Mutagenicity of alkyl glycidyl ethers in three short-term assays
E.D. Thompson 1,,, W.J. Coppinger 1, C.E. Piper 2, N. McCarroll 2, T.J. Oberly 2 and D. Robinson 3
I The Procter and Gamble Company. P.O. Box 39175, Miami Valley Laboratories, Cincinnati, OH 45247; 2 Hazleton Laboratories of America, Vienna, VA; and 3 SRI International. Menlo Park, CA (U.S.A.)
(Received 19 June 1980) (Revision received 6 April 1981 )
(Accepted 5 June 1981)
Summao
The mutagenic potentials of glycidol and 7 alkyl glycidyl ethers having straight alkyl chains of 2, 4, 6, 8, 10, 12, and 14 carbon atoms were examined in a battery of in vitro assays. The battery consisted of the Salmonella/mammalian microsome assay, the L5178Y mouse lymphoma assay, and unscheduled DNA synthesis using WI38 cells. The mutagenic potential of the compounds ranged from strongly mutagenic to non-mutagenic; glycidol exhibited the greatest activity. All the ethers through C-4 showed a definite response while the C-8 or higher ethers showed very weak or no responses. Dose-response curves were obtained by all 3 assays for those compounds that exhibited mutagenic activity. The sensitivity of each assay is discussed, as are the effects of the liver microsome systems used for metabolic activation.
Alkyl epoxides were first shown to be mutagenic in the Salmonella/mammalian microsome assay by McCann et al. (1975). Subsequent studies by Wade, D.R., et al. (1978) and Wade, M.J., et al. (1979) have extended these earlier results. Wade, M.J., et al. (1979) showed that not all epoxides were mutagenic in the Salmonella system, the most notable exception being dieldrin. In a study designed to examine the effects of substitution around the oxirane ring, Wade, D.R., et al. (1978) showed that a definite decrease in the mutagenic potential of epoxides occurred with increased substitution.
* To whom reprint requests should be sent. Abbreviations: DMN, dimethylnitrosamine; DMSO, dimethylsulfoxide; HU, hydroxyurea; 4NQO, 4-
nitroquinoline-N-oxide; UDS, unscheduled DNA synthesis.
The mutagenicity of a specific group of epoxides, the glycidyl ethers (R-O-CH~-CH-CH 2 ), has been examined by Pullin and Legator (1978). 7 glycidyl
- ~ O Z
ethers were examined for mutagenicity by 6 different assays. All 7 compounds showed at least some activity in the Salmonella/mammalian microsome assay; butyl glycidyl ether gave a moderate response whereas a mixture of C-12 and C-14 alkyl glycidyl ethers gave weak responses. None of the compounds were positive in the host-mediated assay or the micronucleus test, and only 2 showed any response in mouse urine analysis. 3 of the glycidyl ethers, including butyl glycidyl ether, induced unscheduled DNA synthesis in human white blood cells. Butyl glycidyl ether was reported to be positive in a dominant lethal assay: however, a subsequent test did not support the initial result.
Because the alkyl glycidyl ethers gave a wide range of mutagenic responses, we decided to test a larger group of them. Glycidol and 7 additional alkyl glycidyl ethers with linear alkyl groups of C-2, C-4, C-6, C-8, C-10, C-12, and C-14 were either purchased or synthesized for the study.
Each compound was subjected to a battery of in vitro assays composed of the Salmonella/mammalian microsome assay, the L5178Y mouse lymphoma assay, and unscheduled DNA synthesis in W138 cells. These assays were selected because each has had some validation and because each has a different genetic endpoint.
The Salmonella assay has been extensively validated and shown to function as expected with epoxides (McCann et al., 1975, Purchase et al., 1978). A total of 5 strains each mutated somewhere in the histidine operon, were used in this study. Strains TA1535 and TA100 have a base-pair substitution in the hisG gene, and they respond primarily to base-pair substitution-type mutagens. Strain TA1537 has a mutation in the hisC gene and strains TA1538 and TA98 have a mutation in the hisD gene. The latter 3 strains respond primarily to frame-shift-type mutagens. Thus, both base-pair substitution and frame-shift-type mutagens are detected in this assay by determining the extent of back-mutation to histidine prototrophy.
The L5178Y mouse lymphoma assay has been used less extensively but has been validated (Clive et al., 1979) and is in use in many laboratories. It was selected as part of the battery because it employs mammalian cells and is a forward mutation assay, the endpoint being the loss of function of thymidine kinase. The loss of thymidine kinase activity is due to a mutation in the structural gene coding for the polypeptides which comprise the enzyme. Presumably the mutational event could range from a single base-pair substitution to a deletion event encompassing the entire gene.
Unscheduled DNA synthesis in WI38 cells was selected because it uses a human cell line (San and Stich, 1975; Rasmussen and Painter, 1966). The endpoint of this assay is the demonstration of increased DNA repair in cells that have been treated with a mutagen. In this study DNA repair was determined by monitoring [3H]thymidine incorporation into the DNA of G o phase cells that had been exposed to the alkyl glycidyl ethers.
The purpose of the study was to examine the effects of alkyl chain length on the mutagenic potential of glycidyl ethers, and to examine the relative sensitivities of 3 in vitro assays, each of which has a different endpoint.
215
Methods and materials
Organisms Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, and TA98
were obtained from Dr. B.N. Ames, University of California at Berkeley. The L5178Y mouse lymphoma cells were obtained from Dr. D. Clive, Burroughs Welcome Co. WI38 cells were obtained from the American Type Culture Collection.
Synthesis of the alkyl glycidyl ethers Glycidol and the alkyl glycidyl ethers with even numbered alkyl groups ranging
from C-0 (glycidol) to C-14 (tetradecyl glycidyl ether) were obtained in high purity by the following procedures. Glycidol, ethyl glycidyl ether, and butyl glycidyl ether were purchased and purified by fractional vacuum distillation. The longer chain length species were synthesized by reacting the high-purity fatty alcohol ( > 97% from Conoco Chemicals) with epichlorohydrin in the presence of SnC14. The resulting chlorohydrin compounds were treated with excess NaOH to give the alkyl glycidyl ethers. Pure alkyl glycidyl ethers were obtained by fractional vacuum distillation.
The ethers were characterized by gas liquid chromatography on a Hewlett Packard instrument equipped with a 2-m stainless steel column packed with SP2100 (Supelco). Oxirane levels were determined as described by Walker (1978). Free epichlorohydrin was determined by gas liquid chromatography of the head space. Table 1 shows analytical data for the glycidyl ethers.
Salmonella~mammalian microsome assay A preliminary toxicity assay was performed by the following procedure to
determine the highest testable level for each alkyl glycidyl ether. 5 serial 10-fold dilutions beginning at 100 mg/ml were prepared in dimethylsufloxide (DMSO). A single plate was prepared from each dilution according to the standard plate assay procedure of Ames et al. (1975) with strain TA100. After 48 h of incubation at 37°C, the number of revertant colonies was determined and the background lawn was examined through a dissecting microscope. The concentration of chemical which reduced the background lawn by 50-75% compared to the solvent control was selected as the highest testable dose. A 50-75% reduction of the background lawn was usually accompanied by a slight reduction in the number of spontaneous revertant colonies for non-mutagenic compounds.
The Salmonella/mammalian microsome plate assay was performed as described by Ames et al. (1975). 6 concentrations of each compound decreasing by 3-fold dilutions from the highest testable level were tested in the presence and absence of an Aroclor-1254-induced rat liver microsome activation system ($9) using strains TA1535, TAI00, TA1537, TA1538, and TA98. The test compounds were dissolved in DMSO and 0.1-ml portions were added to the 2.0-ml overlay agar. Both solvent (0.1 ml DMSO) and water negative controls were prepared. 3 plates were prepared for each dose level and controls.
The following positive control compounds were evaluated in parallel with the
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217
alkyl glycidyl ethers. In the absence of the $9 metabolic activation system, N-methyl- N'-nitro-N-nitrosoguanidine (5/*g/plate) was used as a positive control for TA1535 and TA100, 2-nitrofluorene (50/*g/plate) was used as a positive control for TA1538 and TA98, and 9-aminoacridine (125 / ,g /p la te) was used as a positive control for TA1537. 2-Aminoanthracene (5 / ,g /p la te ) was used as a positive control for all 5 strains in the presence of the $9 metabolic activation system.
L5178 Y mouse lymphoma assay The L5178Y mouse lymphoma assay was performed as described by Clive and
Spector (1975) and later modified by Clive et al. (1979). A toxicity assay was performed on each compound to determine suitable levels for testing. A series of dilutions (usually a 1 000-fold range) was prepared for each compound. Cell toxicity was determined in the presence and absence of $9 activating systems. Liver homo- genates were prepared from Aroclor-1254-induced Sprague-Dawley rats by the method of Ames et al. (1975). Non-induced liver homogenate was prepared in the same manner from Sprague-Dawley rats injected with corn oil (vehicle used with Aroclor 1254). The $9 mix consisted of 25 ml of fiver homogenate added to 75 ml F~0P * medium containing 600 mg NADP, and 1125 mg DL-isocitric acid. The $9 mix was prepared on the day of the experiment, passed through a 0.45-micron membrane filter, and kept on ice until used. The toxicity assay was performed by placing 6 million precleansed TK +/ cells in 6 ml of F10P medium into a 50-ml plastic centrifuge tube (Corning No. 25330). A 0.1-ml volume of test compound and either 4 ml of $9 mix or 4 ml of FioP medium were added, the tubes were gassed with a mixture of 5% CO 2 in air, sealed, and incubated at 37°C in a roller drum at 60 rpm for 4 h. The cells were removed by centrifugation at 500 × g for 10 min, washed twice with FioP medium, and resuspended in 20 ml of Fl0P medium. The tubes were gassed with 5% CO 2 in air, sealed, and incubated at 37°C in a roller drum at 60 rpm. At 24 and 48 h the total cell number and viability were determined in the presence of trypan blue in a hemocytometer. After the initial 24-h incubation the viable cell count was adjusted to 3 X 105 cel ls /ml in all cultures that had grown above this level. The concentration of test chemical which gave approx. 50% inhibition of growth was identified. A narrow range of concentrations around this level was selected for further testing.
5 or more concentrations were used for the mutation assay. The highest dose was set at twice the level that killed 50% of the organisms in the toxicity assay. The mutagenicity assay was performed as described for the toxicity assay. After the 48-h incubation period, the cells were resuspended in F~0P at a concentration of 1 × 10 6
viable cel ls /ml as determined by trypan blue exclusion. A portion of the cells was diluted (10-4 dilution) and 1 ml was plated onto non-selective medium for viable cell determinations (approx. 100 cells/plate), while 0.5 ml of the original cell suspension (approx. 0.5 × 10 6 cells) was plated in the presence of trifluorothymidine (TFT, 1/~g/ml). These plates were incubated for 10-12 days and the number of
* Fisher's medium for leukemic cells of mice supplemented with sodium pyruvate and 10% (v/v) horse s e r u r I ~ .
218
mutant colonies (TFT plates) and the number of viable colonies (viability plates) were determined. A single tube was prepared for each dose level, and the treated cells were cloned in triplicate.
Ethyl methanesulfonate (620/~g/ml) was employed as the positive control in the assay without metabolic activation. Dimethylnitrosamine (74 ~g/ml) was employed as the positive control with the uninduced rat liver $9, and 2-acetylaminofluorene (100/~g/ml) was employed in the assay with Aroclor-1254-induced rat liver $9.
Unscheduled DNA synthesis • WI38 cells grown in T25 tissue culture flasks were used for each UDS assay.
Replicate cultures of these cells were initiated in Eagle's Basal Medium containing 10% (v/v) fetal bovine serum. The cells were grown to confluency and maintained in medium containing 0.5% serum for 5 -6 days preceding the UDS assays. This produced synchronous cultures of contact-inhibited cells in the G o phase of the mitotic cycle. To reduce the possibility that tritiated thymidine (3 H-TdR) might be incorporated by an occasional S-phase cell that had escaped-the contact-inhibition synchrony which would obscure measurements of UDS, the cultures were incubated for 1 h with 10 2 M hydroxyurea (HU) before each assay, and each subsequent step was performed in the presence of 10-2 M HU. Immediately before each assay, the compounds were diluted in DMSO to the appropriate test concentrations. The final concentration of DMSO present in the assay was 1% or less, thus minimizing the possibility of a cytotoxic effect in response to, the solvent. A preparation of a 9000 × g supernatant ($9) of liver homogenate (250 mg of liver per ml) from adult male Swiss-Webster mice was used for the metabolic activation studies. The following cofactors were added to the $9: nicotinamide (3.05 mg/ml), glucose-6- phosphate (16.1 mg/ml) , MgCI2.6 H20 (5.08 mg/ml) , and NADP (0.765 mg/ml) .
The positive controls were 4-nitroquinoline-N-oxide (4NQO), a compound that induces UDS in the absence of metabolic activation, and dimethylnitrosamine (DMN), a compound that induces UDS in vitro only with metabolic activation. The negative control was DMSO diluted in culture medium.
Preliminary tests covered a broad range of chemical concentrations to establish the appropriate dose levels. The preliminary test results were used to determine the maximal testable level of each compound. The maximal testable level was set just below the level which produced cytotoxicity as demonstrated by a decrease in the amount of [3H]thymidine incorporated into the DNA. The procedure for the preliminary assay was the same as described for the UDS assay.
UDS assays The contact-inhibited WI38 cells were incubated with the compound at 37°C with
gentle rocking in the presence of 1/~Ci/ml of 3H-TdR (spec. act., 6.7 Ci/mmole). For testing in the absence of metabolic activation, the cells were exposed simulta- neously to the compounds and to 3H-TdR for 3 h. For testing with metabolic activation, the cells were incubated with the compound, 3H_TdR, and the metabolic activation preparation for 1 h and then with only 3H-TdR in culture medium for an additional 3 h. (The shorter exposure time for metabolic activation keeps the liver
219
homogenate preparation from exhibiting cytotoxic effects to the WI38 cells.) A modification of the PCA-hydrolysis procedure of Schmidt and Thannhauser (1945) was used to extract D N A from the cells. 1 aliquot of the D N A solution was used to measure the D N A content after reaction with diphenylamine (Richards, 1974), and a 2nd aliquot was used to determine the extent of incorporation of 3H-TdR by scintillation spectrometry. Results were expressed as disintegrators per min (dpm) of incorporated 3H-TdR per unit of DNA. Results are the average of 6 replicate samples at each dose level.
Results
The results of the Salmonel la /mammal ian microsome assays with strains TA 1535 and TA100 are shown in Table 2. The results of the assays with strains TA1537, TA1538, and TA98 were negative for all 8 glycidyl ethers and are not shown. For ease of comparison with the other assays, a mutation index was calculated for both strains. The mutation index is the average number of revertant colonies on the test plates divided by the average number of revertant colonies on the solvent control plates. A number of 1 or less indicated no mutagenicity, whereas for strain TA1535, numbers greater than 2.0, when associated with a dose response over at least 3 concentrations, represent a mutagenic response. For strain TA100, dose-related increases in the mutation index over at least 3 concentrations with the highest response at least equal to 2 were considered positive. Positive controls were run in parallel with each assay, but, for brevity, the data are not shown here. In all cases the positive controls exhibited at least a 10-fold increase in the number of revertant colonies (mutation index > 10):
From Table 2~ it is clear that glycidol, ethyl, butyl, and hexyl glycidyl ethers were positive with and without metabolic activation, although enhancement of the re- sponse was seen (especially noticeable with TA1535) in the presence of the $9 fraction. The octyl, decyl, and dodecyl glycidyl ethers induced a weak but demon- strable mutagenic response only in the presence of the $9 fraction. Tetradecyl glycidyl ether was negative in this assay.
The results of the mouse lymphoma assay are shown in Table 3. The assays were first performed over a wide range of concentrations, then rerun over the narrow ranges shown in the table to demonstrate dose response. The relative growth column is an index of the amount of toxicity to the cells. In most cases the range of concentrations was restricted to 5-fold or less due to toxicity. The mutation frequency was calculated by dividing the number of colonies appearing in the selective plates (TFT) by the number of surviving cells as determined by plating a sample of cells in the absence of the selective agent. The mutation index was calculated by dividing the mutation frequency of the test results by the mutation frequency of the solvent control. This index is thus comparable to the mutation indices shown for the Salmonel la /mammal ian microsome assays. For this particular set of experiments, we considered a compound positive if dose-related increases in the mutation index over at least 3 concentrations with the highest response at least
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9
a D
PM
//~
g
DN
A.
b R
esp
on
se i
nd
ex =
rat
io o
f te
st/c
on
tro
l.
c W
ith
met
abo
lic
acti
vat
ion
-dim
eth
yln
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sam
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(5×
10
-2 M
). W
ith
ou
t m
etab
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c ac
tiv
atio
n-4
-nit
roq
uin
oli
ne
ox
ide
(10
5 M
).
a O
il d
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lets
fo
rmed
on
the
su
rfac
e o
f th
e m
ediu
m.
228
equal to 3.0 were obtained. The mouse lymphoma assays were performed without metabolic activation, with metabolic activation by non-induced rat liver microsome preparation, and with an Aroclor-1254-induced rat liver microsome preparation.
In general, the highest mutagenic responses were obtained in the assays without metabolic activation. Responses obtained were slightly reduced in the assays that used the non-induced $9 preparations, whereas much lower responses were obtained with the Aroclor-induced $9 preparations. Cell toxicity was reduced proportionally, i.e. the same dose of chemical produced a more toxic response in the absence of any $9 and a less toxic response in the presence of the $9.
Glycidol and the ethyl, butyl, and hexyl glycidyl ethers gave distinctly positive responses either with one of the $9 preparations or without activation. On a molar basis glycidol was considerably more potent than any of the glycidyl ethers. Decyl glycidyl ether was marginally positive without metabolic activation according to the above definition, but octyl, dodecyl, and tetradecyl glycidyl ethers were negative.
The results of the unscheduled DNA synthesis assays are shown in Table 4. As in the Salmonella and mouse lymphoma assays, preliminary tests were performed to define the maximal testable levels of the chemicals. The response index was calculated by dividing the amount of thymidine incorporation in the test results by the thymidine incorporation in the solvent control.
We considered a compound positive in this assay if a dose-related increased in the amount of [3H]thymidine incorporated into DNA over at least 3 concentrations with the highest response equal to at least twice the solvent control was attained. Positive responses were obtained for glycidol and ethyl glycidyl ether in the presence of the $9 activating system. Demonstrable, although not considered positive,responses were obtained for butyl glycidyl ether in the presence of $9 and ethyl glycidyl ether in the non-activated assay. The remainder of the assays were negative.
Discussion
This investigation had two purposes. The first was to determine the effect of varying the alkyl group on the mutagenic potential of glycidyl ethers. The second was to compare the results of different in vitro assays when applied to a group of related chemicals. Glycidol and a series of 7 alkyl glycidyl ethers with alkyl groups ranging from C-2 to C-14 were selected for this investigation. Although glycidol is not a glycidyl ether, it served as a reference "glycidyl epoxide", and enabled us to examine differences in mutagenic potentials between the alcohol and ether linkage.
Since the alkyl glycidyl e thers are prepared by treating fatty alcohols with epichlorohydrin, which is known to be a mutagen (Elmore et al., 1976; Kucerova et al., 1976), it is essential that the ether be freed of residual epichlorohydrin, lest its presence confound the results. Table 1 shows that all of the alkyl glycidyl ethers were more than 97% pure, with the unreacted fatty alcohol comprising the bulk of the remaining material. The epichlorohydrin level in 5 of the glycidyl ethers was shown to be below 5 ppm which is below the detection limits of the mutagenicity assays. It was not possible to determine the epichlorohydrin level in the short chain glycidyl
229
ethers, but since they were purified in the same manner as the longer chain species, it is believed the epichlorohydrin was also low in these compounds.
The tests selected for the study were the Salmonella/mammalian microsome assay (Ames et al., 1975), the L5178Y mouse lymphoma assay (Clive et al., 1979), and unscheduled DNA synthesis with WI38 cells (San and Stich, 1975). These assays were selected because each measures a different type of mutagenic endpoint. The diversity in the battery allowed us to observe how each assay functions when tested on the same compound.
The results of the Salmonella assay appear straightforward. The epoxides are active only with the strains that detect base-pair substitution mutagens as has been previously reported (McCann et al., 1975; Wade et al., 1978, 1979). Glycidol was by far the most mutagenic compound. Calculation of the number of revertants//~mole, as proposed by McCann et al. (1975), reveals a mutagenic potential of roughly 0.4 compared to the 0.58 reported by McCann et al. (1975). Such close agreement demonstrates the interlab reproducibility of this assay.
Similar calculations on the alkyl glycidyl ethers show two major "breaks" in mutagenic potential. Glycidol is approx. 5 times more potent than the ethyl, butyl, and hexyl glycidyl ethers which all have roughly equivalent mutagenic potencies on a molar basis. The octyl, decyl, and dodecyl glycidyl ethers are only active in the presence of metabolic activation and are much less potent than the intermediate chain-length glycidyl ethers. The tetradecyl glycidyl ether is negative in this assay.
It is interesting that the mutagenic potential of the ethers increases in the presence of the $9 fraction, which reduces the mutagenic potential of glycidol. The epoxide hydrase present in induced $9 fractions has been reported to lower the mutagenic potential of an epoxide by converting it to the diol (Ortiz De Montellano and Boparai, 1978). The stimulatory effect appears to be enzymatic since it is not brought about by $9 that has been inactivated by heat. Since microsome enzymes can cleave glycerol ether linkages (Snyder, 1972), it is possible that the ether linkages of the glycidyl ethers are being cleaved, and that the mutagenic responses elicited by the octyl, decyl, and dodecyl ethers are due to liberated glycidol.
The results of the mouse lymphoma assay both complement and contradict the Salmonella data. This assay, like the Salmonella assay, shows the longer chain-length species to be weakly or non-mutagenic. It appears that the assay may detect lower levels of the highly mutagenic species such as glycidol and the ethyl and butyl glycidyl ethers, and thus is more sensitive than the Salmonella assay. However, it appears that the octyl, decyl, and dodecyl glycidyl ethers are non-mutagenic in this assay and that for them at least the assay is less sensitive than the Salmonella test. As shown in Table 3, the data for the longer chain-length glycidyl ethers are erratic, probably because of their poor solubility and their toxic effects on the mouse of lymphoma cells.
Unscheduled DNA synthesis was induced by glycidol and ethyl glycidyl ether in the presence of an uninduced mouse liver $9 fraction. The effect of $9 in this assay was just the opposite of that seen in the mouse lymphoma assay, i.e. $9 appears to increase the amount of UDS whereas it reduced the mutagenic response in the mouse lymphoma assay. The decrease in mutagenic potency in the presence of the
230
metabolic activation system in the mouse lymphoma assay was probably due to inactivation of the epoxide group by epoxide hydrase in the induced rat liver $9 or by endogenous metabolism by the cells. It has been shown that rat liver $9 contains higher levels of epoxide hydrase than $9 from mice and that induction with Aroclor further increases the epoxide hydrase levels (Oesch, 1972).
A more difficult result to explain was why glycidol induced UDS only in the presence of the $9. While the level of epoxide hydrase is low in mouse $9 compared to rat $9, demonstrable levels of the enzyme are present, and one would expect the conversion of gtycidol to glycerol. The longer chain-length glycidyl ethers are not very soluble in aqueous solutions as evidenced by the presence of oil droplets on the surface of the medium. The protein and lipids present in $9 could enhance the solubility of these compounds thus increasing the exposure of the epoxides to the cells. However, glycidol is totally miscible in water at these concentrations and should be able to reach the cells whether or not the $9 is present. It appears that something in the $9 mix is interacting with the cells to enhance the uptake of glycidol. Whether this is a direct effect on the cells by the liver homogenate or an effect by the cofactors in the $9 mix is not known, but some type of interaction is occurring which results in increased UDS in these cells.
In summary, the study examined the mutagenicity of glycidol and 7 alkyl glycidyl ethers using 3 short-term assays. The chemicals ranged from moderately mutagenic to non-mutagenic and each assay demonstrated dose-related responses. The results generally agreed, but differences in sensitivities of the assays clearly show the need for using a battery approach to mutagenesis testing.
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