The behavioral effects of pesticides in male mice

Neurotoxicologyand Teratology, Vol. 11, pp. 45-50. ~ PergamonPress plc, 1989. Printedin the U.S.A.

0892-0362/89 $3.00 + .00

The Behavioral Effects of Pesticides in Male Mice J. A. M I T C H E L L , S. F. L O N G , M . C. W I L S O N t A N D M . J. K A L L M A N

Department of Pharmacology, School of Pharmacy University of Mississippi, University, MS 38677 R e c e i v e d 8 July 1987

MITCHELL, J. A., S. F. LONG, M. C. WILSON AND M. J. KALLMAN. The behavioral effects of pesticides in male mice. NEUROTOXICOL TERATOL 11(l) 45-50, 1989.--Male Swiss mice, 25-30 g, were utilized to define some of the behavioral effects of the herbicides Lasso [alachlor 43%; (A)], Basalin [fluchloralin 45%; (F)], Premerge 3 [dinoseb 51%; (D)], and the fungicide Maneb-80 [maneb 80%; (M)]. These compounds were tested for their effects on locomotor activity and for their ability to establish a conditioned taste aversion following oral or dermal exposure. Individual and grouped (N=5) activity measures were assessed immediately following the dermal administration of the commercially available pesticide formulations. Grouped activity measures were also assessed following the oral administration of the compounds. Total activity was significantly (p<0.05) increased over vehicle controls in both grouped and individual subjects by A, F, and D following dermal administration. Grouped activity measures were also increased by A, F, D, and M following the oral administration of the compounds. Similar subjects were tested in a conditioned taste aversion paradigm using a normally preferred 0.3% saccharin solution. Animals were given 30 min access to the saccharin solution followed immediately by the administration of the pesticide or control solution. Twenty-four hours later, animals were given the choice of 2 solutions, one containing water and the other the 0.3% saccharin solution. The percent saccharin consumed and the total fluid intake were calculated for each group (N = 8/group). A, F, and D produced a significant aversion to (N = 8/group) the saccharin following both oral and dermal administration. Oral administration of M, but not dermal exposure, also resulted in a flavor aversion. Total fluid intake, however, was not altered by any of the treatments. These data include the acute dermal as well as oral exposure to commonly used herbicide formulations can result in behavioral alterations. Agrichemicals

Conditioned taste aversion

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BETWEEN the years 1980 and 1985, herbicides surpassed insecticides in total volume of production and sales both in the United States and in world markets. The yearly growth of production of herbicides during this time was 1.9% as compared to 0.9% for insecticides (9). Use of chemical herbicides has almost totally supplanted mechanical cultivating and weeding in U.S. agriculture. In addition, herbicides are now widely employed in vegetation control along highways, railways, and utility lines. It is this use along such right-of-ways, that has provoked concern with respect to increased incidence of accidental exposure to these compounds. Many herbicides and fungicides are considered to be highly specific to their target organisms, and exhibit little or no toxicity to nontarget organisms. However, some pesticidal compounds are considered to be a threat to the environment because they are stable and persistent. Increased mammalian toxicity is usually associated with such environmental persistence and low biodegradability (7). In spite of their widespread use, few reports investigating potential mammalian toxicity associated with exposure to herbicides or fungicides exist in the scientific literature. In this study, the behavioral effects of exposure to three .herbicides and one

Behavior

fungicide have been examined. In all cases, the commercially available formulated product was utilized because accidental exposure to mammals would most likely result from the application of the commercial product rather than exposure to the technical grade material. The dermal route of exposure was chosen because it is a likely route of accidental exposure to the compounds. In order to determine if the effects noted following the dermal exposure were systemic rather than local, subjects were also tested following oral exposure to the compounds. Earlier preliminary data from this laboratory examined the behavioral effects of dermal application of 0.15 ml of the formulated products alachlor, fluchloralin, dinoseb, maneb, bentazon, chlorthalonil, and atrazine (11). These formulated pesticides were tested for their effects on locomotor ataxia, activity, and for their ability to produce a conditioned taste aversion to a normally preferred 0.3% saccharin solution. Increases in activity, as measured by the total number of activity counts over a 4-hr session, and a significant aversion to the saccharin solution resulted from the application of the pesticides alachlor, dinoseb, and fluchloralin as compared to xylene vehicle controls. Locomotor ataxia was assessed using a rotating rod (Rotorod, Omnitech Electronics, Columbus, OH). No differences from control were

~Requests for reprints should be addressed to M. C. Wilson.

45

MITCHELL ET AL.

46

noted following any treatment. In the present study, we have examined the behavioral effects of 3 doses of the pesticides alachlor, fluchloralin, dinoseb, and maneb. These compounds were chosen based on their activity in the preliminary testing, with maneb utilized as an example of a pesticide not formulated in an organic hydrocarbon based vehicle, thus limiting absorption following dermal exposure. The behavioral parameters examined in this study included a determination of the effects of the pesticides in a conditioned taste aversion paradigm, and also on ambulatory and nonambulatory activity. Reiter et al. have reported data which suggest that activity provides a sensitive measure for evaluating the behavioral effects of a pyrethroid pesticide, deltamethrin, at doses that did not cause the characteristic neurotoxicological syndrome (12). The conditioned taste aversion procedure employed involved the association of a normally preferred 0.3% saccharin solution with the administration of the pesticide or appropriate control solution. The procedure was utilized because it provides a sensitive method for the measurement of the aversive properties of a compound. Animals will avoid taste stimuli when prior ingestion has been associated with the administration of an aversive compound (13). In order to determine whether the doses of the pesticides utilized were sublethal, 96-hr lethality measures were recorded for all treatment groups.

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METHOD

Conditioned Taste Aversion Following Dermal Exposure One hundred twenty male Swiss mice, (Charles Rivers Laboratories), 20-25 g, were utilized to determine the effects of dermal exposure to the pesticides in a conditioned taste aversion paradigm. Subjects were individually housed in hanging stainless steel cages and provided with ad lib access to food throughout the study. Lighting was maintained on a 12 hr: 12 hr light:dark cycle, and the temperature maintained at 2 2 _ 2°C. The procedure used to produce the conditioned taste aversion was identical to a procedure previously used to assess halogenated hydrocarbons (6,8). During a 7-day acclimation period (days 1-7), the subjects were allowed unlimited access to two 10 ml drinking syringes, located on the right and left front comers of the home cage, containing water. On days 8 and 9, access to the two syringes was limited to 1 hr/day, followed by 4 days of 30 rain/day limited access to the water (days 10-13). On day 12, the portion of each subjects back immediately caudal to the skull was shaved with electric clippers to prepare the animals for dermal exposure to the compounds. On the conditioning day (day 14), the subjects were allowed 30-min access to a drinking syringe containing the 0.3% saccharin solution, and the volume consumed during this period was recorded. Immediately following access to the saccharin solution, one of the test compounds or control solutions was applied via tuburculin syringe to the shaved back of each animal. The 15 groups (N = 8/group) received either alachlor (A), fluchloralin (F), dinoseb (D), or maneb (M), (20, 200, or 2000 mg/kg), water, or xylene (Mixture of isomers). Xylene was utilized as a vehicle control for A, F, and D, while water served in this capacity for M. The classical skin irritant, oil of mustard (0.5%), obtained from Kalsec Inc. Kalamazoo, M1, was utilized as the positive control (2). Twenty-four hr following exposure to the compounds, the subjects were given 30-min choice access to two drinking syringes, one containing water and the other, 0.3% saccharin solution. The syringe containing the saccharin solution was randomly assigned to either the right or the left side of the cage to counterbalance for any position preference. The amount of each solution consumed was recorded, and the mean percent saccharin

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FIG. 1. Mean-+ S.E.M. percent saccharin intake in animals following oral and dermal exposure to fluchloralin, expressed as a function of log dose fluchloralin (top panel) and dinoseb (bottom panel). M=Oil of Mustard (0.5%); W=Water; X=Xylene; *Significantly (p<-0.05) reduced from water control; +Significantly (p-<0.05) reduced from xylene control.

intake and total fluid intake were calculated for each group.

Conditioned Taste Aversion Following Oral Exposure One hundred twelve male Swiss mice, (Charles River Laboratories), 20-25 g, were randomly assigned to one of 14 groups in order to determine if the oral administration of the pesticides would produce a conditioned taste aversion to the preferred saccharin solution. The subjects were individually housed in hanging stainless steel cages and provided with ad lib access to food. Water was also available on an ad lib basis during a 7-day acclimation period. Beginning on day 8, subjects were placed on a limited access schedule of water availability. Drinking syringes filled with water were available for 1 hr/day on days 8 and 9, followed by 4 days of 30-rain limited access. Also on day 8, subjects began receiving a daily gavage with 0.15 ml water immediately following access to the drinking syringes in order to familiarize the animals with the gavage procedure. On the conditioning day (day 14), the subjects were given 30-rain access to a drinking syringe filled with the 0.3% saccharin solution. Immediately following access to the saccharin solution, the subjects were gavaged with 0.15 ml of the appropriate pesticide or control solution. Treatments included A, F, D, or M (0.4, 4.0, or 40

BEHAVIORAL EFFECTS OF PESTICIDES

47

TABLE 1 MEAN +_ S.E.M. 4-HR ACTIVITY COUNTS FOLLOWING THE DERMAL APPLICATION OF THE PESTICIDES IN GROUP TESTED SUBJECTS (6 SETS OF 5 SUBJECTS/TREATMENT) Treatment (mg/kg) Water Xylene Alachlor 20 Alachlor 200 Alachlor 2000 Fluchloralin 20 Fluchloralin 200 Fluchloralin 2000 Dinoseb 20 Dinoseb 200 Dinoseb 2000 Maneb 20 Maneb 200 Maneb 2000

4-Hr Activity 7666.2 12025.7 10453.8 19797.5 31123.8 14040.8 21995.8 31268.3 9513.3 23025.0 32710.8 10666.7 15030.0 23069.2

z 688.1 ± 668.0 - 649.2? --+ 2403.0*? -+ 2300.1" ? --+ 1023.47 "- 1428.0"? --- 2811.2"? ± 1213.1 -- 2595.2*? --- 1809.3"? --- 808.2 --- 2099.2? ± 1640.8"?

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mg/kg), water, or xylene. Twenty-four hr following the administration of the test compound via gavage, the subjects were given 30-min access to two drinking syringes, one containing water and the other the 0.3% saccharin solution. The amount of each solution consumed was recorded, and the total fluid intake and the mean percent saccharin intake were calculated for each treatment group.

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Activity Following Dermal Exposure Male Swiss mice, 20-25 g, were utilized in a procedure to determine the effects of dermal exposure to the pesticides on both grouped (N = 5/group) and individual total 4-hr activity. Subjects utilized in the grouped activity testing procedure were grouphoused (5/cage) in plastic cages and provided with ad lib access to both food and water. Following a 7-day acclimation period, the subjects were randomly assigned to one of 14 treatments ( N = 30/treatment) and tested in groups of 5 subjects for the effects of the pesticides on total 4-hr activity. Five group-housed subjects were tested simultaneously in each session, with each dose tested in 6 groups of subjects. The back of each animal was prepared as described above 24 hr prior to exposure to the pesticides. The subjects were exposed to A, F, D, or M (20,200, or 2000 mg/kg), water, or xylene in a volume of 0.15 ml. Testing was initiated immediately following the administration of the compounds. Activity measures were objectively assessed in a Digiscan optical animal activity monitor (Omnitech Electronics, Columbus, OH). Measurements reflect the mean total 4-hr activity count for each group of five animals. The effects of the pesticides on total activity (ambulatory plus nonambulatory ) were compared to the water and xylene control values. For the assessment of individual activity, subjects were individually housed in hanging stainless steel cages and allowed ad lib access to both food and water. Subjects were assigned to one of 14 groups ( N = 10/group) and tested individually in the activity monitor over a 4-hr session as described above. For individually tested subjects, both ambulatory and nonambulatory activity measures were separately recorded. Ambulatory activity was recorded when a subject sequentially disrupted at least two adjacent infared beams; whereas, nonambulatory activity was

FIG. 2. Mean + S.E.M. percent saccharin intake in animals following oral and dermal exposure to alachlor, expressed as a function of log dose alachlor (top panel) and maneb (bottom panel). M = Oil of Mustard (0.5%); W=Water; X=Xylene; *Significantly (p-0.05) reduced from water control; ?Significantly (p-
recorded when a subject sequentially broke the same beam.

Activity Following Oral Exposure Male Swiss mice were tested in the activity monitor to determine the effects of the oral administration of the pesticides on grouped 4-hr activity. Prior to testing, the subjects were grouphoused (5/cage) in plastic cages and provided ad lib access to food and water. Following a 7-day acclimation period, the subjects were randomly assigned to one of 14 groups (N = 35/group). Daily gavaging with 0.15 ml water was initiated and continued for 4 days prior to testing in order to condition the animals to the procedure. On the test day, the subjects were administered the appropriate pesticide (A, F, D, or M: 0.4, 4.0, or 40 mg/kg), water, or xylene via the gavage tube in a volume of 0.15 ml. The subjects were immediately placed in the activity monitor in groups of 5, and the total activity counts were recorded over a 4-hr session.

96 Hour Lethality Subjects were monitored at 24-hr intervals for lethality following each treatment. Food and water were available on an ad lib basis following the conclusion of the behavioral testing, and the

MITCHELL ET AL.

48

TABLE 2 MEAN -+ S.E.M. 4-HR ACTIVITY COUNTS IN INDIVIDUALLYTESTED SUBJECTS (N = 10) FOLLOWING THE DERMAL APPLICATION OF THE PESTICIDES 4-Hr Activity Treatment mg/kg Water Xylene Alachlor 20 Alachlor 200 Alachlor 2000 Fluchloralin 20 Fluchloralin 200 Fluchloralin 2000 Dinoseb 20 Dinoseb 200 Dinoseb 2000 Maneb 20 Maneb 200 Maneb 2000

Nonambulatory 1876.3 2182.4 2565.0 3116.0 3886.2 2372.4 2965.8 3376.4 2074.3 2486.5 3075.4 1974.6 2188.5 2376.7

+ 262.9 --+- 243.8 ± 290.2t ± 279.6*? ± 252.9"? ± 218.7 ± 318.1"+ ± 287.3*+ ± 318.2 ± 278.6 +- 248.4*# + 218.3 ~ 238.7 _ 263.8

Ambulatory 1074.3 1110.8 1705.0 1781.6 1834.4 1636.3 1875.4 1966.3 1087.5 1346.3 1710.6 1042.3 1042.3 1178.2

+ 113.6 ± 97.9 ± 171.6"+ + 150.9"? ± 122.0'? ± 107.6"t ± 133.7'+ +-- 168.4"f ± 93.2 ± 122.3+ +__ 137.8"t ± 101.7 z 101.7 _ 135.6

*Significantly increased (p<0.05) over xytene vehicle control. tSignificantly increased (p<0.05) over water control. TABLE 3 MEAN _+ S.E.M. 4-HR ACTIVITYCOUNTS FOLLOWING ORAL ADMINISTRATION OF THE PESTICIDES IN GROUP TESTED SUBJECTS (6 SETS OF 5 SUBJECTS/TREATMENT) Treatment (mg/kg)

4-Hr Activity

Water Xylene Alachlor 0.4 Alachlor 4.0 Alachlor 40 Dinoseb 0.4 Dinoseb 4.0 Dinoseb 40 Maneb 0.4 Maneb 4.0 Maneb 40

8265.1 6234.4 7799.0 13749.0 14524.1 8062.2 9540.2 12752.0 8353.6 9124.2 13053.0

+- 491.9 -+ 302.9 --- 568.5 - 752.8"+ +- 792.8"t ± 515.3 _+ 366.1" +_ 874.8*+ _+ 427.5 + 432.9 _+ 788.7"t

*Significantly increased (p<0.05) over xylene vehicle control. #Significantly increased (p<0.05) over water control.

the data in Figs. 1 and 2. F (200 and 2000 mg/kg) Fig. 1 top panel, D ( 2 0 , 2 0 0 and 2000 mg/kg) Fig. 1 bottom panel, and A (200 and 2000 mg/kg), Fig. 2 top panel, produced an aversion to the saccharin solution that was significantly greater (p<0.05) than that resulting from exposure to either the water or xylene vehicle controls. Administration of M did not result in a significant reduction in percent saccharin intake as compared to the controls. Total fluid intake did not differ significantly from either control solution for any of the treatment groups. Since no changes in this parameter resulted from the various treatments, these data were not illustrated. Administration of the 0.5% oil of mustard positive control produced a reduction in percent saccharin intake that differed significantly from both the water and xylene controls (p~0.05). No differences between water and xylene controls were observed.

Conditioned Taste Aversion Following Oral Administration Oral administration of the pesticides by gavage resulted in a significant reduction (p<-0.05) in the percent saccharin intake as compared to the xylene and water controls (Figs. 1 and 2). Administration of A and F (0.4, 4.0, and 40 mg/kg), D (4.0 and 40 mg/kg), and M (40 mg/kg) resulted in a percent saccharin intake significantly reduced (p--<0.05) from both the water and xylene vehicle controls. D (0.4 mg/kg) also significantly reduced ( p ~ 0 . 0 5 ) the percent saccharin intake as compared to the water control. Total fluid intake was not reduced from the controls by any treatment, and no differences between water and xylene controls were observed.

Activity Following Dermal Exposure The effects of dermal exposure to the pesticides on 4-hr activity counts in grouped and individual subjects are shown in Tables 1 and 2. In grouped subjects (Table 1), treatment with A (200 and 2000 mg/kg), D (200 and 2000 mg/kg), F (200 and 2000 mg/kg), and M (2000 mg/kg) resulted in activity counts that were significantly increased over both the water and xylene vehicle controls (p--<0.05). Dermal administration also significantly increased (p--<0.05) activity over the vehicle controls in individually tested subjects (Table 2). A and F (200 and 2000 mg/kg) and D (2000 mg/kg) significantly (p-<0.05) increased both ambulatory and nonambulatory activity. The lowest dose of A and F tested (20 mg/kg) also resulted in a significant increase in ambulatory activity measures in individual subjects as compared to both xylene and water controls. No differences between the water and xylene controls were observed.

Activity Following Oral Administration subjects were monitored for lethality every 24 hr for 96 hr.

Statistical Analyses All data presented in the tables and figures are in terms of the mean-+ the standard error. Data were analyzed using analyses of variance procedures. Individual groups were compared to the water and vehicle (xylene) controls using the Dunnett's modification of the t-test. An alpha level of 0.05 was used to determine significant differences from the control values. RESULTS

Conditioned Taste Aversion Following Dermal Exposure The ability of dermal exposure to the pesticides to produce an aversion to a normally preferred saccharin solution is depicted by

The effects of the oral administration of the pesticides on grouped activity are shown in Table 3. A, F, (4.0 and 40 mg/kg), D and M (40 mg/kg) significantly (p<-0.05) increased total activity as compared to both the water and xylene controls. D (4.0 mg/kg) also significantly increased activity over the xylene control. Administration of xylene resulted in a significant decrease in total 4-hr activity as compared to the water control.

96-Hour Lethality Ninety-six-hr lethality figures are shown in Table 4. Dermal treatment with A and F (2000 mg/kg) resulted in a 16.7% lethality. Deaths occurred between 24 and 48 hr posttreatment. Lethality was also noted following oral treatment with the herbicides: A (40 mg/kg; 20% lethality, and 40 mg/kg; 10% lethality) F (40 mg/kg; 10% lethality), and D (40 mg/kg; 10% lethality).

BEHAVIORAL EFFECTS OF PESTICIDES

49

TABLE 4 PERCENT LETHALITY OVER THE 96 HR TIME PERIOD IMMEDIATELY FOLLOWING APPLICATION OF THE PESTICIDES BY BOTH THE ORAL AND DERMAL ROUTES Oral Treatment (mg/kg) Water Xylene Alachlor 0.4 Alachlor 4.0 Alachlor 40 Dinoseb 0.4 Dinoseb 4.0 Dinoseb 40 Maneb 0.4 Maneb 4.0 Maneb 40

Dermal Lethality

Treatment (mg/kg)

Lethality

0 0 0 20% 10% 0 0 10% 0 0 0

Water Xylene Alachlor 20 Alachlor 200 Alachlor 2000 Dinoseb 20 Dinoseb 200 Dinoseb Maneb 20 Maneb 200 Maneb 2000

0 0 0 0 16.7% 0 0 0 0 0 0

DISCUSSION The results of this study indicate that dermal exposure to the pesticides alachlor, fluchloralin, dinoseb, and maneb can affect certain behavioral parameters in male mice. Compounds which produce a conditioned taste aversion are generally believed to do so because of an aversive reaction in the animals tested. Furthermore, this behavioral response apparently results from an unpleasant effect induced by the treatment (4). In the present study, the effects of the pesticides in the conditioned taste aversion paradigm following dermal administration may have been due to a localized sensory effect at the site of application. Oil of mustard, a known skin irritant, produced a similar effect in the conditioned taste aversion procedure after dermal administration. Whereas, there were definite signs of oil of mustard induced irritation, these were not seen following herbicide administration which suggests that the herbicide effect on conditioned taste aversion was not the result of an irritant action. The dermal administration of maneb did not result in an aversion to the saccharin solution. Of the compounds tested, maneb was the only formulation that was not tested in a xylene-based vehicle. Maneb (80% formulation) is sold as a wettable powder soluble in water. Because of this solubility profile, it is not likely that a significant amount of the dose was absorbed into the circulation following dermal application. However, it is possible that the effect noted in the conditioned taste aversion procedure following dermal application of the other

pesticides may have been due to systemic effects following absorption of the pesticides. This hypothesis is supported by the reduction in percent saccharin intake observed following the oral administration of the pesticides, including maneb. The effects observed in the conditioned taste aversion testing were specific for the normally preferred 0.3% saccharin solution, since total fluid intake was not altered by any of the treatments. This fact would suggest a lack of general toxicity at the time of testing. The effects of the pesticides on activity also support the hypothesis that these agents may affect the central nervous system (1, 3, 5, 10, 12). The increases in activity observed in this study following both dermal and oral administration of the pesticides were not specific for either ambulatory or nonambulatory activity. Both ambulatory and nonambulatory activity measures were affected by the treatment. The increase observed in grouped activity measures following the dermal administration of maneb may have resulted from ingestion of the compound by reciprocal licking of subjects in the test group. This explanation is supported by the finding that maneb given dermally did not increase activity in individually tested animals. In all instances, the commercially available formulations of the products were tested in this project. The effects of technical grade alachlor, dinoseb, and fiuchloralin may be qualitatively and/or quantitatively different from the formulation as a result of interactions of the pesticide with the organic hydrocarbon vehicle in which they are formulated. These substances may enhance absorption of the active material due to their lipophilicity. Further experimentation is necessary in order to compare the effects of the active pesticidal ingredient (i.e., technical grade material) with the formulation and vehicle. A determination of the kinetics following dermal application or oral administration is necessary in order to more accurately define the mechanism of the alterations in activity and development of the conditioned taste aversion which occurred following treatment with these compounds. The results of this study suggest that at least some herbicides, in addition to pyrethrins, organophosphate, and carbamate pesticides, can produce behavioral manifestations following accidental exposure. The effects responsible for the conditioned aversion await further exploration as does the generalization of these effects across herbicides. ACKNOWLEDGEMENTS The authors wish to express their appreciation to Dr. Larry Lane, Mississippi State University, for supplying the formulated pesticides used in this study, and also Kalsec, Inc., Kalamazoo, MI, for donating the oil of mustard. We would also like to express appreciation to the Vicksburg Hospital Medical Foundation for supplying us with the Rotorod used in the ataxia studies. This research was supported in part by the Research Institute of Pharmaceutical Sciences, and was presented in part at the 1986 Society of Toxicology meeting in Washington, D.C.

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9. Marquis, J. K. Herbicides. In: Homburger, F., ed. Contemporary issues in pesticide toxicology and pharmacology. New York: Karger Inc.; 1986:87-95. 10. Mitchell, J. A.; Wilson, M. C. Behavioral effects of dermally applied herbicides. Toxicologist 6(1):219; 1986 abstract. 11. Reiter, L. W.; McPhail, R. C.; Ruppert, P. A.; Eckerman, D. A. Animal models of toxicity: Some comparative data on the sensitivity

M I T C H E L L ET AL.

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