Series: ‘Current Issues in Mutagenesis and Carcinogenesis’

Mutatton Research, 260 (1991) 1-4 © 1991 Elsewer Science Pubhshers B V. 0165-1218/91/$03.50 ADONIS 016512189100072A MUTGEN 01654

Series: 'Current

Issues in Mutagenesis

and Carcinogenesis'

No. 24. Caprolactam: black, white or grey? Keywords Caprolactam

Introduction A recent special issue of Mutation Research (224 (1989) 319-404) was devoted to consideration of the genotoxicity in vitro and in vivo of caprolactam. In a subsequent issue of the B I B R A Bulletin, James Hopkins presented an assessment of those data that was different to that offered by Ashby and Shelby. We consider that the comments by James are worthy of general consideration. We are grateful to Dr. Sharat Gangolh, Director of BIBRA, for permission to reproduce the following article. J. Ashby M.D. Shelby

Macclesfwld, Ches. Research Trtangle Park, N C

Caprolactam: black, white or grey? Early optimism that short-term tests (STTs) would provide a reliable and economic means of divining a chemical's carcinogenic potential (or at least be able to discriminate between genotoxic and non-genotoxic carcinogens) has gradually faded over the years. The genotoxicity community has responded to these disappointments, notably by way of some rather heavywetght articles in specialized journals such as Mutatton Research. At the same time that the practitioners have increasingly come to accept the difficulties faced by their science, we have observed an ever-increasing tendency by the regulators to use STT results to judge chemical acceptability. This paradox raises a number of important issues, some of which are addressed in this article. We have selected caprolactam to illustrate the problems that testers and evaluators face. Used in the manufacture of nylon,

Correspondence" Mr James Hopkans, The British Industrial B~ological Research Assocmt~on, Woodmansterne Road, Carshalton, Surrey SM5 4DS (Great Britain).

caprolactam has been fairly recently tested, in high quality carcinogenicity bloassays and, as part of an IPCS * programme, a range of STTs. Caprolactam has a unique cancer status as the only compound so far considered by the International Agency for Research on Cancer as worthy of inclusion in Group 4 - - ' p r o b a b l y not carcinogenic to humans'. International collaboratwe studies have been a common feature of m a n y genotoxicology laboratories in the last 20 years, and the IPCS caprolactam studies are one part of the latest research effort. These endeavours were in fact generated by an earlier IPCS p r o g r a m m e that had entailed an examination of caprolactam's activity in a series of in vitro STTs. A number of the 'positive' results seen in this first phase were weak and non-reproducible, and were therefore dismissed as being random technical errors of the test systems. Other results were not as easily undermined, in particu* International Programme on Chermcal Safety.

lar the chromosome damage seen in human lymphocytes and the somatic mutations in the fruit-fly Drosophda melanogaster. The new studies on caprolactam were an attempt to discover whether this m vitro actwity would be seen in vivo. The findings are published in a special issue of Mutation Research (224 (1989) 319-404). Before describing the outcome of these studies let us go back a step or two and briefly explain the bas~s of caprolactam's IARC status. Unique it may be in Group 4, but its testing career ~s not particularly extensive, there being no human data for example. Its reputation as a non-carcinogen is, in fact, based on the results of long-term oral studies in rats and mice, carried out under the auspices of the National Toxicology Program in the U.S.A. An IARC Working Group described these negative studies as providing "evidence suggesting lack of carcinogenicity". Only two other compounds, ferric oxide and methyl parathion, have been honoured with this same conclusion in the 20-year history of IARC evaluative activity. Despite all three being negative in cancer bioassays, fernc oxide and methyl parathion - - unlike caprolactam - - have not yet managed to escape from an overall Group 3 (not classifiable as to ~ts carcinogenicity to humans) label. According to IARC, there are insufficient supporting data to allow their classification into Group 4, which presumably meant that, of the three, caprolactam had the most clearly negative genotoxicity profile. Now let us see whether this relatively clean profile survived the rigours of the second IPCS programme. First of all, the in vitro pronouncements were shown to travel reliably both in time and place. Confirmation of chromosome damage in human lymphocytes in culture was provided by all three examining laboratories (E. Kristiansen and D. Scott, Mutatton Res., 224 (1989) 329; H. Norppa and H. J~irventaus, Mutation Res., 224 (1989) 333; T. Sheldon, Mutatton Res., 224 (1989) 325). Furthermore, the induction of sister-chromatid exchange, a more subtle effect on the chromosome, was also reported in Chinese hamster ovary cells (Norppa and J~irventaus, loc. ot.). In all these investigations, the effects seen were fairly weak and were found only at high concentrations of caprolactam. In Drosophila, caprolactam again proved active, being weakly mutagenlc both to

somatic and germ cells (E.W. Vogel, Mutatton Res., 224 (1989) 339). What about the in vivo score? Negative results were generated by studies looking for DNA-strand breaks and unscheduled D N A synthesis in the liver (E. Bermudez et al., Mutatwn Res., 224 (1989) 361) and unscheduled D N A synthesis m the sperm cells (P.K. Working, Mutation Res., 224 (1989) 365) of rats given caprolactam by stomach tube. Similarly, there were no increases in the extent of DNA fragmentation in the liver of rats and mice treated by intraperitoneal injection (S. Parodi et al., Mutanon Res., 224 (1989) 379), or in abnormal sperm in mice treated by stomach tube (M.F. Salamone, Mutatton Res., 224 (1989) 385). Caprolactam's potential to cause chromosome damage in the intact animal was examined in four separate laboratories. No increases in sister-chromatid exchange or chromosome damage were seen in B6C3F~ mice given a near fatal intraperitoneal dose of 700 m g / k g body weight (A. McFee and K. Lowe, Mutatton Res., 224 (1989) 347), chromosomal damage did not occur in 1C3F 1 mice given 1 g / k g body weight by stomach tube (I.-D. Adler and I. Ingwersen, Mutanon Res., 224 (1989) 343), and micronuclel were not induced in I C R / J C L mice given up to 500 m g / k g body weight by intrapentoneal injecUon (M. Ishidate and Y. Odagiri, Mutatton Res., 224 (1989) 357). The fourth study (T. Sheldon, Mutation Res., 224 (1989) 351) gave some indications of micronuclei induction in the bone marrow of C 5 7 B L / 6 J mice. In the first of two experiments, the numbe,rs of micronuclei (per 1000 cells) 24 h following a maximum tolerated oral dose of 700 m g / k g body weight were increased (statistically significant at the 5% level) in both the males and females. In the second experiment, an effect was apparent at a higher level of statistical sigmficance (the 1% level) in the males, but no effect was evident in the females. A statistically slgmficant effect was still apparent in the males when the shdes were reassessed in more detail by scoring 5000 cells per ammal. The paper also presented historical control data for eleven other experiments carried out at the same laboratory. The reassessed value for caprolactam-treated males did not differ significantly from the mean of the eleven historical controls, though it was higher than ten of these.

The use of historical control values poses some well known difficulties of its own. In this particular case, none of the historical 'controls' had been treated with olive oil, the vehicle used for the caprolactam studies. In addition to these signs of activity in the mouse micronucleus test, another m v1vo system, the mouse spot test, did manage to come up with slightly clearer positive results. The spot test ~s one of the assays recommended in the latest CoM guidelines (BIBRA Bull., 28 (1989) 291) to complement the in VlVO bone-marrow tests. It involves the treatment of pregnant mice of a defined genetic character such that specific chemically induced mutations will produce changes in the pigmentation pattern of the pup's coat. The initial caprolactam study revolved three separate experiments and used an embryotoxic intraperitoneal maternal dose of 400 or 500 m g / k g body weight (R. Fahrig, Mutation Res., 224 (1989) 373). An increased frequency of colour spots was noted in each of the caprolactam-treated groups, but the increase was statistically s~gnificant in only one experiment. The combined result was 39 spots in 1374 F 1 pups compared with six in the 407 controls ( P = 0.081). A more detailed microscopic examination of the hair pigmentation, thus allowing a more confident insight into the type of action being seen, led Fahrig to suggest that caprolactam was more a mitotic recombinogen than a straight mutagen. Stronger evidence of caprolactam's activity in the mammalian spot test was generated by further studies (A. Neuh~iuser-Klaus and W. Lehmacher, Mutation Res., 224 (1989) 369). The mouse strain used in these experiments had seven sensitive loci whereas the strain used by Fahrlg had four. Individual experiments were conducted on four successive weeks, and each involved treatment with a single intraperltoneal dose of 500 m g / k g body weight. There was a higher incidence of the appropriate spots in the coats of the offspring of all four groups of treated mice. An essentially negative result was seen in the fifth experiment conducted one year later. On this last occasion, a single dose of 700 m g / k g body weight was used. In only one of the first four experiments did the observed increase achieve statistical significance ( P = 0.02). Nevertheless, a statistical approach invoking the pooling of data from all experimental

groups in this study gave a clearer indication of caprolactam's activity ( P = 0.036), allowing the investigators to conclude that caprolactam be classified "as mutagenlc in the spot test". So, we arrive at the point where a p r o g r a m m e of work has suggested genotoxic activity for caprolactam In two in VlVO assays. Do we conclude that we have identified a non-carcinogenic chemical that is an in vivo mutagen, or would this mean we are taking the wrong message from the STTs? At this stage, ~t is difficult to reach a verdict. If the former is true, it is disappointing for those who believe there may be a reasonable correlation between genotoxic carcinogemcity and in vivo STT results, since they would expect caprolactam to lack activity in the intact animal. The second possibility is that caprolactam's apparent activity in two in vivo STTs is merely fortuitous. False positives (and, indeed, false negatives) are, of course, nothing new in investigative toxicology. Unfortunately, this possibility can only be resolved by retesting caprolactam in the same assays until a definite conclusion can be drawn with confidence. In the meantime, the evaluator(s) can only work with the data that are available. A third possibility is worth considering. Could the non-carcinogemoty verdict on caprolactam be wrong? To note that caprolactam has been tested in good quality long-term studies in rats and mice is reassuring. To note that the animals were both inbred strains and would thus represent a limited range of D N A variations sounds less so. Though caprolactam has not demonstrated any evidence of carcinogenic potential, there remains the possibility that in some other strains of rat or mouse, or indeed in another species of laboratory animal, caprolactam might be able to reduce cancer. If this does occur, however, the observed tumours would possibly be the result of a non-genotoxic rather than a directly genotoxic pathway since one major characteristic of non-genotoxic carcinogens is their tendency to be active only in a specific strain, sex or species. In any case, it must be said that the current weight of negative evidence - clear findings of non-carcinogenicity in two high quality long-term studies - - offers a high level of reassurance. D u n n g the early 1970s, demonstration of the efficiency of STTs for predicting carcinogenicity dominated the research effort in

genotoxicology. A closer look at some of the early correlation papers of negative STT verdicts with non-carcinogeniclty could often uncover a very easy qualification for the latter I an equivocal result in the only avadable long-term cancer study was sometimes sufficient to warrant the desired " - " in the appropriate box in the 2 x 2 matrix of carcinogenic/mutagenic activity. Taking the available data on face value, we believe that the caprolactam findings can provide scientific support for two independent conclusions. The first is that if genotoxlns are still assumed to be some (maybe small) subset of the chemical universe, then the current regulatory favoured STTs may not be a totally reliable means of detecting them. More fundamentally, if the activity in the two in vivo assays is genuine, it could support the view that genotoxicity, just like other types of toxicity and indeed carcinogenicity, may eventually be shown to be a property found

with most (and maybe all) chemicals, and it is just a matter of finding the set of experimental conditions under which this potential can be realised. Under this paradigm caprolactam could, without great anxiety, simply be described as having weak genotoxic activity at high doses, and the IPCS findings be taken to indicate that the currently favoured battery of in vivo tests are reliable indicators of a weak genotoxic potential. Undoubtedly, there will be those who will persist m the view that genotoxicity is a rare property, while others will tend to the view that such activity is a common characteristic. Until the matter is settled with a reasonable degree of certainty, we will continue to face the difficult task of evaluating STT indications of weak genotoxicity, often without the help of any data from carcinogenicity bioassays. James Hopkins