Return to Submission of Objections to EPA's Final Rule on Sulfuryl fluoride

APPENDIX G. Fluoride's adverse effects on the Male Reproductive System March 23, 2004, Submission to US EPA from Fluoride Action Network: Objections and Request for a Hearing RE: US EPA's January 23, 2004, Final Rule for Sulfuryl Fluoride.

The following Tables:

Table 1. RAT - 16 studies

Table 2. MOUSE - 12 studies

Table 3. RABBIT - 6 studies

Table 4. GERBIL and BANK VOLE - 2 studies

Table 5. MISCELLANEOUS - HUMAN - 6 studies

Table 6. RAM - 1 study

Table 1. RAT
F Dose Effect	Abstract
2002 RAT 20mg/kg/day for 29 days oral gavage exerts an adverse effect on the male reproductive system significant diminution in the relative wet weight of the testis, prostate, and seminal vesicle Epididymal sperm count was decreased significantly	Testicular toxicity in sodium fluoride treated rats: association with oxidative stress. Ghosh D et al. Reprod Toxicol 2002 Jul;16(4):385 This study examined the effect of sodium fluoride, a water pollutant important through the world, including India, on testicular steroidogenic and gametogenic activities in relation to testicular oxidative stress in rats. Sodium fluoride treatment at 20mg/kg/day for 29 days by oral gavage resulted in significant diminution in the relative wet weight of the testis, prostate, and seminal vesicle without alteration in the body weight gain. Testicular Delta(5),3beta-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD activities were decreased significantly along with significant diminution in plasma levels of testosterone in the fluoride-exposed group compared to the control. Epididymal sperm count was decreased significantly in the fluoride-treated group and qualitative examination of testicular sections revealed fewer mature luminal spermatozoa in comparison to the control. The seminiferous tubules were dilated in treated animals. Fluoride treatment was associated with oxidative stress as indicated by an increased level of conjugated dienes in the testis, epididymis, and epididymal sperm pellet with respect to control. Peroxidase and catalase activities in the sperm pellet were decreased significantly in comparison to the control. The results of this experiment indicate that fluoride at a dose encountered in drinking water in contaminated areas exerts an adverse effect on the male reproductive system and this effect is associated with indicators of oxidative stress.
2000 RAT 150 mg/L NaF GSH-Px activities in the tissues of testis and epididymis were observed in ascorbic acid and fluoride group	[The primary study of antagonism of selenium on fluoride-induced reproductive toxicity of male rat] [Article in Chinese] Zhu XZ, Ying CJ, Liu SH, Yang KD, Wang QZ. Chung-Kuo Kung Kung Wei Sheng (China Public Health) 2000 Aug;16(8):697-8 Abstract from Toxline at Toxnet Abstract: The protective effect of ascorbic acid at dose level of 1.0 mg/L in drinking water against the fluoride-induced damage on reproductive system of rat was studied. 150 mg/L sodium fluoride (NaF) in drinking water of male rat can cause the significant decrease of sperm count and mobility, the increase of serum and testicular lipid peroxides (LPO) contents, and the adenosine triphosphatase (ATPase) activity depression of epididymis. All of those effects are reversible by adding adequate ascorbic acid in drinking water simultaneously. The effects of ascorbic acid against fluoride-induced damages are similar to those produced by 2.0 mg/L Na2SeO3 in the drinking water of rats. However, no significant recovery of fluoride-induced effects on GSH-Px activities in the tissues of testis and epididymis were observed in ascorbic acid and fluoride group. The mechanism of ascorbic acid on fluoride-induced damage of male reproductive system need to be further studied.
1997 RAT 10 mg NaF/kg BW for 30 days the protein profile was disturbed more in testis than in cauda epididymis, whereas phospholipids and gluthathione levels were affected more in cauda than in testis.	Fluoride toxicity on rat testis and cauda epididymal tissue components and its reversal Chinoy NJ, Shukla S, Walimbe AS, Bhattacharya S Fluoride 1997; 30(1):41-50 Summary: The toxic effects were evaluated of sodium fluoride (NaF) ingestion on the physiology of tissue components of testis and epididymis of adult, male albino rats, and the possible reversal of the effects by use of some antidotes. The results revealed that the testis and cauda epididymal proteins were altered, with disappearance of some proteins and induction of some new ones. This remained unaltered during NaF ingestion, but a significant decrease occurred in phosphatidlyethanolamine, phosphatidylserine and phosphatidylinositol. Significantly low levels of glutathione after 30 days of treatment were also obtained. On comparing the alterations in protein profile, phospholipds and glutathione in both tissues, it was evident that the protein profile was disturbed more in testis than in cauda epididymis, whereas phospholipids and gluthathione levels were affected more in cauda than in testis. The investigations into reversibility showed that ascorbic acid (vitamin C) and calcium could ameliorate fluoride toxicity.
1995 RAT NaF 10 mg/kg BW for 30 and 50 days A significant reduction in electrolyte levels of sperm also occurred which would also affect their viability. The protein levels in cauda epididymal sperm suspension, vas deferens, seminal vesicle and prostate were significantly decreased after NaF administration The results, corroborated by earlier data from our laboratory, show that fluoride has a definite effect on male reproduction and fertility.	Amelioration of fluoride toxicity in some accessory reproductive glands and spermatozoa of rat Chinoy NF, Narayana MV, Dalal V, Rawat M, Patel D Fluoride 1995; 28(2):75-86 Summary: Sodium fluoride (NaF) at a dose of 10 mg/kg body weight was administered orally to male rats (Rettus norvegicus) daily for 30 and 50 days to evaluate the effect of the physiology of some sex accessory glands and sperm functions. The effects of withdrawal upon cessation of NaF ingestion, and of administering ascorbic acid (AA) and/or calcium (Ca++) along with NaF, were also investigated. The results revealed that the NaF treatment caused a significant elevation in serum fluoride levels with a simultaneous rise in Ca++ levels. This could be attributed to the formation of a calcium fluoride complex leading to calcium accumulation. The treatment resulted in structural and metabolic alterations in sperm, leading to low sperm motility, a low sperm mitochondrial activity index (SMAI), reduced viability (live:dead ratio), and changes in sperm membrane phospholipids (particularly phosphatidylinositol, phosphatidylserine and phosphatidylethanolamine, which would affect hormone receptor interaction and their functions). A significant reduction in electrolyte levels of sperm also occurred which would also affect their viability. The protein levels in cauda epididymal sperm suspension, vas deferens, seminal vesicle and prostate were significantly decreased after NaF administration, which may be due to altered protein metabolism by interference of fluoride ions. The changes in epididymal protein profile, with absence of some proteins and induction of some new ones, were probably a result o the "stress proteins" in NaF-treated rats affecting the structural and functional integrity of sperm. Glycogen accumulation in vas deferens and a decrease in fructose in seminal vesicles and vas deferens indicated disturbances in carbohydrate metabolism in these organs. However, withdrawl of treatment resulted in partial recovery. A significant recovery from NaF-induced toxic effects occurred following administation of ascorbic acid and/or calcium, while combined treatment (AA + Ca++) for 70 days manifested a synergistic effect. The transient fluoride-induced effects were reversible. The results, corroborated by earlier data from our laboratory, show that fluoride has a definite effect on male reproduction and fertility. Ascorbic acid and calcium are proposed as therapeutic agents in endemic populations for ameiloration of effects of fluoride.
1995 RAT NaF in drinking water 100 mg/L, and 200 mg/L for 2, 4, and 6 weeks. Results suggest that fluoride may have some harmful effects on the reproductive system in male rats.	The influence of fluoride on the content of testosterone and cholesterol in rat Zhao ZL, Wu NP, Gao WH Fluoride 1995; 28(3):128-130 Summary: Fifty four Wistar male rats were randomly divided into three groups, drinking water containing 0.6 mg/L (control group). 100 mg/L, and 200 mg/L sodium fluoride, respectively. Rats were killed at the second, fourth and sixth weeks after experiment initiation, respectively. The levels of serum testosterone, testis cholesterol, and hepatic tissue cholesterol were determined. Results showed that the serum testosterone level had decreased with time in rats drinking water containing 100 and 200 mg/L fluoride. While testis cholesterol level did not change, it was significantly decreased in the liver at the fourth and sixth week when compared with the control group. Results suggest that fluoride may have some harmful effects on the reproductive system in male rats.
1994 RAT NaF 10 mg/kg BW for 50 days The histomorphometric studies revealed significant change in the Leydig cell diameter in correlation with the androgen levels. These results indicate that fluoride does interfere with steroidogenesis in short-term low-dose exposures in rats.	Effect of fluoride on rat testicular steroidogenesis MV Narayana and NJ Chinoy Fluoride 1994; 27(1):7-12 Summary: In view of reports of infertility among human populations in fluorosis prevailing regions, we investigated the effect of fluoride ingestion on testicular steroidogenesis in rats. Sodium fluoride (NaF) was administered to the rats orally at a daily dose of 10 mg/kg bodyweight for 50 days. The treatment did not cause significant change in testicular cholesterol levels, indicating that metabolism was not altered and that there was no hypo/hypercholesterolemic effect. In addition, activities of the intermediary enzymes in adrongenesis, viz, 3B- and 17B-hydroxysteriod dehydrogenase were only modestly decreased by NaF ingestion. Subsequently, the determination of circulating androgen levels was similar in NaF-treated rats showed a downward trend compared to those of the control group, suggesting alteration in testosterone concentration. The histomorphometric studies revealed significant change in the Leydig cell diameter in correlation with the androgen levels. These results indicate that fluoride does interfere with steroidogenesis in short-term low-dose exposures in rats.
1994 RAT (21-24 days old) NaF 10 mg/kg BW for 30 days changes resulted in a significant decrease in sperm motility and thereby fertility rate.	Beneficial effects of ascorbic acid and calcium on reproductive functions of sodium fluoride-treated prepubertal male rats Chinoy NJ, Reddy VVPC, Michael M Fluoride 1994; 27(2):67-75 Summary: The therapeutic effects of ascorbic acid and calcium (Ca2+) supplementation on reproductive functions of fluoride-treated (10 mg/kg body weight) male rats were investigated. Sodium fluoride treatement resulted in a decrease in almost all parameters studied except concentration of testicular cholesterol, which implies that androgen synthesis might not be affected by NaF treatment. Succinate dehydrogenase activity decreased in testis suggesting that its oxidative metabolism was altered by NaF treatment. Adenosine triposphatase activity, protein, and sialic acid levels in caput and cauda epididymides also showed a decrease. All these changes resulted in a significant decrease in sperm motility and thereby fertility rate. Glycogen concentrations in vas deferens were altered, probably due to impaired metabolic turnover. The fructose levels in vas deferens and seminal vesicle as well as the acid phosphatase activity in ventral prostate were also decreased significantly by NaF treatment. On the other hand, simultaneous treatment of NaF along with ascorbic acid or calcium resulted in recovery in all the affected parameters studied. The recovery was more significant after treatment with ascorbic acid than with calcium. Therefore, ascorbic acid and calcium may be useful for amelioration of fluoride toxicity in endemic areas.
1994 RAT NaF 10 mg/kg BW 50 days sperm acrosomal hyaluronidase and acrosin were reduced low sperm motility and count	Reversible effect of sodium fluoride ingestion on spermatozoa of the rat. Narayana MV, Chinoy NJ. International Journal of Fertility 39 (6) 337-346. 1994. The effects of ingestion of sodium fluoride (NaF), 10 mg/kg body weight for 50 days, on the structure and metabolism of sperm of albino rats (Rattus norvegicus), were investigated. In different groups of rats, the reversible effects upon withdrawal of NaF treatment and by administering some therapeutic agents, viz., ascorbic acid and calcium alone and in combination with NaF (50 and 70 days), on sperm structure and metabolism were also studied. The results revealed that the sperm acrosomal hyaluronidase and acrosin were reduced after 50 days of NaF treatment. Sperm stained with acidic alcoholic silver nitrate revealed acrosomal damage and deflagellation, which might be causative factors for the reduced activity of the enzymes. These alterations also resulted in a decline in sperm motility. The cauda epididymal sperm count was decreased, perhaps because of spermatogenic arrest. Thus, the low sperm motility and count ultimately contributed toward reduction in fertility by NaF treatment. However, withdrawal of NaF treatment for 70 days produced incomplete recovery, while administration of ascorbic acid and calcium, individually and in combination, brought about significant recovery of fluoride-induced effects. Thus, the effects of fluoride on sperm structure and metabolism of rats are transient and reversible.
1991 RAT No abstrac	Effects of fluoride ingestion on the physiology of reproductive organs of male rats Chinoy NJ, Pradeep PK, Sequeira E. Journal of Environmental Biology 13 (1) 55-61. 1991.
1989 RAT 100 or 200 ppm NaF 60 days dose-related decrease in reproductive performance decrease in serum testosterone at 200 ppm	Effect of high fluoride on the reproductive performance of the male rat. Araibi AA, Yousif WH et al. J Biol asci Res 1989: 20(1):19-30. Sodium fluoride administered to male rats at dietary concentrations of 100 or 200 ppm for 60 days caused a dose-related decrease in reproductive performance; a decrease in serum testosterone levels was observed with the higher dose.
1986 RAT NaF is an inhibitor of testicular dolichyl phosphate phosphatase activity	Dolichol kinase activity in the developing rat testis. Berkowitz L, Nyquist SE. Biol Reprod 1986 Apr;34(3):518-26 Dolichyl phosphate concentrations, a primary factor in regulating the rate of N-glycosidically linked glycoprotein synthesis, are dependent upon a cytidine triphosphate (CTP)-dependent dolichol kinase. This study examines dolichol kinase in rat testicular microsomes and defines assay conditions. As with dolichol kinases from other tissues, addition of 2-mercaptoethanol increased activity 60%. Inclusion of NaF, an inhibitor of testicular dolichyl phosphate phosphatase activity, also resulted in a 38% increase in activity. Triton X-100 was necessary for phosphorylation of both endogenous and exogenous dolichol; however, concentrations of detergent in excess of 0.25-0.35% were inhibitory. A 2- to 5-fold stimulation of kinase activity was obtained by addition of 50-100 microM exogenous dolichol. The high level of nucleoside triphosphatase activity in testicular microsomes mandated the inclusion of high levels of uridine triphosphate (UTP) to protect the [gamma-32 P] CTP. Increasing UTP concentrations up to 50 mM resulted in increased product formation. A clear requirement for divalent cations was observed; 5 mM ethylenediaminetetraacetate (EDTA) abolished activity. The following order of cation effectiveness was observed: Mn greater than or equal to Ca greater than Cd greater than Zn much greater than Mg. Ten mM optima were established for Ca2+ and Mn2+; the presence of UTP, however, results in significantly reduced concentrations of free Ca2+. Ion combination studies demonstrated interactive inhibitory effects between Ca2+ and other stimulatory divalent cations. Addition of 2 microM brain calmodulin, in the presence of 10 mM Ca2+, resulted in a 75-100% stimulation of activity.(ABSTRACT TRUNCATED AT 250 WORDS)
1984 RAT NaF 5.0 mg/kg glutathione-S-transferase activity increased 4-fold in the testis NaF at 20.0 mg/kg decrease in lipid peroxidation in testes	Alterations in drug metabolising enzymes and lipid peroxidation in different rat tissues by fluoride. Soni MG, Kachole MS, Pawar SS. Toxicol Lett 1984 May;21(2):167-72 Sodium fluoride at a dose level of 5.0 mg/kg enhanced aminopyrine N-demethylase and NADPH cytochrome c reductase activities and cytochrome P450 and cytochrome b5 levels in rat liver, kidney, lung, intestine and testis, whereas acetanilide hydroxylase activity remained unchanged in kidney and lung and was increased in liver, intestine and testis. Sodium fluoride at 20.0 mg/kg caused a decrease in aminopyrine N-demethylase, acetanilide hydroxylase and NADPH cytochrome c reductase activities and cytochrome P450 and cytochrome b5 levels in all tissues, except for an increase in NADPH cytochrome c reductase activity in the intestine and testis. Fluoride at both dose levels produced only marginal changes in glutathione-S-transferase activity except for a 4-fold increase in the testis at 5.0 mg/kg. Sodium fluoride at 5.0 mg/kg increased lipid peroxidation in all tissues studied. At 20.0 mg/kg there was a decrease in lipid peroxidation in liver, lung and testis and an increase in kidney and intestine.
1978 RAT stimulated adenylate cyclase activity at all Mg2+ concentrations	Properties of particulate and detergent-solubilized adenylate cyclase of rat testis. Effects of follitropin stimulation. Abou-Issa H, Reichert LE Jr. Biochim Biophys Acta 1978 Oct 12;526(2):613-25 Basal, fluoride and follitropin stimulated activities of adenylate cyclase have been studied in the testes of immature rats. The enzyme was maximally activated (about twice the basal activity) by low concentrations of follitropin, with an apparent Km of about 9 . 10(-10) M. Both Mg2+ and Mn2+ activate the enzyme but the apparent Ka for Mg2+ is about 10 times that for Mn2+. However, the apparent Km values for MgATP2- and MnATP2- are nearly the same (1.4 . 10(-4) M) and the cation activation of the enzyme is mainly through an increase in V. Ca2+ inhibited all expressions of testicular adenylate cyclase activity. Follitropin and fluoride stimulated adenylate cyclase activity at all Mg2+ concentrations but did not significantly affect the apparent Ka for Mg2+ or the Km for the substrate (MgATP2-). The stimulatory effect of the hormone or fluoride is mainly through an increase in V. Testicular adenylate cyclase could be solubilized with Triton X-100 or Lubrol-PX. The detergent-solubilized enzyme exhibited Km for substrate and Ka values for divalent cations similar to those of the membrane-bound enzyme and remained responsive to stimulation by fluoride. The stimulatory effect of follitropin, however, was lost. Responsiveness to follitropin was also lost by membrane-bound adenylate cyclase after treatment with phospholipase, although the fluoride effect was unchanged. These results reflect the essential role of lipids in the regulation of the follitropin-specific response.
1978 RAT NaF increased frequency of occurrence of various seminiferous tubules containing spermatids The mechanism of action of NaF may be hypothetical, but it probably consists of direct action on the seminiferous epithelium level.	The influence of human menopausal gonadotropin, natrium fluoride and cyproterone acetate on the spermatogenesis in immature rats. Kula K. Andrologia 1978 May-Jun;10(3):223-33 Human menopausal gonadotropin (HMG), natrium fluoride (NaF) and cyproterone acetate (CYA) were applied to immature male rats between the 26th and 32nd day of life and histological examination was performed in testes of 33rd-day-old rats. HMG treatment alone slightly influenced the spermatid completion process, while NaF evidently increased frequency of occurrence of various seminiferous tubules containing spermatids. CYA alone damaged the spermatid completion process (especially the cap-phase of spermiogenesis), produced a degeneration of meiotic spermatocytes, and decreased the tubular diameter. Those changes were prevented by addition of HMG to the CYA treated animals. The results suggest a possible regulatory role of FSH at the end of the first meiosis, and, also, in the progression of spermiogenesis. The mechanism of action of NaF may be hypothetical, but it probably consists of direct action on the seminiferous epithelium level.
1977 RAT androgen-binding protein (ABP) synthesis is inhibited at 0 degrees C or in the presence of cycloheximide, puromycin or sodium fluoride. Immature (17-25-day-old rat) testes showed a higher rate of ABP synthesis per 100 mg tissue than adult rat testes during 'baseline' conditions	In vitro synthesis of rat testicular androgen-binding protein (ABP). Ritzen EM, Hagenas L, Ploen L, French FS, Hansson V. Mol Cell Endocrinol 1977 Oct;8(4):335-46 Testicular tissue from immature and adult rats shows in vitro synthesis of androgen-binding protein (ABP). The ABP synthesis is dependent on a complete tissue culture medium, the incubation temperature and the age of the rats. ABP synthesis is inhibited at 0 degrees C or in the presence of cycloheximide, puromycin or sodium fluoride. Immature (17-25-day-old rat) testes showed a higher rate of ABP synthesis per 100 mg tissue than adult rat testes during 'baseline' conditions (no additions to the medium). Addition of NIH-FSH-S10 or testosterone to the medium increases the production of ABP by the testicular minces. The in vitro techniques have proved to be useful for studies of direct hormonal influence on the Sertoli cell protein synthesis.
1976 RAT The enzyme of the spermatozoa from the cauda epididymidis was more sensitive to inhibition by ouabain and fluoride	Changes in surface ATPase of rat spermatozoa in transit from the caput to the cauda epididymidis. Chulavatnatol M, Yindepit S. J Reprod Fertil 1976 Sep;48(1):91-7 Rat spermatozoa from the cauda epididymidis were found to have a lower activity of the surface ATPase than the spermatozoa from the caput region. The enzyme from spermatozoa of both regions had the same Michaelis constant (Km) for ATP of 5 X 10(-4) M. It was partly inhibited by ouabain and fluoride, but strongly inhibited by Cu2+, Zn2+,p-chloromercuribenzoate, 8-anilino-1-naphthalenesulphonate Triton X-100, Lubrol-PX, urea, guanidine hydrochloride, sodium dodecyl sulphate and glycerylphosphorylcholine. The enzyme of the spermatozoa from the cauda epididymidis was more sensitive to inhibition by ouabain and fluoride but less sensitive to inhibition by Cu2+ than that of the cells form the caput region. The Arrhenius plot of the temperature dependence of enzymatic activity varied for the cells from the caput and cauda epididymidis. The differences in the enzyme properties of spermatozoa from the two regions of the epididymis suggested that the decline in the activity during epididymal maturation may reflect changes in the lipids and sulphydryl groups of the sperm membrane.

Table 2. MOUSE
F Dose Effect	Abstract
2000 MOUSE 100, 200 and 300 ppm NaF drinking water for 4 or 10 weeks Fertility was significantly reduced at all three concentrations by exposure for 10 weeks results indicate that long-term ingestion of NaF adversely affects fertility in male mice	Fertility effects of sodium fluoride in male mice Ahmed Elbetieha, Homa Darmani, Ahmad S Al-Hiyasat. Fluoride 2000; 33(3):128-134. Full report at: http://www.fluoride-journal.com/00-33-3/333-128.pdf SUMMARY. Sexually mature male Swiss mice were exposed at 60 days of age to 100, 200 and 300 ppm sodium fluoride (NaF) in their drinking water for 4 weeks or 10 weeks. The effect of NaF exposure on fertility was assessed by breeding these males with untreated female mice after the exposure periods. Fertility was significantly reduced at all three concentrations by exposure for 10 weeks but not for 4 weeks. The number of implantation sites and viable fetuses was significantly reduced in females mated with males that had ingested NaF at a concentration of 200 ppm for 10 weeks. Relative weights of seminal vesicles and preputial glands were significantly increased in mice exposed to 200 and 300 ppm NaF for 4 weeks but not in mice exposed for 10 weeks. These results indicate that long-term ingestion of NaF adversely affects fertility in male mice.
2000 MOUSE NaF 10 mg NaF/kg BW The reduced activity of the enzymes as well as the structural and metabolic alterations in the sperm led to a significant decrease in sperm count, and motility and live:dead ratios but an increase in abnormal sperm which ultimately lead to a poor fertility rate. It is concluded that fluoride has a definite effect on male reproduction and fertility.	Reversal of fluoride-induced alteration in cauda epididymal spermatozoa and fertility impairment in male mice. Chinoy NJ and Sharma A Environmental Sciences: an International Journal of Environmental Physiology and Toxicology. 2000; 7(1):29-38 [Dart Special at Toxnet] Abstract: The effects of sodium fluoride (NaF) ingestion (10 mg NaF/kg body weight) and the possible therapeutic effects of ascorbic acid (AA, 15 mg/animal/day) and/or calcium phosphate (Ca, 25 mg/animal/day) on the reproductive functions and fertility of male mice were investigated. NaF-ingestion brought about a significant decline in sperm acrosomal acrosin and hyaluronidase. Cauda epididymal sperm stained with alcoholic acidic silver nitrate reagent revealed acrosomal damage and deflagellation. However, sperm nuclear integrity was not affected by the treatment. The reduced activity of the enzymes as well as the structural and metabolic alterations in the sperm led to a significant decrease in sperm count, and motility and live:dead ratios but an increase in abnormal sperm which ultimately lead to a poor fertility rate. The cessation of NaF-treatment was not conducive to bringing about a complete recovery. However, the administration of AA or Ca to NaF-treated mice revealed significant recovery from fluoride toxicity in all the above parameters. The recovery was more pronounced in the animal group treated with both AA and calcium in combination, thus indicating a synergistic or additive action. It is concluded that fluoride has a definite effect on male reproduction and fertility. However, the effects are transient and reversible with the administration of AA and Ca. Therefore, AA and Ca are proposed as therapeutic agents for populations in endemic areas for the amelioration of fluoride effects on reproductive functions.
1999 MOUSE Fed a protein-deficient diet treated with NaF 5, 10, 20 mg/kg BW for 30 days caused a significant decrease in protein levels in testeis, cauda epididymis, and vas deferens. levels of cholesterol in testis and glycogen in the vas deferens were significantly enhanced as compared to controls.	Effects of protein supplementatin and deficiency on fluoride-induced toxicity in reproductive organs of male mice NJ Chinoy and Dipti Mehta Fluoride 1999; 32(4):204-214 Summary: Feeding a protein-deficient diet to male mice treated for 30 days with NaF (5, 10, 20 mg/kg body weight) caused a significant decrease in protein levels in testeis, cauda epididymis, and vas deferens. The activity of testicular SDH and 3B- and 17B-HDS as well as ATPase in cauda epididymis and vas deferens also decreased as compared to controls fed a normal protein diet. The decrease was more significant in mice treated with 10 and 20 mg NaF/kg than with 5 mg/kg. By contrast, levels of cholesterol in testis and glycogen in the vas deferens were significantly enhanced as compared to controls. A protein-supplemented diet fed along with NaF in the same three doses did not cause any change in these parameters, which remained the same as the controls. These results clearly indicate that protein supplementation is beneficial to overcome to toxic effects of fluoride on testicular steroidogenesis, protein, carbohydrate, and energy and oxidation metabolisms in the reporductive organs of male mice. Protein deficiency, on the other hand, aggravates fluoride toxicity. A protein-supplemented diet might therefore substantially mitigate certain fluoride-induced health hazards in humans living in endemic areas.
1999 MOUSE NaF 10 mg NaF/kg BW The data obtained suggest that fluoride treatment induced significant metabolic alterations in the epididymides, vas deferens and seminal vesicles of mice.	Amelioration of fluoride toxicity by Vitamins E and D in reproductive functions of male mice NJ Chinoy and A Sharma. Fluoride 1998; 31(4):203-216. Full report at: http://www.fluoride-journal.com/98-31-4/314-203.htm SUMMARY. Studies on the beneficial effects of vitamins E and D supplementation on functions of caput and cauda epididymides, their spermatozoa, vas deferens and seminal vesicle of sodium fluoride (NaF) treated (10 mg/kg body weight) male mice (Mus musculus) were carried out. The NaF treatment resulted in significant decrease in the body and epididymis weight but those of vas deferens and seminal vesicle were not affected. NaF treatment brought about alterations in epididymal milieu as elucidated by the significant decrease in levels of sialic acid and protein as well as activity of ATPase in epididymides. As a result, the sperm maturation process was affected leading to a significant decline in cauda epididymal sperm motility and viability. This caused a significant reduction in fertility rate. The cauda epididymal sperm count was also significantly reduced. The data obtained suggest that fluoride treatment induced significant metabolic alterations in the epididymides, vas deferens and seminal vesicles of mice. The withdrawal of NaF treatment (30 days) produced incomplete recovery. On the other hand, supplementation of vitamins E or D during the withdrawal period of NaF treated mice was found to be very beneficial in recovery of all NaF induced effects, thus elucidating their ameliorative role in recovery from toxic effects of NaF on the reproductive functions and fertility. On the whole, a combination of vitamins E and D treatment was comparatively more effective than that with vitamin E or D alone. Therefore, vitamin therapy could be beneficial for the amelioration of fluoride induced changes in reproductive functions.
1998 MOUSE NaF 10 mg/kg BW GET LENGTH significant decrease epididymis weight significant decline in cauda epididymal sperm motility and viability significant reduction in fertility rate. The cauda epididymal sperm count was also significantly reduced	Amelioration of fluoride toxicity by Vitamins E and D in reproductive functions of male mice NJ Chinoy and A Sharma Fluoride 1998; 31(4):203-216 SUMMARY: Studies on the beneficial effects of vitamins E and D supplementation on functions of caput and cauda epididymides, their spermatozoa, vas deferens and seminal vesicle of sodium fluoride (NaF) treated (10 mg/kg body weight) male mice (Mus musculus) were carried out. The NaF treatment resulted in significant decrease in the body and epididymis weight but those of vas deferens and seminal vesicle were not affected. NaF treatment brought about alterations in epididymal milieu as elucidated by the significant decrease in levels of sialic acid and protein as well as activity of ATPase in epididymides. As a result, the sperm maturation process was affected leading to a significant decline in cauda epididymal sperm motility and viability. This caused a significant reduction in fertility rate. The cauda epididymal sperm count was also significantly reduced. The data obtained suggest that fluoride treatment induced significant metabolic alterations in the epididymides, vas deferens and seminal vesicles of mice. The withdrawal of NaF treatment (30 days) produced incomplete recovery. On the other hand, sup-plementation of vitamins E or D during the withdrawal period of NaF treated mice was found to be very beneficial in recovery of all NaF induced effects, thus elucidating their ameliorative role in recovery from toxic effects of NaF on the reproductive functions and fertility. On the whole, a combination of vitamins E and D treatment was comparatively more effective than that with vitamin E or D alone. Therefore, vitamin therapy could be beneficial for the amelioration of fluoride induced changes in reproductive functions.
1992 MOUSE NaF 10 mg 20 mg/kg BW for 30 days. significant decrease in sperm count and motility large numbers of deflagellated spermatozoa, with acrosomal, midpiece and tail abnormalities The treatment caused loss of fertility rate when normal cycling female mice were mated with treated males.	Reversible fluoride induced fertility impairment in male mice NJ Chinoy and E Sequeira Fluoride 1992; 25(2):71-76 Summary: Sodium fluoride (NaF) fed to adult male albino mice at a dose of 10 mg and 20 mg/kg body weight, caused a significant decrease in sperm count and motility. Scanning electron microscopy and silver nitrate staining showed large numbers of deflagellated spermatozoa, with acrosomal, midpiece and tail abnormalities. The treatment caused loss of fertility rate when normal cycling female mice were mated with treated males. Withdrawal of treatment for a period of 2 months resulted in a significant recovery in sperm count and sperm motility as well as fertility rate.
1989 MOUSE NaF 10 mg 20 mg/kg BW for 30 days. NaF treatment caused severe disorganization and denudation of germinal epithelial cells of seminiferous tubules with absence of sperm in the lumina. epithelial cell nuclear pyknosis and absence of luminal sperm were observed.	Effects of fluoride on the histoarchitecture of reproductive organs of the male mouse. Chinoy NJ, Sequeira E. Reprod Toxicol 1989;3(4):261-7 The effects of sodium fluoride (NaF) ingestion in two doses (10 and 20 mg/kg body weight) for 30 days on histology and histocytometry of reproductive organs of the adult male mouse were investigated. In order to study reversibility, treatment was withdrawn for one and two months. The testes, epididymides, vas deferens, prostate, and seminal vesicle were utilized for the study by standard hematoxylin-eosin staining and an ocular eye piece and micrometer scale. NaF treatment caused severe disorganization and denudation of germinal epithelial cells of seminiferous tubules with absence of sperm in the lumina. The Leydig cell and nucleus diameters were not affected. The caput epididymis showed fewer changes than the cauda. However, epithelial cell nuclear pyknosis and absence of luminal sperm were observed. A reduction in epithelial cell height, nuclear pyknosis, denudation of cells, and absence of sperm occurred in the cauda epididymis. The vas deferens epithelium showed nuclear pyknosis, clumped stereocilia, and cell debris but no sperm in the lumen and an increase in the lamina propria. The prostate and seminal vesicles were not affected by treatment. Withdrawal of treatment caused marked recovery in the histoarchitecture of these organs. The effects of NaF treatment are therefore transient and reversible.
1989 MOUSE NaF 10 mg 20 mg/kg BW for 30 days. testis succinic dehydrogenase levels decreased, in the epididmides sialic acid and ATPase levels decreased; in the vas deferens glycogen levels increased, seminal vesicles fructose levels increased in the prostate glands, acid phosphatase and total protein levels increased.	Fluoride induced biochemical changes in reproductive organs of male mice NJ Chinoy and E Sequeira Fluoride 1989; 22(1):78-85 Summary: Adult male albino mice were given 10 mg and 20 mg/kg body weight of NaF for 30 days. NaF caused a decrease in body weight, but no change in organ weight, except for the prostate gland and seminal vesicles. No significant change in testis cholesterol and serum testosterone levels occurred. However, in the testis succinic dehydrogenase levels decreased, in the epididmides sialic acid and ATPase levels decreased; in the vas deferens glycogen levels increased, seminal vesicles fructose levels increased in the prostate glands, acid phosphatase and total protein levels increased. After withdrawal of treatment for a period of two months the levels of these substances returned to normal.
1989 MOUSE NaF 10 mg 20 mg/kg BW for 30 days. NaF treatment caused severe disorganization and denudation of germinal epithelial cells of seminiferous tubules with absence of sperm in the lumina. epithelial cell nuclear pyknosis and absence of luminal sperm were observed.	Effects of fluoride on the histoarchitecture of reproductive organs of the male mouse. Chinoy NJ, Sequeira E. Reprod Toxicol 1989;3(4):261-7 The effects of sodium fluoride (NaF) ingestion in two doses (10 and 20 mg/kg body weight) for 30 days on histology and histocytometry of reproductive organs of the adult male mouse were investigated. In order to study reversibility, treatment was withdrawn for one and two months. The testes, epididymides, vas deferens, prostate, and seminal vesicle were utilized for the study by standard hematoxylin-eosin staining and an ocular eye piece and micrometer scale. NaF treatment caused severe disorganization and denudation of germinal epithelial cells of seminiferous tubules with absence of sperm in the lumina. The Leydig cell and nucleus diameters were not affected. The caput epididymis showed fewer changes than the cauda. However, epithelial cell nuclear pyknosis and absence of luminal sperm were observed. A reduction in epithelial cell height, nuclear pyknosis, denudation of cells, and absence of sperm occurred in the cauda epididymis. The vas deferens epithelium showed nuclear pyknosis, clumped stereocilia, and cell debris but no sperm in the lumen and an increase in the lamina propria. The prostate and seminal vesicles were not affected by treatment. Withdrawal of treatment caused marked recovery in the histoarchitecture of these organs. The effects of NaF treatment are therefore transient and reversible.
1987 MOUSE NaF 10, 20, 40 mg/kg Different assays were used Incidence of micronucleus and sperm abnormality increased with dose. ... Of all the assay results in the present study, the sperm abnormality was highest ...	Genotoxic effect of an environmental pollutant, sodium flouride, in mammalian in vivo test system Pati P and Bhunya Sr Caryologia, 40:1-2; 79-87 Genotoxicity of Sodium fluoride was evaluated in mice in vivo with the help of different cytogenetic assays. The frequency of chromosome aberration was dose - and time - dependent but not exactly route-dependant. Fractionated dosing induced less aberration. Incidence of micronucleus and sperm abnormality increased with dose. Relative sensitivity of the three assays has been found to be: Sperm abnormality > Chromosome aberration > Micronucleus. The present results have revealed mutagenic property of NaF. ... Of all the assay results in the present study, the sperm abnormality was highest and incidence of MN was least. Such result is not unexpected since greater sucess rate of sperm abnormality over other assay results in detecting agents that are active in vivo has been reported for many chemicals.
1981 MOUSE cytochemical alterations in Leidig cells and in the basal parts of the Sertoli cells	Fluorosis: geographical pathology and some experimental findings AA Zahvoronkov and LS Strochkova Fluoride 1981; 14(4):182-191 Excerpts: ... A.P. Tarinsky (4) revealed a 2-3 fold increase of symptoms of oligospermia and azoospermia in male workers suffering from industrial fluorosis compared with healthy men of the same age. Tokar (5, 6) found an association between fluorosis and hypogonadism. These data made it necessary to study the changes in synthetic processes in the testes of mice in experimental fluorosis. A total scintillation method of probe computing on the counter revealed certain shifts of RNA and protein metabolism in this organ, but the data were not statistically significant. More informative, was the cytochemical investigation of certain cell types in the testes on the separate stages of spermatogenesis, particularly on the 7th stage. The cells of spermatogenic epithelium with farily stable DNA content -Sertoli cells, spermatogones of A type, spermatides and also interstitial cells of Leidig - were studied. Between the second and third week afer the beginning of the experiment there was a decrease of rRNA in the basal part of the Sertoli cells, in Leidig cells and in spermatides. In the spermatogones this index was not significantly changed (Fig. 6a). During the course of hyperfluoridation a decrease of the dry weight appeared in Leidig cells and in spermatides (Fig. 6b). An adaptation of the cells to the toxic influence of fluoride is suggested since at the termination of the experiment a tendency to normalization of the rRNA content developed and the dry weight of the spermatides and the interstitial cells reached almost the control level. It is known that Sertoli cells constitute a part of the hematotesticular barrier and are actively attacked by fluoride ions, which is perhaps the cause of the decrease of rRNA in these cells. On the other hand, these cells play a trophic role for spermatides and probably supply them with rRNA. Consequently the decrease of the rRNA content and of the dry weight in spermatides is caused by the disturbance of the metabolims in Sertoli cells and therefore presents a secondary event. The noted cytochemical alterations in Leidig cells and in the basal parts of the Sertoli cells reflect the disturbances in the protein synthesising system of these cells in fluorosis and to a certain degree explain the hormonal imbalance in this disease, since Leidig cells synthesize testosterone and Sertoli cells produce protein-binding androgens. The high resistance of the spermatogones to the influence of fluoride in comparison with Sertoli cells is difficult to explain since in most cases the unfavorable factors affect primarily the sperm cells.
1980 MOUSE NaF 500 and 1000 ppm in drinking water for 3 months lack of maturation and differtiation of spermatocytes spermatogenesis had stopped and seminiferous tubules became necrotic.	.     Histological Finding of Mice Testes Following Fluoride Ingestion Kour K, Singh J. Fluoride 1980; 13(4):160-162 SUMMARY: The study was designed in order to assess the relationship between infertility and histological structure of testes following administration of varying doses of sodium fluoride. One hundred adult male albino mice were fed 10 ppm (Group A), 500 ppm (Group B) and 1000 ppm (Group C) of sodium fluoride in drinking water. The Group A animals were sacrificed at the end of one month, Group B after two and Group C after three months. The testes were removed and, after being processed in the usual manner, they were stained with hematoxylin and eosin. In Groups B and C, the higher dosage groups, there was a lack of maturation and differentiation of spermatocytes. In animals sacrificed at the end of three months, spermatogenesis had stopped and the seminiferous tubules had become necrotic. A definite relationship between fluorosis and damage to the testes has, therefore, been established by this study.

 

Table 3. RABBITT
F Dose Effect	Abstract
1995 RABBIT 10 mg NaF/kg BW/day for 20 and 23 months The structural changes observed in the caput and cauda ductus epididymis might adversely affect the maturation of spermatozoa.	Effects of chronic fluoride toxicity on the morphology of ductus epididymis and the maturation of spermatozoa of rabbit. Kumar A, Susheela AK. Int J Exp Pathol 1995 Feb;76(1):1-11 This study used light and scanning electron microscopy to observe the effect of chronic fluoride toxicity on the structure of the ductus epididymis, testis and spermatozoa in rabbit. The rabbits were treated with 10 mg NaF/kg body weight/day for 20 and 23 months. Serum fluoride was estimated by the fluoride ion-specific electrode method. Fluoride levels in the sera of both 20 and 23-month treated rabbits were significantly increased (P < 0.001). Loss of stereocilia, significant decrease (P < 0.001) in the height of the pseudostratified columnar epithelium and significant increase (P < 0.001) in the diameter of both the caput and cauda ductus epididymis were observed only in the 23-month fluoride treated rabbits. The decreases in the epithelial cell height (P < 0.01) and the tubular diameter (P < 0.001) of the testis were significant only in 23-month treated animals. Spermatozoa in the lumen of the testis of both treated groups of animals and in the caput and cauda ductus epididymis of 20-month treated animals appeared normal, but spermatozoa in the caput and cauda ductus epididymis of 23-month treated animals were fragmented. In the 23-month fluoride treated rabbits, the weights of the caput and cauda epididymis were significantly reduced (P < 0.025) and there was also a reduction in the number of secretory granules in these organs. The structural changes observed in the caput and cauda ductus epididymis might adversely affect the maturation of spermatozoa.
1994 RABBIT 10 mg NaF/kg BW daily for 18 months The abnormalities observed render the sperm nonfunctional and ineffective, and thus there is a possible role of fluoride in causing infertility	Ultrastructural studies of spermiogenesis in rabbit exposed to chronic fluoride toxicity. Kumar A, Susheela AK Int J Fertil Menopausal Stud 1994 May-Jun;39(3):164-71 Abstract: OBJECTIVE--To address the role of fluoride in causing defects to spermatids and epididymal spermatozoa. METHODS--Male rabbits were treated with 10 mg NaF/kg body weight daily for 18 months and maintained under identical laboratory conditions along with the control rabbits not given NaF. Testis and epididymis (caput) were investigated for ultrastructural details of spermatids and spermatozoa. RESULTS--A wide variety of structural defects were observed in the flagellum, the acrosome, and the nucleus of the spermatids and epididymal spermatozoa of fluoride-treated rabbits. Abnormalities included absence of outer microtubules, complete absence of axonemes, structural and numeric aberrations of outer dense fibers, breakdown of the fibrous sheath, and structural defects in the mitochondria of the middle piece of the flagellum. Detachment and peeling off of the acrosome from the flat surfaces of the nucleus were also observed. CONCLUSION--The abnormalities observed render the sperm nonfunctional and ineffective, and thus there is a possible role of fluoride in causing infertility.
1992 RABBIT NaF 5, 10, 20, and 50 mg/kg BW/day abnormal accumulation of lipids in testes.	Biochemical effects of fluoride on lipid metabolism in the reproductive organs of male rabbits A Shashi Fluoride 1992; 25(3):149-154 Summary: The effect of fluoride on testicular lipid metabolism was assessed in male albino rabbits in experimental fluorosis. Fifty male albino rabbits were administered sodium fluoride (5, 10, 20, and 50 mg/kg body weight/day) subcutaneously for 100 days. The control animals were given 1 cc distilled water/kg body weight over the same period. Compared with controls, the experimental animals, especially those given 50 mg NaF/day/kg of body weight, showed abnormal accumulation of lipids in testes. Hyperphospholipidemia, hypertriglyceridemia, and hypercholesterolemia in testes indicate enhanced lipid biosynthesis in response to fluoride toxicosis. A progressive significant (p<0.001) increase in amount of free fatty acids was observed in testes of fluoridated animals. The increase of concentration of all lipid classes except free fatty acids in testes was directly correlated with the increase in dosage of fluoride administered.
RABBIT 10 mg NaF/kg BW for 18 or 29 months. In animals treated for 29 months, the spermatogenic cells in the seminiferous tubules were disrupted, degenerated and devoid of spermatozoa. Spermatogenesis ceased only in animals treated for 29 months.	A study of the effect of high concentrations of fluoride on the reproductive organs of male rabbits, using light and scanning electron microscopy. Susheela AK, Kumar A. J Reprod Fertil 1991 Jul;92(2):353-60 Fluoride was orally administered to rabbits at 10 mg NaF/kg body weight for 18 or 29 months. The animals were then killed and the structure of the testis, epididymis and vas deferens studied under light and scanning electron microscopes. In animals treated for 29 months, the spermatogenic cells in the seminiferous tubules were disrupted, degenerated and devoid of spermatozoa. In animals treated for 18 or 29 months, loss of cilia on the epithelial cells lining the lumen of the ductuli efferentes of the caput epididymidis and of stereocilia on the epithelial cells lining the lumen of the vas deferens was observed. In some regions of the epithelial lining of the lumen of the ductuli efferentes and vas deferens, the boundaries of the cells were not clear and appeared to be peeled off. Mucus droplets were abundant in the vas deferens of control animals, but absent in both the treated groups. Spermatogenesis ceased only in animals treated for 29 months. The difference in the structural changes observed in the testes of the 2 treated groups may have been due to the blood-testis barrier. It is concluded that ingestion of high concentrations of fluoride has harmful effects on the male reproductive system.
1991 RABBIT NaF 20 and 40 mg/kg BW for 30 days Reduction in sperm motility, count, and changes in their morphology and metabolism led to the significant decline in fertility of the treated animals.	Effects of vitamin C and calcium on the reversibility of fluoride-induced alterations in spermatozoa of rabbits Chinoy NJ , Sequeira E, Narayana MV Fluoride 1991; 24(1):29-39 Summary: The present study was designed to investigate the effects of fluoride on the metabolism and functions of cauda epididymal spermatozoa of rabbits. The studies on reversibility of fluoride-induced effects by fluoride withdrawal, Vitamin C (ascorbic acid, AA) or Calcium (Ca+2) and combined AA + CA+2 ingestion were also investigated. Adult Male rabbits, Group II and III, were fed 20 and 40 mg/kg body weight sodium fluoride, respectively, for 30 days. Afterwards, cauda epididymal spermatozoa were obtained by micropuncture technique. Alterations in the activities of some specific androgen-dependent enzymes on sperm namely, ATPase, ACP, SDH, and protein, as well as reduction in Na+ and K+ levels in the spermatozoa, might be due to altered and hostile internal milieu of the epididymis in NaF-treated rabbits. Reduction in sperm motility, count, and changes in their morphology and metabolism led to the significant decline in fertility of the treated animals. After withdrawal of NaF treatment for 30 days (Group IV), no recovery was obtained in all the parameters which were altered, including fertility rates...
1990 RABBIT NaF 5, 10, 20 and 50 mg/kg/day for 100 days Deficient maturation and differentiation of the spermatocytes and an increase in the amount of interstitial tissue were found in the experimental animals. In the higher dosage groups, spermatogenesis stopped and the seminiferous tubules became necrotic.	Histopathological changes in rabbit testes during experimental fluorosis. Shashi. Folia Morphol (Praha) 1990;38(1):63-5 The aim of the study was to evaluate relationship between infertility and the histological structure of the testes following the subcutaneous administration of different doses of sodium fluoride (5, 10, 20 and 50 mg/kg/day), for 100 days, to groups of six male albino rabbits; the six control animals were given 1 cc distilled water/kg b.w./day for the same length of time. Deficient maturation and differentiation of the spermatocytes and an increase in the amount of interstitial tissue were found in the experimental animals. In the higher dosage groups, spermatogenesis stopped and the seminiferous tubules became necrotic. The study thus established the existence of a definite relationship between fluorosis and testicular damage.

 

Table 4. GERBIL and BANK VOLE
F Dose Effect	Abstract
1997 GERBIL High fluoride (HF) pups = 2.3 ug F/g BW/day from birth to 24 days whereafter food contained 37 mg F/kg. Low fluoride (LF) pups: from 24 days old food contained 7 mg F/kg. At 16 weeks: Mean testes weight of High fluoride group significantly less than Low fluoride group	The effect of fluoride on the physiology of the pinal gland Jennifer Anne Luke A dissertation submitted to the School of Biological Sciences, University of Surrey, in fulfilment of the requirements for the Degree of Doctor of Philosophy. Guildford 1997. Abstract: The purpose was to discover whether fluoride (F) accumulates in the pineal gland and thereby affects pineal physiology during early development. The [F] of 11 aged human pineals and corresponding muscle were determined using the F-electrode following HMDS/acid diffusion. The mean [F] of pineal was significantly higher (p<0.001) than muscle: 296 ± 257 vs. 0.5 ± 0.4 mg/kg respectively. Secondly, a controlled longitudinal experimental study was carried out to discover whether F affects the biosynthesis of melatonin, (MT), during pubertal development using the excretion rate of urinary 6-sulphatoxymelatonin, (aMT6s), as the index of pineal MT synthesis. Urine was collected at 3-hourly intervals over 48 hours from two groups of gerbils (Meriones unguiculatus), low-F (LF) and high-F (HF) (12 f, 12 m/group): under LD: 12 12, from prepubescence to reproductive maturity (at 9-12 weeks) to adulthood, i.e., at 7, 9, 11 1/2 and 16 weeks. The HF pups received 2.3 ug F/g BW/day from birth until 24 days whereafter HF and LF groups received food containing 37 and 7 mg F/kg respectively and distilled water. Urinary aMT6s levels were measured by radioimmunoassay. The HF group excreted significantly less aMT6s than the F group until the age of sexual maturation. At 11 1/2 weeks, the circadian profile of aMT6s by the HF males was significantly dimished but, by 16 weeks, was equivalent to the LF males. In conclusion, F inhibits pineal MT synthesis in gerbils up until the time of sexual maturation. Finally, F was associated with a significant acceleration of pubertal development in female gerbils using body weights, age of vaginal opening and accelerated development of the ventral gland. At 16 weeks, the mean testes weight of HF males was significantly less (p<0.002) than that of the LF males. The results suggest that F is associated with low circulating levels of MT and this leads to an accelerated sexual maturation in female gerbils. The results strengthen the hypothesis that the pineal has a role in pubertal development.
1996 BANK VOLE 200 micrograms F/ml drinking water for 4 months histopathologic changes in the germinal epithelium.	Photoperiodic elevation of testicular zinc protects seminiferous tubules against fluoride toxicity in the bank vole (Clethrionomys glareolus). Krasowska A, Wlostowski T. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 1996 Jan;113(1):81-4 Recent work has shown that a high fluoride (F) intake in rodents leads to histopathologic changes in the germinal epithelium of testes and to zinc deficiency in the testis and several other organs. The purpose of the present study was to determine whether an elevation of testicular zinc concentration during fluoride exposure could protect the testes of bank vole from damage. The elevation of testicular zinc was achieved by exposing the bank voles to a long photoperiod (16 hr light/8 hr dark). The zinc concentration in the testes of bank voles kept under the long photoperiod was 38% higher than that in animals exposed to a moderate photoperiod (12 hr light/12 hr dark). Fluoride exposure (200 micrograms F/ml drinking water) during 4 months decreased additionally (p < 0.05) zinc concentration in the testes of bank voles kept under the moderate photoperiod. The same animals also exhibited histopathologic changes in the germinal epithelium. By contrast, these disturbances were not observed in animals maintained in the long photoperiod. This experiment suggests that an increase in testicular zinc due to a long photoperiod prevents seminiferous tubules from a damage induced by fluoride in bank voles. The protective effects of zinc (or a long photoperiod) did not appear to be related to a decrease in testicular fluoride accumulation or lipid peroxidation.

 

Table 5. MISCELLANEOUS STUDIES - HUMAN
F Dose Effect	Abstract
2003 Human population study 3-27mg/day induces a subclinical reproductive effect toxic effect in both Sertoli cells and gonadotrophs. 2-13mg/day serum levels of inhibin-B	Fluoride-induced disruption of reproductive hormones in men. Ortiz-Perez D et al. Environ Res 2003. Sep;93(1):20-30. Fluoride-induced reproductive effects have been reported in experimental models and in humans. However, these effects were found in heavily exposed scenarios. Therefore, in this work our objective was to study reproductive parameters in a population exposed to fluoride at doses of 3-27mg/day (high-fluoride-exposed group-HFEG). Urinary fluoride levels, semen parameters, and reproductive hormones in serum (LH, FSH, estradiol, prolactin, inhibin-B, free and total testosterone) were measured. Results were compared with a group of individuals exposed to fluoride at lower doses: 2-13mg/day (low-fluoride-exposed group-LFEG). A significant increase in FSH (P<0.05) and a reduction of inhibin-B, free testosterone, and prolactin in serum (P<0.05) were noticed in the HFEG. When HFEG was compared to LFEG, a decreased sensitivity was found in the FSH response to inhibin-B (P<0.05). A significant negative partial correlation was observed between urinary fluoride and serum levels of inhibin-B (r=-0.333, P=0.028) in LFEG. Furthermore, a significant partial correlation was observed between a chronic exposure index for fluoride and the serum concentrations of inhibin-B (r=-0.163, P=0.037) in HFEG. No abnormalities were found in the semen parameters studied in the present work, neither in the HFEG, nor in the LFEG. The results obtained indicate that a fluoride exposure of 3-27mg/day induces a subclinical reproductive effect that can be explained by a fluoride-induced toxic effect in both Sertoli cells and gonadotrophs. • Defintion: Inhibin There are two kinds of inhibin: inhibin A and inhibin B. Inhibin inhibits pituitary secretion of follicle-stimulating hormone (FSH) but it does not effect luteinizing hormone (LH) release. Inhibin is released by the Sertoli cells in the testes and granulosa cells in the ovary. Androgens stimulate the release of inhibin.
1996 Serum testosterone concentrations in patients with skeletal fluorosis Circulating serum testosterones in skeletal fluorosis patients were significantly lower than those of Control 1 at p < 0.01.	Circulating testosterone levels in skeletal fluorosis patients. Susheela AK, Jethanandani P. Ref: J Toxicol Clin Toxicol 1996;34(2):183-9 OBJECTIVE: The present study focuses on serum testosterone concentrations in patients with skeletal fluorosis, in order to assess the hormonal status in fluoride toxicity. METHODS: Serum testosterones were compared for patients afflicted with skeletal fluorosis (n = 30) and healthy males consuming water containing less than 1 ppm fluoride (Control 1, n = 26) and a second category of controls (Control 2, n = 16): individuals living in the same house as the patients and consuming same water as patients but not exhibiting clinical manifestations of skeletal fluorosis. RESULTS: Circulating serum testosterones in skeletal fluorosis patients were significantly lower than those of Control 1 at p < 0.01. Testosterone concentrations of Control 2 were also lower than those of Control 1 at p < 0.05 but were higher than those of the patient group. CONCLUSIONS: Decreased testosterone concentrations in skeletal fluorosis patients and in males drinking the same water as the patients but with no clinical manifestations of the disease compared with those of normal, healthy males living in areas nonendemic for fluorosis suggest that fluoride toxicity may cause adverse effects in the reproductive system of males living in fluorosis endemic areas.
1994 Human spermatozoa The altered lysosomal enzyme activity and glutathione levels together with morphologic anomalies resulted in a significant decline in sperm motility with an effective dose of 250 mM	In vitro fluoride toxicity in human spermatozoa. Chinoy NJ, Narayana MV Reprod Toxicol 1994 Mar-Apr;8(2):155-9. Abstract: Effects of sodium fluoride (NaF) on washed, ejaculated human spermatozoa at doses of 25, 50, and 250 mM were investigated in vitro at intervals of 5, 10, and 20 min. Sodium fluoride (NaF) did not affect the extracellular pH of sperm, except that a slight acidification was caused by the 250 mM dose only. The treatment caused a significant enhancement in acid phosphatase (ACPase) and hyaluronidase activities after 5 and 10 min. However, the decrease in the lysosomal enzyme activity after 20 min treatment could have been due to the gradual increase in fluoride accumulation by spermatozoa leading to membrane damage. Silver nitrate staining of sperm revealed elongated heads, deflagellation, and loss of the acrosome together with coiling of the tail. Sperm glutathione levels also showed a time-dependent decrease with complete depletion after 20 min indicating rapid glutathione oxidation in detoxification of the NaF. The altered lysosomal enzyme activity and glutathione levels together with morphologic anomalies resulted in a significant decline in sperm motility with an effective dose of 250 mM.
1978 Human spermatozoa Adenylate cyclase from ejaculated human spermatozoa was inhibited by fluoride	Inhibitors of adenylate cyclase from ejaculated human spermatozoa. Haesungcharern A, Chulavatnatol M. J Reprod Fertil 1978 May;53(1):59-61 Adenylate cyclase from ejaculated human spermatozoa was inhibited by fluoride, Cu2+, Zn2+, Ni2+ and several carboxylic acids.
1977 Human males suffering from fluorosis Compared to healthy controls, testosterone content proved to be decreased and FSH content elevated in patients with fluorosis	[Effect of inorganic fluorine compounds on the functional state of the pituitary-testis system] [Article in Russian] Tokar' VI, Savchenko ON. Probl Endokrinol (Mosk) 1977 Jul-Aug;23(4):104-7 The radioimmunological method was applied to the study of blood testosterone, LH, and FSH content in 41 men suffering from fluorosis, aged from 33 to 45 years. Nineteen men who had no contact with fluorine compounds served as control. In comparison with healthy individuals testosterone content proved to be decreased and FSH content elevated in patients with fluorosis. Blood LH content was increased only in those patients with fluorosis who had long contact with fluorine compounds (over 15 years). The changes in the blood hormone concentration were connected with disturbances of the hormonal, and, possibly, of the germenative function of the testes. The hypophysis proved to suffer less, and the changes of its function were apparently secondary in character.
1972 Patient with endemic fluorosis bilateral calcification of the vas deferens	Cacification of the vas deferens in a patient with endemic fluorosis Case report SPS Teotia and M Teotia Fluoride 1972; 5(2):86-88 Summary: A case of endemic skeletal fluorosis with probable secondary hyperparathyroidism is presented in which bilateral calcification of the vas deferens occurred. No symptoms were associated with calcifications of the vas deferens. They were incidental findings on X-rays of the pelvis taken for other reasons.

 

Table 3. RAM
F Dose Effect	Abstract
2002 Ram semen 5 hr incubation at 381⁄4C 0.38; 1.9; 3.8 ppm F changes undoubtedly affect the physiological functions of the sperm.	In vitro influence of sodium fluoride on ram semen quality and enzyme activities Zakrzewska H, Udala J, Blaszczyk B Fluoride 2002; 35(3):153-160 Summary: The percentage of spermatozoa in ram semen with intact acrosomes and the level of spermatozoa motility decreased significantly after dilution and after 5 hr incubation at 381⁄4C. Both indices decreased significantly in the presence of NaF at concentrations ranging from 20 ugmol/L to 0.1 mol/L. The activities of androgen-dependent enzymes - acid phosphatase (ACP), lactate dehydrogenase (LDH), and gamma-glutamyl transferase (y-GT-10S) - decreased significantly when the ejaculate was treated with NaF at concentrations of 20, 100, 200 ugmol/L (0.38; 1.9; 3.8 ppm F), but they returned to the initial value of the control at 0.1 mol/L (1900 ppm F). The activity of asparate transaminase (AspAT) displayed a large increase with the increasing lower F- concentration. These changes undoubtedly affect the physiological functions of the sperm.
2002	Brain Res 2002 Feb 1;926(1-2):126-36 SSeCKS immunolabeling in rat primary sensory neurons. Siegel SM, Grove BD, Carr PA. Department of Anatomy and Cell Biology, University of North Dakota, 501 North Columbia Road, Grand Forks, ND 58202, USA. SSeCKS (src suppressed C kinase substrate) is a protein kinase C substrate that may play a role in tumor suppression. Recently described in fibroblasts, testes and mesangial cells, SSeCKS may have a function in the control of cell signaling and cytoskeletal arrangement. To investigate the distribution of SSeCKS throughout the nervous system, representative sections of brain, spinal cord and dorsal root ganglia were processed using immunofluorescence. Labeling of central axonal collaterals of primary sensory neurons was observed in the dorsal horn at all spinal levels. SSeCKS-immunoreactivity was also observed in the cerebellum, medulla and sensory ganglia (including trigeminal ganglia). The pattern and distribution of anti-SSeCKS labeling in dorsal root ganglia and the dorsal horn of the spinal cord was similar to that observed for other markers of small primary sensory neurons. Therefore, the coexistence of SSeCKS with substance P, CGRP and acid phosphatase was examined in sections of sensory ganglia, spinal cord and medulla using double immunofluorescent labeling for SSeCKS and substance P/CGRP or sequential SSeCKS immunofluorescence and acid phosphatase/fluoride-resistant acid phosphatase enzyme histochemistry. A small portion of the SSeCKS-labeled cell bodies appeared to represent a subpopulation of substance P (4.8%) and CGRP (4.7%) containing neurons, while 45.0% contained fluoride-resistant acid phosphatase reactivity. These results indicate that SSeCKS has a restricted distribution within the nervous system and that expression of this protein may reflect the specific signaling requirements of a distinct population of nociceptive sensory neurons.

 

Table 2. Twelve Chinoy et al. studies cited in 1997 for effects on the structural and functional integrity of reproductive organs in male and female rodents and guinea pigs which leads to the loss of fertility.
5. 1991	1991 Male Adult Rat A single microdose (50 micrograms/50 microL) injection of NaF into the vasa deferentia found to affect reproductive function and fertility rate.	Microdose vasal injection of sodium fluoride in the rat. Chinoy NJ, Rao MV, Narayana MV, Neelakanta E Reprod Toxicol 1991;5(6):505-12 Abstract: A single microdose (50 micrograms/50 microL) injection of sodium fluoride (NaF) into the vasa deferentia of adult male albino rats (Rattus norvegicus) caused arrest of spermatogenesis and absence of spermatozoa in the lumina of the seminiferous tubules of the testes, which consequently led to a decline in the sperm count in the caudae epididymides. Scanning electron microscopy of cauda and vas deferens sperm revealed deflagellation and tail abnormalities. This is probably related to the alterations in the internal milieu of these organs which rendered the spermatozoa immotile and consequently caused fertility impairment in the experimental animals. Thus microdoses of sodium fluoride were found to affect reproductive function and fertility rate.
9. 1992	No abstract	Fluoride toxicity in female mice and its reversal. Chinoy NJ In: Saxena AK, Ramamurthy R, Srirama Reddy G, Saxena VL. (Eds) Recent Advances in Life Sciences. Manu Publications, Kanpur India 1992. pp 39-50.
10. 1991	No abstract	Effects of fluoride on physiology of some animals and human beings Chinoy NJ Indian Journal of Environmental Toxicology 1 (1) 17-32. 1991
11. 1991	No abstract	Effects of fluoride on some organs of rats and their reversal. Chinoy NJ Proceedings of Zoological Society; Calcuta 44 (1) 11-15. 1991
12. 1991	No abstract	Effects of fluoride ingestion on the physiology of reproductive organs of male rats Chinoy NJ, Pradeep PK, Sequeira E. Journal of Environmental Biology 13 (1) 55-61. 1991.