideline Wattenberg,

1972 J Natl Cancer Inst; 48: 1425

- 1430 Inhibitory effects ETX inhibited the carcinogenicity of benzo(a)pyrene or 7,12

- dimethylbenz(a)anthracene (DMBA) on the foresto mach of the mouse. It also inhibited mammary tumor formation produced by oral administration of DMBA to female Sprague

- Dawley rats. ETX given to female rats 1 hr before oral administration of 30 mg DMBA prevented the adrenal necrosis occurring in anima ls not given the antioxidant
Non

guideline Tsuda et al

,

1984 Carcinogenes is, 5: 525

- 531 Tumor promotion &

inhibition ETX inhibited the development of liver foci induced (initiated) by N

- ethyl

- N

- hydroxyethylnitrosamine (EHEN) but enhanced the appearance of preneoplastic and neoplastic induced lesions in the kidney of rats
Non

guideline Thamavit et

al , 1985 Cancer
Letters 27: 295

- 304 Inhibitory Effect of ETX. Preneoplastic lesions (foci) in rat liver initiated by diethylnitrosamine (DEN, single oral dose 200 mg/kg body wt) and partial hepatotectemy were decreased (number and area of foci) by subsequent ETX diet (0.5%, 0.25% and 0.125%) in a dose dependent fashion.
Non

guideline Takahashi et

al , 1986 Cancer Letters, 30: 161

- 168 Tumor promotion, male

rats r eceived 100 mg of
N

- methyl

- N'

- nitro

- N

- nitro soguanidine (MNNG)/L of drinking water for 8 wks followed by 1% ETX diet for 32 wks or 0, or 1% ETX only for 40 weeks. No rats given ETX alone developed tumors in the glandular stomach. The incidence of gastric t umors was significantly increased in animals given ETX after MNNG treatment compared to the group given MNNG alone. Changes were observed in the kidneys of animals given ETX and were especially marked for animals receiving ETX after MNNG administration.

Non

guideline Masui et al

,

1986 Japanese Journal of
Cancer Research, 77: 231

- 237 Inhibitory Effect of ETX.

Male rats injected 200 mg/kg diethylnitrosamine (DEN), followed by 0.02% of 2

- acetylaminofluorene (AAF) in the diet for 2 to 8 weeks. Partial hepa tectomy 3rd week. Week 10 were fed 0.8% ETX diet for 26 weeks, killed at week 40 and necropsied. Hepatocellular carcinomas were observed in 88.2% of the animals initially treated with DEN and AAF. Rats treated subsequently with ETX showed incidences of hepatocellular carcinoma of 57.1%, significantly lower than that of the positive controls. Rats treated with ethoxyquin showed significant decreases in the number of hepatocellular carcinomas per unit liver, compared with the positive controls. Only the ethoxyquin treated animals showed significant decreases in tumor size. No metastases to other organs or primary tumors in other organs were seen.

Non

guideline Manson et al

,

1987 Carcinogenes is ; 8:723

- 728 Inhibitory Effect of ETX.

Rats pretreated with 0.5% ETX diet followed by aflatoxin B1 ETX completely prevented the formation of AFB1

- induced preneoplastic liver lesions. However, ETX alone caused severe damage to the kidney. In addition, many hyperplastic and putativeEthoxyquin Registration Review Human Health Assessment Scoping Document



### Page 21 of 28 Table A2.2. Ethoxyquin Subchronic, Chronic and Other Toxicity Profile

GL # MRID Study Type Results and Classification preneoplastic tubules were visib le, suggesting that ETX may be exerting a carcinogenic effect in the kidney.
Non

guideline Kensler et al

,

1986 Cancer Res 46: 3924

- 31 Inhibitory Effect of ETX.

Rats fed 0.4 % ETX diet for 1 wk, gavaged 250 ug aflatoxin B1/kg, 5x/wk for 2wks, then normal diet for 4 months. ETX reduced > 95% both area and volume of liver

gamma

- glutamyl transpeptidase

- positive foci. ETX produced a dramatic reduction in the binding of aflatoxin B1 to hepatic DNA: 18

- fold initially and
3

- fold at the end of the dosing period. Apparently, related to induction of detoxication enzymes. Rats fed 0.4% ETX for 7 days showed a 5

- fold increase ETX in hepatic cytosolic glutathione S

- transferase (GST)

- specific activities. Correspondingly, biliary elimination of aflatoxin B1

- glutathio ne conjugate was increased 4.5

- fold in animals on the ETX diet.
Non

guideline Hirose et

al ,1986 Cancer Letters, 30:169

- 174. Promotion study, male rats

treated with 0.25% ETX diet for 2 wks, and during wk 2, 1% butylated

- hydroxyanisole (BHA). Pronounced epithelial damage: hyperkeratosis and ulcer formation was seen in the forestomach in the animals treated with ETX and BHA. Mild hyperplasia induced by BHA was increased by ETX .

Non

guideline Ito, 1986,

NTP, 1990 Food and Chemical Toxicology.
24:1071

- 1 082 Cancer promotion study in

male rats.
N

- ethyl

- N

- hydroxyethyl nitrosamine (EHEN), 0.1% in drinking water for 2 wk, then 0.8% ETX diet for 29 weeks The incidence of renal

- cell adenomas was significantly increased in ETX groups (p<0.01) compared to contro ls. The author concluded that ETX promoted induction of EHEN

- initiated kidney adenomas

Non

guideline Ito et al

,

1986, Toxicologic Pathology, 14: 315

- 323 Cancer promotion study in

male rats.
N

- butyl

- N

- (4

- hydroxybut yl)nitrosamine (BBN), 0.05% in drinking water for 4 weeks, then 0.8% ETX diet for 32 wk Administration of ETX significantly increased (p<0.001) the incidence of papillary or nodular hyperplasia.

Non

guideline Fukishima et

al , 1984 Cancer Letters, 23(1): 29

- 37 Cancer promotion study in

male rats. 0.05%
N

- butyl

- N

- (4

- hydroxybut yl)nitrosamine (BBN), in drinking water for 4 weeks, then an 0.8% ETX diet for 32 weeks ETX rats weighed less than the controls at the end of 36 weeks. Lesions of the urinary bladder were significantly higher in the ET X group pretreated with BBN (p<0.001). Incidence and number of cancers were slightly increased in this group compared to controls, but not significantly. The authors concluded that ETX promotes urinary bladder carcinogenesis.
Non

guideline Fukushima et

al ., 1986; Japan Journal Cancer Research. 77: 1074

- 1082 Cancer promotion study in

male rats. Groups fed a basal diet containing a tumor promoter, one group was fed basal diet with 0.8% ethoxyquin plus a control group At week 4, body weight was less i n the group receiving ETX compared to controls. In ETX treated rats, the lumina surface of the urinary bladder had irregular

- shaped foci with a slight elevation of cells, giving the mucosa a cobblestone appearance. Scanning electron microscopy revealed pl eomorphic microvilli, short uniform microvilli and ropy or leafy microridges.
Non

guideline Fukushima et

al ., 1987; Cancer Lett; 34 (1): 83

- 90 Cancer promotion study in

male rats.
N

- butyl

- N

- (4

- hydroxybut yl)nitrosamine (BBN), 0.05% in drinking water Mean body weights of ETX rats were lower than controls after week 2, with a slight reduction in food intake compared to controls. Incidence of esophageal papillomas was significantly higher (p<0. 05) in ETX rats versus controls. The authors concluded that ETXEthoxyquin Registration Review Human Health Assessment Scoping Document



### Page 22 of 28 Table A2.2. Ethoxyquin Subchronic, Chronic and Other Toxicity Profile

GL # MRID Study Type Results and Classification
for 4 weeks, followed by a diet of 0, or 0.8% ETX for 36 weeks. significantly enhanced the induction of esophageal papillomas by DBN

Non

guideline Fukushima et

al ., 1987; Cancer Research, 47: 2113

- 2116 Cancer promotion study in

male rats.
N

- butyl

- N

- (4

- hydroxybut yl)nitrosamine (BBN), 0.05% in drinking water for 2 wk, followed by a diet of ETX for 22 wk A dose

- dependent decrease in pre

- neoplastic lesions in the bladder of BBN treated rats was seen in
ETX

- exposed animals. The authors report that "no promot ing activity or induction of pre

- neoplastic lesions in the urinary bladder occurred even at the highest ethoxyquin dose (0.5%).

Non

guideline Miyata et al

,

1985 Jpn J Cancer Res); 76: 828

- 834 Cancer promotion study in

male rats. BBN, 0.05% in drinking wa ter for 2 weeks, followed by a diet of 0, or 0.8% ETX for 22 wk. Incidences and quantitative values of hyperplasia significantly higher in ETX group (p<0.05) indicating significant promoting activities in the induction of

pre

- neoplastic lesions of the urin ary bladder.

Non

guideline Manson et al

,

1997; Carcinogenes is 18: 1729

- 1738 Kelly et al,
2000 Cancer Res. 60:957

- 969 Enzyme Induction ETX is a phase II enzyme inducer

Non

guideline Yamano &

Morita, 1993 Arch. of Env Contam and Tox. 25:
271

- 278 Enzyme Induction ETX at 10

- 7

- 10

- 3 M was a potent inhibitor of NADPH/Fe+2 lipid peroxidation in isolated rat liver microsomes. It also significantly reduced hepatocellualr glutathione concentrations at 10

- 4M

Non

guideline Hayes et al

,

1991 & 1998 Biochem J;
27 9: 385

- 398 Chemico

- Biol ogical Interactions,
111

- 112: 51

- 67 Enzyme induction ETX induces glutathione S

- transferase (GST) isoenzymes in rat liver that contribute to ethoxyquin

- induced chemoprotection against AFB1.

Non

guideline Ellis et al

,

1993 Proc Natl Acad Sci 90:
10350

- 10354 Enzyme induction Dietary ETX induces an aldehyde reductase (AFB1

- AR). The mRNA encoding this enzyme is markedly elevated in rat liver fed ETX diet.

Non

guideline Bock et al

,

1980 Arch Pharmacol 310: 249

- 52 Enzyme induct ion 0.5% ETX in diet enhanced rat hepatic
UDP

- glucuronosyltransferase activities and
3

- Methylcholanthrene

- and phenobarbital

- inducible glucuronidation reactions

Non

guideline Buetler et al

,

1995 Toxicology and Applied Pharmacol. 135: 45

- 57 Enzyme induction Phase I and Phase II enzymes are induced by ETX

Non

guideline Wilson et al

,

1979 Enzyme induction A single oral dose of ETX 200 mg/kg to rats inhibited the hepatic microsomal hydroxylation ofEthoxyquin Registration Review Human Health Assessment Scoping Document



### Page 23 of 28 Table A2.2. Ethoxyquin Subchronic, Chronic and Other Toxicity Profile

GL # MRID Study Type Results and Classification

Xenobiotica 9: 343

- 51 thiabendazole (TBD) (65%), aniline (40%) and biphenyl (40%) in vitro at 1 h after dosing; inhibition was less at 5 h. TBD 100 mg/kg administered orally 30 min after oral ETX (400 mg/kg) or TBD 200 mg/kg administered orally 30 min after oral desmethylimipramine (DMP) (80 mg/kg) dela yed absorption of the TBD and resulted in markedly decreased plasma concentration of the anthelmintic. Simultaneous administration of ETX (300 mg/kg) potentiated the anthelmintic effect of TBD (750 mg/kg) on the helminth parasite, Nematospiroides dubius, i n the mouse. DMP showed no similar potentiation
Non

guideline Kahl, 1980

Cancer Letters, 8: 323

- 328 Enzyme induction ETX feeding to rats increases epoxide hydratse activity in liver microsomes. I.p treatment of mice with ETX also increases it.

Non

guid eline Mandel et al

,

1987 Cancer Res 47:5218

- 5223

. Enzyme induction ETX increased microsomal cytochrome P

- 450s, phenobarbital inducible, and the in vitro rate of metabolism of AFB1. ETX increased the glutathione
S

- transferase activity of the liver cytosol fractions.

gamma

- Glutamyl transpeptidase was induced in the periportal areas of the liver lobule. Reduced in vivo binding of [3H]AFB1 to DNA of liver and kidney was found to result from ETX treatment.
Non

guideline Zenser &

Davis, 1978 Metabolism, 27: 227

- 233 In vitro Study, renal

function. Rat inner medula slices incubated with ETX and assayed for prostoglandins E2 (PGE2), prostoglandin F2 (PGF2) and cyclic AMP levels. ETX (0.01 and 0.001 mM) caused a 50% significant (p<0.05) reduction in PGE2. Also, ETX decreased (p<0.05) PGE2 production in the presence of arachidonic acid. ETX also inhibited PGF2 synthesis significantly (p<0.05) at concentrations of 0.01, and 0.1 mM. Prostoglandins within the kidney play an important role in increased rate of urin e flow and sodium excretion and thus this inhibition may be significant to the renal function.
Non

guideline Derbel et al

,

1993 Biochimica biophysica acta; 1158 (2): 175

- 180 Enzyme induction ETX induces various subunits of renal and hepatic glutathione S

- trnasferase.

Ethoxyquin

- Immune Effects
Non

guideline Travis et al

,

1989 Immunol Cell Biol; 67 (1):
83

- 84 Subchronic Study

- Female

mice (10/group) were given diets containing 0.005 ppm Se or 1 ppm Se or 1 ppm Se and 0.02% ETX or 1 ppm Se and 0.25% ETX for 60 days. The 0.005 ppm Se diet is selenium deficient and the 1.0 ppm Se diet is selenium sufficient for immunosuppression or maintenance of normal immune function in mice. Immunosupression was As measured by the plaque

- forming cell assay (PFC) the Se deficient diet led to immunosuppression (p<0.05) compared to the Se sufficient diet. The addition of 0.02% ETX to the Se sufficient diet had no significant effect on the immune response while the addition of 0.25% ETX reduced (p<0.05) the immune response to the level of animals receiving the Se deficient diet. Food consumption and body weights were comparable among all groups. GSH

- Px levels, generally regarded as indicating the ‘Se status’ were statistically equal in the three groups receiving the 1.0 ppm of SE and significantly elevated (p<0.01) over the Se de ficient group. The serum Se levels (~30 ug/100 ml) were equal in the 1 ppm Se groups with or without the 0.25% ETX. The liver DNA,Ethoxyquin Registration Review Human Health Assessment Scoping Document



### Page 24 of 28 Table A2.2. Ethoxyquin Subchronic, Chronic and Other Toxicity Profile

GL # MRID Study Type Results and Classification measured by the plaque

forming cell assay (PFC). RNA and protein values were similar among all the dietary groups. Ethoxyquin: Mutagenicity Studies 870.5100 MRID

05000385 bacterial reverse mutation Not mutagenic up to 1000 ug/plate in Salmonella typhimurium strains in ± microsomal S9 activating system. 870.5100 Hedenstedt11

982 MUTAT RES 97:191 bacterial reverse mutation Mutagenic in Salmonella typhimurium strains TA98, 100 & 1538 with S9 activating system (concentration not specified).

870.5100 Rannung et

al , 1984 Industrial Hazards of Plastics and Synthetic Elastomers (1984), pp. 407

- 419 bacterial reverse mutation Weak mutagen at or above 50 mg/plate in Salmone lla typhimurium strains with S9 activating system

870.5100 Ohta et al

,

1980
Mut. Res. 77: 21

- 30 bacterial reverse mutation Ethoxyquin was negative in the rec

- assay. Non

- mutagenic to E. coli and all strains of S. typhimurium tested at concentrations up to 5 mg/plate

Non

guideline Gille et al,

1991 Mutagenesis 6(4):313

- 8 Clastogenic effects ETX potentiates the clastogenic effects of hyperoxia (80

- 90% O2

- induced chromosomal aberrations in Chinese hamster ovary cells)

Non

guideline Renner, 1984

Mutation
Re s./Genetic Toxicology 135:125

- 129 Bone morrow

chromosomal test: To test a possible antimutagenic activity of ethoxyquin (EQ) in bone

- marrow cells, 3 cytogenetic tests with distinct genetic end

- points were applied (SCE, micronucleus test, or chromosome aberration test). Cyclophosphamide (CPA), as test mutagen, and EQ were administered immediately following each other to Chinese hamster by stomach tube. The CPA dose was constant, the EQ doses were increased up to ratio of CPA/EQ = 1 : 50. The formation of SCEs induced by CPA was not influenced by EQ

- even at the highest dose. In the micronucleus test, however, EQ drastically reduced the micronucleus rate even at the lowest dose applied (20 mg/kg) and abolished the CPA effects at a dose of 100 mg/kg. This action of EQ against CPA was also found in the rat and mouse in the micronucleus test system. Two inbred strains of mouse showed similar reactions. In the chromosome aberration test, EQ also showed a distinct anticlastogenic response. The anticlastogenic effect of EQ was quantitatively similar in the micronucleus and chromosome aberration tests. It was concluded that ETX is antimutagen per se (a factor interfering with spontaneous or induced mutagenesis at a cellular level) in contrast to desmutagen (inactivator or destroyer of mutagen) or a metabolism inhibitor
Non

guideline Katz and Raj,

1985 Chromosomal Breakage The ability of ETX to inhibit chromosome breakage by genotoxic agents was studied in female mice pretreated with ETX 24 hours to 1 week before being injected ip with 7, 12

- dimethylbenz(a)anthraceneEthoxyquin Registration Review Human Health Assessment Scoping Document



### Page 25 of 28 Table A2.2. Ethoxyquin Subchronic, Chronic and Other Toxicity Profile

GL # MRID Study Type Results and Classification (DMBA), benz(a)pyrene (BaP), cyclophosphamide (CP) or mitomycin

- C (MMC). The mice were killed at various times up to 96 hours after treatment, and bone marrow samples were taken and scored for polychromatic erythrocytes (PCEs) and micronucleated polychromatic erythrocytes (MNPCEs). ETX inhibited PCE and MNPCE induction by all agents except BaP.Ethoxyquin Registration Review Human Health Assessment Scoping Document



### Page 26 of 28 Attachment 3. Ethoxyquin Toxicological Endpoints and Doses for Risk Assess ment

Table A3.1. Summary of Toxicological Doses and Endpoints for Ethoxyquin Human Health Risk

Assessments (D310495 and D342695).
Exposure/ Scenario Point of

Departure (mg/kg/day) Uncertainty/

FQPA Safety Factors RfD, PAD, Level of

Concern for Risk Assessment Study and Toxicological

Effects

Dietary Risk Assessments

Acute Dietary (Females 13

– 49 years old) NOAEL = >3

mg/kg/day UF A = 10

UF H = 10 FQPA SF = 1 Acute RfD = 0.03

mg/kg/day
aPAD = acute RfD FQPA SF = 0.03 mg/kg/day Developmental study in rabbits

where the maximum dose tested of 3 mg/kg/day had no developmental effects. Isenstein RS . 1970. Ethoxyquin in rabbit feed: study of relationship to abortion and early neonatal death Am J Vet Res 31:907

- 909

Chronic Dietary (All Populations) NOAEL = 2

mg/kg/day UF A = 10

UF H = 10 FQPA SF = 1 Chronic RfD = 0.02

mg/kg/day
cPAD = chronic RfD FQPA SF = 0.02 mg/kg/day 90

- day subchronic study in

dogs, MRID 44148901* LOAEL = 4 mg/kg/day based

on elevated liver enzymes and microscopic findings in the liver (cytoplasmic vacuolation and minimal hepatocellular necrosis). The dog is highly sensitive compared to rats

Non

- Dietary Risk Assessments

Incidental Oral Short

- Term (1

- 30 Days): Not Applicable

Dermal (All durations) NOAEL = 2

mg/kg/day (Oral)

100% absorption is assumed UF A = 10

UF H = 10 FQPA SF = 1 Occupational

LOC for MOE =
100 90

- day subchronic study in

dogs, MRID 44148901* LOAEL = 4 mg/kg/day based

on elevated liver enzymes and microscopic findings in the liver (cytoplasmic vacuolation and minimal hepatocellular necrosis). The dog is highly sensitive compared to rats

Inhalation (All durations) NOAEL = 2

mg/kg/day (Oral) UF A = 10

UF H = 10
UF DB = 10** FQPA SF = 1 Occupational

LOC for MOE = 1000 90

- day subchronic study in

dogs, MRID 44148901* LOAEL = 4 mg/kg/day based

on elevated liver enzymes and microscopic findings in the liver (cytoplasmic vacuolation and minimal hepatocellular necrosis). The dog is highly sensitive compared to rats

Cancer There are no guideline studies assessing this effect in animals. It is structurally similar to

Flectol H which showed evidence of carcinogenicity (kidney tumors) in male rats.Ethoxyquin Registration Review Human Health Assessment Scoping Document



### Page 27 of 28 Table A3.1. Summary of Toxicological Doses and Endpoints for Ethoxyquin Human Health Risk

Assessments (D310495 and D342695).
Exposure/ Scenario Point of

Departure (mg/kg/day) Uncertainty/

FQPA Safety Factors RfD, PAD, Level of

Concern for Risk Assessment Study and Toxicological

Effects

Ethoxyquin is also a nephrotoxin in male rats. A theoretical upper b ound Q 1 * for ethoxyquin was calculated to be 0.04 (mg/kg/day)

- 1
* The JMPR established an ADI for ethoxyquin of 0

- 0.005 mg/kg body weight/day on the basis of the minimal effect level of 2.5 mg/kg/day in the multigeneration study in dogs and a 500

- fold safety factor to account for the lack of a NOAEL in this study and for the incompleteness of the database. However, we find the subchronic study in dogs better conducted and reported, and is more reliable in deriving an endpoint for human risk assessment. Ethoxyquin was more toxic in dogs compared to rats. (Gilman, M.R. & Voss, W.R (1995) Reproduction/chronic toxicology study of ethoxyquin with beagle dogs. Unpublished report HRP

MI #203

- 1.1from HRP Inc for Monsanto, St Louis, Missouri, USA. Submitted t o WHO by the Oregon, Washington and California Pear Association).
** Until the required inhalation study is submitted and found acceptable (or alternative information is provided to reduce the uncertainty), a 10X database uncertainty factor will be applie d for the inhalation route (TXR# 0053945). 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 environmental ly relevant human exposures. NOAEL = no observed adverse effect level. LOAEL = lowest observed adverse effect level. UF = uncertainty factor. UF A = extrapolation from animal to human (interspecies). UF H = potential variation in sensitivity among membe rs of the human population (intraspecies). UF L = use of a LOAEL to extrapolate a NOAEL. UF S = use of a short

- term study for long

term risk assessment. UF DB = to account for the absence of key data (i.e., lack of a critical study). FQPA SF = FQPA Safety Factor. PAD = population adjusted dose (a = acute, c = chronic). RfD = reference dose. MOE = margin of exposure. LOC = level of concern. N/A = not applicable.Ethoxyquin Registration Review Human Health Assessment Scoping Document



### Page 28 of 28 Attachment 4. Ethoxyquin International Residue Limits

Table A4.1. Ethoxyquin (055501; 6/23/14) Summary of US and International Tolerances and Maximum Residue Limits Residue Definition : US Canada Mexico 1 Codex 2
40 CFR 180.178: Plant: ethoxyquin (1,2

- dihydro

- 6

- ethoxy

2,2,4

- trimethylquinoline) 1,2

- dihydro

- 6

- ethoxy

2,2,4

- trimethyl

quinoline polymer Ethoxyquin

Commodity Tolerance (ppm) /Maximum Residue Limit (mg/kg)
US Canada Mexico 1 Codex 2

Pear 3 3 3 Po Completed: M. Negussie; 06/25/14

1 Mexico adopts US tolerances and/or Codex MRLs for its export purposes.

2 * = absent at the limit of quantitation; Po = post

- harvest treatment, such as treatment of stored grains. PoP = processed post

- harvest treated commodity, such as processing of treated stored wheat. (fat) = to be measured on the fat portion of the sample. MRLs indicated as proposed have not been finalized by the CCPR and the CAC.