Immunotoxicity ( 2011 ) Page 1 of 16

Paraquat Dichloride / 061601 OPPTS 8 70.7800 / DACO 4.8 / OECD None

EPA Reviewer: Ayaad Assaad, D.V.M , Ph.D. Signature :

RAB IV

, Health Effects Division (7509P ) Date :

EPA Secondary Reviewer : Yung Yang , Ph.D

. Signature :

RAB V I

, Health Effects Division (7509P ) Date :

Template version 0 2 /0 6

TXR # : 0056276

DATA EVALUATION RECORD

STUDY TYPE : Immunotoxicity [feeding ]

- in mice ; OPPTS 870.7800

PC CODE : 061601 DP BARCODE : D 396804

TEST MATERIAL (PURITY) : Paraquat Dichloride (99.9%)

SYNONY MS : N

, N ′

- dimethyl

- 4,4′

- bipyridinium dichloride

, Methyl viologen dichloride;

Crisquat; Dexuron; Esgram; Gramuron; Ortho Paraquat CL; Para

- col; Pillarxone

.

CITATION : Author : Eapen, A ( 2011 )

. Title: Paraquat Dichloride, Paraquat

- A 28

- Day Dietary

Immunotoxicity Study in B6C3F1 Female Mice. Laboratory name : WIL Research

Laboratories, LLC, Ashland, OH, USA. Laboratory report number : WIL

- 639105

, full study date: November 2, 2011, MRID # 48667301

. Unpublished.

SPONSOR : Syngenta Crop Prote ction, LLC, Greensboro, NC, USA.

EXECUTIVE SUMMARY :

In an immunotoxicity study (MRID # 48667301 ), Paraquat Dichloride (99.9%, Lot # ASJ10083

-

03 ) was administered to 10 female B6C3F1 mice /dose in diet at dose levels of 0, 25, 75, or 100

ppm (0, 6.9, 19.9 , 27.3 mg/kg bw/day). Two subsets were used in the study. Ten mice/group

(Subset A) were used for the splenic Antibody

- Forming Cell (AFC) assay and 10 mice/group

(Subset B) were used for the Natural Killer Cell (NKC) assay. Mice in the AFC positive contr ol

group (Group 5A) were administered the positive control substance, cyclophosph amide (CPS)

via intraperitoneal injection (50 mg/kg/day) once daily for 4 consecutive days (study days 24

through 27). All AFC group mice (Groups 1A

- 5A) were immunized with a n intravenous or

intraperitoneal injec tion of sheep red blood cells (S RBC) on study day 24, approximately 96

hours prior to the scheduled necropsy. Mice in the NKC positiv e control group (Group 6B) were

administered the positive control substance (anti a sial o GM1) via a single intravenous injection

(0.2 mL/animal) on study day 27, approximately 24 hours prior to the scheduled necropsy.

All animals were observed twice daily for mortality and morbidity. Detailed physical examinations

were performed once weekly. Individual bo dy weights were recorded twice weekly and food

consumption was recorded approximately weekly. Animals were sacrificed by inhalation of carbon

dioxide on day 28 th of the study, followed by blood sample collection for possible future cl inical

pathology and IgM antibody analyses

.Immunotoxicity ( 2011 ) Page 2 of 16

Paraquat Dichloride / 061601 OPPTS 8 70.7800 / DACO 4.8 / OECD None

Complete necropsies were conducted on all animals. The kidney, lung, and spleen were weighed from

all animals assigned to the study. In addition, t he thymus was weighed from all NKC

- designated

animals. Spleen s cell suspensions were prepared, spleen cell counts were performed, and the number

of specific IgM antibody

- form ing cells directed towards the sheep red blood cells (SRBC) antigen

were determined for the AFC

- designated animals to measure the humoral immu ne response. In

addition, s pleens from the NKC

- designated animals were prepared to measure innate immunity using

the NKC assay.

All animals survived to the scheduled necropsy, except for a 75 ppm single animal

. Th ere were

no treatment

- related clinical obs ervations or effects on body w eights, food consumption, organ

weights, or macroscopic findings for either the AFC or NKC assay animals. In the AFC assay,

there were no effects attributed to paraquat dichloride on the specific activity or total activity o f

splenic IgM AFC t o the T cell

- dependent antigen SRBC. In the NKC assay, there were no effects

attributed to paraquat dichloride on NKC activity. It was concluded that treatment of female

B6C3F1 mice with paraquat dichloride on a continuous basis in the d iet for a minimum of 28

conse cutive days at 25, 75, and 100 ppm resulted in no suppression of the humoral (AFC) or

innate (NKC) components of the immune system.

The LOAEL for immunotoxicity for both AFC and NKC assays is not established

. The

NOAEL for i mmunotoxicity is 100 ppm (27.3 mg paraquat dichloride/kg body weight/day ).

The systemic LOAEL for for both AFC and NKC assays is not established

. The systemic

NOAEL is 100 ppm (27.3 mg paraquat dichloride/kg body weight/day ), the highest dose

tested.

This immunotoxicity study is classified acceptable, guideline, and satisfies the guideline

requirement for an immunotoxicity study (OPPTS 870.7800) in mice.

COMPLIANCE : Signed and dated GLP, Quality Assurance, and Data Confidentiality

stateme nts were provided.Immunotoxicity ( 2011 ) Page 3 of 16

Paraquat Dichloride / 061601 OPPTS 8 70.7800 / DACO 4.8 / OECD None

I. MATERIALS AND METHODS

A. MATERIALS :

1. Test m aterial : Paraquat dichloride

Description: T ech nical, light gray, course powder

Lot #: ASJ10083

- 03 [WIL ID nos. 09013F and 110018]

Purity: 99.9 % a.i.

Compound Stabili ty: Stable at room temperature for 12 days.

CAS # of TGAI: 1910

- 42

- 5

Structure :

2. Vehicle and/or positive control :

Treatment vehicle: PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002

(meal)

. Positive control for AFC group : Cyclophosphamide monohydrate (100%), Lot #: 0 79K1569

Positive control for NKC group : Anti asialo GM1, Lot # AGH 5324.

Positive controls vehicle for both AFC & NKC : PBS solution prepared on premises.

Antigen: 7.5xl0 7 (sheep red blood cells) S RBC in 0.2 mL of buffer.

3 Test animals :

Species : Mice

Strain: B6C3F1

Age/weight at study initiation: 51 days, 18.1 g to 21.4 g for the AFC groups and 18.2 to 21.5 g for

the NKC groups at randomization.

Source: Charles River Laboratories, Inc., Raleigh, NC

Housing: Individually in clean, stainless steel, wire mesh cages suspended

above cage board.

Diet: PMI Nutrition Inter national, LLC, Certified Rodent LabDiet® 5002

(meal) ad libitum

Water: Reverse osmosis

- treated (on

- site) ad libitum

Environmental conditions: Temp erature:

Humidity:

Air changes:

Photoperiod: 22 ± 3°C

44.5% to 46.9%

10 /hr

12 hrs dark/12 hrs light

Acclimation period: 13

- day

B. STUDY DESIGN :

1. In life dates

- Start: 2 February 2011 End: 17 March 2011

2

. Animal assignment : Animals were ass igned to the study using a computerized

randomization procedure based on body weight stratification in a block design. Individual

body weights at randomization were within ± 20% of the group mean as noted in Table 1.Immunotoxicity ( 2011 ) Page 4 of 16

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Table 1. Experimental des ign a

a Data extracted from page 24, MRID # 48667301

. PQ

- C1 2 = Paraquat dichloride

Subset A= Mice designated for the AFC assay

Subset B= Mice designated for the NKC assay

3. Dose selection :

The dose levels were selected based on the results from previ ous dietary toxicity studies with

paraquate in mice (Maita et al, 1980; Sotheran et al, 1979a; Sotheran et al, 1979b; Sotheran

et al., 1979c; Sotheran et al., 1981). The US EPA DART (Paraquat: Report of the US EPA

DART, TXR No. 0055562 dated 8 Decembe r 2010) agreed with these dose levels. The

proposed high dose of 100 ppm (paraquat dichloride) was anticipated to be the highest

dietary concentration that would not potentially induce excessive lung damage and thus

possibly compromise interpretation o f study findings.

4. Diet preparation and analysis : (Appendix I ).

Analyses for homogen eity of the test substance was conducted at 25 and 100 ppm concentrations

prior to dosing . Following preparati on , samples were collected from the top, middle, and bot tom

strata of each form ulation

. The top and bottom strata were used for homogeneity analysis, while

t he middle stratum was used for stability determinations following room temperature storage

under laboratory conditions for up to12 days (Tables. 2 & 3)

.

For concentration analysis, d uplicate samples were collected from the top, middle, and bottom

strata of each prepared test substance diet admix formulation during study weeks 0 and 3. One set

of samples was transferred to the a nalytical laboratory fo r concentration analysis. Analyses of diet

admix formulations during the dosing period were conducted pri or to dosing. The remaining

samples were stored frozen at approximately

- 20°C as back

- up samples

. All analyses were

conducted using a validated high performance liquid chromatography method with mass

spectrometric detection (Table. 4)

.Immunotoxicity ( 2011 ) Page 5 of 16

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Results :

Homogeneity a nalysis: Test diet homogeneity was within the protocol

- specified acceptability limits

(90% to 110% of the target concentration and had a relati ve standard deviation [RSD] < 5%).

Table. 2 Homogeneity analysis a

a Data extracted from page 29 , MRID # 48667301.

S tability a nalysis: Samples were stable following 12 days of room temperature storage (90% to

110% of initial value).

Table. 3 Stability analysis a

a Data extracted from page 29 , MRID # 48667301.

Concentration a nalysis: The concentration of the test substance in all of the diet admix

formulations prepared on 14 February 2011 and the 75 (99.2%) ppm diet prepared on 7 March

2011 were within the protocol

- specified range (90% to 110% of ta rget concentration with a R2

>0.99). The 75 ppm diet admix was continued during the last week of test diet administration to

groups 3A/3B

.

The 25 and 100 ppm diet admix formul ations prepared on 7 March 201 1 had mean

concentrations that were 71.3% and 12 5%, respectively, of the target concentration; therefore, all

diets, including the control group, were re formulated on 10 March 2011.

However, the mean concentrations from the 25, 75, and 100 ppm diet admix formulations

prepared on 10 March 2011 were 117%, 132%, and 119%, respectively, of the target

concentration.

Therefore, t he test diets prepared on 7 March 2011 were used for test diet administration from 7

March 2011 until 10 March 2011 (groups 2A/2B & 4 A/4B) , and the test diet prepared on 10

March 2011 was used from 10 March 2011 until 16 March 2011 for groups (2A/2B & 4A /4B)

.Immunotoxicity ( 2011 ) Page 6 of 16

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In addition, the relative standard deviation (RSD) for the mean concentration of the diet admix

formulations prepared on 7 ( grou ps 2A/2B & 4A/ 4B) and 10 March 2011 (all groups) were not

within the protocol

- specified range (< 5%).

With the exception of the 25 ppm test diet (Group 2A/2B) administered from 7 March 2011 until

10 March 2011, the overall mean concentration of the analyz ed te st diet admix formulations met

or slight ly exceeded the range for concentration acceptability (i.e., ± 15% of the target

concentration). In addition, the mean concentration for each stratam (i.e., top, middle, bottom)

from each dietary adm ix formulati on prepared on 7 March 2011 and 10 March 2011 > 83% of

the target concentration

.

Administration of the out of specification 25 ppm dietary admix did not impact the integrity

or interpretation of the data for this study due to the limited administration o f the 25 ppm diet

admix formulation (i.e., approximately 3 days), since th e 25 ppm group was not used for

determination of the no

- observed

- effect

- level (NO A EL), and considering that the other analyzed

diet admix formulations met or exceeded the protoco l

- sp ecified acceptability range for

concentrations.

Table. 4 Concentration analysis a

a Data extracted from page 30 , MRID # 48667301.Immunotoxicity ( 2011 ) Page 7 of 16

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5. Statistics :

Table. 5 Statistical analysis a

Parameter(s) Statistical technique

1. Comparing paraquat

- treated grou ps to the vehicle

control group (Group 1A or IB). 1. Two

- tailed tests (except as noted otherwise) for

minimum significance levels of 1% or 5%.

2. Body weight, body weight change, and food

consumption. 2

. Parametric one

- way ANOVA to determine intergr oup

differences, followed by Dunnett's test to compare

treated groups (AFC Groups 2A

- 4A and NKC Groups

2B

- 4B) to the vehicle control group (Groups 1A and IB).

3 . The positive control group data (AFC Group 5A

[CPS] and NKC Group 6B [anti asialo GM1])

. 3. S tudent's t

- Test and compared to the respective

vehicle control group (Groups 1A and IB).

4. Absolute organ weights. 4. ANOVA followed by Dunnett's test

.

5. The adjusted organ weights based on terminal body weight. 5. ANCOVA followed by Dunnett's test

.

6. AFC data and NKC data. 6. Data were first tested for homogeneity of variances

using the Bartlett's Chi Square Test.

a. Homogenous data were evaluated using parametric

one

- way ANOVA followed by Dunnett's test

. b. Non

- homogenous data were evaluated u sing a

nonparametric analysis of variance

.

7. Dose

- related trends across the vehicle control and

treatment groups. 7. Jonckheere's Test.

a Data extracted from page 29

- 30 , MRID # 48667301.

C. METHODS :

1. Observations :

All animals were observed twice daily, once in the mo rning and once in the afternoon for

signs of toxicity and mortality . Detailed physical examinations were conducted on all animals

approximately weekly.

2. Body weight :

Animals were weighed twice weekly throughout the study, begi nning approximately 1 week

prior to randomization, on study day 0, and prior to the scheduled necropsy. Mean body

weights and mean body weight changes were calculated for the corresponding intervals.

3. Food/water consumption and compound intake :

Individual food consum ption was recorded w eekly during the pretest period and throughout

th e study, beginning 1 we ek prior to randomization and ending just prior to the scheduled

necropsy. Food intake was calculated as g/animal/day for each interval. The mean amounts of

paraquat dichloride consumed (mg/kg body weight/day) by each treatment group were

calculated from the mean f ood consumed (g food/kg of body weight/day using the average of

the first and last body weight during the week) and the appropri ate target concentration of

paraquat dichloride in th e food (mg test substance/kg of food). Food efficiency was not

calculated.Immunotoxicity ( 2011 ) Page 8 of 16

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4. Sacrifice and p athology

Animals were sacrificed by inhalation of carbon dioxide on day 28 th of the study, followed by

b lood sample collection for possible future clinical pathology and IgM antibody analyses

.

a. Gross necropsy :

A complete necropsy was conducted on all animals, except for the 75 ppm (Group 3) animal

(no. 6647) that escaped. The necropsies included, but were not limited to, examination of the

external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities,

including viscera.

The following tissues and organs were collected from animals at the scheduled necropsy

and placed in 10% neutral

- buffered formalin for future histopathological examination

except for the spleen which was p laced in EBSS/HEPES buffer :

• Peyer's patches (GALT)

• Spleen (p laced in EBSS/HEPES buffer; prepared for AFC or NKC assays )

• Liver

• Thymus

• L ymph node (mesenteric)

The following organs were weighed from all animals at the scheduled necropsy

, and o rgan to

final bod y weight ratios were calculated :

• Lung (AFC and NKC groups)

• Spleen (AFC and NKC groups)

• Kidney (AFC and NKC groups)

• Thymus (NKC grou ps)

b. Tissue preparation/histopathology : Was not conducted.

c. IgM Antibody Analysis (AFC Groups):

Blood samples (approximately 0.75 mL) were collected following euthanasia via the caudal

vena cava, and processed to obtain serum samples and stor e d frozen at approximately

- 70°C

. The analysis was not performed in this study.

5. Immunotoxicity :

a. Antibody plaque

- forming cell (A FC) assay :

Spleens were collected from all AFC group animals at the scheduled necropsy. Individual

spleens were placed into individual preweigh t ed tubes containing BBSS with 15 mM

HEPES and gentamicin as a bacteriostat. Each tube was then weighe d to provide a "wet"

weight for each spleen. T he spleens were accessioned and processed into single

- cell

suspen sions prepared in accordance with the performing lab’s SOP for receipt and

processing of mouse spleen. The cell suspensions were centr ifuged and resuspended in

EBSS with HEPES. Spleen cell counts were performed using a Model Zl Coulter

Counter

. Viability of splenocytes was determined using propidium iodide and theImmunotoxicity ( 2011 ) Page 9 of 16

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Coulter® EPICS® XL

- MCL Flow Cytometer. The AFC assay served to determine the

number of specific IgM antibody

- forming cells directed towards SRBC.

b

. NK cell Assay:

Spleens were collected from all NKC group animals at the scheduled necropsy. Individual

spleens w ere placed into individual pre

- weighted tubes containing EBSS with 1 5 mM

HEPES and gentamicin as a bacteriostat. Each tube was then weighed to provide a "wet"

weight for each spleen. T he spleens were accessioned in accordance with the performing

lab’s SOP for receipt and processing of mouse spleen. The spleen samples were

processed into single

- cell suspensions , and t he cell suspensions were centrifuged and

r e

- suspended in BBSS with HEPES. Spleen cell counts were performed using a Model Zl

Coulter Counter®. Viability of splenocytes was determined using propidium iodide and

the Coulter® EPICS® XL

- MCL Flow Cytometer. The NKC assay was performed to

measure innate immunity

.

II. RESULTS :

A. OBSERVATIONs :

1. Clinical signs of toxicity : There were no paraquat dichloride

- related clinical observations.

All clinical findings in the paraquat

- treated groups were noted with similar incidence in the

con trol group, were limited to single animals, were not noted in a dose

- related manner, and/or

were common findings for laboratory mice of this age and strain.

2. Mo rtality : There were no treatment

- related mortalities. All animals survived to the

schedule d necropsy, except for a 75 ppm animal (no. 6647) that escaped during the study

which was euthanized and discarded.

B. Body weight and weight gain : Body weights were unaffected by treatment. Statistically

significant differences in mean body weight and body weight gain were probably due to

biological variability and were not considered treatment

- related

.

C. FOOD/WATER CONSUMPTION AND COMPOUND INTAKE :

1. Food consumption : Food consumption was not affected by treatment. Occasionally noted

statistica lly significant differences in food consumption were probably due to biological

variability and were not considered treatment

- related

.

2. Water consumption : Was not measured.

3. Compound consumption : Average compound consumptions (mg/kg of body weight/ day)

over the entire study for the AFC and NKC groups were based on target dietary

concentrations of paraquat dichloride (Table. 6).Immunotoxicity ( 2011 ) Page 10 of 16

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Table. 6

Average Paraquat Dichloride Consumptions (mg/kg/day) b

a = AFC and NKC Groups combined.

b Data obtai ned from page 33, MRID # 48667301.

4. Food efficiency : was not calculated.

D. GROSS NECROPSY : There were no treatment

- related macroscopic findings. A single 100

ppm NKC group animal had dark red area(s) on the spleen; however, this finding was not

co nsidered to be treatment

- related because it was only noted in a single animal in both the

AFC and NKC groups.

1. Organ weight : There were n o effects of paraquat administration on organ weights.

Statistically significantly higher mean absolute kidney we ight in the 25 ppm AFC group

compared to the vehicle control group was not considered to be test treatment

- related due to

the lack of a dose

- response effect and the lack of similar findings in the 25 ppm NKC group.

S tatistically significantly lower sple en weights were observed in the AFC positive control

group as expected (Table. 7 A & 7B ).

TABLE 7A (AFC GROUPS) a

SUMMARY OF ORGAN WEIGHTS AND ORGAN WTS. RELATIVE TO BODY WTS.

a Data obtained from page 67

, MRID # 48667301.Immunotoxicity ( 2011 ) Page 11 of 16

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TABLE 7A (Cont. AFC GROUPS) a

SUMMARY OF ORGAN WEIGHTS AND ORGAN WTS. RELATIVE TO BODY WTS.

a Data obtained from page 68

, MRID # 48667301.

TABLE 7B (NKC GROUPS) a

SUMMARY OF ORGAN WEIGHTS AND ORGAN WTS. RELATIVE TO BODY WTS.

a Data obtained from page 69

, MRID # 48667301.Immunotoxicity ( 2011 ) Page 12 of 16

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TABL E 7B (Cont. NKC GROUPS) a

SUMMARY OF ORGAN WEIGHTS AND ORGAN WTS. RELATIVE TO BODY WTS.

a Data obtained from page 70

, MRID # 48667301.

2. Histology : Not performed.

E. IMMUNOTOXICITY TESTS :

a. Antibody plaque

- forming cell (A FC) assay : There were no e ffects of paraquat

dichloride on spleen cell numbers in the AFC groups when compared to control group

. As expected, statistically significantly lower spleen cell numbers were observed in the

positive control group (CPS). In the functional evalu ation of the IgM AFC response,

exposure to paraquat dichloride did not suppress the humoral immune response when

evaluated as either specific activity (AFC/10 6 spleen cells) or total spleen activity

(AFC/spleen). As anticipated, statistically s ignificantly lower spleen cell numbers (63%),

specific activity (100%), and total spleen activit y (100%) was noted in the positive

control group (CPS) group (Fig. 1)

.Immunotoxicity ( 2011 ) Page 13 of 16

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Figure 1

IgM Antibody

- Forming Cell Responses to Sheep Erythrocytes in Female B6C3F1

Mice Exposed to Paraquat Dichloride via Diet for 28 Days

- Day 4 Response a

a Data obtained from page 146

, MRID # 48667301.

b. NK cell a ssay: There were no effects of paraquat dichloride on NKC activity in the NKC

groups when co mpared to control. Statistically significantly lower NKC activity was

noted in the positive control group (anti asialo GM1) at all effector: target ratios when

compared to the vehicle control group (Fig. 2).

Figure 2

Natural Killer Cell Activity i n Female B6C3F1 Mice Exposed to Paraquat Dichloride

via Diet for 28 Days a

a Data obtained from page 147

, MRID # 48667301.Immunotoxicity ( 2011 ) Page 14 of 16

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III. DISCUSSION AND CONCLUSIONS :

A. INVESTIGATORS CONCLUSIONS :

The investigators concluded

, b ased on the results of this st udy, that treatment of female

B6C3F 1 mice with paraquat dichloride on a continuous basis in the diet for a minimum of 28

consecutive days a t 25, 75, and 100 ppm resulted in no suppression of the humoral (AFC) or

innate (NKC) components of the immune sys tem. The no

- observed

- effect level (NO A EL) for

both the AFC assay (humoral immunity) and the NKC assay (innate immunity) was 100 ppm

(27.3 mg paraquat dichloride/kg body weight/day ), the highest dose examined.

B. REVIEWER COMMENTS :

In an immunotoxicity study (MRID # 48667301 ), Paraquat Dichloride (99.9%, Lot #

ASJ10083

- 03 ) was administered to 10 female B6C3F1 mice /dose in diet at dose levels of 0,

25, 75, or 100 ppm (0, 6.9, 19.9, 27.3 mg/kg bw/day). Two subsets were used in the study.

Ten mice/gro up (Subset A) were used for the splenic Antibody

- Forming Cell (AFC) assay

and 10 mice/group (Subset B) were used for the Natural Killer Cell (NKC) assay. Mice in the

AFC positive control group (Group 5A) were administered the positive control substance,

cyclophosph amide (CPS) via intraperitoneal injection (50 mg/kg/day) once daily for 4

consecutive days (study days 24 through 27). All AFC group mice (Groups 1A

- 5A) were

immunized with an intravenous or intraperitoneal injec tion of sheep red blood cell s (S RBC)

on study day 24, approximately 96 hours prior to the scheduled necropsy. Mice in the NKC

positiv e control group (Group 6B) were administered the positive control substance (anti

asial o GM1) via a single intravenous injection (0.2 mL/animal) on study day 27,

approximately 24 hours prior to the scheduled necropsy.

All animals were observed twice daily for mortality and morbidity. Detailed physical

examinations were performed once weekly. Individual body weights were recorded twice weekly

an d food consumption was recorded approximately weekly. Animals were sacrificed by

inhalation of carbon dioxide on day 28 th of the study, followed by blood sample collection for

possible future clinical pathology and IgM antibody analyses

.

Complete ne cropsies were conducted on all animals. The kidney, lung, and spleen were

weighed from all animals assigned to the study. In addition, the thymus was weighed from all

NKC

- designated animals. Spleens cell suspensions were prepared, spleen cell counts were

performed, and the number of specific IgM antibody

- forming cells directed towards the sheep

red blood cells (SRBC) antigen were determined for the AFC

- designated animals to measure the

humoral immune response. In addition, spleens from the NKC

- designa ted animals were prepared

to measure innate immunity using the NKC assay.

All animals survived to the scheduled necropsy, except for a 75 ppm single animal

. Th ere

were no treatment

- related clinical observations or effects on body w eights, food

consum ption, organ weights, or macroscopic findings for either the AFC or NKC assay

animals. In the AFC assay, there were no effects attributed to paraquat dichloride on the

specific activity or total activity of splenic IgM AFC to the T cell

- dependent antigen SRBC.

In the NKC assay, there were no effects attributed to paraquat dichloride on NKC activity. It

was concluded that treatment of female B6C3F1 mice with paraquat dichloride on a

continuous basis in the diet for a minimum of 28 consecutive days at 2 5, 75, and 100 ppmImmunotoxicity ( 2011 ) Page 15 of 16

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resulted in no suppression of the humoral (AFC) or innate (NKC) components of the

immune system.

The LOAEL for immunotoxicity for both AFC and NKC assays is not established

. The NOAEL for immunotoxicity is 100 ppm (27.3 mg paraquat dichloride/kg body

weight/day ).

The systemic LOAEL for for both AFC and NKC assays is not established

. The

systemic NOAEL is 100 ppm (27.3 mg paraquat dichloride/kg body weight/day ), the

highest dose tested.

This immunotoxicity study is classi fied acceptable, guideline, and satisfies the guideline

requirement for an immunotoxicity study (OPPTS 870.7800) in mice.

C. STUDY DEFICIENCIES:

No major deficiencies were detected in the study to influen ce the outcome or the end

- point

selection.Immunotoxicity ( 2011 ) Page 16 of 16

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Appe ndix I

Analyses of Diet Admix Formulations Report * :

* Analyses of diet admix formulations report was copied from pages 30

- 31, MRID # 48667301.