neonatal animal testing paradigms and their suitability for testing infant formula

57

Introduction

Conventional wisdom is that the optimal diet, nutritionally and developmentally, for infants, is breast milk. However, various factors such as health, work demands, and other personal issues can make breast feeding impractical or impossible. Manufacturers of infant formulas understand this and are constantly formulating new ingredients for infant formulas in order to better mimic natural breast milk. If the mother’s milk is unavailable, infant formula will serve as the sole source of nutrition that the infant will receive for the first 6 months or so of life. The infant formula consumed must promote growth and normal development. Historically, adult animal studies for the most part have been used to determine the safety of new substances for infant formulas, mostly due to the dif-ficulty of orally dosing neonatal animals. The purpose of

this investigation is to determine if there are appropriate neonatal experimental models which could be used to either confirm or alleviate toxicological concerns pertain-ing to a new substance being added to an existing infant formula.

Methods

This investigation utilized three different areas or forms of media, which will be reported on separately prior to the conclusion. The first informational source is a publication from the IOM entitled: Infant Formula Evaluating the Safety of New Ingredients (2004). The second form of informational data utilized was simple YAHOO and Google Scholar searches on the internet (Key Words: Neonatal, Toxicology Studies, Infant Formula, Oral, Gavage, Autosow,

RevIew ARtIcle

Neonatal animal testing paradigms and their suitability for testing infant formula

Edwin G. Flamm

Division of Biotechnology and GRAS Notice Review, Office of Food Additive Safety, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, MD, USA

AbstractDue to the ever increasing number of substances added to infant formula, and the fact that the majority of data determining the safety of these substances has been derived from adult animals, a search of the available data was performed to determine if an appropriate neonatal model could be found that could be used for performing toxicological safety studies. This exercise utilized three different forms of media. The first informational source is from a publication from the Institute of Medicine (IOM) of the National Academies. The second form of informational data utilized was from simple YAHOO and Google Scholar searches on the internet. The third source of information was from the U.S. Food and Drug Administration (FDA), more specifically, the Center for Drug Evaluation and Review (CDER) preclinical guidance document. Following the examination of the above informational sources, it became apparent that neonatal rats and pigs have been the most utilized of the neonatal models. Following the evaluation of the papers, the experimental paradigm which appears to be the most appropriate for testing substances new to infant formula, and could be used as a pivotal study was the neonatal pig utilizing the automated feeding device called the Autosow.Keywords: Autosow, colostrum, gavage, “pup-in-a-cup”

The opinions expressed in this article are the authors’ personal opinions and do not necessarily reflect those of FDA, DHHS, or the Federal Government.

Address for Correspondence: Edwin G. Flamm, Division of Biotechnology and GRAS Notice Review, Office of Food Additive Safety, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 5100 Paint Branch Parkway, College Park, MD 20740, USA. Tel: 240-402-1285. E-mail: [email protected]

(Received 16 July 2012; revised 13 August 2012; accepted 24 August 2012)

Toxicology Mechanisms and Methods, 2013; 23(2): 57–67© 2013 Informa Healthcare USA, Inc.ISSN 1537-6516 print/ISSN 1537-6524 onlineDOI: 10.3109/15376516.2012.725108

Toxicology Mechanisms and Methods

23

2

57

67

16July2012

13August2012

24August2012

1537-6516

1537-6524

© 2013 Informa Healthcare USA, Inc.

10.3109/15376516.2012.725108

2013

Neonatal animal testing paradigms

E. G. Flamm

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Tec

hnis

che

Uni

vers

iteit

Ein

dhov

en o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.

58 E. G. Flamm


“Pup-in-a-Cup” and the particular animal species). The third source of information was from the FDA/CDER (2006) guidance document entitled: Guidance for Industry Nonclinical Safety Evaluation of Pediatric Drug Products.

Results and discussion

IOMChapter 5 of the IOM (2004) publication entitled: Testing Ingredients with Preclinical Studies was the most relevant chapter pertaining to this investigation. Biological and physiological considerations which must be considered before selecting an animal model was discussed in this chapter. An important criterion in choosing a model is that it should be at a similar developmental stage as the intended human consumer, in this case, an infant. In addition the model chosen should have similar meta-bolic and digestive characteristics as a human infant. Several different animal models were considered in the IOM publication with the bulk of the emphasis on pri-mates, swine, rats, and mice. The following is a summa-tion of the IOM publication pros and cons for these four animal models.

PrimatesThis animal model tends to have a similar diet compared to humans and has no problem consuming infant for-mulas. Primates are highly comparable to humans, and were reported to be a good model for neurological stud-ies. The IOM reported the stage of development between the human brain at 4 months of age and the primates at 1 month of age to be equivalent. The drawbacks of using primates are two-fold, extremely expensive, and many researchers are not willing to sacrifice primates espe-cially if the researcher is only interested in one or two parameters.

SwineLike primates swine can consume high-fat infant formu-las, and they also metabolize fat in the same manner as do humans. Another similarity to humans is that piglets and neonatal humans are similar in size. According to the IOM publication pigs make an excellent model for lipid metabolism, hypoxia, and endocrinology studies. Pigs have also been used to investigate or study biochemi-cal (fatty acids) aspects of the neuronal membranes and myelin sheath of the developing brain. Unlike primates, pigs can be more readily sacrificed in order to perform necessary analyses. The author of this article would like to add that since the pig is mostly thought of as a farm animal an millions of pounds of pork are consumed every year it is reasonable to conclude that researchers would be less hesitant to sacrifice swine than primates. The one drawback of using piglets at the present time according to the IOM is that they are not a good model for perform-ing behavioral or neurodevelopmental toxicity testing, because they are hard to work with. Therefore it can be difficult to determine if an adverse effect occurred or not.

RodentsThe pros and cons for rodents, specifically rats and mice are as follows. There is a vast amount of physiological, anatomical, biological, and behavioral information on the rat. One example given, directly related to neonatal testing, made the correlation between the neonatal rat brain and the structural similarities at a given age to the neonatal human brain. For example the IOM publication reported that the maturity of the rat brain at postnatal day 7 is structurally similar to a 34 week premature human brain, and at postnatal day 12 the maturity of the rat brain was reported to be structurally similar to the brain of a human that went full term. The rat was also reported to be a good behavioral model. The mouse was reported to be an excellent genetic model, due to our current knowl-edge of the genome of this species. The IOM publication reported that 45–55% of the calories consumed by the human infant are consumed as fat whereas the rat diet is normally around 5% fat. Therefore the rat is an inappro-priate model for assessing the toxicological significance or lack thereof when altering fat levels. The mouse also consumes a low fat diet, and was reported to exhibit dif-ferent tissue and liver lipid metabolism than humans. Feeding and dosing new substances for use in infant formulas to neonatal rodents is difficult. There was a procedure reported that is used to artificially rear neona-tal rats, however, the procedure was reported to require gastrostomy placement. This procedure was reported to be invasive and difficult to maintain for very long. This procedure and variations thereof will be discussed later.

Internet searchesThe next step of the investigation was to search the lit-erature for different neonatal testing paradigms using YAHOO and Google Scholar. Since infant formulas are consumed orally the reported data is mostly on oral and intragastric paradigms with a couple of exceptions. This is not to be thought of as an all inclusive list of oral neonatal studies, but a good representation of current neonatal methodologies. These are the relevant stud-ies that were found using YAHOO and Google Scholar. Table 1 illustrates the different neonatal testing methods that are being used at this time, and it gives a sense of which species are the most utilized.

DogsFrom the results of the literature search it is clear that there is a lack of orally dosed neonatal dog studies, at least published ones. Two orally dosed juvenile dog studies (not neonatal) were located. The first one was in juvenile dogs and was reported in Japanese with English abstract and tables (Sawada et al. 2001). The second one was an abstract and 8-week-old puppies were utilized (Clarke et al. 2001). In addition one very short abstract from Covance Laboratories (Grainger and Blakey 2000) was located. The abstract reported that Covance has been uti-lizing immature “neonatal” dogs, when testing pediatric drugs and biological substances. However, no particular study was reported.

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Tec

hnis

che

Uni

vers

iteit

Ein

dhov

en o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.

Neonatal animal testing paradigms 59


Table 1. Current testing methodologies.Species Stage at Initiation Method ReferencesDog Juvenile Diet Sawada et al. (2001)

Oral not specified Clarke et al. (2001)Monkey Neonate Gavage Stegink et al. (1975)

Bottle-Fed Champoux et al. (2002)Jeffrey et al. (2002)

Rat Neonate Gavage Dorman et al. (2000)Fukuda et al. (2004)Pan and Chen (2007)Flores et al. (1996)Pollack et al. (1987)Hsiao et al. (2001)

Artificial Rearing Li et al. (2004)Ismail-Beigi et al. (2006)Wainwright et al. (1999)Srinivasan et al. (2000)Srinivasan et al. (2003)Kinirons et al. (2003)Patel and Srinivasan (2002)Aalinkeel et al. (1999)West et al. (1982)West (1993)Flores et al. (1996)Hall (1975)Potsic et al. (2002)

Injected into Jejunum Kraehenbuhl and Campiche (1969)Mice Neonate Gavage Eriksson et al. (2002)

Sand et al. (2004)Viberg et al. (2006)

Gavage (Modified) Butchbach et al. (2007)Artificial Rearing Beierle et al. (2004)

Pig Neonate Intravenous Wykes et al. (1993)Kansagra et al. (2003)

Injected into Jejunum Kraehenbuhl and Campiche (1969)Bowl Pond et al. (1971)Bottle-Fed/Feeder Hrboticky et al. (1990)Bottle-Fed Craig-Schmidt et al. (1996)

Arbuckle et al. (1994)Wedig et al. (2002)

Gavage Stegink et al. (1973)Autosow Lecce (1969)

Lecce (1971)Lecce (1975)Lecce and Coalson (1976)Lecce et al. (1979)Lecce et al. (1982)Jones et al. (1977)Rhoads et al. (1990)Rhoads et al. (1992)Gomez et al. (1995)Gomez (1997)Ulshen et al. (1991)Garthoff et al. (2002a)a

Garthoff et al. (2002b)Guinea pig Neonate Gavage-fed/Bowl Weaver et al. (1987)

Intravenous Lavoie et al. (2004)Lambs Neonate (1–4 weeks) Bottle-fed/Injected Jones et al. (1993)Rabbits Neonate Injected into Jejunum Kraehenbuhl and Campiche (1969)

Gavage Lee et al. (2000)aThe Garthoff et al. (2002 a,b) are on different aspects of the same experiment.

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Tec

hnis

che

Uni

vers

iteit

Ein

dhov

en o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.

60 E. G. Flamm


PrimatesThree neonatal “infant” monkey studies were located. The first one gavaged monkeys ranging in age from 30 minutes to 17 days old with monosodium glutamate (Stegink et al. 1975). Gavaging primates can have significant drawbacks in that it may cause them to vomit as occurred to a limited extent in the monosodium glutamate study. If vomiting does occur it has to be determined if the animal should be dosed again and with what portion of the first dose. This would depend on when the animal vomited (how much time had passed since exposure) and volume of vomit. Either way, it will be difficult to know exactly how much of the compound was retained by the animal. The follow-ing two studies were bottle-fed at least when the studies first started and both dealt with docosahexaenoic acid and arachidonic acid added to formula (Champoux et al. 2002; Jeffrey et al. 2002). The formula used was a mixture of Similac and a commercial monkey formula. One of the studies indicated that the monkeys were hand-fed until they could feed themselves and that was reported to be usually by 3 days of age (Champoux et al. 2002). If you can afford to maintain primates for example, “monkeys” and are not concerned with the ethical issues that can arise from sacrificing primates, and you have the staff to bottle-feed primates, then the testing paradigm utiliz-ing bottle-fed neonatal primates or “monkeys” would be a viable method for testing new substances for infant formula. This dosing procedure is non-invasive and pri-mates are able to consume formula high in fat. However, as stated above there are significant drawbacks to using primates.

Rodents (Mice and Rats)The differences in nutritional requirements between rats, mice and humans along with the difficulty in feeding and dosing neonatal rats and mice makes these two species inadequate for testing infant formula where assurance of normal growth and development is of paramount importance. Two different methodologies and variations thereof were found that attempt to alleviate the dosing and feeding issue; unfortunately the techniques may lead to more questions than answers. Not all of the listed neonatal rodent references are on substances pertaining to infant formula and are only referenced as a means of illustrating the dosing procedure.

GavageThe first methodology is to gavage the neonates (Dorman et al. 2000; Fukuda et al. 2004; Pan and Chen 2007; Flores et al. 1996; Pollack et al. 1987; Hsiao et al. 2001; Eriksson et al. 2002; Sand et al. 2004; Viberg et al. 2006; Butchbach et al. 2007). Neonates can be gavaged with just the test substance (no milk) and then returned to the dam for nursing, or the test substance can be mixed with a rat milk substitute or in the case of the mice studies, the vehicle was a 20% fat emulsion and gavaged in this manner (Eriksson et al. 2002; Sand et al. 2004; Viberg et al. 2006). In the second case the neonates

would most likely be gavaged additional milk and they may even be returned to the dam for additional nurs-ing. An article based on conclusions of the International Life Sciences Institute Risk Science Institute (ILSI RSI) Expert Working Group (Moser et al. 2005) reported that orally gavaging neonatal rodents can be done success-fully with practice. The ILSI RSI Expert Working Group also reported on several factors which must be taken into consideration so that a negative maternal or neo-natal response does not occur. For example, the techni-cians must wear gloves when handling the neonates and take measures to insure that the gavaged pups smell the same as the rest of the litter. Additionally, neonates that are injured in anyway or showing adverse health effects may be rejected by the dam. The article also reports that the volume being administered must be carefully con-sidered along with the viscosity of the vehicle in which the test substance is being administered. Moreover, gavaging neonates large volumes of a test substance and/or a highly viscous vehicle can interfere with nurs-ing. Thus, dosing volumes should be much less than normal milk intake. One article reported on what was referred to as a novel way of orally dosing neonatal mice (Butchbach et al. 2007). The technique is a modified gavaging method which was reported to be less invasive than the typical gavaging method. The emphasis was on reporting a novel way of delivering drug compounds not infant formula. Neonatal mice 4 days old or younger are held gently by the skin on the back of the neck. A curved 25.4 mm long, 24-gauge feeding needle with a ball (rather than needlepoint) 1.25 mm in diameter is inserted into the mouth of the mouse. When the ball gets to the pharynx the compound is delivered slowly using a Hamilton microsyringe. The needle is then gently removed from the mouth of the neonate. The neonate is sometimes mildly sedated with isoflurane a gaseous anesthetic, prior to performing this procedure. This method was reported as being minimally invasive because the needle does not go down the esophagus and therefore can not cause any damage to the esophagus as normal gavaging can. In addition the procedure with a properly trained technician should only take 10 seconds, and only minimal effects were reported to have been observed in feeding and grooming behaviors between the neonates and the dams. The article reported that out of 955 neonatal mice being dosed in this manner only 46 died due to the procedure (4.8%). This is a very good success record, however even this modified gavaging method is not completely non-invasive. In addition, it was reported that neonatal mice as young as 2 days of age have been dosed in this manner, but the risk of mor-tality is higher than in neonates at 4 days of age. The ILSI RSI Monograph (Zoetis and Walls 2003) reported on a procedure that sounds similar to the above mouse study (Butchbach et al. 2007). In this case, neonatal rats are orally administered a compound but just to the back of the mouth. The ILSI RSI Monograph (Zoetis and Walls 2003) reported this procedure to be relatively safe for

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Tec

hnis

che

Uni

vers

iteit

Ein

dhov

en o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.



the neonate but due to taste aversion or uncooperative pups, some of the dose that was administered may be lost (no particular study was referenced).

Artificially reared rodentsThe second methodology and variations thereof is to artificially rear rodent pups. This method is often called the “pup-in-a-cup” model (Flores et al. 1996; Hall 1975; Potsic et al. 2002; Li et al. 2004; Ismail-Beigi et al. 2006; Wainwright et al. 1999; Srinivasan et al. 2000; Srinivasan et al. 2003; Kinirons et al. 2003; Patel and Srinivasan 2002; Aalinkeel et al. 1999; West et al. 1982; West 1993). Neonatal rats are reared either by intragastric cannulation where a feeding/dosing tube is inserted down the esophagus and into the stomach, or by gastrostomy placement where a feeding/dosing tube is surgically inserted into the stom-ach. Neonatal rats are then placed in Styrofoam cups and floated in a water bath which is kept at a constant temperature. Pumps which are on timers artificially feed and dose the animals. The “pup-in-a- cup” method is normally done on neonatal rat pups, however, one article was located (Beierle et al. 2004) which performed the “pup-in-a-cup” method by surgically inserting a feeding tube into neonatal mice. Due to the complexity of the technique this method requires very experienced, well-trained technicians as noted in the ILSI RSI monograph (Zoetis and Walls 2003), and the procedure is not recom-mended for standard toxicological testing. This is also a time-consuming technique because the neonates must be removed from the cup every day so that the anogenital region can be stimulated by wiping it with a moist tissue so that the animals defecate and urinate. In addition, the tubing and litter must be cleaned and changed every day. The “pup-in-a-cup” procedure using the gastrostomy placement procedure to feed and dose the neonates is invasive because of the surgical procedure. The “pup-in-a-cup” method using the intragastric cannulation just like the gavaging methods can be stressful to the neonates and is an unnatural feeding method. One of the articles pointed out an additional problem to this tech-nique and that is the possible problem of bloat (Kinirons et al. 2003). If the rate of flow is too great the neonates will bloat. In order to reverse bloating the flow must be reduced. However, in reducing the flow to combat the bloat the body weights of the neonates can be adversely affected. Due to differences in nutritional requirements and metabolism between rats and humans, and the stress that can be caused by the “pup-in-a-cup” method, it would be difficult to extrapolate the results using these procedures to the human condition and it would be even less likely to be a pivotal study that would confirm or alleviate toxicological concerns when dealing with substances for use in infant formula. However, the “pup-in-a-cup” methodology may have more research utility when testing non-nutritive substances such as alcohol especially pertaining to the brain. Due to the compara-tive nature and structural characteristics of the neonatal rat brain and neonatal human brain at different stages

of development which was reported on earlier from the IOM publication.

Non-OralFor the most part only oral studies were considered since infant formula would be consumed orally. The substance in this case went directly into the gastrointestinal tract in this immunological article (Kraehenbuhl and Campiche 1969) therefore it was included. Rats, rabbits and pigs were injected with immunoglobulin G directly into the jejunum. This procedure is acceptable for an immu-nological study. However, it would not be a practical method for dosing new substances for infant formula.

RabbitsIn addition to the immunological study which included the rabbit an additional study was located in which new-born rabbits were gavaged (Lee et al. 2000). Newborn New Zealand white rabbits were placed in an incubator on the day of delivery. The newborn rabbits were fed and dosed by gavage Lactobacillus GG twice a day for 2 days either alone or with E. coli (Escherichia coli) K1A and then were sacrificed on day 3 so that tissues could be harvested. The gavaging technique used in this study caused little trauma to the animals overall. However, it was reported that 2% of the neonates were excluded from the data either due to mortality, or trauma caused by gavaging which was evident by the appearance of regur-gitated blood.

Swine (biology and physiology)There is another neonatal animal model which has similar nutritional requirements and is anatomically and physiologically similar to humans. This animal model is the neonatal pig. Piglets (non-miniature) and human neonates are comparable in size at birth. The piglet how-ever is slightly lighter at approximately 1.4 kg, but by day 8 the piglet is approximately 3 kg, and the body weight of the pig can increase up to 1000% from its initial birth weight by week 6 (Miller and Ullrey 1987; Ramirez et al. 1963). The significance of this rapid rate of growth is that nutritional deficiencies that can occur in both pigs and humans will make themselves apparent more rapidly. One example of this pertains to the lack of iron content in mother’s milk of both humans and pigs which is approxi-mately 1 ppm (Miller and Ullrey 1987). Due to the rapid growth of the piglets iron deficiency anemia is observed in piglets at 2–4 weeks of age if they do not receive any additional iron. Humans infants also suffer from iron deficiency anemia if they do not receive any other source of iron, but it takes 4–24 months to be evident (Book and Bustad 1974). The paper entitled: Information Resources on Swine in Biomedical Research 1990–2000 reports on the physiology and anatomy of the pig and can be found at www.nal.usda.gov/awic/pubs/swine/swine.htm. This report was published by the US Department of Agriculture and its agencies including: the Agricultural Research Service, the National Agricultural Library

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Tec

hnis

che

Uni

vers

iteit

Ein

dhov

en o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.

62 E. G. Flamm


and the Animal Welfare Information Center. This paper reports on the anatomical similarities and differences of the organ systems between swine (non-miniature) and miniature breeds (Yucatan, Sinclair Hormel, Hanford and Göttingen to name a few) compared to humans. The main difference of course between these two different types of swine is size. Swine (non-miniature) at 4 months of age are approximately 100 kg and miniature swine are only half or less of this body weight depending on the breed. The difference in body weight gain would suggest that non-miniature piglets would be more appropriate for shorter term studies. Swine (non-miniature) also tend to have larger litters (8–12) than the miniature breeds (4–6). Epiphysial closure occurs by the time swine (non-minia-ture) are 3–4 years of age, this occurs in miniature swine approximately 1–2 years earlier. Organ systems discussed included but was not limited to the heart, gastrointesti-nal tract, kidney, skin and brain. The swine heart and the human heart are similar with one exception, the pig has a left azygous vein humans do not. There are anatomi-cal differences in the gastrointestinal tract. However, the gastrointestinal tract of the pig has been used as a clas-sic model for studying digestive phenomena in humans. The paper reports that the neonatal development of the gastrointestinal tract is similar to humans and that the physiological characteristics of the gastrointestinal tract are also similar, most likely due to the fact that both pigs and humans are omnivores. The anatomy and physiol-ogy of the pig kidney is reported to be very similar to the human. The skin of the pig is thicker, less vascular and has no sweat glands. The cutaneous blood supply how-ever in pigs is similar to humans, and pigs have been used to study wound healing and certain plastic surgery procedures. Due to the massive bone structure of the skull the brain of the pig has not been studied much. However, the vascular structure of the brain is compa-rable to humans. The ILSI RSI Monograph (Zoetis and Walls 2003) reported that the neonatal pig is an ideal model for studying infant formula due to the similarities in nutrients needed and the physiology of the diges-tive tract of the neonatal pig compared to the human infant. Notable differences between piglets and neonatal humans are: Fat reserves are more limited in piglets than human infants, pigs lack the ability to sweat and have a higher metabolic rate and body temperature (Book and Bustad 1974). There has been a long held believe that piglets are born without circulating immunoglobulins due to the placental barrier. There is no doubt that pig-lets will receive the majority of immunoglobulins from the colostrum that is consumed while nursing. However, an article was located that reported low levels of immu-noglobulins in the blood and certain organs of fetal pigs (Butler et al. 2001). The possibility of maternal contami-nation was discussed. Regardless if piglets are born with or without immunoglobulins colostrum consumption is essential for the survival of the piglet in a non-protected environment. This same article (Butler et al. 2001) shows that IgG serum levels are higher than IgA or IgM in the

fetal pig. However, fetuses with the highest IgG levels are still lower than a 1-day-old neonate after nursing for a day by 1000-fold. Another article by the same lead author (Butler et al. 2009) reported the immunoglobulin level (IgM, IgG and IgA) in the blood after 70 days gestation for fetal pigs to be small at 10–27 µg/mL.

Swine methodologies other than the AutosowThe first neonatal pig methodology to be discussed mim-ics a low birth weight infant that cannot take food orally. This method has been developed to study substances administered by total parenteral nutrition (Wykes et al. 1993; Kansagra et al. 2003). Silastic catheters are surgi-cally inserted and the neonatal pigs are intravenously fed compounds typically used in clinical settings through these catheters. This type of study design would take very experienced personnel, however, if you had a substance that was to be used in this manner, such a study as this may be worthwhile. Three additional oral neonatal study methodologies utilized one of the following: a bowl, (Pond et al. 1971) bottle-feeding, (Craig-Schmidt et al. 1996; Arbuckle et al. 1994; Wedig et al. 2002) or gavage (Stegink et al. 1973). The concern with feeding a liquid diet from a bowl is spillage and determining what was actually consumed. However, the study that utilized bowls did not report that any spillage occurred. Bottle-feeding neonates is a very viable way of dosing; however, it is time consuming. Gavaging is stressful and as reported earlier with the monkey study, the animals may vomit making it difficult to determine how much of the dose was retained. This particular study gavaged monosodium glutamate to neonatal pigs and did not report on any of the animals vomiting. One additional study utilized two different dosing procedures, hand-fed (assuming bottle-fed) and a feeder which was used to feed the piglets after 2 days of hand-feeding (Hrboticky et al. 1990). This also seems to be a viable way of dosing piglets and is an intro-duction to the next experimental feeding paradigm.

AutosowNorth Carolina State University in Raleigh has been studying neonatal pigs for years and several articles on the subject have been written by individuals associated with this university (Lecce 1969, 1971, 1975; Lecce and Coalson 1976; Lecce et al. 1979, 1982; Jones et al. 1977; Rhoads et al. 1990, 1992; Gomez et al. 1995; Gomez 1997; Ulshen et al. 1991). The current name of the laboratory facility at North Carolina State University is the Grinnells Intensive Swine Research Laboratory. This facility uses an automated feeding machine called the Autosow (gen-eral term for this type of machine) for dosing piglets. The Autosow works like a surrogate mother which not only feeds the piglets but also administers a specific amount of test substance. The idea of an automated system for raising piglets is not a new one. In 1949 the Wall Street Journal reported on a methodology where 2-day-old piglets would be taken from their mothers and raised on synthetic sow’s milk in what was referred to as “hog

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Tec

hnis

che

Uni

vers

iteit

Ein

dhov

en o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.



hatcheries” (Lecce 1975). This was important due to the fact that many piglets did not survive because they were trampled by their mothers. Now instead of nursing her young the sow can have another litter. Pigs as noted ear-lier are born lacking sufficient circulating immunoglobu-lins and therefore are immunocompromised. The piglets acquire immunoglobulins when they consume colostrum which they receive when they first start to nurse from the sow. However, piglets are infected with pathogens from the sow during birthing and while nursing. Therefore the longer the piglets are left with the sow to nurse, the effects of E. coli and Rotavirus infections will manifest them-selves in the piglets as diarrhea. There are three basic ways of starting a neonatal pig study before the piglets are placed on the Autosow. These procedures differ based on how critical it is for the researcher to minimize diarrhea. Of course, there can be variations in the duration of time the piglets are left with the sow to nurse and the time taken to prepare the sow before giving birth. The first pro-cedure starts out with the sow delivering her piglets in an unsanitary environment. The piglets would be left with the sow for 1–2 days to consume the colostrum needed. Piglets started in this manner will most likely have more diarrhea than the following 2 methods. The second pro-cedure starts with taking the sow to a clean area 4–5 days before giving birth and bathing her in an iodine solution. The area in which the sow and her piglets are in would be cleaned regularly. The piglets would remain with the sow for 1–2 days in order to receive the colostrum needed. Even though this procedure is more sanitary, it does not mean that the piglets will not have diarrhea. It is a normal phenomenon for piglets to have diarrhea up to 3 weeks of age, hopefully it is reduced using this method. The third procedure again starts with placing the sow in a clean area 4–5 days before giving birth and bathing her in an iodine solution. In this procedure the piglets are caught in sterile towels. The adhering membranes are removed and the umbilical cord is cut and the piglet is placed in a clean container and taken directly to the clean room which contains the Autosow. Now you have a colostrum-deprived piglet which has had minimum exposure to the pathogens which cause diarrhea. The piglet in this case should not only have a much reduced frequency of diarrhea but can be used in studies in which an animal model lacking immunity is required. In all cases a heat source should be provided for the piglets if the room tem-perature is not at least 30–35°C (Zoetis and Walls 2003). Additional steps that could be taken in order to reduce the occurrence of diarrhea further would be to take the neonatal pigs either by cesarean section or by hyster-ectomy as discussed in an article on the use of piglets in immunological studies (Butler et al. 2009). Studies using these procedures will require both highly techni-cal personnel and special facilities and therefore would be more expensive. Pertaining to E. coli derived neonatal diarrhea, two articles (Isaacson et al. 1980; Morgan et al. 1978) were located which reported on vaccinating the sows in the hopes that after colostrum consumption the

piglets would benefit and not experience diarrhea and/or death from these pathogens. The piglets did benefit when exposed to the same E. coli strain which possess the same pili (hair-like structures which facilitate coloni-zation and deoxyribonucleic acid transfer) that the sows were vaccinated with, but did not benefit when exposed to a different stain of E. coli. Concerning the number of groups and the number of piglets per group the author of this article will only suggest that all piglet studies have either a vehicle control group or a sow fed control group in addition to a vehicle control group. Current knowledge of the test compound, and the emphasis of the study will be determining factors pertaining to the number of animals used.

FDA swine studyMost neonatal pig studies using the Autosow have one Autosow fed control group, one sow fed control group and the dosed groups. This was also the case in a study which was done by FDA investigators on trypsin inhibitors at what was the Beltsville Research Facility in Beltsville Maryland (Garthoff et al. 2002a; Garthoff et al. 2002b). Minature swine from FDA’s breeding colony were utilized (Hormel-Hanford). Following birth, the piglets were left with the sow for 3 days in order to consume the necessary colostrum. Following 3 days of nursing 37 male piglets (Autosow control group 18, test group 19) were taken to the Autosow where they were weaned and exposed to the test substance until 6 weeks of age. The sow fed control group used 6 male piglets. When weaning was completed, the pigs were removed from the Autosow and were fed a liquid-based diet which contained in the exposed group trypsin inhibitor followed by a regular chow diet. The pigs were sacrificed when they were 39 weeks of age. This study was performed on trypsin inhibitors to study previ-ously reported adverse pancreatic effects in rat studies done mainly on 21-day and 10-week-old animals along with a 2-year study. These studies raised concerns about soy-based infant formula. However, there were questions about extrapolating the rat data to neonatal humans due to the age of the rats used. Also, the rat has cholecysto-kinin-A receptors predominantly in the pancreas which were shown to mediate pancreatic preneoplastic lesions when azaserine was administered. Humans and pigs on the other hand have predominantly cholecystokinin-B receptors. This is another indication that the pig may be a more suitable animal model to assess the potential for human effects (Garthoff et al. 2002a). Swine were chosen because of the developmental stages which can be determined by reproductive and central nervous system development (neonate, infancy, childhood, and adolescence) of the pig could be more easily correlated to similar stages in humans and, because of the physi-ological and anatomical similarities between humans and swine in infancy. However, it was noted that the sequence of miniature swine development is some what different than humans. An example was given report-ing that miniature swine reach sexual maturity unlike

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Tec

hnis

che

Uni

vers

iteit

Ein

dhov

en o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.

64 E. G. Flamm


humans long before epiphysial closure occurs (Garthoff et al. 2002a). The neonatal pig trypsin inhibitor study was published in two separate articles, the first one dealt more with the methodology and the second one dealt more with the results. No neoplastic effects on the pan-creas were observed in the swine study. This result rep-licates what was observed in every other species tested with the exception of the rat. The article also reported that the study provided added assurance that there is no increased risk of pancreatic neoplasia, from consuming foods which contain natural trypsin inhibitors (Garthoff et al. 2002b). Neonatal piglet studies will most likely be more expensive than rodent studies. However, since the results of the swine studies will be more relevant in the case of testing new compounds to be used in infant for-mula this must be taken into consideration. The author of this article believes that the Autosow will help to reduce the cost of maintaining piglets due to the fact that there should be less hands on activity concerning feeding and dosing. Due to the anatomical and physiological similari-ties between pigs and humans and the non-invasiveness of the Autosow feeding method, it would be the author’s choice for testing new substances for infant formulas.

Guinea pigThe guinea pig is a very interesting and underutilized animal model. Guinea pigs share certain lipoprotein and cholesterol metabolism characteristics with humans. The most notable is that both species carry the majority of plasma cholesterol as low density lipoprotein cho-lesterol and less as high density lipoprotein cholesterol. Another similarity is a nutritional requirement, in that both humans and guinea pigs require vitamin C in their diet. The third trimester is a time of rapid development for the guinea pig thus, it may only take 2 days for weaning to be complete (Fernandez 2001). Like humans, guinea pigs are also born with a functional immune system by acquiring immunoglobulins in utero (Weaver et al. 1987). The first methodology removed the young from the mother and placed them with a non-lactating surrogate for rearing. (Weaver et al. 1987). The young were gavage fed (with a syringe) up to 8 times a day for a few days (the exact number of days was dependent on when they could feed themselves). The guinea pigs were then fed from a bowl after only a few days of gavage feeding. The next guinea pig study (Lavoie et al. 2004) administered the substances intravenously through indwelling catheters. Unless the species is anatomically and physiologically very similar to humans and the substance is going to be used in this manner this is not an effective paradigm. Due to the lipoprotein similarities between guinea pigs and humans, this species could fill an experimental void left by rats and mice when testing high-fat substances.

Ruminates (Lamb)The final animal model reported here is on lambs. The following article (Jones et al. 1993) reported that the lambs were bottle-fed. Catheters were surgically inserted

in order to monitor blood pressure. Different substances were injected in order to determine if there would be an effect on blood pressure during feeding. Lambs, of course, can be bottle-fed and therefore can be exposed to infant formulas in a non-invasive manner. However, the practicality of using a ruminate for the toxicological testing of infant formula is questionable due to the differ-ences in anatomy and physiology of the digestive system compared to human infants.

FDA/CDER nonclinical guidance documentThe FDA/CDER (2006) guidance document pertaining to nonclinical studies used to test the safety of pediatric drugs was taken into account. This document is entitled: Guidance for Industry Nonclinical Safety Evaluation of Pediatric Drug Products. The document gives recommen-dations on the implementation and usage of immature animal models. Below is a summation of the guidance document with some direct quotes including those per-taining to study design. This document was developed for drugs, therefore not every aspect of the document will be relevant to the evaluation of infant formula, but useful comparisons can be made.

Pediatric drug safety testing in the past has been sup-ported for the most part by adult clinical studies which have been based on nonclinical adult animal studies. In order to utilize only adult studies the assumption has to be made that pediatric patients will respond in a similar manner as do adult patients. However, it is clear that some drugs can have different pharmacological characteristics in the developing pediatric patient than in the adult patient, and therefore the pediatric patient may react differently to the drug than the adult patient. Juvenile animal studies may be helpful in cases where postnatal developmental parameters have not been adequately tested in reproductive studies and if safety concerns can-not be satisfied clinically. Since certain organ systems continue to develop postnatally there is additional con-cern regarding how these organ systems may respond to certain pediatric drugs, therefore juvenile animal studies could add to our toxicological knowledge of a given drug better than adult animal studies.

The guidance document states within the Long-Term Exposure in Pediatric Subjects paragraph that: “When designing juvenile animal studies, the age of the pediatric population for which the drug is intended is important. Neonates, infants, and older children are at very different developmental stages, and appropriate nonclinical data should support the drug’s use in the intended pediatric population.” Other considerations are stated under the heading Issues to Consider Regarding Juvenile Animal Studies in the guidance document and they are: “(1) the intended or likely use of the drug in children; (2) the tim-ing of dosing in relation to phases of growth and devel-opment in pediatric populations and juvenile animals; (3) the potential differences in pharmacological and toxicological profiles between mature and immature sys-tems; and (4) any established temporal developmental

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Tec

hnis

che

Uni

vers

iteit

Ein

dhov

en o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.



differences in animals relative to pediatric populations. We also recommend that endpoints relevant to identify-ing target organ toxicity across species be included in the juvenile animal study design.”

Rats and dogs have been the traditional animals of choice, however there are times when other species may be more appropriate. When choosing a juvenile animal model the similarities and differences of the animal model and humans should be taken into account; these include: metabolism, the stage of organ development and the sensitivity to certain toxicological observations. The guidance document within the paragraph on Species, gives an example of when a particular species may not be appropriate: “For example, when drug metabolism in a particular species differs significantly from humans, and alternative species (e.g., minipigs, pigs, monkeys) may be more appropriate for testing.” When discussing the route of administration the guidance document reported within the first paragraph under Route of Administration and Dosage Formulation the following: “When perform-ing nonclinical studies, the intended clinical route of administration and dosage formulation should be used unless an alternate route of administration and dosage formulation provides greater exposure or is less invasive with adequate exposure.” The guidance document also refers to the formulation in a footnote: “We recommend safety evaluations of inactive ingredients be conducted to determine potential adverse effects in pediatric subjects. The type of testing is dependent on the extent to which this information is already well understood.” The dosing protocol should be similar in frequency, when possible, to that anticipated in a clinical setting. The dose levels administered should demonstrate a clear dose-response with the high dose indicating a clear toxicological effect, the mid-dose demonstrating some toxicity and signifi-cant toxicological effects should not be demonstrated in the low dose so that a clear no-observed-adverse-effect level (NOAEL) can be determined. (Of course, this does not pertain to new substances in infant formula in which the hope would be that no adverse toxicological effects would be observed, however if a toxicological effect was observed having it demonstrated in a dose-related man-ner is preferable for determining a NOAEL.) Important parameters which should be included in the study are: overall growth, organ weights, gross pathology and histo-pathology, clinical observations, neurobehavioral, repro-ductive, and any other pertinent parameters.

Useful comparisons between drugs and substances new to infant formula can be made particularly when dealing with the study design. Animal models should be chosen that mimic the stage of development of the intended consumer, in this case a neonate or infant. The biological and physiological similarities and differences of the animal model should be taken into account for the substances being administered. The route of administra-tion and the matrix containing the test substance in the test animal model should be as similar as possible to the diet naturally consumed by a human infant. The same

parameters as for drugs are important when assessing substances new to infant formula, particularly overall growth.

Summary and conclusion

The optimal diet nutritionally and developmentally for infants is believed to be natural breast milk. For this very reason, producers of infant formula add new substances to infant formulas in order to more closely mimic natural breast milk. In the past, these substances for the most part were tested using adult animal studies. The reason for this review was to determine whether or not there is an appropriate neonatal animal testing model. Utilizing three different forms of media: IOM (2004) publication, simple YAHOO and Google Scholar searches, and the FDA/CDER (2006) guidance document, neonatal testing paradigms were examined and evaluated. The literature search clearly indicates that neonatal rats and pigs are the major neonatal animal models utilized at the present time. Neonatal rats and mice are extremely difficult to dose (but can be done with practice), and due to dietary differences and the inability to metabolize a high-fat diet in a manner similar to humans; rodent models are inap-propriate for testing new substances in infant formula. Neonatal monkeys are a more appropriate model and can consume a high-fat diet. However, bottle-feeding is time consuming. In addition, due to the high cost of maintaining primates and the ethical questions raised in sacrificing primates, many researchers would hesitate or are unwilling to use them. Reiterating, the advice of the IOM (2004) publication which stated that the animal model chosen should demonstrate similar metabolic and digestive characteristics compared to the human infant. In addition to the FDA/CDER (2006) guidance document which recommends that the test substances be adminis-tered to the test animal in a similar matrix as it would be consumed by a human.

The neonatal pig seems to be the most appropriate neonatal model at this time for testing new substances in infant formula for the following reasons. The neonatal pig can be dosed and fed from the Autosow which is a non-invasive dosing method. Pigs have no problem consum-ing and metabolizing a high-fat diet. Researchers tend to be less hesitant to sacrifice pigs compared to primates. In addition to the anatomical and physiological similarities pertaining to many of the organ systems of both humans and swine particularly the gastrointestinal tract.

Acknowledgements

The author would like to acknowledge David Hattan, Ph.D. for his assistance and guidance during the process of writing this article.

Declaration of interest

The author report no declaration of interest.

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Tec

hnis

che

Uni

vers

iteit

Ein

dhov

en o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.

66 E. G. Flamm


ReferencesAalinkeel R, Srinivasan M, Kalhan SC, Laychock SG, Patel MS.

1999. A dietary intervention (high carbohydrate) during the neonatal period causes islet dysfunction in rats. Am J Physiol 277:E1061–E1069.

Arbuckle LD, MacKinnon MJ, Innis SM. 1994. Formula 18:2(n-6) and 18:3(n-3) content and ratio influence long-chain polyunsaturated fatty acids in the developing piglet liver and central nervous system. J Nutr 124:289–298.

Beierle EA, Chen MK, Hartwich JE, Iyengar M, Dai W, Li N, Demarco V, Neu J. 2004. Artificial rearing of mouse pups: development of a mouse pup in a cup model. Pediatr Res 56:250–255.

Book SA, Bustad LK. 1974. The fetal and neonatal pig in biomedical research. J Anim Sci 38:997–1002.

Butchbach ME, Edwards JD, Schussler KR, Burghes AH. 2007. A novel method for oral delivery of drug compounds to the neonatal SMNDelta7 mouse model of spinal muscular atrophy. J Neurosci Methods 161:285–290.

Butler JE, Sun J, Weber P, Ford SP, Rehakova Z, Sinkora J, Lager K. 2001. Antibody repertoire development in fetal and neonatal piglets. IV. Switch recombination, primarily in fetal thymus, occurs independent of environmental antigen and is only weakly associated with repertoire diversification. J Immunol 167:3239–3249.

Butler JE, Lager KM, Splichal I, Francis D, Kacskovics I, Sinkora M, Wertz N, Sun J, Zhao Y, Brown WR, DeWald R, Dierks S, Muyldermans S, Lunney JK, McCray PB, Rogers CS, Welsh MJ, Navarro P, Klobasa F, Habe F, Ramsoondar J. 2009. The piglet as a model for B cell and immune system development. Vet Immunol Immunopathol 128:147–170.

Champoux M, Hibbeln JR, Shannon C, Majchrzak S, Suomi SJ, Salem N Jr, Higley JD. 2002. Fatty acid formula supplementation and neuromotor development in rhesus monkey neonates. Pediatr Res 51:273–281.

Clarke DO, Connelly CS, Hall RL, Leedle RA, Miller EM, Williams JM, Sarazan RD, Ponsler GD, Sauer JM, Tizzano JP. 2001. Nonclinical pediatric testing of tomoxetine: Toxicity study in young beagle dogs. (abstract) Neurotoxicol Teratol 23:294–295.

Craig-Schmidt MC, Stieh KE, Lien EL. 1996. Retinal fatty acids of piglets fed docosahexaenoic and arachidonic acids from microbial sources. Lipids 31:53–59.

Dorman DC, Struve MF, Vitarella D, Byerly FL, Goetz J, Miller R. 2000. Neurotoxicity of manganese chloride in neonatal and adult CD rats following subchronic (21-day) high-dose oral exposure. J Appl Toxicol 20:179–187.

Eriksson P, Viberg H, Jakobsson E, Örn U, Fredriksson A. 2002. A brominated flame retardant, 2,2’,4,4’,5-pentabromodiphenyl ether: uptake, retention, and induction of neurobehavioral alterations in mice during a critical phase of neonatal brain development. Toxicol Sci 67:98–103.

FDA/CDER. 2006. Guidance for Industry Nonclinical Safety Evaluation of Pediatric Drug Products. [Online] Available at: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm079247.pdf (Last accessed in YAHOO (07/16/2012).

Fernandez ML. 2001. Guinea pigs as models for cholesterol and lipoprotein metabolism. J Nutr 131:10–20.

Flores CA, Hu C, Edmond J, Koldovsky O. 1996. Milk carnitine affects organ carnitine concentration in newborn rats. J Nutr 126:1673–1682.

Fukuda N, Ito Y, Yamaguchi M, Mitumori K, Koizumi M, Hasegawa R, Kamata E, Ema M. 2004. Unexpected nephrotoxicity induced by tetrabromobisphenol A in newborn rats. Toxicol Lett 150:145–155.

Garthoff LH, Henderson GR, Sager AO, Sobotka TJ, O’Dell R, Thorpe CW, Trotter WJ, Bruce VR, Dallas HL, Poelma PL, Solomon HM, Bier JW, O’Donnell MW Jr, Chi RK, Chirtel SJ, Barton CN, Brown LH, Frattali VP, Khan MA. 2002a. The Autosow raised miniature

swine as a model for assessing the effects of dietary soy trypsin inhibitor. Food Chem Toxicol 40:487–500.

Garthoff LH, Henderson GR, Sager AO, Sobotka TJ, Gaines DW, O’Donnell MW Jr, Chi R, Chirtel SJ, Barton CN, Brown LH, Hines FA, Solomon T, Turkleson J, Berry D, Dick H, Wilson F, Khan MA. 2002b. Pathological evaluation, clinical chemistry and plasma cholecystokinin in neonatal and young miniature swine fed soy trypsin inhibitor from 1 to 39 weeks of age. Food Chem Toxicol 40:501–516.

Gomez GG, Sandler RS, Seal E Jr. 1995. High levels of inorganic sulfate cause diarrhea in neonatal piglets. J Nutr 125:2325–2332.

Gomez GG. 1997. The colostrum-deprived, artificially-reared, neonatal pig as a model animal for studying rotavirus gastroenteritis. Frontiers in Bioscience 2:471–481.

Grainger S, Blakey S. 2000. Safety assessment studies in neaonatal dogs. (abstract). Toxicol Lett 116:88.

Hall WG. 1975. Weaning and growth of artificially reared rats. Science 190:1313–1315.

Hrboticky N, MacKinnon MJ, Innis SM. 1990. Effect of a vegetable oil formula rich in linoleic acid on tissue fatty acid accretion in the brain, liver, plasma, and erythrocytes of infant piglets. Am J Clin Nutr 51:173–182.

Hsiao SH, Acevedo JL, DuBois DW, Smith KR, West JR, Frye GD. 2001. Early postnatal ethanol intubation blunts GABA(A) receptor up-regulation and modifies 3alpha-hydroxy-5alpha-pregnan-20-one sensitivity in rat MS/DB neurons. Brain Res Dev Brain Res 130:25–40.

IOM. 2004. Infant formula evaluating the safety on new ingredients. (Chapter 5) Testing ingredients with Preclinical studies. PP 70–97.

Isaacson RE, Dean EA, Morgan RL, Moon HW. 1980. Immunization of suckling pigs against enterotoxigenic Escherichia coli-induced diarrheal disease by vaccinating dams with purified K99 or 987P pili: antibody production in response to vaccination. Infect Immun 29:824–826.

Ismail-Beigi F, Catalano PM, Hanson RW. 2006. Metabolic programming: fetal origins of obesity and metabolic syndrome in the adult. Am J Physiol Endocrinol Metab 291:E439–E440.

Jeffrey BG, Mitchell DC, Hibbeln JR, Gibson RA, Chedester AL, Salem N Jr. 2002. Visual acuity and retinal function in infant monkeys fed long-chain PUFA. Lipids 37:839–848.

Jones EE, Coalson JA, Lecce JG. 1977. Soy flour as a source of protein for baby pigs. J Anim Sci 45:1073–1078.

Jones SA, Langille BL, Frise S, Adamson SL. 1993. Nonadrenergic noncholinergic autonomic mediation of pressor response to feeding in lambs. Am J Physiol 265:R530–R536.

Kansagra K, Stoll B, Rognerud C, Niinikoski H, Ou CN, Harvey R, Burrin D. 2003. Total parenteral nutrition adversely affects gut barrier function in neonatal piglets. Am J Physiol Gastrointest Liver Physiol 285:G1162–G1170.

Kinirons SA, Shall MS, McClung JR, Goldberg SJ. 2003. Effect of artificial rearing on the contractile properties and myosin heavy chain isoforms of developing rat tongue musculature. J Neurophysiol 90:120–127.

Kraehenbuhl JP, Campiche MA. 1969. Early stages of intestinal absorption of specific antibiodies in the newborn. An ultrastructural, cytochemical, and immunological study in the pig, rat, and rabbit. J Cell Biol 42:345–365.

Lavoie JC, Rouleau T, Chessex P. 2004. Interaction between ascorbate and light-exposed riboflavin induces lung remodeling. J Pharmacol Exp Ther 311:634–639.

Lecce JG. 1969. Rearing colostrum-free pigs in an automatic feeding device. J Anim Sci 28:27–33.

Lecce JG. 1971. Rearing neonatal piglets of low birth weight with an automatic feeding device. J Anim Sci 33:47–51.

Lecce JG. 1975. Rearing piglets artificially in a farm environment: a promise unfulfilled. J Anim Sci 41:659–666.

Lecce JG, Coalson JA. 1976. Diets for rearing colostrum-free piglets with an automatic feeding device. J Anim Sci 42:622–629.

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Tec

hnis

che

Uni

vers

iteit

Ein

dhov

en o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.



Lecce JG, Armstrong WD, Crawford PC, Ducharme GA. 1979. Nutrition and management of early weaned piglets: liquid vs dry feeding. J Anim Sci 48:1007–1014.

Lecce JG, Balsbaugh RK, Clare DA, King MW. 1982. Rotavirus and hemolytic enteropathogenic Escherichia coli in weanling diarrhea of pigs. J Clin Microbiol 16:715–723.

Lee DJ, Drongowski RA, Coran AG, Harmon CM. 2000. Evaluation of probiotic treatment in a neonatal animal model. Pediatr Surg Int 16:237–242.

Li N, Liboni K, Fang MZ, Samuelson D, Lewis P, Patel R, Neu J. 2004. Glutamine decreases lipopolysaccharide-induced intestinal inflammation in infant rats. Am J Physiol Gastrointest Liver Physiol 286:G914–G921.

Miller ER, Ullrey DE. 1987. The pig as a model for human nutrition. Annu Rev Nutr 7:361–382.

Morgan RL, Isaacson RE, Moon HW, Brinton CC, To CC. 1978. Immunization of suckling pigs against enterotoxigenic Escherichia coli-induced diarrheal disease by vaccinating dams with purified 987 or K99 pili: protection correlates with pilus homology of vaccine and challenge. Infect Immun 22:771–777.

Moser VC, Walls I, Zoetis T. 2005. Direct dosing of preweaning rodents in toxicity testing and research: deliberations of an ILSI RSI Expert Working Group. Int J Toxicol 24:87–94.

Pan HZ, Chen HH. 2007. Hyperalgesia, low-anxiety, and impairment of avoidance learning in neonatal caffeine-treated rats. Psychopharmacology) 191:119–125.

Patel MS, Srinivasan M. 2002. Metabolic programming: causes and consequences. J Biol Chem 277:1629–1632.

Pollack PF, Goda T, Colony PC, Edmond J, Thornburg W, Korc M, Koldovský O. 1987. Effects of enterally fed epidermal growth factor on the small and large intestine of the suckling rat. Regul Pept 17:121–132.

Pond WG, Snyder W, Walker EF Jr, Stillings BR, Sidwell V. 1971. Comparative utilization of casein, fish protein concentrate and isolated soybean protein in liquid diets for growth of baby pigs. J Anim Sci 33:587–591.

Potsic B, Holliday N, Lewis P, Samuelson D, DeMarco V, Neu J. 2002. Glutamine supplementation and deprivation: effect on artificially reared rat small intestinal morphology. Pediatr Res 52:430–436.

Ramirez CG, Miller ER, Ullrey DE, Hoefer JA. 1963. Swine hematology from birth to maturity. III. Blood volume of the nursing pig. J Anim Sci 22:1068–1074.

Rhoads JM, Keku EO, Bennett LE, Quinn J, Lecce JG. 1990. Development of L-glutamine-stimulated electroneutral sodium absorption in piglet jejunum. Am J Physiol 259:G99–107.

Rhoads JM, Keku EO, Woodard JP, Bangdiwala SI, Lecce JG, Gatzy JT. 1992. L-glutamine with D-glucose stimulates oxidative metabolism and NaCl absorption in piglet jejunum. Am J Physiol 263:G960–G966.

Sand S, von Rosen D, Eriksson P, Fredriksson A, Viberg H, Victorin K, Filipsson AF. 2004. Dose-response modeling and benchmark calculations from spontaneous behavior data on mice neonatally exposed to 2,2’,4,4’,5-pentabromodiphenyl ether. Toxicol Sci 81:491–501.

Sawada T, Karaki K, Hayashi T, Yoneyama S, Mizushima Y, Moriyama T, Nishimura K, Kimura Y, Nakano M, Kato I. 2001. [Toxicity study of cefmatilen hydrochloride hydrate (S-1090) (7)–Three-month repeated oral dose toxicity study in juvenile dogs]. J Toxicol Sci 26 Suppl 1:135–147.

Srinivasan M, Aalinkeel R, Song F, Lee B, Laychock SG, Patel MS. 2000. Adaptive changes in insulin secretion by islets from neonatal rats raised on a high-carbohydrate formula. Am J Physiol Endocrinol Metab 279:E1347–E1357.

Srinivasan M, Laychock SG, Hill DJ, Patel MS. 2003. Neonatal nutrition: metabolic programming of pancreatic islets and obesity. Exp Biol Med (Maywood) 228:15–23.

Stegink LD, Brummel MC, Boaz DP, Filer LJ Jr. 1973. Monosodium glutamate metabolism in the neonatal pig: conversion of administered glutamate into other metabolites in vivo. J Nutr 103:1146–1154.

Stegink LD, Reynolds WA, Filer LJ Jr, Pitkin RM, Boaz DP, Brummel MC. 1975. Monosodium glutamate metabolism in the neonatal monkey. Am J Physiol 229:246–250.

Ulshen MH, Lecce JG, Stiles AD, Lyn-Cook LE. 1991. Effects of nursing on growth and development of small bowel mucosa in newborn piglets. Pediatr Res 30:337–341.

USDA. 2000. Information resources on swine in biomedical research 1990–2000. Smith CP (Eds.) [Online] Available at: www.nal.usda.gov/awic/pubs/swine/swine.htm (Last accessed in YAHOO 07/16/2012).

Viberg H, Johansson N, Fredriksson A, Eriksson J, Marsh G, Eriksson P. 2006. Neonatal exposure to higher brominated diphenyl ethers, hepta-, octa-, or nonabromodiphenyl ether, impairs spontaneous behavior and learning and memory functions of adult mice. Toxicol Sci 92:211–218.

Wainwright PE, Xing HC, Ward GR, Huang YS, Bobik E, Auestad N, Montalto M. 1999. Water maze performance is unaffected in artificially reared rats fed diets supplemented with arachidonic acid and docosahexaenoic acid. J Nutr 129:1079–1089.

Weaver LT, Laker MF, Nelson R, Lucas A. 1987. Milk feeding and changes in intestinal permeability and morphology in the newborn. J Pediatr Gastroenterol Nutr 6:351–358.

Wedig J, Christian MS, Hoberman A, Diener RM, Thomas-Wedig R. 2002. A study to develop methodology for feeding 24-hour-old neonatal swine for 3 weeks. Int J Toxicol 21:361–370.

West DB, Diaz J, Woods SC. 1982. Infant gastrostomy and chronic formula infusion as a technique to overfeed and accelerate weight gain of neonatal rats. J Nutr 112:1339–1343.

West JR. 1993. Use of pup in a cup model to study brain development. J Nutr 123:382–385.

Wykes LJ, Ball RO, Pencharz PB. 1993. Development and validation of a total parenteral nutrition model in the neonatal piglet. J Nutr 123:1248–1259.

Zoetis T, Walls I. (Eds.) 2003. Principles and practices for direct dosing of pre-weaning mammals in toxicity testing and research. A report of the ILSI Risk Science Institute expert working group.

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Tec

hnis

che

Uni

vers

iteit

Ein

dhov

en o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.

neonatal animal testing paradigms and their suitability for testing infant formula

Documents