PROCEEDINGS SERIES ί ^5&, J

BIOLOGICAL EFFECTS OF N E U T R O N I R R A D I A T I O N

P R O C E E D I N G S O F T H E / S Y M P O S I U M O N T H E E F F E C T S O F N E U T R O N I R R A D I A T I O N

U P O N C E L L F U N C T I O N O R G A N I Z E D B Y T H E

I N T E R N A T I O N A L A T O M I C E N E R G Y A G E N C Y A N D H E L D I N N E U H E R B E R G ( M U N I C H ) , 2 2 - 2 6 O C T O B E R 1 9 7 3

I N T E R N A T I O N A L A T O M I C E N E R G Y A G E N C Y V I E N N A , 1 9 7 4

CONTENTS

N E U T R O N SOURCES, S P E C T R O M E T R Y A N D D O S I M E T R Y (Sessions 1 and I I )

N e u t r o n sources avai lable for r a d i o b i o l o g i c a l r e s e a r c h ( I A E A - S M - 1 7 9 / 3 5 , Inv i ted paper) 3 G. B u r g e r , J . J . B r o e r s e

Fas t neutron spectra i n standard seed i r r a d i a t i o n f a c i l i t i e s f o r T R I G A r e a c t o r s ( I A E A - S M - 1 7 9 / 2 0 ) 21 J . K r a j n i k , M . N a j z e r , M . P a u k o , J . R a n t D i s c u s s i o n 28

Resonance energy neutron beams f r o m r e a c t o r s f or rad iob io l ogy studies ( I A E A - S M - 1 7 9 / 9 ) 31 K . R . B r y a n t , S. R. B u l l D i s c u s s i o n 47

Use of a c y c l o t r o n as a fast neutron source i n plant breeding and genetics ( I A E A - S M - 1 7 9 / 1 6 ) 49 J . H u c z k o w s k i , E. G a z e k , H . W o j c i e c h o w s k i D i s c u s s i o n 56

C a r a c t e r i s a t i o n d 'un faisceau de neutrons pour des etudes radiob io log iques ( I A E A - S M - 1 7 9 / 2 1 ) 59 Nico le P a r m e n t i e r , A . B r i d i e r , M . C h e m t o b , P. F a c h e , R. L a g u e r r e , V . D . N g u y e n , A . R i c o u r t , R. S o u l i e , C. V e a u , A . D e b a u c h e , P, L e l e u x , J . P. M e u l d e r s , C. P i r a r t , A . W a m b e r s i e , M . W i n a n t D i s c u s s i o n 80

I n t e r m e d i a t e neutron- induced slow r e c o i l p a r t i c l e s applied i n rad iob io logy ( I A E A - S M - 1 7 9 / 4 ) 83 R. O. B e r g m a n Dis cuss i on 95

The b i o l o g i c a l effect of fast neutrons not i n c h a r g e d - p a r t i c l e e q u i l i b r i u m ( I A E A - S M - 1 7 9 / 5 ) 97 D . K . B e w l e y , N . J . M c N a l l y , B . C . P a g e D i s c u s s i o n 106

Neutron d o s i m e t r y and s p e c t r o m e t r y methods used i n c e l l m u t a t i o n exper iments ( I A E A - S M - 1 7 9 / 1 4 ) 109 P . D . H o l t , S .J . B o o t D i s c u s s i o n 118

Energy depos i t ion on a m i c r o s c o p i c scale , re levant to the b i o l o g i c a l effects of fast neutrons : Report on the F o u r t h E U R A T O M Symposium on M i c r o d o s i m e t r y ( I A E A - S M - 1 7 9 / 3 7 , I n v i t e d paper) 119 J . B o ο ζ

N E U T R O N E F F E C T S A T S U B C E L L U L A R AND C E L L U L A R L E V E L S (Sessions I I I and I V )

Observations re l evant to the mechanism of R B E effects i n the k i l l i n g of ce l l s ( I A E A - S M - 1 7 9 / 2 ) 133 T i k v a h A l p e r Discuss ion 14.7

A t h e o r e t i c a l analysis of r a d i a t i o n sens i t i v i ty i n c e l l s f o l l owing neutron i r r a d i a t i o n ( I A E A - S M - 1 7 9 / 1 8 ) 151 H. P. L e e n h o u t s , K . H . C h a d w i c k Discuss ion 161

D i f f e r e n t i a l effects of e lectrons and neutrons on c e l l k i l l i n g and ab i l i ty of D N A - m e m b r a n e complex to synthesize D N A i n E_. c o l i B / r and B s - 1 ( I A E A - S M - 1 7 9 / 1 1 ) 165 W . A . C r a m p , P . E . B r y a n t Discuss ion 175

Recovery and r e p a i r i n yeast ce l ls a f ter i r r a d i a t i o n w i t h densely i o n i z i n g p a r t i c l e s ( I A E A - S M - 1 7 9 / 2 3 ) 177 W . E . P o h l i t , M . S c h ä f e r Discuss ion 182

The influence of e x p e r i m e n t a l end po in ts , dose, dose r a t e , neutron energy, n i t rogen i ons , hypoxia , c h r o m o s o m e vo lume and ploidy l e v e l on RBE i n Tradescant ia s tamen h a i r s and pol len ( I A E A - S M - 1 7 9 / 3 1 ) 185 A . G. U n d e r b r i n k , A . H. S p a r r o w Discuss ion 213

R B E of the 1 0 B ( n , o ' ) 7 L i capture reac t i on of t h e r m a l neutrons inside the green alga C h l o r e l l a pyrenoidosa ( I A E A - S M - 1 7 9 / 1 7 ) . . 215 H . - U . K n o o p , Μ. P a s c h k e

Ef f i cac i te r e l a t i v e de d i v e r s rayonnements m i x t e s g a m m a , neutrons pour l ' i n d u c t i o n i n v i t r o d 'anomal ies c h r o m o -somiques dans les lymphocytes humains ( I A E A - S M - 1 7 9 / 7 ) 221 M a r i e - T h e r e s e B i o l a , R. L e G o , G. V a c c a , Ginette D u c a t e ζ , Janine D a c h e r , Μ i r e i l l e B o u r g u i g n o n Discuss ion 2 36

The effect of d i f f e r i n g neutron energies on mutagenes is i n c u l t u r e d Chinese ham ster ce l l s ( I A E A - S M - 1 7 9 / 1 5 ) 237 M a r g a r e t R i c h o l d , P . D . H o l t Discuss ion 243

L e t h a l effects of 14 -MeV fast neutrons of f r o g eggs and cu l tures m a m m a l i a n ce l l s ( I A E A - S M - 1 7 9 / 3 0 ) 245 K. T a k e s h i t a , S. S a w a d a Discuss ion 255

Modi f i ca t i on of neutron effects upon ce l l s by r e p a i r , and by phys i ca l and c h e m i c a l means ( I A E A - S M - 1 7 9 / 3 9 , I n v i t e d p a p e r ) . . 257 R . J . B e r r y Discuss ion 2 70

N E U T R O N E F F E C T S ON M U L T I C E L L U L A R SYSTEMS (Sessions V and VI)

^ ' R e s p o n s e of mouse l e u k a e m i c ce l l s after i r r a d i a t i o n w i t h X - r a y s and fast neutrons ( I A E A - S M - 1 7 9 / 8 ) 275 J . J . B r o e r s e , P i a M . v a n O o s t e r o m D i s c u s s i o n 281

Ef fets hemato log iques compares des rayons γ du cobalt et des rayonnements e m i s au cours d'une excurs ion c r i t i q u e ( I n s t a l l a t i o n CRAC) ( I A E A - S M - 1 7 9 / 1 9 ) 283 J . L . Μ a l a r b e t , R. L e G o , J . P r u d h o m m e , Lucet te G e n e s t D i s c u s s i o n 297

Ef fects of i n t r a c a v i t a r y i r r a d i a t i o n by c a l i f o r n i u m - 2 5 2 ( I A E A - S M - 1 7 9 / 2 9 ) 299 M . F . S u l l i v a n , J . L . B e a m e r , T . D . M a h o n e y D i s c u s s i o n 310

Regeneration of g a s t r i c g landular e p i t h e l i u m i n adult m i c e a f ter exposure to 1 -MeV fast neutrons ( I A E A - S M - 1 7 9 / 1 2 ) 311 J . A . G. D a v i d s D i s cuss i on 323

S u r v i v a l of s p e r m a t o g o n i a l s t e m ce l l s i n the mouse after exposure to 1 . 0 - M e V fast neutrons ( I A E A - S M - 1 7 9 / 2 5 ) 325 Anke L . de R u i t e r - Β o o t s m a , Μ. F . K r a m e r , D . G. de R o o i j , J . A . G . D a v i d s D i s c u s s i o n 333

Use of the wound response to compare r e c o v e r y of neutron or X - i r r a d i a t e d i n t e r m i t o t i c ( G 0 ) c e l l s of the r a t lens e p i th e l iu m ( I A E A - S M - 1 7 9 / 2 4 ) 335 E. F . R i l e y , B . T . A u s t i n , A l i c e L . L i n d g r e n , R. C. M i l l e r D i s c u s s i o n 346

RBE values of 4 0 0 - M e V and 1 4 - M e V neutrons us ing var i ous b i o l og i ca l ef fects ( I A E A - S M - 1 7 9 / 6 ) 349 M a r i l e n a B i a η c h i , J . B a a r l i , A . H . S u l l i v a n , M . D i P a o l a , M . Q u i η t i l i a n i D i s c u s s i o n 356

V" Late effects of n e u t r o n o r gamma i r r a d i a t i o n i n mice ( I A E A - S M - 1 7 9 / 1 ) 359 E. J . A i n s w o r t h , R. J . M . F r y , D . G r a h n , F . S. W i l l i a m s o n , P a t r i c i a C. B r e n n a n , S. P h y l l i s S t e a m e r , Α. V . C a r r a no , J . H . R u s t

_ i D i s cuss i on 378 Τ Neutron - induced m a m m a r y neoplasms (1AEA-SM-179 /32 ) 381

Η. H . V o g e l , J r . D i s c u s s i o n 388

Rat m a m m a r y carc inogenes i s f o l l o w i n g neutron or X - r a d i a t i o n ( I A E A - S M - 1 7 9 / 2 6 ) 391 C . J . S h e i l a b a r ge r , R . D . B r o w n , A . R. R a o , J . P. S h a n l e y , V . P. B o n d , A . M . K e l l e r e r , H . H . R o s s i , L . J . G o o d m a n , R. E. M i l l s D i s cuss i on 400

USE OF NEUTRONS I N P L A N T B R E E D I N G (Session V I I )

Re la t i on between m u t a t i o n y i e l d and c e l l l e t h a l i t y o v e r a wide range of X - r a y and f i s s i o n neutron doses i n m a i z e ( I A E A - S M - 1 7 9 / 2 8 ) 405 H . H . S m i t h , H . H . R o s s i , A . M . K e l l e r e r D i s c u s s i o n 415

The ef fectiveness of f i s s i on neutrons , 14. 7 - M e V monoenerget i c neutrons and 6 0 C o gamma r a d i a t i o n on seed l ing g r o w t h r e d u c t i o n and induct ion of c h l o r o p h y l l - d e f i c i e n t m u t a t i o n s i n bar ley ( I A E A - S M - 1 7 9 / 1 0 ) 417 Β. V . C o n g e r , M . J . C o n s t a n t i n D i s c u s s i o n 432

Radio s e n s i t i v i t y of plant seeds to neutrons and i t s m o d i f i c a t i o n ( I A E A - S M - 1 7 9 / 2 7 ) 433 A . R. G o p a l - A y e n g a r , N . S. R a o , Β. B . S i n g h D i s c u s s i o n 43 8

C o m p a r a i s o n des effets physiologiques et genetiques provoques par des i r r a d i a t i o n s aux neutrons et aux rayons gamma de boutures d ' o i e l l e t Dianthus caryophy l lus L . ( I A E A - S M - 1 7 9 / 2 2 ) 441 P. P e r e a u - L e r o y D i s cuss i on 449

O T H E R P R A C T I C A L I M P L I C A T I O N S OF N E U T R O N R A D I A T I O N BIOLOGY (Session V I I I )

Fundamenta l and p r a c t i c a l aspects of the a p p l i c a t i o n of fast neutrons i n c l i n i c a l rad i o therapy ( I A E A - S M - 1 7 9 / 3 ) 453 G. W. B a r e n d s e n , J . J . B r o e r s e D i s cuss i on 458

Genetic effects of neutrons i n m a m m a l s and t h e i r i m p l i c a t i o n s f o r r i s k assessment i n man ( I A E A - S M - 1 7 9 / 3 8 , I n v i t e d paper) . . . 461 A . G. S e a r l e D i s cuss i on 471

C h a i r m e n of Sessions and Secre tar ia t 473 L i s t of P a r t i c i p a n t s 475 A u t h o r Index 483

I A E A - S M - 1 7 9 / 2 6

R A T M A M M A R Y CARCINOGENESIS F O L L O W I N G NEUTRON OR X - R A D I A T I O N *

C . J . S H E L L A B A R G E R , R . D . BROWN, A . R. R A O f J . P. S H A N L E Y , V . P. BOND Brookhaven Nat ional Laboratory, Upton

A . M . K E L L E R E R , H . H . ROSSI , L . J . G O O D M A N , R. E . M I L L S C o l l e g e of Physicians and Surgeons, C o l u m b i a University , New York , Ν. Υ . , United States of A m e r i c a

Abstract

R A T M A M M A R Y C A R C I N O G E N E S I S F O L L O W I N G N E U T R O N OR X - R A D I A T I O N . F e m a l e 61 to 6 3 - d a y - o l d S p r a g u e - D a w l e y rats were exposed o n c e to a single dose of e i ther 0 . 4 3 - M e V

neutrons o r 2 5 0 - k V X - r a y s . For neutrons 23 rats were exposed i n p l a s t i c tubes rotated around and 31 c m from a w a t e r - c o o l e d t r i t i u m i m p r e g n a t e d target bombarded w i t h 2 . 4 5 - M e V protons from a V a n de G r a a f f generator . T h e m e a n k e r m a was measured at the rat l o c a t i o n by integrat ing the response of a r a t - s i z e d homogeneous tissue e q u i v a l e n t i o n i z a t i o n c h a m b e r of m i n i m u m m a s s . T h e rat io between absorbed dose and k e r m a is under invest igat ion and i s a n t i c i p a t e d to be a p p r o x i m a t e l y 0 . 7 . A c o m p e n s a t e d G M g a m m a - r a y dos imeter i n d i ­c a t e d that the g a m m a - r a y doses were 3. 5% of the total dose. A l l rats were e x a m i n e d w e e k l y for the presence of breast tumours and these were r e m o v e d , f i x e d , s tained and v e r i f i e d h i s t o l o g i c a l l y as m a m m a r y n e o p l a s m s . At 10 months after exposure 98<7ο of the rats were a l i v e . T h e neutron k e r m a , the per c e n t of rats w i t h m a m m a r y n e o p l a s i a , and the n u m b e r of rats w e r e , r e s p e c t i v e l y : 0 . 1 2 5 rads, 8.2°}o, 182 ; 0 . 5 rads, 9.0^0, 8 9 ; 2 rads, 2 0 . 6 , 6 8 ; and 8 rads, 3 1 . 1 % , 4 5 . T h e X - r a y results w e r e : 30 R, 1.4% 9 5 ; 60 R, 2 7 . l°Io, 4 8 ; and 90 R, 3 5 . 4 % , 4 8 . A 3. O^o i n c i d e n c e was found in 167 c o n t r o l rats . A t 10 months after exposure the m a m m a r y n e o p l a s t i c response after 8 rads of neutrons corresponds a p p r o x i m a t e l y to that after 60 - 90 R of X - r a y s . S i m i l a r l y , the response after 2 rads of neutrons was i n t e r m e d i a t e between 30 and 60 R of X - r a y s and the response after 0 . 1 2 5 and 0. 5 rads of neutrons was s i m i l a r to that after 30 R of X - r a y s . T h i s demonstrates that the RBE for 0 . 4 3 - M e V neutrons is m u c h lower at high doses than at low doses. D e t e r m i n a t i o n of the c o n f i d e n c e l i m i t s for the d o s e - R B E dependence and d o s e - i n c i d e n c e r e l a t i o n s h i p w i l l be d e t e r m i n e d as a d d i t i ­o n a l data are c o l l e c t e d .

INTRODUCTION

Both the United Nations (1) and the United States National Academy of Sciences - National Research Council (2) have reviewed recently the relative biological effectiveness (RBE) of neutrons for the induction of mammary neoplasia in the female ra t . The UN report, page 387, states "Mammary tumors in Sprague-Dawley rats have also been studied extensively. These data are more d i f f i c u l t to interpret since most of the x- and gamma-ray data come from one laboratory and the data for fission neutrons from another. In either case the RBE approaches a value of one between 350 and 400 rads of χ rays and increases with decreasing dose." The NA.S-NRC report,

* R e s e a r c h c a r r i e d out at Brookhaven N a t i o n a l Laboratory under c o n t r a c t w i t h the U S A t o m i c Energy C o m m i s s i o n . Also based on work performed under c o n t r a c t A T - ( l l - l ) - 3 2 4 3 for U S A E C and U S P H S r e s e a r c h contract No. C A - 1 2 5 3 6 from N C I .

391

392 S H E L L A B A R G E R et a l .

page 141, states, "Experimental studies with rats have shown an RBE of approximately 2 for fast neutrons, for the induction of mammary gland tumors following exposure at relatively high doses. The RBE value for exposure at lower doses i s higher, approximately 10 to 20."

Because the relationship between dose and RBE i s not well known for radiation-induced mammary neoplasia of the r a t , a study was begun to compare the incidence of mammary neoplasia, in a single experiment, over the entire life-span of rats irradiated with either x-rays or neutrons. The purpose of this communication i s to present interim results up to 14 months after exposure.

MATERIALS AND METHODS

Weanling, female, Sprague-Dawley rats were purchased from Sprague-Dawley Inc., Madison, Wisconsin and maintained on commercial rat chow and water ad libJLtum in temperature controlled (72° + 3° F) and humidity con­trolled (55% + 10%) animal rooms under conditions of 12 hours of floures-cent light per day. When they were 61-63 days old they were exposed to either neutron-radiation, x-radiation or sham-irradiation. Five rats were then assigned to a single cage on a random basis independently of treat­ment. Each rat has be-en examined frequently, and when breast tumors were noted by palpation, the tumors have been removed surgically under ether anesthesia and the rat then returned to the experiment. No animal has been ki l l e d unless death appeared imminent. Individual records are kept for each r a t , and the time of appearance and the anatomical location of the breast tumors are recorded. Those mammary neoplasms which occurred at dif­ferent locations in the same animal were considered to be separate neo­plasms. I f mammary neoplasms were noted at closely related anatomical s i t e s , they were considered to be different neoplasms only i f they were of a different pathologic type or, i f the same pathologic type, i f at least six months had elasped between removal of the f i r s t neoplasm and detection of the second neoplasm. The mammary neoplasms were given a pathologic cla s s i f i c a t i o n of either adenocarcinoma, or a combined cla s s i f i c a t i o n of adenofibroma-fibroadenoma, according to the c r i t e r i a published previously ( 3 ) .

Neutron irradiations were accomplished as follows. Twenty-three rats were rotated in a vertical carrousel or Ferris wheel 30 cm from a water-cooled tritium target. Each rat was placed in a 5 cm diameter cylinder 17.5 cm long, made of 0·3 cm thick lucite and weighted so that the rat remained in i t s normal, horizontal, standing position. A Van de Graaff generator was operated to bombard the target with 2.43 MeV protons to pro­duce neutrons with an energy of 0.43 MeV at the mid-point of each cylinder. Calculations of kinematic conditions indicated an energy spread over the extent of the rat of +287. to -14% of the mid-point energy. A l l rats were reversed in their cylinders at mid-dose to reduce nonuniformities in the energy and absorbed dose distributions. The mean tissue kerma in free a i r was measured at the rat locations by integrating the response of a rat-sized, homogeneous tissue equivalent ionization chamber of minimal mass placed in the 24th cylinder. A compensated Geiger-Muller dosimeter in­dicated gamma-ray kefmas 3,5% of the total kermas. Depth-dose measurements have been made to determine the ratio of absorbed dose to neutron-kerma. For this purpose a \ inch spherical tissue equivalent multiplication chamber was inserted* into a nylon rat phantom at 4 radial depths. Subject to additional calculations, the absorbed dose to neutron-kerma ratio varies between 0.75 and 0.88 ovör the entire presumed volume of the mammary tissue of the r a t . We have chosen, for the purpose of this report, to use a fac­tor of 0.8 to convert neutron-kerma to rad. The neutron kerma-rate was approximately 4 rads per hour. The rats were exposed to total kermas of 0.125, 0.50, 2.0, of B.O which i s considered to be the equivalent of 0.10, 0.40, 1.6, or 6.4 rads of. absorbed dose respectively.

Table I . MORTALITY AND MAMMARY NEOPLASTIC RESULTS ~~~~ Months

Radiation Measure 1 2 3 4 5 6 7 8 9 10 11 12 13 14 % 0.43 MeV Number of rats 182 182 182 180 180 180 180 179 178 177 175 173 172 169 92.9 Neutrons Rats with tumors 2 2 6 6 8 15 16 24 28 28 17.6 0.1 rad A l l tumors 3 3 7 7 9 16 18 26 32 36

Adenocarcinomas 1 1 1 1 1 2 2 3 0.43 MeV Number of rats 89 89 89 89 89 89 89 88 88 88 86 85 85 85 95.5 Neutrons Rats with tumors 1 2 2 2 3 6 6 8 9 10 10 16 18.0 0.4 rad A l l tumors 1 2 2 2 3 7 7 9 11 11 11 17

Adenocarcinomas 1 2 2 2 2 2 2 3 3 3 3 4 0.43 MeV Number of rats 68 68 68 68 68 68 68 68 68 68 68 68 68 67 98.5 Neutrons Rats with tumors 1 4 5 7 7 11 14 14 16 20 21 31.0 1.6 rad A l l tumors 1 4 5 7 7 11 14 14 19 24 25

Adenocarcinomas 1 2 2 2 2 2 2 2 2 2 2 0.43 MeV Number of rats 45 45 45 45 45 44 42 42 42 42 42 39 38 36 80.0 Neutrons Rats with tumors 1 1 1 3 4 5 9 12 14 18 19 22 26 57.8 6.4 rad A l l tumors 1 1 1 3 4 5 12 15 18 26 28 31 38

Adenocarcinomas 1 1 1 1 2 3 5 6 6 8 8 8 8 None Number of rats 167 167 167 167 167 167 167 167 167 165 163 163 162 159 95.2

Rats with tumors 2 2 5 8 13 17 20 12.0 A l l tumors 2 2 5 8 14 18 21 Adenocarcinomas 0

250 kVp Number of rats 95 95 95 95 95 95 95 95 94 93 92 92 91 89 93.7 x-rays Rats with tumors 1 1 2 3 4 5 7 7 8 13 17 18 18.9 28 rad A l l tumors 1 1 2 3 4 5 7 8 10 15 20 23

Adenocarc inomas 1 1 1 1 1 1 2 2 2 2 2 2 250 kVp Number of rats 48 48 48 48 48 48 48 48 48 48 47 47 47 46 95.8 x-rays Rats with tumors 1 3 5 6 7 10 12 13 15 20 23 24 50.0 56 rad A l l tumors 1 3 5 6 7 10 12 14 16 24 27 30

Ad enocarc inomas 1 3 4 4 4 4 4 4 4 5 5 5 250 kVp Number of rats 48 48 48 47 46 46 46 45 45 45 45 45 45 42 86.7 x-rays Rats with tumors 3 6 7 8 12 17 20 20 22 22 45.8 85 rad A l l tumors 3 6 8 9 14 19 25 27 31 33

Adenocarcinomas 1 1 2 2 3 3 3 3 4 4

394 S H E L L A B A R G E R et a l .

X-ray exposures were accomplished by using a conventional, therapy x-ray machine. Exposure conditions were: target to specimen distance=100 cm; 0.5 mm Cu + 1.0 mm Al; maximum tissue-like backseatter; 250 kVp; dose rate=30 R per minute measured with a 100 R Victoreen chamber at the dorsal-ventral midpoint of the r a t s . The rats were exposed to either 30, 60, or 90 R. These values were converted, by multiplying by a factor of 0.94 to 28, 56 or 85 rads of absorbed dose respectively.

RESULTS and DISCUSSION

There was l i t t l e mortality during the 14 month follow-up period except in the groups that received either 6„4 rads of neutrons or 85 rads of x-rays where the mortality reached 20,0% and 13.3% respectively (Table I ) . The number of rats with mammary neoplasia, the number of mammary neoplasms, and the number of mammary adenocarcinomas is given for each group, month by month, as well as the percent incidence of rats with mammary neoplasia at 14 months in Table I . The percent of rats with mammary neoplasia at the end of 1 thru 14 months has been plotted in F i g . 1. Similarily, tumors per r a t , that i s , the number of mammary neoplasms divided by the i n i t i a l or starting number of r a t s , has been plotted in Fig, 2. Inspection of these data indicate that a dose of 6.4 neutron rads was followed by a mammary neoplastic response that was similar to the response following 85 rads of x-rays. However, 0,1 or o,4 rads of neutrons was followed by a mammary neoplastic response that was similar to the response following 28 rads of x-rays. Thus, i t i s suggested that 0.43 MeV neutrons show a much higher RBE at lower doses that at higher doses.

The curves in Figs. 1 and 2 are not corrected for mortality. This leads to no serious errors because mortality during the f i r s t 14 months has been insignificant in a l l but two groups. As the experiment continues, mortality w i l l , however, be more extensive, and accurate analysis as well as rigorous definition of the quantities of interest are therefore essen­t i a l .

The mean number of tumors per animal at time t after irradiation w i l l in the following be designated by R ( t ) , The probability of an animal to have at least one tumor, i.e., the cumulative incidence, i s related to R(t) by the equation

I ( t ) = 1 - e -R^ (1

>

From this equation i t follows that the cumulative incidence, I ( t ) , and the mean number, R ( t ) , of tumors per animal have nearly the same value whenever the two quantities are small as compared to 1; this can also be seen from a comparison of Figs. 1 and 2, A third quantity of interest i s the tumor rate r ( t ) , i.e., the probability for the development of a tumor per unit time and per animal. The tumor rate i s the time derivative of the mean number of tumors per animal:

r ( t ) = Lmi r w

dt (2)

and accordingly R(t) i s the time integral or the tumor rate: t

R(t) = 1 r ( T ) dT (3)

I A E A - S M - 1 7 9 / 2 6 395

R(t) can therefore be estimated from the observed incidence in a finite sample of irradiated animals as:

η

ι=1 x

ι

where t. are the times of incidence of the η observed tumors, and N(t.) are the numbers of animals s t i l l under observation at these times. A

L

more detailed discussion of the estimation of I ( t ) and R(t) can be found in references 4-6.

The cumulative incidence, I ( t ) , i s determined by the resulting value of R(t) according to 1. A condition for the validity of this equation i s that a l l scored tumors are primary tumors, that i s , that the occurrence of a tumor neither increases nor decreases the probability of additional tumors being found in the same r a t . In order to avoid this assumption

396 S H E L L A B A R G E R et a l .

concerning the S t a t i s t i c a l independence of multiple tumors, one can derive the quantities I ( t ) and R(t) by only scoring f i r s t tumors and by excluding a l l animals with a second tumor from further consideration. Both types of analyses have been performed and no systematic differences in the resulting values have been found. The assumption that one deals with independent primary tumors is therefore v a l i d , and the complete analysis based on a l l observed tumors i s preferable because i t leads to slightly smaller statis­t i c a l inaccuracies.

The quantity R(t) i s plotted in Figs. 3 and 4 for the different ex­perimental groups. A comparison with F i g . 2 shows that the results are

I A E A - S M - 1 7 9 / 2 6 397

nearly identical except at the highest doses. The logarithmic scale has been chosen in order to obtain a clearer representation of the tumor fre­quency at small doses. One should note that the abscissa scale in f i g s . 3 and 4 i s twice as wide as the ordinate scale. A straight line of slope 1 corresponds therefore to the equation:

R(t) = k t2

(5)

and accordingly to a linear increase of the tumor rate with time after irradiation:

r ( t ) = 2k t (6)

I t appears that the results are not inconsistent with such a dependence, but an exact analysis w i l l have to be based on more complete results as the observations continue.

Standard deviations are not given in Figs. 3 and 4, but approximate values of the standard deviations of R(t) are obtained by the equation:

σ = R(t)/A("t) (7)

where n(t) i s the cumulative number of tumors at time t as given in Table I . There are various ways to deduce dose-effect relations from the ob­

served time dependence of tumor incidence. An obvious way i s to plot the cumulative incidence or the mean number of tumors per rat at a given time as a function of dose. Such a procedure has, however, the disadvantage that i t u t i l i z e s only part of the experimental information, and i t i s therefore sensitive to s t a t i s t i c a l fluctuations. One can avoid this by plotting the time integral of R(t) as a function of dose. In doing this one effectively averages over the whole observation period. The procedure is also j u s t i f i e d because tumors are then weighed according to their time of incidence. The integral:

t

ct) = I P(t) = J R (T) dT (8) ο

can be considered as an "effect period", namely the mean number of tumors per rat multiplied by the number of days the rat i s bearing these tumors. The "effect period" minus i t s control value, in units of "tumor-days" i s plotted in Fig. 5 as a function of neutron and x-ray dose. One finds that, in agreement with an earlier analysis (7) the dose-effect relation for neutrons has a slope considerably less than 1, that i s , the effect of neutron irradiation i s proportional to a power of absorbed dose less than 1. The dose-effect curve for x-rays i s considerably steeper, and i t ap­pears that the RBE of neutrons approaches values near 100 at low doses while at 6.4 rad of neutrons i t i s closer to 10.

A separate s t a t i s t i c a l assesment of the dose dependence of the RBE of neutrons has been performed by comparing the effect of each x-ray dose with that of each neutron dose. These comparisons have been based on a s t a t i s t i c a l evaluation of the f u l l time dependence of tumor incidence in the groups of animals which are compared. Details of this approach have been described earlier (8, 9 ) . One finding of particular interest i s that

398 S H E L L A B A R G E R et a l .

200

k 5 0

ο £

η Q

20

π 1 « Γ

NEUTRONS / /

/ /

X-RATS

Ol I RA D 10 100

F I G . 5. E f f e c t - p e r i o d , i n units of t u m o u r - d a y s , c a l c u l a t e d as the t i m e i n t e g r a l of R(t) , m i n u s its c o n t r o l v a l u e (see text) as a funct ion of neutron or X - r a y dose; l o g - l o g plot . T h e observations e x t e n d to 400 days after i r r a d i a t i o n . T h e broken l i n e s h a v e no m a t h e m a t i c a l s i g n i f i c a n c e and only serve to i n d i c a t e the trend of the d a t a .

1000

ÜJ CD

100

RAD NEUTRON F I G . 6. T h e d e p e n d e n c e of RBE on neutron dose. V e r t i c a l bars i n d i c a t e the ranges of RBE that are e x c l u d e d on a l e v e l of s t a t i s t i c a l s i g n i f i c a n c e e x e e d i n g 95Pjo, wedges i n d i c a t e n o n - s i g n i f i c a n t d i f ferences . T h e s t a t i s t i ­c a l a n a l y s i s [ 8 , 9 ] is based on the current data for neutrons and X - r a y s and on e a r l i e r results for X - r a y s [ 1 0 ] .

the effect of 0 .1 rad of neutrons i s larger on a 95% level of s t a t i s t i c a l significance than the control levels and that i t also exceeds on the same level of s t a t i s t i c a l significance the earlier observed effect (10) of 15 rad of gamma rays. Because only a limited number of x-ray doses i s applied in the present experiments, the earlier data obtained with gamma rays are also utilized in the s t a t i s t i c a l analysis. The results are represented in Fig. 6. In this representation the vertical bars cover those ranges of RBE of neutrons which are excluded on a level of s t a t i s t i c a l significance ex­ceeding 95%c The solid curve of slope - 0 . 5 i s the best estimate of the dependence of neutron RBE on dose. This relation i s in agreement with the dose-RBE dependence observed in a large number of higher organisms ( 1 1 ) . As in the case of lens opacification by 0 .43 MeV neutrons (8) and in the induction of yg2 - mutations by fission neutrons (12) the RBE at low doses approaches values exceeding 100.

I A E A - S M - 1 7 9 / 2 6 399

The observation that the effect of neutron irradiation increases with a power of absorbed dose which i s less than 1 has been discussed earlier ( 7 ) , and i t has been concluded from this observation that the tumor induc­tion can not be solely determined by lesions in individual c e l l s . I t i s necessary to postulate interaction between a number of affected c e l l s . I t is significant that in this system the same dose-RBE relation i s found which has been postulated on general biophysical grounds for a l l radiation effects on higher organisms. The finding i s also of interest because the induction of mammary tumors in the Sprague-Dawley rat presents a special case insomuch as the spontaneous incidence becomes very large as the age of the rats increases. The mammary neoplastic response to neutrons and to x-rays in Sprague-Dawley rats can therefore not be used to predict the radiation induction of other tumors in other systems. However, the cur­rent observations confirm that even in a complicated system the dose-RBE dependence agrees with the dependence predicted by the theory of dual radiation action (11).

The data here reported are in general agreement with the reports of Vogel (13-15) although there are some important differences in experimental techniques. The current data are based on 0.43 MeV neutrons, which would be expected to be more efficient biologically than the fission neutrons used by Vogel. The current data come from animals that had their tumors removed while Vogel allows the tumor to remain in place. Tumor removal should allow for more tumors to be reported because a tumor left in place may "overgrow" subsequently arising tumors and may tend to k i l l the tumor bearing rat sooner than i f the tumor were removed. The present experiment used rats that were born on the same day, were from the same shipment, and were housed together. Vogel, on the other hand, began his studies with rather large neutron doses, and over a period of years, added additional neutron doses and x-ray controls. Since J u l l (16) has shown that mammary tumor incidence may be changed signif­icantly by housing conditions, we tend to place more confidence in data col­lected from a single experiment than from experiments that were done at dif­ferent times and under different housing conditions.

SUMMARY

Sprague-Dawley female r a t s , 61-63 days of age, were given a single dose of either 0.10, 0.40, 1,6, or 6.4 rads of 0.43 MeV neutrons, or 28, 56, or 85 rads of 250 kVp x-rays, or no irradiation in a single experiment. The interim results, either in terms of percent of rats with mammary neoplasia or mean number of mammary neoplasms per ra t , at the end of 14 months after irradiation, indicate that a dose of 0,10-0.40 rads of neutrons was followed by a mammary neoplastic response much like that following a dose of 28 rads of x-rays. The mammary neoplastic response to either a dose of 6,4 rads of neutrons or 56-85 rads of x-rays was similar. Thus the RBE for neutrons is much larger at low doses of neutrons than at high doses.

REFERENCES

(1) IONIZING RADIATION; LEVELS and EFFECTS, A report of the United Nations Scientific Committee on the Effects of Atomic Radiation to the General Assembly, with annexes. Volume I I . Effects. (1972).

(2) THE EFFECTS on POPULATIONS of EXPOSURE to LOW LEVELS of IONIZING RADIATION. Division of Medical Sciences, National Academy of Sciences, National Research Council, Washington, D.C.,(1972).

(3) SHELLABARGER, C.J., CRONKITE, E.P., BOND, V.P., and LIPPINCOTT, S.W. The occurrence of mammary tumors in the rat after sublethal whole-body irradiation. Rad. Res. 6 (1957) 501.

400 S H E L L A B A R G E R et a l .

(4) KAPLAN, E.L., and MEIR, P, Nonparametric estimation from incomplete observations. Amer. S t a t i s t i c a l Assoc. J . 53 (1958) 485.

(5) ROSENBLATT, L.S., HETHERINGTON, N.H., GOLDMAN, Μ., and BUS TAD, L.K. Evaluation of tumor incidence following exposure to internal emitters by application of the logistic dose-response surface. Health physics 21 (1971) 869.

(6) KELLERER, A.M. Quantification of radiation induced carcinogenesis. Annual Report on Research Project, USAEC-C00-3243-2(1973).

(7) ROSSI, H.H., and KELLERER, A.M. Radiation carcinogenesis at low doses. Science 175 (1972) 200.

(8) BATEMAN, J.L., ROSSI, H.H., KELLERER, A.M., ROBINSON, C.V., and BOND, V.P. Dose-dependence of fast neutron RBE for lens opacifica­tion in mice. Rad. Res. 51 (1972) 381.

(9) KELLERER, A.M., and BRENOT, J . Nonparametric determination of modi­fying factors in radiation action. Rad. Res. (1973) in press.

(10) SHELLABARGER, C.J., BOND, V.P., CRONKITE, E.P., and APONTE, G.E. Relationship of dose of total-body 60co radiation to incidence of mammary neoplasia in female r a t s . Radiation-Induced Cancer, IAEA-SM-118/9 (1969).

(11) KELLERER, A.M., and ROSSI, H.H. The theory of dual radiation action. Curr. Topics Radiat. Res. 8 (1972) 85.

(12) SMITH, H.H., ROSSI, H.H., and KELLERER, A.M. Relation between mu­tation yield and c e l l lethality over a wide range of x-ray and fission

neutron doses in maize. IAEA/SM-179/28, this symposium.

(13) VOGEL, Η.Η.,Jr. Mammary gland neoplasms after fission neutron i r ­radiation. Nature 222 (1969) 1279.

(14) VOGEL, H.H., J r . , and ZALDIVAR, R. Experimental mammary neoplasms; a comparison of effectiveness between neutrons, x-and gamma-radia­tion. AEC-tr-7387, ORNL (1969) 207.

(15) VOGEL, H.H., J r . , and ZALDIVAR, R. Neutron-induced mammary neo­plasms in the r a t . Can. Res. 32 (1972) 933.

(16) JULL, J.W. The effect of infection, hormonal environment and genetic constitution of mammary tumor induction in rats by 7,12-dimethylbenz-anthracene. Can. Res. 26. (1966) 2367.

D I S C U S S I O N

V . K O F R A N E K . Have you any r a d i o b i o l o g i c a l l y convincing explana­t i o n f o r the wide ly discussed be l i e f that the incidence of m a m m a r y neoplasms increases w i t h i n c r e a s i n g dose?

C. J . S H E L L A B A R G E R . : I have ne i ther the confidence nor the i n c l i n a t i o n to defend the K e l l e r er and Ross i t h e o r y of dual r a d i a t i o n act ion, RBE and the p r i m a r y mechan ism of r a d i a t i o n act ion . I w i l l leave that to K e l l e r e r and Ross i ; but I t h i n k that t h e i r hypotheses do apply t o r a t m a m m a r y carc inoge ­nesis due t o l o w - a n d h i g h - L E T r a d i a t i o n s .

I A E A - S M - 1 7 9 / 2 6 401

L o l a S. K E L L Y : Would you care to say something about the p o s s i b i l i t y of a v i r u s being present i n the Sprangue-Dawley and Long Evans r a t s and absent i n the case of the r a t s that do not get the t u m o u r ? M u l t i p l e c e l l i n t e r ­act ions m i g h t be due t o v i r u s in fect ion or re lease .

C. J . S H E L L A B A R G E R : I know of no r e a l evidence that a v i r u s p a r t i c i ­pates in the development of a r a t breast t u m o u r . However , a negative f ind ing does not prove that a v i r u s does not p a r t i c i p a t e i n m a m m a r y carc inogenesis .

G. W. BARENDSEN: I f one wishes t o i n t e r p r e t y o u r data i n t e r m s of a r a d i o b i o l o g i c a l m e c h a n i s m , i t w i l l be of in te res t t o note that the dose d i s t r i ­but ion on a m i c r o d o s i m e t r i c scale, w i t h secondary short h i g h - L E T t r a c k s of protons f r o m neutrons and less densely i on i z ing e lectrons f r o m X - r a y s , may account f o r the high R B E . The p r o t o n t r a c k s are much m o r e l i k e l y to damage two adjacent ce l l s than the e lec t rons , so i f i n t e r a c t i o n between two damaged ce l ls is r e q u i r e d f o r t u m o u r induct ion , protons may be expected to be much m o r e ef fect ive because of t h e i r m i c r o d o s i m e t r i c p r o p e r t i e s .

That i n t e r a c t i o n between two damaged cel ls is r e q u i r e d was also i n d i ­cated by exper iments wh i ch I c a r r i e d out w i t h D r . K l e i n i n R i j s w i j k , when mal ignant t r a n s f o r m a t i o n was studied i n c u l t u r e d ce l ls d e r i v e d f r o m a h a m s t e r embryo : The addi t ion of heav i ly i r r a d i a t e d ' feeder ' ce l l s to the ce l ls to be t r a n s f o r m e d increased the frequency of t r a n s f o r m a t i o n cons ider ­ably. Th i s indicates that some type of i n t e r a c t i o n enhances t r a n s f o r m a t i o n .

A . G. S E A R L E : I n connection w i t h the quest ion whether m o r e t h a n one c e l l is involved i n t u m o u r induct ion , I should l i k e to point out that t h e r e is now s t rong evidence that c e r t a i n spontaneous t u m o u r s are der ived f r o m a single c e l l in females heterozygous for p a r t i c u l a r s e x - l i n k e d m a r k e r s , such as glucose 6-phosphate def ic iency. I wonder i f s i m i l a r w o r k has been done on induced t u m o u r s .

C . J . S H E L L A B A R G E R : As f a r as I know, w o r k along these l ines has not been c a r r i e d out on r a t breast t u m o u r s . I t would be a w o r t h - w h i l e approach.

Η. H. V O G E L , J r . : Would you care to comment on the r e l a t i o n s h i p between low doses of neutrons and induced m a m m a r y t u m o u r s i n the female Sprague-Dawley ra t ? Do you t h i n k t h e r e could be a l i n e a r dose-ef fect r e l a ­t i o n at v e r y low doses?

C . J . S H E L L A B A R G E R : The dose-effect r e l a t i o n s h i p could w e l l be l i n e a r , of course , but our own data are not good enough to es tab l i sh that w i t h any degree of s t a t i s t i c a l confidence. M o r e o v e r , since the 1 e f fect ' changes w i t h t i m e a f ter i r r a d i a t i o n , one must be care fu l t o specify the t i m e i n r e l a t i o n to the dose-ef fect curve .