ML13220A065

LTR-13-0660 - Edward Calabrese, University of Massachusetts Email Two Papers Entitled Origin of the Linearity No Threshold Dose-Response Concept and How the Us National Academy of Sciences Misled the World Community on Cancer Risk Assessmen

ML13220A065
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Issue date:	08/07/2013
From:	Calabrese E Univ of Massachusetts - Amherst
To:	Macfarlane A NRC/Chairman
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Joosten, Sandy From: Edward <edwardc@schoolph.umass.edu>

Sent: Wednesday, August 07, 2013 10:41 AM To: CHAIRMAN Resource

Subject:

new papers Attachments: Archiv Toxicol Origin LNT-l.pdf; Arch. Tox. NAS-l.pdf

Dear Chairperson:

I am attaching two papers; one is on the origin of the LNT and the second deals with the documentation of how leading radiation geneticists deceived the scientific community on LNT, leading to the US National Academy of Sciences' (NAS) recommendation to adopt the LNT model for risk assessment.

These assessments may be of some interest to you.

Sincerely, Ed Calabrese 1

Arch Toxicol DOl 10.1007/s00204-013-1104-7 REVIEW ARTICLE Origin of the linearity no threshold (LNT) dose-response concept Edward J. Calabrese Received: 15 March 2013 I Accepted: II July 2013

©Springer-Verlag Berlin Heidelberg 2013 Abstract This paper identifies the origin of the linear- and recommended by national/international advisory com-ity at low-dose concept [i.e., linear no threshold (LNT)] mittees for risk assessment of ionizing radiation-induced for ionizing radiation-induced mutation. After the dis- mutational damage/cancer from the mid-1950s to the pre-covery of X-ray-induced mutations, Olson and Lewis sent. The LNT concept was later generalized to chemical (Nature 121(3052):673-674, 192H) proposed that cosmic/ carcinogen risk assessment and used by public health and terrestrial radiation-induced mutations provide the prin- regulatory agencies worldwide.

cipal mechanism for the induction of heritable traits, pro-viding the driving force for evolution. For this concept to Keywords Ionizing radiation

• Linearity

• Dose be general, a LNT dose relationship was assumed, with response

• Risk assessment

• Threshold dose response

• genetic damage proportional to the energy absorbed. Sub- Target theory

• Eugenics

• LNT sequent studies suggested a linear dose response for ioniz-ing radiation-induced mutations (Hanson and Heys in Am Nat 63(686):201-213, !929; Oliver in Science 71:44-46, Introduction l ()30), supporting the evolutionary hypothesis. Based on an evaluation of spontaneous and ionizing radiation-induced In 1956, the US National Academy of Sciences (NAS) mutation with Drosophila, Muller argued that background Committee on Biological Effects of Atomic Radiation radiation had a negligible impact on spontaneous muta- (BEAR I)/Genetics Panel issued the most far reaching tion, discrediting the ionizing radiation-based evolution- recommendation in the history of risk assessment that ary hypothesis. Nonetheless, an expanded set of mutation genomic risks associated with exposure to ionizing radi-dose-response observations provided a basis for collabo- ation should be evaluated with a linear dose-response ration between theoretical physicists (Max Delbruck and model, no longer via the threshold dose-response model Gunter Zimmer) and the radiation geneticist Nicolai that had long been the "gold" standard for medicine and Timofeeff-Ressovsky. They developed interrelated physical physiology (Calabrese 2005, 2009a, 20 l l ). The Genetics science-based genetics perspectives including a biophysical Panel members believed that there was no safe exposure model of the gene, a radiation-induced gene mutation target to ionizing radiation for reproductive cells with the muta-theory and the single-hit hypothesis of radiation-induced tion risk being increased even with a single ionization mutation, which, when integrated, provided the theoreti- (Hamblin .2007). The LNT concept was generalized in cal mechanism and mathematical basis for the LNT model. 1958 to somatic cells and cancer risk assessment by the The LNT concept became accepted by radiation geneticists National Committee for Radiation Protection and Meas-urement (NCRPM) (Whittemore 1986). Quickly thereaf-ter, other national and international advisory committees E. J. Calabrese (C8:1) and organizations adopted such judgments for ionizing Department of Public Health, Environmental Health Sciences, radiation (Calabrese 2009b ). In 1977, the Safe Drinking University ofMassachw;etts, Morrill I, N344, Amherst, MA 01003, USA Water Committee (SDWC) of the US NAS extended the e-mail: edwardc@schoolph.umass.edu linear dose-response risk assessment model of the BEAR/

Published online: 26 July 2013 ~Springer

Arch Toxicol Biological Effects of Ionizing Radiation (BEIR) commit- Given the central importance of evolution in biology tees to chemical carcinogens, a recommendation that was and underscoring the intensity of the competition to be the soon adopted and implemented by the Environmental Pro- first to demonstrate inducible heritable changes, Muller tection Agency (EPA). On a parallel track, similar LNT ( 1927) provided only an initial "discussion" of his muta-risk assessment procedures were adopted by the Food and genicity findings with no data in his now famous Science Drug Administration (FDA) in 1977 concerning animal paper that led to his Nobel Prize in 1946. This was done carcinogen drug residues. in order to secure recognition of being the first to report Despite the fact that the LNT model has been of central induction of heritable mutations by an environmental agent importance in chemical and ionizing radiation regulatory (i.e., X-rays). The supporting data were published the next risk assessment, its origin is not within the environmentaV year in a conference proceeding of very limited distribution occupational risk assessment domain. The current paper based on the World Cat database (Muller 192Xa) and also provides a novel historical assessment of the scientific ori- within the Proceedings of the National Academy of Sci-gin of the LNT. It will show that the LNT was first applied ences (PNAS) (Muller 1928b). Not only were the findings to the field of biology in 1928 to explain the occurrence of mutation significant so too was the fact that the mutation of genetic variation that would serve as the "biological rate was increased by about 150-fold at the highest dose engine" for evolution. The paper will also demonstrate how tested.

the linear dose-response model as proposed by Olson and Muller speculated that naturally occurring ionizing radi-Lewis ( 1928), which soon afterward became transformed ation might be a significant explanatory factor for genetic into a "Proportionality Rule" by Muller ( 1930), became variation and may drive the evolution process. However, mechanistically framed within the context of a single-"hit" Muller was cautious in making the mutation-evolution link hypothesis based on the target theory by Timofeeff-Resso- as the doses he had used to induce mutation were extremely vsky et a!. ( 1935) in a unique collaborative effort between high, exceeding background by about 200,000-fold, caus-leading theoretical physicists and radiation genetics. This ing sterility or mortality in a substantial proportion of the paper extends two earlier publications within Archives of fruit flies tested. In addition, the dose response was not Toxicology concerning historical foundations of the LNT linear but closer to a square root function due to a mod-concept (Calabrese, 2009b) and threshold/hormetic (Cala- est decline from linearity at the highest dose (Muller 1927, brese 2009a) models. 1928a). If the true dose response for ionizing radiation-induced gene mutation was linear at low dose, as a general condition, then it may have explanatory implications for Evolution and LNT an evolution mechanism. Consequently, he soon directed several members in his laboratory to assess the topic of Since the publication of the Origin of Species in 1859 by dose response more fully than he did in his groundbreak-Darwin and the rediscovery of the works of Mendel on ing mutation discovery. While the follow-up research by gene inheritance, there was intense interest in the biologi- Muller's group was being undertaken, Axel R. Olson and cal community to determine the cause of genetic change or the prestigious physical chemist Gilbert N. Lewis (1928) novelty that would be subject to natural selection, thereby of the University of California/Berkeley published a pro-providing an important mechanism of evolution. As noted posal on April 28, 1928, in Nature that natural radioactiv-by Patterson ( 1933). a well-known colleague of Hermann ity was likely a significant cause of mutation that could J. Muller at the University of Texas/Austin, "the important generate variability from the parent generation and affect question in biology is the problem of evolution" referring the process of evolution. These authors based this suppo-to the need to understand the mechanism of evolution at sition on a report of January 1, 1928, in PNAS by Good-the gene level. Despite the fact that the gene was more of speed and Olson on X-ray-induced heritable changes in a concept than a physical entity during the early decades of tobacco. These authors claimed that the tobacco plant stud-the twentieth century, it was widely believed that the gene ies were specially planned to facilitate a direct comparison was the basic unit of heredity and that the driving force of mutation rates between the artificial X-rays and "natu-for evolutionary change must be via the induction of herit- rally occurring radiations." Olson and Lewis ( 1928) also able genetic changes or mutations at the gene level (Mul- stated that "since the rays can only be effective when they ler l 922). This perspective provided the basis for intense are absorbed, and this produces ionizations, it seems safe to interest by numerous genetics researchers in the second assume that the various rays will produce biological effects and third decades of the twentieth century to induce altera- in proportion to the ionization which they cause" (emphasis tions in heritable traits by environmental (e.g., temperature) added), a perspective based on the emerging target theory alterations, physiological stressors (e.g., starvation), as well for radiation-induced biological effects proposed by leaders as toxic chemicals and ionizing and non-ionizing radiation. in the physics community (Glocker !927; Crowther 1924).

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Arch Toxicol Olson and Lewis ( 1928) then utilized a simple linear math- This conclusion was justified on the belief that the response ematical model to derive a mutation estimate at a selected is linear at low dose, with there being no threshold for a natural background radiation dose. With this method, they mutation response. This relationship was stated as holding estimated the number of variants (mutants) induced per true for all types of high-energy radiation (e.g., gamma, year by natural radiation. These authors concluded that "it beta, X-rays and probably ultra-violet rays). Thus, Oliver seems, therefore not altogether extravagant to assume that ( 1931) concluded that "by inference it can be added that such variations as actually occur in nature are due largely the cosmic and the terrestrial radiations also are capable of to the radioactivity of the environment." The involve- producing mutations in proportion to their power of ioniza-ment of Gilbert Lewis in this activity, while unexpected, tion." Oliver ( 1931) also extended the concept of propor-was derived from his research in the 1920s in the area of tionality to chromosomal inversions and translocations radiation physics (Coffey 2008). Furthermore, his eclectic further arguing for the support of a background radiation research activities had also drawn him toward evolution- influence. For example, Muller and Altenburg ( 1930) noted ary theory, the subject of his major presentation (i.e., Silli- that translocations are induced at a similar frequency as man Lecture) at Yale, just preceding the development of the gene mutations. Given these circumstances, Oliver ( 1931)

LNT paper in Nature (Lewis 1926). This lecture followed noted that "one would expect each of the classes of changes that of Thomas Hunt Morgan of Columbia University in considered to occur with the same frequency when the indi-1925, Muller's Ph. D. advisor and 1936 Nobel Prize recipi- viduals are subjected only to the natural conditions, if natu-ent. The perspective of Olson and Lewis ( 192r\) was also ral radiation can account for all mutations ... " Despite this independently advanced by Muller in a paper read before interpretation of environmental radiation-induced genetic the National Academy of Sciences on April 24, 1928, and changes, Oliver ( 19 31) concluded that "some other condi-published on September 14, 1928. The statement of Mul- tion must, therefore, enter in order to explain the difference ler ( 1928b) was principally conceptual, lacking the detailed in non-radiated material, between the frequency of gene formulation of Olson and Lewis (I 928). mutation and that of the other type of genetic changes."

The following year, Babcock and Collins ( l929a, b) (p. 34) tested the hypothesis of Olson and Lewis ( 1928). They Even though Muller dismissed natural radiation as pro-found a location in which the natural radiation was twice viding a quantifiably significant mutational influence to that found in their University of California/Berkeley labo- derive genetic novelty for evolutionary change, he still ratory. Using the CIB strain sex-linked recessive Dros- retained his belief in the linear dose-response relationship ophila assay, they reported an increase in mutation that (p. 238) (Muller 1930) based on the findings of Hanson corresponded in the same proportion as the difference and Heys ( 1929, I 930) and Oliver ( 1930). Even though the in background radiation, supporting the proportionality hypothesis of Olson and Lewis ( 1928) did not maintain sig-hypothesis. Detailed experimental methods including the nificant support for long within the scientific community, actual radioactivity levels were never published, although Muller and other leaders of the radiation genetics commu-such data were promised to be provided in a subsequent nity became strong advocates of the LNT model to account paper. In 1930, Hanson and Heys provided further support for genomic mutations and the occurrence of cancer.

for the hypothesis that "natural radiation may be responsi- It may seem difficult to understand in retrospect why ble for the mutations that are the grist of the natural selec- prominent scientific leaders such as Gilbert N. Lewis, Her-tion mill with the resulting evolution of new forms." Their mann J. Muller and others so quickly adopted a belief in findings were based on a study of fruit fly mutations in an linearity at low dose. In the case of Muller, he was fully abandoned carnotite (i.e., uranium) mine. Such interpreta- committed to this view after the publication of only three tions were initially supported by commentaries by various studies (Hanson and Heys 1929, 19~0; Oliver 19.i0) in authors (Lind 1929; Dixon 1929, 19.~0). which the lowest cumulative dose was roughly 285 r, In 1930 Muller and Rice University physicist, Matt- administered in an acute manner, the rough approxima-Smith, challenged this LNT evolution perspective by tion of 1,000 modem chest X-rays in 3.5 min or 5 chest reporting that natural radiation, which was of such a low- X-rays/s.

dose rate, could only account for about Ill ,300 of the gene In his rather copious publications during this period of mutations that occurred spontaneously in Drosophila mela- "belief'/concept formulation, Muller never addressed con-nogaster, assuming a linear dose response. The authors temporary publications that did not support a linear inter-concluded that other causes must explain the origin of most pretation (Patterson 1928; Weinstein ! 92X; Stadler I 9:10, mutations that spontaneously occur. Nonetheless, in his dis- I tn I). Yet, he was well aware that the lowest doses in the sertation, under the direction of Muller, Oliver ( 1931) stated Hanson and Heys ( 1929, 1930) and Oliver ( 1930) papers that cosmic and terrestrial radiations must account for some were acute studies that grossly exceeded background radia-proportion of the spontaneous mutations (see Muller I 930). tion exposure. To think within a linear dose-response term

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Arch Toxicol framework ran counter to pharmacological and chemical molecule would not result in observable effects (Delbruck toxicological experience at that time. As Zimmer ( 1966) J 940). The energy of ionizing radiation was assumed to be reflectively wrote, toxic chemicals in the early decades essentially transformed into a genetic effect. According to of the twentieth century demonstrated "no effect up to a the physicist turned biologist Max Delbruck (1969 Nobel threshold dose and then climbed steeply up to 100 %." Prize recipient in Biology and Medicine), the proportion-Muller and others argued that the genetic response to ion- ality rule that was proposed earlier by Muller, based on izing radiation demanded a different evaluative framework. the research of Hansen and Heys ( 1929) and Oliver ( 1930, 19 31) and supported in experimental research by Timofe-eff-Ressovsky et al. ( l 935), provided the basis of the sin-Target theory and LNT gle-hit mechanism interpretation and the calculation of the size of the gene (Delbruck l9..J.O). Table I provides a list-A likely explanation for Muller's (and possibly Gilbert N. ing of quotes in which the early conceptual framing of the Lewis's) acceptance of the LNT in the absence of convinc- dose-response proportionality concept occurred. The trans-ing dose-response data may be found within the scientific forming of a dose-response hypothesis based on a very culture at the time. X-ray-induced mutational effects were limited amount of data into a biological "Rule" by Muller placed within the context of what was called the radia- was done without significant discussion of the concept, its tion target theory. This theory was quantitative and dosi- possible mechanisms as well as the recognition of data that metric, with mathematical calculations related to quantum may contradict this "Rule."

mechanics, reflecting the leadership of prestigious theo- Although Muller was a geneticist, he was drawn quickly retical physicists (von Schwerin 2010). The formation of a toward the physics-mutation interface, accepting significant physics-based target theory was established prior to the dis- elements of target theory for radiation-induced mutational covery of inducible mutations by Muller ( 1927) by medi- effects, including the important assumptions that dam-cal physicists such as Dessauer ( 1922), Glocker ( J927) and age was proportional to the energy absorbed, linear dose-Crowther (!924, [926, 1927), setting the stage for a novel response modeling and that effects were cumulative and scientific framing of the mutational data in the 1930s. The deleterious (Muller et al. 1936). Muller knew Timofeeff-mutation findings of Muller ( 1927) were a major scientific Ressovsky, having met him in the Soviet Union in 1922, advance that easily fit into the target theory concept while encouraging him and his colleagues to transform his labo-also markedly advancing the scientific standing of target ratory to one of the Drosophila genetics. Muller renewed theory itself. contact with Timofeeff-Ressovsky during the 5th Inter-The radiation target theory as applied to mutations was national Congress on Genetics in 1927. From November formulated by the detailed interactions and collaborations 1932 to September 1933, Muller researched in Berlin with of leading radiation geneticists and theoretical physicists Timofeeff-Ressovsky. He also participated in the physics-during the mid-1930s. During this time, radiation geneti- biology/mutation discussions in Copenhagen in 1936, cists, lead by Nicolai Timofeeff-Ressovsky, and physi- engaging Niels Bohr and other leading physicists. Experi-cists, including Niels Bohr, with a profound interest in the ments of radiation geneticists during this period were often interface of physics and biology, would meet each year, designed within the context of this target theory framework.

typically in Copenhagen and Belgium for extensive discus- This was also the case for critical studies performed a dec-sions. From these exchanges developed the seminal con- ade later under the aegis of the Manhattan Project at the ceptual paper by Timofeeff-Ressovsky and the physicists University of Rochester under the direction of Curt Stern Max Delbruck and Kevin Gunter Zimmer (Timofeeff-Ress- (with Muller serving as a consultant) (Spencer and Stern ovsky et al. 1935) that would establish a conceptual frame- 1948; Caspari and Stern 1948).

work for gene structure, target theory for the induction of mutations via ionizing radiation, the single-hit mecha-nism hypothesis to account for the shape of the LNT dose The hit hypothesis response and the application of this dose-response model for what was to become modern cancer risk assessment. As noted above, in his Nobel Prize research, Muller The genetic target theory saw mutation as a purely physical reported that the induction of mutations was not directly action following an all or none Jaw in which a single ioni- proportional to the X-ray dose, but rather to the square root zation or energy absorption produces the mutational effect of the dose (Muller J 927). Based on discussion with the independent of all other ionizations and energy absorptions. physicist and future Nobel Prize winner Irving Langmuir This linearity feature stands in contrast to normal physi- (1932 Nobel Prize in Chemistry), Muller ( 1927) stated ology that invariably deals with large numbers of mol- that this observation suggested that the induction of muta-ecules of each kind, and where the elimination of a single tion was not caused directly by a single quantum of energy.

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Arch Toxicol Table 1 Documentation of the introduction of the proportionality rule concept into the mutation literature, 1929-1960 References Quote Hanson and Heys (I '129) "It is only to be expected that the number of mutations be directly proportional to the number of rays to which the organisms are exposed." Page 207 Muller ( 1930) "Since then Hanson, using radium, and Oliver in our laboratories using X-rays; have both found that the fre-quency of mutations produced is exactly proportional to the energy of the dosage absorbed ... There is, then, no trace of a critical or threshold dosage beneath which the treatment is too dilute to work." Page 236 Oliver ( 19 30) "That is there is a direct proportionality between the percent of lethals and the length of time of treatment may be seen more readily by a comparison of the t 1 values calculated from the results for each of the given doses."

Page 45 Stadler ( 19:~0) "Mutation frequency increased approximately in direct proportion to dosage." Page 13 Hanson et al. ( 1931) "Taking the amount of ionization in air as a measure, the mutation rate seems to vary approximately in direct proportion to the intensity." Page 142 Oliver (1931) "By inference it can be added that the cosmic and the terrestrial radiations of higher energy content also are capable of producing mutations in proportion to their power of ionization." Page 480 Oliver(l93l) "The relation of proportionality to the dosage applies not merely to the lethals in general, but, more specifically, to the lethal gene mutations." Page 485 Oliver (I ,I) " ... [gene mutations and gene rearrangements] all probably occur in direct proportion to the dosage, no matter how small a dose is used." Page 486 Patterson ( Fn I) "In general their results [i.e., Hanson and Heys I <J2R and Oliver 19:10] justify the conclusion that the rate is directly proportional to the dosage employed." Page 133 Hanson and Heys (I ()C\2) "Further evidence of the proportionality rule from a study of the effects of equivalent doses differently applied."

Page 335 Hanson and Heys (I <JC\2) "Experiments planned with a view to determining within what limits the proportionality rule holds show again a strict correspondence existing between the amount of radium administered and the consequent biological effect, the induced mutation frequency obtained varying directly with the dosage." Page 343 Hanson ( 19.'\.<) "The rate seems to be directly proportional to the dosage. Muller has named this the 'proportionality rule.' For example, when all other factors are kept constant, doubling the time of exposure also doubles the number of lethal mutations." Page 486 Oliver (!()34) "The frequency of induced mutations is directly proportional to the intensity of the treatment" Page 391 Delbruck ( 19+0) "The proportionality rule gave the basis for the single-hit interpretation ... " Page 359 Stern ( 1950) "The proportionality rule has been proven to hold over a wide range. Figure 155 shows that, for Drosophila, the relation is essentially linear over the range from 25 r to several thousand r. It has further been shown that the frequency of induced mutations is independent of the time over which the radiation is applied." Page 433 Stern ( 1960) "It has been established for a variety of experimental organisms that the number of mutations induced by radia-tion is proportional to the dose. This proportionality has been proven to hold over a wide range of dosages."

Page491 However, subsequent exposure experiments by Hanson and Based on hypotheses about what constituted a hit, statisti-Heys ( 1929), Oliver ( !930, 193 I) and later by Timofeeff- cal models were used to construct dose-response relation-Ressovsky et a!. ( 1935), even though all experiments were ships. If there was only a single hit on a single target, the at very high dose, supported a proportionality relationship, dose response was linear. As the number of assumed hits which was consistent with the "hit" theory of mutation in increased, a more threshold like the dose response would which the X-ray treatment excites an electron in the target appear. In a practical sense, the mathematical model-gene. This excitation was proposed to affect a permanent derived dose response based on an assumed number of hits change or mutation to a different molecular structure. Ion- could be visually matched against the laboratory-obtained izing irradiation was the only effective way to induce muta- dose-response curve. Using this direct and simplified tions; it showed no threshold, suggesting that the absorp- approach, researchers like Muller, Timofeeff-Ressovsky tion of radiation is a quantized and additive process (von and participating physicists decided the theoretical number Schwerin 20 l 0). A "quantum-jump" was considered to be of hits. This type of target theory was especially strong in the physical process caused by a hit on a target, resulting Germany, with support from leaders such as Boris Rajew-in mutation. Treatment effects induced by a physical agent sky (Director of the KWI for biophysics, 1936), Timofeeff-like ionizing radiation were believed to be caused by one or Ressovsky and others (von Schwerin 20 I 0). This concep-several discrete biophysical events, that is, hits on a target. tual framework led to the conclusion that mutation was a

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Arch Toxicol single-hit process, proceeding from a single ionization, stability due to the function of the hydrogen bond. Muta-from a quantum of ionizing radiation in a specific sensitive tions of such genes could only occur following the absorp-zone of the gene. tion of high energies as from ionizing radiation, not from This theoretically based perspective became not only heat under physiological conditions. In fact, a modest a workable model but a firm belief within the radiation increase in vibrational energy was estimated to increase genetics community even though there was no knowl- the atomic stability, decreasing mutational risk. Since a edge of the physical nature of the gene. As coauthor of the transaction in an atom can be affected by a single digit eV Timofeeff-Ressovsky et a!. ( !9 35) paper, Delbruck subse- and that the initial impact of an X-ray can be several fold quently noted in his Nobel Prize lecture that it was thought greater, it was believed that any gene would be at risk for that genes were very stable and, therefore, showed charac- mutation from radiation. Since the initial energy of impact teristics of molecules. However, the gene concept at that exceeds a threshold energy of activation, ionizing-radiation time was simply that of Mendelian algebraic rates, lacking should affect not only the induction of a localized mutation structural chemistry insight. There was much speculation but also that of a broad range of gene targets.

of gene structure including that of submicroscopic steady- The mutation hit theory was challenged by Caspari state systems or even an entity not readily analyzable in and Stem ( 19-+R) in a chronic, very low-dose rate study, chemistry as proposed by Bohr ( !933 ). leading to the hypothesis that either a threshold exists or The paper of Timofeeff-Ressovsky et a!. (1935), as multiple independent primary actions are required for a noted above, was striking in its collaboration between mutation to occur, or that a recovery or* repair effect/pro-physics and genetics, its proposed chemical nature of the cess occurred at a very low-dose rate (Howarth et a!. 1950; gene, size of the gene and in the proposal of a "hit" hypoth- Key 1951). Over the next several decades, the dominance esis as the foundation of the linear dose response for ion- of the physics-based target theory would yield to improved izing radiation-induced mutation. While the gene structure chemical/biological/physiological understandings of the and size framework would be bypassed and replaced by mutation process, including such modified target theory the DNA structure of Watson and Crick ( 1953), the hit the- effects of ionizing radiation as DNA repair (in reproduc-ory component of Timofeeff-Ressovsky et a!. (! 935) was tive and somatic cells), adaptive response, the bystander accepted and implemented by the radiation genetics com- effect as well as the recognition that the biological effects munity. The term "hit hypothesis" became commonly used of ionizing radiation are principally due to the genera-in the lexicon of radiation genetics, including those com- tion of hydroxyl radicals/hydrated electrons from cellu-prising the BEAR I Committee/Genetics Panel that recom- lar water and their migration to cellular targets (Collinson mended changing to a linear model from a threshold model eta!. !962; Czapski and Schwartz !962; Weiss 19-1-4). In for assessing mutation risks from ionizing radiation (Cala- fact, even as the target theory was being applied to muta-brese 2013). tion by Timofeeff-Ressovsky et a!. (!935), the recognition The impact of this 1935 article was facilitated by the of repair processes, including DNA repair, were emerging actions of Timofeeff-Ressovsky who sent reprints to key (Hanawalt 199-1-). Such challenges to the hit theory would researchers. However, the overall immediate impact of the eventually be brought to the BEAR Committee by Russell paper was very limited as it was published in an obscure ( J 956, 1963) from Oak Ridge, but only after the BEAR I Gottingen journal that was not cited in any leading index Committee made its linearity recommendation.

with only four issues being printed before ceasing publica- Edward Lewis (J 957a), another radiation geneticist tion. This paper, which provides the origin of the single-hit Nobel Prize (1995) recipient, published a very influential hypothesis to support a linear dose-response model, was Science article in 1957, strongly supporting a linear rela-not even cited in the BEAR I report that implemented the tionship for cancer, relying on linearity data in the Uphoff concept. Yet, the term "hit" hypothesis and target theory and Stem ( 1949) paper. In subsequent Congressional Tes-became commonly used, even if credit was not often given timony, Lewis (I 957h) would argue that the dose response to the original paper (Timofeeff-Ressovsky et a!. l 93:'i). was linear, regardless of the mechanism, and should be Nonetheless, this paper did receive a major endorsement accepted as such whether or not a mechanism could even be in the 1944 book "What is Life" by Erwin Schrodinger, discerned. These comments of Lewis suggested that he rec-a Nobel Prize physicist (1933), raising its visibility in the ognized the growing mechanistic challenge to the single-physics community. hit theory as well as new conceptual problems (e.g., mul-The concept of the gene and its striking stability sug- tiple biological processes could yield a linear relationship gested it must have a unique atomic composition. Dei- that did not require a single-hit process) emerging from bruck (I 970) believed that such stability might be due to the physics and genetics communities, including Zimmer each atom of a gene being fixed in its mean position and ( 1941 ), a coauthor of the Timofeeff-Ressovsky eta!. ( 19 35) electron-stable, sunk in an energy well, now seen having paper and radiation biologists/geneticists (Haas et a!. 1950;

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Arch Toxicol Kimball I 952). However, the time period within which 2000a, b; Sankaranarayanan and Wassom 2008 (see Lyon Muller's mutation fi~dings were produced was one of the 2003 for an alternative view)].

cultural scientific dominance of physics. Association with In the case of somatic effects, cancer risks were esti-the leadership of the physics community served to enhance mated via the use of a linear dose-response model. Assum-the significance of the mutational findings and its assumed ing linearity to zero, it was estimated that exposure of one linearity at low dose, as well as providing Muller with an rem to one million people each year would cause one to expanded scientific and cultural context that recognized his two new cases of leukemia on an annual basis for first dec-achievements and enhanced his scientific reputation. ade of life (ICRP 1962; Sowby 1965; UNSCEAR 1962, The influence of the hit concept of Timofeeff-Ressovsky 1964). As with chemical carcinogenesis risk assessment, et al. (1 935) was facilitated via subsequent publications of therefore, the foundations of the LNT modeling for ioniz-Lea ( 1940, 1946), which offered further justification for the ing radiation-increased cancer risks are directly traced back target theory-based LNT-single-hit hypothesis for muta- to Lea, Timofeeff-Ressovsky et al. and ultimately to Mul-tion. The publications of Lea were not only authoritative ler's proportionality rule.

extensions of Timofeeff-Ressovsky et a!. ( 1935) but more readily available than the Timofeef-Ressovsky et al. ( 1935) Chemical carcinogens paper with its publication in a defunct journal.

Five years after the publication of the BEAR I report, Mantel and Bryan ( 196!) published their influential paper Regulatory agency actions entitled "Safety' Testing of Carcinogenic Agents" based on the probit dose-response model in order to estimate tumor Ionizing radiation incidence for carcinogens. Biostatistical estimates of can-cer risks were first provided by Bryan and Shimkin (1943)

In the radiation risk assessment area, two endpoints were when they applied the probit model to estimate the cancer adopted to which linearity was applied: germ cell muta- risk of three carcinogenic hydrocarbons (i.e., 20-methyl-tions and cancer. In the case of germ cell mutations, based cholanthrene; I ,2,5,6-dibenzanthracene; 3,4-benzpyrene) in on several publications in the early I950s by Muller ( 1951, strain C 3H male mice.

1954), the BEAR I Genetics Panel (1956) proposed to limit The motivation for Mantel and Bryan to develop the exposure to ionizing radiation such that exposure would not biostatistical model for predicting carcinogen risk was due exceed doubling of background mutations from concep- to the fact that Mantel, a biostatistician at the US National tion through the first 30 years of life. The panel assumed Cancer Institute (NCI), was asked by the Director of the that exposure to ionizing radiation could cause mutations NCI to develop guidelines for the number of laboratory to germ cells in a linear manner and had the potential to animals that would be needed to establish the safety of a cause adverse genetic effects in individuals and future gen- test agent within the context of a hazard assessment. This erations. The panel derived a risk assessment methodol- response followed a request, after the Thanksgiving cran-ogy for application to both first-generation offspring and berry scare of I959, by the Secretary of the Department total genetic risk, including future generations. The panel of Health, Education and Welfare (HEW) to the NCI. The derived a doubling dose method (i.e., the dose of ionizing cranberry scare was a public relations nightmare in which radiation, assuming linearity at low dose, that would equal trace residues of a cancer-causing herbicide [i.e., amitrole the number of mutations resulting from background expo- (3-amino-1 ,2,4-triazole)] were detected in some sources of sure), to estimate population-based risks. This doubling cranberries just before the holiday. The secretary of HEW dose methodology would predict the number of genetic recommended against buying cranberries that year, lead-diseases based on three parameters: the assumed doubling ing to a consumer panic that threatened the industry. In dose, the proposed exposure limit and the background inci- order to avoid such situations in the future, the secretary dence of genetic disease. Based on this risk assessment of HEW requested the NCI to provide guidance on which framework, the panel recommended a "uniform national cancer-causing substances were "safe" and at what dosage standard" such that the members of the general popula- levels.

tion would not receive more than a cumulative dose of I OR Mantel and Bryan ( 1961) noted the generality of their from conception through 30 years. This basic method of the modeling approach and proposed the concept of a virtually BEAR I Committee, using the doubling dose/linear frame- safe dose with an estimated risk of 1/100 million. Some work, has been refined with recent advances allowing one 12 years later, the FDA would propose the use of the Man-to integrate between rates of radiation-induced mutation tel-Bryan (!961) model and recommend the 1/100 million based on mouse studies and the risk of inducible genetic safety guide in their July I9, I973 risk assessment proposal disease in people [Sankaranarayanan and Chakraborty in the Federal Register. When the rule was finalized in

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Arch Toxicol 1977, the Mantel-Bryan probit model was retained but with adopted the linear no threshold model (LNT) of the Atomic several modifications and with the acceptable (de minimus) Energy Commission (AEC) that had been applied to esti-risk being reduced to 1/million. This value was considered mating risks from fallout from atomic weapon tests. The as the level below which no additional regulatory action LNT model was attractive to EPA since it was very simple would be taken within the context of the safety of animal to apply; all that was needed in a toxicological sense was to carcinogen residues. The finalized Mantel-Bryan model of identify the lowest dose of agent that induced a statistically the FDA was the first quantitative risk assessment model significant response and draw a straight line to the origin of approved by a regulatory agency. Two years later, the FDA the graph for the dose versus cancer incidence. Its biologi-( 1979) significantly revised the cancer risk assessment cal plausibility was based on the linearity of mutation dose policy, replacing the modified Mantel-Bryan model with response within the framework of target theory. He noted a linear dose-response model based on multiple factors, that "any difference between chemical carcinogens and including its more conservative risk estimation and ease of ionizing radiation could be waived aside as they both cause calculations (Anonymous, ! 979). In the low-dose zone, the genetic damage ... "

one-hit model discussed above is closely approximated by Statisticians would argue that the straight line extrapo-a simple linear model. lation to zero from the lowest statistically significant The US EPA strategy for assessment and regulation of response ignored data at the high doses. Thus, during a carcinogens displayed a profound evolution during the meeting of leading statisticians called by the CAG, a deci-1970s. Based on expert testimony during pesticide hear- sion was made to change from the single-hit model to the ings, EPA attorneys developed a legal brief that embodied multi-stage model since it used all the data, while retain-

"cancer principles" (NAS 1983). These "principles" sug- ing linearity at low dose and being compatible with the gested that carcinogen exposures should be prevented. As concept of cancer being a multi-stage process. Consistent the concept of "banning" carcinogenic agents was soon with this assessment, the NAS Safe Drinking Water Com-seen as unrealistic, EPA quickly adopted non-regulatory mittee ( 1977) recommended the adoption of LNT modeling guidelines for a general risk assessment process (EPA for risk assessment using a multi-stage model. However, in 1976). This process advocated the use of quantitative risk 1982, the Safe Drinking Water Committee (SDWC) was assessment as a means to differentiate risks among chemi- skeptical about LNT modeling for chemicals and rescinded cals and engineering processes. The guidance was very its endorsement of the LNT model noting " ... more confi-general, being limited to less than a page within the Fed- dence could be placed in mathematical models for extrap-eral Register. These guidelines were followed by a paper olation if they incorporated biological characteristics of from the EPA Carcinogen Assessment Group (CAG) the animal studies ... since the users of this volume will (Albert et a!. !977), which provided a strong endorsement be likely to favor different varieties of the conventional of the LNT concept, arguing that linearity was supported extrapolation models or will have access to some of the by human epidemiological studies (e.g., ionizing radiation newer developmental methodologies, it is premature at this and cigarette smokiPg related lung cancer) and mutagenic- stage to recommend any single approach by selecting it for ity studies that were also claimed to follow a linear dose calculations ... " (p 8). However, since LNT modeling was response and believed to be the underlying mechanisms of already in use by EPA, in 1983, the SDWC again endorsed carcinogenesis. In a March 15, 1979, Federal Register, the the LNT model and its subsequent use became the default EPA Administrator Douglas Castle stated that "Risk assess- methodology for chemical cancer risk assessment. Accord-ment from animal data is performed using the 'one-hit' ing to Albert ( 1994), none of the possible models (single model" based on the 1976 Interim Guidelines (EPA 1976). hit, multi-hit, logit, probit, multi-stage, others) were biolog-He went on to state that "the one-hit model was endorsed ically credible. The agency simply needed one that would by the four agencies in the Interagency Regulatory Liai- be acceptable. The agency applied LNT risk assessment son Group" based on its highly conservative nature and the methods using the multi-stage model for the regulation of uncertainties in extrapolating from animal data to human trihalomethanes in drinking water in a November 29, 1979, responses and the possibility that humans may be more sus- notice in the Federal Register (EPA Environmental Protec-ceptible than the animal model, because of broad human tion Agency (US EPA) 1979a, b), a process that would be interindividual variability in exposures and "other unknown followed in subsequent EPA cancer risk assessments.

factors". The strongly clarifying and underlying statement The parallel, yet converging linear dose-response strate-of the administrator was due in part to the fact that EPA had gies of the EPA and FDA represent the regulatory origin used other cancer risk assessment models under other regu- of current cancer risk assessment practices throughout the latory acts and by other US federal agencies. world. They are directly traced back to the efforts of Lea According to Albert ( 1994), Chair of the EPA Can- (I 946) and Timofeeff-Ressovsky et a!. ( 19:15), all of which cer Assessment Group (CAG) during the 1970s, the EPA stemmed from the "Proportionality Rule" of Muller (1930).

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Arch Toxicol Eugenics subgroups or other targeted populations to a humanistic framework that would reduce mutational risks to entire While the LNT concept for mutation was born within the populations.

intellectual and scientific framework of the physics-based radiation target theory, its applications also found sup-portive resonance within the philosophical, ideological Evolution and endogenous mutations and political frameworks of eugenics. German eugenicists expressed considerable concern that ionizing radiation may The LNT had its start in an attempt to explain evolution, hurt the German germ plasm (Proctor !999; Martius 1931). finding other outlets in the world of eugenics and later Educational programs based on these concerns cautioned public health regulatory policies. While Muller was a against exposures to ionizing radiation that might adversely leader in these activities, he did not abandon his quest to affect future generations of Germans. Recommendations determine those underlying factors that served to provide as early as 1927 by the Bavarian Society for Pediatrics and the novel mutations for natural selection. In fact, prior Gynecology stated that women receiving excess X-rays to his discovery of X-ray-induced mutations in 1927, during pregnancy should abort their fetuses. Pushing this Muller reported that temperature increases enhanced concept even further, in 1930, Eugene Fisher, director of the mutation rate by about two-fold (Muller !921-\c).

the Kaiser Wilhelm Institute for Anthropology, argued However, the temperature hypothesis was placed on the that women exposed to X-rays should be permanently pre- research back burner when high doses of X-rays were vented from having children (Proctor 1999). Muller's own found to markedly enhance mutation frequency. Muller history is replete with his highly visible association with would return to the temperature-evolution hypothesis national and international activities advancing eugenics some three decades later, completing an intellectual and philosophy and agenda. Even as late as 1955, Muller gave professional circle, reflected in the comments of Plough a strong eugenics advocacy presentation in Germany, test- and Ives ( !934), his former colleagues at Amherst Col-ing such ideas with a large audience of Nobel Prize winners lege (1940-1945) who noted that "since Muller and (The Lindau Mediatheque !955). Mott-Smith conclude that natural radiation is inadequate The biophysical concept of the gene had important to account for mutations in nature, it seems possible eugenics implications. Since mutations could be induced to suggest that ubiquitous temperature variations may by ionizing radiation in a linear at low-dose manner, this play that role". If Muller had lived into the decades of concept provided the principal foundation that all ioniz- the 1980s (he died in the 1967), he would have begun ing radiation-whether via medical diagnosis/treatment to appreciate the so-called other conditions suggested or industrially-was a concern for "genetic health". The by Oliver ( !93 l) as the cause of the overwhelming pro-genetic toxicology studies of Timofeeff-Ressovsky et al. portion of spontaneously occurring mutations is now

(! 935) transformed these above-cited radiation health con- believed to be derived from endogenous metabolism, for cerns, providing biophysical models and the LNT-single-hit which complex and integrative DNA repair processes model risk assessment paradigm. Such actions provided have been selected for via natural selection (De Bont a key vehicle by which eugenics would focus on radia- and van Larebeke 200-l; Lindahl 1996).

tion protection for preventing the occurrence of genetic defects. In fact, the development and activities of the genet-ics department of the Kaiser Willheim Institute under the Summary direction ofTimofeeff-Ressovsky was affected by such per-spectives (Gausemeier 20 I 0). The LNT concept was initially proposed to account for The concept of LNT for ionizing radiation-induced evolutionary change and then later applied for the assess-mutation was, therefore, built upon a scientific/cultural ment of risks for some genetic diseases and cancer inci-framework and applied to a range of health-related poli- dence (Table 2). The initial data upon which the LNT cies, especially those of eugenics during the early dec- concept was based were limited to a few studies of an ades after the discovery of X-ray-induced mutations. acute nature and at very high doses. Within a decade, the In fact, the eugenics area would serve as an intellectual LNT dose-response model was provided with a mecha-training ground for how ideas such as LNT could be nistic foundation via the integration of the single-hit "softened", humanized and successfully integrated within concept within target theory. The LNT-single-hit model a post-World War II society. Some aspects of eugenics was then used by radiation geneticists to frame the intel-advocacy and the LNT concept would morph into mod- lectual debate on low-dose ionizing radiation risk to the ern regulatory policy for carcinogen regulation, evolving human genome. It provided the basis for the recommen-from that of preserving the gene pool of certain racial dations of the US NAS BEAR I Committee in 1956 for

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Arch Toxicol Table 2 LNT history: the temporal sequence leading to the LNT dose-response model for cancer risk assessment References Specific temporal events Muller ( 1927) Mutation findings-X-rays induce mutations in fruit flies

.JJ.

Olson and Lewis ( 1928) LNT model proposed to account for evolutionary changes following Muller's discovery that X-rays can induce mutations in fruit fly germ cells

.JJ.

Muller ( 1930) Develops proportionality rule (i.e., linear dose response) for ionizing radiation-induced muta-genicity

.JJ.

Timofeeff-Ressovsky et al. (I 9:1~) Application of radiation target theory for mutagens. Used target theory to propose a hit theory for ionizing radiation-induced mutation. The hit mechanism was used to explain the LNT dose response

.JJ.

BEAR I 1956 (Biological Effects of Atomic Proposes the use of the linear dose-response model for germ cell mutation, using the "doubling Radiation Committee, Genetics Panel) rule"

.JJ.

Mantel and Bryan ( l% I) Develops carcinogen risk assessment model based on the probit model. This activity was undertaken to advise US governmental agencies on chemical risk assessment

.JJ.

FDA (!973) Proposes a probit-based quantitative risk assessment method for cancer risk based on the Man-tel and Bryan 1961 paper. The proposal stated that an acceptable risk was 1/100 million

.JJ.

EPA ( 197(i) (see Albert et al. ( 1977), Proposed guidelines for carcinogen risk assessment based on quantitative risk assessment.

Anonymous ( 1979) Recommended a linear dose-response model

.JJ.

FDA (1977) FDA rule finalized, retaining the Mantel-Bryan model with some modifications. The acceptable risk value was changed to Ill million (10- 6)

.JJ.

U.S. NAS Safe Drinking Water Committee Recommended that EPA adopt LNT for carcinogen risk assessment. This recommendation was

( 1977) profoundly significant given the widespread multimedia regulatory functions of EPA. Within 2 years of the recommendation, EPA applied the LNT to the regulations of trihalomethanes (e.g., chloroform) in drinking water

.JJ.

FDA (1979) Replaced the modified Mantel-Bryan model with the LNT model for carcinogen risk assess-ment, based on the following reasons: I. Linear procedure is least likely to underestimate risk. 2. Linear extrapolation does not require complicated mathematical procedures. 3. No arbitrary slope is needed to carry out linear extrapolation. 4. Several significant limitations were found with the application of the Mantel-Bryan model (Anonymous 1979)

.JJ.

EPA (l979a, b) EPA established a national drinking water standard for trihalomethanes (including chloroform) based on an LNT methodology as recommended by the US NAS Safe Drinking Water Com-mittee ( l 977) the switch from a threshold to a linear dose-response LNT model by the FDA in 1979, the same year that EPA model for estimating ionizing radiation-induced germ applied the LNT for the regulation of carcinogens (i.e.,

cell mutation using the doubling dose concept. The LNT- trihalomethanes) in drinking water. The LNT model and single-hit model was soon generalized to the process of its single-hit explanation/mechanism theory, therefore, cancer risk assessment and adopted by national and inter- can be traced back to the concept of radiation-induced national committees concerned with ionizing radiation by mutation target theory as proposed by Timofeeff-Resso-the late 1950s and early 1960s. Five years later, Mantel vsky et a!. ( 1935), which was founded on the proportion-and Bryan ( 1961 ), researchers at the US National Can- ality rule of Muller ( 19 JO) which itself had its origins in cer Institute, proposed a probit model-based cancer risk the 1928 paper of Olson and Gilbert that created the LNT assessment method. It was the Mantel and Bryan ( 196 t) concept following the seminal findings of Muller ( 1927) model that was proposed by the FDA in 1973 for can- that ionizing radiation could induce mutation in the germ cer risk assessment procedures, being replaced with a cells of fruit flies.

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Arch Toxicol Acknowledgments The research on the topic of hormesis has been Dixon HH ( 1929) Cosmic radiations and evolution. Nature 123(3113):

supported by awards from the US Air Force and ExxonMobil Founda- 981 tion over a number of years. Sponsors had no involvement in study Dixon HH (1930) The mechanism of variation. Nature 125(3165):

design, collection, analysis, interpretation, writing and decision to 992-993 submit. Environmental Protection Agency (US EPA) (1976) Health risk and economic impact assessments of suspected carcino-gens. Interim Procedures & Guidelines. [FRL 548-2] Fed Reg 41:21402-21405 References Environmental Protection Agency (US EPA) (1979) Water quality criteria. Request for Comments. Notice. [FRL 1062-5] Fed Reg Albert RE (1994) Carcinogen risk assessment in the U.S. Environ- 44(52): 15926-15931 mental Protection Agency. Crit Rev Toxicol 24(1 ):75-85 Environmental Protection Agency (US EPA) (1979) Control of Albert E, Train E, Anderson E (1977) Rationale developed by the trihalomethanes in drinking water. National Interim Pri-Environmental Protection Agency for the assessment of carci- mary Drinking Water Regulations. [FRL 1312-2] Fed Reg nogenic risks. J Nat Cancer Inst 58:1537-1541 44(231 ):68624-6871 0 Anonymous (1979) Scientific bases for identification of poten- FDA (US Food and Drug Administration) (1973) Compounds used tial carcinogens and estimation of risks. J Nat Cancer Inst in food-producing animals. Procedures for determining accept-63(1 ):241-268 ability of assay methods used for assuring the absence of resi-Babcock EB, Collins JL ( 1929a) Does natural ionizing radiation con- dues in edible products of such animals. Proposed rule. Fed Reg trol rate of mutation. Proc Nat Acad Sci 15(8):623-628 19: 19226-19230 Babcock EB, Collins JL (1929b) Natural ionising radiation and rate of FDA (U.S. Food and Drug Administration) (1977) Chapter I-Food mutation. Nature 124(3119):227-228 and Drug Administration, Department of Health, Education, Bohr N (1933) Light and life. Nature 131(421--423):457--459 and Welfare. Subchapter A-General. Subchapter E-Animal Bryan WR, Shimkin MB (1943) Quantitative analysis of dose- Drugs, Feeds, and Related Products. Chemical compounds in response data obtained with three carcinogenic hydrocarbons in food producing animals. Criteria and procedures for evaluating strain C3H male mice. J Nat Cancer Inst 3(5):503-531 assays for carcinogenic residues in edible products of animals Calabrese EJ (2005) Historical blunders: how toxicology got the [Docket No. 77 N-0026] Fed Reg 42(35):10412-10437 dose-response relationship half right. Cell Mol Bioi 51 :643-654 FDA (U.S. Food and Drug Administration) (1979) Department of Calabrese EJ (2009a) Getting the dose response wrong. Why horme- Health, Education, and Welfare. Chemical Compounds in Food sis became marginalized and the threshold model accepted. Producing Animals. Criteria and Procedures for Evaluating Arch Toxicol 83:227-247 Assays for Carcinogenic Residues [Docket No. 77 N-0026] Fed Calabrese EJ (2009b) The road to linearity: why linearity at low doses Reg 44(55): 17070-17114 became the basis for carcinogen risk assessment. Arch Toxicol Gausemeier B (2010) Genetics as a modernization program: biologi-83:203-225 cal research at the Kaiser Wilhelm Institutes and the political Calabrese EJ (2011) Toxicology rewrites its history and rethinks its economy of the Nazi state. Hist Stud Nat Sci 40(4):429--456 future: giving equal focus to both harmful and beneficial effects. Glocker R (1927) Das Grundgesetz der physikalischen Wirkung von Environ Toxicol Chern 30(12):2658-2673 Rontgenstrahlen verschiedener WellenHinge und seine Bezie-Calabrese EJ (2013) How the US National Academy of Sciences mis- hung zum biologisched Effekt. Strahlentherapie 26:147-155 led the world community on cancer risk assessment: new find- Goodspeed TH, Olson AR ( 1928) The production of variation in nico-ings challenge historical fundations of the linear dose response. tiane species by x-ray treatment of sex cells. Proc Nat Acad Sci Arch Toxicol (in press) 14:66-69 Caspari E, Stern C (1948) The influence of chronic irradiation with Haas Fl, Clark JB, Wyss 0, Stone WS (1950) Mutations and muta-gamma rays at low dosages on the mutation rate in Drosophila genic agents in bacteria. Am Nat 84(817):261-274 melanogaster. Genetics 33:75-95 Hamblin JD (2007) A dispassionate and objective effort: negotiating Coffey P (2008) Cathedrals of Science: The Personalities and Rival- the first study on the biological effects of atomic radiation. J ries that Made Modern Science. Oxford University Press, Hist Biol40:147-177 Oxford Hanawalt PC (1994) Evolution of concepts in DNA repair. Environ Collinson E, Dainton FS, Smith DR, Tazuke S (1962) Evidence for Mol Mut 23(Suppl 24 ):78-85 unit negative charge on hydrogen atom formed by action of ion- Hanson FB (1933) Radiation-genetics. Phys Rev 13(4):466--496 ising radiation on aqueous systems. Proc Chern Soc 140-144 Hanson FB, Heys F ( 1928) Effects of radium in producing lethal muta-Crowther JA (1924) Some consideration relative to the action of tions in Drosophila melanogaster. Science 68(1753): 115-116 x-rays on tissue cells. Proc Roy Soc Sect B 96:207-211 Hanson FB, Heys F (1929) An analysis of the effects of the different Crowther JA (1926) The action of X-rays on Colpidium colpoda. Proc rays of radium in producing lethal mutations in Drosophila. Am Roy Soc SerB 100(704):390--404 Nat 63(686):201-213 Crowther JA (1927) A theory of the action of X-rays on living cells. Hanson FB, Heys F (1930) A possible relation between natural (earth)

Proc Camb Phi los Soc 23:284-287 radiation and gene mutations. Science 71 (1828):43--44 Czapski G, Schwarz HA (1962) The nature of the reducing radical in Hanson FB, Heys F (1932) Radium and lethal mutations in Dros-water radiolysis. J Phys Chern 66:471--479 ophila. Further evidence of the proportionality rule from a study De Bont R, van Larebeke N (2004) Endogenous DNA damage in of the effects of equivalent doses differently applied. Am Nat humans: a review of quantitative data. Mutagenesis 19(3): 169-185 66(705):335-345 Delbruck M (1940) Radiation and the hereditary mechanism. Am Nat Hanson FB, Heys F, Stanton E ( 1931) The effects of increasing x-ray 74(753):350-362 voltages on the production of lethal mutations in Drosophila Delbruck M ( 1970) A physicist's renewed look at biology: twenty melanogaster. Amer Nat 65(697):134-143 years later. Science 168(3937): 1312-1315 Howarth JL, Miller H, Walter J (1950) Some measurements of Dessauer F (1922) Uber einige Wirkungen von Strahlen I. Z. Physik gamma-ray doses received by a radiotherapist during radium 12:38 operations. Br J Radio! 23(268):245-255

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Arch Toxicol International Commission on Radiological Protection (ICRP) (1962) National Academy of Sciences Safe Drinking Water Committee (NAS Radiation protection; Recommendations of the Commission (as SDWC) ( 1977) Drinking water and health, vol I. National amended 1959 and revised 1962). Pergamon Press, Inc., New Academy of Sciences, Washington York National Academy of Sciences Safe Drinking Water Committee (NAS Key JM (1951) Neutron and X-ray experiments in barley. Hereditas SDWC) (1982) Drinking water and health, vol 2. National 37(3):421-464 Academy of Sciences, Washington Kimball RF (1952) Genetic effects of radiation. Ann Rev Nucl Sci Oliver CP ( 1930) The effect of varying the duration of x-ray treatment 1:479-494 upon the frequency of mutation. Science 71 :44-46 Lea DE (1940) A radiation method for determining the num- Oliver CP ( 1931) An analysis of the effect of varying the duration of ber of genes in the X chromosome of Drosophila. J Gen x-ray treatment upon the frequency of mutations. Ph.D. Thesis.

39(2): 181-188 University of Texas, Austin Lea DE (1946) Actions of radiations on living cells. Cambridge Uni- Oliver CP (1934) Radiation genetics. Quart Rev Biol9(4):381-408 versity Press, Cambridge Olson AR, Lewis ON (1928) Natural reactivity and the origin of spe-Lewis ON (1926) The anatomy of science. Silliman Lectures. Yale cies. Nature 12 1(3052):673-674 University Press, New Haven Patterson JT ( 1928) The effects of x-rays in producing mutations in Lewis EB (1957a) Leukemia and ionizing radiation. Science the somatic cells of Drosophila. Science 68:41-43 125(3255):965-972 Patterson JT (1931) Continuous versus interrupted irradiation and the Lewis EB ( 1957b) Testimony: Statement of Dr. Edward Lewis, Califor- rate of mutation in Drosophila. Bioi Bull61(2):133-138 nia Institute of Technology. Hearings before the Special Subcom- Patterson JT (1933) The mechanism of mosaic formation in Drosoph-mittee on Radiation of the Joint Committee on Atomic Energy, ila. Genetics 18(1):0032-0052 Congress of the United States. 85th Congress, I st session, Part I. Plough HH, Ives PT (1934) Heat induced mutations in Drosophila.

United States Government Printing Office, Washington Proc Nat Acad Sci 20:268-273 Lind SC ( 1929) The chemical effects of radium radiation. Am J Proctor RN (1999) The Nazi War on cancer. Princeton University Roentgen Rad Ther 21 :480-483 Press, Princeton Lindahl T (1996) The Croonian lecture, 1996: endogenous damage to Russell WL (1956) Comparison of x-ray-induced mutation rates in DNA. Phil Trans Bioi Sci 351 (1347): 1529-1538 Drosophila and mice. Am Nat 90(suppl):69-80 Lyon MF (2003) James Nee! and the doubling dose concept. Mut Res Russell WL (1963) Genetic hazards of radiation. Proc Am Phil Soc 543:115-120 107(1):11-17 Mantel N, Bryan WR (1961) Safety testing of carcinogenic agents. J Sankaranarayanan K, Chakraborty R (2000a) Ionizing radiation and Nat Cancer Jnst 27(2):455-470 genetic risks XI. The doubling dose estimates from the mid-Marti us H (1931) Keimschadigung durch Rontgenstrahlen. Strahlen- 1950s to the present and the conceptual change to the use of therapie 41:47-66 human data on spontaneous mutation rates and mouse data on Muller HJ (1922) Variation due to change in the individual gene. Am induced mutation rates for doubling dose calculations. Mut Res Nat 56:32-50 453:107-127 Muller HJ (1927) Artificial transmutation of the gene. Science Sankaranarayanan K, Chakraborty R (2000b) Ionizing radiation and 66(1699):84-87 genetic risks. XII. The concept of "potential recoverability cor-Muller HJ (1928a) The problem of genic modification. Supplement- rection factor" (PRCF) and its use for predicting the risk of band 1 der Zeitschrift fur lnduktive Abstammungs und Ver- radiation-inducible genetic disease in human live births. Mut erbungslehre Manuscript Department, Lilly Library. Indiana Res 453:129-179 University, Bloomington, pp 234-260 Sankaranarayanan K, Wassom JS (2008) Reflections on the impact Muller HJ (1928b) The production of mutations by x-rays. Proc Nat of advances in the assessment of genetic risks of exposure to Acad Sci 14(9):714-726 ionizing radiation on international radiation protection recom-Muller HJ (1928c) The measurement of gene mutation rate in Dros- mendations between the mid-1950s and the present. Mut Res ophila, its high variability, and its dependence upon tempera- 658:1-27 ture. Genetics 13:279-357 Sowby FD (1965) Radiation protection in Canada. Part VII. Set-Muller HJ (1930) Radiation and genetics. Am Nat 64(692):220-251 ting standards for radiation protection. Can Med Assoc J Muller HJ (1951) Radiation damage to the genetic material. In: Bait- 92:505-507 sell GA (ed) Science in progress, vol 7. Yale University Press, Spencer WP, Stern C (1948) Experiments to test the validity of the New Haven, pp 93-177 linear R-dose/mutation at low dosage. Genetics 33:43-74 Muller HJ ( 1954) The manner of dependence on the permissible dose Stadler LJ ( 1930) Some genetic effects of x-rays in plants. J Heredity of radiation on the amount of genetic damage. Acta Radio! 21:3-19 41:5-20 Stadler LJ (1931) Chromosome number and the mutation rule in Muller HJ, Altenburg E (1930) The frequency of translocations pro- avena and triticum. Proc Nat Acad Sci 15:876-881 duced by x-rays in Drosophila. Genetics 15:283-331 Stern C ( 1950) Principles of human genetics. WH Freeman and Com-Muller HJ, Mott-Smith LM (1930) Evidence that natural radioactivity pany, San Francisco is inadequate to explain the frequency of "natural" mutations. Stern C (1960) Principles of human genetics, 2nd edn. WH Freeman Proc Nat Acad Sci 16:277-285 and Company, San Francisco Muller HJ, Timofeeff-Ressovsky N, Delbruck M, Bohr N (1936) The Lindau Mediatheque (1955) 5th Lindau Nobel Laure-Summary of discussions on mutations. Copenhagen 28-29. ate meeting. The effect of radiation and other present Manuscripts Department-Muller manuscripts, The Lilly day influences upon the human genetic constitution.

Library, Indiana University, Bloomington htlp://www.mcdial hcquc. lindau-nohd.org/#/Vickn 'lid= 102 National Academy of Sciences (1983) Risk Assessment in the Federal Timofeeff-Ressovsky NW, Zimmer KG, Delbruck M (1935)

Government. Managing the process. NAS Press, Washington Uber die Natur der Genmutation und der Genstruktur.

National Academy of Sciences (NAS)/National Research Coun- Nachrichten von der Gesellschaft der Wissenschaften zu cil (NRC) (1956) The biological effects of atomic radiation Gottingen:Mathematische-Physikalische Klass, Fachgruppe VI, (BEAR): a report to the public. NAS/NRC, Washington DC Biologie 1(13):189-245. [English translation: On the Nature

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Arch Toxicol of Gene Mutation and Gene Structure. Reprinted in Sloan PR, Weinstein A (1928) The production of mutations and rearrangements Fogel B (editors). (2011). Creating a Physical Biology. The of genes by x-rays. Science 67:376-377 three-man paper and early molecular biology. The University of Weiss J (1944) Radiochemistry of aqueous solutions. Nature Chicago Press, Chicago.] 153:748-750 United Nations Scientific Committee on the Effects of Atomic Radia- Whittemore GF (1986) The national committee on radiation protec-tion (UNSCEAR) (1962) Report, 17th session, Supplement No. tion, 1928-1960: From professional guidelines to government 16, New York regulation. Ph.D. Dissertation. Harvard University, Cambridge, United Nations Scientific Committee on the Effects of Atomic Radia- Massachusetts tion (UNSCEAR) (1964) Report, 19th session, Supplement No. Zimmer KG (1941) Ergebnisse und Grenzen der treffertheoretischen 14, New York Deutung von strahlenbiologischen Dosis-Effekt-Kurven. Bioi Uphoff DE, Stem C (1949) The genetic effects of low intensity in Zentral 63:78 irradiation. Science 109:609-610 Zimmer KG (1966) The target theory. In: Cairns J, Stent GS, Wat-Von Schwerin A (2010) Medical physicists, biology, and the physiol- son JD (eds) Phage and the origins of molecular biology. Cold ogy of the cell ( 1920-1940). Ident Mut 231-258 Spring Harbor Laboratory Press, New York, pp 33-42 Watson JD, Crick FHC (1953) Molecular structure of nucleic acids. A structure for deoxyribose nucleic acid. Nature 171 (4356):737738

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Arch Toxicol DOl 10.1007/s00204-013-1105-6 2 How the US National Academy of Sciences misled the world 3 community on cancer risk assessment: new findings challenge 4 historical foundations of the linear dose response 5 Edward J, Calabrese 6 Received: 24 April2013 I Accepted: II July 2013 7 ©Springer-Verlag Berlin Heidelberg 2013 a Abstract This paper extends several recent publi- Introduction 32 9 cations indicating that Hermann J. Muller: (1) Made 10 deceptive statements during his Noble Prize Lecture It was recently discovered that the 1946 Nobel Prize Lec- 33 11 on December 12, 1946, that were intended to promote ture for Biology and Medicine by Laureate Hermann 34 12 the acceptance of the linear dose-response model for J. Muller misled the audience on the nature of the dose 35 13 risk assessment for ionizing radiation and (2) that such response in the low-dose zone concerning the effects of 36 14 actions of Muller were masked by a series of decisions ionizing radiation on germ-cell mutagenicity to advance 37 15 by Muller's long-time colleague and esteemed radia- an ideologically motivated risk assessment policy (Cala- 38 16 tion geneticist Curt Stern, affecting key publications in brese 20 l Ia, b, 20! 2). Evidence to support this conclu- 39 17 the mutation literature. Such actions further enhanced sion is found in Muller's own words from letters he sent 40 18 acceptance of the linearity dose-response model while to Professor Curt Stern of the University of Rochester, an 41 19 preventing Muller's deceptions from being discovered. expert in radiation genetics. Stern sent Muller a manu- 42 20 This paper provides documentation that Muller rein- script by Ernst Caspari and himself on November 6, 1946, 43 21 forced such practices within the scientific literature in for review as Muller was a paid consultant to the project 44 22 the early 1950s, by supporting scientifically question- (Calabrese 20 I Ic). This manuscript demonstrated sup- 45 23 able actions of Stern. Detailed documentation is provided port for a threshold dose response, while challenging the 46 24 that demonstrates how these actions affected national and linear dose-response single-hit mutagenicity mechanism 47 25 international risk assessment policy for ionizing radiation model, based on an extensive study of ionizing radiation on 48 26 and chemical carcinogens via the recommendations of mutation in the germ cells of male fruit flies. On Novem- 49 27 the National Academy of Sciences Biological Effects of ber 12, 1946, Muller acknowledged receipt, noting that the 50 28 Atomic Radiation committee in 1956, to adopt the linear findings strongly challenged the linearity dose-response 51 29 dose-response model. concept and, given their importance, needed to be rep- 52 licated as soon as possible (Calabrese 20 II c). This long- 53 30 Keywords Mutation

• Linearity

• Dose response

• Risk term study used the lowest ionizing radiation dose rate yet 54 31 assessment

• History of science

• Muller reported. Despite this new information, Muller would go 55 on to deliver his Nobel Prize Lecture some 5 weeks later 56 (December 12, 1946), proclaiming that one could no longer 57 consider the possibility of a threshold dose response for 58 germ-cell mutagenicity. The only option, he argued, was to 59 switch to a linearity dose-response model for risk assess- 60 ment (Muller l946a). 61 A1 E. J. Calabrese (121) Muller, of course, made these public claims while know- 62 A2 Department of Public Health, Environmental Health Sciences, ing that the most extensive and relevant testing supported 63 A3 University of Massachusetts, Morrill I, N344, Amherst, MA A4 01003, USA a threshold interpretation. A letter from Muller to Stern 5 64 A5 e-mail: edwardc@schoolph.umass.edu weeks after the Nobel Prize Lecture (January 14, 1947) 65

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Arch Texico!

66 confirmed his support for study replication, that he had no Part 1-Stern's plan to promote linearity 119 67 technical criticisms of the Caspari study, and supported 68 publication especially in view of the caveats worked into Curt Stem was a long-time supporter of the idea that ion- 120 69 the discussion, hopefully preventing acceptance of a thresh- izing radiation affected germ-cell mutation in a linear dose- 121 70 old interpretation (Calabrese 20 12; Lilly Library 1947 a, response manner. He expected that this would be observed 122 71 January 14 Jetter). In effect, Muller told the Nobel Prize in studies he was directing under the aegis of the Manhat- 123 72 Lecture audience one story while in private correspond- tan Project using fruit flies. While a linearity dose-response 124 73 ence he revealed a profoundly different view. According to was reported in acute studies with X-rays (Spencer and 125 74 his former student, friend, and colleague, Crow (1995), it Stem 1948), the most significant test would take place 126 75 was well known that Muller would try to win arguments by with the research of Ernst Caspari when gamma radiation 127 76 exaggeration and overstatement. Crow found this behavior would be administered up to a 13,200-fold lower rate than 128 77 exasperating as Muller would often end up hurting his case in the Spencer research. In a troubling development, Cas- 129 78 by unnecessarily misrepresenting facts and circumstances, pari reported to Stem that his findings did not support a 130 79 incorrectly thinking it would help him win his argument. linear interpretation but rather a threshold dose response. 131 80 This same behavioral trait was evident at the Nobel Prize Based on Jetter correspondence between Stem and Caspari, 132 81 Lecture. Stem initially refused to accept this interpretation, arguing 133 82 Before his Nobel Prize Lecture, Muller sought to that the mutation threshold response was most likely due 134 83 raise concern over the public health implications of ion- to unusually high control group values (i.e., spontaneous 135 84 izing radiation and to change the risk assessment process mutations in sperm stored in the spermatheca of the female 136 85 for ionizing radiation from the use of a threshold dose- for 3 weeks) which masked a radiation-induced treatment 137 86 response model to the far more conservative linear dose effect (Calabrese 20 l I b). Cas pari then researched this 138 87 response. This goal was essentially shared by the entire issue by exploring the literature and obtaining substantial 139 88 radiation geneticist community. Following his Lecture, unpublished data on this specific issue from Muller based 140 89 Muller would now have two goals: Protecting his reputa- on research during his appointment at Amherst College 141 90 tion by ensuring that his misleading comments would not (1940-1945). Caspari argued that his control group muta- 142 91 be discovered while still aggressively pushing acceptance tion data were not aberrant but consistent with the litera- 143 92 of the linearity agenda. Both goals were entangled; being ture and Muller's data for aged sperm whether stored in 144 93 such an important scientist and leader any fall in Muller's the spermatheca of the female or in the male. As a result 145 94 status would have a devastating impact on the acceptance of the Caspari analysis, Stern withdrew his objection and 146 95 of the linearity dose response, especially if it involved an accepted the conclusion that the control group spontaneous 147 96 ideological misrepresentation about the linearity concept. mutation values were within the normal range. Since Stem 148 97 Muller achieved both goals due to decisions of Stern that could not dismiss the findings of Caspari due to the con- 149 98 discredited the findings of his colleague and co-author trois, he then opted for an alternative but bizarre strategy to 150 99 Ernst Caspari, thus saving Muller from criticisms about marginalize the threshold dose-response conclusion. Stem 151 100 his Nobel Prize Lecture while supporting the question- directed the manuscript discussion to explain why these 152 101 able findings of Delta Uphoff, another co-author. Mul- data should not be accepted and utilized until it was deter- 153 102 ler's misleading comments and the Stem's apparent data mined why Caspari's findings differed from those of Spen- 154 103 obfuscations would not be revealed for more than 60 years cer and Stem's acute study which they claimed supported 155 104 while the linearity acceptance goal by regulatory agencies linearity. It was this manuscript of Caspari that was sent to 156 105 worldwide was attained. The present paper extends the Muller for review just prior to his Noble Prize Lecture. 157 106 recent reports of Calabrese (20! l a, b, 2012) with newly It is odd that investigators reporting on striking new 158 107 discovered findings that demonstrate a carefully focused findings, using the most advanced methods and the low- 159 108 and timed set of inexplicable scientific judgments by Mul- est dose rate yet studied, would demand the reader not take 160 109 ler concerning the nature of the dose response. These the data seriously. Stem placed no such restriction upon the 161 110 actions reinforced his Nobel Prize Lecture comment~ and Spencer paper, a study with considerable methodological 162 111 the actions of Stern that enhanced the goal of achieving limitations [e.g., inadequate control groups, inappropriate 163 112 a switch from threshold to linearity. This paper also dem- data combining for statistical analysis, lack of adequate 164 113 onstrates the profound impact of the Stem/Muller actions X-ray instrumentation calibration, poor temperature con- 165 114 on the radiation genetics community based on the scien- trol, and dose rates differing by as much as 10-fold (10 166 115 tific publication record and dose-response recommenda- and 100 r/min) between treatments, thereby creating two 167 116 tions/conclusions supporting a linearity dose-response risk experimental variables within one experiment] (Calabrese 168 117 assessment model by the highly influential NAS BEAR I 201! b). Furthermore, there were at least two dozen signifi- 169 118 Committee, Genetics Panel. cant methodological differences between the two studies 170

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Arch Toxicol 111 making them not directly comparable. Stern published the mechanistic foundation for the LNT dose-response model. 216 112 manuscript (Caspari and Stern 1948) with its misdirected Given his goals and ideology, Stern had little choice. 217 173 discussion, without apparent independent, peer review in Another experiment was not going to be practical as Uphoff 218 174 the journal for which he was the editor, that is, Genetics. would leave for a position with the NIH. In the absence of 219 new data, Stern decided upon a new strategy to "save" the 220 175 Comment single-hit linearity dose response. In order to achieve this 221 goal, he would have to do two things: (1) Reverse his posi- 222 176 Based on this temporal sequence, it would appear that the tion on the Uphoff control group data, declare that they 223 177 principal driving force to challenge the Caspari findings are normal, not aberrant, making the Uphoff experiments 224 178 that supported a threshold interpretation was his advisor now interpretable and (2) challenge further the credibil- 225 179 and co-author, Curt Stern. It was Muller who indicated ity and acceptance of the Caspari study (i.e., beyond the 226 180 that the findings of Caspari needed to be replicated since misdirected discussion of the Caspari!Stern paper). Stern 227 181 they were contrary to a linear single-hit dose-response took the bold action of asserting that the Uphoff control 228 182 interpretation. Of particular note, however, was that the group data were part of the normal distribution. He offered 229 183 only changes made to the Caspari manuscript following no explanation or assessment of the literature to justify 230 184 the review of Muller was to add the name of Muller to the this conclusion. This would not be difficult as only very 231 185 acknowledgments section and to remove the statement few people would have known about his earlier concerns 232 186 from the conclusion that the findings supported a tolerance with the Uphoff control group data, since the manuscript 233 187 or threshold interpretation (Calabrese 20 l I b). (Uphoff and Stern 1947) detailing such concerns was never 234 submitted for publication but was placed in the Atomic 235 Energy Commission (AEC) archives, initially as a classi- 236 188 Part 2-the replication studies fied manuscript. Thus, the written critique of the Uphoff 237 control group data and letter communications on this topic 238 189 Since Ernst Caspari and Warren Spencer were no longer were generally not known or available. 239 190 available to continue experimentation, Stern engaged the The Uphoff and Stern (I 949) paper also raised anum- 240 191 services of a Master's student, Delta Uphoff, to assess why ber of doubts about the Caspari paper such as whether its 241 192 the Caspari study did not support a linear interpretation. non-treatment effect/threshold finding was the result of 242 193 The results of the initial experiment were deemed by Stern "errors in sampling." Given standard professional proto- 243 194 as not usable as her control group spontaneous mutation col, the "errors in sampling" hypothesis was a surprising 244 195 rate was strikingly low, being outside the expected range and unexpectedly harsh challenge to the work of Caspari, 245 196 for aged sperm (-40 % lower than expected); no conclu- a University of Rochester team member, especially since 246 197 sions could be drawn from the study (Uphoff and Stern this criticism had never been raised previously by Stern, 247 198 1947). A similar very low control group spontaneous muta- Muller, or others in previous detailed evaluations. In fact, 248 199 tion rate response for aged sperm in her second experi- there was never any documentation to support this possi- 249 200 ment would also make such data uninterruptable. In her bility. Further, Stern also raised the specter of the Caspari 250 201 third and final experiment, Uphoff reported control values control being elevated by unnecessarily stating that his 251 202 in the normal range for aged sperm but the radiation treat- control group was higher than each of the controls of the 252 203 ment response was itself aberrant, far exceeding predicted three Uphoff experiments. Stern neglected to state that two 253 204 responses assuming low-dose linearity (Calabrese 20 l ! b). of the Uphoff studies had aberrantly low control group val- 254 ues based on the published literature and Muller's data. 255 This decision by Stern would now make the Uphoff experi- 256 205 Stern: What to do next mental data "interpretable," whereas several months before 257 he judged it as "uninterpretable." Also, the third Uphoff 258 206 Finding a way to support linearity was the prevailing experimental control data were indistinguishable statisti- 259 207 theme. For example, when Caspari had shared his data with cally from the Caspari control (0.2489 vs. 0.2352 %). Such 260 208 the Head of Genetics at the Brookhaven National Labora- actions helped to achieve the above-stated goals of enhanc- 261 209 tory and future member of the BEAR I Committee/Genet- ing the credibility of the Uphoff data while marginalizing 262 210 ics Panel, Milislav Demerec, he wrote to Caspari asking the Caspari findings. 263 211 what can be done to save the single "hit" linearity dose- The Uphoff and Stern ( 1949) paper changed the way the 264 212 response paradigm (Calabrese 20 I I b; American Philosoph- Caspari data (Caspari and Stern l94R) were perceived and 265 213 ical Society 19471', September 25). The "hit theory" for accepted by members of the scientific community. Below 266 214 ionizing radiation-induced mutation was first postulated by are quotes from several papers (Higgins 195!; Singleton 267 215 Timofeeff-Ressovsky et al. (! 935), providing a theoretical l954a, b) and a dissertation (Jolly 2004) that address very 268

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Arch Toxicol 269 clearly how the Uphoff and Stern ( 1949) paper marginal- relationship with Stern with whom he had been awarded 321 270 ized the research of Caspari. Of particular significance is a National Research Council post-doctoral fellowship at 322 271 that the judgments drawn by each of these papers were fac- the Kaiser Wilhelm Institute in Berlin (Erk 2009). Since 323 272 tually and interpretationally incorrect. Glass was an expert on Drosophila radiation genetics, it 324 273 Higgins ( 195!) stated that "Uphoff and Stern is likely that he oversaw the evaluation of the manuscript. 325 274 (! 949) ... concluded that low-level radiation does produce One must also question to what extent Muller/Stern may 326 275 mutations in fruit-fly sperm and that the apparent inconsist- have exploited their relationship with Glass to facilitate 327 276 encies of previous results were due to different experimen- the publication of such a limited paper and used the jour- 328 277 tal techniques and errors in sampling" (page 10, column 1). nal to advance an ideological perspective. 329 278 Singleton ( 1954a) stated that "Cas pari and Stern ( !948) 279 studying chronic gamma radiation found no increase over 280 controls for doses of 2.5 r/day for 21 days. However, it was Muller's post Nobel Prize dose-response comments 330 281 later documented by Uphoff and Stern ( 19*~9) that the con- about the Caspari and Stern (1948) study 331 282 trois used by Caspari and Stern had an abnormally high sex 283 linked lethal frequency and that actually there was an effect Muller's statement 332 284 of the chronic gamma radiation of 2.5 r/day." (page 599) 285 Jolly (2004) stated (1) that "Stern and Cas pari initially In his 1950 article entitled "Some present problems in 333 286 detected no significant difference in the mutation rates on the genetic effects of radiation" in the Journal of Cellular 334 287 the controls and the irradiated flies, though later they cor- and Comparative Physiology Muller ( l950a) provided an 335 288 rected for experimental errors and got a statistically signifi- explicit characterization of the Cas pari and Stern ( 1948) 336 289 cant difference." (pages 78-79) (2) "The results of Stern's findings. Muller stated on page I 0 "A recent paper by 337 290 initial experiment failed to support the linear hypothesis for Spencer and Stern ....... extends the principle (i.e., one- 338 291 genetic injury. Assuming that something must have been hit principle) down to total doses of 50 r and 25 r." In the 339 292 wrong with the experiment, he eventually identified experi- next paragraph, he stated: "It is true, in a parallel paper... 340 293 mental errors, which, when corrected for, supported linear- .Caspari and Stern have reported results somewhat deviat- 341 294 ity." (pages 80-81). ing from the above." 342 295 Caspari's control group data were therefore once again 296 challenged by Stern; the once aberrantly low controls Comment 343 297 of Uphoff were now seen as being in the normal range.

298 With these changes, the dose response of the collective Muller trivialized the significant challenge of the Caspari 344 299 grouping of the Stern Drosophila experiments would study to the linearity dose-response paradigm. The key 345 300 appear linear. This is the conclusion of what Uphoff Muller phase concerning the Caspari data is "somewhat 346 301 and Stern published in their one-page technical note in deviating". The Spencer and Stern ( 19-1-8) study involved an 347 302 the 1949 Science article summarizing the Spencer and acute exposure, that is, all doses of radiation were admin- 348 303 Stern ( !94~) and Cas pari and Stern ( 1941\) papers and istered within a few minutes to a few hours. In contrast, 349 304 the three Uphoff experiments. This 1949 paper, as noted the .Caspari and Stern (1948) study provided the same 350 305 above, did not include mention that the previous conclu- total dose as in the Spencer and Stern study but spread 351 306 sions (Uphoff and Stern 1947) about the Caspari and the over 21 days, at a dose rate up to 13,200-fold lower. The 352 307 Uphoff control groups that had been reversed by Stern "somewhat deviating" results were such that at the lower 353 308 and the role of the Muller data assessment in the deci- dose rate of the Caspari and Stern study, the data supported 354 309 sion-making process. Since the Uphoff and Stern (! 949) a threshold interpretation, not the expected linear propor- 355 310 brief technical paper lacked any information on research tionality response. Muller was quite concerned with the 356 311 methods and other relevant data, the authors promised Caspari study as it represented a potentially significant 357 312 a detailed follow-up publication to correct this critical challenge to linearity, repeating this perspective in letters 358 313 limitation, a promise never fulfilled. Given the lack of (Lilly Library 1947a, January 14; American Philosophi- 359 314 information provided in the Science paper and the pres- cal Society 1946, November 12) to Stern and emphasizing 360 315 tige of this journal, it raises a question about the circum- the need to replicate this study, despite the requirement for 361 316 stances surrounding its publication within this context. additional funding and the efforts of multiple scientists and 362 317 It should be noted that Hermann J. Muller's first gradu- staff for about 1 year. It is also important to note that Mul- 363 318 ate student (i.e .. H. Bentley Glass) became an editor at ler never mentioned any of the numerous methodological/ 364 319 Science in 1948, only months prior to the submission analysis limitations/flaws of the Spencer and Stern ( 1948) 365 320 of the Uphoff and Stern manuscript. Glass also had a in any of his publications. 366

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Arch Toxicol 367 Muller's statement found a weekly increase of about 0.07 % for sex-linked 417 lethals in various stocks kept at 25 °C. At 18 °C, the temper- 418 368 In footnote I on page 10 of the above-cited article, Mul- ature used for aging in the laboratory, the weekly increases 419 369 ler ( 1950a) stated that "Uphoff and Stem have published may be assumed to be slightly less, perhaps 0.05 %. Taking 420 370 a report of further work, with doses as low as 50 r, given a value of 0.10 %, similar to that of Spencer and Stem's 421 371 an intensity as low as 0.0165 r per minute. The results control rate, for sperm before aging, the expected control 422 372 obtained are entirely in conformity with the one-hit prin- rate after aging should be approximately 0.25 %. This fig- 423 373 ciple. A consideration of these results, together with the ure is much closer to the control rate observed by Caspari 424 374 early work, leads to the conclusion that the deviation first and Stem than to that found in the present work." In their 425 375 referred to (the Caspari and Stem l 94R findings) was acknowledgments of this manuscript, Uphoff and Stem 426 376 caused by a value for spontaneous mutation rate that hap- stated that "we are very grateful to Dr. H. J. Muller for his 427 377 pened to be unusually high." permission to quote from his unpublished data." Thus, Mul- 428 ler would have known that his research was used to evalu- 429 378 Comments ate the reliability of the Caspari and Uphoff control groups. 430 The control group response of Uphoff and Stem ( 194 7) 431 379 Muller claims that the research of Delta Uphoff and Curt was sufficiently low such that they stated that the data were 432 380 Stem is "entirely in conformity with the one-hit principle" uninterpretable (i.e., "a final interpretation of these results 433 381 (Timofeeff-Ressovsky et a!. l9:i5). What Muller neglected cannot be offered."). Uphoff and Stem ( !94 7) explicitly 434 382 to state was: (1) Uphoff's first experiment displayed an raised the possibility that the low control group values 435 383 aberrantly low control group response based on Muller's "may reflect a personal bias of the experimenter." The man- 436 384 own extensive data involving some 200,000 fruit flies uscript did not identify whether the bias concern statement 437 385 (Muller l946b ). A letter from Curt Stem to Ernst Cas pari was directed to Stem, Uphoff or both, or the type of bias. 438 386 (undated) (American Philosophical Society Undated, circa (2) Uphoff's second experiment also displayed a similarly 439 387 July-Aug 1947) addressed the control group issue. It states: aberrant low control group response, likewise affecting the 440 388 "The radiation data continues to be puzzling. Delta's dif- possible utility of the data. (3) The third (and final) Uphoff 441 389 ference between control and exper[imental group] appears experiment obtained control values in the normal range but 442 390 to be due mainly to a much lower control group value than an aberrantly high treatment response, even assuming a 443 391 yours. However, Muller informs me that his data give an linearity dose response (see Calabrese 20! ! a for a detailed 444 392 aged control value close to yours. Thus, my first idea that evaluation). "Appendix" section provides the temporal let- 445 393 your results could be "explained away" by assuming that ter exchange between Stem and Muller on the key ques- 446 394 your control value happened to be unusually high, seems tion of control group mutation frequency upon which the 447 395 unlikely. Rather does Delta's control appear too low. Well, acceptance of the Cas pari and Uphoff studies are based. 448 396 we'll have to meet." Muller provided this information to Muller ( l950b) discredits the conclusion of Cas pari 449 397 Stem twice in letters dated February 3, 1947, and August and Stem ( 1948) by asserting that the control group val- 450 398 4, 1947 (Lilly Library l947b, c). It should be noted that ues were unusually high. (1) Muller failed to state that 451 399 the occurrence of increased mutations in aged sperm in the "high" control value of Caspari and Stem ( 1948) was 452 400 the control group as reported by Caspari was not a new first put forward as a criticism by Stem in the fall of 1946, 453 401 concept to Stem. In fact, when Timofeeff-Ressovsky first when Caspari informed Stem that his findings supported a 454 402 presented such data in the late 1930s, Stem corresponded threshold, rather than a linearity interpretation. (2) He also 455 403 with Demerec specifically addressing these findings. These did not report that Caspari successfully rebutted Stem by 456 404 letter exchanges reveal not only Stem's knowledge of the presenting data on control group responses from published 457 405 findings, but also of his knowledge that the findings had studies in the literature and from unpublished data provided 458 406 been subsequently replicated (Lilly Library 1938a, b, c). by Muller himself. Muller failed to state that he had pub- 459 407 The report of Rajewski and Timofeeff-Ressovsky (I 939) lished a summary of the *mutation rate of sperm stored in 460 408 on this topic would most likely have considerable scientific the spermatheca for several weeks (Muller 1945). This is 461 409 weight as Timofeeff-Ressovsky was on par with Muller for the information that he sent to Stem that supported the reli- 462 410 scientific reputation in the area of radiation genetics. ability of the Caspari control group data and marginalized 463 411 In the Atomic Energy Commission (AEC) manuscript the Uphoff study control group (see "Appendix" section). 464 412 by Uphoff and Stem ( 1947) concerning her replication of Later studies by Muller and his student Helen L. Byers at 465 413 the Caspari study, the low response control group issue the University oflndiana also supported the Caspari control 466 414 was explicitly addressed as follows in their "Discussion" group mutation frequency (Byers 1954; Byers and Mul- 467 415 section. "In his extensive studies on the effect of aging on ler l 952). Nonetheless, Muller ( l 954h) would inexplica- 468 416 the mutation rate in sperm, H.J. Muller (unpublished) has bly continue his criticism of the Caspari and Stem (194?\) 469

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Arch Taxi col 470 study, repeating the "unusually high control frequency" In his 1950 papers, Muller never addressed any of these 523 471 (page 476) conclusion as a basis to reject its challenge to critical issues that might affect a decision on the nature of 524 472 linearity. The question may be raised as to why Muller the dose response (Muller l950a, b). He also failed to state 525 473 would directly contradict himself on such a serious mat- that the Uphoff and Stem (1949) paper was only a one-page 526 474 ter and never be exposed to criticism. While any answers summary, has very low control group values, no presenta- 527 475 to this question must be speculative, Sankaranarayanan tion of research methods and that Uphoff and Stem ( !949) 528 476 and Wassom (2008) unequivocally state that Muller was an promised to publish a detailed paper with all the missing 529 477 "unquestioned authority," suggesting that it would be quite methods and data but had not (and never did). By discredit- 530 478 difficult to challenge him or even consider doing so. ing the Caspari and Stem ( 1948) paper and restoring the 531 479 It should be noted that in early 1949, Muller became Uphoff data, Muller was able to protect his scientific repu- 532 480 concerned that Robley Evans of MIT was publishing a tation, his ethical standing and to give strong support to the 533 481 paper in the journal Science on the mutagenic effects of linearity single-hit theory dose-response model. 534 482 ionizing radiation and the nature of the dose response in the In a second paper in 1950 entitled Radiation Damage 535 483 low-dose zone. Muller had reviewed the manuscript prior to the Genetic Material in the American Scientist, Mul- 536 484 to publication and was upset that Evans had given credibil- ler ( 1950b) used the findings of Stem and his colleagues 537 485 ity to the Caspari and Stem ( 194~) paper. Muller wrote to to extend "the principle of proportionality of mutation fre- 538 486 Stem (Lilly Library I 949, February 5) requesting that Stern quency to dose down to doses of 50 r and 25 r and of Jess 539 487 contact Evans and try to convince Evans to withdraw his than 0.001 r per minute, with a time-intensity relation dif- 540 488 support for the Cas pari and Stem (I 948) findings. There fering by over 400,000 times from that of our high intensity 541 489 is no evidence that Stern did this based on correspondence dose." 542 490 records. However, it is possible that the subsequent attack 491 of Muller (1950a, b) on the Caspari and Stem (1948) find- Comment 543 492 ings was stimulated by this Evans paper (! 949) which 493 would need to be "neutralized." By using the now revitalized data of Uphoff, Muller made 544 494 Muller (1 954b) also further criticized the Cas pari and the claim of linearity over a 400,000-fold dose range. This 545 495 Stem ( 1948) paper in a vague manner as being "more was a major conclusion as it gave an assertion of linearity 546 496 doubtful than the others on some other grounds" (page at low dose by a Noble Prize winner who had great author- 547 497 476), which he never clarified. Such criticism may have ity within the field. Furthermore, Stem ( 1960) continued to 548 498 referred to the fact that Uphoff and Stem ( 1947) introduced affirm the findings of Uphoff and Stem ( 1949) in the sec- 549 499 a modified method of counting sex-linked recessive lethals, ond edition of his acclaimed genetics textbook, published 550 500 one that was different than reported by Caspari and Stem in English, German, Japanese, Polish, Russian, and Spanish 551 501 ( !948) and also different than Spencer and Stem ( 1911\). (American Philosophical Society 1973, November) (auto- 552 502 Uphoff and Stem ( 1947) recounted (i.e., adjusted) the Cas- biographical statement), by stating that the dose rate had no 553 503 pari and Stern ( !948) data with the new counting method impact on the mutation incidence in Drosophila, whether 554 504 in order for it to be as directly comparable to their study administered acutely or given "slowly and continuously, that 555 505 as possible. The results of those adjustments were deemed is, 'chronically,' given over a long period." In order for Stem 556 506 by Uphoff and Stem to be insignificant in their 1947 paper, ( 1960) to have reached this conclusion, he had to diminish 557 507 resulting in control and treatment responses that were, in the findings of Cas pari and Stem ( l94S) and accept those 558 508 fact, even more similar than before the adjustment (i.e., of Uphoff and Stem ( 1949). A further note is that the Mul- 559 509 without a treatment effect). The published paper of Caspari ler ( l950b) paper contradicted his 1950a paper on the dose 560 510 and Stern ( l94S) did not incorporate this adjustment (per- rate: The two papers used a different lowest dose rate: 0.001 561 511 haps resulting in the veiled criticism of Muller 195-ta, b), r/min (Muller l950b) versus 0.00165 r/min (50 r/30240 min 562 512 whereas the Uphoff and Stern ( !94 7) manuscript presented in 21 days) (Muller 1950a)-a 65-fold difference. Muller 563 513 the original and adjusted data; only these adjusted data (1950b) rounded down the 0.00165 r/min rate to 0.001 r/ 564 514 were used for the Cas pari and Stern ( !94S) data as sum- min, increasing the extrapolation range from approximately 565 515 marized in the 1949 paper in Science by Uphoff and Stem. 250,000- to 400,000-fold. Why Muller rounded the num- 566 516 Regardless, the adjustment for differing lethality estimation bers down is not known, nor was it necessary. Secondly, if 567 517 techniques did not affect the study interpretation. In a letter rounding was to occur it would normally have been rounded 568 518 on February 9, 1949, to Caspari in anticipation of the Sci- up to 0.002 r/min. This action of Muller reveals an effort 569 519 ence publication, Stem (American Philosophical Society to exaggerate the linear extrapolation range. Third, Muller 570 520 1949, February 9) stated that "It will be shown below (the ( 1950b) makes an error in his statement that the linearity 571 521 Science manuscript) that the difference in defining a lethal was shown with a dose rate "less than 0.001 r per minute" 572 522 is of no significance in the evaluation of the results." when the actual value was 0.00165 r/min. 573

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Arch Toxicol Table 1 Hermann J Mulier and Curt Stem quotes on low-dose linearity References Quote Muller ( l94X) Page 462

" ... the frequency of the mutations induced will be proportional to the total dose of radiation received over an unlimited period of time."

"There is then absolutely no threshold dose, unlike what is true of many other biological effects of radiation, and even the most minute dose carries a definite chance of producing mutations-a chance exactly proportional to the size of that dose."

Muller ( 1952) Page 317 "In making our calculations it is safe, as both the earlier (6-10) and the more recent (11-15) works have agreed, to accept the principle that the frequency of the gene mutations produced is simply (linearly) proportional to the amount of the total accumulated dose received, as expressed in r units. Moreover, as some of these same studies show, this relation holds within wide limits, regardless of how short and concentrated or dilute and protracted the exposure may have been, or whether it was given in one treatment or many."

"There are good theoretical grounds for inferring that these principles hold true no matter how small the total dose, or the dose per unit time. Of course, such a sweeping conclusion necessarily involves an extrapolation from actual data. Not until recently has it been possible, because of technical difficulties, to test the mutagenic effectiveness of doses lower than about 13 r per day, totaling 400 r (11-13), and even the most recent work goes down no lower than about 2.5 r per day, totaling 25 r (14, 15)."

Stem ( 1950) Page 433 "The proportionality rule has been proven to hold over a wide range. Figure 155 shows that, for Drosophila, the relation is essentially linear over the range from 25 r to several thousand r. It has further been shown that the frequency of induced mutations is independent of the time over which the radiation is applied."

Stem ( 1960) Page 491 "It has been established for a variety of experimental organisms that the number of mutations induced by radiation is propor-tional to the dose. This proportionality has been proven to hold over a wide range of dosages. Figure 202 shows that, for Drosophila, the relation is essentially linear over the range 25-12,500 r (insects, unlike mammals, can survive after exposure to many thousands of roentgens). It would be desirable to extend the data toward dosages lower than 25 r, for instance, to 10 r, 5 r, and still lower. Since, however, the expected differences are small between the rate of mutations in not-artificially irradiated control organisms and that in organisms exposed to low artificial doses, it is difficult to obtain significant results even with large experiments."

574 Impact of the Stern and Muller deceptions support the conclusion that there was a generally consist- 596 ent view that the nature of the dose response in the low- 597 575 Effect on the radiation genetics literature/community dose zone for mutations was linear. Most of these quotes 598 directly cite the research of Stem and his colleagues as pro- 599 576 In the aftermath of his Nobel Prize Lecture, Muller pub- viding the key evidence supporting linearity, especially that 600 577 lished his Lecture in the Journal of Heredity in 1947 of Spencer and Stern (I 948) and Uphoff and Stem ( 1949). 601 578 (Muller 19!7), assuring its broader distribution. Within This demonstrates the significance and success of the Stem 602 579 4 months of the Noble Prize Lecture, he gave a lecture mediated manipulation of the Caspari and Uphoff studies 603 580 to the New York Academy of Medicine during which he in affecting mutation dose-response beliefs of key research 604 581 affirmed his Nobel Prize Lecture message, stating that leaders of the radiation genetics community. 605 582 there was "absolutely no threshold dose" for mutations 583 and that induced mutational response was proportional to Effect on the BEAR I Committee/Genetics Panel 606 584 the total dose (Table I). This presentation was published in 585 the Academy's journal (Muller !948) soon thereafter. Stem Crow (I 995) noted the following in his historical recount- 607 586 (! 950) also cited Spencer and Stern ( 1948) and Uphoff and ing of the BEAR I Committee Genetics Panel: "the debate 608 587 Stem ( 1949) in his acclaimed textbook, emphasizing that over the nature of the dose response for ionizing radiation 609 588 the dose response for mutations was linear (Table ! ). and mutations had been decided before the convening of the 610 589 These follow-up activities by Stern and Muller had an BEAR Committee in November 1955." The accepted view 611 590 impact on other leading radiation geneticists influencing was clear and unified; the answer for the dose response 612 591 them to adopt the linearity dose-response interpretation. question for mutagenicity was "linearity at low dose." 613 592 Table 2 provides a series of quotations from subsequent When reading the transcripts of the BEAR I Committee 614 593 publications of leading contemporary radiation geneticists. Genetics Panel, one is struck by the absence of debate and 615 594 The quotes are numerous, varied, and a fair representa- even discussion on the issue of dose response (e.g., linear- 616 595 tion of what each author stated. These comments strongly ity vs. threshold). To illustrate the fact that the decision on 617

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Arch Toxicol Table 2 Radiation genetics quotations about the mutation dose-response following Hermann J Muller's Nobel Prize and Curt Stem's (with Spencer, Caspari and Uphoff) mutagenicity papers References Quotes Catcheside ( 1950) Page 592 "The induced mutation is proportional to the total dose over the whole range investigated, down to total doses as small as 25 r. There is good reason to conclude that there is no threshold dose, i.e., no dose so small that it gives no muta-tional effect. Also, the intensity of the radiation appears to be without effect on the frequency of mutation induced by a given total dose. A dose of 50 r given in a fraction of a minute appears to give no greater effect than the same dose given in the course of a few weeks. There is no threshold, no time factor, and no recovery, the effects being cumula-tive."

Glucksmann ( 1950) Page 42 "The induction of gene mutations is linearly proportional to dose even down to levels of 25 r (Spencer and Stern l94X)."

Lefevre ( !950) Page 341 "It has been amply verified that the number of mutations produced by X-rays is linearly proportional to the total dose applied, even when the total dose received is very small (see Spencer and Stern !9-tR). Further, the number of muta-tions produced is independent of the rate of dosage (Uphoff and Stern 1949)."

Sax (J'JSO) Page 332 "The early work by Muller and by Timofeeff-Ressovsky showed a linear relationship between X-ray dosage and muta-tion frequency in Drosophila. It was also found that the induced mutation rate was independent of radiation intensity.

From these observations it was concluded that the X-ray-induced mutations are produced by single 'hits,' and that there is no threshold effect. Spencer and Stern (2) found no increase over the spontaneous mutation rate by irradiating Drosophila for 21 days at 2.5 r/day, but later experiments by Uphoff and Stern (3) indicated that low intensities are effective."

Higgins (I 951) Page9 "As a result of exhaustive experiments on the genetics of the fruit fly, of mice and of many plants, it is held that the number of induced mutations bears a linear relationship to the total amount of radiation absorbed by the sensitive volume of the cell and is independent of either the duration or the intensity of exposure. Consequently, a long exposure to low-level radiation would have the same genetic effect as shorter exposure to a higher level. Experiments of Spencer and Stern ( J948) on the fruit fly show that the percentage of sperm containing a sex-linked lethal mutation is increased about .002 per r of radiation exposure and that 50 r exposure is required to double the natural mutation rate."

"Spencer and Stern (I.e.) conclude their exhaustive study of the validity of the linear relationship between radiation exposure and mutation frequency with the statement (p. 64): ' ... for radiation with X-rays, dosages as low as 25 r produce mutations as drastic in their effects and in the same proportion to the dosage as do exposures to high dosages.

If an extrapolation is permissible, one may assume that there exists no tolerance dose below which mutations are not induced."

"The classical hit theory of induction of mutations, particularly the linear relation between dosage at low levels and mutation rate, has been questioned by Caspari and Stern ( 19~R), who found no significant difference in mutation rates in the sperm of the fruit fly between controls and experimentals exposed to 2.5 r per day for 21 days. Uphoff and Stern ( l 949), however, after further tests, concluded that low-level radiation does produce mutations in fruit-fly sperm and that the apparent inconsistencies of previous results were due to different experimental techniques and errors in sampling."

Stone (!952) Page 657 "There is no threshold for genetic mutations ... " (cited Muller reference 1950, J Cell Comp Physiol 35(suppl I ):9-70.)

Singleton ( 1954a) Page 598 (Discussion)

"That a non-linear relationship exists between dose rate of chronic gamma radiation and mutation rate of endosperm characters seems to have been well established by these experiments. This was shown quite conclusively by dispro-portionately higher mutation rates at the higher dosages, and was definitely indicated by the fact that there seems to be a threshold of dosage required to raise the mutation rate from the spontaneous level to a detectable increase over that level."

Page 599 "These data (i.e., data shown in Singleton 195*la study) showing a definite threshold are in contrast to the Drosophila data of Spencer and Stern ( 194S), where no threshold was indicated even when low doses of radiation were used.

In their experiments the effects of acute radiation were studied. Caspari and Stern ( 19.fR), studying chronic gamma radiation, found no increase over the controls for doses of 2.5 r/day for 21 days. However, it was later demonstrated by Uphoff and Stern ( 1949) that the controls used by Caspari and Stern had an abnormally high sex linked lethal frequency and that actually there was an effect of the chronic gamma radiation of 2.5 r/day."

Kelner et al. ( !955) Page 36 "The linear mutation-dose curve indicated for X-ray induced drosophila lethals (Lethals-Dros:X) is perhaps best exemplified by the data of Spencer and Stem (53) for sex linked Jethals and may be considered as the classical type of mutation-dose relation. Interpreted within the target theory, the linear relation indicates that a single hit is sufficient to produce a mutation."

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Arch Toxicol Table 2 continued References Quotes Nybom et a!. ( 19'i6) Page 81 "In this connection references may be made to the concordant results of Uphoff and Stem ( 1949) who did not find any threshold in Drosophila after low dose rates. A similar result was published by Sax (I 950) using chronic irradiation of Tradescantia pollen."

Lewis (!9:'i7) Page 971 (columns 2 and 3)

(This Science article "Gene mutation has long been known to show a linear relationship with respect to dose of ionizing radiation from stud-was reprinted in ies with Drosophila. This linearity has been extended by Spencer and Stem (43) to doses of 50 and 25 roentgens. Gene Congressional mutation is also known to be directly proportional to the accumulated dose of radiation, even when the radiation is Testimony) chronically administered at a relatively low dose rate, as in the studies of Uphoff and Stem (44)."

Norwood (19)8) Page 1929 "Several geneticists 4 have sketched the background which has lead to the concern of this study. Briefly, realization that radiation increases the mutation rate dates back 30 years to Muller's experiments with fruit flies4e. Spencer and Stem. 5 using more than 50 million flies, showed that genetic damage was proportional to dosage in the important range of 25 to 50 r. Concern has been heightened by recent findings 41 that exposure of mice to a given quantity of radiation increases the mutation rate by about 15 times as much as does an equal exposure of Drosophila, which had formerly served as the sole basis for inferring* human risks."

Spear ( 195R) Page 20 "There is general agreement, however, that mutations can be produced with very low dosage down to a level which approaches natural background (Uphoff and Stern 19-W)."

Newcombe ( 1960) Page 331 "One basic premise which has not so far been seriously challenged is that the number of gene mutations resulting from irradiation varies in direct proportion to the dose. In other words, there is no threshold level of radiation below which the mutations will not be produced."

"In the fruitfly the curve has, by dint of considerable work, been pushed to within 25 roentgens of the origin (Caspari and Stem I'!48; Spencer and Stern 19*+11; Uphoff and Stem !949) (3, 4, 5)."

618 LNT had already been settled prior to the creation of the to a profoundly extended and highly unrealistic exposure 645 619 BEAR I Committee, there was no discussion of the scien- period. In effect, the study was a chronic exposure to a cell 646 620 tific foundations of the LNT, including any documenting type that has only a very short developmental stage. The 647 621 of its theoretical basis and experimental support, including basic concept of the study was not appropriate for a chronic 648 622 its strengths and limitations. As noted above, the Genet- exposure with risk assessment application. The BEAR I 649 623 ics Panel placed a high priority on the chronic exposure Committee incorrectly accepted Stem and Muller's concept 650 624 experiments published under the leadership of Curt Stem. of "chronic" for risk assessment purposes as did the entire 651 625 Yet these studies, even ignoring the control group problems field and regulatory agencies. 652 626 of the Uphoff and Stem experiments, had little or no risk While the BEAR I committee relied upon the findings 653 627 assessment relevance. That is, these were sex-linked reces- of the Drosophila research directed by Curt Stem, it failed 654 628 sive lethality studies in which the spermatozoa were depos- to cite other similarly large-scale Drosophila studies (Bon- 655 629 ited in the spermatheca of the female. The females were nier and LUning 1949; Bonnier et a!. 1949) in which the 656 630 then placed into a type of specialized experimental "hiber- lowest total dose was 8 r, below the lowest dose (25 r) of 657 631 nation" in which there was a profound alteration of the the Spencer and Stem ( l94R) findings. These papers docu- 658 632 diet and a lowering of the temperature, changes designed mented the response of several single genetic loci (e.g., 659 633 to prevent egg production. The females (with the deposited white and forked loci) to which their detailed statistical 660 634 spermatozoa) were then exposed for 21 days (24 h/day) to analysis for mutational studies was applied. The analysis 661 635 gamma irradiation. After the 21 days, the dietary and envi- revealed a linear dose response in the dose range of 700-- 662 636 ronmental conditions were changed to permit egg laying 2,800 r, whereas the linearity response was not observed 663 637 so that the testing for sex-linked recessive lethal mutations in the low-dose range (8-16 r), where the data were sup- 664 638 could take place. In effect, Stem exposed the spermatozoa portive of a threshold response. The authors also sug- 665 639 to ionizing radiation for the equivalent of an entire lifespan, gested that the difference in the shape of the dose response 666 640 something comparable to a 70-80-year human lifespan. between high and low doses was indicative of differing 667 641 The spermatozoa are known to be highly compromised, dose-dependent mechanisms. At the high doses, the lin- 668 642 having lost much of their normal repair capability. The ear dose response was consistent with the target theory of 669 643 study represented a worse case exposure scenario, that is, Timofeeff-Ressovsky et a!. ( 1935), whereas at lower doses 670 644 selection of a very susceptible developmental stage linked mutational effects could be due to the effects of chemical 671

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Arch Toxicol 672 mutagens (i.e., hydroxyl radicals from the hydrolysis of was misled on the published findings of Sparrow and Sin- 725 673 water). The dose-dependent mechanism-based hypothesis gleton as the data did not support the pre-determined linear 726 674 of Bonnier and colleagues (Bonnier and Liining !949; Bon- dose-response conclusion. This analysis also suggests that 727 675 nier et al. 1949) was soon supported with experimental data the BEAR I Committee/Genetics Panel was very selec- 728 676 (Haas et al. 1950; MacKey 195 I; Liining 1954; Barron tive in their choice of what data to consider and that such 729 677 1954). According to Barron (1954), "it is dangerous, how- decisions reveal a prevailing bias supportive of LNT model 730 678 ever, to extrapolate from experimental data with large doses acceptance. 731 679 of radiations to what might take place with small doses. In Since 0.41 r per day of radiation in the Sparrow and Sin- 732 680 biological systems the effect of ionizing radiations differs gleton (1953) hypothesis study is more than 1,000 times 733 681 qualitatively when the radiation dose is changed. Small greater than the naturally occurring intensity, these data 734 682 doses act by indirect action and produce mainly oxidations. do not support the theory that the spontaneously occurring 735 683 Large doses act by two mechanisms," that is, free radical micronuclei are produced by naturally occurring ionizing 736 684 formation via water hydrolysis and by a direct collision, radiation. The findings of Sparrow and Singleton (! l)5l) 737 685 which is consistent with the target theory. were similar to that of Giles (I ')40) from Harvard who 738 686 The Bonnier and Liining ( 1949) (Bonnier et al. 1949) showed that when Tradescantia were "subjected to irradia- 739 687 papers were also critical of the use of sex-linked recessive tion 1,000 times that due to natural radiation .... no increase 740 688 lethal experiments for estimating responses in the low-dose in aberration was found." Other experiments by Giles indi- 741 689 zone due to the "impossibility of differentiating between cated that even using ionizing radiation at some 1,800-fold 742 690 true lethals and semi lethals, and the fact that there are sev- above background no impact on the occurrence of sponta- 743 691 era! hundreds of targets per chromosome ready for lethal neous mutations occurred. 744 692 mutations ... " The lack of target specificity would repre- It is possible to obtain a sense of the personal views of a 745 693 sent an important limitation in the interpretation of dose- number of the members of the BEAR I Committee/Genet- 746 694 response relationships and their potential application to a ics Panel on the matter of dose response via two contempo- 747 695 mechanism-based risk assessment process. Bonnier et al. rary publication avenues: Testimonies at a 1957 Congres- 748 696 (1949) also provided a detailed statistical reanalysis of sional Hearings (Table 3) and journal publications in the 749 697 the Spencer and Stern ( 1948) data challenging the broadly open literature (Table 4) such as a special issue of Scientific 750 698 accepted conclusion that the linearity response applied American on ionizing radiation and several other journals. 751 699 across the entire dose-response range, including the lower Based on these collective comments, it follows that the 752 700 dose range. None of these fundamental technical issues BEAR I Committee/Genetics Panel report and an article in 753 701 were discussed by the BEAR I committee. the journal Science (Table 5) summarizing the report of the 754 702 Another relevant aspect of the discussion on the nature Genetics Panel were replete with statements asserting lin- 755 703 of the mutation dose response involved the research earity at low dose. 756 704 of Arnold H. Sparrow and W. Ralph Singleton of the 705 Brookhaven National Laboratory. Chairman Warren 706 Weaver introduced their research and its relevance to the Placing the new Muller and BEAR I Genetics Panel 757 707 BEAR I Committee/Genetics Panel (Weaver W., Febru- developments in perspective 758 708 ary 5-6, 1956, see page 110-Transcript) (BEAR I 1956).

709 The discussion of the Sparrow and Singleton data was then The story of Muller's Nobel Prize Lecture is important 759 710 led by Committee member Berwind D. Kaufmann, who for its history of science implications, as well as its role 760 711 claimed to have copied several tables from their paper. in affecting the decision of the US National Academy of 761 712 He stated that Sparrow and Singleton showed that 0.41 r Sciences (NAS) to recommend a linearity dose-response 762 713 per day yielded a modestly elevated (i.e., less than twice policy for assessing risks to the genome from ionizing 763 714 the control values) but statistically significant effect on radiation, replacing the threshold dose-response model. 764 715 micronuclei formation. What Kaufmann failed to inform This formal recommendation initiated a series of advi- 765 716 the Committee was that Sparrow and Singleton ( !953) spe- sory and regulatory dominoes in essentially all countries 766 717 cifically stated that a threshold response had been observed to adopt linearity and apply it to somatic effects, that is, 767 718 at a lower dose. In fact, there was no discussion concern- cancer risk assessment, for ionizing radiation and later for 768 719 ing their threshold dose-response statement by the BEAR chemical carcinogens (Calabrese 2009). The linearity deci- 769 720 I Committee/Genetics Panel. The data in Table 2 (page 35) sion of the NAS BEAR I Committee/Genetics Panel was 770 721 of the published paper by Sparrow and Singleton ( 1953) strongly championed by Muller, the titular leader of radia- 771 722 show that 0.084 r per day caused no significant increase in tion geneticists and with strong ties to all radiation geneti- 772 723 micronuclei. This recounted activity of the BEAR I Com- cists on the BEAR I Committee/Genetics Panel. In fact, the 773 724 mittee/Genetics Panel demonstrates that it either ignored or switch to linearity, which was ushered into the international 774

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Arch Toxicol Table 3 BEAR I Committee Genetics Panel member quotes at Joint Committee on Atomic Energy-1957 References Quotes Muller ( l 956) Page 392 "In material of varied kinds, but more especially in Drosophila, there is good evidence that over a considerable range of dose (in Drosophila, from some 50 r to more than I ,000 r, a more than 20-fold range) the frequency of point muta-tions (like that of chromosome breaks) is directly proportional to dose."

Crow ( l957a) Page 1013 "4. Evidence from experimental animals, principally Drosophila, indicates that the number of mutations produced is strictly proportional to the amount of radiation received. There are departures from this straight-line relationship at high doses, but these are too high to be likely to be encountered in any ordinary human situation. It is technically impossible to test this relationship for the very lowest doses, but the straight-line relation holds down to the smallest amounts that have been studied."

"For these reasons a simple proportionality between the amount of radiation and the number of mutations is fully accepted by geneticists."

"The proportionality between dose and mutation production holds irrespective of the intensity or spacing of the dose."

Representative Holifield (page 1013) questions Dr. Crow:

"This, then, would establish as far as the majority of the geneticists are concerned the principle of linear progression in deleterious effects of radiation regardless of amount?"

Dr. Crow answers:

"That is correct. A nonthreshold situation, to put this in yesterday's vocabulary."

"This means that there is no such thing as a safe dose of radiation to the population. Any amount of radiation, however, small, that reaches the gonads-testes or ovaries-<>f a person who may later reproduce, involves a risk proportional to that amount."

Glass ( 19:'i7a) Page 1030 "The data are most extensive for the fruitfly and the lowest dose that has actually been studied is 25 r."

Page 1031 "Because a mutation can be produced by a single ionization in the right place, there is no threshold below which the amount of radiation is too small to produce mutations-that is, every dose produces mutations with a probability equal to its magnitude."

"This is to repeat what Dr. Crow said, that there is no safe dose of mutation. This curve continues down without any threshold until it hits the zero point. .. "

Muller ( 1957a) Page 1052 "In respect to the fact that probably there is no threshold, that these effects are proportional to the dose, in this respect these effects of radiation-and also the leukemia-<>n the exposed individual himself resemble those produced by the radiation in weakening descendants."

"You have heard Dr. Glass and Dr. Crow say that geneticists are convinced that there is no threshold for the genetic effects and that others, too, now accept that principle for the genetic effects."

"If this is true of these other effects, and it is certainly time we knew whether it was-I think the evidence is convincing that it is-then this important resemblance between the effects on later generations and on the exposed generation is probably not an accidental resemblance. For there is growing reason to infer that this shortening of life and the other long delayed damage done to an exposed individual have their basis in damage done to the genetic material-the chromosomes and their contained genes-<>f the body's ordinary cells, those of the blood, skin, glands, and so forth, similar to the damage done in his reproductive cells that is passed on to later generations."

Page 1056 "Through work on the fruitflies where we have the most exact knowledge to date, unless Dr. Russell has more exact knowledge on mice now, we can get a kind of minimum estimate of the amount of damage to the children by a given amount of irradiation of the parents."

Muller ( 1957b) Page 1066 "Since there is much evidence indicating a linear relation between the radiation dose and the frequency of the induced point mutations, even at extremely low doses, and the exactly cumulative nature of these radiation effects, it becomes possible to arrive at probable estimates of the minimum damage done to subsequent generations by any given chronic or acute exposure of parents."

Page 1067

" ... leukemia and some other malignancies, the induction of which may also be linearly dependent upon radiation dose ... "

Joint Committee Page 12 on Atomic Energy " ... geneticists believe that the direct proportion applied down to zero dose-that is, that there exists no safe "threshold"

( 1957) below which the dose produces no damage, and that damage occurs from any irradiation of the genetic cells, no mat-ter how small the dose."

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Arch Toxicol Table 4 BEAR I Committee Genetics Panel member quotes on low-dose linearity in journals after the BEAR I Committee References Quotes Crow ( 1957h) Page 19 (column 2)

"2. The number of mutations produced is directly proportional to the dose in roentgens. The linear propor-tionality over wide dose ranges has been shown in several organisms, especially in Drosophila."

"Experimental verification in Drosophila has been carried to as low as 25 r ... "

Page 20 (column I)

"The proportionality between dose and mutation production holds irrespective of intensity or spacing, ... "

Page 20 (column 2)

"The conclusions of the previous section imply that there is no such thing as a "safe" dose. Any increase in radiation, however, small, involves a risk proportional to that amount."

Glass (1957h) Page 956 "Our present evidence indicates that the frequency of these point mutations always increases linearly with the radiation dose (Fig. I). In Drosophila studies this holds over the range from 25 r to 6,000 r. In some plants, the linear range has been extended down to about 5 r. In mice, the linearity in relation to dose holds over the range from 300 r to 600 r, and there is no sign that it does not hold at lower doses. This linear proportionality to dose, over and above the spontaneous frequency of mutation, implies that (a) as long as dosage is measured in terms of roentgens, that is, in terms of the ionization produced by the radiation, absorbed quanta do not interact to produce effects, but are individually effective; and (b) there is no sign of a threshold dose below which mutations are not produced. Rather, even the lowest doses are proportionally mutagenic, and all doses, however, distributed, are additive or cumulative in effect."

Beadle ( 1959) Pages 225 and 226

" ... thus there is probably no threshold below which radiation will produce no mutations. Since there is no repair mechanism, once the mutation process is complete, mutations induced at different times will tend to accumulate in a line of descent. .. "

Hollaender and Stapleton (I 959) "In sum, cell studies have served to elucidate the basic mechanism by which ionizing radiation damages the living organism. They have provided no evidence that there is a true threshold of dosage below which ionizing radiation produces no harmful effects ... "

775 community by the BEAR I Committee Genetics Panel, is spontaneous mutation rate, there is no evidence that he 801 776 the most significant action in regulatory environmental disputed the control group mutation rate reversal decision 802 777 public health history with ever expanding social, political, of Stem barely a year later and of Muiler's equaiiy strange 803 778 economic, and public health implications (Hamblin :2007). affirmation of Stem's position as weii (Muiier I950a, b). 804 779 The present paper provides the first documentation of A January 27, 1949, Jetter from Cas pari to Stem supported 805 780 how Muiler (Muiier 1950a, b, l954a, b) himself used the the publication of the Uphoff and Stem ( 1949) paper now 806 781 carefuiiy constructed activities of Stem (described in detail adopting part of the mantra of Stem, that is, that there is 807 782 in Calabrese 20 I I b) to enhance the concept of linearity and considerable variability in the mutagenic frequency of 808 783 to protect his reputation. Muiler lent credibility to the tech- sperm prolongedly stored in the spermatheca. This conclu- 809 784 nical note of Uphoff and Stem ( 1949) while further mar- sion provided the opportunity to rehabilitate the inexplic- 810 785 ginalizing the Caspari and Stem study results (Caspari and itly low control group values of Uphoff. Caspari, however, 811 786 Stem 1948). The stakes were high on multiple levels and would not go so far as to also state that his control values 812 787 these core individuals knew it. Stem and Muiier needed were unusuaiiy high. At the time of the Uphoff and Stem 813 788 to prevent the acceptance of the Caspari and Stem (I 94R) ( 1949) article, there were only two papers published in the 814 789 study findings in order to sustain the single-hit linearity literature (Rajewski and Timofeeff-Ressovsky !939; Kauf- 815 790 model. They also needed any criticisms of the Spencer and mann 1947) on aged sperm and mutation and the published 816 791 Stem (1948) and Uphoff and Stem ( 1949) papers to be abstract of Muiier ( 1946b). Each supported the mutation 817 792 muted. They were successful as other leaders of the radia- frequency of Caspari. These findings are consistent with 818 793 tion genetics community simply failed to address the seri- subsequent mutation frequencies in aged sperm stored in 819 794 ous limitations of the Spencer and Uphoff findings while the spermatheca of female Drosophila (Byers 1954; Byers 820 795 incorrectly asserting that the Cas pari and Stem ( 1948) and Muiier 1952; Rinehart 1969; Graf 1972; Muiier et a!. 821 796 paper suffered from an aberrantly high control value, sim- 1961 ). Muiler et a!. ( 1961) stated that "The data clearly 822 797 ply re-stating the demonstrably incorrect, but authoritative showed a rise in mutation frequency (averaging some .06 823 798 conclusion of Muiier ( J950a). percent of recessive lethals in the X chromosome per week) 824 799 Despite the fact that Caspari had successfuiiy rebutted resulting from storage of the mature spermatozoa in the 825 800 the first challenge of Stem concerning the control group female" (page 213). Note the striking similarity of how 826

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Arch Toxicol Table 5 Low-dose linearity quotation in the journal Science from article summarizing the findings of the BEAR I Committee Genetics Panel References Quotes BEAR I (1956) Page 1159 (column 2)

" ... the genetic damage done, however, felt and, however, measured, is roughly proportional to the total mutation rate."

Page 1160 (column I)

"3) Any radiation dose, however, small, can induce some mutations. There is no minimum amount of radiation dose, that is, which must be exceeded before any harmful mutations occur."

Page 1160 (bottom column I)

"The probable number of additional induced mutations occurring in an individual over a period of time is by and large proportional to the total dose of extra radiation received, over that period, by the reproductive organs where the germ cells are formed and stored."

Page 1160 (top column 2)

"The total dose of radiation is what counts, this statement being based on the fact that the genetic damage done by radiation is cumulative."

Page 1162 (column 2)-how harmful are radiation-induced mutations?

"I) Thus the first and unanimous reply to the question posed by the title to this section is simply this: Any radiation is genetically undesirable, since any radiation induces harmful mutations. Further, all presently available scientific informa-tion leads to the conclusion that the genetic harm is proportional to the total dose ... This tells us that a radiation dose of 2X must be presumed to be twice as harmful as a radiation dose of X ... "

Page 1164 (column I)

" ... for there is no such figure other than zero." [referring to whether there is an amount of radiation which is genetically harmless (preceding phase))

Page 1164 (column I)

"As geneticists we say: keep the dose as low as you can."

Page 1165 (last sentence)

"From the point of view of genetics, they are all bad." (referring to the effect of exposures to ionizing radiation) 827 Uphoff and Stem (I 947) characterized Muller's data some and radiation genetics domains, the committee as a whole 854 828 14 years earlier, "a weekly increase of about 0.07 % ... " lacked extensive experience in conducting low-dose, 855 829 The 0.06 % increase would yield an estimated 0.28 % dose-response studies. Only two of the members had 856 830 (i.e., 0.06 % x 3 weeks + 0.10% background = 0.28 %) extensive direct experimental dose-response experience 857 831 mutation incidence after 3 weeks, consistent with the Cas- (i.e., Demerec and Russell) up to the time of the BEAR I 858 832 pari and Stern ( 1948) findings, the logic used in Uphoff meetings. This experience was essential for evaluating the 859 833 and Stern ( l94 7) and with the Muller ( 1946b) statement nature of the dose response in the low-dose zone. Of these 860 834 that "spermatozoa aged several weeks in the female may two, Demerec had the most extensive and varied experi- 861 835 contain several times as many mutations as they originally ence having dealt with multiple models and agents as well 862 836 had." Furthermore, the reported inter-study variability for as different types of radiation. His research experience 863 837 mutations of aged sperm and/or stored sperm aged in the on dose response was spread over a 25-year period start- 864 838 spermatheca appears modest with 95 % confidence inter- ing about 1931. Nonetheless, his dose-response experience 865 839 vals typically being about +/-25-30 % of the mean. The with Drosophila was limited to only a few high dose stud- 866 840 attempt by Stern, therefore to assert that the very low val- ies during the 1930s, a key limitation. Despite his signifi- 867 841 ues of Uphoff reflected a highly variable response endpoint cant and prolonged career at Oak Ridge, Russell was rela- 868 842 was not supported in the contemporary and subsequent lit- tively new to the dose-response research area, with about 869 843 erature. Stern never argued his case by a comparative data 5-6 years experience at the start of the BEAR I Commit- 870 844 assessment nor did he address the apparent contradiction tee in 1955. In the case of Russell, his developing research 871 845 with the Muller data and comments which he (i.e., Stem) findings with mice were still somewhat premature, having 872 846 previously used when he concluded that the Caspari data little impact on BEAR I Committee/Genetics Panel con- 873 847 were credible while those of Uphoff were not. He simply elusions. Among the remaining members of the commit- 874 848 made an authoritative declaration that was accepted without tee, Muller's principal dose-response experience is found 875 849 question or comment by the radiation genetics community. in the research of Hanson and Heys (! 929), and Oliver 876

( 1930, 1931) at the University of Texas and Ray-Chaudhuri 877 850 BEAR I Committee/Genetics Panel ( 1944) at Edinburgh (completed in 1939), as well as his 878 consultant role with Stem from 1943 to 1946. Limited rei- 879 851 The BEAR I Committee/Genetics Panel was comprised evant low dose-response research based on the publication 880 852 of outstanding scientists and national leaders. Despite record experience was found for Berwind Kaufmann. Alex- 881 853 their significant individual accomplishments in scientific ander Hollaender, PhD in physical chemistry, had made 882

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Arch Toxicol 883 important contributions on the effects of UV wavelengths of the same order of magnitude as that found by Timofe- 936 884 specificity on mutation in bacteria and fungi. He became eff and Kaufmann." In fact, Caspari and Stern ( 1948) cited 937 885 the director of radiation biology research at Oak Ridge, hir- a 1947 paper by Kaufmann as support for control group 938 886 ing Russell. Hollaender had no experience with Drosophila values of their study. Muller and Kaufmann, both BEAR I 939 887 research. H. Bently Glass' low-dose experimental research committee members, therefore, reported research on muta- 940 888 experience was quit~ limited during BEAR I, becoming far tion incidence of Drosophila aged sperm findings con- 941 889 more extensive only after BEAR I. Importantly, very lim- sis tent with the findings of the Cas pari and Stern ( 1948) 942 890 ited to no meaningful dose-response research experience is paper. Thus, the BEAR I Committee/Genetics Panel should 943 891 apparent for the remaining 11 members [George W. Bea- have been informed on the issue of control group valid- 944 892 die, Charles W. Cotterman, James F. Crow, Gioacchino ity by Demerec, Kaufmann, and/or Muller as it related to 945 893 Failla, Clarence C. Little, James V. Nee!, Tracy M. Sonne- the research of the Caspari and Uphoff studies. However, 946 894 born, Alfred H. Sturtevant, Sewall Wright, Warren Weaver based on the transcripts of the BEAR I Committee/Genet- 947 895 (Chair), and Shields Warren] of the BEAR I Committee/ ics Panel, Demerec, Kaufmann and Muller did not provide 948 896 Genetics Panel. This situation resulted in the "senior" dose- this information. Knowledge of the mutation rates in aged 949 897 response experience to reside with Demerec and Muller, Drosophila sperm should have led to a reconsideration of 950 898 two individuals on record to save the "hit" model. the Caspari and Stern (I 948) paper as well as generated 951 899 The geneticists on the BEAR I committee were princi- serious questions about the findings and interpretations 952 900 pally basic researchers; their experimental approaches were of the Uphoff and Stern ( 1949) data. This was a key issue 953 901 neither dose response nor risk assessment oriented. Even affecting which study would be relied upon by the BEAR I 954 902 Muller ( l 950a, h) claimed that the work of Spencer and committee. By their actions, the BEAR I committee Genet- 955 903 Uphoff (with Stern) at low doses would markedly extend ics Panel came to the erroneous conclusion that the Cas- 956 904 his and his students' (e.g., Hanson and Oliver) research pari study was unreliable due to its "unusually high control 957 905 conducted at very high doses. Further, in the detailed com- group value." 958 906 ments that Muller sent to Stern about the Spencer (Lilly The future of ionizing radiation risk assessment was 959 907 Library ! 946, September 13) and Caspari (Lilly Library largely determined by the actions of a few, by the failure of 960 908  ! 947a, January 14) manuscripts, nearly all dealt with fun- the scientific community, especially the radiation genetics 961 909 damental biological/genetic questions with little direct community, to probe deeper into the key findings of Stern 962 910 relevance to risk asse.ssment. Multiple study design issues and his colleagues and journals such as Science that pub- 963 911 and other methodological/analysis problems documented in lished influential but poorly documented findings (Uphoff 964 912 Calabrese (20 1I b) for the Spencer and Stern ( 1948) paper and Stern 1949). As has been pointed out, the linear- 965 913 were not identified by Muller (Lilly Library 1946, Septem- ity paper of Spencer and Stern ( 1948) was burdened with 966 914 ber 13). The members of the BEAR I Committee/Genetics numerous methodological limitations that only recently 967 915 Panel looked to Muller for leadership on matters related to have been documented, as well as statistical analysis limita- 968 916 the dose-response. However, Muller displayed critical lim- tions that challenged the conclusion of linearity at low dose 969 917 itations in assessing such studies based on his written state- (Bonnier and LUning 19-f9; Bonnier et a!. 1949) while the 970 918 ments. Thus, the methodological and analysis limitations of Cas pari and Stern ( 1948) findings supporting a threshold 971 919 the Spencer and Stern ( 1948) paper and the serious flaws perspective were unfairly marginalized (Calabrese 20 I I b). 972 920 of the Uphoff and Stern (! 949) paper were missed by the Furthermore, the BEAR I Committee/Genetics Panel failed 973 921 radiation genetics community and the BEAR I Committee/ to require Stern to provide the promised detailed account- 974 922 Genetics Panel, a condition that continues (Lipshitz 2005). ing for the Science article (Uphoff and Stern I 949) upon 975 923 Of further note is that Muller ( I946b) and Kaufmann which they so heavily relied. 976 924 ( l 94 7) published findings on the control group mutation According to Muller (J 950a, b), by 1950, the radiation 977 925 rate of aged Drosophila sperm that supported the findings genetics community had accepted the linearity risk assess- 978 926 of Caspari and Stern ( 1948). Kaufmann worked closely ment paradigm (Table 2). Their belief was based largely 979 927 with and under the direction of Demerec at Cold Spring on the fruit-fly work of Stern and his associates as well as 980 928 Harbor at that time. Furthermore, an October 7, 1947, letter the leadership, prestige, and authority of Muller, as few of 981 929 (i.e., 6 weeks before submitting his paper to Genetics) from the geneticist members of the BEAR I Committee/Genet- 982 930 Caspari to Stern (American Philosophical Society 1947g, ics Panel had relevant experience with low-dose research. 983 931 October 7) stated that "I have discussed the paper (the By the time, the National Academy of Sciences BEAR I 984 932 Caspari/Stern manuscript) with Demerec and Kaufmann. Committee/Genetics Panel convened, therefore, the deci- 985 933 Both did not find very much to suggest ...... Both Demerec sion over the nature of the response in the low-dose zone 986 934 and Kaufmann were impressed by the amount of material had been decided by the radiation genetics community 987 935 which we have. The ageing effect in our experiments is as there was no dispute or even debate within the BEAR 988

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Arch Toxicol 989 I Committee/Genetics Panel over the adoption of linearity porting the "uninterpretable" data of Uphoff to achieve 1008 990 to replace the threshold model for germ-cell mutagenic- a linearity interpretation. The bases of these actions are 1009 991 ity (Crow 1995). The actions of Stern and Muller had led documented in this paper. 1010 992 the way, assuring that the ends (i.e., linearity) justified the 3. The paper shows how the actions of Stern and Muller 1011 993 means (i.e., unfair/improper scientific evaluation). In fact, it affected numerous publications and the dose-response 1012 994 is from this heritage and upon this foundation that regula- beliefs of leaders of the radiation genetic community 1013 995 tory cancer risk assessment theory and practice in the USA and the NAS BEAR I Committee/Genetics Panel, 1014 996 and throughout the world was built. affecting the adoption of linearity at low dose for ion- 1015 izing radiation-induced mutation and eventually for 1016 carcinogen risk assessment for ionizing radiation and 1017 997 Conclusions chemical carcinogens. 1018

4. The findings demonstrate that the adoption of the LNT 1019 998 1. This paper provides specific documentation of how model for risk assessment lacked a proper scientific 1020 999 Hermann J. Muller supported and extended the like foundation, yet was accepted by regulatory and public 1021 1000 actions of Curt Stern to prevent the scientific com- agencies worldwide. 1022 1001 munity from discovering Muller's Nobel Prize lecture 1002 deception and to promote his ideological goal of line-1003 arity at low dose for ionizing radiation risk assessment Unresolved issues 1023 1004 (Table 6).

1005 2. Muller strengthened the questionable actions of Stern 1. Why didn't Stern publish the follow-up detailed paper 1024 1006 in key publications in early 1950s while improperly containing the entire methodology for all the relevant 1025 1007 discrediting the threshold findings of Caspari and sup- data for the Uphoff three experiments? 1026 Table 6 A summary concerning Muller's actions that affected the discrediting of Caspari's findings and acceptance of the Uphoff and Stem conclusions A five-page detailed letter sent from Muller to Stern dated January 14, 1947, concerning scientific strengths and limitations of the Caspari and Stem manuscript provided no comment on the control group lethality data Muller was actively researching the area of spontaneous mutations in sex-linked recessive lethality studies using aged sperm stored in the sper-matheca of female fruit flies. This was the research method of the Caspari and Stem paper. Muller had been doing extensive research on this topic since the early 1940s. He was a leading authority on the topic Muller provided his spontaneous control group data to Stem ("Appc~ndix" section) in order to address the concern that Stem expressed about the apparently high control group values of Caspari Based on the data of Muller, Uphoff and Stem ( l9*I7) determined that the average weekly spontaneous mutation rate in Drosophila sperm stored in the spermatheca of the female was about 0.07 %, yielding an additional mutation increase in about 0.21 %by 3 weeks, the length of the Caspari sperm storage time. The 0.21 %increase would be added to a background value of about 0.10 %, yielding an estimated control group value of about 0.31 %. The 95 %confidence intervals were about +/-0.07 %, with an approximate range of 0.24-0.38 %. The values were obtained when studies were conducted at about 25 °C. At the lower temperature of 18 oc used by Caspari, it was estimated by Stem (and Uphoff) that the rate of increase might be reduced to 0.05 % per week. This would result in an estimated value for the Caspari control of about 0.25 %, nearly identical to his final adjusted value (i.e., 0.2489%)

Based on these data, Uphoff and Stem ( 19*17) concluded that the Muller data supported the Caspari conclusion that his control data were well within the normal range and not unusual or aberrant. The Muller data lead Uphoff and Stem ( 1947) to conclude the Uphoff findings were uninterpretable Continued research in the area of spontaneous mutation in sperm stored in the spermatheca by Muller and his graduate students at the University of Indiana were consistent with this conclusion and quantitative assessment (Byers 1954; Byers and Muller 195~.; Graf 1972). These findings were also consistent with that published by other researchers as well (Kaufmann I 9-1 7; Rinehart 1969)

Based on this information, the statements of Muller that Caspari's control group data were unusually high are inconsistent with: (I) His own data and that published by other researchers; (2) his previously detailed assessment of the Caspari data; (3) how Uphoff and Stern (I <J-17) evaluated the Muller data, an evaluation that Muller was knowledgeable of, based on an acknowledgment in the Uphoff and Stem ( 19.:\7) paper, and (4) internal written correspondence between Stem and Caspari This assessment indicates that Muller's statements that Caspari's control group data were unusually high and adversely affected Caspari's thresh-old interpretation are contradicted by the body of evidence While Muller repeatedly challenged the credibility of the Caspari findings by attacking his control group data, he made no statement about the reliability of the extremely low control group data of Uphoff. In fact, he would consistently cite the Uphoff and Stem ( 1')-19) paper as being a critical reference to support a linearity perspective The collective findings on these matters indicate that Muller displayed compromised scientific judgment, having a significant impact on the scientific literature and national and international risk assessment policy that continues to the present

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Arch Toxicol 1027 2. Why didn't the radiation geneticist community demand mutation rate under various physiological conditions. May 1075 1028 that Stern publish these findings? I therefore ask you two questions and will you permit me to 1076 1029 3. Why didn't Stern address the scientific basis, if any, use your answers in a report which I am just preparing for 1077 1030 of why he reversed his position on the Uphoff control the Manhattan Project? Obviously, full credit for it would 1078 1031 group data? be given. The questions are: (I) What is the spontaneous 1079 1032 4. Why didn't Caspari challenge any of the multiple mutation rate in sperm derived from Canton-special males 1080 1033 papers that claimed that the Caspari control group data of from 3- to 6 days old? (2) What is the weekly increase in 1081 1034 were unusually/abnormally high or that their paper mutation rate of sperm from such males stored in females?" 1082 1035 displayed "different techniques" or had "errors in sam- On August 4, 1947 (Lilly Library 19l7c), Muller 1083 1036 piing" that accounted for their threshold-like findings? responds "When sperm were stored in females, there was a 1084 1037 5. Why did Muller agree to let Uphoff and Stem ( 19-1-7) weekly increase in the mutation frequency of about 0.07 %, 1085 1038 acknowledge the use of his aged sperm data that sup- on the average." On August 7, 1947 (American Philosophi- 1086 1039 ported the Caspari control groups findings and then cal Society l947c), Stern cabled Muller asking him the 1087 1040 repeatedly claim that Caspari's control group values temperature used and on August 8, 1947 (American Philo- 1088 1041 were unusually high, adversely affecting the credibility sophical Society 1947d), Muller answered via cable indi- 1089 1042 of this paper? eating "25 °C." A subsequent undated letter, but most likely 1090 prior to September 9, 1947 (American Philosophical Soci- 1091 1043 Acknowledgments The research on the topic of hormesis has been ety 19-+ 7.; ), Muller noted "A recalculation of my data gives 1092 1044 supported by awards from the US Air Force and ExxonMobil Founda-the figure of 0.08 % instead of 0.07 % as the frequency 1093 1045 tion over a number of years. Sponsors had no involvement in study 1046 design, collection, analysis, interpretation, writing, and decision to of lethal accumulating in mature sperm per week." Since 1094 1047 submit. Uphoff and Stern ( 19-+ 7) did not include this correction in 1095 their report to the AEC it suggests that this undated letter 1096 1048 Conflict of interest The author declares that there is no conflict of was received after submittal of their report to the AEC. 1097 1049 interest.

The control value therefore used by Uphoff and Stem 1098

( 1947) of 0.07 % for the estimated mutation rate of the 1099 sperm stored in the spermatheca was based on the earlier 1100 1050 Appendix letter correspondence-supplied estimates of Muller (Lilly 1101 Library l9+7b, c, February 3 and August 4) which Muller 1102 1051 Stem-Muller temporal letter exchange concerning the later clarified as being slightly in error. 1103 1052 aged-stored sperm control mutation rate (Source: Lilly The Caspari and Uphoff studies used Drosophila mela- 1104 1053 Library, Stem-Muller correspondence) nogaster fruit flies, breeding Canton-wild-type (S). males 1105 with Muller-5 females. Muller claimed (Lilly Library 1106 1054 Curt Stem wrote a letter to Hermann J. Muller on January 1947c, August 4) that he never conducted mutation experi- 1107 1055 22, 1947 (American Philosophical Society 194 7a), inform- ments with aged males of the Canton-wild-type stock. 1108 1056 ing him that "At the present time it looks as if our new con- Muller stated that he had tested the aged sperm mutation 1109 1057 trol data (probably the results of the first 3 months of the frequency in "a number of different stocks (of Drosophila 1110 1058 first Uphoff experiment; note that her first month's reading males) without finding any difference." The rate of increase 1111 1059 was an especially low mutation rate of 0.005 %) for aged on a weekly basis was said to be 0.07 % on average. This 1112 1060 sperm are considerably below those of Caspari's." He then value of 0.07 % is believed to be prior to the correction 1113 1061 asked Muller to "send me your figures on rate of sex-linked to 0.08 %. This suggests that Muller did not observe sig- 1114 1062 lethal in sperm aged several weeks, (most desirably, if you nificant inter-stock variation in mutation rates of the stored 1115 1063 have them, data on 3 weeks), in comparison to control data sperm. 1116 1064 from non-aged sperm?" Stern seems to have completed his Uphoff and Stern 1117 1065 On February 3, 1947 (Lilly Library 19-l?b, February 3), ( 19!7) paper for the Manhattan Project during August, 1118 1066 Muller answered by stating that" .... sperm of males which 1947. Stern knew that Uphoff's mean mutation fre- 1119 1067 are about a week old and have been copulating freely (as in quency was 0.1682 % (0.1365-0.2097 %). This suggests 1120 1068 Caspari's experiment) during that period have only about a weekly mean increase in mutation rate of 0.0227 % 1121 1069 .07 or .08 % of lethal. Thus, the latter sperm, after 3 weeks, (0.0122-0.0366 %), far lower than the 0.07 or 0.08 % 1122 1070 should contain something like .28 % of lethal." mean weekly increase in Muller. When Stern wrote to 1123 1071 On July 23, 1947 (American Philosophical Society Muller on September 9, 1947, he stated that for the Can- 1124 1072 1947b), Stem writes Muller again stating that "I have mis- ton-special stock " ... the weekly increase is considerably 1125 1073 laid your letter of some months ago (February 3, 1947, let- less than that found by you and others. It seems to be 1126 1074 ter) in which you gave me some details of your own on the much more of the order of 0.03-0.05." This September 9, 1127

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Arch Toxicol 1128 1947, letter was written probably just after the submission Barron ESG (1954) The effect of x rays on systems of biological 1182 importance. In: Hollaender A (ed) Radiation biology, volume I: 1183 1129 of the Uphoff and Stem (I 947) paper to the AEC, and high energy radiation, chapter 5. McGraw-Hill Book Company, 1184 1130 definitely before the submission of the Caspari and Stern NewYork,pp283-314 1185 1131 ( 194S) paper for publication by Genetics (i.e., November Beadle GW (1959) Ionizing radiation and the citizen. Sci Am 1186 1132 25, 1947). Thus, the judgments of Uphoff and Stern that 201:219-232 1187 1133 found that Uphoff's data were "uninterpretable" and that BEAR I (1956) Genetic effects of atomic radiation. Science 1188 124:1157-1164 1189 1134 supported the reliability of the Caspari control data were Bonnier G, Luning HG (1949) Studies of X-ray mutations in the 1190 1135 made with the information provided by Muller during the white and forked loci of Drosophila melanogaster. I. A Statistical 1191 1136 summer of 1947. The apparent argument that Stern seems analysis of mutation frequencies. Hereditas 35:116-189 1192 1137 to be suggesting in his September 9, 1947, letter to Muller Bonnier G, Luning HG, Perje AM (1949) Studies of X-ray muta- 1193 tions on the white and forked loci of Drosophila melanogaster. 1194 1138 is that the Canton-wild-type stored sperm in the female II. A study of the formation of Gynandromphs and other kinds of 1195 1139 may yield uniquely lower control mutation values. The mosaics. Hereditas 35:301-336 1196 1140 argument is tenuous as the far higher weekly rate was Byers L (1954) Thermal effects on the spontaneous mutation rate in 1197 1141 consistently shown by multiple investigators, and with mature spermatozoa of Drosophila melanogaster. Caryologia 1198 Suppl 1(6):694-696 1199 1142 multiple Drosophila stocks, only being low in two Uphoff Byers L, Muller HJ (1952) Influence of ageing at two different tern- 1200 1143 experiments. In fact, significant inter-strain differences on peratures on the spontaneous mutation rate in mature sperrnato- 1201 1144 the frequency of dominant lethal mutations as induced by zoa of Drosophila melanogaster. Genetics 37(5):570-571 1202 1145 radiation were not reported in various Drosophila strains, Calabrese EJ (2009) The road to linearity: why linearity at low doses 1203 became the basis for carcinogen risk assessment. Arch Toxicol 1204 1146 including the Canton-special wild-type strain (Demerec 83:203-225 1205 1147 and Fano !944; Stromnaes 195! ). This suggestion by Calabrese EJ (2011 a) Toxicology rewrites its history and rethinks its 1206 1148 Stern was not included in the Uphoff and Stern ( 194 7) future: giving equal focus to both harmful and beneficial effects. 1207 1149 report. Environ Toxicol Chern 30(12):2658-2673 1208 Calabrese EJ (2011 b) Key studies to support cancer risk assessment 1209 1150 This letter exchange between Stern and Muller fails to questioned. Environ Mol Mutagen 52(8):595-606 1210 1151 provide support for the later statements of Muller that Cas- Calabrese EJ (20IIc) Muller's Nobel lecture on dose-response 1211 1152 pari's control group was unusually high. The Muller data for ionizing radiation: ideology or science? Arch Toxicol 1212 1153 and statements also do not provide support for the con- 85(12): 1495-1498 1213 Calabrese EJ (2012) Muller's Nobel Prize lecture: when ideology pre- 1214 1154 elusion that the low Uphoff control data were in a normal vailed over science. Toxicol Sci 126(1 ): 1-4 1215 1155 range. None of this information was provided by Stern in Caspari E. Stem C (1948) The influence of chronic irradiation with 1216 1156 his Science publication to permit the scientific community gamma rays at low dosages on the mutation rate in Drosophila 1217 1157 to better evaluate the Uphoff and Caspari control group melanogaster. Genetics 33:75-95 1218 Catcheside DG (1950) Radiations and genetics. Practitioner 1219 1158 data. 165(990):590--593 1220 Crow JF (1957a) Testimony-Statement of Dr. James F. Crow, Pro- 1221 fessor of Genetics and Zoology, University of Wisconsin. Hear- 1222 ings before the Special Subcommittee on Radiation of the Joint 1223 1159 References Committee on Atomic Energy, Congress of the United States. 1224 85th Congress, I st session, part I. United States Government 1225 1160 American Philosophical Society (1946) Muller letter to Stem. Stem Printing Office, Washington DC 1226 1161 Papers, Muller File-Box 16, 12 Nov Crow JF (1957b) Genetic considerations in establishing maximum 1227 1162 American Philosophical Society (1947a) Stem letter to Muller, 22 Jan radiation doses. Radiology 69(1):18-22 1228 1163 1947 Crow JF (1995) Quarreling geneticists and a diplomat. Genetics 1229 1164 American Philosophica~ Society (1947b) Stem letter to Muller, 23 140:421-426 1230 1165 July 1947 Demerec M, Fano U (1944) Frequency of dominant lethal induced 1231 1166 American Philosophical Society (1947c) Stem letter to Muller by radiation in sperms of Drosophila melanogaster. Genetics 1232 1167 (cable), 7 Aug 1947 29:348-360 1233 1168 American Philosophical Society (1947d) Muller letter to Stem, 8 Aug Erk FC (2009) Biographical memoirs. H. Bentley Glass. Proc Am 1234 1169 1947 Philos Soc 153(3):327-339 1235 1170 American Philosophical Society (1947e) Stem letter to Muller, 9 Sept Evans RD (1949) Quantitative inferences concerning the genetic 1236 1171 1947 effects of radiation on human beings. Science 109:299-304 1237 1172 American Philosophical Society (1947f) Caspari letter to Stem. Stem Giles N (1940) Spontaneous chromosome aberrations in Tradescantia. 1238 1173 Papers, 25 Sept 1947 Genetics 25:69-87 1239 1174 American Philosophical Society (1947g) Caspari letter to Stem. Stem Glass B (1957a) Testimony-Statement of Dr. Bentley Glass, Profes- 1240 1175 Papers, Box 21, 7 Oct sor of Biology, the Johns Hopkins University. Hearings before the 1241 1176 American Philosophical Society (Undated) Stem letter to Caspari, Special Subcommittee on Radiation of the Joint Committee on 1242 1177 July-Aug 1947 Atomic Energy, Congress of the United States. 85th Congress, 1st 1243 1178 American Philosophical Society (1949) Stem letter to Caspari. Stem session, part I. United States Government Printing Office, Wash- 1244 1179 Papers, 9 Feb 1949 ington DC 1245 1180 American Philosophical Society (1973) A geneticists' journey, pp Glass B (1957b) The genetic basis for the limitation of radiation 1246 1181 1-19, Stem Nov 1973 exposure. Am J Roentgen Radium Ther Nucl Med 78(6):955-960 1247

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Arch Toxicol 1248 Glucksmann A (I 950) Cytological aspects of protection from ionizing Muller HJ (1950a) Some present problems in the genetic effects of 1314 1249 radiations. Br J Radio! 23(265):41-45 radiation. J Cell Comp Physiol 35(suppl 2):9-70 1315 1250 Graf U (1972) Spontaneous mutations in Drosophila melanogaster. Muller HJ (1950b) Radiation damage to the genetic material. Am Sci 1316 1251 Humangen Hum Genet 16(1):27-32 38(1):32-59, 126 1317 1252 Haas FI, Clark JB, Wyss 0, Stone WS (1950) Mutations and muta- Muller HJ (1952) Genetic effects of cosmic radiation. Chapter XVII. 1318 1253 genic agents in bact;:ria. Am Nat 84(8 I 7):261-274 (Reprinted from Physics and Medicine of the Upper Atmosphere) 1319 1254 Hamblin JD (2007) A dispassionate and objective effort: negotiating Muller HJ (1954a) The nature of the genetic effects produced by radi- 1320 1255 the first study on the biological effects of atomic radiation. J Hist ation. In: Hollaender A (ed) Radiation biology. Volume 1: high 1321 1256 Bioi 40(1):147-177 energy radiation, chapter 7. McGraw-Hill Book Company, New 1322 1257 Hanson FB, Heys F (1929) An analysis of the effects of the different York, pp 351-473 1323 1258 rays of radium in producing lethal mutations in Drosophila. Am Muller HJ (1954b) The manner of production of mutations by radia- 1324 1259 Nat 63:201-213 tion. In: Hollaender A (ed) Radiation biology. Volume 1: high 1325 1260 Higgins E (1951) Atomic radiation hazards for fish. J Wild! Manag energy radiation, chapter 8. McGraw-Hill Book Company, New 1326 1261 15(1):1-12 York, pp 475-626 1327 1262 Hollaender A, Stapleton GE (1959) Ionizing radiation and the living Muller HJ (1956) How radiation changes the genetic constitution. In: 1328 1263 cell. Sci Am 201(3):94-100 Proceedings of the international conference on the peaceful uses 1329 1264 Joint Committee on Atomic Energy (1957) 85th Congress, 1st session. of atomic energy. Volume II, biological effects of radiation, held 1330 1265 Summary-analysis of hearings 27-29 May, and 3-7 June, 1957 in Geneva Aug 1955. United Nations, New York, NY, pp 387-399 1331 1266 on the nature of radioactive fallout and its effect on man. United Muller HJ (1957a) How radiation changes the genetic constitution: 1332 1267 States Government Printing Office, Washington DC, Aug 1957 congressional testimony [reprinted from the Bulletin of the 1333 1268 Jolly JC (2004) Thresholds of uncertainty: radiation and responsibil- Atomic Scientists, Nov 1955] 1334 1269 ity in the fallout controversy. Ph.D. Dissertation. Oregon State Muller HJ (1957b) Potential hazards of radiation----congressional 1335 1270 University, Corvallis testimony. Hearings before the Special Subcommittee on Radia- 1336 1271 Kaufmann BP (1947) Spontaneous mutation rate in Drosophila. Am tion of the Joint Committee on Atomic Energy, Congress of the 1337 1272 Nat 81:77-80 United States. 85th Congress, 1st session, part I. United States 1338 1273 Kelner A, Bellamy WD, Stapleton GE, Zelle MR (1955) Symposium Government Printing Office, Washington DC 1339 1274 on radiation effects on cells and bacteria. Bacteriol Rev 19:22-44 Muller HJ, Carlson E, Schalet A (1961) Mutation by alteration of the 1340 1275 Lefevre G Jr (1950) X-ray induced genetic effects in germi- already existing gene. Genetics 46:213-226 1341 1276 nal and somatic tissue of Drosophila Melanogaster. Am Nat Newcombe B ( 1960) Genetic effects of ionizing radiation. Can J Bio- 1342 1277 84(818):341-365 chem Physiol 38(3):330-337 1343 1278 Lewis EB (1957) Leukemia and ionizing radiation. Science Norwood WD (1958) Common sense approach to the prob- 1344 1279 125(3255):965-972 !em of genetic hazard due to diagnostic radiology. JAMA 1345 1280 Lilly Library (1938a) Indiana University, Bloomington, IN. Stem Iet- 167(16):1928-1935 1346 1281 ter to Demerec, 21 Jan 1938 Nybom N, Gustafsson A, Granhall I, Ehrenberg L (1956) The 1347 1282 Lilly Library (1938b) Indiana University, Bloomington, IN. Demerec genetic effects of chronic-gamma irradiation in barley. Hereditas 1348 1283 letter to Stem, 16 Mar 1938 42(1-2):74-84 1349 1284 Lilly Library (1938c) Indiana University, Bloomington, IN. Stem Iet- Oliver CP (1930) The effect of varying the duration of x-ray treatment 1350 1285 ter to Demerec, 31 Mar 1938 upon the frequency of mutation. Science 71:44-46 1351 1286 Lilly Library (1946) Indiana University, Bloomington, IN. Muller Iet- Oliver CP ( 1931) An analysis of the effect of varying the duration of 1352 1287 ter to Stem, 13 Sept 1946 x-ray treatment upon the frequency of mutations. Doctor of Phi- 1353 1288 Lilly Library (1947a) Indiana University, Bloomington, IN. Muller Iosophy Thesis, University of Texas. Austin, Texas 1354 1289 lettertoStem, 14Jan 1947 Rajewski BN, Timofeeff-Ressovsky NW (1939) Hohen-Strahlung 1355 1290 Lilly Library (1947b) Indiana University, Bloomington, IN. Muller und die Mutationsrate von Drosophila melanogaster. ZIAV 1356 1291 letter to Stem, 3 Feb 1947 77:488-500 1357 1292 Lilly Library (1947c) Indiana University, Bloomington, IN. Muller Ray-Chaudhuri SP (1944) The validity of the Bunsen-Roscoe law in 1358 1293 letter to Stem, 4 Aug 1947 the production of mutations by radiation of extremely low inten- 1359 1294 Lilly Library (1949) Indiana University, Bloomington, IN. Muller Iet- sity. Proc R Soc Edinb 62:66-72 1360 1295 ter to Stem, 5 Feb 1949 Rinehart RR (1969) Spontaneous sex-linked recessive lethal frequen- 1361 1296 Lipshitz HD (2005) From fruit flies to fallout: Ed Lewis and his sci- cies from aged and non-aged spermatozoa of Drosophila meta- 1362 1297 ence. Dev Dyn 232:529-546 nogaster. Mutat Res 7:417-423 1363 1298 Liining KG (1954) Effect of oxygen on irradiated males and females Sankaranarayanan K, Wassom JS (2008) Reflections on the impact 1364 1299 of Drosophila. Hereditas 40(3-4):295-312 of advances in the assessment of genetic risks of exposure to 1365 1300 MacKey J (1951) Neutron and x-ray experiments in barley. Hereditas ionizing radiation on international radiation protection recom- 1366 1301 37(3):421-464 mendations between the mid-1950s and the present. Mutat Res 1367 1302 Muller HJ (1945) Age in relation to the frequency of spontaneous 658:1-27 1368 1303 mutations in Drosophila. Yearb Am Philos Soc 150-153 Sax K (1950) The cytological effects of low-intensity radiation. Sci- 1369 1304 Muller HJ (1946a) The production of mutations. Nobel Lecture, 1946. ence 112(2908):332-333 1370 1305 Nobleprize.org. http:www.nohclpriJ.C.i>rg/nobel-pri;.e,inlc~dicind Singleton WR (1954a) The effect of chronic gamma radiation on 1371 1306 laure;Hc,/1946 endosperm mutations in maize. Genetics 39:587-603 1372 1307 Muller HJ (1946b) Age in relation to the frequency of spontane- Singleton WR (1954b) Radiation effect on living systems. J Hered 1373 1308 ous mutations in Drosophila. In: Year book I Jan 1945, 31 Dec 45:58-64 1374 1309 1945. The American Philosophical Society, Philadelphia, PA, pp Sparrow AH, Singleton WR (1953) The use of radiocobalt as a source 1375 1310 150-153 of gamma rays and some effects of chronic irradiation on grow- 1376 1311 Muller J ( 1947) The production of mutations. J Hered 38(9):259-270 ing plants. Am Nat 87(832):29-48 1377 1312 Muller HJ (1948) Mutational prophylaxis. Bull N Y Acad Med Spear FG (1958) Some biological aspects of experimental radiology. 1378 1313 24(7):447-469 A historical review. Intern Rev Cytol 7:1-77 1379

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Arch Toxicol 1380 Spencer WP, Stem C (1948) Experiments to test the validity of the Timofeeff-Ressovsky NW, Zimmer KG, Delbruck M (1935) Uber die 1390 1381 linear R-dose/mutation at low dosage. Genetics 33:43-74 Natur der Genmutation und der Genstruktur. Nachrichten von der 1391 1382 Stem C (1950) Principles of human genetics. WH Freeman and Com- Gesellschaft der Wissenschaften zu Gottingen:Mathematische- 1392 1383 pany, San Francisco Physikalische Klass, Fachgruppe VI, Biologie 1(13):189-245 1393 1384 Stem C (1960) Principles of human genetics, 2nd edn. WH Freeman Uphoff DE, Stern C (1947) Influence of 24-hour gamma-ray irradia- 1394 1385 and Company, San Francisco tion at low dosage on the mutation rate in Drosophila. MDDC- 1395 1386 Stone RS (1952) The concept of a maximum permissible exposure. 1492, US Atomic Energy Commission, pp 1-6. Hathi Trust Digi- 1396 1387 Radiology 58(5):639-661 tal Library. Available at hop:f/www.hathitrust.org 1397 1388 Stromnaes 0 (1951) X-ray induced lethal mutations in several strains Uphoff DE, Stem C (1949) The genetic effects of low intensity in 1398 1389 of Drosophila melanogaster. Hereditas 37:533-559 irradiation. Science 109:609-610 1399 1400

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