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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 VI, Health Effects Division (7509P) Date:

Template version 02/06

TXR #: 0056276

DATA EVALUATION RECORD

STUDY TYPE: Immunotoxicity [feeding]-in mice; OPPTS 870.7800

PC CODE: 061601 DP BARCODE: D396804 

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

SYNONYMS: 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 Unpublished.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 Protection, 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 control 

group (Group 5A) were administered the positive control substance, cyclophosphamide (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 injection of sheep red blood cells (SRBC) on study day 24, approximately 96 

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

administered the positive control substance (anti asialo 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 and food 

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

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

pathology and IgM antibody analyses.
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Complete necropsies 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-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. There were 

no treatment-related clinical observations or effects on body weights, 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 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 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 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 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 

statements were provided. 
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I. MATERIALS AND METHODS

A. MATERIALS:

1. Test material: Paraquat dichloride

Description: Technical, light gray, course powder

Lot #: ASJ10083-03 [WIL ID nos. 09013F and 110018]

Purity: 99.9% a.i.

Compound Stability: 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 #: 079K1569

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

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

Antigen: 7.5xl07 (sheep red blood cells) SRBC 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 International, LLC, Certified Rodent LabDiet® 5002 

(meal) ad libitum

Water: Reverse osmosis-treated (on-site) ad libitum

Environmental conditions: Temperature:

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 assigned 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. 
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Table 1. Experimental designa

a

Data extracted from page 24, MRID # 48667301.

PQ-C12= 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 previous 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 December 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 of study findings.

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

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

prior to dosing. Following preparation, samples were collected from the top, middle, and bottom 

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

the middle stratum was used for stability determinations following room temperature storage 

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

For concentration analysis, duplicate 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 analytical laboratory for concentration analysis. Analyses of diet 

admix formulations during the dosing period were conducted prior 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). 
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Results:

Homogeneity analysis: Test diet homogeneity was within the protocol-specified acceptability limits 

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

Table. 2 Homogeneity analysisa

a

Data extracted from page 29, MRID # 48667301.

Stability analysis: Samples were stable following 12 days of room temperature storage (90% to 

110% of initial value).

Table. 3 Stability analysisa

a

Data extracted from page 29, MRID # 48667301.

Concentration analysis: 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 target 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 formulations prepared on 7 March 2011 had mean 

concentrations that were 71.3% and 125%, respectively, of the target concentration; therefore, all 

diets, including the control group, were reformulated 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, the test diets prepared on 7 March 2011 were used for test diet administration from 7 

March 2011 until 10 March 2011 (groups 2A/2B & 4A/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).
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In addition, the relative standard deviation (RSD) for the mean concentration of the diet admix 

formulations prepared on 7 (groups 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 analyzed test diet admix formulations met 

or slightly 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 admix formulation 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 of the 25 ppm diet

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

determination of the no-observed-effect-level (NOAEL), and considering that the other analyzed 

diet admix formulations met or exceeded the protocol-specified acceptability range for 

concentrations.

Table. 4 Concentration analysisa

a

Data extracted from page 30, MRID # 48667301.
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5. Statistics:

Table. 5 Statistical analysisa

Parameter(s) Statistical technique

1. Comparing paraquat-treated groups 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 intergroup 

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. Student'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 using 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 morning 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, beginning 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 consumption was recorded weekly during the pretest period and throughout 

the study, beginning 1 week 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 food consumed (g food/kg of body weight/day using the average of 

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

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

calculated.
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4. Sacrifice and pathology

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

blood 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 placed in EBSS/HEPES buffer:

• Peyer's patches (GALT)

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

• Liver 

• Thymus

• Lymph node (mesenteric)

The following organs were weighed from all animals at the scheduled necropsy, and organ to 

final body weight ratios were calculated:

• Lung (AFC and NKC groups)

• Spleen (AFC and NKC groups)

• Kidney (AFC and NKC groups)

• Thymus (NKC groups)

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 stored frozen at approximately -70°C.

The analysis was not performed in this study.

5. Immunotoxicity:

a. Antibody plaque-forming cell (AFC) assay:

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

spleens were placed into individual preweighted tubes containing BBSS with 15 mM 

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

weight for each spleen. The spleens were accessioned and processed into single-cell 

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

processing of mouse spleen. The cell suspensions were centrifuged 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 the 
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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 were placed into individual pre-weighted tubes containing EBSS with 15 mM 

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

weight for each spleen. The 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 the cell suspensions were centrifuged and

re-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 

control 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. Mortality: There were no treatment-related mortalities. All animals survived to the 

scheduled 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 

statistically 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).
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Table. 6 

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

a = AFC and NKC Groups combined.

b

Data obtained 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 

considered 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 no effects of paraquat administration on organ weights.

Statistically significantly higher mean absolute kidney weight 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.

Statistically significantly lower spleen weights were observed in the AFC positive control 

group as expected (Table. 7A & 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.
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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.
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TABLE 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 (AFC) assay: There were no effects 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 evaluation of the IgM AFC response, 

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

evaluated as either specific activity (AFC/106spleen cells) or total spleen activity 

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

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

control group (CPS) group (Fig. 1).
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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 Responsea

a

Data obtained from page 146, MRID # 48667301.

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

groups when compared 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 in Female B6C3F1 Mice Exposed to Paraquat Dichloride

via Diet for 28 Daysa

a

Data obtained from page 147, MRID # 48667301.
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III.DISCUSSION AND CONCLUSIONS:

A. INVESTIGATORS CONCLUSIONS:

The investigators concluded, based on the results of this study, that treatment of female 

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

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

innate (NKC) components of the immune system. The no-observed-effect level (NOAEL) 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/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 control group (Group 5A) were administered the positive control substance,

cyclophosphamide (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 injection of sheep red blood cells (SRBC) 

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

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

asialo 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 

and food consumption was recorded approximately weekly. Animals were sacrificed by

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

possible future clinical pathology and IgM antibody analyses.

Complete necropsies 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-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. There 

were no treatment-related clinical observations or effects on body weights, 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 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 25, 75, and 100 ppm 
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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 classified 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 influence the outcome or the end-point

selection.
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Appendix I

Analyses of Diet Admix Formulations Report*: 

*

Analyses of diet admix formulations report was copied from pages 30-31, MRID # 48667301.