The cytotoxic, mutagenic and clastogenic effects of chromium-containing compounds on mammalian cells in culture

55

Mutation Research, 67 (1979) 55--63 © Elsevier/North-Holland Biomedical Press

THE CYTOTOXIC, MUTAGENIC AND CLASTOGENIC E F F E C T S OF CHROMIUM-CONTAINING COMPOUNDS ON MAMMALIAN CELLS IN C U L T U R E

R.F. NEWBOLD, J. AMOS and J.R. CONNELL Institute of Cancer Research: Pollards Wood Research Station, Chemical Carcinogenesis Division, Nightingales Lane, Chalfont St. Giles, Buckinghamshire HP8 4SP (Great Britain) (Received 21 July 1978} (Accepted 22 December 1978)

Summary Examples of chromic and chromate salts have been examined for their effects on a cultured Chinese hamster cell line. The responses studied were cytotoxicity, mutagenesis and clastogenesis. Chromate (hexavalent chromium) salts of both high and medium water solubility were active in producing all three classes of response, whereas an insoluble chromate salt and a soluble chromic (trivalent chromium) salt were inactive. In addition to illustrating the value of using mammalian cells in culture for screening chemicals for biological activity, the results of this study reinforce current views regarding the genotoxic properties o f chromates.

The widespread use of chromium-containing materials in the pigments and and plating industries is dependent u p o n associated industries involved in the large-scale production of such c o m p o u n d s from naturally occurring chrome ores. Since the 1930s, many surveys [1--3,5,6,8,16,17,19,25] carried out in Germany, the United States and Great Britain have revealed that workers from various chromium industries have an increased risk of developing lung cancer. However, while such epidemiological evidence appears conclusive, the individual chromium compounds responsible for this occupational hazard have, in many cases, not yet been identified. Experiments to determine the carcinogenicity of chromium c o m p o u n d s in laboratory animals have shown that certain chromate (hexavalent chromium) salts -- primarily those of low or medium water solubility -- are u n d o u b t e d l y carcinogenic in several strains of rats and mice b y several routes of administration [ 11,13,15,16,18,29,31,36--38]. Conversely, there is less evidence suggesting that chromic (trivalent chromium) compounds are carcinogenic, although it

56 must be said that the chromic salts which have been tested were chiefly those of high water solubility. Chromates have also been found to possess moderate mutagenic activity in bacteria [35,42] and yeast [4], and are capable of inducing morphological transformation [10,41] and chromosome damage [40,41] in cultured mammalian cells. Where tested, chromic c o m p o u n d s proved ineffective in these systems. Despite such progress, however, the mechanism by which chromates exert their genotoxic properties is far from clear. In order to extend current knowledge of the genetic toxicity of chromium compounds, we have, in the present investigation, examined the cytotoxic and mutagenic effects of some chromic and chromate salts on cultured Chinese hamster (V79) cells. The induction of clones resistant to the purine analogue 8-azaguanine was used as a marker for mutagenesis. With the most active compound -- potassium dichromate -- a more detailed study was carried out in which cytotoxicity, mutagenicity and clastogenicity were assayed simultaneously in each population of treated cells. The results of this work are discussed with particular reference to the somatic mutation theory of carcinogenesis, and an attempt is made to explain previously observed relationships between the valency or the solubility of chromium compounds and their carcinogenic activity. Materials and m e t h o d s

Chemicals The chromium c o m p o u n d s used in this study are listed in Table 1 along with details of their chromium valency and water solubility. Where possible they were, immediately before an experiment, dissolved in distilled water at 300 times the required treatment concentration and the resulting solutions membrane filter (Millipore) sterilised. The insoluble or partially soluble c o m p o u n d s (lead and zinc chromates respectively) were autoclaved as solids and then prepared as fine suspensions in distilled water. Cell line The advantages of the Chinese hamster cell line V79/4 for studies of mammalian-cell mutagenesis have been discussed previously [32,33]. A clone of V79/4, designated V79/4-K,, selected for its high plating efficiency and its TABLE l CHROMIUM COMPOUNDS UNDER INVESTIGATION Compound a

Formula

Chromium valency

Water s o l u b i l i t y

Potassium dichromate Zinc c h r o m a t e Lead c h r o m a t e Chromic acetate

K2Cr 207 ZnCrO 4 PbCrO 4 ( C H 3 C O O ) 3 Cr

VI VI VI III

highly soluble slightly soluble v e r y l o w s o l u b i l i t y ( 0 . 2 mg/1 H 2 0 ) h i g h l y soluble

a All c h r o m i u m c o m p o u n d s w e r e o b t a i n e d f r o m British Drug H o u s e s and w e r e o f B D H L a b o r a t o r y Rea g e n t grade e x c e p t f o r K 2 C r 2 0 7 w h i c h w a s o f A n a l y t i c a l R e a g e n t grade.

57 ability to form diffuse microcolonies, was used throughout the course of this work. The latter characteristic is desirable if efficient selection of mutants is to be achieved [ 33]. Cell culture and treatment with chromium compounds Cells were cultured in Dulbecco's modified MEM supplemented with 10% foetal calf serum (Gibco-Biocult Ltd.); stock cultures were carried in the logarithmic phase of growth in 75-cm 2 culture flasks (Nunc, UK). Culture conditions were maintained at 37 + 0.5°C and pH 7.2 + 0.2 b y incubating medium and cells in an atmosphere of 10% CO2 : 90% air. Suspensions of single cells, when required, were prepared b y incubating monolayer cultures with a mixture of equal parts 0.25% trypsin (1/250 Difco) and 0.5 mM versene at 37°C for 5--10 min. For experiments 1 X 106 cells were plated in 75-cm 2 flasks and incubated for 24 h prior to the addition of the appropriate stock treatment solution (0.1 ml). Cell survival and mutagenesis assay ~ After 24 h treatment at 37°C the cells were detached from each flask with trypsin/versene and the density of the resulting single-cell suspension estimated using a haemocytometer. For the determination of induced toxicity 1 X 102 cells, and for mutagenesis 5 X 104 cells, were plated in 5- and 9-cm plastic tissue culture dishes {Sterilin) respectively. Mass selection of mutants defective in the purine salvage enzyme, hypoxanthine--guanine phosphoribosyl transferase was effected b y adding 8-azaguanine to the plates such that its final concentration in the medium was 30 pg/ml. To achieve maximum recovery of mutants a complete expression time curve [9,34] was constructed for each dose of chromium compound. In practice, 10--15 replicates were treated with 8-azaguanine at each of 5 expression time points ranging from 0--96 h after plating. Survival plates were stained after 7 days incubation, mutation plates 3 days later, and colonies of more than a b o u t 50 cells were scored macroscopically. Representative values for mutation frequency were taken as being those at peak-expression times, which ranged from about 48 h t minimally lethal doses to 72 h for more toxic treatments. Control frequenc,=s never exceeded 1.6 X 10 -s per survivor. A discussion of the various pitfalls encountered in this t y p e of experiment has been published previously [33]. Assay o f cells for chromosome damage Clastogenic effects of potassium dichromate were investigated using the same cell populations as those from which samples had been removed from the mutation assay. 1 X 106 treated cells were seeded in 75-cm 2 flasks and incubated for 24 h. Metaphase cells were harvested following a 2-h treatment with colcemid (0.1 pg/ml; Gibco-Biocult) centrifuged, and resuspended in a h y p o t o n i c solution {0.075 M) of KC1. The cell pellet obtained after further centrifugation was fixed with fresh 3 : 1 methanol : acetic acid. Conventional air-dried slide preparations were stained with Giemsa (2% solution: pH 6.8) for 3 min. From each sample 100 metaphases were scored for chromosome and chromatid aberrations.

58 Results

Cytotoxicity and mutagenicity o f a series o f chromium compounds A summary of the results obtained with a series of chromium compounds of different chromium valency and water solubility is shown in Table 2. Two chromate (hexavalent chromium) salts -- potassium dichromate (highly soluble) and zinc chromate (sparingly soluble) were c y t o t o x i c and mutagenic in a dosedependent manner. With most doses, statistical analysis of the m u t a t i o n results in the form of a t-test revealed a highly significant difference (p < 0.01--0.001) between the mean colony numbers on treated and those on control dishes. When the two compounds were compared in terms of applied dose, potassium dichromate was about 5-fold more effective than zinc chromate; doses higher than 0.8 pg/ml K2Cr207 or 5 gg/ml ZnCrO4 were too toxic to assay. At doses lower than these, c y t o t o x i c i t y was manifest as a reduction in colony-forming efficiency and a reduction in the size of colonies obtained on survival plates after 7 days incubation. In addition, marked vacuolation of the cells was evident immediately after the 24-h t r e a t m e n t period. Chromic acetate (soluble trivalent chromium salt) and lead chromate (insolu-

TABLE 2 INDUCTION OF 8-AZAGUANINE-RESISTANT V79/4 CELL MUTANTS TAINING COMPOUNDS: NUMBERS OF MUTANT COLONIES SCORED Experiment

1 Potassium dichromate (K2Cr207)

2 Potassium dichromate ( K 2 C r 2 0 7)

3 Chromic acetate (CH3COO)3Cr

4 Zinc chromate (ZnCrO4) Lead Chromate (PbCr04)

Dose of chromium compound (/lg/ml)

0

BY

CHROMIUM-CON-

% Survival

Mean number o f 8-azg r colonies per 9-cm dish a

t b

I00

0.6

-1.24

0.35

85.9

0.4 0.5

80.2 83.2

i.i 1.4 2.2

0.76 0.77 0.78

59.6 40.3 38.6

0 0.5 0.6 0.65 0.7 0.75

p

--

1.75 3.12

>0.I <0.1 <0.01

2.8 3.0 1.8

3.70 4.30 2.80

<0.01 <0.001 <0.01

100 75.0 77.3 65.2 68.9 52.2

0.4 2.5 3.2 2.9 3.8 3.9

-3.59 4.64 5.00 5.08 5.00

-<0.01 <0.001 <0.001 <0.001 <0.001

0 50 100 200

100 85.0 82.8 95.5

0.8 1.5 1.0 1.4

-1.18 1.00 1.14

->0.1 >0.1 >0.1

0 1 4

100 99.2 37.7

0.5 1.2 3.2

-1.75 5.46

-0.1 <0.001

5 10

100 80.2

0.7 0.5

0.72 0.93

>0.1 >0.1

a At the peak-expression time. b T h e s i g n i f i c a n c e o f t h e d i f f e r e n c e b e t w e e n c o n t r o l a n d t r e a t e d m e a n s w a s e s t a b l i s h e d u s i n g a t-test. V a l u e s o f p w e r e o b t a i n e d f r o m p u b l i s h e d t a b l e s o f t h e d i s t r i b u t i o n o f t.

59 ble hexavalent salt) were inactive in this system at 200 times and 10 times respectively the maximum dose at which cell survival was measurable with potassium dichromate.

Dose--response with potassium dichromate Survival of V 7 9 / 4 cells following treatment with a series of doses of K2Cr207 is shown in Fig. 1. There existed a clearly defined dose threshold (0--0.7 #g/ml) b e y o n d which survival dropped rapidly. At doses higher than 0.8 pg/ml it became very difficult to obtain reproducible survival data. This t y p e of dose-response curve is usually associated with agents which have a generalised poisoning effect on cells, rather than those which tend preferentially to attack a more specific cellular target such as DNA. The mutational response at the same doses of K2Cr207 is shown in Fig. 2; in this case mutation frequency has been plotted against the logarithm of survival rather than dose to illustrate more clearly the range o f survival within which mutagenesis could be measured. Mutation frequency increased in a linear fashion with increasing lethality when only sub-threshold (minimally lethal) doses were considered. At higher doses it became increasingly difficult to recover mutants, presumably because of the overall poor health of the cells. The last point on the graph shows this drop in mutation frequency just beginning to occur.

100q

• m 15 o r X

> 10

10

o~

g

Q

o/ o//o /•

/o

o.8 Dose K2Cr2O 7 OJg/ml)

7b % Survivol

F i g . 1. S u r v i v a l o f V 7 9 / 4 cells f o l l o w i n g e x p o s u r e t o v a r i o u s d o s e s o f K 2 C r 2 0 7 . A f t e r a 2 4 - h t r e a t m e n t 1 0 0 cells w e r e p l a t e d in e a c h o f 1 0 r e p l i c a t e 5 e m p e t r i - d l s h e s a n d t h e s e w e r e i n c u b a t e d f o r 7 d a y s b e f o r e st~tn!ng and scoring colonies. Different symbols represent different experiments. F i g . 2. M u t a g e n e s i s in V 7 9 / 4 cells f o l l o w i n g e x p o s u r e t o v a r i o u s d o s e s o f K 2 C t 2 0 7 . A f t e r a 24-11 t r e a t m e n t p e r i o d 5 X 1 0 4 cells w e r e p l a t e d i n r e p l i c a t e 9 ~ m p e t r i d i s h e s . M u t a n t s w e r e s e l e c t e d a t a series o f expression times by adding 8-azaguanine (30/~g/ml) to batches of 10--15 dishes. Plates were stained and n u m b e r s o f c o l o n i e s s c o r e d 1 0 d a y s a f t e r s e e d i n g cells; m u t a t i o n f r e q u e n c i e s w e r e c a l c u l a t e d p e r s u r v i v i n g cell. T h e v a l u e s s h o w n i n t h e f i g u r e are t h o s e o b t a i n e d a t p e a k ~ x p r e s s i o n t i m e s . D i f f e r e n t s y m b o l s incHeate different experiments.

6O TABLE 3 I N D U C T I O N O F C H R O M O S O M E D A M A G E IN V 7 9 / 4 C E L L S BY P O T A S S I U M D I C H R O M A T E Dose K2Cr207 (pg/ml) 0 O 0.35

Chromatid breaks

Iso-chromatid deletions

Gaps

Chromatid ring a n d fragments

Chromatid exchanges

Total number of ceils a w i t h aberrations

1 1 4

1 1

1

1 2 5

0.4 0.5 0.6

5 10 19

1 4 6

1 4

1 2 2

7 16 24

0.65 0.7 0.75

25 34 40

3 6 3

10 5 4

3 5 3

30 32 34

0.76 0.77 0.78 0.8

36 36 29 71

9 9 9 33

8 4 7 1

1 3 4 8

39 33 34 66

3

2

a F o r c o n t r o l a n d t r e a t e d V 7 9 cells, 1 0 0 m e t a p h a s e s w e r e c o u n t e d .

Clastogenicity of potassium dichromate The results of the analysis o f dichromate-treated cells for chromosome damage are shown in Table 3. At all doses K2Cr207 behaved as a very powerful clastogenic agent, producing a highly significant increase in chromosome aberrations; the predominant aberrations were chromatid breaks. These results are in accord with those o f Tsuda and Kato [40,41]. Discussion

Acceptance of the somatic mutation t h e o r y of carcinogenesis has recently b e c o m e so widespread that the mutagenicity of an external agent is now considered a reliable indication of its potential carcinogenicity. Indeed, good correlations have been established b e t w e e n mutagenicity and carcinogenicity with diverse groups of chemicals [12,26,27,32]. Inorganic carcinogens, however, have received less attention in this respect than other classes of carcinogen. The present study of carcinogenic and non-carcinogenic chromium c o m p o u n d s was c o n d u c t e d in an effort to further knowledge in this direction. In experiments with laboratory animals, some chromium salts have proved to be moderately p o t e n t carcinogens while others are weak or inactive. Two main factors -- chromium valency and water solubility -- appear to influence the carcinogenic p o t e n c y of these compounds. In general, chromates (hexavalent chromium) appear to be active whereas chromic (trivalent chromium) c o m p o u n d s are inactive; moreover, only chromates of medium or low water solubility are carcinogenic [14]. The latter condition has been shown not to apply in the case of in vitro tests for biological activity, b o t h in the present investigation and in previous work b y others with bacteria [42]. We have shown here that chromates of high (e.g. K2Cr20~) as well as those of medium (e.g. ZnCrO4) water solubility are p o t e n t c y t o t o x i c and mutagenic agents. The

61 obvious explanation for the lack o f carcinogenicity of high-solubility chromates is that, even though they may be p o t e n t mutagens, they disappear in solution from the site of application very rapidly, and are excreted. Our experiments failed to detect biological activity in lead chromate, a highly insoluble salt which is known to be a fairly p o t e n t carcinogen [11,29]. In view of the low water solubility of this c o m p o u n d (approx. 0.2 #g/ml water) and the relatively short exposure time used (24 h) this result was perhaps not too surprising. Significant exposure of cells to chromate ions from PbCrO4 would, on the other hand, be expected in carcinogenicity tests where the period of exposure is very much longer and the material persists at the site of application. There is some d o u b t as to whether trivalont chromium compounds are carcinogenic in laboratory animals. Although a few reports exist describing the induction of small numbers of injection-site sarcomata by chromic salts [13,15] results have, in the main, been negative. However, as the c o m p o u n d s tested were of high water solubility, no fundamental conclusions concerning the biological activity of trivalent chromium should be drawn from these experiments. Nevertheless, in vitro tests, where solubility is not a complicating factor, have also failed to reveal any activity in chromic salts. The results obtained in the present study are no exception; chromic acetate proved inactive at a dose range 200 times greater than that used with potassium dichromate. Various mechanisms have been proposed for the biological activity of chromates [7,30,39]. The most recent and perhaps the most notable of these is d o c u m e n t e d in a series of publications b y Levis and collaborators [20--23,28]. These workers suggest that the effects of potassium dichromate on mammalian cells are attributable b o t h to the action of hexavalent chromium at the plasmamembrane level on the mechanisms involved in nucleoside uptake, and, at the intracellular level, to the interaction of reduced trivalent chromium with DNA. However, as the authors themselves stress, this is only a tentative hypothesis. It is clear that further knowledge of the nature of the interaction of hexavelent and trivalent chromium with intra- and extra-cellular components is required before the question of chromate mutagenesis can be resolved. Acknowledgements We thank Dr. S. Venitt for helpful discussion and Mrs. M. White for excellent technical assistance throughout the course of this work. The work was supported b y NIH (U.S.A.) contract No. I-CP-33367 and, in part, b y grants to the Institute of Cancer Research from the Medical Research Council and the Cancer Research Campaign. J.R. Connell is a Gallaher Research Fellow. References 1 B a e t j e r , A . M . , P u l m o n a r y c a r c i n o m a in chromate workers, I, A r e v i e w of literature and report of cases, A r c h . I n d . H y g . , 2 ( 1 9 5 0 ) 4 8 7 . 2 B a e t j e r , A . M . , P u l m o n a r y c a r c i n o m a in chromate workers, II, I n c i d e n c e o n basis of hospital records, Arch. I n d . H y g . , 2 ( 1 9 5 0 ) 5 0 5 . 3 B i d s t r u p , P . L . , a n d R . A . M . Case, Carcinoma of the l u n g in w o r k m e n in the bichromate-producing

62 i n d u s t r y i n G r e a t Britain, B r i t . J . I n d . M e d . , 1 3 ( 1 9 5 6 ) 2 6 0 . 4 B o n a t t i , S., M. M e i n i a n d A. A b b o n d a n d o l o , G e n e t i c e f f e c t s of p o t a s s i u m d i c h r o m a t e , M u t a t i o n R e s . , 38 (1976) 147. 5 Brinton, H.P., E.E. Frasier and A.L. Koren, Morbidity and mortality experience among chromate workers, Public Health Rep., 67 (1952) 835. 6 B r o w n i n g , E., C h r o m i u m , In: T o x i c i t y o f I n d u s t r i a l M e t a l s , B u t t e r w o r t h s , L o n d o n , 1 9 6 9 , p. 1 1 9 . 7 C h a m p y - H a t e m , S., C a n c e r s d u c h r o m e e t c o m p l e x c h r o m e - i m i d a z o l e , C . R . A c a d . Sci., 2 5 4 ( 1 9 6 2 ) 3267. 8 Davies, J . M . , L u n g - c a n c e r m o r t a l i t y o f w o r k e r s m a k i n g c h r o m e p i g m e n t s , L a n c e t , ( 1 9 7 8 ) 3 8 4 . 9 D u n c a n , M . E . , a n d P. B r o o k e s , T h e i n d u c t i o n o f a z a g u a n i n e r e s i s t a n t m u t a n t s in c u l t t t r e d C h i n e s e h a m s t e r cells b y r e a c t i v e d e r i v a t i v e s o f c a r c i n o g e n i c h y d r o c a r b o n s , M u t a t i o n R e s . , 21 ( 1 9 7 3 ) 1 0 7 . 1 0 F r a d k i n , A., A. J a n o f f , B.P. L a n e a n d M. K u s c h n e r , In v i t r o t r a n s f o r m a t i o n o f B H K 2 1 cells g r o w n in the presence of calcium chromate, Cancer Res., 35 (1975) 1058. 11 F u r s t , A . , M. S c h l a u d e r a n d D . P . S a s m o r e , T u m o r i g e n i c a c t i v i t y o f l e a d c h r o m a t e , C a n c e r R e s . , 36 (1976) 1779. 1 2 H u b e r m a n , E . , a n d L. S a c h s , C e l l - m e d i a t e d m u t a g e n e s i s o f m a m m a l i a n cells w i t h c h e m i c a l c a r c i n o gens, Int. J. Cancer, 13 (1974) 326. 1 3 H u e p e r , W . C . , E n v i r o n m e n t a l e a r c i n o g e n s i s a n d c a n c e r s , C a n c e r R e s . , 21 ( 1 9 6 1 ) 8 4 2 . 1 4 H u e p e r , W . C . , a n d W.D. C o n w a y , in: C h e m i c a l C a r c i n o g e n e s i s a n d C a n c e r , T h o m a s , I L , 1 9 6 5 , p. 3 8 7 . 1 5 H u e p e r , W . C . , a n d W.W. P a y n e , E x p e r i m e n t a l s t u d i e s in m e t a l c a r c i n o g e n e s i s , C h r o m i u m , n i c k e l , i r o n , arsenic, Arch. Environ. Health, 5 (1962) 445. 16 International Agency for Research on Cancer, Chromium and inorganic chromium compounds, I.A.R.C. Monographs, 2 (1973) 100. 1 7 L a n g a r d , S., a n d T. N o r s e t h , A c o h o r t s t u d y o f b r o n c h i a l c a r c i n o m a s in w o r k e r s p r o d u c i n g c h r o m a t e pigments, Brit. J. Ind. Med,, 32 (1975) 62. 1 8 L a s k i n , S., M. K u s c h n e r a n d R . T . D r e w , S t u d i e s in p u l m o n a r y c a r c i n o g e n e s i s , in: I n h a l a t i o n c a r c i n o genesis, U.S. A t o m i c E n e r g y C o m m i s s i o n S y m p o s i u m Series N o . 1 8 ( 1 9 7 0 ) 3 2 1 . 1 9 L e t t e r e r , E . , K . N e i d h a r d t a n d H . K l e t t , C h r o m a t l u n g e n k r e b s e s u n d C h r o m a t s t a u b e l u n g e , E i n e klinische, pathologisch-anatomische und gewerbehygienische Studie, Arch. Gewerhepathol. Gewerbehyg., 12 (1944) 323. 2 0 Levis, A . G . , a n d M. B u t t i g n o l , E f f e c t s o f p o t a s s i u m d i c h r o m a t e o n D N A s y n t h e s i s in h a m s t e r f i b r o blasts, Brit. J. Cancer, 35 (1977) 496. 21 Levis, A . G . , M. B u t t i g n o l a n d L. V e t t o r a t o , I n h i b i t i o n o f D N A s y n t h e s i s in B H K f i b r o b l a s t s t r e a t e d in vitro with potassium dichromate, Experientia, 33/1 (1976) 62. 2 2 Levis, A . G . , V. B i a n c h i , G. T a m i n o a n d B. P e g o r a r o , C y t o t o x i c e f f e c t s of h e x a v a l e n t a n d t r i v a l e n t c h r o m i u m o n m a m m a l i a n cells in v i t r o , B r i t . J. C a n c e r , 37 ( 1 9 7 8 ) 3 8 6 . 2 3 Levis, A . G . , M. B u t t i g n o l , V. B i a n c h i a n d G . S p o n z a , E f f e c t s o f p o t a s s i u m d i c h r o m a t e o n n u c l e i c a c i d a n d p r o t e i n s y n t h e s i s a n d o n p r e c u r s o r u p t a k e in B H K f i b r o b l a s t s , C a n c e r R e s . , 3 8 ( 1 9 7 8 ) 1 1 0 . 2 4 L e v y , L . S . , a n d S. V e n i t t , C a r c i n o g e n i c a n d m u t a g e n i c a c t i v i t y o f c h r o m i u m - c o n t a i n i n g m a t e r i a l s , Brit. J . C a n c e r , 3 2 ( 1 9 7 5 ) 2 5 4 . 2 5 M a e h l e , W., a n d F. G r e g o r i u s , C a n c e r o f t h e r e s p i r a t o r y s y s t e m in t h e U n i t e d S t a t e s c h r o m a t e - p r o ducing industry, Public Health Rep., 63 (1948) 1114. 2 6 M c C a n n , J . , E. C h o i , E. Y a m a s a k i a n d B.N. A m e s , D e t e c t i o n o f c a r c i n o g e n s as m u t a g e n s in t h e Salm o n e l i a / m i c r o s o m e t e s t : A s s a y o f 3 0 0 c h e m i c a l s , P r o c . N a t l . A c a d . Sei. ( U . S . A . ) , 7 2 ( 1 9 7 5 ) 5 1 3 5 . 2 7 M c C a n n , J . , N . E . S p i n g a m , J . K o b o r i a n d B.N. A m e s , D e t e c t i o n o f c a r c i n o g e n s as m u t a g e n s : B a c t e r i a l t e s t e r s t r a i n s w i t h R . - f a c t o r p i a s m i d s , P r o c . N a t . A c a d . Sci. U . S . A . , 7 2 ( 1 9 7 5 ) 9 7 9 . 2 8 M a j o n e , F . , E f f e c t s o f p o t a s s i u m d i c h r o m a t e o n m i t o s i s o f c u l t u r e d m a m m a l i a n cells, C a r y o l o g i a , 3 0 (1977) 469. 2 9 M a l t o n i , C., O c c u p a t i o n a l c h e m i c a l c a r c i n o g e n s i s : n e w f a c t s , p r i o r i t i e s a n d p e r s p e c t i v e s , I n : E x c e r p t a Med. Int. Cong. Ser., 322 (1974) 19. 3 0 M e r t z , W., C h r o m i u m o c c u r r e n c e a n d f u n c t i o n in b i o l o g i c a l s y s t e m s , P h y s i o l . R e v . , 4 9 ( 1 9 6 9 ) 1 6 3 . 31 N e t t e s h e i m , P., M . G . H a n n a J r . , D . G . D o h e r t y , R . F . N e w e l i a n d A . H e l l m a n , E f f e c t of c a l c i u m c h r o m a t e d u s t , i n f l u e n z a v i r u s , a n d 1 0 0 R w h o l e - b o d y X - r a d i a t i o n o n l u n g t u m o u r i n c i d e n c e in m i c e , J . Natl.~Cancer Inst., 47 (1971) 1129. 3 2 N e w b o l d , R . F . , T h e v a l u e o f m a m m a l i a n cell m u t a t i o n m e t h o d s in a n e v a l u a t i o n o f t h e p o t e n t i a l earc i n o g e n i c i t y o f c h e m i c a l s , I n d . T o x i c o l . , ( 1 9 7 8 ) in press. 3 3 N e w b o l d , R . F . , P. B r o o k e s , C . F . A r l e t t , B . A . B r i d g e s a n d B. D e a n , T h e e f f e c t o f v a r i a b l e s e r u m f a c tors and clonal morphology on the ability to detect hypoxanthine--guanine phosphoribosyl transf e r a s e ( H P R T ) d e f i c i e n t v a r i a n t s in c u l t u r e d C h i n e s e h a m s t e r cells, M u t a t i o n R e s . , 3 0 ( 1 9 7 5 ) 1 4 3 . 3 4 N e w b o l d , R . F . , C.B. W i g l c y , M . H . T h o m p s o n a n d P. B r o o k e s , C e l l - m e d i a t e d m u t a g e n e s i s in c u l t u r e d C h i n e s e h a m s t e r cells b y c a r c i n o g e n i c p o l y c y c l i c h y d r o c a r b o n s : n a t u r e a n d e x t e n t of t h e a s s o c i a t e d hydrocarbon--DNA reaction, Mutation Res,, 43 (1977) 101. 3 5 N i s h i o k a , H . , M u t a g e n i c a c t i v i t i e s of m e t a l c o m p o u n d s in b a c t e r i a , M u t a t i o n R e s . , 31 ( 1 9 7 5 ) 1 8 5 .

63 36 Payne, W.W., Production of cancers in mice and rats by c h r o m i u m c o m p o u n d s , Arch. Ind. Health, 21 (1960) 530. 37 Payne, W.W., The role of roasted chromite ore in the p r o d u c t i o n of cancer, Arch. Environ. Health, 1 (1960) 20. 38 Roe, F.J.C. and R.L. Carter, Chromium carcinogenesis : calcium chromate as a p o t e n t carcinogen for the subcutaneous tissues of rat, Brit. J. Cancer, 23 (1969) 172. 39 Schoental, R., C h r o m i u m carcinogenesis, formation of epo xy-a l de hyde s and tanning, Brit. J. Cancer, 32 (1975) 403. 40 Tsuda, H., and K. Kato, Potassium dichromate-induced c h r o m o s o m e aberrations and its c ont rol w i t h sodium sulfite in hamster e m b r y o n i c cells in vitro, Gann, 67 (1976) 469. 41 Tsuda, H., and K. Kato, Chromosomal aberrations and morphological t r a n s f o r m a t i o n in hamster e m b r y o n i c cells treated with potassium dichromate in vitro, Mutation Res., 46 (1977) 87. 42 Venitt, S., and L.S. Levy, Mutagenicity of chromates in bacteria and its relevance to chromate carcinogenesis, Nature (London), 250 (1975) 493.