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Genotoxicity Tests ond -Tagatose
Regulatory Toxicology and Pharmacology 29, S36 –S42 (1999) Article ID rtph.1998.1263, available online at http://www.idealibrary.com on
Genotoxicity Tests on D-Tagatose Claire L. Kruger, 1 Margaret H. Whittaker, and Vasilios H. Frankos Environ Corporation, 4350 N. Fairfax Drive, Arlington, Virginia
D-Tagatose
is a low-calorie sweetener that tastes like sucrose. Its genotoxic potential was examined in five standard assays: the Ames Salmonella typhimurium reverse mutation assay, the Escherichia coli/mammalian microsome assay, a chromosomal aberration assay in Chinese hamster ovary cells, a mouse lymphoma forward mutation assay, and an in vivo mouse micronucleus assay. D-Tagatose was not found to increase the number of revertants per plate relative to vehicle controls in either the S. typhimurium tester strains or the WP2uvrA2 tester strain with or without metabolic activation at doses up to 5000 mg/plate. No significant increase in Chinese hamster ovary cells with chromosomal aberrations was observed at concentrations up to 5000 mg/ml with or without metabolic activation. D-Tagatose was not found to increase the mutant frequency in mouse lymphoma L5178Y cells with or without metabolic activation up to concentrations of 5000 mg/ml. D-Tagatose caused no significant increase in micronuclei in bone marrow polychromatic erythrocytes at doses up to 5000 mg/kg. D-Tagatose was not found to be genotoxic under the conditions of any of the assays described above. © 1999 Academic Press
INTRODUCTION D-Tagatose is a ketohexose in which its fourth carbon is chiral and is a mirror image of the respective carbon atom of the common D-sugar, fructose. D-Tagatose is not digested or absorbed to any large extent and, thus, most of the sugar passes into the colon where it is fermented by the colonic microflora. It tastes like sucrose and is useful as a low-calorie sweetener. Its genotoxic potential was assessed using the following standard assays: the Ames Salmonella typhimurium reverse mutation assay, the Escherichia coli/mammalian microsome assay, the chromosomal aberrations in Chinese hamster ovary cells assay, the mouse lymphoma forward mutation assay, and the in vivo mouse micronucleus assay.
MATERIALS AND METHODS
Ames S. typhimurium Reverse Mutation Assay The mutagenic potential of D-tagatose was evaluated in the Ames S. typhimurium reverse mutation assay using 1
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the histidine auxotrophs TA98, TA100, TA1535, TA1537, and TA1538, with and without metabolic activation. Testing was performed by Covance Laboratories, Inc. (Madison, WI). The tester strains were exposed to Dtagatose via the plate incorporation methodology originally described by Ames et al. (1975) and Maron and Ames (1983). The liver microsomal enzyme mixture (S9 mix) was purchased from Molecular Toxicology, Inc. (Annapolis, MD). The positive controls consisted of 2-aminoanthracene (Sigma Chemical Co., practical grade), 2-nitrofluorene (Aldrich Chemical Co., 98%), sodium azide (Sigma Chemical Co., practical grade), ICR-191 (Polysciences, Inc., .95% pure), and 4-nitroquinoline-N-oxide (Sigma Chemical Co., practical grade). The vehicle control consisted of deionized water. The negative control consisted of a 100-ml aliquot of the appropriate tester strain and a 500-ml aliquot of S9 mix (when appropriate), on selective agar. Responses observed in the assays were evaluated as follows: (a) for a test article to be considered positive in tester strains TA98 and TA100, at least a twofold increase was required in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article. (b) For a test article to be considered positive in tester strains TA1535, TA1537, and TA1538, it had to produce at least a threefold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article. The final test was conducted in duplicate using three plates per dose in the presence and absence of the S9 mix. The final doses of D-tagatose were selected based on the results of a range-finding study using strain TA100 both in the presence and absence of S9 mix. For the final test, six doses of the test article at concentrations from 100 to 5000 mg/plate were tested along with the appropriate vehicle controls, positive controls and negative controls. E. coli/Mammalian-Microsome Reverse Mutation Assay The mutagenic potential of D-tagatose was evaluated in the E. coli/Microsome Reverse Mutation Assay using
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GENOTOXICITY TESTS ON
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TABLE 1 Salmonella typhimurium Mean Revertant Colonies (Number of Revertant Colonies on Each Plate) TA98 Test I
Dose/plate Vehicle control Negative control D-tagatose 100 mg 333 mg 667 mg 1000 mg 3330 mg 5000 mg Positive control a Positive control b
TA100 Test II
Test I
TA1535 Test II
Test I
TA1537
Test II
Test I
TA1538
Test II
Test I
S9 (1)
S9 (2)
S9 (1)
S9 (2)
S9 (1)
S9 (2)
S9 (1)
S9 (2)
S9 (1)
S9 (2)
S9 (1)
S9 (2)
S9 (1)
S9 (2)
S9 (1)
S9 (2)
20
18
25
11
100
105
103
85
12
11
14
7
6
6
8
7
21
15
27
11
112
87
91
83
9
13
12
13
7
4
17
24 29 25 25 31 18
13 16 15 15 16 11
24 24 28 21 17 20
13 18 13 13 16 8
98 95 104 99 108 116
93 93 87 96 98 99
104 115 116 115 113 105
76 84 85 86 84 85
7 10 12 10 9 13
9 10 10 12 10 12
12 8 13 13 11 13
11 10 6 11 8 9
6 7 7 5 10 9
8 5 5 7 5 8
5 8 6 6 5 6
976
—
789
—
1235
—
913
—
109
—
109
—
160
—
129
—
—
136
—
160
—
477
—
421
—
340
—
332
—
300
—
220
S9 (1)
Test II
S9 (2)
S9 (1)
S9 (2)
15
8
18
8
6
15
8
8
9
4 3 4 7 5 4
17 15 19 15 15 16
9 15 10 11 8 10
15 17 19 16 15 14
5 8 7 9 8 9
1209
—
998
—
—
225
—
228
Positive control (all strains): 2-aminoanthracene (2.5 mg/plate). Positive control: TA98, 2-nitrofluorene (1.0 mg/plate); TA100; sodium azide (2.0 mg/plate); TA1535, sodium azide (2.0 mg/plate); TA1537, ICR-191 (2.0 mg/plate); TA1535, 2-nitrofluorene (1.0 mg/plate). a b
the tryptophan auxotroph WP2uvrA-, with and without metabolic activation. Testing was performed by Covance Laboratories, Inc. The liver microsomal enzyme mixture (S9 mix) was purchased from Molecular Toxicology, Inc. Positive control chemicals consisted of 2-aminoanthracene (Sigma Chemical Co., practical grade) and 4-nitroquinoline-N-oxide (Sigma Chemical Co., practical grade). The vehicle control consisted of deionized water. Based on the results of a dose range-finding study with tester strain WP2uvrA2 in the presence and absence of metabolic activation (S9 mix), six doses were selected for the final assay at concentrations of 100 to 5000 mg/plate. For a test article to be considered positive, it had to produce at least a twofold increase in the mean revertants per plate over the mean revertants per plate of the appropriate vehicle control. The increase in the mean number of revertants per plate also had to demonstrate a positive dose response. The final assay was performed in duplicate and was conducted using three plates per dose in the presence and absence of the S9 mix. Chromosomal Aberrations in Chinese Hamster Ovary Cells This in vitro assay measured the potential of D-tagatose to cause chromosomal aberrations in Chinese hamster ovary cells with and without metabolic activation. Testing was performed by Covance Laboratories, Inc. The Chinese hamster ovary cells used in this assay were from a permanent cell line and were origi-
nally obtained from the laboratory of Dr. S. Wolff, University of California, San Francisco. Mitomycin C (MMC) served as the positive control for the direct (nonactivated) assays. Cyclophosphamide (CP) served as the positive control for the metabolically activated assays. Both were purchased from Sigma Chemical Corp. Based on the results of an initial range-finding assay with and without metabolic activation, four concentra-
TABLE 2 Escherichia coli Tester Strain WP2uvrA2 Mean Revertant Ccolonies (Number of Revertant Colonies per Plate) Test I
Dose/plate Vehicle control Negative control D-Tagatose 100 mg 333 mg 667 mg 1000 mg 3330 mg 5000 mg Positive control a Positive control b a b
Test II
S9 (1)
S9 (2)
S9 (1)
S9 (2)
18 24
24 14
18 22
12 16
21 23 21 27 22 21 281 —
23 20 17 24 15 8 — 276
23 16 20 19 18 14 386 —
21 18 13 19 23 17 — 516
Aminoanthracene (both tests): (25.0 mg/plate). 4-Nitroquinoline-N-oxide (both tests): (10.0 mg/plate).
13 7 5 10 5 8 7 14 2 5 6 7
2 2 2 2 2 1 1 1 1 1 1
25
200 200 200 200
200
25
200 200 200 200
TG
1
1
1
1
1
1
SG
2 1 1
1
1
2
UC
Not computed
–
S9 mix
200
Cells scored
1
1
7
2
TB
1
1
4
SB
Simple
2
DM
6
4
ID
4
1
1
2
TR
2
6
QR
CR
1 1 1 1
1
3 2 2
3
D
Complex
1
R
1
1
CI
DF
1
Other: GT
0.01 0.01 0.01 0.01
0.96
0.01
0.02 0.02 0.01 0.02
0.96
0.04
No. of aberrations per cell
1.0 0.5 1.0 0.5
52.0*
0.5
1.5 1.5 1.0 1.0
52.0*
2.5
% of cells with aberrations
0.0 0.0 0.0 0.0
12.0*
0.0
0.0 0.0 0.0 0.5
28.0*
1.0
% of cells with .1 aberrations
Note. MMC, mitomycin c; CP, cyclophosphamide; TG, chromatid gap; SG, chromosome gap; UCC, uncoiled chromosome; TB, chromatid break; SB, chromosome break; DM, “double minute” fragment; ID, interstitial deletion; TR, triradial; QR, quadriradial; CR, complex rearrangement; D, decentric; R, ring; CI, chromosome interchange; DF, dicentric with fragment; GT, cell with .10 aberrations. * Significantly greater than the pooled negative and solvent controls, P , 0.01.
Negative and solvent controls (McCoy’s 5a and sterile deionized water) Positive control: MMC 1.0 mg/ml D-Tagatose 1250 mg/ml 2500 mg/ml 3750 mg/ml 5000 mg/ml Negative and solvent controls Positive control: CP 25.0 mg/ml D-tagatose 1250 mg/ml 2500 mg/ml 3750 mg/ml 5000 mg/ml
Compounds/dose (mg/ml)
Number and type of aberration
TABLE 3 Effect of D-Tagatose on Chromosome Aberrations in CHO Cells (Results Pooled from Duplicate Cultures)
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TABLE 4 L5178Y TK 1/2 Mouse Lymphoma Forward Mutation Assay: Without S9 Activation
Test article
Daily cell counts (cells/ml, 10 5 units)
Vehicle control e Vehicle control e Vehicle control e
14.1 15.3 17.5
16.2 15.4 16.7
MMS 10 nl/ml MMS 15 nl/ml
11.4 10.1
14.7 10.6
14.3 14.5 16.0 13.7 15.1 10.5
16.0 15.9 14.2 17.0 17.2 19.3
D-Tagatose
500 mg/ml 1000 mg/ml 2000 mg/ml 3000 mg/ml 4000 mg/ml 5000 mg/ml
Suspension growth a 25.4 26.2 32.5 Avg. vehicle control (28.0) 18.6 11.9 Relative to vehicle control (%) 90.8 91.5 90.2 92.4 103.1 80.4
Total mutant colonies
Total viable colonies
96 70 89
621 616 524
517 511
352 280
90 107 87 84 67 85
562 612 593 523 454 530
Cloning efficiency b 103.5 102.7 87.3 Avg. vehicle control (97.8) 58.7 46.7 Relative to vehicle control (%) 95.8 104.3 101.1 89.1 77.4 90.3
Relative growth (%) c
Mutant frequency (10 26 units) d
100.0 100.0 100.0
39.9 20.3
30.9 22.7 34.0 Avg. vehicle control (29.2) 298.8 f 365.0 f
87.0 95.4 91.2 82.3 79.8 72.6
32.0 35.0 29.3 32.1 29.5 32.1
Note. Decimal is moved to express the frequency in units of 10 26. a Suspension growth 5 (Day 1 count/3) 3 (Day 2 count)/(3 or Day 1 count if not split back). b Cloning efficiency 5 (Total viable colony count/number of cells seeded) 3 100. c Relative growth 5 (Relative suspension growth 3 relative cloning efficiency)/100. d Mutant frequency 5 (Total mutant colonies/total viable colonies) 3 (2 3 10 24). e Vehicle control 5 10% H 2O; MMS, methyl methanesulfonate; MCA, methylcholanthrene. f Mutagenic, exceeds minimum criterion of 58.4 3 10 26.
tions were selected for the main assay (1250, 2500, 3750, and 5000 mg/ml). In the main assay without metabolic activation, cultures were initiated by seeding approximately 1.5 3 10 6 cells per 75-cm 2 flask into 10 ml of complete McCoy’s 5a medium. One day after culture initiation, the cultures were treated with Dtagatose at predetermined doses for 7.42 h. The cultures were then washed with buffered saline and reincubated in complete McCoy’s 5a medium with 0.1 mg/ml Colcemid present for the last 2.5 h of incubation. The cells were harvested and air-dried slides were made. The slides were then stained in 5% Giemsa solution for the analysis of chromosomal aberrations. In the main assay with metabolic activation, cultures were initiated by seeding approximately 1.5 3 10 6 cells per 75-cm 2 flask into 10 ml of complete McCoy’s 5a medium. One day after culture initiation, the cultures were incubated at 37°C for 2 h in the presence of the test article and the S9 mixture in McCoy’s 5a medium without fetal calf serum. After the 2-h exposure period, the cells were washed twice with buffered saline and complete McCoy’s 5a medium was added to the cells. Cells were incubated for an additional 7.83 h with 0.1 mg/ml Colcemid present during the last 2.5 h of incubation. The cultures were then harvested, and slides were prepared and stained in 5% Giemsa solution. Cells were selected for good morphology and only cells with the number of centromeres equal to the modal number (21 6 2) were analyzed. One hundred cells from each replicate culture at four dose levels of the
test article and from each of the negative and solvent control cultures were analyzed for the different types of chromosomal aberrations (Evans, 1962). At least 25 cells were analyzed for chromosomal aberrations from one of the positive control cultures. For control of bias, all slides except for the positive controls were coded prior to analysis. Chromatid and isochromatid gaps, if observed, were noted in the raw data and were tabulated. They were not, however, considered in the evaluation of the ability of D-tagatose to induce chromosomal aberrations because they may not represent true chromosomal breaks and may possibly be induced by toxicity. Statistical analysis included use of Fisher’s exact test with an adjustment for multiple comparisons (Sokal and Rohlf, 1981) to compare the percentage of cells with aberrations in each treatment group with the results from the pooled solvent and negative controls. A linear trend test (Armitage, 1971) of increasing number of cells with aberrations with increasing dose was also performed. Test article significance was established where P , 0.01. Mouse Lymphoma Forward Mutation Assay The objective of this in vitro assay was to evaluate the ability of D-tagatose to induce forward mutations at the thymidine kinase (TK) locus in the mouse lymphoma L5178Y cell line. Testing was performed by Covance Laboratories, Inc. The assay was based on
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KRUGER, WHITTAKER, AND FRANKOS
TABLE 5 L5178Y TK 1/2 Mouse Lymphoma forward Mutation Assay: With S9 Activation
Test article
Daily cell counts (cells/ml, 10 5 units)
Vehicle control e Vehicle control e Vehicle control e
15.1 18.1 14.9
14.4 14.5 15.5
MCA (2 mg/ml) MCA (4 mg/ml)
7.7 6.4
12.9 8.7
14.3 16.0 14.6 15.4 15.4 16.3
17.9 15.0 13.8 12.3 15.0 13.6
D-Tagatose
500 mg/ml 1000 mg/ml 2000 mg/ml 3000 mg/ml 4000 mg/ml 5000 mg/ml
Suspension growth a 24.2 29.2 25.7 Avg. vehicle control (26.4) 11.0 6.2 Relative to vehicle control (%) 107.7 101.0 84.8 79.7 97.2 93.3
Total mutant colonies
Total viable colonies
130 92 99
606 566 611
755 587
491 200
93 87 93 98 112 139
501 534 641 645 547 635
Cloning efficiency b 101.0 94.3 101.8 Avg. vehicle control (99.0) 81.8 33.3 Relative to vehicle control (%) 84.3 89.9 107.9 108.6 92.1 106.9
Relative growth (%) c
Mutant frequency (10 26 units) d
100.0 100.0 100.0
34.4 7.9
42.9 32.5 32.4 Avg. vehicle control (35.9) 307.5 f 587.0 f
90.8 90.8 91.5 86.6 89.5 99.7
37.1 32.6 29.0 30.4 41.0 43.8
Note. Decimal is moved to express the frequency in units of 10 26. a Suspension growth 5 (Day 1 count/3) 3 (Day 2 count)/(3 or Day 1 count if not split back). b Cloning efficiency 5 (Total viable colony count/number of cells seeded) 3 100. c Relative growth 5 (Relative suspension growth 3 relative cloning efficiency)/100. d Mutant frequency 5 (Total mutant colonies/total viable colonies) 3 (2 3 10 24). e Vehicle control 5 10% H 2O; MMS, methyl methanesulfonate; MCA, methylcholanthrene. f Mutagenic, exceeds minimum criterion of 71.9 3 10 26.
that reported by Clive and Spector (1975), Clive et al. (1979), Amacher et al. (1980), and Clive et al. (1987). The mouse lymphoma L5178Y cell line, heterozygous at the TK locus was obtained from Dr. D Clive (Burroughs Wellcome Co., Research Triangle Park, NC). Methyl methanesulfonate (MMS) (purchased from Kodak Chemical Co.) was used as a positive control chemical for the nonactivated assays. 3-Methylcholanthrene (MCA) (purchased from Sigma Chemical Co.) was used as a positive control chemical for the activated assays at 2.0 and 4.0 mg/ml. The in vitro metabolic activation system consisted of two parts: (a) rat liver enzymes that had been obtained from male Sprague–Dawley rats treated with 500 mg/kg of Aroclor 1254 (S9 mix) and (b) an energy producing system (CORE) consisting of nicotinamide adenine dinucleotide phosphate (NADP, sodium salt) and isocitrate. Doses were selected based on the results of a dose range-finding test that involved doses ranging from 9.77 to 5000 mg/ml, performed with and without metabolic activation. The assay conditions consisted of three vehicle controls, two positive controls, and six different test material dose levels using one culture per dose level. A standard expression period of two days was used to allow recovery, growth and expression of the TK 2/2 phenotype. For both the activated and nonactivated assay, a total sample size of 3 3 10 6 cells was suspended in selection medium to selectively recover mutants. Each total sample was distributed into three
100-mm dishes so that each dish contained approximately 1 3 10 6 cells. All of the dishes were placed in a humidified incubator at approximately 37°C with approximately 5% CO 2 and 95% air. After 10 to 14 days in the incubator, the colonies were counted on an Arteck Model 880 colony counter. Test articles were evaluated on the basis of a combination of a minimum increase in mutant frequency and a series of assay evaluation criteria that take into account individual assay variability. First, the minimum criterion considered necessary to demonstrate mutagenesis was a mutant frequency that was $ 2 times the concurrent background mutant frequency. The background mutant frequency was defined as the average mutant frequency of the vehicle control cultures. Second, a dose-related or toxicity-related increase in mutant frequency must have been observed; however, treatments that induced less than 10% relative growth were not used as primary evidence for mutagenicity. In Vivo Mouse Micronucleus Assay The objective of the in vivo mouse micronucleus assay was to determine whether D-tagatose induced micronuclei in polychromatic erythrocytes from bone marrow of CD-1 (ICR) mice. Testing was performed by Covance Laboratories, Inc. This assay was conducted using modifications of the procedures suggested by Heddle et al. (1983). Male and female CD-1 mice were
GENOTOXICITY TESTS ON
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D-TAGATOSE
TABLE 6 Micronucleus Assay % of micronucleated PCEs (mean of 1000 per animal 6 SE) Treatment
Dose
Sterile deionized water CP D-Tagatose
10 ml/kg 80 mg/kg 1250 mg/kg
2500 mg/kg
5000 mg/kg
Ratio PCE:NCE (mean 6 SE)
Harvest time (h)
Males
Females
Total
Males
Females
24 24 24 48 72 24 48 72 24 48 72
0.04 6 0.02 1.88 6 0.17* 0.06 6 0.04 0.02 6 0.02 0.04 6 0.02 0.02 6 0.02 0.04 6 0.02 0.04 6 0.02 0.04 6 0.02 0.02 6 0.02 0.04 6 0.02
0.04 6 0.02 2.32 6 0.52* 0.06 6 0.02 0.04 6 0.02 0.04 6 0.04 0.06 6 0.02 0.00 6 0.00 0.04 6 0.04 0.00 6 0.00 0.02 6 0.02 0.02 6 0.02
0.04 6 0.02 2.10 6 0.27* 0.06 6 0.02 0.03 6 0.02 0.04 6 0.02 0.04 6 0.02 0.02 6 0.01 0.04 6 0.02 0.02 6 0.01 0.02 6 0.01 0.03 6 0.02
0.87 6 0.18 0.49 6 0.15 0.70 6 0.07 0.63 6 0.09 0.63 6 0.13 0.94 6 0.08 0.76 6 0.03 0.57 6 0.14 0.96 6 0.12 0.74 6 0.12 0.49 6 0.12
0.63 6 0.17 0.42 6 0.12 0.96 6 0.40 0.59 6 0.10 1.09 6 0.14 0.69 6 0.15 0.78 6 0.13 0.75 6 0.10 0.59 6 0.13 1.00 6 0.03 1.00 6 0.03
Note. CP, cyclophosphamide. * Significantly greater than the corresponding vehicle control, P , 0.05.
administered D-tagatose via oral gavage. CP was used as a positive control at 80 mg/kg, whereas sterile deionized water was used as the vehicle control at 10 ml/kg. D-Tagatose dose levels of 1250, 2500, and 5000 mg/kg were selected based on the results of an acute oral toxicity study that indicated mice could tolerate doses as high as 10,000 mg/kg. The D-tagatose-dosed animals were euthanized approximately 24, 48, and 72 h after test article administration. The positive and vehicle control animals were euthanized approximately 24 h after administration of the control articles. At the appropriate harvest time, the animals were euthanized with CO 2 and the adhering soft tissue and epiphyses of both femora were removed. Aspirated marrow was mixed with approximately 0.1 ml fetal calf serum to form a suspension. The cells were then placed on slides and air-dried, fixed in methanol, and stained in May–Grunwald solution followed by Giemsa (Schmid, 1975). The slides were then scored for micronuclei and the polychromatic (PCE) to normochromatic (NCE) cell ratio. One thousand PCEs per animal were scored. The frequency of micronucleated cells was expressed as a percentage of micronucleated cells based on the total PCEs present in the scored optic field. The criteria for the identification of micronuclei were those of Schmid (1976). The unit of scoring was the micronucleated cell, not the micronucleus; the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The criteria for determining a positive response involved a statistically significant dose-related increase in micronucleated PCEs, or the detection of a reproducible and statistically significant positive response for at least one dose level. The analysis of the data was performed using an analysis of variance (ANOVA) on the square root of the arcsine transformation that was performed on the pro-
portion of cells with micronuclei per animal (square root arcsine proportion). Once the ANOVA had been performed, Tukey’s Studentized range test (HSD) with adjustment for multiple comparisons (Sokal and Rohlf, 1981) was used at each harvest time to determine which dose groups, if any, were significantly different (P , 0.05) from the vehicle control. RESULTS
As detailed in Tables 1 and 2, D-tagatose was not found to increase the number of revertants per plate relative to vehicle controls in either the S. typhimurium tester strains or the WP2uvrA2 tester strain in the presence or absence of the S9 mix. Adequate increases in the number of reverse mutation colonies were identified in the positive controls relative to the vehicle control in the absence of the S9 mix in both the S. typhimurium tester strains and the WP2uvrA2 tester strain, demonstrating that the tester strains were capable of identifying a mutagen. Adequate increases in the number of reverse mutation colonies were identified in the positive controls relative to the vehicle control in the presence of the S9 mix in both the S. typhimurium tester strains and the WP2uvrA2 tester strain, demonstrating the S9 mix was capable of metabolizing a promutagen to its mutagenic form(s). As detailed in Table 3, no significant increase in cells with chromosomal aberrations was observed at the concentrations analyzed with or without metabolic activation. The sensitivity of the cell culture for induction of chromosomal aberrations was shown by the increased frequency of aberrations in the cells exposed to the positive control agents, mitomycin and cyclophosphamide. D-Tagatose was not found to increase the mutant frequency in mouse lymphoma L5178Y cells in
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KRUGER, WHITTAKER, AND FRANKOS
the presence or absence of S9 mix at any of the dose levels. These data are presented in Tables 4 and 5. D-Tagatose caused no significant increase in micronuclei in bone marrow polychromatic erythrocytes at any of the dose levels. These data are presented in Tables 5 and 6. CONCLUSION D-Tagatose was not found to be genotoxic under the conditions of any of the assays described above.
REFERENCES Amacher, D. E., Paillet, S. C., Turner, G. N., Ray, V. A., and Salsburg, D. S. (1980). Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. II. Test validation and interpretation. Mutat. Res. 72, 447– 474. Ames, B. N., McCann, J., and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res. 31, 347–364. Armitage, P. (1971). Statistical Methods in Medical Research. Wiley, New York.
Clive, D., Johnson, K. O., Spector, J. F. S., Batson, A. G. and Brown, M. M. M. (1979). Validation and characterization of the L5178Y/ TK1/2 mouse lymphoma mutagen assay system. Mutat. Res. 59, 61–108. Clive. D., Caspery, W., Kirby, P. E., Krehl, R., Moore, M., Mayo, J., and Overly, T. J. (1987). Guide for performing the mouse lymphoma assay for mammalian cell mutagenicity. Mutat. Res. 189, 143–156. Clive, D., and Spector, J. F. S. (1975). Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells. Mutat. Res. 31, 17–29. Evans, H. J. (1962). Chromosomal aberrations produced by ionizing radiation. Int. Rev. Cytol. 13, 221–321. Heddle, J. A., Hite, M., Kirkhart, B., Larsen, K., MacGregor, J. T., Newell, G. W., and Salamone, M. F. (1983). The induction of micronuclei as a measure of genotoxicity. Mutat. Res. 123, 61–118. Maron, D. M., and Ames, B. N. (1983). Revised methods for the Salmonella mutagenicity test. Mutat. Res. 113, 173–215. Schmid, W. (1975). The micronucleus test. Mutat. Res. 31, 9 –15. Schmid, W. (1976). The micronucleus test for cytogenic analysis. In Chemical Mutagens: Principles and Methods for Their Detection (A. Hollaender, Ed.), Vol. 4, pp. 31–53. Plenum, New York. Sokal, R. R., and Rohlf, F. J. (1981). Biometry, 2nd ed. Freeman, New York.
Genotoxicity Tests on D-Tagatose Claire L. Kruger, 1 Margaret H. Whittaker, and Vasilios H. Frankos Environ Corporation, 4350 N. Fairfax Drive, Arlington, Virginia
D-Tagatose
is a low-calorie sweetener that tastes like sucrose. Its genotoxic potential was examined in five standard assays: the Ames Salmonella typhimurium reverse mutation assay, the Escherichia coli/mammalian microsome assay, a chromosomal aberration assay in Chinese hamster ovary cells, a mouse lymphoma forward mutation assay, and an in vivo mouse micronucleus assay. D-Tagatose was not found to increase the number of revertants per plate relative to vehicle controls in either the S. typhimurium tester strains or the WP2uvrA2 tester strain with or without metabolic activation at doses up to 5000 mg/plate. No significant increase in Chinese hamster ovary cells with chromosomal aberrations was observed at concentrations up to 5000 mg/ml with or without metabolic activation. D-Tagatose was not found to increase the mutant frequency in mouse lymphoma L5178Y cells with or without metabolic activation up to concentrations of 5000 mg/ml. D-Tagatose caused no significant increase in micronuclei in bone marrow polychromatic erythrocytes at doses up to 5000 mg/kg. D-Tagatose was not found to be genotoxic under the conditions of any of the assays described above. © 1999 Academic Press
INTRODUCTION D-Tagatose is a ketohexose in which its fourth carbon is chiral and is a mirror image of the respective carbon atom of the common D-sugar, fructose. D-Tagatose is not digested or absorbed to any large extent and, thus, most of the sugar passes into the colon where it is fermented by the colonic microflora. It tastes like sucrose and is useful as a low-calorie sweetener. Its genotoxic potential was assessed using the following standard assays: the Ames Salmonella typhimurium reverse mutation assay, the Escherichia coli/mammalian microsome assay, the chromosomal aberrations in Chinese hamster ovary cells assay, the mouse lymphoma forward mutation assay, and the in vivo mouse micronucleus assay.
MATERIALS AND METHODS
Ames S. typhimurium Reverse Mutation Assay The mutagenic potential of D-tagatose was evaluated in the Ames S. typhimurium reverse mutation assay using 1
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the histidine auxotrophs TA98, TA100, TA1535, TA1537, and TA1538, with and without metabolic activation. Testing was performed by Covance Laboratories, Inc. (Madison, WI). The tester strains were exposed to Dtagatose via the plate incorporation methodology originally described by Ames et al. (1975) and Maron and Ames (1983). The liver microsomal enzyme mixture (S9 mix) was purchased from Molecular Toxicology, Inc. (Annapolis, MD). The positive controls consisted of 2-aminoanthracene (Sigma Chemical Co., practical grade), 2-nitrofluorene (Aldrich Chemical Co., 98%), sodium azide (Sigma Chemical Co., practical grade), ICR-191 (Polysciences, Inc., .95% pure), and 4-nitroquinoline-N-oxide (Sigma Chemical Co., practical grade). The vehicle control consisted of deionized water. The negative control consisted of a 100-ml aliquot of the appropriate tester strain and a 500-ml aliquot of S9 mix (when appropriate), on selective agar. Responses observed in the assays were evaluated as follows: (a) for a test article to be considered positive in tester strains TA98 and TA100, at least a twofold increase was required in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article. (b) For a test article to be considered positive in tester strains TA1535, TA1537, and TA1538, it had to produce at least a threefold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article. The final test was conducted in duplicate using three plates per dose in the presence and absence of the S9 mix. The final doses of D-tagatose were selected based on the results of a range-finding study using strain TA100 both in the presence and absence of S9 mix. For the final test, six doses of the test article at concentrations from 100 to 5000 mg/plate were tested along with the appropriate vehicle controls, positive controls and negative controls. E. coli/Mammalian-Microsome Reverse Mutation Assay The mutagenic potential of D-tagatose was evaluated in the E. coli/Microsome Reverse Mutation Assay using
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TABLE 1 Salmonella typhimurium Mean Revertant Colonies (Number of Revertant Colonies on Each Plate) TA98 Test I
Dose/plate Vehicle control Negative control D-tagatose 100 mg 333 mg 667 mg 1000 mg 3330 mg 5000 mg Positive control a Positive control b
TA100 Test II
Test I
TA1535 Test II
Test I
TA1537
Test II
Test I
TA1538
Test II
Test I
S9 (1)
S9 (2)
S9 (1)
S9 (2)
S9 (1)
S9 (2)
S9 (1)
S9 (2)
S9 (1)
S9 (2)
S9 (1)
S9 (2)
S9 (1)
S9 (2)
S9 (1)
S9 (2)
20
18
25
11
100
105
103
85
12
11
14
7
6
6
8
7
21
15
27
11
112
87
91
83
9
13
12
13
7
4
17
24 29 25 25 31 18
13 16 15 15 16 11
24 24 28 21 17 20
13 18 13 13 16 8
98 95 104 99 108 116
93 93 87 96 98 99
104 115 116 115 113 105
76 84 85 86 84 85
7 10 12 10 9 13
9 10 10 12 10 12
12 8 13 13 11 13
11 10 6 11 8 9
6 7 7 5 10 9
8 5 5 7 5 8
5 8 6 6 5 6
976
—
789
—
1235
—
913
—
109
—
109
—
160
—
129
—
—
136
—
160
—
477
—
421
—
340
—
332
—
300
—
220
S9 (1)
Test II
S9 (2)
S9 (1)
S9 (2)
15
8
18
8
6
15
8
8
9
4 3 4 7 5 4
17 15 19 15 15 16
9 15 10 11 8 10
15 17 19 16 15 14
5 8 7 9 8 9
1209
—
998
—
—
225
—
228
Positive control (all strains): 2-aminoanthracene (2.5 mg/plate). Positive control: TA98, 2-nitrofluorene (1.0 mg/plate); TA100; sodium azide (2.0 mg/plate); TA1535, sodium azide (2.0 mg/plate); TA1537, ICR-191 (2.0 mg/plate); TA1535, 2-nitrofluorene (1.0 mg/plate). a b
the tryptophan auxotroph WP2uvrA-, with and without metabolic activation. Testing was performed by Covance Laboratories, Inc. The liver microsomal enzyme mixture (S9 mix) was purchased from Molecular Toxicology, Inc. Positive control chemicals consisted of 2-aminoanthracene (Sigma Chemical Co., practical grade) and 4-nitroquinoline-N-oxide (Sigma Chemical Co., practical grade). The vehicle control consisted of deionized water. Based on the results of a dose range-finding study with tester strain WP2uvrA2 in the presence and absence of metabolic activation (S9 mix), six doses were selected for the final assay at concentrations of 100 to 5000 mg/plate. For a test article to be considered positive, it had to produce at least a twofold increase in the mean revertants per plate over the mean revertants per plate of the appropriate vehicle control. The increase in the mean number of revertants per plate also had to demonstrate a positive dose response. The final assay was performed in duplicate and was conducted using three plates per dose in the presence and absence of the S9 mix. Chromosomal Aberrations in Chinese Hamster Ovary Cells This in vitro assay measured the potential of D-tagatose to cause chromosomal aberrations in Chinese hamster ovary cells with and without metabolic activation. Testing was performed by Covance Laboratories, Inc. The Chinese hamster ovary cells used in this assay were from a permanent cell line and were origi-
nally obtained from the laboratory of Dr. S. Wolff, University of California, San Francisco. Mitomycin C (MMC) served as the positive control for the direct (nonactivated) assays. Cyclophosphamide (CP) served as the positive control for the metabolically activated assays. Both were purchased from Sigma Chemical Corp. Based on the results of an initial range-finding assay with and without metabolic activation, four concentra-
TABLE 2 Escherichia coli Tester Strain WP2uvrA2 Mean Revertant Ccolonies (Number of Revertant Colonies per Plate) Test I
Dose/plate Vehicle control Negative control D-Tagatose 100 mg 333 mg 667 mg 1000 mg 3330 mg 5000 mg Positive control a Positive control b a b
Test II
S9 (1)
S9 (2)
S9 (1)
S9 (2)
18 24
24 14
18 22
12 16
21 23 21 27 22 21 281 —
23 20 17 24 15 8 — 276
23 16 20 19 18 14 386 —
21 18 13 19 23 17 — 516
Aminoanthracene (both tests): (25.0 mg/plate). 4-Nitroquinoline-N-oxide (both tests): (10.0 mg/plate).
13 7 5 10 5 8 7 14 2 5 6 7
2 2 2 2 2 1 1 1 1 1 1
25
200 200 200 200
200
25
200 200 200 200
TG
1
1
1
1
1
1
SG
2 1 1
1
1
2
UC
Not computed
–
S9 mix
200
Cells scored
1
1
7
2
TB
1
1
4
SB
Simple
2
DM
6
4
ID
4
1
1
2
TR
2
6
QR
CR
1 1 1 1
1
3 2 2
3
D
Complex
1
R
1
1
CI
DF
1
Other: GT
0.01 0.01 0.01 0.01
0.96
0.01
0.02 0.02 0.01 0.02
0.96
0.04
No. of aberrations per cell
1.0 0.5 1.0 0.5
52.0*
0.5
1.5 1.5 1.0 1.0
52.0*
2.5
% of cells with aberrations
0.0 0.0 0.0 0.0
12.0*
0.0
0.0 0.0 0.0 0.5
28.0*
1.0
% of cells with .1 aberrations
Note. MMC, mitomycin c; CP, cyclophosphamide; TG, chromatid gap; SG, chromosome gap; UCC, uncoiled chromosome; TB, chromatid break; SB, chromosome break; DM, “double minute” fragment; ID, interstitial deletion; TR, triradial; QR, quadriradial; CR, complex rearrangement; D, decentric; R, ring; CI, chromosome interchange; DF, dicentric with fragment; GT, cell with .10 aberrations. * Significantly greater than the pooled negative and solvent controls, P , 0.01.
Negative and solvent controls (McCoy’s 5a and sterile deionized water) Positive control: MMC 1.0 mg/ml D-Tagatose 1250 mg/ml 2500 mg/ml 3750 mg/ml 5000 mg/ml Negative and solvent controls Positive control: CP 25.0 mg/ml D-tagatose 1250 mg/ml 2500 mg/ml 3750 mg/ml 5000 mg/ml
Compounds/dose (mg/ml)
Number and type of aberration
TABLE 3 Effect of D-Tagatose on Chromosome Aberrations in CHO Cells (Results Pooled from Duplicate Cultures)
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TABLE 4 L5178Y TK 1/2 Mouse Lymphoma Forward Mutation Assay: Without S9 Activation
Test article
Daily cell counts (cells/ml, 10 5 units)
Vehicle control e Vehicle control e Vehicle control e
14.1 15.3 17.5
16.2 15.4 16.7
MMS 10 nl/ml MMS 15 nl/ml
11.4 10.1
14.7 10.6
14.3 14.5 16.0 13.7 15.1 10.5
16.0 15.9 14.2 17.0 17.2 19.3
D-Tagatose
500 mg/ml 1000 mg/ml 2000 mg/ml 3000 mg/ml 4000 mg/ml 5000 mg/ml
Suspension growth a 25.4 26.2 32.5 Avg. vehicle control (28.0) 18.6 11.9 Relative to vehicle control (%) 90.8 91.5 90.2 92.4 103.1 80.4
Total mutant colonies
Total viable colonies
96 70 89
621 616 524
517 511
352 280
90 107 87 84 67 85
562 612 593 523 454 530
Cloning efficiency b 103.5 102.7 87.3 Avg. vehicle control (97.8) 58.7 46.7 Relative to vehicle control (%) 95.8 104.3 101.1 89.1 77.4 90.3
Relative growth (%) c
Mutant frequency (10 26 units) d
100.0 100.0 100.0
39.9 20.3
30.9 22.7 34.0 Avg. vehicle control (29.2) 298.8 f 365.0 f
87.0 95.4 91.2 82.3 79.8 72.6
32.0 35.0 29.3 32.1 29.5 32.1
Note. Decimal is moved to express the frequency in units of 10 26. a Suspension growth 5 (Day 1 count/3) 3 (Day 2 count)/(3 or Day 1 count if not split back). b Cloning efficiency 5 (Total viable colony count/number of cells seeded) 3 100. c Relative growth 5 (Relative suspension growth 3 relative cloning efficiency)/100. d Mutant frequency 5 (Total mutant colonies/total viable colonies) 3 (2 3 10 24). e Vehicle control 5 10% H 2O; MMS, methyl methanesulfonate; MCA, methylcholanthrene. f Mutagenic, exceeds minimum criterion of 58.4 3 10 26.
tions were selected for the main assay (1250, 2500, 3750, and 5000 mg/ml). In the main assay without metabolic activation, cultures were initiated by seeding approximately 1.5 3 10 6 cells per 75-cm 2 flask into 10 ml of complete McCoy’s 5a medium. One day after culture initiation, the cultures were treated with Dtagatose at predetermined doses for 7.42 h. The cultures were then washed with buffered saline and reincubated in complete McCoy’s 5a medium with 0.1 mg/ml Colcemid present for the last 2.5 h of incubation. The cells were harvested and air-dried slides were made. The slides were then stained in 5% Giemsa solution for the analysis of chromosomal aberrations. In the main assay with metabolic activation, cultures were initiated by seeding approximately 1.5 3 10 6 cells per 75-cm 2 flask into 10 ml of complete McCoy’s 5a medium. One day after culture initiation, the cultures were incubated at 37°C for 2 h in the presence of the test article and the S9 mixture in McCoy’s 5a medium without fetal calf serum. After the 2-h exposure period, the cells were washed twice with buffered saline and complete McCoy’s 5a medium was added to the cells. Cells were incubated for an additional 7.83 h with 0.1 mg/ml Colcemid present during the last 2.5 h of incubation. The cultures were then harvested, and slides were prepared and stained in 5% Giemsa solution. Cells were selected for good morphology and only cells with the number of centromeres equal to the modal number (21 6 2) were analyzed. One hundred cells from each replicate culture at four dose levels of the
test article and from each of the negative and solvent control cultures were analyzed for the different types of chromosomal aberrations (Evans, 1962). At least 25 cells were analyzed for chromosomal aberrations from one of the positive control cultures. For control of bias, all slides except for the positive controls were coded prior to analysis. Chromatid and isochromatid gaps, if observed, were noted in the raw data and were tabulated. They were not, however, considered in the evaluation of the ability of D-tagatose to induce chromosomal aberrations because they may not represent true chromosomal breaks and may possibly be induced by toxicity. Statistical analysis included use of Fisher’s exact test with an adjustment for multiple comparisons (Sokal and Rohlf, 1981) to compare the percentage of cells with aberrations in each treatment group with the results from the pooled solvent and negative controls. A linear trend test (Armitage, 1971) of increasing number of cells with aberrations with increasing dose was also performed. Test article significance was established where P , 0.01. Mouse Lymphoma Forward Mutation Assay The objective of this in vitro assay was to evaluate the ability of D-tagatose to induce forward mutations at the thymidine kinase (TK) locus in the mouse lymphoma L5178Y cell line. Testing was performed by Covance Laboratories, Inc. The assay was based on
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KRUGER, WHITTAKER, AND FRANKOS
TABLE 5 L5178Y TK 1/2 Mouse Lymphoma forward Mutation Assay: With S9 Activation
Test article
Daily cell counts (cells/ml, 10 5 units)
Vehicle control e Vehicle control e Vehicle control e
15.1 18.1 14.9
14.4 14.5 15.5
MCA (2 mg/ml) MCA (4 mg/ml)
7.7 6.4
12.9 8.7
14.3 16.0 14.6 15.4 15.4 16.3
17.9 15.0 13.8 12.3 15.0 13.6
D-Tagatose
500 mg/ml 1000 mg/ml 2000 mg/ml 3000 mg/ml 4000 mg/ml 5000 mg/ml
Suspension growth a 24.2 29.2 25.7 Avg. vehicle control (26.4) 11.0 6.2 Relative to vehicle control (%) 107.7 101.0 84.8 79.7 97.2 93.3
Total mutant colonies
Total viable colonies
130 92 99
606 566 611
755 587
491 200
93 87 93 98 112 139
501 534 641 645 547 635
Cloning efficiency b 101.0 94.3 101.8 Avg. vehicle control (99.0) 81.8 33.3 Relative to vehicle control (%) 84.3 89.9 107.9 108.6 92.1 106.9
Relative growth (%) c
Mutant frequency (10 26 units) d
100.0 100.0 100.0
34.4 7.9
42.9 32.5 32.4 Avg. vehicle control (35.9) 307.5 f 587.0 f
90.8 90.8 91.5 86.6 89.5 99.7
37.1 32.6 29.0 30.4 41.0 43.8
Note. Decimal is moved to express the frequency in units of 10 26. a Suspension growth 5 (Day 1 count/3) 3 (Day 2 count)/(3 or Day 1 count if not split back). b Cloning efficiency 5 (Total viable colony count/number of cells seeded) 3 100. c Relative growth 5 (Relative suspension growth 3 relative cloning efficiency)/100. d Mutant frequency 5 (Total mutant colonies/total viable colonies) 3 (2 3 10 24). e Vehicle control 5 10% H 2O; MMS, methyl methanesulfonate; MCA, methylcholanthrene. f Mutagenic, exceeds minimum criterion of 71.9 3 10 26.
that reported by Clive and Spector (1975), Clive et al. (1979), Amacher et al. (1980), and Clive et al. (1987). The mouse lymphoma L5178Y cell line, heterozygous at the TK locus was obtained from Dr. D Clive (Burroughs Wellcome Co., Research Triangle Park, NC). Methyl methanesulfonate (MMS) (purchased from Kodak Chemical Co.) was used as a positive control chemical for the nonactivated assays. 3-Methylcholanthrene (MCA) (purchased from Sigma Chemical Co.) was used as a positive control chemical for the activated assays at 2.0 and 4.0 mg/ml. The in vitro metabolic activation system consisted of two parts: (a) rat liver enzymes that had been obtained from male Sprague–Dawley rats treated with 500 mg/kg of Aroclor 1254 (S9 mix) and (b) an energy producing system (CORE) consisting of nicotinamide adenine dinucleotide phosphate (NADP, sodium salt) and isocitrate. Doses were selected based on the results of a dose range-finding test that involved doses ranging from 9.77 to 5000 mg/ml, performed with and without metabolic activation. The assay conditions consisted of three vehicle controls, two positive controls, and six different test material dose levels using one culture per dose level. A standard expression period of two days was used to allow recovery, growth and expression of the TK 2/2 phenotype. For both the activated and nonactivated assay, a total sample size of 3 3 10 6 cells was suspended in selection medium to selectively recover mutants. Each total sample was distributed into three
100-mm dishes so that each dish contained approximately 1 3 10 6 cells. All of the dishes were placed in a humidified incubator at approximately 37°C with approximately 5% CO 2 and 95% air. After 10 to 14 days in the incubator, the colonies were counted on an Arteck Model 880 colony counter. Test articles were evaluated on the basis of a combination of a minimum increase in mutant frequency and a series of assay evaluation criteria that take into account individual assay variability. First, the minimum criterion considered necessary to demonstrate mutagenesis was a mutant frequency that was $ 2 times the concurrent background mutant frequency. The background mutant frequency was defined as the average mutant frequency of the vehicle control cultures. Second, a dose-related or toxicity-related increase in mutant frequency must have been observed; however, treatments that induced less than 10% relative growth were not used as primary evidence for mutagenicity. In Vivo Mouse Micronucleus Assay The objective of the in vivo mouse micronucleus assay was to determine whether D-tagatose induced micronuclei in polychromatic erythrocytes from bone marrow of CD-1 (ICR) mice. Testing was performed by Covance Laboratories, Inc. This assay was conducted using modifications of the procedures suggested by Heddle et al. (1983). Male and female CD-1 mice were
GENOTOXICITY TESTS ON
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D-TAGATOSE
TABLE 6 Micronucleus Assay % of micronucleated PCEs (mean of 1000 per animal 6 SE) Treatment
Dose
Sterile deionized water CP D-Tagatose
10 ml/kg 80 mg/kg 1250 mg/kg
2500 mg/kg
5000 mg/kg
Ratio PCE:NCE (mean 6 SE)
Harvest time (h)
Males
Females
Total
Males
Females
24 24 24 48 72 24 48 72 24 48 72
0.04 6 0.02 1.88 6 0.17* 0.06 6 0.04 0.02 6 0.02 0.04 6 0.02 0.02 6 0.02 0.04 6 0.02 0.04 6 0.02 0.04 6 0.02 0.02 6 0.02 0.04 6 0.02
0.04 6 0.02 2.32 6 0.52* 0.06 6 0.02 0.04 6 0.02 0.04 6 0.04 0.06 6 0.02 0.00 6 0.00 0.04 6 0.04 0.00 6 0.00 0.02 6 0.02 0.02 6 0.02
0.04 6 0.02 2.10 6 0.27* 0.06 6 0.02 0.03 6 0.02 0.04 6 0.02 0.04 6 0.02 0.02 6 0.01 0.04 6 0.02 0.02 6 0.01 0.02 6 0.01 0.03 6 0.02
0.87 6 0.18 0.49 6 0.15 0.70 6 0.07 0.63 6 0.09 0.63 6 0.13 0.94 6 0.08 0.76 6 0.03 0.57 6 0.14 0.96 6 0.12 0.74 6 0.12 0.49 6 0.12
0.63 6 0.17 0.42 6 0.12 0.96 6 0.40 0.59 6 0.10 1.09 6 0.14 0.69 6 0.15 0.78 6 0.13 0.75 6 0.10 0.59 6 0.13 1.00 6 0.03 1.00 6 0.03
Note. CP, cyclophosphamide. * Significantly greater than the corresponding vehicle control, P , 0.05.
administered D-tagatose via oral gavage. CP was used as a positive control at 80 mg/kg, whereas sterile deionized water was used as the vehicle control at 10 ml/kg. D-Tagatose dose levels of 1250, 2500, and 5000 mg/kg were selected based on the results of an acute oral toxicity study that indicated mice could tolerate doses as high as 10,000 mg/kg. The D-tagatose-dosed animals were euthanized approximately 24, 48, and 72 h after test article administration. The positive and vehicle control animals were euthanized approximately 24 h after administration of the control articles. At the appropriate harvest time, the animals were euthanized with CO 2 and the adhering soft tissue and epiphyses of both femora were removed. Aspirated marrow was mixed with approximately 0.1 ml fetal calf serum to form a suspension. The cells were then placed on slides and air-dried, fixed in methanol, and stained in May–Grunwald solution followed by Giemsa (Schmid, 1975). The slides were then scored for micronuclei and the polychromatic (PCE) to normochromatic (NCE) cell ratio. One thousand PCEs per animal were scored. The frequency of micronucleated cells was expressed as a percentage of micronucleated cells based on the total PCEs present in the scored optic field. The criteria for the identification of micronuclei were those of Schmid (1976). The unit of scoring was the micronucleated cell, not the micronucleus; the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The criteria for determining a positive response involved a statistically significant dose-related increase in micronucleated PCEs, or the detection of a reproducible and statistically significant positive response for at least one dose level. The analysis of the data was performed using an analysis of variance (ANOVA) on the square root of the arcsine transformation that was performed on the pro-
portion of cells with micronuclei per animal (square root arcsine proportion). Once the ANOVA had been performed, Tukey’s Studentized range test (HSD) with adjustment for multiple comparisons (Sokal and Rohlf, 1981) was used at each harvest time to determine which dose groups, if any, were significantly different (P , 0.05) from the vehicle control. RESULTS
As detailed in Tables 1 and 2, D-tagatose was not found to increase the number of revertants per plate relative to vehicle controls in either the S. typhimurium tester strains or the WP2uvrA2 tester strain in the presence or absence of the S9 mix. Adequate increases in the number of reverse mutation colonies were identified in the positive controls relative to the vehicle control in the absence of the S9 mix in both the S. typhimurium tester strains and the WP2uvrA2 tester strain, demonstrating that the tester strains were capable of identifying a mutagen. Adequate increases in the number of reverse mutation colonies were identified in the positive controls relative to the vehicle control in the presence of the S9 mix in both the S. typhimurium tester strains and the WP2uvrA2 tester strain, demonstrating the S9 mix was capable of metabolizing a promutagen to its mutagenic form(s). As detailed in Table 3, no significant increase in cells with chromosomal aberrations was observed at the concentrations analyzed with or without metabolic activation. The sensitivity of the cell culture for induction of chromosomal aberrations was shown by the increased frequency of aberrations in the cells exposed to the positive control agents, mitomycin and cyclophosphamide. D-Tagatose was not found to increase the mutant frequency in mouse lymphoma L5178Y cells in
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KRUGER, WHITTAKER, AND FRANKOS
the presence or absence of S9 mix at any of the dose levels. These data are presented in Tables 4 and 5. D-Tagatose caused no significant increase in micronuclei in bone marrow polychromatic erythrocytes at any of the dose levels. These data are presented in Tables 5 and 6. CONCLUSION D-Tagatose was not found to be genotoxic under the conditions of any of the assays described above.
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Clive, D., Johnson, K. O., Spector, J. F. S., Batson, A. G. and Brown, M. M. M. (1979). Validation and characterization of the L5178Y/ TK1/2 mouse lymphoma mutagen assay system. Mutat. Res. 59, 61–108. Clive. D., Caspery, W., Kirby, P. E., Krehl, R., Moore, M., Mayo, J., and Overly, T. J. (1987). Guide for performing the mouse lymphoma assay for mammalian cell mutagenicity. Mutat. Res. 189, 143–156. Clive, D., and Spector, J. F. S. (1975). Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells. Mutat. Res. 31, 17–29. Evans, H. J. (1962). Chromosomal aberrations produced by ionizing radiation. Int. Rev. Cytol. 13, 221–321. Heddle, J. A., Hite, M., Kirkhart, B., Larsen, K., MacGregor, J. T., Newell, G. W., and Salamone, M. F. (1983). The induction of micronuclei as a measure of genotoxicity. Mutat. Res. 123, 61–118. Maron, D. M., and Ames, B. N. (1983). Revised methods for the Salmonella mutagenicity test. Mutat. Res. 113, 173–215. Schmid, W. (1975). The micronucleus test. Mutat. Res. 31, 9 –15. Schmid, W. (1976). The micronucleus test for cytogenic analysis. In Chemical Mutagens: Principles and Methods for Their Detection (A. Hollaender, Ed.), Vol. 4, pp. 31–53. Plenum, New York. Sokal, R. R., and Rohlf, F. J. (1981). Biometry, 2nd ed. Freeman, New York.