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Evaluation of the genotoxic potential of alkylalkanolamines
Mutation Research 393 Ž1997. 7–15
Evaluation of the genotoxic potential of alkylalkanolamines Hon-Wing Leung ) , Bryan Ballantyne Union Carbide Corporation, 39 Old Ridgebury Road, Danbury, CT 06817-0001, USA
Abstract Three alkylalkanolamines, N,N-dimethylethanolamine, N-methyldiethanolamine, and tert-butyldiethanolamine, were evaluated for potential genotoxic activity using the Salmonellarmicrosome reverse gene mutation test, the CHOrHGPRT forward gene mutation test, a sister chromatid exchange test in cultured CHO cells, and an in vivo peripheral blood micronucleus test in Swiss–Webster mice. None of the three alkylalkanolamines produced any significant or dose-related increases in the frequencies of mutations, sister chromatid exchanges or micronuclei. These results indicate that N,N-dimethylethanolamine, N-methyldiethanolamine, and tert-butyldiethanolamine are not genotoxic in the tests conducted. q 1997 Elsevier Science B.V. Keywords: Alkylalkanolamine; Salmonella reverse gene mutation test; Chinese hamster ovary HGPRT forward gene mutation test; Sister chromatid exchange test; Micronucleus test; Mouse
1. Introduction The alkylalkanolamines are industrial chemicals with widespread applications including flocculant monomers, urethane catalysts, natural gas and boiler water treatment, corrosion inhibitors, can coatings, ion exchange resins, textile lubricants, and pharmaceuticals. Besides some limited mutagenicity data in a bacterial system w1x, no further study of the mutagenic or clastogenic potentials of the alkylalkanolamines has been performed. The present study was undertaken to evaluate the genotoxic potential of three alkylalkanolamines, N,N-dimethylethanolamine ŽDMEA., N-methyldiethanolamine ŽMDEA., and tert-butyldiethanolamine Ž t BDEA.. These com-
) Corresponding author. Tel.: q1 Ž203. 794-5755; fax: q1 Ž203. 794-5275.
pounds were evaluated in the Salmonellarmicrosome reverse gene mutation test, the CHOrHGPRT forward gene mutation test, a sister chromatid exchange test in cultured CHO cells, and an in vivo micronucleus test in Swiss–Webster mice.
2. Materials and methods 2.1. Chemicals The physicochemical data of the three alkylalkanolamines tested are shown in Table 1. Commercial samples were obtained from the Union Carbide Corporation, South Charleston, WV. All samples were analyzed and their chemical purity determined by capillary gas chromatography. Water from a Millipore Milli-Q w purification system was used to prepare solutions and media. Chemicals used as positive
1383-5718r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 7 . 0 0 1 0 4 - 6
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H.-W. Leung, . Ballantyner Mutation Research 393 (1997) 7–15
Table 1 Physicochemical data of alkylalkanolamines tested for genotoxicity potential Alkylalkanolamine
CAS no.
Chemical formula
MW
Purity
N, N-Dimethylethanolamine N-Methyldiethanolamine t-Butyldiethanolamine
108-01-0 105-59-9 2160-93-2
ŽCH 3 . 2 NC 2 H 4 OH CH 3 NŽC 2 H 4 OH. 2 ŽCH 3 . 3 CNŽC 2 H 4 OH. 2
89.1 119.2 161.3
99.9% 99.8% 99.9%
a
a Analytical characterization by capillary gas chromatography for all samples. In addition, the identity of t-butyldiethanolamine was characterized with mass spectrometry and nuclear magnetic resonance spectroscopy.
controls were obtained from Sigma Chemical Company ŽSt. Louis, MO.. 2.2. Salmonellar microsome reÕerse gene mutation test The method described by Ames et al. w2x was followed. Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 were used in this test. At least 4 concentrations spaced at about half-log intervals were tested. The highest concentration was selected from a cytotoxicity test in strain TA100 which produced either a significant ŽG 50%. reduction in the number of revertant colonies or a significant inhibition of background lawn growth. The highest concentration for non-cytotoxic test substances was 10 mgrplate. The activation-independent positive controls were 4-nitrophenylenediamine, sodium azide, and 9-aminoacridine, and the activation-dependent control was 2-aminoanthracene. Frozen S9 liver homogenate, isolated from Aroclor w 1254-induced rats ŽMicrobiological Associates, Bethesda, MD., was thawed and diluted in buffer each day of testing. To a sterile tube containing 2 ml of top agar, 100 ml each of bacterial culture and test chemical were added. Either 0.5 ml of S9 or phosphate-buffered saline was added for tests with and without metabolic activation, respectively. The top agar mixture was then poured onto a VB-E plate. Each concentration was tested in triplicate. The plates were incubated at 378C in the dark for 48–72 h. The numbers of colonies per plate were counted and the background lawn was examined. If toxicity was observed as an inhibition of growth of the background lawn, the plate was not counted, and was recorded as toxic. A test chemical was considered a bacterial mutagen if the number of revertant colonies was at
least twice the solvent control for at least one concentration and there was evidence of a concentration-related increase in the number of revertant colonies w3x. 2.3. Chinese hamster oÕary (HGPRT) forward gene mutation test The methods described by O’Neill et al. w4x for determining colony-forming ability and the frequency of mutants resistant to 6-thioguanine were followed. Chinese hamster ovary cells with the designation CHO-K1-BH4-Žsubclone D1. were used. Metabolic activation employed S9 homogenate prepared from Aroclor w 1254 induced rat liver. Modified F12 cell-culture medium ŽGibco, Grand Island, NY. supplemented to 5% Žvrv. with heat-inactivated fetal bovine serum was used as the growth medium for test chemicals without metabolic activation. Identical medium without serum was used for treatments incorporating an S9 metabolic activation system. Dimethylnitrosamine and ethylmethane sulfonate were used as positive control agents to assure the sensitivity of the test system for detecting metabolic activation-dependent and independent mutagens, respectively. Approximately 20–24 h prior to the mutation test, 5 = 10 5 cells were inoculated into each of two 25-cm2 culture flasks containing F12-D5 medium, and incubated at 378C in a 5% CO 2 atmosphere. On the day of testing, appropriate concentrations of the test agent were added to duplicate cultures of cells, and cultures were treated for 5 h at 378C. The cells were allowed a period of 18–24 h of recovery from treatment before chemical-induced cytotoxicity was determined. Treatment of cells in the presence of an S9 metabolic activation system was performed identically, with the exception that F12
H.-W. Leung, . Ballantyner Mutation Research 393 (1997) 7–15
medium without serum was used. The colony-forming potential of 100–200 treated cells was used as the measure of treatment-induced cytotoxicity. At 2to 3-day intervals after treatment, cells were subcultured. After a period of at least 7 days to allow expression of the mutant phenotype, cells were dissociated with 0.075% trypsin, counted and plated. The colony-forming ability determined by the viable fraction of the plated cells was used to correct the mutant frequency for the individual treated cultures and to detect variations in the growth ability of the cells. Statistical analysis of the mutation data for this test has been described by Slesinski et al. w5x. 2.4. Sister chromatid exchange test The procedure described by Latt et al. w6x was followed. Between 2 and 3 = 10 5 CHO cells were plated in F12-D5 medium about 40–48 h before treatment and then incubated at 378C in a 5–6% CO 2 atmosphere. Test chemicals and 3 mgrml bromodeoxyuridine ŽBrdU; Sigma Chemical Company, St. Louis, MO. were added. Cells were incubated for 2 and 5 h for testing chemicals with and without a rat S9 metabolic activation system, respectively, then washed with physiological salt solution, and fresh medium containing 3 mgrml BrdU was added for 24 h of additional incubation. Colchicine Ž0.5 mgrml. was added 1–2 h before harvesting to arrest cells in mitosis. Cells were then removed by centrifugation after a 20- to 25-min incubation with 0.01% trypsin ŽDifco, Detroit, MI. in 0.075 M KCl, fixed with methanolracetic acid Ž3:1 vrv., and chromosome spreads were prepared. Chromosomes were stained with Hoechst 33258 dye for 20 min, immersed in Sorenson’s buffer and exposed to a high intensity sunlamp for 15–30 min. Irradiated chromosomes were stained in Gurr’s giemsa. The number of chromosomes and the number of SCEs in a minimum of 25 cellsrdosed culture were scored. Concentrations which markedly inhibited cell division or SCE differentiation were recorded as cytotoxic and SCEs were not counted. The data for quantitative continuous variables were intercompared for the dose and control groups by Levene’s test for equality of variances, analysis of variance ŽANOVA., and t-tests. The t-tests were used following a significant ANOVA
9
to delineate which groups differed from the control group. If Levene’s test indicated homogeneous variances, the groups were compared by an ANOVA for equal variances followed, when appropriate, by pooled variance t-tests. If Levene’s test indicated heterogeneous variances, the groups were compared by an ANOVA followed by separate variance t-tests. The criteria for evaluation of a positive or negative response depended on the level of statistical significance and subjective analyses of concurrent and historical control data. Clearly positive responses would include any of the following: Ž1. doubling in the SCE frequency by any single concentration; Ž2. statistically significant responses of p - 0.01 with one or more consecutive concentrations; and Ž3. a statistically significant, concentration-related increase in the number of SCE. Equivocal results were defined as random statistical indications of positive increases, but which did not meet the criteria defined as a positive test result. 2.5. Micronucleus test in mice The test was based on the procedures developed by Schmid w7x and adapted to screen chemicals for clastogenic potential in peripheral blood erythrocytes w8x. Five male and 5 female young adult Swiss– Webster mice Ž6–8 weeks old. per dose group were used. Test doses were selected from a preliminary range-finding test to determine the approximate toxicity of the test materials. Mice were dosed by a single intraperitoneal injection and observed for mortality for 72 h. Three dose levels of about 80, 50 and 25% of the LD50 were selected for the micronucleus test. Triethylenemelamine or cyclophosphamide monohydrate was used as the positive control agent. Blood samples were collected from the tail vein at 24 or 30, 48 and 72 h after dosing. Slides of blood smears were stained with Gurr’s R-66 Giemsa diluted in phosphate buffer w9x, coded and read without knowledge of treatment group to prevent bias. The polychromaticrnormochromatic erythrocyte ratio for approximately 1000 total cells for each animal was calculated to provide an estimate of cytotoxicity. Excessive cytotoxicity was defined by a polychromaticrnormochromatic erythrocytes ratio of 0.01 or lower. A minimum of 1000 polychromatic erythro-
10
Table 2 Results of the Salmonella mutagenicity test Chemical
mgrplate
TA98 yS9
100 0.01 0.01 0.06 0.0025 0.1 0.3 1 3 5 10 100 0.01 0.01 0.06 0.01 0.01 0.03 0.1 0.3 1 3 10 50 0.01 0.01 0.06 0.0025 0.1 0.3 1 3 10
22"5 683"43
14"5
yS9 123"24
TA1535 qS9 127"27
1087"60
yS9 10"2
TA1537 qS9 11"2
yS9 7"2
TA1538 qS9 6"2
yS9 8"2 794"50
qS9 11"4
917"194 240"33
26"3 19"4 18"3 22 ŽS.
1890"311 21"6 18"1 19"0 15"2 15"3
Toxic 21"8 960"26
31"5
87"13 113"20 106"13 80"6 Toxic 98"20
1757"164 124"17 104"4 102"6 101"16 100"11 90"15
1525"114
15"12 13"5 10"4 8 ŽS. Toxic 13"4
123"10 11"4 13"3 11"2 5"2 11"1 8"3
11"6 4"2 6"3 4"2 Toxic 8"3
299"6 5"2 5"3 5"2 4"2 3"1
8"1 12"3 8"1 7 ŽT.
1655"109 13"3 10"4 13"5 10"4 16"1
10"4
Toxic 9"4 1228"14
19"3
1269"101 121"53
669"106 17"1 24"6 22"6 24"6 16"4
25"2 485"8
32"9 27"4 16"4 12"4 32"4
388"67 88"5 90"9 102"6 97"8 84"13
81"13
91"6 99"9 82"6 S, T 135"13
944"64
64"50 17"1 14"2 20"6 11"3 14"3
13"4
11"2 10"3 8"2 16 ŽT. 12"4
60"30 5"4 7"3 7"2 8"3 7"2
11"4
11"4 8"3 8 ŽS. 4 ŽT. 6"3
103"59 7"2 9"4 7"1 6"2 6 ŽS, T.
12"2 759"32
19"4 22"3 12"6 16 ŽT. 16"5
962"84 331"25
22"2 24"2 18"5 21"2 22"5
670"132 28"3 27"2 25"3 30"2 25"7
99"3 91"20 99"4 97"13 97"14
1127"33 107"10 130"2 109"8 121"36 136"9
12"1 14"5 11"4 12"3 14"6
53"4 10"2 14"4 10"5 13"1 12"3
10"5 6"1 8"2 7"3 6"2
56"15 8"2 9"3 7"1 9"3 9"3
10"4 8"3 8"5 7"2 7 ŽS.
593"61 16"1 23"3 12"2 13"4 9"3
4-NPD, 4-nitrophenylenediamine; NaN3 , sodium azide; 9-AA, 9-aminoacridine; 2-AA, 2-aminoanthracene. Toxic: clearing of background lawn or average number of colonies less than half of the solvent control value. S, sparse growth of background lawn in one or two of the triplicates tested. Counts not included in the calculation of mean and standard deviations not calculated. T, toxic to one or two of the triplicates tested.
H.-W. Leung, . Ballantyner Mutation Research 393 (1997) 7–15
Water 4-NPD NaN3 9-AA 2-AA MDEA MDEA MDEA MDEA MDEA MDEA Water 4-NPD NaN3 9-AA 2-AA DMEA DMEA DMEA DMEA DMEA DMEA DMEA Water 4-NPD NaN3 9-AA 2-AA t BDEA t BDEA t BDEA t BDEA t BDEA
TA100 qS9
H.-W. Leung, . Ballantyner Mutation Research 393 (1997) 7–15
cytes for each animal was scored for the presence of micronuclei unless the cytotoxicity of the test substance prevented this. Data were compared for significant differences using the Fisher’s Exact Test. A positive result was concluded if at least one statistically significant increase above vehicle control was observed with an indication of a dose-related effect.
3. Results and discussion MDEA, DMEA and t BDEA were evaluated for mutagenicity in the Salmonellarmicrosome reverse gene mutation test. Responses from individual sets of concurrent solvent and positive controls were all within the historical control data for this laboratory covering 80 to 100 individual experiments. No mutagenic activity was observed in any of the 5 strains tested in the absence or the presence of S9 activation, either by evidence of a dose–response relationship or a doubling of the mean number of colonies over the vehicle control value ŽTable 2.. Therefore, MDEA, DMEA and t BDEA were considered not mutagenic under the conditions of this in vitro mutagenicity test. The finding that MDEA and DMEA are negative in the Ames test is consistent with the results reported by Zeiger et al. w1x. In addition, two
11
other alkylalkanolamines, N-methylethanolamine ŽCAS 109-83-1. and N, N-diethylethanolamine ŽCAS 100-37-8. were also reported to be not mutagenic in this bacterial gene mutation test w1x. MDEA, DMEA and t BDEA were also tested for their potential to increase the frequencies of gene mutations and sister chromatid exchanges in the CHO cells. A preliminary cytotoxicity test was conducted to select the appropriate dose ranges ŽTable 3.. For the gene mutation test, the maximum concentration was one that produced about 90% inhibition of cell culture growth. Table 4 shows the results of the CHO gene mutation test. While the positive control substances, ethylmethane sulfonate and dimethylnitrosamine, both produced increased mutation frequencies, MDEA treatment did not produce a reproducible dose-related increase in the number of mutants over the range of concentrations tested either with or without an S9 metabolic activation system. Some small and sporadic numerical increases in the mutation frequencies were observed, but these increases were within the historical control range of variability for this test system and they were not statistically different from the concurrent control. Therefore, MDEA was not considered to be mutagenic in this in vitro gene mutation test. Likewise, DMEA did not produce any statistically significant
Table 3 Cytotoxicity determination for the Chinese hamster ovary gene mutation and sister chromatid exchange tests Chemical Žmgrml.
Culture medium 0.0003 0.001 0.003 0.01 0.03 0.1 0.3 0.6 1 3 5 10
% Survival relative to control Žfinal cell density= 10 5 . MDEA
DMEA
t BDEA
y S9
qS9
y S9
qS9
y S9
qS9
100 Ž44.4.
100 Ž40.9.
100 Ž45.8.
100 Ž44.0.
100 Ž23.6. 108 115 113 98 109 134 96
100 Ž34.4. 76 86 85 87 90 85 80
cytotoxic cytotoxic cytotoxic
60 cytotoxic cytotoxic
85.0 92.2 57.5 34.4 cytotoxic cytotoxic
85.4 96.4 93.0 82.0 12.6 cytotoxic
96.2 89.1 72.4 84.2
93.7 77.1 89.9 87.7
cytotoxic cytotoxic
67.5 cytotoxic
cytotoxic
cytotoxic
Cell density was determined approximately 18–24 h after treatment. The initial cell density was 1 = 10 5 cellsrflask. Cytotoxic: all cells lysed or detached from monolayer.
H.-W. Leung, . Ballantyner Mutation Research 393 (1997) 7–15
12
increases in the incidence of mutations of CHO cells with or without an S9 metabolic activation system, although there were some increases which were 2–3 times greater than the concurrent controls. These increases, however, were not dose-related and not repeatable in a duplicate culture at the same corresponding doses. All the mutant indices were within the typical range of variation for this test based on historical negative control variability. Given the random nature of these increases, DMEA was not judged to be positive in this in vitro gene mutation test.
Among t BDEA-treated CHO cells, no significant or dose-related increases in the frequencies of mutation were observed in the absence or in the presence of metabolic activation. Therefore, t BDEA was not considered mutagenic in this in vitro gene mutation test. For the SCE test, a dose level in the cytotoxicity test ŽTable 3. which was moderately toxic, but permitted a cell population increase of at least 40–50%, was selected as the maximum dose. Table 5 shows the results of the SCE test in CHO cells. DMEA did
Table 4 Mutant induction in Chinese hamster ovary gene mutation test Chemical
MDEA
DMEA
y S9 C.E.
M.F.
79
0 8.5 140.0
Culture medium EMS DMN 0.1 mgrml
C.E.
89
M.F.
94
4.2 0
0 16
0.8 1.0
85 78
98
0 7.1
83
0 6.8
0 37.8 2.6 6.4 0 8.1
85
1
228.4 0.9 0 2.7 5.5
C.E.
M.F. 7.5 0
55
75.4
112
0.9 0.8 3.9 3.8
107
8.8 16.2 97 0 6.0
109 103
71
1.5
83
2.3 0 68.2
85
84
1.3
M.F.
102
88
qS9
C.E.
1.0 7.1
85
91
M.F.
6.5 23.5 26.3 16
5.1 12.3 71
94
C.E.
5
97
0.4 0.6
85
15.8 12.3 189.8
95
0.2 0.3
M.F.
78.3
0 1.7
y S9
qS9
C.E.
0 1.5 37
tBDEA
y S9
qS9
12.3 14.9 12.4 15.1
78
2.4 1.8 0 2.0 0 0.9 0 0
104
0 1.8
105
1.1 10.0
102
1.1 0
T 14.4
2.0 33.0
94
1.1 0
71 66
3.7 0.9 0 8.1
T
EMS, ethylmethane sulfonate Ž0.2 mgrml.; DMN, dimethylnitrosamine Ž0.2 mgrml for DMEA and t BDEA, but 0.15 mgrml for MDEA.. C.E., cloning efficiency Ž% of combined negative controls.. About 100 cells inoculated into each plate. M.F., mutation frequencys total number of mutants per million clonable cells corrected for the viable fraction. 2 = 10 5 cells inoculated in each of 5 plates Ž1 = 10 6 total cells.. T, cytotoxic Žall cells lysed or detached from monolayer.. Statistical difference above control: no superscript indicates p ) 0.05; data for positive and negative controls were compared to historical ranges, but were not analyzed statistically. Historical negative control is 3–4 mutantsr10 6 viable cells, but a 95 percentile range of 0–25 mutantsr10 6 viables cells is typical.
H.-W. Leung, . Ballantyner Mutation Research 393 (1997) 7–15
not produce any statistically significant increase in SCEs above control values in tests both with or without the incorporation of an S9 metabolic activation system. Statistically significant increases in the mean number of SCEsrchromosome were observed in one culture at 0.3 mgrml MDEA and 0.4 mgrml t BDEA, and in duplicate cultures at 0.6 and 1.0 mgrml MDEA in the absence of S9 metabolic activation. These increases were small, being less than 1.5-fold those of untreated controls, and they were not dose-related. In the presence of S9 metabolic activation, both MDEA and t BDEA failed to produce any statistically significant increase in SCEs in comparison to concurrent controls. There were no increases in the numbers of first division cells which suggests that the dose ranges used were appropriate Ždata not shown.. Therefore, DMEA, MDEA and t BDEA was not considered to induce reciprocal
13
chromatid exchanges under the condition of this in vitro test. In the mouse micronucleus test, there were no major gender differences in mortality responses. The LD50 Žcombined sexes. for MDEA, DMEA and t BDEA were about 696, 1074 and 302 mgrkg, respectively. Selection of the top test doses was based on the lethality response rather than on bone marrow suppression. There were no significant differences in the polychromatic erythrocyte to normochromatic erythrocyte ratios at any dosages for all three alkylalkanolamines tested ŽTable 6.. Furthermore, no significant increases in the incidence of micronucleated polychromatic erythrocyte were observed at any sampling time ŽTable 6.. Therefore, MDEA, DMEA, and t BDEA were not considered to be inducers of micronuclei under the condition of this in vivo test. The results from the present study show that
Table 5 Induction of sister chromatid exchange in Chinese hamster ovary cells in vitro by alkylalkanolamines Chemical
Number of SCE per chromosome Žmean " SD. MDEA
Culture medium EMS Ž0.1 mgrml. DMN Ž0.3 mgrml.
DMEA qS9
y S9
qS9
y S9
qS9
0.41 " 0.13 0.43 " 0.18 1.08 " 0.31 d
0.47 " 0.14 0.51 " 0.14
0.52 " 0.16 0.50 " 0.15 1.36 " 0.37
0.54 " 0.15 0.52 " 0.15
0.34 " 0.16 0.30 " 0.12 0.88 " 0.31 c
0.57 " 0.17 0.66 " 0.17
1.66 " 0.49
d
d
2.38 " 0.48
0.59 " 0.18 0.54 " 0.15 0.57 " 0.17 0.52 " 0.16
d c
0.44 " 0.13 0.46 " 0.15
0.58 " 0.18 0.59 " 0.18
0.52 " 0.21 0.43 " 0.16
0.57 " 0.16 0.57 " 0.13 0.51 " 0.18 0.57 " 0.18
0.8 0.50 " 0.12 0.52 " 0.16
b c
1.5 2.0
1.91 " 0.80
0.44 " 0.11 b 0.40 " 0.12
0.4
1.0
d
0.56 " 0.20 0.52 " 0.15 0.65 " 0.28 0.54 " 0.19
0.2
0.6
t BDEA
y S9
0.1 mgrml
0.3
)
0.39 " 0.10 0.39 " 0.18 0.31 " 0.13 0.39 " 0.16 0.20 " 0.11 0.22 " 0.10
0.52 " 0.23 0.51 " 0.20
Cells were treated for 5 h without S9; 2 h with S9. EMS, ethylmethane sulfonate; DMN, dimethylnitrosamine. a Determined in duplicate culture from the values of the individual cells examined. Statistical significance above culture medium control: b p - 0.05; c p - 0.01; d p - 0.001.
b
0.63 " 0.16 0.59 " 0.23
0.61 " 0.21 0.75 " 0.18
0.65 " 0.21 0.75 " 0.21
c
H.-W. Leung, . Ballantyner Mutation Research 393 (1997) 7–15
14
Table 6 Induction of micronucleus in peripheral erythrocytes of Swiss–Webster mice injected intraperitoneally with alkylalkanolamines Sex
Mean PCEr1000 NCE Ž"SD.
Mean MN-PCE per 1000 PCE Ž"SD.
Water
Water
30 h postdosing M 52 " 24 F 35 " 10 48 h postdosing M 43 " 13 F 31 " 6 72 h postdosing M 39 " 7 F 32 " 9
TEM
36 " 8 36 " 9
MDEA Žmgrkg. 175
350
560
55 " 17 41 " 8
64 " 11 48 " 9
70 " 12 41 " 14
5.4 " 3.2 2.6 " 1.7
45 " 17 34 " 18
43 " 12 32 " 12
36 " 11 38 " 4
38 " 12 31 " 11
36 " 14 39 " 9
32 " 14 24 " 7
Mean PCEr1000 NCE Ž"SD. Water
30 h postdosing M 20 " 7 F 21 " 8 48 h postdosing M 26 " 7 F 36 " 8 72 h postdosing M 32 " 6 F 35 " 5
TEM
17 " 1 20 " 9
24 h postdosing M 31 " 4 F 33 " 7 48 h postdosing M 28 " 5 F 27 " 5 72 h postdosing M 32 " 8 F 32 " 7
CP
22 " 4 26 " 5
MDEA Žmgrkg. 175
350
560
4.8 " 2.4 4.2 " 2.8
6.6 " 2.9 3.0 " 0.7
4.8 " 3.5 1.6 " 1.5
3.8 " 3.1 2.4 " 1.5
4.2 " 2.8 3.8 " 2.4
5.0 " 4.4 2.2 " 1.3
3.0 " 2.0 2.6 " 2.7
5.0 " 2.0 3.2 " 1.1
2.6 " 2.1 2.2 " 1.3
7.0 " 5.6 3.0 " 2.8
2.7 " 1.2 2.8 " 1.9
42.0 " 8 b 43.0 " 15 b
Mean MN-PCE per 1000 PCE Ž"SD.
DMEA Žmgrkg.
Water
270
540
860
22 " 3 32 " 8
22 " 5 27 " 7
22 " 8 28 " 9
3.2 " 2.3 1.4 " 1.5
19 " 5 42 " 14
20 " 6 30 " 8
18 " 4 32 " 9
30 " 3 32 " 8
33 " 3 33 " 8
36 " 15 33 " 10
Mean PCEr1000 NCE Ž"SD. Water
TEM
TEM
DMEA Žmgrkg. 270
540
860
3.8 " 1.5 2.4 " 2.1
3.8 " 2.4 1.2 " 0.8
1.6 " 1.1 2.0 " 1.4
3.6 " 1.5 1.4 " 1.1
3.2 " 1.9 0.8 " 0.8
4.0 " 2.6 1.4 " 1.1
3.4 " 2.3 1.0 " 1.4
2.8 " 1.5 1.6 " 1.1
2.8 " 1.3 1.8 " 1.5
3.2 " 1.1 1.8 " 2.1
3.2 " 1.9 0.8 " 1.1
20.8 " 3.6 24.8 " 8.7
b b
Mean MN-PCE per 1000 PCE Ž"SD.
t BDEA Žmgrkg.
Water
75
150
240
30 " 5 29 " 4
29 " 1 30 " 7
27 " 6 34 " 12
1.2 " 1.1 1.6 " 1.3
31 " 10 25 " 3
33 " 6 32 " 7
31 " 3 27 " 5
2.2 " 1.1 1.8 " 1.3
32 " 7 30 " 5
33 " 9 30 " 8
26 " 5 30 " 8
1.8 " 0.5 1.6 " 1.1
CP
11.2 " 1.5 11.0 " 1.2
t BDEA Žmgrkg.
a a
75
150
240
1.4 " 0.9 2.4 " 1.7
2.2 " 1.1 1.4 " 1.7
2.4 " 2.1 1.0 " 1.4
2.0 " 1.0 1.8 " 0.8
2.0 " 0.7 1.0 " 0.7
1.0 " 0.8 0.3 " 0.5
2.0 " 1.9 1.8 " 1.3
2.2 " 1.3 1.6 " 1.5
1.0 " 1.2 1.3 " 1.5
Water Ž10 mlrkg.; TEM, triethylenemelamine Ž0.3 mgrkg.; CP, cyclophosphamide Ž15 mgrkg.. PCE, polychromatic erythrocytes; NCE, normochromatic erythrocytes; MN-PCE, micronucleated polychromatic erythrocytes. Statistical analysis employed the Fisher’s Exact Test Ž1-tailed.: a p - 0.01; b p - 0.001.
MDEA, DMEA, and t BDEA consistently gave negative responses in a battery of in vitro and in vivo tests to assess the potential to produce gene mutation or DNA damage. Thus, it can be concluded that these alkylalkanolamines do not represent a genotoxic hazard. Similarly, the alkanolamines, mo-
noethanolam ine, diethanolam ine and tri ethanolamine, were also reported to be non-mutagenic w10x. The overall evidence therefore suggests that the family of ethanolamines or amino ethanols, whether alkyl substituted or not, as a whole is unlikely to be genotoxic.
H.-W. Leung, . Ballantyner Mutation Research 393 (1997) 7–15
References w1x E. Zeiger, B. Anderson, S. Haworth, T. Lawlor, K. Mortelmans, W. Speck, Salmonella mutagenicity tests: III. Results from the testing of 255 chemicals, Environ. Mutagen. 9 ŽSuppl. 9. Ž1987. 1–110. w2x B.N. Ames, J. McCann, F. Yamasaki, Methods for detecting carcinogens and mutagens with the Salmonellarmammalian-microsome mutagenicity test, Mutation Res. 31 Ž1975. 347–364. w3x F.D. deSerres, M.D. Shelby, Recommendations on data production and analysis using the Salmonellarmicrosome mutagenicity test, Mutation Res. 64 Ž1974. 159–165. w4x J.P. O’Neill, P.A. Brimer, R. Machanoff, G.P. Hirsch, A.W. Hsie, A quantitative test of mutation induction at the hypoxanthine-guanine phophoribosyl transferase locus in Chinese hamster ovary cells ŽCHOrHGPRT. system: development and definition of the system, Mutation Res. 45 Ž1977. 91– 101. w5x R.S. Slesinski, P.J. Guzzie, W.C. Hengler, P.G. Watanabe,
w6x
w7x w8x
w9x
w10x
15
M.D. Woodside, R.S.H. Yang, Assessment of genotoxic potential of ethylenediamine, in vitro and in vivo studies, Mutation Res. 124 Ž1983. 299–314. S.A. Latt, J. Allen, S.E. Bloom, A. Carrano, E.D. Falke, P. Kram, E. Schneider, R. Schreck, R. Tice, B. Whitfield, S. Wolff, Sister chromatid exchanges: a report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res. 87 Ž1981. 17–62. W. Schmid, The micronucleus test, Mutation Res. 31 Ž1975. 9–15. J.T. MacGregor, C.M. Wehr, D.H. Gould, Clastogen-induced micronuclei in peripheral blood erythrocytes: the basis of an improved micronucleus test, Environ. Mutagen. 2 Ž1980. 509–514. B.B. Gallapudi, O.P. Kamra, Application of a simple Giemsa-staining method in the micronucleus test, Mutation Res. 64 Ž1974. 45–46. J.B. Knaak, H.W. Leung, W.T. Stott, J. Busch, J. Bilsky, Toxicology of mono-, di-, and triethanolamine, Rev. Environ. Contam. Toxicol. 149 Ž1997. 1–86.
Evaluation of the genotoxic potential of alkylalkanolamines Hon-Wing Leung ) , Bryan Ballantyne Union Carbide Corporation, 39 Old Ridgebury Road, Danbury, CT 06817-0001, USA
Abstract Three alkylalkanolamines, N,N-dimethylethanolamine, N-methyldiethanolamine, and tert-butyldiethanolamine, were evaluated for potential genotoxic activity using the Salmonellarmicrosome reverse gene mutation test, the CHOrHGPRT forward gene mutation test, a sister chromatid exchange test in cultured CHO cells, and an in vivo peripheral blood micronucleus test in Swiss–Webster mice. None of the three alkylalkanolamines produced any significant or dose-related increases in the frequencies of mutations, sister chromatid exchanges or micronuclei. These results indicate that N,N-dimethylethanolamine, N-methyldiethanolamine, and tert-butyldiethanolamine are not genotoxic in the tests conducted. q 1997 Elsevier Science B.V. Keywords: Alkylalkanolamine; Salmonella reverse gene mutation test; Chinese hamster ovary HGPRT forward gene mutation test; Sister chromatid exchange test; Micronucleus test; Mouse
1. Introduction The alkylalkanolamines are industrial chemicals with widespread applications including flocculant monomers, urethane catalysts, natural gas and boiler water treatment, corrosion inhibitors, can coatings, ion exchange resins, textile lubricants, and pharmaceuticals. Besides some limited mutagenicity data in a bacterial system w1x, no further study of the mutagenic or clastogenic potentials of the alkylalkanolamines has been performed. The present study was undertaken to evaluate the genotoxic potential of three alkylalkanolamines, N,N-dimethylethanolamine ŽDMEA., N-methyldiethanolamine ŽMDEA., and tert-butyldiethanolamine Ž t BDEA.. These com-
) Corresponding author. Tel.: q1 Ž203. 794-5755; fax: q1 Ž203. 794-5275.
pounds were evaluated in the Salmonellarmicrosome reverse gene mutation test, the CHOrHGPRT forward gene mutation test, a sister chromatid exchange test in cultured CHO cells, and an in vivo micronucleus test in Swiss–Webster mice.
2. Materials and methods 2.1. Chemicals The physicochemical data of the three alkylalkanolamines tested are shown in Table 1. Commercial samples were obtained from the Union Carbide Corporation, South Charleston, WV. All samples were analyzed and their chemical purity determined by capillary gas chromatography. Water from a Millipore Milli-Q w purification system was used to prepare solutions and media. Chemicals used as positive
1383-5718r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 7 . 0 0 1 0 4 - 6
8
H.-W. Leung, . Ballantyner Mutation Research 393 (1997) 7–15
Table 1 Physicochemical data of alkylalkanolamines tested for genotoxicity potential Alkylalkanolamine
CAS no.
Chemical formula
MW
Purity
N, N-Dimethylethanolamine N-Methyldiethanolamine t-Butyldiethanolamine
108-01-0 105-59-9 2160-93-2
ŽCH 3 . 2 NC 2 H 4 OH CH 3 NŽC 2 H 4 OH. 2 ŽCH 3 . 3 CNŽC 2 H 4 OH. 2
89.1 119.2 161.3
99.9% 99.8% 99.9%
a
a Analytical characterization by capillary gas chromatography for all samples. In addition, the identity of t-butyldiethanolamine was characterized with mass spectrometry and nuclear magnetic resonance spectroscopy.
controls were obtained from Sigma Chemical Company ŽSt. Louis, MO.. 2.2. Salmonellar microsome reÕerse gene mutation test The method described by Ames et al. w2x was followed. Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 were used in this test. At least 4 concentrations spaced at about half-log intervals were tested. The highest concentration was selected from a cytotoxicity test in strain TA100 which produced either a significant ŽG 50%. reduction in the number of revertant colonies or a significant inhibition of background lawn growth. The highest concentration for non-cytotoxic test substances was 10 mgrplate. The activation-independent positive controls were 4-nitrophenylenediamine, sodium azide, and 9-aminoacridine, and the activation-dependent control was 2-aminoanthracene. Frozen S9 liver homogenate, isolated from Aroclor w 1254-induced rats ŽMicrobiological Associates, Bethesda, MD., was thawed and diluted in buffer each day of testing. To a sterile tube containing 2 ml of top agar, 100 ml each of bacterial culture and test chemical were added. Either 0.5 ml of S9 or phosphate-buffered saline was added for tests with and without metabolic activation, respectively. The top agar mixture was then poured onto a VB-E plate. Each concentration was tested in triplicate. The plates were incubated at 378C in the dark for 48–72 h. The numbers of colonies per plate were counted and the background lawn was examined. If toxicity was observed as an inhibition of growth of the background lawn, the plate was not counted, and was recorded as toxic. A test chemical was considered a bacterial mutagen if the number of revertant colonies was at
least twice the solvent control for at least one concentration and there was evidence of a concentration-related increase in the number of revertant colonies w3x. 2.3. Chinese hamster oÕary (HGPRT) forward gene mutation test The methods described by O’Neill et al. w4x for determining colony-forming ability and the frequency of mutants resistant to 6-thioguanine were followed. Chinese hamster ovary cells with the designation CHO-K1-BH4-Žsubclone D1. were used. Metabolic activation employed S9 homogenate prepared from Aroclor w 1254 induced rat liver. Modified F12 cell-culture medium ŽGibco, Grand Island, NY. supplemented to 5% Žvrv. with heat-inactivated fetal bovine serum was used as the growth medium for test chemicals without metabolic activation. Identical medium without serum was used for treatments incorporating an S9 metabolic activation system. Dimethylnitrosamine and ethylmethane sulfonate were used as positive control agents to assure the sensitivity of the test system for detecting metabolic activation-dependent and independent mutagens, respectively. Approximately 20–24 h prior to the mutation test, 5 = 10 5 cells were inoculated into each of two 25-cm2 culture flasks containing F12-D5 medium, and incubated at 378C in a 5% CO 2 atmosphere. On the day of testing, appropriate concentrations of the test agent were added to duplicate cultures of cells, and cultures were treated for 5 h at 378C. The cells were allowed a period of 18–24 h of recovery from treatment before chemical-induced cytotoxicity was determined. Treatment of cells in the presence of an S9 metabolic activation system was performed identically, with the exception that F12
H.-W. Leung, . Ballantyner Mutation Research 393 (1997) 7–15
medium without serum was used. The colony-forming potential of 100–200 treated cells was used as the measure of treatment-induced cytotoxicity. At 2to 3-day intervals after treatment, cells were subcultured. After a period of at least 7 days to allow expression of the mutant phenotype, cells were dissociated with 0.075% trypsin, counted and plated. The colony-forming ability determined by the viable fraction of the plated cells was used to correct the mutant frequency for the individual treated cultures and to detect variations in the growth ability of the cells. Statistical analysis of the mutation data for this test has been described by Slesinski et al. w5x. 2.4. Sister chromatid exchange test The procedure described by Latt et al. w6x was followed. Between 2 and 3 = 10 5 CHO cells were plated in F12-D5 medium about 40–48 h before treatment and then incubated at 378C in a 5–6% CO 2 atmosphere. Test chemicals and 3 mgrml bromodeoxyuridine ŽBrdU; Sigma Chemical Company, St. Louis, MO. were added. Cells were incubated for 2 and 5 h for testing chemicals with and without a rat S9 metabolic activation system, respectively, then washed with physiological salt solution, and fresh medium containing 3 mgrml BrdU was added for 24 h of additional incubation. Colchicine Ž0.5 mgrml. was added 1–2 h before harvesting to arrest cells in mitosis. Cells were then removed by centrifugation after a 20- to 25-min incubation with 0.01% trypsin ŽDifco, Detroit, MI. in 0.075 M KCl, fixed with methanolracetic acid Ž3:1 vrv., and chromosome spreads were prepared. Chromosomes were stained with Hoechst 33258 dye for 20 min, immersed in Sorenson’s buffer and exposed to a high intensity sunlamp for 15–30 min. Irradiated chromosomes were stained in Gurr’s giemsa. The number of chromosomes and the number of SCEs in a minimum of 25 cellsrdosed culture were scored. Concentrations which markedly inhibited cell division or SCE differentiation were recorded as cytotoxic and SCEs were not counted. The data for quantitative continuous variables were intercompared for the dose and control groups by Levene’s test for equality of variances, analysis of variance ŽANOVA., and t-tests. The t-tests were used following a significant ANOVA
9
to delineate which groups differed from the control group. If Levene’s test indicated homogeneous variances, the groups were compared by an ANOVA for equal variances followed, when appropriate, by pooled variance t-tests. If Levene’s test indicated heterogeneous variances, the groups were compared by an ANOVA followed by separate variance t-tests. The criteria for evaluation of a positive or negative response depended on the level of statistical significance and subjective analyses of concurrent and historical control data. Clearly positive responses would include any of the following: Ž1. doubling in the SCE frequency by any single concentration; Ž2. statistically significant responses of p - 0.01 with one or more consecutive concentrations; and Ž3. a statistically significant, concentration-related increase in the number of SCE. Equivocal results were defined as random statistical indications of positive increases, but which did not meet the criteria defined as a positive test result. 2.5. Micronucleus test in mice The test was based on the procedures developed by Schmid w7x and adapted to screen chemicals for clastogenic potential in peripheral blood erythrocytes w8x. Five male and 5 female young adult Swiss– Webster mice Ž6–8 weeks old. per dose group were used. Test doses were selected from a preliminary range-finding test to determine the approximate toxicity of the test materials. Mice were dosed by a single intraperitoneal injection and observed for mortality for 72 h. Three dose levels of about 80, 50 and 25% of the LD50 were selected for the micronucleus test. Triethylenemelamine or cyclophosphamide monohydrate was used as the positive control agent. Blood samples were collected from the tail vein at 24 or 30, 48 and 72 h after dosing. Slides of blood smears were stained with Gurr’s R-66 Giemsa diluted in phosphate buffer w9x, coded and read without knowledge of treatment group to prevent bias. The polychromaticrnormochromatic erythrocyte ratio for approximately 1000 total cells for each animal was calculated to provide an estimate of cytotoxicity. Excessive cytotoxicity was defined by a polychromaticrnormochromatic erythrocytes ratio of 0.01 or lower. A minimum of 1000 polychromatic erythro-
10
Table 2 Results of the Salmonella mutagenicity test Chemical
mgrplate
TA98 yS9
100 0.01 0.01 0.06 0.0025 0.1 0.3 1 3 5 10 100 0.01 0.01 0.06 0.01 0.01 0.03 0.1 0.3 1 3 10 50 0.01 0.01 0.06 0.0025 0.1 0.3 1 3 10
22"5 683"43
14"5
yS9 123"24
TA1535 qS9 127"27
1087"60
yS9 10"2
TA1537 qS9 11"2
yS9 7"2
TA1538 qS9 6"2
yS9 8"2 794"50
qS9 11"4
917"194 240"33
26"3 19"4 18"3 22 ŽS.
1890"311 21"6 18"1 19"0 15"2 15"3
Toxic 21"8 960"26
31"5
87"13 113"20 106"13 80"6 Toxic 98"20
1757"164 124"17 104"4 102"6 101"16 100"11 90"15
1525"114
15"12 13"5 10"4 8 ŽS. Toxic 13"4
123"10 11"4 13"3 11"2 5"2 11"1 8"3
11"6 4"2 6"3 4"2 Toxic 8"3
299"6 5"2 5"3 5"2 4"2 3"1
8"1 12"3 8"1 7 ŽT.
1655"109 13"3 10"4 13"5 10"4 16"1
10"4
Toxic 9"4 1228"14
19"3
1269"101 121"53
669"106 17"1 24"6 22"6 24"6 16"4
25"2 485"8
32"9 27"4 16"4 12"4 32"4
388"67 88"5 90"9 102"6 97"8 84"13
81"13
91"6 99"9 82"6 S, T 135"13
944"64
64"50 17"1 14"2 20"6 11"3 14"3
13"4
11"2 10"3 8"2 16 ŽT. 12"4
60"30 5"4 7"3 7"2 8"3 7"2
11"4
11"4 8"3 8 ŽS. 4 ŽT. 6"3
103"59 7"2 9"4 7"1 6"2 6 ŽS, T.
12"2 759"32
19"4 22"3 12"6 16 ŽT. 16"5
962"84 331"25
22"2 24"2 18"5 21"2 22"5
670"132 28"3 27"2 25"3 30"2 25"7
99"3 91"20 99"4 97"13 97"14
1127"33 107"10 130"2 109"8 121"36 136"9
12"1 14"5 11"4 12"3 14"6
53"4 10"2 14"4 10"5 13"1 12"3
10"5 6"1 8"2 7"3 6"2
56"15 8"2 9"3 7"1 9"3 9"3
10"4 8"3 8"5 7"2 7 ŽS.
593"61 16"1 23"3 12"2 13"4 9"3
4-NPD, 4-nitrophenylenediamine; NaN3 , sodium azide; 9-AA, 9-aminoacridine; 2-AA, 2-aminoanthracene. Toxic: clearing of background lawn or average number of colonies less than half of the solvent control value. S, sparse growth of background lawn in one or two of the triplicates tested. Counts not included in the calculation of mean and standard deviations not calculated. T, toxic to one or two of the triplicates tested.
H.-W. Leung, . Ballantyner Mutation Research 393 (1997) 7–15
Water 4-NPD NaN3 9-AA 2-AA MDEA MDEA MDEA MDEA MDEA MDEA Water 4-NPD NaN3 9-AA 2-AA DMEA DMEA DMEA DMEA DMEA DMEA DMEA Water 4-NPD NaN3 9-AA 2-AA t BDEA t BDEA t BDEA t BDEA t BDEA
TA100 qS9
H.-W. Leung, . Ballantyner Mutation Research 393 (1997) 7–15
cytes for each animal was scored for the presence of micronuclei unless the cytotoxicity of the test substance prevented this. Data were compared for significant differences using the Fisher’s Exact Test. A positive result was concluded if at least one statistically significant increase above vehicle control was observed with an indication of a dose-related effect.
3. Results and discussion MDEA, DMEA and t BDEA were evaluated for mutagenicity in the Salmonellarmicrosome reverse gene mutation test. Responses from individual sets of concurrent solvent and positive controls were all within the historical control data for this laboratory covering 80 to 100 individual experiments. No mutagenic activity was observed in any of the 5 strains tested in the absence or the presence of S9 activation, either by evidence of a dose–response relationship or a doubling of the mean number of colonies over the vehicle control value ŽTable 2.. Therefore, MDEA, DMEA and t BDEA were considered not mutagenic under the conditions of this in vitro mutagenicity test. The finding that MDEA and DMEA are negative in the Ames test is consistent with the results reported by Zeiger et al. w1x. In addition, two
11
other alkylalkanolamines, N-methylethanolamine ŽCAS 109-83-1. and N, N-diethylethanolamine ŽCAS 100-37-8. were also reported to be not mutagenic in this bacterial gene mutation test w1x. MDEA, DMEA and t BDEA were also tested for their potential to increase the frequencies of gene mutations and sister chromatid exchanges in the CHO cells. A preliminary cytotoxicity test was conducted to select the appropriate dose ranges ŽTable 3.. For the gene mutation test, the maximum concentration was one that produced about 90% inhibition of cell culture growth. Table 4 shows the results of the CHO gene mutation test. While the positive control substances, ethylmethane sulfonate and dimethylnitrosamine, both produced increased mutation frequencies, MDEA treatment did not produce a reproducible dose-related increase in the number of mutants over the range of concentrations tested either with or without an S9 metabolic activation system. Some small and sporadic numerical increases in the mutation frequencies were observed, but these increases were within the historical control range of variability for this test system and they were not statistically different from the concurrent control. Therefore, MDEA was not considered to be mutagenic in this in vitro gene mutation test. Likewise, DMEA did not produce any statistically significant
Table 3 Cytotoxicity determination for the Chinese hamster ovary gene mutation and sister chromatid exchange tests Chemical Žmgrml.
Culture medium 0.0003 0.001 0.003 0.01 0.03 0.1 0.3 0.6 1 3 5 10
% Survival relative to control Žfinal cell density= 10 5 . MDEA
DMEA
t BDEA
y S9
qS9
y S9
qS9
y S9
qS9
100 Ž44.4.
100 Ž40.9.
100 Ž45.8.
100 Ž44.0.
100 Ž23.6. 108 115 113 98 109 134 96
100 Ž34.4. 76 86 85 87 90 85 80
cytotoxic cytotoxic cytotoxic
60 cytotoxic cytotoxic
85.0 92.2 57.5 34.4 cytotoxic cytotoxic
85.4 96.4 93.0 82.0 12.6 cytotoxic
96.2 89.1 72.4 84.2
93.7 77.1 89.9 87.7
cytotoxic cytotoxic
67.5 cytotoxic
cytotoxic
cytotoxic
Cell density was determined approximately 18–24 h after treatment. The initial cell density was 1 = 10 5 cellsrflask. Cytotoxic: all cells lysed or detached from monolayer.
H.-W. Leung, . Ballantyner Mutation Research 393 (1997) 7–15
12
increases in the incidence of mutations of CHO cells with or without an S9 metabolic activation system, although there were some increases which were 2–3 times greater than the concurrent controls. These increases, however, were not dose-related and not repeatable in a duplicate culture at the same corresponding doses. All the mutant indices were within the typical range of variation for this test based on historical negative control variability. Given the random nature of these increases, DMEA was not judged to be positive in this in vitro gene mutation test.
Among t BDEA-treated CHO cells, no significant or dose-related increases in the frequencies of mutation were observed in the absence or in the presence of metabolic activation. Therefore, t BDEA was not considered mutagenic in this in vitro gene mutation test. For the SCE test, a dose level in the cytotoxicity test ŽTable 3. which was moderately toxic, but permitted a cell population increase of at least 40–50%, was selected as the maximum dose. Table 5 shows the results of the SCE test in CHO cells. DMEA did
Table 4 Mutant induction in Chinese hamster ovary gene mutation test Chemical
MDEA
DMEA
y S9 C.E.
M.F.
79
0 8.5 140.0
Culture medium EMS DMN 0.1 mgrml
C.E.
89
M.F.
94
4.2 0
0 16
0.8 1.0
85 78
98
0 7.1
83
0 6.8
0 37.8 2.6 6.4 0 8.1
85
1
228.4 0.9 0 2.7 5.5
C.E.
M.F. 7.5 0
55
75.4
112
0.9 0.8 3.9 3.8
107
8.8 16.2 97 0 6.0
109 103
71
1.5
83
2.3 0 68.2
85
84
1.3
M.F.
102
88
qS9
C.E.
1.0 7.1
85
91
M.F.
6.5 23.5 26.3 16
5.1 12.3 71
94
C.E.
5
97
0.4 0.6
85
15.8 12.3 189.8
95
0.2 0.3
M.F.
78.3
0 1.7
y S9
qS9
C.E.
0 1.5 37
tBDEA
y S9
qS9
12.3 14.9 12.4 15.1
78
2.4 1.8 0 2.0 0 0.9 0 0
104
0 1.8
105
1.1 10.0
102
1.1 0
T 14.4
2.0 33.0
94
1.1 0
71 66
3.7 0.9 0 8.1
T
EMS, ethylmethane sulfonate Ž0.2 mgrml.; DMN, dimethylnitrosamine Ž0.2 mgrml for DMEA and t BDEA, but 0.15 mgrml for MDEA.. C.E., cloning efficiency Ž% of combined negative controls.. About 100 cells inoculated into each plate. M.F., mutation frequencys total number of mutants per million clonable cells corrected for the viable fraction. 2 = 10 5 cells inoculated in each of 5 plates Ž1 = 10 6 total cells.. T, cytotoxic Žall cells lysed or detached from monolayer.. Statistical difference above control: no superscript indicates p ) 0.05; data for positive and negative controls were compared to historical ranges, but were not analyzed statistically. Historical negative control is 3–4 mutantsr10 6 viable cells, but a 95 percentile range of 0–25 mutantsr10 6 viables cells is typical.
H.-W. Leung, . Ballantyner Mutation Research 393 (1997) 7–15
not produce any statistically significant increase in SCEs above control values in tests both with or without the incorporation of an S9 metabolic activation system. Statistically significant increases in the mean number of SCEsrchromosome were observed in one culture at 0.3 mgrml MDEA and 0.4 mgrml t BDEA, and in duplicate cultures at 0.6 and 1.0 mgrml MDEA in the absence of S9 metabolic activation. These increases were small, being less than 1.5-fold those of untreated controls, and they were not dose-related. In the presence of S9 metabolic activation, both MDEA and t BDEA failed to produce any statistically significant increase in SCEs in comparison to concurrent controls. There were no increases in the numbers of first division cells which suggests that the dose ranges used were appropriate Ždata not shown.. Therefore, DMEA, MDEA and t BDEA was not considered to induce reciprocal
13
chromatid exchanges under the condition of this in vitro test. In the mouse micronucleus test, there were no major gender differences in mortality responses. The LD50 Žcombined sexes. for MDEA, DMEA and t BDEA were about 696, 1074 and 302 mgrkg, respectively. Selection of the top test doses was based on the lethality response rather than on bone marrow suppression. There were no significant differences in the polychromatic erythrocyte to normochromatic erythrocyte ratios at any dosages for all three alkylalkanolamines tested ŽTable 6.. Furthermore, no significant increases in the incidence of micronucleated polychromatic erythrocyte were observed at any sampling time ŽTable 6.. Therefore, MDEA, DMEA, and t BDEA were not considered to be inducers of micronuclei under the condition of this in vivo test. The results from the present study show that
Table 5 Induction of sister chromatid exchange in Chinese hamster ovary cells in vitro by alkylalkanolamines Chemical
Number of SCE per chromosome Žmean " SD. MDEA
Culture medium EMS Ž0.1 mgrml. DMN Ž0.3 mgrml.
DMEA qS9
y S9
qS9
y S9
qS9
0.41 " 0.13 0.43 " 0.18 1.08 " 0.31 d
0.47 " 0.14 0.51 " 0.14
0.52 " 0.16 0.50 " 0.15 1.36 " 0.37
0.54 " 0.15 0.52 " 0.15
0.34 " 0.16 0.30 " 0.12 0.88 " 0.31 c
0.57 " 0.17 0.66 " 0.17
1.66 " 0.49
d
d
2.38 " 0.48
0.59 " 0.18 0.54 " 0.15 0.57 " 0.17 0.52 " 0.16
d c
0.44 " 0.13 0.46 " 0.15
0.58 " 0.18 0.59 " 0.18
0.52 " 0.21 0.43 " 0.16
0.57 " 0.16 0.57 " 0.13 0.51 " 0.18 0.57 " 0.18
0.8 0.50 " 0.12 0.52 " 0.16
b c
1.5 2.0
1.91 " 0.80
0.44 " 0.11 b 0.40 " 0.12
0.4
1.0
d
0.56 " 0.20 0.52 " 0.15 0.65 " 0.28 0.54 " 0.19
0.2
0.6
t BDEA
y S9
0.1 mgrml
0.3
)
0.39 " 0.10 0.39 " 0.18 0.31 " 0.13 0.39 " 0.16 0.20 " 0.11 0.22 " 0.10
0.52 " 0.23 0.51 " 0.20
Cells were treated for 5 h without S9; 2 h with S9. EMS, ethylmethane sulfonate; DMN, dimethylnitrosamine. a Determined in duplicate culture from the values of the individual cells examined. Statistical significance above culture medium control: b p - 0.05; c p - 0.01; d p - 0.001.
b
0.63 " 0.16 0.59 " 0.23
0.61 " 0.21 0.75 " 0.18
0.65 " 0.21 0.75 " 0.21
c
H.-W. Leung, . Ballantyner Mutation Research 393 (1997) 7–15
14
Table 6 Induction of micronucleus in peripheral erythrocytes of Swiss–Webster mice injected intraperitoneally with alkylalkanolamines Sex
Mean PCEr1000 NCE Ž"SD.
Mean MN-PCE per 1000 PCE Ž"SD.
Water
Water
30 h postdosing M 52 " 24 F 35 " 10 48 h postdosing M 43 " 13 F 31 " 6 72 h postdosing M 39 " 7 F 32 " 9
TEM
36 " 8 36 " 9
MDEA Žmgrkg. 175
350
560
55 " 17 41 " 8
64 " 11 48 " 9
70 " 12 41 " 14
5.4 " 3.2 2.6 " 1.7
45 " 17 34 " 18
43 " 12 32 " 12
36 " 11 38 " 4
38 " 12 31 " 11
36 " 14 39 " 9
32 " 14 24 " 7
Mean PCEr1000 NCE Ž"SD. Water
30 h postdosing M 20 " 7 F 21 " 8 48 h postdosing M 26 " 7 F 36 " 8 72 h postdosing M 32 " 6 F 35 " 5
TEM
17 " 1 20 " 9
24 h postdosing M 31 " 4 F 33 " 7 48 h postdosing M 28 " 5 F 27 " 5 72 h postdosing M 32 " 8 F 32 " 7
CP
22 " 4 26 " 5
MDEA Žmgrkg. 175
350
560
4.8 " 2.4 4.2 " 2.8
6.6 " 2.9 3.0 " 0.7
4.8 " 3.5 1.6 " 1.5
3.8 " 3.1 2.4 " 1.5
4.2 " 2.8 3.8 " 2.4
5.0 " 4.4 2.2 " 1.3
3.0 " 2.0 2.6 " 2.7
5.0 " 2.0 3.2 " 1.1
2.6 " 2.1 2.2 " 1.3
7.0 " 5.6 3.0 " 2.8
2.7 " 1.2 2.8 " 1.9
42.0 " 8 b 43.0 " 15 b
Mean MN-PCE per 1000 PCE Ž"SD.
DMEA Žmgrkg.
Water
270
540
860
22 " 3 32 " 8
22 " 5 27 " 7
22 " 8 28 " 9
3.2 " 2.3 1.4 " 1.5
19 " 5 42 " 14
20 " 6 30 " 8
18 " 4 32 " 9
30 " 3 32 " 8
33 " 3 33 " 8
36 " 15 33 " 10
Mean PCEr1000 NCE Ž"SD. Water
TEM
TEM
DMEA Žmgrkg. 270
540
860
3.8 " 1.5 2.4 " 2.1
3.8 " 2.4 1.2 " 0.8
1.6 " 1.1 2.0 " 1.4
3.6 " 1.5 1.4 " 1.1
3.2 " 1.9 0.8 " 0.8
4.0 " 2.6 1.4 " 1.1
3.4 " 2.3 1.0 " 1.4
2.8 " 1.5 1.6 " 1.1
2.8 " 1.3 1.8 " 1.5
3.2 " 1.1 1.8 " 2.1
3.2 " 1.9 0.8 " 1.1
20.8 " 3.6 24.8 " 8.7
b b
Mean MN-PCE per 1000 PCE Ž"SD.
t BDEA Žmgrkg.
Water
75
150
240
30 " 5 29 " 4
29 " 1 30 " 7
27 " 6 34 " 12
1.2 " 1.1 1.6 " 1.3
31 " 10 25 " 3
33 " 6 32 " 7
31 " 3 27 " 5
2.2 " 1.1 1.8 " 1.3
32 " 7 30 " 5
33 " 9 30 " 8
26 " 5 30 " 8
1.8 " 0.5 1.6 " 1.1
CP
11.2 " 1.5 11.0 " 1.2
t BDEA Žmgrkg.
a a
75
150
240
1.4 " 0.9 2.4 " 1.7
2.2 " 1.1 1.4 " 1.7
2.4 " 2.1 1.0 " 1.4
2.0 " 1.0 1.8 " 0.8
2.0 " 0.7 1.0 " 0.7
1.0 " 0.8 0.3 " 0.5
2.0 " 1.9 1.8 " 1.3
2.2 " 1.3 1.6 " 1.5
1.0 " 1.2 1.3 " 1.5
Water Ž10 mlrkg.; TEM, triethylenemelamine Ž0.3 mgrkg.; CP, cyclophosphamide Ž15 mgrkg.. PCE, polychromatic erythrocytes; NCE, normochromatic erythrocytes; MN-PCE, micronucleated polychromatic erythrocytes. Statistical analysis employed the Fisher’s Exact Test Ž1-tailed.: a p - 0.01; b p - 0.001.
MDEA, DMEA, and t BDEA consistently gave negative responses in a battery of in vitro and in vivo tests to assess the potential to produce gene mutation or DNA damage. Thus, it can be concluded that these alkylalkanolamines do not represent a genotoxic hazard. Similarly, the alkanolamines, mo-
noethanolam ine, diethanolam ine and tri ethanolamine, were also reported to be non-mutagenic w10x. The overall evidence therefore suggests that the family of ethanolamines or amino ethanols, whether alkyl substituted or not, as a whole is unlikely to be genotoxic.
H.-W. Leung, . Ballantyner Mutation Research 393 (1997) 7–15
References w1x E. Zeiger, B. Anderson, S. Haworth, T. Lawlor, K. Mortelmans, W. Speck, Salmonella mutagenicity tests: III. Results from the testing of 255 chemicals, Environ. Mutagen. 9 ŽSuppl. 9. Ž1987. 1–110. w2x B.N. Ames, J. McCann, F. Yamasaki, Methods for detecting carcinogens and mutagens with the Salmonellarmammalian-microsome mutagenicity test, Mutation Res. 31 Ž1975. 347–364. w3x F.D. deSerres, M.D. Shelby, Recommendations on data production and analysis using the Salmonellarmicrosome mutagenicity test, Mutation Res. 64 Ž1974. 159–165. w4x J.P. O’Neill, P.A. Brimer, R. Machanoff, G.P. Hirsch, A.W. Hsie, A quantitative test of mutation induction at the hypoxanthine-guanine phophoribosyl transferase locus in Chinese hamster ovary cells ŽCHOrHGPRT. system: development and definition of the system, Mutation Res. 45 Ž1977. 91– 101. w5x R.S. Slesinski, P.J. Guzzie, W.C. Hengler, P.G. Watanabe,
w6x
w7x w8x
w9x
w10x
15
M.D. Woodside, R.S.H. Yang, Assessment of genotoxic potential of ethylenediamine, in vitro and in vivo studies, Mutation Res. 124 Ž1983. 299–314. S.A. Latt, J. Allen, S.E. Bloom, A. Carrano, E.D. Falke, P. Kram, E. Schneider, R. Schreck, R. Tice, B. Whitfield, S. Wolff, Sister chromatid exchanges: a report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res. 87 Ž1981. 17–62. W. Schmid, The micronucleus test, Mutation Res. 31 Ž1975. 9–15. J.T. MacGregor, C.M. Wehr, D.H. Gould, Clastogen-induced micronuclei in peripheral blood erythrocytes: the basis of an improved micronucleus test, Environ. Mutagen. 2 Ž1980. 509–514. B.B. Gallapudi, O.P. Kamra, Application of a simple Giemsa-staining method in the micronucleus test, Mutation Res. 64 Ž1974. 45–46. J.B. Knaak, H.W. Leung, W.T. Stott, J. Busch, J. Bilsky, Toxicology of mono-, di-, and triethanolamine, Rev. Environ. Contam. Toxicol. 149 Ž1997. 1–86.