Arch Toxicol(1992) 66:368-371
Archives of
Toxdcology 9 Springer-Veflag1992
Short communication
Structure-toxicity relationship of ethylene glycol ethers Hideji Tanii, Sanae Saito, and Kazuo Hashimoto Department of Hygiene,School of Medicine, Kanazawa University,Kanazawa,Japan Received 14 November 1991/Accepted 12 February 1992
Abstract. The ultimate purpose of the present study was to evaluate correlations between acute in vivo and in vitro toxicity and log P (P is n-octanol-water partition coefficient). The in vitro toxicity to cloned cells (neuroblastoma N18TG-2 and glioma C6) in culture (EDs0) and the in vivo toxicity to mice (LDs0) of ethylene glycol ethers were studied in terms of the structure-activity relationship. The test ethers showed a wide range of EDs0 values in both cells. LD50 was determined under two conditions: LDs0-cont. was estimated in mice pretreated with olive oil and LDs0-CC14 in CCl4-pretreated mice. Multiple regression analyses revealed a significant correlation between log 1/LDs0 and log P as follows: log (1/LDso-cont.) = 0.120 (log P)2+0.4871og P-1.182, and log (1/LDs0-CC14) = -0.128 (log P)2+0.5661og P-1.157. There was no significant correlation either between EDs0 and LDs0 or between EDs0 for N18TG-2 and EDs0 for C6. The results suggest that metabolic activation might not occur during acute toxicity from the ethers, and that hydrophobicity, expressed as log P, plays an important role in acute toxicity.
Key words: Ethylene glycol monomethyl e t h e r - Ethylene glycol monoethyl ether - Ethylene glycol monoisopropyl ether - Ethylene glycol monoallyl ether - Ethylene glycol mono-n-butyl ether - Ethylene glycol monoisobutyl ether - Ethylene glycol mono-t-butyl ether - Ethylene glycol mono-n-hexyl ether - Ethylene glycol monophenyl ether Glioma - Mouse - Neuroblastoma - Structure-toxicity relationship
range of industrial and domestic use. Previous studies have shown that ethers have a wide spectrum of toxicity in laboratory animals, including developmental, hematologic and reproductive toxicity (Nagano et at. 1979; Nelson eta[. 1984; Hardin and Eisenmann 1987; Ghanayem et al. 1989). Qualitative structure-toxicity studies have also been carried out to ascertain their developmental (Nagano et al. 1979) and hematologic (Ghanayem et al. 1989) toxicity. In the past in an attempt to predict the toxicity of various chemicals, we have studied the structure-toxicity relationship of several, including acrylates and methacrylates (Tanii and Hashimoto 1982), nitriles (Tanii and Hashimot0 1984a), acrylamide and derivatives (Tanii and Hashimot0 1984b) and monoketones (Tanii et al. 1986). In these studies, the partition coefficient (P, n-octanol-water partition coefficient) has been proved to be one of the most important determinants in estimating their toxicity. In the present study, we examined the relevance of the P in determining the toxic effects of ethylene glycol ethers. Two types of toxicity were subjected to analysis: ED50 values for cloned cells in culture and LDs0 values for mice. In order to evaluate the effect of biotransformation of the ethers, LDs0 was determined under two conditions: LD50-CC14 in CCL4-prctreated mice and LDs0-cont. in olive oil-pretreated animals. CC14 is known to inactivate the hepatic microsomal monooxygenase system (Head et at. 1981; Fander et al. 1982), and may influence the detoxication of the ethers. CC14 also inhibits hepatic non-microsornal enzymes (Hjelle et al. 1983), including alcohol dehydrogenase which has been shown to be involved in the metabolic activation of the ethers (Rowe and Wolf 1982).
Introduction Materials and methods Ethylene glycol ethers are a class of chemicals which, because of their useful physical properties, have a wide Offprint requests to: Hideji Tanii, Department of Hygiene, School of Medicine, Kanazawa University, 13-1, Takara-machi, Kanazawa 920, Japan
All ethylene glycol ethers were purchased from Tokyo Kasei Co. (Tokyo),polyethyleneglycol 20 M on UniportB from Gasukuro Kogyo Co. (Tokyo), Dulbecco's Modified Eagle Medium (DMEM), fetal calf serum, penicillin and streptomycinfrom GIBCO (New York), and other materials from Wako Chemical Industries (Osaka). Cloned cells (N18TG-2 and C6) were the generousgifts of Dr. H. Higashida (Depart-
369
Table 1. Conditions for determining the partition coefficient Compound
Ethylene glycol monomethyl ether Ethylene glycol monoethyl ether Ethylene glycol monoisopropyl ether Ethylene glycol monoallyl ether Ethylene glycol mono-n-butyl ether Ethyleneglycol monoisobutyl ether Ethylene glycol mono-t-butyl ether Ethyleneglycol mono-n-hexyl ether Ethyleneglycol monophenyl ether
Structure
CHzOHCHzOCH3 CH2OHCH2OCH2CH3 CH2OHCH2OCH(CH3)2 CH2OHCH2OCH2CH=CH2 CH2OHCH20(CH2)3CH3 CH2OHCH2OCH2CH(CH3)z CH2OHCH2OC(CH3)3 CH2OHCH20(CH2)sCH3 CH2OHCH2OC6H5
Gas chromatography a Temperature (~
Volume for partitioning (ml)
Column
Detector
Water
n-Octanol
140 140 160 160 170 160 160 240 240
160 160 180 180 190 180 180 260 260
1.0 1.0 3.0 1.0 6.0 5.0 3.0 70.0 10.0
5.0 5.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
a Analysis was carried out using a Hitachi Model 163 gas chromatograph equipped with a flame ionization detector and glass column packed with polyethylene glycol 20 M The carrier gas was N2
mentof Biophysics, Neuroinformation Research Institute (NIRI), School ofMedicine, Kanazawa University). Activity of microsomal mixed function oxidase system is known to be high in liver and very low in the nervous system (Hodgson and Dauterman 1980). To our knowledge, no information was available on the microsomal mixed function oxidase system of either neuroblastoma NISTG-2 or glioma C6. Since both cell lines are derived from the nervous system, it may be expected that they lack in or have little of the metabolic activating systems found in liver. Thus, we used the two cell linesin order to evaluate the direct toxic effect of the ethers in vitro. Cells (0.5 • 106 for both N 18TG-2 and C6) were plated in Falcon plastic dishes (3002, 60 mm) and cultured in DMEM supplemented with 5% fetal calf serum, penicillin (100 units/ml) and streptomycin (100 units/ml) at 37 ~C in a humidified atmosphere of 10% CO2. The test chemicals, except ethylene glycol mono-n-hexylether which was insoluble in the medium, were added at various concentrations to the medium containing the ceils, at the beginning of culture. Six days after culturing, cells were collected fromthe dishes by trituration in calcium and magnesium-free phosphate buffered saline. Cells were counted with a hemocytometer and their viabilitieswere determined by the trypan blue dye exclusion method. The incubationtime was based on a preliminary experiment, where cells were culturedin the absence of test chemicals and their growth was examined. Maximal cell density was found 6 days after culturing for both types of cells, when the effect of test chemicals was examined. The EDso value was calculated after a Probit transformation of the cell viability, obtained at various concentrations of each test chemical. Male ddY mice (Japan SLC Co., Shizuoka, Japan) weighing about 25 g were used for determining the acute toxicity of the test chemicals in vivo.They were maintained on a 12-h light/dark cycle with free access to laboratorychow and water. Based on a previous report (Tanii and Hashimoto 1984a), the animals were given either 2.0 g/kg CCI4 dissolved in olive oil or olive oil only intraperitoneally, in a volume of 0.16 ml per 25 g, 24 h prior to receiving the test ethers, intraperitoneally. This dosage 0fCC14alone caused no death to animals during the experiment. The oral LDs0was determined according to Weil (1952) using four animals per doselevel, four different doses and 1 week observation period. The n-octanol-water partition coefficients of the test ethers were determinedaccording to the previous report (Tanii and Hashimoto 1982). Table 1 shows the conditions for gas-liquid chromatography and partitioning.Under these conditions, the ethers were clearly separated from n-octanol which was dissolved in water. The correlation coefficient was analyzed by Pearson's method and the structure-toxicity relationship by Hansch's model (Hansch and Fujita 1969), using log P. P values less than 0.05 were considered significant.
Results and discussion T h e c y t o t o x i c i t y (EDs0) for N I 8 T G - 2 and C 6 cells, acute toxicity (LDs0-cont. and LDs0-CCi4) in m i c e and h y d r o p h o b i c i t y (log P) o f the test ethers are s h o w n in T a b l e 2. ED5o values for N 1 8 T G - 2 ranged f r o m 0.51 to 10.4 m M , and those for C 6 f r o m 1.51 to 19.5 m M . There was no significant correlation b e t w e e n ED50 values for both cell types (r = 0.304). A significant difference in ED50 b e t w e e n N 1 8 T G - 2 and C6 was noted for e t h y l e n e g l y c o l m o n o i s o p r o p y l ether and e t h y l e n e g l y c o l m o n o a l l y l ether. LD50-cont. ranged f r o m 5.04 to 32.18 m m o l / k g , and LD50-CC14 f r o m 3.48 to 32.18 m m o l / k g . A l t h o u g h there was a g o o d correlation b e t w e e n the two t y p e s o f LD50 (r = 0.985, P
Archives of
Toxdcology 9 Springer-Veflag1992
Short communication
Structure-toxicity relationship of ethylene glycol ethers Hideji Tanii, Sanae Saito, and Kazuo Hashimoto Department of Hygiene,School of Medicine, Kanazawa University,Kanazawa,Japan Received 14 November 1991/Accepted 12 February 1992
Abstract. The ultimate purpose of the present study was to evaluate correlations between acute in vivo and in vitro toxicity and log P (P is n-octanol-water partition coefficient). The in vitro toxicity to cloned cells (neuroblastoma N18TG-2 and glioma C6) in culture (EDs0) and the in vivo toxicity to mice (LDs0) of ethylene glycol ethers were studied in terms of the structure-activity relationship. The test ethers showed a wide range of EDs0 values in both cells. LD50 was determined under two conditions: LDs0-cont. was estimated in mice pretreated with olive oil and LDs0-CC14 in CCl4-pretreated mice. Multiple regression analyses revealed a significant correlation between log 1/LDs0 and log P as follows: log (1/LDso-cont.) = 0.120 (log P)2+0.4871og P-1.182, and log (1/LDs0-CC14) = -0.128 (log P)2+0.5661og P-1.157. There was no significant correlation either between EDs0 and LDs0 or between EDs0 for N18TG-2 and EDs0 for C6. The results suggest that metabolic activation might not occur during acute toxicity from the ethers, and that hydrophobicity, expressed as log P, plays an important role in acute toxicity.
Key words: Ethylene glycol monomethyl e t h e r - Ethylene glycol monoethyl ether - Ethylene glycol monoisopropyl ether - Ethylene glycol monoallyl ether - Ethylene glycol mono-n-butyl ether - Ethylene glycol monoisobutyl ether - Ethylene glycol mono-t-butyl ether - Ethylene glycol mono-n-hexyl ether - Ethylene glycol monophenyl ether Glioma - Mouse - Neuroblastoma - Structure-toxicity relationship
range of industrial and domestic use. Previous studies have shown that ethers have a wide spectrum of toxicity in laboratory animals, including developmental, hematologic and reproductive toxicity (Nagano et at. 1979; Nelson eta[. 1984; Hardin and Eisenmann 1987; Ghanayem et al. 1989). Qualitative structure-toxicity studies have also been carried out to ascertain their developmental (Nagano et al. 1979) and hematologic (Ghanayem et al. 1989) toxicity. In the past in an attempt to predict the toxicity of various chemicals, we have studied the structure-toxicity relationship of several, including acrylates and methacrylates (Tanii and Hashimoto 1982), nitriles (Tanii and Hashimot0 1984a), acrylamide and derivatives (Tanii and Hashimot0 1984b) and monoketones (Tanii et al. 1986). In these studies, the partition coefficient (P, n-octanol-water partition coefficient) has been proved to be one of the most important determinants in estimating their toxicity. In the present study, we examined the relevance of the P in determining the toxic effects of ethylene glycol ethers. Two types of toxicity were subjected to analysis: ED50 values for cloned cells in culture and LDs0 values for mice. In order to evaluate the effect of biotransformation of the ethers, LDs0 was determined under two conditions: LD50-CC14 in CCL4-prctreated mice and LDs0-cont. in olive oil-pretreated animals. CC14 is known to inactivate the hepatic microsomal monooxygenase system (Head et at. 1981; Fander et al. 1982), and may influence the detoxication of the ethers. CC14 also inhibits hepatic non-microsornal enzymes (Hjelle et al. 1983), including alcohol dehydrogenase which has been shown to be involved in the metabolic activation of the ethers (Rowe and Wolf 1982).
Introduction Materials and methods Ethylene glycol ethers are a class of chemicals which, because of their useful physical properties, have a wide Offprint requests to: Hideji Tanii, Department of Hygiene, School of Medicine, Kanazawa University, 13-1, Takara-machi, Kanazawa 920, Japan
All ethylene glycol ethers were purchased from Tokyo Kasei Co. (Tokyo),polyethyleneglycol 20 M on UniportB from Gasukuro Kogyo Co. (Tokyo), Dulbecco's Modified Eagle Medium (DMEM), fetal calf serum, penicillin and streptomycinfrom GIBCO (New York), and other materials from Wako Chemical Industries (Osaka). Cloned cells (N18TG-2 and C6) were the generousgifts of Dr. H. Higashida (Depart-
369
Table 1. Conditions for determining the partition coefficient Compound
Ethylene glycol monomethyl ether Ethylene glycol monoethyl ether Ethylene glycol monoisopropyl ether Ethylene glycol monoallyl ether Ethylene glycol mono-n-butyl ether Ethyleneglycol monoisobutyl ether Ethylene glycol mono-t-butyl ether Ethyleneglycol mono-n-hexyl ether Ethyleneglycol monophenyl ether
Structure
CHzOHCHzOCH3 CH2OHCH2OCH2CH3 CH2OHCH2OCH(CH3)2 CH2OHCH2OCH2CH=CH2 CH2OHCH20(CH2)3CH3 CH2OHCH2OCH2CH(CH3)z CH2OHCH2OC(CH3)3 CH2OHCH20(CH2)sCH3 CH2OHCH2OC6H5
Gas chromatography a Temperature (~
Volume for partitioning (ml)
Column
Detector
Water
n-Octanol
140 140 160 160 170 160 160 240 240
160 160 180 180 190 180 180 260 260
1.0 1.0 3.0 1.0 6.0 5.0 3.0 70.0 10.0
5.0 5.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
a Analysis was carried out using a Hitachi Model 163 gas chromatograph equipped with a flame ionization detector and glass column packed with polyethylene glycol 20 M The carrier gas was N2
mentof Biophysics, Neuroinformation Research Institute (NIRI), School ofMedicine, Kanazawa University). Activity of microsomal mixed function oxidase system is known to be high in liver and very low in the nervous system (Hodgson and Dauterman 1980). To our knowledge, no information was available on the microsomal mixed function oxidase system of either neuroblastoma NISTG-2 or glioma C6. Since both cell lines are derived from the nervous system, it may be expected that they lack in or have little of the metabolic activating systems found in liver. Thus, we used the two cell linesin order to evaluate the direct toxic effect of the ethers in vitro. Cells (0.5 • 106 for both N 18TG-2 and C6) were plated in Falcon plastic dishes (3002, 60 mm) and cultured in DMEM supplemented with 5% fetal calf serum, penicillin (100 units/ml) and streptomycin (100 units/ml) at 37 ~C in a humidified atmosphere of 10% CO2. The test chemicals, except ethylene glycol mono-n-hexylether which was insoluble in the medium, were added at various concentrations to the medium containing the ceils, at the beginning of culture. Six days after culturing, cells were collected fromthe dishes by trituration in calcium and magnesium-free phosphate buffered saline. Cells were counted with a hemocytometer and their viabilitieswere determined by the trypan blue dye exclusion method. The incubationtime was based on a preliminary experiment, where cells were culturedin the absence of test chemicals and their growth was examined. Maximal cell density was found 6 days after culturing for both types of cells, when the effect of test chemicals was examined. The EDso value was calculated after a Probit transformation of the cell viability, obtained at various concentrations of each test chemical. Male ddY mice (Japan SLC Co., Shizuoka, Japan) weighing about 25 g were used for determining the acute toxicity of the test chemicals in vivo.They were maintained on a 12-h light/dark cycle with free access to laboratorychow and water. Based on a previous report (Tanii and Hashimoto 1984a), the animals were given either 2.0 g/kg CCI4 dissolved in olive oil or olive oil only intraperitoneally, in a volume of 0.16 ml per 25 g, 24 h prior to receiving the test ethers, intraperitoneally. This dosage 0fCC14alone caused no death to animals during the experiment. The oral LDs0was determined according to Weil (1952) using four animals per doselevel, four different doses and 1 week observation period. The n-octanol-water partition coefficients of the test ethers were determinedaccording to the previous report (Tanii and Hashimoto 1982). Table 1 shows the conditions for gas-liquid chromatography and partitioning.Under these conditions, the ethers were clearly separated from n-octanol which was dissolved in water. The correlation coefficient was analyzed by Pearson's method and the structure-toxicity relationship by Hansch's model (Hansch and Fujita 1969), using log P. P values less than 0.05 were considered significant.
Results and discussion T h e c y t o t o x i c i t y (EDs0) for N I 8 T G - 2 and C 6 cells, acute toxicity (LDs0-cont. and LDs0-CCi4) in m i c e and h y d r o p h o b i c i t y (log P) o f the test ethers are s h o w n in T a b l e 2. ED5o values for N 1 8 T G - 2 ranged f r o m 0.51 to 10.4 m M , and those for C 6 f r o m 1.51 to 19.5 m M . There was no significant correlation b e t w e e n ED50 values for both cell types (r = 0.304). A significant difference in ED50 b e t w e e n N 1 8 T G - 2 and C6 was noted for e t h y l e n e g l y c o l m o n o i s o p r o p y l ether and e t h y l e n e g l y c o l m o n o a l l y l ether. LD50-cont. ranged f r o m 5.04 to 32.18 m m o l / k g , and LD50-CC14 f r o m 3.48 to 32.18 m m o l / k g . A l t h o u g h there was a g o o d correlation b e t w e e n the two t y p e s o f LD50 (r = 0.985, P
