An international validation study of a Bhas 42 cell transformation assay for the prediction of chemical carcinogenicity

Mutation Research 725 (2011) 57–77

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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres

An international validation study of a Bhas 42 cell transformation assay for the prediction of chemical carcinogenicity Ayako Sakai a,∗ , Kiyoshi Sasaki a , Kumiko Hayashi a , Dai Muramatsu a , Shoko Arai a , Nobuko Endou a , Sachiko Kuroda a , Albrecht Poth b , Susanne Bohnenberger b , Thorsten Kunkelmann b , Masumi Asakura c , Hideki Hirose d , Nana Ishii d , Fukutaro Mizuhashi e , Sawako Kasamoto e , Miho Nagai e , Kamala Pant f , Shannon W. Bruce f , Jamie E. Sly f , Shojiro Yamazaki a , Makoto Umeda a , Noriho Tanaka a a

Hatano Research Institute, Food and Drug Safety Center, Kanagawa, Japan Harlan Cytotest Cell Research GmbH, Rossdorf, Germany Japan Bioassay Research Center, Kanagawa, Japan d Mitsubishi Chemical Medience Corporation, Tokyo, Japan e Biosafety Research Center, Foods, Drugs and Pesticides, Shizuoka, Japan f BioReliance Corporation, Rockville, MD, USA b c

a r t i c l e

i n f o

Article history: Received 7 February 2011 Received in revised form 2 June 2011 Accepted 12 July 2011 Available online 21 July 2011 Keywords: Cell transformation Bhas 42 cells v-Ha-ras Tumor initiator Tumor promoter

a b s t r a c t The Bhas 42 cell transformation assay is a sensitive short-term system for predicting chemical carcinogenicity. Bhas 42 cells were established from BALB/c 3T3 cells by the transfection of v-Ha-ras gene and postulated to have acquired an initiated state in the two-stage carcinogenesis theory. The Bhas 42 cell transformation assay is capable of detecting both tumor-initiating and tumor-promoting activities of chemical carcinogens. The full assay protocol consists of two components, the initiation assay and the promotion assay, to detect the initiating activity and the promoting activity, respectively. An international study was carried out to validate this cell transformation assay in which six laboratories from three countries participated. Twelve coded chemicals were examined in total and each chemical was tested by three laboratories. In the initiation assay, concordant results were obtained by three laboratories for eight out of ten chemicals and in the promotion assay, concordant results were achieved for ten of twelve chemicals. The positive results were obtained in all three laboratories with the following chemicals: 2-acetylaminofluorene was positive in both initiation and promotion assays; dibenz[a,h]anthracene was positive in the initiation assay; sodium arsenite, lithocholic acid, cadmium chloride, mezerein and methapyrilene hydrochloride were positive in the promotion assay. o-Toluidin hydrochloride was positive in the both assays in two of the three laboratories. d-Mannitol, caffeine and l-ascorbic acid were negative in both assays in all the laboratories, and anthracene was negative in both assays in two of the three laboratories except one laboratory obtaining positive result in the promotion assay. Consequently, the Bhas 42 cell transformation assay correctly discriminated all six carcinogens and two tumor promoters from four non-carcinogens. Thus, the present study demonstrated that the Bhas 42 cell transformation assay is transferable and reproducible between laboratories and applicable to the prediction of chemical carcinogenicity. In addition, by comparison of the present results with intra-laboratory data previously published, within-laboratory reproducibility using the Bhas 42 cell transformation assay was also confirmed. © 2011 Elsevier B.V. All rights reserved.

1. Introduction No single short-term test can adequately identify all classes of chemical carcinogens and thus, genotoxicity test batteries are

∗ Corresponding author. Tel.: +81 463 82 4751; fax: +81 463 82 9627. E-mail address: [email protected] (A. Sakai). 1383-5718/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2011.07.006

generally employed as pre-screening tools for this purpose. However, a significant number of carcinogens are considered to be non-genotoxic [1] and as such, genotoxicity tests alone cannot be expected to detect such agents. In vitro cell transformation assays can supplement commonly used short-term test batteries and provide for the ability to identify chemical carcinogens that fail to induce genotoxic responses and would otherwise yield false negative results in genotoxicity tests [2–5]. A cell transformation assay is

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A. Sakai et al. / Mutation Research 725 (2011) 57–77

range of each chemical was designated for the assay in advance, although the chemical names were coded [16]. In the current validation study, the participating laboratories were international and each determined the chemical concentrations for the transformation assays independently according to the dose finding procedures prescribed in the protocol.

Initiation assay 4 x 103 cells/well Chemical treatment Stationary phase

Growth phase Promotion assay 14 x 103 cells/well

2. Materials and methods 2.1. Cells

Chemical treatment Growth phase

Day 0

1

Stationary phase

4

7

11

14

21

Fig. 1. Initiation assay and promotion assay in the Bhas 42 cell transformation assay.

an in vitro assay measuring the phenotypic conversion from normal to malignant characteristics in cultured mammalian cells exposed to test chemicals and capable of detecting non-genotoxic as well as genotoxic carcinogens [6–8]. Among the available cell transformation assays, focus formation assays using BALB/c 3T3 cells or C3H/10T1/2 cells have been noted as possible screening methods for chemical carcinogens [9], but have not yet been adopted by regulatory authorities for routine use in chemical regulation. The Bhas 42 cell transformation assay is a sensitive short-term system that has reduced associated cost and labor compared with those associated with the conventional BALB/c 3T3 cell transformation assay. The Bhas 42 cells were developed from the BALB/c 3T3 cells through transfection with a plasmid pBR322 containing Ha-MuSV-DNA, clone H1 (v-Ha-ras) [10–12] and presumed to be initiated toward transformation by the introduced ras sequence [13]. Using the Bhas 42 cells, Ohmori et al. developed a shortterm cell transformation assay to identify tumor promoters [14]. Asada et al. improved the Bhas 42 cell transformation assay such that it was capable of detecting tumor-initiating activity as well as tumor-promoting activity of chemicals [15]. The current protocol consists of two assay components, the initiation assay and the promotion assay, to detect the tumor-initiating activity and the tumor-promoting activity of chemicals, respectively. The assay components comprising the Bhas 42 cell transformation assay protocol are illustrated in Fig. 1. In the initiation assay, the cells are inoculated at a low density and treated with a test chemical in the beginning of the assay period so that the target cells can undergo cell division several times before contact inhibition of growth at confluence, thereby fixing any induced DNA damage. In the promotion assay, the cells are seeded more densely than in the initiation assay, and the treatment with a test chemical is started at sub-confluence and continued longer than in the initiation assay. Tanaka et al. previously conducted an inter-laboratory collaborative pre-validation study of the Bhas 42 cell transformation assay and demonstrated that it was applicable to the detection of chemical carcinogens [16]. Recently, the Bhas 42 cell transformation assay has been applied to 98 chemicals including carcinogens and noncarcinogens, and it has been confirmed that its performance for the prediction of chemical carcinogenicity is superior or equivalent to that of conventional genotoxicity assays and that the assay is capable of detecting Ames-negative and Ames-discordant carcinogens in addition to Ames-positive carcinogens [17]. This study was performed to validate the Bhas 42 cell transformation assay in an inter-laboratory study. Six laboratories joined in this study from three countries. Twelve coded chemicals were examined in total and each chemical was tested by three laboratories. In the pre-validation study conducted by Tanaka et al., the participating laboratories were domestic and the concentration

Bhas 42 cells were established from the v-Ha-ras-transfected BALB/c 3T3 cells by Sasaki et al. in the National Institute of Health Sciences, Japan [10]. The cells were propagated and at present are stored at the Hatano Research Institute (HRI), Food and Drug Safety Center (FDSC; Hadano, Japan) and Japanese Collection of Research Bioresources (JCRB) Cell Bank, National Institute of Biomedical Innovation (Osaka, Japan). Those cells have been confirmed to be free from mycoplasma and adequate for transformation assays. They are distributed to worldwide laboratories through Health Science Research Resources Bank (HSRRB, Osaka, Japan) (http://cellbank.nibio.go.jp/cellbank e.html). In the present study, frozen Bhas 42 cells from the same cell pool at passage 17 were distributed by HRI to the participating laboratories. We have confirmed that Bhas 42 cells at passage 18 retain the transfected v-Ha-ras gene and express its mRNA at a level similar to that of c-Ha-ras gene [manuscript in preparation]. 2.2. Cell culture The distributed Bhas 42 cells were thawed, expanded and cryopreserved in aliquots so as to generate a large batch of that cell pool in the respective laboratories for use in the transformation assays. The cells were cultivated in Eagle’s minimum essential medium (GIBCO Laboratories, Grand Island, NY, USA) supplemented with 100 units/mL of penicillin (GIBCO), 100 ␮g/mL of streptomycin (GIBCO) and 10% fetal bovine serum (FBS; Moregate Biotech, Bulimba, Australia) (M10F) in a humidified 5% CO2 incubator at 37 ◦ C and when the cultures reached about 70% confluence they were subcultured using 0.25% trypsin (GIBCO Laboratories) in order to maintain a sub-confluent state. Within two passages after thawing, the cultured cells were suspended at 5 × 105 cells/mL in fresh M10F containing 5% dimethyl sulfoxide (DMSO), frozen in 0.5 mL volumes at −80 ◦ C and stored in liquid nitrogen. The transformation assays were always started from this frozen stock. Dulbecco’s modified Eagle’s medium/Ham’s F12 (GIBCO Laboratories) supplemented with 100 units/mL of penicillin, 100 ␮g/mL of streptomycin and 5% FBS (DF5F) was used for the transformation assays. The batch of FBS used throughout this validation study by all participating laboratories was prescreened by HRI to produce a minimal number of transformed foci in the Bhas 42 cells treated with the solvent control, DMSO, and a substantially elevated number of transformed foci in the cells treated with the positive control, 3-methylcholanthrene (MCA, 1 ␮g/mL) or 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.05 ␮g/mL) [3,9]. 2.3. Chemicals The chemicals tested were purchased and coded by the chemical repository supervisor of HRI, who was independent of the validation study group, and distributed by him to the chemical repository officers of participating laboratories. The test chemicals were decoded after all the laboratories submitted their data and all the data were inspected. They were 2-acetylaminofluorene [53–96–3] (Sigma, St. Louis, MO, USA), o-toluidine hydrochloride [636–21–5] (Aldrich, Milwaukee, WI, USA), dibenz[a,h]anthracene [53–70–3] (Wako Pure Chemical Industries, Osaka, Japan), sodium arsenite [7784–46–5] (Wako), lithocholic acid [434–13–9] (Sigma), cadmium chloride [10108–64–2] (Aldrich), mezerein [34807–41–5] (Sigma), methapyrilene hydrochloride [135–23–9] (Sigma), anthracene [120–12–7] (Aldrich), d-mannitol [69–65–8] (Aldrich), caffeine [58–08–2] (Aldrich), l-ascorbic acid [50–81–7] (Sigma). Test chemicals were dissolved or suspended in DMSO (Sigma) or sterile distilled water. Which vehicle to be adopted was decided by each laboratory depending on its respective solubility test. The vehicles used and their final concentrations in the medium are presented in the tables of assay results (Tables 1–12). MCA [56–49–5] [Aldrich] and TPA [16561–29–8] (Sigma) dissolved in DMSO were included as positive controls for the initiation assay and the promotion assay, respectively, and the same lots were purchased by each participating laboratory. 2.4. Cell growth assay The cell growth assays, using a crystal violet (CV) staining method, were performed prior to the transformation assays to determine the doses applicable to the Bhas 42 cell transformation. Cell growth assays were also performed concurrently with every transformation assay (every initiation and promotion assay) to estimate the effect of each treatment on the cell growth and survival. In the cell growth component of the initiation assay, a cell suspension at 2 × 103 cells/mL in DF5F was distributed into each well of 6-well micro-plates in

A. Sakai et al. / Mutation Research 725 (2011) 57–77

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Table 1 Results of transformation assay on 2-acetylaminofluorene. Concentration (␮g/mL)

Lab I

(a) Initiation assay 0c (0.1% DMSO) 0c (0.5% DMSO) 10 12 14 15 16 18 20 25 30 35 40 50 100 300 MCA 1d (0.1% DMSO) MCA d 1 (0.5% DMSO)

a b c d * †

CG

Foci/well

100

3.8 ± 3.4

100

4.5 ± 1.6

118

4.5 ± 2.3

101

5.5 ± 2.3

7.0 ± 1.7

135 91 46

10.0 ± 3.7* 14.5 ± 1.9* 14.3 ± 1.6*

22 19 14 82

8.8 ± 5.7* 5.2 ± 2.4 7.8 ± 3.5 50.0 ± 15.6†

Lab I

(b) Promotion assay 0c (0.1% DMSO) 0c (0.5% DMSO) 0.03 0.1 0.3 1 2.5 3 5 10 15 20 25 30 35 40 50 TPA 0.05d (0.1% DMSO) TPA 0.05d (0.5% DMSO)

Lab VI

Foci/wellb

130

Concentration (␮g/mL)

Lab IV

CGa

104

CG

Foci/well

100

1.0 ± 0.6

103 86

2.7 ± 2.1 5.7 ± 2.5*

87 60 45 20 20

9.2 ± 3.5* 6.3 ± 4.1* 4.2 ± 2.3 1.8 ± 1.5 Toxic

9

Toxic

53

23.7 ± 4.6†

5.8 ± 3.1

94 41 26 18 17

8.7 ± 2.6* 12.5 ± 1.9* 12.3 ± 2.2* 14.2 ± 2.9* 13.5 ± 3.7*

72

38.5 ± 4.1†

Lab IV

Lab VI

CG

Foci/well

CG

Foci/well

100

9.0 ± 2.0

100

6.2 ± 2.6

103 103 104 101

5.7 ± 1.0 4.2 ± 1.9 6.3 ± 2.3 9.2 ± 3.1

102

6.8 ± 2.6

85 83 71

13.0 ± 3.6* 19.5 ± 3.9* 19.3 ± 3.4*

125

38.5 ± 5.0†

100 98 89 85 78 72 78 76

9.5 ± 1.9* 10.8 ± 3.2* 12.0 ± 2.1* 9.7 ± 1.6* 10.0 ± 2.5* 12.7 ± 1.6* 18.2 ± 1.7* 1.3 ± 1.0

219

24.7 ± 3.3†

CG

Foci/well

100

2.2 ± 1.3

98

6.0 ± 3.6

79 88 58 61 49

10.2 ± 2.9* 15.5 ± 2.1* 11.7 ± 4.5 5.3 ± 3.8 Toxic

47

Toxic

104

17.5 ± 3.7†

% of cell growth compared to that of solvent control. Average number of transformed foci/well ± SD. Solvent control: final solvent concentration of the working culture media in parentheses. Positive control: final solvent concentration of the working culture media in parentheses. p < 0.05; one-sided Dunnett test, vs corresponding solvent control. p < 0.05; one-sided t-test or Aspin–Welch test, vs corresponding solvent control.

a 2 mL volume (4000 cells/well, day 0). Three wells were prepared for each treatment group. At 24 h after seeding, the culture medium was replaced with a medium containing a test chemical at various concentrations, or a test chemical dissolved in DMSO or sterile distilled water was added directly to the culture medium in the well without medium exchange. On day 4, the medium containing the test chemical was replaced with the fresh DF5F. On day 7, the cells were fixed with 10% formalin (Sigma) or methanol and stained with a 0.1% CV (Sigma) solution in 5% ethanol. CV was extracted from the stained cells with a solution containing 0.02 mol/L hydrochloric acid in 50% ethanol. The optical density of extract was measured at a wavelength between 540 and 570 nm. The relative cell growth of cultures treated with a chemical was calculated as follows:

 the relative cell growth =

(At − Ab) (Ac − Ab)

 × 100

where At was the absorbance of CV extract from a well with the chemical-treated cells, Ac was the absorbance of CV extract from a well with the solvent-treated cells and Ab was the absorbance of CV extract from a well with the medium only. In the cell growth component of the promotion assay, a cell suspension at 7 × 103 cells/mL was seeded into each well (14,000 cells/well, day 0), and on day 4, the culture medium was replaced with a medium containing a test chemical. On

day 7, the cells were fixed and stained with CV, and the CV extracted from cells was measured as above.

2.5. Dose setting for transformation assay Five or more concentrations were selected based on the results of cell growth assays. In the initiation assay, the concentrations used covered a range from little or no toxicity to a toxicity level that resulted in less than 20% survival compared to the control cultures. In practice, the concentrations generally employed included one dose below the no-effect level (NOEL), two doses between the NOEL and the 50% inhibitory concentration (IC50 ) and two doses between the IC50 and the 90% inhibitory concentration (IC90 ). In the promotion assay, for the chemicals that exhibited marked growth enhancement, the test concentrations were selected to cover a range from little effect on cell growth to growth enhancement. In practice, the concentrations generally employed included one dose below the NOEL, three doses resulting in growth enhancement, and one more higher dose that produces weak growth inhibition. For the chemicals that did not induce marked growth enhancement, the test concentrations were selected to range from a dose two or three levels lower than the no-effect concentration to that showing a survival less than 50%. In practice, the concentra-

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Table 2 Results of transformation assay on o-toluidine HCl. Concentration (␮g/mL)

(a) Initiation assay 0c (0.5% DMSO) 0c (5% Water) 100 150 250 300 450 500 600 700 750 1000 1250 1500 2000 3000 5000 0d (0.1% DMSO) MCA 1e (0.1% DMSO) MCA 1e (0.5% DMSO) Concentration (␮g/mL)

(b) Promotion assay 0c (0.5% DMSO) 0c (5% Water) 10 12.5 25 30 100 250 300 500 700 1000 1500 2000 0d (0.1% DMSO) TPA 0.05e (0.1% DMSO) TPA 0.05e (0.5% DMSO) a b c d e * †

Lab I

Lab V

Lab VI

CGa

Foci/wellb

CG

Foci/well

100 98

1.8 ± 1.3 2.8 ± 1.8

100 92

2.0 ± 2.4 2.5 ± 2.5

79

6.8 ± 3.2*

77

2.5 ± 1.5

61

3.2 ± 2.5

29

2.8 ± 1.8

18

1.8 ± 1.6

3 3 100 53

Toxic Toxic 1.7 ± 0.8 19.3 ± 3.9†

Lab I

50

0

9.5 ± 4.0*

-6 -4

Toxic Toxic

100 33

2.3 ± 0.8 43.7 ± 6.0†

Lab V CG

Foci/well

100 94

4.5 ± 1.6 5.5 ± 2.0

100 94

16.3 ± 4.1 27.0 ± 4.6*

65

4.8 ± 1.7

50 13

Toxic Toxic

100 127

7.8 ± 2.2 41.0 ± 3.3†

100

1.0 ± 1.5

123

2.0 ± 1.8

89 109

1.7 ± 1.2 1.8 ± 1.3

73

4.5 ± 2.7*

64 29 13 9

3.7 ± 1.0* 2.0 ± 1.1 0.3 ± 0.5 0.3 ± 0.8

40

21.7 ± 6.3†

Lab VI

Foci/well

4.3 ± 1.5 3.7 ± 1.6

Foci/well

7.0 ± 2.0*

CG

87 83

CG

CG

Foci/well

100

0.7 ± 0.8

128 65

0.5 ± 0.5 2.5 ± 2.6

92 75 55

31.0 ± 1.7* 26.7 ± 7.4* 27.8 ± 4.6*

91

6.2 ± 2.8*

42

28.0 ± 6.4*

48

8.0 ± 3.5*

33

6.0 ± 4.3

68 28 21

0.5 ± 0.8 Toxic Toxic

100 134

14.8 ± 5.5 43.7 ± 8.4† 104

15.5 ± 2.2†

% of cell growth compared to that of solvent control. Average number of transformed foci/well ± SD. Solvent control: final solvent concentration of the working culture media in parentheses. Solvent control for the positive control: final solvent concentration of the working culture media in parentheses. Positive control: final solvent concentration of the working culture media in parentheses. p < 0.05; one-sided Dunnett test, vs corresponding solvent control. p < 0.05; one-sided t-test or Aspin–Welch test, vs corresponding solvent control.

tions generally employed included two doses below NOEL, two doses between NOEL and IC50, and one dose above IC50 . For a chemical which caused a sharp decline of cell growth within a narrow concentration range, one or two additional doses outside the established concentration range were set up as a precaution against the fluctuation of cell response among experiments. The highest concentration of test chemical in the medium was 5 mg/mL in this study.

cells were treated by the addition of a test chemical solution or the vehicle alone to the cultures, or by complete replacement of the medium containing either the test chemical or the solvent vehicle. The treatment in the initiation phase was continued for 72 h. Following the exposure period, all treatment media were removed and the cells were refed with medium without the test chemical (day 4) and subsequently cultured in the normal medium until day 21, receiving medium exchanges on day 7, day 11 and day 14. The cells were then fixed with methanol and stained with Giemsa’s solution. Each assay also included MCA (1 ␮g/mL) as the positive control.

2.6. Transformation assay The transformation assay was performed using the same procedures as described previously [17]. 2.6.1. Initiation assay to examine initiating activity One tube of the frozen Bhas 42 cells was rapidly thawed and grown in M10F up to about 70% confluence and then subcultured in DF5F to reach about 70% confluence again. Thereafter, cells were cultured in DF5F. The cells were trypsinized and suspended at a density of 2000 cells/mL and seeded into each well of 6-well micro-plates in 2 mL volumes (4000 cells/well, day 0). Nine wells were prepared per concentration, of which six wells were reserved for the transformation assay and three were reserved for the concurrent cell growth assay. At 24 h after seeding, the

2.6.2. Promotion assay to examine promoting activity The promotion assay was carried out in the same manner as the initiation assay except for the following steps. The cells were seeded at a density of 7000 cells/mL (14,000 cells/well, day 0) and cultured for 4 days without a medium exchange. On day 4, day 7, and day 11, the culture medium was replaced with a fresh medium containing a test chemical or vehicle alone and the treatment in the promotion phase was continued until day 14 (for a total of 10 days). The cells were then cultured in the normal medium without the test chemical for one week until day 21. Each assay also included TPA (0.05 ␮g/mL) as the positive control.

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61

Table 3 Results of transformation assay on dibenz[a,h]anthracene. Concentration (␮g/mL)

(a) Initiation assay 0c (0.1% DMSO) 0 (0.2% DMSO) 0.0033 0.01 0.03 0.033 0.04 0.05 0.1 0.3 0.33 1 3 3.3 7 10 0d (0.1% DMSO) MCA 1e (0.1% DMSO) Concentration (␮g/mL)

(b) Promotion assay 0c (0.1% DMSO) 0c (0.2% DMSO) 0.001 0.0033 0.005 0.01 0.03 0.033 0.05 0.1 0.2 0.3 0.33 1 3 3.3 7 10 0d (0.1% DMSO) TPA 0.05e (0.1% DMSO) a b c d e * †

Lab I

Lab II

CGa

Foci/wellb

100

1.5 ± 1.2

113 110

6.0 ± 2.5 8.7 ± 2.9*

84 63

15.0 ± 4.2* 24.2 ± 3.9*

58 54

24.2 ± 3.3* 17.8 ± 4.5*

57 58

17.2 ± 3.5* 17.5 ± 3.5*

79

20.0 ± 4.1†

Lab I Foci/well

100

3.3 ± 1.6

74

Foci/well

100

0.8 ± 0.8

99 80

2.5 ± 1.5 6.2 ± 2.4*

71 65 53

7.3 ± 3.3* 8.3 ± 2.9* 11.2 ± 3.9*

30

12.7 ± 2.9*

38 100 53

9.3 ± 2.4* 2.0 ± 1.3 16.7 ± 3.4†

Lab II

CG

83 80

Lab V

CG

2.2 ± 2.1 2.3 ± 2.0

0.8 ± 1.6

66 66

1.0 ± 1.3 1.7 ± 0.8

71 66

0.7 ± 1.0 1.0 ± 0.9

Foci/well

100 81

2.8 ± 1.5 1.5 ± 0.5

82 70

2.5 ± 1.5 11.2 ± 3.1

76

2.7 ± 1.9

69

1.5 ± 1.0

71

0.2 ± 0.4

118

17.7 ± 4.2†

74 100 128

Foci/well

100

2.8 ± 1.9

89 73

8.0 ± 1.4 13.3 ± 3.1*

37

22.5 ± 3.0*

11

18.5 ± 4.1*

3 4

22.7 ± 7.1* 16.3 ± 4.2*

7

17.5 ± 4.8*

1 100 23

17.3 ± 7.8* 1.2 ± 0.4 41.3 ± 4.9†

Lab V

CG

2.7 ± 1.2

65

CG

1.0 ± 1.3 2.5 ± 2.1 8.8 ± 3.2†

CG

Foci/well

100

8.7 ± 2.3

83 94

9.0 ± 2.1 11.8 ± 3.3

70

11.5 ± 4.4

67

10.5 ± 3.0

59 71

5.0 ± 4.1 2.3 ± 2.4

68

3.2 ± 1.2

136

43.5 ± 4.1†

% of cell growth compared to that of solvent control. Average number of transformed foci/well ± SD. Solvent control: final solvent concentration of the working culture media in parentheses. Solvent control for the positive control: final solvent concentration of the working culture media in parentheses. Positive control: final solvent concentration of the working culture media in parentheses. p < 0.05; one-sided Dunnett test, vs corresponding solvent control. p < 0.05; one-sided t-test or Aspin–Welch test, vs corresponding solvent control.

2.6.3. Counting of transformed foci and statistical analysis The transformed foci were judged on the basis of the morphological characteristics: (a) more than 100 cells, (b) spindle-shaped cells whose morphology was distinctly different from the contact-inhibited monolayer cells, (c) deep basophilic staining, (d) random orientation of cells, especially at the edge of foci, (e) dense multilayering of cells, and (f) invasive growth into the monolayer of surrounding contact-inhibited cells. The number of transformed foci in every well was recorded and a statistical analysis for the increase in the number of the transformed foci produced by a test chemical was performed by multiple comparison using the one-sided Dunnett test (p < 0.05). The statistical significance of positive controls were evaluated by t-test or Aspin–Welch test depending on the result of F-test for homoscedasticity (homogeneity of variance). “Toxic” was recorded for wells which were not confluent at the end of transformation assay because of cytotoxicity resulting from chemical treatment. 2.6.4. Judgment The results in the Bhas 42 cell transformation assays were judged positive when there existed two or more sequential doses that induced statistically significant increases in the number of transformed foci, and negative when there was no dose showing statistically significant increase of foci. When the statistically significant increase was at only one dose, the assay result was regarded as equivocal, and

then the initiation or promotion assay together with the concomitant cell growth assay was repeated and included the positive dose in the first assay. The chemical was judged to be positive if a statistically significant increase in the number of transformed foci resulted at one or more concentrations in the second assay.

3. Course of study Six laboratories participated in this validation study: four from Japan, one from USA and one from Germany. The Japanese laboratories had participated in the pre-validation study on the Bhas 42 cell transformation [16] and the two foreign laboratories participated only in the present validation study. Prior to initiating the validation study, a workshop was held to enlighten and train the participants in the experimental procedures and in assessing the morphological aberrations characteristic of transformed foci. Those laboratories started to assay the test chemicals after enough training using positive controls, MCA and TPA.

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Table 4 Results of transformation assay on sodium arsenite. Concentration (␮g/mL)

(a) Initiation assay 0c (0.5% DMSO) 0c (5% Water) 0.01 0.025 0.05 0.075 0.1 0.11 0.12 0.123 0.13 0.14 0.15 0.159 0.2 0.207 0.269 0.3 0.35 0.4 0.455 0.5 0.592 0.6 0.7 0.769 0.8 0.9 1 2 0d (0.1% DMSO) MCA 1e (0.1% DMSO) MCA 1e (0.5% DMSO) Concentration (␮g/mL)

(b) Promotion assay 0c (0.5% DMSO) 0c (5% Water) 0.0158 0.025 0.05 0.1 0.15 0.158 0.2 0.25 0.3 0.4 0.5 0.7 0.75 1 1.3 1.58 1.7 5 0d (0.1% DMSO) TPA 0.05e (0.1% DMSO) TPA 0.05e (0.5% DMSO) a b c d e f * † ‡

Lab I, 1st run

Lab I, 2nd run

Lab III, 1st run

Lab III, 2nd run

Lab VI

CGa

Foci/wellb

CG

Foci/well

CG

Foci/well

CG

Foci/well

CG

Foci/well

100

0.8 ± 1.0

100

2.0 ± 2.0

100

2.8 ± 1.3

100

1.5 ± 0.5

100

1.5 ± 1.8 99 99 96 108 101 100 96

2.3 ± 1.6 1.2 ± 1.2 2.2 ± 1.9 1.0 ± 1.3 1.8 ± 1.7 0.0 ± 0.0 2.2 ± 2.5

104 100 98

2.2 ± 1.7 0.5 ± 0.8 1.5 ± 1.4

79

12.0 ± 3.5†

113

1.7 ± 1.2

103

114

120

0.7 ± 0.5

96

1.5 ± 0.8

107 101

1.3 ± 0.5 2.2 ± 1.6

103 108

2.7 ± 2.0 1.8 ± 1.5

110

2.8 ± 1.3

102

3.0 ± 2.5

103

3.3 ± 1.6*

84

2.5 ± 2.2

91

1.3 ± 1.0

61

2.5 ± 2.3

74

3.5 ± 1.0*

35

6.3 ± 2.7*

35

2.2 ± 1.3

6

4.7 ± 3.8

22f

15.0 ± 3.3†

100 21

3.3 ± 1.2 9.7 ± 3.6†

2.3 ± 1.8

3.3 ± 1.6

99

2.3 ± 1.5

104

2.3 ± 1.4

103

1.7 ± 2.3

97

3.0 ± 1.3

103 106

1.0 ± 1.1 1.7 ± 1.4

49

5.0 ± 3.0*

96 96

1.2 ± 1.5 1.2 ± 1.2

16 3 100 60

2.5 ± 1.9 0.8 ± 1.0 3.0 ± 1.7 41.3 ± 2.3†

Lab I

100 47

2.8 ± 1.3 32.0 ± 6.7†

Lab III, 1st run

Lab III, 2nd run

Lab VI

CG

Foci/well

CG

Foci/well

CG

Foci/well

CG

Foci/well

100

1.7 ± 1.5

100

4.5 ± 1.8

100 107

2.5 ± 1.4 2.5 ± 2.1

100

0.7 ± 0.8

94 101

6.3 ± 2.6 13.0 ± 2.2*

106

5.2 ± 1.2*

104 118 104 102

2.5 ± 1.4 4.7 ± 1.6* 2.8 ± 1.5* 3.7 ± 1.2*

109 103 105

6.5 ± 1.9* 7.3 ± 1.8* 11.8 ± 2.9*

109

6.2 ± 2.6* 112 125 127

3.5 ± 2.3* 1.7 ± 1.0 0.2 ± 0.4

101

13.7 ± 2.7*

96 96 95

1.5 ± 0.8 0.7 ± 0.8 0.0 ± 0.0

80

Toxic

109

14.3 ± 5.4†

99

9.0 ± 0.9*

86

1.7 ± 0.8

83 85

Toxic Toxic

78

Toxic

100 128

5.0 ± 2.4 36.7 ± 3.3†

97

0.3 ± 0.8

77

Toxic

26

Toxic

133f

13.7 ± 6.8‡

100 198

% of cell growth compared to that of solvent control. Average number of transformed foci/well ± SD. Solvent control: final solvent concentration of the working culture media in parentheses. Solvent control for the positive control: final solvent concentration of the working culture media in parentheses. Positive control: final solvent concentration of the working culture media in parentheses. % of cell growth compared to that of 5% water. p < 0.05; one-sided Dunnett test, vs corresponding solvent control. p < 0.05; one-sided t-test or Aspin–Welch test, vs corresponding solvent control. p < 0.05; one-sided t-test or Aspin–Welch test, vs 5% water.

2.5 ± 1.0 18.2 ± 2.7†

A. Sakai et al. / Mutation Research 725 (2011) 57–77

63

Table 5 Results of transformation assay on lithocholic acid. Concentration (␮g/mL)

(a) Initiation assay 0c (DMSO 0.1%) 0c (DMSO 0.5%) 7 10 14 15 17.5 20 22 22.5 25 27.5 28 28.25 28.5 28.75 29 29.25 29.5 29.75 30 40 56 80 MCA 1d (0.1% DMSO) MCA 1d (0.5% DMSO) Concentration (␮g/mL)

(b) Promotion assay 0c (DMSO 0.1%) 0c (DMSO 0.5%) 2.5 5 10 12 15 18 20 25 30 35 40 42.5 45 50 TPA 0.05d (0.1% DMSO) TPA 0.05d (0.5% DMSO) a b c d * †

Lab I

Lab IV

Lab VI

CGa

Foci/wellb

CG

Foci/well

100

6.3 ± 3.9

100

5.7 ± 1.0

105

5.5 ± 1.9

92 94 96

5.7 ± 2.3 4.5 ± 3.3 5.7 ± 2.4

106 108 104

4.5 ± 2.3 2.0 ± 1.4 3.7 ± 2.3

85

3.7 ± 1.9

107 89 56

2.7 ± 0.5 4.3 ± 2.5 5.5 ± 3.7

11

73

112

3.2 ± 1.2

3 0 1 57

4.2 ± 0.8 Toxic Toxic 39.0 ± 2.6†

3.0 ± 1.4

44.2 ± 3.7†

Lab I

Lab IV Foci/well

CG

Foci/well

100

7.2 ± 3.5

100

2.3 ± 1.6

93 89 80

9.3 ± 2.6 15.7 ± 5.1* 27.3 ± 4.2* 44.8 ± 9.8*

75 86 16

45.7 ± 5.8* 12.2 ± 2.4 Toxic

124

42.3 ± 1.8†

Foci/well

100

1.3 ± 1.4

75

1.8 ± 1.5

10 9 7 8 11 9 8 8 9

0.2 ± 0.4 0.5 ± 0.5 0.8 ± 0.8 0.5 ± 0.8 0.0 ± 0.0 Toxic Toxic Toxic Toxic

56

10.5 ± 4.2†

Lab VI

CG

72

CG

86

6.3 ± 4.1*

86

13.5 ± 2.1*

89

14.0 ± 1.8*

84

Toxic

5 136

Toxic 9.5 ± 3.4†

CG

Foci/well

100

2.7 ± 0.8

83

19.7 ± 5.2*

83

33.2 ± 4.9*

106 50 38 19 14 11

Toxic Toxic Toxic Toxic Toxic Toxic

145

16.8 ± 4.1†

% of cell growth compared to that of solvent control. Average number of transformed foci/well ± SD. Solvent control: final solvent concentration of the working culture media in parentheses. Positive control: final solvent concentration of the working culture media in parentheses. p < 0.05; one-sided Dunnett test, vs corresponding solvent control. p < 0.05; one-sided t-test or Aspin–Welch test, vs corresponding solvent control.

The test chemicals were purchased, coded and distributed to participating laboratories by the chemical repository supervisor of HRI under the conditions that participants in the study were blind to the identity of those chemicals. Every chemical was tested by three laboratories. Each laboratory determined the appropriate solvent and concentrations of a given test chemical for the transformation assay by conducting a solubility test and a cell growth assay. However, the solvent (DMSO) of dibenz[a,h]anthracene and mezerein was instructed in advance and their highest concentrations were also designated to be ≤10 and ≤0.01 ␮g/mL, respectively, in the medium. These amounts were predetermined because these chemicals were expensive and their availability was limited, and, therefore, solubility pretesting was not performed.

Chemical names were decoded after the last data were submitted. 4. Results The results of Bhas 42 cell transformation assays including the respective concurrent cell growth assays are presented in Tables 1–12 and in Figs. 2–13, chemical by chemical. The Data of positive and negative controls are illustrated in Fig. 14. 4.1. 2-Acetylaminofluorene The results for 2-accetylaminofluorene are shown in Table 1 and Fig. 2. All three laboratories judged 2-acetylaminofluorene as pos-

64

A. Sakai et al. / Mutation Research 725 (2011) 57–77

Table 6 Results of transformation assay on cadmium chloride. Concentration (␮g/mL)

Lab I

(a) Initiation assay 0c (5% Water) 0.1 0.2 0.4 0.402 0.482 0.579 0.6 0.694 0.8 0.833 1 1.2 1.4 1.44 1.5 1.6 1.73 1.8 2 0d (0.1% DMSO) MCA 1e (0.1% DMSO) Concentration (␮g/mL)

(b) Promotion assay 0c (5% Water) 0.001 0.01 0.05 0.0625 0.1 0.125 0.2 0.25 0.275 0.3 0.35 0.4 0.45 0.5 0.6 0.8 1 1.2 1.4 1.6 2 0d (0.1% DMSO) TPA 0.05e (0.1% DMSO) a b c d e f * † ‡

Lab II

Lab III

CGa

Foci/wellb

CG

Foci/well

CG

Foci/well

100

4.8 ± 2.5

100 111 104 96

1.5 ± 1.4 1.7 ± 1.4 0.8 ± 1.2 2.0 ± 1.4

100

3.0 ± 1.7

110 105 106

1.7 ± 1.0 1.3 ± 1.4 1.7 ± 1.6

95

1.3 ± 1.5

72 55 7

1.3 ± 1.2 1.3 ± 1.0 0.3 ± 0.5

5

1.0 ± 0.6

-1

0.3 ± 0.8

14f

9.2 ± 2.0†

93

1.0 ± 1.1

88

2.7 ± 0.8

72

0.7 ± 1.2

82 80 44

3.3 ± 0.5 2.3 ± 1.8 1.3 ± 1.2

41 13

0.7 ± 0.8 0.5 ± 1.2

4

0.2 ± 0.4

21

0.7 ± 0.5

6 3 100 74

0.8 ± 1.2 0.3 ± 0.5 4.3 ± 2.1 43.5 ± 3.8†

1.0 ± 0.9 15.8 ± 4.4†

Lab I, 1st run

Lab I, 2nd run

Lab II, 1st run

Lab II, 2nd run

Lab II, 3rd run

Lab III, 1st run

Lab III, 2nd run

CG

Foci/well

CG

Foci/well

CG

Foci/well

CG

Foci/well

CG

Foci/well

CG

Foci/well

CG

Foci/well

100

5.3 ± 2.3

100

2.5 ± 0.8

100

2.6 ± 1.1

100 92 100 97

1.3 ± 0.8 1.0 ± 0.6 0.3 ± 0.5 1.7 ± 1.4

100

1.3 ± 1.5

100

2.0 ± 0.6

100

1.2 ± 0.8

98

1.2 ± 1.6

97

2.2 ± 0.8 102

1.3 ± 0.5

99

2.3 ± 0.8 108

3.7 ± 1.6

108

4.0 ± 1.8* 109

4.2 ± 2.7*

114

4.0 ± 2.0*

112 110

5.3 ± 2.7* 2.3 ± 1.2

100 166

3.8 ± 1.3 20.3 ± 4.2†

101 104

114

21.8 ± 4.4*

126 122 113 96 70 54

14.7 ± 5.6* Toxic Toxic Toxic Toxic Toxic

100 125

9.0 ± 3.4 38.7 ± 5.6†

110 108 114 117 118 123

100 132

2.0 ± 0.9

102

4.2 ± 1.0

11.0 ± 4.7* 13.5 ± 4.0* 13.3 ± 5.4* 14.5 ± 2.2* 14.2 ± 3.1* 14.2 ± 5.6*

5.0 ± 3.3 19.3 ± 5.6†

116

106

4.0 ± 2.2

121

Toxic

117 114 90 67 52 31

Toxic Toxic Toxic Toxic Toxic Toxic 2.3 ± 1.6 8.3 ± 3.1‡

119

100 133

0.8 ± 1.3 2.8 ± 1.9

4.7 ± 1.9*

2.0 ± 0.9 11.5 ± 2.7†

110 124 109 120 123 131 126 138

100 134

1.3 ± 1.4 1.8 ± 1.7 2.7 ± 1.8 3.5 ± 1.2 5.3 ± 2.7* 3.3 ± 2.3 4.0 ± 3.2 2.5 ± 1.4

1.8 ± 0.4 9.5 ± 2.7†

109

3.2 ± 1.8

68

Toxic

25

Toxic

155f

15.7 ± 4.5†

% of cell growth compared to that of solvent control. Average number of transformed foci/well ± SD. Solvent control: final solvent concentration of the working culture media in parentheses. Solvent control for the positive control: final solvent concentration of the working culture media in parentheses. Positive control: final solvent concentration of the working culture media in parentheses. % of cell growth compared to that of 5% water. p < 0.05; one-sided Dunnett test, vs corresponding solvent control. p < 0.05; one-sided t-test or Aspin–Welch test, vs corresponding solvent control. p < 0.05; one-sided t-test or Aspin–Welch test, vs 5% water.

itive both in the initiation assay and in the promotion assay since it induced statistically significant increases in the number of transformed foci at two or more sequential concentrations in both assays in all laboratories. 4.2. o-Toluidine hydrochloride Table 2 and Fig. 3 represent the results of o-toluidine hydrochloride. Two laboratories, Lab V and Lab VI, gave positive results both

in the initiation assay and in the promotion assay. The other laboratory, Lab I, gave negative results in both assays, although acceptable responses were obtained with the positive controls.

4.3. Dibenz[a,h]anthracene The results for dibenz[a,h]anthracene are presented in Table 3 and Fig. 4. All three laboratories reported that

A. Sakai et al. / Mutation Research 725 (2011) 57–77

65

Table 7 Results of transformation assay on mezerein. Concentration (␮g/mL)

(a) Initiation assay 0c (0.1% DMSO) 0.000010 0.000039 0.0001 0.000156 0.0003 0.0005 0.000625 0.00063 0.001 0.0013 0.0025 0.003 0.005 0.01 MCA 1d (0.1% DMSO) Concentration (␮g/mL)

(b) Promotion assay 0c (0.1% DMSO) 0.000005 0.000020 0.000026 0.000064 0.000078 0.0001 0.00016 0.0003 0.000313 0.0004 0.0005 0.001 0.00125 0.0025 0.003 0.005 0.01 TPA 0.05d (0.1% DMSO) a b c d * †

Lab I

Lab III

Lab IV

CGa

Foci/wellb

CG

Foci/well

CG

Foci/well

100

1.7 ± 1.5

100 104 102

2.5 ± 1.4 1.8 ± 1.0 2.3 ± 0.8

100

7.8 ± 2.0

94

1.7 ± 1.6 110

1.5 ± 1.5

94 93

1.5 ± 1.0 2.2 ± 0.8 113

1.7 ± 1.2 94

7.7 ± 1.5

94

111 141 150 56

1.5 ± 1.4

1.7 ± 0.8 1.3 ± 1.2 3.2 ± 1.9 22.2 ± 4.7†

Lab I

140

3.0 ± 0.6

109 130

7.8 ± 3.2 7.3 ± 2.3

202 28

13.3 ± 2.9* 9.2 ± 3.5†

195 208 71

10.2 ± 2.6 13.2 ± 1.7* 45.7 ± 3.6†

Lab III

Lab IV

CG

Foci/well

CG

Foci/well

CG

Foci/well

100

5.7 ± 1.9

100 100 104

3.2 ± 1.8 2.2 ± 2.1 4.5 ± 1.5

100

3.3 ± 2.3

100 102

5.3 ± 2.1 4.3 ± 2.0

113

8.2 ± 1.6*

139

18.3 ± 2.2*

208

40.2 ± 4.1*

236

75.2 ± 5.4*

142

9.8 ± 3.1†

107 103

17.5 ± 5.4*

121

37.2 ± 4.2* 143

142 184

31.8 ± 6.0*

46.5 ± 4.4* 62.7 ± 2.1* 257

181 183 193 124

9.2 ± 2.6

Toxic Toxic Toxic 35.3 ± 5.6†

46.5 ± 7.4*

206

37.2 ± 7.9*

143

12.2 ± 6.1†

% of cell growth compared to that of solvent control. Average number of transformed foci/well ± SD. Solvent control: final solvent concentration of the working culture media in parentheses. Positive control: final solvent concentration of the working culture media in parentheses. p < 0.05; one-sided Dunnett test, vs corresponding solvent control. p < 0.05; one-sided t-test or Aspin–Welch test, vs corresponding solvent control.

dibenz[a,h]anthracene was clearly positive in the initiation assay, but negative in the promotion assay. 4.4. Sodium arsenite Table 4 and Fig. 5 show the assay results for sodium arsenite. In the first initiation assay of sodium arsenite by Lab I, there was a statistically significant increase in the number of transformed foci at a dose of 0.8 ␮g/mL. In accordance with the protocol, Lab I repeated the initiation assay, but there was no significant increase of transformed foci in the second assay. Thus, sodium arsenite was judged as negative in Lab I. Since Lab III first obtained statistically significant increases of transformed foci at two non-sequential doses, 0.455 and 0.769 ␮g/mL, they performed a second initiation assay and again obtained a significant increase at 0.769 ␮g/mL. Consequently, sodium arsenite was judged to be positive in the initiation assay by Lab III. Lab VI carried out initiation assay only once and observed no increase of transformed foci. However, sodium arsenite did not produce any cytotoxicity at the tested concentrations in

the concurrent cell growth assay. In view of its high solubility in the solvent (water) and its lack of cytotoxicity at the lower concentration initially used, sodium arsenite should have been retested by Lab VI at higher concentrations in order to achieve cytotoxicity. Therefore, the initiation assay conducted on sodium arsenite by Lab VI was judged to be incomplete because of inadequate dosing. In the promotion assay on sodium arsenite, all three laboratories reported positive results. Despite having obtained statistically significant increases of transformed foci at two sequential doses in the first assay, however, Lab III repeated the promotion assay. The compound was considered positive and the repeat was not necessary. Lab III’s decision was the result of having lost cultures at the two highest concentrations in the transformation assay because of cytotoxicity, although the cell growth rates at respective concentrations were 77% and 26% of control in the concurrent cell growth assay. The second assay was voluntarily carried out in a lower and narrower concentration range and confirmed the positive results obtained in the first assay.

66

A. Sakai et al. / Mutation Research 725 (2011) 57–77

Table 8 Results of transformation assay on methapyrilene HCl. Concentration (␮g/mL)

(a) Initiation assay 0c (5% water) 53 75 100 110 150 200 210 220 240 250 260 280 300 350 400 450 0d (0.1% DMSO) MCA 1e (0.1% DMSO) Concentration (␮g/mL)

(b) Promotion assay 0c (5% Water) 5 10 20 25 40 50 75 80 100 160 200 300 400 500 0d (0.1% DMSO) TPA 0.05e (0.1% DMSO) a b c d e * †

Lab I

Lab II

Lab IV

CGa

Foci/wellb

CG

Foci/well

CG

Foci/well

100

2.7 ± 1.6

100

2.0 ± 1.4

100 120 149

6.8 ± 1.8 6.2 ± 1.2 7.2 ± 3.1

109

3.3 ± 1.5

86 71

2.8 ± 1.0 3.2 ± 2.1

124 129

6.2 ± 1.5 6.5 ± 2.6

132

5.8 ± 1.5

56

32 16 6 4 100 45

45

1.8 ± 0.8

23 13

1.0 ± 0.6 1.7 ± 1.2

5 3 1

0.8 ± 0.8 1.3 ± 0.8 0.5 ± 0.8

64

4.8 ± 1.3

100 54

1.8 ± 1.2 17.5 ± 3.1†

100 70

5.5 ± 1.5 41.8 ± 3.4†

3.0 ± 1.9

2.0 ± 1.4 1.0 ± 0.9 1.7 ± 1.0 1.3 ± 1.5 3.0 ± 1.4 28.3 ± 3.6†

Lab I

Lab II

Lab IV

CG

Foci/well

CG

Foci/well

CG

Foci/well

100

5.7 ± 2.3

121

10.5 ± 2.3

100 104 116

2.3 ± 1.8 3.0 ± 1.5 4.5 ± 2.6

100 111 116 126

2.7 ± 1.2 6.7 ± 1.5 9.2 ± 5.2* 16.0 ± 1.4*

130

16.2 ± 2.6* 140

29.7 ± 4.8*

151 168

35.7 ± 5.6* 46.0 ± 8.0*

159

18.0 ± 4.8* 199

52.3 ± 7.3*

184

33.8 ± 4.5*

190

24.7 ± 6.2* 217

34.5 ± 2.3*

164 139 114

5.3 ± 1.9 0.7 ± 0.8 Toxic

161

1.0 ± 1.3

72 100 126

5.0 ± 1.5 39.7 ± 4.3†

Toxic 2.7 ± 1.6 8.7 ± 2.8†

2.5 ± 0.8 18.7 ± 4.2†

% of cell growth compared to that of solvent control. Average number of transformed foci/well ± SD. Solvent control: final solvent concentration of the working culture media in parentheses. Solvent control for the positive control: final solvent concentration of the working culture media in parentheses. Positive control: final solvent concentration of the working culture media in parentheses. p < 0.05; one-sided Dunnett test, vs corresponding solvent control. p < 0.05; one-sided t-test or Aspin–Welch test, vs corresponding solvent control.

4.5. Lithocholic acid The results obtained with lithocholic acid are shown in Table 5 and Fig. 6. The data from all laboratories revealed that lithocholic acid was negative in the initiation assay and positive in the promotion assay. 4.6. Cadmium chloride The results for cadmium chloride are presented in the Table 6 and Fig. 7. The chemical gave negative results in the initiation assays as determined by all three laboratories. The promotion assay was repeated by all three laboratories. The reason for the repetition was the cell death caused by cytotoxicity at high concentrations of the test chemical. All three laboratories performed the second assay in a lower concentration range. In the first run of the promotion assay, Lab I obtained statistically significant increases in the number of transformed foci at the lowest two

concentrations, 0.4 and 0.6 ␮g/mL, but the chemical was toxic at the higher 5 doses. In the second run of the assay, the positive results were confirmed at concentrations ≤0.6 ␮g/mL. In Lab II, the 7 highest of 8 doses tested were completely cytotoxic in the first run of the assay. However, in the second run of the assay, which employed lower doses, the number of transformed foci at one dose (the highest dose tested, which was also the lowest dose tested in the first run) was statistically significantly increased. Lab II again repeated the assay for a third time and obtained a significant increase at a single dose again. Thus, based upon the response criteria, cadmium chloride was judged to be positive in the Lab II. Since Lab III set the test concentrations over a wide range, cell killing was caused at only two doses in the first promotion assay. Meanwhile, Lab III obtained a statistically significant increase in transformed foci at a single dose in that first assay. In the second assay that was carried out using a lower and narrower concentration range, there were the significant increases of foci at 3 consecutive doses. Therefore, cadmium chloride was judged positive by Lab III.

A. Sakai et al. / Mutation Research 725 (2011) 57–77

67

Table 9 Results of transformation assay on anthracene. Concentration (␮g/mL)

(a) Initiation assay 0c (0.5% DMSO) 1.25 1.56 2.5 3.13 5 6.25 10 12.5 20 25 50 0d (0.1% DMSO) MCA 1e (0.1% DMSO) MCA 1e (0.5% DMSO) Concentration (␮g/mL) (b) Promotion assay 0c (0.5% DMSO) 1.25 1.56 2.5 3.13 5 6.25 10 12.5 20 25 50 0d (0.1% DMSO) TPA 0.05e (0.1% DMSO) TPA 0.05e (0.5% DMSO) a b c d e * †

Lab I

Lab II

Lab VI

CGa

Foci/wellb

CG

Foci/well

CG

Foci/well

100

4.2 ± 2.6

100

2.5 ± 0.8

100 95

1.3 ± 1.2 1.5 ± 1.0

107

5.8 ± 2.7

99

2.0 ± 0.6 86

2.2 ± 1.2

102

5.5 ± 2.9

116

2.3 ± 1.5 95

2.5 ± 1.6

109

4.0 ± 1.9

112

3.5 ± 2.6 93

1.3 ± 0.8

106

2.8 ± 2.2

105

2.7 ± 1.6 100

2.5 ± 1.0

110 107 100 48

5.7 ± 2.1 6.3 ± 2.2 2.7 ± 0.8 24.3 ± 5.6†

110

1.8 ± 1.3

100 62

3.0 ± 1.5 16.8 ± 1.5† 59

14.3 ± 2.6†

Lab I CG

Foci/well

Lab II CG

Foci/well

100

2.3 ± 1.2

100

2.3 ± 1.0

96

2.7 ± 2.0

106

2.7 ± 0.8

94

2.0 ± 0.9

102

3.8 ± 2.8

97

2.7 ± 0.8

108

4.7 ± 1.5

98

2.8 ± 0.8

93

3.5 ± 2.5

98 96 100 131

3.0 ± 1.4 1.3 ± 0.8 3.7 ± 2.0 17.5 ± 6.5†

96

2.7 ± 1.2

100 137

3.7 ± 2.2 10.8 ± 1.6†

Lab VI CG

Foci/well

100 104

1.8 ± 1.3 5.5 ± 2.3

97

6.7 ± 2.3*

105

6.7 ± 4.4*

96

9.8 ± 4.4*

97

10.2 ± 3.5*

136

14.7 ± 2.9†

% of cell growth compared to that of solvent control. Average number of transformed foci/well ± SD. Solvent control: final solvent concentration of the working culture media in parentheses. Solvent control for the positive control: final solvent concentration of the working culture media in parentheses. Positive control: final solvent concentration of the working culture media in parentheses. p < 0.05; one-sided Dunnett test, vs corresponding solvent control. p < 0.05; one-sided t-test or Aspin–Welch test, vs corresponding solvent control.

4.7. Mezerein

4.8. Methapyrilene hydrochloride

Table 7 and Fig. 8 show the results of mezerein. All three laboratories reported clearly positive results in the promotion assay. The chemical induced marked and dose-dependent increases in the number of transformed foci relative to the solvent control in all cases. In the initiation assay performed by Lab I, mezerein did not induce statistically significantly increases in the number of transformed foci at any concentration up to 0.01 ␮g/mL, which was the maximum concentration used in the present study. In the initiation assays performed by Lab III and Lab IV, a statistically significant increase in the number of transformed foci was observed only at the maximum (preset) concentration used. The results of the initiation assay by both laboratories remained inconclusive since neither laboratory repeated the assay at higher concentrations (due to limited availability) in an attempt to verify the initial results. Rather, they considered that the promotion assay had established that the chemical was clearly positive in the Bhas 42 cell transformation assay and that it was not necessary to further differentiate the initiation vs. promotion properties of the compound.

The results for methapyrilene hydrochloride are shown in Table 8 and Fig. 9. All three laboratories demonstrated methapyrilene hydrochloride to be negative in the initiation assay and positive in the promotion assay.

4.9. Anthracene The results for anthracene are exhibited in Table 9 and Fig. 10. All three laboratories obtained negative results in the initiation assay. In the promotion assay, it was negative in the two laboratories, Lab I and Lab II, but positive in Lab VI. Inhibition of cell growth or focus formation by cytotoxicity was not observed at the concentrations tested in both initiation and promotion assays in all three laboratories. However, anthracene is poorly soluble in DMSO and other usable organic solvents and insoluble in water. The assays by three laboratories were conducted up to the limit of solubility in DMSO or beyond by using a chemical suspension in the vehicle. Thus, the dosing employed in these assays was considered to be sufficient

68

A. Sakai et al. / Mutation Research 725 (2011) 57–77

Table 10 Results of transformation assay on d-mannitol. Concentration (␮g/mL)

(a) Initiation assay 0c (5% Water) 15.8 50 100 158 300 500 1000 1300 1580 1800 2500 3500 5000 0d (0.1% DMSO) MCA 1e (0.1% DMSO) Concentration (␮g/mL)

(b) Promotion assay 0c (5% Water) 15.8 50 100 158 300 500 880 1000 1300 1580 1800 2500 3500 5000 0d (0.1% DMSO) TPA 0.05e (0.1% DMSO) a b c d e f † ‡

Lab I

Lab III

Lab IV

CGa

Foci/wellb

CG

Foci/well

CG

Foci/well

100

2.7 ± 1.0

100 103 98

2.7 ± 1.4 1.0 ± 0.9 1.5 ± 0.8

100

1.2 ± 1.3

107

1.5 ± 1.0 102

2.7 ± 1.6

105

2.0 ± 2.4 99

1.3 ± 1.2

111

0.8 ± 1.2 108

0.8 ± 1.0

110

0.8 ± 0.8 111 114 109 107

1.8 ± 0.8 1.2 ± 0.8 1.8 ± 1.2 1.5 ± 1.6 1.0 ± 0.9 40.5 ± 7.9†

106

3.2 ± 0.8

109 100 64

2.5 ± 1.0 2.7 ± 1.6 33.7 ± 5.0†

Lab I

102

1.8 ± 1.8

33f

14.0 ± 4.4†

Lab III

Lab IV

CG

Foci/well

CG

Foci/well

CG

Foci/well

100

5.8 ± 2.1

100 101 93

2.8 ± 1.5 3.5 ± 1.6 3.0 ± 1.4

100

1.5 ± 1.0

101

6.3 ± 4.5 102

2.2 ± 1.9

101

7.2 ± 2.0 101

2.8 ± 2.3 103

1.7 ± 0.5

107

1.5 ± 0.8

99 110 101 99

1.8 ± 1.7 1.7 ± 1.9 1.3 ± 0.8 1.2 ± 1.2 2.3 ± 1.0 10.2 ± 2.1†

102

9.2 ± 3.2 104

103

5.2 ± 2.6

103 100 134

3.7 ± 2.0 8.2 ± 3.6 50.2 ± 4.6†

1.7 ± 0.5

106

4.3 ± 1.5

157f

8.7 ± 3.6‡

% of cell growth compared to that of solvent control. Average number of transformed foci/well ± SD. Solvent control: final solvent concentration of the working culture media in parentheses. Solvent control for the positive control: final solvent concentration of the working culture media in parentheses. Positive control: final solvent concentration of the working culture media in parentheses. % of cell growth compared to that of 5% water. p < 0.05; one-sided t-test or Aspin–Welch test, vs corresponding solvent control. p < 0.05; one-sided t-test or Aspin–Welch test, vs 5% water.

4.10. d-Mannitol

4.12. l-Ascorbic acid

Table 10 and Fig. 11 represent the results for d-mannitol. Both in the initiation assay and in the promotion assay, d-mannitol was consistently negative in the three laboratories up to the maximum concentration (5 mg/mL) tested.

Table 12 and Fig. 13 show the results for l-ascorbic acid. All three laboratories reported l-ascorbic acid to be negative in both the initiation and promotion assays. Lab II voluntarily repeated the promotion assay. In the first promotion assay, Lab II observed that the transformed focus formation was reduced in the cultures treated with ascorbic acid and the number of foci diminished to 0 at the highest three concentrations. Obvious cell death due to cytotoxicity was not observed in either the transformation assay or the concurrent cell growth assay. In the second promotion assay using a lower concentration range, Lab II confirmed that ascorbic acid decreased the number of transformed foci as compared to the control. Ascorbic acid also inhibited transformed focus formation in the promotion assays performed by the other two laboratories.

4.11. Caffeine The results for caffeine are shown in Table 11 and Fig. 12. Two laboratories (Lab I and Lab V) presented convincing results that caffeine was negative in the initiation assay. Likewise, in the initiation assay reported by the other laboratory (Lab VI), no statistically significant increase in the number of transformed foci was reported, although cytotoxicity was also not induced at any of the concentrations of caffeine in the concurrent cell growth assay. Therefore, it was concluded that the initiation assay by Lab VI was incomplete because of inadequate dosing. Caffeine was judged negative by all three laboratories in the promotion assay.

4.13. Positive and negative controls The numbers of transformed foci in the positive and their negative (DMSO) controls are shown in Tables 1–12. Fig. 14 shows these

A. Sakai et al. / Mutation Research 725 (2011) 57–77

69

Table 11 Results of transformation assay on caffeine. Concentration (␮g/mL)

(a) Initiation assay 0c (0.5% DMSO) 0c (5% Water) 6.25 12.5 25 50 100 200 300 400 500 600 700 800 0d (0.1% DMSO) MCA 1e (0.1% DMSO) MCA 1e (0.5% DMSO) Concentration (␮g/mL)

(b) Promotion assay 0c (0.5% DMSO) 0c (5% Water) 6.25 10 12.5 25 30 50 100 200 300 500 0d (0.1% DMSO) TPA 0.05e (0.1% DMSO) TPA 0.05e (0.5% DMSO) a b c d e †

Lab I

Lab V

Lab VI

CGa

Foci/wellb

CG

Foci/well

100

2.3 ± 1.6

100

2.7 ± 1.2

114

1.8 ± 1.2

92 51 23 13 11 11 100 47

1.7 ± 1.0 2.0 ± 1.3 1.7 ± 1.0 2.5 ± 1.9 1.0 ± 1.3 1.8 ± 1.5 1.3 ± 1.2 19.7 ± 4.0†

Lab I

104 100 76 38 18

3.0 ± 2.5 4.7 ± 2.1 5.2 ± 3.3 5.8 ± 2.3 3.7 ± 2.3

100 54

3.3 ± 1.8 41.3 ± 9.4†

Lab V

CG

Foci/well

100

1.8 ± 1.5

87 89 97 86 90

2.2 ± 1.2 1.7 ± 0.8 0.5 ± 0.8 2.5 ± 1.8 4.0 ± 3.0

56

17.2 ± 3.5†

Lab VI

CG

Foci/well

CG

Foci/well

100

5.0 ± 1.9

100

4.2 ± 2.3

93

2.0 ± 1.4

80

1.5 ± 1.0

87

4.0 ± 2.4

82 70 57 49 39 100 121

3.5 ± 1.0 1.5 ± 0.8 0.2 ± 0.4 0.0 ± 0.0 0.2 ± 0.4 6.7 ± 2.3 35.5 ± 6.1†

73

2.2 ± 2.1

35 33 26 27 100 106

2.5 ± 1.6 2.3 ± 1.0 1.7 ± 1.6 0.5 ± 0.5 2.7 ± 1.8 21.5 ± 3.5†

CG

Foci/well

100

4.0 ± 1.8

88

3.3 ± 1.2

94 85

2.5 ± 1.4 4.3 ± 1.4

88 78

1.5 ± 1.0 1.8 ± 1.5

141

16.5 ± 4.8†

% of cell growth compared to that of solvent control. Average number of transformed foci/well ± SD. Solvent control: final solvent concentration of the working culture media in parentheses. Solvent control for the positive control: final solvent concentration of the working culture media in parentheses. Positive control: final solvent concentration of the working culture media in parentheses. p < 0.05; one-sided t-test or Aspin–Welch test, vs corresponding solvent control.

data plotted pairwise, assay by assay, and clustered by a laboratory. The number of transformed foci were statistically analyzed by t-test or Aspin–Welch test and there were significant differences between negative and positive controls in all the initiation and promotion assays (p < 0.05), although the number of foci induced by each positive control varied between laboratories and sometimes within laboratories. 5. Discussion This international validation study of the Bhas 42 cell transformation assay using 6-well micro-plates was carried out on twelve chemicals in total for the initiation and promotion assays. Each chemical was examined by three laboratories. The judgments of the results are summarized in Table 13. In the initiation assay, concurring conclusions were reached by the three laboratories testing the same respective compounds with 8 out of 10 chemicals. Since the testing of two of the compounds, sodium arsenite and caffeine, were considered inadequate/incomplete, those two chemicals were excluded from this ratio. In the promotion assay, concurrent conclusions were reached for 10 of 12 chemicals. When the majority judgments in the initiation and promotion assays were

integrated into an overall judgment in the Bhas 42 cell transformation assay (Table 13), the six carcinogens and the two tumor promoters were all positive and all the non-carcinogens were negative. That is, carcinogens except o-toluidine hydrochloride and tumor promoters were positive in the initiation or promotion assay or in both assays in all three laboratories, and o-toluidine was positive in both assays in two of the three laboratories: non-carcinogens except anthracene were negative in both assays in all laboratories and anthracene was negative in both assays in two of the three laboratories. Thus, the present inter-laboratory validation study demonstrated that the Bhas 42 cell transformation assay is reproducible between laboratories and applicable to the prediction of chemical carcinogenicity. In the previously reported pre-validation study, nine chemicals had been examined [16]. The chemicals tested in the pre-validation study and the present study are listed in Table 14. Certain chemicals were tested in both the pre-validation and the present validation studies, i.e. lithocholic acid, mezerein, methapyrilene hydrochloride and anthracene. The same conclusions for these four chemicals were reached in the present study and in the pre-validation study, which further substantiates the reproducibility of Bhas 42 cell transformation assay.

70

A. Sakai et al. / Mutation Research 725 (2011) 57–77

Table 12 Results of transformation assay on l-ascorbic acid. Concentration (␮g/mL)

Lab I

(a) Initiation assay 0c (5% Water) 10 25 50 75 100 110 150 200 210 250 300 340 380 400 420 600 0d (0.1% DMSO) MCA 1e (0.1% DMSO) Concentration (␮g/mL)

(b) Promotion assay 0c (5% Water) 0.1 1 10 50 100 110 150 200 210 300 400 420 500 600 700 800 900 1500 0d (0.1% DMSO) TPA 0.05e (0.1% DMSO) a b c d e †

Lab II

Lab IV

CGa

Foci/wellb

CG

Foci/well

CG

Foci/well

100

2.7 ± 2.2

100 108

2.0 ± 1.9 2.2 ± 1.0

100

4.2 ± 1.5

91 87

1.7 ± 1.2 1.7 ± 1.5 96

4.7 ± 2.0

78

2.7 ± 1.0

63 46

2.0 ± 1.1 2.5 ± 1.4

101 95

4.8 ± 1.8 4.8 ± 1.0

91

3.7 ± 1.6

25 14

2.8 ± 0.8 2.3 ± 1.9

73

3.2 ± 1.2

4

2.5 ± 1.4

5 1

2.8 ± 2.0 Toxic 5.5 ± 1.9 43.7 ± 3.0†

2.3 ± 1.5 26.7 ± 5.3†

100 48 Lab I

2.2 ± 1.5

85

73 52 32 6 0

1.0 ± 0.6 1.3 ± 1.0 0.2 ± 0.4 0.5 ± 0.8 0.2 ± 0.4

100 55

1.3 ± 1.0 15.8 ± 5.3†

Lab II, 1st run

Lab II, 2nd run

Lab IV

CG

Foci/well

CG

Foci/well

CG

Foci/well

CG

Foci/well

100

4.5 ± 1.6

100

1.8 ± 1.2

2.2 ± 0.8

1.2 ± 1.2 1.5 ± 1.0

1.0 ± 1.1 0.5 ± 0.5 0.3 ± 0.5 0.2 ± 0.4 0.0 ± 0.0 0.0 ± 0.0

100

92 96

100 99 103 95 102 82

101

0.2 ± 0.4

92

0.0 ± 0.0

104 100

0.5 ± 0.5 0.7 ± 0.5

95

0.2 ± 0.4

101 107

1.5 ± 1.0 0.8 ± 1.0

111

1.7 ± 1.2

39

Toxic

92

1.5 ± 0.8

91 90

0.8 ± 0.4 2.2 ± 2.0

89 86

Toxic Toxic

41

Toxic

100 123

7.5 ± 4.0 37.2 ± 4.7†

103 102 92 78 36 100 144

0.5 ± 0.8 0.3 ± 0.5 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 3.2 ± 1.9 11.2 ± 0.8†

100 156

1.0 ± 1.3 11.5 ± 1.9†

2.7 ± 1.4 20.0 ± 3.3†

% of cell growth compared to that of solvent control. Average number of transformed foci/well ± SD. Solvent control: final solvent concentration of the working culture media in parentheses. Solvent control for the positive control: final solvent concentration of the working culture media in parentheses. Positive control: final solvent concentration of the working culture media in parentheses. p < 0.05; one-sided t-test or Aspin–Welch test, vs corresponding solvent control.

Lab I had previously examined in-house 2-acetylaminofluorene, dibenz[a,h]anthracene, sodium arsenite, lithocholic acid, cadmium chloride, mezerein, methapyrilene hydrochloride, anthracene, dmannitol, caffeine and l-ascorbic acid without coding in the Bhas 42 cell transformation assay [17]. In the present validation study, in which all compounds were coded, Lab I reported the same judgments in the initiation and promotion assays for these same 11 chemicals with the exception of sodium arsenite, which was equivocal in the initiation assay of the in-house study but negative in that of the present study despite concordant result (positive) in the promotion assay between both studies. Overall, these results demonstrate excellent intra-laboratory reproducibility for the Bhas 42 cell transformation assay. o-Toluidine hydrochloride had not been tested in-house by Lab I but free o-toluidine had. There was a discrepancy in that the free o-toluidine was positive in the promotion assay of in-house study but the hydrochloride was negative in

the promotion assay of the present validation study. This is the only incongruity among the 12 chemicals tested both in the in-house study and in the present study by Lab I. In this validation study, the choice of vehicle (DMSO or distilled water) to dissolve a test chemical was left to each laboratory, considering the conditions of actual practice in an assay field. Consequently, different solvents were chosen among laboratories for 3 chemicals, o-toluidine, sodium arsenite and caffeine. However, the vehicles seemed not to affect the judgment of transforming activity for these chemicals, since the judgments obtained in the initiation and promotion assays were not split between DMSO and water (Tables 2, 4 and 11) and there is no information in literatures that these chemicals are unstable in either solvent. However, the vehicle should be designated in advance in a validation study, if a test chemical is unstable in or reactive with certain solvents.

A. Sakai et al. / Mutation Research 725 (2011) 57–77

71

Table 13 Summary of results. Compound

Laboratory

Bhas 42 cell transformation

Carcinogenicity in vivo

Initiation assay

2-Acetylaminofluorene

o-Toluidine HCl

Dibenz[a,h]anthracene

Sodium arsenite

Lithocholic acid

Cadmium chloride

Mezerein

Methapyrilene HCl

Anthracene

d-Mannitol

Caffeine

l-Ascorbic acid

a b c d

I IV VI I V VI I II V I III VI I IV VI I II III I III IV I II IV I II VI I III IV I V VI I II IV

Promotion assay

Integrated judgmentb

Judgment within lab

Agreement between labs

Majority judgmenta

Judgment within lab

Agreement between labs

Majority judgment

+ + + − + + + + + − + Incompletec − − − − − − − ± ± − − − − − − − − − − − Incomplete − − −

3/3

+

3/3

+

+

+

2/3

+

2/3

+

+

+

3/3

+

+ + + − + + − − − + + + + + + + + + + + + + + + − − + − − − − − − − − −

3/3

−

+

+

3/3

+

+

+

3/3

+

+

TPd

3/3

+

+

+

3/3

+

+

TP

3/3

+

+

+

2/3

−

−

−

3/3

−

−

−

3/3

−

−

−

3/3

−

−

–

0/2

3/3

−

3/3

−

−

3/3

−

3/3

−

3/3

−

2/2

−

3/3

−

Judgment by majority rule among three laboratories. Judgment into which judgments in initiation and promotion assays were integrated. Unable to judge results due to inadequate dosing. Tumor promoter.

Table 14 Comparison of the results between the pre-validation study [16] and the present study. Compound

Pre-validation study Initiation

2-Acetylaminofluorene o-Toluidine HCl Dibenz[a,h]anthracene Sodium arsenite Lithocholic acid Cadmium chloride Mezerein Methapyrilene HCl Anthracene d-Mannitol Caffeine l-Ascorbic acid N-Methyl-N’-nitro-N-nitrosoguanidine Benzo[a]pyrene Benz[a]anthracene Pyrene Phorbol a b

Promotion

Present study a

Transformation

−

+

+

− − −

+ + −

+ + −

+ + + + −

− − + + −

+ + + + −

Carcinogenicity in vivo

Initiation

Promotion

Transformation

+ + + +/− − − − − − − − −

+ + − + + + + + − − − −

+ + + + + + + + − − − −

+ + + + TPb + TP + − − − − + + + −

Integrated judgment in the overall Bhas 42 cell transformation assay in which the judgments for the initiation and promotion assays were assimilated. Tumor promoter.

A. Sakai et al. / Mutation Research 725 (2011) 57–77

30

120

25

100 20 80

* ** * **

15

* * * * *

40

10

*

10

120

30

100

25

*

80

*

20

*

*

60

*

40

15

* *

*

* * *

10

* 5

20

0 300

0

20 0 0

Lab I Lab IV Lab VI

No. of foci/well

140

60

Promotion assay

Lab I Lab IV Lab VI

No. of foci/well

Relative cell growth (%)

Initiation assay

Relative cell growth (%)

72

30 100 Concentration (µg/mL)

5

0

1

3 10 Concentration (µg/mL)

30

0 50

Fig. 2. Graphic view of the results of transformation assay and concurrent cell growth assay on 2-acetylaminofluorene. *p < 0.05; one-sided Dunnett test.

Lab I Lab V Lab VI 30 25

100 20

80

15

60 40

*

* *

0

5

-20 0

100

100

40

80

* *

*

*

*

30

60 20 40

*

*

20

10

Relative cell growth (%)

120

50

120

No. of foci/well

Relative cell growth (%)

140

Lab I Lab V Lab VI

Promotion assay

No. of foci/well

Initiation assay

300

1000

20

0 3000 5000

0

*

*

0

10

Concentration (µg/mL)

10

0 1000 2000

100 Concentration (µg/mL)

Fig. 3. Graphic view of the results of transformation assay and concurrent cell growth assay on o-toluidine HCl. *p < 0.05; one-sided Dunnett test.

Initiation assay

30

100

25

80

20

60

15

40

10

5

20

5

0 10

0

80

* *

60

*

*

* 40

*

20 15

* 10

20

0.01

** * * *

*

* * * *

0

25

*

0.1

1

Concentration (µg/mL)

Relative cell growth (%)

*

No. of foci/well

Relative cell growth (%)

30

*

0

Promotion assay 120

120 100

Lab I Lab II Lab V

0 0.001

0.01

0.1

1

No. of foci/well

Lab I Lab II Lab V

0 10

Concentration (µg/mL)

Fig. 4. Graphic view of the results of transformation assay and concurrent cell growth assay on dibenz[a,h]anthracene. *p < 0.05; one-sided Dunnett test.

A. Sakai et al. / Mutation Research 725 (2011) 57–77

25

80

20

60

15 10

40 20

*

* * *

0 0 0.01

0.1 Concentration (µg/mL)

1

Promotion assay

120

25

100 20 80 15

*

*

60

*

5

20

0

0

*

*

* * * *

* * 0

2

10

*

40

0.03

0.1

No. of foci/well

Relative cell growth (%)

100

Lab I Lab III, 1st Lab III, 2nd Lab VI 30

140 Relative cell growth (%)

Initiation assay 120

No. of foci/well

Lab I, 1st Lab I, 2nd Lab III, 1st Lab III, 2nd Lab VI 30

73

5

0.3

1

3

0 5

Concentration (µg/mL)

Fig. 5. Graphic view of the results of transformation assay and concurrent cell growth assay on sodium arsenite. *p < 0.05; one-sided Dunnett test.

Initiation assay 120

that show marked growth enhancement should be assayed at the concentrations covering the region of growth enhancement. For the chemicals that do not induce marked growth enhancement, the promotion assay should be performed up to the concentration resulting in more than 50% growth inhibition in the cell growth assay. It has also been reported that other typical tumor promoters, lithocholic acid and okadaic acid, induce the cell transformation at concentrations showing growth inhibition [14,19]. Another feature in the promotion assay between the cell growth and the transformation was revealed in the examination on cadmium chloride and sodium arsenite; the promotion assay for cadmium chloride had to be repeated by all the three laboratories (Table 6) and that of sodium arsenite by one of the laboratories (Table 4). The reason for these repetitions was the extensive cytotoxicity induced by those chemicals at the high concentration ranges in the transformation assays. In this validation assay, the concentration ranges used for the transformation assay were determined independently by each laboratory, according to the results of the prior cell growth assay as described in Section 2.5. The cell growth assays concurrent with the transformation assays showed no toxicity, or from weak to moderate toxicity at the

Lab I Lab IV Lab VI 30

120

*

80

20

Relative cell growth (%)

25 No. of foci/well

Relative cell growth (%)

* 100

100

40

10

20

5

20

0 100

0

10

30 Concentration (µg/mL)

* 30

*

15

0

40

80

60

0

Lab I Lab IV Lab VI 50

Promotion assay

60

*

20

*

40

*

* 10

*

0

3

10 Concentration (µg/mL)

No. of foci/well

In the initiation assay the cells were treated with a test chemical by the direct addition of chemical solution to the culture medium or by the replacement of existing medium with a fresh medium containing the chemical, although in the promotion assay the chemical treatment was always carried out by periodic exchanges of medium containing a test chemical, as described in Section 2.6. The choice of treatment procedures in the initiation assay was left to each laboratory. Only Lab IV out of 6 participating laboratories treated the cells by the medium exchange. There was no difference in the judgment of initiation assay for the particular 6 chemicals, 2-acetylaminofluorene, lithocholic acid, mezerein, methapyrilene hydrochloride, d-mannitol and l-ascorbic acid, between two treatment procedures (Table 13). In the promotion assay, mezerein and methapyrilene hydrochloride markedly increased the cell density in all the three laboratories, as measured by the concurrent cell growth assay. The cell growth enhancement was parallel with the induction of cell transformation. A group of tumor promoters such as phorbol esters and mezerein have been reported to promote the transformation of Bhas 42 cells in the concentration range where they induce cell growth enhancement [14,19]. Thus, the chemicals

30

0 50

Fig. 6. Graphic view of the results of transformation assay and concurrent cell growth assay on lithocholic acid. *p < 0.05; one-sided Dunnett test.

A. Sakai et al. / Mutation Research 725 (2011) 57–77

Lab I Lab II Lab III

Promotion assay

30

140

100

25

120

80 20

60 15 40 10 20

Relative cell growth (%)

120

No. of foci/well

Relative cell growth (%)

Initiation assay

Lab I, 1st Lab I, 2nd Lab II, 1st Lab II, 2nd Lab II, 3rd Lab III, 1st Lab III, 2nd 30 25

*

100

20 80

* * ** * *

60

15

* 10

40 5

0 -20

0

0.1

0.3 1 Concentration (µg/mL)

* * * ** *

20

0

0

2

No. of foci/well

74

0

0.1

5

0.3 1 Concentration (µg/mL)

2

0

Fig. 7. Graphic view of the results of transformation assay and concurrent cell growth assay on cadmium chloride. *p < 0.05; one-sided Dunnett test.

Lab I Lab III Lab IV 30

20 150 15

**

100

10 50

Relative cell growth (%)

25

200

100

250

No. of foci/well

Relative cell growth (%)

250

Lab I Lab III Lab IV

Promotion assay

*

60

150

*

* *

* *

100

*

*

50

5

80

*

200

*

40

No. of foci/well

Initiation assay

20

* 0

0 0.00001

0.0001

0.001

0 0.01

0 0

Concentration (µg/mL)

0.00001

0.0001

0.001

0 0.01

Concentration (µg/mL)

Fig. 8. Graphic view of the results of transformation assay and concurrent cell growth assay on mezerein. *p < 0.05; one-sided Dunnett test.

Initiation assay

25

120 20

100 80

15

60

10

Relative cell growth (%)

140

No. of foci/well

Relative cell growth (%)

Promotion assay

30

160

250

100

200

80

60

150

* * 100

* *

40 5

20

Lab I Lab II Lab IV

50

**

* *

*

*

40

No. of foci/well

Lab I Lab II Lab IV

20

* 0 0

50

100

300

Concentration (µg/mL)

0 500

0 0

10

30

100

0 300 500

Concentration (µg/mL)

Fig. 9. Graphic view of the results of transformation assay and concurrent cell growth assay on methapyrilene HCl. *p < 0.05; one-sided Dunnett test.

A. Sakai et al. / Mutation Research 725 (2011) 57–77

100

25

80

20

60

15

40

10

Promotion assay 120

30

100

25

80

20

60

15

0 0

3

10

30

5

20

0 50

0

*

*

40

* 20

Lab I Lab II Lab VI

No. of foci/well

30 Relative cell growth (%)

Relative cell growth (%)

Initiation assay 120

No. of foci/well

Lab I Lab II Lab VI

75

10

* 5

0

3

Concentration (µg/mL)

10

30

0 50

Concentration (µg/mL)

Fig. 10. Graphic view of the results of transformation assay and concurrent cell growth assay on anthracene. *p < 0.05; one-sided Dunnett test.

100

25

80

20

60

15

40

10

20

5

0

0

100 1000 Concentration (µg/mL)

0 5000

120

30

100

25

80

20

60

15

40

10

20

5

0

0

No. of foci/well

30

Lab I Lab III Lab IV

Promotion assay

Relative cell growth (%)

Relative cell growth (%)

120

No. of foci/well

Lab I Lab III Lab IV

Initiation assay

0 5000

100 1000 Concentration (µg/mL)

Fig. 11. Graphic view of the results of transformation assay and concurrent cell growth assay on d-mannitol.

25

80

20

60

15

40

10

20 0

0

10

30

100

300

Concentration (µg/mL)

100

25

80

20

60

15

40

10

5

20

5

0 800

0

No. of foci/well

Relative cell growth (%)

120

0

10

30

100

0 300 500

Concentration (µg/mL)

Fig. 12. Graphic view of the results of transformation assay and concurrent cell growth assay on caffeine.

No. of foci/well

100

Lab I Lab V Lab VI 30

Promotion assay

Relative cell growth (%)

120

Lab I Lab V Lab VI 30

Initiation assay

A. Sakai et al. / Mutation Research 725 (2011) 57–77

100

25

80

20

60

15

40

10

20 0

0

10

30 100 300 Concentration (µg/mL)

Promotion assay 120 100

25

80

20

60

15

40

10

5

20

5

0 600

0

No. of foci/well

Relative cell growth (%)

Initiation assay

Lab I Lab II, 1st Lab II, 2nd Lab IV 30

0 0.1

1

10

100

No. of foci/well

120

Lab I Lab II Lab IV 30 Relative cell growth (%)

76

0 1000

Concentration (µg/mL)

Fig. 13. Graphic view of the results of transformation assay and concurrent cell growth assay on l-ascorbic acid.

Initiation assay Negative control (DMSO) Positive control (MCA)

60

No. of foci/well

50 40 30 20 10 0

I

II

III IV Laboratory

V

VI

Promotion assay Negative control (DMSO) Positive control (TPA)

60

No. of foci/well

50 40 30 20 10 0

I

II III IV Laboratory

V

VI

Fig. 14. Number of transformed foci in the positive and negative controls in the Bhas 42 cell transformation assay. Positive and negative controls are paired assay by assay and the pairs are clustered by a laboratory. Bars indicate standard deviation.

concentrations where those chemicals caused cell-killing in the transformation assays. This fact indicated that the doses for the promotion assays were adequately determined according to the protocol. In the present protocol the cultures in the cell growth assay were treated with a test chemical for 3 days, whereas the cultures in the transformation assay were subjected to chemical treatment for 10 days. Thus, it seemed apparent that the longer

exposure to those chemicals caused the cell-killing observed at high concentrations [18]. Similar increase of cell-killing was also observed in the promotion assay of lithocholic acid performed by Lab IV and Lab VI at concentrations where little cytotoxicity was observed in the cell growth assay. In the promotion assay, ascorbic acid reduced focus formation compared to the control in all the three laboratories. Although ascorbic acid was demonstrated not to be carcinogenic in a twoyear oral carcinogenesis bioassay conducted by the United State National Toxicology Program (NTP) [20], it is known that ascorbic acid and sodium ascorbate show pleiotrophic effects on chemical carcinogenesis depending on the particular carcinogen, dosage, animal species and target organs [21]. Ascorbic acid and sodium ascorbate exert both genotoxic effects in several mutagenicity testing systems and anti-mutagenic effects in other test systems [22]. They have shown inhibitory effects on radiation- and MCA-induced cell transformation in C3H/10T1/2 cells [23,24] and inhibited both spontaneous and MCA-induced focus formation in the two-stage BALB/c 3T3 cell transformation assay [25]. All assays were performed including negative and positive controls (Tables 1–12 and Fig. 14). The increases in the number of transformed foci in the positive controls were always statistically significant when compared with the corresponding negative (DMSO) controls (t-test or Aspin–Welch test; p < 0.05). However, there were differences between some laboratories in the numbers of foci produced in the positive (MCA or TPA) control and also in the negative (DMSO) control, as statistically analyzed by the Tukey–Kramer test (p < 0.05: The details of analysis are not shown. The statistical analysis for water control was not carried out because the number of samples was small.). There was a tendency that the laboratories where negative-control values were low gave low positive-control values and the laboratories where negative-control values were high produced high positivecontrol values. That is, in the average numbers of foci produced in individual laboratories, there were a weak positive correlation between the negative and positive controls in the initiation assay (y = 5.7755x + 10.719, R2 = 0.2922), and a substantial correlation in the promotion assay (y = 4.0197x + 4.0308, R2 = 0.9081). The correlations were clear in the analysis without Lab III and Lab V where the sample number was few (initiation assay; y = 7.0694x + 7.1858, R2 = 0.9418: promotion assay; y = 5.2871x + 0.2308, R2 = 0.9196). In this validation study, the limit value of vehicle control was not set. Lab V submitted exceptionally large numbers of foci formed

A. Sakai et al. / Mutation Research 725 (2011) 57–77

in water (16.3 foci/well) and DMSO (14.8 foci/well) controls for the promotion assay of sodium arsenite, as shown in Table 2b. However, there was still a statistically significant difference in focus number between the TPA (43.7 foci/well) and DMSO controls (t-test, p < 0.05). In conclusion, the present international validation study demonstrated that the Bhas 42 cell transformation assay is reproducible both within and between laboratories The overall assay results of test chemicals agreed with their in vivo carcinogenicity. Its proficiency in predicting carcinogenic potential and its capability of detecting Ames-negative and Ames-discordant carcinogens have been shown previously [17]. The authors recommend its inclusion as a standard component of the prevailing genotoxicity test battery in order to improve the accuracy of chemical carcinogen predictivity. The present study was carried out using 6-well micro-plates. We have since developed the Bhas 42 cell transformation assay using 96-well micro-plates that has the potential to be utilized for high throughput automated applications [manuscript in preparation]. The validation study on the 96-well method has been finished and is being prepared for publication. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgement This research was conducted under a New Energy and Industrial Technology Development Organization (NEDO) Project in Japan addressing “Chemical Risk Analysis Technologies”. We thank the Bhas 42 Cell Transformation Assay Advisory Committee and Validation Management Team members for their guidance and efforts. These included Drs. Makoto Hayashi (Biosafety Research Center, Foods, Drugs and Pesticides, Japan), Hajime Kojima (Japanese Center for the Validation of Alternative Methods, Japan), Raffaella Corvi (European Center for the Validation of Alternative Methods, Italy), Takeshi Morita (National Institute of Health Sciences, Japan), William Stokes (Interagency Coordinating Committee on the Validation of Alternative Methods, USA), Leonard Schechtman (Innovative Toxicology Consulting, USA), Abby Jacobs (Food and Drug Administration, USA) and Sebastian Hoffmann (seh consulting + services, Germany). References [1] L.G. Hernández, H. van Steeg, M. Luijten, J. van Benthem, Mechanisms of nongenotoxic carcinogens and importance of a weight of evidence approach, Mutat. Res. 682 (2009) 94–109. [2] R.J. Pienta, J.A. Poiley, W.B. Lebherz III, Morphological transformation of early passage golden Syrian hamster embryo cells derived from cryopreserved primary cultures as a reliable in vitro bioassay for identifying diverse carcinogens, Int. J. Cancer 19 (1977) 642–655. [3] K. Kakunaga, H. Yamasaki (Eds.), Transformation Assay of Established Cell Lines: Mechanisms and Application, IARC Scientific Publication No. 67, IARC, Lyon, 1985. [4] R. Montesano, H. Bartsch, H. Vainio, J. Wilbourn, H. Yamasaki (Eds.), Long-term and Short-term Assays for Carcinogens: A Critical Appraisal, IARC Scientific Publication No. 83, IARC, Lyon, 1986. [5] A. Sakai, BALB/c 3T3 cell transformation assays for the assessment of chemical carcinogenicity, AATEX 13 (Special Issue) (2008) 367–373.

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