Magnesium fluoride

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Read-across justification is provided in Appendix 3 of the CSR.

The available toxicological data on the target and source substances is outlined in the data matrix (Table 8).
The toxicological properties of the target substance are related to the magnesium cations (Mg2+) and fluoride anions (F-). Thus, data on other magnesium salts (mainly magnesium chloride) and inorganic fluorides (mainly sodium fluoride) are justified in the human health hazard assessment.
Humans take in between 250 and 350 mg/day of magnesium and need at least 200 mg, but the body deals very effectively with this element, taking it form food when it can, and recycling what we already have when it cannot. There is no evidence that magnesium produces systemic poisoning although persistent over-indulgence in taking magnesium supplements and medicines can lead to muscle weakness, lethargy and confusion.
Chloride is relatively not toxic to human health but does pose organoleptic issues. Accordingly, EPA has set the potable limit for chloride in drinking water at 250 mg/L.

Experimental data obtained with the magnesium chloride indicate that it has low oral toxicity after repeated exposure. The available data does not show any test item related adverse effects on the highest doses (> 2000 mg/kg bw/day) tested. Sodium fluoride administration did not cause test item related adverse effects in rats dosed up to 175ppm for 2 years. Only exceptions were abnormalities in the teeth of rats in the two highest dose groups (100 and 175 ppm sodium fluoride).

Sodium is a compound of many foodstuffs, for instance of common salt. It is necessary for humans to maintain the balance of the physical fluids system. Sodium is also required for nerve and muscle functioning. Thus, there is no evidence that sodium produces systemic poisoning at reasonably possible exposures.

Fluoride has both positive and negative effects on human health, but there is a narrow range between intakes that are associated with these effects. Exposure to all sources of fluoride, including drinking-water and foodstuffs, is important There is little information to characterize the dose–response relationships for the different adverse effects. In particular, there are few data on total exposure, particularly with respect to intake and fluoride absorption. The most serious effect is the skeletal accumulation of fluoride from long-term excessive exposure to fluoride and its effect on non-neoplastic bone disease — specifically, skeletal fluorosis and bone fractures. There is clear evidence from India and China that skeletal fluorosis and an increased risk of bone fractures occur at total intakes of 14 mg fluoride/day and evidence suggestive of an increased risk of bone effects at total intakes above about 6 mg fluoride/day.
The data of repeated dose toxicity and reproduction toxicity are proposed to be used for read-across according to the information requirements of Annex VIII.

Cross-reference

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Read-across justification is provided in Appendix 3 of the CSR.

The available toxicological data on the target and source substances is outlined in the data matrix (Table 8).
The toxicological properties of the target substance are related to the magnesium cations (Mg2+) and fluoride anions (F-). Thus, data on other magnesium salts (mainly magnesium chloride) and inorganic fluorides (mainly sodium fluoride) are justified in the human health hazard assessment.
Humans take in between 250 and 350 mg/day of magnesium and need at least 200 mg, but the body deals very effectively with this element, taking it form food when it can, and recycling what we already have when it cannot. There is no evidence that magnesium produces systemic poisoning although persistent over-indulgence in taking magnesium supplements and medicines can lead to muscle weakness, lethargy and confusion.
Chloride is relatively not toxic to human health but does pose organoleptic issues. Accordingly, EPA has set the potable limit for chloride in drinking water at 250 mg/L.

Experimental data obtained with the magnesium chloride indicate that it has low oral toxicity after repeated exposure. The available data does not show any test item related adverse effects on the highest doses (> 2000 mg/kg bw/day) tested. Sodium fluoride administration did not cause test item related adverse effects in rats dosed up to 175ppm for 2 years. Only exceptions were abnormalities in the teeth of rats in the two highest dose groups (100 and 175 ppm sodium fluoride).

Sodium is a compound of many foodstuffs, for instance of common salt. It is necessary for humans to maintain the balance of the physical fluids system. Sodium is also required for nerve and muscle functioning. Thus, there is no evidence that sodium produces systemic poisoning at reasonably possible exposures.

Fluoride has both positive and negative effects on human health, but there is a narrow range between intakes that are associated with these effects. Exposure to all sources of fluoride, including drinking-water and foodstuffs, is important There is little information to characterize the dose–response relationships for the different adverse effects. In particular, there are few data on total exposure, particularly with respect to intake and fluoride absorption. The most serious effect is the skeletal accumulation of fluoride from long-term excessive exposure to fluoride and its effect on non-neoplastic bone disease — specifically, skeletal fluorosis and bone fractures. There is clear evidence from India and China that skeletal fluorosis and an increased risk of bone fractures occur at total intakes of 14 mg fluoride/day and evidence suggestive of an increased risk of bone effects at total intakes above about 6 mg fluoride/day.
The data of repeated dose toxicity and reproduction toxicity are proposed to be used for read-across according to the information requirements of Annex VIII.

Reason / purpose for cross-reference:

data waiving: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Principles of method if other than guideline:

The study design is comparable to a standard two-generation reproductive toxicity study, however this paper focuses on investigations of the effects on spermatogenesis in male rats following administration over two generations.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

Test was conducted on a read-across substance sodium fluoride (CAS 7681-94-4; EC 231-667-8). Obtained from Sigma Chemical Co., MO, USA, Lot no. 109F0102. No trace element impurities were detected in the sample.

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CD VAF+

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female rats were obtained from Charles River Laboratories (Raleigh, NC, USA), 22 days old on arrival and quarantined for approximately 1 week. Individuals were identified by ear tags. Rats were housed under standard controlled temperature (19.4-23.3 degrees celcius), humidity (40-70%) and light (12 hour light:dark cycle). Rats were fed a low fluoride NIH-07 diet (7.95 ppm fluoride). The diet was prepared by Ziegler Bros, Inc.

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

Sodium fluoride was dissolved in the rats drinking water, which was provided ad libitum. The water was obtained by filtering house-distilled water through a water purification system. The fluoride concentation in this water was determined to be less than 0.2 ppm.

Details on mating procedure:

Mating took place over a 3 week period. Pregnancy was determined by the presence of sperm plugs in the cage and the presence of sperm in the vagina.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sodium fluoride concentrations for both the control and treated groups were performed at the FDA by potentiometric titration of the fluoride ion with a fluoride ion electrode. Sodium fluoride concentrations for both control and treated groups were determined using an EA 940 pH/ISE meter with appropriate electrodes and filling solutions for fluoride analysis. Sodium fluoride concentrations were determined each time dosing solutions were prepared for any treatment group including the control.

Duration of treatment / exposure:

Approximately 14 weeks per generation.

Frequency of treatment:

Daily. Drinking water was available ad libitum.

Details on study schedule:

The parental (P) generation received sodium fluoride in their drinking water (provided ad libitum) for approximately 14 weeks; 10 weeks pretreatment, 3 weeks mating (to non-siblings), and 1 week post-mating. Pregnant P females continued to be exposed from gestation day 0 until the end of lactation. On post-partum day 4, litters were culled to 10 pups per litter (5 males and 5 females) where possible using a random number table. At day 21, males were randomly selected to represent the F1 generation from as many litters as possible. The weanlings remained in the same treatment group as their parents and were exposed to sodium fluoride for approximately 14 weeks.

Remarks:

0, 25, 100, 175 or 250 ppm

No. of animals per sex per dose:

P generation: 0 ppm/12, 25ppm/13, 100 ppm/13, 175 ppm/12, 250 ppm/14, total 64 rats
F1 generation: 0 ppm/12, 25ppm/12, 100 ppm/12, 175 ppm/12, 250 ppm/12, total 60 rats

Control animals:

yes, concurrent vehicle

Details on study design:

P generation rats were assigned to treatment groups by weight using a random experimental stratified procedure. On post-partum day 4, F1 litters were culled to 10 pups per litter (5 males and 5 females) where possible using a random number table. At day 21, males were randomly selected to represent the F1 generation from as many litters as possible.

Parental animals: Observations and examinations:

After 1 week of post mating NaF treatment, testicular tissues were collected. Body weights were recorded at the time of tissue collection.

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

The left testis was homogenised and the number of homogenisation-resistant spermatids per testis determined. Spermatid numbers were expressed as either numbers of homogenisation-resistant spermatids per testis, spermatids per testis, spermatid numbers per gram of testis, or spermatid numbers per gram of testis per day.

Postmortem examinations (parental animals):

The right testis was perfusion fixed whilst the rat was anaesthetised (after removal of the left testis), see below for method. Testicular histopathology was assessed in the control and high dose groups. 10 sections were evaluated per animal per group. Seminiferous tubules were examined to determine the effects of fluoride on Sertoli cells, germ cells undergoing spermiogenesis, and spermatocytogenesis or meiosis. The boundary tissue of the seminiferous tubules was examined for signs of infolding. The intertubular space was examined to determine whether Leydig cells were affected, whether cells not normally found in the interstitial space were present, and if there was an increase in cells normally found in low numbers.
Blood collected from each animal's right ventricle (under anaesthesia) was allowed to clot at room temperature for approximately 1 hour. The blood was assayed for levels of LH, FSH and serum testosterone.

The epididymides, heart, spleen, liver, kidneys, adrenals, and seminal vesicles/prostates were weighed.

Postmortem examinations (offspring):

Examinations in the F1 males were identical to those in the P males.

Statistics:

Two-way ANOVA was performed for all response variables. ANCOVA was used for organ weights with body weight as the covariate. One-way ANOVA was used to check for differences at dose levels, followed by an LSD t-test. P values equal to or below 0.05 were considered significant.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not examined

The results of examinations performed on P-generation rats are discussed together with F1-generation rats in the section "Any other information".

Dose descriptor:

NOAEC

Effect level:

250 ppm (nominal)

Sex:

male

Basis for effect level:

other: Spermatogenesis and endocrine function

Clinical signs:

no effects observed

Mortality / viability:

not examined

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

not examined

Histopathological findings:

no effects observed

The results of examinations performed on F1-generation rats are discussed together with P-generation rats in the section "Any other information".

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

250 ppm (nominal)

Sex:

male

Basis for effect level:

other: Spermatogenesis and endocrine function

Key result

Reproductive effects observed:

no

The mean body weights of the P and F1 generations from all sodium fluoride treated groups were not statistically different from their respective controls. F1 generation male body weights were higher than those of the P generation, but not significanly different.

There were no dose related effects on testis weight. There were no significant differences in mean testis weights between treated groups and controls in either generation. The right testis weight and the paired testis weights from the F1 generation controls were significantly higher than the P generation controls. The left testis weight of the F1 generation males was significantly lower than that of the P generation males in the 25ppm group. The right testis weight of the F1 generation males was significantly higher than that of the P generation males in the 100ppm group. Statistically significant differences in mean epididymal weights were not observed between control and treatment groups of the P generation. Within the F1 generation the right epididymal weight of the 175ppm group was significantly lower than the F1 control. No dose-related effects were observed. The weight of the right epididymis from the F1 generation was significantly lower than that of the P group when epididymal weights for the 175ppm group were compared. Prostate/seminal vesicle weights were not significantly different between treated and control rats in either generation.

There were no significant differences in spermatid numbers between controls and treated rats in either generation, or between generations. There were no significant differences in serum testosterone, LH and FSH concentrations between treated and control rats in either generation or between generations. Liver weight in the 250ppm group (P generation) was significantly lower than the control group. Spleen weights in the 175 and 250ppm groups were significantly higher than the control group. The authors considered these events to be random and not treatment related because no toxic effects were observed. Adrenal weights in the F1 generation were significantly lower than adrenal weights in the P generation at all dose levels. No dose-related toxic effects were observed and the authors report that weight differences can arise from the removal procedure. There were no treatment related effects on the histopathology of the testis; the histological appearance of the testicular tissue from the control group was indistinguishable from that of the high dose group in both generations. There were no differences between the generations in the high dose groups.

Conclusions:

Prolonged exposure to sodium fluoride in drinking water did not adversely affect spermatogenesis or endocrine function in two generations of male rats.

Executive summary:

The potential of sodium fluoride (NaF) to affect spermatogenesis and endocrine function was assessed in P and F1 generation male rats. Male and female rats received sodium fluoride in their drinking water at 0, 25, 100, 175 or 250 ppm. P generation rats were exposed for 10 weeks, then for 3 weeks during mating. Reproductive tissues were collected from P males 1 week after mating (after approximately 14 weeks of NaF treatment). Pregnant females (P) were exposed to NaF during gestation and lactation. F1 weanling males were exposed to NaF for 14 weeks, at which time reproductive tissues were collected. Dose-related effects were not observed within the P and F1 treatment groups in testis weights, prostate/seminal vesicle weights, non-reproductive organ weights, testicular spermatid counts, sperm production per gram of testis per day, sperm production per gram of testis, LH, FSH or serum testosterone concentrations. Histopathological changes in testicular tissues were not observed. Prolonged exposure to NaF in drinking water up to a dose of 250 ppm does not adversely affect spermatogenesis or endocrine function in P and F1 generation male rats.

Data source

Reference

Reference Type:

publication

Title:

Testing the potential of sodium fluoride to affect spermatogenesis: a morphometric study

Author:

Sprando RL, Collins TFX, Black T, Olejnik N & Rorie J

Year:

1998

Bibliographic source:

Food and Chemical Toxicology 36: 1117-1124

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Principles of method if other than guideline:

The study presents quantitative morphometric information on the testes of F1 generation rats exposed to one of four NaF concentrations in utero, during lactation, and for 14 weeks post weaning.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium fluoride

EC Number:

231-667-8

EC Name:

Sodium fluoride

Cas Number:

7681-49-4

Molecular formula:

FNa

IUPAC Name:

sodium fluoride

Specific details on test material used for the study:

Test was conducted on a read-across substance sodium fluoride (CAS 7681-94-4; EC 231-667-8). Obtained from Sigma Chemical Co., MO, USA, Lot no. 109F0102. No trace element impurities were detected in the sample.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CD VAF+

Sex:

male/female

Details on test animals or test system and environmental conditions:

The male rats were from the larger two-generation reproduction study (Sprando et al. 1997). Male and female rats were obtained from Charles River Laboratories (Raleigh, NC, USA), 22 days old on arrival and quarantined for approximately 1 week. Individuals were identified by ear tags. Rats were housed under standard controlled temperature (19.4-23.3 degrees celcius), humidity (40-70%) and light (12 hour light:dark cycle). Rats were fed a low fluoride NIH-07 diet (7.95ppm fluoride). The diet was prepared by Ziegler Bros, Inc.

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

Sodium fluoride was dissolved in the rats drinking water, which was provided ad libitum. The water was obtained by filtering house-distilled water through a water purification system. The fluoride concentation in this water was determined to be less than 0.2ppm.

Details on mating procedure:

Mating took place over a 3-week period. Pregnancy was determined by the presence of sperm plugs in the cage and the presence of sperm in the vagina.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sodium fluoride concentrations for both the control and treated groups were performed at the FDA by potentiometric titration of the fluoride ion with a fluoride ion electrode. Sodium fluoride concentrations for both control and treated groups were determined using an EA 940 pH/ISE meter with appropriate electrodes and filling solutions for fluoride analysis. Sodium fluoride concentrations were determined each time dosing solutions were prepared for any treatment group including the control.

Duration of treatment / exposure:

Approximately 14 weeks per generation

Frequency of treatment:

Daily

Details on study schedule:

The parent (P) generation received sodium fluoride in their drinking water (provided ad libitum) for approximately 14 weeks; 10 weeks pretreatment, 3 weeks mating (to non-siblings), and 1 week post-mating. Pregnant P females continued to be exposed from gestation day 0 until the end of lactation. On post-partum day 4, litters were culled to 10 pups per litter (5 males and 5 females) where possible using a random number table. At day 21, males and females were randomly selected to represent the F1 generation from as many litters as possible. The weanlings remained in the same treatment group as their parents and were exposed to sodium fluoride for approximately 14 weeks.

Doses / concentrations

Remarks:

0, 25, 100, 175 or 250 ppm

No. of animals per sex per dose:

P generation total: 64 male rats (0 ppm n=12, 25 ppm n=13, 100 ppm n=13, 175 ppm n=12, 250 ppm n=14); F1 generation total: 60 male rats (12 rats per dose). The same number of female rats were used, but no examinations were conducted on these rats.

Control animals:

yes, concurrent vehicle

Details on study design:

P generation rats were assigned to treatment groups by weight using a random experimental stratified procedure. On post-partum day 4, F1 litters were culled to 10 pups per litter (5 males and 5 females) where possible using a random number table. At day 21, males were randomly selected to represent the F1 generation from as many litters as possible.

Examinations

Parental animals: Observations and examinations:

Reported in Sprando et al 1997

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Reported in Sprando et al 1997

Postmortem examinations (parental animals):

Reported in Sprando et al 1997

Postmortem examinations (offspring):

The male F1 rats were weighed prior to anaesthesia. Trunk blood was collected from the right ventricle. The left testis and epididymis were removed. The left epididymis was weighed then discarded. The left testis was homogenised and the number of homogenisation-resistant spermatids per testis determined. The right testis was perfusion fixed.

Statistics:

All the variables except body weight were analysed using ANCOVA, with body weight as a covariate. Body weight data were analysed in an ANOVA. P values equal to or less than 0.05 were considered significant.

Reproductive indices:

Not examined

Offspring viability indices:

Not examined

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

not examined

Body weight and weight changes:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Details on results (P0)

Results from the P generation are reported in Sprando et al 1997.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

250 ppm (nominal)

Sex:

male

Basis for effect level:

other: Spermatogenesis and endocrine function

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not examined

Histopathological findings:

no effects observed

Details on results (F1)

A significant difference in body weight was observed between the controls and the treated rats in the 100, 175ppm and a borderline significant effect was observed in the 250 ppm group, however this decrease in body weight was not dose related. Statistically significant differences in testis weight, volume or specific gravity were not observed between treated and control rats. There was a statistically significant reduction in the volume composition of the lymphatic endothelium in the 175 and 250ppm groups, and in the testicular capsule of the 100ppm group, compared to controls. No other significant differences relating to volumetric composition of the testis were found. There were no significant differences in the mean numbers of Sertoli cell nuclei counted per cross-sectioned seminiferous tubules, the seminiferous tubule diamters, the height of the seminiferous epithelium, the mean absolute seminiferous tubule lengths or mean absolute surface areas.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

250 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

The significant reduction in the absolute volume of the testicular capsule in the 100 ppm group was not considered treatment related because no dose relationship was observed, the authors conclude it is likely to be a sampling error. The absolute volumes of nearly all testicular components examined (seminiferous tubule, tubular lumen, interstitium, Leydig cells, blood vessels) were not sigificantly different than control values.

Applicant's summary and conclusion

Conclusions:

The quantitative information obtained in this study confirms earlier findings (Sprando et al 1997), that sodium fluoride does not affect spermatogenesis in the rat up to a dose of 250 ppm.

Executive summary:

A quantitative examination of the testes of F1 generation males was made, following prolonged exposure to sodium fluoride in the drinking water at 0, 25, 100, 175 or 250ppm. The rats were exposed in utero, during lactation, and directly in their drinking water for 14 weeks after weaning. At the end of the exposure period testicular tissues were perfusion fixed and examined. There were no statistically significant differences between controls and treated rats in almost all the parameters evaluated. A significant decrease in the absolute volume and volume percent of the lymphatic endothelium was observed in the 175 and 250ppm groups, and in the testicular capsule in the 100ppm group. The authors report that the significance of this finding is not clear, and overall the results suggests that exposure to NaF does not adversely affect testis structure or spermatogenesis in the rat.