### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

i SCIENTIST FINAL REPORT

PHARMACOKINETIC EVAL UATION OF THE EQUIVA LENCY OF ORAL ROUTES OF

MANGANESE EXPOSURE I N F344 RATS

A UTHORS 

### :

David C. Dorman, DVM, PhD, DABVT, DABT

Melanie L. Foster, BS, DABT

Thomas Bartnikas, MD, PhD

P ERFORMING 

### L ABORATOR IES



### College of Veterinary Medicine Pathology and Laboratory Medicine

North Carolina State University Brown University

1060 William Moore Drive 70 Ship St.

Raleigh, NC 27607 Providence, RI 02912

USA USA

L ABORATORY 

### P ROJECT 

### ID: (1940) 2013

- 2727

S TUDY 

### S PONSOR 

### : A FTON 

### C HEMICAL 

### C ORPORATION



### 500 S PRING 

### S T



### , R ICHMOND



### , VA 23219

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

ii T ABLE OF 

### C ONTENTS



### S ECTION 

### P AGE



### A BSTRACT 

### 1

I NTRODUCTION 

### 2

- 3

P URPOSE AND 

### S PECIFIC 

### A IMS 

### 3

S TUDY 

### D ESIGN 

### O VERVIEW 

### 4

K EY 

### S TUDY 

### P ERSONNEL 

### 5

M ATERIALS AND 

### M ETHODS 

### 5

- 10

 Chemicals 5

 Animals 5

- 6

 Animal Husbandry 6

- 7

 Veterinary C are 7

 Manganese Exposure 7

- 8

 Necropsy and Tissue Collection 8

- 9

 Chemical Analyses 9

 Statistical Analyses 10

R ESULTS 

### 10

- 18

 C LINICAL 

### E FFECTS 

### 10

- 11

 E STIMATED 

### O RAL 

### M ANGANESE 

### E XPOSURE 

### 12

- 13

 T ISSUE 

### M ANGANESE 

### C ONCENTRATIONS 

### 13

- 16

 T ISSUE 

### I RON



### , C OPPER



### , AND 

### Z INC 

### C ONCENTRATIONS 

### 17

- 18

D ISCUSSION 

### 19

- 24

A CKNOWLEDGEMENTS 

### 24

R EFERENCES 

### 25

- 29

A PPENDIX 

### 1: I NDIVIDUAL 

### A NIMAL 

### D ATA 

### 30

- 67

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

iii T ABLE OF 

### C ONTENTS 

### (C ONTINUED 

### )

F IGURE 

### N UMBER 

### P AGE



### Figure 1. Experimental design. An abnormally high (200 ppm) diet

- was

intended to result in increased biliary manganese excretion and likely

increased tissue manganese concentration. 4

Figure 2. Mean body weights (g) in rats exposed for 61 day s to a 10

- ppm

manganese diet (Control), 200

- ppm manganese diet (High Diet), drinking

water exposure (High Water), and gavage exposure (Gavage). 11

Figure 3. Average daily feed (A) and water (B) consumption in animals in the

61

- day exposure group. High

- dose manganese treatments began on study

day 10. Peaks in water consumptions seen were most commonly associated

with cage change days and reflect sp illage. 12

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

iv T ABLE OF 

### C ONTENTS 

### (C ONTINUED 

### )

T ABLE 

### N UMBER 

### P AGE



### Table 1. Assignment of animals to the 7

- day exposure day group.

Individual and mean (± SEM) body weights are also provided. 6

Table 2. M ean (± SEM) absolute liver and brain weights and hemato crit

following 61 exposure days

. 11

Table 3

. Mean (± SEM) tissue manganese concentrations (μg Mn/g)

following 7 exposure days. Number in parentheses = number of samples

(n). 14

Table 4

. Mean (± SEM) tissue manganese concentrations (μg Mn/g)

following 61 exposure days. Number in parentheses = number of samples

(n). 14

Table 5 . Tissue manganese concentrations (μg Mn/g) following control diet

(10 ppm) or a high dose oral manganese exposure. Exposure duration did

not have a statistically significant effect so data from the 7

- and 61

- day

exposure groups were pooled (mean ± SEM; number in parentheses

represent total number of samples analyzed). 15

Table 6 . Tissue manganese concentrations (μg Mn/g) following 7

- or 61

-

day s of exposure to either a control diet (10 ppm) or excess manganese in

diet (200 ppm Mn), drinking water, and gavage (mean ± SEM; number in

parentheses represent total number of samples analyzed). 16

Table 7

. Mean (± SEM) tissue iron concentrations (μg Fe/g) following 7 or

61 exposure days. Number in parentheses = number of samples (n). 17

Table 8

. Mean (± SEM) tissue copper concentrations (μg Cu/g) following 7

or 61 exposure days. Number in parentheses = number of samples (n). 18

Table 9

. Mean (± SEM) tissue z inc concentrations (μg Zn/g) following 7 or

61 exposure days. Number in parentheses = number of samples (n). 18

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

v T ABLE OF 

### C ONTENTS 

### (C ONTINUED 

### )

A PPENDIX 

### : INDIVIDUAL ANIMAL DA TA



### T ABLE 

### N UMBER 

### P AGE



### Table A1. A ssignment of animals to the 7

- day exposure day group.

Individual and mean (± SEM) initial and terminal body weights (g) are also

provided. 31

Table A2. Assignment of animals to the 61

- day exposure day group.

Individual and mean (± SEM) body weights ar e also provided. 32

Table A3. Individual and mean absolute liver and brain weights and

hematocrit following 61 exposure days. 33

Table A4. Individual animal body weights (g) in the 61 exposure day

groups. 34

- 35

Table A5. Individual animal feed consumption (g/day)

– 7

- day exposure

group. 36

- 37

Table A 6 . I Individual animal water consumption ( ml /day)

– 7

- day exposure

group. 38

- 39

Table A 7 . Individual animal feed consumption (g/day)

– 61

- day exposure

group. 40

- 4 9

Table A 8 . Individual animal water consumption (ml/day)

– 61

- day exposure

group. 50

- 5 9

Table A 9 . Individual animal tissue manganese concentrations (μg Mn/g

tissue wet weight) following 7 exposure days. 60

- 61

Table A 10 . Individual animal tissue manganese concentrations (μg Mn/g

tissue wet weight) following 61 exposure days. 62

- 63

Table A 11 . Individual animal tissue metal concentrations (μg /g tissue wet

weight) following 7 exposure days. 64

- 65

Table A1 2 . Individual animal tissue metal concentrations (μg /g tissue wet

weight) following 61 exposure days. 66

- 6 7s

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

1 A BSTRACT



### Concerns exist as to whether individuals may be at greater risk for neurotoxicity

following increased oral intake of manganese. This study assessed the impact of oral

dose rate on the pharmacokinetics of manganese. Adult male rats were allocated to

contr ol diet (10 ppm), high manganese diet (200 ppm), manganese

- supplemented

drinking water, and manganese gavage treatment groups. Animals in the drinking water

and gavage groups were given the 10 ppm manganese diet and supplemented with

manganese chloride (Mn Cl 2 

### ) in drinking water or once daily gavage to provide a daily

manganese intake equivalent to that seen in the high manganese diet group. No

statistically significant difference in body weight gain or terminal body weights was seen.

Rats were anesthetized with ketamine and x ylazine following 7 and 61 exposure days

and samples of bile and blood collected. Rats were then euthanized and striatum,

olfactory bulb, frontal cortex, cerebellum, liver, spleen, and femur samples were

collected for chemical analysis. Hematocrit was unaffected by manganese exposure.

Liver and bile manganese concentrations were elevated in all treatment groups (day 61

,

relative to controls). In addition, increased cerebellum manganese concentrations were

seen in animals from the high m anganese diet and gavage groups (day 61 ). Increased

femur, striatum, and olfactory bulb manganese concentrations were also seen followin g

gavage exposure (days 7 and 61 ) suggesting that dose rate is an important factor in the

pharmacokinetics of oral mang anese. Th ese data will be used to refine physiologically

based pharmacokinetic models to extend their utility for manganese risk assessment to

include multiple dietary exposures. High dose manganese exposure was also

associated with secondary effort s on t issue iron, copper, and zinc concentrations. For

example, we found that high dose manganese exposure (diet, drinking water, and

gavage) was associated with increased liver copper and elevated spleen zinc

concentrations after 7 exposure days. High dose ma nganese g avage was also

associated with increased liver iron and zinc concentrations (day 7) and increased

frontal cortex (day 61) iron

, copper , and zinc concentrations after 61 exposure days.

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

2 I NTRODUCTION



### Manganese is an essential mineral that is found at low levels in virtually all diets

(ATSDR, 2012). Ingestion is the principal route by which most people are exposed to

manganese, although toxicologically significant inhalation exposures also occur. The

gastrointestinal tract and hepatobiliary systems play an important regulatory role in

maintaining tissue manganese within a relatively narrow physiologic range (Aschner and

Aschner, 2005). For example, a moderate increase in manganese dietary intake is

ac companied by a compensatory decrease in gut absorption, increased liver manganese

concentra tion , and elevated biliary excretion in order to maintain normal manganese

concentrations in the brain and other extrahepatic tissues (Aschner and Aschner, 2005;

Dor man et al., 2001). Excessive exposure to manganese or hepatic injury can

overwhelm these normal homeostatic controls resulting in elevated tissue manganese

and toxicity (Guilarte, 2010).

In humans, manganese

- induced neurotoxicity is of primary concern and is thought to be

the most sensitive endpoint. An extreme case of neurotoxicity caused by manganese is

known as manganism, which is usually caused by chronic inhalation of high levels of

manganese

. Hallmarks of manganism include behavioral changes, ex trapyramidal

motor dysfunction, and neurochemical and neuropathological changes in the basal

ganglia and globus pallidus (Roels et al., 2012). Manganese

- induced cognitive deficits

have also been reported in workers with exposure to manganese

- based welding fumes

(Bowler et al., 2006; Chang et al., 2010; Ellingsen et al., 2008; Park et al., 2009).

Affected brain regions included the striatum, globus pallidus, and other dopaminergic

and γ

- aminobutyric acid (GABA)

- containing mid

- brain structures which control motor

functions (Guilarte, 2013).

In contrast to the numerous reports describing manganese toxicity following

occupational inhalation exposure in humans, there are relatively few reports of

manganism arising from water or dietary sources. In part, this trend is due to the

relatively low levels of manganese found in these media. For example, most diets in

North America result in manganese intakes below 10 mg/day (Finley and Davis, 1999).

Water concentrations of manganese typically range from 1 to 100 μg/ l with most values

below 10 μg/l (Keen and Zidenberg

- Cherr, 1994). On rare occasions, manganese

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

3 toxicity results from ingestion. For example, Kawamura and coworkers (1941) and

Kondakis et al. (1989) documented outbreaks of manganism in Japan and Greece due

to the consumption of water from wells contaminated with extremely high levels of

manganese (1.8 to 14 mg Mn/l). There has also been increasing concern regarding the

role of environmental manganese exposure and children ’s health (Zoni and Lucchini,

2013) . For example, several community studies have purported a link between

manganese content in drinking water and effects on children’s IQ (Wasserman et al.,

2006; Bouchard et al., 2011; Khan et al., 2012) , although questions remain as to the

validity of the se studies

.

Many questions remain as to the conditions under which ingestion of manganese can

result in an increased incidence of human neurological disease (Boyes, 2010).

Experimental animal studies remain an important tool for the study of manganese

pharmacokinetics and neurotoxicity (Dorman et al., 2012). Animal studies examining the

pharmacokinetics and toxicity of ingested manganese often rely on three exposure

methods: diet, drinking water, or gavage. Most dietary studies rely on the use of a

de fined rodent diet to which different amounts of manganese have been added.

Drinking water studies often use manganese chloride (MnCl 2 

### ) or other soluble salts or

insoluble manganese oxides (e.g., MnO 2 

### ). Gavage studies rely on the oral ‘injection’ of

a bolus dose of manganese that is typically dissolved in an aqueous media. Both

drinking water and dietary exposure studies mimic adult human exposures and result in

slower and repeated daily exposures to manganese. Although these different exposure

methods are used, little data is available to assess the impact of either oral dose rate

(e.g., gavage versus drinking water) or manganese intake form (e.g., drinking water

versus diet) on the pharmacokinetics of manganese.

P URPOSE AND 

### S PECIFIC 

### A IMS



### The overall objective of this study was to determine whether dose rate influences the

pharmacokinetics of manganese and evaluate whether dietary and drinking water

manganese intake result in equivalent tissue manganese concentrations.

 Specific Aim 1. Determine whether dose rate influences the pharmacokinetics of

manganese.

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

4  Specific Aim 2. Determine whether dietary and drinking water manganese intake

result in equivalent tissue manganese concentrations.

S TUD Y 

### D ESIGN 

### O VERVIEW



### This study built on our laboratory’s previous research experience evaluating the

pharmacokinetics of manganese following inhalation and oral exposures (reviewed in

Dorman et al., 2012). Figure 1 provides an overview of the experimental design.

Figure 1. Experimental design. An abnormally high (200 ppm) diet was intended

to result in increased biliary manganese excretion and likely increased tissue

manganese concentration.

Four treatment groups were used: Control (AIN

- 93G diet containing 10 ppm Mn); high

dose dietary manganese (AIN

- 93G diet supplemented with 200 ppm Mn); high dose

drinking water manganese (AIN

- 93G diet containing 10 ppm manganese and drinking

water to provid e a daily manganese intake equivalent to the 200 ppm diet group); and

high dose manganese gavage (AIN

- 93G diet containing 10 ppm manganese and once

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

5 daily gavage to provide a daily manganese intake equivalent to the 200 ppm diet group).

Exposures were cond ucted 7 days/week for 7 or 6 1 exposure days.

K EY 

### S TUDY 

### P ERSONNEL



### David C. Dorman, DVM, PhD, DABVT, DABT: Principal Investigator. Oversight for all

phases of the research project.

Melanie L. Foster, BS, DABT : Co

- Investigator/Research Associate

. Involved in all

facets of the study.

Lucia Lazarowski BA , M A : Research Specialist. Assisted with necropsies.

Laura C. Johnson, BA : NCSU Summer Intern. Involved in all facets of the study.

Carol A. Bobbitt, AAS, VMT, LATG , The Hamner Institutes. Assisted with necropsies

and bile collection

Thomas Bar t nikas, MD, PhD : Brown University. Chemical analyses.

Carolina Herrera , BS : Research Assistant, Brown University. Chemical analyses.

Michael A. Pettiglio , BS : Research Assistant, Brown University. Chemical analyses.

M ATERIALS AND 

### M ETHODS



### Chemicals

Manganese (Mn +2 

### ) chloride (MnCl 2 

### •4 H 2 

### O), was obtained from Sigma Aldrich Chemical

Corporation (Milwaukee, WI). The material is a crystalline powder that contains 27.3%

manganese by weight and is relatively soluble in water.

Chemical Name CAS Registry Number

Manganese chloride tetrahydrate 13446

- 34

- 9

Animals

Male 5

- 6 week old F344 rats (Charles River, Raleigh, NC) were used. Assignment of

animals to treatment groups was based upon a weight randomization procedure.

Animals were acclimated for approximately 14 days prior to the start of the manganese

exposure. Our goal was to have a minimum of 8 rats per treatment group available for

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

6 tissue collection at the end of the study. To this end, the dietary and drinking water

groups s tarted with 9 rats/group while the gavage group started with 11 rats. Each lot of

feed (n = 1/diet ) and gavage solutions (n = 3) were analyzed for manganese content.

 Animal welfare oversight: The experimental protocol was reviewed and approved

by the NCSU Institutional Animal Care and Use Committee (IACUC). NCSU

IACUC approval occu rred on December 20 , 201 3 (IACUC # 1 3

- 0140

- B ).

 NCSU animal facility: Research was performed within the CVM’s Laboratory

Animal Resources (LAR) unit. The facility is inspected semiannually by the

NCSU IACUC, and the CVM is accredited by the Association f or the Assessment

and Accreditation of Laboratory Animal Care International (AAALAC).

Table A 1 Provides animal identification, assignment to treatment groups, and weight at

randomization. Table 1 provides a summary of the group means at randomization.

Table 1

. Group m ean ( ± SEM) body weights at randomization for the 7

- and 61

- day

exposure groups.

Group Mean Body weight (g)

Day 7

Control 130.6 ± 4.5

High diet 129.4 ± 3.1

High Water 131.1 ± 4.3

Gavage 128.6 ± 3.6

D ay 61

Control 138.9 ± 4.4

High diet 135.4 ± 4.2

High Water 136.9 ± 3.1

Gavage 137.4 ± 4.1

Animal Husbandry

 Animal rooms and exposure chambers were maintained at daily temperatures of

22 ± 4°C, relative humidity of 30

- 70%, and an air flow rate sufficient to provide

10

- 15 air changes per hour. Fluorescent lighting was controlled by automatic

controls (lights on a pproximately 0700

- 1900).

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

7  Rats were individually housed in polycarbonate cages.

 On arrival, rats were given their previous Purina 5001 diet (Purina Mills, St.

Louis, MO) for the first week during the acclimation period. Afterwards, pelleted,

semi

- purifi ed AIN

- 93G certified diet from Bio

- Serv (Frenchtown, NJ) formulated

to contain 10 ppm manganese and 35 ppm iron was fed to all animals.

 Food and water was available to all animals ad libitum.

 Body weights were measured and recorded at the end of quaran tine when they

were randomized, at least weekly throughout the study, and on the day of

necropsy.

Veterinary C are

Each animal received a phys ical examination upon arrival (26 Feb 2014 [7

- day]; 12 Mar

201 4 [ 61

- day ] ) at the NCSU facility. A veterinarian was available at all times, including

after

- hours/holiday/weekend, and reviewed all Veterinary Services reports.

Manganese Exposure

Four treatment groups were used:

 Control

o Diet: AIN

- 93G diet containing 10 ppm Mn

 High dose dietary manganese

o Diet: AIN

- 93G diet supplemented with 200 ppm Mn

 Began 1

- 2 days after randomization

o A staggered start was used to allow us to determine dietary manganese

intake of the animals in the high manganese diet group.

 High dose drin king water manganese:

o Diet: AIN

- 93G diet containing 10 ppm manganese

o Drinking water: manganese chloride in drinking water to provide a daily

manganese intake equivalent to the 200 ppm diet group

o Mean dietary data (200 ppm Mn group) was used to adjust weekl y Mn

intake rates for drinking water.

 High dose manganese gavage

o Diet: AIN

- 93G diet containing 10 ppm manganese

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

8 o Gavage: once daily gavage with manganese chloride in water to provide

a daily manganese intake equivalent to the 200 ppm diet group

 Gavage e xposures (volume = 2 ml/kg body weight) occurred

between 0800 and 1000 each day using manual restraint and a

rodent gavage tube (18 gauge)

 Mean dietary data (200 ppm Mn group) was used to adjust weekly

intake rates for gavage dose

 Exposures were conducted 7 days/week for 7 or 61 exposure days.

 Assignment of animals to treatment groups was based upon a weight

randomization procedure that occurred at the end of the two

- week acclimation

period.

 A single lot of diet was used, it was stored under refrigeratio n, and feed changed

at least weekly.

 Exposures began on 11, 13, and 15 MAR 2014 (7

- day exposure group) and 28,

29, and 31 MAR 2014 (6 1

- day exposure group).

o Animals from the 200

- ppm diet groups were started on the first day, the

drinking water group start ed on day two, and the gavage group began on

the third day.

Necropsy and Tissue Collection

Necropsies were performed 4

- 6 hr following the gavage exposure (or equivalent) after 7

and 61 exposure days. Animals were anesthetized to effect with ketamine (80

- 100

mg/kg, ip) and xylazine (5

- 10 mg/kg, ip) and ~ 1

- ml samples of bile and blood (serum)

were collected using previously described methods (Dorman et al., 2001). Immediately

afterwards, all ra ts were euthanized by exsanguination and the following tissues

collected: striatum, olfactory bulb, frontal cortex, liver, spleen, and proximal portion of the

left femur for tissue manganese determination. Samples of the following tissues were

also collect ed: pancreas and carcass, but were not analyzed further

. Plastic syringes

fitted with a hypodermic needle were used for blood collection. Blood samples (~ 1

- 2 ml)

were placed into heparinized tubes (Becton Dickinson, Franklin Lakes, NJ) containing

~15

– 20 IU heparin/ml blood. A small (~ 50

- 80 μl) aliquot of blood was used to determine

the packed cell volume. Tissue samples were weighed and transferred to plastic vials,

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

9 frozen in liquid nitrogen, and stored at approximately

- 80 ̊C until manganese analyses

w ere completed.

Necropsies were performed on 18, 20, and 21 MAR 2014 (7

- day exposure group) and

28, 29, and 30 MAY 2014 ( 61

- day exposure group). Control animal necropsies were

split across the three days. Animals from the 200

- ppm diet groups were necropsied on

the first day, animals from the drinking water group on the second day, and animals from

the gavage group on the third day.

Chemical A nalyses

Liver (10

- 100 mg), food (150

- 300 mg) and gavage (25 μl) samples were thawed then

digested in 1 mL 70% Optima grade nitric acid (Fisher Scientific, Pittsburgh, PA) at 65°C

for 2 hours, then diluted 25

- fold with MilliQ

- purified water (EMD Millipore, Billerica, MA)

and analyzed in triplicate by inductively coupled plasma atomic emission spectrometry

(ICP

- AES) using a JY2000 Ultrace spectrometer (Horiba Scientific, Edison, NJ). 0.1 mL

serum and bile and 10

- 100 mg spleen, femur, frontal cortex, striatu m, olfactory bulb and

cerebellum were digested using a stepwise protocol: digestion in 0.2 mL 70% nitric acid

and heating at 85°C until dry, resuspension in 0.2 mL 30% Optima grade hydrogen

peroxide (Fisher Scientific, Pittsburgh, PA) and heating at 85°C u ntil dry, repeated

resuspension in hydrogen peroxide and heating until dry, resuspension in 0.2 mL 70%

nitric acid and heating until dry, then a final resuspension in 1 mL 2% nitric acid. Spleen,

serum, and frontal cortex were analyzed by ICP mass spectro metry (ICP

- MS) using an

XSeries2 (Thermo Scientific, Waltham, MA) and femur, bile, striatum, olfactory bulb and

cerebellum by graphite furnace atomic absorption spectrometry (GF

- AAS) using an

AAnalyst 600 spectrometer (Perkin Elmer, Waltham, MA). Referenc e standards were

analyzed repeatedly during each run to ensure run consistency. ICP

- AES and GF

- AAS

analyses were performed at the Environmental Chemistry Facility at Brown University

using previously described methods (Herrera et al., 2014). ICP

- MS analy ses were

performed in the Department of Earth, Environmental and Planetary Sciences at Brown

University.

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

10 Statistical Analyses

Individual data that appeared to be outliers were critically evaluated using a Dixon

- type

test for discordancy for an upper outlier (Barnett and Lewis, 1984). For comparing

change in weight across treatment groups, a multivariate analysis of variance mode l

(MANOVA) was used with treatment group as a fixed factor. Following an assessment

for homogeneity of variance (Levene’s test), the issue manganese concentration data

were initially analyzed using an analysis of variance (ANOVA) to evaluate whether either

manganese exposure or exposure duration were significant factors. When appropriate,

additional analyses of pooled data were also performed when a significant main effect

(treatment or exposure duration) was not discovered. Tissue manganese concentration s

at each time point were also inter

- compared for the three manganese exposur e groups

by ANOVA or a Wel ch ANOVA for unequal variances followed by either a Dunnett’s or a

Tukey’s honestly significant difference test to perform pair

- wise multiple comparisons

.

Statistical analyses were performed using SAS Statistical Software (JMP, SAS Institutes,

Inc., Cary, NC). A probability value of 0.01 was used for Levene’s test, while < 0.05 was

used as the critical level of significance for all other statistical test s. Unless otherwise

noted, data presented are mean values ± standard error of the mean (SEM).

R ESULTS



### Clinical Effects

Clinical signs were not seen in any exposure group. There were no treatment related

effects on terminal body weight (Table A1), daily feed consumption (Table A5), or daily

water consumption (Table A6)

.

One animal from the 61

- day gavage group (#70) died due to a gavage error. Terminal

body weights (day 61 exposure group) were 256.8 ± 9.8, 244.3 ± 7.9, 233.7 ± 6.5, and

228.2 ± 7. 1 g for the control, high manganese diet, drinking water, and gavage exposure

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

11 groups, respectively. This decrease (vs. controls) was not statistically significant (p =

0.071, ANOVA).

Growth curves for the 61

- day exposure groups are presented in Figure 1. Repeated

exposure ( 61 day ) to elevated levels of manganese did not decrease body weight gain

when compared with control animals given the 10

- ppm manganese diet (p = 0.128,

MANOVA). Individual animal data are provided in Table A2

.

Figure 2. Mean body weights (g) in rats exposed for 61 day s to a 10

- ppm manganese

diet (Control), 200

- ppm manganese diet (High Diet), drinking water exposure (High

Water) , and gavage exposure (Gavage)

.

There were no treatment related effects (day 61 expo sure group) on terminal body

weight, hematocrit

, or absolute liver or brain weights ( Table 2 ).

Table 2. M ean (± SEM) absolute liver and brain weights and hemato crit following 61

exposure days

.

Group Liver weight (g) Brain weight (g) Hematocrit (%)

Control 6.87 ± 0.49 1.87 ± 0.02 47.7 ± 2.7

High diet 7.00 ± 0.42 1.85 ± 0.06 49.5 ± 1.8

Drinking water 6.38 ± 0.26 1.84 ± 0.02 4 5.1 ± 1.9

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

12 Gavage 6.24 ± 0.31 1.83 ± 0.04 46.8 ± 2.6

p value 0.418 0.922 0.613

Estimated O ral M anganese E xposure

Nominal manganese and iron concentrations in control diet were 10 and 35 ppm,

respectively while ICP

- AES analysis revealed 12.2 and 44.8 ppm, respectively. Nominal

manganese and iron concentrations in the high manganese diet were 200 and 35 ppm,

respectiv ely while ICP

- AES analysis revealed 202.4 and 39.2 ppm, respectively.

Measured gavage solution manganese concentrations were within 4 to 15% of nominal

concentrations.

Overall daily mean feed consumption rates in the 61

- day exposure group were 11.0 ±

0 .5, 10.9 ± 0.4, 10.1 ± 0.3, and 10.0 ± 0.3 g/day for the control, high manganese diet,

drinking water, and gavage exposure groups, respectively. Overall daily mean water

consumption rates were 13.6 ± 0.5, 13.9 ± 0.6, 13.1 ± 0.4, and 13.3 ± 0.4 ml/day for the

control, high manganese diet, drinking water, and gavage exposure groups, respectively.

Table s A 5 and A6 give individual daily feed and water consumption rates , respectively

for the 61

- day exposure group

. Figure 3 shows mean daily feed and water consumption

rates for the four treatment groups. There was no effect of exposure group on daily feed

or water consumption.

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

13 Figure 3. Average d aily feed (A) and water (B) consumption in animals in the 61

-

day exposure group. High

- dose manganese treatments began on study day 10.

Peaks in water consumptions seen were most commonly associated with cage

change days and reflect spillage.

Overall mean body weights in the 61

- day exposure group were 213.1 ± 7.5, 2 06.1 ± 5.8,

197.6 ± 5.0, and 193.6 ± 5.6 g for the control, high manganese diet, drinking water, and

gavage exposure groups, respectively. Based on nominal dietary manganese

concentrations and overall mean body weight and feed consumption rates, rats in th e

control and high d iet groups ingested approximately 0.52 and 10.6 mg Mn/kg/day,

respectively. Daily manganese exposures (mg Mn/kg/day) with the high manganese

drinking water and gavage groups were within 5% of the high dose manganese diet

group.

Tissue Ma nganese C oncentrations

Individual animal manganese concentrations are presented in Tables A 7 (day 7) and A8

(day 6 1 )

. Tables 3 and 4 show mean tissue manganese concentrations for the 7

- and

61

- exposure day groups, respectively.

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

14 Table 3

. Mean (± SEM) tissue manganese concentrations (μg Mn/g) following 7

exposure days. Number in parentheses = number of samples (n).

Tissue Control Diet Water Gavage

Cerebellum 0.42 ± 0.01 (9) 0.50 ± 0.04 (9) 0.47 ± 0.01 (9) 0.59 ± 0.03 (11)

Olfactory bulb 0.42 ± 0.02 (9) 0.51 ± 0.03 (9) 0.48 ± 0.02 (9) 1.52 ± 0.30 (9)

Striatum 0.31 ± 0.01 (9) 0.35 ± 0.01 (9) 0.34 ± 0.01 (9) 0.55 ± 0.08 (11)

Frontal cortex 0.39 ± 0.07 (8) 0.35 ± 0.02 (9) 0.36 ± 0.01 (9) 0.48 ± 0.02 (10)

Bile 6.61 ± 1.57 (7) 231.35 ± 78.42 (7) 61.90 ± 16.83 (5) 142.94 ± 43.83 (8)

Serum 0.74 ± 0.14 (9) 1.35 ± 0.26 (8) 1.10 ± 0.15 (6) 1.55 ± 0.29 (10)

Femur 0.35 ± 0.02 (9) 0.42 ± 0.05 (9) 0.38 ± 0.02 (9) 0.53 ± 0.04 (11)

Spleen 0.19 ± 0.03 (9) 0.19 ± 0.01 (8) 0.19 ± 0.01 (9) 0.24 ± 0.02 (11)

Liver 2.45 ± 0.06 (9) 3.31 ± 0.12 (9) 3.04 ± 0.07 (9) 4.11 ± 0.27 (10)



### Bold text: p < 0.05; versus controls, significant ANOVA or Welch ANOVA followed by

Dunnett’s test

Table 4

. Mean (± SEM) tissue manganese concentrations (μg Mn/g) following 61

exposure days. Number in parentheses = number of samples (n).

Tissue Control Diet Water Gavage

Cerebellum 0.46 ± 0.01 (9) 0 .56 ± 0.03 (9) 0.52 ± 0.02 (9) 0.68 ± 0.02 (10)

Olfactory bulb 0.49 ± 0.03 (9) 0.49 ± 0.01 (9) 0.50 ± 0.05 (9) 1.12 ± 0.13 (10)

Striatum 0.36 ± 0.01 (9) 0.43 ± 0.01 (9) 0.44 ± 0.01 (9) 0.75 ± 0.07 (10)

Frontal cortex 0.25 ± 0.02 (8) 0.29 ± 0.01 (9) 0.27 ± 0.02 (9) 0.73 ± 0.08 (9)

Bile 1.48 ± 0.20 (8) 34.49 ± 5.68 (8) 38.63 ± 11.74 (9) 67.38 ± 7.9 1 (9)

Serum 0.66 ± 0.27 (9) 0.73 ± 0.16 (9) 0.42 ± 0.04 (9) 0.46 ± 0.03 (10)

Femur 0.32 ± 0.01 (9) 0.40 ± 0.02 (8) 0.40 ± 0.03 (9) 0.66 ± 0.07 (10)

Spleen 0.13 ± 0.02 (9) 0.16 ± 0.01 (9) 0.13 ± 0.01 (9) 0.15 ± 0.02 (10)

Liver 2.62 ± 0.11 (8) 3.30 ± 0 .17 (9) 2.94 ± 0.05 (9) 3.36 ± 0.09 (10)



### Bold text: p < 0.05; versus controls, significant ANOVA or Welch ANOVA followed by

Dunnett’s test

No statistically significant effect of manganese treatment: Increased manganese

exposure was not associated with changes in either serum or spleen manganese

concentrations (when compared with controls) . Serum manganese concentrations were

1.12 ± 0.13 and 0.56 ± 0.08 μg Mn/g for the 7 and 61 exposure day groups, respectively

(data pooled across treatments) . Spleen manganese concentrations were 0.20 ± 0.01

and 0.14 ± 0.01 μg Mn/g for the 7 and 61 exposure day groups, respectively. Serum

and spleen manganese concentrations were significantly lower at d ay 61 when

compared with values obtained in younger animals after 7 exposure days.

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

15 No statisticall

y significant effect of exposure duration : Olfactory bulb, frontal cortex,

femur, and liver manganese concentrations were not affected by exposure duration so

data for the day 7 and 6 1 time points were pooled (Table 5 ). Liver manganese

concentrations were elevated in all high

- dose manganese treatment groups (relative to

controls). Liver manganese concentrations seen in animals from the gavage exposure

group were significantly higher than those seen following either the high manganese diet

(200 ppm) or drinking water exposure groups. Increased olfactory bulb, frontal cortex,

and femur manganese concentrations were only seen in animals from the gavage

exposure group.

Table 5 . Tissue manganese concentrations (μg Mn/g) following control diet (10 ppm) or

a high dose oral manganese exposure. Exposure duration did not have a statistically

significant effect so data from the 7

- and 61

- day exposure groups were pooled (mean ±

SEM; number in parentheses represent total number of samples analyzed).

Exposure Group

Tissue Control (10 ppm) Diet (200 ppm) Drinking water Gavage

Olfactory bulb 0.46 ± 0.02 (18) 0.50 ± 0.02 (18) 0.49 ± 0.02 (18) 1.31 ± 0.16 (19) †

Frontal cortex 0.32 ± 0.04 (16) 0.32 ± 0.01 (18) 0.31 ± 0.02 (18) 0.60 ± 0.05 (19) †

Femur 0.33 ± 0.01 (18) 0.41 ± 0.03 (18) 0.39 ± 0.02 (18) 0.60 ± 0.04 (21) †

Liver 2.53 ± 0.06 (17) 3.30 ± 0.10 (18) 2.99 ± 0.04 (18) 3.73 ± 0.16 (20) †

Bold text: p < 0.05, versu s control, ANOVA or Wel ch ANOVA and Tukey ’s HSD

† Increased when compared with all other groups (p < 0.05, ANOVA or Wel ch ANOVA and

Tukey ’ s HSD)



### Statistically significant effect s of exposure duration and manganese treatment :

Cerebellum, striatum, and bile manganese concentrations were dependent upon

exposure duration and manganese treatment conditions (Table 6 ). Increased

cerebellum manganese concentrations were seen in animals from the high manganese

diet group after 61 expo sure days. Increased cerebellum and striatum manganese

concentrations were also seen in animals from the gavage group after 7 and 61

exposure days, and both were significantly higher than concentrations seen in either the

high manganese diet (200 ppm) or drinking water exposure groups. Increased bile

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

16 manganese concentrations were seen in animals from the high manganese diet (200

ppm) and gavage groups after 7 exposure days. Bile manganese concentrations were

also elevated in all high

- dose manganese treat ment groups (relative to controls)

following 61 exposure days.

Table 6 . Tissue manganese concentrations (μg Mn/g) following 7

- or 61

- day s of

exposure to either a control diet (10 ppm) or excess manganese in diet (200 ppm Mn),

drinking water, and gavage (mean ± SEM; number in parentheses represent total

number of samples analyzed).

Exposure Group

Tissue Control (10 ppm) Diet (200 ppm) Drinking water Gavage

Day 7

Cerebellum 0.42 ± 0.01 (9) 0.50 ± 0.04 (9) 0.47 ± 0.01 (9) 0.59 ± 0.03 (11) †

Striatum 0.31 ± 0.01 (9) 0.35 ± 0.01 (9) 0.34 ± 0.01 (9) 0.55 ± 0.08 (11) †

Bile 6.61 ± 1.57 (7) 231.35 ± 78.42 (7) 61.90 ± 16.83 (5) 142.94 ± 43.83 (8)

D ay 61

Cerebellum 0.46 ± 0.01 (9) 0.56 ± 0.03 (9) 0.52 ± 0.02 (9) 0.68 ± 0.02 (10) †

Striatum 0.36 ± 0.01 (9) 0.43 ± 0.01 (9) 0.44 ± 0.01 (9) 0.75 ± 0.07 (10)) †

Bile 1.48 ± 0.20 (8) 34.49 ± 5.68 (8) 27.95 ± 15.51 (8) 67.38 ± 7.91 (9) †

Bold text: p < 0.05, versus control, ANOVA or Wel ch ANOVA and Tukey ’s HSD

† Increased when compared with all other groups (p < 0.05, ANOVA or Wel ch ANOVA and

Tukey ’ s HSD)

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

17 Tissue Iron, Copper, and Zinc C oncentrations

Individual animal iron, copper, and zinc tissue concentrations are presented in Tables A9

(day 7) and A10 (da y 61). Tables 7, 8, and 9 show mean tissue i ron, copper, and zinc

tissue concentrations, respectively. High dose manganese gavage exposure was

associated with changes in liver (day 7), frontal cortex (day 61), and spleen (day 61) iron

concentrations (Table 7).

Table 7 . Mean (± SEM) tissue iron concentrations (μg Fe/g) following 7 or 61 exposure

days. N umber in parentheses = number of samples (n).

Tissue Control Diet Water Gavage

Day 7

Frontal cortex 10. 6 3 ± 0. 49 ( 9 ) 9.52 ± 0. 45 (9) 10.03 ± 0. 32 (9) 1 0. 50 ± 0. 39 (1 1 )

Liver 96.4 ± 4.8 (9) 101.1 ± 5.3 (9) 111.2 ± 8.7 (9) 118.1 ± 3.7 (11)

Spleen 220.6 ± 6.6 (9) 2 18.9 ± 18.5 ( 9 ) 234.5 ± 10.8 (9) 256.5 ± 9.9 (11)

Serum 203.0 ± 69.5 ( 8 ) 356.7 ± 76.1 ( 8 ) 268.1 ± 71.7 (9) 247.9 ± 61.1 (11)

Day 61

Frontal cortex 10.88 ± 0. 58 (8) 10.86 ± 0.61 (9) 9.71 ± 0.64 (9) 14.94 ± 1.33 (9 )

Liver 179.1 ± 16.9 (9) 162.9 ± 12.1 (9) 169.7 ± 8.3 (9) 168.9 ± 6.1(10)

Spleen 1142.6 ± 59.3 (9) 948.4 ± 65.8 ( 9 ) 1065.3 ± 45.2 (9) 1259.5 ± 45.1 (10 )

Serum 219.0 ± 22.1 (9) 167.7 ± 21.3 (8) 220.4 ± 27.8 ( 9 ) 1 99.2 ± 16.7 (10)



### Bold text: p < 0.05; versus controls, significant ANOVA or Welch ANOVA followed by

Tukey’s HSD test.

High dose manganese exposure was associated with changes in liver and spleen

copper concentrations after 7 exposure days (Table 8). High dose manganese gavage

exposure was also associated wit h increased frontal cortex copper concentrations after

61 exposure days (Table 8). High dose manganese drinking water exposure was

associated with increased spleen copper concentrations after 61 exposure days (Table

8 ).

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

18 Table 8. Mean (± SEM) tissue copper (μg Cu/g) concentrations following 7 or 61

exposure days. Number in parentheses = number of samples (n).

Tissue Control Diet Water Gavage

Day 7

Frontal cortex 1.28 ± 0. 06 ( 9 ) 1 .30 ± 0.06 (9 ) 1. 37 ± 0.0 2 (9 ) 1. 43 ± 0.0 5 (11 )

Liver 4.57 ± 0. 14 ( 9 ) 6.08 ± 0. 20 (9) 6.53 ± 0.17 (9) 7.08 ± 0. 21 (1 1 )

Spleen 1.15 ± 0.0 3 (9) 1.0 5 ± 0.0 3 ( 9 ) 1.03 ± 0.0 2 (9) 0. 9 9 ± 0.0 2 (11)

Serum 63.8 ± 4.2 (9) 69.6 ± 9.3 (9) 71.0 ± 6.7 ( 8 ) 81.5 ± 5.4 (11 )

Day 61

Frontal cortex 1.53 ± 0. 05 (8) 1.62 ± 0. 04 (9 ) 1. 48 ± 0.0 3 (9 ) 2.10 ± 0.15 ( 9 )

Liver 4.68 ± 0.19 (9) 4.77 ± 0.2 6 (9) 4.31 ± 0.10 (9) 4.71 ± 0. 13 (1 0 )

Spleen 0.92 ± 0.04 (9 ) 1.01 ± 0.03 ( 9 ) 1.13 ± 0.06 ( 9 ) 1 .05 ± 0.02 ( 10 )

Serum 69.0 ± 2.4 (9) 71. 9 ± 3. 4 ( 9 ) 68.2 ± 1.8 (9) 65.6 ± 1.7 (10)



### Bold p < 0.05; versus controls, significant ANOVA or Welch ANOVA followed by Tukey’s

HSD test

.

High dose manganese exposure was associated with increased spleen zinc

concentrations after 7 exposure days (Table 9). High dose manganes e gavage

exposure was also associated with increased liver zinc concentrations after 7 exposure

days and increased frontal cortex zinc concentrations after 61 exposure days (Table 8).

Table 9. Mean (± SEM) tissue zinc concentrations (μg Zn/g) following 7 or 61 exposure

days. Number in parentheses = number of samples (n).

Tissue Control Diet Water Gavage

Day 7

Frontal cortex 11.69 ± 0. 36 ( 9 ) 11.16 ± 0. 38 (9) 11.85 ± 0. 19 (9) 12.18 ± 0. 25 (11 )

Liver 27.09 ± 0. 6 3 ( 9 ) 29.52 ± 0. 61 (9) 29.41 ± 1.05 (9) 30.74 ± 0. 89 (11 )

Spleen 17.21 ± 0. 49 (9) 1 9.01 ± 0. 35 ( 9 ) 19.39 ± 0. 48 (9) 19.25 ± 0. 4 3 (11)

Serum 136.0 ± 16.5 (9) 135.1 ± 7.8 (8 ) 119.6 ± 8.9 (9) 124.6 ± 5.8 (11 )

Day 61

Frontal cortex 10.88 ± 0. 58 (8) 10.86 ± 0. 61 (9) 9.71 ± 0. 64 (9) 14.94 ± 1.33 (9)

Liver 29.94 ± 1.17 (9) 29.20 ± 1.55 (9) 2 8.51 ± 0.64 (9) 27.90 ± 0. 32 (10 )

Spleen 16.83 ± 0.48 ( 9 ) 1 8

. 4 5 ± 0.58 (9 ) 18.14 ± 1 .15 (9 ) 1 7.35 ± 0.71 ( 10 )

Serum 76.1 ± 2.0 (9) 74.6 ± 4.1 (9) 71.1 ± 4.4 (9) 65.6 ± 2.3 (10)



### Bold p < 0.05; versus controls, significant ANOVA or Welch ANOVA followed by Tukey’s

HSD test.

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

19 D ISCUSSION

The overall goals of this study were to determine whether the intake form (e.g., diet

versus drinking water) or dose rate (e.g., drinking water versus gavage) of ingested

manganese affect tissue manganese concentrations. This experiment used three forms

of oral manganese administration (diet, drinking water, and gavage) an d was designed

to produce similar manganese intake levels between these methods. This was

accomplished by using a staggered start to the exposures; which allowed us to initially

determine the daily dietary intake of animals on a 200

- ppm manganese diet. Da ily

manganese exposures (mg Mn/kg/day) in the high manganese drinking water and

gavage groups were then adjusted to be within 5% of the high dose manganese

exposure group’s average daily exposure.

During the course of the study, animals in the 200

- ppm m anganese diet group ingested

approximately 10.6 mg Mn/kg body weight/day. The dietary manganese exposures

used in this study are quite high when compared with typical dietary intake in people.

For example, Yamada and coworkers (2014) reported that adult Japanese men and

women ingested 4.9 to 5.1 mg Mn/day or approximately 0.08 to 0.09 mg Mn/kg/day.

Dietary intake levels reported by Yamada are somewhat higher than those seen in

Western populations which range from 0.9 to 5.0 mg Mn/day for women and 1.0 to 5.2

mg Mn/day for men (Bautista et al., 2005; Nkwenkeu et al., 2002; Patterson et al., 1984).

Based on data reported by Yamada and coworkers (2014) we estimate that the daily

manganese intake rate (mg/kg/day) for rats receiving the 200

- ppm manganese diet in

our study were approximately 125

- fold higher than those seen in adult humans on a

Japanese diet.

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

20 A number of physiological mechanisms exist to maintain relatively constant tissue

manganese concentrations despite wide fluctuations in oral manganese intake

(Schroeder et al., 1966). When dietary manganese levels are high, adaptive changes

often include reduced gastrointestinal absorption of manganese, enhanced manganese

liver metabolism, and increased biliary and pancreatic excretion of manganese

(revi ewed in Aschner et al., 2005). Thus, the fraction of ingested manganese retained by

the body is tightly regulated in order to maintain normal tissue manganese

concentrations under different dietary conditions. The 200

- ppm manganese diet was

selected with t he intent of altering manganese homeostasis as evidenced by changes in

liver and bile manganese concentrations without producing overt signs of toxicity.

Although oral manganese intakes in our study were extremely elevated

, the animals did

not develop out ward clinical signs or statistically significant changes in body weight gain,

feed consumption, or water consumption. We did observe changes in liver manganese

concentrations that were unaffected by the duration of the exposure but were dependent

on the m anganese exposure. Animals with elevated manganese intake (> 10 mg

Mn / kg/day) developed a statistically significant 18.2 to 47.4% increase in liver

manganese concentrations when compared with rats given a control diet that provided

approximately 0.5 mg Mn /kg/day. Bile manganese concentrations were dependent upon

both exposure duration and manganese exposure. Statistically significant increases in

bile manganese concentrations were seen after 7 exposure days. For example, rats fed

the 200

- ppm manganese d iet developed a statistically significant increase in bile

manganese concentration that was approximately 35

- fold higher than that seen in

control animals given a diet with a 20

- fold lower manganese concentration. Increased

biliary manganese elimination p ersisted throughout the study. Rats given the 200

- ppm

manganese diet for 60 exposure days developed a statistically significant 23

- fold

increase in bile manganese concentrations when compared with control animals. Biliary

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

21 excretion of manganese also demo nstrated a dose

- rate dependency since rats given >

10 mg Mn/kg/day by any route (diet, drinking water, and gavage) developed a

statistically significant 1.7

- to 2

- fold increase in bile manganese concentrations when

compared with animals receiving similar a mounts of manganese by either diet or

drinking water.

Another goal of this project was to determine whether the rate at which manganese is

ingested could influence systemic delivery of this metal to the brain and other tissues . In

particular we were in terested in determining whether oral gavage could result in altered

systemic delivery of manganese when compared with drinking water or dietary

exposure. The toxicokinetic profiles of several chemicals have been shown to differ

substantially in animals tr eated via gavage versus administration via other oral routes.

G avage administration in rodents bypasses the highly absorptive buccal surface

membranes and in some cases (e.g., with the drug donepezil) lead to reduced systemic

delivery (Atcha et al., 2010). In contrast, Yuan and coworkers (1995) showed that

gavage administration of benzyl acetate to rats was associated with higher benzoic acid

plasma concentrations when compared with those seen following similar dietary

administration. Similarly, oral gava ge resulted in higher tissue concentrations of

sulindac and lovastem in rats when compared with animals receiving comparable dietary

exposure (Kapetanovic et al., 2006; Martín

- Jiménez et al., 2008 ). These types of

observations have led to concerns being raised regarding the appropriateness of gavage

administration for the testing of chemicals for endocrine disruptor effects (Vandenber g et

al., 2014). In our study, we found multiple examples where tissue manganese

concentrations in animals from the gavage treatment group were statistically higher than

those seen in the high dose diet and drinking water exposure groups . For example,

gav age exposure was associated with striatal, olfactory bulb , femur, and liver

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

22 manganese concentrations that were respectively

, approximately 1.6

- , 2.6

- , 1.4

- , and

1.1

- fold higher when compared with animals receiving similar amounts of manganese by

either the diet or in drinking water. These results suggest that bolus gavage

administration of manganese could be associated with higher brain manganese

concentrations and as a consequence a higher risk of neurotoxicity.

Our study also shows that equivalent diet ary and drinking water exposure of a high

- dose

of manganese (approximately 10.6 mg Mn / kg/day) resulted in very few significant

differences in tissue manganese concentrations. Ingestion of a high dose manganese

diet was associated with statistically higher bile manganese concentrations after 7

exposure days when compared with animals given excess manganese by drinking

water. However, this difference was no longer seen following 60 exposure days. High

dose dietary, but not drinking water, manganese exposur e was associated with elevated

cerebellum manganese concentrations when compared with animals receiving the 10

-

ppm manganese control diet for 60 exposure days. However, cerebellum manganese

concentrations in animals given the 200

- ppm diet and drinking wat er groups were

statistically equivalent. Likewise, liver manganese concentrations and bile manganese

concentrations following 60 days of exposure were also statistically equivalent between

the 200

- ppm diet and drinking water groups. Our findings suggest that drinking water

and dietary exposures result in toxicologically comparable tissue manganese

concentrations, especially in the striatum

, a known target for manganese neurotoxicity

(Guilarte, 2010). This observation could prove useful to toxicologists c onsidering

whether dietary studies may help inform the human health risk assessment of

manganese in drinking water.

An important limitation of the current study is the use of a single high dose of

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

23 manganese for the three different types of oral exposure s as opposed to using a low,

middle and high dose of manganese . One future goal of our research team is to use the

data from this study to help refine existing human physiologically-based pharmacokinetic

(PBPK) models (Andersen et al., 1999; Schroeter et al., 201 1 ; Yoon et al., 2011) to more

thoroughly include oral exposure to manganese. A significant strength of PBPK models

is that they can be used to extrapolate between exposure doses and durations

(Andersen et al., 2009) thus reducing the impact of this experimental limitation.

Increased consideration of oral exposure in PBPK models is also important from a risk

assessment perspective. Current human PBPK models predominantly focus on

inhalation exposures with secondary consideration of oral routes of a dministration

(Taylor et al., 2012). Refined PBPK models developed with data obtained from this

study could be used to explore the biological plausibility of any purported effects of

manganese in drinking water, and could therefore play an important role as regulatory

agencies reexamine manganese drinking water standards.

A secondary effort of this project was to evaluate whether high dose manganese

exposure altered tissue concentrations of other essential metals, namely iron, zinc, and

copper. Mangane se and iron are subject to complex homeostatic control mechanisms

(Farina et al., 2013). Metal

- metal interactions are k nown to occur between manganese,

iron , and other divalent metals ( Fitsanakis et al., 2010; Roth and Garrick , 2003).

Excessive exposure to manganese can overwhelm normal homeostatic controls

resulting in altered tissue concentrations of other divalent metals . The exact hepatic

metallo

- regulatory proteins used to sense and appropriately adjust manganese , i ron , and

other d ivalent metal uptake, storage, and elimination pathways remain poorly

understood.

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

24 In our study, we found that high dose manganese exposure (diet, drinking water, and

gavage) was associated with increased liver, but decreased spleen, copper

concentrations after 7 exposure days. High dose manganese exposure was also

associated with increased spleen zinc concentrations after 7 exposure days. Gavage

exposure to a high manganese dose was also associated with several additional

changes in other divalent metal s . These included increased liver copper and zinc

concentrations after 7 exposure days and increased frontal cortex iron , copper, and zinc

concentrations after 61 exposure days

. Future studies will investigate alterations in

transferrin and other cellul ar responses involved with these changes ( Herrera et al.,

2014 )

.

A CKNOWLEDGMENTS



### The authors would like to thank Lucia Lazarowski, Carol Bobbitt, and the staff of the

NCSU Laboratory Animal Resources facility for their contributions. We also thank Drs.

Mel Andersen, Harvey Clewell, Jr, and Miyoun g Yoon for valuable discussions related to

the design of this study and possible applications of our results to PBPK modeling

efforts. We also thank Drs. David Murray, Joseph Orchardo and Soumen Mallick for

assis tance with manganese measurements. This publication is based on a study

sponsored and funded by the Afton Chemical Corporation in satisfaction of registration

requirements arising under Section 211(a) and (b) of the Clean Air Act and

corresponding regulati ons at 40 C. F. R. Subsections 79.50 et seq

.

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

25 R EFERENCES



### Agency for Toxic Substances and Disease Registry (ATSDR) (2012). Toxicological

Profile for Manganese, U.S. Department Of Health And Human Services Public

Health Service. Available at:

http://www.atsdr.cdc.gov/toxprofiles/tp.asp?id=102&tid=23 [Accessed October 20,

2014].

Andersen, M. E., Dorman, D. C., Clewell, H. J. 3rd, Taylor, M. D., and Nong, A. (2010).

Multi

- do se

- route, multi

- species pharmacokinetic models for manganese and their

use in risk assessment. J Toxicol Environ Health A 73, 217

- 234.

Andersen, M. E., Gearhart, J. M., and Clewell, H. J., III. (1999). Pharmacokinetic data

needs to support risk assessment s for inhaled and ingested manganese.

Neurotoxicology 20 , 161

– 171.

Aschner, J. L., and Aschner, M. (2005). Nutritional aspects of manganese homeostasis.

Mol Aspects Med. 26, 353

- 362.

Aschner, M., Erikson r, K. M., and Dorman, D. C. (2005). Manganese dos imetry: species

differences and implications for neurotoxicity. Crit Rev Toxicol. 35, 1

- 32.

Atcha, Z., Rourke, C., Neo, A. H. P., Goh, C. W. H., Lim, J. S. K., Aw, C

- C., Browne, E.

R., and Pemberton, D. J. (2010). Alternative method of oral dosing for ra ts. J Am

Assoc Lab Anim Sci 49, 335

- 343.

Barnett, V., and Lewis, T. (1984). Discordancy tests for outliers in univariate samples: In

Outliers in Statistical Data (V. Barnett and T. Lewis, Eds.), pp. 52

– 125, 292. John

Wiley & Sons, Inc., Chichester.

Baut ista, L. E, Herrán, O. F., and Pryer, J. A. (2005). Development and simulated

validation of a food

- frequency questionnaire for the Colombian population. Public

Health Nutr. 8, 181

- 188.

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

26 Bowler, R. M., Gysens, S., Diamond, E., Nakagawa, S., Drezgic, M., and Roels, H. A.

(2006). Manganese exposure: neuropsychological and neurological symptoms and

effects in welders. Neurotoxicology 27, 315

– 326.

Boyes, W. K. (2010). Essentiality, toxicity, and uncertainty in the risk assessment of

manganese. J Toxicol Environ Health A. 73, 159

- 165.

Chang, Y., Lee, J. J., Seo, J. H., Song, H. J., Kim, J. H., Bae, S. J., Ahn, J. H., Park, S.

J., Jeong, K. S., Kwon, Y. J., Kim, S. H., and Kim, Y. (2010). Altered working

memory process in the manganese

- exposed brain. Neur oimage 53, 1279

- 1285.

Dorman, D. C., Andersen, M. E., Roper, J. M., and Taylor, M. D. (2012). Update on a

pharmacokinetic

- centric Aternative Tier II program for MMT

- Part I: Program

implementation and lessons learned. J Toxicol. 2012, 946742.

Dorman, D. C ., Struve, M. F., James, R. A., McManus, B. E., Marshall, M. W., and

Wong, B. A. (2001). Influence of dietary manganese on the pharmacokinetics of

inhaled manganese sulfate in male CD rats. Toxicol Sci. 60, 242

- 251.

Ellingsen, D. G., Konstantinov, R., Ba st

- Pettersen, R., Merkurjeva, L., Chashchin, M.,

Thomassen, Y., and Chashchin, V. (2008). A neurobehavioral study of current and

former welders exposed to manganese. Neurotoxicology 29, 48

– 59.

Farina, M., Avila, D. S., da Rocha, J. B. T., and Aschner, M (2013). Metals, oxidative

stress and neurodegeneration: a focus on iron, manganese and mercury.

Neurochem. Int.

. 62, 575

– 594.

Fitsanakis

, V

. A

. , Zhang

, N

. , Garcia

, S

. , and Aschner

, M. (2010). Manganese (Mn) and

iron (Fe): interdependency of transpor t and regulation. Neurotox. Res

. 18, 124

– 131.

Guilarte, T. R. (2013). Manganese neurotoxicity: new perspectives from behavioral,

neuroimaging, and neuropathological studies in humans and non

- human primates.

Front Aging Neurosci 5, 23.

Guilarte, T. R. (2010). Manganese and Parkinson's disease: a critical review and new

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

27 findings. Environ Health Perspect. 118, 1071

- 1080.

Herrera, C., Pettiglio, M. A., and Bartnikas, T. B. (2014). Investigating the role of

transferrin in the distribution of iron, manganese, copper, and zinc. J. Biol. Inorg.

Chem

. 19, 869

– 877.

Kapetanovic, I. M., Krishnaraj, R., Martin

- Jimenez, T., Yuan, L., van Breemen, R. B., and

Lyubimov, A. (2006). Effects of oral dosing paradigm s (gavage versus diet) on

pharmacokinetics and pharmacodynamics. Chem Biol Interact 164, 68

- 75.

Kawamura, R., Ikuta, H., Fukuzumi, S., Yamada, R., Tsubaki, S., Kodama, T., and

Kurata, S. (1941). Intoxication by manganese in well water, Arch. Exp. Med., 18

,

145

– 169.

Keen, C. L. and Zidenberg

- Cherr, S. (1994). Manganese toxicity in humans and

experimental animals. In Manganese in Health and Disease (D.J. Klimis

- Tavantzis,

Ed.), pp. 193

– 205. CRC Press, Boca Raton.

Khan, K., Wasserman, G.A., Liu, X., Ahmed, E., Parvez, F., Slavkovich, V., Levy, D.,

Mey, J., van Geen, A., Graziano, J. H., and Factor

- Litvak, P. (2012). Manganese

exposure from drinking water and children's academic achievement.

Neurotoxicology 33, 91

- 97.

Kondakis, X. G., Makris, N., Leotsinidis, M., Prinou, M., and Papapetropoulos, T. (1989).

Possible health effects of high manganese concentration in drinking water. Arch.

Environ. Health 44, 175

– 178.

Lucchini, R. G., Martin, C. J., and Doney BC (2009 ). From manganism to manganese

induced parkinsonism: a conceptual model based on the evolution of exposure.

Neuromolecular Med 11, 311

- 321.

Martín

- Jiménez, T., Lindeblad, M., Kapetanovic, I. M., Chen, Y., and Lyubimov, A.

(2008). Comparing pharmacokinet ic and pharmacodynamic profiles in female rats

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

28 orally exposed to lovastatin by gavage versus diet. Chem Biol Interact. 171, 142

-

151.

National Research Council (NRC) (2011). Guide for the Care and Use of Laboratory

Animals . 8 th 

### Edition. National Academy P ress, Washington, D.C., 220 pp.

Nkwenkeu, S. F., Kennedy, G., Philippe, S., and Zayed, J. (2002). Oral manganese

intake estimated with dietary records and with direct chemical analysis. Sci Total

Environ. 287, 147

- 53.

Patterson, K. Y., Holbrook, J. T., B odner, J. E., Kelsay, J. L., Smith, J. C. Jr, and Veillon,

C. (1984). Zinc, copper, and manganese intake and balance for adults consuming

self

- selected diets. Am J Clin Nutr. 40, S1397

- 1403.

Roels, H. A., Bowler, R. M., Kim, Y., Claus Henn, B., Mergler, D. , Hoet, P., Gocheva, V.

V., Bellinger, D. C., Wright, R. O., Harris, M. G., Chang, Y., Bouchard, M. F., Riojas

Rodriguez, H., Menezes

- Filho, J. A., and Téllez

- Rojo, M. M. (2012). Manganese

exposure and cognitive deficits: a growing concern for manganese n eurotoxicity.

Neurotoxicology 33, 872

- 880.

Roth

, J

. A

. , and Garrick

, M

. D. (2003). Iron interactions and other biological reactions

mediating the physiological and toxic actions of manganese. Biochem. Pharmacol..

66, 1

– 13.

Schroeder, H. A., Balassa, J. J., and Tipton, I. H. (1966). Essential trace metals in man:

Manganese, a study in homeostasis. J. Chronic. Dis. 19, 545

– 571.

Schroeter, J. D., Nong, A., Yoon, M., Taylor, M. D., Dorman, D. C., Andersen, M. E., and

Clewell, H. J. 3rd. (2011). Analysis of manganese tracer kinetics and target tissue

dosimetry in monkeys and humans with multi

- route physiologically based

pharmacokinetic models. Toxicol Sci. 120, 481

- 498.

Taylor, M. D., Clewell, H. J. 3rd, Andersen, M. E., Schro eter, J. D., Yoon, M., Keene, A.

M., and Dorman, D. C. (2012). Update on a pharmacokinetic

- centric alternative Tier

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

29 II program for MMT

- part II: Physiologically based pharmacokinetic modeling and

manganese risk assessment. J Toxicol. 2012, 791431.

Vande nberg, L. N., Welshons, W. V., Vom Saal, F. S., Toutain, P. L., and Myers, J. P.

(2014). Should oral gavage be abandoned in toxicity testing of endocrine disruptors?

Environ Health

. 13, 46.

Wasserman, G. A., Liu, X., Parvez, F., Ahsan, H., Levy, D., Factor

- Litvak, P., Kline, J.,

van Geen, A., Slavkovich, V., LoIacono, N. J., Cheng, Z., Zheng, Y., and Graziano,

J. H. (2006). Water manganese exposure and children's intellectual function in

Arai hazar, Bangladesh. Environ Health Perspect. 114, 124

- 129.

Yamada, M., Asakura, K., Sasaki, S., Hirota, N., Notsu, A., Todoriki, H., Miura, A., Fukui,

M., and Date, C. (2014). Estimation of intakes of copper, zinc, and manganese in

Japanese adults using 16

- day semi

- weighed diet records. Asia Pac J Clin Nutr

. 23,

465

- 472.

Yoon, M., Schroeter, J. D., Nong, A., Taylor, M. D., Dorman, D. C., Andersen, M. E., and

Clewell, H. J. 3rd. (2011). Physiologically based pharmacokinetic modeling of fetal

and neonatal m anganese exposure in humans: describing manganese homeostasis

during development. Toxicol Sci. 122, 297

- 316.

Yuan, J. H., Goehl, T. J., Abdo, K., Clark, J., Espinosa, O., Bugge, C., and Garcia, D.

(1995). Effects of gavage versus dosed feed administrati on on the toxicokinetics of

benzyl acetate in rats and mice. Food Chem Toxicol 33, 151

- 158.

Zoni, S., and Lucchini, R. G. (2013). Manganese exposure: cognitive, motor and

behavioral effects on children: a review of recent findings. Curr Opin Pediatr 25,

255

- 260.

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

30 

# APPENDIX 1

INDIVIDUAL ANIMAL DATA

Note: Bold data were analyzed to be outliers and

were excluded from statistical analysis (Dixon

test) . Data where feed became wet or water

bottle spillage occurred is considered not

determined (ND).

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

31 Table A1. Assignment of animals to the 7

- day exposure day group. Individual and

mean (± SEM) initial and terminal body weights (g) are also provided.

Rat

ID Pre

random ID Cage

Card # Group Day 0 Body

Weight (g) Terminal Body

Weight (g)

1 20a 136281 Control 115 142

2 3a 136264 Control 130 159

3 2a 136263 Control 140 175

4 15a 136276 Control 120 159

5 35a 136296 Control 150 190

6 37a 136298 Control 130 184

7 6a 136267 Control 145 198

8 33a 136294 Control 135 169

9 21a 136282 Control 110 146

Mean 130.6 ± 4.5 1 69.1 ± 6.4

10 17a 136278 High Diet 140 166

11 24a 136285 High Diet 145 174

12 11a 136272 High Diet 115 139

13 28a 136289 High Diet 130 164

14 12a 136273 High Diet 130 156

15 22a 136283 High Diet 120 150

16 18a 136279 High Diet 125 151

17 5a 136266 High Diet 130 170

18 34a 136295 High Diet 130 170

Mean 129.4 ± 3.1 1 60.0 ± 3. 9

19 30a 136291 High Water 145 192

20 32a 136293 High Water 140 179

21 25a 136286 High Water 130 166

22 8a 136269 High Water 145 182

23 23a 136284 High Water 125 165

24 14a 136275 High Water 145 188

25 31a 136292 High Water 115 134

26 9a 136270 High Water 115 146

27 4a 136265 High Water 120 173

Mean 131.1 ± 4.3 1 69.3 ± 6 .3

28 26a 136287 Gavage 150 195

29 1a 136262 Gavage 115 144

30 13a 136274 Gavage 135 165

31 19a 136280 Gavage 145 184

32 16a 136277 Gavage 120 161

33 38a 136299 Gavage 135 182

34 27a 136288 Gavage 120 162

35 7a 136268 Gavage 120 151

36 10a 136271 Gavage 115 149

37 36a 136297 Gavage 135 167

38 29a 136290 Gavage 125 161

Mean 128.6 ± 3.6 1 65.3 ± 4.8

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

32 Table A2. Assignment of animals to the 61

- day exposure day group. Individual and

mean (± SEM) body weights (g) are also provided.

Rat ID Pre

- random ID CC # Group Day 0 body

weight (g)

39 68a 76595 Control 146.2

40 69a 76596 Control 130.2

41 59a 76586 Control 110.5

42 57a 76584 Control 149.1

43 50a 76577 Control 129.9

44 40a 76567 Control 150.6

45 53a 76580 Control 149.4

46 43a 76570 Control 142.9

47 41a 76568 Control 141.0

Mean 138.9 ± 4.4

48 39a 76566 High Diet 128.6

49 42a 76569 High Diet 120.9

50 66a 76593 High Diet 143.2

51 60a 76587 High Diet 132.3

52 61a 76588 High Diet 129.7

53 56a 76583 High Diet 121.3

54 74a 76601 High Diet 157.9

55 75a 76602 High Diet 134.3

56 44a 76571 High Diet 150.4

Mean 135.4 ± 4.2

57 70a 76597 High Water 150.6

58 49a 76576 High Water 144.9

59 51a 76578 High Water 141.6

60 76a 76603 High Water 119.9

61 64a 76591 High Water 128.8

62 71a 76598 High Water 142.3

63 63a 76590 High Water 135.4

64 67a 76594 High Water 130.8

65 45a 76572 High Water 137.7

Mean 136.9 ± 3.1

66 48a 76575 Gavage 148.6

67 58a 76585 Gavage 148.8

68 55a 76582 Gavage 135.4

69 62a 76589 Gavage 117.1

70 52a 76579 Gavage 127.5

71 65a 76592 Gavage 138.4

72 46a 76573 Gavage 147.2

73 47a 76574 Gavage 154.6

74 73a 76600 Gavage 118.8

75 72a 76599 Gavage 127.4

76 54a 76581 Gavage 137.9

Mean 137.4 ± 4.1

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

33 Table A3. Individual and mean a bsolute liver and brain weights and hematocr it following

61 exposure days.

Rat # Group Liver weight (g) Brain weight (g) Hematocrit (%)

39 Control 7.982 1.845 40.0

40 Control 6.066 1.779 56.0

41 Control 5.695 1.777 53.0

42 Control 4.376 1.914 48.5

43 Control 5.902 1.838 51.0

44 Control 8.799 1.98 30.0

45 Control 7.744 1.862 53.0

46 Control 8.459 1.923 48.7

47 Control 6.894 1.87 49.5

48 High Diet 7.364 2.122 42.5

49 High Diet 5.248 1.621 50.0

50 High Diet 6.02 1.888 48.7

51 High Diet 7.987 2.115 60.0

52 High Diet 5.254 1.795 54.5

53 High Diet 6.7 1.854 45.0

54 High Diet 8.366 1.853 47.5

55 High Diet 7.521 1.767 52.7

56 High Diet 8.531 1.613 44.3

57 Drinking Water 5.707 1.666 40.5

58 Drinking Water 6.121 1.871 38.0

59 Drinking Water 6.566 1.848 44.5

60 Drinking Water 5.505 1.845 45.5

61 Drinking Water 5.729 1.645 53.5

62 Drinking Water 6.754 1.955 51.3

63 Drinking Water 6.429 1.928 46.5

64 Drinking Water 6.504 1.811 37.0

65 Drinking Water 8.14 1.967 49.0

66 Gavage 6.203 1.862 44.5

67 Gavage 6.427 1.872 45.5

68 Gavage 5.134 1.853 48.0

69 Gavage 4.39 1.794 51.3

70 Gavage ND ND ND

71 Gavage 7.139 1.838 25.0

72 Gavage 6.484 1.863 52.5

73 Gavage 7.693 1.852 49.0

74 Gavage 7.038 1.887 49.0

75 Gavage 5.59 1.708 53.7

76 Gavage 6.262 1.779 49.5

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

34 Table A4

. Individual animal body weights (g) in the 61 exposure day groups.

Rat ID Group Day 0 Day 8 Day 15 Day 22 Day 29 Day 36 Day 43 Day 50 Day 57 Day 64 Necropsy Overall mean

39 Control 146.2 170.2 192.8 209.9 226.3 235.9 249.4 257.2 267.7 278.1 279.7 228.49

40 Control 130.2 150.8 167.9 185.9 197.2 206.8 217.3 224.2 229.5 236.4 236.4 198.42

41 Control 110.5 126.5 143.6 160.4 169.8 175.8 183.7 194.6 200.3 205 205.4 170.51

42 Control 149.1 170.3 195.2 214.1 218.3 225.2 236.5 245.8 257.8 266.9 266.6 222.35

43 Control 129.9 140.7 159.4 177.6 192 203.2 204.5 209.8 216.2 219.1 221.9 188.57

44 Control 150.6 175.6 198.8 219.3 239.8 253.9 265.9 281.3 289.6 294.4 295.9 242.28

45 Control 149.4 171 196.9 215.9 227.5 232.6 245.9 253.7 256.9 265.1 267.2 225.65

46 Control 142.9 164.8 188.4 208.1 215.7 229 241.9 253.9 262.1 268.3 276.4 222.86

47 Control 141.0 161.5 186.7 202.7 220.8 232.2 240.1 244.1 251.6 259.7 261.5 218.35

48 High Diet 128.6 151.3 174.3 194.9 214.2 228.6 236.4 250.3 253.5 262.3 261.8 214.20

49 High Diet 120.9 135.4 156.5 179.5 188 196.6 203.8 205.6 206.2 212.3 211.7 183.32

50 High Diet 143.2 163 186.2 207.2 220.5 225.6 232.3 234.7 234.6 240.2 239.6 211.55

51 High Diet 132.3 152.8 170.3 184.1 191.3 196.1 200.6 205.2 212.6 218.6 222.3 189.65

52 High Diet 129.7 144.8 166.9 176.8 192 195.4 201.7 207.7 215.8 220.7 218.2 188.15

53 High Diet 121.3 137.1 156.7 178.5 192.9 208.3 217.3 230.6 231.2 240.4 239.1 195.76

54 High Diet 157.9 182.9 200.6 216.9 225.2 232.3 243 255.5 259.4 269.2 268.6 228.32

55 High Diet 134.3 149.4 176 200.6 213.4 222.2 235.6 246.4 252.8 262.2 262.8 214.15

56 High Diet 150.4 174.3 194.5 210 224.7 238.4 249.3 264.2 267.9 276.3 274.8 229.53

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

35 Table A4 . Individual animal body weights (g) in the 61 exposure day groups. Continued

Rat ID Group Day 0 Day 8 Day 15 Day 22 Day 29 Day 36 Day 43 Day 50 Day 57 Day 64 Necropsy Overall mean

57 High Water 150.6 171.3 181.3 194.2 202.6 206.3 212.7 219.7 221.8 226.2 228.2 201.35

58 High Water 144.9 161.2 172.5 182.3 191.5 199.4 202.8 209.5 217.6 221.6 224.5 193.44

59 High Water 141.6 158.9 176.4 194.1 208.5 213.4 223.8 231.3 237.1 240 238.8 205.81

60 High Water 119.9 138.3 149.8 168 180 189.4 195.6 196.8 201.3 213.7 215.5 178.94

61 High Water 128.8 144.6 158.1 167.1 176.4 184.5 193.9 204.8 210.3 213.8 214.7 181.55

62 High Water 142.3 152.2 166 186.4 202.3 211.4 219.2 225.3 229.5 234.2 235.6 200.40

63 High Water 135.4 156.7 174.8 191.9 206.4 211.2 216.8 224.8 232.3 238.6 240.6 202.68

64 High Water 130.8 145.5 159 168.8 179.3 190.9 199.8 212.3 217.9 221.3 225.6 186.47

65 High Water 137.7 161.6 189.1 210.2 223.4 239.6 250.8 262.8 273.2 281.7 279.9 228.18

66 Gavage 148.6 170.2 188.3 202.3 210.1 214.6 219.2 227.8 232.9 229.4 234.5 207.08

67 Gavage 148.8 166.2 188.2 200.7 203.8 212.2 216.5 222.2 229.2 236.4 239.8 205.82

68 Gavage 135.4 155 168.9 169.8 186.3 190.8 196.2 199.3 205 211 212.8 184.59

69 Gavage 117.1 132.8 140 149.3 157.9 162.4 164.5 170.8 176.5 179.1 181.7 157.46

70 Gavage 127.5 146.8 166.5 177.5 189.1 192.8 203.8 206.9 Dead Dead Dead 176.36

71 Gavage 138.4 155.4 172.6 181.3 194.8 207.9 219.3 222.7 230.5 239.8 241.5 200.38

72 Gavage 147.2 163.3 182.6 185.1 192 198.9 212 225.3 231.9 237.6 241.4 201.57

73 Gavage 154.6 171.8 192.1 203.3 215.6 224.9 222.1 230.7 241 254.7 261 215.62

74 Gavage 118.8 138.7 162.7 178.5 189 190 199.7 208.8 215.7 219.6 223.5 185.91

75 Gavage 127.4 138.9 153.7 159.3 168.2 175.5 186.2 192.6 199.7 204.6 207 173.92

76 Gavage 137.9 161.7 183.6 190.8 200.9 209.6 215.3 227.5 230.1 238.6 238.7 203.15

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

3 6 Table A 5 . Individual animal feed consumption (g/day)

– 7

- day exposure group.

Study Day

Rat ID Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1 Control 9.1 8.4 9.9 10.0 11.2 8.9 10.7 11.0 11 9.9 10.7 10.6 12.6 10.0 9.1

2 Control 10.2 11.5 13.4 12.6 11.9 11.8 13.4 10.8 13 12.1 11.9 14.6 12.6 11.3 10.2

3 Control 9.6 10.5 11.0 13.1 12.0 9.7 13.4 12.9 13 12.1 13.1 12.0 12.4 14.3 9.6

4 Control 9.1 9.3 8.9 10.0 10.1 13.4 8.8 14.8 10 12.0 7.9 10.2 8.7 11.7 9.1

5 Control 9.7 13.4 12.9 12.4 14.8 11.7 13.3 13.7 14 14.3 13.4 15.7 10.7 14.7 9.7

6 Control 11.7 11.1 13.8 12.1 11.4 10.7 13.6 12.6 12 15.8 11.3 13.8 15.3 13.7 11.7

7 Control 11.9 14.5 11.3 12.5 12.8 14.7 13.4 13.4 16 10.6 12.9 15.0 15.4 12.6 11.9

8 Control 9.3 9.7 8.6 10.2 11.6 8.5 12.8 6.6 10 7.2 9.2 9.6 10.0 7.7 9.3

9 Control 10.0 8.1 9.5 7.8 9.9 12.2 9.2 9.5 12 10.9 7.3 10.6 11.6 10.1 10.0

10 High Diet 10.9 9.9 10.5 13.4 10.3 15.2 10.3 11.5 13 14.8 9.4 12.9 9.4 13.3 10.9

11 High Diet 10.4 12.2 12.5 12.6 12.7 14.4 15.2 13.5 13 14.8 12.9 13.6 11.8 15.3 10.4

12 High Diet 7.8 10.8 8.1 9.2 7.0 8.8 11.8 9.4 13 8.8 12.4 9.7 8.9 12.4 7.8

13 High Diet 9.9 8.3 9.2 10.7 12.2 8.7 13.4 10.9 12 13.6 12.9 14.9 11.0 15.3 9.9

14 High Diet 8.4 10.1 9.6 10.4 11.9 8.2 12.3 9.9 11 10.9 9.1 11.8 8.6 11.9 8.4

15 High Diet 9.7 8.7 9.5 8.7 9.7 11.2 10.9 10.2 13 11.7 10.4 13.3 10.2 11.4 9.7

16 High Diet 9.3 8.5 8.1 10.4 7.9 8.7 14.6 11.1 14 13.3 9.1 12.2 8.1 14.1 9.3

17 High Diet 9.5 9.7 10.2 9.8 10.8 9.8 11.8 13.8 10 13.2 9.7 12.7 12.4 11.9 9.5

18 High Diet 11.2 11.7 12.8 12.6 11.9 12.0 14.2 13.3 14 14.9 13.0 15.5 14.9 16.5 11.2

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

37 Table A 5

. Individual animal feed consumption ( g /day)

– 7

- day exposure group. Continued.

Study Day

Rat ID Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

19 High Water 10.3 11.8 13.8 17.8 13.0 11.0 13.9 13.3 14 14.6 11.9 16.6 11.3 13.8 10.3

20 High Water 9.6 10.8 12.5 10.7 10.7 11.6 14.4 11.3 12 11.7 11.4 13.3 10.9 14.5 9.6

21 High Water 9.5 11.9 10.9 10.2 11.6 9.8 12.2 11.2 14 11.1 9.5 13.9 10.8 14.7 9.5

22 High Water 9.6 11.1 9.9 12.5 11.9 10.5 12.1 13.1 12 13.0 14.4 13.6 9.1 15.1 9.6

23 High Water 9.0 9.2 8.2 10.1 9.9 12.3 8.7 15.3 15 11.8 10.8 12.8 11.1 14.9 9.0

24 High Water 12.4 10.0 12.0 14.8 11.3 13.4 14.9 15.3 10 13.9 10.6 14.1 10.2 17.1 12.4

25 High Water 8.5 9.9 10.7 10.0 9.4 11.1 9.4 8.7 12 6.6 7.8 8.9 6.9 7.9 8.5

26 High Water 7.8 9.9 8.5 11.6 9.6 10.7 9.9 12.3 12 10.7 6.1 12.8 4.3 11.4 7.8

27 High Water 9.1 10.3 10.9 10.1 11.1 11.2 13.1 11.3 14 12.3 13.4 11.8 13.5 12.5 9.1

28 Gavage 9.7 13.7 10.3 11.5 11.1 12.7 14.7 13.6 13 10.5 13.6 12.1 14.5 9.7 9.7

29 Gavage 8.4 9.1 8.8 10.5 10.6 9.4 13.6 11.8 10 14.2 9.3 15.2 9.6 13.6 8.4

30 Gavage 9.2 10.7 7.9 10.5 8.4 11.7 10.6 15.2 8 10.1 8.3 11.3 10.1 12.8 9.2

31 Gavage 11.2 11.7 12.0 9.8 12.8 15.0 12.6 12.7 17 12.8 12.2 13.3 13.7 7.8 11.2

32 Gavage 9.0 8.7 9.7 10.1 8.4 8.8 9.7 11.4 11 11.1 10.1 12.7 11.8 13.8 9.0

33 Gavage 8.0 10.0 10.9 11.9 8.5 12.1 12.4 10.7 14 10.1 11.4 14.1 11.9 13.5 8.0

34 Gavage 9.9 9.6 10.0 11.6 9.8 9.4 10.8 10.4 11 10.7 12.4 13.2 11.5 11.2 9.9

35 Gavage 8.7 10.2 8.7 8.5 10.2 9.8 8.3 10.9 9 13.8 5.1 10.8 8.0 10.4 8.7

36 Gavage 10.8 9.0 8.1 10.2 8.1 12.2 7.9 13.3 8 9.5 9.7 8.5 5.6 10.5 10.8

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

38 Table A6. Individual animal water consumption (ml/day)

– 7

- day exposure group.

Study Day

Rat ID Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1 Control 20.00 0.00 20.00 20.00 20.00 0.00 20.00 40.00 15.00 15.00 10.00 19.00 12.70 13.60 16.30

2 Control 20.00 20.00 0.00 20.00 0.00 20.00 0.00 20.00 15.00 15.00 10.00 18.20 13.60 12.00 17.40

3 Control 20.00 20.00 0.00 20.00 0.00 20.00 20.00 20.00 30.00 10.00 15.00 14.90 12.00 14.80 15.50

4 Control 20.00 20.00 20.00 0.00 20.00 0.00 20.00 20.00 15.00 15.00 10.00 17.60 11.10 11.30 10.80

5 Control 20.00 20.00 20.00 0.00 20.00 20.00 15.00 25.00 15.00 15.00 15.00 22.10 15.90 17.00 14.70

6 Control 20.00 20.00 20.00 20.00 0.00 20.00 25.00 35.00 30.00 15.00 15.00 20.10 16.30 23.60 13.60

7 Control 20.00 20.00 20.00 0.00 20.00 20.00 20.00 25.00 15.00 15.00 15.00 20.50 17.30 12.20 15.90

8 Control 0.00 0.00 20.00 0.00 20.00 20.00 20.00 20.00 35.00 10.00 15.00 16.30 11.20 12.50 9.40

9 Control 0.00 20.00 0.00 20.00 20.00 0.00 15.00 25.00 15.00 5.00 10.00 16.90 10.10 13.00 9.80

10 High Diet 40.00 20.00 20.00 0.00 20.00 20.00 20.00 35.00 10.00 15.00 10.00 15.20 12.00 13.10 15.50

11 High Diet 20.00 0.00 20.00 20.00 20.00 20.00 5.00 35.00 20.00 15.00 10.00 21.60 13.30 16.30 17.20

12 High Diet 40.00 0.00 0.00 20.00 20.00 0.00 20.00 15.00 15.00 10.00 10.00 16.10 10.10 15.80 12.50

13 High Diet 20.00 0.00 20.00 20.00 20.00 0.00 20.00 20.00 25.00 15.00 10.00 17.80 12.00 14.90 15.10

14 High Diet 40.00 40.00 20.00 0.00 20.00 20.00 20.00 25.00 10.00 10.00 10.00 17.50 10.40 13.20 ND

15 High Diet 20.00 0.00 20.00 20.00 0.00 20.00 5.00 15.00 15.00 10.00 15.00 13.00 12.90 13.00 16.60

16 High Diet 20.00 20.00 20.00 0.00 20.00 0.00 20.00 35.00 15.00 15.00 10.00 21.00 11.80 14.20 19.30

17 High Diet 0.00 0.00 0.00 20.00 20.00 0.00 20.00 40.00 10.00 15.00 10.00 18.50 12.90 14.50 18.10

18 High Diet 20.00 0.00 20.00 20.00 0.00 20.00 20.00 40.00 25.00 15.00 15.00 21.70 13.50 17.00 22.80

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

39 Table A6. Individual animal water consumption ( ml /day)

– 7

- day exposure group. Continued.

Study Day

Rat ID Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

19 High Water 20.00 20.00 20.00 0.00 40.00 0.00 15.00 25.00 15.00 15.00 15.00 20.90 12.20 6.90 13.90

20 High Water 20.00 20.00 20.00 20.00 20.00 0.00 25.00 35.00 20.00 15.00 15.00 15.40 12.90 16.70 13.70

21 High Water 0.00 20.00 20.00 0.00 20.00 20.00 5.00 15.00 15.00 10.00 15.00 14.30 9.90 15.80 12.20

22 High Water 20.00 20.00 20.00 0.00 20.00 20.00 0.00 15.00 15.00 10.00 10.00 15.80 12.40 21.80 14.20

23 High Water 20.00 20.00 20.00 0.00 20.00 20.00 10.00 30.00 30.00 10.00 10.00 12.20 10.90 6.90 10.90

24 High Water 20.00 0.00 20.00 20.00 20.00 0.00 20.00 30.00 15.00 15.00 15.00 17.30 12.00 15.70 15.40

25 High Water 20.00 20.00 0.00 20.00 20.00 0.00 15.00 25.00 15.00 15.00 5.00 15.70 8.50 15.80 12.20

26 High Water 20.00 20.00 0.00 20.00 0.00 0.00 20.00 40.00 10.00 15.00 10.00 13.90 8.90 17.20 12.80

27 High Water 20.00 20.00 20.00 0.00 20.00 0.00 20.00 30.00 15.00 15.00 15.00 14.00 25.10 80.90 51.30

28 Gavage 20.00 20.00 20.00 0.00 20.00 20.00 15.00 25.00 10.00 15.00 15.00 13.10 14.30 12.60 14.20

29 Gavage 20.00 20.00 20.00 20.00 20.00 0.00 0.00 30.00 10.00 15.00 10.00 ND 11.00 11.30 12.00

30 Gavage 20.00 40.00 0.00 20.00 0.00 20.00 0.00 30.00 10.00 15.00 10.00 19.20 9.60 6.20 7.50

31 Gavage 20.00 0.00 20.00 20.00 20.00 0.00 25.00 35.00 35.00 15.00 15.00 20.00 15.10 19.90 12.30

32 Gavage 20.00 20.00 20.00 0.00 20.00 0.00 20.00 25.00 15.00 10.00 10.00 16.40 10.30 13.30 11.40

33 Gavage 20.00 20.00 20.00 20.00 0.00 20.00 25.00 35.00 25.00 10.00 20.00 18.40 15.00 11.60 14.20

34 Gavage 20.00 20.00 20.00 0.00 20.00 20.00 10.00 30.00 20.00 15.00 15.00 23.50 15.70 10.80 15.00

35 Gavage 40.00 20.00 20.00 0.00 20.00 20.00 0.00 35.00 15.00 15.00 10.00 22.20 13.30 9.50 10.80

36 Gavage 20.00 0.00 20.00 0.00 20.00 0.00 20.00 15.00 10.00 15.00 10.00 15.20 12.10 22.70 11.50

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

40 Table A 7

. Individual animal feed consumption (g/day)

- 61

- day exposure group. Days 1

- 14.

Day

Rat # Treatment Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14

39 Control 12.6 10.1 11.9 10.8 13.5 12.4 11.6 13.8 13.4 8.7 16.9 12.9 9.4 12.2

40 Control 11.0 8.9 11.4 10.6 12 .0 9.9 10.3 9.9 13.5 8.7 13.4 8.0 9.6 8.7

41 Control 6.1 9.3 7.4 8.8 9.4 8.3 7.7 10.7 7.0 7.6 12 .0 7.7 8.4 9.6

42 Control 12.1 10.5 12.5 12.2 13 .0 11.6 13.3 10.1 14.8 9.5 13.6 12.1 13.8 10.8

43 Control 2.7 11.5 6.4 9.2 12.5 9.6 10.0 10.4 10.3 8.5 11.4 12.6 8.7 6.4

44 Control 14.5 10.5 12.5 15 .0 11.8 13.4 13.3 11.8 15.8 10.6 13.2 15.3 12.4 12.7

45 Control 12.3 10.6 12.5 14.3 15.1 11.2 15.2 12.1 13.5 10.9 14.8 14.8 12.6 14.3

46 Control 12.6 9.0 11.9 11 .0 9.2 12.6 11 9.5 12.7 10.6 9.7 13.7 9.6 9.8

47 Control 13.1 11.3 12.6 11.1 11.7 14 .0 13.7 8.6 13.6 9.4 12.5 12.7 13.2 13.1

48 High Diet 10.0 9.0 10.5 8.6 11.2 11.3 10.4 10.1 12.8 9.5 9.2 10.1 12.4 8.9

49 High Diet 13.5 9.0 10.6 11.5 10.1 10.1 11.6 7.8 10.5 7.7 8.6 9.6 11.3 10.1

50 High Diet 8.5 11 12.5 12.7 9.1 11.1 13.3 13.5 11 .0 11.4 11.4 10.0 11.6 11.3

51 High Diet 7.9 10 .0 9.7 10.6 10.6 10.6 9.7 10.5 14.3 10.1 11.7 11.3 12.3 11.6

52 High Diet 7.4 8.8 10.5 9.5 10.5 9.2 12.3 7.2 13.2 8.3 11.9 10.0 11.6 8.5

53 High Diet 9.4 9.9 7.9 12.3 9.2 7.4 11.0 7.0 10.0 9.0 8.6 11.5 9.4 11.6

54 High Diet 15.2 11 .0 11 .0 13.1 14.3 10.3 15.9 11.4 14.4 12.4 13.6 14.4 15.7 11.3

55 High Diet 5.5 8.5 7.1 10.2 12.9 7.2 13.0 8.0 8.2 8.0 10.2 10.2 10.2 9.7

56 High Diet 12.8 11.5 12.1 12.4 12.5 13.6 16.3 10.6 14.1 12.3 15.3 12.0 11.5 14 .0

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

41 Table A 7 . Individual animal feed consumption (g/day)

- 61

- day exposure group . Days 1

- 14.

Day

Rat # Treatment Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14

57 High Water 8.3 11.7 11.4 14.2 13.4 13.1 15.4 14.8 18.4 12.6 13.7 12.5 11.8 11.2

58 High Water 12.6 9.7 8.9 12.2 13.4 8.8 15.1 10.0 14.8 9.1 11.3 12.7 10.7 7.1

59 High Water 11.0 10.4 9.2 ND 11.9 12.3 10.0 6.9 10.4 9.8 9.0 10.2 13.5 7.7

60 High Water 9.6 10.2 9.1 10.3 8.2 9.5 12.9 8.7 9.9 9.4 10.8 10.6 12.1 6.3

61 High Water 9.1 12.0 10.6 11.6 9.2 11.9 8.9 10.2 12.8 9.0 9.7 9.7 11.2 11.1

62 High Water 7.7 8.1 10.2 8.9 12.0 8.4 14.2 9.7 10.4 8.5 10.8 9.2 12.8 6.0

63 High Water 12.9 7.4 11.3 11.3 8.8 10.8 10.5 11.4 12.0 9.3 15.2 9.2 10.5 10.2

64 High Water 9.2 10.8 9.9 11.2 10.5 11.9 12.1 7.2 11.9 5.6 12.6 9.1 9.1 11.0

65 High Water 8.9 7.9 9.4 10.7 13.1 8.3 13.2 10.1 10.9 10.0 7.8 11.8 12.7 10.1

66 Gavage 15.4 11.7 12.2 13.1 10.1 11.9 14.3 8.5 16.0 12.1 11.8 12.2 10.9 13.3

67 Gavage 10.3 10.8 9.4 12.3 13.8 11.0 15.2 10.2 15.5 10.0 10.7 13.3 9.2 10.8

68 Gavage 12.3 11.2 8.8 11.3 13.1 11.1 10.2 10.1 16.7 4.2 13.9 12.5 11.4 12.2

69 Gavage 7.7 10.7 8.2 11.0 8.7 7.7 13.0 5.8 11.0 9.6 10.8 13.7 10.6 8.5

70 Gavage 9.3 9.5 11.7 12.7 13.5 7.3 12.4 9.6 13.0 9.8 9.4 8.6 10.3 11.8

71 Gavage 11.3 8.6 12.1 12.1 10.8 10.7 14.5 9.6 9.2 9.2 12.7 11.6 10.5 14.7

72 Gavage 12.3 8.7 11.9 15.3 10.4 12.8 13.7 9.0 10.1 11.3 11.6 8.3 12.2 14.1

73 Gavage 13.3 11.4 12.2 13.4 15.2 11.8 14.1 10.9 16.1 10.3 13.2 11.5 11.6 17.4

74 Gavage 7.9 10.1 8.1 10.0 12.4 9.5 10.1 7.3 11.1 9.1 11.2 8.9 11.5 12.9

75 Gavage 6.1 7.5 8.5 9.3 8.9 6.6 11.8 5.1 10.6 9.5 7.6 8.8 8.8 10.3

76 Gavage 13.5 10.7 12 .0 12.5 11.3 13.9 11.1 7.9 17.4 8.2 16.0 9.3 13.9 11.6

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

42 Table A 7 . Individual animal feed consumption (g/day)

- 61

- day exposure group . Days 15

- 28.

Day

Rat # Treatment Group 15 16 17 18 19 20 21 22 23 24 25 26 27 28

39 Control 13.8 10.0 13.6 15.2 8.1 11.8 10.8 10.8 16.2 7.9 10.9 9.6 9.3 13.4

40 Control 9.2 8.9 10.0 11.2 8.8 10.7 12.4 10.6 10 11.2 11.2 10.5 9.6 8.6

41 Control 15.8 10.3 9.2 5.3 8.0 9.7 8.6 8.1 9.6 7.3 9.2 9.3 9.6 7.8

42 Control 13.6 4.8 15.8 13.8 13.9 16.1 10.0 10.4 13.7 12.6 13.6 12.2 14.9 9.1

43 Control 10.6 12.1 6.1 8.0 8.6 12.1 7.6 11.0 11.2 9.1 12 10.7 10.4 10.3

44 Control 13.1 11.8 15.1 10.1 11.3 13.4 12.5 13.1 13.7 11.6 13.7 12.4 14.6 14.0

45 Control 14.4 12.5 11.0 14.0 12.4 15.2 13.1 17.5 14.3 10.2 15.2 12.0 14.7 12.5

46 Control 12.2 9.2 14.0 10.4 11.2 11.7 12.1 10.6 8.9 10.4 11 11.9 13.8 11.1

47 Control 14.4 10.8 12.8 11.2 12.5 13.2 11.5 16.0 10.3 10.4 14.3 12.5 12.1 12.1

48 High Diet 14.3 8.9 11.5 9.5 12.0 13.3 9.9 13.2 13.5 13.2 12.3 11.7 12.0 16.0

49 High Diet 12.3 8.8 11.5 9.1 11.2 13 12.6 11.9 11.4 13.8 12.1 12.6 14.4 10.1

50 High Diet 14.2 12.2 15.7 8.5 13.2 14.2 15.3 11.9 12.8 10.3 15.8 12.1 12.8 10.2

51 High Diet 11.3 11.5 13.1 11.2 9.3 12.2 10.8 10.7 12.5 8.3 8.5 9.3 10.5 6.2

52 High Diet 12.4 9.0 13.0 10.2 10.4 14.4 6.4 11.3 8.9 9.0 11.1 4.8 10.8 8.6

53 High Diet 7.6 9.4 10.4 7.7 7.6 11.8 7.8 12.9 10.6 11.2 12.6 11.8 11.2 12.5

54 High Diet 13.1 13.9 9.9 13.1 15.1 11.4 11.9 9.0 16.1 10.1 16.6 10.6 14.0 11.5

55 High Diet 12.7 8.9 13.7 11.3 11.6 13.8 13.6 14.6 12.2 11.3 12.8 10.8 15.2 13.6

56 High Diet 11.9 7.9 17.3 9.1 12.5 12.7 11.1 11.5 14.2 12.9 11.9 12.1 13.3 14.1

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

43 Table A 7 . Individual animal feed consumption (g/day)

- 61

- day exposure group . Days 15

- 28.

Day

Rat # Treatment Group 15 16 17 18 19 20 21 22 23 24 25 26 27 28

57 High Water 9.7 9.5 9.2 9.2 9.5 10.4 16.0 8.0 12.7 9.4 10.6 11.1 11.5 11.5

58 High Water 11.8 5.7 9.0 7.6 9.3 11.9 8.4 9.0 9.9 9.9 10.4 7.0 9.6 9.3

59 High Water 15.8 7.4 9.8 10.6 12.3 10.7 14.8 13.7 12.5 10.1 14.5 8.9 14.9 13.0

60 High Water 11.1 10.4 7.6 9.2 8.6 11.2 10.5 10.9 14.2 7.5 11.0 10.3 9.8 10.0

61 High Water 11.3 9.2 8.7 9.7 9.7 10.4 10.1 11.4 9.7 8.2 11.8 6.4 7.9 12.2

62 High Water 9.7 9.6 8.2 8.2 10.6 9.0 9.6 12.4 9.5 8.9 11.9 12.9 11.1 9.7

63 High Water 11.1 9.7 10.9 10.2 10.8 10.2 12.8 8.4 13.1 9.1 12.3 12.3 11.0 13.7

64 High Water 13.6 8.1 8.2 7.4 7.7 9.1 8.2 6.7 9.4 9.2 9.8 6.8 8.5 7.1

65 High Water 13.9 10.4 11.7 12.6 12.2 11.9 11.5 12.8 11.3 12.2 13.3 12.1 11.8 13.9

66 Gavage 6.2 12.8 12.8 12 13.2 16 13.8 11.9 12.5 10.1 15.0 10.7 13.9 16.8

67 Gavage 11.8 13.3 11.1 11.9 8.9 12.8 11.3 12.6 10.5 8.8 7.3 10.7 10.9 8.1

68 Gavage 11.9 10.9 10.1 9.3 13.6 11.1 3.9 10.0 10.3 2.4 5.9 8.7 11.1 10.2

69 Gavage 7.2 11.7 6.5 5.5 7.8 8.6 6.0 7.5 9.1 9.8 8.1 9.4 8.5 9.1

70 Gavage 9.9 13.8 11.3 9.7 14.6 10.7 10.6 11.4 10.8 9.0 11.4 6.5 10.0 7.5

71 Gavage 9.1 12.8 7.7 13.4 9.4 11.6 9.2 9.5 10.5 9.9 10.8 3.7 8.6 8.3

72 Gavage 6.7 17.5 12.6 11.6 13.2 12.6 11.7 10.8 10.2 8.9 7.7 4.7 7.9 12.7

73 Gavage 11.3 13.0 13.1 13.3 13.6 10.7 15.6 12.8 8.4 13.3 14.9 9.6 10.0 10.4

74 Gavage 8.5 12.7 11.4 9.9 11.2 13.2 11 12 11.6 10.9 13.4 7.8 11.0 11.3

75 Gavage 8.2 14.2 7.6 4.5 12.5 9.5 8.6 9.2 12.9 8.5 12.1 4.8 5.1 5.9

76 Gavage 12.5 15.9 12.0 9.1 14.0 10.9 12.1 12.3 11.7 8.0 8.4 3.2 12.3 4.6

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

44 Table A 7 . Individual animal feed consumption (g/day)

- 61

- day exposure group . Days 29

- 42.

Day

Rat # Treatment Group 29 30 31 32 33 34 35 36 37 38 39 40 41 42

39 Control 11.1 13.8 10.3 12.0 9.3 12.0 14.5 12.3 11.5 7.2 11.5 11.5 8.6 12.0

40 Control 10.5 9.1 12.7 11.5 12.3 9.4 12.7 9.7 9.6 7.8 9.1 11.1 9.7 11.3

41 Control 9.2 9.9 8.1 6.6 5.6 7.7 10.5 5.5 6.3 7.3 8.2 8.8 5.6 10.1

42 Control 7.5 11.6 10.0 11.9 11.1 11.2 10.8 9.6 12.2 10.8 8.1 9.8 10.9 10.6

43 Control 10.3 9.2 12.3 10.0 6.0 9.4 9.6 11.3 7.3 10.4 10.2 9.4 12.7 9.1

44 Control 12.5 14.1 16.1 14.5 13.3 12.5 14.8 16.3 11.5 10.4 16.7 17.1 11.3 15.0

45 Control 9.4 13.8 15.2 10.9 10.0 13.5 13.3 14.8 12.1 6.8 9.4 9.8 6.6 13.1

46 Control 8.0 12.0 10.8 7.9 9.5 11 13.2 11.2 9.3 12.6 8.8 13.2 11.5 12.8

47 Control 13.5 16.6 12.5 14.2 11.9 17.1 12.4 15.6 11.3 12.6 11.3 13.1 10.7 9.1

48 High Diet 10.2 8.7 16.1 9.9 11.8 12.1 12.4 9.8 11.9 9.7 13.1 9.8 16.4 10.2

49 High Diet 12.0 12.5 12.9 8.5 11.6 7.9 8.8 9.9 9.5 7.7 10.7 8.1 11.2 8.7

50 High Diet 13.3 11.6 14.1 8.5 11.3 14.6 10.9 10.8 13 8.2 12.1 14.3 10.0 12.4

51 High Diet 11.7 7.9 13.3 4.6 8.3 10.2 7.7 8.8 11.9 8.8 8.6 7.8 8.2 11.7

52 High Diet 11.4 9.9 12.4 9.8 8.1 11.1 7.7 8.9 9.6 6.0 7.7 8.2 9.4 10.7

53 High Diet 12.1 10.1 12.5 10.1 9.5 10.5 11.2 12.3 12.2 7.5 11.4 8.0 13.5 8.8

54 High Diet 11.8 10.8 11.3 11.9 10.2 11.7 14.8 10.3 10.0 9.3 12.4 13.6 9.4 12.3

55 High Diet 11.6 11.8 12.7 9.8 14.7 12.0 14.7 14.5 9.7 10.6 12.2 14.6 7.4 11.1

56 High Diet 12.8 14.3 15.0 12.1 11.2 14.4 10.5 14.2 9.4 11.9 6.7 11.8 12.2 10.2

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

45 Table A 7 . Individual animal feed consumption (g/day)

- 61

- day exposure group . Days 29

- 42.

Day

Rat # Treatment Group 29 30 31 32 33 34 35 36 37 38 39 40 41 42

57 High Water 12.0 10.5 12.4 6.7 10.1 10.9 12.0 9.0 7.2 6.1 8.6 9.0 9.3 10.5

58 High Water 14.2 7.5 10.8 8.5 7.1 9.7 10.3 11.6 9.6 9.3 7.9 10.3 10.0 11.2

59 High Water 15.3 9.7 14.6 7.8 12.5 6.2 10.5 10.5 11.7 6.6 8.3 11.2 8.3 14.0

60 High Water 13.7 6.8 12.9 6.2 6.1 8.9 9.3 9.8 14.6 5.8 8.3 10.2 8.8 13.3

61 High Water 10.2 6.2 9.7 10.8 7.0 8.2 10.3 9.9 8.9 6.9 10.2 9.1 6.4 9.5

62 High Water 13.5 8.8 13.7 8.2 7.8 11.9 8.7 11.6 9.2 11.5 8.3 12.8 11.9 10.7

63 High Water 13.2 11.4 14.4 9.5 9.7 10.9 10.8 11.3 12.6 7.8 8.3 8.6 8.8 10.6

64 High Water 11.1 4.5 11.9 9.2 6.8 8.8 9.2 10.2 11.4 8.7 7.5 9.7 8.3 11.4

65 High Water 13.8 12.1 12.7 13 12.3 10.3 17.6 10.7 13 11.2 11.9 11.7 16.1 14.8

66 Gavage 3.8 11.2 10.6 10.9 6.9 8.7 10.7 13.1 11.2 7.2 10 6.9 6.2 8.5

67 Gavage 13.1 7.2 10.5 7.8 9.7 7.2 8.3 11.5 8.7 4.9 9.9 9.0 11.6 6.3

68 Gavage 14.0 9.3 12.7 13.7 8.8 11.2 8.4 9.7 8.7 7.1 6.5 8.8 7.7 8.4

69 Gavage 7.1 7.6 8.8 5.7 7.3 7.5 7.4 7.6 10.3 5.5 10.7 6.8 7.0 11.4

70 Gavage 9.7 12.2 14.8 10.4 11.3 11.0 13.3 10.1 8.2 7.9 6.2 9.9 9.4 11.2

71 Gavage 11.4 10.8 13.3 11.1 8.6 10.0 10.0 10.3 12.0 9.6 8.5 11.5 7.8 10.3

72 Gavage 8.3 7.1 13.3 9.4 9.1 5.3 8.9 10.6 8.5 9.4 7.5 11.3 9.1 13.6

73 Gavage 11.4 15.7 10.3 13.9 11.7 10.3 11.5 9.3 13.2 10.3 8.1 11.4 13.5 7.2

74 Gavage 10.4 10.6 10.7 11.3 9.7 11.4 11.2 11.4 8.0 4.2 8.7 7.2 8.2 10.4

75 Gavage 8.7 9.6 3.8 7.8 7.8 10.1 8.3 4.5 12.3 2.5 8.7 7.3 11.0 9.7

76 Gavage 11.3 10.8 12.9 10.0 12.3 10.3 10.1 13.1 11.8 12.6 8.9 10.7 8.0 10.0

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

46 Table A 7 . Individual animal feed consumption (g/day)

- 61

- day exposure group . Days 43

- 56.

Day

Rat # Treatment Group 43 44 45 46 47 48 49 50 51 52 53 54 55 56

39 Control 11.4 16.7 12.1 14.8 9.1 11.8 13.6 7.1 17.0 9.8 12.9 10.8 9.2 11.0

40 Control 11.3 13.0 7.4 9.4 9.4 9.2 11.3 9.0 9.4 8.3 9.0 11.9 8.8 11.5

41 Control 6.8 8.4 9.0 11.0 6.6 7.2 8.9 7.8 11.0 7.0 10.2 6.2 5.2 8.9

42 Control 10.7 12.1 10.1 11.4 10.1 11.2 11.0 9.3 13.9 8.4 11.3 10.7 12.2 10.7

43 Control 7.6 11.1 8.6 7.8 10.0 8.4 12.1 9.0 6.1 7.9 11.3 8.1 7.9 9.7

44 Control 14.4 18.4 15.2 12.7 15.9 9.6 14.4 13.1 12.9 15.0 9.8 13.7 11.6 10.9

45 Control 12.2 12.6 12.1 7.0 10.5 13.1 13.7 9.4 10.0 9.7 10.0 11.0 13.1 9.8

46 Control 13.3 12.1 10.8 10.3 8.2 12.2 12.9 14.3 7.3 11.6 8.6 8.7 12.7 9.2

47 Control 9.9 11.6 12.2 9.0 10.4 10.8 9.2 8.5 12.3 10.3 4.9 11.8 9.8 8.6

48 High Diet 14.5 10.6 12.4 10.7 10.0 14.0 11.9 11.3 13.2 14.4 12.5 10.4 15.2 9.5

49 High Diet 14.9 11.8 6.4 7.2 8.7 12.9 8.5 10.5 10 6.9 8.8 6.1 7.9 9.3

50 High Diet 11.1 10.7 10.2 11.6 9.9 11.3 10.7 9.2 11.7 10.6 8.1 9.8 8.3 4.8

51 High Diet 12.4 4.7 7.6 7.5 9.6 5.5 12.2 8.7 8.8 9.0 6.1 6.7 6.9 8.4

52 High Diet 8.5 8.5 6.6 12.3 5.0 6.3 11.9 8.0 8.3 9.7 7.7 10.5 7.0 11.1

53 High Diet 16.2 9.5 12.5 11.7 10.7 9.1 13.0 8.9 12.4 12.3 11.8 12.1 13.3 9.1

54 High Diet 11.1 14.7 10.5 11.3 10.2 13.4 12.8 8.6 14.3 9.7 12.4 12.8 11.0 8.3

55 High Diet 12.8 12.9 10.5 10.6 13.3 12.6 14.2 8.7 13.3 11.4 12.2 12.0 10.4 12.8

56 High Diet 12.6 7.8 10.7 12.7 10.8 13.4 15.7 10.7 10.9 13.9 12.0 13.5 11.4 10.9

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

47 Table A 7 . Individual animal feed consumption (g/day)

- 61

- day exposure group . Days 43

- 56.

Day

Rat # Treatment Group 43 44 45 46 47 48 49 50 51 52 53 54 55 56

57 High Water 7.9 11.9 12.1 7.5 7.7 9.0 10.2 8.2 12.8 4.6 10.5 8.2 10.4 15.3

58 High Water 4.5 10.6 10.1 8.0 4.1 9.6 10.1 5.9 12.2 8.7 9.3 6.0 8.2 9.2

59 High Water 7.9 15.6 10.3 9.8 7.7 8.1 10.1 9.9 13.2 8.9 11.6 7.1 11.1 11.9

60 High Water 7.7 9.1 8.0 7.1 6.7 6.0 10.5 5.3 9.7 7.1 6.6 6.8 6.8 7.4

61 High Water 9.7 11.0 9.9 8.8 7.7 9.5 15.2 9.2 11.8 6.2 9.9 11.7 9.9 8.6

62 High Water 9.1 8.4 10.6 9.7 8.6 10.8 12.4 9.0 6.3 9.5 8.5 7.8 11.2 9.9

63 High Water 9.9 9.5 9.8 6.3 5.7 6.7 10.6 8.9 11.6 10.4 9.9 10.5 9.5 11.8

64 High Water 9.6 9.8 7.9 10.4 7.0 8.1 9.6 8.4 8.8 11.4 8.2 8.8 12.6 8.9

65 High Water 12.8 14.7 10.6 13.4 9.9 13.0 14.6 12.0 11.0 13.8 9.5 12.9 11.0 14.4

66 Gavage 10.1 10.2 6.6 8.6 7.4 11.1 10.8 10.1 11.8 8.8 11.9 13 10.1 10.6

67 Gavage 8.3 12.4 9.9 5.7 7.7 12 10.7 5.9 11.6 9.5 11.5 7.8 7.6 10.4

68 Gavage 7.4 9.3 7.9 5.9 5.8 7.8 11.1 6.4 8.5 7.7 7.6 10.9 8.0 8.7

69 Gavage 3.6 6.9 5.3 5.4 5.2 6.1 9.1 5.3 9.7 9.1 4.1 7.8 8.4 6.8

70 Gavage 11.2 12.8 7.0 10.8 10.7 10.3 9.9 6.3 13.2 7.5 10.2 10.1 6.9 12.8

71 Gavage 11.4 12.9 8.0 7.4 9.3 13.6 10.1 6.7 11.6 8.8 11.2 9.5 9.4 10.9

72 Gavage 9.6 15.6 8.4 12 8.5 12.9 11.4 8.6 16.6 11.4 14.6 8.9 10.2 11.2

73 Gavage 5.3 6.2 10.0 6.3 8.6 12.7 12.9 9.9 11.8 10.8 10.6 11.4 12.3 13.1

74 Gavage 8.0 11.3 10.5 10.7 11.5 10.7 14.1 9.6 10.5 8.0 9.7 11.4 11.3 8.2

75 Gavage 8.2 10.5 9.0 9.9 9.3 11.2 5.7 9.8 10.3 9.1 9.8 8.8 7.2 10.6

76 Gavage 11.0 8.8 8.6 9.9 8.1 8.1 12.7 8.9 13.8 7.8 10.1 11.1 12.9 9.1

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

48 Table A 7 . Individual animal feed consumption (g/day)

- 61

- day exposure group . Days 57

- 70.

Day

Rat # Treatment Group 57 58 59 60 61 62 63 64 65 66 67 68 69 70

39 Control 11.1 13.2 14 11.5 11.1 11.0 10.6 14.6 14.3 11.1 9.7 9.8 11.7 11.3

40 Control 7.4 11.9 8.9 8.5 10.1 9.4 13.7 9.4 14.6 5.9 8.4 8.6 11.8 9.8

41 Control 7.4 5.2 8.3 7.3 6.5 9.0 7.8 9.3 10.1 6.9 10.5 6.2 8.7 8.8

42 Control 14.2 13.8 10.2 9.8 12.3 11.3 10.5 11.6 15.0 9.7 13.3 10.5 11.3 12.7

43 Control 7.4 11.7 7.9 7.7 8.4 8.1 11.3 6.5 11.4 6.6 7.1 6.5 7.9 9.8

44 Control 12.0 12.3 13.5 12.5 12.5 16.4 12.9 11.7 15.3 12.5 11.4 12.0 12.0 11.0

45 Control 13.4 5.2 11.3 12.5 12.3 8.7 12.7 12.3 13.6 9.6 11.8 11.7 10.6 11.4

46 Control 15.3 9.5 11.4 9.3 8.4 12.8 6.8 11.6 13.6 9.3 11.2 12.7 10.7 15.7

47 Control 12.1 12.0 11.0 9.7 11.8 12.4 9.9 13.7 14.3 8.5 11.6 12.2 10.2 12.9

48 High Diet 10.3 11.5 11.6 10.6 12.9 8.3 10.5 14.9 10.1 10.6 9.7 13.8 11.0 10.3

49 High Diet 7.9 10.0 6.9 8.3 8.9 6.4 8.1 11.5 10.2 6.5 9.7 6.1 8.3 8.6

50 High Diet 11.3 9.5 6.7 10.0 6.9 13.4 5.6 11.5 13.8 9.8 9.3 11.6 5.7 10.5

51 High Diet 9.6 9.1 9.7 7.0 8.2 10.5 7.7 12.9 11.7 8.4 6.3 12.3 7.6 13.2

52 High Diet 10.4 12.1 8.7 7.6 8.3 6.5 7.8 10.5 9.5 8.5 7.7 7.1 8.9 5.6

53 High Diet 10.5 11.4 10.1 10.2 10.2 9.6 10.8 14.1 12.5 10.8 10.4 12.7 10.5 9.1

54 High Diet 12.1 10.4 9.3 10.8 10.6 10.3 11.7 12.6 16.1 10.8 12.8 11.6 15.1 10.5

55 High Diet 11.8 11.3 11.8 10.7 10.5 11.1 11.8 13.1 12.3 13.8 11.9 12 11.8 11.0

56 High Diet 13.7 11.5 11.8 10.1 11.6 15.2 10.4 15.2 12.6 14.6 9.7 10.6 11.9 9.6

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

49 Table A 7 . Individual animal feed consumption (g/day)

- 61

- day exposure group . Days 57

- 70.

Day

Rat # Treatment Group 57 58 59 60 61 62 63 64 65 66 67 68 69 70

57 High Water 6.4 9.7 7.3 6.7 11.0 7.3 10.1 10.7 15.1 7.8 8.1 8.0 7.6 9.8

58 High Water 9.6 11.2 7.3 11.0 8.6 8.6 7.0 8.8 12.2 10.7 6.3 7.3 11.5 7.7

59 High Water 8.1 13.1 5.7 7.9 5.7 12.6 7.2 9.6 10.7 8.8 9.3 9.6 9.9 7.4

60 High Water 9.6 11.3 4.9 8.9 9.7 8.4 8.2 11.7 12.3 8.5 9.6 11.9 7.1 9.4

61 High Water 9.4 12.6 9.5 6.9 9.6 12.0 11.5 9.9 13.8 8.5 6.9 6.5 8.7 8.0

62 High Water 9.3 8.4 10.2 10.5 8.2 11.9 9.1 9.2 10.4 7.7 8.9 10.9 7.9 9.3

63 High Water 11.7 9.3 9.3 10.7 7.0 9.5 9.7 11.7 9.5 8.4 6.7 8.8 8.2 9.9

64 High Water 10.1 9.3 11.1 10.9 8.1 7.6 8.5 9.4 11.7 8.6 7.3 8.1 9.6 8.2

65 High Water 13.8 10.9 12.8 11.4 14.7 13.9 11.2 14.1 15.5 11.7 11.2 12.5 13.3 11.3

66 Gavage 10.3 14.2 5.3 9.3 11.0 9.8 8.4 8.7 10.6 3.7 7.7 3.7 4.8 9.8

67 Gavage 8.0 10.1 9.0 9.6 6.7 9.6 7.9 7.5 9.8 10.9 8.7 11.0 9.9 8.6

68 Gavage 7.5 9.3 7.8 8.1 9.6 5.5 9.2 8.8 9.4 8.3 6.8 7.7 8.9 8.4

69 Gavage 8.3 10.2 4.0 5.3 5.6 10.4 8.6 2.9 11.1 6.0 7.9 7.5 4.4 8.2

70 Gavage 6.7 11.4 1.6 Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead

71 Gavage 11.0 13.7 10.5 6.5 8.9 12.3 9.6 10.6 12.7 10.2 12.0 7.4 11.3 9.4

72 Gavage 10.1 8.6 9.6 6.7 8.7 12.6 8.7 10.5 15.9 8.3 11.7 10.5 10.3 11.5

73 Gavage 11.4 11.1 10.2 13.1 13.1 10.3 12.6 14.0 15.4 10.0 11.7 13.7 13.5 10.7

74 Gavage 10.9 9.4 10.1 10.2 10.2 10.5 10.9 6.3 12.7 9.4 9.1 5.2 8.9 7.0

75 Gavage 11.4 7.6 3.7 10.1 7.1 11.0 4.8 10.3 12.0 7.5 7.2 9.7 9.0 7.9

76 Gavage 9.3 10.1 7.1 8.1 8.8 14.0 10.7 10.9 13.8 9.9 10.0 11.2 11.8 9.6

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

50 Table A 8 . Individual animal water consumption ( ml /day)

- 61

- day exposure group

. Days 1

- 14.

Day

Rat # Treatment Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14

39 Control 4.2 7.6 10.5 10.2 12.2 13.0 15.9 17.9 21.2 12.5 13.3 12.8 13.4 10.9

40 Control 9.1 10.7 11.8 14.0 15.5 12.8 15.7 29.7 20.1 9.8 12.6 10.2 12.6 12.2

41 Control 6.2 7.9 8.9 10.9 10.5 12.1 11.5 25.0 17.5 7.3 10.3 9.7 8.8 9.5

42 Control 9.0 9.2 13.4 14.0 13.8 14.1 16.5 35.1 17.3 12.1 13.1 14.2 14.0 12.7

43 Control 5.4 9.4 9.9 13.4 11.8 10.7 17.9 17.2 17.2 10 .0 11.6 11.3 11.8 13.1

44 Control 8.3 10.8 12.9 12.5 15.1 16.4 16.9 34.5 22.7 12.6 14.6 15.1 16.7 16.3

45 Control 7.5 8.6 13.2 12.5 14.4 12.1 14.7 21.2 19.7 14.5 14.3 14.4 15.0 15.5

46 Control 9.6 7.7 9.4 12.5 12.2 10.1 14.2 19.5 21.6 12.8 13 .0 11.8 14.4 11.6

47 Control 6.2 9.7 11.1 12.1 12.5 14.7 16.9 19.0 19.2 11 .0 13.2 13.8 14.7 14.8

48 High Diet 7.8 9.0 11.2 11.3 10.6 13.9 19 25.8 24.3 9.7 12.6 12.1 13.1 12.6

49 High Diet 10.7 9.4 10.3 10.7 11.7 12.5 12.4 20.7 21.4 10.8 12 .0 11.5 11.5 12.1

50 High Diet 10.2 11.6 13.3 12.6 13.9 14 .0 18.8 19.7 20 .0 12.3 14.2 14.1 15.1 14 .0

51 High Diet 5.6 9.3 9.1 11.3 10.3 10.8 17.7 20.2 19.4 9.6 13.4 10.9 12.3 11.2

52 High Diet 7.3 9.2 10.2 10.2 12.9 11.4 15.6 23.4 16 .0 9.3 12.1 10.4 12.8 12 .0

53 High Diet 4.0 8.9 9.8 12.6 14.1 11.6 18.6 19.5 18.5 12 .0 13.8 14.3 13.2 13.6

54 High Diet 9.6 11.9 13.7 14.7 17.4 15.6 23.1 29.8 22.1 14.2 15.1 17.1 17.6 17.7

55 High Diet 10.6 8.6 9 .0 10.7 12.8 11.8 14.3 27.6 16.1 10.9 11.5 11.7 12.8 11.7

56 High Diet 8.8 11.2 14.7 18.4 15.5 16.6 17 .0 31.3 25 .0 16.2 16.8 16.3 16.8 17.0

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

51 Table A 8 . Individual animal water consumption (ml/day)

- 61

- day exposure group

. Days 1

- 14.

Day

Rat # Treatment Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14

57 High Water 13.0 13.8 13.9 15.1 19.2 15.7 20.1 26.6 22.4 14.6 10.6 12.5 11.5 11.3

58 High Water 8.9 7.9 10.2 11.3 12.1 12.3 15.6 20.7 19.7 13.1 10.2 13.4 14.6 12.2

59 High Water 12.3 10.3 10.1 12.2 12.6 13.4 15.3 21.8 20.9 12.2 9.3 10.6 9.3 10.4

60 High Water 7.5 10.8 12.2 13.4 14.1 13.3 18.6 24.4 17.4 10.9 9.5 12.3 13.0 10.0

61 High Water 10.4 8.8 11.8 12.4 12.0 12.7 15.7 21.7 16.1 8.5 6.4 9.5 9.7 8.8

62 High Water 6.7 8.7 10.6 11.6 12.8 14.6 16.7 24.8 16.4 10.6 8.4 10.4 11.4 9.1

63 High Water 8.4 9.7 12.7 12.4 14.6 13.2 14.3 34.4 16.9 11.9 13.0 12.4 14.4 12.6

64 High Water 9.7 8.3 10.4 11.3 12.0 13.1 13.8 24.7 15.1 9.4 9.4 10.5 9.8 8.3

65 High Water 8.9 10.8 11.7 11.9 12.9 13.0 16.2 15.4 21.7 10.5 10.5 13.4 13.2 11.5

66 Gavage 6.2 10.3 10.6 12.4 13.3 14.2 18.3 20.8 18.1 11.6 13.2 12.0 12.9 11.3

67 Gavage 5.6 10.0 10.9 14.4 14.6 13.2 20.3 22.6 18.9 11.1 11.5 12.4 10.8 12.9

68 Gavage 9.4 9.8 9.8 11.4 14.5 12.9 14.0 24.7 15.6 10.8 12.9 10.3 13.5 15.6

69 Gavage 7.0 9.9 11.1 12.3 11.8 11.9 17.7 25.5 15.8 11.1 11.2 12.4 11.5 12.6

70 Gavage 9.2 10.1 11.8 14.8 14.8 14.9 20.7 17.7 20.5 12.2 13.4 12.0 11.9 12.6

71 Gavage 8.5 9.8 12.2 13.3 12.5 13.1 18.7 25.3 20.2 13.3 13.6 10.6 13.9 14.4

72 Gavage 7.1 9.7 10.4 14.3 13.3 13.8 15.0 28.1 24.1 11.8 13.9 13.0 13.0 12.9

73 Gavage 6.9 12.5 12.9 16.2 15.2 15.7 18.0 29.4 27.1 13.6 17.2 14.4 16.7 16.3

74 Gavage 5.4 8.8 9.0 9.8 12.9 12.3 15.5 19.3 19.1 12.8 15.7 12.5 15.6 16.3

75 Gavage 7.5 8.2 12.3 9.6 11.4 11.5 16.7 15.8 19.4 11.3 12.6 11.1 11.8 11.1

76 Gavage 8.1 9.5 11.9 13.9 15.5 13.9 17.4 22.8 26.1 13.3 17.1 13.6 14.6 15.8

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

52 Table A 8 . Individual animal water consumption (ml/day)

- 61

- day exposure group

. Days 1 5

- 28

.

Day

Rat # Treatment Group 15 16 17 18 19 20 21 22 23 24 25 26 27 28

39 Control 15.8 15.3 18.8 14.2 11.9 13.0 11.3 12.6 15.5 20.9 11.4 12.0 13.1 20.1

40 Control 12.7 14.5 25.8 11.3 14.6 14.4 11.2 11.8 13.9 22.3 11.9 11.4 12.9 23.6

41 Control 13.2 13.8 18.8 8.9 11.8 12.1 11.4 10.3 15.0 15.9 7.5 9.3 10.4 21.5

42 Control 16.0 15.3 19.9 10.1 11.3 15.1 11.1 10.4 14.6 17.4 11.3 12.8 14.4 24.6

43 Control 13.3 16.2 19.2 11.3 12.7 15.8 12.0 10.7 15.5 32.1 12.5 10.8 11.8 25.4

44 Control 17.6 19.8 21.5 14.3 14.5 17.7 13.5 16.1 18.0 26.4 20.1 23.5 18.1 31.9

45 Control 17.9 21.5 17.0 12.3 12.6 13.2 13.0 13.3 16.8 17.7 12.5 11.6 14.6 21.1

46 Control 15.0 16.5 17.1 11.2 12.2 15.2 13.2 12.4 18.3 16.4 15.6 12.6 14.6 21.5

47 Control 17.5 23.3 22.3 13.2 14.2 16.1 15.5 17.3 18.3 18.0 13.0 11.7 12.0 22.6

48 High Diet 15.3 17.6 15.7 9.6 12.9 13.3 11.0 12.6 13.5 19.1 13.2 12.6 12.5 23.2

49 High Diet 14.2 18.9 17.6 11.2 11.9 15.0 12.0 13.2 16.7 18.0 9.1 9.9 14.9 19.4

50 High Diet 18.9 18.6 19.7 14.2 13.9 15.8 13.4 12.7 18.4 17.5 14.4 12.6 15.5 22.2

51 High Diet 13.1 18.5 16.1 10.1 12.4 12.9 11.1 12.0 14.2 15.8 10.5 9.8 10.1 18.5

52 High Diet 15.3 18.6 17.8 10.3 13.4 14.5 10.3 13.2 12.3 18.4 10.8 11.3 12.4 19.1

53 High Diet 14.1 28.7 22.3 12.0 11.9 15.2 11.0 12.8 13.5 19.9 16.2 13.6 15.6 24.9

54 High Diet 20.1 26.4 ND 13.7 17.1 20.4 18.8 ND 20.6 16.3 14.3 11.9 15.4 20.6

55 High Diet 15.6 17.0 22.0 12.9 16.1 15.4 14.2 15.6 16.2 18.1 14.8 12.0 14.6 20.3

56 High Diet 18.9 25.0 21.2 15.1 19.0 17.3 15.5 16.8 17.5 19.4 12.9 14.4 15.3 30.1

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

53 Table A 8 . Individual animal water consumption (ml/day)

- 61

- day exposure group

. Days 1 5

- 28

.

Day

Rat # Treatment Group 15 16 17 18 19 20 21 22 23 24 25 26 27 28

57 High Water 13.0 21.8 12.3 13.3 15.1 15.8 14.9 12.5 16.7 18.0 12.8 12.3 12.8 18.0

58 High Water 15.3 23.5 15.1 11.6 15.3 13.1 12.7 15.3 16.0 17.5 11.3 12.3 11.3 18.0

59 High Water 13.4 18.1 13.1 10.7 13.5 13.1 11.9 13.1 15.1 16.8 12.4 9.9 12.4 19.7

60 High Water 12.9 21.9 12.7 10.0 11.9 12.7 12.5 10.7 13.7 16.2 12.3 9.6 12.4 21.4

61 High Water 11.6 14.3 10.1 8.9 9.9 11.1 9.8 9.9 12.1 12.7 10.2 7.6 10.6 13.3

62 High Water 9.9 15.9 11.4 11.5 12.8 13.5 13.2 12.9 16.0 12.9 11.8 11.2 10.8 19.1

63 High Water 15.8 25.9 16.2 11.3 12.1 13.5 12.7 13.0 15.6 15.2 11.6 12.5 12.5 24.0

64 High Water 11.7 16.3 10.3 10.8 9.8 12.7 10.1 9.4 11.5 15.4 9.5 8.1 10.1 15.4

65 High Water 16.1 20.6 15.2 11.7 15.6 14.5 14.9 12.8 16.7 19.9 14.4 13.5 13.9 23.5

66 Gavage 8.0 16.1 20.6 10.5 10.8 13.7 10.9 11.1 10.7 20.7 12.2 10.6 11.0 21.6

67 Gavage 12.5 19.4 19.6 11.7 10.9 12.0 11.4 11.9 14.9 23.2 9.9 10.6 11.2 21.4

68 Gavage 11.4 26.9 17.3 10.2 11.8 11.7 6.6 11.4 14.2 14.6 8.1 10.3 11.4 22.2

69 Gavage 11.9 19.3 14.9 9.7 9.3 10.5 10.0 9.2 11.6 22.6 17.7 10.1 10.5 21.5

70 Gavage 14.9 19.0 24.9 11.2 13.3 13.4 15.5 10.9 12.5 27.4 11.2 9.7 10.3 18.8

71 Gavage 10.7 19.9 21.9 11.3 10.8 11.5 11.5 10.7 10.8 17.7 16.1 7.1 10.9 20.8

72 Gavage 11.6 20.0 32.0 12.6 14.9 15.0 15.1 12.3 13.2 27.0 12.1 9.2 13.3 23.7

73 Gavage 18.0 23.6 20.2 12.3 15.2 15.9 14.1 15.1 11.9 30.3 19.2 11.5 11.1 23.0

74 Gavage 15.3 20.2 20.5 13.2 12.4 14.8 13.0 14.9 16.8 25.1 22.1 12.3 14.8 25.6

75 Gavage 12.9 18.6 17.7 5.7 12.6 13.6 7.4 12.5 12.3 20.4 18.0 5.8 9.2 23.0

76 Gavage 17.9 22.4 23.1 12.9 16.9 16.0 14.7 15.3 15.2 31.4 14.4 9.4 14.2 ND

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

54 Table A 8 . Individual animal water consumption (ml/day)

- 61

- day exposure group

. Days 29

- 42.

Day

Rat # Treatment Group 29 30 31 32 33 34 35 36 37 38 39 40 41 42

39 Control 10.2 8.4 10.7 9.9 10.3 11.4 13.3 11.1 15.3 17.9 12.4 12.4 10.5 13.1

40 Control 71.0 8.2 10.5 10.4 11.7 10.0 13.2 10.6 12.9 10.2 8.9 9.0 9.0 10.6

41 Control 7.2 6.6 8.8 7.9 7.0 10.1 7.8 7.8 9.6 11.8 6.6 9.6 9.4 8.5

42 Control 8.7 8.0 10.2 9.0 10.8 12.3 9.7 11.6 11.5 10.7 9.0 8.9 8.6 8.8

43 Control 5.8 8.8 7.3 11.6 9.0 11.0 9.3 9.4 15.7 10.2 10.5 13.4 10.1 8.3

44 Control 11.1 12.1 16.0 16.1 14.0 15.2 12.8 13.8 14.2 14.8 13.9 18.3 14.7 14.8

45 Control 6.7 10.0 10.6 10.1 11.3 13.4 11.2 12.4 14.1 12.1 11.4 9.7 7.7 11.3

46 Control 10.6 9.7 8.9 10.4 9.8 9.7 12.7 11.6 12.2 12.8 12.9 14.8 10.9 14.1

47 Control 9.2 10.8 9.1 9.8 10.0 12.0 10.2 12.2 13.7 11.9 9.4 12.1 10.7 10.4

48 High Diet 14.1 12.3 14.0 12.0 13.6 17.0 15.2 13.9 18.9 14.0 7.8 11.0 12.3 10.9

49 High Diet 14.7 10.5 12.0 11.9 12.0 13.6 12.1 12.4 13.4 10.4 8.0 8.1 9.2 8.0

50 High Diet 13.9 12.9 13.1 13.2 15.8 16.3 14.1 17.2 16.4 10.2 8.9 9.8 11.1 8.2

51 High Diet 9.5 7.9 10.1 7.8 10.1 9.9 10.6 7.7 12.3 8.7 7.9 7.4 6.7 8.5

52 High Diet 10.8 10.9 9.9 9.4 11.0 11.3 9.5 11.2 10.3 11.4 6.6 9.6 8.6 9.0

53 High Diet 13.4 11.7 14.2 13.6 15.5 14.1 14.7 12.7 15.3 10.5 9.6 10.3 10.0 11.7

54 High Diet 14.3 13.6 16.5 16.6 18.0 16.7 16.9 17.4 18.1 12.0 14.6 13.2 15.6 12.8

55 High Diet 12.1 12.9 14.1 11.1 13.7 15.1 14.8 12.9 19.4 14.5 37.8 ND 12.0 13.1

56 High Diet 16.1 14.6 16.0 13.6 15.8 16.5 15.2 17.5 17.6 13.2 11.7 13.4 13.7 10.7

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

55 Table A8 . Individual animal water consumption (ml/day)

- 61

- day exposure group

. Days 29

- 42.

Day

Rat # Treatment Group 29 30 31 32 33 34 35 36 37 38 39 40 41 42

57 High Water 19.1 13.6 13.2 14.3 13.5 12.2 10.2 12.4 13.9 20.7 10.6 11.9 10.5 11.3

58 High Water 19.2 12.3 15.2 9.8 11.6 12.3 10.4 11.7 14.4 17.8 12.0 13.7 12.4 12.5

59 High Water 15.2 11.9 14.8 9.8 11.1 12.9 11.2 11.1 15.6 18.0 10.9 12.6 11.4 13.1

60 High Water 15.4 11.1 13.3 10.5 9.1 11.5 11.2 10.6 14.7 16.6 10.5 13.6 11.6 12.1

61 High Water 11.7 10.3 11.0 7.7 7.7 8.4 9.5 7.8 11.6 12.4 9.0 10.4 8.5 8.6

62 High Water 18.1 12.1 13.6 11.0 12.0 11.0 11.6 12.1 12.9 19.1 13.3 14.3 12.1 14.2

63 High Water 15.3 11.6 15.9 11.1 14.6 13.1 12.8 12.0 18.3 20.3 11.4 13.4 11.5 11.7

64 High Water 13.9 9.1 11.7 8.9 10.0 11.1 10.8 10.8 16.1 16.1 9.8 12.2 9.7 10.9

65 High Water 16.1 14.2 16.1 12.6 12.8 12.4 14.6 13.4 19.0 17.2 14.6 12.7 13.8 12.8

66 Gavage 9.5 11.9 11.4 7.7 11.3 13.8 9.1 11.7 14.6 19.1 15.8 10.8 8.8 10.9

67 Gavage 12.8 8.4 9.5 8.6 10.3 9.3 12.1 9.3 12.1 15.8 10.7 11.7 11.0 10.1

68 Gavage 10.2 9.6 11.4 10.7 9.9 11.0 11.2 9.8 14.2 19.8 14.6 12.0 9.0 11.2

69 Gavage 10.5 10.2 10.2 8.8 10.6 12.8 9.8 11.5 15.1 21.5 10.3 7.5 9.9 11.1

70 Gavage 12.5 12.2 12.7 12.5 12.1 13.7 13.7 13.7 16.0 18.1 9.8 11.6 10.3 10.6

71 Gavage 11.9 11.1 13.3 11.9 11.2 12.4 12.5 11.7 16.7 22.3 11.6 12.9 11.8 10.0

72 Gavage ND 14.1 13.2 10.9 10.7 11.6 10.3 13.0 14.3 18.5 7.4 14.1 10.5 12.6

73 Gavage 15.1 13.5 11.4 13.0 13.9 14.0 15.9 12.0 18.5 19.9 9.2 16.3 10.9 12.7

74 Gavage 13.2 10.8 13.6 10.3 12.6 14.6 14.0 14.2 11.6 19.4 13.2 10.9 11.0 13.4

75 Gavage 8.9 10.7 9.1 9.4 11.0 13.2 11.6 11.1 17.4 20.3 11.8 12.8 13.6 11.1

76 Gavage 17.8 14.1 15.7 13.5 13.4 15.0 13.1 ND 16.2 23.1 7.7 14.6 13.0 13.2

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

56 Table A 8 . Individual animal water consumption (ml/day)

- 61

- day exposure group

. Days 43

- 5 6

.

Day

Rat # Treatment Group 43 44 45 46 47 48 49 50 51 52 53 54 55 56

39 Control 12.0 11.1 20.5 12.7 11.4 13.3 11.5 9.8 11.2 21.9 11.3 18.7 25.7 25.5

40 Control 8.6 8.9 21.2 10.9 12.0 10.4 11.5 9.9 10.7 21.7 10.9 10.9 11.3 12.3

41 Control 7.2 8.9 13.9 9.5 10.5 9.3 9.0 8.7 10.6 18.8 11.1 9.1 11.7 10.3

42 Control 9.8 11.0 19.2 10.2 13.1 11.6 13.4 11.4 10.9 20.1 13.4 9.8 12.7 13.4

43 Control 8.4 12.8 16.4 9.6 11.1 11.5 11.9 7.9 12.8 16.5 11.1 9.3 11.1 12.5

44 Control 14.5 15.9 17.9 14.9 15.1 14.7 17.4 17.6 18.1 25.1 15.0 13.2 16.0 13.1

45 Control 9.3 11.9 23.0 11.2 11.8 13.8 13.0 12.7 13.8 15.8 10.0 10.2 11.3 11.7

46 Control 11.5 13.0 22.8 13.2 12.1 14.1 13.1 12.0 15.4 17.6 12.5 11.2 12.6 10.9

47 Control 10.8 11.6 20.1 14.8 13.6 16.8 11.4 10.5 16.5 18.5 11.7 12.2 14.3 12.7

48 High Diet 11.1 12.3 21.5 11.3 13.9 12.8 14.2 12.2 14.6 19.2 13.8 12.1 14.8 14.9

49 High Diet 8.0 9.4 17.8 9.9 8.7 11.5 10.3 11.1 10.2 18.2 11.2 10.5 11.6 11.5

50 High Diet 10.8 10.6 20.9 12.0 9.9 14.6 13.2 10.9 12.5 17.9 10.7 8.7 10.5 10.2

51 High Diet 9.4 7.4 20.9 10.0 10.6 11.6 10.9 11.2 13.3 16.5 8.4 9.6 10.3 10.3

52 High Diet 9.0 9.8 21.6 12.0 9.8 12.7 13.5 10.4 14.5 18.4 10.2 10.1 10.1 12.4

53 High Diet 11.2 12.1 27.7 15.5 14.4 14.5 15.3 16.3 18.1 20.2 13.6 14.2 14.8 13.2

54 High Diet 10.4 14.7 25.5 13.0 14.4 15.5 18.1 16.2 16.7 20.8 11.2 13.5 14.9 11.3

55 High Diet 11.6 14.7 26.4 13.9 13.6 15.2 16.4 14.7 18.3 22.2 13.1 14.6 14.7 16.7

56 High Diet 12.2 14.4 21.9 12.8 13.1 18.9 17.0 17.0 20.1 ND 14.7 14.7 16.0 12.0

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

57 Table A 8 . Individual animal water consumption (ml/day)

- 61

- day exposure group

. Days 43

- 56.

Day

Rat # Treatment Group 43 44 45 46 47 48 49 50 51 52 53 54 55 56

57 High Water 11.8 10.8 22.1 10.7 10.8 12.0 11.8 12.5 27.2 8.4 10.7 10.9 11.0 13.3

58 High Water 11.2 14.5 20.3 11.6 10.5 11.3 13.9 13.0 28.5 9.3 10.4 11.0 12.8 10.0

59 High Water 12.1 22.2 11.6 20.1 11.1 12.4 10.5 11.5 21.4 20.3 18.2 16.2 16.0 14.6

60 High Water 10.6 12.5 8.5 24.6 11.4 11.9 10.4 11.7 23.1 11.8 11.4 9.8 10.6 13.4

61 High Water 8.2 10.9 8.6 21.1 11.5 11.1 12.1 11.1 22.0 8.6 9.5 11.3 9.9 10.4

62 High Water 12.2 12.9 13.8 22.8 11.7 14.2 13.4 11.0 25.4 10.0 11.9 10.1 10.9 10.7

63 High Water 13.2 13.4 9.3 24.3 12.2 12.7 11.2 11.6 25.5 10.9 12.3 9.9 11.6 13.6

64 High Water 10.8 11.8 7.9 18.0 9.1 11.3 10.5 8.3 20.7 ND 11.1 10.4 11.0 12.2

65 High Water 13.1 15.7 10.2 20.7 11.9 12.2 11.6 10.2 25.1 ND 10.1 10.3 11.6 12.0

66 Gavage 9.6 14.0 8.8 14.1 11.4 9.4 10.0 9.3 11.5 17.0 8.7 10.0 10.7 10.6

67 Gavage 8.8 13.1 7.9 19.9 11.3 12.7 13.5 9.9 13.1 17.2 10.3 9.0 10.9 9.2

68 Gavage 13.3 9.7 9.8 19.7 9.8 11.5 13.3 8.3 14.3 23.8 13.1 10.7 9.7 15.0

69 Gavage 9.5 10.9 6.7 15.8 8.3 9.4 8.8 8.7 11.1 18.1 9.1 8.7 10.5 10.9

70 Gavage 10.2 13.6 7.6 23.0 11.2 11.8 12.5 11.2 15.0 17.9 10.3 10.9 10.2 13.1

71 Gavage 13.4 14.4 8.0 23.1 12.1 11.4 13.7 10.1 15.5 21.1 10.4 10.9 10.0 11.4

72 Gavage 9.7 15.5 9.7 22.0 10.1 12.8 13.4 11.2 18.1 17.5 13.2 9.7 12.9 11.5

73 Gavage 6.0 14.3 12.1 18.4 12.5 12.4 13.4 12.8 17.3 21.1 11.1 12.2 11.0 14.6

74 Gavage 11.5 14.1 10.9 24.0 18.8 13.1 15.6 11.0 18.4 19.6 11.5 11.9 11.6 14.1

75 Gavage 12.6 16.9 10.2 20.6 14.1 12.8 12.1 13.6 14.3 19.0 11.2 11.2 12.3 13.7

76 Gavage 12.8 13.9 10.8 24.3 13.4 11.6 13.7 12.1 10.7 19.7 10.3 11.8 10.7 10.6

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

58 Table A8 . Individual animal water consumption (ml/day)

- 61

- day exposure group

. Days 57

- 70.

Day

Rat # Treatment Group 57 58 59 60 61 62 63 64 65 66 67 68 69 70

39 Control 25.1 28.1 21.1 12.3

- 16.2 44.9 13.5 13.4 14.7 20.2 12.8 12.2 11.1 12.4

40 Control 12.9 16.8 20.4 10.9 10.3 13.0 13.0 13.3 13.2 20.4 12.5 10.5 9.4 12.0

41 Control 10.4 14.7 16.0 9.9 10.6 11.7 10.0 13.5 13.7 19.2 8.4 8.4 9.6 9.9

42 Control 14.1 17.0 18.0 11.4 11.0 11.2 11.1 13.4 13.1 19.1 10.5 12.0 13.5 11.6

43 Control 11.2 15.8 16.5 10.7 11.5 12.3 13.3 9.6 12.2 19.5 9.5 10.3 9.7 10.2

44 Control 15.3 18.3 24.2 18.6 14.8 15.3 15.3 15.4 16.4 24.5 13.3 12.4 11.9 13.3

45 Control 11.6 15.0 14.9 12.7 11.1 9.6 11.0 11.5 12.8 21.8 11.8 10.5 11.5 12.5

46 Control 12.7 14.7 17.0 8.6 10.3 11.2 10.1 10.4 11.7 17.7 10.4 11.9 11.7 11.5

47 Control 11.8 19.5 20.1 12.9 60.5 ND ND 19.9 25.0 26.7 11.1 12.3 12.4 13.0

48 High Diet 11.5 16.3 16.6 11.3 10.7 9.9 10.7 11.9 12.4 20.9 11.8 12.3 12.1 12.4

49 High Diet 11.4 16.1 15.3 9.6 10.8 8.4 10.2 10.1 10.3 19.8 10.1 9.1 10.2 10.1

50 High Diet 10.5 15.5 16.2 12.6 10.2 12.4 11.0 13.5 12.7 22.4 10.3 12.9 12.1 12.4

51 High Diet 10.6 12.8 16.1 9.7 8.9 11.5 10.7 10.1 12.3 18.1 8.6 8.6 10.9 9.9

52 High Diet 10.8 17.5 16.7 10.0 10.9 13.2 10.3 10.4 10.7 21.3 10.4 8.9 12.8 10.1

53 High Diet 13.2 18.6 20.3 12.5 13.4 12.9 10.7 12.4 15.1 21.5 13.1 11.8 13.8 14.3

54 High Diet 15.9 12.7 20.6 14.0 12.5 13.4 15.4 12.4 15.5 19.9 12.3 12.4 15.2 12.4

55 High Diet 15.5 19.0 15.9 12.3 13.9 13.6 13.7 13.2 16.2 21.3 13.6 14.0 13.0 15.9

56 High Diet 17.4 17.7 19.2 13.8 12.0 13.9 13.4 13.3 14.5 19.5 14.2 11.0 13.4 12.5

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

59 Table A8 . Individual animal water consumption (ml/day)

- 61

- day exposure group

. Days 57

- 70.

Day

Rat # Treatment Group 57 58 59 60 61 62 63 64 65 66 67 68 69 70

57 High Water 9.9 14.0 14.3 11.1 10.8 9.8 12.0 10.4 11.1 18.2 10.8 10.8 9.6 10.5

58 High Water 11.1 16.8 22.7 13.4 12.6 16.1 11.2 15.7 13.2 20.1 11.1 10.7 13.0 16.0

59 High Water 11.1 20.3 17.9 10.5 10.9 12.1 12.3 11.5 13.7 19.6 11.6 11.8 12.8 12.7

60 High Water 12.3 15.5 16.4 10.3 9.0 11.5 11.5 11.1 13.3 15.5 10.0 13.2 11.8 11.2

61 High Water 11.6 14.9 12.1 6.8 10.6 11.1 8.8 12.2 9.5 13.8 9.8 8.7 9.2 10.0

62 High Water 11.4 14.5 16.2 8.5 12.3 11.6 12.5 11.9 13.3 18.9 11.2 9.8 13.6 12.4

63 High Water 13.2 15.4 16.6 9.2 10.9 9.6 10.9 9.7 12.4 24.4 11.9 12.1 12.6 11.6

64 High Water 11.4 16.4 16.2 9.5 9.1 10.6 8.8 9.7 11.3 20.8 9.6 20.3 11.8 13.5

65 High Water 13.3 16.6 16.0 11.2 9.5 11.4 10.4 12.8 14.4 18.0 12.6 11.5 11.3 12.0

66 Gavage 10.9 14.0 14.1 8.9 8.5 9.1 8.2 9.0 12.5 17.9 9.4 7.3 8.6 11.1

67 Gavage 12.2 15.3 11.7 9.8 7.4 9.7 7.5 8.4 12.3 20.1 8.0 10.7 11.1 8.3

68 Gavage 13.2 19.4 12.6 8.6 8.4 10.5 8.3 9.0 11.6 16.0 8.9 10.7 10.0 12.7

69 Gavage 11.3 11.7 11.9 8.3 8.2 7.8 8.3 9.0 10.2 16.6 8.1 8.1 9.3 9.0

70 Gavage 10.8 16.0 11.2 Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead Dead

71 Gavage 12.4 17.6 16.3 9.1 11.7 10.8 12.2 13.3 13.5 15.2 12.1 9.6 9.5 10.4

72 Gavage 12.1 15.0 16.4 9.7 10.5 11.3 9.8 10.8 13.9 15.0 12.3 11.9 11.3 24.1

73 Gavage 13.6 18.7 18.4 10.7 11.0 13.1 12.2 12.1 16.3 19.5 11.3 11.1 14.0 ND

74 Gavage 13.6 16.1 18.4 10.4 9.9 10.0 9.8 12.2 13.3 16.6 9.3 5.8 10.0 ND

75 Gavage 11.6 15.8 15.7 11.4 9.2 10.7 12.1 12.5 12.2 18.6 11.8 11.4 12.5 ND

76 Gavage 11.9 14.5 14.9 11.4 10.9 13.3 13.2 13.2 16.4 22.1 10.5 11.6 12.5 16.2

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

60 Table A 9 . Individual animal tissue manganese concentrations (μg Mn/g tissue wet weight) following 7 exposure days.

Rat # Treatment Group Spleen Serum Femur Liver Bile Olf. bulb Cerebellum Cortex Striatum

1 Control 0.176 0.44 0.356 2.42 3.86 0.308 0.385 0.358 0.320

2 Control 0.192 1.35 0.373 2.61 5.47 0.379 0.401 0.262 0.285

3 Control 0.164 0.42 0.286 2.53 5.76 0.391 0.383 0.285 0.291

4 Control 0.169 0.36 0.326 2.41 6.11 0.451 0.414 0.332 0.280

5 Control 0.423 0.7 0.469 2.59 NS 0.473 0.389 0.224 0.313

6 Control 0.140 0.74 0.335 2.12 NS 0.461 0.427 6.897 0.374

7 Control 0.165 0.70 0.278 2.31 6.26 0.343 0.441 0.514 0.313

8 Control 0.166 1.47 0.328 2.69 3.20 0.441 0.472 0.844 0.297

9 Control 0.148 0.48 0.357 2.37 15.64 0.542 0.467 0.330 0.291

10 High Diet 0.183 2.85 0.433 3.92 118.24 0.655 0.459 0.241 0.347

11 High Diet 4.142 0.66 0.378 3.48 NS 0.613 0.500 0.315 0.333

12 High Diet 0.175 1.21 0.799 3.43 521.03 0.441 0.507 0.359 0.328

13 High Diet 0.176 0.81 0.302 2.92 16.94 0.385 0.454 0.359 0.345

14 High Diet 0.184 0.87 0.370 2.97 122.48 0.453 0.473 0.427 0.311

15 High Diet 0.168 6.76 0.433 3.71 64.83 0.578 0.456 0.468 0.355

16 High Diet 0.213 1.99 0.311 3.00 271.81 0.452 0.452 0.360 0.335

17 High Diet 0.174 1.15 0.391 3.25 504.12 0.498 0.780 0.313 0.459

18 High Diet 0.214 1.22 0.376 3.07 3.86 0.471 0.449 0.325 0.328

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

61 Table A 9 . Individual animal tissue manganese concentrations (μg Mn/g tissue wet weight) following 7 exposure days.

Rat # Treatment Group Spleen Serum Femur Liver Bile Olf. bulb Cerebellum Cortex Striatum

19 High Water 0.247 1.00 0.366 2.71 61.74 0.500 0.462 0.370 0.376

20 High Water 0.170 1.83 0.371 3.12 16.59 0.370 0.450 0.374 0.318

21 High Water 0.179 24.13 0.432 3.32 115.97 0.485 0.453 0.299 0.366

22 High Water 0.178 0.86 0.484 3.00 NS 0.466 0.457 0.313 0.321

23 High Water 0.183 14.68 0.343 3.00 NS 0.473 0.525 0.372 0.357

24 High Water 0.167 16.57 0.350 3.20 75.66 0.472 0.451 0.365 0.325

25 High Water 0.193 0.76 0.341 3.06 39.55 0.515 0.512 0.417 0.332

26 High Water 0.209 1.06 0.365 3.17 NS 0.553 0.457 0.358 0.335

27 High Water 0.175 1.11 0.369 2.74 NS 0.471 0.483 0.390 0.339

28 Gavage 0.216 8.22 0.524 3.69 79.54 1.656 0.514 0.411 0.441

29 Gavage 0.282 2.27 0.781 4.62 184.95 12.444 0.763 0.609 0.996

30 Gavage 0.284 3.32 0.655 5.70 1609.60 10.396 0.775 10.729 0.857

31 Gavage 0.191 2.50 0.600 3.37 409.28 3.522 0.623 0.511 0.900

32 Gavage 0.319 0.62 0.517 13.58 4368.66 1.765 0.560 0.546 0.409

33 Gavage 0.216 1.99 0.400 5.00 144.93 0.826 0.535 0.551 0.224

34 Gavage 0.182 0.65 0.631 3.46 148.13 1.484 0.535 0.414 0.611

35 Gavage 0.227 1.24 0.442 4.76 136.94 0.548 0.600 0.374 0.325

36 Gavage 0.388 0.84 0.414 3.63 NS 0.666 0.504 0.443 0.325

37 Gavage 0.159 1.42 0.402 2.94 35.86 1.751 0.559 0.404 0.385

38 Gavage 0.186 0.68 0.520 3.88 3.86 1.481 0.578 0.511 0.562

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

62 Table A 10 . Individual animal tissue manganese concentrations (μg Mn/g tissue wet weight) following 61 exposure days.

Rat # Treatment Group Spleen Serum Femur Liver Bile Olf. bulb Cerebellum Cortex Striatum

39 Control 0.163 0.31 0.320 2.56 1.39 0.486 0.463 0.286 0.391

40 Control 0.137 0.45 0.340 3.26 2.67 0.511 0.463 0.303 0.362

41 Control 0.136 0.33 0.280 2.80 NS 0.450 0.461 0.276 0.345

42 Control 0.143 0.47 0.310 2.72 1.73 0.439 0.480 0.170 0.364

43 Control 0.119 2.83 0.320 5.06 1.19 0.562 0.507 NS 0.389

44 Control 0.260 0.28 0.370 2.42 1.42 0.423 0.466 0.242 0.353

45 Control 0.062 0.38 0.380 2.32 1.60 0.471 0.459 0.217 0.377

46 Control 0.103 0.47 0.250 2.27 0.77 0.672 0.380 0.316 0.364

47 Control 0.074 0.41 0.300 2.64 1.07 0.422 0.486 0.170 0.339

48 High Diet 0.154 0.47 0.470 3.71 38.21 0.547 0.647 0.329 0.405

49 High Diet 0.179 0.40 0.560 3.75 40.60 0.443 0.621 0.268 0.438

50 High Diet 0.155 0.48 0.370 3.65 70.57 0.529 0.530 0.278 0.467

51 High Diet 0.232 1.90 0.360 2.14 NS 0.481 0.520 0.299 0.447

52 High Diet 0.192 0.77 0.360 3.31 25.86 0.464 0.510 0.285 0.394

53 High Diet 0.100 0.88 0.410 3.32 29.55 0.458 0.478 0.319 0.381

54 High Diet 0.118 0.55 0.420 3.25 25.31 0.483 0.506 0.331 0.424

55 High Diet 0.171 0.45 0.320 3.56 24.11 0.451 0.713 0.227 0.417

56 High Diet 0.160 0.65 0.370 2.99 21.74 0.545 0.480 0.247 0.507

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

63 Table A10 . Individual animal tissue manganese concentrations (μg Mn/g tissue wet weight) following 61 exposure days.

Rat # Treatment Group Spleen Serum Femur Liver Bile Olf. bulb Cerebellum Cortex Striatum

57 High Water 0.132 0.48 0.430 3.00 9.52 0.460 0.497 0.223 0.419

58 High Water 0.127 0.63 0.400 2.88 51.60 0.465 0.388 0.244 0.391

59 High Water 0.137 0.37 0.300 2.98 34.32 0.470 0.498 0.327 0.446

60 High Water 0.205 0.34 0.560 3.25 48.67 0.428 0.553 0.216 0.469

61 High Water 0.110 0.40 0.400 3.11 14.84 0.878 0.532 0.266 0.516

62 High Water 0.134 0.32 0.440 2.77 21.54 0.451 0.576 0.247 0.448

63 High Water 0.115 0.35 0.410 2.88 25.72 0.467 0.498 0.226 0.416

64 High Water 0.140 0.59 0.300 2.75 124.11 0.463 0.569 0.255 0.428

65 High Water 0.071 0.29 0.370 2.85 17.37 0.417 0.550 0.396 0.461

66 Gavage 0.143 0.48 0.960 3.14 87.65 2.015 0.758 0.576 1.328

67 Gavage 0.097 0.37 0.520 3.09 67.47 1.160 0.581 0.571 0.595

68 Gavage 0.109 0.34 0.680 3.76 100.32 0.896 0.621 0.618 0.573

69 Gavage 0.109 0.46 0.530 3.58 54.26 0.785 0.595 0.546 0.555

70 Gavage Dead Dead Dead Dead Dead Dead Dead Dead Dead

71 Gavage 0.109 0.44 0.599 3.25 47.03 0.776 0.647 9.447 0.627

72 Gavage 0.142 0.57 0.860 3.70 102.70 0.829 0.639 0.520 0.760

73 Gavage 0.154 0.55 0.730 3.09 45.01 1.501 0.794 0.708 0.758

74 Gavage 0.114 0.39 0.280 3.39 41.31 1.487 0.679 1.279 0.755

75 Gavage 0.361 0.43 0.990 3.55 58.71 0.782 0.760 0.910 0.793

76 Gavage 0.123 0.61 0.490 3.00 48.98 1.018 0.708 0.803 0.759

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

64 Table A 11 . Individual animal tissue metal concentrations (μg /g tissue wet weight) following 7 exposure days.

Frontal cortex Liver Spleen Serum

Rat # Group Fe Cu Zn Fe Zn Zn Fe Cu Zn Fe Cu Zn

1 Control 12.84 1.50 102.72 0.320 28.72 220.1 1.19 18.39 3041.66 68.47 192.98

2 Control 10.45 1.17 119.58 0.285 26.71 215.4 1.25 18.87 667.57 67.98 201.41

3 Control 11.30 1.28 89.12 0.291 26.79 197.2 1.08 16.32 183.84 67.77 138.89

4 Control 10.98 1.29 102.45 0.280 28.97 265.1 1.17 18.27 123.11 55.86 102.49

5 Control 8.08 0.93 81.47 0.313 27.82 215.4 1.29 18.16 246.63 70.58 147.39

6 Control 8.77 1.26 72.16 0.374 23.03 210.4 1.08 14.17 145.71 59.58 123.39

7 Control 11.61 1.26 100.71 0.313 25.78 211.6 1.13 17.08 91.31 79.49 155.92

8 Control 11.39 1.47 109.11 0.297 28.89 237.6 1.11 17.37 112.61 69.76 126.65

9 Control 10.21 1.36 90.62 0.291 27.14 212.9 1.04 16.30 53.41 35.14 35.27

10 High Diet 6.90 0.97 127.19 0.347 33.43 221.9 1.19 18.31 1695.24 135.63 363.96

11 High Diet 8.83 1.25 94.66 0.333 27.56 205.3 1.18 21.38 255.36 57.89 138.85

12 High Diet 11.28 1.45 13.07 97.58 0.328 29.56 240.5 0.98 17.96 661.96 60.37 113.48

13 High Diet 10.44 1.31 10.65 107.07 0.345 27.64 347.9 1.08 19.89 405.69 67.97 159.32

14 High Diet 9.94 1.26 11.81 109.39 0.311 29.04 233.9 1.02 19.00 107.94 54.67 119.41

15 High Diet 10.94 1.59 11.32 91.21 0.355 30.10 203.3 1.14 18.55 386.19 59.80 144.12

16 High Diet 8.74 1.40 9.57 117.04 0.335 30.78 191.7 0.98 18.88 667.41 49.32 131.33

17 High Diet 9.78 1.35 11.56 91.76 0.459 29.34 163.3 0.93 18.18 175.30 48.84 105.68

18 High Diet 8.84 1.10 12.09 74.33 0.328 28.20 162.6 1.00 18.91 193.55 91.90 168.64

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

65 Table A 11 . Individual animal tissue m etal concentrations (μg /g tissue wet weight) following 7 exposure days.

Frontal cortex Liver Spleen Serum

Rat # Group Fe Cu Zn Fe Cu Zn Fe Cu Zn Fe Cu Zn

19 High Water 10.51 1.35 12.78 64.02 6.16 24.13 232.4 1.09 20.97 97.29 62.24 80.45

20 High Water 10.67 1.34 12.38 105.90 6.55 29.51 216.8 1.07 18.43 223.44 61.96 110.76

21 High Water 9.11 1.35 8.85 141.30 6.48 31.97 275.5 1.11 19.16 148.47 111.26 96.17

22 High Water 9.09 1.22 10.88 111.84 5.90 33.38 226.9 1.01 19.06 196.10 62.30 106.83

23 High Water 11.09 1.46 11.53 85.58 6.30 26.74 171.4 0.91 18.10 743.83 345.55 170.05

24 High Water 10.72 1.41 12.14 126.02 6.72 28.51 231.7 0.99 18.53 130.49 88.77 139.47

25 High Water 8.24 1.41 10.02 132.98 6.88 31.13 268.8 1.02 20.19 510.00 58.87 128.06

26 High Water 10.13 1.44 10.99 137.24 7.60 32.68 265.4 1.12 22.16 163.07 59.93 136.65

27 High Water 10.67 1.37 11.83 95.93 6.23 26.65 221.3 0.99 17.95 200.11 62.64 108.29

28 Gavage 9.25 1.34 12.33 92.74 6.03 25.98 270.2 0.97 19.98 104.92 68.54 146.14

29 Gavage 11.37 1.36 11.89 121.28 8.38 34.11 209.6 1.04 19.17 619.20 99.35 109.88

30 Gavage 11.40 1.55 11.35 126.14 6.78 29.69 314.5 1.00 18.65 134.99 95.25 127.16

31 Gavage 10.33 1.46 11.40 105.38 7.08 27.96 237.4 0.91 17.17 179.27 116.98 127.43

32 Gavage 9.40 1.29 11.72 123.21 7.92 33.36 201.3 0.96 22.40 529.99 59.46 77.50

33 Gavage 12.00 1.77 12.10 126.70 7.27 35.04 247.6 1.00 18.95 82.80 69.26 147.50

34 Gavage 9.73 1.44 11.86 118.69 7.27 29.71 283.2 1.00 19.14 110.88 69.28 135.75

35 Gavage 8.25 1.20 11.86 138.49 7.26 32.53 265.4 1.10 19.65 139.57 97.73 121.61

36 Gavage 11.96 1.44 11.38 116.58 6.67 31.90 246.4 1.03 20.27 117.55 67.35 132.49

37 Gavage 11.95 1.57 13.18 108.26 6.17 27.13 273.7 0.92 17.22 524.44 78.07 123.41

38 Gavage 9.88 1.29 11.78 121.10 7.04 30.74 272.5 1.00 19.20 182.79 75.02 121.78

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

66 Table A 1 2 . Individual animal tissue m etal concentrations (μg /g tissue wet weight) following 61 exposure days.

Frontal cortex Liver Spleen Serum

Rat # Group Fe Cu Zn Fe Cu Zn Fe Cu Zn Fe Cu Zn

39 Control 9.87 1.47 130.04 5.33 31.77 1177.1 1.13 19.20 147.21 69.64 73.30

40 Control 13.03 1.49 223.79 5.43 36.05 1271.3 1.03 17.15 191.34 59.65 69.95

41 Control 12.17 1.56 263.34 4.66 33.13 1493.6 1.06 18.80 212.13 63.34 80.30

42 Control 9.00 1.52 199.53 4.53 31.66 1053.6 0.91 15.61 205.50 76.90 79.86

43 Control 215.82 3.69 28.98 1116.4 0.88 15.88 382.48 61.83 74.01

44 Control 9.82 1.50 108.79 5.05 28.69 1255.5 0.92 15.35 217.46 67.39 73.10

45 Control 11.74 1.52 135.12 4.35 25.68 923.4 0.76 15.46 234.63 70.20 73.34

46 Control 12.48 1.83 161.98 4.13 25.28 1009.0 0.82 17.55 202.24 82.22 89.61

47 Control 8.95 1.36 173.93 4.96 28.22 983.6 0.78 16.44 177.68 69.78 71.64

48 High Diet 13.32 1.69 123.50 5.15 29.38 709.2 0.91 18.82 115.22 64.80 62.81

49 High Diet 11.51 1.59 231.68 5.85 37.69 1207.5 1.11 22.29 89.11 62.89 59.78

50 High Diet 12.62 1.71 186.54 5.07 27.76 1105.6 0.98 19.34 246.98 63.93 70.26

51 High Diet 12.79 1.88 174.79 3.34 19.71 1108.6 1.02 17.91 791.07 79.05 71.81

52 High Diet 10.58 1.52 174.54 4.59 28.88 1118.8 1.00 18.24 233.56 71.95 71.42

53 High Diet 8.03 1.50 173.52 4.15 29.64 691.5 0.89 16.41 162.73 81.41 99.81

54 High Diet 9.52 1.69 113.05 5.07 27.81 945.2 1.13 18.35 190.04 60.48 74.02

55 High Diet 9.09 1.61 136.26 5.56 30.52 747.7 0.96 16.54 102.64 72.03 74.15

56 High Diet 10.30 1.45 152.42 4.14 31.37 901.8 1.11 18.18 201.67 90.54 87.56

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

67 Table A1 2 . Individual animal tissue manganese concentrations (μg Mn/g tissue wet weight) following 61 exposure days.

Frontal cortex Liver Spleen Serum

Rat # Group Fe Cu Zn Fe Cu Zn Fe Cu Zn Fe Cu Zn

57 High Water 7.63 1.45 174.59 4.46 30.32 1131.0 1.58 27.07 167.64 65.87 68.00

58 High Water 9.10 1.58 178.52 4.20 25.74 807.9 0.99 15.82 187.48 76.74 88.71

59 High Water 10.38 1.49 158.21 3.72 27.76 1154.0 1.11 16.81 186.79 66.55 61.51

60 High Water 9.03 1.50 155.46 4.51 25.76 1135.9 1.06 17.50 126.97 69.97 56.10

61 High Water 13.53 1.54 215.47 4.53 30.18 996.7 1.20 18.51 193.65 74.42 92.86

62 High Water 8.51 1.33 142.51 4.33 27.34 1083.3 0.97 16.38 336.05 66.47 73.21

63 High Water 7.98 1.39 201.13 4.00 29.80 1259.8 1.23 17.72 307.84 70.55 74.99

64 High Water 9.44 1.45 147.99 4.57 29.30 1095.3 1.11 17.38 337.06 59.05 55.74

65 High Water 11.79 1.58 153.36 4.45 30.42 923.7 1.00 16.06 139.71 63.96 68.45

66 Gavage 7.67 1.39 160.77 4.20 27.83 1528.0 1.10 18.18 108.20 67.32 69.28

67 Gavage 9.83 1.64 175.03 4.82 28.45 1209.6 1.02 15.66 190.16 62.96 61.63

68 Gavage 17.21 1.96 168.44 5.38 29.16 1288.4 0.98 15.55 142.54 59.82 53.63

69 Gavage 15.81 1.98 214.76 4.65 27.31 1210.6 1.03 16.47 252.60 62.08 57.39

70 Gavage

71 Gavage 17.62 2.37 171.79 3.94 28.33 1087.2 1.02 16.65 176.64 59.63 59.84

72 Gavage 13.50 1.93 167.12 4.95 27.14 1265.5 1.04 16.07 205.21 59.53 68.65

73 Gavage 16.73 2.45 149.47 4.83 28.50 1135.1 1.00 15.30 186.01 69.89 76.88

74 Gavage 18.36 2.16 155.28 4.71 27.43 1302.3 1.11 17.07 202.44 73.13 65.80

75 Gavage 15.20 2.65 148.12 4.75 29.03 1115.9 1.04 20.62 290.23 70.64 73.27

76 Gavage 20.12 2.75 178.37 4.91 25.79 1452.3 1.18 21.90 238.09 70.94 70.12

### NCSU College of Veterinary Medicine Project (1940) 2013

- 2727

68 

# P AGE 

# L EFT



# I NTENTIONALLY 

# B LANK