Model predicting the teratogenic potential of retinyl palmitate, using a combined in vivo/in vitro approach

Model Predicting the Teratogenic Potentialof Retinyl Palmitate, Using a CombinedIn Vivo/In Vitro ApproachH.E. RITCHIE,1* W.S. WEBSTER,2 C. ECKHOFF,3 AND D.J. OAKES2

1Department of Biomedical Sciences, University of Sydney, Sydney 2141, Australia2Department of Anatomy and Histology, University of Sydney, Sydney 2006, Australia3Hoffmann-La Roche, Inc., Nutley, New Jersey 07110

ABSTRACT Retinyl palmitate (RP) is a knownlaboratory animal teratogen inducing abnormalities ofthe second visceral arch when administered on day 9 ofgestation in the rat. However, there are significantproblems when attempting to extrapolate this result tothe human. A combined in vivo/in vitro model wasdeveloped to assist in human risk assessment. The invitro teratogenic threshold concentration of a numberof retinyl palmitate metabolites was established. Serumconcentrations of retinyl palmitate metabolites follow-ing a single teratogenic dose of RP in the pregnant ratwere also measured. These dosed sera were also usedto culture rat embryos. Our hypothesis was that malfor-mations would only be induced by the dosed sera invitro if the threshold concentration(s) of one or moremetabolites was exceeded. Using this approach, it wasdetermined that the teratogenicity of the sera werebest predicted by serum retinol levels, with someindication that all-trans-retinoic acid and 4-oxo-all-trans-retinoic acid could be involved in some cases. Theavailable human data suggest that threshold concentra-tions of these retinoids were unlikely to be exceededfollowing vitamin A supplements of 25,000 IU/day.While the proposed model does not take into accountspecies differences, protein binding, and transfer to theembryo, it does have potential for human risk assess-ment. Teratology 58:113–123, 1998. r 1998 Wiley-Liss, Inc.

A major challenge in teratology is to be able to relatethe results of animal studies to the human. Nowhere isthis more evident than in the current debate aboutvitamin A. Vitamin A has been shown to be teratogenicin all species in which it has been tested, and at someunknown dose it will undoubtably be teratogenic in thehuman. The question is, at what dose?

A recent study by Rothman et al. (’95) suggested thata daily intake of vitamin A supplements greater than10,000 IU was associated in humans with an increasedrisk of malformations of neural crest origin. The au-thors did not relate their findings to animal studies,perhaps reflecting the difficulty of comparing animaldoses with human doses.

The lowest teratogenic dose of retinyl palmitate (RP)in rats, established in segment II developmental toxic-

ity testing, is ,163,000 IU/kg/day, while in the rabbit itis ,9,000 IU/kg/day (Kamm, ’82). For the most sensi-tive species, the rabbit, the no-effect dose is 3,600IU/kg/day. For a 50-kg pregnant woman, this wouldrepresent a daily intake of 180,000 IU/day (or 76,500IU/day based on surface area). In an attempt to extrapo-late animal data to the human, regulators often applysafety factors to animal doses: 10-fold for species differ-ences and a further 10-fold for interhuman differences.If this common, but arbitrary, 100-fold safety factor isapplied, a daily intake of 1,800 IU for a 50-kg woman isthe upper safe limit. This is below the recommendeddaily vitamin A intake of 2,498 IU/day for pregnantwomen (National Health and Medical Research Councilof Australia, ’91), and well below the average dailyintake of 8,358 IU (Commonwealth Department ofHealth and National Heart Foundation, ’86).

A different approach to the problem would be toconsider what metabolite or metabolites of vitamin Aare responsible for its teratogenic action in experimen-tal animals and at what serum concentrations theseeffects occur. It should then be possible to give humanvolunteers specific doses of vitamin A to determine ifthese metabolites are present in human serum and atwhat concentrations.

The present study describes a series of in vitro and invivo experiments in the rat designed to establish theteratogenic metabolites of retinyl palmitate and theirteratogenic and no-effect serum concentrations. Theresults are discussed in relation to published data onmetabolite levels in the human after specific dosingwith vitamin A either as supplements or in food.

MATERIALS AND METHODS

In vivo teratology

Female Sprague-Dawley rats were housed with malesovernight. The presence of sperm the next morning was

Current address for C. Eckhoff is Cerep, Inc., Redmond, WA 98052.

*Correspondence to: H. Ritchie, Department of Biomedical Sciences,University of Sydney, Lidcombe 2141, Australia.

Received 26 March 1998; Accepted 18 June 1998

TERATOLOGY 58:113–123 (1998)

r 1998 WILEY-LISS, INC.

designated day 0 of pregnancy. The dams were dosedwith retinyl palmitate on day 9 of gestation at 9 PM. Thistime was selected on the basis of preliminary experiments,which established the most appropriate time of dosing withRP for the induction of external ear (or second visceralarch) malformations in rats. Twelve dams were given asingle oral dose of either 300,000 IU RP/kg or 600,000 IURP/kg. The RP was administered by gavage in 1 ml ofblended vegetable oil. Ten control rats were dosed with oilonly. Half of the treated and control rats were killed after48 hr, and half continued until day 21 of gestation.

Gestation day-11 embryos were assessed for growthand development by the degree of embryonic turning,presence of a heartbeat, somite number, and crown-rump length. Gross structural malformations of head,visceral arches, and central nervous system develop-ment were also recorded. Resorptions and dead fetuseswere recorded, and live fetuses were weighed andexamined for malformations. Gestation day-21 fetuseswere examined externally for gross structural malforma-tions. Fetuses with severe craniofacial malformationswere dissected (but not sectioned), and then examinedfor defects of thoracic viscera, particularly great vesseland thymic anomalies, and their brains were examined forcerebellar and gross cerebral defects. Quantitative datawere analyzed using ANOVA, and qualitative data by x2

test (Sokal and Rohlf, ’81). Significance was taken at 95%.

Serum collection

Part I. RP metabolites in vitro. Untreated adultmale rats were anesthetized with ether and bled. Thecollected blood was immediately centrifuged, the clotwas removed, and resultant serum was heat-inacti-vated at 56°C before storage at 220°C (New, ’73).

Part II. Dosed serum in vitro. A total of 47pregnant rats was given a single oral dose of either300,000 IU/kg or 600,000 IU RP/kg at 9 PM on day 9 ofgestation. The rats were anesthetized with ether, andat least three rats were bled at 1, 2, 4, 8, or 16 hr afterdosing. The rats were killed by cervical dislocation afterbleeding. Control sera were collected from pregnantrats dosed with 1 ml of vegetable oil at 9 PM on day 9 ofgestation. At least one control rat was used at each timeperiod indicated above.

The collected blood was immediately centrifuged(New, ’73). Approximately 5 ml of serum were availablefrom each time of bleeding. This was divided: 200 µlwere used for HPLC analysis for retinol and retinylpalmitate/oleate, and 200 µl were used for HPLCanalysis for polar metabolites of retinyl palmitate. Thissecond sample was transported, frozen, in dry ice to C.Eckhoff (Roche Dermatologics, Nutley, NJ). Separateanalyses were performed for the different metabolitesto ensure maximum recovery. The remaining serumwas stored for embryo culture at 220°C. Prior toembryo culture, 2.5 ml of each serum sample wereremoved and heat-inactivated at 56°C.

Rat embryo culture

For rat embryo culture, female Sprague-Dawley ratswere housed with males overnight and examined forpresence of sperm in the vaginal smear the nextmorning. Embryos were explanted during the after-noon of day 9 of gestation and cultured by the techniqueof New (’73). Only those embryos at mid-to-late head-fold stage were used. Embryos were cultured at a ratioof 1 embryo/ml serum (100% heat-inactivated rat se-rum, 50 µg/ml streptomycin and 30 µg/ml penicillin G)in a roller bottle. In part I (RP metabolites in vitro), 5embryos were routinely cultured. In part II (dosedserum in vitro), because of the small amount of serumavailable, only 2 embryos were cultured in each 2 ml ofserum. To prevent photodecomposition of retinoids, theculture bottles were either biotinic or completely en-closed in aluminium foil.

The cultures were initially equilibrated with a gasmixture of 5% O2, 5% CO2, and 90% N2. After 16 hr, thegas mixture was changed to 20% O2, 5% CO2, and 75%N2. Bottles were regassed with this mixture at 22 hrand after 40 hr with a mixture of 40% O2, 5% CO2, and55% N2.

All cultured embryos were allowed to develop for 48hr. At the end of this culture period, the embryos wereexamined using a dissecting microscope. The param-eters used to assess embryonic development includedthe presence of heartbeat and yolk-sac circulation,turning, arch development, crown-rump length, andsomite number. Somite counts were only performed onembryos which had turned completely and assumed aC-shaped position. A normal embryo was defined as onewhich had completed turning, had two normal pairs ofvisceral arches, and a closed neural tube of normal size.

The data on frequency of arch malformations, normalturning, and vitality were examined using x2. Data oncrown-rump length and somite number were examinedusing ANOVA. Results for control embryos were pooled,as there was no difference between controls for thedifferent treatment groups. In each case, significancewas taken as 0.05, as the risk of a type I error wasconsidered minimal.

Part I. RP and metabolites in vitro study. Thefollowing retinoids were tested: retinyl palmitate (RP),all-trans retinol (R), all-trans retinoic acid (tRA), and13-cis retinoic acid (13cRA). Retinyl palmitate (Sigma,Sydney, Australia) was dissolved in chloroform (500mg/ml) and then diluted with absolute ethanol (chloro-form:ethanol, 1:3) to make a stock solution (125 mg/ml).Experimental embryos were grown in media containing2,500, 25,000, and 250,000 ng/ml. A maximum volumeof 10 µl of this mixture was added to each 5 ml of serum.Control bottles received 10 µl of the chloroform:ethanol(1:3) mixture.

Retinol (all-trans, Sigma) was dissolved in absoluteethanol to give a stock solution of 2.5 mg/ml. Experimen-tal embryos were grown in media containing 187.5, 375,750, and 1,500 ng/ml. A maximum of 10 µl of retinol

114 H.E. RITCHIE ET AL.

solution was added to each 5 ml of serum. Controlbottles received 10 µl of absolute ethanol.

All-trans retinoic acid (Sigma) was dissolved in abso-lute ethanol to give a stock solution of 6,670 µg/ml.Experimental embryos were grown in media containing3.125, 6.25, 12.5, 50, and 100 ng/ml. A maximum of 10µl was added to each 5 ml of serum. Control bottlesreceived 10 µl of absolute ethanol.

13-cis retinoic acid (Roche) was dissolved in sun-flower oil to give a stock solution of 125 µg/ml. Amaximum of 80 µl was added to each 5 ml of serum.Embryos were grown in media containing 500, 1,000, or2,000 ng/ml. Control bottles did not receive added oil, asother studies have shown that a concentration of oil upto 2.5% has no effect on development (Kitchin andEbron, ’84).

For each experiment, the retinoid to be tested wasadded to the culture bottle at the beginning of culture,prior to embryos being added.

Part II. Dosed serum in vitro study. Embryoswere added directly to the heat-inactivated serumcollected from pregnant rats, following dosing with RP.

HPLC

Retinol and retinyl palmitate/oleate. All proce-dures were carried out under subdued lighting.

Preparation of standard curve. Prior to the prepara-tion of a standard curve, stock solution concentrationswere checked by measuring the UV-absorbance (325nm) of a 1 in 20 dilution. On the basis of estimated stockconcentrations as determined above, a standard curvefor retinol and RP was prepared using spiked untreatedrat serum. The standard curves were linear over therange used.

Extraction of retinoids. Retinol, retinyl acetate, andretinyl palmitate/oleate were extracted, based on themethod described by Catignani and Bieri (’83). To 100µl of sera, 10 µl of internal standard (retinyl acetate)were added. Isopropanol (300 µl) was added and theserum vortexed for 20 sec. The sample was then spun at11,000g in a Heraeus benchtop centrifuge (Foss Elec-tric, Sydney, Australia) for 2 min. The isopropanol layerwas transferred into a clean Eppendorf tube and evapo-rated under a gentle stream of nitrogen. Immediatelyprior to HPLC analysis, the dried serum extract wasresuspended in 100 µl chloroform:90% methanol (1:1),vortexed for 2 sec, and spun at 11,000g in a benchtopcentrifuge (Foss Electric, Sydney, Australia) for 2 min.The extracted serum was finally transferred to Watersbrown glass autosampler vials and capped.

HPLC assay. A reversed-phase HPLC procedure wasadapted to separate retinol, retinyl acetate, and retinylesters (Rudy et al., ’89). A 5-µm, 4.5 3 50 mm guardcolumn (Alltech) and a fully capped 5-µm, 6 cm 3 4.6mm Spherisorb-ODS 2 (C 18) (Alltech) analytical col-umn were used. The sample was developed with aninitial mobile phase of methanol:water (90:10, v/v)running to ethyl acetate:isopropanol (90:10, v/v) by alinear gradient within 10 min at a flow-rate of 1 ml/min.

This second solvent was maintained for a further 4 min,followed by a 3-min linear gradient back to the initialsolvent, which was maintained for a further 7 min toallow reequilibration of the column, ready for the nextsample to be injected. An ICI LC1600 autosamplerinjecting 20-µl samples was used in conjunction with aGrant LTD6 cooling bath (Selby, Sydney, Australia) setat 0°C.

Detection monitoring was at 325 nm (UV variablewavelength absorbance, ICI LC1200 UV/Vis detector).Retention times for retinol, retinyl acetate, and retinylpalmitate/oleate were 7 min, 9 min, and 14 min,respectively. Retinyl palmitate and oleate peaks couldnot be chromatographically resolved under these condi-tions. Other retinyl esters such as retinyl stearate andlinolate were not specifically measured. Quantitation ofretinol and RP/oleate (RP/O) was by peak area, and therecovery was calculated on area ratios using retinylacetate as the internal standard. Plasma retinol andRP/O were measured in duplicate for each sample ateach time interval. The mean for each time point wascalculated. Mean recovery of retinol and RP/O was 86%and 71%, respectively.

Retinoic acid compoundsPreparation of standard curve. Calibration was per-

formed by analyzing duplicates of calibration samples(prepared with rat control serum) containing four con-centrations of known amounts of retinoid referencecompounds.

Extraction of retinoids. Serum (150 µl) was deprotein-ized with isopropanol (250 µl) containing acitretin (Ro10-1670) as internal standard. The sample was thencentrifuged. The supernatant was diluted with 0.5%acetic acid (500 µl), and solid-phase extraction wascarried out on a Varian AASP C2 (Varian, Darmstadt,Germany) cartridge, an automated device. Extractionwas followed by HPLC analysis.

HPLC assay. A reversed-phase HPLC procedure wasused, which included a 3-µm, 4.0 3 120 mm Spherisorb-ODS 2 analytical column. Column temperature wascontrolled at 42.5°C. The cartridge was developed with60 mM aqueous ammonium acetate (pH 6.8):methanol(1.5:8.5) by a linear gradient within 10 min, at aflow-rate of 1.1 ml/min.

Time-programmed UV detection was used (0–6.5min, 360 nm; 6.5–8 min, 340 nm). Quantitation ofretinoic acid compounds was by peak height ratios,using the internal standard. The limit of detection ofretinoic acid was 0.5 ng/ml.

Student’s t-test was used to determine if there weresignificant differences at individual time points for thetwo different doses used.

RESULTS

In vivo studies

Pregnant rats were treated with an oral dose of RP(300,000 or 600,000 IU/kg) on day 9 of gestation andkilled either 48 hr or 12 days later, along with theirrespective controls. After 48 hr (Fig. 1a,c,e), the most

TERATOGENIC POTENTIAL OF RETINYL PALMITATE 115

Fig. 1. In vivo rat embryos. a, b: Control 11-day-old and 21-day-old rat fetuses, respectively. c, d: Treated11-day-old and 21-day-old rat fetuses, respectively, following maternal dosing with 300,000 IU RP/kg onday 9 of gestation. Note thin second visceral arch in c and reduced external ear in d. e, f: Treated11-day-old and 21-day-old rat fetuses, respectively, following maternal dosing with 600,000 IU RP/kg onday 9 of gestation. Note absent second visceral arch in e and severely reduced external ear in f. Expectedposition of second visceral arch opposite otic placode is indicated by white arrowheads. Expected positionof external ear is indicated by black arrow.


common embryonic malformation from treated ratswas a reduction in size of the second visceral arch,which ranged from moderate to severe (Table 1). Anopen neural tube was seen in 19% of the high-doseembryos. This was not reflected in a high number ofneural tube defects in fetuses killed on day 21 ofgestation. However, this is perhaps not unexpected, asthe observed open neural tubes occurred over the fourthventricle and not the anterior neuropore. There was nosignificant difference in crown-rump length, number ofimplantations, or mean litter size. However, one litterof controls had a large number of resorptions, resultingin a significant difference in this parameter betweencontrol and treatment groups.

When the treated rats were killed on day 21 ofgestation (Fig. 1b,d,f), 46% of fetuses from the low-doseanimals and 69% from the high-dose animals hadexternal ear defects (Table 2). The defects ranged fromanotia to microtia with or without ears in both treat-ment groups, and it is concluded that the second-archdefects led to the permanent external ear defects seenin the late-gestation fetuses. However, it is likely thatnot all embryos which were identified on day 11 ashaving moderate second-arch defects would go on todevelop visible external ear malformations at term. Forthis reason, only severe second-arch defects are reportedfor later experiments. There was no evidence of decreasedthymus size or great-vessel anomalies in any of the dis-sected fetuses. Cleft palate was observed in 32% of thehigh-dose fetuses. The fetuses were not growth-retardedcompared to controls, and there were few resorptions.

In vitro studies

Part I. RP metabolites in vitro. Nine-day ratembryos were cultured for 48 hr in rat sera containing

various retinoids. At the end of the culture period theywere examined for external malformations. The resultsare presented in Table 3.

Retinyl palmitate/oleate. Rat embryos grown in2,500 ng/ml were indistinguishable from controls. At aconcentration of 25,000 ng/ml or higher, many embryosfailed to turn, and some embryos had an opening overthe fourth ventricle, but all of the embryos had normalvisceral arches.

Retinol. The no-effect concentration was 187.5 ng/ml. At 375 ng/ml, 11% had severely reduced secondvisceral arches. At 1,500 ng/ml, all embryos had re-duced second arches. Although some growth param-eters were reduced, the embryos were not significantlygrowth-retarded except at the highest concentration.

All-trans-retinoic acid. The no-effect concentra-tion was 6.25 ng/ml. At 12.5 ng/ml, 27% of the embryoshad second-arch defects. This increased to 80% at 100ng/ml. At this high concentration, the embryos wereseverely retarded, with a high incidence of poorlydeveloped yolk sacs.

13-cis-retinoic acid. The no-effect concentrationwas not established in this study, but was shown to be250 ng/ml from a previous study (Webster et al., ’86).The lowest teratogenic concentration was 500 ng/ml.

Part II. Dosed serum in vitro. Rat embryos at 9days of gestation were cultured for 48 hr in sera fromrats treated with either 300,000 or 600,000 IU RP andbled 1–16 hr later. The embryos were examined forexternal malformations at the end of the culture period(Table 4).

Embryos cultured in sera from pregnant ratsdosed with 300,000 IU RP/kg. Embryos grown incontrol sera were normal in appearance. Sera collected1–4 hr after dosing with RP were increasingly terato-

TABLE 1. Effect of 300,000 IU/kg or 600,000 IU/kg retinyl palmitate on day 9 of gestation on the growthand development of rat embryos in vivo*

Treatment(IU RP/kg)

No.litters

No.imp

Mean littersize

Resorptions(%)

CRL(mm 6 SD)

Turn(%)

Somites(no. 6 SD)

Second visceralarch defect (%)

OpenNTD (%)M S

Control 5 79 15.8 20.0 3.6 6 0.3 100 25.3 6 1.5 0 0 0300 6 66 11.0 6.0* 3.3 6 0.4* 100 23.8 6 1.8* 71 5** 2600 6 62 10.3 0.0* 3.6 6 0.3 98 25.4 6 2.3 39 53** 19**

* Embryos were examined on day 11 of gestation. CRL, crown-rump length; Turn, C-shaped position; NTD, neural tube defect;imp, implantations; M, moderate; S, severe.**Statistically different from control (P , 0.05).

TABLE 2. Effect of 300,000 IU RP/kg or 600,000 IU RP/kg on day 9 of gestationon the growth and development of rat embryos in vivo*

Treatment(IU kg/rat)

No.litters

No.fetuses

Mean littersize

Resorption(%)

Weight(g 6 SD)

Abnormalexternalear (%)

Cleftpalate (%)

Control 5 80 16.0 5.8 4.7 6 0.4 0 0300 6 71 11.8 12.7 5.1 6 0.5 46** 4600 6 77 12.8 3.5 4.7 6 0.5 69** 32**

*Embryos were examined on day 21 of gestation.**Statistically different from control (P , 0.05).


genic, with the 4-hr sera causing second-arch defects inmost embryos. The 8- and 16-hr sera did not inducesevere second-arch defects.

Embryos cultured in sera from pregnant ratsdosed with 600,000 IU RP/kg. At all time intervalsthe effects were more severe than those seen with300,000 IU/kg sera. Second-arch defects were caused byall sera samples.

HPLC studies

The retinoid content of the sera obtained from the RPdosed rats was determined by HPLC. As expected,RP/O levels rose rapidly after dosing, reaching at 2 hr amean maximum of 6,568 ng/ml for the low dose and8,438 ng/ml for the high dose (Fig. 2a). The concentra-

tions gradually decreased over the next 14 hr but werestill much higher than control values (132 6 20.6 ng/ml). Retinol concentrations also increased from controlvalues of 223.1 6 17.7 ng/ml to a maximum of 584 ng/mlat 4 hr for the low dose and 756.5 ng/ml at 8 hr for thehigh dose (Fig. 2b; Table 5). The levels approachedcontrol values after 16 hr. Serum concentrations ofretinol exceeded in vitro threshold concentrations for6.8 hr after the low dose and 12.4 hr after the high dose.All-trans retinoic acid was not detected in sera fromcontrol rats but reached a maximum of 6.8 ng/ml at 2 hrin the low-dose rats and 13.4 ng/ml at 2 hr in thehigh-dose rats (Fig. 2c; Table 5). tRA was not detectable8 hr after dosing. Serum concentrations exceeded invitro threshold concentrations for 0.4 hr after the low

TABLE 3. Effect of retinoids on growth and development of rat embryos in vitro*

TreatmentNo.

embryosYSC(%)

CRL(mm 6 SD)

Turn(%)

Somites(no. 6 SD)

Visceral archdefect (%)

NTD(%)

Control 10 100 3.4 6 0.2 100 25.5 6 0.9 0 10RP2,500 ng/ml 10 100 3.4 6 0.4 100 25.5 6 1.2 0 025,000 ng/ml 9 100 3.3 6 0.2 56** 25.8 6 0.8 0 22250,000 ng/ml 10 90 3.3 6 0.2 40** 24.0 6 1.4** 0 30Retinol187.5 ng/ml 10 100 3.9 6 0.3** 100 26.0 6 0.8 0 0375 ng/ml 9 100 3.8 6 0.3 100 25.7 6 1.0 11** 0750 ng/ml 11 100 3.5 6 0.3 73** 24.3 6 1.3 82** 101,500 ng/ml 10 90 3.6 6 0.5 90 23.4 6 2.1** 100** 40**tRA6.25 ng/ml 10 90 3.4 6 0.5 100 26.6 6 1.5*** 0 012.5 ng/ml 22 90 3.5 6 0.3 83 24.8 6 1.8 23** 525 ng/ml 30 83 3.3 6 0.5 94 25.9 6 1.0 43** 050 ng/ml 11 73** 3.1 6 0.6** 65** 24.0 6 1.4 58** 0100 ng/ml 15 7** 3.0 6 0.4** 38** 23.7 6 1.6** 80** 713cRA500 ng/ml 48 63** 3.3 6 0.5** 89 22.9 6 4.2** 77** 31,000 ng/ml 26 15** 2.5 6 0.4** 12** 22.3 6 0.6** 100** 02,000 ng/ml 9 56** 2.6 6 0.7** 11** 20.0 6 0.0** 100** 20

*YSC, yolk sac circulation; CRL, crown-rump length; Turn, C-shaped position; NTD, neuraltube defect.**Statistically different from control (P , 0.05).

TABLE 4. Effect of sera collected after an oral dose of retinyl palmitateon the growth and development of rat embryos in vitro*

Duration (hr)No.

embryosYSC(%)

CRL(mm 6 SD)

Turn(%)

Somites(no. 6 SD)

Second archdefect (%)

NTD(%)

0 11 92.3 3.8 6 0.3 100 26.2 6 0.7 0 0300,000 IU/kg1 10 100 3.5 6 0.3** 100 24.4 6 2.9** 20** 302 10 60 2.8 6 0.9** 80 22.0 6 5.1** 60** 204 12 67 3.1 6 0.7** 75 23.3 6 2.4** 92** 178 6 100 3.8 6 0.2 100 26.3 6 0.5 0 3316 10 90 3.5 6 0.4** 100 25.2 6 1.7 0 0600,000 IU/kg1 7 100 3.6 6 0.5 71 25.5 6 1.0 29** 142 10 60 2.8 6 0.6** 80 21.7 6 2.9** 80** 50**4 6 67 3.2 6 0.6** 100 20.5 6 4.6** 100** 83**8 8 75 3.2 6 0.6** 75 25.0 6 1.1** 63** 50**16 10 80 3.4 6 0.5** 90 24.9 6 1.1** 40** 20

*YSC, yolk sac circulation; CRL, crown-rump length; Turn, C-shaped position; NTD, neuraltube defect.**Statistically different from control (P , 0.05).


Fig. 2. Serum retinoid concentrations after 300,000 (ª)ª) or 600,000 (—♦—) IU RP/kg (ng/ml). A:Serum retinyl palmitate concentration. B: Serum retinol concentration. C: Serum tRA concentration. D:Serum 4oxotRA concentration. E: Serum 13cRA concentration. F: Serum 4oxo13cRA concentration.


dose and 2.7 hr after the high dose. 4oxotRA was notdetectable in control rat sera, but showed almost thesame levels and time distribution as all-trans RA in thedosed sera (Fig. 2d). 13cRA was not detectable incontrol sera but reached a maximum of 13.6 ng/ml after4 hr in the low-dose sera and 21.3 ng/ml in thehigh-dose sera (Fig. 2e). Serum levels were still ele-vated after 16 hr. 4oxo13cRA was not detectable incontrol sera, but reached 14.1 ng/ml at 4 hr in thelow-dose sera and remained elevated at 16 hr (Fig. 2f).A similar pattern was seen in the high-dose sera. For allof the measured retinoids there were large variationsbetween similarly treated rats.

DISCUSSION

Animal studies

The initial in vivo studies described in this paperwere partly undertaken to attempt to establish areliable biomarker for in vitro studies. The most com-mon external malformation seen in human offspringexposed in utero to the vitamin A analogue, isotreti-noin, is reduced and abnormal external ears (Lammeret al., ’85). As shown in the present study, thesemalformations can be induced in rat fetuses by dosingpregnant rats with 300,000 or 600,000 IU RP/kg on day9 of gestation. It was also observed that the predomi-nant malformation in embryos, 48 hr after dosing, wasunderdevelopment of the second visceral arches. Thismakes biological sense, as the second visceral archgives rise to much of the external ear (Wood-Jones andChuan, ’34). Since it is possible to culture rat embryosfrom day 9 for 48 hr, underdevelopment of the secondvisceral arches in the in vitro studies was taken as abiomarker of a significant teratogenic effect of thetested compounds.

Retinyl palmitate did not cause second-arch defectswhen tested in vitro, even at 250,000 ng/ml. This resulthas been reported elsewhere, and it is thought to beRP’s metabolites that are responsible for its teratogenicactivity in vivo (Morriss and Steele, ’74). All of the othertested metabolites caused second-arch defects: retinol

at 375 ng/ml, tRA at 12.5 ng/ml, and 13cRA at 500ng/ml. Previous studies using the same model hadshown that 4oxo13cRA caused arch defects at 500 ng/ml(Webster et al., ’86).

The normal metabolism of a dose of RP is relativelywell-understood. RP is broken down to retinol withinthe stomach and reesterified within the intestinalmucosa, and incorporated into chylomicra (Blomhoff etal., ’90). The chylomicra containing the retinyl estersenter the systemic circulation via the lymph (Hendrikset al., ’88). The esters are stored in the liver and areonly converted to retinol and released into the circula-tion in response to tissue demand. Essentially allretinol released from the liver is bound to retinolbinding protein (RBP) and its levels seem to be tightlycontrolled (Blomhoff et al., ’90).

Studies in humans (Eckhoff et al., ’91; Buss et al., ’94;Arnhold et al., ’96) and experimental animals (monkey:Eckhoff et al., ’91; rat: Collins et al., ’94) have shownthat the ingestion of a large dose of RP causes a rapidand prolonged increase in the blood concentration of RPand other retinyl esters. Eventually the esters arestored in the liver as RP. At the same time, a variety ofvitamin A metabolites appear in the blood and mayremain there for prolonged periods.

In the present study, there was a large and rapidincrease in serum RP/O following a teratogenic dose ofRP. There was also a significant 2–3-fold increase inserum retinol levels. The prolonged elevation of retinolseen after a teratogenic dose of RP in the present studywas unexpected and implies a loss of homeostaticcontrol. Unfortunately, it is not known if the increasedretinol levels were due to the presence of ‘‘unboundretinol,’’ perhaps absorbed from the intestine, or to‘‘RBP-bound retinol’’ released from the liver.

Retinol is teratogenic in vitro, as shown in thepresent and previous studies (Morriss and Steele, ’77).Some of the retinol added to the embryo culture systemmay be specifically bound if there is a molar excess ofRBP in the culture serum. However, it is presumed thatthe majority of the added retinol remains unbound.

TABLE 5. Pharmacokinetic parameters of retinoids following oral dose of retinyl palmitate in the rat

Dose RP/O Retinol tRA 13cRA 4oxotRA 4oxo13cRA

0Cmax (ng/ml)* 132.0 6 20.6 223.1 6 17.7 ND**** ND ND ND300,000 IU/kgCmax (ng/ml) 6,568.4 6 1,935.2***** 584.2 6 65.1 6.8 6 5.8 13.6 6 2.8***** 6.7 6 3.7 14.1 6 0.9Tmax (hr)** 2 hr 4 hr 2 hr 4 hr 2 hr 8 hrDuration (hr)*** 0 hr 6.8 hr 0.4 hr 0 hr 0.3 hr 0 hr600,000 IU/kgCmax (ng/ml) 8,438.7 6 1,621.9***** 756.5 6 110.3***** 13.4 6 5.2***** 21.3 6 5.8***** 14.9 6 3.7***** 24.2 6 6.8*****Tmax (hr) 2 hr 8 hr 2 hr 4 hr 2 hr 4 hrDuration (hr) 0 hr 12.4 hr 2.7 hr 0 hr 2.8 hr 0 hr

*Mean 6 SE.**Time of peak (hr).***Duration above NOEC.****ND, Below limits of detection.*****Statistically different from control (P , 0.05).


Regardless of the degree of binding (if any), addedretinol is clearly teratogenic in vitro.

Other retinoid metabolites were also detected in ratsera after a teratogenic dose of RP. For the high dose,tRA reached a maximum concentration of 13.4 ng/ml,13cRA 21.3 ng/ml, 4oxotRA 14.9 ng/ml, and 4oxo13cRA24.2 ng/ml. These compounds were not detected incontrol sera (below the level of detection of 0.5 ng/ml),although other studies have reported very low concen-trations of tRA (0.49 ng/ml) and 13cRA (0.42 ng/ml) incontrol rat sera (Collins et al., ’94). All of these metabo-lites have been shown to have teratogenic activity invitro, and together with unbound retinol may collec-tively or individually be responsible for the teratogeniceffects of an oral dose of RP.

The next part of the investigation was to culture ratembryos in each of the serum samples taken up to 16 hrafter dosing with a teratogenic dose of RP. Embryosdeveloped with abnormal second arches when grown inany of the serum samples taken after the high dose andin the 1-, 2-, and 4-hr samples taken after the low dose.This shows that these samples contained above-threshold amounts of one or more of the teratogenicretinoids. If the elevated retinol levels observed weredue to ‘‘unbound retinol,’’ this could be the majorcontributor to the observed teratogenicity. Maximumelevations in retinol levels were 361 ng/ml for the lowdose and 533 ng/ml for the high dose. This exceeds the‘‘effect’’ concentration for ‘‘unbound retinol’’ in embryoculture, which the in vitro study established was be-tween 187.5–375 ng/ml.

Other metabolites may also be involved. The ‘‘effect’’concentration for tRA was between 6.25–12.5 ng/ml.Levels within and exceeding this range were seen in

some of the serum samples. 4oxotRA was seen atsimilar levels and may show similar teratogenic po-tency (Creech Kraft et al., ’89), although it was notspecifically tested in this study. 13cRA and 4oxo13cRAare both teratogenic in vitro at much higher concentra-tions, i.e., between 250–500 ng/ml. This is far in excessof the concentrations observed in the serum samplesand suggests that these metabolites make little contri-bution to the observed teratogenicity of RP. This is incontrast to humans dosed with isotretinoin, where13cRA serum concentrations are in the 180–210 ng/mlrange, and 4oxo13cRA at 600–800 ng/ml (Brazzell et al.,’83). These two metabolites undoubtedly cause humanisotretinoin embryopathy.

Hence, the results suggest that the observed terato-genicity of RP in rats may be due to the combined effectof elevated concentrations of retinol, tRA, and 4oxotRA.The involvement of retinol depends on whether or not itis bound to RBP. A small additional effect could resultfrom the elevated levels of 13cRA and 4oxo13cRA, andperhaps other unmeasured metabolites such as 9-cis-RA.

Human studies

A number of authors have studied the serum concen-trations of RP metabolites following pharmacologicaldoses of RP in humans. These studies vary in the sex ofvolunteers, the dose, frequency, and duration of dosing,and the form of RP (capsule or meal), as well as whichmetabolites were measured (Table 6).

When volunteers were given 500,000 IU of RP as acapsule or in fried calf liver, serum retinol levelsincreased by 260–280 ng/ml (Buss et al., ’94). Again, itwas not established if this was due to free retinol. Forthe 500,000-IU capsule there were also dramatic in-

TABLE 6. Mean maximum serum concentration and factor increase above endogenous levels of retinoidsfollowing an oral dose of retinyl palmitate in the human*

Dose RP/O Retinol tRA 13cRA 4oxotRA 4oxo13cRA

500,000 IU oral*** 3,100 6 2,700**(ND)

980 6 31(1.33)

87.4 6 60.6(62.43)

67.9 6 28.8(48.53)

17.3 6 7.1(ND)

63.6 6 14.7(30.33)

500,000 IU liver*** 2,300 6 1,500(ND)

940 6 26(1.43)

4.1 6 1.0(2.93)

30.9 6 7.4(22.13)

6.2 6 4.9(ND)

43.0 6 10.7(20.53)

Up to 346,500 IU oral**** NM NM 4.7 6 0.8(3.93)

12.8 6 1.8(8.53)

NM NM

250,000 IU liver***** 3,540 6 1,736(109.93)

800 6 105(1.23)

2.0 6 0.5(2.53)

21.5 6 4.3(19.53)

0.8 6 0.2(ND)

32.1 6 4.9(13.43)

50,000 IU oral****** 2,472 6 1,007(73.63)

,467(no increase)

3.5 6 1.6(2.73)

10.4 6 2.1(7.53)

ND 18.8 6 3.8(5.23)

10,000 IU oral******* NM NM 1.7 6 0.2(1.23)

1.9 6 0.4(1.63)

ND 3.4 6 0.7(1.83)

25,000 IU oral******* NM NM 2.1 6 0.4(1.43)

2.4 6 0.8(2.23)

ND 4.8 6 0.9(2.53)

*ND, undetectable or regularly below limits of detection; NM, not measured; in parentheses, factor increase above endogenouslevel.**Cmax (ng/ml), mean 6 SE.***Buss et al. (’94); oral, single dose of retinyl palmitate (Hoffmann-La Roche) or fried calf liver.****Tang and Russell (’91); oral, single dose of oily retinyl palmitate (Hoffmann-La Roche).*****Arnhold et al. (’96); liver, single dose of fried turkey liver.******Eckhoff et al. (’91); oral, 20 daily doses of oily retinyl palmitate (A-Vicotrat oleosum forte).*******Chen et al. (’96); oral, 60 daily doses of vitamin A supplement (Hudson Corp., Bohemia, NY).


creases in tRA, 13cRA, and 4oxo13cRA (30–60-foldincreases). However, these large increases were notseen when the same dose was given in the form of friedcalf liver. Less dramatic increases were recorded inanother two studies, where slightly lower doses of RPwere administered as a capsule (Tang and Russell, ’91)or as turkey liver (Arnhold et al., ’96).

It is evident from these studies that there is widevariation in individual responses to large doses of RP. Itis also clear that in some individuals, a single oral doseof RP of 500,000 IU can cause very large increases intRA plasma concentrations up to 7 times the terato-genic concentration of tRA in vitro (Buss et al., ’94).

Perhaps of equal interest is the response followinglower doses of RP. Human volunteers were dosed for 60days with supplements of 10,000 or 25,000 IU ofvitamin A, and their plasma concentrations of retinoidswere monitored (Chen et al., ’96). Their meals werestandardized for low vitamin A. For both doses, plasmatRA concentrations were similar to baseline levels. Forthe 25,000-IU dose, 13cRA and 4oxo13cRA steady-stateconcentrations were initially elevated to approximately3.7 and 6.9 ng/ml, respectively, and trough levels re-mained elevated at approximately 2.5 and 6.0 ng/mlthroughout the 60 days. These concentrations are insig-nificant compared to the plasma levels of these com-pounds associated with the teratogenicity of isotreti-noin in the human. Unfortunately, retinol levels werenot measured in this study. However, in a similar studyin which volunteers were dosed with 50,000 IU ofRP/day for 20 days, there was no measurable increasein plasma retinol levels, and RBP remained in a molarexcess (Eckhoff et al., ’91). Hence, from the limited dataavailable, it would appear that 25,000 IU per day do notcause elevations in retinoid levels that are likely to beassociated with teratogenesis.

Human risk assessment

The above information would suggest that vitamin Asupplements of 25,000 IU/day are unlikely to be terato-genic, while 500,000 IU/day would present a verysignificant risk. However, there are pitfalls with thissimple extrapolation. Teratogenic concentrations deter-mined in vitro in rat serum cannot be automaticallyapplied to the human, as there may be significantspecies differences in the protein binding of the testcompound and subsequent transfer to the embryo.However, in the case of human isotretinoin embryopa-thy, there is a good correlation between teratogenicconcentrations of 13cRA and 4oxo13cRA determined invitro, and the maternal serum concentration (Brazzellet al., ’83; Webster et al., ’86).

There is another parameter to be considered thatmay affect risk assessment. For what duration ofembryonic development must threshold levels of terato-genic retinoids be exceeded to cause teratogenicity? In aprevious in vitro study, it was shown that to obtainabnormal second visceral arches in rat embryos, it was

necessary to expose embryos to teratogenic concentra-tions of 13cRA for between 3–6 hr during the sensitiveperiod (Ritchie and Webster, ’91). Exposure for lessthan 3 hr did not cause abnormal arches, even if theconcentration of 13cRA was greatly increased. It is notknown if this finding applies to other retinoids. Sincehuman development is considerably slower than that ofthe rat, a similar period of development in the humanmay be between 12–24 hr. This would imply thatteratogenic retinoids would need to exceed thresholdlevels for this period in the human to cause teratogenic-ity. This is characteristic of isotretinoin exposure in thehuman, where plasma 13cRA and 4oxo13cRA are con-tinuously elevated due to the twice-daily dosing regi-men. This repeated exposure in the human is associ-ated with teratogenicity.

The necessary duration of exposure in the human isunknown, but it must be an important parameter. Inthe past, the area under the curve has been used todetermine teratogenicity, but this variable may beinappropriate without consideration of the duration ofexposure. In order to determine potential teratogenic-ity, two threshold variables must be exceeded, i.e.,concentration and duration. Only then may malforma-tions be induced.

Perhaps the only valid way to extrapolate to an invivo situation is to compare the concentration of thetest chemical in human embryonic tissues with that inexperimental embryos. However, this is currently im-practical during the human organogenic period. Atpresent, extrapolation from maternal pharmacokineticdata has the greatest potential for human risk assess-ment.

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Model predicting the teratogenic potential of retinyl palmitate, using a combined in vivo/in vitro approach