A Review of the Studies of the Safety ofPolydextrose in Food

G. A. BURDOCK1,* and W. G. FLAMM2

1Burdock & Associates, 622 Beachland Blvd, Suite B, Vero Beach, FL 32963, and 2Flamm Associates,622 Beachland Blvd, Suite A, Vero Beach, FL 32963, USA

(Accepted 3 July 1998)

SummaryÐPolydextrose (CAS no. 68424-04-4) is a water-soluble polymer of glucose that provides tofoods the bulk and texture of sucrose. There are two main forms of polydextrose, an acidic form (PD-A) and a neutralized potassium salt (PD-N). Polydextrose is resistant to mammalian metabolic and mi-crobial degeneration, rendering it both low in caloric value and non-cariogenic. Little polydextrose isabsorbed intact although some is metabolized by caecal/colonic bacteria. At high enough levels ofingestion, this bacterial metabolism results in ¯atus, bloating, loose stools and ultimately a frank diar-rhoea. Microbial metabolism also produces some volatile fatty acids that are absorbed by the animaland have calorigenic value. The species and dose threshold for persistent loose stools/watery diarrhoeadetermines the degree of electrolyte loss by the animal. In the dog, an obligate carnivore, sodium-spar-ing activity by the kidney and concomitant and obligatory calcium reuptake result in a well-de®nedaetiology of hypercalcaemia and subsequent nephrocalcinosis, particularly for PD-N. Of the speciestested, the dog was the most sensitive to this carbohydrate with a no-e�ect level of 2000 mg/kg bodyweight/day. Omnivores, including the rat, mouse and monkey, have a no-e�ect level ranging from 2500to 10,000 mg/kg body weight/day. No toxicity has been demonstrated in man, although the dose forlaxation (to be distinguished from diarrhoea) is approximately 90 g/day (v. sorbitol at 70 g/day). Poly-dextrose did not show any reproductive toxicity, teratology, carcinogenesis, mutagenicity or genotoxi-city. Polydextrose has been approved for food additive use (21 CFR 172.841) in the US, and an ``ADInot speci®ed'' by the Joint WHO/FAO Expert Committee on Food Additives (JECFA, 1987). It hasbeen approved in over 50 countries around the world and has been used extensively in the diet forover15 years. Speci®cation monographs are published in the Food Chemicals Codex (FCC) (NAS, 1996)and the FAO Compendium (JECFA, 1995). This review provides an overview of the studies and salientdata, not previously reported in the scienti®c literature, which had been submitted to regulatoryagencies in support of these approvals. # 1999 Elsevier Science Ltd. All rights reserved

Keywords: polydextrose; carbohydrate; safety.

Abbreviations: BUN = blood urea nitrogen; DMNA= dimethylnitrosamine; 6-MP= 6-mercaptopur-ine monohydrate; 4NQO= 4-nitroquinoline-N-oxide; PB-A = polydextrose, acid form; PD-N = poly-dextrose, neutralized form; RBC = red blood cell.

Introduction

Polydextrose (CAS no. 68424-04-4) is a water-sol-

uble polymer of glucose which provides to foods

the bulk and texture of sucrose, but at only one-

quarter the caloric value.$ It is produced by vac-

uum-melt condensation of glucose in the presence

of sorbitol and catalytic amounts of citric or phos-

phoric acid, resulting in a randomly linked polymer.

It is available in several forms: the more often

referred to and herein generically, as polydextrose

(also PD-A or acid form), is an amorphous, slightly

acidic, fusible powder. Another, designated poly-

dextrose-N (PD-N or neutralized form), is a practi-

cally neutralized, light-yellow product available as a

70% aqueous solution, which is obtained by ad-

dition of KOH or K2CO3 to a solution of PD-A.

There are also trace amounts of 5-hydroxymethyl

furfural and levoglucosan (1,6-anhydroglucose)

(Anonymous, 1981; Beereboom, 1981; Burdock,

1997; Mitchell, 1996; Setsu, 1989; Torres and

Food and Chemical Toxicology 37 (1999) 233±264

0278-6915/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. Printed in Great BritainPII S0278-6915(98)00113-6

*Corresponding author.$Polydextrose was discovered and patented by scientists at

P®zer Inc., Groton, CT, USA in 1970. P®zer sold thepolydextrose product line to Cultor Food Science in1996.

Thomas 1981). Additional forms, trade named

Litesse1

, are more puri®ed forms of polydextrose

prepared primarily by ion exchange post-treat-

ment.*

Polydextrose is a randomly-linked (1,6-glycosidic

bonds predominate), high molecular weight polymer

(99% has a mol. wt <15,000 and 90% has a mol.

wt <5000) such that it is resistant to mammalian

metabolism and somewhat less so to microbial

degradation. These characteristics endow polydex-

trose with its favourable technological properties

(e.g. as a bulking agent functionally similar to

sucrose) and consumer appeal as a non-cariogenic

calorie-sparing foodstu�, with 25% of the calories

of sucrose.

Figdor and Rennhard (1981) elucidated the meta-

bolic disposition of polydextrose through a series of

experiments in the rat. These authors established

®ve points to the understanding of polydextrose dis-

position: First, they determined that there is limited

endogenous metabolism of PD, because following

an intravenous injection of 14C-PD into the rat, ap-

proximately 90% was excreted in the urine within

the ®rst 24 hr. Metabolism was minimal with only

1.16% of the dose exhaled as the metabolic end-

product (14CO2). Caloric utilization was only 1.9%

of the dose. Some small amount (approx. 4.5%)

passed into the faeces. Second, an oral dose was

primarily excreted in the faeces (approx. 60%), with

21% exhaled as 14CO2 and less than 0.24% of the

dose excreted as unchanged polydextrose in the

urine; giving a calculated caloric utilization of ap-

proximately 35%. Third, the primary source of

metabolic end-product was not through upper gas-

trointestinal digestive processes, but via microbial

fermentation in the lower gut. In the gut, volatile

fatty acids (including, but not limited to, acetic,

propionic and n-butyric acid) are formed as the

result of fermentation and absorbed and utilized by

the host. There may be some source for error in

their estimates, as non-caloric 14CO2, produced as

the result of fermentation in the gut, would also be

absorbed. Fourth, once the rat has excreted poly-

dextrose, catabolism is largely complete, because

``recycling'' previously excreted polydextrose yields

only one-quarter of the 14CO2 and caloric utiliz-

ation of ``virgin'' polydextrose. Fifth, there is no

``induction'' of metabolism of polydextrose, because

animals on a polydextrose-containing diet excreted

the same amounts and proportions of 14C-labelled

end-products as naive rats. These data suggested acaloric value of approximately 1 kcal/g or 4.2 kJ/g.

A polydextrose caloric value of approximately1 kcal/g has been accepted for labelling purposes inmost countries around the world (Auerbach, 1998).

Polydextrose has been approved as a direct foodadditive (21 CFR 172.841) by the US Food andDrug Administration for use as a nutrient sup-

plement, texturizer, stabilizer or thickener, formu-lation aid and humectant. The FDA estimated theper capita individual consumption of polydextrose

for currently approved uses to be 14.3 g/day or0.24 g/kg body weight/day, based on MRCA 5-yearmenu census (1982±87) with 1987±88 USDA por-tion sizes (DiNovi, 1992). Polydextrose was also

approved by the Joint FAO/WHO ExpertCommittee on Food Additives (JECFA) for an`ADI not speci®ed' in 1987. It has been approved in

over 50 countries around the world and has beenused extensively in the diet for over 15 years.Speci®cation monographs are published in the Food

Chemicals Codex (FCC) (NAS, 1996) and the FAOCompendium of Food Additive Speci®cations(JECFA, 1995). The purpose of this review is to

provide an overview of the core animal studies onwhich these approvals were based, accompanied bysalient data and ®ndings not previously published.

Materials and Methods

Test substance and study sites

In this following series of studies, polydextrose

Type A and/or Type N (occasionally referred to asModi®ed Polydextrose Type A or Type N), as indi-cated, was produced by P®zer, Inc. (Groton, CT,USA). The studies were all carried out by various

investigators at P®zer research facilitiesÐeitherP®zer Central Research, Groton, CT, USA, orP®zer Research Centre, Amboise, FranceÐbetween

1972 and 1979.

Test animals and treatment

The single dose and LD50 studies. The acute oraltoxicity of PD-A in male (Crl:COBS1 CD11-(ICR)BR) mice was determined using a 50% or70% aqueous solution. The acute oral toxicity of

PD-A and PD-N in male (Crl:COBS1 CD1

(SD)BR) rats was determined using a 70% aqueoussolution. Acute toxicity of PD-A was also studied

by gavage or capsule and PD-N by intravenous ad-ministration in dogs. Administration followed anovernight fast and all test animals were observed

for signs of toxicity and mortality for 7 days afterdosing. The rodents were necropsied for gross in-ternal pathological changes at sacri®ce on day 7.

The dogs were remanded to stock.3-Month gavage study in monkeys with PD-N.

Eight rhesus monkeys of each sex were divided intoa control and three treatment groups to receive 0

*Litese III is a reduced form of polydextrose wherein allanomeric/reducing glucose endgroups are converted tosorbitol by catalytic hydrogenation to give a more col-our-stable product. These endgroups are present in32% of the polymer of PD-A and PD-N, as deter-mined by careful chemical analyses (P®zer internalreport). Thus, 68% of the polymer dosed in the safetystudies reported herein was the composition of LitesseIII, and the studies are applicable to Litesse III at 68%of the doses reported.

G. A. Burdock and W. G. Flamm234

(distilled water), 1, 2 or 10 g/kg body weight/day

PD-N by gavage, 7 days/week for 91 days. Bodyweights were recorded weekly and individual doseswere adjusted accordingly. The 10 g/kg body

weight/day dose was administered as 5 g/kg bodyweight twice daily. The 10 g/kg body weight/dayanimals were dosed with a 50% solution and the 1

and 2 g/kg body weight/day animals were dosedwith a 70% solution. Haematology, serum chem-

istry and urinalysis examinations were made oneach monkey twice prior to the start of treatmentand at 29, 57 and 85 days. The haematologic par-

ameters evaluated consisted of erythrocyte (RBC),leucocyte (WBC) and WBC di�erential counts; hae-moglobin concentration; haematocrits; and whole

blood clotting times. Clinical chemistry determi-nations consisted of fasting blood sugar, blood urea

nitrogen (BUN), serum glutamic±pyruvic andserum glutamic±oxaloacetic transaminases (SGPTand SGOT), serum alkaline phosphatase, total bilir-

ubin, serum lactic dehydrogenase (LDH), serumcreatinine phosphokinase (CPK), serum sodium(Na+), serum calcium (Ca+), serum creatinine and

uric acid levels.Ophthalmoscopic examinations and electrocardio-

graphic tracings were made twice prior to treatmentand on days 29, 57 and 85. All animals remainingat the end of the test were sacri®ced and necropsied

on day 92, 24 to 48 hours after the 91st dose.3-Month feeding study in dogs with PD-A at 50%

of the diet. 10 male beagle dogs (Marshall Farms,

North Rose, NY, USA) of about 10 months oldwere assigned to two groups (six treated, four con-

trols). On day 1 of the study, the test animals weregiven PD-A in their diet at a level of 50% of thedry weight of the entire ration (100 g Wayne canned

dog food, 150 g Purina dog chow, 17.5 g anhydrouscalcium phosphate dibasic and 1 ml Vi-Daylin vita-min drops). On day 35 the food was increased to

120 g canned + 185 g chow; PD was increased to210 g in the test group to maintain the 50% rate.

Calcium and vitamin supplement remainedunchanged. Each dog was allowed a 1-hour feedingperiod starting at 09.00 hours and water was made

available ad lib.All animals were observed twice daily. Body

weights were taken daily for the ®rst 3 days and

then weekly thereafter. Water consumption wasmeasured for all animals on days 1±3 and 6±9.

Clinical pathology pro®les were performed on allanimals twice prior to the onset of treatment andagain on days 1, 2, 3, 15, 30, 43, 57, 71, 85 and 97.

Serum chemistry determinations were done onsamples obtained at 07.00, 11.00 and 15.00 hours.Necropsies were conducted at sacri®ce and the stan-

dard tissues were taken with H&E stains of allorgans. Liver and kidney were also subjected to

periodic acid Shi� Perls reaction and Oil Red Ostains were made of cryostat sections of theseorgans.

6-Month feeding study in dogs with PD-N, fol-

lowed by PD-A at 50% of the diet. Eight male bea-gle dogs (Marshall Farms) were assigned to twogroups of four animals each. On day 1 of the study,

the test animals were given PD-N in their diet at alevel of 50% of the dry weight of the entire ration(approx. 12.5 g/kg body weight/day). On day 35, all

dogs were given dietary supplements including cal-cium (equivalent to 5.2 g). Beginning on day 136,

PD-A was substituted for PD-N in the diet andcontinued for the duration of the study (days 136±195, inclusive). The size of the ration was increased

periodically to accommodate for the growth of theanimals. Each dog was allowed a 1-hour feedingperiod starting at 09.00 hours and water was made

available ad lib.All animals were observed twice daily. With the

exception of weekends and holidays, body weightswere taken daily until day 126 and then weeklythereafter. Water consumption was measured for all

animals on day 9 and daily on days 13±17 and174±177. Clinical pathology samples were taken at07.00, 11.00 and 15.00 hours each sampling day for

a total of 33 days, and once per sampling day forhaematology and urine samples via catheterization.

Necropsies were conducted at sacri®ce and the stan-dard tissues were taken with H&E stains of allorgans. Liver and kidney were also subjected to

periodic acid Schi� Perls reaction and Oil Red Ostains were made of cryostat sections of theseorgans.

A 13-month feeding study in beagle dogs with PD-A. 15 male and 15 female beagle dogs (Marshall

Farms), approximately 9 months old, were assignedto three groups of ®ve dogs per sex to receive 0,16.7% or 33% (approx. 4 and 8 g/kg body weight/

day, respectively) of a standard laboratory rationdiet as PD-A (PD). Each day the diet was hand-mixed for each dog and all animals were allowed

1 hour of feeding beginning at 09.00. All animalswere observed at least twice daily and body weights

were recorded weekly during the study. Ophthalmo-scopic examinations using a hand-held direct oph-thalmoscope were performed once prior to dosing,

at 6 and 12 months and at study termination. ECG,blood pressure and vital signs (heart rate, respirat-ory rate and rectal temperature) were obtained

from all animals twice prior to the start of thestudy and on days 93, 176, 267, 358 and 410. Water

consumption was measured for all animals twiceprior to the start of the study, during the ®rst weekof compound administration and then at approxi-

mately 3-month intervals.Clinical pathology pro®les (serum chemistry, hae-

matology and urinalysis) were performed on all ani-

mals twice prior to the ®rst PD administration andon days 2 (urinalysis only), 15, 29, 58, 85, 113, 141,

169, 204, 232, 260, 288, 330, 351, 379 and 407. Oneach of these days, serum chemistry determinationswere performed on samples obtained at 07.00 hours

Safety of polydextrose 235

(2 hr before feeding), 11.00 hours (2 hr post-feeding)

and 15.00 hours (6 hr post-feeding). These consisted

of the following parameters: sodium, potassium,

calcium, glucose, blood urea nitrogen, uric acid,

creatinine, total bilirubin, alkaline phosphatase, lac-

tic dehydrogenase, aspartate and alanine amino-

transferase (SGOT and SGPT), total protein,

albumin, globulin, and albumin/globulin ratio.

Haematology parameters were also evaluated on

samples taken at the same intervals and consisted

of WBC, RBC, haemoglobin concentration, haema-

tocrit, MCV, MCH and MCHC. WBC di�erential

counts were performed on the 11.00 hour sample

days 15, 29, 85, 169, 204, 232, 260, 288, 330, 351,

379 and 407. Urine was obtained from the male

dogs by catheterization at 09.00 hours (sample dis-

carded), 11.00 hours and 13.00 hours and combined

into a single 4-hour sample. The female dogs were

catheterized at 11.00 hours and again at 13.00

hours only if the volume of the ®rst sample was

insu�cient to perform all analyses. Urine from all

dogs was analysed for volume, pH, speci®c gravity,

sodium, potassium and calcium. Urinary sodium

and potassium are expressed as meq and urinary

calcium is expressed as mg in the total daily collec-

tion.

On day 15 of the study, a high-dose male dog

was observed to exhibit lipaemic serum at 11.00

and 15.00 hours. Both retrospective and prospective

analysis revealed this to be characteristic of this dog

alone and not related to treatment. This dog was

replaced with another whose values were normal

and it remained on study for 271 days.

All animals were anaesthetized on days 414±416

by intravenous injection of sodium pentobarbital

and exsanguinated by cardiac puncture. Internal

organs were examined and absolute weights

recorded for kidneys (combined), liver and right

and left testicle (individual). Tissues from the fol-

lowing organs were placed in 10% neutral bu�ered

formalin: kidney, liver, heart (left auricle and ventri-

cle), aorta, lung (right and left lobes), spleen, thyr-

oid, adrenal, pituitary, gall bladder, thymus,

mesenteric lymph node, pancreas, mandibular sali-

vary gland, urinary bladder, mammary gland with

skin, oesophagus, stomach, small and large intes-

tine, testis, epididymis and prostate gland, or ovary,

body and cornu of uterus, brain, spinal cord, eye,

sternum, bone (femur) with bone marrow, and sec-

tions of any other grossly altered tissues. After ®x-

ation, tissues were trimmed, embedded in para�n,

cut at 5 m and stained with H&E. Sections of kid-

ney and liver were also stained by periodic acid±

Schi� (PAS)±Perls' method.

For statistical analysis, Student's t-test was used

to determine the signi®cance of di�erences between

means of the treated and control groups. P values

ofE0.05 were considered signi®cant.

24-Month toxicity study in beagle dogs with PD-N

at 10% and 20% of the diet. Six dogs (source:CERM-RIOM, France) per sex per group were al-located to receive 10% or 20% of the diet as PD-N

or 10% as sucrose or untreated diet. The animalswere 11±14 months old at the start of the trial andwere on test for 24 months, with an interim sacri®ce

of two animals per sex per group at month 12. Theamounts of either PD-N or sucrose administered

were calculated as a percentage of the total dailydiet, expressed in terms of dry weight. The com-pounds, in solution, were mixed with 100 g (dry

weight 25 g) of proprietary canned meat. When thishad been eaten, 175 g of food pellets was given.Each dog thus received 200 g of dry food per day.

For an average dog weighing 10 kg, the dose levelswere therefore 2 and 4 g/kg body weight/day PDand 4 g/kg/day sucrose. The dogs were allowed

water ad lib.Body weights were taken weekly and clinical

symptoms recorded daily. An ECG was performedon all survivors at month 24 and eye examinationswere performed at 0, 6, 12, 18 and 24 months on all

animals.Haematology determinations were made at the

beginning of the study and at months 1, 3, 4, 6, 18and 24. The assays were haemoglobin, RBC, hae-matocrit, platelet count, WBC, di�erential count,

plasma ®brinogen, partial thromboplastin time andprothrombin time. In addition, bone marrowsmears (femur) were taken from all animals autop-

sied at 24 months.Clinical chemistries (unless otherwise noted) were

analysed at 0, 3, 4, 6, 12, 18 and 24 months for allsurviving animals. Chemistries consisted of sodium,potassium, chloride, calcium (from month 4), cho-

lesterol, triglycerides, glucose, urea, SGOT, SGPT,alkaline phosphatase, protein, bilirubin, g-glutamyltransferase (GGT), total protein and albumin at the

time of sacri®ce. Urinalysis was performed at 24months on survivors and consisted of osmolality,glucose, ketone bodies, urobilin, proteins, blood

and microscopy of sediment. Liver analyses wereperformed on all survivors at 24 months and con-

sisted of triglycerides, cholesterol and phospholi-pids. Supplementary analysis at 1 month wasconducted for plasma sodium, potassium, chloride,

proteins and calcium.Post-mortem analyses included body weight and

organ weights of heart, spleen, liver, kidney, testisand adrenal. Tissue sections were taken and his-tology performed on brain (cortex, rhinencephalon,

thalamus, hypothalamus, mesencephalon and cere-bellum), spinal cord (cervical, thoracic and lumbar),peripheral nerve and surrounding muscle, eye,

heart, artery, vein, cervical lymph node, mesentericlymph node, thymus, spleen, bone, bone marrow,cartilage, skin, salivary gland, tongue, oesophagus,

stomach, duodenum, ileum, colon, pancreas,liver, gall bladder, pituitary, thyroid, parathyroid,

G. A. Burdock and W. G. Flamm236

adrenal, larynx, trachea, principal bronchi, lung,

kidney, ureter, urinary bladder, urethra, ovary, fal-lopian tube, uterus, vagina, mammary gland, testis,epididymis, prostate and any grossly observed

tumour and any lesion found in tissues not listedabove.24-Month toxicity study in beagle dogs with PD-N

at 50% of the diet. Six dogs (source: CERM-RIOM, France) per sex per group were allocated to

receive 50% of the diet as PD-N or sucrose oruntreated diet. The animals were 14±15 months oldat the start of the trial and were on test for 18

months, followed by 6 months' withdrawal. Theamounts of either PD-N or sucrose administeredwere calculated as a percentage of the total daily

diet, expressed in terms of dry weight. The com-pounds, in solution, were mixed with 100 g (dryweight 25 g) of proprietary canned meat. When this

had been eaten, 225 g of food pellets were given.Each dog thus received 250 g of dry food per day.

The dogs were allowed water ad lib.Body weights were taken weekly and clinical

symptoms recorded daily. An ECG was performed

on all dogs at pre-trial and months 12, 18 and 24.Eye examinations were performed at 0, 6, 12, 18and 24 months on all animals.

Haematology determinations were made at thebeginning of the study and at months 3, 6, 9, 12,

15, 18, 21 and 24. The assays were haemoglobin,RBC, platelet count, WBC, packed cell volume,di�erential count, plasma ®brinogen, thromboplas-

tin time and prothrombin time. In addition, bonemarrow smears (femur) were taken from all animalsautopsied.

Clinical chemistries were analysed at 0, 3, 6, 9,12, 15, 18, 21 and 24 months for all surviving ani-

mals. Chemistries consisted of sodium, potassium,chloride, calcium, cholesterol, triglycerides, glucose,urea, SGOT, SGPT, alkaline phosphatase and total

proteins. Blood acid balance was also measured onday 216. Post-mortem analyses included bodyweight and organ weights of heart, liver, kidney,

testis and adrenal.18-Month carcinogenicity study in mice with PD-

A. Charles River (France) CD-1 mice (50/sex/group) were fed from weaning for 18 months (562days) with a commercial powdered diet mixed with

constant concentrations (5 and 10%) of PD-A.Positive controls received the same diet sup-plemented with 10% sucrose and a control group

received the basal diet only. Animals were housedindividually. Body weights were recorded weekly.

Food consumption was determined on selected ani-mals 1 day/week every week for the ®rst 31 daysthen and until day 365, food consumption was

determined for one dose level per week during eachmonth on a rotating basis and from day 366 tosacri®ce, food consumption was recorded once

every 8 weeks. Clinical observations were recordedweekly at the weighing interval. Eye examinations

were conducted on untreated controls, sucrose and

PD 10% groups on days 0, 365 and 548 and on allsurviving animals. Blood samples for clinical chem-istry and haematology and tissue samples were

taken from all moribund animals and all survivinganimals at termination. Haematologies consisted ofWBC and di�erential counts. Clinical chemistries

included sugar, urea, SGOT, alkaline phosphataseand calcium. Post-mortem body and organ weights

(liver, kidney and testis) were taken from all surviv-ing animals at termination. The customary tissuesamples were taken for histology in addition to any

grossly observed tumour and any lesions found atnecropsy.3-Month dietary study in rats with PD-A. 40

(Charles River CD) rats of each sex were dividedinto control and three treatment groups to receive0, 1, 2 or 10 g/kg body weight/day PD-A in the diet

for 92 days. Body weights and food consumptionwere recorded weekly and PD-A levels in the feed

were adjusted accordingly. Haematology and urin-alysis examinations were conducted prior to treat-ment and on days 29, 57 and 85. Evaluations

included RBC, WBC and di�erential counts, hae-moglobin, haematocrit and clotting time. Clinicalchemistry determinations were made on two rats/

sex/dose level on days 36 and 64, and on six ratsper sex per dose level at day 92. The parameters

evaluated consisted of blood glucose, total bilirubin,BUN, alkaline phosphatase, SGOT and SGPT, cre-atine, uric acid, sodium, potassium and calcium.

Slit-lamp ophthalmoscopic examinations were madeon each animal once prior to commencing treat-ment and on days 29, 57 and 85.

24-Month carcinogenicity study in rats with PD-A.Rats used were (CD-COBS1, Sprague±Dawley ori-

ginated) born in the performing laboratory as theF1 generation of a three-generation reproductionstudy whose parents (F0) were purchased from

Charles River Breeding Laboratories (France). 50males and 50 females (with an additional 20/sex forsacri®ce at day 40) were allocated to one group

each for treatment with 0, 5%, 10% PD-A orsucrose (10%) in the diet. The animals were treated

from birth for a total of 775 days, and were housedsingly starting at the time of weaning. Body weightwas recorded once per week; food consumption

once a week during the ®rst 3 months, then once amonth on all surviving animals. The animals wereobserved for clinical symptoms and recorded weekly

at weighing time. Eye examinations were conductedon days 21, 365, 548 and 730 on all surviving ani-

mals. Haematology and clinical chemistry sampleswere taken from all groups, but limited to 20 ani-mals per sex at day 40, 10 per sex at day 391, 10

per sex at day 560 and all animals at termination.Blood was drawn under light ether anaesthesia viapuncture of the orbital sinus. Haematology par-

ameters were haemoglobin, WBC, RBC, di�erentialcount and bone marrow smears. Clinical chemistry

Safety of polydextrose 237

samples were taken for glucose, urea, proteins,

SGOT, SGPT, alkaline phosphatase, cholesterol,sodium, potassium, triglycerides and calcium.Terminal sacri®ce post-mortem body weights

were taken and organ weights of liver, kidney andtestis recorded. Histology samples included brain,heart, mesenteric lymph node, spleen, stomach,

duodenum, ileum, colon, pancreas, liver, pituitary,thyroid, adrenal, lung, kidney, urinary bladder,

ovary, testis, epididymis, mammary gland, femoralbone marrow, uterus, prostate, salivary gland, ster-num and any grossly observable tumour with re-

gional lymph nodes and any lesion found in tissuesnot listed above. The various data were analysed,when possible, by analysis of variance, Student's t-

test and chi-square test.Segment I study in rats (fertility and general

reproductive performance). 30 female and 15 maleSprague±Dawley (CD-COBS1) albino rats wererandomly divided into ®ve groups to 0, 1, 2 or 4 g

PD per day or 4 g sucrose per day. These dailydoses corresponded to approximately 5, 10 and20% of the daily food consumption of the rat. The

animals were gavaged with distilled water solutionsof PD or sucrose daily which included 79 days

before mating for the males (two cycles of sperma-togenesis). The females were treated from the 14thday before mating to day 13 post-coitus or through-

out gestation (mean length of treatment 27 or 35days). In each group, two females were exposed toone male during 10 consecutive nights. Time zero

of pregnancy was observation of sperm or copula-tory plug.Segment II study in rats (pregnancy and foetal

development). Primiparous pregnant-dated femalerats of the CD COBS1 albino strain from Charles

River France S.A., were divided into 20 females pergroup to receive 0, 1, 2 or 4 g per day per animalPD or 4 g per day per animal sucrose in distilled

water. The animals were dosed via gastric intuba-tion from day 6 to 15. On day 20 post-coitus (pc),the rats were killed by cervical dislocation. Uteri

and ovaries were carefully removed and the follow-ing observations were made: number of corpora

lutea; number and position in the uterus of live foe-tuses, dead foetuses (with no visible degenerationand normal size); late resorptions (recognizable foe-

tus undergoing degeneration regardless of size);early resorptions (evidence of placentation but norecognizable foetal structure) and decidual reaction

of the uterine mucosa. The uteri were stained (Sale-wski method) to reveal very early dead implants,

which were carefully examined for external malfor-mations and weighed. Approximately 50% of thefoetuses were placed in 70% ethanol, their skeletons

stained with Alizarin Red and their soft tissuescleared using the re®ned Staples and Schnellmethod. The remaining foetuses were ®xed in

Bouin±Allen ¯uid and were serially sectioned by theslicing technique of Wilson.

Statistical comparisons were performed on the

number of implantations and corpora lutea, litter

size and the weight of foetuses.

Segment III study in rats (study of the action of

PD on the perinatal and postnatal development of the

rat). The purpose of this study was to examine the

e�ects of PD and sucrose when administered during

the last third of pregnancy and the period of lacta-

tion, on the last stages of foetal development,

labour and delivery, lactation, neonatal and post-

natal viability, growth and development of new-

borns.

Virgin female rats of the CD COBS1 albino

strain (Sprague±Dawley originated) were mated at

the Charles River Breeding Laboratories, observed

for vaginal plug and judged pregnant (day 0) on

positive vaginal examination. The mean body

weight of the 100 animals received at the perform-

ing laboratory was 202 g, corresponding to an age

of 65 days. These animals were evenly divided

between ®ve groups and received once daily intuba-

tions of 0, 1, 2 or 4 g per day PD or 4 g per day of

sucrose in water from day 15 pc, throughout lacta-

tion and up to weaning. The following parameters

were measured in the females: body weight every

3 days from day 3 of gestation to weaning of pups,

plus day 1; examination of the external genital tract

on day 10, 11 and 12 (period of abortion); length of

gestation; parturition when possible (dystocia, pro-

longed and/or delayed labour); lactation; staining of

uteri with Salewski method at sacri®ce (non-littering

females on day 25 pc, littering females at weaning

of pups). The following parameters were measured

in the pups: number of newborns (dead or alive);

sex; external abnormalities; body weight at 24

hours, 4 and 21 days; suckling during the ®rst days

(in case of agalactia or cleft palate); growth tests

(turning re¯ex, pinching re¯ex, grasping re¯ex, time

of opening of the external auditory meatus, pinna

re¯ex, time of opening of the palpebral ®ssures);

ophthalmological examination (controls, sucrose

and 4 g per day and other groups if lesions appear

at the high dose level); sacri®ce of all pups on

weaning day with gross necropsy of ®ve per sex and

level.

Three-generation study in rats. Polydextrose A

(PD-A; powdered form) or sucrose (as a positive

control) were mixed into the diet of rats at concen-

trations of 5 and 10% of the diet for PD and at

10% of the diet for sucrose. The controls were fed

untreated diet. Treatment was continuous from the

mean age of 23 days to sacri®ce for the initial gen-

eration (F0) and during the remainder of life for the

following generations (F1, F2 and F3). When the

animals were mated, they had been treated for

about 60 (F0) or 70 days (F1 and F2). The F0 gener-

ation consisted of CD-COBS1 rats (Sprague±Daw-

ley origin) from Charles River Breeding

Laboratories (France).

G. A. Burdock and W. G. Flamm238

In each generation the rats were randomly allo-

cated into four groups, each consisting of 20 malesand 20 females. The initial groups of immature rats(F0 generation) were presented the diets for 60 days

prior to mating and each female was then caged for7 days with a male from the same dose level. Theappearance of sperm or copulatory plug was con-

sidered time zero of pregnancy and the female wasplaced in a cage alone. On the ®rst day after birth,

litters were culled to 10 pups (®ve per sex, if feas-ible) and no fostering was permitted. This was theF1 generation.

To produce the F2 generation, 20 rats of each sexwere selected at random for each dose level fromthe F1 animals and were mated after 70 days of

treatment to produce the F2 generation. The F3

generation was subsequently produced from the F2

generation in a similar manner. The F0 and F2 gen-erations were sacri®ced at weaning of their o�-spring. The F1 were saved for use in a

carcinogenicity study.The rats were observed daily for general health

and behaviour. Body weights and food consump-

tion were determined weekly. After 100 days oftreatment, eye examinations were performed on the

F0 generation controls, sucrose and 10% PD treat-ment groups.At birth, all o�spring were examined for gross

physical abnormalities. The number of live anddead pups in each litter was recorded. The pupswere culled to 10 per litter on day 1 after birth. Eye

examinations were performed on opening of palpe-bra and before weaning in 50 pups of the control,

PD 10% and sucrose groups in the F1, F2 and F3

generations. Gross necropsy was conducted on ®veweanlings per sex and level in each generation.

Standard assessments of reproductive perform-ance were conducted. The fertility index (percentage

of pregnant females over the number of matings)and the mating index (percentage of pregnantfemales over the number of e�ective copulations),

measured the ability of a pair to produce a litter ofany quantity. The gestation index (percentage ofviable litters over the number of pregnancies) rep-

resented the viability in utero of the o�spring.Because of the equalization of litters to 10 pups perlittering dam on the ®rst day after birth, the de®-

nition of the survival indices was slightly modi®edand became the percentage of surviving pups on

days 4 or 21 after birth over the number of pupsalive on day 1. These indices measured the abilityof pups to survive until weaning. The same applied

to the lactation index (percentage of pups alive onday 21 over the number of pups alive on day 4)which also measured the ability of dams to breed

and feed their o�spring. Sex ratios were calculatedto estimate a possible sex related toxicity.

The various data were analysed, when possible,by analysis of variance, Student's t-test orWilcoxon's rank sum test.

Segment II study in rabbits (pregnancy and foetaldevelopment). Random-bred virgin New Zealand

white rabbits from an accredited breeder (Bellefon-taine Abbey), were divided into 15 females pergroup to receive 0, 3, 6 and 12 g per day per animal

PD or 12 g per day per animal sucrose in distilledwater. The animals were dosed via gastric intuba-tion from day 7 to 18. The rabbits were observed

daily throughout the experimental period for indi-cations of ill-health, abnormal behaviour or reac-tion to the treatment. Maternal body weights were

recorded every 3 days, from day 1 pc to the day ofsacri®ce.The rabbits were sacri®ced on day 28 post-insem-

ination by intravenous injection of an overdose of

pentobarbitone. Uterus and ovaries were carefullyremoved and the following observations were made:number of corpora lutea; number and position in

the uterus of live foetuses, dead foetuses (with novisible degeneration and normal size); late resorp-tions (recognizable foetus undergoing degeneration

regardless of size); early resorptions (evidence ofplacentation but no recognizable foetal structure)and decidual reaction of the uterine mucosa. All

foetuses were carefully examined for external mal-formations and weighed. Approximately 50% of thefoetuses were placed in 70% ethanol, their skeletonsstained with Alizarin Red and their soft tissues

cleared with a modi®cation of the original Staplesand Schnell method. The other 50% of the foetuseswere ®xed in Bouin±Allen ¯uid and were serially

sectioned by the slicing technique of Wilson.Statistical comparisons were performed on num-

ber of implantations and corpora lutea, litter size

and the weight of foetuses.

Genotoxicity studies

Spot test using Salmonella typhimurium. Sub-stances can be evaluated non-quantitatively formutagenic activity by applying crystals or solutions

of substances to the surface of a minimal agar plateseeded with a lawn of histidine-requiring cells of S.typhimurium. After incubation for 48 hours at 378Cand 18 hours at 258C, the plates are examined. Thepresence of a ring of revertant colonies surroundingthe point of application of the substances indicatesthat the substance has induced point mutations in

the bacterial cells (Ames, 1971).Quantitative plate assay using Salmonella typhi-

murium. Tests for microbial point mutations were

performed with both base-substitution and frame-shift histidine auxotrophic mutants of S. typhimur-ium using, with slight modi®cations, the technique

described by Ames et al. (1973). Triplicate plateswere prepared at each level, incubated for 60 hoursat 378C, and the number of revertant colonies per

plate recorded. Mutagenic activity induced by thecompound was detected by reversion to prototropy(histidine independence). The average number ofcolonies per plate with test compound was com-

Safety of polydextrose 239

pared with the average number of spontaneous

revertant colonies per control plate. A threefoldincrease over control value was considered a posi-

tive response.Host-mediated assay with PD. The host mediated

assay was performed using base-substitution orframe shift mutant strains of S. typhimurium and

the CD-1 strain (HAM/ICR) of random-bred

albino mice. Assays were performed by injecting2 ml Salmonella cells (107 cells/ml) intraperitoneally.

Polydextrose was administered in three equal dosesat 0, 1 and 2 hours after implanting the bacteria

(four mice per test group). 30 min after the lastdose, 1.5 ml saline±citrate solution was administered

intraperitoneally to each mouse and its abdomen

gently massaged. Each mouse was sacri®ced by cer-vical dislocation and ¯uid recovered after aseptic

exposure of the peritoneal cavity. The ¯uid contain-ing the cells from animals of the same group was

pooled. Aliquots were impinged on membrane ®l-ters, washed in situ with 0.9% NaCl, and the mem-

branes placed on semi-solid minimal medium. A

viable count was made using complete medium oneach pool of peritoneal ¯uid. The plates were incu-

bated for 48 hours at 378C and the number of colo-nies per membrane recorded. This value and the

results of the viable count were used to calculate amutation frequency (revertants/108 cells) which was

compared with the mutation frequency of the ¯uid

from the control animals to detect any signi®cantincreases.Cytogenetic analyses. As part of an assessment of

the mutagenic potential of PD-A and PD-N, cyto-

genetic studies were conducted both in vivo inmouse bone marrow and in vitro in human lympho-

cytes.For the in vivo studies, groups of ®ve CD-1 male

mice (6±8 weeks old, 30±40 g) were given oral dosesof 2 g PD-A /kg or PD-N. Mice were sacri®ced at

6, 12, 24, 48 or 72 hours post-treatment. Treatment

consisted of 1 g/kg/day for 7 days with sacri®ce at24 hours after the last dose. Compound was dis-

solved in distilled water immediately prior to use.Solvent-treated controls were run simultaneously

with treated mice.

At 3 hours prior to sacri®ce, each animal receivedcolchicine (1 mg/kg, ip). Animals were sacri®ced

and femur bone marrow was ¯ushed into 0.65%

KCl at 378C for hypotonic pretreatment for 15 min.

This was followed by three ®xations in methanol:acetic acid (3:1, v/v) for 30 min, and stained in 2%

lactoacetoorcein. Coverslips were mounted withEuparol. 50 metaphase ®gures were examined forchromosome damage from each mouse.

In vitro studies were conducted on human lym-phocytes cultured in GIBCO Chromosome Medium1A. Cells from normal donors were seeded in cul-

tures and incubated at 378C for 48 hours. At thistime, PD-A and N was added at 500 or 1000 mg cul-ture medium/ml. Control cultures were treated at

the same time with diluent. Incubation was contin-ued for an additional 24 hours with Colcemid(0.02 mg/ml) present the ®nal 3 hours. Cells wereharvested using 1% sodium citrate for 15 minutes

as hypotonic pretreatment and methanol:acetic acid(3:1, v/v) as ®xative. Cells were stained and scoredas described above for mouse bone marrow. 50 or

100 metaphase ®gures were examined for structuralaberrations from each culture.Dominant lethal assay in mice. Polydextrose (PD-

N) was evaluated in the dominant lethal assay usinga subacute dosing regimen. 8-week-old male andfemale mice of the CD-1 strain (HAM/ICR) were

employed in this study. PD-N was administeredorally as a solution in distilled water to male miceat a level of 1.0 g/kg/day for 7 days. Both controland compound-treated groups contained 15 male

mice which were caged with three virgin femaleseach on the 7th day of dosing. These females werereplaced at 7-day intervals for an 8-week period.

All females were autopsied 11 days after removalfrom the mating cages. A positive control groupwas dosed orally with 200 mg ethyl methanesulfo-

nate (EMS)/kg daily for 7 days and then mated tovirgin females by the same protocol as used withthe test compound.

Results

Safety studies

Acute dose studies. As can be seen in Table 1,both PD-A and PD-N at the levels administered in

these studies showed no acute toxicity in mouse, ratand dog. No species was particularly susceptible, allexhibiting oral LD50s of 18 g/kg or above. At thehighest doses, all animals exhibited diarrhoea or

soft stools soon after administration, followed by

Table 1. Acute toxicity studies of polydextrose types A and N

Species Sex Administration Substance (% in aqueous sol.) LD50 (g/kg)

Mouse M Gavage Type A±50% >30.5 g/kgMouse M Gavage Type A±70% >47.3 g/kgRat M Gavage Type N±70% >18.8 g/kgRat M Gavage Type A±70% >18.9 g/kgDog M/F Gavage Type A±70% >20 g/kgDog M/F Capsule Type A±powder >20 g/kgDog F IV Type N±70% >2 g/kg

G. A. Burdock and W. G. Flamm240

recovery within 24 hours and an uneventful obser-

vation period. The LD50 for intravenous adminis-tration in the dog was also markedly high at morethan 2 g/kg.

3-Month gavage study in monkeys with PD-N.There were no mortalities during the study. Allhigh-dose animals exhibited diarrhoea from day 1

until sacri®ce. Loose stools did not occur in thecontrol group and rarely occurred at the two lowerdoses. There were no other clinical signs suggestive

of a treatment-related e�ect. With the exception ofone control female, all animals gained weight

during the study. One of four monkeys at the 10 g/kg level on day 57 and three or four on day85 showed decreases in serum calcium levels. One

of four animals at the 2 g/kg level had decreasedcalcium levels on days 29 and 57. One high-doseanimal showed haematuria and trace proteinuria on

days 29 and 85.Ophthalmoscopic, physical and electrocardio-

graphic ®ndings were not remarkable. Post mortem,all high-dose animals had moderate dilatation ofthe colon and focal areas of accumulation of

macrophages containing haemosiderin in the colo-nic mucosa. The accumulation of haemosiderin wasconsidered by the investigators to be a re¯ection of

prolonged hyperaemia of the colonic mucosa in re-sponse to the protracted diarrhoea. The investi-

gators concluded that there were no othertreatment-related ®ndings in any of the treatedgroups.

3-Month feeding study in dogs with PD-A at50% of the diet. All animals survived this study. AllPD-treated dogs lost weight during the study (0.6±

2.2 kg) while 75% of the control dogs also lostweight (0.5±1.8 kg). Treated dogs had loose

unformed to partially formed and occasionallywatery stools 3±6 hours after feeding for the dur-ation of the study, and drank 2±2.5 times more

water than the controls. Except for stool consist-ency, all treated dogs appeared to be clinically unaf-fected by PD feeding.

In general, mean serum calcium concentrationof all dogs increased from 07.00 to 11.00 hour

samples, although the increase was slightly greaterin the PD-treated dogs. At no time during thestudy was the mean serum calcium concentration

of the PD-treated dogs elevated above normal.While the PD-treated dogs appeared to excreteless sodium in the urine than the control dogs

during the 09.00±13.00 hour collection period, thedi�erences were statistically signi®cant only on

days 43 and 71. In general, urinary calcium ex-cretion of PD-treated dogs was greater than incontrol dogs.

Haematological parameters for all dogs werewithin normal limits. There were no signi®cant

di�erences in histopathological parameters or absol-ute or relative organ weights of liver, kidney ortestes.

6-Month feeding study in dogs with PD-N, fol-

lowed by PD-A at 50% of the diet. There were nomortalities during the course of the study. All fourcontrol dogs and one PD-treated dog lost between

0.5 and 1.9 kg body weight during the study period,while the remaining PD-treated dogs gained 0.2 to1.0 kg body weight. The control animals had gener-

ally normal-appearing stools, while the PD-treateddogs commonly exhibited watery diarrhoea during

the 135 days they were fed PD-N. The diarrhoeawas observed within 2 to 3 hours after feeding andovernight with stools returning to normal by early

morning of the following day. As soon as PD-Awas substituted for PD-N, the stools became lesswatery and of a thicker, although still unformed

consistency.As early as day 9, when water consumption was

®rst measured, the PD-treated dogs drank two to

three times as much water as the four control dogs.On days 174 to 177, when water consumption was

determined again, control dogs drank between 400and 1200 ml/day while PD-treated dogs drank 1300to 2750 ml.

Haematology results were unremarkable. No stat-istically signi®cant di�erences between control and

test animals were detected.Clinical chemistry data exhibited important el-

ements of both periodicity in response to compound

administration and reaction to the di�erent types ofPD. For calcium values, control dogs showed nor-mal ¯uctuations and never exceeded 12 mg/dl. All

four PD fed dogs exhibited daily rhythms withrespect to serum calcium concentrations with peak

values 2 hours post-prandially. Serum calcium con-centrations in these four dogs usually increased byas much as 0.5±1.7 mg/litre and declined to pre-

prandial values again by 15.00 hours or certainly bythe 07.00 sampling the following morning. Onetreated dog exceeded 12 mg/dl at day 71 (post-pran-

dially) and at least two sampling intervals later atthe same post-prandial sampling increased. In gen-eral, all treated dogs showed an increase in calcium

levels over the course of the study.Individual total serum protein values of the four

control dogs showed small variations with no con-sistent pattern. Serum protein values of the fourtreated dogs would generally peak immediately

after eating. Once the diet was changed from TypeN to Type A, there was a marked reduction in

amplitude of the postprandial peaks, with the daily¯uctuations similar to control values.During the 135 days of PD-N feeding, serum pot-

assium values of the four treated dogs displayedmarked transient increases at the postprandialsampling. During that time, serum potassium values

often exceed 6 mg/dl and even reached 7.0 mg/dl atone sampling. Once the switch to PD-A was made,

the potassium values resembled controls.Although mean serum sodium values of the PD

fed dogs at 11.00 hours were often statistically sig-

Safety of polydextrose 241

ni®cantly higher than the corresponding control

mean values, the di�erences in individual valueswere small and all serum sodium values were wellwithin the normal ranges for the beagle dog.

Statistically signi®cant di�erences in serum sodiumwere observed only sporadically during the periodof PD-A feeding.

Serum phosphorous concentrations followed adaily rhythm with peaks occurring at 15.00 hours (6

hours post-prandially), irrespective of treatmentgroup, PD or control.BUN values generally showed large variations in

both treated and control dogs, but was statisticallysigni®cant at the two post-prandial sampling inter-vals (11.00 and 15.00 hours). Although a markedly

elevated BUN occasionally suggests compromisedrenal function, a possible reaction to PD, BUN was

decreased in these animals. A possible explanationfor this may lie in the occurrence and periodicity ofthe decreases. That is, the ®rst decrease occurred on

day 1 and each sampling interval thereafter, butonly at the post-prandial sampling intervals (i.e.only at 11.00 hours and 15.00 hours, never at 07.00

hours). Changes in BUN were not seen followingthe switch to PD-A.

Other changes included glucose, LDH, CO2 andtotal protein which were irregularly elevated in thetreated animals. Most of these changes were abated

mostly, if not entirely, following the switch to PD-A.

Urinary sodium in control dogs generally exhib-ited daily peaks between 09.00 and 13.00 hours andreached values as high as 13.3 meq per 4-hour urine

samples. In contrast, the PD-fed dogs exhibitedonly small peaks in urine sodium excretion, whichnever exceeded 6 meq per sample. Following the

switch to PD-A, the treated animals again displayedperiodically spiking sodium excretion, similar to the

controls.Urinary calcium excretion of PD-N treated dogs

appeared equivalent to control dogs, but when PD-

A was administered, calcium excretion increasedsomewhat over control values. Urine potassium andphosphate levels exhibited wide variation without

any pattern, suggesting an e�ect from polydextrosefeeding.

The only compound-related lesion occurred inthe kidneys of two PD-treated dogs and was typi-cal of calcium nephropathy lesions. The renal

lesion was characterized by linear to wedge-shaped areas of tubular dilatation, accompaniedby mild to moderate scarring. The lesions were

located primarily in the cortex and alternate withareas of normal renal parenchyma. Focal areas of

mineralization that appeared to be obstructingtubules were present in the medulla below thecorticomedullary junction.

13-Month feeding study in beagle dogs with PD-N. There were no mortalities during the study.Three control dogs lost weight (ÿ0.9±1.7 kg)

while the remaining seven gained 1.0±3.4 kg. One

low-dose-level dog lost 0.2 kg, one showed nogain and the others gained 0.8±3.8 kg. One high-dose dog had a slight loss (ÿ0.2 kg), one was

unchanged while the remaining ones gained 0.1±4.5 kg.All control dogs had stools of normal formed

consistency that only rarely were observed to besoft. The dogs receiving dietary levels of 33% PD

usually had soft, unformed and occasionally waterystools within 3 hours after feeding; at 6 hours afterfeeding, the consistency of the stools was always

more watery. By the following morning, the stoolswere no longer watery but were still of an unformedconsistency. Occasionally, dogs among the high-

dose group experienced debilitating diarrhoea andwere taken o� PD diet for a few to several daysuntil the stools regained normal consistency and

then the animals were returned to the PD/diet mix-ture.

The dogs receiving 16.7% PD generally hadloose, unformed stools 3±6 hours after feeding.The stools were occasionally watery but the inci-

dence was much less than was observed in thehigh-dose-level dogs. The low-dose-level dogs also

had smaller amounts of unformed stool overnight.Blood-tinted stools were infrequently observed inseveral PD-treated dogs at both dose levels. The

high-dose-level dogs drank approximately 1.5times more water than the controls. No increasein water consumption was apparent in the low-

dose-level group.ECG tracings, indirect systolic blood pressure

recordings and vital signs were all unremarkable.No ocular lesions were observed in any of the dogs.Individual serum calcium values of all control

and low-dose-level PD treated dogs showed nor-mal ¯uctuations and never exceeded 12 mg/dl.One high-dose-level female had a serum calcium

value of 12.6 mg/dl on day 29 (15.00 hours) anda value of 12.3 on day 58 (15.00 hours).

Thereafter, this dog never reached a value of12 mg/dl. One high-dose-level male exceeded avalue of 12 mg/dl on day 85 (12.1 mg/dl at 07.00

hours), day 141 (12.6, 12.9 and 12.8 mg/dl, re-spectively at 07.00, 11.00 and 15.00 hours), day169 (13.1, 13.1 and 12.2) and day 204 (12.9, 12.9

and 12.7 mg/dl). This dog never again reachedcalcium values of 12 mg/dl. A second high-dose-

level male dog on several occasions had serumcalcium values greater than 13 mg/dl and on oneoccasion reached a level of 14 mg/dl. The el-

evations occurred on days 169 (13.4, 12.3 mg/dlat 07.00 and 11.00 hours, respectively), 260 (13.3,12.8, 13.1 mg/dl, respectively, at 07.00, 11.00 and

15.00 hours), 288 (12.9 mg/dl at 07.00), 330 (13.7,12.6, 12.6 mg/dl), 379 (13.8, 13.0, 12.7 mg/dl), and407 (14.0, 13.2 and 13.8 mg/dl). The remaining

high-dose-level dogs never reached calcium valuesof 12 mg/dl.

G. A. Burdock and W. G. Flamm242

Several dogs in all three experimental groups fre-

quently had BUN values in the range of 30 to39 mg/dl at the 15.00 hr sampling time. One high-dose male was consistently high at the last two

sampling intervals (days 379 and 407) reachinglevels of 50+ at the last interval.

With the exception of two high-dose males, allother PD-treated and control dogs rarely excretedurine with a speci®c gravity of less than 1.020.

Urinary volume and pH appeared una�ected by PDadministration.

Individual urinary sodium values exhibited alarge amount of variation and the mean values indi-cated little apparent di�erence between PD treated

and control dogs. Mean urinary calcium excretionof PD-fed female dogs at both dose levels was con-

sistently greater than that of the corresponding con-trols. The increase in calcium excretion thatoccurred in a high-dose female at two intervals co-

incided with serum calcium values greater than12 mg/dl in this dog.

The low-dose-level and control males excretedapproximately equivalent amounts of calciumthrough day 288. The gradual increase in mean

calcium excretion in the low-dose males from day288 until the end of the study is accounted forby an increased excretion that occurred in only

one dog. The mean calcium excretion in the high-dose-level male dogs showed a more or less

steady increase from day 2 onwards with valuesbecoming substantially elevated above control, butthis was attributable to two dogs (one more so

than the other) skewing the results with higher in-dividual values.

Urine potassium levels exhibited a large amountof variation in both treated and control animalswithout displaying any pattern to suggest an e�ect

of polydextrose consumption.There were no e�ects on haematology par-

ameters.Two high-dose male dogs, which had exhibited

high serum calcium levels and low speci®c gravity

of their urine, had relevant post-mortem ®ndings as

well. These two dogs had macroscopic focal areas

of pale discoloration of the kidney cortex with oc-casional linear white streaks running through cortexand medulla. Microscopically, this lesion consisted

of segmental areas of tubular dilatation with ¯at-tened tubular epithelium, in some regions the epi-

thelium was totally absent. Mineralized foci withinrenal tubules were prominent at the corticomedul-lary junction. There was extensive interstitial ®bro-

sis with focal areas of lymphocytic in®ltration.Calcium nephropathy was diagnosed in both ani-mals. The lesion was much more severe in the dog

which exhibited more or less consistently elevated(>13.0E14.0 mg/dl) calcium.

One low-dose female had a small focal area ofchronic interstitial nephritis consisting of interstitial

®brosis, dilated collecting ducts and absence of thinloops. The localized nature of this lesion, coupledwith the relatively small area of involvement, indi-

cates that the lesion was di�erent in type to thatdescribed for the males and was therefore unrelatedto PD treatment. Kidneys from all other animals

were essentially normal.Thyroid interfollicular cell hyperplasia was seen

in animals from all groups (5/10 high dose, 6/10low dose and 8/10 control). The most severe casewas in a control female, where nearly half of the

examined section consisted of interfollicular cellsand a few small thyroid follicles.

Chronic lymphocytic prostatitis was seen in sev-eral males from all treatment groups and was

most likely related to repeated catheterization pro-cedures used to obtain urine samples. One high-dose and four low-dose males also had multiple

epithelial cysts in the epididymal tubules. Thislesion may also have been related to catheteriza-tion procedures but was not seen in any of the

controls.24-Month toxicity study in beagle dogs with PD-N

at 10% and 20% of the diet. Compared with con-trols, there were no statistically signi®cant changesin mean body weights of PD treated animals

(Table 2). The male and female sucrose-treated

Table 2. 24-Month multidose study in dogsÐbody weights (kg)

Control 10% PD 20% PD 20% Sucrose

MalesPretrial 10.9320.61 10.9320.46 10.9320.39 10.9320.4913 weeks 10.8020.34 10.9720.29 10.9020.53 11.6020.5126 weeks 10.7320.37 10.9320.23 10.6720.57 11.9720.5652 weeks 10.2720.40 11.0720.32 10.6820.81 12.5220.54*78 weeks 11.0020.55 11.2820.61 11.2520.26 13.3720.66*104 weeks 11.6020.43 11.5020.49 11.5820.50 14.7720.59**

FemalesPretrial 9.7020.76 9.7320.59 9.6720.55 9.7020.7113 weeks 9.8720.61 9.6720.49 9.8320.55 10.8020.7426 weeks 10.2720.49 9.5720.38 10.3720.54 11.5020.8352 weeks 10.7220.46 10.2320.26 11.0520.55 12.8221.02*78 weeks 10.9020.96 10.8520.53 11.3020.85 13.6321.53104 weeks 11.6521.17 11.7020.38 11.9321.30 14.3821.91

*P < 0.05; **P < 0.01.

Safety of polydextrose 243

groups both showed an increase in mean body

weight throughout. In the males, the increase wasstatistically signi®cant at all periods after 9 monthsand in the females at 9 and 12 months. Individu-

ally, two dogs (1 M, 1 F) in the 20% PD group lostweight.Only qualitative assessments of the e�ects of

the compound on food consumption were made.In only a few instances did a dog fail to consume

its paà te of meat/polydextrose. The incidence ofdogs not eating granular diet was more frequent,especially in females given sucrose in their diet.

This e�ect was noticeable from 9 monthsonwards. In all animals, the occurrence of par-tially reduced appetite was distributed evenly

throughout the groups.Neither PD-N nor sucrose had an e�ect on beha-

viour. Females given 20% sucrose exhibited charac-teristic body fat deposition, while this e�ect was notas pronounced in males. All animals receiving PD

at both dose levels had diarrhoea. Attempts to clas-sify the diarrhoea into mild, moderate or severe,showed that animals receiving the higher dose level

were only marginally more adversely a�ected thanthe lower dose level animals. The incidence of

severe diarrhoea was greatest during the ®rst 2weeks of administration. For the rest of the studyall animals at both PD levels had only mild to mod-

erate diarrhoea. The e�ect was not seen in controlor sucrose-dosed animals.In some animals of all groups, the appearance of

blood in the stools was noted, but was mostmarked in two males, both receiving 20% PD in

their diet. Invariably, the blood was bright red incolour and was perhaps due to an irritation of thelower alimentary tract, exacerbated by the diar-

rhoea.Vomiting of food occurred sporadically in some

dogs in all groups, including controls. The fre-

quency was low and was never a serious pro-blem.

At 0, 12 and 24 months, all dogs were examinedindividually as follows: pulmonary and cardiac aus-cultation; palpation of abdomen and lymph nodes;

inspection of teeth, skin, urogenital area; testing ofosteotendinous re¯exes; and recording of ECG. Theonly abnormality in treated animals, which was not

found in controls, was galactorrhoea in two femalesat 20% PD and one in the sucrose group.

Galactorrhoea is a relatively common occurrence inbitches, especially following the oestrus period, andis occasionally associated with ``pseudopregnancy.``

The fact that the condition was only found in trea-ted animals is therefore ascribed to chance. Therewere no compound-related changes in any ECG

recording.On day 458, a male sucrose-group dog showed

an adverse reaction to routine anthelmintic treat-ment, and was found dead 1 hour after adminis-tration of 20 mg pyrantel palmoate/kg and 30 mg

morantel tartrate/kg. The animal had been in

good condition, and had not shown any reaction

to earlier treatment with the same compounds.This dog was found to have aspirated the anthel-

mintics, therefore the death was not treatment re-

lated.

Ophthalmologic examinations included ®ndings

in the corneas, lens and retinas of the dogs. No

lesion of the eye was observed which could be

ascribed to the daily administration of PD orsucrose. The observed lesions were post-embryonic,

traumatic and spontaneous age related. They

occurred at similar rates in control and treated ani-

mals.

There was a progressive fall in haemoglobin,

RBC and haematocrit in males of the 20% PDgroup, leading to anaemia in 3/4 animals of this

group at month 24. In females of the same group,

1/4 showed a progressive anaemia. One male of this

group showed evidence of bone marrow regener-

ation, which would be expected in an anaemic state.In other groups the values were in the normal

range. However, the female group receiving sucrose

showed a trend towards an increase in the three

tests, compared with their values at month 0 and tothe female control group.

Platelet counts, WBC, ®brinogen, partial thrombo-plastin time and prothrombin time were generally

within normal limits and no treatment-related e�ect

was noted.

As can be seen in Table 3, the frequency of

plasma calcium values of 115 mEq/litre or above

was increased in the PD groups only. Thisphenomenon was observed in males (20% PD)

from month 4 onwards and in females (20% PD)

at months 18 and 24. Mean values were raised

only in the PD groups and reached levels above

the threshold 115 mEq/litre at the top dose levelonly. Despite the scatter of individual values,

these increases were signi®cant (P <5%) in the

PD 20% male group throughout the trial, and in

the PD 20% female group and PD 10% malegroup at month 24 only.

Table 3. 24-Month multidose study in dogsÐplasma calciumvalues above 115 mEq/litre

Month

Group 4* 6* 12* 18** 24**

MalesControl 0 0 0 0 0PD 10% 0 0 0 0 1PD 20% 4 2 2 3 4Sucrose 2 0 0 0*** 0***

FemalesControl 1 0 0 0 0***PD 10% 0 1 1 2 1PD 20% 0 0 1 2 4Sucrose 0 0 0 1 0

*n = 6/sex/dose.**n = 4/sex/dose (except where noted).***n = 3.

G. A. Burdock and W. G. Flamm244

No treatment-related variations in sodium were

noticed until month 24, when numerous increaseswere observed in all groups. These ®ndings are ofdoubtful biological signi®cance since they could

only be correlated with the laboratory's use of anewer, more sensitive ¯ame photometer.

No treatment-related variations were observeduntil month 18 for urea. At month 24, increases

were observed in the 20% PD groups: values ran-ged from 45 to 233 mg/100 ml in males and 43 to

109 mg/100 ml in females (normal range E50 mg/100 ml).

Large individual variations in triglyceridesoccurred throughout the trial. The di�erencesobserved between control and treated groups at

months 3 and 4 disappeared at months 12 and 18.At month 24, increased levels were noted in the

male PD 20% groups and in both female PDgroups, but these values were still within the normal

range for this laboratory. These variations couldnot be related to PD or sucrose administration.Urines were collected at month 24 by puncture of

the urinary bladder. No treatment-related abnorm-alities were noted for glucose, ketone bodies, urobi-

lin, proteins, blood and sediments. A loweredosmolality was observed in the PD 20% treated

males and in both female groups. Owing to themode of urine collection, urinary volumes per dayare unknown and it is thus not possible to explain

this observation. The laboratory reported no obser-

vations of biological signi®cance for liver lipids (tri-

glycerides, cholesterol or lipid phosphorus).

Post mortem, there were no di�erences observed

in the organ weights of animals killed 12 and 24

months after treatment with PD when compared

with those from the control animals. Macroscopic

changes, the result of chronic in¯ammation, were

seen in the intestines, liver, lungs and kidneys of all

animals; only the pulmonary and renal lesions seen

after 2 years of PD or sucrose administration had

any signi®cance.

The single important histopathological ®nding

was a treatment-related lesion in the kidneys of ani-

mals treated for 2 years with PD. Evidence of other

renal disease was also present in some of the PD-

treated animals as well as some of the sucrose trea-

ted animals and the controls. Virtually all the dogs

showed evidence of calci®cation within the collect-

ing tubules in the renal papilla, but degree of calci®-

cation did not distinguish treated from control

groups, because renal disease and papillary calci®-

cation, especially, are common ®ndings in normal

dogs (Hottendorf and Hirth, 1974). However, when

the comparisons were made of kidney pathology,

plasma calcium and blood urea, a convincing pat-

tern of pathology emerged (Table 4). Parasitic

lesions were noted in the lungs of some PD and

Table 4. 24-Month multidose study in dogsÐrenal lesions related to plasma calcium levels (760 days of treatment)

Dose group Animal no.

Chronicin¯ammation

(focal)Papillary

calci®cation Nephrocalcinosis

Plasmacalcium(mg/litre)

Blood urea(mg/100 ml)

Control M1 ± + ± 99 33M2 ± + ± 94 26M4 ± + ± 98 30M5 + + ± 103 35F51 ± - ± 104 33F52 + + ± 97 13F53 ± + ± 95 25F56 ++ ± ± 97 15

Sucrose 20% M41 ± + ± 101 24M45 ± + ± 106 29M46 + + ± 98 17F91 + + ± 96 36F94 ± + ± 102 34F95 ± ++ ± 109 18F96 ± + + + ± 103 19

PD 10% M12 ± + + + + 116 58M13 ± + + 106 26M14 ± + + 113 44M15 ± ++ + 106 34F62 ± ++ + 113 38F64 ++ + + + + 127 31F65 ± + + 111 17F66 ± + + 107 56

PD 20% M22 ± + + + + 121 134M23 ± ++ + + + 127 46M24 ± + + + ++ 141 47M26 ± + ++ 123 111F71 ± + + 115 52F73 ++ + + + + 154 110F75 ± ++ ++ 121 80F76 ± + ++ 116 43

+minimal.++ moderate.+ + + marked.

Safety of polydextrose 245

sucrose-treated dogs, although these were not con-

sidered treatment related.24-Month toxicity study in beagle dogs with PD-N

at 50% of the diet. One dog in the treatment groupdid not seem able to tolerate the dosed feed; it lost

weight and became cachectic. PD was withdrawn

from its feed at month 5 and the dog was renum-bered and fed control diet until month 24. A repla-

cement dog was added to the PD group at this timeand carried through to the end. This dog was

sampled at the same intervals as the other dogs, but5 months in arrears, and autopsied after 1 year. Its

individual body weight and clinical chemical and

haematological values were not included in thegroup means.

One sucrose-treated dog died at day 349.Anorexia and weight loss occurred during the pre-

vious month. On the morning of its death, it wasobserved to be in a coma with convulsive crisis and

dehydration. An ECG showed tachycardia anddecreased QRS wave amplitudes, and histopatholo-

gical examination revealed severe acute broncho-

pneumonia. One control dog showed anorexia,asthenia and hypothermia on day 224, but recov-

ered in 1 week.

PD-treated dogs showed no consistent weightchanges during the 18 months of compound admin-

istration. In contrast, the controls gained weightsteadily during the ®rst year and weighed 3±4 kg

more than PD-treated dogs from weeks 50 to 82.After PD withdrawal, treatment groups gained

weight steadily but did not reach the mean value of

the controls at time of autopsy. Sucrose-treateddogs steadily gained more weight than controls

until about week 40, when they were approximately2.5 kg heavier. These dogs showed a temporary loss

in mean body weight at the beginning of the secondyear because of severe individual weight losses in

one of four males and two of four females. Aftersucrose was withdrawn, these dogs progressively

lost weight. At the end, males matched controls

while females still exceeded them in body weight.With the exception of the smallest female, all

controls ate all the food o�ered. All PD-treated

dogs except for two females showed some variabledegree of anorexia and reduced appetite. When this

became severe, the dosing regimen was changed: thePD±meat mixture was stirred into the dry pellets

before feeding. This method improved food con-

sumption for a time, but anorexia eventually

returned until PD was withdrawn at 18 months.

After this, no further anorexia was seen. Sucrose-

treated dogs also showed some degree of anorexia.

Females were more a�ected than males and anor-

exia remained steady (rather than ¯uctuating as in

the PD group) until compound was withdrawn at

month 18. Several other isolated instances of severe

anorexia, correlated with sporadic weight loss, were

seen in all groups, and were presumably due to

intercurrent subchronic infection.

All PD-treated dogs had daily diarrhoea in the

morning following the ®rst dietary administration,

and this was present during the 18 months of treat-

ment. There was no evidence of GI tract irritation

since traces of blood were only rarely found in the

faeces. Diarrhoea stopped within 3 days of com-

pound withdrawal.

Vomiting was extremely rare in all groups. Two

dogs in the sucrose group had epileptic crises (one

male three times, one female once). This frequency

was typical for this colony of dogs. PD and sucrose

did not alter the normal frequency and duration of

oestrus periods. In ECG recordings, abnormal ST

segments (depression, concavity) were seen predo-

minantly in the male PD-treatment group. These

e�ects were believed to be due to the hypercalcae-

mia observed in these dogs.

Ophthalmic examinations showed no treatment-

related eye lesions. Those observed were of trau-

matic, postembryonic and age-related origin.

PD or sucrose administration to the dogs for 574

days had no signi®cant e�ect on the plasma concen-

trations of sodium, potassium, chloride, glucose,

protein, SGOT, SGPT and triglycerides. Blood pH,

pCO2 and HCO3ÿ measured on day 216 were unaf-

fected. Plasma calcium levels were raised in nine of

12 PD-treated dogs throughout the study, but

Table 5. 18-Month carcinogenicity study in miceÐsurvival rates atstudy termination

Males Females

Controls 86% 68%PD 5% 90% 72%PD 10% 88% 76%Sucrose 10% 80% 61%

Table 6. 18-Month carcinogenicity study in miceÐbody weight gain (g)

Controls PD 5% PD 10% Sucrose 10%

MalesDay 1 21.4 21.3 21.4 22.4Day 557 43.5 43.1 44.5 44.4Gain (%) +22.1 (103) +21.8 (102) +23.1 (108) +22.0 (98)

FemalesDay 1 18.7 19.0 19.1 19.3Day 557 36.5 36.0 36.7 38.8Gain (%) +17.8 (95) +17.0 (89) +17.6 (92) +19.6 (102)

G. A. Burdock and W. G. Flamm246

promptly returned to normal on withdrawal oftreatment. Urea levels were raised in some animals,

mainly in PD groups; these did not completelyreverse in males on withdrawal. Urea levels dropped

approximately 30% in the sucrose groups. This wasfully reversed on sucrose withdrawal, and may have

been related to reduction of aminoacid catabolism.

No change in plasma alkaline phosphatase was seenin the PD groups, but was signi®cantly elevated in

the sucrose groups; this returned to normal onsucrose withdrawal. A mild polycythaemia was

noted in sucrose females and was probably second-ary to obesity. Haematology results were otherwise

unremarkable.

Some variations in organ weights were seenwithin and between groups. These re¯ected di�er-

ences in body weights due to the diet, except for thereductions in kidney weights, which were related to

the histopathological ®ndings. These observations

(kidney lesions in the PD groups and renal diseasein some dogs of all groups) were the same as those

described in the earlier 24-month dog study(above).

18-Month carcinogenicity study in mice with PD-A. Clinical symptoms recorded were common, spon-

taneous and associated with the ageing of the ani-

mals. No observations could be related totreatment. At the time of study termination, survi-

val was slightly better in the PD-treated groupsthan in the control or sucrose treated groups, but

the di�erences between groups of the same sex werenot signi®cant when compared with a chi-square

test (Table 5). No deaths could be related to treat-

ment. All deaths were the result of accident,unknown cause or sacri®ce in extremis.

The mean body weights of the animals at in-itiation and termination are given in Table 6. The

average growth rates (mean of the ratio of individ-ual body weights at two periods) were calculated

for the di�erent groups and compared to t-test

analysis from start to end of the study. For males,growth was comparable in all groups. Body weight

gain was slightly higher in the PD 10% group, butthere were no statistically signi®cant di�erences in

mean growth rates among the di�erent groups. Infemales, the sucrose group gained signi®cantly more

weight than the control or PD-treated groups, butthere was no di�erence in growth rate between any

of the other groups.

Relative food intake of the sucrose-treated maleswas slightly lower than in other groups except at

months 6, 7 and 8. There were no large variationsbetween the values of any of the other groups. The

food consumption did not vary among female

groups.Ophthalmological observations included dystro-

phies of the corneas in all groups and the incidence

increased with age. Keratitis was observed in onlytwo animals, one in the sucrose group and one in

the high-dose PD group. There were no remarkable®ndings in the irises and changes in lenses appeared

only to be senile and traumatic in nature. The ocu-lar lesions recorded in this study were not related to

the administration or either PD or sucrose. Thelesions seen were either of traumatic, post-embryo-

nic or senile origin.

Table 8. 18-Month carcinogenicity study in miceÐhaematology

WBC (�103/mm3) Di�erential count (%)

Males Females Males Females

Control 15.4322.064 6.4522.322 31.2221.977 35.2622.171PD 5% 6.5022.018** 6.3822.256 36.0021.887 39.0822.110PD 10% 7.9522.041* 6.2222.196 33.9821.909 34.7622.081Sucrose 10% 7.9022.114* 9.1422.514 33.8321.977 41.0722.393

*P < 0.05; **P < 0.01%.

Table 7. 18-Month carcinogenicity study in miceÐterminal body and organ weights (g)

Controls PD 5% PD 10% Sucrose 10%

MalesBody wt 43.1920.795 41.9320.777 43.9820786 44.1220.814(no. of animals) (n = 43) (n = 45) (n = 44) (n = 41)

Liver 2.2120.077 1.9220.075** 2.0120.076 2.1920.079Right kidney 0.3820.008 0.3920.008 0.3920.008 0.3920.008Left kidney 0.3720.008 0.3820.008 0.3920.008 0.9720.008Right testis 0.1420.026 0.1420.025 0.1520.026 0.1820.027Left testis 0.1420.007 01420.007 0.1220.007 0.1320.007

FemalesBody wt (n) 35.2420.894 34.4420.869 35.6120.846 37.5020.952

(n = 34) (n = 36) (n = 38) (n = 30)Liver 1.6520.087 1.5820.084 1.5820.082 1.6520.092Right kidney 0.2520.009 0.2520.009 0.2420.009 0.2620.010Left kidney 0.2420.009 0.2420.009 0.2420.009 0.2420.10

Safety of polydextrose 247

No signi®cant changes were noted in terminal

mean body weights, and the single change in organ

weights was a decrease in the livers of the 5% PD

group (Table 7). There were no gross ®ndings at

necropsy that could be related to treatment. There

was no evidence of an increase in either the inci-

dence or total of malignant tumours, or of a

decrease in the latency of tumour appearance in the

treated animals compared with the controls.

At study termination, the highest average values

of WBC were recorded in male controls (��15.43)and female sucrose groups (��9.14), obviously in re-

lation to the presence of leukaemic mice with

marked leucocytosis (Table 8). In the other groups,

the average values ranged from 6.22 to 7.95. All of

these means were consistent with the historical con-

trols of the performing laboratory. It should be

noted that the lowest mean values of WBC were

recorded in the PD-treated groups. This was not re-

lated to the number of mice with malignant haemo-

pathies. When mice with malignant haemopathies

are excluded from the calculations, the mean WBC

of the PD groups remains below that of the con-

trols but well within the normal range for mice of

this strain.

No treatment-related increases in either benign or

malignant tumours were seen prior to or at termin-

ation of the study (Tables 9±10).

Blood was drawn at month 18 on the surviving

animals of all groups, but due to the low amount of

plasma available, only ®ve assays were carried out:

calcium, glucose, urea, SGOT and alkaline phos-

phatase. No signi®cant variations were noted from

control values in any of the parameters tested.

3-Month dietary study in rats with PD-A. Body

weights of treated animals were depressed in a ran-

dom manner that showed no dose±e�ect relation-

ship. There were no remarkable physical, clinical

pathological, ophthalmological or histopathological

changes in rats receiving PD-A in the daily ration

Table 10. 18 Month carcinogenicity study in miceÐnumerical summary of malignant tumours

Untreatedcontrol PD (5%) PD (10%)

Sucrose(10%)

Time (months) M F M F M F M F

Number examined 18 43 34 45 36 44 38 41 30Prior to termination* 6 15 5 13 6 10 8 17

Total 49 49 50 49 50 48 49 47TumourAdenomatosis 18 11 4 5 1 6 5 11 6

Prior to termination 1 1 1 1 1 2Total 12 4 6 2 6 6 12 8

Adenocarcinoma 18 1 2Prior to termination

Fibrocarcinoma 18 1Prior to termination 2

Haemangiosarcoma 18 1Prior to termination 1

Leiomysarcoma Prior to termination 2Rhabdomyosarcoma Prior to termination 1Undi�erentiated sarcoma 18 1

Prior to termination 1

*Those animals found dead or sacri®ced in extremis prior to scheduled termination of study.

Table 9. 18-Month carcinogenicity study in miceÐnumerical summary of benign tumours

Untreatedcontrol PD (5%) PD (10%)

Sucrose(10%)

Time (months) M F M F M F M F

Number examined 18 43 34 45 36 44 38 41 30Prior to termination* 6 15 5 13 6 10 8 17

Total 49 49 50 49 50 48 49 47TumourAdenoma 18 1 2 1Adenomatous polyp 18 1Benign liver cell tumour 18 5 9 8 8

Prior to termination 1 1Fibroadenoma 18 1Haemangioma 18 1 1 1 1 1 1

Prior to termination 1 1Morphology unknown 18 1Interstitial cell tumour 18 1 1 2

Prior to termination 1

*Those animals found dead or sacri®ced in extremis prior to scheduled termination of study.

G. A. Burdock and W. G. Flamm248

at dose levels of 1, 2 or 10 g/kg body weight/day

for 3 months.24-Month carcinogenicity study in rats with PD-A.

The general health of the rats was una�ected by

treatment with PD. The only treatment-related

symptom was soft and dark faeces when

animals had eaten PD or sucrose for a few days.

Non-treatment-related observations common to this

type of study included, but were not limited to,

mammary masses, papillomatous formations of the

skin, paresia, epistaxis, chromodacryorrhoea, vagi-

nal bleeding and palpable abdominal masses.

During the week preceding test for the ®nal samples

(day 758), all animals exhibited symptoms of sialo-

dacryoadenitis and although no spontaneous mor-

talities occurred at this time, the animals did exhibit

weight loss, anorexia, an increase in the size of the

submaxillary salivary glands and other symptomol-

ogies associated with this contagious disease.

There was no mortality related to treatment and

no statistically signi®cant di�erences between

groups in mortality (Table 11). Unplanned mortal-

ities were observed in similar rates in di�erent

groups due to causes commonly seen in ageing rats

in the performing laboratory.

As animals were bred in the performing labora-

tory as part of a reproductive study and weaned at

intervals varying from 1 to 7 days, signi®cant di�er-

ences existed in the initial mean weights of the ani-

mals and are the result of age dispersal. There was

a nearly universal decrease in mean body weights

from day 758 to 775 which was probably due to

stress from disease and blood withdrawal.

The average growth rates (mean of the di�erences

of individual body weights at two periods) were cal-

culated from the di�erent groups and compared by

t-test analysis. For males, all groups exhibited a fall

in body weight after each mating period, and

growth was slightly lower than controls during the

®rst 2 months of the trial in the high-dose PD and

sucrose groups (P < 0.01) and was probably due to

lower mean body weight at the start of the trial.

After the mating periods (month 6), growth was

normal in all groups until the ®nal sacri®ces. For

females, all growth peaked during gestation, growth

was slightly faster during the ®rst 2 months of the

trial in the 10% PD group (P < 0.01) and after

mating periods (month 6), growth was normal in all

groups until the ®nal sacri®ces (Table 12).

As compared with the untreated controls, the rela-

tive food intake was lower with sucrose and higher

with PD, but the di�erences were slight and non-sig-

ni®cant. The individual values increased in females

in all groups during gestation and lactation. Because

the concentration of PD or sucrose in the diet

remained the same during the entire study, animals

received varying amounts, that is, mean compound

intake of female groups was generally higher (up to

60%) than male groups and doubled during lacta-

tion. Further, from month 1 to the ®nal sacri®ce,

there was a continuous decrease in mean compound

intake by both male and females. The changes of

Table 11. 24-Month carcinogenicity study in ratsÐsurvival ratesat study termination

Males Females

Controls 60% 64%PD 5% 60% 80%PD 10% 66% 62%Sucrose 10% 78% 72%

Table 12. 24-Month carcinogenicity study in ratsÐbody weights(g)

Controls PD 5% PD 10% Sucrose 10%

MalesMonth 1 90 87 84 85Month 12 668 669 674 641Month 24 747 751 755 756

FemalesMonth 1 89 83 80 81Month 12 358 357 357 355Month 24 469 471 459 498

Table 13. 24-Month carcinogenicity study in ratsÐterminal body weights and organ weights (g)

Controls PD 5% PD 10% Sucrose 10%

MalesBody weight 687.73 666.77 679.39 669.87(no. of animals) (n = 30) (n = 30) (n = 33) (n = 39)

Liver 21.54 18.89 19.87 21.24Left kidney 2.01 2.00 1.95 1.86Right Kidney 2.05 2.05 1.88 1.89Left testis 1.57 1.77 1.64 1.67Right testis 1.69 1.85 1.69 1.61

FemalesBody weight 436.31 443.83 418.23 459.31(no. of animals) (n = 32) (n = 40) (n = 31) (n = 36)

Liver 1.323 12.31 12.52 13.20Right kidney 1.27 1.22 1.21 1.23Left kidney 1.30 1.23 1.24 1.25

*P < 0.05; **P< 0.01.

Safety of polydextrose 249

three- to fourfold result from reduced food intake on

a kg body weight basis as the animals grew larger.

Ophthalmic examinations were made at 0, 12, 18

and 24 months of all survivors. The lesions

observed in this study were localized on the cornea,

iris, lens and vitreous humour. The lesions were

classi®ed as having various origins, but limited to

traumatic, post-embryonic, congenital and senile;

no lesions could be attributed to the administration

of PD or sucrose.

Clinical chemistries at day 0 revealed only a few

individual values outside the normal laboratory

ranges. On day 391 and thereafter, there appeared

to be a decrease in cholesterol levels among treated

groups, but statistical signi®cance was not achieved.

No other variations observed for the other determi-

nations could be related to either PD or sucrose ad-

ministration. In some cases, mean values were

modi®ed due to only one or two abnormal values

per group.

At the time of weaning, there was a statistically

signi®cant increase in haemoglobin and RBC in

both males and females in the PD 10% group.

There was also a decrease in WBC mean for

females. At 12 months, the only observation was a

persistence of a slight decrease in WBC mean in the

male sucrose and 10% PD groups, but which was

only statistically signi®cant in the former, not the

latter. At months 18 and 24, there was no notice-

able di�erence between means of controls and any

treated group. There was, however, at month 24, a

slight decrease in all means of all groups because of

an intercurrent disease, with a few (three rats from

various groups) exhibiting frank anaemia. Means of

WBC at months 18 and 24 stayed in the normal

range and showed no di�erence between control

and treated groups. Some cases of leucocytosis or

leucopaenia were scattered among all groups, with-

out relation to treatments. Di�erential counts were

normal in all groups at month 18. At month 24,

relative and absolute di�erentials showed cases of

neutrophilia in all groups (male and female). Means

of absolute neutrophilia reached the upper limit in

all groups. Neutrophilia was most probably related

to the suspected epidemic sialodacryoadenitis.

Autopsy ®ndings were unremarkable. No signi®-

cant changes in organ weights (liver, kidney and

testes) were noted (Table 13). A review of the

microscopic pathology produced no evidence of an

increase in either the incidence of total or of malig-

nant tumours, or a decrease in the latency of

tumour appearance in the treated animals compared

Table 15. 24-Month carcinogenicity study in ratsÐmammary tumours in females

IntervalUntreatedcontrol

PD(5%)

PD(10%)

Sucrose(10%)

Benign Prior to termination* 5 3 5 3At termination 10 14 6 16

Malignant Prior to termination 7 1 4 2At termination 3 5 6 8

Total 25 23 21 29

*Those animals found dead or sacri®ced in extremis prior to scheduled termination of study.

Table 14. 24-Month carcinogenicity study in ratsÐsummary of relevant kidney ®ndings

Untreatedcontrol PD (5%) PD

(10%)

Sucrose(10%)

Interval M F M F M F M F

Animals at termination 30 32 30 40 33 31 39 36Calcium deposition Prior to termination* 4 1 1 2

At termination 1 4 4 1 3Chronic glomerulonepthritis Prior to termination

At terminationChronic in¯ammation foci Prior to termination 7 4 5 1 9 1 4

At termination 22 8 6 13 19 5 24 2Nephrosis Prior to termination 2

At termination 1Cyst Prior to termination 1 2

At termination 1 10 1MedullaÐfocal necrosis Prior to termination

At termination 1PapillaÐfocal necrosis Prior to termination 1

At termination 1 1Renal tubuleÐcast Prior to termination 4 2 1 1 2 2 1

At termination 7 9 8 3 7 5Renal tubuleÐdilatation Prior to termination 2 1 3 1 3

At termination 3 2 3 2 1 8

*Those animals found dead or sacri®ced in extremis prior to scheduled termination of study.

G. A. Burdock and W. G. Flamm250

with the controls. Neither was there evidence of any

treatment-related histopathological change found in

the kidney or elsewhere (Table 14). Mammary

tumours (excluding hibernomas) were the most fre-

quent tumours seen and they are summarized in

Table 15.

Segment I study in rats (fertility and general

reproductive performance). Two accidental deaths

occurred during the course of treatment and were

attributed to dosing errors. The males at the high

dose of PD exhibited soft faeces, which persisted

until the end of the study. There was no signi®cant

di�erence in the average of the ratios of the body

weights of individual rats at various periods of

treatment (Table 16). Three treated males (one each

in the sucrose and mid- and high-dose PD groups)

failed to inseminate females, two of the males

exhibited spontaneous testicular lesions. Insemina-

tions (copulation rates) were highly successful with

no di�erence between groups. The pregnancy rate,

which is, in part, a measure of sperm viability, was

very high and greater than the historical value for

the performing laboratory (mean pregnancy rate of

85%). No signi®cant di�erence in the litter size and

viability was noticed between the groups. A higher

mortality rate was noted in the 1 g PD/day group

due to the death of an entire litter as the result of a

laboratory accident. A slightly better growth rate of

the pups was observed in the PD-treated animals at

4 g/day.

Among the females, there were no clinical signs

or abnormal behaviour. Eight deaths occurred

among females during the course of the study, but

were not treatment related. No large di�erences

Table 17. Segment I study in ratsÐgrowth and fertility of females

Parameter Controls Sucrose (4 g/day) PD (1 g/day) PD (2 g/day) PD (4 g/day)

Growth ratiosDay 0/day 15* 1.13 1.12 1.12 1.12 1.13Day 13/Day 0 1.24 1.22 1.22 1.22 1.18

Growth of littering females (ratios)Day 0/day 15 1.12 1.12 1.11 1.12 1.15Day 21/day 0 1.58 1.55 1.60 1.47 1.54

Copulation rate 26/30 (87%) 26/29 (90%) 28/29 (96%) 25/28 (98%) 23/28 (82%)Pregnancy rate 26/26 (100%) 25/26 (96%) 28/28 (100%) 24/25 (96%) 22/23 (96%)

*Day ÿ15 indicates 15 days prior to conception.

Table 18. Segment I study in ratsÐreproduction data of pregnant females sacri®ced on day 13 post coitus

Parameter Controls Sucrose (4 g/day) PD (1 g/day) PD (2 g/day) PD (4 g/day)

Corpora lutea per female (��2SD) 15.922.75 17.022.61 17.123.51 16.922.08 18.021.79Implantation e�ciency 194/207 193/221 224/256 157/169 184/198

(94%) (87%) (88%) (93%) (93%)Implants (��2SD) 14.922.10 14.823.21 14.923.81 15.722.45 16.721.62Embryomortality rate 17/194 16/193 23/224 11/157 13/184

(8.76%) (8.29%) (10.27%) (7.01%) (7.06%)Viable foetuses (��2SD) 13.621.8) 13.623.2 13.424.1 14.622.6 15.522.1

Table 16. Segment I study in ratsÐresults of male rat fertility study

Parameter Controls Sucrose (4 g/day) PD (1 g/day) PD (2 g/day) PD (4 g/day)

Copulation rate 20/26 (77%) 21/26 (81%) 25/26 (96%) 24/28 (86%) 21/28 (75%)Pregnancy rate 19/20 (95%) 19/21 (90%) 24/25 (96%) 22/24 (92%) 21/21 (100%)Length of gestation, days (range) 21.9 (21±23) 22.1 (21±23) 21.9 (21±22) 21.9 (21±22) 22.0 (21±23)Litter size at birth 14.8422.93 14.7721.88 13.7523.84 14.2322.20 13.0023.35Live birth index 278/282 276/280 318/330 310/313 272/273

(98.6%) (98.6%) (96.4%) (99.0%) (99.6%)Number of viable pups at birth (��2SD) 14.623.71) 14.621.92 13.223.91 14.122.24 13.023.37Mortality during lactation period4 day survival 269/278 271/276 316/318 307/310 270/272index (birth to day 4) (96.8%) (98.2%) (99.4%) (99.0%) (99.3%)Lactation index 268/269 270/271 305/316 301/307 265/270(day 4 to 21) (99.6%) (99.6%) (96.5%) (98.0%) (98.1%)21 day survival 268/278 270/276 305/318 301/310 265/272index (96.4%) (97.8%) (95.5%) (97.1%) (97.4%)Growth of pups (ratio of mean body wts on day 21 and 1)Males 6.14 5.90 5.92 6.15 6.29Females 6.15 5.98 6.07 6.10 6.43Sex ratio (M/F)24 hours 87 101 95 104 11921 days 94 100 93 101 123

Safety of polydextrose 251

Table 20. Segment I study in ratsÐmalformations and lesions

Treatmentgroup Litter Observation

At birthPD 2 g/day F108 Five pups presented ``kinky tail'' syndrome: kinky tail, protruding tongue, syndactyly and cleft palate.

Three showed ablepharia. The ®ve pups presented agenesia of the kidneys and ureters, two withundi�erentiated gonads, one with agenesia of the left testis.

F110 One pup showed diaphragmatic hernia with dextrocardia and atrophic lungs, haemiencephaly associatedwith slight hydrocephaly

PD 4 g/day F124 One foetus had an interventricular septum defect associated with a hypertrophic right atrium andprotruding tongue.

At weaningPD 2 g/day Two females showed gross lesions, one female had a shrunken pericardium and one presented a slight

dilatation of the left kidney pelvis.

Table 19. Segment I study in ratsÐprogeny of littering females

Parameter Controls Sucrose (4 g/day) PD (1 g/day) PD (2 g/day) PD (4 g/day)

Length of gestation (days) (��) 21.4 21.5 21.2 21.7 21.4Litter size at birth (��2SD) 14.323.66 12.723.00 13.823.65 12.423.06 13.722.49Live birth index 185/186 139/140 178/180 142/149 147/151

(99.5%) (99.3%) (98.9%) (95.3%) (97.3%)Number of live pups at birth (��2SD) 14.223.72 12.623.14 13.723.97 11.823.16 13.422.20Viability of pupsSurvival index 185/195 139/139 178/178 140/142 147/147day 1 (94.89%) (100%) (100%) (98.6%) (100%)Survival index 183/185 139/139 176/178 139/140 147/147day 4 (98.9%) (100%) (98.9%) (99.3%) (100%)Lactation index 176/183 138/139 175/176 138/139 146/147

(96.2%) (99.3%) (99.4%) (99.3%) (99.3%)Growth of pups*Males 6.36 6.56 6.12 6.58 6.40Females 6.44 6.12 5.70 6.27 6.60

Sex ratio (M/F)24 hours 85 83 109 89 9921 days 83 75 92 86 106

*Ratio of the mean body weights at days 21 over 1.

Table 21. Segment II study in ratsÐmaternal and foetal data

Parameter Controls Sucrose (4 g/day) PD (1 g/day) PD (2 g/day) PD (4 g/day)

Mortality 0/20 0/20 1/20 0/20 0/20(0%) (0%) (5%) (0%) (0%)

Nidation rate* 17/20 14/20 16/19 17/20 18/20(85.0%) (70.0%) (84.2%) (85.0%) (90.0%)

Copora lutea/pregnancy (��2SD) 14.423.18 14.522.38 13.423.07 14.922.63 15.124.04(n = total) (n = 245) (n = 203) (n = 214) (n = 254) (n = 271)

Implantation site/pregnancy (��2SD) 11.922.50 12.921.82 11.024.30 11.923.93 12.223.66(n = total) (n = 202) (n = 181) (n = 176) (n = 212) (n = 220)

Pre-implantation loss rate** 43/245 22/203 38/214 42/254 51/271(17.55%) (10.84%) (17.76%) (16.54%) (18.82%)

Embryomortality***IMP-VF/IMP 17/202 9/181 8/176 10/212 16/220

(8.4%) (5.0%) (4.5%) (4.7%) (7.3%)VF/litter (��2SD) 10.922.42 12.321.94 10.524.35 11.923.93 11.323.82

Foetal weightMales (��2SD) 3.8620.64 3.7720.37 3.8620.27 3.8621.27 3.8920.31(n = total) (n = 96) (n = 89) (n=83) (n = 105) (n = 116)Females (��2SD) 3.7220.64 3.5620.29 3.6920.36 3.6920.24 3.6920.41(n = total) (n = 89) (n = 83) (n = 85) (n = 97) (n = 88)

Sex ratio (M/F) 1.08 1.07 0.98 1.08 1.32

*Number of animals with implants/number of animals sacri®ced.**[(CL-IMP)/CL� 100] where CL = corpora lutea in all survivingfemales and IMP the number of implantation sites on stained uteri in surviving nidating females.***[(IMP-VF)/IMP�10] whereIMP = number of stained implantations in pregnant females and VF = number of viable foetuses.

G. A. Burdock and W. G. Flamm252

were seen between the mean growth rate of the var-

ious groups (Table 17). The growth of the sucrose

and PD treated groups was slightly delayed during

the gestation period, with the exception of the 1 g/

day PD littering females. Not all females were

copulated in all groups; the lowest copulation rate

was seen in high-dose PD-treated animals, but rates

of low- and mid-dose PD-treated animals and the

sucrose-treated group were higher than the control.

Pregnancy rates were similar between groups.

For those females sacri®ced on day 13 post-coitus

(Table 18), there were no statistically signi®cant

di�erences between groups in number of corpora

lutea, implantation e�ciency, number of implants,

embryomortality rate or mean number of viable

foetuses in a litter.

For females brought to term (Table 19), the

mean length of gestation was similar between

groups and there was no statistically signi®cant

di�erence between groups in litter size at birth, live

births or number of live pups at birth. Further,

there was no treatment-related mortality of the

pups during the lactation period. Pups of the trea-

ted groups (sucrose and PD) were, in general,

slightly heavier than controls.

No treatment-related lesion or malformation was

observed (Table 20). The few cases of abnormalities

occurred at such low rates that the investigators

considered them spontaneous. The kinky tail syn-

drome is known as a cyclic congenital malformation

of CD rats (Palmer, 1972).

Table 22. Segment II study in ratsÐfoetal examinations

Parameter Controls Sucrose (4 g/day) PD (1 g/day) PD (2 g/day) PD (4 g/day)

External abnormalities1 48/185 61/172 25/168 43/202 45/204(25.9%) (35.4%) (14.8%) (21.2%) (22.0%)

Subcutaneous haematoceles 48 57 25 42 43(25.9%) (33.1%) (14.8%) (20.7%) (21.0%)

Coelsomia + club feet 0 0 0 0 0(0.98%)

Protrusion of the tongue, kinky tail4 0 4 0 0 0(2.32%)

Subcutaneous oedema5 0 0 0 1 0(0.49%)

Internal abnormalitites1 2/92 7/87 2/84 12/100 8/103(2.17%) (8.04%) (2.38%) (12.0%) (7.76%)

Dilatation of kidney pelvis and/or ureter 1 3 1 5 4(1.09%) (3.45%) (1.19%) (5.0%) (3.88%)

Intrathoracic haemorrhage 1 1 0 5 4(1.09%) (1.15%) (5.0%) (3.88%)

Ectopic kidney with rudimentary ovary 0 0 1 0 0(1.19%)

Stomachal haemorrhage 0 0 0 1 0(1.0%)

Multiple neural and cardiac defects2 0 0 0 1 0(1.0%)

Absence of kidneys or aplastic kidneys3 0 3 0 0 0(3.45%)

Skeletal abnormalitiesVertebral bodies 5/95 (5.4%) 6/85 (7.0%) 2/84 (2.4%) 8/102 (7.8%) 3/101 (3.0%)

1/93 0/85 1/84 8/1026 2/101Delayed ossi®cation (1.1%) (0.0%) (1.2%) (7.8%) (2.0%)

1Total abnormalities/total foetuses examined.2Associated with agnathia, absence of palate, spina bi®da.3The three foetuses presented also a kinky tail and a protrusion of the tongue and ablepharia.4Associated with ablepharia in three cases.5Associated with spina bi®da and agnathia.6In the same litter.

Table 23. Segment III study in ratsÐmaternal data

Parameter Controls Sucrose (4 g/day) PD (1 g/day) PD (2 g/day) PD (4 g/day)

Theoretical 17/20 17/20 19/20 19/20 17/20nidation rate1 (85.0%) (85.0%) (95.0%) (95.0% (85.0%)

Nidation rate2 17/20 17/20 18/19 18/19 17/20(85.0%) (85.0%) (95.0%) (95.0%) (85.0%)

Parturition rate3 17/17 17/17 17/18 17/18 16/17(100%) (100%) (94%) (94%) (94%)

Length of 21.5 21.3 20.8 21.2 21.2gestation days (range) (20±22) (20±23) (20±22) (20±23) (20±22)

1Number of animals with implants/all mated animals.2Number of animals with implants/number of sacri®ced animals. 3Number of ani-mals delivering/number of pregnant animals.

Safety of polydextrose 253

Segment II study in rats (pregnancy and foetal

development). No adverse symptoms or abnormal

behaviour was noted. One animal (1 g/day PD) died

as the result of a gavage accident on day 8 pc; there

were no implantation sites at autopsy. Mean growth

curves of the groups appeared normal and all other

groups being equal, the sucrose group demonstrated

a slightly smaller body weight during the last third

of pregnancy.

The nidation rate is the number of females show-

ing implantation sites over the number of females

sacri®ced on day 20 pc. Aborting females were

included in the calculation for the nidation rate

when they survived until day 20 pc (Table 21). The

pregnancy rate is the ratio of the females with at

least one viable foetus, over the number of females

sacri®ced on day 20 pc. In this study, because there

were no abortions or deaths of the whole of any lit-

ter, nidation and pregnancy rates were the same.

The non-pregnant females presented no corpora

lutea. The low fertility rate observed in the sucrose

group was a consequence of the randomization and

was not treatment related. There were no signi®cant

di�erences between groups in pre-implantation

losses, number of corpora lutea or implantation

sites. The authors did not comment on any di�er-

ences between PD- or sucrose-treated and control

animals in foetal weight and sex ratio.

External abnormalities (Table 22) included a

number of haematoceles, but for those observed in

the sucrose group, the values were below that

usually seen by the investigators (30.1%). Other

external anomalies observed are listed in the table.

Internal abnormalities (Table 22) were noted

among the 466 foetuses so examined and are listed

in the table. Some of the abnormalities such as dila-

tation of the kidney pelvis or ureters are considered

as common variants when they appear at a low

rate. In the CD strains of rats, the incidence in

45,000 foetuses lies between 6 and 7% (Palmer,

1972). The sample applies to minor anomalies such

as internal haemorrhage, but it is doubtful whether

these lesions can be considered as congenital

defects. Another minor abnormality was noticed in

the 1 g PD/day group: ectopic kidneys with rudi-

mentary ovaries in a small foetus. The same foetus

presented a slight dilatation of cerebral ventricles.

All these observations were described as a delay in

development.

The common skeletal variants included unossi-

®ed, reduced or bipartite sternebrae, and supernu-

merary ribs, all of which were observed at similar

rates in control and treated groups. Some foetuses

presented one or several bipartite or unossi®ed ver-

tebral bodies (8th to 12th thoracic vertebra in most

cases). This indicates a slight delay in ossi®cation,

as well as the rudimentary cranial ossi®cation

noticed in some foetuses.

Segment III study in rats (study of the action of

PD on the perinatal and postnatal development of the

rat). No substance-related adverse symptoms or

abnormal behaviour was observed among the

Table 24. Segment III study in ratsÐpostnatal development

Parameter Controls Sucrose (4 g/day) PD (1 g/day) PD (2 g/day) PD (4 g/day)

No. of viable pupsAt birth 10.623.22 9.023.00* 11.622.32 9.523.61* 12.522.20At 4 days 10.323.39 8.723.09* 11.422.29 9.423.70* 12.522.17At 21 days 10.123.56 8.723.09* 11.222.28 9.223.83* 12.522.17

Viability of pupsLive birth index1 181/182 153/167 197/197 161/161 188/192

(99.45%) (91.62%) (100%) (100%) (97.92%)4 day survival index2 175/181 148/53 194/197 159/161 187/188

(96.69%) (96.73%) (98.48%) (98.76%) (99.47%)Lactation index3 171/175 148/148 194/194 157/159 187/187

(97.71%) (100%) (100%) (98.74%) (100%)21 day survival index4 171/181 148/153 191/197 157/161 187/188

(94.47%) (96.73%)* (96.95%)* (97.51%)* (99.47%)*Groups of pups (��2SD)Day 1Males 7.520.86 6.821.07 6.720.91 7.121.16 6.820.59Females 7.120.82 6.921.25 6.420.90 6.620.69 6.620.61

Day 4Males 11.221.62 10.421.46 10.421.17 10.722.15 9.920.82Females 10.521.41 10.822.07 9.821.13 9.821.19 9.420.76

Day 21Males 50.428.06 49.326.50 46.227.27 48.329.18 42.924.23Females 47.827.20 48.924.89 42.328.40 43.528.28 41.1024.44

Growth rate5

Males 6.72 7.25 6.90 6.80 6.30Females 6.73 7.09 6.61 6.59 6.21

*Statistically di�erent from control (P < 0.05).1(Number pups born alive/total number pups born)� 100.2(Number of pups alive at day 4/number of pups born alive)�100.3(Number of pups alive at weaning time/number of pups alive at day 4)�100.4(Number of pups alive at day 21/number of pups born alive)� 100.5Mean growth rate of pups between day 1 and 21 (in g per g body weight at day 1).

G. A. Burdock and W. G. Flamm254

mothers during the treatment period. Body weight

gain during pregnancy appeared to be homogenous.

Some variations observed between the groups were

not compound related as most of them occurred

before treatment had started on day 15 pc. Two

deaths occurred during the course of the study, one

each in 1 and 2 g/day PD, and both due to gavage

accidents.

Maternal data are presented in Table 23. When

dead females were excluded from the calculations,

the nidation rate was similar to the theoretical nida-

tion rate, the values of both of which were satisfac-

tory. With regards to the fertility, the batch of

females was considered as homogenous, since only

small variations were seen between the groups.

Most of the surviving nidating females maintained

their pregnancy until parturition. The nidating

females, which failed to litter normally, presented

only a trace of implantation on the day of sacri®ce

(day 25 pc). This indicated a very early death of

implants at the beginning of implantation or an

early abortion around days 10±12 pc. Both

phenomena occurred before the polydextrose ad-

ministration had started. The length of pregnancy

was similar in the sucrose and polydextrose groups

and appeared slightly shorter than that of control.

Postnatal development data are presented in

Table 24. The mean number of pups in various groups

at three periods of time (1, 4 and 21 days after birth) is

presented below. The signi®cantly di�erent lower

values observed in the sucrose and PD 2 g/day treated

groups were due to low individual values of some

females in these groups; however, the authors con-

cluded that these di�erences represented only biologi-

cal variation and were not treatment related. The

lower value of the live birth index seen in the sucrose

group was due to 14 stillbirths, eight of them within

the same litter. Other values were satisfactory and

indicated a very low mortality. The perinatal mor-

tality (4-day survival index) was low in the polydex-

trose treated groups, but still greater than in the

sucrose and control groups, although viability in all

groups during the ®rst 4 days of life remained satisfac-

tory. The lactation viability was satisfactory in all

groups, indicating a good lactation in all the females

and no adverse e�ect of sucrose or PD on the suckling

pups. In the 21-day survival index, there was a better

viability in the polydextrose and sucrose groups com-

pared with controls. The survival rate increased in

correlation with the concentration of PD. The mean

growth rate for both sexes in the sucrose group was

better than in the control and PD groups. The smallest

growth rate was recorded in the PD 4 g/day group.

The growth rate was slightly higher for the males and

slightly lower for the females of the PD 1 and 2 g/day

groups when compared with the controls. Postnatal

development of the pups measured by the appearance

of some re¯exes or anatomical modi®cations was sat-

isfactory (data not shown). No di�erence was seen in

the date of appearance of the earliest re¯exes (righting

re¯ex and pinching re¯ex). A slight delay (about 12

hours) in the appearance of other criteria was

observed in the PD-treated groups when compared

with the control and sucrose groups.

None of the abnormalities or lesions recorded

(Table 25) was considered as treatment related.

Hydrocephaly, as well as the associated malfor-

mations: protruded tongue, kinky tail, agenesis of

the kidneys, is known to be a congenital cyclic

anomaly in the Charles River strain of rat.

Three-generation study in rats with PD-A. The gen-

eral health of the rats was not a�ected by the adminis-

tration of PD or sucrose. Only two treatment-related

observations were noted: (1) most of the treated ani-

mals, in all generations, had soft and dark faeces after

having eaten PD or sucrose for a few days, although

diarrhoea was not observed; (2) traces of blood on the

face and forepaws, without chromodacryorrhoea,

were seen in 60% of the sucrose-treated females of the

F0 generation during the second week of gestation

(days 73 to 79 of treatment). This latter observation

was transitory, and was never observed later in the in-

itial or subsequent generations.

Two deaths occurred during the study, both in the

F0 generation, one in the sucrose group and one in

Table 25. Segment III study in ratsÐabnormalities and pathology of the pups

Interval Observations

Pups dead in the course of the trial Controls: one stillbirth and two dead pups were examined. No internal abnormality wasfound at autopsy.Sucrose 4 g/day: One of the stillbirths presented the following associated malformations:subcutaneous thoracic oedema, hare lip and cleft palate, left diaphragmatic hernia, agensia ofleft kidney, atresia of right kidney and ectopic left kidney. One dead pup in another litter, butno speci®c macroscopic lesion was noted.PD 1 g/day: one dead pup presented a slight hydronephrosis.PD 2 g/day: no speci®c lesion was seen in a dead pup.PD 4 g/day: Three stillbirths were examined. Two of them presented with protruded tongue,ablepharia of the left eye, twisted tail, syndactylia, diaphragmatic hernia, agenesia of kidneys.

Pups sacri®ced at weaning time Five pups per sex and group were sacri®ced at weaning time. No lesion was seen at autopsyexcept for once case (PD 1 g/day) of hydrocephaly in one male associated with ectopic lefttestis.

Pathology PD 1 g/day: two cases of hydrocephaly.PD 2 g/day: One case of cataract was seen in the right eye of a pup.No pathological lesion was recorded in the control, sucrose or PD 4 g/day groups.

Safety of polydextrose 255

the 10% PD group. No macroscopic lesions were

noted and the litters of both animals were euthanized.

In general, growth for each sex and generation

was similar and exhibited a familiar pattern com-

mon to all reproduction studies with cycles of

breeding, etc. Within each generation, di�erences

were recorded between the groups. In the F0 gener-

ation, the high-dose PD and sucrose animals were

slightly but not signi®cantly lighter than the con-

trols. There was no di�erence between female

groups of the F0 generation. In the F1 generation,

both sexes exhibited slight variations between the

groups, similar to those described for the F0 gener-

ations. When compared at the mean age of 93 days,

the di�erences between the mean body weights of

the groups within the same sex were not statistically

signi®cant. In the F2 generation at 100 days, no sig-

ni®cant di�erence between the mean body weights

of either males or females from the four groups was

seen. Male rats of the 5% and 10% PD groups

weighed slightly less than those in control and

sucrose groups. The growth rate of females was

similar in all the groups.

In food consumption for the F0 generations, the

male food intake was slightly higher in treated

groups than controls. For females of the F0 gener-

ation, intake was similar in all the groups, between

the 4th and 7th weeks of treatment. A reduced food

intake was observed in the 10% PD group, which

the investigators could not explain. In the F1 gener-

ation, food intake was similar between groups,

although males from treated groups showed higher

food consumption than controls. Female PD trea-

ted animals tended to eat slightly more than con-

trols, while the sucrose group ate slightly less than

controls.

Copulation rate and mating behaviour appeared

to be normal. All the inseminated (e�ective copu-

lation) females were pregnant except for a few in

the F0 generation. The values of the mating index

in this generation were controls, 85%; sucrose,

100%; 5% PD, 85%; 10% PD, 95%. These vari-

ations were not treatment related. Mating indices

for the F1 and F2 generations were 100%. The ferti-

lity index and gestation index were una�ected by

dose (Table 26). The length of gestation was not

Table 26. Three generation study in ratsÐmaternal data

Parameter ControlsSucrose(10%)

PD(5%)

PD(10%)

Fertility index as a %F0 85% 100% 90% 95%F1 100% 100% 100% 100%F2 90% 75% 100% 95%

Length of gestation(days)F0 21.3 21.2 21.8 21.7

(19±23) (19±23) (21±34) (21±23)F1 21.4 21.5 21.8 21.4

(21±22) (21±22) (21±22) (21±23)F2 21.5 21.5 21.8 21.6

(21±22) (21±23) (21±23) (21±22)

Table 27. Three-generation study in ratsÐo�spring data

Parameter Controls Sucrose (10%) PD (5%) PD (10%)

Mean number of live pups per litterAt birthF1 12.9 13.2 12.3 13.0F2 12.9 13.8 12.9 13.8F3 12.9 12.9 11.4 13.7

Day 4 post partum1

F1 9.6 9.6 10.0 10.0F2 9.7 9.8 9.9 9.9F3 9.9 9.9 9.7 9.9

Day 21 post partum1

F1 9.1 9.5 9.9 10.0F2 8.7 9.6 9.0 9.7F3 9.1 9.8 9.4 9.7

Number and (%) of stillbirths per dose levelF1 3/222 9/272 3/225 0/247

(1.4%) (3.3%) (1.3%) (0%)F2 6/264 3/279 6/264 5/281

(2.3%) (1.1%) (2.3%) (1.8%)F3 5/237 6/200 7/236 2/262

(2.1%) (3.0%) (3.0%) (0.8%)4-Day survival index

F1 96.43% 95.94% 100.00% 100.00%F2 100.00% 100.00% 99.49% 99.50%F3 98.98% 97.18% 97.28% 98.95%

Lactation indexF1 94.44% 96.74% 99.42% 100.00%F2 89.74% 97.46% 92.31% 97.99%F3 92.13% 100.00% 97.76% 98.40%

21-Day survival indexF1 91.07% 95.19% 99.42% 100.00%F2 89.74% 97.46% 91.84% 97.46%F3 91.11% 97.18% 95.11% 97.18%

1After litters were culled to 10 pups/litter on day 1 after birth.

G. A. Burdock and W. G. Flamm256

modi®ed by treatment in any generation and was

consistent with historical values.

Extreme individual gestation values (19 and 24

days) were recorded in two females. As the mean

weight of their o�spring at birth was similar to that

of the other litters, it was thought that the observer

had missed the real time of fertilization. The par-

turitions observed were normal.

The number of live pups per litter at birth was

not statistically di�erent between the various dose

levels (Table 27). Within the same dose level,

the mean number of pups born alive was not

Table 28. Three-generation study in ratsÐo�spring data (contd)

Parameter Controls Sucrose (10%) PD (5%) PD (10%)

Sex ratios at weaningF1 1.04 0.98 1.02 1.02F2 1.08 1.13 1.04 1.14F3 1.08 0.97 1.08 1.26

Mean body weight of male pups (g)F1

Day 1 6.8 6.6 7.1 7.2Day 4 10.1 9.9 10.7 11.3Day 21 46.2 44.4 47.0 46.7

F2

Day 1 6.5 6.6 6.9 6.6Day 4 10.1 10.3 10.8 10.3Day 21 39.2 39.6 41.4 36.2

F3

Day 1 6.7 6.5 6.9 6.7Day 4 10.2 10.5 10.7 10.9Day 21 45.6 42.4 45.6 44.3

Mean body weight of female pups (g)F1

Day 1 6.6 6.4 6.7 6.8Day 4 10.0 9.4 10.2 10.8Day 21 45.9 43.3 44.8 44.8

F2

Day 1 6.2 6.1 6.6 6.4Day 4 9.8 9.9 10.5 10.0Day 21 37.8 38.7 39.9 36.0

F3

Day 1 6.3 6.0 6.7 6.4Day 4 9.8 10.0 10.3 10.4Day 21 44.6 41.5 44.0 43.1

Table 29. Segment II study in rabbitsÐmaternal and foetal data

Parameter Controls Sucrose (12 g/day) PD (3 g/day) PD (6 g/day) PD 12 g/day)

Mortality 3/15 1/15 4/15 0/15 1/15(20.0%) (6.66%) (26.66%) (0%) (6.66%)

Nidation rate* 6/12 12/14 10/11 12/15 11/14(50%) (85.7%) (90.9%) (80.0%) (78.6%)

Pregnancy rate 4/15 10/15 9/15 11/15 10/15(26.66%) (66.66%) (60.0%) (73.33%) (66.66%)

Corpora lutea/pregnancy 12.523.70 15.323.02 13.222.54 11.821.60 12.122.02(n = 50) (n = 153) (n = 119) (n = 130) (n = 121)

(��2SD) (n = total)Implantation site/pregnancy 8.722.06 8.324.00 7.822.54 7.722.93 8.823.05

(��2SD) (n= total) (n = 35) (n = 83) (n = 70) (n = 85) (n = 88)Pre-implantation loss rate** 15/50 70/153 49/119 45/130 33/121

(30.00%) (45.75%) (41.17%) (34.16%) (27.27%)Embryomortality***IMP-VF/IMP 2/35 10/83 0/70 4/85 7/88

(5.7%) (12.0%) (4.7%) (7.9%)VF/litter (��2SD) 8.321.50 7.323.89 7.822.54 7.423.11 8.122.81

Foetuses and annexesFoetus weight 32.424.38 27.126.04 33.524.92 30.726.26 33.325.31(��2SD) (n) (n = 33) (n = 73) (n = 70) (n = 81) (n = 81)Placental weight 6.321.06 5.421.26 6.821.20 6.021.59 6.721.17(��2SD)Amniotic ¯uid 2.020.89 2.020.99 2.521.47 2.221.88 2.321.46(��2SD)

*Number of animals with implants/number of animals sacri®ced. **[(CL-IMP)/CL� 100] where CL = corpora lutea in all survivingfemales and IMP the number of implantation sites on stained uteri in surviving nidating females.***[(IMP-VF)/IMP�100} whereIMP = number of stained implantations in pregnant females and VF = number of viable foetuses.

Safety of polydextrose 257

signi®cantly modi®ed along the successive

generations.

For each generation and dose level, the rate of

observed stillbirths was low and consistent with his-torical data at this laboratory. When compared

with a Wilcoxon rank sum test, the values of this

trial, within each generation, did not di�er signi®-

cantly.

There was no mortality in the F1 generation.

Only a few deaths occurred in the F2 and F3 gener-ations. The perinatal mortality (4-day survival

index) was very low in all the groups and gener-

ations. There was no signi®cant di�erence betweenthe values of the index for the various doses of the

same generation or between the generations within

the same dose.

There was no signi®cant di�erence between dose

levels and generations in the lactation index, when

estimated with the Wilcoxon rank sum test. Thevalues were slightly better in the treated groups,

particularly the 10% PD and sucrose groups, than

in the controls for the three generations.

The 21-day survival index values resulted from

data previously analysed under ``4-day survivalindex'' and ``Lactation index.`` The mortality in

the di�erent groups and generations was low (0±

10.3%) between days 1 and 21 after birth. There

were more surviving pups in the treated groups

than in the controls, but the di�erences between

dose levels and generations were not statisticallysigni®cant.

After equalization of litters to 10 on day 1 afterbirth, the sex ratios in the various groups did not

equal 1.00, but were relatively close to this value.

The sex ratios at weaning showed that there was no

sex-related mortality. The observed variationsbetween dose levels and generations were the conse-

quence of the initial di�erences on day 1 after birth

(Table 28).

On day 1 after birth and before culling of litters

to 10, the mean body weights of male and female

pups from PD treated groups were slightly higherthan that of controls in any generation. Sucrose-

treated pups of both sexes weighed slightly less than

controls (except for males of F2 o�spring). The

values recorded were consistent with the historicaldata of the performing laboratory. The di�erences

between the groups still existed on day 4 after birth

in all generations, except for the sucrose group

males (F2 and F3 generations) and females (F3 gen-eration) which were slightly heavier than controls.

At weaning, the mean body weights of male and

female pups were similar to those of controls except

for the 10% PD group of the F2 generations (the

Table 31. Quantitative plate assay of PD-A and PD-N in Salmonella typhimurium

Salmonella typhimurium strains

Average number of revertant colonies per plate

Substance mg/plate TA1535 TA1536 TA1537 TA1538 C340

None ± 21 1 14 32 12EMS 1.0 33 1 6 40 329-Aminoacridine 0.1 ± 20 >500 ± ±Sodium nitrite 1.0 >500 2 13 33 12Atabrine 1.0 20 >1000 >1000 153 7Hycanthone 0.5 Inhib. 6 Inhib. >1000 11PD-A 10 24 1 13 28 10PD-N 10 25 Contam. 17 42 15

Table 30. Segment II study in rabbitsÐfoetal examinations

Parameter Controls Sucrose (12 g/day) PD (3 g/day) PD (6 g/day) PD (12 g/day)

External abnormalities1

Subcutaneous 2.7% 3.7% 1.2%haematoceles

Internal abnormalities1 0/17 1/36 0/35 6/41 2/39(2.77%) (14.63%) (5.13%)

Dilatation of cerebral 1/41ventricles (2.44%)Dilatation of kidney 2/41pelvis (4.88%)Spina bi®da with other 1/41abnormalities (2.44%)Ectopic left kidney 1/41

1 (2.78%) (2.44%)Slight atrophic liver with 2/39hypertrophic gall bladder (5.13%)Cyst near the left 1/41epididymis (2.44%)

1Total abnormalities/total foetuses examined.

G. A. Burdock and W. G. Flamm258

lower mean was due to a very small growth rate of

three litters in this group which might have been

due to hypogalactia). Growth was slightly better in

the 5% PD group than in the 10% PD in relation

to litter size at weaning, which was larger in the

10% PD group due to a better survival rate.

Growth of pups in the sucrose group was slightly

delayed v. controls.

Examination of stillborn animals and autopsies

of pups dying during lactation and autopsies of

weanlings did not indicate any treatment-related

condition. All changes appeared incidental. No

treatment-related lesions were revealed as the result

of ophthalmological examinations. Lesions

appeared at similar rates among dose groups and

were of congenital origin or residues of embryonic

structures.

Segment II study in rabbits (pregnancy and foetal

development). Among the 66 surviving females, six

cases of diarrhoea were observed: one each in the

control and high-dose PD group and two each in

the sucrose and mid-dose PD group. One of the

same animals in the sucrose and high-dose PD

group also aborted. In those animals in which diar-

rhoea appeared after a period of constipation, it

was of brief duration and not treatment related.

This was not wholly unexpected because of the deli-

cate gastrointestinal physiology of the rabbit. They

commonly react to stress by diarrhoea, anorexia,

etc. An increase of water consumption was noted,

but not measured quantitatively in the PD-treated

groups during and after the treatment period.

There were nine deaths during the course of the

study (Table 29), which were due to latent infection

aggravated by stress. There were no statistical

di�erences in the growth of the pregnant females,

due to the large degree of variability among ani-

mals. A lower weight gain was noted in the sucrose

group between day 0 and day 28 due to a growth-

depressant e�ect on foetal development. A slight

decrease observed in all the groups on the last day

of gestation indicated a daily variation in relation

to food intake.

The nidation rate is the number of females show-

ing implantation sites over the number of females

sacri®ced on day 28. Aborting females, when they

survived until day 28, were included in the calcu-

lation for the nidation rate. The pregnancy rate is

the ratio of the females with at least one viable foe-

tus over the number of females sacri®ced on day

28.

The low values for embryomortality rate recorded

in this study did not di�er signi®cantly. There was

no treatment-related di�erence between control and

test groups in the number of viable foetuses.

Among foetal and placental weights, a statistically

signi®cant di�erence was noted between sucrose-

treated animals and control. No di�erence was

noted in amniotic ¯uid weights. It is likely that

sucrose delayed foetal and annexial development, as

previously noted in the literature (Bender and

Damji, 1971).

In describing external abnormalities (Table 30),

the investigators noted the percentages of haemato-

celes, but the e�ect was not dose responsive. They

also noted that 2/8 foetuses in a single group (6 g/

PD/day) presented with severe abnormalities, for

example, one foetus with spina bi®da, club feet and

atrophic tail; and another with similar symptoms

and the absence of a tail.

Internal abnormalities consisted of one foetus

(6 PD g/day) with spina bi®da, absence of tail,

hypertrophic cerebellum with lengthening of the

brain, ectopic left kidney and dilation of the kidney

pelvis. No other cases of a major abnormality was

found in any of the other groups.

In the sucrose group, skeletal abnormalities con-

sisted of delayed sternebral ossi®cation and were re-

lated to the retarded body development of foetuses

in this group. Only one case of major skeletal

abnormality was found in the PD groups (6 g/day)

Table 32. Quantitative plate assay of PD-N in Salmonella typhi-murium strain TA1536

Salmonella typhimurium

Substance mg/plate

Average numberof revertant colonies

per plate

None ± 69-Aminoacridine 0.25 15Hycanthone 0.05 10PD-N 20.0 5

Table 33. Host-mediated assays of PD-A and PD-N

Salmonella typhimurium strains

Mutation frequency (revetants/108 cells) for each run?

Substance mg/kg G46 C207 C3076 C340 TA1534

Saline ± 0.8, 0.7, 1.6 1.3, 0.8 1.3, 2.0 4.0, 5.5 0.2, 0.2, 0.2PD-A 2000 3.5, 0.9, 0.3 0.4, 0.8 1.1, 1.4 0.7, 1.3 NA, 0.2, 0.1PD-N 2000 0.4, 0.5, 0.2 0.8, 0.8 1.9, 1.6 1.0, 2.7 1.2, 0.2, NADMNA 100 59.4, 43.0 8.4, 10.86-MP 28 34.54-NQO 300 33.1, 31.9,2.7

NA = not applicable, no run in this series.

Safety of polydextrose 259

Table 35. Cytogenetic analyses in vivoÐPD-N mouse cytogenetic assay

No. of cellsanalysed

Chromatidbreaks

Chromosomebreaks

Rearrangements Abnormalcells (%)

Control6 hr 50 0 0 0 0.012 hr 50 2 0 0 4.024 hr 50 0 1 0 2.048 hr 50 0 0 0 0.072 hr 50 0 0 0 0.0Subacute 50 2 0 0 4.0Pooled 300 4 1 0 1.71

PD-N (2 g/kg)6 hr 2502 4 1 0 2.012 hr 250 0 1 0 0.424 hr 250 2 0 0 0.848 hr 250 5 2 1 2.472 hr 250 1 0 0 0.4PD-N 1 g/kg/day�7 d (Subacute) 250 3 1 0 1.2

1The frequency of abnormal cells in the pooled experimental controls is not signi®cantly di�erent at (P < 0.01) from the historical controlupper bound of 2.01% abnormal cells in a population of 28,500 controls cells. Against this historical control, a level of 4.4% abnor-mal cells in a sample population of 25 cells is required to achieve signi®cance at P < 0.01. The results in the treated cultures aretherefore not statistically signi®cant.2All PD values are pooled cells for n = 5.

Table 36. Cytogenetic analyses in vitroÐe�ect of polydextrose on human lymphocyte chromosomes in vitro. Compound added to culturemedium 24 hr prior to harvest

No. of cellsanalysed

Chromatidbreaks

Chromosomebreaks Rearrangements

Abnormalcells (%)

Control50 0 1 0 2.050 1 0 0 2.050 2 0 0 4.0

Pooled 150 3 1 0 2.61

PD-A500 mg/ml 50 0 0 0 0.01000 mg/ml 50 2 0 0 4.01000 mg/ml 50 2 0 0 4.01000 mg/ml 50 2 0 0 4.0(Pooled)

PD-N500 mg/ml 50 0 0 0 0.01000 mg/ml 100 6 0 0 6.0

1The frequency of abnormal cells in the pooled experimental controls is not signi®cantly di�erent at (P < 0.01) from the historical controlupper bound of 2.09% abnormal cells in a population of 4700 control cells. Against this historical control, a level of 10% abnormalcells in a sample population of 50 cells (or 7% in 100 cells) is required to achieve signi®cance at P < 0.01. The results in the treatedcultures are therefore not statistically signi®cant.

Table 34. Cytogenetic analyses in vivoÐPD-A mouse cytogenetic assay

No. of cellsanalysed

Chromatidbreaks

Chromosomebreaks

Rearrangements Abnormalcells (%)

Control6 hr 50 0 0 0 0.012 hr 50 0 0 0 0.024 hr 50 1 0 0 2.048 hr 50 1 0 0 2.072 hr 50 0 1 0 2.0Subacute 50 0 0 0 0.0Pooled 300 2 1 0 1.01

PD-A (2 g/kg)6 hr 2502 2 1 0 1.212 hr 250 1 2 1 1.224 hr 250 2 0 0 0.848 hr 250 6 1 1 2.872 hr 250 7 3 0 3.2PD-A 1 g/kg/day� 7 d (Subacute) 250 3 1 0 1.2

1The frequency of abnormal cells in the pooled experimental controls is not signi®cantly di�erent at (P < 0.01) from the historical controlupper bound of 2.01% abnormal cells in a population of 28,500 controls cells. Against this historical control, a level of 4.4% abnor-mal cells in a sample population of 25 cells is required to achieve signi®cance at P < 0.01. The results in the treated groups are there-fore not statistically signi®cant.

2All PD values are pooled cells for n = 5.

G. A. Burdock and W. G. Flamm260

with one foetus (already described under ``externalabnormalities'') presenting a spina bi®da with no

squama occipitalis, no ossi®cation of the basis ofthe interparietal bones and others.

Genotoxicity studies

Spot test using Salmonella typhimurium. Crystals

of PD-A and PD-N were placed on the lawns ofthe following strains of S. typhimurium strainsTA1535, TA1536, TA1537, TA1538 and TA1978.

At least two separate tests were made with eachstrain. No mutagenic activity was seen with anystrain.Quantitative plate assay using S. typhimurium. As

can be seen from Table 31, neither PD-A nor PD-Nat 10 mg/plate produced signi®cant increases in thenumber of revertant colonies per plate. The positive

controls reacted in the manner expected.When PD-N was assayed at double the concen-

tration (20 mg/plate) using TA1536, there were no

increases above the spontaneous rate of revertantcolonies per plate (Table 32). The positive controlsresponded in a matter identical with the ®rst assay.Host-mediated assay with PD. As can be seen in

Table 33, PD-A produced no signi®cant increase inmutation frequency except for the ®rst assay withG46. Two succeeding assays with G46 failed to

show this activity and for this reason, the investi-gators considered this initial assay to be spurious.PD-N did not produce a signi®cant increase in mu-

tation frequency except for the ®rst assay with TA1534. A second assay failed to repeat this result.Dimethylnitrosamine (DMNA), 6-mercaptopurine

monohydrate (6-MP) and 4-nitroquinoline-N-oxide(4NQO) were included as positive controls whereappropriate and performed as expected.Cytogenetic analyses. The results of the in vivo

studies are shown in Tables 34 and 35. In all of thetreatment regimens, there was no indication of com-pound induced chromosome breakage over that

observed in the experimental controls. In addition,statistical comparison of experimental controls ortreated groups with an historical control for this

laboratory and strain of mouse was uniformly nega-tive.Polydextrose also did not produce any evidence

of genetic toxicity in the in vitro assessments

(Table 36). Chromosome damage in the treated cul-tures was not statistically elevated over controls.These studies indicate that polydextrose does not

cause chromosome damage either in vivo in mice orin vitro in human lymphocyte cultures at the dosestested.

Dominant lethal assay in mice. As shown inTable 37, polydextrose did not produce evidence ofdominant lethality at a level of 1 g/kg. The number

of dead implants/pregnant females was not elevatedin a statistically signi®cant manner compared withcontrols in any of the 7-week mating periods of thestudy. The ethyl methanesulfonate positive control

Table

37.Dominantlethalassayin

mice

No.pregnant

Totalim

plants

Totalim

plants/

pregnantfemales

Deadim

plants/

pregnantfemales

Percentdead

implants

Liveim

plants/

pregnantfemales

Week

CT

CT

CT

CT

CT

CT

PD-N

10

00

00.00

0.00

0.00

0.00

0.00

0.00

±±

1g/kg�7days

238

36

475

471

12.50

13.08

1.00

0.64

8.00

4.88

11.50

12.44

339

43

519

575

13.31

13.37

1.18

0.93

8.86

6.96

12.13

12.44

439

40

503

520

12.90

13.00

1.26

0.88

9.74

6.73

11.64

12.12

538

35

502

413

13.21

11.80

1.11

0.83

8.37

7.02

12.11

10.97

637

35

472

430

12.76

12.29

0.86

0.89

6.78

7.21

11.89

11.40

733

33

443

449

13.42

13.61

0.91

1.03

6.77

7.57

12.52

12.58

837

28

498

363

13.46

13.00

0.76

0.82

5.62

6.59

12.70

12.14

EMS

10

00

00.00

0.00

0.00

0.00

0.00

0.00

±±

200mg/kg

238

0475

012.50

0.00

1.00

0.00

8.00

0.00

11.50

±3

39

24

519

233

13.31

9.71

1.18

4.17

**

8.86

42.92

12.13

5.54

439

35

503

429

12.90

12.26

1.26

1.60

*9.74

13.05

11.64

10.66

538

32

502

399

13.21

12.47

1.11

1.13

8.37

9.02

12.11

11.34

637

38

472

476

12.76

12.53

0.86

1.34

*6.78

10.71

11.89

11.18

733

29

443

377

13.42

13.00

0.91

0.55

6.77

4.24

12.52

12.45

837

33

498

433

13.46

13.12

0.76

0.70

5.62

5.31

12.70

12.42

*Signi®cance

level

0.05.*

*Signi®cance

level

0.01.

Safety of polydextrose 261

group produced a signi®cant increase in dominantlethality in week 3 as expected in this type of

dosage regimen. The elevation in week 6 was unex-pected and not typical of other experiments usingthis compound. A level of 200 mg/kg/day EMS for

7 days produced sterility in males during week 2 asevidenced by the lack of pregnant females.

Discussion

As may be seen by the data cited above, polydex-trose (Type A or Type N) has an extremely lowacute toxicity. The only adverse ®nding was diar-

rhoea, seen following administration of high doses.Since the time of these tests, the threshold for diar-rhoea in rats has been established at 60 g/kg of the

diet (Kanauchi et al., 1997).The low toxicity, coupled with a low digestibility

and fermentation products by caecal/colonic bac-

teria tending to produce a watery (osmotic) diar-rhoea, are characteristic of a number of modi®edstarches, sorbitol, xylitol and polydextrose and pro-duced a well-characterized pattern of pathological

sequelae as described by Newberne et al. (1988).This pattern is characterized as a watery diarrhoea(and electrolyte loss) with immediate renal sodium

sparing e�ort, which also concurrently and obliga-torily retains calcium. If ingestion of the substancescontinues, it results in a slowly progressing hyper-

calcaemia that produces a calcium nephropathy.This response to these carbohydrates is seen in ani-mals and in man (Newberne et al., 1988), but is to

be distinguished from ``milk-alkali syndrome'' inman, in which hypercalcaemia produced calciumnephropathy as the result of increased intestinalabsorption of calcium (Brunton, 1996). Increased

intestinal absorption of calcium does not occur inresponse to oral administration of polydextrose(P®zer, unpublished data). The calcium nephropa-

thy is described by Newberne et al. (1988) as fol-lows:``An intrarenal hydronephrosis and acute tubular

damage with varying degrees of mineralization. Thechronic lesion consists of wedge-shaped areas ofscarring, alternating with areas of normal renal par-enchyma. The apex of the wedge scar was in the

medulla just below the corticomedullary junction.Tubular dilatation was often present in the corticalportion of the lesion, but in¯ammatory response

was minimal. Moderate mineralization was mostprominent in the medulla''.Therefore, on the basis of what Newberne et al.

(1988) have described, there are speci®c thresholds(i.e. persistent diarrhoea and persistent hypercalcae-mia) to be surmounted prior to observation of

sequelae. For example, there was no diarrhoeanoted in the 3-month rat dietary study at a maxi-mum dose of 10% PD-A in the diet (approx. 10 g/kg body weight/day) and subsequently, a lack of

®ndings at the completion of the study. There was,

however, in the low- and high-dose (but not mid-dose) females, less weight gain than control females.The decreased weight gain by high-dose females

might be explained by caloric dilution of the diet,but this would not account for the decreased weightgain in the low-, but not mid-dose females or the

lack of departure from control values by the high-dose males, but a similar loss by low-dose males.

Therefore, the lack of diarrhoea, the describedweight changes and the lack of other changes inclinical chemistries (including serum calcium) and

lack of pathological change in the kidney, mandatea conclusion that the threshold for change was notmet in the rat.

The three-generation reproductive study alsore¯ects the lack of a toxicologically signi®cant e�ect

of polydextrose and although the rats were noted ashaving soft stools, frank diarrhoea was not seen.Further, despite the addition of polydextrose at as

much as 10% of the diet (approximately 10 g/kgbody weight/day) no treatment-related e�ects werenoted in maternal fertility and gestation parameters

in F0, F1 or F2 generations. Nor was there a treat-ment-related e�ect in the o�spring (F1, F2 or F3) in

number of pups per litter or stillbirths, survivalpost partum at 4 or 21 days, sex ratio or meanbody weights of pups. Further, the F1 generations

from this reproduction study was employed for usein the 24-month carcinogenicity study, producingan in utero exposure and, as described previously,

no e�ects were noted.Rats were also used in a Segment I study, where

unfortunately the animals were dosed with a ®xedamount per day (to theoretically equal the amountof compound they would have eaten in food per

day). Therefore, only a range of doses of polydex-trose can be estimated and for the high dose wouldhave ranged from 8.3 to 22.2 g/kg body weight/day

for males and 9.0 to 16.6 g/kg body weight/day forfemales. However, even at these exaggerated doses,there was no e�ect on copulation rate, pregnancy

rate or length of gestation or litter size in theparents. The o�spring fared equally well with no

di�erences from control in litter size, live birthindex, number of viable pups at birth or mortalitiesduring the lactation period, growth or sex ratio. In

addition, no treatment-related abnormalities couldbe found.

Segment II (teratology) studies were conducted inboth rats and rabbits. Again, no e�ects were notedin the performance or parameters assessed in the

parents or o�spring. Despite the fact the rabbits ex-perienced a brief bout of diarrhoea, it was insu�-cient to a�ect the outcome of the study.

Segment III studies, to test the perinatal andpostnatal development of the rat, were conducted at

the same dose levels. No treatment-related e�ectswere seen in maternal parameters or postnataldevelopment of the pups.

G. A. Burdock and W. G. Flamm262

This lack of threshold attainment is reinforced by

the 24-month study in rats, wherein again, only soft

stools and not diarrhoea was observed in the high-

dose animals (10% of the diet or approximately

10 g/kg PD). This study was uneventful and showed

no e�ect on body or growth rate, clinical chemistry,

haematology or gross or microscopic pathology

that could be attributed to administration of poly-

dextrose. There were no treatment-related lesions or

trends for incidence of a particular type of kidney

lesion pathognomonic of hypercalcaemic nephrosis.

Nor was there an indication of oncogenic e�ect

resulting from administration of polydextrose.

Therefore, on the basis of what has been dis-

cussed above, rats may repeatedly consume up to

10 g/kg body weight/day PD without untoward

e�ects. Sensitivity to the osmotic e�ects of PD var-

ies with species, however, as exempli®ed by the 3-

month gavage study in monkeys. In this study, the

monkeys were gavaged with PD-A at 1, 2 and 10 g/

kg body weight/day. Diarrhoea was observed in the

high-dose animals on a daily basis, and which only

rarely occurred, if at all, in the two lower PD doses

and not at all in the controls. However, in this

study the diarrhoea was apparently not severe

enough and/or of such a prolonged duration to

cause nephrotoxicity. There was, however, patho-

logical change in the colon indicative of persistent

diarrhoea and similar to the description of caecum/

colon sections in similarly treated animals described

by Newberne et al. (1988).

The most susceptible species to the diarrhoeal

e�ects of PD is the dog. In a 6-month study using

PD-N followed by PD-A at one dose level, the dogs

responded almost immediately on consumption of

PD-N, with watery diarrhoea, low urinary sodium

and hypercalcaemia, typical of the response

described by Newberne et al. (1988). Interestingly,

there was a post-prandial decrease in BUN and not

an increase, although this was probably not due to

renal impairment. Finco (1989) noted that BUN

may decrease with severe diarrhoea or vomiting.

This hypothesis is supported by the fact that BUN

was never decreased prior to a meal, but only after

meals. Following the switch from PD-N to PD-A,

many of the symptoms and their periodicity abated,

including a return to spikes of urinary sodium ex-

cretion. While many of the clinical parameters were

reversed on the switch to PD-A, the pathology of

calcium nephropathy remained. A similar pathology

(clinical and microscopic) was observed in the 13-

month dog study at the high dose (8 g/kg body

weight/day), with a possible no-adverse-e�ect level

at the low dose of 4 g/kg body weight/day.

The e�ect of chronic administration of PD-N in

dogs was clearly illustrated in the 24-month dog

studies with PD-N added to the diet at doses of 2.5

and 5 g/kg body weight/day. While the intensity

and frequency of diarrhoea reported in higher dose

studies (i.e. 50% of the diet) was not seen in these

animals even at the high dose, there was an oc-

casional incidence of elevated calcium, and to a les-

ser extent, BUN. However, even though the clinical

symptomology would not have seemed to have been

as clearly expressed in these animals as in the other,

higher dose studies in dogs, nephrocalcitosis was

apparent during histopathological examination.

These ®ndings also correlated well with the

increased levels of blood calcium. The ®nding of

polycythaemia in the sucrose females is likely to be

secondary to their obesity.

A review of the results of the genotoxicity studies

shows that polydextrose is not genotoxic or muta-

genic in vitro or in vivo.

This presentation of data represents the ®rst com-

prehensive publication of the animal safety data

supporting the use of polydextrose in food. The

conclusions relevant to this work are that omni-

vores such as rats, mice and monkeys are relatively

refractory to the e�ects of polydextrose with a no-

e�ect level for systemic toxicity at 2500 to

10,000 mg/kg body weight/day (Table 38). Further,

Table 38. Mammalian studiesÐe�ect levels

Species Route StudyLowest e�ect level

(mg/kg)Highest no-e�ect level

(mg/kg)

Monkey Gavage 3-Month gavage study in monkeys with PD-N 10,000Dog Diet 3-month feeding study in dogs with PD-A 10,0001

Dog Diet 6-Month feeding study in dogs with PD-N, followed by PD-A 10,0001

Dog Diet 13-Month feeding study with polydextrose in beagle dogs 3340Dog Diet 24-Month toxicity study in beagle dogs with PD-N 4000 2000Dog Diet 24-Month toxicity study in beagle dogs with PD-N 10,0001

Mouse Diet 18-Month carcinogenicity study in mice with PD-A 15,0002

Rat Diet 3-Month dietary study in rats with PD-A 10,000Rat Diet 24-Month carcinogenicity study in rats with PD-A 5000 2500Rat Diet Segment I study in rats 10,0002

Rat Gavage Segment II study in rats 10,0002

Rat Gavage Segment III study in rats 10,0002

Rat Diet Three generation study in rats 10,0002

Rabbit Gavage Segment II study in rabbits 3,0002

1Lowest e�ect level in this single dose study.2Highest level tested.

Safety of polydextrose 263

it is apparent from the data above that the dog isthe most sensitive species, because as a carnivore, it

is unable to tolerate large amounts of fermentablecarbohydrate. The presence of potassium in thePD-N enhances its pathological e�ect. However,

even this most sensitive species had a no-e�ect levelof 2000 mg/kg body weight/day.The relevance of these studies to the use of poly-

dextrose in food addresses the level at which thee�ect becomes apparent in humans. Several clinicalstudies suggest that a laxative e�ect (not diarrhoeal

e�ect) was found at an average of 90 g/day in adulthumans, compared with 70 g/day for sorbitol. Themaximum dose in children of 1 g/kg body weight/day only provoked increased ¯atus (Torres and

Thomas, 1981). Despite the lack of systemic toxicityand the amount required to cause a noticeable laxa-tive e�ect in humans, the FDA in its regulation (21

CFR 172.841), imposed a labelling requirement that`` . . .a single serving of which would be expected toexceed 15 grams of the additive [polydextrose] shall

bear the statement: Sensitive individuals may ex-perience a laxative e�ect from excessive consump-tion of this product''.

AcknowledgementsÐThe authors gratefully acknowledgethe work of N. J. Beutler, P. S. Bourdois, J. J. Burgun,J. F. Dooley, P. C. Estes, T. O. King, H. V. Levinsky,M. F. Pele, J. Perraud and H. Reinert of P®zer Inc. in car-rying out the studies herein, and the assistance of HerbertBlumenthal, Joseph Borzelleca and Michael Auerbach inreviewing and proofreading the manuscript.

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G. A. Burdock and W. G. Flamm264