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dium oitriloJ renal effects tv. R. (1979. A in Canadian nges in renal ion of nitnlo- etabolism of nut. 18. 407. .i of trisodium .ism of selected srch Council ' Animals. III. of Niiniotria. Trisodium ssibl; Carci- uiicatioa No. R. J (19721. nitnlotnace3barmac. 21. Dillon. P. J_ '"I Ecological Report on - :h Advisory non ni sialus of the nitrilotriaceI 3. 375. ad for serial 64. 112. S.. Haygood. ic excretion lustidine. or xicit). fn The 91. Vol. II. emic Press. ll I 4 I 1 11 t FJ C'mrf Tnxutd Vol IV pft 6)1 10 656. PnmcO m Gteai Bnum All rifhu rewvcd 0015-6264 *| 05A65I /Wtyi'fU 0 Copyr.|ht O **** Perga**** Prpt U4 FAILURE OF CHLOROFORM TO INDUCE CHROMOSOME DAMAGE OR SISTER-CHROMATID EXCHANGES IN CULTURED HUMAN LYMPHOCYTES AND FAILURE TO INDUCE REVERSION IN ESCHERICHIA COLI D. J. Kirkland and K. L. Smith Toxicol Laboratories Limited. Bromyard Road. Ledbury. Herefordshire HRS ILC and N. J. Van Abbe Beecham Products. Randalls Road. Leatherhead. Surrey KT22 7RX. England (Received 4 December 1980) Summary--Two strains of Escherichia coli. WP2p and WP2uvrA`p. were treated with chloroform in the plate incorporation assay, and in liquid pre-incubation tests, and in both cases the chemical did not induce reversions. Chloroform, with metabolic activation (addition of S-9 mix! also failed to induce chromosome breakage or sister-chromatid exchanges in human lymphocytes, but benzo(a]pyrene. with and without S-9 mix. induced significant chromosome breakage and sister-chromatid exchanges in lymphocytes from (he same donor. The relevance of these results in the light of other negative in atro tests on chloroform, and positive and negative carcinogenicity tests, is discussed. PLAINTIFF'S EXHIBIT Introduction Chloroform has been widely used over the last cen tury as an anaesthetic, solvent, preservative and fla vour additive, and an evaluation of the safety to humans of such a ubiquitous chemical is clearly im portant. Eschenbrenner A Miller (1945) reported induction of hepatomas in female Strain A mice after repeated oral administration of chloroform in oliveoil solution, but only when the doses were large enough to produce liver necrosis. The results of a study carried out by the National Cancer Institute (1976) on chloroform, given as a solution in corn oil. indicated a significant increase in kidney epithelial tumours in male Osborne-Mende! rats, and a signifi cant increase in hepatocellular carcinomas in male and female B6C3F, mice. However, long-term admin istration of chloroform in a toothpaste vehicle to mice (Roe. Palmer. Worden A Van Abbe. 19791, rats (Palmer. Street Roe. Worden A Van Abbe. 1979) and dogs (Heywood. Sortwell. NoeL Street Prentice, Roe. Wadsworth. Worden A Van Abbe. 1979) produced no significant increase in neoplasms, except for a doserelated sex- and strain-specific increase in mainly benign kidney tumours in mice. Chloroform has also been tested in in vitro muta genicity tests. It was not mutagenic in Chinese ham ster V79 cells at the 8-azaguanine locus (Sturrock. 1977). Neither was it mutagenic in Escherichia coli K.12 or Salmonella typhimurium TA1535 or TA1538 (Gretm. Bitnboes. Egert Goggeimann A Kramer. 1977V nor in TA1537. TA98 or TA100 (Simmon. Kauhanen A TardifL 1977V nor in TA1535. TA1537, TA1538. TA98 or TAIOO (Daniel RichokL Allen. Jones. Roe. Uttley A Van Abbe. 1980V The purpose of the study reported in this paper was to extend the in vitro mutagenicity tests to include mutation in two other strains of . cofi. WP2p and WP2uvrA'p, using both plate incorporation and liquid pre-incubation, and to assess the damage done to human lymphocyte chromosomes and the induc tion of sister-chromatid exchanges by chloroform in the presence of rat-liver micxosomes (S-9 mix). Experimental Reversion in . coli. Two strains of . coli, WP2p and WP2uvrA"p, kindly supplied by Dr S. Venitt of the Institute of Cancer Research. London, were used. Both are tryptophan auxotrophs and both carry the pKMIOl Amptdllin resistance plasmid (McCann. Spingam, Kobori A Ames. 197SV WP2p is fully DNA-repair proficient whereas WP2uvrA ~ p is deficient in excision repair at the uvrA locus. Both strains are reverted to prototrophy either by base change at the site of original alteration or by base change else where in the genome so that the original defect is suppressed. These two strains were treated with chloroform (Analar grade. BDH Chemicals Ltd. Poole. Dorset) in plate incorporation and pre-incubation tests both with and without rat-liver microsomes (S-9) prepared from Aroclor 1254-induced PVG/Ola Hooded rats. In each case chloroform was administered at 10.000, 1000, 100. 10, I or 0-1 ug/plate using acetone as the diluent In the plate incorporation tests, the method was essentially that of Ames. McCann A Yamasaki (1975) for S. typhimurium in that to 2 ml of molten lop agar (66% Lab M agar containing 0-5% NaCl 5. stan- 651 ??l*rfl RowVrK 0!tv.( 652 D J Kirkland. K. L. Smith and N. J Van Abbe dard nutrient broth and 5/ig L-tryptophan/tnl) at 45'C were added 01 ml of a dilution of chloroform plus 01 ml (approximately 10* organisms) of an over night broth culture of the tester bacteria, and. where appropriate. 05 ml of S-9 mix containing 10% S-9 with standard co-factors (Ames et al. 1975). These ingredients were rapidly mixed on a Whirlimixer and poured onto prepared Vogel-Bonner agar plates (Vogel & Bonner. 1956). Each treatment was carried out in triplicate, and the three plates for each dose either with or without S-9 were separately packed in gas-tight containers to avoid leakage of the volatile substances. The plates were incubated at 37CC for 48 hr. after which revertant colonies were counted on a Biotran II automatic colony counter (New Bruns wick Scientific Edison. NJ, USA). In the pre-incubatton tests. 0-5 ml of a dilution of chloroform was mixed with .05 ml of an overnight bacterial culture and. where appropriate 2-5 ml of 10% S-9 mix. -These mixtures were shaken at 200 oscillations/min for 20 min at room temperature on a Braun orbital shaker, and aliquots were added to top agar: 02 ml were taken from non-S-9 tubes and 07 ml from S-9 tubes, in order to give the same final volumes and concentrations as in the plate incorpor ation test. The top agar mixtures were poured on to Vogel-Bonner plates (three per treatment), incubated at 37C for 48 hr and then the revertants were counted. Negative controls were included in each experiment by replacing the chloroform with acetone and posi tive controls were also included: /V-methyl-N'-mtroN-mtrosoguanidine (MNNG: Sigma London Chemi cal Co.. Poole Dorset) was used at 1 or 10/rg/plate as a positive control without S-9 mix. and 2-aminoanthracene (2AA: Sigma Chemical Co.) was used at 5 or 50 (ig/plate with 10; S-9 mix as a control chemical requiring metabolic conversion. Both plate incorpor ation and pre-incubation tests were repeated on sep arate days with fresh cultures, solutions and controls. Chromosome breakage in human lymphocytes. Peri pheral bloiod from a young, healthy volunteer with no history of chromosome fragility, no recent X-ray ex posure and no recent virus infection was cultured in Hepes-bufTered RMPl 1640 medium with glutamine. 20*; foetal calf serum, and 100 units/ml each of peni cillin and streptomycin (Gibco Europe Ltd. Paisley. Renfrewshire). Cultures (10 ml) were established in Sterilin disposable universal bottles and contained 04 ml blood. 01 ml phytohaemagglutinin (PHA: The Wellcome Foundation Ltd. Beckenham. Kent) and Ol ml preservative-free lithium heparin (Sigma Chemical Co.) The cultures were incubated at iTC for 24 hr. duplicate cultures were then treated with 01-ml ali quots of chloroform in acetone to give final concen trations of 50. 100. 200 and 400 pgjtn\ and Ot ml 10*; S-9 mix was added to each culture. The treated cul tures were incubated for 2 hr at iTC. This method of treatment is a slight modification of that described by Thomson &. Evans (1979) At the end of the treatment the cells were centrifuged at 800 rpm for 10 min. resuspended in fresh medium without PHA. and rencubated for a further 22 hr to bring first cycle cells to mitosis after a total of 48 hr culture. One hour before harvest, colcemid (Gibco Europe Ltd) was added to each culture at a final concentration of 0-5 /Jg/ml. Cells were collected and fixed, and slides prepared, stained with Giemsa and mounted using conventional methods. Negative cpntrols were cultures treated with 0-1 ml acetone and 0-1 ml S-9 mix. Positive controls were not included since the donor's lymphocytes had pre viously shown dose-related chromosome breakage after treatment with benzo[u]pyrene in the presence of S-9 mix. Slides were coded by an independent observer, and 100 well-spread metaphases with 46 or more chromo somes were scored from each culture, making a total of 200 cells per treatment. Abnormalities were classi fied as previously reported (Kirkland. Lawler & VenitL 1978). and using the system of Bauchinger. Schmid Einbrodt & Dresp (1976) each abnormality was expressed in terms of the theoretical number of breaks necessary to produce a given lesion: a chroma tid break, a chromosome break, or an acentric frag ment were designated as one break, a dicentric or a rearrangement was designated as two breaks, except where a dicentric and acentric occurred in the same cell, when the total number of breaks equalled two and not three. Sister-chromatid exchanges (5C) in human lympho cytes. Cultures of peripheral blood from the same donor were established as above, but contained 50 pM-bromodeoxyuridine (BUdR) and were kept in the dark to avoid photolysis of BUdR-substituted DNA. Treatments were again for 2 hr in the presence of S-9 mix. but were begun 48 hr after establishment of the cultures, fresh BUdR medium without PHA being used to replace the treatment medium for the final 22 hr. The levels of chloroform used were 25. 50. 75. 100. 200 and 400^g/ml final concentration. The negative control cultures contained acetone, and posi tive controls were not included since the donor's lym phocytes had previously shown a dose-related in crease in SCE after treatment with benzo[a]pyrene in the presence of S-9 mix. Accumulation of metaphase spreads, harvesting of cells and preparation of slides were as above. The dried slides were stored in the dark for at least 7 days after which the chromatids were differentiated by the method of Perry & Wolff (1974) The slides were again coded by an independent observer and. where possible. 50 well-differentiated metaphases with 46 or more chromosomes were scored from each culture, making a possible total of 100 ceils per treatment. Results Rerersion in E. coli The results of the plate incorporation tests are shown in Table 1. It is dear that with chloroform all counts fall in the usual range expected for control counts. An analysis of variance on these figures confinned that there was no dose-response. The bacteria and S-9 were not the cause of this lack of response since the positive control chemicals gave satisfactory reversion rates even though there was wide variation between the two experiments. The results of the pre-incubation tests are shown in Table 2. With chloroform this method of treatment was more toxic than the plate incorporation method. mv-i S2t00f RowVerK rentration of d. and slides runted using i with 0-1 ml ols were not es had prene breakage the presence >bserver, and ore chromoking a total were dassi- Lawler & Bauchinger. abnormality -I number of n: a chromaacentric fragcentric or a eaks. except in the same equalled two non iympho<m the same n contained 'ere kept in -substituted the presence :stablishment thout PHA lum for the . were 25. 50, -itration. The -e. and posiionor's lym-related in>[jjpyrene in arvesting of above. The least 7 days uated by the slides were and. where is with 46 or :ach culture, atment. f 9 I I i I 4 1 i t i l f t i t tests are ..oroform all for control figures conhe bacteria >f response satisfactory de variation e shown in of treatment ion method. Mutagenicity studies with chloroform Table I. Plate incorporation assay of chloroform and positive control chemicals using Escherichia coli strains W P2p and WP2uvrA~ p Mean number of revertants/plaie in strain WP2p WP2uvrA "P Chemical Amount (jig/plate) Without S-9 mix With 10% S-9 mix Without S-9 mix With 10% S-9 mix Chloroform MNNG 2AA Chloroform MNNG 2AA 0 0-1 1 10 100 1000 10.000 0 I 10 0 5 50 0 0-1 1 10 100 1000 10.000 0 1 10 0 5 so Experiment 1 87 5-0 7-7 70 11-0 47 113 8-7 83 3-7 5-0 40 9-0* 6-7 57-3 47* -- -- 498-3 -- -- -- 80 153 185 Experiment 2 117 150 83 83 8-0 17-7 9-3 14-7 10-3 17-7 6-0 21 3 16-0* 12-0 2300 1873-7 -- -- 13 5* -- -- -- 277 21 3 -- 34-7 53 63 63 5-3 70 16-7 63* 6-7 585 6440 -- -- -- 4-3 9-7 30 67 7-3 60 40 14-7 202-3 1091-7 -- -- -- 40 1-7 3-7 30 83 40 3_3* _ 22-5 2890 3860 11-3 20-3 25-3 17 3 15-7 130 14-0* -- -- -- 51 7 268 7 752-3 MNNG N-Methyl-Af'-nitro-N-nitrosoguanidine 2AA - 2-Aminoanthrscene The background lawn was sparse or absent on some or all of the plates. Values are means for three plates. 653 and the control counts are generally higher, but there is no evidence of mutagenic activity. The positive con trols were again satisfactory, but although there is variation between the experiments, both strains show better responses than the controls for the plate incor poration assay. Chromosome breakage in human tysnphocytes The response of the donor's lymphocytes to chromo some breakage by benzo{a]pyrene treatment, with and without addition of S-9 mix. is shown in Fig 1, in which all abnormalities have been calculated as theor etical lesions. It is dear that there is a dose-related increase in breakage with benzo[a]pyrene treatments, and this ts confirmed by calculating correlation coeffi cients. which are 0962 (without S-9) and 0891 (with S-9). The response of the same donor's lymphocytes to chloroform indicated random variation around the control value (Fig 2). The highest breakage level was at 200/ig chloroform/ml with 8 breaks/100 cells com pared with 5-5 breaks/100 cells in the control. How ever. comparing these levels by using the chi-square test with Yates correction shows that this difference is not significant (/J 0635. P > 025). The random nature of the breakage around control levels is con firmed by a correlation coefficient of 0248. SCE in human lymphocytes The response of the donor's lymphocytes to SCE induction by benzofdjpyrene is shown in Fig 3. The control levels of SCE are quite high for this donor, being 16-375 SCE/cell without S-9 mix and 18-23 SCE/ceil with the addition of S-9 mix. However, there is a significant increase in SCE with beruoCu]pyrene treatment and reasonable dose-response trends giving correlation coefficients of 0782 (without S-9) and 0995 (with S-9) over the range 0-10pg/ml. The response of the same donor's lymphocytes to chloroform is shown in Fig 4. The control level of SCE (18-35 SCE/cell) closely resembles that seen in the benzo[a]pyrene control with S-9 mix, but at only two treatment levels are there more SCE/cell than in the control. At one of these (SO jig chloroform/ml) the SCE frequency is significantly different from the con trol (y1 -- 1178, P < 005 with five degrees of free dom), but there is no dose-response trend, and at some doses (e.g 25 pg/ml) the SCE frequency is sig nificantly lowered Or " 26-13, P < OOOl with five degrees of freedom). This lack of dose-response is con- s- i w?:> c ?1" 0 RowVerK 654 D J Kirkland. K L. Smith and N. J Van Abbe Table 1 Pre-incuhation uswn- of chloroform and posnice control chemicals using Escherichia coll strains WP2p and WP2uirA-p Mean no of revertants/plate in strain WP2p WP2uvrA~p Chemical Amount (pg/plate) Without S-9 mix With io*/; S-9 mix Without S-9 mix With 10*/. S-9 mu Chloroform MNNG 2AA Chloroform MNNG 2AA 0 0-1 1 10 100 1000 10.000 0 1 10 0 5 50 0 01 1 10 100 1000 10.000 0 1 10 0 5 50 Experiment 1 24-0 17 7 25 7 183 17-7 17-7 22-7 18 3 NC* 160 NC* 19-7 NC* 21-0 8780 1403-3 -- -- -- NC* -- -- -- 26-3 520 60-3 Experiment 2 16-7 460 21-3 32-3 19-3 19-7 220 27-7 240 303 NC* 15-3* NC* 18-3 4820 2209-7 -- -- NC* -- -- -- 357 81 7 -- 102-3 333 26-7 2-3* NC* NC* NC* NC* 58 3 455-7 15510 -- -- 450 48-7 47-3 530 530 NC* NC* 840 4630 1672-7 -- -- -- 400 403 405 423 440 527 NC* -- -- -- 15-3 152-3 1239-3 77-3 600 113 7 56-3 517 107* NC* -- -- -- 139-3 13607 19900 NC Not counted MNNG - /V-Methyl-/V'-niiro-N-nitrosoguanidme 2AA a 2-Aminoanthracene The background lawn was sparse or absent on some or all of the plates. Values are means for three plates. firmed by the correlation coefficient which is -0206 for the whole dose range and is only 0596 for the range 0-75 pg/mi. _ In this study chloroform has been shown not to be mutagenic in three in citro tests with different genetic endpoints. Since the start of this work, we have learned that chloroform was one of the substances BaneoCeS pyrene, pQ/m\ Fig. I. Dove-related induction of chromosome breakage in human lymphocytes after treatment with benzoa]pyrtne with I4i and without () metabolic activation (addition of S-9 mill. Abnormalities are expressed as the theoretical number of lesions necessary to produce the abnormalities according to the method of Bauchinger ef at. 11926). Fig 2. Lack of (fleet of treatment with chloroform plus S-9 mu oo chromosome breakage in human lymphocytes, obtained from the same donor as for Fig 1. a. i Q??001 R owVer K OH I 4 1 > I f * 4 * -0-206 for the to be genetic e have stances 0 i i 5 *00 Mus $-9 tocyie*. Mutagenicity studies with chiorororm 65} related (Moore. Chasseaud. Majeed. Prentice. Roe & Van Abbe. 1980) to the nephrotoxic action of chloro form at high doses. This may explain why with lower doses of chloroform that are insufficient to cause liver or kidney regeneration, there was no increase in tumours in the rats, mice and dogs treated with tooth paste (Heywood et al. 1979; Palmer e( al. 1979; Roe er al. 1979). An additional feature of the work reported here is that this appears to be the first experimental investi gation of the response of human cells to chloroform. The view given in the IARC monograph on chloro form (1ARC, 1979) that the compound should, for practical purposes, be regarded as if it presented a carcinogenic risk to humans, is not supported by the results of the studies reported here. Fig. 3. Significant and dose-related induction of sistcrchromaud exchanges (SCE) in human lymphocytes after treatment with betuofa]pyrene with (A) and without () S-9 mix. assessed in the international programme for the evaluation of short-term tests for carcinogens (de Senes A Ashby, 1981), and out of 40 assays, 33 vere negative, and seven gave positive results which were unreproducible, giving negative results on repeat These tests included bacterial tests with Salmonella and E. coli, DNA repair in bacteria, mammalian cell transformation, unscheduled DNA synthesis in HeLa cells, the dominant lethal test, the micronudeus test and SCE induction in CHO cells. The results reported here confirm the non-mutagenicity of chloroform in . coli, even after pre-incubation. and extend the negative cytogenetic observations to breakage and SCE induction in human lympho cytes. This tout failure of chloroform to cause any type of mutagenic event in vitro raises serious ques tions in relation to the prediction of cancer risk, par ticularly because in three animal studies (Eschenbrenner & Miller, I94S; National Cancer Institute, 1976; Roe ef al. 1979) signs of cardnogenitity were observed when the dose levels greatly exceeded normal ex posure levels in human use. However, in two of these studies, liver toxicity was observed and it may be that chloroform only acts as a carcinogen when it causes sufficient tissue damage to simulate partial hepatectomy. The development of kidney tumours under cer tain experimental conditions may likewise be directly MM ISO MO ISO 100 HO 400 CMorofOfm, pq/ml Fig. 4. Lack of effect of chlproforns fius S-9 mix on the induction qf'S>Mw<hro(Mtid aChange! (SCE) m human lymphocytes obtained from the same donor as for Fig 3. REFERENCES Ames, B. N, McCann. J. A Yamasaki. E. (I97S). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity tat Muta tion Res. 31, 347. Bauchinger. M,, Schmid. E,, Einbrodl H. J. A Dresp. i. (1976k Chromosome aberrations in lymphocytes after occupational exposure to lead and cadmium. Mutation Res. 40. 57. Daniel M. R_ Richold. M,, Allen. 3. Jones. E,, Roc F. J. C. Uttley, M. A Van Abbe. N. J. (1980k Bacterial muta genicity and cell transformation studies with chloroform. Toxicology Lett. Special Issue p. 247. de Series, F. J. A Ashby. J. (Editors) (1981k Evaluation of Short-term Tent for Carcinogens. Repoet of the Inter national Collaborative Program. Progress in Mutation Research. Vol. I. Elsevier/North-HoUand Inc. New York. Eschenbrenner, A. B. A Miller. E. (I94SI Induction of hepatomas in mice by repeated oral administration of chloroform with observations on sex differences. J. natn. Cancer Inst. 3. 231. Gretm, H_ Bunboes. D, Egert G,, Goggeimann. W. A Kramer. M. (1977). Mutagenicity and chromosomal aberrations as an analytical tool for in vitro detection of mammalian enzyme-mediated formation of reactive metabolites. Arckt ToxicoL 39, 139. Heywood. R,, Sortwcll R. J, Noel P R. B_ Street A. E,, Prentice D. E, Roc F. J. C. Wadsworth. P. F. Worden, A. N. A Van Abbe, N. J. (1979). Safety evaluation of toothpaste containing chloroform III. Long-term study in beagle dogs. J. enetr. Path. ToxicoL 2. 833. IARC Working Group (1979). IARC Monographs on the Evaluation of the Corcutogemc Risk of Chemicals to Humans. Some Halogenated Hydrocmhons. VoL 20. p. 401. International Agency for Research on Cancer. Lyon. Kirkland. D. J. Lawler. S. D. A Venitt S. (1978k Chromo somal damage and hair dyes. Lancet IL 124. McCann. J.. Spingam, N. E_ Koboti J. A Amec B. N. (1975V Detection of carcinogens as mutagens: bacterial tester strains with R factor plasmids. Proc. natn. Acad. Sci. US.A. 72.979. Moore. D. Chasseaud. L F. Majeed. S. K, Prentice D. E,, Roc F. J. C A Van Abbe N. J. (1910k Effect of dose and vehicle on early tissue damage and regenerative activity after chloroform administration. Toxicology Leu. Special Issue p. 234. National Cancer Institute (1976V Report on Carcinogenesis Bioassay of Chloroform. Palmer. A. K, Street A. E. Roe F. J. C, Worden. A. N. A Van Abbe N. J. (1979V Safety evaluation of toothpaste containing chloroform II. Long-term studies in rau. J. enotr. Path ToxicoL 2.121. toluol tfowVerK 0(> 656 D. J. Kirkland. K. L. Smith and N J Van Abie Perry. P. & Wolff. S. (1974). New Giemsa method for the differential staining of sister chromatids. Nature. Load. 2S1. 156 Roe. F. J. C_ Palmer. A. K,, Worden. A. N. & Van Abbe. N. i. (1979). Safety evaluation of toothpaste containing chloroform I. Long-term studies in mice. J. emir. Path. Toxicol. 2. 799. Simmon. V. F, Kauhancn. K. St Tardiff. R. G. (1977). Mutagenic activity of chemicals identified in drinking water. In Progress in Genetic Toxicology, p. 249. Else- vier/North-Holland Biomedical Press. Amsterdam. Slurrock. J. (1977). Lack of mutagenic effect of halothane or chloroform on cultured cells using the azaguaninc test system. Br. J. Anaetth. 49. 207. Thomson. V.ti Evans, H. i. (1979). Induction of sisterchromatid exchanges in human lymphocytes and Chinese hamster cells exposed to aflatoxin B, and Nmethyl-N-nitrosourea. Mutation Res. 67, 47. Vogel. H. J. St Bonner. D. M. (1956). Acetylornithase of Escherichia coli: partial purification and some proper ties. J biot. Chem. 218. 97. ?21001 RowVerh 0!,V(1 u r