DATA EVALUATION RECORD
ACEPHATE
Study Type: OCSPP Non-Guideline; Cholinesterase Inhibition and Pharmacokinetics in Humans
EPA Contract No. 68HERC22D0017
Task Assignment Form No. 5540-3-008 (MRID 45388301)
Prepared for
Health Effects Division
Office of Pesticide Programs
U.S. Environmental Protection Agency
2777 South Crystal Drive
Arlington, VA 22202
Prepared by:
PB&A/
CSS JV
1151 Lost Creek Boulevard
Primary Reviewer:
Scott D. Studenberg, Ph.D., DABT
Secondary Reviewer:
Michael E. Viana, Ph.D.
Quality Assurance:
Steven Brecher, Ph.D.
Project Manager:
Scott D. Studenberg, Ph.D., DABT
Austin, TX 78746
Signature:
Date:
Signature:
Date:
Signature:
Date:
Signature:
Date:
10/14/2022 ~
10/21/2022
10/25/2022
10/25/2022
This Data Evaluation Record may have been altered by the Health Effects Division subsequent to
signing by PB&A/CSS-JV personnel. Contractor's role did not include establishing Agency
policy.
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Cholinesterase Inhibition and Pharmacokinetics in Humans (2001) / Page 1
ACEPHATE / 103301 OCSPP Non-Guideline
EPA Reviewer: Ruthanne Louden Signature:
Risk Assessment Branch I, HED (7509T) Date:
EPA Secondary Reviewer: Abiy Mohammed Signature:
Risk Assessment Branch I, HED (7509T) Date:
Template version 02/06
DATA EVALUATION RECORD
STUDY TYPE: Cholinesterase Inhibition and Pharmacokinetics - Human;
OCSPP Non-Guideline.
PC CODE: 103301; 101201
TXR#: 0058561
DP BARCODE: D465948
SUBMISSION: 1086785
TEST MATERIALS (PURITY): Acephate technical, 99.0%
SYNONYMS: (XY-dimethyl A-acetylphosphoramidothioate; methamidophos ((%Y-dimethyl
phosphoramidothioate [metabolite])
CITATIONS: Freestone, S., and McFarlane, P. (2001) A single oral dose study with acephate
technical in humans - Report Amendment 2. Inveresk Research, Elphinstone
Research Centre, Tranent, Scotland. Laboratory Project ID: ICR 013072,
March 23, 2001. MRID 45388301. Unpublished.
SPONSOR: Valent USA Corporation, 1333 North California Blvd., Suite 600, P.O. Box
8025, Walnut Creek, CA
EXECUTIVE SUMMARY: In a non-guideline, double blind, placebo controlled,
pharmacokinetic and cholinesterase (ChE) inhibition study (MRID 45388301), fifty (40 male; 10
female) human volunteers were administered a single oral dose of acephate technical
(Lot No. 80121; purity 99.0%) in a hard gelatin capsule at 0.35 (7 males), 0.70 (7 males), 1.00 (7
males and females, respectively), or 1.25 mg/kg (7 males). Three male volunteers at each
treatment level were administered lactose in a hard gelatin capsule (placebo). An additional 3
female subjects were administered placebos for the 1.00 mg/kg treated group. All subjects were
housed in a clinical setting through 72 hours post-dose administration. Clinical signs were
monitored up to 336 hours (Day 14) post-dose and vital signs were monitored up to 24 hours
post-dose. Blood samples for hematology and clinical chemistry evaluations and urine samples for
clinical urinalysis were collected at 24 hours post-dose. Blood samples for plasma and red blood
cell (RBC) ChE activity determinations were collected up to Day 14 post-dose, in addition to
multiple pre-dose collections to determine baseline activity values. Additional blood samples were
collected up to Day 14 post-dose for evaluation of systemic acephate and methamidophos
(metabolite) exposure. Urine collections also were conducted through 48 hours post-dose for
determination of excreted acephate and methamidophos concentrations in urine.
There were no treatment-related effects on clinical signs, vital signs, or clinical pathology
findings.
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The acephate plasma concentration vs. time profiles were consistent with oral dose
administration with no evidence of a significant lag phase. Mean time of maximum plasma
concentration (Tmax) estimates ranged from 1.3-2.7 hours. Acephate concentrations declined
rapidly up to 24 hours post-dose, with mean elimination half-life (ti/2ei) estimates of
4.4-5.4 hours. Mean clearance (CL/F) estimates were generally slow, with values of 77-105
mL/hour/kg. Overall systemic exposure demonstrated generally dose-related increases (slightly
greater than dose proportional). There were no sex-related differences in pharmacokinetic
parameters at 1.00 mg/kg (the single dose level in female subjects).
Plasma concentration data were sparser for the metabolite, methamidophos, but valid Tmax,
maximum plasma concentration (Cmax), and area under the curve (AUCo-t) estimates were
determinable at >0.70 mg/kg acephate, with valid estimates for the remaining parameters at
>1.00 mg/kg acephate. Mean Tmax estimates ranged from 3.3-4.3 hours. Methamidophos
concentrations declined rapidly up to 12 hours post-dose, with mean ti/2ei estimates of 6.7-8.3
hours. Mean CL/F estimates were rapid, with values of 3377-4439 mL/hour/kg. Overall
systemic exposure demonstrated generally less than dose-proportional increases. There were no
sex-related differences in pharmacokinetic parameters at 1.00 mg/kg.
The total recovery of acephate and methamidophos (as acephate equivalents) in urine through
48 hour post-dose ranged from 25% to 62% in males and 12% to 51% in females, with most of
the recovery occurring within the initial 12-hour post-dose period. The fractions of the
administered acephate dose recovered as methamidophos were <1% AD and <2% of the total
recovered dose and were independent of dose or sex. Urine concentrations of methamidophos
(systemic exposure) generally decreased in parallel to acephate at >1.00 mg/kg acephate and
accumulation of methamidophos would not be expected.
Mean plasma ChE activities showed statistically significant decreases (as compared to baseline
values) of 11% in low dose males (0.35 mg/kg) at 12 hours post-dose, and 9-13% at 12, 24, 48,
and 72 hours post-dose in the high dose (1.25 mg/kg) males. Non-statistically significant
decreases were observed at 0.70 (J, 10%) in males at 1 hour and 12 hours post-dose as well. Mean
plasma ChE activities were decreased by 10-13% at 8, 12 and 24 hours post-dose in females
(1.00 mg/kg), these changes were also statistically significant.
For RBC ChE activities, decreases of 7% and 11% were noted in the 1.25 mg/kg males and the
1.00 mg/kg females, respectively, at 12 hours post-dose (both showing statistical significance). A
non-statistically significant decrease of 8% was also observed at 8 hours post-dose in the 1.25
mg/kg males. A 12% non-statistically significant decrease in mean RBC ChE was observed in
the 1.00 mg/kg females at 72 hours post-dose, without statistical significance.
In male subjects, statistically significant decreases in both plasma and RBC ChE activities were
noted only at 12 hours post-dose in the 1.25 mg/kg group. In female subjects, statistically
significant decreases in both plasma and RBC ChE activities were also noted only at 12 hours
post-dose in the 1.0 mg/kg group. All changes in plasma ChE were within the variability of
baseline values at each dose level (with the exception of the 13% change at 12-hours in the 1.25
mg/kg males compared to an 11% CV at baseline at this dose), as well as within the variability of
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placebo group controls. There was less variability in the results for RBC ChE, however, results
were still within variability of baseline values for males, and slightly outside for females (11%
change at 1.0 mg/kg compared to baseline CV of 9%). Due to the variability of the data and no
clear dose response for AChE inhibition, there is currently no intention to use these AChE data
for risk assessment purposes, including future physiologically-based pharmacokinetic (PBPK)
modeling.
Analytical methods for the determination of acephate and methamidophos in human blood and
urine were described and validated. Storage and stability were also verified. The analytical
methods (No. 6696 for human blood/plasma, and No. 6695 for human urine), were validated for
the determination of acephate and methamidophos at a concentration range of approximately 10
to 1,000 ng/mL.
There were several results that fell outside of the method validated range of 10 to 1,000 ng/mL
for both human blood/plasma and urine. Mean urine concentration data for acephate and
methamidophos were outside of the method validated concentration range at the with
exceedances of 1,000 ng/mL for acephate at 0-12 hours (all dose levels, both sexes), 12-24 hours
(all dose levels, both sexes), and 24-48 hours (1.00 and 1.25 mg/kg dose levels (males)). For
methamidophos, concentrations were below the limit of quantification of 10 ng/mL at 24-48
hours for the 0.70 mg/kg (males) and 1.0 mg/kg (females). Mean plasma concentration data for
acephate and methamidophos were also outside of the method validated concentration range of
10-1000 ng/mL at several dose levels and timepoints. For acephate, mean plasma concentrations
were above 1000 ng/mL at 1.0 and 1.25 mg/kg in males at 1, 2, and 4 hours post-dose, and at 1.0
mg/kg in females at 2 and 4 hours post-dose. For methamidophos, mean plasma concentrations
were below 10 ng/mL at 0.70 mg/kg (2, 8, and 12 hours post-dose), 1.0 mg/kg (1 and 12 hours
post-dose), and 1.25 mg/kg (1 and 12 hours post-dose) in males, and at 1.0 mg/kg (1 and 12
hours post-dose) in females.
This study is classified acceptable/non-guideline. Although there were data that fell outside of
the method validated range for concentrations of acephate and methamidophos in plasma and
urine, information from the study is still considered useful for improving predictability of future
PBPK modeling.
COMPLIANCE: Signed and dated Data Confidentiality, GLP Compliance, and Quality
Assurance statements were provided.
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I. MATERIALS AND METHODS
A. MATERIALS
1. Test compounds:
Non-Radiolabeled TGAI:
Description:
Lot No.:
Purity:
CAS No. of TGAI:
Expiration/storage:
Structure:
Acephate technical (two portions)
White powder
80121
99.0%
30560-19-1
August 5, 1999 and July 21, 2000/-20°C
o-CH,
H,C
NH N:
S—CH,
2. Placebo: Lactose (J.M. Loveridege [Batch No. 28811] and Thornton and Ross [Batch
No. 18TJ])
3. Subjects:
Species:
Sex/Age:
Age/weight at administration:
Source:
Housing:
Diet:
Human
Males and females/ 18-50 years old (study criterion)
Placebo: 18-45 years/61.1-81.6 kg (12 males)
0.35 mg/kg: 27-41 years/62.7-94.8 kg (7 males)
0.70 mg/kg: 24-41 years/60.5-81.8 kg (7 males)
1.00 mg/kg: 29-46 years/70.8-83.7 kg (7 males)
1.25 mg/kg: 20-48 years/54.7-78.9 kg (7 males)
Placebo: 26-37 years/59.5-71.1 kg (3 females)
1.00 mg/kg: 20-46 years/51.3-81.6 kg (7 females)
Volunteers registered at ICR and screened within 21 days of dosing
Volunteers were housed in the treatment clinic for the initial 72 hours post-dose.
A standard breakfast was provided prior to the single dose administration. Alcohol
and caffeine consumption were not permitted during the initial 72 hours post-dose.
4. Selection criteria: Inclusion/exclusion criteria, pre-study screening parameters,
restrictions, and actions resulting in removal from the study were detailed in the study report
(MRID 45388301). The study protocol and volunteer information were submitted to the
Independent Ethics Review Committee of Inveresk Research for review and approval prior
to study initiation. The main criteria for inclusion were healthy male or female volunteers
between the ages of 18-50 years old, additional details can be found in Appendix 111 of this
DER.
5. Preparation of study capsules: The test item or placebo were administered in size zero
hard gelatin capsules. The required doses were prepared by direct weighing of the bulk
acephate technical or lactose into capsules based on the screening weights of each individual
study subject. Acephate technical reference capsules analyzed at the end of the study
contained the stated amounts of the test item and placebo capsules did not contain any test
item. No decomposition of the test item was observed during the course of the study.
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B. STUDY DESIGN AND METHODS
1. Group arrangements: Study design details are presented in Table 1. The study report
indicates that the objective of the study was to determine the highest no observed effect
level (NOEL) and/or the lowest observed effect level (LOEL) of acephate causing a slight
inhibitory effect on blood cholinesterase concentration in human volunteers. The NOEL is
defined as the highest dose tested at which no inhibition of plasma and red blood cell
cholinesterase activity occurs. The study was conducted as a double-blind, placebo-
controlled study. The body weight for each subject was determined prior to capsule
preparation. Dose administration was staggered with each treatment level evaluated in a
single subject prior to administration to the remaining subjects per level (n = 6). The group
of female volunteers were evaluated in the final session (Session 6) with all seven subjects
as the 1.00 mg/kg dose level had been previously evaluated in male volunteers. The in-life
portion of the study was initiated on June 9, 1999 and completed on August 23, 1999. A
subject number was assigned for all subjects who qualified for the study in accordance with
the inclusion/exclusion criteria. Subject numbers were assigned sequentially using a
computer generated randomization starting at 001.
TABLE 1. Dosing and sampling schedule after a single oral dose of acephate technical up to 1.25 mg/kg to human
volunteers.a
Session
Sex
Number of subjects
Sampling Schedule
0
mg/kg
0.35 mg/kg
0.70 mg/kg
1.00 mg/kg
1.25 mg/kg
1
Male
1
1
Baseline plasma and red blood cell
(RBC) cholinesterase concentrations
were determined three times from
Day -10 to -3, as well as Days -2,
-1, and 30 minutes prior to dose
administration. Blood samples were
collected post-dose at 1, 2, 4, 8,12,
24, 48, and 72 hours (in clinic) and
on Days 7 and 14 post-dose. Urine
samples were collected from
-12-0 hours pre-dose and 0-12,
12-24, and 24-48 hours post-dose.
2
Male
2
6
1
3
Male
3
6
1
4
Male
3
6
1
5
Male
3
6
6
Female
3
7
a Data were obtained from Table 1 on page 26 and pages 38-42 of MRID 45388301.
2. Dosing, safety evaluations, and sample collection
a. Dose selection: Each dose was evaluated for safety in a single subject (lead dose design)
prior to administration to the remaining volunteers for that dose level and evaluation of the next
highest dose level. The acephate dose levels were selected based on previous animal and human
studies with acephate, including rat metabolism, subchronic and chronic studies in rats,
subchronic study in primates, subchronic oral study in humans, a production plant worker
exposure study, genetic toxicity studies, acute and subchronic neurotoxicity studies in rats,
delayed neurotoxicity study in hens, carcinogenicity studies in rats and mice, and reproductive
toxicity studies.
b. Dosing: The capsules (placebo or test item) for each dose session were prepared based on
the individual body weight of each scheduled test subject. On the morning prior to
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administration, the test subjects were fed a standard breakfast. The subjects were
administered their respective capsules approximately five minutes after completion of
breakfast with 150 mL water to aid swallowing of the capsule.
c. Safety evaluations: Blood pressure and heart rate was determined pre-dose and at 2, 4, 8,
and 24 hours post-dose. Oral temperature was determined pre-dose and at 2, 4, and 24 hours
post-dose. Electrocardiography (ECG) was determined pre-dose and 2, 4, 8, and 24 hours
post-dose by 12-lead ECG. Single-channel continuous ECG monitoring was also conducted
from 30 minutes pre-dose to 4 hours post-dose.
d. Sample collection
i. Blood: Blood collection for hematology and clinical chemistry evaluations was conducted
pre-dose (30 minutes prior to dose administration) and at 24 hours post-dose. Samples were
collected into EDTA-coated (3 mL) or heparinized (5 mL) tubes, respectively.
Baseline plasma and red blood cell (RBC) cholinesterase (ChE) concentrations were
determined from blood collections (4.5 mL) at pre-screening, three times from Day -10 to
-3, and Days -2, -1, and 30 minutes prior to dose administration. Blood samples were
collected at 1, 2, 4, 8, 12, 24, 48, and 72 hours (in clinic) and on Days 7 and 14 post-dose.
Daily blood samples (excluding all samples on Day 1, except the 1 hour post-dose
collection) were collected in the morning and at approximately the same time each day. The
blood samples were collected into EDTA and centrifuged to separate the plasma and RBC
fractions. Samples up to 4 hours post-dose on Day 1 were stored at -20°C prior to transfer
on wet ice for plasma/RBC ChE analyses that same day. Further samples were stored at
-20°C overnight.
Additional blood samples (10 mL) were collected into lithium heparin at 0 hours (pre-dose)
and at 1, 2, 4, 8, 12, 24, 48, and 72 hours (in clinic) and on Days 7 and 14 post-dose,
centrifuged to harvest plasma, and stored at approximately -80°C for analysis of acephate
and methamidophos (metabolite) concentrations.
Repeat samples were collected from four subjects, whose initial RBC ChE measurements
were >20% from their baseline value (no other explanation provided):
• Subject 028 (Male; 1.25 mg/kg dose) - 72 hour RBC ChE measurement repeated
• Subject 036 (Male; placebo) - 48 hour RBC ChE measurement repeated
• Subject 038 (Male; placebo) - 72 hour RBC ChE measurement repeated
• Subject 043 (Female; 1.0 mg/kg) - 8 hour RBC ChE measurement repeated
ii. Urine: A collection of urine for clinical urinalysis was conducted at 24 hours post-dose.
Urine samples for analysis of acephate and methamidophos (metabolite) concentrations
were collected from -12-0 hours pre-dose and 0-12, 12-24, and 24-48 hours post-dose.
Total urine volume was measured for each collection and a 20-mL portion was retained at
approximately -80°C.
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3. Sample analysis
a. Plasma and RBC ChE: Plasma and RBC ChE concentrations were determined by the
method of Ellman (1961)1.
b. Acephate and methamidophos - plasma: Acephate and methamidophos concentrations in
human plasma were determined by gas chromatography with flame photometric detection
after dilution with acetone containing polyethylene glycol (PEG). The method was
validated over a concentration range of 10-1000 ng/mL. The limit of detection (LOD;
3x background) was 6 ng/mL for acephate and 1.5 ng/mL for methamidophos. The limit of
quantification (LOQ) was 10 ng/mL for both compounds.
c. Acephate and methamidophos - urine: Acephate and methamidophos concentrations in
human urine were determined by gas chromatography with flame photometric detection
after dilution with acetone containing polyethylene glycol (PEG). The method was
validated over a concentration range of 10-1000 ng/mL. The LOD was 7 ng/mL for
acephate and 4 ng/mL for methamidophos. The LOQ was 10 ng/mL for both compounds.
4. Analytical methodology: The gas chromatography system consisted of a Hewlett Packard
(HP) 6890 plus gas chromatograph (GC), HP 7683 injector and autosampler, and HP flame
photometric detector with an RTX 200, 30 m x 0.53 mm i.d. 1.0 [j,m film thickness (Restek)
analytical column. The GC system conditions were as follows:
mm MM: iWChoMUBrOmto
mm 20"C.mln*'
Ffcwfc ISTCMitef
inhrfcy 23PC
M UWWI » w * w
Dataeioc litre
Marton Hydrogen lOOmUntn"1
Air lOOni-mltf1
Mmm 50 mLmtn'1
Cantor Hpfcoam Mml-Mirf1
Ht* 0.5 mh
Bow 3.0 irtmtn'*
6 id (• P*cfcad infection iiw«h(x*l beuMd>
Ub6y»tiim Vn MuHcHrom 2 V«wton 2.0
Data obtained from page 1124, Appendix T of MRID 45388301.
5. Statistical methods: With a ratio of active subjects relative to placebo subjects of 2.33, a
total of ten subjects (7 active and 3 placebo) were required per dose level to achieve 80%
power to detect a difference of 20% change from baseline ChE concentrations. A between
1 Ellman, G.L. (1961) Biochem. Pharmacol. 7:78.
TemparthiNM:
Put®;
tniadion VetttMix
Oata Handing:
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subject standard deviation (SD) of 8.7 and a 5% significance level were used for this
calculation. Therefore, a total of 50 subjects (10 per dose level) were enrolled in the present
study.
The objective of the statistical analyses was to investigate the clinical tolerability and safety
of the test material. Statistical analyses were only conducted on the plasma and RBC ChE
concentration data. Mean concentration (± SD) were reported at each time point, including
the change from baseline. The baseline value was determined as the mean of all available
pre-dose values, except the initial screening value.
The percentage change from baseline for plasma and RBC ChE concentration values were
analyzed with a repeated measures analysis of variance (ANOVA), including dose level,
time point, and dose level x time point interaction. The subject was included as a random
effect. A test for linear trend with dose was conducted at each time point separately by
using a linear contrast (data from males only). In addition, pairwise comparisons between
placebo and each dose level were conducted for each time point with Student's t-distribution
by using the error variance from the ANOVA. At each time point, if the test for linear trend
was significant (p<0.05), the pairwise comparisons were not adjusted for multiple
comparisons. If the test for linear trend was not significant (p>0.05), a Bonferroni
adjustment was applied to the pairwise comparisons at that time point {i.e., each comparison
was tested at the 1.25% significance level). Adjusted means for each dose level were
presented together with the significance level of each pairwise comparison and the test for
linear trend. If applicable, the significance level of the pairwise comparisons after the
Bonferroni adjustment was also provided.
Normality was assessed with a Shapiro-Wilk test and homogeneity of variance was assessed
by plotting the residuals against the predicted values for the model. If significant
non-normality could not be resolved by data transformation, the data were excluded as
outliers. If the omission of an outlier did not affect the conclusion, results from the full data
set were reported. Descriptive statistical methods were used to report other data.
HED's Chemistry and Exposure Branch (CEB) conducted a data analysis of the plasma and
red blood cell cholinesterase to verify whether the statistical analyses conducted were
appropriate. Additional information is provided in Appendix I to this DER.
6. Pharmacokinetic analysis: Individual plasma concentration profiles of acephate and
methamidophos vs. actual sampling times were generated for each subject. Mean
pharmacokinetic parameter estimates were determined from the individual parameter
estimates for each dose level. Pharmacokinetic parameter estimates were determined with
WinNonlin non-compartmental analysis model 200 (extravascular dosing; WinNonlin
version 1.1, Pharsight Corp.).
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Acephate AUQo-oo) data were analyzed for dose proportionality by ANOVA techniques with
the male subject data only.
log(AUC(0 — co)) = + p * log(Dose)
or
AUC(0 — oo) = a * Dosep
The estimate obtained for P is a measure of dose proportionality; proportionality requires
that P = 1. Dose independent parameters are implied if P = 0.
7. Method Validation and Storage/Stability
Plasma
Analytical method validation and storage stability results for plasma and study data tables for
acephate and methamidophos in plasma can be found in Appendix T of the study report
(MRID 45388301). Upon receipt, samples were immediately stored at -80°C until analyzed
using validated Analytical Method 6696 (Iveresk Project No. 366968).
This analytical method evaluated: 1) system suitability, 2) assay specificity, 3) assay
linearity, 4) assay limit of detection, 5) assay limit of quantification, 6) intraday and interday
assay accuracy and precision, and 7) determination of acephate and methamidophos in
human plasma from spiked human blood.
Storage stability analyses for acephate and methamidophos in human plasma when stored for
a period of 8 weeks at approximately -80°C were conducted.
Urine
Analytical method validation for the determination of acephate and methamidophos in
human urine, and storage stability results, can be found in Appendix U of the study report
(MRID 4538801). Samples were stored at -80° C until analyzed using validated Analytical
Method 6695 (Iveresk Project No. 366952).
In the method, aliquots of urine (1 mL) are spiked with acephate and methamidophos (for
calibration standard and quality control samples). Samples are diluted with the addition of
acetone containing polyethylene glycol (PEG). Aliquots of the diluted samples are analyzed
by gas chromatography and flame photometric detection. The analytical method evaluated: 1)
system suitability, 2) assay specificity, 3) assay limit of detection, 4) assay limit of
quantification, 5)assay linearity, 6) intraday and interday assay accuracy and precision, and
7) determination of acephate and methamidophos in human urine samples over the
concentration range of approximately 10 to 1000 ng/mL.
Storage stability analyses for acephate and methamidophos in human urine when stored for a
period of 12 weeks at approximately -80°C were conducted.
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II. RESULTS
A. ANALYTICAL/METHOD VALIDATION - PLASMA:
Analytical Method No. 6696 is acceptable in terms of system suitability, assay specificity,
assay linearity, assay limit of detection and intraday and interday assay accuracy and
precision, and therefore validated for the determination of acephate and methamidophos in
human plasma in the concentration range of approximately 10-1000 ng/mL. The assay limit
of quantification for both acephate and methamidophos is approximately 10 ng/mL. The
analytical method validation report and storage stability results are located in Appendix T of
the study report (MRID 45388301).
TABLE 2. Evaluation of gas chromatography critical parameters for assay of plasma acephate and methamidophos
Parameter
Acephate
Methamidophos
Column efficiency
143000
53000
(# of theatrical plates)
Tailing factor
1.11
1.26
Precision (%)a
2.8 (at 25.15 ng/mL)
4.1 (at 25.10 ng/mL)
0.9 (at 503.0 ng/mL)
1.8 (at 502 ng/mL)
Plasma limit of detection (ng/mL)
6 ng/mL (3 pg on-column)
1.5 ng/mL (0.75 pg on-column)
Plasma limit of quantification (ng/mL)
10 ng/mL
10 ng/mL
Linearity of detector response (%
+3.7%; 49.65
+2.2; 50.10
deviation; actual amount of acephate in
-5.9; 124.7
-2.8; 125.3
pg on-column)
-5.4; 249.3
-3.7; 250.5
-3.9; 496.5
-5.7; 501.0
-2.5;1247
+1.8; 1253
+7.3; 2493
+4.3; 2505
+6.8; 4985
+3.8; 5010
Intraday and Interday Overall
Accuracy (mean, %)
25.13 ng/mL
+0.8%
-5.8%
100.5 ng/mL
+0.3%
-1.6%
753.8 ng/mL
+0.3%
+1.5%
Overall Intraday and Interday
Assay Precision (%)
25.13 ng/mL
5.8%
4.6%
100.5 ng/mL
7.1%
5.3%
753.8 ng/mL
9.8%
4.8%
a Coefficient of variation based on 10 samples
Data taken from tables beginning on page 1096 of MRID 45388301.
B. ANALYTICAL/METHOD VALIDATION - URINE:
Analytical method validation and storage stability results for the determination of acephate
and methamidophos in human urine is located in Appendix U of the study report (MRID
45388301). Analytical method no. 6695 is satisfactory in terms of system suitability, assay
specificity, assay linearity, assay limit of detection and intraday and interday assay accuracy
and precision, and is validated for the determination of acephate and methamidophos in
human urine over the concentration range of approximately 10 to 1000 ng/mL. The assay
limit of quantification for both analytes was found to be approximately 10 ng/mL. Over-
range human urine samples prepared around 2000 and 5000 ng/mL acephate and
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methamidophos were successfully diluted with control human urine and extracted without
loss of accuracy or precision.
TABLE 3. Evaluation of gas chromatography critical parameters for assay of urine acephate and methamidophos
Parameter
Acephate
Methamidophos
Column efficiency
160000
44900
(# of theatrical plates)
Tailing factor
1.42
1.23
Precision (%)a
10.3 (at 24.93 ng/mL)
4.4 (at 25.05 ng/mL)
5.4 (at 498.5 ng/mL)
3.5 (at 501 ng/mL)
Plasma limit of detection (ng/mL)
7 ng/mL (3.5 pg on-column)
4.0 ng/mL (2.0 pg on-column)
Plasma limit of quantification (ng/mL)
10 ng/mL
10 ng/mL
Linearity of detector response (%
+0.1%; 49.65
-0.7; 50.10
deviation; actual amount of acephate in
-0.2; 124.7
-2.5; 125.3
pg on-column)
NC; 249.3
+11.5; 250.5
-0.2; 496.5
-5.3; 501.0
+2.4; 1247
+1.3; 1253
-5.8; 2493
-6.5; 2505
+3.8; 4985
+2.3; 5010
Intraday and Interday Overall
Accuracy (mean, %)
25.13 ng/mL
-3.9%
-2%
100.5 ng/mL
-0.4%
-5.1%
753.8 ng/mL
-2.1%
-5.6%
Overall Intraday and Interday
Assay Precision (%)
25.13 ng/mL
6.1%
6.1%
100.5 ng/mL
8.6%
7.0%
753.8 ng/mL
8.4%
6.2%
Effect of Dilution on Accuracy and
-6.7% (at 2515 ng/mL)
+1.0% (at 2510 ng/mL)
Precision (mean % deviation from
-6.3% (at 5030 ng/mL)
+0.8 (at 5020 ng/mL)
actual)
a Coefficient of variation based on 10 samples
NC = not calculated
C. STORAGE STABILITY IN HUMAN PLASMA: Results of the storage stability study are
shown in Table 4. For acephate in plasma, all analyses revealed that stability remained within
15% of the original concentration. For methamidophos in plasma: after 1 week of storage, 2
out of 5 of the replicate samples at 25 ng/mL and 1 of the 5 replicates at 750 ng/mL were
outside of the ±15% of the actual concentration; after 8 weeks of storage, 1 replicate (of 5) at
100 ng/mL was outside the ±15% of the actual concentration. Overall, the mean calculated
concentrations for methamidophos in plasma were within ±15% of the actual concentration
and coefficients of variation (CV) were <15% at each concentration. These results indicate
acceptable stability for both acephate and methamidophos in plasma when stored at
approximately 80°C for a period of approximately 8 weeks.
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TABLE 4. Storage stability of acephate and methamidophos in human plasma at -80°C
Test Material
Storage Duration
Mean Calculated3
Mean Percent of Actual
(Concentration)
(Weeks)
Concentration (ng/mL)
Concentration/C V
(ng/mL)
Acephate
0
24.8
98.5/6.9
(25.13 ng/mL)
1
25.7
102.1/5.6
4
25.0
99.5/8.1
8
25.9
103.2/3.3
Acephate
0
99.5
99.0/2.6
(100.5 ng/mL)
1
104
103.5/5.1
4
100.7
100.2/3.1
8
98.2
97.7/4.0
Acephate
0
746
98.9/6.1
(753.8 ng/mL)
1
746
99.0/3.3
4
739
100.2/3.1
8
710
94.2/2.1
Methamidophos
0
27.4
109.1/3.0
(25.13 ng/mL)
1
21.6
86.1/3.0
4
24.8
98.1/2.4
8
23.2
92.5/4.7
Methamidophos
0
104
103.2/2.2
(100.5 ng/mL)
1
87.5
87.1/1.4
4
88.7
86.3/3.0
8
86.4
85.9/1.9
Methamidophos
0
750
99.6/2.6
(753.8 ng/mL)
1
643
85.3/1.1
4
657
87.1/1.6
8
872
89.0/1.4
a Values are means of five samples
CV = coefficient of variation
Data taken from Tables 1-6 from p. 1158- 1163 of MRID 45388301.
D. STORAGE STABILITY IN URINE: Results of the storage stability in human urine are
shown in Table 5. The results indicate that acephate and methamidophos are stable in human
urine when stored for a period of 12 weeks at approximately -80°C.
Acephate: At -100 ng/mL, 3 out of 5 replicates were outside of the acceptance criteria for
accuracy after 8 weeks of storage and the mean calculated concentration was outside of 100
± 15% of the actual concentration. The CV was <15%. At -750 ng/mL, 3 out of 5 replicates
were outside of the acceptance criteria for accuracy after 8 weeks storage and the mean
calculated concentration was outside of 100 ± 15% of the actual concentration. The CV was
<15%. All other mean results were within 100 ± 15% of the actual concentration, including
all results following 12 weeks of storage at each concentration. This indicates that the results
that were outside of 15% of the actual concentration at 8 weeks do not significantly show
instability.
Methamidophos: All mean results were within 100 ± 15% of the actual concentrations and
the CVs for each storage period were <15%.
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Overall, results indicate acceptable stability of acephate and methamidophos in human urine
at concentrations of approximately 25, 100, and 750 ng/mL for a period of 12 weeks when
stored at -80°C.
Table 5. Storage stability of acephate and methamidophos in human urine at -80°C
Test Material
Storage Duration
Mean Calculated3
Mean Percent of Actual
(Concentration)
(Weeks)
Concentration (ng/mL)
Concentration/C V
(ng/mL)
Acephate
0
24.2
96.4/8.8
(25.13 ng/mL)
1
28.6
113.8/8.4
4
23.4
93.0/12.2
8
22.4
89.0/3.2
12
24.7
98.2/7.7
Acephate
0
99.3
98.9/2.8
(100.5 ng/mL)
1
102.5
102.1/4.1
4
91.8
91.4/2.1
8
82.7
82.3/7.2
12
100
99.5/6.3
Acephate
0
742
98.4/2.1
(753.8 ng/mL)
1
746
99.0/3.2
4
754
100.0/5.7
8
830
83.6/4.5
12
718
95.0/3.5
Methamidophos
0
26.1
104/3.5
(25.13 ng/mL)
1
23.8
94.5/2.1
4
28.1
111.9/3.0
8
26.6
105.7/2.4
12
25.3
100.7/3.6
Methamidophos
0
94.4
94/2.9
(100.5 ng/mL)
1
91.3
90.8/3.2
4
89.2
88.7/2.6
8
109
108.3/6.9
12
97.9
97.4/3.5
Methamidophos
0
701
93.0/1.7
(753.8 ng/mL)
1
892
91.8/2.2
4
741
98.3/1.0
8
835
110.8/3.3
12
740
98.2/2.5
a Values are means of five samples
CV = coefficient of variation
Data taken from Tables 1-6 from p. 1279-1284 of MRID 45388301.
E. DISPOSITION OF SUBJECTS: A total of 63 males and 12 females were screened for
inclusion in the present study. Forty male and 10 female volunteers were selected for
inclusion and all 50 subjects selected completed the study. The mean age for male subjects
was 32.3 ± 7.8 years; all were white, except for a single male of Asian origin. The mean age
for female subjects was 32.2 ± 7.3 years; all were white.
F. CLINICAL SIGNS: There were no adverse clinical signs that were considered related to
treatment. There were no adverse clinical events in males dosed at 0.35 or 1.25 mg/kg or in
the females administered the placebo. A single male in the placebo group (No. 038) had
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dyspepsia at 30 minutes post-administration that was considered related to the change in diet
in clinic. A single male at 0.70 mg/kg (No. 020) experienced leg pains at 30 hours
post-dose that were considered related to muscle cramps. In the 1.00 mg/kg group, a single
male (No. 027) reported a headache at 22 hours post-dose that was considered unrelated to
treatment. In addition, the same subject reported a cough and sore throat at 9.5 and 25 hours
post-dose, respectively, that were considered probably due to a viral infection. In the
females administered 1.00 mg/kg, a single subject (No. 045) reported dizziness at 52 hours
post-dose that was considered unrelated to treatment. No serious adverse events were
reported.
G. VITAL SIGNS: There were no treatment-related effects on vital signs, ECG readings, or
physical examinations during the present study.
H. CLINICAL PATHOLOGY FINDINGS: There were no treatment-related or clinically
significant effects on hematology, clinical chemistry, or urinalysis parameters during the
course of the study.
I. PHARMACOKINETIC ANALYSES: The concentration-profiles of acephate and
methamidophos in plasma after a single oral dose of acephate at 0.35-1.25 mg/kg to male
volunteers and at 1.00 mg/kg to female volunteers are presented in Table 6 and shown
graphically in Figures 1-4. The mean pharmacokinetic parameter estimates derived from
these concentration-time profiles are presented by compound and sex in Table 7.
No quantifiable concentrations of acephate or methamidophos were noted in samples from
the placebo-treated subjects or in any pre-dose samples from any acephate treatment group.
The acephate plasma concentration vs. time profiles were consistent with oral dose
administration with no evidence of a significant lag phase (measurable concentrations at
1 hour post-dose at all dose levels) with mean Tmax estimates of 1.3-2.7 hours (range: 1 to
4 hours, except for a single value of 8 hours at 0.70 mg/kg). Acephate concentrations
declined rapidly up to 24 hours post-dose with mean ti/2ei estimates of 4.4-5.4 hours (range:
3.5-6.6 hours). Mean oral clearance (CL/F) was generally slow, with mean values of 77-105
mL/hour/kg (range: 62-136 mL/hour/kg). Overall systemic exposure (AUC) demonstrated
generally dose-related increases (slightly greater than dose proportional). There were no
sex-related differences in pharmacokinetic parameters at 1.00 mg/kg.
Plasma concentration data were sparser for the metabolite, methamidophos, at <0.70 mg/kg
acephate but valid Cmax, Tmax, and AUCo-t estimates were determinable at >0.70 mg/kg
acephate, with valid estimates for the remaining parameters at >1.00 mg/kg acephate.
Similar to acephate, mean Tmax estimates of 3.3-4.3 hours (range: 1 to 4 hours, except for a
single value of 8 hours at 0.70 mg/kg). Methamidophos concentrations declined rapidly up
to 12 hours post-dose, with mean ti/2ei estimates of 6.7-8.3 hours (range: 3.5-11.6 hours).
Mean oral clearance (CL/F) was rapid, with mean values of 3377-4439 mL/hour/kg (range:
2503-7800 mL/hour/kg). Overall systemic exposure (AUC) demonstrated generally less
than dose-proportional increases. There were no sex-related differences in pharmacokinetic
parameters at 1.00 mg/kg.
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Mean plasma concentration data for acephate and methamidophos were also outside of the
method validated concentration range of 10-1000 ng/mL at several dose levels and
timepoints. For acephate, mean plasma concentrations were above 1000 ng/mL at 1.0 and
1.25 mg/kg in males at 1, 2, and 4 hours post-dose, and at 1.0 mg/kg in females at 2 and 4
hours post-dose. For methamidophos, mean plasma concentrations were below 10 ng/mL at
0.70 mg/kg (2, 8, and 12 hours post-dose), 1.0 mg/kg (1 and 12 hours post-dose), and 1.25
mg/kg (1 and 12 hours post-dose) in males, and at 1.0 mg/kg (1 and 12 hours post-dose) in
females.
TABLE 6. Mean (± SD) plasma concentration-time profiles (ng/mL) of acephate and methamidophos after a
single oral administration of acephate to human volunteers.a
Time
(hours)
Concentration (ng/mL)
Male (mg/kg)
Female (mg/kg)
0.35
0.70
1.00
1.25
1.00
Acephate
Pre-dose
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
1
482.9 ± 122.1
641.3 ±275.7
1043.3 ±584.1
1014.9 ±444.8
792.7 ±714.3
2
434.0 ±47.5
842.3 ± 340.3
1320.0 ± 179.2
1598.6 ±240.9
1205.9 ±406.3
4
309.7 ±29.6
744.0 ±208.2
1087.4 ± 158.8
1441.4 ±220.0
1309.3 ±243.5
8
151.1 ± 17.4
433.0 ± 104.7
658.1± 68.6
858.7 ± 134.6
701.1 ± 162.0
12
73.0 ± 12.8
235.1 ±87.1
363.3 ±47.4
462.4 ± 94.5
382.7 ± 120.8
24
12.1 ±8.5
51.4 ± 17.6
83.4 ±24.3
99.9 ±29.4
75.0 ±38.7
48
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
72
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
168 (Day 7)
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
336 (Day 14)
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
Methamidophos
Pre-dose
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
1
0.0 ±0.0
0.0 ±0.0
5.64 ±7.28
3.99 ±6.81
4.63 ±7.95
2
0.0 ±0.0
8.06 ±5.57
13.74 ± 1.78
17.81 ±3.56
11.23 ±8.52
4
0.0 ±0.0
10.79 ±5.17
19.21 ±3.33
24.91 ±2.99
21.76 ±3.14
8
0.0 ±0.0
3.57 ± 6.10
14.87 ±3.54
19.14 ±3.93
14.86 ±6.87
12
0.0 ±0.0
1.63 ± 4.31
4.89 ±6.10
8.64 ±6.29
7.19 ±6.80
24
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
48
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
72
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
168 (Day 7)
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
336 (Day 14)
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
a Data were obtained from Table Q1.2 on pages 967-971 of MRID 45388301; n = 7 sample/time point.
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TABLE 7. Mean (± SD) pharmacokinetic parameter estimates derived from the plasma concentration-time profiles
(ng/mL) after a single oral administration of acephate to human volunteers.a
Time (hour)
Concentration (ng/mL)
Male (mg/kg)
Female (mg/kg)
0.35
0.70
1.00
1.25
1.00
Acephate
Cmax (ng/mL)
506.3 ± 86.6
921.9 ± 156.8
1451.4 ±206.0
1688.6 ±262.3
1515.7 ± 319.6
Tmax (hour)
1.29 ±0.49
2.71 ±2.36
2.00 ± 1.00
2.43 ± 1.13
2.71 ± 1.25
tl/2el (hour)
4.39 ± 0.65
5.00 ±0.34
5.42 ± 0.72
5.21 ±0.52
4.83 ±0.68
AUCo-t (hour ng/mL)
3277.6 ±514.1
8063.3 ± 954.0
12,327.8 ± 667.4
15,473.9 ±2143.6
12,847.3 ±2288.1
AUCo-°° (hour ng/mL)
3407.1 ± 507.5
8439.4 ± 1050.2
13,000.0 ± 812.8
16,243.2 ±2398.5
13,400.9 ±2595.8
CL/F (mL/hour/kg)
105.0 ± 17.5
84.0 ± 10.5
77.3 ±5.10
78.6 ± 12.9
77.0 ± 15.7
Methamidophos b
Cmax (ng/mL)
0.0 ±0.0
12.7 ± 1.98
19.1 ±3.50
25.2 ±2.74
22.0 ±2.65
Tmax (hour)
0.0 ±0.0
4.29 ± 1.80
3.29 ± 1.25
3.71 ±0.76
3.57 ± 1.14
ti/2 el (hour)
0.0 ±0.0
8.30 ± 4.63 c
6.66 ±2.21
6.92 ±2.30
6.65 ±2.77
AUCo-t (hour ng/mL)
0.0 ±0.0
84.0 ±59.2
204.8 ±79.2
281.3 ±76.6
236.2 ± 87.3
AUCo-°° (hour ng/mL)
0.0 ±0.0
222.2 ± 81.3 c
252.8 ±94.3
331.2 ±89.6
284.1 ± 104.4
CL/F (mL/hour/kg)
0.0 ±0.0
3376.5 ±1235.3 c
4438.9 ± 1513.6
4090.1 ± 1375.2
4110.6 ± 1951.1
a Data were obtained from Tables Ql.1.1-1.1.6 on pages 961-966 and Q2.1 on pages 972-976 of MRID
45388301; n = 7 sample/time point, unless noted otherwise,
b Mean ± SD pharmacokinetic parameter estimates for methamidophos were calculated from the individual
data by the Reviewers,
c n = 2.
Plana Concentration* on • Logarlthalc Sola
Acapnata concentration
Main Valuaa: Halae
£
s
100 -
10-
Traataant OOP 0.38 M6/KB
tlaa Point
0.7 MB/K6 O O -O 1.0 H8/K8
1.2B MG/KQ
FIGURE 1. Plasma concentration-time profiles (ng/mL) of acephate after a single oral administration of acephate to
human male volunteers. Figure obtained from page 879 (MRID 45388301).
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ACEPHATE /103301
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nmm Conew\tP»tlam on » kBftcItimle leal*
Ac«en»t> CaneaMrMtan
Man vcluw. #«aal«s
!
1000"
10# *
»-
t-Q
T*
*12 M
*84 M
' - I '
+** H
~72 «
OAY 7
mjm
DAY 14
Tr«aUm*fc
Ti»t Potftt
1,0 M6/K8
FIGURE 2. Plasma concentration-time profiles (ng/mL) of acephate after a single oral administration of acephate to
human female volunteers. Figure obtained from page 881 (MRID 45388301).
Mmmmt G&ncwmtpmtimm on a §«•!•
ttothMidoprtoc CvncMtrtt&an
mmn M»l#»
100-
ti»« Point
Trmt««nt M-B o.as m/m *-*-* i .o mo/ks «-«-• i n M/xa
FIGURE 3. Plasma concentration-time profiles (ng/mL) of methamidophos after a single oral administration of acephate
to human male volunteers. Figure obtained from page 883 (MRID 45388301).
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PIMM ConcantratIons on ¦ Logwlthale Sella
Mathaaidoohoa Concentration
Ha an Valuaa: Foiln
100 ¦
Tiaa Point
TraatMnt BOO 1.0 MS/KS
FIGURE 4. Plasma concentration-time profiles (ng/mL) of methamidophos after a single oral administration of acephate
to human female volunteers. Figure obtained from page 885 (MRID 45388301).
J. URINE CONCENTRATION-TIME PROFILES: The concentration-profiles of acephate
and methamidophos in urine after a single oral dose of acephate at 0.35-1.25 mg/kg to male
volunteers and at 1.00 mg/kg to female volunteers, and the percentage recovery estimates of
the administered dose of acephate (as acephate and methamidophos) are presented in
Table 8.
The total recovery of acephate and methamidophos (as acephate equivalents) in urine
through 48 hours post-dose ranged from 25.0% to 61.1% in males and 12.1% to 50.6% in
females with most of the recovery occurring within the initial 12-hour post-dose period.
The mean acephate recovered at 1.0 mg/kg for males was 51.7%, versus 25.7% for females.
The fractions of the administered acephate dose or the total recovered test material (acephate
and methamidophos [as acephate-equivalents]) recovered as methamidophos ranged from
0.16-0.91% administered dose (AD). The fate of the unrecovered administered acephate
doses is unknown but might be due to incomplete absorption or additional metabolism. The
lower recoveries in females vs. males was attributed to decreases in urine collection
volumes and fractions of unchanged parent observed in females compared to males.
Urine concentrations of methamidophos (systemic exposure) generally decreased in parallel
to acephate at >1.00 mg/kg acephate during the 12-48 hour collection period. Accumulation
of methamidophos would not be expected due to this similarity in systemic
clearance/elimination.
Mean urine concentration data for acephate and methamidophos were outside of the method
validated concentration range of 10-1000 ng/mL at several dose levels/timepoints, including
exceedances of 1000 ng/mL for acephate at 0-12 hours (all dose levels, both sexes), 12-24
hours (all dose levels, both sexes), and slightly above the range at 24-48 hours (1.00 and
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1.25 dose levels in males). For methamidophos, concentrations were below the limit of
quantification of 10 ng/mL at 24-48 hours for the 0.70 mg/kg (males) and slightly below at
1.0 mg/kg (females).
TABLE 8. Mean (± SD) urine concentration-time profiles (ng/mL) of acephate and methamidophos after a single oral administration of
acephate to human volunteers. a
Time (hour)
Concentration (ng/mL)
Male (mg/kg)
Female (mg/kg)
0.35
0.70
1.00
1.25
1.00
Acephate
Pre-dose (-12-0)
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0-12
9535.7 ±3106.5
21,457.1 ± 12,422.4
34,957.1 ±21,973.3
27,371.4 ±8151.6
31,000.0 ± 16,744.2
12-24
1488.1 ±679.7
5344.3 ±2549.9
9694.3 ±4541.4
9328.6 ±2113.6
7967.1 ±4777.1
24-48
105.1 ±43.9
545.7 ± 193.3
1249.3 ±643.8
1020.7 ±206.8
688.0 ±388.8
Acephate recovered (% AD;
minimum/maximum)b
25.0/61.1
27.6/59.0
35.5/60.6
35.3/52.5
12.1/50.6
Acephate recovered
(% AD; mean ± SD)
42.8 ± 12.8
49.6 ± 10.3
51.7 ± 9.3
43.4 ±5.3
25.7 ± 14.0
Methamidophos
Pre-dose (-12-0)
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0.0 ±0.0
0-12
82.7 ±24.4
187.0 ±91.7
295.7 ± 141.3
324.6 ±71.0
283.3 ± 133.4
12-24
22.5 ±8.58
78.2 ±41.7
148.7 ±55.9
169.7 ±38.6
118.5 ± 73.3
24-48
0.0 ±0.0
4.73 ±5.90
15.3 ±9.26
17.8 ±2.90
7.30 ±7.19
Methamidophos recovered (%
AD of acephate;
minimum/maximum)b
0.32/0.70
0.32/0.83
0.50/0.91
0.61/0.90
0.16/0.69
Methamidophos recovered
(%AD of acephate; mean ±
SD)
0.52 ±0.15
0.67 ± 0.18
0.68 ±0.15
0.75 ±0.12
0.34 ±0.19
Total recovery (% AD;
minimum/maximum)b
25.4/61.8
27.9/59.8
36.0/61.3
35.9/53.3
12.2/51.3
a Data were obtained from Table Q3.1 on pages 995-996 of MRID 45388301; n = 7 sample/time point,
b Calculated by the Reviewers from the individual data (see Appendix II).
AD Administered dose.
K. PLASMA AND RBC CHOLINESTERASE: The activities of plasma and RBC ChE after
a single oral dose of acephate at 0.0-1.25 mg/kg to male volunteers and at 0.0 or 1.00 mg/kg
to female volunteers are presented in Table 9 and shown graphically in Figures 5-8.
Plasma ChE activities were decreased (p<0.01) by 9-13% at 12, 24, 48, and 72 hours
post-dose in the 1.25 mg/kg males with significant (p<0.01) linear trend tests at 48 and
72 hours post-dose. Plasma ChE activities were decreased (p<0.05) by 10-13% at 8, 12 and
24 hours post-dose in the 1.00 mg/kg females. There were no treatment-related effects on
plasma ChE activities at <1.00 mg/kg in the male subjects. Decreases in plasma ChE
activities in males at 0.35 mg/kg at 12 and 72 hours post-dose demonstrated no relationship
to dose or time. For RBC ChE activities, decreases (p<0.01) of 7% and 15% were noted in
the 1.25 mg/kg males and 1.00 mg/kg females, respectively, at 12 hours post-dose.
Increases (p<0.05) of 8-9% at 48 and 72 hours post-dose in the 0.35 mg/kg males were
considered unrelated to dose; there were no other treatment-related effects on RBC ChE
activities at <1.00 mg/kg in the male subjects.
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In male subjects, concurrent decreases (p<0.01) in both plasma and RBC ChE activities
were noted only at 12 hours post-dose in the 1.25 mg/kg group, with decreases of 13% and
7%, respectively. The further decreases (p<0.01) in plasma ChE activities at 24, 48, and
72 hours in the 1.25 mg/kg males were considered not relevant toxicologically as they were
<10%, occurred concomitantly with low or non-detectable plasma concentrations of
acephate, and occurred without concomitant effects on RBC ChE concentrations. In female
subjects, the concurrent decreases (p<0.05) in both plasma and RBC ChE activities were
noted only at 12 hours post-dose at 1.00 mg/kg, with decreases of 12% and 15%,
respectively. The further decrease (p<0.001) in plasma ChE activity at 24 hours was
considered not relevant toxicologically as it occurred concomitantly with a decreasing
plasma concentration of acephate and occurred without concomitant effects on RBC ChE
activity. Additionally, RBC ChE measurements are generally preferred over plasma
measures of ChE because data on RBCs may provide a better representation of the
inhibition of the neural target enzyme, AChE2.
There was variability in the data at baseline and within the placebo groups. CVs for plasma
ChE at baseline were 28%, 16%, 23%, and 11% at 0.35, 0.70, 1.0, and 1.25 mg/kg in males,
respectively. The CV for plasma ChE at baseline in the 1.0 mg/kg females was 21%.
Changes and statistical significance for each dose group were compared to baseline values at
each dose level (not to placebo group controls). In most instances (except for the 1.25 mg/kg
males), all changes observed were within the CV of baseline values for plasma ChE. In the
placebo groups, CVs at baseline were approximately 13% in males, and 14% in females.
Therefore, all changes in plasma ChE were within the variability of baseline values at each
dose level (with the exception of the 13% change at 12-hours in the 1.25 mg/kg males), as
well as within the variability of placebo group controls. The difference between the baseline
value for the placebo group and the baseline value for the 1.25 mg/kg males was 8%.
There was less variability in the results for RBC ChE, however, results were still within
variability of baseline values for males, and slightly outside for females (11% change at 1.0
mg/kg compared to baseline CV of 9%). Changes observed in RBC ChE in males were also
mostly within the variability of placebo group baseline of 8% (there was a 9% increase in
RBC ChE at 72 hours in the low dose males, but increases aren't toxicologically relevant).
The placebo baseline in females had a CV of 4%, so the 11% change in RBC ChE in
females at 12 hours, which was also statistically significant, was outside of the CV of
baseline for that dose group, as well as the placebo baseline.
2 "Office of Pesticide Programs Science Policy on The Use of Data on Cholinesterase Inhibition for Risk
Assessments of Organophosphorous and Carbamate Pesticides". OPP, USEPA, 2000.
https://www.epa.gov/sites/default/files/2015-07/documents/cholin.pdf.
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TABLE 9. Mean (± SD) plasma and RBC ChE concentrations (IU/L) after a single oral administration of acephate to human volunteers.a
Time (hour)
Male (mg/kg) Female (mg/kg)
0.0 b
0.35
0.70
1.00
1.25
O
©
1.00
Plasma ChE
Baseline
5440.3 ± 690.7
5525.4 ± 1526.9
(CV=28%)
5151.9 ±824.5
(CV=16%)
5776.3 ± 1332.5
(CV=23%)
5002.7 ± 565.6
(CV=11%)
4310.7 ±608.3
4702.4 ± 979.3
(CV=21%)
1
5038.3 ±703.2
5078.7 ± 1588.3
4631.3 ± 935.0
(|10)
5450.9 ± 1253.7
4691.9 ± 569.7
4116.7 ± 617.2
4276.0 ± 907.8
2
5152.5 ±712.7
5145.4 ± 1505.1
4664.9 ±962.8
5505.3 ± 1258.6
4774.4 ± 660.7
4182.3 ±754.7
4338.3 ±934.7
4
5085.8 ±670.7
5186.7 ± 1616.9
4804.9 ±824.9
5515.1 ± 1400.6
4683.7 ± 567.0
4290.3 ±679.0
4406.9 ±981.9
8
5070.6 ± 629.7
5028.7 ± 1458.0
4743.7 ±930.8
5500.4 ± 1255.2
4459.1 ±568.7
4286.7 ± 1140.6
4111.1 ±938.3*e
(|13)
12
5160.4 ±630.6
4942.6 ± 1430.4*
(|ll)d
4641.6 ±881.7
(|10)
5613.7 ± 1351.9
4365.1 ±563.4**
(|13)
4211.3 ± 671.5
4141.0 ±930.7*
(|12)
24
5371.5 ±800.9
5239.7 ± 1613.2
4934.7 ± 1002.1
5695.0 ± 1361.5
4550.4 ± 475.8** (|9)
4467.3 ±741.1
4222.9 ± 1004.7***
(|10)
48
5403.7 ±621.5
5597.4 ± 1596.5
4900.3 ±757.1
5548.6 ± 1337.5
4544.3 ± 502.5*** (|9)
4604.0 ± 749.7
4716.1 ± 1048.3
72
5494.4 ±618.4
5296.7 ± 1353.0(|4)
5106.3 ±744.2
5765.7 ± 1326.5
4579.1 ± 646.0*** (|9)
4363.3 ±714.0
4529.0 ± 1036.5
168 (Day 7)
5297.8 ±691.8
5336.9 ± 1462.6
5191.1 ±685.7
5531.3 ± 1295.4
4758.4 ± 490.0
3986.7 ±562.6
4388.4 ± 1037.7
336 (Day 14)
5303.8 ±736.8
5270.4 ± 1544.8
5169.0 ±789.3
5892.7 ± 1503.6
4987.3 ± 440.2
4287.7 ± 1022.4
4961.6 ± 1598.0
RBC ChE
Baseline
12493.0 ±1176.1
12026.7 ± 1156.6
(CV=10%)
11647.0 ± 775.9
12389.7 ± 728.6
12014.4 ± 964.3
(CV=8%)
12072.0 ± 464.1
12099.4 ± 1075.6
(CV=9%)
1
12419.4 ± 1359.2
12363.7 ± 1486.5
11616.4 ± 1245.3
12686.6 ± 1112.4
12371.7 ± 1097.3
11011.3 ± 397.8
11549.0 ± 1191.8
2
12902.3 ± 1396.4
12472.7 ± 1378.8
11881.4 ± 1240.5
12460.0 ±929.5
12293.1 ± 1026.4
11583.0 ±686.6
11828.3 ±1106.4
4
12725.6 ± 1042.2
12822.3 ± 1655.7
11413.1 ± 1182.6
12914.1 ± 1340.7
12056.3 ± 1212.3
12090.0 ±211.5
11562.4 ± 1077.8
8
12670.3 ± 1540.9
11936.9 ± 1509.7
11658.7 ±940.7
12439.6 ± 1274.7
11089.4 ±788.0
11790.3 ±867.7
12052.4 ± 1760.2
12
12793.2 ± 1498.9
12116.7 ± 1333.7
12603.0 ± 1637.9
12099.7 ± 1536.9
11189.3 ± 1432.7** (|7)
11875.7 ±356.6
10746.1 ± 1247.4** (|11)
24
12535.8 ± 1397.5
12337.7 ± 1439.9
11437.9 ±922.9
11590.4 ±985.2
11644.6 ±989.5
11055.0 ±577.7
11483.1 ±829.2
48
12493.2 ± 1303.4
12978.3 ± 1556.6* (|8)
11235.9 ±864.4
12193.0 ± 1630.0
11624.9 ±826.5
11829.3 ± 119.3
11427.4 ± 1125.3
72
12100.9 ± 1478.8
13026.0 ± 1159.6*** (|9)
10603.9 ±686.7
11768.3 ±2140.3
11887.7 ± 1043.3
10409.0 ± 1087.4
10647.0 ± 1386.6 (112)
168 (Day 7)
12325.4 ±937.4
12079.7 ± 1561.5
12263.3 ± 1114.0
12561.9 ± 1095.8
11457.6 ± 1273.0
10843.0 ± 572.8
10796.6 ± 1158.5
336 (Day 14)
12573.3 ± 1327.7
12399.1 ± 1574.2
11511.3 ± 819.4
11969.4 ± 1976.4
12636.4 ± 1017.4
12139.0 ±625.3
12133.6 ± 1344.9
a Data were obtained from Tables 5,6,1, and 8 on pages 74, 75, 77, and 79 and Tables M2.1 and M2.2 on pages 703-716 of MR TP 45388301; n = 7 sample/time point, unless
noted otherwise,
b n = 12.
c n = 3.
d Percent changes and statistical analysis are calculated from baseline,
e Statistically significant when outliers were not excluded.
* Significantly different from control (baseline for each dose group): p<0.05 (*); p<0.01 (**); p<001 (***).
CV = coefficient of variation.
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Plasma Cho 1 lnestaras* (iu/l)
Change From Basel ine
Mim Values; Halts
t
I
m
i
I
18-
10-
a-
s-
-9-
-*»-
-19-
~1H MK
~II H
*34 M
Treatment
Time Point
PL*C1B0 38 mfm
1.0 m/m * * * 1 . 33 MG/KG
0.7 MB/ICS
FIGURE 5. Plasma cholinesterase concentration-time profiles (IU/L) after a single oral administration of acephate to
human male volunteers.
Plasms Cholinesterase flu/IJ
Percentage Change Prom Baseline
man Valu*a: FemaJea
1
1
i
?
ti-
lt-
•-
•-
-«•
*T"
~3 M
T~~
~4 H
*i h
*18 H
Tlnw Point ¦
Trootment B-m-B PLACEBO 1.0 MG/KB
FIGURE 6. Plasma cholinesterase concentration-time profiles (IU/L) after a single oral administration of acephate to
human female volunteers.
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SBC Choi lnesterasa fiu/1)
Percentage Change From Baseline
Mean value*: Males
3
I
#¦»
i
'
m-
M-
s-
9'
-»#-
-I8»
« H
1
~4 H
*9 *
~ M H
TiM Point
Traat»ent »«-B PLACEBO 0.3S MG/KG
~ > » i.o m/tm *-*-» i.25 mo/ks
0.7 MS/KG
FIGURE 7. Red blood cell cholinesterase concentration-time profiles (IU/L) after a single oral administration of acephate
to human male volunteers.
ROC Choilnaataraae (lu/ll
Percentage Change Fro* Baseline
Me#r» Values: Females
§
I
i
?
?
IS *
»*
»•
o-
<¦
-»•
M
+4 H
*• H
~ If M
*» H
Tr«0t»ent
Tins# Point
PLACEBO 1,0 MS/KG
FIGURE 8. Red blood cell cholinesterase concentration-time profiles (IU/L) after a single oral administration of acephate
to human female volunteers.
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III. DISCUSSION AND CONCLUSIONS
A. INVESTIGATORS' CONCLUSIONS: There were no observed effects of acephate
technical administration noted in any subject and no clinically-relevant decreases in plasma
or RBC ChE activities were observed. Therefore, the NOEL for single-dose oral
administration of acephate to human volunteers was 1.25 mg/kg in males and 1.00 mg/kg in
females.
B. REVIEWER COMMENTS: There were no treatment-related effects on clinical signs,
vital signs, or clinical pathology findings.
The acephate plasma concentration vs. time profiles were consistent with oral dose
administration with no evidence of a significant lag phase. Mean Tmax estimates ranged
from 1.3-2.7 hours. Acephate concentrations declined rapidly up to 24 hours post-dose
post-Cmax, with mean ti/2ei estimates of 4.4-5.4 hours. Mean CL/F estimates were generally
slow, with values of 77-105 mL/hour/kg. Overall systemic exposure demonstrated
generally dose-related increases (slightly greater than dose proportional). There were no
sex-related differences in pharmacokinetic parameters at 1.00 mg/kg (the single dose level
in female subjects).
Plasma concentration data were sparser for the metabolite, methamidophos, but valid Cmax,
Tmax, and AUCo-t estimates were determinable at >0.70 mg/kg acephate, with valid estimates
for the remaining parameters at >1.00 mg/kg acephate. Mean Tmax estimates ranged from
3.3-4.3 hours. Methamidophos concentrations declined rapidly up to 12 hours post-dose
post-Cmax, with mean ti/2ei estimates of 6.7-8.3 hours. Mean CL/F estimates were rapid, with
values of 3377-4439 mL/hour/kg. Overall systemic exposure demonstrated generally less
than dose-proportional increases. There were no sex-related differences in pharmacokinetic
parameters at 1.00 mg/kg.
The total recovery of acephate and methamidophos (as acephate equivalents) in urine
through 48 hour post-dose ranged from 25% to 62% in males and 12% to 51% in females,
with most of the recovery occurring within the initial 12-hour post-dose period. The
fractions of the administered acephate dose recovered as methamidophos were <1% AD and
<2% of the total recovered dose and were independent of dose or sex. Urine concentrations
of methamidophos (systemic exposure) generally decreased in parallel to acephate at
>1.00 mg/kg acephate and accumulation of methamidophos would not be expected.
Plasma ChE activities were decreased (p<0.01) by 9-13% at 12, 24, 48, and 72 hours
post-dose in the 1.25 mg/kg males with significant (p<0.01) linear trend tests at 48 and
72 hours post-dose. Plasma ChE activities were decreased (p<0.05) by 10-13% at 8, 12 and
24 hours post-dose in females (1.00 mg/kg). For RBC ChE activities, decreases (p<0.01) of
7%> and 11% were noted in the 1.25 mg/kg males and the 1.00 mg/kg females, respectively,
at 12 hours post-dose. In male subjects, decreases (p<0.01) in both plasma and RBC ChE
activities were noted only at 12 hours post-dose in the 1.25 mg/kg group. In female
subjects, the decreases (p<0.05) in both plasma and RBC ChE activities were noted only at
12 hours post-dose.
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Analytical methods and storage/stability for the determination of acephate and
methamidophos in human blood and urine were described and validated. The analytical
methods (6696 for human blood/plasma, and 6695 for human urine), were validated for the
determination of acephate and methamidophos at a concentration range of approximately 10
to 1,000 ng/mL.
There were several results that fell outside of the method validated range of 10 to 1,000
ng/mL for both human blood/plasma and urine. Mean urine concentration data for acephate
and methamidophos were outside of the method validated concentration range at the with
exceedances of 1,000 ng/mL for acephate at 0-12 hours (all dose levels, both sexes), 12-24
hours (all dose levels, both sexes), and 24-48 hours (1.00 and 1.25 dose levels (males). For
methamidophos, concentrations were below the limit of quantification of 10 ng/mL at 24-48
hours for the 0.70 mg/kg (males) and 1.0 mg/kg (females). Mean plasma concentration data
for acephate and methamidophos were also outside of the method validated concentration
range of 10-1000 ng/mL at several dose levels and timepoints. For acephate, mean plasma
concentrations were above 1000 ng/mL at 1.0 and 1.25 mg/kg in males at 1, 2, and 4 hours
post-dose, and at 1.0 mg/kg in females at 2 and 4 hours post-dose. For methamidophos, mean
plasma concentrations were below 10 ng/mL at 0.70 mg/kg (2, 8, and 12 hours post-dose),
1.0 mg/kg (1 and 12 hours post-dose), and 1.25 mg/kg (1 and 12 hours post-dose) in males,
and at 1.0 mg/kg (1 and 12 hours post-dose) in females.
This study is classified acceptable/non-guideline.
The following deficiencies were identified:
• Several values for both acephate and methamidophos in urine and blood/plasma were
outside of the method validated range of 10-1,000 ng/mL. However, information from
the study is still considered useful for improving predictability of future PBPK
modeling.
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Appendix I
Statistical Summary
Data analyses of plasma cholinesterase (ChE) and red blood cell (RBC) ChE measurements in
this study were reviewed by CEB/HED and summarized below (Nguyen, J., D466156, 10-FEB-
2023; TXR# 0058543).
The registrant used "a repeated measures analysis of variance (ANOVA) including terms for
dose level, timepoint (i.e., 1, 2, 4, 8, 12, 24, 48, 72 hours, day 7, and day 14) and dose level by
timepoint interaction" to analyze the percent change from baseline of plasma ChE and RBC
ChE, where each individual subject's plasma ChE (or RBC ChE) baseline value was the average
of measurements before dosing. Both a linear trend test of dose response and then pairwise
comparisons of dose groups vs. the placebo were performed at each timepoint to determine the
timepoint at which statistically significant increasing dose-response trends were observed and/or
statistically significant different responses between dose groups and the placebo group were
observed. The result of the linear trend test was used to determine whether the pairwise
comparisons of the dose groups vs. placebo at each timepoint were adjusted for multiple
comparisons. More specifically, if the p-value of the linear trend test at a specific timepoint was
> 0.05 indicating that observed dose-response trend was not statistically significant, then the
registrant used the Bonferroni method to adjust for multiple comparisons when performing the
pairwise comparisons at that timepoint; otherwise, if p-value of the linear trend test at a
timepoint was < 0.05, no adjustment was applied to the p-values of multiple pairwise
comparisons performed at that timepoint.
Reviewer's comments: While the registrant indicated "a repeated measures analysis of variance
(ANOVA)" was used to analyze the data, it was unclear what variance-covariance matrices the
registrant considered, or which were selected for the models. Also, CEB believes the Dunnett
test would be a more appropriate method to adjust for multiple comparisons of dose groups vs.
the placebo group than the Bonferroni method used by the registrant, because the Bonferroni
method severely sacrifices statistical power to maintain the family-wise error rate.
While reviewing the data, CEB statisticians also discovered that four subjects had extra
measurements of RBC ChE (subject "028" at 72 hours, subject "036" at 48 hours, subject "038"
at 72 hours, and subject "043" at 8 hours). In response to an EPA query regarding the rationale
surrounding these extra measurements, the registrant stated in a responding email that the repeat
measurements were taken when initial RBC ChE measurements were >20% from their baseline
value. However, there were several other measurements that met this criterion and did not have
repeat measurements taken. The initial measurements from the four subjects with repeat
measurements were also within the "normal" ranges provided in the study report for 18-50 year
old humans (page 152 of study report, MRID 45388301).
Given the unclear variance-covariance matrices being considered and selected by the registrant,
the use of the Bonferroni adjustment method, and the unclear reasons for including some
replicate measurements, CEB reanalyzed the plasma and RBC ChE data. CEB statisticians used
mixed effects models to analyze the percent change from the baseline of plasma and RBC ChE
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data. Dose group, timepoint, and the interaction between dose group and timepoint were included
by CEB in the models as fixed effects. In the models, each subject was set as an experimental
subject in the study design (i.e., random effect). For each specific plasma ChE or RBC ChE data
of each gender, CEB explored the following variance-covariance matrices: compound symmetric
(CS), CS by dose group, unstructured (UN), spatial linear (SP(LIN)), spatial power ((SP(POW)),
and SP(POW) by dose group with the intent of selecting that for which the lowest Bayesian
Information Criterion (BIC) value was found. Note that an autoregressive lag-l(AR(l)) variance-
covariance was not considered because the time intervals between the consecutive repeated
measurements were not consistent (i.e., unequal time intervals between consecutive timepoints).
A linear trend test at each timepoint was implemented through an "ESTIMATE" statement in
SAS using appropriate linear coefficients. Dunnett test (not Bonferroni) was used to compare
dose groups to the placebo group at each timepoint.
Given the confusing and inconsistent rationale regarding the extra RBC ChE measurements of 4
subjects, CEB statisticians conducted two separate analyses for the percent change from baseline
of RBC ChE as a sensitivity analysis: (i) one analysis that used the initial (first measured)
replicate values and (ii) an alternative analysis that used the second replicate values. CEB elected
to specifically NOT use the averages of the two replicates in the analysis since doing so would
result in analyses that would be biased. This is because the four subjects and the timepoints were
not randomly selected for obtaining second measurements, rather additional measurements were
taken depending on the value of the initial measurement. Within each data analysis, there were a
few outliers (studentized residuals > 3); given the small number of outliers, CEB statisticians
only performed analyses that included the outliers. All comparisons were done with two-sided
test at a significance level a = 0.05. CEB analysis was done using SAS 9.4.
A spatial power (SP(POW)) variance-covariance matrix was selected for the male plasma ChE
data, and a compound symmetric (CS) variance-covariance matrix was selected for the male
RBC ChE data based on BIC values. For female plasma ChE and RBC ChE, a spatial power
(SP(POW)) variance-covariance by dose group was selected for all datasets.
Overall statistical conclusions based on CEB's analyses (based on initial values only):
• Male Plasma ChE: there was evidence of acephate effect on blood plasma ChE in the
1.25 mg/kg (the percent change from baseline was significantly different from the
placebo at 12, 24, 48, and 72 hours; point estimates of reduction from baseline plasma
ChE were >10% at 12 hours and close to 10% at 24, 48 and 72 hours). Because there
were no significant differences in the percent change from baseline plasma ChE between
the dose groups 0.7 mg/kg or 1.0 mg/kg and the placebo group, the statistically
significant difference between the 0.35 mg/kg dose group and the placebo at 12 hours
was discounted even though the point estimate of reduction from baseline plasma ChE
was > 10%).
• Male RBC ChE: there was no evidence of acephate effect on the blood RBC ChE, even
at the highest dose level 1.25 mg/kg (note that the percent change from baseline RBC
ChE of the 1.25 mg/kg at 12 hours was significantly different from that of the placebo
group; however, the point estimate of RBC ChE reduction was <10%).
• Female Plasma ChE: there was evidence of acephate effect on the blood plasma ChE in
the dose group 1.0 mg/kg (the percent change from baseline was significantly different
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from the placebo at 8, 12, and 24 hours; point estimates of reduction from baseline
plasma ChE were > 10% at 8, 10, and 12 hours). Female RBC ChE: there was no
evidence of acephate effect on the blood RBC ChE in the dose group 1.0 mg/kg.
Conclusion
In conclusion, CEB identified issues with the registrant's approach and performed a more robust
statistical analysis using mixed models. In terms of statistical significance, the results of both the
registrant's analysis and CEB's analysis were similar. OPP will not use the ChE data from this
study to establish a NOAEL or LOAEL. Rather, the results of CEB's analysis effectively
reproduce the results of the registrant's statistical analyses and thus serve as positive
confirmation of the quality of the data for other regulatory purposes. If AChE data would be
considered in the future, CEB recommends that only the initial ChE measurement for each
subject be used.
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Appendix II
Calculations for the recovery of acephate and methamidophos in urine (%AD calculations)
from Table 8.
Subject
dose(mg/kg)
wt (kg)
mg dosed
ng dosed
2
0.35
82.8
28.98
28980000
3
0.35
67.4
23.59
23590000
5
0.35
70.5
24.675
24675000
6
0.35
93.4
32.69
32690000
7
0.35
94.8
33.18
33180000
9
0.35
62.7
21.945
21945000
11
0.35
72
25.2
25200000
min
max
0.7
63.9
44.73
44730000
12
0.7
81.8
57.26
57260000
15
0.7
74.2
51.94
51940000
16
0.7
60.5
42.35
42350000
18
0.7
60.7
42.49
42490000
20
0.7
79.2
55.44
55440000
21
0.7
60.9
42.63
42630000
min
max
17
76.2
76.2
76200000
23
77.8
77.8
77800000
25
79.9
79.9
79900000
26
78.4
78.4
78400000
27
70.8
70.8
70800000
29
83.7
83.7
83700000
31
73.8
73.8
73800000
min
max
28
1.25
67.6
84.5
84500000
32
1.25
78.9
98.625
98625000
33
1.25
78.3
97.875
97875000
34
1.25
74
92.5
92500000
35
1.25
54.7
68.375
68375000
37
1.25
71
88.75
88750000
40
1.25
62.2
77.75
77750000
methamidophos (mg-eq) meth (ng) metabolite (% AD) metabolite (% recovered) % acephate % meth eq Total
16.844
8.369
9.717
14.129
20.28
5.494
9.354
22.896
30.164
24.662
22.615
23.772
32.6
44.458
35.024
45.651
45.419
25.151
50.745
34.69
44.336
34.801
44.777
39.709
29.252
39.605
30.994
16844000
8369000
9717000
14129000
20280000
5494000
9354000
22896000
30164000
24662000
22615000
23772000
32686000
11778000
44458000
35024000
45651000
45419000
25151000
50745000
34690000
44336000
34801000
44777000
39709000
29252000
39605000
30994000
0.19339
0.08077
0.12917
0.1955
0.23144
0.07085
0.12469
0.35037
0.47491
0.34653
0.24892
0.2878
0.45193
0.13566
0.47158
0.387
0.62439
0.71535
0.36749
0.6022
0.54028
0.72361
0.60806
0.80512
0.82918
0.56063
0.54146
0.51163
193390
80770
129170
195500
231440
70850
124690
350370
474910
346530
248920
287800
451930
135660
471580
387000
624390
715350
367490
602200
540280
805120
829180
560630
541460
511630
mm
max
0.667322291
0.342390844
0.523485309
0.598042215
0.697528632
0.322852586
0.494801587
0.322852586
0.697528632
0.783299799
0.829392246
0.667173662
0.587768595
0.677335844
0.815169553
0.318226601
0.318226601
0.829392246
0.618871391
0.497429306
0.78146433
0.912436224
0.519053672
0.719474313
0.732086721
0.497429306
0.912436224
0.856343195
0.616537389
0.822600255
0.896410811
0.819934186
0.610095775
0.658045016
0.610095775
0.896410811
1.135091701 58.1228433 0.6673223 58.79017
0.955884006 35.476897 0.3423908 35.81929
1.31188066 39.3799392 0.5234853 39.90342
1.364794583 43.2211686 0.5980422 43.81921
1.128345938 61.1211573 0.6975286 61.81869
1.273169987 25.0353156 0.3228526 25.35817
1.315477139 37.1190476 0.4948016 37.61385
0.955884006 25.0 0.32 25.4
1.364794583 61.1 0.70 61.8
1.50720306
1.550022504
1.385647217
1.0887022
1.196186169
1.363784642
1.138692895
1.0887022
1.550022504
1.04959806
1.092880743
1.349291708
1.550579991
1.440093046
1.172800075
1.533567147
1.04959806
1.550579991
1.605894947
1.717244118
1.766306613
2.045429765
1.880511716
1.348711692
1.623931977
1.348711692
2.045429765
51.1871227
52.679008
47.4817097
53.4002361
55.9472817
58.9574315
27.6284307
27.6
59.0
58.343832
45.0179949
57.135169
57.932398
35.5240113
60.6272401
47.0054201
35.5
52.4686391
35.286185
45.7491699
42.9286486
42.7817185
44.6253521
39.8636656
35.3
52.5
0.7832998 51.97042
0.8293922 53.5084
0.6671737 48.14888
0.5877686 53.988
0.6773358 56.62462
0.8151696 59.7726
0.3182266 27.94666
0.32
0.83
0.6188714
0.4974293
0.7814643
0.9124362
0.5190537
0.7194743
0.7320867
0.50
0.91
0.8563432
0.6165374
0.8226003
0.8964108
0.8199342
0.6100958
0.658045
0.61
0.90
27.9
59.8
58.9627
45.51542
57.91663
58.84483
36.04306
61.34671
47.73751
36.0
61.3
53.32498
35.90272
46.57177
43.82506
43.60165
45.23545
40.52171
35.9
53.3
Female
Subj
ect dose(mg
wt (kg)
mg dosed
ng dosed
acephate (mg)
acephate(ng)
methamidophos (mg-eq)
meth (ng) metabolite (% AD)
metabolite (% recovered)
%acephate
% meth eq
Total
41
66.9
66.9
66900000
15.872
15872000
0.23792
237920
0.355635277
1.476854013
23.7249626
0.35563528
24.0806
42
51.3
51.3
51300000
18.352
18352000
0.22514
225140
0.438869396
1.211919596
35.7738791
0.4388694
36.21275
43
81.6
81.6
81600000
9.835
9835000
0.14857
148570
0.182071078
1.488145022
12.0526961
0.18207108
12.23477
45
53.6
53.6
53600000
15.259
15259000
0.20997
209970
0.391735075
1.357362513
28.4682836
0.39173507
28.86002
47
52.6
52.6
52600000
7.011
7011000
0.0897
89700
0.170532319
1.263255735
13.3288973
0.17053232
13.49943
49
79.5
79.5
79500000
12.451
12451000
0.12511
125110
0.157371069
0.994822723
15.6616352
0.15737107
15.81901
50
78.6
78.6
78600000
39.798
39798000
0.54332
543320
0.691246819
1.34680769
50.6335878
0.69124682
51.32483
min
0.157371069
0.994822723
12.1
0.16
12.2
max
0.691246819
1.488145022
50.6
0.69
51.3
-------
ACEPHATE /103301
Cholinesterase Inhibition and Pharmacokinetics in Humans (2001) / Page 30
OCSPP Non-Guideline
Appendix III. Study Selection Criteria.
Inclusion/exclusion criteria, pre-study screening parameters, restrictions, and actions resulting in
removal from the study were detailed in the study report (MRID 45388301, starting on page 30
of the PDF) and are provided below.
Inclusion criteria:
To be entered into the study, subjects had to fulfill the following criteria:
(a) Males and females 18-50 years of age.
(b) No clinically important abnormal physical findings at screening examination.
(c) No clinically relevant abnormalities in the results of laboratory screening evaluation
including plasma and RBC cholinesterase (Appendix C of the protocol).
(d) Normal ECG.
(e) Normal arterial pressure (BP) and heart rate (HR). Normal BP was taken to be 100 to 150
mm Hg systolic and 50 to 90 mm Hg diastolic. Normal HR was taken to be 50 to 90 beats
per minute (b.p.m.). Normal erect HR (after standing for 1 min) was taken to be 50 to 100
b.p.m.
(f) Body weight between 50 and 100 kg and within +/- 15% ideal body weight as shown in
Appendix D of the protocol (pg 154-155 of study report PDF).
(g) Able to communicate well with the study investigator and to comply with the
requirements of the entire study.
(h) Provision of written informed consent to participate as shown by a signature on the
volunteer consent form.
Exclusion criteria:
Any of the following would exclude a subject from the study:
(a) Administration of any investigational drug (or other test compound) in the period 0-3
months before entry to the study (0-4 months if previous investigational drug or other test
compound was a new chemical entity).
(b) A need for any medication during the period 0-5 days before entry to the study.
(c) Existence of any surgical or medical condition which, in the judgement of the clinical
investigator, might interfere with the absorption, distribution, metabolism or excretion of
the test compound.
(d) Presence or history of allergy requiring treatment.
(e) Donation or loss of greater than 400 mL of blood in the period 0-12 weeks before the
study.
(f) Serious adverse reaction or hypersensitivity to any drug.
(g) Inability to communicate or co-operate with the investigator because of a language
problem, poor mental development, or impaired cerebral function.
(h) Objection by the subject's general practitioner to his/her patient's participation in the
study.
(i) Females of childbearing potential who were not taking adequate contraceptive
precautions.
(j) Females with a positive urine pregnancy test.
-------
ACEPHATE /103301
Cholinesterase Inhibition and Pharmacokinetics in Humans (2001) / Page 31
OCSPP Non-Guideline
(k) Smokers who could not abstain from smoking from 2 h pre-dose to 8 h post dose.
(1) Any subject with a resting pulse of <45 b.p.m., a systolic BP of <100 mm Hg or a PR
interval on ECG of >210 ms.
(m) Any subject who had exposure to anti-cholinesterases (including home pest control
products) within one month of dosing.
(n) All agricultural workers or pest control applicators.
Pre-studv screen:
The screening examination consisted of:
1. Medical history.
2. Complete physical examination and vital signs (pulse rate, respiratory rate and blood
pressure).
3. 12-lead ECG recording.
4. Haematology, clinical chemistry, plasma and RBC cholinesterase and urinalysis.
5. Hepatitis B: Hbs-Ag
Hepatitis C: Hep Cab.
HIV infection: HIV antibody
6. Urine screening for drugs, including drugs of abuse (including cannabis).
Restrictions:
Volunteers were not permitted alcohol or other drugs whilst resident in the clinic and were
advised to limit their alcohol intake to no more than 2 units daily until after the last blood sample
had been withdrawn on Day 14. Caffeine was not permitted whilst the volunteers were resident
within the clinic. No concomitant medications (apart from paracetamol or other medications used
to treat adverse events) were allowed from 5 days before the study start or during the study
unless approved by the Study Director.
Actions resulting in Removal:
A subject could be withdrawn from the study in any of the following circumstances:
1. Serious adverse events.
2. Major violation of the protocol.
3. Withdrawal of consent.
4. Termination of the study by the Sponsor.
No subject was withdrawn or withdrew from the study.
------- |