UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY AND

 POLLUTION PREVENTION

MEMORANDUM

DATE: December 16, 2015

SUBJECT: Science Review of “Davis et al., 2008. Assessing Intermittent Pesticide Exposure 

from Flea Control Collars Containing the Organophosphorus Insecticide 

Tetrachlorvinphos” for HSRB Consideration

PC Code: 083701 DP Barcode: D430707

Petition No.: NA Registration Nos.: NA

Risk Assessment Type: Science Review Regulatory Action: NA

TXR No.: NA Case No.: NA

MRID No.: NA CAS No.: 22248-79-9

FROM: Wade Britton, MPH, Environmental Health Scientist

Risk Assessment Branch IV (RABIV)

Health Effects Division (HED; 7509P)

Office of Pesticide Programs

THROUGH: Michael Metzger, Chief

RABV and RABVII/HED (7509P)

And

Jeffrey L. Dawson, Acting Deputy Director

Immediate Office (IO/HED; 7509P)

TO: Neil Anderson, Branch Chief

Risk Management and Implementation Branch I (RMIBI)

Pesticide Re-evaluation Division (PRD; 7508P)

Office of Pesticide Programs

This memorandum presents EPA’s science review of the following journal article which measured 

tetrachlorvinphos (herein referred to as TCVP) human exposure in adults and children from contact with 

dogs wearing TCVP pet collars in residences, cholinesterase inhibition in treated dogs, and also 

transferable residues from the fur of treated dogs. The agency is currently planning to use the journal 

article as a source of pet fur transferable residue data for a residential post-application exposure and risk 

assessment for adults and children who could be exposed from contact with dogs wearing TCVP pet 

collars.
Page 2 of 14

CITATION: Study Authors: M. Keith Davis, J. Scott Boone, John E. Moran, 

John W. Tyler and Janice E. Chambers 

Title: Assessing Intermittent Pesticide Exposure from Flea 

Control Collars Containing the Organophosphorus 

Insecticide Tetrachlorvinphos. Journal of Exposure 

Science and Environmental Epidemiology

Report Date: Published January 2008 

Performing Laboratories: Mississippi State University, Mississippi State, MS

TEST MATERIAL: Hartz Control Ultimate Flea Collar - 14.55% active ingredient (TCVP); EPA 

Reg. No.: 2596-139 

SYNONYMS: Tetrachlorvinphos (TCVP): [(Z)-2-chloro-1-(2,4,5-trichlorophenyl) vinyl 

dimethyl phosphate]

SPONSORS: Mississippi Agriculture and Forestry Experiment Station (MAFES) and the 

College of Veterinary Medicine, Mississippi State University. Research also 

supported by grants from the US Environmental Protection Agency’s Science to 

Achieve Results (STAR) grant program (Grant nos. R825170 and R828017). 

INTRODUCTION

The Agency is currently planning to use the Davis et al paper only as a source of transferable pet fur 

residue data in order to complete a residential post-application exposure and risk assessment for adults 

and children who could be exposed from contact with dogs wearing TCVP pet collars. The residue 

measurements would be used as a basis for calculations which are described in the Agency’s SOPs for 

Residential Exposure Assessment.1

 The study was conducted in 2 parts both of which measured 

transferable residues from the fur of treated dogs. Other phases of the study were conducted which

measured the impact of cholinesterase activity on treated dogs (Study 1 only), dermal exposure from 

children living with the treated dogs using passive dosimetry in the form of t-shirts (Study 2 only), and 

internal dose based on residues in urine of children and adults living with treated dogs (Study 2 only). 

The transferable residue data have been determined to be acceptable for regulatory purposes given that 

the sample collection method was the scientific standard at the time the study was conducted and all 

phases of the sample collection and analysis appear adequate for those samples. Also, importantly, the 

risks predicted are higher than previously calculated for TCVP pet collar products which is an important 

consideration related to EPA’s use of the data as discussed in the Agency’s ethics review (Lydon 2015)

which indicates: 

“40 CFR Subpart Q, §26.1703, prohibits EPA from relying on data from any research subject to 

this subpart involving intentional exposure of any human subject who is a pregnant woman (and 

therefore her fetus), a nursing woman, or a child. §26.1706 provides an exception. Under 40 

CFR §26.1706, EPA can only rely on this research if it is crucial to making a decision to impose a 

more stringent regulatory restriction than could be justified without the data. If EPA’s Office of 

Pesticide Programs (OPP) decides to rely on the TCVP glove residue data, under 40 CFR 

 1 http://www.epa.gov/pesticide-science-and-assessing-pesticide-risks/standard-operating-procedures-residentialpesticide 
Page 3 of 14

§26.1706, OPP must first complete three required steps. EPA must obtain the views of the 

Human Studies Review Board, provide an opportunity for public comment, and publish a full 

explanation of its decision to rely on the data, including a thorough discussion of the ethical 

deficiencies of the underlying research and the full rationale for finding that EPA met the 

standard in 40 CFR §26.1706 (c) (i.e., that the research is essential to a more stringent regulatory 

action to improve protection of public health).” 

EPA is not planning on using the other results from the research in EPA’s risk assessment based on the 

following: 1) the t-shirt dosimetry data likely underestimate TCVP exposures to children and result in a 

negligible impact on estimated risks 2) pharmacokinetic data are lacking to reliably quantify exposures 

based on the urinary measures of the TCVP metabolite, 2,4,5-trichloromandelic acid (TCMA) and 3) the 

dog plasma ChE activity measures are not human relevant.

EXECUTIVE SUMMARY:

Davis et al., 2008, investigated the exposures to TCVP that could occur in children and adults from the 

use of a TCVP-containing collar on pet dogs. A single product was tested, Hartz Mountain Ultimate Flea 

Collar, which is composed of 14.55% TCVP. Two separate studies were conducted with the test product 

as a part of the journal article. Both were conducted in Oktibbeha County, Mississippi, with volunteer 

households having pet dogs. 

Study 1: The first study was about 4 months (112 days) and it evaluated the time course of TCVP residue 

dissipation using a transferable residue collection method published by Boone et al., 20012, which at the 

time of the study was the scientific standard for collecting residues that could be transferred from treated 

pets. In this method technicians use white cotton gloves to pet a treated dogs’ fur in a rigorous, 

reproducible manner. Twenty three dogs of different breeds and weights were treated with a TCVP flea 

collar in study 1. Dogs were rubbed by technicians continuously for a 5 minute period with use of cotton 

glove following a defined protocol that required contact with 1) the neck with collar, 2) the neck without 

collar) and 3 along the tail region for each dog. Study 1 also analyzed plasma cholinesterase (ChE)

activity from blood samples taken from each dog at the same time as the rubbing samples. Pre-- and postcollar application samples were collected for the evaluation of residue transfer to gloves and the dogs 

blood ChE activity over the course of the 112 day sampling period. 

Significant increases in transferable TCVP residues were observed compared to pretreatment 

concentrations as would be anticipated. In study 1, transferable residues from all three sampling locations 

decreased (86% decline) throughout the 112 days following a peak at day 7 post-collar application, 

24,000 ± 4,000 μg/glove over the collar. Similar trends were also observed for residues characterized as 

around the neck without the collar in place and in the tail region where there were 94% and 71% 

decreases, respectively. Mean glove residues for all sampling times were 14,300 μg/glove over the collar, 

4,300 μg/glove on the neck with the collar removed, and 130 μg/glove in the tail region. 

No significant changes in dog plasma ChE activities from pretreatment levels were observed except for 

one increase of 10% at 84 days post-collar application.

 2 Boone J.S, Tyler J., and Chambers J.E., Transferable residues from dog fur and plasma cholinesterase inhibition in 

dogs treated with a flea control dip containing chlorpyrifos. Environmental Health Perspectives. 2001: 109(11): 

1109-1114. 
Page 4 of 14

Study 2: The second, subsequent study was conducted on the basis that results from study 1 indicated that 

TCVP residues peaked and then suddenly dropped within 3 weeks of collar placement. Therefore, the 

second study was conducted over a 3 week (21 day) period. The research plan included additional 

transferable residue measures, but it also included human biomonitoring of the TCVP metabolite, 2,4,5-

trichloromandelic acid (TCMA), in urine of adults and children. In addition, potential TCVP exposures 

were measured using dosimetry to quantify the potential exposures of children (i.e., cotton t-shirts).

In study 2, TCVP residues obtained over the collar and around the neck without the collar in place 

decreased (30% decline) from 5 to 12 days post-collar application, while residues obtained from the tail 

region remained fairly constant (81 μg/glove at 5 days and 82 μg/glove at 12 days). The peak 

transferable residues collected over the collar at 5 days post-collar application were of a similar 

magnitude to those observed in study 1. Mean residues (for all gloves analyzed) post-collar application 

were 19,000 μg/glove over the collar, 8,000 μg/glove on the neck with the collar removed, and 80 

μg/glove in the tail region. 

The average amount of TCVP residues detected on children’s t-shirts on sampling days 7-11 post-collar 

application was 1.8 ± 0.8 μg/shirt, with no significant differences among the sampling days. These 

exposures were significantly greater than the mean pre-treatment residue of 0.03 ± 0.006 μg/shirt as 

would be expected. 

Urine samples collected from children generally contained more urinary TCMA than that from the adults 

with significant differences between the ages occurring on only 1 of the 5 sampling days (day 11). No 

significant differences in urinary TCMA concentrations were observed among the adults or among the 

children throughout the study. The ranges of TCMA concentrations were large; 1.4 - 582 ng/ml urine for 

adults, and 2.1 - 1,558 ng/ml urine in children. The urinary TCMA concentrations were all adjusted for 

creatinine content; however, there were no differences in outcomes and, as a result, reported values were 

unadjusted. 

No significant correlations were identified among t-shirt TCVP residues, the amount of time spent with 

treated dogs, and urinary TCMA concentrations. 

As described in the Introduction, the Agency is currently planning to use the Davis et al paper only as a 

source of transferable pet fur residue data in order to complete a residential post-application exposure and 

risk assessment for adults and children who could be exposed from contact with dogs wearing TCVP pet 

collars. EPA is not planning on using the other results in risk assessment based on the following: 1) the 

t-shirt dosimetry data likely underestimate TCVP exposures to children and result in a negligible impact 

on estimated risks 2) pharmacokinetic data are lacking to reliably quantify exposures based on the urinary 

measures of the TCVP metabolite, TCMA, and 3) the dog plasma ChE activity measures are not human 

relevant.

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Material: The test material used is the TCVP pet collar, Hartz Control Ultimate Flea Collar 

(EPA Reg. No.: 2596-139) which contains 14.55% active ingredient. 
Page 5 of 14

2. Relevance of Test Material: The test material used is a pet collar product that was in active usage 

at the time of the conduct of the study. The journal article reports that the product was purchased 

from a local department store. 

B. STUDY DESIGN

The study was conducted (two separate studies/parts) to investigate the exposures to TCVP that could occur 

in children and adults from the use of a TCVP-containing collar on pet dogs. The evaluation of transferable 

residues was conducted by means of Mississippi State technicians rubbing the back of treated dogs at 3 

locations: at the neck with the collar in place, at the neck with the collar removed, and near the base of the 

tail. Children’s potential exposure to the TCVP pet collar were estimated by passive dosimetry (t-shirts)

worn for at least 4 hours at selected time points following collar application. TCVP exposures were also 

measured by means of the collection and analysis of adult’s and children’s urine for the TCVP metabolite, 

TCMA. 

1. Site Description: Study 1 and 2 were both conducted in Oktibbeha County, Mississippi, with 

volunteer households having pet dogs. 

2. Animal(s) Monitored: The dogs which were monitored included a variety short and long hair breeds 

and ranged from 8-85 lbs. All dogs were healthy adults and were of both genders; no pregnant 

females were used. All procedures were approved by the Mississippi State University Animal Care 

and Use Committee. The care and use of dogs were in accordance with the Guide for Care and Use 

of Laboratory Animals (1996). The dogs were used during both studies were monitored by the 

project veterinarian. 

3. Physical State of the Formulation as Applied: Ready-to-use pet collar formulation3

. Hartz Control 

Ultimate Flea Collar - 14.55% active ingredient (TCVP); EPA Reg. No.: 2596-139. 

4. Application Rates and Regimes: The Davis study reports that the amount of active ingredient in the 

TCVP pet collars used was 4,800 mg of active ingredient. The pet collar product used in the Davis 

study, EPA Reg. No. 2596-139 - 14.55% TCVP. No information was provided as to how the pet 

collar was applied to the dogs, or whether any remaining length of the collar was removed following 

fitting. 

5. Sampling Procedures For All Media:

 

Transferable Residues 

Method and Equipment: Volunteer technicians from the College of Veterinary Medicine, Mississippi 

State University, Mississippi, rubbed in marked areas on the dogs with use of a clean, cotton glove for a 

continuous 5 minute period as previously described in the Chambers et al., 20074

, publication. The 

 3 There is uncertainty as to whether the TCVP pet collar, which remains a currently registered product, is a dust or 

liquid formulation. That is, whether TCVP releases from the pet collar matrix onto the treated animal as a liquid or 

dust form. The Agency intends to seek additional information from the product registrant to address this 

uncertainty. 

4 Janice E. Chambers, J. Scott Boone, M. Keith Davis, John E. Moran, and John W. Tyler. Assessing transferable 

residues from intermittent exposure to flea control collars containing the organophosphate insecticide chlorpyrifos. 

Journal of Exposure Science and Environmental Epidemiology (2007) 17, 656-666. 
Page 6 of 14

gloves used for rubbing were 100% cotton. Each glove was washed once with laundry detergent, and 

three times without detergent, pre-extracted for 8 hours with methylene chloride, dried, and stored in glass 

jars that were washed and pre-rinsed three times with water and acetone. All samples were collected by 

technicians in the participants’ homes. 

Sampling Procedure: Dogs were rubbed in a marked 10 x 4 inch area with cotton gloves for a continuous 

5 minute period. Three samples were taken in the following order to avoid cross contamination: 1) near 

the base of the tail 2) at the neck with the collar removed and 3) at the neck with the collar in place, 

rubbing over the collar. Rubbing was with firm pressure in both anterior and posterior directions (backand-forth motions), but not one that would cause discomfort to the dog. The gloves were then inverted 

and removed and placed into a clean, labeled glass jar. 

Times of Sampling: For study 1, rubbing samples were obtained prior to collar placement (day 0) and at 4 

hours, and 3, 7, 14, 28, 56, 84, and 112 days post-collar application, and for study 2, prior to collar 

placement, and again at 5 and 12 days following collar placement. 

Surface Area Sampled: An individual sample was acquired for a marked 10 x 4 inch area (40 square 

inches) of each of the following: 1) near the base of the tail 2) at the neck with the collar removed and 3) 

at the neck with the collar in place.

Sampling Time: For each of the 3 samples acquired from each dog, including the tail and two neck 

samples, a 5 minute rubbing phase was repeated. 

Replicates per Sampling Time Period: For study 1, 23 dogs were each sampled 3 separate times (tail and 

two neck samples) per dog over 9 time periods (prior to collar placement and at 4 hours, and 3, 7, 14, 28, 

56, 84, and 112 days post-collar application) for a total of 69 samples per time period and a grand total of 

621 individual samples. For study 2, 22 dogs were each sampled 3 times per dog over 3 time periods 

(prior to collar placement, and again at 5 and 12 days post-collar application) for a total of 66 samples per 

sample time period and a grand total of 198 samples. 

Plasma Cholinesterase Assay Phase (Study 1)

Method and Equipment: Blood samples were collected from each dog during each rubbing sample. These 

samples were centrifuged after collection to obtain plasma, which was stored at 4°C overnight, and ChE 

activity was determined within 24 hours after collection. No further information was provided regarding 

sample collection.

Sampling Procedure and Timing: Blood samples were taken from each dog at the same time as rubbing 

samples. Samples were collected for study 1 prior to collar placement and at 4 hours, and 3, 7, 14, 28, 56, 

84, and 112 days post-collar application, and for study 2 prior to collar placement, and again at 5 and 12 

days post-collar application. 

T-Shirt Study Phase (Study 2)

Method and Equipment: Child participants (ages and genders were not reported) were supplied with new, 

laundered cotton t-shirts to wear on each pre- and post-collar application. The t-shirts were prepared in 

the same manner as described for the gloves, except for the methylene chloride step which was not
Page 7 of 14

conducted. Each child was instructed no to alter his/her normal behavior with respect to the dog. At the 

end of each sampling day, a 100 cm2 section from the chest area was cut out and used for extraction of the 

t-shirt. These sections were placed into a solvent-washed glass bottle for subsequent extraction. This 

region was used because it was indicated it is the most likely region of the shirt to contain residues from 

the child’s interaction with the dog. 

Sampling Procedure: Children were instructed to wear the supplied t-shirts on sampling days for a 

minimum of 4 hours. T-shirts were collected by researchers at the end of each sampling day for analysis. 

Time of Sampling: T-shirts were worn and collected on the day before treatment (collar application) and 

on each of the day 7-11 post-collar placement. Each t-shirt was worn during the afternoon and evening of 

the sampling day for an average 4-hour period. 

Urine Sampling Phase (Study 2) 

Method and Equipment: Urine samples were collected from children wearing t-shirts and from one adult 

in the same household and analyzed for the TCVP metabolite, TCMA. Because TCMA was unable to be 

prepared due to a lack of commercial starting material, the structurally similar 2,3,6-trichloromandelic 

acid analog was synthesized from 2,3,6-trichlorobenzaldehyde and used as an internal standard.

Sampling Procedure and Timing: First morning urine samples (entire void) were collected prior to the 

day of treatment and then again on each of days 8-12 post-collar placement. 

6. Sample Handling

Gloves (Studies 1 and 2) and T-Shirts (Study 2) Analysis

Following sampling, the gloves were placed into a clean, labeled glass jar and the t-shirts were placed into 

a solvent-washed glass bottle for subsequent extraction. During method development, gloves and t-shirts 

used for petting the dogs were quantified and various concentrations were applied to different gloves to 

check for recovery rates and extraction parameters. After collection, the sample and spiked gloves and tshirts were stored at 4°C until extraction. 

Plasma ChE Assay (Study 1) 

Blood samples were collected from each dog at each sample time. These samples were centrifuged after 

collection to obtain plasma, which was stored at 4°C overnight until ChE activity was conducted (within 

24 hours). 

Urine Sampling (Study 2) 

Following collection, urine samples were brought to Mississippi State laboratories. There a subsample of 

3 ml was removed from each sample and both the original and subsamples were placed in an upright 

freezer and held at -20°C. The subsamples were shipped frozen for analysis. 
Page 8 of 14

7. Analytical Methodology

Gloves (Studies 1 and 2) and T-Shirts (Study 2) Analysis

Extraction Method(s): The entire glove was analyzed for TCVP. For the t-shirts, a 100 cm2 section 

from the front chest area of each shirt was cut out and used for extraction, as this region is the most likely 

region of the shirt to contain residues from the child’s interaction with the dog. After sampling, the 

gloves and t-shirts were extracted with acetone using an Accelerated Solvent Extractor (ASE) by Dionex. 

The operating conditions were heat for 5 minutes at 75◦C and 1500 psi; static for 2 minutes; flush 50% of 

volume; static for 2 minutes; purge with nitrogen for 150 seconds; and a final purge for 60 seconds. For 

every 20 samples, three spiked gloves were included at the time of sampling and extracted with the 

samples. After the gloves were extracted using an ASE, the extract was evaporated under a nitrogen 

stream using an N-EVAP (1–3 mL), transferred to graduated test tubes, and adjusted to 10 mL with 

acetone.

Detection Method(s): All samples were analyzed on an HP5890 gas chromatograph equipped with an 

electron capture detect (ECD). Separation of the analyte was achieved by using an RTX-5 Amine column 

(30m_0.53mm inside diameter/1.0 m film thickness, Restek, Bellefonte, PA, USA). The oven 

temperature was ramped at a rate of 3°C/min from 205°C to 225°C and held for 5 minutes, followed by a 

second ramp of 5°C/min to a final temperature of 290°C. The ECD injector and detector temperatures 

were set at 290°C and 325°C, respectively. 

Method validation: The limit of detection (LOD) was 2 ppb and the limit of quantification (LOQ) was 6 

ppb. 

Plasma ChE Assay (Study 1) 

Extraction Method(s): Cholinesterase (ChE) determinations were done using the following combinations

of inhibitors and substrates to investigate plasma enzymes: butyrylcholinesterase (BChE) using 

butyrylthiocholine (BTCh) iodide as a substrate and tetraisopropyl pyrophosphoramide (iso-OMPA) as an 

inhibitor, and acetylcholinesterase (AChE) using acetylthiocholine (ATCh) iodide as a substrate and 

eserine sulfate as an inhibitor. All assays were a modification of the procedure described in Chambers and

Chambers (1989), which is based on the spectrophotometric method of Ellman et al. (1961). Protein 

concentration was determined by the method of Lowry et al. (1951) for standardization.

Urinalysis (Study 2) 

Urine samples were thawed at ambient room temperature, vortexed, and aliquoted (1.0 ml) to 1.8 ml glass 

autosampler vials, and fortified with the 2,3,6-trichloromandelic acid internal standard. Sufficient volume 

of concentrated (12 N) hydrochloric acid (typically 0.1 ml) was then added to each sample to achieve a 

final volume dilution of 1N. Each vial was then sealed with a PTFE/rubber-lined aluminum crimp cap, 

vortexed to mix, and placed in a water bath maintained at 80°C for a period of 1 hour, after which they 

were allowed to cool to ambient temperature before being analyzed.

Samples were analyzed by reverse-phase liquid chromatography mass spectroscopy using an Applied 

Biosystems/MDS SCIEX API 3000 triple quadrupole mass spectrometer and AnalystTM version 1.4 

software. Analyses were performed in the negative ionization mode using Turbo IonsprayTM ionization. 

Two MS/MS transitions were monitored: 253-209 and 255-211, which are associated with a loss of

CO2 from the corresponding molecular ions [M-H] at 253 AMU (35Cl) and its associated 37Cl at 255 AMU. 

Quantitation was by integration of peak area for the single fragmentation at m/z 209.
Page 9 of 14

Separation of the two analytes (TCMA and 2,3,6-trichloromandelic acid) from each other was achieved 

by reverse-phase C-18 liquid chromatography. The target analyte, TCMA, was identified by monitoring 

the two MS/MS transitions previously mentioned and observing the presence of a similar ion-intensity 

ratio of approximately 1:1 (for the fragment masses 209:211) as observed for the internal standard. 

Because of an interfering, co-eluting component detected in a large number of urine hydrolysates, further 

identification of TCMA at the retention time of 13.9 minutes was confirmed by its anticipated longer 

retention time relative to that of the 2,3,6-trichloromandelic acid internal standard, which eluted at 12.4 

minutes. The longer retention of TCMA can be attributed to the differences in polarity (TCMA is less 

polar than the 2,3,6-trichloromandelic standard).

A typical injection sequence for sample analysis involved an initial acetonitrile solvent blank followed by 

a 20 ng/ml internal standard quality control (prepared in acetonitrile) and 10 experimental samples. This 

sequence was then repeated until all samples were analyzed.

Method Validation: The linear response of the liquid chromatography mass spectroscopy was verified by 

sequential injections of calibrants prepared in acetonitrile at nominal concentrations. Quantitation of the 

target analyte (TCMA) was done by comparing the peak areas of the internal standard (2,3,6-

trichloromandelic acid) and TCMA. The LOD was 0.63 ppb and LOQ was 2 ppb. Samples that were 

below the LOQ were assigned the value of the LOQ for the purpose of sample analysis. Two percent of 

the adult samples were below the LOQ, and 10% of the children’s samples were below the LOQ. Except 

for pretreatment samples, none of the samples were below the LOD.

8. Quality Control

Please note that the following quality control information presented are drawn directly from the 

publication. No further information were provided. 

Gloves (Studies 1 and 2) and T-Shirts (Study 2) Analysis

During method development, gloves and t-shirts used for petting the dogs were quantified and various 

concentrations (0.5–2500 mg) were applied to different gloves to check for recovery rates and extraction 

parameters. The limit of detection LOD was 2 ppb and the limit of quantification was 6 ppb. The percent 

recovery obtained during these tests ranged from 85% to 102%, with a mean of 95%. 

Urinalysis (Study 2) 

The accuracy of the method was evaluated by examining the relative error of triplicate concentrations for 

a selected analysis (family no. 110). Relative error was calculated as (experimentally determined 

concentration-theoretical concentration) x 100 ÷ theoretical concentration. Magnitudes of relative errors 

were less than 17% at the lowest concentration, less than 12% at the midlevel concentration, and less than 

5% at the highest concentration. The percent recoveries were in the range of 88.5% to 116.3%, with a 

mean of 102.1%.

9. Statistics

Analysis of variance calculations for glove and t-shirt residues were performed with the GLM procedure 

of the SAS® System for Windows, Version 9.1, using the 0.05 level of significance. When significant 

differences between treatment groups were found, means were separated using the least significant 

difference test. The clinical importance of the differences was assessed using confidence intervals 
Page 10 of 14

(Braitman, 1991). Each data set (gloves, t-shirts and adjusted urine) was analyzed separately for each 

collar or age group using one-way analysis of variance for a randomized complete block design 

(household is the blocking factor). The glove and t-shirt data were also examined for the presence of 

statistically significant correlations using Spearman’s correlation coefficient. The calculation was 

performed using the CORR procedure of the SAS System for Windows, Version 9.1 (SAS Institute Inc., 

Cary, NC, USA) at 0.05 level of significance.

Urinary TCMA data were statistically analyzed to compare the amount of TCMA between adults and 

children. The analyses were implemented in SAS System for Windows, Version 9.1.3 using PROC 

MIXED with the REPEATED statement and the autoregressive order one (AR 1) covariance structure. 

The AR 1 was a covariance structure with the desired property of correlations being larger for nearby 

time than for far-apart times, which was a better fit in this statistical model than the compound symmetric 

or unstructured covariance.

II. RESULTS

Rubbing Glove Study Phase (Studies 1 and 2)

Significant increases in transferable TCVP residues were observed from cotton gloves used to pet dogs 

compare to pretreatment concentrations. In study 1, transferable residues from all three sampling 

locations decreased (86% decline) throughout the 112 days following a peak at day 7 post-collar 

application, 24,000 ± 4,000 μg/glove over the collar. Similar trends were also observed in detectable 

residues around the neck without the collar in place and in the tail region where there were 94% and 71% 

decreases, respectively. Mean glove residues for all sampling times were 14,300 μg/glove over the collar, 

4,300 μg/glove on the neck with the collar removed, and 130 μg/glove in the tail region. 

In study 2, TCVP residues obtained over the collar and around the neck without the collar in place 

decreased (30% decline) from 5 to 12 days post-collar application, while residues obtained from the tail 

region remained fairly constant (81 μg/glove at 5 days and 82 μg/glove at 12 days). The peak 

transferable residues collected over the collar at 5 days post-collar application were of a similar 

magnitude to those observed in study 1. Mean residues (for all gloves analyzed) post-collar application 

were 19,000 μg/glove over the collar, 8,000 μg/glove on the neck with the collar removed, and 80 

μg/glove in the tail region. 

Pet fur residue transfer studies conducted around the time of the Davis study typically used a repeated 

petting motion to a single sample collection area for a defined number of strokes (e.g., 5 to 10 strokes per 

animal per sample time point) and/or for a defined period of time. (e.g., 5 to 10 minutes per sampling 

period). Therefore, the Davis study collection methodology is consistent with other pet fur residue 

transfer studies conducted around the same time. Further, the Davis study sampled from the neck area, as 

well as the tail, and is not expected to underestimate residue transfer when compared to other residue 

transfer studies conducted around the same time which typically sampled only from one area of the body.

The analytical methods used for analysis of the rubbing glove phase of the study are scientifically valid. 

Each glove was washed once with laundry detergent, and three times without detergent, pre-extracted for 

8 hours with methylene chloride, dried, and stored in glass jars that were washed and pre-rinsed three 

times with water and acetone. Gloves were stored after sampling at 4 ̊C for an undisclosed period of time 

until analysis. Storage at 4°C until analyzed is sufficient assuming samples were not stored for more than 

2 weeks. Samples were extracted using ASE, which is a technique known to be successful for extraction 

of pesticides, including organophosphates, from most matrices. The TCVP standard used was of 

acceptable purity. The study authors verified that the method extraction conditions were sufficient since 
Page 11 of 14

they obtained a recovery ranging from 85% to 102%, after spiking blank gloves with different amounts of 

TCVP. Glove samples were analyzed on an HP5890 gas chromatograph equipped with an ECD for the 

detection of TCVP. Detection of TCVP using GC-ECD is acceptable and achieves the required detection 

limits. 

Study authors performed analysis of variance method for glove and tee shirt residues with the GLM 

procedure of the SAS System for Windows, version 9.1, using 0.05 level of significance. The glove data 

were also examined for the presence of statistically significant correlations using Spearman’s correlation 

coefficient. The calculations were performed using the CORR procedure of the SAS System for 

Windows, version 9.1 at 0.05 level of significance.

The study authors did not specify whether any transformation of residue data was conducted before 

performing analysis of variance approach. Although MIXED procedure in SAS using AR(1) covariance 

matrix was applied for modeling TCMA data it was not explained in the article why the same procedure 

with the similar correlation structure was not used for modeling glove exposure data. It should be noted 

that MIXED procedure in SAS offers a richer selection of variance-covariance structures for modeling 

longitudinal data than GLM procedure. For example autoregressive covariance structure cannot be 

implemented using PROC GLM but it can be implemented using PROC MIXED. EPA is proposing 

using only the estimate of mean glove transferable residues for all sampling times. Although the selection 

of variance-covariance structure will impact the estimation of standard error, test statistic, and p value, it 

will not significantly influence the estimation of overall mean from all sampling times.

For purpose of risk assessment, the mean transferable residue measures from study 1 and 2 are used to 

quantify a fraction of application rate applied (FAR) value. The FAR value was determined by dividing 

the sum of the mean residues measured from the fur over the collar and from the tail region, by the 

amount of active ingredient in the pet collar, 4,800 mg. The transferable residue measures from the fur of 

the neck with the collar removed was not summed with the other measures since the collection of residues 

under the collar is inconsistent with TCVP pet product label language that directs users to secure the pet 

collars in place. For study 1, an FAR value of 0.0030 (0.3%) results from the mean residues (112 days) 

reported from study 1 [where (14.3 mg + 0.013 mg)/ 4,800 mg = 0.0030]. For study 2, an FAR value of 

0.0040 (0.4%) results from the mean residues (12 days) reported from study 2 [where (19 mg + 0.08 mg)/ 

4,800 mg = 0.0040]. These FAR inputs are then used as described in EPA’s 2012 Standard Operating 

Procedures (SOPs) for Residential Pesticide Exposure Assessment5 for estimation of post-application 

risks from exposures to adults and children from contact with TCVP treated dogs. 

Plasma Cholinesterase Assay Phase (Study 1)

No significant changes in dog plasma ChE activities from pretreatment levels were observed except for 

one increase of 10% at 84 days post-collar application; this statistical difference was not view as 

biologically significant. 

The analytical methods used for determination of dog plasma ChE inhibition were conducted adequately. 

The ChE determinations used a combination of inhibitors, butyrylcholinesterase (BChE) and 

acetylcholinesterase (AChE), both of which are contained in dog plasma and are inhibited by OPs, 

including TCVP. The assay methods used measured the hydrolysis of enzyme-specific substrates and 

separated out the relative activities of the separate enzymes by using specific inhibitors of each. This 

method gives a true measure of inhibition of each cholinesterase.

 5 Available: http://www2.epa.gov/pesticide-science-and-assessing-pesticide-risks/standard-operating-proceduresresidential-pesticide
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T-Shirt Study Phase (Study 2)

The average amount of TCVP residues detected on children’s t-shirts on sampling days 7-11 post-collar 

application was 1.8 ± 0.8 μg/shirt, with no significant differences among the sampling days. Transferable 

residues were significantly greater than the mean pre-treatment residue of 0.03 ± 0.006 μg/shirt. 

The use of passive clothing dosimetry (t-shirts) is consistent with post-application exposure monitoring 

guidelines, for measuring dermal exposures via passive dosimetry. The guidelines recommend wholebody dosimetry for measurement of residential indoor exposures such as evaluated in the Davis study. 

While it would have been preferred that the entire t-shirt be analyzed for transferable residues of TCVP, 

the measurement of the 100 cm2 section is comparable to patch dermal dosimetry which described by the 

guidelines as an acceptable method of residue collection. However, there are clear limitations with this 

approach. By sampling only one isolated section of the t-shirt, it is possible that other areas of the t-shirt 

that may have been more highly exposed or subject to greater residue transfer. If the full t-shirt, or more 

100 cm2 sections of each individual t-shirt had been analyzed, it is likely that greater residue transfer 

would have been measured. Therefore, with use of this approach it is likely that exposures to children 

wearing the t-shirts could have been underestimated. 

Further, the resulting measures from the t-shirt samples, a mean of 1.8 μg/shirt, is well below the mean 

values reported for the residue transfer phase of the study, 14,300 μg/glove over the collar (study 1) and 

19,000 μg/glove over the collar (study 2). Accordingly, the consideration of the t-shirt data results in a 

negligible impact on the risks from adult and child exposures to pets treated with a TCVP pet collar.

The analytical methods used for analysis of the t-shirt study phase samples are the same as those 

described above (Section II, Results) for the rubbing glove phase of the study which were determined to 

be scientifically valid. The statistical methods used for t-shirt sampling (also the same as described for 

the rubbing glove phase) resulted in a determination that the MIXED procedure in SAS, which was used 

for urinalysis, offers a richer selection of variance-covariance structures for modeling longitudinal data 

than the GLM procedure of the SAS System for Windows, version 9.1, used. This determination is of no 

consequence since EPA does not intend to rely on the t-shirt data for assessment of risks due to the 

limitations described above. 

Urine Sampling Phase (Study 2) 

Urine samples collected from children generally contained more urinary TCMA than that from the adults 

with significant differences between the ages occurring on only 1 of the 5 sampling days (day 11). No 

significant differences in urinary TCMA concentrations were observed among the adults or among the 

children throughout the study. The ranges of TCMA concentrations were large; 1.4 - 582 ng/ml urine for 

adults, and 2.1 - 1,558 ng/ml urine in children. The urinary TCMA concentrations were all adjusted for 

creatinine content; however, there were no differences in outcomes and, as a result, reported values were 

unadjusted. No attempt was made by researchers to estimate an internal dose based upon the urinary 

TCMA measures. 

The study authors stated that the TCVP metabolite TCMA cannot be readily prepared due to lack of 

commercial starting material. Instead, the structurally similar 2,3,6-trichloromandelic acid analog was 

synthesized from 2,3,6-trichlorobenzaldehyde and used as an internal standard which was added to urine 

samples. TCMA was determined in urine using LC-MS/MS. Quantitation of the TCMA was done by 

comparing the peak areas of the internal standard (2,3,6- trichloromandelic acid) and TCMA. In this case,
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TCMA (2,4,5-isomer) was quantitated against a calibration curve of another compound (2,3,6-isomer). 

Although both compounds are very close in structure, their responses in the mass spectrometer may be 

slightly different. TCMA eluted at a longer retention time (RT), 13.9 minutes, as compared to the 2,3,6-

isomer internal standard which eluted at 12.4 minutes. The difference in retention time when analyzed by 

MS is expected, although quite little to negligible. The difference is also expected to be insignificant 

since the amounts of TCMA equivalent detected vary from one volunteer to another and that a small error 

in the calculated amount may be accepted. For more accuracy the study authors could have referred to 

TCMA detected as TCMA equivalent. Regardless, the detection method is acceptable for use.

Urinary TCMA data were statistically analyzed to compare the amount of TCMA between adults and 

children. The analyses were implemented in SAS System for Windows, version 9.1.3 using PROC 

MIXED with the REPEATED statement and autoregressive order one (AR 1) covariance structure. The 

authors stated that the AR(1) was a covariance structure with the desired property of correlations being 

larger for nearby time than for far apart times, which was a better fit in this statistical model than the 

compound symmetric or unstructured covariance.

 

While the urine sampling phase of the study was conducted in a manner scientifically valid, to rely on 

these data would require on a full understanding the pharmacokinetics of TCVP, including an adequate 

justification for reliance on the metabolite, TCMA, to document the time course of uptake, metabolism, 

and the proportion of TCVP converted to TCMA following dermal and incidental oral exposures to a 

controlled dose of TCVP. Without this information, EPA cannot accurately back-calculate a TCVP dose 

from exposures to TCVP treated pets. 

III. DISCUSSION AND CONCLUSIONS

Rubbing Study Phase (Studies 1 and 2)

For purpose of risk assessment, EPA is proposing that the transferable residue data from the Davis study 

be used to assess the potential from residential post-application exposures to adults (non-cancer and 

cancer) and children (non-cancer only) from actively registered TCVP pet collars. The transferable 

residue data have been determined to be acceptable for regulatory purposes given that the sample 

collection method was the scientific standard at the time the study was conducted and all phases of the 

sample collection, analysis, and recoveries appear adequate for those samples. The residue transfer data 

resulting from the Davis study (both study 1, 112 day mean and study 2, 12 day mean) result in greater 

FAR values that with the best exposure data currently available. 

T-Shirt Study Phase (Study 2)

For purpose of risk assessment, EPA does not intend to rely on the results of the t-shirt study phase data. 

While the use of passive clothing dosimetry is consistent with post-application exposure monitoring 

guidelines there are clear limitations with this approach. The t-shirt sampling method employed, 

collection and analysis of 100 cm2 sections only, likely underestimates TCVP exposures to children 

wearing the t-shirts. Further, inclusion of the t-shirt data would result in a negligible impact on the risks 

from adult and child exposures to pets treated with a TCVP pet collar. 

Urine Sampling Phase (Study 2) 

 

For purpose of risk assessment, EPA does not intend to rely on the results of the urine sampling for risk 

quantitation. To rely on these data would require on a full understanding the pharmacokinetics of TCVP, 

including an adequate justification for reliance on the metabolite, TCMA, to document the time course of 

uptake, metabolism, and the proportion of TCVP converted to TCMA following dermal and incidental 
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oral exposures to a controlled dose of TCVP. Without this information, EPA cannot accurately backcalculate a TCVP dose from exposures to TCVP treated pets. 

Plasma Cholinesterase Phase (Study 1)

While the dog plasma ChE activity analysis was conducted adequately, and the data are relevant for 

determining that no significant changes in dog plasma ChE activities from pretreatment levels were 

observed, these data are not human relevant and, therefore, are not proposed for use in assessing human 

health from TCVP pet collar exposures.