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THE DEVELOPMENTAL TOXICITY OF MECLIZINE USING THE FROG EMBRYO TERATOGENESIS ASSAY-XENOPUS (FETAX).

ABSTRACT

Meclizine, an antihistamine used to treat nausea and vomiting, was proven a potent teratogen for rats, causing abnormalities such as cleft palate, small mouth, short limbs, receded lower jaw, and unclacified vertebral bodies in rat offspring (King, 1963). Frog Embryo Teratogenesis Assay-Xenopus (FETAX) has been proposed to serve as an indicator of potential human teratogenic hazards (Bantle et al., 1994). The purpose of this research was to determine how well FETAX could predict the known mammalian developmental toxicity of meclizine. The procedure involved the exposure of varying concentrations of meclizine to early life stages of Xenopus laevis, a South African clawed frog, for 96 hours. An average lethal concentration to cause 50% death in the test population (96 hour-LC50) was calculated as 4.62 mg/L. In addition to the 96 hour-LC50, an average concentration to induce 50% malformation (96 hour-EC50) was calculated as 0.974 mg/L. The average teratogenic index (TI) for meclizine was calculated as 4.74. Acco rding to ASTM standards (ASTM, 1991), a TI (96 hour-LC50/ 96 hour-EC50) of 1.5 or greater indicates teratogenic risk. Therefore, findings in FETAX with meclizine are consistent with the mammalian literature.

INTRODUCTION

Meclizine is a piperizine derivative antihistamine (Figure 1. Haraguchi et al., 1997) used commonly to treat nausea and vomiting related to motion sickness (Drug Info. American Hospital Formulary Service, 1998; Merck Index, 1996). It can also be used to treat vertigo associated with diseases of the vestibular system (Physicians Desk Reference, 1995). The mechanism of action for meclizine is not precisely known, but is "thought to be related to its central anticholinergic action. It is also believed that an action on the medullary chemoreceptive trigger zone may be involved in the antiemetic action of the drug" (Drug Information for the Health Care Provider, 1987).

Meclizine has an oral LD50 of 1600 mg/kg and an intraperitoneal LD50 of 659 mg/kg (Sigma Chemical Company, Material Safety Data Sheet). Meclizine is a class B pregnancy drug. This means as long as benefits outweigh the potential risks, pregnant women may take the drug. Meclizine is generally considered a "safe" drug for pregnancy (Drug Info. American Hospital Formulary Service, 1998), but some epidemiological studies have suggested that meclizine could pose potential risk to the unborn fetus (Lione and Scialli, 1996).

Meclizine has been a "suspect" teratogen for years, as indicated by Schardein (1993). Some human studies in the United Kingdom showed that women treated with meclizine (plus vitamin B12) had offspring with defects, while other studies showed meclizine having no teratogenic effect (Schardein, 1993). In one case, a woman medicated with meclizine plus pyridoxine (vitamin B12) in the fourth week of gestation, had an infant with a unilateral limb defect (Jaffe et al., 1975). In another case cited by Walker (1974) a woman took meclizine and another antiemetic and had a child with iniencephaly and spina bifida. Bokesoy er al. (1983), cited a case in which a woman treated with meclizine throughout pregnancy, but primarily in the first trimester, had a child with severe micrognathia (abnormally small jaw), small mouth, widened nasal bridge, receded lower lip, cleft palate, and hypoplastic tongue and mandible.

King (1963), analyzed meclizine-induced malformations such as cleft palate, micromelia (short limbs), microstomia (small mouth), brachygnathia (receded lower jaw), and uncalcified vertebral bodies in rat offspring. According to Schardein (1993), "animal species successfully demonstrate the potential for teratogenic effect for all known human teratogens." With this in mind, meclizine could very well be considered a potential human teratogen. More studies need to be performed, however, to further demonstrate the teratogenicity of the drug meclizine.

A conserved genetic program controls embryonic development, therefore in animal assays such as FETAX data can be extrapolated to other species (Bantle, 1995). Bantle (1995) indicated that FETAX was initially designed as an indicator of potential human' developmental hazards. This assay is well suited for complex mixtures and has been ranging from the effects of pond water from Minnesota and Vermont (Fort et al., 1999) to the benefits of ascorbic acid in preventing paraquat embryotoxicity (Vismara et al., 2001).

The in vitro assay performed by Bantle et al. (1994), established that FETAX could be used to detect teratogens in mammals. This research was performed to determine if FETAX could correctly determine the developmental hazard of meclizine. This further validation of FETAX for the determination of developmental health hazards demonstrated its use in predicting the teratogenic hazards of chemicals.

MATERIALS AND METHODS

Frogs were obtained from Xenopus I (Dexter, MI). The drug meclizine, CAS Registry number 569-65-3, was obtained from Sigma Chemical Co. (St. Louis, MO). FETAX assay procedure followed the ASTM Standard Guide for the conduct of FETAX (ASTM, 1991). A reconstituted, mineralized water (FETAX solution) was used as the diluent, as described in the ASTM guide. Two replicates of 20 embryos each were placed into covered plastic Petri dishes for each test concentration. Four control plastic Petri dishes contained FETAX solution only. Embryos used were from the same clutch for each individual experiment. Three definitive experiments were run using meclizine with FETAX.

Stock solutions of meclizine were made by dissolving 10 mg of meclizine in 1 L of FETAX solution by stirring and gentle heating. Afterwards, pH was adjusted (between 6-8) as necessary, then diluted into appropriate concentrations. Control and test solutions were then transferred to plastic Petri dishes and 20 frog embryos in small cell blastula stage were placed into plastic dishes with 8 mLs of test solution. Embryos were first examined under a dissecting microscope to be certain they were viable.

Embryos were placed in an incubator at 24[degrees]C [+ or -] 1. Dead embryos were removed and solutions were changed at 24, 48, and 72 hour intervals. After changing solutions, remaining embryos were counted. At the 96 hour interval surviving embryos were sedated with MS222, counted, and examined for defects then placed in formaldehyde solution, and later in isopropanol for length measurements.

Length measurements of the embryos were made using Image Pro Analysis System. Values for 96 hour-LC50 (concentration required to kill 50%), 96 hour-EC50 (concentration where 50% of the embryos are malformed) and TI values (96 hour-LC50/96 hour-EC50) were also calculated using Litchfield-Wilcoxon probit analysis. Minimum Concentration to Inhibit Growth (MCIG) was determined using a grouped T-test.

RESULTS

Table 1 indicates the control results from the three experiments, which averaged 4.16 % mortality and 10.88% malformation. Mortality rate was below 5% for each of the three experiments. Malformation rate was more variable than mortality. However, only experiment 2 had a higher malformation rate than acceptable by the ASTM guide and all three experiments demonstrated similar results.

Table 2 contained the 96 hour-LC50, 96 hour-EC50, and TI of meclizine. The average 96 hour-LC50 and 96 hour-EC50 was 4.62 and 0.974 respectively, in mg/L. The average teratogenic index (TI) was 4.74. For experiment 1 no mortality above 50 % was achieved, so the 96 hour-LC50 and the TI was estimated not calculated. The averages for the 96 hour-LC50 and TI did not include experiment 1.

The concentration-response curves represented in panel A of figures 2, 3, and 4 showed probit graphs of the test response for each of the three experiments. For Figure 2 panel A the mortality curve was different from the other two experiments because concentrations higher than 5 mg/L were not tested. Figures 3 and 4 panel A show extremely repeatable results from different clutches of embryos. Both mortality and malformation show a good linear probit concentration-response. The slopes for all malformation curves were approximately the same.

Growth curves shown in panel B of figures 2, 3, and 4 indicated that growth is significantly inhibited between 90 and 155% of the 96 hour-LC50 (MCIG). At low concentrations there was apparently no effect on growth. Experiment 1 is the only experiment in which growth increased at low concentrations, however, this growth was not significant. The Minimum Concentration to Inhibit Growth (MCIG), shown in panel B, usually indicated the highest concentration, and was the only concentration significantly different from control.

Figure 2. Panel A is the concentration-response mortality (o) and malformation (O) curves for meclizine from Experiment 1. Response is given in probit values-- a probit of 5=50%. Panel B is embryo length measurements at 96 hours after exposure to meclizine-- concentration is expressed as % of average LC50 value length is expressed as % of mean control embryo length. * indicates significant difference on T-test (MCIG) p[less than or equal to] 0.05.

Figure 3. Panel A is the concentration-response mortality (o) and malformation (O) curves for meclizine from Experiment 2. Response is given in probit values-- a probit of 5=50%. Panel B is embryo length measurements at 96 hours after exposure to meclizine-- concentration is expressed as % of average LC5O value length is expressed as % of mean control embryo length. * indicates significant difference on T-test (MCIG) p[less than or equal to] 0.05.

Figure 4. Panel A is the concentration-response mortality (o) and malformation (O) curves for meclizine from Experiment 3. Response is given in probit values-- a probit of 5=50%. Panel B is embryo length measurements at 96 hours after exposure to meclizine-- concentration is expressed as % of average LC50 value length is expressed as % of mean control embryo length. * indicates significant difference on T-test (MCIG) p[less than or equal to] 0.05.

Figure 5 contained embryos from the same test in three different concentrations. Embryo A was a control embryo with no malformation. Embryo B was from a 0.5 mg/L test concentration of meclizine, and embryo C was from a 5 mg/L test concentration. Figure 6 portrayed an embryo from a test concentration similar to that of embryo C in figure 5, these two embryos had similar malformations. The malformations seen such as gut and eye malformations, and multiple edema were remarkably similar in each experiment, indicating that meclizine causes specific types of abnormalities at increasing concentrations.

DISCUSSION

The averages of all three experiments fell well within the acceptable ranges for the ASTM guide for the conduct of FETAX. The malformation percentage for experiment 2, however, was above acceptable limits. A possible cause for the discrepancy in the malformation averages could be explained by the subjective nature of the malformation scoring process. Each embryo was examined under a microscope and judging malformations was purely up to the person looking at the embryo. Experiment 2 may have been judged more severely than the other two experiments, or the embryos may have had a high malformation rate. The other two experiments had percentages well within the 10% range set forth by ASTM guidelines, showing that malformations in the meclizine test concentrations were not naturally occurring. The increased control mortality in experiment 2 did not appear to be a problem, as results from all three experiments were very similar as indicated by figures 2, 3, and 4.

For experiment 1, fifty- percent mortality was not achieved, so a 96 hour-LC50 or TI could not be calculated for this experiment. An estimation was taken for the two values based upon the highest concentration tested. The 96 hour-LC50 was greater than 5 mg/L and the TI was estimated by taking the ratio of the estimated 96 hour-LC50 to the calculated 96 hour-EC5O, giving a TI of greater than 6.8. In experiment 2, the 96 hour-LC5O was 6.02 mg/L, a number in agreement with the estimation from experiment I. The 96hr-LC5O for test 3 was a little lower. More death occurred than expected in this experiment, however 95% confidence intervals were overlapping. The 96 hour-EC5O for all three experiments were within the same range, a good indication that the malformations noted are caused by meclizine, and not a one-time occurrence. Table 2 also shows that all numbers from the 96 hour-LC50 and 96 hour-EC5O fall within the ninety-five percent confidence limits for each of the three experiments. The average TI for the thr ee experiments was 4.74, not far below the estimated TI for experiment 1. For all three experiments, the TI was above 1.5, showing that meclizine has potential to be a teratogen (ASTM, 1991). A TI of 4.74 indicated a moderate to high teratogenic risk for this drug.

In figure 5 the first embryo, A, was a control embryo. It had no edema, normally set eyes, properly coiled gut, and a straight tail. Embryo B was from an intermediate concentration. It had some edema, eye malformations, and though not visible in the picture, it also had some gut miscoiling, and tail abnormality. The third embryo, C, from the highest test concentration, was severely malformed, with a great amount of edema, small eyes, miscoiled gut, and a curved axial alignment. The malformations of the embryo in figure 6 were very similar to the malformations of the third embryo (C) in figure 5. This property of repeatable results confirmed that meclizine was a teratogen in FETAX.

CONCLUSION

Meclizine causes severe malformations, gut and eye malformations, and multiple edema in FETAX, as evidenced by the embryos in figures 5 and 6. The data presented in this paper support literature that meclizine is a potential direct-acting human developmental toxicant. Lastly, these results are further validation that FETAX can serve as a good indicator of potential human teratogenic risk.

ACKNOWLEDGEMENTS

We thank Jacksonville State University Student-Faculty Research Grant for supporting this research. We also thank Randa Aladdin, Heather Howell, and Darryl Pendergrass for their technical assistance.

LITERATURE CITED

American Society for Testing Materials (ASTM). 1991. Standard Guide for Conducting the Frog Embryo Teratogeneisis Assay-Xenopus (FETAX). E 1439-91. Philadelphia, PA.

Bantle, J. 1995. FETAX-A Developmental Toxicity Assay Using Frog Embryos. In: Fundamentals of Aquatic Toxicology, Effects, Environmental Fate, and Risk Assessment. 2nd Edition. Ed. Gary Rand, Taylor and Francis. Washington D.C.

Bantle, J., D. Burton, D. Dawson, J. Dumont, R. Finch, D. Fort, G. Linder, J. Rayburn, D. Buchwalter, A. Gaudet-Hull, M. Maurice, S. Turley. 1994. FETAX Interlaboratory Validation Study: Phase II Testing. Environmental Toxicology and Chemistry Vol. 13, No. 10:1629-1637.

Bokesoy, I, C. Aksuyek, E. Deniz. 1983. Oromandibular Limb Hypogenesis/Hanhart's Syndrome: Possible drug Influence on the Malformation. Clinical Genetics. 24(1):47-49.

Drug Info. American Hospital Formulary Service. 1998. p. 2410-2411.

Drug Information for the Health Care Provider. 17th ed. 1987. p. 1146-1147.

Fort, D. T. Propst, B. Stover, J. Helgen, R. Levey, K. Gallagher, J. Burkhart. 1999. Effects of Pond Water, Sediment, and Sediment extracts from Minnesota and Vermont, USA, on Early Development and Metamorphosis of Xenopus. Environmental Toxicology and Chemistry. Vol. 18, issue 12:2305-2315.

Haraguchi, K, K. Ito, H. Kotaki, Y. Sawada, T. Iga. 1997. Prediction of Drug-Induced Catalepsy Based on Dopamine D1, D2, and Muscarinic Acetylcholine Receptor Occupancies. Drug Metabolism and Disposition. 25:675-684.

Jaffe, P, M. Liberman, I. McFadyen, H. Valman. 1975. Incidence of Congenital Limb-reduction Deformities. Lancet. 1:526-527.

King, C. 1963. Teratogenic Effects of Meclizine Hydrochloride on the Rat. Science. 141:353-355.

Lione, A. and A. Scialli. 1996. The Developmental Toxicity of the H1 Histamine Antagonists. Reprod. Toxicol. 10(4): 247-255.

Merck Index. 1996. p. 984.

Physicians Desk Reference-Generics. 1st ed. 1995. p. 1779-1780.

Schardein, J. 1993. Chemically Induced Birth Defects. 2nd ed. Revised and expanded. pp. 71, 347, 446, 448-450.

Vismara C., G. Vailati, and R. Bacchetta. 2001. Reduction in Paraquat Embryotoxicity by Ascorbic Acid in Xenopus laevis. Aquatic Toxicology. Vol. 51, issue 3:293-303.

Walker, F. 1974. Familial Spina Bifida Associated with Antiemetic Ingestion in the First Semester. Birth Defects, 10:17-21.

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Table 1. Meclizine Control Results


Test # Mortality Malformation

 1 5% 9.2%
 (4/80) (7/76)

 2 3.75% 19.5%
 (3/80) (15/77)

 3 3.75% 3.95%
 (3/80) (3/77)

Average 4.16% 10.88%
 (10/240) (25/230)
Table 2. Meclizine Test Results


 Test# 96 hour-LC50 96 hour-EC50 TI
 (mg/L) (malformation)
 (mg/L)

 1 [greater than]5 0.732 [greater than]6.8
 0.13-4.2

 2 6.02 1.18 5.1
 0.86-41.98 0.044-31.62

 3 3.21 1.01 3.2
 0.36-28.33 0.16-6.30

Average 4.62 [*] 0.974 4.74 [*]



(*)Does not includes test 1.

(*)Does not includes test 1.
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Author:Wolfe, Andrea; Rayburn, James
Publication:Journal of the Alabama Academy of Science
Article Type:Statistical Data Included
Geographic Code:1U6AL
Date:Jan 1, 2001
Words:2708
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