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In vitro and in vivo evaluation of the genotoxic potential of 2-ethyl-1,3-hexanediol
Toxicology, 53 (1988) 179--198 Elsevier Scientific Publishers Ireland Ltd.
IN V I T R O A N D IN VIVO E V A L U A T I O N O F T H E G E N O T O X I C POTENTIAL OF 2-ETHYL-I,3-HEXANEDIOL*
R.S. SLESINSKIa'**~P.J. GUZZIE~, D.L. PUTMAN b and B. BALLANTYNE~ aBushy Run Research Center, Export, PA, b Microbiological Associates, Inc., Bethesda, M D and cUrdon Carbide Corporation Danbury, C T (U.S.A.)
(Received February 8th, 1988) (Accepted April 16th, 1988)
SUMMARY 2-Ethyl-l,3-hexanediol ( E H D ) has intentional h u m a n exposure because of its application to skin as an insect repellent and its use in various skin care products. Genotoxicity studies on E H D were conducted to determine mutagenic and clastogenic potential using in vitro and in vivo test systems. In vitro tests were conducted both with and without an Aroclor-induced, rat-liver $9 metabolic activation system and within a range of cytotoxic to non-cytotoxic doses. E H D did not produce dose-related positive increases in gene mutations in the Salmonella (Ames) test or in the C H O / H G P R T forward mutation test. N o statistically significant or dose-related increases in sister chromatid exchanges indicative o f D N A damage were produced by E H D in C H O cells. Small but statistically significant increases in chromosome aberrations were produced in C H O cells only in tests with $9 activation. However, no evidence of clastogenicity of E H D was obtained in vivo in a mouse peripheral blood micronucleus test or in 2 rat bone marrow chromosome aberration studies using single or repeated dosing procedures. T h e overall negative pattern of mutagenic and clastogenic results in the majority of tests conducted suggests that E H D is unlikely to pose significant hazard as a genotoxic agent or to possess carcinogenic initiating activity in animals.
K e y words: 2-Ethylhexane 1,3-diol; Genetic toxicology; Mutagenicity; Genotoxic-
ity; Insect repellents; CAS No. 94-96-2 *Presented in part at the 26th Annual meeting of the Societyof Toxicology, February 1987. **Address all correspondence and reprint requests to: Dr. Ronald S. Slesinski, Bushy Run Research Center, R.D. No. 4 Mellon Road, Export, PA 15632, U.S.A. Abbreviations: CHO, Chinese hamster ovary; DMN, dimethylnitrosamine; EMS, ethylmethanesulfonate; mPCE, micronucleated polychromaticerythrocyte; NCE, normochromatic (mature) erythrocyte; SCE, sister chromatid exchange. 0300-483X/88/$03.50 © Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
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INTRODUCTION 2 - E t h y l - l , 3 - h e x a n e d i o l ( E H D ) is a colorless to pale-yellow liquid which acts as a solvent and blending agent for latex paints and printer's ink; at elevated t e m p e r atures, it is used as a reactive c o m p o n e n t in various urethane coatings. H u m a n exposure to E H D typically results f r o m its use as an emollient in cosmetic creams and lotions and as the active ingredient in insect repellent formulations (e.g. " 6 12"). Ballantyne et al. [1] reported on the acute toxicity of E H D to rats and showed that it was only slightly toxic by peroral and percutaneous routes (LDso >~ 5 ml/kg) and that no deaths and only signs of m i n o r irritation occurred following 6-h exposures to E H D aerosol at a dynamic concentration of 3.8 rag/1. T h e y noted only a mild degree of skin irritation after a 4-h occluded exposure, but mild to m o d e r a t e conjunctivitis, iritis and corneal injury were produced by volumes of E H D as low as 0.005 ml. L o w degrees of acute toxicity of E H D were also reported in earlier studies by Draize et al. [2] and S m y t h et al. [3]. Repeated 90day exposures of rats to E H D in drinking water revealed no remarkable toxicity except for growth retardation at 0.7 g/kg/day, which was approximately 1/4 of the L D s 0 dose [4]. I n this same report, only a low degree of toxicity was noted in a 90day garage study with dogs dosed with 0.5 ml/kg/day. E H D was concluded to lack toxic and carcinogenic potential in life-time skin application studies [5], although this study has been questioned because of apparent inconsistencies in tabulation of t u m o r incidences [6]. N o published information on genotoxic potential of E H D was discovered in an extensive search of c o m p u t e r data files. Because of the apparent lack of genotoxicity information on E H D , and in view of its widespread availability and h u m a n exposure, a battery of in vitro and in vivo tests was conducted to determine mutagenic and clastogenic potential of E H D . T h i s report presents data on the evaluation of E H D for effects upon gene mutations in Salmonella and C H O cells, D N A damage (SCE test with C H O cells) and c h r o m o s o m e damage in vitro and in vivo. MATERIALS AND METHODS Chemicals E H D (Synonym: Octylene glycol; CAS No. 94-96-2) was obtained from U n i o n Carbide Corporation, South Charleston, WV. E H D has a molecular weight of 146.2, specific gravity of 0.942, vapor pressure of <0.01 m m H g at 20°C, and a chemical formula of ( C ~ H T ) C H ( O H ) C H ( C 2 H s ) C H 2 O H . T h e purity of the E H D used for all studies, determined by gas chromatographic analyses, was 99.18°.:o (by wt) E H D , with 0.36°/,, m o n o b u t y r a t e ester isomers and less than 0.10% each of other unidentified impurities. E H D is stable and freely soluble in organic solvents with limited solubility to 4.2')~, in water. Ethyl alcohol, corn oil or cell-culture m e d i u m were used to prepare dilutions of E H D , and the vehicle control substances for the various test systems are specified in respective tables. Positive control chemicals used to verify the sensitivity of the various assays were all obtained f r o m Sigma Chemical Co., St. Louis, M O , except for: ethyl methanesulfonate ( E M S ; Eastman Organics, Rochester, NY), N - n i t r o s o -
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dimethylamine (DMN; Aldrich Chemical Co., Milwaukee, WI) and triethylenemelamine ( T E M , Polysciences, Inc., Warrington, PA). Water used to prepare media and solutions was obtained from a Millipore Milli Q® high purity system. Positive and negative control agents were the highest available commercial purity.
Animals Micronucleus test. Four-week-old, Swiss-Webster mice, obtained from Hilltop Lab Animals, Inc. (Scottdale, PA), were acclimated for 5--6 days prior to dosing. Five mice/sex/cage were housed in polycarbonate cages with wire tops and AbSorb-Dri ® bedding. T h e animal room used for the micronucleus test had a controlled environment with HEPA-filtered air, a 12-h light/dark photoperiod, temperature range of 19--22°C (66--72°F) and relative humidity range of 4 0 - 60%. Water and certified rodent chow (Agway Pro-Lab R M H 3000) were provided ad libitum. Animals were identified with unique laboratory animal numbers using ear tags. Cytogenetic study. Sprague--Dawley rats, 6 - - 8 weeks old, were obtained from Charles River Breeding Laboratories, Inc., Kingston, N.Y. Animals were quarantined for a 10--14-day period prior to dosing and evaluated daily for signs of illness or poor health. Rats were housed 3 - - 4 per cage during the quarantine period and singly thereafter in plastic shoe-box type cages with wire tops and hardwood bedding chips. Animals were provided with certified rodent chow and water ad libitum. Ear tags with sequential animal numbers were used for animal identification. T h e animal room had a controlled environment with a temperature range of 23 + 3°C (74 + 6°F), 50 + 20% relative humidity and a 12-h light/dark cycle. In vitro metabolic activation For all tests conducted with metabolic activation, an $9 liver homogenate from Sprague--Dawley male rats, induced with Aroclor 1254, was obtained from Microbiological Associates (Bethesda, MD) or Hazleton Biotechnologies (Kensington, MD). Each lot of $9 was screened by the supplier for metabolic activity. In addition, toxicity, metabolic activity and appropriate amounts of $9 for each test system were also determined in our laboratory. Cofactor concentrations in the $9 activation mixture were those recommended by Ames et al. [7], except that the mixture for tests employing CHO cells also contained 8 - - 1 0 # m o l of calcium chloride/ml [8]. T h e amounts of $9 homogenate for the various in vitro tests were: Ames test - - 50/A/plate; C H O / H G P R T test - - 50 #1/5 ml of culture medium; SCE test - - 15/d/5 ml of culture medium; and CHO chromosome aberration tests ~ 50 and 100/~1/5 ml of culture medium. Metabolic activation mixtures were freshly prepared prior to addition to the test system(s). Salmonella/microsome mutation (Ames) test S. typhimurium strains were obtained from Dr. B.N. Ames, University of California, Berkeley, CA. Overnight cultures (approx. 16-h) were grown in Oxoid broth No. 2 (Oxoid, USA, Columbia, MD) and the plate test was performed following standard procedures [7,9] and as described in previous reports by the authors [10--12]. E H D was tested up to cytotoxic concentrations, based upon
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preliminary dose-finding experiments, using triplicate plates/dose. A positive effect was considered to be a dose-related increase which was at least twice the concurrent solvent control value for at least 2 consecutive doses [13]. Animal cell culture procedures C H O cells, C H O - K I - B H 4 (Clone D 1), were a gift from Dr. A. Hsie, University of Texas, Galveston. Cells were grown in H a m ' s F12 medium (Grand Island Biological Co., G r a n d Island, NY) which lacked both hypoxanthine and antibiotics and medium was supplemented with 10% dialyzed fetal calf serum (FCS) for culture maintenance and 5% F C S for mutation determinations. Cells were incubated at 37°C with 5 - - 6 % C O 2 atmosphere and approximately 98% humidity as described previously [10--12]. C H O cells used for these studies have been repeatedly shown to be free of Mycoplasma contamination using fluorescence assays [14] in our laboratory and by culture procedures at commercial laboratories. C H O / H G P R T mutation test system T e s t methods have been described previously [ 10--12] and they were essentially identical to the procedures published by O'Neill et al. [15] and O'Neill and Hsie [16]. T h e range of test concentrations was chosen to include biologically effective doses based upon evidence of cytotoxic inhibition of cell division in preliminary tests. C H O cells were treated with E H D and control agents for 5 h, cultures were rinsed with phosphate-buffered physiological saline, refed with fresh medium and cell viability was determined after an 18-h recovery period. T h e percentage o f clonable cells and the incidence o f mutants resistant to 6-thioguanine (2/~g/ml) were determined after periodic replating o f 3 - - 5 x 105 cells (at 2 - - 3 - d a y intervals) during a 9-day mutation expression period. A positive effect was defined as evidence of a dose-related and statistically significant (P <~0.05) increase above the concurrent solvent control value [17]. S C E test Cell culture and chemical treatment procedures were essentially identical to methods described previously [10--12]. Chromosomes were stained for SCE differentiation by the F P G method o f Perry and Wolff [18]. Approximately 2.0 x 105 C H O cells were transferred to 75-cm 2 culture flasks 4 0 - - 4 8 h prior to treatment with the test agents to provide cells in exponential growth during testing. Cells were treated with test and control materials for 2 h with $9 activation and 5 h without activation in medium with 3/~g bromodeoxyuridine (BrdU)/ml. Chromosomes were harvested for SCE staining following a total culture interval of 2 4 - - 2 8 h in medium with BrdU. T e s t concentrations were determined from cytotoxicity data from preliminary cytotoxicity tests of cell growth inhibition. Cultures treated with the highest 3 concentrations which did not produce excessive cytotoxicity, evident by low mitotic indices or cell lysis, were evaluated for incidence of SCEs using coded slides to prevent bias. Data were analyzed by A N O V A and Duncan's Multiple Range T e s t to determine significance of differences from concurrent control values. A positive effect was defined as either a reproducible, statistically
182
significant increase for the duplicate cultures with at least 1 dose or as a significant, concentration-related increase in the SCE index. I n vitro cytogenetic test
C H O cell cultures in exponential growth were prepared by seeding 5 x 105 cells/75-cm 2 culture flask 3 6 ~ 4 8 h prior to the test procedure. Appropriate concentration ranges for E H D were determined by preliminary determination o f growth inhibition or decreases in mitotic indices. Cells were exposed to E H D and controls for the entire period until chromosome harvest in tests without metabolic activation (6 or 12 h). A 2-h exposure period to the test and control chemicals was used in tests with $9 activation to minimize cytotoxicity of liver homogenate to C H O cells. Various chromosome harvest times from 6 to 12 h were employed to assure an optimum detection of chromosome damage. Data from the SCE test indicated this sampling period was appropriate because no cytotoxic inhibition of mitotic progression was produced by E H D . Slides were coded to prevent observer bias and 50 cells/culture were examined for aberrations. A positive effect was defined by a statistically significant difference in the incidence of abnormal cells in comparison to concurrent control values and a concentration related trend in the dose-effect values. Fisher's Exact T e s t (one tailed) was used for statistical comparisons to the control using the combined duplicate-culture values. Micronucleus test with mice
Male and female, 4-week-old, Swiss-Webster mice were used to evaluate in vivo clastogenic potential of E H D using the peripheral blood test procedure [19]. Body weight ranges were 2 4 m 2 9 g for males and 2 0 m 2 3 g for females. A range of test concentrations were determined by a preliminary study to assess the toxicity of E H D (dissolved in 50% ethyl alcohol) using the intraperitoneal exposure route and 5 males and females/dose group. T h e L D s o (3-day holding period) was calculated by the Moving Average M e t h o d [20,21]. T h e volume of 50% ethanol vehicle for E H D was 0.05ml/mouse (0.9--1.1mg/kg) and this dose did not cause overt toxicity nor alter the frequency of micronuclei in comparison to undosed controls. Doses of 80%, 50% and 25% of the respective LDso dose were used in the micronucleus test. Females were also exposed to 1 additional dose calculated as 80% of the relatively higher male LDso value. A total of 2500 polychromatic erythrocytes (PCE)/concentration/sex was examined for incidence of mPCEs. T h e PCE to N C E ratio was determined for 1000 cell/animal to assess bone marrow cytotoxicity. A positive effect was defined by a dose-related trend in the incidence of m P C E s with at least 1 statistically significant (P < 0.01; Fisher's Exact T e s t - - one tailed) indication of a difference from the vehicle control. Incidences of m P C E s from males and females were pooled for statistical analyses if no significant sexrelated differences were indicated by statistical comparisons. Bone m a r r o w cytogenetic tests
Male and female, S p r a g u e - - D a w l e y rats, 6 - - 8 weeks o f age, were used for preliminary dose-finding and cytogenetic studies. Five males and females per dosage group were dosed by i.p. injection with test and control materials. F o r the 183
cytogenetic studies, males ranged in weight from 191 to 230 g and females ranged f r o m 141 to 170 g. One or two extra animals were added to the highest dosage groups to provide sufficient group sizes in case of deaths. T h e m a x i m u m tolerated dose was determined in preliminary tests as the highest concentration which produced weight loss or clinical signs of toxicity without producing death. T h e corn oil vehicle and E H D - v e h i c l e mixture was given at a constant volume of 5 ml/kg. Colchicine (1.0 mg/kg) was given by i.p. injection at 2 - - 4 h prior to sacrifice o f the animals. C h r o m o s o m e s were prepared by flushing bone m a r r o w f r o m femurs and fixation by standard procedures [22]. All slides were coded and 100 cells/animal were evaluated randomly. C h r o m o s o m e s were sampled at 12, 24 and 48 h after acute dosing and 6 h after the last dose in the repeat dose study. D a t a were c o m p a r e d for positive increases above concurrent controls using ~(2 analysis and P ~< 0.05 was considered a significant positive effect.
Penetration of bone marrow by E H D Ability of E H D to enter bone m a r r o w ceils was determined using male S p r a g u e - - D a w l e y rats ( 2 8 0 m 3 1 0 g) and 1,3,[14C]EHD (Spec. act 5.4 m C i / m m o l ) . A 600 m g / k g dose of E H D , identical to that used for the bone m a r r o w cytogenetic test, contained 10/~Ci/kg of [14C]EHD and was administered in corn oil by i.p. injection. Animals were sacrificed at 0.5, 1, 2, 4 and 8 h post injection and radioactivity in blood and bone m a r r o w f r o m 2 animals/sacrifice was quantified b y liquid scintillation spectrometry. Good laboratory practices T h e genotoxicity studies on E H D were carried out in full compliance with the Environmental Protection Agency and F o o d and D r u g Administration G o o d L a b o r a t o r y Practices requirements. RESULTS
Ames test D a t a summarized in T a b l e I show that E H D did not produce positive increases in the n u m b e r s of histidine revertants in tests with and without a rat-liver $9 metabolic activation system. N o dose-related increases in the incidence o f m u t a n t s was observed with any of the 5 tester strains and none of the quantitative increases attained a doubling over the respective solvent control value. T h e test was conducted to a cytotoxic concentration limit and cytotoxicity was evident b o t h b y the relative decline in the m u t a n t values at the highest dose and by inhibitory effects u p o n the background lawn in this and a preliminary test. Positive control agents produced highly elevated n u m b e r s of m u t a n t s above the values for the ethanol (100%) vehicle. T h e vehicle control m u t a n t values were within typical ranges for this test f r o m data at our laboratory and others [9]. C H O / H G P R T gene mutation Evaluation of mutagenic potential of E H D to C H O cell cultures is s u m m a r i z e d in T a b l e I I . T h e survival data in the first column indicate that biologically effective
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doses were tested and that the cytotoxic effects were detected despite the 18 h recovery period before determination of colony-forming ability. T h e mean percentage of clonable cells in the second column shows that after 9 - - 1 0 days of expression for the m u t a n t phenotype, the colony-forming potential of most E H D exposed cultures was essentially the same as the solvent control values. In the last column, the mutation index corrected for the respective colony-forming percentages did not reveal a dose-related trend in the n u m b e r of m u t a n t colonies in the tests conducted with or without a rat-liver activation system. However, in the test without $9 activation, 2 statistical indications of an increase above the solvent control values were produced b y non-consecutive doses of 1.0 and 4.0 mg/ml. T h i s statistical indication was not considered a positive test result because of the absence of a dose-related trend, the lack of agreement in duplicate cultures and the values were within the historical negative control range for this test at our laboratory. T h e positive control data showed that the cells were responsive to known mutagens, E M S and D M N , and mutants increased in relation to D M N concentration in tests with $9. With negative control cultures, the low m u t a n t values obtained are typical for this particular subclone [10--12]; although higher values up to 25 mutants/106 clonable cells are seen occasionally and are considered m a x i m u m acceptable limits for negative control data in our laboratory. S C E test T h e data for the S C E evaluation of D N A damage potential of E H D are shown in T a b l e I I I . In the test of direct genotoxic potential without $9 activation, none of the highest 3 of 5 exposure doses evaluated for SCEs produced a significant increase in SCEs above the concurrent negative control values. N o evidence of a doserelated trend relative to E H D concentration was apparent, and all values for treated cultures were essentially identical to the solvent control value. T h e 3.0 m g / m l dose produced excessive cytotoxicity and could not be evaluated for SCEs because of a low mitotic index. With rat-liver $9 activation, essentially identical negative results were seen as in the test without $9 activation. T h e shorter exposure time of 2 h used for the test with $9 did not produce the excessive cytotoxicity seen with the 3.0 m g / m l dose in the 5 h exposure for the test without $9. For both tests, the respective positive control agents E M S and D M N produced highly increased and statistically significant n u m b e r s of SCEs relative to controls which demonstrated the responsiveness of the test system. N o indication of a dose-related cytotoxic inhibition of mitosis was evident in the relative ratios of cells in the first, second or third r o u n d of cell division. E H D appeared to lack D N A - d a m a g e potential u n d e r conditions of this test system. In vitro chromosome damage E H D did not increase the incidence of c h r o m o s o m e aberrations in C H O cells when evaluated for direct clastogenic activity without $9 (Table IV). T h e top doses used for the tests with and without $9 activation were chosen from preliminary test data which showed respective decreases of 4 0 - - 6 0 % in culture growth and reduced mitotic indices. Essentially identical, non-significant differences f r o m control values were observed after continuous exposure to E H D and for sampling times o f
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6, 8, 10 and 12 h. Values for 8- and 12-h sampling times are shown in T a b l e IV; the 6- and 10-h data showed even lower relative increases over respective controls and were not included in T a b l e I V for brevity. With metabolic activation, using 2 concentrations of $9 homogenate, statistically significant increases in c h r o m o s o m e aberrations were observed only within a short time span of c h r o m o s o m e sampling. Positive effects were seen in 1 test with the 8 h but not 12-h blood sample. A second test was conducted with a higher and o p t i m u m $9 concentration of 100/~1 $9/5 ml m e d i u m which was determined in a preliminary study using 3.0 m g / m l of E H D . Positive, statistically significant increases were observed at 10 h but values were only marginally positive at 12 h. T h e 6- and 10-h E H D values in this test using 100/~1 of $9 were not statistically different f r o m controls and are not shown in T a b l e I V for brevity. T h e majority of the c h r o m o s o m e damage in the samples with positive effects was simple chromatid and c h r o m o s o m e breakage, which are likely lethal cellular events. N o remarkable evidence of potentially inheritable damage such as chromatid and c h r o m o s o m e exchanges was observed at any sampling time for tests with and without $9 activation. M i c r o n u d e u s test
T a b l e V shows the production of micronucleated polychromatic erythrocytes ( m P C E s ) in peripheral blood samples of mice exposed to a range of E H D concentrations f r o m 18.75 to 120 pg/kg. T h i s range of doses was based u p o n the determination that the i.p. L D s 0 of E H D to males was 1 5 4 m g / k g (95% C . I . - - 1 0 3 - - 2 1 3 ) and 7 5 m g / k g (95% C.I. = 3 5 - - 1 2 0 ) for females. Following injection of E H D , sedation, lethargy and periocular encrustation were observed with the highest 2 doses. N o significant or dose-related increases in m P C E s were p r o d u c e d by E H D in cells collected at blood sampling times of 24, 48 or 72 h. E H D did not produce remarkable bone m a r r o w cytotoxicity which was evident by similar P C E / N C E ratios for all treated and control samples (data not shown in T a b l e V). A few animals died at higher dose levels which indicated that a m a x i m u m tolerated dose was achieved (or exceeded) but no positive effects upon micronucleus frequencies were produced by these high levels of E H D exposure. T h e positive control substance T E M produced elevated and significant increases of m P C E s indicating the appropriate sensitivity o f the test animals and suitable test prodedures. B o n e m a r r o w cytogenetic tests
F o r acute and repeated-dose protocols, rats were dosed by i.p. injection with 60, 200 and 6 0 0 m g E H D / k g . Animals appeared sedated by the highest dose and moderate loss of weight ( - 4 - - - - 6 % ) was observed on the day of sacrifice in both the acute and repeated dose study. Clinical signs of toxicity were essentially identical with b o t h single and multiple injections of E H D , which indicated a lack o f cumulative toxicity. H o w e v e r , 1 male and 2 females repeatedly dosed with 600 m g / k g died and were replaced with extra dosed animals in order to assure complete group sizes. T h e incidence of c h r o m o s o m e aberrations did not differ between males and females, thus values were pool,ed for statistical analyses. N o significant increases in c h r o m o s o m e damage were observed regardless of sampling
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time or following single or multiple injections with E H D (Table VI). T E M produced a highly significant increase in chromosome aberrations and the corn oil controls were in a low and acceptable range for a valid test. N o evidence o f clastogenic potential in vivo was observed in either test even at the high range o f doses used for this study. Penetration of bone marrow by E H D Measurements of radioactivity in bone marrow and plasma samples from animals dosed by i.p. injection of [14C]EHD in corn oil (Table V I I ) showed that penetration into bone marrow was very rapid and detectable at the first sacrifice (0.5 h) after dosing. T h e radioactivity in bone marrow was detectable for at least 8 h post dosing which was the final sacrifice. T h e bone marrow/plasma ratios were essentially the same between 2 and 8 h indicating that a steady-state condition between radioactive uptake and elimination was established. These data strengthened the conclusion of negative clastogenicity of E H D by demonstration o f the potential for exposure of the bone marrow cells to E H D which did not lead to positive effects in the bone marrow cytogenetic studies. DISCUSSION T h e battery of genotoxicity tests conducted in this study was prompted by the apparent lack of short-term test data on E H D and by the desire to develop a weightof-evidence consensus from multiple genetic end-points. T h e results from the Ames assay, C H O / H G P R T gene mutation and the SCE tests (Tables I, II, I I I ) uniformly indicated that E H D lacked mutagenic and D N A damage potential in tests conducted with and without $9 activation. I n contrast, positive indications of clastogenic potential were obtained reproducibly in 2 in vitro chromosome damage tests with C H O cells when performed in the presence, but not in the absence, of a rat-liver $9 metabolic activation system (Table IV). T h e positive effects were evident only for a brief time span following dosing in cells sampled at either 8 or
TABLE VII DETERMINATION OF POTENTIAL FOR ENTRY OF RADIOACTIVITY FROM [14C]EHD INTO RAT BONE MARROW Sampling time (h) 0.5 1.0 2.0 4.0 8.0
Plasma(p)l
Bone marrow (BM)a
DPM/g
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DPM/g
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75,373 99,585 162,058 101,848 35,730
946 1,250 2,035 1,279 449
52,261 57,016 64,266 50,895 15,904
656 716 807 639 200
BM/P ratio 0.69 0.57 0.46 0.50 0.45
"Mean values of bone marrow and plasma samples from 2 animals except at 2 h where 1 animal was incorrectly dosed and not included.
195
10 h (depending upon $9 concentration), following the start of the 2 h exposure. Cells sampled +_2 h or greater from this optimum time with each respective $9 concentration did not reveal remarkable clastogenicity. Also, the positive increases attained a plateau value which did not increase with higher doses. T h e limited effective time span and narrow range of doses producing the statistically significant increases p r o m p t e d further in vivo studies to determine the significance of the in vitro suggestions of clastogenicity. Evaluation of E H D by the peripheral blood micronucleus method in mice did not show positive or dose-related increases in micronuclei at doses of 80% or higher than the respective 3-day male and female LDs0 do~es. Blood was sampled at 3 different time periods but no significant increases in m P C E s were observed at 24, 48 or 72 h after dosing. Although recent reports [23] suggest that micronucleus determinations with higher numbers o f m P C E than evaluated here provide less sampling variance, it is unlikely that counting more cells would alter the conclusion from this test because of the essential similarity of the m P C E values for E H D treated animals and controls. Also, preparation of uniform blood smears may have less variability than bone marrow slides because of the relatively greater homogeneity of blood samples. T h e absence of in vivo clastogenic potential of E H D was confirmed by an independent bone-marrow cytogenetic assay conducted by 1 o f the authors ( D L P ) in a second laboratory. Again, the results of this bone-marrow study showed that no significant increases in chromosome damage were evident following acute or repeated doses of E H D , even with doses which killed some of the animals. Detection of radioactivity in bone marrow cells of rats administered [14C]EHD by i.p. injection strengthened the conclusion on negative clastogenicity by demonstrating that the test chemical (and/or metabolites) had the opportunity for interaction. T h e rapid appearance of radioactivity in bone marrow and steady-state bone marrow: plasma ratios up to 8 h indicated that there was ample time available for expression of clastogenic potential. T h e absence of genotoxic activity for E H D in the majority of the tests conducted is in agreement with the lack of carcinogenic activity following lifetime dermal applications to mice (Stenbiick and Shubik, 1974). Also, the related molecule 2ethylhexanol, which differs only by absence of 1 hydroxyl group on the third carbon, was shown to lack genotoxicity in several studies employing a variety of test systems [24--27]. T h e reason for the positive effects of E H D in the in vitro chromosome damage test, in view of the majority of negative tests, is not known. However, there are many other examples o f chemicals which produce unexpected or singular positive effects when subjected to a battery of sensitive screening tests [28]. Possible explanations for the transient clastogenic activity observed in this study may include conversion of E H D into an active but unstable metabolite by the in vitro $9 activation system. However, the possibility that the positive clastogenicity may be an artefact of the in vitro test conditions cannot be excluded. For example, Galloway et al. [29] found that spurious positive increases in chromosome aberrations in C H O cells in vitro can be produced by high con3entrations and osmolalities of apparently inocuous substances such as KC1, NaC1 and sucrose. Notably, they found that the quantitative responses to these agents was in the same range of 0.13--0.16 aberrations/cell that we obtained with E H D in these studies. In
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c o n c l u s i o n , a l t h o u g h w e a k l y p o s i t i v e i n c r e ases w e r e seen in c h r o m o s o m e a b e r r a t i o n s in v i t r o , t h e lack o f g e n o t o x i c i t y i n t h e m a j o r i t y o f in v i t r o a n d in v i v o tests s u g g e s t s t h a t E H D is u n l i k e l y to pose a significant g e n o t o x i c h a z a r d o r to b e an i n i t i a t i n g a g e n t in a n i m a l c a r c i n o g e n i c i t y tests. ACKNOWLEDGEMENTS W e t h a n k W . C . H e n g l e r for t e c h n i c a l assistance w i t h s o m e o f t h e in v i t r o s t u d i e s an d M s . K a t h y M c C a b e for assistance in p r e p a r a t i o n o f t h e m a n u s c r i p t . T h e p e r f o r m a n c e o f t h e [ 1 4 C ] E H D b o n e m a r r o w e x p e r i m e n t s b y D r . C.B. J e n s e n is g r a t e f u l l y a c k n o w l e d g e d . T h i s w o r k was s p o n s o r e d by t h e U n i o n C a r b i d e Corporation. REFERENCES 1 B. Bailantyne, D.R. Klorme, R.C. Myers and D.J. Nachreiner, The acute toxicity and primary irritancy of 2-ethyl-l,3-hexanediol. Vet. Human Toxicol., 27 (1985) 491. 2 J.H. Draize, E. Alvarez, M.F. Whitesell, G. Woodard, E.C. Hagan and A.A. Nelson, Toxicological investigations of compounds proposed for use as insect repellents. J. Pharmacol. Exp. Ther., 43 (1948) 26. 3 H.F. Smyth, Jr., C.P. Carpenter and C.S. Weil, Range-finding toxicity data: List IV. Arch. Ind. Hyg. Occ. Med., 4 (1951) 119. 4 H.F. Smyth, C.P. Carpenter and C.S. Weil, Ninety-day repeated oral doses of 2-ethylhexanediol1,3 to dogs. Report 17-37 Carbide and Carbon Chemicals, unpublished report (1954) by Mellon Institute of Industrial Research, Pittsburgh, PA. 5 F. Stenb~ickand P. Shubik, Lack of toxicity and carcinogenicity of some commonly used cutaneous agents. Toxicol. Appl. Pharmacol., 30 (1974) 7. 6 Environmental Protection Agency, 2-Ethyi-l,3-Hexanediol, Pesticide Registration Standard. Office of Pesticides and Toxic Substance (1981) pp. 34--35. 7 B.N. Ames, J. McCann and E. Yamasaki, Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res., 31 (1975) 347. 8 E.L. Tan and A.W. Hsie, Effect of calcium phosphate and alumina ~C gells on the mutagenicity and cytotoxicity of dimethylnitrosamine as studied in the CHO/HGPRT system. Mutat. Res., 84 (1981) 147. 9 D.M. Maron and B.N. Ames, Revised methods for the Salmonella mutagenicity test. Murat. Res., 113 (1983) 173. 10 R.S. Slesinski, W.C. Hengler, P.J. Guzzie and K.J. Wagner, Mutagenicity evaluation of glutaraldehyde in a battery of in vitro bacterial and mammalian test systems. Food Chem. Toxicol., 21 (1983,a) 621. 11 R.S. Slesinski, P.J. Guzzie, W.C. Hengler, P.G. Wantanabe, M.D. Woodside and R.S.H. Yang, Assessment of genotoxic potential of ethylenediamine: in vitro and in vivo studies. Murat. Res., 124 (1983,b) 299. 12 R.S. Slesinski, P.J. Guzzle, W.C. Hengler, R.C. Myers and B. Ballantyne, Studies on the acute toxicity, primary irritancy and genotoxic potential of 1,3,5-triacryloylhexahydro-s-triazine (TAHT). Toxicology, 40 (1986) 145. 13 K.C. Chu, K.M. Patel, A.H. Lin, R.E. Tarone, M.S. Linhart and V.C. Dunkel, Evaluating statistical analyses and reproducibility of microbial mutagenicity assays. Mutat. Res., 85 ( 1981) 119. 14 T.R. Chen, In situ detection ofmycuplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell Res., 104 (1977) 255. 15 J.P. O'Neill, P.A. Brimer, R. Machanoff, G.P. Hirsch and A.W. Hsie, A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary ceils (CHO/HGPRT System): development and definition of the system. Murat. Res., 45 (1977) 91. 197
16 J.P. O'Neill and A.W. Hsie, Phenotypic expression time of mutagen induced 6-thioguanine resistance in Chinese hamster ovary cells ( C H O / H G P R T system). Mutat. Res., 59 (1979) 109. 17 J.D. Irr and R.D. Snee, Statistical evaluation of mutagenicity in the C H O / H G P R T system. Proc. of the Cold Spring Harbor - - Banbury Conference II (1979) pp. 263--274. 18 P. Perry and S. Wolff, New Giemsa method for differential staining of sister chromatids. Nature, 252 (1974) 156. 19 J.T. MacGregor, C.M. Wehr and D.H. Gould, Clastogen-induced micronuclei in peripheral blood erythrocytes: the basis of an improved micronucleus test. Environ. Mutagen. 2 (1980) 509. 20 W.R. Thompson, Use of moving averages and interpolation to estimate median effective dose. Bact. Rev., 11 (1947) 115. 21 C.S. Weil, Tables for convenient calculation of median-effective dose (LD50 or ED50) and instructions for their use. Biometrics, 8 (1952) 249. 22 W.W. Nichols, R.C. Miller and C. Bradt, In vitro anaphase and metaphase preparations in mutation testing, in B.J. Kilbey, M. Legator, W. Nichols and C. Ramel (Eds.), Handbook of Mutagenicity Test Procedures, Elsevier Publ. Co., NY, 1979, p. 225. 23 J. Ashby and R. Mohammed, Slide preparation and sampling as major sources of variability in the mouse micronucleus assay. Mutat. Res., 164 (1986) 217. 24 P.E. Kirby, R.F. Pizzarello, T.E. Lawlor, S.R. Haworth and J.R. Hodgson, Evaluation of di-(2ethylhexyl)-phthalate and its major metabolites in the Ames test and LSI78Y mouse lymphoma assay. Environ. Mutagen. 5 (1983) 657. 25 B.J. Phillips, T.E.B. James and S.D. Gangolli, Genotoxicity studies of di(2-ethylhexyl)phthalate and its metabolites in CHO cells. Mutat. Res., 102 (1982) 297. 26 D.L. Putman, W.A. Moore, L.M. Schechtman and J.R. Hodgson, Cytogenetic evaluation of di-(2ethyl-hexyl)phthalate and its major metabolites in Fischer 344 rats. Environ. Mutagen. 5 (1983) 227. 27 E. Zeiger, S. Haworth, K. Mortelmans and W. Speck, Mutagenicity testing of di(2-ethylhexyl)phthalate and related chemicals in Salmonella. Environ. Mutagen., 7 (1985) 213. 28 F.J. de Serres and J. Ashby, Evaluation of short-term tests for carcinogens. Report of the International Collaborative Program. Progress in Mutat. Res. 1, Elsevier/North Holland, N.Y., 1981, p. 112. 29 S.M. Galloway, C.L. Bean, M.A. Armstrong, D. Dcasy, A. Kraynak and M.O. Bradley, False positive in vitro chromosome aberration tests with non-mutagens at high concentrations and osmolalities. Environ. Mutagen., 7 (Suppl. 3) (1985) 48 (abstract).
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IN V I T R O A N D IN VIVO E V A L U A T I O N O F T H E G E N O T O X I C POTENTIAL OF 2-ETHYL-I,3-HEXANEDIOL*
R.S. SLESINSKIa'**~P.J. GUZZIE~, D.L. PUTMAN b and B. BALLANTYNE~ aBushy Run Research Center, Export, PA, b Microbiological Associates, Inc., Bethesda, M D and cUrdon Carbide Corporation Danbury, C T (U.S.A.)
(Received February 8th, 1988) (Accepted April 16th, 1988)
SUMMARY 2-Ethyl-l,3-hexanediol ( E H D ) has intentional h u m a n exposure because of its application to skin as an insect repellent and its use in various skin care products. Genotoxicity studies on E H D were conducted to determine mutagenic and clastogenic potential using in vitro and in vivo test systems. In vitro tests were conducted both with and without an Aroclor-induced, rat-liver $9 metabolic activation system and within a range of cytotoxic to non-cytotoxic doses. E H D did not produce dose-related positive increases in gene mutations in the Salmonella (Ames) test or in the C H O / H G P R T forward mutation test. N o statistically significant or dose-related increases in sister chromatid exchanges indicative o f D N A damage were produced by E H D in C H O cells. Small but statistically significant increases in chromosome aberrations were produced in C H O cells only in tests with $9 activation. However, no evidence of clastogenicity of E H D was obtained in vivo in a mouse peripheral blood micronucleus test or in 2 rat bone marrow chromosome aberration studies using single or repeated dosing procedures. T h e overall negative pattern of mutagenic and clastogenic results in the majority of tests conducted suggests that E H D is unlikely to pose significant hazard as a genotoxic agent or to possess carcinogenic initiating activity in animals.
K e y words: 2-Ethylhexane 1,3-diol; Genetic toxicology; Mutagenicity; Genotoxic-
ity; Insect repellents; CAS No. 94-96-2 *Presented in part at the 26th Annual meeting of the Societyof Toxicology, February 1987. **Address all correspondence and reprint requests to: Dr. Ronald S. Slesinski, Bushy Run Research Center, R.D. No. 4 Mellon Road, Export, PA 15632, U.S.A. Abbreviations: CHO, Chinese hamster ovary; DMN, dimethylnitrosamine; EMS, ethylmethanesulfonate; mPCE, micronucleated polychromaticerythrocyte; NCE, normochromatic (mature) erythrocyte; SCE, sister chromatid exchange. 0300-483X/88/$03.50 © Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
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INTRODUCTION 2 - E t h y l - l , 3 - h e x a n e d i o l ( E H D ) is a colorless to pale-yellow liquid which acts as a solvent and blending agent for latex paints and printer's ink; at elevated t e m p e r atures, it is used as a reactive c o m p o n e n t in various urethane coatings. H u m a n exposure to E H D typically results f r o m its use as an emollient in cosmetic creams and lotions and as the active ingredient in insect repellent formulations (e.g. " 6 12"). Ballantyne et al. [1] reported on the acute toxicity of E H D to rats and showed that it was only slightly toxic by peroral and percutaneous routes (LDso >~ 5 ml/kg) and that no deaths and only signs of m i n o r irritation occurred following 6-h exposures to E H D aerosol at a dynamic concentration of 3.8 rag/1. T h e y noted only a mild degree of skin irritation after a 4-h occluded exposure, but mild to m o d e r a t e conjunctivitis, iritis and corneal injury were produced by volumes of E H D as low as 0.005 ml. L o w degrees of acute toxicity of E H D were also reported in earlier studies by Draize et al. [2] and S m y t h et al. [3]. Repeated 90day exposures of rats to E H D in drinking water revealed no remarkable toxicity except for growth retardation at 0.7 g/kg/day, which was approximately 1/4 of the L D s 0 dose [4]. I n this same report, only a low degree of toxicity was noted in a 90day garage study with dogs dosed with 0.5 ml/kg/day. E H D was concluded to lack toxic and carcinogenic potential in life-time skin application studies [5], although this study has been questioned because of apparent inconsistencies in tabulation of t u m o r incidences [6]. N o published information on genotoxic potential of E H D was discovered in an extensive search of c o m p u t e r data files. Because of the apparent lack of genotoxicity information on E H D , and in view of its widespread availability and h u m a n exposure, a battery of in vitro and in vivo tests was conducted to determine mutagenic and clastogenic potential of E H D . T h i s report presents data on the evaluation of E H D for effects upon gene mutations in Salmonella and C H O cells, D N A damage (SCE test with C H O cells) and c h r o m o s o m e damage in vitro and in vivo. MATERIALS AND METHODS Chemicals E H D (Synonym: Octylene glycol; CAS No. 94-96-2) was obtained from U n i o n Carbide Corporation, South Charleston, WV. E H D has a molecular weight of 146.2, specific gravity of 0.942, vapor pressure of <0.01 m m H g at 20°C, and a chemical formula of ( C ~ H T ) C H ( O H ) C H ( C 2 H s ) C H 2 O H . T h e purity of the E H D used for all studies, determined by gas chromatographic analyses, was 99.18°.:o (by wt) E H D , with 0.36°/,, m o n o b u t y r a t e ester isomers and less than 0.10% each of other unidentified impurities. E H D is stable and freely soluble in organic solvents with limited solubility to 4.2')~, in water. Ethyl alcohol, corn oil or cell-culture m e d i u m were used to prepare dilutions of E H D , and the vehicle control substances for the various test systems are specified in respective tables. Positive control chemicals used to verify the sensitivity of the various assays were all obtained f r o m Sigma Chemical Co., St. Louis, M O , except for: ethyl methanesulfonate ( E M S ; Eastman Organics, Rochester, NY), N - n i t r o s o -
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dimethylamine (DMN; Aldrich Chemical Co., Milwaukee, WI) and triethylenemelamine ( T E M , Polysciences, Inc., Warrington, PA). Water used to prepare media and solutions was obtained from a Millipore Milli Q® high purity system. Positive and negative control agents were the highest available commercial purity.
Animals Micronucleus test. Four-week-old, Swiss-Webster mice, obtained from Hilltop Lab Animals, Inc. (Scottdale, PA), were acclimated for 5--6 days prior to dosing. Five mice/sex/cage were housed in polycarbonate cages with wire tops and AbSorb-Dri ® bedding. T h e animal room used for the micronucleus test had a controlled environment with HEPA-filtered air, a 12-h light/dark photoperiod, temperature range of 19--22°C (66--72°F) and relative humidity range of 4 0 - 60%. Water and certified rodent chow (Agway Pro-Lab R M H 3000) were provided ad libitum. Animals were identified with unique laboratory animal numbers using ear tags. Cytogenetic study. Sprague--Dawley rats, 6 - - 8 weeks old, were obtained from Charles River Breeding Laboratories, Inc., Kingston, N.Y. Animals were quarantined for a 10--14-day period prior to dosing and evaluated daily for signs of illness or poor health. Rats were housed 3 - - 4 per cage during the quarantine period and singly thereafter in plastic shoe-box type cages with wire tops and hardwood bedding chips. Animals were provided with certified rodent chow and water ad libitum. Ear tags with sequential animal numbers were used for animal identification. T h e animal room had a controlled environment with a temperature range of 23 + 3°C (74 + 6°F), 50 + 20% relative humidity and a 12-h light/dark cycle. In vitro metabolic activation For all tests conducted with metabolic activation, an $9 liver homogenate from Sprague--Dawley male rats, induced with Aroclor 1254, was obtained from Microbiological Associates (Bethesda, MD) or Hazleton Biotechnologies (Kensington, MD). Each lot of $9 was screened by the supplier for metabolic activity. In addition, toxicity, metabolic activity and appropriate amounts of $9 for each test system were also determined in our laboratory. Cofactor concentrations in the $9 activation mixture were those recommended by Ames et al. [7], except that the mixture for tests employing CHO cells also contained 8 - - 1 0 # m o l of calcium chloride/ml [8]. T h e amounts of $9 homogenate for the various in vitro tests were: Ames test - - 50/A/plate; C H O / H G P R T test - - 50 #1/5 ml of culture medium; SCE test - - 15/d/5 ml of culture medium; and CHO chromosome aberration tests ~ 50 and 100/~1/5 ml of culture medium. Metabolic activation mixtures were freshly prepared prior to addition to the test system(s). Salmonella/microsome mutation (Ames) test S. typhimurium strains were obtained from Dr. B.N. Ames, University of California, Berkeley, CA. Overnight cultures (approx. 16-h) were grown in Oxoid broth No. 2 (Oxoid, USA, Columbia, MD) and the plate test was performed following standard procedures [7,9] and as described in previous reports by the authors [10--12]. E H D was tested up to cytotoxic concentrations, based upon
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preliminary dose-finding experiments, using triplicate plates/dose. A positive effect was considered to be a dose-related increase which was at least twice the concurrent solvent control value for at least 2 consecutive doses [13]. Animal cell culture procedures C H O cells, C H O - K I - B H 4 (Clone D 1), were a gift from Dr. A. Hsie, University of Texas, Galveston. Cells were grown in H a m ' s F12 medium (Grand Island Biological Co., G r a n d Island, NY) which lacked both hypoxanthine and antibiotics and medium was supplemented with 10% dialyzed fetal calf serum (FCS) for culture maintenance and 5% F C S for mutation determinations. Cells were incubated at 37°C with 5 - - 6 % C O 2 atmosphere and approximately 98% humidity as described previously [10--12]. C H O cells used for these studies have been repeatedly shown to be free of Mycoplasma contamination using fluorescence assays [14] in our laboratory and by culture procedures at commercial laboratories. C H O / H G P R T mutation test system T e s t methods have been described previously [ 10--12] and they were essentially identical to the procedures published by O'Neill et al. [15] and O'Neill and Hsie [16]. T h e range of test concentrations was chosen to include biologically effective doses based upon evidence of cytotoxic inhibition of cell division in preliminary tests. C H O cells were treated with E H D and control agents for 5 h, cultures were rinsed with phosphate-buffered physiological saline, refed with fresh medium and cell viability was determined after an 18-h recovery period. T h e percentage o f clonable cells and the incidence o f mutants resistant to 6-thioguanine (2/~g/ml) were determined after periodic replating o f 3 - - 5 x 105 cells (at 2 - - 3 - d a y intervals) during a 9-day mutation expression period. A positive effect was defined as evidence of a dose-related and statistically significant (P <~0.05) increase above the concurrent solvent control value [17]. S C E test Cell culture and chemical treatment procedures were essentially identical to methods described previously [10--12]. Chromosomes were stained for SCE differentiation by the F P G method o f Perry and Wolff [18]. Approximately 2.0 x 105 C H O cells were transferred to 75-cm 2 culture flasks 4 0 - - 4 8 h prior to treatment with the test agents to provide cells in exponential growth during testing. Cells were treated with test and control materials for 2 h with $9 activation and 5 h without activation in medium with 3/~g bromodeoxyuridine (BrdU)/ml. Chromosomes were harvested for SCE staining following a total culture interval of 2 4 - - 2 8 h in medium with BrdU. T e s t concentrations were determined from cytotoxicity data from preliminary cytotoxicity tests of cell growth inhibition. Cultures treated with the highest 3 concentrations which did not produce excessive cytotoxicity, evident by low mitotic indices or cell lysis, were evaluated for incidence of SCEs using coded slides to prevent bias. Data were analyzed by A N O V A and Duncan's Multiple Range T e s t to determine significance of differences from concurrent control values. A positive effect was defined as either a reproducible, statistically
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significant increase for the duplicate cultures with at least 1 dose or as a significant, concentration-related increase in the SCE index. I n vitro cytogenetic test
C H O cell cultures in exponential growth were prepared by seeding 5 x 105 cells/75-cm 2 culture flask 3 6 ~ 4 8 h prior to the test procedure. Appropriate concentration ranges for E H D were determined by preliminary determination o f growth inhibition or decreases in mitotic indices. Cells were exposed to E H D and controls for the entire period until chromosome harvest in tests without metabolic activation (6 or 12 h). A 2-h exposure period to the test and control chemicals was used in tests with $9 activation to minimize cytotoxicity of liver homogenate to C H O cells. Various chromosome harvest times from 6 to 12 h were employed to assure an optimum detection of chromosome damage. Data from the SCE test indicated this sampling period was appropriate because no cytotoxic inhibition of mitotic progression was produced by E H D . Slides were coded to prevent observer bias and 50 cells/culture were examined for aberrations. A positive effect was defined by a statistically significant difference in the incidence of abnormal cells in comparison to concurrent control values and a concentration related trend in the dose-effect values. Fisher's Exact T e s t (one tailed) was used for statistical comparisons to the control using the combined duplicate-culture values. Micronucleus test with mice
Male and female, 4-week-old, Swiss-Webster mice were used to evaluate in vivo clastogenic potential of E H D using the peripheral blood test procedure [19]. Body weight ranges were 2 4 m 2 9 g for males and 2 0 m 2 3 g for females. A range of test concentrations were determined by a preliminary study to assess the toxicity of E H D (dissolved in 50% ethyl alcohol) using the intraperitoneal exposure route and 5 males and females/dose group. T h e L D s o (3-day holding period) was calculated by the Moving Average M e t h o d [20,21]. T h e volume of 50% ethanol vehicle for E H D was 0.05ml/mouse (0.9--1.1mg/kg) and this dose did not cause overt toxicity nor alter the frequency of micronuclei in comparison to undosed controls. Doses of 80%, 50% and 25% of the respective LDso dose were used in the micronucleus test. Females were also exposed to 1 additional dose calculated as 80% of the relatively higher male LDso value. A total of 2500 polychromatic erythrocytes (PCE)/concentration/sex was examined for incidence of mPCEs. T h e PCE to N C E ratio was determined for 1000 cell/animal to assess bone marrow cytotoxicity. A positive effect was defined by a dose-related trend in the incidence of m P C E s with at least 1 statistically significant (P < 0.01; Fisher's Exact T e s t - - one tailed) indication of a difference from the vehicle control. Incidences of m P C E s from males and females were pooled for statistical analyses if no significant sexrelated differences were indicated by statistical comparisons. Bone m a r r o w cytogenetic tests
Male and female, S p r a g u e - - D a w l e y rats, 6 - - 8 weeks o f age, were used for preliminary dose-finding and cytogenetic studies. Five males and females per dosage group were dosed by i.p. injection with test and control materials. F o r the 183
cytogenetic studies, males ranged in weight from 191 to 230 g and females ranged f r o m 141 to 170 g. One or two extra animals were added to the highest dosage groups to provide sufficient group sizes in case of deaths. T h e m a x i m u m tolerated dose was determined in preliminary tests as the highest concentration which produced weight loss or clinical signs of toxicity without producing death. T h e corn oil vehicle and E H D - v e h i c l e mixture was given at a constant volume of 5 ml/kg. Colchicine (1.0 mg/kg) was given by i.p. injection at 2 - - 4 h prior to sacrifice o f the animals. C h r o m o s o m e s were prepared by flushing bone m a r r o w f r o m femurs and fixation by standard procedures [22]. All slides were coded and 100 cells/animal were evaluated randomly. C h r o m o s o m e s were sampled at 12, 24 and 48 h after acute dosing and 6 h after the last dose in the repeat dose study. D a t a were c o m p a r e d for positive increases above concurrent controls using ~(2 analysis and P ~< 0.05 was considered a significant positive effect.
Penetration of bone marrow by E H D Ability of E H D to enter bone m a r r o w ceils was determined using male S p r a g u e - - D a w l e y rats ( 2 8 0 m 3 1 0 g) and 1,3,[14C]EHD (Spec. act 5.4 m C i / m m o l ) . A 600 m g / k g dose of E H D , identical to that used for the bone m a r r o w cytogenetic test, contained 10/~Ci/kg of [14C]EHD and was administered in corn oil by i.p. injection. Animals were sacrificed at 0.5, 1, 2, 4 and 8 h post injection and radioactivity in blood and bone m a r r o w f r o m 2 animals/sacrifice was quantified b y liquid scintillation spectrometry. Good laboratory practices T h e genotoxicity studies on E H D were carried out in full compliance with the Environmental Protection Agency and F o o d and D r u g Administration G o o d L a b o r a t o r y Practices requirements. RESULTS
Ames test D a t a summarized in T a b l e I show that E H D did not produce positive increases in the n u m b e r s of histidine revertants in tests with and without a rat-liver $9 metabolic activation system. N o dose-related increases in the incidence o f m u t a n t s was observed with any of the 5 tester strains and none of the quantitative increases attained a doubling over the respective solvent control value. T h e test was conducted to a cytotoxic concentration limit and cytotoxicity was evident b o t h b y the relative decline in the m u t a n t values at the highest dose and by inhibitory effects u p o n the background lawn in this and a preliminary test. Positive control agents produced highly elevated n u m b e r s of m u t a n t s above the values for the ethanol (100%) vehicle. T h e vehicle control m u t a n t values were within typical ranges for this test f r o m data at our laboratory and others [9]. C H O / H G P R T gene mutation Evaluation of mutagenic potential of E H D to C H O cell cultures is s u m m a r i z e d in T a b l e I I . T h e survival data in the first column indicate that biologically effective
184
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doses were tested and that the cytotoxic effects were detected despite the 18 h recovery period before determination of colony-forming ability. T h e mean percentage of clonable cells in the second column shows that after 9 - - 1 0 days of expression for the m u t a n t phenotype, the colony-forming potential of most E H D exposed cultures was essentially the same as the solvent control values. In the last column, the mutation index corrected for the respective colony-forming percentages did not reveal a dose-related trend in the n u m b e r of m u t a n t colonies in the tests conducted with or without a rat-liver activation system. However, in the test without $9 activation, 2 statistical indications of an increase above the solvent control values were produced b y non-consecutive doses of 1.0 and 4.0 mg/ml. T h i s statistical indication was not considered a positive test result because of the absence of a dose-related trend, the lack of agreement in duplicate cultures and the values were within the historical negative control range for this test at our laboratory. T h e positive control data showed that the cells were responsive to known mutagens, E M S and D M N , and mutants increased in relation to D M N concentration in tests with $9. With negative control cultures, the low m u t a n t values obtained are typical for this particular subclone [10--12]; although higher values up to 25 mutants/106 clonable cells are seen occasionally and are considered m a x i m u m acceptable limits for negative control data in our laboratory. S C E test T h e data for the S C E evaluation of D N A damage potential of E H D are shown in T a b l e I I I . In the test of direct genotoxic potential without $9 activation, none of the highest 3 of 5 exposure doses evaluated for SCEs produced a significant increase in SCEs above the concurrent negative control values. N o evidence of a doserelated trend relative to E H D concentration was apparent, and all values for treated cultures were essentially identical to the solvent control value. T h e 3.0 m g / m l dose produced excessive cytotoxicity and could not be evaluated for SCEs because of a low mitotic index. With rat-liver $9 activation, essentially identical negative results were seen as in the test without $9 activation. T h e shorter exposure time of 2 h used for the test with $9 did not produce the excessive cytotoxicity seen with the 3.0 m g / m l dose in the 5 h exposure for the test without $9. For both tests, the respective positive control agents E M S and D M N produced highly increased and statistically significant n u m b e r s of SCEs relative to controls which demonstrated the responsiveness of the test system. N o indication of a dose-related cytotoxic inhibition of mitosis was evident in the relative ratios of cells in the first, second or third r o u n d of cell division. E H D appeared to lack D N A - d a m a g e potential u n d e r conditions of this test system. In vitro chromosome damage E H D did not increase the incidence of c h r o m o s o m e aberrations in C H O cells when evaluated for direct clastogenic activity without $9 (Table IV). T h e top doses used for the tests with and without $9 activation were chosen from preliminary test data which showed respective decreases of 4 0 - - 6 0 % in culture growth and reduced mitotic indices. Essentially identical, non-significant differences f r o m control values were observed after continuous exposure to E H D and for sampling times o f
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6, 8, 10 and 12 h. Values for 8- and 12-h sampling times are shown in T a b l e IV; the 6- and 10-h data showed even lower relative increases over respective controls and were not included in T a b l e I V for brevity. With metabolic activation, using 2 concentrations of $9 homogenate, statistically significant increases in c h r o m o s o m e aberrations were observed only within a short time span of c h r o m o s o m e sampling. Positive effects were seen in 1 test with the 8 h but not 12-h blood sample. A second test was conducted with a higher and o p t i m u m $9 concentration of 100/~1 $9/5 ml m e d i u m which was determined in a preliminary study using 3.0 m g / m l of E H D . Positive, statistically significant increases were observed at 10 h but values were only marginally positive at 12 h. T h e 6- and 10-h E H D values in this test using 100/~1 of $9 were not statistically different f r o m controls and are not shown in T a b l e I V for brevity. T h e majority of the c h r o m o s o m e damage in the samples with positive effects was simple chromatid and c h r o m o s o m e breakage, which are likely lethal cellular events. N o remarkable evidence of potentially inheritable damage such as chromatid and c h r o m o s o m e exchanges was observed at any sampling time for tests with and without $9 activation. M i c r o n u d e u s test
T a b l e V shows the production of micronucleated polychromatic erythrocytes ( m P C E s ) in peripheral blood samples of mice exposed to a range of E H D concentrations f r o m 18.75 to 120 pg/kg. T h i s range of doses was based u p o n the determination that the i.p. L D s 0 of E H D to males was 1 5 4 m g / k g (95% C . I . - - 1 0 3 - - 2 1 3 ) and 7 5 m g / k g (95% C.I. = 3 5 - - 1 2 0 ) for females. Following injection of E H D , sedation, lethargy and periocular encrustation were observed with the highest 2 doses. N o significant or dose-related increases in m P C E s were p r o d u c e d by E H D in cells collected at blood sampling times of 24, 48 or 72 h. E H D did not produce remarkable bone m a r r o w cytotoxicity which was evident by similar P C E / N C E ratios for all treated and control samples (data not shown in T a b l e V). A few animals died at higher dose levels which indicated that a m a x i m u m tolerated dose was achieved (or exceeded) but no positive effects upon micronucleus frequencies were produced by these high levels of E H D exposure. T h e positive control substance T E M produced elevated and significant increases of m P C E s indicating the appropriate sensitivity o f the test animals and suitable test prodedures. B o n e m a r r o w cytogenetic tests
F o r acute and repeated-dose protocols, rats were dosed by i.p. injection with 60, 200 and 6 0 0 m g E H D / k g . Animals appeared sedated by the highest dose and moderate loss of weight ( - 4 - - - - 6 % ) was observed on the day of sacrifice in both the acute and repeated dose study. Clinical signs of toxicity were essentially identical with b o t h single and multiple injections of E H D , which indicated a lack o f cumulative toxicity. H o w e v e r , 1 male and 2 females repeatedly dosed with 600 m g / k g died and were replaced with extra dosed animals in order to assure complete group sizes. T h e incidence of c h r o m o s o m e aberrations did not differ between males and females, thus values were pool,ed for statistical analyses. N o significant increases in c h r o m o s o m e damage were observed regardless of sampling
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time or following single or multiple injections with E H D (Table VI). T E M produced a highly significant increase in chromosome aberrations and the corn oil controls were in a low and acceptable range for a valid test. N o evidence o f clastogenic potential in vivo was observed in either test even at the high range o f doses used for this study. Penetration of bone marrow by E H D Measurements of radioactivity in bone marrow and plasma samples from animals dosed by i.p. injection of [14C]EHD in corn oil (Table V I I ) showed that penetration into bone marrow was very rapid and detectable at the first sacrifice (0.5 h) after dosing. T h e radioactivity in bone marrow was detectable for at least 8 h post dosing which was the final sacrifice. T h e bone marrow/plasma ratios were essentially the same between 2 and 8 h indicating that a steady-state condition between radioactive uptake and elimination was established. These data strengthened the conclusion of negative clastogenicity of E H D by demonstration o f the potential for exposure of the bone marrow cells to E H D which did not lead to positive effects in the bone marrow cytogenetic studies. DISCUSSION T h e battery of genotoxicity tests conducted in this study was prompted by the apparent lack of short-term test data on E H D and by the desire to develop a weightof-evidence consensus from multiple genetic end-points. T h e results from the Ames assay, C H O / H G P R T gene mutation and the SCE tests (Tables I, II, I I I ) uniformly indicated that E H D lacked mutagenic and D N A damage potential in tests conducted with and without $9 activation. I n contrast, positive indications of clastogenic potential were obtained reproducibly in 2 in vitro chromosome damage tests with C H O cells when performed in the presence, but not in the absence, of a rat-liver $9 metabolic activation system (Table IV). T h e positive effects were evident only for a brief time span following dosing in cells sampled at either 8 or
TABLE VII DETERMINATION OF POTENTIAL FOR ENTRY OF RADIOACTIVITY FROM [14C]EHD INTO RAT BONE MARROW Sampling time (h) 0.5 1.0 2.0 4.0 8.0
Plasma(p)l
Bone marrow (BM)a
DPM/g
/zg equiv./g
DPM/g
,ug/equiv./g
75,373 99,585 162,058 101,848 35,730
946 1,250 2,035 1,279 449
52,261 57,016 64,266 50,895 15,904
656 716 807 639 200
BM/P ratio 0.69 0.57 0.46 0.50 0.45
"Mean values of bone marrow and plasma samples from 2 animals except at 2 h where 1 animal was incorrectly dosed and not included.
195
10 h (depending upon $9 concentration), following the start of the 2 h exposure. Cells sampled +_2 h or greater from this optimum time with each respective $9 concentration did not reveal remarkable clastogenicity. Also, the positive increases attained a plateau value which did not increase with higher doses. T h e limited effective time span and narrow range of doses producing the statistically significant increases p r o m p t e d further in vivo studies to determine the significance of the in vitro suggestions of clastogenicity. Evaluation of E H D by the peripheral blood micronucleus method in mice did not show positive or dose-related increases in micronuclei at doses of 80% or higher than the respective 3-day male and female LDs0 do~es. Blood was sampled at 3 different time periods but no significant increases in m P C E s were observed at 24, 48 or 72 h after dosing. Although recent reports [23] suggest that micronucleus determinations with higher numbers o f m P C E than evaluated here provide less sampling variance, it is unlikely that counting more cells would alter the conclusion from this test because of the essential similarity of the m P C E values for E H D treated animals and controls. Also, preparation of uniform blood smears may have less variability than bone marrow slides because of the relatively greater homogeneity of blood samples. T h e absence of in vivo clastogenic potential of E H D was confirmed by an independent bone-marrow cytogenetic assay conducted by 1 o f the authors ( D L P ) in a second laboratory. Again, the results of this bone-marrow study showed that no significant increases in chromosome damage were evident following acute or repeated doses of E H D , even with doses which killed some of the animals. Detection of radioactivity in bone marrow cells of rats administered [14C]EHD by i.p. injection strengthened the conclusion on negative clastogenicity by demonstrating that the test chemical (and/or metabolites) had the opportunity for interaction. T h e rapid appearance of radioactivity in bone marrow and steady-state bone marrow: plasma ratios up to 8 h indicated that there was ample time available for expression of clastogenic potential. T h e absence of genotoxic activity for E H D in the majority of the tests conducted is in agreement with the lack of carcinogenic activity following lifetime dermal applications to mice (Stenbiick and Shubik, 1974). Also, the related molecule 2ethylhexanol, which differs only by absence of 1 hydroxyl group on the third carbon, was shown to lack genotoxicity in several studies employing a variety of test systems [24--27]. T h e reason for the positive effects of E H D in the in vitro chromosome damage test, in view of the majority of negative tests, is not known. However, there are many other examples o f chemicals which produce unexpected or singular positive effects when subjected to a battery of sensitive screening tests [28]. Possible explanations for the transient clastogenic activity observed in this study may include conversion of E H D into an active but unstable metabolite by the in vitro $9 activation system. However, the possibility that the positive clastogenicity may be an artefact of the in vitro test conditions cannot be excluded. For example, Galloway et al. [29] found that spurious positive increases in chromosome aberrations in C H O cells in vitro can be produced by high con3entrations and osmolalities of apparently inocuous substances such as KC1, NaC1 and sucrose. Notably, they found that the quantitative responses to these agents was in the same range of 0.13--0.16 aberrations/cell that we obtained with E H D in these studies. In
196
c o n c l u s i o n , a l t h o u g h w e a k l y p o s i t i v e i n c r e ases w e r e seen in c h r o m o s o m e a b e r r a t i o n s in v i t r o , t h e lack o f g e n o t o x i c i t y i n t h e m a j o r i t y o f in v i t r o a n d in v i v o tests s u g g e s t s t h a t E H D is u n l i k e l y to pose a significant g e n o t o x i c h a z a r d o r to b e an i n i t i a t i n g a g e n t in a n i m a l c a r c i n o g e n i c i t y tests. ACKNOWLEDGEMENTS W e t h a n k W . C . H e n g l e r for t e c h n i c a l assistance w i t h s o m e o f t h e in v i t r o s t u d i e s an d M s . K a t h y M c C a b e for assistance in p r e p a r a t i o n o f t h e m a n u s c r i p t . T h e p e r f o r m a n c e o f t h e [ 1 4 C ] E H D b o n e m a r r o w e x p e r i m e n t s b y D r . C.B. J e n s e n is g r a t e f u l l y a c k n o w l e d g e d . T h i s w o r k was s p o n s o r e d by t h e U n i o n C a r b i d e Corporation. REFERENCES 1 B. Bailantyne, D.R. Klorme, R.C. Myers and D.J. Nachreiner, The acute toxicity and primary irritancy of 2-ethyl-l,3-hexanediol. Vet. Human Toxicol., 27 (1985) 491. 2 J.H. Draize, E. Alvarez, M.F. Whitesell, G. Woodard, E.C. Hagan and A.A. Nelson, Toxicological investigations of compounds proposed for use as insect repellents. J. Pharmacol. Exp. Ther., 43 (1948) 26. 3 H.F. Smyth, Jr., C.P. Carpenter and C.S. Weil, Range-finding toxicity data: List IV. Arch. Ind. Hyg. Occ. Med., 4 (1951) 119. 4 H.F. Smyth, C.P. Carpenter and C.S. Weil, Ninety-day repeated oral doses of 2-ethylhexanediol1,3 to dogs. Report 17-37 Carbide and Carbon Chemicals, unpublished report (1954) by Mellon Institute of Industrial Research, Pittsburgh, PA. 5 F. Stenb~ickand P. Shubik, Lack of toxicity and carcinogenicity of some commonly used cutaneous agents. Toxicol. Appl. Pharmacol., 30 (1974) 7. 6 Environmental Protection Agency, 2-Ethyi-l,3-Hexanediol, Pesticide Registration Standard. Office of Pesticides and Toxic Substance (1981) pp. 34--35. 7 B.N. Ames, J. McCann and E. Yamasaki, Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res., 31 (1975) 347. 8 E.L. Tan and A.W. Hsie, Effect of calcium phosphate and alumina ~C gells on the mutagenicity and cytotoxicity of dimethylnitrosamine as studied in the CHO/HGPRT system. Mutat. Res., 84 (1981) 147. 9 D.M. Maron and B.N. Ames, Revised methods for the Salmonella mutagenicity test. Murat. Res., 113 (1983) 173. 10 R.S. Slesinski, W.C. Hengler, P.J. Guzzie and K.J. Wagner, Mutagenicity evaluation of glutaraldehyde in a battery of in vitro bacterial and mammalian test systems. Food Chem. Toxicol., 21 (1983,a) 621. 11 R.S. Slesinski, P.J. Guzzie, W.C. Hengler, P.G. Wantanabe, M.D. Woodside and R.S.H. Yang, Assessment of genotoxic potential of ethylenediamine: in vitro and in vivo studies. Murat. Res., 124 (1983,b) 299. 12 R.S. Slesinski, P.J. Guzzle, W.C. Hengler, R.C. Myers and B. Ballantyne, Studies on the acute toxicity, primary irritancy and genotoxic potential of 1,3,5-triacryloylhexahydro-s-triazine (TAHT). Toxicology, 40 (1986) 145. 13 K.C. Chu, K.M. Patel, A.H. Lin, R.E. Tarone, M.S. Linhart and V.C. Dunkel, Evaluating statistical analyses and reproducibility of microbial mutagenicity assays. Mutat. Res., 85 ( 1981) 119. 14 T.R. Chen, In situ detection ofmycuplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell Res., 104 (1977) 255. 15 J.P. O'Neill, P.A. Brimer, R. Machanoff, G.P. Hirsch and A.W. Hsie, A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary ceils (CHO/HGPRT System): development and definition of the system. Murat. Res., 45 (1977) 91. 197
16 J.P. O'Neill and A.W. Hsie, Phenotypic expression time of mutagen induced 6-thioguanine resistance in Chinese hamster ovary cells ( C H O / H G P R T system). Mutat. Res., 59 (1979) 109. 17 J.D. Irr and R.D. Snee, Statistical evaluation of mutagenicity in the C H O / H G P R T system. Proc. of the Cold Spring Harbor - - Banbury Conference II (1979) pp. 263--274. 18 P. Perry and S. Wolff, New Giemsa method for differential staining of sister chromatids. Nature, 252 (1974) 156. 19 J.T. MacGregor, C.M. Wehr and D.H. Gould, Clastogen-induced micronuclei in peripheral blood erythrocytes: the basis of an improved micronucleus test. Environ. Mutagen. 2 (1980) 509. 20 W.R. Thompson, Use of moving averages and interpolation to estimate median effective dose. Bact. Rev., 11 (1947) 115. 21 C.S. Weil, Tables for convenient calculation of median-effective dose (LD50 or ED50) and instructions for their use. Biometrics, 8 (1952) 249. 22 W.W. Nichols, R.C. Miller and C. Bradt, In vitro anaphase and metaphase preparations in mutation testing, in B.J. Kilbey, M. Legator, W. Nichols and C. Ramel (Eds.), Handbook of Mutagenicity Test Procedures, Elsevier Publ. Co., NY, 1979, p. 225. 23 J. Ashby and R. Mohammed, Slide preparation and sampling as major sources of variability in the mouse micronucleus assay. Mutat. Res., 164 (1986) 217. 24 P.E. Kirby, R.F. Pizzarello, T.E. Lawlor, S.R. Haworth and J.R. Hodgson, Evaluation of di-(2ethylhexyl)-phthalate and its major metabolites in the Ames test and LSI78Y mouse lymphoma assay. Environ. Mutagen. 5 (1983) 657. 25 B.J. Phillips, T.E.B. James and S.D. Gangolli, Genotoxicity studies of di(2-ethylhexyl)phthalate and its metabolites in CHO cells. Mutat. Res., 102 (1982) 297. 26 D.L. Putman, W.A. Moore, L.M. Schechtman and J.R. Hodgson, Cytogenetic evaluation of di-(2ethyl-hexyl)phthalate and its major metabolites in Fischer 344 rats. Environ. Mutagen. 5 (1983) 227. 27 E. Zeiger, S. Haworth, K. Mortelmans and W. Speck, Mutagenicity testing of di(2-ethylhexyl)phthalate and related chemicals in Salmonella. Environ. Mutagen., 7 (1985) 213. 28 F.J. de Serres and J. Ashby, Evaluation of short-term tests for carcinogens. Report of the International Collaborative Program. Progress in Mutat. Res. 1, Elsevier/North Holland, N.Y., 1981, p. 112. 29 S.M. Galloway, C.L. Bean, M.A. Armstrong, D. Dcasy, A. Kraynak and M.O. Bradley, False positive in vitro chromosome aberration tests with non-mutagens at high concentrations and osmolalities. Environ. Mutagen., 7 (Suppl. 3) (1985) 48 (abstract).
198