-d

United Nations Scientific Committee
on the Effects of Atomic Radiation

1977 report to the General Assembly, with annexes

NOTE
The report of the Committee without its annexes appears as Official Records of the General
Assembly, Thirty-second Session, Supplement No. 40 (A/32/40).
In the text of each annex, Arabic numbers in parentheses are references listed at the end.
The designations employed and the presentation of material in this publication do not imply theexpression of any opinion whatsoever on the part of the Secretariat of the United Nations concerning
the legal status of any country, territory, city or area, or of its authorities, or concerning thedelimitation of its frontiers or boundaries.

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ANNEXJDevelopmental effects of irradiation in utero

CONTENTSParagraphs

Paragraphs

INTRODUCTION . . . . . . . . . . . . . . . . . . .

Litter size . . . . . . . . . . , . . .
Resorptions . . . . . , , . . • . . .
Mortality in utero . . . . . . . . .
Neo-natal mortality . . . . . . . .
Post-natal and long-term mortality

85-89
90.93
94.97
98-101
102-108

B.

Disturbances of growth . . . . . . . , .
1.
Intra-uterine growth . • . . . . . .
2.
Post-natal growth . . . , • . . . . .

109-120109-115
116-120

C.

Malformations . . . . . . . . . . . . . .
General . • . . . . . . . . . . . . .
2.
External head abnormalities
3. Malformations of the eye . • . . .
4. Malformations of the central ner·vous system . . . . . . . . . . . . .
5. Microcephaly . . . . . . . . . . . .
6. Malformations of the skeleton
7.
Malformations of the extremities .
8. Other malformations . . . . . . . .
9.
All malformations . . . . . . . . .

121-175
121-123
124-128
129-134
135-140
141-146147-156
157-164
165-167
168-175

Other effects

176-180

1-10

2.

3.
A.
B.
C.

Definition of malformations . . . . . . . . . .
Previous reviews . . . . . • . . . . . . . . . . ,Present review . . . . . . . . . . . . . . . . . ,

3.5
6
7-10

I. METHODOLOGY AND TECHNIQUES . . .
A.. General discussion . . . . . . . . . . . .
B.
Biological factors . . . . . . . . . . . . .
1. Genetic homogeneity . . . . . . .
2. Maternal factors . . • . . . . . . .
3. Homozygous and heterozygous
animals . . . . . . . . . . . . . . .
4. Timing of pregnancy . . . . • . . . .
5. Periods of maximum sensitivity ..
C.
Comparative mammalian embryology , .
D. Irradiation techniques . . . . . . . . . .
E.
Lethal effects . . . . . . , . . . . . . . .
F.
Disturbances of growth . . . . . . . . .
G. Malformations . . . . . . . . . . . . . .
1. Observation time . . . . . . . . . .
2. Techniques of observation
H. Statistical evaluation . . . . . . . . . . .
I.
Methodology and techniques in humaninvestigations • . . . . . , . . . . . . . .
J.
The main phases of pre-natal develop·
ment . . . . . . . . . . . . . • . . . . . .

11-52
11-12
13-29
13-14
15-17

4.

5.
6.

1.

18-25
26-27
28-29
30.32

33
34-36
37-38
39-44
39-41
42-44
45
46-49

D.

IV. THE FOETAL PERIOD . • . . . . . . . . . .

181-242A.

Lethal effects . . . . . . . . • . . • . . .
1. Mouse . . . . . . , . . . . . . . . .
2.
Rat . . . . . . . . . . . . . . . . . .
3.
Human experience . . . . . . . . .

181-195
181-185
186-191
192-195

B.

Disturbances of growth . . . . . . . . .
1.
Mouse . . • . . . . . . . . . . . . .
2.
Other animals . . . . . . . . . . . .
3.
Human data . . . . . . . . . . . . .

196-208
196-199200-201202-208

C.

Malformations . . . . . . . . . . . . . .
1.
General . . . . . . . . . . . . . . .
2.
Animal data . . . . . . . . . . . . .
3.
Human data . . . . . . . . • . . . .

209-220209-211212-216217-220

D.

Other effects . . . . . . . . . . . . . . .
1. Animal data . . . . . . . . . . . . .
Human data . . . . . . . . . . . . .
2.

221-242221-233234-242

50.52

II. THE PRE-IMPLANTATION PERIOD . . • .
A. Lethal effects . . . . . . . . . . . .
1. Intra-uterine death . . . . . . . ..
2.
Post-natal death . . . . . . . . . .
3.
Mechanisms . . . . . . . . . . . . .
B. Disturbances of growth . . . . . . . . .
C.
Malformations . . . . . . . . , . . . . .
D. Otl1er effects . . . . . . . . . . . . . . .

53-83
53-70
53-63
64-65
66-70
71.74
75.79
80-83

III. THE PERIOD OF MAJOR ORGANOGENESISA.
Lethal effects . . . . . . . . . . . . • . .
1.
Failure of pregnancy . . . . . . . .

84-18084-10884.............. .

655

Paragraphs

Paragraphs

V. INTERNAL IRRADIATION . . . . . . . . .
A. General
B. Tritium . . . . . . . . . . . . . . . . . .
c. Phosphorus-32 . . . . . . . . . . . . . .
D. Strontium-89 and strontium-90 . . . . .
E. Iodine- 131 . . . . . . . . . . . . : . . . .
F. Other nuclides . . . . . . . . . . . . . .
G. Conclusions . . . . . . . . . . . . . . . .
VI. MECHANISMS OF RADIATION TERATOGE?\'ESIS . . . . . . . . . . . . . . . . . . . .
A. Influence of the maternal organism
B.
Nature of the primary damage
C.
Dose-effect relationships . . . . . . . . .
D. The problem of the threshold . . . . . .
1.
Lowest teratogenic doses . . . . .
2.
Experimental tests . . . . . . . . .
E.
Factors modifying the teratogenic response . . . . . . . . . . . . . . . . . . . .

243-262
243-244245-251
252-254
255-256257-258259-260261-262263-334
265-270271-283284-287288-295
290-291292-295

F.

Radiation quality and LET . . . .
Dose rate and fractionation . . . .
The effect of oxygen . . . . . . . .
Radioprotective
andradiosensitizing drugs . . . . . . . . . .
Significance of the experimental data tomanVII. SUMMARY AND CONCLUSIONS
A. General . . . . . . . . . . . . . . . . . .
B. Methodology and techniques . . . . . .
C.
The pre-implantation period
D. The period of major organogenesis ...
E. The foetal period . . . . . . . . . . . . .
F. Internal irradiation . . . . . . . . . . . .
G. Mechanisms of radiation teratogenesis .
H. Research needs . . . . . . . . . . . . . .

297-300
301-321322-325326-330331-334
335-353
335
336
337
338-341342-345346
347-352353
Page

296-330

Introduction
1.
Since its 1962 report (344), the Committee has
not comprehensively reviewed the data on pathologicaland teratological effects induced by irradiation of the
mammalian embryo and foetus, with the exception of
the effects on the developing central nervous system,
which were considered separately in the 1969 report of
the Committee (345). Much information has accumulated in the meantime: obtained mainly in animal
experiments, it could be of help, if not for theprediction, at least for the interpretation, of the humanexperience. Additionally, reassessments of earlier observations and some new data in man have become
available. This annex therefore reviews the effects
observed in various mammalian species following
exposure in utero, with the aim of extracting
information relevant to the assessment of radiation
effects in man. It deals exclusively with somatic effects.
2.
Radiation exposure of the mammalian conceptus
in utero gives rise to a large variety of short- andlong-term deleterious effects. For many of them,especially those following irradiation during latepregnancy, the pathogenesis is similar to that of lesions
seen after irradiation of animals in the extra-uterine life.
There are, however, effects which may be viewed as the
typical sequelae of irradiation of a developing organism,
which is characterized by major processes of differentiation leading to the structural and functional organizationof organs and tissues. The complex morphological andphysiological events leading to the development of amature animal may be disturbed or disrupted byradiation, and the consequence is the production of
malformations of different nature and degree. Some ofthese defects are so profound as. to be incompatible withthe animal's life in zitero; some can easily be recognizedmorphologically at birth; others cannot even be scoredat the microscopic level and are only manifested at alater stage as functional deficiencies.

A. DEFINITION OF MALFORMATIONS
3.
There is some lack of uniformity in thedefinition of congenital malformations owing to thelarge variety of these lesions. Brent (27) regards as
malformations all permanent defects (morphological,
656

1.
2.
3.
4.

References . • . . . . . . . . . . . . . • . • . . . •711

histological or biochemical) which an organism cannot
repair during the course of its normal growth anddevelopment. Warkany (356), on the other hand, draws
a distinction between congenital anomalies and congenital malformations: he regards the first type as abroad category covering all microscopic, biochemicaland functional disturbances seen at birth, and reserves
the second term only for the gross structural effects inthe new-born animal. According to Stevenson (334), thecharacteristic features of congenital malformations are
their production during intra-uterine life (even though
they may be recognized only at a later stage) and theirmacroscopic anatomical nature.
4.
It is not surprising that this absence of uniformity
in the definition is reflected in different estimates of
induction rates as a function of time or radiation dose. It
is largely a matter of definition whether growth
retardation of an otherwise normal animal should or
should not be regarded as a malformation, or whether
malformations should comprise changes in the life-spanof animal populations, or whether one should include
the malformations seen in animals dying in utero or instillborn animals and which therefore are never scored inlive animals.
5.
There is no doubt that when these problems areapproached experimentally the area of concern should
be carefully defined, so that the design, execution andinterpretation of the work may be carried out properly.
However, when reviewing a large body of experimental
data, the acceptance a priori of firm definitions mightlead to the exclusion of important information. Since itis the Committee's intention to consider all evidence at
hand which might be of help in interpreting the scanty
information available for man, it follows that no
information should be disregarded. Data on genetic
damage are covered in another section of theCommittee's report (Annex H) and data on behaviouraleffects of irradiation in utero have also been reviewed
previously (345).
B. PREVIOUS REVIEWS

6.
Taking such a wide perspective does not imply
that the present contribution attempts to review
extensively most of the work in radiation teratogenesis.

which would obviously be an impossible task. It does
not consider, for example, much of the early work on
animals and man, which made important qualitative
contributions but was often limited to very few
observations or lacked quantitative definitions of therelevant physical and biological parameters. Nor does it
include the work on animals that are phylogenetically
far from the mammals, such as birds, amphibia, fishes
and marine organisms. The subject of embryonicirradiation has been discussed with great care andscientific authority in many published reviews (52, 260,
262, 264, 362, 295, 230, 27, 109, 39, 127, 121, 370.
136, 170, 303, 92). An updated bibliographic
compilation of most published contributions was also
made available to the Committee (336), as well as tworeports on the teratogenic action of external (178) andinternal (178a) radiation sources, summarizing relevant
work in the Union of Soviet Socialist Republics.
Previous reports of the Committee have also considered
general (344) or specialized aspects of radiation
teratogenesis (345). All these publications may beconsulted for background information.

C. PRESENT REVIEW
7.
The developing embryo has some inherent
biological characteristics that have always appealed to
the radiation biologist (295). Differentiation of majororgans and tissues takes place in an orderly sequence atwell defined times after copulation or conception. thetime of which can be established to a fairly good
approximation (see paragraphs 26 and 27). Waves of cell
division, differentiation and migration occur duringorganogencsis at certain times and, since radiation isparticularly effective at these times, it is possible toinduce selectively malformations in organs or anatomical
regions. In addition, the dependence of the mammalianembryo on ·the mother allows the analysis ofabnormalities up to very advanced stages of developmentthat would not be compatible with life in other lower
animals.
8.
Although radiation-induced malformations are
non-specific, in the sense that they can be produced byother teratogenic agents, the use of radiation inexperiments offers some advantages over, for example,
chemical agents. In fact, radiation from external sources
can be administered to the embryo in precisely
measurable quantities within very short times. whereas
chemical substances must cross the placental barrier, andtheir kinetics of uptake and clearance result in unevenand less controlled exposures to the effective drug level.
Furthermore. there is no selective concentration
phenomena since radiation is distributed uniformly overthe irradiated embryo: the selectivity of.the teratogenic
response can therefore be related to the sensitivity of thedifferent structures at the time of irradiation. As withchemical substances, radiation may not be administered
to the embryo without concomitant exposure of themother or, at least, of the pregnant uterus. However, theindirect effects on the embryo, in the case of radiation,
are relatively minor (see paragraphs 265-270).
9.
For all these reasons the irradiated mammalianconceptus has also been a favourite biological system forthe experimental embryologist. It is quite clear thatmost of the work reviewed has actually been carried out

with the ultimate purpose of studying the mechanismsof development. rather than of analyzing the effect ofthe main radiobiological variables. There is insufficientquantitative information on dose-effect relationships andon the influence of dose rate and radiation quality. notonly for the irradiated human embryo, as might be
expected, but for experimental mammals as well. Suchinformation lies often buried in large collections ofsystematic data on kinetics and morphology ofmalformed tissues.
10. Contrary to most previous reviews. this one will
therefore attempt to extract the relevant radiobiologicalinformation from the, published experimental work,
giving particular emphasis to the data of prospective use
in the assessment of radiation effects in the humanembryo.

I. METHODOLOGY AND TECHNIQUES
A. GENERAL DISCUSSION
11. Before reviewing experimental data in detail itseems appropriate to examine some general questions ofmethodology and technique relevant to the study of theteratogcnic effects of irradiation. Consideration of thesequestions is essential for the following purposes: (a) toillustrate some of the variables or conditions known to
influence the induction of malformations and theirscoring in laboratory animals: (b) to point out theconfidence limits and the degree of precision of the dataobtainable: (c) to warn against generalizations which
may not apply to all biological materials andexperimental conditions: and (d) to set in an appropriate
perspective the scanty knowledge available in man.
12. In regard to the animals in which experimentation
has been performed. the mouse appears by far the mostthoroughly studied, followed by the rat, the rabbit, thehamster and the dog. Data on other species are only
occasional. The data in humans represent a separate
category and will be treated as such, not only because of
their importance but also because little control of theexperimental variables can be exerted in this case.
Among the biological variables, the embryonic andfoetal ages at irradiation have been investigated very
extensively in many species. The applicability of thesedata to man, through a comparative evaluation of thetimes at which certain important stages of development
occur. is also an important issue for discussion. The
physical variables affecting the teratogenic effect ( dose,
dose rate, fractionation, protraction, type and energy of
the radiation used) have only been examined in a few
special cases. The conditions of exposure which apply tothe human case and to animal experimentation should
therefore be discussed. in order to ascertain if reasonably
uniform sets of data are being compared.
B. BIOLOGICAL FACTORS

I. Genetic homogeneity
13. Certain characteristics are required for ~eselection of the animals used in embryologicalexperiments. According to Jacobson (J 36) these are657

genetic homogeneity, high fertility, large litter size, anda high degree of sensitivity to the teratogenic agent.
These requirements may in some cases be in contrast toeach other, so that the optimum experimental conditionin any given case is often the best compromise between
the various needs.

14. The high fertility and large litter size areconditions obviously favouring the rapid collection of
statistically large experimental samples; they are
inherent characteristics of the animal species and strainsthat are desirable choices for experimentation. Concerning genetic homogeneity, even in the absence ofknowledge on the relative importance of constitutionaland environmental factors in the induction of
malformations, there is certainly an advantage inselecting the genetic background as uniform as possible.
It follows therefore that the animals should be as
homogeneous as possible in their genetic constitution,although some inbred lines may show a high natural
incidence of specific malformations. The susceptibility
to radiation-induced teratogenic effects is very high in
inbred strains, but this advantage is often balanced bydecreased reproductive and somatic vigour. In such cases
hybrid animals obtained by crossing inbred strains may
be the solution of choice to combine vigour and genetic
homogeneity. Several approaches to· this problem, allequally justifiable. appear to have been followed in thework reviewed.

2. Maternal factors

15. Quite aside from the response of the embryo to
any teratogenic agent. there are certain biologicalcharacteristics of the animals used that must be taken
into account when planning experiments. The quantitative features of reproductive performance and theirvariability are the most obvious ones. and some data are
available on these points. Analyzing the interstrain
differences in the breeding capacity of six inbred strains,
Ehling (80) has drawn attention to the fact that the
intraspecies and interspecies variabilities are of comparable degree. In the mouse, for example, the mean
number of offspring per fertile female ranged from 20.5
to 73.4 in various genotypes examined; the embryonicsurvival varied between 57.1 and 75.6 per cent and themean number of litters per female between 5 .9 and 11.2.
Differences in reproductive performance between
F 1 hybrid females from reciprocal crosses appearing
after irradiation at 50 R 1 have been attributed to a
genetically controlled maternal factor (81 ).

16. But variability in reproductive performance is very
high even within the same strain. According to Rugh andWohlfromm (291), the first litter is always the smallest
and the litter size tends to increase in the following threelitters. The average litter size is greater among
multiparous than among primiparous females ofcomparable ages and the incidence of abnormal
offspring, although never very high in control mothers,
tends to increase in successive litters. Many authors
1
Throughout this Annex, the radiation units quoted arethe_ roentgen CR) or the rad, as they appear in the papers
rev~e,~ed. No attempt has been made to express the frequency ofradiation effects on a common basis of unit absorbed dose.

658

therefore tend to standardize experimental conditions
by using second-litter animals for analysis of teratogenic
responses, since the first litter usually has high mortality
and small size. Correlations have been reported (I 36)
between litter size at first delivery and litter size,
conceptus number and weight of offspring of the second
pregnancy. These correlations could to some extent
guide the experimenter in the selection of suitable
experimental animals in second litters on the basis of theoutcome of the first.
17. Regarding the incidence of malformations after
irradiation, the previous reproductive history of themother appears to be of some impmiance. Thus, theincidence of intra-uterine deaths and resorpti6ns after
200 rad received at 8.5 days post-conception (p.c.) isalmost double among breeders 14 months old thanamong primiparous young females (287). Similar
observations have also been made after doses of 2-32 rad(175). The incidence of gross aberrations of the central
nervous system, which is 30 per cent higher in old
females, is reduced after correction for in utero
mortality, but a slight excess of malformations of theorder of 3 per cent is still evident in offspring from oldmothers (287). In another report (290) primiparous
females, even though of the same age as the multiparous,
were shown to produce more abnormal embryos. Theseand other data emphasize the need for careful control ofthe mother's age and of her past breeding experience inorder to ensure good experimental material anduniformity in the teratogenic response. It may be added.
however. that in a careful and extensive study by
Jacobsen (136) of all these maternal factors. they wereassigned a relatively minor influence, in any case notsufficient to result in gross misinterpretations of thedose-effect relationships for the induction of malformations. It may also be mentioned that the radiation
history of the female prior to mating may alter some of
the reproductive parameters, sometimes with an increaseof the Jitter size (104). Other factors, e.g., immunological incompatibility between the mother and foetus, areknov./11 to alter the reproductive performance of
mammalian species. but their effect, particularly inregard to the induction of malformations, has not been
made the object of special consideration in this Annex.
3. Homozygous and heterozygous animals

18. The influence of genetic constitution in the
production of malformations and the relative susceptibility of homozygous versus heterozygous animals havereceived some attention. particularly in mice, where theavailability of inbred strains make such analyses easier.
Russell and Russell (303) tested the relative sensitivityof three different but genetically uniform mouse
populations ((CS? X NB)F 1 , BALB/C and 129). taking
as the end-point the incidence of animals having 13.
more than 13 or less than 13 ribs on each side ;f th~
body. They concluded. on the basis of the variation
between and within strains of the control animals. thatthe expression of this character results fro,m acontinuous array of variation of the underlying process.
TI1ey therefore emphasized the need for great caution in
drawing conclusions about relative sensitivity withregard to characters having a continuous variability. In
fact. particularly under such conditions. a change in theend-result would not necessarily indicate a corresponding difference in primary radiation damage (seeparagraph 287).

lethality (neo-natal and early post-natal). In all cases
inbred genotypes responded to a greater degree thanhybrids.

19. Dagg ( 68) examined the teratogenic response of
three strains (BALB/cGn, C57BL/ks and 129/RI) andsome of their hybrids, showing that the differences inmalformation response between strains observed after
irradiation at 11 days p.c. is not found when theirradiation occurs at 10 days p.c. or when the embryos
are irradiated at approximately equal crown-rumplength. Thus, the differences observed may be due todifferent developmental (as opposed to chronological)
age of the embryos at the time of exposure rather thanto an intrinsic difference in susceptibility.

23. It may thus be concluded that the genetic
constitution has an influence on the degree of theobserved lethal and teratogenic responses. But just how
much of the differential response might be due tointrinsic variation of sensitivity or repair in various
species and strains and how much, on the other hand,might be attributed to small variations of thedevelopmental age at irradiation remains unclear.
Variations of the rate of development are also
genetically controlled, b.ut their effect is indirect anddue to rapid changes in embryonic sensitivity in thecourse of differentiation. The heterozygous conditions
seem invariably to result in a higher resistance tolethality and malformations. but the need to irradiatesimilar developmental stages for meaningful quantitative
comparisons remains valid. Finally, the type of effectscored may affect the conclusions about relative
sensitivities, especially when it is based on somecontinuous variable rather than on discrete morphological traits.

20. Nash and Gowen (218) followed the post-natal
growth response of three inbred strains (Ba, K, and S)
and all their hybrids, including reciprocal crosses. atseveral gestational ages and after various doses. They
found evidence that developmental differences are understrong genetic control. These differences may to someextent be related to even minor strain-related changes ofthe developmental age at the time of irradiation, butthey can be exerted in part through the ability of single
cells and organs to repair radiation damage. A similar
influence of the genetic background was also found withrespect to the duration of life-span (219); here again
the genotype was shown to condition embryologicaldevelopment at irradiation. and the differences between
strains were particularly evident at high irradiation doses
given at late pregnancy stages.
21. In a review centred on the question of outbred
versus inbred animal strains in experimental teratology,
Smithberg (327) has considered all relevant problems
exhaustively. He concluded that the selection of themost appropriate experimental animals cannot be madeon the basis of general principles, but rather depends on
the specific aims to which research is directed; indifferent instances and for different purposes. either
genetically pure or outbred animals may be chosen withequally good justification.

'J

22. The available reports almos.t unanimously showthat the heterozygous condition confers a higher degreeof resistance to the teratogenic action and to the killing
effect of radiation in utero. To cite just some of theevidence, Rugh and Wohlfromm (288) have reported anofhybridembryos
increasedradioresistance(C57BL/6 X CFl) exposed to 200 Rat 8.5 days p.c., bycomparison with the parental strains. At 18.5 days, infact. only 5 per cent of the implants of the C57BL
animals were normal. while for the CF 1 and the hybrid
mice the respective values were 38 and 44 per cent. The
small difference between CFl and hybrids was
attributed to heterosis of the hybrids. Dagg (68)reported that hybrids from crosses of C57BL and
BALB/C were more resistant to lethal effects and to
deforming effects in the foot, palate and tail thanhomozygous C57BL embryos. Jacobsen ( 136) refers also
to a higher degree of sensitivity of homozygous HC micerelative to (HC X C3H) hybrids. Finally, a differential
response between inbred and hybrid mice of three
genetic stocks (BALB. K and S) was shown by Nash
(217) in regard to both induction of malformations and

24. The position of the implants in the uterus is ofrelatively minor importance in relation to the occurrenceof abnormalities, a question that has been examined by
Hashima (114), Trasler (342) and Jacobsen (136).
Hashima reported that offspring in the ovarian positionof the uterine horn had a significantly reduced weight.
Trasler found that embryos in the same position had an
incidence of cleft palate which was higher than in allother positions. On the contrary. Jacobsen showed thatembyros in the intermediate position were apparently at
a disadvantage when the weight of offspring and theplacental weight were taken as indicators of the optimal
uterine environment. The incidence of skeletal malformations and resorptions were unrelated to the uterine
position. These results, although contradictory in thevarious experimental conditions tested, show nevertheless that the position of the embryos in the uterus may
perhaps affect the developmental potentialities and themalformation incidence of the irradiated embryos.
25. Important quantitative differences between
experiments performed in summer and in winter werealso reported by Jacobsen (136). In the experience of
this author. the seasonal variability was so high that it
would have masked the effect of doses up to 100 rad
had the results not been evaluated separately for the two
seasons. In general, summer samples were found to beless radiosensitive. It follows that experimental data
must be tested against control groups run at the sametime.
4. Timing of pregnancy
26. There is one aspect of breeding technique that
requires special attention: the accurate timing of mating
and fertilization. In rodents. successful mating isfollowed by the appearance of the vaginal plug; the timeof scoring of this sign is often the starting point forcalculations of the embryonic and foetal ages, especiallyin mice (266). However. many workers consider thistime as day O and others as day I after fertilization. thus659

creating inconsistencies among their experimental series.
In rats, the presence of sperm in the vaginal canal is also
used as an indication of mating (32). Useful andup-to-date information on the reproductive physiology
of various experimental animals for an accurate timingof pregnancy may be found in the following references:mouse, 328, 266: rat, 223; rabbit, 109: dog, 6; monkey,
249.
27. Owing to differences in mating techniques used byvarious laboratories and because of the large time
variability between ovulation and fertilization, there areusually differences in the estimation of the conception
time and therefore of the gestational age. In someinstances, gestational age is measured from the observedor presumed time of coitus and in other cases from thepresumed time of fertilization or conception. calculated
from the time of coitus or estimated back from the timeof scoring the vaginal plug. However, the sacrifice ofsome experimental animals at irradiation time followedby direct examination of the conceptus, measurementsof the length of embryos or observation of external
morphological features, affords a better timing of thedevelopmental stage at the time of irradiation (299).
Such procedure is, however, carried out only rarely.
Owing to the uncertainties referred to above, mostestimates of embryonic age should not be taken to be
more accurate than about ± 0.5 day, even under the
best-controlled conditions. It should also be mentionedthat marked differences in apparent age may be seen
within embryos of the same litter, especially at theearlier ages (241, 4), and that there is therefore a limit to
the improvement of timing control (135).
5. Periods of maximum sensitivity
28. The need for accurate control of the gestationalage in experimental radiation teratogenesis stems fromthe evidence that there are certain well defined "critical
periods" in embryonic development when some types ofmalformations can be produced more readily thanothers. The concept of critical period was first developedin 1911 from observations on fertilized amphibian eggs
(14). Although repeatedly discussed in the following
years (337), its importance was not fully and
immediately realized. Actually, many of the earliest
observations did not sufficiently specify the age at which
the embryonic or foetal material had been exposed. The
first systematic investigation of this concept in mammals
was made by Job et al. ( 139) in 1935. They showed that
exposing rats to 90 R at day 9 of gestation produced
mainly hydrocephalus, whereas defects of the eye andthe jaw were preferentially induced by irradiation on
day I O and I I , respectively. In this work thecorrelations between teratological effects produced atsome specified times and the stage of differentiation of
the relevant embryonic tissues at such times were clearly
established. It was also pointed out that rather low doses
are needed to obtain ·'specific" teratogenic responses
and that "specificity" does not imply that some
malformations can only be produced at certain times but
rather that a given dose is more effective at some stages.
29. Later experience has shown that some malformations are more likely produced at certain times inpregnancy and that the period of sensitivity may be
660

broadened by raising the dose. These observations havealso indicated that susceptibility to malformationinduction is not sharply defined in time but extends to alesser degree before or after the critical period and that
accurate mapping of these critical periods cannot bemade at high doses (295). The data of Russell (295.
296), Rugh (261) and Rugh and Grupp (278) showed
conclusively that these basic concepts hold generally
true for many types of abnormalities in the mouse;
Warkany and Schraffenberger {357), Wilson (362) and
Brent {27) extended this notion to the rat. It is now
commonly accepted that there are "stages of maximal
sensitivity" in the teratogenic response to radiation(137). This new terminology recognizes that such
periods cannot be defined for all doses of radiation andis in agreement with the definition of the "critical
period" for teratogenic changes given by Russell (295) as
the "developmental interval during which radiation must
be applied to produce that change if the dose ofradiation is the lowest one that gives a detectable
incidence of that change".

.

J

.

..'1,;

C. COMPARATIVE MAMMALIAN EMBRYOLOGY
30. In view of the existence of periods of highsensitivity for the induction of certain malformations,which can be traced back to important stages in theorganization of the relevant structures, it is important tostudy the comparative embryonic development of thedifferent experimental animals. This comparative study
is necessary to be able to generalize experimental
findings and also to extrapolate results from one species
to another. Such an exercise in comparative embryology
has been attempted many times but the results arenecessarily approximate and imprecise.
31. The general pattern of embryonic differentiation,
which is qualitatively similar in all mammals, makes it
possible to specify the approximate time of occurrenceof certain easily recognizable ontogenic events, as shownin table I. This table summarizes pre- and post-implantation events in six mammalian species used in radiation
teratogenesis on the basis of numerous publications. It
will be noted that the occurrence of certain events may
be timed rather precisely, especially at the very early
embryonic stages. However, as pregnancy proceeds andthe structure of the embryo becomes increasingly
complex, there is no single embryonic event for which acomparative analysis can be performed on the basis ofobjective parameters. The number of sornites may be
used as a possible reference of staging, but it remains
true that the time of appearance of each structure in theembryo has a mean that is difficult to measure and avariance that probably increases with the age at which
the structure originates {241).
32. Besides the inherent variability of the onset of thevarious structures, their development in various animal
species may be advanced or retarded with respect to thedevelopment of the whole embryo. Therefore. theappearance of single structures cannot be accepted as avalid reference. It is instead necessary to make theassessment on the basis of several relative measurements
referring to various systems (nervous, digestive, heart
and circulatory), a procedure adopted. for example, byl't

TABLE 1. DEVELOPMENT OF EMBRYOS IN SOME MAMMALIAN SPECIES
Age
(days
p.c.) Mouse

1
1.5

2
2.5
3
3.5
4
4.5

2 cells
4 cells
5-8 cells
9-16 cells
blastocyst

8
8.5
9
9.5
10
10.5

4 cells
8 cellsmorulablastula

early blastocyst
late blastocyst
implantation

RabbirMan

2 cells
4 cells
8 cells
16 cells
morulaearly blastocyst

5-8 cells
9-16 cells

late blastocyst

blastula

2 cells
4 cells

implantation

implantation
primitive streak
5 somites
neural groove

first somites

egg cylinder

liver diverticulum
anterior limb bud

primitive streak
neural plate
fust somitesorganogenesis
optic vescicle
20 somites

proamniotic cavity
neural groove
1-4 somitesorganogenesis
heart formationneural tube closed
torsion of embryo

early blastocyst

limb budformation

posterior limb
budanterior limb bud
10 somites
open eyelid
closed eyelid

forelimb budslate blastocyst
germ layers, extra~mbryonicmembranes

foetal period
foetal periodbirthfoetal I period
separation ofdigitsprimitive streak
implantation

17
18

head process
first somites

19
19.5
20
20.5
21
21.5
22
22.5
23
25
28

Dog

implantation
organogenesis
primitive streakhead process

11
11.5
12.5
13

14
15
16

Hamsrercleavage

implantation

5
5.5
6
6.5
7
7.5

Rar

palpebral fissure
closedbirthprimitive streak
foetal II period
early somites
birthlimb buds

15 somites
tail budend of majororganogenesis

30
31.5
32

appearance of heartneural groove
circulation of blood
liver diverticulurn
optic sulcus
torsion of heart tube
primary brain, G.!.
tract and derivatives
optic vesciclebirthmajor organization ofheart structures, limb buds
optic vescicle into optic
cup, lens formation,
lung buds

34
35
36
37

early foetal stage
thickening of retinal layers
arm, forearm and hands
recognizable
outline of digits (hand)eyelids appear
definite limbs established

40
43
60
63

birth

,I.

Sources: 3, 6, 22, 59, 92, 102, 105, 108,195,223,225,241,245,247,262,266,295,328.

Otis and Brent (241 ). Following such a rationale, theseauthors have constructed a table and a graph for theestimation of equivalent ages in mouse and humanembryos, showing that the rates of development in thetwo species cannot be related by any simple continuousfunction. The estimates are certainly affected bysubstantial uncertainties but provide some average
estimate of approximate age equivalence based on theactual developmental stages in various embryos. Rugh
(266) has reported some data on the age equivalence ofmouse and rat' embryos. Using this information, it ispossible to establish correlations of embryonic ages
661

,
I

between the species as shown in table 2. It should not be
expected that such correlations would apply to minor
details of the embryonic development; on the otherhand. they certainly serve the useful purpose of allowing
rough comparisons of pregnancy stage and embryonicage between these species. The approximate embryonicdimensions of the three species are also given in table 2
for reference purposes.
TABLE 2. EMBRYONIC AGE AND GROWTH DATA IN SOME
MAMMALIAN SPECIES
Mouse
Age

Length(days p.c.) (mm)Age

Length(days p.c.) (mm)Age

Length(days p.c.) (mm)

1

0
1
2
3
4
4.5

2

2
3.25
4

5
5.5
6
6.5
7
1.5
8
8.5
9
9.5
10
10.5
11
11.S
12
12.5
13
13.5
14.5
14
15.5
16
16.5
17-19

Man

Rat

2.0
2.2
3.3
3.8
5.2
6.2
7.2
8.9
9.4
9.8
11.2

5
6
6.75
7.25
1.5
7.75
8.5
9
10
10.5

3

0.13
0.17
0.23

0.5-0.6
0.8
2.0

11

6.0

11.5
12.125
12.5

12.8

13
13.5
14.5

14.0

9.0

36. The present report does not discuss the merits or
disadvantages of the different ways of measuring
lethality and their possible relationships; they are part ofthe experimental methodology under which thereviewed observations have been made. It should
however be realized that even apparently minor
variations of technique may yield quite different
numerical estimates, even though they might reflect
basically the same end-effect, namely radiation-induced
lethality. For example, scoring dead offspring prior to or
after birth may lead to quite different conclusions, since
parturition trauma and mother cannibalism may modify
the results profoundly. When reviewing the experimental
data, the above comments should be kept in mind toevaluate correctly the reported death rates.

38
47

12.0
17.0

F. DISTURBANCES OF GROWTH

65

40.0

4

5-6
7-8
9-10
11-13
14-17
18-20
20.5

0.15
0.40
1.5

22

25.5
26
27
28.S
31
33.5
36
36.5

2.4

3.0
4.2
4.5
7.0

15.5
16

19.0

17-18

29-32
84.5

19-22

Sources: Same as table I.

D.

IRRADIATION TECHNIQUES

33. The usual recommended procedures for theexperimental irradiation of small and medium-size
animals (130) are fully applicable to experiments withpregnant animals. In special instances, irradiation of thepregnant uterus or of a single uterine horn with shieldingof the rest of the mother's body or parts of it has been
adopted in mice (240) and rats (362, 32), particularly toelucidate the direct action of ,radiation on the concept us,
as opposed to possible indirect effects mediated byirradiation of the mother. Such procedure allows also aproper comparison between irradiated and control
animals belonging to the same litter. Specific problems
encountered in the irradiation of the foetus by internal
emitters are discussed in chapter V.

E. LETHAL EFFECTS
34. Death of the offspring appears to be one of themost widely reported effects following irradiation in

662

-~

35. Pre-natal mortality has been studied by means ofseveral indicators, including the complete interruption ofpregnancy, the percentage of resorbed conceptuses, thepercentage of embryos alive pre- or post-partum, and theaverage litter size before or after birth. In a few instances
tentative estimates of the LD 5 0 at or just before term
have been reported; these estimates can more readily becompared with data on post-natal whole-body irradiation. Neo-natal mortality and in some instances thelethality within the first month of delivery have also
been reported. Finally, there are data from animals
irradiated in utero and followed for the rest of theirpost-natal life for the purpose of estimating long-term
parameters related to the time and mode of death.

17.6

13.7
16.1
19.8

utero, and the work reviewed covers embryonic, foetal,
and neo-natal mortality in several animal species.

37. Although there may be doubts as to whether or
not abnormal growth or growth retardation should be
included among the radiation-induced congenital malformations, these effects have been widely described inanimals and man following intra-uterine exposure. As inprevious reviews of the Committee (344, 345), theseeffects are dealt with in the present report.
38. A review of the foetal, maternal and environmental factors that could affect foetal development has
been published by Brent and JeJ}sh (37). Thispublication deals mainly with growth retardations in thehuman foetus and discusses at some length theapplicability to man of data obtained in polytocous
experimental animals. It also reviews the relationshipbetween teratogenesis and growth retardation. pointing
out that although teratogenesis may be viewed as adifferential growth disturbance of some tissues and both
effects are often elicited by the same teratogenic agent,
there are reasons to consider them separately at thepresent stage of knowledge. Whatever the etiology or thepathogenesis of growth retardation may be, it can beessentially defined as the failure of the foetus tomaintain its normal expected growth potential at any
gestational age. as measured by some parameter of foetal
growth such as its weight or length. Frequently foetal
growth retardation leads to growth defects in theextra-uterine life. This is particularly the case whenretardation is induced by radiation.

r

G. MALFORMATIONS

I. Observation time
39. The time at which teratological effects should be
examined is of great methodological relevance and isobviously dependent on the effect under study. Thereare effects that can only be observed under certain
conditions and, in general, the magnitude of the effects
is critically related to the observation time.
40. If lethality during the intra-uterine stages (pre- or
post-implantation) is the end-point of interest, mortality
must definitely be scored before birth. Mortality
immediately after birth cannot be expected to reflectmortality in utero because the trauma of delivery andcannibalism may seriously affect the estimates. Alternatively, neo-natal, 30-day or long-term mortality may
be the relevant end-points, in which case theobservations should be conducted at the appropriate
times. If growth retardation is the end-point, theselection of suitable conditions of scoring must take into
account that the percentage weight reduction increases
during the first few davs after irradiation (362) and thatthe time for maximum effect is probably a dosedependent variable (294, 362).
41. When studying the incidence of malformations,some authors score them after delivery (294, 280), but
most workers prefer to sacrifice the mother just before
term and to observe the foetuses in situ, in order tocollect information that would otherwise be lost
{position of the foetuses, presence of resorptions,placental form and weight, presence of intra-uterine
bleeding etc.). Also, cannibalism practised preferentiallyon malformed offspring would alter quantitative
estimates. The technique of sacrificing the motherbefore birth is adequate and accurate for the study ofmost types of malformations. However, there is no
general rule for selecting the best observation time.
which depends on the particular end-point under study.
2. Techniques of observation
42. Quite different data may result from theexperiments, depending on the technique of examining
the embryos. For this reason few papers among thosereviewed are entirely comparable. Some investigators
have examined, in more or less detail, only the externalfeatures of the foetus. Others have dealt particularly
with special types of malformations (e.g., of the nervous
or skeletal system) or have given elaborate morphological descriptions of their findings, sometimes with few
numerical incidence data. Others again have been mainlyinterested in the pathogenesis of malformations and havetherefore examined histologically the embryos soonafter irradiation, often without relating the histologicalchanges to the induction rate of malformations at laterstages.
43. The combined effect of the large variety ofexperimental techniques, the differences between species
and strains. the difference of gestational age at
irradiation and the wide range of types of radiation anddoses utilized, render any quantitative comparison of all
the data virtually impossible. Reviews must therefore

rely on the internal consistency of a single experimental
series in order to prove certain points of interest. Under
these conditions, only generalizations of gross qualitative
observations are possible.
44. Detailed procedures for autopsy of irradiatedfoetuses have been published (294, 297). In general,they involve necropsy of the mother with careful
examination of the uterus, foetal membranes andresorptions and examination and weighing of theplacenta. The external examination of each foetus
consists of a number of morphological characterizations
and measurements: foetus shape and length; tail shapeand length; morphology pf anus and genitalia: abnormal
posture of limbs: number, shape and position of digits:
shape of head, jaw and mouth: exencephaly; position,
size and morphology of ears, eyes, nostrils and nasal
septa; presence of spina bifida, exomphalos and ventral
hernia; etc. Each foetus is then eviscerated carefully forthe macroscopic study of morphology and position ofthoracic and abdominal viscera. If needed for examina·tion of skeletal changes, the foetus may subsequently beprocessed by fixation, staining and clearance of the
carcass. A large variety of morphological observations
have been carried out on the embryo and foetus. fromthe simplest measurements of length and weight andconventional light microscopy to the most refinedelectron microscopical observations.

H. STATISTICAL EVALUATION
45. Statistical treatment of the data obtained inradiation teratology experiments does not usuallyrequire sophisticated techniques. Unless subtle questionsof correlations between types and degrees of abnormalities are examined. simple statistical techniques forcomparison of means and distributions are adequate.
The papers reviewed only seldom refer to experiment
design on the basis of knowledge of the experimental
material or of the previous pilot work in relation to theexperimental variables to be examined. In many cases
the number of observations is small and, as aconsequence, the statistical significance of the data israther weak. This may be explained by the difficulties in
obtaining appropriately timed and controlled embryonicmaterial for irradiation, requiring a special organizationof the animal quarters, special training of personnel andhighly specialized techniques of observation. But it may
also be attributed in part to the predominantly
descriptive character of traditional embryological research. Experimental data often include extremely
detailed recording of minute abnormalities, reported
sometimes without appropriate homogeneous grouping
and often compounding major malformations withtrivial changes. For these reasons the Committee feelsthat information of a more gross nature, such as thefractional incidence of grossly malformed foetuses,
might often be more informative for the purpose of
assessments of the effects of radiation.

I. METHODOLOGY AND TECHNIQUES IN
HUMAN INVESTIGATIONS
46. The wealth of information provided by wellplanned research on experimental animals far outweighs
the information derived from observations in man. Many663

of the human data are inadequate on account of one ormore of the following reasons: (a) insufficientknowledge of the developmental stage at irradiation; (b)Jack of precise estimates of the absorbed dose in theembryo; (c) malformations can be scored only after
birth and on the basis of medical examinations andlaboratory data; (d) the numerical limitations of theobserved samples, often consisting of single observations,and (e) the difficulty in obtaining adequate control
groups. For all of these reasons, most of the quantitative
conclusions derived from human data are rather
uncertain.
4 7. The embryonic or foetal age at irradiation is only
rarely known precisely: the majority of the publications
reviewed refer to irradiation performed during the first,
second or third trimester of pregnancy, and in very few
cases is the age of the sample under study specified bymonth. In addition, Nishimura et al. (226) pointed out alarge difference between the developmental ages
according to Streeter's horizon and the cluonologicalages estimated from ovulation in human embryos,
showing a remarkable variation of morphological age fora given chronological age. It is therefore difficult tocompare the stage sensitivity between man and other"
species on the basis of developmental scales such as thatgiven in table 2, and this fact greatly limits theusefulness of the available observations. Dealing
specifically with malformations, the most interesting
period would be the second to the sixth week of thehuman embryonic life, because the induction rate ofmalformations is expected to be the highest on the basisof extrapolation from animal data. However, mosthuman surveys refer to the last two trimesters or to thesecond half of pregnancy, where growth disturbances,rather than teratological defects, are the most common
findings (74, 235).

(248, 371, 372). Critical re-evaluations of the doses
received by the exposed individuals were performed atvarious times (8, 115), but only the most recent reports
include reasonably satisfactory dose evaluations (196).
Other methodological and technical problems encountered in the analysis of the human experience are
discussed in other sections of this report.

J. THE 1WN PHASES OF PRE-NATAL
DEVELOPMENT
50. The development of the conceptus in mammals is
usually divided into three major phases: the pre-implantation phase, the period of major organogenesis and thephase of foetal development. Implantation of the early
embryo into the uterine mucosa marks the separation
between the first two periods. Although implantation is
given in the reports at a certain day p.c., it should be
remembered that the complete process, from the initial
contact of the blastocyst with the uterine wall to its firm
attachment through the erosion and invasion of theuterine epithelium by the trophoblast, may last
1-1.5 days in the mouse (266). In man the whole process
may take a few days. A clear-cut separation between theembryonic and the foetal periods is even more difficult
since the transition is marked by the end ofdifferentiation and the growth of the newly formed
organs in an animal which has attained the characteristic
morphological features of the species. Thus, theconventional limits of the three developmental periods,
which are given for various species in table 3, are only
rough approximations.
TABLE 3. APPROXIMATE TIME OF THE BEGINNING AND
E:ND OF THE MAJOR DEVELOPMENTAL PERIODS IN
SOME MAMMALIAN SPECIES{Days p.c.)

48. The estimation of radiation dose in humaninvestigations is also qualified by large uncertainties. In
many cases the doses have been assessed indirectly. The
earliest reports on children irradiated in utero, in thecourse of radiotherapeutical treatments of the mothers,
are generally of clinical nature; furthermore, theimportance of recording the physical parameters of theirradiation was then not fully recognized. Dekaban (71)examined over 200 literature cases of pelvic x irradiationof pregnant women and found that only about 10 per
cent of them provided sufficient information (half-value
layer, energy, distance, size of fields, current, irradiation
time etc.) to allow crude estimation of doses. Even in
the most recent and best planned epidemiologicalsurveys (235), where the same apparatus and standardized techniques were involved and the average per caput
dose in the exposed group was therefore accurately
known. the individual doses showed a fairly large
variation.
49. Most of the early Hiroshima and Nagasaki data
were analyzed in terms of the distance of the irradiatedpersons from the hypocentre (371, 372) and theirlocation with respect to various types of shielding
materials (220). In addition, clinical symptoms of earlyradiation reaction shown by the mother helped to define
the possible range of doses received by the child in utero

664

Pre·

Species

implantation

Hamster
Mouse

Rat
RabbitGuinea-pig
Dog
Man

0-5
0-5
0-7
0-5
0-8
0-17
0-8

Major
organogenesis
6-12
6-13
8-15
6-15
9-25
18-30
9-60

Foetal pen·oa
13-16.5
14-19.5
16-21.5
16-31.5
26-63
31-63

60-270Sources: Same as table I.

51. The division in three phases mentioned above
corresponds approximately to the most significant
developmental events in the embryo and is also suitable
for the description of radio-embryological effects, which
are very different in nature and degree in the tluee
periods (302, 303). For example, pre-natal death ischaraGteristic of the pre-implantation phase (seechapter II) while neo-natal death and malformations areassociated particularly with organogenesis (see chapter III). Irradiation during the foetal stage does not leadto gross malformations but rather to defects of growth,particularly in the central nervous system and gonads.
or, at high doses. to post-natal death (see chapter IV).
Carcinogenic effects following irradiation in utero are
considered in Annex I.

52. This Annex deals in tum with the three periods ofintra-uterine development. Within each period. it
considers the effects observed in different animal
species. Many of the experiments reviewed were actuallydesigned to test the comparative effects of irradiation
during different developmental stages. In general, theseexperiments will be described and discussed in thesection dealing with the period when the mostsignificant observations were actually made, in order toavoid unnecessary repetition.

II. THE PRE-IMPLANTATION PERIOD
A. LETHAL EFFECTS

I. Intra-uterine death
53. Russell and Russell (300) and Russell (295) havestudied the lethal effects in pre-implantation mouseembryos after 200 rad administered each day between
0.5 and 4.5 days p.c. The embryos were observed at 10.5
and 13.5 days after mating, or 6 to 13 days
post-irradiation. Embryos at the very early stages(0.5-2.5 days) were found, on the whole, to be moresensitive than those 3.5-4.5 days old. Death occurred
long before the time of the first observation, so that any
abnormality potentially present in these embryos could
not have been recognized. Three modes of death wereidentified: (a) the death of entire litters before
implantation, which was inferred from the excess of
non-pregnant females over non-irradiated controls at thetime of observation. This modality of death wasestimated to account for about one third of all irradiatedembryos: (b) individual deaths, also at the earliest
pre-implantation stages, manifested by the decreased
number of implants per pregnant female; (c) individual
post-implantation <lea~, assessed from the decreased
percentage of living embryos in the irradiated samples.
54. Most of these conclusions were confirmed by thework of Rugh and Wohlfromm (286), giving roughestimates of the intra-uterine LD 5 0 for embryos
irradiated at various stages of pregnancy and observed at
18 days p.c. The greatest sensitivity was shown to occur
at 0, 2 and 3 days p.c. with a relatively resistant phaseon day 4 p.c. These early embryos, irradiated prior to
implantation, rarely lasted sufficiently long to die as
foetuses and were generally resorbed. The estimated
LD 5 0 for the pre-implantation stage is shown in table 4.

TABLE 4. ESTIMATES OF PRE-NATAL LD 50 AND POST-NATAL LD 50 / 30 FOR IRRADIATION OF MICE AJ>m RATS IN UTERO
Mice

(rad)

(days p.c.)

2
3
4
5
6
7
8
9

10
11

e
lt
1e

:h
:e
is

~e
re
.pad
ill,
is,
{).
ue

12
13
14
15
16
17
18
19
20
21
Reference

Post-natal
LD,o/3oPre-natal LD 50

Age

0
1

Rats

100
350
125
140
330
>200
356
380
200
165
225
} <200255
425} 200-300 560
700>700} >300
>750
>800
>800

Pre-natal LD, 0

Post-natal LD 50130
(rad)

(rad)

(rad)

1,~120-150-200

80-160

400
200
155
140
120
160
175
235
275
315
365

140
150
215
250

120
120
160
210
240

>270
}

>110

185

} ) 110-220 214
>220

500

600

273
295

286

68

217

289

55. A very careful analysis of the sensitivity of themouse embryo to lethal damage during the first 48
hours of development was performed by Russell andMontgomery (299). Within the first 24 hours and with
exposures of I 00 R, very large variations of sensitivity
were observed between developmental stages separated
only by a few hours. With exposures of 200 R, thesensitivity, as shown by the pre-implantation loss. wasfound to be extremely high shortly after sperm entry
and during the earlier pronuclear stages. At the two-cell

362

27

324

212

253

322

stage it remained lowest to increase again at the
beginning of second cleavage. Death occurred at random
about the time of implantation and there were no lateembryonic or foetal deaths.
56. Rugh and Grupp (278) and Rugh (263) studied
the dose-effect relationship for embryonic death in CF I
mice irradiated at 0.5 or 1.5 days p.c., using fiveexposure levels ranging between 5 and 25 R. Under these
conditions, the percentage of resorbed embryos showed665

related increases of the rate of resorption, but the author
states that, although the increase of the rate ofresorption is statistically significant in both theirradiated groups, the difference between the two groups
is short of significance. If a linear regression analysis iscarried out for the two sets of data obtained at 0.5 daysp.c., the calculated average death increase is found to be
1.06 per cent per roentgen (P<0.01) for Rugh andGrupp's series (278) and 1.13 per cent per roentgen(P<0.10) for Ohzu and Makino's data (232). The two
estimates are therefore in very good agreement.

a regular increase at all doses in the 0.5-day series: in the
1.5-day series, the trend with dose was irregular but, on
the average, the percentage was higher than in thecontrols (fig. I). Ohzu and Makino (232) performed ao.

per Citnl

5 dayt P.C.
CJ
• 1.5 days p.c.

40

0 0.5 dayt p.c.
o 1.5 days p.c.

]
~ 20
a:
/

1

r--

J

12781

J

12321

------ -•----/.

57. Dose-effect relationships for embryonic deathfollowing pre-implantation irradiation were also reported
in two recent publications. One publication (91) reports
the results of experiments in which several doses ofneutrons and x rays were given to pregnant mice, withembryos in the pronuclear zygote stage. Survival data
were deduced from the examination of the conceptuses
at 16 days p.c. A single exponential relation between thefraction of surviving embryos and absorbed dose wasfound by the authors to fit the experimental points
closely up to a dose of 20 rad of neutrons or 100 rad of
x rays. The values of Do (the dose necessary to reduce
survival to e -i or 37 per cent) were found to be 19 radfor neutrons and 87 rad for x rays. These doses would
correspond to an average increase in mortality rate of
the order of 0.8 per cent per rad in the x-ray series andof about 3 per cent per rad for the neutron series. The
authors concluded that embryonic death could beconsidered a single-event process for both radiations andthat the neutron RBE was about 4.5 (fig. II). The data
can also be reasonably fitted by a linear relation, and the

----

I
10

1'

I'.,"' .,"....------- ........

....

...._

___________ _0 L.--"""T"---r----r-----.-----.-----,-JO A
25
10
15

20

EXPOSURE

Figure L Intra-uterine mortality after pre-imp Ian tation irr:idiation (mouse)similar experiment on ( dd X CBA )F 1 mice and also
showed, for irradiation at 0.5 days p.c., an increase of
the percentage of dead foetuses as a function of dose.
Mortality values higher than control, with a plateau
between 5 and 25 R, were observed at 1.5 days (fig. I).
In a later publication, Ohzu (231) reported the samevalues for irradiation at 0.5 days, but different ones forirradiation at 1.5 days. Both series show exposureper cent

BO

60

I
O L2-MeV neutrons
0 JOO.kV x tilVJ

10

20

30

40

50

60

70

BO

90

J

(Bl)

100

rad

DOSE

Figure II. Intra-uterine mortality after pre-implantation irradiation (mice exposed to neutrons and x rays)dose to inactivate 50 per cent of the embryos would be.
under this assumption, about 60 rad for x rays and
12 rad for neutrons. The agreement between theseestimates of embryonic sensitivity to lethal damage andthose discussed previously (278, 232) is indeed
remarkable. Other results in the paper concerning thestage at which embryonic death takes place. are inaccordance with results already discussed (see paragraph 53). Dose fractionation experiments, which werealso reported in this paper. will be discussed in paragraph311.
58. The other publication. by Dufrain and Casarett(77), reports the results of irradiation in vitro of zygotes
666

from superovulated mice, the embryos having been
cultured in microdrops of a chemically defined medium.
The exposure required to inhibit 50 per cent of theembryos from reaching the two-cell stage was estimatedto be about 760 R, and from reaching 50 per cent of the
expanded blastocyst stage, about 130 R. As would be
expected, the second value is higher than that estimatedby Friedberg et al. (91) for the development of theembryo up to 16 days. This observation is also
consistent with the well established fact that the
majority of embryos die at cleavage but some can be lost
after implantation has taken place. The in vitro culture
technique also allows quantification of the cleavage
delay observed after irradiation of the zygote. Other

'T
I

1

data by Goldstein et al.
paragraph 69.

(98) are reviewed in

59. For the rat there appears to be conflicting
evidence in regard to the death of entire litters. Some
data show that such an effect may also occur in thisspecies without signs of malformations after exposuresof 400 R during the first week of development (123).
On the other hand, other findings would question thepresence of this phenomenon in the rat (33). thoughconfmning a very high incidence of resorptions and anincreased mortality of the implanted embryos after
150 R administered on the first day of pregnancy (32).
There is agreement, however, that malformations are notseen in surviving conceptuses irradiated at thepre-implantation stages, even though the possible
incidence of malformations in resorbed embryos
remains, of course. unknown.

°

60. In hamsters exposed to 200 R of 6 Co gamma
rays from the ovulation to the late pronuclear ( 110), andfrom the pronuclear to the early blastocyst. stages(0.5-3.5 days p.c. (111 )). Harvey and Chang found anincrease in pre-implantation lossess only when irradiation was performed at 0.5 day p.c. Post-implantation
losses, on the contrary, were increased after irradiations
at all times p.c. No malformations were observed. Other
data concerning the irradiation of unfertilized ova of
mice and hamsters and the subsequent development ofembryos can be found in 173, 112, and 113. Femalemongolian gerbils x-irradiated with various exposuresfrom 200 to 1200 R on day 2 p.c. (two-cell stage of thefertilized egg) and examined on day 18 suffered severe
pre-implantation loss down to 400 R but very few
abnormalities were observed in the foetuses (189).
61. From the data of Chang et al. (59, 61) it can beconcluded that the embryonic rabbit irradiated in situ
before implantation (day 2-6 p.c.) with 400 R of gamma
rays is very sensitive to radiation lethality. Irradiation of
the fertilized ova in vitro followed by transplantation
into recipient animals only resulted in increased
mortality at exposures larger than 400 R. Surviving
embryos were apparently normal and there was noevidence of differential sensitivity among the various
stages of development such as that seen after irradiationin utero. In the work of Inman and Markivee (128)
rabbit blastocysts of 3.5 days p.c. were exposed to 150
or 200 R of x rays and observed at 9.5 days ofpregnancy. These exposures increased pre-implantationmortality from 7 to 18 per cent (average of bothgroups); post-implantation mortality contributed comparatively little to total mortality.

°

62. In the dog, irradiation with 150 R of 6 Co gamma
rays at 5, 15 and 18 days p.c. ( early blastocyst, lateblastocyst and implantation stages, respectively) resulted
in a total pre-natal mortality of 18, 24 and 48 per centin the stages indicated, while the mortality for
non-irradiated embryos was 18 per cent, showing thatthe dog embryo is most sensitive at implantation. At
8 days p.c .. post-implantation mortality was low and it
increased with increasing developmental age at which
irradiation was carried out (245).

63. Thus, each species appears to have a period of highsensitivity to pre-implantation death in the very earlystages. The mouse, rat and hamster are relatively
resistant at implantation. The fact that the dog issensitive at implantation could possibly be attributed to
the late occurrence of implantation in this species, andcoincides with the formation of the primitive streak,which is another period of high sensitivity in all species.
2. Post-natal death
64. Some data on neo-natal and post-natal death inanimals irradiated at the 'pre-implantation stages are alsoavailable, mostly for the mouse. Rugh and Wohlfromm(289) reported that mouse embryos irradiated during thefirst 5 days of development with exposures from 125 to
400 R of x rays, had a heavy mortality in utero; theanimals which survived and came to term had good
survival during the first 30 days of post-natal life. In
another experiment (275), mice irradiated at 0-5 days
p.c. with previously determined {289) intra-uterine
LD 5 0 doses, showed less survival than the controls
during the first 30 days of post-natal life. On the other
hand, the long-term follow-up of animals surviving later
than the first month showed an absence of life-span
shortening, indicating that there was little permanent
damage after the neo-natal period. Konermann et al.
(157) performed an experiment in which mice were
subjected to daily x-ray exposures of 40, 60 and 80 R
during the main phases of pre-natal development. It was
shown that irradiation during the period 1-5 days p.c.
caused a high incidence of pre-natal death but had noeffect (or little effect at the highest daily exposure) on
the post-natal (3 weeks) survival of the offspring thatwere actually born. In dogs exposed to 100 R of x rays
at day 13 and 16 p.c., neo-natal mortality increased
from 34 per cent (control) to 45 and 57 per cent,respectively. A still higher mortality was observed.
however. with irradiations at day 19 and 22 p.c.,corresponding to the time of early organogenesis (245).
65. It appears, in conclusion, that pre-implantation
death leading to implantation failure is the majormechanism of lethality in all species after irradiation at
the early embryonic stages. Pre-implantation death may
affect single embryos in polytocous animals or all themembers of a litter. Intra-uterine death at later
developmental stages is comparatively less important,and the survival of the animals which come to birth ispractically unaffected by irradiation during the preimplantation stages. There are quite substantial variationsof the susceptibility to lethal damage, particularly during
the early segmentation of the fertilized egg. The
sensitivity at the time of implantation is usually lower
than at the blastomeric stages, but differences between
species can be observed. They may be explained by thedifferent developmental organization reached by theembryos at the time of implantation and may notnecessarily reflect intrinsic differences of the susceptibility of the different animal species. Dose-survival
experiments in the mouse, in the range 5-100 R, show
consistently that -the increment in embryonic loss soonafter fertilization may be of the order of 10- 2 R -i . The
data on survival at different doses for early segmentation
667

stages are fitted well by simple linear or exponential
relationships, which are not incompatible with relatively
simple inactivation kinetics.

3. Mechanisms
66. Concerning the cause of pre-implantation deathand the underlying mechanisms, Russell and Russell(302) reported that a high proportion of mouse embryos
exposed to 200 R at second cleavage appeared toundergo a rapid disintegration of the nuclei, resulting inlethal damage, whereas irradiation outside cell division
allowed further development of the conceptus, often up
to implantation. Chromosomal damage in all cells
derived from an injured blastomere gave rise tounbalanced chromosome complements. Such damage tothe genetic material, together with large radiation
sensitivity differences in the cell-division cycle, wereconfirmed for the first cleavage of mouse embryos(299). After irradiation. a high proportion of embryos
showed subnuclei, suggesting that aneuploidy is
responsible for most if not all, of the lethal damage. It
was also shown that the state of the nucleus at the timeof irradiation affects the production of aneuploidy, afact which would ·account for the differences insensitivity as a function of the cell cycle.
67. Rugh and Grupp (276, 277) reported results ofextensive morphological studies of the damage induced
by low doses of radiation in mouse embryos. Exposures
as low as 5 R, at 0.5 or 1.5 days p.c., often resulted infragmented embryos; the entire egg was broken up into
globules, the further development of which appeared
doubtful. Anomalies of cytoplasm and of nuclei,
pycnosis and complete dissolution of the blastomeres
were seen as early as 6 hours after irradiation {281, 282).
Other histological studies, performed at 9 or 10 days p.c.
on embryos irradiated at several pre- and post-implantation stages, showed that the damage, especially to themesenchyme, was highest when embryos were irradiatedearly (310).
68. Kirkpatrick {149) followed the radiation induced
morphological anomalies by means of in vitro cultures ofmouse embryos during the two-cell stage. Also with thistechnique the irradiated embryos showed fragmentation,
disintegration, granulation, nuclear degeneration andpycnosis, or cytoplasmic vacuolization. A distinct
correlation was shown to exist between morphologicalabnormalities and· embryonic death. The sensitivity ofembryos irradiated in vivo or in vitro was found to bevery similar, the sensitivity having been assessed fromthe dose required to stop the development of theembryo before the blastocyst in 50 per cent of cases(150, 77).
69. The experiments of Goldstein et ·at. (98) aresimilar in many respects to those just reported. They
analyzed the radiosensitivity of pre-implantation mouseembryos by culturing them in vitro up to earlypost-implantation stages. X-irradiation was also performed in vitro at various doses when the embryos wereat the 4-cell. 8-cell, morula and blastocyst stages, after
which they were returned to culture and scored for theappearance of further events such as the formation of668

blastocyst, hatching, growth of the trophoblast or of
the inner cell mass. Taking blastulation as the end-point,
an increase in sensitivity was observed between the 2and the 4-cell stage, with a decrease at the 8-cell stage.
Radiation did not prevent blastulation when administered during the morula stage, but it impaired theoccurrence of further events and the efficiency of this
impairment was higher the further removed the specificend-point was from the irradiation stage. Growth of theinner cell mass appeared on the whole as the mostsensitive indicator of the damage. Up to the 8-cell stage
pre- and post-implantation death was responsible fordevelopmental failure, whereas irradiation at the morulaand blastocyst stages resulted predominantly in postimplantation lethality. In spite of minor deviations,these findings appear to be in fair agreement with theother data in vitro reviewed previously (150, 77) andalso with in viva experiments (91. 299, 278, 232). All
these data contribute to strengthen our knowledge ofsensitivity and mechanisms of damage of the earlymammalian embryo.
70. The relationships between the damage to single
blastomeres and death of the whole embryo have also
been studied. On the assumption that the probability ofkilling a blastomere is independent of the number of
blastomeres in the early pre-implantation embryo,
Russell (295) calculated that embryos surviving irradiation at these stages of development included probably
some in which one or more blastomeres were injured.
Since virtually all surviving embryos appeared to benormal, a considerable degree of totipotency of theblastomeres was inferred. On the other hand, it seems
logical to believe that the proportion of damaged
blastomeres over the total number present at the various
developmental stages, together with their capacity for
regulation, may influence the survival of the embryo. It
may be of interest in this context that recovery fromlethal x-ray damage (300 or 500 R) of the embryonic rat
during hormonally induced delayed implantation has
been observed by Ward et al. (355).

B. DISTURBANCES OF GROWTH
71. The data available on growth disturbances induced
by pre-implantation irradiation of mice refer mostly topost-natal observations. Stunting has been reported in
the mouse during pre-natal growth (276, 277), but it
seems difficult to envisage the existence of a growth
disturbance unconnected with other effects. such aslethality or malformations.
·72.

Regarding post-natal life, an exposure of 100 R of

x rays at 0-5 days p.c. did not produce loss of weight or
reduction of skeletal size within 4 months of age, while
the same exposure at later pregnancy stages produced
appreciable stunting {271 ). Pre-implantation mouseembryos subjected to LD 5 0 doses of x rays and whichsurvived to an age of 24 months showed no gross adverse
effects on weight. In some instances. the animals with anin utero radiation history were even heavier thancontrols, probably because of the reduced average litter
size. These mice also showed no permanent defect ofskeletal growth, as determined by radiographs of
selected bones performed at the age of 2 months (275).

The offspring at term of mothers exposed daily to 40 to
80 R during the pre-implantation period did not show
any loss of weight with respect to controls, but actually
showed a significant post-natal overgrowth (157).
73. Brent and Bolden (32) and Brent (31) reported
that in the rat the growth of the surviving embryo
exposed to 150 R during pre-implantation was not
retarded. but this finding contradicted the data of Skreb
et al. (326), who showed a statistically significant loss ofweight in rats irradiated at 5 days p.c. and examined at
15 days of pregnancy. Rabbits irradiated with 150 or
250 Rat 3.5 days p.c., and weighed at 9.5 days. showeda reduction of body size observable in spite of thevariability of body size among controls, since lighter
foetuses in the controls were not as numerous orseverely affected as in the irradiated groups (128).
74. It may be concluded that reduction of body
growth due to irradiation during the pre-implantation
period is not an effect so clearly and universally
recognized as embryonic death, even at exposures inexcess of 100 R.
C. MALFORMATIONS
75. The appearance of malformations following
irradiation of early pre-implantation embryos has beenreported in a few instances. Such malformations involve
particularly the central nervous system and the skeletal
system. In the mouse, a maximum of 6 per centexencephaly was produced (277) by irradiation with 50
or 200 R of x rays during pre-cleavage, when no organ
primordia but only the three primary germ layers arepresent in an embryo. In another report (278) the
dose-effect relationship for this abnormality was
examined in embryos of 0.5 or 1.5 days p.c. within therange 5-25 R. The occurrence of the cerebral malformation was confirmed, but no obvious trend with dose
was established. The authors believe that damage of thesensitive neuroblasts, which is the cause of exencephalyduring organogenesis (see paragraphs 135-140). is not
the orJy possible mechanism of production of thismalformation. At early stages of development (gastrula),
irradiation could interfere with the cellular movements
in a way that alters the process of cephalization of theembryo, causing the appearance of abnormalities of thehead structures.
'
76. The presence of a few cases of exencephaly was
also conformed by Ohzu and Makino (232) and Ohzu
(231 ). These authors irradiated fertilized mouse eggs at
0.5 and 1.5 days p.c. and reported, in addition to
exencephaly. an average incidence of forelimb polydactilia of 28 per cent and 21 per cent, respectively. The
incidence seemed to be unrelated to exposure in theinterval 5-25 R. Hind-foot digits were practicallyunaffected. Since forelimb polydactilia was also
observed in 14 per cent of the non-irradiated mice, it
was concluded that the increased incidence after
irradiation could be indirectly due to an effect on theuterine environment {subsequent, for example, to heavy
lethality of the pre-implantation embryos) rather than toa direct radiation effect on the blastocysts. It should be
mentioned that exencephaly also occurs in normal mice(in about 1.5 per cent of the embryos of the CD-1
strain). and that irradiation during the mononuclearzygote stage (91) does not consistently modify its
incidence. Also, chromosomal imbalance may frequently
lead to exencephaly in the mouse. Therefore. therelationships between an aberrant cytogenic constitution
and the appearance of gross malformations could be
investigated in greater depth to establish precise
pathogenetic mechanisms.

77. Brent and Bolden (32, 33) reported data forembryo rats exposed on the first day of gestation to
150 R, with various combinations of ovary, oviduct anduterine shielding. They were unable to find any
malformation in more than 1,000 offspring examined. A
small number of irradiated rabbit blastocysts gave rise toembryos that were severely stunted and grossly
abnormal in body proportions or retarded in differentiation 9 .5 days p.c. The degree of the malformations was
such that these animals would probably not havesurvived long enough to be scored as malformed at birth(128).
78. In the dog (245), developmental malformations
were usually not seen following pre-implantation
exposure, and the only two cases of severe cranio-facialabnormality seen after an exposure of 150 R at 15 days
p.c. were attributed to uncertainty of the foetal age at
irradiation.
79. In conclusion, the few reported malformations inmouse foetuses irradiated during the pre-implantation
stages (notably exencephaly) represent a very improbable event, particularly in comparison with the frequent
occurrence of aberrations during organogenesis. Thesemalformations can be explained embryo logically, andtheir occurrence does not contradict the general viewthat the death of the embryo, rather than the inductionof malformation, is by far the highest risk of irradiation
during the pre-implantation stages.
D. OTHER EFFECTS
80. A variety of other effects have been reported,
mainly in the mouse, as a result of in utero irradiation
during the pre-implantation stages. They are mentioned
in this report for the sake of completeness but areclearly too scanty for meaningful generalizations. The
sex ratio of mice irradiated in utero with x rays at
different times and doses (289. 292) was equal to that ofcontrols. Similarly, there was no shift of this ratio in the
animals dying during the first 30 days post-partum.
These observations show that radiation is not selectivewith respect to sex in inducing mortality.
81. Rugh, Wohlfromm et al. {292) reported asignificant increase of the incidence of gross lens
opacities (sclerosis of the nucleus and overt cataract)
particularly in male mice irradiated with 100 R of x rays
during the fertilized pre-cleavage stage and followed up
to 18 months of age; however this increase was smaller
than some values reported earlier {270, 275). It wassuggested that the pathogenesis of the radiation-induced
cataract might be related to a damage of thedevelopmental processes that was insufficient to be
669

w
I

lethal. Another effect observed in these mice after
irradiation prior to implantation and survival for up to
two years, was a slight increase in the white-cell countand a small reduction in the red-cell count (275).

observations of Jacobsen {136), who noted thevariability of the estimates and the absence of effects
with exposures of 100 R at 7 .5 and 10.5 days p.c.

82. Data on post-natal fertility of the male mouse
x-irradiated at various gestational ages from conception
to 18.5 days p.c. were also reported, showing that
exposures up to 100 R had little effect (288).

2. Litter size.,

85. The decrease in litter size at or just before birthreflects the death of individuals in a litter due tomechanisms which could in principle operate before orafter implantation. Jacobsen (136) pointed out that,
although this indicator is defined as the ratio between
the number of offspring and the number of mothers, its
exact specification depends on the choice of which
mothers are considered for the ratio: mothers with one
or more offspring at the observation time, all pregnant
females including those aborting and dead, or mothers
that completed pregnancy, including those having
offspring or resorptions. With each choice, the indicator
has a different biological meaning, but, nevertheless, inall cases essentially similar dose-effect relationships arefound.

83. Sex-chromosome losses were studied in theprogeny of mice irradiated within 48 hours after
copulation. Marked changes of sensitivity to this effectwere shown to occur within a few hours, in parallel withsimilar oscillations of sensitivity to lethal damage (299).

III. THE PERIOD OF MAJOR
ORGANOGENESIS
A. LETHAL EFFECTS

86. The change in the average litter size has beenstudied as a function both of developmental age at thetime of irradiation (fixed dose) and of dose. This
indicator is an easy one to measure and relevant data
may be found in many papers. Only some of them arereviewed here. Rugh, Duhamel et al. (270) have reported
in CFI mice the values of litter size at birth after
exposure to 100 R at different times p.c. The data show
(fig. III) a reduction in litter size of the order of 40 per
cent with irradiation soon after copulation. For
irradiations at a later time, a stabilization of theindicator at about l O per cent below normal is found,but a further decrease occurs at around 7 days p.c.,followed by a return, with oscillations, towards control
values. The observations reported by Dekaban (72),
obtained by exposing Webster Swiss mice to 200 R at
different post-implantation times, are qualitatively
similar (fig. III). The average litter size is about 30 per
cent of control for irradiation at 7 days p.c.; after
irradiation at later times, the indicator reaches thecontrol value by day 12 p.c. and remains unchanged
until day I 8 p.c. These data consistently show that the

1. Failure of pregnancy
84. The reduction in the percentage of copulations
that result in pregnancy due to irradiation at various
stages of pre-natal development is a measure of mortality
involving the complete loss of a litter. This indicator israrely mentioned in the recent literature but was used in
the earlier papers (see Russell (295) for a review). In
mice, Russell (295) has shown that complete interruption of pregnancy, which is the frequent result of
pre-implantation exposure (see paragraph 53), is acomparatively rare event after post-implantation irradiations. As an example, exposures up to 400 R between
9.5 and 13.5 days p.c., when pregnancy may bediagnosed externally, result in almost l 00 per centlitter-bearing females, while an exposure of 200 R,
between 0.5 and 8.5 days p.c., when pregnancy is notreadily apparent, clearly decreases that yield. From thesedata it also appears that this indicator of lethality is notsensitive for the case of exposures in the post-implantation stages. Such a conclusion is supported by the

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e:-:pos:.:re nre: 60 R/min
ob~crvatlo:i a: t9 days p.c.

41

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obiet','ltior. a: birth0·------.,...-----r----.------.----,----,------~
6
10
8
12
14
16
18

days p.c.

lrradia1ion tim~

Figure III. Effect of irradiation at various p.c. times on mouse litter size

670..........

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. I

sensitivity to radiation lethality, as measured by litter
size, is maximal immediately after conception andduring the early stages of organogenesis and decreases atlater times to low and constant values during the foetal
stages of development.
87. In the rat, early data by Job et al. (139) and more
recent data by Murphree and Pace (212) show that
exposures of 110 to 220 R in the period 13 to 20 days
p.c. have no effect on litter size at birth. The slightchanges in litter size in the dog reported by Phemister etal. (245) are of doubtful significance with an exposureof 100 Rat 16 and 19 days p.c .

not consider this effect as a particularly sensitive
indicator of lethality in his mouse strain. There is adiscrepancy between the linearity of the response foundby Jacobsen and the shoulder-type relationships
obtained by Russell (295) and by Kriegel et al. (160),
although the stages of pregnancy at irradiation and theexposure conditions in the two experiments seem tohave been comparable. Nash (217) has also studied thelitter size at birth in mice of various genetic
backgrounds, with four different x-ray exposures (20.
80, 160 and 320 R) at four development ages (6.5, 10.5,
14.5, and 17.5 days p.c.) (fig. V). There appears to be

j

I
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88. An analysis of the available data on litter-size
reduction as a function of dose shows that different
dose-effect relationships are valid in the different
embryona1 and foetal stages examined. The data ofRussell (295) obtained on embryos in the pre-implantation. post-implantation and foetal stages are shown infigure IV. For irradiations in the period 0.5-4.5 days

8

irradiation at
O 6.5 dayi p.c. ]• 10.5 day, p.c. 12171

..
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Ii
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17.5..days p.c .

~

300

400

A

EXPOSURE\

Figure V. Dose-effect relationships for mouse litter size at birth
after irradiation at various p.c. times

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irudia1ion 41t

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14.5 days p.c.

•0'-----~--~---~--~-100

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• 0-5- 4.5 dav, p.c.
D 5.5- 8.5 d•v, p.c. 12951• 9.S-15.5 davs p.c. ~

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a 9.0-14.0dav,o.c. 1891

little or no effect :it all ages up to 160 R, and the only
point significantly different from the control is that at
320 R in the 10.5 days p.c. series. It should be
mentioned. however. that in this series all animals weredead soon after birth.
3. Resorptions

0 '----~--~----- - - 100
200
300
400
A
EXPOSURE

Figure IV. Dose-effect relationships for mouse litter size atbirth after irradiation at various p.c. times

p.c., a monetonically decreasing relation is found up to
exposures of about 200 R, followed possibly by aconstant tail. For irradiation during organogenesis, there
seems to be an absence of effect with exposures up to
125 R: at higher exposures, the effect increases
substantially with exposure. The relationship for
irradiation during the foetal stages show much smaller
effects in the same dose range.
89. The data of Fraser and Hall (89), obtained byirradiation during the embryonal and early foetal ages,
show a much more important decrease in the litter sizeat the tested exposures (fig. N) but are of little help forestablishing the form of the relation since they cover
only the interval between 250 and 350 R. The
low-exposure region is adequately covered by Jacobsen's
observations (136), which show an apparently linear
exposure-response curve between 5 and I 00 R in
7.5-day-old embryos (fig. IV); however, Jacobsen did

90. Special attention should be paid to the phenomenon of pregnancy resorption. which is rather
commonly described in experiments with rodents andobserved even during normal pregnancy in a sizeable but
variable percentage. As shown by Jacobsen (136) in themouse, the incidence of resorptions increases drasticallywith the age of the mother and is very sensitive toseasonal influences and to weak extrinsic teratogenic
stimuli, including radiation. Jacobsen (I 36) broadlyclassifies the resorption types observed in the mouse. His
classification may not be sufficient for an accurate
description of mechanisms, but it does make possible
broad evaluations of the stage of pregnancy in which
resorptions could have occurred. On the basis of thework of Kameyama (142) and his own observations.
Jacobsen ( 136) considers resorption to be ·a very
sensitive indicator in the case of radiation exposure,provided that the conditions under which it is scored arerigorously specified and the strain characteristic andextrinsic factors are properly controlled or allowed for.
91. Since implantation is a prerequisite of resorption,a radiation-induced change in resorption frequency
should essentially reflect post-implantation mortality.
671

nussmg embyros were about 20 and 30 per cent,respectively, while the control loss was less than 10 per
cent. Kriegel et al. (160) exposed pregnant mice to
200 R of x rays at different times between 5 and 15
days p.c. and scored resorptions on day 18 p.c. Under
those conditions, they were able to show (fig. VI) that

There are data on this effect in the mouse coveringvarious days p.c. around the time of implantation orvarious doses at fixed gestation times. Murakami andKameyama (204) have reported no change of embryonicloss in mice with exposures up to 50 R at 8 days p.c.;with exposures of 100 R, the fractions of resorbed andper cent

80

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abHn,stio•»t 18 days p.c.

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Figure VI. Effect of irradiation at various p.c. times upon resorption incidence in themouse

the incidence of resorptions, which was around control
level (I 2 per cent) with irradiation at 5 days p.c.,increased substantially with developmental age at
irradiation, reaching a peak of 70 per cent at 7 .5 daysp.c.; for older ages at irradiation, the incidence decreased
to the control level at about day I O p.c.. The embryonal
lethality was also examined as a function of exposure in.---- ..---·

perc»m100

per cent

irradiation at 7.5 days p.c. (136)observation at 19 days p.c.

60

~

40

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a.

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92. These data (160) are at variance with theapparently linear dose-effect relationship between 5 and100 R reported by Jacobsen (136) for a different strainof mice (fig. VIII), but under comparable experimental

a::

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irradiation atI

O 5 d.lys p.c. ] 11601• 8 d.lys p.c.

50
DOSE

75

100

rad

Figure VIII. Dose-effect relationship for resorption incidence inthe mouse

observation at 18 days p.c.

o~--....---~--~----,.---.,...-100

200

.

JOO

400

500

R

EXPOSURE

Figure VIL Dose-effect relationships for resorption incidence inthe mouse

the range of 50 to 400 R, with two irradiation ages (5and 8 days p.c.). The curves (fig. VII) show marked
differences in that they begin to rise steeply at
widely different doses. being fairly parallel from thenon; the maximum effect occurs between 250 and
400 R at 5 days p.c. and between I 00 and 250 rad at
8 days p.c.

conditions (irradiation at 7.5 days p.c., scoring at 19
days p.c.) Even allowing for differences in the technique,the fact that in Jacobsen's experiment (136) an exposureof I 00 R increased the incidence of resorptions from thecontrol value of 10 per cent to 40 per cent shows that
there is a large variability of sensitivity to radiation
among animal strains. Moreover, the different shape ofthe dose-effect curves (fig. VII) supports the existence ofa variation of sensitivity between embryonal stages, as
shown already by the timing experiments previously
discussed ( 160).
93. The data of Skreb and Bijelic (325) in the rat(fig. IX) establish the existence of a stage-differential
sensitivity to the induction of resorption, with amaximum at 8.5 days, in good agreement with the

672
I

. '

-~

mouse data. The work of Skalko (324) with animals of
11 days p.c. exposed to 135 or 270 R confirms that at
this gestational age the resorption effects are weakly
correlated to the level of exposure.
prr cent
180•

dose: 100 rad'

observation at 16 d•ys p.c.

r'
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95. Wilson (362) irradiated Wistar rats of 8 to 11 days
p.c. with exposures in the range of 12.5 to 600 R. In thisexperiment (fig. XI) 8-d~y-old embryos were apparently

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for the C57BL strain there was little indication of such a
response. However, when irradiation took place at thesame developmental age in both strains, both survival
curves became similar, even though the BALB/C mice
showed a somewhat higher sensitivity. For both strains
there was a threshold for the lethal effect of the order of
140 R. The mouse strain 129, on the other hand, washighly resistant to exposures up to 200 R; differenceswere also seen in hybrid crosses between these inbred
strains.

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per cent

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100

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control rangeO'--~----.----.----~---98
lr,._.jiation time

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10.5

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Figure IX. Effect of irradiation at different p.c. times on
resorption incidence in the rat

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4. Mortality in utero
94. The percentage of embryos alive before term is themost straightforward and sensitive indicator forevaluating the lethal action of radiation on single
embryos and foetuses. It measures the induced global
impairment of the complex sequential process that leads
to a fully viable individual. Dagg (68) irradiated inbred
strains of mice in utero (fig. X) with exposures in theI/

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"' 10 days p.c.
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(3621

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0 L<:ettl--.---~--~--~--~----.--100

200

300

400

500

600

EXPOSURE

Figure XI. Dose-effect relationships for intra-uterine mortality
in the rat

per cent

100

resistant up to 100 R but reached 100 per cent mortality
at 200 R. At increasing embryonic age, the sensitivity toradiation lethality tended to decrease. giving rise tomortality curves that were less steep.

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A CSJBL x CSJSL irra 1at1on at
ays: p.c.
0 BALB.

x SALB

irradiation at 10.5 dly1, p,c.

~

100

200

300

R

DOSE

Figure X. Dose-effect relationships for mortality in utero forvarious inbred mouse strains

range of 100 to 200 R and at times between 10 and
11 days p.c. and scored live and dead offspring at
18 days p.c. With irradiation at 11 days p.c .. theincidence of dead embryos in the BALB/C strain
increased with exposure in the range of 100 to 175 R,

96. In the hamster, an exposure of 200 R during
pre-implantation causes a moderately high mortality. The
same exposure at implantation results in low mortality andat 6.5 days p.c. (time of primitive streak formation), in
the highest mortality. At older developmental ages.
mortality gradually decreases to the control value duringorganogenesis and foetal growth (fig. XII). At the timeof peak sensitivity. an exposure of only 100 R induces
about 70 per cent degenerating foetuses, the 100-percent effect being produced by 200 R (I 11, 59). The data
for the rabbit reported by the same authors (59) showthat mortality following an exposure of 400 R is 100
per cent if irradiation takes place at the time of
implantation and tends to fall if irradiation is carried out
later during organogenesis and foetogenesis. There is alsoa fairly complete experimental series with beagle dogs
exposed to 150 R (245), in which pre- and postimplantation mortality has been studied as a function ofirradiation time from 8 to 35 days p.c. The results showthat post-implantation and total mortality are significantly higher than control values (fig. XIII) for
irradiations at a gestational age of 18 to 22 days p.c.
673

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5. Neo-natal mortality

per cer.t

100

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11

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• Post•implantation loues, 200 R expgsure ] f1 l1)
O pre•implantation losses. 200 R expasure

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- - prc•im-piantation control lell'el- - post·implantation control le"81o'--~-~--~-~~-~--~-~-IMPLANTATION

2

4

6

8

10

12

14

16

18

20

daY5P,C.

lrradbtion time

Figure XII. Effect of irradiation at various p.c. times on
intra-uterine mortality in the hamster

per cer,t

exposure: 150 R

60

(2451

O died before implantation• died ahcr implantationo. total prenatal mortality

40

98. Russell (295) reported data on neo-natal mortality
in mice from which it is quite apparent that exposures of
I 00 R or lower have no effect on survival at birth; 200 R
induce a peak of mortality when received between 7 .5
and 11.5 days p.c. (early organogenesis); 300 R at thesame times induces 100-per-cent mortality, the effectfalling off steeply with irradiation at times up to
14.5 days, in a manner apparently parallel to the 200 R
curve. It can be concluded that around 50-per•centmortality, the slope of the curve is very steep for any
given developmental stage.
99. In a more recent report (217) on mice of threedifferent genetic strains and their crosses, the pooleddata shows that 20 or 80 R at any age between 6.5 and17 .5 days p.c. does not cause any neo-natal mortality.
while 160 R causes a peak mortality of up to 75 per centat 10.5 days p.c., and 320 R results in a mortality of
100 per cent when received at 10.5 days p.c .. 23 percent at 14.5 days p.c. and zero at 17.5 days p.c. These
data are in very good agreement with those citedpreviously. Neo-natal mortality in beagle dog puppies
exposed to 100 R of x rays during a period from justbefore to just afer implantation is highest for irradiationsat 19 and 22 days p.c., corresponding to earlyorganogenesis (fig. XIV).
per cent

Control ·;alues:

20

100

~

:,

•
4

8

12

16
20
24
Irradiation time

28

32

36

80

OXP<"c•e: 100 R (2451

days p.c.

value£ shown are mean and~tand,rd error

I·
Figure XIII. Effect of irradiation at various p.c. times on
intra-uterine mortality in the dog(during the phases of implantation, primitive streak andearly somite formation), in good agreement with thedata for rabbits.
97. In conclusion, it appears that pre-natal mortality
in many species is highest for irradiations after
implantation and during the stages of early organogenesis, and that subsequently mortality decreases withthe advancement of embryonic differentiation. From theavailable data in rats and mice it would appear that theshape of the dose-lethality relationship for in urero
mortality might change substantially with developmental
stage and that, in the large majority of cases, at all
post-implantation ages tested a sigmoid relationshipwould apply with an increase in the slope occurring in
the region of a few tens of rads. At the later stages ofpregnancy, the decrease of sensitivity is mainly reflectedby lower slopes of the mortality curvPs. There isinsufficient information to identify the possible
mechanisms of foetal death at these early postimplantation stages.

j-~

20

implantation

0'------,----,-----r---,--'-!-.---,.----,-4

8

12

16

20

24

28

d,ys p,e,

Irradiation time

Figure XIV. Effect of irradiation at various p.c. times on
neonatal mortality in the dog

100. In conclusion, therefore, at exposures below about
100 R there is no (or little) increase of neo-natal
mortality, but the effect becomes manifest at higher
exposures, with an increasing slope of the dose-effectrelationship. As differentiation proceeds, the sensitivityof the embryo and foetus gradually decreases.
101. There is information available for two rodent
species on the LD 5 0 for irradiation in utero and on thevariation during embryonic and foetal development
shown in table 4. The variability among species andstrains found in different experiments is very large, theidentification of general trends being correspondingly
difficult. It appears. however, that sensitivity is very high(with LD 5 0 corresponding to exposures lower than674•

150 R) in the pre-implantation, implantation stages andearly organogenesis stages. In the single case wheretiming was accurate, the fine structure of theradiosensitivity of the mouse embryo showed considerable variability, but a large share of this variation could
be attributed to the technical conditions under which
the observations were made rather than to intrinsic
changes in the sensitivity of the embryo. LD 5 0 data forthe mortality in utero reported for the period between
day 6 and 13 range from 165 to 700 rad. At the latestages of organogenesis, the LD 5 0 begins to rise
gradually to reach during foetal development values
comparable to the post-natal LD 5 0 levels.
6. Post-natal and long-term mortality
102. The mortality after birth of animals irradiated inutero was studied by Rugh and Wohlfromm (289), who
irradiated CF 1 mice at 6-18 days p.c. to establish theradiation dose that would kill, within 30 days after
birth, 50 per cent of the mice born alive. The LDso/JO
values (table 4) were found to fluctuate with developmental age. The lowest values were at 8-10 days p.c., thetime when the most active differentiation of the nervous
system takes place. On days 6, 7 and 8 p.c., the LD 50 inutero (286) was the same as the LD 5 0130 , which implies
that exposures of about 400, 200 and 150 R at theseages, respectively. kill half of the offspring in utero andhalf of those born alive within 30 days. At later
exposure times (see table 4) the LDso/JO wereconsistently and substantially lower than the LD 5 0 in
utero, showing that, while the embryo cannot easily bekilled before term, it can readily be damaged in a way
which renders it unable to survive the first month after
birth. Early post-natal deaths were more frequent among
litters irradiated during early gestational ages.
103. The observations of Nash (217) are in generalagreement with these data, in spite of minor technicaldifferences. He reported that the survival to 60 days ofmice still alive 12 hours after birth was maximum after
irradiation at 6.5 days p.c. Irradiation at 10.5 days p.c.
had the most severe consequences, while exposures at
14.5 or 17 .5 days p.c. produced intermediate effects.
For example, after 80 R at 10.5 days p.c., only 55 percent of the mice alive soon after birth were still surviving
at 60 days. while 62 and 78 per cent survived if the same
exposure occurred at 14.5 or 17.5 days p.c., respectively. No consistent pattern of survival differences
between sexes, doses or embryological stages wasobserved. Inbred mice appeared to survive less thanhybrids, especially immediately after birth, and in thegroups where the increase in post-natal mortality wasmost significant, inbred strains showed a larger responseto the radiation insult than hybrid genotypes.
104. An unexplained and still not confirmed conditioning effect has been reported by Rugh and Wolff(293), whereby an exposure of 10 R of x radiation on
day 15.5 p.c. enabled mouse foetuses to survive better
when irradiated as adults with doses around theLDso/JO· This "beneficial" effect was also apparent at
14.5 and 16.5 days p.c., while higher exposures (25 to
300 R) led invariably to deleterious effects in adult life.

105. The data of Nash and Gowen (219) extended theobservations ~o the whole life span of mice, showing thatthe reduction in longevity after 20 to 320 R at four
gestational ages depended on genetic constitution, sex,
gestational age at irradiation and exposure. The
influence of these factors was evaluated in multifactorial experiments in which data on mature life spansof 647 animals were collected.
106. In the rat, the neo-natal death rate wassignificantly higher than control (6 per cent) when thelitters were exposed to 110 R on day 13-15 p.c. (27 percent) and on day 15-18 p.c. (13 per cent), to 150 Ron
day 14 p.c. (98 per ~ent). and to 220 R on day
12-16 p.c. (77 per cent) (118). Sikov. Resta and
Lofstrom (322) carried out an analysis of the long-term
mortality of rats surviving at weaning, with exposures of
20 or 100 R at 10 days p.c. and of 50 or 185 Rat 15
days p.c. Sex differences were also found in this study;
life-span shortening in females appeared to be greaterthan in males. in which the effects observed were of
doubtful significance and in any case not very
substantial.
I 07. It can be concluded that the lethal effects ofirradiating animals in utero are not exhausted withpre-natal mortality but at appropriate doses may also
become manifest in post-natal life. Thus, a dose ofradiation insufficient to kill an animal in utero may stillbe lethal after birth. Post-natal LD 50130 values
calculated on live-born animals in the region of150-300 rad have been reported for the rat and mouse
irradiated between day 6 and 13 of intra-uterine life.
Mortality after birth takes place essentially during thefirst month after birth. Long-term reductions in life span
may also become evident for exposures in excess of
200 R in rodents, depending on a number of geneticallycontrolled factors of the strain. A detailed discussion oftumour induction in animals irradiated in utero will befound in Annex I.
108. In the human species there are no data relatedwith certainty to the lethal effects of irradiation during
embryonic stages. Since there are great difficulties intracing the pregnancy stage at irradiation in most of thereported experiments and since most of the experiencerefers to irradiation in the foetal stage, this subject will
be reviewed in connection with the irradiation of the
foetus (paras. 192-195).

B. DISTURBANCES OF GROWTH
1. Intra-uterine growth109. Russell (294) reported foetal growth defects inmice irradiated between 0.5 to 13.5 days p.c. with
exposures of 200, 300 and 400 R. The mean weight atbirth of these animals was not significantly below
control for irradiation times up to 7.5 days p.c. The
effect was considerable with irradiations at later stagesof development. The response curves as a function ofirradiation time for the three exposures were found to
be roughly parallel, with maximum effects between l 0.5
and 11.5 days p.c. A complete and apparently linear
dose-effect curve for heterozygous but genetically675•uniform animals irradiated at 11.5 days p.c. wasreported by Russell, Russell and Major (305) covering
the interval 0-300 rad. The resulting average weight
reduction was estimated to be 0.22 g per 100 rad within
this dose range. in fair agreement with the previously
reported data. Jacobsen (136), irradiating homozygous
HC mice at 7.5 days p.c. (a relatively less sensitive stage.
according to the data of Russell (294)), reported a
reduction of 0.12 g per 100 R, for animals sacrificed at
19 days p.c.
110 .. In view of the experimental differences between
the two sets of data it is difficult to draw a clear
conclusion. but it seems possible to assume that weight
reduction is a more sensitive indicator of radiation
damage if irradiation occurs at 10.5 and 11.5 days p.c.
rather than earlier or later in foetal life. Considerable
strain variations are, however, to be expected (136,266). This view is also supported by more recent workby Konermann (155), who studied experimentally the
relative sensitivity of the mouse conceptus to irradiation
during blastogenesis, organogenesis and foetal stages,
with fractionated exposures of 10 to 80 R per day.
Animals examined 18 days p.c,. appeared to be mostsensitive when irradiated during organogenesis. When
irradiation continued during the foetal growth, a still
higher effect was produced, with weight reductions up
to 50 per cent at exposures of 80 R per day.
111. In a study of radiation effects on embryonic
growth, Yoshizawa and Ueda (373) irradiated dd-Y micein utero at various gestational ages (7-17 days p.c.) andat various exposure levels (0-300 R) and exposure rates(1-100 R/min) of a 137 Csgamma source. The bodyweight of the foetuses and the growth of the caudal
vertebrae were taken as indicators of the effects to bestudied; they appeared to show similar dependencies on
the experimental variables. The gestational ages 8-11 and16 days p.c. were found to be sensitive stages for the
inhibition of bone growth, and they appeared to berelated to inhibition of the cartilage bud and to damageof the ossification processes, respectively. The delay inthe degree of ossification and the decrease in bodyweight were insensitive to exposure rate in the interval
indicated. However, both effects were clearly enhanced
by increasing exposure. In this case (irradiation at
11 days and sacrifice at 18 days p.c.) the weight
reduction was of the order of 0.23 g/100 R, in good
agreement with the data above (para. 109).
112. In the rat, Wilson (362) examined growth
retardation after exposures of 12.5-200 Rat days 8, 9,
10, 11 p.c. The effect was assessed as weight loss atvarious times after irradiatio~, according to a fairly
complete experimental schedule. The degree of retardation tended clearly to increase with increasing
exposure (although not in direct proportion) at allembryonal ages tested. However, the exposure to obtaina given effect was larger in older embryos, showing thatthe embryo becomes increasingly resistant to growth
retardation. In fact, the weight retardation effect per
100 rad may be estimated to be 14-30 per cent( depending on the observation day) of the control in the
8-day embryo, about 17 per cent in the 9-day embryo.
6-14 per cent in the 10-day embryo and about 2 per centin embryos aged 11 days.

113. More recent data have added little to these general
conclusions. Campi et al. (58) made similar observations
after 100 rad of x rays given 10 days p.c. and showedthat the longitudinal and transversal embryonic diameters. as well as the weight of the foetuses. wereconsiderably reduced 7 days later. Skreb et al. (326)exposed embryos between 5 and 10 days p.c. to 100 Rof 85-kVp x rays. In reasonable agreement with the dataof Wilson (362), the weight of the embryos on day 15
was up to 30 per cent smaller than control, particularly
if irradiation took place 8.5-9 days p.c., during the onsetof mesoderm formation.
114. It is difficult however to draw more quantitative
estimates from experiments where the sacrifice wascarried out, not at a fo..:ed time or at maximum weight
Joss, but at variable times post-irradiation. Weight
reductions of 7 per cent after 135 R and 28 per centafter 270 R were reported by Skalko (324) on rats
irradiated at 11 days p.c. and observed at term. Brent
and Bolden (32) obtained a weight reduction of 11 to
16 per cent at term after 150 R at 6 days p.c., but wereunable to show any effect at the same exposure given on
the first day p.c. Growth retardation was also reported
in mongolian gerbils given 200-400 R of x rays at theprimitive streak stage (189). In the rabbit, irradiated at
3.5 days p.c. and observed 6 days later, 150 or 200 R
significantly reduced the size of the embryo, in a mannerroughly proportional to exposure (128).
115. The data reviewed show that in several species theperiod of major organogenesis is particularly sensitive toradiation-induced growth disturbances, possibly morethan the foetal period but particularly more than thepreceding blastogenesis phase. The size of this indicatorvaries with the time at which the dose is administeredduring organogenesis and with the interval between
irradiation and observation. In all species there areinsufficient data to evaluate the net effect of a dose at a
fixed post-irradiation time or at the time giving
maximum expression of the damage.

2. Post-natal growth116. Radiation-induced growth disturbances may
persist during post-natal life. Levy et al. (171) havereported significant reductions of the dimensions of thefemoral, parietal and mandibular bones of the mouse.
following an exposure of 300 R of x rays at 15 .5 days
p.c. This effect lasted at least up to 240 days of age,when the last samples were taken. Rugh. Duhamel et al.
(271) made a complete skeletal study of animals
exposed to 100 R in utero at different times of gestation(0-18 days p.c.) and which were alive at the age of
4 months. They showed that, both in male and female
offspring, stunting in all bones examined was more
pronounced if irradiation had taken place between I I
and 13 days p.c., the time when the embryo undergoes
the earliest skeletal differentiation. Additionally. asignificant reduction of body weight occurred in males
irradiated on day 5 p.c. and on day 9-18 p.c., and infemales irradiated on day 8-18 p.c., the most drastic
reduction being the result of inadiation on day
11-14 p.c. This shows that the second half of gestation is
the most significant period for radiation-induced

676

-~

stunting. Stunting persists during the whole life, sincesimilar results are obtained if the anrage weight between
3 and 35 months of age is taken as the indicator instead of
single measurements at foced times (272).
117. In other experiments by Nash and Gowen (218),
three genetically different strains of mice were exposed
to 20-320 R at different times between 6.5 to 17 .S daysp.c., and then their post-natal growth was followed for aperiod up to 75 days of age. The observed body weight
reductions were found to depend on dose, gestational
age and post-natal age. Reduction was maximum formice irradiated at 10.S days p.c. and minimum for those
irradiated at 6.5 days p.c., the other two embryologicalages tested (14.S and 17.5 days p.c.) giving anintermediate response. Weight reduction reached its
maximal expression when the animals were about
40 days old and remained constant afterwards. There
was a marked difference in response between strains,which could be attributed to the difference in thecapacity for recovery from the radiation insult and also
to variations in developmental age at the time ofirradiation.
118. Results available for the rat lead essentially to thesame conclusions. Alexandrovskaya (2) observed that
16-month-old rats which received a dose of 150-200 rad
on day 12 p.c. had pronounced hypoplasia of the central
nervous system. Murphree and Pace (212) reported thatrats exposed to 110-220 R at various gestational ages
from 13 to 20 days p.c. had average weaning weights
lower than the controls. The weights of the body and of
the testes in adult males which had been irradiated werealso significantly lower than control, and the testicularweight depression was dependent on dose and age at
irradiation. Sikov, Resta and Lofstrom (322) showedthat an exposure of 20 or 100 R at 10 days p.c. andsimilarly an exposure of 50 or 185 R at 15 days p.c.
reduced the weight at birth in all groups, except at thelowest dose. Growth depression persisting to older ages
up to about 80 weeks was only evident in groups
irradiated at 15 days p.c., and this effect was manifest
also for single organs, particularly for the brain and thespleen, with differences between sexes. It seems likelythat the reduction of body and organ weights, often
associated with histopathological changes and depressionof hormonal levels, cannot be entirely explained bystunting, being probably also the result of complex
physiopathological deficiencies.
119. Significant reductions of the interzygomatic
distance and of the skull lenr,h were observed in adultdogs expose_d in utero to 6 Co irradiation during theorganogenesis and foetal stages but not during the
pre-implantation period (167). A linear dose-effectrelationship seemed to apply between 20 and 330 R, andthe fractional reduction per 100 R was estimated to be
4-5 per cent for the interzygomatic distance and 1.7-2.S
per cent for the skull length. Irradiation at the foetal
stages was slightly more efficient ( 167). The volume ofthe eye in these animals was also smaller than in controls(168).
120. Growth disturbances observed in humans irradiated in the embryonic stages will be discussed inparagraphs 202-208. together with the description of

growth deficiencies following foetal exposure. This formof presentation is consistent with the fact that growthchanges (as opposed to malformations) are the most
likely outcome of irradiation during the foetal stages.
C. MALFORMATIONS

I. General
121. Some qualifying considerations are necessary forthe analysis of malformations produced by irradiation
during the major organogenesis period. A majordifficulty is the lack of standardized nomenclature ofmalformations, each author having followed his own
criteria for classification. This difficulty is compoundedby the fact that malformations of adjacent but
embryologically unrelated structures are often found in
the same animals, complicating substantially thesystematic treatment of the data.
122. As most of the published work was performed forthe purpose of embryological research, it is very hard to
extract from these publications the information relevantto the study of radiation effects. Sufficiently complete
analyses of dose-effect relationships for various malformations have b~en performed in only few cases; mostobservations have been made at a single or very few
doses. In addition, if one considers the variability of
species and strain, the very different criteria under which
the malformations have been scored and the fact thatexperiments differed in irradiation conditions, it is not
surprising that the various experimental series can hardly
be correlated.
123. The Committee believes, nevertheless, that some
general conclusions can be derived from consideration ofsuch material. The main emphasis is obviously on
reported data that could be used for assessing dose-effectrelationships, and for that reason the following review
should be regarded as selective and by no meanscomplete.
2. External head abnormalities
124. External head abnormalities described in connection with embryonic irradiation comprise: (a} abnormal
shapes of the head (dome-shaped or vaulted cranium:
narrow, long head); (b) abnormalities of nostrils and thesnout (tapered, beak-like): (c) abnormal shape of themaxilla, jaw and mouth; (d} cleft lip or harelip and cleft
palate and face; and (e) abnormalities of the external
ear. Cranial blisters are described below in subsection 4,
malformations of the central nervous system (paras.
135-140), and microcephaly is treated in a special
section (subsection 5, paras. 141-146) in view of its
importance in human radiation biology.
125. In the mouse, the maximum sensitivity for
inducing malformations with exposures of about 200 R
occurs between 7.5 and 10.S days p.c. (294) or between
7 .5 and 13.5 days p.c. (72), although each malformation
probably recognizes a specific "critical period" withsmaller exposures (209). According to Jacobsen (136),
exposures in the range 5-100 Rat 7.S days p.c. induce
few malformations of the head, jaw and mouth. These677

results are in accordance with those of Nash (217),
showing that vaulted cranium is induced only at
exposures of at least 160 R (at 10.5 days p.c.) and alsowith the observations of Dagg (68), where cleft palate
occurred only at exposures of at least 100 R (at 11 or
10.5 days p.c.). Murakami et al. (209) found abnormallyshaped heads, harelips and cleft palates following
exposures of 200 R on day 9 p.c. The same authors(210) reported malformations of snout, maxilla,mandible, harelip and cleft palate following exposures of
150 to 300 R in the period 7-12 days p.c. It appears thatin the mouse 50 R is the lowest exposure at whichoccasional abnormalities of this type are found (204).
126. Studies on the morphogenesis of the palate and on
mechanisms of cleft palate induction after 300 R of
x rays administered to two strains of mice at 11 days
p.c. were reported by Callas (56) and by Callas andWalker (57). The progress in palate development wasevaluated by morphological rating in relation to thechronological age and to the embryonic weight. Thesestudies showed that the primary cause of themalformation could be traced to a delay of the
movement of the palatine shelf during embryogenesis.
The A/Jax mouse appeared to be more susceptible thanthe C57BL to the induction of this malformation.
127. Warkany and Schraffenberger (357) describedmalformations in the rat, following exposures up to1120 R at 9-15 days p.c. These malformations included
defects of the skull with partial protrusion of the brain,cleft palate and lip, and maxillary and jaw abnormalities.
Their incidence depended markedly on exposure andtime of irradiation, but the data are insufficient for ananalysis of the dose-effect relationships. More recently,
Rajtova and Horak (251) reported malformations of thechrondrocranium in animals exposed to 200 R at 12-15
days p.c. The maximum effects were seen with
irradiation at 13 days p.c.; they included degenerative
processes and defects of the nasal wall and septum, of
the turbinate primordia and of the lamina cribrosa. The
incidence of malformations observed with irradiation at
15 days p .c. was not greatly different than in controls.
I 28. Cleft palate and cleft lip were also found in
hamsters (200 Ron day 7.5 p.c. (111)), in monogolian
gerbils (200-400 R on day 9 p.c. (189)) and in rabbits(400 Ron days 9-11 p.c. (59, 61)). Severe cranio-facial
malformations, harelip and cleft palate were observed indogs exposed to 100-150 R immediately preceding or
following implantation. They were frequently accompanied by major abnormalities of the brain and eyes(245). Cleft palate and micrognatia were also seen inmonkeys following 250 R of x rays during majororganogenesis (249).

3. Malformations of the eye
129. Russell (294) reported malformations of the eye inmice exposed to 200-400 R at 7.5-10.5 days p.c. They
included various degrees of microphthalmia including
anophthalmia. coloboma of different types, and narrowing of the pigmented ring of the iris. Eyelids open
at birth is also a pathological condition in this species.
Ru$sell's data are insufficient for establishing relevant

dose-effect relationships. Anophthalmia and microphthalmia were also seen by Murakami and Kameyama(204) in mice exposed on the day 8 p.c. to 100-150 R,
but not if exposed to 25 or 50 R. Majima (181) was thefirst to report a dose-effect relationship for gross eye
malformations. applicable to irradiation at 8.5 days p.c.
(fig. XV). This relationship had a threshold of 25 R, in
per cent
30

p

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I

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I

,.i:1',, , /,,/"""
0 0--0-""-~-c:;-,;:/~-----.,---~--~--40
80
120
160
R

EXPOSURE

Figure XV. Dose-effect relationship for induction of anophthalmia and microphthalmia in the mouse (181)the sense that no increase of abnormalities was seen at
this dose. The defects were invariably associated withmalformations of the central nervous system, and theauthor described the histopathogenic changes responsible for the malformations. In a second paper (182),
Majima irradiated mice 8.5 days p.c. with a fixed dose of
200 R and studied the embryos by gross andmicroscopical observations at invervals of 1 day between
11 and 19 days p.c. Histology showed the presence of
microscopic changes a few days after irradiation.
I 30. About 29 per cent of eye malformations in ddN
mice were reported by Murakami et al. (209) following
exposure to 200 Rat 9.5 days of pregnancy. Murakami
et al. (210), in their report dealing specifically withmalformations of the extremities, include also data on
the incidence of microphthalmia and anophthalrnia in
ddN mice exposed to 150, 200 and 300 R on day 7-11
p.c. From these data it is very difficult to identify theperiod of maximum sensitivity. Murakami (203) founddifferences between ddN and CFl mice in this critical
period for the induction of eye abnormalities. using
exposures of 200 and 300 R (fig. XVI).
131. Sakurai (307) confirmed interstrain differences andthe presence of two sensitivity peaks in one of the twomouse lines examined. In addition, he studied
microscopic morphogenetic changes at various intervals
within 24 hours of irradiation. Experiments on eye
malformations in mice were also reported by Badtke etal. (250) (130 R at various p.c. times between 7 and 11days), showing a sensitivity peak between 7 and 8 days.
and by Degenhardt and Franz (70) (140 Ron day 6-11p.c.). who obtained essentially the same type of
information on the same mouse strain. Other data on
eye abnormalities in the mouse are also to be found in
papers by Jacobsen (136) and Nash (217), where
exposures of 5-100 R did not have clear effects.
132. The most recent and complete set of data on thehistopathology of radiation-induced eye malformations

678

.'

pe: cent

C200Rj- CF1• JOO R
strain100

O 200 R main ddN•JOOR...1lJ

per cent

1203)

•001
80 -

60
O all ocular malformations

irradiation at 9 days p.c.
>100R/min•40

all ocular malformations

J-

(364)

l
J

irradiation at 10 days p.c. 1365)>IOOR/min_a .all ocular malformations
irradiation at 11 days p.c.

1338)~,-'-- - - - - - ~ ,--~,-3-.3-R-/m-in---r-- 1
1
50
Irradiation time tdays p.cJ

100

150

200

R

EXPOSURE

Figure XVI. Effect of irradiation at various p.c. times on theincidence of gross ocular malformations in the mouse

Figure xvn. Dose-effect relationships for the induction ofgross ocular malformations in the rat

in the mouse is that of Christensen ( 65. 66). He
performed extensive investigations on the C3H mouse
between 7 and 14 days p.c. at exposures of 55 to 255 R.
Animals were examined before birth and gross,stereomicroscopical and histological changes were recorded accurately. The amount of data provided is verylarge in respect to many effects, but, limiting the present
comments to eye malformations, the maximum response
was found at 8-11 days p.c. with 222 R. By far thegreatest number of malformations were observed after
this dose and only a few after 55 and 166 R. Some welldefined effects (coloboma, microphthalmia and mild
palpebral defects) were found to be stage-specific. Since
the study was meant especially as a histopathologicalone, with a particular view to stage-specificity, the dataon dose-effect relationships are not as informative as
they might have been otherwise. However the data
showed that severe microscopic changes were present ineyes that would be classified as normal on gross orstereomicroscopical examination. Other histologicaleffects in various mammalian species were also reported
by Rugh and Skaredoff (284). Lucas (174) and Hicks etal. ( 122), as well as in many of the previously cited
papers on malformations of the eye.

dose-rates of 1, 3.3, and 47 R/min; 3.3 R/min appeared
to be the most effective exposure-rate (see also
paragraph 318) and produced at 50 R a 7-per-cent
incidence of animals with defective eyes (fig. XVII).
These curves might be described as roughly curvilinear.

133. In the rat, the eye has been reported as the mostconsistently affected - of all organs. Anophthalmia,
microphthalmia and other malformations are foundfollowing exposure at 9 or 10 days p.c. {362, 364. 365).
Dose-effect relationships (in the range 25-200 R) havebeen deduced for irradiation at these times. Even thelowest exposure produced about 5 per cent ocularmalformations if delivered at 9 days p.c. (fig. XVII)(364, 365). It appears that, with exposures up to 100 R,
microphthalmia has the highest incidence. while for
200 R anophthalmia prevails. A 59-per-cent incidence ofanophthalmia was reported by Skalko (324) in animals
exposed to 270 R of 6 °Co radiation at 11 days p.c.
Strange and Murphree (338) have examined theexposure-rate dependence of eye malformations in thisspecies (11 days p.c.), providing reasonably complete
exposure-incidence curves in the range of 50-200 R at

134. Microphthalmia, anophthalmia and other malformations of the eye were induced in hamsters. The
incidence was 38 per cent with exposures of 100 R at
6.5 days p.c., 52 per cent with 200 R at 7.5 days p.c.,and 65 per cent with 200 R at 8.5 days p.c. (111). Eye,brain and visceral malformations were seen in rabbits
exposed to 130 or 250 R of x rays on the 10th day ofpregnancy (353). Finally, ocular abnormalities have also
been reported in dogs following exposures of 100-150 R
before or after implantation (13-38 days p.c.) (245).

4. Malformations of the central nervous system
135. The gross malformations of the central nervous
system (CNS) resulting from irradiation of embryos
comprise exencephaly (or pseudencephaly), anencephaly, encephalocele, hydrocephaly (dysgenetic hydrocephaly) and spinal cord abnormalities (flexion, hernia,hydromyelia, myeloschisis). They are often accompanied
by other malformations of the bones and structures of
the head. The "cranial blisters" that some authors
describe as skeletal abnormalities are in fact small brain
hernias.
136. In mouse experiments with 150-200 R, the peak
incidence of exencephaly is observed with irradiation at
8 to 9 days p.c. (fig. XVIII) (276, 277, 210, 72), while
the sensitive period for hydrocephaly extends to 10 days
p.c. (fig. XIX) (210, 72).
137. A detailed classification of the abnormalities of
the CNS was published by Kameyama (142) and by
Murakami and Kameyama (204). The mice were
irradiated on day 8 p.c.andexamined on day 13, and the
x-ray exposures were in the range 25-150 R. Occasional679')

exposure: 150 R (276) (277)

per cent

40
a.,

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C:

It

"'

"'O

·;:;

I \
I

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,

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ft

20
,

I

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I

/

I

,•
,

'

6

5

7'

I

i'

,,

'
'

I

9

8

10

11

days p.c.

Irradiation time
Figure XVIII. Effect of irradiation at various p.c. times on
induction of exencephaly in the mouse

per cent

O exposure; 150 R

exposure: 200 R• exposure: 300 R

660• exposure: 200 R

J- 12101

138. Wilson (362) summarized the observations on
abnormalities of the CNS in rats reported by Wilson andKarr (365) and Wilson et al. (364, 363). The rat brain
appears to be affected differently depending on time ofirradiation. between 9 and 11 days p.c. On day 9 p.c.,several brain malformations were produced, resulting
from the fusion of the brain wall with the ectoderm. On
day 10 p.c., the effects were hypoplasia and growthirregularities of various parts of the forebrain. while on
day 11, eversion of the choroid plexus and other minor
distortions were induced. From the few data relatingdose and malformation incidence quantitatively. it
would appear that exposures as low as 50 R on day 9
p.c. result in an observable incidence increase. With
irradiation at older ages p.c .. embryo CNS-abnormalities
appear only with higher exposures. Skalko (324)reported an incidence of 81 per cent of hydrocephaly
following an exposure of 270 R on day 11 p.c.

per cent

80
1721

irradiation time:0•"

9 days p.c.J

10 days p.c.
11 days p.c.

(362)

developmental age in ref. 72
is reckoned from one day
~

a;
'O

earlier than in ref. 210

40

'1l

E

.

1l

.,C:

'O

Ti

20

C:

er0
6

7

9

11

13

14

15

days p.c.

Irradiation time

20

Figure XIX. Effect of irradiation at various p.c, times on
induction of hydrocephaly in the mouseo'-----~---~---~---~-50

malformations (exencephaly, hydrocephaly, flexion of
the spinal cord) were observed at exposures as low as
25 R. the incidence increasing progressively at higher
exposures. Exposure-effect data for exencephaly arereported also in two papers by Rugh and Grupp (276,277) for the exposure range 25-200 R. Pooling together
all abnormalities seen at each exposure level in the fourgroups of mice irradiated between 7.5 and 9 days p.c., it
can be shown that the incidence is very low at exposuressmaller than I 00 R. while at exposures of 150 and
200 R the incidence is 17 per cent and 16 per cent.
respectively. Murakami (203) and Murakami et al. (206)observed the incidence of exencephaly and hydrocephaly in mice of two strains exposed to 200 or 300 R
at 8-13 days p.c. The observations were at 19 days p.c.,and showed some difference between the strains. The
same authors also described the histopathogenesis ofthese malformations in animals sacrificed serially withthe purpose of correlating the malformations with thedevelopmental events of the embryonic nervous systemat the time of irradiation. Histopathological observations
have also been reported by several authors (204, 276,
23, 72, 73, 237, 238. 239, 116, 126). Irradlating on day
7 .5 p.c., Jacobsen (136) did not find cases of
exencephaly with exposures of 20 and 100 R, although a
few cases did appear in the control groups.
680

100

150

200

R

EXPOSURE

Figure XX. Dose-effect relationships for the induction of brain
malformations in the rat

139. In reference 121 Hicks and D'Amato summarized
their own previous work (1 18. I 19, 122) and resultsfrom other sources. The summarized work related
mainly to CNS malformations and correlates malformations with embryological stage rather than withradiation dose. Histopathological studies show that
abnormalities of the microscopic architecture of thebrain are seen in general at rather low doses. Riggs et al.
(257) and Hicks and D' Amato (120) have reported
results on various alterations of the nerve cell and of thecerebral cortex: cortex alterations can be induced byexposures as low as 10 to 40 R. It may be said, inconclusion, that although more observations have been
carried out in rats than in mice, they are usually
insufficient for dose-effect studies and their importancelies mainly in the establishment of correlations between
time of irradiation and type of resulting malformation,owing to the differentiation of the relevant structures at
the time of irradiation.

5. Microcephaly140. In hamsters, exencephaly was seen at exposures of
200 R on day 6.5 and 7 .5 p.c. ( 111 ). In dogs, exposuresof 100 to 150 R on day 13-38 p.c. caused CNS
malformations. which were reported in a single classwith ocular defects by Phernister et al. (245). Marmosets
irradiated with 250 R of x rays during organogenesis
developed cranioschisis (249).

141. In the mouse, the peak of sensitivity for inducing
microcephaly with 200 R is at day 10-11 p.c. (210, 206,72); in the rat, on the other hand, it is at day 12-13 p.c.
(206) (fig. XXI). Very few dose-response data are
availat1i for the mouse. Murakami et al. (210) report forper cent

60

I I

C

40

I

I

20

\
',
\

,

D

200 A (206)

I \
\
I
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I
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:
:

O 200 A (72)• 200 A (210)• 300 A {2061

~

,,,,'
I \I

~

'G

Rat:
exposure:

I\
I \
I \

0

11

Mouse:

expo:sure:

II\=J;~L.-~'_\-<J,~---~--

0 ._____ _ _
6

7

B

9

10

11

12

13

14

15

16

17

days p.c.

Irradiation time

Figure XXI. Effect of irradiation at l'arious p.c. times on the induction of
microcephaly in the mouse and rat

irradiation at peak sensitivity no incidence of microcephaly with 150 R, about 15 per cent with 200 R andabout 17 per cent with 300 R. In another publication(206). the incidence with 300 R is reported to be about
55 per cent, an incidence similar to Dekaban's estimate(72) of 55 per cent for exposures around 200 R. It is
justified to assume that the technique of scoring themalformation may have influenced the estimates in thesecases. since the head diameter is a continuous variable. It
is doubtful (206) that the microcephalic condition in themouse {which is accompanied by abnormalities inmorphogenesis) could be compared to the "genuine"
microcephaly seen in man. Ballardin and Metalli ( 13)have exposed (C57BL X C3H)F I mice to 50-200 R on
day 9 and 16 p.c. The net weight of the brain was
estimated post-natally at the age of 3 months. A
statistically non-significant decrease of 1.5 per cent of
the brain mass was shown following 50 R on day 9 p.c.,while I 00 R on day 16 p.c. resulted in a very significant
decrease, amounting to 9.6 per cent in the male. and 7.5
per cent in the female, offspring.
142. The induction of malformations in the CNS of thehuman embryo {particularly microcephaly with associated mental retardation) has been the object of several
reviews (95, 96, 97, 121, 213. 294, 370). These reviews
considered the earlier work, which consisted mainly of
isolated observations, mostly of a qualitative nature. Morerecently. Dekaban in his 1968 review (71) and theCommittee in its 1969 report have analyzed in detail thewhole subject of CNS abnormalities in man (345).
143. Dekaban (71), studying more than 200 literaturecases of therapeutic pelvic irradiation of pregnant
women. selected 26 cases as being sufficiently infor-

mative and estimated the probable doses received by theembryos. Having established that in no case was the
exposure of the embryo lower than 250 R, Dekaban
attempted to correlate the type of damage observed withthe gestational age at irradiation {from about 2 to about
25 weeks). The most frequently observed abnormalities
in this series were these: small size at birth and stunted
post-natal growth: microcephaly, often associated withmental retardation: microphthalmia: pigmentary degeneration of the retina; genital and skeletal malformations:and cataract. Dekaban was able to conclude thatincidence in man followed patterns similar to thoseestablished in experimental animals. and that thepresence and type of abnormalities depended upon thegestational age at irradiation. Irradiation during the
pre-implantation stages or at times not exceeding
4 weeks of gestation can lead to high lethality, but thechildren that survive are likely to develop normally.
Between about 4 and 11 weeks many organs and systems
appear to be sensitive to irradiation, as shown by thevariety of malformations produced. The brain appears to
be one of the organs most consistently affected,
particularly with production of microcephaly. At
12-16 weeks, stunted growth and microcephaly is still
induced. but gross abnormalities of other organs are notseen. Finally, microcephaly, mental retardation andstunted growth are induced, in a mild form, with
irradiation at 16-19 weeks; irradiation in the period
20-25 weeks causes no obvious abnormalities but only
minor defects of haemopoiesis and of the skin. It should
be pointed out that large doses can be lethal to theembryo at any gestational age.
144. In the review carried out by the Committee in its
1969 report (345), a rough estimation is given of the
681

.

•
possible incidence of ment~ retardatio~ asso:~ated \~ith
microcephaly. The value 1s about 10 rad , denved
from observations at high acute doses (in excess of
50 rad). The report stressed the uncertainties involved in
extrapolating these findings to smaller doses,_ due to_theabsence of suitable human data and to msufficientknowledge of the mechanisms responsible for the
production of malformations.
145. A recent report by Miller and Blot (196)considered the dose-effect relationships for microcephaly on the basis of the Hiroshima and Nagasaki
experience and updated dose estimates. A progressi~eincrease of the incidence with dose was observed mchildren exposed in utero before the 18th week ofgestation and examined at 10 years of age (388 children
in Hiroshima and 99 in Nagasaki). The Hiroshima data,
which were more abundant, revealed a higher frequency
and severity of the condition when exposure occurred
within 3 to 17 weeks of gestation, but some effect waspresent with irradiation at older stages. While the lowest
effective total kerma in Hiroshima was between 10 and
19 rad, in Nagasaki no effect "".as found up to a total
kerma of 150 rad. It should also be pointed out thai in
Hiroshima the neutron kerma amounted to one fifth of
the total kerma. and therefore the fraction of theabsorbed dose due to neutrons would have been
substantially lower than that due to gamma rays, In
Nagasaki, the contribution of neutrons to the absorbed
dose was much lower still. These facts suggest that theefficiency of neutrons for induction of microcephaly
must be high.
146. Mental retardation, which is a consequence of
substantial brain cell depletion at high doses, wassimilarly examined as a function of dose by Blot andMiller (24) in a fixed sample cohort of 1613 children
exposed within 2 km of the hypo centre in the two cities.
When they reached 19 years of age, they were tested formental deficiency. The controls were children not
exposed or exposed at distances of 3-5 km. Gestational
ages between 6 and 15 weeks appeared to be mostsensitive. Expressing the increase of mental retardation
incidence as the ratio of the incidence observed in thesample to that observed in the two control proportions,the increase in Hiroshima in the total kerma range
200-299 rad was more than I 00, while in Nagasaki it was
only 12 in the same total kerma range and 53 for valuesof kerma higher than 300 rad. The difference between
the two cities may have been due to the neutroncomponent of the irradiation, which was almost absent
in Nagasaki, or perhaps to other environmental
differences between the two cities at the time of the
explosions.

6. Malformations of the skeleton
147. In contrast with the paucity of quantitative data
for many types of malformations, exhaustive descriptions of radiation-induced malformations of theskeleton of the mouse at several dose levels have beenreported by Russell (296, 297) and Jacobsen (136). A
less complete set of data on these types ofmalformations was reported by Novel (229).
682

148. The skeletal system is particularly suited forteratological experiments since bone embryonic primordia are sensitive to radiation and the defects are
relatively easy to diagnose and quantify by staining
procedures ( 136). Russell (296) has also pointed outthat data on skeletal abnormalities could be used as anindicator of malformations of other important systems,
such as the central nervous system.
149. It should be realized that the phenotypicalvariability of the skeletal system gives rise toquantitatively defined defects, even in normal unirradiated animals. This variability depends on a number ofgenetic and exogenous factors, to which radiation isadded as a supplementary teratogenic agent. Malformations of the skeleton are usually classified according
to (a) the anatomical region affection (skull, vertebral
column, thorax etc.), (b) the topographic location(cer1ical, thoracic etc.), (c) the numbers of ossificati~ncentres and (d) their form (fusion, splitting, constnctions etc.). Complex systems of classification can bederived from the interplay of all these variables andexhaustive lists of possible defects are given in the papers
cited above.
150. From the work of Russell (296) it appears that thedevelopmental period of the mouse where skeletal
abnormalities are induced extends from 6.5 to 13 .5 days
p.c. Each abnormality has its own period of maximum
sensitivity. At the lowest effective dose, the period foreach type of lesion lasts for only l or 2 days; for most
malformations increasing the dose usually results inlonger induction periods. Often biphasic or unusuallylong periods of maximum sensitivity are observed,
probably indicating that developing organ primordia
have a multitude of targets and that the same type of
malformation may occasionally be produced bydifferent pathogenetic mechanisms.
151. The results of Russell in 1956 (296) covered theexposure interval of 200 to 400 R and the whole of theperiod of major organogenesis in the mouse. From theseresults it was quite clear that the shape of thedose-response curves obtained at the period of peaksensitivity was different for each malformation.
152. Subsequent work (297) with BALB/C embryos
irradiated on day 7 .5 and on day 9.5 p.c., extended theexposure range downwards (25-100 R). At these
exposures it was possible to differentiate better between
those malformations which had a very high incidence athigh doses and to search for the presence of thresholds.
Concerning the day -7.5 series, the incidence of all
malformations (of any degree) increased as a function of
exposure. as shown in figure XXII. The author pointed
out the difficulties in the interpretation of these curves
due to the heterogeneity and the different degree of
expression of the malformations. Even at 25 R, there
was an increment in the frequency of abnormalities
normally found, and furthermore, types of abnormalities
normally not present in the controls were also observed.
The significance of the incidence increment was assessedon the basis of two extreme and oppo~ite assumptions:(a) that the malformations were completely uncorrelatedwith each other (i.e., by comparing the number ofmalformations obtained versus those expected in theirradiation at 7.5 days p.c.

s.o...I{2971• jumbling of vertical centers
O ribs fusion• absence of vertical centen
O splitting of lumbJr centers

irradiation at 7.5 days p.e.

{1361

o defects of the ar'Jical verut:ral column• defects of the thoracic vertebral column• defects of the lumbar vertebral a,h.imn
O detects of the sternum and ribs;;, 1I
4.0

80-,

-:,

·;:

~

!. 3.o ...
;

~ I
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~

1

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I

.§

I

0

00~

1 2.0

~

0

l

z

1.0

:,

0

I,.,
.40

'

60

80

100

R

EXPOSURE

Figure XXII. Dose-effect relationships for incidence of skeletal
malformations in t11e mouse

control and in the 25 R group) and (b) that themalformations were completely correlated (i.e., bycomparing the number of mice with one or more
malformations in the two groups). The increment was
shown to be significant under both assumptions, in spiteof the small sample size, comprising 39 control and 30
irradiated animals. Similar considerations applied to four
quantitative skeletal characters with irradiation at
8.5 days p.c.

I

i

i

100

20
20

I 53. Malformations of the vertebral column, withspecial regard to induction time, were studied by
Murakami and Kameyama (205) on mice of the strains
CFI and ddN given a single 200-R exposure of x rays at
8-13 days of pregnancy. These authors reported ahead-to-tail shift in the occurrence of malformations
with advanced pregnancy stage (see also (208)) and theoccurrence of two peaks of sensitivity in the vertebral
abnormalities. The first peak was attributed to a
disturbance of the early somitization. the second to a
direct effect on the vertebral primordium itself.
Interstrain differences in malformation frequency werenoted. probably related to the rate of differentiation of
the relevant structures. Other differences regarded theincidence of supernumerary or ectopic ribs, which were
attributed to the genetic constitution.
154. The problem of the dose-effect relationship forskeletal malformations was the object of a thoroughstudy by Jacobsen (136), who extended the range down
to 5 R, thus covering the interval 5-100 R. After adetailed analysis of the abnormalities observed indifferent bones, where 5 and 20 R produced several
defects, Jacobsen plotted, as a function of dose, thecombined number of all malformations found in a given
skeletal region (fig. XXIII). In all regions, an increase of
exposure was associated with a rise of incidence. The
lowest exposure tested, 5 R, produced an increase which
was statistically significant at the 5-per-cent confidence
level of the malformation incidence observed in all
skeletal regions, when such increase was expressed byR

EXPOSURE

Figure XXIII. Dose-effect relationships for incidence of skeletal
malformations in the mousecomparison with the appropriate controls. The experiment therefore did not establish the existence of athreshold, which might imply that exposures of less than
5 R could conceivably entail a risk of teratogenic
damage (see also paragraphs 293 and 294).
I 55. Irradiation of the rabbit embryo (250 R on day
8- I Op.c.) has been reported to induce damage to theaxial skeletal system (69). Dose-effect data for skeletal
abnormalities in lambs were reported by Mcfee et al.
(188). The experiments were carried out in the exposure
range I 00400 R at day 23 p.c. The same paper includes
a few data on cattle exposed on day 32 p.c. to
I 00-300 R. and also on the pig, with an average dose of
367 rad of neutrons received at different times( 15-27 days p.c.).
156. Concerning information on humans, a roentgenographic survey of a small sample of 74 children exposed
in utero during the Nagasaki explosion was performed in
1951 and 1952 by Sutow and West (339). These
children had been exposed within 2 km of the
hypocentre: the skeletal findings were compared withthose of 91 children exposed within a distance of
4-5 km. There was no significant difference between thetwo groups. Furthermore, the occurrence of thoseskeletal anomalies which are found in animal experiments could not be documented in this human group.
7. Malformations of the extremities
157. The peak of sensitivity for the induction of
malformations of the extremities by exposures in theinterval 150.300 R, is found at 12 days p.c. in themouse: the peak spreads over more days at higher
exposures (fig. XXIV) (210). Sensitivity for digital
malformations at 200 R is a maximum at 12 days ofpregnancy (227), the morning after copulation being
considered as day 1 of pregnancy. Considering the same
morning as day 0, a careful series of experiments (343)
has established the sensitivity peak to be at about
273 hours after mating, that i~ early on day 11
683

exposures:

per centi

O
150RJ• 200 R (210)100

A 300 R

occurs on day 9 p.c. (210). When the exposure isincreased to 200 R. the peak is on day 9-11 (210, 72).
and with 300 R it extends to day 13 (210) (fig. XXVI).

per centI100~

t:r:.posure1:b.

150 A].

• 200 R (2101"' 300 A

O 200 R 1721

60
BO

dev:elopment 1ge 111 ref. 72
is reckoned horn one day
earlier thin in , ef, 21 O

60

20

40

20

I

b.

I '

,/J/
\\ .
.,"'-..1,.

lrradiatton time_A

Figure xxrv. Incidence of malformations of the extremities in
the mouse after irridiation at various times p.c.

~-"'=.--,---,-"'-.,.--,--~--,---

0 l-.-,---,-~t-·

6

1

a

9

10

11

12

13

14

15

c1avs p.c.

Irradiation time

(fig. XXV). The critical period for inducing abnormal
development of the forelimb appears earlier and is longer
than the corresponding period for the hind limb (294).
~:.oc,sure: 200 R

per cent•(227)

O (34310dnelopmmt age in ref. 343
is rwckoned from one day*Mlier than in ref. 227

l

,,

;\
I'

''

J

I
I

60

\

I
I

I'

II

I'

II

f

I

I

\
I
I'
I

0

I
•

I

\

\
\
I
I
I

~ - . O - -•C > - ~ - . - - '----,---=---~-~-,
6
7
B
9
10
11
12
13
14
15
I

doys p.c.

lrndiatkm time

Figure XXV. Incidence of digital malformations in the mouseafter irradiation at various times p.c.

158. Erickson and Murphree (82) described abnormallimb development in cattle, sheep and swine exposed inutero at various gestational' ages to 200 R of6
°Co gamma rays. The period of maximum sensitivity
was approximately day 32 p.c. for the cattle and day
23 p.c. for the sheep, in good correlation with therudder stage of limb development. As in other species,
the critical period for forelimb malformations occurred
0.5-1 day earlier than that for pelvic limb malformations.
159. Tail malformations are induced in the mouse byirradiation at a time between day 9 and 13 p.c.
Following an exposure of 150 R, the peak sensitivity
684

Figure XXVL Incidence of tail abnormalities in the mouse after
irradiation at various times p.c.

With larger exposures, therefore, the sensitivity peak
becomes wider. Tail length is a parameter of interest
because it can be associated with the number ofvertebrae and may be scored by external inspection.
Russell {294) reported results of experiments with
exposures of 200-400 R at 4.5-13.5 days p.c .. showingthat the relative tail length is altered by irradiation on
day 9-13 of pregnancy. Abnormal tail shape was ingeneral correlated with tail-length effects. Jacobsen(136) found that, exposing on day 7.5 p.c. to levels in
the range 5-100 R, the modal distribution of tail length
shifted towards lower values with increasing exposure.
160. Warkany and Schraffenberger (357) describedvarious types of skeletal abnormalities in the rat, among
which malformations of the extremities were dominant.
Depending on the radiation dose, these abnormalities
were induced from 9-16 days p.c. On the whole .
malformations of the long bones of the extremities
occurred with irradiation at earlier stages, while toe andfinger malformations were more common for later
exposures. Similar observations were also reported by
Wilson (362). Fused digits in forelimbs and hind limbs
were observed in hamsters with an exposure to 200 R at
9.5 and 10.5 days p.c. {l 11) and in rabbits following
400 R on day 12-14 p.c. (59). Digital malformations
appeared also in dogs following 150-200 R in the period
25-28 days p.c. (245).
161. Malformations of the tail. the extremities andvarious other abnormalities were examined as a functionof exposure {150-300 R. 170-kV x rays) by Murakami etal. (210), irradiating mice at 8-13 days p.c. Dose-effectrelationships of the threshold type were obtained for tailmalformations; the highest yield was induced on day
12 p.c. With irradiation at this time, the induction
increase in the range 150-200Rwasabout 1.310- 2 R- 1
(fig. XXVII), similar to that observed by Dagg (68).

i
I

r

I

'
I
r

i.

'.

'.

par Clffllt

•J~A

too-;

II

/'jl<f;/

: "

80~

I

it

• I

II
I I

60

~

l

I
I

I
I

O 129/RT irudio1ech1 10 days p,:.
• BALB/Cirradl11od •t 10 d1y1 p.c.
"' C57BL irradilUd It 10 days p.c.
(681
• BALB/Ci,radiarftl ar 10.5 days p.c. .J

t

I
I
I

20

D ddN irradiated at 10 doYi p.c.
• ddN irradiated at 11 diY1 p.c.

+ ddN irr ldilUd 1t 12 days p.c.

J/,.,,,,,]{2101• inbrl'Ct mice irradilttd at 10.Sdays p.c. (217)"'o~-----~-----~-------r--R
JOO
200
uio

EXPOSURE

Figure XXVU. Dose-effect relationships for induction of digital malformations in the mouse

162. Malformations of the extremities were seen mainly
in mice irradiated 11 and 12 days p.c., particularly
following exposures of 200 or 300 R. A very detailed
study of digital malformations in mice of the ddN and
CF! strains was performed by Nogami (227). Variables
examined were the pregnancy time (8-13 days p.c. at a
fixed exposure of 200 R), the exposure (150-300 Rat a
fixed time of 12 days p.c.) and the genotype. The
incidence of individuals with malformations was
proportional to exposure in the interval 150-300 R, at
the rate of about 5 10-3 R- 1 but below 150 R no data
were obtained, which did not allow the assessment of a
possible threshold (fig. XXVIII). Concerning the
per cent

100

D •ff "9ral m.dfornwrion,
0 ectrDdllclylilm• brldlvdlcty~,m"' syndlCIYlitm
80

~

8
e

-8

!

~
20.:
I

I
ol
100
Figure XXYUI. Dose-effect relationships for various forms ofdigital malformations in the mouse (227)
The incidence of all digital malformations (0) was calculated
over total living foetuses whereas the incidence of ectrodactylism(o), brachydactylism (•) and syndactylism (A) were calculated
over foetuses with digital malformations only.

different types of malformations. ectrodactylism incidence clearly increased with dose following a thresholdtype relationship, while brachy- and syn-dactylism
displayed a decreasing frequency with dose (227).
163. Dagg ( 68) studied the induction of tail malfor·
mations and hyperphalangia of hind feet in three inbred

strains and some reciprocal crosses. The defects werescored at 18 days p.c. on live foetuses that had been
irradiated at 10, 10.5 or 11 days p.c. with exposures of
250-kV x rays in the range 75-200 R. Disregarding somedifferences between strains due to the variability indevelopmental age, the exposure-response curves for tailmalformation were in general of the threshold type; theresponse increased linearly with exposures from about
75 to about 150 R (fig. XXVII). In this range theinduction frequency can be calculated to be approximately 1.2 10- 2 R- 1 , a value roughly applicable to thethree strains used. The dose-effect relationship formalformations of the hind foot was more complicated.
However, if the percentage of live litters with one ormore hyperphalangous feet is plotted against exposure.
the plot is consistent with a non-threshold linear
relationship with a slope of about 5 10-3 R- 1 • Other
plotting criteria could suggest curvilinear relationships.
Mechanisms to explain the linear relationship have beensuggested (68).
164. Nash (217) also observed a threshold-type
dose-effect relationship for tail malformations in inbred
mice following irradiation at 10.5 days p.c. with
exposures in the range 80-320 R (fig. XXVII)." Differences with respect to hybrid mice were also noted. No
abnormalities of the feet were observed by external
examination at exposures below 80 R, but at higher
exposures overgrowth or reduction of the feet wereclearly manifest. A different result was found by
Jacobsen (I 36). who analyzed the degree of ossificationof the paws. using alizarin-stained skeletons, and foundeffects at exposures as low as 5 R. He believed that the
number of stainable centres of ossification in theextremities might be a suitable indicator, in sufficiently
large samples, for studying the effect of small radiation
doses. Limb malformations were reported to occur inmarmosets given 250 R of x irradiation at 25 days ofpregnancy. during the period of major organogenesis(249).
8. Other malformations
165. Malformations of the heart and great vessels of the
rat were the subject of some early reports (295, 362).
More recently. they were studied by Okamoto et al.
685

(233). The incidence of these malformations r-eached 85
per cent after a dose of 130 rad of 14.1-MeV neutrons,
on day 8 p.c. Histopathological, cellular and kinetic
changes of the relevant structures were described inother publications (199). Cardiac malformations were
induced in dogs by exposures to 100-150 R at 21-22
days p.c. (245).
166. Malformations of the anus (small or imperforate)
and of the urogenital system (hydronephrosis. convoluted or distorted ureter) were observed by Russell (294)
in a small percentage of mice exposed to 200-400 Rat
9.5 and 10.5 days p.c. In rats, Wilson (362) reported theoccurrence of kidney agenesis after exposure to 100 or
200 R on day 9 p.c., and horseshoe kidney after
irradiating on day 10 p.c. Both types of malformations
were induced also on day 11 p.c. A 77-per-cent
incidence followed an exposure of 200 R. Urogenital
malformations (unilateral or bilateral kidney agenesis)
with a peak sensitivity just before the appearance of themetanephric kidneys were reported in the dog (245).
167. Thoracic and ventral hernia in mice were describedby several authors. Murakami et al. (209) obtained anincidence of about 5 per cent with 200 R on day 9 p.c.
In a subsequent experiment (210) a maximum incidenceof 44 per cent was obtained with 300 R on day 8 p.c.
Jacobsen (136) described an incidence of about 2 percent following an exposure of 100 R at 7.5 days p.c. In
hamsters, Harvey and Chang (111) reported a 12-percent incidence of hernia and spina bifida following
200 Ron day 7 .5 p.c.
9. All malformations
168. In spite of the great interest for risk assessments of
the overall induction of malformations, very few authors
have given figures for the total percentage of malformed
conceptuses in their experiments. Taking all malformations together, the peak sensitivity to induction
occurs on day 9 p.c. in the rat embryo (325). In the dog
the time immediately following implantation (corresponding to the primitive streak stage and earlyorganogenesis) appears to be the most sensitive forthe production of malformations (245). Concerning
dose-effect relationships (fig. XXIX), Murakami andirradiation time100+

8 dav, p.c.

O•

8 day,
9 dayi p,c,

1204]

p.c.1

~ ~~~~~(210)

O 12 days p.c.
• 13duv~P.c._.

+------

60

//

/+

+
al___
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
200
300 R
100
EXPOSURE

Figure XXIX. Dose-effect relationships for induction of grossmalformations in the mouse

686

Kameyama (204) reported the incidence of grossly
abnormal mouse embryos (living and dead on day
13 p.c.) following irradiation with various exposures(25-150 R) on day 8 of pregnancy. From this study the
induction can be calculated to be about 5 10-3 R- 1
without any apparent threshold. In a later report (210),
Murakami gave the incidence of abnormal foetusesreaching 19 days p.c. for an exposure range of150-300 R, with irradiation occurring in the period 8-13
days p.c. The induction in this range (discardin~ deadfoetuses) appears to vary between 1.9 10-3 R - and4.5 10- 3 R -t. depending on age at irradiation.
169. Recent human studies relevant to all types ofmalformations deserve attention. Kinlen and Acheson(148) published a retrospective study of the irradiation
history of 379 women hospitalized for spontaneousabortions and of 605 women who delivered malformed
children. Their radiation history was compared with thatof a control group. No significant difference was found.
The sample size was clearly too small to determine theeffects of doses at the diagnostic level. The authors
concluded that, under the conditions of observation,
there was no evidence that irradiation could play animportant part in the etiology of abortions requiring
hospitalization or in the induction of congenital
malformations. Hagstrom et al. (103) made observationsin about 700 women who received 5 9 Fe during
pregnancy for metabolic studies, resulting in an
estimated dose to the foetus of 5-15 rad and also in
about 700 controls. Their findings were essentiallynegative for the induction of malformations in theoffspring.
170. More recently, Diamond et al. (74) reported on aprospective study of about 20 OOO white and blackchildren who were exposed in utero over the period
1947-1959 in the course of abdominal irradiations of
their mothers. mainly for pelvimetry, placentograms or
simple radiography of the abdomen. The children weretraced in 1961-1967 to determine their mortality andthe cause of death according to the death certificates.
The group was compared with a non-irradiated cohort ofapproximately 36 OOO children matched by hospital andtime of birth, sex, parity and race. A sample of about
152 OOO person-years in the exposed group and 28 OOO
person-years in the control were thus collected andexamined. The study was mainly concerned withleukaemogenesis. but data on malformations were alsoavailable and have been summarized by Lilienfeld (172).
No statistically significant difference in the mortality
from congenital malformations was seen between
exposed and control children. Concerning the sensitivityof different gestation periods, there was no difference inthe malformation frequency between two groups
defined by the trimester at which they were irradiated.
Within each trimester, the irradiated children had aslightly higher incidence of abnormalities than thecontrols. in both sexes and races. In white children, themortality of the exposed group was almost twice that ofcontrols (183 per 10 5 person-years against 98 per 10 5 ).
In black children. on the contrary, the same mortality
was observed in exposed and control groups (212 per
10 5 person-years and 229 per 105 person-years,
respectively). The difference could not be attributed tocongenital malformations.

-~--

171. In a prospective study (235), the morbidity andmortality of about l OOO white children exposed in utero(average foetal dose about 700 mrad) during routine
diagnostic pelvimetry were followed for 15 years,
compared to an adequate control group. The data(which had been reported previously by Griem et al. (99,l 00)) were most carefully reanalysed and compared withprevious studies, but were insufficient to associate
radiation conclusively with any effect. It should bepointed out that only 3 children were exposed during
the first 50 days of gestation and only 24 during the firsttrimester, while 87 per cent were irradiated during thesecond half of pregnancy. With this sample size,radiation effects could be detected only if they were
induced in excess of 1 per cent, which is at least an orderof magnitude higher than might be expected for themost frequently radiation-induced abnormality in man,namely microcephaly (see paragraph 144). In theopinion of the authors. the study is therefore of value in
excluding the possibility that doses of the order of 1 rador less could produce effects exceeding 10 times theincidence observed at high doses.
172. Oppenheim et al. (236) reported a comparison
between studies in which the exposure to radiation was
the result of medical indications (74) and studies
involving non-selective exposure to radiation (100, 145,
235). Significant discrepancies were found, suggestingthat the data reported in the first instance could be
attributable to the bias introduced by the medical
conditions requiring the exposure, rather than to the
exposure itself. These new data therefore cast somedoubt on the validity of the studies of diagnostic
exposures and suggest that such exposures may actually
be less harmful than previously assumed (236).
173. Neumeister (222) reported most recently on aseries of 37 cases of embryonic exposure between week
1 and week 10 p.c. up to a maximum of 20 rad. These
cases had been referred to a specialized unit of clinicalradiobiology for expert opinions concerning a possible
abortion. Of the 35 cases that were conducted to term,
only 15 children were examined at 1-3 years of age. In
all these cases the pregnancy had proceeded normally,
the weight and body size and the subsequentdevelopment of the children were found to be normal
and no difference was noted between the exposed
individuals and their brothers or sisters. In one casewhere the exposure had been less than 1 R at 4-6 weeks
p.c. a talipes calcaneus was observed, but a causal
relationship with irradiation was excluded. A secondcase (exposure 3-5 R at 2 weeks) showed talipes
calcaneus and a lumbar myelomeningocele: a relationship with the previous exposure was estimated to be
improbable. In spite of the accuracy in the estimates of
exposure time and of dose calculations, the size of thisseries is clearly too small to be of any generalsignificance.
174. In connection with the possible effects of very low
doses of radiation. some correlations have been
attempted between the overall incidence of malformations in man and the level of natural radioactivity. In
what they regarded as a feasibility study, Kratchman andGrahn (158) tabulated the incidence of deaths fromcongenital malformations for the years 19 52-19 5 6 invarious areas of the United States of America andgrouped these data by geologic provinc Th
to show a higher mortality from alfe. ey were able
· them . ormat'ions mgeologic provinces containing ma_ior
uraru
deposits, uraniferous waters or high her
um oretrations. It should be pointed out that no mm concen·
1 1
measurementsof ra di ation eve s were made and also that1Y crude
·
·
al
·
d
d
statisticm exes were use , without correon.
ct·10ns f or1birth rate,. materna age, death rate or other fa ct ors t h at
could influence the observed correlation Th da·etahowever, were considered by the authors t 0 b'
sufficiently provocative to justify further investioa
ti·o ns.eo175. In a highly speculative paper Wesley (361), took adifferent approach: he analysed the fraction of
malformed births over the earth as a function ofgeomagnetic latitude, which is associated with different
doses from cosmic rays. The relation between theworld-wide incidence of congenital malformations
obtained from vital statistics and the geomagnetic
latitude appeared to be highly significant. The lines of
equal incidence of malformations were said to match thecorresponding lines of the magnetic field of the earth.
According to the author. all congenital malformations
could be attributed to "background" radiation (presumably its extraterrestrial component). It should,
however, be pointed out that both the backgroundradiation data and the epidemiological data on whichmuch of this paper rests have been seriously questioned(46, 330).

D. OTHER EFFECTS
176. Fertility and fecundity. Male CFl mice were
exposed to 25 or 100 R in the period 0.5-18.5 days p.c.
and .tested for reproductive potential at ages ranging
from 2 to 8 months. With the exception of a reductionof· the reproductive quotient in animals exposed to
100 R at 10.5-12.5 days p.c., no significant effect on
fertility was documented. In the opinion of the authors.
the small reduction of the reproductive quotient could
reflect a general disability of the animals, rather than areduction of their true reproductive potential (288).
177. Concerning effects in the female. some rather
incomplete data ae available for mice and rats. Female
mice were exposed during the first and the second halfof the organogenesis to 20-80 R per day for 5
consecutive days. Their reproductive capacity was thentested in a successive generation. Irradiation on day
11-15 p.c. was shown to have the greatest effect and thegroups exposed to 60 and 80 R per day were unable toreproduce further. Animals exposed to 40 R per day
produced litters with very high mortality. and thenumber of litters produced depended both on exposure
and on gestational age at irradiation. The frequency oflitters was particularly small among animals irradiated in
the embryonic stage. The average litter size, on thecontrary, was not greatly influenced by irradiation, thedifferences in the reproductive capacity being mainlyreflected by litter frequency rather than by litter size(166). Rat females exposed in utero to 110 R on day
15 p.c., to 150 Ron day 18 or 20 p.c., or to 220 Ron
day 17-20 p.c. were subsequently mated to normal
males. These irradiations had no effect on the incidence
of infertility and on the number of offspring produced.
687

The only effect consisted in a significant (20 per cent)reduction of the ovulation rate, which was dosedependent (212).

178. Cataract formation has been reported as a result of
exposing mice to about 100 R during the embryonicstages (270, 292). The induction of tumours following
irradiation in utero is discussed in Annex I.
179. A significant decrease of the in vivo and in vitro
incorporation of labelled aminoacids into proteins, 24hours after irradiation. has been shown in embryo rats asa result of a dose of 180 rad on day 13 p.c. (313).
Enzyme changes appearing post-natally in the brain andthe liver of rats irradiated in utero (25 or 50 R on day
14 p.c.) have also been described (214, 215, 216).
Concerning haemopoiesis, an arrest of cell divisionaccompanied by alterations of the haemoglobin
synthesis in the embryonic erythroid cells has been
. shown to occur in the mouse after a single x-ray dose of
120 rad received on day 10 p.c. (85).
180. Ultrastructural effects induced by 50 R in the liver
cells of the rat embryo on day 13 p.c. and consisting
essentially in changes of the aggregation of polysomes
and in a delay in the appearance of the smooth
endoplasmic reticulum provide some insight into thechain of molecular, biochemical and structural events
taking place after pre-natal irradiation ( 62). Someinfonnation concerning the effects of radiation on theregenerative capacity of the muscle are also available.
The disorders observed were more pronounced inanimals irradiated during the second half of pregnancy,
when histogenesis of the muscle occurs (163).

IV. THE FOETAL PERIOD
A. LETHAL EFFECTS

1. Mouse

181. The foetal period in the mouse begins approximately at 13 days p.c. It should be pointed out thatmany of the experiments on the induction of mortality
during the major organogenesis period reviewed inparagraphs 84-108 covered some of the later stages aswell. As the foetal development proceeds, the dosenecessary to kill animals before or after birth increases
gradually, particularly the pre-natal LD 5 0 • The percentage of abnormal animals with a given dose, on theother hand, tends to decrease with foetal age.
182. Rugh and Wohlfromm (286) established that the
pre-natal LD 50 for irradiations at day 14-17 p.c. ishigher than 700 rad (see table 4), with a clear tendency
to increase with foetal age. Their experiments show thatkilling the foetus in utero requires very high doses, mostof the lethality at lower doses being seen at birth or justafter birth. Irradiation of the foetus results in fewer
macroscopic anomalies than irradiation of the embryo,being instead consistently associated with stunting.
These data are similar to the results of Dekaban (72),
who exposed foetal mice to 200 R and scored the688

percentage of surviving and apparently normal animals.
This indicator was shown to rise from 38 per cent forirradiation on day 13 p.c. to around I 00 per cent forirradiations on days 17 and 18 p.c. The mean litter size,
serverely affected by the exposures of the same
magnitude during the embryonic period, gradually
increased to normal for irradiations during the foetal
stage. Experiments with fractionated (152, 155) or
chronic irradiation ( 154) also show clearly that lesslethality is induced during the foetal development thanin earlier stages.

183. The study of post-natal lethality induced byirradiation in the foetal stage is a necessary complement
to the experiments on in utero mortality. Rugh andWohlfromm (289) extended their previously reported
observations (286) to th!! frrst 30 days of life: They
showed that a gradual increase of the post-natal
LD 50130 takes place with increasing post-conception age
at irradiation from about 250 Ron day 13 p.c. to about
600 R on day 18 p.c. The second day after birth showed
the highest death rate. From these data it can beconcluded that the late foetus is at least as radioresistantas the mature animal. Although limited to only 2 days inthe foetal stage (14.5 and 17.5 days p.c.), the data of
Nash (217) are also useful to compare the resulting preand post-natal viability with that observed with
irradiation at earlier stages. They show a decline ofsensitivity with increasing foetal age for induction of
pre-natal and post-natal lethality. together with a slightly
higher resistance of hybrid. as compared to inbred,
genotypes. No differences of sex could be shown.
Similarly, the data of Konermann et al. (15 5) on
fractionated exposures at 40-80 R per day during themain developmental phases show that post-natal survival
is only slightly affected. This observation is particularly
striking when compared with the effect of the same
exposures during organogenesis. Again. the first week
after birth is the time when most deaths are observed.
184. Long-term survival was studied by Upton et al.
(347), who reported that exposure to 300 R at 14.Sdays p.c. produced much more life-span shortening inmales than in females. Minimal reductions of longevity
after exposure at later stages of gestation were reported.
Nash and Gowen (219) also emphasized the effect of sex
in the survival as adults of mice irradiated during the
foetal stage.
185. Rugh and Wolff (293) claimed that an exposure of
10 R during the foetal stage increased the survival ofmice receiving a mid-lethal dose at an age of 4 months.
The age dependence of this beneficial effect is however
uncertain, and higher exposures (25-300 R) to foetusesof any age are detrimental to survival after test doses.
Christensen and Jackson (64) exposed mice 14.5 days
p.c. to 150 R and subsequently determined their
LD 50130 at the age of 8 weeks. The data suggested thatbetween 30 per cent and 53 per cent of the foetal
exposure had been repaired by that time. The authors
also concluded that the foetal stage was considerably
more sensitive than the adult in terms of irreparable
radiation damage, probably of the haemopoietic system.
1l1ese results should be compared wit'1 the data of Sikov
et al. (322) and others in the rat (see paragraphs I 89 to

191).

·-! ..

.·.

~

2. Rat

3. Human experience

186. In the rat the foetal period begins approximately
16 days after conception. The data in this species are farless complete and often limited to a few ages in the
foetal period (see table 4). They are, however,self-consistent and in agreement with the results
obtained in the mouse.

192. Some studies have been made on the terminationof pregnancy in women irradiated in the pelvic region
because of various pathological conditions or fortherapeutic abortion. These studies provide qualitative
information on the mortality of irradiated humanembryos or foetuses. The early observations of Murphy(213) are not very relevant for this purpose, but the dataof Harriss (106) on therapeutic abortion are somewhat
more significant. These data covered 138 cases ofwomen irradiated at 6-18 weeks of pregnancy with
exposures of 510 R of 200-kV x rays in the uterine
fundus. The treatment caused interruption of pregnancy
in 129 cases, within an average time of 4 weeks. The
foetuses were grossly macerated and in one case a cyst of
the brain was found. Non-specific degenerative changes
in the placenta were also noted. Mayer et al. {187)reported that a single exposure of 360 R in the uterus issufficient to cause abortion in the large majority ofcases.

187. Murphree and Pace (212) irradiated with a few
selected exposures (110, 150, 220 R) at ages from 13 to
20 days p.c. Their data show an increased resistance topost-natal lethality as a function of age. The majority of
post-natal deaths were observed, as in mice, within thefirst week. Reineke et al. (253, 254) determined
carefully the LD 50130 of rats irradiated S days or 1 day
before birth, confirming the increase of radioresistanceof the foetus with age, and the absence of any sex effect.
Sikov et al. (322) examined the effect of SO or 185 Ron
day IS p.c., showing that the incidence of still birth andneo-natal mortality was larger with the highest exposure,and that most live-born animals died within the first
week. The post-natal mortality of the rat foetus was alsostudied by Ader and Deitchman (1), who used exposuresof 200 Ron day 16 p.c. It should be pointed out that inspite of similar values of LD 50130 for the adults in both
species. the LD 50130 for the foetal rat is between 200
and 300 R and therefore consistently lower than that for
the foetal mouse, which appears to reach 600 R during
the late gestational ages.
188. In 1954, Russell (295) summarized the information on the mode of death of various animal species
exposed during the foetal period. From the summary it
may be concluded that the syndrome of haemopoietic
failure is the most common cause of death at doses
around the LD 50 • More recent reports do not add
further quantitative data on this subject.
I 89. Reineke et al. (254) studied long-term survival
following foetal irradiation. They found that exposure
to 220 R S days before birth resulted in 53 per centsurvival after I month of life, and that the subsequent
survival, up to about 4 months, was practically the same
( 48 per cent). The long-term survival of these animals
was not significantly different from that of controls.
190. Sikov et al. (322) reported that the post-weaningmortality of female rats exposed to 185 R on day
15 p.c. was significantly higher than that of controls,
while the median life span of males was not significantly
affected, thus pointing again to a dependence of thelong-term mortality on sex. An interesting observation inthese experiments is that the LD 5 0130 of an irradiation
at 100 days of age decreases as a linear function of thedose of a pre-natal irradiation at all developmental ages.
In the opinion of the authors. such a linear dependence
would imply a general decrement of fitness as a result of
pre-natal exposure.
191. According to Maisin et al. (180) the radiosensitivity of rats surviving an exposure of 70 R at
10 days p.c. is lower than that of non-irradiated control
animals. Although a tentative interpretation wasadvanced. based on the larger elimination of the moresensitive animals by the first dose. no definite conclusion
was drawn.

193. Autopsies of irradiated human foetuses are rare.
Driscoll et al. (76) reported two cases where the mothers
had been treated with radium for cancer of the cervix.
The foetuses, measuring 15 and 21 cm, had been
exposed to about 800 and 1600 R. respectively. One was
examined 2 days and the other 10 days after the
beginning of the irradiation. Damages to the nervous andthe haemopoietic systems were observed, and thealterations of the mesenchymal cells extended to regionsof the body where the estimated exposures had been of
the order of 50 to 100 R. This pattern of damage is notunlike that described for other species.
194. The data on mortality of children irradiated inutero as a result of the Hiroshima and Nagasaki
explosions are of epidemiological nature and are possibly
more informative. Yamazaki et al. (371, 372) reported
on the outcome of pregnancy of 98 women exposed in
Nagasaki. Among 30 women with major signs of theradiation syndrome who were within 2 km of the
hypocentre, 7 (- 23 per cent) foetal deaths and 6(20 per cent) neo-natal and infant deaths were observed.
Of the 17 surviving children, 4 were mentally retarded.
The overall morbidity and mortality was therefore about
60 per cent in this group. In another 68 mothers
exposed within the same distance but with no signs ofradiation syndrome, the overall mortality was 10 per
cent, only slightly above the mortality of the control
group (6 per cent). In the second and third trimester ofgestation, the foetal, neo-natal and infant mortalities
were significantly elevated among the offspring of
mothers who suffered from the radiation sickness. The
evaluation of these data is, however, very difficult,owing partly to the absence of proper dosimetry andpartly to the possible effect of the blast and thermalradiation, as well as to the indirect effects of maternal
haemorrhagia and infection.
195. Kato (145) studied the mortality at later stages oflife, following for 24 years 1300 children exposed inutero in Hiroshima and Nagasaki. He showed adose-related increase of mortality during the first year oflife, an absence of any relationship between mortalityand dose from 1 to 10 years. and again a dose-related689bincrease at later ages. These increases of mortality withdose were not attributable to concomitant variables,such as age of parents, birth order, socio-economic
factors etc. Mortality after I O years was not associatedwith any specific cause of death and, contrary to whatmight have been expected from the animal data,radiation mortality was only apparent among children
exposed in the third trimester, possibly because of ahigher rate of abortions and still births of embryos
exposed earlier. Dosimetric uncertainties concerning
exposure in utero do not allow more definite
conclusions.
B. DISTURBANCES OF GROWTH
I. Mouse
196. Generalized and local growth disturbances areprobably the most commonly described effects ofirradiation during the foetal stages. The following review
is rather arbitrarily limited to those reports paying
special attention to growth defects.
197. Duplan and Izadian (78) studied pre-natally the
body and liver weights of foetal mice exposed between
day 14 and 18 p.c. to 200. 500 or 800 Rand sacrificed
1-4 days after irradiation. The body weight was lowest
for the higher exposures and for irradiation at early age
p.c., the effect being more marked with long observation
times. The liver weight, on the contrary, was mostdepressed when the interval between irradiation andsacrifice was short. suggesting that the effect was
temporary.
198. In the experiments of Rugh, Duhamel et al. (271 ),mouse stunting was studied systematically with exposures of I 00 R at various times from fecundation to
18 days p.c. The average weight of the animals at an ageof 4 months was minimum for irradiations at 12 or
13 days p.c. (females and males, respectively). Althoughmature weight tended to increase with increasing foetal
age at irradiation, it never reached control values ineither sex. When followed to 35 months of age, theaverage weight of the animals was still depressedcompared to controls. The dependence of thisdepression on gestational age at irradiation was very
similar to the weight depression observed at 4 months ofage (272). A "linear" decrease of body weight one day
after birth with dose in the range 120-240 rad forirradiations on day 15 p.c. was reported by Hazzard andBudd (117). The body weight of the rats, averaging 6-8 gin the control new-boms, decreased to about 3-6 g with
240 rad. However, if the control weight is taken into
consideration, the trend of the curve is clearly
curvilinear.
199. With respect to fractionated irradiation, Konermann et al. {155) reported that the average post-natal
weight of mice exposed to 40, 60 or 80 R per day during
the foetal period was always below control in the
3 weeks after birth, with a maximum depression in thefirst week. The effect of the exposure rate appears to beminor. Post-natal growth measurements referring specifically to the skeleton are also available in the mouse,
where stunting of several bones may be traced until
sexual maturity (271 ).

2. Other animals
200. Sikov et al. (322) reported a small decrease in thebirth weight of rats after exposure to 50 or 185 Ron
day 15 p.c. This difference persisted to an age of 80 to
100 weeks. both in male and female animals, and wasmore pronounced with the higher exposure. The weightsof the brain, spleen, thymus and kidney were also
depressed in the irradiated animals. Martin (186. 183,
184, 185) carried out a series of experiments on the
response of the whole-body and organ growth in animals
exposed at 18 days of gestation to 160 or 220 R of
gamma radiation. Depression of the whole-body weight
relative to controls was maximum at 21 days, but
persisted up to 1 year of age, a time at which the weight
was about 70 per cent of that of the controls. Althoughmost organs were lighter in irradiated animals, thosewhich were consistently different from controls {whenreferred to the body weight) were the brain, testes,
pituitary and adrenals. The number of cells in thevarious organs, between 7 and 60 days, expressed as percent of control, indicated that the kidney and liver
followed in the main the body weight changes. Spleen
and thymus, on the contrary, followed different
patterns, reflecting presumably the sensitivity of thespecific cell populations and their recovery kinetics.
Other results of experiments on body weight of rats canbe found in Ader and Deitchman (I). They used
exposures of 200 R at 16 days p.c. and followed the
body weight during 60 days of age.
201. Rugh, Duhamel et al. (273) followed the bodyweight during 23 months of life of two monkeys which
had been exposed in the foetal stage to 200 and 300 R.
The organ weight at the autopsy and some skeletal
measurements were also reported. All the measured
indicators failed to attain the normal values.
3. Human data
202. Only the general effects on human post-natal
growth are reviewed in this section, the special case of
microcephaly having been already considered inparagraphs 141-146. The data available regard theexposures of A-bomb survivors in Japan and, to a lesser
degree. medical exposures.
203. About 1700 Japanese children exposed in utero toradiation from the atomic explosions we.re followed on
an annual basis {366, 367. 368). More than 80 per centof these children were subjected to anthropometricmeasurements and various other tests when they reached
17 years of age. They were subdivided into three major
comparison groups: those exposed between O and 2 km
from the hypocentre, those exposed between 2 and
5 km from it, and unirradiated controls. The groupnearest to the hypocentre was further classified
according to the estimated dose to the mothers (morethan 25 rad and less than 25 rad (18)), and according to
the presence of acute irradiation symptoms. In those
exposed within 1.5 km. a reduction of mean headcircumference (I cm, or 2 per cent) was observed;
additionally, the height and body weight, in both sexes
and in both cities. were also reduced by 2.3 cm and 3 kg,
respectively. In most cases the incidence of the effects
was significant. Other indicators. such as the chest

690

,,·

circumference and the intercristic diameter. were also
affected. although more irregularly. The analysis ofinduction by trimester of· gestation revealed no
consistent pattern.

204. In another study, 286 adolescents who had been
exposed in utero at Nagasaki, were examined for growthand developmental defects (48). The indicators were theage at menarche, the degree of epiphyseal closure in thewrist and several other body measurements, such as headcircumference, standing and sitting height, and weightand chest circumference. Numerous differences were
found that were consistent with a postulated radiation
effect. With regard to pregnancy stages at irradiation, no
significant differences were observed in the males. but
almost all measurements in the females exposed during
the first trimester were significantly greater than in those
exposed during the last trimesters. Head size was the
single anthropometric indicator most significantly
affected.
205. Another study, by Russell et al. (306), specificallyconcerns bone maturation. Here, 556 subjects exposedto the A-bomb explosions over Hiroshima and Nagasaki
were examined to record the age at which epiphyseal
closure of the wrist bones occurred. In both boys andgirls, closure was found to take place 6-9 months laterthat in other Japanese children and in American
children. Possible contributory factors were discussed.
However. contrary to previous i;eports (48). nocorrelation was seen between the dose to the mother andthe time of closure of the children exposed i11 utero.
206. Shohoji and Pasternak (312) used an elaborate
Gompertz function to characterize and analyse thegrowth of about 900 children exposed in utero in Japanand to test for differences between sexes, cities,
trimester of exposure and exposure groups. Differences
observed between groups exposed at various postconception times were found almost without exception
to be non-significant. Differences between exposuregroups were greater in males than in females, a fact
pointed out earlier (368), but there was no consistent
relationship between growth impairment and exposuregroup. These new findings provide some support for thepreviously reported retardation in stature observed in
pre-natally exposed subjects ( 48, 368), but the authors
believe that this support is inconclusive.
207. A comprehensive evaluation of all these studies
indicates beyond doubt that reductions of body size andgrowth can be induced by radiation in humans.
Moriyama et al. (200), Belsky et al. (20) and Blot (23)have identified the following effects on growth as
characteristic of in utero exposure of children: (a)reduced height and reduced head and chest circumference. effects which are especially pronounced at highdoses and persist to maturity; (b) microcephaly andmental retardation, which are more frequent in children
exposed within the first trimester and for which thedose-effect relationships were discussed in paragraphs
141-146.
208. Regarding medical exposures, Nokkentved (228)reported on 152 Danish children who had been
irradiated within the first 4 months p.c. in the course ofdiagnostic procedures, compared with 143 non-exposed
siblings. Several types of x-ray procedures were involved,
only a few being simple pelvirnetries. In general, theirradiations were delivered in the course of only one
examination. but in about 40 cases multiple exami·
nations were performed, up to five in 2 cases. It was
found that 15 of the irradiated children (9.9 per cent)and 13 of the controls (9.2 per cent) presented a variety
of malformations, including heart malformations. spina
bifida, inguinal hernia, microcephaly etc. Both theradiation dose estimated by the number of radiographs
and the time at irradiation could not be clearlycorrelated with the incidence and type of malformations. However, the height of the children irradiatedduring the second month of gestation was clearly smaller
than control, and this finding could not be explained bya number of biological, medical or social factors taken
into consideration. Furthermore, the weight at birth ofchildren irradiated during the second month appeared to
be low, although the information was incomplete
because this indicator was not determined in all children.
This study confirms therefore the existence of a
growth-retarding effect of irradiation during the second
month of pregnancy. However, it does not contribute toimprove the information on the incidence of malformations, because of the relatively small sample size.

C. MALFORMATIONS
1. General

209. It is widely accepted that following the terrnination of major organogenesis, the effects of irradiation
become gradually more subtle and more difficult to
document, at least macroscopically. The large number ofreports on malformations induced during the embryonicstages (see chapter III, section C), compared with thefew papers on teratogenesis during foetal development,
is in itself a demonstration of that fact.
210. However, that statement. like any general
assertion, needs to be qualified. In fact. the distinction
usually drawn at the whole-body level between
embryogenesis (implying differentiation of new structures) and foetal development (mainly involving the
growth through cellular division of the newly differentiated structures) is hardly tenable at the level of single
systems or organs. At any given time, new structures canstill be formed while others are simply expanding: someorgans may still be undergoing differentiative processes
while others are enlarging. In addition, the period of
maximum sensitivity to induce malformations of certain
embryological structures, which at low doses ischaracteristically confined to the time of max.imurn
differentiation, can be longer with sufficiently highdoses (see paragraphs 28-29). Therefore. some malformations are in fact induced with irradiation at ages
conventionally regarded as part of the foetal stage.

211. In general, the malformations appearing withincreasing foetal age are more subtle and resulting
functional defects gradually less pronounced. That
influences the fate of the malformed offspring. sincemajor abnormalities produced during organogenesis may
be lethal during the intra- or the early extra-uterine life,
691

it,

while less serious defects. produced at the foetal stages,
may be compatible with life, although more or lessseriously disabling.

external examination of the embryos. The incidence of
these deformities also decreases as the maturation of the
foetus proceeds to completion.

2. Animal data

3. Human data

212. The considerations made in paragraphs 121-123
naturally apply to malformation induction during
foetogenesis, but the extreme paucity of data does notallow any analysis of the time and dose dependence ofthe effects. Very few ,of the reports examined inchapter III. section C, cover also the induction ofteratogenic effects in the foetal period.

217. In man. the malformation most consistently
reported for irradiation during the foetal stages is
microcephaly. Its production however, is not specific to
the foetal period; on the contrary, the experience inanimals shows that the time of maximum sensitivity
occurs during embryogenesis. For that reason, both theanimal and the human data relevant to this malformation were reviewed in paragraphs 141-146. It is
however appropriate to point out that, particularly withregard to man, most of the information reviewed inthose paragraphs applies to foetal irradiation as well.

213. Concerning external head abnormalities, Dekaban(72) reported that an exposure of 200 R in the mouse
induced small or deformed heads in 14 per cent or 4 percent of cases, if irradiation took place at 14 or 15 daysp.c., respectively. No effect was seen for irradiation atlater ages. Few observations have been reported on the
induction of ocular defects in the foetal stage. Hicks et al.
(122) carried out experiments with a few rats irradiatedon days 16-20 p.c., showing a red1,1ction of the ocular
diameters and microscopical retinal changes (rosetteformation).
214. Relatively more abundant are the data on
malformations of the central nervous system. In themouse, the induction of gross abnormalities byirradiation with 200 R after day 13 p.c. is less common
than in previous stages but still present {72, 143).
Microscopically, however, malformations can be ascertained far more accurately, and most of the brains prove
to be abnormal with heterotopias, deformities andarchitectural alterations of various kinds, for irradiation
at times at least up to 17 days p.c., although with
progressively decreasing incidence (72, 73). The papers
by Hicks et al. (121,122) provide a good morphologicaldescription of the cerebral lesions in rats following an
exposure of 200 R during the foetal st~ges.
215. These data, together with those of Satow and
Miyahara (308), Kameyama and Hoshino {143), Hoshino
et al. (126), Hayashi et al. (I 16), Mullenix and Norton{202), are of great interest for establishing thepathogenesis of the malformations or their relation to
behavioural disturbances, but are of limited value for the
purpose of establishing dose-effect relationships. Thereis, however, unanimity on the significant fact that thedeveloping structures of the central nervous systemremain highly sensitive to teratogenic damage until latein gestational life, when gross malformations of otherorgans may no longer be induc~d.
216. There are very few data for the induction of othertypes of malformations by irradiation of other mammals
during the foetal stage. In conclusion, it appears thatsome gross malformations can be produced byirradiation during the foetal stages. particularly in theearliest phases and with doses of a few hundred rad.
With increasing age, however, the foetus becomes
progressively more resistant to their induction. Microscopic analysis of the anatomical structures, particularly
the brain, shows the presence of lesions or architecturaldefects which would otherwise be undetectable by692

218. A special case of malformation reported in thehuman after irradiation during the foetal period issegmental heterochromia of the iris. Lejeune et al. (169)preliminarily described their findings in a group of about2800 children, including controls and children of
mothers who during pregnancy had been irradiated for
diagnostic reasons in the abdomen or the pelvis.
Considering the whole population observed, 8 out of
567 irradiated children and 2 out of 2276 controls hadheterochromia of the iris. When the comparison was
limited to two smaller groups, 3 out of 421 irradiatedand 1 out of 448 control children were foWld with thiscondition. It appeared that the critical period of theinduction of heterochromia might be the 4th and 5thmonths of gestation.
219. Cheesman and Walby (63) retrospectively studied
7813 children. 4-7 years old, who entered school in
Belfast in 1961. Of these, 67 (0.86 per cent) had
heterochromia of some sectors of the iris. No significant
difference in incidence was found between children who
were reported by their mothers to have been irradiatedfor diagnostic purposes in utero (1.17 per cent) and thenon-irradiated controls (0.81 per cent). Among
irradiated boys the prevalence of heterochromia wasslightly, though not significantly, higher than among thenon-irradiated boys. That situation was not observed ingirls. Eight affected boys had been irradiated in utero.
and in 7 of these the irradiation had been performed
during the 7th month of gestation.
220. Jacobsen and Mellemgaard (138) studied theoffspring of 201 female patients irradiated in the
gonadal region in 1924-1930 for radiological urinary
examinations or for assessment of the position of the
foetus. The offspring of these patients were subdivided
into 250 born prior to irradiation of their mothers, 46
exposed in utero and 169 conceived after irradiation. No
significant differences were found in the number ofabortions suffered by the irradiated mothers, and in themorbidity. sex ratio and incidence of gross congenital
malformations of the children. However, in 184 casesthat were examined directly for ocular defects 4 out of
42 individuals irradiated in utero and 7 out of 142
children conceived after irradiation were found to carrysevere ocular anomalies such as coloboma, atrophy of
the iris, choroidal coloboma, strabismus etc. Although............ no statistical sie:nificance in the incidence of suchconditions could be demonstrated between the twogroups and the doses delivered to the foetus were notknown, 11 cases of eye malformations in a small sample
of 184 subjects were thought to be an unusually highnumber, and the authors could not rule out thepossibility that irradiation in utero (or before conception) might induce other and more serious
malformations of the eye, in addition to heterochromia.

D. OTHER EFFECTS
1. Animal data
221. A number of papers deal with the damage of
haemopoietic tissues during the foetal stages. Many ofthe earlier reports on foetal irradiation of various
animals ( 11, 139, 295) refer to disturbances of
haemopoiesis shortly after irradiation. Among the more
recent contributions, Reineke (252) showed that rat
embryos receiving 270 rad on the 18th day of pregnancy
had a lower peripheral blood count which was at its
minimum 8-12 days after the irradiation. This wasfollowed by an over-recovery with a peak at 50 days, thehaemopoietic regenerative capacity of the surviving
animals being similar to that of normal adult animals.
Rats irradiated 15 days p.c. with 120-240 rad showed asignificant decrease of the white blood cell count (WBC)
1 day after birth. The haematocrit and the red blood
cell count (RBC) were, on the other hand, unaffected(117).
222. In the case of mice exposed 15 days p.c. to 50 and
100 R, the WBC at day 17 p.c. was higher than controls,
while the RBC was lower. This response is in contrast to
the adult reaction. The platelet level was also depressed,
but all the haematologic values returned to normal by
the third week of age (285).
223. These data regarding the peripheral blood picture,
as well as the data on the intrinsic radiosensitivity of the
foetal haemopoietic precursors (323), show that thereaction of the foetal haemopoietic cells is _compatible
with the available information on sensitivity and cell
kinetics of the blood-forming system of the rodent.
These data are relevant to the post-natal survival ofirradiated animals, discussed in paragraphs 181-195, andconfirm the transient character of the haemopoietic
changes induced by sublethal doses of radiation. The
data of Christensen and Jackson (64) would imply. on
the other hand, that the fraction of irreparable damage
to the haemopoietic system of the foetal animal is higher
than in the adult.
224. Kusarna and Yoshizawa (165) studied the effects3
of 1 7 Cs gamma radiation ( 5-100 R) on the mitotic
index of the ddY mouse foetal liver cells (14-16 daysp.c.). Following an analysis of the dependence on timepost-irradiation of the cellular changes at the various
doses tested, they concluded that the threshold exposurein regard to the decrease of the mitotic index and to thefrequency of prophase cells was between 5 and l O R.
Similar - conclusions were reached by Hoshino et al.
(126a) in an analysis of the mitotic delay as a functionof radiation exposure in the matrix cells of the mousetelencephalon at 13 days p.c.

225. Studying the effects on male fertility, Rugh andJackson (283) observed a 45-per-cent reduction of litter
production in mice exposed to 200 R at 15.5 and16.5 days p.c. Rugh and Wohlfromm (288) reported on
the effects of lower exposures (25 or 100 R) at
0.5-18.8 days p.c. The male offspring were tested fortheir reproductive potential when their ages were
2-8 months. In contrast with irradiation during theembryonic stages, where some reduction of the
reproductive potential was observed, the doses usedduring the foetal stages were insufficient to produce any
reduction of fertility.
226. In females, Lang~ndorff and Neuman (166)showed that exposure for 5 consecutive days to 20-80 R
per day during blastogenesis, the frrst and second half oforganogenesis. or the foetal period reduced thefrequency, size and reproductive capacity of the litters.
The greatest reduction appeared in animals irradiatedbetween 1 I and 15 days p.c.; with exposure rates higher
than 60 R per day the offspring had no further
descendants. The number of litters produced depended
on the dose and the phase of gestation at irradiation, thedifferences of the reproductive capacity depending morecritically on the frequency than on the size of the litters.
227. Ershoff (84a) described the effects of irradiationon the development and morphology of the reproductive
system of male and female rats. Male animals exposed to
gamma rays (300 R) at the rate of 17.92 R/min on day
18 p.c. or exposed to the same dose at the rate of
0.03 R/min over the period 13-20 days p.c. had asignificant degree of gonadal damage. Females, incontrast had normal-size ovaries following the acuteexposure but significantly smaller ovaries and an absenceof follicles and corpora lutea following the long-term
exposure.
228. Testicular atrophy in adult rats exposed to as little
as 50 R on the 18th day of their foetal life was also
reported by Brent (29) and cited as an unusual
phenomen of an organ showing two widely separate
peaks of sensitivjty at 9 and 18 days of pre-natal
development. Very recent data of Erickson and Martin(84) have revealed important discrepancies between the
rat and the pig in regard to effects on the germ cells
following continuous exposure throughout gestation.
229. Further information about the effects on theovaries of the foetal mouse and rat as a function ofgestational age (8-15 days p,c.) and radiation exposure(50-100 R) can be found in Mintz {197), Beaumont (15,16). and Be.aumont and Mandi (17). Other data on thefertility and reproductive capacity of rats exposed inutero have also been reported by Murphree and Pace(212) and Baev et al. ( 10).
230. Erickson and Martin (84) reported experiments on
the pig irradiated continuously for 108 days (thegestation period in this animal lasts 112 days) at dose
rates of 1.5, 3, 9 and 20 rad per 22-hour day with a
6°Co gamma source. Foetal dose rates were 0.5, 1, 3 and
7 rad per day. At all dose levels the health of themothers, the number of live births and their post-natal
viability appeared to be normal. At birth and again at 70
and 150 days of age the irradiated offspring were tested
for body weight and organ development. Weight and
693

...

_j
'

growth of the body were unaffected by dose rates of
3 rad per day or less. At this dose rate only the weight ofthe brain was decreased, and at I rad per day or less only
the weight of the gonads was affected. Sterility wasobserved both in male and female animals down to 3 rad
per day, and an appreciable reduction of the germ cellnumber was observed in both sexes even at 0.5 rad perday. Parallel experiments conducted on the rat undersimilar conditions of chronic irradiation (21 days of a22-day gestation period) revealed an important interspecies difference in that the dose of I rad per day did
not produce apparent effects on the germ cell
population in either sex.
231. Cataract formation in the mouse irradiated during
the foetal stages has been described by Rugh, Duhamel
et al. (270) and Rugh, Wohlfromm et al. (292).
232. Two monkeys (Macaca mu/atta), irradiated duringfoetal development (200 R on day 80 p.c. and 300 Ron
day 60 p.c.), were followed for 23 months of their lives.
The studies covered the changes in the electroencephalographic, electrocardiographic and electroretinographic recordings. The brain appeared functionally
impaired, concomitantly with signs of microcephaly.
Depression of light reactions documented a reduced
retinal function. while electrocardiographic changes wererelatively minor (274).
233. Slightly but distinctly higher levels of urea
nitrogen were detected in the blood of beagle dogs
irradiated with 20 or 100 R in utero 55 days p.c. (341).
In the interval 270435 R of 6 °Co radiation (delivered at
the same time or soon after birth) high incidences ofrenal failure were reported, particularly in male animals(246).
2. Human data
234. Data have also been reported concerning thehaematology of humans exposed in utero at Hiroshima(340). The sample size varied during the period of
observation, 1951-1958, totalling up to 1020 exposedchildren and an adequate number of controls. The
sample was divided by year of examination, gestation
time at irradiation, sex, and distance from the
hypocentre. Little indication was given of the actual
doses received. apart from the remark that the exposure
beyong 2 km was probably less than 20 R. No changes
peculiar to the irradiated group were observed. A
homogeneous subsample of 63 children, exposed during
the first semester of gestation, was analysed. No
correlation was found between leukocyte, erythrocyte
and haemoglobin values and dose, since the changes
associated with distance we're comparable with thevariation within each distance group. The progressively
decreasing count of total leukocytes with age, which
qualitatively is a common finding in man, appeared to befaster than normal, but this fact could not be attributedwith certainty to any specific cause.
235. Haematological findings in adolescents exposed inutero at Nagasaki were also negative (47), in spite of agood assessment of the doses to the mother. It may
therefore be concluded that, whatever haematologicchanges might have taken place soon after exposure,694

there was complete haematological recovery by the ageof 5 years. Furthermore, the profound changes of
endocrine activity during adolescence did not bring tolight any haematological deficiency in the exposed
individuals.
236. It has been reported that parasitic infestation
occurred at a significantly greater rate in children whose
mothers were within 2 km of the hypocentre, a finding
which, in the view of the authors, is probably to be
associated more with dietary, living and socio-economic
habits than with radiation ( 4 7).
'237. Determination of butanol-extractable iodine inserum performed on 249 15-year-old children. some
exposed in utero at Nagasaki and some unexposed,
revealed no statistically significant differences between
them with respect to dose and trimester of gestation at
irradiation ( 49).
238. The urine analyses of children {9-16 years old)
exposed in utero at Hiroshima or Nagasaki werecompared with a suitable control group (90). An
increased proteinuria was detected at various ages in
Hiroshima boys and in all girls whose mothers were
within 1.5 km of the hypocentre. Although in some of
the groups the differences reached significant levels, thebiological significance of these findings remain to be
explained.
239. Two conflicting reports are available concerning
the sex ratio of babies born from mothers exposed inutero. These reports should be examined with the
purpose of analyzing the response of germ cells as afunction of their stage of development at the time of
exposure. It is known that in the human female theoogonial divisions stop about 5 months p.c., when allcells enter meiotic prophase. Meyer et al. (190, 191)
reported on the sex ratio in children from a group of
black women born in Baltimore in 1947-1949, about
one third of whom had been exposed to x rays in utero
in the course of diagnostic pelvimetry. No precise
estimates of the radiation doses were given, but they
may be assumed to be 1-5 rad in mc:ist instances (133).
Each exposed person was matched with two controls byhospital of birth, parity and birth data. By 1966, 1993
members of this population had given birth to children
and had experienced a number of foetal and neo-natal
deaths and abortions. The rates of these events wereindistinguishable in the exposed and control groups. A
number of variables related to pregnancy, delivery andclinical history of the babies were also identical, showingthat matching had really resulted in comparable groupsof exposed and unexposed mothers. Under theconditions of exposure examined. it clearly appears thatthe reproductive capacity of the individuals had notbeen affected, at least up to 17-19 years of age.
However, when the exposed group was subdivided as afunction of gestation time at irradiation. it was possible
to show that mothers exposed early (0-29 weeks) intheir own foetal life had an unusually high andstatistically significant excess of male babies. Thissuggests. in the view of the authors, an excess of
pre-natal loss of female conceptuses, which might occur
as a result of non-disjunction in the irradiated oocytes.
because XXY males would be I 0-30 times more viable
than XO females.

240. The conclusions reached by Meyer et al. (190,
191 j are indeed difficult to reconcile with the data of
Jablon and Kato (133) based on the experience ofJapanese A-bomb survivors. No effect on the sex ratio of
the offspring was found, irrespective of the dose groupor the trimester of gestation at irradiation. The data
were completely negative, despite the larger radiation
doses and sample sizes involved in the Japanese series.
The question of possible alterations in the sex ratio ofchildren born to parents which had been exposed inutero must therefore remain open for the time being.
241. Another epidemiological study was publishedrecently (192) concerning 1458 black women exposed inutero to diagnostic doses (1-5 rad) of x rays and thesame number of carefully matched controls. The
reproductive patterns of the two groups were investigated as a test for possible injuries to the ovaries andoocytes irradiated during foetal life. To this end, thenumber of live births and foetal deaths in the offspringof the two groups were ascertained and compared. A
10-15 per cent increase in the fertility of exposed
women was found. It was statistically significant.
(P =0.011) and remained so even after adjustment
between exposed and controls for socio-economic andmedical factors. There was some evidence that thishigher fertility might be decreasing with time and that aparallel rise in the rates of spontaneous abortions andfoetal deaths might occur in the exposed group. If thistrend is confirmed, it is possible that the total
reproductive capacity of the exposed individuals may
eventually be less than that of the control sample. A
prevalence of other minor health problems was also
found in the follow-up of the exposed women.
242. In a publication by Blot et al. (25), various
reproductive indices were determined up to 1973 for acohort of 2457 persons exposed in utero to the A-bombs
in 1945 and for another 63 persons in a different sample
who had a small head-circumference. It was found thatthe marriage rate was significantly lower in persons
heavily exposed in utero (more than 100 rad maternaldose) than in unexposed or more lightly exposed
individuals. This difference was attributed in part to thelesser marriage ability of the mentally retarded, whowere significantly more numerous among the heavily
exposed, and in part to other unknown variables,
including possible social discrimination against survivorsof the bombing. However, among those married, thefrequency of childless marriages, the number of births
and the interval between marriage and first birth showed
no consistent relation with exposure.

V. INTERNAL IRRADIATION
A. GENERAL
243. The first reported observations on the effect ofinjected radioisotopes in the foetal mammal are those of
Bagg (11). He injected radon solutions in amounts oftens of rnillicuries into pregnant rats at different
gestation times and described post-implantation deathand various types of malformations in the offspring.
More recently. some data on teratogenic effects ofinternal irradiation have been published, but information
on any one nuclide is still very scanty. It does not seem
possible to draw general conclusions, and the main valueof the existing data is the indication of possible levels of
toxicity for the various radioisotopes. The available data
will be reviewed according to the nuclide tested. but
only for those nuclides for which there seems to besignificant information. It was felt that the present stateof knowledge in this field would not justify any attempt
to express the data in terms of the radiation dose
actually received by the conceptus or its organs andtissues. Exposure or dose data are therefore given
according to the way they were report~d in the original
publication.
244. Two .documents have been submitted to. theCommittee summarizing the effects on foetus andprogeny of mothers exposed to radionuclides before·.conception ( 177) and reviewing the various effects inanimals treated with radioactive substances in the courseof intra-uterine development ( 178); both of them dealt
with work carried out in the USSR. An indexedbibliography dealing specifically with the transfer
through the placenta and into the foetus of radioactive
substances injected to mothers is also of interest forreference to published data on this subject {335). The
emphasis of the presentation to follow, in accordancewith the objects specified in the introduction, will
however be centred on the teratological effects
themselves induced by different doses of the administered nuclides, rather than on the mechanisms and rateof transfer of such nuclides from the mother to the
foetus. For these the reader is referred to the specialized
literature included in the previously mentioned publications.

B. TRITIUM
245. Concerning the passage of tritium administeredunder the form of tritiated water from the motherthrough the placenta and into the foetus, Moskalev et al.
(201) reported that placental accumulation depended
only very slightly on the gestational age at the time oftreatment: it was found to be 0.38 per cent at 15 days
and 0.45 per cent at 19-21 days of pregnancy. In the
foetus, 0.38-0.40 per cent of tritium was found on day
13-15 p.c. and 2.0 per cent on day 19-21. Istomina et al.
(132) studied the transfer of tritium from the mother tothe offspring by milk. Lyaginskaya (176) investigated
the relationships between the pregnancy stage at thetime of treatment with HTO in the rat and the ensuing
effects in post-natal development. For a dose of
0.3 µCi/g, little relationship was found among thesevariables, although treatment at implantation did cause asomewhat increased pre-natal death of the embryos andadministration during the foetal stages resulted in greater
post-natal death. However, at this dose no teratologicaleffects were manifest. LD 5 0 values for foetal mortality
in rats after single HTO treatments at various doses were0.1 µCi/g (corresponding to an estimated dose to the
foetus of 100 rad) at 4 days p.c.; 10 µCi/g (400 rad) at
9 days p.c.; and 100 µCi/g {1000 rad) at 17 days p.c.
(178).
246. Cahill and Yuile (53. 54) evaluated the resulting
foetal tissue doses in rats, the body water of which was695maintained throughout pregnancy at a constant tritium
level in the range 1-100 µCi/ml. This was achieved byadjusting the ingestion of tritiated water. Dose rates inembryos and foetuses were calculated to range from 0.3
to 30 rad per day. Most mothers were sacrificed before
birth for observation of the conceptuses, but in somecases the observations were carried out on born offspring
and followed to their adulthood. Several statisticallysignificant effects were found at various HTO levels, in
no apparent relationship with dose. These included
microcephaly, sterility, stunting, reduction of the Jitter
size and increase in the resorption frequency. Stunting
appeared at 20 µCi/ml and its degree increased at higher
concentrations in direct relation to dose. Organ weightdecreased in proportion to dose. The incorporation oftritium into foetal organs was directly proportional tothe maternal HTO activity during gestation andamounted to 20-30 per cent of this activity. After
180 days of life, stunting only persisted in males withconcentrations in the range 50-100 µCi/ml.
247. A paper by Dobson (75) dealt specifically with theRBE of tritium relative to 6 ° Co gamma radiation in
long-term exposures. To this end, tritiated water invarious doses was administered in drinking water topregnant mice throughout gestation and lactation for a
total of 33 days, the specific activity of the body water
being checked by the radio-assay of urine samples. Other
groups of mice were exposed to 6 °Co gamma radiation
at various dose rates for the same time lapse. The
number of oocytes in the progeny of these animals wascounted at the end of treatment, and it was found thatoocyte survival decreased exponentially with tritium
concentration in body water with an LD 5 0 level ofabout 2 µCi/ml body water, corresponding to aneffective dose to the foetus and the new-born offspringof about 6.5 rad. The survival curve of oocytes withrespect to 6 °Co gamma rays was convex upward,
indicating a decreased killing effectiveness of the
gamma-ray treatment at low doses. By comparing
individual gamma-ray and tritium experiments it was
shown that tritium was more effective, and limiting RBE
values were found to vary between 2.5 and 4.2 with alikely maximum value of about 3 at doses approachingzero. Short-term and protracted exposures were also
compared by the same end-point, and higher RBEs for
protracted irradiations were obtained.
248. Recent attempts were made by Cahill et al. (55) toassess by a number of different end-points (morphological, biochemical and functional) the effects on ratsof a simultaneous long-term administration of lead andtritiated water. The experiments allowed the important
general conclusions that combination of the treatments
resulted in less-than-additive effects and that asignificant reduction of brain v.Jeight by tritium exposure
was apparent at dose rates of the order of 300 mrad per
day continuously from conception to 180 days of age.
Haemopoietic disturbances (anisocytosis, leukopenia,
thrombopenia) in the progeny of rats given tritiatedwater at doses of 0.008-0.3 µCi/g were described by
Zhukova (reported in (178)). Animals from mothers
treated at 0.3 µCi/g on day 4 p.c. were the most severely
affected. Finally, Lyaginskaya ( 178) reported a decreasein the life-span of three subsequent generations of rats
irradiated in utero with tritiated water on day 4, 11 and17 p.c., particularly upon treatment on day 4 p.c.
696249. Concerning administration of tritium in the formof tritiated thymidine, it has been possible to study anumber of effects on both the mother and the foetus
induced by a continuous perfusion of tritiatedthymidine (3HTdR) to pregnant rats from day 9-22 p.c.
(88. 101). Activities of 1.6 µCi/g per day were in general
well tolerated; but higher levels, from 3.2 to 6.4 µCi/g
per day, produced a marked bone-marrow syndrome in
the mother. Although the litter size at birth did not
appreciably change at these levels, the percentage ofstill-born offspring increased with the activity injected,
while the number of offspring surviving more than
12 hours after birth decreased in proportion to that
activity. With 8 µCi/g per day, no rat was born alive.
Retardation of growth and macroscopic and microscopic
malformations of the head, brain, eyes, ears, mouth andextremities were observed at these high levels. With
6.4 µCi/g per day, no gross external abnormalities wereseen, but the general development and the haemopoietic
system of the animals were severely impaired inproportion to the administered activity. The post-natal
growth and weight of surviving animals also showed
activity-related pathological changes.
250. In another paper (311), the incorporation oftritium from 3 HTdR in developing rats was studied bybiochemical techniques. The total incorporated activity
and the DNA specific activity showed a direct relation to
the activity of the tritium injected in the mother. The
distribution of radioactivity between the DNA and thelow-molecular-weight fraction was independent of theadministered activity, and the time variation of the DNA
specific activities was characteristic for each organ.
Because of all these facts, the system of continuous
perfusion was recognized as a suitable procedure forstudying 3 HTdR toxicity in the embryo.
251. Mouse embryos were also grown in vitro from the2-cell stage to the blastocyst stage in the presence of3
HTdR (329). Concentrations of tritium above
0.1 µCi/ml were definitely lethal, and concentrations
between 0.01 and 0.1 µCi/ml caused a highly significant
reduction of the number of cells in the blastocyst. The
latter effect could be largely accounted for by selective
cell death occurring at the 16-cell stage. As the cells in
the inner mass were most susceptible to killing, it waspossible to obtain blastocysts composed entirely oftrophoblast.

C. PHOSPHORUS-32
252. Burstone (50) described the effects of 3 2 P on thedevelopment of embryonic tooth primordia in mice,
showing that injections of 5-17 µCi/g in pregnant
animals 2-6 days before birth gave rise to developmental
disturbances of teeth. observable within about one
month after birth. He also reported that activities above
10 µCi/g produced general post-natal growth retardationand that this effect was more evident when 32 P wasgiven early in pregnancy.
253. Sikov and co-workers carried out a series ofsystematic quantitative studies on the consequences of32
P irradiation throughout gestation. Sikov and Noonan(320) gave several IV injections of the nuclide to female

rats on day 6, 8, 9 and 10 p.c. They then established thevalues of the LD 5 0 , for lethality of the foetuses before
day 14 p.c. These LD 5 0 values'increased with the age ofthe embryos at treatment. from 0.46 mCi on day 6 p.c.
to 1.29 mCi on day 10 p.c. The uptake of the isotope by
the foetus was assessed, and from this the doses to theembryos were calculated for each day of gestation. At
the LD 5 0 levels mentioned above, the doses varied fromabout 6000 rad on day 6 p.c. to about 10 200 rad on
day 10. The uptake appeared to be proportional to theactivity injected. indicating that there was no majorinterference with the phosphorus metabolism. A
reduction of embryonic weight was found at the time ofobservation. the reduction being greater when embryos
were irradiated earlier.
254. Growth retardation (321) and skeletal defects(316, 317) were also studied in rats, following theadministration of 0.6-2.0 mCi of 3 2 P on days 14 and17 p.c. The study of specific defects and themeasurements of various bones were carried out using
serial sacrifice from injection to birth. A dose-related
decrease of the foetal size and of the bone sizes wasobserved and this decrease was found to be greater inanimals tested at the younger developmental age. The
same pattern applied to skeletal malformations. whichwere also more frequent and severe in the group injectedon day 14 p.c. Differences with respect to previous
experience with x irradiation were noted (357). With theaid of a dosimetric study (314), such differences couldin part be attributed to the pattern of accumulation of
the radiation doses at the two ages tested. Other possible
explanations related to the effects of the dose rate or of
the LET of the radiations compared (315).
D. STRONTIUM-89 AND STRONTIUM-90
255. Most of the early papers on the effects of 89 Sr and90
Sr dealt with the transfer of the nuclides through theplacental barrier (244, 360, I 24. 221). Finkel (86)concluded that, in the mouse, negligible amounts of89
Sr were transferred through the placenta during thefirst 1S days of gestation. She described an increase in
the percentage of still births, some growth retardation,bone fragility and anaemia in mice treated in utero. In
the rat, the retention of 9 0 Sr in the placen_ta wasestimated to be 0.02 per cent of the amount injected
and was found to be rather independent of thegestational age at treatment. However, the passage of thenuclide to the foetus was such, that for early gestational
ages its content equalled that in the placenta(0.01-0.02 per cent) but at 16-19 days was about 20
times higher (201). Hopkins and Baxter (125) reported
that the fraction of the 90 Sr injected into pregnant
female rats at 17 days p.c. which was retained at birthby the offspring decreased with increasing maternal age
at injection, although the ratio of offspring body burden
to maternal content was independent of maternal ·age
under the specific conditions tested. There was nomarked difference in the percentage of the dose of 9 0 Sr
retained by new-born offspring in the dose range
24-382 µCi.
256. Single injections of 90 Sr to pregnant rats at
various dose levels were used to establish the values of
LD 5 0 in the offspring. They were found to be 0.4 µCi/g(corresponding to a calculated foetal dose of 3.5 rad) at
4 days p.c. and 0.8 µCi/g (10 rad) at 15-19 days p.c.
(data reported in (178a)). A dose of 10-15 µCi/kg of90
Sr injected IV in pregnant dogs at 1-9 days before
birth produced no obvious malformations (87).
Burykina et al. (51) concluded that in their experiments
the chronic introduction of 90 Sr (0.02 µCi/kg per day inthe cou~se of 3-3.5 years) produced skeletal doses ofabout SO rad/year in the parents. Under these
conditions. disturbances in the development of theprogeny were observed, together with a decrease of their
viability. A reduction in the number of oocytes was
described by Nilsson and Henricson (224) in mice after
IV injection of the pi;egnant females with 20 µCi of90
Sr. The effect was more pronounced with injections at
day 16 than at day II p.c. Finally. Kincade et al. (147)
described changes in the haemopoietic and immunopoietic cell populations of the foetal liver, marrow andspleen after 90 Sr treatment.

E. IODINE-131
257. After single parenteral injections of 131 I (4 µCi/ g)
at 1-16 days p.c. in the rat, pre-implantation embryos
were found more susceptible than those at later stages:
for treatment at day I p.c. a three-fold increase in foetal
intra-uterine mortality compared with controls was
obtained (164). Oral administration of 1.0 µCi of thesame nuclide on day 13 produced an accumulation of
0.035 per cent in the placenta and 0.01 per cent of theadministered activity in the foetus. By day 20 or 21, theamount in the placenta was increased only slightly while
the foetal retention was 28-40 times that at day 13. This
increase was related to the increase in the mass of the
foetal thyroid and to an improvement of its ability toco~centrate the isotope (179).
258. Chronic treatment at the level of 65 µCi per rat
daily throughout pregnancy led also to a significant
increase of the death rate (about 41 per cent), of which
10 per cent could be accounted for by pre-implantation,and 31 per cent by post-implantation, mortality. Delayin the development of the progeny, microcephaly andliver damage (chromosomal aberrations) were also
apparent ( 164). Iljin et al. ( data in 178a) also reported
on repeated treatment of rats with 1 31 I at I-7, 8-14 and14-21 days p.c. for a total of about 30 µCi per rat given
at progressively lower doses. According to the various
treatment schedules, an increase in mortality at birth ofabout 15-25 per cent was seen. A delay in thyroid
development was also apparent. Finally, histologicalchanges in adult mouse thyroids following treatment of
the mothers with 131 I at 18 days p.c. were described byWalinder and Sjoden (352) but no reference was made to
teratogenic effects induced by the treatment.

F. OTHER NUCLIDES
259. Female rats were exposed to monomeric or
polymeric B 9 pu on day 15 or 19 p.c., and the
distribution of the nuclide in various portions of the
foetoplacental unit was studied by counting and byautoradiography (319). At the above gestational ages,
50 µCi produced no pre-natal mortality. On the other697

hand. a substantial difference m the mortality at 14 days
following an injection on day 9 p.c. was observed:
2-12 µCi induced about 60 per cent mortality and 25 or
SO µCi produced death of all embryos. None of thesurviving foetuses showed gross morphological malformations. In view of the high activities administered, thevalue of these experiments is purely theoretical andwithout applicability to risk estimation.
260. In order to establish the LD 50130 of 241 Am forthe rat foetus at various stages of development, thepregnant females received IV injections of the nuclide at
10, 13, 16 and 19 days p.c. The concentrations of thesolutions were increased stepwise from 1.2 to 7.6 percent to cover the required range of doses, while thevolume injected was kept approximately constant. Since
the placenta behaved like an inconstant barrier (the ratioof the maternal to foetal concentrations of 241 Am
varied from 6:1 to.2:1 (201)), the LD 50 values wereexpressed as the doses to the mother that produced
SO-per-cent mortality of the foetuses at different ages,
followed by the calculated cumulative rad doses to the
foetuses: 10 days p.c., 0.03 µCi/g (100 rad): 14 daysp.c., 0.01 µCi/g (800 rad): 19 days p.c., 40.2 µCi/g(1000 rad) (375).
G. CONCLUSIONS
261. It seems impossible at present to draw any precise,
general conclusions about the action of radionuclides inthe development of the conceptus because of thepaucity of data on dose-effect relationships for the
nuclides tested. None of the effects observed up to now
appears to be specific to internal irradiation, and theseeffects may be viewed as the end-points of a continuing
action exerted during the various stages of embryonal
and foetal life. Their precise interpretation andevaluation must be based on a precise knowledge of thedose accumulated by the mother and the foetus at any
particular stage and in any particular sensitive structure
under all possible conditions of administration. The
information we have at present is far from being goodenough for that.
262. In spite of some attempts to compound the known
data on the placental transfer of the various nuclides
into a general model of transport and accumulation (242,332, 333), the information is still too fragmentary, notonly with respect to the dosimetry, but particularly inregard to the effects. Careful consideration of the data
reviewed allows only a schematic enumeration of themain variables that have been shown to interact in the
production of the end-effects. They are (a) the nature of
the radionuclide in respect to its physical characteristics
and to the different organottopic behaviour; (b) the
chemical form in which the nuclide is administered, in so
far as it may influence its pattern of accumulation anddeposition; (c) the route of administration and the
dosage schedule, particularly because the rate of passage
and accumulation from the mother into the conceptus
may be critical in relation to the rate at which the
ontogenic events take place in a rapidly-growing anddifferentiating organism; (d) the developmental age at
irradiation, since periods of maximum sensitivity are to
be expected in the case of internal as well as for external
irradiation, even though they may be difficult to resolve698

owing to the continuous action of the deposited
nuclides; (e) the animal species. not only in regard to thepeculiarities of the differentiation pattern. but also inrespect to all physiological differences in the absorption
and deposition that could alter the incidence of thebiological end-points: ([) the age of the pregnant animals
at treatment, for which information is virtually absent:(g) the possible role of the maternal irradiation. Further
extensive experimentation in all these fields, at least forthe most hazardous nuclides, is clearly called for.

VI. MECHANISMS OF RADIATIONTERATOGENESIS
263. The present chapter has two purposes:(a) It tries to identify some common features
and trends among the data and effects described. This
information may explain the origins of malformations
and the likely pathways involved in their manifestation;(b) It discusses the scanty information available
on the role of some radiobiological variables (LET, dose
fractionation etc.) and of some of the modifiers of theteratogenic response (oxygen effect. chemical protectionetc.).

264. Since the mechanisms of the lethal action in utero
have already been discussed (paras. 66-70, 94-97,
186-191, 221-224), the discussion will be limited
essentially to the induction of growth disturbances andmalformations. A few remarks about the applicability to
the human case of data obtained experimentally inanimals are also included.
A. INFLUENCE OF THE MA TERN AL
ORGANISM
265. One issue concerning mechanisms which has
attracted much attention, particularly in the older
literature. is the influence that the irradiated maternalorganism might have on the appearance of foetal
abnormalities. The reasons for such a continued interest
can hardly be explained, since it stems mainly fromexperiments of doubtful significance and it is known
that malformations can also be induced by irradiation in
oviparous species. The close relation of the conceptus tothe mother in mammals is not by itself a sufficientreason to postulate any abscopal mechanisms. In a
critical evaluation of the data, Russell (295) observed in
1954 that two facts stand against such a hypothesis: (a)the existence of sharply defmed periods of sensitivity inthe embryo, which cannot be easily reconciled with therelatively Jong-lasting effects of radiation in the mother,and (b) the extremely short time between irradiationand the appearance of embryonic lesions, which would
require the improbable existence of very rapidmechanisms.
266. Among the more recent contributions, Chang andHunt (60) have reported experiments in which
pre-implantation rabbit embryos irradiated in utero weretransplanted into non-irradiated recipients or into
animals exposed to 400 R at different times after aninduction of ovulation by hormonal treatment. Under
these conditions, embryonic mortality was increased by
the irradiation of both the ovum and the mother, but
the relative contribution of the two factors was found todepend on time elapsed since ovulation. Brent and
Bolden (32) raised an objection to these experiments on
account of the comparatively high doses used, whichmight actually have resulted in an increased foetal
mortality (though not necessarily in an increased yieldof malformations). They also criticized the lack ofsynchronization between development of the embryoand of the decidua, which is critical for proper embryo
implantation. Experiments similar in many aspects to
those of Chang and Hunt (60) were reported at the sametime by Issatchenko ( 131) who transplanted 2-day-old
rabbit embryos from irradiated animals (250 R) to
normal females and vice versa. Under these conditions anincreased mortality of the embryos was observed abovethat obtained by irradiation of the embryo alone, thus
confirming the presence of an indirect effect of theirradiated maternal organism.
267. Duplan and Monnot (79) irradiated pregnant mice(200 and 400 R of x rays) with shielding of the foetuses
and observed a dose-related increase of pre- andpost-natal mortality in the offspring. Also, protection of
the mother's abdomen resulted in a higher percentage
survival of the non-irradiated offspring when comparedwith the shielding of the head and neck. Valentini and
Hahn (349) studied the survival of rat embryos in theright uterine horn irradiated with 200-400 R at day 1.5
or 4.5 p.c. and found a significant increase when the left
lower body quadrant was concomitantly shielded. These
results would indicate that irradiation of the mothermight be a contributing factor in pre-implantationembryonic mortality.
268. A series of papers by Brent and co-workers havecontributed greatly to clarifying this issue. These authors
were able to show that exposures of 200 R in rat
embryos at 9 days p.c. were definitely lethal (38). Whenthe embryo was shielded while the mother was exposed
to x rays at 400 R of whole-body irradiation or to over1000 R of partial-body irradiation, no embryonicmalformations were seen in embryos at term. Foetal
mortality, on the contrary, was higher than in control
animals, presumably as a result of radiation sickness inthe mother. When the placenta was exposed to 400 R(28), while both the mother and the conceptus wereshielded, there were no lethal effects, growth disturbances or induction of malformations.
269. In another paper dealing with pre-implantationembryos (32), Brent and Bolden used shielding
techniques. to differentiate the results of ovary, oviduct,
uterus and zygote irradiation (150 R). Treatment on day
1 p.c. showed that uterine and ovarian irradiation had noeffect on foetai mortality. which instead was caused byirradiation of the oviduct containing the fecundated ova.
Malformation incidence was very low, in accordancewith the observations reported in paragraphs 75-79.
Although this experiment does not rule out possible
effects of oviduct irradiation, it strongly suggests thatthe major and primary action is caused by direct
irradiation of the ova. Brent and Bolden (34) showedalso that embryonic rats in the first day of development

irradiated with doses of 150 R were resorbed in 60-70
per cent of the cases when irradiated directly, while
irradiation of the mother and shielding of the embryos
did not appreciably increase the resorption level above
control. The same experiment with a dose of 400 R
resulted in a 25-per-cent resorption of the unirradiatedova, showing a moderate indirect effect at the higher
doses.
270. It may be concluded that irradiation of the motheris likely to be of very minor importance for the
induction of malformations, particularly at low doses.
On the other hand, it is conceivable that mortality of theembryo and foetus may be indirectly affected by theirradiation of the mother'. especially at doses of a few
hundred rads.

B. NATURE OF THE PRIMARY DAMAGE
271. In the mid 1950s, Russell and her collaborators(295, 302, 303) presented a complete analysis of thenature of the primary damage which develops into
teratological effects. In the more recent literature, there
have been few systematic treatments of the subject,
although some special subjects have been dealt withmore frequently (318, 255).
272. The materials reviewed in the preceding sections of
this report show that the developing embryo iscomposed of a variety of dividing, differentiating,interacting and migrating populations of cells, arranged
in complex and ever-changing patterns. Unlike the adult,
the embryo is characterized by the absence of cell
populations in steady state, and it is this circumstancethat may be related to the variable radiation response as
a function of dose and time. Molecular embryology is
beginning to elucidate some of the intracellularmechanisms of gene regulation and some of the possible
intercellular interactions taking place in orderly
sequence in the various embryonic cell lines. However,
the occurrence of certain ''macroscopical" events indeveloping embryos cannot yet be related with certainty
to specific molecular changes (19, 12, 26).
273. Nevertheless, two qualitative features of embryogenesis are important in relation to the effect ofradiation. The first is the sequential character ofdevelopment, the occurrence of certain events appearing
to trigger the initiation of other events taking place atlater times. The second is the constancy of the timing of
the events, which occur within rather narrow intervals oftime from conception. The first of these features
explains the peculiar character of the radiation-induced
malformations, which often appear to be the consequence of an arrest of development at some rudimentarystage; the second explains the specificity of irradiation atcertain well defined times in the production of given
types of malformations.
274. Conceptually, the mechanism of radiation-induced
malformation may be examined at different levels of
biological organization: (a) the subcellular level, where
the problems of concern are the distribution of primary
events within the cell and the resulting changes insubcellular structures: (b) the cell as a separate unit699

d

reacting to the lesions of its components; (c) the cell
population level, including the damage and repopulationof the compartments originating from common precursors and also the relations with other cell
populations; and finally (d) the developmental pathways
that lead from the injury of single cellular precursors to
the final malformative ohange.
275. The information available at all levels is extremely
limited. For some of the issues discussed in the following
paragraphs there is no experimental data at all, andtherefore the problems can only be formulated in very
general terms.
276. At the subcellular level, it seems quite likely thatthe primary events taking place in embryonic cells are
qualitatively equal to those occurring in other cell types.
The differences between embryonic tissues with respect
to water content, oxygen tension, concentration andproperties of radicals seem insufficient to justify
important differences of the primary effects (309).
Substantial knowledge has developed in recent years
about the effects of radiation on DNA and the action of
reparative enzymes in mammalian cells (5). This
information is regarded as of great radiobiologicalimportance because of the essential role of DNA in cell
division and differentiation. However, there are no direct
data in embryonic or foetal cells.
277. Direct quantitative data on embryonic or foetal
cells and tissues are at the present time few andscattered. Those available concern changes in thesynthesis of DNA (198, 183, 184,185,324,311, 199,
53, 54), RNA (198, 183, 184, 185) and proteins (198,183, 184, 185, 313, 85); the disturbances in the
ontogenesis of particular enzymes or enzyme systems(351, 216, 215, 214, 233, 163, 94); the ultrastructuralchanges of cytoplasmic organelles (233, 258, 259, 62);
the analysis of kinetic changes of specific cell lines ortissues (83, 186, 185, 84, 165, 40, 41, 42, 199, 126,
107, 234). The species differences and the nonsystematic character of the observations in regard toirradiation time, dose delivered, heterogeneity of
end-points etc. preclude the formation of any coherent
picture of the fundamental pathways through whichgross teratological effects become expressed.
278. It seems logical to postulate nevertheless that theobservation in irradiated mammalian cells in vitro and invivo apply as well to cells irradiated in utero (309). It isalso reasonable to assume, as a first approximation, thatdamage to the nuclear structures of the cells should be
preferentially considered. In this context, two possible
mechanisms of damage of the genetic structures havebeen proposed as the initiating. processes: gene mutationand chromosomal aberration.
279. The role of gene mutations (365, 363, 364) is
probably insignificant. in view of the very low frequencyof these events at doses shown to be teratogenic, andalso because of the low degree of expression of thesemutations in diploid cells (295). In analogy withobservations made on irradiated somatic cells of theadult (5), it seems quite reasonable to assume therefore
that the major component of cellular damage isattributable to chromosomal abberations (295).
700

Chromosomal aberrations are likely to impair cell
reproduction owing to the chromosomal imbalance ofthe daughter cells or possibly to mechanical disturbancesof the mitotic process.
280. It is generally agreed that cell inactivation can be
the common denominator of all types of malformations.
Microscopical evidence of cellular damage is, in many
instances, the first change observed in irradiatedembryonic cells of the nervous system (258. 120. 121,
161, 237, 238, 239, 308, 21,207.144), the eye (162,284), the skeleton (295, 210). the urogenital system(243, 121) and many other tissues.
281. This uniform pattern of cell reaction is consistent
with the great similarity of the malformative response
obtained in any given primordium or stage (295). It
should, however, be reconciled with the observed
selectivity of the damage, which appears to develop
preferentially in certain structures at certain times.
282. Mechanisms of differential sensitivity to radiation
may be invoked to explain selectivity. Furthermore, in
accordance with what is observed in adult tissues, it ispossible to postulate a different intrinsic cell sensitivity
to inactivation in different phases of the mitotic cycle,
and also a differential sensitivity of tissues having
different kinetic structures. Repair phenomena at the
intracellular level and repopulation of the tissuecompartments may also contribute to the specificsensitivity of the embryonic primordia during certain
phases of their development when cell division anddifferentiation are most active. The normal sequence of
these cellular periods is strictly dependent on some
unknown regulatory mechanisms. and indirect damage
to these mechanisms may also operate in the inductionof abnormalities.
283. The loss of reproductive integrity of a cell isreflected in the immediate descendants of the affected
precursor cell. It is also conceivable that damage of agiven structure may result in secondary effects on otherstructures or processes which are functionally dependent
on the originally damaged precursor and which may, inturn, be additionally affected by the radiation exposure.
It should not therefore be expected. that the large variety
of processes involved can be directly inferred from a
simple analysis of the malformative end-point (303).

C. DOSE-EFFECT RELATIONSHIPS
284. In the absence of · direct information about
fundamental mechanisms it appears nonetheless possible
to formulate simplified mathematical models and to test
their compatibility with observed data on dose-effectrelationships. Such an exercise has been carried out byRussell (303); the model is illustrated in the following
paragraphs.
285. If it is assumed that all reproductive cells in a givenembryological structure have, as a first approximation.
the same probability p of being inactivated by a given
radiation treatment, and if it is further assumed that p isa function of dose, the number of inactivated cells in thestructure will be distributed according to a binomial
distribution of the typeP(x) = (:) p-'' (1 - p'r-x

where P{x) is the probability of encountering x
inactivated cells, and n is the number of reproductive
cells in the structure. It may also be postulated that
there is a threshold xT of the number of inactivated cells
below which no teratological damage will be apparent.
This threshold could be different for each structure andpresumably related to its repair capacity. If the damage
inflicted on the structure exceeds the value of thethreshold, a malformation would be induced and its
severity would be in proportion to the number of cells
damaged above the threshold. The probability of
inducing a malformation would then bep=xtT(:) i'

(1 -

Pr- x

286. Under these hypotheses. Russell {303) has shown
that, irrespective of the value of n specifically assumed
for each structure, a dose increase will produce anincrease in both the number and the severity of themalformations. Furthermore, even with fLxed values ofp
and n. owing to the variability introduced by cell
division of the cells retaining their capacity for
proliferation and of their descendants, some variability
in the malformative end-point will be expected. Thisvariability will operaie even under conditions of good
homogeneity of the genetic background of the animals
and of the experimental conditions under which a given
dose-effect relationship might be determined. Other
causes of variability, discussed at length in previous
sections of this Annex, will obviously add considerably
to these inherent causes of variation.

287. It follows therefore that the derivation of theactual values of the parameters mentioned above
through the simple mathematical analysis of measured
dose-effect relationships would. give highly uncertain
results of purely speculative value. It would not bejustifiab.le to inte~pret such . data in terms of simple
underlymg mechamsms, even m the most simple case of
an apparently linear response.

D. THE PROBLEM OF THE THRESHOLD

288. The frequently encountered sigmoid nature ofdose-effect relationships for damage to the embryo andfoetus (see chapter III, section C), the action of repair
systems at the subcellular and tissue levels (paras.
271-283) and the theoretical notions to be gained bymodels of the type described in paragraphs 284-287
strongly suggest the existence of non-linear components
in the dose-effect relationships or possibly the existenceof true thresholds.
289. Two approaches have been followed in regard tosuch problems. One is the determination of the lowest
doses at which various abnormalities have actually been
observed (lowest teratogenic doses). The other consistsof experimental tests aimed at establishing the existenceof thresholds.
1. Lowest teratogenic doses

290. The literature contains many tabulations of the
mm1mum doses at which different types of malformations have been observed in various experimental
animals (27, 92, 370). Table 5 presents an updated

TABLE 5. SYNOPSIS OF THE LOWEST TERATOGENIC DOSES OBSERVED IN MICE AND RATS
Gestationalage

Exposure

Species(days p.c.J(R)

Effects observed

Mouse

0.5
0.5-1.5
0.5-1.5
7.5
7.5
7.5
7.5
7.5
7.5-8.5
8

5

278
276
277
231

25
25

8.5

50

Increase in resorption frequency
Exencephaly
Polydactilia
Increase in resorption frequency
Skeletal malformations
Decrease in litter weight (winter sample)
Hydramnios
Reduced tail length
Malformations of the axial skeleton
Hydrocephalus, flexion of the spinal cord,architectural changes of ependymal cells
Eye defects

0-8
8
8-9
9
9
9
9

5-25
12.5
36-40
25
50
12.5
100

27
362
139
365
364
93

9
9
16-22

50
25
10-40

Growth disturbances
Growth disturbances
Ocular and cerebral malformations
Growth disturbances
Increase in resorption frequency
Ocular and cerebral malformations
Heart and aortic, face, and urinary tract
malformations
Brain and spinal cord malformations
Microphthalmia, anophthalmia
Permanent alterations of nerve cells andcortical architecture of the brain

Rat

15-20

5
5
5
5
5
5

Reference

136

297
204
181

362
362
362
120

701

summary for the mouse and rat, in which species
sufficiently low doses have been tested. Doses of theorder of 5 rad, administered during the period of majororganogenesis, have definitely been shown to induce
malformations, particularly in the skeleton of themouse, together with some lethal effects and growth
disturbances. There appears to be no systematicinvestigations with rats at the same dose level, but it
seems likely that for doses of a few rads, careful
experiments might reveal teratogenic effects, particularly
growth disturbances and ocular defects. The table also
shows that in the nervous system, where cell division anddifferentiation proceed to very late foetal age, doses of a
few tens of rads can produce microscopic lesions of thenerve cells and malformations of the cerebral architecture. The observation accow1ts for the fact thatmicrocephaly and mental retardation can be induced
throughout foetal life with comparatively high frequency (see paragraphs 141-146).
291. The fact that teratogenic effects have not beenshown in humans with doses of the same order ofmagnitude in the course of pelvimetry or otherdiagnostic procedures (see paragraphs 202-208 and 217)
could possibly be attributed to the developmental
heterogeneity of the irradiated samples and to the late
age at irradiation, which in the majority of cases wasafter the organogenesis period. It can be concluded,
however, that in those animal species where low doses ofirradiation have been thoroughly tested at the time ofthe sensitivity peak, there is evidence of some effect at
doses as low as 5 rad.
2. Experimental tests
292. Much of the work concerning the experimental
demonstration of threshold is insufficient to permit
drawing definite conclusions. The usual reasons are thesmall number of animals used, the absence of wide-range
dose-response data, the relatively high doses employed
and, in some cases, technical imperfections. Thresholdproblems have been particularly considered by Jacobsen( 136). His experiments concerned skeletal abnormalities
induced in 7.5-day-old mouse embryos by exposures of0, 5, 20 and 100 R. The data are limited to one type of
malformation and to a single embryonic age (in order toimprove the precision of the estimates), but thelimitations are largely compensated by the number andaccuracy of the observations. However, it is impossible
to predict to what extent his conclusions would apply toother malformations and embryonic ages.
293. Jacobsen concluded that dose-effect relationships
for single malformations are of little practical relevancefor an overall evaluation of the teratogenic effects,
mainly because of the low frequency of occurrence ofsuch events. He therefore grouped the scored malformations according to skeletal region. The effects were
found to be linear or almost linear with dose, within theconfidence limits, for the control. 5-R and 100-R
groups, while the 20-R group was in most cases slightly
below the expected value. This pattern was found to
apply to most of the skeletal regions examined and also
to other traits not involving the skeleton. It was found insummer as well as in winter experimental samples.
702

294. In spite of the slight departure from linearitv
however, the author concluded that there was ~~suggestion of threshold, since both the 5-R and 20-R
points were significantly above the values of control.
Clearly these experiments do not exclude the possibility
that thresholds might exist at exposures below 5 R, but
experiments at lower doses would require a much greaternumber of animals and result in having, in principle, thesame problem, but within a lower dose range.
295. It may be concluded that the most refined
experiments so far exclude the existence of threshold for
exposures as low as 5 R at 1east under the specificconditions tested. The low-dose effects (at 25 R or
below) which have been recognized in animal experiments (I 36, 204, 231, 297) may be regarded as anincreased incidence of some types of malformations oreffects occurring spontaneously in the species tested. It
may be very difficult to establish threshold doses forthese effects, since the incidence of spontaneous
malfomrations and minor anomalies is controlled by thegenetic constitution of the strain or of the individual,
which has an intrinsic variability.

E. FACTORS MODIFYING THE TERATOGENICRESPONSE·'.,

296. In addition to the factors examined systematicallyin the other sections of the Annex with regard to eachtype of malformation, there are other factors that canmodify the teratogenic response to a given radiation
dose. These include physical factors such as LET anddose rate and the modifying effect of oxygen andchemical protectors.

1. Radiation quality and LET

297. The type and energy of radiation, as reflected by
the ionization density and the LET, are among the mostimportant radiobiological parameters affecting thedegree of response (256). There are, however, very few
experiments on LET effects on the induction of
malformations in mammalian embryos. Furthermore,these experiments are not systematic and, in particular,
the data do not include the effect of a range of LET
values for various abnormalities at different embryological ages.
298. Some very early reports (11, 52, 106) are hardly
amenable to quantitative analysis. Sikov and Lofstrom(315) reported on the RBE of 60 co gamma rays and
250-kV x rays in rats irradiated at various gestational
ages (9-10.5 days p.c.). The exposures (110-450 R) weredelivered either acutely every 12 hours or intermittently
throughout most of the half-day interval between
exposures. Embryos were examined at 14 days p.c.:mortality and malformations of the mandible. eye,maxilla, extremities and tail were considered. It was
found, in general, that gamma radiation produced lesseffect than x radiation, giving rise in most instances to
RBE values in the range of 0.80-0.95. However, formalformations of the eye, maxilla and mandible induced
at 9.5 days p.c., substantially lower RBE were obtained.
The slopes of the dose-effect curves for mortality were..

the same for both radiation schedules used, regardless ofembryonic age and protraction. Concerning malformations, similar dose-effect curves were produced by thetwo radiations but sometimes protraction resulted incurves of smaller slope; a reduced magnitude of someeffects and an increased value of the RBE were induced
by protraction. The effects were interpreted as
depending on the differences of recovery rates ofembryonic structures. especially when the change ofRBE by fractionation was particularly evident.
299. Friedberg et al. (91) irradiated mouse embryos in
the pronuclear-zygote stages with 300-kV x rays (67rad/rnin) or with fission neutrons (2 rad/min) andexamined them for embryonic survival of the uterine
content of the mother 16 days later. Non-thresholdexponential dose-survival relationships were folll!d to
apply to both x rays and neutrons in the range of 0-100
rad and,.0,19.5 rad, respectively. A comparison of theslopes resulted in an RBE of 4.5. With increasing dose,the pre-natal mortality increased, and the embryos diedat earlier development stages.
300. These data are clearly insufficient to allow
generalization, although they appear to be compatible
with our knowledge of RBE for other somatic effects in
mammalian systems. More systematic investigation forother radiations and LET spectra are required to cover
the subject adequately.

2. Dose rate and fractionation
301. In contrast to the paucity of data on the effects ofradiation quality, there are comparatively numerous
studies on the effects of dose rate and fractionation. The
reason for this interest is the possibility that protraction
might not reduce the effect in a system like the embryo.
In view of the complex pattern of cell types, each with adifferent time-related pathway of division, differentiation and interaction, a protracted dose could in
principle produce more adverse effects in the embryothan an acute dose of the same magnitude. However, it
should be pointed out that it is extremely difficult tocontrol these experiments, mainly because of the rapidchanges in sensitivity taking place as a function of timein the various structures and of the changing pattern ofmalformations specific to each developmental period.
302. The report by Auerbach (7) did in fact show someincreased hazard in mammals of fractionated, as
compared to single-dose, irradiation. Hybrid mouseembryos at 9.5 days p.c. were exposed to either 300 R
or to three 100-R fractions delivered at 30-min intervals
and were examined by uterine dissection 3-6 days after
irradiation. The criteria of damage used were theincidence and degree of lumbosacral spina bifida andcoloboma of the eye. While the total incidence ofmalformations was not affected by fractionation, theirseverity increased: this effect was seen in bothabnormalities, in spite of their different developmental
pathogeneses.
303. The observations of Rugl1 and Grupp (279)covered the period from Oto 8.5 days p.c., and included
almost 3600 embryos exposed to 50 R in two equal

fractions separated by different times. Exencephaly was
the malformation particularly studied. When the dose
fractions were separated by less than 4 hours. there wasno difference compared with the effect of a single
combined total exposure. Longer fractionation intervals
seemed to reduce the incidence of anomalies but not theincidence of early intra-uterine death.
304. Russell, Badgett and Saylors (298) carried out
rather more elaborate experiments. studying different
effects (mortality, weight loss, long-term survival,
fertility and morphology). They tested the effects of (a)171 R (0.096 R/rnin) accumulated during the first 2
weeks of pre-natal develQpment; (b) 200 R given acutely(83 R/min) at one of seven stages of pregnancy (0.5, 1.5,
4.5, 7.5, 9.5, 11.5, 13.5 days p.c.); (c) 191 R
administered in 15 daily acute fractions of 12.8 R within
the same time. As expected (see chapters II and lll),
acute irradiation produces a great variety of incidence
and severity of effects, strikingly dependent for their
type and degree on the stage at irradiation. Chronic
irradiation, on the other hand, induced no special effect,
except for shortening of the fertility period in females.
Daily acute fractions had no more adverse effects thancontinuous exposure.
305. On the likely hypothesis that cell killing might be
the main cellular effect responsible for the initiation of
malformations (see paragraphs 280-283), these results
were interpreted to mean that cell killing in embryos islower with chronic than with acute irradiation. The factthat single doses of radiation given at the sensitive
periods were effective in causing abnormalities, while
lower doses within the same sensitive periods were not,is compatible with the model reviewed in paragraphs 284-287, assuming that the total dose administered during these periods was insufficient to inactivate
more than the threshold number of cells required for the
production of abnormalities. The difference to Auerbach's findings (7) could be explained by the assumptionthat cyclical periods of higher sensitivity could havebeen responsible for the potentiation of the effects by
fractionation.
306. More recently Yoshizawa and Ueda (373)submitted six different groups of ddY mice at 11 days
p.c. to 200 R given at the rate 1-100 R/min. The bodyweight and the delay in ossification of the caudal
vertebrae scored at 18 days of pregnancy were assumed
as indicators of whole-body and systemic damage. Boththese parameters were found to be in good relationshipto increasing exposure, but they were insensitive to the
exposure rate within the range indicated.
307. The work of Kriegel and Langendorff (159) does
not allow a direct comparison of the effects of acute andfractionated doses. It gives, however, information on theincidence of malformations and of lethal effects inducedby fractionated, daily x-ray exposures administeredduring the total pregnancy period of the mouse. Levelsof 2.5-10 R per day had an effect on embryologicaldevelopment. At 20 R per day a small increase inresorption rate and in malformation incidence wasobserved together with a decreased weight of theconceptus. Fractionation regimes of 80 R per day caused
the interruption of pregnancy. Another series with 80,
703

120, and 160 R per day for limited pregnancy periods ofdays 11-16, 11-13 and 14-16p.c. was reported by
Kriegel and Reinhardt (161). At 18 days of gestation
striking effects on growth and malformations of the tailand the extremities were obtained.
308. Konermann (152, 153, 154, 155) published aseries of papers dealing specifically with the effects offractionated and chronic irradiation. In the first report(152), the differential stage sensitivity was particularly
examined at exposure regimens of 5-100 R per day. It
was shown that the developmental stage, rather than thedose, affected the type and severity of the morphological damage. Studying the effects on foetal weight
and the teratogenic effects as a function of dose andtime, the paper concluded that limited processes ofrestitution may correct in part the primary radiationinduced lesions. The second paper (153) dealt withskeletal malformations and their relations to external
morphological abnormalities.
309. The third contribution (154) is of particularimportance for comparing the effects of fractionatedand continuous irradiation during blastogenesis, organogenesis and foetogenesis, and also during the whole
period of gestation. Resorptions, intrauterine death,malformations and foetal weight were the end-points
studied. The degree of effect for a· given fractionateddose was compared with the effect of the same dose
under continuous irradiation. The minimum dose, theSO-per-cent dose and the maximum effective dose forlethal and te~atogenic actions were compared, and it was
concluded that continuous exposure required 1.5 times
higher doses than fractionated exposure for the same
degree of effect. This factor was considered to be small
compared to the ratios between the dose rates of thetwo exposure types (2 10 3 to 2 lcf), thus confirming thatthe stage sensitivity of the embryo is by far the mostimportant factor determining the final outcome ofirradiation.
310. The last paper (155) examined the recovery
mechanisms in the embryo by the use of the
exposure-fractionation technique. Groups of mice
irradiated with daily fractions (10-80 R per day) on day
1-5 p.c. or 6-13 p.c. were compared with other groups
receiving a second series of doses on day 14-18 p.c. The
foetuses were examined on day 18 p.c. The percentage
of normal, abnormal and dead animals and their weight
showed that sensitivity during the foetal period was notenhanced in the dose range studied by previous
irradiation during blastogenesis. On the other hand, mostparameters studied, and particularly weight loss, showed
an effect of potentiation, by pre-irradiation duringorganogenesis.
311. Observations on mice irradiated continuously
throughout gestation were also reported by Warren andGates (358). A recent paper by Friedberg et al. (91)dealt with the survival of mice irradiated in the
pronuclear-zygote stage with single or fractionated dosesof x rays (60 rad or 30 rad + 30 rad) and fission
neutrons (14 rad or 7 rad+ 7 rad). The intt:rval between
the two doses was 4 hours. No significant differenceswere observed between the single-dose and the split-dosegroups. In view of the changes in sensitivity during thefractionation interval (see also reference 299), the effectof the split doses could be interpreted assuming that thefirst fraction blocks the embryo from proceeding
towards a less sensitive stage.
312. There are also experiments on the effects of dose
rate and fractionation in other animal species. In rats,
Sikov and Lofstrom (315), using the experimental
schedule described in paragraphs 297-300, produced
evidence of a reduction of the teratogenic action by do·se
protraction. Brown et al (45) bred female rats undergamma radiation levels of 2-10 R per day. They foundno essential difference in the litter size and in thenumber of offspring born in six successive litters of themothers living in the continuous radiation field.
However, at 20 R per day, the fifth litter was drasticallyreduced in size and the sixth litter had no offspringborn. Malformations were not seen. but there wasevidence of some weight reduction. Animals born fromthe third litter were tested for fertility and found to benormal up to 5 R per day but sterile at I O or 20 R perday.
313. In a series of papers on morphological damage ofbrain structures induced by fractionated irradiation,
Brizzee et al. ( 42, 134) reported the results of exposuresof 13.5 and 14-day embryos singly to 150 R or tovarious fractionation patterns within the same half-day
period. The damage to brain structures shown at
19.5 days p.c. was higher after single exposures anddecreased progressively when two 75-R fractions wereadministered at intervals of 1, 3, 6, 9 or 12 hours.
Subdivision of the dose into 2, 3, 5 or 9 equal fractions
over the same time also progressively decreased theeffect, which was less in these animals than in those of
the previously mentioned variable-interval group. It was
concluded that, for a fixed dose over a given time, thesize of individual fractions is more important than thenumber of fractions in determining the final effect.
314. In another experiment (41) a group of embryo rats
was irradiated on day 13 p.c. with single whole-body
exposures of 12.5-200 R. A second group received
100 R on day 13 p.c. and, after 9 hours, a second
exposure varying of 12.5-125 R. The damage to theolfactory lobes and the cerebral hemispheres, as seen on
day 19 p.c., was small for the exposures between 12.5
and 87.5 R in the single irradiation groups and for
exposures between 12.5 and 50 R in the split-irradiationgroups. At higher exposures, the damage manifestly
increased in both groups.
315. The logarithm of the mean depth of the corticalzone was plotted versus the radiation exposure. The plot
showed a shoulder up to 62.5 R, followed by an
exponential decrease to 200 R. Some shoulder (up to50 R) was also evident in the split-dose series, which
could indicate the presence of some degree of cellrecovery with time between the two doses. Treatment of
the animals with hypothermia to 20°C during thefractionation interval (40, 43) reversed to some extent
the decreased effect of the split dose, indicating a
possible inhibition of the repair processes.
316. In the experiments of Martin (184), an exposure
to 160 R on day 18 p.c., at rates of 1, 3 or 47 R/minproduced some effect on the pre-natal and post-natal

704

"·

.........

body weight. The amount per cell of protein and RNA
in the brain were not affected consistently by theirradiation. The cerebral cortex of the irradiated animals,
however, weighed less than that of controls and had alower content of DNR, RNA and proteins, and thiseffect was apparently not influenced by the exposurerate. The other portions of the brain showed partial
recovery of the total DNA, compared to the control
value. Exposure rate had some effect, since recovery was
higher in the 1-R/min group and progressively lower athigher exposure rates. After 2 months of life, the brainof the controls and of the 1-R/min group contained
more DNA than those of the groups irradiated at higher
exposure rates.
317. Reviewing the information on the influence of
exposure rate, Brent (30) concluded that exposure rates
below 5 R per day did not produce observabledeleterious effects in the surviving offspring. He also
reported results of experiments in which rat embryos
were exposed to 150 R on day 9 .5 p.c. at different rates
from 0.5 to 100 R/min. These results indicated that
protraction reduced the resorption and the growth
retarding effects. Higher exposure rate groups had ahigher incidence of malformations and were more
severely malformed. Malformations like anencephaly,
microcephaly, microstomia, evisceration, renal agenesis
and absent pinna did not appear in animals irradiated at
low rates. However, any malformation with an incidence
higher than 45 per cent in the high exposure-rate groups
was also seen following protracted irradiation.

the total number of implants was not affected but anincrease of mortality was seen at day 12 p.c., the great
majority of lethal effects occurring before day 15 p.c.
The pre-natal growth of the embryos was greatly delayed
and many types of abnormalities. including microcephaly,
anophthalmia and skeletal malformations, were also
found.
320. Malformative defects of the limb possibly
associated with a specific dose rate have been describedin irradiated cattle, sheep and swine by Erickson andMurphree (82) and Mcfee et al. (188). Murphree andGraves (211) studied the effect of exposure andexposure rate on lambs, irradiated on day 23 p.c. with
6
°Co gamma rays. Pregnant ewes were exposed to 282 R
at a rate of either l or 47 R/min. About 15 per cent of
the ewes exposed to 1 R/min and 38 per cent of those
exposed to 47 R/min died about 23 days after
irradiation. The foetuses recovered from these deadanimals were all smaller than nonnal. In the 1-R/min
group, 4 ewes produced 4 defonned lambs; in the
47-R/min group, 1 ewe had twins, one of which was
defonned. Of the 5 defonned lambs, 3 had ulnar aplasia
and 2 aplasia of the ulnae and femora. A second series
with a total exposure of 188 R was perfonned. Out of
28 lambs born in the 1-R/min group, 10 had deformitiesof the extremities, eyes and head. In contrast, only I outof the 29 lambs born in the 47-R/min had leg
deformities.
321. In conclusion, the majority of the data available
for most species indicate a decrease of the cellular andmalformative effects by lowering the dose rate or by
fractionating the dose. However, examples of deviations
from this trend have been well documented in a few
instances and are not inconsistent with the knowledge
about mechanisms of the teratogenic effects. It istherefore impossible to assume that dose rate andfractionation factor have the same influence on all
teratological effects.

318. After obtaining an exposure-effect curve(50-200 R gamma radiation) for ocular malformations inrats at 11 days p.c. to establish a suitable level for
exposure-rate studies, Strange and Murphree (338)irradiated these embryos with 100 R given at I, 2, 3.3,
5, 10, 25 and 47 R/min. Grading the ocular damage
observed at 30 days of age, they showed an increase ofeye abnormalities up to 3.3 R/min, a gradual but notsignificant further rise up to 10 R/min and a significant
drop at 25 and 47 R/min below the levels observed at
3.3 and 10 R/min. The increase in susceptibility of thissystem with increasing rate of exposure falls within theusual pattern found in dose-rate experiments and iscompatible with the commonly accepted notion thatany end-effect results from competing phenomena ofdamage and repair, both of which are time-dependent.
However, the fall at the high exposure rates is asurprising finding. The authors suggest that there may be
a limited and short period of sensitivity in the life of thetarget cells and that the probability of hitting thissensitive stage with an effective dose might decrease atvery high exposure rates. Whatever the explanation may
be, these data represent further evidence that thecomplexities in the division and differentiation
mechanisms of a developing embryo, by comparison
with a more conventional cell population at equilibrium,
may justify unusual· findings with respect to specificend-points.

322. There has been some research on the influence ofoxygen pressure on the induction by radiation ofteratogenic effects in mammalian embryos. Russell et al.
(305) (also reported in Russell (295)) showed thathypoxia resulted in substantial protection in mouseembryos exposed to 100400 R on day 11.5 p.c.
Hypoxia was induced by breathing a mixture of 5 percent oxygen and 95 per cent helium. A number of
end-points were scored at birth, such as the mean birthweight, mortality, tail length and shape and footmalfonnations. Irrespective of the shape of thedose-effect curves for the indicators examined, themagnitude of the protection afforded by the low oxygenpressure was approximately the same for all indicators.
Oxygen seemed to act as a simple dose-modifying agent,
with an oxygen-enhancement ratio of 2-3.

319. Continuous low-intensity gamma irradiation wasgiven to pregnant rats at 50 R per 20-hour day, startingon the first day of pregnancy (67). Embryos, foetuses
and uteri were examined at 10-20 days p.c. for growth
and the presence of abnormalities. At this exposure level

323. Rugh and Grupp (280) treated pregnant females(8.5 days p.c.) with a mixture of 6 per cent oxygen and
94 per cent nitrogen for 8-13 min, and then exposed
these animals while still unconscious to 200 R.
Non-irradiated animals kept at low oxygen pressure·

3. The effect of oxygen

705

served as controls. The foetuses were observed byuterine dissection on day 18.5 p.c. Anoxia alone had noeffect on the embryos. Low oxygen pressure in
combination with radiation resulted in 71 per centnormal embryos, while the same dose at normal oxygenpressure would yield only 41 per cent normal embryos.
Resorbed embryos and exencephaly in foetuses, after
irradiation in. hypoxia, was recorded as 16 and 13 percent against 34 and 21 per cent in the corresponding
controls. Therefore anoxia produced some degree ofprotection against both lethal and teratogenic damage.
Since there are insufficient data to draw a dose-effectcurve, the numerical factor to be attached to thisprotective action cannot be evaluated with precision.
324. Uterine vascular clamping (35) is an alternative
procedure for producing anoxia of the embryo. When
this technique was applied to one uterine horn of the rat
for 45 min on day 8 or 9 p.c., it had minimal
detrimental effect on foetal growth and survival. This
technique has been shown to protect against radiationinduced lethality and weight loss (36). Another methodof producing hypoxia indirectly, by inducing hypoglycemia with insulin, was found to be totally ineffectivein the mouse (280).
325. It may be concluded that the oxygen effect in theembryo is similar to that observed in the irradiation ofother cellular systems of the adult animal.
4. Radioprotective and radiosensitizing drugs
326. There are numerous reports on the effect ofradioprotective drugs upon the induction of embryonicand foetal damage by radiation. The protective effect ofcysteinarnine (269) and cysteamine (369) had beenshown in irradiated foetal mice with respect to survival,
growth rate, weight and induction· of malformation.
Rugh and Grupp (280) examined the effect of thesesubstances on CFl embryos exposed to 200 R on day
8.5 p.c. When 3 mg of cysteinamine were administered
IP 30 min prior to exposure, the incidence of normal
foetuses increased from the expected 41 per cent to73 per cent, resorption decreased from 34 per cent to
8 per cent and exencephaly incidence decreased from21 per cent to 16 per cent. In the case of cysteamine(3 mg IP withing 30 min of exposure) the shifts were:normal animals, from 41 per cent to 80 per cent;resorptions, from 34 per cent to 12 per cent; andexencephaly, from 21 per cent to 8 per cent. These two
drugs therefore appeared to act beneficially, increasing
survival and reducing the incidence of malformations in
the irradiated embryo. However, these experiments witha single dose of radiation do not allow the derivation ofgood estimates of the protection factor. AET did notcounteract the radiation effects.
327. Numerous other treatments (hypo tonic andhypertonic solutions, dextrose, alcohol, chlorpromazine,
and homogenates of spleen, marrow and liver) tested forpossible action in the same experiments (280) were also
found ineffective. Konermann (156) reported complete
dose-exposure curves, in the range 150-450 R deliveredin single irradiations at 3, 6, 9, 12 and 15 days p.c., withand without cysteinamine (a single amount of
706

200 mg/kg given 8 min prior to irradiation). The
end-points, checked by uterine dissection on day 19 p.c.,
were morphologically normal and abnormal animals,
malformed and dead foetuses, frequency of embryonic
deaths, foetal and placental weight. and skeletal
malformations. Marked differences were observed in therelative protective effect of the drug for various
radiation doses and various stages irradiated. Protection
was shown to exist both in the pre-implantation and in
the organogenesis period, and the value of the protection
factor was different for different malformations.
328. In the rat, Starkie (331) reported a partial
inhibition by cysteamine of the radiation effects on thetestis. Animals were irradiated at 17 and 21 days p.c.
with doses of 50-150 rad and the testes examinedhistologically at the age of 25 days. Kalinina (140)tested the effects of a number of possible radioprotective substances. such as phenatine, magnesium
sulphate, chlorpromazine and mercaptamine, injected inappropriate doses 15-20 min before irradiation(200.300 R on day 11 p.c.). Embryo and placental
weight, mortality and malformations were scored on day
22 p.c. The mortality rate, and to some extent theincidence of malformations, were lower in the injectedgroups.
329. Baev et al. (9) tested the protective effect ofcysteinamine and cysteamine in the case of fractional
exposure to radiation throughout gestation ( 40 R per
day for 20 days). The irradiated offspring were followed
to adulthood, and the changes in litter size, post-natal
death and body weight were noted. While protection wasafforded by both drugs in regard to litter size andweight, the post-natal death rate was unchanged. Twenty
females and 24 male offspring surviving after drug
treatment and tested when sexually mature (10) showed
greatly reduced reproductive performance, sterility andincreased frequency of dominant lethal mutations. A
protective effect of cysteinamine, most pronounced
after completion of organogenesis, was also described by
Kirushenkov (151 ). Treatments of pregnant females withproteolytic enzymes (348) or with progesterone (353)
were also reported to act beneficially in protecting the
foetus from radiation-induced malformations. In
contrast to that result, small doses of iodacetamide{194) and miracil D (193) were shown to act
synergistically in potentiating the teratogenic action ofradiation.
330. It may be concluded that many of the known
radioprotective drugs which are active in modifying theradiation response of adult systems are also effective
against the damage caused by irradiation in utero. In thelatter case there is evidence that the protection factor
may vary according to the stage irradiated and to theparticular type of damage analysed (lethality, growth
disturbance and type of malformation).
F. SIGNIFICANCE OF THE EXPERIMENTAL
DATA TOMAN
331. The majority of authors who have contributed
experimental information on teratogenic effects ofradiation have considered, directly or implicitly, theapplicability of their findings to the human species. A
number of papers have also specifically dealt with thissubject (see for example 295,297,301,304,261,265,
268, 27, 136, and 345). The following paragraphs
discuss the opinions expressed by the authors andsummarize the views oft.he Committee regarding present
knowledge and the need for future research in radiation
teratogenesis.
332. It is clear from all the preceding sections that for
teratogenesis, more than for any other early or latesomatic effects of radiation, data on humans are
extremely limited. Apart from growth disturbances,microcephaly, mental retardation and a few otherdefects of less importance, there are very few systematic
data applicable to man, particularly at the low doses ofinterest. However, the scanty data available for otherabnormalities are probably sufficient to permit theassumption that the effects observed in animal
experiments also occur in man. The animal experience
therefore appears to be of unique and special importancefor the assessment, even if only qualitative, of
malforrnative effects in man.
333. Animal research has established two importantqualitative facts: (a) there is a uniform pattern in thesequence and timing of the early developmental stages in
the mammalian species tested (see chapters I-III); and(b) there is a general similarity among the various species
with regard to the type of malformations induced andthe teratogenic mechanisms (see chapters III-VI).
However, these facts are in themselves insufficient tojustify any direct quantitative extrapolation of effects
between species for a number of reasons: (a) the great
specificity of the malformations induced at comparable
stages in different species and even among different
strains of the same species (see chapter II); (b) the
species difference in the duration of the foetal period(see table 3). which causes marked discrepancies in the
final state of the malformations and causes serious
obstacles in comparative studies between animals andman as, for example, with the central nervous system(141); (c) the extremely variable form of the dose-effectrelationships in different species (see chapter III).
334. For all the above reasons there is general consensus(304, 136, 267, 268, 344 and 345) that data applicableto man can only be derived from human epidemiologicalstudies. These studies are, however, not available atpresent, at least on the scale required and at the low
doses of interest. The Committee believes that this pointshould be particularly emphasized so as to discourage
numerical extrapolations not sufficiently justified bypresent knowledge.

VII. SUMMARY AND CONCLUSIONS
A. GENERAL

335. Scientific contributions concerning the somaticeffects on experimental animals irradiated in utero havebeen reviewed with the aim of extracting information
relevant to the assessment of similar effects in man. Dataon embryological effects in the human have also beenreconsidered and updated. In addition to lethal effects,

effects on growth and development, and miscellaneous
pathological consequences of the exposure, malformations have been particularly examined as typicalsequelae of irradiation in utero. The amount of data
directly or indirectly related to this subject is indeed
rather large, but most of it has been produced with theultimate object of analysing developmental mechanisms.
Thus. the quantitative information to be gained on themain radiobiological variables {dose, dose rate. fractionation, LET etc.) is definitely insufficient, particularly in regard to the human embryo. The Committeeemphasizes, however, that appropriate evaluation ofwhatever data is available may help to set the scanty
knowledge on man into a better perspective. It may also
strengthen the qualitative extrapolation of effects
between species, pending the quantitative assessment ofradiological hazards which must of necessity be carriedout in man.

B. METHODOLOGY AND TECHNIQUES
336. Among the variables or conditions affecting theinduction of embryological effects in mammals, thegenetic constitution, the breeding characteristics of thespecies and the reproductive history of the mother havebeen shown to influence the degree of the lethal andteratogenic responses of the conceptus; other variables
have a relatively minor but not trivial importance. There
are well defined periods of maximum sensitivity during
gestation for the induction of different types ofmalformations; these periods are related to the stage ofmajor differentiation of the relevant embryonicstructures. It is possible to reconstruct developmental
tables in animals and man to compare the approximate
critical times for the occurrence of certain recognizable
ontogenic events and to facilitate rough intercomparisons of pregnancy stage and of embryonicdifferentiation. In animals, a number of variables
affecting the scoring of lethal, developmental andmalformative effects have been recognized, and theirimportance for correct radiobiological assessments has
been discussed. Regarding the human experience,
insufficient knowledge of the conception time, lack ofprecise dose estimates, paucity of irradiated groups anddifficulties in the selection of control groups prevent any
precise estimate of hazard. Following an analysis of themain periods of pre-natal development in various
mammals, a systematic review of embryological effects isprovided.
C. THE PRE-IMPLANTATION PERIOD
337. Killing of the embryo prior to implantation stands
out during this period as the most conspicuous effect ofirradiation, which may be expressed in polytocous
animals as death of single embryos or death of the entire
litter. Post-implantation and post-natal death are
regarded as relatively less important hazards of the
exposure during the pre-implantation stages. In addition
to considerable differences in sensitivity between animal
species. there have been described substantial changes inthe susceptibility to this type of damage within eachspecies, particularly during the early segmentation of thefertilized egg. In the mouse, the species where the most707

data have been obtained, a number of reports point out,
in good agreement, that the absolute increment ofembryonic loss soon after fertilization could be of theorder of 10 -z R -i. Chromosomal damage to theirradiated blastomeres followed by degeneration of theprimitive embryo cells appears to be the majormechanism responsible for embryonic death. During thisstage, the reduction of body growth is not an effectclearly and universally recognized. The malfonnations
documented. particularly in the nervous system of therodent, may also be regarded as less frequent events, incomparison with their higher frequency during thefollowing stage of organogenesis.

D. THE PERIOD OF MAJOR
ORGANOGENESIS
338. Lethal effects during this period may be studied
through several indicators, such as the complete failureof pregnancy. the decrease of the average litter size, themortality in utero of the irradiated embryo and the
neo-natal and long-tenn survival of the live-born animals.
Concerning pre-natal mortality, the sensitivity of theembryo is highest soon after implantation and during
early organogenesis, with a subsequent decrease at latertimes. The shape of the exposure-lethality function
appears to change substantially with the developmental
stage, and the majority of data point to curvilinear
functions. There is some infonnation on the effective
doses, particularly in rodent species, in regard to preand neo-natal death. They have shown LD 5 0 values
lower than 150 R at the pre-implantation and earlyorganogenesis stages, after which these values invariably
increase to attain during the foetal stages levels
comparable to the post-natal LD 5 0 • Long-tenn reduction of life span may also be documented in rodents for
exposures of a few hundred roentgens; these effects
strongly depend on a number of genetic factors.
Infonnation in man is virtually absent.
339. Data obtained from several animal species indicatethat the period of major organogenesis is very sensitive
to growth disturbances, probably more than thefollowing foetal period but definitely more than thepreceding pre-implantation phase. When growth defects
are scored prior to birth, the magnitude of the growth
deficit appears to vary with the interval between
irradiation and observation. Only in very few cases are
there sufficient data to evaluate the net effect of thedose after a fixed post-irradiation time or at the time of
maximum effect. Growth disturbances induced byirradiation of the embryo but persisting in adult life andexpressed at the whole-body or organ level have also
been described in the experimehtal animal.
340. In spite of the great wealth of information on
malformations induced during organogenesis in various
animals, it appears extremely difficult to summarize
these data into any coherent quantitative conclusion for
the following reasons: the species and strain variability;
the scarcity of complete dose-effect series; the different
criteria of scoring the induced aberrations: and thedifferent embryological and physical conditions underwhich irradiation has been performed. A few generalstatements may however be justified:
708(a) With due regard to developmental andanatomical differences betweert species, similar classes
and types of malformations occur upon irradiation atcomparable developmental ages. This finding is sufficiently general to suggest that man might not represent
an exception in this respect, even though no quantitative
comparisons between experimental animals and humanspecies would be warranted at present:(b) Within each species there is a well definedtime at which each malformation may be induced.
Increasing the dose usually results in a spread of thesensitivity peak and in an increase of the malformation
incidence. In some cases, biphasic periods of sensitivity
are observed, related presumably to different mechanisms of induction resulting in similar malformative
end-points;(c) The time specificity of each malformation
usually coincides with the major phase of differentiationand organization of the relevant structure. Cell killing
followed by an arrest of organ development at somerudimentary stage may be identified as the mechanism
initially responsible for the malformative event;(d) Data are too few and their variability too
high to allow any firm conclusion about the relative
radiation susceptibility of different embryologicalstructures within each species and, even less, between
various mammals, but malformative responses have in a
few cases been described down to exposures of 5 R:(e) In the vast majority of cases the malformation incidence has a curvilinear trend with dose,
implying relatively less effect per unit dose at low thanat high doses. However, linear or quasi-linear dose-effectrelationships are not uncommon. particularly whenembryologically or topographically related malformations are grouped together in such a way to express
the malforming effects as the ratio of malformed tonormal animals, irrespective of which type or number ofmalformations might affect the malformed conceptuses.
Such cases have been documented only in animals; it isconceivable that in the case of animals absolute increases
in the incidence of malformed foetuses of the order of 5
10- 3 R- 1 of low-LET radiation delivered at high dose
rates might occur. It should however be emphasized thatsuch estimates are very tentative and their projection toother species and particularly to man is unwarranted;([) In man the best documented type of
malformation is microcephaly, which in its mostextreme cases is accompanied by mental retardation.
Following high acute doses its incidence has beententatively estimated at around 10-3 rad -t , with thelowest effective doses ranging between 10 and 150 rads
under different conditions of exposure;(g) Other epidemiological surveys in man
following doses in the region of 1.20 rad have given
either negative or non-significant answers; they could
only be of some value in excluding the possibility that atthese doses the human embryo could be 10 times moresensitive than the incidence of malformations at higher
doses would imply.

341. Among other miscellaneous effects of irradiation
during embryogenesis, changes in fertility and fecundity,
cataract, and relatively minor defects have also beenstudied in animals.

E. THE FOETAL PERIOD
342. Lethal effects have been described in the rodent
for irradiation during this period. Although limited to
selected ages, these data show consistently a reductionof sensitivity with advancement of foetal age. There isalso some information in man obtained in cases ofradiation-induced therapeutic abortion arid, with somedosimetric uncertainties, following the A-bomb explosions in Japan. This information refers to both pre- andpost-natal radiation lethality and to long-term effects on
survival.
343. Generalized and local growth disturbances arecommonly described effects of irradiation of the foetus,
and they are seen in many cases to persist throughout
the extra-uterine life. In man numerous studies agree inpointing out a growth-retarding effect of irradiation
manifested by a reduction of various body parameters,in addition to microcephaly and mental retardation.
344. It is commonly accepted that as foetogenesis
proceeds the malformative effects of radiation become
more difficult to document macroscopically since, forthe same radiation dose, the size of the anatomical
structures involved becomes progressively smaller andthe resulting functional defects less important. It should
however be expected that these subtle malformations,rather than the major defects induced at organogenesis,
would be the ones more likely to survive and to result inthe main social burden. Data in animals relateparticularly to effects on the eye, the nervous system andthe gonads. In man, besides microcephaly, heterochromia of the iris has also been described.
345. Other pathological effects on the foetus regard thehaemopoietic system at the bone-marrow and peripherallevel; haemopoietic failure at this stage is closely related
to the whole-body lethal effect. Laboratory investigations carried out on samples of children and adolescents
irradiated in utero have shown a good capacity forrecovery of whatever haematological damage might havebeen induced soon after exposure. The question of a
possible alteration of the sex ratio of children born toparents exposed in utero is still controversial.

F. INTERNAL IRRADIATION
346. The interpretation and evaluation of the developmental effects of irradiation in utero by incorporated
radionuclides must be based on a precise knowledge ofdoses received at specified dose rates by the mother andthe conceptus at any given developmental stage and inany sensitive structure under various conditions oftreatment. The information available falls far short ofthis objective. The effects observed are similar to thosedescribed for external irradiation; they are, however. lessspecific owing to the fact that exposure to the nuclides
usually extends throughout embryonic and foetal stages.
Among the main variables known to interact in the
production of these effects, the following have been
identified: the physical nature of the nuclide and thechemical form in which it is administered, the route andschedule of administration, the dose, the developmental
age at irradiation, the animal species, and the possible
role of the irradiated maternal organism.

G. MECHANISMS OF RADIATIONTERATOGENESIS
347. Most of the data support the contention thatindirect effects of the mother's irradiation do not play amajor role in the induction of malformations,particularly at the low doses of interest for radiation
protection. On the other hand, it is conceivable that highdoses given to the mo~er might also influence themortality of the embryo and foetus.
348. Malformations are typical sequelae of damage
inflicted by radiation on a developing organism. They
are produced through a disturbance or disruption of theorderly sequence of ontogenic events that result in adeveloped foetus. At the subcellular level it appears
quite unlikely, even in the absence of much direct
information, that the primary biophysical events taking
place in embryonic tissues may grossly differ from thoseoccurring in adult tissues. At the cellular level
chromosomal aberrations are thought to be responsible
for the loss of reproductive capacity of the irradiatedembryonic tissues. This notion is in agreement with what
is observed in adult tissues and has been substantiatedfor many embryological structures as well. Selectivity ofthe damage with respect to different tissues which areseen to respond at different times is postulated todepend on changes in the intrinsic radiosensitivity ofcells, on the variable kinetic parameters of the different
embryonal tissues and on the capacity for repair andrepopulation of each tissue. Radiation damage toregulatory mechanisms could also be regarded as acomponent of the teratological damage, but up to thepresent all these processes have only been consideredtheoretically and have never been experimentally
documented. Simple mathematical models for tlie
development of malformative damage have also been
proposed and they are not incompatible with theobserved dose-effect relationships. The analysis of such
relationships in order to gain a better knowledge of
mechanisms would however be unwarranted in theabsence of further experimental evidence.
349. Regarding the possible existence of thresholds in
dose-effect relationships there is consistent evidence
showing that doses as low as S rad may still be effective
in inducing selected malformations. Direct experimental
tests of the absence of thresholds in this dose region
would tend to exclude their existence at even lower
doses. Theoretically, the possibility does exist thatthresholds might occur at even lower doses. but
experiments of sufficient precision to reveal them would
be technically difficult or even impossible for statisticalreasons.
350. Data on the effect of radiation quality and energy
in respect to teratogenic damage are extremely rare anddefinitely insufficient for a more than speculativeknowledge of the relevant RBE factors. Within theselimitations it appears, however, that present information
is not incompatible with the notion that higher RBE
709

factors might apply to the more densely ionizing
radiations and to the case of dose protraction. Morerefined information on RBE factors relating to different
malformations is absent.
351. Data in many species would indicate somedecrease of the malformative effects induced at low dose
rates or with dose fractionation. However, the nature of
the embryonic material is such that under special
conditions a potentiation, rather than a reduction, ofeffect might be possible: a few examples of such effects
have indeed been found. In any case, it seems impossible
to assume that the same reduction factors for low dose
rate or fractionation might apply to all effects inducedby radiation in embryonic tissues.
352. Anox.ia and treatment with radiosensitizing andradioprotective drugs have been shown consistently to
act as modifiers of the damage to the embryo in thesame sense and by about the sam~ factors as for adulttissues. The protection afforded is variable and depends
on the age of the embryo and the type of damage
observed.
H. RESEARCH NEEDS
353. The qualitative value of animal experimentation
for the assessment of human effects in no way limits-its
usefulness, particularly in view of the lack of precise
information for many types of teratogenic effects inman. The Committee has identified several fields where
further information would be highly desirable. The
fields, which relate to both human observations andanimal experimentation, are as follows:(a) Studies of stochastic2 versus non-stochastic
effects occurring in the embryo and foetus at various
stages of development would appear to be of great value
for their implications on risk assessments in man. Thesestudies would imply the ascertainment of some cellularparameters which have been postulated in some models(see paragraphs 284-287) but have never been determined experimentally;(b) Too little is known about whether, and towhat degree, the mechanisms and kinetics of inactivation, recovery and repair of adult somatic cells andtissues apply also to embryonic and foetal systems (seechapter VI, section B). A better understanding of the
primary effects might also be of help in developing morerefined models of teratogenic damage, reflecting theeffects of the main radiobiological variables (dose, doserate, LET, fractionation etc.). For example, the conceptsof microdosimetry ( 146) have ~o far not been extended
to damage in utero, and such an approach in this field(see chapter VI, section C) might lead to a re-evaluationof old data or to the design of new and more precise
experiments:
2
In the present context the terms "stochastic" and"non-stochastic" are used as defined for the purpose in question
by the International Commission on Radiological Protection:"stochastic effects are those for which the p,obability of aneffect occurring, rather than its severity, is regarded as a functionof dose, without threshold. 'Non-stochastic' effects are those forwhich the severity of the effect varies with the dose, and forwhich a threshold may therefore occur" (129a).

710(c) The study of pathogenetic mechanisms anddevelopment patterns of malformations are thought bysome (267) to be of little value at present, while actionsin the fields of etiology and prevention are advocated as
the future major steps. This opinion could be supported
in regard to accumulating further descriptive work on
malformations. However, the Committee believes that
extensive data on specific malformations as a function ofdose (and particularly at low doses) and in several animal
species would be highly desirable and would considerably increase our confidence in interpreting theestimates in man (136);(d)
In planning and performing such experiments, the co-operation of radiobiologists and embryologists is essential to ensure that such large and costly
undertakings would eventually be of value for humanassessments. In addition to the technical points
underlined in chapter I, the selection of relevant
end-points for experimentation should reflect the factthat relatively minor morphological and functional
defects in man may be more important than extensive
malformations which would in most cases be incompatible with extra-uterine life;(e)
One area where data are extremely scarceis the influence of radiation quality on malformationinduction (see chapter VI, section D.1). More systematic
experiments, especially at low neutron doses, should be
performed with various LET spectra and at different
stages of embryonic development to accumulate more
precise data on the RBE values for different types ofmalformations, which at present are only largely
estimated on the bases of inferential evidence (256):(!)
Effects with dose fractionation and low dose
rate also deserve better attention, in view of thepossibility that special regimes of fractionation or
particular dose rates may be more harmful than single
acute doses (see chapter VI, section D.2);(g)Internal irradiation by various nuclides has
only been episodically explored. It will be necessary toextend present experience to the potentially mosthazardous and important nuclides with studies of
uptake, distribution, clearance, dosimetry and effects on
both the mother and the foetus:(h) There should be more surveys to evaluate
the type and frequency of x-ray examinations of thelower abdomen of pregnant women and the resulting
doses to embryos (see Annex I). Such data have alreadybeen obtained in some cases (44, 359, 361), but theyshould be extended to different socio-economic andmedical situations in order to obtain representative
estimates of the risks involved;(i)Finally, no occasion should be lost for
accumulating data on irradiated human populations;only on the basis of such data are refined risk estimates
possible. It is essential, therefore. to continue the studyof the irradiated populations already surveyed. Epidemiological surveys on other population groups yet tobe identified will also be required to improve the present
incomplete knowledge, at least to the point of excluding
the possibility that exposures might be more hazardous
to man than to experimental animals.

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725

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