### Page 2 of 18



## II. BACKGROUND

In 2005, a revised HED human health risk assessment for formetanate HCl was issued . The

dose and endpoint for acute dietary (general population) exposure scenarios were based on

female pup ChE inhibition observed in a comparative ChE study in rats (MRID 46618901)

. However, a re

- evaluation of the ChE assay methodologies and ChE data in November 2008

concluded that t he study was Unacceptable/Non

- Guideline for risk assessment purposes

( D356185; TXR 0055042 ). The re

- evaluation noted a significant number of assay errors in the

measurement of ChE activity which likely resulted in t he underestimation of the true ChE

inhibit ion. Fur the rmore, the difficulty in consist ently and accurately measuring ChE activity

likely resulted in the poor dose

- response in both sexes of both pups and adults that was reported

in the original DER of this study (D320464, TXR 0053305).

In 2009

, post

- natal day 17 ( PND17 ) rat pup ChE data bec a me available from the EPA Office of

Research Development (MRID 47692902 )

. This study examined both brain and red blood cell

( RBC ) ChE in male PND17 rat (Long Evans) pups (female pups not examined) 4 0 minutes after

a single dose of 0, 0.1, 0.3, 0.75, or 1.5 mg/kg/day. A n a nalysis of these data was performed

with an exponential benchmark dose ( BMD ) modeling program written in R language , and the

acute dietary point of departure ( PoD ) for formetanate w as re

- eval uated on the basis of the 2009

BMD results ( D 364562

, TXR 00555179 ) . A 10x FQPA safety factor, however, was retained

based on the lack of acceptable PND11 ChE data for the acute dietary risk assessment. This

FQPA factor was consistent with other N

- methyl c arbamates without acceptable PND11 ChE

data.

In 2010, Gown submitted to the Agency a new formetanate CCA study ( MRID 48298401 ) that

was conducted at Charles River Laboratories, the same laboratory which performed the 2005

formetanate CCA study. The new C CA study was reviewed by HED and the male PND11 ChE

data (both RBC and brain) were considered unaccept able for use in risk assessment ( D 383678,

TXR0055523 ). As in the 2005 CCA study, there were numerous assay errors that likely resulted

in the underestima tion of the true brain and RBC ChE activity in the PND11 male pups

. The

ChE data from the PND11 female pups and male and female adults, however, are considered

acceptable since the same level of assay errors were not present in these groups

. Since both C hE

compartments are now available for formetanate in PND11 female pups, a new PoD was

determined along with a n appropriate FQPA safety factor to be consistent with the other N

methyl carbamates

. T he discussion below provides details regarding the weight

- o f

- evidence

(WOE) development of both the PoD as well as the FQPA safety factor for formetanate based on

the results of the acceptable PND11 and PND17 ChE data.

III. RESULTS/DISCUSSION

Benchmark Dose Analysis

Detailed results of the new BMD analysis (ou tputs) based on the 2010 formetanate CCA study

( MRID 48298401 ) are incl uded in Appendix 1 . A summary of the BMD estimates for all ChE

data with formetanate i s presented in Table 1 below.

### Page 3 of 18 

## Table 1. BMD 1 

## R esults for PND11 and PND17 R ats to F ormetanate

AGE SEX Compartment BMD 10

mg/kg 

## BMDL 10

mg/kg 

## Timepoint

(min) MRID

PND11 Male Brain & RBC NA 2 

## NA NA 48298401

PND11 Female

(BMDS) 2 

## Brain 0.041 0.032 30 48298401

PND11 Female

(BMDS) 2 

## RBC 0.067 0.034 30 48298401

PND17 Male

(BMDS) 2 

## Brain 0.059 0.051 40 47692 902

PND17 Male

(BMDS) 2 

## RBC 0.047 0.037 40 47692902

1 2010 Benchmark dose analysis using the current BMDS exponential model . D381401, TXR0055532

2 NA: Data not available/ not acceptable



## PND11 ChE Results

The RBC and brain ChE data from the male PND11 pups from the new CCA study are

unacceptable due to numerous assay errors that resulted in the underestimation of the true

inhibition of the enzyme. The female PND11 ChE data , however, were considered acceptable

for use in risk assessment (D383678, TXR 0055523 , final pending ). The ChE compartments in

the PND11 females are 1.6x different with the brain BMD 10 

## of 0.041 mg/kg and RBC BMD 10 

## of

0.067 mg/kg. The difference in brain and RBC BMDL 10 

## estimates are 1.1x with the brain

BMDL 10 

## of 0.032 mg/kg and RBC BMDL 10 

## of 0.034 mg/kg.

A comparison of the PND11 ChE data with the PND17 ChE data required the reanalysis of the

PND17 data. The previous BMD analysis of the PND17 data (2009) was performed with an

exponential BMD modeling program written in R lan guage , which was the only available model

at the time ( D 381408, TXR0055532 ). To compare the PND17 with the new PND11 ChE data

(2010) which were analyzed with the BMDS software , the PND17 ChE data were also analyzed

with the BMDS software. The BMDS expone ntial model includes a family of nested models that

provides improved model fitting for all kinds of datasets. Because of the flexibility and

advantages of the EPA BMDS exponential models, the formetanate data were reanalyzed with

this software. The BMDS exponential model result ed in significantly higher BMD 10 

## and

BMDL 10 

## estimates for both PND17 RBC and brain ChE

. The 2010 BMDS analysis suggests that

PND17 brain and RBC are similar in ChE sensitivity with only a 1.2 fold difference between the

BMD 10 

## esti mates. Furthermore, the BMD 10 

## estimates displayed in Table 1 suggest that the

PND11 brain ChE data (BMD 10 

## 0.041 mg/kg) are 1. 4 x more sensitive than the PND17 brain ChE

(BMD 10 

## 0.059 mg/kg) . For RBC ChE, the PND11 BMDL 10 

## (0.034 mg/kg) estimate is similar t o

the PND17 BMDL 10 

## (0.037 mg/kg) estimate

.

### Page 4 of 18



## IV. Endpoint and Point of Departure (PoD) Weight of Evidence (WOE)

A weight of evidence determination was performed for all of the ChE data available for

formetanate. The BMD 10 

## estimates for RBC and brain ChE from PND11 females from the recent

CCA study ( MRID 48298401 ) suggest no significant differences in compartment sensitivity but

brain slightly more sensitive (i.e., lower)

. The BMDL 10 

## estimates are also similar but with brain

ChE again slightly more sensit ive. Therefore, the brain BMDL 10 

## of 0.032 mg/kg from the

PND11 female pups is the most appropriate for use as the PoD in the acute dietary risk

assessment. This PoD is protective and consistent with PoDs established for other NMCs. Since

the slightly mor e sensitive compartment (i.e

. , brain ChE) from the most sensitive subpopulation

(PND11) is the basis of the PoD, an FQPA factor should not be retained. Furthermore, t here are

no sex differences in the adult formetanate data or for the other NMCs to suggest the PND11

females would be less sensitive than PND11 males to formetanate. Therefore, the acute dietary

aPAD of 0.0003 2 mg/kg is based on a PoD of 0.0 32 mg/kg (PND11 female brain BMDL 10 

## ), per

a 1 x FQPA safety factor

, 10x intraspecies factor

, and 10x inte rspecies factor.

Table 2. Acute Dietary Endpoints for Formetanate Human Health Risk Assessment

Exposure/Scenario Point of Departure Uncertainty/FQPA

Factor RfD, PAD, Level of

Concern for risk

assessment Study and

Toxicological

Effects

Acute Dietary

(ge neral population,

including infants and

children) BMDL 10 

### = 0.032

mg/kg UFA= 10x

UFH= 10x

FQPA = 1x aRfD = 0.00032

mg/kg/day

aPAD= 0.00032

mg/kg/day Comparative

Cholinesterase Study

( MRID 48298401 )

Acceptable RBC and

brain ChE data in

PND11 female pups

Brai n BMD 10 

### :

0.041mg/kg

Brain BMDL 10 

### :

0.032 mg/kg



## V. CONCLUSIONS

The single chemical assessments for the recently updated NMCs have consistently evaluated

compartment sensitivity , especially in PND11 pups

. Formetanate was therefore evaluated in a

consistent f

ashion to the other NMCs. Reliable RBC and brain ChE data are available in PND11

females for formetanate. The PoD of 0.032 mg/kg for the acute dietary assessment is based on

the brain ChE BMDL 10 

## in PND11 female pups. Since the PoD is based upon the mos t sensitive

subpopulation and the most sensitive ChE compartment

, retention of the FQPA factor is not

warranted. The aPAD of 0.00032 mg/kg is based on the PoD of 0.032 mg/kg, with a 1x FQPA,

10x intraspecies factor, and 10x interspecies factor.

### Page 5 of 18 

# APPENDIX 1

RBC PND11

- female

### Page 6 of 18



## formetanate new

CCA ACUTE NONGUIDELINE PND11 RBC F 30 min

High dose dropped

Exponential

Model var

10 bmr

====================================================================

Exponential Model. (Version: 1.7; Date: 12/10/2009)

Input Data File: C:/Usepa/BMDS212/Data/exp_Testrunscontinuous_Setting.(d)

Gnuplot Plotting File:

Tue Nov 16 09:01:34 2010

====================================================================

BMDS Model Run

~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

The form of the response function by Model:

Model 2: Y[dose] = a * exp{sign * b * dose}

Model 3: Y[dose] = a * exp{sign * (b * dose)^d}

Model 4: Y[dose] = a * [c

- (c

- 1) * exp{

- b * dose}]

Model 5: Y[dose] = a * [c

- (c

- 1) * exp{

- (b * dose)^d}]

Note: Y[dose] is the median response for exposure = dose;

sign = +1 for increasing trend in data;

sign =

- 1 for decreasing trend.

Model 2 is nested within Models 3 and 4.

Model 3 is nested within Model 5.

Model 4 is nested within Model 5.

Dependent variable = Mean

Independent variable = Dose

Data are assumed to be distributed: normally

Variance Model: ex p(lnalpha +rho *ln(Y[dose]))

The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)

Total number of dose groups = 4

Total number of records with missing values = 0

Maximum number of iterations = 250

Relative Function Con vergence has been set to: 1e

- 008

Parameter Convergence has been set to: 1e

- 008

MLE solution provided: Exact

Initial Parameter Values

Variable Model 2 Model 3 Model 4 Mod el 5

-------- ------- ------- ------- -------

lnalpha

- 4.20227

- 4.20227

- 4.20227

- 4.20227

rho 3.0041 3.0041 3.0041 3.0041

a 0.996322 1.1328 2.2449 2.2449

b 2.90384 1.82258 2.99935 2.99935

### Page 7 of 18 c

-- -- 0.000334982

0.000334982

d

-- 3

-- 1

Parameter Estimates by Model

Variable Model 2 Model 3 Model 4 Model 5

-------- ------- ------- ------- -------

lnalpha

- 4.26902

- 4.34669

- 4.26902

- 4.34669

rho 3.04507 3.06327 3.04507 3.06327

a 2.22012 2.06288 2.22012 2.06288

b 2.97577 2.79384 2.97577 2.79384

c

-- -- 0 2.19711e

- 024

d

-- 1.34119

-- 1.34119

Table of Stats From Input Data

Dose N Obs Mean Obs Std Dev

----- --- ---------- -------------

0 5 2.138 0.386

0.04 8 1.874 0.345

0.12 8 1.67 0.235

0.36 7 0.752 0.081

Estimated Values of Interest

Model Dose Est Mean Est Std Scaled Residual

------- ------ ---------- --------- ----------------

2 0 2.22 0.3984

- 0.4609

0.04 1.971 0.3324

- 0.8252

0.12 1.553 0.2313 1.425

0.36 0.7605 0.07798

- 0.2901

3 0 2.063 0.345 0.4869

0.04 1.957 0.3181

- 0.7341

0.12 1.638 0.2422 0.3792

0.36 0.753 0.0737

- 0.03665

4 0 2.22 0.3984

- 0.4609

0.04 1.971 0.3324

- 0.8252

0. 12 1.553 0.2313 1.425

0.36 0.7605 0.07798

- 0.2901

5 0 2.063 0.345 0.4869

0.04 1.957 0.3181

- 0.7341

0.12 1.638 0.2422 0.3792

0.36 0.753 0.0737

- 0.03665

Other models for which likelihoods are calculated:

Model A1: Yij = Mu(i) + e(ij)

Var{e(ij)} = Sigma^2

Model A2: Yij = Mu(i) + e(ij)

Var{e(ij)} = Sigma(i)^2

Model A3: Yij = Mu(i) + e(ij)

Var{e(ij)} = exp(lalpha + log(mean(i)) * rho)

Model R: Yij = Mu + e(i)

### Page 8 of 18 Var{e(ij)} = Si gma^2

Likelihoods of Interest

Model Log(likelihood) DF AIC

------- ----------------- ---- ------------

A1 23.99972 5

- 37.99945

A2 30.61741 8

- 45.23483

A3 30.45202 6

- 48.90404

R 1.940927 2 0.118145

2 28.98214 4

- 49.96429

3 30.08243 5

- 50.16485

4 28.98214 4

- 49.96429

5 30.08243 6

- 48.16485

Additive constant for all log

- likelihoods =

- 25.73. This constant added to the

above values gives the log

- likelihood including the term that does not

depend on the model parameters.

Explanation of Tests

T est 1: Does response and/or variances differ among Dose levels? (A2 vs. R)

Test 2: Are Variances Homogeneous? (A2 vs. A1)

Test 3: Are variances adequately modeled? (A2 vs. A3)

Test 4: Does Model 2 fit the data? (A3 vs. 2)

Test 5a: Does Mod el 3 fit the data? (A3 vs 3)

Test 5b: Is Model 3 better than Model 2? (3 vs. 2)

Test 6a: Does Model 4 fit the data? (A3 vs 4)

Test 6b: Is Model 4 better than Model 2? (4 vs. 2)

Test 7a: Does Model 5 fit the data? (A3 vs 5)

Test 7b: Is Mode l 5 better than Model 3? (5 vs. 3)

Test 7c: Is Model 5 better than Model 4? (5 vs. 4)

Tests of Interest

Test

- 2*log(Likelihood Ratio) D. F. p

- value

-------- ------------------------ ------ --------------

Test 1 57.35 6 < 0.0001

Test 2 13.24 3 0.004154

Test 3 0.3308 2 0.8476

Test 4 2.94 2 0.23

Test 5a 0.7392 1 0.3899

Test 5b 2.201 1 0.138

Test 6a 2. 94 2 0.23

Test 6b 0 0 N/A

Test 7a 0.7392 0 N/A

Test 7b

- 7.105e

- 015 1 N/A

Test 7c 2.201 2 0.3328

The p

- value for Test 1 is less than .05. There appears to be a

difference between response and/or variances among the dose

levels, it seems appropriate to model th e data.

The p

- value for Test 2 is less than .1. A non

- homogeneous

variance model appears to be appropriate.

### Page 9 of 18 The p

- value for Test 3 is greater than .1. The modeled

variance appears to be appropriate here.

The p

- value for Test 4 is greater than .1. Model 2 seems

to adequately describe the data.

The p

- value for Test 5a is greater than .1. Model 3 seems

to adequately describe the data.

The p

- value for Test 5b is greater than .05. Model 3 does

not seem to fit the data better than Model 2.

The p

- value for Test 6a is greater than .1. Model 4 seems

to adequately describe the data.

Degrees of freedom for Test 6b are less than or equal to 0.

The Chi

- Square test for fit is not valid.

Degrees of freedom for Test 7a are less than or equal to 0.

The Chi

- Square test for fit is not valid.

The p

- value for Test 7b is less than .05. Model 5 appears

to fit the data better than Model 3.

The p

- value for Test 7c is greate r than .05. Model 5 does

not seem to fit the data better than Model 4.

Benchmark Dose Computations:

Specified Effect = 0.100000

Risk Type = Relative deviation

Confidence Level = 0.950000

BMD and BMDL by Model

Model BMD BMDL

------- ------------ ----------

2 0.0354061 0.0322295

3 0.0668501 0.0339757

4 0.0354061 0.029042

5 0.0668501 0.0339757

### Page 10 of 18

### Page 11 of 18

### Page 12 of 18 

# APPENDIX 2

brain PND11

- female

### Page 13 of 18



### formetanate new

CCA ACUTE NONGUIDELINE PND11 BRAIN F 30 min



## Exponential

Constant var

10 bmr

===================================================== ===============

Exponential Model. (Version: 1.7; Date: 12/10/2009)

Input Data File: C:/Usepa/BMDS212/Data/exp_Testrunscontinuous_Setting.(d)

Gnuplot Plotting File:

Tue Nov 16 09:12:53 2010

================================== ==================================

BMDS Model Run

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

The form of the response function by Model:

Model 2: Y[dose] = a * exp{sign * b * dose}

Model 3: Y[dose] = a * exp{sign * (b * dose)^d}

Model 4: Y[dose] = a * [c

- (c

- 1) * exp{

- b * dose}]

Model 5: Y[dose] = a * [c

- (c

- 1) * exp{

- (b * dose)^d}]

Note: Y[dose] is the median response for exposure = dose;

sign = +1 for increasing tren d in data;

sign =

- 1 for decreasing trend.

Model 2 is nested within Models 3 and 4.

Model 3 is nested within Model 5.

Model 4 is nested within Model 5.

Dependent variable = Mean

Independent variable = Dose

Data are a ssumed to be distributed: normally

Variance Model: exp(lnalpha +rho *ln(Y[dose]))

rho is set to 0.

A constant variance model is fit.

Total number of dose groups = 5

Total number of records with missing values = 0

Maximum number of iterat ions = 250

Relative Function Convergence has been set to: 1e

- 008

Parameter Convergence has been set to: 1e

- 008

MLE solution provided: Exact

Initial Parameter Values

Variable Model 2 Mode l 3 Model 4 Model 5

-------- ------- ------- ------- -------

lnalpha

- 1.21046

- 1.21046

- 1.21046

- 1.21046

### Page 14 of 18 rho(S) 0 0 0 0

a 2.40207 2.40207 5.58705 5.58705

b 1.25126 1.25126 4.03036 4.03036

c

-- -- 0.256887

0.256887

d

-- 1

-- 1

(S) = Specified

Parameter Estimates by Model

Variable Model 2 Model 3 Model 4 Model 5

-------- ------- ------- ------- -------

lnalpha

- 0.949194

- 0.949193

- 1.20882

- 1.20921

rho 0 0 0 0

a 5.10881 5.10881 5.34674 5.33365

b 1.67587 1.67587 3.5434 3.58703

c

-- -- 0.260103 0.265245

d

-- 1

-- 1.02562

Table of Stats From Input Data

Dose N Obs Mean Obs Std Dev

----- --- ---------- -------------

0 7 5.321 0.48

0.04 8 4.862 0.529

0.12 8 3.961 0.765

0.36 8 2.494 0.672

1 6 1.507 0.293

Estimated Values of Interest

Model Dose Est Mean Est Std Scaled Residual

------- ------ ---------- --------- ----------------

2 0 5.109 0.6221 0.9024

0.04 4.778 0.6221 0.3838

0.12 4.178 0.6221

- 0.9871

0.36 2.795 0.6221

- 1.366

1 0.9561 0.6221 2.169

3 0 5.109 0.6221 0.9024

0.04 4.778 0.6221 0.3838

0.12 4.178 0.6221

- 0.9871

0.36 2.795 0.6221

- 1.366

1 0.9561 0.6221 2.169

4 0 5.347 0.5464

- 0.1246

0.04 4.824 0.5464 0.197

0.12 3.977 0.5464

- 0.08025

0.36 2.495 0.5464

- 0.007527

1 1.505 0.5464 0.008554

5 0 5.334 0.5463

- 0.06125

0.04 4.83 4 0.5463 0.1473

0.12 3.986 0.5463

- 0.1317

0.36 2.483 0.5463 0.05721

1 1.511 0.5463

- 0.01793

Other models for whi ch likelihoods are calculated:

### Page 15 of 18 Model A1: Yij = Mu(i) + e(ij)

Var{e(ij)} = Sigma^2

Model A2: Yij = Mu(i) + e(ij)

Var{e(ij)} = Sigma(i)^2

Model A3: Yij = Mu(i) + e(ij)

Var{e( ij)} = exp(lalpha + log(mean(i)) * rho)

Model R: Yij = Mu + e(i)

Var{e(ij)} = Sigma^2

Likelihoods of Interest

Model Log(likelihood) DF AIC

------- ----------------- ---- ------------

A1 3.893561 6 4.212879

A2 7.109295 10 5.78141

A3 3.893561 6 4.212879

R

- 33.05449 2 70.10897

2

- 0.939906 3 7.879812

3

- 0.939906 3 7.879812

4 3.863087 4 0.2738264

5 3.870359 5 2.259282

Additive constant for all log

- likelihoods =

- 34. This constant added to the

above values gives the log

- likelihood including the term t hat does not

depend on the model parameters.

Explanation of Tests

Test 1: Does response and/or variances differ among Dose levels? (A2 vs. R)

Test 2: Are Variances Homogeneous? (A2 vs. A1)

Test 3: Are vari ances adequately modeled? (A2 vs. A3)

Test 4: Does Model 2 fit the data? (A3 vs. 2)

Test 5a: Does Model 3 fit the data? (A3 vs 3)

Test 5b: Is Model 3 better than Model 2? (3 vs. 2)

Test 6a: Does Model 4 fit the data? (A3 vs 4)

Test 6b: Is Model 4 better than Model 2? (4 vs. 2)

Test 7a: Does Model 5 fit the data? (A3 vs 5)

Test 7b: Is Model 5 better than Model 3? (5 vs. 3)

Test 7c: Is Model 5 better than Model 4? (5 vs. 4)

Tests of Interest

Test

- 2*log(Likelihood Ratio) D. F. p

- value

-------- ------------------------ ------ --------------

Test 1 80.33 8 < 0.0001

Test 2 6.431 4 0.1692

Test 3 6.431 4 0.1692

Test 4 9.667 3 0.02162

Test 5a 9.667 3 0.02162

Test 5b

- 3.058e

- 011 0 N/A

Test 6a 0.06095 2 0.97

Test 6b 9.606 1 0.001939

Test 7a 0.0 464 1 0.8294

### Page 16 of 18 Test 7b 9.621 2 0.008146

Test 7c 0.01454 1 0.904

The p

- value for Test 1 is less than .05. There appears to be a

difference between response and/or variances among the dose

levels, it seems appropriate to model the data.

The p

- value for Test 2 is greater than .1. A homogeneous

variance model appears to be appropriate here.

The p

- value for T est 3 is greater than .1. The modeled

variance appears to be appropriate here.

The p

- value for Test 4 is less than .1. Model 2 may not adequately

describe the data; you may want to consider another model.

The p

- value for Test 5a is less than .1. Model 3 may not adequately

describe the data; you may want to consider another model.

Degrees of freedom for Test 5b are less than or equal to 0.

The Chi

- Square test for fit is not valid.

The p

- value for Test 6a is grea ter than .1. Model 4 seems

to adequately describe the data.

The p

- value for Test 6b is less than .05. Model 4 appears

to fit the data better than Model 2.

The p

- value for Test 7a is greater than .1. Model 5 seems

to adequately describe the data.

The p

- value for Test 7b is less than .05. Model 5 appears

to fit the data better than Model 3.

The p

- value for Test 7c is greater than .05. Model 5 does

not seem to fit the data better than Model 4.

Benchmar k Dose Computations:

Specified Effect = 0.100000

Risk Type = Relative deviation

Confidence Level = 0.950000

BMD and BMDL by Model

Model BMD BMDL

------- ------------ ----------

2 0.0628692 0.0512403

3 0.0628692 0.0512403

4 0.0409787 0.0317504

5 0.0427913 0.0317731

### Page 17 of 18

### Page 18 of 18