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Dairy Science to tiio mammalian rans Roy Soc B uals in young -e tween radiation 'male mice. Proc female mice and .ion in women. nicity of ethyl 's t and their ', Kondo S, Takebe nogens (Proceeon Environmental Tokyo Press, Alan JW, Maddux SC, iws othylnitro>e mouse. Proc al control of 1:75. of translocations stages in male imnssion. Mutat of the ovary and i. Vale, New . Problems and pringer , p 301. Mutagens in Our Environment, pages 149-168 o 1982 Alan R. Lias, Inc., 150 Fifth Ave., New York, NY 10011 THE PREDICTABILITY OF BIOASSAYS Diana Anderson, SSc., MSc, PhD. DipEd, MIBiol. British Industrial Biological Research Association Woodmansterne Road Carshalton, Surrey, UK. INTRODUCTION Between three and forty percent of cancers are assumed to be due to occupational exposures (Bridbord, et al. 1978; Cole 1977; Cairns 1981; Higginson, Muir 1976; Wynder, Gori 1977) but the unequivocal identification of a human mutagen or carcinogen depends upon epidemiological studies of exposed populations. Epidemiological approaches cor detecting mutagens and carcinogens. Some industrial agents and processes have been identified as carcinogenic in epidemiological studies but because of mixed exposure to carcinogens and long latency periods for cancer development it is difficult to adapt epidemiological surveys as a means of establishing occupational health policy. Possible long term harmful effects of chemicals are cancer, congenital malformation and spontaneous abortion. The similarities between cancer and teratogenesis have been well reviewed (Harbison 1978; Kaltor 1975; Miller 1977) and mutations appear Lo be a possible common mechanism between them and also a factor in spontaneous abortions (Carr 1977). Many aborted fetuses appear to be malformed (Carr 1977; Kline, et al. 1977; Sentrakul, Potter 1966). The potential ities and Limitations of cancer epidemiology have been topics of concern for many years but those of the epidemiology of heritable defects have not and the literature on the subject is greatly Lacking. This is probably because until recentlv there was Little evidence to suggest that there was a 1 sufficient scaleenvironmental t^rotuu1 Lo cmtn^'n:, t or the effects to~5e detected (Bridges 1980). Chromosome anomalies are present in about i0% of all aborted foteses (lloih.kuv 19/7; St t in, et al . 197 5) and increased abortion rates are Implied in cigarette smokers (flimmelberger, ot al, 1978) tamale anesthetists, operating room nurses (K.n il 1-Jones , et al . 1977; Rosenberg, Kirves 1979; Cohen, et al. 1974) laboratory technicians (Strandberg, 1978) and copper smelter female employees (Nordstrom, el al. % 1978; 1979). The e.Laim that wives of vinyl chloride poly merisation workers are similarly affected (Infante 1976; Infante, et al. 1976a-c) has been questioned (Paddle 1976). Spontaneous abortions may form the basis of useful monitor ing procedures since they are frequent enough to allow large sample sizes. In the total number of births, childhood cancer is about 0.2Z, the incidence of major congenital malformations about 1% (not aLl genetic in origin (Hakasalo 1973) and spontaneous abortion is up to 15% in retrospective studies (Hemminki, et al. 1979). Table 1 (adapted from * " Hemminki, et al. 1979) shows the relationship of cancer, i abortions and malformations of exposed persons and experi mental data of some occupationally important chemicals. Dominant mutational diseases occur in approximately a further 0.2%, sex-linked diseases in around 0.1% and re cessive autosomal mutations become visible only over many generations (Bridges 1980). Dominant and sex-linked diseases are expressed in the first or second generation after in duction and as such also may be amenable to human screening. <1 However, only ver) few dominant changes with unambiguously identifiable phenotypes could be used in monitoring. Suggested marker phenotypes include dominant disorders such it as retinoblastoma(frequency 0.05/1000) or achondroplasia (frequency 0.1/1000) (Hemminki, et al. 1979) or sex-linked disorders such as the Lesch-Nyhan syndrome. There is evidence to suggest a rising frequency of chromosomal abnormalities causing such anomalies as Down's syndrome (trisomy 21), and Pacou's syndrome (trisomy 13) (Hook 1978). Screening exposed populations for induced genetic effects, may have advantages over screening for cancer in that effects may be manifest earlier since for such changes there would be little~trc no latent period and they may thus refLect current exposure levels to chemicals. o5 uo o o ir. In a <a < a< su W02 sW Cl, 35 WX O i r--l (A nc 65 C --* 6s u ^o JoS --< CO 2 O CO aUJ CL, uai CO oa* x u c 2UU3 3 Hwco rc C2O o H < W 0 < CO 2 oCO & SI UQCO Ou 2 U 2 m , f-- :r i-- r, -- o r~ i^i rr C C G \i # T "D 7? JU. 0 2 3 -- "O Cj G A, r-- - h** vO O*' 0 G 3 o 01 r-- rr ^4 . t-- rr ti rr P G O'1 *< er --* nn ' 1 j g <3 03 1 *--` 03 ft cm 1 TABLE (After Hemminki et al. 1979) RELATIONSHIP BETWEEN EXPOSED PERSONS AND EXPERIMENTAL DATA OF SOME OCCUPATIONALLY IMPORTANT CHEMICALS INCREASE IN EXPOSED PERSONS Chemical Compound Mal Cancer Abort ions formations Chromo somal Aberrations in Lymphocytes POSITIVE EXPERIMENTAL DATA Muta- Care ino- Terato- genes is genesis genesis Acrylonitrile (+) Anesthetic gases (+) Arsenic - Ashes Cos Benzene + Chloroprene Epichlorohydrin (+) *- (+ ) + (+) -+ + + ++ ++ ++ Chromium + ++ Ethylene Oxide Lead Styrene Vinyl Chloride + + *` + <+) + (*) (+) + + + ++ ++ + = positive data; { + ) ** limited data; = c:ontradictory data; no data. + ++ ++ + ++ + + + 4- ++ + (+) ++ = negative data; SL 097398 .................................. I - -i t 4 * 'r ( \'A r-J t * 1 -rtj CD 152 / Anderjpn The public in recent years are becoming more aware of possible threats to health. There are about 25,000 occup ational chemicals with potential mutagenic hazard (Loprieno 1977) and to date about 7,000 chemicals have been tested for carcinogenicity in animals and 1,300 arc suspected carcino gens. The world capacity for adequate animal testing of chemicals for carcinogenicity is about 500 a year (Maugh 1978 a,b). Obviously many more chemicals could be tested using short term tests for mutagenicity. Whilst cancer is of immediate concern to an individual increased mutational risk may have serious cumulative implications for human genetic health. The majority of known individual animal and human carcinogens are of the classic type giving rise to electrophilic metabolites with mutagenic properties. Even though other factors may affect the metabolism preceding the formation of the electrophiles, accepted models for carcinogensis imply that DNA damage is the initial step in the carcinogenic process. Thus both for mutagenesis and careinogenes is it might be useful to calibrate human effects in terms of DMA damage and cellular response. THE PREDICTION OF HUMAN GENOTOXICITY For genotoxins, Bridges (1980) has suggested that the calibration of human response will involve the linking of epidemiological and laboratory investigations into both the calibration of human response against tissue exposure and the calibration of man against rodents for the same tissue exposures, (Figure 1). APPLIED DOSE BIOLOGICALLY SIGNIFICANT DOSE MARKERS OF GENETIC DAMAGE " GENETIC, CARINOGF.NIC EFFECT Concentration in gonads, blood, urine, etc. Extent of react ion with DNA or markers such as hemoglobin Chromosome breakage,SCE's or gene mutations in peripheral lymphocytes or other tissues Epidemiology : Spontaneous fetal loss, congenital abnormalities, perinatal mortality, cancer MAN 7s ANIMAL -) MAN 7s -> ANIMAL -9 -) ANIMAL -> MAN T' ANIMAL Fig. 1. Suggested linkage of Laboratory and epidemiology studies necessary for the calibration of human response to agents which damage DNA. $ ino obta care orgai agen type phosi phosj ac t ii hist whicl ogenc direi SL 097399 Gr. WiTVr?*- mo re aware of 25 ,000 occupiza rd (Lopr i eno be en tested fo: Tee ted earcinoL t est ing of ye ar (Maugh ild be tested (Is t c ancer is ied mu rational j for human Ldual animal : giving rise iroperties. sbolism preceding models for litial step in igenesis and Le human effects ?sted that the le linking of s into both the exposure and ie same tissue -lEXETIC, CARINQ.11-MIC EFFECT Epidemiology : Spontaneous fetal loss, congenital abnormalities, perinatal aortality, ancer ------- > MAN 7^ -----) ANIMAL upidemiology .in response to Predictability of Bioassays / 153 ra l inf o rma t ion is gen e rally di fficult to of expo sure (i.e . the dose) to a mutagen or ed that can be a pp 1 icd directly for comparative purposes to man, and labo ratory animals or in vitro systems. This ma; L ake t he f o rm o f meas urements of the conGenerations of the agent in body fluids, gonads or in other organs, but presents problems where there is a mixture of agents being investigated or exposure is of low level chronic type. One way is to measure the DNA repair independent phosphotriesters formed when the reaction occurs with the phosphate groups of DNA, Another way is to measure the re action of electrophilic species with amino acids such as histidine in hemoglobin. Such reactions involve indicators which are themselves not involved in mutagenesis or carcin ogenesis but reaction with these molecules may be used directly for interspecific comparative purposes. The measurement of some change in the cells of exposed persons resulting from DNA damage provides other information and confirms dosage or exposure and takes into account human metabolism. Peripheral lymphocytes from exposed work forces have been used to measure chromosome damage both as con ventional aberrations and sister chromatid exchanges (e.g. Purchase et al. 1978 J,b; Anderson,et al. 1980 and 1981) and also for the measurement of gene mutation using the radioautographic method (Strauss, Albertini 1979; Strauss, et al, 1979) to detect hypoxanthine-guanine phosphoribosy1 transferase (HGPRT) deficient variants. The detection of HGPRT mutants in cultured ceLls is well established for screening purposes (e.g. Arlett 1977). Seminal fluid can be used for detecting sperm abnormalities in human populations and the mouse (Wyrobek, Bruce 1978). Sperm abnormalities may be inherited (Topham 1980) and reflect the action of mutagens but can also occur as a response to physiological differences. The measurement of human fetal loss detects /dominant lethals arising from male and female exposure and ( also teratogenic effects as a result of exposure in utero. ! Dominant lethalitvis thought to result from gross chromosomal aberrations although it is kn.o_wn_t.hat it can arise from non genet ic events and is thus not a heritabie hazar.LlPJ man__ (Bateman , Epstein 1972). However, it is a germ line effect which may be an indicator for less easily detectable serious heritable chromosome abnormalities. Changes in the sex ratio may arise due to a preferential loss of male conccptuses resulting from recessive lethal mutations on X chromo somes but this method was of little use in detecting SL 097400 __ 154 / Anderson rad l at i in1 effects among Hiroshima de.scenr.K-.nL? (bridge? 1980). The mea?i;reien! s dt. at r i bed above are best suited to substance? where elertrophiLic reactive species are systemically distributed throughout the body and for direct acting carcinogens it might be possible to proceed in a similar way to that described above for mutagens. Some populations are systemicalIv exposed to carcinogens and mutagens e.g. cigarette smokers who inhale have a mutagenic urine (Yamasaki , Ames 1977) with carcinogenicity of organs remote from the lung and this indicates the likelihood of distribution throughout the body. Cigarette smoke mutagens reach the testes (Wyrobek. Bruce 1978) and chromosomal exchanges (Obe, Herha 1978) and sister chromatid exchanges (Lambert et al. 1978) are found in peripheral blood lymph ocytes. There is a dose-related increase in perinatal mortality and the incidence of congenital abnormalities in the children of smoking fathers (Man, ."setter 1974). The linking of aLl these factors could provide the calibration of response of human spermatogonia to chemical mutagens. The already existing information about cancer incidence in cigarette smokers could also be linked with estimates of tissue reaction and cellular response particularly for data (other than bronchial carcinoma) for cancers at sites remote from the lung which can then be attributed to the carcinogens carried in the blood (Bridges 1980). Human mutagenicity? Infante and co-workers reported an increase in fetal wastage amongst vinyl chloride exposed workers (Infante 1976; Infante,et al , 1976 a-c). This study was thought to provide evidence of human chemical mutagenicity. It compared proand post-employment reproductive history of vinyl chloride workers and compared them with workers in other parts of the chemical industry. However no dose-response relation ship was established since go exposure history was available. The survey was carried out as a consequence of questioning the fathers which produces less than accurate data on re productive history, the data were corrected for paternal age when maternal age has the biggest eEfect on fetal wastage, and only a small number of workers were interviewed amongst the total available for interview. Congenital abnprma_y.ti.es_ did .not Lnc reae._alongs ide fetal wastage. Because of these considerations it is not proven definitively that vinyl chloride is causally associated with such_effects in man (Put anesthetic increase tl In these c, as cone 1 us There detecting situation mutagenic influence but becau? Human cart The . genicity regenerated mutations thought t Answers t chemica1 and its t Cart provided gen (as i the chem particul as posit test in non-carc assessir various al. 1979 produce tests on test to results the abil the pro d) Pred among t both tb the pre being t :nts (Sridges 1980). best suited to >ecies are system] for direct acting sd in a similar Some populations nd mutagens e.g. enic urine city of organs he likelihood of tte smoke mutagens nd chromosomal omatid exchanges oral blood lymph in perinatal abnormalities in .ter 1974) . The le the calibration mical mutagens, inter incidence in ith estimates of rticularly for data jers at sites remote ed to the carcinogens increase in fetal orkers (Infante 1976; s thought to provide It compared pre' of vinyl chloride n other parts of -response relationlistory was available, nice of questioning curate data on re tted for paternal ffcet on fetal kers were interviewed ew. Congenital e fetal wastage, ot proven definitively ted with such effects __ 4 Predictability of Bioassays /. 155 in man (Purchase 1980b). Exposure to lead (Rom 1976) and anesthetic-gases (Cohen,et al. 1974) has been reported to increase the abortion rate in wives of exposed workers. Tn these eases also Lhe evidence cannot yet be considered as conclusive. There are a wide variety of test systems available for detecting mutagenic and carcinogenic potential but the situation for carcinogenesis can be more complex than for mutagenic effects, however, not only because of the influence of promotors or other non-mutagenic substances but because of the phenomenon of organ specificity. Human carcinogenicity? The method of assessment of potential human carcino-'^ genicity other than by epidcmiologically relies on data generated from laboratory studies such as the production of mutations or the induction of tumors in rodents which are thought to be indicators of potential human carcinogenicity. Answers that the tests might provide are whether the chemical is a carcinogen, the potency of Chat carcinogen and its organ specificity (Purchase 1980a). Carcinogen or not? A qualitative answer can be provided as to whether the chemical J^s or is not a carcino gen (as opposed to quantitative answer which implies that the chemical will produce a carcinogenic effect at a particular dose). It is possibLe to classify a chemical as positive or negative in a short-term test or long-term test in a given species and either as a human carcinogen or non-carcinogen. The efficiency of the testing systems in assessing potential carcinogenicity can be judged using various criteria (Purchase, et al. 1978c,1980a; Cooper, et al. 1979): a) Reproducibility: the ability of the test to produce qualitative and quantitative results during repeat tests on a chemical; b) Sensitivity: the ability of the test to detect carcinogens L.e. the proportion of positive results among the carcinogens tested; c) Specificity: (he ability of the test not to detect non-carcinogens or the proportion of negative results among the non-carcinogens; d) Predictive value: this is the proportion of carcinogens among the chemicals positive in the test. It depends on both the sensitivity and the specificity of the test and on the proportion of carcinogens in the group of chemicals being tested. Because of such dependence, predictive values SL 097402 156 / Anderson __-4" c in only bn estimated if a knowledge of the composLtion of the group of chemicals in terms ot proportion of carcinogens is available. When testing chemtcaLs of unknown activity s this information is not available and therefore weight most ne given to the comparison of the structure of the chemical with established carcinogens or non-careinogens. It can be assumed, however, that there is a particular value for the proportion or percentage of carcinogens in the group of chemicals being tested (e.g. 10%) and the predictive valuefPV) can be expressed at that proportion. Thus the PVio would be the proportion of carcinogens among the chemicals found to be positive wheal 10% of the group submitted for testing are care inogens. There are many short-term tests available for carcino genicity and these are based on end-points other than the production of cancer. Many of them are tests for mutation i e.g. when considering reproducibility the international collaborative study (dc Serres, Ashby 1981) revealed that 5 there are a number of chemicals with differing results in the Ames test in different laboratories. When considering quantitative reproducibility where the same doses of the same compound were used in reference and 'in-house' strains in the i international Genetic Drift study (draft in preparation) i variability in 38 interlaboratory comparisons was revealed even with standardisation of details in techniques. (A t significant difference in mutability was found between 'in- house' and reference cultures in a number of cases suggesting genetic drift. These effects, however, were less significant overall than the lack of reproducibility with the reference culture). The sensitivity, specificity and PVio values for the six tests used by Purchase,et al. 1978 are shown in Table 2 and it is seen that the Ames and cell transformation tests have the greatest sensitivity and specificity. Further increases in these parameters can be obtained by using both tests together when they give the same result but predictions of carcinogenicity become less accurate when the tests do not give the same result. This has also been demonstrated for microbial assays by Rosenkranz and Mermelstein (1980) who point out that chemical choice and carcinogenicity criteria greatly influence the description of the validity of a study. Sensitivity g~presumably most important in a screening programme designed to identify potential carcinogens even at the risk of scoring false positives. However, from an industrialist's point of view this poses a problem. A text devoted wholly to the predictive value of short-term screening in care inogen ici 1980). TABLE 2. THE SENS IT IM SHOR' TEST Ames Cell transforms Degranulation Sebaceous gland Tetrazolium red Subcutaneous im Ames and cell t 1. Chemicals fo tests gave s 2. All chemical positive Long-term which potential definition of a studies. To va are compared wi due to the lac! not be validatt are known to pi are also anima Table 3. This predictive val carcinogenic ci by epidemiology mice are the me studies and the tested in both interspecies cc idea of the ef` may be obtaine species i.e. t to predict the considered. T i SL 097403 on of inogen s vi ty it must lemical can be nr the of value(PV) vould be and to ing are care mon the cation nal that ts in Jering the same in the ion) vealed (A en 'inuggest ing gnifleant ference ues for 1 in "ormation . Further ing both rediet ions sts do not Led for J) who criteria t" a study, n ing s even at an A text m screening Predictability of Bioassays / 157 in carcinogenicity evaluation is available (Williams, et al. 1980). TABLE 2. THE SENSITIVITY, SPECIFICITY AND PREDICTIVE VALUE OF SHORT-TERM TESTS FOR CARCINOGENICITY TEST SENSITIVITY SPECIFICITY PV10 Ames Cell transformation Degranulation Sebaceous gland suppression Tetrazolium reduction Subcutaneous implant Ames and cell transformation 1. Chemicals for which both tests gave same result 2. All chemicals - one test positive 91 91 71 67 AO 37 96 99 94 63 97 77 71 21 64 17 73 14 95 45 100 100 91 55 Long-term animal studies provide the main data base on which potential carcinogenicity for man is judged. The definition of a human carcinogen depends upon epidemiological studies. To validate long-term animal studies the results are compared with known human carcinogens. For mutagenicity, due to the lack of known human mutagens, animal studies can not be validated in the same way. There are 26 agents which are known to produce cancer in man of which 19 chemicals are also animal carcinogens; examples are as shown in Table 3. This information is insufficient to calculate the predictive value and suffers because the majority of the carcinogenic chemicals were identified as carcinogens firstly by epidemiology and only later tested in animals. Rats and mice are the most commonly used animals for carcinogenicity studies and there are a number of chemicals which have been tested in both species. Extrapolation to man is by an interspecies comparison of man with the test animal. An idea of the effectiveness of animal carcinogenicity studies may be obtained by comparing the results from the two species i.e. the ability of carcinogenicity studies in rats to predict the outcome in mice and vice versa can be considered. The information under consideration is from SL 097404 ' t': 3lLhs S TABLE 3. SOME CHEMICALS ASSOCIATED WITH CANCER INDUCTION IN HUMANS AND RODENTS (IARC 1978) Compound MAN Route Target organ RAT Target Route organ MOUSE Target Route Organ Pre diction human ta r get org an by ra t or mouse Af]a toxins Dietary Liver ip/co Live r (No good positive study) Yes Benzene ih top po Humopoie tic system (No good animai mode 1) Diethylstilboestrol po Uterus, vagina sc Mammary gland, po sc Mammary gland pituitary cervix, vagina, Yes testis 2-NaphthyLamine ih top po B Ladder (no positive study) po sc I, ive r i oca I (dog, monkey, hams ter b i adder No Vinyl chloride ih top Liver, ih brain, lung Liver, brain Zymbal gland kidney ih Lung, mammary gland, liver Yes ip = indraperitoneal; po = oral; sc = subcutaneous; ib = inhalation; ib = intrabronchial; top = topical err* Qft" OftDCD* DfOl f3t ejt &. C/s 50 b> rr *7* E? c w (A rr C <s> (ft Cl cr m o3 uH* h3 * Oft _* ro V& O 2S 3Cl rtJaft 50 o *n H n rr "O rr > CD P* f-l CO n o r( ftr1 n c QO ftm K c 3 (JO 3 O 3 in JfOt rr 3ft O C `-r r( SL 097405 m t r a p e r it o n e a l; po - o r a l; sc => s u b c u ta n e o u s ; ih = in h a la t io n ; ib = in t r a b r o n c h ia l; to p ic a l __ -* Predictability of Bloassays / 159 that of Purchase (1980a) and the data were obtained from three sources; the IARC monograph series, the NCI Bioassay programme and by assessing information in the literature through the "Survey of Chemicals which have been tested for carcinogenicity". The results are. shown in Table 4. TABLE CARCINOGENICITY STUDIES IN RATS AND MICE NUMBER OF CHEMICALS RESULT IN MOUSE RESULT IN RAT 109 Positive Positive 98 Negative Negative 21 Positive Negative 17 Negative Positive 5 Negative Positive other species 250 Using these data the reproducibility, specificity, sensitivity, and predictive value can be considered as shown in Table 5. TABLE 5. THE SENSITIVITY SPECIFICITY AND PREDICTIVE VALUE OF RAT OR MOUSE STUDIES AS PREDICTORS OF CARCINOGENICITY IN RATS OR MICE SENSITIVITY SPECIFICITY PVio Mouse study Rat study 87 82 35 84 85 33 The short-term tests actuaLly appear to have a higher sensitivity and specif icy than animal studies and have a --correspondingly higher PVio. However, in the case of animal studies results are defined in terms of one species but for the short-term tests any species was taken as the end-point. Since the predictive value of a test is partly dependent on the proportion of carcinogens in the chemicals being tested as the proportion of carcinogens decreases so it has a greater influence on the PV. For a single chemical SL 097406 160 / Anderson the FV is difficult to estimate but structural comparisons with known carcinogens and non-carcinogens provide an opinion which effectively replaces the need to estimate the PV provided a sensitive and specific test is utilised. Ihe potency of the carcinogen. Potency correlations have been attempted with short-term tests. Meselson and Russell (1977) presented a comparison of the potency of a number of chemicals in the Salmonella plate incorporation assay and animal carcinogenicity studies. They deduced a fixed relationship between the mutagenicity to Salmonella and the potency of carcinogens in animals. The relation ship is dependent, however, on the values obtained for a few chemicals at either end of the potency scale and there are notable exceptions to the relationship. A fixed quantitative relationship is improbable for several reasons. Small modifications in the protocol of the Salmonella test can have a considerable influence in the quantitative results obtained (Ashby> Styles 1978). Comparison of the dose-response relationships requires expression both of the response at a given dose and the slope of the dose response curve. When the slopes are omitted as in this correlation, slope divergence could make a difference to the quantitative expression of the relationship. In the Ames assay generally Aroc.lor induced rat liver S-9 is used and is probably unlikely to be quantitatively representative of the metabolism for all carcinogens in vivo at all target organs. False positive and negative results belie a quantitative relationship between in vitro and in vivo results. The studies of Coombs,et al. (1976) and Kameswar Rao,et al. (1979) evaluated the polycyclic aromatic hydro carbons and aliphatic nitrosamines respectively by comparing Salmonella results with ^_n vivo data and did not confirm the relationship. More comparative data would be needed before a quantitative relationship can be proven. The use of animal carcinogenicity data for quantitative extrapolation to man shouLd be more satisfactory for potency considerations than the use of short-term tests because of the greater similarity of the laboratory model to the human. However, a number of assumptions are made to allow quantitative extrapolation of animal carcinogenicity data to man 1) it must be assumed that there are only small species differences in susceptibility. However of the 250 chemicals shown in Table 4 there is evidence that in 43 there is no qu suggests differen literatu laborato fold mor 500-fold dibromid example (Bridges data pro used for is no tf threshol several 'response Servatir cell mut and are are base or on pr threshol property proper a a suitat 1980) re take acc EDoi Tat induce j a singlt and refs concile vers ibit Also for mechanis usually assumed in anima the sami in susci specif i1 S and are extrapo to thos' SL 097407 comparisons /ide an estimate the _ilised. irrelations selson and ateucy of a corporation y deduced a Salmons l la e relationined for a le and there . fixed veral reasons. monel La test itative irtson of ssion both of the dose is in this 'ference to ip. In the r S-9 is used representative at all target belie a d _in vivo and Kameswar omatic hydroly by comparing not confirm d be needed veil. or quantitative ocy for potency ,ts because of l to the human. le to allow ^enicity data mly small ;er of the 250 that in 43 there -# Predictability of Bioassays / 161 is no qualitative agreement between rat and mouse which suggests that there are probably large quantitative differences in susceptibility. There are examples in the literature of differences in susceptibility of men and laboratory animals e.g. to diethy1stilboestrol man is 50fold more suceptible, to aflatoxin 50-fold less susceptible, 500-fold to vinyl chloride and several 100-fold to ethylene dibromide (Ramsey, et al. 1979). Cigarette smoke is an example where man is similar to the laboratory animal (Bridges,et al. 1979). 2) It must also be assumed that data produced in high dosage experiments in animals can be used for extrapolation to very low doses in man i.e. there is no threshold. It is usual to assume that there is no threshold for chemically induced carcinogenesis based on several concepts: a) that mathematical expression of dose response data show no threshold b) that this is a con servative way to respond to the data and c) somatic or germ ceil mutations can be induced by a single lesion in the DNA and are then fixed in the genome. The mathematical models arc based on a one-hit model which is linear at low dosages or on probit/log dose extrapolation. Neither allows for a threshold and thus do not take account of a basic biological property. However a conservative approach to the data is a proper approach with the Current lack of knowledge to provide a suitable basis for extrapolation. The ED<u study (Cairns 1980) re-examination reports now present models which might take account af possible thresholds (Members of the SOT. CDoi Task force 1981). As low doses as 1 rad of X-rays can induce genetic damage which lends support to the dogma that a single lesion can cause mutation (Gaulden, Read 1978 and references therein). Such a dogma is difficult to re concile with two stage models of carcinogenesis and the re versibility or redifferentiation of some neoplastic cells. Also for those chemicals which act through a different mechanism of action other Chan the DNA a threshold can usually be established (Kroes 1979). 3) It has also to be assumed that the same mechanism of cancer induction applies in animals and humans over a wide dose range i.e. they have the same pharmacokinetics etc. The quantitative differences in s use ept i! spec it i c ity S ince and are not ext r apo iat ii to t hos c situations when the biological assumptions are known 162 / Anderson -t PREDICTION OF THE TARGET ORGAN Short-term tests arc not yet able to predict organ specLtio i ty but the use of S-9 homogenates other than from the liver hold promise. Preliminary investigations using this approach with the oesophageal carcinogen N'-ni t roso-.',' metnyLan ilino (NMA) however have not shown oesophageal S-9 mix to activate this compound when rats were pretreated with Aroclor or NMA (Anderson, t-1 nl. unpublished). The data are shown in Table 6 after NMA pretreatment. TABLE 6. ORGAN SPECIFICITY STUDIES WITH SAdy'S.'.L'LLA STRAIN TA 98 TREATED WITH NITRO S 0M THYI,AN ILINE (NMA' (rats pretreated with 100 mg/kg NMA i.p.) COMPOUND REVERTANTS PER PLATE NMA NORHARMAN (pmoles/ml incubate) 00 50 5 1 .2 00 0.5 0 10 20 50 roo LIVER OESOPHAGUS SALIVARY GLAND (50 pi 25% S-9 mix) 40 9975 ;) (25 til) 27 29 39 32 48 38 60- 1 10* 21 200* 41 38 39 37 * = +ve result (Anderson D., Craddock V.M., Blowers S.D.) Comparison of carcinogenicity data in rat and mouse allows a determination of whether carcinogenic chemicals have the same organ specificity in these two species (Table 7). A few carcinogens have exactly the same target organs in both species and many carcinogens affect more than one organ. There is a common target organ in rat or mouse of only tl out of the 19 human carcinogens i.e, 58% of the 109 carcinogens from the various sources 70 have at least one or more organs as a common site in both rat and mouse. Vinyl chloride is an example of a chemical with a common target organ i that organ spe well substanti TABLE 7. THE C No. of chemic; positive in r. and mouse No. of chemic with at least one common si itrfSflth speci CONCLUSION The moi and earcinogi quantitative systems is n< require a fu the laborato and distribu the receptor available an is possible, variabi1ity accross spec that do not way in man. should be un extrapolatio Anderson D, WGF (1980) workers. initial s Anderson D, ML (1981) workers; SL 05740s organ than from >ns using iitroso-M lageal S-9 :reated ) . The 3 (nma' -P-) E SALIVARY GLAND 39 37 blowers S.D.) and mouse chemicals ecies (Table rget organs e than one r mouse of % Of the e at least and mouse, i a common Predictability of Bioassays t 163 target organ in man and animals but in general the belief that organ specificity can be predicted from animals is not well substantiated. TABLE 7. THE ORGAN SPECIFICITY OF CHEMICAL CARCINOGENS NCI DATA IARC DATA LITERATURE TOTALS No. of chemicals positive in rat and mouse No. of chemicals with at least one common site in both species 26 15 (58%) 60 40 (67%) 23 109 15 (65%) 70 (64%) CONCLUSION The models available for assessing human mutagenicity and carcinogenicity have many inadequacies. To extrapolate quantitatively to man from experimental data from these systems is not generally feasibLe. Such extrapolation would require a full appreciation of the mechanism of action in the Laboratory model and man by understanding the metabolism and distribution of the chemical and how it interacts with the receptor organ. For man such information is not always available and information from Laboratory models is all that is possible. Thus it is important to understand the variability of these models. 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